bluuebunny/crossref_metadata_2025 huggingface dataset:
B-060 Measurement of Infliximab Clearance using a Bayesian Forecasting Tool and Impact of Albumin and Immunization Status During Maintenance Treatment of Inflammatory Bowel Disease
Authors: McLachlan O , Schwalbe M , McFarland J , Pham D , Hughes P , Dervieux T
Published: September 2023

Background

Accelerated Clearance of monoclonal antibodies such as Infliximab (IFX) is a known predictive factor of pharmacokinetic origin that associates with poor outcome in patients with Inflammatory bowel diseases (IBD). We have implemented a Bayesian forecasting tool for IFX (PredictrPK® IFX) and calculated its Clearance. Our objective was to evaluate the impact of Albumin (ALB) and antibodies to IFX (ATI) on Clearance and exposure from patient specimens with IBD and submitted for testing in the routine clinical pharmacokinetic laboratory.

Methods

IFX concentration and ATI status were determined from serum using a homogenous mobility shift assay. Serum ALB concentration was determined using nephelometry. Clearance was estimated using inputted IFX concentration, ATI status, ALB, dose and interdose interval, and nonlinear mixed effect modeling with Bayesian priors. Patient specimens were collected anytime 20 days after the infusion and submitted for testing. Median Clearance (expressed as L/day) and estimated IFX trough concentrations by ATI status and lower Albumin levels (<4.0 g/dL) was calculated. Statistical analysis consisted of nonparametric Mann-Whitney tests.

Results

The prevalence of ATI was 9.1% in this cohort of patient specimens. Median Clearance was 0.246 L/day (IQR: 0.191–0.323 L/day) and median IFX Trough concentration was 11.9 µg/mL (IQR: 6.5–19.9 µg/mL). ATI status associated with lower IFX Trough concentrations and higher Clearance (P < 0.01). Lower ALB concentration regardless of ATI status also associated with lower IFX Trough concentrations (P < 0.01). There was twofold higher Clearance and twelvefold lower IFX Trough concentration in the presence of ATI and lower ALB. Results are presented in Table 1.

Conclusion

The data are consistent with the notion that IFX immunization and lower ALB impact Clearance and exposure to IFX.
B-067 Performance Assessment of a Newly Developed Lateral Flow Assay for the Quantification of Faecal Pancreatic Elastase
Authors: Afonso J , Schneider M , Guschin D , Gerhold C , Ricken B
Published: September 2023

Background

Pancreatic exocrine insufficiency (PEI) can be defined as an insufficient secretion of pancreatic enzymes and bicarbonate to maintain a normal digestion (1). PEI is associated to fat malabsorption, weight loss and malnutrition-related complications. Due to its important role in the digestive process, as a proteolytic enzyme and the high stability of the enzyme during intestinal transit, fecal pancreatic elastase (fPELA) is an ideal biomarker for the assessment of pancreatic function in patients suffering from PEI. The aim of this work was to develop and optimize a new rapid test for the quantification of fPELA.

Methods

A quantitative lateral flow assay was designed for the selective measurement of elastase antigen concentrations from stool extracts. The BÜHLMANN Quantum Blue® fPELA is a sandwich immunoassay. A monoclonal detection antibody is conjugated to gold nanoparticles. Elastase present in the sample binds to this gold conjugate. A polyclonal capture antibody, highly specific for elastase, is immobilized on the analytical membrane and captures the complex of elastase bound to the anti-elastase gold conjugate, resulting in a coloring of the test line. The remaining free anti-elastase gold conjugate binds to the control line. The signal intensities of the test line and the control line are measured quantitatively by the Quantum Blue® Reader. Pancreatic elastase is quantified in extracts of human stool samples collected with the BÜHLMANN CALEX® Cap device. Several analytical studies, such as sensitivity, linearity, High Dose Hook Effect (HDHE), were performed during optimization phase to assess the performance of the assay. Moreover, a method comparison with the commercially available high throughput turbidimetric BÜHLMANN fPELA® turbo assay was also performed.

Results

Both analytical and clinical performance were evaluated. Preliminary results of the assay performance were obtained during the optimization phase. Analytical sensitivity was determined as 12.8 µg/g LoB, 16.6 µg/g LoD and 17.2 µg/g LoQ. The linearity evaluation revealed a linear range of 10 to 472 µg/g. No HDHE was observed in native samples up to 4000 µg/g. Method comparison of the new lateral flow assay with BÜHLMANN fPELA® turbo assay, with 80 clinical samples, revealed good analytical agreement with a slope of 1.04 (Passing-Bablok) and a bias at cutoff (200 µg/g) of 5.6%. Relative bias (Bland-Altman) of the two assays was 6.9%. Considering the turbidimetric assay as the reference method, the sensitivity and specificity were 92% and 83%, respectively.

Conclusion

The newly developed quantitative lateral flow assay, BÜHLMANN Quantum Blue® fPELA, is an accurate, non-invasive and rapid test to determine fPELA levels in fecal extracts. The lateral flow assay is an attractive alternative to ELISA with a turn-around time of only 15 minutes enabling patients’ management in a more patient near clinical environment.
B-070 Performance Assessment of an Immunoassay for Detection of APOE4 Carriers
Authors: Shields A G , Shields M S , Jiménez-González I , Rodríguez-Martín A , Ito S , Verbel D , Snyder J
Published: September 2023

Background

The APOE gene corresponds to apolipoprotein E, a glycoprotein involved in lipid metabolism. There are three different versions of the APOE gene, e2, e3, and e4 where e3 is the most common allele. APOE4 has been associated with Alzheimer’s Disease (AD) and other neurological diseases. The presence of a single APOE4 allele increases the risk of AD by 3–5 fold and two copies, increase the risk by 8–12 fold. Detection of the APOE4 allele is commonly performed using genetic test methods. Studies were undertaken to evaluate the analytical performance of an immunoassay that detects the presence of APOE4 protein in and assess the discrimination of APOE4 gene carriers from non-APOE4 gene carriers.

