Download Vice President Global Coverage and Reimbursement Global Health Economics

Joshua Ofman, MD, MSHS
Vice President
Global Coverage and Reimbursement
Global Health Economics
Global Government Affairs
601 Thirteenth Street, NW
12th Floor
Washington, DC 20005
www.amgen.com
December 20, 2010
Maria Ellis
Executive Secretary for MEDCAC
Centers for Medicare & Medicaid Services
Office of Clinical Standards and Quality,
Coverage and Analysis Group
C1-09-06
7500 Security Boulevard
Baltimore, MD 21244
Re: Medicare Program; Meeting of MEDCAC, January 19, 2011, on Erythropoiesis
Stimulating Agents (ESA) in Anemia Related to Kidney Disease
Dear Ms Ellis:
Amgen Inc. (Amgen) is writing to comment on the topics to be addressed at the Medicare
Evidence Development and Coverage Advisory Committee (MEDCAC) January 19, 2011
meeting on Erythropoiesis Stimulating Agents (ESA) in anemia related to chronic kidney
disease (CKD), which the Centers for Medicare & Medicaid Services (CMS) published notice of
on October 26, 2010 on the website, http://www.cms.gov/mcd/viewtrackingsheet.asp?id=245.
ESAs are indicated for the treatment of anemia associated with chronic renal failure (CRF),
including patients on and not on dialysis and Amgen’s comments will be provided separately for
these two patient populations.
As a science-based, patient-care driven company, Amgen is committed to using science and
innovation to dramatically improve people’s lives and is vitally interested in improving access to
innovative drugs and biologicals for Medicare beneficiaries.
Attached, you will find our detailed written submission addressing the effects of ESAs on health
outcomes in adult CKD patients, both on dialysis and not on dialysis. In particular, we call your
attention to Appendix A, which specifically addresses the voting questions posted on the CMS
website on November 24, 2010, and Appendix C, which provides an independent review
commissioned by Amgen of the clinical literature specific to the effects of blood transfusions on
renal transplant graft survival.
By way of additional background, Amgen recently developed and submitted the following
materials containing important clinical information regarding the overall effects of ESAs on
health outcomes in adult CKD patients:
•
March 24, 2010 MEDCAC Meeting - Erythropoiesis Stimulating Agents (ESA) in Anemia
Related to Kidney Disease;
http://www.amgen.com/media/amgen_medcac_esa_meeting.html
•
October 18, 2010 US Food and Drug Administration’s Cardiovascular and Renal Drugs
Advisory Committee (CRDAC) meeting on the benefits and risks of ESAs in patients with
CKD, not on dialysis;
http://www.amgen.com/media/statement_on_briefing_docs_TREAT_review.html.
Amgen appreciates the opportunity to provide this important information and looks forward to
the opportunity to discuss the evidence for ESAs in patients with CKD at the upcoming
MEDCAC. If you have any questions or need additional information, please do not hesitate to
contact me.
Regards,
Joshua J. Ofman, MD, MSHS
Vice President,
Global Coverage and Reimbursement
and Global Health Economics
Page 3 of 290
MEDCAC Background Information
Page 1
BACKGROUND INFORMATION
FOR
THE MEDICARE EVIDENCE DEVELOPMENT AND COVERAGE ADVISORY
COMMITTEE
JANUARY 19, 2011
BRIEFING MATERIALS DESCRIBING CURRENTLY AVAILABLE
EVIDENCE ON THE USE OF ERYTHROPOIESIS STIMULATING AGENTS
(ESAs) TO MANAGE ANEMIA IN PATIENTS WHO HAVE CHRONIC
KIDNEY DISEASE (CKD)
Submitted: DECEMBER 20, 2010
Amgen Inc.
One Amgen Center Drive
Thousand Oaks‚ CA 91320-1799
Page 4 of 290
MEDCAC Background Information
Page 2
Table of Contents
1.
EXECUTIVE SUMMARY.......................................................................................... 7
2.
BACKGROUND AND INTRODUCTION ................................................................ 10
2.1
Patients with CKD Requiring Dialysis Differ Substantially From
Patients Who Do Not Require Dialysis ....................................................... 10
3.
ANEMIA IN DIALYSIS PATIENTS ......................................................................... 11
3.1
Anemia is Severe, Nearly Universal, Highly Symptomatic and
Associated with Significantly Decreased Physical Functioning,
Exercise Tolerance and Health Related Quality of Life in Dialysis
Patients....................................................................................................... 11
3.2
Severe Anemia Requires Therapeutic Intervention to Alleviate
the Substantial Symptoms .......................................................................... 12
3.3
Transfusions Are Only Transiently Effective In Dialysis Patients
Who Are Chronically Unable To Produce Sufficient Red Blood
Cells, And Transfusions Carry A Range Of Acute And Chronic
Risks ........................................................................................................... 12
3.3.1
Transfusions Are Recognized As Having Intrinsic
Risks .......................................................................................... 14
3.3.2
Transfusions Can Transmit Infectious Diseases ........................ 14
3.3.3
Transfusions Can Cause Volume Overload,
Hyperkalemia, Transfusion Reactions, And
Transfusion-Related Acute Lung Injury (TRALI) ........................ 15
3.3.4
Transfusions Can Cause Iron Overload ..................................... 16
3.3.5
Transfusions Can Result In Sensitization, Which
Compromises Transplant Eligibility And Graft Survival .............. 17
3.3.5.1
Transplant Is The Preferred Treatment
Strategy For End-Stage Renal Disease:
Delayed Transplantation Decreases Both
Patient And Graft Survival........................................ 17
3.3.5.2
Techniques In Characterizing Immunologic
Impediments To Successful
Transplantation ........................................................ 19
3.3.5.3
The Role Of Transfusions In The
Development Of Allosensitization ............................ 23
3.3.5.4
Impact Of Transfusions And
Allosensitization On Transplant
Opportunities And Outcomes ................................... 24
3.3.5.5
Donor-Specific Transfusions And
Transplant Outcomes Among Living Donor
Kidney Transplants .................................................. 27
3.3.5.6
Management Of Patients With High Levels
Of Donor-Specific Antibodies ................................... 27
MEDCAC Background Information
3.4
4.
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Page 3
ESA Therapy Reduces The Need For Recurrent RBC
Transfusions And Their Attendant Risks By Effectively Raising
And Maintaining Hemoglobin Concentrations And Improving
Physical Function, Exercise Tolerance And The Symptoms Of
Anemia ....................................................................................................... 28
3.4.1
ESA therapy reduces the need for transfusions when
used to raise Hb ≥ 10 g/dL and maintain it between
10-12 g/dL .................................................................................. 28
3.4.2
ESAs Reduce The Frequency And Impact Of
Transfusion Related Risks ......................................................... 30
3.4.3
ESAs Improve Physical Function and Exercise
Tolerance When Used to Raise Hb ≥ 10 g/dL and
Maintain it Within the Approximate Range of 10-12
g/dL ............................................................................................ 31
3.4.4
ESAs Should be Considered in the Context of their
Labeled Risks ............................................................................. 35
3.4.5
Substantial Evidence Supports the ESA Labeled
Hemoglobin Range of 10-12 g/dL in Dialysis Patients ............... 36
3.4.5.1
The Need for Transfusions Decreases
Substantially when Hb Concentrations are
Raised ≥ 10 g/dL and Maintained Within
the Range of Approximately 10-12 g/dL
with ESA Therapy .................................................... 36
3.4.5.2
A Two Gram Hb Range is Appropriate
when Titrating ESA Doses to Maintain Hb
Concentrations Above 10 g/dL................................. 38
3.4.5.3
ESAs Have a Broad Dose-Response and
There is no Evidence Supporting a Single
Maximum Dose ........................................................ 39
3.4.5.4
The Current ESA Label Provides
Conservative Dosing Guidance for
Patients Who Do Not Adequately Respond
to ESA Therapy........................................................ 39
3.4.6
There are No New Data in Dialysis Patients to Support
a Change in the Labeled Hemoglobin Range of 10-12
g/dL and Data from Clinical Trials and Over 20 years
of Clinical Experience Support this Range ................................. 40
3.4.7
The Prospective Payment System (PPS) will Impact
the Use of ESAs ......................................................................... 41
3.4.8
Conclusion: ESAs are an Essential Therapy for
Dialysis Patients ......................................................................... 42
ANEMIA MANAGEMENT IN CKD PATIENTS NOT ON DIALYSIS ....................... 43
4.1
CKD-NOD Patients are Heterogeneous with Respect to Kidney
Function, Prevalence and Severity of Anemia, and there is a
Sub-Population who May Require Anemia Management ........................... 43
4.2
Transfusions are Not Uncommon in CKD-NOD Patients with
Significant Anemia ...................................................................................... 44
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MEDCAC Background Information
4.3
4.4
4.5
4.6
Page 4
RBC Transfusions Carry Similar Risks in CKD-NOD and Dialysis
Patients....................................................................................................... 44
ESAs are an Effective Therapy for Managing Anemia in CKDNOD Patients.............................................................................................. 45
ESAs should be considered in light of their labeled risks ........................... 46
Conclusion: ESA Therapy in CKD-NOD Patients is an Important
Treatment Option in Those With Significant Anemia and for
Whom Transfusion Avoidance is a Meaningful Clinical Outcome .............. 46
5.
CONCLUSION ....................................................................................................... 47
6.
REFERENCE LIST................................................................................................. 48
Appendix A - CMS Questions and Amgen's Responses ..................................................60
Appendix B - Select References ......................................................................................90
Appendix C - Independent Review of the Clinical Literature ..........................................251
Appendix D - Guidelines for Blood Transfusion .............................................................287
List of Figures
Figure 1. Pattern of Transient Improvements in Hb Concentrations Following
Transfusions in a Dialysis Patient ............................................................ 13
Figure 2. Rate of Hospitalization or Emergency Room Evaluation for Heart
Failure Immediately Preceding and Following an Outpatient
Transfusion Event in Hemodialysis, CKD-NOD and Non-CKD
Medicare Patients Between 2003 and 2007. ........................................... 16
Figure 3. Adjusted Five-Year Survival Among Dialysis and Kidney Transplant
Patients70 ................................................................................................. 18
Figure 4. Long-Term Survival of Patients Receiving a Kidney Transplant
Before (Pre-Emptive) and After (Non Pre-Emptive) the
Initiation of Dialysis 51............................................................................... 18
Figure 5. Unadjusted Graft Survival in 21,836 Recipients of Living
Transplants by Length of Dialysis Treatment Before Transplant ............. 19
Figure 6. (a) Unadjusted Graft Survival in 56,587 Recipients of Cadaveric
Transplants by Length of Dialysis Treatment Before Transplant
(b) Unadjusted Graft Survival in 21,836 Recipients of Living
Transplants by Length of Dialysis Treatment Before Transplant ............. 19
Figure 7. Effect of Transfusion on Graft Survival ........................................................... 23
Figure 8. Relationship Between the Number of Transfusions and the Risk of
Allosensitization ....................................................................................... 25
Figure 9. (a) Long-term (10-year) graft survival of cadaver kidney transplants
according to pre-transplant allo-sensitization (measured as
PRA), and (b) 10-year follow-up of kidney grafts from HLAidentical sibling donors 61. ........................................................................ 26
MEDCAC Background Information
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Figure 10. Percent of Patients Receiving a Transfusion at Baseline and in
Weeks 1-12 and 13-24 for Patients Randomized to Epoetin
Alfa and Placebo Treatment 96. ................................................................ 29
Figure 11. Outpatient Transfusion Rate in US Dialysis Patients in Each
Quarter Over Time. .................................................................................. 30
Figure 12. The proportion of US patients between 1991 and 2007 (a) who
were transplanted and received previous transfusions, and (b)
who had a PRA=0% while on the transplant wait list71. ........................... 31
Figure 13. Improvements in Exercise Tolerance and Physical Function
Observed when Hemoglobin Levels were Increased with
Epoetin Alfa Compared to Placebo-Treated Patients. ............................. 32
Figure 14. Summary of Studies Examining Changes in Exercise Tolerance
(VO2peak) in Dialysis Patients following ESA treatment12 ........................ 33
Figure 15. Summary of Studies Examining Change in the Karnofsky
Performance Scale (KPS) following ESA treatment ................................ 33
Figure 16. Improvements in Fatigue Observed when Hemoglobin Levels
were Increased with Epoetin Alfa Compared to PlaceboTreated Patients 96, 102. ............................................................................. 34
Figure 17. Mortality rate and hemoglobin levels preceding and following the
introduction of EPOGEN® ........................................................................ 35
Figure 18. Transfusion Risk by the Previous Month’s Hemoglobin Level in
the Lower Hemoglobin Arm of the Normal Hematocrit Cardiac
Trial (NHCT)............................................................................................. 36
Figure 19. Hemoglobin Levels and Transfusion Rates between 1991 and
2007 ......................................................................................................... 37
Figure 20. Transfusion Rates by Number of Months with an Outpatient
Hemoglobin Below 10 g/dL and 11 g/dL; Medicare
Hemodialysis Patients, 2004.................................................................... 37
Figure 21. The Prevalence of Significant Anemia (Hb < 10 g/dL) According to
Level of Kidney Function (Estimated Glomerular Filtration Rate
[eGFR]; Lower eGFR Indicates More Severe Disease) ........................... 43
Figure 22. Annual Transfusion Rates in CKD-NOD Patients in the Absence
of ESA Therapy (2002-2007) ................................................................... 44
Figure 23. Hemoglobin Levels and Associated Kidney Disease
Questionnaire (KDQ) Scores for Physical Symptoms, Fatigue,
Depression, Relationship with Others, Frustration, and Overall
KDQ (Clinically Meaningful Change in KDQ is 0.5) 108, 139, 143,
144
. ............................................................................................................ 46
MEDCAC Background Information
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Page 6
List of Abbreviations
Abbreviation or Term
Definition/Explanation
AABB
American Association of Blood Banks
AMR
Antibody Mediated Rejection
CESG
Canadian Erythropoiesis Study Group
CKD
Chronic Kidney Disease
CMS
Centers for Medicare & Medicaid Services
CRDAC
Cardiovascular and Renal Drugs Advisory Committee
CV
Cardiovascular
DAC
(FDA) Drug Advisory Committee
DSA
Donor-Specific Antigen
EMP
Erythropoietin Monitoring Policy (National Claims Monitoring
Policy for ESAs in Hemodialysis Patients)
ESA
Erythropoiesis-Stimulating Agent
ESRD
End Stage Renal Disease
Hb
Hemoglobin
HLA
Human Leukocyte Antigens
KDQ
Kidney Disease Questionnaire
MEDCAC
Medicare Evidence Development and Coverage Advisory
Committee
NHCT
Normal Hematocrit Cardiac Trial
NOD
CKD-Not On Dialysis
PPS
Prospective Payment System
PRA
Panel Reactive Antibody
PRCA
Pure Red Cell Aplasia
PRO
Patient Reported Outcomes
QIP
Quality Incentive Program
RBC
Red Blood Cells
RCT
Randomized Controlled Trial
SD
Standard Deviation
SIP
Sickness Impact Profile
USPI
United States Prescribing Information
USRDS
United States Renal Data System
VA
Veteran’s Administration
MEDCAC Background Information
1.
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Page 7
EXECUTIVE SUMMARY
Amgen has prepared this document in response to the Medicare Evidence Development
and Coverage Advisory Committee (MEDCAC) meeting scheduled by the Centers for
Medicare & Medicaid Services (CMS) to review the currently available evidence
regarding the effects of Erythropoiesis Stimulating Agents (ESAs) on health outcomes in
adult patients with chronic kidney disease (CKD). In particular, CMS has asked the panel
to review the available evidence on the impact of ESAs on renal transplant graft survival
in patients who have CKD (pre-dialysis and dialysis) and anemia.
Patients with CKD requiring dialysis are distinct from those who do not require dialysis
(referred to as CKD-NOD patients) in that dialysis patients have significantly greater comorbidity, are hospitalized more frequently, have higher mortality rates, and require
more intensive clinical management. Additionally, the prevalence and severity of
anemia, due to insufficient production of erythropoietin by the kidney, is greater in
dialysis patients, and is compounded by the substantial blood loss associated with the
dialysis procedure. Anemia symptoms, which include fatigue, decreased energy,
reduced physical function, weakness, and cognitive impairment, can have a significant
adverse impact on the lives of CKD patients. This document separately reviews the
management of anemia in these two distinct populations, with a focus on the risks of
transfusions on transplant access and viability, and the appropriate use of ESAs to
manage anemia in order to reduce transfusions and improve anemia symptoms.
Anemia Management in CKD Patients on Dialysis
The severity of anemia in dialysis patients necessitates therapeutic intervention. Prior to
the development of ESAs, the primary treatment for anemia was RBC transfusions.
RBC transfusions provide only transient elevations in hemoglobin and have intrinsic
risks. ESAs have demonstrated effective management of anemia, thereby reducing the
need for transfusions and improving physical function, exercise tolerance, and
symptoms of anemia. Additionally, transfusions expose patients to numerous risks, such
as blood-borne diseases, iron overload, and allosensitization. Allosensitization is a
uniquely important transfusion-related risk for CKD patients as it can delay or preclude a
kidney transplant and shorten kidney graft survival. As such, transfusions are not
appropriate therapy for management of chronically anemic dialysis patients.
Kidney transplant is the preferred treatment modality for CKD patients with end-stage
renal disease (ESRD), as it removes the dependency on dialysis to sustain life. Patients
with kidney transplants have superior overall survival, better quality of life, and lower
MEDCAC Background Information
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Page 8
health resource utilization and cost as compared to patients receiving dialysis. The
earlier that a kidney transplant is performed, including prior to the initiation of dialysis,
the better the overall patient and kidney graft survival.
Allosensitization is the development of antibodies to foreign antigens, which can lead to
an immunologic attack on a transplanted organ. Allosensitization can be measured by
the panel reactive antibody (PRA) test, which reflects the percentage of the
representative organ donor pool to which the potential recipient has alloantibodies (i.e.,
allosensitized). Higher PRA levels reflect greater levels of sensitization and fewer
potential compatible organs. There are three principle ways in which patients can
become allosensitized: 1) blood transfusion, 2) prior organ transplantation, and 3)
pregnancy; of these, exposure to transfusions is the most readily modifiable factor.
Studies suggest that the risk of allosensitization from RBC transfusions is cumulative, a
particular concern for dialysis patients who are more likely to have been allosensitized
by previous transfusions or failed kidney transplants. It has been estimated that prior to
the availability of ESAs, patients on dialysis received, on average, 5-10 units of
transfused blood annually. A large body of evidence shows that increasing PRA levels
are associated with: 1) increasing wait time on the transplant waiting-list, 2) decreasing
likelihood of finding a compatible organ, and 3) if a patient receives a transplant, shorter
survival of the transplanted kidney.
While many advances have been made in the field of transplantation and various
strategies to reduce allosensitization have been and continue to be investigated,
allosensitization persists as a key obstacle to transplantation. In recognition of this,
transplant centers across the US recommend that patients on the transplant waiting-list
avoid transfusions, if at all possible. The risks related to RBC products is recognized by
the Circular of Information for the Use of Human Blood and Blood Components, which is
published jointly by the American Association of Blood Banks (AABB), American Red
Cross, America’s Blood Centers, and the Armed Services Blood Program, and states:
“Red cell containing components should not be used to treat anemias that can be
corrected with specific hematinic medications such as iron, vitamin B12, folic acid, or
erythropoietin.”
ESAs offer the clinically significant and unequivocal benefit of transfusion reduction, as
well as improvements in physical function and exercise tolerance, in patients receiving
dialysis. Transfusion avoidance protects against the acute risks of volume and
MEDCAC Background Information
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potassium overload, the cumulative risks of iron overload, and exposure to viral and
other infectious agents, as well as sensitization to foreign antigens
The benefits of ESA therapy have been demonstrated in registrational trials and in over
20 years of clinical practice. Since their introduction into the dialysis population, ESAs
have been an important therapeutic intervention for the effective management of anemia
and have led to substantial reductions in transfusions and significant improvements in
physical function, exercise tolerance, and anemia symptoms. Along with the decline in
transfusions, exposure to transfusion-related risks has also markedly declined, as
evidenced by the near-absence of iron overload and a doubling of the proportion of
patients on the transplant waiting-list who are not currently allosensitized. These
benefits were achieved with ESA therapy used to raise and maintain hemoglobin
concentrations above 10 g/dL and within the range of approximately 10-12 g/dL.
The totality of available evidence and the current FDA-approved label supports a Hb
range of 10-12 g/dL for ESA therapy in dialysis patients, which reduces transfusions and
improves physical function and exercise tolerance, while accommodating Hb variability
and avoiding target Hb levels of ≥ 13 g/dl where risks have been observed. There are no
new clinical data regarding the benefits of ESAs in dialysis to support changes in the
recommended hemoglobin range of 10-12 g/dL.
Anemia Management in CKD-NOD Patients
While the prevalence of anemia is lower in CKD-NOD patients, anemia can be severe in
some patients, particularly those nearing dialysis. Transfusions in these patients are not
uncommon, and CKD-NOD patients who receive transfusions are vulnerable to the
same risks as patients on dialysis, including the risk of allosensitization and its potential
impact on transplant eligibility and graft survival. This is particularly relevant for CKDNOD patients nearing dialysis since up to 15% of all transplants are performed before
dialysis initiation. ESAs are an important therapy for anemic CKD-NOD patients in
whom transfusion avoidance is a meaningful clinical goal. Based on recently completed
studies in CKD-NOD patients, Amgen has proposed label changes to limit the use of
ESAs in CKD-NOD patients to those with significant anemia, who are at high risk for
transfusion and in whom transfusion avoidance is clinically meaningful.
At the end of this review there are four appendices which contain the following:
Appendix A - CMS Questions and Amgen's Responses
Appendix B - Select References
MEDCAC Background Information
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Appendix C - Independent Review of the Clinical Literature
Appendix D - Guidelines for Blood Transfusion
2.
BACKGROUND AND INTRODUCTION
The approval of the first recombinant erythropoiesis-stimulating agent (ESA), Epoetin
alfa, in 1989 represented an important scientific breakthrough in medicine and
revolutionized the care of patients with anemia of chronic kidney disease (CKD). The
ability to produce erythropoietin (the hormone produced by the kidneys to stimulate red
blood cell [RBC] formation) is impaired in CKD patients and this impairment is the
primary cause of anemia in this disease. Amgen Inc. (Amgen) developed ESAs as
therapies to stimulate RBC production in order to elevate and maintain hemoglobin (Hb)
concentrations in patients with CKD and anemia, to avoid RBC transfusions and improve
the symptoms of anemia. In the US, Epoetin alfa is approved and marketed under the
trade names EPOGEN® by Amgen for the treatment of anemia in dialysis, and
PROCRIT® by Centocor Ortho Biotech Inc. for the treatment of anemia in CKD not on
dialysis (NOD). Darbepoetin alfa is marketed under the trade name Aranesp® by Amgen
for both CKD-NOD and dialysis. EPOGEN® and Aranesp® are approved for the
treatment of anemia associated with CKD, which includes patients receiving and not
receiving dialysis.
The Centers for Medicare & Medicaid Services (CMS) has called this meeting to discuss
the evidence available regarding the effects of ESAs on health outcomes in adult CKD
patients (pre-dialysis and dialysis). Specifically, CMS has asked the panel to review the
available evidence on the impact of transfusion on renal (kidney) transplant graft survival
in patients who have CKD with anemia. This briefing book discusses anemia in CKD in
patients on and not on dialysis, and reviews the management of anemia in these two
distinct populations, with a focus on the risks of transfusions on transplant access and
viability, and the appropriate use of ESAs to manage anemia in order to decrease
transfusions and improve anemia symptoms.
2.1
Patients with CKD Requiring Dialysis Differ Substantially From
Patients Who Do Not Require Dialysis
Patients with CKD requiring dialysis are distinct from those patients who do not require
dialysis (referred to as CKD-NOD patients) in that dialysis patients have significantly
greater co-morbidity, are hospitalized more frequently, have higher mortality rates, and
require more intensive clinical management 1. Importantly, these populations also differ
in the prevalence and severity of anemia 1. Anemia is severe and almost universal in
patients receiving dialysis, due to their lack of renal mass and relative erythropoietin
MEDCAC Background Information
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insufficiency for their level of anemia 2, 3. Compounding the anemia, patients receiving
dialysis are subject to continuous blood loss from the dialysis procedure 4. In dialysis
patients, if left untreated or undertreated, anemia can be severe and have a significant
effect on patients’ lives with symptoms including decreases in physical functioning, lower
energy, and cognitive impairment.
In contrast, the CKD-NOD patient population is heterogeneous with respect to the
degree of impairment of kidney function, presence of co-morbidities and severity of
anemia. The prevalence of significant anemia (Hb < 10 g/dL) is relatively low in patients
with early stage renal disease, but increases to approximately 20-30% in the subset of
patients approaching dialysis 5, and is more common among women and ethnic
minorities, especially African Americans 6. In the subset of CKD NOD patients with
advanced kidney disease and significant anemia, patients may be symptomatic and
treatment of anemia may be warranted.
3.
ANEMIA IN DIALYSIS PATIENTS
3.1
Anemia is Severe, Nearly Universal, Highly Symptomatic and
Associated with Significantly Decreased Physical Functioning,
Exercise Tolerance and Health Related Quality of Life in Dialysis
Patients
Dialysis patients experience severe anemia almost universally 2. In the 1980s, before
the development of ESAs, a typical dialysis patient had a mean Hb of approximately
7 g/dL 7, and often required chronic transfusions to maintain Hb concentrations at this
level. For reference, healthy adults have, on average, Hb concentrations in the range of
13-17 g/dL in men and 12-16 g/dL in women 8. In addition, there are sub-groups within
the dialysis population in whom anemia is more common and more severe (e.g., African
Americans) 6, 9.
The severity of anemia in dialysis patients is largely driven by the relative deficiency of
endogenous erythropoietin production, and is compounded by the blood loss during
each dialysis procedure along with a shortened RBC survival time 4. Dialysis patients
lose blood by way of the dialysis procedure itself, require frequent blood sampling to
monitor laboratory parameters, have an increased tendency for bleeding attributable to
anticoagulation therapy administered during dialysis,4 and have an increased risk of
gastrointestinal bleeding and re-bleeding, all of which contribute to substantial and
ongoing blood loss 10, 11. In total, blood loss is estimated to be between 2.5 and 5.1 L (510 Units) of blood annually per hemodialysis patient 4, roughly equivalent to the
circulating blood volume in a healthy adult.
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Anemia is associated with lethargy, weakness, shortness of breath, decreased physical
functioning and decreased exercise tolerance 12. These symptoms contribute to a poor
quality of life, and decreased productivity.
3.2
Severe Anemia Requires Therapeutic Intervention to Alleviate the
Substantial Symptoms
The severity of anemia in dialysis patients often necessitates therapeutic intervention. In
the 1980s, before the development of ESAs, the available treatment options for anemia
were limited to RBC transfusions, and to a smaller extent, androgen and iron therapy 2.
Androgens and iron therapy conferred only modest efficacy but had substantial side
effects 13, 14, leaving RBC transfusions as the mainstay of anemia therapy in the pre-ESA
era.
There are two approaches to the treatment of anemia: replacing blood through
transfusion of RBCs or stimulating RBC production, which can be accomplished through
correction of deficiencies such as iron, vitamin B12 or erythropoietin. Administration of
ESAs is used to treat the anemia of CKD, which is primarily caused by inadequate
production of erythropoietin because of damage to the kidney. Transfusions and ESAs
are distinct in their efficacy, characteristics and clinical profile, and they are not
interchangeable. Amgen is unaware of any RCTs which have evaluated the efficacy or
quality of life of transfusions as a randomized treatment compared to ESA therapy, for
the treatment of anemia in CKD patients. Today, nearly all dialysis patients who require
chronic anemia therapy receive ESAs and intravenous iron to support erythropoiesis.
Also, as there is a time lag between ESA administration and Hb response 15, ESAs are
unsuitable as the sole therapy for anemia requiring urgent treatment. Therefore,
transfusions are generally reserved for acute situations requiring immediate anemia
treatment, whereas ESAs are more appropriate for routine maintenance of Hb.
3.3
Transfusions Are Only Transiently Effective In Dialysis Patients Who
Are Chronically Unable To Produce Sufficient Red Blood Cells, And
Transfusions Carry A Range Of Acute And Chronic Risks
Transfusions are transiently effective and insufficient for maintaining elevated Hb
concentrations to alleviate the symptoms of anemia in the chronically anemic dialysis
patient 16. Figure 1 depicts a typical pattern of transient improvement in Hb
concentrations following transfusion in a dialysis patient, as well as the consequent
reliance upon repeated transfusions to elevate Hb concentrations 16.
The placebo arm of the Canadian Erythropoiesis Study Group (CESG) study 7 is
illustrative of the limitations of the transient nature of transfusion therapy for the
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MEDCAC Background Information
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management of anemia in dialysis patients. The placebo arm employed usual care,
including transfusions guided by normal medical practice. The resulting achieved Hb
concentration was between 7-8 g/dL in the placebo arms of this and other trials; in
contrast, the ESA arms of the CESG study had Hb concentrations between 10 and 12
g/dL. There was a demonstrable improvement in measurements of exercise tolerance,
physical functioning and other domains of Quality of Life in the ESA treated groups
compared to the group treated with placebo with normal transfusion practice.
Figure 1. Pattern of Transient Improvements in Hb Concentrations Following
Transfusions in a Dialysis Patient
45
14
Hct (%)
10
25
8
6
15
5
↑ ↑ ↑ ↑ ↑ ↑ ↑
Hb (g/dL)
12
35
4
Transfusions
4
8
12
16
Weeks
Furthermore, blood transfusions also constitute an additional burden for dialysis patients,
both from a time and resource perspective. Before a blood transfusion can be infused, a
blood sample is drawn to match with the blood intended for the transfusion. A
transfusion generally requires several hours to complete. Currently, most dialysis
centers do not have the capability of administering transfusions, thereby requiring the
patient to visit an outpatient transfusion center, or be referred for hospitalization.
Transfusions performed in non-dialysis outpatient infusion clinics necessitate venous
access and as such increase the possibility that patients’ large veins may be
compromised, thereby impairing opportunities for dialysis fistulas. This is a significant
issue as dialysis vascular access complications are the most common cause for
hospitalization 17. Lastly, while serious transfusion reactions are rare, non-hemolytic
febrile reactions may still occur and produce symptoms that negatively impact patients.
MEDCAC Background Information
3.3.1
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Page 14
Transfusions Are Recognized As Having Intrinsic Risks
Prior to the availability of ESAs, transfusions were chronically administered but used with
restraint given their known risks. These risks include transmission of blood-borne
diseases, transfusion reactions, acute volume and potassium overload, and more
chronically, iron overload and sensitization to foreign antigens 2, 18-21. Management of
many of these complications may require hospitalization, which can further add to the
risks, burden, and costs of transfusions 22, 23.
Almost all US states have laws recognizing transfused blood as intrinsically hazardous
24
. The FDA also recognizes that the risks of blood transfusions can never be
eliminated: “FDA is responsible for ensuring the safety of the Nation's blood supply.
While a blood supply with zero risk of transmitting infectious disease may not be
possible, there are several measures taken by FDA to protect and enhance the safety of
blood products” 25.
The Circular of Information for the Use of Human Blood and Blood Components is
prepared jointly by American Association of Blood Banks (AABB), the American Red
Cross, America’s Blood Centers, and the Armed Services Blood Program 26,and the
Food and Drug Administration (FDA) recognizes this Circular of Information as an
acceptable extension of container labels. This document provides under the
contraindication section the following statement:
Red-cell-containing components should not be used to treat anemias that can be
corrected with specific hematinic medications such as iron, vitamin B12, folic acid,
or erythropoietin. RBCs or Whole Blood should not be used solely for volume
expansion or to increase oncotic pressure of circulating blood.
Thus, even with the substantial improvements in blood safety, transfusions of blood
products are recognized as intrinsically dangerous, and thus are not to be used when
alternatives are available.
3.3.2
Transfusions Can Transmit Infectious Diseases
Over the past 20 years, the blood supply has become safer with regard to transmission
of infectious diseases including hepatitis B, hepatitis C, HIV, and other infections, due to
the implementation of sensitive donor-screening strategies. Nonetheless, blood-borne
pathogens including viral, protozoan, and parasitic diseases can still be transmitted by
transfusion 27, 28,18.
Known and emerging blood-borne pathogens are a significant concern in the blood
supply. The AABB’s Transfusion Transmitted Diseases Committee recently conducted a
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Page 15
review to identify actual or potential risks of current transfusion transmission. Sixty-eight
infectious agents were identified, and with few exceptions, these agents did not have
available interventions to reduce the risk of transmission 29. These agents and diseases
include Creutzfeldt-Jakob disease, Dengue viruses, Babesia, Cytomegliovirus (CMV),
HGV, malaria, leishmaniasis, Lyme disease, Human Herpesvirus 8 (HHV-8),
toxoplasmosis, and cryoglobulinemia 30. More recently, Xenotropic Murine Leukemia
Virus-Related Virus (XMRV), a virus linked to chronic fatigue syndrome, has been
identified as a possible agent which could be transmitted via blood transfusion. In 2010,
the AABB issued a recommendation that until further definitive data are available its
member blood collectors should discourage potential donors diagnosed with chronic
fatigue syndrome from blood donation 31. The effectiveness of these efforts is unknown.
Usually interventions are not implemented until it is clear that transfusion transmission
has occurred. Consequently, transfusions continue to expose patients to the risk of
blood-borne diseases, some known, some emerging, and, some as yet unidentified.
3.3.3
Transfusions Can Cause Volume Overload, Hyperkalemia,
Transfusion Reactions, And Transfusion-Related Acute Lung Injury
(TRALI)
In contrast to the general population, CKD patients have a diminished ability to excrete
fluid and electrolytes due to their impaired renal function 32, and thus are particularly
vulnerable to volume overload (heart failure exacerbation) and hyperkalemia 18-21. These
particular risks related to transfusions in CKD patients were identified over three
decades ago32. The frequency and impact of these transfusion-related risks were
examined in a recent analysis of US Medicare data; this analysis showed that in the
days immediately following an outpatient transfusion event, hospitalization or emergency
room visits with a diagnosis for heart failure occurred at a rate 4- to 7-fold higher than in
the days immediately preceding the transfusion event 33 (Figure 2). Similar results were
observed for hospitalizations for hyperkalemia. Thus, the CKD population demonstrates
particular susceptibilities to blood transfusion that may not be present in the general
population.
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Page 16
Heart Failure Rate per 100,000 Transfusions
Figure 2. Rate of Hospitalization or Emergency Room Evaluation for Heart Failure
Immediately Preceding and Following an Outpatient Transfusion Event in
Hemodialysis, CKD-NOD and Non-CKD Medicare Patients Between 2003 and 2007.
800
600
CKD
400
200
HD
Non-CKD
0
Day Prior Day After
Day 2
Day 3
Day 4
CKD = CKD-NOD, HD = Dialysis
Additional acute but rare consequences of transfusions include transfusion reactions and
TRALI, the latter of which can be severe or fatal 34.
3.3.4
Transfusions Can Cause Iron Overload
Iron overload can occur in transfusion-dependent patients since each unit of RBCs
contains approximately 200-250 mg of iron, which is ~100 times greater than what is
absorbed through daily dietary absorption in healthy adults 35. The body’s ability to
excrete iron is extremely limited and excess iron accumulates in several organs
including liver, spleen, heart, and vasculature. Iron accumulation in tissues can be
detected, quantified, and localized via magnetic resonance imaging (MRI) of affected
organs 35. The accumulation of iron in the tissues can cause damage or failure of
multiple organs 35, liver cirrhosis with its associated risk of hepatocellular carcinoma,
diabetes mellitus, and cardiac failure 36. In dialysis patients, studies performed before the
introduction of ESAs also suggest that iron overload may increase a patients’
susceptibility to bacterial infections 37-39. The risks of iron overload are well-described in
the literature and were known as early as the mid 1970s32, 40-43. Iron overload can occur
after approximately 20 units of transfused blood 44. To contextualize this burden of
transfusions, prior to the availability of ESAs, transfused dialysis patients were reported
to have received approximately 5-10 transfusions a year 7.
The frequency of iron overload in the CKD population has decreased since the
introduction of ESAs and the consequent reduction in the use of transfusions 14.
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Page 17
However, even today, chronic transfusion therapy for the treatment of anemia can still
result in iron overload. Iron overload is a significant clinical problem in other diseases
that require chronic transfusion therapy, such as myelodysplastic syndrome (MDS),
sickle cell anemia, and thalassemia major. In these patients, transfusion-related iron is
associated with substantial morbidity and mortality 45-48. Given these findings and the
data from the pre-ESA era, the evidence suggests that iron overload would once again
be a significant problem if transfusions become more prevalent.
3.3.5
Transfusions Can Result In Sensitization, Which Compromises
Transplant Eligibility And Graft Survival
For CKD patients, potentially the most important transfusion-related risk is the
development of sensitization to foreign antigens, which can increase time spent on the
transplant waiting-list or even preclude transplant eligibility, and for patients who receive
a transplant, can shorten graft survival 49.
3.3.5.1
Transplant Is The Preferred Treatment Strategy For End-Stage Renal
Disease: Delayed Transplantation Decreases Both Patient And Graft
Survival
Kidney transplant is the preferred CKD treatment modality as it removes the dependency
on dialysis to sustain life, and effectively replaces kidney function in most cases.
Patients with kidney transplants have superior overall survival (Figure 3), better quality of
life, and have significantly lower health resource utilization and cost as compared to
patients receiving dialysis50.
Delays in transplantation decrease both patient and allograft survival. As compared to
patients who receive a transplant following initiation of dialysis, patients who receive a
transplant prior to initiating dialysis (pre-emptive transplant) have a 52% reduction in the
risk of allograft failure during the first year after transplant (p = 0.002) and a more than
80% reduction yearly 51. Similarly, a study examining the fate of paired donor kidneys
(i.e., two kidneys from the same deceased donor) demonstrated a decreased 10-year
graft survival of kidneys transplanted into patients who had received 24 months of
dialysis as compared to patients who had received 0-6 months of dialysis prior to
transplant (Figure 4) 52. These authors also reported a similar relationship between
duration of dialysis and overall graft survival for both cadaveric and living donor
transplants (Figures 5 and 6). Because the length of time on dialysis prejudices against
both patient and graft survival, the preference is to perform the transplant as early as
possible, including before the initiation of dialysis.
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Figure 3. Adjusted Five-Year Survival Among Dialysis and Kidney Transplant
Patients70
Survival Probability
100
80
60
40
Dialysis
20
Transplant
0
1
2
3
4
5
Years
Proportion of Allografts Surviving
Figure 4. Long-Term Survival of Patients Receiving a Kidney Transplant Before
(Pre-Emptive) and After (Non Pre-Emptive) the Initiation of Dialysis 51.
1.00
Preemptive
0.75
Nonpreemptive
0.50
0.00
No. at Risk
Preemptive
Nonpreemptive
0
365
730
1095
1460
Allograft Survival (days)
1819
6662
1778
6430
1336
4519
877
2543
271
786
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Figure 5. Unadjusted Graft Survival in 21,836 Recipients of Living Transplants by
Length of Dialysis Treatment Before Transplant
100
% event free survival
90
0–6
80
70
>2
60
4m
on
m on
th s
50
78%
th s
on d
ialy
sis
on
58%
dia
63%
lys
is
40
29%
30
20
0
12
24
36 48 60 72 84
months post-transplant
96 108 120
Figure 6. (a) Unadjusted Graft Survival in 56,587 Recipients of Cadaveric
Transplants by Length of Dialysis Treatment Before Transplant (b) Unadjusted
Graft Survival in 21,836 Recipients of Living Transplants by Length of Dialysis
Treatment Before Transplant
b.
100
100
90
90
80
preemtive
70
60
50
0-6 months
40
6-12 months
12-24 months
24+ months
30
20
0
3.3.5.2
12 24 36 48 60 72 84 96 108 120
months post-transplant
% event free survival
% event free survival
a.
80
preemtive
70
0-6 months
60
6-12 months
12-24 months
24+ months
50
40
30
20
0
12 24 36 48 60 72 84 96 108 120
months post-transplant
Techniques In Characterizing Immunologic Impediments To
Successful Transplantation
With the exception of transplants between identical twins, transplantation poses the risk
of immunological attack on the transplanted organ that may lead to immediate, shortterm, or long-term loss of the transplant through both cell-mediated and antibodymediated rejection 53. Given that the presence of recipient antibodies directed against
donor organs negatively affects the outcome of transplantation 54, antibody mediated
rejection is the focus of discussion in this document.
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There are specific immunological tests that are traditionally performed prior to
transplantation: ABO blood type testing, Human Leukocyte Antigen (HLA) typing, and
Panel Reactive Antibody (PRA) evaluation. Transplants are generally not performed
across ABO blood type boundaries, although there are some experimental approaches
to transplantation across this barrier 55. Greater degrees of HLA mismatching generally
correlate with worse transplant outcomes and affect waiting list priority for organ
allocation 56. However, even in the presence of high degrees of HLA type mismatch,
renal transplants may proceed, and thus HLA matching is not determinative.
The PRA test has traditionally been used to inform the likelihood of a positive crossmatch and to appropriately manage organ allocation, and is described in greater detail in
subsequent sections. Patients with elevated PRA are considered to be sensitized and
sensitized patients may experience:
•
Increased wait time for a suitable donor kidney
•
Inability to ever find a suitable donor kidney
•
Greater chance of both acute rejection and of long-term organ loss
The importance of the PRA and HLA matching notwithstanding, the ultimate test that
determines whether a transplant for an individual candidate can proceed is the
crossmatch test. The crossmatch, which is performed between the potential recipient
and the donor cells at the time of transplant, is a specific determination of whether the
kidney transplant recipient has antibodies directed towards the specific donor’s cells. If
the crossmatch test is positive at time of transplant, there is a very high probability of
acute antibody mediated rejection57 and will generally preclude transplant of the organ
(other than in special and investigational circumstances). Thus, the presence of a
negative crossmatch is the determining factor in deciding whether to perform a
transplant between a donor and recipient pair. A more detailed discussion of
crossmatching is provided in Section 3.3.5.4.
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It is important to note that even in the presence of a negative crossmatch, recipients with
a high PRA, indicating the presence of broad allosensitization, are at greater risk for
early acute antibody-mediated rejection and have worse short- and long-term
outcomes 58, 59. The precise mechanism for this is unknown, but is hypothesized to be
related to both low-level pre-existing antibodies that are not detected by crossmatch, the
occurrence of post-transplant sensitization 60, and the possibility that non-HLA directed
antibodies may promote late graft loss 61.
Historical Development of Immunological Matching Techniques
In 1964, Terasaki, et al demonstrated that the presence of recipient antibodies directed
toward organ donor tissue strongly predicted immediate transplant failure 57. This finding
was the basis of the pre-transplant crossmatch test, which is still used today. When a
potential donor organ is identified, the prospective recipient’s serum is mixed with the
donor’s cells to determine if the recipient has antibodies against the donor tissue. If a
recipient has antibodies which react against the donor cells, known as a positive
crossmatch, the probability of rejection is high, and the transplant will generally not
proceed. The performance of the crossmatch remains the gold standard of organ
compatibility.
In a seminal advancement in 1969, Patel and Terasaki demonstrated that when a
patient’s serum contained antibodies to a broad range of donor cells, randomly derived
from different individuals in the general population, the patient had a much higher
probability of experiencing acute organ rejection following transplantation 62. This was
the basis for the panel reactive antibody test, which measures a patient’s level of
sensitization to a representative pool of donor HLA antigens. The PRA, expressed as a
percentage, reflects the percentage of the likely organ donor pool for which the potential
recipient has alloantibodies (is allosensitized). For example, a PRA of 70% suggests
that 70% of donors will likely be unacceptable for the tested patient due to the presence
of anti-HLA antibodies against donor antigens. Thus, the higher the percent PRA, the
more ‘allosensitized’ a patient is to the general donor pool, and the more difficult to find a
suitable donor. Allosensitization is often categorized as a PRA of 0%, indicating no
sensitization; <10% or < 20%, indicating a low level of sensitization; < 79% indicating a
moderate degree of sensitization; and ≥ 80% indicating a high-degree of sensitization.
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Evolving Techniques in Determining Allosensitization
While the role of antibodies in hyper-acute rejection has been known for decades 57, it
was not until the introduction of specific and sensitive anti-HLA antibody testing
techniques that the role of antibody-mediated rejection in short and long-term graft
outcome was fully recognized 54, 63, 64. In recent years, solid phase assays have been
introduced that improve upon the cell-based assays for HLA antibody screening and
have refined our understanding of sensitization 63, 65. These assays are more sensitive
than previous methods in detecting HLA antibodies, and fall into two categories, ELISAbased and HLA antigen-coated beads used in either a Flow Cytometry system or a
Luminex platform. The Luminex platform-based assay is now the standard methodology
for assessing sensitization and determining organ allocation in the US 63.
These tests allow the determination of a calculated PRA (cPRA). The cPRA is based
upon HLA antigens to which the patient has been sensitized and which, if present in a
donor kidney, would represent an unacceptable risk of rejection; these are referred to as
unacceptable antigens. The cPRA is computed from HLA antigen frequencies among
approximately 12,000 kidney donors in the United States between 2003 and 2005 and
thus represents the percentage of actual organ donors that express one or more of
those unacceptable HLA antigens 64. Since 2009, transplant waiting list patients’ cPRA
and their specific unacceptable antigens are required to be reported to UNOS in an effort
to increase the chances that an organ offered to a highly sensitized patient, who has
likely spent considerable time on the transplant waiting list, will prove to be crossmatch
negative.
It is important to clarify what these newer tests accomplish:
•
They may more closely approximate the results of the pre-transplant cross-match
•
They improve the efficiency of organ allocation
•
They reduce the chances that a highly sensitized patient will be offered an organ
that will be found to have a positive crossmatch
It is also important to clarify what the newer tests do not accomplish:
•
They do not alter the likelihood that a suitable donor will be found
•
They do not alter the length of time required to find a donor
•
They do not alter the fact that highly sensitized patients may never find a suitable
organ
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MEDCAC Background Information
•
Page 23
They do not alter the fact that even if an organ is found, higher PRAs predict a
higher risk of both acute rejection and long-term organ loss
3.3.5.3
The Role Of Transfusions In The Development Of Allosensitization
There are three principle ways in which patients can become allosensitized: blood
transfusion, a prior organ transplantation, and pregnancy 66, 67 . Of these, exposure to
transfusions is the most readily modifiable factor. The role of transfusion in sensitization
has been described since the 1960s, with the initial description of the PRA test 62.
Analyses of the relationship between prior transfusions and transplant survival revealed
that pre-transplant transfusions are detrimental to graft survival (Figure 7) 68.
Figure 7. Effect of Transfusion on Graft Survival
100
100
Percent Graft Survival
PRA 0–10%
PRA >10%
90
90
80
70
60
80
Number of
transfusions
N
0
18,086
1–5
8,126
6–10
993
>10
577
0
1
Number of
N
transfusions
3,816
0
2,871
1–5
427
6–10
333
>10
70
2
3
60
0
1
2
3
Years Posttransplant
Following the demonstration of the negative effects of transfusion on transplant outcome
68
, numerous studies have evaluated the impact of transfusions on the development of
allosensitization 69, 70 and the impact of allosensitization on the time on transplant
waiting-list, ability to receive a transplant, and transplant graft survival 3, 61, 71.
Studies suggest that anti-HLA antibodies can be observed after one episode of
transfusion 72, 73. In a study of over 600 non-sensitized female patients receiving a pretransplant transfusion, 21% became sensitized 69. Data from approximately 70,000
transplanted patients show that the patients who receive transfusions have higher risk of
sensitization 3 and the risk of allosensitization from RBC transfusion is cumulative, as the
number of previous transfusions increases, so does the risk of elevated PRA levels 74
(Figure 8a). The likelihood of sensitization following transfusion is greatest among
multiparous females and previous transplant patients 3, 68, 71, 75. Each transfusion
introduces the possibility of sensitization to more HLA-antigen subtypes, thereby
reducing the pool of suitable donor organs for the patient 3, 68, 75. Importantly, since
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dialysis patients are more likely to have been allosensitized by previous transfusions 68,
69, 71
, or previous failed kidney transplants
67
, they are at particular risk for an increase in
allosensitization following transfusion.
Leukoreduction in Transfusions
A proposed mitigation strategy to reduce the sensitizing effect of transfused blood is
leukoreduction, a process that removes leukocytes from the donor blood prior to
transfusion. In the oncology population, where patients receiving chemotherapy also
receive multiple transfusions, the development of HLA-antibodies may reduce the
effectiveness of platelet transfusions. Leukoreduction of transfused platelets has been
shown to successfully reduce the relative risk of developing anti-platelet antibodies by
approximately 50% 76, thereby increasing the effectiveness of platelet transfusions.
However, transfusing leukoreduced blood has not been shown to mitigate the risk of
developing HLA antibodies in the context of renal transplantation 53, 77. Karpinski, et al 78
have shown that after a Canadian province uniformly adopted leukoreduction, there was
no decrease in the level of allosensitization following RBC transfusion among kidney
transplant candidates. Consequently, the authors concluded: “...transfusions continue to
be an important cause of allosensitization for potential kidney transplant recipients.” The
precise reason for the differential impact of leukocyte reduction in reducing
allosensitization associated with transfusions in the oncology setting, but not in the renal
setting, is not clear. One hypothesis that has been proposed is that there is a difference
in the level of immune suppression in patients with cancer receiving chemotherapy (as in
the TRAP study76) in comparison to transplant-eligible CKD patients 53. Additionally,
studies in the surgical setting have shown that HLA sensitization persists after
transfusion of leukoreduced blood 79, 80.
3.3.5.4
Impact Of Transfusions And Allosensitization On Transplant
Opportunities And Outcomes
The relationship between PRA levels and transplant outcomes has been extensively
described in large population studies and in review articles and textbooks of
transplantation medicine 3, 61, 68. The presence and degree of allosensitization can
adversely impact transplant outcomes in multiple ways, including the availability of fewer
suitable organs, increased wait time for transplant, inability to receive a transplant, and
shortened graft survival over the short and long term 3, 61, 68.
USRDS data indicate that moderately to highly sensitized patients have the longest
median wait times for a kidney transplant (Figure 8b), and the longer wait times are
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MEDCAC Background Information
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associated with greater likelihood of death (Figure 8c). For patients who are highly
sensitized (PRA > 80%), there is no median time to transplant as the majority are never
transplanted 81. This is even more pronounced among African Americans who have a
higher likelihood of being sensitized by transfusions and a lower probability of finding a
suitable matching organ 71, 74.
Figure 8. Relationship Between the Number of Transfusions and the
Risk of Allosensitization
(a)
ref
10
Number of Previous Transfusions
(c)
PRA
Likelihood of dying
while awaiting transplant
8
6
Probability
Median Wait Time in Years
Odds Ratio
Relationship between
transfusion and PRA
(b)
4
2
Years of Listing
Years of Listing
(a) Relationship between the number of transfusions and the risk of allosensitization (measured as panel
reactive antibody [PRA] > 50%; PRA measures anti-human antibodies in the blood) (b) The median time
spent on the transplant waiting-list by the level of allosensitization (measured as PRA < 10% versus > 10%)
(c) The likelihood of dying while waiting for transplant 50, 74.
The longer a patient remains on the transplant waiting-list, the higher the likelihood the
patient will receive additional transfusions, further exposing the patient to the risk of
increasing levels of sensitization. Recent estimates suggest that more than 30% of
patients on the transplant waiting list receive one or more blood transfusions within 3
years of being listed 71. A recent analysis of USRDS data suggests that blood
transfusion is associated with a 28% reduction in the likelihood of receiving a transplant
in up to 8 years of follow up 71. This is important as the longer a patient is on a
transplant waiting list the higher the likelihood the patient will die prior to receiving a
transplant (Figure 8c). Additionally, as discussed previously, kidney allograft survival
has been shown to be substantially shorter the longer a patient remains on dialysis
before transplantation 51. The disadvantages of long wait times have a significant impact
on the lives of dialysis patients.
A key concept regarding allosensitization in kidney transplantation is that once a suitable
organ is found, (i.e., an organ with a negative crossmatch) the presence of pretransplant allosensitization can still compromise the overall function and longevity of the
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transplanted kidney 61, 82. A recent study of over 100,000 transplants showed that
increasing levels of sensitization are associated with significantly shorter graft survival (3
years shorter on average for highly sensitized compared to non-sensitized patients) 68
(Figure 9a). Similar findings were also observed among HLA-identical sibling
transplants (Figure 9b) 61, which is notable since there is a reduced intrinsic
immunologic barrier to transplantation between HLA-identical siblings. This relationship
is further supported by a recent study in nearly 70,000 kidney transplant recipients which
shows that increasing levels of sensitization are associated with significantly higher risks
of allograft failure and death 3. In addition to HLA antigens, there may be other non-HLA
immunologic factors that explain these findings 61. The relationship between graft failure
and higher levels of PRA as determined with older assays, has been shown in numerous
studies to be consistent with data using the current solid phase techniques for detecting
antibodies 82, 83.
Figure 9. (a) Long-term (10-year) graft survival of cadaver kidney transplants
according to pre-transplant allo-sensitization (measured as PRA), and (b) 10-year
follow-up of kidney grafts from HLA-identical sibling donors 61.
(b)
100
100
90
90
Grafts surviving (%)
Grafts surviving (%)
(a)
80
70
60
50
p<0·0001
No PRA
40
0
1–50% PRA
>50% PRA
0
2
4
6
Time (years)
8
10
Number of transplants
116562 83720 62516 44887 30819 20674
No PRA
1–50% PRA 36314 25005 18402 12842 8590 5586
>50% PRA
7610 4712 3582 2579 1817 1242
80
No PRA
70
1–50% PRA
60
p<0·0001
>50% PRA
50
40
0
0
2
4
6
8
Time (years)
Number of transplants
No PRA
3001 2495 1929 1418
1–50% PRA 803
647 514 362
>50% PRA
244
192 149
111
989
249
84
10
687
158
65
Of the three ways in which allosensitization occurs, transfusions are the most easily
avoided. Thus, the principle of transfusion avoidance is widely accepted and is
integrated into pre-transplant management protocols at most transplant centers
(Appendix B).
MEDCAC Background Information
3.3.5.5
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Donor-Specific Transfusions And Transplant Outcomes Among
Living Donor Kidney Transplants
Donor-specific transfusions (DSTs) were proposed as a method of inducing immune
tolerance based on experimental considerations 84, and first reported as potentially
beneficial by Salvatierra in 1980 85. DSTs are a therapy consisting of transfusions of 13 units of blood from the donor who is offering the kidney. These transfusions are
administered to the recipient prior to the kidney transplant, with the intent of inducing
immune tolerance to the donor kidney and improving the long-term kidney allograft
survival 85. DSTs, which can only be employed in the context of living donor kidneys, are
distinctly different from therapeutic transfusions used for the treatment of anemia;
therapeutic blood transfusions expose the recipient to blood from many unselected
donors for the purpose of treating anemia. For context, in the pre-ESA era, dialysis
patients received on average 5-10 units of transfused blood annually, and in the
process may have been exposed to the blood of a large number of donors in the course
of receiving multiple therapeutic transfusions 7.
There is evidence supporting superior graft survival among living donor transplants in
which DSTs were employed 86-90. However, it has also been reported that contrary to the
intended purpose of inducing tolerance, up to 30% of prospective transplant recipients
administered DSTs develop antibodies to the donor organ, thus precluding the organ
transplant 91. While the patients who are allosensitized by DSTs remain eligible for other
donor transplantation, living or deceased, the sensitization induced by the DST can
compromise subsequent outcomes. Because of uncertainty about whether a DST will
induce tolerance and improve graft outcomes, or induce sensitization which can
preclude a living donor transplant and reduce the transplant options for the patient, DST
is no longer widely advised 69, 92, and has been abandoned by most transplant centers 53.
3.3.5.6
Management Of Patients With High Levels Of Donor-Specific
Antibodies
For many highly allosensitized transplant candidates, an acceptable donor is never
identified and the patient remains on dialysis indefinitely 55. In an attempt to offer the
possibility of a transplant, protocols are being developed to permit transplants in highly
sensitized patients with a positive crossmatch against a prospective living donor
kidney 55. These protocols aim to acutely lower donor-specific antigen (DSA) activity to
below the level that causes immediate renal allograft injury, and to maintain this reduced
level during the first weeks to months after transplantation. The most commonly used of
these experimental approaches involves the use of pre- and post-transplant
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immunosuppression, including intravenous immunoglobulin, with or without
plasmapheresis, or the use of anti-CD20 monoclonal antibodies 93. However, these
approaches are resource intensive 94, do not eliminate the risk of sensitization, are not
FDA approved for this indication, and some are still considered experimental. A number
of newer immunological reagents are under investigation for this application 55.
With the exception of intravenous immunoglobulin (IVIG), desensitization protocols
remain an experimental approach and do not reduce donor specific antibodies to a level
allowing transplantation in all patients. Among patients whose antibody levels have
been reduced sufficiently to allow a transplant to be performed, acute antibodymediated rejection (AMR) still occurs in 20 to 50% of transplants despite
desensitization 55. Thus high levels of sensitization continue to persist as a key obstacle
to transplantation 82.
3.4
ESA Therapy Reduces The Need For Recurrent RBC Transfusions
And Their Attendant Risks By Effectively Raising And Maintaining
Hemoglobin Concentrations And Improving Physical Function,
Exercise Tolerance And The Symptoms Of Anemia
Given the substantial risks associated with transfusions, the approval of ESAs markedly
improved the management of anemia in CKD patients receiving dialysis. ESAs are
approved for raising and maintaining Hb concentrations and reducing the need for
transfusions. The Epoetin alfa label95 also describes improvements in physical
functioning and exercise tolerance, and the literature supports other findings regarding
the improvement in symptoms of anemia with ESA therapy. The replacement of chronic
transfusions with ESA therapy aligns with recent recommendations on the use of RBC
components by the AABB and the American Red Cross which state26:
“Red-cell-containing components should not be used to
treat anemias that can be corrected with specific hematinic
medications such as iron, vitamin B12, folic acid, or
erythropoietin.”
3.4.1
ESA therapy reduces the need for transfusions when used to raise
Hb ≥ 10 g/dL and maintain it between 10-12 g/dL
The primary registration trials used for the approval of Epoetin alfa demonstrated
improvement of anemia and the virtual elimination of transfusions (> 90% reduction) in
patients treated with ESAs to a hematocrit target of 35% ± 3% (Hb of 11.7 ± 1 g/dL) 96.
While the Epoetin alfa treated patients became nearly transfusion independent, placebo
treated patients remained severely anemic and continued to receive multiple
transfusions. Once patients randomized to placebo were crossed over to receive Epoetin
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alfa, they experienced a reduction in transfusions similar to that seen in the patients
initially randomized to Epoetin alfa treatment 96 (Figure 10).
Figure 10. Percent of Patients Receiving a Transfusion at Baseline and in Weeks
1-12 and 13-24 for Patients Randomized to Epoetin Alfa and Placebo Treatment 96.
100%
Percent (SE) of Subjects
Receiving Transfusions
80%
Placebo
Epoetin alfa
Placebo Δ Epoetin alfa
72%
58%
63%
60%
Epoetin alfa
from Week 13-24
40%
17%*
17%
20%
0%
0%*
Baseline
Weeks 1-12
Weeks 13-24
N = 32 (placebo); N = 36 (Epoetin alfa); *p < 0.05 placebo vs Epoetin alfa
Baseline rates are based on the 6 months before the start of the study.
Placebo Δ Epoetin alfa group: Transfusion requirements for subjects originally randomized to receive
placebo in Study 8701 who began to receive Epoetin alfa after week 12.
Almost immediately following the introduction of EPOGEN® into clinical practice in 1989,
the outpatient transfusion rate among US hemodialysis patients fell sharply. This is
shown in an analysis of USRDS data, which provides near-complete monitoring of all
dialysis patients including medications, biochemical parameters, hospitalization events
and deaths (Figure 11).
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Patients Transfused Per Quarter (%)
Figure 11. Outpatient Transfusion Rate in US Dialysis Patients in Each Quarter
Over Time.
Use of ESAs Introduced
20
16
12
8
4
0
78
80
82
84
86
88
90
92
94
96
Year
Footnote: Only pre-1996 outpatient transfusion data are represented as method for transfusion data
collection changed after 1996.
Adapted from USRDS ADR 2010
By 1992 almost 90% of US dialysis patients received ESA therapy and this treatment
prevalence of > 90% continues today 97. Between 1991 and 2000, the mean population
Hb increased from ~9.8 g/dL to ~11.2 g/dL and the total transfusion rate (inpatient plus
outpatient) was significantly reduced 98.
3.4.2
ESAs Reduce The Frequency And Impact Of Transfusion Related
Risks
The availability of ESAs and the ability to raise Hb concentrations above 10 g/dL and
maintain them within the range of approximately10 to 12 g/dL, has significantly reduced
the frequency of transfusions, resulting in reduced patient exposure to transfusionrelated risks. For example, with regard to allosensitization, USRDS data show that
between 1991 and 2008, the population mean Hb concentration increased, the
proportion of transplanted patients who had a prior transfusion decreased from 49% to
15%, and the proportion of patients with no sensitization (PRA levels = 0%) increased
from 24% to nearly 50% (Figures 12a and 12b).
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Figure 12. The proportion of US patients between 1991 and 2007 (a) who were
transplanted and received previous transfusions, and (b) who had a PRA=0%
while on the transplant wait list71.
Pre-transplant transfusion
80.0
80.0
70.0
70.0
60.0
12.00
11.00
10.00
50.0
40.0
30.0
20.0
9.00
8.00
3.4.3
10.0
0.0
199 2 199 4 199 6 199 8 200 0 200 2 200 4 200 6 200 8
PRA = 0% Among Transplant
Waitlist Patients
13.00
Mean Hb concentration
Pre-transplant transfusion (%)
Mean Hemoglobin (g/dL)
14.00
60.0
50.0
40.0
30.0
20.0
10.0
0.0
199 2 199 4 199 6 199 8 200 0 200 2 200 4 200 6 200 8
ESAs Improve Physical Function and Exercise Tolerance When
Used to Raise Hb ≥ 10 g/dL and Maintain it Within the Approximate
Range of 10-12 g/dL
Statistically significant and clinically meaningful improvements in exercise tolerance and
physical functioning were observed in registrational trials of dialysis patients receiving
Epoetin alfa compared to placebo, and in numerous single arm and observational
studies. In one registrational study, after 6 months of Epoetin alfa treatment, mean Hb
increased from 7.1 g/dL to greater than 10 g/dL and improvements in exercise tolerance
were demonstrated by objective measurements of time and distance walked using the
modified Naughton Treadmill Test99, 100 and the six-minute walk test (Figure 13). In the
same study, patient-reported physical functioning improved by approximately 61% using
the Sickness Impact Profile (SIP) physical function scale, and 40% using the Kidney
Disease Questionnaire (KDQ) Physical Symptoms domain 100.
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Figure 13. Improvements in Exercise Tolerance and Physical Function Observed
when Hemoglobin Levels were Increased with Epoetin Alfa Compared to PlaceboTreated Patients.
Minutes Walked
Placebo (n = 40)
Sickness Impact Profile (SIP)
Physical Functioning
Epoetin alfa (n = 78)
Hb Level (g/dL)
7.2 7.1
7.0 10.7
7.4 11.0
7.4 11.0
17.0
16.9
17.2
15
10
11.9
13.4
12.8
12.7
13.2
5
0
Baseline
2
4
6
5.0
Mean (SE) Improvement
From Baseline
Minutes Walked
20
4.0
3.9
3.0
2.8
2.9
2.0
1.0
0.0
1.1
2
1.1
4
Months
Months
P < 0.001 placebo vs Epoetin alfa
Epoetin alfa (n = 78)
Placebo (n = 40)
P < 0.001 placebo vs Epoetin alfa
Additional evidence supporting the labeled PRO benefits of improvement in exercise
tolerance and physical function comes from a recent systematic review and metaanalysis 12. Findings from this meta-analysis show a 23.8% increase in VO2peak (a
measure of exercise tolerance) and 10.5% increase in Karnofsky Performance Score
(functional outcomes) following initiation of erythropoietin therapy in dialysis patients
(Figure 14; Figure 15). A more recent systematic review and meta-analysis of
randomized controlled trial data related to the SF-36 short-form also found statistically
significant improvement in physical function domains 101.
0.7
6
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Figure 14. Summary of Studies Examining Changes in Exercise Tolerance
(VO2peak) in Dialysis Patients following ESA treatment12
Box reflects mean change and whiskers represent 95% CI
Figure 15. Summary of Studies Examining Change in the Karnofsky Performance
Scale (KPS) following ESA treatment
Box reflects mean change and whiskers represent 95% CI
In addition to improvements in exercise tolerance and physical function which are
described in the FDA-approved label, statistically significant and clinically meaningful
improvements in other anemia symptoms including fatigue, depression, energy, and
weakness were observed in registrational studies comparing Epoetin alfa to placebo.
After 6 months of ESA treatment patient-reported fatigue improved by 24% (Figure 16),
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depression improved by 10.4 %, energy improved by 37.1%, and weakness improved by
55.9% when using the KDQ 96, 102.
Figure 16. Improvements in Fatigue Observed when Hemoglobin Levels were
Increased with Epoetin Alfa Compared to Placebo-Treated Patients 96, 102.
% Improvement from Baseline
Kidney Disease Questionairre (KDQ)
Fatigue
30.0%
Placebo (n=40)
Epoetin alfa (n=78)
25.0%
20.0%
23.8%
21.4%
23.8%
15.0%
10.0%
5.0%
0.0%
4.4%
4.4%
2
4
Month
0.0%
6
P<0.0001 placebo versus Epoetin alfa (repeated measures mixed model)
There were significantly greater improvements in energy in patients treated with Epoetin
alfa compared to placebo, in a registrational RCT, as measured by the National Kidney
Dialysis Kidney Transplantation Symptom Checklist (NKDKTS) Energy item (p = 0.006)
and a single item Energy PRO (p < 0.001). A recent systematic review of patientreported fatigue demonstrated that raising Hb above 10 g/dL to within the 10-12 g/dL
range yielded an average fatigue improvement of 29% 96.
A recent meta-analysis of randomized control trial data related to the SF-36 also found
statistically significant improvements in mental health and social function domains when
Hb concentrations are raised and maintained in the 10-12 g/dL range using ESAs 101.
Also measured by the SF-36, cognitive function has been found to be significantly
improved by using ESA treatment 103.
Statistically significant improvements were observed in patient-reported health status,
with more than a 50% increase in the number of subjects rating their health as “Good” or
“Excellent”, and more than a 30% increase in health satisfaction using the WHO QOL
questionnaire, when average Hb concentrations increased to above 10 g/dL and were
maintained to within the 10-12 g/dL range 104. Similar findings were observed in two
additional randomized, placebo-controlled registrational studies, which showed
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statistically significant and clinically meaningful improvements in patient-reported health
status 96.
3.4.4
ESAs Should be Considered in the Context of their Labeled Risks
Safety considerations associated with ESAs have been discussed extensively at the
September 2007 CRDAC meeting and at the October 2010 CRDAC meeting.
Hypertension and thrombotic events are known adverse reactions of ESAs. Additional
but rare risks include hypersensitivity reactions and pure red cell aplasia (PRCA). An
increased risk of cardiovascular (CV) events and mortality was observed in clinical trials
that used ESAs to target Hb concentrations ≥ 13 g/dL (above the labeled Hb range) 105107
. Based on the results of these trials, the United States Prescribing Information
(USPI) 95 boxed warning describes that in clinical trials patients experienced greater
risks for death, serious cardiovascular events, and stroke when administered ESAs to
target Hb levels of 13 g/dL or above. In addition to clinical trial data, surveillance data on
nearly all US hemodialysis patients is available through the United States Renal Data
System (USRDS). These data show that since the introduction of ESAs into clinical
practice where Hb levels are lower, the overall mortality rate in the dialysis population
has not increased (Figure 17).
Figure 17. Mortality rate and hemoglobin levels preceding and following the
introduction of EPOGEN®
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3.4.5
Page 36
Substantial Evidence Supports the ESA Labeled Hemoglobin Range
of 10-12 g/dL in Dialysis Patients
There is a large body of data from registrational trials and 20 years of clinical experience
regarding the use of ESAs and their clinical benefits in dialysis patients. This evidence
is summarized in the following sections.
3.4.5.1
The Need for Transfusions Decreases Substantially when Hb
Concentrations are Raised ≥ 10 g/dL and Maintained Within the
Range of Approximately 10-12 g/dL with ESA Therapy
Evidence from RCTs and general clinical practice strongly supports the need to raise
and maintain Hb concentrations above 10 g/dL and within the range of 10-12 g/dL to
reduce the need for transfusions 50, 96, 108. A post-hoc analysis of RCT data in the dialysis
population calculating the likelihood (relative risk) of transfusion in the month following
Hb measurement indicates that relative to a Hb of 10-11 g/dL, transfusion risk doubles if
the Hb is between 9-10 g/dL and doubles again when the Hb is less than 9 g/dL
(Figure 18)96.
Figure 18. Transfusion Risk by the Previous Month’s Hemoglobin Level in the
Lower Hemoglobin Arm of the Normal Hematocrit Cardiac Trial (NHCT)
8
7
6
The Risk of Transfusion by the Previous Month's Hemoglobin Level
5
Hazard Ratio (95% CI)
4
3
2.5
2
1.5
1.2
1
0.8
0.6
0.4
<9
9 - < 10
10 - < 11
11 - < 12
>=12
Hemoglobin level (g/dL)
Data Source: NHCT study, data on file
Likewise, in the clinical setting, the transfusion rate has declined substantially in the
dialysis population as the proportion of patients with a Hb ≥ 10 g/dL has increased. A
plateau was seen when the mean population Hb concentration reached 11.2 g/dL 71
(Figure 19).
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Figure 19. Hemoglobin Levels and Transfusion Rates between 1991 and 2007
Hb
Transfusion
15.0
12.5
14.0
12.0
13.0
11.5
12.0
11.0
11.0
10.5
10.0
10.0
9.0
9.5
8.0
9.0
7.0
1991
1993
1995
1997
1999
2001
2003
2005
Proportion Transfused (%)
Mean Hemoglobin (g/dL)
13.0
2007
The likelihood of transfusion also increases the longer the patient's Hb remains low 109.
An analysis of approximately 160,000 Medicare hemodialysis patients shows that the
transfusion rate increases substantially the longer Hb concentrations remain below 10
g/dL during consecutive months (Figure 20).110 These analyses emphasize the
cumulative likelihood of transfusions when Hb concentrations remain below 10 g/dL.
Figure 20. Transfusion Rates by Number of Months with an Outpatient
Hemoglobin Below 10 g/dL and 11 g/dL; Medicare Hemodialysis Patients, 2004.
Thus, the benefits in ESA therapy, when used to raise Hb concentrations above 10 g/dL
to within the 10-12 g/dL range, have been demonstrated in numerous clinical trials and
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through surveillance of nearly the entire dialysis population (approximately 350,000
patients in 2008) 71. The importance of maintaining Hb concentrations above 10 g/dL
and in the range of approximately 10-12 g/dL is recognized by the nephrology
community, as well as CMS, and is currently incorporated as a quality metric by which
dialysis units are evaluated.
3.4.5.2
A Two Gram Hb Range is Appropriate when Titrating ESA Doses to
Maintain Hb Concentrations Above 10 g/dL
Variations in Hb concentrations within individuals over time (intra-subject variability)111
are inherent. In dialysis patients, Hb variability is common and is further complicated by
the frequent hospitalization events, inflammation, and other co-morbidities that patients
experience, and the receipt of multiple interventions 111, 112. Additionally, there is a lag
between ESA dosing and Hb changes.15 Therefore, it is extremely difficult to maintain
Hb concentrations within a narrow range in most patients.111 In clinical practice, the
population mean intra-patient Hb standard deviation (SD) is approximately 1.0 g/dL 113,
therefore, at any given point in time a substantial fraction of patients will not be within a 2
g/dL range. Both the original registrational trials and the current FDA-approved ESA
labels refer to a 2 g/dL Hb range of approximately 10-12 g/dL. This range is consistent
with:
i.
the recognized need to maintain Hb levels above 10 g/dL to avoid transfusion
ii.
the inherent variability in patient Hb levels and the time lag between ESA
administration and Hb response; and
iii.
avoidance of targeting Hb levels of ≥ 13 g/dL which has been associated with
risk
The influence of Hb variability on the management of anemia in dialysis patients is well
recognized by CMS and has been incorporated into its National Claims Monitoring Policy
for ESAs in hemodialysis patients (EMP) 114 . The EMP recognizes that in administering
ESAs to achieve and maintain the Hb 10-12 g/dL Hb range, its reimbursement policy
should account for this inherent variability 115. The importance of this range is further
reinforced by the established Hb concentration levels used in the proposed Quality
Incentive Program (QIP) under the Prospective Payment System (PPS) (10 and 12 g/dL)
116
. Thus, the Hb range of 10-12 g/dL enables physicians to effectively manage Hb
concentrations with ESA therapy according to patient needs in order to avoid
unnecessary RBC transfusions and improve physical function and exercise tolerance.
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3.4.5.3
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ESAs Have a Broad Dose-Response and There is no Evidence
Supporting a Single Maximum Dose
ESA therapy has been shown to be effective at raising and maintaining Hb
concentrations across a wide range of doses. In the original EPOGEN® registrational
program, phase II studies demonstrated a Hb response across all dosage levels 96. In
the subsequent phase III studies, a 40-fold range in the dose was required to raise and
maintain Hb concentrations within the target range of 10.7 to 12.7 g/dL 117. These
results along with evidence from current practice highlight the marked variability in ESA
doses required to raise and maintain Hb concentrations at a given level across individual
patients.
There is a wide range of doses needed to raise and maintain Hb concentrations within
the CKD population, and the preponderance of available evidence does not support an
increased risk of adverse events with higher doses. Initially, higher ESA doses were
associated with increased CV and mortality risk 118, 119, however, it is now more widely
understood that these observations were largely attributable to patient characteristics,
worsening clinical status, and poor Hb response rather than ESA dose 120-122. Patients
requiring the highest ESA doses are those most likely to have a greater CV disease
burden, have more inflammation and malnutrition, be hospitalized more frequently, be
poorly responsive to ESA therapy, and have low Hb levels 121, 123. The correlation of
these prognostic factors with higher ESA doses can produce significant confounding 124.
Numerous analyses using multiple, different analytical techniques to address the
confounding have found that higher ESA dose is not associated with an increased risk of
mortality 120, 124-127.
3.4.5.4
The Current ESA Label Provides Conservative Dosing Guidance for
Patients Who Do Not Adequately Respond to ESA Therapy
The management of anemia in patients with CKD who respond poorly to ESA therapy
remains a clinically relevant question. ESA hyporesponsiveness has been associated
with poor clinical outcomes 122, however, no clinical trials have explicitly examined the
question of whether the morbidity experienced by ESA hyporesponsive patients is a
result of the ESA or the hyporesponsive state itself. Thus, as a prudent and cautious
measure, the ESA USPIs were updated in 2007 to provide conservative dosing guidance
for the management of patients with CKD with poor response to ESA therapy. This
guidance was added limiting the number of ESA dose increases to no more than three
for patients who do not attain a Hb level within the range of 10-12 g/dL despite the use of
appropriate ESA dose titrations over a 12-week period 95.
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3.4.6
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There are No New Data in Dialysis Patients to Support a Change in
the Labeled Hemoglobin Range of 10-12 g/dL and Data from Clinical
Trials and Over 20 years of Clinical Experience Support this Range
The benefits of ESA therapy, when used to manage anemia to a Hb range of 10-12 g/dL,
have been demonstrated in numerous clinical trials and in clinical practice. RCTs
targeting hemoglobin levels ≥ 13 g/dL 105, 106, 128, first reported in 1998, have shown
increased CV and mortality risk, and therefore, the USPI reflects these risks and
recommends not targeting these Hb levels. The most recent of these studies was
completed in 2009 and was conducted in CKD-NOD patients and does not inform the
benefits of ESA therapy in dialysis patients.
The long-standing approach to anemia management, consistent with the USPI (treating
to a Hb of 10-12 g/dL), has resulted in a marked decrease in transfusions, which are
now reserved for the acute treatment of anemia (Figure 11). Treating to a lower Hb
target would almost certainly increase the proportion of patients with Hb < 10 g/dL, which
is known to be associated with a greater severity of anemia symptoms and a greater
need for RBC transfusions. Since the intended benefit of ESA therapy includes
transfusion avoidance, there is no rationale for lowering the target and accepting a
higher transfusion rate.
ESA therapy has been discussed in great depth at recent FDA and CMS advisory
committee meetings. These meetings examined the benefits and risks of ESA therapy
focusing on the appropriate therapeutic Hb range and patient populations. Select
highlights include:
•
The September 2007 Cardiovascular and Renal Drugs Advisory Committee
(CRDAC) panel reviewed the totality of available evidence from RCTs and 20
years of clinical practice data. Following the meeting, the USPI was revised and
one of the important revisions included the re-establishment of the recommended
therapeutic Hb range of 10-12 g/dL.
•
At the March 2010 MEDCAC meeting, the panel was asked to review a set of
questions regarding the use of ESAs. The last of these questions asked if the
CKD patient’s status (requiring dialysis or not requiring dialysis) would impact the
assessment of risk and benefits related to ESA use. The panel acknowledged
the significant differences between dialysis and CKD-NOD patients, and thus,
decided to review the available evidence for the two populations separately.
MEDCAC Background Information
•
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Most recently, the October 2010 CRDAC panel reviewed the results of a large
RCT of moderately anemic diabetic patients with moderate CKD-NOD, which
demonstrated an increased risk of stroke with ESA treatment to a Hb target of 13
g/dL. In light of this recently completed study, the committee re-examined the
benefits and risks of ESAs in CKD patients, on and not on dialysis. As this was
the most recent review of the totality of evidence on the benefits and risks of
ESAs, the specific questions and results of the panel votes from this meeting are
provided below 129. Following the CRDAC panel meeting, discussions with FDA
are ongoing.
3.4.7
The Prospective Payment System (PPS) will Impact the Use of ESAs
Current ESA use has been effective at treating anemia and dramatically reducing the
need for transfusions. However, forthcoming policy changes have the potential for
reducing ESA use in the dialysis population. The new Medicare ESRD PPS is the most
significant change to the way ESRD services are reimbursed since Congress mandated
coverage of dialysis services in 1972, and was the result of considerable research and
discussion within the nephrology community. The projected impact of the ESRD PPS
removes incentives for overutilization of services and promotes increases in efficiencies
in the management of patients.
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The concern has been raised that the inclusion of ESAs in the PPS may result in
undesirable decreases in Hb concentrations as a result of inadequate ESA dosing, and
may lead to an increase in blood transfusions, particularly among subpopulations with
greater ESA dose requirements, such as African Americans 130, 131 and patients who
exhibit poor Hb response 122. CMS recognized this concern specifically:
“While the inclusion of any item or dialysis service in the payment bundle
provides an incentive for dialysis facilities to maximize profits by skimping
on the provision of that item or service, we point out that an important part
of our Quality Incentive Program (QIP) is the monitoring of hemoglobin
levels among dialysis patients to ensure that target levels are met, and
that anemia management does not deteriorate under the ESRD PPS”
(section II.M. of the final PPS rule).
The QIP specifically penalizes providers who allow Hb concentrations to remain below
10 g/dL or above 12 g/dL, which is aligned with the Hb range in the FDA approved ESA
labels. Additionally, CMS intends to monitor the incidence of transfusions and ensure
that effective anemia management with ESAs is not replaced with transfusions.
The registrational trials and the use of ESAs over the past 20 years in clinical practice
have demonstrated a marked reduction in transfusions when ESAs are used to a
therapeutic Hb range of 10-12 g/dL. Using ESAs to lower therapeutic Hb ranges would
reduce the seminal benefit of ESAs.
3.4.8
Conclusion: ESAs are an Essential Therapy for Dialysis Patients
ESAs are an essential therapy in the management of anemia in dialysis patients.
Evidence from RCTs, as well as observational data, has strongly supported that
maintaining Hb levels above 10 g/dL and within the range of approximately 10-12 g/dL
with ESAs is essential for reducing transfusions and improving physical function and
exercise tolerance. Additionally, by reducing exposure to transfusions, ESA therapy
reduces the acute and long-term risks associated with transfusions, including volume,
iron and electrolyte overload, infectious complications, and allosensitization. The use of
ESAs to reduce transfusions is aligned with the FDA-approved label for red blood cell
products 26. Avoidance of transfusions and consequent sensitization is a particularly
relevant issue for patients who are, or may ever become, transplant candidates, in order
to preserve the opportunity for successful transplantation and remove the dependency
on dialysis. Treating to a Hb range of 10-12 g/dL with ESAs has been reviewed and is a
reasonable therapeutic range for treatment of anemia and reduction of transfusion, while
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avoiding the high Hb targets where risks have been observed. There are no new data
that alter the established benefits of ESAs in dialysis patients or support changes to the
therapeutic Hb range of 10-12 g/dL for dialysis patients. Reducing the Hb target range
would increase the rate of transfusion and expose patients to numerous transfusionrelated risks including allosensitization. There are no off-setting benefits to this strategy.
