Development and evaluation of an external quality control and internal quality control containing real-time RT-PCR assay for the detection of o'nyong-nyong virus.

O'nyong-nyong fever is a mosquito-borne tropical viral disease while few molecular diagnostic tools have been established for its surveillance until now. In the current study, a single-step, dual-color real-time reverse transcription polymerase chain reaction (RT-PCR) assay which contained both external quality control (EQC) and internal quality control (IQC) prepared by armored RNA technique was developed and evaluated for the detection of o'nyong-nyong virus (ONNV). Results showed that the assay was established successfully without cross-reaction with genetically related or symptom-alike diseases, which showed high specificity of the assay. The coefficient of variation of the assay was 0.97%, far less than 5%, indicating good repeatability of the assay. The lower limit of detection of the assay could reach as low as 100 copies of genome equivalent. During evaluation, the assay could correctly detect ONNV from spiked human serum samples and Anopheles species mosquito samples, while no ONNV positive was observed either from serum samples of patients with acute febrile illness or from local Anopheles species mosquitoes, suggesting no ONNV had been transmitted locally. In conclusion, the assay could potentially provide a valuable platform for ONNV molecular detection, which may improve the preparedness for future o'nyong-nyong fever outbreaks.

Report from the HarmoSter study: impact of calibration on comparability of LC-MS/MS measurement of circulating cortisol, 17OH-progesterone and aldosterone.

OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended for measuring circulating steroids. However, assays display technical heterogeneity. So far, reproducibility of corticosteroid LC-MS/MS measurements has received scant attention. The aim of the study was to compare LC-MS/MS measurements of cortisol, 17OH-progesterone and aldosterone from nine European centers and assess performance according to external quality assessment (EQA) materials and calibration. METHODS: Seventy-eight patient samples, EQA materials and two commercial calibration sets were measured twice by laboratory-specific procedures. Results were obtained by in-house (CAL1) and external calibrations (CAL2 and CAL3). We evaluated intra and inter-laboratory imprecision, correlation and agreement in patient samples, and trueness, bias and commutability in EQA materials. RESULTS: Using CAL1, intra-laboratory CVs ranged between 2.8-7.4%, 4.4-18.0% and 5.2-22.2%, for cortisol, 17OH-progesterone and aldosterone, respectively. Trueness and bias in EQA materials were mostly acceptable, however, inappropriate commutability and target value assignment were highlighted in some cases. CAL2 showed suboptimal accuracy. Median inter-laboratory CVs for cortisol, 17OH-progesterone and aldosterone were 4.9, 11.8 and 13.8% with CAL1 and 3.6, 10.3 and 8.6% with CAL3 (all p<0.001), respectively. Using CAL1, median bias vs. all laboratory-medians ranged from -6.6 to 6.9%, -17.2 to 7.8% and -12.0 to 16.8% for cortisol, 17OH-progesterone and aldosterone, respectively. Regression lines significantly deviated from the best fit for most laboratories. Using CAL3 improved cortisol and 17OH-progesterone between-method bias and correlation. CONCLUSIONS: Intra-laboratory imprecision and performance with EQA materials were variable. Inter-laboratory performance was mostly within specifications. Although residual variability persists, adopting common traceable calibrators and RMP-determined EQA materials is beneficial for standardization of LC-MS/MS steroid measurements.

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.

INTRODUCTION: The 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. METHODS: We 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. RESULTS: As 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. CONCLUSION: The 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.

[The use of intralaboratory and external quality control of immunohistochemical studies to improve the detection of estrogen and progesterone receptors]. / Primenenie vnutrilaboratornogo i vneshnego kontrolya kachestva immunogistokhimicheskikh issledovanii dlya uluchsheniya vyyavleniya retseptorov estrogenov i progesteronov.

