Past Present and Future of Antibody Testing

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Past Present and Future of Antibody Testing

• Patricia Campbell University of Alberta. Edmonton

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Objectives

• Past technology
• Present current assays and strategies
• Future

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HOW IT ALL STARTED
PATEL AND TERASAKI, NEJM 280735,1969
Courtesy of H. Gebel
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Evolution of HLA Antibody Detection
Cytotoxicity
Anti-HLA Antibody
C1
Membrane Attack Complex
Dye
Ly
Courtesy of H Gebel
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HOW IT ALL STARTED
PATEL AND TERASAKI, NEJM 280735,1969
Adapted from a slide of H. Gebel
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Evolution of HLA Antibody Detection
Cytotoxicity Enhanced
Cytotoxicity
Anti-HLA Antibody
Anti-Human Globulin
C1
Membrane Attack Complex
Membrane Attack Complex
Dye
Dye
Ly
Ly
Courtesy of H Gebel
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CADAVERIC RENAL ALLOGRAFT SURVIVAL AMONG PRIMARY
RENAL ALLOGRAFT RECIPIENTS
NIH AHG NEG NEG POS
82 67 (124/151)
(10/15) plt0.01
81 (134/166)
Kerman et al. Transplantation 51316-320 1991
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HLA ANTIBODIES and ACUTE REJECTION
Abve Ab-ve p value
(n14) (n11)
Days to AR 4.2 days
13.9 days lt0.001 Dialysis with AR
13 (92.8) 2 (18.2)
lt0.001 Graft loss lt3 Months 9 (64.3)
1 (9) lt0.001
Repeat crossmatch 9/14 (64.3)
1/30(3) ve
control group, not Ab-ve pts
Trpkov et al Transplantation, Volume
611586,1996.
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CDC Assays

• Class I only
• Doesnt distinguish IgM from IgG
• Less sensitive than solid phase assays
• Cell panel viability
• Interference from thymoglobulin, OKT3, campath.
• High PRA cannot determine specificities

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AHG T-Cell Final Crossmatches 1o vs 2o Renal
Allograft Recipients
Survival (years post Tx)
1 5 7
AHG - Primary
93 78 72
AHG - Regrafts
71 55 45
Bryan, C.F., et al Transplantation 661827,
1998.
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Flow Cytometry
ELISA
Suspension Array
Anti-IgG
HLA alloantibody
Anti-IgG-PE
Anti-IgG-FITC
PRA 78
Gebel and Bray. Transplantation Reviews 20
189-194, 2006
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Bray, et al. AJT 62307-2315, 2006
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• Goal of antibody screening is immunological risk
  assessment and crossmatch prediction
• Important to have the antibody screening
  technique as sensitive as the crossmatch
• If antibody screening is much less sensitive than
  the crossmatch unexpected positives will occur

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Antibody Screens Cell vs Solid Phase
Zeevi A et al Immunol Res 2006
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Interpretation of Antibody Screen

• Is antibody present? (Antibody screen PRA)
• Is it HLA? (method)
• HLA vs non HLA (solid phase HLA)
• Class I, class II or both?
• What are the strong antibodies? (unacceptable
  antigens)
• What are the low-moderate risk antibodies
• Define acceptable mismatches (are there antigens
  that are clearly negative)

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vPRA HGebel cPRA http//www.unos.org/resources/
frm_CPRA_Calculator.asp?index78
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A2-B44-DR4-DQ2

vPRA HGebel
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Virtual Crossmatch

• Using the single antigen beads detailed antibody
  analysis is possible
• Assign strong, moderate and weak antibodies and
  define unacceptable and acceptable mismatches.
• Crossmatch Prediction Virtual Crossmatch

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Virtual Crossmatch

• Panel tested does it cover all antigens ie Cw,
  DP
• Frequency of testing- when was the last test?
• Sensitizing events- recent transfusions/pregnancie
  s/withdrawal of immunosuppression can result in
  new HLA specificities.

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How does Virtual Crossmatch compare with Final
Crossmatch?

• Use of cPRA in UNet requires entry of
  unacceptable antigens
• Organs will not be offered to recipients if UA
  listed for that donor
• Reviewed two scenarios
• 1. listing strong and moderate antibodies as
  unacceptable
• 2. listing strong antibodies only as
  unacceptable.

