Reagent Lots Research Articles

B-142 Using Big Data Analytics to Measure the Multi-year Consistency of Sigma metrics for Vitros Cardiac Assays

Abstract Background Sigma metrics assess lab quality by incorporating precision, accuracy and total error allowed (TEa). Sigma metrics report quality on a 0-6 scale, with 6 representing World-Class quality. Due to logistical constraints, Sigma metrics are often calculated with limitations such as a small number of analyzers, single reagent lot or short duration. QuidelOrtho developed a novel approach using big data analytics for the objective, automated calculation of Sigma metrics across thousands of Vitros(R) analyzers running in real lab conditions. This approach was previously used to report the Sigma metric performance for 115 assays using data from 2022. Of particular interest is the consistency of quality levels for Vitros cardiac assays assessed in the study. Methods The methods follow the previous approach using the same Python algorithm to extract QC data from Jan. to Nov. 2023 (inclusive). A QC fluid lot from the study period was selected for each analyte to align with the fluid level reported in the 2022 study, which was based on medical relevance. Metrics were calculated for each set of QC data that originated from a single analyzer, reagent lot and calibration curve; the median Sigma metric is reported in the Results. Proficiency testing limits were utilized as TEa limits where available, with preference to CLIA 2024 limits. Results The following assays reported Sigma metrics as >6 using both 2022 and 2023 data: apolipoprotein A1, apolipoprotein B, CK-MB, dLDL, LDH, myoglobin, NT-proBNP, and triglycerides. Assays with higher metrics using 2023 data: CK (5.2 vs 5.5), dHDL (5.8 vs >6), hsTnI (4.1 vs 4.3), and NT-proBNP2 (4.2 vs >6). Assays with lower metrics using 2023 data: cholesterol (5.8 vs. 5.3) and homocysteine (>6 vs 5.9). CKMB2 (>6) is a newer assay that did not have metrics reported in 2022. Vitros cardiac assays report metrics within 0.5 Sigma of the 2022 metrics. An exception is NT-proBNP2, which changed from 4.2 to >6. However, the 2023 analysis has almost double the number of Vitros analyzers (51 vs 90), representing new use of NT-proBNP2. Conclusions Vitros cardiac assays report consistent metrics when using 2023 data compared to 2022. All Vitros cardiac assays assessed report Sigma metrics of 4 or greater, indicating they achieve results within the corresponding allowable error.

B-037 Zeon original coating technology COP microplates for cell imaging

Abstract Background Zeon has developed high quality microplate made of Cyclo Olefin Polymer (COP) and special coat reagent for cell culture for Aurora microplates. The new 96 well-microplates have best-in-class properties for analysis of cultured cells can reduce the background of fluorescence images absolutely and excellent bottom flatness for observation of target. This enables to obtain high-precision data for cell analysis. Also, we developed Zeon original coating (ZOC) reagent specialized for COP surface. It is suitable for various cells including neurons derived from human induced pluripotent stem cells (iPSC), primary cells, general cell lines, and so on. ZOC coated COP microplates (ZOC plates) are fully-precoated and ready-to-use. Methods We evaluated the following points regarding ZOC plates. The cells used were derived from human iPSC. [1. Storage stability] ZOC plates were stored at room temperature for a long time. After that, cardiomyocyte cells were cultured for 3 days on the plates. The cell adhesion and beat rate were evaluated. [2. Neurite extension] Dopaminergic neurons were cultured for 14 days, then the neurite extension was evaluated. For comparison, a conventional poly-D-lysine* (PDL) coating was used. [3. Lot-to-lot variation] COP microplates coated with different lots of ZOC reagents were prepared. Glutamatergic neurons were cultured for 14 days on those plates, then the neurite extension and cell morphology were evaluated. *The typical coating reagent applied for neuronal culture. Results [1.] ZOC plates stored over 12 months at room temperature showed excellent cell adhesion, and the beat rate was equivalent to prepared just before use. [2.] ZOC plates tended to extend neurites more than PDL coated plates. [3.] No variation between lots was observed on ZOC plates regarding the neurite extension and cell morphology. Conclusions We suggest applying the combination of high accuracy for cell imaging using COP microplates and high performance for cell culture using ZOC can provide high precision data at bioassay research. This would be a significant value for development of drug screening processes at pharmaceutical company.

