Laboratory Workflow Research Articles

Illuminating the Path to Thyroid Disorder Management Using Artificial Intelligence: A Narrative Review

Background: Dysregulation of thyroid function, manifesting as hyperthyroidism or hypothyroidism, can profoundly impact an individual's overall health. In this context, artificial intelligence (AI) applications have the potential to revolutionize diagnostic approaches, treatment strategies, and patient monitoring. Objectives: This study comprehensively reviews the latest literature on AI applications in thyroid functional and autoimmune disorders. Methods: An online search was conducted on databases using search queries crafted with MeSH terms related to AI and thyroid disorders. After screening, studies aligned with our research focus were selected for this narrative review. Results: Multiple studies have explored the use of AI technologies, including machine learning (ML) and deep learning (DL), to enhance laboratory workflows for thyroid function tests (TFT) and improve the accuracy of TFT interpretation by incorporating clinical data. In imaging, DL-based models have demonstrated the potential to assist less experienced radiologists in interpreting scintigraphy and ultrasound images. Artificial intelligence has also provided valuable insights into identifying diagnostic genes for thyroid-related autoimmune disorders and understanding the effects of environmental factors, such as chemicals, on thyroid gland function. Some ML models have been developed to predict the risk of hypothyroidism following radioiodine therapy (RAI). Furthermore, AI has shown promise in personalized levothyroxine dose adjustments, predicting treatment responses, and accurately diagnosing complications such as thyroid-associated ophthalmopathy (TAO). Finally, ML-based models forecasting the risk of suicide attempts in patients with major depressive disorder (MDD) and predicting pregnancy outcomes, such as gestational diabetes mellitus (GDM) and preterm delivery, based on TFT results, appear beneficial in addressing these significant health issues. Conclusions: The current state of AI in diagnosing and treating thyroid function disorders is promising, with applications primarily focused on improving diagnostic accuracy, consistency, and personalized treatment approaches. However, challenges remain that prevent these models from fully substituting professionals. Addressing these challenges is crucial to ensure AI effectively contributes to the management of patients with thyroid diseases.

Automation of multiplex biochemical assays to enhance productivity and reduce cycle time using a modular robotic platform

Pharmaceutical and biotechnology companies are increasingly being challenged to shorten the cycle time between design, make, test, and analyze (DMTA) compounds. Automation of multiplex assays to obtain multiparameter data on the same robotic run is instrumental in reducing cycle time. Consequently, an increasing need in automated systems to streamline laboratory workflows with the goal to expedite assay cycle time and enhance productivity has grown in industrial and academic institutions in the past decades. Herein, we present a customized robotic platform with operational modularity and flexibility, designed to automate entire assay workflows involving multistep reagent dispensing, mixing, lidding/de-lidding, incubation, centrifugation, and final readout steps by linking spinnaker robot with various peripheral instruments. Compared to manual workflows, the system can seamlessly execute processes with high efficiency, evaluated by standard assay validation protocols for robustness and reproducibility. Furthermore, the system can perform multiple, independent protocols in parallel, and has high-throughput capacity. In this publication, we demonstrate that the modular robotic platform can fully automate multiplex assay workflows through ‘one-click-and-run’ solution with tremendous benefits in liberating manual intervention, boosting productivity while producing high-quality data combined with reduced cycle time (>20%), as well as expanding the capacity for higher throughput.

Full characterization of unresolved structural variation through long-read sequencing and optical genome mapping

Structural variants (SVs) are important contributors to human disease. Their characterization remains however difficult due to their size and association with repetitive regions. Long-read sequencing (LRS) and optical genome mapping (OGM) can aid as their molecules span multiple kilobases and capture SVs in full. In this study, we selected six individuals who presented with unresolved SVs. We applied LRS onto all individuals and OGM to a subset of three complex cases. LRS detected and fully resolved the interrogated SV in all samples. This enabled a precise molecular diagnosis in two individuals. Overall, LRS identified 100% of the junctions at single-basepair level, providing valuable insights into their formation mechanisms without need for additional data sources. Application of OGM added straightforward variant phasing, aiding in the unravelment of complex rearrangements. These results highlight the potential of LRS and OGM as follow-up molecular tests for complete SV characterization. We show that they can assess clinically relevant structural variation at unprecedented resolution. Additionally, they detect (complex) cryptic rearrangements missed by conventional methods. This ultimately leads to an increased diagnostic yield, emphasizing their added benefit in a diagnostic setting. To aid their rapid adoption, we provide detailed laboratory and bioinformatics workflows in this manuscript.

