Proficiency Testing Samples Research Articles

Development and Validation of a Loop-Mediated Isothermal Amplification (LAMP) Method for the Rapid, Sensitive, and Specific Detection of Hazelnut as an Allergen in Food Matrix.

Analytical verification of hazelnut in food supports risk-based approaches for proper allergen labeling to prevent unwanted allergic reactions or to quality control diagnostic or therapeutic allergen preparations for allergic subjects. We present the development and validation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of hazelnut by targeting the internal transcribed spacer 2 gene. The qualitative method requires neither sophisticated analytical equipment nor antibodies, allowing an easy-to-use application with no ethical concern related to the use of animals. It demonstrated a limit of detection at or below 10 mg/kg hazelnut in various food matrices, making it also suitable for verifying hazelnut at levels of clinically relevant eliciting doses. Validation against proficiency test samples and testing of applicability with commercial food items confirmed its usefulness in processed foods. The simplicity of the method, including visual colorimetric detection, combined with high specificity and sensitivity, represents an advancement over existing qualitative methods.

Laboratory performances during proficiency tests for the analysis of active substances in pesticide formulations during 2021–2023

During the period 2021–2023, the Italian National Institute of Health (ISS) organised, in collaboration with the Italian Health Ministry, proficiency testing (PTs) for about 22 Italian and other EU laboratories involved in the monitoring programmes for Plant Protection Products (PPPs). The aim of the PTs was to determine the amount of active ingredient in various formulations of PPPs. Overall, the type of formulations to be analysed were soluble concentrate (SL), suspension concentrate (SC), capsule suspension (CS), emulsion water oil (EW), soluble water granule (SG), dustable powder (DP), water dispersible granule (WDG) and emulsion concentrate (EC). In 2021, the ISS organised PTs for the determination of Imazamox, Fenhexamid and Trinexapac-ethyl in three different PPPs as soluble concentrate (SL), suspension concentrate (SC), and capsule suspension (CS), respectively. In 2022, PTs for the determination of Bentazone, Spinosad and Tau-Fluvalinate were organised. For this PTs, commercial PPPs were obtained from the Italian market as SC, emulsion oil water (EW) and soluble water granule (SG). In 2023, the PT was organised to determine the active ingredient Trifloxystrobin, Deltamethrin and Cyprodinil in the commercial product as water dispersible granule (WDG), dustable powder (DP) and emulsion concentrate (EC), respectively. Homogeneity and stability, performed for all PT samples, fulfilled the acceptability criteria. The Jarque–Bera test was used to verify the hypothesis of normality of the data distributions before to calculate the z-score values. Laboratory performances, expressed in terms of z-scores, were satisfactory for almost all participants for all substances.

A-085 Comparing CO2 stability in patient samples and proficiency testing material

Abstract Background Total carbon dioxide (tCO2) measurements are important in the assessment of acid-base disorders. The collection and storage conditions of specimens are important preanalytical factors that can influence the accuracy of tCO2 measurements. Proficiency testing (PT) materials are used to assess the accuracy and reliability of a laboratory’s test performance. Unsuccessful performance on PT challenges can be an indicator of procedural issues. However, the stability of tCO2 in PT material compared to physiologic specimens is unknown and no guidance is provided to laboratories on how to handle PT samples for tCO2 measurement. This study aims to compare tCO2 stability in PT material with that of plasma samples when exposed to ambient air at room temperature. Methods Using enzymatic endpoint measurement on Vitros 4600 Chemistry System, tCO2 was measured in PT material (n = 5) and patient samples (n = 6) at baseline and after exposure to air for 15, 30, 60, 90, and 120 minutes. 500μL of each sample was aliquoted into EZ-nest tube adapters and baseline measurements were immediately analyzed. Average percent difference from baseline was calculated and >-10% bias was considered acceptable. Results The average percent difference from baseline in PT samples exceeded -10% as early as 30 minutes; whereas -3% bias was observed in patient samples at 60 minutes. Results are summarized in the table below. Conclusions The assessment of tCO2 stability ensures the reliability of laboratory measurements. Our results indicate tCO2 in PT material is not stable when exposed to air beyond 15 minutes, however tCO2 in most patient samples remained stable beyond 120 minutes. Understanding the stability limits of tCO2 in PT material allows us to devise workflows that prevent sample degradation during PT events. By establishing best practices for maintaining tCO2 integrity in PT material, laboratories can enhance the accuracy of proficiency testing results.

