Matched Samples Research Articles

Iatrogenic emboli during mechanical thrombectomy for acute ischemic stroke: comparison between stent retriever technique and contact aspiration-a retrospective case-control study.

Mechanical thrombectomy (MT) is an effective treatment for acute ischemic stroke from large vessel occlusion (LVO). While embolization to a new territory (ENT) after MT is well-documented, data on embolization in the same distal territory (EDT) are limited. Achieving modified Treatment In Cerebral Infarction (mTICI) 3 reperfusion presents significant clinical benefits over mTICI 2b/2c, necessitating strategies to reduce both ENT and EDT. Previous studies suggest higher rates of EDTs with contact aspiration compared with stentrievers. However, comprehensive comparison studies in clinical practice are scarce. This study compares the rates of overall clot emboli (OCE) between these MT strategies. A retrospective, multicenter observational study was conducted at four university hospitals in France from January 2015 to November 2019. Adult patients (≥18 years) with acute ischemic stroke due to LVO, treated with either contact aspiration (ADAPT, A Direct Aspiration First Pass Technique) or stentrievers, specifically using the Embotrap device to maintain sample homogeneity, were included. Digital subtraction angiography was used for imaging, with two independent, blinded reviewers assessing OCE post-first MT pass. Propensity score full matching and independent sample testing were employed to evaluate OCE after the first MT pass. A significant difference in OCE rates was observed between contact aspiration and stentriever techniques, with the stentriever technique resulting in fewer embolic events compared with ADAPT, based on a propensity score analysis that accounts for key confounding factors. A statistically significant reduction in embolic events was observed with the stentriever technique compared with contact aspiration. These results suggest that the stentriever method may offer a safer profile in terms of embolic risk for LVO interventions, and should be considered over contact aspiration when embolic risk is a primary concern, while also considering individual patient factors.

Identification of single-cell blasts in pediatric acute myeloid leukemia using an autoencoder.

Pediatric acute myeloid leukemia (AML) is an aggressive blood cancer with a poor prognosis and high relapse rate. Current challenges in the identification of immunotherapy targets arise from patient-specific blast immunophenotypes and their change during disease progression. To overcome this, we present a new computational research tool to rapidly identify malignant cells. We generated single-cell flow cytometry profiles of 21 pediatric AML patients with matched samples at diagnosis, remission, and relapse. We coupled a classifier to an autoencoder for anomaly detection and classified malignant blasts with 90% accuracy. Moreover, our method assigns a developmental stage to blasts at the single-cell level, improving current classification approaches based on differentiation of the dominant phenotype. We observed major immunophenotype and developmental stage alterations between diagnosis and relapse. Patients with KMT2A rearrangement had more profound changes in their blast immunophenotypes at relapse compared to patients with other molecular features. Our method provides new insights into the immunophenotypic composition of AML blasts in an unbiased fashion and can help to define immunotherapy targets that might improve personalized AML treatment.

Estimated Plasmodium 18S ribosomal RNA prevalence in asymptomatic blood donors from three African countries.

The World Health Organization (WHO) African Region accounts for 94% of malaria cases globally, with variability recognized within endemic regions. To determine the detection rate of Plasmodium RNA in blood donors resident in malaria-endemic areas, samples from three African countries were tested using a Plasmodium nucleic acid test. Whole blood (WB) samples collected from routine donors in Cameroon, Madagascar and Mali were shipped frozen to the United States. Samples were tested individually from WB lysates with the Procleix Plasmodium assay (transcription-mediated amplification [TMA]). Reactive samples were considered either repeat reactive or initial reactive only, depending on TMA-retest results. When available, matching plasma samples were tested for Plasmodium antibodies by enzyme immunoassay (EIA). Plasmodium repeat reactivity ranged from 41% (91/223 tested) in Cameroon to 12% (26/216) in Mali and 1% (3/249) in Madagascar. Initially reactive samples, where reactivity did not repeat, were identified from Cameroon (5/223; 2%) and Mali (2/216; 1%). The matched-plasma subgroup had EIA reactivity ranging from 86% (113/131 tested) in Cameroon to 59% (10/17) in Mali and 27% (68/248) in Madagascar. Plasmodium ribosomal RNA (rRNA) detection and antibody rates varied greatly in the three countries studied. Detection of Plasmodium rRNA can provide an additional tool to address malaria risk in blood donors.

