Sensitive Assay Research Articles

Clinical performance of a new lateral flow immunoassay for xylazine detection.

Xylazine is a potent sedative used in veterinary medicine. Recently, recreational drugs such as fentanyl havebeen found to contain xylazine, increasing the risk of respiratory depression and death. Despite a similar presentation to opioid overdose, patients who ingest xylazine donot respond to treatment with Narcan. Therefore, rapid detection of xylazine could improve patient management and prevent adverse outcomes. We evaluated the XYL500 one-step xylazine drug of abuse test for its ability to detect xylazine in 152 urine samples from patients on chronic opioid therapy for pain management or in treatment for substance use disorder. Results were compared to LC-MS/MS as the reference method. Precision, cross-reactivity, interference and stability studies were performed. Pooled patient samples were consistently negative or positive when tested five times on the same day and over three days of testing. The diagnostic sensitivity, specificity and accuracy of the XYL500 assay were 74, 98, and 82 % respectively, as compared with LC-MS/MS. XYL500 detected 77 of the 104 LC-MS/MS positive samples identified in our initial evaluation, including some that contained low levels of xylazine (n=8), <10 ng/mL. Minimal cross-reactivity with other opioid analgesics and commonly encountered drugs was seen with only one false positive result. Interferences by common urine contaminants were negligible. Specimens were stable up to 160 days refrigerated and up to 80 days at room temperature. XYL500 allows for rapid detection of xylazine, illustrating its utility in monitoring patients who ingested recreational drugs containing the additive, xylazine, and its potential to improve patient management.

SPECC1 as a pan-cancer biomarker: unraveling its role in drug sensitivity and resistance mechanisms

Previous studies have shown a relationship between SPECC1 and the prognosis of breast cancer, indicating a potential function for SPECC1 in the initiation and progression of cancer. However, the role played by SPECC1 in other tumors is not yet known. Therefore, we used bioinformatics techniques to conduct a thorough investigation into the possible mechanism of SPECC1 in pan-cancer, analyzing data reported in the literature as well as databases such as GTEx and CCLE, cBioportal, TCGA, and UCSC XENA. Comparing the results with matching normal tissues, the majority of cancers, including pancreatic adenocarcinoma (PAAD) and breast invasive carcinoma (BRCA), exhibited higher levels of SPECC1, while hepatocellular carcinoma (HCC) showed lower expression levels. SPECC1 was also found to be genetically mutated in endometrial cancer, sarcoma, and esophageal cancer. The prognosis of lung adenocarcinoma, kidney papillary cell carcinoma, and breast cancer is highly correlated with dysregulation of SPECC1 expression. This work helps guide clinical therapy by highlighting the sensitivity of tumor-treating medicines and the prognostic importance of SPECC1 in various malignancies. KEGG pathway enrichment analysis revealed focused adhesion, collagen-containing extracellular matrix (collagen), and the primary enrichment domains for SPECC1-related genes. These findings were obtained through gene annotation (GO) examination of SPECC1 expression. Primary mediators of the cytokine-cytokine receptor interaction include PICOC1-associated genes, cell-substrate junction genes, and extracellular matrix containing collagen. PICOC1-associated genes primarily mediate the PI3K-AKT signaling pathway. Drug sensitivity assay showed that SPECC1 high-expressing cell lines were more sensitive to docetaxel, doxorubicin, etc. In conclusion, the current study shows how SPECC1 is expressed in different cancers and how this expression relates to the prognosis of the tumor. It also revealed the mutations and copy number variations of SPECC1 in various tumors and its potential involvement in cellular pathway regulatory networks and cytological processes. This study examines the relationship between immune genes, cellular infiltration, and immunological scores in the tumor microenvironment, which explain the severity of the disease. This study looks at the response of SPEC1 expression to anticancer therapy. Explains the prognostic significance and drug response of SPECC-1.

