Cobas Assay Research Articles

Two Thyroid Stimulating Hormone assays correlated in clinical practice show disagreement in subclinical hypothyroidism patients

Thyroid Stimulating Hormone (TSH) levels are related to the pituitary gland's ability to detect thyroid hormone concentration. Many studies have analyzed the correlation between TSH and T4, demonstrating a complex system correlation. This complex system may vary among different TSH levels and patients. ObjectivesThe main purpose of this study is to assess the correlation and agreement of serum TSH measured with two assays in different settings. Design & methodsWe evaluated healthy individuals as well as subclinical or overt hypothyroid patients. Eighty participants had TSH levels measured by Cobas Roche Elecsys 600 (Roche Diagnostics) and Abbott Architect I 2000 (Abbott Diagnostics). The TSH methods correlations were established with Pearson's correlation, and the strength of the agreement was determined by the McBride scale. The paired Student's t-test was applied to evaluate TSH values from both methods. The one-sample t-test was used to evaluate the difference between TSH values. The agreement was also assessed by a Bland-Altman plot. A regression analysis was applied to the correlation between TSH and T4. ResultsThere was a significant difference in TSH values measured by the two methods (p<0.01). Our results demonstrated a poor correlation for TSH in the euthyroid (r: 0.888, p<0.01) and the subclinical hypothyroid (r:0.886, p<0.01) range. The Bland-Altman plot demonstrates that the majority of the TSH values fell between the lines of equality. There were few differences in the values in the normal upper range and slightly above that range (from a TSH: 3.25 to 6.36mUI/L). The level of correlation between TSH assays remains high in all scenarios for age (r≥0.951), BMI (r≥0.962), anti-TPO antibodies (r: 0.977) or levothyroxine use (r: 0.970). ConclusionsTSH measurement is essential to access thyroid function. Although the overall agreement between the methods is substantial, there was a poor agreement in the normal upper range and close above. The disagreement observed reinforces the difficulty in using different assays in clinical practice. The better correlation with fT4 and the reference range used by Cobas assay allowed the best clinical performance.

Performance evaluation of the Cobas TaqMan MTB assay on respiratory specimens according to clinical application

ObjectiveTo evaluate the performance of the Cobas TaqMan MTB assay (Cobas assay) with respect to its clinical application. MethodsThis was a retrospective analysis of 1154 results from 1034 patients for whom mycobacterial cultures and the Cobas assay were performed simultaneously. Based on the patient medical records, two categories of clinical application were defined: (1) the diagnosis of patients with a high probability of pulmonary tuberculosis according to clinical and radiological features (n=128), and (2) the exclusion of tuberculosis in clinically indeterminate patients (n=1026). Standard culture was used as the reference method. ResultsThe sensitivity of the Cobas assay for the detection of Mycobacterium tuberculosis was 70.4% (95% confidence interval (CI) 49.7–85.5%) for category 1, but only 25.0% (95% CI 4.5–64.4%) for category 2. The specificity was ≥95.0% for both categories. The positive predictive value was 79.2% (95% CI 57.3–92.1%) for category 1 and 33.3% (95% CI 6.0–75.9%) for category 2, while the negative predictive value was 92.3% (95% CI 85.0–96.4%) for category 1 and 99.4% (95% CI 98.7–99.8%) for category 2. ConclusionsThe results of this study indicate that Cobas assay results must be interpreted carefully according to the clinical purpose of the assay.

Abstract 4651: Multiple platform evaluation of KRAS codon 12/13 reference material

