Reference Measurement Procedure Research Articles

Using mass spectrometry to overcome the longstanding inaccuracy of a commercially-available clinical testosterone immunoassay

Accurate measurement of testosterone is important for the diagnosis of gonadal disorders in men, women, and children. Testosterone measurement has limited accuracy at low concentrations by most commercially available immunoassays. We aimed to develop an LC-MS/MS assay to address the inaccuracy of the in-house immunoassay observed over the past decade and to replace it with the new assay. Testosterone in serum/plasma was extracted with commercial supported liquid extraction plates. Method validation was performed following the CLSI C62-A guideline. A total of 126 samples were used for method comparison between the Beckman UniCel DxI immunoassay and LC-MS/MS. Results by immunoassay were 20% lower compared with LC-MS/MS and had minimal correlation (R2 = 0.403) with LC-MS/MS below 100 ng/dL. When comparing specimens from the Accuracy-Based Survey from the College of American Pathologists, the newly developed assay agreed well with the CDC reference measurement procedure. In summary, immunoassay measurement of testosterone can be significantly inaccurate, especially at low concentrations. The newly developed LC-MS/MS assay provides accurate results across the entire measurable range.

Preparation of Three New Certified Reference Materials for Food and Environmental Analysis and Certification Using Laboratory Intercomparison as well as Primary Reference Measurement Procedures

Three new reference materials: MODAS-3 Herring Tissue (M-3 HerTis), MODAS-4 Cormorant Tissue (M-4 CormTis), and MODAS-5 Cod Tissue (M-5 CodTis) were prepared and certified on the basis of results of a worldwide intercomparison exercise. Independently of our proven method of establishing the certified and information values, the content of several essential and toxic elements was additionally determined by the use of ratio primary reference measurement procedures (definitive methods) based on radiochemical neutron activation analysis (RNAA) in the case of As, Cd, Co, Cr, Fe, Mo, Se, and U and isotope dilution mass spectrometry (IDMS) in the case of Hg, respectively. Good agreement of the established certified values and the results obtained by ratio primary reference measurement procedures confirmed the validity of the certification procedure. The total number of elements which could be certified amounted to 30, 21, 18 in M-3 HerTis, M-4 CormTis, and M-5 CodTis, respectively. The relative frequency of use of individual analytical techniques in this intercomparison campaign was calculated and discussed. Inductively coupled plasma mass spectrometry (ICP-MS) is now a dominant technique, followed by atomic absorption spectroscopy (AAS), NAA, and emission spectroscopy (ES). The decreasing share of NAA as compared to several earlier intercomparison exercises should be noticed. NAA is the only method in the array of highly sensitive methods of inorganic trace analysis, which is essentially free from blank. The lack of this method in the foreseeable future may be an obstacle in the prospective certification campaigns and may endanger the implementation of quality assurance in trace analysis.

Interlaboratory comparison of 25-hydroxyvitamin D assays: Vitamin D Standardization Program (VDSP) Intercomparison Study 2 - Part 2 ligand binding assays - impact of 25-hydroxyvitamin D2 and 24R,25-dihydroxyvitamin D3 on assay performance.

An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of ligand binding assays (Part 2) for the determination of serum total 25-hydroxyvitamin D [25(OH)D]. Fifty single-donor samples were assigned target values for concentrations of 25-hydroxyvitamin D2 [25(OH)D2], 25-hydroxyvitamin D3 [25(OH)D3], 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3], and 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] using isotope dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS). VDSP Intercomparison Study 2 Part 2 includes results from 17 laboratories using 32 ligand binding assays. Assay performance was evaluated using mean % bias compared to the assigned target values and using linear regression analysis of the test assay mean results and the target values. Only 50% of the ligand binding assays achieved the VDSP criterion of mean % bias ≤ |± 5%|. For the 13 unique ligand binding assays evaluated in this study, only 4 assays were consistently within ± 5% mean bias and 4 assays were consistently outside ± 5% mean bias regardless of the laboratory performing the assay. Based on multivariable regression analysis using the concentrations of individual vitamin D metabolites in the 50 single-donor samples, most assays underestimate 25(OH)D2 and several assays (Abbott, bioMérieux, DiaSorin, IDS-EIA, and IDS-iSYS) may have cross-reactivity from 24R,25(OH)2D3. The results of this interlaboratory study represent the most comprehensive comparison of 25(OH)D ligand binding assays published to date and is the only study to assess the impact of 24R,25(OH)2D3 content using results from a reference measurement procedure.

