Dear Haywood we developed an improved method for detecting CMV Ag by flow cytometry by using the Caltag Fix&Perm kit (remeber that fixation IS the crucial turning point for this analysis either on slide or by flow) and the DAKO CMV Ag antibody (diluited 1:5); after 30' incubation at 4°C in dark , a secondary FITC-conjugated anti-mouse Ab is added (DAKO diluted 1:100). All samples were run together with a control for autofluorescence (that's higher for PMN) by using the DAKO secondary antibody alone. In our hands , after severals samples analyzed with flow and standard immunophluorescence with optical microscopy, we found a perfect correlation of data. I've heard that now a new Ab for CMV (with better specificity) is available from Serotec. Gating a bitmap on the PMN region you must analyze a very large number of cells since sometimes the parasitemia is very low : if you have hardware troubles (low RAM) you can avoid to save your data as listmode files. If you need some further informations feel free to contact me . Edoardo Colombo MD Director Flow Lab Dept. of Pathology Valduce Hospital COMO , Italy coledoco@tin.it
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