Abstract An assay’s ability to detect the presence of a KRAS driver mutation is critical when attempting to determine whether a tumor may respond to anti-EGFR therapy. Currently, there are two FDA approved companion diagnostics that can be used to assess whether there are mutations in KRAS codons 12 and 13, and both include their own internal controls. However, there is a need for external reference materials that allow laboratories to validate such assays - especially, at levels near their limits of detection (LODs). We describe the design of the Seraseq™ FFPE Tumor KRAS Reference Material. This reference material is composed of 7 different 5-micron FFPE curls that contain the 7 different KRAS mutations detected by the companion diagnostics: G12A, G12C, G12D, G12R, G12S, G12V and G13D. Each curl contains a mixture of KRAS wildtype GM24385 cells and a KRAS mutant cell line in sufficient quantity for most assays. KRAS mutant frequencies of about 5% were targeted and subsequently verified by digital PCR. A 5% mutant frequency was selected because it is near the LOD for the existing companion diagnostics, and is close to the minimum reported frequency of many next generation sequencing (NGS)-based assays that test for somatic variants. The reference material was evaluated using different methods, which include digital PCR, the Roche cobas® KRAS Mutation Test, the QIAGEN therascreen® KRAS RGQ PCR Kit and several NGS-based assays. Testing revealed general compatibility with these assays in that sufficient DNA could be extracted, and concordance of the results with the expected calls was high. For example, apparent DNA yields by Nanodrop with the cobas assay exceeded the required amount by more than 3-fold and all 7 curls led to a “mutation detected” result in duplicate testing. Interestingly, not all variants were detected with the therascreen assay, which may be due to some variants being present at slightly below the claimed LODs, however DNA yields were sufficient and the correct variants were identified. Finally, variant frequencies obtained by NGS were consistent with those observed by digital PCR. We conclude that the Seraseq FFPE Tumor KRAS Reference Material may be useful as part of KRAS codon 12/13 assay validation. Citation Format: Russell Garlick, Yves Konigshofer, Farol L. Tomson, Dale Yuzuki, Bharathi Anekella. Multiple platform evaluation of KRAS codon 12/13 reference material [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4651. doi:10.1158/1538-7445.AM2017-4651

Validation of the denaturing capillary electrophoresis (DCE) assay for non-invasive liquid biopsy in lung carcinoma: A study of concordance with the cobas EGFR mutation test v2.

e23040 Background: The detection of circulating tumor DNA (ctDNA), referred to as “liquid biopsy” is becoming a preferred option for some patients with lung adenocarcinomas. The key for a suitable technology is high sensitivity enabling to detect small fractions of mutated alleles, the method has to be solid against false-positives. The aim of this work was to compare our method based on capillary electrophoresis under denaturing conditions (DCE) to FDA and CE IVD approved commercial kit - cobas EGFR Mutation Test v2. Methods: A total of 44 plasma samples were acquired from patients with clinicaly confirmed stage IV lung adenocarcinoma. There were samples from patients prior to receiving therapy (with a known tissue EGFR genotype) as well as several ones showing progression on a previously administered antiEGFR-therapy (with no tissue info available at the time of plasma sampling). DNA was isolated from multiple 2 mL aliquots of each plasma. The first aliquot was run on cobas z480 real-time instrument using unmodified manufacturer's protocol. Another 2ml aliquot was run by DCE assay (accredited method under ISO 15189:2012), which is based on a PCR followed by a fragment analysis on an ABI capillary analyzer (Sanger sequencer). Results: The mutual concordance was 84.1% (37/44). cobas assay identified 11 EGFR-positive plasma samples (6 x Ex19del, 3 x L858R, 1x T790M combined with Ex19del and 1x T790M combined with L858R). DCE identified 8 EGFR-positive samples (6 x Ex19del, 1 x L858R, 1xT790M combined with L858R). DCE missed 5 of cobas-positive samples (2x Ex19del, 2x L858R and 1x T790M) while in turn identifying 2x Ex19del plasma samples that were concordant with tissue, but missed by cobas. Overall concordance with tissue was 73.3% (22/30) for cobas, 70.0% (21/30) for DCE assay and 80.0% (24/30) for both combined. Conclusions: DCE approach is reliable for low levels of mutations in plasma of lung cancer patients and comparable (and complementary) to cobas. Its advantage is simplicity, low cost and a universal, straighforward adaptability to virtually any mutation at any site allowing to detect oncogenic as well as tumor supressor mutations in ctDNA. Supported by grant 17-30748A.

