Intra-assay Precision Research Articles

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-074 A Laboratory Developed Serum Anti-Mullerian Hormone Electrochemiluminescent Immunoassay: Performance and Retrospective Analysis of Over 2.2 Million Tests in Females and Males Across Lifespan

Abstract Background The quantification of serum Anti-Mullerian hormone (AMH), a glycoprotein dimer produced by granulosa cells of the ovarian follicle in females and by Sertoli cells of the testes in males, has clinical utility across lifespan in both females and males. AMH is most frequently ordered to assess fertility in females of reproductive age, where it correlates with ovarian reserve. The clinical applications of serum AMH beyond ovarian reserve assessment, especially in males, pediatric, and postmenopausal populations, include the investigation of peri-menopause transition, delayed or precocious onset of puberty, differential diagnosis of disorders of sexual development, the evaluation of male gonadal function, and as an ovarian tumor marker. The objective was to establish and validate a highly sensitive assay to quantify AMH and to use our database of more than 2 million tests to understand the clinical utility of measuring AMH across sex and lifespan. Methods A validated two-step electrochemiluminescence assay was used for the quantification of serum AMH. The analytical performance of the assay was assessed, including accuracy, precision, and dilutional linearity. ICD-10 codes ordered between 2012 and 2023 were retrospectively analyzed for each subgroup based on age and sex to determine test usage. The analysis includes results from 2,229,845 female and 10,362 male patients across lifespan. Results The AMH analytical measurement range (AMR) was determined to be 0.015 - 15.0 ng/mL with a maximum reportable concentration of 960 ng/mL when run on dilution. Both inter- and intra-assay standard accuracy and precision were ≤ ±15.4% bias and ≤ 11.0% CV for all levels. Inter- and intra-assay sample precision was ≤ 9.6% CV across the AMR. Samples diluted linearly and recovered within 80 - 120% of the expected values when diluted up to 64X. Analytical interference caused by icterus, lipemia, or hemolysis was not observed. The most common ICD-10 associated with AMH orders was for fertility testing in females of reproductive age. AMH was also ordered in females for evaluation of ovarian cancer, menstrual disorders, and PCOS. In males, common ICD-10 codes associated with AMH include delayed puberty, hypoplasia of the penis, fertility testing, hypogonadism, and childhood short stature. Conclusions This study demonstrates a highly sensitive assay for the quantification of serum AMH with clinical utility across sex and lifespan. Using retrospective data, we determined that in addition to being used primarily in fertility testing, AMH is also commonly used to evaluate and manage other endocrine disorders and malignancies, including disorders of sexual development, disorders of puberty, PCOS, male gonadal dysfunction, and granulosa cell tumors. This assay can aid in defining the clinically relevant age and sex-specific levels of AHM in non-fertility-related conditions.

A-320 Determination of Guanidinoacetate Methyltransferase Enzyme Biomarkers in Newborn Dried Blood Spot by Tandem Mass Spectrometry-A Two Tier Approach

Abstract Background Guanidinoacetate methyltransferase enzyme (GAMT) deficiency is a rare, inherited metabolic disorder that affects the synthesis of creatine. Creatine is an essential metabolite for regenerating energy, particularly in the brain and muscles. Creatine is synthesized from guanidinoacetate (GUAC) by the GAMT enzyme. Mutations in the GAMT gene result in a shortage of the GAMT enzyme that causes a deficiency of creatine and accumulation of GUAC in the body, which is a neurotoxin. Following a recommendation by the Recommended Uniform Screening Panel and in line with California State statute, all California newborns will be screened for GAMT deficiency beginning July 2024. Here, we developed and validated a quantitative two-tier tandem mass spectrometry (MS/MS) assay for the detection of GAMT biomarkers (GUAC, creatine and creatinine) in newborn dried blood spot (DBS). Methods This method punches a 3.2 mm disc of controls and newborn patient DBS into a 96-well plate. 100 μL of extraction solution, containing internal standards of GAMT biomarkers, is added to each well and incubated at 40°C for 45 minutes. The extract is transferred, evaporated, and derivatized into a 3N-butanol-HCl solution at 60°C for 30 minutes. The excess HCl is evaporated to dryness. The conversion of GUAC and creatine to their butyl esters has improved their detection sensitivity by MS/MS. The butyl esters are reconstituted into the mobile phase and shaken for 10 minutes at 27°C. The final 10 μL extract was analyzed by flow injection analysis (FIA)-MS/MS in multiple reaction monitoring (MRM) positive ion mode with a total run time of 1.5 minutes per sample. The presumptive positive samples from initial analysis are prepared following the same method, and a 5 μL extract is resolved through an ultra-performance liquid chromatography (UPLC) column and analyzed by UPLC-MS/MS in MRM positive ion mode with a total run time of 3.5 minutes per sample. The validation was performed by assessing the linearity, accuracy, imprecision, carryover, limit of detection (LOD) and lower limit of quantitation (LLOQ), matrix effect, analytical measurement range (AMR), percent total allowable error (TEa), and reference range. Results This method is linear for GUAC, creatine and creatinine over a measured range of 1.17-40.00, 121.20-857.36 and 41.34-387.46 µmol/L respectively. The R2 values=1.000, 0.997, 0.9998; slope=0.999, 0.9945, 0.9982; intercept=0.0398, 4.881, 1.7027; mean recoveries=99-104%, 100-104%, 100-104%; intra-assay precision (n=20) =4.05-5.95%, 4.62-5.31%, 3.27-5.19%, inter-assay precision (n=40)=3.7-12.6%, 4.3-7.6%, 3.8-7.4%; TEa=6-21%, 7-17%, 6-17%; LOD=0.12, 0.54, 1.11 µmol/L; AMR=0.33-1300, 1.11-2700, 3.50-4500 µmol/L; median patient results observed from 2293 normal patient=1.48, 494.02, 89.97 μmol/L for GUAC, creatine, and creatinine respectively. The tier-1 and tier-2 assay screening cutoff for GUAC at the population 95th percentile is 2.53 and 4.55 μmol/L respectively. Conclusions The tier-1 and tier-2 methods has been developed and validated to determine GAMT biomarkers in newborn DBS, making it suitable for routine screening of ∼2000 newborn specimens per day by tier-1 and followed by more specific tier-2 to separate GAMT biomarkers from potential isobaric interfering compounds. This two-tier approach greatly improved the test performance and significantly reduced false positive rate.