4.
ANEMIA MANAGEMENT IN CKD PATIENTS NOT ON DIALYSIS
4.1
CKD-NOD Patients are Heterogeneous with Respect to Kidney
Function, Prevalence and Severity of Anemia, and there is a SubPopulation who May Require Anemia Management
Unlike patients on dialysis, who by definition have end-stage renal disease, and are
almost universally anemic and require ESA treatment to maintain Hb levels, the
population of patients with CKD not receiving dialysis is heterogeneous in terms of renal
impairment and the prevalence and severity of anemia 6. The prevalence of significant
anemia (Hb < 10 g/dL) is low in the early stages of renal disease and increases to
approximately 20-30% in the small sub-population of patients in the later stages of the
disease (Figure 21) 5. It is for this small population that treatment with ESA therapy can
be beneficial.
Figure 21. The Prevalence of Significant Anemia (Hb < 10 g/dL) According to
Level of Kidney Function (Estimated Glomerular Filtration Rate [eGFR]; Lower
eGFR Indicates More Severe Disease)
100
Patients with anemia (%)
90
80
70
60
50
40
30
20
10
0
> 60
30-<60 15-<30
eGFR (mL/min/1.73m2)
<15
MEDCAC Background Information
4.2
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Transfusions are Not Uncommon in CKD-NOD Patients with
Significant Anemia
Among Medicare patients with CKD-NOD and anemia, the annual transfusion rate is
approximately 20-25%, three-fold higher than in CKD-NOD patients without anemia, and
10-fold higher than in those without CKD17. In 2004, there were approximately 400,000
anemic CKD-NOD patients covered by Medicare and an estimated 60,000-100,000
transfusion events annually 17. An analysis of approximately 83,000 CKD-NOD patients
with anemia in the Veteran’s Administration (VA) healthcare system shows a similar
transfusion burden in patients not receiving ESA therapy. In this population, the annual
transfusion rate ranges from 20-40% depending on the severity of anemia (Figure 22),
with a 1-year transfusion rate substantially higher in patients with a baseline Hb < 10
g/dL compared to Hb ≥ 10 g/dL 132.
Figure 22. Annual Transfusion Rates in CKD-NOD Patients in the Absence of ESA
Therapy (2002-2007)
4.3
RBC Transfusions Carry Similar Risks in CKD-NOD and Dialysis
Patients
In the CKD-NOD patient population, RBC transfusions carry similar risks to those in
dialysis patients. The risks include transmission of viral disease, transfusion reactions,
as well as transfusion-related complications such as acute volume overload and
hyperkalemia; the volume and potassium-related risks occur at lower frequency than in
dialysis patients 2, 18-21. Most important to the long-term outcome of these patients, RBC
transfusions result in sensitization to foreign antigens 58, 71 that can be enduring and
delay or preclude future kidney transplantation50 and impact overall graft survival in
transplanted patients 61. Successfully transplanted patients have superior survival, and
quality of life and incur lower health care costs 133. Therefore, avoidance of transfusion
to maintain transplant eligibility is of critical importance in CKD-NOD patients, as the
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need for a transplant may occur prior to, or following, dialysis initiation 50. Currently,
15% of transplants occur in CKD-NOD patients near to but before initiation of dialysis 50.
USRDS projects that ~700,000 patients will be receiving dialysis or be transplanted by
the year 2020 50. The largest group progressing to dialysis is projected to be 45-64 yearolds, all of whom will become Medicare beneficiaries upon progressing to ESRD and
many will be candidates for renal transplantation.
4.4
ESAs are an Effective Therapy for Managing Anemia in CKD-NOD
Patients
Data from multiple trials and clinical practice have demonstrated that ESAs raise and
maintain Hb concentrations in anemic CKD-NOD patients 106, 107, 134, 135. In the original
registrational studies with EPOGEN® in dialysis patients, transfusions were avoided
when Hb concentrations were raised above 10 g/dL 117. Transfusion reduction was
demonstrated in an open-label single-arm study of anemic CKD-NOD patients 135, where
transfusion events were reduced by ~65% when Hb concentrations were raised above
10 g/dL and maintained to within the range of 10-12 g/dL. Transfusion reduction with
ESA therapy has also been observed in general clinical practice in CKD-NOD patients.
In the Medicare population with CKD-NOD and anemia, transfusion rates have declined
as the proportion of patients receiving ESA treatment has increased 17.
While not an FDA labeled benefit, a number of studies in CKD-NOD patients have
shown varying degrees of improvement in PROs with ESA therapy 107, 136-138. Studies
which initiated treatment at Hb < 10 g/dL and treated to targets > 10 g/dL have
demonstrated more improvement in PROs than studies in which treatment was initiated
when Hb > 10 g/dL 96. Several studies have shown that as Hb concentrations rise above
10 g/dL, CKD-NOD patients experience clinically meaningful improvement in physical
function and vitality scores, as measured by various PRO instruments 139, 140 (Figure 23).
Similar improvements have been demonstrated in scales measuring fatigue and activity
levels 137, 141, 142.
Page 48 of 290
MEDCAC Background Information
Page 46
4.5
6.0
5.8
5.6
5.4
5.2
5.0
4.8
4.6
4.4
4.2
4.0
3.8
3.6
3.4
3.2
3.0
Spearman Correlation Coefficients
r = 0.46; P < 0.0001
Physical Symptoms
r = 0.42; P < 0.0001
Fatigue
r = 0.24; P < 0.0001
Depression
Relationship with others r = 0.29; P < 0.0001
r = 0.21; P < 0.0001
Frustration
r = 0.38; P < 0.0001
Overall KDQ
35
30
25
20
15
Overall KDQ Score
KDQ Score
Figure 23. Hemoglobin Levels and Associated Kidney Disease Questionnaire
(KDQ) Scores for Physical Symptoms, Fatigue, Depression, Relationship with
Others, Frustration, and Overall KDQ (Clinically Meaningful Change in KDQ is 0.5)
108, 139, 143, 144
.
10
5
7
8
9
10
11
12
Hemoglobin level (g/100 mL)
13
14
0
ESAs should be considered in light of their labeled risks
The USPI describes risks of ESAs and prescriber warnings. See section 3.4.4 for a
description of these risks 95, 145.
4.6
Conclusion: ESA Therapy in CKD-NOD Patients is an Important
Treatment Option in Those With Significant Anemia and for Whom
Transfusion Avoidance is a Meaningful Clinical Outcome
The prevalence of anemia requiring treatment is lower in CKD-NOD patients compared
to that in dialysis patients. However, in some CKD-NOD patients, particularly those at
the low end of the GFR spectrum nearing dialysis or being considered for pre-emptive
transplant, anemia can be severe and transfusions are not uncommon. Anemic CKDNOD patients who receive transfusions are vulnerable to the same risks of transfusions,
particularly the risk of sensitization, and its potential impact on transplant eligibility and
graft survival, as patients on dialysis. Based on recently completed studies in CKD-NOD
patients, Amgen has proposed label changes to limit the use of ESAs in CKD-NOD
patients to those with significant anemia, who are at high risk for transfusion, and in
whom transfusion avoidance is clinically meaningful.
MEDCAC Background Information
5.
Page 49 of 290
Page 47
CONCLUSION
There are significant differences in the clinical characteristics of CKD patients requiring
dialysis compared to CKD patients who do not. Dialysis patients, by definition, are at the
end stage of renal disease and are reliant on the dialysis procedure to sustain life.
Additionally, dialysis patients are almost universally anemic, with low Hb levels attributed
to insufficient endogenous erythropoietin levels and ongoing blood loss which has been
estimated to be 2.5-5 L annually, roughly equivalent to the blood volume of a normal
adult. Anemia carries a significant burden causing symptoms such as fatigue,
decreased energy, reduced physical function, and cognitive impairment, all of which can
be severe and life-altering. Before ESA therapy was available, the treatment options for
severe anemia were primarily limited to RBC transfusions. Dialysis patients were
dependent on chronic transfusions and their quality of life remained poor. Transfusions
were, and remain, only transiently effective in raising Hb concentrations in dialysis
patients who are chronically unable to produce sufficient RBCs, and they have
significant acute and long-term risks. The acute risks include volume overload,
hyperkalemia, and transfusion reactions, and the long-term risks include iron overload,
transmission of infectious diseases, and allosensitization to foreign antigens.
Allosensitization, the development of antibodies to foreign antigens, is an important risk
of transfusions for the CKD patient as it negatively impacts the likelihood and success of
kidney transplantation, which is the optimal therapy for patients with end-stage renal
disease. Allosensitization increases wait-time for a kidney transplant, reduces the
likelihood of receiving a transplant, and increases the risk of acute graft rejection and
long-term graft loss for those who do receive a transplant. The benefits of ESA therapy,
when used to raise and maintain Hb concentrations above 10 g/dL to within a
therapeutic range of approximately10-12 g/dL, have been demonstrated in registrational
trials and in over 20 years of clinical practice. With the availability of ESAs, Hb
concentrations increased, anemia symptoms improved, and transfusions decreased
dramatically. With the decrease in transfusions, patients have had less exposure to
transfusion-related risks and the proportion of patients on the renal transplant waiting list
who have no sensitization has more than doubled. The totality of available evidence
supports the current Hb range of 10-12 g/dL for ESA therapy in dialysis patients, which
reduces transfusions and improves anemia symptoms, while accommodating Hb
variability.
In some CKD-NOD patients, particularly those at the low end of the GFR spectrum
nearing dialysis or being considered for pre-emptive transplant, anemia can be severe
MEDCAC Background Information
Page 50 of 290
Page 48
and transfusions are not uncommon. Anemic CKD-NOD patients who receive
transfusions are vulnerable to the same risks of transfusions, particularly the risk of
sensitization, and its potential impact on transplant eligibility and graft survival, as
patients on dialysis. ESAs are an important therapy for this subgroup of significantly
anemic CKD-NOD patients at high risk for transfusion and in whom transfusion
avoidance is a meaningful clinical goal.
6.
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Cotter DJ, Stefanik K, Zhang Y, et al. Hematocrit was not validated as a
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119.
Zhang Y, Thamer M, Stefanik K, et al. Epoetin requirements predict mortality in
hemodialysis patients. Am J Kidney Dis. 2004;44(5):866-76.
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Bradbury BD, Danese MD, Gleeson M, Critchlow CW. Effect of epoetin alfa dose
changes on hemoglobin and mortality in hemodialysis patients with hemoglobin
levels persistently below 11 g/dL. Clin J Am Soc Nephrol 2009;4(3):630-7.
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Bradbury BD, Wang O, Critchlow CW, et al. Exploring relative mortality and
epoetin alfa dose among hemodialysis patients. Am J Kidney Dis. 2008;51(1):6270.
122.
Kilpatrick RD, Critchlow CW, Fishbane S, et al. Greater epoetin alfa
responsiveness is associated with improved survival in hemodialysis patients.
Clin J Am Soc Nephrol. 2008;3(4):1077-83.
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Solid CA, Foley RN, Gilbertson DT, Collins AJ. Perihospitalization hemoglobinepoetin associations in U.S. hemodialysis patients, 1998 to 2003. Hemodial Int
2007;11(4):442-7.
124.
Bradbury BD, Brookhart MA, Winkelmayer WC, et al. Evolving statistical methods
to facilitate evaluation of the causal association between erythropoiesisstimulating agent dose and mortality in nonexperimental research: strengths and
limitations. Am J Kidney Dis 2009;54(3):554-60.
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Bradbury BD, Do TP, Winkelmayer WC, et al. Greater Epoetin alfa (EPO) doses
and short-term mortality risk among hemodialysis patients with hemoglobin levels
less than 11 g/dL. Pharmacoepidemiol Drug Saf 2009;18(10):932-40.
126.
Wang O, Kilpatrick RD, Critchlow CW, et al. Relationship between Epoetin alfa
dose and mortality: findings from a marginal structural model. Clin J Am Soc
Nephrol 2010;In press.
127.
Zhang Y, Thamer M, Cotter D, et al. Estimated effect of epoetin dosage on
survival among elderly hemodialysis patients in the United States. Clin J Am Soc
Nephrol 2009;4(3):638-44.
MEDCAC Background Information
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128.
Pfeffer MA, Burdmann EA, Chen CY, et al. A Trial of Darbepoetin Alfa in Type 2
Diabetes and Chronic Kidney Disease. N Engl J Med 2009;30:30.
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FDA. Summary of the minutes of the Cardiovascular and renal Drugs Advisory
Committee www.fda.gov/downloads/AdvisoryCommittees/.../Drugs/.../UCM236323.pdf.
2010.
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Ishani A, Guo H, Arneson TJ, et al. Possible effects of the new Medicare
reimbursement policy on African Americans with ESRD. J Am Soc Nephrol
2009;20(7):1607-13.
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Lacson E, Jr., Wang W, Lazarus JM, Hakim RM. Hemodialysis facility-based
quality-of-care indicators and facility-specific patient outcomes. Am J Kidney Dis
2009;54(3):490-7.
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Lawler EV, Gagnon DR, Fink J, et al. Initiation of anaemia management in
patients with chronic kidney disease not on dialysis in the Veterans Health
Administration. Nephrol Dial Transplant 2010;25(7):2237-44.
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Jofre R, Lopez-Gomez JM, Moreno F, et al. Changes in quality of life after renal
transplantation. Am J Kidney Dis. 1998;32(1):93-100.
134.
Nissenson AR, Berns JS, Sakiewicz P, et al. Clinical evaluation of heme iron
polypeptide: sustaining a response to rHuEPO in hemodialysis patients. Am J
Kidney Dis. 2003;42(2):325-30.
135.
Provenzano R, Garcia-Mayol L, Suchinda P, et al. Once-weekly epoetin alfa for
treating the anemia of chronic kidney disease. Clin Nephrol. 2004;61(6):392-405.
136.
Rossert J, Levin A, Roger SD, et al. Effect of early correction of anemia on the
progression of CKD. Am J Kidney Dis. 2006;47(5):738-50.
137.
Alexander M, Kewalramani R, Agodoa I, Globe D. Association of anemia
correction with health related quality of life in patients not on dialysis. Curr Med
Res Opin 2007;23(12):2997-3008.
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Gandra SR, Finkelstein FO, Bennett AV, et al. Impact of erythropoiesisstimulating agents on energy and physical function in nondialysis CKD patients
with anemia: a systematic review. Am J Kidney Dis 2009;55(3):519-34.
139.
Lefebvre P, Vekeman F, Sarokhan B, et al. Relationship between hemoglobin
level and quality of life in anemic patients with chronic kidney disease receiving
epoetin alfa. Curr Med Res Opin 2006;22(10):1929-37.
140.
Revicki DA, Brown RE, Feeny DH, et al. Health-related quality of life associated
with recombinant human erythropoietin therapy for predialysis chronic renal
disease patients. Am J Kidney Dis 1995;25(4):548-54.
141.
Benz R, Schmidt R, Kelly K, Wolfson M. Epoetin alfa once every 2 weeks is
effective for initiation of treatment of anemia of chronic kidney disease. Clin J Am
Soc Nephrol. 2007;2(2):215-21. .
142.
Kleinman KS, Schweitzer SU, Perdue ST, et al. The use of recombinant human
erythropoietin in the correction of anemia in predialysis patients and its effect on
renal function: a double-blind, placebo-controlled trial. Am J Kidney Dis.
1989;14(6):486-95.
143.
Laupacis A. Changes in quality of life and functional capacity in hemodialysis
patients treated with recombinant human erythropoietin. The Canadian
Erythropoietin Study Group. Semin Nephrol. 1990;10(2 Suppl 1):11-9.
MEDCAC Background Information
Page 59 of 290
Page 57
144.
Jaeschke R, Singer J, Guyatt GH. Measurement of health status. Ascertaining
the minimal clinically important difference. Control Clin Trials 1989;10(4):407-15.
145.
Amgen. ARANESP(R) (Darbepoetin alfa) Prescribing Information. Amgen Inc.,
Thousand Oaks, CA.
Page 60 of 290
Appendix A - CMS Questions and Amgen's Responses
Appendix A - CMS Questions and Amgen's Responses
Page Page 61 of 290
Appendix A - CMS Questions and Amgen’s Responses
Page 1
MEDCAC –January 19, 2011 QUESTIONS
Erythropoiesis Stimulating Agents (ESAs) for Treatment of Anemia in Adults with CKD Including Patients on Dialysis and
Patients not on Dialysis: The Impact of ESA Use on Renal Transplant Graft Survival
ESAs are used with the intention of reducing the need for red blood cell transfusion and thereby minimize immune sensitization as
detected by panel reactive antibody (PRA) assays. PRA may be predictive of renal transplant graft survival. Some have proposed,
therefore, that ESAs increase the survival of renal transplant grafts.
For the voting questions, use the following scale identifying level of confidence - with 1 being the lowest or no confidence and 5
representing a high level of confidence. Please consider the questions in light of the following descriptive model.
1
Low
confidence
2
3
Intermediate
confidence
4
5
High
confidence
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Appendix A - CMS Questions and Amgen’s Responses
Page 2
1. How confident are you that there is adequate evidence to determine whether or not current panel reactive antibody (PRA) assays predict
renal transplant graft survival for individual patients in contrast to populations?
Confidence
Level
4
Answer
Supporting Evidence
There is a high level of evidence indicating that PRA methods (assays) predict: 1) time on wait-list,
2) probability of never receiving a transplant, and 3) renal transplant graft survival. Although PRA
methods have evolved, fundamentally, all PRA assays provide information regarding the presence
and level of antibodies to foreign antigens (allosensitization).
1. Cecka et al. AJT 2010 ;
10 : 26-29
2. Gloor J et al. Nat Rev
Nephrol. 2010;6:297306
3. USRDS 2010 Annual
Data Report
4. UNOS 2009 Annual
Report
5. Opelz et al., Lancet.
2005;365:1570-1576.
6. Ibrahim et al., Clin
Transplant [in press]
7. Patel R et al. N Eng J
Med, 1969 735-739
8. Tait et al., Nephrology
2009;14:247-254
9. Gloor J and Stegall M.
Nat Rev Nephrol.
2010;6:297-306
10. Cecka et al. AJT 2010 ;
10 : 26-29
11. McKenna et al,
Transplant
2000;69:319-326
12. Terasaki PI. Am J
Transplant 2003;3:665673
The level of evidence used to guide therapies for individual patients is always established at the
population level, through randomized controlled trials and/or large scale observational studies. For
example, while it is not possible to predict how an individual will respond to lowering cholesterol levels
with lipid lowering agents or lowering of blood pressure with anti-hypertensive therapies, the population
benefit of these therapies has been sufficiently demonstrated in RCTs, and in so doing, allow physicians
to apply them at the individual level. Similarly, observational studies have demonstrated associations
between tobacco use and lung cancer and while it is not possible to predict whether any single individual
who smokes will develop lung cancer, the evidence from large observational studies are used to guide
recommendations that individuals should abstain from tobacco use.
1
The PRA test measures a patient’s level of sensitization to a representative pool of donor HLA antigens .
The PRA, expressed as a percentage, reflects the percentage of the representative organ donor pool for
which the potential recipient has alloantibodies (i.e. allosensitized)2. For example, a PRA of 70%
suggests that 70% of donors will likely be unacceptable for the tested patient due to the presence of antiHLA antibodies against donor antigens. Thus, the higher the percent PRA, the more ‘allosensitized’ a
patient is to the general donor pool, and the more difficult to find a suitable donor. Levels of
allosensitization are often categorized and interpreted as no sensitization (PRA=0%), low degree of
sensitization (0-<10% or < 20%), moderate degree of sensitization (20-<79%), and high degree of
sensitization (≥ 80%).
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Appendix A - CMS Questions and Amgen’s Responses
Regardless of the method used to measure PRA, high PRA levels are strongly associated with: 1)
longer time on the transplant wait-list, 2) higher probability of never receiving a transplant, and 3)
worse kidney graft survival. Even when the crossmatch test is negative, a high pre-transplant PRA
level is associated with shorter kidney survival. The evidence supporting these associations
comes from complete population registries of transplant wait-list and transplanted patients
(United Network for Organ Sharing [UNOS] and United States Renal Data System [USRDS]), and
from multiple studies of over 50,000 transplanted patients 3-6.
Historical PRA methods
In 1969, Patel and Terasaki7 demonstrated that when a patient’s serum contained antibodies to a broad
range of donor cells, randomly derived from different individuals in the general population, the patient had
a much higher probability of experiencing acute organ rejection following transplantation. This was the
basis for the panel reactive antibody test, which measures a patient’s level of sensitization to a
representative pool of donor HLA antigens. The PRA, expressed as a percentage, reflects the
percentage of the likely organ donor pool for which the potential recipient has alloantibodies (i.e.
allosensitized).
Current PRA methods
In recent years, solid phase assays have been introduced that improve upon the cell-based assays for
HLA antibody screening and have refined our understanding of sensitization8. These assays are more
sensitive than previous methods in detecting HLA antibodies, and fall into two categories, ELISA-based
and HLA antigen-coated beads used in either a Flow Cytometry system or a Luminex platform. The
Luminex platform-based assay is now the standard methodology for assessing sensitization and
determining organ allocation in the US 9.
These tests allow the determination of a calculated PRA (cPRA). The cPRA is based upon HLA antigens
to which the patient has been sensitized and which, if present in a donor, would represent an
unacceptable risk for the candidate; these are referred to as unacceptable antigens. The cPRA is
computed from HLA antigen frequencies among approximately 12,000 kidney donors in the United States
between 2003 and 2005 and thus represents the percentage of actual organ donors that express one or
more of those unacceptable HLA antigens10. Since 2009, transplant wait-list patients’ cPRA and their
specific unacceptable antigens are required to be reported to UNOS in an effort to manage more fairly the
allocation of organs for highly sensitized patients, who have likely spent considerable time on the
transplant wait-list and cannot easily find a crossmatch negative (i.e.no donor specific antibodies) kidney
due to the high levels of alloantibodies and broad sensitization.
Page 3
13. Buscaroli A et al.
Transplant Int.
1992;5:S54-S57
14. Terasaki PI et al. AJT
2010;4:438-443
15. Lefaucheur C et al. J
Am Soc Nephrol.
2010;21:1398-1406
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Appendix A - CMS Questions and Amgen’s Responses
Crossmatch Test
A cross-match between a recipient and selected donor measures the presence of specific antibodies in
the recipient’s blood that react with the donor’s cells. The crossmatch test is the gold standard for
ultimately determining if a transplant will occur. A positive crossmatch corresponds with the presence of
detectable specific anti-donor antibodies and is generally a contraindication to proceed with
transplantation from that donor. In the face of a negative crossmatch, a transplant can proceed8.
Evidence supporting the impact of PRA levels on graft outcomes at the population level:
Terasaki and colleagues 11-12, in a series of papers, has summarized the evidence and synthesized the
current view regarding the importance of antibodies in both acute and chronic graft rejection
(approximately 50 independent publications).
• In 2000, he reviewed 23 pubs in which the presence of HLA antibodies was associated with acute
and chronic rejection11.
• In 2003, he reviewed 35 studies, again showing an association between HLA antibodies and graft
rejection12.
Opelz et al.5 presents data from the Collaborative Transplant Study Group (N = 116,562) on the effect of
PRA on cadaver renal transplant graft survival. At 10 years, the proportion of graft survival was 72.4%
(no PRA), 63.3% (PRA 1-50%), and 55.5% (PRA > 50%). Patients with 1-50% pre-transplant PRA had
increased relative risk of graft loss compared to non-sensitized patients (PRA=0%) (RR 1.29; 95% CI
1.09-1.53; P = 0.0033). The relative risk of graft loss was even higher in patients with pre-transplant PRA
> 50% compared to non-sensitized patients (RR 1.87; 95% CI 1.47-2.37; P < 0.0001). Transplants from
HLA-identical sibling donors do not provide a target for antibodies to HLA antigens and should therefore
not be affected by PRA. However, higher PRA levels were strongly associated with long-term graft loss
even in kidney transplants between siblings found to be HLA-identical by the transplant center prior to
transplant.
In a separate study of nearly 70,000 transplanted patients, Ibrahim et al.6 showed that PRA at the time of
transplant was associated with a significantly elevated risk of death with graft failure or death with
function. Those with a PRA of 20%-79% were at elevated risk compared to those with PRA=0%
(HR=1.18, 95% CI: 1.11-1.26), after adjusting for differences in case-mix; this risk was even greater for
those with a PRA > 80% (HR=1.30, 95% CI: 1.17-1.45).
Buscaroli et al.13 evaluated intermediate sensitization (PRA 30% - 60%) compared to lower levels of
sensitization (PRA < 30%) on kidney graft outcomes. Patients with intermediate sensitization had
significantly lower 1-year graft survival than patients with lower levels of sensitization (79.3% vs. 90.4%).
Page 4
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Appendix A - CMS Questions and Amgen’s Responses
Page 5
Terasaki et al.14 conducted a prospective trial in 23 kidney transplant centers (n=2231) to determine
whether HLA antibodies could predict kidney transplant failure within 1 year. Among the 500 patients who
had HLA antibodies, 6.6% failed compared to 3.3% among the 1778 patients without antibodies (p =
0.0007).
Lefaucheur et al.15, evaluated the occurrence of acute antibody-mediated rejection and survival in kidney
transplant patients with preexisting donor-specific HLA antibodies (HLA-DSA). The results showed
patients with HLA-DSA had significantly lower 8-year graft survival compared to sensitized patients
without HLA-DSA or non-sensitized (61% vs. 93% vs. 83.6%, P < 0.001).
For a more comprehensive list of the publications cited in the McKenna11 and Terasaki12 publications,
which evaluate the relationship between HLA antibodies, PRA methods, and kidney graft outcomes, see
the reference list in Appendix B.
2. If the result of Question 1 is at least Intermediate (mean vote ≥ 2.5) how confident are you that current PRA assays predict renal transplant
graft survival for individual patients?
Confidence
Level
4
Answer
There is a high level of confidence that there is adequate evidence to determine that PRA assays
predict 1) time on wait-list, 2) probability of never receiving a transplant, and 3) renal transplant
graft survival. While the newer cPRA methods in use have higher sensitivity than previous
methods, they do not alter the fact that there is a relationship between allosensitization and renal
transplant graft survival.
The level of evidence used to guide therapies for individual patients or investigate risk factors for
adverse clinical outcomes is always established at the population level by way of RCTs and/or
observational research. These findings are then applied to individuals.
Regardless of the method used to measure PRA, high PRA levels are strongly associated with: 1)
longer time on the transplant wait-list, 2) higher probability of never receiving a transplant, and 3)
worse kidney graft survival. Even when the crossmatch test is negative, a high pre-transplant PRA
level is associated with shorter kidney survival.
Supporting Evidence
1. USRDS 2010 Annual
Data Report
2. UNOS 2009 Annual
Report
3. Opelz et al., Lancet.
2005;365:1570-1576.
4. Ibrahim et al., Clin
Transplant [in press]
5. USRDS 2009 Annual
Data Report
6. Meier-Kriesche et al.,
Transplantation
2002 ;74 :1377-81.
7. Buscaroli A et al.
Transplant Int.
1992;5:S54-S57.
Page 66 of 290
Appendix A - CMS Questions and Amgen’s Responses
Page 6
The evidence supporting the relationship between higher PRA levels and longer time on the wait-list,
greater probability of never receiving a transplant and worse renal transplant graft survival comes from
complete population registries of transplant wait-list and transplanted patients (United Network for Organ
Sharing [UNOS] and United States Renal Data System [USRDS]), and from multiple studies of over
50,000 transplanted patients 1-4. The following figures illustrate these relationships.
Higher PRA levels associated with longer wait-time5
Median Wait Time In Years
10
PRA <10
PRA 10+
8
6
4
2
91
95
99
03
07
Years of Listing
Longer time on the wait-list associated with greater likelihood of dying5
8. Terasaki PI et al. AJT
2010;4:438-443
9. Lefaucheur et al. JASN
2010;21:1398-1406.
10. OPTN Annual Report
2009
11. Susal C.
Transplantation
2009;87: 1367–1371
12. Lachmann N.
Transplantation.
2009;87(10):1505-13
13. Horovitz D et al.
Transplantation
2009;87: 1214–1220
14. Yabu et al.,
Transplantation 2010 [in
press]
15. Burns et al. AJT
2008;8:2684-2694
16. Mauiyyedi et al. JASN
2001;
17. Sayegh et al. NEJM
2003;348:1033-1044
18. Campos et al. AJT
2006;6:2316-2320
19. Gibney et al. NDT
2006;21:2625-2629
20. Karpinski et al. JASN
2001;12:2807-2814;
21. Mizutani et al. AJT
2005;5 :2265-2272
22. Mizutani et al. AJT
2007;7 :1027-2031
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Appendix A - CMS Questions and Amgen’s Responses
Kidney graft survival in 21,836 recipients of living transplants by length of dialysis treatment
before transplant6
Higher PRA levels associated with shorter graft survival in a) all transplanted patients and b)
transplants between HLA-identical sibling donor-recipient pairs3
Page 7
23. Panigrahi et al. Hum
Immunol 2007;68:362367
24. Smith et al. AJT
2007;7:2809-2815
25. McKenna et al,
Transplant
2000;69:319-326
26. Terasaki PI. Am J
Transplant 2003;3:665673
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Appendix A - CMS Questions and Amgen’s Responses
Evidence supporting the impact of PRA levels on graft outcomes at the population level
Opelz et al.3 presents data from the Collaborative Transplant Study Group (N = 116,562) on the effect of
PRA on cadaver renal transplant graft survival. At 10 years, the proportion of graft survival was 72.4%
(no PRA), 63.3% (PRA 1-50%), and 55.5% (PRA > 50%). Patients with 1-50% pre-transplant PRA had
increased relative risk of graft loss compared to non-sensitized patients (PRA=0%) (RR 1.29; 95% CI
1.09-1.53; P = 0.0033). The relative risk of graft loss was even higher in patients with pre-transplant PRA
> 50% compared to non-sensitized patients (RR 1.87; 95% CI 1.47-2.37; P < 0.0001). Transplants from
HLA-identical sibling donors do not provide a target for antibodies to HLA antigens and should therefore
not be affected by PRA. However, PRA reactivity was strongly associated with long-term graft loss even in
kidney transplants between siblings found to be HLA-identical by the transplant center prior to transplant.
In a separate study of nearly 70,000 transplanted patients, Ibrahim et al.4 showed that PRA at the time of
transplant was associated with a significantly elevated risk of death with graft failure or death with
function. Those with a PRA of 20%-79% were at elevated risk compared to those with PRA=0%
(HR=1.18, 95% CI: 1.11-1.26), after adjusting for differences in case-mix; this risk was even greater for
those with a PRA > 80% (HR=1.30, 95% CI: 1.17-1.45).
Buscaroli et al.7 evaluated intermediate sensitization (PRA 30% - 60%) compared to lower levels of
sensitization (PRA < 30%) on kidney graft outcomes. Patients with intermediate sensitization had
significantly lower 1-year graft survival than patients with lower levels of sensitization (79.3% vs. 90.4%).
Terasaki et al.8 conducted a prospective trial in 23 kidney transplant centers (n=2278) to determine
whether HLA antibodies could predict kidney transplant failure within 1 year. Among the 500 patients who
had HLA antibodies, 6.6% failed compared to 3.3% among the 1778 patients without antibodies (p =
0.0007).
Lefaucheur et al.9 evaluated the occurrence of acute antibody-mediated rejection and survival in kidney
transplant patients with preexisting donor-specific HLA antibodies (HLA-DSA). The results showed
patients with HLA-DSA had significantly lower 8-year graft survival compared to sensitized patients
without HLA-DSA or non-sensitized (61% vs. 93% vs. 83.6%, P < 0.001).
Data supplied by the Organ Procurement and Transplantation Network’s (OPTN)10 Scientific Registry of
Transplant Recipients (SRTR) showed disparities in graft survival for patients with elevated PRA at time of
transplant. 1-year kidney graft survival rates were 91.7% (n = 36) for patients with PRA of > 80%, while
patients with a PRA of 0-9% had a 96.2% (n = 1,469) graft survival rate.
Page 8
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Appendix A - CMS Questions and Amgen’s Responses
Page 9
Susal et al.11 conducted a prospective multicenter study of over 1100 recipients of deceased donor kidney
transplants demonstrated that human leukocyte antigen (HLA) class I antibodies present before
transplantation were associated with a higher rate of delayed graft function and acute rejection episodes
during the first 3 months after transplantation. This ultimately resulted in increased risk of graft loss by 3
years.
Lachmann et al.12 showed with serial monitoring of HLA antibodies after transplantation using Luminex
technology that patients with elevated antibody levels have lower 5-year graft survival compared to those
without antibodies (79% vs. 95%).
Horovitz et al13 evaluated graft outcomes from deceased donors comparing third renal transplants (TRTR)
to primary renal transplants (PRTR). Patients with TRTR had higher PRA levels than the PRTR (24% vs
7%). TRTR patients experienced greater delayed graft function (46% vs 22%; P = 0.05) and biopsyproven rejection episodes (50% vs 29%; P = 0.01) compared to PRTR, despite greater frequency of
induction therapy (74% vs 35%; P = 0.004).
Evidence supporting the impact of PRA levels on graft outcomes at the individual level
Several case studies have reported the association between HLA antibodies measured with cPRA
methods and renal transplant graft survival. Higher levels of circulating antibodies have been associated
with histological evidence of rejection in the transplanted kidney. Lowering the measured antibody levels
with treatment has reversed the lesions and improved graft outcomes 14-24.
For a more comprehensive list of the publications cited in the McKenna25 and Terasaki26 publications,
which evaluate the relationship between HLA antibodies, PRA methods, and kidney graft outcomes, see
the reference list in Appendix B.
Discussion Question:
2a. How do PRA assays relate to more specific tests of HLA sensitivity and whether titer levels predict specific organ HLA sensitivity?
Answer
Regardless of the assay, high PRA levels indicate high levels of sensitization and predict a high likelihood of
specific organ HLA sensitivity (a positive crossmatch). The crossmatch is the ultimate test done prior to
transplantation to determine donor and recipient compatibility, and is the gold standard. A positive crossmatch
corresponds with the presence of detectable specific anti-donor antibodies and is generally a contraindication to
proceed with transplantation from that donor. In addition, the cPRA method is calculated from information on the
presence of anti-HLA antibodies to specific antigens.
Supporting Evidence
1. USRDS 2010 Annual
Data Report
2. UNOS 2009 Annual
Report
3. Opelz et al., Lancet.
2005;365:1570-1576.
Page 70 of 290
Appendix A - CMS Questions and Amgen’s Responses
The higher the level (PRA percentage) of sensitization, the less likely a patient is able to achieve a negative
crossmatch. Regardless of the method used to measure PRA and even in the face of a negative crossmatch, high
PRA levels predict: 1) longer time on the transplant wait-list, 2) higher probability of never receiving a transplant,
and 3) worse kidney graft survival worse graft survival. The evidence supporting these associations comes from
complete population registries of transplant wait-list and transplanted patients (United Network for Organ Sharing
[UNOS] and United States Renal Data System [USRDS]), and from multiple studies of over 50,000 transplanted
patients 1-4.
cPRA methods are more sensitive than previous methods. PRA evaluations were performed using lymphocyte cytotoxicity
(antiglobulin-enhanced, complement-dependent cytotoxicity [AHG-CDC]) or assays (enzyme-linked immunosorbent assay
[ELISA]; flow cytometry) in which solubilized HLA molecules were affixed to solid phase matrices (n = 264 samples).
Results among the three methods were concordant for 83% of these sera. Discordant results occurred with 32 samples and
demonstrated a distinct hierarchy in the sensitivity of the three techniques to detect alloantibodies. None of the 32 sera
were positive by AHG-CDC, 20/32 were positive by ELISA, and 32/32 were positive by flow cytometry5.
UNOS convened a meeting of laboratory directors and transplant physicians in March 2008 to identify the problems of
using solid phase testing. Evidence was presented from proficiency testing which revealed excellent concordance in
identifying specific antibodies, even in complex antisera6.
The development of PRA is cumulative7. The higher the PRA level, the greater the degree of sensitization and the greater
the likelihood of a positive crossmatch. It is for this reason that patients with high PRAs have longer wait time on the list and
may never find a suitable organ. Importantly, even if a patient with elevated PRA levels is able to find a suitable cross
match negative kidney, these broadly sensitized patients have poorer graft survival than those who are not sensitized.
Page 10
4. Ibrahim et al., Clin
Transplant [in press]
5. Gebel H and Bray R.
Clin Transplant.
2000;69:1370-1374.
6. US Department of
Health and Human
Services HHDoT. 2009
Annual Report of the
U.S. Organ
Procurement and
Transplantation
Network (OPTN) and
the Scientific Registry of
Transplant Recipients:
Transplant Data 19992008.
7. USRDS 2004 Annual
Data Report
Discussion Question:
2b. Are the various proprietary PRA assays clinically interchangeable, i.e. would the treating physician’s management of the patient differ
depending on the specific assay?
Answer
PRA methods are not interchangeable but provide the same information: PRA methods measure the presence and
level of antibodies to foreign antigens. In addition, the cPRA method is calculated from information on the level of
anti-HLA antibodies to specific HLA antigens.
Supporting Evidence
1. USRDS 2010 Annual
Data Report
2. UNOS 2009 Annual
Report
3. Opelz et al., Lancet.
2005;365:1570-1576.
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Appendix A - CMS Questions and Amgen’s Responses
Regardless of the method used to measure PRA, high PRA levels are strongly associated with: 1) longer time on
the transplant wait-list, 2) higher probability of never receiving a transplant, and 3) worse kidney graft survival.
Even when the crossmatch test is negative, a high pre-transplant PRA level is associated with shorter kidney
survival. The evidence supporting these associations comes from complete population registries of transplant
wait-list and transplanted patients (United Network for Organ Sharing [UNOS] and United States Renal Data
System [USRDS]), and from multiple studies of over 50,000 transplanted patients 1-4.
Page 11
4. Ibrahim et al., Clin
Transplant [in press]
Discussion Question:
2c. Do current PRA assays provide the same clinical information as older assays, i.e. do historical data on the performance of PRA assays
apply to currently available assays?
Answer
Yes, all PRA assays fundamentally provide information regarding the presence and level of antibodies to foreign
antigens. The newer methods (cPRA) are calculated from information on the level of anti-HLA antibodies to
specific HLA antigens.
Older PRA methods were based on reacting the patient’s sera to a broad range of donor cells. In recent years, solid phase
assays have been introduced that improve upon the cell-based assays for HLA antibody screening and have refined our
understanding of sensitization1-3. These assays are more sensitive than previous methods in detecting HLA antibodies, and
fall into two categories, ELISA-based and HLA antigen-coated beads used in either a Flow Cytometry system or a Luminex
platform. The Luminex platform-based assay is now the standard methodology for assessing sensitization and determining
organ allocation in the US2.
Regardless of the method used to measure PRA, high PRA levels predict 1) longer time on wait-list, 2) greater
probability of never receiving a transplant, and 3) worse kidney graft survival. Even when the crossmatch test is
negative, a high pre-transplant PRA level predicts shorter kidney survival. This is supported by substantial
evidence from multiple studies over many years using the older and current PRA methods.4-8
Supporting Evidence
1. Gloor J and Stegall M.
Nat Rev Nephrol.
2010;6:297-306;
2. Cecka JM. A JT
2010;10:26-29.
3. Tait et al. Nephrology
2009;14:247-254
4. USRDS 2010 Annual
Data Report
5. UNOS 2009 Annual
Report
6. Opelz et al. Lancet.
2005;365:1570-1576.
7. Ibrahim et al., Clin
Transplant [in press]
8. Lefaucheur et al. JASN
2010;21:1398-1406.
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Page 12
3. Donor-specific blood transfusions were frequently employed prior to renal transplantation for immune modulation and improved graft
survival. These differ from therapeutic blood transfusions, which are performed for anemia/blood loss management. How confident are you that
there is adequate evidence whether or not therapeutic blood transfusions decrease renal transplant graft survival?
Confidence
Level
4
Answer
There is a substantial body of evidence demonstrating that red blood cell (RBC) transfusions
administered for the therapy of anemia in CKD patients increase allosensitization, as measured by
PRA levels. There is also substantial evidence demonstrating that higher PRA levels are strongly
associated with: 1) longer time on the transplant wait-list, 2) higher probability of never receiving a
transplant, and 3) worse kidney graft survival. The evidence supporting these associations comes
from complete population registries of transplant wait-list and transplanted patients (United
Network for Organ Sharing [UNOS] and United States Renal Data System [USRDS]), and from
multiple studies of over 50,000 transplanted patients1-4.
DSTs are distinctly different from therapeutic transfusions administered for anemia/blood loss
management
Donor specific transfusions (DSTs) were proposed as a method of inducing immune tolerance based on
experimental consideration5, and first reported as potentially beneficial by Salvatierra in 19806. DST is a
therapy consisting of transfusions of 1- 3 units of blood from the donor who is offering the kidney. These
transfusions are administered to the recipient prior to the kidney transplant, with the intent of inducing
immune tolerance to the donor kidney and improving the long-term kidney allograft survival7. DSTs, which
can only be employed in the context of living donor kidneys, are distinctly different from therapeutic
transfusions used for the treatment of anemia; therapeutic blood transfusions expose the recipient to
blood from many unselected donors for the purpose of treating anemia.
There is evidence supporting superior graft survival among living donor transplants in which DSTs were
employed 8-12. However, it has also been reported that contrary to the intended purpose of inducing
tolerance, up to 30% of prospective transplant recipients administered DSTs develop antibodies to the
donor organ, thus precluding the organ transplant. While the patients who are allosensitized by DSTs
remain eligible for other donor transplantation, living and deceased, the sensitization induced by the DST
can compromise subsequent outcomes.
Supporting Evidence
1. USRDS 2010 Annual
Data Report
2. UNOS 2009 Annual
Report
3. Opelz et al., Lancet.
2005;365:1570-1576.
4. Ibrahim et al. Clin
Transplant [in press]
5. Opelz G et al.
Transplant Proc.
1973;5:253-259.
6. Salvatierra et al. Ann
Surg 1980;192:534–52
7. Cecka JM. AJT
2010;10:26-29.
8. Marti et al. Transpl Int
2006
9. Mackie F. Nephrol.
2010;15:S101-S105;
10. Okazaki H et al,
Transplant Proc.
1997;29(1-2):200-2.
11. Anderson et al.
Transplant Proc.
1995;27:991-4.
12. Aalten et al. NDT
2009;24:2559-2566
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Page 13
Because of uncertainty about whether a DST will induce tolerance and improve graft outcomes or induce
sensitization which can preclude a living donor transplant and reduce the transplant options of the patient,
DST is no longer widely advised 13, 16, and has been abandoned by most transplant centers15
13. Sharma et al;
Nephron.1997;75:20-4
14. Karpinski et al; JASN
2004;15:818-24
15. Alexander et al.
Transplantation.
1999;68:1117-24.
4. If the result of Question 3 is at least Intermediate (mean vote ≥ 2.5) how confident are you that therapeutic blood transfusions decrease
renal transplant graft survival?
Confidence
Level
4
Answer
There is a high level of confidence that therapeutic blood transfusions decrease renal transplant
graft survival. This has been shown in numerous large scale studies of transplant recipients.
Analyses of the relationship between prior transfusions and transplant survival revealed that pretransplant transfusions are detrimental to graft survival. The figure below shows that graft survival is
shortened as the number of pre-transplant transfusions increases, and this is evident for patients with
lower (0-10%) and higher (>10%) levels of sensitization at the time of transplant1.
Supporting Evidence
1. Hardy et al. Clin
Transplants. 2001;271227.
2. O’Brien et al. American
Society of Nephrology
Congress Abstract 2010
3. USRDS 2010 Annual
Data Report
4. UNOS 2009 Annual
Report
5. Opelz et al. Lancet
2005;365:1570-1576.
6. Ibrahim et al., Clin
Transplant [in press]
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Page 14
100
100
Percent Graft Survival
PRA 0–10%
PRA >10%
90
90
80
70
60
80
Number of
transfusions
N
0
18,086
1–5
8,126
6–10
993
>10
577
0
1
Number of
N
transfusions
3,816
0
2,871
1–5
427
6–10
333
>10
70
2
3
60
0
1
2
3
Years Posttransplant
O’Brien et al. showed in a single centre retrospective cohort study that patients who received one blood
transfusion had 52% graft survival at ten years, while those that received four transfusions had 41% graft
survival at ten years. The effect of transfusion on graft survival was not changed after adjustment for
donor and recipient age, and acute rejection not resulting in graft failure.
2
There is substantial evidence demonstrating that transfusions increase levels of allosensitization,
and that higher PRA levels are strongly associated with: 1) longer time on the transplant wait-list,
2) higher probability of never receiving a transplant, and 3) worse kidney graft survival. The
evidence supporting these associations comes from complete population registries of transplant
wait-list and transplanted patients (United Network for Organ Sharing [UNOS] and United States
Renal Data System [USRDS]), and from multiple studies of over 50,000 transplanted patients3-6.
Evidence supporting that transfusions increase allosensitization and this effect is cumulative at the
population level:
USRDS 2004 data7 show that in patients on the transplant wait list (n~50,000), greater numbers of
previous transfusions were significantly associated with PRA levels > 50%.
7. USRDS 2004 Annual
Data Report
8. Soosay et al. Ir Med J.
2003;96:109-112
9. Ling et al. American
Society of Nephrology
Congress 2010 Poster.
10. Miller et al. Lancet
1975;895-893.
11. Moore et al. Vox Sang.
1984;47:354-361.
12. Aalten et al. NDT
2009;24:2559-2566
13. Opelz et al. Transplant
Proc. 1973;5:253-259.
14. Lefaucheur et al. JASN
2010;21:1398-1406
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Recently, a study by Ibrahim et al.6 of approximately 70,000 transplant recipients showed that higher PRA
levels were strongly and positively associated with previous transfusion in both males and females.
Soosay et al.8 evaluated the relationship between transfusion and the risk of allosensitization. All highly
sensitized patients (PRA > 80%) had received at least 1 unit of blood and there was a clear increase in
the degree of sensitization with increasing numbers of units transfused, independent of other sensitizing
events.
Ling et al.9 assessed PRA levels in 778 patients on the kidney transplant wait list in a single center and
determined that 198 patients were sensitized. Sixty-seven of 198 patients (33.8%) were sensitized by a
single event (transfusion, prior transplantation, or pregnancy), of which 31.3% (n = 21) had received one
or more blood transfusions and 47.8% (n = 32) had prior transplantation. Of the patients who had multiple
events (n = 113), 98.2% had transfusions.
Miller et al.10 showed that between 15% and 52% of patients who received blood products developed
allosensitization.
Moore et al.11 demonstrated that 77% of men and 86% of nulliparous women developed allosensitization
(PRA of 1-10%) following one or more transfusions.
Aalten et al.12 showed in a study of over 600 non-sensitized female patients, 20% became sensitized
following receipt of a pre-transplant, donor-specific transfusion.
Evidence supporting that allosensitization is associated with worse renal transplant graft survival at the
population level:
Opelz et al.13 presents data from the Collaborative Transplant Study Group (N = 116,562) on the effect of
PRA on cadaver renal transplant graft survival. At 10 years, the proportion of graft survival was 72.4%
(no PRA), 63.3% (PRA 1-50%), and 55.5% (PRA > 50%). Patients with 1-50% pre-transplant PRA had
increased relative risk of graft loss compared to non-sensitized patients (PRA=0%) (RR 1.29; 95% CI
1.09-1.53; P = 0.0033). The relative risk of graft loss was even higher in patients with pre-transplant PRA
> 50% compared to non-sensitized patients (RR 1.87; 95% CI 1.47-2.37; P < 0.0001). Transplants from
HLA-identical sibling donors do not provide a target for antibodies to HLA antigens and should therefore
not be affected by PRA. However, PRA reactivity was strongly associated with long-term graft loss even in
kidney transplants between HLA-identical siblings
Page 15
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Page 16
In a separate study of nearly 70,000 transplanted patients, Ibrahim et al.6 showed that PRA at the time of
transplant was associated with a significantly elevated risk of death with graft failure or death with
function. Those with a PRA of 20%-79% were at elevated risk compared to those with PRA=0%
(HR=1.18, 95% CI: 1.11-1.26), after adjusting for differences in case-mix; this risk was even greater for
those with a PRA > 80% (HR=1.30, 95% CI: 1.17-1.45).
Lefaucheur et al.14 evaluated the occurrence of acute antibody-mediated rejection and survival in kidney
transplant patients with preexisting donor-specific HLA antibodies (HLA-DSA). The results showed
patients with HLA-DSA had significantly lower 8-year graft survival compared to sensitized patients
without HLA-DSA or non-sensitized (61% vs. 93% vs. 83.6%, P < 0.001).
Discussion Question:
4a. The relative roles of sensitization as opposed to underlying co-morbid conditions in affecting renal transplant graft survival.
Answer
There are multiple factors that may influence graft survival in addition to PRA levels including patient
demographic characteristics and co-morbid conditions. The available evidence suggests that PRA remains an
independent predictor of graft failure.
In a recent study of nearly 70,000 transplanted patients, Ibrahim et al.1 show that higher PRA levels were associated with
significantly elevated risk of death or graft failure with death, even after adjusting for various clinical characteristics
including demographics, blood type, dialysis modality, pre-listing duration on dialysis, and multiple co-morbid conditions.
Those with a PRA of 20%-79% were at elevated risk compared to those with PRA=0% (HR=1.18, 95% CI: 1.11-1.26), after
adjusting for differences in case-mix; this risk was even greater for those with a PRA > 80% (HR=1.30, 95% CI: 1.17-1.45).
Supporting Evidence
1. Ibrahim et al. Clinical
Transplantation 2010 [in
press]
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Page 17
Discussion Question:
4b. The adequacy of the evidence based on the relationship if any between the number of units transfused and renal transplant graft survival.
For example, is there a threshold number of units that predict renal transplant graft survival or is there a linear or exponential relationship
between the number of units transfused that predict renal transplant graft survival?
Answer
Supporting Evidence
Analyses of the relationship between prior transfusions and transplant survival show that pre-transplant transfusions are
detrimental to graft survival. The figure below shows that graft survival is shortened as the number of pre-transplant
transfusions increases, and this is evident for patients with lower (0-10%) and higher (>10%) levels of sensitization at the
time of transplant1.
100
100
Percent Graft Survival
PRA 0–10%
PRA >10%
90
90
80
0
1–5
6–10
70
60
>10
0
80
N
18,086
8,126
993
70
577
1
0
1–5
6–10
2
3
60
N
3,816
2,871
427
>10
0
333
1
2
3
Years Posttransplant
O’Brien et al.2 showed in a single centre retrospective cohort study that patients who received one blood transfusion had
52% graft survival at ten years, while those that received four transfusions had 41% graft survival at ten years. The effect
of transfusion on graft survival was not changed after adjustment for donor and recipient age, and acute rejection not
resulting in graft failure.
Data from approximately 70,000 transplanted patients show that the patients who receive transfusions have higher risk of
sensitization3 and the risk of allosensitization from RBC transfusion is cumulative: as the number of previous transfusions
increases, so does the risk of elevated PRA levels4.
1. Hardy S, et al. Clin
Transplants. 2001;271278.
2. O’Brien et al. American
Society of Nephrology
Congress Abstract 2010
3. Ibrahim et al., Clin
Transplant [in press]
4. USRDS 2004 Annual
Data Report
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Page 18
Discussion Question:
4c. The relative roles of blood transfusions, pregnancy, prior renal transplant, and other factors that cause sensitization.
Answer
There are only three recognized sources of HLA sensitization, of which, blood transfusions are the only clinically
modifiable event. The ability of transfusions to induce sensitization can be amplified in the presence of previous
sensitization.
Hardy et al.1 stated in 2001: “rejection of a kidney was the most powerful means by which patients became sensitized.
Transfusions were next in ability to sensitize followed by pregnancies.”
Evidence supporting the relative role of blood transfusions on sensitization:
Soosay et al.2 evaluated the relationship between transfusion and the risk of allosensitization. All highly sensitized patients
(PRA > 80%) had received at least 1 unit of blood, in addition to other sensitizing events (ie, pregnancy (32%; prior
transplantation 74%). In addition, there is a clear increase in the degree of sensitization with increasing number of units
transfused, independent of other sensitizing events.
Ling et al.3 assessed PRA levels in 778 patients on the kidney transplant wait list in a single center and determined that 198
patients were sensitized. Sixty-seven of 198 patients (33.8%) were sensitized by a single event (transfusion, prior
transplantation, or pregnancy), of which 31.3% (n = 21) had received one or more blood transfusions and 47.8% (n = 32)
had prior transplantation. Of the patients who had multiple events (n = 113), 98.2% had transfusions.
Miller et al.4 showed that allosensitization by HLA antibodies ranged between 15% to 52% of patients who received blood
products.
Moore et al.5 demonstrated that 77% of men and 86% of nulliparous women developed allosensitization (PRA of 1-10%)
following one or more transfusions.
O’Brien et al.6 showed in a single centre retrospective cohort study that patients who received one blood transfusion had
52% graft survival at ten years, while those that received four transfusions had 41% graft survival at ten years. The effect
of transfusion on graft survival was not changed after adjustment for donor and recipient age, and acute rejection not
resulting in graft failure.
Supporting Evidence
1. Hardy et al. Clin
Transplants. 2001;271278
2. Soosay et al. Ir Med J.
2003;96:109-112
3. Ling et al. American
Society of Nephrology
Congress 2010 Poster.
4. Miller et al. Lancet
1975;895-893.
5. Moore S et al. Vox
Sang. 1984;47:354-361.
6. O’Brien et al. .
American Society of
Nephrology Congress
Abstract 2010
7. Vaidya S. Transplant
Proc. 2005;37:648-649.
8. Rebibou et al.
Transplant Immunology
2000;8:125-128
9. Scornik et al.,
Transplantation
1984;38:594-8.
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Page 19
Evidence supporting that the relative role of pregnancy on sensitization:
In a retrospective study by Vaidya7, multiple pregnancies and ≥ 2 prior transfusions were both strongly associated with
greater levels of sensitization.
Rebibou et al.8 showed that in patients with previous pregnancies, transfusions induced sensitization in 36% of patients.
Evidence supporting the relative role of prior renal transplant on sensitization:
Rejection of a prior kidney transplant was the most powerful means by which patients became sensitized. Transfusions
were next in ability to sensitize, followed by pregnancies1.
Scornik et al.9 prospectively examined the development of sensitization following transfusion in patients who experienced
graft failure without immediate sensitization. Among these patients, 18 received subsequent blood transfusion, and of
those, over 70% were subsequently sensitized.
5. How confident are you that there is adequate evidence to determine whether or not ESA use for anemia/blood loss management improves
renal transplant graft survival?
Confidence
Level
Answer
Supporting Evidence
4
ESAs are approved for the management of anemia in CKD patients. There is substantial evidence
from registrational studies and data from near-universal capture of the US dialysis population that
ESAs, when used to raise and maintain Hb within the range of ~10-12 g/dL, reduce the need for
RBC transfusions.
The level of confidence behind this conclusion is 5.
1. Eschbach et al. Ann Int
Med.1989;111:9921000.
®
2. Aranesp USPI
3. Provenzano et al. Clin
Nephrol. 2004;61:392405
4. USRDS 2007 Annual
Data Report
5. Ibrahim et al. Nephrol
Dial Transplant
2009;24: 3138–3143
6. USRDS 2004 Annual
Data Report
7. Ibrahim et al., Clinical
Transplant [in press]
Reduction in transfusions reduces patient exposure to transfusion-related risks, including
allosensitization, which is strongly associated with 1) longer time on wait-list, 2) higher probability
of never receiving a transplant, and 3) shortened renal transplant graft survival. The level of
confidence behind this conclusion is 4.
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Page 20
Evidence supporting that ESA therapy reduces the need for transfusions:
The clinical benefit of transfusion avoidance in anemic dialysis patients was demonstrated in the
registrational clinical trials with EPOGEN® and was the basis of approval1. Raising hemoglobin
concentrations was the key outcome for approval of ESAs in the non-dialysis setting. The Aranesp®2
clinical trial program evaluated hemoglobin response in CKD-NOD subjects and demonstrated that
hemoglobin levels could be raised and maintained within a targeted hemoglobin range and approval for
this indication was granted by the FDA in 2001 (Amgen).
In the original registration studies with EPOGEN® in dialysis patients, transfusion avoidance occurred
when hemoglobin levels were raised above 10 g/dL 1, and maintained within a 2 g/dL range of
approximately 10-12 g/dL. These trials demonstrated a virtual elimination of transfusions (> 90%
reduction) in patients treated with ESAs compared to patients treated with placebo. While the Epoetin alfa
treated patients became nearly transfusion independent, placebo treated patients remained severely
anemic and continued to receive multiple transfusions. Similar efficacy was demonstrated in an open-label
single-arm study of anemic CKD-NOD patients 3. In this study, hemoglobin levels were raised above
10 g/dL with ESA therapy and transfusion events were reduced by ~70%.
Patients Transfused Per Quarter (%)
Once ESA therapy was introduced into the US dialysis population, there was a dramatic decline in
outpatient transfusions which persisted over time, as shown in the figure below from USRDS4.
16
12
8
4
0
78
80
82
84
86
88
Year
90
9.
10.
11.
12.
13.
14.
Soosay et al. Ir Med J.
2003;96:109-112
Ling et al. American
Society of Nephrology
Congress 2010 Poster
Miller et al. Lancet
1975;895-893.
Moore et al. Vox Sang.
1984;47:354-361.
Aalten et al. NDT 2009;
24: 2559-2566
O’Brien et al. American
Society of Nephrology
Congress Abstract
2010
Opelz et al. Lancet.
2005;365:1570-1576;
15. Buscaroli et al.
Transplant Int.
1992;5:S54-S57
16. Terasaki et al. AJT
2010;4:438-443
17. Lefaucheur et al. JASN
2010;21:1398-1406.
Use of ESAs Introduced
20
8.
92
94
96
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Appendix A - CMS Questions and Amgen’s Responses
In the Medicare population with CKD-NOD and anemia, transfusion rates have declined from 30% to 15%
among patients treated with ESAs between 1995 and 20045.
Evidence supporting transfusions are associated with allosensitization:
USRDS 2004 data show that in patients on the transplant wait-list (n~50,000) greater numbers of previous
transfusions were significantly associated with PRA levels > 50%6.
Recently, a study by Ibrahim et al.7 of approximately 70,000 transplant recipients showed that higher PRA
levels were strongly and positively associated with previous transfusion in both males and females.
Soosay et al.8 evaluated the relationship between transfusion and the risk of allosensitization. All highly
sensitized patients (PRA > 80%) had received at least 1 unit of blood and there is a clear increase in the
degree of sensitization with increasing number of units transfused, independent of other sensitizing
events.
Ling et al.9 assessed PRA levels in 778 patients on the kidney transplant wait list in a single center and
determined that 198 patients were sensitized. Sixty-seven of 198 patients (33.8%) were sensitized by a
single event (transfusion, prior transplantation, or pregnancy), of which 31.3% (n = 21) had received one
or more blood transfusions and 47.8% (n = 32) had prior transplantation. Of the patients who had multiple
events (n = 113), 98.2% had transfusions.
Miller et al.10 showed that between 15% to 52% of patients who received blood products developed
allosensitization.
Moore et al.11 demonstrated that 77% of men and 86% of nulliparous women developed allosensitization
(PRA of 1-10%) following one or more transfusions.
Aalten et al.12 showed in a study of over 600 non-sensitized female patients, 20% became sensitized
following receipt of a pre-transplant, donor-specific transfusion.
O’Brien et al.13 showed in a single centre retrospective cohort study that patients who received one blood
transfusion had 52% graft survival at ten years, while those that received four transfusions had 41% graft
survival at ten years. The effect of transfusion on graft survival was not changed after adjustment for
donor and recipient age, and acute rejection not resulting in graft failure.
Page 21
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Evidence supporting allosensitization is associated with poor graph survival
Opelz et al.14 presents data from the Collaborative Transplant Study Group (N = 116,562) on the effect of
PRA on cadaver renal transplant graft survival. At 10 years, the proportion of graft survival was 72.4%
(no PRA), 63.3% (PRA 1-50%), and 55.5% (PRA > 50%). Patients with 1-50% pre-transplant PRA had
increased relative risk of graft loss compared to non-sensitized patients (PRA=0%) (RR 1.29; 95% CI
1.09-1.53; P = 0.0033). The relative risk of graft loss was even higher in patients with pre-transplant PRA
> 50% compared to non-sensitized patients (RR 1.87; 95% CI 1.47-2.37; P < 0.0001). Transplants from
HLA-identical sibling donors do not provide a target for antibodies to HLA antigens and should therefore
not be affected by PRA. However, PRA reactivity was strongly associated with long-term graft loss even in
kidney transplants between HLA-identical siblings.
In a separate study of nearly 70,000 transplanted patients, Ibrahim et al.7 showed that PRA at the time of
transplant was associated with a significantly elevated risk of death with graft failure or death with
function. Those with a PRA of 20%-79% were at elevated risk compared to those with PRA=0%
(HR=1.18, 95% CI: 1.11-1.26), after adjusting for differences in case-mix; this risk was even greater for
those with a PRA > 80% (HR=1.30, 95% CI: 1.17-1.45).
Buscaroli et al.15 evaluated intermediate sensitization (PRA 30% - 60%) compared to lower levels of
sensitization (PRA < 30%) on kidney graft outcomes. Patients with intermediate sensitization had
significantly lower 1-year graft survival than patients with lower levels of sensitization (79.3% vs. 90.4%).
Terasaki et al.16 conducted a prospective trial in 23 kidney transplant centers (n=2278) to determine
whether HLA antibodies could predict kidney transplant failure within 1 year. Among the 500 patients who
had HLA antibodies, 6.6% failed compared to 3.3% among the 1778 patients without antibodies (p =
0.0007).
Lefaucheur et al.17 evaluated the occurrence of acute antibody-mediated rejection and survival in kidney
transplant patients with preexisting donor-specific HLA antibodies (HLA-DSA). The results showed
patients with HLA-DSA had significantly lower 8-year graft survival compared to sensitized patients
without HLA-DSA or non-sensitized (61% vs. 93% vs. 83.6%, P < 0.001).
Page 22
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Page 23
6. If the result of Question 5 is at least Intermediate (mean vote ≥ 2.5) how confident are you that there is adequate evidence to conclude that
ESA use to maintain hemoglobin levels >10 g/dl is necessary to improve renal transplant graft survival?
Confidence
Level
Supporting Evidence
There is a high level of confidence that raising and maintaining hemoglobin concentrations > 10
g/dL and within the range of 10-12 g/dL significantly reduces the need for RBC transfusions 1,3.
The level of confidence behind this conclusion is 5. Registrational trials in dialysis patients
demonstrated that ESAs effectively raise Hb concentrations and significantly decrease the need
for RBC transfusions3.
Data from clinical trials and surveillance of the entire US dialysis population indicate that the
likelihood of transfusions increases substantially when Hb concentrations fall below 10 g/dL, and
this risk increases the longer Hb concentrations remain below 10 g/dL. The following figures
support these conclusions. The level of confidence behind this conclusion is 5.
Higher likelihood of transfusion when preceding month’s Hb is < 10 g/dL6.
8
7
6
The Risk of Transfusion by the Previous Month's Hemoglobin Level
5
4
Hazard Ratio (95% CI)
4
Answer
3
2.5
2
1.5
1.2
1
0.8
0.6
0.4
<9
9 - < 10
10 - < 11
11 - < 12
Hemoglobin level (g/dL)
Data Source: NHCT study, data on file
>=12
1. EPOGEN® USPI
2. Aranesp® USPI
3. Eschbach et al. Ann Int
Med.1989;111:9921000
4. Provenzano et al. Clin
Nephrol. 2004;61:392405;
5. Ibrahim et al. AJKD
2008;52:1115-1121.
6. Amgen Data on File
7. USRDS 2009 Annual
Data Report
8. Ibrahim et al., NDT
2009;24:3138-3143
9. USRDS 2004 Annual
Data Report
10. Ibrahim et al., Clinical
Transplant [in press]
11. Soosay et al. Ir Med J.
2003;96:109-112
12. Ling et al. American
Society of Nephrology
Congress 2010 Poster.
13. Miller et al. Lancet
1975;895-893.
14. Moore et al. Vox Sang.
1984;47:354-361.
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Transfusion rate increases substantially as the number of months with a Hb < 10 g/dL increases
(Medicare hemodialysis data)6
Transfusion rate has declined significantly over time in dialysis patients as the mean hemoglobin
concentration has increased to above 10 g/dL7
Page 24
15. Aalten et al. NDT 2009;
24: 2559-2566
16. O’Brien et al. .
American Society of
Nephrology Congress
Abstract 2010
17. Opelz et al. Lancet.
2005;365:1570-1576
18. Buscaroli et al.
Transplant Int.
1992;5:S54-S57.
19. Terasaki et al. AJT
2010;4:438-443
20. Lefaucheur et al. JASN
2010;21:1398-1406.
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Appendix A - CMS Questions and Amgen’s Responses
By raising and maintaining Hb concentrations above 10 g/dL and within the range of 10-12 g/dL,
the risks related to transfusions are reduced. One of these risks is allosensitization, which has
been consistently shown to impair the outcome of renal transplant including increased wait time
for transplant as well as decreased renal transplant graft survival. This is shown in the figure
below. The level of confidence behind this conclusion is 4.
Evidence supporting that ESA therapy used to maintain hemoglobin concentrations > 10 g/dL and within
the range of 10-12 g/dL decreases transfusions and their associated risks, including sensitization:
The clinical benefit of transfusion avoidance in anemic dialysis patients was demonstrated by the
registrational clinical trials with EPOGEN® and established hemoglobin as the key outcome for approval of
ESAs in the non-dialysis setting. The Aranesp® clinical trial program evaluated hemoglobin response in
CKD-NOD subjects and demonstrated that hemoglobin levels could be raised and maintained within a
targeted hemoglobin range and approval for this indication was granted by the FDA in 2001 (Amgen).
In the original registration studies with EPOGEN® in dialysis patients, transfusion avoidance occurred
when hemoglobin levels were raised above 10 g/dL3, and maintained within a 2 g/dL range of
approximately 10-12 g/dL. These trials demonstrated a virtual elimination of transfusions (> 90%
reduction) in patients treated with ESAs compared to patients treated with placebo. While the Epoetin alfa
treated patients became nearly transfusion independent, placebo treated patients remained severely
anemic and continued to receive multiple transfusions. Similar efficacy was demonstrated in an open-label
single-arm study of anemic CKD-NOD patients4. In this study, hemoglobin levels were raised above
10 g/dL with ESA therapy and transfusion events were reduced by ~70%.
Data from the entire US hemodialysis patient population indicate that as Hb levels have increased over
time with ESA therapy to a mean above 10 g/dL (~11.2 g/dL), transfusions have declined significantly and
the proportion of un-sensitized transplant candidates has more than doubled from 24% to 49%5. In the
Medicare population with CKD-NOD and anemia, transfusion rates have declined from 30% to 15%
among patients treated with ESAs between 1995 and 20048.
Evidence supporting that transfusions are associated with allosensitization:
USRDS 2004 data9 show that in patients on the transplant wait list (n~50,000), greater numbers of
previous transfusions were significantly associated with PRA levels > 50%.
More recently, a study by Ibrahim et al.10 of approximately 70,000 transplant recipients showed that higher
PRA levels were strongly and positively associated with previous transfusion in both males and females.
Page 25
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Appendix A - CMS Questions and Amgen’s Responses
Soosay et al.11 evaluated the relationship between transfusion and the risk of allosensitization. All highly
sensitized patients (PRA > 80%) had received at least 1 unit of blood and there is a clear increase in the
degree of sensitization with increasing number of units transfused, independent of other sensitizing
events.
Ling et al.12 assessed PRA levels in 778 patients on the kidney transplant wait list in a single center and
determined that 198 patients were sensitized. Sixty-seven of 198 patients (33.8%) were sensitized by a
single event (transfusion, prior transplantation, or pregnancy), of which 31.3% (n = 21) had received one
or more blood transfusions and 47.8% (n = 32) had prior transplantation. Of the patients who had multiple
events (n = 113), 98.2% had transfusions.
Miller et al.13showed that between 15% to 52% of patients who received blood products developed
allosensitization.
Moore et al.14 demonstrated that 77% of men and 86% of nulliparous women developed allosensitization
(PRA of 1-10%) following one or more transfusions.
Aalten et al.15 showed in a study of over 600 non-sensitized female patients, 20% became sensitized
following receipt of a pre-transplant, donor-specific transfusion.
O’Brien et al.16 showed in a single centre retrospective cohort study that patients who received one blood
transfusion had 52% graft survival at ten years, while those that received four transfusions had 41% graft
survival at ten years. The effect of transfusion on graft survival was not changed after adjustment for
donor and recipient age, and acute rejection not resulting in graft failure.
Evidence supporting allosensitization is associated with poor graph survival:
Opelz et al.17 presents data from the Collaborative Transplant Study Group (N = 116,562) on the effect of
PRA on cadaver renal transplant graft survival. At 10 years, the proportion of graft survival was 72.4%
(no PRA), 63.3% (PRA 1-50%), and 55.5% (PRA > 50%). Patients with 1-50% pre-transplant PRA had
increased relative risk of graft loss compared to non-sensitized patients (PRA=0%) (RR 1.29; 95% CI
1.09-1.53; P = 0.0033). The relative risk of graft loss was even higher in patients with pre-transplant PRA
> 50% compared to non-sensitized patients (RR 1.87; 95% CI 1.47-2.37; P < 0.0001). Transplants from
HLA-identical sibling donors do not provide a target for antibodies to HLA antigens and should therefore
not be affected by PRA. However, PRA reactivity was strongly associated with long-term graft loss even in
kidney transplants between HLA-identical siblings.
Page 26
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Appendix A - CMS Questions and Amgen’s Responses
Page 27
In a separate study of nearly 70,000 transplanted patients, Ibrahim et al.10 showed that PRA at the time of
transplant was associated with a significantly elevated risk of death with graft failure or death with
function. Those with a PRA of 20%-79% were at elevated risk compared to those with PRA=0%
(HR=1.18, 95% CI: 1.11-1.26), after adjusting for differences in case-mix; this risk was even greater for
those with a PRA > 80% (HR=1.30, 95% CI: 1.17-1.45).
Buscaroli et al.18 evaluated intermediate sensitization (PRA 30% - 60%) compared to lower levels of
sensitization (PRA < 30%) on kidney graft outcomes. Patients with intermediate sensitization had
significantly lower 1-year graft survival than patients with lower levels of sensitization (79.3% vs. 90.4%).
Terasaki et al.19 conducted a prospective trial in 23 kidney transplant centers (n=2278) to determine
whether HLA antibodies could predict kidney transplant failure within 1 year. Among the 500 patients who
had HLA antibodies, 6.6% failed compared to 3.3% among the 1778 patients without antibodies (p =
0.0007).
Lefaucheur et al.20 evaluated the occurrence of acute antibody-mediated rejection and survival in kidney
transplant patients with preexisting donor-specific HLA antibodies (HLA-DSA). The results showed
patients with HLA-DSA had significantly lower 8-year graft survival compared to sensitized patients
without HLA-DSA or non-sensitized (61% vs. 93% vs. 83.6%, P < 0.001).
7. What significant evidence gaps exist regarding the clinical criteria, including hemoglobin level, of patients who should receive blood
transfusions for chronic anemia with the intent of improving renal transplant graft survival?
Answer
There is no evidence gap on the question of use of therapeutic transfusions for the treatment of anemia with the
intent of improving graft survival; they should not be used for this purpose.
There is a substantial body of evidence demonstrating that red blood cell (RBC) transfusions administered for the therapy
of anemia in CKD patients increase allosensitization, as measured by PRA levels. There is also substantial evidence
demonstrating that higher PRA levels are strongly associated with decreased renal transplant survival. The evidence
supporting the relationship between PRA methods and worse renal transplant graft survival comes from multiple studies of
over 50,000 transplanted patients1.
Supporting Evidence
1. Cecka et al. AJT.
2010;10:26-29.
2. Opelz et al. Lancet.
2005;365:1570-1576
3. Ibrahim et al., Clinical
Transplantation [in
press
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Therapeutic Blood Transfusions
The evidence linking therapeutic transfusions with allosensitization is unequivocal in CKD patients: allosensitization
significantly worsens renal transplant graft survival2,3.
This has been established in multiple large studies and from surveillance of the entire dialysis and transplant populations
(USRDS4 and UNOS5).
• RBC transfusions increase allosensitization (as measured by PRA levels);
• Higher PRA levels decrease the likelihood of finding a suitable donor, increasing the wait-time for a renal
transplant, resulting in:
o Increased probability of future transfusion and allosensitization
o Increased probability of death while waiting for the transplant
o Decreased probability of graft survival following transplantation.
• Higher PRA levels increase acute and chronic graft rejection and shorten graft survival (time spent not on dialysis),
even for patients with a negative cross-match.
6
A recent review article by Cecka concluded:
"With the highly effective immunosuppressive drugs currently available, deliberate administration of donor transfusions
represents an indefensible risk of sensitization and delay of transplantation without compensatory benefits."
A recent review by the Circular of Information for the use of human blood and blood components7 concluded the following
about the use of RBC transfusions:
“Red-cell-containing components should not be used to treat anemias that can be corrected with specific hematinic
medications such as iron, vitamin B12, folic acid, or erythropoietin.”
Page 28
4. USRDS 2010 Annual
Data Report
5. UNOS 2009 Annual
Report
6. Cecka JM. Annu Rev
Med 2000;51:393-406
(p. 401).
7. AABB, Circular of
Information for the use
of human blood and
blood components,
2009
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Appendix A - CMS Questions and Amgen’s Responses
Page 29
8. What significant gaps exist regarding the relationship, if any, of number of units transfused, screening PRA assays, more specific HLA
assays, immune suppressive regimen, and the timing of rejection to determine the role various factors in transplant graft survival outcomes?
Answer
The highly sensitized patient remains a challenge to clinical transplantation. There is irrefutable evidence that transfusions,
pregnancy and previous transplant can lead to sensitization and that sensitization predicts worse graft survival.
Additional research in the following areas may be helpful:
• PRA methods
• Desensitization approaches
• Optimal immunosuppressive regimen and approach to post-transplant management of highly sensitized patients
• Identification of methods to prevent allosensitization
• Identification of patients who are more likely to be sensitized
Supporting Evidence
Appendix B - Select References
Appendix B - Select References
Page 90 of 290
Page Page 91 of 290
CIRCULAR OF
INFORMATION
FOR THE USE OF HUMAN BLOOD AND BLOOD COMPONENTS
This Circular was prepared jointly by AABB, the American Red Cross, America’s Blood
Centers, and the Armed Services Blood Program (August 2009, revised December 2009). The
Food and Drug Administration recognizes this Circular of Information as an acceptable
extension of container labels.
The online version of this Circular of Information is provided for educational purposes. It may
not be modified in any way without the express permission of the AABB, ARC, ABC, and
ASBP. Printed copies of the Circular are intended to accompany blood and blood components,
and can be ordered through the AABB sales department or the online Bookstore. Please refer to
this Web site’s “Terms of Use” for additional information.
Federal Law prohibits dispensing the blood and blood components described in this circular
without a prescription.
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Page 92 of 290
Table of Contents
Notice to All Users............................................................................................................. 1
General Information for Whole Blood and All
Blood Components ......................................................................................................... 1
Donors ............................................................................................................................ 1
Testing of Donor Blood.................................................................................................. 1
Blood and Component Labeling..................................................................................... 2
Instructions for Use ........................................................................................................ 3
Side Effects and Hazards for Whole Blood and All
Blood Components ......................................................................................................... 4
Immunologic Complications, Immediate ....................................................................... 4
Immunologic Complications, Delayed ........................................................................... 5
Nonimmunologic Complications.................................................................................... 5
Fatal Transfusion Reactions ........................................................................................... 7
Components Containing Red Cells.................................................................................. 7
Overview ........................................................................................................................ 7
Components Available ................................................................................................. 10
Plasma Components........................................................................................................ 12
Overview ...................................................................................................................... 12
Fresh Frozen Plasma..................................................................................................... 13
Plasma Frozen Within 24 Hours After Phlebotomy ..................................................... 14
Plasma Cryoprecipitate Reduced.................................................................................. 15
Liquid Plasma Components.......................................................................................... 15
Cryoprecipitated Components....................................................................................... 17
Overview ...................................................................................................................... 17
Components Available ................................................................................................. 18
Platelet Components ....................................................................................................... 18
Overview ...................................................................................................................... 18
Components Available ................................................................................................. 21
Granulocyte Components............................................................................................... 22
Further Processing.......................................................................................................... 23
Leukocyte Reduction.................................................................................................... 24
Further Testing to Identify CMV-Seronegative Blood................................................. 24
Irradiation ..................................................................................................................... 25
Washing ........................................................................................................................ 25
Volume Reduction........................................................................................................ 26
References........................................................................................................................ 26
Tables
Table 1. Contents of Anticoagulant-Preservative Solutions........................................... 8
Table 2. Content of Additive Solutions (in mg/100 mL)................................................ 8
Table 3. Coagulation Factor Activity of Thawed
Plasma Derived from FFP......................................................................................... 15
Table 4. Summary Chart of Blood Components .......................................................... 34
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Page 93 of 290
1
Notice to All Users
The Circular of Information for the Use of Human Blood and Blood Components (hereafter
referred to as Circular) is an extension of container labels, as the space on those labels is limited.
Blood and blood components are biologic products and, in the form of cellular products, living
human tissue intended for use in patient treatment. Professional judgment based on clinical
evaluation determines the selection of components, dosage, rate of administration, and decisions
in situations not covered in this general statement.
This Circular, as a whole or in part, cannot be considered or interpreted as an expressed or
implied warranty of the safety or fitness of the described blood or blood components when used
for their intended purpose. Attention to the specific indications for blood components is needed
to prevent inappropriate transfusion.
Because of the risks associated with transfusion, physicians should be familiar with
alternatives to allogeneic transfusion. Blood banks and transfusion services are referred to the
AABB Standards for Blood Banks and Transfusion Services for additional information and
policies, especially in the areas of recipient sample identification, compatibility testing, issue and
transfusion of blood and blood components, investigation of transfusion reactions, and proper
record-keeping practices. Transfusionists are referred to the AABB Technical Manual for
applicable chapters on adult and pediatric transfusion.
The specific product manufacturer’s package insert should be reviewed for instructions
pertaining to use of transfusion devices (eg, filters, blood administration sets, and blood warmers).
This Circular is supplied to conform with applicable federal statutes and regulations of the Food
and Drug Administration (FDA), United States (US) Department of Health and Human Services.
The blood components in this Circular marked with the symbol “Ω ” are blood components for
which FDA currently has not received data to demonstrate that they meet prescribed
requirements of safety, purity, and potency, and therefore are not licensed for distribution in
interstate commerce.
General Information for Whole Blood and All Blood Components
Donors
Blood and blood components described in this Circular have been collected from volunteer
blood donors for use in other patients (allogeneic transfusions) or from patients donating for
themselves (autologous transfusions). The donors have been questioned about risk factors for
transmissible infectious agents, have satisfactorily completed a health assessment that includes a
questionnaire on past and present illnesses, have satisfied minimum physiologic criteria, and may
have had the opportunity to confidentially exclude their donation from transfusion.
Testing of Donor Blood
Testing of a sample of donor blood is performed before units of blood or blood components are
distributed for routine transfusion. The donor’s ABO group and Rh type have been determined,
including testing for the presence of weak D antigen.
A sample from each donation intended for allogeneic use has been tested by FDA-licensed
tests and found to be nonreactive for antibodies to human immunodeficiency virus (anti-HIV1/2), hepatitis C virus (anti-HCV), human T-cell lymphotropic virus (anti-HTLV-I/II), and
hepatitis B core antigen (anti-HBc), and nonreactive for hepatitis B surface antigen (HBsAg).
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2
Licensed nucleic acid tests (NAT) for HCV ribonucleic acid (RNA), HIV-1 RNA, and West Nile
virus (WNV) RNA have been performed and found to be nonreactive. A serologic test for
syphilis has been performed and found to be nonreactive.
For units labeled “FOR AUTOLOGOUS USE ONLY,” infectious disease testing
requirements vary depending on whether the unit will be drawn in one facility and infused in
another facility and whether the unit might be made available for allogeneic transfusion.
Infectious disease testing may be omitted for autologous units drawn, stored, and infused at the
same facility. Autologous units for which testing has not been performed are labeled “DONOR
UNTESTED.” Autologous units with reactive test results may be used for transfusion to the
donor-patient with appropriate physician authorization. A biohazard label will be applied to
autologous units that are tested for evidence of infection as listed above and determined to be
reactive. If the units labeled “FOR AUTOLOGOUS USE ONLY” are infused at a different
facility, at a minimum the first donation from the donor-patient in each 30-day period is tested
for evidence of infection as listed above. Subsequent units that are not tested will be labeled as
“DONOR TESTED WITHIN THE LAST 30 DAYS.” If an establishment allows any autologous
donation to be available for allogeneic transfusion, or ships autologous donations to any
establishment that does, the collecting establishment must test each donation for evidence of
infection as listed above. This includes units labeled “FOR AUTOLOGOUS USE ONLY.”
