Rapid and sensitive detection of viable bacteria in contaminated platelet concentrates using a newly developed bioimaging system.
BACKGROUND
Rapid and sensitive methods for the detection of bacteria in platelet concentrates (PCs) are required as well as inactivation techniques to decrease the transfusion-associated risk of infection from bacterially contaminated PCs. In this study, a rapid microbiologic method for the sensitive counting of viable bacteria in PCs was developed by combining a fluorescent staining technique and a bioimaging system.


METHODS
An esterase indicator, carboxyfluorescein diacetate, was used to detect physiologically active bacteria. Treatment was optimized to selectively remove platelets (PLTs). Bacterial cells trapped on a filter were automatically discriminated from other particles or PLT debris and counted by a bioimaging system. The sensitivity, rapidity, and recovery rates were evaluated using PCs spiked with 14 reference bacterial strains and clinical isolates.


RESULTS
Lysis treatment with enzyme and detergent was effective to remove PLTs and white blood cells. Two buffers for fluorescent vital staining were needed for highly sensitive detection of pathogenic bacteria. Fewer than 100 cells spiked in 5-mL PCs were detected by the bioimaging system after treatment and fluorescent staining, and this result shows that PLTs are selectively digested by the treatment. Bacterial cells spiked in 25-mL PCs were detected within 45 minutes (treatment, 15 min; filtration and fluorescent staining, 15 min; automated counting and precise image analysis, 10-15 min).


CONCLUSIONS
The microbiologic method described here is rapid and sensitive, and this method has potential for the screening of PCs contaminated with bacterial cells. Furthermore, this method could contribute to further evaluation of inactivation techniques.
Motoyama
Yasuo
Y
Asahi Breweries, Ltd., Minami-Aoyama, Minato-ku, Tokyo, Japan.
Yamaguchi
Nobuyasu
N
Matsumoto
Mami
M
Kagami
Noboru
N
Tani
Yoshihiko
Y
Satake
Masahiro
M
Nasu
Masao
M
D016470:Bacteremia / Q000517:prevention & control / Q000635:transmission; D001419:Bacteria / Q000302:isolation & purification*; D001792:Blood Platelets / Q000382:microbiology*; D002021:Buffers; D003199:Computer Systems; D004867:Equipment Design; D005452:Fluoresceins / Q000032:analysis; D005456:Fluorescent Dyes / Q000032:analysis; D006801:Humans; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D010784:Photomicrography / Q000295:instrumentation / Q000379:methods*; D017713:Platelet Transfusion / Q000009:adverse effects; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013997:Time Factors; D014584:User-Computer Interface
D002021:Buffers; D005452:Fluoresceins; D005456:Fluorescent Dyes; C093616:6-carboxyfluorescein diacetate; C084577:5-carboxyfluorescein diacetate