Clinical Laboratory Research Articles

A-150 Extended Pre-Analytical Stability Validation for Coagulation Tests in Brazilian Clinical Laboratory

Abstract Background The transport of samples for coagulation tests is a challenge for clinical laboratories, due to their limited stability and short time for processing according to the manufacturer's recommendations. The objective of this study was to evaluate the possibility of extending the stability of samples collected for Prothrombin Time and Activity (PTA), activated Partial Thromboplastin Time (aPTT) and Fibrinogen tests for up to 10 hours at room temperature, in comparison with the stability described in the literature and by the manufacturer of the reagents (4 hours for aPTT, 8 hours for PTA and Fibrinogen). We verified the acceptability of the samples with a longer period between collection and analysis, including its impact on the clinical management Methods Four tubes of 3.2% sodium citrate from 40 volunteers were analyzed at times of up to 4 h, 6 h, 8 h and 10 h. The results obtained were compared with the dosage performed within 4 hours (recommended by manufacturer) and the Total Error obtained in the analysis of the samples was evaluated. The tubes remained at controlled room temperature of 15 to 25°C and were centrifuged at 1500 g for 15 minutes. Then, the analyses were performed by coagulometric method. Results The results showed safety in the analysis of samples for coagulation tests even within 10 hours after collection at ambient temperature and were within the Maximum Allowable Total Error (Carmen Ricós): 95% of the PTA results (TE 5,3%); 92.2% of aPTT results (TE 4,5%) and 98% of Fibrinogen analyses (TE 13,6%) (Figures 1, 2 and 3). In addition, even the results that exceeded the specified Total Error showed no impact on the clinical decision. Conclusions The study demonstrated that there was no significant loss of stability for the analyses of PTA, aPTT and Fibrinogen performed on samples kept at room temperature within 10 hours after collection. Most of the results showed variation within the specified Total Error and all results showed acceptability from a medical point of view

B-241 Next-generation monitoring: M-inSightclinical application in multiple myeloma

Abstract Background The improvement of multiple myeloma (MM) therapy effectiveness has increased the need for precise, sensitive, and dynamic monitoring of minimal residual disease (MRD). Current MRD assessment methods that are based on bone marrow aspirates, pose limitations due to the invasiveness of the procedure and potential heterogeneous sampling. Recognizing the need for non-invasive, frequent testing, a targeted mass spectrometry, M-InSight®, (MS)-based MRD blood-test was developed. This innovative assay identifies and tracks clonotypic peptides from the patient's monoclonal immunoglobulin, offering a blood-based alternative with equivalent sensitivity to existing bone marrow reliant techniques.Here we employed M-InSight® to sequence and select clonotypic peptides, enabling highly specific, ultra-sensitive quantification of the M-protein. This study examines the correlation between M-protein quantification using M-InSight® and conventional serum protein electrophoresis (SPEP) values routinely obtained in clinical settings. Given the established role of M-protein as a biomarker for MM diagnosis and longitudinal monitoring, ensuring concordance between emerging methodologies and existing standards is vital. Methods Serum samples were obtained from the clinical laboratory at the University of Miami and characterized using SPEP (capillary electrophoresis, Sebia), immunotyping (Sebia), and free light chain quantitation (The binding site). Patients with M-protein concentrations ≥0.2 g/dL by SPEP were included. Longitudinal samples were collected. Subsequently, all samples were analyzed using M-InSight®. The initial sample for each patient underwent de novo sequencing of the M-protein by mass spectrometry and was also used to calibrate the concentration with the known value from SPEP. An in-house bioinformatics algorithm aligned, sequenced, and selected patient-specific clonotypic peptides. These peptides were targeted for the M-inSight® follow up assay allowing the quantification of M-protein. Results A variety of M-protein isotypes were successfully sequenced (IgGK, IgGL, IgAK, IgAL, IgMK, IgML, free K, free L, IgDL, biclonal) at concentrations ranging from 0.2 to 2.9 g/dL. M-protein was successfully sequenced and clonotypic peptides were identified in 57 out of 59 patients using the initial sample. A total of 109 serum samples collected during maintenance or post treatment were analyzed, 50 of them were follow-up samples and were used to correlate M-protein values from both techniques. M-protein quantification by M-Insight showed high concordance with the one obtained by SPEP, with a correlation value of 0.88 (intercept set to 0, slope 0.91. Pearson = 0.886, p-value <0.001). Out of 57 patients whose M-protein was sequenced, 55 were positive by SPEP and were successfully quantified by M-InSight®. Furthermore, M-InSight® detected and quantified M-protein on samples of the 2 remaining patients that showed M-protein values below SPEP detection limits, highlighting the higher sensitivity of M-InSight®. Conclusions M-inSight® assay successfully identified clonotypic peptides in 57 out of 59 patients which were used to quantify M-protein concentration and showed a strong concordance with SPEP and affirming the utility of M-InSight® as a reliable method for routine monitoring of MM. The ability to seamlessly integrate M-InSight® into routine monitoring protocols offers clinicians a valuable tool for MRD monitoring, tracking disease progression and response to therapy with enhanced sensitivity and specificity.

B-021 Impact of Assay Conditions on FT4 Measurement Accuracy of Equilibrium Dialysis LC/MS/MS Clinical Assay