Immunohistochemical study of breast cancer has been practiced for several decades; however, the standardization of this process has not yet been achieved, despite substantial advances in methodology. The paper presents practical guidelines and standards for testing breast carcinomas, which can be used as day-to-day control of the work of an immunohistochemistry laboratory. It considers the concept of external quality control in the immunohistochemical detection of estrogen (ER) and progesterone (PR) receptors, as well as the problems of harmonization in the immunohistochemical analysis of breast carcinomas, by using the nuclear biomarkers, such as ER and PR, as an example. The agreed standard-based external control in determining the optimal result may yield reproducible data for use in clinical practice.

External quality control of phenotypic drug susceptibility testing for Mycobacterium tuberculosis in China.

The aim of our study was to evaluate the performance of conventional drug susceptibility testing (DST) among the tuberculosis (TB)-specialized hospitals in China. A total of 40 hospitals participated in the external quality assurance program for assessment of DST results from each hospital. The sensitivity, specificity, and accuracy of DST were analyzed. The mean accuracy was 96.5% for isoniazid (INH), 95.8% for rifampin (RIF), 97.0% for ethambutol (EMB), 96.8% for ofloxacin (OFX), 97.1% for kanamycin (KAN), 96.1% for amikacin (AMK), and 93.6% for capreomycin (CAP), respectively. Of the 40 participating laboratories, 4 (10.0%) and 6 (15%) failed to achieve 90% accuracy for INH and RIF, respectively. In addition, six hospitals (15%) were confirmed as certified to provide reliable DST results for both first-line and second-line drugs. The certified proportion for DST dropped from 73.9% in the non-western region to 59.2% in the western region. The significant difference was observed in the certified proportion for first-line drugs between the western and non-western region (P = 0.013). Our results demonstrate that the quality of phenotypical DST is frequently unsatisfactory, with approximately one-third of participated laboratories failing to produce certified phenotypical DST results. In addition, the uncertified laboratories majorly come from the western region in China.

External quality control program in screening for infectious diseases at blood banks in Mexico.

INTRODUCTION: Quality control for the detection of infectious markers in blood banks is a necessary activity to ensure the accuracy of donor screening results. Considering that in Mexico blood safety is one of the goals of the National Action Programs, it is essential to evaluate banks through an External Quality Control Program. OBJECTIVE: To analyze one of the evaluations that showed the greatest participation (2014-2/lot46) of banks in the Mexican Republic in the detection of transfusion-transmitted diseases. MATERIALS AND METHODS: A randomized panel of infectious markers of HIV, HCV, HBV, Treponema pallidum and Trypanosoma cruzi was manufactured under high quality standards. The evaluation criteria for each infectious marker were the identification of false positives and false negative results. Additionally, technologies used to detect infectious markers were requested for each bank. RESULTS: Of the 503 banks, only 374 participated in the evaluation. Technologies based on chemiluminescence, immunofluorescence and immunocolorimetry were used to detect viral markers. Even rapid tests for T. pallidum continue to be the methods of choice with 42%. Trypanosoma cruzi was 20% with fast techniques versus 80% with automated tests. Highest incidence of false positives was identified for T. pallidum and HBV, followed by T. cruzi, HIV and HCV. Fourteen (3.74%) false negatives results were identified for T. cruzi, followed by T. pallidum (n = 5/1.33%), HCV (n = 4/1.06) and HVB/HIV (n = 2/0.53%). CONCLUSION: False positive results identified for each infectious marker was considered high. This evidence will allow us to focus on areas of opportunity during serologic screening with greater emphasis on good laboratory practices.

Hepatitis E virus serology and PCR: does the methodology matter?

