Objective: To determine how long fetal nucleated cells can be detected in maternal peripheral blood after delivery in cases of fetomaternal hemorrhage, using fluorescence in situ hybridization or polymerase chain reaction. Methods: Nucleated cells with Y chromosomes were analyzed in 90 blood samples using fluorescence in situ hybridization with a Y-chromosome-specific DNA probe. The samples were obtained from 15 mothers (gravida 1), who were delivered of normal male neonates at University of Tsukuba Hospital, Japan, with fetomaternal hemorrhage 1 and 7 days, and 1, 3, 6, and 12 months after delivery. The 15 mothers were selected if fetal cells were detected above the ratio of one fetal cell per 3000 mothers' cells at 1 day after delivery. Polymerase chain reaction amplifying two different Y-specific fragments (a 102 base-pair fragment in the pHY10 vector, which was a highly repetitive sequence, and a single-copy, Y-specific 198 base-pair fragment) was also carried out for the analysis of genomic DNA from the same samples. Results: Seven days and 1 month after delivery, Y-positive cells were detected in nine and four of 15 samples, respectively. No cells with the hybridization signal were detected in any of the 45 samples obtained 3 months or more after delivery. When we performed the polymerase chain reaction experiment amplifying the single-copy Y-specific fragment, none of the 45 samples after 3 months or more showed the expected band. However, from the same 45 samples, four obtained from different mothers showed positive results by amplification of the fragment in the pHY10 vector. Because multiple samples taken from these mothers before the collection of the positive samples showed negative results in the three experiments, these findings strongly suggest that the observed positive results were false-positives. Conclusion: Fetal nucleated cells in maternal peripheral blood become undetectable 3 months after delivery in most, if not all, cases of fetomaternal hemorrhage.