Abstract Background Reliable FT4 measurements are vital for accurate diagnosis and effective management of thyroid disorders. However, currently used FT4 immunoassays (IAs) lack standardization, leading to significant variations between platforms. Equilibrium dialysis (ED) coupled with LC-MS/MS analysis, considered the gold standard for FT4 measurement, has been adopted as a reference measurement procedure (RMP) for IA standardization. This study aimed to establish a high-throughput version of the routine clinical FT4 assay based on ED-LC/MS/MS technology and assess the impact of altered assay conditions on measurement accuracy. Methods A commercially available micro-ED plate was employed for ED-LC/MS/MS analysis. FT4 extraction from the dialysate was performed by utilizing a 96-well C18 SPE plate, followed by FT4 quantitation via LC-MS/MS in positive ion mode. Certified primary reference material was used to calibrate the assay. To evaluate assay robustness, the effects of minor method modifications were studied, including incubation time of ED, serum/buffer ratios in ED inserts, ED incubation temperature, shaker speed during ED, serum sample dilution ratio, and dialysate sample dilution ratio. Additionally, short-term and long-term stability of serum FT4 were assessed. Results The developed ED-LC/MS/MS assay successfully resolved T4, T3, reverse triiodothyronine (rT3), and other interferences within 4 minutes using C18 chromatography. The assay's linear range (0.5-100 pg/mL) encompassed clinically relevant FT4 concentrations across hypo- and hyperthyroid patient samples. Excellent agreement was observed between the routine assay based on ED-LC/MS/MS and CDC RMP with a Deming regression slope near 1 (95%CI: 0.87-1.06). Furthermore, serum/buffer ratios from 1:1 to 1:2.5 in ED inserts, shaker speeds from 75 to 150 rpm, and ED incubation times from 16 to 20 hours did not significantly affect FT4 measurement. However, elevated ED temperature (38°C) led to a more than 10% increase in FT4 values. Serum FT4 remained stable when exposed to room temperature for 12 hours, five freeze-thaw cycles, and -70°C storage for 1.5 years. Additionally, dilution of sera by up to 3-fold before ED did not alter FT4 measurement results. Diluting dialysate samples at least 3 times prior to SPE extraction proved accurate for samples exceeding the calibration curve range. Conclusions In conclusion, we successfully developed a routine clinical FT4 assay based on ED-LC/MS/MS that generates data comparable to the RMP. The assay exhibits robustness, with several altered conditions not significantly impacting measurement accuracy. This high-throughput and accurate assay demonstrates suitability for both clinical laboratories and large-scale epidemiological studies. Disclaimer: The findings and conclusions in this report are those of the author(s) and do not necessarily represent the official position of the Centers for Disease Control and Prevention/the Agency for Toxic Substances and Disease Registry. Use of trade names is for identification only and does not imply endorsement by the Centers for Disease Control and Prevention, the Public Health Service, and the US Department of Health and Human Services.

A-095 A New CO2 Assay with Concentrated Reagent Having Superior Onboard Stability on Mindray BS-2800M Analyzer

Abstract Background Measurement of total content of CO2 in serum or plasma can be used to assist in the diagnosis of various acid-base disorders. There are small amounts (about 0.04%) CO2 in the earth’s atmosphere, or more in an enclosed clinical laboratory space. The environmental CO2 could be dissolved into the CO2 reagent continuously, in particular at lowered temperature when it is opened and stored on the system, which leads an impact on CO2 assay performance. Presently, competitors mainly use additional inserts or with small opening of the reagent container to reduce the contact with air from reagent, to improve its onboard stability. Here, we designed a new CO2 assay by optimizing the reagent formulation with a new protection mechanism to reduce the amount of CO2 absorption to the reagent and improved its onboard stability. This study evaluated performances of the new CO2 assay on Mindray BS-2800M analyzer. Methods Mindray CO2 assay with concentrated reagent is based on PEPC (Phosphoenolpyruvate Carboxylase) enzymatic method. It is designed with an innovative mechanism of reagent formulations with a protective layer of chemical(s) on top of the reagent that prevents CO2 from entering the reagent and reduced its impact to its on-board stability. The onboard stability was conducted with a constant reagent consumption mode to simulate as it in clinical lab use. The onboard study used two levels of Quality Control materials and serum pools. According to CLSI protocols, the following assay performances were evaluated: onboard stability, precision, method comparison, linearity, and interference. Results On Mindray BS-2800M analyzer, Mindray CO2 assay demonstrated an excellent onboard stability. With uncapped reagent on the system, the stability is for 21 days without recalibration, with less than ± 5% deviation from day zero. In parallel onboard stability studies, with Roche CO2 reagent on Cobas c701 system, the relative deviation from day zero was found to be within ±10%; and other three manufacturers` results with deviation exceeded ±10% vs. day zero in 7 days. The linearity of Mindray CO2 assay was determined as from 2.0 to 50.0 mmol/L. Our CO2 assay correlated well with Roche CO2 on Cobas c701 (Mindray = 1.0032 * Roche - 0.0186, r≥0.99). The repeatability and with-lab CVs were ranged from 0.64% to 3.41%, respectively. There was no significant interference (<±10%), at CO2 concentration of 15 mmol/L with bilirubin up to 60 mg/dL,hemolysate up to 800 mg/dL, Intralipid up to 1000 mg/dL, triglycerides up to 1000 mg/dL. Conclusions On Mindray BS-2800M analyzer, the new CO2 assay demonstrated a good ability against environmental CO2 inference in 21 days of onboard time. The new CO2 assay also shown good performances in precision, correlation (accuracy) and anti-interference from endogenous substances. Therefore, we conclude that the new Mindray CO2 assay is suitable for routine clinical laboratory use.

B-057 Evaluating the Use of Unconcentrated Urine for Protein Electrophoresis and Immunofixation: An Alternative Approach for Efficient Analysis

Abstract Background Protein electrophoresis is a widely employed technique for separating and analyzing proteins based on their charge and size. Traditionally, concentrated urine samples have been utilized to enhance protein detection sensitivity. However, this study explores the feasibility of using unconcentrated urine as an alternative sample type for protein electrophoresis (UEL) and immunofixation (UIF) to streamline the analysis process and reduce costs and turnaround time in clinical laboratories. It offers a more efficient and resource-friendly alternative. Methods Both UEL and UIF were performed on routinely ordered 80 urine specimens (random or 24 Hr.) for patient management and performed by both concentrated (X40) by Helena and unconcentrated using Sebia (HYDRAGEL 9IF gels using HYDRAGEL BJ dynamic mask procedure. The electrophoretic patterns obtained from unconcentrated urine were compared with those from concentrated urine. Various electrophoretic parameters, including resolution, sensitivity, and reproducibility, were evaluated to determine the reliability of unconcentrated urine in protein electrophoresis. In addition, retrospective analysis was performed before and after implementation on all urine specimens ordered to analyze if there were any differences in the monoclonal band distributions in the population. Results All the protein concentrations were correlated well with 95-100% agreement between the methods (Figure 1) clear separation of protein fractions. Serial dilution studies of monoclonal band analyses showed that these bands can be detected at 1.25 mg/dL using an unconcentrated method. Retrospective analysis of patient data before and after the implementation of the new method shows no differences in the identification of monoclonal proteins (Helena [69 of 872 total patients tested (8.0%)] vs. Sebia [90/800 (11.0%)]. Conclusions These results indicate that unconcentrated urine can be successfully utilized for UEL and UIF with comparable outcomes to traditional concentrated samples. Utilizing unconcentrated urine simplifies the sample preparation process and presents a promising alternative.

