Download HLA typing in transfusion and transplantation

Significance of HLA
in Transfusion and
Transplantation
Terry Aly
Clinical Pathology Conference
February 8, 2008
Main Topics
• HLA System Overview
• HLA and Transfusion
– Alloimmunization
• Refractoriness to platelets
• Graft rejection
– Transfusion-Associated Graft-Versus-Host Disease
(TA-GVHD)
– Transfusion-Related Acute Lung Injury (TRALI)
• HLA and Transplantation
– Bone marrow
– Cord blood
– Solid organ
The Human Leukocyte Antigen
(HLA) System
• Essential to immune function: HLA molecules
present peptide antigens to the immune system
(T-cells)
• Important for self versus non-self distinction
HLA Class I Monitors Inside of the Cell
Tapasin
CR
CN
Dr. Brian Freed
HLA Class II Monitors Outside of Cell
Peptides
Extracellular
Proteins
DM
monitors
peptide
specificity
for DR
DM
Dr. Brian Freed
HLA Class I and II Molecules Have a Distinct Structure and Function
•Binds 8-10mers
•Expressed on most
Nucleated cells
•Presents Cytosolic
Proteins to CD8+ T cells
•Binds 13-25mers
•Expressed on APCs,
Macs, B cells, activated
T cells
•Presents Vesicular
Proteins to CD4+ T cells
 
 
2 3
 
Class I
Class II
BDC
Antigen–presenting cells (APCs):
monocytes, macrophages, dendritic cells, B cells
Teaching slides: www.barbaradaviscenter.com
Humoral
Versus
Cellular
Immune
Response
Class I (1.1 Mb)
Class III (0.7 Mb)
Class II (2.2
Mb)
Complement
& cytokines
Gene
low high resolution typing
“subtype”=01
Allele:
Haplotype:
Genotype:
HLA-DRB1*0401
HLA-DRB1*0401
HLA-DQB1*0302
HLA-DRB1*0301
HLA-DQB1*0201
DRB1*02
HLA-DRB1*04
HLA-DQB1*0302
J. Noble
Why care about HLA type for
transfusions?
• Alloimmunization with increased risk for platelet
refractoriness and humoral transplant rejection
– Recipient can make antibody against donor HLA
antigen, most common
• Transfusion-Related Acute Lung Injury
– donor HLA antibodies react against recipient antigens
• Transfusion-Associated Graft-Vs-Host Disease
Alloimmunization
• Alloimmunization: Development of
antibodies after exposure to non-self human
antigen
– HLA on leukocytes or platelet specific antigens
– May occur with transfusions (PRBC>platelet
because PRBC contain more leukocytes),
pregnancies, or transplants
Antigen exposure associated with leukocytes in
transfusion, pregnancy, or transplantation
Alloimmunization
Resolves
Spontaneously
(Transfusion Ab resolve
more than pregnancy Ab)
Refractoriness
to platelets*
Humoral
transplant
rejection
*Antibodies can react with Class I HLA (most common), ABH
antigens, or platelet antigens that exist on surface of platelets
How to prevent alloimmunization?
Trial to Reduce Alloimmunization to Platelets (TRAP) study
• Leukoreduction (LR) effect
– Removes 99.9% of leukocytes, leaving < 5*106 per unit
– Only 19% of AML patients receiving leukoreduced
platelets developed lymphocytotoxic antibodies vs. 45%*
receiving unmodified platelets
*alloimmunization rate likely higher: current methods
detect more alloantibodies than previously
The Trial to Reduce Alloimmunization to Platelets Study Group. N Engl J Med. 1997.
How to prevent alloimmunization?
• UV irradiation was equally effective in TRAP
study (prevents lymphocytes from
proliferating or stimulating), but blood banks
typically use Gamma instead of UV b/c can
penetrate plastic bags
• LR or irradiation decreased alloimmunization
from 13% to 3-4% in previously unexposed
patients
The Trial to Reduce Alloimmunization to Platelets Study Group. N Engl J Med. 1997.
