Run Imprecision Research Articles

B-024 An Evaluation of the Analytical Performance of the New Beckman Coulter DxC 500 AU Clinical Chemistry System

Abstract Background The Beckman Coulter DxC 500 AU clinical chemistry analyzer* is the latest system from Beckman Coulter. It is a fully automated, random-access analyzer, designed for low to medium throughput laboratories, with a throughput of 800 tests/hour including ion selective electrodes. The purpose of this study was to evaluate the analytical performance of the new DxC 500 AU and to compare the performance against the current AU480 and DxC 700 AU analyzers. Methods To assess the performance of the DxC 500 AU, Beckman Coulter assays were selected for evaluation that covered a range of sample types and assay methodologies. Precision was assessed using pooled human samples over 20 days following CLSI guideline EP05-A3. The linear range was assessed following CLSI EP06-A. The DxC 500 AU was compared to the AU480 and DxC 700 AU analyzers using a minimum of 100 samples spanning the dynamic range based on CLSI guideline EP09c-ED3. Results IgG Serum: OSR6x172 Estimates of repeatability and within laboratory precision were assessed following EP05-A3 at multiple critical analyte concentrations. The IgG serum assay exhibits total imprecision of < 6 % with a within run imprecision of ≤ 3.5 %. The IgG serum assay demonstrated acceptable linearity throughout the analytical measuring range of 75 to 3000 mg/dL following CLSI EP06-A. A method comparison study following CLSI EP09c-ED3 compared the IgG serum assay on the DxC 500 AU against the AU480 and DxC 700 AU. Serum patient samples (n > 100) measured across the analytical range were analyzed using Weighted Deming regression and yielded a slope of 1.046, an intercept of −20.2884 mg/dL and correlation coefficient R = 0.9979 against the DxC 700 AU and yielded a slope of 1.005, an intercept of −3.6578 mg/dL and correlation coefficient R = 0.9973 against the AU480. CRP Serum (Highly Sensitive): OSR6x99 Estimates of repeatability and within laboratory precision were assessed following EP05-A3 at multiple critical analyte concentrations. The CRP serum assay exhibits total imprecision of ≤10 %CV or ≤0.02 SD with a within run imprecision of ≤5 %CV or ≤0.02 SD. The CRP serum assay demonstrated acceptable linearity throughout the analytical measuring range of 0.2 to 80 mg/L following CLSI EP06-A. A method comparison study following CLSI EP09c-ED3 compared the CRP serum assay on the DxC 500 AU against the AU480 and DxC 700 AU. Serum patient samples (n > 100) measured across the analytical range were analyzed using Weighted Deming regression and yielded a slope of 0.990, an intercept of 0.0421 mg/dL and correlation coefficient R = 0.9997 against the DxC 700 AU and yielded a slope of 0.995, an intercept of −0.0008 mg/dL and correlation coefficient R = 0.9998 against the AU480. Conclusion The results of the study demonstrated excellent analytical performance of the new Beckman Coulter DxC 500 AU analyzer and confirms comparable performance to the AU480 and DxC 700 AU analyzers. *Product In development. Pending clearance by the United States Food and Drug Administration and achievement of CE compliance. Not currently available for in vitro diagnostic use.

Usklađenost POCT metode sa potpuno automatizovanom metodom za određivanje HbA1c

Previous research suggests that point-of-care (POCT) determination of glycated hemoglobin (HbA1c) is a diagnostic test that can be an adequate alternative to measuring HbA1c in the laboratory. The main goal of this study was to examine the analytical characteristics of the novel INCLIX POCT method for HbA1c determination in order to test its performance before introducing this method into routine use. HbA1c is measured in a duplicate in 44 EDTA blood samples parallel on INCLIX POCT device (Sugitech, Inc.) and using automated turbidimetric immunoinhibition test on Olympus AU400 (Beckman Coulter). The within run imprecision was 7.58%, between runs imprecision was 6.63% and 6.22%, and day-to-day imprecision was 8.80% and 7.51%. Total laboratory imprecision was in agreement with those stated by the manufacturer. A statistically significant Pearson correlation coefficient was calculated (r = 0.871, P < 0.01; linear R2 = 0.757). Using Deming regression analysis, the following equation was obtained: y = - 1.80 + 1.304x. Our results indicate statistically significant correlation, linear relationship, and a significant degree of compatibility between the two analyzed methods. However, the negative bias of the HbA1c values determined on the POCT analyzer compared to the Olympus AU400 was confirmed, highlighting the need to standardize the INCLIX method.

