Clinical Diagnostic Laboratories Research Articles

Medical laboratory staff labor cost analysis and modeling for SARS-CoV-2 RNA PCR-testing: A time-and-motion study

BACKGROUND: Reverse transcription polymerase chain reaction (PCR) is the main method for detecting the viral agent of coronavirus disease-2019 (COVID-19). This analysis is performed in a non-automated mode in many medical laboratories, which implies a significant labor intensity of a test. However, its magnitude, as well as the workload on staff in the context of variability in testing volumes, has not yet been quantified due to the lack of systematic approaches to studying the labor costs of laboratory staff.
 AIM: This study aimed to investigate the structure and magnitude of the labor cost components of laboratory staff when performing manual PCR for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) ribonucleic acid (RNA) and obtain stratified data that characterize the labor intensity of the testing, which are suitable for workload modeling.
 MATERIALS AND METHODS: The study of the labor costs (as labor time) of the PCR laboratory staff was conducted based on the Clinical Diagnostic Laboratory of the Multidisciplinary Medical Center of the Bank of Russia using a time-and-motion study methodology. Hence, the authors method for researching the labor costs of medical laboratory staff was applied.
 RESULTS: The lists of standard manipulations of the time-studied labor process were formed workplaces, the manipulations were classified according to the labor cost components, and the duration of each component was statistically determined. The labor intensity per test under current working conditions was 110.2 s for the reception and registration, 144.6 s for RNA extraction, and 79.4 s for reaction mixture preparation/amplification workplaces. The workload magnitude for the personnel of these workplaces corresponded to 47%, 62%, and 34% of a lab specialists annual working rate budget. The stratification of labor costs into fixed and variable allowed us to model the workload depending on the change in the testing volume and the number of analytical runs, as well as develop nomograms for the annual total labor time calculation.
 CONCLUSION: The proposed approach to labor cost accounting and analysis makes it possible to obtain suitable stratified data for modeling the workload of personnel during manual PCR-testing for SARS-CoV-2 RNA. The developed nomograms based on them can serve the purposes of planning the laboratory work and increasing its efficiency by choosing the optimal schemes for organizing the labor process. The obtained data can be used in other laboratories that perform real-time PCR tests for molecular genetic markers.

Utility and Outcomes of the 2019 American College of Medical Genetics and Genomics–Clinical Genome Resource Guidelines for Interpretation of Copy Number Variants with Borderline Classifications at an Academic Clinical Diagnostic Laboratory

In 2019, the American College of Medical Genetics and Genomics and the Clinical Genome Resource published updated technical standards for the interpretation and reporting of copy number variants (CNVs), introducing a semiquantitative classification system to improve standardization and consistency between laboratories. Evaluation of these guidelines' performance will inform laboratories about the impact of their implementation into clinical practice. A total of 145 difficult-to-classify CNVs, originally assessed by an academic molecular diagnostic laboratory, were re-interpreted/classified according to the American College of Medical Genetics and Genomics-Clinical Genome Resource guidelines. Classifications between interpretation systems were then compared. The concordance rate was 60.7%, and significantly more variants of uncertain significance were obtained when using the guidelines (n=98) versus the laboratory's classification system (n=49; P<0.001). The concordance rate was presumably impacted by the intentionally unclear nature of the selected variants. The difference in variant of uncertain significance rate was largely due to laboratory-specific practices for variant interpretation and reporting and differences in utilization of general population data. Laboratory-specific policies and practices may need to be addressed for true standardization. Challenges to consistent guideline utilization are centered around the general lack of high-quality curated data available for CNV interpretations and the inherent subjectivity in the selection of evidence criteria and application of evidence points. Multiple aspects of the guidelines were highlighted to further improve classification standardization.

Clinically Applicable Assessment of Tisagenlecleucel CAR T Cell Treatment by Digital Droplet PCR for Copy Number Variant Assessment.

