Platelet Allo-Antigens Flashcards
GPIIb/ IIIa
- heterodimeric integrin
- 50,000 - 80,000 copies/ PLT
- involved in aggregation
- have alloantigens on both subunits
— GPIIb = HPA-3
— GPIIIa = HPA-1, HPA-4
HPA - Human Platelet Allo-antigen
GPIa/ IIa
- heterodimeric integrin (a and b)
- 800 - 2800 copies/ PLT
- involved in adhesion & activation; binds collagen
- GP1a subunit = has HPA-5
HPA - Human Platelet Alloantigen
GP1b/V/IX
- heterodimeric integrin
- 12,000 - 25,000 copies/ PLT
- binds vWF for adhesion
- both subunits have HPA alloantigens
– GPIa = HPA-2
– GPIbb = HPA-12
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 to non-self platelet-specific antigens (following transfusion and pregnancy)
- Antibodies to non-self class I HLA may be developed (following pregnancy, transfusion—especially platelet—and transplant)
FNAIT
Fetal/ Neonatal Allo-Immune Thrombocytopenia:
- Mom develops IgG antibodies to 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
- Ab 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
NOTE: HPA 1a is the most abundant platelet alloantigen
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 (secondary Ab) = detects antibodies bound to platelet antigens on the beads
- MFI detected by the Luminex = positive or negative
- Combination of reactive beads = Ab ID
MFI - mean fluorescence intensity
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 = presence or absence of an HPA antigen
qPCR = real-time PCR
Monitoring & Treatment of FNAIT
Monitoring:
- Percutaneous umbilical blood sampling (PUBS) can monitor PLT count in utero
Treatment:
- Transfusion with maternal washed PLTs but HPA-typed donors can be used (if antibody specificity is known)
- Treat mom with IVIg during pregnancy (IVIg therapy in baby has had mixed success)
PTP & Onset
Post-Transfusion Purpura:
- Delayed production of Ab to PLT allo-antigens = acute immune thrombocytopenia 5 to 12 days after transfusion
= PLT <15 x10^9/ L and excessive bleeding
- Rare
Screening & ID Test for PTP
Screening:
-Tests patient serum
- Luminex assay tests patient serum
ID Test:
- HPA genotyping
- HLA antibody-specificity and typing
NOTE: HPA-1a is most common
Treatment for PTP
- IVIg
- PLT-transfusion (HPA-compatible when possible)
- Corticosteroids
- Plasmapheresis
Platelet Transfusion Refractoriness
-
PLTs not increasing as expected after transfusion
ie. Multiple-transfused patients can develop anti-PLT antibodies (50-90%)
NOTE: Most commonly anti-HLA Class I but maybe anti-HPA
Antibody Testing Algorithm for PLT Transfusion Refractoriness
- Class I HLA Antibody screen
- If not HLA antibodies then HPA Antibody screen
If required:
- HLA typing
- HPA genotyping
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
PRA - panel-reactive antigens
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