Trial design for assessing analytical and clinical performance of high-sensitivity cardiac troponin I assays in the United States: The HIGH-US study.

BackgroundHigh-sensitivity cardiac troponin I (hs-cTnI) assays have been developed that quantify lower cTnI concentrations with better precision versus earlier generation assays. hs-cTnI assays allow improved clinical utility for diagnosis and risk stratification in patients presenting to the emergency department with suspected acute myocardial infarction. We describe the High-Sensitivity Cardiac Troponin I Assays in the United States (HIGH-US) study design used to conduct studies for characterizing the analytical and clinical performance of hs-cTnI assays, as required by the US Food and Drug Administration for a 510(k) clearance application. This study was non-interventional and therefore it was not registered at clinicaltrials.gov.MethodsWe conducted analytic studies utilizing Clinical and Laboratory Standards Institute guidance that included limit of blank, limit of detection, limit of quantitation, linearity, within-run and between run imprecision and reproducibility as well as potential interferences and high dose hook effect. A sample set collected from healthy females and males was used to determine the overall and sex-specific cTnI 99th percentile upper reference limits (URL). The total coefficient of variation at the female 99th percentile URL and a universally available American Association for Clinical Chemistry sample set (AACC Universal Sample Bank) from healthy females and males was used to examine high-sensitivity (hs) performance of the cTnI assays. Clinical diagnosis of enrolled subjects was adjudicated by expert cardiologists and emergency medicine physicians. Assessment of temporal diagnostic accuracy including sensitivity, specificity, positive predictive value, and negative predictive value were determined at presentation and collection times thereafter. The prognostic performance at one-year after presentation to the emergency department was also performed. This design is appropriate to describe analytical characterization and clinical performance, and allows for acute myocardial infarction diagnosis and risk assessment.

Capillarys 2 Flex Piercing: Analytical performance assessment according to CLSI protocols for HbA1c quantification.

HbA1c is used for monitoring diabetic balance. In this paper we report an assessment of the analytical performances of Capillarys 2 Flex Piercing (C2FP) for HbA1c measurement using CE (Capillary Electrophoresis). CLSI (Clinical and Laboratory Standard Institute) protocols are used for the evaluation of apparatus performances: precision, linearity, method comparison, trueness and common interferences. HbA1c CVs average in intra-assay was 1.6% between run imprecision CV ranged from 0.1 to 1.8%. The linearity was demonstrated between 4.7 and 15.0%. The comparison study revealed that Bland Altman plot mean difference was equal to -0.03 (CI 95% (-0.05 to -0.0003)) and Passing-Bablok regression intercept was -0.05, CI95%(-0.13 - -0.05); slope: 1.00, CI95%[1.00-1.01]. A strong correlation (r>0.99) was proved. No significant effects of hemoglobin variants were seen with CE on HbA1c measurement. No problem related to sample-to-sample carry over was noted. No interferences of LA1c and cHb were observed. CE allowed quantification of HbA1c even at low level of total hemoglobin (40 g/L) in contrast to HPLC. Furthermore, this analyzer offered the opportunity of quantifying the HbA2 simultaneously with HbA1c . This evaluation showed that C2FP is a convenient system for the control of diabetes and the detection of hemoglobinopathies.

The Roche Total Mycophenolic Acid® assay: An application protocol for the ABX Pentra 400 analyzer and comparison with LC–MS in children with idiopathic nephrotic syndrome

