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Canadian Blood Services
Transfusion Medicine Resident Topic Teaching Session:
Trojan Antibodies and Transplant Malware:
Improbable Sensitizations
&
Hemolytic Passenger Lymphocyte Syndromes
Tuesday, October 5th 2010, 12:00 – 1:00 pm
Conference Dial-In: 1-866-752-7690, Passcode 1775282
www.transfusionmedicine.ca
Christine Cserti-Gazdewich, MD FRCPC
Assistant Professor, University of Toronto
Departments of Laboratory Hematology (Pathology) & Clinical Hematology (Medicine)
Blood Transfusion Laboratory, University Health Network
Toronto, Ontario, Canada
office 416-340-5390, pager 416-790-9597, email [email protected]
NO CONFLICTS OF INTEREST. FUNDING FOR RESEARCH FROM PUBLIC FOUNDATIONS ONLY.
1
Princess
Margaret
Hospital
Toronto
General
Hospital
Toronto
Western
Hospital
University Health Network
Holland
Ortho &
Arthritic
Centre
Ontario Regional Blood
Coordinators
provincial blood management
Sunnybrook
Hospital
Women’s
College
Hospital
Canadian Blood Services
Sunnybrook Health Sciences Centre
St Michael’s Hospital
Hospital for Sick Children
national blood provider
Ontario
Transfusion
Coordinators
provincial blood conservation
Mount Sinai Hospital
TRANSFUSION MEDICINE COLLABORATIVE
University of Toronto teaching hospitals:
Hospital Transfusion Services
Trillium Gift of
Life Network
provincial organ network
2
Goals
•
Re-orient to sensitization biology in transfusion
medicine
•
Appreciate what is known about hemolytic
passenger lymphocyte syndromes (PLS) in
transplantation
•
Note some twists in ABO-incompatible stem cell
transplantation
•
Consider new insights into “accommodation”
3
It All Starts With This One Tube…
plasma
4
ABO Types in the General Population
A donor plasma:
• anti-B IgM
40%
45%
O donor plasma:
• anti-A,B IgG
• anti-A & anti-B IgG & IgM
A
B
AB
O
4%
AB donor plasma:
• no anti-A or anti-B
11%
B donor plasma:
• anti-A IgM
5
30 blood group antigen systems
(ABO, Rh, …) containing structures that are
naturally “polymorphic”
(278 antigens + 38 in “collections/series”)
6
Who Are The Antibody-Makers Anyway?
• anyone whose immune system has seen a foreign red cell
at any other time in their lives
– the parous (85% of child-bearing age females)
• feto-maternal hemorrhage (FMH), especially at parturition
– any transfusion history
• up to 1-3% of the healthy donor population
• up to 1/3rd of those with a hospital admission history
– any tissue grafts or transplants (solid organs,
hematopoietic progenitor [stem] cells), inevitably
contaminated with passenger erythrocytes
7
“Antibody Makers”
• 13% of people are “antibody makers”
• difference between -makers and -others appears, at least in
part, to be absence vs integrity of T-reg-suppressive activity
8
Guy (Gal) Walks In The Door…
What Are The Chances?
