19/02/2018. Transplant Webinar Series: Ep. 3 Donor Selection For Solid Organ Transplants Is A Virtual Crossmatch Better Than Real?

Transcription

1 Transplant Webinar Series: Ep. 3 Donor Selection For Solid Organ Transplants Is A Virtual Crossmatch Better Than Real? 1

2 Future Webinars Histocompatibility testing: From antigens towards epitopes Featuring Prof. dr. Frans H. J. Claas Leiden University Medical Center Eurotransplant Reference Laboratory Leiden, the Netherlands 15 March :30-17:30 IST (11:00-12:00 GMT) 08:00-09:00 EST (13:00-14:00 GMT) 14:00-15:00 EST (19:00-20:00 GMT) Link to register: All Content 2015 Immucor, Inc. 2

3 Handouts Program-Handouts.aspx All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc. 3

4 Continuing Education ABHI, ASCLS/P.A.C.E., Florida and California Credits 1.0 Contact Hour or 0.15 continuing education credits (CECs) awarded Each attendee must register to receive CE credits at: Registration deadline is 16 March 2018 Certificates will be sent via only to those who have registered by 30 March 2018 All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc. 4

5 Presentation Recording Session will be recorded and posted to Immucor s LEARN site. Access information will be sent to each registrant when the recording becomes available CE credits will be issued to anyone who listens to the recording within one year of the original presentation date (today). All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc. 5

6 Questions? You are all muted Q&A following session - Type in questions All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc. 6

7 Course content is for information and illustration purposes only. Immucor makes no representation or warranties about the accuracy or reliability of the information presented and this information is not to be used for clinical or maintenance evaluations. The opinions contained in this presentation are those of the presenter and do not necessarily reflect those of Immucor. All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc. 7

8 Dr Luis G. Hidalgo All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc. 8

9 DONOR SELECTION FOR SOLID ORGAN TRANSPLANTS IS A VIRTUAL CROSSMATCH BETTER THAN REAL? Luis G. Hidalgo, Ph.D., D(ABHI) Associate Director - Histocompatibility Laboratory University of Alberta Hospitals Dept. of Lab. Med. and Pathology 9

10 LEARNING OBJECTIVES HISTORY OF CROSSMATCHES IN TRANSPLANTATION REVIEW OF CURRENT CROSSMATCH METHODS ADVANTAGES AND DISADVANTAGES OF ACTUAL CROSSMATCHES ADVANTAGES AND DISADVANTAGES OF VIRTUAL CROSSMATCHES UNDERSTAND THE LIMITATIONS OF BOTH TYPES OF CROSSMATCHES 10

11 THE HISTORY OF HLA ABS IN TRANSPLANTATION IN THE DECADE FOLLOWING THE FIRST KIDNEY TRANSPLANT (DR. JOSEPH MURRAY 1954), A MAJOR PROBLEM WAS REALIZED HYPERACUTE REJECTION 11

12 THE HISTORY OF HLA ABS IN TRANSPLANTATION HYPERACUTE REJECTION CAN DESTROY THE KIDNEY TRANSPLANT WITHIN MINUTES/ HOURS AFTER REPERFUSION: 12

13 THE HISTORY OF HLA ABS IN TRANSPLANTATION HOWEVER, UP UNTIL THE MID 1960 S A ROLE FOR ANTIBODIES IN TRANSPLANTATION WAS DEBATED: Kissmeyer-Nielsen et. al., Lancet,

14 THE HISTORY OF HLA ABS IN TRANSPLANTATION IN THE LATE 1960 S THE LANDMARK PAPER BY PATEL AND TERASAKI CLEARLY SHOWED THE BENEFIT OF DETECTING DONOR-REACTIVE ANTIBODIES PRE-TRANSPLANT: Dr. Paul Terasaki: Patel et. al., NEJM 1969 THIS MARKED THE BEGINNING OF TESTING FOR DONOR-REACTIVE ANTIBODIES AS A STANDARD PROCEDURE PRE-TRANSPLANT THE BIRTH OF THE FIELD OF HISTOCOMPATIBILITY 14

15 THE CONCEPT OF A CROSSMATCH A CROSSMATCH TESTS FOR REACTIVITY BETWEEN A RECIPIENT S SERUM AND A DONOR S CELLS IN HISTOCOMPATIBILITY, THE ASSUMPTION IS THAT THE REACTIVITY IS DUE TO HLA ANTIBODIES ITS APPLICATION IS NOT RESTRICTED TO TESTING OF HLA ANTIBODIES ALSO USED FOR TESTING OF ANTI-ABO ANTIBODIES (ISOHEMAGLUTINNINS) 15

