Interlaboratory Agreement Research Articles

Proficiency Testing of Standardized Samples Shows High Interlaboratory Agreement for Clinical Next Generation Sequencing-Based Hematologic Malignancy Assays With Survey Material-Specific Differences in Variant Frequencies.

As laboratories increasingly turn from single-analyte testing in hematologic malignancies to next-generation sequencing-based panel testing, there is a corresponding need for proficiency testing to ensure adequate performance of these next-generation sequencing assays for optimal patient care. To report the performance of laboratories on proficiency testing from the first 4 College of American Pathologists Next-Generation Sequencing Hematologic Malignancy surveys. College of American Pathologists proficiency testing results for 36 different engineered variants and/or allele fractions as well as a sample with no pathogenic variants were analyzed for accuracy and associated assay performance characteristics. The overall sensitivity observed for all variants was 93.5% (2190 of 2341) with 99.8% specificity (22 800 of 22 840). The false-negative rate was 6.5% (151 of 2341), and the largest single cause of these errors was difficulty in identifying variants in the sequence of CEBPA that is rich in cytosines and guanines. False-positive results (0.18%; 40 of 22 840) were most likely the result of preanalytic or postanalytic errors. Interestingly, the variant allele fractions were almost uniformly lower than the engineered fraction (as measured by digital polymerase chain reaction). Extensive troubleshooting identified a multifactorial cause for the low variant allele fractions, a result of an interaction between the linearized nature of the plasmid and the Illumina TruSeq chemistry. Laboratories demonstrated an overall accuracy of 99.2% (24 990 of 25 181) with 99.8% specificity and 93.5% sensitivity when examining 36 clinically relevant somatic single-nucleotide variants with a variant allele fraction of 10% or greater. The data also highlight an issue with artificial linearized plasmids as survey material for next-generation sequencing.

Autoantibody Diagnostics in Neuroimmunology: Experience From the 2018 Italian Neuroimmunology Association External Quality Assessment Program.

Background: Neuroimmunology has impressively expanded in the past decade. Novel assays, especially cell-based assays (CBAs) can detect conformational antibodies (Abs) recognizing antigens in their native conformation. Generally, the availability of in-house and of commercial tests has improved the diagnostics, but introduced demanding laboratory tasks. Hence, standardization and quality controls represent a key step to promote accuracy. We report on the results of the 2018 external quality assessment program (EQAP) organized by the Italian Neuroimmunology Association.Methods: EQAP regarded 10 schemes, including oligoclonal bands (OCBs), intracellular-neuronal (ICN)-Abs, neuronal-surface (NS)-Abs, aquaporin-4 (AQP4)-Abs, myelin oligodendrocyte glycoprotein (MOG)-Abs, myelin-associated glycoprotein (MAG)-Abs, ganglioside-Abs, acetylcholine-receptor (AChR)-Abs, and muscle-specific-kinase (MuSK)-Abs, and 34 laboratories. Assays were classified as tissue-based assays (TBAs), solid-phase assays (SPAs), liquid-phase assays (LPAs), and CBAs. Thirty-three samples were provided.Results: Three-quarter of the tests were commercial. Median accuracy for the laboratories was 75% (range 50–100). In 8/10 schemes, at least one sample provided discrepant results. Inter-laboratory “substantial agreement” was found in 6/10 schemes (AChR, MuSK, MAG, AQP4, MOG, and NS-Abs), whereas the worst agreements regarded OCBs and ganglioside-Abs. Both commercial and in-house assays performed better in experienced laboratories.Conclusions: Assays could be divided in (a) robust commercial tests with substantial inter-laboratory agreement (MAG-Abs; AChR- and MuSK-Abs); commercial/“in-house” tests with (b) partial inter-laboratory agreement (AQP4-Abs, MOG-Abs, NS-Abs, ICN-Abs), and (c) with large inter-laboratory disagreement (OCBs, ganglioside-Abs). This real-life snapshot of the neuroimmunology test performances highlights shortcomings attributable to technician-dependent performances, assay structural limitations, and errors in test interpretations.

Vitamin C measurement in critical illness: challenges, methodologies and quality improvements.