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.

B-099 Validation and Clinical Implementation of Organ Procurement Serology Testing: Our Analytical and Regulatory Experience in Implementing a New Donor Testing Program

Abstract Background The Mount Sinai Hospital recently implemented a comprehensive organ donor testing program to service the clinical needs of transplant services in the New York City metro area. Conventional serology testing is not approved by the FDA for donor testing. To support this program, the MSH Chemistry Laboratory implemented and validated a comprehensive donor testing panel on the Abbott Alinity S donor testing platform. We validated donor serology testing for the following assays: HIV-1/2 (HIV), Anti-Hepatitis B Core (Anti-HBc), Hepatitis B Surface Antigen (HBsAg), Human T-lymphotropic virus I and II (HTLV-I/II), Anti-Hepatitis C (Anti-HCV), and Trypanosoma cruzi antibodies (T.Cruzi). The Alinity S System is an automated immunoassay-based analyzer FDA-approved for donor testing. The analyzer consists of two distinct sample ‘paths’ and is effectively two instruments combined in one analyzer. The analyzer is approved for use in living donor serum and plasma and in cadaveric serum. Prior to going live, we found that there was limited guidance on laboratory quality assurance programs related to donor testing. In addition to confirming assay analytical performance, we designed and implemented a comprehensive quality assurance program prior to satisfy compliance with regulatory guidelines, addressing proficiency compliance, sample TAT, and control ranges, amongst other quality indicators. Methods Inter and Intraday precision was assessed through replicate analysis of positive and negative controls, where 10 replicates of both controls were assayed on a single day, and one replicate of each control was assayed daily for 7 distinct days, results were represented as %CV. Result accuracy was assessed through split sample analysis of known serologically positive (n=20) and negative samples (n=40). Each sample was assayed on two distinct lots of reagents, each with its own unique calibration curve. Accuracy was confirmed in both living donor (n=30), and cadaveric samples (n=10). In addition, split sample analysis was performed between the Alinity S and FDA-approved donor testing assays in a reference laboratory (N=7). A carryover study was performed through the analysis of triplicate replicates of low, high, and then low controls. These analyses were performed for all 6 assays. Results Intraday precision using assay general controls afforded the following %CV: HIV: 3.1%, 6.5%; Anti-HBc: 1.3%, 2.6%; Anti-HCV: 3.3%, 11.7%; HTLV-I,II: 2.5%, 5.9%; T.Cruzi: 2.8%, 0%; HBsAg: 3.2%, 11.6%. Interday precision yielded the following in %CV: HIV: 2.9%, 10.0%; Anti-HBc: 3.0%, 5.6%; Anti-HCV: 3.3%, 3.4%; HTLV-I,II: 2.8%, 4.6%; T.Cruzi: 2.4%, 0%; HBsAg: 4.7%, 7.8%. Accuracy studies yielded 100% positive and negative correlation for all 6 assays in both living donor and cadaveric samples. In addition, there was a 100% serological correlation for samples compared to reference laboratories. Carryover studies showed no evidence of sample carryover. Conclusions The Abbott Alinity S shows acceptable assay precision on all 6 validated assays. The assays are serologically accurate in both cadaveric and living donor samples. As such, the assays and platform are acceptable for use in donor screening. We designed a unique quality assurance program including a comprehensive self-proficiency and instrument crosscheck program to ensure analyzer accuracy, as well as adherence to regulatory guidelines.