Chimeric antigen receptor (CAR) T cell therapy is an innovative immunotherapy for treating cancers in both children and adults with proven utility in numerous clinical trials. Significantly, some CAR T cell therapies have now been approved by relevant national regulatory bodies across numerous countries for clinical therapeutic use outside of clinical trials. One such recently licensed product is tisagenlecleucel, a CAR T therapy approved for the treatment of B-cell acute lymphoblastic leukemia (B-ALL) using autologous T cells from the patient. The genetically engineered T cells target a protein called CD19, common to B cells, through a CAR incorporating a 4-1BB costimulatory domain to improve response. Since tisagenlecleucel is now a standard of care treatment for B-ALL, it is clinically essential to be able to accurately monitor these CAR T cells in patients. Assessment of the copy number variant (CNV) of the CAR T cell products allows this within a clinically acceptable timeframe for optimal patient benefit. However, no standardized method with high reproducibility and efficiency has been described within a routine clinical laboratory setting. Here, we demonstrated a novel digital droplet PCR (ddPCR)-based methodology for the study of CNV (ddPCR-CNV) in 4-1BB CD19-specific CAR T cells with universal applicability across clinical diagnostic laboratories.

Analysis of preanalytical errors in a clinical chemistry laboratory: A 2-year study.

Patient safety and medical diagnosis of patients are mainly influenced by laboratory results. The present study aimed to evaluate the errors in the preanalytical phase of testing in a Clinical Chemistry diagnostic laboratory.A review was conducted at the Clinical Chemistry Laboratory of a hospital in Saudi Arabia from January 2019 to December 2020. Using the laboratory information system, the data of all canceled tests and requests were retrieved and evaluated for preanalytical errors.A total of 55,345 laboratory test requests and samples from different departments were evaluated for preanalytical errors. An overall rate of 12.1% (6705) was determined as preanalytical errors. The occurrence of these errors was found to be highest in the emergency department (21%). The leading preanalytical errors were nonreceived samples (3.7%) and hemolysis (3.5%). The annual preanalytical errors revealed an increasing rate in outpatient and inpatient departments, while a decreasing rate was observed in the emergency department. An increased rate of errors was also noted for the 2-year study period from 11.3% to 12.9%.The preanalytical phase has a significant impact on the quality of laboratory results. The rate of error in the study was high and the leading causes were nonreceived samples and hemolysis. An increased occurrence of hemolyzed samples in the outpatient department was noted. Enhanced educational efforts emphasizing specimen quality issues and training in sample collection among hospital staff must be carried out.

ПРАКТИЧЕСКОЕ ПРИМЕНЕНИЕ МЕТОДОВ МАШИННОГО ОБУЧЕНИЯ НА ПРИМЕРЕ ОПРЕДЕЛЕНИЯ АКТИВНОСТИ ТУБЕРКУЛЕЗНОГО ПРОЦЕССА У ЛИЦ С МИНИМАЛЬНЫМИ ТУБЕРКУЛЕЗНЫМИ ИЗМЕНЕНИЯМИ, ВЫЯВЛЕННЫМИ НА РЕНТГЕНОГРАММЕ ОРГАНОВ ГРУДНОЙ КЛЕТКИ

Background: According to the World Health Organization (WHO), over 23% of the human population infected with tuberculosis bacilli – M.tuberculosis. For each infected person, the likelihood of a transition from a state of latent tuberculosis infection (LTBI) to active tuberculosis remains. WHO considers testing and treatment for LTBI in groups at high risk of reactivations as a necessary condition for tuberculosis elimination. Early detection of the transition of LTBI into active tuberculosis presents a certain difficulty due to the absence of clinically and radiographically distinguishable symptoms of the onset of the disease, therefore, immunodiagnostics, including the use of a skin test with a recombinant tuberculosis allergen, and methods of clinical laboratory diagnostics come to the aid of clinicians. The objective of our study was to demonstrate the possibilities of using an artificial intelligence system to identify the level of activity of tuberculosis infection in children with the presence of small tuberculosis changes in the respiratory organs, detected by X-ray. Material and methods The total number of patients registered in anti-tuberculosis institutions enrolled in the study was 489, including: the main group – a training sample consisting of patients with confirmed active tuberculosis (n1 = 369); the control group – a test sample of patients in whom the pathogen was in an inactive form (n2 = 120). As variables for calculations: anamnesis, laboratory parameters and X-ray data, were obtained by routine methods and used in accordance with current national standards of care and clinical guidelines, which did not require additional invasive interventions, equipment and material costs. The above survey results: age, gender, medical history, BCG vaccination, blood biochemical parameters in dynamics, X-ray signs, formalized according to the binary principle (presence / absence), were retrieved from patients files into the study database based on MS Excel spreadsheets for further processing. At the initial stage, the Wolfram Mathematica software package was used for calculations; six classical machine learning (ML) methods were carried out: Logistic Regression, Naive Bayes, Nearest Neighbors, Neural Network and Random Forest. Results: The results of the calculations based on combinations of categorical features did not suit us in terms of the quality of the forecast, and we proceeded to the search for a decision rule based on quantitative features. All spelled above methods predicted the presence of the disease significantly better than its absence. The Random Forest method showed the best results for both categorical and quantitative traits, however, interpretation of its results was not possible for clinical decision making. Convinced of the non-optimality of applying classical ML methods, it was decided to apply the author’s committee machine method with the possibility of minimal correction of conditions for significantly different cardinalities of the separable sets and subsequent geometric interpretation of the results. As a result of the application of the committee machine method, 7 most informative parameters were identified to create a decision rule that makes possible to distinguish patients with inactive pathogen and who do not require treatment in children with suspected tuberculosis. Conclusion: the committee machine method in a geometric formulation lead to localize areas in the feature space that correspond to sick and healthy patients from the training sample. That areas were unambiguously described in the form of a system of inequalities and could be easily explained to clinicians and allow moving from a geometric interpretation to a meaningful description of cause-and-effect relationships between the laboratory parameters in a certain area and the patient’s condition.