Tests for unexpected antibodies against red cell antigens have been performed on samples
from all donors. The results of these tests are negative or have been determined to be clinically
insignificant unless otherwise indicated on the label. Other tests may have been performed on
donor blood as indicated by information that has been provided by the blood bank or transfusion
service on an additional label or tie tag, or in a supplement to this Circular.
Blood and Component Labeling
All blood components identified in this Circular have the ISBT 128 product name listed first and
other recognized component names in parentheses.
Blood and blood component labels will contain the following information:
1. The proper name, whole blood or blood component, including an indication of any
qualification or modification.
2. The method by which the blood component was prepared, either by whole blood or apheresis
collection.
3. The temperature range in which the blood component is to be stored.
4. The preservatives and anticoagulant used in the preparation of the blood or blood
components, when appropriate.
5. The standard contents or volume is assumed unless otherwise indicated on the label or in
Circular supplements.
6. The number of units in pooled blood components and any sedimenting agent used during
cytapheresis, if applicable.
7. The name, address, registration number, and US license number (if applicable) of the
collection and processing location.
8. The expiration date (and time if applicable), which varies with the method of preparation
(open or closed system) and the preservatives and anticoagulant used. When the expiration
time is not indicated, the product expires at midnight.
9. The donation (unit or pool) identification number.
10. The donor category (paid or volunteer, and autologous if applicable).
11. ABO group and Rh type, if applicable.
12. Special handling information, as required.
13. Statements regarding recipient identification, this Circular, infectious disease risk, and
prescription requirement.
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3
Instructions for Use
The following general instructions pertain to Whole Blood and all the blood components
described in this Circular:
1. All blood and blood components must be maintained in a controlled environment and stored
under appropriate conditions as described in the AABB Standards for Blood Banks and
Transfusion Services.
2. The intended recipient and the blood container must be properly identified before the
transfusion is started.
3. Aseptic technique must be employed during preparation and administration. If the container
is entered in a manner that violates the integrity of the system, the component expires 4 hours
after entry if maintained at room temperature (20-24 C), or 24 hours after entry if refrigerated
(1-6 C).
4. All blood components must be transfused through a filter designed to remove clots and
aggregates (generally a standard 170- to 260-micron filter).
5. Blood and blood components should be mixed thoroughly before use.
6. Blood and blood components must be inspected immediately before use. If, upon visual
inspection, the container is not intact or the appearance is abnormal (presence of excessive
hemolysis, a significant color change in the blood bag as compared with the tubing segments,
floccular material, cloudy appearance, or other problems), the blood or blood component
must not be used for transfusion and appropriate follow-up with the transfusion service must
be performed.
7. No medications or solutions may be routinely added to or infused through the same tubing
with blood or blood components with the exception of 0.9% Sodium Chloride, Injection
(USP), unless 1) they have been approved for this use by the FDA or 2) there is
documentation available to show that the addition is safe and does not adversely affect the
blood or blood component.
8. Lactated Ringer’s, Injection (USP) or other solutions containing calcium should never be
added to or infused through the same tubing with blood or blood components containing
citrate.
9. Blood components should be warmed if clinically indicated for situations such as exchange
or massive transfusions, or for patients with cold-reactive antibodies. Warming must be
accomplished using an FDA-cleared warming device so as not to cause hemolysis.
10. Some life-threatening reactions occur after the infusion of only a small volume of blood or
blood components. Therefore, unless otherwise indicated by the patient’s clinical condition,
the rate of infusion should initially be slow.
11. Periodic observation and recording of vital signs should occur during and after the
transfusion to identify suspected adverse reactions. If a transfusion reaction occurs, the
transfusion must be discontinued immediately and appropriate therapy initiated. The infusion
should not be restarted unless approved by transfusion service protocol.
12. Specific instructions concerning possible adverse reactions shall be provided to the patient or
a responsible caregiver when direct medical observation or monitoring of the patient will not
be available after transfusion.
13. Transfusion should be started before component expiration and completed within 4 hours.
14. All adverse events related to transfusion, including possible bacterial contamination of blood
or a blood component or suspected disease transmission, must be reported to the transfusion
service according to its local protocol.
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4
Side Effects and Hazards for Whole Blood and All Blood Components
Immunologic Complications, Immediate
1. Hemolytic transfusion reaction, the destruction of red cells, is discussed in detail in the
section on components containing red cells and in the platelet section.
2. Immune-mediated platelet destruction, one of the causes of refractoriness to platelet
transfusion, is the result of alloantibodies in the recipient to HLA or platelet-specific antigens
on transfused platelets. This is described in more detail in the section on platelets.
3. Febrile nonhemolytic reaction is typically manifested by a temperature elevation of ≥1 C or 2
F occurring during or shortly after a transfusion and in the absence of any other pyrexic
stimulus. This may reflect the action of antibodies against white cells or the action of
cytokines, either present in the transfused component or generated by the recipient in
response to transfused elements. Febrile reactions may occur in approximately 1% of
transfusions, and they occur more frequently in patients receiving non-leukocyte-reduced
platelets and those previously alloimmunized by transfusion or pregnancy. No routinely
available pre- or posttransfusion tests are helpful in predicting or preventing these reactions.
Antipyretics usually provide effective symptomatic relief. Patients who experience repeated,
severe febrile reactions may benefit from receiving leukocyte-reduced components. If these
reactions are caused by cytokines in the component, prestorage leukocyte reduction may be
benefical.
4. Allergic reactions frequently occur as mild or self-limiting urticaria or wheezing that usually
respond to antihistamines. More severe manifestations including respiratory and
cardiovascular symptoms are more consistent with anaphylactoid/anaphylactic reactions and
may require more aggressive therapy (see below). No laboratory procedures are available to
predict these reactions.
5. Anaphylactoid/anaphylactic reactions, characterized by hypotension, tachycardia, nausea,
vomiting and/or diarrhea, abdominal pain, severe dyspnea, pulmonary and/or laryngeal
edema, and bronchospasm and/or laryngospasm, are rare but dangerous complications
requiring immediate treatment with epinephrine. These reactions have been reported in IgAdeficient patients who develop IgA antibodies. Such patients may not have been previously
transfused and may develop symptoms after infusion of very small amounts of IgAcontaining plasma, in any blood component. Similar reactions have also been described in
patients with haptoglobin deficiency. In certain circumstances, patients might benefit from
the use of washed cellular components to prevent or reduce the severity of allergic reactions
not minimized by treatment with medication alone.
6. Transfusion-related acute lung injury (TRALI) is the acute onset of hypoxemia within 6
hours of a blood or blood component transfusion and is the most commonly reported cause of
transfusion-related deaths in the United States. In addition to hypoxemia, criteria for
diagnosis include the presence of bilateral infiltrates on frontal chest radiographs and the
exclusion of transfusion-associated circulatory overload (TACO), or preexisting acute lung
injury. The exact mechanism of TRALI is not known, but hypotheses include donor
antibodies that react against white cell antigens (HLA or human neutrophil antigens) and the
sequestration of neutrophils by the pulmonary endothelium (caused by the recipient’s
underlying condition) that are subsequently activated by the infusion of substances in the
donor plasma such as antibodies or other biologically active substances. In far fewer cases,
antibodies in the recipient that may react with antigens on transfused white cells have been
implicated. Laboratory testing does not alter management of this reaction, which is diagnosed
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5
mainly on clinical and radiographic findings. Treatment of TRALI requires aggressive
respiratory support, frequently requiring mechanical ventilation.
Immunologic Complications, Delayed
1. Delayed hemolytic reaction is described in detail in the section on components containing red
cells.
2. Alloimmunization to antigens of red cells, white cells, platelets, or plasma proteins may occur
unpredictably after transfusion. Blood components may contain certain immunizing
substances other than those indicated on the label. For example, platelet components may
also contain red cells and white cells. Primary immunization does not become apparent until
days or weeks after the immunizing event, and does not usually cause symptoms or
physiologic changes. If components that express the relevant antigen are subsequently
transfused, there may be accelerated removal of cellular elements from the circulation and/or
systemic symptoms. Clinically significant antibodies to red cell antigens will ordinarily be
detected by pretransfusion testing. Alloimmunization to antigens of white cells, platelets, or
plasma proteins can be detected only by specialized testing.
3. Posttransfusion purpura (PTP) is a rare syndrome characterized by the development of
dramatic, sudden, and self-limited thrombocytopenia, typically 7 to 10 days after a blood
transfusion, in a patient with a history of sensitization by either pregnancy or transfusion.
Although the immune specificity may be to a platelet-specific antigen the patient lacks, both
autologous and allogeneic platelets are destroyed. High-dose Immune Globulin, Intravenous
(IGIV) may correct the thrombocytopenia.
4. Transfusion-associated graft-vs-host disease (TA-GVHD) is a rare but extremely dangerous
condition that occurs when viable T lymphocytes in the transfused component engraft in the
recipient and react against recipient tissue antigens. TA-GVHD can occur if the host does not
recognize and reject the foreign transfused cells, and it can follow transfusion of any
component that contains even very small numbers of viable T lymphocytes. Recipients with
severe cellular immunodeficiency (except for HIV infection) are at greatest risk (eg, fetuses
receiving intrauterine transfusions, recipients of hematopoietic progenitor cell transplants,
and selected patients with severe immunodeficiency conditions), but TA-GVHD has also
been reported in recipients receiving fludarabine for oncologic and rheumatologic diseases,
and in immunologically normal recipients who are heterozygous for a tissue antigen
haplotype for which the donor is homozygous. Tissue antigen haplotype sharing is most
likely to occur when the transfused component is from a blood relative or has been selected
for HLA compatibility. TA-GVHD remains a risk with leukocyte-reduced components
because they contain sufficient residual T lymphocytes. Irradiation of the component renders
T lymphocytes incapable of proliferation and is presently the only approved means to prevent
TA-GVHD.
Nonimmunologic Complications
1. Because whole blood and blood components are made from human blood, they may carry a
risk of transmitting infectious agents [eg, viruses, bacteria, parasites, the variant CreutzfeldtJakob disease (vCJD) agent, and, theoretically, the classic CJD agent]. Careful donor
selection and available laboratory tests do not totally eliminate the hazard. Also, septic and
toxic reactions can result from transfusion of bacterially contaminated blood and blood
components. Such reactions are infrequent, but may be life-threatening. This may occur
despite careful selection of donors and testing of blood. Donor selection criteria are designed
to screen out potential donors with increased risk of infection with HIV, HTLV, hepatitis,
and syphilis, as well as other agents (see section on Testing of Donor Blood). These
procedures do not totally eliminate the risk of transmitting these agents.
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6
2.
3.
4.
Cytomegalovirus (CMV) may, unpredictably, be present in white-cell-containing
components from donors previously infected with this virus, which can persist lifelong
despite the presence of serum antibodies. Up to 70% of donors may be anti-CMV positive.
Transmission of CMV by transfusion may be of concern in low-birthweight (≤1220 g)
premature infants born to CMV-seronegative mothers and/or certain other categories of
immunocompromised individuals, if they are CMV seronegative. For at-risk recipients, the
risk of CMV transmission by cellular components can be reduced by transfusing CMVseronegative or leukocyte-reduced components.
For other infectious agents (eg, Babesia spp, Leishmania spp, and Plasmodia spp) there
are no routinely available tests to predict or prevent disease transmission. All potential blood
donors are subjected to screening procedures intended to reduce to a minimum the risk that
they will transmit infectious agents.
Bacterial sepsis occurs rarely but can cause acute, severe, sometimes life-threatening effects.
Onset of high fever (≥2 C or ≥3.5 F increase in temperature), severe chills, hypotension, or
circulatory collapse during or shortly after transfusion should suggest the possibility of bacterial
contamination and/or endotoxin reaction. Although platelet components stored at room
temperature have been implicated most frequently, previously frozen components thawed by
immersion in a waterbath and red cell components stored for several weeks at 1 to 6 C have
also been implicated. Although most apheresis platelets are routinely tested for bacterial
contamination, this does not completely eliminate the risk.
Both gram-positive and gram-negative organisms have been identified as causing septic
reactions. Organisms capable of multiplying at low temperatures (eg, Yersinia enterocolitica)
and those using citrate as a nutrient are most often associated with components containing
red cells. A variety of pathogens, as well as skin contaminants, have been found in platelet
components. Endotoxemia in recipients has resulted from multiplication of gram-negative
bacteria in blood components.
Prompt recognition of a possible septic reaction is essential, with immediate
discontinuation of the transfusion and aggressive therapy with broad-spectrum antimicrobials
and vasopressor agents, if necessary. In addition to prompt sampling of the patient’s blood
for cultures, investigation should include examination of material from the blood container
by Gram’s stain, and cultures of specimens from the container and the administration set. It is
important to report all febrile transfusion reactions to the transfusion service. Follow-through
from the transfusion service to the blood collection facility may facilitate retrieval of other
components associated with the collection.
TACO, leading to pulmonary edema, can occur after transfusion of excessive volumes or at
excessively rapid rates. This is a particular risk in the very young and the elderly and in
patients with chronic severe anemia in whom low red cell mass is associated with high
plasma volume. Small transfusion volumes can precipitate symptoms in at-risk patients who
already have a positive fluid balance.
Pulmonary edema should be promptly and aggressively treated, and infusion of colloid
preparations, including plasma components and the suspending plasma in cellular
components, reduced to a minimum.
Hypothermia carries a risk of cardiac arrhythmia or cardiac arrest and exacerbation of
coagulopathy. Rapid infusion of large volumes of cold blood or blood components can
depress body temperature, and the danger is compounded in patients experiencing shock or
surgical or anesthetic manipulations that disrupt temperature regulation. A blood warming
device should be considered if rapid infusion of blood or blood components is needed.
Warming must be accomplished using an FDA-cleared warming device so as not to cause
hemolysis.
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5.
Metabolic complications may accompany large-volume transfusions, especially in neonates
and patients with liver or kidney disease.
a. Citrate “toxicity” reflects a depression of ionized calcium caused by the presence in the
circulation of large quantities of citrate anticoagulant. Because citrate is promptly
metabolized by the liver, this complication is rare. Patients with severe liver disease or
those with circulatory collapse that prevents adequate hepatic blood flow may have
physiologically significant hypocalcemia after rapid, large-volume transfusion. Citrated
blood or blood components administered rapidly through central intravenous access may
reach the heart so rapidly that ventricular arrhythmias occur. Standard measurement of
serum calcium does not distinguish ionized from complexed calcium. Ionized calcium
testing or electrocardiogram monitoring is more helpful in detecting physiologically
significant alteration in calcium levels.
b. Other metabolic derangements can accompany rapid or large-volume transfusions,
especially in patients with preexisting circulatory or metabolic problems. These include
acidosis or alkalosis (deriving from changing concentrations of citric acid and its
subsequent conversion to pyruvate and bicarbonate) and hyper- or hypokalemia.
Fatal Transfusion Reactions
When a fatality occurs as a result of a complication of blood or blood component transfusion, the
Director, Office of Compliance and Biologics Quality, Center for Biologics Evaluation and
Research (CBER), should be notified within 1 FDA business day (telephone: 301-827-6220; email: [email protected]). Within 7 days after the fatality, a written report must be
submitted to the Director, Office of Compliance and Biologics Quality, HFM-600, CBER, FDA,
1401 Rockville Pike, Rockville, MD 20852-1448. A copy of the report should be sent to the
collecting facility, if appropriate. Updated information about CBER reporting requirements may
be found at http://www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/ReportaProblem/
TransfusionDonationFatalities/default.htm.
Components Containing Red Cells
Overview
Description
Red cells contain hemoglobin and serve as the primary agent for transport of oxygen to tissues.
The primary red-cell-containing transfusion component is Red Blood Cells (RBCs). This
component is prepared by centrifugation or sedimentation of Whole Blood to remove much of
the plasma. RBC components can also be prepared by apheresis methods.
Depending upon the collection system used, a single whole blood donation typically contains
either 450 mL (±10%) or 500 mL (±10%) of blood collected from blood donors with a minimum
hematocrit of 38%, withdrawn in a sterile container that includes an anticoagulant solution
licensed for this component. Occasionally, units of other volumes are collected and those
volumes are stated on the label.
Red-cell-containing components can be stored for an interval (“shelf life”) determined by the
properties of the anticoagulant-preservative solution (see Table 1). Whole Blood units are
prepared in an aseptic manner in a ratio of 14 mL of anticoagulant-preservative solution per 100
mL of whole blood collected. Apheresis components are collected into anticoagulants as
recommended by the manufacturer.
After plasma is removed, the resulting component is Red Blood Cells, which has a hematocrit
of 65% to 80% and a usual volume between 225 mL and 350 mL. Additive solutions (AS) may
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8
be mixed with the red cells remaining after removal of nearly all of the plasma (see Table 2).
The typical hematocrit of AS RBCs is 55% to 65% and the volume is approximately 300 to 400
mL. AS RBCs have a shelf life of 42 days. Descriptions of specific components containing red
cells are given at the end of this section.
Table 1. Contents of Anticoagulant-Preservative Solutions
Citric Acid
Monobasic
Sodium
Phosphate
Dextrose
Adenine
Shelf Life
8.0 g/L
0
24.5 g/L
0
21 days
26.3 g/L
3.27 g/L
2.22 g/L
25.5 g/L
0
21 days
Citrate-phosphate-dextrose-dextrose
(CP2D)
26.3 g/L
3.27 g/L
2.22 g/L
51.1 g/L
0
21 days
Citrate-phosphate-dextrose-adenine
(CPDA-1)
26.3 g/L
3.27 g/L
2.22 g/L
31.9 g/L
0.275 g/L
35 days
Citric
Acid
Shelf Life
Trisodium
Citrate
Anticoagulant citrate-dextrose A
(ACD-A)*
22.0 g/L
Citrate-phosphate dextrose (CPD)
Anticoagulant-Preservative
*ACD is used for apheresis components.
Table 2. Content of Additive Solutions (in mg/100mL)
Additive Solution
(mg/100 mL)
Monobasic
Sodium
Phosphate
Mannitol
Sodium
Chloride
0
750
900
0
0
42 days
30
276
0
410
588
42
42 days
30
0
525
877
0
0
42 days
Dextrose
Adenine
AS-1
(Adsol)
2200
27
AS-3
(Nutricel)
1100
AS-5
(Optisol)
900
Sodium
Citrate
Actions
All RBC components and Whole Blood increase the recipient’s oxygen-carrying capacity by
increasing the mass of circulating red cells. Processing and/or storage deplete the component of
virtually all potential therapeutic benefit attributable to the functions of white cells and platelets;
cellular elements remain in these blood components and may cause adverse immunologic or
physiologic consequences. Residual plasma in the component provides the recipient with volume
expansion and nonlabile plasma proteins to the extent that residual plasma is present in the
preparation. Depending on the method of production, RBCs may contain approximately 20 to
100 mL of residual plasma. RBCs prepared with additive solutions are the most commonly used
red cell product and have limited residual plasma.
Indications
Red-cell-containing components are indicated for treatment of symptomatic or critical deficit of
oxygen-carrying capacity. They are also indicated for red cell exchange transfusion.
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Contraindications
Red-cell-containing components should not be used to treat anemias that can be corrected with
specific hematinic medications such as iron, vitamin B12, folic acid, or erythropoietin.
RBCs or Whole Blood should not be used solely for volume expansion or to increase oncotic
pressure of circulating blood.
Dosage and Administration
Each unit of RBCs or Whole Blood contains enough hemoglobin to increase the hemoglobin
concentration in an average-sized adult by approximately 1 g/dL (increase hematocrit by 3%).
Smaller aliquots can be made available for use with neonatal or pediatric patients, or adults with
special transfusion needs.
The ABO group of all red-cell-containing components must be compatible with ABO
antibodies in the recipient’s plasma. Whole Blood must be ABO identical with the recipient;
RBCs, which contain a reduced volume of antibody-containing plasma, need not be ABO
identical.
Serologic compatibility between recipient and donor must be established before any red-cellcontaining component is transfused. This may be accomplished by performing ABO/Rh typing,
antibody screening, and crossmatching by serologic technique or use of a computer crossmatch.
In cases when delay in transfusion will be life-threatening, uncrossmatched group O RBCs or
ABO group-specific RBCs may be transfused before completion of pretransfusion compatibility
testing.
The initial portion of each unit transfused should be infused cautiously and with sufficient
observation to detect onset of acute reactions. Thereafter, the rate of infusion can be more rapid,
as tolerated by the patient’s circulatory system. It is undesirable for components that contain red
cells to remain at room temperature longer than 4 hours. If the anticipated infusion rate must be
so slow that the entire unit cannot be infused within 4 hours, it is appropriate to order smaller
aliquots for transfusion.
Side Effects and Hazards
Hazards that pertain to all transfusion components are described in the earlier section titled Side
Effects and Hazards for Whole Blood and All Blood Components. Listed below are hazards that
apply specifically to components that contain red cells.
1. Hemolytic transfusion reaction is the immunologic destruction of transfused red cells,
nearly always the result of incompatibility of antigen on the transfused cells with antibody in
the recipient’s circulation (see item 5 below for discussion of nonimmunologic hemolysis).
The most common cause of severe, acute hemolytic reactions is transfusion of ABOincompatible blood, resulting from identification errors occurring at some point(s) in the
transfusion process. Serologic incompatibility undetected during pretransfusion testing is a
much less common cause of acute hemolysis. If a transfusion reaction is suspected, the
transfusion must be stopped and the transfusion service laboratory notified immediately.
Information identifying the patient, the transfusion component, and associated forms and
labels must be reviewed promptly to detect possible errors. A postreaction blood sample,
preferably drawn from a site other than the transfusion access, must be sent to the laboratory
along with the implicated unit of blood and administration set.
Acute hemolytic reactions characteristically begin with an increase in temperature and pulse
rate; symptoms may include chills, dyspnea, chest or back pain, abnormal bleeding, or shock.
Instability of blood pressure is frequent, the direction and magnitude of change depending
upon the phase of the reaction and the magnitude of compensatory mechanisms. In
anesthetized patients, hemoglobinuria, hypotension, and evidence of disseminated
intravascular coagulopathy (DIC) may be the first signs of incompatibility. Laboratory
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2.
3.
4.
5.
findings can include hemoglobinemia and/or hemoglobinuria, followed by elevation of serum
bilirubin. The direct antiglobulin test (DAT) is usually positive, with rare exceptions (ie,
complete hemolysis of incompatible red cells). Treatment includes measures to maintain or
correct arterial blood pressure; correct coagulopathy, if present; and promote and maintain
urine flow. Lack of symptoms does not exclude an acute hemolytic reaction.
Delayed hemolytic reactions occur in previously red-cell-alloimmunized patients in whom
antigens on transfused red cells provoke anamnestic production of antibody. The anamnestic
response reaches a significant circulating level while the transfused cells are still present in
the circulation; the usual time frame is 2 to 14 days after transfusion. Signs may include
unexplained fever, development of a positive DAT, and unexplained decrease in
hemoglobin/hematocrit. Hemoglobinemia and hemoglobinuria are uncommon, but elevation
of lactate dehydrogenase (LDH) or bilirubin may be noted. Most delayed hemolytic reactions
have a benign course and require no treatment.
Hemolytic transfusion reactions in patients with sickle cell anemia may be particularly
severe, with destruction of autologous as well as transfused red cells. In such patients,
serologic investigations may not reveal the specificity of the causative antibody. Prospective
matching for Rh and Kell antigens may decrease risk.
Antigens on transfused red cells may cause red cell alloimmunization of the recipient.
Clinically significant antibodies to red cell antigens will usually be detected in pretransfusion
antibody screening tests. For most patients, red cell antigen matching beyond ABO and Rh is
unnecessary.
TACO, resulting in pulmonary edema, can accompany transfusion of any component at a
rate more rapid than the recipient’s cardiac output can accommodate. Whole Blood creates
more of a risk than Red Blood Cells because the transfused plasma adds volume without
increasing oxygen-carrying capacity. Patients with chronic anemia have increased plasma
volumes and are at increased risk for circulatory overload.
Hemoglobinopathies is a long-term complication of repeated RBC transfusions. Each
transfusion contributes approximately 250 mg of iron. Patients requiring multiple
transfusions for aplastic anemia, thalassemias, or hemoglobinopathies are at far greater risk
than patients transfused for hemorrhagic indications, because blood loss is an effective means
of iron excretion. Patients with predictably chronic transfusion requirements should be
considered for treatment with iron-chelating agents or a program of exchange transfusion
therapy, if applicable.
Nonimmunologic hemolysis occurs rarely, but can result from: 1) introduction of hypotonic
fluids into the circulation, 2) effects of drugs co-administered with transfusion, 3) effects of
bacterial toxins, 4) thermal injury to transfusion components, by either freezing or
overheating, 5) metabolic damage to cells, as from hemoglobinopathies or enzyme
deficiencies, or 6) development of physical or osmotic stresses. Examples of situations
capable of causing nonimmune red cell hemolysis include: exposure to excessive heat by
non-FDA-approved warming methods, mixture with hypotonic solutions, or transfusion
under high pressure through small-gauge or defective needles.
Components Available
1. RED BLOOD CELLS (RED BLOOD CELLS) are prepared from blood collected into any of
the anticoagulant-preservative solutions approved by the FDA, and separated from the
plasma by centrifugation or sedimentation. Separation may be done at any time during the
allowable storage interval (“shelf life”). Red Blood Cells may contain from 160 to 275 mL of
red cells (50-80 g of hemoglobin) suspended in varying quantities of residual plasma.
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2.
3.
4.
5.
6.
7.
8.
9.
RED BLOOD CELLS ADENINE SALINE ADDED (RED BLOOD CELLS ADENINE
SALINE ADDED) are prepared by centrifuging whole blood to remove as much plasma as
possible, and replacing the plasma with usually 100 to 110 mL of an additive solution that
contains some combination of dextrose, adenine, sodium chloride, and either monobasic
sodium phosphate (AS-3) or mannitol (AS-1 and AS-5); the hematocrit is usually between
55% and 65%. Red Blood Cells in an additive solution have lower viscosity than Red Blood
Cells, and flow through administration systems in a manner more comparable to that of
Whole Blood. Red Blood Cells stored with an additive solution have an extended shelf life.
RED BLOOD CELLS LEUKOCYTES REDUCED (RED BLOOD CELLS LEUKOCYTES
REDUCED) are prepared from a unit of Whole Blood (collected in anticoagulant-preservative
solution as noted above) containing ≥1 to 10 × 109 white cells. In general, leukocyte
reduction is achieved by filtration: 1) soon after collection (prestorage) or 2) after varying
periods of storage in the laboratory. Leukocyte reduction will decrease the cellular content
and volume of blood according to characteristics of the filter system used. RBCs Leukocytes
Reduced must have a residual content of leukocytes <5.0 × 106. Leukocyte reduction filters
variably remove other cellular elements in addition to white cells. The leukocyte-reduced
component contains at least 85% of the original red cell content.
APHERESIS RED BLOOD CELLS (RED BLOOD CELLS PHERESIS) are red cells collected
by apheresis. This component must be collected in an approved anticoagulant. The red cell
volume collected and the anticoagulant used are noted on the label. Aside from the
automated collection method used, the component is comparable to whole-blood-derived
RBCs in all aspects. The dosage can be calculated, as for RBCs, from the red cell content of
the product. Apheresis RBCs contain on average 60 g of hemoglobin per unit.
APHERESIS RED BLOOD CELLS LEUKOCYTES REDUCED (RED BLOOD CELLS
PHERESIS LEUKOCYTES REDUCED) are collected by apheresis methods. Leukocyte reduction
is achieved in the manufacturing process resulting in a final product containing <5.0 × 106
leukocytes and at least 85% of the target red cell content.
RED BLOOD CELLS, LOW VOLUME (RED BLOOD CELLS, LOW VOLUME) are
products prepared when 300 to 404 mL of whole blood is collected into an anticoagulant
volume calculated for 450 mL ± 45 mL or when 333 to 449 mL of whole blood is collected
into an anticoagulant volume calculated for 500 mL ± 50 mL. These products reflect a
collection with an altered ratio of anticoagulant to red cells and may not be an indication of a
lower dose of hemoglobin. Plasma and platelet components should not be prepared from lowvolume collections.
WHOLE BLOOD (WHOLE BLOOD) is rarely used for transfusion. In situations where
Whole Blood is indicated but RBCs are used, a suitable plasma volume expander should be
administered. See also General Information for Whole Blood and All Blood Components,
Instructions for Use. All whole blood transfusions must be ABO identical.
FROZEN RED BLOOD CELLS (RED BLOOD CELLS FROZEN) and FROZEN
REJUVENATED RED BLOOD CELLS (RED BLOOD CELLS REJUVENATED FROZEN)
are prepared by adding glycerol to red cells as a cryoprotective agent before freezing. The
glycerol must be removed from the thawed component before it is infused. Frozen RBCs
may be stored for up to 10 years, and for longer intervals if there is particular need for
specific units. Ω Frozen storage is especially suitable for red cells with unusual antigenic
phenotypes.
DEGLYCEROLIZED RED BLOOD CELLS (RED BLOOD CELLS DEGLYCEROLIZED) is
the form in which cryopreserved red cells (Frozen Red Blood Cells) are made available for
infusion. Glycerol is added to red cells as a cryoprotective agent before freezing, and must be
removed from the thawed component before it is infused.
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10.
11.
12.
13.
Deglycerolized RBCs contain 80% or more of the red cells present in the original unit of
blood, and have approximately the same expected posttransfusion survival as RBCs.
Glycerol is removed by washing the cells with successively lower concentrations of Sodium
Chloride, Injection (USP); the final suspension is in 0.9% Sodium Chloride, Injection (USP),
with or without small amounts of dextrose. Small amounts of residual-free hemoglobin may
cause the supernatant fluid to be pink-tinged.
Deglycerolized RBCs provide the same physiologic benefits as RBCs, but their use is
usually restricted to situations in which standard transfusion components are inappropriate or
unavailable. Deglycerolized RBCs may be useful for transfusions to patients with previous
severe allergic transfusion reactions, because the process efficiently removes plasma
constituents.
In addition to the side effects and hazards of RBC transfusion, Deglycerolized RBCs carry a
risk of intravascular hemolysis if deglycerolization has been inadequate.
Deglycerolized RBCs must be transfused within 24 hours after thawing if prepared in an
open system. If prepared in a closed system, they can be infused within a 2-week interval
after thawing.
REJUVENATED RED BLOOD CELLS (RED BLOOD CELLS REJUVENATED) may be
prepared from red cells stored in CPD, CPDA-1, and AS-1 storage solutions up to 3 days
after expiration. Addition of an FDA-approved solution containing inosine, phosphate, and
adenine restores 2,3-diphosphoglycerate and adenosine triphosphate to levels approximating
those of freshly drawn cells. These products must be washed before infusion to remove the
inosine, which may be toxic. Rejuvenated RBCs may be prepared and transfused within 24
hours or frozen for long-term storage.
DEGLYCEROLIZED REJUVENATED RED BLOOD CELLS (RED BLOOD CELLS
REJUVENATED DEGLYCEROLIZED) is the form in which rejuvenated, cryopreserved red cells
(Frozen Rejuvenated Red Blood Cells) are made available for infusion. For additional
information, see sections on Rejuvenated RBCs and Deglycerolized RBCs above.
Autologous Whole Blood and RBCs are drawn from patients who anticipate requiring
blood transfusions. Donor-safety screening criteria and testing procedures applicable to
collection from allogeneic donors do not always apply to these components. Each unit must
be labeled “FOR AUTOLOGOUS USE ONLY.” A biohazard label is required if these units
have a reactive test result. In addition, if these units are untested, they must be labeled as
“DONOR UNTESTED.” Autologous Whole Blood or RBCs can be modified into any of the
components described above. If a facility allows for autologous units to be crossed over for
inclusion in the general blood inventory, the donors and units must be subjected to the same
donor eligibility requirements and test requirements as allogeneic donors and units.
See section on Further Processing for irradiated products.
Plasma Components
Overview
Plasma is the aqueous part of blood and can be derived from the separation of a whole-blood
collection or by apheresis collection. Important elements in plasma include albumin, coagulation
factors, fibrinolytic proteins, immunoglobulin, and other proteins. Once plasma is collected, it
can be stored frozen and subsequently thawed and kept in a liquid state. If Fresh Frozen Plasma
(FFP) is thawed at 1 to 6 C, and the insoluble cryoprecipitate (see Cryoprecipitated Components)
is removed by centrifugation, the supernatant plasma can be refrozen and labeled as Plasma
Cryoprecipitate Reduced. Labile coagulation factor levels vary based upon ABO group, storage
conditions, and/or further processing (see Table 3).
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Table 3. Coagulation Factor Activity of Thawed Plasma Derived from FFP*
Level†
Day 1
Day 2
Day 3
Day 4
Day 5
Mean Change
from Day 1 to
Day 5 (%)
107 ± 26
103 ± 44
70 ± 16
81 ± 9
79 ± 7
90 ± 18
85 ± 13
225 ± 12
76 ± 19
74 ± 37
51 ± 10
81 ± 9
75 ± 8
81 ± 15
84 ± 13
224 ± 13
66 ± 18
71 ± 35
43 ± 10
81 ± 9
71 ± 9
76 ± 15
84 ± 15
224 ± 13
65 ± 17
67 ± 36
43 ± 7
80 ± 10
68 ± 9
72 ± 14
82 ± 11
224 ± 17
63 ± 16
67 ± 33
41 ± 8
80 ± 10
66 ± 9
72 ± 15
80 ± 11
225 ± 12
41
35
41
1
16
20
6
0
Coagulation
Factor
Factor VIII (%)
Blood group A
Blood group B
Blood group O
Factor II (%)
Factor V (%)
Factor VII (%)
Factor X
Fibrinogen
(mg/dL)
p Values
<0.004‡
<0.02‡
<0.001‡
NS
NS
NS
NS
NS
*Reported with permission from Downes KA, Wilson E, Yomtovian R, Sarode R. Serial measurement of clotting factors in thawed plasma for 5
days (letter). Transfusion 2001;41:570.
†
Mean ± SD.
‡
Comparison of Factor VIII activity at Day 1 and that at Day 3 was statistically significant.
Fresh Frozen Plasma
Description
FRESH FROZEN PLASMA (FRESH FROZEN PLASMA) is prepared from a whole blood or
apheresis collection and frozen at –18 C or colder within the time frame as specified in the
directions for use for the blood collection, processing, and storage system. The anticoagulant
solution used and the component volume are indicated on the label. On average, units contain
200 to 250 mL, but apheresis-derived units may contain as much as 400 to 600 mL. FFP contains
plasma proteins including all coagulation factors. FFP contains high levels of the labile
coagulation Factors V and VIII.
FFP should be infused immediately after thawing or stored at 1 to 6 C for up to 24 hours. If
stored longer than 24 hours, the component must be relabeled (see Thawed Plasma) or discarded
depending on the method of collection.
Action
FFP serves as a source of plasma proteins for patients who are deficient in or have defective
plasma proteins.
Indications
FFP is indicated in the following conditions:
1. Management of preoperative or bleeding patients who require replacement of multiple
plasma coagulation factors (eg, liver disease, DIC).
2. Patients undergoing massive transfusion who have clinically significant coagulation
deficiencies.
3. Patients taking warfarin who are bleeding or need to undergo an invasive procedure before
vitamin K could reverse the warfarin effect or who need only transient reversal of warfarin
effect.
4. For transfusion or plasma exchange in patients with thrombotic thrombocytopenic purpura
(TTP).
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5.
6.
Management of patients with selected coagulation factor deficiencies, congenital or acquired,
for which no specific coagulation concentrates are available.
Management of patients with rare specific plasma protein deficiencies, such as C1 inhibitor,
when recombinant products are unavailable.
Contraindications
Do not use this product when coagulopathy can be corrected more effectively with specific
therapy, such as vitamin K, Cryoprecipitated AHF (Antihemophilic Factor), or specific
coagulation factor concentrates.
Do not use this product when blood volume can be safely and adequately replaced with other
volume expanders.
Dosage and Administration
Compatibility tests before transfusion are not necessary. Plasma must be ABO compatible with
the recipient’s red cells. The volume transfused depends on the clinical situation and patient size,
and may be guided by laboratory assays of coagulation function.
Do not use FFP if there is evidence of container breakage or of thawing during storage. FFP
must be thawed in a waterbath at 30 to 37 C or in an FDA-cleared device. If a waterbath is used,
thaw the component in a protective plastic overwrap using gentle agitation.
Side Effects and Hazards
Hazards that pertain to all transfusion components, including FFP, are described in the earlier
section on Side Effects and Hazards for Whole Blood and All Blood Components.
Plasma Frozen Within 24 Hours After Phlebotomy
Description
PLASMA FROZEN WITHIN 24 HOURS AFTER PHLEBOTOMY (PLASMA FROZEN
WITHIN 24 HOURS AFTER PHLEBOTOMY) is prepared from a whole blood collection and must
be separated and placed at –18 C or below within 24 hours from whole blood collection. The
anticoagulant solution used and the component volume are indicated on the label. On average,
units contain 200 to 250 mL. This plasma component is a source of nonlabile plasma proteins.
Levels of Factor VIII are significantly reduced and levels of Factor V and other labile plasma
proteins are variable compared with FFP.
Plasma Frozen Within 24 Hours After Phlebotomy should be infused immediately after
thawing or stored at 1 to 6 C for up to 24 hours. If stored longer than 24 hours, the component
must be relabeled (see Thawed Plasma) or discarded.
Action
This plasma component serves as a source of plasma proteins for patients who are deficient in or
have defective plasma proteins. Coagulation factor levels might be lower than those of FFP,
especially labile coagulation Factors VIII and V.
Indications
See Fresh Frozen Plasma.
Contraindications
See Fresh Frozen Plasma. In addition, this product is not indicated for treatment of deficiencies
of labile coagulation factors including Factors VIII and V.
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Dosage and Administration
See Fresh Frozen Plasma.
Side Effects and Hazards
See Fresh Frozen Plasma.
Plasma Cryoprecipitate Reduced
Description
PLASMA CRYOPRECIPITATE REDUCED (PLASMA, CRYOPRECIPITATE REDUCED) is
prepared from FFP after thawing and centrifugation and removal of the cryoprecipitate. The
remaining product is plasma that is deficient in fibrinogen, Factor VIII, Factor XIII, von
Willebrand factor (vWF), cryoglobulin, and fibronectin. This supernatant plasma must be
refrozen within 24 hours. Proteins such as albumin; ADAMTS13; and Factors II, V, VII, IX, X,
and XI remain in almost the same levels as in FFP [the high-molecular-weight forms of vWF
(multimers) are more thoroughly removed by this process than smaller multimers].
Action
This component serves as a source for plasma proteins except for fibrinogen, Factor VIII, Factor
XIII, and vWF.
Indications
Plasma Cryoprecipitate Reduced is used for transfusion or plasma exchange in patients with
TTP. It may be used to provide clotting factors except fibrinogen, Factor VIII, Factor XIII, and
vWF.
Contraindications
This component should not be used as a substitute for FFP, Plasma Frozen Within 24 Hours
After Phlebotomy, or Thawed Plasma.
Dosage and Administration
See Fresh Frozen Plasma.
Side Effects and Hazards
See Fresh Frozen Plasma.
Liquid Plasma Components
Description
Other plasma components may be made from whole blood collected in all approved
anticoagulants. Levels and activation state of coagulation proteins in these products are variable.
The volume is indicated on the label.
THAWED PLASMA Ω (THAWED PLASMA) is derived from FFP or Plasma Frozen Within
24 Hours After Phlebotomy, prepared using aseptic techniques (closed system), thawed at 30 to
37 C, and maintained at 1 to 6 C for up to 4 days after the initial 24-hour post-thaw period has
elapsed. The volume is indicated on the label. Thawed Plasma contains stable coagulation factors
such as Factor II and fibrinogen in concentrations similar to those of FFP, but variably reduced
amounts of other factors (see Table 3).
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Action
This component serves as a source of plasma proteins. Levels and activation state of coagulation
proteins in thawed plasma are variable and change over time.
Indications
1. Management of preoperative or bleeding patients who require replacement of multiple
plasma coagulation factors except for patients with a consumptive coagulopathy.
2. Initial treatment of patients undergoing massive transfusion who have clinically significant
coagulation deficiencies.
3. Patients taking warfarin who are bleeding or need to undergo an invasive procedure before
vitamin K could reverse the warfarin effect or who need only transient reversal of warfarin
effect.
This component should not be used to treat isolated coagulation factor deficiencies where
other products are available with higher concentrations of the specific factor(s).
Contraindications
See Fresh Frozen Plasma. Do not use liquid plasma components as the treatment for isolated
coagulation factor deficiencies where other products are available with higher concentrations of
the specific factor(s).
Dosage and Administration
See Fresh Frozen Plasma.
Side Effects and Hazards
See Fresh Frozen Plasma.
LIQUID PLASMA (LIQUID PLASMA) is separated no later than 5 days after the expiration date
of the Whole Blood and is stored at 1 to 6 C. The profile of plasma proteins in Liquid Plasma is
poorly characterized. Levels and activation state of coagulation proteins in Liquid Plasma are
dependent upon and change with time in contact with cells, as well as the conditions and
duration of storage.
Action
This component serves as a source of plasma proteins. Levels and activation state of coagulation
proteins are variable and change over time.
Indications
Initial treatment of patients who are undergoing massive transfusion because of life-threatening
trauma/hemorrhages and who have clinically significant coagulation deficiencies.
Contraindications
See Fresh Frozen Plasma. Do not use liquid plasma components as the treatment for isolated
coagulation factor deficiencies where other products are available with higher concentrations of
the specific factor(s).
Dosage and Administration
See Fresh Frozen Plasma.
Side Effects and Hazards
See Fresh Frozen Plasma.
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Cryoprecipitated Components
Overview
Description
Cryoprecipitated Antihemophilic Factor (AHF) is prepared by thawing whole-blood-derived FFP
between 1 and 6 C and recovering the precipitate. The cold-insoluble precipitate is refrozen
within 1 hour. Cryoprecipitated AHF contains fibrinogen, Factor VIII, Factor XIII, vWF, and
fibronectin. Each unit of Cryoprecipitated AHF should contain ≥80 IU Factor VIII units and
≥150 mg of fibrinogen in approximately 5 to 20 mL of plasma.
If the label indicates “Pooled Cryoprecipitated AHF,” several units of Cryoprecipitated AHF
have been pooled. The volume of the pool is indicated on the label and, if used, the volume of
0.9% Sodium Chloride, Injection (USP) added may be separately listed. To determine the
minimum potency of this component, assume 80 IU of Factor VIII and 150 mg of fibrinogen for
each unit of Cryoprecipitated AHF indicated on the label.
Action
Cryoprecipitate serves as a source of fibrinogen, Factor VIII, Factor XIII, vWF, and fibronectin.
Indications
This component is used in the control of bleeding associated with fibrinogen deficiency and to
treat Factor XIII deficiency. It is also indicated as second-line therapy for von Willebrand
disease and hemophilia A (Factor VIII deficiency). Coagulation factor preparations other than
cryoprecipitate are preferred when blood component therapy is needed for management of von
Willebrand disease and Factor VIII deficiency. Use of this component may be considered for
control of uremic bleeding after other modalities have failed. Indications for use as a source of
fibronectin are not clear.
Contraindications
Do not use this component unless results of laboratory studies indicate a specific hemostatic
defect for which this product is indicated. Cryoprecipitate should not be used if virus-inactivated
Factor VIII concentrates or recombinant factor preparations are available for management of
patients with von Willebrand disease or hemophilia A.
Dosage and Administration
Compatibility testing is unnecessary. ABO-compatible material is preferred. Rh type need not be
considered when using this component.
The frozen component is thawed in a protective plastic overwrap in a waterbath at 30 to 37 C
up to 15 minutes (thawing time may need to be extended if product is pooled before freezing).
This component should not be given if there is evidence of container breakage or of thawing
during storage. Do not refreeze after thawing. Thawed Cryoprecipitated AHF should be kept at
room temperature and transfused as soon as possible after thawing, within 6 hours if it is a single
unit (from individual donor, or pooled before freezing or administration using an FDA-cleared
sterile connecting device), and within 4 hours after entering the container (eg, to attach an
administration set or to pool) without using an FDA-cleared sterile connecting device.
Cryoprecipitated AHF may be transfused as individual units or pooled. For pooling, the
precipitate in one or more concentrates should be mixed well with 10 to 15 mL of diluent to
ensure complete removal of all material from the container. The preferred diluent is 0.9%
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Sodium Chloride, Injection (USP). Serial use of each bag’s contents to resuspend the precipitate
into subsequent bags may be used to efficiently pool cryoprecipitate into a single bag.
The recovery of transfused fibrinogen is 50% to 60%. When used to correct
hypofibrinogenemia, Cryoprecipitated AHF may be dosed according to the following formula to
raise plasma fibrinogen by approximately 50 to 100 mg/dL: Number of bags = 0.2 × body weight
in kg. Thrombosis alters fibrinogen kinetics; therefore, patients receiving cryoprecipitate as
fibrinogen replacement in conditions associated with increased fibrinogen turnover should be
monitored with fibrinogen assays.
For treatment of bleeding in patients with hemophilia A when Factor VIII concentrates are not
available, rapid infusion of a loading dose expected to produce the desired level of Factor VIII is
usually followed by a smaller maintenance dose every 8 to 12 hours. To maintain hemostasis
after surgery, a regimen of therapy for 10 days or longer may be required. If circulating
antibodies to Factor VIII are present, the use of larger doses, activated concentrates, porcinederived concentrates, or other special measures may be indicated. To calculate cryoprecipitate
dosage as a source of Factor VIII, the following formula is helpful: Number of bags = (Desired
increase in Factor VIII level in % × 40 × body weight in kg) / average units of Factor VIII per
bag, minimum 80. Good patient management requires that the Cryoprecipitated AHF treatment
responses of Factor VIII-deficient recipients be monitored with periodic plasma Factor VIII
assays.
For treatment of von Willebrand disease, smaller amounts of Cryoprecipitated AHF will
correct the bleeding time. Because the vWF content of Cryoprecipitated AHF is not usually
known, an empiric dose of 1 bag per 10 kg of body weight has been recommended. These
patients should be monitored by appropriate laboratory studies to determine the frequency of
Cryoprecipitated AHF administration.
Side Effects and Hazards
Hazards that pertain to all transfusion components are described in the earlier section on Side
Effects and Hazards for Whole Blood and All Blood Components.
If a large volume of ABO-incompatible cryoprecipitate is used, the recipient may develop a
positive DAT and, very rarely, mild hemolysis.
Components Available
1. CRYOPRECIPITATED AHF (CRYOPRECIPITATED AHF)
2. POOLED CRYOPRECIPITATED AHF (CRYOPRECIPITATED AHF, POOLED)
Platelet Components
Overview
Description
Platelet therapy may be achieved by infusion of either Apheresis Platelets or Platelets (wholeblood-derived platelet concentrates). In either component, platelets are suspended in an
appropriate volume of the original plasma, which contains near-normal levels of stable
coagulation factors that are stored at room temperature. One unit of Platelets derived from a
whole blood collection usually contains no fewer than 5.5 × 1010 platelets suspended in 40 to 70
mL of plasma. Platelets may be provided either singly or as a pool. One unit of Apheresis
Platelets usually contains ≥3.0 × 1011 platelets and is a therapeutic equivalent to 4 to 6 units of
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Platelets. Platelet components may contain a varying number of leukocytes depending upon the
technique used in preparation. Some units may contain more than the trace amounts of red cells
usually present and will appear pink to salmon in color.
Actions
Platelets are essential for normal hemostasis. Complex reactions occur between platelets, vWF,
collagen in the walls of disturbed vasculature, phospholipids, and soluble coagulation factors,
including thrombin. These changes induce platelet adherence to vessel walls and platelet
activation, which leads to platelet aggregation and formation of a primary hemostatic plug. The
therapeutic goal of platelet transfusion is to provide adequate numbers of normally functioning
platelets for the prevention or cessation of bleeding.
Indications
Platelet transfusions may be given to patients with thrombocytopenia, dysfunctional platelet
disorders, active platelet-related bleeding, or serious risk of bleeding (ie, prophylactic use).
Patients with the following medical conditions may require platelet transfusion: leukemia,
myelodysplasia, aplastic anemia, solid tumors, congenital or acquired platelet dysfunction,
central nervous system trauma. Patients undergoing extracorporeal membrane oxygenation or
cardiopulmonary bypass may also need platelet transfusion. Thrombocytopenia is unlikely to be
the cause of bleeding in patients with platelet counts of at least 50,000/μL. Higher transfusion
thresholds may be appropriate for patients with platelet dysfunction. For the clinically stable
patient with an intact vascular system and normal platelet function, prophylactic platelet
transfusions may be appropriate at 5000 to 10,000/μL.
Prophylactic platelet transfusion may not be of therapeutic benefit when thrombocytopenia is
related to destruction of circulating platelets secondary to autoimmune disorders [eg, immune
thrombocytopenic purpura (ITP)]; however, when these patients bleed, platelet therapy is often
useful.
Platelets Leukocytes Reduced or Apheresis Platelets Leukocytes Reduced are indicated to
decrease the frequency of recurrent febrile, nonhemolytic transfusion reaction, HLA
alloimmunization, and transfusion-transmitted CMV infection (see section on Further
Processing).
Contraindications
Do not use this component if bleeding is unrelated to decreased numbers of, or abnormally
functioning, platelets. If platelet function is normal, platelets should not be transfused when the
platelet count is greater than 100,000/μL. Prophylactic transfusion is generally not indicated
when platelet dysfunction is extrinsic to the platelet, such as in uremia, certain types of von
Willebrand disease, and hyperglobulinemia. Patients with congenital surface glycoprotein(s)
defects should be transfused conservatively to reduce the possibility for alloimmunization to the
missing protein(s).
Do not use in patients with activation or autoimmune destruction of endogenous platelets, such
as in heparin-induced thrombocytopenia (HIT), TTP, or ITP, unless the patient has a lifethreatening hemorrhage.
Dosage and Administration
Compatibility testing is not necessary in routine platelet transfusion. Except in unusual
circumstances, the donor plasma should be ABO compatible with the recipient’s red cells when
this component is to be transfused to infants or when large volumes are to be transfused. The
number of platelet units to be administered depends on the clinical situation of each patient. One
unit of Platelets would be expected to increase the platelet count of a 70-kg adult by 5000 to
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10,000/μL and increase the count of an 18-kg child by 20,000/μL. The therapeutic adult dose is 1
unit of Apheresis Platelets or 4 to 6 units of whole-blood-derived platelets, either of which
usually contain ≥3.0 × 1011 platelets. For prophylaxis, this dose may need to be repeated in 1 to 3
days because of the short lifespan of transfused platelets (3-4 days). Platelet components must be
examined before administration. Units with excessive aggregates should not be administered.
Transfusion may proceed as quickly as tolerated, but must take less than 4 hours. Do not
refrigerate platelets.
The corrected count increment (CCI) is a calculated measure of patient response to platelet
transfusion that adjusts for the number of platelets infused and the size of the recipient, based
upon body surface area (BSA)
CCI = (post-count – pre-count) × BSA / platelets transfused
where post-count and pre-count are platelet counts (/μL) after and before transfusion,
2
respectively; BSA is the patient body surface area (meter ); and platelets transfused is the
number of administered platelets (× 1011). The CCI is usually determined 10 to 60 minutes after
transfusion. For example:
2
A patient with acute myelogenous leukemia with a nomogram-derived BSA of 1.40 meter is
11
transfused with a unit of Apheresis Platelets (a platelet dose of 4.5 × 10 ). The pretransfusion
platelet count is 2000/μL. The patient’s platelet count from a sample of blood collected 15
minutes after platelet transfusion is 29,000/μL. The CCI is calculated as (29,000 – 2000) × 1.4 /
11
2
4.5 = 8,400/μL per 10 per m .
In the clinically stable patient, the CCI is typically greater than 7500 at 10 minutes to 1 hour
after transfusion and remains above 4500 at 24 hours. Both immune and nonimmune
mechanisms may contribute to reduced platelet recovery and survival. Along with supportive
serologic test results, a CCI of less than 5000 at 10 minutes to 1 hour after transfusion may
indicate an immune-mediated refractory state to platelet therapy. With nonimmune mechanisms,
platelet recovery within 1 hour may be adequate, although survival at 24 hours is reduced (refer
to Platelet Alloimmunization).
Side Effects and Hazards
Hazards that pertain to all transfusion components are described in the section on Side Effects
and Hazards for Whole Blood and All Blood Components. Listed below are hazards that apply
specifically to components that contain platelets.
1. Bacterial Contamination: Although methods to limit and detect bacterial contamination
have been implemented for most platelet components, they remain the most likely blood
components to be contaminated with bacteria. Gram-positive skin flora are the most
commonly recovered bacteria. Symptoms may include high fever (≥2.0 C or ≥3.5 F increase
in temperature), severe chills, hypotension, or circulatory collapse during or immediately after
transfusion. In some instances, symptoms, especially when associated with contamination by
gram-positive organisms, may be delayed for several hours following transfusion. Prompt
management should include broad-spectrum antibiotic therapy along with cultures from the
patient, suspected blood component(s), and administration set. A Gram’s stain of suspected
contaminated unit(s) should be performed whenever possible. Apheresis Platelets are usually
tested for bacterial contamination before issue.
2. Platelet Alloimmunization: Platelets bear a variety of antigens, including HLA and plateletspecific antigens. Patients transfused with platelets often develop HLA antibodies. The
patient may become refractory to incompatible platelets. When platelets are transfused to a
patient with an antibody specific for an expressed antigen, the survival time of the transfused
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3.
4.
platelets may be markedly shortened. Nonimmune events may also contribute to reduced
platelet survival. It is possible to distinguish between immune and nonimmune platelet
refractoriness by assessing platelet recovery soon after infusion (ie, a 10- to 60-minute
postinfusion platelet increment). In immune refractory states secondary to serologic
incompatibility, there is poor recovery in the early postinfusion interval. In nonimmune
mechanisms (ie, splenomegaly, sepsis, fever, intravascular devices, and DIC) platelet
recovery within 1 hour of infusion may be adequate while longer-term survival (ie, 24-hour
survival) is reduced. Serologic tests may confirm the presence of alloimmunization.
Serologic tests (HLA typing or a platelet crossmatch) may also be helpful in selecting
platelets with acceptable survival.
Red Blood Cell Alloimmunization: Immunization to red cell antigens may occur because of
the presence of residual red cells in Platelets. Red cell compatibility testing is necessary only
if the component is prepared by a method that allows the component to contain 2 mL or more
of red cells, making the unit appear pink to salmon in color. When platelet components from
Rh-positive donors must be given to Rh-negative females of childbearing potential because
of lack of availability of Rh-negative platelets, prevention of D immunization by use of Rh
Immune Globulin should be considered.
Hemolysis: Platelet transfusions that are not ABO identical may contain incompatible
plasma and may cause a positive DAT and possibly, hemolysis. Platelet transfusions from
group O donors with high-titer isohemagglutinins (anti-A or anti-B) may cause acute
hemolytic reactions in susceptible patients.
Components Available
1. PLATELETS (PLATELETS) are a concentrate of platelets separated from a single unit of
Whole Blood. One unit of Platelets should contain no fewer than 5.5 × 1010 platelets
suspended in 40 to 70 mL of plasma. This component is usually provided as a pool. See
below.
2. POOLED PLATELETS (PLATELETS POOLED) are composed of individual platelet units
combined by aseptic technique and have an allowable shelf life as specified in the directions
for use for the blood collection, processing, and storage system. The number of units of
Platelets in the pool will be indicated on the label. To determine the minimum potency of this
component, assume 5.5 × 1010 platelets per unit of Platelets indicated on the label. See the
label for the approximate volume.
3. PLATELETS LEUKOCYTES REDUCED (PLATELETS LEUKOCYTES REDUCED) may be
prepared using an open or closed system. One unit of Platelets Leukocytes Reduced should
10
5
contain 5.5 × 10 platelets and <8.3 × 10 leukocytes. Components prepared using an open
system will expire 4 hours after preparation. Components prepared using a closed system will
have a shelf life as specified in the directions for use for the blood collection, processing, and
storage system. This component is usually provided as a pool. See below.
4. POOLED PLATELETS LEUKOCYTES REDUCED (PLATELETS LEUKOCYTES
REDUCED, POOLED) may be prepared by pooling and filtering Platelets or pooling Platelets
Leukocytes Reduced in an open system that will have a 4-hour shelf life. The number of
units in the pool will be indicated on the label. To determine the minimum potency of this
10
component, assume 5.5 × 10 platelets per unit of Platelets Leukocytes Reduced indicated on
the label and <5 × 106 leukocytes in the pool. See the label for the approximate volume. This
component can also be prepared and pooled using an FDA-cleared system to provide a
product with a 5-day shelf life. Components prepared using this system provide a therapeutic
adult dose of platelets and <5.0 × 106 leukocytes.
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5.
6.
APHERESIS PLATELETS (PLATELETS PHERESIS) are an effective way to harvest a
therapeutic adult dose of platelets from a single donor. Apheresis Platelets should contain
11
≥3.0 × 10 platelets. One unit of Apheresis Platelets may replace 4 to 6 units of Platelets. The
volume of plasma is indicated on the label and varies between 100 and 500 mL. The number
of leukocytes contained in this component varies depending upon the blood cell separator
and protocol used for collection. Apheresis Platelets are supplied in one bag or in two
connected bags to improve platelet viability during storage by providing more surface area
for gas exchange. ACD-A is the anticoagulant solution currently used for the collection and
preservation of Apheresis Platelets.
APHERESIS PLATELETS LEUKOCYTES REDUCED (PLATELETS PHERESIS
LEUKOCYTES REDUCED) can be leukocyte reduced during the collection process or may be
prepared by further processing using leukocyte reduction filters. Apheresis Platelets
Leukocytes Reduced should contain ≥3.0 × 1011 platelets and <5.0 × 106 leukocytes. When
Apheresis Platelets Leukocytes Reduced are prepared by further processing, these may be
labeled Apheresis Platelets Leukocytes Reduced provided the requirement for residual
leukocyte count is met and the platelet recovery is at least 85% of the prefiltration content.
The volume, anticoagulant-preservative, and storage conditions for Apheresis Platelets
Leukocytes Reduced are the same as those for Apheresis Platelets. Apheresis Platelets
Leukocytes Reduced have a shelf life of 5 days, unless the facility is participating in a postmarketing program, which allows a 7-day expiration date.
Granulocyte Components
Description
APHERESIS GRANULOCYTES Ω (GRANULOCYTES PHERESIS) contain numerous leukocytes
and platelets as well as 20 to 50 mL of red cells. The number of granulocytes in each concentrate
is usually >1.0 × 1010. Various modalities may be used to improve granulocyte harvest, including
donor administration of granulocyte colony-stimulating factor and/or corticosteroids. The final
volume of the product is 200 to 300 mL including anticoagulant and plasma as indicated on the
label.
Red cell sedimenting agents approved by the FDA, such as hydroxyethyl starch (HES), are
typically used in the collection of granulocytes. Residual agents will be present in the final
component and are described on the label. Apheresis Granulocytes should be administered as
soon after collection as possible because of well-documented deterioration of granulocyte
function during short-term storage. If stored, maintain at 20 to 24 C without agitation for no
more than 24 hours.
Actions
Granulocytes migrate toward, phagocytize, and kill bacteria and fungi. A quantitative
relationship exists between the level of circulating granulocytes and the prevalence of bacterial
and fungal infection in neutropenic patients. The ultimate goal is to provide the patient with the
ability to fight infection. The infusion of a granulocyte component may not be associated with a
significant increase in the patient’s granulocyte count and is dependent on multiple factors,
including the patient’s clinical condition.
Indications
Granulocyte transfusion therapy is controversial. Apheresis Granulocytes are typically used in
the treatment of patients with documented infections (especially gram-negative bacteria and
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fungi) unresponsive to antimicrobial therapy in the setting of neutropenia [absolute granulocyte
count <0.5 × 109/L (500/μL)] with expected eventual marrow recovery, or neonatal sepsis. A trial
of broad-spectrum antimicrobial agents should be used before granulocyte transfusion therapy is
initiated. If the intended recipient is CMV-seronegative and severely immunosuppressed (eg, a
marrow transplant recipient), serious consideration should be given before administration of
CMV-seropositive granulocytes. In addition to neutropenic patients, patients with hereditary
neutrophil function defects (such as chronic granulomatous disease) may be candidates for
granulocyte transfusion therapy.
Contraindications
Prophylactic use of granulocytes in noninfected patients is not routinely recommended.
Dosage and Administration
Transfuse as soon as possible. A standard blood infusion set is to be used for the administration
of Apheresis Granulocytes. Do not administer using leukocyte reduction filters. Depth-type
microaggregate filters and leukocyte reduction filters remove granulocytes.
The red cells in Apheresis Granulocytes must be ABO compatible. Once granulocyte
transfusion therapy is initiated, support should continue at least daily until infection is cured,
9
defervescence occurs, the absolute granulocyte count returns to at least 0.5 × 10 /L (500/μL), or
the physician in charge decides to halt the therapy.
Because most patients receiving these products are severely immunosuppressed, Apheresis
Granulocytes are usually irradiated to prevent TA-GVHD (see section on Further Processing).
Side Effects and Hazards
Hazards that pertain to all transfusion components are described in the section on Side Effects
and Hazards for Whole Blood and All Blood Components. Listed below are hazards that apply
specifically to Apheresis Granulocytes.
1. Febrile Nonhemolytic Reactions: These reactions are frequently noted in patients receiving
granulocyte transfusions. Fever and chills in patients receiving granulocyte components may
be avoided or mitigated by slow administration and recipient premedication.
2. Allergic Reactions: Allergic reactions to HES and other red cell sedimenting solutions may
occur during granulocyte transfusion.
3. Pulmonary Reactions: Granulocyte transfusion can cause worsening of pulmonary function
in patients with pneumonia, and rarely severe pulmonary reactions, especially in patients
receiving concomitant amphotericin B.
4. Alloimmunization: Immunization to HLA antigens frequently occurs with granulocyte
transfusion and can cause refractoriness to platelet transfusion.
Further Processing
This section addresses further processing of previously described blood components. The
processes described in this section are: Leukocyte reduction, identification of CMV-seronegative
components, irradiation, and washing. A component may undergo one or more of these
processes.
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Leukocyte Reduction
Description
A unit of whole blood generally contains ≥1 to 10 × 109 white cells. Leukocyte reduction may be
achieved by in-process collection or filtration: 1) soon after collection (prestorage), 2) after
varying periods of storage in the laboratory, or 3) at the bedside. The method used in the
laboratory for leukocyte reduction is subject to quality control testing; leukocyte-reduced
components prepared at the bedside are not routinely subjected to quality control testing.
Leukocyte reduction will decrease the cellular content and volume of blood according to
characteristics of the filter system used. Red Blood Cells Leukocytes Reduced, Apheresis Red
Blood Cells Leukocytes Reduced, and Apheresis Platelets Leukocytes Reduced must have a
residual content of leukocytes <5.0 × 106 and Platelets Leukocytes Reduced must have <8.3 × 105
residual leukocytes. Leukocyte reduction filters variably remove other cellular elements in
addition to white cells. Washing is not a substitute for leukocyte reduction. Leukocyte reduction
is not a substitute for irradiation.
Indications
Leukocyte-reduced components are indicated to decrease the frequency of recurrent febrile
nonhemolytic transfusion reactions. They have also been shown to reduce the risk of transfusiontransmitted CMV and to reduce the incidence of HLA alloimmunization.
Contraindications
Leukocyte-reduced components do not prevent TA-GVHD. Leukocyte reduction filters are not to
be used in the administration of Apheresis Granulocytes or Apheresis Granulocytes/Platelets.
Side Effects and Hazards
The use of blood components that are leukocyte reduced at the bedside may cause unexpected
severe hypotension in some recipients, particularly those taking angiotensin converting enzyme
inhibitor medication.
Specific Leukocyte-Reduced Components
RED BLOOD CELLS LEUKOCYTES REDUCED (RED BLOOD CELLS LEUKOCYTES
REDUCED)
APHERESIS RED BLOOD CELLS LEUKOCYTES REDUCED (RED BLOOD CELLS
PHERESIS LEUKOCYTES REDUCED)
PLATELETS LEUKOCYTES REDUCED (PLATELETS LEUKOCYTES REDUCED)
APHERESIS PLATELETS LEUKOCYTES REDUCED (PLATELETS PHERESIS
LEUKOCYTES REDUCED)
Further Testing to Identify CMV-Seronegative Blood
Description
CMV-seronegative blood is selected by performing testing for antibodies to CMV. Transmission
of CMV disease is associated with cellular blood components. Plasma, cryoprecipitate, and other
plasma-derived blood components do not transmit CMV; therefore, CMV testing is not required
for these components.
Indications
Transfusion of CMV-negative blood is indicated in CMV-seronegative recipients who are at risk
for severe CMV infections. These at-risk groups include pregnant women and their fetuses, low
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birthweight infants, hematopoietic progenitor cell transplant recipients, solid-organ transplant
recipients, severely immunosuppressed recipients, and HIV-infected patients. Leukocyte-reduced
components may be an alternative to CMV-seronegative transfusion in some clinical conditions.
Irradiation
Description
Blood components that contain viable lymphocytes may be irradiated to prevent proliferation of
T lymphocytes, which is the immediate cause of TA-GVHD. Irradiated blood is prepared by
exposing the component to a radiation source. The standard dose of gamma irradiation is 2500
cGy targeted to the central portion of the container with a minimum dose of 1500 cGy delivered
to any part of the component.
Indications
Irradiated cellular components are indicated for use in patient groups that are at risk for TAGVHD from transfusion. At-risk groups include: fetal and neonatal recipients of intrauterine
transfusions, selected immunocompromised recipients, recipients of cellular components known
to be from a blood relative, recipients who have undergone marrow or peripheral blood
progenitor cell transplantation, and recipients of cellular components whose donor is selected for
HLA compatibility.
Side Effects and Hazards
Irradiation induces erythrocyte membrane damage. Irradiated red cells have been shown to have
higher supernatant potassium levels than nonirradiated red cells. Removal of residual supernatant
plasma before transfusion may reduce the risks associated with elevated plasma potassium. The
expiration date of irradiated red cells is changed to 28 days after irradiation if remaining shelf
life exceeds 28 days. There are no known adverse effects following irradiation of platelets; the
expiration date is unchanged.
Washing
Description
Washed components are typically prepared using 0.9% Sodium Chloride, Injection (USP) with
or without small amounts of dextrose. Washing removes unwanted plasma proteins, including
antibodies and glycerol from previously frozen units. There will also be some loss of red cells
and platelets, as well as a loss of platelet function through platelet activation. The shelf life of
washed components is no more than 24 hours at 1 to 6 C or 4 hours at 20 to 24 C. Washing is not
a substitute for leukocyte reduction.
Indications
Washing of blood components is indicated to remove unwanted plasma when it contains
constituents that predispose patients to significant transfusion reactions (eg, the removal of IgAcontaining plasma in providing transfusion support for an IgA-deficient recipient or in rare
recipients experiencing anaphylactoid reactions to plasma components).
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Specific Washed Components
WASHED RED BLOOD CELLS (RED BLOOD CELLS WASHED)
WASHED APHERESIS RED BLOOD CELLS (RED BLOOD CELLS PHERESIS WASHED)
WASHED PLATELETS (PLATELETS WASHED)
WASHED APHERESIS PLATELETS (PLATELETS PHERESIS WASHED)
Volume Reduction
Description
Volume reduction is a special manipulation of cellular blood products using centrifugation. The
process involves the aseptic removal of a portion of the supernatant, containing plasma and
storage medium. Volume reduction removes excess plasma, thereby reducing unwanted plasma
proteins, including antibodies. It is more commonly used in pediatric and in-utero transfusions.
There will be some loss of platelet function through platelet activation as a result of volume
reduction. The shelf life of volume-reduced components is no more than 24 hours at 1 to 6 C or 4
hours at 20 to 24 C.
Indications
Reducing the plasma volume of cellular components is indicated in cases where the volume
status of a patient is being aggressively managed, such as in infants with compromised cardiac
function. Volume reduction may be used to reduce exposure to plasma proteins or additives
(such as mannitol), to achieve a specific component concentration, or to reduce exposure to
antibodies targeting known recipient antigens (especially in an Apheresis Platelet unit containing
ABO-incompatible plasma collected from a mother for the treatment of neonatal alloimmune
thrombocytopenia).
Contraindications
Volume reduction is not a substitute for washing or for dosing with small aliquots.
Volume reduction of platelets may result in adverse consequences associated with
overtransfusion of platelets.
Specific Volume-Reduced Components
RED BLOOD CELLS VOLUME REDUCED (VOLUME REDUCED RED BLOOD CELLS)
APHERESIS RED BLOOD CELLS VOLUME REDUCED (VOLUME REDUCED RED
BLOOD CELLS PHERESIS)
PLATELETS VOLUME REDUCED (VOLUME REDUCED PLATELETS)
APHERESIS PLATELETS VOLUME REDUCED (VOLUME REDUCED PLATELETS
PHERESIS)
References
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Page 126 of 290
34
Table 4. Summary Chart of Blood Components
Action/Recipient
Benefit
Not Indicated for
Special
Precautions
Hazards*
Rate of
Infusion
Category
Major Indications
Red Blood Cells;
Red Blood Cells, Low
Volume; Apheresis Red
Blood Cells
Symptomatic anemia.
Increases oxygencarrying capacity.
Pharmacologically
treatable anemia.
Coagulation
deficiency.
Volume expansion.
Must be ABO
compatible.
Infectious diseases.
Hemolytic, septic/toxic,
allergic, febrile reactions.
TACO.
TRALI.
TA-GVHD.
As fast as
patient can
tolerate but
less than
4 hours.
Deglycerolized Red Blood
Cells
See Red Blood Cells.
IgA deficiency with
anaphylactoid
reaction.
See Red Blood
Cells.
Deglycerolization
removes plasma
proteins.
See Red Blood Cells.
See Red Blood
Cells.
See Red Blood Cells.
Hemolysis due to
incomplete
deglycerolization can
occur.
See Red Blood
Cells.
Risk of allergic and
febrile reactions
reduced.
Red Blood Cells Leukocytes
Reduced; Apheresis Red
Blood Cells Leukocytes
Reduced
See Red Blood Cells.
Reduction of febrile
reactions.
See Red Blood Cells
Reduction of
leukocytes reduces
risk of febrile
reactions, HLA
alloimmunization
and CMV
infection.
See Red Blood Cells.
Leukocyte reduction
should not be used to
prevent TA-GVHD.
See Red Blood
Cells.
See Red Blood Cells.
Hypotensive reaction may
occur if bedside
leukocyte reduction filter
is used.
See Red Blood
Cells.
Washed Red Blood Cells
See Red Blood Cells.
IgA deficiency with
anaphylatoid
reaction.
Recurrent severe
allergic reactions to
unwashed red cell
products.
See Red Blood
Cells.
Washing reduces
plasma proteins.
Risk of allergic
reactions may be
reduced.
See Red Blood
Cells.
See Red Blood
Cells.
See Red Blood
Cells.
See Red Blood
Cells.
(Continued)
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35
Table 4. Summary Chart of Blood Components (Continued)
Special
Precautions
Rate of
Infusion
Action/Recipient
Benefit
Not Indicated for
Symptomatic anemia
with large volume
deficit.
Increases oxygencarrying capacity.
Increases blood
volume.
Condition responsive to
specific component.
Treatment of
coagulopathy.
Must be ABO
identical.
See Red Blood Cells.
As fast as
patient can
tolerate but
less than
4 hours.
Fresh Frozen Plasma (FFP)
Clinically significant
plasma protein
deficiencies when no
specific coagulation
factors are available.
TTP.
Source of plasma
proteins, including
all coagulation
factors.
Volume expansion.
Coagulopathy that can
be more effectively
treated with specific
therapy.
Must be ABO
compatible.
Infectious diseases.
Allergic reactions.
TACO.
TRALI.
Less than
4 hours.
Plasma Frozen Within 24
Hours After Phlebotomy
(PF24)
Clinically significant
deficiency of stable
coagulation factors.
Source of nonlabile
plasma proteins.
Levels of Factor
VIII are significantly reduced and
levels of Factor V
and other labile
plasma proteins
are variable
compared with
FFP.
Volume expansion.
Deficiencies of labile
coagulation factors
including Factors VIII
and V.
Must be ABO
compatible.
See FFP.
Less than
4 hours.
Plasma Cryoprecipitate
Reduced
TTP.
Plasma protein
replacement for
plasma exchange
in TTP.
Deficient in fibrinogen, Factor VIII,
vWF, and Factor
XIII.
Deficient in highmolecular-weight
vWF multimers as
compared to FFP.
Volume expansion.
Deficiency of
coagulation factors
known to be depleted
in this product,
fibrinogen, Factors
VIII, vWF, and XIII.
Must be ABO
compatible.
See FFP.
Less than
4 hours.
Category
Major Indications
Whole Blood
Hazards*
(Continued)
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Page 128 of 290
36
Table 4. Summary Chart of Blood Components (Continued)
Action/Recipient
Benefit
Category
Major Indications
Thawed Plasma Ω
Bleeding patients
except consumptive
coagulopathy.
Source of plasma
proteins.
Not indicated as
treatment for isolated
coagulation factor
deficiencies.
Reversal of warfarin
effect.
Levels and
activation state of
coagulation
proteins in thawed
plasma are
variable and
change over time.
Levels and activation
state of coagulation
proteins in thawed
plasma are variable and
change over time.
Initial treatment of
patients undergoing
massive transfusion.
Coagulation support
for life-threatening
trauma/
hemorrhages.
Not indicated as
treatment for isolated
coagulation factor
deficiencies.
The profile of
plasma proteins in
Liquid Plasma is
poorly characterized. Levels and
activation state of
coagulation
proteins are
dependent upon
and change with
time in contact
with cells, as well
as the conditions
and duration of
storage.
The profile of plasma
proteins in Liquid
Plasma is poorly
characterized. Levels
and activation state of
coagulation proteins
are dependent upon
and change with time
in contact with cells, as
well as the conditions
and duration of
storage.
Liquid Plasma
Not Indicated for
Special
Precautions
Hazards*
Rate of
Infusion
Must be ABO
compatible.
See FFP.
Less than
4 hours.
Must be ABO
compatible.
See FFP.
Less than
4 hours.
(Continued)
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37
Table 4. Summary Chart of Blood Components (Continued)
Hazards*
Rate of
Infusion
Infectious diseases.
Allergic reactions.
Less than
4 hours.
Should not use
some filters
(check
manufacturer’s
instructions).
Infectious diseases.
Septic/toxic, allergic,
febrile reactions.
TACO.
TA-GVHD.
TRALI.
Less than
4 hours.
See Platelets.
See Platelets.
See Platelets.
See Platelets.
See Platelets.
Reduction of
leukocytes reduces
risk of febrile
reactions, HLA
alloimmunization,
and CMV infection.
See Platelets.
Leukocyte reduction
should not be used to
prevent TA-GVHD.
See Platelets.
See Platelets.
See Platelets.
Provides granulocytes
with or without
platelets.
Infection responsive to
antibiotics, eventual
marrow recovery not
expected.
Must be ABO
compatible.
Should not use
some filters
(check
manufacturer’s
instructions).
Infectious diseases.
Hemolytic, allergic, febrile
reactions.
TACO.
TRALI.
TA-GVHD.
One unit over
2-4 hours.
Closely observe
for reactions.
Action/Recipient
Benefit
Category
Major Indications
Cryoprecipitated AHF; Pooled
Cryoprecipitated AHF
Hypofibrinogenemia.
Factor XIII deficiency.
von Willebrand disease.
Hemophilia A.
Provides fibrinogen,
vWF, Factor XIII,
and Factor VIII.
Deficiency of any plasma
protein other than those
enriched in Cryoprecipitated AHF.
Platelets; Pooled Platelets
Bleeding due to
thrombocytopenia or
platelet function
abnormality.
Prevention of bleeding
from marrow
hypoplasia.
Improves hemostasis.
Plasma coagulation
deficits.
Some conditions with
rapid platelet destruction
(eg, ITP, TTP) unless
life-threatening
hemorrhage.
Apheresis Platelets
See Platelets.
See Platelets.
May be HLA (or other
antigen) selected.
Platelets Leukocytes Reduced;
Pooled Platelets Leukocytes
Reduced; Apheresis Platelets
Leukocytes Reduced
See Platelets.
Reduction of febrile
reactions.
Reduction of HLA
alloimmunization.
Apheresis Granulocytes Ω ;
Apheresis Granulocytes/
Platelets Ω
See Platelets.
Neutropenia with
infection,
unresponsive to
appropriate antibiotics.
Not Indicated for
Special
Precautions
(Continued)
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Page 130 of 290
38
Table 4. Summary Chart of Blood Components (Continued)
Category
Major Indications
Action/Recipient
Benefit
Not Indicated for
Special
Precautions
Hazards*
Rate of
Infusion
See component.
See component.
See component.
See component.
Further Processing:
Irradiated Components
See component.
Increased risk for TAGVHD (eg, congenital
immunodeficiencies,
HLA-matched platelets or transfusions
from blood relatives).
Donor lymphocytes
are inactivated
reducing risk of
TA-GVHD.
*For all cellular components there is a risk the recipient may become alloimmunized and experience rapid destruction of certain types of blood products. Red-cell-containing components and thawed plasma
(thawed FFP, thawed PF24, or Thawed Plasma) should be stored at 1-6 C. Platelets, Granulocytes, and thawed Cryoprecipitate should be stored at 20-24 C. Disclaimer: Please check the corresponding section of
the Circular for more detailed information.
TACO = transfusion-associated circulatory overload; TRALI = transfusion-related acute lung injury; TA-GVHD = transfusion-associated graft-vs-host disease; CMV = cytomegalovirus; TTP = thrombotic
thrombocytopenic purpura; AHF = antihemophilic factor; ITP = immune thrombocytopenic purpura; vWF = von Willebrand factor.
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Page 131 of 290
39
093011
August 2009*
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Page 132 of 290
American Journal of Transplantation 2010; 10: 26–29
Wiley Periodicals Inc.
Special Feature
C 2009 The Author
C 2009 The American Society of
Journal compilation Transplantation and the American Society of Transplant Surgeons
doi: 10.1111/j.1600-6143.2009.02927.x
Calculated PRA (CPRA): The New Measure of
Sensitization for Transplant Candidates
J. M. Cecka
UCLA Immunogenetics Center, Los Angeles, CA
Corresponding author: J. Michael Cecka,
[email protected]
The ways we measure whether a patient is sensitized to HLA antigens and to what extent sensitization
affects access to transplantation have changed remarkably during the past decade. What we mean by sensitized and broadly sensitized today is heavily dependent upon the sensitivity of the test that is used to
measure antibodies. Because we provide additional allocation points for broadly sensitized patients in the
United States kidney allocation system in an effort to
compensate for their biological disadvantage, some
consistency and accountability are required. The calculated panel-reactive antibody, which provides an estimate of the percentage of deceased organ donors
that will be crossmatch incompatible for a candidate
provides both consistency and accountability.
Key words: Access to transplantation, anti-HLA antibodies, crossmatching, donor-specific antibodies,
histocompatibility, PRA
Received 21 August 2009, revised 24 September 2009
and accepted for publication 05 October 2009
Sensitization remains a formidable barrier to transplantation. Patients who have preformed antibodies against
HLA antigens are at risk for hyperacute rejection, accelerated acute rejection, antibody-mediated rejection, delayed graft function and longer term complications when
transplanted from a donor expressing the target HLA antigens. We avoid donor-specific antibodies by crossmatching all potential donors and recipients before transplantation, and as a result, sensitized transplant candidates
have limited access to transplantation in proportion to how
broadly their anti-HLA antibodies react with the potential
donor population. In the case of renal transplant candidates, those who are broadly sensitized (80+% panelreactive antibody; PRA) received additional points in the
UNOS allocation system to compensate for their biological disadvantage. This is a strategy that did not work
very effectively because over time, the most broadly sen26
sitized patients with a combination of long accumulated
waiting time and four additional sensitization points appeared in the same order at the top of the match run
for each blood group compatible donor. Although preliminary crossmatches eliminated most of these patients
from consideration, many laboratories used a more sensitive test for their final than their preliminary crossmatch
and positive final crossmatches were common among the
broadly sensitized patients. To facilitate timely placement
of organs, a limited number of broadly sensitized patients
would be crossmatched and those patients would be the
most likely to be crossmatch incompatible. UNOS implemented a new strategy on October 1, 2009, using unacceptable HLA antigens and a calculated PRA (CPRA)
to award sensitization points that fundamentally changes
how sensitized renal candidates are ranked for kidney
offers.
PRA has been the measure of sensitization since the
recognition that catastrophic hyperacute rejection was
associated with anti-donor HLA antibodies in the mid1960s (1). This landmark paper by Patel and Terasaki also
described a simple surrogate test that could identify sensitized patients and estimate their likelihood of finding
a crossmatch-compatible donor using a panel of normal
blood donors as representative of the potential local organ
donor pool. PRA was simply the percentage of this pool
of donors to which a patient had reactive antibodies. A
patient with 80% PRA would be crossmatch incompatible
with 80% of donors.
