title: 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.
authors.affiliation: Department of Hematogenetics, Institute of Immunohaematology, Parel, India.
authors.forename: Edna
authors.initials: E
authors.lastname: D'Souza
authors.forename: Pratibha M
authors.initials: PM
authors.lastname: Sawant
authors.forename: Anita H
authors.initials: AH
authors.lastname: Nadkarni
authors.forename: Ajit
authors.initials: A
authors.lastname: Gorakshakar
authors.forename: Dipika
authors.initials: D
authors.lastname: Mohanty
authors.forename: Kanjaksha
authors.initials: K
authors.lastname: Ghosh
authors.forename: Roshan B
authors.initials: RB
authors.lastname: Colah
mesh_terms: D000911:Antibodies, Monoclonal; D003362:Cost-Benefit Analysis; D004900:Erythroblasts; D005260:Female; D005312:Fetal Blood; D005820:Genetic Testing; D005914:Globins / Q000235:genetics*; D006453:Hemoglobinopathies / Q000175:diagnosis*; D006801:Humans; D007194:India; D018598:Minisatellite Repeats; D009154:Mutation; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000191:economics / Q000379:methods*
chemical_list: D000911:Antibodies, Monoclonal; D005914:Globins