Maximizing Laboratory Turnaround Time Efficiency: Workflow Optimization in Resource-Limited Settings

Laboratory Turnaround Time (TAT) is a critical metric in assessing the quality of laboratory services, directly impacting diagnostic speed and patient outcomes. Many laboratories in resource-constrained environments face challenges such as inefficiencies in workflow, resource limitations, and administrative redundancies, which hinder optimal TAT performance. This research aims to optimize laboratory workflow to improve TAT efficiency in a resource-constrained environment, addressing workload variability, inefficient station grouping, and administrative overlap. The research, which was conducted at the Tanah Abang Puskesmas Laboratory from May to July 2022, consisted of three stages: observation and data collection, workflow optimization, and evaluation. Interventions included task reallocation, workflow segmentation, and administrative streamlining. TAT improvement was assessed from the Complete Blood Count (CBC) and Random Blood Glucose (RBG) tests using pre- and post-intervention analysis. The results showed that after optimization, the mean TAT for the CBC test decreased significantly from 36 minutes (SD = 13.4) to 25.6 minutes (SD = 4.1), p-value <0.05. For the RBG test, the TAT increased from 88.6 minutes (SD = 4.6) to 81.1 minutes (SD = 1.4), p-value < 0.01. This intervention demonstrates the feasibility and effectiveness of lean management strategies in a resource-constrained environment. This research has implications for emphasizing the importance of systematic workflow optimization to improve laboratory efficiency. This research highlights practical and scalable solutions that can be adopted in similar settings globally, and future efforts should focus on integrating advanced technologies such as Laboratory Information Systems (LIS) to further improve accuracy, efficiency and patient outcomes.

Clinical laboratory evaluation of the Hologic Panther Aptima BV and CV/TV assays for the diagnosis of vaginitis in Dunedin, Aotearoa New Zealand.

Vaginitis presentations are common, but traditional diagnostic methods are imperfect. Molecular methods for bacterial vaginosis (BV) and vulvovaginal candidiasis (CV) are increasingly available but not commonly utilized in Aotearoa New Zealand. We evaluated the Hologic Aptima BV and CV/Trichomonas vaginalis (TV) assays against our current methods (Gram stain, yeast culture, and Hologic Aptima TV assay) and performed a retrospective BV clinical audit. The BV Aptima assay performed well with high sensitivity (97.5%) and specificity (96.3%) when the indeterminate BV category was excluded. BV indeterminate samples were almost evenly split between positive and negative results when tested on the Aptima BV assay. BV Gram stain interpretation was error prone, with 20% of samples discordant on duplicate examination. Although the Aptima CV assay was highly sensitive, it lacked specificity compared with Gram stain (83.5%) but was similar to culture (91.2%). Our BV clinical audit showed that patients with a BV indeterminate result were less likely to be treated for BV than those with a positive result, meaning more women may be treated for BV if this assay were implemented. Overall, implementation may improve laboratory workflow and consistency of reporting, but cost may be a barrier. The clinical impact of changing methods needs to be considered.IMPORTANCEIn this paper, we evaluate the performance of the Aptima molecular assays against current Gram stain and culture methods, as well as a clinical audit to determine the potential clinical impact of implementation. Although molecular methods are increasingly used in other countries, New Zealand has not yet adopted this approach. Importantly, we found Gram stain for bacterial vaginosis (BV) to be error prone, with 20% of Gram stain results discordant on repeat examination. We show the potential for molecular methods to increase BV diagnoses and improve reproducibility and consistency of reporting which, according to our clinical audit results, would lead to more women being treated for this dysbiosis condition overall.

Genomic Surveillance of Foodborne Pathogens: Advances and Obstacles

Context: The genomic surveillance of foodborne pathogens in the United States has grown exponentially in the past decade, grounded in a powerful combination of novel sequencing technologies, bioinformatic approaches, data-sharing networks, and metadata harmonization efforts. This practice report examines recent advances in genomic epidemiology as applied to food safety programs and delineates State, Tribal, Local, and Territorial infrastructure necessary for continued life-saving improvements in public health. Program: National databases of foodborne pathogen genomes, along with data sharing and evaluation networks such as GenomeTrakr and PulseNet, have transformed how connections are made among isolates and how root causes of outbreaks are determined, allowing much more timely interventions to protect public health. Freely available bioinformatics tools such as GalaxyTrakr and the National Center for Biotechnology Information Pathogen Detection database have allowed laboratories with limited local computing resources to participate in surveillance efforts and contribute to traceback investigations. Implementation: In this report, we describe advances in genomic epidemiology that have occurred over the past decade and examine obstacles to fully implementing this technology within State, Tribal, Local, and Territorial public health systems. Evaluation: Despite a clear return on investment from governmental expenditures on genomic surveillance of foodborne pathogens, we identify significant obstacles to further sustained progress. These obstacles include workforce gaps, ineffective data sharing, and lack of constitutive and sustained funding. Discussion: Many public health laboratories face major obstacles to widespread and routine adoption of genomic surveillance technologies. While whole genome sequencing has become an integral part of routine public health microbiology, the seamless integration of these protocols into the existing practices, laboratory workflows, and information systems remains challenging. Centralized efforts to address these issues include (1) support through the Food and Drug Administration Laboratory Flexible Funding Model, (2) training and proficiency assessments, (3) open-source, standardized protocols for collecting high-quality genomic data, and (4) open access informatics software.

