Intra-laboratory Reproducibility Research Articles

Verification of analytical performance of Creatine Kinase isoenzyme MB (CK-MB) on the Alinity c® Experience from the Biochemistry Laboratory of Mohammed VI University Hospital in Oujda

The verification of analytical methods is a requirement of the NF EN ISO 15189 standard. It involves evaluating the performance of an analytical method according to a well-defined protocol and comparing it to pre-established analytical objectives. In this study, we present the results of the verification protocol for the measurement method of creatine kinase isoenzyme MB (CK-MB) on the Alinity c® analyzer, which is employed for functional exploration of the heart. Our study is conducted according to the criteria of Scope A detailed in the Verification/Validation Guide for Methods in Medical Biology, following the SH GTA 04 recommendations from COFRAC. The verification focused on the CK-MB assay on the Alinity c® analyzer using International Federation of Clinical Chemistry (IFCC) method based on immunoinhibition principle for an assessment of analytical performance in terms of repeatability and intermediate precision. these results were aligned with the manufacturer's specifications. The results obtained for various CK-MB assay verification criteria on the Alinity c® analyzer demonstrate satisfactory repeatability across one level with CV = 1.29%. Intra-laboratory reproducibility was satisfactory for one level with CV = 2.58%. The coefficient of variation (CV) values obtained in our study were compared with manufacturer's specifications, no CV reference values were established by French Society of Clinical Biology SFBC. The findings from this study have allowed us to verify the performance of the CK-MB assay method and to compare them with the objectives set in the accreditation process to which our laboratory is committed.

Verification of the analytical performance of ASO (AntiStreptolysin O) Assay on the Abbott Alinity ci®: Experience of the Biochemistry laboratory of Mohammed VI University Hospital in Oujda/Morocco

ASO (AntiStreptolysin O) is an antibody directed against streptolysin O which is an exotoxin secreted by Streptococcus pyogenes. The assay is mainly used for the diagnosis of post-streptococcal complications (AAR, nephropathy, etc.), False positives may be observed in cases of hyperlipemic serum, or liver disease. The objective of our work is to evaluate the analytical performance of the ASO assay on the Abbott Alinity ci® at the Biochemistry laboratory of the Mohammed VI University Hospital in Oujda. The methodology of this work is based on the evaluation of reproducibility and repeatability, based on the technical accreditation guide (SH GTA 04) of the French Accreditation Committee (COFRAC). The results obtained for the different ASO dosage verification criteria show satisfactory repeatability for both levels (1: low / 2: medium), Intra-laboratory reproducibility was also satisfactory for both levels. By comparing these results with the CV used by the CLSI, we see that the results are consistent with and below the tolerated limits. This study shows that the Biochemistry laboratory of the Mohammed VI University Hospital in Oujda is able to provide accurate and precise results which regards with the requirements of learned societies for the determination of ASO.

An Efficient LC–HRMS-Based Approach to Evaluate Pesticide Contamination in Water Bodies with Measurement Uncertainty Considerations

Over recent decades, the global occurrence of pesticide residues in aquatic environments has been a pivotal issue; however, their trace-level concentrations necessitate the establishment of ultra-sensitive and reliable analytical approaches. To this end, the present study describes the optimization and validation of an LC-HRMS-based method for the accurate determination of 18 pesticides in river and sea water, accompanied by a measurement uncertainty estimation. This method was applied to analyze 17 real samples from agriculture and aquaculture-impacted areas in Greece and Albania. Different solid-phase extraction (SPE) protocols were tested. For the analysis, cutting-edge Orbitrap MS technology and MS/MS fragmentation, along with the use of matrix-matched calibration curves, provided unprecedented accuracy (<5 ppm) and sensitivity for the confirmation of positive detections. Regarding method performance, exceptional linearity was obtained; the limits of quantification ranged from 1.7 ng L−1 to 90 ng L−1, recoveries varied from 61% to 96% in river water, while slightly higher recoveries (60–111%) were observed in seawater. In all cases, repeatability and intra-laboratory reproducibility were below 15%. The measurement expanded uncertainty (U′, k = 2) was estimated considering precision and bias. MU% values were lower than 50% in all cases, as recommended in SANTE guidelines and applied to the quantified results. The matrix effect study exhibited negative values (<20%) for all compounds. Application to real samples showed a low pesticide contamination load that should not be underestimated.

