Serum Samples Research Articles

Circulating tumor DNA predicts recurrence and survival in patients with resectable gastric and gastroesophageal junction cancer.

Gastric and gastroesophageal junction (GEJ) cancer represents a significant global health challenge, with high recurrence rates and poor survival outcomes. This study investigates circulating tumor DNA (ctDNA) as a biomarker for assessing recurrence risk in patients with resectable gastric and GEJ adenocarcinomas (AC). Patients with resectable gastric and GEJ AC, undergoing perioperative chemotherapy and surgery, were prospectively enrolled. Serial plasma samples were collected at baseline, after one cycle of chemotherapy, after preoperative chemotherapy, and after surgery. ctDNA was assessed by a ddPCR test (TriMeth), which targets the gastrointestinal cancer-specific methylation patterns of the genes C9orf50, KCNQ5, and CLIP4. ctDNA analysis was performed on 229 plasma samples from 86 patients. At baseline, ctDNA was detected in 56% of patients, which decreased to 37% following one cycle of chemotherapy, 25% after preoperative chemotherapy and 15% after surgical resection. The presence of ctDNA after one cycle of chemotherapy was associated with reduced recurrence-free survival (RFS) (HR = 2.54, 95% confidence interval (CI) 1.33-4.85, p = 0.005) and overall survival (OS) (HR = 2.23, 95% CI 1.07-4.62, p = 0.032). Similarly, ctDNA after surgery was associated with significantly shorter RFS (HR = 6.22, 95% CI 2.39-16.2, p < 0.001) and OS (HR = 6.37, 95% CI 2.10-19.3, p = 0.001). Multivariable regression analysis confirmed ctDNA after surgery as an independent prognostic factor (p < 0.001). ctDNA analysis has the potential to identify patients at elevated risk of recurrence, thus providing personalized treatment strategies for patients with resectable gastric and GEJ cancer. Further validation in larger cohorts and ctDNA-guided interventions are needed for future clinical use.

Comparative study of oxidative stress in cancer patients occupationally exposed to the mixture of pesticides.

Cancer isa leading cause of death worldwide, accounting for nearly 10 million deaths in 2020.Reviews indicated a positive relationship between exposure to pesticides and the development of cancers. In the present study, we have estimated the level of oxidative stress markers in serum samples of pesticide exposure and unexposed cancer patients as compared to normal control. We have found a significant decrease in peroxygenase (PON) and arylesterase (ARE) activity and substantial increases in homocysteine levels in both cancer groups. The level of heme biosynthesis rate-limiting enzymesdelta-aminolevulinic acid dehydratase(δ-ALA-D)also significantly decreased compared to control. The statistical comparison between the cancer groups does not show significant changes. We concluded the involvement of oxidative stress in carcinogenesis in both cancer group patients. However, more study is needed to put homocysteineas a novel marker for a variety of diseases on a single platform.

Predictive value of IL-8 for mortality risk in elderly sepsis patients of emergency department

BackgroundSepsis significantly impacts morbidity and mortality, particularly among older adults. Despite extensive research, early recognition and prognosis prediction of sepsis remain challenging. IL-8, a chemokine produced by inflammatory cells like monocytes and endothelial cells, has shown potential in predicting mortality in sepsis patients, though its role in elderly sepsis remains unexplored. ObjectivesThe present study aimed to explore the predictive ability of interleukin-8 (IL-8) for mortality risk in elderly septic patients. Methods220 elderly sepsis patients were included in the present study. Serum samples were obtained within 1 h of admission to assess serum IL-8, white blood cell (WBC), procalcitonin (PCT), C-reactive protein (CRP), and lactic acid (LAC) levels. The Sequential Organ Failure Score (SOFA) and Acute Physiological and Chronic Health Assessment II (APACHE II) were recorded. Logistic regression analysis was employed to identify independent predictors of mortality within 28 days for elderly patients diagnosed with sepsis. Further, the capacity of these factors to predict 28-day mortality within this patient cohort was evaluated. ResultsSOFA score, APACHE II score, LAC, and IL-8 were all significant independent predictors for 28-day mortality in elderly sepsis patients (P < 0.05). The AUC of the ROC curve for IL-8 was calculated to be 0.701, indicating a moderately predictive performance. In comparison, the AUC for LAC was marginally higher at 0.708. Nevertheless, the results of the statistical analysis revealed no significant difference in the predictive value between IL-8 and LAC. Moreover, the present findings indicate that the combined assessment of IL-8 and SOFA score demonstrated superior predictive value for mortality compared to using IL-8 alone. ConclusionsIL-8 LAC, APACHE II, and SOFA can be considered independent predictors factors for mortality of elderly sepsis patients. Utilizing the combination of IL-8 and SOFA demonstrates a heightened predictive capability compared to using any single index alone.

Noncoding RNAs and diabetic kidney disease.

