Total Coefficient Of Variation Research Articles

Comparison of Air Sampling Methods for Aerosolized Spores of B. anthracis Sterne

Bacillus anthracis Sterne spores were aerosolized within a chamber at concentrations ranging from 1×103 to 1.7×104 spores per cubic meter of air (particles (p)/m3) to compare three different sampling methods: Andersen samplers, gelatin filters, and polytetrafluoroethylene (PTFE) membrane filters. Three samples of each type were collected during each of 19 chamber runs. Chamber concentration was determined by an aerodynamic particle sizer (APS) for the size range of 1.114–1.596 μm. Runs were categorized (low, medium, and high) based on tertiles of the APS estimated air concentrations. Measured air concentrations and recovery efficiency [ratio of the measured (colony forming units (CFU)/m3) to the APS estimated (particles/m3) air concentrations] for the sampling methods were compared using mixed-effects regression models. Limits of detection for each method were estimated based on estimated recovery efficiencies. Mean APS estimated air concentrations were 1600 particles/m3, 4100 particles/m3, and 9100 particles/m3 at the low, medium, and high tertiles, respectively; coefficient of variation (CV) ranged from 25 to 40%. Statistically significant differences were not observed among the three sampling methods. At the high and medium tertiles, estimated correlations of measured air concentration (CFU/m3) among samples collected from the same run of the same type were high (0.73 to 0.93). Among samples collected from the same run but of different types, correlations were moderate to high (0.45 to 0.85); however, correlations were somewhat lower at the low tertile (–0.31 to 0.75). Estimated mean recovery efficiencies ranged from 0.22 to 0.25 CFU/particle with total CVs of approximately 84 to 97%. Estimated detection limits ranged from 35 to 39 particles/m3. These results will enable investigators to conduct environmental sampling, quantify contamination levels, and conduct risk assessments of B. anthracis.

Serum cholesterol measured by isotope dilution liquid chromatography tandem mass spectrometry

Accurate cholesterol measurements are essential for the prevention and management of cardiovascular diseases. Quality assurance of cholesterol measurements requires reference methods. An isotope dilution liquid chromatography mass spectrometric (ID/LC/MS) method was developed. Serum samples were sampled volumetrically using automated dilutors, treated with potassium hydroxide and equilibrated with 3,4-(13)C(2) cholesterol. The natural cholesterol and the internal standard were extracted with hexane and oxidized to cholest-4-en-3,6-diones with chromic acid. The oxidation products were separated on reversed phase LC and detected by tandem MS. The method was calibrated using aqueous cholesterol calibrators and the calibration function was established with a polynomial regression. The correlation coefficients of the calibration curves were always >0.9999. The coefficients of variation (CV) of the volumetric sampling and the LC/MS analysis averaged 0.22% and 0.50%, respectively, and the total measurement CV was 0.60%. Other sources of measurement uncertainty were minor. Results on certified reference materials agreed within 1% of the certified values. An ID/LC/MS method for serum cholesterol has been developed. The method is simple and accurate and may be used as a candidate reference method.

Performance Characteristics of Six Intact Parathyroid Hormone Assays

The aim of this study was to evaluate the performance characteristics of 6 intact parathyroid hormone assays: Access 2 (Beckman Coulter, Fullerton, CA), ARCHITECT i2000(SR) (Abbott Diagnostics, Abbott Park, IL), ADVIA Centaur (Siemens Healthcare Diagnostics, Deerfield, IL), Modular E170 (Roche Diagnostics, Indianapolis, IN), IMMULITE 2000 (Siemens Healthcare Diagnostics), and LIAISON (DiaSorin, Stillwater, MN). Sample collection tubes and storage conditions were compared. Imprecision studies were performed using commercial quality control materials. Linearity was assessed using pools prepared from samples. For method comparison, serum and EDTA plasma samples were tested by all methods, and the ARCHITECT was used as the comparison method. Reference intervals were determined using various vitamin D cutoffs. The types of collection tubes and storage conditions are more important for some methods than others. Total coefficients of variation were 10.9% or less. The maximum deviation from the target recovery for linearity ranged from 5.0% to 82.2%. Bland-Altman plots demonstrated percentage biases ranging from -36.3% to 24.4%. The lower limit of the reference interval was not influenced by vitamin D status, whereas the upper reference limit was affected.