Detection of cervical precancerous lesions with Aptima HPV assays using SurePath preservative fluid specimens

SurePath specimens from women referred to colposcopy were treated with Aptima Transfer Solution (ATS) before testing in Aptima HPV (AHPV) and Aptima HPV 16, 18/45 (AHPV-GT) assays. Untreated SurePath specimens were tested with the cobas HPV test. PreservCyt specimens were assessed for cytology and tested with AHPV. High-grade cervical intraepithelial neoplasia lesions served as the reference standard. Excellent agreement (95.5%; k=0.91) was observed for ATS-treated SurePath specimens between Tigris and Panther systems and between the PreservCyt and ATS-treated SurePath specimens (91.1%, k=0.81) with the AHPV assay on Tigris. Agreement between the AHPV and cobas assays with SurePath specimens was substantial (89.9%, k=0.80). AHPV sensitivity for CIN2+(n=147) was 91.2% for SurePath and PreservCyt. Cobas HPV sensitivity was 93.9% for SurePath specimens. AHPV testing of SurePath specimens was more specific (59.4%) than cobas (54.7%) (p<0.001). Detection and genotyping showed similar absolute and relative risks. ATS-treated SurePath specimens tested with AHPV and AHPV-GT assays showed similar performance with greater specificity than cobas HPV on SurePath specimens. Similar overall results were seen using a CIN3 disease endpoint.

Impact of season and different vitamin D thresholds on prevalence of vitamin D deficiency in epidemiological cohorts-a note of caution.

We investigated the impact of different cut-offs on the prevalence of 25-hydroxyvitamin D [25-(OH)D] deficiency. We used baseline data of 4149 participants (45-75 years, 50% women) of the population-based Heinz Nixdorf Recall study. Serum 25-(OH)D was measured with the Roche Cobas assay. Quartiles (p25, p50, and p75) were calculated. Data were stratified by months, sex, and age. According to the recommendations of 'Dachverband Osteologie', Endocrine Society and National Institute of Health we used 25-(OH)D thresholds of 12, 20, and 30 ng/ml to estimate vitamin D deficiency. Overall the median of 25-(OH)D was 19.8 ng/ml (p25 = 14.4 ng/ml, p75 = 26.6 ng/ml), with highest concentrations in July (p50 = 23.8 ng/ml, p25 = 18.2 ng/ml, and p75 = 31.2 ng/ml) and lowest in March (p50 = 15.8 ng/ml, p25 = 11.5 ng/ml, and p75 = 20.6 ng/ml). Prevalence of vitamin D deficiency rose from 16, 51 up to 83% using the cut-offs of <12, <20 ng/ml, and <30 ng/ml, respectively. With respect to seasonal variance, prevalence of vitamin D deficiency rose to 92% in February/March using the cut-off <30 ng/ml (<12: 28%, <20 ng/ml: 71%) whereas in June/July prevalence of vitamin D deficiency decreased to 71% (<12: 6%, <20 ng/ml: 30%). The chance to attest the diagnosis of vitamin D deficiency for cut-off 12 ng/ml in March is 6.4-fold higher than in June, for cut-off 20 ng/ml, 5.5-fold higher and for cut-off 30 ng/ml, 3.1-fold higher. Guidelines to define vitamin D deficiency revealed extremely different prevalence rates ranging between 6 and 92%. Accounting for collection time and antecedent sun exposure are important to reduce bias in research studies and improve decision-making in clinical care. Vitamin D thresholds have to be rethought.

Clinical applicability and cost of a 46-gene panel for genomic analysis of solid tumours: Retrospective validation and prospective audit in the UK National Health Service.