B-182 Development and validation of an LC-MS/MS assay for simultaneous measurement of serum androstenedione and 17-hydroxyprogesterone in serum

Abstract Background Androstenedione and 17-hydroxyprogesterone are measured via immunoassays or high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) in clinical laboratories. Immunoassays have limited sensitivity and specificity for certain steroid hormones testing. We aimed to develop and validate a sensitive, accurate, and specific in-house LC-MS/MS assay to simultaneously measure both 17-hydroxyprogesterone and androstenedione in serum. Methods 13C-labeled androstenedione and 17-hydroxyprogesterone internal standards were added to calibrator, control, and patient samples. A hexane/ethyl acetate solution was used in a liquid-liquid extraction procedure. The organic layer was then removed and dried followed by reconstituting the extracts in 2.5 mM ammonium formate in 50% methanol. The HPLC mobile phase gradient began with 47%, 2.5 mM ammonium formate in water and 53%, 2.5 mM ammonium formate in methanol. This progressed to 56% of the methanol phase over three minutes. The mixtures were separated though an Agilent 1260 Infinity HPLC system with a Poroshell 120 EC-C18 analytical column (2.1 × 50 mm × 2.7 µm). The separated HPLC eluents then entered an Agilent 6460C QQQ mass spectrometer in positive ion mode through an electrospray ionization source where protonated androstenedione (m/z = 287.2) with the protonated androstenedione-[2, 3, 4-13C3] internal standard (m/z = 290.2) and protonated 17-hydroxyprogesterone (m/z = 331.2) with the protonated 17-hydroxyprogesterone-[2, 3, 4-13C3] (m/z = 334.2) were mass-selected for collision-induced dissociation. For androstenedione, respective quantifier and qualifier m/z values of 287.2 > 97.1 and 287.2 > 109.1 were used while respective quantifier and qualifier m/z values of 331.2 > 97.1 and 331.2 > 109.1 were used for 17-hydroxyprogesterone. Validation studies were performed including imprecision, lower limit of quantitation (LLOQ), analytical measurement range (AMR), interference, specificity, carryover, extraction recovery, matrix effect, and comparison with a reference laboratory LC-MS/MS method. The total allowable error limits used in this study were 23.5% for androstenedione and 30.2% for 17-hydroxyprogesterone. Results Total imprecision results across three concentrations yielded a maximum CV of 4.6 % for androstenedione and 6.5% for 17-hydroxyprogesteron. The AMRs of androstenedione and 17-hydroxyprogesterone were 0.2 to 13.4 and 0.1 to 22.0 ng/mL without significant carryovers. No significant interferences were observed from 11-deoxycorticosterone (700 ng/dL), testosterone (2500 ng/dL), DHEA (1000 ng/mL), bilirubin (30.1 mg/dL), hemoglobin (1032 mg/dL), or triglycerides (1858 mg/dL). The extraction recoveries of androstenedione and 17-hydroxyprogesterone were 98% and 81%, respectively. No significant matrix effects were identified. In comparison to the reference lab LC-MS/MS method, the in-house LC-MS/MS assay demonstrated a 0.523% bias for androstenedione and a -3.77% bias for 17-hydroxyprogesterone with both exhibiting excellent correlations with the reference lab measurements. More specifically, our results (as a function of values obtained from the reference lab) followed the relationship of y = 1.039x - 0.092 (R = 0.9951) for androstenedione and y = 0.957x + 0.01 (R = 0.9974) for 17-hydroxyprogesterone. Conclusions An LC-MS/MS assay is developed for simultaneous detection of serum androstenedione and 17-hydroxyprogesterone. The assay demonstrates acceptable imprecision, AMR, and accuracy. No significant carryover and interference from other structurally similar steroid hormones and other common interferents are identified.

B-165 The EXENT System reliably distinguishes daratumumab from endogenous monoclonal immunoglobulins in multiple myeloma patients