Gene Expression Changes in CAD Patients Due to Smoking Using Matched Samples

Abstract Background Smoking is a well-known risk factor for coronary artery disease (CAD). However, the effects of smoking on gene expression in the blood of CAD patients in Hungary have not been extensively studied. Aim To identify differentially expressed genes associated with smoking in CAD patients. Methods Eleven matched samples, based on age and gender, were selected for analysis in this study. All patients were non-obese, non-alcoholic, non-diabetic, and non-hypertensive and had moderate to severe stenosis of one or more coronary arteries, confirmed by coronary angiography. Whole blood samples were collected using PAXgene tubes. Next-generation sequencing was employed using the NextSeq 500 system to generate high-throughput sequencing data for transcriptome profiling. The differentially expressed genes were analyzed using the R programming language. Results The median age of patients was 67 years (range: 54-75). RNA sequencing was performed on two groups: smokers and non-smokers. After quality control and filtering, gene expression data were obtained for all samples. Using DESeq2, we identified 279 differentially expressed genes with a p-value ≤ 0.05 and a log2 fold change ≥1. Of these genes, 160 were upregulated in the smokers, and 119 were downregulated compared to non-smokers. Gene ontology analysis revealed that the upregulated genes were enriched for pathways related to immune responses and activities (FDR< 0.03). Specifically, upregulated genes were involved in keratinocyte differentiation, cornification, and epidermis development. The downregulated genes were enriched for cell-cardiac muscle cell adhesion (FDR= 0.004) and epithelium development (FDR= 0.001) pathways. Conclusions This research illuminates smoking’s biological effects, aiding personalized medicine for predicting and treating smoking-related diseases. Key messages • Smoking alters gene expression in CAD patients’ blood, identifying 279 differentially expressed genes, revealing smoking’s biological effects. • The study underscores gene expression profiles’ role in personalized medicine, predicting smoking-related disease risks and tailoring CAD patient treatments.

Novel role of human epicardial adipocyte-derived SPARCL1 in suppression of postoperative atrial fibrillation in patients undergoing cardiovascular surgery

Abstract Background Postoperative atrial fibrillation (POAF) occurs in 20-50% after cardiovascular surgery and is associated with the short and long-term morbidity/mortality. The epicardial adipose tissue (EAT) may have a potential role to regulate the incidence of POAF. Purpose We explored the possible role of human epicardial adipocyte-derived adipokines in the regulation of the myocardial redox state and POAF. Methods We prospectively evaluated 149 patients without known AF who underwent scheduled open-heart cardiovascular surgery between May 2020 and November 2022. They were divided into POAF or Non-POAF group and followed up after discharge (476.6 ± 290.0 days). POAF was defined as AF lasting more than 30 seconds after the surgery. Human EAT samples were obtained from these patients during surgery and the preadipocytes were isolated. Preadipocytes were terminally differentiated to mature adipocytes and mRNAs were extracted. Genome-wide expression profiling of 6 vs. 6 matched samples of the POAF and Non-POAF groups was performed using Microarray analysis, and the results were validated with qPCR in all the cohort samples. The effects of secreted adipokines on the cardiomyocytes were assessed in terms of oxidative stress. Superoxide and whole reactive oxygen species (ROS) were evaluated with luminometry and live-cell fluorescent probes respectively. As the oxidative stress associated signalling, the phosphorylation of ERK and p38-MAPK were evaluated with western blotting. Angiotensin II (Ang-II) was used to induce oxidative stress and phosphorylation of MAPK. Results POAF was observed in 53 patients (36%). Microarray analysis (n = 6) revealed that there were 132 down- or up-regulated cording genes in epicardial adipocytes of the POAF group compared to the Non-POAF group. Of these genes, 11 genes cording secretable molecules were validated by qPCR (n = 69), showing that only SPARCL1 had significantly downregulated in the POAF group compared to the Non-POAF group (P = 0.005). Superoxide and ROS were induced by Ang II (P < 0.01) and attenuated by SPARCL1 dose-dependent manner (P < 0.01) in neonatal rat cardiomyocytes. The phosphorylation of ERK and p38-MAPK were induced by Ang II (P < 0.01) and attenuated by SPARCL1 (P < 0.01) dose-dependently. Co-culture of human induced pluripotent stem cell-derived atrial cardiomyocytes (iPS-ACM) and human epicardial adipocytes revealed that the adipocytes derived from Non-POAF group suppressed the superoxide (P = 0.002) and ROS (P = 0.02) in iPS-ACM compared to POAF group. Kaplan-Meier survival analysis revealed that the POAF group (P = 0.01) and low SPARCL1 expression in epicardial adipocytes (P = 0.018) were associated with a higher incidence of long-term adverse cardiovascular events after surgery. Conclusion: SPARCL1 derived from epicardial adipocytes may play an important role in the suppression of POAF and long-term cardiovascular events via improving the myocardial redox state.

