bluuebunny/crossref_metadata_2025 huggingface dataset:
B-128 Delivering Predictable Operational efficiency gains at our core central reference laboratory with strategic deployment of navify® Lab Operation informatics automation solution
Authors: Christy A L , Gohil S , Lad A , Alangadan K , Shah N , Bhakta S , Manjure A , Shaikh S
Published: October 2024

Background

Metropolis Healthcare Limited (‘MHL’) operates a network of 200 clinical labs in India, our Global Reference Lab (GRL) in Mumbai processes over 25000 tests in a 17 hour daily operation. We are an ISO15189 and CAP accredited laboratory. Our strategic goals have been to bring operational standardization across the network & Improve speed of service with a predictable 90 percentile production TAT. To achieve this goal, we chose digitalization of our laboratory operation with the deployment of navify ® Lab Operations informatics automation solution in. This poster explains our journey of planning, implementing, and delivering on our goal of predictable operational efficiency. The digital solution is at the forefront of all digitalization IQC processes, auto validation and auto approval of patient results passing through a framework of autoverification rules and complete traceability of processes.

Methods

The poster will explain the scope, planning and implementation approach of this digital automation project at Global reference Lab in 5 stepsHL7 order structure for communication between LIS and navify ® Lab Operations22 analyzers from 11 different manufacturers being connected to the Informatics solution. the corresponding testingTest & IQC (Internal Quality Control) Master with specific ranges and rules10 steps AV (Auto verification) Rules decision framework of concentric sieves across lab disciplines (155 AV Algorithms have been configured at GRL)Structured user acceptance testing framework before Go Live.

Results

This poster discusses the outcomes that we have seen on deployment of this digital solution.Over 25,000 test results are subject AV engine daily with AV rate of 75 % of all results in Biochemistry, Immunoassays.66 % of all Complete Blood Count (CBC) reports 95 % of all HbA1c results 93 % of all ESR resultsOver 1750 QC results are subject to QC rules dailyThere is an improvement of 4 hours observed in production TAT

Conclusions

Laboratory Informatics solution has enhanced lab performance in terms of TAT & efficiency of review. The staff satisfaction (both technical and medical) has increased and we see an impact on production outcome. The poster will also show an incremental effectiveness analysis by comparing key performance areas such as predictability in production, staff time saved, before and after technology deployment.
B-130 Time-of-day dependence of running averages for some components of metabolic panels at our hospital: relation to strategy for patient-based quality control
Authors: Stickle D , Karaseva A O
Published: October 2024

Background

Our hospital system recently reorganized its lab services so as to restrict in-house testing almost exclusively to inpatient testing. This disruption in patient mix caused us to reassess properties of running averages for our most common analytes, for potential use of such data in real-time patient-based quality control (PBQC). In particular, we examined whether there were analytes for which there was significant dependence of running averages on 24-h clock time (time-of-day, TOD).

Methods

We performed a retrospective analysis of 3-months' data (FY24, Q2) for patient results of 13 components of the Basic Metabolic Panel (BMP) and Comprehensive Metabolic Panel: Albumin*, Alkaline phosphatase, Alanine amino transferase, Aspartate aminotransferase*, Calcium*, Chloride*, CO2*, Creatinine, Potassium*, Total Protein*, Sodium, Total Bilirubin, and Urea. Running averages for 20 consecutive results (20-mers) were computed for data restricted to results within reference intervals. This produced an overall mean (X) and standard-deviation (SD) of 20-mers for each analyte. We then computed the average 20-mer result (Y) reported within 1-h bins across 24-hour clock time (t). Y(t) was regarded as having TOD-dependence if: (a) there was an 8-h interval of contiguous results for Y that were strictly either below or above X; and (b) either nadir or apex values for |Y-X| exceeded 0.5 SD.

Results

Seven analytes (*, above) demonstrated TOD-dependence of running means for 20-mers according to criteria given above. An example of TOD-dependent Y(t) is shown in Figure (Albumin).

Conclusions

For our hospital, TOD-dependence of running means was identified for 7 of 13 metabolic panel analytes. TOD-dependence of running means is a circumstance in which use of only a fixed target for running means in PBQC would lead to inherent TOD-dependent variation in capability for systematic error detection. Use of TOD-dependent targets for PBQC would be appropriate in these cases.
B-131 The fallacy of average sigma levels from mixed sample levels
Authors: BROOKS Z
Published: October 2024

Background

Sigma measures the number of SDs (z-value) from the existing sample mean to the nearest analytical performance standard or allowable error limit. Authors and software programs often measure sigma metrics for each QC sample but use an average sigma to compare methods and select QC strategies. That practice leads to dramatic over or under-estimation of the number of errors reported and the selection of inappropriate QC strategies.

Methods

Data samples were created to produce sigma values of 3.0, 4.5, and 6.0. Microsoft Excel function NORMSDIST was used to convert sigma to percent to number of errors per million patients. NORMSINV was used to convert the number of errors per million patients to sigma.

Results

A. Six sigma represents a method with a failure rate of 0.000001% or 0.001 failures of ASP/TEa per million patients. B. Three sigma represents a method with a failure rate of 0.135 percent or 1,350 failures of ASP/TEa per million patients. C. While the average sigma value of samples A and B was 4.5s, the average error rate was 0.0675 percent or 675 failures of ASP/TEa per million patients. D. An error rate was 0.0675 percent converts with the NORMSINV function to a sigma of 3.21. E. A true 4.5 sigma method would have a failure rate of 0.00034 percent or 3.4 failures of ASP/TEa per million patients.

