External quality assessment programmes for detection of HCV RNA, HIV RNA and HBV DNA in plasma: improved proficiency of the participants observed over a 2-year period.

BACKGROUND AND OBJECTIVES: Two External Quality Assessment Programmes (EQAPs) were run in 2008 and 2009 to evaluate the proficiency of blood centres in detecting, by nucleic acid amplification techniques (NAT), the possible contamination of plasma with hepatitis C virus (HCV), human immunodeficiency virus (HIV) and hepatitis B virus (HBV). MATERIALS AND METHODS: In the EQAP-2008, three customized panels were designed; each containing positive samples with a viral nominal concentration for the three viruses of about three times the 95% DL of the respective commercial NAT assay. In the EQAP-2009, the proficiency of the participants was evaluated with a single panel, independently on the NAT method used. RESULTS: While 84% (102/122) of the participants in the EQAP-2008 correctly identified the positive and negative samples of the panels, in the EQAP-2009 the percentage of proficient laboratories increased to 97% (118/122). Most importantly, in this 2-year experience, we observed a decrease in the number of pre-/postanalytical errors, from 14 in 2008 to two in 2009. CONCLUSIONS: The design of these two EQAPs allowed participants to assess the performance of the NAT methods applied in their routine screening of blood donations, not only with respect to analytical errors but also to human errors that, despite the high level of automation reached by NAT methods, can still occur.

External quality assessment for the detection of HCV RNA, HIV RNA and HBV DNA in plasma by nucleic acid amplification technology: a novel approach.

BACKGROUND AND OBJECTIVES: In this EQA study a novel approach was used to assess the performance of blood centres and blood product manufacturers in detecting the possible contamination of plasma with HCV, HIV and HBV by NAT. MATERIALS AND METHODS: A panel of 12 samples, three negative and three positive for each virus, was distributed to the EQA participants. The positive samples were prepared, using the respective WHO standards, in order to obtain a viral concentration of about three times the 95% DL of the methods most commonly used by laboratories involved in blood screening by NAT. Participants were requested to test each sample of the panel on different days, possibly by different operators using their routine NAT assay. RESULTS: Overall, the participants' performance was satisfactory. In particular, 49 of the 59 participants (83%) were able to correctly identify all samples. Regarding the remaining 10 laboratories, in three cases a deviation from the laboratory's procedure that could be attributed to an operator's mistake was observed, in two cases a possible cross-contamination occurred while in the remaining five cases the failure to detect the positive samples couldn't be ascribed to any relevant deviation in the laboratory's procedure. CONCLUSIONS: The novel design of this EQA study allowed participants to verify their day by day activity as the study was carried out in the context of their routine testing. Under these conditions, it was demonstrated that, despite the high level of automation reached by NAT assays, human errors can still occur.

External quality assessment for the detection of blood-borne viruses in plasma by nucleic acid amplification technology: the first human immunodeficiency virus and hepatitis B virus studies (HIV EQA/1 and HBV EQA/1) and the fifth hepatitis C virus study (HCV EQA/5).

BACKGROUND AND OBJECTIVES: This External Quality Assessment (EQA) study was aimed at assessing the proficiency of blood centres and blood product manufacturers in detecting, by nucleic acid amplification technology (NAT), the possible contamination of plasma with hepatitis C virus (HCV), human immunodeficiency virus (HIV) and hepatitis B virus (HBV). MATERIALS AND METHODS: Three independent panels, one for each virus, were prepared at the Istituto Superiore di Sanità (ISS) by diluting the respective reference preparations. NAT methods used by the EQA participants included polymerase chain reaction (PCR) assays by Roche, transcription-mediated amplification (TMA) assays by Chiron and in-house PCR assays. RESULTS: Forty-three of the 45 participants (95.6%) in the HCV EQA/5 who used a validated method were consistently able to detect a nominal concentration of 100 IU/ml for all six major genotypes. In the case of the HIV EQA/1, all 35 participants detected the samples containing 1000 IU/ml HIV, while five (14.3%) did not identify the samples containing 100 IU/ml HIV. With respect to the HBV EQA/1, all 16 participants correctly identified the positive samples containing either 1000 IU/ml or 100 IU/ml HBV. No false-positive results were observed with any of the three panels. CONCLUSIONS: The HCV EQA/5 showed an improved proficiency of laboratories as compared with the HCV EQA/4. In fact, HCV genotypes 1, 2, 3 and 5 were correctly identified in 100% of the assays and genotypes 4 and 6 in 97.8% of the assays. While most of the participants in the HIV EQA/1 showed a good level of proficiency, an excellent performance was shown by all participants in the HBV EQA/1.

Ubiquitin-mediated stress response in a rat model of brain transient ischemia/hypoxia.

Ubiquitin (Ub) is a small 76-residue protein, involved in intracellular protein degradation through a specific ATP-dependent system, which uses Ub as a tag to label proteins committed to be hydrolyzed by a specific 26 S protease. PGP-9.5 is another important component of the Ub system, i.e. a neuron-specific carboxyl-terminal hydrolase, which recycles Ub from Ub-polypeptide complexes. We have investigated the expression of Ub and PGP-9.5 in rat hippocampal neurons in an early phase of reperfusion in a model of transient global brain ischemia/hypoxia (bilateral occlusion of common carotid arteries for 10 min accompanied by mild hypoxia-15% O2-for 20 min), by means of immunohistochemical methods using light and electron microscopy. The intensity of Ub and PGP-9.5 immunoreactivity was evaluated by image analysis. We have detected a marked increase of Ub immunoreactivity (UIR) in neurons of CA1, CA2, CA3, CA4, and dentate gyrus subfields 1 hr after ischemia/hypoxia (but not after hypoxia only), statistically significant as confirmed by image analysis. Such increase in immunoreactivity in ischemic/hypoxic rats was localized essentially in the nuclei of hippocampal neurons. There were no changes in PGP-9.5 immunoreactivity. The data suggest that in the present model of rat brain ischemia/hypoxia Ub is involved in the neuronal stress response.

Gas-liquid-chromatography of trimethylsilyl derivatives from whole cell methanolysates of Leptospira : preliminary evaluation of its applicability to the taxonomy of the genus.

Gas-liquid-chromatography of trimethylsilyl derivatives from whole cell methanolysates was investigated as a supplmentary means for taxonomical classification within the genus Leptospira. Reproducibility of this technique was assessed through the peak height variations occurring in chromatograms of strain Patoc 1, serotype patoc, when samples either from the same or different batches of culture were used. From each chromatogram seven peaks were selected. Their heights were measured and calculated as percent values of the seven peaks total height. The values of relative standard deviation reported show that the reproducibility of this technique lies within the usual limits of biological methods. Four out of seven different serotypes analyzed gave elution patterns dissimilar enough to allow a clear distinction among them by the simple visual examination. Differentiation of the other three had to be done comparing the relative heights of the seven selected peaks. One not yet classified new strain was submitted to this technique; results seemed to confirm available serological information about it. Data reported encourage further research in order to evaluate the potential of GLC as an useful aid in the taxonomy of genus Leptospira.