Detection of antibiotic-induced platelet dysfunction in whole blood using flow cytometry

Using flow cytometry and activation-dependent monoclonal antibodies, we have developed a technique based on forward angle-light scatter (FALS) and immunofluorescence that simultaneously detects human platelet activation, secretion, and aggregation in whole blood. To detect the effects of cefotetan and latamoxef, both of which contain an N-MTT side chain, and of free N-MTT and cefoxitin, which does not contain the N-MTT side chain, on platelet activation and secretion, platelets were stained by the indirect method using a murine-produced platelet specific activation-dependent monoclonal antibody, S12, and a goat anti-mouse IgG fluorescein-conjugated antibody. S12 binds to a 140kd alpha granule membrane protein (GMP-140) that is expressed during secretion. Single parameter, 256 channel, log integrated green fluorescence histograms were generated, and negative and positive fluorescent populations were defined. Latamoxef and cefotetan reduced the number of platelets expressing S12 by more than 43%. In contrast, cefoxitin reduced the number of platelets expressing S12 by only 13.5%. The inhibition of GMP-140 expression per platelet was calculated by converting the log data to linear fluorescence intensity. Latamoxef and cefotetan inhibited expression of GMP-140 by 88% and 87% respectively. Free N-MTT inhibited its expression by 68%. In contrast cefoxitin reduced GMP-140 expression per platelet by only 45%.