External Quality Assessment Surveys Research Articles

Longitudinal analysis of 20Years of external quality assurance schemes for PCR/NAAT-based bacterial genome detection in diagnostic testing.

Quality control (QC), quality assurance, and standardization are crucial for modern diagnostic testing in the field of medical microbiology. The need for efficient QC to ensure accurate laboratory results, treatment, and infection prevention has led to significant efforts in standardizing assay reagents and workflows. External quality assessment (EQA) schemes, like those offered by INSTAND, play a vital role in evaluating in-house and commercial routine diagnostic assays, regarded as mandatory by national and global guidelines. The recent impact of polymerase chain reaction/nucleic acid amplification technology (PCR/NAAT) assays in medical microbiology requires that high-performing assays be distinguished from inadequately performing ones, especially those made by inexperienced suppliers. The study assesses the evolving diagnostic performance trends over 2decades for the detection of EHEC/STEC, Borrelia (B.) burgdorferi, and MRSA/cMRSA. It explores the historical context of assay utilization, participant engagement, and rates of correct results in EQA schemes. The research seeks to identify patterns in assay preferences, participant proficiency, and the challenges encountered in detecting emerging variants or clinical strains. The study highlights the decline in in-house PCR assay usage, the emergence of new diagnostic challenges, and educational aspects within EQA schemes. Specific examples, such as the inclusion, in certain EQA surveys, of EHEC strains carrying stx-2f or B. miyamotoi, highlight the role of EQAs in increasing awareness and diagnostic capabilities. Advancements in MRSA detection, especially through the adoption of commercial assays, demonstrate the impact that technology evolution has had on diagnostic performance. Achieving excellence in diagnostic molecular microbiology involves a multifaceted approach, including well-evaluated assays, careful instrumentation selection, and structured training programs. EQA schemes contribute significantly to this pursuit by providing insights into the evolving diagnostic landscape and identifying areas for improvement in the diagnostic workflow as well as in PCR/NAAT assay design.

Quality assessment of glucose measurement with regard to epidemiology and clinical management of diabetes mellitus in Germany.

During the last decade, Germany has seen an increased prevalence and a redistribution from undetected to diagnosed diabetes mellitus. Due to this substantial epidemiological development, the number of people with documented type 2 diabetes was 8.7 million in 2022. An estimated two million undiagnosed subjects are to be added. Beyond that, the life expectancy of diabetic subjects is increasing due to more responsive health systems in terms of care. Possible reasons include improved screening of at-risk individuals, the introduction of HbA1c for diagnosis in 2010, and the higher use of risk scores. Additionally, quality aspects of the laboratory methodology should be taken into consideration. Epidemiology and clinical management of diabetes in Germany are presented in the light of publications retrieved by a selective search of the PubMed database. Additionally, the data from German external quality assessment (EQA) surveys for the measurands glucose in plasma and HbA1c in whole blood, reviewed from 2010 until 2022, were evaluated. Above this, data concerning the analytical performance of near-patient glucometer devices, according to the ISO norm 15197:2013, were analyzed. Two laboratory aspects are in good accordance with the observation of an increase in the diabetes mellitus prevalence when retrospectively reviewing the period 2010 to 2022: First, the analytical performance according to the ISO norm 15197:2013 of the glucometer devices widely used by patients with diabetes for the glucose self-testing, has improved during this period. Secondly, concerning the EQA program of INSTAND, the number of participating laboratories raised significantly in Germany. The spreads of variations of the specified results for plasma glucose remained unchanged between 2010 and 2022, whereas for HbA1c a significant decrease of the result scattering could be observed. These retrospectively established findings testify to an excellent analytical quality of laboratory diagnostics for glucose and HbA1c throughout Germany which may be involved in a better diagnosis and therapy of previously undetected diabetes mellitus.

