Reagent Lots Research Articles

A non-Aqueous Potentiometric Titration Method for Validation of Secnidazole from Pharmaceutical Dosages

A sensitive non-aqueous potentiometric titration method was developed for quantitative determination of secnidazole from pharmaceutical dosage form. The titration was carried out using standardized 0.1 N perchloric acid. The proposed method was found to be precise with % RSD 0.999) between 20% to 100% of 500 mg of drug substance weight. The percentage recovery of secnidazole in the optimized method was between 99.643 to 100.505%. The method is also found to be rugged when checked by different analysts and using different lots of reagents and different makes of titrators.

Validation of a Modified ANSR® for E. coli O157:H7 Method for Detection of E. coli O157:H7 in Select Foods.

Background: The ANSR method is based on isothermal nucleic acid amplification technology. The modifications to assay components improve sensitivity of the assay and robustness of the internal positive control. Objective: A Performance Tested MethodSM validation study was conducted to assess performance of a modified version of the ANSR® for Escherichia coli O157:H7 method. Methods: The validation study included inclusivity/exclusivity, matrix, robustness, accelerated stability, and independent laboratory testing. Results: In inclusivity testing of 55 strains of E. coli O157:H7 and E. coli O157:NM variants, all strains produced positive results. In exclusivity testing of 41 strains including E. coli of other serotypes and bacteria of closely related genera, all strains produced negative results. In matrix testing of beef trim, raw ground beef, spinach, and sprout-irrigation water, ANSR method performance was compared with that of the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook or the U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture procedures. Conclusions: all trials, ANSR method performance was not statistically different from that of the reference methods. Results of independent laboratory testing of ground beef corroborated those of internal testing. Introducing modest changes to three assay operating parameters did not materially affect ANSR method performance. Finally, accelerated stability testing results of three independently manufactured lots of ANSR reagents support a shelf-life of 1 year when stored at 2-8°C.

Analytical validation of a prognostic prostate cancer gene expression assay using formalin fixed paraffin embedded tissue

BackgroundThere is a clear need for assays that can predict the risk of metastatic prostate cancer following curative procedures. Importantly these assays must be analytically robust in order to provide quality data for important clinical decisions. DNA microarray based gene expression assays measure several analytes simultaneously and can present specific challenges to analytical validation. This study describes the analytical validation of one such assay designed to predict metastatic recurrence in prostate cancer using primary formalin fixed paraffin embedded tumour material.MethodsAccuracy was evaluated with a method comparison study between the assay development platform (Prostate Disease Specific Array) and an alternative platform (Xcel™ microarray) using 50 formalin-fixed, paraffin-embedded prostate cancer patient samples. An additional 70 samples were used to establish the assay reportable range. Determination of assay precision and sensitivity was performed on multiple technical replicates of three prostate cancer samples across multiple variables (operators, days, runs, reagent lots, and equipment) and RNA/cDNA inputs respectively using the appropriate linear mixed model.ResultsThe overall agreement between the development and alternative platform was 94.7% (95% confidence interval, 86.9–98.5%). The reportable range was determined to be 0.150 to 1.107 for core needle biopsy samples and − 0.214 to 0.844 for radical prostatectomy samples. From the precision study, the standard deviations for assay repeatability and reproducibility were 0.032 and 0.040 respectively. The sensitivity study demonstrated that a total RNA input and cDNA input of 50 ng and 3.5 μg respectively was conservative.ConclusionThe Metastatic Assay was found to be highly reproducible and precise. In conclusion the studies demonstrated an acceptable analytical performance for the assay and support its potential use in the clinic.

