Demonstration of spontaneously dividing male fetal cells in maternal blood by negative magnetic cell sorting and fish

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Abstract
We investigated a case of massive feto‐maternal bleeding by using negative magnetic cell sorting (MACS) and fluorescent in situ hybridization (FISH). A 37‐year‐old pregnant woman had an uncomplicated amniocentesis for advanced maternal age at 16 weeks' gestation. The fetal karyotype was 46, XY. At 19 weeks' gestation, she had a minor car accident and slight vaginal bleeding. A subsequent Kleihauer‐Betke test showed a 140 ml feto‐maternal haemorrhage. Serial sonographic examinations indicated a normal fetus and placenta. We performed FISH analysis on maternal peripheral blood at 25 weeks. Anti‐CD45 and MACS were used to deplete maternal leucocytes, enriching the proportion of fetal nucleated erythrocytes present. The isolated cells were analysed by using dual‐colour FISH with X and Y specific probes. Approximately 65 800 nucleated cells were obtained after MACS depletion. A total of 234 cells were analysed by FISH. The results revealed that 70 of the nucleated cells (30 per cent) were male with one X and one Y signal. Among these cells, six male metaphases were observed in spontaneously dividing cells.