First results using automated epifluorescence microscopy to detect Escherichia coli and Staphylococcus epidermidis in WBC-reduced platelet concentrates

BACKGROUND: Many methods have been tested for the detection of bacterial co ntamination in platelets. However, only those using molecular biology or ce ll culturing consistently detect contamination at levels below 105 bacteria per mL. This report describes the initial investigation into an alternativ e method that offers the possibilities of high sensitivity and rapid respon se while using available laboratory equipment and supplies. This method rel ies on a fluorescent nucleic acid stain, which preferentially stains bacter ia but not platelets, and automated epifluorescence microscopy for rapid an alysis. Measurements in WBC-reduced platelet concentrates (PCs) contaminate d with bacteria are reported at concentrations between 10(3) and 10(6) bact eria per mL. STUDY DESIGN AND METHODS: Staphylococcus epidermidis or Escherichia coli wa s inoculated into aliquots of WBC-reduced PCs on Days 2 through 5 of storag e. Bacterially inoculated and control PCs were stained, platelets and resid ual WBCs were lysed, and 200 muL of sample was filtered onto black polycarb onate filters. All preparations were done in triplicate. An automated epifl uorescence microscope examined approximately 2 percent of the area of each filter and used image analysis to select the fluorescent particles that sho uld be counted as bacteria. RESULTS: Samples containing 3 to 5 x 10(3) bacteria per mL produced about t hree times as many fluorescent particles classified as bacteria as the cont rols. Lower concentrations of S. epidermidis were detected because of highe r fluorescence intensity, Simultaneous preparation of six samples requires about 35 minutes. Analysis of each prepared sample takes 10 minutes, for a total preparation and analysis time of about 95 minutes for 6 samples. CONCLUSION: Low concentrations (<5 x 10(3) bacteria/mL of deliberately inoc ulated S. epidermidis or E coli can be measured quickly in WBC-depleted PCs by using a fluorescent nucleic acid stain, differential lysis, and automat ed microscopy. Continued refinement of the method, studies employing other bacterial strains, and further vali.