In transition to the next generation reference materials and reference measurement procedures for apolipoprotein standardization

Background and Aims: Global standardization of conventional tests and emerging biomarkers is key to enable exchange of test results and to apply universal clinical decision limits across hospitals. Therefore, calibration hierarchies should be established in line with ISO 17511:2020. For apolipoprotein (apo) A-I, apoB and apo(a), the existing WHO-IFCC Reference Materials (RM) are running out of stock and the Reference Measurement Procedure (RMP) is no longer in place. Technological advances enable SI-traceability through direct protein quantitation using mass spectrometry (MS).

Candidate reference measurement procedure based on HPAEC-PAD for the development of certified reference materials for monosaccharides in serum.

To achieve the measurement reliability of monosaccharides used as diagnostic markers in clinical fields, it is essential to establish certified reference materials (CRMs). The purpose of this study is to develop a serum CRM by adopting high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as a new candidate reference measurement procedure for the measurement of glucose and galactose, common diagnostic markers of diabetes and galactosemia, respectively. Using various monosaccharides as internal standards, the accuracy of the HPAEC-PAD method was tested by measuring glucose CRM following treatment with three different deproteinization methods: ultrafiltration, protein precipitation by trichloroacetic acid (TCA), and protein precipitation by acetonitrile. Results showed that ultrafiltration and 5% TCA provided good accuracy with every tested monosaccharide as the internal standard. Accordingly, serum samples in this study were treated by ultrafiltration after adding 2-deoxy-D-glucose and arabinose, which were selected as internal standards for galactose and glucose, respectively. Both intra- and inter-day recovery tests showed good precision and accuracy within 2%. From the serum CRM batches prepared at two levels, 11units were analyzed by exact-matched calibration methods, and the mass fractions of galactose and glucose were determined via HPAEC-PAD. The between-unit relative standard deviations were not more than 1.5%, showing homogeneity. The expanded uncertainties (%) of galactose and glucose for both levels were less than 3.6% and 2.3% at 95% confidence. The HPAEC-PAD method presented in this study can significantly improve the accuracy and precision of simultaneous monosaccharide analysis, allowing for the development of further serum CRMs for monosaccharides. Graphical abstract.

Recommendations for proficiency testing criteria for hemoglobin A1c based on the Shanghai Center for Clinical Laboratory's study.

The US Centers for Medicare & Medicaid Services proposed in 2019 that glycated hemoglobin A1c (HbA1c) be a CLIA'88 regulated analyte. People who commented expressed concerns that the proposed acceptance limit (AL, HbA1c in NGSP unit) ±10% for proficiency testing (PT) would be unable to maintain already improved analytical performance and guarantee the clinical utility of HbA1c testing. Assessing impact of various ALs on PT performance is needed to provide scientific evidence for adopting an appropriate AL. Ten patient EDTA-whole blood specimens were distributed to 318 and 336 laboratories in the 2018 and 2019 PT events organized by Shanghai Center for Clinical Laboratory (SCCL). HbA1c concentrations were measured by participants using various methodologies commonly used in the USA and China. Targets were determined using secondary reference measurement procedures (SRM) at SCCL. "Failed Results" were those outside the SRM-defined target±AL (5% through 10%). Laboratories with Failed Results≥2 out of five samples per PT event obtained Event Unsatisfactory Status. HbA1c target values ranged 33.3mmol/mol (5.2 NGSP%)-102.2mmol/mol (11.5 NGSP%) for 2018 event, and 33.3mmol/mol (5.2 NGSP%)-84.7mmol/mol (9.9NGSP%) for 2019 event. Overall Laboratory Event Unsatisfactory Rates were 11.3-12.2%, 4.8-5.3%, 0.9-3.1%, 0.6-2.2%, 0.6-1.4% and 0.6-1.4%, at AL of ±5, ±6, ±7, ±8, ±9 and ±10%, respectively. The AL (in NGSP unit) of ±6% or ±7% for PT evaluation of HbA1c results would be appropriate, with satisfactory event scores for about 95% of participant laboratories in a PT event.