A-059 Validation of self-collected capillary dried blood spot collection for clinical HbA1c testing

Abstract Background The increased prevalence of diabetes in socially disadvantaged populations requires solutions that are amenable to these communities. Unmanaged diabetes can cause cardiovascular disease, kidney damage, blindness, and limb amputation. Diabetes is traditionally diagnosed using fasting/oral glucose tolerance or hemoglobin A1c (HbA1c) tests; both of which require access to medical and laboratory facilities. Self-collected capillary dried blood spot (DBS) testing for HbA1c could alleviate health disparities by offering access to low cost and minimally invasive remote collection. The objective of this study was to validate self-collected capillary DBS specimens for clinical HbA1c testing. Methods DBS HbA1c was measured using the hemolysate application of the Roche cobas c513 Tina-quant Hemoglobin A1cDx Gen.3 assay. Calibration was performed according to the manufacturer’s package insert. Patient samples were prepared by collecting two 3mm DBS punches. Non-biological standards were prepared by placing two blank 3mm DBS punches into an empty tube and adding 10uL of standard. After 24 hours, samples were resuspended in 750uL of hemolyzing reagent and incubated for 75 minutes at room temperature (RT) while shaking at 800 RPM. Preliminary data showed that non-biological standards spotted onto DBS cards did not behave like real patient samples. A correction factor (CF) was derived (n=134) and challenged (n=20) to account for the systematic bias observed between matched patient DBS and venous specimens. Imprecision was established by running two patient DBS specimens as controls. Accuracy was evaluated using residual College of American Pathologists (CAP) proficiency testing (PT) material (n=32). Linearity was confirmed by running 5 levels of Maine Standards Linearity HbA1c Kit in triplicate. Stability of the DBS specimen and the hemolysate supernatant extracted from the DBS specimen were observed at RT. Results Specimens tested between venous and DBS HbA1c to derive the CF (n=134) had a bias of 0.26% (4.32%) and a linear equation of (y=0.938x +0.631; R=0.996). The resulting CF is: reported HbA1c% = (DBS HbA1c% - 0.631)/0.938. Matched venous and DBS specimens that challenged the CF (n=20) had a bias of -0.10% (-1.83%) and a linear equation of (y=1.06x -0.41; R=0.988). Imprecision was 2.3% and 1.3% for patient DBS control specimens with mean concentrations of 5.3% and 5.9%, respectively. Residual CAP PT samples (n=32) had an average bias of -0.05% (-0.40%). DBS HbA1c concentrations from 4.2-15.5% were linear (y=0.903x +1.15; R=0.999). The DBS specimen is stable for 60 days at RT and the hemolysate supernatant is stable for at least 10 hours at RT. Conclusions Self-collected capillary DBS specimens are acceptable for the clinical measurement of HbA1c, and effectively increase accessibility to diabetes screening and monitoring. Applying a CF to DBS patient samples removes the systematic bias observed between matched venous and DBS samples. Non-biological standards and capillary whole blood do not behave the same on DBS cards. Alternative PT for DBS specimens should be investigated as at-home collection advances.

Analytical performance of publicly dispensed glucometers in primary health care in a southern Brazilian city

AimsThis study aimed to assess the use of glucometers by patients and the analytical performance of glucometers provided by the primary care services. MethodsThe analytical performance of 48 glucometers Accu-Chek® Active, was assessed through quintuplicate analyses of one Roche and one PNCQ (National Quality Control Program) control sample at different concentrations; 31 were also evaluated by a single proficiency testing sample. The evaluation metrics included imprecision, bias, and total error and were measured according to quality specifications based on biological variation (QSBV). Glucometer users answered a questionnaire regarding their experience. ResultsAmong the 48 glucometers evaluated with internal control samples, 17 met precision criteria at both control levels according to QSBV, while 24 met the criteria at only one control level. Of the 31 glucometers further evaluated through proficiency test, 11 met accuracy criteria according to QSBV, and only one device showed an unacceptable result. Out of these 31, only 15 demonstrated a total error within the acceptable maximum limits based on QSBV. ConclusionsOverall, our findings showed that patients had a good understanding of glucometer usage and suggested that some glucometers should be replaced, as they sometimes failed to meet even the manufacturer’s acceptable variation limits, and/or did not meet QSBV.

Determination of 7 nitroimidazoles compounds in meat and natural casing using modified QuEChERS combined with HPLC Orbitrap MS: impact of meat processing approaches on the analytes residue

