Assessment of bacterial growth in leukoreduced cold-stored whole blood supports overnight hold at room temperature prior to filtration: A pilot study.

BACKGROUND AND OBJECTIVES: Whole blood (WB) transfusion has regained attention to treat trauma patients. We reported no significant changes in in vitro quality through 21 days of cold storage for leukoreduced WB (LCWB) when time to filtration was extended from 8 to 24 h from collection. This study evaluated the impact of extended WB-hold at room temperature (RT) prior to leukoreduction on proliferation of transfusion-relevant bacteria. MATERIALS AND METHODS: WB units were spiked with suspensions of Klebsiella pneumoniae, Streptococcus pyogenes, Staphylococcus aureus and Listeria monocytogenes prepared in saline solution (SS) or trypticase soy broth (TSB) to a concentration of ~0.2 CFU/ml (N = 6). Spiked units were held at RT for 18-24 h before leukoreduction and cold-stored for 21 days. Bacterial growth was determined on days 2, 7, 14 and 21. In vitro quality of WB inoculated with unspiked diluents was assessed. RESULTS: K. pneumoniae and S. pyogenes proliferated in WB prior to leukoreduction reaching concentrations ≤102 CFU/ml. These bacteria, however, did not proliferate during the subsequent cold storage. S. aureus did not survive in WB while L. monocytogenes reached a concentration of ~102 CFU/ml by day 21. LCWB in vitro quality was not affected by SS or TSB. CONCLUSION: Extended WB-hold prior to leukoreduction allowed proliferation of bacteria able to resist immune clearance, although they did not grow to clinically significant levels. While L. monocytogenes proliferated in LCWB, clinically relevant concentrations were not reached by day 21. These data suggest that transfusing LCWB may not pose a significant bacterial contamination safety risk to transfusion patients.

Evaluating the 4-hour and 30-minute rules: effects of room temperature exposure on red blood cell quality and bacterial growth.

BACKGROUND: A 30-minute rule was established to limit red blood cell (RBC) exposure to uncontrolled temperatures during storage and transportation. Also, RBC units issued for transfusion should not remain at room temperature (RT) for more than 4 hours (4-hour rule). This study was aimed at determining if single or multiple RT exposures affect RBC quality and/or promote bacterial growth. STUDY DESIGN AND METHODS: Growth and RT exposure experiments were performed in RBCs inoculated with Serratia liquefaciens and Serratia marcescens. RBCs were exposed once to RT for 5 hours (S. liquefaciens) or five times to RT for 30 minutes (S. marcescens) with periodic sampling for bacterial counts. Noncontaminated units were exposed to RT once (5 hr) or five times (30 min each) and sampled to measure in vitro quality variables. RBC core temperature was monitored using mock units with temperature loggers. Growth and RT exposure experiments were repeated three and at least six times, respectively. Statistical analysis was done using mixed-model analysis. RESULTS: RBC core temperature ranged from 7.3 to 11.6°C during 30-minute RT exposures and the time to reach 10°C varied from 22 to 55 minutes during 5-hour RT exposures. RBC quality was preserved after single or multiple RT exposures. Increased growth of S. liquefaciens was only observed after 2 hours of continuous RT exposure. S. marcescens concentration increased significantly in multiple-exposed units compared to the controls but did not reach clinically important levels. CONCLUSION: Single or multiple RT exposures did not affect RBC quality but slightly promoted bacterial growth in contaminated units. The clinical significance of these results remains unclear and needs further investigation.

Bacterial contamination in platelets: incremental improvements drive down but do not eliminate risk.

BACKGROUND: Bacterial contamination of platelet components (PCs) remains an important cause of transfusion-associated infectious risk. In 2004, Canadian Blood Services (CBS) implemented bacterial testing of PCs using the BacT/ALERT 3D system (bioMérieux). This system has been validated and implemented and continuous monitoring of culture rates allows gathering of data regarding true and false positives as well as false negatives. STUDY DESIGN AND METHODS: National data gathered between March 2004 and October 2010 from 12 CBS sites were analyzed to compare bacterial contamination rates across three platelet (PLT) preparation methods: apheresis, buffy coat, and PLT-rich plasma. Data were compared before and after implementation of protocol changes that may affect bacterial detection or contamination rates. RESULTS: Initial positive rates among the three production methods were significantly different, with apheresis PCs being the highest. The rates of confirmed positives among production methods did not differ significantly (p = 0.668). Increasing sample testing volumes from 4 to 6 mL to 8 to 10 mL significantly increased the rate of initial positives, while confirmed positives increased from 0.64 to 1.63 per 10,000, approaching significance (p = 0.055). Changing the skin disinfection method from a two-step to a one-step protocol did not significantly alter the rate of confirmed positives. During the period of data analysis, eight false-negative cases were reported, with five implicated in adverse transfusion reactions. CONCLUSION: Bacterial testing of PCs and implementation of improved protocols are incrementally effective in reducing the risk of transfusion of bacterially contaminated PLT concentrates; however, the continued occurrence of false-negative results means the risk has not been eliminated.

Assessment of biofilm-forming ability of coagulase-negative staphylococci isolated from contaminated platelet preparations in Canada.

BACKGROUND: Coagulase-negative staphylococci (CoNS) are the most prevalent bacterial contaminants of platelet (PLT) preparations and have been implicated in adverse transfusion reactions worldwide. The most frequently identified contaminant is Staphylococcus epidermidis, which is noted for its ability to maintain chronic hospital-acquired infections by forming biofilms as a chief virulence mechanism. STUDY DESIGN AND METHODS: Strains of S. epidermidis isolated from contaminated PLT preparations in Canada were distinguished via gene-specific polymerase chain reaction (PCR) with divIVA as a marker. Biofilm-forming ability was assessed by the presence of the gene icaD, slime production on Congo red agar, and biofilm formation on polystyrene surfaces. Production of polysaccharide intercellular adhesin (PIA) was resolved by immunofluorescence. RESULTS: Eight of the 13 (62%) CoNS isolates under study were identified as S. epidermidis. Of these, four strains (50%) were classified as strong biofilm producers. Three of the four biofilm-positive strains (75%) produced slime, harbored the icaD gene, and had positive expression of PIA. CONCLUSIONS: Despite the presumable commensal origin of the CoNS isolates, a large proportion of S. epidermidis strains demonstrated a potential for enhanced virulence. Identification of contaminant staphylococci as biofilm producers is thus relevant and informative with regard to treatment approach in the circumstance of inadvertent infection of a PLT recipient.