Limit Of Quantitation Research Articles

6703 Age-Related Reference Intervals for 11-Oxygenated Androgens in Children

Abstract Disclosure: L. IJsselstijn: None. R. Zellerer: None. R. Oude Engberink: None. B. van Zelst: None. S.A. van den Berg: None. Context: 11-Oxygenated androgens have emerged as major components of several disorders of androgen excess. Levels of 11-keto androstenedione (11KA4), 11β-hydroxy androstenedione (11OHA4), 11-keto testosterone (11KT) and 11β-hydroxy testosterone (11OHT) have been mainly studied in adults and in relation to various disorders. Although it is known that 11-oxygenated androgens are pivotal in adrenarche and pubarche, well established reference intervals for children are lacking. Objective: To establish age-related reference intervals for 11-oxygenated androgens in children. Methods: Serum samples were collected from apparently healthy children with ages ranged 0-18 years (n=186, 108 boys and 78 girls). Concentrations of 11KA4, 11OHA4, 11KT and 11OHT were determined by isotope dilution mass spectrometry. Before measurement the serum was deproteinized and purified by solid-phase extraction. The lower limit of quantitation for 11KA4, 11KT and 11OHT were 3.0 ng/dL (0.10 nmol/L), and for 11OHA4 9.1 ng/dL (0.30 nmol/L). Results: For 11KA4 a difference between boys and girls was observed until the age of 6. In girls 11KA4 was undetectable where in boys concentration up to 9.61 ng/dL (0.32 nmol/L) were measured. From the age of 6 an increase of 11KA4 was observed with increasing age. From the age of 13 concentrations stabilized at 4.21-28.5 ng/dL (0.14-0.95 nmol/L) without a difference between boys and girls. 11OHA4 was detectable at all ages and showed no differences between boys and girls, except for an apparently slightly higher concentration for boys until the age of 4. The concentration 11OHA4 increased with age in an almost linear way. At the age of 18 concentrations were observed of 60.5- 296.4 ng/dL (2.00-9.80 nmol/L). For 11KT a similar relation between the concentration and age was observed until the age of 14. At the age of 14 a plateau seems to be reached at concentrations of 14.5-74.4 ng/dL (0.48-2.46 nmol/L), which slightly decreased from the age of 17. 11OHT was undetectable in both boys and girls up to the age of 9 years. After the age of 9 the concentration range increased by age up to 30.4 ng/dL (1.00 nmol/L). From the age of 12 11OHT was detectable in all girl samples, where as in boys samples with undetectable concentrations were present at all ages. Conclusions: Age-related reference ranges were established for 11KA4, 11OHA4, 11KT and 11OHT. Overall no differences were observed between boys and girls, except for 11KA4 at a young age. The concentrations measured at the age of 18 correspond to the adult values known from literature for all 4 11-oxygenated androgens. Presentation: 6/1/2024

