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

OBJECTIVES: 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. METHODS: 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. RESULTS: 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 8.0 g/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. CONCLUSIONS: The G6PD assay with automated lysis is acceptable for patient testing. Several measures ensured the robustness of this workflow in a hospital laboratory.

Matrix-matched calibrators are necessary for robust and high-quality dried blood spots lead screening assays by inductively coupled plasma-mass spectrometry.

Background and aims: Reliable lead screening methods are necessary to support early identification of lead exposure in children. Sample collection using dried blood spots (DBS) offers advantages compared to traditional venipuncture and capillary collection. Here, we describe and compare three lead DBS inductively coupled plasma-mass spectrometry (ICP-MS) methods for lead screening. Materials and methods: Lead was extracted from Whatman 903 protein saver cards punches and analyzed by ICP-MS across three independent clinical laboratories. Each laboratory evaluated the performance of aqueous and matrix-matched DBS calibrators using external quality control samples (WI State of Laboratory of Hygiene Program). Leftover patient samples (n = 39) were used for an interlaboratory comparison of lead DBS. Lead DBS results were compared to whole blood methods. Results: The DBS ICP-MS methods using matrix-matched DBS calibrators had superior performance to the aqueous calibrations. There was a strong correlation between lead measured in DBS (matrix-matched) and whole blood for the three methods evaluated. Conclusion: Lead can be measured accurately by ICP-MS in DBS samples when matrix-matched calibrators are used. External quality control programs are valuable to assess the performance of DBS methods. DBS lead ICP-MS methods are a robust analytical option for lead screening even though the limitations of DBS are well recognized.

The Androstenedione Roche Elecsys immunoassay has superior comparability to the LC-MS/MS assay than the Siemens Immulite immunoassay.

Androstenedione (ASD) is a biomarker used in the diagnostic workup of hyperandrogenism, congenital adrenal hyperplasia, premature adrenarche, and polycystic ovary syndrome (PCOS). The Elecsys ASD competitive electrochemiluminescence immunoassay (Roche Diagnostics, Indianapolis, IN) is a new assay recently available in the US. Objective: This study evaluated the analytical and clinical performance of the Elecsys ASD assay. Design & Methods: We evaluated the linearity/analytical measuring range (AMR), precision, and accuracy of the Elecsys ASD assay on the cobas e601 analyzer. ASD was measured in serum/plasma in the Elecsys ASD, Immulite (Siemens Medical Solutions USA, Inc. Malvern, PA), and LC-MS/MS assays. Reference intervals (RI) were evaluated across genders, menopausal status, and in children. Statistical analysis was performed using EP evaluator and R program. Results: The Elecsys ASD assay had a linear response across the AMR. The intra- and inter-assay coefficients of variation at various concentrations were ≤4.5%. The Elecsys ASD assay had a mean difference of -0.04 ng/mL (-1.7%) with the LC-MS/MS assay, whereas the Immulite assay had a mean difference of 1.17 ng/mL (66%) and -1.22 ng/mL (-38%) compared to the LC-MS/MS and Elecsys ASD assays, respectively. The Roche recommended RIs for healthy men (0.280-1.52 ng/mL) and postmenopausal women (0.187-1.07 ng/mL) were successfully verified. The RIs for children were adopted from published data. For pre-menopausal women, a RI of <1.60 ng/mL was established. The ASD concentrations in women with and without PCOS overlapped. Conclusions: The Elecsys ASD assay has superior comparability to the LC-MS/MS assay than the Immulite assay.

