Precision Studies Research Articles

B-034 Performance Evaluation of the Total Bile Acids (TBA) Assay on Mindray BS-2800M Analyzer

Abstract Background Bile acids (BA) are synthesized in the liver and secreted into bile. The function of BA is to promote absorption of lipids including fat-soluble vitamins. During liver diseases such as viral hepatitis and liver injury by hepatotoxic drugs, and cholestasis condition, BA secretion to bile is impaired, causing serum BA level to rise. Therefore, the determination of serum BA can be used as a sensitive indicator of liver function. TBA reagent contains NADH that requires pH ≥ 9 to be stable that is usually difficult to be maintained during reagent opened and stored on the analyzer. Meanwhile, 3α-Hydroxysteroid Dehydrogenase (3α-HSD) is required for the reaction. 3α-HSD is sensitive to pH condition. Minor changes in pH results in significant changes of enzyme activity. We formulated our TBA reagent with prolonged stability when reagent is onboard on the analyzer. This study evaluated the performance of the new Mindray TBA assay on Mindray BS-2800M. Methods TBA assay was based on the method with coupled Thio-NAD and NADH reaction for BA. The R1 contains Thio-NAD. The R2 contains NADH and 3α-HSD. R1 is incubated with serum sample containing BA for 5 min at 37°C and R2 is then added. During further incubation in the presence of Thio-NAD, 3α-HSD converts BA to 3-ketosteroids and Thio-NADH. The reaction is reversible, and 3α-HSD can convert 3-keto steroids and Thio-NADH to BA and Thio-NAD. In the presence of excess NADH, enzyme cycling occurs efficiently and the rate of formation of Thio-NADH is determined by measuring specific change of absorbance at 412 nm. Two-point-line calibration is used by this assay. The TBA concentrations for the test samples are obtained by the absorbances of test samples from the calibration line. The performances of precision, linearity, onboard stability, interference (hemolysis, lipid and bilirubin), correlation are evaluated following CLSI protocols. Results The precision study was run according to EP05A3 with two commercial controls (about 21 and 36μmol/L) and two serum sample pools(about 9.9 and 24.2 μmol/L)on the Mindray BS-2800M system. The repeatability and within-lab CVs ranged from 0.74% to 1.97%, respectively. The linearity of the assay was determined as from 2.0 μmol/L to 180 μmol/L. The reagent onboard stability is for 28 days without recalibration that is superior to TBA reagents from other manufacturers. The interference of hemolysis, lipid and bilirubin are with less than ±10% impact with hemolysate Hemoglobin up to 500 mg/dL, Intralipids up to 500 mg/dL, and Conjugated bilirubin up to 30 mg/dL, Unconjugated bilirubin up to 30mg/dL. This assay (Y) also correlated well with SEKISUI TBA assay (x), (Y = 0.9965X + 0.2694, r= 0.9993). Conclusions From this evaluation, we concluded that Mindray TBA assay can measure serum TBA precisely and accurately with good performances, especially for onboard stability. It is suitable for use in routine clinical laboratories.

B-340 Evaluation of the Analytical Performance of Three Major Immunoglobulins Assays on the Atellica CI Analyzer

Abstract Background The Atellica® CI Analyzer is an automated, high-throughput integrated chemistry and immunoassay analyzer utilizing both Atellica® CH and Atellica® IM Assays. This study evaluated the analytical performance of the the Atellica CH Immunoglobulin A_2 (IgA_2), Immunoglobulin G_2 (IgG_2), and Immunoglobulin M_2 (IgM_2) Assays on the Atellica CI Analyzer. Methods Precision studies were performed according to CLSI EP05-A3 using native and contrived human serum samples. One aliquot of each sample was tested in duplicate in two runs per day ≥2 hours apart on each analyzer for ≥20 days. Precision was evaluated using three reagent lots on one system. Method comparison studies were performed using three reagent lots according to CLSI EP09c. Individual native and contrived human serum samples were analyzed using the Atellica CH IgA_2, IgG_2, and IgM_2 Assays on the Atellica CH Analyzer and the Atellica CI Analyzer. LoB and LoD were determined as described in CLSI EP17-A2. Results Representative precision and method comparison results for each assay are listed in the table. For the three assays tested, repeatability and within-lab %CVs were <1.8% and <3.9%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1. Detection capability for the Atellica CH IgA_2 Assay on the Atellica CI Analyzer was estimated at 1.6 mg/dL (0.02 g/L) and 3.6 mg/dL (0.04 g/L) for LoB, and LoD. For the Atellica CH IgG_2 Assay, LoB and LoD were determined at 7 mg/dL (0.07 g/L) and 21 mg/dL (0.21 g/L). For the Atellica CH IgM_2 Assay, LoB and LoD were determined at 5.0 mg/dL (0.05 g/L) and 8.0 mg/dL (0.08 g/L). Conclusions Evaluation of the Atellica CH IgA_2, IgG_2, and IgM_2 Assays using the Atellica CI Analyzer demonstrated good precision and equivalent performance compared to the same assays on the Atellica CH Analyzer.