ABSTRACT Validation of a High-Resolution Orbitrap (HR-Orbitrap) method for the simultaneous identification, confirmation, and quantification of seven 5-Nitroimidazoles (5-NDZs) in meat and casing for the first time to our knowledge. The method was validated under Commission Decision (CD) 2002/657/EC and satisfied its requirements for the Minimum Method Performance Requirements (MMPRs) of 1 µg/kg for these Compounds in meat and casing. The samples were extracted using a modified QuEChERS method. The analyte recovery ranging 75.4–104.8% for meat and 72.44–103.53% for casing, with a relative standard deviation (RSD) of ≤ 19.0% for meat and ≤ 19.83% for casing. The decision limits (CCα) encompass values between 0.06 to 0.67 µg/kg and 0.06 to 0.6 µg/kg respectively for meat and casing, while the detection capabilities (CCβ) range from 0.05 to 0.61 µg/kg and 0.05 to 0.57 µg/kg respectively for meat and casing. The method’s accuracy was confirmed by evaluating a proficiency testing sample (PT), and the findings demonstrated that the results ranging from 5.15 to 11 µg/kg which located in the acceptable range of Z-scores (from −1.2 to −2), indicating their validity. A total of 100 meats and 100 casing real samples were gathered and subjected to testing for the investigated compounds. None of the samples exhibited any traces of the studied compounds. Additionally, a study was conducted to assess how different cooking methods, affect the behavior of these substances in meat. The study introduced 3 µg/kg of 5-NDZs contamination into the meat samples and examined the subsequent changes. The study observed reduced compound concentrations through different cooking methods, with reductions ranging from −5% to −91%. Boiling was especially effective, showing an expected reduction range of −61% to −100% for ronidazole. This study presents a highly effective method for detecting residues of 5-NDZs in meat and casing. The findings are expected to contribute to the development and regulation of a national monitoring program.

Analysis of over 250 novel synthetic opioids and xylazine by LC-MS-MS in blood and urine.

Novel Synthetic Opioids (NSO) are frequently found in postmortem (PM) and human performance (HP) forensic toxicology casework, resulting in impairment and fatal overdoses. Developing a broad NSO method benefits public health, as it can be used to identify trends in potent opioid use to develop risk management programs. This project aimed to design a comprehensive, rapid and routine method for the selective analysis of over 250 novel synthetic opioids in blood and urine. This method rapidly extracted 150 µL of blood or urine via protein precipitation followed by size-exclusion filtration, evaporation and reconstitution. Separation and data acquisition were achieved on a 12 min LC-MS-MS method using an F5 column. Data processing was expedited with a custom built-in query created in-house that automated processing and enhanced quality assurance. Validation according to ASB/ANSI Standard 036 was performed and applicability of the method was assessed using proficiency test and authentic casework samples. Assessed in blood and urine qualitatively were 261 unique analytes including fentanyl analogs (fentalogs), nitazenes and other miscellaneous synthetic opioids. As 59 isomeric target analytes were placed into groups due to co-elution, there were 202 distinct acquired targets or target - groups. To demonstrate applicability, 27 proficiency test blood samples received over an approximate 4-year period were analyzed with 126 expected results assessed comprising 25 unique target analytes. Additionally, 617 fatal accidental overdoses within San Francisco in 2022 were retroactively analyzed by this method with almost 10% of cases containing a new NSO substance(s). Such trends and NSO substances were previously unknown in this community.

Improvement of laboratory testing services by implementation of quality assurance scheme

Introduction: The National AIDS Control Organization (NACO), Government of India and the Uttar Pradesh State AIDS Control Society (UPSACS) established the State Referral Laboratory (SRL) in the Department of Microbiology, Motilal Nehru Medical College, Prayagraj for HIV testing and monitoring of linked HIV Counselling and Testing Services (HCTS) in Uttar Pradesh.Methods: This is a retrospective study conducted from 2008 to 2021. In this evaluation period 10144 serum samples (9580 HIV negative, 555 HIV positive and 9 discordant) were received for retesting at SRL. The Quality Control serum samples received from testing sites consisted of 5% negative and 20% positive from all tested samples collected in the first seven days in the External Quality Assurance Scheme (EQAS) quarter month i.e. January, April, July and October. All samples were tested using rapid diagnostic kits procured by NACO/UPSACS with NACO testing strategy. All linked centres with SRL received 4 coded serum samples for proficiency testing (PT) which were tested similarly.Results: Of 10144 serum samples, 99.92% reported correct results and 0.08% reported discrepancy due to various issues. Similarly in the proficiency testing programme, of 4072 samples tested between 2010 to 2021 by testing sites, 4061 (99.07%) reported concordant results and the remaining14 (0.34%) samples reported indeterminate results. Of the 4072 samples, 14 shows discordant results by three different rapid tests based on different principles. Hands on training were provided for technicians who reported indeterminate results in PT or retesting.Conclusion: The tremendous increase in quality testing at peripheral centrally linked centres increase the reliability of HIV diagnosis at ground level. The study also concluded that continuous monitoring and mentoring of technicians of the diagnostic laboratories under EQAS improves their quality for HIV diagnosis.