BackgroundSingle gene tests to predict whether cancers respond to specific targeted therapies are performed increasingly often. Advances in sequencing technology, collectively referred to as next generation sequencing (NGS), mean the entire cancer genome or parts of it can now be sequenced at speed with increased depth and sensitivity. However, translation of NGS into routine cancer care has been slow. Healthcare stakeholders are unclear about the clinical utility of NGS and are concerned it could be an expensive addition to cancer diagnostics, rather than an affordable alternative to single gene testing.Methods and findingsWe validated a 46-gene hotspot cancer panel assay allowing multiple gene testing from small diagnostic biopsies. From 1 January 2013 to 31 December 2013, solid tumour samples (including non-small-cell lung carcinoma [NSCLC], colorectal carcinoma, and melanoma) were sequenced in the context of the UK National Health Service from 351 consecutively submitted prospective cases for which treating clinicians thought the patient had potential to benefit from more extensive genetic analysis. Following histological assessment, tumour-rich regions of formalin-fixed paraffin-embedded (FFPE) sections underwent macrodissection, DNA extraction, NGS, and analysis using a pipeline centred on Torrent Suite software. With a median turnaround time of seven working days, an integrated clinical report was produced indicating the variants detected, including those with potential diagnostic, prognostic, therapeutic, or clinical trial entry implications. Accompanying phenotypic data were collected, and a detailed cost analysis of the panel compared with single gene testing was undertaken to assess affordability for routine patient care.Panel sequencing was successful for 97% (342/351) of tumour samples in the prospective cohort and showed 100% concordance with known mutations (detected using cobas assays). At least one mutation was identified in 87% (296/342) of tumours. A locally actionable mutation (i.e., available targeted treatment or clinical trial) was identified in 122/351 patients (35%). Forty patients received targeted treatment, in 22/40 (55%) cases solely due to use of the panel. Examination of published data on the potential efficacy of targeted therapies showed theoretically actionable mutations (i.e., mutations for which targeted treatment was potentially appropriate) in 66% (71/107) and 39% (41/105) of melanoma and NSCLC patients, respectively. At a cost of £339 (US$449) per patient, the panel was less expensive locally than performing more than two or three single gene tests.Study limitations include the use of FFPE samples, which do not always provide high-quality DNA, and the use of “real world” data: submission of cases for sequencing did not always follow clinical guidelines, meaning that when mutations were detected, patients were not always eligible for targeted treatments on clinical grounds.ConclusionsThis study demonstrates that more extensive tumour sequencing can identify mutations that could improve clinical decision-making in routine cancer care, potentially improving patient outcomes, at an affordable level for healthcare providers.

Rapid Sputum Multiplex Detection of the M. tuberculosis Complex (MTBC) and Resistance Mutations for Eight Antibiotics by Nucleotide MALDI-TOF MS

The increasing incidence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis (MTB) adds further urgency for rapid and multiplex molecular testing to identify the MTB complex and drug susceptibility directly from sputum for disease control. A nucleotide matrix-assisted-laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS)-based assay was developed to identify MTB (MTBID panel) and 45 chromosomal mutations for resistance to eight antibiotics (MTBDR panel). We conducted a 300 case trial from outpatients to evaluate this platform. An MTBID panel specifically identified MTB with as few as 10 chromosome DNA copies. The panel was 100% consistent with an acid-fast stain and culture for MTB, nontuberculous mycobacteria, and non-mycobacteria bacteria. The MTBDR panel was validated using 20 known MDR-MTB isolates. In a 64-case double-blind clinical isolates test, the sensitivity and specificity were 83% and 100%, respectively. In a 300-case raw sputum trial, the MTB identification sensitivity in smear-negative cases using MALDI-TOF MS was better than the COBAS assay (61.9% vs. 46.6%). Importantly, the failure rate of MALDI-TOF MS was better than COBAS (11.3% vs. 26.3%). To the best of our knowledge, the test described herein is the only multiplex test that predicts resistance for up to eight antibiotics with both sensitivity and flexibility.

Mutations of the Epidermal Growth Factor Receptor Gene in Triple-Negative Breast Cancer.

PurposeEpidermal growth factor receptor (EGFR) is considered a potential therapeutic target for anti-EGFR therapy in triple-negative breast cancer (TNBC). However, the frequency of EGFR gene mutation in TNBC is low and varies with ethnicity. This study aimed to investigate the incidence of EGFR gene mutation in TNBC.MethodsEGFR protein expression was evaluated by immunohistochemistry in tissue microarrays of 493 TNBC cases using four different primary antibodies, which included mutation-specific antibodies. For cases with an immunoreactivity level ≥1+, we performed pyrosequencing analysis for EGFR gene mutation. A case was considered mutation-positive when its mutation frequency minus its limit of detection (LOD) was >10%. Cases with mutation frequency higher than LOD were assessed for EGFR gene mutation status using the Cobas assay and by peptide nucleic acid-mediated polymerase chain reaction (PNA-clamping).ResultsAmong 493 TNBCs, 148 (30.0%) exhibited staining ≥1+ for EGFR, including 78 with 1+, 49 with 2+, and 21 with 3+. Positive EGFR expression (≥2+) was significantly associated with lymphovascular invasion (p=0.010), but not with overall survival (p=0.444) or disease-free survival (p=0.388). None of the 493 TNBCs harbored an EGFR gene mutation. Among 148 cases with an EGFR staining result ≥1+, five (3.4%) showed mutation frequencies (4.4%–10.9%) higher than LOD (2.6%–4.3%) in exons 19 (L747_P753>Q) or 21 (L858R and L861Q) as determined by pyrosequencing. However, Cobas and PNA-clamping failed to detect the presence of EGFR gene mutation in these five cases.ConclusionNo activating mutation of EGFR gene of clinical significance was observed in 148 TNBC cases using three commercially available methods. Thus, EGFR gene mutation appears to be an extremely rare event in patients with TNBC.

