Quantification of fetomaternal hemorrhage: a compariative study of the HbF dual staining and RhD single staining by flow cytometry

Background and ObjectivesThe quantification of fetal cells in the maternal circulation is an important goal to determine the amount of anti-D for prevention of active immunization of a D-negative mother giving birth to a D-positive baby. The aim of this study was to evaluate two flowcytometric staining technics for determination of fetal erythrocytes in maternal blood and to recognize the best.Materials and MethodsIn this experimental study, 34 adult D-negative blood samples were spiked with six serial dilutions of D-positive cord blood (0.125, 0.25, 0.5, 1, 5 and 10%) which was representative of 99.9% of the clinical fetomaternal hemorrhage (FMH); they were stained for flow cytometric analysis. The Fetal Cell Count Kit was used for HbF dual staining and monoclonal anti-D for RhD single staining.ResultsA comparison between RhD and HbF percentages and FMH volume in spiking samples was performed. A significant correlation between two different parameters in flowcytometric percentages was observed (anti-HbF versus anti-D, r= 0.897, p<0.05). FMH volume was calculated and a significant correlation between expected and observed FMHs in RhD and HbF was obtained (p<0.05, r=0.984, r=0.874). The anti-HbF flowcytometric dual staining allowed better distinction between fetal RBCs (HbF+,CA-), F cells (HbF+,CA+), and adult RBCs (HbF-,CA+). Although Rh-D showed better correlation, but higher values with the RhD in suspensions lower than 10% were noted.ConclusionsData showed that anti-HbF labeling is significantly much more accurate than RhD labeling. Quantification of FMH using these two techniques described allows precise dosage of RhD immunoglobulin for protection against anti-D allo-immunization.