Accurate quantitation of D+ fetomaternal hemorrhage by flow cytometry using a novel reagent to eliminate granulocytes from analysis

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Quantitation of fetomaternal hemorrhage (FMH) is performed to determine the dose of prophylactic anti-D (RhIG) required to prevent D immunization of D– women. Flow cytometry (FC) is the most accurate method. However, maternal white blood cells (WBCs) can give high background by binding anti-D nonspecifically, compromising accuracy.


Maternal blood samples (69) were sent for FC quantitation of FMH after positive Kleihauer-Betke test (KBT) analysis and RhIG administration. Reagents used were BRAD-3–fluorescein isothiocyanate (FITC; anti-D), AEVZ5.3-FITC (anti–varicella zoster [anti-VZ], negative control), anti-fetal hemoglobin (HbF)-FITC, blended two-color reagents, BRAD-3-FITC/anti-CD45-phycoerythrin (PE; anti-D/L), and BRAD-3-FITC/anti-CD66b-PE (anti-D/G). PE-positive WBCs were eliminated from analysis by gating. Full blood counts were performed on maternal samples and female donors.


Elevated numbers of neutrophils were present in 80% of patients. Red blood cell (RBC) indices varied widely in maternal blood. D+ FMH values obtained with anti-D/L, anti-D/G, and anti-HbF-FITC were very similar (r = 0.99, p < 0.001). Correlation between KBT and anti-HbF-FITC FMH results was low (r = 0.716). Inaccurate FMH quantitation using the current method (anti-D minus anti-VZ) occurred with 71% samples having less than 15 mL of D+ FMH (RBCs) and insufficient RhIG calculated for 9%. Using two-color reagents and anti-HbF-FITC, approximately 30% patients had elevated F cells, 26% had no fetal cells, 6% had D– FMH, 26% had 4 to 15 mL of D+ FMH, and 12% patients had more than 15 mL of D+ FMH (RBCs) requiring more than 300 μg of RhIG.


Without accurate quantitation of D+ FMH by FC, some women would receive inappropriate or inadequate anti-D prophylaxis. The latter may be at risk of immunization leading to hemolytic disease of the newborn.

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