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2019 Transplant Webinars

Link to register: https://immucor.webinato.com/register


learn.immucor.com


Continuing Education

• PACE, Florida and California DHS

• 1.0 Contact Hours

• Each attendee must register to receive CE at:

https://www.surveymonkey.com/r/HLAtoolbox

• Registration deadline is March 29, 2019

• Certificates will be sent via email only to those

who have registered April 12, 2019



Questions?

• You are all muted

• Q&A following session - Type in questions


• 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.

HLA DEPOT: WHAT’S IN YOUR TOOLBOX?

Anne Halpin & Esmé DijkeUniversity of Alberta

Alberta Transplant InstituteEdmonton, Alberta

DISCLOSURE

This presentation may contain Canadian content

EVOLUTION IN TOOLS

In the beginning, there were sera, complement, and isolation of cells in straws.

And it was good.

http://www.polysciences.com Pinshow Straw Co

EVOLUTION IN TOOLS

But as with all great tools, even better tools follow….

EVOLUTION IN TOOLS

And now these tools are part of the standard of care in the HLA laboratories

http://beckmanflow.com https://www.luminexcorp.com/

Flow cytometry for crossmatch (FCXM)

Luminex technology (for typing and antibody detection)

EVOLUTION OF HLA TYPING TOOLS

Serology Typing

Molecular Typing• SSO• SSP• rSSO• qPCR

Sequencing• SBT• NGS

EVOLUTION OF CROSSMATCH TOOLS

• Flow crossmatch still has many possible sources of variability

• Pronase use

• Cell isolation method

• “Halifaster” XM is widely used across Canada (courtesy of Dr. Rob Liwski*)

• Additional solid phase XM tools are also available

Serology XM ELISA XMFlow Cytometry

Crossmatch

*Liwski et al Hum Immunol. 2018 Jan;79(1):28-38

SINGLE ANTIGEN BEAD TOOLS

• Single antigen beads have revolutionized our ability to detect HLA

antibodies, such as:

• Ability to define HLA specificities in high PRA patients

• Increased sensitivity as compared to CDC

• Class II antibodies are much more easily characterized

• BUT…

• Non-specific reactivity can be observed

• PRA can no longer be defined in the same way

CALCULATED PANEL REACTIVE ANTIBODY (CPRA)

• The use of single antigen beads allow us to to define antibody specificity in a

new way BUT created a need to re-define PRA values

• UNET created a cPRA tool based on antigen frequencies in the donor pool

• The Canadian Transplant Registry (CTR) created a similar tool

CPRA UNET

• The UNOS/UNET cPRA

calculator has evolved to

include many alleles

• cPRA is derived from U.S.

deceased donor antigen

frequencies

CPRA CANADIAN

TRANSPLANT

REGISTRY

• Unacceptable antibody is captured in a

different field

• cPRA is calculated based on Canadian

deceased donor antigen frequencies

EPITOPE TOOLS

• Epitopes were initially defined by serological methods

• Rodey ’CREG’

• HLA Matchmaker was first described in 2001

• ’Reflections on HLA Epitope-Based Matching for Transplantation’

Rene J. Duquesnoy, Front. Immunol., 28 November 2016

• Today there are many epitope tools available such as

• HLA Matchmaker http://www.epitopes.net

• Epitope Registry http://www.epregistry.com.br

• PIRCHE https://www.pirche.com/pirche/#/

http://www.epitopes.net/

http://www.epregistry.com.br/

https://www.pirche.com/pirche/#/

HOW DO WE USE THESE EPITOPE TOOLS?

• Epitopes can be used for:

• HLA matching

• Transplant risk assessment

• Investigating antibody patterns

ADDITIONAL TOOLS

• Tools to investigate allele and haplotype frequencies

• Haplostat

https://www.haplostats.org/haplostats?execution=e1s1Allele

• Allele frequencies

http://www.allelefrequencies.net

• IMGT

https://www.ebi.ac.uk/ipd/imgt/hla/

https://www.haplostats.org/haplostats?execution=e1s1Allele

http://www.allelefrequencies.net/

https://www.ebi.ac.uk/ipd/imgt/hla/

HOW DO WE USE THESE TOOLS?

CASE STUDY EXAMPLES FROM OUR TRANSPLANT CENTRE

CASE 1

• 48-year old male with chronic renal failure

• Received first kidney transplant in summer 2015

• Patient typing: A29 B72 Cw12 DR8 DR52 DQ7 DP04:02

A30 B39 DQ4 DP02:01

• Donor typing: A24 B18 Cw17 DR17 DR52 DQ7 DP02:01

A25 B44 Cw11 DR11 DQ2

• Pre-transplant cPRA = 48%, no DSA detected by SAB

• HLA antibodies in cPRA calculation include B37 and DR53

• Both pre-transplant T and B cell flow crossmatch were negative

CASE 1

• 3 ½ years post-transplant patient developed consistent positive urinalysis for

protein and hemoglobin (creatinine is stable)

• HLA antibody testing was performed at the time of biopsy for cause as per local

centre protocol

• No Class I antibody was detected

• Questionable Class II antibody detected → required further investigation

CASE 1 Luminex SAB Class II (OLI*): strong reactivity to 1 DQ6 bead: DQB1*06:01

Is this a real antibody?

