Multilaboratory Study Research Articles

Results of the 2023 Rapid DNA Multi-Laboratory Study – I-Chip

Identical sensitivity and mixture sample sets were provided to six external laboratories to evaluate the recent enhancements to the Rapid DNA technology for crime scene-type samples. Data were returned to the Federal Bureau of Investigation and the National Institute of Standards and Technology for review and analysis. Manual interpretation (modified Rapid DNA analysis) was conducted on all samples to assess the success of genotyping for both full profiles and the 20 Combined DNA Index System (CODIS) core STR loci using 18 total cellular DNA input amounts ranging from 102.4ng to 0.025ng. Two-person and three-person mixtures at varying ratios for two total DNA inputs were analyzed and observed allele percentages were compared to expected allele percentages.

Results of the 2023 Rapid DNA Multi-Laboratory Study – RapidINTEL Plus Sample Cartridge

Identical sensitivity and mixture sample sets were provided to six external laboratories to evaluate the recent enhancements to the Rapid DNA technology for crime scene-type samples. Data were returned to the Federal Bureau of Investigation and the National Institute of Standards and Technology for review and analysis. Manual interpretation (modified Rapid DNA analysis) was conducted on all samples to assess the success of genotyping for both full profiles and the 20 Combined DNA Index System (CODIS) core STR loci using 18 total cellular DNA input amounts ranging from 102.4ng to 0.025ng. Two-person and three-person mixtures at varying ratios for two total DNA inputs were analyzed and observed allele percentages were compared to expected allele percentages.

Testing the relationship between preferences for infant-directed speech and vocabulary development: A multi-lab study.

From early on, infants show a preference for infant-directed speech (IDS) over adult-directed speech (ADS), and exposure to IDS has been correlated with language outcome measures such as vocabulary. The present multi-laboratory study explores this issue by investigating whether there is a link between early preference for IDS and later vocabulary size. Infants' preference for IDS was tested as part of the ManyBabies 1 project, and follow-up CDI data were collected from a subsample of this dataset at 18 and 24 months. A total of 341 (18 months) and 327 (24 months) infants were tested across 21 laboratories. In neither preregistered analyses with North American and UK English, nor exploratory analyses with a larger sample did we find evidence for a relation between IDS preference and later vocabulary. We discuss implications of this finding in light of recent work suggesting that IDS preference measured in the laboratory has low test-retest reliability.

Housing and Husbandry Factors Affecting Zebrafish (Danio rerio) Novel Tank Test Responses: A Global Multi-Laboratory Study.

The reproducibility crisis in bioscience, characterized by inconsistent study results, impedes our understanding of biological processes and global collaborative studies offer a unique solution. This study is the first global collaboration using the zebrafish (Danio rerio) novel tank test, a behavioral assay for anxiety-like responses. We analyzed data from 20 laboratories worldwide, focusing on housing conditions and experimental setups. Our study included 488 adult zebrafish, tested for 5 min, focusing on a variety of variables. Key findings show females exhibit more anxiety-like behavior than males, underscoring sex as a critical variable. Housing conditions, including higher stocking densities and specific feed types, influenced anxiety levels. Optimal conditions (5 fish/L) and nutritionally rich feeds (e.g., rotifers), mitigated anxiety-like behaviors. Environmental stressors, like noise and transportation, significantly impacted behavior. We recommend standardizing protocols to account for sex differences, optimal stocking densities, nutritionally rich feeds, and minimizing stressors to improve zebrafish behavioral study reliability.