B-281 Performance Characteristics Evaluation of the ARK Diagnostics Immunoassay for Xylazine/4-HydroxyXylazine in Urine

Abstract Background Xylazine exposure is currently common in some US cities, but routine laboratory testing is not available. We evaluated the ARK Diagnostics immunoassay for qualitative detection of xylazine/4-hydroxy Xylazine in urine, prior to its anticipated release as a Forensic Use Only assay. Evaluation of performance characteristics of the ARK assay were conducted at two university hospitals, using either the semi-quantitative application of the assay, as performed on the Roche Cobas 503 analyzer (Institution A), or the qualitative application of the assay, as performed on the Beckman Coulter AU480 analyzer (Institution B). Methods Xylazine (X) and 4-hydroxy Xylazine (4-OHX) were obtained from Cayman Chemical (Ann Arbor, MI). Pooled xylazine-free urine was spiked either with X or 4-OHX in the range of 0-2000 ng/mL. De-identified, to-be-discarded patient urine samples were obtained from among those submitted for routine drugs-of-abuse panel testing. X measurements on all samples were performed by LC-MS/MS; samples were segregated into X-NEGATIVE (X<10 ng/mL) and X-POSITIVE (X>=10 ng/mL). The ARK assay was conducted according to manufacturer’s instructions, either as a semi-quantitative assay (Institution A, Roche analyzer) or as a qualitative assay (Institution B, Beckman Coulter analyzer), with 10 ng/mL as the manufacturer-specified cutoff for ARK POSITIVE, using manufacturer-supplied calibration standards ranging from 0-500 ng/mL X. Results The ARK semi-quantitative analysis (Roche analyzer) showed essentially equivalent curvilinear response curves for either X or 4-OHX at concentrations below 1000 ng/mL; for the semi-quantitative ARK assay, we use “>1200 ng/mL” for X-ARK >1200 ng/mL. By mixing studies (Roche), there were neither positive nor negative interferences observed as assessed for common drugs of abuse or for common therapeutic drugs. The presence of hemoglobin, bilirubin or moderate lipemia were devoid of assay interference. Precision studies for manufacturer-supplied controls were as follows: intra-assay CVs were 6.9% for the NEGATIVE control (5.1±0.35 ng/mL) and 5.9% for the POSITIVE control (15.7±0.53 ng/mL) (n=20); interassay CVs were 7.8% for the NEGATIVE control (5.1±0.40 ng/mL) and 5.7% for the POSITIVE control (15.9±0.90 ng/mL) (n=20). For the qualitative ARK assay as performed on the Beckman analyzer, POSITIVE and NEGATIVE controls ran invariably according to their designations. For the semi-quantitative assay application (Roche analyzer): among 78 X-NEGATIVE samples by LC-MS/MS, there were 0% ARK-POSITIVE results (false-positive rate=0%); among 74 X-POSITIVE samples by LC-MS/MS, there was one ARK-NEGATIVE result (false-negative rate=1.4%); this sample (X=65 ng/mL) was found to have a high amorphous crystalline content, and was unable to be reanalyzed. For the qualitative assay application (Beckman Coulter analyzer): among 78 X-NEGATIVE samples there was one ARK-POSITIVE sample (false-positive rate=1.3%) for a sample with xylazine=8.8 ng/mL by LC-MS/MS, with undetectable 4-OHX (<10 ng/mL); among 74 X-POSITIVE samples, there were no ARK-NEGATIVE results (false-negative rate=0%). Conclusions The ARK xylazine immunoassay demonstrated acceptable analytical performance with respect to detection of xylazine in urine at 10 ng/mL. With additional consideration of the assay’s cross reactivity with the major xylazine metabolite, 4-OH-xylazine, the assay was judged to be highly suitable for routine use as a screening test for detection of xylazine exposure via urine testing.

