Practicability of prenatal testing using lectin-based enrichment of fetal erythroblasts

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.

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.

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.

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.