BackgroundFor TDM of mycophenolate acid (MPA), the Roche Total Mycophenolic Acid® assay based on the inhibition of recombinant inosine monophosphate dehydrogenase (IMPDH) has been shown to be a simple and reliable alternative to chromatographic methods. We have adapted this assay on the ABX Pentra 400 analyzer (HORIBA). ObjectiveTo investigate the analytical performances of the Roche Total Mycophenolic Acid® assay on the ABX Pentra 400 and to compare it to an LC-MS method using samples from children with nephrotic syndrome treated with mycophenolate mofetil (MMF). Material and methodsConfiguration of the open-channel on the ABX Pentra 400 was based on the Roche MPA assay package insert. Precision was determined as described in the CLSI protocol EP5-A2. Comparison with the LC-MS method was performed using 356 plasma samples from 42 children with nephrotic syndrome (8h pharmacokinetic profiles). ResultsThe enzymatic assay demonstrated high precision. The %CV for Within Run Imprecision ranged from 5.5% at 1.2mg/L to 1.5% at 14.1mg/L and Total Imprecision ranged from 9.3% to 2.5%. The method comparison with plasma samples from children yielded overall a good correlation and a good agreement between both methods. The Passing Bablok regression analysis showed the following results: [Roche MPA assay]=1.058 [MPA LC-MS] −0.06; rho=0.996. ConclusionThe Roche Total Mycophenolic Acid® assay is adaptable to the ABX Pentra 400 analyzer, and demonstrates accurate and precise measurement of MPA in plasma obtained from children with nephrotic syndrome.

Analytical and clinical performance of a new molecular assay for Epstein-Barr virus DNA quantitation

Quantitation of EBV DNA has been shown to be a useful tool to identify and monitor patients with immunosuppression and high risk for EBV-associated disease. In this study, the analytical and clinical performance of the new Realquality RS-EBV Kit (AB Analitica, Padova, Italy) was investigated. The clinical performance was compared to that of the EBV R-gene (bioMerieux, Varilhes, France) assay. When the accuracy of the new assay was tested, all results except of one were found to be within ±0.5log10 unit of the expected panel results. Determination of linearity showed a quasilinear curve, the between day imprecision ranged from 18% to 88% and the within run imprecision from 16% to 53%. When 96 clinical EDTA whole blood samples were tested, 77 concordant and 19 discordant results were obtained. When the results for the 69 samples quantifiable with both assays were compared, the new assay revealed a mean 0.31log10 unit higher measurement. The new assay proved to be suitable for the detection and quantitation of EBV DNA in EDTA whole blood in the routine diagnostic laboratory. The variation between quantitative results obtained by the assays used in this study reinforces the use of calibrators traceable to the existing international WHO standard making different assays better comparable.

Evaluation of the Sysmex XT‐4000i for the automated body fluid analysis

SummaryIntroductionBody fluid (BF) analysis is traditionally performed using microscopy, which is time‐consuming and labor‐intensive and has wide interobserver variability. Automation has been suggested to improve these elements.MethodsBody fluid mode of the Sysmex XT‐4000i was used to analyze 51 cerebrospinal fluid (CSF) and 81 BF samples. white blood cell (WBC) count (cell/μL), WBC differential (MN% and PMN%), and turnaround time (TAT) were compared with manually performed microscopic counting.ResultsWithin‐run imprecision of BF (CV%) was 2.8–19.3. Functional sensitivity was 18 cell/μL (CV% 19.3). The accuracy of WBC count was moderate with CSF samples (rs = 0.55) and good with BF samples (rs = 0.95). The TAT of BF and CSF samples decreased with samples without high HF‐BF count when analyzed with XT‐4000i.ConclusionThe BF mode of Sysmex XT‐4000i was found to be accurate and precise with samples where the WBC count was >200 cell/μL. Our results indicate that Sysmex XT‐4000i can help to reduce TAT of BF sample analysis and decrease work effort in a laboratory. However, automatically performed cell counting cannot replace microscopic analysis in samples with abnormal findings.

Comparison of three commercial calibrators for alpha-tocopherol using liquid chromatography–tandem mass spectrometry