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-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-065 The CDC HoSt Certification Requirements Have Been Met for More Than Four Years by the Atellica IM, ADVIA Centaur Testosterone II and Dimension Vista LOCI Total Testosterone Immunoassays

Abstract Background The CDC Hormone Standardization (CDC HoST) Certification Program, is traceable to the primary testosterone Standard National Measurement Institute (NMI) M914. The Atellica® IM, ADVIA Centaur® Testosterone II (TSTII) and Dimension Vista® LOCI Total Testosterone (TTST) assays are enrolled in the program. Certification is a yearlong process of 40 blinded samples from the CDC, 10 for testing each quarter. If 4 consecutive quarters meet bias acceptance criteria of ± 6.4%, CDC certification is achieved. Methods Initially, to standardize the assay approximately 120 samples were obtained from CDC with CDC HoST ID-LC-MS dose values to value assign master curve standards. Multiple ADVIA Centaur TSTII reagent lots were tested with CDC patient samples. Internal master curve standards were assayed as unknowns and dose values assigned using the RLU (system generated units-Relative Light Units) to CDC ID-LC-MS dose relationship. For TSTII (2019- 2023), after standard value assignment, at least 179 CDC blinded samples were tested across all platforms. Each quarter, 10 blinded samples were tested in duplicate over 2 days. Similar procedures were followed for TTST. Results were analyzed by the CDC. Results Correlation between the CDC assigned values and Atellica IM, ADVIA Centaur, and ADVIA Centaur CP TSTII assay results was strong across all quarters from 2019 to 2023 combined. TTST results were strong for 2023. Mean bias between TSTII and TTST results and CDC reference values was consistently <6.4%. This enabled the TSTII assays to be CDC certified for a fourth consecutive year, and TTST for 2023. The table presents TSTII (2019-2023) and TTST (2014-2023) results. Conclusions The TSTII assay consistently achieved CDC certification from 2019 to 2023, while the TTST assay was initially certified in 2014. Siemens Healthineers is the only testosterone immunoassay manufacturer to be currently certified, the TSTII assay has achieved certification for four consecutive years.

Analytical validation of the LungLB test: a 4-color fluorescence in-situ hybridization assay for the evaluation of indeterminate pulmonary nodules

BackgroundEvaluation of indeterminate pulmonary nodules (IPNs) often creates a diagnostic conundrum which may delay the early detection of lung cancer. Rare circulating genetically abnormal cells (CGAC) have previously demonstrated utility as a biomarker for discriminating benign from malignant small IPNs in the LungLB assay. CGAC are identified using a unique 4-color fluorescence in-situ hybridization (FISH) assay and are thought to reflect early cell-based events in lung cancer pathogenesis and the anti-tumor immune response. LungLB is a prognostic tool that combines the CGAC biomarker and clinical features to aid in IPN evaluation by improving the stratification of patient risk of malignancy.MethodsHerein we describe the analytical performance of the LungLB blood test. Analytical validation was performed according to Clinical and Laboratory Standards Institute (CLSI) guidelines with adaptations for rare cell-based assays. Multiple operators, reagent lots, and assay runs were tested to examine accuracy, precision, reproducibility, and interfering factors.ResultsThe FISH probes used in the LungLB assay demonstrate 100% sensitivity and specificity for their intended chromosomal loci (3q29, 3p22.1, 10q22.3 and 10cen). LungLB demonstrates analytical sensitivity of 10 CGAC per 10,000 lymphocytes analyzed, 100% analytical specificity, and high linearity (R2 = 0.9971). Within run measurements across 100 samples demonstrated 96% reproducibility. Interfering factors normally found in blood (lipemia, biotin) and exposure to adverse temperatures (-20ºC or 37ºC) did not interfere with results. Sample stability was validated to 96 hours.ConclusionThe analytical performance of LungLB in this validation study successfully demonstrates it is robust and suitable for everyday clinical use.

ASPYRE-Lung: validation of a simple, fast, robust and novel method for multi-variant genomic analysis of actionable NSCLC variants in FFPE tissue.