External quality assessment survey for SARS-CoV-2 nucleic acid amplification tests in clinical laboratories in Tokyo, 2021

IntroductionNucleic acid amplification tests (NAATs) play a pivotal role in clinical laboratories for diagnosing COVID-19. This study aimed to elucidate the accuracy of these tests. MethodsIn 2021, an external quality assessment of NAATs for SARS-CoV-2 was conducted in 47 laboratories in Tokyo, Japan. In open testing, where the laboratories knew that the samples were intended for the survey, a simulated nasopharyngeal swab suspension sample was used, featuring a positive sample A with a viral concentration of 50 copies/μL, positive sample B with 5 copies/μL, and a negative sample. Laboratories employing real-time RT-PCR were required to report cycle threshold (Ct) values. In blind testing, where the samples were processed as normal test samples, a positive sample C with 50 copies/μL was prepared using a simulated saliva sample. ResultsOf the 47 laboratories, 41 were engaged in open testing. For sample A, all 41 laboratories yielded positive results, whereas for sample B, 36 laboratories reported positive results, 3 laboratories reported “test decision pending”, 1 laboratory reported “suspected positive”, and 1 laboratory did not respond. All 41 laboratories correctly identified the negative samples as negative. The mean Ct values were 32.2 for sample A and 35.2 for sample B. In the blind test, six laboratories received samples. Sample C was identified as positive by five laboratories and negative by one laboratory. ConclusionsThe nature of the specimen, specifically the saliva, may have influenced the blind test outcomes. The identified issues must be meticulously investigated and rectified to ensure accurate results.

Results of a Pilot External Quality Assessment Scheme for Genetic Testing of Newborns with Spinal Muscular Atrophy.

This study aims to evaluate the ability of laboratories to perform spinal muscular atrophy (SMA) genetic testing in newborns based on dried blood spot (DBS) samples, and to provide reference data and advance preparation for establishing the pilot external quality assessment (EQA) scheme for SMA genetic testing of newborns in China. The pilot EQA scheme contents and evaluation principles of this project were designed by National Center for Clinical Laboratories (NCCL), National Health Commission. Two surveys were carried out in 2022, and 5 batches of blood spots were submitted to the participating laboratory each time. All participating laboratories conducted testing upon receiving samples, and test results were submitted to NCCL within the specified date. The return rates were 75.0% (21/28) and 95.2% (20/21) in the first and second surveys, respectively. The total return rate of the two examinations was 83.7% (41/49). Nineteen laboratories (19/21, 90.5%) had a full score passing on the first survey, while in the second survey twenty laboratories (20/20, 100%) scored full. This pilot EQA survey provides a preliminary understanding of the capability of SMA genetic testing for newborns across laboratories in China. A few laboratories had technical or operational problems in testing. It is, therefore, of importance to strengthen laboratory management and to improve testing capacity for the establishment of a national EQA scheme for newborn SMA genetic testing.

A-214 Modified CDC Beta-Quantification Reference Method for HDL-C and LDL-C Determinations Using Isotope-Dilution Mass Spectrometry—Pilot Report

Abstract Background It is known that there is average positive 1.6% bias of total cholesterol by modified Abell-Levy-Brodie-Kendall (AK) colorimetric method, which have been the traceable reference measurement procedure (RMP) used by most of clinical guidelines, comparing to those by isotope-dilution mass spectrometry (ID-MS), the most accurate RMP. The CDC beta-quantification (BQ) reference method is currently the only RMP registered JCTLM database for high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) using the AK colorimetric assay after ultracentrifugation. We compared the modified BQ method using ID-MS assay after ultracentrifugation with the original CDC BQ reference method of HDL-C or LDL-C. Methods For this study, 18 commutable serum pools according to the CLSI 37A and 6 external quality assessment survey materials operated by Cholesterol Reference Method Laboratory Network (CRMLN) were used. We used the same ultracentrifugation procedure as in the original CDC BQ reference method, and the bottom fraction cholesterol (BFC) and HDL-C from supernatant after apo B-containing lipoprotein precipitation using heparin/manganese were measured for each sample using both AK and ID-MS methods, then LDL-C was calculated as the difference between BFC and HDL-C. The comparability analysis between methods at multiple medical decision levels (MDL), and Passing-Bablok regression analysis for each analyte were performed. Results Each Passing-Bablok regression equation of modified CDC BQ reference method vs. original CDC BQ reference method for three fractions, BFC, HDL-C, and LDL-C, were as follows; BFC was y (IDMS) = 0.996x (AK) - 0.342 (R = 0.999), for HDL-C was y (IDMS) = 1.007x (AK)-1.037 (R = 0.995), and for LDL-C was y (IDMS) = 0.990x (AK) + 0.860 (R = 0.999). Both cholesterol results by two methods showed excellent correlation and agreement of the three fractions showing regression R larger than 0.995 and slope or intercept containing 1 and 0 in their 95% confidence interval. The mean differences of IDMS against AK method were −0.56% for BFC, −0.51 mg/dL for HDL-C, and −0.35% for LDL-C, respectively. When the regression prediction at MDLs, 40/50/60 mg/dL for HDL-C and 100/130/160 mg/dL for LDL-C were estimated, all the differences were confirmed to be within allowable level claimed by CRMLN member laboratory accreditation criteria, ±1mg/dL for HDL-C and ±2.0% for LDL-C, showing equivalent results against CDC BQ reference method, but average negative bias of −0.72%, −1.59%, and −0.28% for BFC, HDL-C, and LDL-C, respectively are still present. These results of the modified BQ method using ID-MS assay may be promising, because the tedious and less accurate AK-assay can be switched to the ID-MS assay, not sacrificing trueness LDL-C or changing clinical guidelines, but quite large negative bias of HDL-C for some samples should require further investigation. Conclusion We got comparable HDL-C and LDL-C results using modified BQ method using ID-MS assay with those by original CDC BQ reference method using AK colorimetric assay. The quite large negative bias of HDL-C for some samples require large-scale additional further multi-center study among CRMLN network laboratories to evaluate the results observed in our study.