The crossmatch tests used today often are more sensitive than those that were used in the past. Even more
importantly, the technologies available for identifying and
measuring anti-HLA antibodies have undergone remarkable changes in the past decade and particularly in the
past 5 years since the introduction of solid-phase tests
using single HLA antigens produced by recombinant DNA
technologies. The diversity of HLA antibody tests being performed by HLA laboratories has increased as these newer,
more sensitive and more precise technologies have become available. Lacking organized guidelines for laboratories to indicate which PRA should be reported, many labs
and transplant centers chose the highest PRA value among
their test platforms because broadly sensitized patients receive four extra points. With diverse test platforms and
sensitivities now many fold higher than previous lymphocytotoxicity tests permitted (2), sensitization estimates can
Page 133 of 290
Sensitized Patients, PRA and CPRA
40
9000
8000
30
Luminex
Flow PRA
25
20
15
10
5
0
1994
CPRA (%)
80+
7000
Registrations
Percent 80+% PRA
35
1-20
5000
0
3000
2000
Transplanted
1000
*68%
*50%
0
0
1999
2004
*90%
4000
Waiting
Added
21-79
6000
2009
1-20
21-80
PRA for Allocation
80+
*concordance
Year
Figure 1: Escalating sensitization levels and correlation with
more sensitive test platforms. The percentage of sensitized patients who had 80+% PRA added to the UNOS waitlist, remaining
on the waitlist at the end of each year and transplanted each year
increased because the introduction of microparticle solid-phase
tests using purified or recombinant HLA antigens, most notably
following the introduction of Luminex technology for HLA antibody identification in 2002 (based on OPTN waitlist data as of July
3, 2009 and OPTN recipient histocompatibility data as of July 3,
2009).
vary widely depending upon the method used to identify
antibodies.
Figure 1 shows that levels of sensitization reported to
UNOS have escalated during the past 15 years. The percentage of sensitized patients with 80+% PRA who were
added to the UNOS waitlist each year, who were on the
waitlist at the end of each year or who were transplanted
each year all increased by about 10% during this period.
There was a clear rise in the percentage of broadly sensitized waitlist candidates and transplant recipients beginning in 2002, the year when solid-phase antibody tests using purified HLA antigens on the luminex platform were
introduced. Although some centers may have become
more adroit at transplanting their broadly sensitized patients through desensitization or transplantation in the face
of a positive crossmatch (3–5), it seems more likely that the
2002 introduction and widespread use of solid-phase tests
was a contributing factor in escalating PRA levels.
The UNOS Histocompatibility Committee crafted a proposal to bring some accountability to PRA reporting and,
at the same time, to take advantage of the new and evolving technologies. The calculated CPRA is based upon unacceptable HLA antigens to which the patient has been
sensitized and which, if present in a donor, would represent an unacceptable risk for the candidate or the transplant program. The CPRA is computed from HLA antigen
frequencies among approximately 12,000 kidney donors
in the United States between 2003 and 2005 and thus
American Journal of Transplantation 2010; 10: 26–29
Figure 2: Correlation between PRA and CPRA. Among 19,046
active registrations on the UNOS Kidney waiting list with a
CPRA value, this figure shows the distribution of CPRA values calculated within each PRA group. In the group with 1–20%
PRA, 50% also had 1–20% CPRA. Concordance was 68% for the
21–79% group and 90% for the 80+% PRA group. In the lower
PRA groups, CPRA tended to be higher, whereas some patients
in the 80+% PRA group did not have sufficient unacceptable antigens reported to warrant this CPRA level. CPRA was rounded to
zero when only unacceptable antigens with a frequency less that
1% were listed (based on OPTN data as of June 12, 2009 and
reported to the UNOS Histocompatibility Committee at its July 15
meeting).
represents the percentage of actual organ donors that express one or more of those unacceptable HLA antigens.
What adds accountability is that entering an unacceptable
antigen for a patient means that kidneys from donors expressing that antigen will not be offered for that patient.
The higher the CPRA, the fewer offers would be received.
The proposal was approved by the UNOS Board of Directors and the initial phase was implemented in December
2007. During the first phase, the CPRA value appeared on
the UNET waitlist form together with the traditional peak
and current PRA values determined by the laboratories. At
least one unacceptable antigen had to be entered for a patient to receive PRA points based on the traditional PRA.
In the second phase, which began on October 1, 2009,
the CPRA replaced peak and current PRA and sensitization
points are now awarded based upon the CPRA.
The UNOS Histocompatibility Committee monitored the
first phase of CPRA during the past year and noted rapid
acceptance. By March 2009, only 13 of the 256 U.S. kidney
transplant programs—most small—had not entered unacceptable antigens for any of their patients. Figure 2 shows
a comparison between the PRA value programs had designated for use in allocation with the patient’s CPRA. Concordance was high, with 90% of active renal candidates
with a PRA 80% or higher having a CPRA in the same
range. At the time of the analysis, about 12% of candidates
who would have received points for 80+% PRA would not
have gotten any points based upon their CPRA. The actual
27
Page 134 of 290
Cecka
number of patients at risk of losing sensitization points was
presented during Fall 2009 at each of the UNOS Regional
meetings (tailored for each Region) to increase awareness
and to encourage communication between transplant programs and their laboratories on the assignment of unacceptable HLA antigens. It may be that these patients had
an inflated PRA that could not be justified based on the
frequencies of the antigens to which they were sensitized.
On the other hand, nearly 20% of active candidates whose
PRA was 21–79% would receive points based on their
CPRA. In fact, concordance was generally lower among
the lower PRA groups due to underestimation of these patients’ sensitization levels using traditional PRA. CPRA provides a more accurate estimate of sensitization because it
includes both class I and class II HLA specificities in the
calculation, a major departure from traditional PRA, where
class I and class II specificities are measured separately.
Even B-cell panels, which express both class I and class II
antigens are generally constructed to cover the class II HLA
antigens and are not representative of HLA distributions in
the general population.
The new accountability built into the CPRA calculation requires a change in how we regard sensitization. A high
traditional PRA value meant a high probability of a positive crossmatch, but because CPRA is based on unacceptable antigens that will prevent offers from those donors to
which the patient is most highly sensitized, an offer for a
patient with a high CPRA value should mean a high probability of a negative crossmatch. Previously, the same highly
sensitized patients would appear at the top of each match
run for donors with their blood group, and OPOs and centers were reluctant to set up final crossmatches for more
than a few highly sensitized patients for fear of not placing the kidneys. The order of sensitized patients on the
match run now is dictated by the donor’s HLA type and different sensitized patients will be ranked first for different
donors, increasing their chances for a transplant. Indeed,
several programs have reported a higher rate of transplantation for sensitized patients using the ‘virtual’ preliminary
crossmatch (6–8). OPOs and centers that avoid broadly
sensitized patients should abandon the practice of limiting
final crossmatches for sensitized patients.
Defining unacceptable antigens was left to the transplant
programs and their laboratories. Some programs may be
more aggressive and willing to assume the risks associated with donor-specific HLA antibodies, while others
may not have the experience or resources to provide the
more intensive and aggressive treatments for these patients. Communication between histocompatibility laboratories and the transplant programs they serve is a critical
element for the success of this new method for assessing
sensitization levels.
The lack of standards for identifying anti-HLA antibodies
and defining unacceptable antigens initially raised some
concerns. A meeting between Laboratory Directors and
28
Transplant Physicians was held in Chicago in March 2008
to identify the problems of using solid-phase testing and
to develop some solutions. There was general agreement that laboratories were quite good at defining strong
antibodies—evidence from proficiency testing revealed
excellent concordance among laboratories in identifying
specific antibodies, even in complex antisera, that were
present in high quantities. There was less agreement
among laboratories for weaker antibodies and a major issue
was a lack of data on the clinical relevance of antibodies
that could only be detected by the very sensitive solidphase tests. The participants identified several strategies
to improve and monitor uniformity in solid-phase antibody
testing that have been or are about to be implemented,
including comparisons of raw test results as well as interpretations among several laboratories using the same test
specimens and collection of raw data by providers of proficiency testing in which all accredited laboratories must participate. Many laboratories had already begun to correlate
the solid-phase test results with their crossmatch results
and were able to eliminate their preliminary crossmatch
tests with a ‘virtual’ preliminary crossmatch based on antibody strength and specificity to define what was unacceptable. Since that meeting, a number of laboratories have
reported more detailed strategies to define unacceptable
antigens and to avoid predictably positive crossmatches
(9–14). Weak anti-HLA antibodies appear to have little clinical importance (15–17). Some have suggested that it is
important that laboratories use multiple tests on different
platforms when initially assigning unacceptable antigens
to reduce the potential pitfalls of anomalous results from
a single test (10,14). The solid phase tests have very fine
sensitivity and not every antibody that can be detected is
important. Although listing every HLA antigen to which a
patient has detectable antibody as unacceptable will eliminate positive crossmatches, it may also eliminate all potential donor offers. The best strategy may be to begin
predicting very strong positive crossmatches and tighten
the thresholds to reduce the incidence of unacceptable
crossmatches.
There are some remaining problems with predicting crossmatches based upon antibody strength and specificities.
We do not know whether multiple weak antibodies may
have additive or synergystic effects on crossmatches or
transplants. The single HLA antigen solid-phase tests also
allow for detection of antibodies that react with antigens
that are not always typed in deceased donors. Antibodies
to the HLA-Cw, the DQ alpha chain and DP antigens may
prevent accurate crossmatch prediction (18) for some patients. Extensive data on the role of these antibody specificities in transplantation are not available yet, but the antibodies may cause positive crossmatches when the target
antigen is present in the donor (19,20). These antigens do
not contribute to the CPRA calculation at present, because
too few donors had been typed for these antigens to estimate their frequencies. The C-locus antigens are being
revisited in the current update of frequency tables, but
American Journal of Transplantation 2010; 10: 26–29
Page 135 of 290
Sensitized Patients, PRA and CPRA
donors typed for DQ alpha chains and DP antigens are still
extremely rare. Allele-specific antibodies are sometimes
detected in the single antigen tests, which may cause
problems because donor HLA alleles are rarely typed and
because when an antibody detected against an allele of
the patient’s own HLA antigen is detected, it cannot be
listed as unacceptable.
Despite these limitations, laboratories can accurately predict many incompatible crossmatches, streamlining allocation and providing more options for broadly sensitized
patients. Like PRA, CPRA is a tool for characterizing and
monitoring sensitization. Unlike PRA, the CPRA provides a
meaningful estimate of transplantability for most patients,
because it is calculated from unacceptable HLA antigens
that will preclude offers from predictably crossmatch incompatible donors.
The change to CPRA represents a paradigm shift in many
ways. Although the concepts of unacceptable antigens and
virtual crossmatches have been with us for many years,
their widespread use and formal incorporation into the system for kidney allocation in the United States is unprecedented. As our ability to predict crossmatches and compatibility of donors for sensitized patients improves, it should
be possible to encourage wider geographical sharing of
deceased donor kidneys for this disadvantaged group beyond the current practice of sharing zero-HLA mismatched
kidneys for sensitized patients. Accurate crossmatch prediction is a critical aspect of paired living-donor kidney exchanges as these increasingly involve patients and donors
at different transplant centers. Finally, although the current focus is on their role in renal transplant allocation,
unacceptable antigens and CPRA are also important tools
for many thoracic programs that have begun using virtual
crossmatches for distant donors to broaden the opportunities for their sensitized patients.
Acknowledgments
Dr. Cecka is currently Chair of the UNOS Histocompatibility Committee.
The data analyses presented in this article were supported in part by Health
Resources and Services Administration contract 234-2005-370011C. The
content is the responsibility of the author alone and does not necessarily reflect the views or policies of the Department of Health and Human
Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
I thank Ann Harper and Anna Kucheryavaya for their excellent contributions
of OPTN/UNOS data and analyses performed for the Histocompatibility
Committee.
3.
4.
5.
6.
7.
8.
9.
10.
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13.
14.
15.
16.
17.
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2. Pei R, Lee JH, Chen T, Rojo S, Terasaki PI. Flow cytometric detec-
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Zachary AA, Montgomery RA, Ratner LE et al. Specific and durable
elimination of antibody to donor HLA antigens in renal transplant
patients. Transplantation 2003; 76: 1519–1525.
Vo AA, Lukovsky M, Toyoda M et al. Rituximab and intravenous
immune globulin for desensitization during renal transplantation.
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Gloor J, Cosio F, Lager DJ, Stegall MD. The spectrum of antibodymediated renal allograft injury: Implications for treatment. Am J
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Bray RA, Nolen JD, Larsen C et al. Transplanting the highly sensitized patient: The Emory Algorithm. Am J Transplant 2006; 6:
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86: 1052–1059.
Bingaman AW, Murphey CL, Palma-Vargas J, Wright F. A virtual
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Vaidya S. Clinical importance of anti-human leukocyte antigenspecific antibody concentration in performing calculated panel reactive antibody and virtual crossmatches. Transplantation 2008;
85: 1046–1050.
Zachary AA, Montgomery RA, Leffell MS. Defining unacceptable
HLA antigens. Curr Opin Organ Transplant 2008; 13: 405–410.
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of crossmatch results in highly sensitized patients. Transplantation
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Tambur AR, Ramon DS, Kaufman DB. Perception versus reality?:
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logistic limitations. Am J Transplant 2009; 9: 1886–1893.
Reinsmoen NL, Lai CH, Vo A et al. Acceptable donor-specific antibody levels allowing for successful deceased and living donor
kidney transplantation after desensitization therapy. Transplantation 2008; 86: 820–825.
Zachary AA, Sholander JT, Houp JA, Leffell MS. Using real data
for a virtual crossmatch. Human Immunol 2009; 70: 574–579.
Phelan D, Mohanakumar T, Ramachandran S, Jendrisak MD. Living donor renal transplantation in the presence of donor-specific
human leukocyte antigen antibody detected by solid-phase assay.
Human Immunol 2009; 70: 584–588.
Aubert V, Venetz JP, Pantaleo G, Pascual M. Low levels of human leukocyte antigen donor-specific antibodies detected by solid
phase assay before transplantation are frequently clinically irrelevant. Human Immunol 2009; 70: 580–583.
Ho EK, Vasilescu ER, Colovai AI et al. Sensitivity, specificity and
clinical relevance of different cross-matching assays in deceaseddonor renal transplantation. Transplant Immunol 2008; 20: 61–67.
Tait BD, Hudson F, Cantwell L et al. Luminex technology for
HLA antibody detection in solid organ transplantation (Review).
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29
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Hematol Oncol Clin N Am 21 (2007) 147–161
HEMATOLOGY/ONCOLOGY CLINICS
OF NORTH AMERICA
Transfusion Risks
and Transfusion-related
Pro-inflammatory Responses
George John Despotis, MDa,b,*, Lini Zhang, MDa,
Douglas M. Lublin, MD, PhDb
a
Department of Anesthesiology, Box 8054, Washington University School of Medicine,
660 South Euclid Avenue, St. Louis, MO 63110, USA
b
Department of Pathology and Immunology, Box 8118, Washington University School
of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110, USA
A
pproximately 14.2 million red cell units and 1.6 million platelet transfusions (>80% single donor apheresis platelet units and the rest pools of
usually six random donor platelet units) are administered in the United
States each year [1,2,3]. Transfusion-related adverse events can occur with 10%
of transfusions, and serious adverse events have been estimated to less than
0.5% of transfusions. Early estimates indicated that transfusion-associated adverse events could lead to a short-term (ie, not including disease transmission-related deaths) mortality of 1 to 1.2 deaths per 100,000 patients, or
approximately 35 transfusion-related deaths/year in the United States [1,2].
More recent estimates suggest transfusion-related deaths are under-reported,
and that long-term or total (ie, including disease transmission-related deaths)
mortality is probably closer to one death per every 37,000 platelet or
130,000 red cell units administered, or approximately 220 transfusion-related
deaths per year in the United States [1]. Even these estimates, however, may
be underestimating transfusion-related mortality. For example, there were only
21 transfusion-related acute lung injury (TRALI)-related fatalities reported in
2003 [4], while projections based on an incidence of 1:5,000 transfusions with
a 6% mortality rate indicate that this syndrome can account for at least 300
deaths annually in the United States. With respect to the leading causes of
death, reports to the Food and Drug Administration (FDA) from 2001 to
2003 indicated that TRALI (16% to 22%), ABO Blood Group hemolytic transfusion reactions (12% to 15%), and bacterial contamination of platelets (11% to
18%) accounted for 40% to 50% of all transfusion-related deaths [5].
*Corresponding author. Department of Pathology and Immunology, Box 8118, Washington
University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110. E-mail
address: [email protected] (G.J. Despotis).
0889-8588/07/$ – see front matter
doi:10.1016/j.hoc.2006.11.002
ª 2007 Elsevier Inc. All rights reserved.
hemonc.theclinics.com
148
Page 137 of 290
DESPOTIS, ZHANG, & LUBLIN
The composite risk of transmission of lipid-enveloped viruses such as HIV
(1:1,400,000 to 2,400,000 U), human T-lymphotropic virus HTLV-I/II
(1:250,000 to 2,000,000 U), hepatitis B (1:58,000 to 1: 149,000 U), hepatitis C
(1:872,000 to 1,700,000 U) is estimated to be 1:83,000 U [2]. A substantial
decline in the risk for transfusion-related viral transmission has occurred
over the past 15 years related to implementation of donor screening and test
strategies. This improvement came from immunoassays of increased sensitivity, and more recently from nucleic acid testing procedures that can detect viral
RNA/DNA during the window period.
Fifty percent of patients who acquire the hepatitis C virus (HCV) develop
liver disease (although symptoms can be apparent within 2 weeks to 6 months,
most patients are asymptomatic); 20% develop cirrhosis within 20 years, and
1% to 5% subsequently develop hepatocellular carcinoma. In contrast, transmission of hepatitis A or E, both enteric forms of hepatitis, is rare, and not associated with chronic infection. Other blood-borne, infectious diseases such as
syphilis, Epstein-Barr virus, leishmaniasis, Lyme disease, brucellosis, B-19 parvovirus (increased prevalence in hemophiliacs), tick-borne encephalitis virus,
Colorado tick fever virus, severe acute respiratory syndrome (SARS), West
Nile virus, human herpes viruses, parasitic diseases (eg, malaria, babesiosis,
toxoplasmosis, and Chagas’ disease), and variant Creutzfeldt–Jakob disease
(vCJD) can be transmitted by means of transfusion, although many of these
agents are rare in blood donors in the United States.
Febrile, nonhemolytic transfusion reactions (NHTR) consisting of fever
(>1 C) with a transfusion, occurs with 0.5% to 1.5% of red cell transfusions
and can be related to one of several potential mechanisms. Preformed cytokines
within the stored unit and host antibodies to donor (ie, graft) lymphocytes are
generally self-limiting. The incidence of febrile NHTR may decrease by perhaps 50% with the use of prestorage leukoreduced blood components, and
these reactions often can be prevented by pretreatment with acetaminophen.
Although the estimated death rate related to HIV and hepatitis is declining,
death related to transfusion caused by sepsis secondary to bacterial contamination of platelets is estimated to be at 20 deaths per million units of transfused
platelets [2]. This is concerning, based on the substantially increased use of platelet transfusions in the United States to support cardiac surgery, oncology, and
peripheral blood stem cell (PBSC) transplantation programs. The infusion of
bacterially contaminated blood is an uncommon cause (0.0002% to 0.05%) of febrile transfusion reactions, occurring with 0.0001% to 0.002% of red blood cell
(RBC) products stored at 4 C (the organism is often Yersinia enterocolitica) and
at a much higher frequency with platelets stored at 20 C (ie, 0.05%) [1,2]. It, however, can lead to sepsis in 17% to 25% of patients transfused with contaminated
blood, with an associated mortality rate of 26% [1]. Additionally, it accounts for
at least 16% of transfusion-related fatalities previously reported to the FDA [6].
Bacterial growth more commonly occurs in components stored at room temperature (1:2,000 per apheresis platelet unit), especially if the storage interval is
greater than 5 days, which has led to the current FDA limit for platelet out-date
Page 138 of 290
TRANSFUSION-RELATED PRO-INFLAMMATORY RESPONSES
149
of 5 days. Very recently, the FDA licensed systems for storage of apheresis
platelets for up to 7 days when bacterial cultures are performed on the product
before release. Some form of bacterial quality control screening is performed
for all platelet products, but it is not required that the method be as sensitive
as culture systems. Transfusion of bacterially contaminated blood should be
suspected when patients manifest one or more of the following symptoms or
complications: high fever, chills, hemodynamic perturbations (eg, tachycardia,
hypotension, shock), gastrointestinal (GI) symptoms (eg, emesis, diarrhea),
hemoglobinuria, disseminated intravascular coagulation (DIC), or oliguria. Before transfusion, units should be examined for signs of bacterial contamination
(eg, discoloration or dark color, bubbles).
Transfusion-associated respiratory distress can be related to one of the following in order of decreasing frequency: fluid overload (transfusion-associated
circulatory overload or TACO), allergic reactions, or TRALI. Although the exact incidence of circulatory overload related to transfusion is unknown (eg, 1 in
every 200 to 10,000 U) [7], it is more likely in older patients with a history of
congestive heart failure. Estimated prevalence rates of TRALI range from 1 in
432 U to 1 in 88,000 U of transfused platelets and 1 in 4,000 U to 1 in 557,000 U
of red blood cells [8]. These ranges for transfusion associated circulatory
overload (TACO) and TRALI reflect the clinical difficulty of diagnosis and
the under-reporting of these transfusion reactions. TRALI can occur when
anti-HLA (human leukocyte antigen) or anti-HNA (human neutrophil antigen) antibodies (more commonly observed in units from multiparous donors)
and possibly neutrophil-activating lipid mediators within transfused units attack
circulating and pulmonary leukocytes and stimulate complement activation and
pulmonary injury [7]. This hypothesis, however, cannot explain all cases of
TRALI, and a two-hit hypothesis was proposed previously [9]. The first event
involves priming of neutrophils by some underlying condition (eg, trauma, infection, or surgery), which is followed by the infusion of substances by transfusion (eg, anti-HLA or anti-HNA antibodies, biologically active lipids). This leads
to TRALI. This syndrome is characterized by acute (<6 hours after transfusion)
onset of severe hypoxemia, bilateral noncardiogenic pulmonary edema, tachycardia/hypotension, and fever [10,11]. With ventilatory and hemodynamic supportive management, most patients recover within 48 to 96 hours. The
prevalence of TRALI or development of acute respiratory compromise during
or after transfusion has been advocated to be much more common in a recent
Canadian consensus meeting [4]. In fact, with increased reporting to the FDA,
the incidence of TRALI-related deaths (5% to 25% of patients who develop
this syndrome) may be much higher than previously thought (as high as 18%
of all deaths reported between 2001 and 2003), which places it close to the other
leading causes of death (ie, acute hemolytic reactions, or bacterial contamination
of platelets) [5,12,13]. Analysis of recent publications indicates that this syndrome is under-reported, because there were only 21 fatalities reported in
2003 [4], while low-end projections (ie, incidence of 1:5,000 transfusions with
a 6% mortality rate) indicate that this syndrome can account for as many as
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300 deaths annually in the United States. In addition, if TRALI is not diagnosed
correctly, treatment of these patients with therapy designed to manage cardiogenic pulmonary edema (ie, diuretic administration) can lead to adverse outcomes [10]. The pathophysiology of TRALI is still being elucidated, and it is
uncertain whether the mechanisms will expand beyond anti-HLA and antiHNA antibodies and lipid mediators [14]. The understanding of the exact
role of transfusion in the development of acute lung injury in susceptible patients
with endothelial dysfunction (eg, trauma, cardiac surgery, sepsis) who also
develop other end-organ dysfunction as part of multiorgan system failure is
evolving.
Hemolytic transfusion reactions can be immediate and life-threatening or delayed with minimal resulting clinical consequences (eg, serologic conversion).
Current estimates indicate that the wrong unit of blood is administered 1 in every 14,000 U, of which transfusion of 1:33,000 U involves ABO incompatibility [2,15,16]. Catastrophic, acute hemolytic transfusion reactions (HTRs) are
rare (ie, 1 in every 33,000 U to 1 in every 500,000 to 1,500,000 U). They
can be fatal in 2% to 6% [1,2,6,15] of cases, however, and they account for
at least (ie, these events are probably under-reported) 16 deaths every year
(ie, 1:800,000 U transfused) in the United States [2,6]. Based on transfusion
of 14.2 million units of red cells annually in the United States, there are approximately 1,000 nonfatal and 20 to 60 fatal mis-identification errors each year.
This is in contrast to the 131 deaths (or 37% of the total deaths) related to
ABO-incompatible transfusion reported between 1976 and 1985 [6,15]. Data
from the United Kingdom for serious hazards of transfusion (SHOT) between
1996 and 2003 revealed that there were 2087 errors (1:11,000 transfusions), of
which 24% resulted in major morbidity or death [15,16]. This report also revealed that in 50% of these events there were multiple errors in the process,
that 70% of the errors occurred in clinical areas, and that the most frequent error (27% in 2003) involved a failure to link the unit to the patient at the bedside
[16].
Catastrophic acute HTR initiates a sequence of responses, including complement and hemostatic system activation and neuroendocrine responses, which
occur predominantly when host antibodies attach to red cell antigens on incompatible donor red cells. Generally, catastrophic acute HTR involves preformed
IgM antibodies to ABO antigens, which lead to hemolysis by means of complement fixation and formation of immune complexes. As little as 10 to 15 mL of
ABO-incompatible blood can initiate symptoms consistent with a severe, acute
HTR such as:
Fever in 48% (cytokine-related)
Hypotension in 15% (secondary to bradykinin, mast cell histamine/serotonin
and other vasoactive amines)
Diffuse microvascular bleeding (secondary to hemostatic system activation or
DIC)
Complement-mediated acute intravascular hemolysis (eg, acute anemia,
hemoglobinemia/hemoglobinuria in 87%)
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Acute renal insufficiency secondary to alpha-adrenergic vasoconstriction or
deposition of antibody-coated stroma within the renal vasculature [17]
The diagnosis can be confirmed with detection of free hemoglobin within the
blood and urine in the setting of a positive direct antiglobulin test (DAT) with
a mixed-field pattern on post-transfusion but not pretransfusion specimens.
Additional tests that should be ordered include:
Repeat ABO/Rhesus (Rh) testing of the unit
Repeat cross-match and antibody detection on the patient’s pre- and postreaction samples and on blood from the unit
Haptoglobin
LDH
Serial hemoglobin/hematocrit on patient specimens
Examination of the blood remaining in the unit for hemolysis [18]
Treatment is generally supportive and involves resuscitation to maintain organ perfusion using volume and vasopressor, which preferably do not vasoconstrict the renal bed (eg, low dose dopamine), maintenance of good renal urine
output (>100 mL/h 24 hours) with intravenous crystalloids and diuretics,
and on occasion transfusion support with hemostatic blood products in the
setting of DIC and clinical bleeding.
In contrast, most reactions to non-ABO antigens involve IgG-mediated extravascular clearance within the reticuloendothelial system (RES). They often are
delayed (ie, 2 to 10 days), and they are not detected by pretransfusion testing,
because they represent an anamnestic response. An exception to this pattern is
Kidd antibodies, which are strong complement activators that can result in
acute intravascular HTR. Finally, nonimmune HTR also can occur related
to temperature (eg, overwarming with blood warmers, use of microwave
ovens), use of hypotonic solutions for dilution of packed red blood cells
(PRBCs), and mechanical issues during administration (ie, pressure infusion
pumps, pressure cuffs, and small-bore needles). In addition, normal saline
should be used to dilute the red cell units (calcium-containing solutions should
be avoided), and units should be examined for large clots before transfusion.
Because clerical or misidentification errors, which occur 1 in every 14,000 U,
cause most immediate immune-mediated HTR [19], this potentially lethal
complication can be prevented by diligent confirmation of patient and unit
identification by individuals who initiate transfusion intraoperatively (ie, the
anesthesiologist and circulating nurse). First, the blood bank confirms that the
unit identification number and the ABO/Rh type on the unit of blood match
the label attached to the unit. Most importantly, two clinical transfusionists
must confirm that three pieces of patient identification (eg, patient name, hospital
identification number, birth date, or social security number) on the hospital identification band or surrogate (eg, patient name plate imprint on the anesthesia
record) needs to match the respective parameters on the unit of blood.
To obtain a thorough understanding of hemolytic reactions, red cell antigen
systems and serologic diagnostic tests are reviewed. Red cell antigen systems
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include the ABO and related carbohydrate antigens (ie, H, P, I, and Lewis
blood groups), the 48 Rh system antigens (including RhD) and over 200 other
non-ABO/Rh antigens. The ABO carbohydrate and Rh polypeptide molecules
reside on the surface of red blood cells with a US population frequency distribution (O: 44%, A: 43%, B: 9%, AB: 4%; RhDþ: 84%). ABO molecules express specific antigenic activity after individual sugar moieties are added to
short sugar chains (ie, oligosaccharides) by several genetically determined glycosyltransferase enzymes. The ABO antigens are linked to cells (ie, red cells
and other cells) by means of their association with membrane-bound proteins
(ie, glycoproteins) or ceramide residues (ie, glycosphingolipids). Antibodies to
the A and B antigens generally are thought to form as a result of exposure to
other sources of antigen (ie, on bacteria) after the first few months of life. Blood
group A and B individuals produce predominantly IgM antibodies (ie, anti-B
and anti-A, respectively), whereas blood group O individuals produce both
anti-B and anti-A IgG/IgM antibodies. Antibodies to Lewis and P1 antigens
are generally clinically insignificant.
Although there are 49 identified Rh antigens, the five principal antigens, D,
C, E, c and e, and corresponding antibodies account for more than 99% of clinical issues involving the Rh system. The Rh system antigens are nonglycosylated, fatty-acylated polypeptides that traverse the red cell membrane 12
times. Although individuals who lack the D antigen do not form antibodies
without blood exposure, the D antigen is highly immunogenic, and 80% of individuals who lack the D antigen will form anti-D once exposed through transfusion, or, at a lower frequency of approximately 15% through pregnancy.
Over 200 other non-ABO/Rh, glycoprotein antigens can be identified on red
cells, and some of these antigens also are expressed on other cells and body
fluids. These non-ABO/Rh antigens frequently are subdivided into common
(ie, MNS, Kell, Duffy, and Kidd systems) and uncommon antigen systems
(eg, Lutheran, Diego, Yt, Xg, and Scianna). Antibodies to most of the common
antigens can cause both clinically significant immediate and delayed HTR, but
do not usually result in catastrophic, complement-mediated hemolysis, although this can occur with Kidd, Duffy, and S antibodies. Severe delayed
HTRs are particularly common with anti-Kidd antibodies. Another important
factor is the relative immunogenicity (ie, antibody formation), which can vary
substantially between non-ABO antigens (eg, anti-D in 80%, anti-K in 10%, and
anti-Fya in 1% of exposures).
Several blood bank procedures (type, screen, and cross-match) are employed
routinely to ensure transfusion of compatible blood. Patient ABO type is determined using direct agglutination of red cells and involves use of forward (ie,
using the patient’s red cells with anti-A and anti-B reagents) and reverse (using
the patient’s sera with reagent A1 and B cells) typing. Only forward typing is
accurate in newborns or infants younger than 4 to 6 months based on transfer
of maternal IgG molecules and lack of anti-A or anti-B production before 4 to
6 months of age. An antibody screen (ie, indirect antiglobulin or Coombs test)
determines whether unexpected antibodies against common non-ABO red cell
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antigens are present, These antibodies are found in 0.2% to 0.6% of the general
population [20], 1% to 2% of hospitalized patients, or in 8.3% of surgical patients. The antibody screen is performed using reagent red cells (ie, two or three
screening cells) and a cross-linking antibody (rabbit/mouse antihuman globulin
or Coombs reagent) that enhances the IgG-mediated agglutination of red cells.
Sera is tested routinely only for antibodies to the common antigens, because the
uncommon non-ABO antigens infrequently (ie, <0.01%) result in cross-match
incompatibility of ABO compatible units. In the setting of a negative result on
the antibody screen, the final cross-match can be done by a Coombs test, an immediate spin cross-match, or an electronic cross-match. The latter two procedures simply confirm the ABO compatibility of the donor unit and require
less time. An elective procedure for which a type and screen or cross-matched
blood has been requested should never commence until the antibody screen
has been determined to be negative, or in the setting of a positive antibody
screen, with antibodies and cross-matched compatible blood identified.
Because availability of blood for same-day and urgent surgery is of critical
importance, understanding a generally applicable timetable is important [20].
O negative (in some settings O positive for males) RBCs are generally immediately (<5 minutes) available, whereas type-specific RBCs are available within
15 minutes after receipt of the patient specimen. Cross-matched RBCs are generally available within 45 to 60 minutes by means of a type and cross-match
(T&C) procedure using an immediate spin cross-match, which can be done if
no antibodies are detected during the antibody screen. With a positive antibody screen, additional time (1 to several hours or even days) may be required
to determine the antibodies and identify and cross-match blood that is antigennegative. In the event that antibodies are detected from the screen, the probability of finding compatible units can be calculated from the frequency of
antigens for those preformed antibodies (eg, an Aþ individual with anti-c,
anti-Fya antibodies will be compatible with 0.18 0.34 ¼ 0.06 or 6 of 100
Aþ units in the blood bank). Accordingly, obtaining cross-match compatible
blood is also difficult when a patient has antibodies to a very common antigen
(eg, k, in which case only 1 in 500 units is compatible). Clinicians also may be
faced with an inability to obtain cross-match compatible blood in patients who
have a warm auto-antibody. In this setting, more extensive serologic analysis
using absorption techniques is required to identify alloantibodies; alternatively,
if the patient has not been transfused recently, the partial phenotype can be
determined to provide antigen-negative red cells.
Single-donor, apheresis platelets (which now constitute >80% of platelet
transfusions) are generally available immediately, whereas pooled platelet concentrates may take 10 to 15 minutes to process. The time required to obtain
plasma or cryoprecipitate varies from 5 minutes to 30 minutes and is dependent on whether an inventory of thawed plasma units is maintained and the
availability of a rapid thawing system.
Mild urticarial symptoms (eg, rash, hives, or itching) occur with 1% of transfusions [21]. They are generally self-limiting and may improve with or be
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prevented by antihistamine prophylaxis. More significant allergic transfusion
reactions can occur with 0.1% to 0.3% units and are most likely related to reactions to other soluble transfusion constituents (eg, complement or other
plasma proteins, drugs, or soluble allergens). Severe anaphylactic reactions,
which occur infrequently (ie, 0.005% to 0.0007%), may be accompanied by
IgE-mediated symptoms involving the respiratory (eg, dyspnea, bronchospasm), GI (eg, nausea, diarrhea, cramps) or circulatory (eg, arrhythmias, hypotension, or syncope) systems. IgA deficiency, which occurs in 1 of every 800
patients (only 30% of whom have preformed anti-IgA), is an uncommon cause
of transfusion-associated anaphylaxis, and this diagnosis should be considered
in any patient exhibiting anaphylaxis. Other potential causes of hemodynamic
perturbations during or after a transfusion include:
Citrate-related hypocalcemia (ie, with rapid infusion)
Inadvertent intravenous air embolus (particularly with autologous blood
recovery and reinfusion)
Cytokine-mediated effects
Bradykinin activation by leukoreduction filters, which may be aggravated by
inadequate clearance in patients on angiotensin-converting enzyme inhibitors
(80 reports to the FDA)
Metabolic consequences of transfusion include coagulopathy, hypothermia
(ie, with inadequate warming of refrigerated PRBC units) and hyperkalemia,
because potassium concentration increases with the storage interval of PRBC
units (eg, 42 mEq/L at 42 days of storage, or approximately 6 mEq total in
a unit of PRBCs).
In addition to development of alloantibodies to red cell antigens, several other
immune-related phenomena can occur subsequent to transfusion. Antigens
of the HLA system are determined by genes on the major histocompatibility
complex on the short arm of chromosome 6. HLA gene products are cell–
surface glycoproteins on all cells except mature red cells (class I comprised of
HLA-A, B, or C antigens) or on B lymphocytes and cells of monocyte/macrophage lineage (class II comprised of HLA-DR, DQ, or DP gene cluster codes).
Because they contribute to the recognition of self versus non-self, they are important with respect to rejection of transplanted tissue and long-term survival
after solid organ and bone marrow transplantation. Alloimmunization to
HLA antigens, which occurs commonly (ie, 20% to 70% of the time) in transfused and multiparous patients, can lead to immune-mediated platelet refractoriness (ie, insignificant or inadequate rise in platelet count not related to DIC,
amphotericin, or splenomegaly), and febrile NHTR. Alloimmunization can be
associated with development of autoantibodies, leading to autoimmune hemolytic anemia and development of post-transfusion purpura (ie, severe thrombocytopenia secondary to platelet-specific antibodies, usually anti-HPA-1a/PLA1
antibodies) 5 to 10 days after transfusion. Transfusion-associated immune system modulation has been shown to have beneficial effects, including improved
renal allograft survival, reduced risk of recurrent spontaneous abortion, and
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155
reduced severity of autoimmune diseases such as rheumatoid arthritis. Proposed detrimental effects of transfusion-associated immune system modulation
include increased cancer recurrence, perioperative infections, multiorgan system failure, and overall mortality, but these effects are controversial [22].
Transfusion, however, potentially can attenuate the immune response based
on one of several potential mechanisms, including:
a reduction in CD8 suppressor T cell function and number
CD4 T helper cell number
NK cell number and function
Macrophage number and function,
MLC response
Response to mitogen,
Cell-mediated cytotoxicity [23]
Although several studies have demonstrated an independent effect (ie, using
multivariate statistical models) of transfusion on increased perioperative infection ratesfour to five times) in numerous different surgical populations (ie,
trauma [24–27], hip arthroplasty [25,28], spinal [29], colorectal [30–36] and cardiac [37–39]), the immune–modulatory effect of transfusion on the incidence of
perioperative infection remains controversial. In addition, a recent meta-analysis involving review of 20 peer-reviewed articles and 13,152 patients revealed
that transfusion was associated with perioperative infection (odds ratio of
3.45, range 1.43 to 15.15) [27]. Accordingly, four recent studies have demonstrated that administration of leukoreduced units may reduce perioperative infection in patients undergoing either colorectal [31] or GI, [40], or cardiac
surgery [41,42], This has not been confirmed by other studies, however [43–
45]. Another recent retrospective analysis demonstrated a reduction in perioperative complications and mortality when leukoreduced units were used
[44–46]. Rarely, transfusion-associated graft-versus-host disease, a syndrome
manifested by several symptoms (ie, fever, dermatitis or erythroderma, hepatitis/enterocolitis, diarrhea, pancytopenia, or hypocellular bone marrow) may occur and be secondary to transfusion of cellular blood components that contain
HLA-compatible T-lymphocytes, This occurs more frequently with transfusions from related individuals, and it can be prevented by standard irradiation
of the blood product.
Several recent studies have demonstrated that transfusion has an association
with multiorgan system failure (MOSF) in the perioperative setting [47–49]. Although the exact mechanisms of the potential effect of transfusion on the incidence of this complication have not been elucidated, it is postulated that in
patients who are at high risk (eg, trauma, long CPB intervals) for developing
endothelial dysfunction, that either white cell lytic enzymes or other cellular debris injure an already dysfunctional endothelium. These studies also have demonstrated that there is an effect imposed by the age of the PRBC units and
a load effect (ie, a direct relationship between the number of PRBCs units administered and MOSF rates). The prevalence of TRALI is expanding, in part
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based on improved reporting and potential overlap between the diagnoses of
TRALI versus MOSF in the high-risk patients. In addition, a recent study
by van de Watering [41] demonstrated that mortality related to MOSF was reduced by 90% when patients undergoing cardiac surgery received leukoreduced PRBC units.
Excessive bleeding requiring transfusion to correct anemia or hemostatic
defects also may result in other complications such as stroke and may affect
long-term mortality. In a large (n ¼ 16,000), recently published analysis [50],
transfusion of more than 4 U of PRBC was the strongest (odds ratio ¼ 5)
independent predictor with respect to perioperative stroke; it is not clear from
this analysis whether transfusion support was a causative factor versus a predictor [50]. Another recent publication demonstrated a strong relationship
between perioperative platelet transfusion and both stroke and death [51].
This was supported by another recent retrospective analysis that demonstrated
that the death rate in a large series of patients undergoing cardiac surgery was
much higher in patients who received platelets using multivariate statistical modeling [52]. Accordingly, a retrospective analysis demonstrated that long-term
mortality may be doubled in patients who receive transfusion [53]. Because of
their retrospective design, these studies cannot definitively link transfusion of
either PRBC or platelet components with stroke or increased mortality, which
may be reflecting colinearity or a statistical passenger effect with other comorbidities such as excessive bleeding. These studies, however, do help explain why
agents such as aprotinin, which has been shown to reduce blood loss and transfusion by 50% to 90% and re-exploration rates by 70% in several large, randomized, placebo-controlled trials [54–57], also is associated with a 60% to 70%
reduction in perioperative stroke [58] and reduced mortality [59]. Whether the
beneficial effects of this agent are related to a reduction in the incidence of anemia
and hypoperfusion related to bleeding in patients who also receive multicomponent transfusion or if they are the indirect effects of this agent on reducing transfusion in the bleeding patient with a concomitant reduction in transfusion-related
sequelae remains unclear. Iron overload (ie, accumulation and deposition of iron
within the vital organs) can occur in chronically transfused individuals such as
patients with hemoglobinopathies and other susceptible patients.
Emerging techniques to reduce disease transmission and hemolytic transfusion reactions are under active investigation and implementation. The introduction of nucleic acid technology (NAT) testing procedures can minimize
blood-borne disease transmission by detecting viral RNA/DNA during the serologic window period. Inactivation of viral and bacterial RNA/DNA by photochemicals (eg, psoralen) with UVB irradiation is under investigation. Other
techniques to reduce hemolytic transfusion reactions are under investigation
such as conversion of A, B, or AB red cell units to O by means of enzymatic digestion of A and B antigens or generation of AB equivalent plasma by means of
adsorption of anti-A and anti-B from plasma. In addition, new patient identification systems (eg, bar coding of identification bands and blood and the Bloodloc
Safety System [Novatek Medical, Effingham, Illinois]) are being implemented to
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reduce transfusion of incompatible blood, in part based on a recent Joint Commission on Accreditation of Healthcare Organizations high-priority directive to
enhance patient safety by means of improved patient identification.
Although the on-going interface between transfusion medicine and perioperative services is an important topic, it is reviewed only briefly. The transfusion
medicine service can provide assistance with respect to patients with unique
clinical problems (eg, patients with cold agglutinin disease), use of specialized
blood components, and implementation and monitoring of one of several nonpharmacologic blood conservation strategies such as preautologous donation
[60,61], normovolemic hemodilution and cell salvage techniques [62], and
other technical blood conservation methods [63]. Several pharmacologic agents
(eg, tranexamic acid, epsilon amino caproic acid, or aprotinin) can be used to
reduce bleeding and transfusion after cardiac, orthopedic, and liver transplantation procedures. Aprotinin, however, is the only agent that is FDA-approved
for patients undergoing cardiac revascularization. This is also the only agent
with established efficacy and safety based on multiple prospective, randomized
(placebo-controlled) trials [54–57].
Other important interactions between transfusion medicine and perioperative services include establishment and monitoring of standardized transfusion
protocols for red cells, hemostatic components, and emerging and off-label indications for factor concentrates (eg, factor VIIa) as important blood management strategies. Although several case reports have indicated that off-label use
of activated factor VII can reverse life-threatening bleeding, cost and risk of
thrombosis preclude routine use. Because any factor concentrate potentially
can lead to life-threatening thrombotic complications in a subset of high-risk patients (ie, patients with congenital or acquired thrombotic disorders or systemic
activation of the hemostatic system such as with DIC or after cardiac surgery),
large clinical trials evaluating the efficacy and safety of rFVIIa are needed
before any widespread use can be recommended [64].
Use of point-of-care or laboratory-based coagulation results when coupled to
a standardized approach (ie, algorithm) for managing bleeding after cardiac surgery has been shown to result in a 50% reduction in total donor exposures in all
but one [65] of eight published studies [66–72]. Other studies also have demonstrated that certain patient subgroups may benefit from off-label use of DDAVP
with respect to reduced bleeding and transfusion such as patients who have:
Type I von Willebrand’s disease
Uremia-induced platelet dysfunction
A platelet defect after cardiac surgery as identified using point-of-care platelet
function tests [73,74]
Use and monitoring of point-of-care diagnostics to guide transfusion and
pharmacologic management of bleeding also can be enhanced by means of a collaborative approach with the transfusion medicine service with respect to implementation, quality control monitoring, and regulatory compliance (eg, Joint
Commission or College of American Pathologists). Future availability of blood
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substitutes may be critical in unique clinical situations such as in patients with
multiple antibodies, with Jehovah’s Witness patients, and in trauma settings.
These agents also may enhance blood conservation techniques or organ preservation because of their ability to enhance tissue oxygenation.
Despite improvements in blood screening and administration techniques, serious adverse events related to transfusion continue to occur, albeit at a much lower
incidence. In addition to the development and implementation of new screening
and blood purification/modification techniques, the incidence and consequences
of transfusion reactions can be reduced by a basic understanding of transfusionrelated complications. Although acute hemolytic transfusion reactions, transfusion-associated anaphylaxis, sepsis, and TRALI occur infrequently, diligence
in administration of blood and monitoring for development of respective
signs/symptoms can minimize the severity of these potentially life-threatening
complications. In addition, emerging blood banking techniques such as psoralen-UV inactivation of pathogens and use of patient identification systems may
attenuate the incidence of adverse events related to transfusion. With respect
to optimizing blood management by means of pharmacologic and nonpharmacologic strategies, the ability to reduce use of blood products and to decrease operative time or re-exploration rates has important implications for not only disease
prevention, but also for blood inventory and costs and overall health care costs.
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Page 151 of 290
Recombinant Human Erythropoietin in Anemic Patients
with End-Stage Renal Disease
Results of a Phase I11 Multicenter Clinical Trial
Joseph W. Eschbach, MD; Mohamed H. Abdulhadi, MD; Jeffrey K. Browne, PhD;
Barbara G. Delano, MD; Michael R. Downing, PhD; Joan C . Egrie, PhD; Roger W. Evans, PhD;
Eli A. Friedman, MD; Stanley E. Graber, MD; N. Rebecca Haley, MD; Stephen Korbet, MD;
Sanford B. Krantz, MD; A. Peter Lundin, MD; Allen R. Nissenson, MD; David A. Ogden, MD;
Emil P. Paganini, MD; Barbara Rader, MEd.; Edwin A. Rutsky, MD; John Stivelman, MD;
William J. Stone, MD; Paul Teschan, MD; John C . Van Stone, MD; David B. Van Wyck, MD;
Kenneth Zuckerman, MD; and John W. Adamson, M D
Study Objective: To determine the effectiveness and safety
of recombinant human erythropoietin (rHuEpo) .
Patients: Hemodialysis patients (333) with uncomplicated anemia (hematocrit < 0.30). All received rHuEpo intravenously, three times per week a t 300 or 150 U / k g body
weight, which was then reduced to 75 U/kg and adjusted to
maintain the hematocrit a t 0.35 k 0.03 (SD).
Results: The baseline hematocrit (0.223 k 0.002) increased to 0.35, more than 0.06 over baseline within 12
weeks in 97.470 of patients. Erythrocyte transfusions (1030
within the 6 months before rHuEpo therapy) were eliminated in all patients within 2 months of therapy. Sixty-eight
patients with iron overload had a 39% reduction in serum
ferritin levels after 6 months of therapy. The median maintenance dose of rHuEpo was 75 U/kg, three times per week
(range, 12.5 to 525 U / k g ) . Nonresponders had complicating causes for anemia: myelofibrosis, osteitis fibrosa, osteomyelitis, and acute or chronic blood loss. Adverse effects
included myalgias, 5%; iron deficiency, 43 %; increased
blood pressure, 35%; and seizures, 5.4%. The creatinine,
potassium, and phosphate levels increased slightly but significantly. The platelet count increased slightly but there was
no increase in clotting of vascular accesses.
Conclusions: The anemia of hernodialysis patients is corrected by rHuEpo resulting in the elimination of transfusions, reduction in iron overload, and improved quality of
life. Iron stores and blood pressure must be monitored and
treated to maintain the effectiveness of rHuEpo and to minimize the threat of hypertensive encephalopathy.
Annals oflnternal Medicine. 1989;111:992- 1OOO.
From the University of Alabama at Birmingham, Birmingham, Alabama; the University of Arizona, Tucson, Arizona;
The Cleveland Clinic, Cleveland, Ohio: the Downstate Medical Center, New York, New York; the University of Missouri at Columbia, Columbia, Missouri; Rush-PresbyterianSt. Luke’s Medical Center, Chicago, Illinois; the University
of California at Los Angeles, Los Angeles, California:
Vanderbilt University and the Nashville Veterans Affairs
Medical Center, Nashville, Tennessee; and the University of
Washington/Northwest Kidney Center, Seattle, Washington. For current author addresses. see end of text.
992
0 1 9 8 9 American College of Physicians
Severe anemia is one of the major limitations to rehabilitation in patients with end-stage renal disease. The
anemia is primarily due to the inability of the diseased
kidney to secrete adequate amounts of erythropoietin
(1). In late 1986 and early 1987, the results of the
initial phase 1-11 clinical trials with recombinant human erythropoietin (rHuEpo) in severely anemic patients on hemodialysis were reported, including 25 patients in the United States ( 2 ) and 10 in the United
Kingdom ( 3 ) . All responded with an effective increase
in erythropoiesis, cessation of transfusion requirements, normalization of hemoglobin and hematocrit,
and improvement in general well-being. These studies
showed the clinical benefits of treatment. In addition,
five potential issues related to patient management
were identified. First, approximately 25% of the patients developed an increase in blood pressure which
required initiation or modification of antihypertensive
therapy. Second, two seizures occurred in the first 35
patients treated with rHuEpo (2, 3 ) . Third, functional
or absolute iron deficiency appeared to limit the effectiveness of the drug. In two-thirds of the patients, supplemental iron was required to restore or maintain a
full erythropoietic response to rHuEpo ( 2 ) . Fourth,
there were concerns that dialysis would become less
efficient in patients with higher hematocrits and lead
to fatal complications such as hyperkalemia ( 2 ) . Finally, there was the possibility that vascular thromboses in general, and access thrombosis in particular,
might increase when the hematocrit increased. Increased heparin doses during dialysis were required in
some patients.
After the initial studies, a phase I11 multicenter
clinical trial of rHuEpo in patients on hemodialysis
was initiated in the United States in the autumn of
1986. The trial was designed to determine the overall
effectiveness of rHuEpo and its effect on patient quality of life, evaluate the safety of the drug, and determine what medical consequences, if any, might result
from near-normalization of the hematocrit in a larger
population of patients with end-stage renal disease. As
reported in the earlier studies, rHuEpo was found to
be effective in virtually all anemic patients on hemodialysis and, although the management of blood pres-
Page 152 of 290
sure and iron status in these patients will require close
attention by physicians, the drug is well tolerated and
has proved to be safe.
Patients and Methods
Patients
The study was approved by the Human Subjects Review
Committees of the nine participating centers and all patients
gave informed consent. The centers participating in the
phase 111clinical trial included the University of Alabama at
Birmingham, the University of Arizona, the Cleveland Clinic, Downstate Medical Center in New York, the University
of Missouri at Columbia, Rush-Presbyterian-St. Luke’s
Medical Center in Chicago, the University of California at
Los Angeles, Vanderbilt University and the Nashville VA
Medical Center, and the University of Washington/Northwest Kidney Center.
To be included in the study, patients met the following
criteria: They had a hematocrit of 0.30 or less; were medically stable on hemodialysis and known to the participating
dialysis center for a minimum of 3 months; had a life expectancy estimated to be at least 6 months; had no hemolysis or
blood loss; had a serum ferritin level greater than 100 pg/L
with a percent-transferrin saturation of 20 or more; were 18
years or older; and had stable liver enzyme tests including
alanine aminotransferase no greater than twice the upper
limit of normal for the reporting center’s laboratory. Exclusion criteria included poorly controlled hypertension with
diastolic blood pressures persistently greater than 100 mrn
Hg, history of a seizure disorder, systemic hematologic disease or unexplained acquired microcytosis, active inflarnmatory disease, therapy with immunosuppressive drugs, or concurrent diseases that might impair the response to rHuEpo.
A total of 333 patients were entered into the study: 159
men and 174 women, with a mean age of 5 1 years (range, 18
to 8 1) . The renal diseases included chronic glomerulonephritis, 23%; hypertension, 23%; diabetes mellitus, 18%;
obstructive uropathy, 5 % ; polycystic kidney disease, 4%;
interstitial nephritis, 2%; pyelonephritis, 2%; miscellaneous,
15%; and unknown, 8%.
Recombinant Human Erythropoietin
The rHuEpo was provided as Epogen by Amgen Inc., Thousand Oaks, California, and was purified from the growth
medium of Chinese hamster ovary cells into which the human erythropoietin gene had been transfected and expressed
(4, 5). The rHuEpo preparation was formulated in a sterile
buffered saline solution containing 0.25 70human serum albumin.
by a Coulter counter (Coulter Electronics, Inc., Miami,
Florida); and by the decrease in erythrocyte transfusion requirements. The leukocyte count and differential, platelet
count, serum iron, total iron binding capacity, and serum
ferritin were also measured serially. In addition, a questionnaire designed to evaluate quality of life (6, 7 ) was completed by the patients before initiating rHuEpo therapy and approximately 6 and 10 months later. Potential adverse organ
effects of rHuEpo and adequacy of dialysis were monitored
by serial liver function tests, serum albumin, electrolyte, and
mineral panel including sodium, potassium, chloride, CO2,
blood urea nitrogen, creatinine, uric acid, calcium, and
phosphorus and by blood coagulation tests. Laboratory values are expressed as mean k standard error. A physical examination, chest roentgenogram, and electrocardiogram
were done on patients before initiation of therapy, after the
target hematocrit was attained, and every 3 months thereafter. Blood was measured initially and then periodically to
determine antibodies to rHuEpo using a radioimmunoprecipitation assay (2).
Results
Effectiveness of Recombinant Human Erythropoietin
We evaluated the efficacy of rHuEpo by increases in
hematocrit, decreases in transfusion requirements, and
changes in patient quality of life. Treatment with
rHuEpo caused dose-dependent increases in hematocrit (Figure 1). The 35 patients receiving 300 U/kg
three times per week during the acute phase of study
all responded, achieving the target hematocrit of 0.35,
and a group average hematocrit increase of 0.13 above
baseline within 6 to 8 weeks. The 201 patients who
received an acute-phase dose of 150 U/kg had a hematocrit rate of rise that was approximately 55% that of
the 300 U/kg group. By study week 10 their group
average hematocrit was within the target range (0.32
to 0.38). The remaining 97 study patients initially received an acute-phase rHuEpo dose of 300 U/kg,
which was subsequently reduced to 150 U/kg. Of the
309 patients who received treatment for at least 4
1
Study Design
Recombinant human erythropoietin was administered as an
intravenous bolus directly into the venous return line or into
the arteriovenous access after the dialyzer was disconnected,
three times a week. On the basis of results of the initial
clinical study ( 2 ) , a starting dose of 300 U/kg body weight
was chosen for the initial (acute) phase of the study. The
initial dose was reduced to 150 U/kg several months after
the beginning of the study, and those patients receiving 300
U/kg had their dose changed to 150 U/kg. When the hematocrit reached 0.35, patients entered the maintenance phase
of the protocol, when the rHuEpo dose was reduced to 75
U/kg. A stable hematocrit of 0.32 to 0.38 (0.35 & 0.03) was
maintained by adjusting the three times weekly dose as necessary in increments of 12.5 to 25 U/kg. If the hpmatocrit
exceeded 0.40,rHuEpo was withheld until the hematocrit
fell below 0.38.
Responses to rHuEpo were monitored by serial measurements of the reticulocyte count (corrected for the hematocrit); hemoglobin and hematocrit, which were determined
15 December 1989
0.22
k
B
/I
2
4
6
I
8
I
1
10 12
Week on study
1
14
16
I
18
Figure 1. The mean hematocrit values a t biweekly intervals for 35 patients receiving 300 U r H u Ep o A g body weight (circles) or 201 patients
receiving 150 U (dimonds) rHuEpoAg. The r H u E p was given intravenously three times per week.
Annals oflnfernalMedicine
Volume 11 1
Number 12
993
Page 153 of 290
Table 1. Effmtsof Recolnbinant Hvman Evthropoietin on Hematologic Values
Variable
Baseline
Number
of
Patients
(fSE)
Hematocrit
Hemoglobin, mmoUL
WdL)
Reticulocytes, corrected
Mean cell volume, fz.
Platelet count, x 1@/L
Leukocyte count, X 1@/L
Serum iron, pmoUL
( P d W
Transferrin, pmol/L
(FddL)
Transfemn saturation, %
304
0.223
0.002
4.65 (7.5)
0.06 (0.1)
0.01 1
304
298
O.OOO4
90.7
1.3
224.0
4.6
6.5
0.1
17.7 (98.6)
0.8 (4.4)
43.3 (241.7)
0.7 (3.9)
41.1
303
303
304
285
286
286
1.5
Ferritin, p&L
962.2
89.4
280
Baseline
(fSE)
Maintenance
Number
of
Patients
0.343
0.002
6.95 (1 1.2)
0.06 (0.1)
0.026
O.OOO9
90.6
0.5
252.9
5.6
6.6
0.1
15.3 (85.6)
1.9 (10.7)
43.2(241.2)
1.5 (8.5)
30.1
1.2
628.5
75.7
P Value.
304
< 0.0005
304
< 0.0005
298
< 0.0005
303
NS
303
< 0.0005
304
NS
285
NS
286
NS
286
< 0.0005
280
< 0.0005
Compared with baseline, using paired t-test. NS = not significant.
weeks, the hematocrit of 295 patients increased to 0.35
or rose a minimum of 0.06 over baselinean effective
response rate of 95.5%. Six more patients met these
criteria by the end of 18 weeks of therapy, for a total
response rate of 97.4%. Most patients achieved the
target hematocrit range within the first 12 weeks of
treatment.
The 333 study patients required a total of 1030
erythrocyte transfusions during the 6 months before
initiation of rHuEpo therapy (an average of 0.52 units
per patient per month) in order to maintain a hematocrit at a level that permitted usual daily activities. After therapy, transfusion requirements decreased progressively (Figure 2) and after month 2 of treatment
virtually all patients were transfusion-independent and
have remained so to date. Occasional transfusions
have been administered following blood losses due to
surgery or dialysis, or when rHuEpo therapy was interrupted. In addition, iron overload, arbitrarily defined as a serum ferritin greater than lo00 ng/mL, and
present in 68 patients at baseline, was reduced. In
these patients, serum ferritin levels decreased a mean
of 39% after 6 months of rHuEpo therapy
(3179 f 258 pg/L compared with 1949 f 213 pg/
L).
Of the eight evaluable patients who failed to achieve
a hematocrit of 0.35, or 0.06 or more over baseline,
one each had myelofibrosis, thalassemia minor, osteomyelitis, and both acute and chronic blood loss. The
other four patients were treated with rHuEpo for less
than the minimum 12-week evaluation period before
withdrawing from the study.
Figure 3 shows the distribution of doses required to
maintain the hematocrit in the target range. The median dose was 75 U/kg but doses of as little as 12.5 U/
kg or as high as 525 U/kg were required for occasional
patients. The distribution of maintenance doses is
994
15 December 1989
Annals oflntemaf Medicine
skewed: 17% of the patients required more than 150
U/kg given intravenously three times weekly to maintain a stable hematocrit. Patient maintenance doses
have remained relatively stable, and no patient has developed resistance to rHuEpo. In addition, no evidence of antibody formation to rHuEpo has been detected t o date.
Table 1 summarizes the pertinent hematologic data
observed at baseline, when patients began maintenance
rHuEpo therapy, and after 6 and 10 months of maintenance rHuEpo therapy. Significant increases in hematocrit, hemoglobin, and corrected reticulocyte
O-7
Weeks
Figure 2. Transfusion requirements (mits/patient) per month for 6
months before initiation of rHuEpo therapy ( p ~and
) at Cweek intervals thereafter. At week 52, one patient autodonated three units in the
previous month for elective hip surgery.
Volume 1 1 1
Number 12
Page 154 of 290
Table 1. (Continued)
6
(*
Months
Number of
Patients
Maintenance
P Value*
(kSE)
SE)
0.338
0.003
6.89( 11.1)
0.06 (0.1)
0.019
0.0007
89.6
0.6
241.0
5.4
6.7
0.2
11.6 (65.0)
0.6 (3.2)
39.8(222.4)
0.6 (3.5)
30.3
1.4
538.6
71.2
233
< 0.0005
233
< 0.0005
228
< 0.0005
232
10
Months
0.353
0.004
7.14(11.5)
0.06 (0.1)
0.02 1
0.0013
91.9
1.o
232.0
7.0
6.7
0.3
12.3 (68.4)
0.8 (4.6)
38.1(212.8)
0.8 (4.5)
33.6
2.4
796.7
145.7
NS
23 1
< O.oOO5
233
0.03
210
< 0.0005
21 1
< 0.0005
209
< 0.0005
204
< 0.0005
count occurred with rHuEpo therapy. There were no
significant alterations in the mean erythrocyte volume,
total leukocyte count, or serum transferrin level. The
leukocyte differential remained normal except for an
increase in the percent of monocytes (5.6 f0.2 at
baseline to 6.7 f0.2 6 months after entry into the
maintenance phase, P < 0.01).
Most patients reported an increase in their quality
of life as manifested by increased exercise tolerance
and activity levels, higher energy levels, improved
sleep-wake cycles, improved appetite, increased body
warmth, and an enhanced perception of their health
status. A quantitative assessment of the effect of nearcorrection of anemia with rHuEpo therapy on the
quality of life is summarized in Table 2. Of 130 patients completing questionnaires at baseline and after
approximately 6 and 10 months of rHuEpo therapy,
there was almost a two-fold increase in the percentages of patients who had no complaints and were able to
carry on normal activity (26%, 45%, and 44%, respectively, per Karnofsky scoring). Conversely, 46%
of patients complained of low or no energy at baseline,
whereas only 23% and 22% had these complaints during the immediate and later maintenance treatment
phases, respectively.
Of perhaps greater clinical significance are the reports of energy level as determined by the Nottingham
Health Profile. A score of 100 on this profile indicates
complete limitation; a score of 0 indicates the absence
of limitations. Energy level improved from a baseline
score of 47 to 32 after the hematocrit was acutely corrected and then to 28 at further follow-up. In response
to objective quality-of-life questions, particularly those
relating to energy and activity levels, the patients reported a statistically significant and sustained improvement between baseline and both follow-up time
periods.
15 December 1989
Number of
Patients
P Value*
104
< 0.0005
104
< 0.0005
101
< 0.0005
104
0.006
104
NS
104
0.0 1
96
< 0.0005
97
< 0.0005
96
< 0.0005
94
< 0.0005
Patient Study Status
Of the 333 patients beginning rHuEpo therapy, 266
were on therapy as of 30 November 1987, 13 months
after the start of the trial. The reasons for patient
withdrawal from the study included renal transplantation in 15 patients; death, 22 patients; possible toxicity, 9 patients; voluntary patient withdrawal, 13
patients; and other causes, 8 patients. Of the 7 who
60
50
-
v)
I-
z
W
F 40-
8
B
I"i
20
"'1
*
0 0
400 ,600
DOSE OF rHuEPO (U/kg)
Figure 3. The distribution of maintenance doses of rHuEpo required to
maintain the hematocrit between 0.32 and 0.38. The dose level refers to
the upper value within each 25 U/kg dose range. The rHuEpo was given
intravenously three times per week.
Annals ofhternaf Medicine
Volume 1 1 1
Number 12
995
Page 155 of 290
Table 2. Effect of Recombinant Human Erythropoietin on
Patients’ Functional Impairment, Energy, and Activity
Level (n = 130)
Baseline
0.237
Hematocrit, mean
Functional impairment
Normal, no complaints;
able to carry on normal
activity (Karnofsky),
% ofpatients
25.9
Activity level
Very or mostly
active, % of
pa tients
19.8
Energy level
Patient reporting
very full of
energy or fairly
energetic most
of the time, %
25.9
Patients reporting
low energy or no
energy at all, % 46.2
Nottingham
Health Profile
47.0
score6
Second
Evaluation*
Third
Evaluation?
0.342
0.339
44.53
43.5s
37.3$
35.5s
45.4$
48.1$
23.23
22.2s
31.5f
27.71
* Approximately 6 months after initiation of rHuEpo therapy.
t Approximately 10 months after initiation of rHuEpo therapy.
1 P 5 0.01 compared with baseline.
4 Scores, 100 = complete limitation; 0 = no physical limitation.
withdrew because of possible toxicity, 5 had seizures, 2
had diffuse myalgias, 1 had a cardiac arrest, and 1 a
decrease in vision. Of the 15 who voluntarily withdrew
from therapy, 2 failed to respond to rHuEpo, 2 became hypertensive, and 2 developed headaches. Isolated causes comprised the balance. Miscellaneous
non-toxicity-related reasons for withdrawal included
protocol violation, 4 patients; moving to another location, 2 patients; and loss to follow-up, l patient.
The mean age of the 22 patients who died was
60 f 4 years (range, 19 to 79). The causes of renal
failure in these patients were diabetes mellitus in 10
patients; hypertension, 6 patients; chronic glomerulonephritis, 4 patients; and miscellaneous, 2 patients.
The causes of death included myocardial infarction,
arrhythmias, cardiopulmonary arrest in 11 patients;
sepsis, 6 patients; cerebral hemorrhage, 2 patients
(one from trauma); hyperkalemia, 1 patient; liver failure from hemochromatosis, 1 patient; and voluntary
withdrawal from dialysis, 1 patient. The duration of
rHuEpo therapy before death ranged from 26 to 329
days.
Adverse Effects and Patient Management Issues
The commonest adverse effect temporally related to
rHuEpo administration was reported by 15 patients
who had myalgias and a flu-like syndrome. These
symptoms were usually mild. Two patients, however,
discontinued rHuEpo therapy; in the other 13 patients
these symptoms did not persist with continued admin996
15 December 1989
AnnalsofInfernalMedicine
istration of rHuEpo, and they remained on therapy.
Ten patients developed injected conjunctivae.
In 25 1 patients there were sufficient data to evaluate
changes in blood pressure and antihypertensive medications after 3 months of rHuEpo therapy. Of these
patients, 88 (35%) developed an increase in the diastolic blood pressure of 10 mm Hg or more or required
an increase in blood pressure medication or both within 3 months of rHuEpo therapy. Of the 251 patients,
180 (72%) had baseline hypertension and 57 (32%)
had an exacerbation which required increased antihypertensive medication. Of the 71 patients who were
not hypertensive before rHuEpo therapy, 31 (44%)
had an increase in diastolic blood pressure of 10 mm
Hg or more and 23 (32%) required initiation of antihypertensive treatment. Increase in blood pressure was
thus unrelated to baseline levels.
Seizures occurred in 18 of 333 (5.4%) patients. In
ten instances, the seizure activity occurred within the
first 3 months of rHuEpo therapy when the hematocrit was increasing and was sometimes related to the
onset of uncontrolled hypertension. The risk of seizure
in the total group of patients was 1 per 13 patient
years.
Of 333 patients, 142 (43%) who began treatment
with rHuEpo developed evidence of absolute or functional iron deficiency as defined by a serum ferritin of
less than 30 pg/L and a percent transferrin saturation
of less than 20, or by a percent transferrin saturation
less than 20 with a normal serum ferritin, respectively
( 2 ) . Despite receiving intravenous iron dextran (Imferon, Fisons Corporation, Bedford, Massachusetts)
or oral iron medication, the serum ferritin often decreased, particularly during the acute phase of therapy, Serum ferritin levels decreased from a mean baseline value of 962 f 89 pg/L to 629 f 76 pg/L by the
entry into the maintenance phase of the study (Table
1)The adequacy of dialysis was determined by following predialysis creatinine, blood urea nitrogen, phosphorus, and potassium concentrations. As seen in Table 3, there was a slight but statistically significant
increase in predialysis serum creatinine, potassium,
and phosphorus levels when the hematocrit increased
as a result of rHuEpo therapy.
Sixty thrombotic events involving vascular accesses
were observed in a total of 39 patients, a rate of 0.3
thrombotic events per patient year. Most of the vascular accesses were polytetrafluoroethylene or bovine
grafts. This incidence of access clotting is no greater
than that observed in 1 1 1 1 patients on hemodialysis
not treated with rHuEpo (0.5 clotting events/
patient . year; Downing M. Unpublished observations). Although the mean platelet count increased
from 224 f 5 to 253 k 6 x lO9/L (P < 0.0005)
during the acute phase, there was no further increase
after 10 months of maintenance therapy (Table 1).
Although statistically significant, the increase in platelet count was still within the normal range. Prothrombin and partial thromboplastin times and fibrinogen
levels did not change over the course of rHuEpo theraPY.
Volume 111
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Page 156 of 290
Discussion
Severe chronic anemia is a major impediment to the
rehabilitation of patients with end-stage renal disease.
The major contributor to the anemia is a relative or
absolute deficiency of erythropoietin production by
the diseased kidneys. Although modest reductions in
erythrocyte survival and putative inhibitors of erythropoiesis could contribute to the anemia, their effect
cannot be great given the uniform and rapid response
of essentially all patients on hemodialysis in clinical
trials of rHuEpo. The results of this multicenter clinical trial involving 333 patients confirm the results reported by the smaller initial trials (2, 3, 8-14). A total
of 323 patients have been previously reported from
these other studies and all had significant erythropoietic responses to rHuEpo. Virtually all patients in
this multicenter trial responded to rHuEpo in a dosedependent manner. Therapy with rHuEpo thus resulted in a correction of the anemia with achievement of
target hemoglobin and hematocrit levels for more than
97% of patients, virtual elimination of the need for
erythrocyte transfusions, and the reduction of iron
overload, if present. In addition, in response to a series
of questionnaires, patients reported significant increases in their energy and activity levels and health status,
resulting in a substantial improvement in their quality
of life (15).
Doses of rHuEpo required to maintain the hematocrit at 0.35 & 0.03 varied from 12.5 to 525 U/kg, but
83% of the patients required 150 U/kg or less. The
reason why a small percentage of these patients
(17%) required higher doses of rHuEpo in order to
maintain a stable hematocrit is unknown.
Therapy with rHuEpo was well tolerated and many
of the adverse effects of drug administration are likely
coincidental. Among the direct adverse effects which
were noted most frequently, myalgias or flu-like syndrome or both, infected sclera, headache, and flank
pain were the most prominent. These symptoms have
been reported previously in association with rHuEpo
therapy (3, 9, 16). Whereas we observed a 5% incidence of flu-like myalgias, 17% incidence of headaches, and a 14% incidence of flank pain, the European Cilag multicenter trial using the same rHuEpo
observed a 770, 2770, and 30% incidence of these adverse effects, respectively (17). The role of rHuEpo in
these complaints is uncertain but, they were generally
mild and usually did not persist nor preclude continued treatment with, or an effective response to the
drug.
Therapy with rHuEpo had no detectable adverse organ effects during the course of this trial. Liver function remained unchanged and cholesterol and triglyceride levels, blood glucose, serum calcium, and serum
albumin levels did not change with rHuEpo therapy.
The overall prorated yearly mortality rate for the patients on study was 9.4%, which is slightly less than
the first-year mortality rate (12%) observed for US.
patients on dialysis ages 45 to 54 years during 1983
through 1986 (18).
These results clearly show that rHuEpo is effective
15 December 1989
in correcting the anemia of hernodialysis patients and
that few, if any, side effects can be directly attributed
to its use. Equally important is the fact that antibodies
have not developed against the recombinant protein.
Three of the five major issues of patient management-hypertension, seizures, and functional-absolute
iron deficiency-were confirmed and detailed by the
present study. Hypertension is a frequent complication
of chronic renal failure; hypertension was the major
mechanism that resulted in renal failure in 23% of the
study patients. In this clinical trial 35% of all patients
developed an increase in blood pressure which was defined as an increase in diastolic blood pressure of 10
mm Hg or more, whereas 25% of the patients required
initiation or increase in antihypertensive medication.
Although 72% of the rHuEpo-treated patients had existing hypertension at baseline, they were at no greater
risk for exacerbation of hypertension than those patients who were not originally hypertensive (32% and
32%, respectively). Blood pressure increased at a similar incidence in patients receiving 150 or 3 0 0 U/kg as
the initial doses of rHuEpo. In a few instances, hypertensive crises were associated with seizure activity.
Careful monitoring and control of blood pressure in
rHuEpo-treated patients, particularly during the acute
phase of treatment, is essential. Some patients will require more antihypertensive medication than others as
their anemia is being corrected.
The development or exacerbation of hypertension in
patients on hemodialysis responding to rHuEpo is
probably caused by a reversal in the peripheral arteriolar vasodilation that occurs with anemia. Preliminary
data from several groups suggest that the increased
hematocrit results in a decrease in peripheral vasodilation with an increase in total peripheral resistance (19,
20). These observations are consistent with earlier
studies by Neff and coworkers (21). They studied six
hemodialysis patients who were transfused to a hematocrit of 0.40 over a 2-week period and noted that
correction of the anemia (that is, a doubling in the
hematocrit) was associated with a twofold increase in
peripheral vascular resistance as well as a decrease in
cardiac index.
Although increased seizure activity is well recognized among patients with end-stage renal disease, the
precise frequency in patients similar in age, sex, and
clinical status to those in the rHuEpo study is not
known. The incidence of seizure activity in 1 1 1 1 patients not treated with rHuEpo was 8% (Downing M.
Unpublished observations), which is similar to that
observed in those patients receiving rHuEpo. Approximately 50% of the seizures in rHuEpo-treated patients
occurred during the initial 12 weeks when the anemia
was being corrected. Although not statistically significant, it is possible that the frequency of seizures seen
during this period in this study exceeds that of the
background seizure activity in the end-stage renal disease population in general. Whether or not this is true, it
is clear that careful monitoring of blood pressure and
control of hypertension will be an important management issue as rHuEpo becomes available for routinely
treating anemia in patients with end-stage renal disease.
Annals ofZnternalMedicine
Volume 1 1 1
Number 12
997
Page 157 of 290
Table 3. Effectof Recombinant Human Erythropoietin on Biochemical Values
--
Solutes
Creatinine, ,umol/L
(mddL)
Blood urea nitrogen, mmoII/L
(mg/dl;)
Potassium, mmol/L
(mEg/L)
Bicarbonate, mmoI/L
(mEq/L)
Uric acid, p n o l / L
(mg/dL)
Albumin, g / L
WdL)
Phosphorus, mmoI/L
fm,ddL)
1167 (13.2)
18
(0.2)
28.8 (80.8)
0.4 (1.2)
5.1 (5.1)
0.04 (0.04
20.7 (20.7)
0.2 (0.2)
393
(6.6)
0.59 (0.1)
38.0 (3.8)
0.3 (0.03
1.78 (5.5)
0.03 (0.1)
* Compared with baseline, using paired t-test. NS
298
298
297
296
241
260
292
15 December 1989
P Value+
298
< 0.0005
298
0.0 14
297
0.012
296
< 0.0005
24 1
< 0.0005
260
0.002
292
< 0.0005
= not significant.
A third management issue involves the availability
of iron for rHuEpo-stimulated erythropoiesis, particularly during the treatment-induction phase, as the hematocrit is rising. Functional or absolute iron deficiency occurred in 43% of all patients during the early
treatment phase. The incidence of iron deficiency
would have been greater if 20% of the patients had not
been iron overloaded. Studies from one of our centers
suggest that most severely anemic patients on hemodialysis treated with rHuEpo will eventually require
supplemental iron unless their baseline serum ferritin
is greater than lo00 pg/L (22). Iron deficiency,
whether relative or absolute, will reduce the effectiveness of rHuEpo and unnecessarily add to the cost of
treatment and delay rehabilitation of the patient. Furthermore, iron deficiency may lead to confusion about
the responsiveness of the patient to the drug. On the
basis of our experience of the phase I11 clinical trial,
we feel that transferrin saturations of less than 20%
indicate impaired iron availability for hemoglobin synthesis and suggest the need for either oral or intravenous iron replacement. If use of oral iron supplementation does not maintain a percent saturation greater
than 20, intravenous iron dextran may be required periodically for optimal erythropoiesis.
Underdialysis and an increase in clotting of arteriovenous grafts at higher hematocrits were potential
(23), but unproven, concerns. As noted in Table 3,
the mean predialysis creatinine values, although statistically significantly higher, were only 4.5% higher after the hematocrit had increased from 0.22 to 0.34.
Although dialyzer clearance of creatinine decreases as
the hematocrit rises (24), the magnitude of this
change did not result in a need to either lengthen dialysis times or increase dialyzer membrane surface area.
The mean blood urea nitrogen values did not increase,
consistent with the data that suggest that urea diffuses
instantly from the erythrocyte and that urea clearance
is not altered by a change in the hematocrit (25). Predialysis serum phosphate levels also increased slightly,
but significantly. Although dialyzer clearance of phosphate may decrease as dialyzer plasma volume de998
1220 (13.8)
27
(0.3)
27.7 (77.5)
0.5 (1.4)
5.2 (5.2)
0.06 (.06
19.0 (19.0)
0.2 (0.2)
434
(7.3)
0.43 (0.1)
39.0 (3.9)
0.3 (0.03
2.03 (6.3)
0.06 (0.2)
Number
of
Patients
Annals ofznternafMedicine
creases, it is not clear what role that increased dietary
phosphate may have contributed to the observed
change. Serum potassium levels did increase minimally, but significantly. The small change seen could be
contributed by any combination of increased dietary
intake, decrease in dialyzer clearance, or increased
erythrocyte potassium release, because the increased
erythrocyte mass will release proportionately more potassium at the time of erythrocyte death. Although the
clinical significance of slight increases in predialysis
serum creatinine and phosphate concentration is probably minimal, the effects of hyperkalemia are of greater concern. Serum potassium levels should be monitored carefully in all patients and potassium should be
eliminated from the dialysate if medically indicated.
In this multicenter trial, 39 patients experienced 60
thrombotic events that involved the arteriovenous access, most of which were polytetrafluoroethylene
grafts. When these events were corrected for the total
number of patients responding to rHuEpo, 12% of
patients per year experienced clotting in their vascular
access. In previous studies of hemodialysis patients not
receiving rHuEpo, polytetrafluoroethylene arteriovenous grafts were associated with a greater frequency of
thrombosis than was observed in natural (Cimino) fistulae (26, 27). These authors reported that 49% and
29% of 163 and 219 patients, respectively, had recurrent polytetrafluoroethylene graft failure, primarily
due to thrombosis requiring surgical revisions every
0.5 years. Therefore, there is a subgroup of patients
not treated with rHuEpo who clot their arteriovenous
grafts an average of one to two times per year. In
addition, in another survey (Downing M. Unpublished data), the incidence of access clotting in over
lo00 hernodialysis patients not treated with rHuEpo
was approximately twice the incidence of access clotting in our study. Therefore, we feel that rHuEpo
treatment does not increase the incidence of vascular
access clotting. There was no increase in cerebrovascular, coronary or peripheral arterial thromboses when
compared to over lo00 hemodialysis patients not
treated with rHuEpo (Downing M. Unpublished
Volume 111
Number 12
Page 158 of 290
Table 3. (Continued)
6 Months
(fSE)
1246 (14.1)
27
(0.3)
29.4 (82.4)
0.5 (1.5)
5.2 (5.2)
0.05 (0.05)
18.7 (18.7)
0.3 (0.3)
428
(7.2)
0.43 (0.1)
39.0 (3.9)
0.3 (0.03)
1.91 (5.9)
0.03 (0.1)
Number of
Patients
Maintenance
P Value*
224
0.0013
224
NS
225
0.003
222
< 0.0005
177
< 0.0005
189
NS
220
< 0.0005
data.) Three centers noted an increased need for heparin in some patients with near-normal hematocrit levels to adequately clear the dialyzer at the end of dialysis; this problem was easily managed by increasing the
primary dose of heparin by lo00 units.
Patients who previously required transfusions became transfusion-independent with the advent of
rHuEpo therapy. If this group of patients is representative of patients with end-stage renal disease as a
whole, the savings in blood nationally would be projected to be 500 OOO units a year. This figure is based
on the fact that the mean transfusion need per month
for the patients in this clinical trial was 0.52 and assumes that all patients with end-stage renal disease
who required transfusions will receive rHuEpo, that
all patients will respond and become transfusion-independent, and that the patients in this study are representative of the approximately 75% of the 100 OOO patients with end-stage renal disease in the United States
who are candidates for rHuEpo therapy. The alleviation of transfusion requirements will have not only a
positive effect on the nation’s blood supply, but also on
nursing time, on the potential exposure of patients and
health personnel to infection, the risks of iron overload
in selected patients, and the number of potential kidney transplant recipients since there will be less transfusion-induced sensitization.
Use of rHuEpo had no predictable effect on other
hematopoietic lineages; its major effect appears to be
specific for erythropoiesis. Nevertheless, the mean
platelet count in patients treated with rHuEpo rose
significantly. Whether this reflects a minor effect of
rHuEpo on megakaryocyte maturation and platelet
production, as has been reported previously (8, 11, 16,
28, 29), is not known. It is equally conceivable that
some patients who developed either absolute or relative iron deficiency during the course of rHuEpo therapy also developed reactive thrombocytosis. Thus,
iron deficiency might account for the marginal increase in platelet count but would not be considered a
direct effect of the hormone on megakaryocytopoiesis.