Intra‐individual short‐term variability of prostate‐specific antigen and other kallikrein markers in a serial collection of blood from men under evaluation for prostate cancer

Diagnostic (exploratory cohort). 2b. To assess variation of total prostate-specific antigen (tPSA), free PSA (fPSA), percent fPSA, human glandular kallikrein 2 (hK2) and intact PSA measured three times within 2 weeks. Knowledge of the variation in an individual's PSA level is important for clinical decision-making. Study participants were 149 patients referred for prostate biopsy, of which 97 had benign disease and 52 had prostate cancer. Three blood samples were drawn with a median of 4 h between first and second samples and 12 days between first and third samples. Variability was described by absolute differences, ratios and intra-individual coefficients of variation. Total PSA, fPSA, hK2 and intact PSA were measured in anticoagulated blood plasma. At baseline, the median tPSA was 6.8 (interquartile range, 4.5-9.6) ng/mL. The intra-individual variation was low for all biomarkers, and lowest for tPSA. For 80% of participants, the ratio between first and second time points for tPSA was in the range 0.91-1.09 and the ratio for percent fPSA was in the range 0.89-1.15. Total coefficients of variation between time 1 and 2 for tPSA, fPSA, percent fPSA, hK2 and intact PSA were 4.0%, 6.6%, 6.0%, 9.2% and 9.5%, respectively. The measurements taken several days apart varied more than those taken on the same day, although the variation between both time points was not large. The intra-individual variation for all the kallikrein-like markers studied was relatively small, especially for samples drawn the same day. Few cases are reclassified between the time points. This indicates the high short-term biological and technical reproducibility of the tests in clinical use.

Qualitative detection of the Marburg I alloenzyme of factor VII-activating protease by an immunoassay and its comparison to PCR testing

The Marburg I (MRI) single nucleotide polymorphism (SNP) of the factor VII-activating protease (FSAP) gene has been associated with thrombophilia, thromboembolism, atherosclerosis, and the incidence and progression of carotid stenosis. At present, MRI SNP testing is mainly performed using costly nucleic acid analysis. The ratio between FSAP activity and antigen concentrations in citrated plasma has been used to assess the FSAP genotype. This article describes the development of a prototype ELISA for the detection of the MRI FSAP alloenzyme, and its correlation to FSAP genotypes to assess whether a positive MRI FSAP ELISA result may be used as a surrogate marker for the presence of the MRI SNP. ELISA results were correlated with FSAP genotypes from 523 blood donors measured using PCR. Diagnostic sensitivity and specificity of the assay for determination of the genotype were 100% (95% confidence interval [CI]: 93.36-100) and 99.79% (95% CI: 98.80-99.96), respectively. Maximum run-to-run, within-run, and total coefficients of variation were 7.8%, 7.9%, and 9.9%, respectively. No cross-reactivities with homologues of the MRI FSAP alloenzyme were observed. Test performance was not affected by typical interfering compounds. The data demonstrate that an immunoassay applying antibodies specific to the MRI FSAP alloenzyme can provide sufficiently accurate detection of the MRI SNP. This will significantly simplify MRI FSAP testing, particularly in large cohorts.

An on-line solid phase extraction procedure for the routine quantification of urinary methylmalonic acid by liquid chromatography-tandem mass spectrometry

The goal of this study was to develop and to validate an improved isotope-dilution-liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of methylmalonic acid (MMA) in urine. A previously described sample preparation protocol requires two solvent extraction steps, including evaporation. The first extraction is to extract the analyte from the sample, and second occurs following derivatization of the extract. In the method described here, the second evaporation step was substituted by on-line solid phase extraction employing column-switching and a permanent co-polymer based extraction cartridge. A standard validation protocol was applied to investigate the performance of the method. The method was found to be linear in the clinically relevant range of concentrations (6-100 μmol/L). Total coefficients of variation were below 10% and inaccuracy was <10% for quality control samples at three concentrations. By omitting one evaporation step, the semi-automated method described in this article enables for more convenient work-flow in the quantification of urinary MMA compared to the previous protocol. This is of relevance for MMA measurement in the routine clinical laboratory setting. Validation demonstrated acceptable analytical performance.

