Assessment of WHO 07/202 reference material and human serum pools for commutability and for the potential to reduce variability among soluble transferrin receptor assays.

OBJECTIVES: The clinical use of soluble transferrin receptor (sTfR) as an iron status indicator is hindered by a lack of assay standardization and common reference ranges and decision thresholds. In 2009, the WHO and National Institute for Biological Standards and Controls (NIBSC) released a sTfR reference material (RM), 07/202, for assay standardization; however, a comprehensive, formal commutability study was not conducted. METHODS: This study evaluated the commutability of WHO 07/202 sTfR RM and human serum pools and the impacts of their use as common calibrators. Commutability was assessed for six different measurement procedures (MPs). Serum pools were prepared according to updated CLSI C37-A procedures (C37) or non-C37 procedures. The study design and analyses were based on Parts 2 and 3 of the 2018 IFCC Commutability in Metrological Traceability Working Group's Recommendations for Commutability Assessment. WHO 07/202 and serum pools were used for instrument/assay and mathematical recalibration, respectively, to determine if their use decreases inter-assay measurement variability for clinical samples. RESULTS: The WHO 07/202 RM dilutions were commutable for all 6 MPs assessed and, when used for instrument calibration, decreased inter-assay variability from 208 to 55.7 %. Non-C37 and C37 serum pools were commutable for all 6 MPs assessed and decreased inter-assay variability from 208 to 13.8 % and 4.6 %, respectively, when used for mathematical recalibration. CONCLUSIONS: All materials evaluated, when used as common calibrators, substantially decreased inter-assay sTfR measurement variability. MP calibration to non-C37 and C37 serum pools may reduce the sTfR IMPBR to a greater extent than WHO 07/202 RM.

An updated protocol based on CLSI document C37 for preparation of off-the-clot serum from individual units for use alone or to prepare commutable pooled serum reference materials.

Manufacturers of in vitro diagnostic medical devices, clinical laboratories, research laboratories and calibration laboratories require commutable reference materials that can be used in the calibration hierarchies of medical laboratory measurement procedures used for human specimens to establish metrological traceability to higher order reference systems. Commutable materials are also useful in external quality assessment surveys. In order to achieve these goals, matrix-based reference materials with long-term stability, appropriate measurand concentrations and commutability with individual human specimens are required. The Clinical and Laboratory Standards Institute (CLSI) guideline C37-A (now archived) provided guidance to prepare commutable pooled serum reference materials for use in the calibration hierarchies of cholesterol measurement procedures. Experience using the C37-A guideline has identified a number of technical enhancements as well as applications to measurands other than cholesterol. This experience is incorporated into this updated protocol to ensure the procedure will continue to meet the needs of the medical laboratory. The updated protocol describes a procedure for preparing frozen human serum units or pools with minimal matrix alterations that are likely to be commutable with individual human serum samples. The protocol provides step-by-step guidance for the planning phase, collection of individual serum units, processing the units, qualifying the units for use in a pool and frozen storage of aliquots of pooled sera to manufacture frozen serum pools. Guidance on how to perform quality control of the final product and suggestions on documentation are also provided.

Practical considerations for accurate determination of free thyroxine by equilibrium dialysis.

Background: Free thyroxine (FT4) measurement is one of the most requested tests in patient care for diagnosing and treating thyroid-related illnesses. Equilibrium dialysis (ED) is considered the "gold standard" for FT4 measurement; however, several factors have a profound effect on the reliability of FT4 assays and require special consideration. Methods: In the current study, we focused on evaluating critical factors that could contribute to reporting errors, such as adsorption of thyroxine (T4) to labware surfaces, stability of serum samples, stock solutions, and calibrator storage conditions, as well as the solvents used to prepare T4 solutions. Results: The adsorption of T4 in ethanolic solutions and dialysates to labware surfaces can be reduced with the careful selection of pipette tips, test tubes, and 96-well plates. Adding pH modifiers to neat T4 solutions can improve its stability. FT4 in serum samples remains stable after exposure to four freeze-thaw cycles, 5 °C for 18-20 h, or -70 °C for a minimum of three years. Conclusion: The presented study has demonstrated that the loss of analyte due to pre-analytical and analytical factors during operation of the FT4 reference measurement procedure (RMP) can be minimized by careful selection of all labware for sample preparation. It was found that the accuracy and imprecision of FT4 assays can be influenced by different types of dialysis devices, but acceptable alternatives to ED membranes were identified. This study demonstrates approaches to establish a FT4 method that is independent from specific suppliers and addresses critical pre-analytical and analytical factors important for FT4 measurements.

Development of an equilibrium dialysis ID-UPLC-MS/MS candidate reference measurement procedure for free thyroxine in human serum.

