Analytical Measurement Range Research Articles

Evaluation of an Alternate Diluent for Tests to Ensure Timely Patient Management

Abstract Introduction/Objective Recently, backorder of Multi-diluent (MD) for various assays on ADVIA Centaur analyzer (Siemens Healthineers) led to delay in reporting numeric values to the clients when results above the analytical measurement range (AMR) had to be reported as “> value”. The samples were stored frozen (-20 degrees Celsius or - 80 degrees Celsius) for over 2 weeks until the diluent became available, then thawed and re-analyzed upon appropriate dilution. Delay in reporting results beyond AMR may impact patient management; therefore, this study evaluated saline as an alternative diluent for serum folate, carbohydrate antigen 19-9 (CA 19-9), and carbohydrate antigen 27-29 (CA 27.29). Methods/Case Report For each test, samples from 5 patients were tested in duplicate, and average of the results was considered the target value (results within undiluted AMR). Subsequently, the samples were diluted 20-fold with either MD or saline, tested in duplicate and average of the results was compared to the target value. The diluent was considered acceptable for use if the difference or % difference between diluted vs target value was less than the Total Allowable Error (TEA). Results (if a Case Study enter NA) For serum folate and CA 19-9, the bias between diluted and target value for each diluent (saline and Multi-diluent) was < TEA, and the average bias was also < TEA. For CA 19-9, the average difference from target for saline was smaller than that for Multi-diluent, suggesting it performs better (p-value of 0.0035). However, saline was not an acceptable diluent for CA 27.29 as the bias exceeded TEA. Conclusion Saline was an acceptable alternative diluent for serum folate and CA 19-9 but not for CA27.29.

Validation of Serum Tetrahydrocannabinol Assay and Its Clinical Application

Abstract Background The widespread use of marijuana across all age groups in the United States, driven by the legalization of recreational and medical cannabis, has underscored the need for accurate measurement of tetrahydrocannabinol (THC) and its metabolites in blood specimens. This requirement spans both legal and medical contexts, prompting the development of precise diagnostic tools. Methods We developed a high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay, utilizing a straightforward protein precipitation extraction method to quantify THC and its metabolites: THC, 11-OH-THC, THC-COOH, cannabidiol (CBD), and THC-COOH glucuronide. The assay was rigorously validated for precision, linearity, stability, carryover, and quality assurance to ensure its reliability and accuracy for clinical use. Results Precision studies demonstrated a coefficient of variation ranging from 1.7% to 4.9%. The analytical measurement range was established at 1-100 ng/mL for THC, CBD, and 11-OH-THC, 5-500 ng/mL for THC-COOH, and 10-1000 ng/mL for THC glucuronide, accommodating the broad spectrum of THC/metabolite concentrations observed in our patient cohort. Carryover from all analytes were negligible (<0.01%). Sample stability was confirmed for up to two weeks at -20°C and through three freeze-thaw cycles. Despite a median THC concentration of 5 ng/mL in a limited sample size (N=44), high concentrations of THC and its metabolites correlated with trauma-related incidents and a history of polysubstance abuse in emergency department admissions. Conclusions The LC-MS/MS assay offers superior sensitivity and specificity compared to traditional immunoassays for cannabinoid detection, effectively distinguishing between THC and its metabolites. Its implementation will offer an advanced approach for analyzing the prevalence and clinical implications of THC and metabolite levels in various patient populations, including trauma, pediatric patients, and organ transplant candidates.

Who Keeps Changing My Results? - A Case of Heterophile Antibody Interference

Abstract Introduction/Objective Heterophile antibodies are known to cause interference in immunoassays leading to misleading results causing diagnostic confusion and unnecessary testing and/or treatment. Many clinically important analytes are tested using antibody-based assays, as such, these are prone to interference. Methods/Case Report We describe a case of a 73 year old male who was referred with history of hypocalcemia and his previous lab tests were suggestive for idiopathic hypoparathyroidism, Hashimoto’s disease and Addison’s disease raising physician concern for a possible Type 1 autoimmune endocrinopathy. One of his results (cortisol) was outside the analytical measurement range and hence the sample was diluted and tested again, but the result was much less than what was expected, prompting an investigation. Investigation for an interfering substance included serial dilution and analysis of the same sample for the same analytes by different methods (different immunoassay, mass spectrometry or both). Based on non-parallelism in serial dilution results, discordant results with other methods, and loss of interference after dilution it was determined that the most likely cause was a heterophile antibody. Results (if a Case Study enter NA) NA Conclusion Awareness of the possibility that interference by heterophile antibodies can lead to erroneous laboratory results is important to prevent unnecessary testing and inappropriate diagnosis/management.