Hepatitis E virus (HEV) genotype 3 is an emerging pathogen in the developed world. As the clinical manifestations and routine laboratory parameters are often nonspecific, accurate diagnostic tests are crucial. In the current study, the performance of six serological assays and three PCR assays for the detection of HEV was evaluated. In the setting of the Ghent University Hospital, patients with clinically suspected HEV infection were tested for the presence of HEV IgM and IgG as well as HEV RNA. Serology was performed using six commercial HEV ELISA assays: Biorex, Wantai and Mikrogen IgM and IgG. HEV RNA was detected using one commercial assay (Altona RealStar®), and two optimized in-house real-time RT-PCR assays (according to Jothikumar et al., 2006 and Gyarmati et al., 2007). In addition, all three PCR assays were performed on 16 external quality control (EQC) samples. In a period of 39 months (January 2011-April 2014), 70 patients were enrolled. Using different ELISA assays, the prevalence of antibodies varied from 5.7% to 14.3% for HEV IgM and from 15.7% to 20.0% for IgG. All but two of the results of the PCR assays performed on clinical samples agreed. However, 10 out of 16 EQC samples results showed major discrepancies. We observed important differences in the performance of various serological and PCR assays. For this reason, results of both serological and molecular tests for HEV should be interpreted with caution.

[Analysis of the results of the SEIMC External Quality Control Program. Year 2014]. / Análisis de resultados del Programa de Control de Calidad Externo SEIMC. Año 2014.

The External Quality Control Program of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) include controls for bacteriology, serology, mycology, parasitology, mycobacteria, virology and molecular microbiology. This article presents the most relevant conclusions and lessons from the 2014 controls. As a whole, the results obtained in 2014 confirm the excellent skill and good technical standards found in previous editions. However, erroneous results can be obtained in any laboratory and in clinically relevant determinations. Once again, the results of the SEIMC program highlighted the need to implement both internal and external controls in order to assure the maximal quality of the microbiological tests.

[Analysis of the results of the HIV-1, HCV and HBV viral load of SEIMC External Quality Control Program. Year 2014]. / Análisis de resultados del Programa de Control de Calidad Externo de carga viral del VIH-1, del VHC y del VHB. Año 2014.

Human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV) and hepatitis C virus (HCV) viral load determinations are among the most relevant markers for the follow up of patients infected with these viruses. External quality control tools are crucial to ensure the accuracy of results obtained by microbiology laboratories. This article summarizes the results obtained from the 2014 SEIMC (Spanish Society of Infectious Diseases and Clinical Microbiology) External Quality Control Programme for HIV-1, HCV, and HBV viral loads. In the HIV-1 program, a total of 5 standards were sent. One standard consisted in seronegative human plasma, while the remaining 4 contained plasma from 3 different viremic patients, in the range of 2-5 log10 copies/mL; 2 of these standards were identical aiming to determine repeatability. A significant proportion of the laboratories (30.8% on average) obtained values out of the accepted range (mean ± 0.25 log10 copies/mL), depending on the standard and on the method used for quantification. Repeatability was excellent, with up to 95.8% of laboratories reporting results within the limits (Δ < 0.5 log10 copies/mL). The HBV and HCV program consisted of 2 standards with different viral load contents. Most of the participants, 83.7% in the case of HCV and 87.9% in the HBV, obtained all the results within the accepted range (mean ± 1.96 standard deviations log10 IU/mL). Data from this analysis reinforce the utility of proficiency programmes to ensure the quality of the results obtained by a particular laboratory, as well as the importance of the post-analytical phase on the overall quality. Due to the remarkable interlaboratory variability, it is advisable to use the same method and the same laboratory for patient follow up.

[Analysis of the results of the SEIMC External Quality Control Program. Year 2013]. / Análisis de resultados del Programa Externo de Control de Calidad SEIMC. Año 2013.

The External Quality Control Program of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) include controls for bacteriology, serology, mycology, parasitology, mycobacteria, virology, molecular microbiology and HIV-1, HCV and HBV viral loads. This manuscript presents the analysis of results obtained of the participants from the 2013 SEIMC External Quality Control Programme, except viral loads controls, that they are summarized in a manuscript abroad. As a whole, the results obtained in 2013 confirm the excellent skill and good technical standards found in previous editions. However, erroneous results can be obtained in any laboratory and in clinically relevant determinations. Once again, the results of this program highlighted the need to implement both internal and external controls in order to assure the maximal quality of the microbiological tests.

