Pooled Samples Research Articles

Intergroup variability of the khakas based on non-metric cranial traits data

Introduction. The formation of the gene pool of modern Khakas people occurred through the interaction of various groups of ancient inhabitants of the Minusinsk Basin. This article is focused on the inter-group analysis of craniological series representing different Khakassian subethnic groups. Materials and Methods. The cranial series of the Kachins, Koibals, Sagais, and Beltirs was studied using the battery of 36 cranial non-metric characteristics. The obtained data was compared with data on other populations of Southern and Western Siberia: Shors, Tuvans, Telengits, Selkups, Khants, and Mansis. The analysis of the biological diversity of populations was carried out using Smith's mean measure of divergence (MMD) followed by multidimensional scaling and cluster analysis. Results. The Kachins are the most distant from the other Khakas subethnic groups, forming a cluster with the Telengits and Tuvans. The Koibals and Beltirs were positioned quite close to the Shors. The Sagais occupied a separate position in the Khakas cluster, presumably due to a greater proportion of Europoid admixture compared to other Khakas groups. The pooled Khakas sample shares similar cranial non-metric characteristics with Turkic-speaking ethnic groups of Southern Siberia: Shors, Telengits, and Tuvans. Discussion. The nature of phenetic differentiation of the Khakas sub-ethnic groups presumably reveals their complex population history. The position of the Kachins outside the Khakas cluster based on non-metric traits resulted from specificity of their phenofund, which is also supported by molecular genetic data. The similarity in the phenofund of the Khakas groups to those of the Tuvans and Telengits may result from gene flow between Khakas populations and the peoples of Southern Siberia or/and from their common episodes in their ethnogenesis. The similarity between pooled Khakassian sample and the Shors presumably suggests involvement of common genetic components in the gene pools of these peoples, which is consistent with molecular genetic data. Conclusion. The correspondence of the obtained results with genetic data suggests the possibility of using cranial non-metric traits to identify genetic relationships between ancient populations in the absence of direct genetic information.

Multiplexed chromatin immunoprecipitation sequencing for quantitative study of histone modifications and chromatin factors.

ChIP-seq is a widely used technique for studying histone post-translational modifications and DNA-binding proteins. DNA fragments associated with a specific protein or histone modification epitope are captured by using antibodies, sequenced and mapped to a reference genome. Albeit versatile and popular, performing many parallel ChIP-seq experiments to compare different conditions, replicates and epitopes is laborious, is prone to experimental variation and does not allow quantitative comparisons unless adequate spike-in chromatin is included. We present a detailed protocol for performing and analyzing a multiplexed quantitative chromatin immunoprecipitation-sequencing experiment (MINUTE-ChIP), in which multiple samples are profiled against multiple epitopes in a single workflow. Multiplexing not only dramatically increases the throughput of ChIP-seq experiments (e.g., profiling 12 samples against multiple histone modifications or DNA-binding proteins in a single experiment), but also enables accurate quantitative comparisons. The protocol consists of four parts: sample preparation (i.e., lysis, chromatin fragmentation and barcoding of native or formaldehyde-fixed material), pooling and splitting of the barcoded chromatin into parallel immunoprecipitation reactions, preparation of next-generation sequencing libraries from input and immunoprecipitated DNA and data analysis using our dedicated analysis pipeline. This pipeline autonomously generates quantitatively scaled ChIP-seq tracks for downstream analysis and visualization, alongside necessary quality control indicators. The entire workflow requires basic knowledge in molecular biology and bioinformatics and can be completed in 1 week. MINUTE-ChIP empowers biologists to perform every ChIP-seq experiment with an appropriate number of replicates and control conditions, delivering more statistically robust, exquisitely quantitative and biologically meaningful results.

Identification of Potential Growth-Related Proteins in Chick Vitreous during Emmetropization Using SWATH-MS and Targeted-Based Proteomics (MRMHR).

