Multinational proficiency tests for EGFR exon 20 insertions reveal that the assay design matters.

Insertion mutations in exon 20 of the epidermal growth factor receptor gene (EGFR exon20ins) are rare, heterogeneous alterations observed in non-small cell lung cancer (NSCLC). With a few exceptions, they are associated with primary resistance to established EGFR tyrosine kinase inhibitors (TKIs). As patients carrying EGFR exon20ins may be eligible for treatment with novel therapeutics-the bispecific antibody amivantamab, the TKI mobocertinib, or potential future innovations-they need to be identified reliably in clinical practice for which quality-based routine genetic testing is crucial. Spearheaded by the German Quality Assurance Initiative Pathology two international proficiency tests were run, assessing the performance of 104 participating institutes detecting EGFR exon20ins in tissue and/or plasma samples. EGFR exon20ins were most reliably identified using next-generation sequencing (NGS). Interestingly, success rates of institutes using commercially available mutation-/allele-specific quantitative (q)PCR were below 30% for tissue samples and 0% for plasma samples. Most of these mutation-/allele-specific (q)PCR assays are not designed to detect the whole spectrum of EGFR exon20ins mutations leading to false negative results. These data suggest that NGS is a suitable method to detect EGFR exon20ins in various types of patient samples and is superior to the detection spectrum of commercially available assays.

The development and implementation of a proficiency testing program for SARS-CoV-2 using dried tube specimens in resource-limited countries.

INTRODUCTION: When COVID-19 hit the world in 2019, an enhanced focus on diagnostic testing for SARS-CoV-2 was essential for a successful pandemic response. Testing laboratories stretched their capabilities for the new coronavirus by adopting different test methods. The necessity of having external quality assurance (EQA) mechanisms was even more critical due to this rapid expansion. However, there was a lack of experience in providing the necessary SARS-CoV-2 EQA materials, especially in locations with constrained resources. OBJECTIVE: We aimed to create a PT (Proficiency testing) programme based on the Dried Tube Specimens (DTS) method that would be a practical option for molecular based SARS-CoV-2 EQA in Low- and Middle-Income Countries. METHODS: Based on previous ISO/IEC 17043:2010 accreditation experiences and with assistance from the US Centers for Disease Control and Prevention, The Supranational Reference Laboratory of Uganda (adapted the DTS sample preparation method and completed a pilot EQA program between 2020 and 2021. Stability and panel validation testing was conducted on the designed materials before shipping to pilot participants in six African countries. Participants received a panel containing five SARS-CoV-2 DTS samples, transported at ambient conditions. Results submitted by participants were compared to validation results. Participants were graded as satisfactory (≥ 80%) or unsatisfactory (< 80%) and performance reports disseminated. RESULTS: Our SARS-CoV-2 stability experiments showed that SARS-CoV-2 RNA was stable (-15 to -25 °C, 4 to 8 °C, (18 to 28 °C) room temperature and 35 to 38 °C) as well as DTS panels (4 to 8 °C, 18 to 28 °C, 35 to 38 °C and 45 °C) for a period of 4 weeks. The SARS-CoV-2 DTS panels were successfully piloted in 35 test sites from Zambia, Malawi, Mozambique, Nigeria, and Seychelles. The pilot results of the participants showed good accuracy, with an average of 86% (30/35) concordance with the original SARS CoV-2 expectations. CONCLUSION: The SARS-CoV-2 DTS PT panel is reliable, stable at ambient temperature, simple to prepare and requires minimal resources.

Sources of variability in Luminex bead-based cytokine assays: Evidence from twelve years of multi-site proficiency testing.