USING RETROSPECTIVE DATA TO ASSESS THE UNCERTAINTY OF INDICATORS OF BIOLOGICAL SAFETY OF FOOD AND FEED RAW MATERIALS

The results of the analysis of the advantages and disadvantages of the use of retrospective data for the implementation of the evaluation of uncertainty in the measurement of indicators of biological safety of food and feed raw materials are given. The model measurement procedure used quantitative determination of genetically modified organisms by means of polymerase chain reaction in real time 250 measurement results of certified reference material with a content of 0.1% genetically modified soybean line obtained under conditions of intra-laboratory reproducibility and 10 results of relevant inter-laboratory comparisons with the participation accredited provider. The suitability of retrospective data was assessed by the Shapiro-Wilk criterion and statistical controllability analysis using standardized score charts and modified Shewhart charts, uncertainty by 4 methods described in general and branch international guidelines and regulations. The results of using a combination of modified Shewhart maps demonstrate the acceptability of this approach for establishing the suitability of retrospective measurement results as input data for uncertainty assessment. By integrating additional "fitness limits" to a set of standardized control limits, this analysis acquires the ability to simultaneously establish not only stability, through the interpretation of geometric patterns, but also compliance with specific criteria. Using this approach, objective evidence of the suitability of retrospective data for estimating measurement uncertainty was obtained. The distribution of retrospective data obtained under conditions of intralaboratory reproducibility is assumed to be normal. As a result of the assessment of statistical controllability and compliance with the established criteria, objective evidence of the suitability of the data for uncertainty assessment was obtained. Analysis of uncertainty intervals of subgroups with the volume of 25 measurements, using 4 assessment methods, demonstrated significant variability in the results of measuring biological safety indicators. The uncertainty interval could narrow or widen by almost 100% of the value within fifty measurements. Median values obtained using different uncertainty estimation methods were 2.69%, 22.69%, 39.23, and 43.08%. This variability is due to the limited coverage of sources of uncertainty, which is inherent in each of the analyzed methods. The most optimal method is the evaluation based on the routine measurement of the certified reference material. Retrospective results of routine measurement of certified reference material and interlaboratory comparisons are an effective source of input data for assessing the uncertainty of the results of measuring biological safety indicators of agricultural raw materials. There is a need for further research to establish an assessment model that will provide an opportunity to take into account the optimal number of influential sources of uncertainty that are inherent in biological systems and cannot be simultaneously taken into account using existing approaches offered by standard assessment methods.

Performance Characteristics of the Quantitative Method for Staphylococcus aureus in Food Products Correspond to ISO 16140-3: 2021

Staphylococcus aureus is a pathogenic bacterium that poses significant health risks when present in food products. High levels of Staphylococcus aureus in food can indicate poor hygiene and handling practices during food production, processing, or storage. In order to maintain food safety and quality, verification of the quantitative method of Staphylococcus aureus is very important. Method verification represents a crucial stage in guaranteeing that all employed procedures conform to the necessary criteria for dependable and precise data. Following the guidelines established in ISO 16140-3:2021, method verification is accomplished through the calculation of the Intralaboratory Reproducibility Standard Deviation (SIR) for implementation validation and the eBias value for verifying the suitability of the method for assessing specific food items. This research was conducted with the objective of confirming the ISO 6888-1:2021 as the designated reference method for quantifying Staphylococcus aureus in food products. For the purpose of confirming the implementation of the method, Cheese was utilized as the test food item. In the context of verifying the method's applicability to different food products, a range of items including condensed milk, margarine, baby porridge, cassava chips and ready-to-eat sausage were examined as challenging foods. Every chosen food item was artificially contaminated with Staphylococcus aureus WDCM 00034. The SIR value obtained was 0.04 ≤ 2× 0.11 (the lowest mean of SR value from ISO 6888-1:2021) which indicated that the National Quality Control Laboratory of Drug and Food (NQCLDF), Indonesian FDA was able to implement the method very well. In addition, the eBias value for all types of food tested was below 0.5log10, which showed that the quantitative method for coagulase-positive Staphylococci (Staphylococcus aureus and other types) could be applied in the NQCLDF laboratory for the extensive scope of food.

The Sansure® Human Papillomavirus DNA Diagnostic Kit offers excellent reproducibility performance for the detection of high-risk HPV.