The vitreous humor (VH) is a transparent gelatin-like substance that occupies two-thirds of the eyeball and undergoes the most significant changes during eye elongation. Quantitative proteomics on the normal growth period in the VH could provide new insights into understanding its progression mechanism in the early stages of myopia. In this study, a data-independent acquisition (SWATH-MS) was combined with targeted LC-ESI-MS/MS to identify and quantify the relative protein changes in the vitreous during the normal growth period (4, 7, 14, 21 and 28 days old) in the chick model. Chicks were raised under normal growing conditions (12/12 h Dark/light cycle) for 28 days, where ocular measurements, including refractive and biometric measurements, were performed on days 4 (baseline), 7, 14, 21 and 28 (n = 6 chicks at each time point). Extracted vitreous proteins from individual animals were digested and pooled into a left eye pool and a right pool at each time point for protein analysis. The vitreous proteome for chicks was generated using an information-dependent acquisition (IDA) method by combining injections from individual time points. Using individual pool samples, SWATH-MS was employed to quantify proteins between each time point. DEPs were subsequently confirmed in separate batches of animals individually on random eyes (n = 4) using MRMHR between day 7 and day 14. Refraction and vitreous chamber depth (VCD) were found to be significantly changed (p < 0.05, n = 6 at each time point) during the period. A comprehensive vitreous protein ion library was built with 1576 non-redundant proteins (22987 distinct peptides) identified at a 1% false discovery rate (FDR). A total of 12 up-regulated and 26 down-regulated proteins were found across all time points compared to day 7 using SWATH-MS. Several DEPs, such as alpha-fetoprotein, the cadherin family group, neurocan, and reelin, involved in structural and growth-related pathways, were validated for the first time using MRMHR under this experimental condition. This study provided the first comprehensive spectral library of the vitreous for chicks during normal growth as well as a list of potential growth-related protein biomarker candidates using SWATH-MS and MRMHR during the emmetropization period.

B-355 Assessment of Calcitonin Cutoff Values for Post-Thyroidectomy Surveillance in Patients with Medullary Thyroid Carcinoma: Findings from a Single-Center Investigation

Abstract Background Patients with sporadic or hereditary Medullary Thyroid Carcinoma (MTC) often undergo total thyroidectomy and lymph node dissection, with postoperative surveillance relying on serum calcitonin levels and ultrasound examinations. According to American Thyroid Association (ATA) guidelines, detectable levels of serum calcitonin and Carcinoembryonic Antigen (CEA) post-thyroidectomy suggest potential disease persistence or recurrence, necessitating frequent monitoring for early detection. This study sought to reassess the role of postoperative calcitonin levels in identifying possible tumor recurrence. Methods A retrospective analysis was conducted using Siemens Atellica to test calcitonin levels in 562 samples collected between 09/27/2022 and 08/11/2023, at least 3 months post-total thyroidectomy. Imaging studies performed within 6 months of the calcitonin assessment were also reviewed. Sample pools with calcitonin concentrations near 3.00 and 5.00 pg/mL were utilized to determine the limit of quantification (LoQ). CEA results were included for comparison. Additionally, 16 serum samples were analyzed using the Roche Cobas system at a reference laboratory. Results Precision analysis around 3.00 and 5.00 pg/mL revealed coefficients of variation (CVs) of 16.49% and 8.87%, respectively. Comparison of calcitonin levels between Siemens Atellica and Roche Cobas systems demonstrated consistent levels &amp;lt; 5.00 pg/mL in 15 out of 16 samples, indicating alignment and reliability between the platforms. A calcitonin cutoff of 1.89 pg/mL yielded 43% sensitivity and 67% specificity, while a cutoff of 5.00 pg/mL resulted in 0% sensitivity and 100% specificity. Conclusions Our findings suggest that a 5 pg/mL calcitonin cutoff on the Siemens Atellica platform may be suitable for identifying tumor persistence or recurrence in post-thyroidectomy patients within our institution. However, individual laboratories should establish their LoQ criteria when interpreting calcitonin levels for postoperative monitoring of tumor recurrence.

A-136 Icteric Interference is not Resolved with Sample Dilution in Serum Creatinine and Total Protein Assays