B-260 Prevalence and Relative Distributions of Major Phosphatidylethanol (PEth) Homologs in a Large-Scale Clinical Study

Abstract Background Ethanol abuse remains a significant public health challenge in the United States. Detection of ethanol exposure is crucial for preventive, diagnostic, and therapeutic endeavors. Phosphatidylethanol (PEth) is a specific and direct ethanol biomarker. Compared to other metabolites, such as ethyl glucuronide and ethyl sulfate in urine (average 2-3 days), PEth in blood has a longer window of detection (average 1-2 weeks). Among the known 48 PEth homologs, PEth 16:0/18:1 (POPEth) and PEth 16:0/18:2 (PLPEth) are the most abundant species accounting for 37% to 46% and 26% to 28%, respectively. PLPEth is synthesized at a greater rate than POPEth, but POPEth has a longer half-life than PLPEth. The purpose of this study was to understand the prevalence and levels of POPEth and PLPEth in a large number of clinical specimens. Methods Briefly, lavender-top whole blood specimens were diluted with isotopically labeled internal standard (IS) in an organic buffer to precipitate proteins on an automated Hamilton platform. The mixture was centrifuged, and a supernatant portion isolated for injection onto a Sciex QTRAP 5500 liquid chromatography tandem mass spectrometry (LC-MS/MS) system using electrospray ionization (ESI) in negative mode. The assay was fully validated under CLIA regulations for lab developed tests: the analytical measurement range (AMR) was 10-400 ng/mL; the administrative reporting threshold for PEth positivity was 20 ng/mL based on clinical literature and a consensus among other United States laboratory methods. Results Precision of 4 QC levels was evaluated with between-run CV% (n=25) ranging from 1.6-6.3%, and accuracy across the AMR was achieved with 87-110%. No significant matrix effects were observed; variation in different whole blood specimens was corrected with IS normalization. No significant interferences were observed from 137 commonly seen prescription and nonprescription drugs and 6 other PEth homologs. Of the clinical specimens, most (20,833, 71.3%) were negative (<20 ng/mL) for both analytes. Meanwhile, 8,222 specimens (28.3%) were positive with either POPEth, or PLPEth or both ≥ 20ng/mL. There were 150 specimens excluded from the statistics due to interference challenges. Interestingly, 757 specimens (2.6%) were PLPEth negative and POPEth positive (mean value 40.1 ng/mL). Conversely, 62 specimens (0.2%) were POPEth negative and PLPEth positive (mean value 24.6 ng/mL). Among the specimens with both POPEth and PLPEth levels within the AMR, the average POPEth/PLPEth ratio was 1.4 supporting the overall prevalence of higher POPEth concentrations than PLPEth. However, the POPEth/PLPEth ratio varied widely, from 0.3 to 8.8, reflecting patient differences in PEth metabolism patterns. Conclusions To help verify probable ethanol abstinence, we have developed, validated, and deployed a high-throughput clinical assay for the major PEth homologs, POPEth and PLPEth. In a retrospective analysis of 29,205 clinical specimens: 71.3% of results were consistent with abstinence; 28.3% of the results were positive suggesting moderate alcohol consumption within the last 1-2 weeks. With POPEth most prevalent, yet variation observed, there were 62 specimens with PLPEth positive and negative for POPEth. This clinical PEth study including both POPEth and PLPEth provides greater sensitivity to identify ethanol exposure than only testing a single homolog.

A-133 Trusted Test Goes Awry: Investigating Dilution Aberrations for Total Bilirubin

Abstract Background A patient with acute decompensated cirrhosis was hospitalized due to lightheadedness and jaundice, undergoing evaluation for a liver transplant. The attending physician brought to the laboratory’s attention a perplexing pattern in the patient's total bilirubin results. Initially, the patient displayed a consistent trend of elevated total bilirubin levels above the upper limit of linearity, ULOL (> 30 mg/dL), which then plummeted to under the limit of quantification (< 0.1 mg/dL) before surging back to > 30 mg/dL. Methods The total bilirubin in heparinized plasma was measured on the AU5800 by Beckman Coulter. Samples > ULOL are diluted automatically 1:3 utilizing the instrument’s on-board dilution feature. The final test results are reported automatically to an electronic medical record. Following the reported incident, an investigation and troubleshooting plan was developed, encompassing the following steps: Repeat patient testing with total bilirubin concentration > 30 mg/dL and <0.1 mg/dL; Rule out the previously reported potential interferences, including Immunoglobin M and acetaminophen; Conduct a 90-day quality control (QC) review; Perform split test analysis on two instruments within the laboratory and at a reference laboratory; Conduct patient chart reviews to identify discrepancies based on previously reported values; Complete a root cause analysis and develop corrective/preventive action plan. Results Patient sample < 0.1 mg/dL was tested on an alternative instrument, resulting > 30 mg/dL. Patient with normal IgM of 161 mg/dL didn’t reveal any other potential drug interferences in chart review. The 3-month QC review found consistent with peer performance. Split comparison of 6 patients revealed discrepancies for samples > 30 mg/dL on one of the two instrument units. When total bilirubin > 30 mg/dL, the malfunctioning unit erroneously reported values < 0.1 mg/dL after dilution. Review of 34 patient charts showed the impact dates were spanned for 39 days. Since the on-board dilution feature is utilized by 10 other analytes, 892 diluted samples were analyzed for discrepancies. 36 discrepant results were identified among 4 affected assays including total bilirubin, direct bilirubin, creatine kinase, and alanine aminotransferase. These results were corrected, and providers were notified. Conclusions The root cause was traced to a malfunction in one instrument unit, despite no malfunction flags. The laboratory QC concentrations fell within the analytical measurement range, missing the dilution issue during the daily QCs. During an extensive repair period, the on-board dilution was disabled on the affected unit and samples were re-routed to alternative instrument for testing. Policy changes and operator training were initiated for a new result verification process, impacting the laboratory productivity. Autoverification of diluted sample results was disabled for all chemistry analyzers. The vendor identified the issue as sample transfer unit crane misalignment in the up/down direction. It is worth noting that proficiency testing or QCs often do not extend beyond the analytical measurement ranges, leading to undetected incidents. Additional quality measures like periodic patient chart reviews, patient sample comparisons, and other quality assurance tools can aid in on-going dilution performance verification. Open communication between the physicians and laboratories is crucial for prompt issue identification and resolution.