ObjectivesAlpha-tocopherol is the predominant form of vitamin E in plasma and is routinely measured to assess vitamin E status. Agreement between vitamin E assays is essential to provide consistent result interpretation. Lack of agreement among calibrators is potentially a significant obstacle to method harmonization. The aim of this study was to ascertain the agreement between commercial secondary calibrators for analysis of serum/plasma alpha-tocopherol using two methods of isotope dilution liquid chromatography–tandem mass spectrometry (LC–MSMS). Design and methodsThree commercial single level calibrators (Bio-Rad Laboratories, Chromsystems Diagnostics and RECIPE) were prepared in quintuplicate in conjunction with an in-house calibrator set for alpha-tocopherol. Samples were analyzed by two methods using an Agilent-6490 LC–MSMS. ResultsThe linearity of both methods ranged from at least 4 to 70μmol/L. The expanded within run imprecision of both LC–MSMS methods was ±6% at 95.4% confidence interval across the assay range. The percentage observed difference for the commercial calibrators was calculated from the observed mean against the given value of the calibrator: Bio-Rad (bias +1.4% in both methods); Chromsystems (bias +5.4% [first method] and +5.0% [second method]); and RECIPE (bias −8.9% [first method] and −9.8% [second method]). ConclusionsResults demonstrate an overall discordance between the commercial calibrators that is greater than the assay uncertainty. It is observed that the greatest deviation of the three calibrators is traceable to a different standard reference material. This lack of harmonization means that results from different laboratories may not be comparable.

Analytical validation of therapeutic drug monitoring (TDM) on AxSYM Abbott analyzer

Careful monitoring of drug concentration (therapeutic drug monitoring, TDM) is essential for a large number of drugs. The aim of this study was to evaluate analytical performance of Abbott AxSYM analyzer for therapeutic drug monitoring of theophylline, carbamazepine, phenobarbital and valproic acid. For the purpose of analytical validation following parameters were determined for all analytes: inaccuracy (bias), within-run and between-run imprecision and measurement uncertainty. Additionally, concentration of valproic acid was compared with the previously used analytical system (TDx FLx Abbott analyzer) for 30 patients’ samples. Inaccuracy results (bias) were as follows: for theophylline from –3.66% to –5.84% ; for carbamazepine –0.46% to 1.00% ; for phenobarbital –1.83% to –8.08% and for valproic acid from – 1.01% to –5.65%. The highest coefficient of variation (CV) for within run imprecision was observed for phenobarbital (7.07%) and the lowest for theophylline (2.71%). The highest CV for between run imprecision was observed for carbamazepine (4.73%) and the lowest for theophylline (2.94%). The highest measurement uncertainty was observed for phenobarbital assay (21.7%) and the lowest for carbamazepine (10.7%). Passing-Bablock regression analysis of valproic acid comparison on two analyzers showed statistically significant, but clinically insignificant deviation in slope of the regression equation (b = 1.121 ; 95% CI = 1.028– 1.197) ; however the Cusum linearity test proved that there was a linear relationship between two methods. In conclusion, analytical validation fulfilled all previously established criteria and could be implemented in a routine laboratory work.

Detection and quantitation of Epstein-Barr virus (EBV) DNA in EDTA whole blood samples using automated sample preparation and real time PCR

Detection and quantitation of Epstein-Barr virus (EBV) DNA in EDTA whole blood samples has gained significance in the routine diagnostic laboratory. In this study, the analytical and clinical performance of the artus EBV RG PCR kit in conjunction with automated sample preparation on the QIAsymphony SP instrument was evaluated. When the accuracy of the new test system was tested, all results were found to be within +/-0.5 log(10) unit of the expected panel results. Determination of linearity showed a quasilinear curve over 4 log units. The lower limit of detection was determined to be 391 EBV DNA copies/mL in EDTA whole blood. The between day imprecision ranged from 18% to 66%, and the within run imprecision ranged from 11% to 50%. When clinical samples were tested and the results compared with those obtained with the routinely used easyMAG sample preparation and EBV R-gene test system, 60 samples tested positive and 31 samples tested negative by both assays. Nineteen samples were found to be positive using the QIAsymphony sample preparation and artus EBV RG PCR test system only, and no samples tested positive with the routinely used test system only. The QIAsymphony sample preparation and artus EBV RG PCR test system is suitable for the detection and quantitation of EBV DNA in EDTA whole blood in the routine diagnostic laboratory.