Green synthesis of self-oriented flower-like Ag@Ag2O nanostructures functionalized with L-Tryptophan for colorimetric simultaneous determination of ultra-trace level of thiamin and riboflavin

The study focuses on the green synthesis of Ag@Ag2O nanostructures using Padina algae extract and functionalizing them with L-tryptophan to enhance their properties as a colorimetric sensor for simultaneous detection of ultra-trace levels of thiamin and riboflavin. The nanostructures are characterized using techniques like XRD, FESEM, FTIR, TEM, AFM, and DLS to understand their morphology, structure, and interactions with target molecules. FESEM analysis revealed the hierarchical flower-like Ag@Ag2O nanostructures. The TEM image shows the formation of core-shell nanostructures. Also, DLS analysis and surface zeta potential spectra illustrated the aggregated nature of fabricated nanocomposites in the presence of vitamins. The study is the first to report simultaneous determination of thiamin and riboflavin using a colorimetric sensor based on Ag@Ag2O-L-Try nanocomposites using partial leas square (PLS). The dynamic range of thiamin and riboflavin was achieved in 0.1 mol L−1 acetate buffer pH 4 and the ratio Ag@Ag2O: L-try 1:1. The Ag@Ag2O-L-Try sensor exhibited two linear ranges of 0.1- 1.0 and 3-350 µMol L− 1 for riboflavin and a linear range 3.0–60 µMol L− 1 for thiamin. Also, low detection limit of 1.92 µMol L− 1 and 0.048 µMol L− 1 was obtained for riboflavin and thiamin, respectively. The results indicated that the success of the method depends on the selective and sensitive colorimetric assay of the sensor along with the simultaneous determination by the PLS algorithm. Hence, the proposed technique can be used for the accurate and precise determination of vitamins in different pharmaceutical syrup and tablet samples.

DNA demethylation triggers cell free DNA release in colorectal cancer cells

BackgroundLiquid biopsy based on cell-free DNA (cfDNA) analysis holds significant promise as a minimally invasive approach for the diagnosis, genotyping, and monitoring of solid malignancies. Human tumors release cfDNA in the bloodstream through a combination of events, including cell death, active and passive release. However, the precise mechanisms leading to cfDNA shedding remain to be characterized. Addressing this question in patients is confounded by several factors, such as tumor burden extent, anatomical and vasculature barriers, and release of nucleic acids from normal cells. In this work, we exploited cancer models to dissect basic mechanisms of DNA release.MethodsWe measured cell loss ratio, doubling time, and cfDNA release in the supernatant of a colorectal cancer (CRC) cell line collection (N = 76) representative of the molecular subtypes previously identified in cancer patients. Association analyses between quantitative parameters of cfDNA release, cell proliferation, and molecular features were evaluated. Functional experiments were performed to test the impact of modulating DNA methylation on cfDNA release.ResultsHigher levels of supernatant cfDNA were significantly associated with slower cell cycling and increased cell death. In addition, a higher cfDNA shedding was found in non-CpG Island Methylator Phenotype (CIMP) models. These results indicate a positive correlation between lower methylation and increased cfDNA levels. To explore this further, we exploited methylation microarrays to identify a subset of probes significantly associated with cfDNA shedding and derive a methylation signature capable of discriminating high from low cfDNA releasers. We applied this signature to an independent set of 176 CRC cell lines and patient derived organoids to select 14 models predicted to be low or high releasers. The methylation profile successfully predicted the amount of cfDNA released in the supernatant. At the functional level, genetic ablation of DNA methyl-transferases increased chromatin accessibility and DNA fragmentation, leading to increased cfDNA release in isogenic CRC cell lines. Furthermore, in vitro treatment of five low releaser CRC cells with a demethylating agent was able to induce a significant increase in cfDNA shedding.ConclusionsMethylation status of cancer cell lines contributes to the variability of cfDNA shedding in vitro. Changes in methylation pattern are associated with cfDNA release levels and might be exploited to increase sensitivity of liquid biopsy assays.

On-Site Visualization Assay for Tumor-Associated miRNAs: Using Ru@TiO2 as a Peroxidase-like Nanozyme.

