Use of the Kleihauer test to detect fetal erythroblasts in the maternal circulation

Non-invasive prenatal diagnosis of aneuploidies on fetal nucleated erythroc ytes present in the maternal circulation is hampered by the extremely small cell number of uncertain origin (70% of erythroblasts circulating during p regnancy have a maternal origin). Therefore, a method allowing selection of the fetal cells among the maternal cells is indispensable after the erythr oblast enrichment step. In the present study, after an erythroblast enrichm ent step on a ficoll gradient followed by a positive immune-magnetic select ion with anti-CD71 or anti-GPA antibodies, a rapid, simple and direct chemi cal staining method adapted from the classical Kleihauer test was developed to select fetal cells. Precise differentiation between fetal and maternal erythroblasts is based on the constitutional difference between fetal and a dult haemoglobin (Hb). The fetal cells appear with an intense pink cytoplas mic staining while maternal cells with adult haemoglobin are colourless. Pr eservation of the cytoplasmic integrity allows one to distinguish morpholog ical characteristics and to visualize simultaneously nuclear hybridization signal by FISH (fluorescent in situ hybridization). This approach was teste d by FISH analysis using dual-colour X- and Y-specific DNA probes on blood samples from 15 pregnant women, with the results being compared to cytogene tic or sonographic sex determination. For 12 pregnancies fetal sex was dete rmined successfully (5 XY/7 XX), in two cases in situ hybridization failed, and in one case no fetal erythroblast was observed after the Kleihauer tes t. The selection method was applied to a pregnancy at risk for cystic fibro sis (CF). After a Kleihauer test, fetal erythroblasts were collected by mic rodissection, whole genomic DNA was amplified by primer extension pre-ampli fication (PEP) followed by a nested CF PCR. The fetal genotype was successf ully characterized and confirmed by conventional prenatal diagnosis. Copyri ght, 2001 John Wiley & Sons, Ltd.