EXPOSURE
X
INTRINSIC
SEROCONVERSION
RISK
X
HOW RECENT &
HOW DURABLE
THE ANTIBODY IS
1 – 5 % of the population has a positive red cell antibody screen
9
Targets for
Graft versus Host
Attack
HLA Antigens (Platelets, Tissues)… ie- GVHD
Red Cell Antigens… ie- Passenger Lymphocyte Syndrome (PLS)
10
Diversity (Polymorphisms) Set Up The
Tissue Incompatibility Fight
extensive
variations
therein…
ALLOIMMUNIZATION
11
Diversity (Polymorphisms) Set Up The
RBC Incompatibility Fight
Kell: K vs k, others
ABO
Duffy: Fya vs Fyb
Rh(D)
Kidd: Jka vs Jkb
complete
absence or
presence
ISOIMMUNIZATION
etc: MNS, P, Lutheran, Lewis,
Diego, Scianna, Dombrock,
other Rh(CE) Colton, LW, Chido/Rodgers,
Gerbich, Cromer, Knops, Indian
antigens:
C vs c
E vs e
subtle (eg
SNP)
variations
therein…
ALLOIMMUNIZATION
12
The Vocabulary of Immune
Violence
MAJOR vs MINOR incompatibility
ACTIVE vs PASSIVE immunity
13
Directions of Incompatibility
• MAJOR
• “MINOR”
– incoming donor’s
cellular antigens are the
incompatible target
– host-source immunity
mounts the attack
– donor-source immunity
mounts the attack
– PRODUCT REJECTION
by a fighting recipient
– HOST REJECTION
by a fighting donor
• eg. ABO incompatible RBC
mistransfusion
– A into O: donor A cells
destroyed by host O
plasma
– host’s cellular antigens are
the incompatible target
• eg. GVHD from a solid
organ transplant, a
transfusion, or a BMT
• eg. TRALI
• eg. acute rejection of
organs
14
Active vs Passive Immunity
• ACTIVE
n∞
• PASSIVE
– renewable humoral
(via cellular) fighting
power
all forms of
MAJOR
incompatibility
recipient
plasma cell
– finite (humorally
contained) fighting
power
some forms of
MINOR
incompatibility
passenger
donor
plasma cell
n=x
graft*
most forms of
MINOR
incompatibility
product
15
GVHD: What does it take?
• living passenger lymphocytes
• proliferative stimulus: something recognizably
different in the host to attack
• invulnerability: some way for the passengers to “get
away with it”
– host too immune suppressed
– host “immune oblivious” (eg. homozygous haploidentical
donor indistinguishable from self)
16
passenger
donor
plasma cell
graft
host RBC
Passenger Lymphocyte
Syndromes
minor incompatibility scenario of donororigin (graft-associated) alloimmune
hemolytic anemia
of host RBCS
aka. HUMORAL GRAFT VERSUS HOST DISEASE FOR THE BLOOD BANK
17
PLS – The Implicated RBC Antigen
Spectrum
(Usual Victims)
555-fold
• ABO > Rh > minor RBC antigens:
– probability of minor discrepancy
• 25% for ABO vs <15% for Rh
– presence of preformed Ab or primary historical sensitizations
• ~100% for ABO vs 0.3% for Rh(D)
• Rh: usually from donors who have been alloimmunized
previously
– prior donor alloimmunization “almost necessary”
18
First Reports of Anti-RBC PLS
• 1964 – 1st hemolytic ABO PLS Marchioro, NYAS
• 1985 – 1st hemolytic anti-D PLS Swanson, Transfusion
• 1990 – 1st multiorgan (L, K) PLS Ramsey, aaBB
19
PLS – “Lymphoid” Load Spectrum
(Usual Aggressors)
kidney
(heart-) lung
small bowel,
pancreas
HPC:
PB > BM
liver
• dead or alive…
• graft (usually) unaffected
• at least 10 6 – 10 7
20
Timing For “Serologically
Expressive” Chimerism
• median onset: 9 days (5 – 17)
• median duration: 4.5 weeks (2 – 23)
• undetectable by 3 months
• the records:
– fastest onset: 12 h
– most delayed onset: 45 d
– longest persistence: 2 years for Ab (2 mo for HA)
21
The Role of Incidental HLA Matching
(“graft stealth” by tolerance of
inadvertently HLA-similar passengers)
• borne out in TA- & SOT- GVHD
literature
• anti-RBC PLS may also be more likely
with inadvertent HLA matching
22
Things That May Make it Worse
• density of vulnerable antigen
– group A recipients
• use of non-B-cell suppressive IST
– cyclosporine
• pro-hemolytic ± complement-fixing capabilities of antibody
• inflammation / cytokines
– TNF-α & IL-1 (conditioning or ischemic injuries)
– co-stimulatory interactions: CD40 - CD154, CD28 - B7
– IL-2 RαCD25-dependent clonal expansion with Th1 – CTL
response
– overly receptive RES/MPS
23
Things That May Make it Better
(or Preventatively Subdue it?)
• post- (or pre-?) transplant graft irradiation
• reducing immunosuppression = de-repression revenge
• evolution towards non-hemolytic antibody properties
• promotion of activity of inhibitory FcR
• “non-hemolytic red cell antigen loss”
• senescence of passenger lymphocytes
• Ag-Ab excesses
 negative feedback to memory B cells
24
Transfusion Care in PLS
• issuing antigen-negative blood, compatible with respect to
graft-origin antibodies, and not just the same blood type as
the host
• failure to do so = “Incorrect Blood Component Transfused”
(IBCT) medical error event… +/- ABO mis-transfusion
• top-up transfusions… or therapeutic red cell exchange (TREX)
25
Is There a Role for Apheresis?