In both human clinical and animal experimental studies, the altered expression of regulatory RNAs such as miRNAs, lncRNAs, and circRNAs are reported in diabetes and its complications by investigating various samples of serum, plasma, whole blood, and tissues. These identified ncRNAs are candidates for the disease diagnostic markers, prognostic markers, and also therapeutic targets. In the updates, the recently published ncRNAs involved in the onset and progression of DKD are discussed.

Clinical and laboratory peculiarities in postmenopausal patients with benign ovarian tumors

Objective. To assess clinical and laboratory peculiarities of postmenopausal patients with benign ovarian tumors. Materials and methods. The data of a prospective study of concomitant somatic and gynecological pathology in postmenopausal patients with benign ovarian tumors (basic group, n = 40) and patients without pathologies (comparison group, n = 15) are presented. Clinical and laboratory data were evaluated in both groups as well. Results. Postmenopausal patients with benign ovarian tumors were frequently exposed to myomas, chronic salpingitis and chronic cervicitis, when women with no ovarian neoplasms did not have these pathologies. Patients of the main group had higher levels of FSG, leptin, GBP and lower levels of vitamin D, than patients of the comparison group. Conclusions. Postmenopausal patients with benign ovarian tumors frequently have concomitant gynecological pathology, pronounced vitamin D insufficiency and high levels of FSG, leptin and GBP in serum samples.

A Magnetic Molecularly Imprinted Electrochemical Sensor with Selective and Sensitive Detection Ability towards Methotrexate in Serum Samples

A Magnetic Molecularly Imprinted Electrochemical Sensor with Selective and Sensitive Detection Ability towards Methotrexate in Serum Samples

Liquid biopsy for CNS lymphoma: CSF exosomes and CSF exosomal miR-15a, miR-21, miR-155, miR-210, and miR-19b are promising biomarkers for diagnosis.

Central nervous system lymphoma (CNSL) is a devastating disease with a poor prognosis. Early diagnosis, monitoring of the treatment response, and outcome prediction carry the utmost importance in the management of patients with CNSL. Surgical biopsy is the gold standard for tissue diagnosis, however, this procedure has potential complications. Therefore, there is a need for a method that provides information about diagnosis and patient monitoring to avoid surgical risks. The study aimed to investigate potential diagnostic biomarkers for patients with CNSL. Patients with secondary CNSL were included in this study. Serum and cerebrospinal fluid (CSF) samples were collected before treatment and after completion of the treatment. Cell-free DNA (cfDNA), exosomes, free and exosomal microRNA (miR)-15a, miR-21, miR-155, miR-210, and miR-19b in both serum and CSF were examined, and they were compared with the controls. Also, their levels before and after treatment were compared. Nine patients with the diagnosis of secondary CNSL were reviewed. cfDNA, miR-15a, and miR-155 in serum, and exosome in CSF were found to be significantly higher in CNSL patients compared to the controls. Exosomal miR-15a, miR-21, miR-155, miR-210, and miR-19b in CSF were found to be significantly higher in CNSL patients compared to controls, whereas their levels in serum were not significantly high. Our findings suggested that exosomes and exosomal miR-15a, miR-21, miR-155, miR-210 and miR-19b in CSF would be promising biomarkers for the diagnosis of patients with CNSL. Further studies are needed to confirm our findings.

Site-Specific Stability Evaluation of Antibody-Drug Conjugate in Serum Using a Validated Liquid Chromatography-Mass Spectrometry Method.

Antibody-drug conjugate (ADC) consists of engineered antibodies and cytotoxic drugs linked via a chemical linker, and the stability of ADC plays a crucial role in ensuring its safety and efficacy. The stability of ADC is closely related to the conjugation site; however, no method has been developed to assess the stability of different conjugation sites due to the low response of conjugated peptides. In this study, an integrated strategy was developed and validated to assess the stability of different conjugation sites on ADC in serum. Initial identification of the conjugated peptides of the model drug ado-trastuzumab emtansine (T-DM1) was achieved by the proteomic method. Subsequently, a semiquantitative method for conjugated peptides was established in liquid chromatography-hybrid linear ion trap triple quadrupole mass spectrometry (LC-QTRAP-MS/MS) based on the qualitative information. The pretreatment method of the serum sample was optimized to reduce matrix interference. The method was then validated and applied to evaluate the stability of the conjugation sites on T-DM1. The results highlighted differences in stability among the different conjugation sites on T-DM1. This is the first study to assess the stability of different conjugation sites on the ADC in serum, which will be helpful for the design and screening of ADCs in the early stages of development.