Cervical cancer screening is a cornerstone of cervical cancer elimination. Detection of high-risk human papillomavirus (hrHPV) is recommended as the first step in screening provided that the assay used has been adequately validated. The Sansure® Human Papillomavirus DNA Diagnostic Kit is a new assay designed to detect HPV16, HPV18 and 13 other HPV in aggregate. The study aimed to evaluate the intra- and interlaboratory reproducibility of the assay according to international guidelines. Five hundred and fifty cervical residual cell samples from women attending cervical cancer screening were selected from the biobank of the HPV National Reference Centre in Belgium and used in this study. After DNA extraction, HPV was tested using the Sansure® Human Papillomavirus DNA Diagnostic Kit. The lower 95% confidence limit around the general reproducibility of this assay should be greater than or equal to 87%, with κ ≥ 0.50. Five hundred and thirty-three samples had valid results. The Sansure® Human Papillomavirus DNA Diagnostic Kit demonstrated an excellent intra-laboratory reproducibility of 93.8% (95% confidence interval [CI]: 91.4-95.7, κ = 0.85). The interlaboratory reproducibility was 93.4 (95% CI: 91.0-95.4, κ = 0.84). Intra and interlaboratory reproducibility were also excellent at the genotype level. Excluding HPV53 single infection samples from the analyses also resulted in excellent agreement. These data show that the Sansure® Human Papillomavirus DNA Diagnostic Kit is highly reproducible.

Ion chromatography coupled with optical emission spectrometry (IC-ICP-OES) methodology for the analysis of inositol phosphates in food and feed

This study presents the development of ion chromatography coupled with inductively coupled plasma optical emission spectrometry (IC-ICP-OES) for the simultaneous determination and quantification of inositol phosphates (InsPx). Using a CarboPac PA100 column with a nitric acid-water gradient, 28 InsPx isomers (InsP6 to InsP2) were separated within 33 min. The method eliminates baseline drift and post-column derivatization thereby simplifying detection and quantification. It achieves low detection limits of 63 μg/L P across a range of 63–3200 μg/L P. Various extraction and sample preparation methods for food and feed matrices were tested, including acidic and alkaline agents, C18 SPE and spin concentrators. The analysis shows intra-day and intra-laboratory reproducibility with deviations smaller than 1 % for standard solutions and under 4 % for feed samples (80 % recovery rate of phytate). This methodology is applicable to explore enzymatic degradation pathways and the analysis of InsPx in complex food and animal feed matrices.

Development and validation of a LC-MS method for the quantification of microcystins in fish for their monitoring in environmental and food context

Cyanobacterial blooms in aquatic ecosystems can result in the accumulation of cyanotoxins in other organisms. Fish can potentially accumulate large amounts of cyanotoxins through their direct interaction with contaminated water and the ingestion of cyanobacterial cells. As a human food source, this can result in a potential health risk. Investigating and monitoring this risk requires analytical methods sufficiently robust to be suitable for different fish species. This study, therefore, aimed to optimize and validate an UHPLC-MS/MS method for the quantification of eight microcystin congeners and nodularin in five different fish species. The limits of detection and quantification were 1 and 3 μg kg−1, respectively. The linearity of the method was very good, with calculated R2 values above 0.99. The mean recovery of the method was within the acceptable range of 70.0%–120.0 %, the repeatability was not higher than 12.6 %, and the highest intra-laboratory reproducibility was 18.7 %. The estimated range for the expanded measurement uncertainty was between 29 % and 58 %. Moreover, multiple fish from Flemish waters were analyzed to show the method's applicability. Multiple microcystin congeners were detected (up to 88.3 μg kg−1 sum of analyzed microcystins) in the liver and viscera of perch and sander. Small amounts of MC-LR and MC-YR (up to 6.1 μg kg−1) were also detected in perch muscle tissue.

Method of rapid spectrophotometric determination of iron in dithionite extracts of soils

Amethod for the determination of dithionite iron for soil samples and fractions of the order of 20–25 mg with a spectrophotometric termination has been developed and optimized.Unlike the previously described methods, phenanthroline was used as a chromogen, which made it possible to simplify the analysis by eliminating the combustion stage.The procedure for processing samples with citrate-bicarbonate-dithionite extractant has been optimized.The repeatability and intra-laboratory reproducibility of the technique are investigated.Verification of the proposed methodology by comparison with the standard one showed good convergence of the results.