Detection of Epidermal Growth Factor Receptor Mutations in Lung Adenocarcinoma: Comparing Cobas 4800 EGFR Assay With Sanger Bidirectional Sequencing

IntroductionAccurate detection of epidermal growth factor receptor (EGFR) mutations has a crucial role in the current treatment of patients with lung adenocarcinoma, and identification of clinically relevant mutations would qualify patients for treatment with tyrosine kinase inhibitors. Historically, Sanger sequencing has been used as the reference standard assay for EGFR mutational analysis; however, Cobas 4800 is a relatively new method. In the present study, we compared the performance of the Cobas assay against that of Sanger sequencing. Materials and MethodsA total of 493 consecutive formalin-fixed paraffin-embedded samples of lung adenocarcinoma were simultaneously tested for EGFR mutations using both methods. ResultsAfter exclusion of the invalid results (n = 19), 474 samples from 455 patients were analyzed. The Cobas assay showed a mutation detection rate comparable to that of Sanger sequencing (18.1% vs. 17.9%, respectively; P < .05). Excellent agreement of 98.9% (κ, 0.964) was observed between the 2 methods. ConclusionThe Cobas assay is a fast and diagnostically robust platform with high analytical sensitivity; however, it is limited by its detection range and low tolerance to low DNA quality. Sanger sequencing is mostly affected by its lower analytic sensitivity. Ultimately, a dual testing strategy will be justified to increase the detection of novel mutations and reduce the false-negative results within an acceptable turnaround time.

Detection of EGFR mutations in lung adenocarcinoma; comparing cobas 4800 EGFR assay with Sanger bi-directional sequencing

Background: Accurate detection of EGFR mutation is crucial in management of patients with lung adenocarcinoma and would qualify them for targeted therapy. Historically Snager bi-directional sequencing (SBS) has been used as the gold standard for EGFR mutational analysis, however there are emerging new assays utilizing targeted real time PCR technology. In this study we compared the performance of ‘Cobas 4800’ (COBAS) against Sanger sequencing.Methods: 480 consecutive formalin fixed paraffin embedded samples of lung adenocarcinoma were simultaneously tested for EGFR mutations by SBS and COBAS. Mutational results were catogorised as positive, negative and invalid. Unweighted Kappa test was utilsed to compare the concordance between two assays.Results: After exclusion of invalid results (n=16), 477 samples from 458 patients (47.2% male, 52.8% female) underwent statistical analysis. There was an excellent observed percentage agreement of 98.3% (kappa value 0.944, SE 0.0194) between the two methods. The combined mutation detection rate (19%) was superior to either SBS (18.4%) or COBAS (18%). EGFR mutation frequency was significantly higher in women (23%) compare to men (12%).Discussion: COBAS assay is a diagnostically robust platform comparable with Sanger with very high analytical sensitivity and short turns around time. COBAS failure is due to lower sensitivity to samples with low DNA quality and its limited primer detection range, while Sanger is mostly affected by its lower analytic sensitivity related to low volume of diagnostic material. Consequently, the higher combined mutation detection rate necessitates a dual testing strategy to guarantee detection of novel mutations and mutations outside the COBAS detection range, and avoiding false negative results due to lower analytical sensitivity of SBS. Background: Accurate detection of EGFR mutation is crucial in management of patients with lung adenocarcinoma and would qualify them for targeted therapy. Historically Snager bi-directional sequencing (SBS) has been used as the gold standard for EGFR mutational analysis, however there are emerging new assays utilizing targeted real time PCR technology. In this study we compared the performance of ‘Cobas 4800’ (COBAS) against Sanger sequencing. Methods: 480 consecutive formalin fixed paraffin embedded samples of lung adenocarcinoma were simultaneously tested for EGFR mutations by SBS and COBAS. Mutational results were catogorised as positive, negative and invalid. Unweighted Kappa test was utilsed to compare the concordance between two assays. Results: After exclusion of invalid results (n=16), 477 samples from 458 patients (47.2% male, 52.8% female) underwent statistical analysis. There was an excellent observed percentage agreement of 98.3% (kappa value 0.944, SE 0.0194) between the two methods. The combined mutation detection rate (19%) was superior to either SBS (18.4%) or COBAS (18%). EGFR mutation frequency was significantly higher in women (23%) compare to men (12%). Discussion: COBAS assay is a diagnostically robust platform comparable with Sanger with very high analytical sensitivity and short turns around time. COBAS failure is due to lower sensitivity to samples with low DNA quality and its limited primer detection range, while Sanger is mostly affected by its lower analytic sensitivity related to low volume of diagnostic material. Consequently, the higher combined mutation detection rate necessitates a dual testing strategy to guarantee detection of novel mutations and mutations outside the COBAS detection range, and avoiding false negative results due to lower analytical sensitivity of SBS.