Determination of cerebrospinal fluid cystatin C on Architect ci8200

Background Human cystatin C is a cysteine protease inhibitor produced by all nucleated cells in the body and the protein is present in all body fluids. The concentration in cerebrospinal fluid (CSF) is considerably higher than in plasma. Cystatin C levels seem to influence the development of Alzheimer disease (AD) and low levels in the brain are associated with an increased risk for AD. The aim of this study was to develop a high throughput assay for the quantification of cystatin C in CSF. Methods Antigen excess, imprecision, interference, linearity, recovery, sample stability and reference values were evaluated on Architect ci8200 (Abbott Laboratories, Abbott Park, IL, USA). Results The assay had an antigen-excess limit at 23 mg/L and was linear over the range of 0.84 to 8.33 mg/L. Results > 8.33 mg/L were automatically rerun in a higher dilution. Within-run coefficient of variation (CV) was 1.71, 1.10 and 0.79%, between day CV was 1.71, 0.39 and 1.45%, between-run CV was 0.58, 0.66 and 0.48%, and total CV was 2.49, 1.34 and 1.72% at cystatin C concentrations of 1.39, 3.17 and 6.28 mg/L, respectively. The recovery was 97–102%. No interference at a 7.5% deviation level was observed for 8.5 g/L of hemoglobin or 800 mg/L (1368 μmol/L) of bilirubin. Reference values for cystatin C in cerebrospinal fluid obtained with this method were 2.42–14.33 mg/L.

Determinants of the variability of heart rate measures during a competitive period in young soccer players

Measurements of exercise heart rate (HR(ex)), HR recovery (HRR) and HR variability (HRV) are used as indices of training status. However, the day-to-day variability of these indices throughout a competitive soccer period is unknown. On 14 occasions during a 3-week competition camp, 18 under 15 (U15) and 15 under 17 (U17) years soccer players performed a 5-min submaximal run, followed by a seated 5-min recovery period. HR(ex) was determined during the last 30 s of exercise, while HRR and HRV were measured during the first and last 3 min of the post-exercise recovery period, respectively. U15 players displayed greater HR(ex) (P = 0.02) and HRR (P = 0.004) compared with the U17 players, but there was no difference in HRV (P = 0.74). The mean coefficient of variation (CV) for HR(ex) was lower than that for HRV [3.4 (90% CL, 3.1, 3.7) vs. 10.7 (9.6, 11.9)%, P < 0.001]; both were lower than that for HRR [13.3 (12.2, 14.3)%, P < 0.01]. In contrast to HR(ex) and HRR, the CV for HRV was correlated to maximal aerobic speed (r = -0.52, P = 0.002). There was no correlation between total activity time (training sessions + matches) and CV of any of the quantified variables. The variability of each of these measures and player fitness levels should be considered when interpreting changes in training status.

Emergence of Bluetongue Serotypes in Europe, Part 1: Description and Validation of Four Real-Time RT-PCR Assays for the Serotyping of Bluetongue Viruses BTV-1, BTV-6, BTV-8 and BTV-11

The control of bluetongue virus (BTV) in Central-Western Europe is greatly complicated by the coexistence of several BTV serotypes. Rapid, sensitive and specific assays are therefore needed to correctly identify the currently circulating BTV serotypes in field samples. In the present study, four serotype-specific real-time RT-PCR assays (RT-qPCR) are described for the detection of the BTV-1, BTV-6, BTV-8 and BTV-11 serotypes. The analytical sensitivity of the BTV-1/S2, BTV-6/S2, BTV-8/S2 and BTV-11/S2 serotype-specific RT-qPCR assays is comparable to the earlier described serogroup-specific pan-BTV/S5 RT-qPCR assay. In silico and in vitro analyses indicated that none of the assays cross-react with viruses which are symptomatically or genetically related to BTV and only detect the intended BTV serotypes. All assays exhibited a linear range of at least 0.05-3.80 log(10) TCID(50) ml(-1) and a PCR-efficiency approaching the ideal amplification factor of two per PCR cycle. Both intra- and inter-run variations were found to be low with a total coefficient of variation of 1-2% for clear positive samples and <10% for very weak positive samples. Finally, the performance of the described assays was compared with commercially available kits for the detection of BTV-1, BTV-6 and BTV-8. Three in-house assays gave exactly the same diagnostic result (positive/negative) as the commercial assays and can thus be used interchangeably. Together with the earlier described serogroup-specific pan-BTV/S5, the serotype-specific RT-qPCR assays form a flexible and properly validated set of tools to detect and differentiate the BTV serotypes currently circulating in Central-Western Europe.