204P - Analytical validation of OncoMasTR, a multigene test for predicting risk of distant recurrence in hormone receptor-positive early stage breast cancer

Background: OncoMasTR is a new multigene prognostic test that was discovered via a novel transcriptional network analysis method that identified upstream Master Transcription Regulators (MTRs), which regulate previously identified prognostic biomarkers. The optimised OncoMasTR signature incorporating clinicopathological information has been shown to be significantly prognostic for predicting distant recurrence in two independent cohorts (TransATAC & a subset of TAILORx from participating Irish centres). The analytical performance characteristics of the OncoMasTR test, comprising solely three prognostic MTRs, were determined. Methods: Relative gene expression levels were measured by RT-qPCR. Assay precision and input ranges were determined using a panel of samples representative of low and high recurrence risk tested across a number of runs incorporating different sources of variation. Serial dilutions of a pooled patient RNA sample was used to establish the linear range and efficiency of the individual gene assays. Results: The overall standard deviation of the OncoMasTR risk score was 0.15, which represents less than 2% of the 10-unit risk score range. The majority of the variability in OncoMasTR results was related to within-run variation (78.2%) with other between-run variation sources contributing relatively little (PCR instrument (0.6%), assay operator (5.2%), reagent lots (7.3%) or loading position (8.7%)). Consistent risk scores were measured for individual samples from 40ng down to 500-fold RNA input range corresponding to CT values of 23 – 36, demonstrating the ability of the test to reliably detect low level expression of the OncoMasTR panel. Importantly, PCR efficiencies were similar for the individual MTR and reference gene assays which ranged from 79 – 95%. Conclusions: The OncoMasTR prognostic test displays robust analytical and clinical performance and is being launched as a CE-marked test. The concise nature of the three gene signature and a simplified workflow that can be readily adopted using standard laboratory equipment will enable convenient qualification by local laboratories for decentralised use. (Less)

High-performance nanogap electrode-based impedimetric sensor for direct DNA assays

The rapid and sensitive detection of pathogen DNA (Deoxyribonucleic acid) would be essential for diagnosis and appropriate antibiotic treatment time. Herein, we report a novel direct DNA detectable impedimetric sensor. Direct assay of the amplified target DNA (mecA gene from methicillin-resistant Staphylococcus aureus (MRSA)) was performed using the PCR (polymerase chain reaction) product without any purification. Even though there are lots of PCR reagents and excess salts in sample PCR product, the nanogap electrode-based impedimetric sensor was able to detect DNA amplification fast in 5th PCR cycle which had 260 fM mecA gene in sample originally. The 70 nm gap electrode sensor yielded over 20% signal increase at the 5th PCR cycle and the impedance change grew up to about 60% at 25th in case of sample with 260 fM mecA gene template originally. The increased concentration of target DNA template led to the rise in impedance change such as 60% up at 5th and 120% up at 25th cycle with 260 pM, respectively. It is very outstanding result as compared with the traditional PCR agarose gel. Besides, it is 7-fold superior sensitivity to the microgap electrode. Furthermore, genomic DNA sample extracted from MRSA was detected rapidly. The nanogap electrode-based impedimetric sensor could be a good candidate for a rapid, sensitive, and low-cost electrical biosensor for DNA characterization in diagnostics and disease monitoring.

Abstract 939: Analytical validation of InVisionFirst™, a liquid biopsy assay for high-sensitivity broad molecular profiling of circulating tumor DNA using plasma samples of cancer patients