A survey of the reactivity of in vitro diagnostic bilirubin reagents developed in Japan using artificially prepared bilirubin materials: A comparison of synthetic delta, unconjugated, and taurine-conjugated bilirubin.

In vitro diagnostic bilirubin reagents based on oxidation with bilirubin oxidase or vanadic acid for total and direct-reacting bilirubin are widely used in Japan; however, their reactivity to unconjugated and conjugated bilirubin and delta bilirubin has not been completely disclosed by manufacturers. We used artificially prepared bilirubin materials to investigate the reactivity with four in vitro diagnostic bilirubin reagents. Porcine unconjugated bilirubin solution, chemically synthesized ditaurobilirubin solution, and chemically synthesized delta bilirubin solution were used as surrogates of naturally occurring unconjugated bilirubin, conjugated bilirubin, and delta bilirubin, respectively. The total bilirubin and direct-reacting bilirubin concentrations were measured by three bilirubin oxidase methods and one vanadic acid method, and the observed concentrations were compared with those obtained by the diazo-based reference measurement procedure. The unconjugated bilirubin and delta bilirubin concentrations were similar when any of the four in vitro diagnostic bilirubin reagents were used during total bilirubin measurement. This was consistent with reference measurement procedure and exhibited a converged inter-method variation. Compared with reference measurement procedure, significantly low ditaurobilirubin concentrations were observed by the in vitro diagnostic bilirubin reagents despite the converged inter-method variation. In delta bilirubin measurement, some reagents reacted doubtfully with unconjugated bilirubin, while showed lower ditaurobilirubin concentrations than its corresponding total bilirubin concentration. Reactivity with delta bilirubin was different for each method including reference measurement procedure. Some reagents were developed to react less with delta bilirubin and others to strongly react with delta bilirubin. We revealed the reactivity of IVD-TB and IVD-DB reagents to artificially prepared bilirubin materials, and their consistency with reference measurement procedure. The delta bilirubin data results vary depending on the reagents used.

Assessment of serum total 25-hydroxyvitamin D assay commutability of Standard Reference Materials and College of American Pathologists Accuracy-Based Vitamin D (ABVD) Scheme and Vitamin D External Quality Assessment Scheme (DEQAS) materials: Vitamin D Standardization Program (VDSP) Commutability Study 2.

An interlaboratory study was conducted through the Vitamin D Standardization Program (VDSP) to assess commutability of Standard Reference Materials® (SRMs) and proficiency testing/external quality assessment (PT/EQA) samples for determination of serum total 25-hydroxyvitamin D [25(OH)D] using ligand binding assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A set of 50 single-donor serum samples were assigned target values for 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] using reference measurement procedures (RMPs). SRM and PT/EQA samples evaluated included SRM 972a (four levels), SRM 2973, six College of American Pathologists (CAP) Accuracy-Based Vitamin D (ABVD) samples, and nine Vitamin D External Quality Assessment Scheme (DEQAS) samples. Results were received from 28 different laboratories using 20 ligand binding assays and 14 LC-MS/MS methods. Using the test assay results for total serum 25(OH)D (i.e., the sum of 25(OH)D2 and 25(OH)D3) determined for the single-donor samples and the RMP target values, the linear regression and 95% prediction intervals (PIs) were calculated. Using a subset of 42 samples that had concentrations of 25(OH)D2 below 30nmol/L, one or more of the SRM and PT/EQA samples with high concentrations of 25(OH)D2 were deemed non-commutable using 5 of 11 unique ligand binding assays. SRM 972a (level 4), which has high exogenous concentration of 3-epi-25(OH)D3, was deemed non-commutable for 50% of the LC-MS/MS assays.

