title: Basics of flow cytometry-based sterility testing of platelet concentrates.
abstract: BACKGROUND
  Flow cytometry (FACS) is a common technique in blood banking. It is used, for example, for the enumeration of residual white blood cells in plasma and in cellular blood products. It was investigated whether it can also be applied for sterility testing of buffy coat-derived platelet concentrates (PCs).
  
  
  METHODS
  Plasma-reduced PCs were spiked with bacteria and stored at 20 to 24 or 37 degrees C for various times. The following 10 species were used: Bacillus cereus, Enterobacter cloacae, Escherichia coli, Klebsiella pneumoniae, Propionibacterium acnes, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Serratia marcescens, and Yersinia enterocolitica. Bacterial DNA was stained with thiazole orange. After the platelets were lysed, bacteria were enumerated by FACS.
  
  
  RESULTS
  All bacteria species used were detectable by FACS. The lower detection limit was approximately 100 bacteria per microL, that is, 10(5) per mL. In general, the titers measured were 1.2- to 3-fold higher than those determined by colony forming assay. In one case (K. pneumoniae) in which the dot plot of the bacteria cloud overlapped with that of bacteria debris, they were consistently lower. When PC samples were inoculated with approximately 1 colony-forming unit per mL of bacteria and kept at 37 degrees C, most species were detected within 21 hours or less. Exceptions were E. cloacae and P. acnes, which were detected after 24 to 40 and 64 hours, respectively. At 20 to 24 degrees C, the detection times were strongly prolonged.
  
  
  CONCLUSIONS
  Sterility testing of PCs by FACS is a feasible approach. The present data suggest incubating PC samples for 20 to 24 hours at 37 degrees C before testing. For slow-growing bacteria, the incubation period must be prolonged by 1 to 2 days.
authors.affiliation: Blood Center of the German Red Cross Chapters of NSTOB, Institutes Springe and Gera, Springe and Gera, Germany. hmohr@bsd.nstob.de
authors.forename: Harald
authors.initials: H
authors.lastname: Mohr
authors.forename: Bernd
authors.initials: B
authors.lastname: Lambrecht
authors.forename: Anette
authors.initials: A
authors.lastname: Bayer
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Spengler
authors.forename: Sven-Boris
authors.initials: SB
authors.lastname: Nicol
authors.forename: Thomas
authors.initials: T
authors.lastname: Montag
authors.forename: Thomas H
authors.initials: TH
authors.lastname: Müller
mesh_terms: D001419:Bacteria; D001431:Bacteriological Techniques; D001771:Blood Banks; D001792:Blood Platelets / Q000382:microbiology*; D004340:Drug Contamination / Q000517:prevention & control; D005434:Flow Cytometry; D006801:Humans; D017713:Platelet Transfusion; D012680:Sensitivity and Specificity