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1 Trojan Antibodies and Transplant Malware: Improbable Sensitizations & Hemolytic Passenger Lymphocyte Syndromes Trojan Antibodies and Transplant Malware:

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Presentation on theme: "1 Trojan Antibodies and Transplant Malware: Improbable Sensitizations & Hemolytic Passenger Lymphocyte Syndromes Trojan Antibodies and Transplant Malware:"— Presentation transcript:

1 1 Trojan Antibodies and Transplant Malware: Improbable Sensitizations & Hemolytic Passenger Lymphocyte Syndromes Trojan Antibodies and Transplant Malware: Improbable Sensitizations & Hemolytic Passenger Lymphocyte Syndromes 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 Canadian Blood Services Transfusion Medicine Resident Topic Teaching Session: NO CONFLICTS OF INTEREST. FUNDING FOR RESEARCH FROM PUBLIC FOUNDATIONS ONLY. Tuesday, October 5 th 2010, 12:00 – 1:00 pm Conference Dial-In: 1-866-752-7690, Passcode 1775282

2 2 University Health Network St Michael’s Hospital Holland Ortho & Arthritic Centre Sunnybrook Health Sciences Centre Hospital for Sick Children TRANSFUSION MEDICINE COLLABORATIVE Canadian Blood Services Mount Sinai Hospital University of Toronto teaching hospitals: Hospital Transfusion Services Ontario Regional Blood Coordinators Sunnybrook Hospital Women’s College Hospital Princess Margaret Hospital Toronto General Hospital Toronto Western Hospital Ontario Transfusion Coordinators Trillium Gift of Life Network provincial blood management national blood provider provincial blood conservation provincial organ network

3 3 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”

4 4 It All Starts With This One Tube… plasma

5 5 ABO Types in the General Population O donor plasma: anti-A,B IgG anti-A & anti-B IgG & IgM A donor plasma: anti-B IgM B donor plasma: anti-A IgM AB donor plasma: no anti-A or anti-B

6 6 30 blood group antigen systems (ABO, Rh, …) containing structures that are naturally “polymorphic” (278 antigens + 38 in “collections/series”)

7 7 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

8 8 “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

9 9 Guy (Gal) Walks In The Door… What Are The Chances? 1 – 5 % of the population has a positive red cell antibody screen EXPOSURE INTRINSIC SEROCONVERSION RISK HOW RECENT & HOW DURABLE THE ANTIBODY IS XX

10 10 Targets for Graft versus Host Attack HLA Antigens (Platelets, Tissues)… ie- GVHD Red Cell Antigens… ie- Passenger Lymphocyte Syndrome (PLS)

11 11 extensive variations therein… ALLOIMMUNIZATION Diversity (Polymorphisms) Set Up The Tissue Incompatibility Fight

12 12 ABO Rh(D) Kell: K vs k, others Duffy: Fy a vs Fy b Kidd: Jk a vs Jk b etc: MNS, P, Lutheran, Lewis, Diego, Scianna, Dombrock, Colton, LW, Chido/Rodgers, Gerbich, Cromer, Knops, Indian other Rh(CE) antigens: C vs c E vs e complete absence or presence subtle (eg SNP) variations therein… ISOIMMUNIZATIONALLOIMMUNIZATION Diversity (Polymorphisms) Set Up The RBC Incompatibility Fight

13 13 The Vocabulary of Immune Violence MAJOR vs MINOR incompatibility ACTIVE vs PASSIVE immunity

14 14 Directions of Incompatibility MAJOR –incoming donor’s cellular antigens are the incompatible target –host-source immunity mounts the attack –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 “MINOR” –donor-source immunity mounts the attack –host’s cellular antigens are the incompatible target –HOST REJECTION by a fighting donor eg. GVHD from a solid organ transplant, a transfusion, or a BMT eg. TRALI

15 15 Active vs Passive Immunity ACTIVE –renewable humoral (via cellular) fighting power PASSIVE –finite (humorally contained) fighting power all forms of MAJOR incompatibility most forms of MINOR incompatibility some forms of MINOR incompatibility recipient plasma cell passenger donor plasma cell graft*product n  ∞n = x

16 16 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)

17 17 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 passenger donor plasma cell graft host RBC

18 18 PLS – The Implicated RBC Antigen Spectrum (Usual Victims) 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” 555-fold

19 19 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

20 20 PLS – “Lymphoid” Load Spectrum (Usual Aggressors) kidney liver HPC: PB > BM (heart-) lung dead or alive… graft (usually) unaffected at least 10 6 – 10 7 small bowel, pancreas

21 21 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)

22 22 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

23 23 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

24 24 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

25 25 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)

26 26 Whole blood removal eg. manual red cell exchange, replacing with pRBC ± plasma/NS/albumin (reconstituted whole blood) Red cell replacement of removed plasma (hybrid TPRE) Therapeutic red cell exchange (TREx) Top-up transfusion Is There a Role for Apheresis?

27 27 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

28 28 Other Management Options rituximab graft irradiation splenectomy cytotoxic agents, prednisone adjustments combination therapy: RBC/TREX, IST, apheresis, splenectomy

29 29 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?