UVB-PC: UV B-irradiated, pooled platelet concentrates, random donors
F-PC: Filtered pooled platelet concentrates, random donors
F-AP: Filtered apheresis, single random donor
16%
10%
8%
Leukocyte reduction and ultraviolet B irradiation of platelets to prevent
alloimmunization and refractoriness to platelet transfusions. The Trial to Reduce
Alloimmunization to Platelets Study Group. N Engl J Med. 1997.
Who needs alloimmunization
prevention?
• Give LR or irradiated blood products to
patients who are:
– chronically transfused (20-70% become
refractory)
– immunoincompetent
– pre- and post-transplant
• All transplant patients? Include kidney?
• Time-frame?
– everyone??
Prestorage Universal
Leukoreduction in Canada
• Universal prestorage leukoreduction
(ULR) of red cell and platelet products
has been performed in Canada since
August 1999 (and UK too)
Seftel MD, Growe GH, Petraszko T, et al. Universal prestorage
leukoreduction in Canada decreases platelet alloimmunization and
refractoriness. Blood. 2004 Jan 1;103(1):333-9.
Prestorage Universal Leukoreduction
(ULR)—Canada study
• Retrospective analysis of 13,902 platelet
transfusions in 617 patients undergoing
chemotherapy for ALL or stem cell transplantation
before (n=315) and after (n=302) ULR instituted
• Alloimmunization was significantly reduced (19%
to 7%, P <.001) in the post-ULR group.
• Alloimmune platelet refractoriness was similarly
reduced (14% to 4%, P <.001).
Prestorage Universal Leukoreduction
(ULR)—UK study
• ULR reduced TA-GVHD rate in
immunocompetent individuals from 13 prior to
ULR to 0 after ULR
• ULR reduced post-transfusion purpura rate from
10.3 per year to 2.3 per year
• Alloimmunization was significantly reduced (19%
to 7%, P <.001) in the post-ULR group.
• Alloimmune platelet refractoriness was similarly
reduced (14% to 4%, P <.001).
Refractoriness
• Repeated failure to achieve the
expected increment in platelet count
after 2 or more platelet transfusions
• % Maximum increment =
([pretransfusion count – posttransfusion
count in platelets/µL] X blood volume in
mL) ÷ (number of platelets transfused,
ie, number of U X 6 X 105)
Slichter SJ. Evidence-Based Platelet Transfusion Guidelines Hematology 2007
Refractoriness
• Can be due to:
• Alloimmunization to HLA or platelet antigen
• ABO mismatch
• Non-immune factors such as insufficient
dosing, poor platelet quality, hypersplenism,
accelerated consumption, sepsis, DIC,
GVHD, and bleeding
Slichter SJ. Evidence-Based Platelet Transfusion Guidelines Hematology 2007
Management of Refractoriness
• Stage 1: Check dosing
• Stage II: Try ABO matched platelets <48 hours
old
• Stage III: If urgent, give cross-matched platelets.
Screen for anti-HLA and anti-platelet antibodies
– Antibody-mediated or not? Solid-phase assay
– HLA antibody mediated or not? Panel reactive antibody
(PRA) as general screen for HLA Ab positivity.
– If positive, single antigen testing with microparticle
beads to ID which antigen(s) to avoid in donors
Slichter SJ. Evidence-Based Platelet Transfusion Guidelines Hematology 2007
ABO matching prevents
platelet refractoriness
Table 1 -- Refractoriness and alloimmunization rates after transfusing ABO matched versus mismatched platelets
New antibodies
Platelet transfusions
Enrolled
Female patients
Possible prior sensitization[a]
Platelet transfusions[b]
Platelet refractoriness[c]
ABO matched
13
10 (77%)
9 (69%)
7 (5–9)
1 (8%)
ABO mismatched
13
2 (15%)
4 (31%)
9 (4–30)
9 (69%)
P value
0.001
Data from Carr R, Hutton JL, Jenkins JA, et al. Transfusion of ABO-mismatched platelets leads to early platelet refractoriness. Br J Haematol
1990;75:408–13
Anti A/B[d]
0
7 (54%)
Anti-HLA
Platelet-specific
1 (8%)
1 (8%)
5 (38%)
4 (31%)
Problems with HLA Typing
and Matching
• HLA matching requires the availability of large
numbers of HLA-typed donors. A registry of about
18 000–25 000 HLA-typed people is needed to
provide at least five HLA-A and HLA-B matched
donors for 80% of white patients.