B-177 Development of a Contrived Fecal Matrix for Clinical Validation of a LC-MS/MS Assay to Quantify Microbiota-derived Metabolites in Stool Specimens

Abstract Background The gut microbiota plays a critical role in health and its disruption can impact clinical outcomes in a variety of circumstances, including graft-versus-host disease following hematopoietic stem cell transplantation, Clostridioides difficile infections, and liver disease. Metagenomic and metabolomic analyses of fecal material is frequently performed in the research setting; however, evaluation of the gut microbiota is not yet incorporated into routine patient care. One barrier to its implementation is the lack of matrix-appropriate materials for validation of clinical assays. While surrogate fecal matrices are being developed for metagenomic sequencing, matrices specific for targeted metabolomics are currently unavailable. In this study, we designed a contrived fecal matrix to validate a new LC-MS/MS method for measurement of microbiota-derived metabolites from stool specimens. Methods Common food products and mucin were individually analyzed by LC-MS/MS for quantities of three microbiota-derived metabolites - butyrate, deoxycholic acid (DCA) and taurocholic acid (TCA). Food items with the lowest endogenous metabolite concentrations were selected to create a contrived fecal matrix with a consistency similar to human stool specimens. Calibrators and quality controls (QC) were then created by spiking butyrate, DCA, and TCA into either the contrived fecal matrix or 50% methanol (MeOH). The internal standards (ISTD) used were d7-butyrate, d4-DCA, and d4-TCA, respectively. Three human stool specimens were selected and spiked at a range of metabolite concentrations to assess matrix effects. All samples, calibrators and controls were homogenized in 80% MeOH extraction solvent containing the ISTDs using beadruptor tubes. The suspension was centrifuged and 25 µL of supernatant was derivatized using 12.5 µL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 12.5 µL 3-nitrophenylhydrazine at 40°C for 30 minutes. For optimal separation of isomers, 2 µL of sample was injected and separated on a CORTECS T3 column (1.6 µm, 2.1 x 100 mm, Waters) at 45°C with a flow rate of 0.350 mL/min and run-time of 20 min using the Exion LC AD (Sciex). Analytes were detected using a Sciex QTRAP 6500 mass spectrometer with an electrospray ionization source. Results A contrived fecal matrix containing a mix of avocado, black beans, gluten-free flour, and cornstarch blended in water and mucin was created. The contrived matrix was confirmed to contain minimal to no endogenous levels of the 3 metabolites of interest: butyrate, DCA, and TCA. Validation of the LC-MS/MS assay using the contrived fecal matrix showed a linear analytical measurement range from 25-2000 µM for butyrate, 1-60 µM for DCA, and 0.5-640 µM for TCA. Intra-assay precision at 7 QC levels for all analytes was <10%. The matrix effects from use of the contrived fecal matrix compared to patient fecal samples were 140-148% for d7-butyrate and 101-112% for d4-DCA across multiple spiked analyte concentrations. Conclusions Our LC-MS/MS method quantifies butyrate, deoxycholic acid, and taurocholic acid, metabolites which serve as indicators of microbiota health in liver disease patients. The contrived fecal matrix provides an additional tool for the development and validation of clinical assays to assess the gut microbiota.

B-162 Evaluation of two commercially available stable isotope labeled whole-protein infliximab as internal standard for a clinical infliximab LC-MS/MS assay

