Platelet immunology and NAIT

Platelet immunology and NAIT

Platelet Immunology

 

HLA class I

§   HLA antibodies commonest cause of platelet refractoriness to random donor platelets

 

Platelet specific antigens

§   Account for 5% of platelet refractoriness

§   Also cause NAIT and PTP

 

§   HPA 1-15 a more frequent than b

 

§   GpIIb(HPA 3/9) IIIa (HPA 1/4/6/7/8/10/11)

 

§   GPIb(HPA2/12)/IX/V

 

§   GP Ia(HPA5/13)/IIa

 

HPA1a

§   Present in 97% of Caucasian population

 

HPA5b

§   Present in 20% of Caucasians

 

Platetlet Immunofluorescence test (PIFT)

§   High sensitivity for platelet antibodies / low specificity

§   Uses antihuman globulin to detect antibodies bound to platelets

 

MAIPA

§   ELISA based test

§   Gives specificity to the antibodies

§   Monoclonal mouse antibody to the antigen put into solution then platelets lysed and solution put onto plates with anti mouse antibodies so that only antigen that binds to the mouse monoclonal sticks.  Anti-human monoclonal then used to show whether there are patient antibodies directed against the antigen in question..

 

TAQMAN Allelic Discrimination

§   Allows rapid high through put automated platelet genotyping by PCR-SSP

 

 

 


Platelet refractoriness

§   Defined as increment <10 after at least two transfusions of random donor platelets

§   Counts ideally taken 1h post transfusion (but can be 24h)

 

Increments calculated CCI = (P1-P0 109/L) x Body surface ares / Platelets transfused 1011

 

Causes of refractoriness

§        Non-immune

§        Old platelets / small dose for patient size

§        Splenomegaly / hepatomegaly

§        DIC / sepsis

§        Infection (eg CMV)

§        Antibiotics (amphotericin B, vanc, cipro)

§        Immune

§        HLA antibodies – principally class A or B (C largely ignored)

§        HPA antibodies - uncommon

§        ABO antibodies – due to incompatible platelets being transfused into patients with high titre ABO antibodies

 

Risk factors

§   Non-leukocyte depleted (Canadian study shows that platelet refractoriness reduced by leukodepletion)

§   Previous pregnancy

§   Transfusion (>13 platelet transfusions)

 

Ways of HLA matching

1. Patient HLA typed and matched from a HLA typed donor panel (ideal)

§   Low res typing of both donor and recipient

§   Allows perfect ‘A’ match especially with homozygous HLA type donors (NB must be irradiated)

§   Sometimes have to use ‘B match’ B1= single mismatch / B2 = double mismatch donations also given but need to know patients antibody specificity first so that these antigens can be avoided

§   Can also use ‘epitope sharing’ donations eg HLA B54, 55, 56

2. Patient’s HLA antibody specificity defined and Ag compatible platelets selected = selected platelets

§   HLA antibody screening performed by high through put technology (Luminex) which has different beads each with a different antigen attached – read by laser)

3. Random apheresis platelets crossmatched to select compatible units

 

Management algorithm

1. ABO identical pools

2. Change to apheresis platelets

3. Screen for HLA antibodies

§   Even if negative try HLA matched platelets / but consider non-immune causes carefully

4. Give HLA matched platelets

§   If poor response try ‘A’ matched

§   If still poor response reconsider non-immune causes then look for HPA antibodies

5. HPA antibodies

6. Return to ABO identical platelets

 

Important to measure increments after transfusions

 

HLA typing in transplants

§        Serological techniques

§        Assesses expression

§        Still useful to detect null alleles

§        DNA based techniques

§        PCR SSP – sequence specific primers

§        Rapid 2.5 hours

§        Low-medium resolution

§        Used for deceased donors, HLA matched platelets

§        PCR SSOP – sequence specific oligonucleotide probes

§        Highly automated

§        Used for registry

§        High resolution sequence based typing

§        Sequences DNA – allelic specifity

§        For VUD/ confirmatory

§        Functional assays

§        CTLp – cytotoxic T lymphocyte precursor test

§        Difficult and expensive

§        Can help distinguish between a harmful and acceptable mismatch

 