Reference Protocol to Assess Analytical Performance of Higher Order Structural Analysis Measurements: Results from an Interlaboratory Comparison.

Measurements of protein higher order structure (HOS) provide important information on stability, potency, efficacy, immunogenicity, and biosimilarity of biopharmaceuticals, with a significant number of techniques and methods available to perform these measurements. The comparison of the analytical performance of HOS methods and the standardization of the results is, however, not a trivial task, due to the lack of reference protocols and reference measurement procedures. Here, we developed a protocol to structurally alter and compare samples of somatropin, a recombinant biotherapeutic, and describe the results obtained by using a number of techniques, methods and in different laboratories. This, with the final aim to provide tools and generate a pool of data to compare and benchmark analytical platforms and define method sensitivity to structural changes. Changes in somatropin HOS, induced by the presence of zinc at increasing concentrations, were observed, both globally and at more localized resolution, across many of the methods utilized in this study and with different sensitivities, suggesting the suitability of the protocol to improve understanding of inter- and cross-platform measurement comparability and assess analytical performance as appropriate.

Reference measurement procedure for the determination of mass fraction of fat content in food

Reference measurement procedure for the determination of mass fraction of fat content in food

An isotope dilution liquid chromatography-tandem mass spectrometry candidate reference measurement procedure for aldosterone measurement in human plasma.

Accurate quantitation of aldosterone is clinically important in standardized testing for primary aldosteronism. The results are often variable when performed by clinical immunoassays. To standardize and ensure the accuracy of clinical systems, reference measurement procedures (RMPs) with higher metrological order are required. A simple and reliable isotope dilution LC-IDMS/MS-based measurement procedure for human plasma aldosterone has been developed. This method involved plasma spiked with a deuterium-labelled internal standard, equilibrated for 0.5h, and extracted by liquid-liquid extraction (LLE) without derivatization. Aldosterone and its structural analogues were baseline separated with a C18-packed UHPLC column with gradient elution within 7min. The signal intensity variability and measurement imprecision were reduced by bracketing calibration during plasma aldosterone value assignment. The limit of detection (LoD) was 19.4pmol/L with a signal-to-noise ratio (S/N) > 3. The lowest limit of quantification (LLoQ) was 27.7pmol/L (S/N > 10 and CV < 10.0%). LLE was performed with 1mL of n-hexane/ethyl acetate (3:2, v/v), and the extraction recovery was determined to be 92.15 ± 3.54%. The imprecisions were ≤ 3.18% for samples at 124.8, 867.0, and 2628.5pmol/L. The recoveries were 98.11-101.61%. The relative bias between this candidate RMP and the established RMP was 2.76-1.89%. The linearity response ranged from 27.7 to 2774.4pmol/L with R2 = 0.999. The method performance met the requirements of RMPs (≤ 5% total CV and ≤ 3% bias). Furthermore, the developed method was applied to evaluate immunoassays through 41 patient sample comparisons. The calibration and measurement capability (CMC) of this method were also evaluated by measuring these samples. The candidate RMP can serve as an accurate reference baseline for routine methods and can be used for value assignment for reference materials. Selected ion chromatograms by LC-MS/MS using a C18 column for aldosterone and its structural analogues.

Vitamin D Standardization Program (VDSP) intralaboratory study for the assessment of 25-hydroxyvitamin D assay variability and bias