CSR disclosure, financial performance, and ownership: evidence from China

PurposeThis study investigates the relationship between corporate social responsibility (CSR) disclosure and financial performance (FP), and ascertains whether ownership structure (OS) moderates the CSR disclosure–FP nexus.Design/methodology/approachWe distinctly employed the well-established approach of panel data analysis to examine the comprehensive dataset of Shanghai A-share listed firms from 2008 to 2017 with 20,236-full sample and 4,190-disclosed sample firm-year observations. To test the hypotheses, the study used panel regression analysis. The study used CSR disclosure as an explanatory variable and accounting-based performance measures: return on equity (ROE) and earning per share (EPS) as dependent variables. In addition, we used CSR score to determine the extent of disclosure by each firm and employed matched pair sample analysis to check for the robustness of the earlier obtained results.FindingsThe findings indicate significant positive association among CSR disclosure and CSR score with ROE and EPS. Further, the CSR disclosure–FP nexus is more pronounced when the OS moderates it.Research limitations/implicationsThe results of this study lack generalizability due to its unique setting. A limitation of this paper is that our sample period only covers 2008–2017. Future studies can extend our research to a more recent period to test whether our findings remain valid in other periods.Practical implicationsOur findings suggest stronger CSR disclosure measures to enhance the image of businesses in the eyes of stakeholders. The study findings are consistent and confirm the theoretical basis (stakeholder theory) that Chinese listed firms can be more beneficial from disclosing CSR related information, and they should put more emphasis on the improvement of CSR disclosure.Originality/valueThis research offers empirical evidence that sheds light on the importance of OS as the moderating effect on the nexus of CSR disclosure–FP measures. In doing so, this study’s findings contribute to the literature significantly, along with the regulators and shareholders.

Exploring Dual-Targeted Therapy in the Management of Moderate to Severe Inflammatory Bowel Disease: A Retrospective Study

Abstract Background Inflammatory bowel disease (IBD), including ulcerative colitis (UC) and Crohn's disease (CD), often results in significant morbidity among patients with moderate to severe forms. While biologics and small molecules are effective in inducing remission, many patients experience refractory disease or extra-intestinal manifestations. This study assesses the safety and efficacy of dual-targeted therapy in IBD patients treated at the Inflammatory Bowel Disease Center. Methods This retrospective cohort study examined 79 patients with UC or CD who received dual-targeted therapy at the University from October 2018 to August 2023. Data collected included demographics, disease characteristics, previous treatments, and clinical outcomes. Primary outcomes were endoscopic, radiographic, and patient-reported clinical improvements, with secondary outcomes focusing on safety profiles. Results Among the 79 patients (42 UC, 37 CD), 97 dual-targeted therapy cases were analyzed, primarily involving a biologic combined with a JAK inhibitor (90.7%). The median therapy duration was 39.1 weeks. Endoscopic improvement occurred in 69% of matched samples, with significant differences between pre- and post-dual-targeted therapy Mayo scores for UC (p = 0.002) and SES-CD scores for CD (p = 0.018). The median pre- and post-dual-targeted therapy Mayo scores across matched samples were 3 (range 1-3) and 1 (range 0-3) respectively, and for SES-CD scores were 12 (range 0-36) and 4 (range 0-20) respectively. Clinical improvement was reported by 73.2% of patients, with notable reductions in ESR (median 19 [range 2-124] mm/hr to 9 [range 0-116] mm/hr, p = 0.006), CRP (median 8.0 [range 0.2-78.5] mg/L to 3.0 [range 0.2-68.2] mg/L, p <0.001) , and albumin levels (4.0 [range 2.2-4.9] mg/dL to 4.2 [range 3.4-5.2], p < 0.001). Non-obesity was associated with both more endoscopic improvement (p = 0.002) and clinical improvement (p = 0.007). Adverse events occurred in 37 cases, predominantly upper respiratory tract infections and dermatologic issues, with no thromboembolic events reported. Discussion Dual-targeted therapy demonstrated efficacy in improving clinical and endoscopic outcomes in patients with severe, refractory IBD, and exhibited an acceptable safety profile. Despite the promising results, further research is needed to confirm these findings and determine optimal therapy combinations.

Distant Metastases of Breast Cancer Resemble Primary Tumors in Cancer Cell Composition but Differ in Immune Cell Phenotypes.

Breast cancer is the most commonly diagnosed cancer in women, with distant metastasis being the main cause of breast cancer-related deaths. Elucidating the changes in the tumor and immune ecosystems that are associated with metastatic disease is essential to improve understanding and ultimately treatment of metastasis. Here, we developed an in-depth, spatially resolved single-cell atlas of the phenotypic diversity of tumor and immune cells in primary human breast tumors and matched distant metastases, using imaging mass cytometry to analyze a total of 75 unique antibody targets. While the same tumor cell phenotypes were typically present in primary tumors and metastatic sites, suggesting a strong founder effect of the primary tumor, their proportions varied between matched samples. Notably, the metastatic site did not influence tumor phenotype composition, except for the brain. Metastatic sites exhibited a lower number of immune cells overall, but had a higher proportion of myeloid cells as well as exhausted and cytotoxic T cells. Myeloid cells showed distinct tissue-specific compositional signatures and increased presence of potentially matrix remodeling phenotypes in metastatic sites. This analysis of tumor and immune cell phenotypic composition of metastatic breast cancer highlights the heterogeneity of the disease within patients and across distant metastatic sites, indicating myeloid cells as the predominant immune modulators that could potentially be targeted at these sites.