Novel Serum Markers that Distinguish Behcet’s Disease from Idiopathic Recurrent Aphthous Stomatitis

ABSTRACT Background Behcet’s disease (BD) is a rare and recurrent autoinflammatory disorder characterized by systemic vasculitis, frequently manifested as recurrent aphthous stomatitis (RAS). We aim to identify specific serum proteins to discriminate between BD and idiopathicRAS. Method Peripheral blood was collected from 12 BD patients, 12 idiopathic RAS patients, and 21 healthy volunteers. The serum samples underwent Tandem Mass Tag-based mass spectrometry analysis. Differentially expressed proteins (DEPs) were identified for KEGG pathway enrichment, Gene Ontology (GO), and protein-protein interaction (PPI) analyses. ELISA was utilized to verify two BD-specific DEPs in another cohort consisting of 18 BD patients, 18 idiopathic RAS patients, and 18 controls. Results Compared with RAS serum, BD serum showed 242 DEPs. 49 proteins were differentially expressed in BD but not RAS serum compared to healthy controls. KEGG pathway and GO analyses revealed that DEPs in BD and RAS have similar biological functions and cellular distributions, featuring a significant association with pathways regulating blood coagulation and immune response. When comparing DEPs between BD and RAS, several keratins emerged as markers that distinguish RAS from BD. We also identified multiple DEPs in BD but not RAS patients. PPI analysis uncovered that lipoprotein metabolism regulators serve as hub proteins, indicating their potentially essential roles in BD pathology. In addition, ELISA results confirmed the elevated LRG1 and SOD3 levels in BD, but not RAS patients, compared to healthy donors. Conclusion Our data uncovered novel serum proteins that distinguish BD from RAS, which may potentially be useful in BD diagnosis and treatment.

Tissue transglutaminase immunoglobulin A exceeds endomysial antibody in specificity of celiac diagnosis at ≥10 times the upper limit of normal.

Serologic diagnosis using tissue transglutaminase immunoglobulin A (TTG-IgA) and endomysial antibody (EMA) is being integrated into the care of pediatric patients with positive screening for celiac disease. The purpose of this study was to assess the utility of EMA in pediatric patients being considered for serologic diagnosis. Patients with TTG-IgA testing performed between May 1, 2022 and April 30, 2023 and with subsequent duodenal biopsy within 6 months were included. TTG-IgA serum samples were frozen and sent for EMA testing and titer. EMA was evaluated for positivity and TTG-IgA (normal <15 u/mL) for elevation <10 times (10x) the upper limit of normal (ULN) and ≥10x ULN (≥150 u/mL). Sensitivity and specificity of EMA and TTG-IgA were calculated using biopsy histology as the gold standard. Four hundred and eighty-six patients were included. The sensitivity and specificity of TTG-IgA ≥15 u/mL was 87.5% and 95.4% while EMA was 77.5% and 97.3%. For patients with TTG-IgA ≥10x ULN the specificity was 99.3%. The positive predictive value of TTG-IgA at ≥10x ULN was 91.4% and for EMA was 83.6%. All three patients with false positive TTG-IgA ≥10x ULN also had false positive EMA, and two of these patients had type 1 diabetes mellitus. TTG-IgA has greater sensitivity at the screening threshold of ≥15 u/mL and greater specificity and positive predictive value at ≥10x ULN than EMA. TTG-IgA at ≥10x ULN is superior to EMA for the serologic diagnosis of celiac disease.

A-129 Evaluation of the Analytical Performance of Magnesium and Calcium General Chemistry Assays on the Atellica CI Analyzer

Abstract Background The Atellica® CI Analyzer is an automated, mid-throughput, integrated chemistry and immunoassay analyzer utilizing both Atellica® CH and Atellica® IM assays. This study was designed to evaluate the analytical performance of the Atellica® CH Magnesium (Mg) and Calcium (Ca), Assays on the Atellica CI Analyzer. Methods The Atellica CI Mg and Ca assays use the same reagents and calibrators as the Atellica CH assays. Precision studies were performed according to CLSI EP05-A3 using serum and urine samples. One aliquot of each sample pool was tested in duplicate in two runs per day &amp;gt;2 hours apart on each analyzer for 20 days. MC studies were performed according to CLSI EP09-A3. Individual human urine and serum samples were analyzed using the Atellica CH and Atellica CI Analyzers. LoB, LoD, LoQ were determined according to CLSI EP17-A2. Results Representative precision and MC results observed across indicated sample ranges are listed for each assay in the table below. Over the two assays tested, repeatability and within-lab %CVs were &amp;lt;4.9%. Overall, slopes determined using the Deming linear regression model were approximately equal to 1. LoB, LoD, LoQ for serum Mg were 0.07 mg/dL (0.03 mmol/L), 0.11 mg/dL (0.05 mmol/L), and 0.50 mg/dL (0.21 mmol/L), and for urine Mg, 0.07 mg/dL (0.03 mmol/L), 0.14 mg/dL (0.06 mmol/L), and 1.00 mg/dL (0.41 mmol/L). LoB and LoD for serum Ca were 0.2 mg/dL (0.05 mmol/L) and 0.3 mg/dL (0.08 mmol/L), and for urine Ca, 0.3 mg/dL (0.08 mmol/L) and 0.5 mg/dL (0.13 mmol/L), respectively. Linearity was demonstrated for both assays. Conclusions Atellica CH Mg and Ca Assays on the Atellica CI Analyzer demonstrated good precision and equivalent performance to the same assays on the Atellica CH Analyzer.