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.

A-099 Case Study of a Patient With Heterophile Antibodies Interfering with Both Competitive and Immunometric Assays

Abstract Background Immunoassay results may be affected by the presence of interfering substances including heterophile antibodies. These heterophile antibodies can cause either an increase or decrease in the result based on assay design and antibody target. We report a case study wherein we identified several analytes whose results were either increased or decreased by heterophile antibodies in a patient. Methods 74 year male presented to Endocrinology with history of hypocalcemia and his previous lab tests were suggestive for idiopathic hypoparathyroidism, Hashimoto’s disease and Addison’s disease raising physician concern for a possible Type 1 autoimmune endocrinopathy. Cortisol was ordered (for an ACTH stimulation test for adrenal insufficiency); upon testing the post-stimulation cortisol result was higher than the analytical measurement range (AMR), and so the sample was diluted 1:2 and repeated. The repeat cortisol result after correction for the dilution factor was well within the AMR and prompted an investigation for an interfering substance. Serial dilution of analytes was performed and send-out testing for the same with another immunoassay from a different manufacturer and/or mass spectrometry method where available were done. Results The results for most analytes did not match with the reference lab results. Serial dilution showed non-parallelism in the results. For most analytes, after dilution of the sample by 1:4 or 1:8 times, the interfering substance was diluted enough to obtain a result matching the reference lab result. Conclusions Based on non-parallelism in serial dilution results, discordant results with other methods, and loss of interference after dilution it was determined that the most likely cause was a heterophile antibody. Formal testing using blocking antibodies was not done as it seemed that the reference lab used did not run our method and their assays were not affected by this particular heterophile antibody. Analysis of the assay designs and analyte results showed that competitive assays showed falsely high results, while immunometric assays showed falsely low results. The physician was contacted and the case discussed in detail and it was recommended that further testing on this individual be sent out to a reference lab using the alternate method or to use mass spectrometry based assays for further testing if needed. Today, the patient continues to have low calcium, high phosphate and an undetectable PTH suggestive of idiopathic hypoparathyroidism but his other endocrine functions are within normal limits.

B-297 Tacrolimus Concentrations in Envarsus vs Prograf Patients: An Evaluation of the Clinical Impact of LC-MS/MS and Immunoassay Methods on Tacrolimus measurement

Abstract Background Tacrolimus is a calcineurin inhibitor used as part of an immunosuppressive regimen in kidney and liver transplant patients to prevent graft-versus-host disease (GvHD) and requires therapeutic drug monitoring due to its narrow therapeutic index. Tacrolimus levels are commonly measured using two different methodologies - immunoassay and liquid chromatography tandem mass spectrometry (LC-MS/MS). A global proficiency study to assess and compare the measurement of trough levels by these two methodologies noted a positive bias with the ARCHITECT immunoassay which is likely due to the presence of tacrolimus metabolites. The aim of our study is to assess the impact of two different formulations of tacrolimus on the accuracy of its analytical measurement. While differences between immunoassay and LC-MS/MS results have been evaluated for immediate-release tacrolimus (Prograf), it has yet to be characterized for extended-release formulations such as Envarsus. The discrepancy between immunoassay and LC-MS/MS has clinical implications, as dosage modifications are based on measured concentrations, analytical variation may cause intervals of subtherapeutic or supratherapeutic exposure, increasing the risk of graft rejection or toxicity. Methods Development of an LC-MS/MS method for the simultaneous quantification of tacrolimus and its major metabolite, desmethyl tacrolimus, was performed in whole blood. Traceable calibrators and quality control material for tacrolimus and specimens supplemented with desmethyl tacrolimus were used to determine accuracy and precision. Tacrolimus concentrations were measured by LC-MS/MS and ARCHITECT immunoassay methods in excess whole blood specimens from patients treated with either Prograf or Envarsus. Measurement biases between LC-MS/MS and immunoassay methodologies were determined for both formulations of tacrolimus. Results External calibration curves for both tacrolimus and desmethyl tacrolimus were linear (R2>0.995), and the analytical measurement range (AMR) for tacrolimus spanned from 1.1 - 31.6 ng/ml. Calibrator and Quality control (QC) biases were within 15% of their target values throughout the AMR and within-run imprecision was less than 10% (n=25) for all calibrators and QCs. Between-run imprecision for Low, Mid, and High QC levels over a period of 2 weeks (n=5 days) was less than 10%. Comparative bias of tacrolimus concentrations between immunoassay and LC-MS/MS was significantly lower (P value=0.0074) for Envarsus (n=20 specimens) relative to Prograf (n=32 specimens). Conclusions The differential bias between immunoassay and LC-MS/MS in the measurement of tacrolimus in patients dosed with immediate-release versus extended-release formulations suggests that their distinct pharmacokinetic profile may impact the accuracy of the measurement. Therefore, applying observed measurement biases from different assays derived from Prograf patients to Envarsus patients and vice versa may result in erroneous estimation of tacrolimus concentrations.

