Detection and relocation of cord blood nucleated red blood cells by laser scanning cytometry

Abstract

Background Fetal nucleated red blood cells (NRBC) present in the peripheral blood of pregnant women at low frequency are a potential target for noninvasive prenatal diagnostics. Methods CD71‐enriched cells from male cord blood (CB) were stained for the gamma chain of HbF (Hb‐γ) and cytocentrifuged. Fluorescence in situ hybridization (FISH) was done for the Y chromosome. Following staining of the nucleus with TO‐PRO‐3, laser scanning cytometry was performed. Artificial mixtures of small volumes of male CB and blood drawn from nonpregnant females were analyzed. Results In CB, 59% of events double positive for Hb‐γ and TO‐PRO‐3 were identified as CB‐NRBC. In contamination studies, male fetal CB‐NRBC were identified perfectly on the basis of morphologic characteristics and FISH reactivity following relocation and visual assessment. Mean recovery was 8.7%. Conclusions Laser scanning cytometry of preenriched fetal NRBC may offer a promising way for noninvasive prenatal diagnostics. This is because it provides a virtual enrichment step and the position on the slides of cells visually confirmed to correspond to fetal NRBC is known. Further experimental procedures on well‐defined and located target cells may be feasible. Cytometry 48:87–92, 2002. © 2002 Wiley‐Liss, Inc.

Automatic retrieval of single microchimeric cells and verification of identity by on‐chip multiplex PCR

“…Prior to the analysis, the software was trained to detect fetal erythroblasts (preparation described in Ref. [38]) and cytotrophoblast cells as specific fetal candidate cells. Optimized parameter settings retrieved from training data were stored as classifiers and used for scanning.…”

Laser Scanning Cytometry for selection of green fluorescent protein transgenic mice using small number of blood cells

“…However, because the position of each cell is fixed on the slide and the LSC saves the positional information, they can be relocated and visually analyzed or re-scanned with the LSC after re-staining with conventional stains or fluorescent markers [1,2]. The LSC has already been used for detection of nucleated red blood cells of fetal origin in maternal blood [3,4], which occur at a very low incidence. In this study, we examined the sensitivity of the LSC for detection of low numbers of GFP positive cells in the presence of a large excess of GFP negative cells.…”