• HLA-matched platelets requires the recruitment of
specific donors, such platelets can only be
obtained by apheresis
• Expensive
K Takahashi, T Juji and H Miyazaki, Determination of an appropriate
size of unrelated donor pool to be registered for HLA-matched platelet
transfusion, Transfusion, 1987.
Problem with cross-matching
to find compatible platelets
• Limited to units available for testing
• 5 day life to platelets, future transfusions
will require re-crossmatching
Antibody Specificity Prediction
The antibody specificity prediction (ASP) method of
donor selection for refractory alloimmunized patients
appears as effective as HLA matching or
crossmatching. Far more donors are identified in a
file of HLA-typed donors by the ASP method than by
HLA matching, and this indicates that the ASP
method provides important advantages regarding the
availability of compatible platelet components.
Percentage of platelet recovery
is similar for HLA-matched,
cross-matched, and antibody
specificity prediction method
matched donor platelets
Petz LD. Selecting donors of platelets for refractory patients on
the basis of HLA antibody specificity. Transfusion. 2000.
PRA=panel reactive antibodies
Cross-Reactive Groups
• Systems have been developed to match donor and
recipient by assigning HLA-A and HLA-B
antigens with shared public epitopes to clusters
called cross-reactive groups (CREGs).
• When one or two mismatches of HLA-A or HLAB antigens in CREGs is permitted, a pool of 1000–
3000 donors will meet the transfusion needs of
most white patients
HLA Matchmaker
• Based on the principle that short three-amino acid
sequences characterize polymorphic sites of the
HLA molecules, and are the critical components of
allo-sensitizing epitopes
• Retrospective study has shown that platelets
selected with this algorithm result in higher posttransfusion count rises than those selected using
traditional HLA matching strategies
A Nambiar, RJ Duquesnoy and S Adams et al., HLAMatchmaker-driven analysis
of responses to HLA-typed platelet transfusions in alloimmunized
thrombocytopenic patients, Blood 107 (2006), pp. 1680–1687.
Clinimmune
• Testing available for:
• Clinical Transplantation- solid organ and
hematopoietic stem cell
• Platelet Transfusion support
Clinimmune
• HLA-A, B, Cw, DRB, DQB1, DPB1 loci
– Low to high resolution testing for HLA-A, B, Cw,
DRB1,DRB3/4/5, DQB1
– Intermediate to high resolution testing for HLA-DPB1
– Serological and molecular testing
• HLA Antibody Detection
– PRA=Panel Reactive Antibody, including HLA Specificity
Analysis
• Luminex
• ELISA
• Complement Dependent Cytotoxicity, including AHG (anti-human
globulin)
– Titration/Quantitation studies for pre-transplant immunotherapy
protocols
– Crossmatching for transplant compatibility
• Flow cytometry
• Complement Dependent Cytotoxicity, including AHG (anti-human
globulin)
From Clinimmune website
Other options to treat
refractoriness?
• For highly alloimmunized patients and those with
rare HLA types, finding compatible platelets can
be difficult.
• Several immune-modulatory therapies tried:
intravenous immune globulin; cyclosporin A;
vinblastine; staphyloccal protein A; removal of
HLA antigens with citric acid.