12456 Changes In High-sensitivity Cardiac Troponin I After 12 Months Of Gender Affirming Hormone Therapy

Abstract Disclosure: A.S. Cheung: None. I. Bretherton: None. S. Perera: None. L. Singh: None. C.Y. Chiang: None. Background: Due to differences in cardiac size and physiology between cisgender men and women, sex-specific reference cut-offs are recommended for Abbott high-sensitivity cardiac troponin I (hsTnI) to improve diagnostic accuracy of acute coronary syndrome. There are no prospective studies which examine the impact of gender affirming hormone therapy on hsTnI. We aimed to prospectively evaluate the change in hsTnI in transgender men and women newly commencing gender affirming hormone therapy (GAHT) compared to cisgender men and cisgender women over 12 months. Methods: Transgender men (TM) and transgender women (TW) aged >18 years newly commencing GAHT were recruited from outpatient endocrine clinics. Cisgender men (CM) and cisgender women (CW) were recruited from local community advertisements. Fasting serum collected at baseline and 12 months was frozen at - 80 degrees with batch analysis on the Abbott ARCHITECT i2000 hsTnI assay. One-way ANOVA analysis was performed on age, baseline hsTnI, repeat hsTnI, and change in hsTnI over 12 months between the 4 groups. Chi-square was used to analyze categorical status of detectable hsTnI using a Limit of Quantitation of < 2 ng/L. Data are presented as mean (standard deviation). P value < 0.05 is defined as significant. Results: Baseline samples were available for 263 participants, and 103 had paired baseline and repeat hsTnI samples. The mean age of participants was 31 years ± 12.4 and none had renal impairment. The TM group was younger (n=58, 25.7 ± 6.2 years old, p < 0.005) compared to the other 3 groups (TW n=56, 33.8 ± 15.7 years, CM n=70, 32.7 ± 13.8 years, CF n=79, 31.2 ± 10.7 years). At baseline, CM (2.1 ± 2.4 ng/L) had comparable hsTnI to TW (1.8 ± 2.1, p = 0.9), but higher hsTnI compared to CF (0.8 ±1.1, p < 0.001) and TM (1.1 ± 1.8, p = 0.03). At 12 months follow-up, CM (2.5 ± 2.8ng/L) had higher hsTnI compared to TW (0.9 ± 0.9, p = 0.004), CF (0.6 ±0.6, p < 0.001) and TM (0.9 ± 0.5, p = 0.002). There was a significant decrease in hsTnI after 12 months in TW (-1.4 ± 2.2) compared with CM (0.6 ± 1.9, p = 0.013). Detectable baseline hsTnI became undetectable (<2ng/L) in 73% (8/11) of TW compared to 13% (1/8) CM subjects (p = 0.03). Conclusions: After 12 months of GAHT, hsTnI in transgender women reduced and became comparable to cisgender women and transgender men. Our data supports the use of the cisgender female hsTnI reference cut-off in transgender women and transgender men. Data suggests a possible reduction in cardiac muscle mass or improvement in cardiac physiology occurs after 12 months of GAHT in transgender women. Presentation: 6/1/2024

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.

Development and validation of a stability-indicating HPLC method for assay of tonabersat in pharmaceutical formulations

A stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed to assay tonabersat and assess its stability in pharmaceutical formulations. Chromatographic separation was achieved using a Kinetex® C18 column (2.6 µm, 150 x 3 mm, 100 Å) at 50 °C, with a 20 µL injection volume. A linear gradient of acetonitrile in water (5 – 33.5 %) was applied for 1 min, followed by a gradual increase to 100 % over 26 min at a flow rate of 0.5 mL/min. Tonabersat and its degradation products were detected at 275 nm and 210 nm, respectively. The optimized method was used to evaluate the stability of tonabersat in lipid-based pharmaceutical formulations at 5 ± 3 °C, 25 ± 2°C/60 ± 5 % RH, and 40 ± 2 °C/75 ± 5 % RH over 3 months. The method was validated as per ICH guidelines and demonstrated linearity in the range of 5 – 200 µg/mL (R2 = 0.99994) with good accuracy (98.25 – 101.58 % recovery) and precision (% RSD < 2.5 %). The limits of detection and quantitation were 0.8 µg/mL and 5 µg/mL, respectively. Forced degradation studies showed significant degradation on exposure to alkaline (90.33 ± 0.80 %), acidic (70.60 ± 1.57 %), and oxidative stress (33.95 ± 0.69 %) at 70 °C, but no degradation was observed on exposure to thermal or photolytic stress. No chemical degradation was observed in either formulation on storage. Thus, the method was sensitive, specific, and suitable for stability testing of tonabersat in pharmaceutical formulations.

A-061 Analytical Performance and R55G Mutant Detection Ability of an Improved Thyroid-Stimulating Hormone Assay, TSH3-Ultra II, on the ADVIA Centaur Immunoassay System and the Atellica IM Analyzer