A-017 Clinical Performance Evaluation of the Atellica IM High-Sensitivity Troponin I Assay to Aid in Risk Stratification for All-Cause Mortality and Major Adverse Cardiac Events

Abstract Background Cardiac troponin I (cTnI) is the only troponin isoform present in the myocardium, where it binds to actin and inhibits actin and myosin interaction. Upon myocardial injury, cTnI is released into circulation and serial measurements can be used to aid in the diagnosis of acute myocardial infarction (AMI). In addition, in patients presenting with signs and symptoms of acute coronary syndrome (ACS), elevated cTnI levels provide useful prognostic information relative to short and long-term risk of all-cause mortality (ACM) and major adverse cardiac events (MACE). The objective of this study was to evaluate the clinical performance of the Atellica® IM TNIH assay to aid in risk stratification for ACM/MACE*. Methods Patients from 29 emergency departments (ED) across the United States presenting with signs and symptoms suggestive of ACS were followed for 30-, 90-, and 365-day progression to ACM/MACE (consisting of myocardial infarction, urgent revascularization, cardiac death, or heart failure hospitalization). At each timepoint, the risk (cumulative incidence) and hazard ratios were calculated for populations with baseline cTnI levels ≤ or &amp;gt; the 99th percentile value. Kaplan-Meier curves were generated to display the absolute risk (cumulative incidence) of ACM/MACE outcomes. Analyses were performed for all enrolled patients (entire population) and separately for the entire population excluding those with an adjudicated AMI at presentation. Analyses were performed separately for both serum and plasma (lithium heparin) sample types. Results For the Entire Population cohort, the binary baseline TNIH result (TNIH &amp;gt; overall 99th percentile or ≤ overall 99th percentile) was significantly associated with 30-, 90-, and 365-day cumulative ACM/MACE (p-value&amp;lt;0.0125) for both plasma and serum samples. For plasma samples, risk difference increased from 6.27% at day 30 to 19.62% at day 365. For serum samples, risk difference increased from 6.24% at day 30 to 18.71% at day 365. Cox proportional hazard regression analysis compared the TNIH 99th percentile groups in their hazard of experiencing an ACM/MACE event in the 30-, 90-, and 365-days post ED presentation. For plasma samples, the hazard of ACM/MACE for subjects with cTnI values above the TNIH 99th percentile was relatively constant at each follow-up interval as more than double the rate of subjects below the 99th percentile; hazard ratios ranged between 2.81 (p-value&amp;lt;0.0001) and 3.49-fold (p-value&amp;lt;0.0001) for the three follow up visits. For serum samples, the hazard ratios were between 2.69 (p-value&amp;lt;0.0001) and 3.28-fold (p-value&amp;lt;0.0001) for the three follow up visits. Similar results were obtained for the Entire Population Excluding Subjects with Adjudicated AMI During Initial Presentation cohort. Conclusions The Atellica IM TNIH assay can be used in the detection of myocardial injury and can aid in risk stratification for 30-, 90-, and 365-day all-cause mortality (ACM) and major adverse cardiac events (MACE) in patients presenting with signs and symptoms suggestive of acute coronary syndrome (ACS). *This prognostic claim is not available in the United States.

B-075 Evaluation of patients with monoclonal gammopathy of undetermined significance using Optilite Freelite assays

