A manufacturer's approach to development of matrix robust methods.

Use of matrix robust methods (MRM) to solve the problem of matrix-induced analytic errors appears to be a sound strategy. The development of MRM, however, is in an infancy stage. The principal barrier is the complexity of the matrix effect that involves interactions of the matrix, the analyte, and the technology base of the test method. Each of these three components has its own set of variables. The present article focuses on concepts and tactics to develop the MRM that appear promising on a path-forward basis. The author believes that the current environment favors probability of successful development of MRM. The quality awareness at all functional levels is high, technically feasible models for the design and development of MRM exist, and commercialization of such a method promises the developer a competitive advantage in the marketplace. The optimum strategy for MRM development appears to be evolutionary, ie, starting with a few critical methods and the samples representing the prevalent matrix types. Success in developing MRM also depends on close cooperation between the developers of the MRM, proficiency testing material, the proficiency testing providers, and the regulatory bodies. The research and development program may also include approaches that detect and/or correct the matrix-caused error(s) both in place of, or as an adjunct to, MRM. With respect to the development of genuine MRM, the author has given a typical development scenario comprising the design specifications, specific experimental approaches, evaluation, market introduction, and postintroduction monitoring of its robustness. The crux of the experimental approach is the response surface co-optimization of reaction conditions for the samples of prevalent matrix types such as the proficiency testing materials. The recommended approach is supported by examples of existing methods that exhibit robustness against certain types of matrices. The author believes that addition of MRM to the clinical chemistry methods repertoire is likely to improve the test result quality. It will also improve proficiency testing performance and patient care while boosting the morale of laboratory personnel.

Response-surface co-optimization of reaction conditions in clinical chemical methods.

We describe the theory and applications of the response-surface approach to simultaneous optimization (co-optimization of multiple interdependent variables. The co-optimization experiments are designed according to a selected response-surface model. Computer-assisted analysis includes fitting the data to the model, testing the resulting fit for statistical validity, and plotting contour maps of the model for simple interpretation. Co-optimizations of the reaction parameters of three methods--creatine kinase, lipase, and aspartate aminotransfrease--are discussed to illustrate the application of the approach. In contrast to the commonly used optimization strategies in which each factor is varied in turn while the others are kept constant, the response-surface approach allows study of several responses (reaction rates, sensitivities) and effects (linear, curvature, interaction) at the same time. It also allows determination of accurate optima, which is necessary for the formulation of analytically reliable clinical methods.

Automated enzymic measurement of total cholesterol in serum.

We describe a completely automated enzymic system for measuring total cholesterol in serum. All reagents are contained in an analytical test pack and the test is performed on Du Pont's Automatic Clinical Analyzer (aca), which mixes the sample (20 microliter) and reagents and performs the necessary absorbance measurements and calculations. In the procedure, cholesterol oxidase oxidizes free cholesterol. The oxidation step produces cholest-4-en-3-one and hydrochloride peroxidesterase hydrolyzes cholesterol esters and cholesterol in direct proportion to the amount of cholesterol present. N,N-Diethylaniline hydrochloride and 4-aminoantipyrine react with the hydrogen peroxide to produce a quinoneimine dye (lambda max = 553 nm). Interacting reagents have been optimized simultaneously (coöptimization) utilizing response surface designs coupled with computer analysis of the data. Reagent efficiency is high and analytical performance reliable.

Simple cultural test for relative cellulolytic activity of fungi.

A simple method is described for determining the relative cellulolytic activity of fungi. Opaque columns of an agar medium containing a partially crystalline cellulose preparation were inoculated with the fungi. Depth of the clear zone that developed beneath the growing cultures provided a visual measure of cellulolytic activity on a continuous, cumulative basis. Depth of clearing (DC) was determined for 25 species of fungi differing widely in cellulolytic activity, and compared by correlation analysis with results of three other methods for measuring cellulolytic activity. Relatively high coefficients of correlation (greater than 0.6) were obtained between DC and weight loss of cotton sliver, loss in tensile strength of cotton duck, and carboxymethyl cellulase activity in culture filtrates. In comparison with conventional assay procedures, the clearing method offered several advantages: (i) results were at least as well correlated with the capacity to utilize native cellulose as a substrate; (ii) the method measured activity of growing cultures rather than culture filtrates, thus involving less risk of losses due to product inhibition, binding, or denaturation of enzymes; (iii) repeated measurements were made on the same experimental set up, so that errors due to arbitrarily selected times of harvest were avoided conveniently; and (iv) the method required less working time and very simple equipment, making it convenient for large-scale screening tests.