Methods

The Biocross e4Risk® test is a latex agglutination immunoassay used for the quantitative determination of isoform E4 of the apolipoprotein E (APOE4) in human EDTA plasma samples. The test consists of one reagent with latex particles and another reagent with a mouse anti-ApoE4, which induces the latex particle agglutination when ApoE4 is present in the plasma sample. This agglutination is measured photometrically. The turbidity is proportional to APOE4 content of the sample, which can be used to infer the APOE4 genotype responsible.

The Biocross kit reagents were run on the Siemens Healthineers Atellica® Chemistry Analyzer (open channel). Stored clinical samples from Eisai’s elenbecestat Phase 3 MissionAD Studies were used in the analysis.

Results:

Conclusion

The Biocross e4Risk® test on the Atellica® Chemistry Analyzer (open channel) demonstrated good analytical performance, with sensitivity and precision comparable to manufacturer’s claims. The assay also had very good sensitivity and specificity when compared to known APOE4 genotype results. A simple blood-based robust APOE4 immunoassay test that runs on a routine clinical chemistry instrument may be a more convenient and cost-effective tool to help recruit patients for AD clinical trial programs.
B-078 Prevelance of Autoimmune Factors in Patients with Seizures of Unknown Etiology in Western China
Authors: Guo S , Meng Z , Wang M , Ying B
Published: September 2023

Objective

To delineate the features of autoantibody in patients with seizure of unknown etiology in Western China and to analyze other potential risk factors of an epileptic seizure.

Methods

From January 2017 to June 2022, 639 patients with unexplained seizure were assessed retrospectively. Neuronal autoantibodies were assessed in 471 individuals, including 12 common neural-specific autoantibodies, antibodies against four common pathogens associated with central nervous system infections, or neurophagocytosis, and two thyroid-related autoantibodies.

Results

Sixty-nine patients were positive for neural-specific autoantibodies in CSF and serum. Among the patients, 32 (46.38%) expressed NMDAR-Ab, 14 (20.29%) expressed GABABR-Ab, 10 (14.49%) expressed LGI1-Ab, 7 (10.14%) expressed GFAP-Ab, 3 (4.34%) expressed Yo-Ab, 2 (2.90%) expressed CASP2-Ab, and 1 (1.45%) expressed Hu-Ab. Compared to patients with non-autoimmune epileptic seizure, the positive rates of thyroid-related antibody expression and the neurophagocytosis pathogen levels of HSV-IgG and T. gondii-IgG were significantly different in patients with autoimmune epileptic seizure.

Conclusion

This study elucidated the antibody status of autoimmune epilepsy in western China, allowing for better identification and diagnosis of autoimmune epilepsy in the future.
B-083 Novel High Affinity Monoclonal Antibodies for Specific Detection of Neurofilament Light Protein in Serum and Cerebrospinal Fluid
Authors: Grönholm K , Vesilahti E , Eklin S , Hämäläinen T , Mattila M , Tuomiranta P
Published: September 2023

Background

Neurofilaments are very important protein components of axons, and they maintain the function of nervous system. Cerebrospinal fluid (CSF) and serum neurofilament levels elevate due to damages in nervous system making neurofilaments promising biomarkers for neural diseases and injuries, such as Alzheimer’s disease, traumatic brain injury and Multiple sclerosis. Based on their size, neurofilaments are divided in groups: Neurofilament light (NfL), neurofilament medium (NfM), neurofilament heavy (NfH), α-internexin, and peripherin. Of these, NfL is considered to be the most promising biomarker since it is the most abundant form of neurofilaments.

Methods

We have developed four mouse monoclonal antibodies against human NfL. These antibodies, designated as Anti-h NfL 12601, 12603, 12604 or 12605, were tested as pairs in sandwich fluorescence-based immunoassay (FIA) with NfL spiked serum and buffer to determine linear measuring ranges and the matrix effect. Furthermore, the ability of the antibodies to measure NfL in clinical samples was tested in sandwich FIA by testing 10 CSF samples in the range of 0 to 5 083 pg/mL. The antibodies were also compared against commercially available reference antibodies in FIA. Kinetic parameters were determined with bio-layer interferometry (BLI).

Results

In sandwich FIA, the widest linear measuring range of 10 to 40 960 pg/mL of recombinant human NfL spiked in normal serum was achieved with antibody pair 12603 and 12604. In contrast, the commercial reference antibody pair achieved a linear measuring range of 2560 to 40 960 pg/mL. Matrix effect caused by serum was negligible. With clinical samples, the best correlation with the reference method (NF-light CSF ELISA from Uman Diagnostics) with a coefficient of correlation of 0.986 was achieved with antibody pair 12604 and 12601. This pair also showed a good correlation with the commercial reference pair with a coefficient of correlation of 0.984. In kinetics measurements, none of four antibodies showed dissociation indicating very high affinity. The fastest association of 4.0 × 105 1/Ms was achieved with antibody 12605.