Abstract Background Monoclonal gammopathy of undetermined significance (MGUS) is a pre-malignant disorder with a progression risk to multiple myeloma of 1% per year. Patients with MGUS may produce monoclonal intact immunoglobulins (Ig) and/or free light chains (FLC). The International Myeloma Working Group (IMWG) recommendations for the use of FLC measurements in MGUS are to establish the diagnosis of light chain only MGUS (LC-MGUS) and further to risk-stratify newly diagnosed MGUS. Once identified, MGUS is longitudinally monitored at intervals dependent on the risk of transformation. The IMWG recommends serum protein electrophoresis (SPEP) for monitoring of known MGUS with intact Ig, however others have published benefits of additionally monitoring with FLC. In this study, we evaluated the diagnostic sensitivity and specificity of the Optilite® Freelite® Kappa Free Kit and the Optilite Freelite Lambda Free Kit (The Binding Site, part of Thermo Fisher Scientific) in patients with MGUS, and the monitoring performance of Freelite results measured in serial samples from patients with clinically stable and progressive MGUS. Methods Serum samples from 234 patients with clinically diagnosed MGUS and 140 disease controls, taken at single time points, and serial samples (≥3) from 49 patients with MGUS were tested with the Freelite assays. Intact M-protein presence and isotype was confirmed in MGUS samples with immunofixation electrophoresis (IFE). In patients clinically diagnosed with LC-MGUS, the presence of detectable M protein on IFE was not required. The disease control cohort (non-MGUS) had disorders commonly associated with elevated polyclonal immunoglobulins (e.g autoimmune, liver, and renal diseases), confirmed by total IgG and/or IgA and/or IgM results and the absence of M-protein by IFE. Patients represented a diverse population from both European and US sources. Results The overall diagnostic sensitivity of the Optilite Freelite assays was 59.4%, calculated as the frequency of abnormal free light chain ratio (rFLC) in intact immunoglobulin MGUS, and abnormal rFLC with concurrent elevated involved FLC level in LC-MGUS. The diagnostic sensitivity was: 58.0% in non-IgM MGUS, 41.7% in IgM MGUS, 66.7% in biclonal MGUS and 100% in LC-MGUS. In intact (IgG/IgA/IgM) kappa MGUS, the diagnostic sensitivity was 67.5%, versus 41.5% in intact (IgG/IgA/IgM) lambda MGUS. The diagnostic specificity was 86.4%. In the 49 patients with serial samples, concentrations of involved FLC were stable in 93.3% of patients with clinically stable MGUS, whereas in patients with clinically progressive MGUS an increasing trend was seen in 50% of cases. Conclusions These results demonstrate that the Optilite Freelite Kappa and Lambda assays produce clinically relevant results that are useful in the evaluation of MGUS.

A-048 Reference Interval and Clinical Performance Studies for Vitros® Immunodiagnostic Products Intact PTH II Assay to Aid in Diagnosis of Calcium and Parathyroid Disorders and Intraoperative Testing

Abstract Background Intact parathyroid hormone (iPTH) measurements aid in the differential diagnosis of hypercalcemia, hypocalcemia, and parathyroid disorders. The National Academy of Clinical Biochemistry has established guidelines for intraoperative PTH testing to monitor surgical success during parathyroidectomies. Studies were conducted to define the reference interval and clinical performance of the Vitros Immunodiagnostic Products Intact PTH II (Vitros iPTH II) assay, using samples from healthy individuals and patients with conditions commonly requiring iPTH testing, including intraoperative cases. Methods The reference interval study was conducted in accordance with Clinical and Laboratory Standards Institute (CLSI) guideline EP28-A3c in serum samples from 134 healthy individuals. For the clinical performance study, serum samples from individuals with hypoparathyroidism, primary hyperparathyroidism, chronic renal failure (secondary or tertiary hyperparathyroidism), hypercalcemia, hypercalcemia of malignancies, and hypocalcemia were tested. Intraoperative samples used were collected from parathyroidectomy patients in series at each surgical timepoint along with the standard plasma samples used to determine surgical success. Results The reference samples were composed of 134 serum donors that met all inclusion/exclusion criteria for healthy donors and had a normal test result on calcium, TSH, alkaline phosphatase, creatinine, and phosphate assays. The reference interval for iPTH was determined to be 14.2-75.2 pg/mL. The results in various disease states demonstrated the clinical performance of the Vitros iPTH II assay. Of 32 intraoperative sample series included in the study, three samples demonstrated no intraoperative decrease in iPTH due to surgical failures. Consistent with the goal of the parathyroidectomy procedure, at least a 50% decrease in iPTH was observed between the presurgical and post-excision samples in all other sample series. The clinical concordance with the Roche PTH assay used intraoperatively in these procedures was 100%. Conclusions Defining the reference interval for a normal healthy population and results from the intended use population demonstrated that the Vitros iPTH II assay can be used as an aid in the diagnosis of calcium and parathyroid disorders and intraoperative testing.

B-167 Quantification of Thyroglobulin in Serum for Clinical Research using SISCAPA Workflow Combined with LC-MS/MS