Genomic variant testing of tumors is a critical gateway for patients to access the full potential of personalized oncology therapeutics. Current methods such as next-generation sequencing are costly and challenging to interpret, while PCR assays are limited in the number of variants they can cover. We developed ASPYRE® (Allele-Specific PYrophosphorolysis REaction) technology to address the urgent need for rapid, accessible and affordable diagnostics informing actionable genomic target variants of a given cancer. The targeted ASPYRE-Lung panel for non-small cell carcinoma covers 114 variants in 11 genes (ALK, BRAF, EGFR, ERBB2, KRAS, RET, ROS1, MET & NTRK1/2/3) to robustly inform clinical management. The assay detects single nucleotide variants, insertions, deletions, and gene fusions from tissue-derived DNA and RNA simultaneously. We tested the limit of detection, specificity, analytical accuracy and analytical precision of ASPYRE-Lung using FFPE lung tissue samples from patients with non-small cell lung carcinoma, variant-negative FFPE tissue from healthy donors, and FFPE-based contrived samples with controllable variant allele fractions. The sensitivity of ASPYRE-Lung was determined to be ≤ 3% variant allele fraction for single nucleotide variants and insertions or deletions, 100 copies for fusions, and 200 copies for MET exon 14 skipping. The specificity was 100% with no false positive results. The analytical accuracy test yielded no discordant calls between ASPYRE-Lung and expected results for clinical samples (via orthogonal testing) or contrived samples, and results were replicable across operators, reagent lots, runs, and real-time PCR instruments with a high degree of precision. The technology is simple and fast, requiring only four reagent transfer steps using standard laboratory equipment (PCR and qPCR instruments) with analysis via a cloud-based algorithm. The ASPYRE-Lung assay has the potential to be transformative in facilitating access to rapid, actionable molecular profiling of tissue for patients with non-small cell carcinoma.

Impact of immunoglobulin preparations on anti-HLA antibody specificity analysis

Abstract Background Donor-specific antibodies (DSAs) targeting human leukocyte antigens (HLAs) substantially reduce the longevity of transplanted organs. Desensitization of DSA-positive renal transplant recipients is achieved through intravenous administration of immunoglobulin (IVIg). However, the presence and detectability of anti-HLA antibodies in IVIg preparations following administration are not fully understood. We aimed to assess whether immunoglobulin preparations contain anti-HLA antibodies that can be detected as passive antibodies when administered into the body. Methods We evaluated 3 immunoglobulin preparations from different pharmaceutical companies, using anti-HLA class I and II antibody specificity tests and immunocomplex capture fluorescence analysis (ICFA). Results Direct testing for anti-HLA antibodies resulted in high background errors, particularly for Venoglobulin. Diluting Venoglobulin to physiological concentrations revealed the presence of anti-HLA class I antibodies; however, no common alleles were found between the specificity identification test and ICFA. For Glovenin and Venilon, anti-HLA class I and II antibodies were detected; however, variability was observed across different test reagent lots. Moreover, dilution of the globulin formulation revealed a prozone phenomenon. Conclusion The administration of IVIg complicates the accurate detection of anti-HLA antibodies, underscoring the need for careful interpretation of test results post-IVIg administration.

Quality control for serological testing

ObjectivesThe quality control of serological assays remains controversial. The aim of this project was to describe the problems associated with a working model for controlling these assays and solutions, including using a source of well-defined targets and acceptable limits, a process to identify lot-to-lot reagent variation and an interpretation of the result that accounted for the clinical situation. False-negative results are problematic but can be reduced by identifying and comparing reagent lot variation with previous results. MethodsThe components of the Quality Assurance strategy are the following: Lot-to-lot reagent and calibrator variation assessment; dynamic, big-data approach to determine accurate targets and acceptable limits for manufacturer-provided QC material; negative QC monitoring process; use of commutable EQA with a sufficient method subgroup size to assess bias; clinical assessment of any statistically flagged error; and provision of support to the clinician for the interpretation of results. ResultsThe model described has been used for twelve months, and acceptable variation has been maintained. ConclusionsThe paper presents a solution that emphasizes the early detection of reagent lot variation and patient risk rather than instrument control.Reducing the risk of a false result to patients requires optimal assay quality control and an effective mechanism to support the clinician’s use of these results in diagnosis and monitoring. The problems of serological assays are well-known, but there remain few integrated solutions in the literature.

Recent Recall of Iron Reagent-Investigation of Potential Reagent Contamination and Assay Improvement Strategy.

