Flow cytometric measurement of cold agglutinins for diagnosing Mycoplasma pneumoniae pneumonia

Background: Mycoplasma pneumoniae is associated with community-acquired pneumonia in children and adults. Although conventional cold agglutinin testing (using the manual cold agglutinins [CA] assay) has not been regarded as a valuable tool for diagnosis of M. pneumoniae, we have developed a new and efficient assay, flow CA, for flow cytometric measurement of cold agglutinins. Methods: Flow CA measures cold agglutinins, which bind to group O red blood cells, using immunoglobulin M- (IgM) specific secondary antibodies after incubation of the O cells with serum samples at 4°C for 60 minutes. Once optimal parameters were established for this assay, its diagnostic efficiency was verified using serum samples from groups of individuals with M. pneumoniae (n = 27) and non-M. pneumoniae (n = 68) pneumonia; we then compared the resulting data with those generated by testing using manual CA or the M. pneumoniae IgM enzyme-linked immunosorbent assay (ELISA). Results: For diagnosis of M. pneumoniae pneumonia, flow CA testing showed significantly higher sensitivity than manual CA (78% and 46%, respectively, P =.01) and showed significantly higher specificity than M. pneumoniae IgM ELISA (94% and 66%, respectively, P < 0.001). The diagnostic efficiency of flow CA (90%) was also higher than that of the other two types of assay (82% and 71%, respectively). Conclusion: Flow CA is simpler, more rapid, more sensitive, and more quantitative than the manual CA assay; its results are also more specific than those of M. pneumoniae IgM ELISA. Therefore, flow CA can replace manual CA for use in diagnosis of M pneumoniae pneumonia.