Background There is renewed interest in high-dose vitamin C interventions in clinical medicine due to its antioxidant properties, safe use and cost-effectiveness. Yet, randomised control trials (RCTs) employing these interventions are failing to include robust analytical methodology and proper sample handling and processing techniques. Consequently, comparisons between studies becomes impossible as there is no metrological traceability and results may be prone to pre-analytical errors. Content Through published vitamin C stability studies, method comparison papers and data from vitamin C external quality assurance programs, an assessment was made on the functionality of current methods for critically ill patient samples. Summary Data was obtained from two external quality assurance programs, two papers assessing sample stability and interlaboratory agreement and a publication on vitamin C method comparisons. A shift from spectrophotometric and enzymatic methodologies to high performance liquid chromatography (HPLC) greatly improved the variability and interlaboratory agreement. Therefore, the current analytical performance of vitamin C HPLC methodologies are acceptable for the requirements of a high-dose vitamin C RCTs. Outlook Recommendations across the total testing process of vitamin C have been provided to improve the quality of the results. The harmonisation of sample handling and processing procedures will further improve the reliability of current analytical methodologies.

Interlaboratory Concordance of ProMisE Molecular Classification of Endometrial Carcinoma Based on Endometrial Biopsy Specimens.

Molecular classifiers improve the consistency of categorization of endometrial carcinoma and provide valuable prognostic information. We aimed to evaluate the interlaboratory agreement in ProMisE assignment across 3 dedicated Canadian gynecologic oncology centers. Fifty cases of endometrial carcinoma diagnosed on biopsy were collected from 3 centers and 3 unstained sections were provided to each participating site so that immunohistochemistry for MSH6, PMS2, and p53 could be performed and interpreted at each center, blinded to the original diagnoses and the results from other centers. A core was taken for DNA extraction and POLE mutation testing. Overall accuracy and κ statistic were assessed. MSH6, PMS2, and p53 could be assessed for all 50 cases, with agreement for 140/150 results. There was a high level of agreement in molecular classification (κ=0.82), overall. Cases with a discordant result for one of the features used in classification (n=10) were reviewed independently and the most common reason for disagreement was attributable to the weak p53 staining in 1 laboratory (n=4). Interpretive error in PMS2 (n=1) and MSH6 (n=2) assessment accounted for 3 of the remaining disagreements. Interpretive error in the assessment of p53 was identified in 2 cases, with very faint p53 nuclear reactivity being misinterpreted as wild-type staining. These results show strong interlaboratory agreement and the potential for greater agreement if technical and interpretive factors are addressed. Several solutions could improve concordance: central quality control to ensure technical consistency in immunohistochemical staining, education to decrease interpretation errors, and the use of secondary molecular testing.

Intra- and inter-laboratory agreement of the disc diffusion assay for assessing antimicrobial susceptibility of porcine Escherichia coli

Reliable assessment of the susceptibility of animal bacterial pathogens to antimicrobials is of paramount importance in the fight against antimicrobial resistance. This work aims to estimate the repeatability (intra-laboratory agreement) and reproducibility (inter-laboratory agreement) of the disc diffusion assay in veterinary laboratories to understand further if the assay has a role in the surveillance of antimicrobial resistance in animals. Seven major veterinary laboratories from all States in Australia participated, and each tested the same panel of isolates five times at three to four-week intervals, against six antimicrobial agents using Clinical and Laboratory Standards Institute protocols. The panel consisted of twenty different isolates from porcine Escherichia coli from clinical cases and a single reference strain (ATCC 25922). Laboratories were blinded to the identity of the isolates, replicates, and to each other. In total, 4200 inhibition zone diameters (mm) were collected, and analysed descriptively, graphically, and with linear mixed models. Regardless of the laboratories and isolate/antimicrobial combinations, the overall very major error rate (proportion of isolates classified as susceptible when actual status is resistant) was 1.6%; the major error rate (proportion of isolates classified as resistant when actual status is susceptible) was 1.6%; and the ‘minor error’ rate (proportion of isolates with intermediate susceptibility that measure fully susceptible or resistant or vice versa) was 2.4%. The variation between repeated measurements ranged between 4.4–7.2 mm depending on the antimicrobial agent assessed. The reproducibility was always more variable than the repeatability, which suggested some laboratory effects. The repeatability coefficient of disc diffusion was lowest for tetracycline (4.4 mm, 95% CI: 3.8–5.0 mm) and ampicillin (4.6 mm, 95% CI: 4.2–5.2 mm) and highest for trimethoprim-sulfamethoxazole (6.6 mm, 95% CI: 5.9–7.4 mm). The reproducibility coefficient of disc diffusion was lowest for gentamicin (5.4, 95% CI: 4.0–7.2) and highest for trimethoprim-sulfamethoxazole (7.2 mm, 95%CI: 4.5–11.7 mm). The precision of the disc diffusion assay was deemed satisfactory for use in a national surveillance program for clinical porcine E. coli isolates. However, measurement variation of the disc diffusion assay is of concern for isolates with marginal susceptibility or resistance due to increased risk of misclassification.