Abstract Circulating tumor DNA (ctDNA) analysis enables minimally invasive assessment of somatic genetic alterations for cancer patients. ctDNA analysis is quickly being incorporated into cancer care; notably in profiling patients' tumors to guide treatment decisions. Patients have demonstrated a response to targeted therapies even when the actionable mutations detected in their plasma DNA was at low variant allele fractions (VAFs) (< 0.5%). Here we describe a detailed analytical validation study for InVisionFirst™, an NGS-based assay offering broad molecular profiling with exceptional sensitivity for analysis of ctDNA. The InVisionFirst assay is based on enhanced tagged amplicon sequencing (eTAm-SeqTM) technology and profiles 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations. Analytical validation demonstrated the performance of this assay for detection of point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. Over 100 different contrived samples and 200 plasma samples were analyzed, representing a wide spectrum of genetic aberrations and variant allele frequency (VAF). Analysis was performed by multiple operators, at different times and using different reagent lots. The InVisionFirst assay demonstrated an excellent sensitivity, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33% and 92.46% sensitivity for indels at 0.25% VAF. DNA amplifications for ERBB2, FGFR1, MET and EGFR were also detected with high sensitivity and specificity. Greater than 50% of SNVs were detected down to a few molecules (0.06%-0.08% VAF), with the lower reportable range of 0.0125% for SNVs and indels. This high sensitivity was achieved while still retaining exceptional specificity (99.9997% per base). The assay also demonstrates, for the first time, detection of ALK and ROS1 gene fusions with an amplicon-based ctDNA technology. The novel methodology detected EML4-ALK and SLC34A2-ROS1 breakpoints at a VAF of 0.0625%. Comparison of VAFs between the InVisionFirst assay and ddPCR showed excellent concordance (R2 = 0.965). This analytical validation study has evaluated the performance characteristics of the InVisionFirst assay across a range of genomic alterations, establishing it as a highly sensitive and specific assay that meets the analytical requirements for clinical applications. The InVisionFirst assay can be deployed as a liquid biopsy NGS assay for broad molecular profiling of plasma to aid in the management of cancer patients. Citation Format: Samuel Woodhouse, Vincent Plagnol, Karen Howarth, Stefanie Lensing, Matt Smith, Michael Epstein, Mikidache Madi, Sarah Smalley, Catherine Leroy, Jonathan Hinton, Frank de Kievit, Esther Musgrave-Brown, Colin Herd, Katherine Baker-Neblett, Will Brennan, Peter Dimitrov, Nathan Campbell, Nitzan Rosenfeld, James Clark, Davina Gale, Jamie Platt, John Calaway, Greg Jones, Tim Forshew. Analytical validation of InVisionFirst™, a liquid biopsy assay for high-sensitivity broad molecular profiling of circulating tumor DNA using plasma samples of cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 939.

Analytical validation of a next generation sequencing liquid biopsy assay for high sensitivity broad molecular profiling.

Circulating tumor DNA (ctDNA) analysis is being incorporated into cancer care; notably in profiling patients to guide treatment decisions. Responses to targeted therapies have been observed in patients with actionable mutations detected in plasma DNA at variant allele fractions (VAFs) below 0.5%. Highly sensitive methods are therefore required for optimal clinical use. To enable objective assessment of assay performance, detailed analytical validation is required. We developed the InVisionFirst™ assay, an assay based on enhanced tagged amplicon sequencing (eTAm-Seq™) technology to profile 36 genes commonly mutated in non-small cell lung cancer (NSCLC) and other cancer types for actionable genomic alterations in cell-free DNA. The assay has been developed to detect point mutations, indels, amplifications and gene fusions that commonly occur in NSCLC. For analytical validation, two 10mL blood tubes were collected from NSCLC patients and healthy volunteer donors. In addition, contrived samples were used to represent a wide spectrum of genetic aberrations and VAFs. Samples were analyzed by multiple operators, at different times and using different reagent Lots. Results were compared with digital PCR (dPCR). The InVisionFirst assay demonstrated an excellent limit of detection, with 99.48% sensitivity for SNVs present at VAF range 0.25%-0.33%, 92.46% sensitivity for indels at 0.25% VAF and a high rate of detection at lower frequencies while retaining high specificity (99.9997% per base). The assay also detected ALK and ROS1 gene fusions, and DNA amplifications in ERBB2, FGFR1, MET and EGFR with high sensitivity and specificity. Comparison between the InVisionFirst assay and dPCR in a series of cancer patients showed high concordance. This analytical validation demonstrated that the InVisionFirst assay is highly sensitive, specific and robust, and meets analytical requirements for clinical applications.

Validating critical analytical variables of a multiplexed gene expression assay measuring tumor inflammation designed to predict response to anti-PD1 therapy.