Evaluation of the Clinical Performance of 7 Serological Assays for SARS-CoV-2 for Use in Clinical Laboratories.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) serological assays have emerged as a response to the global pandemic, warranting studies evaluating their clinical performance. This study investigated 7 commercially available SARS-CoV-2 serological assays in samples from noninfected individuals and hospitalized patients. METHODS: SARS-CoV-2 qualitative serological assays by Abbott (IgG), Beckman (IgG), DiaSorin (IgG), EUROIMMUN (IgG and IgA), Roche and Bio-Rad (Total) were evaluated using specimens collected pre-December 2019 (n = 393), from nucleic acid amplification testing (NAAT) negative patients (n = 40), and from 53 patients with COVID-19 by NAAT collected 3-21 days post-onset of symptoms (POS) (N = 83). Negative agreement (NA), positive agreement (PA), and positive and negative predictive values (PPV and NPV) at prevalences of 5% and 10% were calculated. RESULTS: The overall %NA; 95% CI in the negative samples were: Roche 99.8%; 99.3-100.2, Beckman 99.8%; 98.7-100.0, Abbott and Bio-Rad 99.3%; 98.0-99.9, DiaSorin 98.4; 97.2-99.6, EUROIMMUN IgG 97.5%; 95.5-98.7, and EUROIMMUN IgA 79.7%; 75.9-83.5), accounting for positive/equivocal results as false positives. The %PA; 95% CI in samples collected 14+ days POS (n = 24) were: Bio-Rad 83.3%; 68.4-98.2, Abbott and Roche 79.2%; 62.9-95.4, EUROIMMUN IgA 70.8%; 52.6-89.0, Beckman 58.3%; 38.6-78.1, DiaSorin 54.2; 34.2-74.1, and EUROIMMUN IgG 50.0%; 30.0-70.0, accounting for negative/equivocal results as false negatives. NPVs ranged from 97.4%-98.9% and 94.7%-97.7% for prevalences 5% and 10%, respectively. PPVs ranged from 15.5%-94.8% and 27.9%-97.4% for prevalences 5% and 10%, respectively. CONCLUSION: The Roche and Beckman assays resulted in fewer false positives, followed by the Bio-Rad and Abbott assays. While the Bio-Rad assay demonstrated higher antibody detection in COVID-19-positive patients, PA claims cannot be established with a high level of confidence in our sample population.

Comparison of symmetric dimethylarginine with creatinine, cystatin C and their eGFR equations as markers of kidney function.

OBJECTIVES: Symmetric dimethylarginine (SDMA) is a catabolic product of arginine-methylated proteins and is an emerging biomarker for kidney function. A limited number of studies in selected populations have shown good correlation between SDMA and a few known markers of glomerular filtration rate (GFR). However, a comprehensive comparison of SDMA with all existing serum endogenous markers in a population with varied kidney function and against measured GFR is lacking. The objective of this study was to compare the correlations of SDMA, creatinine, cystatin C and their eGFR equations against GFR measured by iothalamate clearance in an adult population with varied kidney function. DESIGN & METHODS: Left-over serum and plasma specimens were collected from 40 adults with normal and reduced kidney function. GFR was measured using a radioactive iothalamate procedure. Creatinine and cystatin C were measured on Roche Cobas 8000. SDMA was measured by a published liquid chromatography-tandem mass spectrometry method. RESULTS: SDMA correlated highly with measured GFR (r=-0.84), which was better than creatinine (r=-0.70) but equivalent to cystatin C (r=-0.86) and the eGFR equations [MDRD and CKD-EPI (separate and combined)]. CONCLUSIONS: SDMA is a strong marker of kidney function and further studies are needed to establish an eGFR formula that includes it for widespread clinical use.

Sensitive, Simple, and Robust Nano-Liquid Chromatography-Mass Spectrometry Method for Amyloid Protein Subtyping.

Amyloidosis is a rare condition characterized by deposits of insoluble proteins in the form of ß-pleated sheets. These deposits interfere with the normal structure and function of varying tissues. Thirty-one amyloid proteins have been identified, and the correct identification is critical due to the varying treatments. Immunohistochemistry, the most routine method for identification of amyloid proteins, suffers from limitations. Mass spectrometry (MS)-based methods offer better sensitivity and specificity. We describe here a sensitive, simple, and robust MS-based method for the identification of amyloid proteins. Amyloid deposits are excised from formalin-fixed tissue by laser microdissection and is put through protein extraction followed by trypsin digestion. The resulting peptides are separated by nano-liquid chromatography and analyzed by high-resolution Orbitrap mass spectrometry. The mass spectrometry data are then searched against a human protein database for identification and semi-quantification.

Quantitation of Free Metanephrines in Plasma by Liquid Chromatography-Tandem Mass Spectrometry.