Effects of Improved 17O Correction on Interlaboratory Agreement in Clumped Isotope Calibrations, Estimates of Mineral‐Specific Offsets, and Temperature Dependence of Acid Digestion Fractionation

AbstractThe clumped isotopic composition of carbonate‐derived CO2 (denoted Δ47) is a function of carbonate formation temperature and in natural samples can act as a recorder of paleoclimate, burial, or diagenetic conditions. The absolute abundance of heavy isotopes in the universal standards VPDB and VSMOW (defined by four parameters: R13VPDB, R17VSMOW, R18VSMOW, and λ) impact calculated Δ47 values. Here, we investigate whether use of updated and more accurate values for these parameters can remove observed interlaboratory differences in the measured T‐Δ47 relationship. Using the updated parameters, we reprocess 14 published calibration data sets measured in 11 different laboratories, representing many mineralogies, bulk compositions, sample types, reaction temperatures, and sample preparation and analysis methods. Exploiting this large composite data set (n = 1,253 sample replicates), we investigate the possibility for a “universal” clumped isotope calibration. We find that applying updated parameters improves the T‐Δ47 relationship (reduces residuals) within most labs and improves overall agreement but does not eliminate all interlaboratory differences. We reaffirm earlier findings that different mineralogies do not require different calibration equations and that cleaning procedures, method of pressure baseline correction, and mass spectrometer type do not affect interlaboratory agreement. We also present new estimates of the temperature dependence of the acid digestion fractionation for Δ47 (Δ*25‐X), based on combining reprocessed data from four studies, and new theoretical equilibrium values to be used in calculation of the empirical transfer function. Overall, we have ruled out a number of possible causes of interlaboratory disagreement in the T‐Δ47 relationship, but many more remain to be investigated.

Collaborative national multicenter for the identification of conversion factors from copies/mL to international units/mL for the normalization of HCMV DNA load

The present multicentric (n = 11 laboratories) study aimed to identify conversion factors from copies/mL to international units (IU)/mL for the normalization of HCMV DNA load using the first WHO International Standard for HCMV nucleic acid amplification techniques and to enhance interlaboratory agreement of HCMV DNA quantification methods. Study protocols for whole blood and plasma (extraction and amplification) were performed to calculate conversion factors from HCMV DNA copy number to IU. The greatest variability was observed in samples with lower HCMV concentrations (3.0 Log10) in both biological matrices. Overall, 73.1% (206/282) of whole blood and 82.2% (324/394) of plasma samples analyzed fell within an acceptable variation range (±0.5 Log10 difference). An average of 0.64 (range 0.21–1.17) was the conversion factor calculated for the HCMV whole blood panel and 0.82 (range 0.39–2.2) for the HCMV plasma panel.