Bead array assays, such as those sold by Luminex, BD Biosciences, Sartorius, Abcam and other companies, are a well-established platform for multiplexed quantification of cytokines and other biomarkers in both clinical and discovery research environments. In 2011, the National Institute of Allergy and Infectious Diseases (NIAID)-funded External Quality Assurance Program Oversight Laboratory (EQAPOL) established a proficiency assessment program to monitor participating laboratories performing multiplex cytokine measurements using Luminex bead array technology. During every assessment cycle, each site was sent an assay kit, a protocol, and blinded samples of human sera spiked with recombinant cytokines. Site results were then evaluated for performance relative to peer laboratories. After over a decade of biannual assessments, the cumulative dataset contained over 15,500 bead array observations collected at more than forty laboratories in twelve countries. These data were evaluated alongside post-assessment survey results to empirically test factors that may contribute to variability and accuracy in Luminex bead-based cytokine assays. Bead material, individual technical ability, analyte, analyte concentration, and assay kit vendor were identified as significant contributors to assay performance. In contrast, the bead reader instrument model and the use of automated plate washers were found not to contribute to variability or accuracy, and sample results were found to be highly-consistent between assay kit-manufacturing lots and over time. In addition to these statistical analyses, subjective evaluations identified technical ability, instrument failure, protocol adherence, and data transcription errors as the most common causes of poor performance in the proficiency program. The findings from the EQAPOL multiplex program were then used to develop recommended best practices for bead array monitoring of human cytokines. These included collecting samples to assay as a single batch, centralizing analysis, participating in a quality assurance program, and testing samples using paramagnetic-bead kits from a single manufacturer using a standardized protocol.

Continued improvement in the development of the SARS-CoV-2 whole genome sequencing proficiency testing program.

Application of whole genome sequencing (WGS) has allowed monitoring of the emergence of variants of concern (VOC) of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) globally. Genomic investigation of emerging variants and surveillance of clinical progress has reduced the public health impact of infection during the COVID-19 pandemic. These steps required developing and implementing a proficiency testing program (PTP), as WGS has been incorporated into routine reference laboratory practice. In this study, we describe how the PTP evaluated the capacity and capability of one New Zealand and 14 Australian public health laboratories to perform WGS of SARS-CoV-2 in 2022. The participants' performances in characterising a specimen panel of known SARS-CoV-2 isolates in the PTP were assessed based on: (1) genome coverage, (2) Pango lineage, and (3) sequence quality, with the choice of assessment metrics refined based on a previously reported assessment conducted in 2021. The participants' performances in 2021 and 2022 were also compared after reassessing the 2021 results using the more stringent metrics adopted in 2022. We found that more participants would have failed the 2021 assessment for all survey samples and a significantly higher fail rate per sample in 2021 compared to 2022. This study highlights the importance of choosing appropriate performance metrics to reflect better the laboratories' capacity to perform SARS-CoV-2 WGS, as was done in the 2022 PTP. It also displays the need for a PTP for WGS of SARS-CoV-2 to be available to public health laboratories ongoing, with continuous refinements in the design and provision of the PTP to account for the dynamic nature of the COVID-19 pandemic as SARS-CoV-2 continues to evolve.

Proficiency of phenotypic drug susceptibility testing for Mycobacterium tuberculosis in China, 2008-2021.

To analyze the results of proficiency testing for anti-tuberculosis drug susceptibility testing (DST) in China. Number of laboratory participating the proficiency testing performed DST, and the sensitivity, specificity, reproducibility, and accordance rate were calculated from data of 13 rounds proficiency testing results for DST from 2008 to 2021. A total of 30 and 20 strains of Mycobacterium tuberculosis with known susceptibility results were sent to each laboratory in 2008 to 2019, 2020 and 2021, respectively. The number of participating laboratories ranged from 30 in 2009 to 546 in 2021. L-J DST was the predominant method. The specificity presented relatively higher than sensitivity. Improvement of specificity were observed for all drugs through the years, while sensitivity did not show improvement for amikacin and capreomycin. Accordance rate of pyrazinamide and kanamycin and reproducibility of capreomycin and pyrazinamide were not significantly improved through the years. Most of the participating laboratories significantly improved the quality of their DST through the consecutive rounds of proficiency testing except for second-line injectable drugs and pyrazinamide. The results highlight the importance of developing novel and/or improving existing methods for phenotypic DST for certain drugs.

Proficiency testing for event-specific quantification of genetically modified maize MON87427: Performance assessment based on the metrologically traceable reference values as assigned values.