Abstract Background Icterus is a common endogenous interference that can cause falsely low results for serum creatinine and total protein. It can be defined by elevated unconjugated (indirect) and conjugated (direct) bilirubin levels and is most commonly associated with liver dysfunction. Recent literature suggests that dilutions can be a reliable method to reduce bilirubin interference. The purpose of this study was to determine whether the vendor’s on-board sample dilution can eliminate icteric interference with creatinine and total protein. Methods Creatinine (enzymatic) and total protein interference studies were performed on an automated chemistry analyzer (Roche Diagnostics, cobas c702). Low, mid, and high sample pools for each analyte were divided into four equal volumes. To achieve an icterus index (II) of approximately 40 (approximately 40 mg/dL of bilirubin), one sample pool was spiked with unconjugated bilirubin, and one pool was spiked with conjugated bilirubin. An equal amount of diluent was added into each control pool and remained at an II of approximately 0. The icteric and control samples were measured for conjugated and unconjugated bilirubin, serum indices, and the target analyte. Using the on-board dilution function, the icteric creatinine and total protein pools were diluted using a 1:4 and 1:3 dilution, respectively. All samples were analyzed in triplicate. Percent variance was found by calculating the difference between the average dilution result and control result and then dividing by the average control result. Results Conclusions Only modest alleviation of icteric interference was noted with dilution. The average bias was 10% for creatinine and 24% with total protein, after dilution. The on-board sample dilution is not effective in eliminating icteric interference when measuring both creatinine and total protein.

A-056 Investigating Iodine Contrast Contamination in Cortisol and Aldosterone Samples

Abstract Background Primary aldosteronism (PA) is a condition characterized by hypertension resulting from excess secretion of aldosterone. PA caused by a unilateral aldosterone-producing adenoma can be treated with adrenalectomy but requires diagnosis by adrenal vein sampling (AVS). In this procedure, intravenous cosyntropin is administered to stimulate adrenal cortical secretion, followed by obtaining samples from the right and left adrenal veins and IVC. Laterization index is calculated by obtaining the aldosterone:cortisol ratio for each side, with a ratio cutoff ≥3 is used for confirmation. Iodinated contrast media facilitates visualization of catheter positioning during AVS and sometimes results in contamination of samples, evidenced by abnormal gel separator flotation (i.e. gel above plasma). This study had two aims: 1) to determine at what concentration contrast dye causes gel flotation and 2) discern whether contrast media interferes with our cortisol or aldosterone assays. Methods Gel flotation studies were performed by spiking pooled plasma samples with iohexol contrast media (Omnipaque 240, GE Healthcare) at concentrations ranging from 3%-10% v/v and aliquoting into fresh lithium heparin plasma separator tubes (BD, PSTs). Samples were then centrifuged and visually inspected for abnormal gel flotation. Contrast interference studies were performed using three pools of low (5 µg/dL), medium, (30 µg/dL), and high (55 µg/dL) cortisol concentrations. Sample pools were diluted with 20% contrast media or saline (control) and subsequently mixed at different ratios to create a range of dye concentrations (0.5%-18% v/v). Each mixture was assayed in triplicate for cortisol and aldosterone on a cobas e602 (Roche Diagnostics) and LIAISON XL (DiaSorin) immunoanalyzer, respectively. The total allowable error (TEa) was ±0.4 μg/dL or ±20% for cortisol and ±2 ng/dL or ±20% for aldosterone. Results Flotation of the gel separator was observed at a dye concentration of 4.5% v/v and greater. For the cortisol interference study, the bias was &amp;lt;5% for the three pools at all contrast media concentrations. Aldosterone was also measured in these pools, as bias could affect ratios calculated for PA. Aldosterone interference occurred at contrast dye concentrations ranging from 12%-20% v/v. An average positive bias of 34% was observed in aldosterone samples spiked with contrast media relative to the saline control. Compared to sample pool controls without added saline or dye, measurements for cortisol and aldosterone were 19%-22% and 16-25% lower in media- and saline-spiked samples (20% v/v), respectively, indicating a logical dilution effect. Conclusions Contrast dye contamination of AVS specimens can cause gel flotation in lithium heparin PSTs, preventing their analysis. The presence of contrast media at certain concentrations interfered with the DiaSorin aldosterone assay, leading to falsely elevated results. No cortisol assay interference occurred in the presence of contrast media but results were falsely decreased due to sample dilution. Samples collected during AVS may be contaminated with contrast dye causing gel flotation to occur. This is indicative of a minimum contrast media concentration of 4.5% v/v. Aldosterone:cortisol ratios may be inaccurate due to the positive aldosterone immunoassay bias with contrast media contamination. Care should be taken when reporting and interpreting AVS results with suspected contamination.

