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Accurate quantitation of D + fetomaternal hemorrhage by flow cytometry using a novel reagent to eliminate granulocytes from analysis
Author(s) -
Kumpel Belinda,
Hazell Matthew,
Guest Alan,
Dixey Jonathan,
Mushens Rosey,
Bishop Debbie,
WrefordBush Tim,
Lee Edmond
Publication year - 2014
Publication title -
transfusion
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.045
H-Index - 132
eISSN - 1537-2995
pISSN - 0041-1132
DOI - 10.1111/trf.12484
Subject(s) - fluorescein isothiocyanate , flow cytometry , medicine , reagent , andrology , fluorescein , immunology , chemistry , physics , quantum mechanics , fluorescence
Background 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. Study Design and Methods Maternal blood samples (69) were sent for FC quantitation of FMH after positive K leihauer‐ B etke test ( KBT ) analysis and RhIG administration. Reagents used were BRAD ‐3–fluorescein isothiocyanate ( FITC ; anti‐ D ), AEVZ 5.3‐ FITC (anti–varicella zoster [anti‐ VZ ], negative control), anti‐fetal hemoglobin ( HbF )‐ FITC , blended two‐color reagents, BRAD ‐3‐ FITC /anti‐ CD 45‐phycoerythrin ( PE ; anti‐ D / L ), and BRAD ‐3‐ FITC /anti‐ CD 66b‐ PE (anti‐ D / G ). PE ‐positive WBCs were eliminated from analysis by gating. Full blood counts were performed on maternal samples and female donors. Results 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 . Conclusion 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.