A-342 Precision Evaluation of the pH, PO2 and PCO2 tests in the i-STAT® G3+ Cartridge using the i-STAT 1 System for Venous, Arterial and Capillary Whole Blood Specimens in Clinical Settings

Abstract Background Precise and accurate measurements of blood gases are important for the diagnosis, monitoring and treatment of respiratory, metabolic, and acid-base disturbances. This study demonstrates the precision of the pH, partial pressure of oxygen (PO2), and partial pressure of carbon dioxide (PCO2) tests in the i-STAT G3+ cartridge with the i-STAT 1 System in arterial, venous, and capillary whole blood specimens in point-of-care or clinical laboratory settings. Methods The precision of the i-STAT pH, PO2, and PCO2 tests in the i-STAT G3+ cartridge was assessed at external clinical sites by end-users using whole blood specimens across the measurement range for each test: pH (6.500 - 7.800 pH units), PO2 (5 - 700 mmHg), PCO2 (5.0 - 130.0 mmHg). The study included venous whole blood samples from ∼140 adult subjects (≥ 18 years) and arterial whole blood samples from ∼140 adult subjects at 3 clinical sites. Each whole blood sample was run in duplicate on the i-STAT G3+ cartridge. In addition, ∼140 specimens were collected in capillary tubes using two fingerstick draws from adult subjects and ∼20 capillary heel-stick specimens were collected into two capillary tubes from neonate subjects (birth - 28 days) at two clinical sites. Specimens used in this study were prospectively collected venous, arterial, or capillary specimens under an Institutional Review Board approved informed consent or were de-identified leftover venous or arterial specimens. For each specimen type, duplicates from each subject were grouped into subintervals based on their mean values. The subintervals included the medical decision levels of 7.300, 7.350 and 7.450 pH units for pH, 30, 45 and 60 mmHg for PO2 and 35, 45, 50 and 70 mmHg for PCO2. The estimates of the pooled standard deviation (SD) and pooled coefficient of variation (%CV), as well as their respective 95% confidence intervals were calculated for each subinterval. Results Across all specimen types (excluding outliers), the pooled SD for pH ranged from 0.00591 - 0.01747 pH units for subintervals 6.500 - 7.300, >7.300 - 7.450, >7.450 - 7.800 pH units. For PO2, the pooled SD ranged from 0.71 - 3.76 mmHg for subintervals 10 - 40 mmHg and >40 - 50 mmHg and the pooled %CV ranged from 1.63 - 8.76 %CV for subintervals >50 - 100 mmHg, >100 - 250 mmHg and >250 - 700 mmHg. For PCO2, the pooled SD ranged from 0.000 - 1.709 mmHg for subintervals 5.0 - 35.0 mmHg, >35.0 - 50.0 mmHg and >50.0 - 62.5 mmHg and the pooled %CV ranged from 1.10 - 3.62 %CV for subinterval >62.5 - 130.0 mmHg. Conclusions The precision study demonstrated that the pH, PO2 and PCO2 tests in the i-STAT G3+ cartridge when tested using the i-STAT 1 System deliver precise results within ∼2 minutes for venous, arterial, and capillary whole blood patient specimens when tested by end users in a point-of-care or clinical laboratory setting. These studies were funded by Abbott Laboratories.

A-029 Evaluation of the Analytical Performance of Four Assays Used for Diabetes on the Atellica CI Analyzer