Red cell
replacement of
removed plasma
(hybrid TPRE)
Whole blood removal
eg. manual red cell exchange,
replacing with pRBC ±
plasma/NS/albumin
(reconstituted whole blood)
Therapeutic red
cell exchange
(TREx)
Top-up
transfusion
26
When Sh/(C)ould Therapeutic Plasma
Exchange Be Offered?
• removal of pathologic antibody
– IgM > IgG
– high titer > low titer
• “detox” of RBC breakdown products
– for extreme cases with intravascular hemolysis
• plasma free hemoglobin: ? danger thresholds ?
– massive acute hemolysis (MAH):
free plasma hemoglobinemia > 60 µmol/L
= > 387 mg/dL (N: 0.5 – 5 mg/dL)
• stromal factors & DIC
27
Other Management Options
• rituximab
• graft irradiation
• splenectomy
• cytotoxic agents, prednisone adjustments
• combination therapy: RBC/TREX, IST, apheresis, splenectomy
28
A Passenger Lymphocyte
Traceback Story…
Why does my D+ patient now have anti-D after transplant?
-passive sensitization?
-partial D with alloimmunization?
-active (passenger lymphocyte-mediated) sensitization?
29
case 2
case I
case 3
30
Case 1:
The good news:
ABO compatibility…
Donor: “A Negative”
Recipient: “A Positive”
The bad news:
the seeming Rh(D)-compatibility isn’t
31
Case 1
• 66F presented for a R SLT for COPD
• group & screen pre-op:
– A, Rh(D)+
– screen negative amid a history of A+ transfusions
• group & screen POD17 in the pre-transfusion
investigations of her new hemolytic anemia:
– A, Rh(D)+
– screen: anti-D & anti-C
– DAT: anti-D & anti-C
32
Case 1 Traceback of Donor
• elderly multiparous female
• group A, Rh(D)-negative (rr)
• screen:
Anti-C Anti-D Anti-E
33
RECIPIENT:
Serologically:
C+ D+ e+
c- E-
Weiner designation: R1 R1
(17% of population)
Anti-C
Anti-D
Anti-E
DONOR:
Serologically:
c+ e+
C- D- E-
Weiner designation: r r
(15% of population)
passenger
lymphocyte
34
anti-D on IAT
216
anti-C on IAT
123
217
anti-D on DAT (eluate)
anti-C on DAT (eluate)
clinically significant hemolytic
anemia (depleted haptoglobin,
increased LDH & bilirubin)
10
therapeutic plasma exchange
67
39
60
36 - 40
110
160
210
days post transplant
35
Case 1: Indirect Attributable Mortality
Due to PLS
• POD 21, Hb 6.0 g/dL:
– massive AMI (“demand-ischemic” with rAF)
– grade IV EF
 left with refractory CHF
 cardiac dysrhythmiae & fluid imbalances
• died day 330 post transplant
= 10 months later
= 113 days after last detectable anti-D
36
Case 2: Recipient of the Contralateral
Donor’s Lung:
• 43 y old female presented for L SLT 9/28/07
• A, R1 r , negative pre-transplant screen
• lifelong transfusion-free history
• screened monthly due to Case 1 patient
37
reaction strength
Case 2:
Novel Serological Course
4
serologically positive
3
120
DAT
IAT
159
2
acute
rejection
1
173
0
0
100
TPE
259 266
200
days post-transplant
300
38
Case 3: It happened again!
RECIPIENT 3: ♂
DONOR 2: ♂
O, R1 R1
( CC DD ee )
anti-C
anti-C
O, r r
anti-D
anti-D
( cc dd ee )
anti-V
0
• 30 year old male presented for DLT for CF
• RBC antibody screen negative pre-operatively
• RBC antibody screen + POD 23
– anti-D on IAT
– anti-D & anti-C on eluate of DAT
• no hemolysis
• ongoing persistence at follow-ups (> 6 mo)
39
Why Did anti-D PLS Happen So Often
In Our Series?