BACKGROUND: Accurate and reliable measurement of human serum free thyroxine (FT4) is critical for the diagnosis and treatment of thyroid diseases. However, concerns have been raised regarding the performance of FT4 measurements in patient care. Centers for Disease Control and Prevention Clinical Standardization Programs (CDC-CSP) address these concerns by creating a FT4 standardization program to standardize FT4 measurements. The study aims to develop a highly accurate and precise candidate Reference Measurement Procedure (cRMP), as one key component of CDC-CSP, for standardization of FT4 measurements. METHODS: Serum FT4 was separated from protein-bound thyroxine with equilibrium dialysis (ED) following the recommended conditions in the Clinical and Laboratory Standards Institute C45-A guideline and the published RMP [20,21,23]. FT4 in dialysate was directly quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. Gravimetric measurements of specimens and calibrator solutions, calibrator bracketing, isotope dilution, enhanced chromatographic resolution, and T4 specific mass transitions were used to ensure the accuracy, precision, and specificity of the cRMP. RESULTS: The described cRMP agreed well with the established RMP and two other cRMPs in an interlaboratory comparison study. The mean biases of each method to the overall laboratory mean were within ±2.5%. The intra-day, inter-day, and total imprecision for the cRMP were within 4.4%. The limit of detection was 0.90 pmol/L, which was sufficiently sensitive to determine FT4 for patients with hypothyroidism. The structural analogs of T4 and endogenous components in dialysate did not interfere with the measurements. CONCLUSION: Our ED-LC-MS/MS cRMP provides high accuracy, precision, specificity, and sensitivity for FT4 measurement. The cRMP can serve as a higher-order standard for establishing measurement traceability and provide an accuracy base for the standardization of FT4 assays.

Biomarkers of Potential Harm among Adult Cigarette and Smokeless Tobacco Users in the PATH Study Wave 1 (2013-2014): A Cross-sectional Analysis.

BACKGROUND: While smokeless tobacco (ST) causes oral cancer and is associated with cardiovascular diseases, less is known about how its effects differ from other tobacco use. Biomarkers of potential harm (BOPH) can measure short-term health effects such as inflammation and oxidative stress. METHODS: We compared BOPH concentrations [IL6, high-sensitivity C-reactive protein, fibrinogen, soluble intercellular adhesion molecule-1 (sICAM-1), and F2-isoprostane] across 3,460 adults in wave 1 of the Population Assessment of Tobacco and Health study (2013-2014) by tobacco use groups: primary ST users (current exclusive ST use among never smokers), secondary ST users (current exclusive ST use among former smokers), exclusive cigarette smokers, dual users of ST and cigarettes, former smokers, and never tobacco users. We estimated geometric mean ratios using never tobacco users, cigarette smokers, and former smokers as referents, adjusting for demographic and health conditions, creatinine (for F2-isoprostane), and pack-years in smoker referent models. RESULTS: BOPH levels among primary ST users were similar to both never tobacco users and former smokers. Most BOPH levels were lower among ST users compared with current smokers. Compared with never tobacco users, dual users had significantly higher sICAM-1, IL6, and F2-isoprostane. However, compared with smokers, dual users had similar biomarker levels. Former smokers and secondary ST users had similar levels of all five biomarkers. CONCLUSIONS: ST users have lower levels of inflammatory and oxidative stress biomarkers than smokers. IMPACT: ST use alone and in combination with smoking may result in different levels of inflammatory and oxidative stress levels.

Development of a Sensitive High-Resolution Mass Spectrometry Approach for Urea Nitrogen Quantitation in Small Volumes of Bronchoalveolar Lavage Fluid (BALF).

A sensitive, selective, and quantitative method incorporating high-resolution mass spectrometry was developed for the determination of blood urea nitrogen (BUN) in bronchoalveolar lavage fluid. The method requires no sample cleanup or derivatization prior to analysis. High-performance liquid chromatography (HPLC) on a Hypersil Gold PFP column (100 × 3 mm, 3 µm particle size) connected to a C18 guard column was employed for a 10 min chromatographic separation. The detection of urea was achieved using a Thermo Scientific Q-Exactive Plus instrument incorporating selected ion monitoring (SIM) modes for the protonated adduct of urea. The urea analytical measuring range for the method is 0.047-17.134 mg/dL, resulting in a BUN analytical measurement range of 0.022-8.007 mg/dL, which allows for quantitation over 3 orders of magnitude (R2 = 0.999). In addition, the method is suitable for small sample volumes (15 µL) with a high level of accuracy, precision, and specificity.

A fully automated high-throughput liquid chromatography tandem mass spectrometry method for measuring creatinine in urine.

Reliable creatinine measurements are important to evaluate kidney function and for creatinine correction to reduce biological variability of other urinary analytes. A high-throughput, accurate liquid chromatography tandem mass spectrometry method for quantitation of human urinary creatinine has been developed and validated. Sample preparation was fully automated including cryovial decapping, sample ID scanning and two serial dilution steps. Quantitation was performed using a stable isotope-labeled internal standard. Multiplexed chromatographic separation of creatinine was achieved within a one-minute analysis and followed by tandem mass spectrometry in positive electrospray ionization mode. The precursor and product ions of creatinine and D3-creatinine were monitored in selected reaction monitoring mode. Method validation results showed reproducibility with within-run precision of 3.59, 3.49 and 2.84% and between-run precision of 4.01, 3.28 and 3.57% for low, medium and high quality control materials prepared from pooled donor urine, respectively. The method showed excellent accuracy with a bias of -1.94%, -0.78% and -1.07% for three levels of certified reference material. The calibration curve was linear throughout a 7.50-300 mg/dL (0.663-26.5 mmol/L) measurement range (R2 = 0.999), with the mean slope of 0.0115 (95%CI, 0.0108-0.0122) and intercept of 0.0027 (95%CI, 0.0003-0.0051). The limit of detection (LOD) of the method was 3.17 mg/dL (0.280 mmol/L). Analytical specificity was achieved by chromatographically separating creatinine from potentially interfering creatine within a one-minute run and monitoring the Quantitation Ion/Confirmation Ion (QI/CI) ratios in samples. A simple, accurate, high-throughput method was successfully developed for measuring creatinine in human urine samples.