Conclusions

Sigma studies that present an average sigma value underestimate the true number of errors reported. It would be more scientifically correct to either report the number of errors reported or to report the average sigma value based on the average number of errors. Laboratory professionals should interpret sigma studies and publications cautiously if a single sigma is used to represent two or more data sets.
B-141 Analysis of risk factors and construction of prediction model for pneumonia in patients with pneumoconiosis based on the clinical laboratory indexes
Authors: Xu Y , Cui X , Guo Y
Published: October 2024

Background

Pneumoconiosis has been the leading industrial disease in China. Pneumonia is one of the common comorbidities for patients with pneumoconiosis, which may lead to the worse condition and poor prognosis. Timely and accurate diagnosis is necessary to retard the pneumonia progression in pneumoconiosis patients. Due to the similarity of clinical features between pneumoconiosis and pneumonia, clinical laboratory tests are needed for assessing the hazard of pneumonia occurrence in patients with pneumoconiosis. Therefore, we aim to explore the risk factors of pneumonia from laboratory test indexes for patients with pneumoconiosis, construct prediction model, and evaluate its feasibility.

Methods

A total of 243 patients with pneumoconiosis from the General Hospital of Xuzhou Mining Group from September 2021 to August 2023 were selected as the modeling set, which was further separated as the non-pneumonia group and the pneumonia group. Nineteen laboratory test indexes related to inflammation, tumor markers, renal function, and blood routine examination were compared between the two groups. The univariate and multivariate Logistic analysis was used to screen out risk factors and construct the prediction model. Two hundred and twenty-eight patients with pneumoconiosis from General Hospital of Xuzhou Mining Group (Yi'an Branch) were enrolled as the validation set. The predictive efficiency of the model on pneumonia for patients with pneumoconiosis was evaluated through Hosmer-Lemeshow(H-L)test and receiver operating characteristic (ROC) curve.

Results

C-reactive protein (CRP), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), β2 microglobulin, creatinine, urea, neutrophil percentage, lymphocyte percentage, and lymphocyte count in pneumonia group were significant different from those in non-pneumonia group (<0.05). Logistic regression analysis suggested that CRP (OR=3.883, <0.001), IL-6 (OR=1.217, =0.003), IL-8 (OR=1.215, =0.002) and β2-microglobulin (OR=2.838, =0.002) were independent risk factors for pneumonia in patients with pneumoconiosis. The prediction model was constructed as Logit (P) =1.357×CRP+0.196×IL-6+0.195×IL-8+1.043×β2-microglobulin-9.772. The H-L test demonstrated that there was no significantly difference between predicted values and the true values. The areas under ROC curves of the model in the modeling set and the validation set were 0.909 and 0.848. The sensitivity was 86.8% and 75.6%, and accuracy 82.1% and 79.7%, respectively.

Conclusions

CRP, IL-6, IL-8, and β2-microglobulin were independent risk factors of pneumonia for pneumoconiosis patients. The risk model constructed by laboratory test indexes showed a good predictive performance, which might provide laboratory evidence for the pneumonia diagnosis in patients with pneumoconiosis.
B-147 Enhancing the Detection of Vitamin D Metabolites by LC-MS/MS Using a Novel Derivatization Kit
Authors: Muramatsu S , Nakagawa T , Yokota H , Wakui M , Matsushita H
Published: October 2024

Background

Vitamin D is crucial for various biological functions and exists in various tissues, including bones, intestines, and kidneys. 25-hydroxyvitamin D serves as a key marker for assessing vitamin D status in the body. Currently, LC-MS/MS is the gold standard for measuring vitamin D levels. The JeoQuant Kit for LC/MS Analysis of Vitamin D Metabolites (JEOL, Tokyo, Japan), which is used for the determination of vitamin D metabolites in serum based on the DAP-PA (Seki et al, 2019), enhances the ionization efficiency in the LC-MS/MS system for four vitamin D metabolites (25(OH)D3, 25(OH)D2, 24,25(OH)2D3 and 3-epi-25(OH)D3), enabling accurate fractionation and quantification. DAP-PA was developed as a caged Cookson-type reagent, demonstrating significantly enhanced stability compared to previous non-caged Cookson-type reagents. Our research aimed to assess whether the kit works practically.

Methods

The JeoQuant Kit contains internal standards of stable isotope-labelled compounds at appropriate concentrations, derivatization reagents, quality control materials derived from human serum and calibration materials. An Acquity H-Class UPLC coupled with an Acquity TQD mass spectrometer (Waters, MA, US) was used for analysis. CAPCELL CORE C18 2.1mm*100mm (Osaka-soda, Tokyo, Japan) was used as the analytical column. For the pretreatment, an Isolute SLE+ column (Biotage, Tokyo, Japan) was used. Eluent of LC-MS/MS was used 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile, with binary gradient.

Results

Using attached control L/H, reproducibility was shown by intra- and inter-assay CVs which were below 10% and 15%, respectively. Storage tests of derivatization agents at -20°C, 4°C, and room temperature for one week showed no significant difference on peak areas (one-way ANOVA). Through this derivatization, it was possible to achieve more than a 20-fold increase in sensitivity compared to measurements without derivatization. Notably, low concentrations of 3-epi-25(OH)D3 were distinctly separated and detected.

