Anti-D quantification by flow cytometry: a comparison of five methods

BACKGROUND Three flow cytometric methods for anti-D quantification have bee n published. All use different cell sensitization and antibody detection co nditions that may lead to varied results. Therefore, a direct comparison of the three methods is timely. STUDY DESIGN AND METHODS: The published flow cytometric methods and two new in-house modifications were compared. Ten serum samples containing anti-D at levels between 11.6 and 915 IU per mt were selected for analysis, and ea ch was tested a minimum of three times. Anti-D bound to cells was detected with fluorescence-labeled anti-human IgG reagents. RESULTS The interassay CV of the standard curves for each of the five metho ds was less than 10 percent. The intra-assay CV was consistently <10 percen t with four out of the five methods, but, by the fifth method, it was >20 p ercent in more than one-third of the tests. In 72 percent of the sample and method combinations, the interassay CV was <25 percent. Plotting of the me an anti-D value for each sample as a percentage of the value determined by an automated technique (AutoAnalyzer) revealed wide variability between the methods. CONCLUSION: Anti-D quantification by flow cytometry is influenced by the se rum antibody characteristics and the method used. The differences between t he flow cytometric and AutoAnalyzer techniques indicate that further valida tion of the flow cytometric method is required before routine use.