Flow‐Cytometric Quantitation of Anti‐D Antibodies

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Abstract
Quantitation of Rh antibodies is important clinically in predicting the risk of hemolytic disease of the newborn. We describe a flow cytometry method for the quantitation of anti-D antibodies that we developed in parallel to a recently described method. As a secondary antibody we used whole IgG instead of Fab molecules. The advantages, besides lower cost, include a strong fluorescence signal with no need for amplification, and the possibility of diluting samples to minimize the risk of agglutination by IgM antibodies. We did extensive studies on reproducibility. Reproducibility was superior to the autoanalyzer method. The two methods were roughly in agreement in estimating low, medium, or high levels of anti-D with a correlation coefficient of 0.89. The autoanalyzer measures the in vitro agglutination of all anti-D antibodies whereas flow cytometry measures the amount of IgG anti-D bound to red cells, which is more like the in vivo situation. Further studies in a clinical setting will show whether flow-cytometric quantitation may improve the diagnostic value of anti-D concentration measurement.