Conclusion

These results demonstrate that the newly developed mouse monoclonal NfL antibodies are promising tools for development of diagnostic immunoassays measuring NfL levels in human serum and CSF samples. Comparison against commercial reference antibodies indicates that higher NfL detection sensitivity and wider detection ranges can be achieved with antibody pairs developed in this study. Additionally, the kinetic measurement results forecast these antibodies to be suitable for fast reaction kinetic assay formats, such as lateral flow assays, which might become a key resolution in acute neural injury diagnostics.
B-094 Vaccination Anti-SARS-Cov2 Scheme Effect on Humoral Responses on Brazilian Cohort
Authors: Bacarov N B , Christofolini D , Sano C L , Dias R H , Bellucco F T , Soane M M , Bianco B , Barbosa C P
Published: September 2023

Background

Due to high spread of SARS-CoV-2, a global emergency vaccination program using diverse and new vaccines was implemented, providing protection mortality of COVID-19. In this way, populational investigation before and after vaccination is essential to understand and monitoring the effectiveness of these efforts.

Methods

Observational non-randomized study was performed using samples of 95 workers from healthcare institutions. They were vaccinated with two homologous doses of Sinovac, ChAdOx1 and Pfizer booster dose. Serum levels of anti-S1 IgA and IgG, anti-NCP IgG and neutralizing antibodies (Nabs) were measured immediately before (T0), two weeks after each dose (T1, T2), 21 days after T2 (T3) and two weeks after boost (T4). Tests were performed through commercial kits of EUROIMMUN.

Results

Results are summarized in Fig. 1. Anti-S1 IgG and IgA titles of CoronaVac samples showed a statistically significant increase after each vaccine dose (T1, T2 and T4). For ChAdOx1, statistically significant difference (SSD) was noticed between T1 and T4. Comparing anti- S1 IgG titles between both vaccines, SSD was observed between all collections, above 2500 BAU/mL on both.Anti-NCP titles increased after each CoronaVac dose, as expected since is an inactivated virus vaccine and SSD was observed from T1 to T4. NAbs at T3 and T4 showed SSD between collection times for each vaccine and between the vaccines. At T3 NAbs titles were higher for ChAdOx1 in comparison to Coronavac vaccinated. However, after the booster dose there was no difference of titles between vaccines and all participants were considered responsive.

Conclusion

The vaccine antibodies titles increased after each vaccine dose. Although there was difference of the diverse antibody titles between vaccines after 1st and 2nd doses, after booster dose all vaccinated were equally highly responsive. Such results reinforce the importance of the booster dose in the vaccination strategy against SARS-CoV-2.
B-118 Evaluation of Thirteen Commercially Available Clinical Chemistry Assays on the Alinity® c System
Authors: Li H , Li M , Wang J , Guo H
Published: September 2023

Background

Expansion of instrument analyte menus is often needed to accommodate country specific needs. To address specific needs in China, thirteen clinical chemistry assays have been modified for evaluation on the Alinity c system. These analytes are used in the investigation of several diseases including liver disease, hepatobiliary disease, cardiovascular and cerebrovascular disease, kidney disease, inflammation, infectious disease, alcoholism and glucose metabolism disease.

Methods

The following analytes were evaluated: a1-Microglobulin (a1-MG), Ischemia modified albumin (IMA), Glutamate Dehydrogenase (GLDH), Fibrin Degradation Products (FDP), Myeloperoxidase (MPO), Fibronectin (FN), Pyruvate (PYR), Monoamine Oxidase (MAO), Heart-type Fatty Acid-Binding Protein (H-FABP), Cholyglycine (CG), Serum Amyloid A Protein (SAA), Ethanol (EtOH) and Ferritin (Fer). Reagents from Beijing Strong Biotechnologies Inc. (BSBE) were evaluated on the ABBOTT Alinity c system. Precision, LoQ and linearity range were evaluated with guidance from CLSI documents EP15-A3, EP17-A2 and EP06-Ed2. Correlation to commercially available assays were performed using human serum/plasma/urine samples across the measuring range.

Results

The table below summarizes key performance of assays.

Conclusions

These initial results are very promising. All the assays tested exhibit good precision, linearity, anti-interference, and LOQ performances on Alinity c system. In addition, they also have excellent correlation with Jinya assays from BSBE on ARCHITECT system except SAA assay, and SAA has excellent correlation with Siemens BNII SAA assay.
B-142 Development and Clinical Validation of Artificial Intelligence Assisted Flow Cytometry Diagnosis for Acuteleukemia
Authors: Lu Z , Lyu Y E , Gao A , Yeager T S , Morita M , Wang S , Wang A , Fan G
Published: September 2023

Background

Flow cytometry is one of the most commonly used diagnostic tools in hematopathology. Manual flow cytometry data analysis is vulnerable to substantial inter-operator variation and typically takes a technologist 20 min to analyze a case. In the last decade, artificial intelligence (AI) technology has advanced dramatically and become widely used in image processing. Previously, we created an AI-assisted workflow for classifying white blood cell subsets in peripheral blood. The goal of this study is to evaluate the automated flow cytometry analysis system for diagnosing acute leukemia.

Methods

A total of 117 cases were retrieved from the archived diagnostic cases, consisting of three diagnostic categories: 20 normal cases, 37 acute lymphoblastic leukemia and 60 acute myeloid leukemia. Flow cytometry data manually analyzed by a pathologist is the gold standard. The same flow cytometry data was processed by the flow cytometry analysis software DeepFlow® using proprietary AI algorithm. The final diagnosis and blast cell count of AI and manual analysis are compared by Pearson correlation coefficient.

