BACKGROUND: The FDA requirement for sensitivity of viral NAT methods used in blood screening is a 95‐percent detection limit of 100 copies per mL, whereas the NAT screening system should have a ...sensitivity of at least 5000 copies per mL per individual donation. According to the Common Technical Specifications of the European Directive 98/79/EC for in vitro diagnostics, viral standard dilutions (calibrated against the WHO standard) should be tested at least 24 times for a statistically valid assessment of the 95‐percent detection limit.
STUDY DESIGN AND METHODS: Viral standard dilution panels (PeliCheck, VQC‐CLB) were prepared for HCV RNA genotypes 1 and 3 and for HIV RNA genotypes B and E. In a multicenter study, 23 laboratories tested the panels all together in 8 to 91 test runs per NAT method.
RESULTS: The following 95‐percent detection limits (and 95% CIs) were found on the HCV RNA genotype 1 reference panels (shown as geq/mL): Gen‐Probe TMA, 85 (64‐118); AmpliScreen, 126 (83‐225); AmpliScreen with NucliSens Extractor, 21 (13‐44); Amplicor with NucliSens Extractor, 69 (50‐102), and Amplicor with Qiagen extraction technology, 144 (74‐102). On HIV RNA genotype B dilution panels, the following 95‐percent detection limits were found (shown as geq/mL): Gen‐Probe TMA, 31 (20‐52); AmpliScreen, 126 (67‐311); AmpliScreen with NucliSens Extractor, 37 (23‐69), and NucliSens QL assay, 123 (51‐566). HIV RNA genotype E panels were detected with equal sensitivity as HIV RNA genotype B panels. In the Gen‐Probe TMA assay, the 50‐percent detection limits on HIV RNA type B and type E were 3.6 (2.6‐5.0) and 3.9 (2.4‐5.8) geq per mL, respectively. The HCV RNA genotype 1 and 3 standards were detected with equal sensitivity.
CONCLUSION: The differences in sensitivity between NAT assays can be explained by the input of isolated viral nucleic acid in the amplification reactions. The FDA requirements for sensitivity of NAT blood screening assays can be met by the Gen‐probe TMA, as well as by the AmpliScreen assays, particularly when combined with the NucliSens Extractor.
Because of its short life span in blood, its low RNA content and its highly condensed nucleus, the granulocyte was initially considered as a terminally differentiated cell unable to express novel ...genes. However, mature granulocytes still contain a variety of mRNAs and may respond to external stimuli by rapid and complex changes in gene expression. The present work was undertaken to provide a wider view of the gene expression profile in unstimulated circulating PMNs. We used serial analysis of gene expression (SAGE) adapted to downsized extracts (SADE) to cope with their small mRNA content. As cell samples of the highest degree of purity were crucial for this project, we adapted a magnetic cell sorting method to reach the required high levels of purity (99.55±0.19%) together with low activation rates (1.37±0.28). We analyzed 20787 tags identifying 8547 different transcripts, of which 47.8% were unknown, 34.6% were transcripts of known genes, and 13.8% matched with expressed sequence tags (EST). Highly expressed genes were those involved in cell mobility, diapedesis, cell signaling and destruction of micro-organisms. In addition, this method led to the identification of genes which had not previously been reported in granulocytes. These results could provide new molecular markers and a reliable reference for the investigation of pathologies involving alteration of the granulocyte gene expression profile.
BACKGROUND:The Pall third-generation enhanced bacterial detection system (eBDS) was recently approved for detection of bacterial contamination in leukoreduced platelets (PLTs). The method is based on ...the measurement of the oxygen content as a marker for bacteria. eBDS incorporates major modifications including removal of the sample-set filter, modification of the culture medium, and incubation with agitation of the sample pouch. STUDY DESIGN AND METHODS:Ten whole blood-derived random-donor PLT units collected on Day 1 after donation and 10 single-donor apheresis PLT units were spiked with low levels of bacteria in three different blood transfusion centers. Inoculation was performed at a final concentration of 5 to 50 colony-forming units per mL with reference strains of five organisms involved in severe transfusion-associated infections. PLT units were stored at 22 degree C for 24 hours before sampling. Six sample sets were then sterile-connected to each unit and placed on a horizontal agitator at 35 degree C for 18 or 24 hours of incubation. RESULTS:No false-positive results were obtained, indicating a 100 percent specificity of the assay. Of 126 spiked sample pouches tested, 61 of 63 (96.82%) and 63 of 63 (100%) were detected positive after 18 or 24 hours of incubation, respectively. In the two missed cases that failed to detect Bacillus cereus, the measured oxygen was slightly above the detection threshold but was markedly different from the negative samples. CONCLUSION:The eBDS method allows definitive testing of PLTs as soon as 42 hours after collection and offers an alternative culture method to the BacT-ALERT system.
Understanding the virus‐host interactions that lead to approximately 20% of patients with acute Hepatitis C Virus (HCV) infection to viral clearance is probably a key towards the development of more ...effective treatment and prevention strategies. Acute hepatitis C infection is usually asymptomatic and therefore rarely diagnosed. Nevertheless, HCV nucleic acid testing carried out on all blood donations detects donors who have resolved their HCV infection after seroconversion. Here we have used SELDI‐TOF‐MS technology to compare, at a proteomic level, plasma samples respectively from donors with HCV clearance, from donors with chronic HCV infection and from unexposed healthy donors (n = 15 per group). A candidate marker of about 9.4 kDa was detected as differentially expressed in the three groups. After purification we identified by nanoLC‐Q‐TOF‐MS/MS this candidate marker as Apolipoprotein C‐III (ApoC‐III). The identification was confirmed by western blot analysis. Levels of ApoC‐III were then determined in the 45 plasma samples by immunoturbidimetric assay. ApoC‐III was found to be higher in donors who had resolved their HCV infection than in donors with chronic infection, results which were consistent with SELDI‐TOF‐MS data. ApoC‐III is the first reported candidate biomarker in plasma associated with the spontaneous resolution of HCV infection.
Symposium S09 Nucleic Acid Testing (NAT 1) Joliette, COSTE; Cees, L VAN DER POEL
Transfusion clinique et biologique : journal de la Société française de transfusion sanguine,
6/2001, Letnik:
8
Journal Article
The prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the population remains uncertain, although it has been estimated that 1 in 2000 people in the United Kingdom are positive for abnormal ...prion protein (PrPTSE) by a recent survey of archived appendix tissues. The prominent lymphotropism of vCJD prions raises the possibility that some surgical procedures may be at risk of iatrogenic vCJD transmission in healthcare facilities. It is therefore vital that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated. A current limitation is the lack of a rapid model permissive to human prions. Here, we developed a prion detection assay based on protein misfolding cyclic amplification (PMCA) technology combined with stainless-steel wire surfaces as carriers of prions (Surf-PMCA). This assay allowed the specific detection of minute quantities (10(-8) brain dilution) of either human vCJD or ovine scrapie PrPTSE adsorbed onto a single steel wire, within a two week timeframe. Using Surf-PMCA we evaluated the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD PrPTSE was lower than expected. Overall, our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of human vCJD PrPTSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions.
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