B-127 The Effect of Hemolysis on Potassium Measurement in a Community Setting: The Marching Error

Abstract Background Potassium is one of the most sensitive analytes to in-vitro hemolysis. To detect this error, modern clinical chemistry analyzers calculate the hemolysis index (HI) to estimate the degree of specimen hemolysis. Recently, Roche Diagnostics lowered the acceptable potassium HI limit from 100 to 20 and recommended laboratories avoid using specimens with an HI&amp;gt;20. The goal of this study was to determine a clinically acceptable HI cut-off for potassium at our community reference laboratory, relying on patient data from routine laboratory operation. Methods Plasma venous potassium, sodium, and HI test results from community patients measured on Roche cobas 8000 ISE and c701 modules between September 2022 to December 2023 were extracted from the Roche Infinity Middleware (n=1,101,897). Data was divided into groups based on HI which ranged from 0 to 300. The sample pool with the lowest level of hemolysis (HI≤10) was defined as the base group. Mean and standard deviation of potassium for each HI group was calculated and the difference in the mean between each group compared to the base group was calculated as both absolute and percent ΔK. Percent changes in critical values for both potassium and sodium as a function of hemolysis were calculated for each HI group. Measurement uncertainty (MU) for potassium was calculated using a top-down approach. Data were analyzed in Rstudio and Microsoft Excel. Results The vast majority of potassium specimens (83.8%) were stratified into the HI base group (HI ≤10) and 96.8 % had an HI ≤ 20, the cut-off recommended by Roche. The mean potassium level for each HI group increased as the degree of hemolysis in the specimens became more elevated. A MU-derived analytically significant shift of ΔK&amp;gt;0.1 mmol/L was observed for the bin HI 21-30. Depending on the employed total allowable error limit, clinically significant shifts in mean potassium levels were observed at HI bin 41-50 (RCPA: ΔK&amp;gt;0.2 mmol/L) or HI bin 101-150 (EFLM-derived reference change value: ΔK&amp;gt;10.1% and CLIA: ΔK&amp;gt;0.5mmol/L. The shift in patient potassium values seen as HI increased led to a change in critical results reporting frequencies. In the base group (HI≤10), 0.06% and 0.07% of results had low (&amp;lt;2.6 mmol/L) or high (&amp;gt;6.2 mmol/L) critical potassium values, respectively. When HI was 101-300, 0.00 and 15.7% of results had low or high critical potassium values. A similar comparison performed with sodium did not show the same shift in reported critical values. Linear regression of ΔK plotted vs HI produced the equation ΔK=0.0051xHI, validating the equation of Martinez-Morillo and Alvarez published previously using paired hemolyzed/non-hemolyzed potassium laboratory data. Conclusions In community patients, in-vitro hemolysis produced analytically significant errors at an HI of 20. Clinically significant errors occurred between an HI of 40-100. Past HI 100, mean ΔKs of 0.5-1.20 mmol/L were seen in our population. This shift in patient values as a function of hemolysis led to a change in critical result reporting, suggesting the generation of both factious critically high potassium results and the masking of critically low potassium results in our patient population.

A-098 Reducing Hemolyzed Specimen Rejection for Aspartate Aminotransferase (AST): A Quality Improvement Initiative to Further Optimize Concentration-Specific H-index Thresholds