Results

The AI-assisted blast cell enumeration showed moderate correlation as compared to manual evaluation (r = 0.8, Fig. 1). Manual adjustment for enumeration of CD34 negative blast population with uncommon immunophenotype is required for 7 cases, including 4 CD34−/CD117+ acute myeloid leukemia and 3 CD34−/TDT+ lymphoblastic leukemia. The AI analysis effectively recognized cell populations and provided correct diagnoses on 94% of cases (110/117). With pathologists’ review, all cases were classified into the correct diagnostic categories.

Conclusion

Our study demonstrated that AI can accurately recognize the flow cytometry pattern of leukemia and make the proper diagnosis. Additional training for CD34 negative blast gating and other uncommon blast immunophenotypes is being conducted. Ultimately, AI will decrease the demand on manual analysis by technologists and pathologists, standardize the analysis process, and provide accurate diagnosis.
B-146 Discriminative Index: A Novel Indicator for Evaluating Machine Learning Algorithms in Laboratory Medicine
Authors: Yu J , Lin W , Lin W , Wang Y , Tseng Y , Wang H
Published: September 2023

Background

In clinical scenarios, incorrect predictions by machine learning (ML) models are inevitable. One way to reduce misleading predictions is to avoid reporting predictions that fall within a predefined “gray zone”. This method improves predictive performance by reporting only the less uncertain cases. However, the cost-effectiveness of applying the “gray zone” rule in an ML model is unclear without massive computation. Thus, this study aims to propose a novel metric to evaluate the effectiveness of using gray zones and validate the metric in real-world ML models.

Methods

This study defined a statistical metric called the “discriminative index” (D-index) for evaluating the effectiveness of gray zones. To calculate the D-index, the predictive outcomes of the ML model are first transformed into two probability distributions based on the truth labels (e.g., positive or negative). The D-index is then derived from the kurtosis of these two distributions. To validate the metric, we applied the D-index to three different antibiotic susceptibility-predicting ML models (namely, convolutional neural network (CNN), random forest (RF), and XGBoost (XGB)) based on mass spectrometry data. We assessed the performance and unpredicted case numbers of each model with different gray zones and correlated the results with the proposed D-index.

Results

The D-index values for the CNN, XGB, and RF models were 4.36, 0.38, and −1.66, respectively. When applying the “gray zone” rule to achieve 90% area under the receiver operating characteristic, the CNN, XGB and RF models retained up to 90%, 68%, and 62% of total cases, respectively. A higher D-index value indicates a more effective application of the gray zone rule.

Conclusion

The D-index is a simple and statistically insightful metric for evaluating the cost-effectiveness of applying gray zone rule in an ML model. This metric has been validated in three mass spectrometry-based predictive models and has shown promising results. The D-index can be a useful tool for comparing and applying different ML algorithms.
B-150 Impact of Clinical Catastrophic Events on Individual Reference Values for 20 Analytes: Results of a Six-Measurement Study.
Authors: de Almeida T F , Ferreira dos Santos Silva I F , Branchi Evans Salvia T , Shoji Hayama D , Carvalho da Silva A , Pitangueira Mangueira C L
Published: September 2023

Reference values are an essential aspect of clinical laboratory testing, providing a basis for comparison of results against established norms for specific patient populations. These reference values are used to evaluate diagnostic test results, monitor disease progression, and assess treatment effectiveness. The accuracy and reliability of these values are crucial to the diagnosis and treatment of disease and illness and also important in the monitoring of disease progression and treatment effectiveness. By tracking changes in test results over time, healthcare providers can assess the effectiveness of treatments and make adjustments as needed. This information is critical in the management of chronic conditions such as diabetes and hypertension. Despite their importance, reference values can be subject to variability due to a variety of factors. These include differences in laboratory testing procedures, variations in patient populations, and differences in the methods used to establish reference ranges. To address these issues, clinical laboratories can use individual references calculated based on previous test results. Nevertheless, individual reference values can be subjected to pathophysiological alterations that can modify the values of specific analytes. The aim of this study was to investigate the impact of a clinical catastrophic event, specifically troponin levels above 1000, on the calculation of individual reference values for 21 analytes. Six measurements were taken before and after the event from a sample of 5 to 450 patients. The results showed that the mean and covariance coefficient of the majority of the analytes remained unchanged despite the presence of the clinical event. Applying a Wilcoxon test to the mean before and after the event 10 out of the 21 analytes differ significantly before and after the event. These analytes included glucose, BNP, chloride, gamma GT, hematocrit, hemoglobin, potassium, C-reactive protein and urea. The mean and covariance of the remaining 11 analytes, which included platelets, creatinine, alkaline phosphatase, magnesium, sodium, AST, ALT, TSH, fibrinogen, prothrombin time, and activated partial thromboplastin time (aPTT), were not significantly impacted by the clinical event. These results suggest that despite the presence of a clinical catastrophic event, individual reference values can still be calculated for the majority of the analytes. This is important for the accurate interpretation of clinical laboratory test results, especially in the presence of acute illnesses or injuries. However, caution should be exercised when interpreting results for the three analytes that were significantly impacted by the event. Further studies are needed to determine if similar results can be obtained for other clinical catastrophic events and analytes.
B-151 Estimation of the Uric Acid Reference Interval by the Indirect Method Using an Unsupervised Machine Learning Tool and R Programming Language
Authors: Canali D G , Lopes A C , Vieira L M , Garoli G Y , Sabino C d , Dias C M , Zampieri G , Muller K G
Published: September 2023

Background

Determining the Reference Intervals (RI) of quantitative clinical laboratory tests is crucial for patient diagnosis, treatment, and monitoring. It is also a concern for laboratories seeking to meet accreditation requirements. Our study aimed to evaluate the manufacturer’s RI for uric acid (3.7–9.2 mg/dL) through indirect methods using a programming tool in R language in a Brazilian laboratory.