Detection by Real-Time PCR of Helicobacter pylori and Clarithromycin Resistance Compared to Histology on Gastric Biopsies.

The global rise in Helicobacter pylori (H. pylori)-related gastric complications is largely driven by increasing antimicrobial resistance and treatment failures. As a result, accurate diagnosis followed by effective treatment is crucial. We analyzed 232 gastric biopsy samples from patients undergoing endoscopy during the method validation phase, followed by 502 samples in the routine evaluation phase. Each sample was tested using the Allplex™ H. pylori and ClariR Assay on a CFX96™ real-time PCR (RT-PCR) system, with results processed through Seegene Viewer software. In the validation phase, RT-PCR results were compared to bacterial culture, while in the routine phase, they were compared to histology. The sensitivity and specificity for H. pylori detection were 100% and 96.05% (95% Confidence Interval [CI]: 93.38-98.73), respectively. For clarithromycin resistance detection, the sensitivity and specificity were 100% and 93.33% (95% CI: 84.4-100). Additionally, RT-PCR identified 11 positive samples (10.89%) that histology failed to detect. Incorporating the Allplex™ H. pylori and ClariR Assay into our laboratory workflow improved efficiency, reduced turnaround time (TaT), and proved to be more sensitive than both culture and histology combined.

Mass spectrometry-based proteomics for source-level attribution after DNA extraction

Biological traces recovered from crime scenes serve as vital evidence in forensic investigations. While DNA evidence is frequently used to address the sub-source level of the hierarchy of propositions, the biological source of the DNA can be highly probative at the source level. Current body fluid detection methods pose certain limitations, such as reports of false positive results from some of the presumptive and/or confirmatory tests in current use. These tests are also individual tests for the detection of one body fluid, meaning that if the sample is suspected to be a mixture of multiple body fluids, then different tests would need to be conducted to confirm the body fluid(s) present, which may exhaust small amounts of available biological trace. Proteomics applications for the identification of body fluids have been previously explored, and potential biomarkers indicative of body fluids discovered from liquid-chromatography tandem mass spectrometry (LC-MS/MS) methods have been reported. This work focuses on developing a mass spectrometry-based proteomics approach for the identification of body fluids by targeting discriminating peptide biomarkers from the non-DNA component left over after DNA extraction of samples. The non-DNA component is typically a waste product but with unappreciated evidential value. Our methodology for the purification of proteins from the post-DNA extraction waste includes an acetone precipitation and single-pot solid-phase-enhanced sample preparation (SP3) technique, microwave-assisted trypsin digestion, and LC-MS/MS analysis of the resultant peptides. Preliminary results from this proof-of-concept study include a list of potentially discriminating proteins and peptides for blood, saliva, and semen developed from the analysis of post-DNA extraction waste. Our method allows for multiple analytes to be targeted simultaneously from a DNA profiling waste stream and we anticipate that it could eventually be incorporated into standard forensic laboratory workflows.

Multi-year comparison of VITEK MS performance for identification of rarely encountered pathogenic Gram-negative organisms (GNOs) in a large integrated Canadian healthcare region.

This multi-year study (2014-2019) compared identification of rare and unusual Gram-negative organisms (GNOs) by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (VITEK MS, bioMérieux, Laval Que.) to 16S rRNA gene sequencing (16S) according to our laboratories routine workflow; 16S is done if initial MALDI-TOF MS gave discordant, wrong, or no results. GNB isolates were first analyzed by standard phenotypic methods and MALDI-TOF MS using direct deposit-full formic acid extraction; proteomics was repeated if no result occurred. Medically approved 16S analyses were done using fast protocols. Isolate sequences were analyzed using the Integrated Database Network System (IDNS3) bacterial database (SmartGene, Lausanne, Switzerland). Three hundred thirty-one GNOs including 251 (76%) aerobic Gram-negative bacilli (GNB), 63 (19%) fastidious Gram-negative coccobacilli (fGNCBs), and 17 (5%) Campylobacterales (CAMPB) isolates were recovered from 304 specimens; >1 isolate was recovered from 19 (6%). GNOs were mainly recovered from blood cultures (31.6%) and lower respiratory specimens (43%) (one-half were isolated from cystic fibrosis patients). Accurate genus vs species identities were obtained for 67.7% and 32.5% aerobic GNBs, 73% and 60% fGNCBs, and 23.5% CAMPB (with no discrepant species), respectively. Wrong or no results were obtained for 81 (32.3%) aerobic GNBs, 17 (27%) fGNCBs, and 13 (72.2%) CAMPB. No results or misidentifications occurred for 33% of aerobic GNBs, 26% of fGNCBs, and 76.5% of CAMPB due to absence of species in the instrument's database. VITEK MS performance remained stable for aerobic GNBs and fGNCBs but improved for CAMPB with addition of Campylobacter rectus and Campylobacter curvus to the database. 16S remains important for identification of GNOs when proteomics fails.IMPORTANCEMatrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has transformed the identification of commonly encountered Gram-negative organisms (GNOs) in the clinical laboratory, but rare and unusual organisms continue to challenge the technology. This study verified performance of VITEK MS for identification of a broad range of rare and unusual clinical GNO isolates by our large reference laboratory workflow over a multi-year period. Although most GNOs were accurately identified by MALDI-TOF MS, a small number of clinical isolates (~1%-6%) required 16S sequencing for identification depending on the GNO category. Approximately one-third of aerobic Gram-negative bacilli (GNBs) and two-thirds of Campylobacterales could not be accurately identified by proteomics due to lack of an organism in the instrument's database. MALDI-TOF MS databases should be continuously updated and validated, and laboratories should have a workflow for identification of unusual or rarely encountered aerobic, fastidious, and Campylobacterales GNOs that includes 16S rRNA gene sequencing whenever proteomics cannot give a definitive identification.