[Analysis of the results of the HIV-1, HCV and HBV viral load of SEIMC External Quality Control Program. Year 2013]. / Análisis de resultados del Programa de Control de Calidad Externo de carga viral del VIH-1, del VHC y del VHB. Año 2013.

Human immunodeficiency virus type 1 (HIV-1) and hepatitis B (HBV) and C virus (HCV) viral load determinations are among the most relevant markers for the follow up of patients infected with these viruses. External quality control tools are crucial to ensure the accuracy of results obtained by microbiology laboratories. This article summarized the results obtained from the 2013 SEIMC External Quality Control Programme for HIV-1, HCV, and HBV viral loads. In the HIV-1 program, a total of five standards were sent. One standard consisted in seronegative human plasma, while the remaining four contained plasma from three different viremic patients, in the range of 2-5 log10 copies/mL; two of these standards were identical aiming to determine repeatability. A significant proportion of the laboratories (25% on average) obtained values out of the accepted range (mean ± 0.25 log10 copies/mL), depending on the standard and on the method used for quantification. Repeatability was excellent, with up to 98.9% of laboratories reporting results within the limits (D < 0.5 log10 copies/mL). The HBV and HCV program consisted of two standards with different viral load contents. Most of the participants, 82% in the case of HCV and 78% in the HBV, obtained all the results within the accepted range (mean ± 1.96 SD log10 UI/mL). Data from this analysis reinforce the utility of proficiency programmes to ensure the quality of the results obtained by a particular laboratory, as well as the importance of the post-analytical phase on the overall quality. Due to the remarkable interlaboratory variability, it is advisable to use the same method and the same laboratory for patient follow up.

[Analysis of the results of the SEIMC External Quality Control Program. Year 2012]. / Análisis de resultados del Programa de Control de Calidad Externo SEIMC. Año 2012.

The External Quality Control Program of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) include controls for bacteriology, serology, mycology, parasitology, mycobacteria, virology and molecular microbiology. This article presents the most relevant conclusions and lessons from the 2012 controls. As a whole, the results obtained in 2012 confirm the excellent skill and good technical standards found in previous editions. However, erroneous results can be obtained in any laboratory and in clinically relevant determinations. Once again, the results of this program highlighted the need to implement both internal and external controls in order to assure the maximal quality of the microbiological tests.

[Analysis of the results of the HIV-1, HCV and HBV viral load of SEIMC External Quality Control Program. Year 2012]. / Análisis de resultados del Programa de Control de Calidad Externo de carga viral del VIH-1, del VHC y del VHB. Año 2012.

Human immunodeficiency virus type 1 (HIV-1) and hepatitis B (HBV) and C virus (HCV) viral load determinations are among the most relevant markers for the follow up of patients infected with these viruses. External quality control tools are crucial to ensure the accuracy of results obtained by microbiology laboratories. This article summarized the results obtained from the 2012 SEIMC External Quality Control Programme for HIV-1, HCV, and HBV viral loads. In the HIV-1 program, a total of five standards were sent. One standard consisted in seronegative human plasma, while the remaining four contained plasma from three different viremic patients, in the range of 2-5 log10 copies/mL; two of these standards were identical aiming to determine repeatability. A significant proportion of the laboratories (22.3% on average) obtained values out of the accepted range (mean±0.25 log10 copies/mL), depending on the standard and on the method used for quantification. Repeatability was excellent, with up to 98.9% of laboratories reporting results within the limits (Δ < 0.5 log10 copias/mL). The HBV and HCV program consisted of two standards with different viral load contents. Most of the participants, 84% in the case of HCV and 88% in the HBV, obtained all the results within the accepted range (mean±1.96 SD log10 UI/mL). Data from this analysis reinforce the utility of proficiency programmes to ensure the quality of the results obtained by a particular laboratory, as well as the importance of the post-analytical phase on the overall quality. Due to the remarkable interlaboratory variability, it is advisable to use the same method and the same laboratory for patient follow up.