203 Background: The development and analytical performance of the Tumor Inflammation Signature (TIS) assay has been described previously. The TIS is an investigational use RNA expression assay on the nCounter Dx Analysis System, which is being evaluated as a patient enrichment biomarker for treatment with pembrolizumab single agent across multiple solid tumor types. Here we describe the analytical validation of the RNA input range and analytical precision starting from RNA isolated from formalin fixed paraffin embedded (FFPE) tissue blocks. Methods: Analytical validation of TIS assay performance across an RNA input range was performed using samples from 11 tumor types. The analytical precision between sites, instruments, reagent lots, and users was measured, using RNA samples isolated from FFPE tissue blocks. Results: The assay was validated across the specified RNA input range with ≥ 94% concordance at the minimum specified RNA input (50ng). The total standard deviation of the TIS score was < 0.04 units across three sites with ≥98% concordance between sites. The 6 users across the three sites did not significantly contribute to the assay variability. There was 100% concordance in biomarker high/low categorization between multiple reagent lots and multiple instruments. Conclusions: The analytical performance of the NanoString TIS assay has been validated to give consistent results across the RNA input range and between site, instrument, assay user, and reagent kit lot. The assay is well suited for decentralized clinical testing and is currently under investigation as a biomarker to enrich for response to anti-PD1 therapy across multiple tumor types.

Multi-site N-Glycan mapping study 2: UHPLC.

In the first part of this publication, the results from an international study evaluating the precision (i.e., repeatability and reproducibility) of N-glycosylation analysis using capillary electrophoresis of APTS-labeled N-glycans were presented. The corresponding results from ultra-high performance liquid chromatography (UHPLC) with fluorescence detection are presented here from 12 participating sites. All participants used the same lot of samples, reagents, and columns to perform the assays. Elution time, peak area and peak area percent values were determined for all peaks ≥0.1% peak area, and statistical analysis was performed following ISO 5725-2 guideline principles. The results demonstrated adequate reproducibility, within any given site as well across all sites, indicating that standard UHPLC-based N-glycan analysis platforms are appropriate for general use.

Approaches to the Assessment of LOT-TO-LOT Variation

Reproducibility of analytical systems over time is essential for monitoring of individual patients, as well as for the application of reference intervals and other medical decision limits. Variation between different manufacturer lots of reagents and calibrators can contribute to significant variation in laboratory measurements and hence to patient results over time. The assessment of reagent and calibrator lots represents a significant challenge for laboratories and different approaches have been taken for acceptance testing of new lots. The Clinical Laboratory Standards Institute (CLSI) has published an evaluation protocol EP26-A for the assessment of new reagents although it has been criticised for being difficult to implement. Other approaches have been published as pragmatic alternatives to the CLSI protocol. The aim of this session will be to briefly review the CLSI protocol and published alternatives for the assessment of lot variation in reagents and calibrators.

Potential of biological phosphorus removal

The purpose of studies was to assess the potential effectiveness of phosphorus compounds removal from waste water by biological method. Phosphorus removal is very important because of strict standards to phosphate concentration in effluent. But removal of phosphorus by chemical method requires a lot of reagents. Moreover objectives of research were to determine actual efficiency of the biological phosphorus removal. The study shows that under certain technological parameters of biological treatment it is possible to meet strict requirement phosphate removal. This technology can be implemented on existing wastewater treatment plants. The technology of biological phosphorus removal can be combined with the technology of nitrification and denitrification.

A novel liquid media mycobacteria extraction method for MALDI-TOF MS identification using VITEK® MS

A low-cost identification method that can be performed directly from a positive liquid medium culture is needed for the diagnosis of mycobacterial infections. Here, we describe a novel, cost-effective, and validated method that allows for direct and rapid identification of mycobacteria from a positive liquid culture using VITEK® MS with a total process duration under 45min. From a liquid mycobacteria culture a 3.0mL aliquot is removed 24–72h post positivity and centrifuged to create a pellet. After decanting, the tube is blotted dry, the pellet is re-suspended in 0.5mL of 70% ethanol and then transferred into a 2.0mL tube containing glass beads. Mycobacteria are disrupted mechanically followed by a 10min. incubation at room temperature to complete inactivation. Inactivated material is pelleted by centrifugation and then re-suspended in 10μL of 70% formic acid and 10μL of acetonitrile. After centrifugation, 1μL of supernatant (protein extract) is deposited onto target slide, allowed to dry, and then 1μL CHCA matrix is added.A seeded study was conducted to demonstrate the reliability of the method, a total of 251 culture samples obtained from automated culture systems (BacT/ALERT® MP bottles, BACTEC MGIT™ 960 tubes, and VersaTREK® Myco bottles), were tested and resulted in 98.8% correct identification. Reproducibility was shown by testing three organisms across three reagent lots, between four laboratory technicians, over the course of five days for three liquid media systems resulting in a total of 180 deposits with an overall correct identification of 98.9% with the remaining results giving no identification. Additional studies were performed including comparison of different mechanical disruption techniques, stability of frozen extracts, and stability of slide deposits to allow for flexibility in a routine clinical workflow.The described method proved to be safe while providing consistent and reproducible results for different species of mycobacteria and is compatible with the three most widely used liquid media medium detection systems.