A rigorous multi-laboratory study of known PDAC biomarkers identifies increased sensitivity and specificity over CA19-9 alone

A blood test that enables surveillance for early-stage pancreatic ductal adenocarcinoma (PDAC) is an urgent need. Independent laboratories have reported PDAC biomarkers that could improve biomarker performance over CA19-9 alone, but the performance of the previously reported biomarkers in combination is not known. Therefore, we conducted a coordinated case/control study across multiple laboratories using common sets of blinded training and validation samples (132 and 295 plasma samples, respectively) from PDAC patients and non-PDAC control subjects representing conditions under which surveillance occurs. We analyzed the training set to identify candidate biomarker combination panels using biomarkers across laboratories, and we applied the fixed panels to the validation set. The panels identified in the training set, CA19-9 with CA199.STRA, LRG1, TIMP-1, TGM2, THSP2, ANG, and MUC16.STRA, achieved consistent performance in the validation set. The panel of CA19-9 with the glycan biomarker CA199.STRA improved sensitivity from 0.44 with 0.98 specificity for CA19-9 alone to 0.71 with 0.98 specificity (p < 0.001, 1000-fold bootstrap). Similarly, CA19-9 combined with the protein biomarker LRG1 and CA199.STRA improved specificity from 0.16 with 0.94 sensitivity for CA19-9 to 0.65 with 0.89 sensitivity (p < 0.001, 1000-fold bootstrap). We further validated significantly improved performance using biomarker panels that did not include CA19-9. This study establishes the effectiveness of a coordinated study of previously discovered biomarkers and identified panels of those biomarkers that significantly increased the sensitivity and specificity of early-stage PDAC detection in a rigorous validation trial.

Determination of Aloin A, Aloin B, and Aloe-Emodin in Raw Materials and Finished Products Using HPLC Multi-Laboratory Validation Study, AOAC 2016.09, Final Action Status.

A multi-laboratory validation study was performed on AOAC Official Method of Analysis (OMA) 2016.09, for final action. Eight different laboratories throughout the world participated in the study tested the same set of 6 different laboratory samples of raw materials (commercial) and formulated products (commercial), and all the laboratories successfully generated results within the acceptance criteria. The intention of the study was to evaluate specificity, precision (variation), linearity/range, system suitability, limit of detection (LOD), and limit of quantitation (LOQ) by multiple laboratories to satisfy the requirement of moving the OMA 2016.09 to the final action status. Accuracy and ruggedness were already validated in earlier work of single laboratory validation (1), and it was not necessary to include these validation parameters in the multi-laboratory validation study. Laboratory samples containing Aloe were sent out to participating laboratories. Each lab followed AOAC 2016.09 (First Action status) to analyze contents of aloin A, aloin B, and aloe emodin using high performance liquid chromatography (HPLC) instrument. The results generated by each laboratory were collected and evaluated. The specificity results show that blank and matrix chromatograms do not contain major interfering peaks on the retention time of aloin A, aloin B, and aloe-emodin. The precision (variation) results of duplicated preparations are not more than 0.05 parts per million (ppm) different. The linearity/range results from six standards (10 parts per billion (ppb), 20 ppb, 40 ppb, 80 ppb, 160 ppb, and 500 ppb) have correlation coefficient (R) value of ≥ 0.999. The system suitability results meet the acceptance criteria to show the instrument validity. The limit of detection (LOD) results show that the signal-to-noise (S/N) ratio of 10 ppb standards for all three components are about 3. The limit of quantitation (LOQ) results show that the S/N ratio of 20 ppb standards for all three components are about 10. The validation parameters (specificity, precision (variation), linearity/range, system suitability, LOD, and LOQ) have been successfully analyzed, and it shows that the test method is suitable for its intended use. The test method has been successfully validated by the eight different laboratories around the world (US, UK, Germany, and Canada). Each of the laboratory is managed independently by the site lab management team. This multi-lab study validated the method of Determination of Aloin A, Aloin B, and Aloe-Emodin in Raw Materials and Finished Products Using high performance liquid chromatography (HPLC), and the method fits for its intended purpose.

Analysis of Bovine Lactoferrin in Infant Formula and Adult Nutritional Products by Optical Biosensor Immunoassay: Collaborative Study, Final Action 2021.07.