Interlaboratory comparison study on lead in blood, in external quality assessment schemes since 1996: a progress report.

In 1992, at the request of the French labor ministry, an External Quality Control for lead in whole blood (F-EQCPbB) came into being. After 15 years (1996-2011), the ministry wished to exploit the database collected with a sufficient number of laboratories. Indeed, the number of participating laboratories had decreased from 73 to 41. However, the key finding pertained to the highly improved performance of the laboratories, which was associated with a spread of the results over the entire range of tested PbB (9 and 700 µg/l). So, it was that in laboratories having participated for >10 years, the good scores rose between 1996 and 2011 from 49% to 93%. To sum up, analysis has shown progressive and highly pronounced diminution of CVs (%) for all the ranges having undergone testing. We have observed increasing use of inductively coupled plasma with mass spectrometry (from 9% in 2005 to 29% in 2011) and decreasing use of electrothermal atomic absorption spectrometry. That said, and provided that they are based on the same degree of expertise in metrology, on all tested concentrations the two analytical techniques yield results that are not statistically different. Thanks to the F-EQCPbB, laboratories have enhanced their proficiency and registered demonstrably improved performance.

External quality control survey on identification of microorganisms using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（ MALDI-TOF MS） is a bacterial typing tool that was approved as a medical device in 2011. However, external accuracy control examination of bacterial typing using mass spectrometry is still only performed on a small scale. In this study, E. faecium and S. maltophilia were selected and tested according to established procedures using Score Values at 228 institutions. The Score Values for MALDI Biotyper were 2.43±0.08 for E. faecium and 2.38±0.08 for S. maltophilia; and those for VITEK MS/PRIME were 99.9±0.0 for E. faecium and S. maltophilia. These results suggest that it is useful to evaluate external accuracy control with Score Values using the procedures we have developed.

Disease-related data patterns in cerebrospinal fluid diagnostics: medical quality versus analytical quantity.

Cerebrospinal fluid (CSF) diagnostics is characterized by the biologically relevant combination of analytes in order to obtain disease-related data patterns that enable medically relevant interpretations. The necessary change in knowledge bases such as barrier function as a diffusion/CSF flow model and immunological networks of B-cell clones and pleiotropic cytokines is considered. The biophysical and biological principles for data combination are demonstrated using examples from neuroimmunological and dementia diagnostics. In contrast to current developments in clinical chemistry and laboratory medicine, CSF diagnostics is moving away from mega-automated systems with a constantly growing number of individual analyses toward a CSF report that integrates all patient data. Medical training in data sample interpretation in the inter-laboratory test systems ("EQA schemes") has become increasingly important. However, the results for CSF diagnostics (EQAS from INSTAND) indicate a crucially misguided trend. The separate analysis of CSF and serum in different, non-matched assays and extreme batch variations systematically lead to misinterpretations, which are the responsibility of the test providers. The questionable role of expensive accreditation procedures and the associated false quality expectations are discussed. New concepts that reintegrate the medical expertise of the clinical chemist must be emphasized along with the positive side effect of reducing costs in the healthcare system.

Quality Control in RT-PCR Viral Load Assays: Evaluation of Analytical Performance for HIV, HBV, and HCV.

Introduction: Quality Control Management (QCM) in clinical laboratories is crucial for ensuring reliable results in analytical measurements, with biological variation being a key factor. The study focuses on assessing the analytical performance of the Reverse Transcription Polymerase Chain Reaction (RT-PCR) system for Human Immunodeficiency Virus (HIV), Hepatitis B (HBV), and Hepatitis C (HCV). Five models proposed between 1999 and 2014 offer different approaches to evaluating analytical quality, with Model 2 based on biological variation and Model 5 considering the current state of the art. The study evaluates the RT-PCR system's analytical performance through Internal Quality Control (IQC) and External Quality Control (EQC). Materials and Methods: The Laboratório Central de Saúde Pública do Estado do Ceará (LACEN-CE) conducted daily IQC using commercial kits, and EQC was performed through proficiency testing rounds. Random error, systematic error, and total error were determined for each analyte. Results: Analytical performance, assessed through CV and random error, met specifications, with HIV and HBV classified as "desirable" and "optimal." EQC results indicated low systematic error, contributing to total errors considered clinically insignificant. Conclusion: The study highlights the challenge of defining analytical specifications without sufficient biological variability data. Model 5 is deemed the most suitable. The analytical performance of the RT-PCR system for HIV, HBV, and HCV at LACEN-CE demonstrated satisfactory, emphasizing the importance of continuous quality control in molecular biology methodologies.

