Immunoglobulin isotype identification in red cell antibodies using flow cytometry

BACKGROUND: Identifying the isotype of an immunoglobulin (IgM vs. IgG) dete cted in a patient sample is especially important in anticipating the risk o f hemolytic disease of the newborn. Currently, 2-mercaptoethanol (2-ME) tre atment of a sample is used in the authors' laboratory to degrade IgM, and t his is followed by retesting. This method has multiple drawbacks. The purpo se of this study was to develop a flow cytometry (FC) assay that would repl ace the 2-ME treatment protocol (2-ME treatment). STUDY DESIGN AND METHODS: A preliminary FC assay was developed, modified, a nd refined through the use of stock antibodies. Then, 10 samples containing antibodies were tested in parallel by the FC assay and 2-ME treatment. RESULTS: When a 10-unit mean channel fluorescence change was used as an ind ex of a positive result, the FC assay detected all isotypes identified by 2 -ME treatment. The FC assay was also able to identify mixtures of isotypes. One antibody that had not reacted in conventional agglutination testing wa s detected by the FC assay. The amount of fluorescence and the agglutinatin g strength of the antibody did not parallel each other. In one case, this d iscrepancy may have reflected an antibody that was primarily IgA. CONCLUSIONS: The FC assay appears to be as accurate as 2-ME treatment in di fferentiating IgG from IgM. The FC assay produces a positive endpoint for b oth isotypes, will identify IgA, requires less sample, and has no odor.