An intralaboratory study assessing assay variability and bias for determination of serum total 25-hydroxyvitamin D [25(OH)D] was conducted by the Vitamin D Standardization Program (VDSP). Thirteen assays for serum total 25(OH)D were evaluated in a single laboratory including 11 unique immunoassays and one liquid chromatography – tandem mass spectrometry (LC–MS/MS) assay. Fifty single-donor serum samples, including eight samples with high concentrations of 25(OH)D2 (> 30 nmol/L), were assigned target values for 25(OH)D2 and 25(OH)D3 using reference measurement procedures (RMP). Using four replicate measurements for each sample, the mean total percent coefficient of variation (%CV) and mean % bias from the target values were determined for each assay using the 50 single-donor samples and a 42-sample subset, which excluded 8 high 25(OH)D2 concentration samples, and compared with VDSP performance criteria of ≤ 10 % CV and ≤ ±5 % mean bias. All 12 assays achieved the performance criterion for % CV, and 9 of the 12 assays were within ≤ ±5 % mean bias. The Fujirebio Inc. assay exhibited the lowest %CV and highest percentage of individual measurements within ≤ ±5 % mean bias. Ten immunoassays exhibited changes in response due to the high 25(OH)D2 samples with Abbott, Biomérieux, DiaSorin, DIAsource, and IDS-iSYS assays having the largest deviations. The Fujirebio Inc. and Beckman Coulter assays were only minimally affected by the presence of the high 25(OH)D2 samples. Samples with high concentrations of 25(OH)D2 provided a critical performance test for immunoassays indicating that some assays may not have equal response or recovery for 25(OH)D2 and 25(OH)D3.

Development of food industry metrological assurance complex

The paper considers the contribution of experts from the Laboratory of Metrology of Moisture Measurement and Reference Materials to the improvement of the food industry metrological assurance system. The information on the results of the development of forty types of reference materials (RM) for the composition of various nutrition, food staples and nutritional supplement in the period 2008-2020 is generalized. The development of each new RM type included preparation of the material, research for its stability and homogeneity, and determination of certified value with the appliance of State primary (GET 173) and secondary (GVET 176-1) standards, as well as primary reference measurement procedures and conducting the interlaboratory experiment for checking the applicability of the RM. The creation in 2008 of GET 173 was accompanied by the development of RMs for the composition of grains and dry milk product. The approval in 2010 of GVET 176-1 allowed to add in the RM the certified value for the mass fraction of nitrogen (protein). As a result of successful participation in comparisons, UNIIM got 6 items of calibration and measurement capabilities on measurements of mass fraction of nitrogen in glycine, milk powder, grain, egg powder, cereal and feed-stuff in the BIPM database. 35 types of RM for the composition of dairy and meat products, egg powder, infant food, oil and starch products together with the development of primary reference measurement procedures for measurement of fat, crude fat, ash and carbohydrates mass fraction were created between 2016 and 2019. Moreover, the RM for the composition of nutritional supplement (glycine, melamine, cystine) and dairy products for metrological assurance of IR-analysers as well as RM for the composition of reconstituted milk were developed for the control of measurement results by the enzyme-linked immunosorbent assay (ELISA). Developed RM were systemized on fields of the appliance of Technical regulations of Customs Union and the sectors of the international food triangle model. These RMs allow providing the measurement uniformity of the identification indicators and the nutritional value of food products and food staples.

EQA/PT scheme to improve the equivalence of enzymatic results between mutual recognition laboratories in Beijing.

BackgroundTo utilize the external quality assessment (EQA)/proficiency testing (PT) scheme to evaluate the equivalence of different clinical enzymatic measuring systems in Beijing.MethodsThe Beijing Center for Clinical Laboratory (BCCL) distributed three investigation samples to mutual recognition clinical laboratories in Beijing including alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ‐glutamyltransferase (GGT), creatine kinase (CK), and lactate dehydrogenase (LDH). These samples were derived from serum pools with values assigned by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) enzymatic reference measurement procedures (RMPs). Each laboratory performed duplicate tests of the samples. Then, the samples at level 1 were used to recalibrate individual measuring systems for repeating the tests. BCCL collected data for evaluation of their analytical quality.ResultsBefore recalibration, the biases of ALT and AST tests were not traceable to the IFCC RMPs, and the bias pass rates of GGT, CK, and LDH tests were only 51.2%, 55.7%, and 48.6% respectively. After recalibration, the pass rates of ALT, AST, GGT, CK, and LDH increased to 95.1%, 82.9%, 95.1%, 97.1%, and 70.0% respectively. The EQA/PT also showed that after recalibration, more than 95% of laboratories met the optimum level specifications of the biological variation for ALT, AST, GGT, and CK tests and the desirable for LDH tests.ConclusionThe enzymatic tests in Beijing need to be further standardized by category 1 or 2 EQA/PT scheme for mutual recognition between clinical laboratories. The criteria of biological variation are more relevant for determining the equivalence of clinical enzymatic tests.