Clinical Utility of Broad Range PCR for Infectious Organisms

Abstract Broad range PCR is used to identify infectious organisms when bacterial, fungal, and mycobacterial cultures are negative despite clinical evidence of infection. We performed a 6-month retrospective chart review to assess the actionability of broad range PCR and identify opportunities to improve the utility of this referral test at our institution. One hundred eleven broad range PCR tests were ordered from July to December 2023. Seventy-five (67.6%) results did not affect patient management, 18 (16.2%) results affected patient management, and the treatment impact of 18 (16.2%) results is unknown because they were from outside consult cases. There were no instances in which antibiotic therapy was completely discontinued due to a negative broad range PCR result. Concurrent in-house microbiology testing yielded a positive result in 15 (13.5%) cases, of which 8 (53%) had a negative broad range PCR result and 2 (13.3%) had a conflicting broad range PCR result. Thirty-eight (34.2%) specimens were formalin fixed paraffin embedded tissue, 29 (26.1%) were fresh tissue, 13 (11.7%) were ESWABS, and the remaining 31 specimens were various fluid types (27.9%). While fresh tissue is the preferred specimen, broad range PCR detected an organism in 20 (53%) formalin fixed paraffin embedded tissue specimens, 11 (37.9%) fresh tissue specimens, 3 (23.1%) ESWAB specimens, and 2 (6.5%) fluid specimens (abscess aspirate and cerebrospinal fluid). Improvements in physician communication and laboratory workflow are necessary to improve the specimen quality and thus the utility of this referral test at our institution.

Detecting and Reporting Extended Spectrum Beta-Lactamases Producers: Evaluation of Three Approaches and the Clinical Impact

Abstract Introduction/Objective Since the COVID-19 pandemic, bacteria producing extended spectrum beta-lactamases (ESBLs) have increased >30%. Laboratories can perform phenotypic microbroth dilution or molecular approaches to identify ESBL producers. However, the gold standard is a laborious double disc synergy test. We evaluated the concordance among the three approaches, the clinical impact, and justification of reporting ESBL producers. Methods/Case Report Seventy-eight ESBL producers identified by microbroth dilution (MicroScan WalkAway, Beckman Coulter, Brea, California, USA) or detection of blaCTX-M gene from blood cultures (BCID2 Panel, BioFire, Biomerieux, Salt Lake City, Utah, USA) were tested using double disc synergy test retrospectively. Concordance among the methods evaluated. Discordant cases were reviewed with subsequent patient chart review. Results (if a Case Study enter NA) Tested isolates included E. coli (n=44, 57%), K. pneumoniae (n=19, 24%), K. oxytoca (n=7, 9%), and P. mirabilis (n=8, 10%). Concordance between microbroth dilution and disk diffusion was 86% (67/78). For isolates with genotypic testing (n=16), concordance with microbroth dilution and disk diffusion was 93% and 88%, respectively. Overall concordance was 88%. There were 12 isolates (E. coli:8, P. mirabilis: 2, and K. pneumoniae:2) with discordant results from one method. The susceptibility profile of discordant isolates revealed resistance to ceftriaxone or ceftazidime (11/12), penicillin (11/12), and aztreonam (6/12); 10/12 isolates were resistant to at least 2 groups of these antibiotics, a phenotype of ESBL producers. Clinical teams acknowledged presence of ESBLs in 83% (10/12) of cases and 58% (7/12) warranted treatment with meropenem or combinational therapy. Infection prevention implemented contact isolate procedures in all cases. Conclusion Our current in-house methods (e.g., microbroth dilution and genotypic testing) are still justified for ESBL testing to optimize patient care in a timely fashion. In the minor cases with discordant results, clinical care was unaffected. Laboratory workflow changes need to take laboratory efficiency, patient outcomes, and actionable clinical changes into consideration.