Abstract Background Measurement of thyroid stimulating hormone (TSH) is a critical tool for the investigation of thyroid disorders. Previous studies have demonstrated that some immunoassays are unable to detect TSH from patients with the R55G mutation in the TSHβ subunit. This study evaluated the analytical performance of the ADVIA Centaur® and Atellica® IM TSH3-Ultra II (TSH3ULII) assay and its ability to detect the R55G TSH mutant. Methods Limit of quantitation (LoQ) and linearity were determined as described in CLSI EP17-A2 and EP06, respectively. Precision and method comparison (MC) studies were performed with three reagent lots according to CLSI EP05-A3 and CLSI EP09c using native and contrived human serum and plasma samples. For precision, one aliquot of each sample was tested in duplicate in two runs per day &amp;gt;2 hours apart on each analyzer for ≥20 days. MC studies were performed by testing native human serum samples using ADVIA Centaur TSH3-UL assay (current TSH assay) and the newer ADVIA Centaur TSH3ULII assay. An additional MC was performed by testing native human serum samples with the TSH3ULII assay on both the ADVIA Centaur XP and the Atellica IM Analyzer. Samples from 61 patients with TSH results below the LoQ when using TSH3-UL were sequenced and confirmed to have the R55G mutant. Each of these samples was tested in duplicate using the ADVIA Centaur TSH3ULII assay. Results LoQ on both the ADVIA Centaur XP and the Atellica IM Analyzer was estimated to be 0.010 μIU/mL (mIU/L). Linearity was verified from 0.010 to 150.000 μIU/mL (mIU/L). ADVIA Centaur TSH3ULII repeatability %CVs ranged from 1.4 to 3.6% at 0.099 and 91.993 μIU/mL (mIU/L), and within-lab %CVs ranged from 2.3 to 6.2% at 4.908 and 91.993 μIU/mL, respectively. Atellica IM TSH3ULII repeatability %CVs ranged from 1.1 to 3.4% at 4.800 and 93.097 μIU/mL, and within-lab %CVs ranged from 2.2 to 4.6% at 0.103 and 93.097 μIU/mL, respectively. For the MC studies, slopes determined by the Passing-Bablock regression model were approximately 1 (0.96-0.97 range) across the sample interval tested. All TSH3ULII results from the 61 mutant patient samples were greater than LoQ, ranging from 0.864 to 23.443 μIU/mL (mIU/L). Conclusions Evaluation of the TSH3ULII assay using the ADVIA Centaur system demonstrated performance comparable to the commercially available TSH3-UL assay for LoQ, linearity, and precision, when using normal patient samples. TSH3ULII results on the Atellica IM Analyzer were comparable to the results obtained on the ADVIA Centaur XP. All 61 TSH mutant samples used in this study that had TSH values below the LoQ with the TSH3-UL assay, were detectable with the TSH3ULII assay. The data presented here demonstrate the ability of the TSH3ULII assay to detect the TSH in samples from patients with the R55G mutation.

B-004 Method Validation of Lactate in the Plasma of Rats

Abstract Background The objective of this study is to validate Lactate (LAC) measurement in rat plasma using the ADVIA 1800 analyzer. Methods LAC was validated in rat plasma using the ADVIA 1800 analyzer. Validation testing included intra-assay and inter-assay precision, accuracy, linearity of dilution, limit of quantitation (LOQ), limit of detection (LOD), reference interval, carry-over, correlation, recovery, and stability. Samples were collected into tubes containing sodium fluoride. Intra-assay precision was determined by analyzing two biological samples and the two quality control levels of Bio-Rad Lyphochek Assayed Chemistry Control 10 consecutive times. Inter-assay precision was determined by analyzing the two quality control levels in duplicate for six days over a 10-day period. Accuracy was calculated using the data obtained from the inter-assay precision testing. For linearity of dilution, Audit MicroControl General Chemistry Linearity Kit and biological samples were diluted and analyzed in duplicate. The LOQ was determined by diluting the calibrator material to produce a value at the low end of the reportable range. Diluted material was tested six times. The LOD was determined by assaying the appropriate blank 10 times. The reference interval was determined by analyzing 21 plasma samples. Correlation between the two ADVIA 1800 analyzers was determined by assaying 10 samples on both analyzers once. The carry-over of the assay was determined by analyzing the high-level quality control material followed by the low-level quality control material. Recovery was determined by analyzing a pair of test samples in duplicate that have been spiked and diluted. The proportional error between the two test samples was calculated. Stability of plasma samples was tested on wet ice, refrigerated, and frozen (at -70°C) for various durations of storage. Results Measurement of LAC in rat plasma met the acceptance criteria for precision, accuracy, linearity of dilution, limit of quantitation, carry-over, recovery, and stability. For intra-assay precision in specimen and control material, the %CV was within ±20%. The inter-assay precision %CV was within ±20%. The total error observed (TEobs) was within ±20% for accuracy and the obtained mean of the control material was within the manufacturer’s acceptable range. For linearity of dilution, the coefficient of determination was ≥ 0.9000, the slope was within 1.00 ± 0.25, and TEobs for each plasma and linearity kit dilution was ≤20%. The LOQ was set at the lowest concentration for which the TEobs was within ±20%. The LOD was calculated by adding the mean concentration obtained and three times the standard deviation. The reference interval was set to the 2.5th to 97.5th percentile interval using Excel software. The carry-over was acceptable at ≤2%. Correlation between the two ADVIA 1800 analyzers was determined to describe any bias in result interpretation. Recovery was acceptable as the proportional error was within ±20%. Stability at all storage conditions was found to be acceptable with a mean %difference within ±20%. Conclusions LAC in rat plasma met the acceptance criteria for all required validation parameters. The ADVIA 1800 analyzer can be used for preclinical studies to test LAC in rat plasma.