• expected:
– (13% risk of D- to D+) x (2% risk of anti-D) x (100% transfer) =
0.3%
• observed:
– 3/92 or 3% [95% CI: 1 – 9 %]
• 100% transfer did indeed occur based on lookback
• 3 donors were found to be D-sensitized
– of a denominator of 12 D- donors, rate was 25% [ 6 – 57%,
95% CI]
(>12x higher than expected… )
40
Update: 10 Sequential Cases at the UHN
Over 3.87 Years of Surveillance (q 25 wk)
Implicated
organ
Donor
antibodies
Cognate recipient
attack
Significant
hemolysis?
Lung
Lung
Lung
Anti- C, D, E
Anti- C, D, E
Anti- C, D, V
C, D
D
C, D
Liver
Lung
Liver
Liver
Anti- B
Anti- B, Jka, N
Anti- D
Anti- B
B
B, Jka, N
D
B
Liver
Liver
Anti- A
Anti- A (IgM)
A
A
yes
yes
Lung
Anti-A
A
yes
yes
no
no
yes
yes
yes * (day 798)
yes
41
The Only Prospective Post-Solid Organ
Tranplant Sensitization Surveillance
Study in the Literature:
aaBB Abstracts 2007
– 27% of the ABOi KTs developed +DAT
• checked q 10d x 3 checks post-operatively
42
Types Of HPCT ABO Incompatibility
R
D
Type
O
O
O
A
B
A
B
AB
AB
AB
Major
A
B
AB
AB
AB
O
O
O
A
B
Minor
A
B
B
A
Both
O
A
B
AB
O
A
B
AB
None
75% random
combinations
(assuming
equal
prevalence of
types) are
incompatible
(12/16)
43
Major Incompatibility 5-ways:
non-O into O, or AB into A or B
recipient
donor
44
Minor Incompatibility 5-ways:
O into non-O, or A or B into AB
recipient
donor
45
All Possible Players…
(or the Worst Case Scenario):
Bidirectional Incompatibility 2-ways:
A to B or B to A
recipient
donor
46
Donor Distributions By Recipient
PROBABILITIES
given:
O:
A:
B:
AB:
0.45
0.40
0.11
0.04
R
D
Type
Ptype specific
O
O
O
O
A
B
AB
O
Major
Major
Major
None
55% major
A
A
A
A
A
O
B
AB
None
Minor
Both
Major
40% compatible
45% minor
11% bidirectional
4% major
B
B
B
B
B
O
A
AB
None
Minor
Both
Major
11% compatible
45% minor
40% bidirectional
4% major
AB
AB
AB
AB
AB
O
A
B
None
Minor
Minor
Minor
4% compatible
45% identical
96% minor
47
Probability of HPCT
ABO Incompatibility Among MUDs
Major
Minor
Bidirectional
Compatible
O
24.75%
n/a
n/a
18.00%
A
1.60%
18.00%
4.4%
16.00%
B
0.44%
4.95%
4.4%
1.21%
AB
n/a
3.84%
n/a
0.16%
P total
26.8%
26.8%
8.8%
37.6%
Recipient
62.5 %
48
Quick Summary of Vagaries of ABOi in HPCT
• MAJOR INCOMPATIBLE
HPCT
– multi-lineage marrow
aplasia/hypoplasia
– delayed (red cell)
engraftment / pure red cell
aplasia (PRCA)
– INCOMING
DONOR CELLS
FOUGHT OFF
(acute hemolysis of passive
contaminant, delayed
establishment of active
production)
• MINOR INCOMPATIBLE
HPCT
– delayed,
potentially severe hemolytic
anemia
– HISTORICAL
RECIPIENT CELLS FOUGHT
OFF
(acute hemolysis of recipient
cells by passive antibody,
delayed hemolysis of
recipient cells by active
antibody)
49
ABOi HPCT and
Increased RBC Alloimmunization?