Electrical characterization intercomparison of high‐efficiency c‐Si modules within Asian and European laboratories

AbstractThis work presents the results of a high‐efficiency (HE) photovoltaic (PV) module round‐robin intercomparison between five Asian and European ISO/IEC 17025 accredited laboratories and one industrial laboratory based in Europe. The scope of the round‐robin was to examine the measurements comparability for this PV technology with respect to ISO/IEC 17025 laboratory conformity assessment and also to examine the accuracy of step‐like methods towards transient errors against already validated methods. The devices under test were four types of HE c‐Si PV modules with efficiencies varying between 16.5% and 19.0%. The results indicate that a satisfactory agreement was achieved with maximum deviations of 1.59% in Pmax, 1.13% in Isc, and 0.64% in Voc for all devices under test. The weighted standard deviations in Pmax per device type, which can be seen as a conservative estimate of interlaboratory agreement for HE c‐Si PV, ranged within 0.82% to 2.23% (k = 2). The accuracy of step‐like methods towards transient errors was evaluated by comparing a second series of results at fixed Isc for each module under test, eliminating the influence of the effective irradiance measurement. This work suggests that the contribution of capacitive errors was in the range (0.47 ± 0.19) % (k = 2). A spectral mismatch sensitivity analysis showed that an accurate measurement of the spectral irradiance and of the involved spectral responsivities together with the punctual correction for the spectral mismatch can reduce the error in the measurement of PV modules performance of about 2% even in the case of c‐Si against c‐Si and class AAA solar simulators.

HPV testing for cervical cancer screening: technical improvement of laboratory logistics and good clinical performance of the cobas 6800 in comparison to the 4800 system

BackgroundEuropean guidelines for cervical cancer screening now recommend the use of clinically validated assays for high-risk HPV-DNA sequences as primary test in women older than 30 years, performed in centralized laboratories, and run on systems providing automated solutions for all steps.MethodsWe conducted a comparison study, according to the international guidelines, nested within the organized population-based cervical screening program, between the cobas 4800 and 6800 systems (Roche Diagnostics), to evaluate accuracy and reproducibility of HPV test results and laboratory workflow. In Italy implementation of HPV cervical screening is under way on a regional basis; in Veneto it started in June 2015, following a piloting phase; the assay in use in the three centralized laboratories is the cobas 4800 HPV test, run on the cobas 4800 system. Comparison of HPV results with a new version of the assay (cobas 6800/8800 HPV) run on the cobas 6800 system, and intra- and inter-reproducibility analyses have been conducted in samples collected in PreservCyt medium (Hologic) from women without and with a subsequent diagnosis of high-grade lesion.ResultsSamples from women older than 30 years attending organized cervical cancer screening were used. Clinical sensitivity and specificity were evaluated on 60 cases and 925 controls, respectively; intra-laboratory reproducibility and inter-laboratory agreement by the 6800 system were evaluated on 593 and 460 specimens, respectively. Our results showed a very high agreement (> 98%) for overall qualitative results between the two systems; clinical sensitivity and specificity of the HPV assay run on 6800 were non-inferior to those of the HPV assay run on 4800 (p = 0,0157 and p = 0,0056, respectively, at the recommended thresholds of 90 and 98%); kappa values of 0.967 and 0.969 were obtained for intra- and inter-laboratory reproducibility analyses in the 6800 system. The 6800 platform displayed several technological improvements over the 4800 system, with higher throughput and laboratory productivity, and lower operator’s hands-on time.ConclusionsThe new cobas 6800/8800 HPV assay run on the 6800 instrument is suitable for use in large centralized laboratories included within population-based cervical cancer screening programs.

Laboratory validation studies in Ki-67 digital image analysis of breast carcinoma: a pathway to routine quality assurance