Human Immunology Oct 2007 K. Nelson et al.
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• Results
• 1. Highly sensitized 12 of list and received 15
  transplants.
• Over 80 accuracy in predicting negative
  crossmatch
• 2. 14 of list and received 20 of the
  transplants
• lt60 accuracy in predicting negative crossmatch.

Human Immunology Oct 2007 K. Nelson et al.
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cPRA and Virtual Crossmatch

• Discrepancies mainly due to donors having
  multiple HLA antibodies that were medium to low
  titer (additive effect) or to antibodies to DP,
  or antibodies to alleles of recipients own HLA

Human Immunology Oct 2007 K. Nelson et al.
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Final Crossmatch

• Whenever possible a final crossmatch is performed
  prior to transplant
• For sensitized recipients of renal transplants it
  should be done prior to transplant
• When cold time prohibits a prospective crossmatch
  the OR proceeds and the crossmatch is set up as
  soon as possible.
• In sensitized individuals the risk of proceeding
  must be determined.

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Crossmatch Target Cells

• Donor lymphocytes are used in the HLA crossmatch
• T cells Class I antigens (A,B,C)
• B cells Class I and II antigens (A,B,C,DR,DQ)

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Evolution of HLA Antibody Detection
Cytotoxicity Enhanced
Cytotoxicity Flow Cytometry
Anti-HLA Antibody
Anti-Human Globulin
Fluorescenated Anti-Human Globulin
C1
Membrane Attack Complex
Membrane Attack Complex
Dye
Dye
Ly
Ly
or
CD19
CD3
Flow Cytometer
Courtesy of Dr H Gebel
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Effect of Sensitive Crossmatch Tests on Early
Rejection (1999-2003)
14
First Tx
Re-Tx
12
plt0.001
plt0.001
10
8
Percent with Rejection
6
4
2
0
CDC
AHG
FLOW
CDC
AHG
FLOW
Crossmatch
Cecka, Clinical Transplants 2004 (p.8)
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Comparative Flow Cytometric and AHG T-Cell
Final CrossmatchesRenal Allograft Studies
Survival (years post Tx)
1 5 7
AHG - Primary
93 78 72
AHG - Regrafts
71 55 45
Flow - Regrafts
88 72 68
Bryan, C.F., et al Transplantation 661827,
1998.
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Fluorescence
Emitted Fluorescence Intensity ??Binding Sites
FITC
FITC
FITC
FITC
FITC
FITC
FITC
FITC
FITC
FITC
Number of Events
Negative positive
Fluorescent Intensity
Courtesy of H Gebel
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Evolving Clinical Paradigm (2007 ?)
Low Risk
High Risk
Surgical Threshold
T and B cell CDC CXM solid-phase -ve
reliable history or AHG-CDC CXM solid-phase -ve
or Flow CXM solid-phase -ve
T and B cell CDC CXM ve Solid Phase ve
or AHG-CDC CXM Solid Phase ve or Flow CXM
Solid Phase ve
Patient
Transplant
Contraindicated
Courtesy of H Gebel
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Kidney Transplants U of A Deceased vs Living Donor
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NIH
AHG
FLOW
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cPRA in patients transplanted vs listed for
kidney transplant
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2008 listed cPRA vs transplanted heart and lung
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Deceased donor kidney transplants
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Deceased donor kidney transplants graft Loss lt4
months
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Causes of graft loss lt4 months Kidney Transplants
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Causes of graft loss lt4 months in kidneys that
never worked.
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• Can detect low levels of HLA antibodies
• Reduce early rejection and graft loss from
  immunological causes.
• Early AMR not seen anymore unless patient
  undergoes desensitization
• Wait list accumulates high PRA retransplants

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Future

• How can we use these tools to improve outcomes

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Locations of Polymorphic Residues on HLA-A, B, C
Molecules
From Kostyu et al. Human Immunology 57, 1-18,
1997
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Immunobiology 5 Janeway, Travers, Walport and
Shlomchik
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El-Awar et al Human Immunology 2007 vol 68 issue 3
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El Awar et al Transplantation 2007 Aug 27
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http//hlamatchmaker.net/powerpoint/perfect08ashi.
ppt27
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Structural Basis of a HLA-B51 Mismatch
Seen by A2,A68 B27,B44
Seen by A2,A24 B7,B8
Seen by A2,A68 B35,B44
Polymorphic Residues on B51
http//hlamatchmaker.net/powerpoint/perfect08ashi.
ppt27
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HLA matching at the triplet level
Donor m.m B18


