title: Fetal cells in maternal blood: a comparison of methods for cell isolation and identification.
abstract: OBJECTIVE
  A variety of methods have been used to select and identify fetal cells from maternal blood. In this study, a commonly used 3-step selection method is compared with selection directly from whole blood. Identification of fetal origin by XY FISH of male cells was also evaluated.
  
  
  METHODS
  Maternal blood was drawn either before invasive chorion villus sampling (pre-CVS) or after (post-CVS) from women carrying a male fetus. Fetal cells were isolated either by density gradient centrifugation succeeded by CD45/CD14 depletion and CD71-positive selection from CD45/CD14-negative cells, or by CD71-positive selection directly from whole blood. The true origin of fetal cells recovered by the two methods was established by two rounds of XY chromosome FISH in reverse colors, in some instances combined with anti-zeta (zeta) or anti-zeta/anti-gamma (gamma) antibody staining.
  
  
  RESULTS
  In blood samples taken post-CVS and enriched by CD71 selection directly from whole blood, fetal cells were identified with a frequency that was almost four orders of magnitude higher than in post-CVS samples enriched by the 3-step method. In blood samples taken pre-CVS and enriched by the 3-step procedure, no fetal cells were identified by reverse color FISH in 371 ml of blood. In similar samples enriched by CD71 selection on whole blood, two fetal cells were identified in 27 ml of blood. Rehybridization with X and Y chromosome probes with reverse colors was necessary to exclude false Y chromosome signals. Not all fetal cells identified by the presence of a true Y chromosome signal stained with anti-zeta antibody.
  
  
  CONCLUSIONS
  Selection of fetal NRBCs from maternal blood by CD71-positive selection directly from whole blood is superior to density gradient centrifugation succeeded by CD45/CD14 depletion and CD71 selection of CD45/CD14-negative cells. Combining two markers for fetal origin is recommended for unambiguously identifying a cell as fetal.
authors.affiliation: Prenatal Research Unit, The Juliane Marie Center, section 4074, H:S Rigshospitalet, Copenhagen, Denmark. b.christensen@rh.dk
authors.forename: Britta
authors.initials: B
authors.lastname: Christensen
authors.forename: John
authors.initials: J
authors.lastname: Philip
authors.forename: Steen
authors.initials: S
authors.lastname: Kølvraa
authors.forename: Lene
authors.initials: L
authors.lastname: Lykke-Hansen
authors.forename: Ilona
authors.initials: I
authors.lastname: Hromadnikova
authors.forename: Dhanesh
authors.initials: D
authors.lastname: Gohel
authors.forename: Thomas
authors.initials: T
authors.lastname: Lorch
authors.forename: Andreas
authors.initials: A
authors.lastname: Plesch
authors.forename: Jens
authors.initials: J
authors.lastname: Bang
authors.forename: Steen
authors.initials: S
authors.lastname: Smidt-Jensen
authors.forename: Jens
authors.initials: J
authors.lastname: Hertz
authors.forename: Henning
authors.initials: H
authors.lastname: Djursing
mesh_terms: D015703:Antigens, CD / Q000032:analysis; D000944:Antigens, Differentiation, B-Lymphocyte / Q000032:analysis; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D015193:Chorionic Villi Sampling; D004900:Erythroblasts / Q000166:cytology / Q000276:immunology; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D005914:Globins / Q000032:analysis; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D017493:Leukocyte Common Antigens / Q000032:analysis; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D008297:Male; D011247:Pregnancy; D011990:Receptors, Transferrin
chemical_list: D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; C068126:CD71 antigen; D018950:Lipopolysaccharide Receptors; D011990:Receptors, Transferrin; D005914:Globins; D017493:Leukocyte Common Antigens