Establishment of electrochemiluminescence immunoassay to detect proinsulin levels in human serum

Objective To establish a electrochemilumineecenee immunoaasay (ECLIA) for detecting proinsulin(PI) levels in human serum. Methods PI ECLIA kit contains biotinylated monoclonal insulin-specificantibody, ruthenium labeled monoclonal C-peptide specific antibody, and streptavidin-coated microparticlea. Its method performance, such as sensitivity, specific, accuracy, was evaluated. Then, the levels of PI in clinical samples were detected by ECLIA, including 44 type 2 diabetes and 60 healthy volunteers. Results Average recovery of ECLIA was 103. 8% with a lower detection limit of 0.38 pmol/L and biologic detection limit between 0.25 pmol/L and 0.5 pmol/L, and the function sensitivity was 0.5 pmol/L. The human insulin and human C-peptide did not cross-react at 500 μU/ml recovery of ECLIA was 103.8% with a lower detection limit of 0.38 pmol/L and biologic detection limit between 0.25 pmol/L and 0.5 pmol/L, and the function sensitivity was 0.5 pmoL/L. The human insulin and human C-peptide did not cross-react at 500 μU/ml and 450 ng/ml respectively. The total coefficient of variation of the ECLIA were leas than 5.0%, and the analytical measure range (AMR) was between 2.22 and 169.68 pmol/L. There was a good correlation (r=0.967,P=0.000) between ECLIA and radio immunoassay (RIA). The 95% reference value for human proinsulin was 1.07-15.54 pmol/L. The plasma levels of PI in type 2 diabetes were significantly higher than those in control healthy group[6.72(3.61-11.92) pmol/L vs 5.72(2.65-8.74) pmol/L, Z=-2.023, P<0.05]. Conclusions The monoclonal-based ECLIA is a sensitive, specific, and rapid method and no radiocontamination. It can be used to detect hanum serum proinsulin in type 2 diabetes. Key words: Proinsulin; Electrochemistry; Luminescence; Immunoassay

Analytical and clinical evaluation of a novel high-sensitivity assay for measurement of soluble ST2 in human plasma — The Presage™ ST2 assay

Background The protein ST2 is a member of the interleukin-1 receptor family. Blood concentrations of the soluble isoform of ST2 (sST2) are increased in inflammatory diseases and in heart disease and are considered a prognostic marker in both. The aim of this study was the analytical and clinical evaluation of the novel Presage™ ST2 assay for the determination of sST2 in human plasma. Methods We evaluated precision and linearity of the assay, analyte stability, and biological variability, determined reference values, performed a method comparison with an established ELISA, and quantified sST2 concentrations in various diseases. Results Within-run and total coefficients of variation were < 2.5% and < 4.0%. The method was linear across the whole measurement range of the assay. The analyte was stable for 48 h at room temperature, for 7 days at 4 °C, and for at least 2 months at − 20 °C and − 80 °C. The reference change value for healthy individuals was 30%. Age-independent reference values were 3–28 U/mL in males, and 2–16 U/mL in females. The method comparison revealed a high proportional bias. sST2 plasma concentrations were increased modestly in heart failure and moderately in pneumonia and chronic obstructive pulmonary disease. Patients with sepsis exhibited highly elevated sST2 values. In patients with chronic renal disease, however, there was no difference compared to healthy individuals. Conclusion The Presage™ ST2 assay meets the needs of quality specifications of laboratory medicine. The results of the clinical assay evaluation are novel with respect to sST2 in various diseases and should initiate further studies.

Hemoglobin A1c Point-of-Care Assays; A New World with a Lot of Consequences!

Point-of-care instruments for the measurement of hemoglobin A1c (HbA1c) may improve the glycemic control of people with diabetes by providing a rapid result if the performance of the instruments used is acceptable. A 0.5% HbA1c difference between successive results is considered a clinically relevant change. With this in mind, the In2it from Bio-Rad and the DCA Vantage from Siemens were evaluated according to Clinical and Laboratory Standards Institute (CLSI) protocols. The CLSI protocols EP-5 and EP-9 were applied to investigate precision, accuracy, and bias. The bias was compared with three certified secondary reference measurement procedures. Differences between capillary and venous blood were investigated by an end-user group consisting of nurse practitioners at a diabetes care center. At HbA1c levels of 5.1 and 11.2%, total coefficients of variation (CV) for the In2it were 4.9 and 3.3%, respectively, and for the DCA Vantage were 1.7 to 1.8% and 3.7 to 5.5% depending on the lot number of the cartridges. Method comparisons showed significant lot number-dependent results for the In2it and the DCA Vantage compared with the three reference methods. No overall difference was observed between capillary and venous blood for both methods. Performance results of the In2it and the DCA Vantage showed variable and lot number-dependent results. To maintain the interlaboratory CV of 5% for HbA1c, the Clinical Laboratory Improvement Amendments rules for waived point-of-care instruments should be revised. An obligation for participating in external quality schemes and taking adequate action should be considered for POC instruments that perform poorly.