Development and extensive sequencing of a broadly-consented Genome in a Bottle matched tumor-normal pair.

The Genome in a Bottle Consortium (GIAB), hosted by the National Institute of Standards and Technology (NIST), is developing new matched tumor-normal samples, the first to be explicitly consented for public dissemination of genomic data and cell lines. Here, we describe a comprehensive genomic dataset from the first individual, HG008, including DNA from an adherent, epithelial-like pancreatic ductal adenocarcinoma (PDAC) tumor cell line and matched normal cells from duodenal and pancreatic tissues. Data for the tumor-normal matched samples comes from thirteen distinct state-of-the-art whole genome measurement technologies, including high depth short and long-read bulk whole genome sequencing (WGS), single cell WGS, and Hi-C, and karyotyping. These data will be used by the GIAB Consortium to develop matched tumor-normal benchmarks for somatic variant detection. We expect these data to facilitate innovation for whole genome measurement technologies, de novo assembly of tumor and normal genomes, and bioinformatic tools to identify small and structural somatic mutations. This first-of-its-kind broadly consented open-access resource will facilitate further understanding of sequencing methods used for cancer biology.

Prediction of homologous recombination deficiency identifies colorectal tumors sensitive to PARP inhibition

The synthetic lethal effect observed with the use of PARP inhibitors (PARPi) with tumors characterized by the loss of key players in the homologous recombination (HR) pathway, commonly referred to as “BRCAness”, is maintaining high interest in oncology. While BRCAness is a well-established feature in breast, ovarian, prostate, and pancreatic carcinomas, our recent findings indicate that up to 15% of colorectal cancers (CRC) also harbor defects in the HR pathway, presenting promising opportunities for innovative therapeutic strategies in CRC patients. We developed a new tool called HRDirect, which builds upon the HRDetect algorithm and is able to predict HR deficiency (HRD) from reference-free tumor samples. We validated HRDirect using matched breast cancer and CRC patient samples. Subsequently, we assessed its efficacy in predicting response to the PARP inhibitor olaparib by comparing it with two other commercial assays: AmoyDx HRD by Amoy Diagnostics and the TruSight Oncology 500 HRD (TSO500-HRD) panel by Illumina NGS technology. While all three approaches successfully identified the most PARPi-sensitive CRC models, HRDirect demonstrated superior precision in distinguishing resistant models compared to AmoyDX and TSO500-HRD, which exhibited overlapping scores between sensitive and resistant cells. Furthermore, we propose integrating HRDirect scoring with ATM and RAD51C immunohistochemical analysis as part of our “composite biomarker approach” to enhance the identification of HRD tumors, with an immediate translational and clinical impact for CRC personalized treatment.

Comparative targeted lipidomics between serum and cerebrospinal fluid of multiple sclerosis patients shows sex and age-specific differences of endocannabinoids and glucocorticoids

Multiple sclerosis (MS) is a complex chronic neuroinflammatory disease characterized by demyelination leading to neuronal dysfunction and neurodegeneration manifested by various neurological impairments. The endocannabinoid system (ECS) is a lipid signalling network, which plays multiple roles in the central nervous system and the periphery, including synaptic signal transmission and modulation of inflammation. The ECS has been identified as a potential target for the development of novel therapeutic interventions in MS patients. It remains unclear whether ECS-associated metabolites are changed in MS and could serve as biomarkers in blood or cerebrospinal fluid (CSF). In this retrospective study we applied targeted lipidomics to matching CSF and serum samples of 74 MS and 80 non-neuroinflammatory control patients. We found that MS-associated lipidomic changes overall did not coincide between CSF and serum. While glucocorticoids correlated positively, only the endocannabinoid (eCB) 2-arachidonoyl glycerol (2-AG) showed a weak positive correlation (r = 0.3, p < 0.05) between CSF and serum. Peptide endocannabinoids could be quantified for the first time in CSF but did not differ between MS and controls. MS patients showed elevated levels of prostaglandin E2 and steaorylethanolamide in serum, and 2-oleoylglycerol and cortisol in CSF. Sex-specific differences were found in CSF of MS patients showing increased levels of 2-AG and glucocorticoids in males only. Overall, arachidonic acid was elevated in CSF of males. Interestingly, CSF eCBs correlated positively with age only in the control patients due to the increased levels of eCBs in young relapsing-remitting MS patients. Our findings reveal significant discrepancies between CSF and serum, underscoring that measuring eCBs in blood matrices is not optimal for detecting MS-associated changes in the central nervous system. The identified sex and age-specific changes of analytes of the stress axis and ECS specifically in the CSF of MS patients supports the role of the ECS in MS and may be relevant for drug development strategies.

