Flow-cytometric quantitation of anti-D antibodies

Objectives: 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. Methods: As a secondary antibody we used whole IgG instead of F-ab molecules. The advantages, besides lower cost, include a stronge 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. Results: 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. Conclusion: Further studies in a clinical setting will show whether flow-cytometric quantitation may improve the diagnostic value of anti-D concentration measurement.