Analytical comparison of three different versions of a high-sensitivity cardiac troponin I assay over 10 years

BackgroundConcerns have been raised on the long-term analytical performance of high-sensitivity cardiac troponin (hs-cTn) assays with respect to different reagent formulations, lots and instrumentation. Our goal for the present study was to compare three different versions of an hs-cTnI assay in two different study populations to evaluate if assay re-formulation over 10years has also affected the analytical results. MethodsBeckman Coulter's CE marked hs-cTnI assay (Access hsTnI, 2017) was tested in 100 lithium heparin plasma samples first tested in 2007 with their prototype hs-cTnI assay and in 100 serum samples tested with their enhanced hs-cTnI assay in 2011 with comparison performed by Passing-Bablok regression. The Beckman Coulter hs-cTnI results from 2017 and 2011 from the serum samples were also compared to the Abbott ARCHITECT i1000 hs-cTnI results (2013) with 3-fold differences used to identify possible outliers. Freeze/thaw stability testing (−20°C) was also performed on normal cTnI (Beckman=4.0ng/L; Abbott=5.3ng/L) and high cTnI concentration (Beckman=77.6ng/L; Abbott=126.1ng/L) lithium heparin plasma pools for both hs-cTnI assays. ResultsAfter 3 freeze-thaws the Beckman hs-cTnI assay yielded minor decreases in concentrations (normal pool −0.7ng/L and high pool −12.6ng/L lower). Regression analyses yielded the following relationship between the Beckman hs-cTnI versions: 2017 hs-cTnI=2.0*(2007 prototype hs-cTnI)-5.1ng/L and 2017 hs-cTnI=1.04∗(2011 enhanced hs-cTnI)-2.5ng/L. Compared to the Abbott 2013 hs-cTnI results, the 2011 Beckman enhanced version had 8 results 3-fold higher, with the 2017 Beckman version yielding 6 results 3-fold lower.Conclusions: The 2017 Beckman hs-cTnI version (Access hsTnI) is closely aligned with the previous enhanced hs-cTnI assay and appears to have reduced the frequency of aberrantly high results.

Analysis of "Seven Year Surveillance of the Clinical Performance of a Blood Glucose Test-Strip Product".

The article titled "Seven Year Surveillance of the Clinical Performance of a Blood Glucose Test-Strip Product" by Setford and coworkers in this issue of Journal of Diabetes Science and Technology is an impressive study showing that over 7 years in three clinics, using multiple reagent lots, a total of 73 600 samples met the ISO 15197 2015 standard with no results in the D or E zones of a Parkes glucose meter error grid. Three requirements are suggested for a clinically acceptable glucose meter. The authors provide strong evidence for meeting two requirements but fail to provide summarized data about the number of nonnumeric results. Finally, the authors overstate some results, called "spin" by some which is not necessary. The superb results should stand on their own.

Assay Stability, the missing component of the Error Budget

Gaining a better understanding of Quality Control (QC) processes is a key requirement to improving performance and reducing patient risk. Detecting analytical error is dependent on a QC strategy that reliably detects a critical shift in a result away from the true value.Recently the concept of Six Sigma has been used by diagnostic laboratories to assess the performance of assays and to assist in the selection of QC rules. The sigma metric is one measure of an assay's ability to perform within specification. However an additional dimension to managing an assay is its stability in bias over time.The concept of long term stability is the same as measured QC drift (SEdrift) which is the effect of numerous calibrations, changes in reagent lots and other conditions i.e. a long term effect. This implies that the standard error budget is wrong because it is modelled on short term QC and misses this SEdrift stability component.We show that SEdrift provides a measure of Assay Stability that should be included in Quality Planning and that by including an allowance for this drift, determining target imprecision appropriate for matched QC algorithms that provide high error detection is as simple as dividing the Allowable Performance Specification by 4, 5 or 6.