DEVELOPMENT AND VALIDATION OF METHODS FOR THE SPECTROPHOTOMETRIC DETERMINATION OF PROSPIDIUM CHLORIDE FOR IDENTIFICATION AND ASSAY ANALYSIS

Objective. The work is devoted to the development and validation of UV-spectrophotometric analysis of propidium chloride, an antitumour drug of the dyspyropiperazine group. The relevance of the study is due to the need to introduce into the practice of quality control of prospidium chloride analysis method characterised by high sensitivity, accuracy, rapidity and availability. Material and methods. The object of the study was a medicinal substance-powder Prospidin (Prospidium chloride, series 271222, expiry date until 12.2027g, produced by «Unitehprom BSU», Minsk). Solutions with working concentration range from 0.05 mol/l to 0.2 mol/l for spectrophotometric analysis were prepared using purified water. Absorption spectra with determination of analytical wavelength (λmax, nm) and value of molar absorption coefficient ε, (l mol-1cm-1) in the range from 200 nm to 400 nm were obtained and analysed on Agilent Cary 60 equipment. Validation parameters were determined in accordance with the regulations of the General Pharmacopoeial Article 1.1.0012.15. Results. Validation evaluation of the developed analytical techniques for the determination of authenticity and active ingredient content by UV spectro-photometry included tests for specificity (in the presence of NaCl 0.9% solution), analytical range (0.05−0.2 mol/L), linearity (r=0.9997), accuracy, preci-sion in terms of repeatability 3.89±0.24 ( ±SD) and intra-laboratory reproducibility (ε=2.1%) and stability. Conclusion. The analysis of electronic spectra of aqueous solutions of propidium chloride demonstrated the presence of R-band absorption (German radikalartig − radical, for systems with unshared electron pairs) with a characteristic wavelength in the far-UV region at λmax=282±0.4 nm, and λmin=255±0.4 nm. It was found that the absorption at λmax in the concentration range from 0.05 mol/L to 0.2 mol/L of a solution of propidine chloride containing nitrogen heteroatoms with unshared electron pairs is due to the implementation of the so-called formally forbidden n→σ* electronic transition involving molecular orbitals. The characteristic appearance of the electronic spectrum with λmax and λmin, compliance with the Beer–Lambert law in the given concentration range, the calculated value of the molar extinction coefficient ε (l mol1cm1) − allow to use the above described spectral characteristics to determine the authenticity and content of the active substance in quality control of prospidium chloride.

Intra‐ and interlaboratory reproducibility evaluation toward international validation status of the AmpFire assay

AbstractTo meet the screening goal of WHO's 90‐70‐90 strategy aimed at eliminating cervical cancer (CC) by 2030, clinical validation of human papillomavirus (HPV) assays is essential to provide accurate and valid results through fulfilling three criteria of the international validation guidelines (IVGs). Previously, the clinical accuracy of the AmpFire® HPV Screening 16/18/HR assay (AmpFire assay) was reported but reproducibility data are lacking. Here, we aim to evaluate the intra‐ and inter‐laboratory reproducibility of the AmpFire assay. The reproducibility of the isothermal AmpFire assay was assessed using 556 cervical cell samples collected from women attending CC screening and biobanked in a Belgian HPV national reference center. This assay detects HPV16, HPV18, and 12 other high‐risk HPV (hrHPV) types (31/33/35/39/45/51/52/56/58/59/66/68) in aggregate. Lower 95% confidence interval bound around the assay's reproducibility should exceed 87%, with κ ≥ 0.50. Additionally, a literature review of the assay's clinical performance was performed. The AmpFire assay showed an excellent intralaboratory (96.4%, 95% CI:94.5–97.8%, κ = 0.920) and interlaboratory (95.3%, 95% CI:93.2–96.9%, κ = 0.897) reproducibility. One study demonstrated noninferior sensitivity of a prototype AmpFire assay targeting 15 hrHPV types (including HPV53) to detect CIN2+. However, clinical specificity became similar to the comparator after removing HPV53 from analyses. The low‐cost and easy‐to‐use AmpFire assay presents excellent reproducibility and—after removing HPV53 from the targeted types—fulfills also clinical accuracy requirements. Inclusion of HPV53, which is not recognized as carcinogenic, comprises clinical specificity of screening assays.