Evaluation of the effects of lyophilization and post-reconstitution stability of plasma samples containing PT, APTT, and Fibrinogen parameters

Objective: To evaluate the effects of lyophilization and post-reconstitution stability of plasma samples containing PT (Prothrombin Time), aPTT (activated Partial Thromboplastin Time), and fibrinogen parameters. Research method: Experimental study was conducted on lyophilized plasma samples containing PT, aPTT, and fibrinogen parameters. The homogeneity of sample set before and after lyophilization was evaluated, then the stability of the sample set was assessed in variable time and temperature reservation conditions by Paired–samples t–test. Results: Plasma samples containing 3 parameters PT, aPTT and fibrinogen were not significantly affected by the lyophilization process, shown through repeated measurements with p-value of 0.912, 0.699 and 0.334 respectively. Lyophilized plasma samples after reconstitution are stored at -20°C to achieve stability for 8 weeks, at 2 - 8°C stable for 24 hours, at laboratory temperature stable for 8 hours with all PT, aPTT, and fibrinogen parameters. Conclusion: The freeze-drying process does not change the values of PT, aPTT, fibrinogen parameters in plasma samples, so it can be applied to produce coagulation proficiency testing samples by lyophilization. The reconstituted sample is stable in different time and reservation conditions, met the requierment of reservation condition of reconstituted sample. Key words: Lyophilization, reconstitution, PT, aPTT, fibrinogen.

A reliable quantification of organophosphorus pesticides in brown rice samples for proficiency testing using Japanese official analytical method, QuEChERS, and modified QuEChERS combined with isotope dilution mass spectrometry.

The objective of the present study is to provide reliable concentration values as assigned values for target pesticides in brown rice samples used in proficiency testing (PT) organized by the Hatano Research Institute (HRI). The test samples for PT were prepared by immersing brown rice in the pesticide solution and using a spray dryer by the HRI. Homogeneity and stability assessments were performed for PT samples, and the relative uncertainties due to inhomogeneity and instability were 0.58 %-0.78 % and 0 %-0.96 %, respectively. Quantification for the assigned values of target pesticides by the National Metrology Institute of Japan (NMIJ) was carried out using the multiple analytical methods including Japanese official analytical method, QuEChERS, and modified QuEChERS, which were combined with isotope dilution mass spectrometry, to ensure the reliability of the analytical results. The NMIJ assigned values were 0.065±0.004 mg/kg for chlorpyrifos, 0.217±0.012 mg/kg for diazinon, 0.138±0.008 mg/kg for fenitrothion, and 0.138±0.008 mg/kg for malathion.

Evaluation of a new Electrochemiluminescence Immunoassay System for Measuring High-Sensitivity Cardiac Troponin T.

The aim of this study was to verify the analytical performance of the UD90DT electrochemiluminescence immunoassay system (the UD90DT system) for measuring high-sensitivity cardiac troponin T (hs-cTnT). According to the Clinical and Laboratory Standards Institute guidelines, the imprecision, linearity, reference interval, limit of blank (LoB), limit of detection (LoD), and functional sensitivity (FS) of hs-cTnT using the UD90DT system were verified. The trueness was validated using the Proficiency Testing (PT) materials. The within-run and between-run coefficients of variations (CVs) of two hs-cTnT levels were 7.2% and 1.5%, and 7.1% and 2.6%, respectively. The biases of the PT samples (n = 6) all fell within the allowable total error. The linearity satisfied the requirements, with a slope of 0.9963 and an R12 value of 0.9998. The hs-cTnT levels of the healthy volunteers (n = 20) ranged from 3.0 ng/L to 7.7 ng/L. All blank calibrator measurements (n = 20) fell within the LoD claim, and none of the samples (n = 25) had a LoB value ≤ 3.0 ng/L. The FS was 5.3 ng/L. Furthermore, a good correlation between the UD90DT system and the Cobas e 601 module was observed for hs-cTnT. The analytical performance of hs-cTnT using the UD90DT system is acceptable and satisfies clinical needs.

Evaluation of an automated real-time transcription-mediated amplification (TMA) assay for detection and quantification of cytomegalovirus DNA in different clinical specimens

BackgroundReliable and fast detection and quantification of human cytomegalovirus (CMV) DNA in various diagnostic specimens is essential for care of immunocompromised or congenitally infected individuals. ObjectivesTo evaluate the analytical and clinical performance of the Panther Aptima® CMV (Hologic) quantitative real-time transcription mediated amplification (TMA) assay. Study designPerformance of the TMA assay run on the Hologic Panther Fusion was analysed for 32 proficiency testing samples and 21 quantitative reproducibility panel samples; additionally, we compared results of TMA assay and routine quantitative real-time PCR assays ("PCR-A"= Biomérieux CMV R-gene® or "PCR-B"= Laboratory-developed CMV-PCR) in 518 diagnostic specimens (254 plasma, 120 EDTA whole blood, 43 urine, 45 amniotic fluid and 56 breast milk) at two university hospital laboratories. ResultsAll proficiency panel samples were correctly identified and quantified by the TMA assay; replicate testing of the reproducibility panel samples showed good reproducibility within and between the two laboratories. Sensitivity in plasma and WB was higher for the TMA assay detecting low-level CMV-DNAemia in samples tested negative by routine PCR. Quantitative CMV-DNAemia values correlated well between TMA and real-time PCR. Similarly, urine, AF and BM specimens showed a high rate of concordant results (91%, 98% and 98%, respectively) among TMA and PCR with good correlation of quantitative values. ConclusionThe performance of the Aptima® CMV TMA assay for viral blood load testing compared well to established real-time PCRs. In addition, it can be useful for diagnostics in urine, amniotic fluid and breast milk specimens.

