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    Mdfund Unit17chapter15

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    Jose Alfonso C
    This document discusses human leukocyte antigen (HLA) typing methods. It begins by describing the structure and role of the major histocompatibility complex (MHC) locus, which contains HLA genes. HLA antigens are identified using serology testing methods like cytotoxicity assays or flow cytometry with antibody-coated beads. More recently, DNA-based methods like sequence-specific oligonucleotide probing, sequence-specific PCR, and sequencing are used for high-resolution HLA typing. These DNA methods allow more precise identification of HLA alleles compared to serology.

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    Mdfund Unit17chapter15

    Uploaded by

    Jose Alfonso C
    48 views24 pages
    This document discusses human leukocyte antigen (HLA) typing methods. It begins by describing the structure and role of the major histocompatibility complex (MHC) locus, which contains HLA genes. HLA antigens are identified using serology testing methods like cytotoxicity assays or flow cytometry with antibody-coated beads. More recently, DNA-based methods like sequence-specific oligonucleotide probing, sequence-specific PCR, and sequencing are used for high-resolution HLA typing. These DNA methods allow more precise identification of HLA alleles compared to serology.

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    This document discusses human leukocyte antigen (HLA) typing methods. It begins by describing the structure and role of the major histocompatibility complex (MHC) locus, which contains HLA genes. HLA antigens are identified using serology testing methods like cytotoxicity assays or flow cytometry with antibody-coated beads. More recently, DNA-based methods like sequence-specific oligonucleotide probing, sequence-specific PCR, and sequencing are used for high-resolution HLA typing. These DNA methods allow more precise identification of HLA alleles compared to serology.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPTX, PDF, TXT or read online from Scribd
    Download as pptx, pdf, or txt
    48 views24 pages

    Mdfund Unit17chapter15

    Uploaded by

    Jose Alfonso C
    This document discusses human leukocyte antigen (HLA) typing methods. It begins by describing the structure and role of the major histocompatibility complex (MHC) locus, which contains HLA genes. HLA antigens are identified using serology testing methods like cytotoxicity assays or flow cytometry with antibody-coated beads. More recently, DNA-based methods like sequence-specific oligonucleotide probing, sequence-specific PCR, and sequencing are used for high-resolution HLA typing. These DNA methods allow more precise identification of HLA alleles compared to serology.

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    of 24

    Chapter 15

    DNA-Based Tissue Typing


    Objectives
     Describe the structure of the MHC locus
    that encodes the HLA antigens.
     Explain the role of these antigens in tissue
    engraftment and rejection.
     Describe the laboratory methods used to
    identify HLA antigens by serology testing.
     Describe the DNA-based testing methods
    that are used for the identification of HLA
    antigens.
    The Major Histocompatability
    Complex (MHC)
     The MHC is located on chromosome 6.

    1 Mb 2 Mb TNF 3 Mb 4 Mb
    baba ba bbbba a b

    HLA- DP DQ DR B C A

    Class II Class III Class I

     The MHC contains the human leukocyte antigen


    (HLA) and other genes.
    Genes of the Major
    Histocompatibility Locus
    MHC region
    Gene products Tissue location Function
    Identification and
    destruction of abnormal
    Class I HLA-A, HLA-B, HLA-C All nucleated cells
    or infected cells by
    cytotoxic T cells
    B lymphocytes,
    monocytes,
    macrophages, dendritic
    Identification of foreign
    Class II HLA-D cells, activated T cells,
    antigen by helper T cells
    activated endothelial
    cells, skin (Langerhans
    cells)
    Defense against
    Class III Complement C2, C4, B Plasma proteins
    extracellular pathogens
    Cytokine Cell growth and
    TNFa, TNFb Plasma proteins
    genes differentiation
    The Human Leukocyte Antigens
    (HLA)
    Human leukocyte antigens, the MHC gene products,
    are membrane proteins that are responsible for
    rejection of transplanted organs and tissues.
    a1 b1 a2 a1

    HLA-D
    a2 b2
    a3 b 2 microglobulin

    Cell membrane

    a chain b chain a chain


    The Human Leukocyte Antigens
    (HLA)
     HLA-gene sequences differ from one individual to
    another.
    a.CGG GCC GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GGT GAG AGC TTC ACA
    CGG GCC GCC GTG GAC ACC TAT TGC AGA CAC AAC TAC GGG GCT GTG GAG AGC TTC ACA

    CGG GCC GCC GTG GAC ACC TAT TGC AGA CAC AAC TAC GGG GCT GTG GNN NNN NNN NNN

     Also written as:


    b.
    CGG GCC GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GGT GAG AGC TTC ACA

    --- --- --- --- --- --- --T --- --- --- --- --- --- -C - -TG --- --- --- ---
    --- --- --C --- --- --- --T --- --- --- --- --- --- -C- -TG -** *** *** ***

     Each sequence is a different allele.


    HLA Allele Nomenclature
     A standard nomenclature has been established by the
    World Health Organization (WHO) Nomenclature
    Committee.
    Gene region Subregion

    HLA-DRB1
    a- or b-chain polypeptide
    Gene locus
     A small “w” is included in HLA-C, HLAB-4, and HLAB-
    6 allele nomenclature: HLA-Cw, HLABw-4, HLABw-6.
    HLA Allele Nomenclature
     HLA-typing at the DNA level requires nomenclature
    for specific DNA sequences.