Emergence des sérotypes de fièvre catarrhale ovine en Europe. Partie I - Description et validation de quatre tests diagnostiques par RT-PCR en temps réel

The control of bluetongue virus (BTV) in Central Western Europe is greatly complicated by the coexistence of several BTV serotypes. Rapid, sensitive and specific assays are there­fore needed to identify correctly the currently circulating BTV serotypes in field samples. In the present study, four serotype-specific real-time reverse transcription - polymerase chain reac­tion (RT-qPCR) assays are described for the detection of BTV serotype 1 (BTV-1), 6, 8 and 11. The analytical sensitivity of BTV-1/S2, BTV-6/S2, BTV-8/S2 and BTV-11/S2 serotype-specific RT-qPCR assays was comparable to the earlier described sero­group-specific pan-BTV/S5 RT-qPCR assay. In silico and in vitro analyses indicated that none of the assays cross-reacted with viruses which were symptomatically or genetically related to BTV, and only detected the intended BTV serotypes. All assays exhibited a linear range of at least 0.05–3.80 log10 TCID50 mL) and a PCR-efficiency approaching the ideal amplification factor of two per PCR cycle. Both intra- and inter-run variations were found to be low with a total coefficient of variation of 1–2% for clear positive samples and &lt; 10% for very weak positive samples. Finally, the performance of the described assays was compared with commercially available kits for the detection of BTV-1, 6 and 8. Three in-house assays gave exactly the same diagnostic result (positive/negative) as the commercial assays and can thus be used interchangeably. Together with the earlier described serogroup-specific pan-BTV/S5, the serotype-specific RT-qPCR assays form a flexible and properly validated set of tools to detect and differentiate the BTV serotypes currently circulating in Central Western Europe.

Therapeutic drug monitoring and drugs of abuse testing on the cobas® 6000 analyzer series: analytical performance under routine-like conditions

The analytical performance of the clinical chemistry module c 501 (cobas 6000 analyzer series) was evaluated for therapeutic drug monitoring and drugs of abuse testing using a spectrum of representative assays. Particular attention was paid to potential interactions between reagents using a simulated routine workload. Within-run and total imprecision were assessed using a selection of representative reagents. Deviation from a consensus mean was tested using samples from a proficiency testing scheme. Method comparison using routine samples was carried out against the MODULAR ANALYTICS SWA and COBAS INTEGRA 800 analysis systems. Total coefficients of variation (CV) ranged from 1.9% to 7.8% for individual drugs, and from 3.2% to 8.6% for drugs of abuse testing. Results from proficiency test samples were between 81% and 125% of the consensus mean for therapeutic drugs. Method comparisons (Passing-Bablok regression) showed overall good comparability to MODULAR ANALYTICS SWA and COBAS INTEGRA 800 systems, with slopes from 0.93 to 1.17 and correlation coefficients r > 0.98. Imprecision in a simulated routine run was tested using a total of 42 methods (10 therapeutic drug monitoring, 9 drugs of abuse testing, 3 enzymes, 12 substrates, 8 specific protein assays). Imprecision in the reference batch run ranged from 0.7% to 5.0% CV for therapeutic drug monitoring assays, except for digoxin (DIG) (7.3%), and from 0.9% to 7.7% for drugs of abuse testing. The CVs of general clinical chemistry and specific protein tests were within the expected limits of 2% and 4%. CV changes in the simulated routine run were within the expected limits for most assays. Negative DeltaCVs (> or = 2%) for DIG, digitoxin (DIGIT), cannabinoids (THC), and phencyclidine (PCP) may indicate improved performance when running these assays in a simulated routine operation. A positive DeltaCV (> or = 3%) was found for amphetamines (AMPHs). In conclusion, the cobas c 501 module seems to be well-suited for routine use as consolidated workstation. Except for a potential interaction with AMPH, as indicated by the positive DeltaCV, no significant interferences from different reagents could be observed during this study.