Development and validation of a simple solid-liquid extraction protocol coupled with LC-ESI-MS/MS for the determination of aflatoxin M1 in products of colostrum-based supplements and whey protein-based sports food

Special categories, such as infants and athletes, rely heavily on milk-based products. However, aflatoxin M1 (AFM1) contamination is a prevalent risk. This study aims to develop and validate a simple solid-liquid extraction protocol coupled with LC-ESI-MS/MS for AFM1 determination in colostrum-based supplements and whey protein-based sports foods. Extracts of acetonitrile-water (3:2, v/v) were directly injected into LC-ESI-MS/MS in a positive-ion mode. Separation was performed on the Agilent Poroshell 120 EC-C18 column with core-shell properties in a total analysis run time of 7.5 min. Among the tested extraction solvents, acetonitrile-water (3:2, v/v) provided an efficient direct extraction without any further treatments. AFM1 was eluted at a tR of 4.20 ± 0.05 min using the Agilent Poroshell column, and a tolerable ME% was successfully achieved. In both commodities studied, the recovery percentage, repeatability, intermediate precision, LOD, LOQ, linearity range, and linear regression coefficient (R2) results were 94.3–104.1% with RSDs of 4.70–8.40%, 4.49–9.7%, 7.89–10.6%, 0.0015 µg/kg, 0.005 µg/kg, 0.005–1.000 ng/mL, and 0.9999, respectively. Applicability was demonstrated on two proficiency testing (PT) samples and 60 domestic samples. Results have confirmed the practicality of the proposed assay protocol, with concentrations ranging from 0.010 to 4.423 µg/kg for positive samples. Out of the tested samples, 4.8% of the whey protein-based sports foods violated the EU's established limits. This validated assay protocol would help increase sample processing capacity while also ensuring effective and regular oversight by national regulatory authorities.

Developing DPYD Genotyping Method for Personalized Fluoropyrimidines Therapy.

Fluoropyrimidine drugs are widely used in chemotherapy to treat solid tumors. However, severe toxicity has been reported in 10% to 40% of patients. The DPYD gene encodes the rate-limiting enzyme dihydropyrimidine dehydrogenase responsible for fluoropyrimidine catabolism. The DPYD variants resulting in decreased or no enzyme activity are associated with increased risk of fluoropyrimidine toxicity. This study aims to develop a pharmacogenetic test for screening DPYD variants to guide fluoropyrimidine therapy. A multiplex allele-specific polymerase chain reaction (AS-PCR) assay, followed by capillary electrophoresis, was developed to detect 5 common DPYD variants (c.557A > G, c.1129-5923C > G, c.1679T > G, c.1905 + 1G > A, and c.2846A > T). Deidentified population samples were used for screening positive controls and optimizing assay conditions. Proficiency testing samples with known genotypes were analyzed for test validation. All variants detected were confirmed by Sanger sequencing. From the deidentified population samples, 5 samples were heterozygous for c.557A > G, 2 samples were heterozygous for c.1129-5923C > G (HapB3), and 1 sample was heterozygous for c.2846A > T. The 20 proficiency samples matched with their assigned genotypes, including 13 wild-type samples, 3 samples heterozygous for c.1679T > G, 2 samples heterozygous for c.1905 + 1G > A, and 2 samples heterozygous for c.2846A > T. One of the 3 patient samples was heterozygous for c.1129-5923C > G (HapB3). All the variants detected by the multiplex AS-PCR assay were concordant with Sanger sequencing results. A robust multiplex AS-PCR assay was developed to rapidly detect 5 variants in the DPYD gene. It can be used for screening DPYD variants to identify patients with increased risk of toxicity when prescribed fluoropyrimidine therapy.