*OLI: One Lambda Thermo Fisher

CASE 1Luminex SAB Class II (LC*): also strong reactivity to DQB1*06:01

*LC: LIFECODES Immucor

Patient has allele-specific reactivity to DQB1*06:01 (not DSA)

• non-antibody verified epitope that is specific to DQB1*06:01 = 3P4

http://www.epregistry.com.br

CASE 1

• 48-year old male with chronic renal failure

• Received first kidney transplant in summer 2015


A30 B39 DQ4 DP02:01


A25 B44 Cw11 DR11 DQ2

• 3 ½ years post-transplant patient developed consistent positive urinalysis for protein

and hemoglobin (creatinine is stable)

• No DSA detected (and biopsy showed no evidence of rejection)

CASE 2

• 56-year old female with coronary artery disease – left ventricular dysfunction

• Listed for heart transplantation

• Sensitizing events: multiple pregnancies, multiple transfusions


A31 B60 Cw15 DR15 DR51 DQ7

• Work up for listing: questionable Class I antibody was detected

→ required further investigation

• No Class II antibody was detected

CASE 2Luminex SAB Class I (OLI*): strong reactivity to Cw5

Is this a real antibody?


CASE 2Luminex SAB Class I (LC*): strong reactivity to Cw5 and Cw8

C*05:01 and C*08:02: share an eplet 138K



CASE 2

Surrogate crossmatch:‘Donor’ typing: A2 B44 Cw5 DR13 DR52 DQ6 DP04:01

A3 B62 Cw9 DR8 DQ4

T-cell flow crossmatch: positiveB-cell flow crossmatch: positive

Patient has strong reactivity to Cw5 (listed as unacceptable antigen)

CASE 2

• 56-year old female with coronary artery disease – left ventricular dysfunction

• Listed for heart transplantation

• Sensitizing events: multiple pregnancies, multiple transfusions


A31 B60 Cw15 DR15 DR51 DQ7


A25 B27 Cw12 DP23

• Both T and B cell crossmatch were negative

• Transplanted in summer 2018

CASE 3

• 37-year old female with cystic fibrosis listed for lung transplantation

• Sensitizing events: none known at time of listing

• Non-specific patterns of reactivity with weak antibodies to

DQ7, DQ8, DQ9 was observed in single antigen bead assay


B58 Cw16 DR1 DP02:01


A26 B56 Cw1 DR11 DQ2 DP10

• Both T and B cell flow crossmatch were NEGATIVE with this previously screened serum

sample BUT were POSITIVE with the immediate pre-transplant serum sample

CASE 3Luminex SAB Class I (OLI*): strong reactivity to multiple HLA-A and –B alleles

Donor-specific antibody

Self antibody?

• Including to A2: A*02:01, A*02:03 and A*02:06 • Patient typing: A*02:02 and A*02:05


CASE 3Luminex SAB Class I (LC*): also strong reactivity to multiple HLA-A and –B alleles

• Including to A*02:01 and A*02:03

• But NOT to A*02:05 and A*02:02

• Reactivity is allele-specific• non-antibody verified epitope = 95V



CASE 3Luminex SAB Class II (OLI*): strong reactivity to DQ7, DQ8, DQ9; moderate reactivity to DQ2

Donor-specific antibody


CASE 3

• 37-year old female with cystic fibrosis

• Listed for lung transplantation

• Sensitizing events: none known at time of listing


B58 Cw16 DR1 DP02:01


A26 B56 Cw1 DR11 DQ2 DP10

• Multiple DSA were observed in immediate pre-transplant sample

• Post transplant monitoring was performed

CASE 3Antibody tracking over time:

• DSA levels declined over time

• Patient is doing well in this early post-transplant phase

SUMMARY OF CASES- WHAT WAS IN OUR TOOLBOX

• To investigate these cases we used:

• Multiple vendor kits for antibody analysis

• Epitope analysis

• Surrogate flow crossmatch

SUMMARY

• We have many tools in our HLA

toolbox

• Our earliest tools were limited

• We now have more tools at our

disposal

• They can be used in combination

https://www.iphonelife.com/blog/31936/taskone-iphone-case-swiss-army-knife-your-phone

IS OUR TOOLBOX COMPLETE?

• The fields of histocompatibility

and transplantation are most

consistent in their ability to be

constantly evolving

• Our toolbox likely will have more

tools in the future! Star Wars: Episode IV – A New Hope

THANK YOU!

QUESTIONS?


Questions?




2019 Advanced Track Webinars



Questions?




Continuing Education

• PACE, Florida and California DHS

• 1.0 Contact Hours

• Each attendee must register to receive CE at:


• Registration deadline is March 29, 2019

• Certificates will be sent via email only to those

who have registered April 12, 2019



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Program-Handouts Program... · •Single antigen beads have revolutionized our ability to detect...

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