Abstract Background Serum aspartate aminotransferase (AST) is a commonly ordered test useful for diagnosing and monitoring hepatocellular damage. The hemolysis (H) index interference threshold for the Roche Cobas AST reagent is H=20, which is the lowest of all tests on Roche chemistry platforms (Cobas c701, c502, and e801). Previous optimization studies supported the implementation of an AST concentration-specific tiered hemolysis interference threshold schema: AST&amp;lt;100 U/L (H=50), AST=100-200 U/L (H=100), AST=201-300 U/L (H=200) and AST&amp;gt;301 U/L (H=500). This schema significantly reduced redraws; however, a significant number of rejected samples were noted to have AST results within the reference interval (RI). The aim of this study was to establish an H-index threshold for samples with AST results within the RI. Methods Twelve pools of residual serum samples were created with AST concentrations ranging from 17-66 U/L. Each pool was split into 2 aliquots, receiving either 10% of a lab-produced hemolysate solution generating H-indices from 62-508 or 10% of a 0.9% saline control. Aliquots were mixed in equal portions to create a series of 5 samples with increasing hemolysis according to CLSI guideline EP-07 and analyzed in EP Evaluator. Median(interquartile range (IQR)) H-index thresholds were calculated for error limits beginning at +5 U/L and increasing incrementally (+1 U/L) to +10 U/L. Clinical cases with an H-index slightly above the hemolysis interference threshold (50≤H≤70) and results within the RIs (Male:8-48U/L; Female:8-43U/L) were reviewed by the Chemistry Resident/Fellow on-call (n=68) over a period of 8 months. Results were released with a comment if risk of clinical misinterpretation due to a falsely elevated result was low. Previous, non-hemolyzed AST results were compared within 45 days, if available, by paired-Wilcoxon test (Graphpad Prism). Results The median(range) of initial AST results in the serum pools was 20(17-66) U/L. The calculated H-index thresholds (median(IQR)) using error limits of +5, +6, +7, +8, +9, +10 U/L were 49(43-53), 56(52-63), 66(61-78), 78(72-81), 91(84-91) and 101 (96-107), respectively. All slightly hemolyzed AST results (50≤H≤70) within the RI were released with a comment upon chart review. Previously reported AST values (n=23) were compared to current (hemolyzed) concentrations and found to be statistically significantly increased relative to previous results from the same patient (median(range) difference: 3.7(-3.6-11)U/L, p=0.009). However, the median difference was &amp;lt;5U/L or 10%, the laboratory’s criteria used to define a clinically significant difference. Conclusions A median H-index limit of up to 100 can be applied to samples when AST is within the RI. The risk of misinterpretation for results with mild-moderate hemolysis was low in the cases reviewed. Sample rejection rates and reporting delays can be diminished by implementing this practice. Interpretation can be further enhanced by appending the following comment to hemolyzed samples: “Specimen was received with hemolysis slightly above the accepted threshold. This degree of hemolysis is associated with a false increase of approximately 5-10 U/L. Interpret in conjunction with other laboratory and clinical findings.”

Retrospective analysis of SARS-CoV-2 RNA detection in pooled saliva samples: An effective cost saving method.

Retrospective analysis of SARS-CoV-2 RNA detection in pooled saliva samples: An effective cost saving method.

Soil nematode community profiling using reference-free mito-metagenomics

Nematodes are ubiquitous and diverse components of soil ecosystems worldwide. The 18S-based metabarcoding is known to have low species-level resolution and introduce bias in PCR. The mito-metagenomics (MMG) approach involves directly sequencing pooled samples, yields numerous mitochondrial reads that can be assembled into full or partial mitogenomes. This method circumvents the challenges associated with PCR-based metabarcoding and hold significant promise in biodiversity and phylogeny study. However, a reference database is typically required to extract mito-reads/contigs and provide taxonomic or phylogenetic context, thereby limiting its applicability. In this study, we introduced a novel reference-free pipeline for MMG assembly and diversity estimation. This pipeline has been integrated into a snakemake workflow, enabling the generation of output that is readily useable for phylogeny reconstruction in a single run. The performance tests have indicated that this new approach surpasses reference-based methods in soil nematode community profiling. We demonstrated that assembly quality improves with increasing sequencing depth, recommending an average of 1–2 Gb per species to achieve acceptable MMG assembly. Our pipeline presents an opportunity to create high-resolution phylogenies and assess diversity for poorly understood taxa, including neglected microscopic eukaryotes.

A-030 Evaluation of the Analytical Performance of Two Reproductive Endocrinology Related Assays on the Atellica CI Analyzer

Abstract Background The Atellica® CI Analyzer is an automated, high-throughput integrated chemistry and immunoassay analyzer utilizing both Atellica® CH and Atellica® IM Assays. This study evaluated the analytical performance of the Atellica IM Sex Hormone Binding Globulin (SHBG) and the total Testosterone II (TSTII) Assays on the Atellica CI Analyzer. Methods Precision studies were performed according to CLSI EP05-A3 using native and contrived human serum samples. One aliquot of each sample pool was tested in duplicate in two runs per day ≥2 hours apart on each analyzer for ≥20 days. Precision studies were evaluated with one reagent lot on two systems. Method comparison (MC) studies were performed using three reagent lots according to CLSI EP09c. Individual native and contrived human serum samples were analyzed using the Atellica IM SHBG and TSTII Assays on the Atellica IM Analyzer and the Atellica CI Analyzer. LoB, LoD, and LoQ were determined as described in CLSI protocol EP17-A2. Results Representative precision and MC results for each assay are listed in the table. For the two assays tested, repeatability and within-lab %CVs were &amp;lt;4.0% and &amp;lt;6.6%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1. Detection capability for the Atellica IM SHBG Assay on the Atellica CI Analyzer was estimated at 1.33 nmol/L (standard international (SI) units; 0.13 μg/mL, common units), 1.60 nmol/L (0.15 μg/mL) and 2.00 nmol/L (0.19 μg/mL) for LoB, LoD, and LoQ respectively. When using the Atellica IM TSTII Assay, detection capability was estimated at 0.09 nmol/L (2.50 ng/dL), 0.17 nmol/L (5.00 ng/dL) and 0.24 nmol/L (7.00 ng/dL) for LoB, LoD, and LoQ, respectively. Conclusions Evaluation of the Atellica IM SHBG and TSTII Assays using the Atellica CI Analyzer demonstrated good precision and equivalent performance compared to the same assays on the Atellica IM Analyzer.