Strategies for validation of a low-volume BCR::ABL p190 assay in the clinical laboratory

Abstract Background The low prevalence of samples positive for the BCR::ABL p190 (e1a2) fusion transcript (<1/week) can be a barrier for molecular test validation. The Cepheid Xpert BCR-ABL Ultra p190 Assay is a single-use cartridge-based RUO assay designed for the quantitative detection of BCR::ABL p190 from fresh specimens. The following study describes our validation strategy to overcome this barrier for implementing a quantitative molecular LDT. Methods This study was conducted in a hospital clinical laboratory setting between 2019 to 2023. RNA was extracted and frozen from peripheral blood (PB) and bone marrow (BM) samples that were sent in parallel to a reference lab for BCR::ABL p190 testing. High positive diagnostic specimens (>10% p190/ABL1) were presumptively identified as BCR::ABL FISH positive and p210 negative. Diagnostic specimens were diluted in known negative PB or BM to achieve %p190/ABL1 in various quantitative ranges. Reverse transcription, amplification, and quantitative real-time PCR were performed in a single reaction format (Cepheid Xpert cartridge®). For accuracy determinations, reported results of clinical samples from the reference lab were compared to the obtained values from our validation using extracted RNA only. Banked proficiency testing (PT) material was used to supplement qualitative and quantitative accuracy. For precision, Invivoscribe (IVS) control material (San Diego, CA) diluted in seven levels (12.5% to 0.0031%; 4-fold increments) and Maine Molecular Quality Controls (MMQCI, Saco ME) at 10%, 1%, 0.1%, 0.02%, 0% were run by at least three technologists and rotated amongst the 16 instrument modules. To evaluate linearity, IVS controls at 15 levels (100% to 0.003%; 2-fold increments) were assayed in triplicate. Limit of detection (LoD) was the minimum concentration at which the detection rate is 100% in 10 independent runs. For analytical specificity, high-positive p210 specimens (>10%) were evaluated for detection of p190. Results a total of 169 samples were analyzed (33 BM, 32 PB, 5 PT samples, 73 IVS controls, and 26 MMQCI controls). We found a correlation (linearity) between reference lab results and clinical/ control samples (R-squared ~0.88, P < 0.01). Overall Cohen’s kappa concordance for qualitative accuracy analysis was 0.80 (CI 95% 0.65-0.95). Sensitivity, specificity, and accuracy were 91%, 92%, and 91%, respectively. This assay showed precision in all levels of dilution (IVS and MMQCI samples) as previously established. LoD was 0.02%. Samples with high level of p210 /ABL remained negative with the p190 assay. Conclusion in the setting of low-volume assays optimized for fresh samples, such as for the detection of BCR::ABL p190, dilutions of clinical samples, banked frozen RNA extracts, PT samples, and commercial controls are important tools for validation of a quantitative LDT.

Performance of a Modified ALLType NGS HLA Typing Assay with the Ion Chef/Ion S5/TypeStream Visual System

Abstract OBJECTIVE S: The commercial ALLType assay (One Lambda, West Hills, CA) enables accurate, high-resolution typing of 11 HLA genes using the Ion Chef/Ion S5/TypeStream Visual (TSV) system. The assay features a multiplexed PCR to enrich all target HLA genes in a sample, and up to 48 samples can be indexed and sequenced in a run. The assay is laborious due to the late pooling of indexed libraries. Early pooling significantly simplifies the workflow but may increase the background noise and typing errors. This project aims to examine the feasibility of early pooling immediately after the indexing step and evaluate the performance of such a modified ALLType protocol. METHODS A total of 75 unique samples with diverse HLA genotypes were included in this study, which comprised 52 proficiency testing samples of three different sources, 10 stem cell recipients, 7 stem cell donors (including 2 buccal swabs), and 6 deceased organ donors. Genomic DNA extracted from these samples was tested in four sequencing runs, with 15-31 samples per run, using the modified ALLType procedure (reagent lot: 15). The results were compared with the reference typings generated by the manufacturer-established protocol. All data were analyzed using TSV (version 3.0) and the IPD-IMGT/HLA library (version 3.47); the default analysis parameters were applied. The primary endpoint of the study was the concordance of 3-field typing results. The secondary endpoint was key health metrics, including allele balance, key-exon coverage, and background noise. We also evaluated the saving on hands-on time and reagents with the modified protocol. RESULTS The concordance rates of 3-field typing results were 100% for HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, and HLA-DRB1, and 96.2% for HLA-DRB3/4/5. The three discrepant HLA-DRB3/4/5 results with the modified protocol were false positive allele calls due to a small number of contaminating reads, which were corrected by increasing the threshold for HLA-DRB3/4/5 in TSV. The allele balance and exon 2 coverage across all samples and genes were comparable between the modified and reference protocols, with R-squared values of 0.83 and 0.81, respectively. High background signals in exons were observed in only 0.6% of all samples with both protocols. The modified protocol reduced the hands-on time by 15%, from 6.5 to 5.5 hours, and reduced the use of library preparation reagents by 30%. CONCLUSION It is feasible to pool indexed libraries early during the library preparation process without increasing the background noise in the sequencing data. The modified ALLType protocol showed typing accuracy and health metrics comparable to the original protocol, with significant savings on time and reagents.