Hematology instruments don’t speak the same language: a comparison study between flagging messages of sysmex XN-1000 and alinity H

Abstract Objectives While manual review is the gold standard, automated hematology analyzers are increasingly used. This study assessed the efficiency of white blood cell (WBC)-related flagging messages from the Sysmex XN-1000 and Alinity hq analyzers compared to peripheral blood smear (PBS) findings and evaluated their inter-platform agreement. Methods K3EDTA blood samples from hospitalized patients were analyzed using the Sysmex XN-1000. Samples triggering a morphology flag were reanalyzed on the Alinity hq, with PBS reviewed per CLSI protocol H20-A2-2007. Results Of 5530 samples, 196 had morphology-related flags requiring PBS review. Sysmex flagged 144 samples with leukocyte-related messages; Alinity flagged 120. The positive predictive value (PPV) for the Left Shift flag was 100 % for Sysmex and 77.5 % for Alinity; for Immature Granulocytes, it was 19.4 % for Sysmex and 94.6 % for Alinity. The Blast Flag’s PPVs were 9.3 % for Sysmex and 17.9 % for Alinity. Left Shift specificities were high (>94 %), but sensitivities varied. Sysmex showed 100 % sensitivity for the Blast flag but moderate specificity (53 %), while Alinity performed well (77–82 %). Agreement between platforms ranged from poor to good. Conclusions Tailored SOPs are crucial for optimizing laboratory workflow based on different flagging performances. Understanding each analyzer’s strengths and limitations improves interpretation and workflow management.

Efficiently Improving Laboratory Workflow with Continuous Sample Loading by Validating the COBAS 5800 for HIV and HCV Viral Loads

Abstract Introduction/Objective A regional veteran affairs medical center (VAMC) was using the COBAS AmpliPrep/COBAS Taqman (Roche, Basel Switzerland) with a throughput of 72 tests/8 hours without continuous loading capability. However, a newer cobas 5800 System (Roche, Basel Switzerland) was validated to allow for continuous loading with better parameters for workflow including 144 tests/per 8 hours with a capacity for continuous loading of samples without batching. As validation experiences between the two instruments is sparse in the literature, the validation is described. Methods/Case Report The validation process included a linearity study using 24 samples with levels (100, 600, 6,000, 60,000, 600,000, 2,000,000, and 6,000,000 copies/mL for HIV and 100, 300, 3,000, 30,000, 300,000, 3,000,000, and 30,000,000 copies/mL for HCV) across the analytical range, a precision study at a low (1,000 copies/mL) and high (100,000 copies/mL for HIV, 1,000,000 copies/ML for HCV) values of a triplicate run over 5 days, an accuracy study based on the precision study, a limit of detection study using 20 control samples at the limit of detection, control monitoring (high and low positive controls) over 9 runs, and a method correlation between the Cobas 5800 and the Cobas AmpliPrep/COBAS Taqman. Results (if a Case Study enter NA) The validation study resulted as expected. For the method correlation, the results were plotted and evaluated with an R2 of 0.937 (HIV from 91 samples) and 0.9646 (HCV from 74 samples). Linearity across the analytical range had an R2 of 0.9967 (HIV) and 0.9950 (HCV). All samples were detected at the limit of detection study. Both the accuracy and precision yielded expected results. The values from running the controls (high and low positive) over 9 runs remained within the expected range and within two standard deviations for all runs. Conclusion The COBAS 5800, which has improved capabilities compared to the COBAS AmpliPrep/COBAS Taqman, was validated for clinical use successfully.

Performance of Gram Stain Characteristics for Predicting Culture Growth in Lower Respiratory Specimens Across Multiple Institutions

