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

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

Frequency of red cell, leukocytic and platelet alloantibodies in patients with hematological diseases

History of multiple transfusions in patients with hematological diseases increases the likelihood of immunization to donor blood cells antigensand immunological complications development. Incidence of alloantibodies development in this patients was assessed in this work. Alloantibodies detection was performed in patients with aplastic anemia, acute leukemia, chronic lymphocytic leukemia, and autoimmune thrombocytopenia. 9696 patients were included in this study. Frequency of alloantibodies to red cell antigens was 3.8 %, with 0.9 % of the antibody belong to the immunoglobulin G, and 2.9 % of the cases – to immunoglobulin M. Most of the IgG antibodies had following specificity:monospecific anti-D antibody (21 cases), anti-DC and anti-DE antibodies (4 cases), anti-C (8 cases), anti-E (15), anti-c (13), and anti-K (11). Anti-e (1), anti-Fya (2), anti-Lea (4), anti-S (2), anti-s (2), anti-Jka (2) antibodies were less common. Granulocytes antibodies were found in 66.7 % of 384 patients, with results dependent on the detection method used. The presence of antiplatelet alloantibodies studied in 285 serum samples, of which antibodies were detected in 99 patients (34.7 %). Specificity of platelet antibodies was determined in three serum samples only: anti-2b, anti-1a, anti-1b. In other patients, probably present antibodies to several antigens simultaneously, and to identify them was not possible.</p

Frequency of red cell, leukocytic and platelet alloantibodies in patients with hematological diseases

History of multiple transfusions in patients with hematological diseases increases the likelihood of immunization to donor blood cells antigensand immunological complications development. Incidence of alloantibodies development in this patients was assessed in this work. Alloantibodies detection was performed in patients with aplastic anemia, acute leukemia, chronic lymphocytic leukemia, and autoimmune thrombocytopenia. 9696 patients were included in this study. Frequency of alloantibodies to red cell antigens was 3.8 %, with 0.9 % of the antibody belong to the immunoglobulin G, and 2.9 % of the cases – to immunoglobulin M. Most of the IgG antibodies had following specificity:monospecific anti-D antibody (21 cases), anti-DC and anti-DE antibodies (4 cases), anti-C (8 cases), anti-E (15), anti-c (13), and anti-K (11). Anti-e (1), anti-Fya (2), anti-Lea (4), anti-S (2), anti-s (2), anti-Jka (2) antibodies were less common. Granulocytes antibodies were found in 66.7 % of 384 patients, with results dependent on the detection method used. The presence of antiplatelet alloantibodies studied in 285 serum samples, of which antibodies were detected in 99 patients (34.7 %). Specificity of platelet antibodies was determined in three serum samples only: anti-2b, anti-1a, anti-1b. In other patients, probably present antibodies to several antigens simultaneously, and to identify them was not possible