Diagnosis of Neonatal Alloimmune Thrombocytopenia: Determination of the Specificity of Antiplatelet Alloantibodies in the Maternal Blood Plasma Using a Molecular and Genetic Method

Abstract

   Background: Thrombocytopenia occurs in 1-5 % of newborns (platelet count < 150 × 109/L). Low platelet count of 50 × 109/L leads to the hemorrhagic syndrome, with one of its causes being neonatal alloimmune thrombocytopenia resulting from incompatibility between the mother and the fetus with human platelet antigens (HPA) inherited from the father and absent in the mother, which leads to the formation of maternal antibodies. Anti-HPA-1a, anti-HPA-5b, anti-HPA-3a, and anti-HPA-3b antibodies are clinically significant as they destroy fetal/neonatal platelets causing severe complications (intracranial hemorrhage in 20 % of cases and prenatal or postnatal death in 10 % of cases). Adequate diagnosis is a key to a successful treatment approach, which largely depends on the thrombocytopenia cause.   Objective: To determine the alloimmune nature of neonatal thrombocytopenia and the specificity of antibodies in the mother’s blood.   Materials and methods: We studied blood samples of parents (21 pairs) of newborns with thrombocytopenia in Saint Petersburg, Russian Federation. We used flow cytometry to determine alloantibodies in the maternal plasma after incubation with paternal platelets and staining with Goat F(ab’)2 Anti-Human IgG-FITC and CD41-PE monoclonal antibodies. Allosensitization index was calculated as the percentage of IgG-positive cells to the number of cells fixing anti-CD41 antibodies. At the value of ≥ 15 %, antiplatelet alloantibodies were considered present in a sample. We used a molecular detection system of the FluoVista analyzer (Inno-Train, Germany) for genetic testing with allele-specific primers. Alleles of genes encoding the expression of HPA-1, HPA-2, HPA-3, HPA-4, HPA-5, HPA-6, HPA-9, and HPA-15 antigens were determined by a real-time polymerase chain reaction using a set of HPA-FluoGene reagents (Inno-Train, Germany). Genomic DNA was isolated using the DNA-sorb-B set (AmpliSens, Russian Federation).   Results: We found that 8 of 21 (38 %) mothers had antibodies against paternal platelets. During genotyping in mother/father pairs, incompatible combinations of platelet antigens were revealed: HPA-1b/HPA-1a in 9 pairs (HPA-1a antigen absent on the maternal platelets and present on the paternal platelets), of which 5 mothers (55%) had antibodies with a probable specificity to anti-HPA-1a; HPA-1a/HPA-1b incompatibility in 4 pairs, with 2 (50 %) mothers having antibodies with an anti-HPA-1b specificity. HPA-3a/HPA-3b incompatibility was observed in 4 pairs, with antibodies (probably anti-HPA3b) in 1 mother (25%). HPA-2a/HPA-2b, HPA-5a/HPA-5b, HPA-15a/HPA-15b, HPA-15b/HPA-15a incompatibilities were detected (1 case each in 21 pairs), with no antibodies found in mothers. The probable specificity of the antibodies was distributed as follows: 62 % for anti-HPA-1a, 25% for anti-HPA-1b, and 13 % for anti-HPA-3a.   Conclusions: We confirmed the immune nature of neonatal thrombocytopenia and determined the probable specificity of maternal alloantibodies in 8 of 21 cases.