A-110 Analytical Performance Evaluation of Vitros® Immunodiagnostic Products Intact PTH II Assay

Abstract Background Intact parathyroid hormone (iPTH) is indicated to aid in the diagnosis of hyperparathyroidism, hypoparathyroidism, differential diagnosis of hypocalcemia or hypercalcemia and for intraoperative measurement of iPTH levels. Studies were conducted to evaluate the analytical performance of the Vitros Immunodiagnostic Products Intact PTH II (Vitros iPTH II) assay by determining limit of detection (LoD), limit of quantitation (LoQ), and precision. A method comparison of the Vitros iPTH II assay against the Roche Elecsys PTH assay was also conducted. Methods The LoD and LoQ were determined consistent with CLSI EP17. Precision was evaluated consistent with CLSI EP05 using six patient pools for 20 days. Repeatability was determined using two replicates per run. Within-lab precision was determined using a single reagent lot and a single calibration. A quantitative comparison between the Vitros iPTH II assay on a Vitros 3600 system and the Roche Elecsys PTH assay on a Roche Cobas 8000 system was conducted in accordance with CLSI guideline EP09 using 206 endogenous patient samples. Samples were selected to cover the full measuring interval and were tested over a total of six non-consecutive days. Results The LoD for the Vitros iPTH II assay was determined to be 1.7 pg/mL. The claimed LoQ was determined using a precision profile approach with a %CV limit of 20% and set at 1.7 pg/mL. For mean parathyroid hormone (PTH) concentrations ranging from 15.9 to 4656 pg/mL, repeatability (within-run) percent coefficient of variation (%CV) ranged from 0.8% to 1.4% and within-lab %CV ranged from 1.5% to 3.0%. The method comparison of the Vitros iPTH II assay versus the Roche Elecsys PTH assay yielded a correlation coefficient of 0.991 and a Passing-Bablok regression slope of 1.01 and intercept of 0.3. Conclusions The Vitros iPTH II assay demonstrated acceptable analytical performance in these studies, with high correlation and close agreement to the Roche Elecsys PTH assay.

A-009 Analytical Characterization of a Novel NT-proBNP Assay on a Central Laboratory Platform

Abstract Background N-terminal pro-B-type Natriuretic Peptide (NT-proBNP) or B-type Natriuretic Peptide (BNP) assay(s) are widely measured with a Class 1, Level of Evidence A recommendation in professional heart failure (HF) guidelines for diagnosis and prognosis. We present the analytical characteristics of the first NT-proBNP assay on Beckman Coulter platforms. Methods Measurements were conducted with the Access NT-proBNP assay, a one-step sandwich immunoenzymatic assay using monoclonal capture and detection antibodies, on the DxI 9000 Immunoassay Analyzer with Lithium Heparin (LiHep) plasma samples. Performance was characterized according to CLSI guidelines for: Limit of Blank (LoB), Limit of Detection (LoD), Limit of Quantitation (LoQ), linearity, imprecision (repeatability, reproducibility) and matrix comparison with LiHep, EDTA and serum. Reference intervals (RI) were evaluated in a healthy, adult U.S. population comprised of 675 subjects with 54.6% females, 79.1% White and 15.1% Black individuals. Exclusion criteria included elevated cardiac troponin, abnormal eGFR and cardiac medications. The 97.5th percentile RI upper limit was defined for age-stratified ranges of &amp;lt;50, 50-75, and &amp;gt;75 years old. Results Performance was LoB=1.1ng/L; LoD=4.8ng/L; LoQ=4.8ng/L; linearity=35,000ng/L. Repeatability ranged from 1.5%-3.5% and reproducibility from 2.3%-7.9% in seven samples ranging from 38ng/L to 23,848ng/L. Matrix measurement regression (n=68) yielded slopes of 1.00, 1.01; intercepts -0.03 to -2.63; r=0.98. Table 1 shows that NT-proBNP values increase with age and women generally have higher values than men. Conclusions The LoB, LoD and LoQ demonstrate highly sensitive low-end characteristics. Repeatability and reproducibility show excellent performance across a wide measurement range. NT-proBNP values are known to be age dependent, and the age-stratified 97.5th percentile RI upper limits were 162 ng/L for &amp;lt;50 years, 311 ng/L for 50-70 years, and 457 ng/L for &amp;gt;75 years. This novel Access NT-proBNP assay offers robust analytical performance as an aid to clinical HF management in acute settings.