Implementation of ISO 16140-3:2021 for Enumeration of Enterobacteriaceae in Food Products

Method verification is one of the steps required to ensure that all methods used meet the standards required for data reliability and accuracy in a laboratory that implements Good Laboratory Practices (GLP). In accordance with ISO 16140-3:2021, the method verification for microbiology testing implemented by calculating the Intralaboratory Reproducibility Standard Deviation (SIR) for implementation verification and eBias value for food item verification. Method verification for enumeration of Enterobacteriaceae in food products using ISO 16140-3 has never been carried out at the Indonesian Food and Drug Authority (FDA) laboratories. Therefore, verification of this method needs to be performed. This study aimed to verify the ISO 21528-2:2017 as reference method for enumeration of Enterobacteriaceae in food products by colony count technique. The selected food item for implementation verification was pasteurized milk while for food item verification, the selected food items were yogurt, traditional preserved egg (telur pindang), sweet and hard candy, seasoning dry powder, worcester sauce and cassava chips. All of selected foods were artificially contaminated with Escherichia coli WDCM 00012. The SIR value obtained was 0.12 ≤ 2× 0,18 (lowest mean value of SR from ISO 21528-2:2017), indicating that the acceptability limit for the implementation verification was met. While the eBias value for all selected food item were less than 0,5 log10, thus the verification study in the National Quality Contol Laboratory of Drug and Food (NQCLDF), Indonesian FDA, meets the acceptance criteria defined in the standard ISO 16140-3:2021.

Matrix uncertainty in feeding-stuffs according to ISO 19036:2019

Abstract The combined measurement uncertainty of quantitative microbiological methods in the area of food and feed is calculated according to ISO 19036:2019 from the technical (intra-laboratory reproducibility standard deviation), the distributional and the matrix uncertainty (umatrix) of the respective sample type. The matrix uncertainty is a property of a certain sample type and independent of the individual laboratory and analysis, respectively. This study broadens the, so far, comparatively small data base on matrix uncertainties for feeding stuffs by investigating 20 representative feed types in 46 sample series. Thereby, difficulties and limitations arising from sample preparation, the selection of different microbial groups (bacteria and fungi) and the nature of different feed types are highlighted. In addition, the applicability with industrially added feed additives (e.g. probiotics) is discussed. In conclusion, the postulated independence and scope of the matrix uncertainty are limited in these respects.

Establishment of Reference Reagents for Single-Radial-Immunodiffusion Assay on the 2022/23 Seasonal Influenza Vaccine in Japan and Their Quality Validation

Potency tests for influenza vaccines are currently performed using a single-radial immunodiffusion (SRID) assay, which requires a reference antigen and anti-hemagglutinin (HA) serum as reference reagents. Reagents must be newly prepared each time a strain used for vaccine production is modified. Therefore, establishing reference reagents of consistent quality is crucial for conducting vaccine potency tests accurately and precisely. Here, we established reference reagents for the SRID assay to conduct lot release tests of quadrivalent influenza vaccines in Japan during the 2022/23 influenza season. The potency of reference antigens during storage was confirmed. Furthermore, we evaluated the cross-reactivity of each antiserum raised against the HA protein of the 2 lineages of influenza B virus toward different lineages of influenza B virus antigens to select a suitable procedure for the SRID assay for accurate measurement. Finally, the intralaboratory reproducibility of the SRID assay using the established reference reagents was validated, and the SRID reagents had sufficient consistent quality, comparable to that of the reagents used for testing vaccines during previous influenza seasons. Our study contributes to the quality control of influenza vaccines.

Development of a simple screening method for analyzing cereulide toxin in fried rice using liquid chromatography-tandem mass spectrometry.