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

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

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

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

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

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

A-034 Evaluation of the Analytical Performance of Five Chemistry Assays on the Atellica CI Analyzer

Abstract Background The Atellica® CI Analyzer is an automated, mid-throughput, integrated chemistry and immunoassay analyzer utilizing both Atellica® CH and Atellica® IM assays. This study evaluated the analytical performance of the Atellica® CH Alpha-1-antitrypsin (AAT), Ammonia (AMM), Amylase_2 (AMY_2), Cholinesterase_2 (CHE_2), Lipase (Lip) Assays on the Atellica CI Analyzer. Methods The Atellica CI assays use the same reagents and calibrators as the Atellica CH assays. Precision studies were performed according to CLSI EP05-A3 using human urine (AMY_2), serum (AAT, AMY_2, CHE_2, Lip), and plasma (AMM) samples. One aliquot of each sample pool was tested in duplicate in two runs per day for 20 days. MC studies were performed according to CLSI EP09-A3. Samples were analyzed using the Atellica CH and Atellica CI Analyzers. Results Across the five assays, repeatability CVs were &amp;lt;5.7% and within-laboratory CVs were &amp;lt;8.2%. Representative precision, and MC results observed for each assay across indicated sample ranges are presented in the table. Slopes determined using the Deming linear regression model were approximately equal to 1. Conclusions Atellica CH AAT, AMM, AMY_2, CHE_2, and Lip Assays on the Atellica CI Analyzer demonstrated good precision and equivalent performance to the same assays on the Atellica CH Analyzer.

A-096 A New Direct Bilirubin (DBIL) Assay with Enhanced Performance in Anti-hemolysis Interference on Mindray BS-2800M Analyzer

Abstract Background Serum bilirubin could be used to assess liver functions. Due to clinical importance, it is highly desired to have an accurate bilirubin test. However, there are circumstances where error could occur in bilirubin test results. In particular, the hemolysis interference is one of the major problems that could mislead the direct bilirubin results because of their relatively high occurring frequencies in the clinical laboratories. To resolve this issue and improve the accuracy for the direct bilirubin, we developed a new reagent formulation for DBIL assay that minimizes hemolysis interference. In addition, this reagent also shown good performance characteristics on Mindray BS-2800M Clinical Chemistry analyzer. Methods The new DBIL assay is a vanadate-oxidation method that bilirubin is gradually transformed to biliverdin because of oxidizing agent vanadate, resulting in decreasing of absorbance at 450 nm. Two-level calibration is used for this assay. DBIL concentrations are obtained from the calibration line with signals from the patient samples in the assay. Following CLSI protocols, basic assay performances were evaluated: interference, precision, linearity, method comparison and low end detection (LOB, LOD, LOQ). Results Hemolysis interference was tested in parallel with DBIL reagents from different IVD companies, Roche, Wako and Chinese domestic main-player companies, A, B, and C. The results showed that the reagents of A and B companies were significantly suffered from hemolysis interference with maximum bias exceeded -30%. Although there are some effects on the anti-hemolysis with DBIL reagents of Wako and C company, the DBIL reagent of Mindray was the best with less than -8% of impact when hemolysate hemoglobin was up to 1000 mg/dL (H index). The Diazo DBIL assay from Roche shown issue with hemolysis interference. The interference to our new DBIL assay from lipid (up to 1000 mg/dL) and ascorbic acid (up to 30 mg/dL) were also with less than ±10% impact. The precision study was run according to EP05A3 with two commercial controls (∼ 10 and 30 µmol/L) and three serum sample pools (∼ 6.8,17.1 and 342 µmol/L). The CVs of repeatability were ranged from 0.54% to 0.58% for controls and 0.24% to 0.88% for serum pools, respectively. The within-lab precision was below 2.5%. The analytical linearity was determined as from 0.66 µmol/L to 439.66 µmol/L. For low-end detection, the LOB, LOD and LOQ were 0.5,1.0 and 1.7 µmol/L, respectively. In addition, the Mindray (y) correlated well with the DBIL assay of Wako (x) reagent on Mindray BS-2800M system (y): y =0.9847 x+0.1301 (r =0.9997, n =400, sample range: 1.08 - 430.00 μmol/L). Conclusions The new Mindray direct bilirubin assay shown a good anti-hemolysis interference performance on BS-2800M system and demonstrated good performances in other assay characteristics. It is therefore suitable for use in clinical laboratories.