Accurate diagnosis of highly aggressive and deadly tumors is essential for effective treatment and improved patient outcomes, and microRNAs (miRNAs) have emerged as crucial biomarkers for their roles in tumor initiation, progression, and metastasis. Herein, we present an on-site visualization colorimetric assay for tumor-associated miRNAs using ruthenium nanoparticle decorated titanium dioxide nanoribbon (Ru@TiO2) as a peroxidase-like (POD) nanozyme. Remarkably, the Ru@TiO2 nanozyme can catalyze the oxidation of chromogenic substrates through its POD-like activity, which is effectively inhibited by pyrophosphate generated during the rolling circle amplification process, thereby enabling miRNA detection through a visible colorimetric readout. This approach provides a highly sensitive and specificity assay for miRNAs in diluted human serum with a detection limit of 100 pM. It shows great potential for clinical diagnostics and biological research, offering a promising tool for early cancer diagnosis and molecular diagnostics.

7588 Coexistent Toxic Nodular Goiter And Primary Hyperparathyroidism (PHPT), A Combo With Diagnostic And Management Implications

Abstract Disclosure: D. Dave: None. A. Bhan: None. S.D. Rao: None. About 17-84% of patients with PHPT have concomitant thyroid disorders, but the prevalence of coexistent hyperthyroidism in PHPT varies 9-14 %. A vast majority had Graves’ disease with only 3 cases of toxic multinodular goiter (MNG) and PHPT reported in the literature as of 2022 ([1]). We present a rare case of coexistent toxic MNG and PHPT. Case Description: A 68y woman presented with recent 23 kg unintended weight loss and palpitations, but no other symptoms. Denied having kidney stones or fractures. On examination she had fine tremors of hands, MNG without exophthalmos. An ultrasound showed multinodular MNG with cystic lesions. A diagnosis of toxic MNG was made. Laboratory results were: TSH (&amp;lt;0.01 uIU/mL), free T4 1,68 ng/dL (normal: 0.61-1.44 ng/ml), high serum Ca (11.0 - 11.4 mg/dl) over 4y, but PTH was unavailable prior to the visit when it was 107 pg/ml and 89 pg/ml after 3 months of antithyroid therapy. Despite clinical and biochemical euthyroidism after 3 months, serum Ca and PTH levels remained high (11.4 mg/dl and 89 pg/ml respectively). Because she had pulmonary embolism a few years ago, we deferred parathyroidectomy until complete remission of hyperthyroidism. Discussion: Hypercalcemia is the biochemical hallmark of PHPT but can occur in 2-10% of patients with hyperthyroidism alone but only 1% of those will have coexistent PHPT. When these two conditions coexist, hypercalcemia is moderate to severe, and an occasional patient may present with hypercalcemic crisis (2). Prior to the availability of PTH assays, it was a challenge to diagnose coexistent PHPT. Hypercalcemia in hyperthyroidism responds to propranolol but not in PHPT, although the response is variable (3). With the availability of sensitive PTH assays, this maneuver is not necessary. In most cases, current PTH assays distinguish hypercalcemia of hyperthyroidism with suppressed PTH from coexistent PHPT in whom PTH level is non-suppressed or elevated. Monitoring serum Ca and PTH levels during therapy of hyperthyroidism would be useful as normalization of serum Ca and PTH has occasionally been reported (4). In our patient, serum Ca and PTH levels remained high with 3-months of methimazole and atenolol. The impact of both conditions on bone and mineral metabolism are profound, with fractures and kidney stones. Since kidney stones are uncommon in hyperthyroidism, presence of kidney stone in a hyperthyroid hypercalcemic patient should alert the possibility of PHPT. Similarly, significant decrease in bone density in such patient also raises the possibility of PHPT. Prompt diagnosis and appropriate treatment of both conditions is necessary to avoid irreversible complications.