Performance and Verification of a Real-Time PCR Assay Targeting the gyrA Gene for Prediction of Ciprofloxacin Resistance in Neisseria gonorrhoeae.

In the United States, 19.2% of Neisseria gonorrhoeae isolates are resistant to ciprofloxacin. We evaluated a real-time PCR assay to predict ciprofloxacin susceptibility using residual DNA from the Roche Cobas 4800 CT/NG assay. The results of the assay were 100% concordant with agar dilution susceptibility test results for 100 clinical isolates. Among 76 clinical urine and swab specimens positive for N. gonorrhoeae by the Cobas assay, 71% could be genotyped. The test took 1.5 h to perform, allowing the physician to receive results in time to make informed clinical decisions.

Comparison of the bioMérieux NucliSENS EasyQ HIV-1 v2.0–HIV-1 RNA quantification assay versus Abbott RealTime HIV-1 and Roche Cobas TaqMan HIV-1 v2.0 on current epidemic HIV-1 variants

BackgroundAn improved version of the bioMérieux NucliSENS® EasyQ® HIV-1 v2.0 has been introduced to overcome the underquantification observed with previous versions, especially with non-B HIV-1 subtypes. ObjectivesComparing bioMérieux NucliSENS® EasyQ® HIV-1 v2.0 versus Roche Cobas CA/CTM v2.0 and Abbott RealTime HIV-1 assays for HIV-1 group M and non-M (N, O, P) viral load measurement. Study designThe three assays were tested in parallel on 103HIV-1 group M plasma samples, and on non-group M HIV-1 culture supernatants. ResultsValues obtained for the 103HIV-1 group M plasma samples tested with bioMérieux assay showed good overall correlation compared to the 2 others. The Roche Cobas assay gave higher values than the bioMérieux assay, while Abbott and bioMérieux both displayed similar results. However, analysis showed a wider dispersion in results when comparing the bioMérieux NucliSENS® EasyQ® HIV-1 v2.0 and the other 2 techniques. All data taken into account, we observed frequent discrepancies in quantification, of the plasma samples and major differences above 1log in 10/72 (13.8%). The quantification of non-M HIV groups in culture supernatant has shown variable results, with better quantification of HIV-O and of HIV-N respectively with the Abbott assay and the bioMérieux assay. ConclusionsThe bioMérieux NucliSENS® EasyQ® HIV-1 v2.0 showed improved sensitivity to non-B HIV-M subtypes compared to previous versions. Notwithstanding, we observed frequent discrepancies and a wide dispersion in results when comparing bioMérieux NucliSENS® EasyQ® HIV-1 v2.0 and the other 2 techniques.

Monitoring the quality of HIV-1 viral load testing through a proficiency testing program using dried tube specimens in resource-limited settings.