Repository of intra- and inter-run variations of quantitative autoantibody assays: a European multicenter study

No reference data are available on repositories to measure precision of autoantibody assays. The scope of this study was to document inter- and intra-run variations of quantitative autoantibody assays based on a real-world large international data set. Members of the European Autoimmunity Standardisation Initiative (EASI) group collected the data of intra- and inter-run variability obtained with assays quantifying 15 different autoantibodies in voluntary participating laboratories from their country. We analyzed the impact on the assay performances of the type of immunoassay, the number of measurements used to calculate the coefficient of variation (CVs), the nature and the autoantibody level of the internal quality control (IQC). Data were obtained from 64 laboratories from 15 European countries between February and October 2021. We analyzed 686 and 1,331 values of intra- and inter-run CVs, respectively. Both CVs were significantly dependent on: the method of immunoassay, the level of IQC with higher imprecision observed when the antibody levels were lower than 2-fold the threshold for positivity, and the nature of the IQC with commercial IQCs having lower CVs than patients-derived IQCs. Our analyses also show that the type of autoantibody has low impact on the assay' performances and that 15 measurements are sufficient to establish reliable intra- and inter-run variations. This study provides for the first time an international repository yielding values of intra- and inter-run variation for quantitative autoantibody assays. These data could be useful for ISO 15189 accreditation requirements and will allow clinical diagnostic laboratories to assure quality of patient results.

Metagenomic DNA sequencing for semi-quantitative pathogen detection from urine: a prospective, laboratory-based, proof-of-concept study

Semi-quantitative bacterial culture is the reference standard to diagnose urinary tract infection, but culture is time-consuming and can be unreliable if patients are receiving antibiotics. Metagenomics could increase diagnostic accuracy and speed by sequencing the microbiota and resistome directly from urine. We aimed to compare metagenomics to culture for semi-quantitative pathogen and resistome detection from urine. In this proof-of-concept study, we prospectively included consecutive urine samples from a clinical diagnostic laboratory in Amsterdam. Urine samples were screened by DNA concentration, followed by PCR-free metagenomic sequencing of randomly selected samples with a high concentration of DNA (culture positive and negative). A diagnostic index was calculated as the product of DNA concentration and fraction of pathogen reads. We compared results with semi-quantitative culture using area under the receiver operating characteristic curve (AUROC) analyses. We used ResFinder and PointFinder for resistance gene detection and compared results to phenotypic antimicrobial susceptibility testing for six antibiotics commonly used for urinary tract infection treatment: nitrofurantoin, ciprofloxacin, fosfomycin, cotrimoxazole, ceftazidime, and ceftriaxone. We screened 529 urine samples of which 86 were sequenced (43 culture positive and 43 culture negative). The AUROC of the DNA concentration-based screening was 0·85 (95% CI 0·81-0·89). At a cutoff value of 6·0 ng/mL, culture positivity was ruled out with a negative predictive value of 91% (95% CI 87-93; 26 of 297 samples), reducing the number of samples requiring sequencing by 56% (297 of 529 samples). The AUROC of the diagnostic index was 0·87 (95% CI 0·79-0·95). A diagnostic index cutoff value of 17·2 yielded a positive predictive value of 93% (95% CI 85-97) and a negative predictive value of 69% (55-80), correcting for a culture-positive prevalence of 66%. Gram-positive pathogens explained eight (89%) of the nine false-negative metagenomic test results. Agreement of phenotypic and genotypic antimicrobial susceptibility testing varied between 71% (22 of 31 samples) and 100% (six of six samples), depending on the antibiotic tested. This study provides proof-of-concept of metagenomic semi-quantitative pathogen and resistome detection for the diagnosis of urinary tract infection. The findings warrant prospective clinical validation of the value of this approach in informing patient management and care. EU Horizon 2020 Research and Innovation Programme.