Rapid and effective protocol to measure glutathione peroxidase activity

Abstract Background Intracellular hydroperoxides are harmful reactive oxygen species that can cause damage. Cells depend on antioxidant enzymes such as glutathione peroxidase (GPx) to prevent their accumulation. GPx is present in all cell compartments, including the cytosol, mitochondria, and peroxisomes. This article will present a rapid and reliable method for measuring GPx activity, which has been rigorously tested and proven dependable. Results The enzyme samples are incubated in a phosphate buffer containing specific concentrations of glutathione (GSH) and peroxide and then incubated for a suitable time to react before Ellman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) is added. This method, designed for ease of use, is effective and straightforward. When GSH reacts with DTNB, mixed disulfides (GS-S-DTNB) are formed, where the thiol group replaces one of the nitrobenzoic acid moieties in DTNB. The release of the nitrobenzoic acid group as nitro-2-thiobenzoate anion (TNB−) creates a yellow anion that can be measured spectrophotometrically at a wavelength of 412 nm. The intensity of the yellow color is directly proportional to the unreacted glutathione concentration. GPx activity was linked to decreased absorbance of the TNB− anion, providing a sensitive and relatively specific method for detecting thiol groups in various biological samples. Conclusion This study utilized a Box–Behnken design (BBD) to apply to response surface modeling (RSM) to determine the best conditions for measuring GPx activity. The efficiency of the new protocol was validated by comparing GPx activity in matched samples using the Bland–Altman plot analysis and reference methods. The new protocol was just as accurate as the reference one, with a correlation coefficient of 0.9991. Graphical abstract

Single nucleus DNA sequencing of flow sorted archived frozen and formalin fixed paraffin embedded solid tumors.

Archived samples, including frozen and formalin fixed paraffin embedded (FFPE) tissues, are a vast resource of clinically annotated materials for the application of high-definition genomics to improve patient management and provide a molecular basis for the delivery of personalized cancer therapeutics. Notably, FFPE tissues are stable, provide repeat sampling of tissues of interest, and can be stored indefinitely at ambient temperature. The development of single cell DNA sequencing (scDNA-seq) technologies provides an unparalleled opportunity for the study of tumor heterogeneity and the identification of often rare subclonal cell populations that drive tumor evolution and progression to advanced therapy resistant disease. However, major limitations to the use of archived tissues for scDNA-seq include the low yields of intact cells in the presence of high levels of subcellular debris in biopsies, and the highly variable quantity and quality of the DNA extracted from samples of interest. The latter is of high significance for the use of FFPE tissues due to the presence of DNA-protein crosslinks. In addition, many samples, notably tumors arising in solid tissues, contain admixtures of reactive stroma, inflammatory cells, and necrosis in immediate contact with tumor cells. To expand their use for translational studies, we optimized flow sorting and sequencing of single nuclei from archived fresh frozen (FF) and FFPE tumor tissues. Our methods, which include isolation of intact nuclei suitable for library preparations, quality control (QC) metrics for each step, and a single cell sequencing bioinformatic processing and analysis pipeline, were validated with flow sorted nuclei from matching FF and FFPE ovarian cancer surgical samples and a sequencing panel of 553 amplicons targeting single nucleotide and copy number variants in genes of interest. Our flow sorting based protocol provides intact nuclei suitable for snDNA-seq from archival FF and FFPE tissues. Furthermore, we have developed QC steps that optimize the preparation and selection of samples for deep single cell clonal profiling. Our data processing pipeline captures rare subclones in tumors with highly variable genomes based on variants in genes of interest.

Employment and retirement among workers who develop vision loss in midlife.

Vision loss increases with age and is thus more likely to happen later in one's career. With more individuals working beyond typical retirement age, the possibility of experiencing vision loss while working has increased. The purpose of this study was to investigate how developing vision loss during midlife affects employment and retirement. Using longitudinal Health and Retirement Study data, we identified a sample of 167 workers, 44 to 64 years old, who developed vision loss and a matched comparison sample of 800 workers who did not. We explored job retention and retirement differences between the groups and differences between people with vision loss who retained jobs versus those who did not. Vision loss was associated with leaving the labor force, although there was a clear trend over time of increasing likelihood of job retention. Occupational category was associated with job retention and people who continued working had more favorable financial situations. Retirees with vision loss were more likely to report involuntary retirement and dissatisfaction with retirement. The decreasing likelihood of leaving the labor market after vision loss is an encouraging finding of this study. Workers who left the labor force after vision loss were more likely to be in precarious financial positions, and retirees did not have positive experiences with retirement. Assisting workers with vision loss to remain in the labor force is of vital importance, and increasing awareness and usage of free services for this population may reduce involuntary retirement and its negative consequences.