Conclusions

This study showed that the JeoQuant Kit effectively increases the sensitivity of vitamin D metabolites through derivatization. It also emphasized the reagent's notable stability. Furthermore, it enabled the detection of 3-epi-25(OH)D3 with less sensitive LC-MS/MS models, offering clear separation from 25(OH)D3. The kit, which is complete with calibrants, internal standards, and quality control samples, simplifies analytical procedures for vitamin D metabolite quantification, potentially aiding in developing Vitamin D- related biomarkers. Efforts to compare analytical performance of this kit with that of other methods are underway to establish its benefits.
B-152 Analysis of carbonyl compounds in exhaled breath for detection of COVID-19
Authors: Fu X , Xie Z , Morris J D , Alksas A D , El-Baz A D , Huang J D , Nantz M H
Published: October 2024

Background

COVID-19 pandemic has caused enormous loss of lives and economic disruption. The global crisis has resulted in more than 700 million infections and 7 million deaths worldwide. Rapid screening and detection of highly infectious respiratory diseases is a key for preventing and curbing future epidemic and pandemic. The objective of this work is to develop a novel breath analysis technique for detection of COVID-19 using chemoselective capture of carbonyl compounds in exhaled breath for analysis by gas chromatography-mass spectrometry (GC-MS). Respiratory viral infection causes oxidative stress and inflammation in its host body which leads to production of higher levels of some carbonyl compounds through oxidation of lipids and could be detected in exhaled breath. This breath analysis technique can be rapidly adapted for screening infectious respiratory diseases for future epidemic and potential pandemic.

Methods

A total of 237 subjects were enrolled in this study. Of these, 142 were COVID-19 positive and 95 were negative. One liter exhaled breath in Tedlar bags from both COVID-19 positive and negative subjects confirmed by reverse transcriptase polymerase chain reaction between April 2022 and September 2023. The collected one liter breath samples were completely evacuated through a cartridge packed with silica particles in 2-3 minutes. Silica particles were coated with a reactive aminooxy agent for capture of carbonyl compounds in the breath samples through oximation reactions. The captured carbonyl compounds were eluted by methanol and the eluted solutions were analyzed by GC-MS for calculation of each compound concentration in the exhaled breath samples. Biostatistics was used to identify significant carbonyl compounds (defined as p value < 0.05) as markers of CPOVID-19 infection. A machine learning algorithm was implemented with the captured carbonyl compounds as input features for differentiation of COVID-19 positive from negative subjects.

Results

A total of more than 30 carbonyl compounds were detected in exhaled breath samples of all subjects. 10 features including 3 individual carbonyl compounds and 7 compound ratios show significant differences (p value <0.05) between COVID-19 positive and negative groups. The developed machine learning and artificial intelligence (AI) algorithms successfully separated COVOD-19 positive from negative with an accuracy of 95.4%, sensitivity of 96.5%, specificity of 93.7%, and AUROC=0.974.

Conclusions

The analysis of carbonyl compounds in exhaled breath by GC-MS has great potential for non-invasive detection of COVID-19. The method could be rapidly adapted for detection of other respiratory infectious diseases with a small sample size and data set for training of the machine learning and AI algorithms.
B-154 Development and Evaluation of an Automated High-Throughput Magnetic Bead-Based pre-treatment Platform for Lipid-Soluble Vitamins in Clinical Mass Spectrometry
Authors: Ma T , Liu H , Yao H , Yang Y , Dong L
Published: October 2024

Background

Mass spectrometry has become a critical tool in clinical diagnostics due to its unparalleled sensitivity, specificity, and ability for multiplex analysis, redefining standards in small molecule detection. However, its efficacy is limited by complex sample preparation requirements, such as purification and enrichment. Traditional pre-treatment methods often involve labor-intensive, error-prone procedures that can dilute samples, thereby necessitating enhanced sensitivity in mass spectrometry. Addressing these challenges, we developed an automated, high-throughput pre-treatment platform using magnetic bead technology to streamline the preparation of small molecules for analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study assesses the platform's effectiveness and reliability using lipid-soluble vitamins as a test case.

Methods

We developed a magnetic bead-based extraction process for lipid-soluble vitamins (Vitamins A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, E, and K1) from clinical serum samples, utilizing Zybio's EXM3000 for automation. Protein-bound vitamins were dissociated using methanol and isopropanol, followed by magnetic bead extraction. The extracted vitamins underwent LC-MS/MS analysis. Comparative analysis with traditional liquid-liquid extraction methods, using 20 clinical samples and commercially available kits, evaluated this novel approach's analytical efficacy.

Results

The method showed excellent linearity (r > 0.99) for the target compounds. The imprecision ranged from 1.5% to 9.3%, and the accuracy (recovery rate) was between 91.5% and 113.4%. The limit of quantification, repeated six times, had a deviation of -8.5% to 13% and a CV of 1.5%-6.4%, meeting industry standards and EP document requirements. Comparisons with commercial kits in 20 clinical samples showed deviations within ±15%, and the 95% confidence interval for expected bias met the testing requirements. The method also demonstrated high correlation with the commercial kits, as evidenced by regression equations for Vitamin A: y = 0.9732x - 1.7985, 25-hydroxyvitamin D2: y = 1.0163x - 0.0055, 25-hydroxyvitamin D3: y = 1.0597x - 0.7968, Vitamin E: y = 1.0569x - 0.2917, and Vitamin K1: y = 1.0055x - 0.0039.

