title: Use of a phycoerythrin-conjugated anti-glycophorin A monoclonal antibody as a double label to improve the accuracy of FMH quantification by flow cytometry.
abstract: The use of flow cytometry for quantifying fetomaternal haemorrhage is increasing, 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 normally gated manually on the basis of forward and side scatter. We investigated whether the use of an antiglycophorin A monoclonal antibody conjugate (red cell specific) in a dual labelling technique would improve the gating of RBC and FMH quantification. Mixes of adult rr and cord R1r RBC were prepared to simulate 1, 0.5, 0.25, 0.12 and 0.06% fetal bleeds. Phycoerythrin-conjugated BRIC 256 (mouse monoclonal antiglycophorin A) was used to label all RBC, and FITC-BRAD-3 monoclonal anti-D was used to determine the proportion of D-positive cells. Results from the dual labelling experiments were compared to those from single labelling of the same mixtures with FITC-BRAD-3 alone, using gated and ungated 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 adult RBC.
authors.affiliation: International Blood Group Reference Laboratory, Southmead Road, Bristol BS10 5ND. paul.lloyd-evans@nbs.nhs.uk
authors.forename: P
authors.initials: P
authors.lastname: Lloyd-Evans
authors.forename: A R
authors.initials: AR
authors.lastname: Guest
authors.forename: E B
authors.initials: EB
authors.lastname: Austin
authors.forename: M L
authors.initials: ML
authors.lastname: Scott
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal; D004912:Erythrocytes / Q000276:immunology; D005260:Female; D005331:Fetomaternal Transfusion / Q000175:diagnosis*; D005434:Flow Cytometry / Q000379:methods*; D006021:Glycophorins / Q000276:immunology*; D006801:Humans; D051379:Mice; D010799:Phycoerythrin; D011247:Pregnancy; D012204:Rh-Hr Blood-Group System / Q000032:analysis
chemical_list: D000911:Antibodies, Monoclonal; D006021:Glycophorins; D012204:Rh-Hr Blood-Group System; D010799:Phycoerythrin