Chapter 15Chapter 15

DNA-Based Tissue Typing

ObjectivesObjectives

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 The Major Histocompatability Complex (MHC)Complex (MHC)

The MHC is located on chromosome 6.

The MHC contains the human leukocyte antigen (HLA) and other genes.

1 Mb 2 Mb 3 Mb 4 Mb

HLA- DP DQ DR B C A

Class II Class III Class I

TNF

Genes of the Major Genes of the Major Histocompatibility LocusHistocompatibility Locus

MHC regionGene products Tissue location Function

Class I HLA-A, HLA-B, HLA-C All nucleated cells

Identification and destruction of abnormal or infected cells by cytotoxic T cells

Class II HLA-D

B lymphocytes, monocytes, macrophages, dendritic cells, activated T cells, activated endothelial cells, skin (Langerhans cells)

Identification of foreign antigen by helper T cells

Class III Complement C2, C4, B Plasma proteinsDefense against extracellular pathogens

Cytokine genes

TNF, TNF Plasma proteinsCell growth and differentiation

The Human Leukocyte Antigens The Human Leukocyte Antigens (HLA)(HLA)

Human leukocyte antigens, the MHC gene products, are membrane proteins that are responsible for rejection of transplanted organs and tissues.

2 microglobulin

1 1

2 2

2 1

3

HLA-D

Cell membrane

chain chain chain

The Human Leukocyte Antigens The Human Leukocyte Antigens (HLA)(HLA) HLA-gene sequences differ from one individual to

another.

Also written as:

Each sequence is a different allele.

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

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 -** *** *** ***

a.

b.

HLA Allele NomenclatureHLA Allele Nomenclature

A standard nomenclature has been established by the World Health Organization (WHO) Nomenclature Committee.

A small “w” is included in HLA-C, HLAB-4, and HLAB-6 allele nomenclature: HLA-Cw, HLABw-4, HLABw-6.

HLA-DRB1Gene region

Gene locus

Subregion

- or -chain polypeptide

HLA Allele NomenclatureHLA Allele Nomenclature

HLA-typing at the DNA level requires nomenclature for specific DNA sequences.

There are over 900 HLA alleles identified so far in all loci.

HLA-DRB1*2503Gene region

Gene locus

Subregion

-or -chain polypeptide

Allele family 25

Third allele

HLA Alleles are Inherited in HLA Alleles are Inherited in Blocks as Blocks as HaplotypesHaplotypes

A24

Cw1B14

DR14

A30

Cw3B7

DR15

A1

Cw1B12

DR5

A6

Cw7B44

DR14

A24

Cw1B14

DR14

A30

Cw3B7

DR15

A1

Cw1B12

DR5

A6

Cw7B44

DR14

A24

Cw1B14

DR14

A30

Cw3B7

DR15

A1

Cw1B12

DR5

A6

Cw7B44

DR14

haplotype

alleles

X

HLA-TypingHLA-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 TypingSerological Typing

Lymphocytes are HLA-typed by crossmatching to panel reactive antibodies (PRA) using the complement-dependent cytotoxicity (CDC) test.

Complementantibody

Negative reaction to antibody:cells survive and exclude dye.

Buffy coatfrom patient

Positive reaction to antibodykills cells. Dead cells pick up dye.

Serological TypingSerological Typing

Recipient antihuman antibodies are assessed by crossmatching to donor lymphocytes.

Recipient serum

Lymphocytes from organ donor or lymphocytes of known HLA types

Positive reaction to antibodykills cells. Dead cells pick up dye.

Negative reaction to antibody:cells survive and exclude dye.

Serological Typing Using Bead Serological Typing Using Bead ArraysArrays

Recipient antihuman antibodies are assessed by crossmatching to known lymphocyte antigens conjugated to microparticles. Results are assessed by flow cytometry.

Positive for antibody

Serum antibodies

(Wash)

Negative for antibody

Beads conjugated to different lymphocyte antigens

Fluorescent reporter antibodies

Other Serological Typing Other Serological Typing MethodsMethods

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 DNA-Based Typing MethodsMethods

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: DNA-Based Typing Methods: SSOPSSOP

Sequence-specific oligonucleotide probe hybridization (SSOP, SSOPH)

TAG CGATATC GCTA

TAG AGATATC TCTA

Specimen 1 (Type A*0203) Specimen 2 Type A*0501

Amplify, denature, and spot onto membranes

Specimen 1

Specimen 2Probe with allele-specific

probes...TAGCGAT..(A*02) ...TAGAGAT…(A*05)

Specimen 1 Specimen 2 Specimen 1 Specimen 2

DNA-Based Typing DNA-Based Typing Methods: Methods: SSP-PCRSSP-PCR

Sequence-specific PCR is performed with allele-specific primers.

SSP

Amplification

Noamplification

SSP

Amplificationcontrols

Allele-specificproduct

SSP= Sequence-specific primer

SSP matches allele

SSP does not match allele

DNA-Based Typing Methods: DNA-Based Typing Methods: SSP-PCRSSP-PCR

Primers recognizing different alleles are supplied in a 96-well plate format.

Amplification control

Allele-specific product

Reagent blank

Agarose gel

DNA-Based Typing Methods: DNA-Based Typing Methods: Sequence-based TypingSequence-based Typing

Sequence-based typing (SBT) is high resolution.

Polymorphic regions are amplified by PCR and then sequenced.

Exon 2 Exon 3

HLA-B

Forward PCR primer

Sequencing primers

Reverse PCR primer

Sequence-based TypingSequence-based TypingIsolate DNA

PCR

clean amplicons

sequenceamplicon

Sequences are compared to reference sequences for previously assigned alleles.

Typing DiscrepanciesTyping 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 Resolution Levels of HLA Typing MethodsMethods

Low resolution methods

Intermediate resolution methods

High resolution methods

CDC (Serology) PCR-SSP PCR-SSP

PCR-SSP PCR-SSOP PCR-SSOP

PCR-SSOP PCR-RFLPSSP-PCR + PCR-

RFLP

SSOP-PCR + SSP-PCR

SBT

Combining Typing ResultsCombining Typing Results

SSP-PCR followed by PCR RFLP. SSOP followed by SSP-PCR. SBT results clarified by serology.

SummarySummary

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.