• Described once by de la Rubia et al in 2001, in a transplant
cohort of 217 transplant patients, of whom 8 had developed
non-ABO RBC antibodies
– ABO blood group incompatibility (p = 0.005) and patient's age (p =
0.02) were the only two variables significantly associated with the
development of RBC alloantibodies
• Took a peak (in 2006 while at BIDMC) at 90 consecutive
transplant patients, of whom 8 had non-ABO RBC antibodies
50
ABOidentical
MUD
transplants,
n=6
ABOincompatible
MUD
transplants,
n = 12
ABO-incompatible MRD transplants, n = 28
(odds 3X higher
for ABOincompatible
HPCTs among
MUD vs MRD)
ABO-incompatible transplants, n = 40 (28 MRD, 12 MUD [30%])
51
MUD transplants, n = 18
MRD transplants, n = 72
(4X as many MRDs as MUDs)
ABO-identical MRD transplants, n = 44
patients post- ABO identical HPCT, n = 50
(0 [0%] new alloimmunizations)
p=0.006
(2-tailed
Fisher’s exact
test)
2/2 of historically alloimmunized
patients experienced antibody
persistence
32,
80%
8,
20%
non-alloimmunized patients
persistent, pre-transplant anti-E
persistent, pre-transplant anti-Jk(a)
new, post-transplant anti-K
new, post-transplant anti-K
new, post-transplant anti-E
new, post-transplant anti-E
new, post-transplant anti-E
new, post-transplant anti-K, E, C(W)
1
1
1
1
1
1
3
10 antibodies
1
8 alloimmunized
patients
0/1 of historically alloimmunized
patients experienced antibody
persistence
patients post- ABO incompatible HPCT, n = 40
(6 [15%] new alloimmunizations)
52
Minor Not So Innocent, Yet Again
ABO minor incompatible HPC transplants
ABO major incompatible HPC transplants
21
15
(1)
(7)
red cell sensitization (n=8):
• 1/3rd of ABO minor incompatible HPC transplants
• 1/15th of ABO major incompatible HPC transplants
Suggests that antibody formation is not only more
frequently observed in ABO-mismatched cases, but
specifically in the minor incompatibility scenario.
53
The Discovery of Non-Hemolytic Red
Cell Antigen Loss
•
after transfusion of Ag+ cells
to Ab+ host…
D
R
1. hemolytic clearance of DAT+
allogeneic cells
D
2. non-hemolytic persistence of
DAT+ cells
D
3. non-hemolytic transformation
of Ag-, DAT- cells
D
R
R
R
54
How Does Loss of Ag Happen Without
Losing the RBC?
• no single mechanism found yet…
– ? synthetic feedback suppression of antigen
– ? post-expression tear-away
– ? partial alteration of antigen
– ? benign antibody binding
D
• you don’t need a spleen, but it won’t happen ex-vivo…
– need a liver with FcγRIII
– need to simultaneously cross-link antibodies that have
separate specificities for parts of the same target antigen
55
A Natural Mechanism,
Neglected Because of Its Silence…
• CR1 (CD35) on RBCs* have been known to transfer immune
complexes to Kupffer cells of the liver without undergoing
hemolysis!
* CR1 also present in B cells, myeloid cells…
• Unnatural clinical precedents, good and bad:
– “organ accommodation”
– CD20+ CLL cells becoming CD20- and thus escaping the
effects of rituximab (anti-CD20)
56
CR1 (CD35) Biology: Expression Implications
genotype
+/+
+/-
-/-
phenotype
high
moderate
low/absent
Normal range: 50 – 1200/cell
20-fold natural variation in healthy people
Low expression: <200 / cell
• C3b/C4b complement receptor = CR1 = CD35, chr 1q32 within RCA
(regulators of complement activation) family
• On red blood cells:
– Primary function: limits activation of complement pathway
• Binds complement cleavage products C3b and C4b, thus acting as a cofactor to
inactivate them to iC3b and iC4b
– Secondary function: provide a removal/clearance function
• Immune complexes (IC) bind to C3b, which is then trafficked on CR1 of RBCs (and
other cells) to the liver and spleen for removal by macrophages…
• On B-cells and macrophages:
– Pro-inflammatory cytokine release in response to immune complexes?
57
Final Aphorisms
• just because something isn’t major incompatible, it doesn’t mean
it’s compatible enough…
sometimes “minor” is anything but
• unexpected antibodies – for a specific self-alloantigen, at a time
when de novo antibody production is assumed to be stymied, tell
us interesting stories
• knowing more about what controls the difference between harmful
versus harmless sensitizations has vast implications for us in
troubleshooting our way through transfusion medicine, transplant
immunobiology, & biologic therapy
58