B-267 Validation of the Newly Developed Fully Automated Chemiluminescent Enzyme Immunoassay (CLEIA) Reagent for Measurement of Cyclosporine in the Blood

Abstract Background Cyclosporine (CsA) is an immunosuppressive drug requiring therapeutic drug monitoring (TDM) for its narrow-ranged medication level. Approximately 60% of CsA in the blood exist as molecular complexes with proteins like cyclophilin in lymphocytes and erythrocytes. Measurement of CsA in the blood by immunoassay or liquid chromatography-tandem mass spectrometry (LC-MS/MS) requires an extraction process for CsA from the whole blood specimen, which is often manual complicated and time-consuming process causing a risk of variation in the measurement values among different operators. In addition, the small (cyclic polypeptide composed of 11 amino acids) and hydrophobic property of CsA, make it difficult to establish antibodies for a sandwich immunoassay. In this study, we have validated a novel fully automated sandwich immunoassay reagent for measurement of blood CsA that used a combination of newly-established CsA antibodies and iTACT® (immunoassay for total antigen including complex via pretreatment) technology. Methods The reagent was developed and validated on a fully automated immunoassay system, LUMIPULSE® L2400 (FUJIREBIO INC). Twenty uL whole blood specimens are sampled after mixed with suction/discharge stirring to prevent that blood cells settle to the bottom of a container on the analyzer, and then treated with a pretreatment solution to obtain free CsA. The treated samples are assayed by a two-step sandwich immunoassay. Limit of Detection (LoD), Limit of Quantitation (LoQ), within-laboratory precision, linearity, no interference, and the correlation of this assay and LC-MS/MS were validated, following the current CLSI guidelines protocols. Data were analyzed using Microsoft Excel statistical tool Analyse-it. Results The LoD and the LoQ were 2.7 ng/mL and 10.8 ng/mL respectively. The within-laboratory precision showed CV 1.6-2.9 %. Linearity was demonstrated over the range 13.1-2381.5 ng/mL. No interference was observed with unconjugated (≤19.7 mg/dL) or conjugated bilirubin (≤20.1 mg/dL), chyle (≤1480 FTU), RF (rheumatoid factor, ≤1000 IU/mL), and HAMA (human anti-mouse antibody, ≤1010 ng/mL). The correlation coefficient and the regression slope of this assay and LC-MS/MS were 1.0 and 1.0, respectively (N=120). Conclusions The performance of our novel CsA assay, which use LUMIPULSE L2400, was satisfactory for routine analysis. A unique fully-automated process, including the pretreatment process of whole blood specimens, for measurement would contribute the low LoQ and the low Within-laboratory CV. In addition, the results are obtained in 35-minute. We expect this novel immunoassay could reduce the burden on operators and turnaround time.

A-090 Performance of GFAP and UCH-L1 Biomarkers for Traumatic Brain Injury in the ARCHITECT i1000SR TBI Test