30 30 case I case 2 case 3

31 31 Case 1: The good news: ABO compatibility… The bad news: the seeming Rh(D)-compatibility isn’t Recipient: “A Positive” Donor: “A Negative”

32 32 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

33 33 Case 1 Traceback of Donor elderly multiparous female group A, Rh(D)-negative (rr) screen: Anti-C Anti-D Anti-E

34 34 RECIPIENT: DONOR: Serologically:C+ D+ e+ c- E- Weiner designation: R1 R1 (17% of population) Serologically:c+ e+ C- D- E- Weiner designation: r r (15% of population) passenger lymphocyte Anti-CAnti-D Anti-E

35 35 therapeutic plasma exchange 36 - 40

36 36 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

37 37 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

38 38 Case 2: Novel Serological Course 120 159 173 serologically positive 259266 acute rejection TPE

39 39 Case 3: It happened again! RECIPIENT 3: ♂ DONOR 2: ♂ O, r r ( cc dd ee ) O, R1 R1 ( CC DD ee ) anti-C anti-D anti-V anti-C 0  anti-D 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)

40 40 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… )

41 41 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? LungAnti- C, D, EC, Dyes LungAnti- C, D, EDno LungAnti- C, D, VC, Dno LiverAnti- BByes LungAnti- B, Jka, NB, Jka, Nyes LiverAnti- DDyes * (day 798) LiverAnti- BByes LiverAnti- AAyes LiverAnti- A (IgM)Ayes LungAnti-AAyes

42 42 The Only Prospective Post-Solid Organ Tranplant Sensitization Surveillance Study in the Literature: – 27% of the ABOi KTs developed +DAT checked q 10d x 3 checks post-operatively aaBB Abstracts 2007

43 43 Types Of HPCT ABO Incompatibility RDType OOOABOOOAB A B AB Major A B AB OOOABOOOAB Minor ABAB BABA Both O A B AB O A B AB None 75% random combinations (assuming equal prevalence of types) are incompatible (12/16)

44 44 Major Incompatibility 5-ways: non-O into O, or AB into A or B recipientdonor

45 45 Minor Incompatibility 5-ways: O into non-O, or A or B into AB recipient donor

46 46 All Possible Players… (or the Worst Case Scenario): Bidirectional Incompatibility 2-ways: A to B or B to A recipient donor

47 47 Donor Distributions By Recipient PROBABILITIES given: O: 0.45 A: 0.40 B: 0.11 AB: 0.04 RDTypeP type specific OOOOOOOO A B AB O Major None 55% major 45% identical AAAAAAAA A O B AB None Minor Both Major 40% compatible 45% minor 11% bidirectional 4% major BBBBBBBB B O A AB None Minor Both Major 11% compatible 45% minor 40% bidirectional 4% major AB O A B None Minor 4% compatible 96% minor

48 48 Probability of HPCT ABO Incompatibility Among MUDs Recipient MajorMinorBidirectionalCompatible O 24.75%n/a 18.00% A 1.60%18.00%4.4%16.00% B 0.44%4.95%4.4%1.21% AB n/a3.84%n/a0.16% P total 26.8% 8.8%37.6% 62.5 %

49 49 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)

50 50 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

51 51 ABO-identical MRD transplants, n = 44 ABO- identical MUD transplants, n = 6 ABO-incompatible transplants, n = 40 (28 MRD, 12 MUD [30%]) ABO-incompatible MRD transplants, n = 28 ABO- incompatible MUD transplants, n = 12 (odds 3X higher for ABO- incompatible HPCTs among MUD vs MRD) MRD transplants, n = 72 (4X as many MRDs as MUDs) MUD transplants, n = 18

52 52 patients post- ABO identical HPCT, n = 50 (0 [0%] new alloimmunizations) patients post- ABO incompatible HPCT, n = 40 (6 [15%] new alloimmunizations) p=0.006 (2-tailed Fisher’s exact test) 0/1 of historically alloimmunized patients experienced antibody persistence 2/2 of historically alloimmunized patients experienced antibody persistence 8 alloimmunized patients 10 antibodies 1 1 1 1 1 1 1 3

53 53 15 21 (7)(1) 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 Minor Not So Innocent, Yet Again Suggests that antibody formation is not only more frequently observed in ABO-mismatched cases, but specifically in the minor incompatibility scenario.

54 54 The Discovery of Non-Hemolytic Red Cell Antigen Loss Ab+after transfusion of Ag+ cells to Ab+ host… 1.hemolytic clearance of DAT+ allogeneic cells 2.non-hemolytic persistence of DAT+ cells 3.non-hemolytic transformation of Ag-, DAT- cells R D D R D R D R

55 55 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 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 D

56 56 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)

57 57 CR1 (CD35) Biology: Expression Implications genotype+/++/--/- phenotypehighmoderatelow/absent Low expression: <200 / cell Normal range: 50 – 1200/cell 20-fold natural variation in healthy people 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?

58 58 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

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