Determination of N-Acetylcysteine and Main Endogenous Thiols in Human Plasma by HPLC with Ultraviolet Detection in the Form of Their S-Quinolinium Derivatives

A new, sensitive, repeatable, and robust high performance liquid chromatography assay for the determination of total N-acetylcysteine, cysteine, cysteinylglycine, glutathione, and homocysteine in human plasma has been developed. The thiol concentrations were measured using precolumn specific derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate, followed by high performance liquid chromatographic reversed phase separation with spectrophotometric detection. The oxidized and protein bound forms of thiols were converted into their reduced forms employing a reductive agent tris(2-carboxyethyl)phosphine. The chromatographic separation was accomplished in 12.5 min; the within run and between run imprecision were all less than 10%. The elution profile was as follows: 0–4 min, 11% B; 4–8 min, 11–30% B; 8–12 min, 30–11% B. The calibration graphs, obtained with the use of normal plasma spiked with growing amounts of analytes, were linear over the concentration ranges, covering most experimental and clinical cases (1–32 µM for homocysteine and glutathione, 1–320 µM for N-acetylcysteine and cysteine, and 1–64 µM for cysteinylglycine). For all analytes, recoveries between 91.2 and 108.6%, were observed.

Évaluation de deux méthodes de dosage de la concentration sanguine de lactate

We evaluated two automated methods for measuring blood levels of lactates with Integra 800 (Roche Diagnostics) and ABL 725 (Radiometer). Our evaluation had shown a within run imprecision of 1% (Intégra 800) and 2% (ABL 725) and a between-assay imprecision of 2% (Intégra 800) and 5% (ABL 725). The two methodologies appeared very well associated. The selective electrode remains expensive but it is very interesting because it saves a blood sample.

Beta-Methylamino-L-Alanine Analysis by Liquid Chromatography Tandem Mass Spectrometry with iTRAQ as the Derivative

Amino acid BMMA is produced by cyanobacteria and has been linked to the development of neurodegenerative diseases. We developed a method for quantitative analysis of BMAA in biological samples and plant extracts. The method is utilizing iTRAQ and LC-MS/MS detection using multiple reaction monitoring mode. The method uses 50 microL of sample and has a limit of quantitation of 300 ng mL(-1), within-run run imprecision below 1%. Using this method we analyzed human serum samples, human cerebrospinal fluid samples and extract of the cycad seed. No BMAA could be detected in the human samples. Content of BMAA in the seed was 50 mg kg(-1).

Performance Evaluation of a New Small-Volume Coagulation Monitor

The SmartCheck INR (Unipath, Bedford, England) is a point-of-care device for professional and patient self-monitoring of oral anticoagulant therapy. It measures the international normalized ratio (INR) and assesses test strip integrity, temperature, and sample quality. No significant differences were found in SmartCheck INR results from 3 different instruments or in 3 test-strip lots. Within run imprecision was 0.89% and 6.36% for low and high control samples, respectively (mean INR, 0.99 and 4.08, respectively). Comparability was assessed in 68 patients receiving warfarin by using PTHS Plus (Instrumentation Laboratory, Lexington, MA) and Innovin (Dade Behring, Marburg, Germany) thromboplastins. Good correlations were observed between the methods (r = 0.89 and r = 0.90, respectively) with no significant differences in means: SmartCheck INR, 2.82; Innovin, 2.75; PTHS Plus, 2.74. Clinical agreement with laboratory methods was 88% for Innovin and 97% for PTHS Plus. The SmartCheck INR was easy to use with no mechanical failures during the evaluation and a low test-strip failure rate of 4.7%. The SmartCheck INR provides accurate and reproducible results and is suitable for routine monitoring of oral anticoagulant therapy in the majority of patients.

Evaluation of an Immunoassay (Abbott-IMX Analyzer) Allowing Routine Determination of Sirolimus: Comparison With LC-MS Method

Background The use of the immunosuppressive agent sirolimus is increasing in renal transplantation but its monitoring often requires high-performance liquid chromatography (HPLC) with ultra-violet (UV) or tandem mass spectrometric (MS-MS) detection. The aim of this study was to compare a new microparticle enzyme immunoassay (MEIA, Microparticle Enzyme Immunoassay) on IMx Abbott Analyser with a liquid chromatography-mass spectometry (LC-MS) method. Method The accuracy of immunoassay analytical performance including within run and between run imprecision and linearity was tested. For comparison studies, sirolimus level was then determined with the two methods on 98 samples from 52 transplant patients. Results Total intra-assay and inter-assay variation coefficients were below 10% at the three levels tested, and the coefficient of linearity was r = 0.99. The values obtained were highly correlated with the LC-MS method (MEIA = 1.02LC-MS + 0.91; r 2 = 0.87). As a result, the immunoassay showed good performance, and clinical sample measurements were not affected by the method. The MEIA may be a useful alternative for routine monitoring of sirolimus.