Conclusions

This study showed excellent performance of a novel automated high-throughput magnetic bead sample preparation platform for small molecules in clinical mass spectrometry. This technology overcomes the complexities associated with sample pre-treatment in clinical mass spectrometry and holds broad clinical application prospects in mass spectrometric analysis.
B-161 Liquid chromatography/mass spectrometry analysis of ethyl glucuronide and ethyl sulfate using a perfluorophenyl stationary phase
Authors: Simms P
Published: October 2024

Background

Ethyl glucuronide (EtG) and ethyl sulfate (EtS) are minor metabolites of ethanol that are excreted in urine. These two metabolites are often tested for to determine if an individual has recently consumed alcohol. EtG and EtS are very polar analytes, which make them difficult to chromatograph using reversed-phase chromatography. These analytes often elute early, which can lead to irreproducible results. When EtG and EtS are analyzed by LC/MS with a mobile phase that contains formic acid, the signal of these two analytes can be suppressed. This leads to increased LOQ and cutoffs. We examined the perfluorophenyl (PFP) stationary phase for its utility in analyzing EtG and EtS. This stationary phase has been shown to have greater retention of polar compounds than C18 reversed-phase columns. In addition, we compared the effect of formic acid to acetic acid on the separation.

Methods

Urine samples containing EtG and EtS were diluted 10:1, using 50mM acetic acid or 0.1% formic acid, into a 96-well microtiter filter plate containing a 0.2micron filter. Internal standards were added to the microtiter plate containing the urine samples. The samples were filtered through the filter plate and analyzed by LC/MS using the PFP column (50 x 2.7micron) with a 50mM acetic acid or 0.1% formic acid/acetonitrile mobile phase. The analytes were detected using negative mode ionization. The total analysis time was 4.5minutes.

Results

EtG and EtS showed improved retention using the PFP column. Retention times for EtG and EtS were 0.75 and 1.20 minutes respectively using the PFP column, compared to 0.4 and 0.3 minutes using the C18 column. The greater retention time improved the LOQ for both analytes.The LOQ for EtS and EtG were determined to be 125 and 250 ng/mL, respectively. Greater retention of EtG was observed by switching the modifier from formic acid to acetic acid. The retention time increased from 0.45 to 0.75 minutes. There was less signal suppression using acetic acid in place of formic acid. Improvement in peak shape was observed when the injection volume was reduced to 5microliters. The method was validated over a range of 250 to 10000 ng/mL for EtG and 125 to 5000 ng/mL for EtS. Acceptable linearity was obtained for both analytes. Accuracy was performed at three concentration levels. All three levels demonstrated acceptable accuracy with the difference between the measured and expected concentration being with in 20% of each other.

Conclusions

The PFP stationary can be used to test EtG and EtS. This stationary phase gave greater retention of EtS and EtG. The use of acetic acid improved the limit of detection by decreasing the ion suppression, which decreased the limit of detection. The assay was accurate and precise for both analytes, demonstrated in the validation.
B-169 Combining the EXENT System and LC-MS for the Detection of Monoclonal Immunoglobulins at Low Levels
Authors: Barnidge D R , Troske D , North S , Wallis G , Perkins M , Harding S
Published: October 2024

Background

The EXENT® system is an automated platform designed to quantify monoclonal immunoglobulins in serum. EXENT® combines immunoprecipitation and MALDI-TOF mass spectrometry for the detection of monoclonal immunoglobulins at levels lower than traditional gel based methods. This study was designed to demonstrate if EXENT® prepared samples that were negative could be reflexed to a more sensitive method based on capillary flow Liquid Chromatography- Mass Spectrometry (LC-MS) without any modifications to the EXENT® prepared sample.

Methods

Patient samples containing a monoclonal immunoglobulin (ranging from 20 to 40 g/L) were diluted into pooled polyclonal serum and the samples were prepared and analyzed by EXENT®. Samples that were negative by EXENT® were reflexed by directly loading EXENT® eluates onto the LC-MS instrument. The abundance of the monoclonal immunoglobulin was defined by the signal-to-noise (S/N) of the light chain peak observed after deconvolution.

Results

Baseline samples were diluted down to two levels, 0.100 g/L and 0.001 g/L. LC-MS detected the same light chain from the monoclonal immunoglobulin as the EXENT® system in all reflexed samples that were negative by EXENT®.

Conclusions

LC-MS can detect monoclonal immunoglobulins that can no longer be detected using the EXENT® system by tracking the unique molecular mass of the monoclonal light chain. Reflexing samples to LC-MS did not require additional sample handling resulting in a faster time-to-result than current NGS, NGF, and clonotypic peptide approaches. Furthermore, monitoring the intact monoclonal light chain circumvents enzymatic cleavage to create a unique clonotypic peptide, alleviating a situation where no unique peptide can be generated as has been shown previously by Bergen et al. As a consequence, this reflex-methodology results in a consistent way of measuring the presence of malignant B-cells with high sensitivity.
B-175 Development of a Rapid and Sensitive LC-MS/MS Assay for Dolutegravir Quantitation in Breastmilk
Authors: Rackow A R , Pandey A , Marzinke M A
Published: October 2024

Background

Approximately 1.5 million HIV positive individuals experience pregnancy annually. The goal of HIV treatment in pregnancy is to achieve viral suppression prior to delivery, minimizing the risk of vertical transmission. Dolutegravir (DTG) is an integrase strand inhibitor that prevents insert of HIV DNA into host cells, thus decreasing viral load. In a clinical trial conducted in conjunction with 60 pregnant women initiating anti-retroviral therapy (ART) in the third trimester, dolutegravir (DTG) achieved viral suppression approximately two months faster than the standard of care at the time (28d vs. 84d). Secondary endpoints from this study demonstrate transplacental and breastmilk transfer of dolutegravir to the infant and delayed clearance. The pharmacokinetics of dolutegravir in infancy remain uncharacterized, with important implications for the development of HIV and resistance if there is intermittent medication compliance and/or barriers to care. The development of a robust DTG breastmilk assay will allow future pharmacokinetic studies to quantify pharmacologic exposure and relate this to infant health, namely HIV transmission and development of resistance. The objective of this study was to develop and validate a rapid LC-MS/MS assay in breastmilk with a dynamic measuring range, suitable for application in pharmacology studies.

