title: First results using automated epifluorescence microscopy to detect Escherichia coli and Staphylococcus epidermidis in WBC-reduced platelet concentrates.
abstract: BACKGROUND
  Many methods have been tested for the detection of bacterial contamination in platelets. However, only those using molecular biology or cell culturing consistently detect contamination at levels below 10(5) bacteria per mL. This report describes the initial investigation into an alternative method that offers the possibilities of high sensitivity and rapid response while using available laboratory equipment and supplies. This method relies on a fluorescent nucleic acid stain, which preferentially stains bacteria but not platelets, and automated epifluorescence microscopy for rapid analysis. Measurements in WBC-reduced platelet concentrates (PCs) contaminated with bacteria are reported at concentrations between 10(3) and 10(6) bacteria per mL.
  
  
  METHODS
  Staphylococcus epidermidis or Escherichia coli was inoculated into aliquots of WBC-reduced PCs on Days 2 through 5 of storage. Bacterially inoculated and control PCs were stained, platelets and residual WBCs were lysed, and 200 microL of sample was filtered onto black polycarbonate filters. All preparations were done in triplicate. An automated epifluorescence microscope examined approximately 2 percent of the area of each filter and used image analysis to select the fluorescent particles that should be counted as bacteria.
  
  
  RESULTS
  Samples containing 3 to 5 x 10(3) bacteria per mL produced about three times as many fluorescent particles classified as bacteria as the controls. Lower concentrations of S. epidermidis were detected because of higher 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.
  
  
  CONCLUSIONS
  Low concentrations (<5 x 10(3) bacteria/mL) of deliberately inoculated S. epidermidis or E. coli can be measured quickly in WBC-depleted PCs by using a fluorescent nucleic acid stain, differential lysis, and automated microscopy. Continued refinement of the method, studies employing other bacterial strains, and further validations of assay performance are warranted.
authors.affiliation: Naval Research Laboratory, Washington, DC 20375, USA. seaver@ccs.nrl.navy.mil
authors.forename: M
authors.initials: M
authors.lastname: Seaver
authors.forename: J C
authors.initials: JC
authors.lastname: Crookston
authors.forename: D C
authors.initials: DC
authors.lastname: Roselle
authors.forename: S J
authors.initials: SJ
authors.lastname: Wagner
mesh_terms: D001331:Automation; D001792:Blood Platelets / Q000382:microbiology*; D015169:Colony Count, Microbial; D004926:Escherichia coli / Q000302:isolation & purification*; D006801:Humans; D007937:Leukapheresis; D008856:Microscopy, Fluorescence; D012016:Reference Values; D013212:Staphylococcus epidermidis / Q000302:isolation & purification*; D013997:Time Factors