Abstract Background The Atellica® CI Analyzer is an automated, high-throughput integrated chemistry and immunoassay analyzer utilizing both Atellica® CH and Atellica® IM Assays. This study evaluated the analytical performance of the Atellica IM C-peptide (CpS) and Insulin (IRI) Assays, and the Atellica CH Enzymatic Hemoglobin A1c (A1c_E) and Fructosamine (Fruc) Assays on the Atellica CI Analyzer. Methods Precision studies were performed according to CLSI EP05-A3 using native and contrived human serum samples, except for the A1c_E assay using native and contrived human whole blood samples. One aliquot of each sample pool was tested in duplicate in two runs per day ≥2 hours apart on each analyzer for ≥20 days. Precision was evaluated using reagent lot and system combination varying between one to three reagent lots and one to three systems depending on the assay tested. Method comparison (MC) studies were performed using three reagent lots according to CLSI EP09c. Individual native and contrived human serum or whole blood (A1c_E) samples were analyzed using the Atellica IM Cps, IRI Assays on the Atellica IM Analyzer, the Atellica CH A1c_E, Fruc Assays on the Atellica CH Analyzer and the four Atellica IM and CH Assays presented on the Atellica CI Analyzer. Results Representative precision and MC results for each assay are listed in the table. For the four assays tested, repeatability and within-lab %CVs were <2.8% and <3.2%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1. Conclusions Evaluation of the Atellica IM Cps, IRI and Atellica CH A1c_E, Fruc Assays using the Atellica CI Analyzer demonstrated good precision and equivalent performance compared to the same assays on their respective Atellica CH and Atellica IM Analyzers.

A-120 Analytical and Clinical Verification of the Elecsys® Alzheimer’s Disease Cerebrospinal Fluid Biomarkers pTau/Abeta42 and Total-Tau/Abeta42

Abstract Background Alzheimer’s disease (AD) is the most common form of dementia characterized by amyloid beta (Abeta) plaques and neurofibrillary tau tangles in the brain. Cerebrospinal fluid (CSF) biomarkers Abeta42, total tau (tTau), and phosphorylated tau (pTau181) offer a high degree of sensitivity and specificity in the diagnosis of AD and have utility in monitoring disease progression. AD biomarker ratios, pTau181/Abeta42 and tTau/Abeta42, correlate with amyloid positron emission tomography (PET) imaging, and several assays have received FDA clearance. This study was designed to evaluate the analytical performance and clinical utility of AD-related Abeta and tau biomarkers on the Roche cobas e601. Methods Precision studies were performed using Roche PreciControl kits. Intra- and inter-precision samples were tested ten times in a single day or twice a day for ten days, respectively. Five levels of Roche Calcheck material were measured in triplicate for linearity. Method comparison was carried out using 39 samples from the Elecsys CSF Correlation Sample Panel from Roche. Qualitative comparisons to amyloid PET Centiloid scores were performed using 35 CSF samples (n=11 PET negative, n=24 PET positive). CSF samples and PET interpretation were obtained from the Cleveland Clinic Institutional Review Board approved Cleveland Alzheimer's Disease Research Center (CADRC) and Cleveland Clinic Lou Ruvo Center for Brain Health Aging and Neurodegenerative Disease Biobank (LRCBH-Biobank). Results Conclusions Evaluation of AD biomarkers on the Roche cobas e601 revealed reliable precision and analytical accuracy. The pTau181/Abeta42 and tTau/Abeta42 ratios demonstrated excellent positive agreement when compared to amyloid PET, but poor negative agreement. This may be due to the small sample size of PET-negative patients. Alternatively, as amyloid PET detects neuritic plaque accumulation, the CSF biomarker-positive/PET-negative patients may have early non-neuritic amyloid plaque pathology not detectable by imaging. Overall, this assay is suitable for use and may serve as a valuable tool in AD diagnosis.

A-166 Performance verification of a polyclonal anti-PF4 immunoassay in evaluation of Heparin Induced Thrombocytopenia (HIT)