CDC Clinical Standardization Programs (CSP) for Free Thyroxine (FT4) to Improve the Accuracy and Reliability of FT4 Measurements in Patient Care and Clinical Research

Reliable FT4 measurement is critical to assess thyroid function and diagnose and treat thyroid disorders. The Partnership for the Accurate Testing of Hormones (PATH) categorizes FT4 as a biomarker in high need for standardization, and currently high inter-assay variability restricts the interpretation of FT4 results in patient care to assay-specific reference intervals. The CDC CSP has partnered with the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) and PATH to create a standardization program for FT4 to improve accuracy, reliability, and comparability of current methods and thus to improve diagnosis, treatment and prevention of thyroidal illnesses. Currently, there are no serum FT4 reference materials available to assess the accuracy and reliability of FT4 assays. CDC has developed an accurate and sensitive reference measurement procedure (RMP) to create commutable serum reference materials with FT4 target values. Assay manufacturers, research and clinical laboratories can use these reference materials to assess their calibration, certify the analytical performance of their measurements, and monitor performance over time by collaborating with CDC CSP. The CDC FT4 RMP uses equilibrium dialysis (ED) with LC-MS/MS based on an internationally recognized ED procedure[1] followed by solid-phase and solvent extractions. Certified primary reference material IRMM468 was used to prepare calibrators. Chromatographic separation is achieved with a gradient of methanol and water with 0.1% formic acid. FT4 is quantified using positive electrospray ionization in positive mode. The intra- and inter-day imprecision of the CDC RMP are 3.0% and 1.1%. A comparison among FT4 RMPs resulted in a +2.5% bias for the CDC RMP to the mean for all labs. The CDC RMP measurement range was 3.02-258 pmol/L and thus suitable for analysis of hypo- and hyperthyroid patients. The CDC FT4 RMP demonstrates good accuracy and precision, and can be used as a viable accuracy base to which routine methods can be compared. An initial comparison study of a commercially available FT4 immunoassay (IA) and the CDC RMP with 24 samples (7.98-109 pmol/L) indicated a mean bias of -37.7%, further indicating a need for standardization. Findings from an IFCC study suggest that alignment of IA measurements to a FT4 RMP can improve comparability and would allow for a uniform reference interval for FT4.[2] CDC CSP established a new standardization program for FT4 to address the needs of the community and assist with improving test comparability and reliability.1. Clin. Chem. Lab. Med. 2011, 49: 1275-81. 2. Clin. Chem. 2017, 63: 1642-52.

Development of High-Throughput Measurement of Free Thyroxine in Serum Using Equilibrium Dialysis in Couple With Liquid Chromatography-Tandem Mass Spectrometry

