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HLA Ab, Donor Reactivity and Risk of Rejection and Graft Loss

HLA Ab, Donor Reactivity and Risk of Rejection and Graft Loss. Ronald H. Kerman, PhD The University of Texas Medical School ~ Houston, TX Division of Immunology and Organ Transplantation. Type: Hyperacute Accelerated Acute Chronic. Mediated by: Abs Abs/cells Cells/Abs

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HLA Ab, Donor Reactivity and Risk of Rejection and Graft Loss

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  1. HLA Ab, Donor Reactivity and Risk of Rejection and Graft Loss Ronald H. Kerman, PhD The University of Texas Medical School ~ Houston, TX Division of Immunology and Organ Transplantation

  2. Type: Hyperacute Accelerated Acute Chronic Mediated by: Abs Abs/cells Cells/Abs Abs/cells/? Time: 0-48 hrs 5-7 days Early/delayed >60 days Allograft Rejection

  3. Responsibilities of the Histocompatibility Laboratory To identify clinically relevant recipient IgG HLA antibodies

  4. Positive crossmatches, due to Abs or other factors not impacting on graft outcome, should not influence the donor-recipient pairing for transplantation.

  5. Detection of Recipient Sensitization Screen sera for reactivity vs target cells by cytotoxicity/fluorescence readouts. Use the most informative sera when performing the recipient vs donor crossmatch (historically most reactive, current and pretransplant sera).

  6. Detection of Immunoglobulin Reactivity • NIH-CDC • AHG-CDC • Flow cytometry Membrane-dependent assays

  7. Complement-dependent Cytotoxicity NIH Assay

  8. Complement-dependent Cytotoxicity NIH Assay

  9. Complement-dependent Cytotoxicity NIH Assay

  10. Anti-human Globulin (Enhancement) Assay

  11. Anti-human Globulin (Enhancement) Assay

  12. Anti-human Globulin (Enhancement) Assay

  13. Flow Cytometry Assay NIH - CDC Negative AHG – CDC Negative Now measuring binding of IgG (absent C’)

  14. Cadaveric Renal Allograft Survival Among 1o CsA-Pred Recipients at 12 months NIH Neg. n=166 81% (134/166) AHG Neg. Pos. n=151 n=15 82% 67% (124/151) (10/15) P<0.01 Kerman et al, Transplantation; 51:316, 1991

  15. Cadaveric Renal Allograft Survival Among 1o CsA-Pred Recipients at 12 months AHG Pos. n=15 67% (10/15) DTE-AHG Neg. Pos. n=12 n=3 83% 0% (10/12) (0/3) P<0.01 Kerman et al, Transplantation; 51:316, 1991

  16. Cadaveric Renal Allograft Survival Among 1o CsA-Pred Recipients at 12 months DTE/AHG XM Neg. n=166 81% FCXM Neg. Pos. n=130 n=36 81% 81% Kerman et al, Transplantation; 51:316, 1991

  17. Neg-NIH Extended XM: FCXM Study T-FCXM Pos. n=148 75% T-FCXM Neg. n=693 82% P<0.01 Ogura et al, Transplantation; 56:294, 1993

  18. Could Ron Kerman have been wrong about his crossmatch results and interpretation?

  19. 50 P=NS 45 40 35 30 25 20 15 10 5 0 Negative (n=56) Positive (n=41) IgG FCXM: Renal Allograft Study Frequency of Rejection in a Single Center % Rejection Kerman et al, Transplantation; 68:1855, 1999

  20. Could Ron Kerman have been wrong about his crossmatch results and interpretation? I don’t think so!

  21. The Cell Surface Is a Jungle HLA

  22. Membrane-dependent Assays • NIH-CDC • AHG-CDC • Flow cytometry Detection of membrane receptors may not be related to HLA!

  23. Membrane-independent Assays ELISA-determined IgG HLA Abs vs MHC-I (pooled platelets) ELISA-determined IgG HLA Abs vs MHC-I/II (PBL cultures) Flow bead PRA-determined IgG HLA vs I/II (soluble HLA I/II antigens on microbeads measured by cytometry)

  24. PRA by Different Methodologies Type: CDC AHG-CDC ELISA Flow Positive 102 116 127 139 Negative 162 148 137 125 Gebel & Bray, Transplantation; 69:1370, 2000

  25. AHG-PRA vs Rejection 493 Consecutive CAD Recipients AHG-PRA Rejection YES NO <10% 134 159 10% 100 100 P=NS

  26. ELISA-PRA and Rejection ELISA-PRA Rejection YES NO <10% 38 168 10% 117 63 P<0.001

  27. Correlation Between % ELISA-PRA and Graft Survival ELISA-PRA Graft Survival (months) 12 24 36 <10% (n=312) 85% 82% 81% >10% (n=181) 74% 70% 67% P<0.01 P<0.01 P<0.01

  28. Sensitivity and sensitization, defining the unsensitized patient Application of membrane-independent assays to identify HLA antibodies Gebel & Bray, Transplantation; 69:1370, 2000