Bovine lactoferrin is increasingly being used as an ingredient in infant formula manufacture to enhance nutritional efficacy through the provision of growth, immunoprotective, and antimicrobial factors to the neonate. To evaluate method reproducibility of AOAC First Action Official Method 2021.07 for compliance with the performance requirements described in Standard Method Performance Requirement (SMPR®) 2020.005. Eight laboratories participated in the analysis of blind-duplicate samples of seven nutritional products. Samples were diluted in buffer, and an optical biosensor immunoassay was used in a direct-assay format to quantitate bovine lactoferrin by its interaction with an immobilized anti-lactoferrin antibody. Quantitation was accomplished by the external standard technique with interpolation from a four-parameter calibration regression. After outliers were removed, precision as reproducibility was found to be within limits set in SMPR 2020.005 (≤ 9%) for six out of seven samples and all had acceptable Horwitz Ratio (HorRatR) values ranging from 1.0 to 2.1. Additionally, comparison with an alternative independent Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) First Action method (heparin cleanup LC-UV), showed negligible difference between results. The method described is suitable for the quantification of intact, undenatured bovine lactoferrin in powdered infant formulas. The SPIFAN Expert Review Panel evaluated the method and accompanying validation data from this multi-laboratory testing (MLT) study in July 2023 and recommended Official Method 2021.07 for adoption as a Final Action Official MethodSM. A multi-laboratory validation study of an automated optical biosensor immunoassay for the determination of intact, undenatured bovine lactoferrin is described.

Effective orifice diameter: a new sizing parameter of surgical valve prostheses to inform valve selection.

The labeled sizes of surgical valve prostheses and their discordance with the physical internal valve orifice sizes has long been a controversy in the cardiac surgery community, leading many to believe it to be a contributing factor in prosthesis-patient mismatch following valvular replacement surgery. In an attempt to address this issue, the International Organization for Standardization (ISO) 5840-2:2021 standard for surgical valve prostheses recommends that a new sizing parameter, namely, the effective orifice diameter, be provided in labeling by all manufacturers as an indicator of the true flow-passing capacity of a prosthetic valve. The ISO Cardiac Valves Working Group conducted a multi-laboratory round-robin study to investigate whether the effective orifice diameter of a prosthetic surgical valve could be derived repeatably and reproducibly through steady forward-flow testing. A total of seven valve models, each with multiple sizes, were tested, including a mechanical heart valve and multiple biological heart valves. The round-robin study confirmed that the steady forward-flow test had good intra-laboratory repeatability and inter-laboratory reproducibility in deriving the effective orifice diameters of surgical valve prostheses. On average, among the participating laboratories, the experimentally derived effective orifice diameter of a prosthetic heart valve was 3-12 mm smaller than its labeled size. The effective orifice diameter provides better characterization of the hydrodynamic characteristics of a surgical valve prosthesis and can be derived using a validated steady forward-flow test method. This new sizing parameter will soon be adopted by surgical valve manufacturers and provided in device labeling to inform valve selection by surgeons.

Screening of 152 Veterinary Drug Residues in Animal Source Foods by LC-MS/MS, Multilaboratory Validation Study: Final Action 2020.04.

The presence of veterinary drug residues in food-producing animals and animal products is regulated through the enforcement of maximum residue limits (MRLs). To answer the need of the food sector to monitor these substances in a wide range of food commodities, stakeholders at AOAC INTERNATIONAL identified the need for a reliable confirmatory screening method. Such a qualitative approach is required for compliance checking and to support product release in manufacturing. Data were collected from five independent laboratories that applied the First Action Official Method 2020.04 to demonstrate adequate performance under reproducibility conditions. The probability of detection (POD) was calculated in blank test samples and test samples spiked at the screening target concentration (STC) level, with the objective to achieve PODs ≤10% and ≥90%, respectively. Additionally, the effectiveness of the screening method was evaluated by participating in 92 proficiency tests. Four streams were optimized to screen for 152 veterinary drug residues by LC-MS/MS in a wide variety of food commodities including milk-based ingredients and related products (e.g., milk fractions, infant formula, infant cereals, and baby foods), meat- and fish-based ingredients and related products (fresh, powdered, cooked, infant cereals, and baby foods), and other ingredients based on eggs, animal fat, and animal byproducts. The four streams covered 105 antibiotic residues, anti-inflammatory and antiparasitic agents (stream A), 23 beta-lactams (stream B), 14 aminoglycosides (stream C), and 10 tetracyclines (Stream D). The multilaboratory validation led to PODs at the STC ≥94% and PODs in the blank ≤9%. Further application of the multilaboratory validated method to 92 proficiency tests provided more than 99% satisfactory submitted results (n = 784). The interlaboratory reproducibility determined for this method met the acceptance criteria defined in AOAC Standard Method Performance Requirement (SMPR®) 2018.010. AOAC has approved the method for Final Action status.