Study and analysis of national current status and problems of anticoagulant proteins assay

Objective: To investigate current status and problems of anticoagulant proteins assay in domestic laboratories so as to provide suggestions for implementing the standardization and quality improvement. Methods: Two hundred and seventy-four laboratories those had developed or prepared to do anticoagulant proteins assay were selected from one thousand and five hundred participants in the national coagulation screening External Quality Assessment(EQA) program by an internet survey and then a questionnaire and quality control materials were sent to them to carry out a further survey. The questionnaire information was analyzed statistically. The results of quality control materials were grouped by the reagents and the average, median, standard deviation(s), coefficient variation(CV) of each group were calculated. The deviations or percentage deviations were determined by comparing the results of each laboratory to the target defined as the peer-group median after exclusion of outliers, and then the pass rates were calculated based on the criterion of RCPA, DGKL and the allowable total error based on biological variation. Results: Two hundred and thirty-five questionnaires were collected. The number of laboratories testing antithrombin(AT), protein C(PC) and protein S(PS) activity were 194, 63 and 50 respectively. The instruments and reagents were mainly from abroad (more than 96%), the matching rate of which were above 94%. For AT, PC and PS activity testing, there were 30.4%, 33.3%, 34.0% of laboratories did not perform verification assays respectively, and 8.8%, 7.9%, 14.0% of laboratories did not renew calibration curve when the reagent lots were changed. 11.3%, 17.5%, 16.0% of laboratories didn't run internal quality control, and 34.9%, 26.9%, 21.4% of laboratories only performed a single level of quality control. 4.1% of laboratories set the reference intervals of AT activity according to different age groups, and the percentage of that of PC and PS activity were 1.6% and 2.0%. 16.0% of laboratories set the reference interval of PS activity by sex. For normal control materials, the CV of AT, PC and PS activity results were 5.7%-12.9%, 4.2%-7.7% and 18.4%-33.1% while the CV for abnormal level were 13.3%-38.3%, 6.1%-14.4% and 31.5%-34.5% respectively. The pass rate was different when it was judged by different criteria. A suitable criterion for each item should be selected according to the concentration level of quality control materials. Conclusion: The comparability between laboratory results are not satisfactory and in order to promote quality improvement, it is necessary to develop guidelines, organize trainings and establish a national EQA scheme.

Clinical performance of the VERIS HCV assay for hepatitis C virus RNA quantification

BackgroundDiagnosis of hepatitis C virus (HCV) infection and treatment monitoring rely on detection/quantification of HCV RNA and real-time polymerase chain reaction (PCR) techniques are expected to equivalently quantify the different HCV genotypes. ObjectiveThe clinical performance of the VERIS HCV assay for HCV RNA quantification was compared to that of the Abbott RealTime HCV assay. Study designQualitative concordance and quantitative comparison were evaluated on a first panel of 286 clinical samples containing HCV genotypes 1–6. Forty additional genotype 4 samples were tested to explore genotype 4 HCV RNA underquantification. ResultsQualitative discrepancies were observed for low viral loads (<2 log10 IU/mL) in patients under antiviral therapy and would not have had any impact on patients’ management with the current guidelines for the monitoring of patients on direct-acting antivirals (DAAs). Quantification results were well correlated (R2=0.89) with an overall minimal quantification bias (mean VERIS − Abbott difference) of −0.09 log10 IU/mL. Quantification agreement for genotypes 1, 2 and 3 samples was excellent, but reached −0.57 log10 IU/mL for 46 genotype 4 samples. A lower quantification bias of −0.24 log10 IU/mL was observed when testing 40 additional genotype 4 samples with a second reagent lot. Underquantification was not associated with 5′ untranslated region (UTR) sequence polymorphisms but could be explained by 5′ UTR RNA molecular modeling. ConclusionHCV RNA quantification by the VERIS HCV assay and the Abbott RealTime HCV assay was well correlated for all HCV genotypes, except genotype 4 where 5′ UTR RNA folding may impact quantification. Nevertheless, this underestimation of HCV RNA levels had no impact on clinical use.