Performance Characteristics of Six Homocysteine Assays

Elevated concentrations of homocysteine (Hcy) are associated with a range of disorders. Linearity, imprecision, interference, method comparison, and accuracy were evaluated on the ADVIA Centaur (Siemens Healthcare Diagnostics, Deerfield, IL), ARCHITECT i2000SR (Abbott Diagnostics, Abbott Park, IL), AxSYM (Abbott Diagnostics), and IMMULITE 2000 (Siemens Healthcare Diagnostics) methods and analyzers and the Catch (Equal Diagnostics, Exton, PA) and Diazyme (Diazyme Laboratories, San Diego, CA) methods, both on the Modular P analyzer (Roche Diagnostics, Indianapolis, IN). All methods were linear with maximum deviations from target recoveries of less than 10%. Total coefficients of variation ranged from 1.7% to 9.4%. The effects of hemolysis, icterus, and lipemia were assessed. Method comparisons were performed using high-performance liquid chromatography as the comparison method. Correlation coefficients were 0.95 to 0.99. Bland-Altman plots demonstrated percentage bias of -29.3% (IMMULITE) to 7.2% (Centaur). Accuracy using the National Institute of Standards and Technology Standard Reference Material 1955 showed varying results with only 1 method within the certified range for all 3 levels. All methods demonstrated acceptable performance except the IMMULITE, which is less precise and accurate. Standardization of most methods seems acceptable, although continuing efforts are warranted.

Comments on the Interpretation of Results from the Restrained Ring Test

Abstract The restrained ring test has recently been standardized (ASTM C1581-04) as a test method to assess the restrained shrinkage cracking susceptibility of a concrete mixture. Unfortunately, there is currently a lack of published information regarding the repeatability of the test. This paper quantifies the variability of the ASTM C1581-04 ring test procedure. To determine the repeatability of the standard test, 24 restrained ring specimens were cast from the same mortar mixture and stored in carefully controlled environmental conditions. Results from the restrained ring test were assessed using the same mixture proportions. The results indicate that a standard deviation of approximately 6 με can be expected in the strain measurements from the same mixture and 8 με from different batches of the same mixture. The total coefficient of variation observed was less than 12 % (within single mixture and between different batches). A probabilistic approach was used to describe the variability associated with the age of cracking and to provide a tool that allows cracking prediction, especially when only a fraction of the ring specimens crack.

Uncertainty of sweat chloride testing: does the right hand know what the left hand is doing?

Although analytical variation in sweat electrolyte testing can be easily estimated, there is limited data on total variation. This study aims to evaluate the total variation of the sweat test by measuring the difference between sweat electrolyte values in specimens obtained simultaneously from two sites. Chloride is recommended in published guidelines as the only discriminant for the diagnosis of cystic fibrosis, and sodium may be measured as a guide to the adequacy of collection and analysis. Both are reported here. Sweat was collected in patients by the Gibson Cooke method from two sites simultaneously. Coefficient of variation in this laboratory is 4.1 and 5% for chloride and sodium, respectively. 295 patients had sufficient sweat collected from both sites for analysis. The values for chloride and sodium were compared between the two sites. The total coefficient of variation (CV(t)) calculated for the whole group between the two sites was 20.2% for chloride and 16.9% for sodium, and the standard deviations 4.3 mmol/L and 4.8 mmol/L, respectively. In patients with intermediate chloride concentrations; in different age groups; and when those tests with a difference between sodium and chloride concentration of more than 15 were excluded, minimal differences in these figures were observed. Use of strictly defined cut-off points to discriminate between normal and intermediate electrolyte values, and between intermediate and raised electrolyte values, does not reflect the variation in sweat electrolyte content found within an individual patient. This has important implications for reporting.

