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


METHODS
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.


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.


CONCLUSIONS
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.
Bristol Institute for Transfusion Sciences, NHS Blood and Transplant, Bristol, UK; International Blood Group Reference Laboratory, NHS Blood and Transplant, Bristol, UK.
Belinda
B
Kumpel
Matthew
M
Hazell
Alan
A
Guest
Jonathan
J
Dixey
Rosey
R
Mushens
Debbie
D
Bishop
Tim
T
Wreford-Bush
Edmond
E
Lee
D015703:Antigens, CD / Q000276:immunology; D015815:Cell Adhesion Molecules / Q000276:immunology; D005260:Female; D005331:Fetomaternal Transfusion / Q000175:diagnosis*; D005434:Flow Cytometry / Q000379:methods*; D058851:GPI-Linked Proteins / Q000276:immunology; D006098:Granulocytes / Q000276:immunology*; D014645:Herpesvirus 3, Human / Q000276:immunology; D006801:Humans; D017493:Leukocyte Common Antigens / Q000276:immunology; D011247:Pregnancy; D017452:Receptors, IgG / Q000276:immunology; D012204:Rh-Hr Blood-Group System / Q000032:analysis*; D018029:Rho(D) Immune Globulin / Q000276:immunology
D015703:Antigens, CD; C504120:CEACAM8 protein, human; D015815:Cell Adhesion Molecules; C428200:FCGR3B protein, human; D058851:GPI-Linked Proteins; D017452:Receptors, IgG; D012204:Rh-Hr Blood-Group System; D018029:Rho(D) Immune Globulin; C026410:Rho(D) antigen; D017493:Leukocyte Common Antigens; C516769:PTPRC protein, human