W7L8 Part 2 - Human Leucocyte Antigen (HLA)

Question 1

HLA Antibodies

Answer 1

For allo-transplantation, antibodies reactive with donor organ may result in hyperacute rejection
For platelet transfusion, recipient HLA antibodies will remove platelets from circulation resulting in haemorrhage

Question 2

HLA Antibodies Tests

Answer 2

Complement dependent cytotoxicity
- sera are tested against a panel of cells of known HLA specificity
- sero-graphs are produced for the identification of reactions, from which the HLA antibody specificity is determined
ELISA
- commercial ELISA trays with specific HLA antigens coated in wells
Luminex fluorescent beads
- colour coded beads with specific purified HLA antigens coated
- reacted with sera, then anti human IgG conjugate (FITC)
- interrogated through a flow cytometry instrument

Question 3

Percent Reactive Antibody (PRA)

Answer 3

The PRA value is a measure of a patient’s level of sensitisation to donor antigens
- percentage of cells from a panel of blood donors against which a potential recipient’s serum reacts
Reflects the percentage of the general population that a potential recipient makes antibodies against
The higher the PRA, the more sensitised a patient is to the general donor pool, and thus the more difficult it is to find a suitable donors
A patient may become sensitised as a result of pregnancy, a blood transfusion, or a previous transplant

Question 4

PRA Testing

Answer 4

Tested by reacting the potential recipients’ serum against a panel of cells (60)
A PRA of 50% indicates that the patient will react against 50% of the donor population
Using ELISA or Luminex the PRA can be calculated using known frequency of HLA antigens in population
Patients on transplant list have blood collected every 2 months for HLA antibody testing
Crossmatches done against patients current serum and peak serum

Question 5

Cross-match for Transplantation - First Thing to Consider

Answer 5

Determine if there are cytotoxic antibodies present in recipient serum that would initiate destruction of donor cells

Question 6

3 Types of Cross-match

Answer 6

Cytotoxicity based crossmatches
Flow cytometry crossmatches
Virtual crossmatches

Question 7

CDC Cross-matches

Answer 7

Autologous cross-match
- cross-match between recipient serum and recipients own lymphocytes
- to determine if patient waiting for transplant has IgM autoantibody reactions to own cells
Allogenic cross-match
- cross-match between recipient serum and donor lymphocytes
- to determine if recipient has cytotoxic antibodies against MHC of donor
- negative allogeneic cross-match required for transplantation

Question 8

Problems with CDC

Answer 8

Reliable antibodies for typing
Treatment to distinguish IgM from IgG
Low sensitivity
Cell viability important
Interference from therapies patient may be on

Question 9

Cross-match by Flow Cytometry

Answer 9

Start with recipient serum
Add donor cells
If recipient has antibodies to HLA type on cells = binding
Add anti-human IgG labelled antibodies
Analyse by flow cytometry

Question 10

Virtual Cross-match

Answer 10

Identify anti-HLA antibodies in recipient
- ELISA or Luminex (bead array)
Determine HLA type of donor cells from DNA
Compare: if recipient has antibodies to donor HLA type = positive

Question 11

Details Required for Transplant

Answer 11

HLA type of both donor and recipients
- recipients need to have x2 HLA typings to go on waiting list
HLA antibody results for recipient
Cross-match results
Other details (health, age, ABO, viruses etc.)

Question 12

Immunosuppression

Answer 12

All allografts receive some immunosuppressive therapy to prevent rejection
Prolong graft survival
Long term therapy
Predisposed to infections, malignancies
Often combination of drugs used

Question 13

Corticosteroids

Answer 13

Anti-inflammatory
Bind intracellular steroid receptors
Effect of down regulation of expression of genes coding for:
- IL-1-5, IL-8, TNFα, adhesion molecules, MHC class II
Toxic
- fluid retention
- bone loss
- thinning of skin
- diabetes
- weight gain

Question 14

Cytotoxic Drugs

Answer 14

Cytotoxic to lymphocytes
Range of toxic effects
- interfere with DNA synthesis of many tissues
- can cause anaemia, leucopenia, thrombocytopenia, gut damage, hair loss

Question 15

Cytokine Inhibitors

Answer 15

Used as supplement to immunosuppression
Bind to immunophilins
- family of intracellular proteins involved in lymphocyte signalling pathways
- this binding causes signals for clonal expansion of lymphocytes to be hindered
Some are nephrotoxic and increase risk of cancer development

Question 16

Bone Marrow Transplantation

Answer 16

Replace genetically abnormal blood forming stem cells with normally functioning stem cells
- restore haematopoietic stem cell function
Can retrieve small numbers of stem cells from blood as well as BM
- known as “stem cell transplantation”

Question 17

Autologous Bone Marrow Transplantation

Answer 17

Stem cells from recipient taken and stored while patient undergoes aggressive chemo or radio therapy, and then replaced

Question 18

Allogeneic Bone Marrow Transplantation

Answer 18

Stem cells taken from BM, blood, umbilical cord blood etc.
Main problem is graft vs host disease
- donor graft cells react against host cells
HLA typing to match, but GVHD due to mHAg
Pre-treatment by removal of T cells from donor cell population

Question 19

Graft vs Host Disease

Answer 19

Patient is immune-compromised due to disease and pre-transplant treatment
Immune-competent T cells transplanted from donor
Host cannot reject the graft due to decreased immunity
Graft T cells perceive recipients tissue as foreign (different HLA type)
Activation of CD4 and CD8 T cells
Inflammation and destruction of host cells leading to death

Question 20

Xenogeneic Transplantation

Answer 20

Across species
Mostly from Pig (farm for organs)
Less T cell response to MHC than for allogeneic Tx

Question 21

Why does the mother not reject the foetus?

Answer 21

The foetus expresses fathers MHC
Mothers produce antibodies to father antigens (anti-Rh antibodies)
Multiparous women produce antibodies to father MHC
Trophoblasts of foetus that contact maternal tissue in placenta do not express polymorphic MHC class I or class II molecules
Trophoblasts express HLA-G (non-polymorphic), binds NK cells of mother preventing killing of foetal cells
Cytokine and complement inhibitory factors

Question 22

HLA Disease Associations

Answer 22

HLA-B27 has strong association with ankylosing spondylitis
HLA-A1, B8, DR3 has strong association with autoimmune disease
- SLE
- autoimmune chronic hepatitis
HLA-A1, B8, DR3 associated with rapid loss of CD4 cells and rapid progression of HIV disease, while HLA-B27 has slower loss of CD4 and slower progression of disease