Development of an accreditation process for analytical methods to measure microplastics in drinking water for regulatory monitoring

Development of a laboratory accreditation program to ensure competency of laboratories performing analytical measurements is a key step in adopting new analytical measurement methods for regulatory decision-making. Here, we describe California's three-part accreditation process for spectroscopically measuring microplastics in drinking water, and show how data from a multi-laboratory method comparison study informed development of accreditation programs for the resulting methods, which can inform analogous future work for other analytes. The first part is periodic performance evaluation (PE) samples, in which laboratories are provided blind samples of known composition to quantify within acceptable performance limits. The second is inspection, or audit, assessing whether the laboratory has the proper equipment to conduct the work and whether it is correctly employing proper procedures. The third is implementation of a quality management system providing documentation that protocols demonstrated during inspection are continuously maintained. These fell into three broad categories: instrument maintenance; laboratory cleanliness, especially important for microplastics and one that must be accompanied by a blanks measurement and correction process; and training so samples are being processed by qualified analysts. An intercomparison exercise among 22 laboratories was necessary to determine what parameter permutations were important for PE samples, and expected accuracy from competent laboratories. The recommended PE sample composition was two size categories (20-50um and 500um-1mm), two polymer types, and two morphologies (fibers and non-fibers). Discussions among intercomparison exercise participants were key in determining the factors that most contributed to laboratory variability, and the focus for both on-site inspections and quality management systems.

Using mice from different breeding sites fails to improve replicability of results from single-laboratory studies

Theoretical and empirical evidence indicates that low external validity due to rigorous standardization of study populations is a cause of poor replicability in animal research. Here we report a multi-laboratory study aimed at investigating whether heterogenization of study populations by using animals from different breeding sites increases the replicability of results from single-laboratory studies. We used male C57BL/6J mice from six different breeding sites to test a standardized against a heterogenized (HET) study design in six independent replicate test laboratories. For the standardized design, each laboratory ordered mice from a single breeding site (each laboratory from a different one), while for the HET design, each laboratory ordered proportionate numbers of mice from the five remaining breeding sites. To test our hypothesis, we assessed 14 outcome variables, including body weight, behavioral measures obtained from a single session on an elevated plus maze, and clinical blood parameters. Both breeding site and test laboratory affected variation in outcome variables, but the effect of test laboratory was more pronounced for most outcome variables. Moreover, heterogenization of study populations by breeding site (HET) did not reduce variation in outcome variables between test laboratories, which was most likely due to the fact that breeding site had only little effect on variation in outcome variables, thereby limiting the scope for HET to reduce between-lab variation. We conclude that heterogenization of study populations by breeding site has limited capacity for improving the replicability of results from single-laboratory animal studies.

Sugar Profile Method by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection in Food, Dietary Supplements, Pet Food, and Animal Feeds: Interlaboratory Validation Study, Final Action 2018.16.