Abstract Background The use of therapeutic monoclonal antibodies (t-mAbs) in the treatment of multiple myeloma (MM) has created challenges in the clinical laboratory. Co-migration of t-mAbs (predominantly IgGK) and the endogenous M-protein during serum protein electrophoresis can result in inability to distinguish between a patient’s M protein and the t-mAb. This can hinder accurate assessment of therapeutic responses or may lead to extensive workup with immunofixation electrophoresis and gel-shift assays for accurate confirmation of a complete response, or to exclude the emergence of a new clone. There is a need for an analytically specific routine method to identify t-mAb interference. MALDI-TOF mass spectrometry is an especially promising tool, as due to its ability to identify M proteins’ molecular mass, it has the potential to recognize any t-mAbs with known amino acid composition. We have used the EXENT® Immunoglobulin Isotypes (GAM) Assay for the EXENT Analyzer (The Binding Site, part of Thermo Fisher Scientific) to assess the reproducibility of daratumumab (anti-CD38, IgGK) detection in an analytical study, and assessed the EXENT System’s ability to detect daratumumab in a cohort of MM patients. Methods The expected mass to charge ratio (m/z) of the +2-charge state of daratumumab’s kappa light chain was calculated based on published information. Daratumumab was spiked into normal human serum at 1.0 g/L, and this material was tested in 5 replicates a day over 5 days. In addition, 109 samples from 34 MM patients receiving daratumumab, carfilzomib, lenalidomide and dexamethasone (Dara-KRd) treatment were also tested. Samples were obtained after 14 or 28 days following daratumumab administration in various treatment cycles, ranging from cycle 4 to end-of-treatment. Results The expected m/z for the +2-charge state of daratumumab’s kappa light chain was calculated as 11,691.01 to 11,693.1. In the reproducibility experiment, the median m/z was 11691.3, within the expected range. The difference between the lowest and highest m/z was 1.3 across the 25 replicates. Daratumumab was detected in 103 out of 109 samples (97.3%): 51/57 (89.5%) in IgGK samples, and in all IgGL (23) and IgA (29) samples. In two IgGK patients, where daratumumab was not detected in 2 out of 4 samples and in 1 out of 3 samples, respectively, the patients’ own M proteins were in the proximity (24.8 and 27.1 m/z) of daratumumab’s m/z. In the other three cases where daratumumab was not modeled by the algorithm, the daratumumab peak overlapped with the endogenous M-protein's adduct peak (estimated m/z 11,688) in one sample; it was masked by the shoulder of a large (8.42 g/L) IgGK M-protein in another sample; and no obvious reason was identified in the third sample. There was no difference in the detection rate between 14 days and 28 days post administration. Conclusions The EXENT Immunoglobulin Isotypes (GAM) Assay has successfully identified the presence of daratumumab in approximately 90% of IgGK patient samples in addition to all other (non-IgGK) MM samples. These results demonstrate the potential of the EXENT Immunoglobulin Isotypes (GAM) Assay for recognizing known t-mAb interference.

A-189 Six Sigma for Thyroid Stimulating Hormone (TSH)

Abstract Background Effective quality control processes within clinical laboratories are crucial for monitoring and assessing the analytical performance in terms of precision and accuracy. The regular measurement of these parameters plays a pivotal role in ensuring patient safety and is often a prerequisite for accreditation by various organizations. Six Sigma metrics have emerged as one of the most efficient tools for evaluating the analytical performance of clinical laboratory systems. This approach integrates Total Allowable Error (TEa), encompassing both random and systematic errors into a single comprehensive index. This study details the establishment of Six Sigma in our laboratory for Thyroid Stimulating Hormone (TSH) for eight different modules of Siemens Atellica IM. Methods A one-year dataset was compiled from the assessment of internal quality control for evaluating the imprecision (expressed as coefficient of variation - CV%) and Bio Rad’s Unity Interlaboratory Program for inaccuracy (quantified as bias%) in TSH test processed across eight modules of Atellica IM (Siemens). The data was categorized into two groups based on QC levels. The TEa utilized in this study was set at 20% according to CLIA standards. Six Sigma metrics were subsequently calculated for each QC level and equipment based on TEa, CV%, and bias%, utilizing the formula Sigma=(TEa - bias%)/CV%. Results Six Sigma values calculated for the QC level 1 ranged from 4.92 to 6.20 across all equipment, while for the QC level 2 Six Sigma varied from 4.87 to 7.59. All values found were classified as Good, Excellent and World Class according to the normalized method decision chart. These results showed that analytical performance of all eight modules of Atellica IM were similar in terms of random and systematic error. Conclusions Six Sigma serves as potent tool for monitoring the analytical performance of the TSH test. It is crucial to independently track different levels of QC in TSH testing for enhanced control over the analytical system. The performance of Siemens TSH test in our laboratory has proven to be very satisfactory.

A-091 Evaluation of the Analytical Performance of the New Prealbumin Assay on the Atellica CH Analyzer

Abstract Background Analytical performance studies for the new Atellica® CH Prealbumin (PALB) Assay were performed to evaluate precision, detection capability, linearity, and method comparison with the Atellica® CH Prealbumin (PreAlb) Assay. Methods Precision was conducted with 3 reagent lots per CLSI EP05-A3. Daily, two runs were performed, separated by >2 hours with 2 replicates/run, for 20 days, using normal human serum, QC material (Bio-Rad Liquid Assayed Multiqual® Level 3), and normal human serum spiked with prealbumin (Cliniqa Prealbumin spike). Reproducibility was determined per CLSI EP05 A3 and internal protocols. Samples were assayed n=5 in 1 run for 5 days using 3 instruments and 3 reagent lots. Method comparison (MC) studies were conducted with 3 reagent lots per CLSI EP09C-ED3. Individual native serum samples were tested in singlicate on two Atellica® CH analyzers. Passing & Bablok regression used PALB assay as test assay and PreAlb assay as predicate assay. Where needed, MC samples were spiked with concentrated prealbumin (Clinqa Prealbumin spike) to cover the assay measuring interval. LoB and LoD testing were conducted with 3 reagent lots per CLSI EP17-A2. For LoB, four blank samples were run (n=6) per day for three days, total 72 per reagent lot. For LoD, and additional four low samples were run (n=6) per day for three days on 3 lots, on one instrument, total 72 per lot. Linearity studies followed CLSI EP06 A; low and high pools were diluted and spiked (9 levels; n=5 per level). Results The linear range was 3.0-70.0 (30-700) mg/dL (mg/L). Results are presented in the table below for detection capability, repeatability, and method comparison. Reproducibility was 2.7-6.5% at 15.0-58.4 mg/dL (150-584 mg/L). Conclusions The PALB assay detection capability, precision, linearity, and method comparison with the PreAlb assay results were acceptable. This assay is fit for use in a clinical laboratory.