A-014 Fifth Generation Troponin T is Unaffected by Extreme Levels of Lipemia and Icterus

Abstract Background The tissue specific cardiac isoform of troponin (cTnT) originates exclusively from the myocardium; therefore, it is used as the primary diagnostic biomarker for myocardial infarction. Lipemia, caused by elevated lipoprotein particles, and icterus, caused by elevated bilirubin, are common endogenous interferences that can lead to erroneous results. According to the instructions for use (IFU), recovery is acceptable when bilirubin levels are ≤25 mg/dL (conjugation not specified) and intralipid levels are ≤1500 mg/dL. However, it is common in large hospital settings to observe higher levels for these potential interferents. The purpose of this study was to determine whether 5th generation cTnT results are negatively impacted by extreme levels of lipemia or conjugated or unconjugated bilirubin. Methods cTnT interference studies were performed on a cobas e602 (Roche Diagnostics). Low, mid, and high cTnT sample pools at approximate concentrations of 20 ng/L, 100 ng/L, and 1000 ng/L were split into target and control pools. To evaluate the effect of lipemia, target pools were spiked with intralipid solution to achieve a lipemia index (LI) ranging from 1500-2000 (correlates poorly with mg/dL triglycerides). Samples were analyzed in triplicate for cTnT and serum indices. To evaluate the effect of icterus, target pools were spiked with conjugated or unconjugated bilirubin to achieve an icterus index (II) ranging from 30-55 (approximately equal to mg/dL bilirubin). Samples were analyzed in triplicate for cTnT, conjugated and unconjugated bilirubin, and serum indices. Interference was determined by calculating the variance between the average target and corresponding control result. Results Conclusions cTnT has a high lipemia and icterus tolerance with recoveries &amp;lt;10% for all three cTnT concentrations.

A-053 Evaluation of the Analytical Performance of Atellica CH Prealbumin and β2-Microglobulin Chemistry Assays on the Atellica CI Analyzer

Abstract Background The Atellica® CI Analyzer is an automated, mid-throughput, integrated chemistry and immunoassay analyzer utilizing both Atellica® CH and Atellica® IM assays. This study was designed to evaluate the analytical performance of the Atellica® CH Prealbumin (PALB) and β2-Microglobulin (B2M) Assays on the Atellica CI Analyzer. Methods The Atellica CI PALB and B2M assays use the same reagents and calibrators as the Atellica CH assays. Precision and method comparison (MC) were used as performance indicators for the Atellica CI Analyzer. Precision studies were performed according to CLSI EP05-A3 using native, QC, and contrived human serum samples. One aliquot of each sample pool was tested in duplicate in two runs per day &amp;gt;2 hours apart on each analyzer for 20 days. Method comparison (MC) studies were performed per CLSI EP09-A3. Individual serum samples were analyzed using the Atellica CH and Atellica CI Analyzers. Linearity and verification of detection capability studies were demonstrated for both assays. Results Representative precision, and MC results are presented in the table. Over the two assays tested, repeatability and within-lab %CVs were &amp;lt;4.0% and &amp;lt;4.6%, respectively. Slopes determined using the Deming linear regression model were approximately equal to 1. The Atellica CH PALB assay is linear from 3.0-70.0 mg/dL (30-700 mg/L); LoB, 1.1 mg/dL (11 mg/L); LoD. 1.9 mg/dL (19 mg/L); LoQ, 3.0 mg/dL (30 mg/L). The Atellica CH B2M assay is linear from 0.25-18.00 mg/L; LoB, 0.10 mg/L; LoD, 0.20 mg/L. Conclusions Atellica CH PALB and B2M Assays on the Atellica CI Analyzer demonstrated good precision and equivalent performance to the same assays on the Atellica CH Analyzer.