Abstract Background Accurate measurement of Thyroglobulin (Tg) using existing immunoassay-based techniques can be challenging in the presence of anti-Tg antibodies (TgAb), which can prevent the binding of Tg to assay antibodies thus leading to non-quantifiable Tg concentrations. The Stable Isotope Standards and Capture with Anti-Peptide Antibodies (SISCAPA™) Workflow combined with LC-MS/MS has been successfully employed to circumvent this problem by digesting the serum sample thus eliminating interfering TgAb and measuring a Tg-specific surrogate peptide instead. Methods Using a Andrew+™ Pipetting Robot, denaturation and reduction buffer was added to 250µL serum samples and incubated at 37°C. Internal standard and trypsin were added and samples incubated at 37°C. The samples were quenched with protease inhibitor and Anti-Tg FSP mAb SISCAPA was added to the serum samples and mixed for 60 minutes. Using a magnetic array plate, the beads were left to pull down, samples were discarded, and the beads were twice washed with PBS/CHAPS prior to elution with acetonitrile with formic acid. Using an ACQUITY™ UPLC™ I-Class PLUS FL System, samples were injected onto a Waters™ XSelect™ HSS T3 Column using a water/acetonitrile/formic acid gradient elution profile and the Tg FSP peptide was quantified using a Waters Xevo™ TQ Absolute Mass Spectrometer. Results The method demonstrated no significant carryover or matrix effects and was shown to be linear from 0.1 - 50 ng/mL. Analytical sensitivity investigations indicate the analytical sensitivity of this method would allow precise quantification (&amp;lt;20%) at 0.1 ng/mL with S/N (PtP) &amp;gt;10:1. Coefficients of variation (CV) for total precision and repeatability on 5 analytical runs for low, mid and high QCs were all &amp;lt; 9.5% (n = 25) for both automated and manual precision assessments. Comparison with samples from the UK NEQAS scheme demonstrated significant method bias of -40% for the developed LC-MS/MS method. Re-assignment of the calibrator concentrations using the EQA samples reduced the bias to -5.5%, indicating differences in the calibration materials used for these measurements. Conclusions A LC-MS/MS clinical research method for serum thyroglobulin was developed using SISCAPA, followed by analysis using ACQUITY UPLC I-Class PLUS FL System and the Xevo TQ Absolute Mass Spectrometer. The method provides analytical sensitivity down to 0.1 ng/mL from 250 µL serum, while providing sufficient sample for re-analysis. The method demonstrates excellent linearity across the calibration range, with no significant carryover, interferences, and matrix effects. Total reproducibility and repeatability of the method was ≤9.5% RSD for manual and automated sample preparation, using the Andrew+ Pipetting Robot. In addition, the Andrew+ Pipetting Robot can minimize user touch-time, allowing a full plate to be prepared within four hours without user intervention.

A-313 Use of the Preeclampsia Ratio in the Prognosis of Preterm Delivery and Adverse Outcomes

Abstract Background Preeclampsia (PE) is a hypertensive disorder of pregnancy that is characterized by high blood pressure and end-organ dysfunction with or without proteinuria after 20 weeks gestation. PE occurs in 6-8% of pregnancies and contributes to significant fetal, neonatal, and maternal morbidity and mortality. The ratio of two angiogenic placental proteins, soluble fms-like tyrosine kinase 1 (sFlt-1) and placental growth factor (PlGF), are used in the management of PE. The purpose of this study was to evaluate use of the sFlt-1/PlGF ratio (PE ratio) generated using the ADVIA Centaur® and Atellica® IM sFlt-1 assay and the ADVIA Centaur and Atellica IM PlGF assays in the prognosis of preterm delivery and adverse outcomes in women presenting with signs and symptoms of PE. Methods A total of 654 females with singleton pregnancies between gestational weeks 20+0 days and 35+6 days and who had signs and symptoms of PE, but without severe features of PE were enrolled at 24 collection sites and followed prospectively for two weeks. Serum samples were collected at enrollment and adverse outcomes that included preterm delivery were prospectively collected within the two weeks after enrollment. Serum samples from approximately half of the population (n=316) were tested in singlicate using the sFlt-1 and PlGF assays on the ADVIA Centaur system in a derivation study that determined the optimal cutoff of the PE ratio in relation to adverse outcome. The remaining serum samples (n=338) were subsequently tested on the ADVIA Centaur system in a cut-off validation study that evaluated the clinical performance (sensitivity [SE], specificity [SP], positive predictive value [PPV], negative predictive value [NPV]) of the PE ratio at the derived cutoff in relation to adverse outcome. Method comparison studies were performed between the ADVIA Centaur versus the Roche Elecsys sFlt-1 and PlGF assays (n=255) and the ADVIA Centaur versus the Atellica IM sFlt-1 and PlGF assays (n=316). Results The rate of maternal adverse outcomes within two weeks was similar in the derivation study (32.3%) and validation study (28.1%). Using a receiver operating characteristic (ROC) curve analysis with the derivation cohort, a derived cutoff of 38 showed optimal clinical performance of the PE ratio in relation to adverse outcome (SE=72.5%, SP=94.7%, PPV=84.6%, NPV=89.5%). Similar clinical performance results of the PE ratio at the cutoff of 38 in relation to adverse outcome were found with the verification cohort (SE=88.42%, SP=87.65%, PPV=73.68%, NPV=95.09%). Weighted Deming regression analysis demonstrated equivalency between the Centaur, Roche, and Atellica PE ratios with correlation coefficients (r) of 0.9242 (Centaur vs Roche) and 0.995 (Centaur vs Atellica). Conclusions Measurement of sFlt-1 and PlGF to determine PE ratio can be performed on either the ADVIA Centaur or Atellica Analyzer and can be used to identify women presenting with PE or signs and symptoms of PE who are at high risk of experiencing an adverse event in the subsequent two weeks. Not available for sale in the U.S.A. The products/features mentioned herein are not commercially available in all countries. Their future availability cannot be guaranteed.