Plasma metanephrines are measured to aid in the diagnosis of pheochromocytomas. In patients with pheochromocytomas there is excessive production of catecholamines and metanephrines. Measurement of plasma free metanephrines is one of the first-line clinical tests that are used for the diagnosis and follow-up of pheochromocytoma. We describe here a liquid chromatography-tandem mass spectrometry method to measure free metanephrines in plasma. Free metanephrine and normetanephrine are extracted via solid-phase extraction. After extraction and evaporation, the reconstituted supernatant is analyzed by high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). The MS/MS is set to selective reaction monitoring mode (180.1 → 148.1 m/z for metanephrine, 183.1 → 168.1 for d3-metanephrine, 166.1 → 134.1 m/z for normetanephrine, and 169.1 → 137.2 m/z for d3-normetanephrine) with positive electrospray ionization. Quantitation is based on peak area ratio of the analyte to its respective deuterated internal standard. The assay is linear from 5.9 to 4090.0 pg/mL for metanephrine and 22.0 to 4386.7 pg/mL for normetanephrine with precision of <6 % over the ranges.

A sensitive and rapid method for quantification of lacosamide and desmethyl lacosamide by LC-MS/MS.

BACKGROUND: Monitoring lacosamide (LCM) helps optimize therapeutic dosing in some clinical settings. We developed a novel LC-MS/MS method for measuring serum LCM and O-desmethyl lacosamide (ODL). RESULTS/DISCUSSION: The sample preparation was protein precipitation with methanol. The total CV was less than 4.7%. Calibration range was 0.95-30.29 µg/ml and 0.95-30.41 µg/ml while the expanded linear range was 0.41-47.49 µg/ml and 0.34-48.17 µg/ml for LCM and ODL, respectively with analytical accuracy of 87.2-106.0%. 45 random serum samples collected from 23 individuals on either 200 mg/day or 400 mg/day of LCM therapy showed concentrations of 2.2-19.8 µg/ml for LCM and up to 2.5 µg/ml for ODL. CONCLUSION: A simple and sensitive LC-MS/MS assay was validated for quantification of LCM and ODL in human serum.

A simple and robust LC-MS/MS method for measuring sirolimus and everolimus in whole blood.

Therapeutic drug monitoring of immunosuppressants sirolimus and everolimus is mandatory and liquid chromatography tandem mass spectrometry (LC-MS/MS) is the preferred technology for the measurement. Due to the high hydrophobicity these analytes bind to reverse-phase columns tightly and need column heating to elute. Column heating not only requires extra instrument preparation but also causes permanent column damage if the heater is left on while elution pumps stop by the end of the run. The primary improvement in the current method was to elute the analytes at room temperature using special buffers. This new LC-MS/MS method has been validated for clinical use and offers improved simplicity and robustness by eliminating column heating yet with high sensitivity, precision and accuracy.

Comparison of a stable isotope-labeled and an analog internal standard for the quantification of everolimus by a liquid chromatography-tandem mass spectrometry method.

BACKGROUND: Everolimus is an immunosuppressant drug used in solid organ transplantation. Immunoassays and liquid chromatography-mass spectrometry (LC-MS) methods have been used for therapeutic drug monitoring of this drug. In LC-tandem mass spectrometry (MS/MS) methods, both 32-desmethoxyrapamycin and everolimus-d4 have been used as internal standards. OBJECTIVES: To compare 2 internal standards (32-desmethoxyrapamycin and everolimus-d4) for the quantification of everolimus by an LC-MS/MS method. METHODS: Both 32-desmethoxyrapamycin and everolimus-d4 were introduced in the method validation process with 2 transitions simultaneously monitored for everolimus (975.6 → 908.7 as the quantifier and 975.6 → 926.9 as the qualifier) by an established LC-MS/MS method. The key performance characteristics were lower limit of quantification, accuracy, precision, and comparison with an LC-MS/MS method offered by another laboratory. RESULTS: The lower limit of quantification (LLOQ) was 1.0 ng/mL using either internal standard with an analytical recovery of 98.3%-108.1% across the linear range. The total coefficient of variation for everolimus was 4.3%-7.2% with no significant difference between the 2 internal standards. In comparison with an independent LC-MS/MS method, though everolimus-d4 offered a better slope (0.95 versus 0.83), both internal standards showed acceptable results and had a coefficient of correlation r > 0.98 in the tested concentration range of 1.2-12.7 ng/mL. CONCLUSIONS: Although everolimus-d4 offered a more favorable comparison with an independent LC-MS/MS method, both everolimus-d4 and 32-desmethoxyrapamycin had acceptable performance as the internal standards for everolimus quantification by the LC-MS/MS method.