Methods

Breastmilk was purchased from commercial vendors and spiked with dolutegravir and an isotopically labeled internal standard. Samples were prepared by protein precipitation and injected into an LC-MS/MS system consisting of a Shimadzu LC-40, a C8 column, and a Sciex QTRAP 5500. Peaks were identified by monitoring two transitions for DTG and the presence of the internal standard. All data analysis was performed in Analyst and Excel.

Results

The DTG breastmilk assay has an analytical measuring range of 0.5-1,000 ng/mL with a final method time of 2.2 minutes. Breastmilk was prepared by protein precipitation with a required sample volume of 30 µL and an injection volume of 3 µL. There was no evidence of ion enhancement or suppression, nor was there any evidence of carryover or crosstalk. DTG was reliably detected across the analytical measuring range and among different lots of breastmilk.

Conclusions

This assay meets performance criteria put forth by the European Medical Association and the Food and Drug Administration. Upon completion of all validation experiments, this assay may be applied to assess the pharmacokinetics of DTG in mother-infant pairs.
B-176 Rapid Diagnosis of 21-Hydroxylase Deficiency Using LC-MS/MS for Urinary Unconjugated Steroid Analysis
Authors: Nakagawa T , Homma K , Shimojo S , Yokota H , Wakui M , Matsushita H
Published: October 2024

Background

Urinary steroid profiling through gas chromatography-mass spectrometry (GC-MS) serves as a potent diagnostic tool for detecting abnormal steroid production linked to congenital enzyme deficiencies and steroid-related diseases. This technique involves deconjugating and extracting both conjugated and unconjugated steroids, followed by derivatization and analysis via GC-MS. It provides comprehensive insights into steroid metabolism, aiding in the identification of genetic disorders and conditions with atypical steroid levels. However, the procedure is intricate and time-consuming, particularly the deconjugation of steroids for analysis. The present study aimed to assess the usability of LC-MS/MS assays measuring unconjugated Δ4- and Δ5-steroids in urine for diagnosing steroid abnormalities.

Methods

The Biotage ISOLUTE SLE+ (Biotage, Sweden) was used for sample preparation, with dichloromethane for elution. LC-MS/MS analysis was performed on an LCMS8060NX (Shimadzu, Kyoto, Japan), using a Shim-Pack Velox Biphenyl column 2.1mm*150mm* 1.8μm (Shimadzu GLC, Tokyo, Japan). The mobile phase consisted of a 0.2mmol/L ammonium fluoride aqueous solution and a 0.2mmol/L ammonium fluoride methanol solution. We targeted 38 steroids, including four steroid medications, and used 19 stable isotope-labeled steroids for quantification. For the measurements, 30 neonatal urine samples were used (IRB Approval Number 20221210), which were suspected of 21-hydroxylase deficiency (21OHD) based on clinical symptoms or screening for 17α-hydroxyprogesterone (17OHP) using Dried Blood Spot. Of these, 18 had been confirmed as 21OHD cases by exceeding the GC-MS diagnostic criterion for Pregnanetriolone (>0.06ng/g Creatinine).

Results

Blood 21-deoxycortisol (21DOF) is a reliable marker for diagnosing 21OHD, which is generated from 17OHP through an alternative biochemical route. In consideration of that, we measured the urinary concentration of unconjugated 21-deoxycortisone (21DOE, precurcer of Pregnanetriolone), into which 21DOF is putatively converted in the kidney. In cases where 21OHD was excluded by GC-MS diagnosis, 21DOE was undetectable, falling below the sensitivity levels. On the other hand, 21DOE was detected in all samples confirmed 21OHD cases, consistent with the conversion of 21DOF to 21DOE in the kidney.

Conclusions

The present study suggested the usability of LC-MS/MS assays measuring urinary unconjugated 21DOE for diagnosing 21OHD. This approach provides a faster, less complex method for diagnosis, eliminating the need for the deconjugation process. To establish the value of urinary unconjugated steroids as diagnostic markers for steroid abnormalities, efforts to accumulate further findings are underway using clinical samples.
B-180 A Component Equation Approach to Resolving Calibration Curve Nonlinearity When Using Stable Isotope-Labeled Internal Standards for Quantifying Amino Acids With Mass Spectrometry
Authors: Kim Y , Miller A , Vicente F , Haymond S
Published: October 2024

Background

Stable isotope-labeled internal standards (SIL-IS) have been widely used with mass spectrometry to accurately quantify analytes. However, over an extended dynamic range, calibration curves calculated with the relative ratio of analytes to their corresponding SIL-IS are typically nonlinear. This makes accurate estimation of sample concentration difficult, especially at lower concentrations. Highly multiplexed clinical mass spectrometry assays routinely face this challenge. The component equation assumes interaction between an analyte and its SIL-IS causes the nonlinearity. Modeling this resulted in a linear calibration curve and high accuracy across the dynamic range. We compared the component equation with other calibration methods to quantify amino acids.

Methods

A total of 47 compounds were serially diluted and added with extraction solution containing a SIL-IS mixture to prepare 7 levels of calibrators. Calibrators were chromatographically separated using a Sciex ExionLC system with an Imtakt Intrada Amino Acid column and injected into a Sciex Citrine triple-quadrupole mass spectrometer. Seven different fitting methods were compared for calibration of each amino acid: non-weighted linear, quadratic and cubic models, linear models with 1/X and 1/X2 weighting, Padé[1,1] approximation, and component equation.

