Reflections and Future Directions HLA Testing in ... –Solid Phase Assay •Virtual crossmatch •

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  • Dr. Robert Liwski, MD, PhD, FRCPC Medical Director, HLA Typing Laboratory Division of Hematopathology Professor, Department of Pathology Dalhousie University, Halifax Medical Director, HLA Laboratory, CBS, Ottawa Robert.liwski@nshealth.ca

    Reflections and Future Directions HLA Testing in Transplantation

  • Disclosure

    • Nothing significant to disclose

  • Disclosure

    • Nothing significant to disclose

  • HLA class I and class II antigens

    • Monomer with non- covalently associated subunit (b2m)

    • Presents antigenic peptides to CD8+ T cells

    • Expressed by all nucleated cells including endothelium

    • Heterodimer

    • Presents antigenic peptides to CD4+ T cells

    • Restricted expression on antigen presenting cells (dendritic cells, B cells, macrophages)

    • Inducible on other cells (endothelium and epithelium)

  • Polymorphism of the Major Histocompatibility Complex in humans - Human Leukocyte Antigen (HLA)

    3492 3111 4358 7 2127 73 940 43 677

    2482 2196 3221 2 1569 33 702 21 555

    68 44 125 2 95 15 22 6 40 Common alleles

    10961

    7899

    237

    3867

    2882

    180

    Effective polymorphism

  • A C B DR DQ DP

    a b1 a1 b1 a1 b1 b3,4,5

    Class I Class II

    A C B DR DQ DP

    maternal

    paternal

  • HLA inheritance

    Mother Father

    Sib 1 Sib 2 Sib 3 Sib 4

    A

    C B

    DR

    DQ

    Approximately 25-30% chance of having an HLA matched sibling

    Patient

  • HLA-A

    HLA-B

    HLA-C

    Polymorphic residues on Class I HLA molecules (polymorphisms are concentrated around peptide binding groove)

    Top view Side view

    Alpha chain conserved aa

    Alpha chain polymorphic aa

    Peptide

    b2 microglobulin

  • HLA antibody development

    Your (“self”) HLA

  • HLA antibody development

    Your (“self”) HLA Donor (“allo”) HLA

  • HLA antibody development

    Your (“self”) HLA Donor (“allo”) HLA

  • HLA antibody development

    Your (“self”) HLA Donor (“allo”) HLA

    Sensitizing events: Pregnancy Transplantation Transfusion

  • • HLA typing and matching of recipient/donor pairs

    • Detection of donor specific HLA antibodies.

    Strategies used to avoid/minimize transplant rejection

  • Evolution of HLA testing

    Past (2010)

    • HLA typing – Serology

    • CDC

    • HLA antibody screening – Lymphocyte panel

    • CDC

    • Lymphocyte crossmatching – CDC

    Present

    • HLA typing – Molecular methods

    • IR SSOP, allele level

    • HLA antibody ID – Solid Phase Assay

    • Virtual crossmatch

    • Lymphocyte crossmatching – Flow cytometry

  • T cell B cell

    3-Color Flow Cytometric Crossmatch

    Bray, Lebeck and Gebel. Transplantation 48:834-840, 1989.

    Donor lymphocytes

    Recipient serum

  • T cell B cell

    3-Color Flow Cytometric Crossmatch

    Bray, Lebeck and Gebel. Transplantation 48:834-840, 1989.

  • T cell B cell

    3-Color Flow Cytometric Crossmatch

    Bray, Lebeck and Gebel. Transplantation 48:834-840, 1989.

  • T cell B cell

    FITC-a-IgG

    3-Color Flow Cytometric Crossmatch

    Bray, Lebeck and Gebel. Transplantation 48:834-840, 1989.

  • T cell B cell

    FITC-a-IgG

    FITC-a-IgG

    Bray, Lebeck and Gebel. Transplantation 48:834-840, 1989.

    3-Color Flow Cytometric Crossmatch

  • T cell B cell

    PerCP-a-CD3 PE-a-CD19

    FITC-a-IgG

    FITC-a-IgG

    Bray, Lebeck and Gebel. Transplantation 48:834-840, 1989.

    3-Color Flow Cytometric Crossmatch

  • T cell B cell PerCP-a-CD3 PE-a-CD19

    Detect fluorescent labels by flow cytometry

    FITC-a-IgG

    FITC-a-IgG

    Bray, Lebeck and Gebel. Transplantation 48:834-840, 1989.

