Evaluation of the use of monoclonal antibodies and nested PCR for noninvasive prenatal diagnosis of hemoglobinopathies in India

Abstract

Our purpose was to develop and evaluate isolation and enrichment of fetal erythroblasts and a nested polymerase chain reaction (PCR) approach using fetal erythroblasts for detecting the beta-globin gene mutations for a noninvasive prenatal diagnosis of hemoglobinopathies. Maternal blood at different periods of gestation was layered on a Percoll density gradient for enrichment of fetal nucleated RBCs (NRBCs). A combination of 3 monoclonal antibodies (CD45-peridinin chlorophyll protein, glycophorin A-phycoerythrin, and anti-hemoglobin F-fluorescein isothiocyanate) was used for flow cytometric sorting of fetal NRBCs from enriched cells. Different nested PCR-based approaches were used for identification of fetal mutations. Owing to heterogeneity of beta-thalassemia mutations in the population of India, we had to screen for 12 mutations and were able to give an accurate diagnosis in 84 (84.0%) of 100 cases when compared with chorionic villus sampling or cordocentesis and DNA analysis.This nested PCR approach enabled amplification of small quantities of DNA from fetal erythroblasts, providing a cost-effective method for noninvasive diagnosis.