Abstract Background Heparin-induced thrombocytopenia (HIT) is a severe complication of exposure to heparin that occurs in a small percentage of patients in anticoagulation therapy. HIT could happen regardless of the dose, exposure, schedule, or route of administration. HIT results from an autoantibody directed against endogenous platelet factor 4 (PF4) that forms complex with heparin. This antibody activates platelets and can cause life threatening arterial and venous thrombosis. Untreated HIT has a mortality rate as high as 20%. However, with appropriate screening, diagnosis and early intervention, mortality rates have been reported to be below 2%. The laboratory evidence in evaluating patients for HIT is crucial for early intervention. There are several assays available to diagnose Heparin Induced Thrombocytopenia (i.e., immunoassay and/or functional assay for HIT antibodies; serotonin release assays). The purpose of this study is to establish the measurement criteria and verify performance characteristics of a polyclonal anti-PF4 immunoassay. The test of interest (anti-PF4 immunoassay) is intended for the qualitative detection of the anti-heparin-platelet factor 4 antibodies in plasma or serum by ELISA. Methods The ASSERACHROM Assay (Diagnostica Stago, Inc. USA) was implemented in the automated Dynex DS2 ELISA Processing System (Dynex Technologies Inc USA). Two Dynex DS2 instrument comparisons along with precision, accuracy and measurement range for qualitative positive and negative results were studied. The reference method comparisons with serotonin release assay and other monoclonal anti-PF4 immunoassays were performed. To evaluate performance of the assay, we conducted precision, accuracy, and qualitative method comparison. We compared the assay of interest with two reference lab immunoassays and the gold standard reference method (serotonin release assay). The statistical analysis was carried out in EP Evaluator and GraphPad Prism. Results The negative control precision demonstrated CV of 9.2% (N=20, mean= 0.11, SD= 0.01). The analyte measurement is expressed as optical density (OD). The positive control precision characteristics showed CV of 8.1% (N=20, mean= 1.62, SD= 0.13). The threshold was defined as reference reagent absorbance (R6OD) times the variance factor divided by 100 ((R6OD* variance factor)/100= threshold (X)). This is used for positive (>the X) and negative (< the X) absorbance cut off for patient samples. The R6 reference material precision study resulted in a CV of 7.95% (N=20, mean= 2.1, SD= 0.17). The R6 variance factor per precision statistics at 3SD level was 23.85%. This is comparable to peer group variance provided in the assay package insert (23.5%). The polyclonal anti-PF4 immunoassay versus a reference monoclonal method (N=40) demonstrated agreement of 90.9% with positive agreement of 100% and negative agreement of 89.5%. When compared with gold standard serotonin release assay (N=40), the assay demonstrated agreement of 75% with positive agreement of 90% and negative agreement of 60%. The two instruments demonstrated agreement of 97.5% with positive agreement of 100% and negative agreement of 93.3% (Cohen's Kappa 94.6%; with Kappa > 75% indicates “high” agreement). Conclusions This anti-PF4 immunoassay is precise, accurate and comparable with three different reference methods. The test is best utilized when 4T score based clinical algorithm is used for evaluation of HIT.

A-128 Evaluation of the Analytical Performance of Three Inflammation Related Assays on the Atellica CI Analyzer

Abstract Background The Atellica® CI Analyzer is an automated, high-throughput integrated chemistry and immunoassay analyzer utilizing both Atellica® CH and Atellica® IM Assays. This study evaluated the analytical performance of the the Atellica CH Complement C3 (C3), Complement C4 (C4), and Alpha-1-acid Glycoprotein (AAG) Assays on the Atellica CI Analyzer. Methods Precision studies were performed according to CLSI EP05-A3 using native and contrived human serum samples. One aliquot of each sample was tested in duplicate in two runs per day ≥2 hours apart on each analyzer for ≥20 days. Precision was evaluated using three reagent lots on one system. Method comparison (MC) studies were performed using three reagent lots according to CLSI EP09c. Individual native and contrived human serum samples were analyzed using the Atellica CH C3, C4, and AAG Assays on the Atellica CH Analyzer and the Atellica CI Analyzer. LoB and LoD were determined as described in CLSI EP17-A2. Results Representative precision and MC results for each assay are listed in the table. For the three assays tested, repeatability and within-lab %CVs were <1.4% and <3.3%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1. Detection capability for the Atellica CH C3 Assay on the Atellica CI Analyzer was estimated at 0.2 mg/dL (0.00 g/L) and 0.4 mg/dL (0.00 g/L) for LoB, and LoD. For the Atellica CH C4 Assay, LoB and LoD were determined at 0.1 mg/dL (0.00 g/L) and 0.2 mg/dL (0.00 g/L). For the Atellica CH AAG Assay, LoB and LoD were determined at 0.9 mg/dL (0.01 g/L) and 2.0 mg/dL (0.02 g/L). Conclusions Evaluation of the Atellica CH C3, C4, and AAG Assays using the Atellica CI Analyzer demonstrated good precision and equivalent performance compared to the same assays on the Atellica CH Analyzer.