B-310 High Through-put Online Solid Phase Extraction of Immunosuppressive Drugs From Whole Blood on Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) in a Tertiary Hospital Laboratory

Abstract Background Cyclosporine A and Tacrolimus are immunosuppressive drugs that are commonly used in solid organ transplant and are characterized by a relatively narrow therapeutic index, potential drug interactions and significant intra- and inter-individual variability. Therapeutic drug monitoring is therefore beneficial for optimizing dosage regimens, preventing rejection of the graft and reducing toxicities in transplant recipients receiving immunosuppressive drug therapy. A commercial assay (RECIPE ClinMass LC-MS/MS Advanced kit) for simultaneous measurement of immunosuppressants in whole blood using LC-MS/MS methodology has been adapted for assay performance evaluation in our laboratory on the Agilent 1290 Infinity II liquid chromatograph coupled to an Agilent 6470 triple quadrupole mass spectrometer. Our study evaluated the analytical performance of Cyclosporine A and Tacrolimus on the RECIPE assay and compared it against our existing in-house LC-MS/MS assay. Methods The RECIPE ClinMass® LC-MS/MS Advanced Immunosuppressants assay kit consist of mobile phase solvents, precipitation reagents, stable isotopic labeled internal standards, solid phase extraction and analytical columns, lyohilized whole blood calibrators and quality controls. Drug analytes were extracted from whole blood using single-step protein precipitation followed by on-line solid phase extraction on the LC, eluted on isocratic chromatography and detected on positive ESI MS ionisation mode. The RECIPE assay run time is under 3 min. Performance validation parameters included within/total assay imprecision, linearity, recovery, limits of detection (LOD)/quantification (LOQ) and carry-over. Method correlation studies using fresh patient whole blood specimens (n = 212 Cyclosporine A; n = 120 Tacrolimus) were performed on existing in-house assay on Agilent 6460 LC-MS/MS and the RECIPE assay on Agilent 6470 LC-MS/MS systems. Results Within run and total imprecision for Cyclosporine A and Tacrolimus on the RECIPE assay were determined to be ≤4.4% and ≤7.5% on the Agilent 6470 LC-MS/MS. The assay demonstrated linearity across the analytical measurement range up to 1500 ug/L for Cyclosporine A and up to 55 ug/L for Tacrolimus. Analytical recoveries obtained for both drugs ranged between 93%–100%. The LODs were assessed to be 13.0 ug/L and 0.70 ug/L for Cyclosporine A and Tacrolimus respectively. LOQs were assessed to be 22.7 ug/L and 1.30 ug/L for Cyclosporine A and Tacrolimus respectively. Results of carry-over studies were insignificant. Participation in the CAP Immunosuppressive Drugs External Quality Assessment Survey showed good agreement with LC-MS/MS peers. Method correlation with in-house assay demonstrated good agreement: (1) Cyclosporine A: Passing Bablok regression RECIPE = 0.94 (In-house) + 17.2, Bland Altman mean bias of +2.0%, Spearman correlation coefficient of 0.985; (2) Tacrolimus: Passing Bablok regression of RECIPE = 1.03 (In-house) - 0.2, Bland Altman mean bias of -0.2%, Spearman correlation coefficient of 0.988 Conclusion The RECIPE ClinMass® LC-MS/MS Immunosuppressants assay performed within manufacturer’s specifications except for LOD/LOQs which were determined at higher concentrations. The assay is adaptable to contemporary LC-MS/MS systems and confer benefits from rapid turnaround time (<3 min), simplified specimen preparation procedures and ready prepared reagents. The advantages allow enhancements in workflow efficiency and ease of staff training in our laboratory and the assay is suitable for routine clinical use in high workload clinical laboratories.