Abstract Background Liquid chromatography tandem mass spectrometry (LC-MS/MS) based quantitation of infliximab has been successfully introduced in the clinical laboratory for therapeutic drug monitoring in recent years. Although stable isotope labeled (SIL)-peptide or non-labeled analog protein internal standards have been utilized, SIL-whole protein infliximab is generally considered to be the ideal internal standard for the correction of variations during sample preparation and analysis. In this study, we compared two commercially available SIL infliximab proteins, Sigma SILu™MAb and Promise Proteomics, for use in a clinical infliximab assay. Methods Both labeled infliximab proteins incorporate [13C6, 15N4]-arginine and [13C6, 15N2]-lysine in their sequence and are designed for use as an internal standard for LC-MS/MS based quantitative analysis of infliximab. For the comparison of their performance, a laboratory-developed LC-MS/MS clinical method was utilized. Linearity at 6 levels in triplicate with Remicade and 3 biosimilars (-abda, -axxq, and -dyyb), intra-assay precision at 2 levels (n=10/level), and method comparison (n=15) experiments were performed using each labeled infliximab as the internal standard. For sample preparation, internal standard was mixed with the serum sample, followed by protein precipitation with ammonium sulfate, protein denaturation, and tryptic digestion. The peptides YASESMSGIPSR (YASE) from infliximab and YASESMSGIPSR [13C6, 15N4] (SIL-YASE) from labeled infliximab were monitored by LC-MS/MS (Transcend TLX and TSQ Atlis, Thermo Scientific) and utilized for the quantitation of infliximab. Results For the linearity experiment (1-50 µg/mL), while utilizing the Promise Proteomics internal standard to assay had an accuracy range of 103.4% to 114.1% for Remicade, 100.6% to 108.2% for Infliximab-axxq, 103.0% to 110.9% for Infliximab-abda, 102.7% to 108.3% for Infliximab-dyyb. For the Sigma SILu™Mab SIL infliximab, the accuracy range was 92.1% to 99.6% for Remicade, 94.5% to 107.2% for Infliximab-axxq, 99.0% to 104.2% for Infliximab-abda, and 105.1% to 112.0% for Infliximab-dyyb. The mean overall error for Remicade and biosimilars was 0.203 µg/mL (2.0%) for Sigma SILu™Mab SIL infliximab and 0.193 µg/mL (2.0%) for Promise Proteomics internal standardization. Intra-day precision using each SIL infliximab was evaluated with Remicade at low and high levels (5 and 20 µg/mL). Using Promise Proteomics, the coefficient of variation (CV) was 2.8 and 2.2%, while using Sigma SILu™Mab the precision was 6.8 and 6.5% for the low and high levels, respectively. The regression analysis for the method comparison experiment had a correlation coefficient (r) of 0.9996 with a slope of 1.050 (95% confidence interval, 1.030-1.070) and intercept of -0.318 (95% confidence interval, -0.842 to 0.205). Conclusions The mean overall error for the linearity experiment was the same using Sigma SILu™Mab or Promise Proteomics as the internal standard. The Promise Proteomics product demonstrated a lower intra-assay CV compared to the Sigma SILu™Mab. The 2 products displayed a strong correlation during the method comparison experiment. Both Sigma SILu™Mab and Promise Proteomics labeled standards showed acceptable performance as an internal standard for LC-MS/MS based quantitative analysis of infliximab. Having both products available increases the robustness of LC-MS/MS clinical assays in the event of supply issues.

B-231 Evaluation of the KingFisher Flex for DNA Isolation From FFPE Tissue Utilizing Autolys M Tubes and the MagMAX FFPE DNA/RNA Ultra Kit

Abstract Background Our institution is facing increasing demand for oncology testing that requires high quality DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. The current DNA extraction workflow involves a time and labor-intensive manual process that limits our ability to accommodate increasing downstream clinical testing volumes. We evaluated the KingFisher (KF) Flex utilizing Autolys M Tubes and the MagMAX FFPE DNA/RNA Ultra Kit as a possible high-throughput, automated DNA isolation workflow option that would decrease hands-on time and be conducive to volume growth without the need for additional staff. Methods DNA from 52 FFPE tissue samples (n=24 unique cases previously extracted by the current “manual method”) were extracted using Autolys M tubes, the MagMAX FFPE DNA/RNA Ultra Kit, and the KF Flex (Thermo Fisher Scientific Waltham, MA) over 4 “automated method” runs. Method modifications to the published procedure were introduced over the runs to determine any effects on DNA quality/quantity. Timings were taken during each run to compare hands-on and total run time against that of the manual method. Precision experiments were performed by extracting 4 replicates of sample on a single run (intra-assay precision) and 2 samples from 2 cases over 2 runs (inter-assay precision). Three cases were processed using varying tissue input levels to assess input-yield correlation. DNA yield was measured using HS dsDNA Qubit (Thermo Fisher Scientific, Waltham, MA). A subset of samples was tested by digital droplet PCR (ddPCR) (n=10), chromosomal microsomal microarray (CMA) (n=1), and next-generation sequencing (NGS) (n=3). Variants and QC metrics of the KF DNA were compared to those of the manual method DNA. Results The average total DNA yield was 3% lower from samples extracted following the manufacturer recommended KF procedure compared to matched samples extracted using the manual method (n=28 samples from 23 unique cases). A CV of 2.8% was observed in an intra-assay experiment (n=4 replicates from 1 case) and 3.0% in an average inter-assay experiment (n=2 replicates from 2 cases). There was a direct correlation between the tissue input amount (4 amounts of varied tissue input) and the total DNA yield (n=3 samples). We observed concordant fractional abundance values for ddPCR samples and concordant variant detection for NGS and CMA samples. We did not see a significant difference in the quality metrics tracked for any of the downstream testing assays. Conclusions This evaluation demonstrated that, as compared to our manual method, the automated method resulted in comparable DNA yields and downstream assay results. The incorporation of this automation would significantly increase our FFPE DNA extraction capacity without requiring additional staff while reducing hands-on time per run by about six hours. We would also be able to reduce ergonomic hazards related to tube cap manipulation, eliminate use of hazardous reagents, and leverage the plate format to better integrate extracted DNA into our clinical sample preparation workflows. These operational benefits highlight the value of automating high-throughput workflows.