Ki-67 proliferative index (PI) has prognostic and predictive value in invasive breast carcinoma (IBC), but clinical uptake has been hampered by suboptimal accuracy, reproducibility and standardisation. Published guidelines have addressed pre-analytical and analytical factors to improve Ki-67 PI utility; however, practicalities of ongoing monitoring of Ki-67 PI quality in IBC reporting have not been established. We aimed to evaluate the internal and external quality of our established digital Ki-67 PI IBC reporting practice at a tertiary institution. In the 5 years since initial validation work, we've completed a series of internal and external quality assurance (QA) projects: (1) an interobserver agreement study, (2) a two site interlaboratory agreement study, (3) determination of the error of our Ki-67 results, (4) an audit of the year-to-year Ki-67 values, (5) an audit of Ki-67 in neoadjuvant chemotherapy (NAC) treated cases, and (6) comparison of our Ki-67 datasets with similar published datasets. There was excellent concordance (intra-class correlation = 0.98) and good agreement [kappa (κ) = 0.76-0.96] between pathologists, excellent concordance [Pearson correlation (R) = 0.94] and very good agreement (κ = 0.80) between laboratories and excellent concordance (R = 0.92-0.95) and good agreement (κ = 0.67-1.0) over time for our Ki-67 results. No significant difference was observed in Ki-67 data from year-to-year. Expected associations with clinico-pathological prognosticators, pathological complete response following NAC and mitotic index were evident. The median Ki-67 values from the overall and NAC treated datasets were within the range reported in other studies, and our data could be separated into similarly proportioned 'high' and 'low' Ki-67 PI groups when dichotomised as per protocols in other studies. Collectively, our work provides evidence of adequate internal and external quality control for our digital Ki-67 PI IBC reporting protocols. Given the paucity of formal Ki-67 QA programs, our approach could be emulated, and results compared between laboratories as a framework for internal and external Ki-67 QA.

Serum vascular endothelial growth factor-D as a diagnostic and therapeutic biomarker for lymphangioleiomyomatosis.

BackgroundIn lymphangioleiomyomatosis (LAM), tuberous sclerosis gene mutations activate the mechanistic target of the rapamycin pathway, resulting in vascular endothelial growth factor-D (VEGF-D) overproduction. While the utility of serum VEGF-D testing for the diagnosis of LAM is outlined in ATS/JRS LAM Guidelines, the assay has not been fully validated for Asian populations. Our aims were to validate serum VEGF-D testing in Japan, by directly comparing measurements in Japan and the U.S., determining the diagnostic cut-off for serum VEGF-D levels among the Japanese women with typical thin walled cystic change on CT, and determining the performance of VEGF-D as a prognostic biomarker.Subjects and methodsWe determined serum VEGF-D levels from 108 LAM patients, 14 disease controls, and 51 healthy volunteers from the Japanese population. Measurements of 61 LAM patients were compared to those from the principal VEGF-D laboratory in the U.S at Cincinnati Children’s Hospital Medical Center. We correlated baseline serum VEGF-D levels with baseline and longitudinal clinical data to determine how pregnancy, sirolimus or gonadotrophin-releasing hormone (GnRH) agonists influence serum VEGF-D levels.ResultsSerum VEGF-D measurements in Japan and the U.S. were very similar. Baseline serum VEGF-D levels effectively distinguished LAM from other diseases and healthy volunteers at a cut-off level of 645 pg/ml and were diagnostically specific at 800 pg/ml, consistent with the recommendations of the ATS/JRS LAM Guidelines. Baseline serum VEGF-D correlated negatively with the DLco baseline % predicted and with the annual decrease in DLco % predicted. There was no significant association between baseline serum VEGF-D level and the outcomes of death or transplant. Serum VEGF-D levels markedly decreased during treatment with sirolimus, but not with GnRH analogues. Serum VEGF-D levels of most LAM patients did not increase over time, and neither pregnancy nor menopause significantly modulated serum VEGF-D levels.ConclusionsSerum VEGF-D is a useful diagnostic and therapeutic biomarker for LAM. Satisfactory precision and international inter-laboratory agreement of the clinical assay support VEGF-D recommendations in the ATS/JRS LAM Guidelines for the Japanese population.

Method-Dependent Epidemiological Cutoff Values for Detection of Triazole Resistance in Candida and Aspergillus Species for the Sensititre YeastOne Colorimetric Broth and Etest Agar Diffusion Methods