Free thyroxine (FT4) measurements are critical in the diagnosis, classification, and treatment of thyroid diseases. It is estimated that about 18 million FT4 tests are requested in the USA per year annually. In clinical laboratories, most of FT4 assays are performed by using immunoassays (I As). However, the significant bias of IAs and large variation between laboratories have been reported. The reference measurement procedures (RMPs) of FT4based on equilibrium dialysis (ED) - liquid chromatography-tandem mass spectrometry (LC-MS/MS) have been established and recognized by the clinical chemistry community. However, the FT4 RMP is relatively low throughput and labor-intensive. Also, an aliquot of 1 mL sample is required for an RMP. A routine FT4 assay high-throughput procedure that is based on ED LC-MS/MS and utilized less sample volume will allow to conduct large biomonitoring studies and establish FT4 levels in US population. In the described method, FT4 in 150 uL of serum was separated from protein-bound T4 at 37.0 oC in 96-well Micro-ED Teflon devices from HTDialysis. The ED conditions suggested by CLSI C45-A guideline were followed. A volume of 150 uL dialysate samples with FT4 was obtained after the ED step. FT4 in the dialysate was purified by extractions before LC-MS/MS analysis. Chromatographic separation of T4 from the sample matrix is achieved on a C18 UPLC column with a gradient of methanol and water with 0.1% formic acid. Quantification of FT4 was performed by using selective reaction monitoring in positive electrospray ionization mode. The IRMM-468 certified primary reference material (JRC, Belgium) of T4 is used for the calibration curves. FT4 concentrations reached equilibrium after 4 hours under current dialysis conditions, which was also observed in the RMP setting. The developed routine FT4 assay 96-well ED system is within 5% bias from the FT4 RMP based on preliminary method comparison study using human serum. The studies to further characterize the FT4 routine method performance are ongoing. In summary, we are developing an analytical method based on a 96-well ED-LC-MS/MS system for the measurements of FT4 in serum. By comparison with RMP, the described method significantly improve throughput and reduce sample volume, which will fulfill the requirement of FT4 routine assay in clinical laboratories and allow for its use in the large biomonitoring studies and other activities in the research and public health settings.

Measurement of Free Testosterone in Serum Using Equilibrium DialysisCoupled With ID-UHPLC-MS/MS: Comparison Between Equilibrium Devices

Free testosterone (FT) has been used as a biomarker in clinical patient care and public health research to assess and manage patients with androgenic abnormalities. The latest Endocrine Society clinical practice guideline for testosterone therapy in men with hypogonadism recommends measuring FT for those with borderline and low total testosterone concentrations, or those who have conditions that change SHBG concentrations, such as some metabolic or hormonal diseases, certain medication use, or SHBG genetic polymorphisms. Measuring FT is technically challenging and shows high variability. The CDC clinical standardization program is developing a high throughput method using the gold-standard equilibrium dialysis (ED) procedure with isotope dilution ultra-high-performance liquid chromatography tandem mass spectrometry (ID-UHPLC-MS/MS).A serum sample was dialyzed against a protein-free HEPES buffer (pH 7.4) at 37 °C until equilibrium. After isolating endogenous FT from protein-bound testosterone by ED, isotope-labeled internal standard (13C3-testosterone) was added to the dialysate for quantification. Certified pure primary reference material (National Measurement Institute M914) was used to prepare calibrators, enabling traceable quantitation and ensuring measurement trueness. FT was further isolated from the dialysate matrix using supported liquid extraction and a chromatographic separation from interfering compounds and quantitation by tandem MS. The dialysis step requires maintaining the endogenous free hormone equilibrium so that results in dialysate reflect FT concentrations in the blood without influence from dilution, temperature, or pH. The dialyzer system has a 1:1 sample-to-buffer volume and has been used in reference measurement procedures for free hormone measurements, serving as the standard for method performance comparison. Four commercially available devices designed for high throughput in a multiple well-plate format, requiring respective sample-to-buffer ratios, were evaluated for their recovery, speed, ease of automation by a liquid handling system, repeatability, and robustness. Preliminary results showed that a device with 1:1 sample-to-buffer volume had the most comparable results to those obtained from the standard dialyzer, with the mean bias less than 15%. The device with the highest sample-to-buffer ratio showed bias as high as 50%. These data suggest that controlling sample-to-buffer ratio is a critical step in ED FT method.

Improving the Accuracy and Reliability of Parathyroid Hormone Levels Through the Mass-Spectrometric Measurement of Full-length PTH and C-Terminal PTH Fragments

