Establishment of a proficiency panel for an external quality assessment programme for the detection of bacterial contamination in platelet concentrates using rapid and cultural detection methods.

BACKGROUND: Platelet concentrates (PCs) are the main focus regarding the residual risk of transfusion-transmitted bacterial infections. Rapid screening methods for bacterial detection in platelets have been optimized over the last decade, but their external evaluation represents a complicated process. We developed a new type of proficiency panel for bacterial detection in PCs using currently available screening methods (especially rapid methods) suitable for external quality assessment programmes (EQAP). METHODS: PC samples were inoculated with different bacteria at two concentrations (10E+03 CFU/ml, 10E+05 CFU/ml) and stored under temperature-controlled conditions (1-5 days). Bacterial growth was further prevented by the addition of 0-20 µg/ml cotrimoxazole. Samples were analysed prior to and after storage using rapid detection methods (Bactiflow (BF), bacteria-generic NAT) and cultural methods to determine the influence of storage and antibiotic treatment on bacterial counts and the result outcome. A pilot EQAP was performed with four participants. RESULTS: Testing under the evaluated conditions demonstrated that bacterial counts remained constant prior to and after storage. The supplementation of 10 µg/ml cotrimoxazole did not influence bacterial detection using the two rapid detection methods BF and NAT. Furthermore, the detection of bacteria using cultural methods is still possible despite of antibiotic supplementation. The pilot EQAP confirmed these results. A storage time of up to 3 days proved practicable, showing no considerable influence on bacterial count and outcome of test results. CONCLUSION: The established proficiency panel provided PC matrix-conform samples with stabilized bacterial counts which can be analysed in parallel by rapid and cultural detection methods.

Blood donor screening with cobas s 201/cobas TaqScreen MPX under routine conditions at German Red Cross institutes.

BACKGROUND: In 1997 the German Red Cross (GRC) blood donor services introduced mini-pool nucleic acid testing (NAT) for human immunodeficiency virus (HIV)-1, hepatitis C virus (HCV) and hepatitis B virus (HBV) to increase blood safety. With the new cobas s 201/cobas TaqScreen MPX, a fully automated extraction method and a multiplex amplification system specifically adapted to the needs of blood donation services is available. METHODS: The cobas s 201 system was evaluated at the GRC BTS locations Hagen, Springe and Frankfurt. In phase A, the analytical sensitivity for the detection of HBV, HCV and HIV-1 was investigated and in phase B, at least 60,000 samples at each test site were screened in parallel with the MPX test on s 201 system and the existing routine mini-pool NAT system to compare the diagnostic specificity and the diagnostic sensitivity. RESULTS: Comparable analytical sensitivities in a range of 1.6-3.6 IU/ml, 4.9-10.9 IU/ml and 14.7-26.6 IU/ml for HBV, HCV HIV, respectively, for the MPX test on s 201 system (95% probability based on probit analysis) were determined at all test sites. The diagnostic sensitivity was 99.8% and the diagnostic specificity was 99.85%. CONCLUSIONS: The MPX test on s 201 system is a fully automated NAT system suitable for routine blood donor screening. The analytical sensitivity as well as the diagnostic sensitivity fulfilled all requirements of the Paul Ehrlich Institute for blood donor screening in mini-pools up to 96 donations per pool. A major benefit of the automated NAT system is the reduced personnel time and the extensive complete barcode-controlled process documentation.

Sterility screening of platelet concentrates: questioning the optimal test strategy.

BACKGROUND: Routine bacterial monitoring of apheresis platelet concentrates (APC) and pooled platelet concentrates (PPC) was introduced in two German blood services using culture and real-time reverse transcriptase (RT)-polymerase chain reaction (PCR). The results of testing are reviewed and used to discuss different strategies for detection of bacterial contamination of PCs. STUDY DESIGN AND METHODS: Two thousand three hundred and sixty-two APCs and 1993 PPCs have been tested by real-time RT-PCR and the BacT/Alert automated culturing system using aerobic and anaerobic culture bottles. After standard processing of PCs and storage of 22-24 h at 20-24 degrees C with agitation, samples were taken under aseptic conditions. Reactive culture bottles were confirmed as positive and bacterial isolates were identified by 16S rRNA analysis and biochemical tests. RESULTS: Seventeen of 2362 tested APCs were reactive in culture and one also in RT-PCR. Of these, 13 APCs were identified as initially positive as Staphylococcus warneri (n = 1, positive in aerobic and anaerobic culture), Propionibacterium acnes (n = 12, positive only in anaerobic culture) and four were initially reactive. Two of 1993 PPCs were initially reactive (anaerobic) and two more were confirmed positive (anaerobic) from a repeat culture and identified as P. acnes. All remaining specimens were tested negative. CONCLUSION: Our study demonstrates that the predominant organisms implicated in platelet bacterial contamination are part of the human skin flora. Inoculating blood culture systems and anaerobic cultivation detects these bacteria after approximately 3-7 days when blood products have been transfused. Based on the presented data different screening strategies are discussed.

Low energy electron energy-loss spectroscopy of CF3X (X=Cl,Br).

We report threshold electron energy-loss spectra for the fluorohalomethanes CF3X (X=Cl,Br). Measurements were made at incident electron energies of 30 and 100 eV in energy-loss range of 4-14 eV, and at scattering angles of 4 degrees and 15 degrees. Several new electronic transitions are observed which are ascribable to excitation of low-lying states as well as are intrinsically overlapped in the molecules themselves. Assignments of these electronic transitions are suggested. These assignments are based on present spectroscopic and cross-section measurements, high-energy scattering spectra, and ab initio molecular orbital calculations. The calculated potential curves along the C-X bond show repulsive nature, suggesting that these transitions may lead to dissociation of the C-X bond. The present results are also compared with the previous ones for CF3H, CF4, and CF3I.

Identification of the nuclear-encoded chloroplast ribosomal protein L12 of the monocotyledonous plant Secale cereale and sequencing of two different cDNAs with strong codon bias.

Two different cDNA clones (SCL12-1 and SCL12-2) encoding precursors of a chloroplast ribosomal protein with homology to L12 from Escherichia coli were isolated from rye leaf cDNA libraries and sequenced. The corresponding polypeptide of rye chloroplast ribosomes was identified. The sequences for the mature proteins of M(r) 13,447 and 13,609 share 85% amino acid identity. The mature polypeptide of clone SCL12-1 has an amino acid identity of 71%, 72% or 44%, respectively, relative to L12 proteins from spinach, tobacco, or E. coli. Codon usage of the rye L12 cDNAs shows a high preference (97% and 82%) for G or C in the third base position.