A sensitive immunoassay for the quantitation of Pig-MAP in pig saliva samples

The purpose of the study is to develop a sensitive assay for the proper quantification of the acute phase protein Pig-MAP in pig saliva samples. A time-resolved immunofluorometric assay (TR-IFMA) was developed using two pig-MAP-specific monoclonal antibodies. The limit of detection of the assay was 4 ng/mL, enough to measure pig-MAP concentration in saliva. Precision was evaluated for saliva samples of low, medium and high concentration, with inter assay CV of 4–14 % and inter-assay CV of 8–20 %. The assay kept linearity under dilution and a method comparison study performed with serum samples showed good correlation with ELISA. Median Pig-MAP concentration in saliva from healthy animals was 19 ng/mL whereas in pigs with different inflammatory conditions was 11 times higher. In the same animals median pig-MAP serum concentrations were 0.72 mg/mL in the healthy group and 4.61 in the diseased group. The Spearman coefficient of correlation between Pig-MAP concentration in serum and saliva was of 0.72. A correlation was also observed between the salivary concentration of pig-MAP and other two acute phase proteins such as haptoglobin (r = 0.62) or C-reactive protein (r = 0.65). The concentration of Pig-MAP in saliva of pigs with severe respiratory disease decreased significantly from a median value of 128 ng/mL at the time of disease detection to 8 ng/mL after 1 day of antibiotic therapy. Studies performed show that pig-MAP is present in saliva and this specimen may be an alternative to serum for pig-MAP quantification.

Clinical Pharmacokinetics of Atropine Administered Ocularly Using an Ultrasensitive Bioanalytical Assay.

Purpose: Previous pharmacokinetic studies conducted on atropine sulfate ophthalmical solution have utilized bioanalytical assays that lacked sufficient sensitivity to fully characterize the complete pharmacokinetic profile. To address these limitations, Pharma Medica Research Inc. has developed and validated an ultrasensitive bioanalytical method capable of accurately quantifying the active enantiomer, L-hyoscyamine, with a very low limit of quantitation of 0.500 pg/mL. The objective of this study was to evaluate the pharmacokinetics of L-hyoscyamine in healthy subjects using a highly sensitive bioanalytical assay. Methods: Ten subjects were administered 0.3 mg of Isopto Atropine solution into the conjunctival sac of the eye. Blood samples were taken as early as 2 min and up to 24 h following administration. The plasma samples were assayed for L-hyoscyamine using a chiral method with an analytical range of 0.500-500 pg/mL. The pharmacokinetic parameters were estimated using both a noncompartmental and compartmental approach. Results: The pharmacokinetics of L-hyoscyamine were fully characterized as there were no samples that were below the limit of quantitation following dosing. Using noncompartmental analysis, the mean Cmax was 467.9 ± 159.4 pg/mL with a median (range) Tmax of 0.5 (0.08-1) h. The mean area under the concentration-time curve was 1668.96 ± 436.02 h·pg/mL and the mean half-life was 3.91 ± 1.16 h. Overall, the study drug was well tolerated and no serious adverse events were reported. Conclusion: Through the utilization of a proprietary ultrasensitive bioanalytical method, a comprehensive investigation into the pharmacokinetics of L-hyoscyamine has been successfully conducted. This advanced method offers significant potential for optimizing study designs and facilitating in-depth examinations of the pharmacokinetics of ocularly administered atropine formulations.

Deep Learning-Enhanced Paper-Based Vertical Flow Assay for High-Sensitivity Troponin Detection Using Nanoparticle Amplification.

Successful integration of point-of-care testing (POCT) into clinical settings requires improved assay sensitivity and precision to match laboratory standards. Here, we show how innovations in amplified biosensing, imaging, and data processing, coupled with deep learning, can help improve POCT. To demonstrate the performance of our approach, we present a rapid and cost-effective paper-based high-sensitivity vertical flow assay (hs-VFA) for quantitative measurement of cardiac troponin I (cTnI), a biomarker widely used for measuring acute cardiac damage and assessing cardiovascular risk. The hs-VFA includes a colorimetric paper-based sensor, a portable reader with time-lapse imaging, and computational algorithms for digital assay validation and outlier detection. Operating at the level of a rapid at-home test, the hs-VFA enabled the accurate quantification of cTnI using 50 μL of serum within 15 min per test and achieved a detection limit of 0.2 pg/mL, enabled by gold ion amplification chemistry and time-lapse imaging. It also achieved high precision with a coefficient of variation of <7% and a very large dynamic range, covering cTnI concentrations over 6 orders of magnitude, up to 100 ng/mL, satisfying clinical requirements. In blinded testing, this computational hs-VFA platform accurately quantified cTnI levels in patient samples and showed a strong correlation with the ground truth values obtained by a benchtop clinical analyzer. This nanoparticle amplification-based computational hs-VFA platform can democratize access to high-sensitivity point-of-care diagnostics and provide a cost-effective alternative to laboratory-based biomarker testing.