Abstract Background Traumatic Brain Injury (TBI) has been recognized as an important cause of death and disability and is a growing public health problem. An estimated 69 million people globally experience a TBI annually. The vast majority (80%) of patients evaluated for TBI in the Emergency Department will receive head CT scans, however less than 10% will have findings of acute traumatic abnormalities. This highlights the need for objective, rapid, accurate tools to help clinicians evaluate and triage patients with suspected TBI and reduce unnecessary radiation exposure. Blood based biomarkers such as glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase L1 (UCH-L1) have shown utility to predict acute traumatic intracranial injury on CT scan after TBI. Methods The ARCHITECT TBI test is a panel of in vitro diagnostic chemiluminescent microparticle immunoassays for the measurement of GFAP and UCH-L1 in plasma and serum. A method comparison study was performed to compare the GFAP and UCH-L1 assays on ARCHITECT i1000SR to the FDA cleared Alinity i assays. Performance characteristics such as detection limits, imprecision, linearity, and measuring interval were established following CLSI guidance. Results The analytical measuring interval (AMI) extends from the limit of quantitation (LoQ) to the upper LoQ and is determined by the range that demonstrates acceptable performance for linearity, imprecision, and bias. The AMI is 6.1 to 42,000 pg/mL for GFAP and 26.3 to 25,000 pg/mL for UCH-L1. Within-laboratory imprecision ranged from 2.2 to 6.2% CV for GFAP and 2.2 to 4.5% CV for UCH-L1. The Overall Reproducibility (includes repeatability, between-run, between-day, between-instrument variance components) results ranged from 2.7 to 6.8% CV for GFAP and 2.4 to 3.9 %CV for UCH-L1. The method comparison study evaluated 123 specimens across the range of 6.5-32,548.6 pg/mL for GFAP, and 51.5-24,127.7 for UCH-L1. The correlation coefficients were 1.0, with a slope of 1.03 and 1.06, an intercept of -0.6 and -6.0, for GFAP and UCH-L1, respectively. The results support use of the established GFAP and UCH-L1 cutoffs and clinical performance findings in a multi-site pivotal study which enrolled 1899 mild TBI (Glasgow Coma Scale score 13 to 15) subjects. 116 of the 120 specimens with a positive CT scan had a positive TBI interpretation (Sensitivity 96.7%). Of the 1779 subjects with negative CT scan results, 713 had a negative TBI interpretation (Specificity 40.1%). The negative predictive value (NPV) of the test was 99.4% (713/717). Conclusions The ARCHITECT TBI test performance was evaluated compared to the Alinity i TBI test across a broad concentration range of GFAP and UCH-L1. The results support equivalence of the ARCHITECT TBI test to the Alinity i TBI test. The established cutoffs were evaluated in a prospective cohort of mild TBI subjects and demonstrated high sensitivity and NPV relative to head CT scan results, supporting the utility to rule out the need for CT scan in mild TBI subjects presenting to the emergency department. Funding This work is in collaboration with the US Army Medical Materiel Development Activity, US Army Medical Research and Development Command CRADA 20-1266-CRA.

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.

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

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

B-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-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-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.

Microfluidic Affinity Selection of B-Lineage Cells from Peripheral Blood for Minimal Residual Disease Monitoring in Pediatric B-Type Acute Lymphoblastic Leukemia Patients.

Assessment of minimal residual disease (MRD) is the most powerful predictor of outcome in B-type acute lymphoblastic leukemia (B-ALL). MRD, defined as the presence of leukemic cells in the blood or bone marrow, is used for the evaluation of therapy efficacy. We report on a microfluidic-based MRD (MF-MRD) assay that allows for frequent evaluation of blood for the presence of circulating leukemia cells (CLCs). The microfluidic chip affinity selects B-lineage cells, including CLCs using anti-CD19 antibodies poised on the wall of the microfluidic chip. Affinity-selected cells are released from the capture surface and can be subjected to immunophenotyping to enumerate the CLCs, perform fluorescence in situ hybridization (FISH), and/or molecular analysis of the CLCs' mRNA/gDNA. During longitudinal testing of 20 patients throughout induction and consolidation therapy, the MF-MRD performed 116 tests, while only 41 were completed with multiparameter flow cytometry (MFC-MRD) using a bone marrow aspirate, as standard-of-care. Overall, 57% MF-MRD tests were MRD(+) as defined by CLC numbers exceeding a threshold of 5 × 10-4%, which was determined to be the limit of quantitation. Above a threshold of 0.01%, MFC-MRD was positive in 34% of patients. The MF offered the advantage of the opportunity for efficiently processing small volumes of blood (2 mL), which is important in the care of pediatric patients, especially infants. The minimally invasive means of blood collection are of high value when treating patients whose MRD is typically tested using an invasive bone marrow biopsy. MF-MRD detection can be useful for stratification of patients into risk groups and monitoring of patient well-being after completion of treatment for early recognition of potential impending disease recurrence.

A-306 High Sensitivity Capability of an Anti-müllerian Hormone Assay on the Beckman Coulter DxI 9000 Immunoassay Analyzer*, a Next Step Toward Enabling Ovarian Failure Staging?

