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

3. 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).

4. 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

5. 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

6. 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).

7. 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

8. 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

9. 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

10. 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.

11. 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

12. 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

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

14. 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

15. https://obgynkey.com/neonatal-thrombocytopenia-2/

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