B-236 New real-time PCR test for APOE genotyping in patients with Alzheimer’s disease before anti-amyloid therapy

Abstract Background In 2023, the US Food and Drug Administration approved LEQEMBI, a monoclonal antibody therapy targeting beta-amyloid plaques, for patients with early-stage Alzheimer’s disease (AD). However, anti-amyloid drug trials demonstrated an elevated risk for amyloid-related imaging abnormalities with cerebral edema (ARIA-E) in carriers of the AD risk allele apolipoprotein E epsilon 4 (APOE-ɛ4), especially in ɛ4/ɛ4 homozygous individuals [1-3]. Here, we report on the evaluation of a new real-time polymerase chain reaction (PCR) test for APOE genotyping. Methods The EURORealTime APOE (EUROIMMUN), which enables simultaneous testing for all six APOE genotypes (ɛ2/ɛ2, ɛ2/ɛ3, ɛ2/ɛ4, ɛ3/ɛ3, ɛ3/ɛ4, and ɛ4/ɛ4) by real-time PCR, was performed using genomic DNA (gDNA) isolated from EDTA blood samples. The PCR amplification results were evaluated using the EURORealTime Analysis Software (EUROIMMUN). Samples from healthy blood donors (HBD) were used to assess intra-assay, inter-assay and between-lot precision (n=6) as well as analytical sensitivity (n=21), with each APOE genotype represented by at least one sample. Assay accuracy was analyzed by comparing genotype assignments to the results of the CE-IVD-marked EUROArray APOE (EUROIMMUN) and bidirectional Sanger sequencing using 110 samples (100 AD, 10 HBD). APOE genotype frequencies were compared between patients (100 AD) and controls (250 HBD). Results Precision testing of the EURORealTime APOE resulted in 100% correct genotype calls. Assessment of the analytical sensitivity revealed valid and correct genotype assignment in all 21 samples, confirming 1 ng/µl as the recommended minimum gDNA concentration. The analytical accuracy amounted to 100% based on agreement with the two reference methods in 110/110 cases. Comparison of the APOE genotype distribution showed a higher proportion of ɛ4 allele carriers (i.e., ɛ2/ɛ4, ɛ3/ɛ4, and ɛ4/ɛ4) in AD patients than in HBD (50.0% vs. 25.2%). The prevalence of the ɛ4/ɛ4 genotype in these cohorts was 25.0% for AD patients compared with 2.8% for HBD. Conclusions The well-established high prevalence of the APOE-ɛ4/ɛ4 genotype in AD patients, together with the increased risk of ARIA-E in these homozygous carriers, substantiates the relevance of APOE genotyping prior to anti-amyloid treatment. The EURORealTime APOE test provides sensitive and reliable determination of APOE alleles, thus presenting a suitable tool to improve risk stratification for potential LEQEMBI recipients.

B-291 Validation of a Simple Qualitative Immunoassay for 21 Drugs of Abuse in Meconium on a Single Biochip Array

Abstract Background Drugs use during pregnancy is a major social and medical issue, which may lead to significant pre- and post-natal complications, such as preterm birth and newborn mortality. Exposed newborns may have long-term behavioral, cognitive, and developmental problems. Accurate detection of in utero drug exposure is crucial for timely medical intervention for the newborn and the mother. Meconium is the ‘gold standard' matrix for detection of drugs in newborn which reflects drug exposure during pregnancy. This study aims to develop and optimize a clinical assay for simultaneous screening of 21 drugs in meconium using a custom biochip array. Methods Custom biochip array (Randox Laboratories Ltd.) included 21 discrete test regions (DTRs) for amphetamine, methamphetamine, opioids/opiates (targeting morphine, oxymorphone, oxycodone, hydrocodone, 6-monoacetyl morphine, tramadol, buprenorphine, fentanyl, and methadone), phencyclidine (PCP), cocaine (targeting benzoylecgonine), benzodiazepines (targeting oxazepam, lorazepam, and clonazepam), zolpidem, barbiturates (targeting phenobarbital), ethyl glucuronide, methylphenidate, and marijuana metabolite (targeting Delta-9-Carboxy-Tetrahydrocannabinol [THC-COOH]). Patient samples were obtained from the residual meconium samples that had been previously used for other tests. Blank meconium samples were spiked at in-house cutoff, negative and positive assay control levels. All samples were extracted in specimen diluent using Biotage® Lysera and centrifuged. The supernatant was analyzed on a fully automated Randox Evidence+® Analyzer. A robotic arm pipetted 60 µL sample on to a biochip and then the analyzer performed a competitive chemiluminescent immunoassay. The results were generated as relative light units (RLUs). Results The sample results were determined to be “positive” or “negative” based on average RLU of cutoff controls for each analyte on an assay. The intra-assay (CV ≤ 15%) and inter-assay (CV ≤ 18%) precision was 100% agreement for positive and negative controls and there was no observed carryover for any of the analytes. The sample recovery was excellent with minimal to no matrix suppression for all analytes. Accuracy was performed by comparing spiked analyte results from Randox Evidence+® Analyzer with the expected results and all the spiked samples matched (positive or negative). When authentic patient or spiked results were compared with external laboratory test results, some discordant results were obtained. However, the discrepancies could be explained based on the different cutoffs (e.g., the Randox assay had lower cutoffs) or the known assay cross reactivities within the analytes of same class. These deviations did not affect clinical utility of the assay. Conclusions We validated a novel and simple qualitative chemiluminescent immunoassay in meconium using a custom biochip array. Unlike other routine screening/ELISA methods, which require separate assays for detection of similar drug targets, the custom biochip array offers a convenient, rapid and efficient method that utilizes only a single sample preparation for the screening of all 21 drug targets. Therefore, this technology has potential to be utilized as high-throughput screening assay. Like all qualitative assays, the limitation of this test is that all positive results will need to be assessed quantitatively for a definitive interpretation.