Abstract Background Measurement of anti-müllerian hormone (AMH) has proven value in the evaluation of ovarian reserve using assays such as the Beckman Coulter Access AMH assay. If improved sensitivity of AMH measurement can be achieved, enhanced clinical utility during the assessment of reproductive aging and ovarian response to enable staging of ovarian failure is possible (Practice Committee of the American Society of Reproductive Medicine 2020, Cappola et al. 2023). The Beckman Coulter DxI 9000 Immunoassay Analyzer* has been designed with new technology that can be leveraged to improve assay sensitivity. Such technological advancements include the new Lumi-Phos PRO chemiluminescent substrate which yields markedly increased signal-to-noise, a new luminometer with increased dynamic range, and improved low-volume pipetting capabilities. Data herein summarize results from analytical verification studies of the Access AMH assay on the DxI 9000 Immunoassay Analyzer. Results demonstrate an example of DxI 9000 technologies enabling sensitivity claims that are up to 20-fold improved. * The official name is DxI 9000 Access Immunoassay Analyzer. Methods A method comparison study was performed based on CLSI guideline EP09c, 3rd ed. to compare results for the Access AMH assay on DxI 9000 to the predicate device, the Access AMH assay on the Access 2 Immunoassay System. Within-laboratory precision was also evaluated following CLSI EP05-A3. Finally, performance at low analyte levels was evaluated following CLSI EP17-A2, and linearity was evaluated following CLSI EP06-Ed2. Results Method comparison of the Access AMH assay on DxI 9000 compared to the Access AMH assay on the Access 2 yielded excellent agreement with a Passing-Bablok slope of 1.02 for N=126 samples. Further, bias estimates for a number of key medical decision points suggest minimal bias (≤ 2%) across the assay range, and minimal non-linearity was observed. 20-day imprecision studies yielded CV’s between 2 and 5% across a broad range of concentrations evaluated. Sensitivity studies yielded estimates of limit of quantitation (LoQ) between 0.001 and 0.003 ng/mL. This represents an approximately 10- to 20-fold improvement in sensitivity claims compared to the AMH assay on the Access 2 and other commercially available automated immunoassay analyzers. Conclusions The data herein present performance data for the Access AMH assay on the DxI 9000 Immunoassay Analyzer. The assay demonstrates excellent accuracy relative to the predicate device, while showing good linearity and imprecision. Of note, the Access AMH assay exhibits exceptional low-end performance on the DxI 9000 analyzer as demonstrated by the ability to enable sensitivity claims that are up to 20-fold improved in comparison to existing immunoassays for the measurement of AMH. The sensitivity capabilities of the Access AMH assay on DxI 9000 suggest an opportunity for the addition of further intended uses for scenarios where extremely low AMH measurements can provide additional value of clinical significance. Further studies are needed to fully understand additional clinical utilities of the Access AMH assay with high sensitivity capability on DxI 9000, including the assessment of reproductive aging for menopause diagnosis or ovarian response for fertility treatment.

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 &amp;gt;2 hours apart for up to &amp;gt;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 &amp;lt;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.

A-309 Multicenter Performance Evaluation of the ProNephro AKITM Assay and Sample Stability in Pediatric Patients

Abstract Background Acute Kidney Injury (AKI), defined by increased serum creatinine or decreased urine volume, is highly prevalent in critically ill pediatric patients. Those who develop AKI experience worsened outcomes, raising the need for accurate and early biomarkers for AKI. Relying in serum creatinine for AKI diagnosis is unreliable due to its diagnostic inaccuracy and delayed rise in the AKI pathophysiology. Neutrophil Gelatinase-Associated Lipocalin (NGAL) is a novel and widely studied biomarker for early identification of AKI. During pathological progression, NGAL is elevated in blood and urine within two hours of the insult, making it useful for early diagnosis and management of kidney injury in critically ill children. This study aims at evaluating the performance characteristics of a quantitative test for NGAL in urine intended for use in critically ill pediatric patients, and investigating the stability of NGAL in pediatric urine. Methods The ProNephroAKITM is a particle-enhanced turbidimetric immunoassay on the Roche cobas® c501 analyzer to measure NGAL quantitatively in urine in pediatric patients 3 months to 21 years of age. These studies were approved by the respective institutional review boards. The limits of blank (LOB), detection (LOQ) and quantitation (LOQ) were determined following CLSI EP17A2. Linearity was assessed across the measurement range (MR) of 50-3000 ng/mL. The assay repeatability, between-run, between-day and total imprecision were determined in pediatric urine samples (n=9, covering the MR) and controls (n=2) following CLSI EP5-03 guidelines. Likewise, multisite reproducibility was evaluated in pediatric urine (n=7, covering the MR) and controls (n=2) in 3 laboratories and alternating operators. The assay specificity was assessed in the presence of several endogenous and exogeneous substances (e.g. drugs, contrast agents), potential related cross-reactant molecules, and several pH-values. The stability of NGAL in urine was evaluated using freshly collected pediatric urine samples covering the MR from 16 consented patients after storage at ambient (15-30°C), refrigerated (2-8°C), and frozen temperatures (≤-70°C), and following 3 freeze/thaw cycles. Results The ProNephroAKITM is linear across the MR of 50-3000 ng/mL. The LOB, LOD and LOQ were 9, 14 and 18 ng/mL, respectively. The coefficients of variation (CVs) for repeatability, between-run, between-day and total precision were ≤5.0%, ≤3.1%, ≤1.7% and ≤4.9%, respectively in urine and control samples. The reproducibility across 3 independent laboratories was ≤6.6%. The recovery of NGAL was not significantly affected by endogenous substances, contrast agents, pH (3.3-10.3), and common and special pharmaceutical compounds (exception: very high Cefepime concentrations may decrease NGAL), and potential cross-reactants. Freshly collected urine samples spanning concentrations from 71-2,373 ng/mL were stable (±10% recovery) for 2 days at ambient, 4 days refrigerated and 13 months frozen, and after 3 freeze/thaw cycles. Conclusions We report the performance characteristics of the ProNephroAKITM assay, including excellent precision and adequate reproducibility across 3 laboratories, sensitivity, specificity, and urine sample stability. The test was determined to meet the analytical performance required for clinical use in critically ill pediatric patients at risk of developing or having persistent AKI.