Although the Sensititre Yeast-One (SYO) and Etest methods are widely utilized, interpretive criteria are not available for triazole susceptibility testing of Candida or Aspergillus species. We collected fluconazole, itraconazole, posaconazole, and voriconazole SYO and Etest MICs from 39 laboratories representing all continents for (method/agent-dependent) 11,171 Candida albicans, 215 C. dubliniensis, 4,418 C. glabrata species complex, 157 C. guilliermondii (Meyerozyma guilliermondii), 676 C. krusei (Pichia kudriavzevii), 298 C. lusitaniae (Clavispora lusitaniae), 911 C. parapsilosis sensu stricto, 3,691 C. parapsilosis species complex, 36 C. metapsilosis, 110 C. orthopsilosis, 1,854 C. tropicalis, 244 Saccharomyces cerevisiae, 1,409 Aspergillus fumigatus, 389 A. flavus, 130 A. nidulans, 233 A. niger, and 302 A. terreus complex isolates. SYO/Etest MICs for 282 confirmed non-wild-type (non-WT) isolates were included: ERG11 (C. albicans), ERG11 and MRR1 (C. parapsilosis), cyp51A (A. fumigatus), and CDR2 and CDR1 overexpression (C. albicans and C. glabrata, respectively). Interlaboratory modal agreement was superior by SYO for yeast species and by the Etest for Aspergillus spp. Distributions fulfilling CLSI criteria for epidemiological cutoff value (ECV) definition were pooled, and we proposed SYO ECVs for S. cerevisiae and 9 yeast and 3 Aspergillus species and Etest ECVs for 5 yeast and 4 Aspergillus species. The posaconazole SYO ECV of 0.06 µg/ml for C. albicans and the Etest itraconazole ECV of 2 µg/ml for A. fumigatus were the best predictors of non-WT isolates. These findings support the need for method-dependent ECVs, as, overall, the SYO appears to perform better for susceptibility testing of yeast species and the Etest appears to perform better for susceptibility testing of Aspergillus spp. Further evaluations should be conducted with more Candida mutants.

External Quality Assessment of 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) assays

The discovery that mutations of the CYP24A1 gene are a cause of idiopathic infantile hypercalcemia (IIH) has revived interest in measuring serum 24,25(OH)2D3. Several studies have also suggested that a high 25-hydroxyvitamin D3(25-OHD3):24,25(OH)2D3 ratio might provide additional diagnostic information in the investigation of vitamin D deficiency. Measurement of 24,25(OH)2D3 is necessarily restricted to laboratories with mass spectrometry methods although cross reactivity of the metabolite in immunoassays for 25-OHD is a potential cause of misleading results. The international External Quality Assessment (EQA) scheme for vitamin D metabolites (DEQAS) was set up in 1989. In 2013 DEQAS became an accuracy based EQA for 25-OHD with ‘target values’ assigned by the National Institute of Standards and Technology (NIST) Reference Measurement Procedure (RMP). A pilot scheme for serum 24,25(OH)2D3 was started in 2015 and participants were asked to measure the metabolite on each of the 5 samples sent out for 25-OHD. Inter-laboratory agreement was poor but this may reflect methodological differences, in particular different approaches to assay standardization. An important potential contribution to reducing variability among assays was the development by NIST of a 24,25(OH)2D3 RMP and its use in assigning values to SRMs 972a, 2973 and 2971, supported by the NIH Office of Dietary Supplements (ODS) as part of the Vitamin D Standardization Program (VDSP) effort.

Validation of the Dual-path Platform chromatographic immunoassay (DPP® CVL rapid test) for the serodiagnosis of canine visceral leishmaniasis.

BACKGROUND Visceral leishmaniasis is a major public health challenge in South America, and dogs are its main urban reservoir. OBJECTIVE Validation of the canine Dual-path Platform immunoassay for canine visceral leishmaniasis (DPP® CVL) for a sample set composed of 1446 dogs from different Brazilian endemic areas. METHODS A well-defined reference standard by means of parasitological culture, immunohistochemistry, and histopathology was used. Animals were classified as asymptomatic, oligosymptomatic, or symptomatic. Sensitivity and specificity were assessed as a single set and in clinical groups. A reproducibility assessment of the tests was conducted using the Kappa (κ) index at three different laboratories (A, B, and C). FINDINGS Overall, 89% sensitivity and 70% specificity were obtained for the entire sample set. Analysis of the clinical groups showed a gradual decrease in the sensitivity and an increase in the specificity with the reduction of clinical signs in the dogs that were assessed, reaching a sensitivity of 75% (42.8-94.5%) among asymptomatic dogs and lower specificity of 56% (46.2-66.3%) among symptomatic dogs. Inter-laboratory agreement was substantial (κAB= 0.778; κAC= 0.645; κCB= 0.711). MAIN CONCLUSIONS The test performance is somewhat dependent on canine symptomatology, but such influence was less evident than in previous studies. Favourable results for sensitivity and specificity can be obtained even in asymptomatic animals; however, caution is needed in these evaluations, and the results suggest that the immunochromatographic test may be further improved for better investigation in asymptomatic dogs. The results obtained confirm the usefulness of DPP® CVL for application in serological surveys.