  29. Correlation of Pre-transplant Abs Detected by Flow PRA with Biopsy-documented Cardiac Rejection Tambur et al, Transplantation; 70:1055, 2000

  30. 50 P=NS 45 40 35 30 25 20 15 10 5 0 Negative (n=56) Positive (n=41) IgG FCXM: Renal Allograft Study Frequency of Rejection in a Single Center Were positive crossmatches due to HLA Abs? % Rejection Kerman et al, Transplantation; 68:1855, 1999

  31. Immunosuppressive Menu: • Neoral - CsA • Steroids • Prograf - FK506 • Cellcept - MMF • Rapamycin - Sirolimus • Thymoglobulin • OKT3, anti-IL-2R, FTY720

  32. If new immunosuppressive therapies reduce the incidence of acute rejection, are pre-Tx HLA antibodies clinically relevant?

  33. RAPA-CsA-Pred treated primary recipients of CAD renal allografts experience fewer acute rejections vs CsA-Pred recipients. We therefore tested their pre-Tx sera for the presence of HLA Abs and correlated the results to the occurrence of rejection during the first 12 months post-transplant.

  34. 147 RAPA-CsA-Pred recipients were studied 48 patients were chosen specifically because they had a rejection episode. 99 patients were chosen because they had not experienced a rejection episode during the first year post-transplant.

  35. PRA Testing Anti-human globulin (AHG) ELISA (One Lambda, Inc. LAT) Flow PRA (One Lambda, Inc.)

  36. Results: AHG-PRA detected 18 reactive sera ELISA-PRA detected 25 reactive sera (11 vs HLA class I, 3 vs II, 11 vs I/II) Flow PRA detected 59 reactive sera (31 vs HLA class I, 9 vs II, 19 vs I/II)

  37. There was no significant correlation between AHG-PRA, ELISA-detected HLA Abs, and Flow PRA HLA class II Abs and rejection. • AHG vs Rejection P=NS • LAT-I vs Rejection P=NS • LAT-II vs Rejection P=NS • LAT-I/II vs Rejection P=NS • F-II vs Rejection P=NS

  38. Flow PRA-1 Rejection NO YES <5% 76 21 5% 23 27 X2=15.7; P<0.001

  39. Day of 1st Rejection 57 ± 34 Flow PRA 0% FCXM Pos. Neg. 2 8 No grafts lost (+) FCXM vs non-HLA Ab

  40. Day of 1st Rejection 55 ± 31 Flow PRA 13 ± 9% FCXM Pos. Neg. - 30 No grafts lost.

  41. Day of 1st Rejection 32 ± 15 Flow PRA 28 ± 9% FCXM Pos. Neg. 12 13 (+) HLA Ab and (-) FCXM: rejection, no grafts lost. (+) HLA Ab and (+) FCXM: rejection, 58% (7/12) grafts lost.

  42. Day of 1st Rejection 17 ± 12 Flow PRA 48 ± 31% FCXM Pos. Neg. 8 7 (+) HLA Ab and (-) FCXM: rejection, no grafts lost. (+) HLA Ab and (+) FCXM: rejection, 63% (5/8) lost to AMR.

  43. Day of Rejection 57 ± 34 55 ± 31 32 ± 9 17 ± 12 % PRA 0 13 ± 9 28 ± 9 48 ± 31 N 75 32 25 15 % Rejection 5% (4/75) 13% (4/32) 100% 100%

  44. 1. Assays that measure binding of immunoglobulin to targets may not represent HLA Ab reactivity. 2. The AHG-XM protects RAPA-CsA-Pred recipients from hyperacute rejection. 3. The Flow PRA assay detects clinically relevant HLA Abs associated with rejection and/or graft loss.

  45. 4. How many antibodies are present may be clinically relevant. 5. The antibody titer may also be important. 6. Patients with pre-Tx (+) HLA Abs and (+) donor reactivity (+ FCXM) are at risk for graft rejection and loss.

  46. We have performed heart transplantation following a negative AHG-XM. We evaluated the clinical relevance of FCXM for heart recipients.

  47. FCXM Results: Heart Recipient IgG FCXM Neg. 1YGS 86% IgG FCXM Pos. 68% P<0.02 Of the 22 IgG FCXM-Pos. Recipients: 7 grafts were lost 15 grafts were successful WHY?

  48. We Flow PRA Tested the IgG FCXM-Pos. Sera 5 sera tested from lost grafts All 5 sera were Flow PRA reactive vs MHC I (Flow PRAs of 36%, 52%, 68%, 50% and 49%) 11 sera tested from successful recipients All 11 sera were Flow PRA non-reactive

  49. FCXM (-) Flow PRA I/II 51% 100% (13/13) FCXM (+) Flow PRA I/II 51% 55% (5/9) Graft Survival 12 mo. 31% (4/13) 89% (8/9) Rejection 0-12 mo. Both comparisons p<0.01

  50. HLA Ab and Donor Specific Reactivity Rank Order of Risk 1. HLA Ab negative, FCXM negative (at risk for reversible, cellular rejection) 2. HLA Ab negative, FCXM positive (non-HLA allo-Ab - at risk for reversible, cellular rejection)

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