Results

Results for an example amino acid, tyrosine, are shown in the table below. Most models yield high accuracy at high concentrations, however, all models recorded significantly higher bias for the lowest calibrator than the component equation (≥55.7% vs 11.8%). The component equation outperformed all other models for mean bias (≥16.6% vs 5.9%). Similar results were obtained for the complete set of analytes.

Conclusions

When compared to other popular calibration methods to quantify a panel of amino acids, the component equation generated a linear curve and the greatest accuracy across all concentrations. The application of this simple equation could overcome the limitations of existing weighting and non-linear fitting approaches when using SIL-IS for quantifying analytes with mass spectrometry.
B-185 A Comparison Between Two Oral Sample Collection Devices on Preserving High-Quality Genomic DNA
Authors: Wood A , Lam V , Wang Y
Published: October 2024

Background

High-quality genomic testing significantly enhances the knowledge on gene-disease association, and greatly benefits future biomedical research and precision medicine. However, widely used saliva sample collection presents challenges with high-quality and quantity of human DNA and high bacterial DNA ratio. In this study, we validated the performance of two commercially available oral collection devices for their ability to preserve high-quality genomic DNA long-term at room temperature.

Methods

Fifty-one buccal samples in total from twelve donors were collected using two kinds of commercially available oral sample collection devices (iSWAB®-DNA (Device 1) and OCD-100 (Device 2)). All of the collected samples were stored at ambient temperature for up to 1 year. Collected samples were extracted with QIAamp Blood Mini Kit (Qiagen) at baseline, 30 days, 60 days, and 1 year. All DNA yields were measured using a Nanodrop One Microvolume UV-Vis Spectrophotometer; TapeStation System was used for determining the integrity of DNA. The bacterial DNA ratio was estimated by quantitative PCR with primers targeting the 16S rRNA gene known to be present in prokaryotes but not eukaryotes.

Results

Device 1 had higher yield (Device 1: 18.59 ± 8.98; Device 2: 6.44 ± 4.62), lower bacterial DNA ratio (Device 1: 2.06 ± 1.93; Device 2: 3.79 ± 4.58), and higher overall DNA integrity (Device 1: 6.1 ± 0.31; Device 2: 5.42 ± 0.96) compared to Device 2. Device 1 demonstrated longer post-collection sample stability at ambient temperature compared with Device 2 regarding DNA yield, bacterial DNA ratio and DNA integrity. No significant changes in DNA yield, integrity, and bacterial DNA ratio were observed after one year of storage in Device 1. No significant changes in DNA yield, integrity, and bacterial DNA ratio were observed after 60 days of storage in Device 2. However, Device 2 showed a significant increase in DNA yield, bacterial DNA ratio, and DNA integrity comparing baseline to 1 year.

Conclusions

Device 1 preserves higher quantity and quality of DNA from oral samples at ambient temperature for up to 1 year. These advantages significantly enhance efficiency and cost-effectiveness in downstream applications such as genotyping and sequencing, making it a valuable tool in genomics research with a commendable success rate.
B-191 Genomic Profile of Circulating Tumor DNA in Advanced Lung Cancer: Comparison with Tissue DNA
Authors: Park J , Choi Y , Jeon S , Kee S , Na E , Shin M
Published: October 2024

Background

The most common cancer in Korea is thyroid cancer, followed by lung cancer, and the most common cancer in men is lung cancer. The cancer with the highest mortality rate is lung cancer. Recently, the precision medical approach is becoming a major treatment strategy for lung cancer. For precision medical approach, the usefulness of next-generation sequencing (NGS) analysis targeting circulating tumor DNA (ctDNA) is being emphasized. Therefore, in this study, NGS analysis was performed on blood and tissues of advanced lung cancer patients to compare and analyze genetic mutation patterns to confirm the usefulness of ctDNA NGS assay.

Methods

A total of 101 patients with advanced lung cancer (stage 3 or 4) were recruited, and ctDNA NGS assays were performed on their plasma using a Pan100 panel (Dxome). Tissues from primary lung cancer, metastatic lymph nodes, or metastatic organs of 22 patients were analyzed using ONCOaccuPanel (NGeneBio). All variants were classified into four tiers based on the level of clinical significance, according to the standards and guidelines established by the Association for Molecular Pathology, American Society of Clinical Oncology, and College of American Pathologists.

Results

In the ctDNA NGS assay of 101 patients, 70 (69.3%) harbored 154 tier 1 or 2 variants. Mutations in TP53 (45, 29.2%) and EGFR (23, 14.9%) were the most frequently detected, followed by mutations in RB1, CHEK2, ATM, JAK2, ATR, DNMT3A, and KRAS. Among the 22 patients whose tissue was examined, tier 1 or 2 variants were detected in both the plasma and tissue of 14 patients and only in the tissue of six patients. All genes for these variants were targeted using both methods. Identical variants were detected in the plasma and tissue of six of the 14 patients, and discordant results were observed in the remaining eight patients. Twenty copy number variations were detected in 13 of the 101 patients, with copy number gain of EGFR being the most common (6, 30%). Fusion genes were detected in five of the 101 patients, two of which were not detected in the tissue. Overall, 74 (73.3%) of the 101 patients had positive ctDNA NGS assay results.