B-153 Improving Accuracy of IgG subclasses Quantitation in Serum Using Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS) on SCIEX 7500

Abstract Background Serum IgG subclasses (IgGSC) are most often measured for identification of selective immunodeficiency disease and IgG4-related disease (IgG4RD). Traditional nephelometric (IN) assays suffer from errors related to antigen excess and cross-reactivity that can impact the accuracy of results. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been the routine testing methodology for serum IgG subclasses at St. Paul’s Hospital since 2015 due to the simplicity and freedom from inter-subclass cross-reactivity problems. In brief, 20 μL of serum sample is denatured, reduced, alkylated and digested, processed semi-automatically on Hamilton Starlet liquid handler. This is followed by analysis on a SCIEX 5500 QTRAP LC-MS/MS. However, when the method was migrated to the SCIEX 7500, it suffered from calibration curve splitting and poor between-device comparability. It was hypothesized that this could be caused by detuning of some high concentration analytes, suboptimal internal standard (IS) concentrations and, suboptimal IS multiple reaction monitoring (MRM) transition choices, and matrix effects from artificial calibrators. Methods Several modifications were made to improve accuracy and robustness: 1) new in-house calibrators were prepared using patient sample pool instead of using Binding Site IgG Subclass Kit calibrators. 2) More IS MRM transitions were added to the method with each analyte MRM quantitated using its own IS MRM using the same fragment ion. The IS concentration was also increased 5-fold for IgG1 and Total IgG. 3) To avoid problems arising from selectively ion detuning, the injection volume was reduced from 10 μL to 1 μL which permitted collision energies (CEs) to be normalized for each analyte/IS MRM pair. Results The new SCIEX 7500 method compares well with the original method (N=168) with Passing Bablok regression slopes of 0.94, 1.1, 1.0, 0.98, and 0.98 for IgG1-4 and IgG total respectively with corresponding intercepts of 0.3, -0.1, 0.0, 0.0, and 0.41 g/L and R^2 of 0.96, 0.95, 0.99, 0.98, and 0.95. Median differences were -1.76%, 1.07%, 1.06%, 6.13% and 1.91%. In a typical batch of 90 patient samples and four sets of calibrators, the calibration curves overlap well. Use of patient-based calibrators allowed calibration values to be centered in the range of typical patient sample values, particularly for IgG4, where the calibration curve was extended to 3.46 g/L compared with the previous high calibrator value of 0.48 g/L. Conclusions The new IgG subclass quantitation method on SCEIX 7500 LC-MS/MS system is robust with excellent reproducibility and between-device comparability.

B-045 Evaluation of the Analytical Performance of Three Sepsis Related Assays on the Atellica CI Analyzer

Abstract Background The Atellica® CI Analyzer is an automated, high-throughput integrated chemistry and immunoassay analyzer utilizing both Atellica® CH and Atellica® IM Assays. This study evaluated the analytical performance of the Atellica IM BRAHMS Procalcitonin (PCT) Assay, and the Atellica CH Revised C-Reactive Protein (RCRP) and Lactate_3 (Lac_3) Assays on the Atellica CI Analyzer. Methods Precision studies were performed according to CLSI EP05-A3 using native and contrived human serum (PCT, RCRP) or plasma and cerebrospinal fluid (Lac_3) samples. One aliquot of each sample pool was tested in duplicate in two runs per day ≥2 hours apart on each analyzer for ≥20 days. Precision studies were evaluated with one to three lots of reagent on one to two systems. Method comparison (MC) studies were performed using three reagent lots according to CLSI EP09c. Individual native and contrived human serum (PCT, RCRP) or plasma and cerebrospinal fluid (Lac_3) samples were analyzed using the Atellica IM PCT Assay on the Atellica IM Analyzer, the Atellica CH RCRP, Lac_3 Assays on the Atellica CH Analyzer and all three Atellica IM and CH Assays listed above on the Atellica CI Analyzer. Results Representative precision and MC results for each assay are listed in the table. For the three assays tested, repeatability and within-lab %CVs were &amp;lt;1.4% and &amp;lt;8.9%, respectively. Slopes determined by the Deming linear regression model were approximately equal to 1. Conclusions Evaluation of the Atellica IM PCT and Atellica CH RCRP, Lac_3 Assays using the Atellica CI Analyzer demonstrated good precision and equivalent performance compared to the same assays on their respective Atellica CH and Atellica IM Analyzers.