HIV-1 viral load (VL) levels are used for monitoring disease progression and antiretroviral therapy outcomes in HIV-infected patients. To assess the performance of laboratories conducting HIV-1 VL testing in resource-limited settings, the U.S. Centers for Disease Control and Prevention implemented a voluntary, free-of-charge, external quality assurance program using dried tube specimens (DTSs). Between 2010 and 2012, DTS proficiency testing (PT) panels consisting of 5 specimens were distributed at ambient temperature to participants. The results from the participants (n≥6) using the same assay were grouped, analyzed, and graded as acceptable within a group mean±3 standard deviations. Mean proficiency scores were calculated by dividing the combined PT scores by the number of testing cycles using a linear regression model. Between 2010 and 2012, the number of participants enrolled increased from 32 in 16 countries to 114 in 44 countries. A total of 78.2% of the participants reported results using 10 different VL assays. The rates of reporting of acceptable results by the participants were 96.6% for the Abbott assay, 96.3% for the Roche Cobas assay, 94.5% for the Roche Amplicor assay, 93.0% for the Biocentric assay, and 89.3% for the NucliSens assay. The overall mean proficiency scores improved over time (P=0.024). DTSs are a good alternative specimen type to plasma specimens for VL PT programs, as they do not require cold chain transportation and can be used on PCR-based assays. Our data suggest that the CDC HIV-1 VL PT program using DTSs positively impacts the testing performance of the participants, which might translate into better and more accurate VL testing services for patients.

Comparison of the Roche Cobas® 4800 HPV assay to Digene Hybrid Capture 2, Roche Linear Array and Roche Amplicor for Detection of High-Risk Human Papillomavirus Genotypes in Women undergoing treatment for cervical dysplasia

BackgroundThe recently FDA (U.S. food and drug administration) approved Roche Cobas® 4800 (Cobas) human papillomavirus (HPV) has limited performance data compared to current HPV detection methods for test of cure in women undergoing treatment for high grade lesions. ObjectiveEvaluation of Cobas HPV assay using historical samples from women undergoing treatment for cervical dysplasia. Study designA selection of 407 samples was tested on the Cobas assay and compared to previous results from Hybrid Capture 2, HPV Amplicor and Roche Linear Array. ResultsOverall, a correlation between high-risk HPV positivity and high grade histological diagnosis was 90.6% by the Cobas, 86.1% by Hybrid Capture 2, 92.9% by HPV Amplicor and 91.8% by Roche Linear Array. ConclusionThe Cobas HPV assay is comparative to both the HPV Amplicor and Roche Linear Array assays and better than Hybrid capture 2 assay in the detection of High-Risk HPV in women undergoing treatment for cervical dysplasia.

BRAF Mutation Testing in Solid Tumors: A Methodological Comparison

Solid tumor genotyping has become standard of care for the characterization of proto-oncogene mutational status, which has traditionally been accomplished with Sanger sequencing. However, companion diagnostic assays and comparable laboratory-developed tests are becoming increasingly popular, such as the cobas 4800 BRAF V600 Mutation Test and the INFINITI KRAS-BRAF assay, respectively. This study evaluates and validates the analytical performance of the INFINITI KRAS-BRAF assay and compares concordance of BRAF status with two reference assays, the cobas test and Sanger sequencing. DNA extraction from FFPE tissue specimens was performed followed by multiplex PCR amplification and fluorescent label incorporation using allele-specific primer extension. Hybridization to a microarray, signal detection, and analysis were then performed. The limits of detection were determined by testing dilutions of mutant BRAF alleles within wild-type background DNA, and accuracy was calculated based on these results. The INFINITI KRAS-BRAF assay produced 100% concordance with the cobas test and Sanger sequencing and had sensitivity equivalent to the cobas assay. The INFINITI assay is repeatable with at least 95% accuracy in the detection of mutant and wild-type BRAF alleles. These results confirm that the INFINITI KRAS-BRAF assay is comparable to traditional sequencing and the Food and Drug Administration-approved companion diagnostic assay for the detection of BRAF mutations.