Proficiency Testing of Standardized Samples Shows Very High Interlaboratory Agreement for Clinical Next-Generation Sequencing-Based Oncology Assays.

Next-generation sequencing-based assays are being increasingly used in the clinical setting for the detection of somatic variants in solid tumors, but limited data are available regarding the interlaboratory performance of these assays. To examine proficiency testing data from the initial College of American Pathologists (CAP) Next-Generation Sequencing Solid Tumor survey to report on laboratory performance. CAP proficiency testing results from 111 laboratories were analyzed for accuracy and associated assay performance characteristics. The overall accuracy observed for all variants was 98.3%. Rare false-negative results could not be attributed to sequencing platform, selection method, or other assay characteristics. The median and average of the variant allele fractions reported by the laboratories were within 10% of those orthogonally determined by digital polymerase chain reaction for each variant. The median coverage reported at the variant sites ranged from 1922 to 3297. Laboratories demonstrated an overall accuracy of greater than 98% with high specificity when examining 10 clinically relevant somatic single-nucleotide variants with a variant allele fraction of 15% or greater. These initial data suggest excellent performance, but further ongoing studies are needed to evaluate the performance of lower variant allele fractions and additional variant types.

Validation of the Dual-path Platform chromatographic immunoassay (DPP® CVL rapid test) for the serodiagnosis of canine visceral leishmaniasis

BACKGROUND Visceral leishmaniasis is a major public health challenge in South America, and dogs are its main urban reservoir. OBJECTIVE Validation of the canine Dual-path Platform immunoassay for canine visceral leishmaniasis (DPP® CVL) for a sample set composed of 1446 dogs from different Brazilian endemic areas. METHODS A well-defined reference standard by means of parasitological culture, immunohistochemistry, and histopathology was used. Animals were classified as asymptomatic, oligosymptomatic, or symptomatic. Sensitivity and specificity were assessed as a single set and in clinical groups. A reproducibility assessment of the tests was conducted using the Kappa (κ) index at three different laboratories (A, B, and C). FINDINGS Overall, 89% sensitivity and 70% specificity were obtained for the entire sample set. Analysis of the clinical groups showed a gradual decrease in the sensitivity and an increase in the specificity with the reduction of clinical signs in the dogs that were assessed, reaching a sensitivity of 75% (42.8-94.5%) among asymptomatic dogs and lower specificity of 56% (46.2-66.3%) among symptomatic dogs. Inter-laboratory agreement was substantial (κAB= 0.778; κAC= 0.645; κCB= 0.711). MAIN CONCLUSIONS The test performance is somewhat dependent on canine symptomatology, but such influence was less evident than in previous studies. Favourable results for sensitivity and specificity can be obtained even in asymptomatic animals; however, caution is needed in these evaluations, and the results suggest that the immunochromatographic test may be further improved for better investigation in asymptomatic dogs. The results obtained confirm the usefulness of DPP® CVL for application in serological surveys.

Validation of intra- and inter-laboratory reproducibility of the Xpert HPV assay according to the international guidelines for cervical cancer screening

BackgroundCervical cancer screening with assays detecting DNA of high-risk human papillomavirus (hrHPV) types is more effective than cytology-based screening. This study completes the diagnostic accuracy assessment conducted previously within the framework of VALGENT-2 (Validation of HPV genotyping Tests) and aims to determine whether the reproducibility of Xpert HPV is in line with international validation criteria.MethodsValidation of new hrHPV DNA assays requires demonstration of good reproducibility and non-inferior clinical accuracy for cervical precancer compared to a standard comparator assay. The international reproducibility criteria are: lower bound of 95% confidence interval of the intra- and inter-laboratory agreement regarding detection of high-risk HPV DNA exceeding 87% with kappa ≥0.5.ResultsThe Xpert HPV assay showed high intra-laboratory reproducibility with an overall positivity/negativity agreement of 96.9% and a kappa of 0.925. Inter-laboratory testing showed an agreement of 97.8% with a kappa of 0.948.ConclusionsThe Xpert HPV assay fulfills the HPV test reproducibility criterion requirement for use in cervical cancer screening.