Conclusions

Although the NGS assay with ctDNA is less sensitive than that with tissue DNA, some genetic alterations are detected only in ctDNA NGS assays; therefore, it is useful to apply the ctDNA NGS assay as a complement.
B-195 Accelerating Molecular Diagnostics: Development of a novel Rapid, Integrated PCR System for Acute Respiratory Infections Detection
Authors: Ma T , Dong L , Chen H , Lv Z , Chen J , Yang Y
Published: October 2024

Background

The COVID-19 pandemic and acute respiratory infections have underscored the necessity for high-throughput and timely molecular diagnostics. Existing molecular diagnostic methods are labor-intensive, a challenge particularly evident in large-scale testing. This study aims to streamline the nucleic acid testing process by integrating extraction and detection in PCR testing, thereby optimizing efficiency.

Methods

We developed 'EXP-160R', an integrated PCR system capable of rapid thermal cycling. This innovation employs advanced temperature control technology to markedly reduce heating, cooling, and annealing-extension times. The PCR reagent-kit (Zybio Inc.) was optimized to complement the instrument's capabilities, aiming for pathogen nucleic acid detection in acute respiratory infections within 25 minutes. The precision of in-tube temperature during rapid amplification was validated by using temperature-sensitive molecular probes in system development stage. Performance evaluation used the WHO International Standard for SARS-CoV-2 RNA to assess sensitivity and detection limits. We also analyzed 500 clinical samples from multiple Chinese hospitals, including 295 COVID-19 positives and 205 negatives, comparing the EXP-160R system's performance with an NMPA-approved PCR kit.

Results

The innovative system significantly improved PCR cycle times, enabling detection of 16 samples in 22 minutes and a throughput of 2400 tests per day, without compromising sensitivity or specificity. Validation confirmed a detection limit of 200 copies/mL, and the system achieved a 100% detection rate for medium and high viral load samples, with a coefficient of variation (CV) for Ct values ≤ 5%. Clinical trial comparisons with clinical standard diagnostics revealed a sensitivity of 98.05%, specificity of 100%, accuracy of 99.2%, and a Kappa coefficient of 0.984. When compared to the established PCR kit, our system showed comparable efficacy, with a sensitivity of 98.50%, specificity of 98.67%, accuracy of 98.6%, and a Kappa of 0.971.

Conclusions

The rapid PCR system represents a significant advancement in molecular diagnostics, offering rapid, high-throughput, and accurate detection of SARS-CoV-2. This system is particularly beneficial in urgent testing scenarios, such as airports and fever clinics. It addresses current pandemic needs and sets a new benchmark for future respiratory and infectious disease diagnostics, enhancing both efficiency and user experience.
B-199 Rapid Molecular Detection of Meningitis Agents in Diverse Brazilian Regions (2019-2023): A Multicenter Study
Authors: de Araujo C M , Kappel H B , Barra G B , Jacomo R , Abdalla L F
Published: October 2024

Background

Meningitis, caused by viral and bacterial agents, is a significant public health issue globally, with a substantial economic impact. In Brazil, the incidence of meningitis varies by region, making the identification of the causative agent crucial for effective treatment. This study aims to evaluate the epidemiology of meningitis agents diagnosed using a rapid molecular detection panel in samples from various hospital units of a nationwide diagnostic and healthcare company.

Methods

Samples from several hospitals across different regions of Brazil were analyzed using the FilmArray® rapid molecular panel for meningitis and encephalitis, capable of identifying up to 14 agents, including bacteria, viruses, and fungi/yeasts. Gender, age group, regional distribution, and agent prevalence from January 2019 to April 2023, were retrieved from the database ensuring compliance with the Brazilian Data Privacy and Protection (LGPD) and local Research Ethics Committee guidelines.

Results

The study included 485 patients from 41 cities across 11 Brazilian states. The age range was 0 to 96 years, with an average age of 45 years, and a majority of female participants (59%). The highest number of test requests occurred in 2022 (17.5%), followed by 2021 (10%), with 2019, 2020, and 2023 accounting for less than 5% of requests each. The overall positivity rate was 10.5% (51/485), with 92.8% (13/14) of the possible agents identified. Viral agents were detected in 60.7% of positive cases, bacterial agents in 39.2%, and one mixed infection (human herpesvirus 6 and Escherichia coli K1) in a 2-year-old child. No fungal/yeast infections were detected. The most common agents were varicella-zoster virus (18.2%), Haemophilus influenzae (12.7%), herpes simplex viruses type 1 and 2 (12.7% each), and enterovirus (12.7%).

Conclusions

The study highlights the importance of rapid molecular diagnostic methods in Brazilian clinical-laboratory practice, offering fast, safe, and sensitive detection of meningitis-causing agents, which can contribute to improved patient outcomes.
B-200 Expediting Identification of Occult Sepsis with a Novel Diagnostic for Patients Presenting to the ED with Possible Infection
Authors: Jagneaux T , Grantham A , Richard K , Thomas C , D’Antonio C , Laperouse M , Scoggins R , O’Neal H
Published: October 2024

Background

In August 2023 Our Lady of the Lake Regional Medical Center implemented a process for sepsis care based on a novel sepsis diagnostic (IntelliSep) in the Emergency Department (ED). As a component of our Sepsis Learning Health Program, we continually evaluate this process.

Methods

A nurse-driven protocol allows for IntelliSep ordering with triage assessment. Dependent upon bed availability and assessment for clinical stability by the triage staff, patients may be referred to the waiting room after blood draw. An IntelliSep Band 3 result is a critical value, initiating a sepsis pathway. Patients in waiting room at the time of a Band 3 result are immediately placed in an ED bed. We evaluated this process for efficacy and efficiency through review of the medical record.