Methods

We performed a data mining study for uric acid using the laboratory’s LIS database from January to December 2021. Outpatients >18 years old, analyzed on the Siemens® platform with results within linearity were included. Exclusion criteria were total cholesterol >200 mg/dL, triglycerides >150 mg/dL, glycemia >126 mg/dL, ferritin >300 ng/mL and creatinine >1.5 mg/dL at any time in the patient history and CRP >10 mg/dL in the same month. We used a tool that integrates different algorithms including preliminary treating, grouping, and statistical reduction of test results. The Expectation-Maximization (EM) algorithm calculates the mean, standard deviation, and weight of the distribution for each cluster. Additionally, it integrates algorithms that determine the global mean/median, modes, antimodes, kurtosis, and skewness, to find values that best represent the reference population’s distribution (Figure). Manufacturer's and estimated RI for Male and Female partitions.

Results

The final database included 49.489 subjects, partitioned in 26.439 women and 23.050 men. Statistically estimated RI were 2.8–6.7 mg/dL for women and 3.8–8.6 mg/dL for men. Considering the RI proposed by the manufacturer, 23.8% of women were outside the RI provided by the manufacturer, all of them below the lower reference value.

Conclusions

Our results confirm the importance of verification/establishment of reference intervals by clinical laboratories and suggest that estimated RI using this software is appropriate for the assisted population, demonstrating consistency with physicians’ concerns. After specialist analysis, the manufacturer´s RI has been replaced by the calculated population-specific RI.
B-152 Estimation of Creatinine Reference Interval (RI) by the Indirect Method using an Unsupervised Machine Learning Tool and R Programming Language
Authors: Canali D G , Lopes A C , Vieira L M , Santos B d , Sabino C d , Dias C M , Zampieri G
Published: September 2023

Background

Creatinine is widely used in diagnostic medicine as a primary renal marker. Clinical laboratory measurement of serum creatinine is not standardized, and the RI varies slightly between different manufacturers. Roche brings as reference ranges 0.5–0.9 mg/DL for women and 0.7–1.2 mg/dL for men. After clinical manifestations of elevated female results without clinical correspondence, the laboratory proceeded with reevaluation of RI.

Methods

We retrospectively studied the creatinine results of the laboratory database of samples run from January to October 2022 in two different technical areas in Brazil (São Paulo and Rio de Janeiro). The laboratory platform was Roche® 502/702, Jaffe method to creatinine. The LabRI tool was used with parametric, non-parametric, and robust statistical treatments to exclude outliers and algorithms in the R Language and latent abnormal values of outpatient patients. The partitions proposed were gender (male and female), age (18–60 years old and over 60 years old). The proposed RI for verification were those proposed by Rosenfeld, LG et al in REV BRAS EPIDEMIOL 2019; 22 (SUPPL2):E190002.SUPL.2 - F: 0.5–0.9 and M: 0.7–1.2. Exclusion criteria were patients with urea >48.5 mg/dL, glycemia >200 mg/dL, 24 h proteinuria >150 mg/24 h, LDL > 130 mg/dL, uric acid > 130 mg/dL, anti-nucleus positive factor, as well as clinical data compatible with chronic renal failure or dialysis, hypertension and diabetes.

Results

After exclusion, retrieved 225 181 individuals 52% female. Between the different sites, the results of the partitions did not undergo significant changes. For women in both age partitions, the values did not vary significantly. For men, there was no significant variation in the lower limit of IR, and we had a variation of 0.20 (18–60: 0.69–1.31 and >60: 0.69–1.41) in the upper limit, mainly for the age partition >60 years old, below the 95% confidence interval of the proposed references.

Conclusions

The adoption of the Roche IR and the Brazilian article was approved for the female gender (0.5–0.9) with 118 100 individuals, and a new IR was defined for the male gender (0.7–1.2) with 107 081 adult individuals, including the elderly over 60 years old, for both genders.
B-156 Impact of Seven Incremental Scenarios of QC Strategies
Authors: Brooks Z C
Published: September 2023

Background

CLSI EP23A states that “At the least, the ability of the QC procedures to detect medically allowable error should be evaluated.” It also defines that “The QC strategy using QC samples should include the following for each measuring system:

Methods

Eighty QC sample mean and SD values were compared to Peer Means and TEa limits for 14 routine chemistry analytes and four cardiac markers tested in December 2022. The number of patient results was normalized to 200 and 60 per day for routine and cardiac; the number reported before the first QC flag after a simulated shift to a 5% error was calculated using CatalystQC software for seven incremental scenarios of quality control beginning with [A] Original QC rules (1-3s/2-2s), chart limits, frequency (once per day), analytical bias & SD; [B] Westgard Rules based on sigma values; [C] Westgard QC frequency; [D] Chart means & SDs set to measured values; [E] Improved bias and/or SD where advised by CatalystQC software; [F] Single CatalystQC™ rules; [G] QC bracketed at the end of each day.

Results

The number of patients at risk decreased with each component of the QC process. Observed and advised number of QC samples per year was inconsistent.