B-117 Rapid Classification of Cleanliness Grades of Vaginal Discharge through Computational Analysis of SERS Spectra via Variational Auto-Encoder Model And Long Short-Term Memory Networks

Abstract Background Classification of cleanliness grades of vaginal discharge plays a pivotal role in clinical laboratories, serving as indicators for the presence of various vaginal infections such as bacterial vaginosis and candidal vulvovaginitis, etc. The conventional method for assessing vaginal discharge cleanliness involves manual microscopy, a technique susceptible to variations based on the examiner's expertise and subjective judgment, hindering the attainment of swift and accurate diagnoses. Methods This study introduces a novel approach by integrating Surface-Enhanced Raman Spectroscopy (SERS) with a deep learning algorithm for the rapid classification of vaginal discharge cleanliness grades. The proposed classification method combines the Variational Auto-Encoder (VAE) and Long Short-Term Memory (LSTM) to process SERS spectra. The quality of the training data is assessed using the signal-to-noise ratio (SNR) as a standard indicator. Results The application of the VAE-LSTM algorithm significantly enhances classification performance, providing swift and accurate results while reducing the required time. Comparative analyses with various classical machine learning algorithms and evaluation metrics reveal the superior predictive capability and time efficiency of the VAE-LSTM algorithm, demonstrating 100% accuracy and a fitting time of 2 seconds. Blind testing of SERS spectra collected from unknown patients further validates the reliability of the proposed method. Conclusions This study presents a promising and practical approach for the rapid classification of vaginal discharge cleanliness grades. The integration of the SERS technique with the VAE-LSTM algorithm not only ensures accuracy and efficiency in clinical diagnosis but also lays the foundation for advancements in non-invasive diagnostic technologies in gynecological healthcare.

A-209 Preanalytical Phase Errors Constitute the Vast Majority of Errors in Clinical Laboratory Testing

Abstract Background Laboratory errors can occur during the preanalytical, analytic, or post-analytic phase of testing and may pose a considerable threat to patient safety. Studies have estimated that pre-analytical error encapsulates ∼45-68% of laboratory errors and occurs in up to 1.52% of specimens. However, limitations of previously published studies include the use of manual error detection, reliance on manual transcription, small study sizes, and exclusion of common errors. Herein, we aim to identify the types and frequency of errors occurring in each phase of clinical laboratory testing using a large retrospective dataset across a broad range of errors detected in clinical laboratories. Methods This study was performed in the Barnes-Jewish Hospital core laboratory which provides testing for a 1,400-bed tertiary care hospital and included all tests performed for clinical chemistry, special chemistry, toxicology, automated hematology, blood gas testing, coagulation, non-infectious serology, infectious disease serology, urinalysis, and send out testing between January 2022 and May 2023. Errors were compiled from two separate sources. The first source of errors was captured from the laboratory information system (LIS, Cerner). These errors were flagged manually by laboratory staff in real time. The second source of error was a daily report manually curated by a trained medial laboratory scientist as part of routine, laboratory operating procedures and standard workflow. Results There were ∼40,000,000 individual results from ∼11,000,000 specimens during the study period and 87,317 errors observed (∼0.8% of specimens). Of these errors, specimens that were hemolyzed sufficiently to impact result but still reportable were the majority (41,047, 47.2%). When excluding minimal hemolysis, there were 46,270 errors documented. Among these, 44,847 (97.0%) were in the preanalytical phase, 451 (1.0%) were in the analytical phase, and 972 (2.0%) occurred in the postanalytical phase. Subcategorization of preanalytical errors revealed that most of the errors were related to hemolyzed samples requiring results to be suppressed (n=19,701), quantity not sufficient samples (n=8,068), clotted specimens (n=5,840), collection errors (e.g., wrong tube type, n=5,780), not on ice (n=1,369) and IV fluid contamination (n=1,122). In the analytical phase, the causes of error were interfering substances (n=279), QC out of range (n=65), instrument problems (n=58) and reagent issues (n=49). Among the postanalytical errors, 581 were comment errors, 216 were results entry errors, 131 were comments errors with potential impact on patient care, and 44 were dilution errors. Conclusions This study demonstrates that the vast majority of lab errors (∼97.0%) occurring during the preanalytical phase. The high incidence of preanalytical errors, especially errors related to sample integrity implies a need for enhanced tools and training to minimizing errors from specimen collections, maintenance of sample stability, transportation, and processing.

B-171 Iohexol Mass Spectrometry Method Validation and Comparison in Clinical Pediatric Laboratory

Abstract Background Accurate assessment of kidney function is important yet challenging in pediatric patients due to changing muscle mass affecting serum creatinine. There are several key methods to assess kidney function in this population, including estimated glomerular filtration rate (eGFR) from serum creatinine, urine creatinine clearance, and eGFR from serum iohexol clearance, which often provides a more reliable estimate. Iohexol is a non-ionic iodinated contrast agent filtered freely through the glomeruli without tubular secretion, making it an ideal GFR marker.In this study, we validated the LC-MS/MS method to quantitatively measure iohexol in clinical pediatric plasma and serum samples. The clinical performance was compared with the novel Verispray method. Methods A series of iohexol standards (Caymen Chemical) were prepared at 10, 100, 500, 1000 µg/mL in serum (UTAK). The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and Verispray method were developed to quantitatively measure iohexol concentrations in spiked pediatric specimens. The internal standard (IS) solution was prepared with Iohexol-D5 (Cayman Chemical) at 9 µg/mL in UPLC grade methanol. Specimen or calibration/QC (50 µL) were mixed with IS solution (450 µL), and then centrifuged at 14,000xg for 3 minutes. The supernatants were analyzed. For LC-MS/MS, 3 µL supernatants were chromatographed on a AccucoreTM Vanquish C18 50 x 2.1mm (Thermo Scientific) using a gradient of water with 0.1% formic acid (FA) against methanol with 0.1% FA. Iohexol and IS were measured by multiple reaction monitoring using a TSQ Quantis Plus Mass Spectrometer (ThermoFisher). For Verispray, 10 µL supernatants were loaded on the Verispray sample plate (ThermoFisher) without LC portion. The samples were extracted from the plate paper using 95% acetonitrile with 5% water and 0.1% FA before directly injected into the mass spectrometer. The LC-MS/MS method was evaluated for linearity, patient comparison, carryover, matrix effect, and stability. 40 patients were analyzed between methods. Results Linearity was assessed over 5 days at 4 calibrator levels, the coefficient of variation (CV) ranged from 3.46 - 5.99%. The patient comparison of 40, 20 serum and plasma respectively, was conducted with Mayo Clinics, and good concordance was observed. Matrix effects were evaluated with spiked samples prepared with 50% mobile phase A and B, the ion enhancement was at 10.7%, within the allowable limits. No significant carryover was seen. The samples were stable at room temperature for 48 hours and 4oC for 7 days. Freezing is not recommended. The comparison between Verispray and LC-MS/MS showed significant bias between and within the performing personnel, the deviation can be up to 40% comparing to LC-MS/MS method. Conclusions The LC-MS/MS method demonstrated stable and consistent analytical performance for iohexol measurement. However, the Verispray method showed significant inter- and intra-user variabilities. As the Verispray sample spot can only be used once, respotting is required for any additional measurement, reducing reproductivity. It is highly recommended to duplicate all samples when using Verispray, to limit preparation to one user per patient run. Overall, our validation indicates the LC-MS/MS method provides reliable performance as a useful alternative to other complex gold standard pediatric tests of kidney function.