Abstract Data from Gram stains performed on clinical specimens can guide clinical decision-making and downstream laboratory workflows. However, the extent to which Gram stain results predict gold-standard culture results, and the generalizability of this performance across sites, warrants additional exploration. We sought to discover which Gram stain features would be associated with clinically significant culture growth (“growth”). To do so, 574 remnant specimens from the lower respiratory tract collected from four institutions were analyzed. A standardized Gram stain and culture protocol was performed across all study sites. Final reference organism identification was assigned using MALDI-ToF MS, and clinically significant growth was defined as any organism reported to the medical record. Gram stains with mixed microorganisms detected were considered concordant if ≥2 organisms of different Gram stain appearances were identified in culture. Growth was observed in 53% of specimens, including 25% of specimens in which Gram stain demonstrated no organisms. The 95% confidence intervals (CI) of the site-wise growth rates were 30-42%, 42-52%, 51-67%, and 60-77% (p < 0.001 by Chi-squared). By Gram stain morphology, 53-69% of specimens with Gram-positive cocci grew any clinically significant bacteria, while 31-49% grew a concordant Gram-positive cocci. 65-85% of specimens with Gram-negative bacilli grew any clinically significant bacteria, while 59-74% grew a concordant Gram-negative bacilli. 63-83% of specimens with mixed microorganisms grew 2 or more clinically relevant bacteria of differing Gram stain morphologies. Specimens from bronchoalveolar lavages (BALs) were less likely to have growth (30-42%) than endotracheal aspirates (66-77%). Polymorphonuclear leukocyte (PMNs) abundance was associated with culture growth, with Gram stains showing no, rare, few, moderate, or abundant PMNs demonstrating growth 28, 44, 52, 60, and 67% of the time (p < 0.001). Specimens with None and few squamous epithelial cells on Gram stain demonstrated growth 38-52% and 59-68% of the time (p < 0.001). No significant difference in growth was observed for red blood cells. Organism abundance on Gram stain was significantly associated with culture growth, with growth rate CIs being 20-32% for no organisms, 44-62% for rare, 58-77% for few, 70-93% for moderate, and 81-95% for abundant organisms seen. The extent to which these associations could guide the development of decision rules or machine learning models that demonstrate acceptable negative predictive value for pathogen growth to alleviate the operational burden of low-utility cultures is an exciting future direction to explore.

The Expression of miR-211-5p in Sentinel Lymph Node Metastases of Malignant Melanoma Is a Potential Marker for Poor Prognosis

Metastatic primary cutaneous melanoma is a frequently fatal disease despite recent therapeutic advances. Biomarkers to stratify patients’ prognosis are lacking. MicroRNAs (miRNAs) are small, non-coding RNAs. We aimed to determine the expression of miR-211-5p in primary tumors and metastases of malignant melanoma and its potential use as a prognostic biomarker. We performed in situ hybridization for miRNA-211-5p on 109 FFPE melanoma samples from 76 patients, including 31 paired primary tumor/metastasis samples. For validation, we performed in silico analyses of TCGA skin cutaneous melanoma (SKCM) cohort. High miR-211-5p expression was more frequent in primary tumors (70.8%) compared to metastases (39.3%). In metastases, it was associated with a significantly worse overall survival. Data from TCGA SKCM cohort confirmed that high miR-211-5p expression in melanoma metastases, but not primary tumors, is associated with worse overall survival. MiR-211-5p expression in metastases is associated with a shorter survival, emphasizing the potential of miR-211-5p as a risk predictor for a less favorable clinical outcome in metastatic disease. In situ hybridization could be implemented in a routine laboratory workflow and can be performed on diagnostic tissue.

A-205 Evaluating the Cost and Importance of Supply Chain Resilience in the Clinical Laboratory

Abstract Background Recent pandemics, epidemics and outbreaks continually underscore the critical importance of resilient supply chains in healthcare, particularly in clinical laboratories where timely access to supplies is essential for patient care. Supply chain disruptions, whether due to global crises or localized challenges, can significantly impact laboratory operations, leading to delayed diagnoses, compromised patient outcomes, and increased costs. It is estimated that 70%-80% of a patients’ EMR is clinical laboratory results. Despite this, supply chain resilience in clinical laboratories remains an under-addressed issue and remains a buzzword. Methods To evaluate the cost and importance of supply chain resilience in the clinical laboratory, we conducted a comprehensive review and interviews with 9 Healthcare Supply Chain experts analyzing the impact of supply chain disruptions on laboratory operations and patient care. Data was also collected from a diverse range of clinical laboratories, including hospitals, independent labs, and research institutions, spanning different geographic regions as well as a survey of 50 Laboratory experts with a 50% response rate. Key metrics assessed included: -Frequency and duration of supply chain disruptions. -Financial costs incurred due to disruptions, including expenses related to expedited shipping, alternative sourcing, and inventory management. -Impact on laboratory workflow, turnaround times, and patient care outcomes. -Strategies employed to mitigate supply chain risks and enhance resilience. Results Our review revealed findings regarding the cost and importance of supply chain resilience in the clinical laboratory: -Supply chain disruptions were frequent, with 85% of laboratories experiencing at least one significant disruption in the past year and 100% in experienced disruptions 2022. -The average duration of disruptions was 2-3 weeks, leading to substantial delays in test processing and patient care. -Financial costs associated with disruptions averaged $50,000 per laboratory per annum, including expenses for rush orders, and premium-priced alternatives. -Common strategies employed were increased inventory or safety stocks, multi-vendor sourcing, and supply chain mapping. Conclusions The findings of this study underscore the critical importance of supply chain resilience in the clinical laboratory. Supply chain disruptions are not just inconveniences; they pose significant risks to patient safety, operational efficiency, and financial sustainability. Investing in supply chain resilience is imperative for laboratories to fulfill their mission of delivering timely and accurate patient results. To continue to enhance supply chain resilience, laboratories must adopt a proactive approach, leveraging data-driven insights and collaborative partnerships. This includes diversifying supplier networks and fostering closer collaboration between laboratory and procurement teams.