A method for sugar profile analysis granted First Action 2018.16 was subjected to a multi-laboratory study. Perform a multi-laboratory study with this method to determine the performance parameters of repeatability and reproducibility against the AOAC Standard Method Performance Requirements (AOAC SMPR 2018.001) for Final Action status. Eleven laboratories from three different countries participated in the study. Each laboratory was provided practice materials for successful method setup. Each laboratory then proceeded with analysis of blind duplicates of 10 different products covering the scope of the method. Results were reported to the study directors with any modifications and assessed following the procedures of Appendix D of the AOAC Official Methods of AnalysisSM (guidelines for collaborative study procedures). The majority of results from the study met the SMPR requirements. The data is presented along with any outlying observations or modifications. The method was proven to be flexible across different instrumentation and laboratories, and the method was updated to provide further system suitability and guidelines to maintain the performance of the method across the large scope of matrixes. The results from the collaborative study supported the method for Final Action status. The Expert Review Panel reviewed and voted to move the method forward to Final Action and was followed by review from the Official Methods Board and granted approval. The method was granted Final Action Official Methods status.

Sulbactam-durlobactam susceptibility test method development and quality control ranges for MIC and disk diffusion tests.

Sulbactam-durlobactam is a β-lactam/β-lactamase inhibitor combination developed to treat hospital-acquired and ventilator-associated bacterial pneumonia caused by Acinetobacter baumannii-calcoaceticus complex (ABC). Durlobactam is a diazabicyclooctane β-lactamase inhibitor with potent activity against Ambler classes A, C, and D serine β-lactamases and restores sulbactam activity against multidrug-resistant ABC. Studies were conducted to establish sulbactam-durlobactam antimicrobial susceptibility testing methods for both broth microdilution minimal inhibitory concentration (MIC) and disk diffusion tests as well as quality control (QC) ranges. To establish the MIC test method, combinations of sulbactam and durlobactam were evaluated using a panel of genetically characterized A. baumannii isolates which were categorized as predicted to be susceptible or resistant based on the spectrum of β-lactamase inhibition by durlobactam. MIC testing with doubling dilutions of sulbactam with a fixed concentration of 4 µg/mL of durlobactam resulted in the greatest discrimination of the pre-defined susceptible and resistant strains. Similarly, the sulbactam/durlobactam 10/10 µg disk concentration showed the best discrimination as well as correlation with the MIC test. A. baumannii NCTC 13304 was selected for QC purposes because it assesses the activity of both sulbactam and durlobactam with clear endpoints. Multi-laboratory QC studies were conducted according to CLSI M23 Tier 2 criteria. A sulbactam-durlobactam broth MIC QC range of 0.5/4-2/4 µg/mL and a zone diameter QC range of 24-30 mm were determined for A. baumannii NCTC 13304 and have been approved by CLSI. These studies will enable clinical laboratories to perform susceptibility tests with accurate and reproducible methods.

Interlaboratory comparison of centrifugal ultrafiltration with ICP-MS detection in a first-step towards methods to screen for nanomaterial release during certification of drinking water contact materials

A key requirement for evaluating the safety of nano-enabled water treatment devices is measuring concentrations of insoluble nanomaterials released from devices into water that may be ingested by consumers. Therefore, there is a need for simple technique that uses commonly available commercial laboratory techniques to discriminate between nanoparticles and dissolved by-products of the nanomaterial (e.g., ionic metals). Such capabilities would enable screening for particulate or dissolved metals released into water from nanomaterial-containing drinking water contact materials (e.g., paint coatings) or devices (e.g., filters). This multi-laboratory study sought to investigate the use of relatively inexpensive centrifugal ultrafilters to separate nanoparticulate from ionic metal in combination with inductively-coupled plasma mass spectrometry (ICP-MS) detection. The accuracy, precision, and reproducibility for the proposed method were assessed using mixtures of nanoparticulate and ionic gold (Au) in a standard and widely utilized model water matrix (NSF International Standard 53/61). Concentrations for both ionic and nanoparticulate gold based upon measurements of Au mass in the initial solutions and Au permeating the centrifugal ultrafilters. Results across different solution compositions and different participating labs showed that ionic and nanoparticulate Au could be consistently discriminated with ppb concentrations typically resulting in <10 % error. A mass balance was not achieved because nanoparticles were retained on membranes embedded in plastic holders inside the centrifuge tubes, and the entire apparatus could not be acid and/or microwave digested. This was a minor limitation considering the ultrafiltration method is a screening tool, and gold concentration in the permeate indicates the presence of ionic metal rather than nanoforms. With further development, this approach could prove to be an effective tool in screening for nanomaterial release from water-system or device materials as part of third-party certification processes of drinking water compatible products.

