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Transplant Webinar Series: Ep. 3Donor Selection For Solid Organ Transplants – Is A Virtual Crossmatch Better Than Real?
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Histocompatibility testing: From antigens towards epitopes
Featuring
Prof. dr. Frans H. J. ClaasLeiden University Medical Center
Eurotransplant Reference LaboratoryLeiden, the Netherlands
15 March 201816:30-17:30 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)
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Continuing Education
• ABHI, ASCLS/P.A.C.E., Florida and California Credits
• 1.0 Contact Hour or 0.15 continuing education credits (CECs) awarded
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• Registration deadline is 16 March 2018
• Certificates will be sent via email only to those who have registered by 30 March 2018
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• 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.All Content © 2015 Immucor, Inc.
Dr Luis G. Hidalgo
DONOR SELECTION FOR SOLID ORGAN TRANSPLANTS –
IS A VIRTUAL CROSSMATCH BETTER THAN REAL?
Luis G. Hidalgo, Ph.D., D(ABHI)
Associate Director - Histocompatibility LaboratoryUniversity of Alberta Hospitals
Dept. of Lab. Med. and Pathology
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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
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
HYPERACUTE REJECTION CAN DESTROY THE KIDNEY TRANSPLANT WITHIN MINUTES/ HOURS AFTER REPERFUSION:
THE HISTORY OF HLA ABS IN TRANSPLANTATION
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HOWEVER, UP UNTIL THE MID 1960’S A ROLE FOR ANTIBODIES IN TRANSPLANTATION WAS DEBATED:
Kissmeyer-Nielsen et. al., Lancet, 1966
THE HISTORY OF HLA ABS IN TRANSPLANTATION
Patel et. al., NEJM 1969
Dr. Paul Terasaki:
IN THE LATE 1960’S THE LANDMARK PAPER BY PATEL AND TERASAKI CLEARLY SHOWED THE BENEFIT OF DETECTING DONOR-REACTIVE ANTIBODIES PRE-TRANSPLANT:
THE HISTORY OF HLA ABS IN TRANSPLANTATION
THIS MARKED THE BEGINNING OF TESTING FOR DONOR-REACTIVE
ANTIBODIES AS A STANDARD PROCEDURE PRE-TRANSPLANT –
THE BIRTH OF THE FIELD OF HISTOCOMPATIBILITY
THE CONCEPT OF A CROSSMATCHA 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)
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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)
THE TYPES OF CROSSMATCHES
CYTOTOXIC CROSSMATCHES - CDC
SUBJECTIVE SCORING
LIMITED SENSITIVITY!
COMPLEMENT-DEPENDENT CYTOTOXICITY (CDC)
THIS IS THE ASSAY THAT PAUL TERASAKI ORIGINALLY DEVELOPED
CYTOTOXIC CROSSMATCHES – AHG-CDC
AN ENHANCEMENT METHOD WAS DEVELOPED TO HELP DEAL WITH THE SENSITIVITY OF THE CDC ASSAY:
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CLINICAL RELEVANCE OF A POSITIVE CYTOTOXIC CROSSMATCH
Gloor JM et al
Am J Transplant (2010)
T CELLAHG-CDC +
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)
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
FOLLOWING THE RELEASE OF THE FIRST COMMERCIAL FLOW CYTOMETER IN 1969, THE FLOW CYTOMETRY CROSSMATCH CAME ABOUT IN THE EARLY 1980’S
THE FLOW CYTOMETRY CROSSMATCH
MORE SENSITIVE
FASTER RESULTS
CAPABLE OF ASSAYING T AND B CELLS IN A SINGLE TUBE
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Recipient serum Donor
cells
Fluorescently- labeled Anti-
human IgG
Fluorescently-labeled
T or B cell markers
T cell
Fluorescent label
Analyze on a flow cytometer
THE FLOW CYTOMETRY CROSSMATCH
WHY STAIN T AND B CELLS IN THE FCXM?
T cell B cell
HLA CLASS I HLA CLASS II
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):
DETECTION OF HLA ABS IN A FCXM
T cellB cell
Anti-class I DSA Anti-class II DSA
++ +++ +++-
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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.
