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

Dian Winkelhorst1,2, Leendert Porcelijn3, Eva Muizelaar3

  • 1Department of Obstetrics, Leiden University Medical Center, Leiden, The Netherlands.

Transfusion
|July 23, 2019
PubMed

Insights

A new, cost-effective assay accurately identifies HPA-1a-negative women, crucial for preventing fetal and neonatal alloimmune thrombocytopenia (FNAIT). This high-throughput method enables widespread screening for FNAIT, improving maternal and infant health outcomes.

Area of Science:

  • Immunology
  • Genetics
  • Obstetrics

Background:

  • Fetal and neonatal alloimmune thrombocytopenia (FNAIT) results from maternal antibodies against fetal human platelet antigens (HPAs), primarily anti-HPA-1a.
  • Population-based screening for FNAIT is debated due to the logistical and financial challenges of HPA-1a typing in pregnant women.
  • A need exists for a high-throughput, low-cost assay for HPA-1a typing to facilitate screening.

Purpose of the Study:

  • To develop and validate a high-throughput, low-cost HPA-1a phenotyping assay.
  • To assess the assay's reliability for population-based screening of pregnant women.
  • To enable efficient identification of HPA-1a-negative women for FNAIT risk assessment.

Main Methods:

  • A sandwich ELISA was developed using anti-GPIIIa coating and HRP-conjugated anti-HPA-1a detecting antibodies.
  • Assay performance was compared against an allelic discrimination PCR assay.
  • Two phases of testing were conducted: Phase I with 506 unselected pregnant women and Phase II with 62,171 samples, including supportive genotyping.

Main Results:

  • The ELISA assay demonstrated 100% sensitivity with a cut-off OD of 0.160.
  • Specificity was determined to be 99.9%, with a false-HPA-1a negative rate of 0.03%.
  • The assay is optimized for stored plasma samples, usable from 3- to 6-day-old samples.

Conclusions:

  • A high-throughput, low-cost, and reliable HPA-1a phenotyping assay has been successfully developed.
  • This assay is suitable for population-based screening to identify women requiring anti-HPA-1a testing.
  • The assay's applicability in suboptimal conditions makes it valuable for widespread FNAIT screening.
Abstract

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