Tissue typing and Platelets

Question 1

Methods of crossmatch for tissue typing

Answer 1

1. CDC
2. Flow crossmatch
3. Virtual crossmatch
4. Endothelial cell crossmatch

Question 2

CDC crossmatch method

Answer 2

Extract lymphocytes (T and B) using Dynabeads
==> T lymphocytes for class I, B lymphocytes for class II
Add patient serum
Add ethidium bromide and acridine orange
Green = no lysis, no antibody-antigen reaction

Question 3

Describe the flow crossmatch method

Answer 3

1. Donor lymphocytes are treated with pronase to get rid of Fc receptors
2. Add patient serum
3. Add anti-CD3, anti-CD20 and anti-IgG FITC labelled antibody
   ===> AHG binds to bound patient/recipient antibody
4. Look for shift in MFI compared to control.

Question 4

How is an antibody screen performed for tissue typing?

Answer 4

Luminex - flow cytometry multiplex method
Purified antigens cover beads
Each bead has a unique dye to allow for identification
Patient serum/plasma is added then anti-IgG-PE is added
Fluorescence if antibody is present

Question 5

What is the significance of antibodies in renal transplants?

Answer 5

A positive T cell crossmatch is a contraindication to transplant if the donor has the corresponding antigen
==> cause hyperacute and chronic rejection of the graft

Question 6

What is high resolution typing?

Answer 6

In HSCT refers to a 10/10 match

==> HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1

Question 7

Definition of platelet refractoriness

Answer 7

Failure to have an adequate response (increment <10 at 1 hour) to random donor platelets on 2 occasions.

Question 8

Causes of platelet refractoriness

Answer 8

Immune
- alloimmune = plt refractoriness from HLA/HPA antibodies
- autoimmune (ITP)
- drug dependent immune destruction
Non-immune
- sepsis
- hypersplenism
- bleeding
- DIC

Question 9

Diagnosis of platelet refractoriness

Answer 9

1. PIFT (sensitive but not specific)
2. PakLx and Luminex testing for HPA and HLA antibodies and ID
3. MAIPA for HPA-15 (not detected by PakLx)
4. Tissue typing

Question 10

Transfusion of platelets in platelet refractoriness

Answer 10

1. ABO matched single donor apheresis platelets in double dose while awaiting testing/results
2. HLA/HPA matched platelets or antigen negative platelets must be irradiated

Question 11

Approach to management of a patient with platelet refractoriness:

Answer 11

1. Confirm diagnosis
   - is platelet refractory (test 30 mins, 2 hrs and 24 hrs post platelet transfusion)
   - exclude non-immune causes
   - platelet and HLA antibody testing
   - tissue typing
2. Assess clinical situation
   - how urgently do they need platelets
   - how long will they need platelets for
3. Give platelets
   - HLA-A and HLA-B matched platelets required (platelets only display class I antigens)
   - Alternatives = antigen negative or antigens in same CREG
4. Assess response
5. Determine ongoing need - need to coordinate donors, storage times etc.

Question 12

Diagnosis of NAIT

Answer 12

1. Severe thrombocytopenia in fetus/neonate
2. Maternal antibodies
   - PIFT, PAKLx and PIFT crossmatch using paternal platelets
3. Demonstrate antigens present on neonate or father that the mother doesn’t have
   HPA-1a phenotyping on mother and father
   HPA genotyping on mother, baby and/or father

Question 13

Transfusion strategy in a confirmed case of NAIT

Answer 13

Need to transfuse the neonate platelets:

1. Antigen negative platelets
2. HPA-1a negative platelets (80% of cases are due to anti-HPA-1a antibodies)
3. Maternal platelets (washed to get rid of antibodies and irradiated)
4. Random donor platelets