B-178 A Sensitive LC-MS/MS Assay for Total Testosterone Quantification

Abstract Background Total testosterone immunoassay is a commonly ordered test for the diagnosis of gonadal disorders. However, immunoassays suffer from low specificity and sensitivity, which leads to inaccuracy especially in the low concentration range (&amp;lt; 100 ng/dL). As a result, commercial testosterone immunoassays have difficulties to accurately measure total testosterone levels for women, children, and hypogonadal men. LC-MS/MS has emerged as the method of choice for the accurate quantitation of steroid hormones. We presented here a sensitive laboratory-developed total testosterone assay, which was validated on two LC-MS/MS instruments. Methods An internal standard containing [2,3,4-13C3]-testosterone was added to each sample. The spiked serum sample was loaded on a supported liquid extraction (SLE) cartridge and eluted using methylene chloride. The elute was dried down under nitrogen and reconstituted in sample reconstitution buffer. Chromatographic separation was achieved using a Restek Ultra C18, 50 x 2.1 mm, 3µm analytical column and a total run time of each sample was 5 minutes. The assay was validated on two triple quadrupole mass spectrometers, API 6500 and API 4500. Results were compared with those of a reference laboratory. Results The assay was linear in the range of 1 to 1200 ng/dL for total testosterone and the AMR was set at 2-1200 ng/dL. The interday and intraday imprecision were less than 8%. Split sample comparison to a reference laboratory demonstrated acceptable accuracy with the slope of 1.053 and correlation coefficient (R2) of 0.991. The limit of detection (LOD) was 0.2 ng/dL and the limit of quantitation (LOQ) was 0.5 ng/dL. Results obtained from two instruments agreed well with the slope of 0.996 and the R2 of 0.999. No carryover or interferences was observed for this assay. Conclusions A LC-MS/MS based method was developed and validated for measuring total testosterone levels for female and children. This sensitive and specific assay will improve the diagnosis of hypogonadism and monitoring of testosterone-suppressing treatment.

A-273 Rapid, Portable Invasive Fungal Infection Diagnostic and Candida Antimicrobial Susceptibility Testing

Abstract Background Traumatic wounds are associated with a higher risk of invasive fungal infections (IFI), which lead to a significant increase in amputation or death. Actionable treatment decisions are hampered because true infections can be difficult to differentiate from external wound contaminates, and tissue biopsy and quantification of fungal load is required to determine if treatment is necessary, which currently takes several days. GeneCapture is developing the CAPTURE 60-minute, portable infection identification platform to detect infectious levels of common fungi including the genera Aspergillus, Fusarium, and Scedosporium, the family Herpotrichiellaceae, and the order Mucorales. The company is also pursuing rapid identification and antimicrobial susceptibility testing for Candida species from multiple infection types. Methods This study seeks to determine the Limit of Detection (LoD), sensitivity and specificity of the CAPTURE ID assay for fungal pathogens. Since tissue biopsy samples must be used to assess the presence of IFI in wounds, GeneCapture has developed a manual process for the crude homogenization of muscle tissue to allow this matrix to be used as the starting sample for the ID assay. Mock samples were tested for inclusivity and exclusivity on a series of IFI ID panels against multiple strains of the included fungi, against fungi not yet specifically detected on the panel, and against bacteria that commonly infect wounds. While the LoD for yeasts may be determined in CFU/mL, there is no similar, easy-to-define metric for molds. GeneCapture has developed a quantitative PCR method to detect the single copy beta-tubulin gene to roughly correlate fungal dry mass and nuclei number to assay signal for molds cultured in Potato Dextrose Broth. Determining beta-tubulin copy numbers for extracts from mock samples then allowed detection of the progression of IFI over the course of infection. Lastly, antimicrobial susceptibility testing (AST) was performed on two species of Candida to validate utilizing GeneCapture’s rapid phenotypic AST method for fungal infections. Results To date, the CAPTURE ID panel has been tested against 27 included or excluded strains of fungi or bacteria with 97% sensitivity and 100% specificity. The current LoD for mold organisms varies from approximately 1,000 nuclei for Aspergillus species, to 100,000 nuclei for Fusarium species. Additional work to understand this range is underway. Lastly, we have correctly determined the susceptibility of Candida species using our novel approach within 4-6 hours. Conclusions GeneCapture’s ID and AST technology offer a unique solution to address the increasing prevalence of fungal infections. Our platform can identify the most common fungal components in a wound within 60 minutes of receiving a tissue biopsy sample. Candida infections occur across multiple matrices and rapidly identifying the species of Candida and then determining its phenotypic susceptibility profile will have a large impact on treating these difficult pathogens.