FCXM ANALYSIS
REACTIVITY WITH
NEGATIVE CONTROL
SERUM
REACTIVITY WITH
RECIPIENT SERUM
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 +++ +++
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
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NON-SPECIFIC FCXM REACTIVITY IS NOT UNCOMMON IN PATIENTS WHO LACK DSA:
THINGS THAT TRANSLATE INTO A POSITIVE FCXM – THAT MATTER
Johnson et. al., Am. J. Transpl. 2016
N = 508
% NON-SPECIFIC FCXM POS = ~11%
MORE IMPORTANTLY, FCXM POSITIVITY IN THE ABSENCE OF
DSA IS CLINICALLY IRRELEVANT
POSITIVE FCXM IN ABSENCE OF DSA FAIL TO AFFECT GRAFT SURVIVAL EVEN IN
HIGHLY SENSITIZED PATIENTS (cPRA>80%):
THINGS THAT TRANSLATE INTO A POSITIVE FCXM – THAT MATTER
Johnson et. al., Am. J. Transpl. 2016
IT IS WITHIN THIS CONCEPT THAT THE TRUE VALUE OF
VIRTUAL CROSSMATCHES IS OBSERVED
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…
THINGS THAT TRANSLATE INTO A POSITIVE FCXM – THAT MATTER
THESE ANTIBODIES ARE NOT DETECTABLE ON T OR B LYMPHOCYTES –THEREFORE CANNOT ACCOUNT FOR A POSITIVE FCXM RESULT
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HLA ANTIBODY TESTING IN SOLID ORGAN TRANSPLANTS
CELL BASED(aka SEROLOGY)
SOLID PHASE
ELISAFLOW
CYTOMETRY SAg BEADS
LUMINEX SAB
C1Q C3d IgG SUBCLASSES
SEROLOGY-BASED XMs (CDC, CDC-
AHG)
FLOW CYTOMETRY
XMs
HLA ANTIBODY TESTING METHODS CAN BE SPLIT INTO TWO MAJOR FAMILIES OF METHODS:
PAN IgG
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:
RECIPIENT SERUM DONOR
CELLS
CYTOTOXICITY
FLOW CYTOMETRY
RECENT LSAB
RESULTS FOR
POTENTIAL RECIPIENT
COMPLETE DONOR TYPING
DETERMINE WHETHER DSA IS DETECTED IN THE RECIPIENT SERUM
VIRTUAL CROSSMATCHVIRTUAL 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-DRB3,4,5
HLA-DQA1 HLA-DQB1
HLA-DPA1 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
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DONOR 1
VIRTUAL CROSSMATCH
DONOR 1 PREDICT: DSA POSPOS CDC-AHG CROSSMATCH
A1A2
B18
B65
DR10
DR11
DQ7
DR52
DQ5
A2 22350
A3 1200
A23 11890
A24 12450
A68 21880
A69 20920
B13 950
B18 14560
B35 15382
B51 12275
B52 11630
B53 13210
DR4 6010
DR53 16000
DQ2 3200
DQ7 21880
DQ8 20680
DQ9 20920
DONOR 2
A1A3
B8
B13
DR15
DR13DR51
DQ6DR52
DONOR 2 PREDICT:DSA POSNEG CDC-AHG AND NEG FCXM CROSSMATCHDONOR 3
A1A25
B8
B44
DR13
DR8DR52
DQ6DQ4
DONOR 3 PREDICT:DSA NEGNEG CDC-AHG AND NEG FCXM 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
THERE IS A GENERAL
CORRELATION BETWEEN
LUMINEX SAB VALUES AND
CROSSMATCH RESULTS:
- AHG-CDC > STRONG POS T
FCXM > MOD. POS T FCXM
CORRELATIONS BETWEEN SAB TESTS AND CROSSMATCH RESULTS
OF INTEREST, FCXM NEG SAMPLES
CAN HAVE DSA SPECIFICITIES THAT
ADD UP TO 3000-8000 MFI RANGE
Gloor JM et al
Am J Transplant 2010
CORRELATIONS BETWEEN SAB AND ACTUAL CROSSMATCHES ARE CRITICAL IF
THE AIM OF THE VIRTUAL CROSSMATCH IS TO PREDICT CROSSMATCH OUTCOME
Reinsmoen N et al Transplantation (2008)
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.
CORRELATIONS BETWEEN SAB TESTS AND CROSSMATCH
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SIMILAR PROBLEMS ARE OBSERVED WHEN B CELL FCXM REACTIVITY IS
CORRELATED TO THE SUM OF CLASS I AND II DSA:
CORRELATIONS BETWEEN SAB TESTS AND CROSSMATCH RESULTS
IT IS EASY TO STATISTICALLY ARGUE FOR A CORRELATION
Ellis et. al., Hum. Immunol. 2012
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!
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:
UNACCOUNTED VARIABLES IN CROSSMATCH RESULTS
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
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:
VIRTUAL CROSSMATCH TO PREDICT CROSSMATCH REACTIVITY
Tambur et. al., Am. J. Transpl. 2016
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VIRTUAL CROSSMATCHES ARE ALSO AFFECTED BY REACTIVITIES AGAINST
SAB DUE TO “CRYPTIC EPITOPES” AND/OR MISFOLDED HLA MOLECULES
Morales-Buenrostro et. al., Transplantation 2008
VIRTUAL CROSSMATCH TO PREDICT CROSSMATCH REACTIVITY
Gombos et. al., Am. J. Transpl. 2013
NON-HLA SPECIFIC
REACTIVITIES AGAINST SAB
INVOLVE MANY COMMON
ANTIGENS:
VIRTUAL CROSSMATCH TO PREDICT CROSSMATCH REACTIVITY
A DANGEROUS ASSUMPTION IS TO ASSUME THAT HIGH MFI SAB REACTIVITIES
ARE ALWAYS REAL:
NON-SPECIFIC SAB REACTIVITY
Surrogate cell typing MCS Interpretation Interpretation Notes
T cell 112 Positive False positive T cell reactivity
B cell 38 NegativeNegative B cell reactivity reflect the actual reactivity by which the
surrogate XM can be assessed
B46, B75
AND CROSSMATCH INTERPRETATION MAY
NOT BE STRAIGHT FORWARD:
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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
DEFINING THE CLINICAL RELEVANCE OF POSITIVE CROSSMATCHES
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
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
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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
QUESTIONS???
All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.
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:
https://www.surveymonkey.co.uk/r/ImmucorTransplantEp3
• Registration deadline is 16 March 2018
• Certificates will be sent via email only to those who have registered by 30 March 2018
19/02/2018
17
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Future Webinars
Link to register: https://immucor.webinato.com/register
Histocompatibility testing: From antigens towards epitopes
Featuring
Prof. dr. Frans H. J. ClaasLeiden University Medical Center
Eurotransplant Reference laboratoryLeiden, the Netherlands
15 March 201816:30-17:30 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)
All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.All Content © 2015 Immucor, Inc.
Thank you!