SAMBA: A Multicolor Digital Melting PCR Platform for Rapid Microbiome Profiling.

During the past decade, breakthroughs in sequencing technology have provided the basis for studies of the myriad ways in which microbial communities in and on the human body influence human health and disease. In almost every medical specialty, there is now a growing interest in accurate and quantitative profiling of the microbiota for use in diagnostic and therapeutic applications. However, the current next-generation sequencing approach for microbiome profiling is costly, requires laborious library preparation, and is challenging to scale up for routine diagnostics. Split, Amplify, and Melt analysis of BActeria-community (SAMBA), a novel multicolor digital melting polymerase chain reaction platform with unprecedented multiplexing capability is presented, and the capability to distinguish and quantify 16 bacteria species in mixtures is demonstrated. Subsequently, SAMBA is applied to measure the compositions of bacteria in the gut microbiome to identify microbial dysbiosis related to colorectal cancer. This rapid, low cost, and high-throughput approach will enable the implementation of microbiome diagnostics in clinical laboratories and routine medical practice.

Mutation spectrum and rates of variants of uncertain significance among African American males undergoing prostate cancer germline testing: Need for equity in genetic testing.

10502 Background: Germline testing (GT) for prostate cancer (PCA) is central to metastatic disease management, PCA screening strategies, and hereditary cancer assessment. African American (AA) males have a higher burden of PCA, yet have lower engagement in germline testing which limits understanding of genetic contribution to PCA. Here we evaluated the germline spectrum of AA and White males with PCA undergoing clinical multigene panel testing (MGPT) to inform germline testing strategies with attention to equity. Methods: Study participants included AA men and White men with PCA who underwent a 14-gene MGPT between April 2012 - December 2020 at a clinical diagnostic laboratory (Ambry Genetics). Exclusions were men with known pathogenic variants reported in their families or who had prior genetic testing. MGPT included: ATM, BRCA1, BRCA2, CHEK2, EPCAM, HOXB13, MLH1, MSH2, MSH6, NBN, PALB2, PMS2, RAD51D, TP53. Sanger or next generation sequencing analysis was performed per standard clinical testing protocol. Variant classification was per ACMG 5-tier system. Descriptive statistics summarized results with counts and percentages for categorical variables and mean and standard deviation for continuous variables. Data were compared with Fisher’s exact, Chi-squared, two proportion z-test, or two sample t-test, as appropriate. Significance level was set a priori at 0.05. Results: The dataset included genetic and clinical data from 427 males who had undergone MGPT: White males (n = 190; 45.5%) and AA males (n = 237; 55.5%). Mean age at diagnosis was 59 + 9.3 years. Among men whose Gleason score was indicated (72%), 47% had Gleason &gt; = 8. Majority of men indicated having a 1st/2nd degree relative with PCA (68.97%) or 1st/2nd degree relative with any cancer (99.2%). In the entire cohort, 8.2% tested positive for a pathogenic/likely pathogenic variant; AA males (n = 14, 5.91%) and White males (n = 21, 11.05%). VUS only was reported in 21.31% of the overall cohort with a significant difference noted between AA and White males (25.32% vs. 16.32%, respectively; p = 0.0238). Mutation spectrum in AA males included: BRCA2 (n = 7), PALB2 (n = 3), ATM (n = 3), and BRCA1 (n = 1). Among White males, a wider spectrum of mutations was observed: BRCA2 (n = 6), ATM (n = 5), HOXB13 (n = 5), CHEK2 (n = 2), TP53 (n = 1), and NBN (n = 2). The proportion of AA males with multiple VUS was significantly higher than for Caucasian males (5.1% vs. 0.53%, p = 0.003). Conclusions: Clinical germline testing among AA males reveals a narrow spectrum of mutations in key DNA repair genes, with important implications for precision therapy and hereditary cancer assessment. Furthermore, AA males had significantly higher rates of multiple VUS, indicating critical need for greater inclusion of diverse populations in genetic studies to discern the pathogenic spectrum contributing to PCA aggressiveness and risk.