Hypoxanthine, Uric Acid and Allantoin as Indicators of in Vivo Free Radical Reactions. Description of a HPLC Method and Human Brain Microdialysis Data

A practical one-step high performance liquid chromatography (HPLC) method was developed for the simultaneous determination of hypoxanthine, uric acid and allantoin in small (4 microL) microdialysis samples. The rationale for this work was the current interest in hypoxanthine as a marker for energy perturbation in hypoxia/ ischemia, in uric acid as an endogenous antioxidant, and in allantoin as a marker for in vivo formation of reactive oxygen species (ROS). The method is based on ion pairing reversed phase chromatography and UV detection. The coefficient of variance for within and between run imprecision was generally below 5%, somewhat higher for concentrations close to the detection limit (0.4-1.0 pmoles). Recoveries ranged from 89 to 102% and linearity was observed in the concentration range from 0.25 to 25.0 mmol/L. There was an excellent correlation between the present method and available reference methods. The method was applied to cerebral microdialysis samples from a patient with severe subarachnoid haemorrhage complicated by secondary ischemia leading to cerebral infarction. The secondary ischemia was associated with an increase of hypoxanthine followed by increasing allantoin and uric acid levels. We submit that this pattern of chemical changes indicates increased ROS formation produced by secondary ischemia. The HPLC method appears to be a useful tool for studying changes of hypoxanthine, uric acid and allantoin levels in microdialysis samples.

The Routine Determination of the Endogenous Thrombin Potential, First Results in Different Forms of Hyper- and Hypocoagulability

The area under the thrombin generation curve (the endogenous thrombin potential; ETP) has been proposed as a parameter for plasma-based hypercoagulability and to monitor anticoagulant treatment. We present an ETP assay for the routine laboratory using a centrifugal analyser. Throughput is 30 samples/h, within and between run imprecision is 4-5.6%. Suitable substrates were developed for the ranges of 10-500% and 2-100% of normal. Independent of tissue factor concentration (if > 4 pM), the normal value of the extrinsic ETP is 384.8 +/- 51.7 nM.min. The intrinsic ETP, triggered by ellagic acid, is 414 +/- 41 nM.min. The ETP is decreased to 15 and 35% of normal by oral anticoagulation (INR 2.5-4.0) and by heparin administration (APTT 1.5-2.5 x control). The ETP is increased in untreated subjects with congenital antithrombin deficiency and in women using oral contraceptives. In deep vein thrombosis (phlebographically confirmed), it is increased by 29.4% (extrinsic) and 53% (intrinsic). In (angiographically assessed) coronary artery disease the increase is by 10% and 17% respectively.

Evaluation of the Hitachi 717 analyser

The selective multitest Boehringer Mannheim Hitachi 717 analyser was evaluated according to the guidelines of the Comisión de Instrumentación de la Sociedad Española de Química Clinica and the European Committee for Clinical Laboratory Standards. The evaluation was performed in two steps: examination of the analytical units and evaluation in routine operation. The evaluation of the analytical units included a photometric study: the inaccuracy is acceptable for 340 and 405 nm; the imprecision ranges from 0.12 to 0.95% at 340 nm and from 0.30 to 0.73 at 405 nm, the linearity shows some dispersion at low absorbance for NADH at 340 nm, the drift is negligible, the imprecision of the pipette delivery system increases when the sample pipette operates with 3 μl, the reagent pipette imprecision is acceptable and the temperature control system is good. Under routine working conditions, seven determinations were studied: glucose, creatinine, iron, total protein, AST, ALP and calcium. The within-run imprecision (CV) ranged from 0.6% for total protein and AST to 6.9% for iron. The between run imprecision ranged from 2.4% for glucose to 9.7% for iron. Some contamination was found in the carry-over study. The relative inaccuracy is good for all the constituents assayed.