16 THE TYPES OF CROSSMATCHES AT LEAST THREE TYPES OF CROSSMATCHES ARE CURRENTLY USED: - CYTOTOXICITY-BASED CROSSMATCHES (1964-PRESENT): - FLOW CYTOMETRY CROSSMATCHES (FCXM) (~1983-PRESENT) - VIRTUAL CROSSMATCHES (POST SAB TESTING, ~2005-PRESENT) 16

17 CYTOTOXIC CROSSMATCHES - CDC COMPLEMENT-DEPENDENT CYTOTOXICITY (CDC) THIS IS THE ASSAY THAT PAUL TERASAKI ORIGINALLY DEVELOPED SUBJECTIVE SCORING LIMITED SENSITIVITY! 17

18 CYTOTOXIC CROSSMATCHES AHG-CDC AN ENHANCEMENT METHOD WAS DEVELOPED TO HELP DEAL WITH THE SENSITIVITY OF THE CDC ASSAY: 18

19 CLINICAL RELEVANCE OF A POSITIVE CYTOTOXIC CROSSMATCH THERE IS NO DOUBT TO THE CLINICAL RELEVANCE OF A T CELL POSITIVE CYTOTOXIC CROSSMATCH: AHG-CDC+ XM PATIENTS WERE DESENSITIZED (PLEX/ IVIG) UNTIL XM NEG + RITUX INDUCTION (N = 52) T CELL AHG-CDC + Gloor JM et al Am J Transplant (2010) 19

20 PROBLEMS WITH CYTOTOXICITY CROSSMATCHES RELIABLY ASSESS ABS TO HLA CLASS I ONLY REQUIRED ADDITIONAL TREATMENT TO DISTINGUISH IgM FROM IgG LOW SENSITIVITY, AHG ENHANCEMENT CAN MAKE NON-COMPLEMENT FIXING ANTIBODIES POSITIVE BIOLOGICALLY ACCURATE? CELL VIABILITY REQUIRED ISSUES WITH DISTANT DONORS INTERFERENCE FROM COMMONLY USED DEPLETION THERAPIES (THYMOGLOBULIN, CAMPATH, RITUXIMAB, AND ANY OTHER HUMANIZED DEPLETION ANTIBODIES) IT IS STILL THE ONLY TRULY FUNCTIONAL ASSAY AVAILABLE CYTOTOXICITY CROSSMATCHES HAVE EXCELLED AT BEING CLINICALLY RELEVANT FOR >50 YEARS, LARGELY DUE TO THEIR INSENSITIVE NATURE 20

21 THE FLOW CYTOMETRY CROSSMATCH FOLLOWING THE RELEASE OF THE FIRST COMMERCIAL FLOW CYTOMETER IN 1969, THE FLOW CYTOMETRY CROSSMATCH CAME ABOUT IN THE EARLY 1980 S MORE SENSITIVE FASTER RESULTS CAPABLE OF ASSAYING T AND B CELLS IN A SINGLE TUBE 21

22 THE FLOW CYTOMETRY CROSSMATCH Recipient serum Donor cells Fluorescentlylabeled T or B cell markers Fluorescently - labeled Antihuman IgG Analyze on a flow cytometer Fluorescent label T cell 22

23 WHY STAIN T AND B CELLS IN THE FCXM? T AND B CELLS HAVE HIGH BASAL HLA EXPRESSION ON THEIR SURFACE AND CAN BE EASILY ISOLATED FROM WHOLE BLOOD (DONORS/ RECIPIENTS) OR SPLEEN/ LN s (DONORS): HLA CLASS I HLA CLASS II T cell B cell 23

24 DETECTION OF HLA ABS IN A FCXM Anti-class I DSA Anti-class II DSA T cell B cell 24

25 FCXM ANALYSIS REACTIVITY WITH NEGATIVE CONTROL SERUM REACTIVITY WITH RECIPIENT SERUM THE FCXM RESULT IS BASED ON A SIMPLE EQUATION: FCXM READOUT = Median fluorescence (sample) Median flourescence (neg control) THIS EQUATION SIMPLY MEASURES HOW MUCH MORE ANTIBODY IS BOUND TO DONOR CELLS BY THE PATIENT SERUM WHEN COMPARED TO THE NEGATIVE CONTROL SERUM. 25