Validation of PVT VIABLE® for Detection of Legionella in Potable and Non-Potable Water: AOAC Performance Tested MethodSM 082303.

Phigenics Validation Test (PVT) VIABLE® (Viability Identification Assay by Legionella Enrichment) is a method to detect viable Legionella bacteria in building water systems. To evaluate PVT VIABLE against the ISO 11731:2017 Water Quality-Enumeration of Legionella reference method for the detection of viable Legionella species in potable and non-potable water. PVT VIABLE was tested for inclusivity (n = 50 strains of Legionella) and exclusivity (n = 30 non-target strains), robustness, and stability. A multi-laboratory instrument variation study was performed to evaluate the PCR data. The matrix study was performed on potable and non-potable water samples inoculated with Legionella pneumophila at a low (n = 20) and high fractional level (n = 5). Samples were analyzed using the PVT VIABLE and confirmed using ISO 11731:2017. Statistical analysis showed no difference between PVT VIABLE and ISO 11731:2017 for 100 mL test portions of potable and non-potable water. PVT VIABLE demonstrated high levels of specificity and sensitivity in the inclusivity and exclusivity study. Results of the robustness and stability studies demonstrated the method was sufficiently robust to handle small method changes and met the method claims of stability. PVT VIABLE allows the end user to obtain presumptive results for viable Legionella spp. contamination of potable and non-potable water in 2-3 days. PVT VIABLE provides viable Legionella results in 2-3 days versus 10-14 days for traditional spread plating. This novel diagnostic tool also differentiates between Legionella pneumophila sg1, Legionella pneumophila sg2-15, and Legionella spp. without the need for additional confirmation steps as outlined in ISO 11731:2017.

Multi-laboratory validation study of a real-time PCR method for detection of Salmonella in baby spinach

The FDA Bacteriological Analytical Manual (BAM) Salmonella culture method takes at least 3 days for a presumptive positive result. The FDA developed a quantitative PCR (qPCR) method to detect Salmonella from 24-h preenriched cultures, using ABI 7500 PCR system. The qPCR method has been evaluated as a rapid screening method for a broad range of foods by single laboratory validation (SLV) studies. The present multi-laboratory validation (MLV) study was aimed to measure the reproducibility of this qPCR method and compare its performance with the culture method. Sixteen laboratories participated in two rounds of MLV study to analyze twenty-four blind-coded baby spinach test portions each. The first round yielded ∼84% and ∼82% positive rates across laboratories for the qPCR and culture methods, respectively, which were both outside the fractional range (25%–75%) required for fractionally inoculated test portions by the FDA's Microbiological Method Validation Guidelines. The second round yielded ∼68% and ∼67% positive rates. The relative level of detection (RLOD) for the second-round study was 0.969, suggesting that qPCR and culture methods had similar sensitivity (p > 0.05). The study demonstrated that the qPCR yields reproducible results and is sufficiently sensitive and specific for the detection of Salmonella in food.

On the same page? A qualitative study protocol on collaboration in a multi-laboratory preclinical study.