The Asia Pacific Metrology Program and the Accreditation Cooperation joint Proficiency Testing (PT) program for the quantification of genetically modified maize MON87427 was organized by the National Institute of Metrology, China, to enhance the measurement accuracy and metrological traceability in the region. Certified reference materials were employed as test samples; metrologically traceable certified reference values served as PT reference values (PTRVs) for evaluating the participants results. The consensus values obtained from the participants were higher than the assigned values, potentially due to the systematic effects of DNA extraction process. The participants' relatively poor overall performance by the ζ-score compared with z-score demonstrates their need to thoroughly investigate quantification bias to elevate the measurement capability of genetically modified (GM) content and deepen their understanding of uncertainty estimation. This program confirmed the importance of using metrologically traceable reference values instead of consensus values as PTRV for reliable performance assessment.

External quality assurance in the era of standardization.

Metrology in clinical chemistry aims to ensure the equivalence of measurement results from different in-vitro diagnostic measurement devices (IVD MD) for use in healthcare. The metrological traceability of measurement results to higher-order references is the cornerstone to achieving equivalent results. However, other fundamentals are also needed, including the commutability of reference materials and external quality assessment (EQA) materials for monitoring the equivalence of measurement results at the end-user level. This manuscript summarizes the findings and opinions expressed at the Joint Community for Traceability in Laboratory Medicine (JCTLM) workshop held on December 4-5, 2023. The workshop explored the relationship between EQA/proficiency testing and metrological traceability to higher-order references. EQA monitors the equivalence of measurement results from end-user IVD MDs. The workshop discussed the role and challenges of using EQA to improve and maintain the equivalence of measurement results. It also elucidated current developments in establishing the clinical suitability of laboratory results expressed as analytical performance specifications (APS).

[Analysis on vitamin B_1 and vitamin B_2 proficiency testing assessment in China disease control and prevention system].

OBJECTIVE: To evaluate the detection ability of vitamin B_1 and vitamin B_(2 )in rice flour in the laboratories of disease control and prevention system, by conducting the proficiency testing(PT)activity. METHODS: Before the vitamin B_1 and vitamin B_2 quality control samples were distributed to the laboratories of disease control and prevention system, the uniformity and stability of samples were analyzed by one-way ANOVO respectively. High performance liquid chromatography(HPLC) method was required to determine vitamin B_1(GB 5009.84-2016: determination of vitamin B_1 in food, first method as reference). HPLC method was also required to determine vitamin B_2(GB 5009.85-2016: determination of vitamin B_2 in food, first method as reference). Robust statistics analysis of proficiency testing result was conducted to evaluate laboratory testing ability through Z score. RESULTS: A total of 43 laboratories completed the proficiency testing. In all of the laboratories participated in the determination of vitamin B_(1 )and vitamin B_2, the total satisfactory rate of vitamin B_1 was 88.4%, while vitamin B_2 was 86.0%. CONCLUSION: The ability of vitamin B_1 and vitamin B_2 detection in disease control and prevention system in China is better than expected, and the testing ability of a few laboratory needs to be improved.

Proficiency testing of diagnosis in histopathology and immunohistochemistry of breast pathology in China: results from a pilot work of National Single Disease Quality Control Program for breast cancer.

AIM: Pathologists are currently supposed to be aware of both domestic and international guidelines for breast cancer diagnosis, but it is unclear how successfully these guidelines have been integrated into routine clinical practice in China. Thus, this national proficiency testing (PT) scheme for breast pathology was set up to conduct a baseline assessment of the diagnostic capability of pathologists in China. METHODS: This national PT plan is designed and implemented according to the "Conformity assessment-General requirements for proficiency testing" (GB/T27043-2012/ISO/IEC 17043:2010). Five cases of breast cancer with six key items, including histologic type, grade, ER, PR, HER2, and Ki67, were selected for testing among 96 participants. The final PT results were published on the website of the National Quality Control Center for Cancer ( http://117.133.40.88:3927/cn/col22/362 ). RESULTS: Our study demonstrated that the median PT score was 89.5 (54-100). Two institutions with scores < 67 were deemed unacceptable. The accuracy of histologic type, ER, PR, HER2, and Ki67 was satisfactory (all > 86%). However, the histologic grade showed low accuracy (74.0%). The unacceptable results mainly included incorrect evaluation of histologic grade (36.7%), inaccurate evaluation of ER/PR/HER2/Ki67 (28.2%), incorrect identification of C-AD as IBC-NST (15.7%), inappropriate use of 1+/2+/3+ rather than staining percentage for ER/PR (6.1%), misclassification of ER/PR < 1% weak expression as positive staining (1.4%), and no evaluation of histologic grade in ILC, MC, and IMC (5.8%). CONCLUSIONS: our nationwide PT program exhibited a satisfactory baseline assessment of the diagnostic capability of pathologists in China. More importantly, we identify some areas for further improvement.