Aggregated data from the same laboratories participating in two glucose external quality assessment schemes show that commutability and transfers of values to control materials are decisive for the biases found.

We report the results of glucose measurements performed during one year by the same measurement procedures (MPs) in 58 Norwegian hospital laboratories using control materials provided by external quality assessment (EQA) schemes from two different providers. The providers used materials with presumed vs. verified commutability and transfers of values using reference material vs. using a highest-order reference MP. Data from six Labquality and three Noklus glucose EQA surveys were aggregated for each MP (Abbott Alinity, Abbott Architect, Roche Cobas, and Siemens Advia) in each scheme. For each EQA result, percent difference from target value (% bias) was calculated. Median percent bias for each MP per scheme was then calculated. The median % biases observed for each MP in the Labquality scheme were significantly larger than those in the Noklus scheme, which uses verified commutable control materials and highest-order reference MP target values. The difference ranged from 1.2 (Roche Cobas, 2.9 vs. 1.7 %) to 4.4 percentage points (Siemens Advia, 3.2 % vs.-1.2 %). The order of bias size for the various MPs was different in the two schemes. In contrast to the Labquality scheme, the median % biases observed in the Noklus scheme for Abbott Alinity (-0.1 %), Abbott Architect (-0.5 %), and Siemens Advia (-1.2 %) were not significantly different from target value (p>0.756). This study underlines the importance of using verified commutable EQA materials and target values traceable to reference MPs in EQA schemes designed for assessment of metrological traceability of laboratory results.

Longitudinal Evaluation of AFP and CEA External Proficiency Testing Reveals Need for Method Harmonization.

The glycoproteins alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) have long been approved as biomarkers for diagnosing and monitoring tumors. International Reference Preparations (IRPs) have been around since 1975. Nevertheless, manufacturer-dependent differences have been reported, indicating a lack of harmonization. This paper analyzes data from 15 external quality assessment (EQA) surveys conducted worldwide between 2018 and 2022. The aim was to gain insight into the longitudinal development of manufacturer-dependent differences for CEA and AFP. In each survey, participating laboratories received two samples with different tumor marker levels. Inter- and intra-assay variability was analyzed and the mean 80% and 90% of the manufacturer collectives were compared to the evaluation criteria of the German Medical Association (RiliBÄK). The median EQA results for CEA revealed manufacturer-dependent differences between the highest and lowest collective of up to 100%; for AFP, the median differences mostly remained below 40%. The coefficients of variation were predominantly low for both markers. We concluded that the current assays for AFP and CEA detection are better harmonized than previously reported. The assays displayed a good robustness; however, a narrowing of the current assessment limits in EQA schemes could further enhance the quality of laboratory testing.

A New Strategy for Evaluating the Quality of Laboratory Results for Big Data Research: Using External Quality Assessment Survey Data (2010-2020).