Medical advancements are slow to reach the patient bedside due to issues with knowledge translation from preclinical studies. Multi-laboratory preclinical studies are a promising strategy for addressing the methodological deficiencies that weaken the translational impact of single laboratory findings. However, multi-laboratory preclinical studies are rare and difficult, requiring strong collaboration to plan and execute a shared protocol. In multiteam systems such as these, collaboration is enhanced when members have cohesive ways of thinking about their goals and how to achieve them-that is, when they have "shared mental models". In this research project, we will examine how members of Canada's first multi-laboratory preclinical study build shared mental models and collaborate in the execution of their study. Six independent labs in Canada will conduct a preclinical study using a common protocol. To investigate mental models and collaboration in this multiteam system we will conduct a longitudinal qualitative study involving interviews at four time points, team observation, and document analysis. We will analyze interview transcripts using deductive coding to produce a matrix analysis of mental model content over time and inductive coding to produce a thematic analysis of members' experiences of collaboration over time. We will also triangulate data sources to "tell the story" of teamwork, capturing events and contextual information that explain changes in mental models and collaboration over time. This study will be one of the most comprehensive longitudinal analyses of a real-world multiteam system, and the first within a preclinical laboratory setting. The results will contribute to our understanding of collaboration in multiteam systems, an organizational form increasingly used to tackle complex scientific and social problems. The results will also inform the implementation of future multi-laboratory preclinical studies, enhancing the likelihood of effective collaboration and improved 'bench to bedside' translation.

A multi-laboratory assessment of clinical exome sequencing for detection of hereditary disease variants: 4441 ClinVar variants for clinical genomic test development and validation

Background and aimsWhole-exome sequencing (WES) technology has become an essential tool in the clinical diagnostic for rare genetic disorders, however, the issues that reduce testing precision, sensitivity, and concordance are not clear under routine testing conditions. The study is to systematically evaluate the comparability of clinical WES testing results in laboratories under routine conditions. MethodsWe designed a multi-laboratory study across 24 participating laboratories in China. We assessed sequencing quality across capture methods and sequencing platforms, benchmarked the impact of coverage and callable regions on detecting single nucleotide variants (SNVs), small insertions and deletions (Indels) under the same computational approaches, and compared the sensitivity, precision and reproducibility on detecting mutations across laboratories. ResultsHigh inter-laboratory variability on variants detection were found across participating laboratories. Sample DNA concentration and sequencing evenness are two major variables that lead to the coverage variation. The difference in bioinformatics tools and computational settings affect the sensitivity and precision of the final output. Besides, copy-number variants (CNVs) identification is less reproducible than SNVs and Indels in the WES testing. We also compiled a list of 4441 low coverage ClinVar variants of 1176 genes from this study, which can be used as a source for creating in silico and synthetic DNA reference materials for clinical genetic disorder detection. ConclusionsThe considerable inter-laboratory variability seen in both sequencing coverage evenness and variants detection highlights the urgent need to improve the precision, sensitivity and comparability of the results generated across different laboratories. The list of low coverage variants can have important implications for the development and validation of clinical genetic disorder tests by laboratories. This study also serves to best practice inform guidelines for detecting clinical genetic disorders by exome sequencing.

Ciliary Beat Frequency: Proceedings and Recommendations from a Multi-laboratory Ring Trial Using 3-D Reconstituted Human Airway Epithelium to Model Mucociliary Clearance

The use of reconstituted human airway (RHuA) epithelial tissues to assess functional endpoints is highly relevant in respiratory toxicology, but standardised methods are lacking. In June 2015, the Institute for In Vitro Sciences (IIVS) held a technical workshop to evaluate the potential for standardisation of methods, including ciliary beat frequency (CBF). The applicability of a protocol suggested in the workshop was assessed in a multi-laboratory ring study. This report summarises the findings, and uses the similarities and differences identified between the laboratories to make recommendations for researchers in the absence of a validated method. Two software platforms for the assessment of CBF were used — Sisson-Ammons Video Analysis (SAVA; Ammons Engineering, Clio, MI, USA) and ciliaFA (National Institutes of Health, Bethesda, MD, USA). Both were utilised for multiple read temperatures, one objective strength (10×) and up to four video captures per tissue, to assess their utility. Two commercial RHuA tissue cultures were used: MucilAir™ (Epithelix, Geneva, Switzerland) and EpiAirway™ (MatTek, Ashland, MA, USA). IL-13 and procaterol were used to induce CBF-specific responses as positive controls. Further testing addressed the impact of tissue acclimation duration, the number of capture fields and objective strengths on baseline CBF readings. Both SAVA and ciliaFA reliably collected CBF data. However, ciliaFA failed to generate accurate CBF measurements above ∼10 Hz. The positive controls were effective, but were subject to inter-laboratory variability. CBF endpoints were generally uniform across replicate tissues, objective strengths and laboratories. Longer tissue acclimation increased the percentage active area, but had minimal impact on CBF. Taken together, these findings support the development and validation of a standardised CBF measurement protocol.