26 COMPARISON OF CROSSMATCH METHODS FLOW CROSSMATCHES HAVE SOME ADVANTAGES OVER CYTOTOXICITY-BASED CROSSMATCHES BUT ARE SIMILARLY HAMPERED BY OTHER FACTORS: CYTOTOXICITY XM FLOW XM SENSITIVITY ABILITY TO EQUALLY ASSESS T AND B LYMPHOCYTES REACTIVITY FROM ANTIBODIES TO NON-HLA TARGETS INTERFERENCE FROM DEPLETION THERAPIES

27 THINGS THAT TRANSLATE INTO A POSITIVE FCXM PRESENCE OF DONOR-SPECIFIC HLA ANTIBODY (DSA) CONSIDER CELL POSITIVITY PATTERN; T POS/ B NEG IS NON-SPECIFIC NON-SPECIFIC REACTIVITY IS OFTEN OBSERVED IN PATIENTS WITH AUTOIMMUNITIES AND VARIES AMONG ORGAN GROUPS: ISLET/ PANCREAS, LIVER > KIDNEY > LUNG > HEART LIKELY REFLECTING THE RESPECTIVE RATES OF AUTOIMMUNITY IN EACH ORGAN GROUP 27

28 THINGS THAT TRANSLATE INTO A POSITIVE FCXM THAT MATTER NON-SPECIFIC FCXM REACTIVITY IS NOT UNCOMMON IN PATIENTS WHO LACK DSA: N = 508 % NON-SPECIFIC FCXM POS = ~11% Johnson et. al., Am. J. Transpl MORE IMPORTANTLY, FCXM POSITIVITY IN THE ABSENCE OF DSA IS CLINICALLY IRRELEVANT 28

29 THINGS THAT TRANSLATE INTO A POSITIVE FCXM THAT MATTER POSITIVE FCXM IN ABSENCE OF DSA FAIL TO AFFECT GRAFT SURVIVAL EVEN IN HIGHLY SENSITIZED PATIENTS (cpra>80%): Johnson et. al., Am. J. Transpl IT IS WITHIN THIS CONCEPT THAT THE TRUE VALUE OF VIRTUAL CROSSMATCHES IS OBSERVED 29

30 THINGS THAT TRANSLATE INTO A POSITIVE FCXM THAT MATTER CURRENT LITERATURE ONLY SUPPORTS POSITIVE FCXMs DUE TO DONOR-SPECIFIC HLA ANTIBODIES AS BEING CLINICALLY RELEVANT WHAT ABOUT NON-HLA ANTIBODIES??? Eg. MIC-A, AT1R, Kα1 tubulin, collagen V, etc THESE ANTIBODIES ARE NOT DETECTABLE ON T OR B LYMPHOCYTES THEREFORE CANNOT ACCOUNT FOR A POSITIVE FCXM RESULT 30

31 HLA ANTIBODY TESTING IN SOLID ORGAN TRANSPLANTS HLA ANTIBODY TESTING METHODS CAN BE SPLIT INTO TWO MAJOR FAMILIES OF METHODS: CELL BASED (aka SEROLOGY) SOLID PHASE SEROLOGY- BASED XMs (CDC, CDC- AHG) FLOW CYTOMETRY XMs ELISA FLOW CYTOMETRY SAg BEADS LUMINEX SAB C1Q C3d IgG PAN IgG SUBCLASSES 31

32 VIRTUAL CROSSMATCH ONCE THE DONOR HLA TYPE IS KNOWN, THE HLA SPECIFICITIES DETECTED BY SAB (USING A RECENT RECIPIENT SERUM SAMPLE) CAN BE CROSS-REFERENCED THIS FORMS THE BASIS FOR A VIRTUAL CROSSMATCH: RECENT LSAB RESULTS FOR POTENTIAL RECIPIENT RECIPIENT SERUM COMPLETE DONOR DONOR CELLS TYPING DETERMINE CYTOTOXICITY WHETHER DSA IS DETECTED FLOW CYTOMETRY IN THE RECIPIENT SERUM 32

33 VIRTUAL CROSSMATCH VIRTUAL CROSSMATCH ACCURACY REQUIRES: LSAB RESULTS THAT ARE RECENT, PARTICULARLY IN PATIENTS AT RISK FOR SENSITIZATION eg. TRANSFUSIONS (PARTICULARLY PLATELET), HOMOGRAFTS - COMPLETE DONOR HLA TYPINGS INCLUDING HLA-A HLA-B HLA-C HLA-DRB1 HLA-DQA1 HLA-DPA1 HLA-DRB3,4,5 HLA-DQB1 HLA-DPB1 ABS TO ANTIGENS WITHIN THESE LOCI CAN GENERATE A POSITIVE CROSSMATCH, BUT THAT IS NOT TO SAY THAT THERE IS CLEAR EVIDENCE FOR CLINICAL RELEVANCE FOR THESE 33