Gelatin medium for preserving of Trichinella spp. for quality control in laboratories - estimation of larvae viability.

Official food control laboratories ensure food safety using reliable, validated methods. Council Regulations (EC) No. 853/2004, 854/2004 and 882/2004 of the European Parliament established hygiene rules the production of food of animal origin, together with requirements for official controls. This leads to detailed requirements for Trichinella control set out in Commission Implementing Regulation (EU) 2015/1375 of 10 August 2015. These regulations require the laboratory to participate in proficiency testing (PT) to confirm their competence and improve the quality of testing, and require the PT Organizer to use methods for the preparation and preservation of parasite larvae in order to evaluate and improve detection. Traditional methods of preparing such larvae expose them to rapid degradation, making it necessary to simultaneously isolate the larvae and place them in meatballs to ensure quality. We developed a technique for preserving of Trichinella spp. for quality control such as PT sample preparation. The procedure protects larvae against toxic oxygen activity and bacterial destruction via a gelatin barrier. To estimate the viability of larvae preserved by this method, gelatin capsules with 10 larvae of T. spiralis in each were stored (4-8 °C) during 45 days of an experiment. Samples were tested at 2 day intervals (3 samples each day of testing). In total, 75 samples were tested. Larvae remained alive up to 3 weeks. The number of living larvae diminished after 27 days through day 43, after which no living larvae were observed. The gelatin medium procedure facilitated easy, high-throughput sample preparation and supported 100% recovery for 3 weeks. The method allows fast, efficient and accurate PT sample preparation.

Analysis of PCDD/Fs in environmental samples by using gas chromatography in combination with high resolution mass spectrometry: optimization of sample preparation

ABSTRACT This study developed a simple, economical and time-saving clean-up procedure based on an in-house packed column system for the determination of the most toxic-relevant 17 PCDD (polychlorinated dibenzo-p-dioxin) and PCDF (polychlorinated dibenzofuran) congeners in environmental samples following accelerated solvent extraction. The extract solution was cleaned up using a manually packed column system consisting of a multi-layer silica gel column and an activated carbon column. After clean-up, dioxins and furans were analysed by using gas chromatography in combination with high-resolution mass spectrometry. Isotopic labelled spiking experiments investigated the recovery of all dioxin and furan congeners. Then, the clean-up procedure for environmental samples was validated by applying the analysis of both clean soil reference material (CRM EDF-5183) and heavily contaminated sediment reference material (CRM EDF-5184) samples, as well as participating in the proficiency test (PT) for sediment samples. The recovery efficiency of isotope-labelled standard compounds in certified reference material (CRM) samples and PT samples ranged from 35% to 101% and between 46.1% and 105%, respectively. The mass concentrations of 17 PCDD/Fs in CRM samples were in the acceptable range. The absolute z-score value for each PCDD/F congener in the PT samples was below 1, confirming the analytical procedure’s high accuracy. The applied clean-up procedure in this study has demonstrated accuracy, repeatability and a significant increase in sample extraction/preparation productivity by reducing the time for sample preparation.

B-203 Development and Validation of an LC/MS/MS Method to Quantify Creatinine in Dried Blood Spots

