Platelet Allo-Antigens Flashcards
GPIIb/ IIIa
- encoded by GP2B and GP3A
- heterodimeric integrin
- 50k - 80k copies per PLT
- involved in aggregation
- have alloantigens on both subunits
— GPIIb = HPA-3
— GPIIIa = HPA-1, HPA-4
WIP GPIa/ IIa
WIP GP1b/V/IX
HPA nomenclature
- more frequent allele is named with an “a” ie. HPA 1a (98%) vs HPA 1b (28%)
- distribution varies by GP complex
Platelet allo-antibodies
- Antibodies (humoural response) to non-self platelet
specific antigens may be developed following exposure to non-self platelet HPA (transfusion and pregnancy) - Antibodies to non-self class I HLA may be developed
following exposure to non-self HLA (pregnancy, transfusion—especially platelet, and transplant)
FNAIT
Fetal/ Neonatal Allo-Immune Thrombocytopenia:
- Mom develops IgG antibodies to the baby’s platelet
allo-antigens
= Passive thrombocytopenia
- in 1/ 1700-5000 live births
NOTE: Major platelet antigens are expressed as early as 18-
19 weeks gestation thus thrombocytopenia can occur in utero
Clinical Aspects of FNAIT
~ 60% detected in first pregnancy
- more frequent in subsequent pregnancies
= Thrombocytopenia for 1-3 weeks post-partum
= 10-30% experience intracranial hemorrhage
Lab Detection of HPA Ab in FNAIT
- Antibody screen in maternal serum for HPA antibodies
- done during gestation if previous pregnancy was affected
— not a quantitative assay but different sample dates can
be tested in parallel for estimate of titre changes - HPA alloantigen typing of mom and dad
- Anti HPA -1a is reported to be the most common antibody detected (HPA 1a is most abundant antigen)
Methods used to detect HPA Antibodies
- PIFT-FFC (Platelet immunofluorescence- read by flow cytometry
- SPAA (Solid phase red cell adherence assay)
- ACE (Antigen capture ELISA)
- MAIPA (Monoclonal antibody-specific immobilization of
platelet antigens) - Luminex
- PakLX assay
Describe Luminex Screening/ ID Test
- Uses phycoerythrin (PE) labelled anti-human IgG to
detect antibodies bound to platelet antigens on the
beads - Amount of fluorescence detected by the Luminex
(MFI) determines whether a bead is positive or negative - Which antibodies are present is determined by the combination of beads that are reacting in the panel
Follow-up testing if antibodies were detected to HPA allo-antigens
HPA genotyping (qPCR)
Follow-up testing if HLA class I antibodies were detected
HLA antibody specificity and typing
Describe HPA-Typing by qPCR
- Specific DNA sequences are amplified while simultaneously quantified by spectrofluorometric
- Sybr Green is a dsDNA binding fluorescent molecule
- Amplified product is then heated to identify melt curve characteristics that determine the presence or absence of an HPA antigen
qPCR = real-time PCR
Treatment of FNAIT
- Transfusion with maternal washed platelets—preferably apheresis (or compatible platelets if antibody specificity is known)
- Unrelated HPA typed donors are often used (Random platelet units may result in successful increments)
- Percutaneous umbilical blood sampling (PUBS) can be performed to monitor platelet count in utero (and transfuse)
- Treatment of mum with IVIg during pregnancy (IVIg therapy in baby has had mixed success)
PTP
Post-Transfusion Purpura:
- Rare event characterized by an acute episode of severe immune thrombocytopenia approximately one week post transfusion
- Amnestic response to previous exposure to
platelet allo-antigens
Post-Transfusion Purpura % and Onset
% = rare; 1 /2million RBCs
- higher incidence in females >50 years
- onset = 5 - 12 days
= PLT <15 x10^9/ L and excessive bleeding
Luminex Screening/ ID Test for PTP
- Same antibody assay for allo antibodies to HPA, GP, HLA Class I
- Using serum from patient
- Anti HPA -1a is reported to be the most common antibody detected however case reports of PTP
due to other HPA exist
If required:
- HPA genotyping
- HLA antibody specificity and typing
Treatment for PTP
- IVIg
- Corticosteroids
- Platelet transfusion (of HPA compatible platelets when possible)
- Plasmapheresis
- In future, use of HPA matched units and IVIg pre-transfusion
Platelet Transfusion Refractoriness
- PLTs not increasing as expected after transfusion
- Multiply transfused patients can develop anti-platelet antibodies with a frequency of 50-90%
- Most common antibody causing refractoriness is anti HLA Class I although there are case reports of platelet transfusion refractoriness due to anti-HPA
Antibody Testing Algorithm for PLT Transfusion Refractoriness
- Class I HLA antibody screen
- If no HLA antibodies detected, HPA antibody may be
done - HLA typing if required
- HPA genotyping if required
Treatment of PLT Transfusion Refractoriness
- Use of HLA matched units
- IVIg?? (Some trials do not show a sustained response following treatment with IVIg)
- Continuous monitoring of PRA following future transfusions
Pro vs Cons of Methods for HLA Antibody Testing in PLT Transfusion Refractory patients
Serology methods: CDC/ CDC-AHG
Pro: Perhaps a more relevant sensitivity?
Con: May not get clear specificity assignment
Solid Phase Assays
Pro: Luminex SAB methods will provide more clarity for antibody specificity
Con: Need to determine relevant threshold for positive reactivity