Recently, a major manufacturer recalled several lots of iron assay reagent due to positive bias of roughly 15%-30% and the cause remains unknown. This study investigated the root cause of this positive bias and evaluated a simple practical approach to improve the assay. Performance comparison of recalled and unimpacted iron assay kits was done utilizing calibrators, quality control (QC) materials, and 42 remnant patient samples. Spectral scan and trace elements analysis of R1 and R2 reagents was performed. Copper (Cu) and thiourea (TU) spiking experiments were utilized to elucidate the cause and prevention of positive bias seen with recalled lots. Iron measurements in QC materials and patient samples using recalled reagents generated a positive bias of 17.5% and 21%, respectively. Correspondingly, the recalled R2 reagents, but not R1, showed a rise in basal absorbance along with an unanticipated presence of Cu (22.7 µg/dL) and lead (7.5 µg/L). Cu spiking to recalled and unimpacted R2 reagent intensified the reagent color besides falsely increasing its absorbance, calibration factor, and patient iron measurements. Interestingly, addition of TU (65 mmol/L) to R2 reagent from unimpacted lot prevented the short-term and prolonged Cu-induced spurious rise in calibration factor and patient iron estimations. We conclude that accidental copper contamination of R2 reagent during manufacturing could be a reason underlying the positive bias in the recalled iron reagent lots. Addition of TU in ferene-containing R2 reagent is a simple and effective means to prevent Cu-induced false elevation in iron values.

A modified quality control protocol for infectious disease serology based on the Westgard rules

When traditional statistical quality control protocols, represented by the Westgard protocol were applied to infectious disease serology, the rejection limits were questioned because of the high rejection probability. We first define the probability of false rejection (Pfr) and error detection (Ped) for infectious disease serology. QC data in 6 months were collected and the Pfr of each rule in the Westgard protocol and Rilibak protocol was evaluated. Then, as improvements, we chose different rules for negative and positive QC data to constitute an asymmetric protocol, furthermore, while reagent lot changes, the mean value of QC protocol is reset with the first 15 QC results of new lot reagent. QC materials and Standard Reference Materials were tested synchronously in the next 6 months, to verify whether the Pfr and Ped of the asymmetric protocol could meet the requirement. Protocol 1 exhibited the higher level of rejection rate among the two protocols, especially after reagent lot changes; Pfr below the lower control limit (LCL) was 1.39–21.78 times higher than the upper control limit (UCL); false rejections were more likely to occur in negative QC data, with Pfr-total of 27–65%. The asymmetric protocol can significantly reduce the proportion of analytes with Pfr by over 20%. Systematic error due to reagent lot changes and random error due to routine QC data variation were considered potential factors for excessive Pfr. Asymmetric QC protocol that can reduce Pfr by different control limits for negative and positive QC data.

Rapid-Test Kit for Cardiac Troponin I: A Reliable Enzyme-Linked-Immuno-Substrate-Assay-Based Biosensor for Daily-Use Naked-Eye Detection and Pharmacokinetic Studies for Myocardial Infarction in Cardiovascular Disease.

Myocardial infarction (MI) is a severe cardiovascular event that can lead to death. Cardiac troponin I (cTnI) is an MI biomarker in the circulation system; however, methods for detecting cTnI protein require substantial time, tedious operations, an expensive reader for translating signals, and a lot of reagents. This study aims to create a cTnI protein test kit with results easily distinguished by color differences, explicitly focusing on the resolution between different concentrations that eyes can discern. These results will aid in creating a commercial, portable, convenient, daily-use rapid-test kit. This study proposes a cTnI biosensor that the naked eye can perceive, performs diagnoses based on pattern color, does not require a reader machine, is easy to operate, and is portable. Our device shortens diagnosis time, has a 0.32-200 ng/mL quantitative analysis range in the human serum matrix, achieves a 0.32 ng/mL limit of detection, and exhibits many advantages compared to a traditional cTnI ELISA plate.

Robust Regression Techniques for Multiple Method Comparison and Transformation.

