An international quality control programme for PRISM chemiluminescent immunoassays in blood service and blood product laboratories.

BACKGROUND: Laboratories screening for blood-borne virus infections in blood and blood products are required by international standards and guidelines to ensure that their testing processes remain within control. An effective means of ensuring this aim is through participation in a quality control programme. Analyses of results from a quality control (QC) programme conducted for the Abbott PRISM (PRISM) assays are reported. MATERIALS AND METHODS: Laboratories participating in the National Serology Reference Laboratory, Australia's PRISM QC programme were provided with aliquots of a multimarker QC sample which were tested regularly in each PRISM subchannel. Test results were submitted to a single database using an Internet-based QC monitoring system, EDCNet. The QC test results submitted between 15 October 2001 and 5 March 2006 for each PRISM instrument and each lot of PRISM reagent were analysed to determine the imprecision and bias in each test system. RESULTS: A total of 157,404 test results from approximately 47,000 test runs submitted into the EDCNet database were analysed. Six batches of the multimarker QC samples were tested in 454 PRISM reagent lots. The coefficient of variation of QC sample test results ranged from 9.17 to 15.83%, 8.29 to 9.44%, 10.50 to 15.38% and 7.05 to 10.32% when tested in the PRISM anti-hepatitis C virus, anti-human immunodeficiency virus, anti-human T-cell lymphotrophic virus and hepatitis B surface antigen assays, respectively. Analysis of QC test results reported from testing in the anti-HTLV assay detected one lot of reagent (10572HN00) which was identified to be an outlier using Tukey's filter. DISCUSSION: Analysis of test results of an external QC sample can be used as a statistical process control through ongoing measurement of imprecision. When laboratories test the same QC sample in the same assay and submit test results to a single database, the results can be compared and a measure of bias can be calculated. The resulting QC programme can offer detection of unexpected variation in the testing processes and the source of variation investigated.

Identification of performance problems in a commercial human immunodeficiency virus type 1 enzyme immunoassay by multiuser external quality control monitoring and real-time data analysis.

In June 2005, a pilot program was implemented in Canadian laboratories to monitor the performance of the Abbott human immunodeficiency virus types 1 and 2 (HIV-1/2) gO enzyme immunoassay (EIA). Two different external quality control (QC) reagents and a "real-time" software analysis program were evaluated. In November 2005, higher-than-expected calibrator rate values in these kits were first reported at the Ontario Ministry of Health (Etobicoke), followed by the Alberta Provincial Public Health Laboratory (Edmonton and Calgary) and others. These aberrations were easily and readily tracked in "real time" using the external QC reagents and the software program. These high calibrator values were confirmed in Delkenheim, Germany, by Abbott, and a manufacturing change was initiated beginning with lot 38299LU00, which was distributed to laboratories in Canada in April 2006. However, widespread reports of calibrator failure by laboratories outside Canada were made in March 2006. In April 2006, Abbott Diagnostics initiated a level III investigation to identify the root cause, which was prolonged storage, under uncontrolled storage conditions, of the raw material used in the manufacture of the matrix cells. To the best of our knowledge, this is the first example of a program in Canada for serological testing that combines a common external QC reagent and a "real-time" software program to allow laboratories to monitor kit performance. In this case, external QC monitoring helped identify and confirm performance problems in the Abbott HIV-1/2 gO EIA kit, further highlighting the benefit of implementing such a program in a national or multilaboratory setting for laboratories performing diagnostic and clinical monitoring testing.

Automation of an absorbed enzyme immunoassay for the detection of Mycobacterium paratuberculosis antibodies for an eradication program.

As part of an ongoing Johne's disease eradication program by the state of Victoria, Australia, the Victorian Veterinary Pathology Services (VVPS) has been involved in testing over 150000 cattle samples per year for the presence of Johne's-specific antibodies. The Parachek kit (CSL, Victoria, Australia) has been used throughout the project. This method was automated using a Rosys 3300 and a Plato 7 (Dade/Behring Diagnostics) to pipette samples and the Behring ELISA Processor 3 (Dade/Behring Diagnostics) to wash EIA plates, add reagents, reads absorbances and perform data reduction of the results. Automation saved about 15h of labour per day, allowing one staff member to analyse up to 2000 samples per 8h shift compared to 800 samples using a manual protocol. Problems that were encountered include pipetting issues, the reading of the Paracheck endpoint, and excessive packaging of the kit. Maintenance, calibration and control of the analysers were an integral part of the process. VVPS, working in conjunction with CSL, suggested modification to the Parachek kit to make automation of the product more convenient. The approach resulted in a change in reagent volume and a reduction in packaging that reduced the cost of the test by 25%.

