EXPRESS: 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

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

Correction to: Determination of serum 25-hydroxyvitamin D status among population in southern China by a high accuracy LC-MS/MS method traced to reference measurement procedure

An amendment to this paper has been published and can be accessed via the original article.

Verification of Low-Density Lipoprotein Cholesterol Levels Measured by Anion-Exchange High-Performance Liquid Chromatography in Comparison with Beta Quantification Reference Measurement Procedure

Background A new lipoprotein testing method based on anion-exchange HPLC (AEX-HPLC) was recently established. We verified the accuracy of LDL-C levels, a primary therapeutic target for the prevention of cardiovascular disease (CVD), measured by AEX-HPLC comparing with LDL-C levels measured by beta quantification-reference measurement procedure (BQ-RMP), homogenous assays, and calculation methods. Methods We compared LDL-C levels measured by AEX-HPLC (adLDL-Ch: LDL-Ch and IDL-Ch) and BQ-RMP using blood samples from 52 volunteers. AdLDL-Ch levels were also compared with those measurements by homogeneous assays and calculation methods (Friedewald equation, Martin equation, and Sampson equation) using blood samples from 411 participants with dyslipidemia and/or type 2 diabetes. Results The precision and accuracy of adLDL-Ch were verified by BQ-RMP. The mean percentage bias [bias (%)] for LDL-C was 1.2%, and the correlation was y = 0.990x + 3.361 (r = 0.990). These results met the acceptable range of accuracy prescribed by the National Cholesterol Education Program. Additionally, adLDL-Ch levels were correlated with LDL-C levels measured by the 2 homogeneous assays (r > 0.967) and the calculation methods (r > 0.939), in serum samples from patients with hypertriglyceridemia. Conclusions AEX-HPLC is a reliable method for measuring LDL-C levels for CVD risk in daily clinical laboratory analyses.

Thrombin: An Approach to Developing a Higher-Order Reference Material and Reference Measurement Procedure for Substance Identity, Amount, and Biological Activities

Thrombin, the proteolytic enzyme that catalyzes the transformation of soluble fibrinogen to the polymerized fibrin clot, participates in multiple reactions in blood coagulation in addition to the clotting reaction. Although reference materials have existed for many years, structural characterization and measurement of biological activity have never been sufficient to permit claims of clear metrological traceability for the thrombin preparations. Our current state-of-the-art methods for protein characterization and determination of the catalytic properties of thrombin now make it practical to develop and characterize a metrologically acceptable reference material and reference measurement procedure for thrombin. Specifically, α-thrombin, the biologically produced protease formed during prothrombin activation, is readily available and has been extensively characterized. Dependences of thrombin proteolytic and peptide hydrolytic activities on a variety of substrates, pH, specific ions, and temperature are established, although variability remains for the kinetic parameters that describe thrombin enzymatic action. The roles of specific areas on the surface of the thrombin molecule (exosites) in substrate recognition and catalytic efficiency are described and characterized. It is opportune to develop reference materials of high metrological order and technical feasibility. In this article, we review the properties of α-thrombin important for its preparation and suggest an approach suitable for producing a reference material and a reference measurement procedure that is sensitive to thrombin’s catalytic competency on a variety of substrates.