A-318 Validation of umbilical cord blood as an alternate sample type for quantitative in vitro detection of glucose-6-phosphate dehydrogenase in neonates

Abstract Background Detection of glucose-6-phosphate dehydrogenase (G6PD) deficiency provides important information for neonatal clinical management. G6PD newborn screening is recommended using umbilical cord blood specimens in vulnerable patient populations.1 Also, cord blood is used by many clinicians instead of peripheral blood for neonatal screening of G6PD.2 Use of cord blood is a modification to the FDA-approved Pointe Scientific G6PD kinetic-340 nm assay. The aim of this study was to validate cord blood as an alternate specimen type for the Pointe Scientific G6PD assay. Methods Hemoglobin (Hgb) results are from Sysmex XN 9100™ analyzers that use SLS methodology with photometric detection. G6PD results are from Roche cobas® c502 analyzers with Pointe Scientific kinetic method and spectrophotometric detection of activity, expressed as U/g Hgb. Neonatal blood specimens were obtained as paired samples from heel sticks and umbilical cord collected in EDTA lavender top tubes. Accuracy acceptance criteria was defined as Deming regression slope between 0.90 and 1.10, r-value of >0.90 and bias ±15% and mixing study recovery of ±10%. Precision (intra-assay and inter-assay), reportable range and linearity, carryover, and stability were established using cord blood samples or control materials sourced from Thermo Fisher Scientific or Sysmex. Reference intervals3, analytical sensitivity, specificity, and clinical validity were transferred to cord blood due to comparability with whole blood FDA-approved method. EP Evaluator software by Data Innovations® was used for calculations. Results Direct comparison of G6PD results (U/g Hgb) from 125 paired samples yielded a 0.9025 correlation coefficient, 1.024 Deming slope, and -0.25% bias. Mixing and recovery of admixed samples gave % recoveries of 102, 103, 101, 102, 100, and 100 for G6PD (U/g Hgb), and 102, 102, 101, 104, 99, and 105 for Hgb (g/dL). Intra-assay precision results were 2.4, 0.6, 0.8, 0.5, 0.4 % CV for G6PD and Hgb, respectively. Inter-assay precision results were 3.1, 3.3, 0.7, 0.7, 0.5 % CV for G6PD and Hgb, respectively. Reportable range and linearity evaluation resulted in a maximum deviation from target recovery of 4%, and a slope of 0.979 for a range of 50 - 2740 U/L, and reportable range for Hgb was confirmed as 0.1 - 26.0 g/dL using controls (1% maximum deviation and 0.991 slope). Carryover was -5.8 (mean concentration) for G6PD and 0% for Hgb after testing 21 replicates of pooled high and low samples in a defined order. Testing 3 samples in 3 replicates at each defined time interval defined stability as 1 week refrigerated or 72 hours room temperature for G6PD, and 3 days refrigerated or 1 day room temperature for Hgb. Conclusions Regression statistics and mixing-recovery studies demonstrated accuracy for detecting G6PD (U/g Hgb) and Hgb (g/dL) from EDTA cord blood. Additional performance characteristics were transferrable for this specimen type from the FDA-approved method for whole blood. Umbilical cord blood is an acceptable alternate specimen type for quantitative detection of G6PD (U/g Hgb) using Pointe Scientific with Sysmex XN-9100™ and Roche cobas® c502 instrumentation.

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

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

Analytical Validation of Esopredict, an Epigenetic Prognostic Assay for Patients with Barrett's Esophagus.