A generalization of Passing-Bablok regression is proposed for comparing multiple measurement methods simultaneously. Possible applications include assay migration studies or interlaboratory trials. When comparing only two methods, the method boils down to the usual Passing-Bablok estimator. It is close in spirit to reduced major axis regression, which is, however, not robust. To obtain a robust estimator, the major axis is replaced by the (hyper-)spherical median axis. This technique has been applied to compare SARS-CoV-2 serological tests, bilirubin in neonates, and an in vitro diagnostic test using different instruments, sample preparations, and reagent lots. In addition, plots similar to the well-known Bland-Altman plots have been developed to represent the variancestructure.

Synthetic abnormal mast cell particles successfully mimic neoplastic mast cells by flow cytometry.

Clinical flow cytometry laboratories require quality control materials for assay development, validation, and performance monitoring, including new reagent lot qualification. However, finding suitable controls for populations with uncommonly expressed antigens or for rare populations, such as mast cells, can be difficult. To that end, we evaluated synthetic abnormal mast cell particles (SAMCP), developed together with, and manufactured by, Slingshot Biosciences. The SAMCP's were designed to phenotypically mimic abnormal neoplastic mast cells: they were customized to have the same light scatter and autofluorescence properties of mast cells, along with surface antigen levels of CD45, CD33, CD117, CD2, CD25, and CD30 consistent with that seen in mast cell disease. We evaluated several performance characteristics of these particles using ARUP's high sensitivity clinical mast cell assay, including limit of detection, off-target activity and FMO controls, precision, scatter properties of the particles utilizing several different cytometer platforms, and particle antigen stability. The phenotype of the SAMCP mimicked abnormal mast cells, and they could be distinguished from normal native mast cells. FMO controls demonstrated specificity of each of the markers, and no off-target binding was detected. The limit of detection of the particles spiked into normal bone marrow was found to be ≤0.003% in a limiting dilution assay. The mast cell particles were found to perform similarly on Becton Dickinson Lyric, Cytek Aurora, and Beckman Coulter Navios and CytoFLEX platforms. Within run and between run precision were less than 10% CV. SAMCP were stable up to 13 days with minimal loss of antigen fluorescence intensity. The SAMCP's were able to successfully mimic neoplastic mast cells based on the results of our high sensitivity mast cell flow cytometry panel. These synthetic cell particles represent an exciting and innovative technology, which can fulfill vital needs in clinical flow cytometry such as serving as standardized control materials for assay development and performance monitoring.

Analytical validation of the Mindray CL1200i analyzer high sensitivity cardiac troponin I assay: MERITnI study.

This study performed an analytical validation study of the Mindray high-sensitivity cardiac troponin I (hs-cTnI) assay addressing limit of blank (LoB), limit of detection (LoD), precision, linearity, analytical specificity and sex-specific 99th percentile upper reference limits. LoB, LoD, precision, linearity and analytical specificity were studied according to Clinical and Laboratory Standards Institute. We used one reagent lot and one CL1200i analyzer. Skeletal troponin I and T, cardiac troponin T, troponin C, actin, tropomyosin, myosin light chain, myoglobin and creatine kinase (CK-MB) were studied for cross-reactivity. Interference with biotin was examined. Lithium heparin samples (one freeze thaw cycle) from healthy males and females were measured to determine the 99th percentiles by using the non-parametric method. Analyses were performed before and after excluding subjects with clinical conditions and/or increased surrogate biomarkers. The Mindray hs-cTnI assay met criteria to be considered as a hs-cTn assay. LoB and LoD was <0.1 ng/L and 0.1 ng/L, respectively. Repeatability had a coefficient of variation 1.2-3.8 %, and within-laboratory imprecision 1.7-5.0 %. The measuring interval ranged from 1.1 to 28,180 ng/L. The analytical specificity was clinically acceptable for the interferents studied. After exclusions, the 99th percentile URLs obtained were 10 ng/L overall, 5 ng/L for females and 12 ng/L for males. Analytical observations of the Mindray hs-cTnI assay demonstrated excellent LoB, LoD, precision, linearity and analytical specificity, that were in alignment with the manufacturer's claims and regulatory guidelines for hs-cTnI. The assay is suitable for clinical investigation for patient-oriented studies.

Methods and reagent-lot comparisons by regression analysis: sample size considerations.