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 <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 < 8% and r > 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 < 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-237 Analytical Performance Evaluation of Cytomegalovirus IgG and Syphilis Assays on the Atellica CI Analyzer

Abstract Background The Atellica® CI Analyzer is an automated, high-throughput integrated chemistry and immunoassay analyzer utilizing both Atellica® CH and Atellica® IM Assays. This study evaluated the analytical performance of the Atellica IM Cytomegalovirus IgG (CMV IgG) and Syphilis (Syph) Assays on the Atellica CI Analyzer. Methods Precision studies were performed according to CLSI EP05-A3 using native and contrived human serum samples. CMV IgG and Syph Assays were evaluated with one reagent lot on two Atellica CI analyzers. One aliquot of each sample was tested in duplicate in two runs per day ≥2 hours apart on each analyzer for ≥20 days. Method comparison studies were performed according to CLSI EP12-A2. Individual native human serum samples were analyzed using the Atellica IM CMV IgG and Syph Assays on the Atellica IM and Atellica CI Analyzers. The results were assessed based on Index values distinguishing reactive (Index ≥cut-off value) and nonreactive (Index <cut-off value) specimens. Results As shown in table below, repeatability and within-lab %CVs for the two assays presented were <3.7% and <7.0%. Negative and positive agreement were 100% for the 111 nonreactive CMV IgG samples and the 169 CMV IgG reactive samples tested. Negative and positive agreement were 100% for the 103 Syph reactive samples and the 126 nonreactive Syph samples tested. Overall clinical agreement between each of the presented assays on the Atellica CI Analyzer and Atellica IM Analyzer was 100%. Conclusions Evaluation of the Atellica IM CMV IgG and Syph Assays using the Atellica CI Analyzer demonstrated acceptable precision and equivalent performance compared to the same assays on the Atellica IM Analyzer.

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 (>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.

A-334 Vital one: a point-of-care platform for rapid, comprehensive, central-lab quality blood testing

Abstract Background Diagnostic tools are fundamental to informed healthcare decisions, but the current state of in vitro diagnostics presents barriers to providing timely access to a wide menu of tests. This poster discusses a method for overcoming these challenges with a novel and miniaturized point-of-care (PoC) solution, the VitalOneTM. While many PoC solutions have limited test menus tailored for specific scenarios, Vital Biosciences introduces a comprehensive test menu in a compact PoC format that uses centrifugal microfluidic workflows to deliver quantitative results for over 50 analytes spanning hematology, clinical chemistry, and immunoassay modalities, ensuring central-lab quality results for a wide variety of use cases in a 20-min time frame (or 15 min for a Comprehensive Metabolic Panel (CMP) lipid panel, and liver panel). The VitalOneTM is designed to perform a complete blood count (CBC), a comprehensive panel with a full array of immunoassays and clinical chemistry assays, returning over 50 results with as little as 300-750 µL of whole blood. This poster provides a comprehensive and transparent view of both our results and underlying methods of operation. By comparing the VitalOneTM system with established benchmarks like Roche, Sysmex, and Beckman Coulter instrument systems, we demonstrate consistent assay performance, resilience to interference, and performance that is compliant with CLIA Total Allowable Error standards. Methods A method comparison analysis of measurements on VitalOneTM platform was performed against predicate instruments that are commonly used in centralized laboratories (Roche Cobas, Beckman AU480, Beckman Coulter DxH500, Beckman Access 2, and Sysmex 140). Additionally, a 5-8 point calibration curve was generated for each analyte across the desired assay measuring range (AMR) using commercially available validation kits on three separate days, resulting in a master calibration curve. To check for sensitivity to potential interferences that may be present in patient samples, endogenous and exogenous interference testing was performed following Clinical & Laboratory Standards Institute (CLSI) guidelines. Lastly, robustness testing was carried out to mimic the extremes of variances in volumes, times, or temperatures that may be encountered when running an assay protocol. Results The method comparison analysis (n = 840 samples) and precision studies revealed that the VitalOne exhibited a strong correlation with the benchmark instruments and were in compliance with CLIA's total allowable error limits, reinforcing the reliability and clinical relevance of our diagnostic system. Assays across Hematology, Clinical Chemistry, and immunoassays demonstrated >95% of data within CLIA TEa% and run-to-run imprecision ≤ 5%. Additionally, linearity studies revealed that each analyte AMR spans across physiologically relevant concentrations, defined by medical decision levels and reference intervals for each analyte. Our robustness and endogenous and exogenous interference testing performed as per CLSI guidelines has demonstrated that the VitalOneTM system closely matches, and in certain parameters, such as biotin interference, even surpasses that of the predicate instruments. Conclusions In summary, the VitalOneTM platform represents a significant advancement in point-of-care diagnostics, offering a comprehensive, accurate, and efficient alternative to traditional laboratory testing. This innovation is poised to transform the way clinicians approach diagnostics, enabling more informed decision-making and ultimately enhancing patient outcomes.