A-162 Improved Laboratory Workflow using Automated Detection of Hemolysis, Icterus, and Lipemia for Coagulation Testing

Abstract Background Managing preanalytical variables is critical to providing quality laboratory test results. However, this can be a challenge for laboratory staff, particularly with manual detection of interfering substances and complicated workflows. For coagulation testing, we assess specimen integrity and acceptability by visual checks and quantification of interfering substance on a chemistry analyzer. To improve this subjective process, we evaluated the technical performance of a new model of coagulation analyzer with automated preanalytical specimen integrity detection. Our objective was to determine if we could reduce the number of specimens requiring visual inspection/off-analyzer interference testing in order to simplify our testing workflow. Methods Citrated plasma specimens received in the Hospital Clinical Laboratory at Mayo Clinic (Rochester, MN) were tested on the TOP ACL 500 or TOP ACL 550 Series Coagulation analyzers (Werfen, Boston, MA) according to manufacturer’s instruction. Specimens tested on the TOP 500 were visually inspected for hemolysis, icterus, and lipemia (HIL). If the technologist detected a possible interferent, an aliquot of plasma was analyzed on our cobas c501 chemistry analyzer (c501) (Roche Diagnostics, Indianapolis, IN) to obtain HIL values. Laboratory-established or manufacturer-defined interference thresholds were applied in determining specimen acceptability. For samples tested on the TOP 550, specimen integrity was assessed by the instrument’s automated interference detection module. The analyzer reported HIL as an “interference cloud” or estimated range, rather than a single value like the c501. HIL values were estimated by visual assessment of the low end of the “interference cloud”. Values were confirmed on the c501 only for specimens with an “interference cloud” at/exceeding the test-specific interference thresholds. We analyzed data from a 2-month period pre- (TOP 500) and post-implementation (TOP 550). The percent of specimens requiring quantitation/confirmation on the c501 was calculated. Statistical significance was calculated using the Chi-squared test where significance was defined as p<0.01. Correlation between c501 and TOP 550 estimated HIL values was evaluated. Results A total of 2.2% (171/7605) and 1.3% (113/8983) of specimens tested on the TOP 500 and TOP 550, respectively, required HIL measurements on the c501 (p<0.001). Of those samples, 8.2% (14/171) and 14.2% (16/113) from the TOP 500 and TOP 550, respectively, had HIL values above the interference thresholds and were rejected by the laboratory. Comparison of the estimated HIL values from the “interference cloud” and from the c501 resulted in slopes (Pearson correlation coefficients) of: H 1.56 (0.77), I 0.67 (0.97), and L 1.35 (0.99). Conclusions In comparison to our workflow with visual assessment of interference, use of the automated interference detection functionality on the TOP 550 instrument resulted in a lower percentage of specimens requiring HIL measurements on the chemistry analyzer. Agreement between interference measurements obtained on the c501 and the TOP 550 was moderate where TOP 550 overestimated the degree of hemolysis and lipemia and underestimated icterus. Implementation of automated interference detection on the TOP 550 analyzer improved our workflow by eliminating the need for tedious and subjective visual checks and reducing the percentage of specimens requiring additional HIL measurements.

A-138 Implications of native protein conformation in the IgM quantification: total Ig vs HLC methods comparison