• Despite anecdotal positive outcomes, these
strategies are usually not successful or practical
Transfusion-associated graft-vshost disease
• Depends on
– immune system function in recipient
– lymphocytes in transfused component
– degree of HLA similarity between donor and
recipient
• Donor lymphocytes not recognized as foreign if
HLA similar
• More common with related donors
– Especially if donors homozygous for shared HLA
haplotype with recipient
Transfusion-associated graft-vshost disease
• Caused by persistent chimerism after blood
transfusion
– Transfused lymphocytes avoid destruction by host
immune system, proliferate, and attack host organs
– Causes skin rash (histo “satellite dyskeratosis”), fever,
liver dysfunction, diarrhea, bone marrow dysplasia
– Occurs 4-30 days post-transfusion, >90% fatal
– In 1980, incidence in patients with hematologic cancers
or lymphoproliferative disorders 0.1-1%, now lower
due to leukoreduction and irradiation; more rare if not
immunocompromised
www.up-to-date.com/TA-GVHD
TA-GVHD more common in
immunocompromised patients receiving
directed donor HLA-matched products,
but can occur in immunocompetent
individuals and can be random donor
with high HLA similiarity
For example……
• Common “ancestral” haplotype
–DR3, B8, A1 haplotype
–In 9% of chromosomes in the
Caucasian population, and 18%
of chromosomes in Caucasians
with diabetes (Alper 2006)
–Diabetic population at risk for
kidney transplantation
Illumina 2: 24 DR3B8A1 (8.1) Homozygous Individuals
Column=individual; Row=SNP; Yellow=Homozygous region for 8.1 alleles
Gray=non-8.1 allele; White=8.1 allele; Dark Gray Bar=1st SNP homozygous for non-8.1 alleles
telomere
25.2 Mb
7.6 Mb
conserved
HLA-F
HLA-A
DQB1
32.8 Mb
A
A
B
A
B
A
B
A
B
A
B
A
A
A
A
B
B
A
B
A
A
B
B
A
B
B
B
B
A
B
B
A
A
A
A
B
B
A
B
A
B
A
B
A
B
A
B
B
A
A
A
-A
-A
B
B
A
B
A
-A
B
B
A
B
A
B
B
A
B
A
-B
-A
B
B
A
B
A
-B
-A
B
B
A
B
A
B
A
A
B
A
A
A
B
A
B
B
A
A
B
A
B
A
A
B
B
A
B
B
B
B
A
B
A
B
A
B
B
A
B
A
B
A
B
A
A
B
A
B
A
B
A
B
A
A
A
B
A
A
B
B
A
A
B
A
B
A
B
A
B
A
B
B
B
A
B
B
A
B
A
A
B
B
A
B
A
B
A
B
B
A
B
A
B
B
A
A
B
A
A
A
B
A
B
A
A
B
A
A
B
A
B
A
B
A
B
B
B
B
A
A
B
A
B
A
B
B
A
B
A
B
A
B
A
B
A
B
B
--A
A
B
A
-A
A
B
A
A
-B
A
A
A
B
-
A
B
B
B
--A
A
B
A
-A
B
B
A
B
B
A
-B
A
-A
B
B
-
-A
A
B
A
B
A
B
A
-A
-B
A
A
B
A
B
-A
B
A
-A
B
A
B
-
-B
B
B
A
B
A
-B
B
A
B
B
-A
B
A
B
A
B
-
A
B
A
B
B
A
B
A
A
B
A
B
A
A
A
A
B
A
A
B
A
B
A
B
B
B
A
B
A
B
B
A
B
A
B
B
A
B
A
B
A
B
A
B
A
B
A
A
B
A
A
B
A
A
A
B
A
A
A
B
B
A
B
A
B
A
B
B
A
A
B
A
A
B
B
B
B
A
B
A
B
A
B
A
B
A
B
B
A
A
B
B
A
B
A
A
B
A
B
A
A
B
B
A
B
B
B
A
B
A
B
B
A
B
B
B
B
A
B
A
B
A
B
A
B
A
B
A
B
B
A
A
A
B
A
B
A
A
B
A
B
A
A
B
B
B
A
B
A
B
B
A
A
B
B
A
B