Patient B7
B52
A33
.
All MM are single HLA B mismatches but very
different no of triplet MM
Claas F presentation http//hlamatchmaker.net/powe
rpoint/frans08efi.ppt
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Examples of HLA Antigens with 0-2 Triplet
Mismatches
Nr of Mismatched Triplets Patient 1 HLA-A2,A30 B18,B27 Cw2,Cw4 Patient 2 HLA-A2,A31 B42,B53 Cw2,Cw7
Zero A69, B64, B65 A32, A74, B8, B35, B54, B55, B56, B59, Cw3, Cw6
One A68, B37, B39, B73, B74, Cw6 A69, B51, B67, B70, B71, B72, B76, B78
Two B61, B70, B71, B72, Cw1, Cw3, Cw6 A33, A68, B7, B38, B39, B46, B52, B58, B64, B65, B75, B77, B81, B82, Cw1, Cw8
http//hlamatchmaker.net/powerpoint/perfect08ashi.
ppt27
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Dankers M et al Transplantation Vol 77 No 8 April
27 2004
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Dankers M et al Transplantation Vol 77 No 8 April
27 2004
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• What are the mismatched epitopes?
• Are they immunological mismatches?
• If we can avoid some key mismatches perhaps the
  degree of sensitization post tx will be less.
• Use HLA matchmaker as a tool to understand
  matching/mismatching
• Ability to match limited due to limited number of
  donors

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• we have the tools to detect low levels of
  antibody
• can we use these to help transplant sensitized
  recipients safely rather than exclude them
• Good at predicting low immunological risk and
  very high risk but poor at predicting when it is
  safe to proceed in the presence of DSA
• Key Factors
• single vs multiple antibodies
• Strength of antibody
• Predicting memory response
• Detecting and treating antibody mediated rejection

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Reinsmoen N et al Transplantation 2008 Vol 86 no 6
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Burns JM et al Am J Transplant 200882684-94
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B cell channel shift
Total DSA
Burns JM et al Am J Transplant 200882684-94
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DR10,, DR12. DR13, DR14, DR15, DR16,
DR17, DR18, DR52
DR11
DR8,DR10, DR11, DR12. DR13, DR14, DR15,
DR16, DR17, DR18,DR52
DR1,DR51
DR52
DR52
DR1
DR52
strong by flow, moderate by luminex
weak, luminex only
Presented to the HLA working group CST 2008
Courtesy of H. Gebel
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Luminex FLOW
DR1,DR51
DR1
DR1
NEGATIVE (for DR1)
DR1
Courtesy of H. Gebel
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Issa N et al Transplantation 2008
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• Class II antibodies associated with transplant
  glomerulopathy
• Currently screening for antibodies at the time of
  dysfunction.
• May be too late to intervene
• Post transplant monitoring would allow for
  earlier detection of antibody

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Biopsy and test for HLA antibodies
Smith RN et al AJT 200881662-1672
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HLA antibody monitoring
Smith RN et al AJT 200881662-1672
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Antibody Monitoring Post Transplant

• To screen for antibodies is very expensive
• follow 1075 patients here
• 143.00 per screen x 1075 153725 per year to
  screen alone
• 64000 for specificity testing (assuming 15
  positive)
• Kidney costs alone
• Need studies to show early intervention prevents
  TG and delays graft loss.

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• Strength of the antibody
• Class I vs class II
• Total DSA
• Consider desensitization for low titre
  especially class I
• Need the resources to monitor closely
• New agents to remove antibody -bortezomib

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Summary

• Pre transplant
• Antibody presence, strength, target (homozygous
  vs heterozygous) class I vs II
• Decide for each recipient acceptable MM,
  unacceptable antigens and which antibodies might
  be amenable to immunomodulation
• Detailed antibody analysis is critical for
  national sharing programs
• Post Transplant
• Tailor immunosuppression based on immunological
  risk and epitope matching
• Monitor for development of HLA antibodies

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5 Year Deceased-Donor Renal Allograft Survival in
Unsensitized PRA Patients with Negative FCXM
pgt 0.05
Graft Survival
n272 n100 n 120
n 58
Months Post Tx
Bray, et al. AJT 62307-2315, 2006
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Thank you to everyone in the HLA lab for all
their hard work and to Sandra Cockfield for
providing the kidney data