Module 11: Transplant Immunology Obj. 3 SA

Question 1

Tissue typing involves testing for(3)

Answer 1

HLA antigens of donor and recipient

HLA Ab present in recipient serum

Question 2

Tissue crossmatch

Answer 2

predicts compatibility of the transplant

Question 3

HLA Ag detection techniques (2)

Answer 3

Serological (Old technique), may then require Mixed lymphocyte culture
Molecular technique

Question 4

Serological technique for HLA Ag detection

Answer 4

Commercial antisera containing known ab
Incubate patient or donor lymphs with known antisera
Add complement
Cells which bind HLA Ab will be damaged due to complement binding
Damaged cells take up dye (then quantitated)

Lymphs must be from LIVE donor

Question 5

Mixed lymphocyte culture

Answer 5

test for Class II MHC Ag

Culture 2 pts cells together
Cell proliferation is detected by uptake of radioactive thymidine (From DNA synthesis)

Low MLC = better success for transplant

Question 6

Molecular techniques fro HLA Ag detection

Answer 6

Sequencing DNA by PCR

Basic steps:

1. Obtain DNA from nuclei of a cell
2. Double stranded DNA is denatured by heat
3. Oligonucleotide primer sequences are then chosen to identify the region of interest
   - if primer chosen is close to region of interest, only that part of the DNA will replicate when DNA polymerase enzyme and deoxyribonucleotide triphosphates are added
   - if a primer is used that is specific to a certain sequence, the product will only be produced if the patient has that particular sequence

Question 7

HLA Ab detection techniques (2)

Answer 7

ELISA

Flow Cytometry

Question 8

What happens if a patient has Ab against certain HLA’s

Answer 8

Ab will attach to corresponding Ag on the transplanted tissue and lead to tissue destruction

Question 9

ELISA

Answer 9

Pure HLA Ag bound to well wall
Binding of Ab from pt is detected by second Anti-IgG Ab labeled with alkaline phosphatase
Addition of enzyme substrate for detection

Question 10

Flow cytometry

Answer 10

2nd Ab is a fluorescent dye-labelled anti human Ab which binds to the pt Ab attached to the reagent T and B lymphs