B-182 Development and validation of an LC-MS/MS assay for simultaneous measurement of serum androstenedione and 17-hydroxyprogesterone in serum

Abstract Background Androstenedione and 17-hydroxyprogesterone are measured via immunoassays or high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) in clinical laboratories. Immunoassays have limited sensitivity and specificity for certain steroid hormones testing. We aimed to develop and validate a sensitive, accurate, and specific in-house LC-MS/MS assay to simultaneously measure both 17-hydroxyprogesterone and androstenedione in serum. Methods 13C-labeled androstenedione and 17-hydroxyprogesterone internal standards were added to calibrator, control, and patient samples. A hexane/ethyl acetate solution was used in a liquid-liquid extraction procedure. The organic layer was then removed and dried followed by reconstituting the extracts in 2.5 mM ammonium formate in 50% methanol. The HPLC mobile phase gradient began with 47%, 2.5 mM ammonium formate in water and 53%, 2.5 mM ammonium formate in methanol. This progressed to 56% of the methanol phase over three minutes. The mixtures were separated though an Agilent 1260 Infinity HPLC system with a Poroshell 120 EC-C18 analytical column (2.1 × 50 mm × 2.7 µm). The separated HPLC eluents then entered an Agilent 6460C QQQ mass spectrometer in positive ion mode through an electrospray ionization source where protonated androstenedione (m/z = 287.2) with the protonated androstenedione-[2, 3, 4-13C3] internal standard (m/z = 290.2) and protonated 17-hydroxyprogesterone (m/z = 331.2) with the protonated 17-hydroxyprogesterone-[2, 3, 4-13C3] (m/z = 334.2) were mass-selected for collision-induced dissociation. For androstenedione, respective quantifier and qualifier m/z values of 287.2 > 97.1 and 287.2 > 109.1 were used while respective quantifier and qualifier m/z values of 331.2 > 97.1 and 331.2 > 109.1 were used for 17-hydroxyprogesterone. Validation studies were performed including imprecision, lower limit of quantitation (LLOQ), analytical measurement range (AMR), interference, specificity, carryover, extraction recovery, matrix effect, and comparison with a reference laboratory LC-MS/MS method. The total allowable error limits used in this study were 23.5% for androstenedione and 30.2% for 17-hydroxyprogesterone. Results Total imprecision results across three concentrations yielded a maximum CV of 4.6 % for androstenedione and 6.5% for 17-hydroxyprogesteron. The AMRs of androstenedione and 17-hydroxyprogesterone were 0.2 to 13.4 and 0.1 to 22.0 ng/mL without significant carryovers. No significant interferences were observed from 11-deoxycorticosterone (700 ng/dL), testosterone (2500 ng/dL), DHEA (1000 ng/mL), bilirubin (30.1 mg/dL), hemoglobin (1032 mg/dL), or triglycerides (1858 mg/dL). The extraction recoveries of androstenedione and 17-hydroxyprogesterone were 98% and 81%, respectively. No significant matrix effects were identified. In comparison to the reference lab LC-MS/MS method, the in-house LC-MS/MS assay demonstrated a 0.523% bias for androstenedione and a -3.77% bias for 17-hydroxyprogesterone with both exhibiting excellent correlations with the reference lab measurements. More specifically, our results (as a function of values obtained from the reference lab) followed the relationship of y = 1.039x - 0.092 (R = 0.9951) for androstenedione and y = 0.957x + 0.01 (R = 0.9974) for 17-hydroxyprogesterone. Conclusions An LC-MS/MS assay is developed for simultaneous detection of serum androstenedione and 17-hydroxyprogesterone. The assay demonstrates acceptable imprecision, AMR, and accuracy. No significant carryover and interference from other structurally similar steroid hormones and other common interferents are identified.

B-287 Comparing a Recently FDA-Cleared Free Phenytoin Assay to a Laboratory Developed Test