The effect of rHuEpo on the quality of life of pa15 December 1989
10 Months
(*SE)
Number of
1299 (14.7)
35
(0.4)
31.1 (87.0)
0.8 (2.3)
5.4 (5.4)
99
0.04
99
NS
99
0.001
99
< 0.0005
81
0.009
74
NS
98
0.037
.08 (0.08)
18.4 (18.4)
0.3 (0.3)
410
(6.9)
0.41 (0.2)
40.0 (4.0)
0.4 (0.04)
1.91 (5.9)
0.03 (0.2)
P Value*
Patients
tients with end-stage renal disease is most impressive
when the objective variables of functional ability, energy level, and health status are assessed. Of the previous studies (6) of quality-of-life assessments in endstage renal disease patients, none has documented a
treatment intervention (other than transplantation)
that enhances quality of life to the magnitude as that
achieved by rHuEpo.
The results of the phase I11 clinical trial with
rHuEpo in patients with anemia and end-stage renal
disease have been evaluated and the initial conclusions
of the phase 1-11 clinical trials have been confirmed.
Recombinant human erythropoietin is effective, safe,
and well tolerated. We recommend that rHuEpo become standard therapy in the management of anemic
patients with end-stage renal disease and believe it will
contribute to the rehabilitation and improved health
status of these patients.
Acknowledgments:The authors thank the registered nurses who coordinated, the staff who administered the study, and the patients who volunteered for the study.
Grant Support: Supported in part by Amgen. Inc., Thousand Oaks, California.
Requests forRephts: Michael Downing, PhD, Amgen, Inc., 1900 Oak
Terrace Lane, Thousand Oaks, CA 91320.
Current Author Addresses: Drs. Exhbach and Haley: Division of He-
matology, RM-10, Department of Medicine, University of Washington,
Seattle, WA 98195.
Drs. Abdulhadi and Paganini: Section of Dialysis and Extracorporeal
Therapy, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland,
OH 44106.
Drs. Browne, Downing, and Egrie: Amgen, Inc., 1900 Oak Terrace
Lane, Thousand Oaks, CA 9 1320.
Drs. Delano, Friedman, and Lundin: Division of Renal Disease, Department of Medicine, State University of New York Health Science Center
at Brooklyn, 470 Clarkson Avenue, Box 52, Brooklyn. NY 11203.
Dr. Evans and Ms. Rader: Battelle, Human Affairs Research Center,
P.O. Box C-5395, 4ooo N.E. 41st Street, Seattle, WA 98105.
Drs. Graber and Krantz: Division of Hematology, Department of Medicine, Vanderbilt University, Nashville, TN 37232.
Dr. Korbet: Section of Nephrology, Department of Medicine, RushPresbvterian-St. Luke’s Medical Center. 1653 West Congress
- Parkwav.
chicigo. IL 60612.
Dr. Nissenson: Division of Nephrology, Department of Medicine,
UCLA School of Medicine, Los Angeles, CA 90024.
Drs. Ogden and Van Wyck: Department of Internal Medicine, Section of
AnnalsofZnternalMedicine
Volume 111
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Page 159 of 290
Renal Disease, The University of Arizona Health Sciences Center, Tucson, AZ 85724.
Dr. Rutsky: University of Alabama at Birmingham, Department of
Medicine/Division of Nephrology, Birmingham, AL 35294.
Dr. Stivelman: Department of Medicine, Emory University School of
Medicine, 69 Butler Street, S.E., Atlanta, GA 30303.
Drs. Stone and Teschan: Nashville Veterans Affairs Medical Center,
1310-24th Avenue, South, Nashville, T N 37212.
Dr. Van Stone: Department of Medicine, Division of Nephrology, University of Missouri Medical School, 18005 E. Walnut, Columbia, MO
65212.
Dr. Zuckerman: University of Alabama at Birmingham, Division of Hematology/Oncology. Department of Medicine, Birmingham, AL 35294.
Dr. Adamson: The New York Blood Center, 310 East 67th Street, New
York. NY 10021.
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Kidney International, Vol. 55, Suppl. 69 (1999), pp. S-35–S-43
Iron overload in renal failure patients: Changes since the
introduction of erythropoietin therapy
JOSEPH W. ESCHBACH and JOHN W. ADAMSON
University of Washington, Minor and James Medical, Seattle, Washington, and Lindsley F. Kimball Research Institute,
New York Blood Center, New York, New York, USA
Iron overload in renal failure patients: Changes since the introduction of erythropoietin therapy. Iron overload was a common
complication in patients with chronic renal failure treated with
dialysis prior to the availability of recombinant human erythropoietin (rHuEPO) therapy. Iron overload was the result of hypoproliferative erythroid marrow function coupled with the need
for frequent red blood cell transfusions to manage symptomatic
anemia. The repetitive use of intravenous iron with or without
the use of red blood cell transfusions also contributed to iron
loading and was associated with iron deposition in liver parenchymal and reticuloendothelial cells; however, there were no
abnormal liver function tests or evidence of cirrhosis unless viral
hepatitis resulted from the transfusions. With rHuEPO therapy, the excess iron stores were shifted back into circulating red
blood cells as the anemia was partially corrected, and red blood
cells were lost from circulation by the hemodialysis procedure.
After several years of rHuEPO therapy, most hemodialysis
patients required iron supplements to replace the continuing
blood losses related to hemodialysis. The potential complications of iron overload (parenchymal iron deposition, permanent organ damage, increased risk of bacterial infections, and
increased free radical generation) are reviewed in the context
of this setting.
mias [4], including the anemia of chronic renal failure
(CRF) [5–7] or from the repetitive injection of parenteral
iron. Hemosiderosis is also referred to as secondary iron
overload.
Traditionally, hemochromatosis and hemosiderosis have
been separated by the concept that in the former, the
iron overload is primarily in tissue parenchymal cells
leading to organ dysfunction and eventually organ failure, whereas in hemosiderosis, the iron is limited to the
RE cell, with no organ dysfunction or failure. However,
in hemosiderosis, these lines of separation may not always be that precise, as noted in a review of iron overload
in various conditions [8], particularly that of CRF [9].
In order to appreciate the changes that recombinant
human erythropoietin (rHuEPO) has made on the ironoverload complication in CRF, it is important to understand what impact, if any, iron overload has had on the
overall health of dialysis patients.
IRON PHYSIOLOGY
Iron stores in normal subjects vary between approximately 800 mg to 1200 mg, depending on body size [1],
although phlebotomy studies suggest that normal iron
stores may be as high as 1200 to 1500 mg [2]. Primary
iron overload, or primary hemochromatosis, is a common
hereditary disorder in which excessive amounts of iron
are absorbed from the gastrointestinal tract, resulting
over many years in the accumulation of massive amounts
of iron (as much as 20 to 40 g) in the parenchymal cells
of various tissues, leading to end-organ damage to the
heart, liver, and pancreas [3]. Hemosiderosis is the accumulation of excess iron, primarily in the reticuloendothelial (RE) cells of the liver, marrow, and spleen, as the
result of the repetitive infusion of iron from red blood
cell (RBC) transfusions for the treatment of severe aneKey words: chronic renal failure, blood, rHuEPO, anemia, dialysis.
 1999 by the International Society of Nephrology
The amount of iron in excess of normal iron stores
required to develop hemosiderosis is not known, but
probably is considerable. One estimate is that the storage
limit of macrophages is exceeded after the accumulation
of 5 g of unexcretable iron [8]. An understanding of iron
transport and kinetics is essential in order to comprehend
the issue of “iron overload.” This is particularly true
since the introduction of rHuEPO therapy, which has
changed our understanding of iron metabolism and its
management in CRF.
Iron that is absorbed from the gastrointestinal tract is
transported in circulation to either the erythroid marrow,
RE cells, or various tissues (for example, myoglobin in
muscle cells or liver in severe iron overload) by the
protein transferrin. Iron that is injected intravenously,
either as RBCs or as an iron compound (iron dextran,
ferric gluconate, or ferric saccharate), is processed by
the RE cell and then transported on one or both ironbinding sites of the transferrin molecule to either the
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Eschbach and Adamson: Iron overload in renal failure
erythroid marrow or to various other tissues. The likelihood that nonerythroid tissues will take up transferrinbound iron is determined by the activity of the erythroid
marrow and by the degree of saturation of transferrin.
Transferrin receptors are probably expressed on all cells,
with the exception of mature RBCs, with the highest
expression of receptors on hemoglobin-synthesizing cells
[10]. One of the few ferrokinetic studies in hemodialysis
patients not treated with rHuEPO noted that the nonerythroid iron turnover was directly related to serum iron
levels and with the percentage transferrin saturation,
with serum iron values of more than 150 mg/dl, and
transferrin saturations of more than 60% associated with
a nonerythroid iron turnover of more than 0.4 mg/100
ml whole blood/day (normal 5 0.16; Fig. 1). This suggests
that when the transferrin saturation is more than 60%
and the serum iron is greater than 150 mg/dl, the likelihood of transferrin-bound iron being shunted to nonerythroid cells is increased in the anemic dialysis patient
not treated with rHuEPO. In the anemic patient treated
with rHuEPO, erythropoiesis is stimulated, and more
iron will be preferentially taken up by the erythroid
marrow, reducing the likelihood of iron deposition in
nonerythroid tissues and mobilizing stored iron in order
to support new hemoglobin synthesis.
IRON OVERLOAD IN CRF PRIOR TO
ERYTHROPOIETIN THERAPY
To appreciate the effect that rHuEPO treatment has
had on iron overload in anemic CRF patients, it is necessary to understand what the experience was with iron
overload prior to the introduction of rHuEPO therapy.
Prior to the availability of rHuEPO therapy in CRF,
iron overload was very common. The natural progression
of the anemia of CRF results in a gradual shift of red
cell iron into storage sites in the RE system (RES) so
that by the time patients start dialysis, if not treated with
rHuEPO, they are often severely anemic (hematocrit
15% to 25%/hemoglobin 5 to 8 g/dl). If external blood
losses have not occurred, then the iron, previously part
of the RBC mass, becomes sequestered in the RES,
resulting in elevated serum ferritin levels even in those
patients who have not received any exogenous iron.
The anemia of CRF is primarily due to insufficient
production of renal erythropoietin. Recently, erythropoietin has been shown to increase transferrin receptor
synthesis and cell surface expression in erythroid cells by
activating the iron regulatory protein 1, thus stabilizing
transferrin receptor mRNA [11]. Without sufficient erythropoietin stimulation of the erythroid cell, the number
of erythroid cell surface transferrin receptors is probably
down-regulated, increasing the likelihood of iron uptake
by nonerythroid tissues, including the liver.
As reflected by in vivo organ counting following 59Fe
Fig. 1. The amount of nonerythroid iron turnover in patients on
chronic hemodialysis related to their serum iron values (A) and percentage transferrin saturation (B; unpublished observations).
administration in dialysis patients, iron deposition often
occurred in the liver when erythroid marrow function
was severely depressed [9]. Liver biopsies in these patients disclosed iron in both hepatocytes and Kupffer
cells (the RE cell of the liver) [9]. Because of the persistent anemia, RBC transfusions were often necessary.
Gradually, as more RBC transfusions were given and
iron intake exceeded iron losses, the transferrin saturation and serum ferritin levels increased, and as the transferrin saturation rose, iron was delivered in increasing
amounts to the liver and the parenchymal cells of other
tissues, including heart, thyroid, and pancreas. This ultimately led to evident tissue iron overload, as demonstrated by bone marrow and liver biopsy and autopsy
studies [6, 7, 9, 12–19]. Some individuals eventually had
transferrin saturations and serum ferritin levels exceeding
80% and 4000 ng/ml, respectively. However, this was not
necessarily an irreversible process. Some dialysis patients
had enough erythroid marrow function to not require
RBC transfusions and did not shunt unincorporated
RBC iron into the liver [9]. Occasionally, erythropoiesis
in some dialysis patients spontaneously improved [9],
particularly if RBC transfusions were avoided. Red cell
transfusions in these patients had resulted in further suppression of the small amount of renal erythropoietin that
was being produced [20]. If, and when, erythropoiesis
improved in these patients, iron overload gradually was
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reduced because of the elimination of RBC transfusions
coupled with the ongoing blood losses related to the
hemodialysis procedure. Prior to rHuEPO therapy, there
evolved the practice of not transfusing except when there
was symptomatic tissue hypoxia. Most patients without
angina could slowly adjust to a hematocrit of 22% to
28% and not require RBC transfusions, although energy
levels and quality of life were diminished.
Nevertheless, although hemodialysis-related iron
(blood) losses were often large (1 to 3 g/year) [21–23],
these losses frequently did not compensate for the amount
of iron previously received from RBC transfusions. Therefore, iron overload persisted. Attempts to reduce iron
overload with desferrioxamine were successful primarily
in those patients who no longer required RBC transfusions. However, in the majority of patients, the amount
of iron chelated and transferred across the dialyzer membrane was less than the amount of iron received intravenously through ongoing RBC transfusions [24–28]. Although no recent studies of dialyzer blood losses have
been published, we believe there are still substantial
iron losses occurring even with the newer dialyzers. For
instance, during 1994, 75% of the 615 hemodialysis patients at the Northwest Kidney Centers (Seattle, WA,
USA) received 0.5 to 3.0 g (1.0 6 0.6 g, mean 6 sd)
of iron dextran i.v., despite ingesting oral iron, just to
maintain serum ferritin levels and transferrin saturations
close to 100 ng/ml and 20%, respectively (J. W. Eschbach,
R. Garth, and C. R. Blagg, unpublished results).
Iron overload also has occurred as the result of the
excessive administration of parenteral iron. Most studies
of iron overload in dialysis patients have included patients who have received both parenteral iron as well as
RBC transfusions [12, 14, 16, 17, 27, 29–31]. There has
been only one report of iron overload in dialysis patients
caused by intravenous iron infusions without any RBC
transfusions: Two groups of dialysis patients with iron
overload were studied. One group received repeated
injections of parenteral iron. The other group received
multiple RBC transfusions [32]. The serum ferritin levels
ranged from just below 1000 ng/ml up to 3000 ng/ml in
the parenteral iron group and less than 1000 ng/ml in
the transfused group. Although iron was present in both
hepatocytes and Kupffer cells on liver biopsies in some
patients, the authors did not attempt to determine if the
liver cell iron deposition was different between the two
groups. Iron overload also has been reported in a subject
with normal renal function who received 52 g of elemental iron intramuscularly over a 20-year period for treatment of an ill-defined anemia [33]. The serum ferritin
was 2840 ng/ml, and liver function tests were normal.
Despite the fact that liver biopsy disclosed large deposits
of iron in the parenchyma with minimal iron deposition
in the Kupffer cells, there was no cirrhosis.
The distinction between iron given parenterally and
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iron given as RBC transfusions may be a critical one. The
hemosiderosis from iron overload secondary to RBC
transfusions may be complicated by the presence of hepatitis B or C acquired in the course of being transfused
[16]. Hepatocellular injury from hepatitis C may favor
iron deposition in liver parenchymal cells [34]. Because
serological testing for hepatitis C was not available during the pre-rHuEPO era when RBC transfusions were
common therapy for the anemia of CRF, it is difficult
to determine the relative contributions of hepatitis and
iron deposition to the abnormal liver biopsy/autopsy
findings and other liver tests performed.
Parenteral infusion of iron, in various forms, has been
studied extensively in animals. The amount of iron these
animals received in different studies ranged between 0.1
and 3.3 g/kg with a follow-up between four weeks and
seven years [35, 36]. Despite these large infusions of iron
(up to 17 g), the kind of parenchymal tissue changes
seen in primary hemochromatosis has not been observed;
that is, cirrhosis and pancreatic fibrosis have not been
found, and tests of liver and cardiac function and of
glucose tolerance have remained normal. The large
amounts of iron were predominantly sequestered in RE
cells without any fibrotic reaction present.
IRON OVERLOAD IN CRF SINCE
rHuEPO THERAPY
There are no good epidemiological data to indicate
the magnitude of iron overload prior to the availability
of rHuEPO, but it must have been common. Approximately 50% of dialysis patients in one study required
more than one RBC transfusion monthly [37]. One center noted that 64 of its 120 hemodialysis patients had
serum ferritin levels of more than 1000 ng/ml [17]. The
longer patients survived and were transfused, the greater
the likelihood that iron overload would develop.
The introduction of rHuEPO has resulted in major
benefits for the patient with the anemia of CRF. One of
these has been the elimination of the need for routine
RBC transfusions and the eventual elimination of iron
overload. Many studies have demonstrated that serum
ferritin levels decrease abruptly with the use of rHuEPO
in CRF [38–40], as well as in normal subjects [41]. This
results from the mobilization of storage iron for incorporation into newly synthesized hemoglobin. Further depletion of iron stores occurs in the hemodialysis patient,
particularly, because of continued dialyzer-related blood
losses [21–23]. Several investigators have shown that iron
overload could be reduced more rapidly if periodic phlebotomy were performed in association with rHuEPO
therapy [13–15, 19]. We noted that of the original 23
hemodialysis patients treated with rHuEPO [37], 17 were
still receiving rHuEPO three years later [42]. Many of
these patients originally had iron overload, as arbitrarily
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Eschbach and Adamson: Iron overload in renal failure
These concerns relate to the following possible complications: (a) parenchymal cell involvement with or without
organ dysfunction, (b) permanent organ damage, that is,
cirrhosis and/or pancreatic fibrosis, cancer, or myocardial
infarction, (c) increased risk of bacterial infections, and
(d) increased free radical generation from free iron causing increased oxidant-mediated tissue injury. None of
these concerns has been proven, but because iron therapy is so essential to the optimum effectiveness of
rHuEPO, it is appropriate to review these concerns in
relation to the extensive degree of iron overload in the
pre-rHuEPO era and whether these complications might
develop today if the use of intravenous iron were more
routine and poorly monitored.
Fig. 2. Decline in serum ferritin levels during three to four years of
rHuEPO therapy in 17 hemodialysis patients [42]. Symbols are: (s)
iron excess, N 5 6; (d) normal stores, N 5 11.
defined by a serum ferritin of more than 1000 ng/ml
(mean 6 sd serum ferritin 1073 6 956 ng/ml). After
three years of rHuEPO therapy, all 17 had normal or
low serum ferritin levels (186 6 196 ng/ml; Fig. 2), and
15 required supplemental iron therapy [41]. Today, very
few dialysis patients have iron overload unless they continue to require RBC transfusions and/or are unable to
be treated with rHuEPO.
Iron deficiency is now far more common than iron overload as a result of the combined significant dialyzer-related
blood losses and the stimulating effect of rHuEPO on
hemoglobin synthesis. As recently as 1996, the United
States Renal Disease System’s Dialysis Morbidity and
Mortality Study noted that more than 50% of 2613 dialysis patients in 1993 had transferrin saturation values of
less than 20%, and 36% had serum ferritin levels of less
than 100 ng/ml [43]. Because of the need to correct iron
deficiency and to maintain iron stores in the dialysis
patient treated with rHuEPO, iron supplementation is
required, and iron given intravenously accomplishes this
task much better than does oral iron [40].
ADVERSE EFFECTS OF CHRONIC IRON
OVERLOAD IN CRF
There has been much concern raised about the potential toxicity of chronic iron exposure in dialysis patients.
Parenchymal iron deposition
In one report of chronically anemic adults with normal
renal function, multiple RBC transfusions resulted in
iron overload. These patients had evidence of hepatomegaly with iron in both the hepatocytes and Kupffer
cells on liver biopsy [4]; however, cirrhosis was not observed except in one patient with a prior history of hepatitis. A similar pattern was observed in another anemic
subject (with normal renal function) who received excessive amounts of intramuscular iron [33]. Cirrhosis also
failed to develop after chronic parenteral iron administration to dogs, although the iron deposits were localized
in the RE cells and not the parenchyma [35]. Iron overload in hemodialysis patients has resulted in increased
iron deposition in liver parenchymal cells as well as the
Kupffer cells [9]. Cirrhosis was observed primarily in
iron-overloaded patients who had a history of hepatitis
B, or perhaps hepatitis C, but there was no way to document the latter disease at that time.
Iron overload in hemodialysis patients has also been
associated with a proximal myopathy [45]. Ten patients
had proximal muscle weakness and serum ferritin levels
of 1030 to 5000 ng/ml. Proximal muscle biopsies (in seven
of 10 patients) disclosed iron deposition in muscle fibers
(five of seven) and macrophages (six of seven). However,
there was no evidence of muscle injury or inflammation,
and the amount of iron deposition did not correlate with
the severity of muscle weakness. All of these patients
had one or more of the “hemochromatosis alleles,” that
is, HLA3, B7, or B14. The authors suggested that patients with the hemochromatosis alleles are at increased
risk to develop iron overload and muscle iron deposition.
Because primary hemochromatosis is a very common
inherited metabolic disorder (1 in 300 whites being homozygous and 1 in 10 being heterozygous) [46], it would
be expected that among the thousands of dialysis patients, many would either have the disease or be a carrier
of the gene for this disorder. One theory is that these
patients would lack the regulatory mechanism that prevents iron absorption in the presence of adequate iron
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Eschbach and Adamson: Iron overload in renal failure
stores, and this would result in the preferential deposition of iron in parenchymal cells, rather than the RES.
A report from Italy suggested that dialysis patients that
had one or more of these three hemochromatosis alleles
were more prone to develop iron overload from RBC
transfusions than those patients without any of these
HLA antigens [47]. However, another report failed to
confirm a correlation between the presence of hemochromatosis alleles in CRF and iron overload [48]. There
are a number of problems with this concept: (a) Only
one center has reported excess iron in muscle cells, and
even that study noted that the myopathy was out of
proportion to the amount of iron present. (b) Myopathy
is not a prominent complication of primary hemochromatosis or hemosiderosis in nondialysis patients, and
(c) patients heterozygous for hemochromatosis do not
absorb excessive amounts of iron.
Permanent organ damage
In dialysis patients with hemosiderosis, there have
been no reports of cirrhosis, pancreatic fibrosis, or cardiac failure caused by iron overload. Cirrhosis is seen in
patients with hepatitis B or C. Pancreatic fibrosis may be
seen in patients with insulin-dependent diabetes mellitus,
and cardiac failure may occur in patients with coronary
artery disease, hypertensive cardiovascular disease, or
other cardiomyopathies unrelated to iron overload. All
of these complications are common in the dialysis population and are not necessarily related to iron overload.
In view of the common prevalence of the gene associated
with primary hemochromatosis, there must be many dialysis patients who are at risk to exhibit findings of this
disorder eventually. Perhaps the constant blood loss of
the hemodialysis procedure prevents the development
of the organ failure associated with primary hemochromatosis. Whether the administration of intravenous iron,
rather than oral iron, will result in an earlier unmasking
of this disorder remains to be determined.
A report from Finland suggested that excess dietary
and body iron was a risk factor for myocardial infarction
[49]. This report has been subsequently contradicted by
a large study from the United States that concludes that
higher transferrin-saturation levels are not associated
with an increased risk of coronary artery disease [50]. The
Finnish study used only serum ferritin levels as a marker
for iron status, which may be inaccurate because ferritin
is also an acute phase reactant and could be falsely elevated in the presence of inflammation or infection.
Cancer was more likely to develop in men, but not
women, when iron stores were elevated as determined
by a statistically significantly lower total iron-binding
capacity and higher transferrin saturation [51]. However,
serum ferritin levels were not measured. The authors
assumed that there was an inverse relationship between
total iron-binding capacity and serum ferritin. Although
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there was a statistically significant difference in the previously mentioned iron values between those men who
did or did not develop cancer, the biological significance
of these differences must be questioned (transferrin saturation 33.1% vs. 30.7%; total iron-binding capacity 61.4
mmol/liter vs. 62.9 mmol/liter).
Increased risk of bacterial infections
There is concern that iron overload promotes the proliferation of microorganisms. In vitro, free elemental iron
is a growth factor for bacteria. Although free iron has
been assumed to be present in iron overload states, there
is no evidence to support this assumption. There is no
free iron in circulation as long as transferrin is less than
fully saturated. Most dialysis patients of 20 to 30 years
ago who had iron overload had transferrin saturations
of less than 95%, and it is rare for the parenteral irontreated patient to have a transferrin saturation chronically greater than 50%. Nevertheless, an increased incidence of infections has been reported in dialysis patients
with iron overload [52–55]. This has been assumed to be
due to the suppression of phagocytosis by iron as studied
in vitro [56–58]. Seifert et al noted that 10 hemodialysis
patients with serum ferritin levels of 1001 to 2000 ng/ml,
caused by multiple RBC transfusions and not treated
with desferrioxamine, had a significantly increased incidence of bacterial infections when compared with 125
“control” hemodialysis patients whose serum ferritin levels were 10 to 330 ng/ml (P , 0.01) [52]. Sixteen other
hemodialysis patients treated with desferrioxamine, whose
mean serum ferritin level exceeded 3000 ng/ml (range
1856 to 6112 ng/ml), also had a significantly increased
incidence of bacterial infections compared with the “control” group mentioned earlier here (P , 0.01). No Yersinia infections were noted. Fourteen of these 16 patients
were women over 60 years of age. The authors emphasize
that the serum ferritin levels were calculated by taking
the mean of several measurements throughout their
study period when no active infection was present, thus
avoiding spurious elevations due to infection per se. However, the inflammatory effect on serum ferritin levels
may persist beyond the period of active infection, and
the immunologic suppressive effects of multiple RBC
transfusions could account for the increased susceptibility to infections.
A second study claiming that iron overload is a risk
factor for bacterial infections in dialysis patients [53]
noted that the most significant factors were a previous
history of infection and the presence of a “central” venous catheter. An elevated serum ferritin, particularly
greater than 500 ng/ml, was also considered a risk factor
for infection, but not as statistically significant (P 5
0.028) as the first two risk factors (P , 0.0001 for both
prior infection and the presence of a central venous dialysis catheter); the mean 6 sd serum ferritin was 521 6
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Eschbach and Adamson: Iron overload in renal failure
775 ng/ml for those with infections (N 5 118) versus
376 6 529 ng/ml for those without infection (N 5 489).
Although the differences in serum ferritin levels between
the two groups may be statistically significant, it does
not appear to be biologically significant in view of the
large standard deviation and considerable overlap in the
values between groups. These authors have recently published a prospective study on the risk factors for bacteremia in hemodialysis patients. There was no difference
between the group with bacteremia and those without
bacteremia as to the serum ferritin level, nor the incidence of iron therapy in the previous six months [59].
A third prospective study reported that the incidence
of bacteremia was 2.92 times higher in hemodialysis patients whose serum ferritin exceeded 1000 ng/ml compared with those with lower serum ferritin levels [54].
There was no difference in the incidence of bacteremia
in those with serum ferritin levels of 500 to 1000 ng/ml
versus those with serum ferritin levels of less than 500
ng/ml. Serum ferritin levels also correlated significantly
with the number of RBC transfusions given. Of the 98
patients studied, bacteremia was documented 29 times
in 20 patients, eight of whom had ferritin levels of more
than 1000 ng/ml. Fourteen patients had 17 episodes of
bacteremia associated with serum ferritin levels of less
than 1000 ng/ml (2 patients had bacteremia at both serum
ferritin levels greater and less than 1000 ng/ml). Despite
more bacteremic episodes occurring in those with serum
ferritin levels less than 1000 ng/ml, the incidence/patient
years of dialysis was 2.92 times greater in those with
serum ferritin levels of more than 1000 ng/ml. There
were no differences between the groups of patients in the
types of microorganisms identified. Presumably, most, if
not all, of these patients were anemic, but no data were
presented. rHuEPO was given to some of the patients
later in their follow-up, but no information is available
about the rates of infection in these patients.
A fourth study of 61 patients reported that there was
a significant increase in bacterial infections and sepsis
in 18 hemodialysis patients whose serum ferritin levels
exceeded 500 ng/ml [55]. When 26 patients with a ferritin
of less than 500 ng/ml were compared with 26 whose
ferritin levels were more than 500 ng/ml, the numbers
of infections were 1 versus 12, respectively (P , 0.005),
and septicemias 1 versus 7, respectively (P , 0.005).
However, when another group of 21 patients with iron
overload was treated for aluminium intoxication with the
iron chelater, desferrioxamine, the incidence of infection
decreased significantly, yet the serum ferritin levels
(mean 6 sem) did not decline (2493 6 219 ng/ml to 2293 6
254 ng/ml). However, when desferrioxamine therapy was
stopped, the incidence of infection rose, whereas the
serum ferritin levels changed little (2444 6 103 ng/ml).
The authors suggested that the benefit of desferrioxamine was to bind “free” plasma iron (not bound to trans-
ferrin). (A single report in dialysis patients claimed that
the amount of “free” iron was greater in patients whose
serum ferritin exceeded 500 ng/ml [60]. However, there
was no correlation between such levels and an increased
incidence of infection.) The mean transferrin saturation
value was 70 6 6% prior to and 62 6 6% after approximately 16 months of desferrioxamine therapy. Whether
binding of “free” iron by desferrioxamine is the reason
for the decreased incidence of infections in these dialysis
patients with iron overload is debatable. Because transferrin was not completely saturated, it is difficult to know
whether there was “free” iron in circulation.
A different study by Kessler et al concluded that bacteremia was more likely in patients with serum ferritin
levels of more than 1000 ng/ml, yet this was true for only
12 of 55 patients, whereas the majority of the bacteremic
(56%) and nonbacteremic (72%) patients had serum
ferritin levels of less than 500 ng/ml [62].
There are a number of in vitro studies indicating that
iron suppresses neutrophil function [56–58, 62]. Phagocytic function was noted to be better in 19 hemodialysis
patients with serum ferritin levels more than 1000 ng/ml
(13 to 950 ng/ml) than in 21 chronically transfused patients with serum ferritin levels of more than 1000 ng/ml
(range 1000 to 14,370 ng/ml; median 3770 ng/ml) [56].
Phagocytosis was assumed to be dysfunctional because
superoxide anion production by polymorphonuclear leukocytes (PMN) after in vitro stimulation with opsonized
zymosan was less than in normal controls or those hemodialysis patients with serum ferritin levels of less than
1000 ng/ml. Although the differences were statistically
significant, there was much overlap between the two
groups of dialysis patients. Another study [57] noted
that 91 patients with iron overload from multiple RBC
transfusions (mean serum ferritin 1901 6 1044 ng/ml)
had decreased PMN phagocytosis as quantitated by the
amount of Yersinia enterocolitica ingested compared
with 91 hemodialysis patients with a mean serum ferritin
of 122 6 100 ng/ml. Although the authors attribute the
increased incidence of bacterial infections in hemodialysis patients to iron overload, there are two reasons to
question their conclusion: The incidence of bacterial infections in the group with iron overload was not stated,
and the liver enzyme activity was greater in that group,
suggesting associated hepatitis.
A recent study reported that various in vitro tests of
neutrophil function, particularly intracellular killing and
the neutrophil oxidative burst, were more impaired in
eight hemodialysis patients whose serum ferritin levels
were more than 650 ng/ml (911 6 69 ng/ml, mean 6
sem) than in patients with lower serum ferritin levels
[62]. This impairment in neutrophil function was similar
to that observed in a group of patients with myelodysplastic syndrome who had secondary iron overload from
RBC transfusions. Myelodysplastic patients are known
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to have an increased incidence of infections, whereas
patients with primary hemochromatosis do not; however,
the latter group also had in vitro abnormalities in neutrophil function in this study. The authors assumed the
dialysis patients with neutrophil dysfunction had functional iron deficiency, however, chronic inflammation
was not ruled out. The eight patients received 10 mg of
iron i.v. with each dialysis, making it unlikely that they
had functional iron deficiency and raising the possibility
that they had an inflammatory disorder to account for
the high serum ferritin and the low transferrin saturation
levels. Furthermore, the clinical relevance of these findings is difficult to assess, as these patients did not have
iron overload. The transferrin saturation values were
low (15.6 6 3.7%, mean 6 sem), which argues against
any “free” iron available to increase the likelihood of
infection.
Although the previously mentioned studies—most
of which were conducted prior to the routine use of
rHuEPO—suggest an association between iron overload
and bacterial infections, factors other than iron overload
may explain this association. Only 12% of patients with
idiopathic hemochromatosis die with pneumonia [63],
which is an incidence that is lower than the 15.5% infectious mortality in United States dialysis patients (who
presumably did not have associated iron overload) for
1996 [64]. Whether the increased incidence of bacterial
infections is due to iron overload or immunosuppression
from established cirrhosis or diabetes mellitus in hemochromatosis is difficult to determine. Thalassemia is
associated with severe iron overload from RBC transfusions. Infections appear to occur mainly in those patients
having had a splenectomy [63].
Anemia (hemoglobin of less than 9.9 g/dl) is associated
with a greater incidence of infection [63], and rHuEPO
reverses the PMN dysfunction in dialysis patients with
iron overload [65, 66]. In the latter study, the hematocrit
was not stated, and the reversal in PMN dysfunction
occurred after an average of six months of rHuEPO
therapy, when the serum ferritin was still markedly elevated (from 1860 6 1492 ng/ml to 958 6 756 ng/ml).
Consequently, it is not clear whether anemia or iron
overload is the stronger determinant of PMN dysfunction. Additionally, RBC transfusions are known to be
immunosuppressive [67], which could also contribute to
the increased incidence of infections.
Increased free radical generation
There is concern that there may be increased free
radical generation from free iron that will cause oxidant
tissue injury. Transferrin, which is present in plasma and
lymph, is normally less than 50% saturated with iron.
Transferrin transports iron from the gut to the marrow
and RE cells and to other tissues requiring iron. Ordinarily, there is no free iron available for the growth of
S-41
microorganisms [68]. However, there remains the theoretical concern that if free iron occurs, it can be reversibly
oxidized or reduced, making it potentially hazardous
because of its ability to participate in the generation of
powerful oxidant species, such as the hydroxyl radical,
thus causing cellular injury. However, in vivo, most of
the iron is bound to heme or nonheme proteins (that is,
myoglobin or transferrin) and does not directly catalyze
the generation of hydroxyl radicals or other oxidants
[69]. An exception to this has been the observation that
cisplatin-induced acute renal failure in rats may be mediated by free iron [70]. It is difficult to ascertain from the
records of those dialysis patients with severe transfusioninduced iron overload whether they exhibited evidence
of tissue injury from iron overload, in contrast to that
related to hepatitis.
In summary, iron overload in the pre-rHuEPO era
was a serious problem with hepatosplenomegaly, hypersplenism, and hyperpigmentation commonly seen. However, unless chronic hepatitis with cirrhosis developed
as a result of transfusion-transmitted infection, there are
insufficient data to conclude that these patients had more
serious bacterial infections or had an increased mortality
compared to patients without iron overload. rHuEPO
therapy, with correction of the chronic anemia, has
greatly reduced the need for transfusions and hence the
likelihood of iron overload. There are no data that iron
overload from medicinal iron increases patient morbidity
or mortality. The use of more intravenous iron to replace
iron (blood) losses associated with the hemodialysis procedure, and to maintain optimal iron stores should not
pose a threat for the development of iron overload as
long as the patient’s iron status is monitored on a regular
basis.
Reprint requests to Joseph W. Eschbach, M.D., Minor and James
Medical, 515 Minor Avenue, Seattle, Washington, 98104, USA.
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BZ, Van Landuyt HW: Iron overload in haemodialysis patients
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Page 169 of 290
2111
CHAPTER 24
Sensitization 2001
Steven Hardy, SUQHui Lee, and Paul 8. Terasaki
Terasaki Foundation Laboratory, Los Angeles, California
The three sources of sensitization as shown by
For the trend study, patients registered in the UNOS
development of HLA antibodies are: rejection of a graft,
Kidney Transplant Registry from 1991-2000 were uti-
transfusion, and pregnancy.
We re-examine here the
lized for analysis . For the remainder of the studies,
effect of these three factors on the outcome of recent
patients transplanted from 1995 through 2000 were
transplants after the introduction of new immunosup-
analyzed . Graft survival was calculated according to
pression (1995-2000). During this same time period,
Kaplan and Meir. Unless otherwise stated, the graft
the more sensitive flow cytometry crossmatching tests
survival was for first transplant patients.
became more widespread. As a result, sensitization ,
per se, has come to have a relatively smaller effect. The
transfusion effect was also re-examined .
RESULTS
The following fjgures summarize the results of
these analyses .
Figure 1. Yearly trend in transfusions.
The numbers of transfusions given to kidney trans-
tients continue to be transfused. Today it is unlikely that
plant patients has decreased from a peak of 64% in
patients are being transfused deliberately to obtain
1992 being transfused down to 36% in 2000 . Even
after the introduction of Epogen, over one-third of pa-
higher graft survival.
Clinical Transplants 2001 , Cecka and Terasaki, Eds. UCLA Immunogenetics Center, Los Angeles, California
Page 170 of 290
272
HARDY, LEE, AND TERASAKI
Figure 2. Transfusions in patients with various diseases.
Patients with polycystic kidney disease received the
least number of transfusions and SLE patients received
the most. In all diseases, females received more transfusions than males.
Figure 3. Sensitization and transfusions in first transplant patients.
tized. This incidence may represent errors in false positive PRA results, possibly due to autoantibodies or in
reporting transfusions, since males without transfusions should not have HLA antibodies. Non-transfused,
in sensitization . With more transfusions, the incidence
of sensitization increased for all groups. Tis increase
was greater in females than in males and in parous
females compared with non-parous females. It should
also be noted that these rates of sensitization do not
nulliparous females likewise should not have HLA an-
reflect the true sensitization rate of any of these factors
tibodies, but 20% were classified as sensitized. Among
females, an additional source of sensitization could be
unrecognized or unreported pregnancies. Pregnancies
without transfusions resulted in only a minor increase
since they represent the rates present in patients who
were transplanted. Many more patients could have been
sensitized, but not transplanted.
Among non-transfused males, 13% were sensi-
Page 171 of 290
273
Figure 4. Sensitization after transfusions in regraft patients.
When regraft patients were examined, the rate of
the fact that these patients had rejected a prior trans-
sensitization was considerably greater than for first graft
plant. Thus rejection of a graft is a greater stimulus for
patients as shown in Figure 3. Also, the effect of in-
production of HLA antibodies than transfusions or preg-
creasing numbers of transfusions was not as great as
nancies.
Figure 5. Sensitization of males and females with various diseases.
In these diseases, some correlation of sensitiza-
where transfusions are more frequently performed tend
tion with the frequency with which patients with these
to have more sensitization . Females again were more
diseases are transfused can be noted by comparing
sensitized than males.
the results to Figure 2. That is, patients with diseases
Page 172 of 290
214
HARDY, LEE, AND TERASAKI
o Male
N
21 ,826
II!l Nulliparous 3,797
Parous
7,742
Figure 6. Effect ofpregnancies on graft survival.
Males, nulliparous females and parous females
who were non-sensitized all had approximately the
lower graft survival, with the parous females having the
lowest survival.
same graft survival. When sensitized, they had a slightly
Figure 7. Effect of transfi.tsion on graft survival.
Among non-sensitized and sensitized patients,
those with no transfusions had the highest graft survival and those with the greatest number of transfusions
had the lowest graft survival. This is opposite to the
earlier "transfusion effect" in which patients with no transfusions had the lowest survival. This complete turnaround in the data appears to have occurred in the past
5 years.
Page 173 of 290
275
For cadaver donor reg raft patients in non-sensitized patients, had
a 3 year graft survival rate which was
only 3% below that for first transplants.
The difference in survival occurs in
the first 6 months after transplantation, suggesting that undetected antibodies may have resulted in early
loss of reg raft patients without antibodies. Also it should be noted that
PRA tests were performed mostly by
cytotoxocity.
Among patients who were sensitized (PRA>10%), the 3 year graft
survival difference was 4% between
first and reg raft patients. Again the
difference developed within the first
6 months after transplantation.
Sensitized and non-sensitized
patients had almost the same graft
survival in living donor transplants.
Adult Diab
%PRA
%PRA
N
• 0-10
11-50
• 0-10 6,002
11-50 894
377
--------------------%PRA
N
.0-10 2,952
011-50 474
A >50
250
%PRA
%PRA
• 0-10
o 11-50
• 0-10 2,056
o 11-50 342
203
N
of sensitization on graft survival ofpatients with various diseases.
In general, for the diseases shown, graft survival
>50%, had a lower graft survival. A statistically signifi-
for unsensitized patients and sensitized patients with
cant lower graft survival was seen for patients with SLE
PRA <50% were the same. Only patients with PRA
( p<0.001) and adult diabetes (p<0.01).
Page 174 of 290
276
HARDY, LEE, AND TERASAKI
Figure 10. Effect of HLA matching in primary graft and regraft patients of various diseases.
The HLA-AB mismatching effect was the same for
HLA-DR mismatches had a greater effect in reg rafts
first and reg raft patients since the difference between
since the difference between 0 and 2 DR mismatches
the best and worst matches was 6% for first grafts and
was 5% for first grafts and 10% for regrafts.
6% for regrafts.
HLA-ABDR mismatches had a difference between
best and worst matches of 7% for first graft and 9% for
regrafts.
Page 175 of 290
277
ABDRMM
N
2,807
5,961
8,164
2,860
ABDRMM
o
0
1-2
3-4
5-6
~=.."...,.,.,.,.,.."..,..,
Figure 11. Effect of HLA matching on first and regrafl patients in living donor transplants.
The most striking effect is the superiority of transplants with 0 AS, 0 DR, and 0 ASDR mismatch in first
and regraft patients. These probably represent HLA
identical sibling donor grafts, that provide essentially
the same graft survival in first and regraft patients.
Page 176 of 290
278
HARDY, LEE, AND TERASAKI
DISCUSSION
period of a decade since the first discovery of the trans-
Rejection of a kidney was the most powerful means
by which patients became sensitized . Transfusions
were next in ability to sensitize, followed by pregnancies. In all these instances, females were more prone
to become sensitized than males, possibly because of
more unreported transfusions or pregnancies . The
greater need for transfusions among females was
noted for those with all the various diseases.
The so-called "transfusion effect" was found to have
completely reversed itself in the recent data. The paradoxical effect of transfusions producing a higher graft
survival was no longer found. Instead, both in sensitized and non-sensitized patients, patients with no transfusions had the highest graft survival and those with the
most transfusions had the lowest survival. For over a
1.
The rate of transfusion decreased from 64% in
1992 to 36% in 2000. This need for transfusions
fusion effect (1) to its disappearance (2), we did not
understand its origin . The situation as we now see it
can be explained on the simple basis of sensitization
by transfusion.
Sensitization, as measured by development of HLA
antibodies is shown here to have become a relatively
small factor influencing the outcome of transplants. Its
largest effect is in reg raft patients, where HLA-oR mismatching was again shown to be the most important
factor (3).
survival than non-sensitized patients. As in the prior
experience, the most effective way of handling sensitization was the use of kidneys from HLA-matched related donors.
5.
continued despite the introduction of
erythropoetin. Females were transfused more
frequently than males. SLE patients were transfused more often than those with other diseases.
2.
Transfusions no longer had a beneficial effect
on the outcome of transplantation, but rather with
6.
Rejection of a kidney transplant had the strongest effect on sensitization, followed by transfu-
7.
sion and then pregnancies. Females were more
susceptible to sensitization than males. Al-
For cadaver donor reg raft patients, HLA-oR mismatch had a greater effect than AB mismatch.
graft survival in cadaver donor reg raft patients
mismatched for 2 OR antigens than mismatched
been sensitized, as many as 13% were reported
to have antibodies. As many as 20% of nullipa-
4.
Patients with polycystic kidney disease had the
There was a 10 percentage point lower 3-year
though non-transfused males should not have
rous females without transfusions also were reported to have antibodies.
SLE patients were most often sensitized among
diseases were more sensitized than males.
Unsensitized reg raft patients had a 3% lower
3-year graft survival than unsensitized first graft
patients. Among sensitized patients, regraft patients had a 4% lower graft survival than sensitized first graft patients.
highest 3-year graft survival in both the sensitized and non-sensitized patients. Sensitization
to a PRA level of less than 50% was not detrimental to patients with all the various diseases.
more transfusions, the graft outcome became
lower, as might be expected.
3.
SLE and adult diabetic patients with PRA
>50% were also shown have significantly lower graft
for 0 OR antigens.
8.
For living donor transplants, regrafts from 0 AB or
o OR mismatched transplants had the same graft
survival as first transPlants)'
patients with various diseases. Females of all
REFERENCES
1.
Opelz G, Sengar DPS, Mickey MR, Terasaki PI. Effect of
blood transfusion on subsequent kidney transplant.
Transplant Proc 1973;4:253-259.
2.
Opelz G for the Collaborative Transplant Study: Effect of
HLA matching in 10,000 cyclosporine-treated cadaver kidney transplants. Transplant Proc 1987; 19:641 -646.
3.
Cecka JM, Terasaki PI. Repeating HLA antigen mismatches
in renal transplants - A second class mistake? Transplantation 1994; 57:515-519.
Page 177 of 290
Blood Transfusions in Kidney Transplant Candidates Are Common and Associated With
Adverse Outcomes
Hassan N. Ibrahim, MD, MS,1 Melissa A. Skeans, MS,2 Qi Li, MS,2 Areef Ishani, MD, MS,1,2
Jon J. Snyder, PhD, MS2
1
Department of Medicine, University of Minnesota, Minneapolis, Minnesota, USA
2
Chronic Disease Research Group, Minneapolis Medical Research Foundation, Minneapolis,
Minnesota, USA
Running head: Transfusions Before Kidney Transplant
Corresponding Author
Hassan N. Ibrahim, MD, MS
Division of Renal Diseases and Hypertension, University of Minnesota
717 Delaware Street SE, Suite 353
Mail Code 1932
Minneapolis, MN 55414 USA
Phone, 612-624-9444; fax, 612-626-3840; [email protected]
1
Page 178 of 290
Ibrahim HI, Skeans MA, Li Q, Ishani A, Snyder JJ. Blood Transfusions in Kidney Transplant
Candidates Are Common and Associated With Adverse Outcomes. Clin Transplant.
Abstract
Surprisingly, there are no data regarding transfusion frequency, factors associated with
transfusion administration in patients on the kidney transplant waiting list, or transfusion impact
on graft and recipient outcomes. We used United States Renal Data System data to identify
43,025 patients added to the waiting list in 1999-2004 and followed through 2006 to assess the
relative risk of post-listing transfusions. In 69,991 patients who underwent transplants during the
same time period, we assessed the association between pretransplant transfusions and level of
panel-reactive antibody (PRA) at the time of transplant, and associations between PRA and
patient outcomes. The 3-year cumulative incidence of transfusions was 26% for patients added to
the waiting list in 1999, rising to 30% in 2004. Post-listing transfusions were associated with a
28% decreased likelihood of undergoing transplant, and a more than 4-fold increased risk of
death. There was a graded association between percent PRA at the time of transplant and
adjusted risk of death-censored graft failure, death with function, and the combined event of graft
failure and death. These data demonstrate that transfusions remain common, and confirm the
adverse association between transfusions and PRA, and high PRA and inferior graft and patient
outcomes.
Keywords: Blood transfusion, kidney transplantation, graft survival, waiting list, panel-reactive
antibody
Corresponding Author: Hassan N. Ibrahim, MD, MS, Division of Renal Diseases and
Hypertension, University of Minnesota, 717 Delaware Street SE, Suite 353, Mail Code 1932,
Minneapolis, MN 55414 USA [email protected]
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Introduction
The kidney transplant waiting list continues to grow, and candidates with high levels of
panel-reactive antibody (PRA) experience an increasingly prolonged waiting time and incur
increased risk of graft failure when they undergo transplant (1-6). This is of major relevance as
39.6% of currently listed transplant candidates have PRA > 10%, 15.8% have PRA > 20%, and
5.1% have PRA > 80% (7). We recently demonstrated surprisingly high frequency of blood
transfusion administration, a major cause of sensitization, in dialysis patients and also in
nondialysis-dependent chronic kidney disease (CKD) patients (8;9). Interestingly, despite the
common and firmly held belief that the relationship between blood transfusions, sensitization,
and adverse outcomes is well established, we found no detailed accounts of how often
transfusions are administered to waitlisted patients. Furthermore, there is no information
regarding the impact of transfusions on kidney transplant outcomes in recent years.
Therefore, we determined the frequency of and factors associated with postlisting blood
transfusions, and the effect of these transfusions on the likelihood of death or undergoing
transplant while on the list and elevated PRA level at the time of transplant. We also assessed the
association of elevated PRA level at the time of transplant with the adjusted risk of adverse
patient and graft outcomes. We hypothesized that transfusions occur much more frequently in
this population than is currently appreciated by the transplant community, and that these
transfusions are not inconsequential.
Materials and Methods
We conducted a retrospective cohort study using the United States Renal Data System
(USRDS) standard analytic files, which contain all Organ Procurement and Transplantation
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Network (OPTN) data related to kidney transplants, including the kidney transplant waiting list
and OPTN form data, which include transplant candidate registration data (candidate
demographic and clinical characteristics recorded at the time of wait listing), transplant recipient
registration data for patients who undergo transplants, and recipient histocompatibility data (PRA
information). The USRDS standard analytical files contain all Centers for Medicare & Medicaid
Services (CMS) end-stage renal disease (ESRD) data, including data from the Medical Evidence
Report (form CMS-2728), the Medicare enrollment database (coverage periods and patient
demographics), the ESRD Death Notification (form CMS-2746), Medicare Part A institutional
claims (inpatient, outpatient, skilled nursing facility, home health, and hospice), and Medicare
Part B physician (inpatient and outpatient) and supplier claims (used to identify blood
transfusions).
Study Population
The study population consisted of 43,025 Medicare patients added to the kidney
transplant waiting list in 1999-2004. Patients listed for combined organ transplants (kidneypancreas, kidney-liver, kidney-lung, kidney-heart, or any other combination), patients with prior
kidney transplants, and patients who were ESRD certified after listing were excluded.
Assessment of trends in transfusion use was limited to patients with Medicare primary coverage
because claims for transfusions are available for them but not for patients without Medicare
coverage. To assess the trends in most recent and peak PRA at the time of transplant and the
impact of PRA on graft and patient outcomes, we studied 69,991 transplant recipients who
underwent transplants during the same time period, 1999-2004 with follow-up through 2006.
Blood transfusion use was assessed after listing for patients with Medicare primary
coverage. Blood transfusions were identified in inpatient, outpatient, skilled nursing, and
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physician/supplier Medicare claims using Current Procedural Terminology and International
Classification of Diseases, Ninth Edition, Clinical Modification procedure codes. For patients
undergoing transplant, OPTN collects current and peak PRA data and supplies them to the
USRDS. More than 90% of PRA values are complete for transplant patients.
Analysis
The cumulative incidence of postlisting transfusions through 3 years after listing was
assessed using the Kaplan-Meier method as 1 minus the Kaplan-Meier estimate and presented as
a percentage. Adjusted hazard ratios for postlisting transfusions were estimated using a Cox
proportional hazards model. The association between the first postlisting transfusion and the
subsequent likelihood of transplant and risk of death were estimated using an adjusted timedependent Cox proportional hazards model with the time to first transfusion entered as a timedependent covariate. The adjusted odds ratios of having PRA ≥ 10%, 20%, and 80% at the time
of transplant by prior transfusion status were estimated using logistic regression for patients who
underwent transplants during 1999-2004. A Cox proportional hazards model was then used to
compute the adjusted hazard ratios for death-censored graft failure, death with function, or the
combined outcome by PRA at the time of transplant. Adjustments were made for sex, prior
pregnancy, age, race, ethnicity, pretransplant dialysis duration, dialysis modality, primary cause
of ESRD, transplant year, body mass index (BMI), recipient and donor hepatitis C and
cytomegalovirus status, number of HLA matches, primary insurance, and presence of congestive
heart failure, ischemic heart disease, cerebrovascular accidents, peripheral vascular disease,
cancer, and tobacco use. Adjustments were also made for the following donor factors: donor
type, cold ischemia time, age, sex, BMI, race, and history of diabetes. Adjusted hazard ratios
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were calculated for a PRA of 1%-19%, 20%-79%, and ≥ 80%, all in comparison to a PRA of 0%
at the time of transplant.
All analyses were conducted using SAS version 9.1.3 (SAS Institute, Cary, NC).
Results
In total, 43,025 US Medicare patients were added to the waiting list in 1999-2004 (Table
1). Men, whites, and patients with diabetic nephropathy accounted for most listings. Regarding
comorbid conditions, 11% had atherosclerotic heart disease, 15% had congestive heart failure,
and 3.6% had a history of cerebrovascular accidents. In 1999, 26% received one or more
transfusions in the first 3 years after listing; that proportion was 30% in 2004. Patients with a
higher likelihood of transfusions while on the list were older, female, and white, with diabetes as
cause of ESRD, lower BMI, and longer dialysis duration before wait listing (Table 1).
Interestingly, peritoneal dialysis patients were more likely to receive transfusions (31% versus
28% for hemodialysis patients, P < 0.0001). As one may expect, candidates with comorbid
conditions were more likely to receive transfusions.
The 1-year cumulative incidence of transfusions while on the waiting list for all patients
added to the list between 1999 and 2004 was 10.8%; 3-year cumulative incidence was 27.7%.
Cumulative incidence of transfusions was highest for patients aged > 65 years (Figure 1).
Erythropoiesis stimulating agent (ESA) use was not entered into the primary model assessing
predictors of transfusions, as 80% of waitlisted patients were receiving ESAs. When ESA use
was entered into the model, however, it was associated with a 2-fold increase in the risk of
receiving a transfusion (hazard ratio [HR] 2.06, 95% confidence interval [CI] 1.86-2.29).
Including ESA use in the regression model predicting transfusions did not change the magnitude
of the associations of other variables with transfusion risk (data not shown).
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In a model adjusting for year of listing, age, sex, race, ethnicity, primary cause of ESRD,
blood type, education, BMI, prelisting dialysis duration, dialysis modality, and comorbid
conditions, receiving a blood transfusion while on the list was associated with a more than 4-fold
increase in risk of death (HR 4.04, 95% CI 3.78-4.31), and a 28% lower likelihood of undergoing
transplant (Figure 2).
Receiving any pretransplant transfusion was associated with higher odds of PRA ≥ 10%,
20%, and 80% at the time of transplant (Figure 3). This risk was highest for multiparous women.
PRA at the time of transplant was associated with a step-wise graded association with
death-censored graft failure, death with function, and the combined outcome (Figure 4). Patients
with PRA of 20% to 79% were 21% more likely (95% CI 1.12-1.32) to experience deathcensored graft failure, 15% more likely (95% CI 1.05-1.26) to experience death with function,
and 18% more likely (95% CI 1.11-1.26) to experience the combined outcome. For highly
sensitized patients (PRA ≥ 80%), the adjusted hazard ratios for these events were 1.41 (95% CI
1.22-1.62), 1.19 (95% CI 1.00-1.41), and 1.30 (95% CI 1.17-1.45), respectively. We also provide
the parallel hazard ratios for the subsets of patients with without transfusion history (Table 2).
We did not include transfusion history as a main effect in the models, as it is on the causal
pathway for PRA elevation. In prior modeling, we found no significant interaction between
transfusion history and PRA category for any of the outcomes.
Discussion
These results demonstrate that a significant proportion of waitlisted kidney transplant
candidates receive blood transfusions after being listed. The transfusions were not
inconsequential, as they were associated with sensitization, excess death, and a significantly
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lower likelihood of undergoing kidney transplant. These data also strengthen the link between
higher PRA and poor graft outcomes, in a regression model that adjusted for many important
donor and recipient factors.
A third of patients received transfusions in the first 3 years after being listed; these data
are very surprising in an era with effective strategies to treat anemia of CKD, heightened
awareness against transfusions, and declining ability to administer transfusions in dialysis units.
Prior studies have shown that introduction of epoetin has resulted in a significant decrease in
blood transfusion incidence among listed candidates, and that ESA use is associated with
markedly reduced sensitization and waiting time (10).
Many of these transfusions are likely given due to acute events such as gastrointestinal
bleeding or surgery, and could not be prevented by anemia treatment. Some transfusions may
also reflect a general feeling in the medical community that patients with cardiovascular disease
need higher hemoglobin levels, despite lack of evidence to support such practice (11). While the
reason for transfusions was not included in the Medicare claims data, the recent observation by
Lawler et al, using data from the Veterans Administration Health Care System, supports the
suggestion that CKD patients with anemia receive transfusions more often than other patients
(12). In this analysis and even after excluding transfusions that occurred as a result of an acute
bleeding event, diagnosis of pernicious or hemolytic anemia, or surgery within the month
preceding the index hemoglobin value that triggered the transfusion, CKD patients, particularly
those not receiving ESAs or iron, received transfusions at an alarming frequency of 22% to 58%
depending on the hemoglobin level (12). As there is little doubt that patients with multiple
comorbid conditions, who would have been excluded from undergoing transplants in the past,
now undergo transplants, these high rates of transfusions may not be entirely surprising. The
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adjustments made in our model attempt to address this issue, but likely cannot address it
completely.
Some blood transfusions given to waitlisted patients may have been given intentionally to
improve graft survival, as has been previously described (13-16). However, the practice of giving
transfusions to potential kidney transplant patients, from their directed donors or from random
donors, has fallen out of favor more recently, as the introduction of modern
immunosuppressants, particularly calcineurin inhibitors, has dramatically reduced the incidence
of acute rejection, and showing benefit of such a practice became difficult. In fact, a recent
analysis of the relationship between pretransplant blood transfusions and transplant outcomes did
not demonstrate any benefit (16). Therefore, this possibility is unlikely to have contributed to the
surprisingly high prevalence of transfusions that we observed.
It is worth mentioning that prelisting transfusions are also common. In fact, over 40% of
kidney transplant candidates received transfusions before being waitlisted before 2001. While
they were not the main focus of the current analysis, we studied prelisting transfusions in the
cohort of patients added to the waiting list in 1995-2001, the timeframe in which the question
regarding “any previous transfusions” was included on the OPTN Transplant Candidate
Registration form, and answers were collected and routinely reported for new transplant
candidates. We found that 41% of patients reported prelisting transfusions in 2001. Within 1 year
of wait-listing, 30.5% of patients who received prelisting transfusions received kidney
transplants, compared with 32.2% of patients who did not receive prelisting transfusions (P <
0.001). Prelisting transfusions were associated with a higher likelihood of receiving transfusions
while on the list and a higher risk of death and lower likelihood of undergoing transplant (data
not shown).
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This analysis has limitations. The demonstrated effect of high PRA on graft outcomes
may be related to development of donor-specific antibodies, which are clearly linked to
antibody-mediated rejection (6,17-19). Our data source does not allow us to distinguish between
high-PRA recipients with and without donor-specific antibodies. Moreover, we did not address
results of cross-match at the time of transplant. Most recently, flow cytometry-based PRA assays
have almost become the standard; whether the association observed with cytotoxic PRA and
outcomes is similar is yet to be seen, but trends are likely to be the same. Identifying blood
transfusions using Medicare claims required that we limit the population studied to the
Medicare-primary-payer population, which makes up only 50% of incident wait listings in any
given year.
Medicare-based data also lack important laboratory values, such as hemoglobin levels at
which transfusions took place. This is an analysis of primary kidney transplant patients only,
who currently account for 83% of all transplant patients. Patients listed for combined kidneypancreas transplants were excluded, possibly reducing the number of diabetic patients studied;
diabetic patients have a high comorbidity burden and not uncommonly receive transfusions.
The adverse impact of postlisting transfusions on patient and graft outcomes may be at
odds with the recent analysis by Scornik et al (20). These investigators found that 45% of their
746 kidney transplant patients received transfusions mainly in the first month after transplant.
Interestingly, the incidence of posttransplant antibodies was similar for patients who received
transfusions and for those who did not, and only one-twelfth of patients who received ≥ 10 units
became sensitized. The incidence of rejection and allograft loss were numerically but not
statistically higher for patients who received transfusions. Conceivably, receiving transfusions
while heavily immunosuppressed may not be as deleterious. One must seriously consider that
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these surprisingly high rates of transfusions may become more common, as ESA use is not
without risks and may harm some patients.
While the transplant community proactively avoids transfusions in potential transplant
candidates, transfusions are clearly common, and this issue needs further study. An additional
challenge involves choosing whether to treat anemic CKD patients, particularly those with type 2
diabetes mellitus, with transfusion or ESA, as ESA use may increase risk of stroke in this
population. This issue is very relevant because type 2 diabetes is a major reason patients are
referred for transplants (21).
In summary, these data reveal a surprisingly high frequency of blood transfusions among
ESRD patients on the kidney transplant waiting list. These transfusions appear to be associated
with higher risk of death, lower likelihood of undergoing transplant, and sensitization with its
attendant inferior long-term graft survival. More studies are needed to understand the reasons for
these transfusions, and efforts directed at minimizing their frequency should be carefully
balanced against the potential adverse consequences of ESA use, particularly in CKD patients
with type 2 diabetes.
Acknowledgments
This study uses data supplied by the United States Renal Data System. The interpretation
and reporting of these data are the responsibility of the authors and in no way should be seen as
an official policy or interpretation of the US government. The authors would like to thank
Chronic Research Group colleagues Shane Nygaard, BA, for manuscript preparation, and Nan
Booth, MSW, MPH, ELS, for manuscript editing. This study was supported by a research
contract from Amgen Inc., Thousand Oaks, California. The contract provides for the authors to
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have final determination of the content of this manuscript. Hassan N. Ibrahim and Areef Ishani
consult for, and Melissa A. Skeans, Qi Li, and Jon J. Snyder are employed by, the Chronic
Disease Research Group.
Author Contributions
Hassan N. Ibrahim, research design, data analysis, preparation of manuscript; Melissa A.
Skeans, data analysis, preparation of manuscript; Qi Li, data analysis, preparation of manuscript;
Areef Ishani; research design, data analysis, preparation of manuscript; Jon J. Snyder, research
design, data analysis, preparation of manuscript.
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Reference List
[1] Andreoni KA, Brayman KL, Guidinger MK, Sommers CM, Sung RS. Kidney and
pancreas transplantation in the United States, 1996-2005. Am J Transplant 2007;7:13591375.
[2] The Scientific Registry of Transplant Recipients: 2007 OPTN/SRTR Annual Report:
Transplant Data 1997-2006. Table 5.2: Time to transplant. Available at:
www.ustransplant.org. Accessed October 4, 2010.
[3] Cardarelli F, Pascual M, Tolkoff-Rubin N, et al. Prevalence and significance of anti-HLA
and donor-specific antibodies long-term after renal transplantation. Transpl Int
2005;18:532-540.
[4] Sautner T, Gnant M, Banhegyi C, et al. Risk factors for development of panel reactive
antibodies and their impact on kidney transplantation outcome. Transpl Int 1992;5 Suppl
1:S116-S120.
[5] Terasaki PI, Ozawa M. Predicting kidney graft failure by HLA antibodies: a prospective
trial. Am J Transplant 2004;4:438-443.
[6] Opelz G, Dohler B. Effect of human leukocyte antigen compatibility on kidney graft
survival: comparative analysis of two decades. Transplantation 2007;84:137-143.
[7] US Renal Data System, USRDS 2004 Annual Data Report: Atlas of End-Stage Renal
Disease in the United States. National Institutes of Health, National Institute of Diabetes
and Digestive and Kidney Diseases, Bethesda, MD, 2004.
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[8] Ibrahim HN, Ishani A, Foley RN, Guo H, Liu J, Collins AJ. Temporal trends in red blood
transfusion among US dialysis patients, 1992-2005. Am J Kidney Dis 2008;52:11151121.
[9] Ibrahim HN, Ishani A, Guo H, Gilbertson DT. Blood transfusion use in non-dialysisdependent chronic kidney disease patients aged 65 years and older. Nephrol Dial
Transplant 2009;24:3138-3143.
[10] Vella JP, O'Neill D, Atkins N, Donohoe J, Walshe J. Sensitization to human leukocyte
antigen before and after the introduction of erythropoietin. Nephrol Dial Transplant
1998;13:2027-2032
[11] Besarab A, Bolton WK, Browne JK, et al. The effects of normal as compared with low
hematocrit values in patients with cardiac disease who are receiving hemodialysis and
epoetin. N Engl J Med 1998;339:584-590.
[12] Lawler E, Bradbury B, Fonda JR, Gaziano J, Gagnon D. Transfusion burden among
patients with chronic kidney disease and anemia. Clin J Am Soc Nephrol 2010;5:667672.
[13] Salvatierra O, McVicar J, Melzer J, et al. Improved results with combined donor-specific
transfusion (DST) and sequential therapy protocol. Transplant Proc 1991;23:1024-1026.
[14] Lockard-Marduel A, Gumbert M, Tomlanovich S, Amend W, Vincenti F, Schralla P,
Melzer J, Feduska NJ, Salvatierra O, Jr., Garovoy MR: Immunologic alterations induced
by donor-specific transfusion. Transplant Proc 1989;21:1171-1172.
[15] Salvatierra O, Jr., Melzer J, Vincenti F, et al. Donor-specific blood transfusions versus
cyclosporine--the DST story. Transplant Proc 1987;19:160-166.
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[16] Aalten J, Bemelman FJ, van den Berg-Loonen EM, et al. Pre-kidney-transplant blood
transfusions do not improve transplantation outcome: a Dutch national study. Nephrol
Dial Transplant 2009;24:2559-2566.
[17] Glotz D, Antoine C, Duboust A. Antidonor antibodies and transplantation: how to deal
with them before and after transplantation. Transplantation 2005;79(Suppl 3):S30-S32.
[18] Vasilescu ER, Ho EK, Colovai AI, et al. Alloantibodies and the outcome of cadaver
kidney allografts. Hum Immunol 2006;67:597-604.
[19] Gebel HM, Bray RA, Nickerson P. Pre-transplant assessment of donor-reactive, HLAspecific antibodies in renal transplantation: contraindication vs. risk. Am J Transplant
2003;3:1488-1500.
[20] Scornik J, Schold J, Bucci M, Meier-Kriesche U. Effects of blood transfusions given after
renal transplantation. Transplantation 2009;87:1381-1386.
[21] Pfeffer M, et al. A trial of darbepoetin alfa in type 2 diabetes and chronic kidney disease.
New Engl. J Med 2009;361:2019.
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Table 1. Factors Associated With Post-Listing Transfusions
Factor
Year of listing
1999
2000
2001
2002
2003
2004
Age at listing, yr
18-34
35-49
50-64
≥ 65
Sex
Men
Women
Race
White
African American
Native American
Asian
Other
Primary cause of
ESRD
Diabetes
Hypertension
GN
Cystic
Other
Unknown
BMI, at listing kg/m2
< 18.5
18.5-24.9
25-29.9
30-34.9
≥ 35
Unknown
Pre-listing dialysis
duration, yr
<1
1-< 2
2- < 3
≥3
% of population
3-year Cumulative
Adjusted hazard
*
(n = 43,025) Transfusion Incidence, % ratio (95% CI)
P
13.9
15.4
15.7
17.0
18.3
19.7
26
27
28
29
28
30
1.00 reference
1.07 (0.99-1.15)
1.04 (0.96-1.12)
0.98 (0.91-1.06)
0.89 (0.82-0.97)
1.02 (0.94-1.10)
15.1
29.4
38.4
17.1
24
25
30
33
1.00 reference
1.02 (0.95-1.09) 0.6695
1.14 (1.06-1.22) 0.0004
1.25 (1.15-1.36) < 0.0001
59.5
40.5
26
31
1.00 reference
1.25 (1.20-1.31) < 0.0001
55.2
36.6
1.7
5.2
1.3
30
26
27
24
25
1.00 reference
0.87 (0.82-0.91) < 0.0001
0.78 (0.67-0.92) 0.0027
0.75 (0.68-0.83) < 0.0001
0.97 (0.82-1.15) 0.7218
35.8
25.7
18.0
4.6
2.5
13.4
32
25
23
23
30
31
1.00 reference
0.89 (0.82-0.96) 0.0015
0.81 (0.75-0.89) < 0.0001
0.72 (0.63-0.82) < 0.0001
1.00 (0.87-1.16) 0.9908
1.06 (0.98-1.16) 0.1581
2.8
34.0
32.3
18.5
9.3
3.1
33
28
28
27
28
31
1.00 reference
0.85 (0.76-0.96) 0.0110
0.79 (0.70-0.90) 0.0002
0.76 (0.67-0.87) < 0.0001
0.81 (0.71-0.93) 0.0021
0.93 (0.79-1.09) 0.3712
38.8
26.5
13.1
21.7
25
28
31
31
1.00 reference
1.10 (1.04-1.16) 0.0006
1.20 (1.12-1.28) < 0.0001
1.32 (1.24-1.39) <.0001
16
0.0918
0.3668
0.6757
0.0099
0.6440
Page 193 of 290
Dialysis modality at
listing
Hemodialysis
Peritoneal dialysis
Unknown
History of ASHD
History of CHF
History of CVA/TIA
History of PVD
History of hypertension
History of diabetes
History of COPD
83.4
12.8
3.8
11.0
15.0
3.6
5.4
74.8
36.4
2.1
28
31
27
36
32
35
37
28
32
39
1.00 reference
1.18 (1.10-1.25) < 0.0001
1.03 (0.92-1.16) 0.5911
1.13 (1.06-1.21) 0.0003
1.05 (0.99-1.12) 0.0795
1.12 (1.01-1.24) 0.0331
1.19 (1.09-1.29) < 0.0001
0.97 (0.92-1.02) 0.1975
1.13 (1.06-1.22) 0.0004
1.20 (1.05-1.37) 0.0093
ASHD, atherosclerotic heart disease; BMI, body mass index; CHF, congestive heart failure; CI,
confidence interval; COPD, chronic obstructive pulmonary disease; CVA/TIA, cerebrovascular
accident/transient ischemic attack; GN, glomerulonephritis; PVD, peripheral vascular disease.
*
Medicare patients added to kidney transplant waiting list 1999-2004, followed through
December 31, 2006.
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Table 2. Hazard Ratios for Panel-Reactive Antibody Category in a
Model Predicting Graft Failure
PRA Category
0%
1-19%
20-79%
80-100%
Patients
Pretransplant
Transfusion History
1.00 (reference)
1.04 (0.97-1.12)
1.16 (1.05-1.28)
1.41 (1.20-1.65)
All
1.00 (reference)
1.05 (1.01-1.09)
1.18 (1.11-1.26)
1.30 (1.17-1.45)
PRA, panel-reactive antibody.
18
No Pretransplant
Transfusion History
1.00 (reference)
1.02 (0.96-1.08)
1.18 (1.07-1.30)
1.39 (1.00-1.43)
Page 195 of 290
Figure Legends
Figure 1. Cumulative incidence of first transfusion post-listing through
3 years, by age at listing.
Figure 2. Association between postlisting transfusions and likelihood
of transplant and death.
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Figure 3. Association between pretransplant blood transfusion and panel-reactive antibody
(PRA) level at time of transplant by sex and pregnancy history.
20
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Figure 4. Association between panel-reactive antibody (PRA) level at time of transplant
and outcomes within 3 years posttransplant. Patients who underwent transplant 1999-2004
(n = 69,991).
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J Am Soc Nephrol 15: 818–824, 2004
Leukocyte Reduction of Red Blood Cell Transfusions Does
not Decrease Allosensitization Rates in Potential Kidney
Transplant Candidates
MARTIN KARPINSKI,* DENISE POCHINCO,† IGA DEMBINSKI,†
WILLIE LAIDLAW,† JAMES ZACHARIAS,* and PETER NICKERSON*†
*Department of Medicine, University of Manitoba, Winnipeg, Manitoba, Canada; and †Immunogenetics
Laboratory, Winnipeg Blood Center, Winnipeg, Manitoba, Canada
Abstract. A significant proportion of potential kidney transplant candidates continue to periodically require blood transfusions that carry a risk of allosensitization. Leukocyte reduction (leukoreduction) of blood products has been proved to
reduce transfusion-associated allosensitization in patients with
hematologic malignancies; however, the effect in potential
kidney transplant candidates is unknown. A total of 112 kidney
transplant candidates who received red blood cell transfusions
while on the transplant waiting list were identified retrospectively. Sixty received a transfusion before leukoreduction (nonLR), and 52 received a transfusion after the local implementation of universal leukoreduction of blood products (LR).
There was no difference in transfusion-associated allosensitization rates in patients who received a transfusion during the
two eras (non-LR 27% [16 of 60] versus LR 33% [17/52]; NS).
Likewise, no difference was observed in subgroups identified
as being at high risk of allosensitization (previous pregnancy,
transplant, or five or more previous transfusions) or at low risk
(no previous allogeneic exposures) (high risk: non-LR 52%
versus LR 55%; low risk: non-LR 10% versus LR 8%). Multivariate analysis revealed previous pregnancy to be the only
significant risk factor associated with transfusion-associated
allosensitization (relative risk, 8.2; 95% confidence interval,
2.4 to 24.0; P ⫽ 0.0001). Leukoreduction, in particular, was
not associated with any protective effect. In summary, leukoreduction of red blood cell transfusions does not confer any
protection against transfusion-associated allosensitization for
potential kidney transplant candidates. Physicians who care for
patients with ESRD must continue to practice careful transfusion avoidance while alternative strategies to minimize transfusion associated allosensitization are sought.
Despite the fact that recombinant erythropoietins have substantially decreased the need for transfusions in patients with
ESRD, United Network for Organ Sharing (UNOS) data indicate that approximately 30% of wait-listed transplant candidates continue to receive red blood cell (RBC) transfusions at
some point before transplantation (1, 2). In the past, some
transplant programs administered deliberate pretransplant
transfusions aimed at optimizing graft outcomes (i.e., the beneficial transfusion effect); however, more recent data indicate
that this beneficial effect is no longer apparent, perhaps as a
result of improving graft outcomes overall (2–5). Concerns of
transfusion-associated allosensitization persist for potential
transplant candidates, and it is likely that the majority of
current transfusions are administered for other clinical
indications.
Allosensitization is associated with significant barriers to
successful transplantation in patients with ESRD, including
prolonged waiting times and inferior graft outcomes (6 – 8).
Accordingly, any measure to limit allosensitization would represent a substantial advance for ESRD patients. Of the three
principal causes of allosensitization—pregnancy, transplantation, and transfusions— only the last is perhaps modifiable.
Leukocyte reduction of blood products (leukoreduction) reduces the transfused load of allogeneic leukocytes and has
been proved to limit transfusion-associated allosensitization in
patients with hematologic malignancies undergoing chemotherapy (9).
The impact of RBC leukoreduction on allosensitization in
ESRD patients is unknown. The few studies that have examined this practice either have been uncontrolled or have
screened for allosensitization using technically inferior antiHLA antibody screening techniques (10, 11). Several recent
studies have highlighted the superior sensitivity of flow cytometric anti-HLA antibody screening (FlowPRA) (12–14). We
thus set out, in this retrospective cohort study, to use sensitive
flow cytometric techniques to determine whether universal
RBC leukoreduction has reduced the incidence of transfusionassociated allosensitization in potential kidney transplant candidates within our center.
Received October 9, 2003. Accepted December 12, 2003.
Correspondence to Dr. Martin Karpinski, University of Manitoba, Room
GE421B, Health Sciences Centre, 820 Sherbrook Street, Winnipeg, MB,
Canada R3A 1R9. Phone: 204-787-1524; Fax: 204-787-3326;
E-mail:[email protected]
1046-6673/1503-0818
Journal of the American Society of Nephrology
Copyright © 2004 by the American Society of Nephrology
DOI: 10.1097/01.ASN.0000115399.80913.B1
Page 199 of 290
J Am Soc Nephrol 15: 818–824, 2004
Leukocyte Reduction and Allosensitization Rates
Materials and Methods
Universal Leukoreduction in Canada
All blood products within Manitoba are distributed by a single
agency, Canadian Blood Services, and since September 1999, all RBC
units distributed within Manitoba have been leukoreduced in compliance with a nationwide Health Canada directive (15). This directive
was issued in response to numerous lines of evidence indicating that
leukoreduction of blood products likely reduces the incidence of
several adverse transfusion reactions, including allosensitization. The
Winnipeg Blood Centre now performs universal prestorage leukoreduction of RBC units with commercially available in-line filtration
systems (Leukotrap WB and RC PL; Pall Medical, East Hills, NY),
and the maximum accepted residual white blood cell (WBC) count is
⬍5 ⫻ 106/unit (normal WBC content approximately 5 ⫻ 109/unit).
Internal quality control testing is applied to at least 1% of all units, and
the actual residual WBC content is observed to be approximately 3 ⫻
105/unit (unpublished data, Canadian Blood Services/Pall Corp.).
Study Procedures
This study was approved by the University of Manitoba Biomedical Research Ethics Board. The study population consisted of patients
who were on the Manitoba renal transplant waiting list and had
received RBC transfusions while wait-listed for transplantation. None
of the transfusions administered was prescribed as deliberate pretransplant transfusions aimed at optimizing graft outcomes. Sera for antiHLA antibody screening on wait-listed patients were collected bimonthly during the period of study as well as 2 to 4 wk after any
transfusion. Serum collection and transfusions are tracked meticulously by local transplant coordinators and Immunogenetics Laboratory technologists. Adult transplant candidates who received RBC
transfusions between January 1996 and June 2003 thus were identified
for retrospective study, and of 112 wait-listed ESRD patients identified, 60 received RBC units before the implementation of universal
leukoreduction and 52 thereafter. Individuals who were broadly sensitized (FlowPRA ⱖ80%) before transfusion were excluded (n ⫽ 3).
Patient data and transfusion records were abstracted from Manitoba
Renal Program database.
Anti-HLA Antibody Screening
Transfusion-associated allosensitization was determined by antiHLA panel reactive antibody (PRA) screening pre- and posttransfusion. Sera were batched and then screened concurrently by both the
anti-human globulin cytotoxicity technique (AHG-CDC PRA) and a
flow cytometric technique (FlowPRA; OneLambda). Both screening
assays were performed in the Immunogenetics Laboratory at the
Winnipeg Blood Centre using standard techniques previously described (13). A patient was considered sensitized before a transfusion
when the AHG-CDC PRA was ⱖ10% and/or when the FlowPRA
assay revealed any detectable anti-HLA antibodies. Transfusion-associated sensitization was defined as the de novo appearance of a
positive FlowPRA or as an increment in the FlowPRA value of
ⱖ10%.
Statistical Analyses
Statistical analysis was performed using Statview 5.0 software
(SAS Institute, Cary, NC). Values are reported as mean ⫾ SEM or,
where indicated, as medians and ranges. The ␹2 test was used for
comparison of categorical variables, whereas the t test was applied to
comparisons of continuous variables. P ⱕ 0.05 was considered to be
significant, and values ⬎0.10 are reported as nonsignificant (NS). In
the multivariate analysis of risk factors for allosensitization, univariate
risk factors associated with the outcome with P ⱕ 0.10 were allowed
into the final model. These included a ⫹ve FlowPRA before transfusion, previous pregnancy, previous transplantation, previous transfusions, and the number of RBC units transfused in the episode under
study. Leukoreduction was considered in the models despite being
found to be nonsignificant in univariate analysis. Pregnancy and
previous transfusions were considered as both categorical and continuous variables in the models analyzed. There was no demonstrable
relationship between increasing numbers of pregnancies or transfusions and an increasing incidence of allosensitization, and the overall
strength of the model was superior when these were considered as
categorical variables. For these reasons, five or more previous transfusions was chosen as the transfusion variable, and this cutoff is also
supported by previous studies (16).
Results
During the period of study, 112 individuals on the renal
transplant waiting list received RBC transfusions and had
appropriate pre- and posttransfusion serum samples collected
for anti-HLA antibody screening. Sixty patients received a
transfusion before universal leukoreduction (non-LR) and 52
thereafter (LR). There were significant baseline demographic
differences between these two groups (Table 1). Patients who
received leukoreduced transfusions were more likely to have
Table 1. Baseline demographicsa
Gender (male/female)
Age when transfused
Pregnancy
No. of pregnancies (median, range)
Previous transplant
Previous RBC transfusion
ⱖ5 Previous RBC transfusions
⫹ve FlowPRA pretransfusion
Units transfused
a
RBC, red blood cell.
819
Pre-leukoreduction
(n ⫽ 60)
Leukoreduction
(n ⫽ 52)
P
39/21
40 ⫾ 2
13
2 (0–7)
6
18 (30%)
8
12 (20%)
3 ⫾ 0.4
28/24
42 ⫾ 2
20
2 (0–12)
8
33 (63%)
18
20 (38%)
3 ⫾ 0.3
NS
NS
NS
NS
NS
⬍0.001
0.008
0.03
NS
820
Page 200 of 290
Journal of the American Society of Nephrology
J Am Soc Nephrol 15: 818–824, 2004
had a previous transfusion, were more likely to have received
five or more RBC transfusions in the past, and were more
likely to be allosensitized before the transfusion episode under
examination (Table 1). Both groups received the same mean
number of RBC units in the transfusion episode under study.
Transfusion-Associated Allosensitization and
Leukoreduction
The overall rates of transfusion-associated allosensitization
were 27% (16 of 60) in the population that received standard
RBC units and 33% (17 of 52) in those who received leukoreduced RBC (NS; Table 2). Of the 33 individuals who met the
definition of transfusion-associated allosensitization, 16 were
previously unsensitized and 17 demonstrated a ⫹ve FlowPRA
before transfusion. Fifteen of the 33 individuals developed
isolated new HLA class I antibodies, six developed isolated
class II antibodies, and 12 developed new class I and II
antibodies. In previously unsensitized patients, the mean HLA
class I and class II FlowPRA posttransfusion became 53 ⫾ 8%
and 34 ⫾ 11%, respectively, whereas for previously sensitized
patients, the mean increment in the class I and class II FlowPRA was 35% and 39%, respectively (class I pre, 44 ⫾ 8%;
post, 79 ⫾ 5% [P ⬍ 0.01]; class II pre, 35 ⫾ 7%; post, 74 ⫾
9% [P ⬍ 0.01]). There was no significant difference in either
the degree (% ⌬PRA) or the nature of allosensitization (HLA
class I and/or class II) that developed in patients who received
standard versus leukoreduced transfusions (data not shown).