An evaluation of the in vitro activity of piperacillin/tazobactam.

Tazobactam is a new, irreversible inhibitor of bacterial beta-lactamases of staphylococci, plasmid-mediated beta-lactamases of the TEM and SHV types found in Escherichia coli and Klebsiella species and beta-lactamases of anerobes such as Bacteroides species. Its combination with piperacillin, a broad spectrum ureido-penicillin, would be expected to improve the activity of piperacillin against staphylococci, TEM and SHV beta-lactamase producing Gram negative bacteria and anerobes. Minimal inhibitory concentrations (MIC) of piperacillin/tazobactam were determined for 1952 individual patient isolates of Gram positive and negative bacteria causing significant infections and compared with MIC values for cefotaxime, ceftazidime, ciprofloxacin, imipenem, ticarcillin/clavulanic acid. MICs were determined by agar dilution (NCCLS 1990 and 1992). Piperacillin/tazobactam had excellent activity against methicillin susceptible staphylococci, Streptococcus pneumoniae, Haemophilus influenzae, enterococci and organisms of the Bacteroides fragilis group. It was also active against the majority of Enterobacteriaceae and Pseudomonas aeruginosa isolates tested. It was not active against extended spectrum beta-lactamase (ESBL) producing Klebsiella species and some high level TEM and SHV beta-lactamase producing E. coli and Klebsiella species. Activity against Gram negative organisms capable of producing chromosomally mediated beta-lactamases was good, since in most organisms tested, the enzymes were not induced in sufficient quantities to cause antibiotic resistance. However some Enterobacter species were derepressed hyperproducing mutants; these isolates showed resistance to piperacillin/tazobactam since tazobactam does not inhibit these Class I beta lactamases. Activity was superior to ticarcillin/clavulanic acid for Gram negative rods. Imipenem was the most active agent against ESBL producing Klebsiella species. Piperacillin/tazobactam has a suitable spectrum of activity in vitro to suggest its use in monotherapy of mixed anerobic infections, mixed respiratory infections such as aspiration pneumonia and, in combination with an aminoglycoside, it would provide Gram positive as well as Gram negative cover of febrile episodes in immunosuppressed patients.

Typing of strains from a single-source outbreak of Pseudomonas pickettii.

Plasmid profiles, genome restriction fragment polymorphisms, carbohydrate oxidation-fermentation reactions, methylumbelliferyl substrate hydrolysis patterns, antimicrobial susceptibilities, and results obtained with the Biolog GN biochemical substrate kit were used to type 19 common-source, but mixed-biotype, outbreak strains and one epidemiologically distinct strain of Pseudomonas pickettii. Biotyping with conventional and methylumbelliferyl substrates failed to distinguish between strains. Plasmid profile testing was found to be inconsistent and not reproducible. The Biolog GN kit allowed greater strain differentiation than restriction fragment polymorphism did (12 biotypes versus 5 biotypes); antimicrobial susceptibility testing yielded 4 biotypes, and oxidation-fermentation tests gave 3 biotypes. Oxidation-fermentation results were consistent with restriction fragment polymorphs in all but 1 of the 20 strains tested. For ease of typing, comprehensive typeability, and reproducibility, oxidation-fermentation tests should be performed initially and followed if necessary by restriction fragment polymorph analysis for the elucidation of P. pickettii infection outbreaks.

Multicenter evaluation of five commercial rubella virus immunoglobulin G kits which report in international units per milliliter.

