Platelet serology in Neonatal Thrombocytopenia Dr C Shivaram Consultant & Head-Transfusion Medicine Manipal Hospital Bangalore

Case Report –Neonatal Thrombocytopenia HISTORY A 13 day late preterm male baby presented with thrombocytopenia with no obvious clinical signs of bleeding. H/o treatment of Infection and respiratory distress. Baby referred for management of respiratory distress . Baby was ventilated for 3 days and weaned off uneventfully. Investigations CBC showed PLT count to be 21000/uL. Hemoglobin, White cells, PT/PTT were normal. CBC on Mother showed normal platelet count. Direct Antiglobulin test in baby was positive Response to Platelets Random donor whole blood derived platelet transfusion was given to correct thrombocytopenia but did not yield a satisfactory response. SDP compatible with baby’s serum yielded a good response. Platelet cross match was done by using Capture-P, Ready screen Platelet- crossmatch kit (IMMUCOR).

Differential Diagnosis Neonatal thrombocytopenia Infection/Sepsis/DIC Could be contributory. Infection was present. PT /PTT was normal. Thrombocytopenia persisted even after treatment of infection ITP Maternal auto-immune Thrombocytopenia R/O due to normal Platelet count in mother Post Transfusion Purpura(PTP) Thrombocytopenia was present before transfusion Chromosomal abnormalities- Trisomy 13, 18, 21, Turner’s syndrome. Congenital disorders Bernard Soulier /Wiskott Aldrich syndrome/Thrombocytopenia with absent Radii. No Clinical evidence. NOT TESTED

Evidence for Anti-Platelet Antibody CROSSMATCH RESULTS Mother’s plasma with Baby’s platelets Incompatible Mother’s plasma with Partner’s platelets Baby’s plasma (containing maternal antibodies) with Father’s platelets NEONATAL ALLOIMMUNE THROMBOCTOPENIA

Platelet Antigens in FNAIT Peterson JA, Pechauer SM, Gitter ML, Szabo A, Curtis BR, Aster RH. The human platelet antigen-21bw is relatively common among Asians and is a potential trigger for neonatal alloimmune thrombocytopenia. Transfusion. 2012;52(4):915-6. Genetic polymorphisms resulting from at least 27 single amino acid substitutions located on 6 different glycoproteins can cause Fetal/NAIT. HPA-1a located on GP IIIa is the most commonly implicated antigen in caucasians and africans followed by HPA-5b (Bra) located on GP1a. HPA-4b located on the same glycoprotein as HPA-1 ie GP III a is the most commonly implicated antigen in Asians.  HPA-21bw is another antigen implicated in Asians. Others: anti HPA-3a (Baka) and anti HPA-1b (PLA2).

Expression of human platelet antigens-HPA1a/1b GLYCOPROTEIN III a Expression of human platelet antigens-HPA1a/1b Leucine in position 33 of GP IIIaHPA-1a Proline is in position 33 of GP IIIa HPA-1b

Pathogenesis of NAIT In Caucasians Only 2% of women are HPA-1a negative (i.e. HPA-1b/b) Predicted Incidence 2:100 or 1:50 However incidence of NAIT is only 1:1000 1: 2500. Mere Incompatibility does not lead to NAIT. Genetic pre-disposition-HLA restriction HPA-1a (PLA1) sensitization Associated with  the DR52a HPA-5b (Bra) sensitization is associated with  DRw6. NAIT Genetic-HLA Restriction HPA-1a: DR52a HPA-5b:DRw6 Platelet Allo-immunization Platelet Antigens HPA-1 :98% HPA-2, 3,4, 5, 9, 15 HPA-4 : Asians

Diagnosis of NAIT Diagnosis of NAIT should be considered in otherwise healthy term infant who presents with severe unexplained thrombocytopenia in the first 24 to 48 hours of life. However, NAIT also needs to be considered in ill-appearing infants, especially if severe thrombocytopenia is present, appears to be out of proportion to the clinical illness, or persists when the clinical illness improves.  NAIT is distinguished from other causes of neonatal thrombocytopenia by the identifying maternal antiplatelet antibodies. Anti-HLA class I antibody mediated FNAIT has been reported 30% of multiparous women have HLA class I antibodies https://www.uptodate.com/contents/causes-of-neonatal-thrombocytopenia#H457204138 https://www.uptodate.com/contents/causes-of-neonatal-thrombocytopenia#H457204138

Laboratory Diagnosis of NAIT Checking the Mother’s Platelet Type: If the mother is HPA-1 negative, the test result will return  HPA-1b/1b Checking the Father’s Platelet Type If the father is HPA-1 positive, his result can be HPA-1a/1a( Homozygous) Or  HPA-1a/1b( Heterozygous) Check for Maternal Antibodies Maternal blood is checked for antibodies to her partner’s ( biological father)platelets Amniocentesis- For Typing Fetal Platelet Antigens Cordocentesis-Fetal blood sampling is reserved for only few cases. Platelet antigen typing : Solid phase adherence assays. DNA assays Platelet antibody assays Solid phase assays. Flow cytometric assays. https://www.naitbabies.org/resources/what-is-nait/

Amniocentesis For Typing Fetal Platelet Antigens Cordocentesis Percutaneous umbilical blood sampling (PUBS): To determine PLT response to therapy Procedure is done after 32 weeks of gestation to document that the fetal platelet response to therapy has been adequate enough to safely permit a vaginal delivery Amniotic fluid surrounding the baby inside the womb is tested after about 15 weeks of pregnancy by inserting a needle into the amniotic sac under ulltrasound guidance. In about half of cases, the baby will be found to HPA-1 negative and there will be no further concerns in the pregnancy. Availability of antigen negative platelets for transfusion is a pre-requisite for Cordocentesis if the fetal platelet count is < 50,000/ul

Father –Homozygous for 1a Mother –Homozygous for 1b Genetic counseling and Platelet antigen studies To Determine Father’s Zygosity Father –Homozygous for 1a Mother –Homozygous for 1b Child- Always Heterozygous 1a1b At Risk for NAIT

One Parent –Heterozygous : 1a/1b Another parent(partner) –Homozygous for 1b Child- 50% chance Anti-HPA- 1a will develop NAIT

Fetal/Neonatal Platelet transfusions Management of FNAIT Prevention Genetic counseling & obstetrical high risk support. Goal of antenatal diagnosis and treatment is to prevent intrauterine ICH. Recurrence of NAIT-90% chance with equal or increasing severity. If the fetus is found to be PLA1 positive cordo centesis to determine the fetal platelet count. A fetus is considered affected if the platelet count is <100,000/uL Maternal Therapy Maternal therapy with IVIG 1 gm/kg/week to improves the fetal thrombocytopenia in 75% of cases. If IVIG / high-dose steroids (60 mg prednisone/day) fail to maintain platelets above 50000/ul Platelet Transfusions Antigen negative Maternal Platelets (RDP/SDP) with low volume of plasma HPA-1b/1b platelets(?) Fetal/Neonatal Platelet transfusions If PLT count is <30,000/ul or if there are signs of bleeding. 50,000/ul in preterm infants or in term infants who are ill or have risk factors (eg, fetal neonatal distress). 50,000/ul or 100,000/ul in infants who have evidence of ICH. Risk of ICH is maximum in the first 72 to 96 hours

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THANK YOU shivaram@manipalhospitals.com