To ensure valid results of big data research in the medical field, the input laboratory results need to be of high quality. We aimed to establish a strategy for evaluating the quality of laboratory results suitable for big data research. We used Korean Association of External Quality Assessment Service (KEQAS) data to retrospectively review multicenter data. Seven measurands were analyzed using commutable materials: HbA1c, creatinine (Cr), total cholesterol (TC), triglyceride (TG), alpha-fetoprotein (AFP), prostate-specific antigen (PSA), and cardiac troponin I (cTnI). These were classified into three groups based on their standardization or harmonization status. HbA1c, Cr, TC, TG, and AFP were analyzed with respect to peer group values. PSA and cTnI were analyzed in separate peer groups according to the calibrator type and manufacturer, respectively. The acceptance rate and absolute percentage bias at the medical decision level were calculated based on biological variation criteria. The acceptance rate (22.5%-100%) varied greatly among the test items, and the mean percentage biases were 0.6%-5.6%, 1.0%-9.6%, and 1.6%-11.3% for all items that satisfied optimum, desirable, and minimum criteria, respectively. The acceptance rate of participants and their external quality assessment (EQA) results exhibited statistically significant differences according to the quality grade for each criterion. Even when they passed the EQA standards, the test results did not guarantee the quality requirements for big data. We suggest that the KEQAS classification can serve as a guide for building big data.

An external quality assessment for the molecular testing of the SARS-CoV-2 virus genome in Zhejiang Province, China

The COVID-19 pandemic has necessitated the rapid expansion of laboratories that conduct SARS-CoV-2 tests. A provincial external quality assessment (EQA) scheme on SARS-CoV-2 tests was organized by Zhejiang Provincial CDC to assess the accuracy of the tests in individual CDC municipal and county laboratories in Zhejiang Province, China. Three positive samples in high, medium, and low concentrations, respectively, were prepared using the serial dilutions from the culture with the viral titer concentration of 1×106.3 TCID50/mL, and one negative sample were included. A total of 93 laboratories participated, contributing results from 36 distinct combinations of nucleic acid extraction methods and PCR reagents. There was 100% concordance among all laboratories for all EQA samples, and no false-positive or false-negative results were observed. The EQA survey provides confidence in the identification of infected individuals or asymptomatic populations and assurance for clinical and public health decision-making based on test results.

A study of quality assessment in SARS-CoV-2 pathogen nucleic acid amplification tests performance; from the results of external quality assessment survey of clinical laboratories in the Tokyo Metropolitan Government external quality assessment program in 2020

IntroductionThe Tokyo Metropolitan Government (TMG) conducted an external quality assessment (EQA) survey of pathogen nucleic acid amplification tests (NAATs) as a TMG EQA program for SARS-CoV-2 for clinical laboratories in Tokyo. MethodsWe diluted and prepared a standard product manufactured by Company A to about 2,500 copies/mL to make a positive control and distribute it with a negative control. The participants reported the use of the NAATs methods for SARS-CoV-2, the name of the real-time RT-PCR kit, the name of the detection device, the target gene(s), nucleic acid extraction kit, Threshold Cycle value in the case of RT-PCR and the Threshold time value and Differential calculation value in the case of Loop-Mediated Isothermal Amplification (LAMP) method. ResultsAs a result, 17 laboratories using fully automated equipment and 34 laboratories using the RT-PCR method reported generally appropriate results in this EQA survey. On the other hand, among the laboratories that adopted the LAMP method, there were a plurality of laboratories that judged positive samples to be negative. ConclusionThe false negative result is considered to be due to the fact that the amount of virus genome contained in the quality control reagent used this time was below the detection limit of the LAMP method combined with the rapid extraction reagent for influenza virus. On the other hand, false positive results are considered to be due to the non-specific reaction of the NAATs. The EQA program must be continued for the proper implementation of the pathogen NAATs.

Serological diagnostic for SARS-CoV-2: an experimental External Quality Assessment Scheme.

Numerous analytical systems, rapidly made available on the market throughout the SARS-CoV-2 pandemic, aim to detect COVID-19, and to continuously update and improve the same systems. Medical laboratory professionals have also developed in-house analytical procedures in order to satisfy the enormous volume of requests for tests. These developments have highlighted the need control the analytical procedures used in order to guarantee patient safety. The External Quality Assessment (EQA) Scheme, an important quality assurance tool, aims to guarantee high standard performance for laboratory and analytical procedures. The aim of the present study was to report on the results collected in an experimental EQA scheme for the serological diagnosis of SARS-CoV-2. All qualitative results collected in the different EQA surveys were summarized in order to identify the percentage of laboratory results in relation to typology of antibodies, results and samples. A total of 4,867 data sets were collected. The analysis of EQA data made, demonstrates a better agreement among laboratories results for total Ig than single immunoglobulins (IgG, IgM, IgA) in the case samples positive for SARS-CoV-2, and a wide divergence between IgM results for positive samples (only 34.9% were correct). Results for negative controls and specificity controls demonstrated a better overall agreement than results for positive samples. Working in collaboration with the IVD manufacturers, laboratory professionals must strive to achieve harmonization of results, and to develop well-defined protocols complying with the ISO 15189 requirements.