A-002 Analytical Validation of the Zybio High Sensitivity Cardiac Troponin I CLIA Assay

Abstract Background High-sensitivity cardiac troponin (hs-cTn) plays a pivotal role in the early diagnosis of Acute Coronary Syndrome (ACS). With technological advancements, the sensitivity of troponin assays has significantly improved. The International Federation of Clinical Chemistry (IFCC) criteria for hs-cTn assays include a total imprecision (Coefficient of Variation, CV) ≤10% at the gender-specific 99th percentile value and detectable levels in ≥50% of a healthy population. This study aims to evaluate the performance of Zybio's hs-cTnI assay against these high-sensitivity standards Methods This study involved 1661 individuals undergoing routine health checks at Chongqing Medical University Third Hospital. Exclusion criteria included recent medication use, abnormal NT-proBNP levels, significant cardiac silhouette changes in chest X-rays, age &amp;lt;20, incomplete data, pregnancy, or history of severe chronic diseases. Participants were classified into apparent healthy and healthy groups based on blood pressure, lipid profiles, and electrocardiogram results. Laboratory examinations included hs-cTn and NT-ProBNP concentrations using Zybio's chemiluminescence instrument EXI2400 and associated reagent kits, with other parameters measured using standard biochemical and high-performance liquid chromatography methods. Results Of the 1247 participants (556 males, 691 females) included in the final analysis, 1157 showed detectable hs-cTnI levels above the limit of detection (LOD), yielding an overall detection rate of 92.78%. Detection rates were 93.71% in males and 92.04% in females. The total imprecision (CV) of hs-cTnI at gender-specific 99th percentile values over 20 days was below 10%, meeting the high-sensitivity criteria. This finding was consistent across lower and higher concentration ranges. Conclusions The Zybio hs-cTnI assay demonstrated a high detection rate in a healthy population, with 92.78% detectability overall, 93.71% in males, and 92.04% in females. The assay met the high-sensitivity criteria of IFCC, with a total imprecision (CV) of less than 10% at the gender-specific 99th percentile levels. These results validate the utility of Zybio's hs-cTnI assay for clinical application in the early diagnosis of ACS.

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.

B-054 Performance Evaluation of the VITROS HIV Combo Assay on the Ortho VITROS 3600 system