Abstract Background For IgM monoclonal gammopathies such as Waldenström Macroglobulinemia, the diagnosis, risk stratification and monitoring response to therapy is based on the detection and quantification of the secreted IgM monoclonal protein. Accurate IgM measurement is challenging due to its complex nature and the occurrence in higher polymeric structures (i.e., pentamers or hexamers), as well as heterogeneity and inherent subjectivity of the selected common methodologies. The recently developed Heavy/Light chain assay (Binding Site, part of Thermofisher Scientific) allows the separate determination of the IgM-Kappa and IgM-Lambda molecules, offering an alternative method to the serum electrophoresis (SPE) with reported higher sensitivity (Ref. Boyle E. et al, Clin Cancer Research 2016). In this study we explore the effect of the molecular structure on 3 different analytical techniques for accurate IgM measurements Methods 214 serum samples from patients (age median 63yo) under follow-up at our hospital were tested for: IgG, IgA, and IgM levels (immunoturbidimetry, either on a Beckman Coulter AU5800 analyser; Nyon, Switzerland; or a Cobas c 702 system, Roche Diagnostics); Hevylite assay (on an Optilite, Binding Site); and SPE and Immunofixation (Capillarys 3 Octa and Hydrasys 2, Sebia Inc.). The effect of the molecular conformation was evaluated through sample depolymerization with N-Acetylcysteine (NAC) according to the manufacturer’s instruction (Sebia Inc.) and IgM quantification by: tIgM (immunoturbidimetric total Ig); red-Ig (idem after NAC reduction); and, HLC-Ig (i. e. sum of the IgM-K+IgM-L heavy/light chain pair measured by Hevylite (Binding Site)). Results Total-Ig, red-Ig, and HLC-Ig returned similar results for IgG and IgA immunoglobulins (Kruskal-Wallis test, p-value > 0.05), and Passing Bablok regression analysis showed good agreement between the three methods, except for higher IgA levels. In the later, both HLC-IgA levels red-IgA values were higher than total-IgA. Regarding the IgM, PB regression revealed a significant proportional bias, with obtained values approx. 2,5x higher by red-IgM and approx. 1,4x by HLC-IgM methods than tIgM (red-IgM=-5.5+2.42*tIgM, Spearman’s r=0.995; HLC-IgM=-0,77+1.34*tIgM, Spearman’s r=0.978; red-IgM=-6.29+1.82*HLC-IgM, Spearman’s r=0.98). So, both red-IgM and HLC-IgM results are higher than total IgM. Of note, 1 sample presented antigen excess, resulting in unusually low IgM levels by tIgM but high red-IgM and high HLC-IgM levels coherent with the patient’s clinical condition. 75 samples had monoclonal protein measurable by SPE, showing good correlation with the three methods, however with greater proportional errors between measurements with other techniques Conclusions IgM molecular conformational status may affect the epitopes exposed and, therefore, available for the different assays’ recognition. Higher IgM complexes depolymerization into monomeric conformation results in higher IgM levels, which agree better with the Hevylite than with the total IgM measured previous NAC reduction, reflecting the impact of the native protein conformation in the IgM precise detection. Although also affected by the conformational status, Hevylite IgM results were closer to the reduced IgM levels than the obtained by the total IgM assay, therefore offering a sensitive assay that does not need a reduction treatment for a more accurate IgM quantification, optimizing the laboratory workflow

A novel method for the simultaneous determination of drugs of abuse, ethyl glucuronide and synthetic opioids in human hair through a single digestion, purification and analysis in LC-MS/MS

Polydrug use is a serious health and social problem worldwide. Over the past several years, there has been an increasing tendency to combine narcotics, alcohol, sedatives, and/or stimulants. To the traditional drugs of abuse and alcohol, an increase of new abuse drugs such as synthetic opioids has been added. In the current study, the development and validation of an innovative and fast analytical procedure has been presented to determine drugs of abuse, ethyl glucuronide and synthetics opioids in 30 mg of human hair through a single digestion, purification and analysis in LC-MS/MS. A combine simple preparation of hair sample followed to a single chromatographic run of 10 min has been proposed. A full validation for 54 target analytes for the parameters of selectivity, linearity, limit of detection, limit of quantification, accuracy, precision, matrix effects, recovery, and dilution integrity was successful completed. The method was linear in different ranges with r values of at least 0.990; the value to the validated LLOQ values were in the range 0.1–100 pg/mg. The method offered satisfactory precisions (CV<15 % and accuracy ± 20 %). In conclusion, a significant reduction in the overall times of the analytical procedure and the reduction of consumables costs make this method extremely advantageous and undoubtedly useful in routine laboratory workflow analyses and open the way to the prospect of a further implementation which also includes other classes of xenobiotics.

Detection and differentiation of low virulence and virulent Orthoavulavirus javaense using a molecular beacon with RT-LAMP

Newcastle disease (ND), an economically important disease in poultry, is caused by virulent strains of the genetically diverse Orthoavulavirus javaense (OAVJ). Laboratories rely on quantitative real-time reverse transcription PCR (qRT-PCR) to detect OAVJ and differentiate between OAVJ pathotypes. This study demonstrates that a fusion cleavage site based molecular beacon with reverse transcription loop mediated isothermal amplification (MB-RT-LAMP) assay can detect and differentiate OAVJ pathotypes in a single assay. Data show that the assay can rapidly identify diverse OAVJ genotypes with sensitivity only one log-fold lower than the current fusion qRT-PCR assay (104 copies), exhibits a high degree of specificity for OAVJ, and the molecular beacon can differentiate mesogenic/velogenic sequences from lentogenic sequences. Further, data show that a two-minute rapid lysis protocol preceding MB-RT-LAMP can detect and differentiate OAVJ RNA from both spiked samples and oropharyngeal swabs without the need for RNA isolation. As the MB-RT-LAMP assay can rapidly detect and discriminate between lentogenic and mesogenic/velogenic sequences of OAVJ within one assay, without the need for RNA isolation, and is adaptable to existing veterinary diagnostic laboratory workflow without additional equipment, this assay could be a rapid primary screening tool before qRT-PCR based validation in resource limited settings.