EsopredictTM is a prognostic assay that risk-stratifies Barrett's esophagus patients to predict future progression to high-grade dysplasia (HGD) or esophageal adenocarcinoma (EAC). Established based on foundational studies at Johns Hopkins University, a risk algorithm was developed and clinically validated in two independent studies (n = 320). EsopredictTM is currently offered as a clinical test under the Clinical Laboratory Improvement Amendments (CLIA) guidelines. Here we present the analytical validation by repeated testing of FFPE tissues (n = 26 patients), cell lines, and contrived DNA controls to determine assay performance regarding analytical sensitivity (as defined by the limit of detection (LOD)), analytical specificity (as defined by the limit of blank (LOB)), accuracy as determined from the average positive and negative agreement, repeatability, and reproducibility. The LOD for the assay at 1.5% DNA methylation was significantly higher than the LOB, as determined by an unmethylated DNA control (0% methylated DNA). Inter- and intra-assay average positive agreement (APA) were 88% and 94%, respectively, while average negative agreement (ANA) values were 90% and 94%, respectively. Average inter- and intra-assay precision were <9% and <5% coefficient of variation (CV), respectively. These results confirm that EsopredictTM is a highly reproducible, sensitive, and specific risk categorization assay for the prediction of progression to HGD or EAC within 5 years.

Analytical and operational considerations of measuring glucose 6-phosphate dehydrogenase (G6PD) activity using a fully automated assay.

This study determined the performance characteristics of a quantitative glucose-6-phosphate dehydrogenase (G6PD) assay with automated lysis and evaluated the robustness of the operational workflow following implementation in a hospital laboratory. The G6PD activity was measured in whole blood using an enzymatic quantitative test on a Roche cobas c501 analyzer with onboard lysis configuration and normalized to hemoglobin (Hb). The performance characteristics of the method and stability of G6PD in whole blood collected in EDTA-containing tubes were evaluated, and the reference interval was established on a population of healthy individuals (n = 279). The robustness of this automated workflow for sample lysis was evaluated during validation and after implementation for routine clinical use for 18 months and in 2,181 patients. The G6PD assay was linear from 0.7 to 16.5 U/g Hb. Inter- and intra-assay precision using control and patient samples was below 12%. The G6PD results correlated well with a reference laboratory method (r = 0.96, y = 0.9615x - 1.222). The reference interval in our population was 9.8 to 15.5 U/g Hb. There were no interferences by lipemia and icteria, although grossly hemolyzed specimens may be affected. The testing workflow requires analyzing samples within minutes from mixing and loading into the instrument to avoid sample sedimentation. Measures to repeat samples with Hb 8.0g/dL or less identified sedimented samples. In our patient population, 10.6% and 5.8% of the total males and females tested were G6PD deficient, respectively. The G6PD assay with automated lysis is acceptable for patient testing. Several measures ensured the robustness of this workflow in a hospital laboratory.

Rheological behavior of the synovial fluid: a mathematical challenge

BackgroundSynovial fluid (SF) is often used for diagnostic and research purposes as it reflects the local inflammatory environment. Owing to its complex composition, especially the presence of hyaluronic acid, SF is usually viscous and non-homogeneous. The presence of high-molar-mass hyaluronan in this fluid gives it the required viscosity for its function as a lubricant. Viscosity is the greatest major hydraulic attribute of the SF in articular cartilage.MethodsEmpirical modeling of previously published results was performed. In this study, we explored the flow of a non-Newtonian fluid that could be used to model the SF flow. Analyzing the flow in a simple geometry can help explain the model’s efficacy and assess the SF models. By employing some viscosity data reported elsewhere, we summarized the dynamic viscosity values of normal human SF of the knee joints in terms of time after injecting hyaluronidase (HYAL) at 25°C. The suggested quadratic behavior was obtained through extrapolation. For accurate diagnosis or prediction, the comparison between three specific parameters (ai, t0, and ln η0) was made for normal and pathological cases under the same experimental conditions for treatment by addition of HYAL and for investigation of the rheological properties. A new model on the variation of viscosity on the SF of knee joints with time after injection of HYAL with respect to normal and postmortem samples at different velocity gradients was proposed using data previously reported elsewhere.ResultsThe rheological behavior of SF changes progressively over time from non-Newtonian to a Newtonian profile, where the viscosity has a limiting constant value (η0) independent of the gradient velocity at a unique characteristic time (t0 ≈ 8.5 h). The proposed three-parameter model with physical meaning offers insights into future pathological cases. The outcomes of this work are expected to offer new perspectives for diagnosis, criteria, and prediction of pathological case types through comparisons with new parameter values treated under the same experimental conditions as HYAL injection. This study also highlights the importance of HYAL treatment for better intra-assay precision.

In vitro CAR-T cell killing: validation of the potency assay

For advanced therapy medicinal products, the development and validation of potency assays are required, in accordance with international guidelines, to characterise the product and obtain reliable and consistent data. Our purpose was to validate the killing assay for the evaluation of autologous anti-CD19 chimeric antigen receptor (CAR) T potency. We used CD4 + and CD8 + lymphocytes or anti-CD19 CAR-T cells as effector cells and REH (CD19 +) or MOLM-13 (CD19 −) cell lines as target cells. After co-culturing target and effector cells (1:1 ratio) for 24 h, samples were labelled with 7-AAD, anti-CD3 and anti-CD19 antibodies and the frequency of CD19 + dead cells was evaluated by flow cytometry. In order to verify the CAR-T specificity for the CD19 + target, the co-culture between CAR-T and REH or MOLM-13 at different effector-to-target ratios was scheduled. Moreover, not transduced CD4 + and CD8 + lymphocytes were tested in comparison with CAR-T from the same donor to demonstrate the assay specificity. Linearity and accuracy were evaluated, and established acceptance criteria were compiled for both parameters (r2 ≥ 0.97 for linearity and average relative error ≤ 10% for accuracy). Furthermore, the method was considered robust when performed between 23 and 25 h of co-culture, and the intra-assay, inter-assay and inter-day precision was obtained. Finally, in order to verify the inter-analyst precision, the test was executed by three different operators and the intra-class correlation coefficient was > 0.4 in both cases. In conclusion, we consider this CAR-T potency assay as validated and usable in all steps of product development and quality control.