B-044 Development and Evaluation of an Assay for the Detection of Human Total IgE in Serum on the Fully Automated Chemiluminescence Immunoassay Platform - Autolumo® 2000 Plus Analyzer

Abstract Background The escalating prevalence of allergic diseases on a global scale, affecting approximately 20% of the world’s population, has become a significant health concern. Clinical determination of total IgE in serum and plasma is used as an aid to the diagnosis of allergic diseases. The tIgE CLIA Microparticles Kit is a chemiluminescent microparticle immunoassay for the quantitative determination of total IgE concentrations in human serum. This kit, integrated with the AutoLumo® 2000 Plus Analyzer, offers an automated, cutting-edge platform for total IgE detection. This streamlined combination provides clinicians with a reliable tool for comprehensive and efficient allergic diseases diagnosis. Methods The performance of tIgE CLIA Microparticles Kit was evaluated on AutoLumo® A2000 Plus automated chemiluminescence analyzer and compared to a well-characterized platform (Elecsys IgE II on Cobas e601, Roche). This assay is based upon the two-step sandwich method. During detection, the patient sample and the anti-IgE coated microparticles solution were added sequentially. Unbound components were washed off after incubation. The formation of a complex occurred with the subsequent addition of a HRP conjugated anti-IgE antibody solution. The catalysis of chemiluminescent substrates by this complex generated a measurable light signal recorded as RLUs (Relative Light Units). A 2-point calibrator was used to analyze the tIgE antibody concentrations in the serum sample. The limit of blank (LoB), limit of detection (LoD), limit of quantitation (LoQ), accuracy, repeatability, linearity, specificity, on-board stability and real-time stability of the assay kit were evaluated. Serum samples (n=220) that had been previously well characterized were used for method comparisons. Results The tIgE CLIA Microparticles assay demonstrated a measurement range of 5-2500 IU/mL, with LoB, LoD, and LoQ values of ≤3 IU/mL, ≤4 IU/mL, and ≤5 IU/mL, respectively. The accuracy of the assay kit was assessed by trueness and precision following CLSI EP15-A2 and EP5-A2 guidelines. The trueness was evaluated using WHO IRP 11/234 as the reference standard, the estimate %bias between the expected and measured mean value was ≤5%. The average %CV of the intra-run and inter-run precision were 5.00% and 3.24% respectively, with a linearity of R≥0.990. The specificity to tIgE is evident, with no cross-reactivity to IgA, IgG, and IgM (≤100 μg/mL), and minimal interference from rheumatoid factor (RF) and anti-nuclear antibodies (ANA) (&amp;lt;10%). Furthermore, based on the CLSI EP7-A2 guideline, it showed no endogenous interference with bilirubin (30 mg/dL), hemoglobin (250 mg/dL), and triglycerides (3000 mg/dL) and no exogenous interferences with 14 commonly used allergy treatment drugs except Omalizumab (360μg/mL). Notably, the assay remains stable on-board for 28 days and exhibits real-time stability for up to 12 months at 2-8ᵒC. Clinical evaluation, involving 220 samples covering the linearity range of 5.0-2500 IU/mL, reveals a strong correlation (R&amp;gt;0.990) and the bias was within 15% when compared to a reference kit according to the CLSI EP9-A2 guideline. Conclusions Autolumo® Analyzers, in conjunction with the tIgE CLIA Microparticles Assay, presents a fully automated chemiluminescence immunoassay platform for accurate and cost-effective screening to aid allergic diseases diagnostics with excellent specificity, precision, and on-board stability.

B-079 Characterization of an isoelectric focusing method for detection of IgG oligoclonal banding in paired cerebrospinal fluid and serum specimens