External quality assessment scheme for sperm DNA fragmentation: a pilot study in China.

BACKGROUND: The aim of this article is to establish an external quality assessment (EQA) scheme for sperm Deoxyribonucleic acid (DNA) fragmentation (SDF) detection, and to assess the feasibility of the scheme. In addition, this article provides some case analysis of abnormal results in order to really help improve the performance of the laboratory. RESULTS: In 2021 and 2022, 10 and 28 laboratories in China volunteered to participate in the EQA program respectively. Two samples were selected for EQA each year, a large spread of results was obtained for the four samples, and the highest values were 13.7, 4.2, 8.0 and 4.0 times the lowest respectively. The coefficients of variation (CVs) were very high for the four samples, at 46.6%, 30.1%, 26.7% and 30.3%, respectively. The CVs of the samples with high SDF values were lower than those of the samples with low SDF values. There was no significant difference between the results of sperm chromatin structure assay (SCSA) and sperm chromatin dispersion (SCD). For the 10 laboratories that participated in EQA in 2021 and 2022, the CVs of low SDF value samples and high SDF value samples decreased from 46.6% and 30.1% in 2021 to 32.5% and 22.7% in 2022, respectively. CONCLUSION: This is the first study to evaluate the EQA program on SDF, which involved a number of laboratories and was demonstrated to be feasible. It is recommended that all laboratories participate in the EQA of SDF to ensure the accuracy of the results.

RéSUMé: CONTEXTES: L'objectif de cet article est d'établir un système externe d'évaluation de la qualité (EEQ) pour la détection de la fragmentation de l'ADN des spermatozoïdes (SDF) et d'évaluer la faisabilité de ce système. En outre, cet article fournit une analyse de cas de résultats anormaux afin d'aider réellement à améliorer les performances du laboratoire. RéSULTATS: En 2021 et 2022, respectivement 10 et 28 laboratoires en Chine se sont portés volontaires pour participer au programme EEQ. Deux échantillons ont été sélectionnés chaque année pour l'EEQ ; un large éventail de résultats a été obtenu pour les quatre échantillons, et les valeurs les plus élevées étaient respectivement de 13,7, 4,2, 8,0 et 4,0 fois les plus faibles. Les coefficients de variation (CV) étaient très élevés pour les quatre échantillons, soit respectivement 46,6 %, 30,1 %, 26,7 % et 30,3 %. Les CV des échantillons avec des valeurs de SDF élevées étaient inférieurs à ceux des échantillons avec de faibles valeurs de SDF. Il n'y avait pas de différence significative entre les résultats du test de structure de la chromatine des spermatozoïdes (SCSA) et ceux de la dispersion de la chromatine des spermatozoïdes (SCD). Pour les 10 laboratoires qui ont participé à l'EEQ en 2021 et 2022, les CV des échantillons à faible valeur de SDF et ceux des échantillons à valeur élevée de SDF ont diminué, passant respectivement de 46,6 % et 30,1 % en 2021 à 32,5 % et 22,7 % en 2022. CONCLUSIONS: Il s'agit de la première étude à évaluer le programme externe d'évaluation de la qualité (EEQ) de l'analyse de la SDF, qui a impliqué un certain nombre de laboratoires, et qui s'est avéré réalisable. Il est recommandé que tous les laboratoires participent à l'EEQ de la SDF afin d'en assurer l'exactitude des résultats.