Abstract Background Phenytoin is an anti-convulsant drug that is commonly prescribed to epileptic patients to control seizures. The drug circulates in two forms: protein-bound (90%), and the free, biologically active form (10%). In certain circumstances, such as protein-binding disruption, hypoalbuminemia, and drug-drug interactions, the total phenytoin serum concentration may not accurately reflect active phenytoin levels. Therefore, a free phenytoin level (i.e. non-protein bound) may be indicated. Until recent FDA-clearance, we utilized an in-house, laboratory-development test (LDT) to deliver these needed results. The purpose of this study was to validate the FDA-cleared assay and compare its performance characteristics to a free phenytoin LDT. Methods Validation of the FDA-cleared assay was performed on an automated chemistry analyzer (Roche Diagnostics, cobas c502), utilizing a kinetic interaction of microparticles in a solution (KIMS) method. Both the FDA and LDT assay used the same free phenytoin reagent, however the calibrators and pipetting parameters for each assay were different. FDA assay used a six-point calibration at concentrations of: 0, 2.5, 5, 10, 20, 40 µg/mL (Preciset TDM I Calibrators), whereas the LDT assay used a six-point calibration at concentrations of: 0.0, 0.5, 1.0, 2.0, 3.0, 4.0 µg/mL (cobas FP Free Phenytoin Calibrators). The following performance characteristics were determined for the FDA-cleared assay and compared to the current LDT: analytical measurement range, precision, accuracy, and maximum dilution. Free forms of the drug were obtained as a filtrate through centrifugation using a molecular weight cut-off filter (30 kDa, Millipore), spun at 3600 rpm for twenty minutes. Results Conclusions The FDA-cleared free phenytoin assay is suitable for use in the clinical laboratory producing similar results to our LDT. Switching to the FDA-cleared assay reduces the regulatory burden and eliminates the need for transferring reagent into open channel cassettes and manual dilutions.

B-112 Regression Analysis Techniques for Assessing Calibration Reproducibility Within and Between Instruments. A Practical Example with Blood Ca²⁺ Assayed with Two cobas® b 221Analyzers

Abstract Background Reproducibility of the calibration curve within the analytical measurement range (AMR) is crucial for ensuring reliable patient values within and between laboratory instruments to detect shifts and trends of clinical significance. In this practical example for ionized calcium (Ca²⁺) assayed with two cobas b221® instruments, the equality of calibration lines and the verification with patient specimen comparison study was assessed with regression analysis techniques. Methods Instruments: Two cobas b 221 (Roche Diagnostics). Calibration material: COOX/MSS™ (Five levels, Roche Diagnostics). This was independently assayed either three or five times in parallel with both instruments. The patient specimens were obtained by venipuncture in Na/Li-heparin BD™ tubes (Becton-Dickinson). Upon receipt, specimens were assayed with both instruments within two minutes. The data were transferred to Minitab® (Version 21, Minitab Inc) statistical software and analyzed with polynomial ordinary least squares (POLS), orthogonal and locally weighted scatterplot smoother (lowess) regression analysis models. The aptness of the model was assessed with standardized residuals (std. res.) diagnostics. For acceptable total error the CLIA’s criterion target value ± 10% was adopted. Results While the POLS model did not show probabilistically significant differences between instruments (P=0.8), it showed probabilistically significant differences between years (P=0.003). However, this was due to a small MSE (= 0.003 mg/dL), which is not clinically significant. The test for linearity showed probabilistically significant lack-of-fit (P<0.0001) due to a small MS pure error (<0.0001 mg/dL). The lowess for the standardized deleted residuals by the fitted value showed a quasi-linear relationship. The multiple comparisons (P=0.3) and the Levene’s (P=0.4) tests did not detect probabilistically significant heteroscedasticity of the variance by reference value. The plot of the std. res. by the leverage showed two possible outliers (std. res.>|3|) and no influential observations (Hi<0.5). These diagnostics proved the aptness of the POLS model. The harmonization between the two instruments was verified with patient specimens. The orthogonal regression model showed in the interval 3-6 mg/dL a quasi-linear relationship between the values as obtained with the two instruments. The orthogonal regression line (y=0.1+1.02 n) and the OLS regression line (y=0.03+601 n) were very similar. There was no probabilistically significant lack-of-fit (P=0.5). The plot of the std. res. by the leverage showed one possible outlier (std.res.>|3|) and no influential observations. Additionally, the plot of the relative differences between the values as obtained with b221#9371 and b221#24796 by the values as obtained with b221#9371 showed only one value exceeding half of the total allowable error. Conclusions The POLS model clearly showed that the calibration lines for the two instruments were quasi-linear in the interval 2-10 mg/dL and reproducible within and between instruments. Additionally, this reliable performance was verified with patient specimens in the interval 3-6 mg/dL. The differences between values as obtained with each instrument were within half of the CLIA’s total allowable error. This clearly showed that equality of calibration lines ensured interchangeable patient values between instruments. Finally, electronic transfer of data and the availability of statistical software like Minitab was crucial for conducting this study.

A-308 Placental Growth Factor Kryptor Immunoassay Reveals Quality Control Imprecision and Drift