A-260 Mapping pediatric urinary tract infections: trends, prevalence and sensitivity to antimicrobials

Abstract Background Urinary tract infections (UTIs) rank among the most prevalent bacterial infections in pediatric populations, with neonates being particularly susceptible. Moreover, UTIs have a propensity for recurrence, posing ongoing health challenges. Prompt diagnosis of pediatric UTIs is crucial for timely and effective treatment, averting potential long-term complications. While clinical signs often suggest UTI diagnosis, additional tests like urinalysis and urine culture are vital to identify the causative pathogen and its antibiotic sensitivity. This study aims to quantify microorganism prevalence in positive pediatric urine tests and assess their antimicrobial resistance profiles. The purpose is, therefore, to offer scientific basis so that there is greater agility in establishing the diagnosis of UTIs in children and indicating the best antibiotic to be used empirically to prevent late consequences. Methods Between January and December 2019, a cross-sectional study was carried out with urine samples from children and adolescents collected in the database of the Clinical Analysis Laboratory of Cuiabá - MT in 2019. Only records of samples collected in the study were included in the study. laboratory of patients of both sexes, age range 1 month to 15 years. Bacterial analyzes were classified according to Gram positive, Gram negative (glucose fermenters or non-fermenters) and the sensitivity profile was standardized according to the Clinical and Laboratory Standards Institute (2019). The data were subjected to descriptive statistical analysis. Results Of the 532 positive urine samples from pediatric patients, enterobacteria were responsible for 435 (81.76%) with E. coli (64.1%) and Proteus mirabilis (15.8%) being the most isolated bacteria, in relation to Gram-positive bacteria 64 (12.03%) main bacteria was Enterococcus faecalis (81.2%) and non-glucose fermenters for 33 (6.2%) corresponding to Pseudomonas aeruginosa. E. coli, with 279 cases, was the most frequently isolated microorganism, representing (64.1%) of the cases. Females were the most affected, in 83.5% there was growth of Enterobacteria, in the Pseudomonas aeruginosa group, 51.5% of cases were female. and in Gram+ girls represented 56.2%. In those tested only for E. coli, the highest sensitivity was observed in fosfomycin (97.82%). Enterobacteriaceae showed high susceptibility to meropenem (99.8%), ertapenem (99.8%), ceftriaxone (93.8%), and ceftazidime (93.6%), but exhibited considerable resistance to sulfamethoxazole/trimethoprim (35.5%), amoxicillin (33.4%) and ampicillin (29.2%). Pseudomonas aeruginosa and gram positive bacteria were sensitive to all antibiotics tested. Conclusions The data indicate that E. coli continues to be the predominant agent in pediatric UTIs, particularly affecting females and the very young age group < 3 years. Regarding sensitivity profiles, bacteria were more sensitive to carbapenems and aminoglycosides. Early diagnosis is crucial to preventing long-term complications associated with UTIs in children. Consequently, this research provides a scientific basis for faster UTI diagnosis and empirical antibiotic selection in pediatrics, which may prevent late adverse outcomes.

A-326 Testing for Multidrug-Resistant Acinetobacter baumannii with a Microfluidic Device

Abstract Background Multidrug-resistant (MDR) Acinetobacter baumannii in hospitals contribute to high mortality rates and entail expensive infection control procedures. Conventionally, species and resistance determination of MDR bacteria is performed by mass spectrometry and labor-intensive phenotypical assays in clinical microbiology laboratories. Some institutions also apply time-consuming and expensive whole genome sequencing for in-depth resistance and transmission analysis. In contrast, faster and more cost-efficient MDR typing can be achieved by on-site application of real-time PCR-based point-of-care tests (POCTs) that target species- and resistance-specific genes and enable rapid screening of large sample sizes. Methods To construct a real-time PCR-based POCT system for species and resistance determination of MDR A. baumannii, we selected two species-specific genes (rpoB, OXA-51-like) and 18 resistance genes found in MDR A. baumannii (OXA-23-like, OXA-24/40-like, OXA-51-like, OXA-58-like, OXA-143-like, mcr, ADC, NDM, GIM, VIM, IMP, GES, PER, TEM, SHV, VEB, CTX-M, KPC) as targets. For each gene, one to six sets of primers and fluorophore-labeled probes that amplify conserved genetic regions were designed and their functionality was tested in vitro. Parallel, a fully automated system comprising microfluidic chips and a bench-top analyzer able to perform DNA extraction and real-time PCR starting from swab eluate was developed. Results All oligonucleotides tested so far reliably detected 100 to 100,000 copies of DNA extracted from MDR bacteria in single- and multiplex experiments in vitro. Tests with the microfluidic system showed that lysis of different bacterial species could be accomplished via sonication on a membrane located on the microfluidic chip. In addition, chip implementation of all PCR reagents like lyophilized master mix as well as primers and probes was successful and can be stored on chip at least one year. So far, the system delivered detectable fluorescence signals within one hour when performing test runs with bacteria eluted from swabs. Conclusions A real-time PCR-based Point-of-Care screening platform was developed for the detection of antibiotic-resistant A. baumannii from various patient sample materials starting directly from swabs, which detects colonization/infection and at the same time the relevant resistance genes within about an hour.

