BACKGROUND Many different cystatin C–based equations exist for estimating glomerular filtration rate. Major reasons for this are the previous lack of an international cystatin C calibrator and the nonequivalence of results from different cystatin C assays. METHODS Use of the recently introduced certified reference material, ERM-DA471/IFCC, and further work to achieve high agreement and equivalence of 7 commercially available cystatin C assays allowed a substantial decrease of the CV of the assays, as defined by their performance in an external quality assessment for clinical laboratory investigations. By use of 2 of these assays and a population of 4690 subjects, with large subpopulations of children and Asian and Caucasian adults, with their GFR determined by either renal or plasma inulin clearance or plasma iohexol clearance, we attempted to produce a virtually assay-independent simple cystatin C–based equation for estimation of GFR. RESULTS We developed a simple cystatin C–based equation for estimation of GFR comprising only 2 variables, cystatin C concentration and age. No terms for race and sex are required for optimal diagnostic performance. The equation, eGFR=130×cystatin C−1.069×age−0.117−7, is also biologically oriented, with 1 term for the theoretical renal clearance of small molecules and 1 constant for extrarenal clearance of cystatin C. CONCLUSIONS A virtually assay-independent simple cystatin C–based and biologically oriented equation for estimation of GFR, without terms for sex and race, was produced.

The IFCC Working Group for the Standardisation of Cystatin C (WG-SCC), in collaboration with the Institute for Reference Materials and Measurements (IRMM), announces the availability of the new certified reference material ERM-DA471/IFCC. The material was characterised using a pure protein primary reference preparation (PRP) as calibrant. The PRP was prepared from recombinant cystatin C, and its concentration measured using dry mass determination. The characterisation of ERM-DA471/IFCC was performed by particle enhanced immuno-nephelometry, particle enhanced immuno-turbidimetry, and enzyme amplified single radial immuno-diffusion. The certified cystatin C mass concentration in ERM-DA471/IFCC, if reconstituted according to the specified procedure, is 5.48 mg/L, the expanded uncertainty (k=2) being 0.15 mg/L.

The albumin levels decreased in the second and third trimesters, whereas the levels of chi2-macroglobulin were unchanged, which is compatible with a virtually unaltered transfer of chi2-macroglobulin between the intra- and extravascular space during pregnancy and a significantly increased extravascular fraction of albumin.

In this study, the maternal plasma levels of beta2-microglobulin, beta-trace protein and cystatin C were all significantly elevated in pre-eclampsia compared to those of healthy pregnant women, and displayed similar diagnostic performance for diagnosing pre-eclampsia. The results indicate that low molecular mass proteins are useful as markers of renal impairment in pre-eclampsia.

Quality-control surveys in recent years, in various parts of the world, have shown poor between-laboratory agreement for measurements of plasma proteins. Despite the existence of international reference materials distributed by the World Health Organization, standards produced by diagnostics manufacturers and professional organizations differ significantly in their ascribed values. The reasons for this are complex but include poor availability of the primary materials, confusion about their use, and the fact that their turbidity on reconstitution precludes their use in modern optical immunoassays. This unfortunate situation led to an important initiative to produce sufficient quantities of a widely available, optically clear secondary reference material for plasma proteins that could be used worldwide by manufacturers, professional organizations, and laboratories. Here we present an overview on how the laboratory community, including manufacturers, clinical laboratories, professional societies, and regulators, has reached what we consider is a successful conclusion to a difficult problem.

BACKGROUND The availability of a suitable matrix reference material is essential for standardization of the immunoassays used to measure serum proteins. The earlier serum protein reference material ERM-DA470 (previously called CRM470), certified in 1993, has led to a high degree of harmonization of the measurement results. A new serum protein material has now been prepared and its suitability in term of homogeneity and stability has been verified; after characterization, the material has been certified as ERM-DA470k/IFCC. METHODS We characterized the candidate reference material for 14 proteins by applying a protocol that is considered to be a reference measurement procedure, by use of optimized immunoassays. ERM-DA470 was used as a calibrant. RESULTS For 12 proteins [α2 macroglobulin (A2M), α1 acid glycoprotein (orosomucoid, AAG), α1 antitrypsin (α1-protease inhibitor, AAT), albumin (ALB), complement 3c (C3c), complement 4 (C4), haptoglobin (HPT), IgA, IgG, IgM, transferrin (TRF), and transthyretin (TTR)], the results allowed assignment of certified values in ERM-DA470k/IFCC. For CRP, we observed a bias between the lyophilized and liquid frozen materials, and for CER, the distribution of values was too broad. Therefore, these 2 proteins were not certified in the ERM-DA470k/IFCC. Different value transfer procedures were tested (open and closed procedures) and found to provide equivalent results. CONCLUSIONS A new serum protein reference material has been produced, and values have been successfully assigned for 12 proteins.

A Primary Reference Preparation has been produced using pure, recombinant, Cystatin C in a solvent of 0.1 mol/L KCl. Dry mass determination of the Primary Reference Preparation resulted in a Cystatin C concentration of 5.20 g/L. Agarose-electrophoresis and SDS-electrophoresis, as well as N-terminal sequencing, verified the purity, homogeneity and identity of Cystatin C in the Primary Reference Preparation. For the Secondary Reference Preparation, a serum pool was collected and stabilized. A pilot batch was made to verify the selected procedure and spiking with the pure, recombinant Cystatin C. The final Secondary Reference Preparation is now produced (4468 vials) and ready for value assignment and further characterization.

Quantitative protein determinations in routine laboratories are today most often carried out using automated instruments. However, slight variations in the assay principle, in the programming of the instrument or in the reagents may lead to different results. This has led to the prerequisite of method optimization and standardization. The basic principles of turbidimetry and nephelometry are discussed. The different reading principles are illustrated and investigated. Various problems are identified and a suggestion is made for an integrated, fast and convenient test system for the determination of a number of different proteins on the same instrument. An optimized test system for turbidimetry and nephelometry should comprise high-quality antibodies, calibrators, controls, and buffers and a protocol with detailed parameter settings in order to program the instrument correctly. A good user program takes full advantage of the optimal reading principles for the different instruments. This implies--for all suitable instruments--sample preincubation followed by real sample blanking, which automatically corrects for initial turbidity in the sample. Likewise it is recommended to measure the reagent blank, which represents any turbidity caused by the antibody itself. By correcting all signals with these two blank values the best possible signal is obtained for the specific analyte. An optimized test system should preferably offer a wide measuring range combined with a wide security range, which for the user means few re-runs and maximum security against antigen excess. A non-linear calibration curve based on six standards is obtained using a suitable mathematical fitting model, which normally is part of the instrument software.