Accurate quantitation of D+ fetomaternal hemorrhage by flow cytometry using a novel reagent to eliminate granulocytes from analysis. Issue 5 (17th November 2013)

• Record Type: Journal Article
• Authors: Kumpel, Belinda; Hazell, Matthew; Guest, Alan; Dixey, Jonathan; Mushens, Rosey; Bishop, Debbie; Wreford‐Bush, Tim; Lee, Edmond
• Is Part Of: Transfusion. Volume 54:Issue 5(2014)
• Page Start: 1305
• Page End: 1316
• Publication Date: 2013-11-17
• Subjects: Hematology -- Periodicals; Blood -- Transfusion -- Periodicals; Blood Group Antigens -- Periodicals; Blood Preservation -- Periodicals; Blood Transfusion -- Periodicals; 615
• DOI: 10.1111/trf.12484
• Languages: English
• ISSNs: 0041-1132

Abstract

<abstract abstract-type="main"> <title> <x xml:space="preserve">Abstract</x> </title> <sec id="trf12484-sec-0001" sec-type="section"> <title>Background</title> <p>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.</p> </sec> <sec id="trf12484-sec-0002" sec-type="section"> <title>Study Design and Methods</title> <p>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.</p> </sec> <sec id="trf12484-sec-0003" sec-type="section"> <title>Results</title> <p>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.</p> </sec> <sec id="trf12484-sec-0004" sec-type="section"> <title>Conclusion</title> <p>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.</p> </sec> </abstract>