B-341 Comparative Study of Free Light Chains in the Framework of the Project Prevalence of Monoclonal Gammopathies in Adult Uruguayan Population

Abstract Background Within the framework of a study to evaluate the Prevalence of Monoclonal Gammopathies in the Adult population of Uruguay through detection monoclonal bands by capillary electrophoresis and free light chains in serum, the aim is evaluating the comparison of quantification of FLC (Free Light Chains) in this group of patients with 2 assays: Freelite Biding Site® versus Sebia® FLC Elisa. Methods Study period: September 2021 to November 2022. Prospective, single-cohort, descriptive study. Approved by the Hospital de Clinicas Ethics committee, Montevideo, Uruguay.A total of 3905 patients were included (≥40 years, without previously known plasma cell disorder).Serum samples were collected from three health centers, and processed in Clinical Chemistry Laboratory at Hospital de Clinicas, following an algorithm designed to perform electrophoretic proteinogram (by capillary electrophoresis); suspicious samples with monoclonal bands were confirmed by immunotyping, and in some cases by immunofixation (difficult interpretation), and quantification FLC by 2 different assays (Sebia FLC: manual sandwich ELISA technique- BIO-TEK wash/reader; Freelite The Binding Site immunoturbidimetric assay : Spa Plus and Optilite instruments. Comparative analysis of FLC is a transversal and descriptive study.For statistical analysis: Spearman test, Passing and Bablok and Bland-Altman plot. Results κFLC, λFLC and ratio κ/ λ were measured in 93 of the 104 positive serum samples for Monoclonal Gammopathies (new diagnoses, prevalence 2.7%), using two groups Sebia FLC and Spa Plus Freelite, and Sebia FLC and Optilite Freelite. During the project, the Spa Plus (n=48) FLC was replaced with Optilite (n=45), since the comparative group was not analytically homogeneous, so the comparison was carried out in two groups. Spearman rank correlation coefficients between first group (N:48) for κFLC, λFLC and ratio κ/ λ were r=0.596, 0,549, 0.709, respectively (p<0.0001); for the second group (N:45) Spearman rank correlation coefficients were r=0.778, 0.714, 0.878, respectively (p<0.0001). The first group showed good Spearman rank correlation for ratio, although less better correlation for kappa and lambda isolated. Bland Altman showed high media differences in assays for values for Sebia. For the second group, there was a better Spearman Correlation for ratio and in kappa and lambda isolated too. Bland-Altman plot revealed, in the second group analysis, that Freelite provides higher values than Sebia in Kappa and ratio, but not in Lambda. Passing Bablock showed differences systematic and proportional in different group of comparatives. Conclusions There is a good agreement; despite this, within high FLC concentrations, there are particularly significant differences between both assays, in both groups. We found in same cases FLC quantifications with Freelite ratios lower than those reported by the Elisa Sebia assay, which in these patients could change the diagnosis and therefore the opportunity for treatment or the time between follow-ups. In other cases, greater amount of ratio FLC assayed by Freelite, which may represent loss of linearity in subsequent dilutions. Falsely high results may occur due to polymerization of light chains.Our results suggest that Sebia FLC is a valid assay alternative, but needs more studies to demonstrate these findings; the prospective follow up of this group promises more.

A-264 Validation of a Novel Real-Time PCR Assay for Identification of Mycobacterium species Isolates Recovered from Clinical Culture

Abstract Background Historically, clinical mycobacteriology laboratories used the nucleic acid hybridization-based AccuProbe assay manufactured by Hologic, Inc., to rapidly rule in or rule out Mycobacterium tuberculosis (MTB) and members of the M. avium-intracellulare complex (MAC). The clinical value of this test was in its ability to rapidly rule in/out MTB from positive broth cultures and allow the initiation of appropriate antimicrobial therapy and infection prevention practices. Recent discontinuation of this commercial assay left a gap in testing available for the rapid identification of these organisms, which allowed us to develop an in-house real-time PCR test. Methods A replacement assay was developed by Labcorp R & D based on real-time PCR amplification of extracted nucleic acids from positive culture growth. Aliquots from positive AFB VersaTREK broth cultures were heat inactivated, and nucleic acids were extracted using the MagNA Pure 24 (MP24) instrument (Roche). Real-time PCR using published primer probe sets (Rocchetti, et al 2016) was performed on the QuantStudio™ 7 (QS7) instrument (Applied Biosystems®). The assay was designed with three independent primer/probe sequence targets: one for MTB Complex (IS6110), one for MAC (ITS), and one pan-target for the Mycobacterium genus (16s rRNA). Results Procedural verification was performed using 100 residual clinical specimens previously identified using the Hologic AccuProbe® assay. Categorical concordance was 97% (97/100). The three discrepant results were identified as MAC positive by PCR; however, these were not identified as MAC on the predicate assay. Analytical sensitivity for the PCR assay was established to be 1,000 CFU/mL for each target. Analytical specificity was assessed in vitro for nine Mycobacterium species, with no cross-reactivity observed. In vitro analysis of target detection in the presence of yeast (Candida glabrata) was also performed, and we saw no inhibition. Analysis of the primer/probe sets for all targets was performed in silico using XploreDB database of microbial genomes, with confirmed 100% matches for both MAC and MTB genomes compared to hundreds of species. By comparison, the PAN-Myco primer set showed decreased performance, having three mismatches (two on forward primer and one on the probe). As this target sequence is used as an Internal Control (IC) this was not problematic for overall assay performance. The assay demonstrated excellent precision (%CV) for each target: MTB = 0.74%, MAC = 1.10%, PAN-Myco = 3.19%. Conclusions Utilizing the depth of molecular methodologies available in our laboratories, we developed a rapid method for identification of MAC and MTB from positive broth cultures, which had excellent performance compared to the predicate assay. The PCR ID LDT test is now available and allows testing to continue with no interruption in patient care.