Conclusion

Selecting Westgard rules and frequency alone will not reduce patient risk significantly. Assigning QC chart values to recent mean and SD and improving analytical bias and SD have the most impact on reduction of patient risk. Catalyst QC single rules are more effective than Westgard rules. Bracketing QC with verified-effective QC will reduce patient risk to zero.
B-157 Metrics to Express Risk in Terms of A. Risk Sources, B. Potential Events, C. Their Consequences and D. Their Likelihood.- As Recommended by ISO 31000
Authors: Brooks Z
Published: September 2023

Risk Metrics to express risk in terms of A. risk sources, B. potential events, C. their consequences and D. their likelihood (as recommended by ISO 31000) compared to sigma metrics.

Background

ISO 31000 Risk Management Guidelines provides guidelines on managing risk faced by all organizations. “Risk is usually expressed in terms of A. risk sources, B. potential events, C. their consequences, and D. their likelihood.” I compared risk metrics to implement these guidelines in medical laboratories to sigma metrics to examine the most effective way to enable risk evaluation as “the process of comparing the estimated risk against given risk criteria to determine the acceptability of the risk” (CLSO EP 23A).

Method

Eighty QC sample mean and SD values were compared to Peer Means and CLIA TEa limits for 14 routine chemistry analytes and four cardiac markers tested on two Ortho Vitros 5600 analyzers in December 2022. Risk evaluation was divided into Current Risk, driven by analytical accuracy and precision, and Potential Risk driven by effectiveness of the daily QC system to detect a simulated shift to a 5% error rate.

Current risk consequences were quantified as the number of failures per million opportunities and per year, and the estimated healthcare cost of lab error based on the costing process established for calcium in 2004.i

Potential risk consequences were quantified as the number of failures reported and healthcare cost of lab error following a simulated shift to create a 5% error rate.

Results

Conclusion

There is a good relationship between sigma and projected failures per year. Sigma is NOT a good indicator of cost of error per year, or the number or cost of failures reported after a simulated failure.

Endnotesi National Institute of Standards and Technology. Planning Report 04.1. The impact of calibration error in medical decision making.
B-158 Application of Statistical Analysis as a Tool to Monitor the Variation of SARS-CoV-2 and Other Respiratory Viruses
Authors: Carvalho G C , Nurmberger J M , Perez B B , Ramadan D R , Tufik S
Published: September 2023

Background

After the emergence of SARS-CoV-2 and its worldwide spread, only a few studies have correlated its co-circulation with other respiratory viral pathogens (RVP). The current report aims to identify the pattern of SARS-CoV-2 positivity in individuals co-infected with influenza A (FluA), influenza B (FluB), or respiratory syncytial virus (RSV) in São Paulo, Brazil.

Methods

Study Population and Characteristics: This study was a retrospective analysis from a clinical laboratory database in São Paulo, Brazil from January 15 to March 10, 2022. Test results for respiratory viral co-infections were recorded for individuals with SARS-CoV-2, FluA, FluB, or RSV. Nasopharyngeal swab samples were evaluated for the molecular diagnosis of respiratory virus infections using a real-time multiplex-PCR panel to search for specific genes for the detection of SARS-CoV-2, FluA, FluB, and RSV. Only samples positive for one or more pathogens (including SARS-CoV-2) were included in the study. We further stratified the specimens by SARS-CoV-2 positivity, comparing those that also tested positive for an RVP in each group. Statistical Analysis: Kruskal-Wallis analysis of variance followed by Dunn’s multiple comparison tests was used. Results with P < 0.05 were considered statistically significant. Georeferencing analysis of SARS-CoV-2 mono-infection or co-infection with another respiratory virus was performed using an online available software application.

Results

A total of 28 932 samples were positive for at least one RVP, meeting the study criteria. Among those, a total of 93.13% were from individuals diagnosed with SARS-CoV-2 only, 2.99% positive for SARS-CoV-2 and FluA, and 0.99% positive for SARS-CoV-2 and RSV. There was no significant difference observed between genders nor individuals with mono-infection or co-infection. The analysis of age group showed a significant variation in positive cases between SARS-CoV-2 vs SARS-CoV-2 and FluA in the 0 to 10-year-old population (P = 0.017) and between SARS-CoV-2 vs SARS-CoV-2 and RSV in the 0 to 5-year-old population (P = 0.018). The highest number of positive samples for both SARS-CoV-2 and RSV was detected in the southern region of São Paulo (55.7%), compared to the north and east regions (44.3%).

Conclusion

Differences in diagnostic patterns of COVID-19 compared to other respiratory viral infections RVP co-infections are important for a better understanding of clinical outcomes, improved patient management, and better public health planning.
B-162 Direct Quantitation of Five Immunosuppressant Drugs in Dried Whole Blood Spots by a Fully Automated DSM-LC-MS System
Authors: Gibson R , Guo J , Samra S
Published: September 2023

Background

Liquid chromatography-mass spectrometry (LC-MS) is increasingly used in clinical research to quantify immunosuppressant drugs in whole blood as it can offer higher sensitivity and selectivity than other analytical techniques. LC-MS systems may be coupled with upstream dried matrix spot modules within one high-throughput, integrated and online workflow, allowing the extraction of matrices such as whole blood from pre-collected sample cards collected in a quick and minimally invasive manner.

In this poster, such a workflow will be utilized to demonstrate the quantitation of five commonly monitored therapeutic immunosuppressants; cyclosporin A, everolimus, mycophenolic acid, sirolimus and tacrolimus.

Methods

Calibration and QC samples were prepared in whole blood. 10 μL of each calibrant was then spotted in triplicate on DBS cards. Immunosuppressants and standards were extracted using a Thermo Scientifc™ Transcend™ DSX-1 Systems via flow-through desorption with a heated clamp. TurboFlow allowed interference removal in the extracted samples prior to analytical separation. An integrated software, Aria MX, controlled each step of sample desorption and separation. Analyte quantitation was performed by a TSQ Altis™ mass spectrometer, and the data was analyzed using TraceFinder™ 5.1 General Quan software.