The presence of cereulide, an emetic toxin produced by Bacillus cereus, in fried rice samples is critical evidence of food poisoning even in situations where B. cereus could not be detected. This study aims to develop a screening method for analyzing cereulide in fried rice using the QuEChERS procedure and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cereulide was identified and quantified in fried rice samples using the QuEChERS extraction method and LC-MS/MS. The accuracies of the methods were determined by analyzing fortified blank samples at two concentrations (10 and 50µg/kg) conducted on three samples daily for five days. The QuEChERS procedure removed matrix compounds from fried rice. Characteristic MS/MS spectra enabled the identification of cereulide. As the matrix effects in seven fried rice samples were within ± 6%, an external solvent calibration curve could be used for quantification. This method exhibited good accuracy ranging from 88 to 89%. The relative standard deviations for both repeatability and intra-laboratory reproducibility were < 4%. These standard deviations satisfied the criteria of the Japanese validation guidelines for residues (MHLW 2010, Director Notice, Syoku-An No. 1224-1). The limit of quantification was 2μg/kg. The applicability of this method was confirmed using the analysis of cereulide in fried rice samples incubated with emetic Bacillus cereus. The QuEChERS extraction procedure described herein showed substantial promise as a reliable screening tool for cereulide in fried rice sample.

Verification of the analytical performance of the serum haptoglobin assay on the Abbott Alinity ci

Introduction: The aim of our study is to evaluate the haptoglobin assay on the Alinity ci automated system. This evaluation is part of an overall approach to verifying the methods used in the central laboratory of the Mohammed VI University Hospital of Oujda , with a view to compiling an accreditation file in accordance with the requirements of standard NF ISO 15189. Materials and methods: The working methodology adapted by our study is based on the recommendations of the protocol of the COFRAC accreditation technical guide SH GTA 04. Verification involved assessing the repeatability and reproducibility of Alinity ci . Results: The results obtained for the various haptoglobin assay verification criteria show satisfactory repeatability for all three levels, with CV1=1.10%, CV2=0.78% and CV3=1.07% respectively. intra-laboratory reproducibility was satisfactory for all three levels, with CV1=1.79%, CV2= 4.2% and CV3=1.62% respectively. Discussion: Verification of an analytical method is an essential step in guaranteeing that the result obtained is as close as possible to the reference value of a sample. Comparing our results with the CV adopted by the SFBC, we can see that the results are in line with and below the tolerated limits. Conclusion: We can therefore conclude that the Abbott Alinity ci system meets the requirements set by scientific societies for the determination of haptoglobin.

Co-Occurrence of Aflatoxin B1, Zearalenone and Ochratoxin A in Feed and Feed Materials in Central Italy from 2018 to 2022.

Mycotoxin contamination of feed and feed materials represent a serious health hazard. This study details the occurrence of aflatoxin B1 (AFB1), zearalenone (ZEN) and ochratoxin A (OTA) in 826 feed and 617 feed material samples, collected in two Italian Regions (Umbria and Marche) from 2018 to 2022 analyzed using a UPLC-FLD platform. The developed method was validated and accredited (ISO/IEC 17025) with satisfactory accuracy and precision data obtained in repeatability and intralaboratory reproducibility conditions. Feed had a higher incidence of contaminated samples (26%) with respect to feed materials (6%). AFB1 was found up to 0.1045 mg/kg in cattle feeds and 0.1234 mg/kg in maize; ZEN was detected up to 6.420 mg/kg in sheep feed while OTA was rarely reported and in lower concentrations (up to 0.085 mg/kg). Co-contamination of at least two mycotoxins was reported in 0.8% of the analyzed samples. The incidence of above maximum content/guidance level samples was 2% for feed and feed materials while almost 3-fold-higher for maize (5.8%) suggesting how mycotoxin contamination can affect some matrices more than others. Obtained data can be useful to improve official monitoring plans and therefore further raise awareness of this issue between agriculture stakeholders, healthcare entities and non-professionals.

Allplex HPV HR Detection assay fulfils all clinical performance and reproducibility validation requirements for primary cervical cancer screening