Abstract Background Qualitative assessment of IgG-specific oligoclonal banding (OCB) in paired serum and cerebrospinal fluid (CSF) samples is useful for evaluating inflammatory conditions of the central nervous system such as multiple sclerosis. Here we characterized the performance of an IgG-specific method using isoelectric focusing on agarose gel matrix for the detection of OCB in paired serum and CSF samples. Methods Specimens of 20 paired, simultaneously collected CSF and serum patient samples were tested on Sebia (Hydrasys 2 System) provided by the National Institutes of Health (NIH) Diagnostic Laboratories. We tested these paired samples using the Helena IgG IFE-20 kit (SPIFE® Touch) method and the results were read by two independent individuals blinded to each other’s assessments. The results were compared to the NIH results for agreement. Positive Percent Agreement (PPA) and Negative Percent Agreement (NPA) were calculated. Analytical sensitivity of the Helena assay was assessed via serial dilutions of a CSF OCB positive sample (total IgG 4.5 mg/dL). Results Figure 1 shows serum and CSF pairs for the control (A), two example positive OCB patients (B &amp; D) and a negative OCB patient (C). The Helena assay was concordant with Sebia for 11/12 positive OCB patients (PPA 91.6%) and 8/8 negative subjects (NPA 100%). 20-fold was the highest dilution that remained positive. Conclusions OCB detection using paired serum and CSF samples and isoelectric focusing electrophoresis is considered the preferred method. The Helena IgG IFE-20 assay is such a method and showed high PPA and NPA concordance with the Sebia System. Thus, the Helena OCB assay can be considered an accurate and analytically sensitive method for assessing OCB in serum and CSF.

Dihydromyricetin ameliorate postmenopausal osteoporosis in ovariectomized mice: Integrative microbiomic and metabolomic analysis

The gut microbiota may help mitigate bone loss linked to postmenopausal osteoporosis by affecting the immune and inflammatory responses and the gut-bone axis. Dihydromyricetin (DMY), a natural flavonoid, has some anti-inflammatory and antioxidant properties. This study aimed to investigate the mechanisms underlying the amelioration of bone loss in ovariectomized (OVX) mice treated with various doses of DMY. Eight-week-old C57/BL6 mice underwent ovariectomy and received varying DMY doses over 8 weeks. Thereafter, femoral bone microarchitecture, serum biomarker levels, and colon samples were analyzed to assess bone metabolism and inflammatory and hormonal responses. Fecal samples were subjected to 16S rDNA sequencing, and short-chain fatty acids were quantified. An untargeted metabolomics approach was applied to both serum and fecal samples to investigate alterations in the intestinal microbiota and metabolic profiles following DMY treatment in the OVX mice. The results show high-dose DMY has anti-osteoporotic effects. Compared to the OVX group, the DMY-treated group showed enhanced bone mineral density and reduced inflammation and colonic damage levels. The DMY treatment altered the gut microbiota composition, including the relative abundances at both the phylum and genus levels. In addition, DMY treatment increased the production of acetate and propionate. Metabolomic analysis revealed differential regulation of 37 and 70 metabolites in the serum and feces samples, respectively, in the DMY-treated group compared to those in the OVX group, affecting the serotonergic signaling, arachidonic acid metabolism, and unsaturated fatty acid biosynthesis pathways. In conclusion, these findings indicate that DMY can ameliorate bone loss in OVX mice via the gut-bone axis.

A-003 Beckman Coulter DxI 9000 Immunoassay Analyzer’s High Sensitivity Troponin I achieves &amp;gt;90% six sigma performance when assessed against clia 2024 performance goals

Abstract Background In July 2024, the Clinical Laboratory Improvement Act (CLIA) proficiency testing (PT) criteria will directly regulate Troponin I performance for the very first time. The new CLIA goal is 0.9 ng/mL or 30%, whichever is greater. CAP previously set a goal of 30% or 3 times the group standard deviation (SD), whichever is greater, a more permissive setting. Estimates of current instrument group performance from an international proficiency testing (PT) survey have shown none of the 5 major diagnostic instruments can achieve the biological minimum goal at a 6-Sigma level, while 4 of the 5 instruments perform at 3 Sigma or below. Performance of these platforms was assessed using the methodology introduced in 2006 by Westgard JO and Westgard SA. The DxI 9000 high sensitivity Troponin I assay was assessed to determine if it could achieve the new CLIA 2024 goals. Methods The DxI 9000 high sensitivity Troponin I assay was assessed with three reagent lots, on both serum and Lithium Heparin (LiHep) samples, following Clinical Laboratory Standards Institute (CLSI) protocols EP05 and EP09 to estimate imprecision and bias. The new CLIA 2024 PT criteria supplied the allowable total error for the standard Sigma-metric calculation: Sigma-metric = (TEa - |bias|) / SD The Sigma-metric predicts not only future problems with PT, but also potential optimization of QC procedures, including fewer Westgard Rules, control levels, even reduced QC frequency which can lead to less cost, time, and materials. Results The majority (91.7%) of data points across the analytical measuring range for DxI 9000’s high sensitivity Troponin I assay from both serum and LiHep samples achieved 6-Sigma performance. For serum, only 8.3% of samples achieved 5-sigma performance. For LiHep, only 4.2% of the performance was 4 Sigma. None of the performance was 3 Sigma or lower. Conclusions The superior precision observed on DxI 9000 high sensitivity Troponin I delivers overwhelming 6-sigma performance when assessed by CLIA’s 2024 goal. This assay is highly unlikely to face PT difficulties and can be optimized for reduced Westgard Rules, reduced control levels leading to a reduction in time, materials, and cost.