title: Practicability of prenatal testing using lectin-based enrichment of fetal erythroblasts.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Center for Maternal-Fetal and Neonatal Medicine, National Center for Child Health and Development, Tokyo, Japan.|Department of Obstetrics and Gynecology, Sanno Birth Center, Tokyo, Japan.
authors.forename: Eriko
authors.initials: E
authors.lastname: Kanda
authors.affiliation: Center for Maternal-Fetal and Neonatal Medicine, National Center for Child Health and Development, Tokyo, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Yura
authors.affiliation: Department of Obstetrics and Gynecology, Sanno Birth Center, Tokyo, Japan.
authors.forename: Michihiro
authors.initials: M
authors.lastname: Kitagawa
mesh_terms: D000906:Antibodies; D002469:Cell Separation / Q000379:methods*; D004900:Erythroblasts / Q000737:chemistry / Q000166:cytology / Q000276:immunology; D005260:Female; D005312:Fetal Blood / Q000737:chemistry / Q000166:cytology / Q000276:immunology; D005690:Galactose / Q000737:chemistry; D005820:Genetic Testing / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry; D037102:Lectins / Q000737:chemistry*; D017493:Leukocyte Common Antigens / Q000276:immunology; D008297:Male; D062145:Maternal Serum Screening Tests / Q000379:methods*; D011247:Pregnancy
chemical_list: D000906:Antibodies; D037102:Lectins; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human; D005690:Galactose
keywords: erythroblast; fluorescence in situ hybridization; lectin; prenatal diagnosis; sex-determining region Y gene