Results

Five commonly therapeutically monitored immunosuppressant drugs were quantified from blood spotted onto DBS cards using a fully automated workflow, resulting in a highly sensitive 5-minute method. Calibration curves were fitted using a weighting factor of 1/x with requirements of R2 > 0.98, RSD/CV < 15%. The calibrants were used to determine the limits of detection (LODs) and quantitation (LOQs) shown in the below table.

Conclusion

A complete, integrated and fully automated workflow has been developed to rapidly and robustly quantify immunosuppressant drugs from dried blood spots. Quick and reproducible extraction of each target analyte and efficient chromatographic separation facilitated a 5-minute method for the quantitation of five of the most commonly therapeutically monitored immunosuppressant drugs.
B-171 Comparison of Intact PTH and PTH Assays using LC-MS/MS Among Non-Dialysis Dependent Chronic Kidney Disease Patients
Authors: Park H , Lee M , Lee K
Published: September 2023

Background

Parathyroid hormone (PTH) is an important regulator of calcium and phosphate homeostasis and bone remodeling. It is metabolized into PTH fragments, which are measured to a different extent by PTH assays of different generations because of differences in fragments recognized and lack of assay standardization. Therefore, we developed an LC-MS/MS method for 1-84 PTH and aimed to compare with immunoassay among patients with chronic kidney disease (CKD) stages 3 to 5 not yet on dialysis.

Methods

Blood samples were collected from 252 patients with CKD stages 3 to 5. PTH concentrations were measured simultaneously by using the second-generation—PTH intact and immunocapture in situ digestion LC-MS/MS spectrometry assays. The severity of the kidney disease was based on the eGFR calculated by using the Modification of Diet in Renal Disease (MDRD) formula.

Results

Serum 1-84 PTH concentrations were strongly correlated but significantly lower than the intact PTH concentrations (r = 0.956, P < 0.0001). This finding was consistent among CKD stages 3 to 5. PTH concentrations by both assays (intact and 1-84 PTH) positively correlated with BUN (r = 0.613, r = 0.61; P < 0.0001, respectively), phosphorus (r = 0.423, r = 0.427; P < 0.0001, respectively) and negatively correlated with blood calcium (r = −0.355, r = −0.421; P < 0.0001, respectively), protein (r = −0.289, r = −0.305; respectively, P < 0.0001) and the estimated glomerular filtration rate (r = −0.468, r = −0.485; P < 0.0001, respectively).

Conclusion

Among patients with CKD stages 3 to 5 not on dialysis, the 1-84 PTH assay detected significantly lower PTH concentrations compared with intact PTH assay. This LC-MS/MS method can provide accurate and precise PTH results in patients with severe chronic renal failure. Additional studies that correlate the diagnosis and management of CKD mineral and bone disorders are needed to determine whether 1-84 PTH assay results are better surrogate markers in these early stages of CKD.
B-174 Simple and Accurate Quantification of Four Direct Oral Anticoagulants in Plasma Using UPLC-MS/MS
Authors: Lin C , Huang Y , Cheng C , Yeh Y , Chang S
Published: September 2023

Background

Direct oral anticoagulants (DOACs) constitute first-line therapy used for many thromboembolic indications, such as prevention and treatment of venous thromboembolism and stroke prevention in atrial fibrillation. This class of medication consists of the direct thrombin inhibitor (dabigatran) or direct factor Xa inhibitors (apixaban, rivaroxaban, and edoxaban). DOACs quantification may be useful in the case of critical clinical situations, including drug accumulation in long-term treatment, overdosage, thrombotic or bleeding events. This study aimed to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the quantification of four DOACs: dabigatran, apixaban, rivaroxaban, and edoxaban in plasma.

Methods

The calibration curve samples were prepared by spiking drug free plasma with DOACs. The stable isotope labeled DOACs was used as an internal standard. After addition of the internal standard and protein precipitation, the supernatant was 10-fold diluted and injected into a chromatography system consisting of a UPLC BEH C18 (2.1 × 50 mm; particle size 1.7 µm) analytical column with gradient made of mobile phase (5 mM ammonium formate pH3.5 in water and methanol). The outlet of the column was connected to a triple quadrupole mass spectrometer with electrospray interface. Ions were detected in the positive multiple reaction monitoring mode. The concentration of analyte was calculated from the calibration curve and ion ratios between the analyte and the internal standard.

Results

The analytical range was linear with a correlation coefficient of over 0.99 in the range of 0–500 ng/mL for each analyte. Intra- and inter-assay imprecision were less than 3.1% (n = 20) and 6.0% (n = 20), respectively. The accuracy was evaluated by spike recovery and the mean recoveries were between 97.2% and 102.1% for all analytes. This assay showed no ion suppression or enhancement and no carryover. The chromatography run time was 4.5 min. The assay was applied to quantitate DOACs in plasma samples from 93 patients. Our data showed that the DOACs concentrations vary markedly individual patients after equal dose, this therapeutic drug monitoring of DOACs is essential to optimized drug efficacy.

