Lecture 13

Question 1

Major Histocompatibility Complex (MHC)

Answer 1

The MHC locus on chromosome 6 codes for human leukocyte antigen (HLA) -membrane
proteins that are responsible for recognition of non-self cells and tissues
-test for organ transplant

Question 2

Human Leukocyte Antigens (HLA’s)

Answer 2

-sequences differ from one individual to another at single base pairs
-transmembrane class 1 - cytotoxic class 2 - helper t cells
*Class 2 contains alpha and beta chain
*class 1 - heavy and light chain with beta2 macroglobulin
*highly polymorphic
* Each sequence is a different allele which are inherited in blocks as haplotypes - combination of polymorphic in HLA region . Youll only get 4 variations

• HLA typing at the DNA level requires nomenclature for specific DNA sequences

HLA:DRB1

HLA- Gene region
D-Gene locus (A,B,C,D)
R - Subregion (P,Q,R )
B- alpha- Beta chain polypeptide

Question 3

HLA Typing

Answer 3

• 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
• HLA Compatibility (matching) 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
  *low resolution - allele family
  *high resolution - allele itself

Question 4

Serological Testing – CDC Test

Answer 4

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

-plates loaded with preknown AB with diff AB in each AB so if the specimen has the AG that binds with that AB then there will be complement dependent cell death which will pick up a dye. look at the number of cells that have taken up the dye - percentages of color

Question 5

Serological Testing – Bead Arrays

Answer 5

• Recipient antihuman antibodies are assessed by crossmatching to known lymphocyte antigens conjugated to microparticles
  *beads with known AG so if patient has the AB it will bind and unbound AB will be washed. A fluorescent labeled 2ndary AB will be added to be detected
• Results are assessed by flow cytometry - more consistent

Question 6

Cytotoxic and noncytotoxic methods

flow cytometry

ELISA

Mixed lymphocyte culture

isoelectric focusing or two-dimensional gel electrophoresis

Answer 6

flow cytometry - Cytotoxic and noncytotoxic with diff AB

Enzyme-linked immunosorbent assay (ELISA) with solubilized HLA antigens for HLA AB

Mixed lymphocyte culture - lymphocytes activated by cross-reactivity - for growth of T lymphs. If they are compatible they will not grow if they are not they will cause an immune reaction and proliferate

isoelectric focusing or two-dimensional gel electrophoresis-HLA-protein mobility differences in gel
-looking at protein products

Question 7

DNA-Based Typing Methods

Answer 7

-Whole-blood patient specimens collected in
anticoagulant used - EDTA
-typing on polymorphic HLA-B, and HLA-DRB

Question 8

DNA-Based Typing Methods: SSOP

Answer 8

-Sequence-specific oligonucleotide probe
hybridization (SSOP, SSOPH)

amplify, denature and bind to membrane and express probes with allele specific probes

pcr at region of interest, amplicon are immobilized on a membrane and labelled complementary probes are added if signal occurs then allele is present

Question 9

DNA-Based Typing Methods: SSOP Bead-Array

Answer 9

Immobilized probes can be used to identify patient alleles by hybridization
-probes on fluorescent beads is the basis for bead-array SSOP

probes are on the membrane instead of the patient sample
patient sample bound to primer and biotin
add amplicon if they bind then probe matches the amplicon and 2andry signal due to biotin

Question 10

DNA-Based Typing Methods: SSP-PCR

Answer 10

Sequence-specific PCR (SSP-PCR) is performed with allele-specific primers in a 96 well plate format

-design a primer that only binds to the allele that youre looking for so you get the product you want - one allele at a time
need primers for each allele- downside
-line in each well and need another line for allele specific product

Question 11

DNA-Based Typing Methods: Sequence-Based
Typing

Answer 11

• Sequence-based typing is high resolution
• Polymorphic regions are amplified by PCR and then sequenced

amplification of class 1 -exons 2, 3, 4 and class 2 is exon 2, 3
purified with gel or capillary ele - making an electropherogram

NGS- more class two alleles with more targets, long range pcr, dye terminator sequencing - ddntp or ion conductance. Pcr product amplified and fragmented with additional primers

Question 12

DNA Typing Discrepancies

Answer 12

• DNA sequence changes do not always affect epitopes. Geno wont match pheno
• Serology does not recognize every allele detectable by DNA
• New antigens recognized by serology may be assigned to a previously identified parent allele by sequence-based testing
• Serology antibodies may be cross-reactive for multiple alleles

bone marrow - high res
heart transplant - low re

Question 13

Class 1

Answer 13

Gene products
HLA-A, HL A-B, HLA-C

Tissue location
All nucleated cells

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

Question 14

Class 2

Answer 14

Gene products
HLA-D

Tissue location
APC cells Langerhans cells

Function
Identification of foreign antigen by helper T cells

Question 15

Class 3

Answer 15

Gene products
Complement C2, C4, B

Tissue location
B Plasma proteins

Function
Defense against extracellular pathogens

Question 16

Cytokine genes

Answer 16

Gene products
TNF-α, TNF-β

Tissue location
B Plasma proteins

Function
Cell growth and differentiation

Question 17

Serological Testing – Crossmatching by CDC

Answer 17

-Recipient antihuman antibodies are assessed by crossmatching to donor lymphocytes

if the AB of recipient kills donor cells it is not a good match