МЕТОДИКА ФОРМУВАННЯ КОМПЕТЕНТНОСТЕЙ ТА РЕЗУЛЬТАТІВ НАВЧАННЯ БАКАЛАВРІВ ТЕХНОЛОГІЇ МЕДИЧНОЇ ДІАГНОСТИКИ ТА ЛІКУВАННЯ У ПРОЦЕСІ ВИВЧЕННЯ ТЕМИ «РОЗРАХУНКИ ТА ПРИГОТУВАННЯ РОЗЧИНІВ ПРИБЛИЗНИХ КОНЦЕНТРАЦІЙ»

The article substantiates the professional significance of the formation of competencies in chemistry and learning outcomes of higher education in the specialty "Technologies of medical diagnostics and treatment" in the process of studying the discipline "Technique of laboratory work". It is proved that this discipline integrates knowledge of the technique of chemical experiment, in particular – the preparation of solutions of certain concentrations, which is of immense importance for the professional activities of laboratory assistants of clinical diagnostic laboratories. Thus, this problem requires special methodological attention. It is determined that the formation of competencies in chemistry and learning outcomes of higher education in the specialty "Technologies of medical diagnostics and treatment" should take place in the conditions of creating a specially organized student-centered educational environment in higher education institutions (HEIs). It enables students to use organizational, intellectual-communicative, educational, methodological, logistical, informational-technological resources of HEIs, as well as to influence the systematic updating of educational programs, thus determining the goals, structure, methods and professional orientation of their education. A list of general and special competencies as well as learning outcomes of higher education seekers, which will be formed in the specified topic of the course "Technique of laboratory work" has been composed. On the example of conducting a practical lesson on the corresponding topic, the author's method of forming and evaluating the learning outcomes of applicants for higher education is given. It is shown that the formation of these learning outcomes begins with the acquaintance of applicants with their content. Algorithms for solving computational problems of preparation of tinctures by methods of sample and dilution solutions are given. Detailed instructions for the preparation of solutions of salts, alkalis and acids and determination of their concentrations using a hydrometer are given. The place of group educational activity in the process of formation of students' learning outcomes is outlined. Methods of control and assessment of students' academic achievements are indicated, namely: online testing using modern educational platforms, self-evaluation, evaluation by group members and assessment based on the results of their experimental work.

Обзор практик применения бережливого производства в клинических лабораториях

The main role of clinical laboratory diagnostics is to obtain reliable, reproducible and timely results that help to make the right clinical decisions. As the literature review shows, there are a number of common problems for many clinical laboratories, such as: long turnaround times, high laboratory costs for consumables, insufficient level of satisfaction with quality of services received by patients and high number of complaints. The use of lean manufacturing can be one way of solving these problems. Purpose of the study is to summarize and analyze the literature regarding the application of lean manufacturing in clinical laboratories. Materials and methods. A literature review was performed using the following abstract databases: Web of Science, Scopus, PubMed, ScienceDirect, Mendeley, and NCBI. The depth of the search according to the time parameter had no limitations. The following keywords were used as search markers: “lean management”, “lean thinking”, “lean six sigma”, “lean tools”, “healthcare”, “health services”, “clinical laboratory”. More than 100 publications were analyzed, from which 28 most representative studies were selected. Results. The implementation of lean production methods provides a reduction in turnaround time, costs and errors in the clinical laboratory, reduces the contamination of biological material at the preanalytical stage and reduces waiting time for services and, as a consequence, reduces the number of patient complaints. Conclusion. The above results indicate that lean production methods can be used in laboratories to solve frequently occurring problems and demonstrate the effectiveness of their application in clinical laboratories.

Analysis of possible markers of effective antitumor cellular immune response before starting therapy with immune check-point inhibitors

The aim of the study. to analyse possible markers of an effective antitumor cellular immune response.Material and methods. using the keywords «checkpoint inhibitors, immunotherapy, t-lymphocytes, exhausted t-lymphocytes, anti-tumor immune response», review and original articles (n=34) published from 2005 to 2020 in the Pubmed, Web Of Science, Elsevier databases were selected.Results. the study revealed possible markers reflecting a high activity of an adaptive immune response based on effective recognition of tumor antigens through MHC molecules, a sufficient number of T-lymphocytes and a predominance of T-cytotoxic cells, as well as a low level of expression of inhibitory receptors and small molecules. the presence of single nucleotide polymorphisms in the HLA-I and HLA-II genes encoding MHC-I and MHC-II proteins, respectively, a high level of lymphocytes, among which the most important is the predominance of CD8+ t cells and a low level of T-regulatory cells (T-reg), as well as the presence of single nucleotide polymorphisms in the genes encoding FcγR receptors of T-lymphocytes showed their predictive significance. the diagnostic significance of determining the expression of inhibitory receptors for T-lymphocytes (TIM3, LAG3, TIGIT), especially in combination with the determination of PD-1 expression, was also revealed.Conclusion. the results obtained may be relevant for applying new methods for the assessment of the functional activity of the T-cell immune response before starting therapy with checkpoint inhibitors, as well as for the development of new diagnostic panels, which may be of interest to employees of clinical diagnostic laboratories and research centers.