False-Positive Total T3 Using the Ortho Vitros Immunoassay in Pediatric Populations.

To the Editor: Total triiodothyronine (T3)1 (TT3) measurements can provide clinical utility elucidating T3-dominant Graves' disease and other thyrotoxicosis conditions. Thyroid hormone assays, including TT3, are susceptible to protein-based interferences, usually caused by endogenous antibodies. Interferences should be considered when the thyroid hormone concentrations are inconsistent with clinical impression (1). We detected an interference with the Ortho® Vitros TT3 assay causing falsely increased concentrations in 20 pediatric patients, some of whom had a history of high TT3 for years, suggesting that the interference was not limited to a single reagent lot. The erroneous results were questioned …

Evaluation of the CLSI EP26-A protocol for detection of reagent lot-to-lot differences

BackgroundVerification of new reagent lots is a required laboratory task. The Clinical and Laboratory Standards Institute (CLSI) EP26-A guideline provides a lot-to-lot verification protocol to detect significant changes in test performance. The aim of this study was to compare the performance of EP26-A with our laboratory reagent lot verification protocol. MethodsProspective evaluations for two reagent lots each for thyroid stimulating hormone (TSH), thyroglobulin (Tg), thyroxine (T4), triiodothyronine (T3), free triiodothyronine (fT3), and thyroid peroxidase antibody (TPOAb) were performed. The laboratory's lot verification process included evaluation of 20 patient samples with the current and new lots and acceptability based on a predefined criteria. For EP26-A, method imprecision data and critical differences based on previously defined lot-to-lot consistency goals were used to define sample size requirements and rejection limits. ResultsEP26-A required the following number of samples: 23 for TSH, 17 for Tg, 33 for T4, 31 for T3, 48 for fT3, and 1 for TPOAb. Our current protocol and EP26-A were in agreement in 9 of the 12 (75%) paired verifications. Of the 3 discrepant verifications, Tg and TSH reagent lots were rejected by EP26-A due to significant differences at medical decision points; whereas TPOAb was rejected by the current laboratory protocol. ConclusionsThe EP26-A protocol arrived at the same conclusions as our protocol in 75% of the evaluations and required more samples for 4 of the 6 analytes tested. Challenges associated with determining rejection limits and the need for increased sample sizes may be critical factors that limit the utility of EP26-A.

An Automated Assay for Growth Differentiation Factor 15.

Growth differentiation factor 15 (GDF-15) can serve as a biomarker for cardiovascular disease burden and risk. We evaluated a new, fully automated electrochemiluminescence immunoassay for measuring GDF-15. Six laboratories independently characterized the Elecsys® GDF-15 assay (Roche Diagnostics) under routine conditions. Within-run precision (repeatability), within-laboratory precision (intermediate precision), and between-laboratory precision (reproducibility) were assessed. Plasma-serum sample correlation, reagent lot-to-lot reproducibility, and instrument comparisons were performed. The Elecsys assay was compared to a research immunoradiometric assay (IRMA) and a commercially available ELISA. GDF-15 concentrations were measured with the Elecsys assay in 739 apparently healthy individuals. CVs for within-run and within-laboratory precision ranged from 0.7% to 7.7% and 1.7% to 8.6%, respectively, for samples containing 670-16039 ng/L. CVs for between-laboratory precision ranged from 7.1% to 8.9% (766-14289 ng/L). Recovery of GDF-15 was comparable for serum, Li-heparin plasma, K2- and K3-EDTA plasma, and citrated plasma, between 2 reagent lots, and on the cobas e 411 and cobas e 601 analyzers (Roche Diagnostics). GDF-15 concentrations in the clinically relevant range (400-3000 ng/L) measured with the Elecsys assay showed a good correlation and agreement with those measured by IRMA or ELISA. GDF-15 concentrations in apparently healthy individuals increased with age but did not vary by sex. The Elecsys GDF-15 assay demonstrates a robust analytic performance under routine conditions and provides an automated method for measuring GDF-15 concentrations in serum and plasma.