Histocompatibility

Question 1

T or F. HLa antigens are protein molecules

Answer 1

T

Question 2

This class of HLA is expressed on all nucleated cells and platelets

Answer 2

Class I HLA

Question 3

each different DNA sequence at a given locus codes for an _______

Answer 3

allele

Question 4

define polymorphic

Answer 4

MANY PPL HAVE A DIFFERENT DNA SEQUENCE FOR THAT GENE

Question 5

polymorphism for HLA molecules are focused around this region

Answer 5

peptide binding region

Question 6

which chromosomes are HLA genes located on

Answer 6

6

Question 7

T or F. One of each HLA gene is inherited maternally and pternally

Answer 7

T, A, B, C, and DR, DQ

Question 8

T or F. HLA antigens are co-dominantly expressd

Answer 8

T

Question 9

HLA is associated with several autoimmune diseases such as:

Answer 9

Celiac
Type 1 diabetes
MS
Ankylosing spondylitis

Question 10

HLA is also associated with drug sensitivities

Answer 10

Abacovir
Carbamazepine

Question 11

why does the HLA typing matter?

Answer 11

typing of the donor allows the potential for allocation according to degree of HLA match

HLA typing also helps to interpret antibody specificities and explain and interpret crossmatch results

Question 12

traditional HLA typing

Answer 12

• serological CDC methodology using lymphocytes
• lymphocytes isolated and tested against numerous anti-sera
• approx. 300 sera are required for a full HLA typing!

Question 13

the standard method for HLA testing now

Answer 13

CDC (complement dependent cytotoxicity)
- lymphocytes incubated with serum (1 uL volumes)
- addition of rabbit complement = cell lysis when Abs attached
- cell lysis/death observed

Question 14

T or F. We test all MHC genes for transplantation

Answer 14

F! only classical HLA

Question 15

complete HLA allele for DR15

Answer 15

DRB1*15:03

DR = antigen
B1 = gene
15 = antigen (DR15 serologically)
03 = third allele described

Question 16

HLA inheritance

Answer 16

• chromosome 6
• one of each (A, B, C, DR, DQ) inherited maternally and paternally
• Mendelian inheritance
• HLA co-dom expressed

Question 17

6 loci of interest fo HLA typing

Answer 17

A,B,C = I
DR, DQ, DP = II

Question 18

these HLA loci are traditionally typed in solid organ transplantation

Answer 18

A, B , DR

Question 19

these HLA loci are more routinely typed

Answer 19

HLA C, DQA, DQB, and DP

Question 20

list the molecular techniques for HLA typing

Answer 20

• SSP
• reverse SSOP (including Luminex based)
• qPCR
• sequence based typing- SBT vs NGS
• Oxford Nanopore

Question 21

SSP

Answer 21

• sequence specific primer
• primers specific for every allele of interest included
• series of primers used almost like antisera
• many primers required to cover common HLA alleles

Question 22

this has replaced SSP typing methods

Answer 22

reverse SSO typing
- loci amplified and many probes used to detect the different alleles
- uses a Luminex instrument for read out
- can run in batches or single samples

Question 23

rSSO workflow

Answer 23

• isolated DNA amplified
• DNA amplified with HLA locus specific primers
• PCR product biotinylated
• PCR product incubated with probe labelled beads
• Streptavidin-PE labelled conjugate is added
• PE fluorescent on the beads is measured

Question 24

advantages of DNA-based typing vs serology CDC

Answer 24

• serology requires viable lymphocytes (dies in -50 weather)
• age of specimen and conditions of transport are much less important
• easier to create a primer for an HLA antigen than to screen antisera for HLA specificities
• DNA samples easy storage for repeat testing
• higher res/more accurate typing
• less subjective readings

Question 25

pitfalls of DNA based typing

Answer 25

- lymphocytes still needed for crossmatching - potential DNA contamination demands stringent lab set-up and training - rare "null" alleles will be detected by DNA but not expressed on the cells - new alleles are always being identified so software must be frequently updated