Triplet-Primed PCR Assays for Accurate Screening of FMR1 CGG Repeat Expansion and Genotype Verification.

Fragile X syndrome and other fragile X-associated disorders are caused by the full-mutation (>200 copies) and premutation (55 to 200 copies) expansion, respectively, of the CGG short tandem repeat in the fragile X messenger ribonucleoprotein 1 (FMR1) gene. Clinical diagnostic laboratories use Southern blot analysis and polymerase chain reaction (PCR)-based tests to detect and/or size the FMR1 CGG repeats. The development of sensitive and high-throughput triplet-primed PCR (TP-PCR) assays has diminished the need to subject all samples to Southern blot analysis, which is both labor- and time-intensive. In this article, we describe two direct TP-PCR (dTP-PCR) assays for the detection of FMR1 CGG repeat expansions. We outline a protocol that is based on melting curve analysis of dTP-PCR amplicons for a rapid and cost-effective first-tier screening and identification of individuals with premutation and full-mutation expansions. We also describe a protocol that employs capillary electrophoresis to resolve the dTP-PCR amplicon fragments and to estimate the repeat sizes of normal (5 to 44 copies), intermediate (45 to 54 copies), and premutation alleles, as well as to detect full mutations and determine the structure of the FMR1 alleles. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Direct triplet-primed PCR master mix preparation and amplification of the FMR1 CGG repeat locus for melting curve analysis Basic Protocol 2: Melting curve analysis of direct triplet-primed PCR amplicons on the Rotor-Gene Q MD × 5plex high-resolution melt platform Alternate Protocol: Melting curve analysis of direct triplet-primed PCR amplicons on the LightCycler 480 system Basic Protocol 3: Generation of direct triplet-primed PCR melting curve analysis profiles Basic Protocol 4: Direct triplet-primed PCR master mix preparation and amplification of the FMR1 CGG repeat locus for capillary electrophoresis Basic Protocol 5: Generation of control FMR1 plasmids for direct triplet-primed PCR melting curve analysis Basic Protocol 6: Sanger sequencing assay to verify FMR1 CGG repeat size and structure of plasmid DNA controls.

Diagnostic value of autoantibodies and novel automated methods of their detection

Autoantibodies (aAb) are human immunoglobulins that can specifically bind to antigenic epitopes of molecules of their own body. Most of AATs have diagnostic value and are serological markers, as well as reflect the main mechanisms of loss of tolerance and inflammation in patients with autoimmune diseases. Indirect immunofluorescence (IIF) was the first method used to detect aAb. During the 1970s and 1980s, there was an evolution in qualitative methods with the introduction of immunoblotting or dot blotting, and second generation quantitative immunometric assays (radioimmunoassay,, enzyme-linked immunosorbent assay (ELISA), fluoroimmunoassay, immunochemiluminescence assay) were suggested. The growing number of aAbs, as well as the growing request for aAb research in general, has fueled the development of automated and multiplex testing. An example is the Chorus trio multi-parameter automatic station (Italy), which combines the ELISA method and the complement fixation reaction (CFR) on one platform. The advantages of this device include a large panel of tests, minimal labor costs, the ability to use the analyzer to perform analyzes in the “cito” mode, perform CFR tests in an automatic mode, high accuracy and reproducibility of results, and a compact design of the analyzer. The Chorus trio multi-parameter automatic station has been implemented into the routine practice of our laboratory for the diagnosis of autoimmune diseases as a confirmatory test. Thus, the effectiveness of new technologies, the possibility of automation and an increase in the speed and quality of testing, as well as the extensive information provided by innovative multi-parameter systems, will bring significant benefits for clinical laboratory diagnostics and clinicians.

