1. DNA-Based Tissue Typing
Chapter 15
DNA-Based Tissue Typing

2. 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.

3. The Major Histocompatability Complex (MHC)
The MHC is located on chromosome 6.
The MHC contains the human leukocyte antigen (HLA) and other genes.
1 Mb Mb Mb Mb
TNF
b a b a b a b b b b a a b
HLA DP DQ DR
B C A
Class II Class III
Class I

4. Genes of the Major Histocompatibility Locus
MHC region
Gene 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 proteins
Defense against extracellular pathogens
Cytokine genes
TNFa, TNFb
Cell growth and differentiation

5. 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. b 2
microglobulin a 1 3
HLA-D
Cell membrane
chain

6. The Human Leukocyte Antigens (HLA)
HLA-gene sequences differ from one individual to another.
Also written as:
Each sequence is a different allele.

7. HLA 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-DRB1
Gene region
Gene locus
Subregion
a- or b-chain polypeptide

8. HLA 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*2503
Gene region
Gene locus
Subregion
a-or b-chain polypeptide
Allele family 25
Third allele

9. HLA Alleles are Inherited in Blocks as Haplotypes

10. 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.

11. Serological Typing
Lymphocytes are HLA-typed by crossmatching to panel reactive antibodies (PRA) using the complement-dependent cytotoxicity (CDC) test.
Complement
antibody
Negative reaction to antibody:
cells survive and exclude dye.
Buffy coat
from patient
Positive reaction to antibody
kills cells. Dead cells pick up dye.

12. Serological 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 antibody
kills cells. Dead cells pick up dye.
Negative reaction to antibody:
cells survive and exclude dye.

13. 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 antibodies
Beads conjugated to different lymphocyte antigens
Positive for antibody
(Wash)
Fluorescent reporter antibodies
Negative for antibody

14. 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.

15. 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.

16. DNA-Based Typing Methods: SSOP
Sequence-specific oligonucleotide probe hybridization (SSOP, SSOPH)
TAG C
GAT
ATC G
CTA A T
Specimen 1 (Type A*0203) Specimen 2 Type A*0501
Amplify, denature, and spot onto membranes
Specimen 1
Specimen 2
Probe with allele-specific probes
...TAGCGAT..(A*02)
...TAGAGAT…(A*05)

17. 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

18. 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

19. DNA-Based Typing Methods: Sequence-based Typing
Sequence-based typing (SBT) is high resolution.
Polymorphic regions are amplified by PCR and then sequenced.
HLA-B
Forward PCR primer
Sequencing primers
Reverse PCR primer

20. Sequence-based Typing
Sequences are compared to reference sequences for previously assigned alleles.

21. 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.

22. Resolution Levels of HLA Typing Methods
Low resolution methods
Intermediate resolution methods
High resolution methods
CDC (Serology)
PCR-SSP
PCR-SSOP
PCR-RFLP
SSP-PCR + PCR-RFLP
SSOP-PCR + SSP-PCR
SBT

23. Combining Typing Results
SSP-PCR followed by PCR RFLP.
SSOP followed by SSP-PCR.
SBT results clarified by serology.

24. 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.