Detection of Bacterial Contamination of Platelets Using the Real-time Polymerase Chain Reaction

Chang, Ho Eun; Kim, Mi Jung; Kwon, So Yong; Song, Sang Hoon; Shin, Sue; Park, Kyoung Un; Song, Junghan; Han, Kyou Sup

Abstract

BACKGROUND: The risk of transfusion-transmitted bacterial infection has been reported in addition to the risk of transmission of viral disease. Especially for platelets that are stored at 20degrees C~24degrees C with agitation to sustain platelet function. This storage method facilitates bacterial proliferation. Therefore, sensitive and rapid detection of bacteria must be considered for stored platelets.
METHODS
Six concentrations of platelets were spiked (1.5 mL) with five different bacteria and were analyzed by the 16S rDNA real-time polymerase chain reaction (PCR) using the ABI PRISM 7500 (Applied Biosystems, Foster, CA, USA) and the LightCycler 2.0 (Roche, Penzberg, Germany). The 16S rDNA gene was analyzed in three lots by real-time PCR reagents with sterile water. Three concentrations of spiked platelets (0.5 mL) with three different bacteria were preincubated in thioglycollate medium at 37degrees C for 8, 12, 16, 20, and 24 hours and then were analyzed by the 16S rDNA real-time PCR. The spiked platelets (0.5 mL) with fast-growing (8 hours of preincubation) and slow-growing (24 hours preincubation) bacteria were analyzed for the minimum incubation time.
RESULTS
The average crossing points (Cps) of the five bacteria were 21.2 with the ABI PRISM 7500 and 22.2 in the LightCycler 2.0. All five bacteria (10(1) bacteria/mL) were detected by both instruments. The average Cps of the three lots by real-time PCR was 29.3, 31.5 and 35.6. The contamination levels of the 16S rDNA were different. Fast-growing and slow-growing bacteria, preincubated at 8 and 24 hours, respectively, were detected at levels of 10(1) bacteria/mL.
CONCLUSION
The results of this study suggest that slow-growing bacteria can be detected at concentrations of 10(1) bacteria/mL in platelets preincubated with thioglycollate medium, at 37degrees C for 24 hours using platelets segment.