A
A
B
A
B
A
B
A
B
A
B
A
A
A
A
A
B
A
B
A
A
B
A
A
A
B
A
B
A
B
B
A
B
B
A
A
B
B
B
B
A
B
B
A
B
A
B
A
A
B
A
A
A
B
A
A
B
A
A
A
-A
B
A
B
A
B
A
B
B
A
B
B
A
A
B
B
A
B
B
-B
A
B
A
B
A
B
A
B
A
B
A
B
A
A
A
A
B
A
B
A
A
B
A
B
A
B
A
B
A
B
B
A
B
A
B
B
A
B
B
B
B
B
A
B
A
B
A
A
B
A
A
A
B
A
A
B
A
A
B
A
-B
A
A
B
B
A
B
B
A
B
A
B
A
A
B
B
A
B
B
A
B
-B
A
A
B
B
A
B
A
B
B
A
A
A
B
A
B
A
A
B
A
B
A
B
A
B
A
B
B
B
A
B
A
B
A
B
A
B
A
B
B
B
A
B
A
B
A
A
B
A
B
A
A
A
A
B
B
A
A
B
A
A
B
A
A
B
B
B
B
B
A
B
A
B
A
B
B
A
B
A
B
A
B
A
B
A
B
A
B
A
A
A
A
A
B
A
A
B
A
A
B
A
B
A
B
B
A
B
B
A
B
A
B
B
B
A
B
B
B
A
B
A
B
A
B
A
B
A
B
B
A
A
A
B
A
B
A
B
A
A
B
A
B
A
B
A
A
B
A
B
A
B
B
A
B
A
B
A
B
B
A
B
B
A
B
A
B
A
A
B
A
B
A
B
B
A
A
B
A
A
A
B
A
A
B
A
B
A
A
B
B
A
B
A
B
B
A
B
B
A
B
B
A
B
B
A
B
A
B
B
A
B
A
B
A
B
A
B
A
A
B
A
B
A
B
A
B
A
A
B
A
B
A
A
B
B
A
B
B
B
A
B
A
A
B
B
A
B
B
A
B
A
B
A
B
B
A
B
A
B
A
A
B
A
A
A
A
B
A
A
B
A
B
A
A
B
B
A
B
B
B
B
A
B
A
B
A
B
A
B
B
B
B
A
B
A
B
A
A
A
B
A
B
A
A
A
B
A
A
B
A
B
A
A
B
A
B
A
B
A
A
B
B
B
B
A
A
B
B
A
B
B
B
B
A
B
A
B
B
A
B
A
A
A
A
A
B
A
B
A
B
A
B
A
A
B
A
B
A
A
B
B
A
B
B
B
B
A
B
A
B
B
A
B
B
A
B
B
A
B
A
B
A
Case study
• 31 yo F receives kidney transplant from her father,
matched for all but one antigen, DR7
• 2 wks later, she needs PRBC transfusion
– Family wants her to receive directed donor transfusion
from father
• Should a directed donor transfusion from her father be
permitted?
• If so, should it be leukoreduced or irradiated?
• If not, should a random donor blood product be leukoreduced
or irradiated?
• Should the guidelines for kidney transplant patients differ from
those of heart, lung or bone marrow transplant patients?
• What if patient is known to have one HLA antibody formed
against transplant?
Follow-up Information
• Received directed donor routine PRBC
given from father (not leukoreduced or
irradiated)
• Humoral transplant rejection diagnosed
• PRA antibody negative prior to transfusion;
DR7 antibody detected post-transfusion
HLA and Transplantation
Bone marrow
• HLA class I and II matching High
resolution
• Aim for at least 9/10 HLA alleles matching
HLA and Transplantation
Cord Blood
• Low resolution HLA class I match; highresolution class II HLA match
• Safer than bone marrow transplant because
cord blood induces less reaction
• Aim for at least 4 out of 6 matching for
transplantation
HLA and Transplantation
Solid organ
• Lung: Class II HLA match
• Heart: Class I HLA match on case-by-case basis,
no class I
• Liver: No HLA typing indicated
• Kidney: Class I and II HLA matching but O.K if
only 1 or 2 out of 6 matching HLA alleles because
prognosis depends more on timing of transplant;
prefer negative cross-matching strategy