Abstract Background The utility and value of determining the soluble fms-like tyrosine kinase-1 to placental growth factor (sFlt-1/PlGF) ratio for predicting the likelihood of progression to pre-eclampsia in pregnant women admitted with hypertension are increasingly becoming widely recognized. A key finding of the multi-center PRAECIS trial (NEJM Evid 2022;1(12)) is the observation that sFlt-1/PlGF ratios ≥ 40 correlate with higher risk for adverse maternal outcomes. The sFlt-1 and PlGF immunoassays performed on the ThermoFisher Scientific B·R·A·H·M·S Kryptor autoanalyzer became the first FDA-approved tests in 2023 for determining sFlt-1/PlGF ratio. In this study, an evaluation of the analytical performance of the sFlt-1 and PlGF KRYPTOR immunoassays was performed, which included analyte measuring range (AMR), accuracy, precision, and stability studies. Methods Calibration and QC materials (consisting of lyophilized recombinant human proteins), test reagents and solutions, and consumables were purchased and provided by ThermoFisher Scientific. Analytical runs were performed according to vendor’s instructions by trained medical laboratory scientists. Between-day precision of our two instruments for both immunoassays was assessed by assaying all three levels of sFlt-1 and PlGF QC materials, as well as patient serum pools with low PlGF concentration, once to twice daily for multiple consecutive days. Results While the analytical performance of the sFlt-1 immunoassay was robust and performed according to expectation (within-day CV <1% and between-day CV <3%), the PlGF immunoassay was surprisingly less reliable. Imprecision and inaccuracy of the PlGF immunoassay at the low end of its analytical measurement range was reflected by both sporadically out-of-control between-day measurements of all three QC levels (with target ranges of 24 - 36 pg/mL, 80 - 120 pg/mL, and 320 - 480 pg/mL), as well as slow downward drifts of particularly the low level QC material. In one instance, a downtrend was observed from 28 pg/mL to 14 pg/mL over 6 days, even with fresh reagents, solutions, and calibration. Such shortened stability of the PlGF assay does not meet the vendor’s claims of calibration stability of 15 days and onboard kit stability of 29 days. Using vendor’s provided material, the AMR of both sFlt-1 and PlGF were adequately verified and showed excellent linearity (r2> 0.99). Conclusions Despite low level QC imprecision and drift, the sFlt-1/PlGF ratios determined using the B·R·A·H·M·S Kryptor analyzers are acceptable for clinical use but with the caveat that we will perform batch testing with bracketing QCs and conduct stringent QC monitoring, frequent calibrations, and reduce the onboard kit stability as defined by ThermoFisher Scientific. Our findings serve as a reminder that FDA approval does not preclude thorough verification studies of any assay prior to implementation for clinical use.

B-105 Farr Radioimmunoprecipitation Method Enhances Serial Monitoring of Quantitative Anti-dsDNA (double-stranded) Antibody Measurements in Lupus

Abstract Background Anti-double stranded DNA (anti-dsDNA) antibodies are an important criterion in the diagnosis, classification, and management of systemic lupus erythematous (SLE). High avidity anti-dsDNA IgG may be more clinically relevant and specific for SLE. As such, radioimmunoassay according to the Farr technique (Farr-RIPA) is often considered more sensitive and quantitative than enzyme-linked immunosorbent assay (ELISA) and Crithidia luciliae immunofluorescence assay (CLIFT) methods. Methods Measurements of anti-dsDNA antibodies in 147,016 serum patient samples were obtained by lab-developed Farr-RIPA. Serum with endogenous antibodies binds to 125I-radiolabelled dsDNA and undergoes subsequent immunoprecipitation under high salt conditions which selects for highly avidity anti-dsDNA antibodies. The analytic measurement range (AMR) of the assay is 5.7 to 85 IU/mL, and dilutional linearity is validated to 68,000 IU/mL for results >85 (ULOQ). Inter-assay precision is <10% CV. Results Of 147,016 measured samples, 5.4% (7800) were positive for anti-ds DNA antibodies (mean 251.7) with numeric values, ranging from 8.0 to 55,399 IU/mL. The numeric distribution of all positive results was such that 76% (5859/7800) were between 8.0 - 60 IU/mL, and the median was 19.4 with an interquartile range (IQR) of 36.1. A small percentage (3.9%) had very high anti-dsDNA levels greater than 1000 IU/mL and 0.4% (35/7800) were greater than 10,000 IU/mL. Serial measurements comprised almost half (45.5%) of all positives, occurring in 1016 unique patients an average of 3.5 levels within a 2-year time frame. A fifth of those patients, 200, were more frequently monitored with at least 4 levels. Of patients with the very highest dsDNA (>10,000), we analyzed the results of 17 unique patients who were frequently monitored an average of 6.1 times. Peak and nadir values yielded a mean delta of 20,751 with a mean of 264 days elapsing between peak and nadir. All except one showed a marked decrease in Anti-dsDNA over time with an average decrease of 15,685 IU/mL. Conclusions Here, we performed 147,016 Anti-dsDNA antibody measurements by Farr-RIPA, a precise method with a broad analytic range as compared to other methods which have a limited upper limit of quantitation or are semi-quantitative. Our 7800 positive Farr-RIPA results exhibited quantitative results from 8.0 to 55,399 IU/mL. The median of 19.4 and IQR of 36.1 showed most results to be <55.5 IU/mL. Very high levels >300 and >1000 IU/mL were seen in 10% and 4% of patient samples, respectively. Serial monitoring was frequent. In patients with the very highest levels >10,000, an-eight fold decrease in peak Anti-dsDNA levels was achieved over a mean of 264 days with more than half (9/17) achieving levels <250. The ability to detect and meaningfully monitor these patients is enhanced by the Farr-RIPA method. In conclusion, in 7800 positive samples, we demonstrated a wide range of inter- and intra-patient numeric Anti-dsDNA concentrations that denote the clinical usefulness of serial monitoring by Farr-RIPA.