Development and interlaboratory agreement of real-time PCR for HPV16 quantification in liquid-based cervical samples

High risk HPV infection is the necessary cause for the development of precancerous and cancerous lesions of the cervix. Among HPV, HPV16 represents the most carcinogenic type. Since the determination of HPV16 DNA load could be clinically useful, we assessed quantitative real-time PCR targeting E6HPV16 and albumin genes on two different platforms. Series of SiHa cells diluted in PreservCyt were used to assess repeatability and reproducibility of two in-house real-time PCR techniques run in two different laboratories to determine HPV16 load. Furthermore, 97 HPV16 positive cervical samples were evaluated to estimate inter-center variability using Bland-Alman plots. As a whole, both techniques presented coefficients of variation for HPV16 load measurement similar to those established for other virus quantification with commercial kits. Moreover, the two real-time PCR techniques showed a very good agreement for HPV16 load calculation. Finally, we emphasize that robust HPV16 DNA quantification requires normalization of viral load by the cell number.

Fosfomycin Etest for Enterobacteriaceae: Interobserver and interlaboratory agreement

ObjectivesThe increasing use of fosfomycin requires reliable susceptibility testing in clinical practice. The reference standard, agar dilution (AD), is rarely used in routine settings. The fosfomycin Etest (BioMérieux) is frequently used, although reading MICs can be hampered by the interpretation of the growth of macrocolonies in the inhibition zone. We investigated the interobserver (IO), interlaboratory (IL), and interobserver-interlaboratory (IOIL) agreement of the fosfomycin Etest and evaluated the agreement with AD. MethodsEtests were performed for 57 extended spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae of four bacterial species (Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca and Enterobacter cloacae) in two laboratories. Photographs of fosfomycin Etests were interpreted by four observers following manufacturer's instructions. ResultsEssential agreement (EA) and categorical agreement (CA) between Etest and AD were 57% and 89% (κ-value 0.68), respectively, with an underestimation of Etest interpretations compared with AD of 0.26 (95% confidence interval [CI] 0.03-0.48) 2-fold dilutions. Between Etest observations, IO-EA and -CA were reached in 82% and 94% of comparisons; IL-EA and -CA in 38% and 85% of comparisons; and IOIL-EA and -CA in 40% and 85% of comparisons, respectively. Agreement of the Etest with AD and between Etests was better for E. coli than for other species. Ignoring all macrocolonies and haze during Etest interpretation improved the agreement with AD (CA κ-value 0.80) and between Etests (CA κ-value from 0.68 to 0.81). ConclusionsIn this study on 57 ESBL-producing Enterobacteriaceae, IOIL agreement was low with an EA of 40% and a CA of 85%, affected most by IL agreement and to a lesser extent by IO agreement.

Interlaboratory Comparison of Six Real-Time PCR Assays for Detection of Bovine Leukemia Virus Proviral DNA

Quantitative real-time PCR (qPCR) is increasingly being used for the detection of bovine leukemia virus (BLV) proviral DNA. Nevertheless, quality control for the validation and standardization of such tests is currently lacking. Therefore, the present study was initiated by three Office International des Epizooties (OIE) reference laboratories and three collaborating laboratories to measure the interlaboratory variability of six already developed and available BLV qPCR assays. For that purpose, an international panel of 58 DNA samples reflecting the dynamic range of the majority of the assays was distributed to six testing centers. Based on qualitative results, the overall agreement among all six laboratories was moderate. However, significant variability in the measurement of the BLV proviral DNA copy number was observed among different laboratories. Quantitative PCR assays, even when performed by experienced staff, can yield large variability in BLV proviral DNA copy numbers without harmonization. Further standardization of different factors (i.e., utilization of unified protocols and unique calibrators) should increase interlaboratory agreement.