Parametric regression analysis is widely used in methods comparisons and more recently in checking the concordance of test results following receipt of new reagent lots. The greater frequency of reagent-lot evaluations increases pressure to detect bias with smallest possible sample sizes (i.e. smallest consumption of time and resources). This study revisits bias detection using the joint slope, intercept confidence region as an alternative to slope and intercept confidence intervals. Four cases were considered representing constant errors, proportional errors (constant CV) and two more complicated error patterns typical of immunoassays. Maximum:minimum range ratios varied from 2:1 to 2000:1. After setting a maximum tolerable difference a series of slope, intercept combinations, each of which predicted the critical difference, were systematically evaluated in simulations which determined the minimum sample size required to detect the difference, firstly using slope, intercept confidence intervals and secondly using the joint slope, intercept confidence region. At small to moderate range ratios, bias detection by joint confidence region required greatly reduced sample sizes to the extent that it should encourage reagent-lot evaluations or, alternatively, transform those already routinely performed into considerably less costly exercises. While some software is available to calculate joint confidence regions in real-life analyses, shifting this testing method into the mainstream will require a greater number of software developers incorporating the necessary code into their regression programs. The computer program used to conduct this study is freely available and can be used to model any laboratory test.

Abstract 7622: ASPYRE-Lung: Validation of a simple, fast, robust and novel method for multi-variant genomic analysis of actionable NSCLC variants in tissue

Abstract Genomic variant testing of tumors is critical for patients to access the full potential of personalized oncology therapeutics. Current methods such as NGS are costly and challenging to interpret, while PCR assays are limited in the number of variants they can cover. We developed ASPYRE® (Allele-Specific PYrophosphorolysis REaction) technology to address the urgent need for rapid, accessible and affordable diagnostics informing actionable genomic target variants of a given cancer. The targeted ASPYRE-Lung® panel for NSCLC covers 114 variants in 11 genes (ALK, BRAF, EGFR, ERBB2, KRAS, RET, ROS1, MET &amp; NTRK1/2/3) to robustly inform clinical management, based on practice guidelines. The assay detects single nucleotide variants, insertions, deletions, and gene fusions from tissue-derived DNA and RNA simultaneously. We tested the limit of detection, specificity, analytical accuracy and analytical precision of ASPYRE-Lung using FFPE lung tissue samples from patients with NSCLC, variant-negative FFPE tissue from tonsil tissue, and FFPE-based contrived samples with controllable variant allele fractions. The sensitivity of ASPYRE-Lung was determined to be ≤ 3% variant allele fraction for single nucleotide variants and insertions or deletions, ≤ 100 copies for fusions, and ≤ 200 copies for MET exon 14 skipping. The specificity was 100% with no false positive results. The analytical accuracy test yielded no discordant variants between ASPYRE-Lung and orthogonal testing (targeted enrichment NGS) or contrived samples, and results were replicable across operators, reagent lots, runs, and real-time PCR instruments with a high degree of precision. The technology is simple and fast, requiring only four reagent transfer steps using standard laboratory equipment (PCR and qPCR instruments) with analysis via a cloud-based analysis algorithm. ASPYRE-Lung has the potential to be transformative in facilitating access to rapid, actionable molecular profiling of tissue for patients with NSCLC. Citation Format: Ryan T. Evans, Elizabeth Gillon-Zhang, Julia Brown, Katherine Knudsen, Candace King, Amanda S. Green, Ana-Luisa Silva, Justyna M. Mordaka, Rebecca N. Palmer, Alessandro Tomassini, Alejandra Collazos, Christina Xyrafaki, Iyelola Turner, Chau Ha Ho, Dilyara Nugent, Jinsy Jose, Simonetta Andreazza, Kristine von Bargen, Eleanor R. Gray, Magdalena Stolarek-Januszkiewicz, AIshling Cooke, Wendy J. Levin, Honey V. Reddi, Barnaby W. Balmforth, Robert J. Osborne. ASPYRE-Lung: Validation of a simple, fast, robust and novel method for multi-variant genomic analysis of actionable NSCLC variants in tissue [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7622.