In a multicenter study, the consistency of international units expressed by five commercially available rubella virus immunoglobulin G kits was evaluated. The linearity and within-run and between-run precision were determined for each kit. All kits demonstrated good linearity and had within-run and between-run precision coefficients of variation ranging from 5.1 to 21.7% and from 9.5 to 51.0%, respectively. To compare the international units expressed, the results from 40 samples tested in duplicate were compared with the results of a reference enzyme immunoassay calibrated with World Health Organization international standard serum and a hemagglutination inhibition test. The results of the kits were plotted against those of the reference tests, and linear regression analysis was applied. The Pearson correlation coefficient ranged from 0.64 to 0.75 when the commercial kit results were compared with those of the reference enzyme immunoassay, indicating only a moderate degree of correlation. Therefore, the international units expressed by the commercial kits are insufficiently consistent to be of practical use in diagnostic clinical microbiology.

Measurement of anti-DNA antibodies by ELISA: a comparative study with Crithidia and a Farr assay.

One hundred and twenty six sera from 116 patients with systemic lupus erythematosus (SLE) and from 51 control patients were assayed for the presence of anti-DNA antibodies, using a commercial enzyme linked immunosorbent assay (ELISA). Fifty three sera (42%) from SLE patients were positive and a further 13 sera (10%) fell in the 'equivocal' positive range. Three control sera were positive. In a standard 14C DNA Farr assay, 67 sera (53%) from SLE patients were positive. One control serum was weakly positive. There was a good linear correlation between absorption in the ELISA and the 14C DNA binding result (r = 0.73). Results in the ELISA and Farr assays were concordant in 96 of the 126 SLE sera, and 47 of 51 control sera. Sequential sera from a further 6 patients with fluctuating clinical activity of SLE showed similar patterns of change of anti-DNA antibodies in both assays. The ELISA was more sensitive than the Crithidia luciliae immunofluorescence assay which detected 44 positive sera (35%) in the SLE group. These results suggest that this ELISA assay may be a useful alternative to the Crithidia assay or an effective screen prior to testing in the more technically difficult and time consuming Farr assay for the measurement of anti-DNA antibodies.

Detection of anti-DNA antibodies: a comparison between two Farr assays, Crithidia luciliae and a human chromosomal substrate assay.

Four commercially available assays were compared with a 14C DNA Farr assay for their ability to detect anti-DNA antibodies in 119 sera from 109 patients with systemic lupus erythematosus (SLE) and 25 control sera. The 14C DNA Farr assay was the most sensitive and specific assay (SLE, 57% positive, controls, 0%). A commercial 125I DNA Farr assay was significantly less sensitive (SLE, 39% positive). The fluorescence human chromosomal preparation assay was as sensitive as the 14C DNA Farr assay (SLE, 58% positive) but less specific (controls, 8% positive). The immunofluorescence Crithidia luciliae assay was specific, but less sensitive (SLE, 37% positive) than the 14C DNA Farr assay. Adaptation of Crithidia to immunoperoxidase did not alter its sensitivity or performance. These results confirm that the 14C DNA Farr assay, locally refined and performed by experienced hands, is the most sensitive and specific assay for anti-DNA antibodies. The 125I DNA Farr was no more sensitive than the Crithidia assay but considerably more laborious. The human chromosomal preparation may be suitable as a rapid screening test for anti-DNA antibodies.

Comparison of immunofluorescence and immunoperoxidase for demonstration of antinuclear antibodies on HEp-2 substrate.

A comparison of antinuclear antibody (ANA) detection on HEp-2 cell substrate, using immunofluorescence (IF) and immunoperoxidase (IP) techniques, was performed on 183 sera from 178 individuals, with and without connective tissue diseases. All sera were number coded and scored blindly by two independent observers. ANA was detected in 61% of sera by IF and 62% of sera by IP. Positive and negative results corresponded between the two techniques in 97% of sera. Patterns and titres corresponded in 85% of positive sera. The two independent observers scored ANA more consistently with IP than with IF. Both methods were technically simple to perform and produced consistent results with control sera. These data show that IP provides results equivalent to the traditional IF technique for demonstrating ANA on HEp-2 substrate. Fewer discordant results between two independent observers using IP suggests that this technique has technical advantages for interpretation of ANA on HEp-2 substrate.