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 < 2% and r of > 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 < 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 < 5% and r > 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 < 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.

A-131 An External Laboratory Validation of the Atellica IM Serum Neurofilament Light Chain Assay

Abstract Background Neurofilament light chain (NfL) in serum is a promising biomarker for axonal injury with potential for use in neurodegenerative disease testing, including multiple sclerosis (MS), Alzheimer’s disease, and amyotrophic lateral sclerosis (ALS). This study validated the Siemens Healthineers Atellica® IM Serum Neurofilament Light Chain (sNfL) assay* for research applications. Methods Precision studies were performed using serum and plasma samples. Intra-assay precision comprised three levels of internal assay quality control (QC) and one plasma or serum pool (consisting of serum spiked with CSF). For plasma, one aliquot of each sample was tested in duplicate in two runs per day >2 hours apart for up to >20 days. Serum inter-assay involved three levels of internal assay QC run over 10 days (1 run/day). Sensitivity studies were performed to evaluate the limit of quantitation (LoQ) for serum and plasma using five low concentration samples. Each concentration was tested in 25 replicates for serum, and 50 replicates for plasma. Linearity studies involved five levels, each run three times in duplicate (plasma) or four times (serum). Method comparison studies were performed according to CLSI EP09-A3 using the Atellica® IM NfL laboratory developed test (LDT) offered by Siemens Healthcare Laboratory, LLC. 50 samples within the measuring interval of the LDT for each sample type (plasma and serum) were tested. Results Intra-assay coefficients of variation (%CVs) for internal QC ranged from 1.7-2.5% for concentrations spanning 12.75 to 147.22 pg/mL. Serum and plasma pool intra-assay %CV were 2.5% and 3.4%, respectively. Inter-assay precision of serum %CVs ranged from 2.9-16.1% for 13.4 to 150.8 pg/mL. Plasma inter-assay %CVs demonstrated a slightly improved precision versus serum across runs with %CV ranging from 2.4-12.8% with plasma concentrations ranging from 13.4 to 158.0 pg/mL. Serum and plasma LoQs, defined as the concentration at which CV is <20%, were 2.5 pg/mL (serum/plasma). The assay was linear between 2.5 to 300 pg/mL for serum and plasma. The Atellica IM sNfL assay method comparison to the Atellica IM NfL LDT was analyzed using a Passing-Bablok linear regression model. The assays exhibited acceptable agreement with slopes in the range of 1.12-1.14 across the sample interval tested, 6.4 to 460 pg/mL for serum/plasma. Conclusions This external validation of the Atellica IM sNfL assay demonstrated performance characteristics suitable for research applications. *For Research Use Only. Not for use in diagnostic procedures.

Incidence and Prevalence of Edema and the Effect of Aprocitentan Treatment Strategy in the PRECISION Study

Incidence and Prevalence of Edema and the Effect of Aprocitentan Treatment Strategy in the PRECISION Study

Efficient Method for Liposome-Based Gene Transfer in Cattle Embryos.

In vitro embryo production (IVP) in cattle is crucial for advancing genetic enhancement and preserving valuable genetic lineages, enabling precise genetic modifications and gene studies through modern techniques. Successful genetic manipulation in cattle embryos requires efficient delivery of exogenous DNA/RNA molecules. This research investigates the efficacy of a single embryo culture system for developing genetically modified zona-free (ZF) embryos and examines the use of liposome-based SAMTOR target siRNA transfer in these individually cultured ZF embryos. The findings indicated that the individual culture system resulted in increased cleavage rates, and blastocyst rates were minimally impacted. The new culture system effectively achieved SAMTOR silencing, with 8-16 cell embryos exhibiting reduction in transcript levels compared to control. Measurement of total protein content in the spent culture media was performed to validate the single-culture approach for further analytical applications. Total protein content analysis demonstrated the system's suitability for comprehensive evaluation of the embryo-media interaction, enhancing the scope for in-depth genetic research and applications. This research sheds light into an innovative method to improve genetic editing techniques in reproduction research.

