immucor transplant ep 3 webinar - main page program handouts/ous...-flow cytometry...

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

All Content © 2015 Immucor, Inc.

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.

Handouts

http://www.immucor.com/en-us/Pages/Educational-Program-Handouts.aspx

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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

• 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


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).


Questions?

• You are all muted

• Q&A following session - Type in questions

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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.


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:


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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


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:


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%):


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…


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


Gombos et. al., Am. J. Transpl. 2013

NON-HLA SPECIFIC

REACTIVITIES AGAINST SAB

INVOLVE MANY COMMON

ANTIGENS:


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???


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

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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)


Thank you!

immucor transplant ep 3 webinar - main page program handouts/ous...-flow cytometry...

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