Optimization of Heparin Monitoring with Anti-FXa Assays and the Impact of Dextran Sulfate for Measuring All Drug Activity.

Heparins, unfractionated or low molecular weight, are permanently in the spotlight of both clinical indications and laboratory monitoring. An accurate drug dosage is necessary for an efficient and safe therapy. The one-stage kinetic anti-FXa assays are the most widely and universally used with full automation for large series, without needing exogenous antithrombin. The WHO International Standards are available for UFH and LMWH, but external quality assessment surveys still report a high inter-assay variability. This heterogeneity results from the following: assay formulation, designed without or with dextran sulfate to measure all heparin in blood circulation; calibrators for testing UFH or LMWH with the same curve; and automation parameters. In this study, various factors which impact heparin measurements are reviewed, and we share our experience to optimize assays for testing all heparin anticoagulant activities in plasma. Evidence is provided on the usefulness of low molecular weight dextran sulfate to completely mobilize all of the drug present in blood circulation. Other key factors concern the adjustment of assay conditions to obtain fully superimposable calibration curves for UFH and LMWH, calibrators’ formulations, and automation parameters. In this study, we illustrate the performances of different anti-FXa assays used for testing heparin on UFH or LMWH treated patients’ plasmas and obtained using citrate or CTAD anticoagulants. Comparable results are obtained only when the CTAD anticoagulant is used. Using citrate as an anticoagulant, UFH is underestimated in the absence of dextran sulfate. Heparin calibrators, adjustment of automation parameters, and data treatment contribute to other smaller differences.

Diagnostic methods for neonatal hyperbilirubinemia: benefits, limitations, requirements, and novel developments.

Invasive bilirubin measurements remain the gold standard for the diagnosis and treatment of infants with severe neonatal hyperbilirubinemia. The present paper describes different methods currently available to assess hyperbilirubinemia in newborn infants. Novel point-of-care bilirubin measurement methods, such as the BiliSpec and the Bilistick, would benefit many newborn infants, especially in low-income and middle-income countries where the access to costly multi-analyzer in vitro diagnostic instruments is limited. Total serum bilirubin test results should be accurate within permissible limits of measurement uncertainty to be fit for clinical purposes. This implies correct implementation of internationally endorsed reference measurement systems as well as participation in external quality assessment programs. Novel analytic methods may, apart from bilirubin, include the determination of bilirubin photoisomers and bilirubin oxidation products in blood and even in other biological matrices. IMPACT: Key message: Bilirubin measurements in blood remain the gold standard for diagnosis and treatment of severe neonatal hyperbilirubinemia (SNH). External quality assessment (EQA) plays an important role in revealing inaccuracies in diagnostic bilirubin measurements. What does this article add to the existing literature? We provide analytic performance data on total serum bilirubin (TSB) as measured during recent EQA surveys. We review novel diagnostic point-of-care (POC) bilirubin measurement methods and analytic methods for determining bilirubin levels in biological matrices other than blood. Impact: Manufacturers should make TSB test results traceable to the internationally endorsed total bilirubin reference measurement system and should ensure permissible limits of measurement uncertainty.

Abstract PS5-46: Introduction and clinical validation of metrology standards for immunohistochemistry (IHC); New tool for standardization of estrogen receptor (ER) IHC assay in breast cancer

