Development of a preparation and staining method for fetal erythroblasts in maternal blood: Simultaneous immunocytochemical staining and FISH analysis

Simultaneous fetal cell identification and diagnosis by epsilon-globin chain immunophenotyping and chromosomal fluorescence in situ hybridization

“…2,4 Previous attempts at reducing or correcting autofluorescence, which involved concomitant use of dyes absorbing certain emitted wavelengths 20 or mathematical manipulations of pixel intensity, 17 met with little success. We chose, instead, to systematically study heme autofluorescence within first trimester fetal erythroblasts.…”

“…Our high chromosomal FISH hybridization efficiency is important if diagnosis is reliant upon few cells only. Conventional immunoenzymatic staining with Vector Blue Substrate does not allow similar hybridization efficiency, 4 possibly because its dense precipitate hampers penetration of FISH probes into the nucleus.…”

“…1 There are currently 3 steps: enrichment of fetal cells in maternal blood, identification of fetal cells among background maternal cells, and diagnosis using fluorescence in situ hybridization (FISH) or single-cell techniques. Antibody directed against the ␥ chain of fetal hemoglobin is commonly used for both the fetal cell enrichment 2,3 and identification 4 steps. There may be a case for using ␥-globin for sorting, favoring yield over purity, but it is not nearly specific enough for accurate fetal erythroblast identification because of increased maternal fetal hemoglobin production in pregnancy 5 and ␤ thalassemia.…”

Optimization of nucleated red blood cell (NRBC) recovery from maternal blood collected using both layers of a double density gradient

“…The cytocentrifugation was carried out at 1000 rpm for 10 min. Afterwards, slides were air-dried overnight before being ®xed in methanol and stained with diaminobenzidin (DAB) (Sigma) to detect the presence of hemoglobin, as well as with hematoxylin, to counterstain the nuclei (Oosterwijk et al, 1998b).…”

Fetal hemoglobin-expressing nucleated red blood cell frequencies in pregnancies with intrauterine growth restriction

“…Immunocytochemical staining for hemoglobin c-chain was performed as described by Oosterwijk et al (1998a). After drying overnight, slides were ®xed in 100% methanol (10 min at x20uC), 100% acetone (10 min at x20uC), rinsed with PBS and then ®xed in 2% formaldehyde in PBS (10 min at room temperature).…”