Development and Clinical Application of an LC-MS-MS Method for Mescaline in Urine

Mescaline (3,4,5-trimethoxyphenylethylamine) is an hallucinogenic psychoactive substance present in several species of cacti. Mescaline has a documented use dating back 5700 years. In more recent years, the interest in hallucinogenic designer drugs such as ecstasy has also triggered interest in the naturally occurring mescaline. This study was undertaken to develop a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the screening and confirmation of mescaline in human urine samples and to apply this method to routine testing in patient samples. For the screening procedure, chromatographic separation was achieved on a 5-microm HyPURITY C(18) column, using a methanol gradient in ammonium acetate buffer. The MS-MS analysis was performed using selected reaction monitoring; the transitions monitored were m/z 212.3 --> m/z 180.3 for mescaline and m/z 221.3 --> m/z 186.3 for the deuterated internal standard (mescaline-d(9)). The detection limit for mescaline in urine matrix was 3-5 microg/L, the upper limit of quantification was 10,000 microg/L, and the total coefficient of variation for spiked samples containing 10 to 1025 microg/L was < 8.5%. The confirmation procedure included a sample clean-up by solid-phase extraction on a C(18) cartridge, and one extra transition for mescaline (m/z 212.3 --> m/z 195.2) was monitored. The LC-MS-MS method was found to be sensitive and specific for the routine detection of mescaline in urine. Among 462 urine samples collected from young people with alcohol or drug problems, 32% were positive for illicit drugs, but none for mescaline.

Tests for the measurement of factor VII-activating protease (FSAP) activity and antigen levels in citrated plasma, their correlation to PCR testing, and utility for the detection of the Marburg I-polymorphism of FSAP

The single nucleotide Marburg I (MRI) polymorphism of the factor VII-activating protease (FSAP) gene, the prourokinase-activating activity of FSAP, and antigen levels of FSAP in plasma have been associated with incidence and progression of carotid stenosis and venous thromboembolism. However, more information on the extent of these associations, potential further ones, and respective clinical utilities remain to be determined. At present, testing is performed mainly by PCR assays based on probes or SYBR Green I. Some studies include testing for antigen levels of total FSAP and its ability to activate prourokinase. To test large cohorts, it is beneficial to rely on assays that are cost-effective, reliable, easy to use, rapid to perform, and that may eventually be automated. In addition, it appears advantageous to use functional tests or tests that determine antigen levels as they may relate more closely to the phenotype than the genotype does. Tests for the measurements of antigen levels of FSAP and its prourokinase-activating activity were improved and performance characteristics assessed. To determine the FSAP genotypes, an amplification created restriction site (ACRS) PCR test was developed. Key performance characteristics of the FSAP activity and antigen tests were as follows: measuring range: 350-1400 mPEU/mL and 1.8-120 ng/mL, total coefficients of variation (CV): 5%-20% and 5%-14%, within-run CV: 4%-11% and 2.3%-12%, and run-to-run CV: 2%-17% and 4.3%-8.3%, respectively. The ratio of the activity and antigen level of FSAP correctly identified the FSAP genotypes of 126 samples tested. The ACRS PCR test is useful for laboratories that do not have the equipment to perform probe or SYBR Green I based real-time PCR. Furthermore, the tests developed for the determination of FSAP activity and antigen levels are convenient for determining clinical correlations, even for large population studies. The ratio of activity and antigen level of FSAP appears to be a promising and efficient alternative to molecular diagnostic techniques to detect the MRI polymorphism of FSAP.

Analytical performance evaluation of the Cobas 6000 analyzer – special emphasis on trueness verification

Consolidation of analyzers is an emerging issue in clinical chemistry. We evaluated the analytical performance of the Cobas 6,000 analyzer (Roche Diagnostics), which is considered a candidate for replacement of current Hitachi 917 analyzers and for consolidation of chemistry and immunochemistry. The precision, accuracy, linearity and correlation with current field methods were evaluated according to Clinical and Laboratory Standards Institute protocols EP5, EP9 and EP10. A total of 31 routine chemistry assays and 18 immunoassays were studied. Accuracy and linearity were verified for 24 chemistry parameters using value-assigned trueness controls from the Dutch External Quality Assessment Scheme organizers. In addition, traceability to methods endorsed by the Joint Committee of Traceability in Laboratory Medicine was examined. All analytes met allowable precision criteria, apart from the low level for sodium and folate. Total coefficients of variation ranged between 0.6% and 4.4% for routine chemistry and between 0.8% and 5.8% for immunochemistry, apart from folate (12% at the low end). The correlation coefficients for comparison to current field methods were >0.975, except for magnesium and for six out of 18 immunochemistries. Recovery experiments indicated high recovery for most of the 24 routine chemistry assays. Considering the excellent precision data and the result equivalence for most assays, it can be concluded that Cobas 6,000 accommodates robust chemistry and immunochemistry, and has good potential for workstation consolidation in medium-sized laboratories.