Canadian Blood Services Transfusion Medicine Resident Topic Teaching Session: Trojan Antibodies and Transplant Malware: Improbable Sensitizations & Hemolytic Passenger Lymphocyte Syndromes

Tuesday, October 5 th 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.

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Princess Margaret Hospital Toronto General Hospital Toronto Western Hospital

University Health Network

Holland Ortho & Arthritic Centre Sunnybrook Hospital Women’s College Hospital

Sunnybrook Health Sciences Centre St Michael’s Hospital Hospital for Sick Children Mount Sinai Hospital

TRANSFUSION MEDICINE COLLABORATIVE University of Toronto teaching hospitals:

Hospital Transfusion Services Ontario Regional Blood Coordinators

provincial blood management

Canadian Blood Services

national blood provider

Ontario Transfusion Coordinators

provincial blood conservation

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% O donor plasma:

• anti-A,B IgG • anti-A & anti-B IgG & IgM

45% 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 1/3 rd 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

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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

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Diversity (Polymorphisms) Set Up The RBC Incompatibility Fight

ABO

Kell: K vs k, others Duffy: Fy a vs Fy b

Rh(D)

complete absence or presence

ISOIMMUNIZATION

Kidd: Jk a vs Jk b other Rh(CE) antigens: C vs c E vs e etc: MNS, P, Lutheran, Lewis, Diego, Scianna, Dombrock, Colton, LW, Chido/Rodgers, Gerbich, Cromer, Knops, Indian subtle (eg SNP) variations therein…

ALLOIMMUNIZATION

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The Vocabulary of Immune Violence

MAJOR vs MINOR incompatibility ACTIVE vs PASSIVE immunity 13

Directions of Incompatibility

• MAJOR – incoming donor’s cellular antigens are the incompatible target – host-source immunity mounts the attack • “MINOR” – donor-source immunity mounts the attack – host’s cellular antigens are the incompatible target – PRODUCT REJECTION by a fighting recipient • eg. ABO incompatible RBC mistransfusion – A into O: donor A cells destroyed by host O plasma • eg. acute rejection of organs – HOST REJECTION by a fighting donor • eg. GVHD from a solid organ transplant, a transfusion, or a BMT • eg. TRALI 14

Active vs Passive Immunity

• ACTIVE • PASSIVE n  ∞ n = x – renewable humoral (via cellular) fighting power – finite (humorally contained) fighting power all forms of MAJOR incompatibility some forms of MINOR incompatibility most forms of MINOR incompatibility recipient plasma cell passenger donor plasma cell graft* 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 donor origin (graft-associated) alloimmune hemolytic anemia of host RBCS

aka. HUMORAL GRAFT VERSUS HOST DISEASE FOR THE BLOOD BANK

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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 – 1 st hemolytic ABO PLS Marchioro, NYAS • 1985 – 1 st hemolytic anti-D PLS Swanson, Transfusion • 1990 – 1 st multiorgan (L, K) PLS Ramsey, aaBB 19

kidney

PLS – “Lymphoid” Load Spectrum (Usual Aggressors)

(heart-) lung small bowel, pancreas liver HPC: PB > BM

• dead or alive… • graft (usually) unaffected • at least 10

6 – 10 7

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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) Therapeutic red cell exchange (TREx) Whole blood removal eg. manual red cell exchange, replacing with pRBC ± plasma/NS/albumin (reconstituted whole blood) 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?

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case I case 2 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

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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 anti-C on IAT anti-D on DAT (eluate) 123 216 217 anti-C on DAT (eluate) clinically significant hemolytic anemia (depleted haptoglobin, increased LDH & bilirubin) 10 therapeutic plasma exchange 39 67 60 36 - 40 110 160 210

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

4 3 2 1 0 0

Case 2: Novel Serological Course

serologically positive 120 159 DAT IAT 173 acute rejection 259 266 100 200 days post-transplant 300

TPE 38

Case 3: It happened again! RECIPIENT 3: ♂

O, R1 R1

( CC DD ee )

0



anti-C anti-D

DONOR 2: ♂

O, r r

( cc dd ee )

anti-C anti-D anti-V

• 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 Lung Lung Lung Liver Lung Liver Liver Liver Liver Lung Donor antibodies Anti- C, D, E Anti- C, D, E Anti- C, D, V Anti- B Anti- B, Jka, N Anti- D Anti- B Anti- A Anti- A (IgM) Anti-A Cognate recipient attack C, D D C, D B B, Jka, N D B A A A Significant hemolysis?

yes no no yes yes yes * (day 798) yes yes yes 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 O O O A B A B AB AB AB A B O A B AB D A B AB AB AB O O O A B B A O A B AB Type Major Minor Both None

75% random combinations (assuming equal prevalence of types) are incompatible (12/16)

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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

O

O O O B B B B A A A A AB AB AB AB D

A

B AB O A O B AB B O A AB AB O A B Type Major Major Major None None Minor Both Major None Minor Both Major None Minor Minor Minor P type specific 55% major

45% identical

40% compatible 45% minor 11% bidirectional 4% major 11% compatible 45% minor

40% bidirectional

4% major 4% compatible 96% minor 47

Probability of HPCT ABO Incompatibility Among MUDs Minor Bidirectional Compatible Major Recipient O A B AB P total 24.75% 1.60% 0.44% n/a 26.8% n/a 18.00% 4.95% 3.84% n/a 4.4% 4.4% n/a 26.8% 8.8% 18.00% 16.00% 1.21% 0.16% 37.6%

62.5 %

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Quick Summary of Vagaries of ABOi in HPCT • M A JOR INCOMPATIBLE HPCT – multi-lineage marrow a pl a sia/hypopl a si a – del a yed (red cell) engraftment / pure red cell aplasia (PRCA) – INCOMING DONOR CELLS FOUGHT OFF (acute hemolysis of passive contaminant, delayed establishment of active production) • M I NOR INCOMPATIBLE HPCT – delayed, potentially severe hemolyt i c anemia – HISTORICAL RECIPIENT CELLS OFF FOUGHT (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

ABO-identical MRD transplants, n = 44 ABO-incompatible MRD transplants, n = 28 ABO identical MUD transplants, n = 6 ABO incompatible MUD transplants, n = 12

(odds 3X higher

for ABO incompatible HPCTs among

MUD vs MRD)

ABO-incompatible transplants, n = 40 (28 MRD, 12 MUD [30%]) 51

patients post- ABO identical HPCT, n = 50 (0 [0%] new alloimmunizations)

0/1 of historically alloimmunized patients experienced antibody persistence (2-tailed test)

patients post- ABO incompatible HPCT, n = 40 (6 [15%] new alloimmunizations)

p=0.006

Fisher’s exact 2/2 of historically alloimmunized patients experienced antibody persistence

32, 80% 8, 20%

1 1 1 1 1 1 1 3

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)

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Minor Not So Innocent, Yet Again

15 (1) 21 (7) ABO minor incompatible HPC transplants ABO major incompatible HPC transplants red cell sensitization (n=8): • 1/3 rd of ABO minor incompatible HPC transplants • 1/15 th 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

D • after transfusion of Ag+ to Ab+ host… cells R 1. hemolytic clearance of DAT+ allogeneic cells 2. non-hemolytic persistence of DAT+ cells 3. non-hemolytic transformation of Ag-, DAT- cells D D 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?

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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