7701 Comparative Analysis of Dried Blood Spot and Serum ELISAs for AMH, FSH, and LH in Patients Seeking IVF Treatment

Abstract Disclosure: T. Kumar: None. R. Chauhan: None. B. Kalra: None. A.S. Patel: None. S. Chauhan: None. A. Kumar: None. Relevance: Dried Blood Spot (DBS) technology has not been widely implemented in the diagnostic industry due to its limitations in obtaining reliable, quantitative data from small blood samples. However, recent advancements in methods, i.e., sensitive antibodies, homogeneous and reproducible paper quality, and painless needles have renewed the interest in the DBS collection technique. Objective: The aim of the study is to validate the hormone measurements using DBS technology and compare the correlation matrix between AMH, LH, FSH, inhibin B, Antral Follicle Counts (AFC), age, and BMI in patients seeking infertility workup. Materials and Methods: Matched serum and DBS samples from thirty-nine patients between the ages of 18 and 45 years were collected on day 3 of the cycle. The subjects were visiting the clinic for initial fertility workup and subjects on medications for ovarian suppression were excluded. The samples were tested on Ansh Labs DBS AMH (AL-129), FSH (AL-187), and LH (AL-190), PCOCheck AMH (N-N terminus, AL-196), picoAMH (C-N terminus, AL-124-r), US Inhibin B (AL-195) ELISAs. Baseline AFC obtained via transvaginal ultrasound, BMI, and age were collected. Spearman’s correlations, and regression analysis using Passing and Bablok were calculated to examine the relationship between AFC, AMH, inhibin B, FSH LH, age, and BMI. Results: DBS samples were highly correlated to their matched serum specimens. The slope and Spearman’s rank correlation (rs) between matched serum and DBS specimens for AMH, FSH, and LH were 1.17x, 1.07x, 1.07x, and 0.98, 0.93, and 0.97, respectively. DBS AMH strongly correlated with AFC. The Spearman’s rank correlation (rs) between AFC and DBS AMH, DBS LH, and DBS FSH were 0.80, -0.11, and -0.31, respectively. A strong correlation was observed between age and AMH, LH and FSH. A weak correlation was observed between BMI and AMH, LH and FSH. The Spearman’s rank correlation (rs) between age and DBS AMH, LH, and FSH were -0.34, 0.28, and 0.25, respectively. DBS AMH, LH, and FSH correlation with BMI were -0.06, 0.05, and 0.09, respectively. Inhibin B had a strong correlation to age (rs -0.33), BMI (rs -0.67), AFC (rs 0.44), DBS AMH (rs 0.40), DBS FSH (rs -0.19), DBS LH (rs-0.11), respectively. AMH method comparison between N-N and N-C terminal ELISA using 39 serum samples yielded slopes of 0.85x and Spearman’s rank correlation of 0.97. Conclusions: DBS and serum measurements for AMH, LH, and FSH are very accurate and precise. They can be used interchangeably. The strong relationship between BMI and Inhibin B will require further investigation and may open new avenues in obesity and reproductive endocrinology. Presentation: 6/1/2024

B-284 Evaluation of Bupivacaine Metabolite Interference With Norfentanyl in a Liquid Chromatography Tandem Mass Spectrometry Assay in Neonatal Urine