SPOT/Dx Pilot Reanalysis and College of American Pathologists Proficiency Testing for KRAS and NRAS Demonstrate Excellent Laboratory Performance.

CONTEXT.­: The Sustainable Predictive Oncology Therapeutics and Diagnostics quality assurance pilot study (SPOT/Dx pilot) on molecular oncology next-generation sequencing (NGS) reportedly demonstrated performance limitations of NGS laboratory-developed tests, including discrepancies with a US Food and Drug Administration-approved companion diagnostic. The SPOT/Dx pilot methods differ from those used in proficiency testing (PT) programs. OBJECTIVE.­: To reanalyze SPOT/Dx pilot data using PT program methods and compare to PT program data.Also see p. 136. DESIGN.­: The College of American Pathologists (CAP) Molecular Oncology Committee reanalyzed SPOT/Dx pilot data applying PT program methods, adjusting for confounding conditions, and compared them to CAP NGS PT program performance (2019-2022). RESULTS.­: Overall detection rates of KRAS and NRAS single-nucleotide variants (SNVs) and multinucleotide variants (MNVs) by SPOT/Dx pilot laboratories were 96.8% (716 of 740) and 81.1% (129 of 159), respectively. In CAP PT programs, the overall detection rates for the same SNVs and MNVs were 97.2% (2671 of 2748) and 91.8% (1853 of 2019), respectively. In 2022, the overall detection rate for 5 KRAS and NRAS MNVs in CAP PT programs was 97.3% (1161 of 1193). CONCLUSIONS.­: CAP PT program data demonstrate that laboratories consistently have high detection rates for KRAS and NRAS variants. The SPOT/Dx pilot has multiple design and analytic differences with established PT programs. Reanalyzed pilot data that adjust for confounding conditions demonstrate that laboratories proficiently detect SNVs and less successfully detect rare to never-observed MNVs. The SPOT/Dx pilot results are not generalizable to all molecular oncology testing and should not be used to market products or change policy affecting all molecular oncology testing.

Impact of Academia-Government Collaboration on Laboratory Medicine Standardization in South Korea: analysis of eight years creatinine proficiency testing experience.

OBJECTIVES: To evaluate the performance of the Academia-Government Collaboration for Laboratory Medicine Standardization in Korea (KR-STDZN) based on data from KR-STDZN proficiency testing (KR-STDZN-PT) for creatinine over eight years (2015-2022). METHODS: We used KR-STDZN-PT data of creatinine tests from 2015 to 2022. Acceptance of the participating institutions' test results was assessed by calculating the acceptance performance as absolute bias (absBias%), total coefficient of variance (tCV%), and total error (TE%) for each sample using six measurements from each institution and true values of each reference material. The test result was considered acceptable when absBias%, tCV%, and TE% were <5.10, <3.20, and <11.40 %, respectively. The proportion of acceptable institutions among all participating institutions in each round was defined as the acceptance rate. Improvements in absBias%, tCV%, and TE% were analyzed using creatinine concentration ranges in samples. RESULTS: The number of participating institutions increased from 2015 to 2017 but remained consistent since 2018. The acceptance rates for absBias% and TE% increased from 52.2 and 77.6 %, in 2015 and to 90.7 and 96.3 %, in 2022, respectively. The acceptance rate for tCV% remained in the 90 % range for eight years. When creatinine <3 mg/dL, mean absBias%, and mean TE% improved significantly in 2021-2022 compared to 2015-2016 (p<0.05). When creatinine >3 mg/dL, acceptance performance did not improve. Mean tCV% remained consistent annually regardless of creatinine concentration. No significant variations in test methods were observed. CONCLUSIONS: The collaboration between academia and the government improved creatinine testing quality. Nevertheless, KR-STDZN must be expanded and refined.