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-254 Analytical and Clinical Performance Evaluation of Plasma pTau217 in a Clinical Diagnostic Laboratory

Abstract Background Blood-based biomarkers provide an accessible and cost-effective tool for diagnosing Alzheimer’s disease (AD). Among these biomarkers, phosphorylated tau 217 (pTau217) shows promise for both AD diagnosis and differential diagnosis, as well as for facilitating access to emerging disease-modifying therapies. The ALZpath SIMOA pTau-217 v2 assay is a digital immunoassay designed for quantifying pTau-217 in human EDTA plasma samples. This study assesses the analytical performance of plasma pTau217 assays as a Laboratory Developed Test (LDT) appropriate for diagnostic application. Methods In this analytical study, we evaluate accuracy, precision, detection limits, linearity, and analyte stability. Plasma pTau217 levels were measured using the ALZpath pTau217 v2 assay on a single Quanterix HD-X SIMOA Analyzer platform at BC Neuroimmunology Lab. Inc. Vancouver, British Columbia, Canada. As a clinical testing laboratory, our validation procedures are in accordance with CAP and CLIA requirements, with a primary focus on adhering to CLSI guidelines for both analytical and clinical performance testing. To determine a reference range and cut-off, samples of Amyloid PET-confirmed cases (n=263) and healthy subjects between the ages of 55 and 95, with a roughly equal ratio of males to females (n=219), were tested. Results The ALZpath pTau217 assay demonstrates an analytical measurement range of 0.0046 - 3.4056 ng/L, with a limit of blank (LoB) of 0.0019 ng/L, a limit of detection (LoD) of 0.0046 ng/L, and a limit of quantification (LoQ) of 0.03 ng/L. Linearity spans from 0.06 to 1.75 ng/L, with repeatability showing intra-laboratory precision above LoQ ≤ 20% CV. Stability is maintained at 4⁰C for ≤ 1 week, at room temperature for ≤ 72 hrs, at -20⁰C for ≤ 2 weeks, and at -70⁰C for ≤ 3 weeks, with freeze/thaw cycles tolerated up to 6 times. Moreover, interference is not detected at the maximum concentrations tested for albumin, unconjugated bilirubin, biotin, hemoglobin, creatinine, and heterophilic antibodies. The reference range was determined using the 95% reference interval, resulting in 0.398 ng/L. ROC analysis yielded an AUC of 0.932 with a Youden index of 0.338 ng/L. At the Youden index, sensitivity was 90.0% with a specificity of 85.6%. Specificity reached 95.5% at the upper cut point of 0.471 ng/L, while sensitivity was 94.8% at the lower cut point of 0.242 ng/L. Conclusions The outstanding analytical performance of pTau217 supports its adoption as a reliable screening and diagnostic tool in clinical practice, aiding clinicians in the accurate diagnosis of AD.

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.

Development of a UPLC-MS/MS Method to Simultaneously Measure 13 Antiepileptic Drugs with Deuterated Internal Standards.

The goal of this study was to develop and validate a UPLC-MS/MS method for simultaneous mea-surement of 13 AEDs, including carbamazepine, oxcarbazepine, lamotrigine, levetiracetam, topiramate, primidone, zonisamide, gabapentin, lacosamide, perampanel, pregabalin, rufinamide, and vigabatrin, in whole blood samples. A UPLC-MS/MS method for simultaneous determination of 13 AEDs in whole blood was developed, and validation was conducted for accuracy, precision, limit of quantification (LOQ), matrix effect, and stability. Our method was compared to two different hospitals using UPLC-MS/MS. All AEDs exhibited linearity across the AMR (analytical measurement range), with R2 values ranging from 0.994 to 1.000. The imprecision and inaccuracy for low and high quality control (QC) levels were within an acceptable range, with the coefficient of variation (CV) < 15%. The LOQ was 0.62 µg/mL for carbamazepine, 1.61 µg/mL for oxcarbazepine, 1.30 µg/mL for lamotrigine, 13.20 µg/mL for levetiracetam, 1.26 µg/mL for topira-mate, 1.01 µg/mL for primidone, 1.59 µg/mL for zonisamide, 1.09 µg/mL for lacosamide, 1.61 µg/mL for gabapentin, 0.50 µg/mL for pregabalin, 0.07 ng/mL for perampanel, 3.00 µg/mL for rufinamide, and 2.06 µg/mL for vigabatrin. All AEDs demonstrated acceptable assay parameters for carryover, stability, and matrix effects. Moreover, the assay showed satisfactory results compared to two different hospitals with a bias of less than 15%. We successfully developed and validated a fast and robust UPLC-MS/MS method for routine therapeutic drug monitoring of thirteen antiepileptic drugs simultaneously.