Abstract Background n 2014, the US CDC updated HIV screening algorithm, recommended any assay capable of reliably detecting p24 antigen and both IgG&amp;IgM antibodies as starting point. In 2021 Chinese FDA approved VITROS HIV Combo assay as early detection of acute HIV infection with fourth-generation Ag/Ab combination test, with several declared earlier seroconversion panel reactivity than the third generation assay. This study records the experiment run to assess clinical performance using prospectively collected HIV-1 and HIV-2 antibody positive samples from Chinese CDC on the VITROS HIV Combo Assay on the VITROS 5600 Integrated System, Methods In total 450 serum/plasma samples, including 164 HIV antibody positive (162 HIV-1 antibody positive and 2 HIV-2 antibody positive) and 286 HIV negative. The HIV-2 antibody positive samples were purchased from Seracare (United States), and the remaining samples were prepared by the Chinese CDC reference laboratory. In the samples prepared in the reference laboratory, all HIV-1 antibody positive samples were tested by HIV antibody confirmation test, and the result was positive; The HIV negative samples were confirmed based on a comprehensive assessment of HIV antibodies, HIV-1 p24 antigen, HIV nucleic acid testing, and follow-up testing results, and window period has been ruled out. Ten linear and sensitivity reference samples were added to the HIV-1 p24 antigen national reference material (batch number 20190505, purchased from the China Academy of Food and Drug Control) . The antigen concentrations from L1 to L10 were 20, 10, 5, 2.5, 1.25, 0.625, 0.313, 0.156, 0.0781, and 0 IU/ml, respectively. Results The sensitivity for this positive population of samples in the VITROS Combo test was calculated as 100.00% (164/164) with an exact 95% confidence interval (Cl) of 99.26% to 100.00%. The specificity for this negative population was 99.58% (Cl 99.38%-99.73%). Conclusions The sensitivity and specificity of VITROS Combo assay are very high. With class leading antigen sensitivity doesn’t sacrifice specificity. This new assay delivers excellent confidence in results, and with more target (HIV-1 p24) being detected, can helps laboratory shortening HIV infection window, improve operators and technicians’ confidence in the outcome of HIV testing.

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.

B-358 Siemens NSE: an assay for measurement of human Neuron-Specific Enolase

Abstract Background Neuron-specific enolase (NSE), a glycolytic enzyme (2-phospho-D-Glycerate hydrolase) can be found in a variety of non-neuroendocrine cells. Determination of NSE is used for monitoring response to therapy and detection of recurrent disease, such as SCLC and neuroendocrine tumors. An assay for NSE on Siemens ADVIA Centaur® XPT System and Siemens Atellica® IM system (Siemens NSE) is being developed and analytical performance is being presented. Methods The Siemens NSE assay (in development) is a one-step chemiluminescent microparticle immunoassay (CMIA) for the quantitative determination of NSE in human serum. The specimen is incubated with paramagnetic microparticles coated with the monoclonal antibody (mAb) NSE21, and acridinium-labeled mAb NSE17 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 NSE in the sample allowing for the quantitative determination of NSE in serum. Results The following performance were determined using 3 unique lots of reagents and calibrators with the calibration range for the assay being 0.0 to 400.0 ng/mL. For the ADVIA Centaur XPT and Atellica IM systems were shown to be equivalent within 2%. The limit of blank (LoB), limit of detection (LoD), and limit of quantitation at 20% CV (LoQ) were determined and met the goal of ≤ 1.6 ng/mL. Linearity according to CLSI EP06 Ed2 was demonstrated for a range of 1.6 through 400.0 ng/mL. A Precision study of 2 controls and 6 panels spanning the range of the assay demonstrated a total %CV ≤ 7% at all levels. Comparison of 50 specimens collected in Red Top serum and Serum Separator Tubes (SST) had a Passing-Bablock slope of &amp;lt; 2% and r of &amp;gt; 0.99 when evaluating samples within the measurement range. Five endogenous substances were evaluated for interference in the Siemens NSE assay. The average percent difference between test and control samples for all endogenous interferents was &amp;lt; 10%. Percent difference in the presence of 38 potentially interfering drugs was ≤ 10.0%. The potential cross-reactant αα enolase was evaluated at concentration of 900 ng/mL and the percent cross-reactivity was ≤ 2%. Conclusions The Siemens NSE assay currently in development is a sensitive and precise assay for the quantitative determination of NSE in human serum.

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.