Conclusion

An accurate, and cost-effective UPLC-MS/MS method for the quantification of DOACs was successfully applied for therapeutic drug monitoring in patients.
B-190 Development of an LC-MS/MS Method for Analysis of 7α-hydroxy-4-cholesten-3-one and Investigation Into its Utility as a Biomarker for Bile Acid Diarrhoea in the United Kingdom
Authors: Starbrook L , Kumar A , Ford C , Brookes M , Gama R
Published: September 2023

Background

Bile Acid Diarrhoea (BAD) is a common gastrointestinal condition estimated to affect 1% of the population. It is, however, significantly under-diagnosed. This is partly due to lack of diagnostic methods. In the United Kingdom (UK) the 75-SeHCAT retention scan is considered the gold-standard diagnostic method and is the only routinely available diagnostic test for BAD. SeHCAT retention of <15% is considered consistent with BAD. The SeHCAT test is, however, expensive, time consuming, and exposes the patient to a dose of radiation. 7-alpha-hydroxy-4-cholesten-3-one (7αC4) is a precursor in the bile acid synthesis pathway and has demonstrated utility as a marker of BAD. There is limited data comparing 7αC4, which is measured in the United States, to the SeHCAT test, as SeHCAT is not licensed in the United States. 7αC4 can be measured by LC-MS/MS, thus can provide a more convenient, low cost and high throughput option for diagnosis of BAD.

Aims

1) To develop and validate an LC-MS/MS method for analysis of 7αC4. 2) To analyse serum samples taken from patients referred for SeHCAT scans. 3) To determine the utility of 7αC4 compared to SeHCAT for diagnosis of BAD.

Methods

The method was adapted from Donato et al. 750 µL of ice-cold acetonitrile containing 50 nmol/L D7-7αC4 as internal standard was added to 250 µL serum. Samples were vortexed for 30 s, centrifuged at 3000g for 10 min, and supernatant transferred into a vial for LC-MS/MS analysis. The LC-MS/MS system utilised a Gemini 5um NX-C18 column coupled to a Sciex 6500 TripleQuad mass spectrometer. Ethical approval was obtained to collect serum on patients undergoing SeHCAT retention scans for investigation of BAD. 40 patients who attended for SeHCAT scans had serum collected and stored at −80 degrees Celsius until analysis for 7αC4. Sensitivity and specificity of 7αC4 was calculated and ROC curve analysis performed.

Results

The 7αC4 method demonstrated an LoQ of 1.5 nmol/L, linearity up to 1095 nmol/L, and intra-assay %CV of 3.0%–7.7%. Using SeHCAT retention of <15% as positive for BAD, ROC curve analysis demonstrated an AUC of 0.765. 7aC4 at a concentration of 175 nmol/L provided a sensitivity of 30% and a specificity of 92%. 7αC4 at a concentration of 50 nmol/L provided a sensitivity of 93% and a specificity of 46%.

Conclusion

The 7αC4 method demonstrated good analytical performance and was suitable for use in a routine laboratory. 7αC4 results of <50 nmol/L suggest BAD is unlikely, and results of >175 nmol/L make a diagnosis of BAD likely. Results in between these two values may be considered indeterminate and referral for SeHCAT scanning may be considered appropriate. The 7αC4 assay provides an opportunity to stratify patients for SeHCAT testing in the UK, improving diagnosis and management of BAD in patients with chronic diarrhoea and streamlining the patient pathway.
B-191 Development of a Simple, Robust, and Accurate LC-MS/MS Method for the Quantification of 17 Therapeutic Drugs: Initial Validation Results
Authors: Payto D A , Tang X , Giles R , Wei R , Colón-Franco J M
Published: September 2023

Background

Therapeutic Drug Monitoring (TDM) of Zonisamide (ZON), Felbamate (FELB), Lamotrigine (LMTR), 10-Hydroxycarbamazipine (OXCARB), Carbamazepine Epoxide (CARBEP), and Pentobarbital (PENTO) is essential in developing the regimen to produce the best clinical outcomes while monitoring for possible toxicity. Historically, both HPLC-UV and LC-MS/MS have been used for the quantification of these types of drugs. HPLC-UV is often subject to interferences and extended run times. Because of this, there has been a shift to LC-MS/MS methods which are often simple, fast, robust, and suffer from less interferences. Our objective was to develop a simple, robust, and accurate LC-MS/MS method to replace the HPLC-UV methodology.

Method

Serum (50 µL) and IS solution (100 µL; Zonisamide-13C6, Lamotrigine-13C,15N4, (±)-10,11-Dihydro-10-hychoxycarbamazepine-13C6, Carbamazepine-10,11-epoxide-13C6, Felbamate-D4, Pentobarbital-D5 in methanol with 0.5% acetic acid) were vortex mixed and centrifuged. Supernatant (50 µL) was mixed with 950 µL of water and vortexed mixed. Extract was injected on a Thermo Accucore C18 column. A quantifier and qualifier transition was monitored for all analytes. Multiple Reaction Monitoring (MRM) was performed in positive mode for all analytes except for PENTO (negative). Total chromatographic time for the 17 analytes is 7.31 min.

Results

No ion suppression, differential matrix effect, or interferences were observed. Analytical Measurement Range (AMR) data for the initial 6 analytes is presented in Table 1. The coefficient of variation (%CV) was <2.8% for ZON, <4.3% for FELB, <7.3% for LMTR, <3.8% for OXCARB, <3.0% for CARBEP, and <3.5% for PENTO. Method comparison with a previously validated method were acceptable (Table 1).

Conclusion

This simple, robust, and accurate LC-MS/MS method has been validated for the initial 6 anlytes and has been determined to be acceptable for clinical use. Validation of the remaining 11 analytes is ongoing (Pregabalin, Gabapentin, Levetiracetam, Ethosuximide, Lacosamide, Rufinamide, Primidone, Lamotrigine, Topiramate, Brivaracetam, and Perampanel).