title: Intact fetal cell isolation from maternal blood: improved isolation using a simple whole blood progenitor cell enrichment approach (RosetteSep).
abstract: Isolation and analysis of intact fetal cells in maternal blood is an attractive method of non-invasive prenatal diagnosis; however, detection levels are not optimal. The poor sensitivity and inconsistent recovery of fetal cells is compounded by small numbers of circulating fetal cells and loss of fetal cells during enrichment procedures. Optimizing selection criteria by utilizing less complicated methods for target cell enrichment is essential. We report here salutary results using a simple density-based depletion method that requires neither MACS (magnetic-activated cell sorting) nor flow cytometric separation for enrichment of progenitor cells. Maternal blood samples (n = 81) were obtained from women prior to invasive prenatal genetic diagnostic procedures and processed randomly within 24 h using one of two density-based enrichment methods. For progenitor cell enrichment, samples (n = 49) were labeled with a RosetteSep progenitor antibody cocktail to remove unwanted mature T-cells, B-cells, granulocytes, natural killer cells, neutrophils and myelomonocytic cells. For CD45-negative cell enrichment, samples (n = 14) were labeled with RosetteSep CD45 antibody to remove unwanted maternal white cells. The desired cellular fraction was collected and analyzed by either fluorescent in situ hybridization (FISH) or real-time PCR for the presence of intact fetal cells and to quantify Y-chromosome-specific DYS1 sequences, respectively. Overall, FISH and real-time PCR correct detection rates for the progenitor cell enrichment approach were 53% and 89% with 3% (1 out of 30 cases) and 0% false-positive detection, respectively. Fetal sequences were detected in the range from 0.067 to 1.167 genome equivalents per milliliter of blood. No fetal cells were detected using the CD45-negative enrichment method. Flow cytometric analysis of cord blood showed that a unique myeloid population of cells was recovered using RosetteSep trade mark progenitor enrichment compared with the CD45-negative enrichment method. Sensitivity of the RosetteSep progenitor enrichment approach for detection of fetal cells in this pilot study shows great promise with recovery of cells that are suitable for FISH and automated microscope scanning. This simple and rapid method may also allow expansion in culture and characterization of the fetal cell type(s) that circulate in maternal blood, hence, greatly improving reliability of non-invasive prenatal diagnosis.
authors.affiliation: Department of Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX 77030, USA. bischoff@bcm.tmc.edu
authors.forename: F Z
authors.initials: FZ
authors.lastname: Bischoff
authors.forename: D A
authors.initials: DA
authors.lastname: Marquéz-Do
authors.forename: D I
authors.initials: DI
authors.lastname: Martinez
authors.forename: D
authors.initials: D
authors.lastname: Dang
authors.forename: C
authors.initials: C
authors.lastname: Horne
authors.forename: D
authors.initials: D
authors.lastname: Lewis
authors.forename: J L
authors.initials: JL
authors.lastname: Simpson
mesh_terms: D001773:Blood Cells / Q000166:cytology*; D002469:Cell Separation / Q000379:methods*; D005260:Female; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D008431:Maternal-Fetal Exchange; D011247:Pregnancy; D011296:Prenatal Diagnosis / Q000379:methods*; D012397:Rosette Formation