B-090 Efficient Hydrophilic Surface Coating with Low Concentrations of Bovine Gamma Globulins

Abstract Background Bovine Gamma Globulin (BGG) is a high purity immunoglobulin fraction of bovine serum. BGG is used as a blocker to prevent non-specific binding in immunoassays and, due to the mixture of immunoglobulins, its blocking capabilities differ from traditional blockers like Bovine Serum Albumin (BSA). Our objective was to evaluate the adsorption characteristics of BGG to In vitro diagnostic relevant hydrophilic surfaces compared to BSA. A bead-based immunoassay was utilized to quantify the mass of protein coating a bead surface and atomic force microscopy (AFM) to analyze physical properties of the protein-surface layer. Methods The mass of BGG bound to hydrophilic beads (Dynabeads M-270 Carboxylic Acid) was determined across varying concentrations ranging from 0.1% to 1.0% in pH 7.0 phosphate buffer. Coated beads were heated in 1x SDS loading dye to dissociate bound protein, then ran on a nonreducing SDS-PAGE gel. The total protein was measured using densitometry against a standard curve, using ImageJ, and analyzed with GraphPad Prism. This methodology was also used for BSA. AFM was used to image a topographical view of BGG binding to a silica surface. Samples were prepared at concentrations from 0.1% to 1.0% in phosphate buffer at pH 7.0. Followed by a series of washes to remove non-bound molecules, the surface was allowed to dry, then scratched with an AFM cantilever. The surface was then analyzed for roughness and thickness of BGG adsorption for each concentration. Results BGG and BSA have notable differences in their coating properties at a concentration of 1.0%. In the bead assay, the protein-surface binding is enhanced ∼4.8-fold for BGG compared to BSA. Interestingly, even at 0.1% BGG bound ∼0.8-fold more than BSA at 1.0%. Despite a 6-fold increase in mass bound to the bead surface with 1.0% BGG compared to 0.1%, AFM revealed a 1.3-fold and 1.8-fold decrease in height and roughness at the lower concentration, while maintaining effective coating. These results suggest that higher BGG concentrations result in thicker, rougher surface coating. This could lead to steric hindrance and obstruction of binding sites, affecting assay performance. Therefore, a lower concentration of BGG may lead to better surface coating and prevent reduced sensitivity and accuracy within immunoassays. Conclusions The bead binding assay demonstrates that BGG displays surface adsorption with comparable or improved results with lower concentrations compared to BSA. At 0.1%, BGG exhibits effective binding to a hydrophilic surface equivalent to that achieved by BSA at 1.0%. Additionally, AFM data suggest that even at 0.1%, BGG effectively coats the silica surface and potentially reduces steric hinderance compared to BGG at 1%. While BSA remains a reliable choice for many applications, our findings suggest that BGG can be an effective blocker for immunoassay applications. The choice of a suitable blocker varies depending on factors such as assay high background, sensitivity, and specificity. By tailoring the choice of blocker to the specific requirements of the assay, researchers can optimize assay performance by improving the accuracy and the reliability of the assay.

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 &amp;gt; 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

Establishment and characterization of 18 Sarcoma Cell Lines: Unraveling the Molecular Mechanisms of Doxorubicin Resistance in Sarcoma Cell Lines

Sarcomas, malignant tumors from mesenchymal tissues, exhibit poor prognosis despite advancements in treatment modalities such as surgery, radiotherapy, and chemotherapy, with doxorubicin being a cornerstone treatment. Resistance to doxorubicin remains a significant hurdle in therapy optimization. This study aims to dissect the molecular bases of doxorubicin resistance in sarcoma cell lines, which could guide the development of tailored therapeutic strategies. Eighteen sarcoma cell lines from 14 patients were established under ethical approvals and classified into seven subtypes. Molecular, genomic, and transcriptomic analyses included whole-exome sequencing, RNA sequencing, drug sensitivity assays, and pathway enrichment studies to elucidate the resistance mechanisms. Variability in doxorubicin sensitivity was linked to specific genetic alterations, including mutations in TP53 and variations in the copy number of genomic loci like 11q24.2. Transcriptomic profiling divided cell lines into clusters by karyotype complexity, influencing drug responses. Additionally, pathway analyses highlighted the role of signaling pathways like WNT/BETA-CATENIN and HEDGEHOG in doxorubicin-resistant lines. Comprehensive molecular profiling of sarcoma cell lines has revealed complex interplays of genetic and transcriptomic factors dictating doxorubicin resistance, underscoring the need for personalized medicine approaches in sarcoma treatment. Further investigations into these resistance mechanisms could facilitate the development of more effective, customized therapy regimens.