Abstract Background Bupivacaine is a local anesthetic commonly used when administering epidurals during labor and delivery. Bupivacaine metabolite, N-desbutylbupivacaine (NDB), has recently been shown to cause isobaric interference in common urine norfentanyl quantifier and qualifier multiple reaction monitoring (MRM) transitions. This can interfere with the detection of prenatal fentanyl exposure in this vulnerable patient population, which is especially concerning if fentanyl is unexpected. A recent review of urine fentanyl reporting at our institution identified that the number of urine norfentanyl results reported as an interference comment is increasing. A chromatographic review determined this is due to the presence of an unidentified peak co-eluting just before norfentanyl in both the qualifier and quantifier MRM transitions. Most of these samples are from neonates. This study aimed to determine if NDB is the cause of the interfering peak in the urine norfentanyl MRM transitions and evaluate if other specimen types are also affected. Methods NDB standards were prepared and tested using the routine urine fentanyl assay performed by LC-MS/MS in our laboratory, using norfentanyl MRM transitions 233.1 --&amp;gt; 84.1 and 233.1 --&amp;gt; 150.1. Chromatography results were compared to patients with the interference. A retrospective data pull was performed to identify neonatal patients with a urine fentanyl result who also had an umbilical cord or meconium testing requested. Referring centers with patients with the interference comment were contacted to determine neonatal urine collection practices and if bupivacaine was used in their labor and delivery departments. Results NDB produced the same chromatographic pattern observed in the patient samples with norfentanyl interference. This confirmed NDB as the source of the interference. Centers with high rates of NDB interference were contacted and confirmed bupivacaine was routinely administered during labor and delivery at their facilities. Retrospective data analysis identified 577 urine samples with either paired umbilical cord (547) or meconium (30) samples. Of those, 478/577 (82.8%) urine samples displayed NDB interference, and norfentanyl could not be reported due to interference; however, 59 samples were positive for parent fentanyl. Of the 478 urine samples with NDB interference, 455 had a matching cord sample, and 23 had a matching meconium sample. Further examination identified 34/455 (7.5%) cord samples with matching urine samples that had NDB interference were positive for fentanyl. This suggests umbilical cord samples may be useful in some cases to resolve whether a neonate was exposed to fentanyl if NDB interference is observed in urine. Of the 23 paired urine/meconium samples, no positive meconium samples were associated with a urine sample with NDB interference. Conclusions Bupivacaine metabolite is commonly seen in the urine of neonates if bupivacaine is administered as an epidural. Chromatographic separation of bupivacaine metabolite from norfentanyl should be evaluated, especially for centers that receive neonatal samples.

A-132 Next generation ammonia reagent with improved reagent composition and assay performance

Abstract Background Ammonia is produced by catabolism of protein and other compounds which contain nitrogen. Elevated ammonia levels are toxic for the central nervous system. High plasma ammonia is a prognostic factor for hepatic and kidney disease or inherited metabolic disorders in urea cycle metabolism. The reliable and accurate quantification of ammonia is influenced by assay, reagent, and sample management. Current commercially available GLDH enzyme-based ammonia assay reagents experience high bias between matched Lithium Heparin and K2-EDTA samples, poor accuracy, imprecision at the low end, large HIL (hemolysis, icterus and lipemia) interferences and shorter shelf life. To improve upon the listed deficiencies, we developed an improved GLDH based ammonia assay in a two-part format with improved reagent composition. Methods Ammonia reagent in 2-part format R1 and R2 were assessed for accuracy, precision, interference by HIL reportable range, sensitivity (LoB, LoD, LoQ), shelf life, matched matrix comparison (Lithium Heparin vs K2-EDTA) and method comparison. All testing were performed using Li Heparin plasma samples in Beckman DxC700AU analyzer. Accuracy was tested using Verichem ammonia standards which are ACS ammonia standard material. Twenty-day precision studies were performed using three levels of ammonia concentrations using Bio-Rad Liquichek Ammonia controls and plasma samples. Interference was tested in two ammonia concentrations (40-50 µmol/L) and (200-250 µmol/L) using commercially available spiking material for clinical chemistry assays such as conjugated and unconjugated bilirubin, Intralipid and in-house prepared hemolysate. Infinity Ammonia Reagent on Beckman Coulter AU Chemistry Analyzers was used for method comparison studies Results Next generation ammonia showed good accuracy with Verichem ammonia standards and acceptable within-run and total precision. Studies showed significant improvement in sensitivity and longer shelf life (&amp;gt;18 months). Next generation ammonia is less affected by interferences from icterus (100 mg/dL), lipemia (1000 mg/dL intralipid) and hemolysis (100 mg/dL). Comparison studies show no significant bias between match Lithium Heparin and K2-EDTA matched samples. Conclusions Next Generation Ammonia reagent showed very good overall performance required for reliable and accurate quantification of ammonia in human plasma sample.

B-357 Siemens CA 72-4: an assay for measurement of Tumor Associated Glycoprotein (TAG) 72