Quantification of treprostinil concentration in rat and human using a novel validated and rapid liquid chromatography-tandem mass spectrometry method: Experimental and clinical applications in ischemia–reperfusion injury

Treprostinil (Remodulin®) is a Food and Drug Administration (FDA) approved prostacyclin analog to treat pulmonary arterial hypertension. Recently, treprostinil has been investigated to reduce ischemia–reperfusion injury (IRI) during transplantation, which currently has no approved treatment. A validated analytical method is necessary to measure treprostinil concentrations in biological specimens. Here, a novel, sensitive, and specific method to measure treprostinil concentrations in rat serum, human serum, and human plasma has been developed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Biological samples were processed by protein precipitation before chromatography and 6-keto Prostaglandin F1α-d4 was used as an internal standard. A gradient method was established with a total run time of 4 min. The assay was linear over the range of 0.25–75.0 ng/ml with accuracy (92.97–107.87 %), intra-assay precision (1.16–3.34 %), and inter-assay precision (1.11–4.58 %) in all biological matrices, which are within FDA acceptance criteria. No significant variation in treprostinil or 6-keto Prostaglandin F1α-d4 concentrations were observed under the investigated storage conditions. This novel, sensitive, and specific LC/MS-MS method is cost-effective and suitable for measuring treprostinil concentrations in animal studies and human biological samples for clinical applications.

Towards a better understanding of idiopathic epilepsy through metabolic fingerprinting of cerebrospinal fluid in dogs

Cerebrospinal fluid metabolomics is a promising research technology in the elucidation of nervous system disorders. Therefore, in this work, a cerebrospinal fluid (CSF) metabolomics method using liquid chromatography coupled to mass spectrometry was optimized and validated to cover a wide range of metabolites. An acceptable coefficient of variance regarding instrumental, within-lab and intra-assay precision was found for 95, 70 and 96 of 102 targeted metabolites, together with 1256, 676 and 976 untargeted compounds, respectively. Moreover, approximately 75% of targeted metabolites and 50% of untargeted compounds displayed good linearity across different dilution ranges. Consequently, metabolic alterations in CSF of dogs with idiopathic epilepsy (IE) were studied by comparing CSF of dogs diagnosed with IE (Tier II) to dogs with non-brain related disease. Targeted metabolome analysis revealed higher levels of cortisol, creatinine, glucose, hippuric acid, mannose, pantothenol, and 2-phenylethylamine (P values < 0.05) in CSF of dogs with IE, whereas CSF of dogs with IE showed lower levels of spermidine (P value = 0.02). Untargeted CSF metabolic fingerprints discriminated dogs with IE from dogs with non-brain related disease using Orthogonal Partial Least Squares Discriminant Analysis (R2(Y) = 0.997, Q2(Y) = 0.828), from which norepinephrine was putatively identified as an important discriminative metabolite.

Point-of-care blood tests using a smartphone-based colorimetric analyzer for health check-up.

A microscale colorimetric assay was designed and implemented for the simultaneous determination of clinical chemistry tests measuring six parameters, including glucose (GLU), total protein (TP), human serum albumin (HSA), uric acid (UA), total cholesterol (TC), and triglycerides (TGs) in plasma samples. The test kit was fabricated using chromogenic reagents, comprising specific enzymes and binding dyes. Multiple colors that appeared on the reaction well when it was exposed to each analyte were captured by a smartphone and processed by the homemade Check6 application, which was designed as a colorimetric analyzer and simultaneously generated a report that assessed test results against gender-dependent reference ranges. Six blood checkup parameters for four plasma samples were conducted within 12min on one capture picture. The assay achieved wide working concentration ranges of 10.45-600 mg dL-1 GLU, 1.39-10.0g dL-1 TP, 1.85-8.0g dL-1 HSA, 0.86-40.0 mg dL-1 UA, 11.28-600 mg dL-1 TC, and 11.93-400 mg dL-1 TGs. The smartphone-based assay was accurate with recoveries of 93-108% GLU, 93-107% TP, 92-107% HSA, 93-107% UA, 92-107% TC, and 99-113% TGs. The coefficient of variation for intra-assay and inter-assay precision ranged from 3.2-5.2% GLU, 4.6-5.3% TP, 4.3-5.3% HSA, 2.8-6.6% UA, 2.7-6.5% TC, and 1.1-3.9% TGs. This assay demonstrated remarkable accuracy in quantifying the concentration-dependent color intensity of the plasma, even in the presence of other suspected interferences commonly present in serum. The results of the proposed method correlated well with results determined by the microplate spectrophotometer (R2 > 0.95). Measurement of these six clinical chemistry parameters in plasma using a microscale colorimetric test kit coupled with the Check6 smartphone application showed potential for real-time point-of-care analysis, providing cost-effective and rapid assays for health checkup testing.