P-615 Lack of analytical interference of dydrogesterone in progesterone immuno-assays: a multi-laboratory spike and recovery study of widespread used assays

Abstract Study question Does dydrogesterone (DYD) and its active metabolite 20-α-dihydrodydrogesterone (DHD) interfere with routinely used progesterone assays? Summary answer This multi-laboratory spike-and-recovery study does not indicate any relevant interference of DYD/DHD within routinely used progesterone assays. What is known already Progesterone (P4), a sex steroid, is measured in serum or plasma by competitive immunoassay or Liquid Chromatography mass spectrometry (LC-MS) in a variety of clinical contexts. One potential limitation of steroid hormone immunoassays is interference caused by compounds with structural similarity to the target steroid of the assay. Dydrogesterone, an orally active close stereoisomer of progesterone, is used for various indications in women’s health, including luteal phase support. To date, the potential interference of DYD/DHD with P4 immunoassays has not systematically been studied. Study design, size, duration The present systematic in-vitro study investigated potential interferences of DYD/DHD in seven widely used, commercially available P4 immunoassays and a LC-MS method over a range of concentrations of DYD/DHD and P4. The study was performed in collaboration with participants in the endocrine section of the Dutch foundation for quality assessments in medical laboratories. For each assay method, each laboratory received a set of nine blinded human plasma samples (containing different P4 and/or DYD/DHD concentrations). Participants/materials, setting, methods Routine human plasma samples were anonymized and pooled to create three graded concentration levels of progesterone (P4 high, P4 medium, P4 low). Each pooled P4 plasma sample (6-7 ml) was spiked at high, medium and “none” DYD/DHD concentration and was divided into 0.5 ml aliquots. The blinded aliquots were analyzed by seven different laboratories with their routine progesterone assay (six different immunoassays and LC-MS assay, respectively). Main results and the role of chance High, medium, and low P4 samples had concentrations of approximately 60 nmol/l progesterone, 20 nmol/l progesterone and &amp;lt;10 nmol/l (male plasma), respectively. The immuno- assays tested were Immulite 200XPI (Siemens Healthineers), Advia Centaur (Siemens Healthineers), Atellica (Siemens Healthineers), Architect I2000 (Abbott Diagnostics), Cobas 8000 (Roche Diagnostics), UniCel DxI 800 (Beckman-Coulter) and LC-MS (Agilent). The sample recovery rate (P4 result obtained by the assays tested for sample spiked with DYD/DHD, divided by the result obtained for the corresponding sample with no DYD/DHD x 100) was within a +/-10% window for the medium P4 and high P4 concentrations, but more variable for the low P4 samples. The latter is, however, attributable to high inter- and intra-method variability, especially at low P4 concentrations. The P4 plasma concentrations generally showed considerable inter-assay variability at each concentration level, with Immunlite 2000 at the lower end and Cobas 8000/801 and Dxi at the higher end of the reported results for the High and Medium P4 samples as compared to LC-MS. The study confirms the inherent method variability of P4 immunoassays casting doubts on the clinical use of P4 cut-offs to guide clinical management, especially at low concentrations. Limitations, reasons for caution Only one sample per concentration level was provided to the laboratories which didn't allow for a systematic quantification of intra-method variability. The concentrations of DYD/DHD haven't been verified by means of a validated bioanalytical method. Measurement of DYD and DHD is to date only feasible by a highly-selective LC-MS method. Wider implications of the findings The present study is strongly indicative that relevant interference of DYD/DHD does not exist. This finding implies that a clinician can reliably distinguish P4 concentrations from DYD/DHD concentrations in a blood sample within a routine care-setting. Trial registration number not applicable