    Gene region Subregion Allele family 25

    HLA-DRB1*2503
    Third allele
    a-or b-chain polypeptide
    Gene locus
     There are over 900 HLA alleles identified so far in all
    loci.
    HLA Alleles are Inherited in
    Blocks as Haplotypes
    A24 A30 A1 A6
    alleles

    Cw1 Cw3 Cw1 Cw7


    haplotype B14 B7 B12 B44
    X
    DR14 DR15 DR5 DR14

    A24 A6 A1 A30 A6 A30 A24 A1

    Cw1 Cw7 Cw1 Cw3 Cw7 Cw3 Cw1 Cw1


    B14 B44 B12 B7 B44 B7 B14 B12

    DR14 DR14 DR5 DR15 DR14 DR15 DR14 DR5


    HLA-Typing
     Every person (except identical twins) has
    different sets of HLA alleles.
     Transplanted organs are allografts, in which the
    donor organ and the recipient are genetically
    different.
     Compatibility (matching) of the HLA of the donor
    and the recipient increases the chance for a
    successful engraftment.
     Matching is determined by comparing alleles.
     Resolution is the level of detail with which an
    allele is determined.
    Serological Typing
    Lymphocytes are HLA-typed by crossmatching to
    panel reactive antibodies (PRA) using the
    complement-dependent cytotoxicity (CDC) test.
    Complement
    antibody

    Positive reaction to antibody


    kills cells. Dead cells pick up dye.

    Buffy coat Negative reaction to antibody:


    from patient cells survive and exclude dye.
    Serological Typing
    Recipient antihuman antibodies are assessed by
    crossmatching to donor lymphocytes.
    Lymphocytes from organ
    donor or lymphocytes of
    known HLA types

    Positive reaction to antibody


    Recipient serum
    kills cells. Dead cells pick up dye.

    Negative reaction to antibody:


    cells survive and exclude dye.
    Serological Typing Using Bead
    Arrays
    Recipient antihuman antibodies are assessed by
    crossmatching to known lymphocyte antigens
    conjugated to microparticles. Results are assessed
    by flow cytometry.
    Serum
    Beads antibodies Positive for antibody
    conjugated to
    different
    lymphocyte (Wash)
    antigens

    Fluorescent Negative for antibody


    reporter
    antibodies
    Other Serological Typing
    Methods
     Cytotoxic and noncytotoxic methods with flow
    cytometry detection.
     Enzyme-linked immunosorbent assay (ELISA)
    with solubilized HLA antigens.
     Mixed lymphocyte culture measuring growth of
    lymphocytes activated by cross-reactivity.
     Measure of HLA-protein mobility differences in
    one-dimensional gel isoelectric focusing or two-
    dimensional gel electrophoresis.
    DNA-Based Typing
    Methods
     DNA typing focuses on the most
    polymorphic loci in the MHC, HLA-B, and
    HLA-DRB.
     Whole-blood patient specimens collected
    in anticoagulant are used for DNA typing.
     Cell lines of known HLA type are used for
    reference samples.
    DNA-Based Typing Methods:
    SSOP
    Sequence-specific oligonucleotide probe
    hybridization (SSOP, SSOPH)
    Specimen 1 (Type A*0203) Specimen 2 Type A*0501
    TAG CGAT TAG A GAT
    ATC G CTA ATC T CTA

    Amplify, denature, and


    spot onto membranes

    Specimen 1
    Specim
    en 2
    Probe with allele-specific
    ...TAGCGAT..(A*02) ...TAGAGAT…(A*05)
    probes

    Specimen 1 Specimen 2 Specimen 1 Specimen 2


    DNA-Based Typing
    Methods: SSP-PCR
    Sequence-specific PCR is performed with
    allele-specific primers. Amplification
    SSP= Sequence-specific primer controls
    Allele-specific
    product
    SSP matches allele
    Amplification
    SSP

    No
    amplification
    SSP
    SSP does not match allele
    DNA-Based Typing Methods:
    SSP-PCR
    Primers recognizing different alleles are
    supplied in a 96-well plate format.
    Reagent blank

    Amplification control

    Allele-specific product

    Agarose gel
    DNA-Based Typing Methods:
    Sequence-based Typing
     Sequence-based typing (SBT) is high
    resolution.
     Polymorphic regions are amplified by
    PCR and then sequenced.
    Reverse PCR primer
    Forward PCR primer
    Exon 2 Exon 3

    Sequencing primers
    HLA-B
    Sequence-based Typing
    Isolate DNA

    PCR

    clean amplicons

    Sequences are
    sequence
    amplicon compared to
    reference sequences
    for previously
    assigned alleles.
    Typing Discrepancies
     DNA sequence changes do not always affect epitopes.
     Serology does not recognize every allele detectable by
    DNA.
     New antigens recognized by serology may be assigned
    to a previously identified parent allele by SBT.
     Serology antibodies may be cross-reactive for multiple
    alleles.
     Due to new allele discovery, retyping results may differ
    from typing performed before the new allele was known.
    Resolution Levels of HLA Typing
    Methods
    Low resolution Intermediate High resolution
    methods resolution methods methods

    CDC (Serology) PCR-SSP PCR-SSP


    PCR-SSP PCR-SSOP PCR-SSOP
    SSP-PCR + PCR-
    PCR-SSOP PCR-RFLP
    RFLP
    SSOP-PCR + SSP-
    PCR

    SBT
    Combining Typing Results
     SSP-PCR followed by PCR RFLP.
     SSOP followed by SSP-PCR.
     SBT results clarified by serology.
    Summary
     The MHC is a polymorphic locus encoding the HLA
    genes.
     Antigens encoded by the HLA genes are responsible for
    allograft tissue and organ rejection. Identifying and
    matching alleles increases the chance of successful
    organ and tissue transplant.
     HLA antigens and their corresponding sequence alleles
    are determined by serological- and DNA- based
    methods.
     Serology identifies functional antigen recognition, while
    sequence analysis identifies genetic alleles with high
    resolution.

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