Establishment and Validation of a Robust Reversed-Phase HPLC Method for the Determination of Calotropis gigantea in Bulk Material and Marketed Product

Objective: The objective of the present work is to develop and validate a novel, specific, precise, and reliable method for the estimation of Calotropis gigantea in bulk and herbal dosage form using the RP-HPLC method. Methods: RP-HPLC analysis was performed using a C18 column of dimension 150×4.6mm, 5 μ. The chromatography system comprised an Agilent 1220 Infinity II LC equipped with a VWD detector and a 1220 Infinity II LC binary pump, wherein the instrument operation was managed through Control Panel software at a flow rate of 0.5 ml/min. Methanol: water in the ratio of 55:45 was used as the mobile phase, and the effluents were analyzed at 275nm. The proposed method was validated for various parameters like linearity, precision, accuracy, robustness, ruggedness, selectivity, limit of detection, limit of quantification, and assay as per the ICH Q2(R1) guidelines. Results: Linearity was noted over a concentration range of 50-250 μg/ml with a correlation coefficient of 0.999. The limit of detection (LOD) and limit of quantification (LOQ) were determined to be 16.02 and 48.56 μg/mL, respectively. The % RSD for interday and intraday precision studies was less than 2%, which was within the official RSD limit. Recovery analysis performed using marketed formulation was found to be in the range of 97-105%. Conclusion: The method developed was validated according to the ICH guidelines. Hence, it is evident that the developed method is novel, sensitive, precise, and reliable, and it can be successfully applied to estimate Calotropis gigantea in bulk material and its herbal dosage form.

Head Space GC-MS/MS Method for Quantification of Five Nitrosoamine- Genotoxic Impurities in Metformin HCl

Background: N-nitrosamines have recently been discovered in metformin hydrochloride and other generic drugs. To quantify the five N-nitrosamines in metformin hydrochloride, we devised sensitive and reliable multiple reactions monitoring mode-based GCMS/ MS technique, particularly, N-nitrosodiethy amine (NDEA), N-nitroso ethyl isopropylamine (NEIPA), N-nitrosodiisopropylamine (NDIPA), N-nitrosodipropylamine (NDPA), as well as N-nitrosodibutylamine (NDBA). Objective: To develop a sensitive, precise, and accurate MRM mode based GC-MS/MS method for the quantification of five N-nitrosamines in metformin hydrochloride and valídate as per ICH guidelines. Methods: The settings for mass spectrometry and gas chromatography were optimized. With the linearity, sensitivity, specificity, accuracy, and precision of the parameter, the procedure was as per the recommendation of ICH: International Council for Harmonization guidelines. Results: N-nitrosamines in metformin hydrochloride had detection and quantification limits of 0.001 ppm and 0.004 ppm, correspondingly. The obtained results were within the sensitivity limitations issued by the US Food and Drug Administration. The calibration curve's regression coefficients for five N-nitrosamines were over 0.99, demonstrating the process's good linearity. The retrievals of N-nitrosamines in metformin hydrochloride between 97.1 – 127.4 %. The RSD (Relative Standard Deviation) was lower than 10 % for both inter-day and intra-day precision studies. Conclusion: The proposed method exhibited a rapid analysis capability, high accuracy, sensitivity, and precision, making it a trustworthy method for monitoring N-nitrosamines in metformin hydrochloride.

Precision studies in charm and new physics searches at BESIII

The investigation of charm physics plays an important role in checking the Standard Model’s description of strong and weak interactions, validating its predictions for particle physics, and exploring new physics beyond the Standard Model. Using a large dataset from [Formula: see text] annihilation, BESIII has conducted comprehensive and extensive studies in the areas of hadronic, leptonic and semileptonic decays. This proceeding presents an overview of the recent researches and discoveries by BESIII on decays of charmed baryons, charmed mesons and charmonium states, highlighting their significance in advancing the understanding of new physics, experimental determination of physical parameters and improvement of measurement precision.