Parathyroid hormone (PTH) is a key biomarker for hypo/hyperparathyrioidism as well as chronic kidney disease, one of the leading health conditions in the USA. As a result, the most recent update of the Kidney Disease Improving Global Outcomes (KDIGO) guideline for CKD-MBD management emphasized the role of PTH as one of the key biomarkers of this disorder. The earlier stages of CKD generate few symptoms and only until the kidney is significantly impaired do patients begin experiencing signs of renal failure. Therefore, the measurement of parathyroid hormone (PTH) in serum and/or plasma is critical not only for the correct detection, diagnosis, and prevention of renal failure, but also calcium, phosphate, and vitamin D disorders. Current laboratory methods for PTH show high variability and inaccuracy, thus creating the need for a reference measurement procedure that can help laboratories and assay manufacturers improve their measurement accuracy and reliability to avoid the misclassification of patients. Due to the short half-life of PTH, this 84 amino-acid polypeptide hormone is produced at low circulating levels in normal conditions ranging from 10 - 65 pg/mL. In addition, N-terminal and C-terminal peptides, which have shown to interfere with clinical analyzer platforms, account for over 80% of all circulating PTH levels. Therefore, a highly specific and sensitive method is needed for the accurate detection of full-length PTH and these PTH fragments. There is clinical relevancy in the ability to measure C-terminal PTH fragments as the ratio of these PTH fragments to full-length PTH has been diagnostic for severe or end-stage renal disease, non-dynamic bone disease, and hyperparathyroid-associated bone loss. Bottom-up proteomics approaches that incorporate enzymatic digestion steps during sample preparation will result in the loss of information for the fragments. Therefore, a new, innovative, top-down proteomics method was developed to measure full-length PTH and its breakdown products (fragments) by mass spectrometry (UHPLC-HRMS). This method enabled, for the first time, the detection of full-length PTH at very low concentrations typically observed in patients with hypoparathyroidism as well as C-terminal fragments that may interfere with regular immunoassays typically used in patient care. The highly specific and sensitive method for PTH and related peptides in CKD patient sera demonstrated no interference from the internal standards and other PTH fragments. Therefore, the optimized method was applied for the screening of normal and CKD serum at various stages of disease progression. Preliminary results demonstrated that certain PTH fragments are correlated with eGFR and different stages of chronic kidney diseases (CKD). This method along with the respective findings from this study will help to improve the diagnosis, treatment, and prevention of CKD-MBD.

Progress in the traceability of tumor marker detection

Tumor markers (TM) detection is of great significance in tumor screening, monitoring and treatment intervention, which puts forward higher requirements for its detection quality. TM traceability is very important in the process of reagent production and clinical laboratory testing, which can help improving the reliability and comparability of TM testing. Based on the current principles and classification system of metrology traceability in the world, this paper reviews the quality requirements of reference materials and reference measurement methods related to protein and nucleic acid of TM, as well as the problems existing in the international convention reference measurement procedure and traceability system of TM, so as to provide a new idea for the quality assurance work of TM detection.

The performance of human cytomegalovirus digital PCR reference measurement procedure in seven external quality assessment schemes over four years.

A candidate digital PCR (dPCR)-based reference measurement procedure for quantification of human cytomegalovirus (hCMV) was evaluated in 10 viral load comparison schemes (seven external quality assessment (EQA) and three additional training schemes) organized by INSTAND e.V. over four years (between September 2014 and March 2018). Four metrology institutes participated in these schemes using the same extraction method and dPCR measurement procedure for the hCMV specific target sequence of UL54 gene. The calibration independent reference measurement procedure results from the metrology institutes were compared to the results of the clinical diagnostic laboratories applying hCMV qPCR measurement procedures calibrated to reference materials. While the criteria for the acceptable deviation from the target value interval for INSTAND's EQA schemes is from -0.8 log10 to +0.8 log10, the majority of dPCR results were between -0.2 log10 to +0.2 log10. Only 4 out of 45 results exceeded this interval with the maximum deviation of -0.542 log10. In the training schemes containing samples with lower hCMV concentrations, more than half of the results deviated less than ±0.2 log10 from the target value, while more than 95% deviated less than ±0.4 log10 from the target value. Evaluation of intra- and inter-laboratory variation of dPCR results confirmed high reproducibility and trueness of the method. This work demonstrates that dPCR has the potential to act as a calibration independent reference measurement procedure for the value assignment of hCMV calibration and reference materials to support qPCR calibration as well as ultimately for routine hCMV load testing.