Comprehensive hygienic assessment of the environmental physical factors at the workplaces of medical workers

Introduction. Occupational health of medical workers is an intensively studied field of hygienic science. This is due to the high incidence of medical workers, which exceeds the average for sectors of the economy. The presented study uses a risk-based approach for a comprehensive assessment of the influence of physical factors on medical workers working with a large number of electronic medical equipment: physiotherapists, clinical laboratory diagnostics doctors, and ultrasound diagnostics doctors. Materials and methods. Studies of adverse factors of working environment factors were carried out in 4 medical institutions of the Altai Territory and the Novosibirsk Region. Based on the obtained results the values of health risk were calculated and a class of working conditions was established for all the studied jobs. Results. At the first stage, a hygienic assessment of working conditions at the workplaces of medical workers was conducted. The largest number of jobs did not meet sanitary standards for the following indicators: air temperature of the working area (55.6% of all jobs), artificial light (84.5%), light pulsation coefficient (46.7%), concentration of aero ions of negative polarity (57,8%), the coefficient of unipolarity (80%). In addition, a class of working conditions was established at each of the studied jobs (obtained in the range from 2 to 3.4). The estimated health risk of workers from exposure to gamma radiation varied from negligible to moderate. Limitations. Exposures to many of the physical factors in the workplace are approximate based on a typical work day for an employee by department and profession. Conclusion. An assessment of the harmful effects of physical factors at various workplaces in medical institutions heterogeneous for their purpose revealed similar violations in terms of microclimate parameters, illumination, air ion composition and electromagnetic fields from computer monitors, which is consistent with data obtained by previous researchers in this area. It seems necessary to implement a system of preventive measures for employees in medical organizations based on the concept of professional risk analysis with a review of the results each year, taking into account the worked experience.

Serotype Features of 17 Suspected Cases of Foodborne Botulism in China 2019-2022 Revealed by a Multiplex Immuno-Endopep-MS Method.

Diagnosis of botulism caused by multiple serotypes of botulinum neurotoxin (BoNT) is still a challenge due to the lack of a reliable detection method. The present study develops a feasible laboratorial method based on an isotope dilution Immuno-Endopep-MS to detect BoNTs and determine their serotypes and activities in clinical samples. Eleven positive foodborne botulism cases out of a total of 17 suspected cases in China, 2019–2022, were determined by the established method. Blood, urine, vomitus, gastric mucosa samples, and food samples were employed and evidenced to be suitable for the detection. Results showed that, although single type A-intoxication was still the first cause among these foodborne botulism cases, other causes involving type E, type B, and their mixed types were also determined, providing a glimpse to the serotype profile of botulism happened in recent years in China. Furthermore, in order to provide insights into in vivo profiles of toxin serotypes, a comprehensive analysis of clinical specimens collected from one family of four patients was performed during a clinically and therapeutically relevant time frame. Serotypes and concentrations of BoNT in specimens revealed a good correlation with symptoms and progresses of disease. Additionally, serum was proved to be more suitable for detection of BoNT/A with a detection window up to 12 days. A urine sample, although rarely reported for foodborne botulism diagnosis, was validated to be suitable for testing BoNTs, with a longer detection window up to 25 days. To the best of our knowledge, this is the first comprehensive analytical research on in vivo profiles of serotypes A, B, and E in different types of specimens from mixed botulism cases. Our method and findings facilitate the toxin detection and identification by clinical diagnostic laboratories.

Occurrence of Cryptosporidium spp. and Giardia spp. Infection in Humans in Latvia: Evidence of Underdiagnosed and Underreported Cases.

Background and Objectives: Protozoan parasites—Cryptosporidium and Giardia—are important causes of diarrhea with an underestimated short-term burden on childhood growth and wellbeing in children under five years of age. The main transmission routes for both parasites are food and drinking water; transmission from person to person; and, due to their zoonotic nature, from domestic or wild animals to humans. The aims of the present study were to summarize the officially reported human cases of cryptosporidiosis and giardiasis in Latvia and to assess the occurrence of Cryptosporidium and Giardia in children within a prospective prevalence study. Materials and Methods: The number of officially reported cases of cryptosporidiosis and giardiasis in the time period of 2000–2020 was collected from the Centre for Disease Prevention and Control of Latvia. Data from a clinical diagnostic laboratory were included in the study in the period from 1 January 2008 to 31 December 2018. Additionally, a prospective study was performed, and fecal samples were collected from unique 0–17-year-old patients from January to February 2021 and tested using fluorescent microscopy. Results: Overall, during the 20-year period, 71 cases (mean per year = 9) of cryptosporidiosis and 1020 (mean per year = 34) cases of giardiasis were officially reported in Latvia. Meanwhile, within the prospective study, we found 35 (6.0%; 95%CI 4.3–8.1) Cryptosporidium and 42 (7.2%; 95%CI 5.3–9.6) Giardia cases. Conclusions: Here, we provide clear proof that both Cryptosporidium and Giardia are underdiagnosed in Latvia, which could also be true for neighboring Baltic and European countries, where a low number of cases are officially reported. Therefore, we highlight the hypothesis that the actual number of cryptosporidiosis and giardiasis human cases in the Baltic states is higher than that officially reported, including in Latvia.