Abstract Traceability of measurement to a higher order reference standard is a foundation of laboratory testing. There is as yet no method for creating reference standards for cellular proteins in situ in an analogous fashion as for soluble analytes. At present, IHC laboratories produce results for breast cancer hormone receptors without connection to a reference standard. Not surprisingly, high rates of testing variation as well as discrepancies among IHC laboratories have been reported. To address this need, we developed a system of measurement traceability using a linked fluorescein tag for creating reference standards for any cellular analyte and, as a first test, validate it for estrogen receptor (ER) testing. In this study, the newly developed ER standard defines and compares the thresholds separating “high positive”, “low positive”, and “negative” tests according to updated ASCO/CAP guidelines as detected by clinical IHC laboratories in a national external quality assessment survey. This reference standard utilizes NIST Standard Reference Material (SRM) 1934 as a universal IHC standard. We calculated ER concentration based on a linked fluorescence measurement traceable to NIST SRM 1934 as each ER is linked to a single fluorescein, and fluorescein concentration equals ER concentration. Each laboratory’s lowest detected ER concentration (i.e. “limit of detection”, LOD) was compared to their results with 80 tumor samples enriched for triple negative breast cases. For the Canadian Immunohistochemistry Quality Control (CIQC) ER proficiency testing run, calibrator sets with peptides for the SP1, EP1, and 6F11 epitopes were created. The various concentrations were pipetted onto histology slides used by CIQC to place its 80-case breast cancer tissue microarray. These slides were stained by participating laboratories using their routine ER IHC protocols and returned to the CIQC. For the purpose of this study, Histology Score and ASCO/CAP categorical scoring recommendation was used for the readout. Results with SP1 clone are reported here because it was employed by overwhelming majority of laboratories. A total of 3,038 readouts were included in the analysis. Most IHC laboratories had a LOD between 10,000 – 25,000 molecules ER per microbead. Highly sensitive ER assays (low LODs) detected more positive cases while those with poorly sensitive assay detected fewer. The LOD correlated with the percent positive cases (R2= -.767, p < 0.0001, Spearman Correlation) as well as with cumulative individual laboratory Histology Score (R2= -.612, p < 0.0001, Spearman Correlation). The tumor samples accounting for this difference were principally “low positive” (ASCO/CAP classification) while ER-high positive tumors were less affected. Although the concept of a non-quantitative IHC LOD has been introduced with critical assay performance controls (iCAPCs, e.g. germinal center cells in the tonsil for ER), there were no tools until now to actually measure LOD for ER IHC testing. With the calibrators introduced here, it is now possible to measure the LOD for ER IHC assays. Furthermore, our data show that ER low positive and some cases of ER high positive breast cancers are highly affected by the variability of analytic sensitivity between clinical IHC laboratories. These data argue for the urgent need to standardize testing to a defined analytic sensitivity range, which is now possible for the first time. Beyond inter-laboratory standardization, known measured LOD also enables: i) methodology transfer from clinical trials to clinical laboratories; ii) determination of optimal analytical sensitivity to produce assays with highest clinical and analytical validity, and iii) daily monitoring of analytical sensitivity for each tested IHC slide. Citation Format: Emina Emilia Torlakovic, Seshi R. Sompuram, Kodela Vani, Lili Wang, Anika K Schaedle, Paul C. DeRose, Steven A. Bogen. Introduction and clinical validation of metrology standards for immunohistochemistry (IHC); New tool for standardization of estrogen receptor (ER) IHC assay in breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-46.

Evaluation of INSTAND e.V.’s external proficiency testing program for tetanus and diphtheria antitoxin detection: Lessons for assessing levels of immunoprotection

ObjectivesThe aim of this study was to evaluate the development and status quo of the quality of high throughput in vitro diagnostic testing for tetanus and diphtheria antitoxin antibody (ATX) concentrations based on external quality assessment (EQA) data. MethodsWe analyzed manufacturer-specific data of 22 EQA surveys—each for the detection of tetanus and diphtheria ATX—to check the diagnostic strength of the corresponding in vitro diagnostic systems. ResultsWhile the results were mostly well aligned, individual surveys showed widely dispersed ATX concentrations. The medians of manufacturer collectives deviated from the overall median by up to 8.9-fold in the case of diphtheria ATX and by up to 3.5-fold in the case of tetanus ATX. Such a distribution in the results is particularly critical in the cut-off range for immunity and may lead to an incorrect assessment of vaccination status. ConclusionThese results were surprising as there are International Standards for both ATX; however, the results may be linked to the high ATX concentration of the reference material, which deviates considerably from clinically significant concentrations. To increase the accuracy and diagnostic strength of both assays, we recommend a recalibration of the test systems and verification of their traceability to the International Standards.

水道水質検査精度管理のための統一試料調査に関する経年分析：平成23～29年度

水道水質検査精度管理のための統一試料調査に関する経年分析：平成23～29年度

The results of external quality assessment programme on urine leukocyte and erythrocyte counting in Poland.