Fifty-two of 112 patients were considered to be at high risk
of transfusion-associated allosensitization on the basis of having had previous allogeneic exposures (previous pregnancy,
previous transplantation, and five or more previous RBC transfusions), whereas 44 of 112 were considered to be at low risk
(no previous allogeneic exposures). No effect of leukoreduction on allosensitization rates was seen in either of these two
subgroups (high risk, 52% non-LR versus 55% LR [NS];
low-risk, 10% non-LR versus 8% LR [NS]; Table 2).
AHG-CDC PRA was positive in only six (19%) of 32
patients who displayed a ⫹ve FlowPRA before transfusion.
Similarly, AHG-CDC PRA detected new anti-HLA antibodies
Table 2. Transfusion-associated allosensitization ratesa
Transfusion-Associated
Allosensitization
P
Preleukoreduction
All patients (n ⫽ 112)
High risk (n ⫽ 52)
(previous pregnancy,
Tx, ⱖ5 tf)
Low risk (n ⫽ 44) (no
previous pregnancy,
Tx, or tf)
a
16/60 (27%)
12/23 (52%)
3/31 (10%)
Tx, transplant; tf, transfusion.
Leukoreduction
17/52 (33%) NS
16/29 (55%) NS
1/13 (8%)
NS
in only 22 (67%) of the 33 patients who developed transfusionassociated allosensitization as determined by FlowPRA.
Risk Factors for Transfusion-Associated
Allosensitization
In univariate analysis, factors that correlated with an increased likelihood of transfusion-associated allosensitization
included a ⫹ve FlowPRA before transfusion, previous pregnancy, and five or more previous RBC transfusions (Table 3).
In multivariate regression analysis, only previous pregnancy
was associated with an increased risk of transfusion-associated
allosensitization (relative risk, 8.2; 95% confidence interval,
2.8 to 24.0; P ⫽ 0.0001). Leukoreduction per se was not found
to be protective in either univariate or multivariate analyses.
Rates of allosensitization were similar for women who had a
history of pregnancy and received either standard or leukoreduced transfusions (9 [69%] of 13 non-LR versus 11 [55%] of
20 LR; NS).
Discussion
A significant proportion of patients with ESRD are denied
the full potential benefits of transplantation as a result of
allosensitization. Allosensitized patients experience longer
waiting times for finding compatible donors and are at risk of
inferior graft outcomes transplanted (6). The barrier created by
allosensitization is exemplified by the fact that approximately
30% of UNOS wait-listed renal transplant candidates are allosensitized yet only approximately 10% of transplants are
performed in sensitized recipients (6). Of the three principal
causes of allosensitization—pregnancy, previous transplantation, and transfusions— only the last is potentially modifiable.
Nephrologists who care for ESRD patients are well aware of
the risk of deleterious allosensitization and are careful to avoid
unnecessary transfusions; however, this patient population remains at risk of periodically requiring allogeneic transfusions.
The UNOS database indicates that approximately 30% of waitlisted transplant candidates continue to require blood transfusions at some point before transplantation (2).
Recently, several groups reported their experience with
novel immunosuppressive protocols incorporating intravenous
immunoglobulin and plasmapheresis to enable the successful
transplantation of sensitized recipients (17–19). Although encouraging, these protocols are available to only a small proportion of sensitized potential recipients and will likely do little
to address the disparity in access to transplantation. Strategies
to prevent allosensitization are likely to have a greater impact
for ESRD patients.
Leukoreduction of blood products reduces the load of allogeneic HLA in transfusions and has been proved to diminish
allosensitization rates in patients who have hematologic malignancies undergoing chemotherapy (9). Several randomized
trials have reported a benefit in this population (20 –27). The
TRAP study, most notably, randomized ⬎200 patients with
acute leukemia to receive leukoreduced RBC and either unmodified platelet preparations or irradiated, filtered, or apheresed platelet concentrates (27). Patients who received any of the
modified platelet products were significantly less likely to
Page 201 of 290
J Am Soc Nephrol 15: 818–824, 2004
Leukocyte Reduction and Allosensitization Rates
821
Table 3. Risk factors for transfusion-associated allosensitizationa
Risk Factors (RR, 95% CI)
FlowPRA ⫹ve pretransfusion
Pregnancy
Previous transplant
ⱖ5 Previous transfusions
Leukoreduction
RBC units given (per unit)
a
Univariate
P
Multivariate
P
4.5 (1.9–11)
7.8 (3.1–19.5)
2.8 (0.9–8.7)
5.1 (2.0–13.1)
0.5 (0.3–1.7)
1.1 (1.0–1.3)
⬍0.001
⬍0.001
0.08
⬍0.001
NS
0.10
2.4 (0.8–7.1)
8.2 (2.8–24)
2.4 (0.6–9.9)
2.6 (0.8–8.8)
2.0 (0.7–6.0)
1.1 (0.9–1.3)
0.10
0.0001
NS
NS
NS
0.10
RR, relative risk; CI, confidence interval.
develop new anti-HLA antibodies and become refractory to
platelet transfusions (17 to 21% versus 45%; P ⬍ 0.001). It
must be noted, however, that studies that have examined leukoreduction and allosensitization have almost exclusively been
performed in this patient population. Patients therein have also
received large absolute numbers of transfusions with both
platelet and RBC preparations (e.g., 14 ⫾ 11 platelet and 15 ⫾
7 RBC transfusions in the TRAP study).
There are comparatively few data on the impact of leukoreduction on allosensitization as a result of RBC transfusions in
patients with ESRD. In the 1980s, SanFilippo et al. (10)
conducted a randomized study transfusing renal transplant
candidates with either standard or leukoreduced RBC units and
found no difference in allosensitization. Importantly, there was
no assessment of the extent and consistency to which leukoreduction was achieved with the techniques applied, and antiHLA antibody screening was performed with the AHG-CDC
technique, which is less sensitive than current flow cytometric
techniques. Christiaans et al. (11) examined potential transplant candidates who were given leukoreduced RBC transfusions and reported that de novo HLA class I antibodies developed in only 6% when screened by flow cytometry. This study,
however, was uncontrolled and examined only low-risk, previously unsensitized patients. Limited literature also exists in
other, nonrenal patient populations. Recently, Van de Watering
et al. (28) randomized ⬎400 patients who were undergoing
cardiac surgery to receive either standard or leukoreduced RBC
transfusions and observed no difference in allosensitization
rates in either unsensitized or previously sensitized patients.
In the current study, we found no significant difference in
the rate of transfusion-associated allosensitization in renal
transplant candidates who received either standard or leukoreduced RBC transfusions (27% versus 33%, respectively; mean,
3 ⫾ 0.3 transfusions). Furthermore, similar rates and degrees
(i.e., ⌬%PRA) of allosensitization were seen in both low-risk
and high-risk patients who received transfusions of leukoreduced blood. The observed rate of allosensitization in high-risk
patients is slightly higher than previously reported, and this is
likely attributable to the superior sensitivity of the flow cytometric screening technique that we used. Studies using CDC
techniques have reported allosensitization rates of approximately 30% in high-risk recipients, in contrast to the approximately 50% rate that we observed with FlowPRA screening
(29 –31). These anti-HLA antibodies detected solely by flow
cytometry are clinically relevant and have been increasingly
associated with adverse outcomes posttransplantation (12–14,
31–34).
We observed previous pregnancy to be the strongest risk
factor for transfusion-associated allosensitization, a finding in
keeping with previous observational studies (35, 36). Other
allogeneic exposures, such as a previous transplant or previous
transfusions, have also been reported to be risk factors for
transfusion-associated allosensitization, although did not reach
statistical significance in multivariate analysis herein (35, 36).
This may represent a limitation of the size of our data set or,
alternatively, an accurate representation of risk factors within
our patient population. Notably, leukoreduction was not associated with any protective effect in either univariate or multivariate analysis.
This study is retrospective, and ideally a randomized, controlled trial would be performed to evaluate the impact of
leukoreduction on allosensitization in potential renal transplant
recipients. This, however, is unlikely to occur. Many blood
distribution organizations have in recent years adopted a policy
of universal leukoreduction of blood products, and although
not without controversy, this practice is now widespread (37,
38). In Canada and most of Europe, universal leukoreduction
has been in place for several years, whereas approximately
70% of U.S. RBC units are currently leukoreduced before
distribution. Organizations that monitor transfusion standards
are unlikely to permit a change to previous blood-handling
procedures; thus, retrospective studies such as this are necessary to investigate the effects of leukoreduction in patient
populations other than those with hematologic malignancies
(39).
Several reasons may underlie why leukoreduction fails to
diminish allosensitization rates in patients with ESRD. Individuals with ESRD are likely more immunocompetent than
those who have hematologic malignancies and undergo treatment with myeloablative chemotherapy. This is supported by
the similar sensitization rates in the two populations, despite
the considerably greater overall exposure to allogeneic blood
products in the latter. The mean number of RBC units transfused in the current study was 3 ⫾ 0.3, whereas patients who
received leukoreduced products in the TRAP trial, in which
approximately 20% became allosensitized, received fivefold
822
Page 202 of 290
Journal of the American Society of Nephrology
this amount of platelet and RBC transfusions (27). Similarly,
the degree of leukoreduction achieved with current techniques
may be inadequate to prevent allosensitization in ESRD patients. Quality control assessments performed for Canadian
Blood Services reveal reliable three to four logfold reductions
in RBC unit leukocyte content. However, it may be that the
residual leukocyte content (approximately 3 ⫻ 105/unit) represents a sufficient residual exposure to allogeneic HLA to
induce an alloimmune response. Current leukoreduction techniques are similar in the degree of leukoreduction achieved,
and there are no clinical data to favor current cutoff standards
of approximately 1 to 5 ⫻ 106/unit for minimizing allosensitization (9, 38, 40 – 42). It is interesting that rodent models
suggest that too great a degree of leukoreduction may in fact
promote allosensitization, although the clinical relevance of
this is unknown (43). Finally, leukocytes are not the sole
source of allogeneic HLA in transfusions as soluble HLA and
even RBC-bound HLA are present as well, and these are not
diminished by leukoreduction (44 – 46).
If standard leukoreduction fails to diminish allosensitization
in potential renal transplant candidates, then alternative approaches must be sought. One approach that is occasionally
used is the prescription of a brief course of immunosuppression
beginning at the time of transfusion (e.g., with azathioprine or
cyclosporine). This strategy has not been evaluated adequately,
and its safety and generalizability are questionable (47– 49).
Many patients who require transfusions are likely too acutely
ill to be prescribed such therapy, which would presumably be
necessary for a number of weeks peritransfusion. HLAmatched transfusions have in the past been successful in preventing allosensitization, and this strategy would be preferable
but is limited by logistic concerns (50, 51). Existing blood
distribution centers may have sizable numbers of HLAmatched donors on file (e.g., in bone marrow donor registries),
but blood from HLA-typed individuals may not necessarily be
on hand and may not be available in the time frame required for
transfusion. Last, there is hope that less allogeneic blood substitutes or modified blood products will become available;
however, this seems unlikely in the near future (52, 53).
In summary, transfusions continue to be an important cause
of allosensitization for potential kidney transplant recipients.
Leukoreduction of RBC transfusions does not seem to reduce
allosensitization rates in this patient population. The need for
periodic transfusions persists for many patients on transplant
waiting lists, and alternative transfusion strategies to prevent
allosensitization must be found.
J Am Soc Nephrol 15: 818–824, 2004
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
Acknowledgments
This study was funded by grants from Canadian Blood Services
(Canadian Blood Services Small Projects Fund) and the Kidney
Foundation of Canada. This work was presented in abstract form at
the 2003 American Transplant Congress in Washington DC, (ATC
2003 abstract #1356).
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Preexisting Donor-Specific HLA Antibodies Predict
Outcome in Kidney Transplantation
Carmen Lefaucheur,* Alexandre Loupy,† Gary S. Hill,† Joao Andrade,‡ Dominique Nochy,†
Corinne Antoine,* Chantal Gautreau,‡ Dominique Charron,‡ Denis Glotz,* and
Caroline Suberbielle-Boissel‡
Departments of *Nephrology and Kidney Transplantation and ‡Immunology and Histocompatibility, Saint-Louis
Hospital, Paris, France; and †Department of Histopathology, Georges Pompidou European Hospital, Paris, France
ABSTRACT
The clinical importance of preexisting HLA antibodies at the time of transplantation, identified by
contemporary techniques, is not well understood. We conducted an observational study analyzing the
association between preexisting donor-specific HLA antibodies (HLA-DSA) and incidence of acute
antibody-mediated rejection (AMR) and survival of patients and grafts among 402 consecutive deceaseddonor kidney transplant recipients. We detected HLA-DSA using Luminex single-antigen assays on the
peak reactive and current sera. All patients had a negative lymphocytotoxic cross-match test on the day
of transplantation. We found that 8-year graft survival was significantly worse (61%) among patients with
preexisting HLA-DSA compared with both sensitized patients without HLA-DSA (93%) and nonsensitized
patients (84%). Peak HLA-DSA Luminex mean fluorescence intensity (MFI) predicted AMR better than
current HLA-DSA MFI (P ⫽ 0.028). As MFI of the highest ranked HLA-DSA detected on peak serum increased,
graft survival decreased and the relative risk for AMR increased: Patients with MFI ⬎6000 had ⬎100-fold
higher risk for AMR than patients with MFI ⬍465 (relative risk 113; 95% confidence interval 31 to 414). The
presence of HLA-DSA did not associate with patient survival. In conclusion, the risk for both AMR and graft
loss directly correlates with peak HLA-DSA strength. Quantification of HLA antibodies allows stratification of
immunologic risk, which should help guide selection of acceptable grafts for sensitized patients.
J Am Soc Nephrol 21: 1398 –1406, 2010. doi: 10.1681/ASN.2009101065
Anti-HLA immunization constitutes an immunogenetic hurdle to transplantation, leading to increasingly protracted waiting times for sensitized
kidney transplant recipients.1–3 In France, 25% of
patients on the waiting list have a panel-reactive
antibody (PRA) level of ⬎5%,4 and in the United
States, 32% of patients awaiting transplantation are
sensitized.1 Despite efforts to diminish the risk for
sensitization by use of recombinant erythropoietin,
leukocyte-depleted transfusions, and the cessation
of pregraft transfusion protocols, the number of
sensitized patients on transplant lists remains substantial. Moreover, loss of a previous graft has become the primary cause of anti-HLA sensitization.
Patel and Terasaki5 in 1969 demonstrated the
efficacy of complement-dependent lymphocytotoxic cross-match (CXM) in defining immunologic
1398
ISSN : 1046-6673/2108-1398
risk in renal transplantation. This became the standard method, still used today, for graft allocation. It
became clear with time that it did not identify all
preexisting donor-specific HLA antibodies (HLADSA). In recent years, techniques for detection of
HLA antibodies have become more sensitive with the
Received November 1, 2009. Accepted April 8, 2010.
Published online ahead of print. Publication date available at
www.jasn.org.
C.S.-B. and D.G. contributed equally to this work.
Correspondence: Dr. Carmen Lefaucheur, Département de
Néphrologie et Transplantation Rénale, Hôpital Saint-Louis, 1
Avenue Claude Vellefaux, 75010 Paris, France. Phone: ⫹33-1-4249-96-08; Fax: ⫹33-1-42-49-96-08; E-mail: carmen.lefaucheur@
wanadoo.fr
Copyright © 2010 by the American Society of Nephrology
J Am Soc Nephrol 21: 1398–1406, 2010
Page 206 of 290
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introduction of solid-phase assays, including ELISA, and multiple
bead– based technology, of which the Luminex-based assays are
the most frequently used. The clinical impact of the antibodies
detected by these more sensitive techniques has yet to be fully
evaluated in terms of graft survival and definition of acceptable
grafts.3 Studies of the clinical relevance of HLA-DSA in patients
who receive a transplant with a negative CXM have been contradictory.6 –9
The ability to quantify these antibodies10 has added a dimension of complexity to the equation. The semiquantitative
ELISA technique was used to advantage by our group in a
cohort of 237 patients with renal transplants, showing an increase in the occurrence of acute antibody-mediated rejection
(AMR) with increasing HLA-DSA levels detected in historic
sera.11 The Luminex technique has been used in recent studies
to choose the type of desensitization according to HLA-DSA
strength12 and to determine acceptable HLA-DSA levels, allowing for successful kidney transplantation after desensitization.13 Forty years after the initial definition of immunologic
risk by Terasaki and Patel, the introduction of these more sensitive techniques revives and carries to a new level the basic
question of the clinical relevance of donor-specific anti-HLA
antibodies and their integration into current strategies of
transplantation. Indeed, no single study has compared the sensitivity, specificity, and positive predictive value (PPV) of classic or flow CXM, ELISA, and Luminex techniques in the prediction of AMR and graft survival.
The objective of this study was to appraise the full clinical
potential of HLA-DSAs detected before transplantation, with
reference to the previously described ELISA single-antigen
technique. We used the capacity of the Luminex technique to
identify with precision and to quantify HLA-specific antibodies to grade increasing immunologic risk. This observational,
single-center study of 402 consecutive deceased-donor kidney
transplant patients examined the impact of
the strength of HLA-DSA detected on historic and current sera on the risk for AMR
occurrence and graft survival in deceaseddonor kidney graft patients. Our graft strategy was the current worldwide strategy
based on a negative National Institutes of
Health lymphocytotoxic CXM test.
RESULTS
Pretransplantation HLA Antibodies in
Kidney Transplant Recipients
Historic (Peak) Sera
Among the 402 renal graft patients, 61
(15.2%) had a PRA level of ⱖ1%. A total of
118 (29.4%) patients had antibodies against
class I or class II HLA on any pretransplantation serum and were considered as sensitized. Of these, 46 (39%) had HLA-DSA
J Am Soc Nephrol 21: 1398 –1406, 2010
CLINICAL RESEARCH
identified by ELISA and 83 (70.3%) had HLA-DSA identified
by single-antigen flow-beads Luminex testing (peak SAFB
HLA-DSA).
Twenty-four (6%) patients presented a remote positive CXM:
Nine patients with IgG T cell complement-dependent cytotoxicity CXM (CDCXM), two patients with IgG T cell antiglobulin
enhanced complement-dependent cytotoxicity (AHG-CDCXM), two patients with B cell CXM, eight patients with IgG T
and B cell CXM, and three patients with IgG B and T cell
AHG-CDCXM. Figure 1 shows the distribution of peak SAFB
HLA-DSAs according to the positivity/negativity of remote
CXM (rCXM).
Current Sera
Seventy-six (18.9%) patients showed HLA-DSA at the time of
transplantation (current SAFB HLA-DSA). The mean of the
highest ranked SAFB HLA-DSA mean fluorescence intensity
(MFI) detected on the current sera was 1293 ⫾ 200, not significantly different from that of HLA-DSAs detected on peak sera
(1137 ⫾ 178; NS). There was no statistically significant intraindividual variation between the maximum SAFB HLA-DSAs
of peak and current sera (P ⫽ 0.67).
Survival Rates of Patients and Grafts
Patients
The mean follow-up time was 51.4 ⫾ 30.6 months (range 1.0 to
132.0 months). Patient 8-year survival was similar in nonsensitized patients, sensitized patients without peak SAFB HLADSA, and patients with peak SAFB HLA-DSA (90.2 versus 91.2
and 90.9%, respectively; P ⫽ 0.98).
Grafts
Five- and 8-year death-censored graft survivals were 89.2 and
83.6% in nonsensitized patients, 92.5 and 92.5% in sensitized
Kidney transplant patients
01/1998-06/2006
N=402
r CXM +
N=24
r CXM N=378
Peak DSA
Luminex +
N=18
Peak DSA
Luminex N=6
Peak DSA
Luminex +
N=65
Peak DSA
Luminex N=313
AMR
N=13
AMR
N=0
AMR
N=16
AMR
N=3
Figure 1. Distribution of donor-specific anti-HLA antibodies in kidney transplant recipients. rCXM⫹, patients with positive remote CDCXM; rCXM⫺, patients with negative remote CDCXM; peak HLA-DSA Luminex⫹, patients with donor-specific HLA
antibodies detected by SAFB Luminex technique on the peak sera; peak HLA-DSA
Luminex⫺, patients without donor-specific HLA antibodies detected by SAFB Luminex
technique on the peak sera.
Preformed Donor-Specific Antibodies
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Figure 2. The presence of HLA-DSAs on the highest rank pregraft serum associates with a significantly decreased graft survival (A),
regardless of whether HLA-DSAs were class I or II (B). (C) The occurrence of acute AMR associates with a significantly decreased graft
outcome. (D) Even in the absence of acute AMR, patients with preexisting HLA-DSAs have poorer graft survival as compared to patients
without preexisting HLA-DSAs. Donor-specific HLA antibodies are detected by SAFB Luminex technique. P values are calculated with
the use of the log-rank test.
patients with no HLA-DSA recognized on peak, and 71.2 and
60.8% in patients with HLA-DSA, detected by Luminex technique on the peak sera. Kaplan-Meier analysis revealed that
patients with peak SAFB HLA-DSA had a significantly lower
graft survival as compared with sensitized patients with no
HLA-DSA recognized and nonsensitized patients (P ⬍ 0.001,
respectively; Figure 2A). There was no difference in graft survival analyzed according to the class of the maximum HLADSA identified on the peak sera (P ⫽ 0.8). Patients with
HLA-DSA had poorer graft survival regardless of whether
the maximum HLA-DSA was class I or II (P ⬍ 0.0002; Figure 2B).
Acute AMR Episodes
vival as compared with patients without HLA-DSA (69.5 versus
84.4% at 96 months respectively; P ⫽ 0.02; Figure 2D).
Peak Sera
Analysis of the PPV, sensitivity, and specificity for AMR of the
various methods of identifying preexisting HLA-DSA is shown
in Table 1. A positive rCXM has a high predictive performance
of 54.2% with a high specificity at 97% but the lowest sensitivity at
40.6%. The Luminex technique has the highest sensitivity of these
techniques (90.6%) but a low PPV of 34.9%, whereas the combination of Luminex and rCXM has the highest PPV of 72.2%.
Current Sera
The presence of SAFB HLA-DSA on the current serum has a
PPV for AMR of 31.6% (sensitivity 75%, specificity 86.2%).
Acute AMR occurred in 8% of kidney transplant patients. The
5- and 8-year graft survivals of patients who
had an episode of AMR were 54.3 and Table 1. Relationship of pretransplantation anti-HLA antibody status to acute
45.5%, respectively, significantly worse AMR occurrence
AMR
than that of the remaining transplant popPositive Negative
ulation (88.5 and 81.9%, respectively; P ⬍
Parameter
Values
Values
PPV Sensitivity Specificity
(n)
(n)
0.0001; Figure 2C). The relative risk (RR)
(%)
(%)
for graft loss for patients who had an epi- PRA ⱖ1%
61
341
36.1
68.8
89.5
sode of AMR was 4.1 (95% confidence in- rCXM
24
378
54.2
40.6
97.0
terval [CI] 2.2 to 7.7) as compared with pa- Peak ELISA HLA-DSA
46
356
41.3
59.4
92.7
83
319
34.9
90.6
85.4
tients without AMR. Even in patients Peak Luminex HLA-DSA
76
326
31.6
75.0
86.2
without any episode of AMR, the presence of Current Luminex HLA-DSA
18
384
72.2
40.6
98.7
SAFB HLA-DSA on the peak serum was still rCXM/peak Luminex HLA-DSA
rCXM/current
Luminex
HLA-DSA
20
382
60.0
37.5
97.8
associated with a significantly lower graft sur1400
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The incidence of AMR was 40% (24 of 60 patients) in patients
with HLA-DSA detectable on both the peak serum and the
current serum, versus 21.7% (five of 23 patients) in those with
HLA-DSAs detectable on the peak serum but not on the current serum.
The receiver operating characteristic (ROC) curve analyses
of HLA-DSA strength in the prediction of AMR showed that
maximum peak HLA-DSA MFI area under the curve (AUC)
was significantly higher than that of maximum current HLADSA MFI (0.94 ⫾ 0.02 versus 0.86 ⫾ 0.04, respectively; P ⫽
0.028; Figure 3).
Graft Survival and Risk for AMR According to
Quantification of Donor-Specific Anti-HLA Antibodies
by SAFB Assays
CLINICAL RESEARCH
Table 2. RR for acute AMR according to the MFI of
highest pregraft ranked DSA detected by Luminex (logistic
regression)
DSA MFImax class
RR (95% CI)
P
ⱕ465
465 to 1500
1500 to 3000
3000 to 6000
⬎6000
1.0
24.8 (4.6 to 134.8)
23.9 (3.5 to 160.8)
61.3 (11.5 to 327)
113.0 (30.8 to 414)
⬍0.001
0.001
⬍0.001
⬍0.001
⬍3000. These results were also valid in patients without AMR
(RR 2.8; 95% CI 1.5 to 16.9; P ⫽ 0.009; Figure 4B). In patients
with MFIs ⬎3000, 57.1% of the graft losses at 1 year were due
to AMR.
ROC curve analysis determined that an MFI of 465 for the
highest single or an MFI of 820 for total HLA-DSA MFI on the
peak serum is associated with maximal specificity and sensitivity regarding the occurrence of AMR (AUC of 0.94 ⫾ 0.02 and
0.91 ⫾ 0.02, respectively; each P ⬍ 0.0001). The following
analysis uses the highest single MFI values.
The prevalence of AMR rises significantly with increasing
MFI of highest pregraft HLA-DSA detected by Luminex technique on peak pregraft serum: 0.9% in patients with MFI
⬍465, 18.7% in those with MFI between 466 and 3000, 36.4%
for MFI between 3001 and 6000, and 51.3% for patients with
MFI ⬎6000 (␹2 ⫽ 138.1, P ⬍ 0.0001). The RR for AMR according to MFI is shown in Table 2.
The 1-, 3-, and 8-year graft survivals decrease progressively
with rising peak HLA-DSA MFI: 95.0, 93.8, and 82.5% in patients with MFI ⬍465; 100.0, 92.1, and 78.4% for patients with
MFIs between 466 and 3000; and 85.0, 75.0, and 60.6% for
patients with MFIs ⬎3000 (P ⬍ 0.001; Figure 4A). The graft
survival in patients with MFIs ⬎3000 was significantly lower
than that of patients with MFIs ⱕ3000 (P ⬍ 0.0001).
The RR for graft loss for patients who underwent transplantation with peak HLA-DSAs ⬎3000 was 3.8 (95% CI 3.5 to
18.4; P ⬍ 0.0001) as compared with those with MFI HLA-DSA
Figure 3. Peak HLA-DSA MFIs are better predictors of acute
AMR than current HLA-DSA MFIs. Peak versus current ROC AUC
was compared using ␹2 test with Bonferroni correction.
J Am Soc Nephrol 21: 1398 –1406, 2010
Figure 4. The graft survival in patients with preexisting HLA-DSA
MFIs ⬎3000 is significantly lower than in patients with HLA-DSA
MFIs ⱕ3000. Kaplan-Meier estimates of graft survival according
to the MFImax of preexisting HLA-DSAs in the entire cohort of
kidney transplant patients (A) and in the subgroup of patients
without acute AMR (B).
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Significance of the Association of SAFB HLA-DSA/
Remote CDCXM
A total of 18 (21.7%) patients with peak SAFB HLA-DSA had a
remote positive IgG T or B cell CXM. Their mean of the maximum HLA-DSA MFI was 7700.6 ⫾ 1139.0, not significantly
different from the mean of the maximum HLA-DSA MFI of
patients with negative rCXM (5782.6 ⫾ 672.3; NS). In patients
with preformed HLA-DSA, the presence of peak complementfixing HLA-DSA on rCXM increased significantly the risk for
AMR (peak HLA-DSA⫹/rCXM⫹ versus peak HLA-DSA⫹/
rCXM⫺; P ⫽ 0.0005) and significantly reduced graft survivals
(P ⬍ 0.01). One- and 8-year graft survivals were 95.6 and
74.5% in patients with peak HLA-DSA⫹/rCXM⫺ and, respectively, 76.4 and 29.1% in patients with peak HLA-DSA⫹/
rCXM⫹.
DISCUSSION
This study shows the clinical relevance of precise immunologic
characterization of patients before transplantation, using single-antigen flow-beads technology. Long-term outcomes of
kidney grafts in patients with preexisting HLA-DSA detected
by SAFB Luminex technique are significantly worse as compared with patients who undergo transplantation without
HLA-DSA, confirming the recent results reported by Amico et
al.9 Our study furthers those observations by showing that
there is a gradation of the risk for AMR and of kidney graft
survival according to the levels of HLA-DSA detected before
transplantation. It also underlines the pertinence and importance of analysis of peak sera by SAFB techniques in defining
the immunologic risk for patients on the waiting list. The high
sensitivity of Luminex permits definition of the cutoff points
above which antibody levels detected before the graft are clinically relevant. We have shown a dramatic increase in the risk
for AMR with increasing levels of preexisting HLA-DSA above
465 as detected by Luminex. We have also shown that patients
who undergo transplantation with HLA-DSA MFIs ⬎3000
have a 3.8 increased risk for graft loss as compared with patients who undergo transplantation with HLA-DSA MFIs
⬍3000.
Our study also refines the current definition of immunologic risk3,9,14 in specifying the importance of the temporal
element (peak and current serum) and of quantification of
HLA-DSAs. The combination of different techniques for detecting HLA-DSA contributes to increasing their predictive
performance. We have shown here that a precise estimate of
immunologic risk before transplantation is possible, much
earlier than any CXM assay and in advance of the point where
grafts are accepted from deceased donors. This permits transfer of the focus from the simple identification of contraindication to transplantation (i.e., the CXM veto) to a personalized
appraisal of immunologic risk and helps define the transplant
strategy for any patient on the waiting list. This strategy should
help in weighing the risk/benefit ratio on the basis of defined
1402
Journal of the American Society of Nephrology
immunologic risk before transplantation. More generally, this
early definition of immunologic risk should allow the transplant community to establish (1) an active policy for the transplantation of immunized patients, with priority programs for
highly sensitized patients and the reduction of unnecessary
shipping of organs, and (2) protocols for immunosuppression
therapy and monitoring adapted to the immunologic risk of
the recipients.
Avoiding important confounding factors in graft survival,
the design of this observational study has permitted an accurate analysis of the clinical relevance of HLA-DSAs. The Luminex analysis was performed retrospectively at the end of the
study, avoiding possible bias in the decision to accept the graft
and in modifications of immunosuppressive treatment. We
have also excluded the patients for whom a pregraft conditioning was performed and patients for whom decisions were made
taking into account sensitive techniques for detecting HLADSA (living donors, kidney-pancreas transplants). These data
are important in the current context of widespread use of sensitive techniques for HLA-DSA detection but with practices
varying considerably from one transplantation center to another.
In recent years, a major change in renal transplantation has
come from the recognition of the importance of AMR, recognized initially only as the cause of hyperacute rejection but now
known to be responsible for acute and chronic lesions.15 Our
study underlines that AMR is a major factor in the evolution of
HLA-incompatible kidney transplants and is associated with
higher rates of graft loss, even though interpretation of the
long-term graft survival of patients with AMR suffers from
variation of the approach to treatment of AMR over time. For
treatment of AMR, we used a specific treatment based on intravenous Ig (IVIg) products known to have powerful immunomodulatory effects.16 Treatment of AMR has evolved from
IVIg-based regimens to combination therapies using plasmapheresis, IVIg, and rituximab, leading to an amelioration of
short-term graft survival of patients with AMR.17–20 The immunologic and histologic profiles of patients with AMR and
poor prognosis have largely been defined.17,21 The concept of
quantification of HLA-DSA posttreatment and the optimization of treatment according to the DSA levels17,22 should allow
the histologic and immunologic evolution of AMR to be better
defined over the long term as well.
Importantly, our study shows that even in the absence of
clinical AMR, the long-term graft course is worse in patients
with preexisting HLA-DSA. The recently described entity of
subclinical AMR23,24 in which progressive morphologic lesions
are found on biopsy in the absence of overt clinical rejection
may account for this different course. A recent study demonstrated that subclinical AMR is a frequent finding in patients
with preformed HLA-DSA (31.1% at 3 months) and is associated with worse GFR at 1 year.25 These progressive lesions lead
to chronic humoral rejection, first described in 200126 and now
recognized to be a distinct cause of late graft dysfunction and
loss.25,27,28
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Our study shows the major impact that pregraft Luminexbased HLA-specific antibody screening is having on the field of
transplantation. The Luminex technique is more sensitive in
detection of HLA-DSAs than the other two techniques analyzed, ELISA and CDCXM. Luminex analysis permits pregraft
characterization of the antibody profiles in sensitized patients
and gives improved definition of safe (antibody-negative) and
at-risk (antibody-positive) HLA specificities. The first step in
the transplant strategy for sensitized patients is to define
whether a graft with minimal immunologic risk is possible.
Whenever possible, kidney transplantation should be performed in the absence of DSAs. Virtual cross-matching, recently promoted by the United Network for Organ Sharing,29
consists of selecting potential donors without HLA specificities
against a recipient’s antibodies, predicting a negative CXM.
The use of the virtual cross-matching permits expanding the
geographic regions from which kidneys can be drawn and reduces waiting time and deaths on the waiting list.30 –32 It is,
furthermore, a good indicator for reducing the risk of AMR.33
In practice, we must evaluate, for each sensitized patient on
the waiting list, whether the listing of forbidden antigens permits a donor pool sufficient to ensure transplantation. For
some highly sensitized patients, use of virtual cross-matching
leads to an insufficient number of donors. Thus, alternatively,
the access to transplantation for these patients can be augmented in three ways: Priority programs, desensitization regimens, or increased immunologic risk of transplantation with
preexisting HLA-DSA.
Priority programs such as the Eurotransplant “Acceptable
Mismatch” program34 raise the probability for a hypersensitized patient to receive a kidney with a negative CXM,35 with
excellent graft survivals.34 Desensitization protocols based on
high-dosage IVIgs36 –38 have been used with success. Other
therapies39,40 can be efficacious in desensitizing patients who
are awaiting transplantation. Transplantation in the presence
of HLA-DSA requires a careful estimation of the immunologic
risk, as assessed by rCXMs and levels of antibodies detected by
Luminex technique. For such patients at high immunologic
risk, specific posttransplantation protocols41– 44 and close
monitoring are indispensable.
Access to and results of transplantation can therefore be
improved in sensitized patients. The data presented in this article emphasize the importance of precisely characterizing the
status of the anti-HLA pretransplantation immunization using
sensitive techniques. This should allow optimization of donor
immunologic selection, immunosuppressive regimen, and
posttransplantation monitoring.
CLINICAL RESEARCH
2006. Patients with multiple transplants (20 patients with combined
liver-kidney transplants and 14 with pancreas-kidney transplants),
living-donor transplants (36 patients), or pregraft conditioning (17
patients) and those for whom historic sera were not available for further research (24 patients) were excluded.
All patients were followed up through January 2009. Data on the
HLA typing of transplant donors and recipients and rCXM results
were recorded on day 0 (day of transplantation). Data on survival of
patients and grafts, AMR episodes, serum creatinine values, and posttransplantation immunosuppressive therapy were obtained at months 3
and 6; at years 1, 3, 5, and 8; and at the end of follow-up.
Criteria for Accepting a Donor
CXMs were performed by complement-dependent cytotoxicity (CDCXM)
and T cell antiglobulin enhanced complement-dependent cytotoxicity (AHG-CDCXM) on lymph nodes and by complement-dependent
cytotoxicity on separated B lymphocytes or spleen cells, according to
National Institutes of Health recommendations. Peak and current
sera were tested for all patients according to European Federation for
Immunogenetics standards. Sera were tested both diluted and undiluted, with and without dithiothreitol. For all kidney transplant recipients, negative current IgG T cell and B cell CDCXMs were required.
CXMs positive only for IgM were not considered as a contraindication to transplantation.
Detection of HLA Antibodies
HLA typing of transplant recipients was performed by molecular biology (Innolipa HLA typing kit; Innogenetics, Gent, Belgium). For all
kidney transplant donors, HLA-A/B/DR/DQ tissue typing was performed using the microlymphocytotoxicity technique with One
Lambda Inc. tissue-typing trays and was controlled by molecular biology. HLA-CW and HLA-DP typing of the donor was performed
when an isolated HLA-CW or HLA-DP HLA-DSA was potentially
present.
All pretransplantation (historic) sera were screened by the most
sensitive routine screening test available at the beginning of the study,
ELISA assays (LAT-M; One Lambda, Canoga Park, CA), to determine
the presence or absence of HLA class I or class II antibodies of the IgG
isotype. HLA class I antibodies were then identified by complementdependent cytotoxicity on a frozen cell tray of 30 selected HLA-typed
lymphocytes (Serascreen FCT30 Frozen Cell Trays; Gen Trak, Liberty,
NC). PRAs of the IgG class directed against HLA class I molecules
were calculated from this complement-dependent cytotoxicity assay.
All patients in whom HLA antibodies were detected were screened
for the presence of HLA-DSA in pretransplantation peak sera by two
techniques: ELISA and SAFB Luminex assays. Peak serum was determined on the basis of the serum’s having the highest % PRA. The
interval between the peak serum values and the actual transplantation
was 29.4 ⫾ 11.8 months.
CONCISE METHODS
Detection of HLA-DSA Using ELISA
Patients
The study included 402 consecutive deceased-donor single-organ
kidney transplants recipients who underwent transplantation in
Saint-Louis Hospital (Paris, France) between January 1998 and June
J Am Soc Nephrol 21: 1398 –1406, 2010
Identification of ELISA HLA-DSA class I was done using a high-definition single-antigen ELISA (LAT-1HD; One Lambda). For ELISA
HLA-DSA class II identification, we performed an ELISA (LAT 2-40;
One Lambda) test, which identified DR and DQ subtypes on a panel
Preformed Donor-Specific Antibodies
1403
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CLINICAL RESEARCH
www.jasn.org
of purified HLA antigens. Both ELISA tests were performed as recommended by the manufacturer and described previously.11 Detection
of HLA-DSA by ELISA on the peak sera was performed retrospectively between June and November 2006.
tients received as additional treatment four plasmaphereses and two
weekly doses of rituximab (375 mg/m2 body surface area; MabThera;
Roche, Meylan, France).17
Statistical Analysis
Detection of HLA-DSA Using SAFB on Luminex Platform
Identification of class I and class II HLA-DSA by Luminex analysis
(One Lambda) uses sets of 96 beads (class I) and 76 beads (class II),
respectively; each bead is coated with a single HLA glycoprotein, permitting precise identification of antibody specificity. Presence of antibodies was detected using a goat anti-human IgG coupled with phycoerythrin. The fluorescence of each bead was detected by a reader
(LABscan) and recorded as the MFI. All beads showing a normalized
MFI ⬎300 were considered positive. For each patient, we recorded the
number of HLA-DSA and the maximum HLA-DSA MFI defined as
the highest ranked donor-specific bead.
All current (day 0) sera were screened for the presence of HLADSA by class I and class II SAFB Luminex assays. Luminex analyses
were performed retrospectively between January and March 2009 for
the peak sera and in November 2009 for the current sera.
Posttransplantation Induction Protocols and
Maintenance Immunosuppressive Therapy
Immunosuppression protocols were defined according to the immunologic risk, determined by the current system using lymphocytotoxic PRAs and T and B cell– based assays. Patients received induction
therapy consisting of rabbit antithymocyte globulin (1.5 mg/kg per d
for 10 days; Thymoglobulin; Genzyme) with maintenance immunosuppression consisting of tacrolimus (Prograf; Astellas) or cyclosporine (Neoral; Novartis), mycophenolate mofetil (CellCept; Roche),
and steroids. Patients with remote positive IgG T and B cell CXM
received IVIg at the time of transplantation (2 g/kg body wt on days 0
to 1, 20 to 21, and 40 to 41).
Results are expressed as mean ⫾ SD for continuous variables, with the
exception of MFIs, or which mean ⫾ SEM is used. Comparisons were
based on the ␹2 test for categorical data and the t test for normally
distributed continuous data. For parameters without Gaussian distribution (MFI), the Mann-Whitney U test was used. For individual
HLA-DSA MFI evolution between peak and current sera, the Wilcoxon
matched-pairs test was used. Death-censored graft survivals were calculated by Kaplan-Meier analysis. Differences between survivals were
calculated by log-rank analysis. P ⬍ 0.05 was regarded as statistically
significant.
For studying the usefulness of HLA-DSA MFIs as a predictor of
AMR, ROC curves were plotted to estimate the cutoff of MFImax
HLA-DSA and total HLA-DSA MFIs in terms of yielding the highest
sensitivity ⫹ specificity/2. We determined the AUC to evaluate its
significance. For peak versus current ROC curves, AUC were compared using ␹2 test with Bonferroni correction.
The association of AMR occurrence and MFI strength classes was
determined by univariate logistic regression. The risk for graft failure
according to the occurrence of AMR and HLA-DSA MFIs was determined using univariate Cox analyses.
All tests were two-sided. All statistical analyses were performed
using STATA 10.0 software (Stata Corp., College Station, TX).
ACKNOWLEDGMENTS
We thank Astellas France for the contribution to the statistical analysis.
Diagnosis and Treatment of Acute AMR Episodes
Among the patients with clinical acute graft dysfunction, three had
episodes of borderline changes, 18 had episodes of acute T cell–mediated rejection (IA for six patients, IB for three patients, IIA for four
patients, IIB for two patients, and III for three patients), and 32 patients had episodes of AMR. All rejection episodes were biopsyproven. Biopsy specimens were evaluated by light microscopy and
immunofluorescence (C4d and Igs). Findings were graded according
to the Banff ’07 classification.45 C4d was detected by a two-step indirect immunofluorescence method using a mAb specific for complement fragment C4d on frozen tissue (Quidel, Santa Clara, CA). All
patients with AMR had characteristic histologic lesions delineated by
the Banff classification of allograft rejection,46 positive C4d staining,
and HLA-DSAs detected by SAFB assays at the time of diagnosis.
All patients with AMR were treated with methylprednisolone
pulses (500 mg/d for 3 days), with switch to tacrolimus for patients
who were previously on cyclosporine and a protocol of high-dosage
IVIg (2 g/kg, repeated every 3 weeks for four administrations). Between January 1998 and December 2003, eight patients received an
additional treatment with plasma exchange (five patients) or muromonab-CD3 (OKT3; three patients). After January 2004, all pa1404
Journal of the American Society of Nephrology
DISCLOSURES
None.
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NEPHROLOGY 2010; 15, S101–S105
doi:10.1111/j.1440-1797.2009.01217.x
Donor-specific transfusions
Date written: June 2007
Final submission: October 2008
Author: Fiona Mackie
GUIDELINES
a. The best designed randomised controlled trial demonstrates no advantage of donor-specific transfusions (DSTs)
in graft survival at 2 years or in the incidence of acute rejection. (Level II evidence)
b. There is randomised controlled trial evidence (in a small trial) for a beneficial effect of DSTs in cyclosporinetreated recipients of one haplotype mismatch living related donations in terms of less acute rejection and lower serum
creatinine in the short term. (Level II evidence)
SUGGESTIONS FOR CLINICAL CARE
(Suggestions are based on Level III and IV evidence)
• The high risk of sensitisation does not justify the
routine use of DSTs (Level III evidence)
• A single pre-transplant DST is as effective as multiple
DSTs. (Level III evidence)
• The potential benefit of DST needs to be weighed
against the risk of sensitisation (approximately 30%) and
infection.
• There is insufficient evidence on the use of DSTs to
assist deletion of donor-specific antibodies pre-transplant
to support their use.
IMPLEMENTATION AND AUDIT
No recommendation.
BACKGROUND
Maximising graft survival from living donors is a major goal
in transplantation given the mismatch between the number
of available donors and the ever-increasing number of
recipients. Blood transfusion from living donors to the
recipient prior to kidney transplantation was introduced
several decades ago with the aim of improving graft
outcome. However, with reduced acute rejection rates
associated with newer immunosuppressive agents and
recombinant erythropoietin use, DST is rarely practised.
Nevertheless, modulating the immune response to the
donor and inducing ‘pseudo-tolerance’ without having to
rely heavily on immunosuppression continues to be a goal of
transplantation medicine.
When reviewing the evidence, it needs to be recognized
that there may be fundamental differences between early
reports of DST use and the DST of today; red blood cells are
now washed and are essentially free of white blood cells –
© 2010 The Author
Journal compilation © 2010 Asian Pacific Society of Nephrology
which may have been an important mediator of the observed
effects. Furthermore, more recent literature suggests that
the beneficial effect of tolerance develops only if the blood
donor and recipient have one HLA haplotype or at least
one HLA-B and one HLA-DR antigen in common.1 Many of
the studies reviewed below do not specify these details.
The purpose of these guidelines is to review the evidence on DST in living kidney donation (LKD) and to
provide recommendations on when and whether its use is
warranted.
SEARCH STRATEGY
Databases searched: MeSH terms and text words for kidney
transplantation and living donor were combined with
MeSH terms and text words for blood transfusion. The
search was carried out in Medline (1966 – September Week
2, 2006). The Cochrane Renal Group Trials Register was
also searched for trials not indexed in Medline.
Date of search: 26 September 2006.
Update search:
Databases searched: MeSH terms and text words for kidney
transplantation were combined with MeSH terms and text
words for living donor and combined with MeSH terms and
text words for open and laparoscopic nephrectomy. The
search was carried out in Medline (1966 – March Week 1,
2009). The Cochrane Renal Group Trials Register was also
searched for trials not indexed in Medline.
Date of searches: 9 March 2009.
WHAT IS THE EVIDENCE?
Level II evidence
The beneficial effect of DST in one haplotype mismatch
living related donors was first suggested by Salvatierra et al.2
Since then, two prospective randomized trials have been
reported.3,4
Page 215 of 290
S102
Alexander et al.3 compared patients given DST 24 hours
prior to transplant and 7–10 days post-transplant (n = 115)
with patients who did not receive DST (n = 97). The immunosuppression regimen was routine triple immunosuppression commenced post-transplant. All patients were -HLA
non-identical (>50% had more than two Class I mismatches
and more than one Class II mismatch). There was a similar
distribution of HLA mismatch between the two groups.
Biopsy-proven rejection episodes were seen more frequently
in the DST group (81 vs 54 in non-DST) but this difference
was not statistically significant. A significantly higher creatinine level was seen in the DST group at 7 and 14 days but
this did not translate into a difference in 1- or 2-year graft
survival. One of the primary outcomes of the study was the
ability to withdraw steroid treatment; no significant difference was seen between the two groups for this outcome.
There was no difference in adverse events between the two
groups. Limitations of this study include the inclusion of a
diverse degree of HLA matches and too small a sample size
to adequately study the effect of DST for the different HLA
matches.
In a smaller prospective trial, Sharma et al.4 randomized
living related recipients (n = 15) to receive DST (one transfusion 24 hours prior to transplant) or no DST (n = 15). All
patients received cyclosporine 3 days prior to transplant and
continued routine triple therapy post-transplant. In addition, all patients received third-party transfusions 2–3 weeks
prior to transplantation to correct anaemia. Sharma et al.
found a significantly greater incidence of acute rejection
in the non-DST group (1.1 vs 0.26 per patient, P < 0.01).
A significantly lower creatinine level was also seen in the
DST group from 3 months to 12 months post-transplant (at
12 months, 1.12 vs 2.02 mg/dL, P < 0.05). However, there
was no difference in graft survival in the short term (1 year).
It is difficult to extrapolate results from this study to current
practice because the degree of HLA match was not specified
and patients in both groups received third-party transfusions
to correct anaemia (prior to standard erythropoietin usage).
Bordes-Aznar et al.5 reported a small randomized trial
comparing the outcome of DST to cyclosporine and prednisone followed by azathioprine in living related recipients
who were haploidentical but who had highly reactive mixed
lymphocyte cultures (MLC). This group traditionally has a
lower graft survival and is considered high risk. There was
no difference in patient or graft survival at 1 year between
the two groups (70% graft survival in both). In the DST
group, 30% of potential donors were not able to be used
because of sensitisation. Immunosuppression was not given
during the transfusion periods. Bordes-Aznar et al. did not
clearly state sample size or immunosuppression regimen, and
the randomization method was not explained.
Level III evidence
In 2006, Marti et al.6 reported a prospective study of 61
potential allograft recipients (adults >16 years), both living
related and unrelated, who received DSTs and compared
them to carefully selected matched controls from the
The CARI Guidelines
Collaborative Transplant Study Group (CTS). The controls
were matched for age, sex, related vs unrelated, original
disease, cold ischemia time, number of transplants, year of
transplant, time on dialysis and HLA match. All patients
were on cyclosporin and prednisone with 31/55 also receiving either azathioprine or mycophenolate. There was no
significant difference in induction therapy between the DST
and matched control group. Although there was a trend to
better allograft survival in the DST group (98% vs. 82%)
this failed to reach statistical significance and when examined on an intention-to-treat basis, the 6-year graft survival
of the DST group was 88.5%. There were no statistically
significant differences in 1-year serum creatinine or treated
acute rejection rate between the two groups. Of concern was
the fact that 10% of patients (n = 6) in the DST group
developed positive T cell crossmatches following the transfusions and living donation did not proceed. This study was
underpowered to look at graft survival differences and historical controls were used. There were more pre-emptive
transplants in the DST group (although time on dialysis was
similar).
Sonoda and Ishibashi7 retrospectively analyzed patients
in the Japanese transplant registry. One HLA haplotype
mismatch living related donor (LRD) patients (n = 1292)
were analyzed in subgroups according to immunosuppression (cyclosporin n = 315; no cyclosporine n = 977) and
DST transfusion (97/315 cyclosporin; 298/977 without
cyclosporin). In the cyclosporin groups, the graft survival
rate at 4 years for those with DST was 93.5%, compared
with 76.2% for those with third-party transfusion (not
DST) and 62.7% for those without transfusion. This
improvement in graft survival was not seen in the noncyclosporin group, where the 4-year graft survival for DST
was 73.3%, 73.2% for third-party transfusion and 69.0% for
those with no transfusion.
Davies et al.8 prospectively (not randomized) compared
three different protocols for DST:
1. multiple pre-transplant DST with azathioprine during
the period and oral cyclosporin post-transplant (n = 34),
2. multiple pre-transplant DST with azathioprine during
this period and oral cyclosporin 1 day pre-transplant
(n = 31), and
3. single pre-transplant DST 24–48 hours prior to transplant with pre-transplant intravenous cyclosporin (n = 21).
All patients were LRD recipients with a 1 haplotype mismatch. There were no significant differences in recipient
age, panel reactive antibodies (PRA) or the number of
third-party transfusions between the three groups. Davies
et al. found no significant differences in the acute rejection
rate or in the 1-year patient or graft survival between the
three groups. There was, however, a significantly greater
incidence of CMV infection in Group 2 compared with
the other groups (16% for Group 2 vs 0% for Groups 1
and 3).
Satoh et al.9 retrospectively examined long term
(3–13 years) graft survival in 52 one-haploidential living
related first renal transplants conducted between 1983 and
1996. Twelve patients received prednisone, azathioprine
and cyclosporin plus DST and 38 received prednisone,
Page 216 of 290
S103
Living Kidney Donor
azathioprine and cyclosporine alone. Recipients received 3
DSTs without immunosuppression. Historical controls were
not extensively matched as in the study by Marti et al.6 and
the DST group had signicantly lower donor age. There was
no significant difference in acute rejection or long-term
graft survival rates between the two groups. Two patients
(16.7%) in the DST group developed donor specific antibodies which were subsequently removed by plasmapheresis
and T and B cell crossmatches became negative. This study
was important in demonstrating that longer term graft survival was not improved by DST, as one of the hypotheses
regarding use of DSTs was that it may reduce chronic rejection and therefore alter long-term outcome.
Otsuka et al.10 retrospectively analyzed 40 potential
recipients of DST and cyclosporine, comparing them to a
historical control who received a one haplotype matched
living related kidney but no DST during the same period
(n = 13). All patients received a calcineurin inhibitor.
Cyclosporin was administered at the time of DST. There was
no significant difference in graft survival rate at 5 and
10 years between the two groups, and no difference in acute
rejection rates within 3 months after transplant. The sensitization rate was 7.5%, and one of the three patients who
developed positive crossmatches could not proceed with
living donation. One patient developed CMV infection as a
consequence of the DST.
Lezaic et al.11 retrospectively compared living related
transplant recipients who had received DST with azathioprine cover (n = 19) to untransfused patients (n = 15) and
25 random polyinfused patients. Post-transplant immunosuppression consisted of azathioprine, cyclosporine and
prednisone. Serum creatinine was significantly higher at 1
and 3 years in the non-transfused group compared with the
DST and the randomly transfused group, despite the fact
that there were no differences in the incidence of acute
rejection or early graft function. There was also no difference in HLA mismatch, MLC reactivity and panel reactivity. This report provides little detail on the patients included
or how the groups were selected and the numbers included
are small. Three patients (15.7%) developed cross-reactivity
with their donors in the DST group.
Flye et al.12 examined the effect of three 200 mL aliquots
of DSTs given biweekly with azathioprine cover in 163 oneor two haplotype-mismatch living related or living unrelated potential renal transplant recipients. Following
transplantation, only prednisone and azathioprine were
given. Their outcome was compared with a group of HLAidentical living recipients (n = 53) and a group of one-or
two haplotype-mismatched living donor recipients (n = 54)
treated with triple immunosuppression and induction
therapy. Permanent T cell crossmatch sensitization occurred
in 11 of the 163 patients (7%). Actual one- and five-year
graft survivals were 94%, 100%, 100% and 72%, 85% and
71% for DST-treated groups with one HLA haplotype mismatched donors (n = 121), two HLA haplotype mismatched
related donors (n = 14) and two haplotype-mismatched
unrelated donors, respectively. This was comparable to the
HLA identical group. No lymphoproliferative or CMV
disease was seen in the DST group.
In a retrospective paediatric study (Leone et al.13), the
results of DST plus post-transplant immunosuppression
with prednisone and azathioprine were compared with a
routine triple immunosuppression group. All received haploidentical grafts. Three of 24 patients treated with DST
had circulating cytotoxic antibodies to the donor. There
was no difference in graft or patient survival at 1 year or in
mean rejection episodes. However, there was less hospitalization and less severe rejection during the first 3 months
in the cyclosporine (non-DST) group. Given the equivalent graft survival and the risk of recipient sensitization,
the authors concluded that routine triple immunosuppression is preferable.
Anderson et al.14 administered donor-specific whole
blood or buffy coat in conjunction with azathioprine immunosuppression in 163 patients. Transient sensitization
occurred in 2% and permanent sensitization in 7%. Over
the 10 year duration, DST + azathioprine graft survival was
similar to the HLA-identical sibling transplantation. The
CMV sepsis rate was 2% and there was no occurrence of
lymphoproliferative neoplasms.
SUMMARY OF THE EVIDENCE
Please refer to the enclosed evidence tables.
WHAT DO THE OTHER GUIDELINES SAY?
Kidney Disease Outcomes Quality Initiative: There is
some evidence that donor-specific transfusion with living
donor transplantation improves survival, but the decision
to perform donor-specific transfusion must still be made on
a case-by-case basis. Blood transfusions can induce antibodies to histocompatibility leukocyte antigens that can
reduce the success of kidney transplantation; thus, transfusions generally should be avoided in patients awaiting a
renal transplant.
UK Renal Association: No recommendation.
Canadian Society of Nephrology: No recommendation.
European Best Practice Guidelines: No recommendation.
International Guidelines: No recommendation.
SUGGESTIONS FOR FUTURE RESEARCH
No recommendation.
CONFLICT OF INTEREST
Fiona Mackie has no relevant financial affiliations that
would cause a conflict of interest according to the conflict of
interest statement set down by CARI.
REFERENCES
1. van Twuyver E, Mooijaart RJ, ten Berge IJ et al. Pretransplantation
blood transfusion revisited. N Engl J Med 1991; 325: 1210–3.
Page 217 of 290
S104
2. Salvatierra O Jr, Vincenti F, Amend W et al. Deliberate donorspecific blood transfusions prior to living related renal transplantation. A new approach. Ann Surg 1980; 192: 534–52.
3. Alexander JW, Light JA, Donaldson LA et al. Evaluation of preand post-transplant donor-specific transfusion/cyclosporine A in
non-HLA identical living donor kidney transplant recipients.
Cooperative Clinical Trials in Transplantation Research Group.
Transplantation 1999; 68: 1117–24.
4. Sharma RK, Rai PK, Kumar A et al. Role of preoperative donorspecific transfusion and cyclosporine in haplo-identical living
related renal transplantation recipients. Nephron 1997; 75: 20–4.
5. Bordes-Aznar J, Odor A, Dib-Kuri A et al. Randomized clinical
trial of cyclosporine or donor specific transfusion in high risk
living-related donor transplantation. Transplant Proc 1987: 19:
2276–7.
6. Marti HP, Henschkowski J, Laux G et al. Effect of donor-specific
transfusions on the outcome of renal allografts in the cyclosporine
era. Transpl Int 2006; 19: 19–26.
7. Sonoda T, Ishibashi M. Donor-specific transfusion: a report of the
Japanese Renal Transplant Registry. Clin Transpl 1987; 257–60.
8. Davies CB, Alexander JW, Cofer BR et al. Efficacy of a single
pretransplant donor-specific transfusion and cyclosporin A admin-
The CARI Guidelines
9.
10.
11.
12.
13.
14.
istered 24 to 48 hours before one-haplotype-mismatched living
related donor kidney transplant. Ann Surg 1992; 215: 618–25.
Satoh S, Sugimura J, Omori S et al. Long-term graft survival with
or without donor-specific transfusion in cyclosporine era in one
haplo-identical living related renal transplant recipients beyond
the first year: a 19 year experience. Tohoku J Exp Med 2002; 197:
201–7.
Otsuka M, Yuzawa K, Takada Y et al. Long term results of donorspecific blood transfusion with cyclosporine in living related transplantation. Nephron 2001; 88: 144–8.
Lezaic V, Jovicic S, Simic S et al. Donor-specific transfusion and
renal allograft outcome. Nephron 2002; 92: 246–7.
Flye MW, Burton K, Mohanakumar T et al. Donor-specific transfusions have long-term beneficial effects for human renal allografts.
Transplantation 1995; 60: 1395–401.
Leone MR, Alexander SR, Melvin T et al. A comparison of 2
protocols for living-related renal transplantation in children:
donor-specific transfusions versus cyclosporine. J Urol 1990; 144:
721–3.
Anderson CB, Brennan D, Keller C et al. Beneficial effects of
donor-specific transfusions on long-term renal allograft function.
Transplant Proc 1995; 27: 991–4.
Page 218 of 290
S105
Living Kidney Donor
APPENDICES
Table 1 Characteristics of included studies
Study ID
(author,
year)
Intervention
(experimental
group)
Intervention
(control
group)
Follow up
(months)
N
Study design
Setting
Participants
Alexander
et al. 19993
212
Randomised
controlled
clinical
trial
8 centres,
US
Non-HLA
identical
living kidney
transplant
recipients
Donor-specific
transfusion
No
intervention
21 months
Sharma
et al. 19974
30
Randomised
controlled
clinical
trial
India
Haplotypematched
living related
renal
transplant
recipients
Donor-specific
transfusion
No
intervention
13 to 18
months
Main conclusions
DST had no
practical effect on
patient or graft
survival for up to
2 yrs, donor-specific
responsiveness was
more frequent in
transfused patients
DST and
cyclosporine
administered 24 h
before Tx is
effective in
improving graft
function and
reducing acute
rejection
Table 2 Quality of randomised trials
Study ID
(author, year)
Method of allocation
concealment*
Alexander
et al. 19993
Sharma
et al. 19974
Blinding
Intention-to-treat
analysis†
Loss to
follow up (%)
(participants)
(investigators)
(outcome assessors)
Central
No
No
No
No
5.8%
Computer generated
No
No
No
Not stated
0.0%
*Choose between: central; third party (e.g. pharmacy); sequentially labelled opaque sealed envelopes; alternation; not specified.
†
Choose between: yes; no; unclear.
Table 3 Results for dichotomous outcomes
Study ID
(author,
year)
Outcomes
Alexander Immunological
et al. 19993 hyporesponsiveness
Mortality at 2 yrs
At least one
rejection
Severe steroidSharma
et al. 19974 resistant rejections
Mortality
Graft loss
Control group
Intervention group (number of patients
(number of patients with events/number
of patients not
with events/number
exposed)
of patients exposed)
Relative risk (RR)
[95% CI]
Risk difference (RD)
[95% CI]
0.15 (95%CI: 03.02, 1.11) -0.16 (95%CI: -0.28, -0.04)
1/37
9/49
4/115
60/115
2/97
44/97
13.69 (95%CI: 0.32, 9.01)
1.15 (95%CI: 0.87, 1.52)
0.01 (95%CI: -0.03, 0.06)
0.07 (95%CI: -0.07, 0.20)
2/15
3/15
0.67 (95%CI: 0.13, 3.44)
-0.07 (95%CI: -0.33, 0.20)
1/15
2/15
2/15
3/15
0.50 (95%CI: 0.05, 4.94)
0.67 (95%CI: 0.13, 3.44)
-0.07 (95%CI: -0.28, 0.15)
-0.07 (95%CI: -0.33, 0.20)
Page 219 of 290
0041-1337/02/7410-1377/0
TRANSPLANTATION
Vol. 74, 1377–1381, No. 10, November 27, 2002
Printed in U.S.A.
Copyright © 2002 by Lippincott Williams & Wilkins, Inc.
WAITING TIME ON DIALYSIS AS THE STRONGEST MODIFIABLE
RISK FACTOR FOR RENAL TRANSPLANT OUTCOMES
A PAIRED DONOR KIDNEY ANALYSIS1
HERWIG-ULF MEIER-KRIESCHE2,3
Background. Waiting time on dialysis has been
shown to be associated with worse outcomes after living and cadaveric transplantation. To validate and
quantify end-stage renal disease (ESRD) time as an
independent risk factor for kidney transplantation,
we compared the outcome of paired donor kidneys,
destined to patients who had ESRD more than 2 years
compared to patients who had ESRD less than 6
months.
Methods. We analyzed data available from the U.S.
Renal Data System database between 1988 and 1998 by
Kaplan-Meier estimates and Cox proportional hazards
models to quantify the effect of ESRD time on paired
cadaveric kidneys and on all cadaveric kidneys compared to living-donated kidneys.
Results. Five- and 10-year unadjusted graft survival
rates were significantly worse in paired kidney recipients who had undergone more than 24 months of dialysis (58% and 29%, respectively) compared to paired
kidney recipients who had undergone less than 6
months of dialysis (78% and 63%, respectively; P<0.001
each). Ten-year overall adjusted graft survival for cadaveric transplants was 69% for preemptive transplants versus 39% for transplants after 24 months on
dialysis. For living transplants, 10-year overall adjusted graft survival was 75% for preemptive transplants versus 49% for transplants after 24 month on
dialysis.
Conclusions. ESRD time is arguably the strongest
independent modifiable risk factor for renal transplant outcomes. Part of the advantage of living-donor
versus cadaveric-donor transplantation may be explained by waiting time. This effect is dominant
enough that a cadaveric renal transplant recipient
with an ESRD time less than 6 months has the equivalent graft survival of living donor transplant recipients who wait on dialysis for more than 2 years.
Kidney transplantation is considered the treatment modality of choice for the majority of patients with end-stage-renal
disease (ESRD). Preemptive transplantation has been advocated over transplantation after a period of dialysis. Initially
this position was motivated by the observation that preemp-
AND
BRUCE KAPLAN2
tive renal transplant recipients were doing significantly better than patients who had undergone longer periods of maintenance dialysis (1, 2). These studies, however, could not
exclude the potential selection bias of lower risk patients who
obtain preemptive transplants and, therefore, could not directly implicate dialysis as a causal factor for the worse graft
survival in transplants after maintenance dialysis. Evidence
that time on dialysis in itself conferred a higher risk for graft
loss after transplantation came initially from a single-center
study by Cosio et al. who showed that increased time on
dialysis before transplantation was associated with decreased patient and graft survival (3). The argument that
time on dialysis itself is an independent risk factor for graft
loss was strengthened by a subsequent retrospective study
that was based on U.S. Renal Data System (USRDS) data
that showed a clear dose effect of the detrimental effect of
dialysis time on transplant outcomes not only for patient and
graft survival but somewhat surprisingly also for death-censored graft survival in both cadaveric and living transplantation (4). In addition, this study found that the dose-dependent detrimental effect of dialysis time was proportional
across different primary disease groups, making an argument against the hypothesis that the risk of increased ESRD
time was only related to cumulative disease burden. Shortly
thereafter, Mange et al. confirmed the better outcomes of
living donated grafts in preemptive transplants versus patients on dialysis for longer periods of time (5).
All previous studies looked at the relative impact of ESRD
time on subsequent renal transplant outcomes, but they did
not quantify this risk factor. In addition, it was difficult to
quantify ESRD time as a risk factor unless proven to be
independent of potential donor-related confounding factors.
It is conceivable that part of the negative effect of ESRD time
is related to poorer kidney grafts going to people who have
been on the waiting list for longer times.
For this reason, we decided to first investigate whether
ESRD time is a risk factor for outcomes after kidney transplantation independent of donor factors and, if so, to subsequently quantify the absolute impact of ESRD time in cadaveric and living transplantation. Identifying ESRD time as a
donor-independent risk factor would be of great significance
because ESRD time would have to be considered a modifiable
risk factor for kidney transplantation.
To ascertain that ESRD time before kidney transplantation is a significant risk factor for graft survival independent
of donor factors, we analyzed 2,405 kidney pairs harvested
from the same donor and transplanted subsequently into one
recipient with short ESRD time and the other in a recipient
with long ESRD time (6). We also assessed overall 5- and
10-year graft survival rates by length of pretransplant dialysis in living versus cadaveric transplants in an attempt to
1
The data reported in this study have been supplied by the U.S.
Renal Data System and the U.S. Scientific Renal Transplant Registry. The interpretation and reporting of these data are the responsibility of the authors and in no way represent an official policy or
interpretation of the U.S. government.
2
Division of Nephrology, Hypertension and Transplantation, University of Florida, Gainesville, FL.
3
Address correspondence to: Herwig-Ulf Meier-Kriesche, M.D.,
Department of Internal Medicine, Division of Nephrology, Hypertension and Transplantation, 1600 SW Archer Road, Box 100224,
Gainesville, FL 32610 – 0224. E-mail: [email protected].
Received 12 June 2002. Accepted 9 July 2002.
DOI: 10.1097/01.TP.0000034632.77029.91
1377
Page 220 of 290
1378
TRANSPLANTATION
quantify the relative impact of ESRD time versus living donation in determining long-term outcomes after transplantation.
TABLE 1. Demographic characteristics of 2,405 recipients of
paired kidneys with short compared to long ESRD
MATERIALS AND METHODS
We retrospectively analyzed data available from the USRDS for
renal transplantations performed between 1988 and 1998 in the
United States. In the database, we identified all cadaveric donors
from whom two kidneys had been available for transplantation. We
limited the analysis to kidney pairs that would go to primary adult,
single-organ, renal transplant recipients. All pairs of which one
kidney went to a six-antigen–matched recipient were excluded from
the analysis. We then identified those kidney pairs that went to one
recipient who had been on dialysis for less than 6 months, including
preemptive transplants, and to the other recipient who had been on
dialysis for more than 2 years.
Study endpoints for this cohort of patients were overall graft
survival, patient survival, death-censored graft survival, and patient
survival with a functioning graft. We compared the study endpoints
between the kidney pairs by Kaplan-Meier analysis and estimated
whether observed differences were significant by the log-rank test.
In addition, we used a Cox proportional hazards model to obtain
adjusted survival rates for short versus long pretransplant ESRD
time. These models were adjusted for known risk factors for graft
and patient survival such as recipient demographics (but not donor
demographics), HLA match, panel reactive antibody (PRA), immunosuppressive regimen, and delayed graft function.
We also identified a second cohort of patients in whom all solitary
adult first renal transplants between 1988 and 1998 were included.
In this cohort of patients, we estimated differences in graft survival
by Kaplan-Meier methods and calculated adjusted graft survival
rates from a Cox proportional hazards model, which adjusted for the
covariates and for the donor demographics. To evaluate the relative
impact of waiting time on dialysis versus the affect of living versus
cadaveric transplantation, we introduced an interaction term between ESRD time and transplant modality in the Cox model.
In addition, we retrospectively analyzed 77,469 patients with
ESRD who had been on the cadaveric renal transplant waiting list
for at least 2 years between 1988 and 1998. We used a Cox nonproportional hazards model that used time to transplantation as the
time-dependent covariate to estimate the risk for death associated
with cadaveric renal transplantation compared to remaining on the
waiting list (7).
A probability of type 1 error less than 0.05 was considered the
threshold of statistical significance. For multiple comparisons, we
used Bonferroni methods to assess statistical significance. Statistical
analysis was performed with SAS version 8.2 and SPSS version 11.0.
Vol. 74, No. 10
N
Donor age (years)
Recipient age (years)
Peak PRA (%)
AB mismatch
DR mismatch
ESRD time (mo)
Cold time
Female recipients
Female donor
AA recipient
AA donor
Mechanical perfusion
Antibody Induction
MMF
Neoral
Prograf
DGF
Acute rejection
ESRD time
⬍6 mo
ESRD time
⬎24 mo
2,405
33.3⫾16.0
44.3⫾12.8
12.0⫾22.9
3.0⫾1.1
1.5⫾0.7
1.1⫾1.9
22.7⫾10.3
40.2%
38.1%
19.1%
10.8%
12.5%
33.3%
13.5%
13.6%
5.7%
20.1%
24.0%
2,405
33.3⫾16.0
47.3⫾12.5
17.3⫾26.7
3.1⫾1.0
1.5⫾0.7
51.2⫾34.6
22.8⫾10.2
41.4%
38.1%
33.2%
10.8%
12.4%
33.2%
14.4%
13.6%
5.6%
31.2%
27.4%
P
nsa
⬍0.01a
⬍0.01a
nsa
nsa
⬍0.01a
nsa
nsb
nsb
⬍0.01b
nsb
nsb
nsb
nsb
nsb
nsb
⬍0.01b
⬍0.01b
a
t test.
Chi-square test.
MMF, mycophenolate mofetil; ns, not significant.
b
By Kaplan-Meier analysis, 5- and 10-year graft survival
rates for paired kidneys (Fig. 1) were significantly worse in
the patients who had undergone more than 24 months of
dialysis (58% and 29%, respectively) compared to the patients who had been on dialysis for less than 6 months before
transplantation (78% and 63%, respectively, P⬍0.001 each).
The 5- and 10-year unadjusted death-censored graft survival
rates for paired kidneys were 86% and 77%, respectively, in
patients who had been transplanted early compared to 77%
and 57%, respectively, in patients who were transplanted
late (P⬍0.001 each).
Five- and 10-year unadjusted overall patient survival for
paired kidneys was 89% and 76%, respectively, in the group
on dialysis less than 6 months compared to 76% and 43%,
RESULTS
Table 1 displays the demographics of recipients of paired
kidneys in the short versus long ESRD time group. Donor
demographics were identical between the groups because of
each kidney pair selected, one kidney went to the short ESRD
time group and one kidney went to the long ESRD time
group. Recipient age was significantly higher in the patients
who had been on dialysis for more than 2 years. Peak percent
PRA before transplantation was significantly higher in patients in the long ESRD time group, but HLA matching was
not significantly different between the groups. Cold ischemia
time was virtually identical between the groups. Recipient
gender distribution was similar, whereas African American
recipients were observed more frequently in the long ESRD
time group. Immunosuppressive therapy was equally distributed between the groups. Acute rejection and delayed graft
function were both significantly more frequent in the long
ESRD time group.
FIGURE 1. Unadjusted graft survival in of 2,405 recipients of
paired kidneys with short compared to long ESRD time.
Page 221 of 290
November 27, 2002
1379
MEIER-KRIESCHE AND KAPLAN
respectively, in the group on dialysis for more than 2 years
(P⬍0.001 each). The 5- and 10-year adjusted graft survival
for paired kidneys was 78% and 60%, respectively, in the
short ESRD time group and 65% and 41%, respectively, in
the long ESRD time group, and the relative risk for graft loss
in the long ESRD time group was 1.73 (confidence interval
1.54 –1.95, P⬍0.001).
The unadjusted graft survival of all cadaveric transplants
between 1988 and 1998 is displayed in Figure 2. The 10-year
overall unadjusted graft survival was 71% in the preemptive
group, 49% in the 0 to 6 month dialysis group, 43% in the 6
to 12 month dialysis group, and 38% in the 12 to 24 month
dialysis group and 35% in the patient group who had been on
dialysis for more than 24 months.
For all living donated kidneys, the 10-year overall unadjusted graft survival (Fig. 3) was 78% in the preemptive
group, 62% in the 0 to 6 month dialysis group, 55% in the 6
to 12 month dialysis group, and 50% in the 12 to 24 month
dialysis group and 48% in the patient group who had been on
dialysis for more than 24 months.
Relative risks for graft loss and 10-year adjusted graft
survival rates in all cadaveric versus living transplants by
ESRD time are displayed in Table 2. Preemptive cadaveric
transplants were assigned the reference group in the interaction model to evaluate the relative impact of waiting time
on cadaveric versus living transplants. Only living preemptive transplants did significantly better than preemptive cadaveric transplants (10-year adjusted graft survival rate of
75% vs. 69%, P⬍0.001). All other categories did significantly
worse. Living transplants performed on patients who had
been on dialysis up to 6 months were associated with a
significantly higher risk of graft loss (relative risk⫽1.4,
P⬍0.001) than preemptive cadaveric transplants with a projected 10-year graft survival of 62% versus 69%, respectively.
Cadaveric transplants performed after more than 2 years of
maintenance dialysis had the worst projected 10-year graft
survival of 39%.
The results of the Cox nonproportional hazards model that
investigated the relative benefit of transplantation versus
dialysis in patients on dialysis for at least 2 years is displayed in Figure 4. Of the 77,469 patients still on the waiting
FIGURE 2. Unadjusted graft survival in 56,587 recipients of
cadaveric transplants by length of dialysis treatment before
transplant.
FIGURE 3. Unadjusted graft survival in 21,836 recipients of
living transplants by length of dialysis treatment before
transplant.
list after 2 years, 15,414 eventually underwent cadaveric
renal transplantation whereas 61,055 remained on the waiting list until the study ended in June 1999. After 5 years,
cadaveric renal transplantation was associated with a relative risk of 0.58 (P⬍0.001) compared to patients who remained on the cadaveric renal transplant waiting list. The
evolution of the risk over time after cadaveric renal transplantation is displayed in Figure 4.
DISCUSSION
Our study demonstrates that waiting time on dialysis before transplantation is quantitatively one of the largest independent modifiable risk factors for graft loss after kidney
transplantation. By analyzing pairs of donor kidneys that
were transplanted in a recipient with short ESRD time and a
recipient with long ESRD time, we can effectively exclude
that part of the elevated risk for graft loss in the recipients
who had undergone dialysis for a prolonged time was a result
of donor characteristics not readily available from the
database.
Because of the national donor policy to share six-antigen–
matched kidneys regardless of waiting time and across organ
procurement organizations, more six-antigen matches can be
found in preemptive cadaveric transplants. To prevent this
potential bias in analyzing graft survival in the paired-kidney analysis, we excluded all kidney pairs of which any
kidney went to a six-antigen–matched recipient. After this
adjustment, the distribution of HLA matches was almost
identical between recipients who received transplants early
and those who received transplants late.
Although we excluded a donor selection bias, it is conceivable that the worse unadjusted graft survival in the long
ESRD time group was to a certain degree a result of higher
risk recipients. On the other hand, higher PRA and more
advanced recipient age are probably intrinsic characteristics
of the patients with prolonged waiting time. When adjusting
in the multivariate analysis for these risk factors, including
African American recipients, we still observed an absolute
difference of 12% worse graft survival in the long ESRD time
recipients at 5 years and 19% worse graft survival at 10
years. This translates into a 15% relative difference in graft
Page 222 of 290
1380
TRANSPLANTATION
Vol. 74, No. 10
TABLE 2. Adjusted overall 10-year graft survival in cadaveric compared to living donor recipients by ESRD timea
Cadaveric donor
Living donor
ESRD time
Preemptive
0–6 mo
6–12 mo
12–24 mo
⬎24 mo
RR (CI)
Graft survival
RR (CI)
Graft survival
1 (Ref)
1.9 (1.8–2.0)
2.0 (1.9–2.1)
2.3 (2.1–2.4)
2.5 (2.3–2.6)
69%
49%
47%
43%
39%
0.84 (0.7–0.9)
1.4 (1.2–1.5)
1.6 (1.5–1.8)
1.8 (1.6–1.9)
2.1 (1.9–2.3)
75%
62%
56%
54%
49%
a
Calculated from Cox model adjusting for donor and recipient demographics, HLA matching, cold ischemia time, and immunosuppressive
regimen.
RR, relative risk; CI, confidence interval; Ref, reference group.
FIGURE 4. Mortality risk of recipients of cadaveric renal
transplants vs. wait-listed patients with ESRD who were on
dialysis for at least 2 years.
survival at 5 years and an overwhelming 32% relative difference at 10 years. These numbers quantify the real affect of
length of ESRD time on graft survival and make ESRD time
the largest potentially modifiable risk factor for renal transplant outcomes.
The magnitude of the impact of ESRD time on outcomes is
also reflected by the multivariate model including all patients, showing a 44% worse 10-year graft survival in cadaveric renal transplant recipients on dialysis for more than 2
years. Even in living donated kidneys, in which a potential
donor selection bias is less likely, the overall adjusted 10year graft survival rate was 35% worse in recipients who had
been on dialysis for prolonged periods of time.
Note that the beneficial effect of a living transplant compared to a cadaveric transplant gradually fades when living
transplants are performed after the patients have spent prolonged times on dialysis. By analyzing ESRD time versus
transplant modality with an interaction term in the multivariate analysis, we were able to evaluate the relative benefit
of living transplantation versus waiting time on dialysis. The
10-year–adjusted living graft survivals for transplants after
more than 2 years of dialysis are similar to 10-year–adjusted
cadaveric graft survival for transplants performed within the
first 6 months of dialysis initiation. In fact, much of the
overall beneficial effects of living donation on graft survival
shown in literature (8, 9) seem to be attributable to the on
average shorter ESRD times in these patients. At any given
wait time, living donor recipients still have better graft survival rates than cadaveric donor recipients; however, this
effect is smaller than the affect of waiting time.
Of the basis of this data, waiting time on dialysis for a
kidney transplant should be considered when determining
the optimal choice of transplant type for a patient with near
ESRD. Also, on the basis of this data, a cadaveric kidney
transplant with an average waiting time of 2 years (U.S.
average) yields a 48% worse 10-year graft survival compared
to a preemptive living transplant. Obviously, waiting times
vary widely across the United States, and pertinent information in regard to the locally expected waiting time and the
resulting adjusted 10-year graft survival rates in living versus cadaveric transplantation can be obtained from Table 2.
Despite the worse outcomes after transplantation in patients who received transplants after prolonged times on
dialysis, the survival advantage of transplantation over dialysis was maintained even in the patients who had been on
dialysis for more than 2 years. This suggests that whatever
ongoing damage occurs to patients while they are on dialysis
may be halted after transplantation. In fact, the relative
long-term mortality benefit of transplantation over dialysis
in this cohort of patients with ESRD times more than 2 years
was similar to the survival benefit shown for the overall
cohort of transplant recipients published by Wolfe et al. (7).
The reason that an increased waiting time on dialysis is
associated with decreased graft and patient survival can not be
discerned from the data that we have presented. One possible
explanation may be that, while dialysis is clearly a life-saving
therapy, it is a less-than-perfect renal replacement modality
and, thus, the longer patients wait on dialysis for a transplant
the longer patients are exposed to the chronic effects of endstage renal failure and dialysis. It is well documented that
patients on dialysis have alterations in the concentration of a
number of substances (e.g., homocysteine, advanced glycosylation end products, and lipoproteins) that may predispose these
patients to both cardiovascular and renal allograft vascular
damages (10 –16). In addition, the poor nutrition, chronic inflammatory state, altered immunologic function, and inadequate clearance that often accompanies patients with ESRD on
dialysis (17, 18) may predispose these patients to poorer tolerance to the immunosuppressive agents after transplantation.
Therefore, patients on long-term dialysis may be at a disadvantaged state when they finally receive their transplant.
CONCLUSION
Transplant waiting time on dialysis is one of the strongest
independent modifiable risk factors for renal transplant outcomes. A large part of the advantage of living versus cadav-
Page 223 of 290
November 27, 2002
1381
BENITO ET AL.
eric transplantation may also be explained by this phenomenon. This effect is dominant enough that a cadaveric renal
transplant recipient with ESRD time less than 6 months has
the equivalent graft survival as living-donor transplant recipients who wait on dialysis for more than 2 years.