A-344 Delivering better and timely diagnosis for critical patients: POC clinical performance versus Central Laboratory assays in key cardiac markers

Abstract Background Cardiac markers are often used in critical settings, such as emergency department and coronary units. Clinica Redsalud Santiago implemented a reference Cardiovascular center to improve patient length of stay and clinical output, supported by diverse procedures and opportune diagnosis. Our aim was to implement POC devices for measuring key Cardiac Markers. Here we validated clinically and analytically the performance of POC devices versus Central Laboratory results for Troponin I, hs Troponin I, BNP and NT-proBNP markers. Methods Forty-nine patients admitted at emergency department were included in the study. Forty samples were obtained from male patients (69.7±9.8 years) and 9 from female patients (67.8±3.9 years). Samples were drawn and processed in parallel using Triage SOB panel, hsTroponin I and NT-proBNP tests, and the other samples were analyzed in a VITROS system, using Troponin I ES, hs Troponin I and NT-proBNP. Results Clinical agreement was assessed for different methodologies, using kappa values. Almost perfect agreement was achieved for Troponin I and NT-proBNP between Triage and VITROS systems (Figure 1). For harmonization results reporting purposes, regression fits and Bland-Altman plots were analyzed for both assays (data not shown). Hs Troponin methods did not show a good linear correlation fit, but it improves significantly on quadratic regression. For NT-proBNP methods, a good linear correlation was achieved. In both comparisons, major differences were observed for high values, while those values near to cut-off or low-to-mid in pathological range were comparable. Conclusions Timely decisions are important but is difficult to find perfect solutions for end-to-end needs. We identified two key cardiac biomarkers used routinely in emergency department and coronary care that can be harmonized for clinicians, in terms of quality and traceability. Based upon results, we can expect value discrepancies between different methodologies, hence the need for creating proper guidance for supporting key decisions for clinicians.

B-205 Performance of a Novel Fluorogenic Assay for Detection of Carbapenemase-producing Enterbacteriaceae

Abstract 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.

Gastroenteritis: A Comprehensive Review

Gastroenteritis, commonly referred to as stomach flu, is an acute inflammation of the gastrointestinal tract, marked by symptoms including diarrhea, vomiting, abdominal cramps, and fever. This review article provides a comprehensive overview of gastroenteritis, addressing its etiology, epidemiology, pathophysiology, diagnosis, management, and prevention strategies. The condition is caused by a variety of infectious agents such as viruses (noroviruses, rotaviruses), bacteria (Campylobacter, Salmonella), and parasites (Giardia lamblia), with transmission typically occurring through contaminated food, water, or person-to-person contact. Globally, gastroenteritis remains a significant public health issue, with high morbidity and mortality rates, particularly in children under five in developing countries. Diagnosis often relies on clinical evaluation and laboratory tests, while management focuses on rehydration therapy and symptomatic relief. Preventive measures include personal hygiene, food safety practices, environmental sanitation, and vaccination, with rotavirus vaccines significantly reducing severe cases in children. Emerging trends in gastroenteritis research aim at developing rapid diagnostic tools, novel therapeutic approaches, and new vaccines, highlighting the importance of a multidisciplinary approach to mitigate the global impact of this disease.

B-198 Rapid Molecular Detection of Respiratory Infections in Brazilian Hospitals and Outpatient Units: Insights from the Diagnostic Medicine and Health Network

Abstract Background Diagnosing agents causing respiratory infections remains a global challenge due to the numerous coexisting agents and similar symptoms. This study evaluates the epidemiology of viral and bacterial agents responsible for major respiratory infections through molecular detection in patients across various Brazilian hospital units served by a clinical diagnostic and health laboratory. Understanding the distribution and prevalence of these pathogens is crucial for effective diagnosis, treatment, and prevention strategies in the face of evolving epidemiological landscapes. Methods Utilizing an internal database, we conducted a comprehensive analysis of tests performed on the FilmArray® system. This system employs multiplex PCR technology, capable of simultaneously identifying 21 respiratory agents in a single sample obtained from nasopharyngeal and bronchial lavage specimens collected from diverse geographical regions across Brazil. The data for the examination of patient samples with respect to gender, age group, geographical regions, and the prevalence of various agents between January 2021 and April 2023 were retrieved from the database and analyzed. To ensure privacy, sensitive and individual data from all patients were meticulously preserved throughout the study. Results The analysis encompassed a diverse sample of 477 patients, originating from 26 cities across 11 Brazilian states, including Goiás, Minas Gerais, Mato Grosso, Pará, Paraná, Rio de Janeiro, São Paulo, Tocantins, Amazonas, Bahia, and Distrito Federal. The distribution of patients was nearly split between hospital settings (39.8%) and outpatient units, with hospital patients predominantly from departments such as internal medicine, cardiology, and nephrology. In contrast, outpatient samples were primarily requested by specialists in pulmonology and infectious diseases. The demographic profile revealed an average age of 36 years, with a majority male representation (54%). However, a notable shift occurred in the first four months of 2023, where females constituted 60.8% of the sample. The overall test positivity rate stood at 42.3%, with a significant detection of 71% (15 out of 21) of the surveyed respiratory agents at some point during the study period. The most frequently identified pathogens were Rhinovirus, Enterovirus, and SARS-CoV-2, collectively accounting for 55.1% of the positive findings. Additionally, mixed infections were observed in 29 (6%) of the patients, with combinations such as SARS-CoV-2 and Rhinovirus, SARS-CoV-2 and H3N2, and SARS-CoV-2 and RSV, among others, highlighting the complexity of respiratory infections. Conclusions The findings from this study underscore the critical role of advanced molecular diagnostics in the Brazilian clinical-laboratory landscape. The utilization of multiplex PCR technology has proven to be a pivotal tool in accurately identifying a wide array of respiratory pathogens, thereby enhancing the precision of diagnosis and treatment strategies. The detection of a high prevalence of agents such as Rhinovirus, Enterovirus, and SARS-CoV-2, along with the identification of mixed infections, highlights the complexity of respiratory infections and the necessity for robust diagnostic tools. This study not only contributes to the understanding of the epidemiology of respiratory infections in Brazil but also emphasizes the importance of continuous advancements in molecular diagnostics for better patient care and public health management.