Direct analysis in real time mass spectrometry (DART-MS/MS) for rapid urine opioid detection in a clinical setting

Background and aimsCurrent laboratory methods for opioid detection involve an initial screening with immunoassays which offers efficient but non-specific results and a subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmation which offers accurate results but requires extensive sample preparation and turnaround time. Direct Analysis in Real Time (DART) tandem mass spectrometry is evaluated as an alternative approach for accurate opioid detection with efficient sample preparation and turnaround time. Materials and methodsDART-MS/MS was optimized by testing the method with varying temperatures, operation modes, extraction methods, hydrolysis times, and vortex times. The method was evaluated for 12 opioids by testing the analytical measurement range, percent carryover, precision studies, stability, and method-to-method comparison with LC-MS/MS. ResultsDART-MS/MS shows high sensitivity and specificity for the detection of 6-acetylmorphine, codeine, hydromorphone, oxymorphone, hydrocodone, naloxone, buprenorphine, norfentanyl, and fentanyl in urine samples. However, its performance was suboptimal for norbuprenorphine, morphine and oxycodone. ConclusionIn this proof-of-concept study, DART-MS/MS is evaluated for its rapid quantitative definitive testing of opioids drugs in urine. Further research is needed to expand its application to other areas of drug testing.

PrecivityAD2™ Blood Test: Analytical Validation of an LC-MS/MS Assay for Quantifying Plasma Phospho-tau217 and Non-Phospho-tau217 Peptide Concentrations That Are Used with Plasma Amyloid-β42/40 in a Multianalyte Assay with Algorithmic Analysis for Detecting Brain Amyloid Pathology.

Alzheimer's disease (AD) is a progressive irreversible neurodegenerative disorder that represents a major global public health concern. Traditionally, AD is diagnosed using cerebrospinal fluid biomarker analysis or brain imaging modalities. Recently, less burdensome, more widely available blood biomarker (BBM) assays for amyloid-beta (Aβ42/40) and phosphorylated-tau concentrations have been found to accurately identify the presence/absence of brain amyloid plaques and tau tangles and have helped to streamline AD diagnosis. However, few BBMs have been rigorously analytically validated. Herein, we report the analytical validation of a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) multiplex method for quantifying plasma phosphorylated-tau217 (p-tau217) and non-phosphorylated-tau217 (np-tau217) peptide concentrations. We combined the p-tau217/np-tau217 concentrations ratio (%p-tau217) and the previously validated LC-MS/MS multiplex assay for plasma Aβ42/40 into a new multianalyte assay with algorithmic analysis (MAAA; PrecivityAD2™ test) that identifies brain amyloid status based on brain amyloid positron emission tomography. We found (a) the %p-tau217 assay is precise, accurate, sensitive, and linear over a wide analytical measurement range, and free from carryover and interference; (b) the pre-analytical specimen collection, processing, storage, and shipping conditions that maintain plasma tau peptide stability; and (c) using the measured analytical imprecision for plasma Aβ42/40 and p-tau217/np-tau217 levels in a worst-case scenario model, the PrecivityAD2 test algorithm for amyloid pathology classification changed for only 3.5% of participants from brain amyloid positive to negative, or from negative to positive. The plasma sample preparation and LC-MS/MS methods underlying the PrecivityAD2 test are suitable for use in the clinical laboratory and valid for the test's intended purpose: to aid in the diagnostic evaluation of individuals aged 55 and older with signs or symptoms of mild cognitive impairment or dementia.

Specimen type validation and establishment of normal cytokine reference intervals in cerebrospinal fluid

Cerebrospinal fluid (CSF) is a critical body fluid to examine in attempts to discover potential biomarkers for neuroinflammatory and other disorders of the central nervous system (CNS). Serum and/or plasma cytokine levels have been associated with a variety of inflammatory conditions, and some have been shown to be actionable therapeutic targets. Less is known, however, about cytokine levels in CSF. Serum and plasma cytokine testing is widely available in clinical and research laboratories, but cytokine testing in CSF is extremely limited and if performed, accompanied by a disclaimer that it is an unvalidated specimen type. In this study, we validate CSF as a suitable specimen type and determine normal reference intervals for multiple cytokines as well as a soluble cytokine receptor. CSF was validated as a specimen type for testing using a laboratory developed multiplexed cytokine assay previously validated to measure 13 cytokines/markers in serum and plasma. Performance parameters including specimen dilution, specimen interference, linearity and precision were examined. Reference intervals were established using 197 normal and control CSF specimens by non-parametric quantile-based methods. CSF cytokine analysis demonstrated within and between run precision of <10% and < 20% CV, respectively and linearity of ±15% for all analytes throughout the analytical measurement range of the assay. Reference intervals for the 13 cytokines/markers were established from 197 normal and control CSF specimens (78 Male; mean 44.8 y ± 21.7 SD, 119 Female; mean 42.8 y ± 20.3 SD). Cytokine concentrations in CSF from normal donors and controls were less than the lower limit of quantitation of our assay for 6 of the 13 measured cytokines/markers. The chemokine IL8 demonstrated the highest concentration of all analytes measured. CSF demonstrated acceptable performance as a specimen type in our multiplexed cytokine assay. By validating CSF as a specimen type and establishing normal reference intervals for cytokine concentrations in CSF, their potential as biomarkers for infectious, autoimmune and other inflammatory CNS disorders can be more appropriately investigated.