Results

Between 01-Sep-2023 and 07-Feb-2024, we performed a total of 2322 IntelliSep tests, with 247 (10.6%) resulting prior to bed assignment, consisting of 124 (50.2%) Band 1, 73 (29.6%) Band 2, and 50 (20.2%) Band 3. The median time to bed assignment (TTB) for these patients was 104 min (Q1-Q3 71-180). TTB for Band 3 (median 73 min, Q1-Q3 58-102) was significantly lower than both Band 1 (146 min, Q1-Q3 86-201 min) and Band 2 (98 min, Q1-Q3 70-174 min), p < 0.0001 and 0.01 respectively. Providers admitted 45 (90%) of Band 3 patients and 62 (50%) of Band 1 patients (p < 0.0001). Discharge diagnosis included infection in 47 (94%) and sepsis in 27 (54%) of Band 3 patients, and infection in 686 (54.8%) and sepsis in 2 (1.6%) of Band 1 patients (p < 0.0001 for infection and sepsis).

Conclusions

An IntelliSep-based process for sepsis diagnosis, implemented at triage, can expedite identification and treatment of patients presenting to the ED with occult sepsis who appear clinically stable by triage staff.
B-204 A Real-time Probe-based PCR Assay for Detection of Pneumocystis Pneumonia
Authors: Zhou Z , Li S , Jiang D , Wang Z , Wang Y , Zhang Y , Wang H , Su Y
Published: October 2024

Background

Pneumocystis jirovecii is an atypical opportunistic fungus with lung tropism and worldwide distribution that continues to be one of the major opportunistic pathogens causing severe Pneumocystis pneumonia (PcP) in individuals with acquired immune deficiency syndrome (AIDS) and patients with immunosuppression due to other causes. Widespread prophylaxis and treatment for P. jirovecii with sulfa drugs have decreased the incidence of PcP, but concerns have been raised about the possible emergence of P. jirovecii isolates resistant to these drugs. Point mutations in this gene have been associated with prior exposure to sulfa drugs in other microorganisms, the polymorphic positions 165 and 171, which lead to an amino acid change at positions 55 (Thr55Ala) and 57 (Pro57Ser) of the polypeptide chain, respectively. Fluorescent PCR technology can detect the nucleic acid fragments of P. jirovecii in patients' serum, alveolar perfusion fluid (BALF) and tissue samples, and further identify drug-resistant mutations, which is of great significance for early clinical and accurate initiation of antifungal therapy. MycoMDx Pneumocystis Jirovecii PCR Assay is the use of PCR-fluorescence probe method, combined with other methods to achieve the auxiliary diagnosis of P. jirovecii infection and resistance to sulfonamides (Fig. 1).

Methods

In this study, we did a performance evaluation of the MycoMDx Pneumocystis Jirovecii PCR Assay (CE-approved), including diagnostic sensitivity and specificity, the limit of detection (LoD), precision and stability. The above PCR assay reagents were developed and produced by Dynamiker Biotechnology (Tianjin) Co., Ltd.

Results

The sensitivity and specificity of the PCR assay reagents were above 90%, the LoD was 1000 copies/mL, and the precision and stability were in accordance with the requirements (CV≤5%).

Conclusions

The MycoMDx Pneumocystis Jirovecii PCR Assay has great clinical value by providing a rapid and reliable test to aid in early diagnosis.
B-205 Performance of a Novel Fluorogenic Assay for Detection of Carbapenemase-producing Enterbacteriaceae
Authors: Zhou Z , Li S , Jiang D , Wang Z , Wang Y , Zhang Y , Wang H , Su Y
Published: October 2024

Background

In recent years, under the selective challenge of antibiotics, the variety and number of drugresistant pathogenic microorganisms have increased significantly, which brings great challenges to clinical diagnosis and treatment, especially the infection caused by carbapenem resistant Enterobacteriaceae (CRE). Carbapenemases production is the main mechanism of drug resistance of Enterobacteriaceae to carbapenems. Drug resistance of Carbapenems can be caused by three mechanisms, resulting in the production of five major Carbapenemases. These are Klebsiella pneumoniae enzyme (KPC), New Delhi metal β-lactamase (NDM), carbapenem hydrolyzed oxalase (OXA-48 like), integrin-encoded metal β- lactamase (VIM) and IMP (Imipenemase). The real-time fluorescence quantitative PCR (qPCR) method based on molecular beacons was combined with the melting curve analysis to identify five drug resistance genes simultaneously by a single PCR reaction, with rapid detection, high sensitivity and strong specificity. Based on this principle, we developed the novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae (Dynamiker Biotechnology (Tianjin) Co., Ltd.).

Methods

We evaluated the performance of the novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae, including the limit of detection (LoD) and cross-reactivity, and compared it with the lateral flow immunochromatography assay (LFA).

Results

The LoD ranged from 75-450 CFU/mL for the five carbapenemase genes. The analytical specificity for target genes was 100%, as assessed with a panel of 15 pathogens, which indicated no cross-reactions. Comparison of qPCR and LFA results from twenty-three CRE clinical isolates with characterized carbapenemase content demonstrated a complete agreement (Table 1).

Conclusions

The novel fluorogenic assay for rapid detection of Carbapenemases in multidrug-resistant Enterobacteriaceae is an accurate and rapid method to identify KPC, NDM, VIM, IMP and OXA-48-like carbapenemases in the clinical microbiology laboratory, which can guide infection control programs to limit the spread of these organisms.