34 VIRTUAL CROSSMATCH VIRTUAL XM MAY BE USED TO DO TWO THINGS TO EXPEDITE DONOR ACCEPTANCE: - DETERMINE WHETHER A PRE-EXISTING DSA IS PRESENT - PREDICT THE OUTCOME OF A GIVEN TYPE OF CROSSMATCH A A A A A A B B B B B B DR DR DQ DQ DQ DQ A2 A1 B65 B18 A25 A1 B44 B8 DONOR 1 DONOR 3 DR10 DR11 DR52 DQ7 DQ5 DR13 DR8 DR52 DQ4 DQ6 DONOR 1 PREDICT: DSA POS POS CDC-AHG CROSSMATCH A3 A1 B13 B8 DONOR 2 DR15 DR13 DR51 DR52 DQ6 DONOR 3 PREDICT: DSA NEG NEG CDC-AHG AND NEG FCXM CROSSMATCH, DONOR 2 PREDICT: DSA POS NEG CDC-AHG AND NEG FCXM CROSSMATCH 34

35 CORRELATIONS BETWEEN SAB TESTS AND CROSSMATCH RESULTS CORRELATIONS BETWEEN SAB AND ACTUAL CROSSMATCHES ARE CRITICAL IF THE AIM OF THE VIRTUAL CROSSMATCH IS TO PREDICT CROSSMATCH OUTCOME THERE IS A GENERAL CORRELATION BETWEEN LUMINEX SAB VALUES AND CROSSMATCH RESULTS: Gloor JM et al Am J Transplant AHG-CDC > STRONG POS T FCXM > MOD. POS T FCXM OF INTEREST, FCXM NEG SAMPLES CAN HAVE DSA SPECIFICITIES THAT ADD UP TO MFI RANGE 35

36 CORRELATIONS BETWEEN SAB TESTS AND CROSSMATCH THERE IS A GENERAL CORRELATION BETWEEN LUMINEX SAB VALUES AND CROSSMATCH RESULTS: - SAMPLES WITH SIMILAR LEVELS OF ABS CAN GREATLY VARY IN FCXM REACTIVITY - THE CLINICAL OUTCOME (ABMR) MAINLY OCCURS IN PATIENTS WITH THE HIGHEST FCXM REACTIVITIES, BUT NOT ALWAYS 60% IN THIS EG. Reinsmoen N et al Transplantation (2008) 36

37 CORRELATIONS BETWEEN SAB TESTS AND CROSSMATCH RESULTS SIMILAR PROBLEMS ARE OBSERVED WHEN B CELL FCXM REACTIVITY IS CORRELATED TO THE SUM OF CLASS I AND II DSA: IT IS EASY TO STATISTICALLY ARGUE FOR A CORRELATION OR LIKEWISE TO DRAW A THRESHOLD BUT WE SHOULD NOT OVERLOOK THE FACT THAT SAMPLES WITH SIMILAR DSA LEVELS CAN VARY BY UP TO 500 MCS! Ellis et. al., Hum. Immunol

38 UNACCOUNTED VARIABLES IN CROSSMATCH RESULTS CROSSMATCHES OF ALL TYPES SUFFER FROM LACK OF STANDARDIZATION MANY KEY PARAMETERS ARE OFTEN OVERLOOKED AND TREATMENTS TO ENHANCE CROSSMATCH REACTIVITY ARE NOT CONSISTENTLY PERFORMED: PARAMETERS OVERLOOKED THAT AFFECT CORRELATIONS TO SAB: - VARIABILITY IN CELL POPULATIONS, SPECIALLY WHEN USING WHOLE BLOOD - FICOLL-BASED ISOLATION VS. IMMUNOMAGNETIC PURIFICATION TO ENHANCE LYMPHOCYTE NUMBERS TESTED - VARIABILITY IN CELL % ASSESSED BY ONE COMMON THRESHOLD - PRONASE TREATMENT - VARIABILITY IN ANTIGEN DENSITY ON THE DONOR CELLS BEING TESTED 38

39 VIRTUAL CROSSMATCH TO PREDICT CROSSMATCH REACTIVITY THE COMMON PREDICTION OF CROSSMATCH REACTIVITY RELIES ON SAB MFI VALUES. HOWEVER, WE KNOW MFI VALUES CAN BE AFFECTED BY SERUM INTERFERENCES AND BEAD SATURATION: Tambur et. al., Am. J. Transpl

