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BACKGROUND: Errors of calibrator-assigned values lead to errors in the testing of patient samples. The ability to estimate the uncertainties of calibrator-assigned values and other variables minimizes errors in testing processes. International Organization of Standardization guidelines provide simple equations for the estimation of calibrator uncertainty with simple value-assignment processes, but other methods are needed to estimate uncertainty in complex processes. METHODS: We estimated the assigned-value uncertainty with a Monte Carlo computer simulation of a complex value-assignment process, based on a formalized description of the process, with measurement parameters estimated experimentally. This method was applied to study uncertainty of a multilevel calibrator value assignment for a prealbumin immunoassay. RESULTS: The simulation results showed that the component of the uncertainty added by the process of value transfer from the reference material CRM470 to the calibrator is smaller than that of the reference material itself (<0.8% vs 3.7%). Varying the process parameters in the simulation model allowed for optimizing the process, while keeping the added uncertainty small. The patient result uncertainty caused by the calibrator uncertainty was also found to be small. CONCLUSIONS: This method of estimating uncertainty is a powerful tool that allows for estimation of calibrator uncertainty for optimization of various value assignment processes, with a reduced number of measurements and reagent costs, while satisfying the requirements to uncertainty. The new method expands and augments existing methods to allow estimation of uncertainty in complex processes.

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A procedure for the purification of human prealbumin, orosomucoid and transferrin as primary protein preparations has been developed. The procedure describes in detail the chemicals, the fractionation equipment and the purification of the three proteins from the same starting material (a 90-donor plasma pool). The fractionation steps involve methods like salting out, various anion and cation exchange chromatographies, preparative electrophoresis, and finally, size chromatography. Only mild and highly reproducible fractionation methods are used in order to obtain high recoveries. All of these provide the best guarantee that no serious subfractionation has taken place. At the same time, high recoveries are a necessity for the pure protein to be representative of the same protein in vivo. The following recoveries were obtained: prealbumin 55%, orosomucoid 70% and transferrin 85%. The pure proteins are produced as liquid calibrators (primary protein preparations) dissolved in one electrolyte (0.1 mol/l KCl) and stored in sealed glass ampoules at -80 degrees C. These three pure proteins were used as primary reference preparations in the certification of the international reference preparation CRM 470.

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Clinical trials to identify patients at risk and to assess new therapeutic agents in the nutritional field are often single-site. The principal advantage of mounting a multicentric trial is that patient accrual is much quicker. Albumin, transthyretin, and C-reactive protein are frequently used biochemical markers of nutritional and inflammation status. However, the different techniques, reagents, and calibrators used to measure these markers introduce wide variations in values among laboratories. This study was carried out as part of a prospective multicentric study in chronic respiratory disease patients to evaluate variability and comparability of results among laboratories for these biochemical markers, and to determine whether centralization is necessary. Thirty enrolled laboratories provided their own range of reference values for those proteins a nd were then requested to process two control samples C1 and C2 blind. The results showed a broad dispersion of values for albumin and transthyretin. In 7% of laboratories, results of albumin for C2 (mean, all techniques: 39.1+/-3 g/l) were <35 g/l, the threshold value indicating a potential risk of malnutrition. When only laboratories using immunonephelemetry were considered, the results were satisfactory (CV<10% for all proteins). Given the possible incorrect classification of patients at risk, measurement should be made per site only if all participants use an immunonephelometric method. Otherwise, a centralizing assay of these biological markers should be considered.

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Serum prealbumin standards for the first and sixth days of life have been determined by immunoprecipitation ascertained with kinetic nephelometry in two samples of healthy newborns, born at term and of normal weight.

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Eight different types of genetic variants of albumin are observed in the French population. The analysis of electrophoretic patterns of sera containing these variants, performed a three different pHs (8.6, 5.0 and 6.9) after addition of a reference protein (transferrin), allows the identification each variant by a quantitative estimation of its relative mobilities. The accuracy and reproducibility of the technique make it a useful reference method, commonly employed for studying European variants. The samples used as references for five genetic variant types, proalbumins Christchurch and Lille, albumins Vanves, B and Reading, were subjected to sequence analysis to determine the nature and localization of their structural change. Together with the mutations of albumins Gent and Roma previously described, the data presented here make available seven reference specimens for which the structural changes are characterized out of the eight variants known to exist in France.

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