Analytical performance evaluation of the Mindray enzymatic assay for hemoglobin A1c measurement

Hemoglobin A1c (HbA1c) plays a crucial role in diabetes management. We aimed to evaluate the analytical performance of a new enzymatic method kit for HbA1c measurement. The performance of the enzymatic method, including precision, accuracy, and linearity, was evaluated. Moreover, the interference effect from conventional interferents, Hb derivatives, Hb variants, and common drugs were assessed. In addition, the agreement of HbA1c results was compared between enzymatic methods, cation-exchange high-performance liquid chromatography (HPLC), and immunoassays. The intra-assay, between-assay, and total precision of HbA1c were all lower than 2%. HbA1c showed good linearity within the range of 3.96–20.23%. The enzymatic assay yielded results consistent with the external quality control samples, with a bias of less than ± 6% from the target values. The enzymatic method showed no interference from bilirubin, intralipid, vitamin C, Hb derivatives, common Hb variants, as well as antipyretic analgesics and hypoglycemic drugs. The HbA1c results of the enzymatic assay showed good agreement and accuracy compared to those obtained from the HPLC method and the immunoassay. The enzymatic method kit performed on the BS-600M chemistry analyzer is a reliable and robust method for measuring HbA1c. It is suitable for routine practice in clinical chemistry laboratories.

A Microfluidic, Multi-Antibody Cell Capture Method to Evaluate Tumor Cells in Cerebrospinal Fluid in Patients With Suspected Leptomeningeal Metastases.

Leptomeningeal disease (LMD) is a clinical sequela of central nervous system metastasis involving the cerebrospinal fluid (CSF), often seen in late-stage solid tumors. It has a grave prognosis without urgent treatment. Standard of care methodologies to diagnose LMD include CSF cytology, magnetic resonance imaging, and clinical evaluation. These methods offer limited sensitivity and specificity for the evaluation of LMD. Here, we describe the analytic performance characteristics of a microfluidic-based tumor cell enrichment and detection assay optimized to detect epithelial cells in CSF using both contrived samples as well as CSF from patients having suspected or confirmed LMD from carcinomas. To demonstrate the feasibility of using a microfluidic, multi-antibody cell capture assay to identify and quantify tumor cells in CSF. An artificial CSF solution was spiked with 34 different human carcinoma cell lines at different concentrations and assayed for the ability to detect tumor cells to assess analytic accuracy. Two cell lines were selected to assess linearity, intra-assay precision, interinstrument precision, and sample stability. Clinical verification was performed on 65 CSF specimens from patients. Parameters assessed included the number of tumor cells, coefficient of variation percentage, and percentage of tumor cell capture (TCC). Among contrived samples, average tumor cell capture ranged from 50% to 82% (261 of 522; 436 of 531), and coefficients of variation ranged from 7% to 67%. The cell capture assay demonstrated a sensitivity of 92% and a specificity of 95% among clinical samples. This assay demonstrated the ability to detect and enumerate epithelial cells in contrived and clinical specimens in an accurate and reproducible fashion. The use of cell capture assays in CSF may be useful as a sensitive test for the diagnosis and longitudinal monitoring of LMD from solid tumors.

#1504 Performance evaluation of MicroVue sC5b-9 for analysis of urine samples

Abstract Background and Aims Complement mediated kidney diseases including C3 glomerulopathy (C3G), atypical uremic syndrome (aHUS), lupus nephritis (LN), and immunoglobulin A nephropathy (IgAN) are chronic inflammatory conditions characterized by dysregulated complement activation. Traditionally, complement activation is measured in plasma. However, patients with kidney disease often have an abundance of complement activation fragments in their urine—including soluble C5b-9 (sC5b-9), a marker of terminal pathway activation—highlighting the potential value of sC5b-9 measurement in urine for diagnosis and risk management in patients with C3G, aHUS, LN, IgAN, and other kidney diseases. Herein we evaluated the analytical performance of the Quidel MicroVue sC5b-9 Plus EIA for measurement of sC5b-9 in urine. Method The Quidel MicroVue sC5b-9 Plus EIA was evaluated using randomly collected healthy urine samples or healthy urine samples spiked with a known concentration of sC5b-9, as well as chronic kidney disease (CKD) urine samples. Results The analytical performance of the Quidel MicroVue sC5b-9 Plus EIA for measurement of sC5b-9 in urine met acceptability criteria for accuracy and precision. Assay accuracy was (100 ± 20%) and intra-assay precision as well as inter-assay precision was ≤ 11% coefficient of variability (CV). sC5b-9 was not detected in healthy urine samples (N = 40), but sC5b-9 was detected in a subset of urine samples from patients with CKD (N = 10). Positive linearity was observed over a wide range of concentrations. sC5b-9 measured in urine was not affected by hemoglobin or triglyceride, and was stable following three freeze-thaw cycles. Lastly, sC5b-9 measured in urine was stable when stored at −20°C and –80°C for 4 weeks. Long term stability testing at –80°C is underway. Conclusion Quidel MicroVue sC5b-9 Plus EIA is an accurate, reliable assay for detection of sC5b-9 in urine samples.