IntroductionUrine particle analysis is an important diagnostic tool. The aim of this study was to evaluate the quality of urine leukocyte (WBC) and erythrocyte (RBC) counting results obtained with manual and automated methods in Polish laboratories participating in the international external quality assessment (EQA) programme.Materials and methods1400 WBC and RBC counting results were obtained from 183 laboratories in EQA surveys organised by Labquality (Helsinki, Finland) from 2017 to 2019. The between-laboratory coefficient of variation (CV), the percentage difference between the laboratories' results and target values (Q-score (%)), as well as modified Youden plots were analysed.ResultsFor automated method groups, the medians of inter-laboratory CVs varied from 14% to 33% for WBC counting and from 10% to 39% for RBC counting. For manual method groups, the medians of CV varied from 53% to 71% (WBC) and from 55% to 70% (RBC), and they were significantly higher, in comparison to CVs for most automated method groups (P < 0.001). The highest percentage of results outside the target limits (36%) and the highest range of Q-score (%) (from - 93% to 706%) were observed for laboratories which participated in the surveys for the first or second time. The percentage of deviating results and the ranges of Q-score decreased with an increased frequency of laboratories’ participation in the surveys.ConclusionsThe quality of manual methods of urine WBC and RBC counting is unsatisfactory. There is an urgent need to take actions to improve laboratories’ performance and to increase harmonisation of the results.

An updated protocol based on CLSI document C37 for preparation of off-the-clot serum from individual units for use alone or to prepare commutable pooled serum reference materials.

Manufacturers of in vitro diagnostic medical devices, clinical laboratories, research laboratories and calibration laboratories require commutable reference materials that can be used in the calibration hierarchies of medical laboratory measurement procedures used for human specimens to establish metrological traceability to higher order reference systems. Commutable materials are also useful in external quality assessment surveys. In order to achieve these goals, matrix-based reference materials with long-term stability, appropriate measurand concentrations and commutability with individual human specimens are required. The Clinical and Laboratory Standards Institute (CLSI) guideline C37-A (now archived) provided guidance to prepare commutable pooled serum reference materials for use in the calibration hierarchies of cholesterol measurement procedures. Experience using the C37-A guideline has identified a number of technical enhancements as well as applications to measurands other than cholesterol. This experience is incorporated into this updated protocol to ensure the procedure will continue to meet the needs of the medical laboratory. The updated protocol describes a procedure for preparing frozen human serum units or pools with minimal matrix alterations that are likely to be commutable with individual human serum samples. The protocol provides step-by-step guidance for the planning phase, collection of individual serum units, processing the units, qualifying the units for use in a pool and frozen storage of aliquots of pooled sera to manufacture frozen serum pools. Guidance on how to perform quality control of the final product and suggestions on documentation are also provided.

Insufficient harmonization of antibiotics assays – Polish experience with an external quality assessment program in the years 2011–2018

IntroductionTreatment with vancomycin and gentamycin requires strict monitoring of its serum concentration for proper dosage optimization. This study aimed to assess the quality and the harmonization of antibiotics assays in Polish laboratories. Materials and methods413 results of vancomycin and 148 results of gentamycin assays obtained from Polish laboratories in 30 international external quality assessment (EQA) surveys carried out from March 2011 to May 2018 were analyzed. ResultsInterlaboratory robust coefficients of variation (rCVs) in particular surveys comprised between 1.3 and 47.2% for vancomycin, and between 1.8 and 34.2% for gentamycin. The percentage of the results with the difference above acceptable limit ±10% from the target value established for own method group was 25.7% for vancomycin and 25.6% for gentamycin. When the difference was established according to target value for all methods, the percentage of results outside the acceptable limit was 2-fold higher on average (54.8% for vancomycin and 43.2% for gentamycin). The comparison of target values for methods revealed statistically significant differences between analytical systems used (p < .0001). The highest difference was 40% for vancomycin and 12% for gentamycin. ConclusionsThe present analysis revealed high dispersion of the antibiotics assays results in Polish laboratories. Moreover, vancomycin and gentamycin results differed significantly in a way dependent on the analytical system used. There appear to be an urgent need for harmonization of methods used for vancomycin and gentamycin measurement.