40 VIRTUAL CROSSMATCH TO PREDICT CROSSMATCH REACTIVITY VIRTUAL CROSSMATCHES ARE ALSO AFFECTED BY REACTIVITIES AGAINST SAB DUE TO CRYPTIC EPITOPES AND/OR MISFOLDED HLA MOLECULES Morales-Buenrostro et. al., Transplantation

41 VIRTUAL CROSSMATCH TO PREDICT CROSSMATCH REACTIVITY NON-HLA SPECIFIC REACTIVITIES AGAINST SAB INVOLVE MANY COMMON ANTIGENS: Gombos et. al., Am. J. Transpl

42 NON-SPECIFIC SAB REACTIVITY A DANGEROUS ASSUMPTION IS TO ASSUME THAT HIGH MFI SAB REACTIVITIES ARE ALWAYS REAL: AND CROSSMATCH INTERPRETATION MAY NOT BE STRAIGHT FORWARD: Surrogate cell typing MCS Interpretation Interpretation Notes T cell 112 Positive False positive T cell reactivity B46, B75 B cell 38 Negative Negative B cell reactivity reflect the actual reactivity by which the surrogate XM can be assessed 42

43 DEFINING THE CLINICAL RELEVANCE OF POSITIVE CROSSMATCHES THE FIXATION OF HLA LABORATORIES TO PREDICT ACTUAL XM REACTIVITY BASED ON SAB NEEDS TO CONSIDER THE FOLLOWING ASSUMPTION: ALL CROSSMATCHES ARE ACCURATE SURROGATES FOR WHAT CAN OCCUR ON THE ENDOTHELIUM OF THE DONOR ORGAN: = WE KNOW VERY LITTLE ABOUT ENDOTHELIAL HLA EXPRESSION, OTHER THAN THAT IT IS DIFFERENTLY REGULATED FROM EXPRESSION ON T AND B CELLS 43

44 SENSITIVITY OF HLA ANTIBODY TESTING METHODS THE HIGH SENSITIVITY OF SAB METHODS HAS IT S OWN SET OF PROBLEMS WITH NON-HLA RELATED REACTIVITY BUT HAS FEWER VARIABLES TO ACCOUNT FOR THAN ACTUAL CROSSMATCHES 44

45 UPCOMING CROSSMATCH METHODS GIVEN THE LIMITATIONS ON BOTH ACTUAL AND VIRTUAL CROSSMATCHES, ARE THERE ANY TOOLS THAT MAY HELP TO ADDRESS THESE ISSUES? CYTOTOXICITY XM FLOW XM LUMINEX XM SENSITIVITY ABILITY TO EQUALLY ASSESS T AND B LYMPHOCYTES N/A REACTIVITY FROM ANTIBODIES TO NON-HLA TARGETS INTERFERENCE FROM DEPLETION THERAPIES ALTHOUGH LIMITATIONS OF EXTRAPOLATION TO ENDOTHELIAL HLA EXPRESSION REMAIN 45

46 SUMMARY CROSSMATCH OUTCOMES HAVE HISTORICALLY INHERITED A CLINICAL RELEVANCE, INTRINSIC TO THE INSENSITIVE NATURE OF THE EARLY ASSAYS VIRTUAL CROSSMATCHES BASED ON SENSITIVE SAB TESTING EXPEDITES THE ACCEPTANCE OF DONOR OFFERS, AS LONG AS SERA ARE PROPERLY TREATED AND SAB DATA IS CORRECTLY INTERPRETED THE RELENTLESS PURSUIT TO ATTEMPT TO PREDICT ACTUAL CROSSMATCH REACTIVITY NEEDS TO TAKE INTO ACCOUNT THE BIOLOGIC LIMITATION OF WHAT INFORMATION AN ACTUAL CROSSMATCH CAN PROVIDE 46

47 QUESTIONS??? 47

48 Continuing Education ABHI, ASCLS/P.A.C.E., Florida and California Credits 1.0 Contact Hour or 0.15 continuing education credits (CECs) awarded Each attendee must register to receive CE credits at: Registration deadline is 16 March 2018 Certificates will be sent via only to those who have registered by 30 March 2018 All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc. 48

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50 Future Webinars Histocompatibility testing: From antigens towards epitopes Featuring Prof. dr. Frans H. J. Claas Leiden University Medical Center Eurotransplant Reference laboratory Leiden, the Netherlands 15 March :30-17:30 IST (11:00-12:00 GMT) 08:00-09:00 EST (13:00-14:00 GMT) 14:00-15:00 EST (19:00-20:00 GMT) Link to register: All Content 2015 Immucor, Inc. 50

51 Thank you! All Content 2015 Immucor, Inc. All Content 2015 Immucor, Inc. 51