Fast and low-cost direct ELISA for high-throughput serological HPA-1a typing.

BACKGROUND: Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is caused by maternal alloantibodies against fetal human platelet antigens (HPAs), mostly caused by anti-HPA-1a. Population-based screening for FNAIT is still a topic of debate. Logistically and financially, the major challenge for implementation is the typing of pregnant women to recognize the 2% HPA-1a-negative women. Therefore, there is need for a high-throughput and low-cost HPA-1a-typing assay. STUDY DESIGN AND METHODS: A sandwich ELISA was developed, using a monoclonal anti-GPIIIa as coating antibody and horseradish-peroxidase-conjugated recombinant anti-HPA-1a, as detecting antibody. The ELISA results were compared to an allelic discrimination PCR-assay. In phase I, samples from unselected consecutive pregnant women were tested with both assays. Phase II was part of a prospective screening study in pregnancy and genotyping was restricted to samples with an arbitrary set, OD < 0.500. RESULTS: The ELISA was optimized to require no additional handling (swirling or spinning) of stored tubes. During phase I, 506 samples were tested. In phase II, another 62,171 consecutive samples were phenotyped, with supportive genotyping in 1,902. In total 1,585 HPA-1a negative and 823 HPA-1a positive women were genotyped. The assay reached 100% sensitivity with a cut-off OD from 0.160, corresponding with a 99.9% specificity and a false-HPA-1a negative rate of 0.03. CONCLUSION: A high-throughput, low-cost, and reliable HPA-1a phenotyping assay was developed which can be used in population-based screening to select samples for testing of presence of anti-HPA-1a. Because plasma from tubes of 3- to 6-days-old samples can be used, this assay is applicable to settings with suboptimal conditions.

Non-invasive Prenatal Diagnosis of Feto-Maternal Platelet Incompatibility by Cold High Resolution Melting Analysis.

Fetal and Neonatal alloimmune thrombocytopenia (FNAIT) is a condition which could occur when pregnant women develop an alloimmunization against paternally inherited antigens of the fetal platelets. Approximately 80 % of FNAIT cases are caused by anti-HPA-1a, about 15 % by anti-HPA-5b and 5 % by other HPA antibodies. Only 2 % of the total population is HPA-1a negative (HPA-1b1b). The HPA-1a allele differs by one single nucleotide from HPA-1b allele, yet it represents around 27 % of total severe thrombocytopenias. HPA-1 was studied in serum cDNA from 12 volunteer pregnant women to determine their HPA-1 genotype by HRM (high resolution melting) PCR. When an homozygous HPA-1 gene was detected in a mother, a COLD HRM was performed to determine whether or not the fetal genotype differs from the mother's.The differences in the melting curve shapes allow us to accurately distinguish the three pregnants genotypes. The fetal heterozygous genotype of homozygous pregnant women was correctly detected by COLD PCR HRM in maternal serum. HPA-1 genotyping by HRM may be a useful aproach for genotyping all pregnant women in inexpensively. Moreover, when HPA-1 homozygosis was detected in a pregnant woman, fetal heterozygosis may be diagnosed by COLD HRM to select pregnancies for preventive monitoring.