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Practicability of prenatal testing using lectin‐based enrichment of fetal erythroblasts
Author(s) -
Kanda Eriko,
Yura Hirofumi,
Kitagawa Michihiro
Publication year - 2016
Publication title -
journal of obstetrics and gynaecology research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.597
H-Index - 50
eISSN - 1447-0756
pISSN - 1341-8076
DOI - 10.1111/jog.12982
Subject(s) - lectin , fetus , microbiology and biotechnology , erythroblast , laser capture microdissection , chromosome , fluorescence in situ hybridization , pathology , stain , medicine , staining , andrology , biology , gene , genetics , pregnancy , gene expression , stem cell , haematopoiesis
Abstract Aim The aim of this study was to investigate the practicability and efficiency of lectin‐based isolation of fetal erythroblasts for clinical use in non‐invasive prenatal testing. Methods Peripheral blood samples were collected from 39 pregnant women. Leukocytes were removed with an anti‐CD45 antibody after density gradient centrifugation. After blood cells were attached to slides by binding to a galactose‐specific lectin and galactose‐bound vinyl polymer, the slides were stained with May–Grünwald–Giemsa stain and cells were classified by automated image analysis based on their size and the nuclear area/cytoplasmic area ratio. In 14 samples from the women with male fetuses, fetal origin of the isolated erythroblasts was confirmed by detecting the Y chromosome using fluorescence in situ hybridization. In eight samples, single erythroblasts were collected by the laser capture microdissection technique for amplification of the sex‐determining region Y gene to confirm fetal origin. Results Panning with an anti‐CD45 antibody achieved stable removal of leukocytes without aggregation. In all samples, erythroblasts were successfully identified by automated image analysis (18–6000/10 mL of blood). The number of slides required to examine 10 mL of blood ranged from one to six, which was reasonable for clinical use. The Y chromosome was detected in 7.5–43.6% of erythroblasts by fluorescence in situ hybridization, and the sex‐determining region Y gene was amplified in seven of eight samples. Conclusion The combination of lectin‐based erythroblast isolation and automated image analysis is a practical and efficient method for isolating fetal erythroblasts as a source of fetal genomes.