Abstract Background Health disparities in diagnosis and treatment of chronic kidney disease (CKD) are apparent with Black, Brown, and socially disadvantaged people experiencing worse outcomes. Improving access to CKD screening may decrease disparities. Blood creatinine concentrations are a mainstay in CKD screening, as it is commonly used to estimate glomerular filtration rate. Creatinine is classically measured in serum/plasma using high-throughput chemistry analyzers. Dried blood spots (DBS) could offer a solution to under-screening, but require an alternative method since extraction from DBS retrieves minimal sample volume and is difficult to scale using conventional automation. The purpose of this study was to develop an LC/MS/MS DBS method to quantify creatinine and ultimately to increase screening access for underprivileged communities. Methods Creatinine was measured using a 100 × 2.1 mm Hypersil Silica column with an ABSciex API4000 MS/MS detector. A 5-point calibration curve was made using creatinine certified reference material (Cerilliant) and blank whole blood (UTAK) (target concentrations: 0.575, 0.975, 4.575, 10.575, 20.575 mg/dL). Calibrators were spotted onto Whatman903 cards and allowed to dry for at least 24-hours. A 6 mm punch was used for analysis of calibration points and patient samples. Once punched, water (50 ul) was added and the samples were incubated at 60°C for 15 min before shaking at 800 RPM. Acetonitrile with 2H-creatinine internal standard (IS; .06 mg/dL) was added and the mixture was shaken and centrifuged. The resulting supernatant was diluted to contain 20% water before injecting 10 uL. Creatinine was separated using an isocratic mobile phase (80% Acetonitrile, 20% DI water, 10 mM Ammonium Formate) and detected using MRM mode (Q1 114.0 m/z; Q3 86.8 m/z (quantifier) and 44.5 m/z (qualifier)). For the IS a Q1 of 117.0 m/z and Q3 of 89.9 m/z was applied and the extraction ratio of endogenous creatinine to IS was calculated to determine the concentration. Linearity was confirmed by running five levels of DBS calibration standards for five days. Imprecision was established by running 3 control levels five times for five consecutive days. Control material was purchased from BioRad and run as liquid. Accuracy was assessed using residual liquid CAP serum creatinine proficiency testing (PT) material in triplicate. DBS creatinine concentration determined by LC/MS/MS was compared to paired venous serum creatinine concentration determined by the Roche Cobas enzymatic creatinine assay (n = 63). Results Creatinine concentrations from 0.2–20 mg/dL were within the allowable nonlinearity of 0.075 mg/dL or 5.0% (y = 1.00x−0.010;R = 0.999). Intraday and interday precision was <2%and <3%, respectively for all levels (mean concentrations 1.00, 2.02, and 7.05 mg/dL). Residual CAP PT samples (n = 4) were within 2.5% of the peer group mean. Compared to venous results, DBS had a bias of −0.03 mg/dL (−3.573%) and a linear equation of (y = .904x +0.049; R = 0.83). Conclusion Preliminary results indicate that DBS are a viable sample source for measuring creatinine. Creatinine concentrations measured by DBS compared favorably with CAP PT. A clinically insignificant negative bias was seen when DBS creatinine concentration was compared to venous blood.

A-341 Development and Validation of an ICP-MS Method to Quantify Lead in Dried Blood Spots

Abstract Introduction The CDC estimates that ∼500 000 children in the US between ages 1–5 have unsafe blood lead concentrations (> 3.5 ug/dL). Lead toxicity can affect all bodily systems and can be detrimental to children's developing brains and nervous systems. Since lead exposure does not always induce obvious and immediate symptoms, screening is an essential tool to public health. Recently, there was a point of care device recalled that was used to measure blood lead across the country. The demise of this method left a gap for lead screening nationally. Dried blood spot (DBS) sampling is an excellent alternative to traditional collections as it is cost effective, less contamination prone, less invasive, and is amenable to non-standard collection locations. The objective of this study was to develop an accurate and precise DBS lead method with the intention of improving access to screening in underserved populations. Methods Lead was detected using an Agilent7800 ICP-MS. DBS were punched (6mm) and lead was extracted into a water based diluent containing EDTA, ammonium pyrrolidine dithiocarbamate, tetramethylammonium hydroxide, Triton X-100, ethanol and iridium. All DBS were sampled from Whatman903 protein saver cards using a 6mm punch. A 6-point calibration curve was made using custom UTAK whole blood lead controls and a diluent blank (target concentrations: 0, 1, 7, 14, 60 and 100 ug/dL). Exact concentrations of the liquid calibrators were determined using a previously validated lead whole blood method. Once the whole blood calibration concentrations were determined, 40 uL of each calibrator was spotted and allowed to dry for at least 24 hours before a 6mm spot was punched and extracted. Precision was determined using three concentrations of UTAK Whole Blood Lead QC (40 uL/spot; 6mm punch). Each level was run in replicates of five for five days. Linearity was confirmed by running five levels of DBS calibration standards for five days. Accuracy was evaluated using Wisconsin DBS Event Proficiency Testing (PT) Samples and results were compared to the peer means. For the method comparison, venous whole blood samples were collected into Royal Blue EDTA metal free vacutainer tubes; DBS samples were spotted directly from the finger (n = 20). Additional comparison samples (n = 10) were derived from the Pennsylvania whole blood PT program by spotting 40 uL. Results The interday precision for the low, mid, and high QC material was 5.0%, 7.5%, and 3.0% at concentrations of 6.8, 20.7, and 35.8 ug/dL, respectively. Lead concentrations from 1.0–100 ug/dL were within the allowable nonlinearity of 1 ug/dL or 5.0% (y = 1.01x-1.6;R = 0.999). Wisconsin DBS PT results (n = 12; range 0.0–38 ug/dL) were accurate within 10% of the peer mean and within acceptability criteria. Comparison of paired whole blood to DBS samples had an average bias of 4.68% (0.31 ug/dL; Y = 1.027x + 0.13(R = 0.999); range 0.1–77.7 ug/dL). Conclusions We developed and validated an accurate and precise lead DBS method using matrix-matched calibrators. This method supports that DBS collection may be an adequate sample to screen for lead exposure and could supplement the current need for additional ways to screen underserved and/or remote populations.