B-043 Q-nab™ Igg kit: verification and validation of a novel cell-free quantitative neutralizing antibody assay

Abstract Background As the world exits the COVID-19 pandemic era, it is very important to assess population-level and individual seroprotectivity against multiple circulating and emerging SARS-CoV-2 strains, especially in vulnerable populations. Current serological measurements for neutralizing antibodies (NAbs) are either done using complex and time-intensive pseudovirus- or micro-neutralization assays or as a high throughput competitive ELISA assay. These assays, by design, cannot be multiplexed to measure NAb titers against multiple strains and suffer from a limited dynamic range. The Q-NAb IgG Tests, which rely on blocking the non-neutralizing antibodies (NNAbs) using a novel fusion protein, were developed and validated to quantitatively measure NAb titer against the SARS-CoV-2 strains used to develop the first two mRNA vaccines viz. ancestral and BA.4/5 strains. Methods The Q-NAb assay measures the NAb titer in a sample by first sequestering the NNAbs using the fusion protein (made by covalently coupling SARS-CoV-2 BA.4/5 RBD and h-ACE2 protein) thereby enriching the NAbs in the sample. Variant-specific NAbs are then detected using an indirect ELISA where the wells are coated with the variant RBD and yield a signal that is directly proportional to the concentration of NAb. Clinical samples from subjects that have either had a SARS-CoV-2 infection (N=27), taken the initial vaccine series (N=123) and/or the bivalent booster (N=151) and pre-pandemic samples (N=29) were commercially sourced. Q-NAb NAb titers were measured using the ancestral and BA.4/5 kits and were compared against corresponding MN50 values using both parametric and non-parametric models. Single-site precision, low-end sensitivity, dilutional linearity, and interference screening of Q-NAb IgG Kits were evaluated in accordance with appropriate CLSI guidelines. Traceability and total uncertainty of the Q-NAb IgG Kits (Ancestral and BA.4/5) to WHO International Standard 21/340 and 21/338 respectively was established using a calibration hierarchy adapted from ISO 17511:2020. Results Both the ancestral and BA.4/5 Q-NAb IgG Test Kits correlated with MN50 titers with a Spearman correlation coefficient of 0.90 and 0.89 respectively. The total uncertainty in traceability of calibration to WHO International standards 21/340 and 21/338 was found to be less than 10% for both tests. Single site precision across the assay range for BA.4/5 kit was found to be 6-10% with a limit of quantitation of 312 IU/mL. Assays exhibited dilutional linearity across their respective dynamic range and had no significant interference from common endogenous and exogenous substances. Conclusions We have successfully validated the novel Q-NAb IgG neutralization assay platform as individual assays for measuring neutralizing titers against ancestral and BA.4/5 SARS-CoV-2 strains. The assays have expanded dynamic range, excellent analytical performance and correlation to accepted gold-standard microneutralization assays. Both assays are calibrated and traceable to WHO international standards, thereby enabling the compilation and comparison of data from multiple clinical cohorts run across multiple laboratories. The design of the Q-NAb IgG Test kit makes it amenable to multiplexing (for example, on an MSD platform) to assess neutralization titers against multiple variants at the same time. Such a multiplexed platform can be used to assess vaccine efficacy and seroprotection against multiple variants including emerging variants.