Organ allocation models geared toward improving outcomes in patients with ESRD will have to take into account
that changes in average waiting time are a major factor in
determining posttransplant graft and patient survival. Because waiting times are increasing as a result of the widening
gap between the increase in the demand for organs and the
increase in organ donations, improvements in cadaveric graft
survival seen over the past decade may be difficult to match
in the coming decade.
7.
8.
9.
10.
11.
12.
Acknowledgment. The authors thank Suzanne C. Johnson who
has helped with the editing and review of the paper.
13.
REFERENCES
1. Roake JA, Cahill AP, Gray CM, et al. Preemptive cadaveric renal transplantation: clinical outcome. Transplantation 1996; 62:1411-1416.
2. Asderakis A, Augustine T, Dyer P, et al. Pre-emptive kidney transplantation: the attractive alternative. Nephrol Dial Transplant 1998; 13:17991803.
3. Cosio FG, Alamir A, Yim S, et al. Patient survival after renal transplantation, I: the impact of dialysis pre-transplant. Kidney Int 1998; 53:767772.
4. Meier-Kriesche HU, Port FK, Ojo AO, et al. Effect of waiting time on renal
transplant outcome. Kidney Int 2000; 58:1311-1317.
5. Mange KC, Joffe MM, Feldman HI. Effect of the use or nonuse of long-term
dialysis on the subsequent survival of renal transplants from living
donors. N Engl J Med 2001; 344:726-731.
6. Mange KC, Cherikh WS, Maghirang J, et al. A comparison of the survival
14.
15.
16.
17.
18.
of shipped and locally transplanted cadaveric renal allografts. N Engl
J Med 2001; 345:1237-1242.
Wolfe RA, Ashby VB, Milford EL, et al. Comparison of mortality in all
patients on dialysis, patients on dialysis awaiting transplantation, and
recipients of a first cadaveric transplant. N Engl J Med 1999; 341:17251730.
Cecka JM. The UNOS Scientific Renal Transplant Registry: 2000. Clin
Transpl 2000:1-18.
Ojo AO, Port FK, Mauger EA, et al. Relative impact of donor type on renal
allograft survival in black and white recipients. Am J Kidney Dis 1995;
25:623-628.
Zimmermann J, Herrlinger S, Pruy A, et al. Inflammation enhances cardiovascular risk and mortality in hemodialysis patients. Kidney Int
1999; 55:648-658.
Lowrie EG. Acute-phase inflammatory process contributes to malnutrition, anemia, and possibly other abnormalities in dialysis patients.
Am J Kidney Dis 1998; 32:S105-S112.
Wanner C, Zimmermann J, Quaschning T, et al. Inflammation, dyslipidemia and vascular risk factors in hemodialysis patients. Kidney Int
Suppl 1997; 62:S53-S55.
Gris JC, Branger B, Vecina F, et al. Increased cardiovascular risk factors
and features of endothelial activation and dysfunction in dialyzed uremic patients. Kidney Int 1994; 46:807-813.
Degenhardt TP, Grass L, Reddy S, et al. The serum concentration of the
advanced glycation end- product N epsilon-(carboxymethyl)lysine is
increased in uremia. Kidney Int 1997; 52:1064-1067.
Hricik DE, Wu YC, Schulak A, et al. Disparate changes in plasma and
tissue pentosidine levels after kidney and kidney-pancreas transplantation. Clin Transplant 1996; 10:568-573.
Friedlander MA, Witko-Sarsat V, Nguyen AT, et al. The advanced glycation endproduct pentosidine and monocyte activation in uremia. Clin
Nephrol 1996; 45:379-382.
Kaufmann P, Smolle KH, Horina JH, et al. Impact of long-term hemodialysis on nutritional status in patients with end-stage renal failure.
Clin Invest Med 1994; 72:754-761.
Descamps-Latscha B, Herbelin A, Nguyen AT, et al. Immune system
dysregulation in uremia. Semin Nephrol 1994; 14:253-260.
0041-1337/02/7410-1381/0
TRANSPLANTATION
Copyright © 2002 by Lippincott Williams & Wilkins, Inc.
Vol. 74, 1381–1386, No. 10, November 27, 2002
Printed in U.S.A.
DIAGNOSIS AND TREATMENT OF LATENT TUBERCULOSIS
INFECTION IN LIVER TRANSPLANT RECIPIENTS IN AN
ENDEMIC AREA
NATIVIDAD BENITO,1,3 OMAR SUED,1 ASUNCIÓN MORENO,1 JUAN PABLO HORCAJADA,1 JULIÀ GONZÁLEZ,1
MIQUEL NAVASA,2 AND ANTONI RIMOLA2
Background. Treatment of latent tuberculosis infection (LTBI) with isoniazid is recommended for transplant recipients with positive tuberculin skin test
(TST). However, TST could be an imperfect identifier
1
Institut Clínic de Infeccions i Inmunologia, Hospital ClínicIDIBAPS Barcelona, University of Barcelona, Barcelona, Spain.
2
Institut Clínic de Malalties Digestives, Hospital Clínic-IDIBAPS
Barcelona, University of Barcelona, Barcelona, Spain.
3
Address correspondence to: Dr. N. de Benito, Infectious Disease
Service, Hospital Clínic, Villarroel, 170, 08036 Barcelona, Spain.
E-mail: [email protected].
Received 8 February 2002. Accepted 9 July 2002.
DOI: 10.1097/01.TP.0000034629.23838.5E
of LTBI in this population. In addition, the risk of
isoniazid hepatotoxicity could be high in liver transplant recipients (LTR). A retrospective cohort study
was performed to evaluate the diagnosis and treatment of LTBI in LTR.
Methods. Charts of all 547 patients who received primary liver transplantation at a University Hospital in
Spain between 1988 and 1998 were reviewed.
Results. TST was performed in 373 patients (71%)
before transplantation. The result was positive in 89
(24%). The median follow-up after transplantation was
49 months. None of the TST-positive patients developed tuberculosis (TB), but 5 out of 284 patients with
negative TST (1.76%) had active TB (Pⴝ0.6). Twenty-
Page 224 of 290
Mechanisms of Disease
Non-HLA transplantation immunity revealed by
lymphocytotoxic antibodies
Lancet 2005; 365: 1570–76
Gerhard Opelz for the Collaborative Transplant Study*
See Comment page 1522
*Centres listed at the end of the
paper
Department of Transplantation
Immunology, Institute of
Immunology, University of
Heidelberg, Im Neuenheimer
Feld 305, D-69120 Heidelberg,
Germany (Prof G Opelz MD)
Correspondence to:
Prof Gerhard Opelz
[email protected]
Summary
Background The presence of panel-reactive antibodies (PRA) against HLA antigens before transplantation is
associated with early rejection of kidney grafts from cadaver donors. Transplants from HLA-identical sibling donors
do not provide a target for antibodies to HLA antigens and should therefore not be affected by PRA.
Methods Data from the Collaborative Transplant Study were used to examine the influence of PRA on graft survival.
Uncensored graft survival and death-censored graft survival were calculated, and the data were analysed by
multivariate Cox’s regression methods.
Findings Among recipients of HLA-identical sibling transplants, 3001 patients with no PRA had significantly higher
10-year graft survival (72·4% [SE 1·1]) than 803 patients with 1–50% PRA (63·3% [2·5]; p=0·0006) or 244 patients
with more than 50% PRA (55·5% [4·0]; p⬍0·0001). The effect of PRA became apparent after the first post-transplant
year and was, therefore, strikingly different from the early steep decline in graft survival during the first year
associated with PRA in recipients of cadaver kidneys. We could not discern whether graft loss was a direct effect of
non-HLA humoral sensitisation or whether PRA served as an indicator of heightened immunity against non-HLA
transplantation antigens.
Interpretation PRA reactivity is strongly associated with long-term graft loss in kidney transplants from HLAidentical sibling donors.
Panel-reactive antibodies
Serum of a potential transplant
recipient is tested for reaction
with a panel of lymphocyte
suspensions, generally obtained
from random blood donors, in a
dye-exclusion assay. The
proportion of donors against
whose lymphocytes a serum
gives positive test reactions is
recorded as a general measure of
the patient’s state of
presensitisation.
Lymphocytotoxicity
Commonly referred to in clinical
transplant immunology as the
serological dye-exclusion assay.
When human lymphocytes are
incubated with antibodies
against HLA antigens in the
presence of rabbit complement,
the lymphocyte membrane is
damaged and rendered
permeable to eosin
(cytotoxicity). In the absence of
antibody, the cell membrane
remains intact (dye exclusion).
HLA
Human leucocyte antigens are
controlled by genetic loci located
on chromosome 6 and expressed
on all nucleated cells of the body.
Part of the major
histocompatibility complex
(MHC). Known to be influential
in transplantation of organs,
bone marrow, and stem cells.
1570
Relevance to practice Our findings suggest that non-HLA immunity has a much stronger role in clinical
transplantation than previously thought. In contrast to immunity against HLA mediated by antibodies present
before transplantation, which leads to early acute graft rejection, non-HLA immunity is associated with chronic graft
loss. The possibility of identifying recipients at increased risk of late graft loss before transplantation could be used
to devise specific immunosuppressive strategies for these patients.
Introduction
Terasaki and colleagues first reported 30 years ago that
kidney-transplant recipients whose serum contained
lymphocytotoxic antibodies before transplantation were
at increased risk of graft rejection.1 Their finding was
confirmed in many subsequent studies, and now
patients awaiting renal transplantation are routinely
tested for lymphocytotoxic panel-reactive antibodies
(PRA).2 The lymphocytotoxicity assay has certain
drawbacks. There is no binding convention about the
size of the cell panel used for testing, although most
laboratories use commercial kits with frozen
lymphocytes from 56 random donors. There is general
acceptance that the results of the test system are
suboptimally reproducible. Nevertheless, risk of
rejection appears to rise as serum reactivity against the
random lymphocyte test panel increases.3,4 PRA-positive
serum samples contain antibodies against HLA antigens
on lymphocytes,5,6 and graft survival in preimmunised
recipients is assumed to be lower as the result of
insufficient sensitivity in the pretransplant lymphocytotoxic cross-match test against donor lymphocytes.
Much effort has therefore been spent on increasing the
sensitivity of the cross-match assay so that weak antiHLA sensitisation can be detected,7,8 and the use of new
techniques for pretransplant antibody testing based on
highly sensitive, strictly HLA-specific ELISAs has been
encouraged.9–11 Patients cannot form antibodies against
their own HLA antigens; therefore they cannot form
anti-HLA against lymphocytes of an HLA-identical
sibling donor. In distinction from genetically identical
twins, who share all genes and therefore do not require
immunosuppression when tissues are transplanted
between them, the common definition of HLA-identical
siblings is that they are matched for both HLA
chromosomes but mismatched at other chromosomes;
thus, they can be of different sex, eye colour, and so on,
as well as age. Since HLA chromosomes are inherited
according to mendelian rules, the likelihood that two
siblings will inherit identical HLA chromosomes from
their parents is 25%. Although many other factors
influence the outcome of kidney transplantation,
transplants from HLA-identical sibling donors are
recognised as a special category. They have significantly
better success rates than transplants from HLAmismatched donors,12 and they are the standard against
which the results of transplantation from other donor
sources are compared. However, since rejection of HLAidentical sibling grafts commonly occurs if no
immunosuppression is given, these recipients are
www.thelancet.com Vol 365 April 30, 2005
Page 225 of 290
Mechanisms of Disease
treated with immunosuppressive drugs, albeit at lower
doses than recipients of grafts from cadaver donors. This
need for immunosuppressive treatment shows that,
aside from HLA, there must be other antigen systems
that can cause transplant rejection. HLA-identical
sibling transplants do not provide a target for anti-HLA,
and PRA reactivity before transplantation should
therefore not influence their success rate.
We studied the influence of pretransplant PRA status
on the long-term outcome of kidney grafts from HLAidentical sibling donors.
reactivity.15 In the analysis of graft survival, all graft
failures irrespective of cause (including death of the
patient) were counted as failures. In the analysis of
functional graft survival, deaths were censored. The
Kruskal-Wallis and Mantel-Haenszel tests were used to
estimate the statistical association between PRA
reactivity and number of pretransplant blood
transfusions, recipient’s sex, and proportion of
retransplants. Immunosuppressive treatment (analysed
by intention to treat) of patients with transplants from
HLA-identical sibling donors included ciclosporin in
Methods
Cadaver kidney transplants
Patients
100
Kidney transplants reported by 245 centres to the
international Collaborative Transplant Study13 were
analysed. Transplants were carried out between 1982,
the year the study was initiated, and 2002. The centres
included in this analysis provided written assurance of
compliance with local ethical and consent guidelines
and of patients’ consent for the use of data for scientific
analysis. When consent from patients could not be
obtained, care was taken to ensure that all data
processing was carried out anonymously.
p⬍0·0001
Grafts surviving (%)
90
80
70
No PRA
1–50% PRA
⬎50% PRA
60
50
40
Procedures
0
0
3
12
116 562
103 234
99 382
95 688
94 252
1–50% PRA
36 314
31 155
29 889
28 827
28 288
⬎50% PRA
7610
6019
5724
5473
5349
9
12
No PRA
HLA-identical sibling transplants
100
90
p=0·0831
80
70
60
50
40
0
0
www.thelancet.com Vol 365 April 30, 2005
9
Number of
transplants
3
Statistical analysis
Graft survival was calculated by Kaplan-Meier analysis.14
Statistical significance was assessed with the log rank
test. Transplant outcome in relation to pretransplant
antibody status was analysed by weighted regression
analysis, in which the dependent variable was survival
proportion, the independent variable was the degree of
antibody reactivity, and the weight factor was the
number of patients who had the given antibody
6
Time (months)
Grafts surviving (%)
A transplant was classed as HLA-identical if recipient
and donor were reported to have identical HLA A, B,
and DR antigens. 3681 first transplants and
367 retransplants from HLA-identical sibling donors
formed the main study population for this analysis.
160 486 cadaver-donor transplants were analysed for
comparison. HLA typing and testing for PRA was done
at participating laboratories and reported to the study
centre. The PRA reactivity of the last pretransplant
serum sample was analysed. For 16 patients (five with
no PRA, five with 1–50% PRA, and six with more than
50% PRA), positive pretransplant lymphocytotoxic crossmatches against lymphocytes of the kidney donor were
reported, whereas all other patients had negative crossmatch results. All 16 patients with positive anti-donor
cross-matches also had positive cross-match results
against their own (autologous) lymphocytes, indicating
that the positive cross-matches were the result of
autoantibodies.
Clinical follow-up was recorded at 3 months,
6 months, and 12 months, and yearly thereafter.
6
Time (months)
Number of
transplants
3001
2914
2864
2774
2765
1–50% PRA
803
774
766
744
741
⬎50% PRA
244
235
229
223
215
No PRA
Figure 1: 1-year graft survival analysis of kidney transplants from cadaver
donors or HLA-identical sibling donors in relation to PRA
1571
Page 226 of 290
Mechanisms of Disease
2712 (67%) of the patients, tacrolimus in 122 (3%), and
regimens without calcineurin inhibitors in 1214 (30%);
there were no significant differences in success rates
between the groups treated with these regimens.
Multivariate Cox’s regression analysis16 was used to
ascertain the effect of the covariates: transplant number
(first or retransplant); year of transplantation;
immunosuppressive regimen (calcineurin inhibitor or
not); age, sex, and race of recipient and donor; original
disease leading to endstage renal failure; number of
pretransplant blood transfusions; and geographical
location of the transplant centre (continent). The
software packages SPSS (version 11.5) and SAS (version
8.2) were used.
Role of the funding source
No source of funding had any role in study design;
collection, analysis, or interpretation of data; or in the
writing of the report. The corresponding author had full
access to all the data in the study and had final
responsibility for the decision to submit for publication.
Results
The differential effect of PRA on survival of transplants
from cadaver donors or HLA-identical sibling donors
during the first year after transplantation is shown in
figure 1. As expected, a significant effect of PRA on graft
survival was evident in cadaver transplants (p⬍0·0001),
but no significant effect was noted in transplants from
HLA-identical sibling donors (p=0·0831). PRA
reactivity in cadaver-transplant recipients was
associated with immunological graft loss rather than
death of the patient. The calculation of death-censored
functional graft survival, which provides an
approximation of the rate of immunological graft
rejection, gave almost the same result as that shown in
figure 1 (p⬍0·0001). By contrast, PRA in HLA-identical
sibling transplants affected neither graft nor functional
survival.
When the analysis was extended to 10 years of followup, however, a significant effect of PRA on graft survival
became apparent in the analysis of HLA-identical
sibling transplants (figure 2). At 10 years, the
proportion of grafts surviving was 72·4% (SE 1·1) in
patients with no PRA, 63·3% (2·5) in those with 1–50%
PRA, and 55·5% (4·0) in those with more than 50%
PRA (regression p⬍0·0001). The result was significant
in both first transplants (p=0·0002) and retransplants
(p=0·0001), and subset analysis showed significant
associations for the transplant periods 1982–90
(p=0·0003) and 1991–2002 (p=0·005). Analysis of
death-censored functional survival showed that the PRA
effect was due to graft loss and not death of patients.
The 10-year functional graft survival proportions were
82·5% (1·0), 75·3% (2·2), and 63·1% (4·0), for patients
with no PRA, 1–50% PRA, and more than 50% PRA,
respectively (p⬍0·0001; figure 3). The proportions of
patients surviving at 10 years were 86·2% (0·9), 81·7%
(2·0), and 81·9% (3·1) respectively (p=0·0266; data not
shown). Multivariate Cox’s regression analysis showed
that, compared with patients who were PRA-negative
before transplantation, recipients with 1–50% PRA had
a significantly increased risk of graft loss (relative risk
1·29 [95% CI 1·09–1·53], p=0·0033) and the risk was
even higher in patients with more than 50% PRA (1·87
[1·47–2·37], p⬍0·0001). In the analysis of functional
100
90
90
Functional grafts surviving (%)
100
Grafts surviving (%)
80
No PRA
70
1–50% PRA
60
p⬍0·0001
⬎50% PRA
50
1–50% PRA
70
60
⬎50% PRA
p⬍0·0001
50
40
40
0
0
0
2
4
6
Time (years)
8
10
0
2
4
6
Time (years)
8
10
Number of
transplants
Number of
transplants
3001
2495
1929
1418
989
687
No PRA
3001
2495
1929
1418
989
687
1–50% PRA
803
647
514
362
249
158
1–50% PRA
803
647
514
362
249
158
⬎50% PRA
244
192
149
111
84
65
⬎50% PRA
244
192
149
111
84
65
No PRA
Figure 2: 10-year follow-up of kidney grafts from HLA-identical sibling
donors
1572
No PRA
80
Figure 3: Death-censored functional graft survival of HLA-identical sibling
transplants
www.thelancet.com Vol 365 April 30, 2005
Page 227 of 290
Mechanisms of Disease
analysis of graft survival for the period after the first
post-transplant year, when grafts subject to early antiHLA immunity had already been rejected (figure 1).
Indeed, when the graft-survival analysis was restarted at
100% at 1 year after transplantation, a long-term effect of
PRA became apparent in cadaver transplants (figure 5).
We ruled out the possibility that the degree of HLA
matching, which is known to affect long-term survival of
cadaver transplants,12 might have caused the separation
of the survival curves. The mean number of HLA A, B,
Cadaver kidney transplants
100
90
p⬍0·0001
80
Grafts surviving (%)
graft survival, the risk was significantly increased for
patients with 1–50% PRA (1·26 [1·02–1·57], p=0·0282),
and the result was highly significant for those with
more than 50% PRA (2·23 [1·70–2·93], p⬍0·0001).
The long-term evolution of the success of cadaver and
HLA-identical sibling transplants differed substantially.
PRA affected the survival proportion of cadaver-donor
transplants primarily in the first few months after
transplantation (figure 4). By contrast, transplants from
HLA-identical siblings were not affected during the first
year and the influence of PRA developed continuously
during the 10-year follow-up, which suggests that
different mechanisms were involved (figure 2).
Owing to the intrafamilial pathway of inheritance of
HLA chromosomes, the definition of HLA-matched
transplants is not difficult in sibling settings. However,
owing to the complexity of the HLA system and
imperfect characterisation of HLA alleles in clinical
typing for renal transplantation, identification of
perfectly matched transplants in a registry series of
cadaver kidney donors is impossible. At the level of
allelic definition of HLA antigens,17 almost all cadaver
transplants included in this analysis must be deemed
HLA mismatched. With the available data, an analysis of
perfectly matched cadaver transplants was therefore not
possible. Nevertheless, one would assume that the nonHLA effect of PRA described in HLA-identical sibling
grafts must also have a role in HLA-mismatched cadaver
transplants, in addition to the anti-HLA effect shown in
figure 1. We addressed this issue by doing a separate
70
No PRA
1–50% PRA
⬎50% PRA
60
50
40
0
0
2
4
6
8
10
Time (years)
Number of
transplants
No PRA
88 389 83 720 62 516
44 887 30 819
1–50% PRA
26 676 25 005 18 402
12 842
8590
5586
2579
1817
1242
⬎50% PRA
5075
4712
3582
20 674
100
HLA-identical sibling transplants
90
100
90
70
80
Grafts surviving (%)
Grafts surviving (%)
80
60
50
p⬍0·0001
No PRA
1–50% PRA
40
⬎50% PRA
70
p⬍0·0001
60
50
0
0
2
4
6
8
40
10
Time (years)
0
Number of
transplants
No PRA
1–50% PRA
⬎50% PRA
0
2
11 6562 83 720 62 516 44 887 30 819 20 674
36 314 25 005 18 402 12 842
7610
4712
3582
2579
8590
5586
1817
1242
Figure 4: Long-term (10-year) survival of cadaver kidney transplants
according to pretransplant PRA
Note the early separation of survival curves in the analysis of cadaver
transplants, in contrast to the late separation in the analysis of HLA-identical
sibling grafts (figure 2).
www.thelancet.com Vol 365 April 30, 2005
4
6
8
10
Time (years)
Number of
transplants
2539
2495
1929
1418
989
687
1–50% PRA
664
647
514
362
249
158
⬎50% PRA
199
192
149
111
84
65
No PRA
Figure 5: Analysis of long-term effect of PRA starting at 1 year after
transplantation
1573
Page 228 of 290
Mechanisms of Disease
Characteristic
Preformed PRA
None (n=3001)
Proportion female
1014 (34%)
Proportion with retransplant
159 (5%)
Mean (SE) pretransplant blood transfusions
3·47 (0·15)
p
1–50% (n=803)
⬎50% (n=244)
361 (45%)
112 (14%)
6·01 (0·43)
154 (63%)
97 (40%)
10·7 (1·12)
⬍0·0001
⬍0·0001
⬍0·0001
Table: Association of preformed PRA with various characteristics of patients
DR mismatches was similar in the groups with no PRA
and 1–50% PRA (2·8 [SD 1·4] and 2·9 [1·4]), and
recipients with more than 50% PRA even showed a
significantly lower number of mismatches (2·3 [1·5])
than the other PRA groups (p⬍0·0001).
Lymphocytotoxic antibodies in potential transplant
recipients can be autoreactive in some cases. Such
autoantibodies are believed to be the result of
autoimmune processes in the recipient; they are
commonly of the IgM subclass and are not generally
associated with graft rejection.18,19 All 16 patients with
positive pretransplant cross-match results had
autoreactive antibodies. 14 of these patients had
functioning grafts at 10 years, which shows that the
antibodies were not involved in rejection. By contrast,
antibodies to HLA antigens are mostly of the IgG class
and the result of alloimmunisation in response to
immunological challenge by pregnancy, blood
transfusions, or rejection of a previous transplant. Our
data suggest that the antibodies associated with late graft
rejection in HLA-identical sibling transplants were
alloantibodies. Although data on previous pregnancies
were not available, we found significant associations of
PRA reactivity with female sex and thus the possibility of
pregnancy (p⬍0·0001), the number of pretransplant
blood transfusions (p⬍0·0001), and the loss of a
previous
non-HLA-identical
kidney
transplant
(p⬍0·0001; table).
Discussion
Minor histocompatibility
antigens
These antigens, less well defined
than HLA, controlled by genes on
other chromosomes, determine
immunogenic epitopes able to
elicit a T-cell response against
transplanted cells or tissue.
Influential in marrow/stem-cell
transplantation but until now no
proven influence on organtransplant survival.
1574
This study showed a highly significant association
between the presence of lymphocytotoxic antibodies
before transplantation and the outcome of kidney grafts
from cadaver donors and HLA-identical sibling donors.
Such an association was previously known in cadaver
kidney transplantation, in which it has been attributed to
unrecognised antibody reactivity against mismatched
HLA antigens. However, the finding of a similar
association for transplantation of organs between HLAidentical siblings was unexpected because the HLAantigen profile of recipient and donor is identical. In this
special setting, no effect would be expected because PRA
reactivity is generally believed to be directed against
HLA antigens. The presence of weakly reactive
antibodies to HLA antigens that went undetected in the
pretransplant cross-match assay cannot explain the
lower graft success rate observed in recipients of
transplants from HLA-identical siblings. An association
of graft rejection with the presence of lymphocytoxic
antibodies before transplantation has been noted
previously in bone-marrow transplantation from HLAidentical sibling donors.20
Apart from the main issue that antibodies to HLA
antigens should not influence the outcome of
transplants between HLA-identical siblings, the
differing evolution of the PRA effect on graft survival
suggests that cadaver and HLA-identical-sibling
transplants were affected by different types of antibodies
or different mechanisms of immunological rejection.
The result for cadaver transplants is compatible with the
concept that HLA antibodies present in the recipient’s
circulation at the time of transplantation reacted with
mismatched HLA antigens on donor tissue. These
antibody reactions were probably weak, because they
went undetected in the pretransplant cross-match assay
and did not lead to immediate graft failure from
hyperacute rejection. Irreversible rejection, however,
occurred within a few weeks or months as shown by the
early decline of graft survival curves in presensitised
patients (figure 1). The survival curves for HLAidentical-sibling transplants, by contrast, declined very
slowly, indicating a much later immunological event
(figure 2). This differentiation in early and late graft loss
also argues against the unlikely possibility that graft
failure in the sibling group might have been due to
incorrect HLA typing; had HLA mismatches been
present among the HLA-identical siblings, early graft
losses as a result of antibody-mediated rejection would
have been expected. All sibling donors and recipients
analysed in this study were identical at the HLA A, B,
and DR loci, the histocompatibility loci established to be
influential in clinical kidney transplantation. Although
the presence of incompatibilities at other loci within the
HLA region (eg, DQ, DP) cannot be excluded, the
likelihood that such incompatibilities existed can be
estimated at less than 3%. Thus, the chance that the
results of this study were influenced to an important
extent is very small.
The targets for antibodies causing late rejections could
be so-called minor histocompatibility antigens, which are
not coded for in the HLA genetic region and have been
shown to influence the outcome of bone-marrow
transplants.21 Antibodies against these antigens might
not lead to acute rejection of kidney grafts but to
protracted chronic rejection. Since decreased graft
survival was associated with PRA reactivity, two possible
hypotheses
are
that
antibodies
to
minor
histocompatibility antigens occur frequently together
with anti-HLA, or that cross-reactivity of anti-HLA with
epitopes on minor histocompatibility antigens might
induce late graft rejection.
Another possibility is that PRA reactivity does not
signal the existence of antibodies against minor
histocompatibility antigens but serves as an indicator of
a generally increased state of immune responsiveness
due to allogeneic preimmunisation, and that
www.thelancet.com Vol 365 April 30, 2005
Page 229 of 290
Mechanisms of Disease
incompatibilities for minor histocompatibility antigens
might exert a strong but protracted graft-damaging
influence in patients with heightened immunity. Aside
from acute rejections of cadaver kidneys mediated by
antibodies to HLA antigens, delayed rejections would
not be directly mediated by the antibodies detected in
pretransplant serum, thus explaining the differential
time profiles of rejection due to “major” HLA
incompatibilities (cadaver transplants with HLA
mismatches) or “minor” non-HLA incompatibilities (a
proportion of HLA-identical-sibling and cadaver
transplants). In cadaver-transplant recipients with
heightened immunological reactivity against both major
and minor incompatibilities, the grafts would fail early.
The finding that a substantial proportion of sibling
transplants failed even in the absence of any detectable
antibody reactivity (figure 2) shows that the process
described here is the cause of only some, not all, graft
failures.
The results presented here have important
fundamental and practical implications. The study has
shown
that
non-HLA
immunity
contributes
substantially to long-term kidney-transplant failure.
When the long-term results for kidney recipients with
PRA were examined over 10 years of follow-up, the
influence of non-HLA-directed immunity was of similar
magnitude to that of antibodies against HLA (figures 2
and 4). The study shows the importance of
characterising the non-HLA antigens that bring about
graft loss. However, because it was based on registry
data, serum and cells from recipients and donors are not
available for in-vitro studies. Prospective collection of
biological material from donors and recipients of HLAidentical sibling transplants will be needed for further
progress on this issue. If future research is successful,
many of the late graft failures attributable to non-HLA
effects might be avoidable. For the time being, the fact
that HLA-identical siblings at increased risk of late graft
loss can be identified before transplantation could be
used to devise specific immunosuppressive strategies
for these patients.
Another interesting point arising from these results is
the implication that the current worldwide trend towards
replacement of pretransplant antibody testing in the
complement-dependent lymphocytotoxicity assay with
strictly HLA-specific ELISA testing might be
counterproductive. ELISA assays have the advantage of
better reproducibility than the complement-dependent
cytotoxicity method, and ELISA testing at increased
sensitivity with strict anti-HLA specificity is widely
expected to lead to better clinical transplant results.9–11
Direct comparison of transplant outcome in relation to
pretransplant antibody testing with lymphocytotoxicity
or HLA-specific ELISA, however, did not show a
convincing advantage for either method and provided
evidence that some serum samples contained ELISAnon-reactive antibodies that were associated with kidney
www.thelancet.com Vol 365 April 30, 2005
graft rejection.22 The results of this study suggest that an
important part of the antibody range might be missed if
pretransplant serum testing were limited to HLAspecific ELISA. Although the introduction of ELISA
techniques has greatly improved ability to characterise
antibodies to HLA, the additional non-HLA reactions
detected in the classic lymphocytotoxicity assay seem to
be clinically highly relevant. Although the exact
immunological mechanisms involved remain to be
discovered, caution should be used in modifying
pretransplant screening procedures without further
knowledge about the effect of lymphocytotoxic
antibodies on long-term outcome. For the time being,
use of both ELISA and cytotoxicity assays in parallel for
pretransplant testing seems wise to allow a separation of
anti-HLA from anti-non-HLA activities. Our results also
suggest that the introduction of highly sensitive, strictly
HLA-specific ELISA-based pretransplant cross-match
assays23 has only a limited potential for improving
transplant outcome. Although these tests can be
expected to lower further the incidence of HLA-antibodymediated rejections, they will not affect the substantial
rate of late rejections attributable to immunity unrelated
to HLA.
Contributors
Gerhard Opelz initiated and coordinated the study and wrote the report.
Participating centres
Argentina—Buenos Aires (3); Mar del Plata; Rosario. Australia—
Adelaide; Brisbane; Heidelberg; Hobart; Melbourne; Newcastle; Perth;
Sydney (7). Austria—Graz; Innsbruck; Linz; Vienna. Belgium—Leuven
(2); Liege. Brazil—Belo Horizonte (2); Maceio; Pato Branco; Porto
Alegre (2); Ribeirao Preto; Rio de Janeiro (2); Sao Paulo (4). Canada—
Quebec; Toronto; Vancouver; Winnipeg. Chile—Santiago (2); Valdivia.
Colombia—Medellin. Croatia—Rijeka; Zagreb. Egypt—Cairo (2);
Mansoura. Finland—Helsinki. France—Lille; Lyon; Nancy; Nantes;
Paris; Poitiers; Reims; Rennes; St Etienne; Toulouse. Germany—
Aachen; Augsburg; Berlin (2); Bochum; Bonn; Bremen; Cologne (2);
Düsseldorf; Erlangen; Frankfurt; Freiburg; Fulda; Giessen; Göttingen;
Halle; Hann-Muenden; Hannover; Heidelberg; Homburg-Saar; Jena
(2); Kaiserslautern; Kiel; Lübeck; Mainz; Mannheim; Marburg; Münster;
Munich; Regensburg; Rostock; Stuttgart; Tübingen; Ulm; Würzburg.
Greece—Thessaloniki. Hong Kong—Hong Kong (8). Hungary—
Budapest; Debrecen; Pecs; Szeged. India—New Delhi. Iran—Shiraz;
Tehran. Ireland—Dublin. Israel—Petach Tikva. Italy—Brescia; Cagliari;
Florence; Genova (2); Milan (4); Padova (2); Treviso; Turin; Udine;
Varese; Verona; Vicenza. Korea—Seoul. Lithuania—Kaunas; Vilnius.
Mexico—Guadalajara (2); Mexico City. Netherlands—Nijmegen. New
Zealand—Auckland; Christchurch; Hamilton; Wellington. Pakistan—
Islamabad; Karachi. Peru—Lima. Philippines—Manila. Poland—
Katowice. Russia—Moscow. Slovakia—Martin. Slovenia—Ljubljana.
South Africa—Cape Town (5). Spain—Badalona; Barcelona (6); Madrid
(5); Oviedo; Pamplona; Santander; Valencia (3). Sweden—Goteborg;
Malmo-Lund; Uppsala. Switzerland—Basel; Bern; Geneva; Lausanne; St
Gallen; Zurich. Turkey—Ankara (2); Antalya; Izmir (2). UK—Aberdeen;
Belfast; Birmingham; Brighton; Bristol; Cambridge; Cardiff; Carshalton;
Coventry; Dresden; Dundee; Edinburgh; Glasgow (2); Kent; Leeds;
Leicester; Liverpool; London (9); Manchester; Newcastle upon Tyne;
Nottingham; Oxford; Plymouth; Portsmouth; Sheffield. USA—
Cincinnati; Dallas (3); Grand Rapids; Kansas City (3); New Orleans;
New York (2); Omaha; Orlando; Portland (2); Shreveport; Stanford;
Valhalla. Venezuela—Caracas; Maracaibo.
Conflict of interest statement
I declare that I have no conflict of interest.
1575
Page 230 of 290
Mechanisms of Disease
Acknowledgments
I thank staff members at 245 transplant centres who provided data for
this study for their generous support, and Bernd Doehler for valuable
assistance with statistical analysis.
References
1
Terasaki PI, Mickey MR, Kreisler M. Presensitization and kidney
transplant failure. Postgrad Med J 1971; 47: 89–92.
2
Terasaki PI, McClelland JD. Microdroplet assay of human serum
cytotoxins. Nature 1964; 204: 998–1000.
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Opelz G, for the Collaborative Transplant Study. Kidney
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4
Katznelson S, Bhaduri S, Cecka MJ. Clinical aspects of
sensitization. In: Cecka JM, Terasaki PI, eds. Clinical transplants
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Fuller TC, Fuller AA, Golden M, Rodey GE. HLA alloantibodies
and the mechanism of the antiglobulin-augmented
lymphocytotoxicity procedure. Hum Immunol 1997; 56: 94–105.
6
Rodey GE, Neylan JF, Whelchel JD, Revels KW, Bray RA. Epitope
specificity of HLA class I alloantibodies: I, frequency analysis of
antibodies to private versus public specificities in potential
transplant recipients. Hum Immunol 1994; 39: 272–80.
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Pettaway CA, Freeman CC, Helderman HJ, Stastny P. Kidney
transplant recipients with long incubation-positive, antiglobulinnegative T-cell crossmatches. Transplantation 1987; 44: 529–33.
8
Talbot D, Givan AL, Shenton BK, et al. The relevance of a more
sensitive crossmatch assay to renal transplantation. Transplantation
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9
Kao KJ, Scornik JC, Small SJ. Enzyme-linked immunoassay for
anti-HLA antibodies: an alternative to panel studies by
lymphocytoxicity. Transplantation 1993; 55: 192–99.
10 Zachary AA, Ratner LE, Graziani JA, et al. Characterization of HLA
class I specific antibodies by ELISA using solubilized antigen
targets: II, clinical relevance. Hum Immunol 2001; 62: 236–46.
11 Pei R, Lee JH, Shih NJ, Chen M, Terasaki PI. Single human
leukocyte antigen flow cytometry beads for accurate identification
of human leukocyte antibody specificities. Transplantation 2003; 75:
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Opelz G, Wujciak T, Döhler B, Scherer S, Mytilineos J. HLA
compatibility and organ graft survival. Rev Immunogenet 1992; 1:
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Cox DR. Regression models and life-tables. J R Stat Soc (B) 1972;
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Marsh SGE, Albert ED, Bodmer WF, et al. Nomenclature for
factors of the HLA system. Tissue Antigens 2002; 60: 407–64.
Barger CF, Shroyer TW, Hudson SL, et al. Successful renal
allografts in recipients with cross-match-positive, dithioerythrioltreated negative sera. Transplantation 1989; 47: 240–44.
Bryan CF, Martinez, J, Muruve N, et al. IgM antibodies identified
by a DTT-ameliorated positive cross-match do not influence renal
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Süsal C, Opelz G. Kidney graft failure and presensitization against
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Page 231 of 290
Copyright
American Journal of Transplantation 2003; 3: 665–673
Blackwell Munksgaard
#
Blackwell Munksgaard 2003
ISSN 1600-6135
Special Article
Humoral Theory of Transplantation
Paul I. Terasaki*
Terasaki Foundation Laboratory, Los Angeles, CA
*Corresponding author: Dr Paul I. Terasaki,
[email protected]
According to the humoral theory of transplantation,
antibodies cause allograft rejection. Publications are
cited showing that antibodies: (1) cause hyperacute
kidney rejection, (2) lead to C4d deposits associated
with early kidney graft failures, (3) are a good indicator
of presensitization leading to early acute rejections,
(4) were present in 96% of 826 patients who rejected a
kidney graft, (5) are associated with chronic rejection
in 33 studies of kidney, heart, lung and liver grafts,
and (6) in three studies, appeared in the circulation
BEFORE evidence of bronchiolitis obliterans in lung
transplants, and BEFORE kidney rejection. In addition,
a prospective cooperative study of 1629 patients in 24
centers demonstrated that antibodies foretold subsequent failures after a follow-up period of 6 months
(p = 0.05). The specificity of antibodies detected in
the serum of rejecting patients were often not donor
specific, presumably because they were absorbed by
the rejecting organ.
If the humoral theory is accepted, even provisionally,
transplanted patients who have antibodies could be
treated with immunosuppression until the antibodies
disappear to determine whether chronic rejection can
be blocked. If successful, in patients who do not have
antibodies, immunosuppression could be reduced
until antibodies appear.
Received 13 November 2002, revised 26 December
2002 and accepted for publication 13 February 2003
Sir Peter Medawar. Another possible reason we do not
hear much of the debate today is that we now ‘KNOW’
that BOTH humoral and cellular mechanisms are involved
in rejection. But is this so?
Of course, antibodies are produced by cells, and in this
sense all rejections are cellular. The critical difference
between the two theories is: (a) are grafts destroyed by
the action of antibodies or (b) by direct cellular cytotoxicity,
as occurs with cytotoxic T cells, NK cells, or DTH reactions.
I will review the accumulating evidence that HLA antibodies can directly cause allograft rejection in humans. This
is not intended to be a ‘balanced’ review, and assumes
that the reader has had sufficient exposure to the abundant literature showing that rejection is produced by cells,
such as DTH reactions and tubulitis lesions produced by
T cells in kidney transplants. Various current immunosuppressive treatments may have successfully suppressed
cellular immunity, leaving humoral reactivity to be dealt
with in the remaining patients.
Gorer first showed that various antibodies in mice could
be detected against the H-2 histocompatibility locus antigens (1). Based on these studies, a large-scale international effort with extensive collaboration in international
workshops resulted in the discovery of antibodies to
HLA antigens in humans. Thus, acceptance of the humoral
theory led to uncovering the antigens against which allograft reaction is directed. Admittedly, some pursued this
line of research purely as a scientific endeavor, but my
work was based from the outset on the idea that the
antibodies are the visible indicators of the transplantation
antigens, and the means by which we can get to the root
of the problem.
Introduction
In the 1950s, the important question of the day was, ‘are
grafts rejected by antibodies (humoral theory) or by cells?’
We do not hear much about this debate today, because
for the past 40 years it has almost been an accepted fact
that the cellular theory is correct. This conclusion was
reached by the sheer force of one man’s personality, for
few would take exception to the overpowering dogma of
Conflict of interest: I am a major shareholder and Chairman of
One Lambda, one of the companies that sells antibody testing
kits. My publications in the humoral theory predate the formation
of the company by 25 years.
HLA antibodies instantly kill a kidney:
hyperacute rejection
The awesome power of HLA antibodies became apparent
with the finding that when a kidney graft is transplanted
into a patient with HLA antibodies directed against antigens of the kidney, the kidney is killed immediately (2).
Thus, an entire organ such as a kidney could be destroyed
by antibodies within minutes. Occasionally, not often, HLA
antibodies directed against B cells produce hyperacute
rejection (3). The particular susceptibility of the lung to
antibody-mediated hyperacute rejections is apparent
665
Page 232 of 290
Terasaki
ence or absence of cytotoxic antibodies (13) (Figure 1).
Patients sensitized before transplantation had lower graft
survival rates than nonsensitized patients. Moreover, this
effect was even more marked in patients who had previously rejected a transplant. In the intervening 30 years,
many publications have reconfirmed the fact that antibodies, as a measure of presensitization, are important
for kidney (14–26), heart (27–30), liver (31–34) and lung (5)
transplants.
from recent experiences (4,5). Hearts are also susceptible
to hyperacute rejection (6).
When the kidney is rejected minutes before closure of
the incision, hyperacute rejection is obvious. However,
we called attention to hidden hyperacute rejection when
the kidney is rejected after the wound is closed, and the
kidney never functions. In 1987, the primary nonfunction
rate of the first cadaver grafts was 8% in 7788 first grafts,
14% in 1471 second grafts, and 20% in 224 third grafts
(7). This large difference in primary nonfunction rate was
almost certainly the result of hidden hyperacute rejection
following sensitization by graft rejection. Today, with
improved crossmatching methods, this large difference in
primary nonfunction has been eliminated, and graft survival
in first and regrafted patients is virtually identical (8).
HLA antibodies are associated with acute
early rejection
For many years, antibodies were sought in biopsies taken
during acute rejections, searching by immunofluorescence
for IgG or IgM antibodies, generally to no avail. A major
break occurred in 1993 when Feucht demonstrated that
an end-product of complement C4d could be demonstrated in peri-tubular capillaries (35). Feucht showed
C4d deposits in 51 of 93 dysfunctional grafts. Among
patients with c4d, 1-year survival was 57% compared
with 90% in those without C4d. Eight years later the
same group provided convincing proof of antibodies producing early graft failures (36). They showed that presence
of C4d in 117 grafts led to significantly lower graft survival
than in 101 grafts without C4d (p ¼ 0.0001) (Figure 2).
Antibodies to lymphoblastoid cell lines of the donor DR
type were found together with C4d in 14 of 18 patients.
Among those patients without antibodies, 11 of 30 had
C4d (p ¼ 0.008), suggesting that C4d detection was a
more sensitive indicator of antibodies than antibody detection in the circulation.
The extraordinary power of antibodies is shown when
high-titered HLA antibodies transfused intravenously can
even kill a patient within hours. The phenomenon of
transfusion-related acute lung injury (TRALI) produced by
sera from highly immunized pregnant women was first
described in 1970 (9), and reviewed in 1985 (10) and
2001 (11).
State of preimmunization is detected by
HLA antibodies
When patients are immunized by the process of rejecting
an allograft, the only current practical way to detect this
state of immunization is by a humoral (not cellular) test:
that is, examining patients for the presence of HLA antibodies. Such antibodies are found in pregnant women,
patients immunized by transfusions, those immunized by
rejection of a previous graft, and patients with cadaveric
venous and arterial allografts (12). Proof that a humoral
test for sensitization is effective was first provided by
survival curves in patients classified according to the pres-
100
A flurry of recent publications by the Munich group
(37,38), Boston group (39–43), Vienna group (44–47),
Basel group (48) and the Vancouver group (49) has reconfirmed the association of antibody detected with C4d
staining in early as well as late failure.
100
Second Tx
First Tx
80
Survival (%)
80
n = 225
Without Cytotoxins
60
60
40
40
With Cytotoxins
n = 72
20
Without
Cytotoxins
n = 19
20
With Cytotoxins
n = 13
0
0
0
4
8
12
16
20
24
28
0
Time (months)
666
4
8
12
16
20
Figure 1: First demonstration that
patients classified by their panel
reactive antibody (PRA) as having
HLA antibodies BEFORE transplantation would have lower graft
survival rates than those without
antibodies (13). The effect is greater in
second transplants than in first grafts. It
is probable that the antibodies
themselves produce graft destruction
early after transplantation.
American Journal of Transplantation 2003; 3: 665–673
Page 233 of 290
Humoral Theory of Transplantation
kidneys (17,79–83), kidney-pancreas (84), heart (30), and
lungs (85,86).
Cumulative Survival (%)
100
75
HLA antibodies are present after almost all
kidney failures
C4d–
(n = 101)
50
p = 0.0001
25
C4d+
(n = 117)
0
0
4
8
12
Post-transplant (years)
16
Figure 2: Evidence that early detection of C4d in biopsies is
associated with early graft failure (37). The effect of antibodies
occurs in the early post-transplantation period.
HLA antibodies are associated with chronic
rejection
Chronic rejection is currently recognized universally as
the main transplantation problem. The term ‘chronic rejection’ may have become too loosely defined, and Halloran
has proposed a new nomenclature (50,51). However,
because this review deals with older studies for which
more precise definitions cannot be substituted, we will
use the term chronic rejection in the old sense, with
special reference to failure resulting from immunologic
rejection.
HLA antibodies found AFTER transplantation were first
associated with failures in 1968 (52) and 1970 (53).
Antibodies to DR were shown to appear after rejection
of grafts in 1978 (54), and were studied in a series of
patients post-transplantation (55).
In 2000, we reviewed 23 publications in which the
presence of HLA antibodies was associated with acute
and chronic rejection (56). There were 12 studies of
antibodies in post kidney transplant patients (19,57–68).
In all the studies, there was a statistically significant
correlation of antibodies with acute rejection, chronic
rejection, or graft survival. Similarly, there were five
studies of heart transplant patients showing that patients
with antibodies had lower graft survival rates than those
without antibodies (59,69–72). Three studies of lung
transplants (73–75), one of liver transplants (76), and
two of cornea grafts (77,78) also showed that graft survival was lower in patients with antibodies than in those
without antibodies.
Since publication of this review, 10 further papers have
been published that show the same type of association for
American Journal of Transplantation 2003; 3: 665–673
If HLA antibodies cause graft failure they should be found
in the serum of all patients who reject a graft. Our first
studies of kidney transplant patients following graft rejection
showed 54% had antibodies (52). After the identification
of Class II antibodies (54), the percentage of patients
with antibodies rose to 72% (55). With further improvements in sensitivity, 82% of patients rejecting a graft
had antibodies (87). Following the introduction of the
more sensitive flow cytometry tests, Harmer found that
95% of 100 patients studied had antibodies (88). We
recently compiled the test results of 826 patients from
five different transplant centers who had rejected their
transplants (89). Approximately 90% of the patients
tested by the AHG enhanced cytotoxicity test for antibodies had antibodies, and when those patients who did
not have antibodies were retested by ELISA or flow
cytometry tests, the percentage of patients with antibodies rose to approximately 96%. Thus, it is certain
that almost all patients who reject a transplant have
HLA antibodies. I am not aware of comparable studies
in which, for example, 100 patients rejecting a graft
have all been shown to have cellular immunity. Although
this does not definitively prove a causal relationship for
antibodies, it is a result that would be anticipated if the
humoral theory is correct. It could, however, be argued
that the antibodies were the result of rejection and not
the cause of rejection. Evidence to the contrary is given
in the next section.
HLA antibodies precede kidney rejection
If HLA antibodies cause chronic rejection they should be
found BEFORE graft rejection. Development of HLA antibodies preceded the development of bronchiolitis obliterans in 10 of 15 patients. Among the 12 patients who did
not develop HLA antibodies, none developed bronchiolitis
obliterans (p < 0.001; 75). The temporal relationship
strongly indicates that antibodies were responsible for
development of bronchiolitis obliterans. In a study of 76
nonsensitized renal transplant recipients, among those
who developed antibodies, 11 of 12 patients (92%) lost
their grafts, whereas only 11% of 64 patients lost their
grafts if they did not produce post-transplant antibodies
(p < 0.001; 81). Thus, antibodies were predictive of
chronic rejection and were found BEFORE graft failures.
In a similar study of 150 renal transplant patients, 25% had
antibodies, and among those who had antibodies six had
graft failure 3 years later, whereas only one patient who
did not have antibodies failed (p < 0.009; 90).
667
Page 234 of 290
Terasaki
0
<6 m
1y
2
3
4
7
6
5
8y
Fail
F
F
F
F
F
F
F
Negative
Positive
F
F
F
F
F
F
Post-transplant antibody
Figure 3: Annual tests for antibodies were carried out on 14
patients who did not have any pretransplant antibodies (91).
Chronic rejection and failure after transplantation often occurred
many years AFTER the appearance of antibodies.
grafts may survive for long periods of time with antibodies
. . . but they eventually fail. We hypothesize that antibody
binds to the endothelium causing a cycle of injury and
repair over many months and even years. The process of
intimal vessel thickening is slow and gradual. It is critical to
understand that all surveys of patients with functioning
grafts show that approximately 30% of the patients
already have antibodies (see section on ‘HLA antibodies
are associated with chronic rejection’). This does not disprove the humoral theory, as the theory postulates that
damage is gradual and all those with antibodies WILL
eventually fail (Figure 3). Unfortunately, to date we are
not aware of studies on the natural history of antibodies;
for example, in some instances they may disappear as a
result of immunosuppression and may then return. Hopefully our prospective study mentioned earlier will gather
information on this issue.
Antibody specificity
The clearest evidence that antibodies PRECEDE rejection
is provided by the studies of Lee (91) (Figure 3). Over a
period of 8 years, Lee tested his patients annually for
development of antibodies. In the subset of 14 patients
who did not have antibodies before transplantation shown
in Figure 3, antibodies appeared in all cases before the
graft failure. In many instances, years had elapsed
between antibody appearance and graft rejection. This
suggests that injury produced by antibodies takes a
variable and sometimes a considerable amount of time
before its action finally manifests.
In a cooperative prospective chronic rejection study
involving 24 international centers, 1629 patients with
functioning allografts, who did not have antibodies when
they were transplanted, were examined for de novo HLA
antibodies. Subsequently, approximately 6 months later,
centers were asked to report on patients whose grafts
had failed since the time of testing. Of 212 patients who
developed antibodies, 3.3% failed compared with a 1.3%
failure rate among 1417 patients who did not develop
antibodies (p ¼ 0.05). If deaths were counted as a failure,
3.8% of those who developed antibodies failed compared
with 1.8% of those who did not develop antibodies
(p ¼ 0.05). Thus, there were significantly more failures in
those who developed antibodies than in those who did
not. This prospective study is ongoing to establish that the
development of antibodies in patients with functioning
grafts presages subsequent failure.
One might ask, ‘why did not 100% of those with antibodies fail?’ The answer is that we have only followed up
these patients for a 6-month period. We are aware of only
one study published to date in which the follow up of
patients who have antibodies has been as long as
8 years. In the publication of Lee et al. (91) cited earlier
and as shown in Figure 3, patients with well-functioning
668
Until now we have referred to antibody as detected by a
panel of cells or antigens. Many studies have not had
available cells from the original donor. With the recent
development of flow cytometry beads having single antigens prepared from recombinant cells (92), it has become
possible to study the specificities present in sera. Interestingly, patients who rejected a kidney transplant had antibodies against specificities other than those to which they
were immunized. This phenomenon has actually often
been encountered in the past. For example, Ceppellini
carefully chose donors and recipients for his planned
immunization program in order to obtain monospecific
typing sera. However, despite great efforts, very few
‘clean’ sera with specificity directly against the donor mismatch were produced. In addition, screening thousands of
pregnancy sera was necessary to find sera that were
monospecific in their reactivity. For reasons still unknown,
extra specificities are often produced upon immunization.
Lee et al. (91) also found that often patients who are
rejecting their grafts do not have antibodies directed specifically against their donor. As such antibodies were
found in sera AFTER rejection (92), we assume that DURING rejection the antibodies directed against the donor are
absorbed by the graft and only the extra antibodies circulate in the peripheral blood. Thus, the strong association of
antibodies with rejection in the studies of Lee et al. (91)
resulted from detection of nondonor specific antibodies.
The nonspecific nature of the response could be explained
by the idea that antibodies are merely indicators of
immune responsiveness, and that they might not be
directly involved in the rejection, as would be postulated
by the humoral theory. After rejection of grafts, donorspecific antibodies were found (92), making it unlikely
that the antibody is simply an indicator of responsiveness.
American Journal of Transplantation 2003; 3: 665–673
Page 235 of 290
Humoral Theory of Transplantation
Consequences of accepting the humoral
theory
An important consequence of adopting the humoral theory
is that we can now treat patients by reducing antibody
levels and monitoring antibodies. Three patients with
acute humoral rejections following live donor transplants
were treated successfully by plasmapheresis and IV IgG
(93). Pre-emptive plasmapheresis and IV IgG treatment of
five patients with a positive flow cytometry crossmatch
was also successful. Immuno-absorption of antibodies
with protein A columns was effective in five of six patients
with a mean PRA of 65% for a mean follow up of
54 months (94).
Intravenous IgG treatment has been shown to reduce HLA
antibody levels (95,96). Desensitization of patients with
antibodies was effective in 13 of 15 patients treated with
IV IgG (97). Although the exact mechanisms by which this
occurs remains unclear (98), it assumes that antibodies
are the principal agents of damage.
Treatment with tacrolimus and mycophenolate mofetil
was effective in four instances of chronic humoral rejection in patients 4–16 years post-transplantation (99). After
treatment, the titers of donor-specific antibodies dropped
dramatically and the serum creatinines stabilized. MMF
was also effective in reducing titers of anti-A and -B red cell
antibodies in patients who had received ABO incompatible
kidneys (100). Whether or not certain drugs influence
humoral immunity can be investigated. In a study of 86
cardiac transplant patients, MMF treatment resulted in less
antibody formation than with azathioprine treatment (101).
Treatment of acute cardiac humoral rejection with antiCD20 monoclonal antibodies directed against B cells was
effective in one patient whose rejection was reversed and
who remained rejection free for at least 1 year (102).
Remaining issues
patients who rejected kidneys, are of particular interest
because they are detected on endothelial cells but not
lymphocytes (109–112). Antibodies were found against
epithelia, monocyte, and endothelial lines in patients
rejecting allografts (113–116). Also, antibodies to endothelial cells may not necessarily be polymorphic, but rather
antibodies that occur secondarily to damage (117). Hyperacute rejection as a result of antibodies to endothelial cells
has been reported (118).
Some antibodies may be helpful to the graft such as
autoantibodies (119). When present before transplantation,
antibodies against Fab have been shown to be beneficial
(120), and more recently IgA anti-Fab autoantibodies have
been shown to improve graft survival (108). They could
possibly counteract the activity of conventional cytotoxic
antibodies. Anti-idiotypic antibodies to HLA may also be
counter reactive (121–123).
A second histocompatibility locus
A second histocompatibility locus, other than HLA, is
important in transplantation. HLA identical sibling donor
grafts are slowly rejected and graft vs. host reactions
occur in bone marrow transplants from HLA-identical
siblings. Antibodies against these minor histocompatibility
loci have not yet been found.
Soluble antigens
The presence of soluble HLA antigens in serum may
complex with HLA antibodies in serum, interfering with
measurement of HLA antibodies (68 124). The presence
of soluble antigens in liver transplant patients may inhibit
antibody action (125).
Mechanism of action
Antibodies are thought to be the key triggering factor in
the humoral theory, but how it produces its damage
through a cascade of events remains to be clarified. Studies of the transducing activation signals in endothelial
cells following adherence of HLA antibodies have been
reported (126 127).
HLA antibody isotypes
The relative importance of Class I and Class II antibodies
remains to be resolved (103). As for the antibody isotype,
most of the tests were performed with IgG antibodies. In
five patients transplanted across an IgM-positive crossmatch, hyperacute rejection was not found and the
patients had good early function (104). Patients who had
IgM-positive crossmatch by flow cytometry had slightly
higher graft survival rates than those who were negative
(105). IgA antibodies pretransplant were associated with
higher graft survival (106–108).
Immunogenic epitopes
Perhaps the most interesting work still pending is the
determination of immunogenic epitopes, or the actual
epitopes against which the antibody response is made.
These immunogenic epitopes should provide us with
more accurate HLA donor/recipient organ matching than
the currently utilized ‘antigens’.
Non-HLA antibodies
Antibodies that affect grafts may be against antigens other
than HLA antigens. MICA antibodies, found in the sera of
I have reviewed accumulated evidence supporting the
humoral theory of transplantation. The purpose of a theory
is to stimulate research proving its validity. My own work
American Journal of Transplantation 2003; 3: 665–673
Conclusion
669
Page 236 of 290
Terasaki
since 1959 (128) has been fueled by this hypothesis.
Although antibodies can kill cells within minutes in vitro,
perhaps the crucial new understanding is that antibodies
may take many months or years to produce the chronic
vascular endothelial thickening that ultimately chokes off
the graft. The presence of antibodies in well-functioning
grafts appears at first sight to prove that they are not
important. Only by a longer follow up period can the
significance of the antibodies be appreciated (see section
on ‘HLA antibodies precede kidney rejection’).
15.
16.
17.
18.
I hope this review will stimulate a parallel review of the
cellular theory. For example, what evidence is there that
direct action of cells causes hypercute or chronic rejection? If cells cause acute rejections, for what fraction do
they account?
Acknowledgment
19.
20.
21.
22.
I thank Dr Michael Cecka for his helpful comments.
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mofetil in renal allograft recipients with chronic rejection. Transplantation 2001; 72: 77–83.
100. Ishida H, Tanabe K, MF et al. Mycophenolate mofetil suppresses the production of anti-blood type antibodies after
renal transplantation across the ABO blood barrier: Elisa to
detect humoral activity. Transplantation 2002; 74: 1187–1189.
101. Rose ML, Smith J, Dureau G, Keogh A, Kobashigowa J.
Mycophenolate mofetil decreases antibody production after cardiac transplantation. J Heart Lung Transplant 2002; 21: 282–285.
102. Aranda JM Jr, Scornik JC, Normann SJ et al. Anti-CD20 monoclonal antibody (rituximab) therapy for acute cardiac humoral
rejection: a case report. Transplantation 2002; 73: 907–910.
103. Susal C, Opelz G. Kidney graft failure and presensitization
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Kam I. IgM antibodies in renal transplantation. Clin Transplant
1997; 11: 558–564.
105. Kerman RH, Susskind B, Buyse I et al. Flow cytometrydetected IgG is not a contraindication to renal transplantation:
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1855–1858.
106. Koka P, Chia D, Terasaki PI et al. The role of IgA anti-HLA class I
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112. Sumitran-Holgersson SWH, Holgersson J, Soderstrom K.
Identification of the nonclassical HLA molecules. MICA, as
targets for humoral immunity associated with irreversible rejection of kidney allografts. Transplantation 2002; 74: 268–277.
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Page 240 of 290
Studies evaluating the association between HLA antibodies and acute and chronic
kidney transplant rejection were reviewed in two recent publications:
• McKenna et al., Transplantation 2000;69:319-326
• Terasaki PI. Am J Transplant 2003;3:665-673.
Below are the abstracts from the 21 papers relating to kidney transplantation that were
reviewed in these two papers.
1. Abe, M., T. Kawai, et al. (1997). "Postoperative production of anti-donor antibody
and chronic rejection in renal transplantation." Transplantation 63(11): 1616-9.
To study the relevance of anti-donor antibody (ADA) to chronic rejection in kidney
transplantation, we retrospectively examined the long-term kinetics of ADA by
flow cytometric analysis.. In the CR group, IgG antibody to donor B cells of the
most current serum was positive in 25 of 29 patients, whereas it was positive in
only 5 patients in the ST group P<0.001. We conclude that the posttransplant
production of IgG antibody to donor B cells seemed to be highly relevant to
chronic rejection.
2. al-Hussein, K. A., B. K. Shenton, et al. (1994). "Characterization of donor-directed
antibody class in the post-transplant period using flow cytometry in renal
transplantation." Transpl Int 7(3): 182-9.
Over the past few years there has been increasing awareness of the importance
of humoral mechanisms in the rejection of renal transplants. In this study we
have monitored the development of antibodies directed against donor T and B
lymphocytes using the sensitive flow cytometric technique. Forty-two cadaveric
renal transplants were studied both before and for a maximum of 14 days after
transplantation. Donor cells were separated from spleen on the day of
transplantation and stored in liquid nitrogen until required. The dual colour flow
cytometric assay was used to detect IgG or IgM directed against donor T or B
lymphocytes. Using AB sera as controls, results were expressed as relative
median fluorescence (RMF) and then correlated with the clinical performance of
the grafts. Significant associations were found between the incidence of donordirected antibodies and the development of clinical rejection. The magnitude of
the rise in antibody levels was also related to graft performance. In patients
showing severe graft rejection, high levels of antibodies of the IgG class
developed before the clinical diagnosis of rejection was made. The routine use of
this test allows the prediction of impending severe rejection to be made and may
have important implications for immunosuppressive therapy.
3. Barr, M. L., D. J. Cohen, et al. (1993). "Effect of anti-HLA antibodies on the long-term
survival of heart and kidney allografts." Transplant Proc 25(1 Pt 1): 262-4.
Study of anti-HLA antibodies in a population of 238 primary renal and 199
primary transplants. The 5-year renal allograft survival was 70% in recipients
without antibodies and 53% in recipients who developed anti-HLA alloantibodies
during the first year following transplantation. Development of antibodies is
associated with acute rejection episodes and probably with the release of soluble
HLA antigens.
Page 241 of 290
4. Christiaans MH, Overhof-de Roos R, Nieman F, van Hooff JP, van den Berg-Loonen
EM. Donor-specific antibodies after transplantation by flow cytometry: relative
change in fluorescence ratio most sensitive risk factor for graft survival.
Transplantation 1998; 65 (3):427
BACKGROUND: There is no consensus on the role of donor-directed antibodies
after renal transplantation detected by complement-dependent cytotoxicity (CDC)
or by flow cytometry (FC).METHODS: Therefore, antibody formation was studied
by FC and correlated with clinical course in a group of patients who received
transplants between 1983 and 1993. All had a negative current CDC crossmatch
and were treated with cyclosporine. Current and posttransplant sera from 143
donor-recipient combinations were studied retrospectively. Antibodies were
considered present in FC if the fluorescence ratio between serum and negative
control was > 2.65.
RESULTS: Of 143 patients, 17 (11.9%) were found to be positive in the
posttransplant FC crossmatch and 126 (88.1%) were negative. Of the positive
patients, 3 were already positive in the current FC crossmatch, whereas 14
demonstrated a positive posttransplant FC crossmatch after a negative current
FC crossmatch. It was noteworthy that, from 16 patients with a positive current
FC crossmatch, 13 turned negative in the posttransplant crossmatch. In 113
recipients (79%), both pre- and posttransplant FC crossmatches were negative.
The development of a positive FC crossmatch after transplantation was a
significant risk factor for graft survival in Cox regression analysis (P = 0.01). The
results were also studied as relative change in fluorescence ratio (RCFR). RCFR
was determined by classifying the recipients in quartiles according to their
change in flow cytometric value from current to posttransplant serum. Quartiles
were defined as follows: quartile 1, decrease > 10%; quartile 2, decrease 0-10%;
quartile 3, increase > 0-30%; and quartile 4, increase > 30%. RCFR proved to be
the only significant risk factor for graft survival (odds ratio for quartile 4 vs.
quartile 1, 3.27; P < 0.02). More rejections were shown for increasing quartile
numbers (P < 0.001).
CONCLUSIONS: Classification of patients by RCFR detected more patients with
unfavorable clinical outcome (25% vs. 11%) than by FC crossmatch.
5. El Fettouh, H. A., D. J. Cook, et al. (2000). "Association between a positive flow
cytometry crossmatch and the development of chronic rejection in primary renal
transplantation." Urology 56(3): 369-72.
This study examined the effect of kidney transplantation against a positive flow
cytometry crossmatch (FCXM) on the subsequent development of chronic
rejection and graft failure.. All of these patients had a negative cytotoxicity
crossmatch. All had a pretransplant FCXM, and patients were divided according
to the results of the FCXM into three categories: FCXM negative, FCXM class I
positive, and FCXM class II positive. RESULTS: We found that a positive FCXM
at the time of transplantation was strongly associated with the ultimate
development of chronic rejection. In FCXM-negative individuals, 16.9%
developed chronic rejection compared with 80% of those with an HLA class I (T
and B-cell) reaction and 40.9% of those with a class II (B-cell-only) reaction (P
<0.001). The 3-year graft survival rate was 93% for FCXM-negative patients
compared with 86% for FCXM class II positive and 80% for FCXM class I positive
Page 242 of 290
patients (P = 0.001). CONCLUSIONS: A strong association between a positive
FCXM and subsequent development of chronic rejection was identified.
6. Halloran, P. F., J. Schlaut, et al. (1992). "The significance of the anti-class I
response. II. Clinical and pathologic features of renal transplants with anti-class I-like
antibody." Transplantation 53(3): 550-5.
Although the ability of preformed anti-class I antibodies to mediate hyperacute
rejection is well established, their pathogenic role in acute rejection remains illdefined. We set out to identify patients with anti-class I against donor cells and to
define the clinical and pathological features of such patients. We collected sera
pretransplant and in the first 3 months posttransplant from 64 renal transplant
recipients (59 cadaver donors and 5 one-haplotype matched living-related
donors). We assayed the sera for class I-like antibody against donor T cells in
complement-dependent microcytotoxicity, with crossmatches against autologous
T cells to exclude auto-antibodies. All pretransplant sera were negative against
donor T cells. Of the 797 sera tested posttransplant, 131/195 sera from 13
patients were positive, and 602 sera from 51 patients were negative. All patients
who formed anti-class I underwent rejections compared with only 41% of patients
with no anti-class I detected (P less than 0.0005). More rejections in patients with
anti-class I were classed as severe (12/15 [80%] compared with 9/28 [32%] P
less than 0.005), and graft loss was significantly higher (5/13 vs. 2/51; P less
than 0.002). Rejections associated with anti-class I occurred earlier; more
frequently developed oliguria (35% versus 10%) and required dialysis (40%
versus 10%) and biopsies (10/13 vs. 6/28); and had a higher rate of rise in serum
creatinine (249 versus 79 microns/L in the first 48 hr). Biopsies during anti-class I
positive rejections more frequently displayed endothelial injury in the
microcirculation, neutrophils in the glomeruli and/or peritubular capillaries, and
fibrin deposition in glomeruli or blood vessels. The biopsies in anti-class I
negative rejection episodes tended to have tubulitis, interstitial infiltration, and
blasts, suggesting that these lesions reflect T-cell-mediated mechanisms. We
conclude that patients with antibody against donor class I had more severe
rejection, probably because anti-class I injuries the endothelium of small blood
vessels of the graft, leading to rapid functional deterioration. We believe that anticlass I may be a major factor in some severe rejection episodes
7. Kerman RH, Susskind B, Kerman DH, et al. Anti-HLA antibodies detected in
posttransplant renal allograft recipient sera correlate with chronic rejection.
Transplant Proc 1997; 29 (1–2):1515.
Chronic rejection is an important cause of graft loss after the first posttransplant
year. Both antigendependent (immune injury) and alloantigen-independent
events contribute to chronic graft dysfunction. Our study focuses on the antigendependent, immune-injury aspects of chronic rejection. Of the several
hypotheses explaining the immune basis of chronic rejection, antibody-mediated
graft damage may predominate. A reliable postoperative monitor of serologic
alloimmunity could identify high-risk patients experiencing rejections and
rejection-related morbidity. Most studies attempting to correlate percent panel
reactive antibodies (% PRA) and specific antidonor reactivity with clinical events
Page 243 of 290
utilized a complement-dependent cytotoxicity (CDC) assay. Because of the
inherent problems related to CDC-PRA assays, the test has been variably
reliable. An ELISA procedure to detect IgG anti-HLA class I antibodies has
recently been reported that determines anti-HLA % PRA and specificity as well.
In the present study we tested pre- and posttransplant sera from renal allograft
recipients for anti-HLA alloimmunity by antihuman globulin (AHG) and ELISA
methodologies, and correlated AHG-PRA and/or ELISA-PRA to rejection. The
data suggest that ELISA-PRA, but not AHG-% PRA,identified recipients at risk
for adverse immunologic and
clinical events.
8. Kerman, R. H., S. M. Katz, et al. (2001). "Posttransplant immune monitoring of antiHLA antibody." Transplant Proc 33(1-2): 402
Although the presence of anti-HLA antibodies in renal transplant recipient sera
has been associated with graft rejection and decreased graft survivals, the role of
these antibodies is not well-understood.[1] A reliable test that monitors serologic
anti-HLA reactivity might identify patients at risk for rejection or graft loss. [2] We
used an enzyme–linked immunosorbent assay (ELISA) to detect IgG anti–HLA
class I and class II antibodies bound to a matrix of soluble HLA antigens (PRASTAT). Sera were collected pre– and serially–posttransplantation for 19 ± 8
months from 123 cyclosporine (CsA)-Prednisone (Pred) treated primary
recipients of a cadaveric donor renal allograft. Sera were collected monthly or
every other month with an average of 15 sera per patients tested. Patients were
transplanted with ABO compatible donor organs following a negative anti-human
globulin (AHG) enhanced complement dependent cytotoxicity crossmatch. No
patient experienced hyperacute or accelerated rejection. One third of the patients
(41 of 123) presented with a pretransplantation IgG anti-HLA PRA ≥ 10% with
67% of the antibody directed against MHC class I and 33% against MHC class I
and II. The rejection frequency of 73% for patients with pretransplantation IgG
anti-HLA PRA ≥ 10% was significantly greater than the 27% for patients with a
pretransplantation PRA < 10% (73% vs 27%, P < .01). Postoperatively, one-third
of the patients did not develop ELISA-detected anti-HLA antibodies, had a 10%
frequency of rejections, had a 90% one-year graft survival, and had only an 8%
frequency of chronic rejection. In contrast, the 67% of the patients with ELISAdetectable IgG anti-HLA PRA ≥ 10% experienced a 55% rejection frequency
(10% vs 55%, P < .001), a 79% 1-year graft survival (90% vs 79%, P < .02), and
a 26% frequency of chronic rejection from one to three years posttransplantation
(8% vs 26%, P < .02). Antibody specificities were directed at both donor and nondonor HLA antigens. Two-thirds of the antibodies were against MHC class I and
one third against MHC class I and II antigens. There was a significantly
consistent monthly ELISA-detected IgG anti-HLA PRA ≥ 10% present in sera 3 ±
4 months prior to the diagnosis of chronic rejection but not present for nonchronic
rejectors (71% vs 18%, P < .02). Our data suggest that patients with
pretransplantation IgG anti-HLA antibody reactivity are at risk to (1) display
posttransplantation anti-HLA antibody (including donor specific reactivity) and (2)
experience early and/or chronic rejection. Patients developing
posttransplantation detectable IgG anti-HLA antibody are at risk for chronic
rejection. Patients without posttransplantation anti-HLA antibody could represent
successfully immunoregulated recipients.
Page 244 of 290
9. Kerman, R. H., C. G. Orosz, et al. (1997). "Clinical relevance of anti-HLA antibodies
pre and post transplant." Am J Med Sci 313(5): 275-8.
Pretransplant histocompatibility testing seeks to select compatible donorrecipient pairs for transplantation. Sera from prospective renal transplant
recipients are screened for the presence of human leukocyte antigen (HLA)
antibodies to determine humoral alloimmunization. Present techniques screen
patient sera using a complement-dependent cytotoxicity assay and express the
results as percent of panel reactive antibody (PRA). However, the standard
assay suffers because it needs viable target cells, a variable sensitivity of cells
for complement, subjective evaluation, a lack of standardized methodology, and
a variable correlation with clinical outcomes. Alternatively, an enzyme-linked
immunosorbent assay (ELISA) methodology can detect IgG anti-HLA reactivity
based on the binding of immunoglobulin to soluble HLA class I antigens. This
method provides increased objectivity and reproducibility, does not require use of
viable target cells, and most importantly, detects immunoglobulin that is reactive
to HLA class I antigens. Data discussed herein suggest that identifying reactive
recipient sera using the enzyme-linked immunosorbent assay (ELISA) (PRASTAT, Sang Stat Med, Menlo Park, CA) methodology may be more informative
clinically than current standard percent of panel reactive antibody (PRA) assays.
10. Martin, S., P. A. Dyer, et al. (1987). "Posttransplant antidonor lymphocytotoxic
antibody production in relation to graft outcome." Transplantation 44(1): 50-3.
Serial serum samples from 266 recipients of primary renal allografts were
monitored posttransplant for the presence of panel reactive lymphocytotoxic
antibodies (PRA). The minimum posttransplant follow-up period was 18 months.
Patients were classified according to whether or not they produced PRA before
and/or after transplantation. The groups were as follows: PRA negative before
and after transplant, -/-, 171; PRA positive before and negative after transplant,
+/-, 5; PRA positive before and positive after transplant, +/+, 27; PRA negative
before and positive after transplant, -/+, 63. Actuarial graft survival at 1 year for
each group was 81.3%, 100%, 70.4%, 47.6%, respectively. Fifty-five of the 63 -/+
recipients were retrospectively crossmatched with posttransplant sera against
stored donor lymphocytes. Of these, 50 (91%) were posttransplant cross match
positive, and 37 (67%) have lost their grafts. In 23 of the 26 cases where an antiHLA specificity was defined, the antibody was directed against antigens present
in the donor but not in the recipient. These results clearly indicate that the
production of PRA in recipients of renal transplants is associated with antidonor
reactivity and poor graft outcome. The fact that these PRA were often directed
against donor HLA antigens emphasizes one of the hazards of mismatching for
HLA at transplantation.
11. Monteiro, F., R. Buelow, et al. (1997). "Identification of patients at high risk of graft
loss by pre- and posttransplant monitoring of anti-HLA class I IgG antibodies by
enzyme-linked immunosorbent assay." Transplantation 63(4): 542-6.
Page 245 of 290
Identification of risk factors influencing graft survival may lead to the development
of models to predict graft outcome. Such models may provide guidance for
immunosuppressive therapy, measure posttransplantation outcome, and
eventually improve graft survival in high-risk patients. A major risk factor
influencing graft survival is allosensitization. However, due to the lack of
standardization of lymphocytotoxicity assays, the detection of alloantibodies
utilizing this current methodology may not correlate with posttransplant events.
Recently, a novel standardized enzyme-linked immunosorbent assay (ELISA) for
the detection of anti-HLA class I IgG antibodies was developed. To evaluate the
predictive value of this diagnostic test, a retrospective analysis of 124 renal
allograft recipients with an 18-month follow-up time was performed. A highly
significant (P=0.01) correlation between pre-transplant ELISA panel reactive
antibody (PRA) results and graft loss was observed. Patients with pre-transplant
ELISA PRA of >10% had a three times higher risk of graft loss compared with
patients who tested negative. No such correlation was observed with
complement-dependent cytotoxicity results independent of the reduction of IgM
antibodies with dithiothreitol. Similarly, a highly significant correlation of ELISA
results with the occurrence of early graft dysfunction was observed. Almost all
patients (88%) with a pretransplant ELISA PRA of >50% required posttransplant
dialysis, compared with 45% of patients with a pretransplant ELISA PRA of 1050% and 27% of patients with a pretransplant ELISA PRA of <10%. No such
difference was observed with complement-dependent cytotoxicity %PRA values.
Analysis of posttransplant specimens by ELISA demonstrated a strong
correlation of assay results with graft rejection and graft dysfunction. In summary,
these results suggest that detection of anti-HLA class I antibodies by ELISA
identifies patients at high risk for graft loss. No other single risk factor of such
magnitude has been identified so far.
12. Monteiro, F., C. Mineiro, et al. (1997). "Pretransplant and posttransplant monitoring
of anti-HLA class I IgG1 antibodies by ELISA identifies patients at high risk of graft
loss." Transplant Proc 29(1-2): 1433-4
NO abstract available – see full article
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VJ0-3VN9BV8SN&_user=1525358&_coverDate=03%2F31%2F1997&_rdoc=1&_fmt=high&_ori
g=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000053540&
_version=1&_urlVersion=0&_userid=1525358&md5=17b5094f2b3dd994e57bcf1
ca8bafff9&searchtype=a
13. Muller-Steinhardt, M., L. Fricke, et al. (2000). "Monitoring of anti-HLA class I and II
antibodies by flow cytometry in patients after first cadaveric kidney transplantation."
Clin Transplant 14(1): 85-9.
While the relevance of pre-formed anti-human leukocyte antigen (HLA)
antibodies has been studied extensively, the role of anti-HLA class I and II
antibodies produced after cadaveric kidney transplantation is still a matter of
discussion. As it has been proposed that they are involved in a considerable
number of cases, it should be investigated whether a post-transplant monitoring
is a sensitive parameter for the early diagnosis of acute rejection episodes.
Page 246 of 290
Additionally, it has been suggested that antibodies are a major cause for chronic
rejection; thus, it would be of interest to correlate antibody detection and graft
survival. We retrospectively investigated 59 patients after a first cadaveric kidney
transplantation without known anti-HLA antibodies (complement-dependent
cytotoxicity [CDC] testing). The panel reactivity was determined with a new highly
sensitive and specific flow-cytometric technique (Flow-PRA Screening Test, One
Lambda, Canoga Park, USA) in sequentially collected serum samples pre- and
post-transplant. In patients with acute rejection episodes during the clinical
course, the last sample prior to rejection, and in patients without rejection, the
last sample prior to discharge, was analyzed. Furthermore, we analyzed 3-yr
graft survival and several clinical parameters such as cold ischemia time (CIT).
Twenty-four of 59 patients (41%) experienced acute rejections during the clinical
course. Five of 59 died with a functioning graft within the first 3 yr. Seven of 54
patients, still alive after 3 yr, lost their graft. Anti-HLA antibodies were detectable
in only 7/59 patients and a correlation between antibody positivity and acute
rejections (p = 0.32 and 0.54 for anti-HLA class I and II, respectively) could not
be identified (sensitivity 12.5 and 8.3%). However, we found a significant
correlation between the detection of anti-HLA class II and graft loss within 3 yr (p
= 0.005, specificity 97.9%). Additionally, anti-HLA class II positive patients had
significantly longer CIT (p = 0.003). Whether the detection of anti-HLA class II
antibodies in the early post-transplant phase is of great value for the identification
of patients at high risk for early graft loss needs additional investigation.
However, we found that anti-HLA antibodies are detectable only in a minority of
unsensitized patients and we conclude that flow-cytometric monitoring with Flow
PRA is not a sensitive parameter for the early diagnosis of acute rejection
episodes in patients after first cadaveric kidney transplantation.
14. Pelletier, R. P., P. K. Hennessy, et al. (2002). "Clinical significance of MHC-reactive
alloantibodies that develop after kidney or kidney-pancreas transplantation." Am J
Transplant 2(2): 134-41.
The purpose of this study was to determine the relationships between acute
rejection, anti-major histocompatibility complex (MHC) class I and/or class IIreactive alloantibody production, and chronic rejection of renal allografts following
kidney or simultaneous kidney-pancreas transplantation. Sera from 277
recipients were obtained pretransplant and between 1 month and 9.5 years posttransplant (mean 2.6years). The presence of anti-MHC class I and class II
alloantibodies was determined by flow cytometry using beads coated with
purified MHC molecules. Eighteen percent of recipients had MHC-reactive
alloantibodies detected only after transplantation by this method. The majority of
these patients produced alloantibodies directed at MHC class II only (68%). The
incidence of anti-MHC class II, but not anti-MHC class I, alloantibodies detected
post-transplant increased as the number of previous acute rejection episodes
increased (p = 0.03). Multivariate analysis demonstrated that detection of MHC
class II-reactive, but not MHC class I-reactive, alloantibodies post-transplant was
a significant risk factor for chronic allograft rejection, independent of acute
allograft rejection. We conclude that post-transplant detectable MHC class IIreactive alloantibodies and previous acute rejection episodes are independent
risk factors for chronic allograft rejection. Implementing new therapeutic
strategies to curtail post-transplant alloantibody production, and avoidance of
Page 247 of 290
acute rejection episodes, may improve long-term graft survival by reducing the
incidence of chronic allograft rejection.
15. Piazza, A., D. Adorno, et al. (1998). "Flow cytometry crossmatch: a sensitive
technique for assessment of acute rejection in renal transplantation." Transplant
Proc 30(5): 1769-71
No abstract available – see attached
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6VJ0-3Y51H4H22&_user=1525358&_coverDate=08%2F31%2F1998&_rdoc=1&_fmt=high&_orig
=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000053540&_
version=1&_urlVersion=0&_userid=1525358&md5=49e716dcf17e36e1b2870b30
39ceebf1&searchtype=a
16. Piazza, A., L. Borrelli, et al. (2000). "Posttransplant donor-specific antibody
characterization and kidney graft survival." Transpl Int 13 Suppl 1: S439-43.