§        Standards require high resolution typing for all but genotypically identical siblings

§        Ideal is match for A, B, C, DRB, DQB

§        Antigenic mismatch worse than alleleic mismatch

§        Donor selection

§        HLA mismatch

§        Class I preferred to class II

§        DR matching is the most important

§        Male or nullip female

§        CMV status

§        Younger donor age, larger donors

§        Blood group

 

§        HLA class I

§        A, B, C

§        HLA class II

§        DRB, DRQ, DRP


Neonatal thrombocytopenia

 

Causes of neonatal thrombocytopenia

§        < 72hours

§        Placental insufficiency

§        Pre-eclampisa

§        IUGR

§        Diabetes

§        NAIT

§        Birth asphyxia

§        Perinatal/ congenital infection

§        Maternal autoimmune

§        Severe rhesus HDN

§        Aneuploidy (21, 18,13)

§        Inherited (wiskott-aldrich, TAR)

§        > 72hours

§        Bacterial and fungal sepsis

§        NEC

§        Congenital infection

§        Maternal autoimmune

§        Inherited

 

Investigation

§        FBC and film

§        Congenital infection screen

§        NAITP screening esp if platelets < 50 and no obvious sepsis

 

 

Fetomaternal Alloimmune Thrombocytopenia (NAIT)

§   1 in 1000 pregnancies

§   50% of cases are in first pregnancy

§   Mortality 14%

 

Pathology

§        Maternal alloantibodies  (IgG) to antigens on fetal platelets

§        Platelet destruction occurs in utero

§        HPA-1a, HPA-5b and HPA-3a

§        HPA-1a (80%) = severe thrombocytopenia

§        HPA-5b (15% ) = mild thrombocytopenia

§        Fetal antigen is inherited from the father

§        HLA DRB3 0101 positive women are 140x more likely to make anti-HPA-1a

 

Clincal features

§   Bleeding may occur in utero

§   Intracranial haemorrhage

§   Thrombocytopenia in the newborn (Platelets may be <30)

§   Normal maternal platelet count

§   Superficial bleeding / petechiae

§   Internal bleeding / intracranial haemorrhage

§   Likely to recur in following pregnancies

 

Diagnosis

Maternal anti-platelet antibodies

§        PIFT to detect the presence of anti-platelet Abs against a panel of different platelets

§        Detection of maternal platelet specific antibodies (MAIPA)

§        Maternal serum tested against a panel of HPA typed platelets

§        Maternal serum also tested against paternal platelets             

§        Detects low frequency or private antigens

Genotyping

§   Done on both parents and the baby

§   TAQMAN PCR SSP - HPA-1, HPA-2, HPA-3, HPA-5 and HPA-15

 

Treatment

§        Plts < 20 – HPA-1a, 5b negative platelets

§        Risk of bleeding highest within first 48hours after delivery

§        All should have a cranial USS

§        Platelet count should recover within  a week

§        If persistent and severe, consider IVIG

§        Suspected cases

§        Monitor platelets for at least 72hours as may continue to fall

 

Antenatal management

§        IUT and fetal blood sampling at weekly intervals or just before delivery

§        Reduce ICH but associated with a high mortality due to haemorrhage, dysrhythmias, premature labour

§        IVIG and or steroids to the mother

§        IVIG alone probably first line

§        Consider starting at 16 weeks if history of antenatal ICH

§        Start at 20-22 weeks if sibling history of severe thrombocytopenia but no antenatal ICH

§        Combination of the above

§        Paternal typing – homozygous v heterozygous (100%v. 50% risk)

 

Counselling

§        Thrombocytopenia in subsequent pregnancies generally as or more severe

§        Best predictors are what happened in the previous pregnancy

§        Antibody specificity and titre are of no value

 

 

Post transfusion purpura

§   Anti HPA1a responsible for 85%; HPA 1b 5%, HPA 3a in 7%

 


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