Human papillomavirus (HPV)-based screening offers better protection against cervical cancer compared to cytology, but HPV screening assays must adhere to validation requirements of the international guidelines to ensure optimal performance. Allplex HPV HR Detection (Allplex) assay, launched in the late 2022, is a fully automated real-time PCR-based assay utilizing innovative technology that enables quantification and concurrent distinction of 14 high-risk HPV genotypes (HPV16,18,31,33,35,39,45,51,52,56,58,59,66 and 68). We assessed the validity of the Allplex for cervical cancer screening purposes, via comparison to a clinically validated comparator assay (Hybrid Capture 2; HC2), and through assessment of intra-laboratory reproducibility and inter-laboratory agreement. A clinical validation panel comprised of 973 residual ThinPrep samples was obtained from women aged 30-64 years participating in the organized Slovenian screening program, of these 863 were from women undergoing their regular screening visit after a previous negative screen test while 110 were from women with underlying cervical intraepithelial neoplasia grade 2 or worse (CIN2+) lesions. The Allplex's relative clinical sensitivity for detection of CIN2+ and CIN3+ were 1.01 (95%CI;0.98-1.04) and 0.98 (95%CI;0.95-1.02), compared to that of HC2. At recommended thresholds of ≥98% and ≥90%, the Allplex's clinical sensitivity and specificity (p=0.0004 and p=0.02, respectively) were non-inferior to HC2. High intra-laboratory reproducibility and inter-laboratory agreement, both overall (98.1% and 97.9%, respectively) and at genotype level (>98.7%) was observed. In addition, analytical genotype-specific performance of Allplex was compared to that of its predecessor Anyplex HPV HR; high overall agreement was observed (96.3%; kappa value 0.88), with some variations in performance. In conclusion, Allplex met all validation criteria described in the international guidelines on sensitivity, specificity and laboratory reproducibility and can be considered clinically validated for primary cervical cancer screening.

ИММУНОФЕРМЕНТНАЯ ТЕСТ-СИСТЕМА ДЛЯ ОЦЕНКИ НАПРЯЖЕННОСТИ ПОСТВАКЦИНАЛЬНОГО ИММУНИТЕТА К ВИРУСУ КЛАССИЧЕСКОЙ ЧУМЫ СВИНЕЙ

Classical swine fever (CSF), being a particularly dangerous transboundary disease of pigs of all breeds and ages, remains one of the most important threats to the global pig industry. An urgent task is the development of new recombinant vaccines that meet the requirements of the DIVA strategy. No less important is the issue of equipping preventive means with accurate and rapid diagnostic test systems that allow assessing the intensity of post-vaccination immunity in target animals. The purpose of this study is to construct an enzyme-linked immunosorbent test system for indicating antibodies to the CSF virus based on a recombinant antigen. Specific components of the test system were obtained using genetic engineering and biochemical methods. Optimization of the ELISA protocol was carried out on a sample of immune and normal pig sera with variations in parameters (time and temperature of sensitization, working concentrations of specific components, reaction time). Determination of the analytical characteristics of the test system included tests for specificity, sensitivity limit, positive-negative threshold, and intra-laboratory reproducibility. It was found that the following parameters are optimal for performing ELISA: sensitizing antigen concentration – 50 ng per well, sensitization time – 3 hours at 37 °C or 12 hours at 4 °C; dilution of the antispecies conjugate is 1:50000, binding time to the enzymatic label is 45 min. The analytical sensitivity limit of the test system was 190 ng/ml for protein, the intra-laboratory reproducibility of results between wells of one plate was no less than 95 %, between individual plates was no less than 93 %. The developed ELISA test system, which has high sensitivity and specificity, can be recommended for assessing the effectiveness of vaccination against CSF.

Metrological characteristic of the pipette method (GOST 12536-2014) for determining soil granulometric composition

The aim of the study. To calculate precision indicators (repeatability and reproducibility) and uncertainty in determining the granulometric (grain) composition of soils using the pipette method. Methods. Using the pipette method the content of the following granulometric fractions was determined in soil samples: 0,1–0,05 mm, 0,05–0,01 mm, 0,01–0,005 mm, 0,005–0,002 mm, 0,002–0,001 mm, less than 0,01 mm, less than 0,001 mm. The determination of pH of the salt extract was carried out by potentiometric technique, and organic matter was measured photometrically. For each aliquot of the sample 16 single measurements were obtained for each soil fraction. Based on these data, precision indicators were assessed under conditions of intra-laboratory repeatability and reproducibility, uncertainty indicators were assessed as well. Main results. The following intralaboratory quality indicators were obtained for the method of determining the granulometric (grain) composition of soils using the pipette method: 1) The value of the intralaboratory repeatability indicator of analysis results (σr) ranged 0,4–1,8%. 2) The relative indicator of intralaboratory precision (σR) was within the 0,7–2,9% range. 3) Expanded uncertainty (U) for different soil fractions ranged from 0,9 to 3,9%. Conclusions. The calculated metrological characteristics of determining the granulometric (grain) composition of soils using the pipette method facilitates the internal control of the measurements quality, promoting the quality and confidence in the laboratory work.