This study was designed to investigate the clinical relevance of donor-specific
antibodies (DS-Abs) and their influence on graft survival. Among 106 patients
who underwent cadaveric kidney donor transplantation and were monitored by
flow cytometry crossmatch (FCXM) during the 1st posttransplantation year, 25
(23.6%) resulted positive for DS-Ab production. During a 2-year follow up only 12
of the 81 FCXM-negative patients (14.8%) suffered rejection vs 17 of 25 FCXMpositive patients (68%; P = 0.00001). Correlating graft loss to DS-Ab production,
9 FCXM-positive patients lost the graft vs only 1 among the FCXM-negative
patients. A worse graft function was evidenced in FCXM-positive subjects who
had also suffered rejection episodes than in those which had acute rejection but
did not produce DS-Abs. A high incidence of HLA-AB mismatches was found in
FCXM-positive subjects which produced anti-class I antibodies. FCXM appears
useful in estimating posttransplant alloimmune response. Moreover our findings
confirm the harmful effects of anti-class I DS-Abs on long-term graft survival.
17. Piazza, A., E. Poggi, et al. (2001). "Impact of donor-specific antibodies on chronic
rejection occurrence and graft loss in renal transplantation: posttransplant analysis
using flow cytometric techniques." Transplantation 71(8): 1106-12.
BACKGROUND: Improvements in immunosuppressive therapy have greatly
reduced acute rejection (ARj) episodes, ensuring better short-term graft outcome,
but have not modified long-term survival in renal transplantation. It is now well
accepted that chronic rejection (CRj) can be determined by both immune and/or
nonimmune mechanisms. The aim of this study was to evaluate the importance
of the posttransplant humoral immune response towards mismatched HLA graft
antigens in CRj occurrence and graft outcome. METHODS: Serum samples from
120 nonpresensitized renal transplant recipients were prospectively screened for
1 year after surgery by means of flow cytometry cross-match (FCXM) and
FlowPRA beads (microbeads coated with purified HLA class I and class II
antigens) assays. All transplants were followed-up for 2 years or until graft
removal. RESULTS: FCXM monitoring identified donor-specific antibodies (DSAbs) in 29 (24.2%) of 120 transplanted patients. Correlation with clinical data
highlighted a higher incidence of ARj in DS-Abs-positive patients compared to
Page 248 of 290
negative patients (62% vs. 13%, P<0.00001). Furthermore, graft failure occurred
more frequently among FCXM-positive patients than among negative patients
(34% vs. 1%, P<0.00001). The deleterious effect of DS-Abs on graft function was
confirmed by serum creatinine levels 2 years after transplantation. These were in
fact higher in subjects producing DS-Abs than in subjects with only ARj (mean
creatinine: 2.5+/-1.3 mg/dL vs.1.7+/-0.5 mg/dL, P=0.04). FlowPRA analysis of
DS-Ab HLA specificity highlighted the presence of anti-HLA class I antibodies in
85% of FCXM-positive patients, who also presented with a higher incidence of
HLA-B mismatches than FCXM-negative patients (1.23+/-0.66 vs. 0.92+/-0.59,
P=0.02). CONCLUSIONS: Flow cytometric techniques are precious tools for
investigating the activation of the humoral response against HLA antigens of the
graft in renal transplantation. DS-Abs production has a worse impact on organ
function and survival than ARj episodes. These findings represent further proof of
the threat posed by DS-Abs on long-term graft function and draw attention to the
need for a specific immunosuppressive therapy aimed at counteracting the
different kinds of immune activation toward graft.
18. Schonemann, C., J. Groth, et al. (1998). "HLA class I and class II antibodies:
monitoring before and after kidney transplantation and their clinical relevance."
Transplantation 65(11): 1519-23.
BACKGROUND: In search of an alternative screening technique, we compared
complement-dependent cytotoxicity (CDC) with PRA-STAT, a commercially
available enzyme-linked immunosorbent assay (ELISA). METHODS: A total of
188 pre- and posttransplant sera from 50 renal allograft recipients were tested
with both methods. RESULTS: A significant correlation was found between both
methods. Discrepant results could be explained by the fact that PRA-STAT
detects both HLA class I and II antibodies (while CDC with peripheral blood
lymphocytes as target cell detects mainly HLA class I reactivity), by the presence
of IgM antibodies (which are not detected by the IgG-specific ELISA test), and by
CDC "false-positive" results due to antibody rejection treatment. The clinical
relevance of antibodies detected by PRA-STAT is suggested by the following. (a)
In eight patients, donor-specific HLA antibodies detected by PRA-STAT (but not
seen by CDC) resulted in severe rejection episodes, which led to graft loss in
four cases. In all but one patient, antibodies were directed against class II or
mixtures of class I and H antigens. Six patients with complications were shown to
have developed de novo antibodies against DQ incompatibilities. (b) Half of the
patients with a positive ELISA test at the moment of crossmatch experienced
complications. Such patients are at a threefold higher risk of suffering from
rejection episodes and/or graft loss than patients who are not sensitized (P<0.05,
Fisher exact test). CONCLUSIONS: Because PRA-STAT is very reproducible,
detects both HLA class I and II antibodies, and is not influenced by rejection
therapy, we consider it an additional tool for pre- and posttransplant monitoring of
kidney allograft recipients.
19. Scornik, J. C., D. R. Salomon, et al. (1989). "Posttransplant antidonor antibodies and
graft rejection. Evaluation by two-color flow cytometry." Transplantation 47(2): 28790.
Page 249 of 290
The posttransplant production of antibodies against cryopreserved donor cells
was studied in 50 consecutive cadaveric kidney graft recipients and in 23
additional patients selected for acute rejection. Serum was obtained twice weekly
during the first 3 weeks posttransplant and then monthly for 6 months. IgM and
IgG anti-T cell Abs were measured by 2-color flow cytometry. Results were
analyzed in conjunction with the patients' demographics, previous sensitization,
HLA-matching, posttransplant blood transfusions, incidence of delayed function,
rejection episodes, and biopsy results. Antidonor antibodies, predominantly IgG,
were detected in 19/48 (40%) of the patients proximate to the time of rejection. In
contrast, antibodies were seen in only 2/22 (9%) of nonrejecting patients, and
these antibodies were exclusively IgM. Younger patients were more likely to have
antibody-mediated rejections. Cytotoxic antibody reactivity against panel cells
developed or increased posttransplant in some patients, but it did not correlate
with rejection. Previous sensitization and posttransplant transfusions favored the
development of posttransplant panel reactivity but not of antidonor antibodies.
Most rejections, including those associated with antidonor antibodies, were
reversed by antirejection therapy. We conclude that antidonor antibodies are
involved in a significant proportion of rejection episodes and that the damage
induced does not necessarily culminate with loss of the graft.
20. Suciu-Foca, N., E. Reed, et al. (1991). "Soluble HLA antigens, anti-HLA antibodies,
and antiidiotypic antibodies in the circulation of renal transplant recipients."
Transplantation 51(3): 593-601.
Chronic rejection represents the major threat to long-term survival of organ
allografts. It is presumed that this form of rejection is mediated by antibodies
against mismatched HLA antigens of the graft. The presence and specificity of
anti-HLA-antibodies in posttransplantation sera are, however, difficult to
document. We have explored the possibility that anti-HLA antibodies form
immune complexes with soluble HLA antigens released from the injured graft
and/or that they are blocked by antiidiotypic, anti-anti-HLA-antibodies. Our data
demonstrate that the long-term survival of renal allografts is significantly lower in
patients who develop anti-HLA-antibodies following transplantation than in
patients who do not form antibodies. Following depletion of soluble HLA antigens
by magnetic immunoaffinity, we could identify anti-HLA-antibodies in 57% of the
sera obtained from patients undergoing chronic rejection of kidney allografts,
compared with 41% prior to antigen depletion. In patients tolerating the graft for 4
years or more, the corresponding frequencies of antibody-positive sera was 2%
and 5% prior and following depletion of HLA antigens. The presence of HLA
antigen/anti-HLA-antibody immune complexes in patients' sera was positively
associated with chronic humoral rejection (P less than 0.0001). Patients who
tolerated the graft in spite of having developed antibodies against one of its
mismatched HLA antigens show specific antiidiotypic (anti-anti-HLA-antibodies).
Such antiidiotypic antibodies were not found in sera from patients with chronic
rejection (P = 0.005). This indicates that antiidiotypic antibodies may delay the
progression of chronic humoral rejection.
Page 250 of 290
21. Trpkov, K., P. Campbell, et al. (1996). "Pathologic features of acute renal allograft
rejection associated with donor-specific antibody, Analysis using the Banff grading
schema." Transplantation 61(11): 1586-92.
Alloantibody frequently appears during the immune response to alloantigens in
renal transplant recipients. We studied whether the presence of antibody against
donor class I antigens correlated with the clinical and pathologic features of acute
rejection episodes. We identified patients who had (1) clinical evidence of acute
rejection, (2) a renal biopsy showing pathologic features of acute rejection,
defined by the Banff criteria, and (3) pre- and posttransplant sera screened
against donor T cells. We divided these patients into those with or without donorspecific alloantibody reactive with donor T cells. Of 44 patients with biopsyproven rejection, 20 were antibody negative (Ab-R) and 24 were antibody
positive (Ab+R). The biopsies from Ab+R patients had a higher incidence of
severe vasculitis (P=0.0009) and glomerulitis (P=0.01). Fibrin thrombi in the
glomeruli and/or vessels, fibrinoid necrosis, and dilatation of peritubular
capillaries were also more frequent in the Ab+R group. Infarction was present in
biopsy specimens from 9/24 Ab+R patients versus none in the Ab-R group
(P=0.002). The Ab+R biopsy specimens more often had polymorphonuclear
leukocytes in the peritubular capillaries (P=0.003). In contrast, specimens of AbR patients showed tubulitis more often than the specimens of Ab+R patients:
moderate and severe tubulitis was present in 19/20 (95%) Ab-R patients versus
12/24 (50%) Ab+R patients (P=0.002). Graft loss was increased in Ab+R
patients, particularly in the first 3 months (12/24 compared with 3/20, P=0.025).
Thus, during biopsy-proven acute rejection episodes, anti-class I antibody
correlates with severe vascular lesions, glomerulitis, and infarction, whereas
more severe tubulitis predominates in rejection episodes without antibody.
Appendix C - Independent Review of the Clinical Literature
Appendix C - Independent Review of the Clinical Literature
Page 251 of 290
Page
Page 252 of 290
Literature Review:
The Impact of
Transfusion on
Transplantation
December 20, 2010
Prepared by: HERON Evidence Development LLC
Page 253 of 290
Literature Review: The Impact of Transfusion on Transplantation
For further details regarding this document please contact:
Amgen
HERON Evidence Development LLC
One Amgen Center Drive
50 Division Street, Suite 503
Thousand Oaks, CA 91320-1799
Somerville, NJ 08776
USA
USA
TEL: +1 805 447 1000
TEL: +1 908 864 6281
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2
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Literature Review: The Impact of Transfusion on Transplantation
AABB
ASH
Acc
AM
AMR
AR
AST
AVR
CDC
CI
CKD
CMS
CMV
CRDAC
DSA
DSG
DST
ELISA
ESRD
ET
FK
HHS
HLA
HSP
IgG
IgM
IvIG
ITT
LAR
mPTF
MedCAC
MMF
MP
NBG
NKF
LAR
OPTN
PRA
PRTR
PTA
PTF
rPTF
RBC
RR
SEM
SRTS
TRIPS
TRTR
TTVS
UNOS
USRDS
Abbreviations
American Association of Blood Banks
American Society of Hematology
Accelerated Rejection
Acceptably Mismatched
Antibody Mediated Rejection
Acute Rejection
American Society of Transplant
Acute Vascular Rejection
Complement Dependent Cytotoxicity
Confidence Interval
Chronic Kidney Disease
Centers for Medicare and Medicaid Services
Cytomegalovirus
Cardio Renal Drugs Advisory Committee
Donor Specific Antibody
Deoxyspergualin
Donor-Specific Blood Transfusion
Enzyme-Linked Immunosorbent Assay
End Stage Renal Disease
Eurotransplant
FK-506 (Tacrolimus)
Health and Human Services
Human Leukocyte Antigen
Highly Sensitized Patient
Immunoglobulin G
Immunoglobulin M
Intravenous Immune Globulin
Intention to Treat
Late Acute Rejection
Matched Pre-Transplant Blood Transfusion
Medicare Evidence Development and Coverage Advisory Committee
Mycophenolate Mofetil
Methylprednisolone
Normalized Background (ratio)
National Kidney Foundation
Late Acute Rejection
Organ Procurement and Transplantation Network
Panel Reactive Antibody
Primary Renal Transplant Recipient
Post Transplant Anemia
Pre-Transplant Blood Transfusion
Random Pre-Transplant Blood Transfusion
Red Blood Cell
Relative Risk
Standard Error of the Mean
Scientific Registry of Transplant Recipients
Transfusion-Related Infections Prospective Study
Third Renal Transplant Recipients
Transfusion-Transmitted Viruses Study
United Network for Organ Sharing
United States Renal Data System
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3
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Literature Review: The Impact of Transfusion on Transplantation
Table of Contents
Project Objectives................................................................................. 7
Project Methodology ............................................................................. 7
1
The relationship between transfusions and panel reactive
antibodies...................................................................................... 8
1.1 Summary ............................................................................................................... 8
1.2 Overview of panel reactive antibodies ...................................................................... 8
1.3 Evidence of the correlation between transfusions and antibody development ............... 9
2
The impact of PRAs on renal transplant matching and
wait times ................................................................................... 12
2.1 Summary ............................................................................................................. 12
2.2 Overview of the correlation between PRAs, HLA cross-matching and difficulties in renal
transplantation ..................................................................................................... 12
2.3 Evidence of the correlation between PRAs and wait times in renal transplantation ...... 13
3
The impact of elevated PRA levels and wait times on renal
transplant success ....................................................................... 15
3.1
3.2
3.3
3.4
3.5
4
The evidence supporting strategies to mitigate the risk of graft
failure .......................................................................................... 24
4.1
4.2
4.3
4.4
5
Summary ............................................................................................................. 24
Leukoreduction of the blood supply and impact on allosensitization........................... 24
Desensitization protocols and experimental induction therapy agents ........................ 25
Variance in post-transplant immunosuppressant protocols and associated costs ......... 26
Key Evidence Gaps ...................................................................... 28
5.1
5.2
5.3
5.4
5.5
5.6
5.7
6
Summary ............................................................................................................. 15
Overview of PRA levels.......................................................................................... 15
Elevated PRA levels and increases in wait time correlate to pre-transplant mortality ... 15
Evidence of elevated PRAs impacting graft survival and post-transplant complications 16
Evidence of blood transfusions impacting graft survival ............................................ 19
Inconsistent definition of highly sensitized patient ................................................... 28
Impact of timing of PRA assays on PRA levels ......................................................... 28
Reporting of exact number of prior transfusions ...................................................... 28
Under-reporting of true sensitization rates .............................................................. 28
Limited data set for post-transplant outcomes in highly sensitized patients ................ 28
Relevance of antibodies detected on newer HLA assays ........................................... 29
Reports of the positive impact of acceptable mismatch programs on graft survival
outcomes in the US .............................................................................................. 29
Discussion ................................................................................... 30
References .......................................................................................... 31
Appendix A
Detailed Search Strategy .............................................. 34
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4
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Literature Review: The Impact of Transfusion on Transplantation
List of Tables
Table
Table
Table
Table
Table
Table
Table
Table
Table
Table
1: Percentage of sensitizing events in each PRA category ................................................ 9
2: Risk factors for transfusion-associated allosensitization .............................................. 10
3: PRA levels in correlation to waiting times.................................................................. 13
4: 10-year graft survival by PRA level, HLA-identical sibling transplants ........................... 18
5: Acute AMR incidence as reported in six separate studies ............................................ 25
6: Immunosuppressive regimens, 1990-2007 (Toki 2009) .............................................. 26
7: Search filters for transfusion.................................................................................... 34
8: String for transplantation ........................................................................................ 34
9: String for antigens and histocompatibility ................................................................. 35
10: String for organ restriction .................................................................................... 35
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Literature Review: The Impact of Transfusion on Transplantation
List of Figures
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
1:
2:
3:
4:
5:
6:
7:
Sensitization and transfusions in first transplant patients, 1995-2000 ......................... 10
Share of transplants performed by PRA status ......................................................... 13
Median transplantation wait time by transfusion status ............................................. 14
Observed and projected median wait times by year of listing and PRA ....................... 14
3-year outcomes of first-time waitlisted patients in 2005 by PRA level........................ 16
Unadjusted graft survival for living and deceased donor transplants, by PRA .............. 17
10-year graft survival according to pre-transplant PRA, cadaver kidney and
HLA-identical sibling transplants ............................................................................ 18
8: Post-transplant complications in highly-sensitized TRTR vs. PRTR patients ................. 19
9: Effect of transfusions on graft survival in non-sensitized (PRA<10%) and sensitized
patients ............................................................................................................... 20
10: Acute rejection rate within 3 months of transplantation in recipients of a pre-transplant
transfusion (rPTF) vs. controls (p < 0.05) .............................................................. 20
11: Death-censored 10-year graft survival in recipients of a kidney from a deceased donor:
rPTF versus no rPTF (p=0.77) ............................................................................... 21
12: Death-censored 8-year graft survival in recipients of a kidney from a living donor:
mPTF versus DST versus no PTF group (p=0.96) .................................................... 21
13: Graft survival after living renal allograft transplantation in patients with and without
DST .................................................................................................................... 22
14: (A) Overall graft survival rates in group A (without DSG prophylaxis) and group B (with
DSG prophylaxis); (B) Graft survival rate with and without accelerated rejection; (C)
Graft survival rate with and without acute rejection (AR); (D) Graft survival rate with
and without late AR (LAR) ..................................................................................... 23
15: Comparison of graft survival between acceptably mismatched patients (AM) and
nonsensitized (<5% PRA), sensitized (5-85% PRA) and highly sensitized (>85% PRA)
patients ............................................................................................................... 29
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Literature Review: The Impact of Transfusion on Transplantation
Project Objectives
This literature review was undertaken t o summarize published and reported evidence
demonstrating t he im pact o f allosensitization a nd p re-transplant transfusions o n transplant
matching, wait times and renal allograft survival.
To enable the presentation of the evidence to support MedCAC discussions, this document follows
an o utline that w ould support examination of the re lationship between transfusions and
transplantation. Each chapter of this document addresses a distinct aspect of this relationship,
which can be summarized as described below.
1. Transfusions elevate levels of panel reactive antibodies (PRAs).
2. Elevated levels of PRAs make finding transplant matching more difficult,
increasing wait times.
3. PRAs and increased wait times both negatively impact the likelihood of successful
transplantation.
4. Sensitization remains a significant challenge in management of the ESRD patient
despite s trategies t o m itigate th e r isk o f sensitization a nd s ubsequent g raft
failure (such as leukoreduction, pre-transplant desensitization, and
immunosuppression protocols).
Project Methodology
HERON conducted an independent, comprehensive review of t he l iterature based on focused
keyword searches of MEDLINE. Studies of interest were restricted to the most recent ten years of
English-language publications. As t his review concerns t he i mpact o f p rior t ransfusion o n
transplantation, particularly in terms of the likelihood of graft rejection and reaction to antigens
on the new tissue, the search facets comprised the following:
•
Transfusion
•
Transplantation and graft survival
•
Antigens and histocompatibility
The overall search strategy was to produce sub-searches for each of these facets, which were
then combined and restricted to date limits and to kidney transplants as the organ of interest.
Further details of the search strategy are presented in Appendix A.
Where literature referenced publicly available and relevant data sources, HERON examined the
most r ecent r eports to gather up -to-date supporting d ata. In a ddition, HERON c onducted
focused s earches within the re fined search re sults t o uncover findings o f i nterest re garding
leukoreduction, d esensitization and i mmunosuppression p rotocols. La stly, HERON i dentified
studies based on reference searching of articles returned by the search.
To identify abstracts of interest in addition to published studies, HERON also searched conference
proceedings from 2008-2010 meetings of The American Association of Blood Banks (AABB), The
American S ociety o f Hematology ( ASH), and T he National Kid ney F oundation ( NKF). La stly,
HERON searched the websites of AABB and The American Society of Transplant (AST) in order to
identify position papers containing relevant data on transfusions and transplantation.
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Literature Review: The Impact of Transfusion on Transplantation
1
The relationship between
transfusions and panel reactive
antibodies
1.1
Summary
•
•
•
1.2
The three p rincipal c auses o f a ntibody d evelopment a re p revious t ransplantation,
pregnancy and transfusions.
Retrospective studies have found that multiple transfusions correspond to increased
antibody development, and that levels of sensitization increase with the number of
units of blood transfused.
Statistical analyses of prior transfusion as a risk factor for sensitization have found
that prior transfusion is not c onsistently identified as a n independent predictor of
hypersensitization; however, va riability in t he num ber o f units t ransfused and
confounding procedures may have influenced these results.
Overview of panel reactive antibodies
A possible complication of transfusion is the development of antibodies. Panel reactive antibody
(PRA) is a test that measures anti-human antibodies in the blood and is routinely performed on
patients waiting for kidney and heart transplants (Opelz 2005). PRA has been the measure of
sensitization since t he re cognition that c atastrophic hyp eracute rejection w as a ssociated with
anti-donor antibodies to human leukocyte antigen (HLA) in the mid-1960s (Cecka 2010).
Patients with elevated PRA levels are often referred to as sensitized. The PRA level is defined as
the p ercentage o f t he p opulation against which a p otential kidney t ransplant re cipient is
sensitized. Although definitions may vary, a PRA level of less than 10 percent indicates that a
potential recipient is not sensitized, whereas a level of greater than 80 percent indicates high
sensitization (Cecka 2010) (US Renal Data System 2010). Proficiency testing of complement
dependent c ytotoxicity ( CDC) assays has shown t hat d ifferent l aboratories c an a ssign widely
varying (5 to 80%) PRA values. Another more reproducible definition of sensitization can be
derived fro m t he num ber o f s pecificities t o H LA a ntibodies o f a p atient i n re lation t o the
frequencies of t he t arget a ntigens i n t he d onor p opulation ( percentage PRA t o percentage
population PRAs) (Claas 2009b).
Despite the variability noted above, CDC assays are currently the gold standard in most transplant
matching programs. Newer solid phase HLA antibody detection assays such as ELISA, Flow-PRA®,
and Luminex® are more sensitive than the standard CDC assay, although the clinical relevance of
the antibodies detected has yet to be established. Therefore, CDC-dependent crossmatch because
of donor HLA-specific antibodies is considered to be a contraindication for transplantation, while
antibodies d etected in t he s olid p hase are c urrently c onsidered ri sk fa ctors ( Claas 20 09a).
Unresolved issues related to newer assays are addressed in Section 5.6.
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Literature Review: The Impact of Transfusion on Transplantation
1.3
Evidence of the correlation between
transfusions and antibody development
The p resence o f a ntibodies t o H LA h as b een a major o bstacle t o t ransplantation of hig hly
sensitized patients (Nikaein 2009). Development of high antibody levels usually results from
multiple b lood t ransfusions, p revious f ailed t ransplants, a nd p regnancies (Jordan 2003) . In
addition, in regraft patients, previous graft rejection appears to be associated with the production
of HLA antibodies and high levels of sensitization (Hardy 2001).
Although nephrologists who treat End-Stage Renal Disease (ESRD) patients are aware of this risk
and may attempt to avoid unnecessary transfusions, a significant proportion of potential kidney
transplant c andidates c ontinue t o p eriodically re quire b lood t ransfusions t hat c arry a ris k o f
allosensitization (Karpinski 2004; Nikaein 2009). The United Network for Organ Sharing (UNOS)
database indicates that approximately 30 percent of waitlisted transplant candidates continue to
require b lood t ransfusions a t some p oint b efore transplantation (Karpinski 2 004). Likewise,
according to the most recently available data from the United States Renal Data System (USRDS),
approximately 30 percent of transplant candidates in 2007 had evidence of at least one blood
transfusion within t hree years o f b eing a dded t o t he l ist (US R enal D ata S ystem 201 0). In
addition, the use of blood transfusions was greater among patients highly sensitized at the time
of transplant (US Renal Data System 2010).
Prior studies and clinical observations have shown that historical anti-HLA antibodies can become
apparent after b lood t ransfusions (Aalten 2009) . In a ddition, p rior t ransfusion ha s been
correlated to highly elevated PRAs in clinical studies, as described below.
In a retrospective study of 244 patients on a renal transplant waiting list, Soosay et al. identified
a relationship between prior transfusion and the risk of allosensitization, as illustrated in Table 1.
Transfusion of one or more units of blood was documented in 173 (71%) of subjects. Of the
highly sensitized patients (HSPs) (n= 31), 100 percent had received transfusion of at least one
unit, in addition to other sensitizing events (Soosay 2003).
Table 1: Percentage of sensitizing events in each PRA category
Sensitizing
Events
Transfusion
Pregnancy
Grafting
Not Sensitized
PRA 0−9%
60
18
8
Sensitized
PRA 10−59%
83
26
43
Significant
Sensitization
PRA 60−79%
80
47
47
Highly
Sensitized
PRA 80−100%
100
32
74
In this same retrospective analysis, the level of sensitization clearly increased with the number of
red blood cell units transfused. Non-sensitized subjects received a mean of 5.65 units with a
standard error of the mean (SEM) of 1.38, while highly sensitized subjects received a mean of
37.8 units (SEM 8.4) (Soosay 2003). Note that the retrospective analysis did not address specific
pre-transplant t ransfusion p rotocols t hat may ha ve b een fo llowed, s o i t i s no t p ossible t o
determine from this study if pre-transplant transfusions continued after patients were identified
as sensitized. The authors conclude from these data that transfusion remains an important cause
of sensitization, despite the perception of reduced need for transfusion in patients with end-stage
renal failure due to the availability of recombinant human erythropoietin (Soosay 2003).
A correlation between number of units transfused and levels of sensitization measured by PRA
assays was also identified in an analysis of patients registered in the UNOS Kidney Transplant
Registry between 1995 and 2000, conducted by Hardy et al. (Hardy 2001). As depicted in Figure
1, Hardy et al. found that the incidence of sensitization increased with the number of transfusions
and was more pronounced in women than in men (Hardy 2001).
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9
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Literature Review: The Impact of Transfusion on Transplantation
Figure 1: Sensitization and transfusions in first transplant patients, 1995-2000
(Hardy 2001)
In addition, the authors note that the rates identified may in fact under-report true sensitization
rates, as sensitization rates were only reported in patients who were transplanted, and did not
include rates present in patients who were not transplanted (Hardy 2001).
A retrospective cohort study of 112 patients conducted by Karpinski et al. sought to identify
independent risk factors for allosensitization, with a focus on evaluating the impact of the practice
of universal leukoreduction in Canada on pre-transplant allosensitization (Karpinski 2004) (see
Section 4.2 for additional information regarding leukoreduction). In addition, prior transfusion
(five or more) was evaluated as an independent risk factor, as illustrated in Table 2 (Karpinski
2004).
Table 2: Risk factors for transfusion-associated allosensitization
Flow-PRA® positive pretransfusion
Pregnancy
Previous transplant
≥5 Previous transfusions
Leukoreduction
RBC units given (per unit)
Risk Factors (RR, 95% CI)
Univariate
P
Multivariate
4.5 (1.9–11)
0.001
2.4 (0.8–7.1)
7.8 (3.1–19.5)
0.001
8.2 (2.8–24)
2.8 (0.9–8.7)
0.08
2.4 (0.6–9.9)
5.1 (2.0–13.1)
0.001
2.6 (0.8–8.8)
0.5 (0.3–1.7)
NS
2.0 (0.7–6.0)
1.1 (1.0–1.3)
0.10
1.1 (0.9–1.3)
P
0.10
0.0001
NS
NS
NS
0.10
Prior transfusion was associated with an increased risk of allosensitization, but did not reach
statistical significance in multivariate analysis in this study. Note that the number of previous
transfusions received w as no t re ported w ithin t his s tudy. T his l imitation i s p otentially o f
importance t o und erstanding o f t he find ings, a s S cornik e t a l. observed that s ensitization i s
infrequent and transient when patients receive fewer than 20 transfusions (Scornik 2009).
A retrospective study of serum samples from 145 patients also reported that more than one prior
transfusion contributed to anti-HLA antibody production, although as in Karpinski et al.
transfusion was not found to be an independent risk factor upon statistical analysis (Vaidya
2005). This study included patients immunized as a result of acute or chronic rejection (n=22), as
well as patients with a history of no more than two prior transfusions (n=20) or pregnancy (n=6)
(Vaidya 2005). Neither a single pregnancy nor two prior transfusions were independent variables
of immunization in primary transplant recipients (p=0.17, and p=0.42, respectively); however,
multiple pregnancies and two prior t ransfusions together were associated w ith a potent
immunogenic stimulus (p=0.00001) (Vaidya 2005). Note that this study did not observe the
same cut-off of five or more prior transfusions as in Karpinski et al.; this cut-off is supported by
Karpinski and by previous studies (Opelz 1981).
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History of transfusion and number of prior transfusions were likewise not found to be predictive
of PRA levels in a cross-sectional study of 98 patients conducted at two dialysis centers by PourReza-Gholi et al (Pour-Reza-Gholi 2005). Pour-Reza-Gholi et al. also found that PRA levels were
higher after dialysis procedures than prior to dialysis (p=0.0003), suggesting that, for reasons not
well understood, dialysis itself may be a confounding procedure impacting PRA results (PourReza-Gholi 2005).
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2
The impact of PRAs on renal
transplant matching and wait
times
2.1
Summary
•
•
•
•
2.2
Patients with performed antibodies against HLA antigens are at risk for hyperacute
rejection, accelerated acute rejection, antibody-mediated rejection (AMR), delayed
graft function and longer-term complications (Cecka 2010).
These risks can be minimized by cross-matching a patient’s specific antibody profile
with the graft donor.
Patients who are sensitized to a high number of HLA specificities have more difficulty
finding an appropriate donor, and typically experience longer wait times as a result.
Patients with PRA of 10 percent or lower usually receive a kidney transplant during
the first year, whereas patients with PRA in excess of 80 percent usually do not
receive a kidney transplant during the first two to three years (Soosay 2003).
Only a small percentage of highly-sensitized p atients r eceive a transplant due to
elevated PRA levels.
Overview of the correlation between PRAs,
HLA cross-matching and difficulties in renal
transplantation
Renal t ransplantation o f a hi ghly s ensitized p atient ( >80% P RA) c an b e d ifficult (DeMeester
2002). The presence of preformed anti-HLA antibodies is a contraindication to transplantation of
allografts bearing these HLA types since this carries the risk of hyperacute rejection (Soosay
2003). In addition, a patient may experience other complications, including accelerated acute
rejection, AMR, delayed graft function and longer-term complications when transplanted from a
donor expressing the target HLA antigens (Cecka 2010).
A s pecific antibody p rofile is often p erformed t o maximize the c hances of hig hly s ensitized
patients receiving a successful graft (Nikaein 2009). Detecting the presence of antibodies prior to
transplantation via cross-matching can help a patient find a suitable donor. However, patients
who are sensitized to a large number of HLA specificities have lower chances of receiving a crossmatch negative donor and will typically experience longer wait times (Soosay 2003) (DeMeester
2002).
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2.3
Evidence of the correlation between PRAs and
wait times in renal transplantation
A recent review reported that, although protocols enabling successful transplantation patients
with donor-specific antibodies can help ensure early to intermediate-term allograft survival, the
identification of donor kidneys for transplant candidates with high levels of circulating antibodies
against HLA is a major challenge and results in prolonged waiting times for transplantation (Gloor
2010).
A retrospective survey of all patients who were active on the Irish renal transplant waiting list
during 1996 demonstrated a clear increase in wait time linked to increased PRA. As illustrated in
Table 3, the modal waiting time for patients with a PRA of 81−100 percent was greater than 35
months, while non-sensitized patients had a modal waiting time of 0-5 months with a median
waiting time of seven months. Only half of highly sensitized patients received a transplant after
five years (Soosay 2003).
Table 3: PRA levels in correlation to waiting times
PRA<10%
0-5*
Waiting times (months)
*modal waiting time
10%≥PRA≤80%
>15
PRA>80%
>35
The same study demonstrated that the majority of highly sensitized patients (52% with PRA of
11−59%; 46% with PRA of 60−79% and 84% with PRA >80%) waited more than fifteen months
on o ther f orms o f re nal replacement t herapy, whereas t he m ajority ( 56%) of no n-sensitized
patients received a transplant within 10 months (Soosay 2003).
A review authored by Jordan et al concluded that patients with PRAs greater than 30 percent
have d ouble t he w aiting t ime b efore t ransplantation (Jordan 2003) . S imilarly, patients w ith
elevated anti-HLA antibodies often wait extended periods of time for a compatible organ (Jordan
2003). Furthermore, one study found that, while 30 percent of UNOS wait-listed renal transplant
candidates are allosensitized, only approximately 10 percent of all transplants are performed in
sensitized recipients (Karpinski 2004).
United Network for Organ Sharing (UNOS) data indicates that patients with elevated PRAs are
less likely to receive a transplant. As illustrated in Figure 2, less than 10 percent of all transplants
performed in 1998 had a PRA of greater than 20 percent. Waiting times for patients with PRAs
greater than 30 percent were double the time for all other patients (Jordan 2003).
Figure 2: Share of transplants performed by PRA status
7
2.5
PRA 0-19%
PRA 20-78%
PRA 80%+
90.5
(Jordan 2003)
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The United States Renal Da ta S ystem ( USRDS) also i llustrates t hat p atients re ceiving blood
transfusions and those with elevated PRAs experience longer median wait times for
transplantation. As illustrated in Figure 3, patients who report a history of transfusion prior to
transplant consistently wait longer for a transplant.
Median waiting time (months)
Figure 3: Median transplantation wait time by transfusion status
(US Renal Data System 2010)
In addition, patients with elevated PRAs have historically experienced longer median waiting
times, as shown in Figure 4.
Figure 4: Observed and projected median wait times by year of listing and PRA
(US Renal Data System 2010)
Note above that data is projected for more recent years, as a median has not yet been observed
(i.e., greater than 50 percent of the patients listed in that year have yet to be transplanted).
Estimates of median wait times for these years were made using a linear regression model.
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3
The impact of elevated PRA levels
and wait times on renal transplant
success
3.1
Summary
•
•
•
•
•
3.2
Although it was once thought that pre-transplant blood transfusions could have a
beneficial effect on the outcomes of transplantation, recent studies have shown that
pre-transplant transfusions have a deleterious effect on mortality and graft survival,
and are associated with increased risk of acute rejection.
Patients awaiting renal transplantation are routinely tested for lymphocytotoxic PRA.
Elevated PRA levels increase the chances of a patient dying or being removed from
the transplant wait list while awaiting a transplant.
Elevated PRA levels also impact long-term graft survival, regardless of the level of
HLA cross-match achieved in the transplant. Patients with an elevated PRA at the
time of transplant have shorter graft half-lives (Jordan 2003).
Patients with renal dysfunction who have not undergone transplantation have lower
survival rates compared to patients who have received kidney transplants (Soosay
2003). In addition, increased morbidity correlates with increased renal dysfunction.
Patients experiencing graft loss will die or return to chronic dialysis due to rejection.
Overview of PRA levels
It has long been known that kidney transplant candidates whose serum contained
lymphocytotoxic PRA before transplantation were at increased risk of graft rejection. This finding
was published over thirty years ago, and has been confirmed in many subsequent studies. Today
patients awaiting renal transplantation routinely undergo PRA testing (Opelz 2005). Patients with
high PRA levels experience longer wait times, which may cause them to be delisted or die while
awaiting a transplant (US Renal Data System 2010). Thus, a significant proportion of patients
with ESRD are denied the benefits of transplantation due to allosensitization (Karpinski 2004).
The percentage of sensitized or highly sensitized patients receiving transplants is comparatively
low. For example, of the 1,761 patients receiving transplants in the U.S. between 2006 and 2007,
1,469 (83%) had a PRA level of 0-9% at transplant, with sensitized (10-79% PRA) and highly
sensitized ( PRA le vel o f 80%+) m aking up o nly 174 ( 10%) a nd 36 ( 2%) o f a ll t ransplants,
respectively (US Department of Health and Human Services 2009).
3.3
Elevated PRA levels and increases in wait time
correlate to pre-transplant mortality
Highly sensitized patients have few options to improve the odds of successful transplantation and
wait extended periods of time on dialysis, which is associated with attendant morbidities and
mortality (Jordan 2003).
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In an Irish study of 244 patients on waiting list for kidney transplant, Soosay et al. found that, of
14 patients dying before receiving transplants, 43 percent of those who died were on the waiting
list for 2-3 years, while 28.6 percent who died were on the list for 1-2 years. Only 4 patients
(28.6%) died within one year of being listed for transplantation (Soosay 2003).
Data supplied by the USRDS also show that patients with high PRA levels were more likely to be
removed from the transplant list or die while waiting for a transplant, as illustrated in Figure 5.
Figure 5: 3-year outcomes of first-time waitlisted patients in 2005 by PRA level
(US Renal Data System 2010)
3.4
Evidence of elevated PRAs impacting graft
survival and post-transplant complications
Transplantation of incompatible organs with positive antibody cross-matches usually results in
severe rejection a nd a llograft loss. I n a ddition, elevated P RA levels a re associated with
significantly shorter g raft ha lf-life in patients re ceiving b oth t ransplants fro m b oth l iving a nd
cadaver sources (Jordan 2003).
Data supplied b y t he Org an P rocurement a nd Transplantation N etwork’s ( OPTN) S cientific
Registry o f Transplant Recipients ( SRTR) il lustrates t he im pact o f e levated P RA a t time o f
transplant on graft survival. As shown in Figure 6, numerically far fewer patients with elevated
(>80%) PRA levels experienced survival of kidney grafts from living and deceased donors at 3
months and one year than patients with low (0-9%) PRA levels (202 vs. 7,181 and 1,272 vs.
15,321, respectively), although the percentage of grafts surviving was similar (98.1% vs. 96.0%
and 95. 4% vs . 95. 1%, re spectively). T he d ifference i n g raft s urvival b etween p atients w ith
elevated and low PRA levels appeared to widen at 10 years, with 52.50% graft survival in highPRA living donor and 40.50% in high-PRA deceased donor recipients, in comparison to 58.40%
and 43.70% in low-PRA living and deceased donor recipients, respectively. These differences
were not reported as having been tested for statistical significance (US Department of Health and
Human Services 2009).
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Figure 6: Unadjusted graft survival for living and deceased donor transplants, by PRA
Unadjusted Graft Survival, Living Donor Kidney Transplants
Survival at 3 Months, 1 Year, 5 Years, and 10 Years
Total
PRA at
Transplant
3 Months
1 Year
5 Years
10 Years
(Tx 2006 - 2007)
(Tx 2006 - 2007)
(Tx 2002 - 2007)
(Tx 1997 - 2007)
N
%
Std. Err.
N
%
Std. Err.
N
%
Std. Err.
N
%
All
12,462
98.10%
0.10%
12,462
96.30%
0.20%
38,350
81.40%
0.30%
62,864
59.40%
0-9%
7,181
98.10%
0.20%
7,181
96.50%
0.20%
20,112
81.70%
0.40%
30,097
58.40%
10-79%
1,141
98.20%
0.40%
1,141
96.30%
0.60%
2,702
79.90%
1.20%
3,632
53.50%
80%+
202
96.00%
1.40%
202
93.50%
1.70%
517
69.20%
3.20%
651
52.90%
Unknow n
3,938
98.10%
0.20%
3,938
96.10%
0.30%
15,019
81.60%
0.40%
28,484
60.50%
Unadjusted Graft Survival, Deceased Donor Kidney Transplants
Survival at 3 Months, 1 Year, 5 Years, and 10 Years
Total
PRA at
Transplant
3 Months
1 Year
5 Years
10 Years
(Tx 2006 - 2007)
(Tx 2006 - 2007)
(Tx 2002 - 2007)
(Tx 1997 - 2007)
N
%
Std. Err.
N
%
Std. Err.
N
%
Std. Err.
N
%
All
20,298
95.30%
0.10%
20,298
91.00%
0.20%
55,513
69.30%
0.30%
94,990
43.30%
0-9%
15,321
95.40%
0.20%
15,321
91.20%
0.20%
42,316
69.60%
0.30%
72,885
43.70%
10-79%
2,970
94.80%
0.40%
2,970
90.10%
0.60%
7,352
67.90%
0.80%
11,839
40.60%
80%+
1,272
95.10%
0.60%
1,272
90.30%
0.80%
3,064
68.80%
1.20%
4,491
40.50%
Unknow n
735
95.00%
0.80%
735
90.40%
1.10%
2,781
67.90%
1.10%
5,775
45.20%
(US Department of Health and Human Services 2009)
It is important to note that reports commenting on the OPTN/SRTR data extracts do not identify
confounding factors in the high-PRA group that received transplants. For example, it is not
possible to evaluate the quality of the renal transplant match in this group, which may have
influenced the results. As the majority of patients with elevated PRAs have difficulty finding an
acceptable match, many are not transplanted as illustrated in Sections 2.3 and 3.3. As a result, it
is possible that those patients in the high-PRA group who were transplanted reflect a biased
sample of patients who received an acceptable match, which may have positively influenced the
data regarding graft survival.
An examination of data from the Collaborative Transplant Study also investigated the influence of
PRA on g raft s urvival. A significant e ffect o f P RA on o ne-year g raft s urvival was e vident in
cadaver transplants (p<0.0001), but no significant effect was noted in transplants from HLAidentical sibling donors (p=0.0831) (Opelz 2005). Immunosuppressive regimens in patients with
transplants fro m H LA-identical s ibling d onors i ncluded c yclosporine, t acrolimus a nd re gimens
without calcineurin inhibitors; however, intent-to-treat analysis found no significant differences in
graft survival rates depending on the regimen used. The differential effect of PRA on survival of
transplants from cadaver donors during the first year after transplantation is shown in Figure 7.
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Figure 7: 10-year graft survival according to pre-transplant PRA, cadaver kidney transplants
(Opelz 2005)
Interestingly, P RA w as strongly a ssociated with l ong-term g raft l oss in HLA-identical s ibling
donors, even though HLA-identical sibling donors do not provide a target for antibodies to HLA
antigens and should therefore not be affected by PRA. Among the patients studied receiving HLAidentical sibling transplants, 3,001 patients with no PRA had significantly higher 10-year graft
survival to the 803 patients with 1-50 percent PRA (p=0.0006) or to the 244 patients with greater
than 50 percent PRA (p<0.0001) (Table 4).
Table 4: 10-year graft survival by PRA level, HLA-identical sibling transplants
PRA=0%
% graft survival
72.4%
(Opelz 2005)
1%≥PRA≤50%
PRA>50%
SE
% graft survival
SE
% graft survival
SE
1.1
63.3%
2.5
55.5%
4.0
The e ffect o f P RA i n H LA-identical sibling t ransplants w as d ifferent fro m t he a cute re jection
associated with PRA in recipients of cadaver kidneys. In addition, the authors comment that for
the HLA-identical grafts, it is difficult to say whether PRA served as an indicator of heightened
immunity against non-HLA transplantation antigens, or whether graft loss was a direct effect of
non-HLA humoral sensitization (Opelz 2005).
Another study comparing graft outcomes in sensitized and less sensitized patients focused on
higher-PRA t hird re nal t ransplant re cipients (TRTR) and lower-PRA p rimary re nal transplant
recipients ( PRTR). T his study d emonstrated th at t he p ercentage of P RA in TRTR and P RTR
patients ( 24%+34% vs . 7% +14%, re spectively, p= 0.03) c orresponded with d elayed g raft
function (defined as the need for dialysis within the first seven days after transplantation) and
rejection episodes. F orty-six p ercent o f s ensitized T RTR p atients e xperienced d elayed g raft
function compared with 22 percent of PRTR patients (p=0.05), while 50 percent of TRTR patients
experienced bio psy-proven rejection e pisodes compared w ith 29 percent of P RTR p atients
(p=0.01), despite greater frequency of induction therapy (74% vs. 35%, respectively, p=0.004)
(Horovitz 2009). The differences in 1- and 5-year patient survival in the TRTR and PRTR patient
groups were not as marked, with both cohorts experiencing similar survival rates (93%, 83% and
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96%, 87%, respectively), and similar renal function. However, as illustrated in Figure 8, highlysensitized TRTR p atients experienced more b acterial i nfections ( 43%) a nd w ound p roblems
(28%) t han t heir P RTR c ounterparts ( 18% a nd 11%, p= 0.001 a nd p= 0.09, r espectively)
(Horovitz 2009).
Percentage of total population
Figure 8: Post-transplant complications in highly-sensitized TRTR vs. PRTR patients
50
45
40
35
30
25
20
15
10
5
0
TRTR
PRTR
(Horovitz 2009)
It is im portant t o no te t hat t he int ent o f t his re trospective a nalysis w as t o i dentify w hether
acceptable medical a nd s urgical o utcomes c ould b e a chieved i n T RTR p atients, d espite t heir
higher im munologic and s urgical risks, i n a ddition t o i ncreased m orbidity i n t his p opulation.
These risks may not be presented in the populations analyzed in the other studies reviewed for
this report.
In a single-center, prospective study involving six highly-sensitized patients treated preoperatively
with protein A immunoadsorption, one-year graft survival was reported as 66 percent (Hickstein
2002). The one patient in whom the graft did not survive beyond 2 months was also the patient
with the highest PRA level reported (80%) following the protein A immunoadsorption procedure
(Hickstein 2002); of note, however, high levels of sensitization among these six patients were
related to either prior transplant or pregnancy rather than transfusion.
3.5
Evidence of blood transfusions impacting graft
survival
In the past, some transplant programs administered pre-transplant transfusions deliberately to
achieve a beneficial “transfusion e ffect” aimed at o ptimizing g raft o utcomes. H owever, m ore
recent data indicate that this beneficial effect is no longer apparent (Karpinski 2004) (Hardy
2001). One analysis of UNOS Kidney Transplant Registry data has even identified the interval
from 1995-2000 as the pivotal period when DST ceased demonstrating a beneficial effect on graft
survival and began to show a deleterious effect (Hardy 2001). As illustrated in Figure 9, this
analysis found that among sensitized and non-sensitized patients alike, those with no transfusions
had the highest graft survival, while those with the greatest number of transfusions had the
lowest survival.
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Figure 9: Effect of transfusions on graft survival in non-sensitized (PRA<10%) and sensitized patients
(Hardy 2001) (Note: p-value not reported)
Note that these data may not be representative of current clinical practices, which may restrict
transplantation if high amounts of DSAs are detected prior to transplant.
One study of 77 living donor living transplants identified the risk factors associated with graft
rejection. E leven p atients l ost their g rafts (6 fro m li ving unre lated d onors and 5 fro m l iving
related donors), seven of which were due to chronic rejection (n=7). Overall 3-, 5- and 10-year
graft survival in live donors was 92.8 percent, 86.6 percent, and 76.9 percent, respectively. The
study found acute rejection episodes, especially 3 or more episodes (risk ratio [RR] = 11.1) and
preoperative multiple transfusion history (RR = 4.2) were the primary factors influencing graft
survival (Park 2004).
One Dut ch study c ompared t he outcomes of re nal t ransplants in p atients w ho ha d r eceived
random pre-transplant blood transfusions (rPTF), matched (mPTF), donor-specific blood
transfusion (DST) and those who received no PTF. The outcomes showed that PTF did not have a
beneficial e ffect o n t he outcomes o f t ransplantation. I nstead, rPTF was associated w ith a
significantly increased risk of acute rejection (Figure 10) and a deleterious effect on 10-year
cadaver-donor renal graft survival (Figure 11), although the latter result did not achieve statistical
significance in this study (Aalten 2009). In addition, as illustrated in Figure 12, DST was not
associated with a significant impact on 8-year living-donor renal graft survival.
Figure 10: Acute rejection rate within 3 months of transplantation in recipients of a pre-transplant
transfusion (rPTF) vs. controls (p < 0.05)
(Aalten 2009)
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Figure 11: Death-censored 10-year graft survival in recipients of a kidney from a deceased donor: rPTF
versus no rPTF (p=0.77)
(Aalten 2009)
Figure 12: Death-censored 8-year graft survival in recipients of a kidney from a living donor: mPTF versus
DST versus no PTF group (p=0.96)
(Aalten 2009)
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Although the above-referenced studies by Park and Aalten have reported a deleterious effect of
prior transfusion on graft survival, others report a beneficial effect of DST in combination with
specific cyclosporine-based immune suppressive protocols.
A study by Marti et al. that sought to evaluate the benefits of a cyclosporine-based pre-transplant
DST protocols reported 6-year graft survival for a cohort of patients who received pre-transplant
DST. The outcomes of 61 patients who had received pre-transplant DST and cyclosporine (the
‘Bern’ group) were compared with subjects from Swiss transplant centers (n=513) and Western
Europe (n=7,024). Additionally, a ‘Matched-Cases’ group (n= 55), a control for relevant factors
influencing outcome, was created to ensure that study findings reflected the effects of DST rather
than patient selection. As illustrated in Figure 13, the study revealed 6-year graft survival rates of
98%, 82%, 84% and 81% in patients in the ‘Bern’, ‘Matched Cases’, ‘Switzerland’ and ‘Western
Europe arms, respectively. On an intent-to-treat basis, 6-year graft survival in the ‘Bern’ group
with pre-transplant DST was 88.5 percent (Marti 2006).
Figure 13: Graft survival after living renal allograft transplantation in patients with and without DST
(Marti 2006) (non-ITT analysis)
Similarly, a retrospective analysis of 64 patients transplanted following a cyclosporine-based pretransplant DST protocol was undertaken by Barbari et al. in order to evaluate the benefits of DST
in light of the availability of cyclosporine A (Barbari 2001). In a combined group of patients
treated with pre-transplant DST and various immune suppressive protocols based on azathioprine
and cyclosporine A (n= 44), rejection rates were found to be lower than in a historical control
group of patients who did not receive pre-transplant cyclosporine A and DST (45% vs. 75%,
p<0.02) (Barbari 2001). However, the finding that the results were most pronounced when
comparing b etween a g roup o f p atients w ith p re-transplant D ST a nd c yclosporine A vs . t he
control g roup w ith no p re-transplant im munosuppression o r DST ( 39% v s. 75 %, p <0.01)
suggests that the benefit may be attributable to pre-transplant cyclosporine A rather than DST.
Amada et al. studied t wo g roups of DS T p atients i n an attempt t o e stablish t he e ffect of
deoxyspergualin (DSG) prophylaxis. A historical control group (Group A) who received
cyclosporine, p rednisolone, a nd antilymphocyte g lobulin w ith ( n= 15) o r w ithout azathioprine
(n=49) was c ompared with DS G-treated p atients ( Group B) receiving immunosuppressive
treatment c onsisting o f c yclosporine, p rednisolone, and a ntilymphocyte g lobulin and
deoxyspergualin (n=76) (Amada 2003). As illustrated in Figure 14, overall five-year graft survival
rates were significantly higher for group B than group A (89.5 vs. 73.4 percent (p= 0.0070).
Subdivision b y re jection type ( accelerated r ejection, A cc; acute rejection, A R; a nd l ate a cute
rejection, LAR), revealed that in non-DSG treated patients (group A) five-year graft survival was
not affected by the presence or absence of Acc (75.0 vs. 73.1%), but was influenced by the
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presence of absence of AR (50.0 vs. 85.7%, p=0.0012) or LAR (46.7 vs. 81.6%, p<0.0001). In
patients receiving DSG, five-year graft survival did not change significantly by the presence or
absence of Acc (100 vs. 88.7%), AR (81.8 vs. 92.6%), or LAR (81.0 vs. 92.7%) (Amada 2003).
Figure 14: (A) Overall graft survival rates in group A (without DSG prophylaxis) and group B (with DSG
prophylaxis); (B) Graft survival rate with and without accelerated rejection; (C) Graft survival rate with and
without acute rejection (AR); (D) Graft survival rate with and without late AR (LAR)
(Amada 2003)
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4
The evidence supporting
strategies to mitigate the risk of
graft failure
4.1
Summary
•
•
•
•
4.2
Strategies to mitigate the risk of sensitization and subsequent graft failure include
leukoreduction, as well as pre-transplant desensitization and immunosuppression at
the time of transplant or shortly thereafter.
Despite universal leukoreduction policies in several countries, sensitization continues
to be identified by sensitive PRA assays.
Pre-transplant d esensitization a nd i mmunosuppression p rotocols a re no t fo llowed
consistently due to lack of clear guidelines supporting their efficacy.
Immunosuppression is associated with significant costs.
Leukoreduction of the blood supply and impact
on allosensitization
Historically reserved for specific patient populations (including dialysis), leukoreduction of red
blood cell (RBC) transfusions has now been recommended for universal adoption in Canada, the
United Kingdom, France and Portugal. In the US, approximately 70 percent of RBC units are
currently leukoreduced prior to distribution (Karpinski 2004).
However, there are comparatively few data on the impact of leukoreduction on allosensitization
as a result of red blood cell transfusions in patients with ESRD (Karpinski 2004). As previously
discussed in Section 1.3, Karpinski et al. evaluated the impact of universal leukoreduction in
Canada on p re-transplant a llosensitization i n a r etrospective cohort study o f 112 p atients
(Karpinski 2004). Multivariate analysis did not identify leukoreduction as a significant risk factor
in t his s tudy ( RR, 2. 0; 9 5% C I, 0. 7-6.0; p = NS), and w as t herefore no t a ssociated w ith a ny
protective effect against transfusion-associated allosensitization for potential kidney transplant
candidates (Karpinski 2004) (see also Table 2).
The findings presented in Sections 0-3 of this report underscore that despite the broad availability
of leukoreduced blood products from the late 1990s, evidence of allosensitization remains an area
of current investigation and clinical significance, particularly for patients awaiting organ
transplantation. Recently, fi ndings fro m a c omparative analysis o f C lass I and C lass I I H LA
antibodies in recipients of non-leukoreduced and leukoreduced blood were presented at the 2010
Annual Meeting of the American Association of Blood Banks (AABB) (Norris 2010). This analysis
tested longitudinal panels at pre- and post-transfusion time points from 29 recipients of nonleukoreduced blood in the Transfusion-Transmitted Viruses Study (TTVS) and 20 recipients of
leukoreduced blood in the Transfusion-Related Infections Prospective Study (TRIPS). While the
analysis did reveal that the development of new HLA antibodies was more frequent in panels
from patients who received non-leukoreduced blood (p<0.01), the authors observed frequent
alloimmunization i n re cipients o f l eukoreduced blo od. Ap plication o f a s ensitive a ssay cutoff
(normalized background (NBG) ratio 2.2) revealed that 31 percent of the leukoreduced blood
recipients were sensitized to Class I HLA antibodies (Norris 2010).
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4.3
Desensitization protocols and experimental
induction therapy agents
Many highly allosensitized patients may never have an acceptable donor identified, and these
patients w ill remain o n d ialysis i ndefinitely. To a ddress t his challenge, strategies h ave b een
developed that will allow positive-crossmatch transplantation via patient desensitization, which
involves the temporary removal of donor specific antibodies (Gloor 2010; Claas 2009b). The goal
of desensitization protocols is to lower donor specific antibody (DSA) activity to avoid immediate
allograft injury, and to maintain reduced levels for the first weeks and months after
transplantation. It appears that this period of desensitization will allow the allograft to develop a
degree of “accommodation,” or relative resistance to AMR (Gloor 2010). Antibody mediated injury
may occur minutes or hours after transplantation, as in the case of hyperacute rejections, or days
after transplantation, when memory B lymphocytes in the bone marrow, spleen and lymph nodes
undergo a reaction causing antibody-secreting cells to produce high levels of DSAs (Gloor 2010).
Desensitization protocols va ry w idely, a nd e ncompass b oth p lasma e xchange a nd i nduction
therapies. Plasma exchange may involve plasmapheresis with low-dose or high-dose intravenous
immunoglobulin (IvIG) and/or immunoabsorption (Claas 2009b). Regarding the latter approach,
Hickstein et al. report superior one-year graft survival results in highly sensitized patients treated
with protein A immunoadsorption immediately prior to surgery (Hickstein 2002). However, the
authors note that prospective multicenter trials would be required to validate this experience
(Hickstein 2002).
Induction immunosuppressive therapies m ay invo lve antithymoglobin, a nti-interleukin-2
antibodies, or alemtuzumab (Campath 1-H) (Claas 2009b). Rituximab may be used in high-risk
patients who become refractory to standard desensitization treatment, or to treat AMR
(Kopchaliiska 2009). Some small studies have reported that bortezomib (VELCADE®), a treatment
approved for use in plasma-derived cancers, has offered some benefit for patients experiencing
AMR, p ossibly b ecause p lasma c ells are re sponsible fo r p roducing H LA a lloantibodies (Claas
2009b).
Although some desensitization regimens have been used for almost 20 years, most protocols are
still associated with significant challenges and costs. Firstly, patients with high DSA levels at
baseline are often resistant to treatment and are unable to proceed with transplantation (Gloor
2010). In addition, as illustrated in Table 5, studies of patients undergoing positive crossmatch
transplantation experience high rates of AMR regardless of the method used to desensitize the
patient. AMR is a recognized risk factor in poor graft outcome in terms of graft survival and
glomerulopathy (Gloor 2010).
Table 5: Acute AMR incidence as reported in six separate studies
Study
Number of patients
(Lefaucheur 2009)
43
(Thielke 2009)
51
(Magee 2008)
28
(Haririan 2009)
41
(Vo 2008)
8
Adapted from (Gloor 2010)
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AMR incidence %
35
32
39
12
31
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Additional efficacy and safety concerns for various interventions have also been identified by
Gloor et al. Plasma exchange, supplemented by IvIG therapy, appears less effective than IvIG
therapy alone in patients receiving a deceased donor transplantation, as patents’ DSA levels will
return to pretreatment levels upon discontinuation of therapy (Gloor 2010). High-dose IvIG is also
associated with s afety c oncerns, i ncluding a dverse a dministration-related ev ents, a naphylactic
transfusion reactions and serious thrombotic events. Administration of large fluid volumes may
also be difficult in anuric patients undergoing renal dialysis (Gloor 2010).
Lastly, each of these interventions is associated with increased resource utilization, as well as
therapy c osts. J ordan e t a l. re ported t hat o ne i nstitution’s a verage p er p atient c ost f or a n
antibody lowering protocol was $35,540 (Jordan 2003).
4.4
Variance in post-transplant
immunosuppressant protocols and associated
costs
Advances in im
munosuppressive t herapies are g enerally c redited w ith c orresponding
improvements in hig her survival ra tes o f ki dney a llografts ( Haberal 2002; T urkowski-Duhem
2005), as further described below.
A retrospective cohort study of 164 patients by Toki et al. observed a statistically significant
difference in the incidence of graft loss according to the era in which the transplantation occurred
(era 1 vs. 2 vs. 3  12 vs. 2 vs. 0%, respectively; p=0.009, chi-square test) (Toki 2009). The
study defined eras by immunosuppressant regimen as described in Table 6.
Table 6: Immunosuppressive regimens, 1990-2007 (Toki 2009)
Era
1
Years
1990-2000
2
3
2001-2004
2005-2007
Regimen(s)
Cyclosporine or tacrolimus (FK)/azathioprine or mizoribine,
or mycophenolate mofetil (MMF)/methylprednisolone
(MP)/deoxyspergualin/antilymphocyte globulin
/splenectomy/irradiation
FK/MMF/MP/splenectomy
FK/MMF/MP/rituximab
Patients (n)
79
37
48
(Toki 2009)
As noted a bove, a preoperative t riple-drug i mmunosuppressive re gimen of seven days was
introduced in 2001 including FK, MMF, and MP. Additionally, three or four sessions of doublefiltration plasmapheresis were routinely performed to remove anti-A/B antibodies before
transplantation, a lthough this w as n ot c onducted postoperatively unle ss a patient d eveloped
acute AMR. Targeted maximal IgG/IgM titers at time of transplantation also differed between
eras: 1:16 until 2000 and 1:32 thereafter (Toki 2009).
A study of 154 renal allograft patients by Lederer et al. investigated the effect of
immunosuppressive drugs on antibody-mediated mechanisms impacting graft survival. The study
compared the production of anti-HLA antibodies and DSAs in kidney and pancreas transplant
patients receiving differing immunosuppressive regimens. Group 1 patients (n=60) had received
MMF since transplantation in combination with either cyclosporin A or tacrolimus and steroids.
Group 2 p atients (n= 29) ha d received an immunosuppressive re gimen of cyclosporin A,
tacrolimus and steroid initially, followed by the later addition of MMF. Group 3 patients (n= 65)
received cyclosporin A in combination with azathioprine or tacrolimus and steroids and no MMF.
Results revealed that 83.3 percent (50/60) of patients in group 1 had not developed HLA class I
or I I a ntibodies, w hereas a l ower p ercentage o f p atients i n g roup 2 a nd 3 d id no t d evelop
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antibodies (72.4 % (21/29) and 69.2% (45/65), respectively). The proportion of patients with
graft rejection episodes (acute and/or chronic rejection) was slightly higher in groups 2 and 3
(34.5% and 36.9%, respectively) than in group 1 (26.6%). However, group 1 patients suffered
from higher rates of cytomegalovirus (CMV) infections (43.3%) than group 2 and 3 (54.5% and
16.9%, respectively) (Lederer 2005).
Another area of investigation is the role of cytotoxic T lymphocytes, which may become resistant
to i mmunosuppressants. A s tudy o f 1 0 p atients b y va n Ka mpen e t a l. fo und a n a ssociation
between the presence of cyclosporine-resistant cytotoxic T lymphocytes and early graft rejection
and between c yclosporine-sensitive cytotoxic T l ymphocytes a nd a g ood graft survival in
transplantation p atients w ith a his torical p ositive c rossmatch. The s tudy c oncluded t hat t he
presence of activated cyclosporine resistant donor specific cytotoxic T lymphocytes was
associated with early graft loss and rejection, whereas cyclosporine-sensitive cytotoxic T
lymphocytes on t he d ay of t ransplantation were associated w ith g ood g raft o utcomes (van
Kampen 2002).
A s tudy p ublished b y G upta e t al. d istinguished b etween m ethods of t reating a cute c ellular
rejection and acute vascular rejection (AVR). While most cases of acute cellular rejection respond
to high dose steroids and/or antilymphocyte globulin, acute vascular rejection (AVR) does not
respond to increased levels of immunosuppression. The small series of 19 patients found that
patients with AVR plasma exchange and monoclonal CD3 antibody responded to treatment and
enjoyed n ormal re nal fu nction a t 4 t o 60 ( 27.8 + 20.1) m onths o f f ollow-up (Gupta 2 001).
However, the authors stipulate that the response noted was only evaluated prospectively in 19
patients, and that larger controlled studies are indicated to examine the relevance and role of
both plasma exchange and monoclonal CD3 antibody therapy (Gupta 2001).
Despite evidence of relative effectiveness of the more recent generation of immune suppressive
drugs, immunosuppresant protocols continue to vary and are marked by experimentation with
new agents. Several studies report using standard triple immunosuppressive protocols involving
prednisone/prednisolone, tacrolimus or cyclosporine, MMF or azathioprine, but these studies differ
in combination or agents used and doses a dministered (Kreijveld 2007) (Haberal 2 002).
Immunosuppression therapy involving monoclonal antibodies, such as rituximab (Rituxan) and
alemtuzumab (Campath 1-H), has also been reported (Kopchaliiska 2009) (Claas 2009b).
In addition, there are known risk factors associated with various immunosuppressive regimens. In
a study of 950 patients, Said et al. identified rare (1.26%) cases of hemolytic uremic syndrome
induced b y calcineurin inhibitors ( Said 2010). Turkowski-Duhem et al. observed th at th e
antiproliferative drugs are associated with hematological toxicities and that anticalcineurin agents
cause some degree of chronic nephrotoxicity, resulting in impaired renal function, both of which
are li nked t o a nemia (Turkowski-Duhem 2005) . In addition, in an e xamination of f actors
predicting post-transplant anemia (PTA), one multivariate analysis identified a significant
association between PTA detected at six months post-transplant and use of sirolimus therapy (OR
12.75 [1.16-140.54]; p= 0.04); note however that this association was not noted at 12-month
follow-up (Turkowski-Duhem 2005).
Moreover, t he effectiveness of a vailable i mmune s uppressive r egimens m ay b e i mpacted b y
medication noncompliance, which has previously been linked to graft failures due to rejection
(Cecka 2000). Cecka et al. hypothesize that the high cost of immunosuppressive drugs may
contribute to medication noncompliance. In the early 2000s, annual costs reported ranged from
$5,700 to $15,000 for standard maintenance immunosuppression (Kasiske 2000). In addition,
the costs of drugs for infection prophylaxis, hypertension and cholesterol may make immune
suppressive regimens cost-prohibitive for treatment centers (Cecka 2000), particularly as ESRD
ancillary drugs move into prospective payment bundle under Medicare.
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Literature Review: The Impact of Transfusion on Transplantation
5
Key Evidence Gaps
The authors of the studies reviewed for this report commented on key gaps in current knowledge
of the impact of transfusion on transplantation, as highlighted below.
5.1
Inconsistent definition of highly sensitized
patient
Various studies report different thresholds to establish the clinical definition of elevated PRAs and
high allosensitization. Soosay et al. 2003 report a threshold of 80 percent, whereas others report
thresholds of 10, 30, or 50 percent.
5.2
Impact of timing of PRA assays on PRA levels
Of the studies included in this report, only Pour-Reza-Gholi et al. 2005 (a single-center study)
reported the timing of the PRA assay in relation to dialysis procedures. To evaluate the potential
impact of the timing of PRA assay testing on levels and graft outcomes, additional studies may be
required.
5.3
Reporting of exact number of prior
transfusions
Of the studies reviewed to date that examine the impact of prior transfusions on PRA levels, only
one (Soosay 2003) specifies the exact number of prior transfusions. While Karpinski et al. cite
Opelz 1981 as support for five prior transfusions as the minimum cut-off to establish relevance,
Karpinski et a l. d o no t ind icate w hether p atients w ho r eceived p rior t ransfusions r eceived
between five and 20 prior transfusions, or more than 20 (the point at which the relationship
between prior transfusions and allosensitization may become more dependent) (Scornik 2009).
5.4
Under-reporting of true sensitization rates
As noted by Hardy et al., true sensitization rates may be under-reported in data sets that only
identify PRA levels for patients who are ultimately transplanted, as most patients with elevated
PRAs are not transplanted (Hardy 2001).
5.5
Limited data set for post-transplant outcomes
in highly sensitized patients
In addition, as a relatively low percentage of patients with elevated PRA receive transplants, there
is limited data to establish post-transplant mortalities, morbidities or overall survival in this subpopulation of interest.
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Literature Review: The Impact of Transfusion on Transplantation
5.6
Relevance of antibodies detected on newer
HLA assays
Donor-specific antibody and antibody subtypes detected on newer assays (e.g. Luminex®, FlowPRA®, and ELISA) are currently the subject of study and discussion (Bartel 2007; Toki 2009).
However, the presence of specific antigen subclasses continues to be considered a risk factor
rather than a contraindication (Claas 2009a). Further studies are necessary to clarify the actual
relevance of these findings since high-sensitivity assays, such as Luminex®, will lead to a higher
number of highly sensitized patients. A recent article commented that two steps should be taken
to resolve the issues presented by newer assays: (1) a general agreement should be reached on
the assignment of positive and negative antibody reactivity in Luminex®, and (2) a multicenter
study should be conducted to define the clinically relevant parameters for antibodies detectable
on Lum inex® (e.g. antibody t iter, i mmunoglobulin s ubclass, a nd c apacity t o fi x c omplement)
(Claas 2009a).
5.7
Reports of the positive impact of acceptable
mismatch programs on graft survival outcomes
in the US
Special p rograms e xist t o he lp hig hly s ensitized p atients fi nd a c rossmatch negative donor.
Examples include the Eurotransplant (ET) Acceptable Mismatch program, Save Our Souls in the
UK, and Regional Organ Procurement program in the US. An acceptable mismatch is defined by
an analysis of the HLA typing of panel donors. Donors carrying antigens to which the patient has
never formed antibodies are considered an acceptable mismatch (Claas 2004). A study of 112
highly sensitized patients receiving a kidney transplant revealed that acceptably mismatched (AM)
patients experience graft survival (87% at two years) identical to that of nonsensitized patients in
the ET program (Figure 15); n ote h owever that t his re port d id no t examine the num ber o f
sensitized patients enrolling and benefiting from similar programs in the US (Claas 2004).
Figure 15: Comparison of graft survival between acceptably mismatched patients (AM) and nonsensitized
(<5% PRA), sensitized (5-85% PRA) and highly sensitized (>85% PRA) patients
(Claas 2004)
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Literature Review: The Impact of Transfusion on Transplantation
6
Discussion
Although nephrologists treating ESRD patients may attempt to avoid unnecessary transfusions,
these p atients c ontinue t o re ceive b oth t herapeutic ( donor-specific) t ransfusions a s w ell a s
transfusions to achieve target hemoglobin levels. Retrospective analyses of patient data (1995–
present) reviewed in Section 1 of this report evidenced an association between pre-transplant
transfusion and high levels of sensitization in waitlisted kidney transplant candidates. While the
strength of the association varied across the studies, the authors consistently commented that
the ris k o f a llosensitization c ontinues to p ose a s ignificant c hallenge to the m anagement o f
waitlisted kidney transplant candidates. To provide evidence to address this ongoing challenge,
further studies may be required to investigate of the relationship between the number of pretransplant transfusions and the risk of allosensitization.
In addition, the current generation of high-sensitivity assays such as Luminex® will lead to a
higher num ber o f p atients id entified a s hi ghly s ensitized in t he fut ure. Because t he c linical
relevance of the specific subclasses of antigens detected on high-sensitivity assays has not been
established in multicenter studies, it is not yet possible to establish a contraindication to kidney
transplant b ased o n t he p resence of these antigens. A s a re sult, assessing t he i mpact o f
sensitization on graft survival will persist as a management challenge in ESRD.
In contrast, c andidates with preformed anti-HLA a ntibodies d etected on CDC assays are
contraindicated for transplant due to the risk of hyperacute rejection of kidney allografts. Pretransplant mortality in these patients (as reported in Section 3.3) means that the pool of available
patients with high PRA levels receiving a transplant is by definition smaller, which makes studying
the im pact o f s ensitization o n g raft survival e ven more d ifficult. De spite t his c hallenge, t he
evidence reviewed for this report (Section 3.4) revealed that highly sensitized patients who did
receive transplants experienced more rejection of allografts and shorter graft life. Differences in
graft survival by level of pre-transplant sensitization were consistently greatest at 10-year followup.
As noted above, some waitlisted kidney transplant candidates continue to receive therapeutic pretransplant (donor-specific) transfusions, despite findings suggesting a deleterious effect of this
practice on graft survival i n s ensitized a nd n on-sensitized patients ( Section 3.5). Ongoing
investigation into the benefits and risks of DST may have been prompted by the availability of
new i mmunosuppressants a nd e xperimentation w ith d ifferent re gimens. As a re sult, f urther
analysis may be required to evaluate whether DST is an independent risk factor for decreased
rejection and/or long-term graft survival.
While the evidence reviewed for this report highlighted gaps in current knowledge of the impact
of transfusion on transplantation, it is important to note that the appropriate management of pretransplant transfusion remains important in the treatment of the ESRD patient. As discussed in
Section 4 of this report, despite broad availability of leukoreduced RBCs, experimentation with desensitization protocols, and growing experience with immune suppressive regimens, sensitization
remains r elevant t o c linical p ractice. T he reduction o f unnecessary transfusions through
conservative management therefore continues to present an opportunity to address a known risk
factor impacting graft and patient survival in the pre-transplant phase.
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and End-Stage Renal Disease in the United States.
Vaidya S. (2005) Synthesis of new and memory HLA antibodies from acute and chronic rejections
versus pregnancies and blood transfusions. Transplant Proc. 37(2): 648-649.
van Kampen CA, Roelen DL, Versteeg-van der Voort Maarschalk MF, Hoitsma AJ, Allebes WA, et
al. (2002) Activated HLA class I-reactive cytotoxic T lymphocytes associated with a positive
historical crossmatch predict early graft failure. Transplantation. 74(8): 1114-1119.
Vo AA, Lukovsky M, Toyoda M, Wang J, Reinsmoen NL, et al. (2008) Rituximab and intravenous
immune globulin for desensitization during renal transplantation. N Engl J Med. 359(3): 242-251.
Confidential. For internal use only.
33
Page 285 of 290
Literature Review: The Impact of Transfusion on Transplantation
Appendix A Detailed Search Strategy
This p roject concerns t he i mpact of p rior t ransfusion o n t ransplantation, p articularly in
terms of the likelihood of graft rejection and reaction to antigens on the new tissue. As
such, the search facets proposed are:
•
Transfusion
•
Transplantation and graft survival
•
Antigens and histocompatibility
The overall search strategy is to produce sub-searches for each of these then combine
them and add any limits identified in the protocol (e.g. date limits).
Transfusion
Transfusion-related terms w ere id entified b y s earching t he MeSH i ndex f or t he t erm
transfusion and adding keyword terms. The transfusion searches are shown in Table 7.
Table 7: Search filters for transfusion
#
1.
2.
3.
Medline
“blood transfusion”[Mesh]
“transfusion”[All Fields]
#1 OR #2
Transplantation and graft survival
Terms were identified through a search of the MeSH index for transplant, cytotoxicity and
graft survival.
Table 8: String for transplantation
#
Medline
1.
“graft survival/immunology”[Mesh]
2.
“Transplants”[Mesh:NoExp]
3.
“Transplantation”[Mesh:NoExp]
4.
“Graft Rejection”[Mesh]
5.
“Transplant”[All Fields]
6.
“Cytotoxicity, Immunologic”[Mesh]
7.
#1 OR #2 OR #3 OR #4 OR #5 OR #6
Antigens and histocompatibility
Table 9 shows the antigens and histocompatibility searches.
Confidential. For internal use only.
34
Page 286 of 290
Literature Review: The Impact of Transfusion on Transplantation
Table 9: String for antigens and histocompatibility
#
1.
2.
3.
4.
Medline
“HLA Antigens/immunology”[Mesh]
“PRA/immunology”[Mesh]
“Histocompatibility Testing”[Mesh]
#1 OR #2 OR #3
Organ restriction
Given that the primary organ of interest is the kidneys, an additional facet was used to
restrict t he o verall s earch t o kid neys. This p rovides a n ind ication o f alternative s earch
approaches.
Table 10: String for organ restriction
#
1.
2.
3.
4.
Medline
“Kidney”[Mesh]
“Kidney”[All Fields]
“Renal”[All Fields]
#1 OR #2 OR #3
Combined search strings
The i ndividual fa cets c ombined p roduce 437 c itations w hen the kid ney r estriction i s
included (792 without kidney restriction).
To future refine results, HERON applied the kidney restriction and further limited to the
search strategy to ensure that the most recent and relevant information is retrieved.
Language limits:
•
English language only (406 articles total)
Date limits for waves of review:
•
First Wave: Past ten years of articles (62 citations)
•
Second Wave: Past twenty years of articles (165 citations)
•
Third Wave: Full history (406 citations)
Waves o f re view, c orresponding t o t he t ime p eriods s pecified above, a llow H ERON t o
concentrate on the most recent citations first. Subsequent wave(s) of review above were
omitted as earlier waves retrieved robust information. Of the 62 citations returned, 50
were identified as relevant upon a first pass detailed review of abstracts, and 40 were
identified as high priority upon second pass. Studies were excluded if the primary focus
was basic science, animal or pediatric research, or if the studies addressed only antibody
screening or diagnostic procedures.
Confidential. For internal use only.
35
Appendix D - Guidelines for Blood Transfusion
Appendix D - Guidelines for Blood Transfusion
Page 287 of 290
Page 287
Page 288 of 290
Appendix D – Guidelines for Blood Transfusion
Page 1
INTER/NATIONAL GUIDELINES
Source
UK Renal
Association-clinical
practice guideline
European Best
Practice Guidelines
(EBPG)
National Kidney
Foundation KDOQI™
Position on Pre-Transplant Blood Transfusions
Website (if applicable)
"We recommend that in patients with anaemia of CKD, especially those in
whom renal transplantation is an option, red blood cell transfusion should
be avoided if possible. (1A)"; "Also transplant recipient sensitisation may
occur following transfusion resulting in longer transplant register waiting
times and difficulty in finding a cross match negative donor. A study from
Ireland looking at causes of sensitisation of potential allograft recipients
showed that the level of sensitisation increased with the number of units
of blood transfused and also demonstrated a direct relationship between
degree of sensitisation and waiting time for transplantation.3 Blood
transfusions can induce antibodies to histocompatibility leukocyte
antigens that can reduce the success of kidney transplantation; thus
transfusions generally should be avoided in patients awaiting a renal
transplant.4"
"Red blood cell transfusions should be avoided, if at all
possible, in
patients with chronic kidney disease (CKD),
especially those
awaiting kidney transplantation.
(Evidence level B)"; "Transfusions
should not be given unless patients have
one or more of the
following:
(1) symptomatic anaemia (fatigue, angina, dyspnoea)
and/or
associated risk factors (diabetes, heart failure,
coronary artery disease,
arteriopathy, old age)
(2) acute worsening of anaemia due to blood
loss
(haemorrhage or surgery) or haemolysis
(3) severe resistance to, or
hyporesponsiveness to, ESA
therapy, e.g. due to the presence of a
haematological
disease or severe inflammatory systemic disease."
"Blood transfusions can induce antibodies to histocompatibility leukocyte
antigens that can reduce the success of kidney transplantation; thus,
transfusions generally should be avoided in patients awaiting a renal
transplant.267 If deemed essential, red blood cell transfusions in this
patient group should be conducted in line with published
recommendations.268"
http://www.renal.org/Clinical/Guideline
sSection/AnaemiaInCKD.aspx
Date
Accessed
12/17/2010
http://ndt.oxfordjournals.org/content/1
9/suppl_2/ii16.full.pdf+html
12/17/2010
http://www.kidney.org/professionals/k
doqi/guidelines_anemia/cpr34.htm
12/17/2010
Page 289 of 290
Appendix D – Guidelines for Blood Transfusion
Page 2
LOCAL GUIDELINES
Source
Position on Pre-Transplant Blood Transfusions
Website (if applicable)
Date Accessed
Northwestern
University, Dr. Dixon
Kaufman, Director of
Pancreas
Transplantation
Multiple random blood transfusions: Once, this was associated with
improved kidney transplant survival in the precyclosporine era.
Currently, transfusion offers no clinical benefit, and the risk of
sensitization is significant. In the setting of living kidney
transplantation, donor-specific transfusion therapy has also has
been almost completely eliminated.
WebMD: Renal transplantation
(Medical): Differential diagnoses &
Workup
12/17/2010
http://emedicine.medscape.com/article/
429314-diagnosis
12/17/2010
UCSF Transplant
Center
On-line policy on transplant procedures: Crossmatches are
obtained several times during preparation for a living-related donor
transplant, particularly if donor-specific blood transfusions are used.
A final crossmatch is performed within 48 hours before the
transplant.
http://www.ucsfhealth.org/conditions/kid
ney_transplant/diagnosis.html
12/17/2010
University of Wisconsin
Letter to the patients: "If you receive a blood transfusion, be sure
the blood is “filtered” to avoid developing antibodies (sensitizes you
for transplant). Call your coordinator if you receive any blood
products."
http://www.uwhealth.org/files/uwhealth/
docs/pdf/Volume5_2008.pdf
12/17/2010
VCU Health System
Hume-Lee Transplant
Center
Desensitization Protocol
About 30% of patients who are waiting for a kidney transplant are
sensitized, meaning that they have developed harmful antibodies in
their blood against foreign tissue. These antibodies can develop
through previous exposure to foreign tissue resulting from
pregnancies, previous transplants, or blood transfusions. This may
cause patients to wait three or four times longer than unsensitized
patients for a compatible deceased kidney.
http://www.vcuhealth.org/upload/docs/T
ransplant/pre_op_kidney_booklet.pdf
12/17/2010
Page 290 of 290
Appendix D – Guidelines for Blood Transfusion
Page 3
LOCAL GUIDELINES
Source
Position on Pre-Transplant Blood Transfusions
Website (if applicable)
Date Accessed
http://www.renalmd.org/legis.aspx?id=1
827
12/17/2010
Many sensitized patients have living donors that are willing to give
them a kidney, but the transplant has little chance of success. The
recipient’s blood, when mixed with the donor’s blood, reacts against
the donor’s cells because of the antibodies. This is a positive
crossmatch, which means that the recipient will likely reject the
kidney immediately following transplant. A negative crossmatch is
needed before a transplant can be performed.
There is a process that allows the antibodies to be removed from
the recipient’s blood called desensitization. This involves the patient
undergoing plasmapheresis treatments to help remove the harmful
antibodies from the blood. Your doctor will discuss this option in
more detail with you if it is needed.
Renal Physician’s
Association
RPA: ESA use for nondialysis CKD patients with Hb<10 gm/dl
reduces the need for transfusions and may improve patient
reported outcomes. Particularly for patients who are candidates for
kidney transplantation, avoidance of blood transfusions may reduce
presensitization and improve the likelihood of finding a good donorrecipient match.