    3-Color Flow Cytometric Crossmatch

  • Negative

    Weak

    positive

    Strong

    positive

    T cell X-match B cell X-match

    Gating strategy

    FITC-a-IgG FITC-a-IgG

    3-Color Flow Cytometric Crossmatch

  • HLA antibody identification by LABScreen® Single Antigen Luminex Assay

    100 different types of beads

    Each bead type coded with different red/infrared dye combination

    Each bead type is coated with different recombinant HLA antigen

    Classification laser (635nm)

    Tells the instrument

    which bead is being

    examined

    Reporter laser (532nm)

    Tells the instrument how

    much PE dye is bound to

    the bead

    2 lasers

    Detection with anti-IgG-PE

  • 1 2 3 4 8 10 9 7 6 5

    HLA antibody identification by LABScreen® Single Antigen Luminex Assay

  • 1 2 3 4 8 10 9 7 6 5

    A1 A2 A3 A11 A23 A24 A25 A26 A29 A30

    HLA antibody identification by LABScreen® Single Antigen Luminex Assay

  • 1

    2

    3

    4

    8 10

    9

    7

    6 5

    A1

    A2

    A3

    A11

    A23 A24

    A25

    A26

    A29

    A30

    2

    A2

    1

    A1

    3

    A3

    4

    A11 5

    A23

    6

    A24

    7

    A25

    8

    A26

    9

    A29

    10

    A30

    HLA antibody identification by LABScreen® Single Antigen Luminex Assay

  • 1

    2

    3

    4

    8 10

    9

    7

    6 5

    A1

    A2

    A3

    A11

    A23 A24

    A25

    A26

    A29

    A30

    2

    A2

    1

    A1

    3

    A3

    4

    A11 5

    A23

    6

    A24

    7

    A25

    8

    A26

    9

    A29

    10

    A30

    HLA antibody identification by LABScreen® Single Antigen Luminex Assay

  • 1

    2

    3

    4

    8 10

    9

    7

    6 5

    A1

    A2

    A3

    A11

    A23 A24

    A25

    A26

    A29

    A30

    2

    A2

    1

    A1

    3

    A3

    4

    A11 5

    A23

    6

    A24

    7

    A25

    8

    A26

    9

    A29

    10

    A30

    HLA antibody identification by LABScreen® Single Antigen Luminex Assay

  • 1

    2

    3

    4

    8 10

    9

    7

    6 5

    A1

    A2

    A3

    A11

    A23 A24

    A25

    A26

    A29

    A30

    2

    A2

    1

    A1

    3

    A3

    4

    A11 5

    A23

    6

    A24

    7

    A25

    8

    A26

    9

    A29

    10

    A30

    HLA antibody identification by LABScreen® Single Antigen Luminex Assay

  • 1

    2

    3

    4

    8 10

    9

    7

    6 5

    A1

    A2

    A3

    A11

    A23 A24

    A25

    A26

    A29

    A30

    2

    A2

    1

    A1

    3

    A3

    4

    A11 5

    A23

    6

    A24

    7

    A25

    8

    A26

    9

    A29

    10

    A30

    PE-a-IgG

    HLA antibody identification by LABScreen® Single Antigen Luminex Assay

  • 1

    2

    3

    4

    8 10

    9

    7

    6 5

    A1

    A2

    A3

    A11

    A23 A24

    A25

    A26

    A29

    A30

    2

    A2

    1

    A1

    3

    A3

    4

    A11 5

    A23

    6

    A24

    7

    A25

    8

    A26

    9

    A29

    10

    A30

    HLA antibody identification by LABScreen® Single Antigen Luminex Assay

  • Patient A3,31 B7,60 DR1,14 (52) DQB5,6

    Previous Donor A1, B8 DR7,17 (53,52) DQB2

    Unacceptable antigens: A1, A36, B8

    HLA Class I antibody analysis

  • Patient A3,31 B7,60 DR1,14 (52) DQB5,6

    Previous Donor A1, B8 DR7,17 (53,52) DQB2

    Unacceptable antigens: DR7, DR53, DQ2

    HLA Class II antibody analysis

  • Canadian PRA calculator

  • Canadian PRA calculator

    Tinckam, Liwski, Pochinco, Mousseau, Grattan, Nickerson and Campbell, AJT 2015

  • Virtual Crossmatching

    Correlation between donor HLA typing and recipient HLA

    antibody testing.

    Presence/Absence of Donor Specific Antibodies (DSA)

  • Class I HLA antibody analysis

    Recipient HLA typing (mother