Use of a phycoerythrin-conjugated anti-glycophorin A monoclonal antibody as a double label to improve the accuracy of FMH quantification by flow cytometry

The use of flow cytometry for quantifying fetomaternal haemorrhage is incre asing, and has been shown to be more accurate than the Kleihauer-Betke test for evaluating larger bleeds of over 4 mL in volume. Red cells are stained with fluorescently labelled monoclonal anti-D. Cells for analysis are norm ally gated manually on the basis of forward and side scatter. We investigat ed whether the use of an antiglycophorin A monoclonal antibody conjugate (r ed cell specific) in a dual labelling technique would improve the gating of RBC and FMH quantification. Mixes of adult rr and cord R(1)r RBC were prepared to simulate 1, 0.5, 0.25 , 0.12 and 0.06% fetal bleeds. Phycoerythrin-conjugated BRIC 256 (mouse mon oclonal antiglycophorin A) was used to label all RBC, and FITC-BRAD-3 monoc lonal anti-D was used to determine the proportion of D-positive cells. Resu lts from the dual labelling experiments were compared to those from single labelling of the same mixtures with FITC-BRAD-3 alone, using gated and unga ted data. The results showed that single labelling with manual gating gave falsely low FMH estimates. We conclude that use of a fluorescently labelled antiglycophorin A antibody improves the accuracy of the FMH measurement by flow cytometry, as manual subjective gating of RBC excludes a higher proportion of fetal than of adul t RBC.