Accuracy-based proficiency testing for estradiol measurements.

OBJECTIVES: Accuracy of estradiol measurements is important but conventional proficiency testing (PT) cannot assess accuracy when possibly non-commutable samples are used and method peer-group means are the targets. Accuracy-based assessment of estradiol measurements is needed. DESIGN AND METHODS: Five serum samples were prepared from single donors, frozen, and distributed overnight to 76 New York State Department of Health (NYSDOH)-certified laboratories. Participants analyzed samples for estradiol. The biases of group means were assessed against the Centers for Disease Control and Prevention (CDC)-defined targets, evaluated using the Hormone Standardization Program (HoSt) E2 performance criterion of ±12.5 %. Each laboratory's performance was evaluated using total allowable error (acceptance limits) of target ±25 % or ±15 pg/mL (55 pmol/L) (whichever was greater, NYSDOH), target ±30 % (Clinical Laboratory Improvement Amendments [CLIA]), and target ±26 % (minimal limit based on biological variation [BV]). RESULTS: The biases (range) were 34 % (-17 % to 175 %), 40 % (-33 % to 386 %), 16 % (-45 % to 193 %), 5 % (-27 % to 117 %), and -4% (-31 % to 21 %), for samples at estradiol of 24.1, 28.4, 61.7, 94.1, and 127 pg/mL, or 89, 104, 227, 345, and 466 pmol/L, respectively. Large positive method/analytical systematic biases were revealed for 9 commonly used method/analytical systems in the United States at low estradiol concentrations. Of the 9 analytical systems, 0, 0, 3, 7 and 6 met the HoSt criterion for the samples with estradiol at the five respective concentrations. PT evaluation showed that 59 %, 69 % and 87 % of laboratories would receive a PT event passing (satisfactory) score when the CDC-defined target and a criterion of NYSDOH, CLIA or BV was used, respectively. However, >95 % laboratories would obtain PT passing score if method peer-group means were used as targets regardless of the criterion used. CONCLUSIONS: Improvement in accuracy of estradiol measurements is needed, particularly at low estradiol concentrations. Accuracy-based PT provides unambiguous information about the accuracy of methods/analytical systems.

Total error in lymphocyte subpopulations by flow cytometry-based in state of the art using Spanish EQAS data.

OBJECTIVES: Flow cytometry analyses of lymphocyte subpopulations (T, B, NK) are crucial for enhancing clinical algorithms and research workflows. Estimating the total error (TE) values for the percentage and absolute number of lymphocyte subpopulations using the state-of-the-art (SOTA) approach with real data from an external proficiency testing (EPT) scheme was performed. A comparison with previously published Biological Variability (BV)-based specifications was carried out. METHODS: A total of 44,998 results from 86 laboratories over 10 years were analysed and divided into two five-year periods (2012-2016) and (2017-2021). Data come from the IC-1 Lymphocytes scheme of the Spanish External Quality Assurance System (EQAS) GECLID Program. This quantitative scheme includes percentages and absolute numbers of CD3+, CD3+CD4+, CD3+CD8+, CD19+, and CD3-CD56+CD16+ NK cells. The percentage of TE was calculated as: |reported value - robust mean|*100/robust mean for each laboratory and parameter. The cut-off for TE is set at 80 % best results of the laboratories. RESULTS: A significant reduction in the SOTA-based TE for all lymphocyte subpopulations in 2017-2021 was observed compared to 2012-2016. The SOTA-based TE fulfils the minimum BV-based TE for percentages of lymphocyte subpopulations. The parameter with the best analytical performance calculated with SOTA (2017-2021 period)-based TE was the percentage of CD3+ (TE=3.65 %). CONCLUSIONS: The values of SOTA-based specifications from external quality assurance program data are consistent and can be used to develop technical specifications. The technological improvement, quality commitment, standardization, and training, reduce TE. An update of TE every five years is therefore recommended. TE assessment in lymphocyte subsets is a helpful and reliable tool to improve laboratory performance and data-based decision-making trust.