Abstract Background The tumor associated glycoprotein (TAG) 72, also known as CA 72-4 is a mucin protein of high molecular weight. CA 72-4 is found on the surface of many cancer cells, including stomach (gastric), ovary, breast, colon and pancreatic cells. CA 72-4 is currently one of the primary markers for the diagnosis of gastric cancer and monitoring the course and efficacy of gastric cancer. In ovarian cancer, it has a higher sensitivity for mucin- type ovarian cancer, and its combination with CA125 for detection can significantly improve the clinical sensitivity. In colorectal cancer, CA 72-4 combined with CEA detection was also found to significantly improve the sensitivity of the initial diagnosis. An assay for CA 72-4 on Siemens Atellica IM (Siemens CA 72-4) is being developed and analytical performance is being presented. Methods The Siemens CA 72-4 assay (in development) is a one-step chemiluminescent microparticle immunoassay (CMIA) for the quantitative determination of CA 72-4 in human serum and plasma. The specimen is incubated with paramagnetic microparticles coated with the monoclonal antibody (mAb) B72.3 and acridinium-labeled mAb CC49 conjugate on the Siemens Atellica IM® System. After incubation and washing, Acid and Base reagents are added. The resulting relative light units (RLUs) are directly proportional to the amount of CA 72-4 in the sample allowing for the quantitative determination of CA 72-4 in the specimen. Results The following performance were determined using 3 unique lots of reagents and calibrators with the calibration range for the assay being 0.00 to 200.00 U/mL. The limit of blank (LoB), limit of detection (LoD), and limit of quantitation at 20% CV (LoQ) were determined and met the goal of ≤ 1.00 U/mL. Linearity according to CLSI EP06 Ed2 was demonstrated for a range of 1.00 through 200.00 U/mL. A Precision study of 2 controls and 5 panels spanning the range of the assay demonstrated a total %CV ≤ 6% for samples ≥ 5.00 U/mL and ≤ 0.25 SD for samples &amp;lt;5.00 U/mL. In the sample tube type study, a matrix comparison of 23 matched serum and plasma samples were evaluated. Passing-Bablock slopes of &amp;lt; 8% and r &amp;gt; 0.99 were observed when evaluating samples within the measurement range for plasma (LiHep, LiHep separator, K2EDTA and K3EDTA) tubes and serum separator tubes (SST) compared to the Red Top serum samples. Five endogenous substances were evaluated for interference in the CA 72-4 assay. The average percent difference between test and control samples for all endogenous interferents was &amp;lt; 10%. Percent difference in the presence of 28 potentially interfering drugs was ≤ 4.0%. Conclusions The Siemens CA 72-4 assay currently in development is a sensitive and precise assay for the quantitative determination of CA 72-4 in human serum and plasma.

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

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

B-356 Siemens CYFRA 21-1: an assay for measurement of cytokeratin fragment 21-1

Abstract Background Cytokeratins are structural proteins forming the subunits of epithelial intermediary filaments. The soluble fragment of cytokeratin 19, cytokeratin fragment 21-1 (CYFRA 21-1), can be found in the blood of lung cancer patients, primarily those with non-small cell lung cancer (NSCLC). CYFRA 21-1 is the preferred biomarker in the management of NSCLC, particularly squamous cell and large cell carcinoma subtypes. An assay for CYFRA 21-1 on Siemens ADVIA Centaur® XPT and Siemens Atellica® IM (Siemens CYFRA) is being developed and analytical performance is being presented. Methods The Siemens CYFRA 21-1 assay (in development) is a one-step chemiluminescent microparticle immunoassay (CMIA) for the quantitative determination of CYFRA 21-1 in human serum and plasma. The specimen is incubated with paramagnetic microparticles coated with the monoclonal antibody (mAb) BM19.21 and acridinium-labeled mAb KS19.1 conjugate on the Siemens ADVIA Centaur XPT System and Siemens Atellica IM System. After incubation and washing, Acid and Base reagents are added. The resulting relative light units (RLUs) are directly proportional to the amount of CYFRA 21-1 in the sample allowing for the quantitative determination of CYFRA 21-1 in the specimen. Results The following performance were determined using 3 unique lots of reagents and calibrators with the calibration range for the assay being 0.00 to 100.00 ng/mL. For the ADVIA Centaur XPT and Atellica IM systems were shown to be equivalent within 4%. The limit of blank (LoB), limit of detection (LoD), and limit of quantitation at 20% CV (LoQ) were determined and met the goal of ≤ 1.00 ng/mL. Linearity according to CLSI EP06 Ed2 was demonstrated for a range of 0.66 through 100.00 ng/mL. A Precision study of 2 controls and 5 panels spanning the range of the assay demonstrated a total %CV ≤ 8% at all levels. In the sample tube type study, a matrix comparison of 61 matched serum and plasma samples were evaluated. Passing-Bablock slopes of &amp;lt; 5% and r &amp;gt; 0.99 were observed when evaluating samples within the measurement range for plasma (LiHep, LiHep separator, K2EDTA and K3EDTA) tubes and serum separator tubes (SST) compared to the Red Top serum samples. Six endogenous substances were evaluated for interference in the CYFRA 21-1 assay. The average percent difference between test and control samples for all endogenous interferents was &amp;lt; 10%. Percent difference in the presence of 20 potentially interfering drugs was ≤ 4.0%. Conclusions The Siemens CYFRA 21-1 assay currently in development is a sensitive and precise assay for the quantitative determination of CYFRA 21-1 in human serum and plasma.