Evaluation of 2 Real-Time PCR Assays for In Vitro Diagnostic Use in the Rapid and Multiplex Detection of EGFR Gene Mutations in NSCLC

Activating mutations of the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer predict for a favorable clinical response to tyrosine kinase inhibitor therapy. Although Sanger sequencing is a conventional method to detect EGFR gene mutations, multiplex real-time allele-specific polymerase chain reaction (PCR) systems are increasingly used in the routine molecular diagnostic setting. We aim to evaluate 2 proprietary real-time PCR assays (cobas and therascreen) against Sanger sequencing in the detection of EGFR gene mutations. The overall concordance rate between cobas and therascreen assays with Sanger sequencing was 89% and 88%, respectively, and increased to 96% and 98%, respectively, if the mutations not covered were excluded. The cobas assay showed a superior coverage of exon 20 mutations, but L861Q was not targeted. The nature of specimen, DNA integrity, and tumor cell content are factors that affect the assay performance. DNA extracted from cell block and clot of pleural fluid gave rise to 1 invalid call and 1 false-negative result by the cobas assay and 1 missed T790M mutation and 1 false-negative result by the therascreen assay. Both assays are around 5 times more expensive compared with Sanger sequencing in terms of reagent cost. We conclude that both assays prove to be a rapid, simple, and validated method in detecting the most common and clinically significant EGFR gene mutations in non-small cell lung cancer. Although less convenient compared with real-time PCR assays, Sanger sequencing is cheaper in terms of reagent cost and allows the detection of rare or novel EGFR gene mutations.

Detection of BRAF V600 Mutations in Metastatic Melanoma: Comparison of the Cobas 4800 and Sanger Sequencing Assays

Detection of BRAF V600 Mutations in Metastatic Melanoma: Comparison of the Cobas 4800 and Sanger Sequencing Assays

Comparison of the Xpert MTB/RIF and Cobas TaqMan MTB Assays for Detection of Mycobacterium tuberculosis in Respiratory Specimens

The Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA) is a fully automated, cartridge-based real-time PCR assay designed to detect Mycobacterium tuberculosis and rifampin resistance within 2 h. The performance of the Xpert assay has been evaluated in various clinical settings. However, there are few data comparing the Xpert assay to the Cobas TaqMan MTB test (Roche Diagnostics, Basel, Switzerland), one of the most widely utilized molecular assays for M. tuberculosis detection. In this prospective study, 320 consecutive respiratory specimens were processed simultaneously using acid-fast bacillus (AFB) staining, mycobacterial cultures with both solid and liquid media, and the Cobas and Xpert assays. The Xpert assay was performed with direct respiratory specimens, while the Cobas assay was done with decontaminated concentrated specimens. Based on the culture as a reference method, the overall sensitivities of the Cobas and Xpert assays were 71.4% and 67.9%, respectively. When AFB smear results were taken into consideration, the sensitivities of the Cobas assay for smear-positive and -negative specimens were 87% and 54%, while those of the Xpert assay were 67% and 69%, respectively. The Cobas assay showed 100% specificity and 100% positive predictive value (PPV) regardless of smear results, while the Xpert assay showed 100% specificity and 100% PPV for smear-positive specimens but 98% specificity and 60% PPV for smear-negative specimens. In conclusion, the Xpert assay showed performance that was slightly inferior to that of the Cobas assay but seems useful for the rapid detection of M. tuberculosis, considering that it was performed without laborious and time-consuming decontamination and concentration procedures.

Thyroid-stimulating hormone and free thyroxine on the ADVIA Centaur immunoassay system: A multicenter assessment of analytical performance

ObjectivesWe assessed the analytical performance of the TSH and FT4 assays on ADVIA Centaur in a multicenter national evaluation. Design and methodsA precision study and a method comparison were performed. Reference values stated by the manufacturer were checked from 379 normal subjects. ResultsFor TSH and FT4, the intra-assay CVs were below 2.3 and 5.2%, respectively, and the inter-assay CVs below 4.4% and 7.2%, respectively. Therefore, the precision and reproducibility were acceptable. Bland–Altman bias plots revealed good correlation and agreement with Cobas assays. TSH and FT4 data yielded reference ranges of 0.64–3.24mIU/L and 10.5–18.9pmol/L, respectively. ConclusionThese assays demonstrate reliable characteristics. The reference ranges obtained can be used for interpretation of thyroid function.