Клініко-анамнестичні характеристики та медикаментозний супровід вагітних жінок із обтяженим акушерським анамнезом і тромбофіліями

The purpose of the study was to determine the clinical and anamnestic features (age characteristics of women and anamnestic features of previous pregnancies) of pregnant women with burdened obstetric history and thrombophilia with the establishment of the main pharmacological mechanisms of therapeutic support. Materials and methods. The study was performed at the Department of Clinical Laboratory Diagnostics of Kharkiv National Medical University and at the Kharkiv Clinical Laboratory Center in the period from 2017 to 2021. We examined 137 pregnant women: the main group (101 women – pregnant women with a burdened obstetric history and thrombophilia aged 29.0 to 37.0 years) and the control group (36 women – pregnant women with a burdened obstetric history without thrombophilia aged 26.0 to 30.0 years). In general, the predominance of one (35.8 %) and two (46.7 %) pregnancies in the history of women with a burdened obstetric history compared to three (17.5 %) was noted. Results and discussion. An increase (U = 706.0; p &lt;0.001) in the chances of developing negative consequences of pregnancy in thrombophilia with increasing age due to negative idiopathic thromboembolic effects due to thrombophilia was noted. A higher mean age of pregnant women with a burdened obstetric history in the presence of thrombophilia (33.0 [29.0; 37.0] years) was determined compared with the control (28.0 [26.0; 30.0] years). The maximum negative manifestations of idiopathic thromboembolic effects due to thrombophilia in the first pregnancy were found. The predominance of cases of fetal death in the first pregnancy of women with two (94.1 %) and three (78.3 %) pregnancies was determined. In general, the need for antiplatelet therapy in thrombophilia was stated. Its use (χ2 = 46,950; p &lt;0.001) was determined in 85.1 % of cases, the majority (65.3 %) of one drug (probably (χ2 = 164.54; p &lt;0.001) more often (52.5 %) acetylsalicylic acid compared with dipyridamole (5.9 %)). It was found that anti aggregate therapy, which included two drugs (combination: acetylsalicylic acid, dipyridamole or klexan) was most often used in the first (40.7%) pregnancy in contrast to the second (11.8%) and third (13.0 %). Conclusion. The study found that antiplatelet therapy with only one drug (acetylsalicylic acid, dipyridamole or klexan) was more commonly used in the second (74.5%) and third (73.9%) pregnancies than in the first one (40.7%). It was naturally determined that the absence of any medication was noted most often by pregnant women in the first (18.5%) pregnancy in contrast to the second (13.7%) and third (13.0%)

EP474: Prenatal exome sequencing analysis in the clinical setting of fetuses with structural anomalies or increased nuchal translucency

Although prenatal screening and diagnosis are generally mostly focused on the detection of aneuploidies and subchromosomal abnormalities, testing for monogenic disorders has become an important prenatal diagnostic test for pregnancies complicated by congenital anomalies. Multiple studies using gene panels and exome sequencing of fetal samples have revealed that pathogenic variants in single genes are an important cause of congenital anomalies. In this study, we reviewed the indications and results of prenatal trio-exome sequencing performed in a clinical diagnostic laboratory.

EP374: Variant classification changes over time at a clinical molecular diagnostic laboratory

As genetic testing increasingly informs clinical decision making across medical specialties, accurate variant interpretation is essential to ensure patients receive appropriate care. Current guidelines for genomic sequence variant interpretation delineate the evidence that should be incorporated in variant assessments as well as how different types of evidence should be weighted. Variants can be re-interpreted and reclassified over time as new evidence accrues. The Advanced Molecular Diagnostics Laboratory (AMDL) at Mount Sinai Hospital in Toronto, Ontario, provides publicly funded multigene panel testing for patients with suspected hereditary disorders.