Production and certification of secondary enzyme reference materials (ERMs). Part 2: The need for meaningful protocols that assure photometric accuracy and a well-defined process for value assignment.

A collaborative study to assign values to two enzyme reference materials (ERMs) was performed by 18 laboratories whose spectrophotometers were checked by us, just before the study. We measured the wavelength accuracy and repeatability, the accuracy and linearity of the absorbance curves, the cuvette pathlength, equilibration time, equilibrium temperature, and a few other variables. Five spectrophotometers exhibited a marked wavelength-dependent nonlinearity. Most instruments were rather slow in bringing the sample to the correct temperature and the final temperature was often too high. In the collaborative study, each participant performed the same manual, well-described methods on four occasions in triplicate, using reagents prepared locally. The relation between the photometric checks and the analytical results is discussed, as well as the treatment of outliers and the effects on the variances. Suggestions are made about various facets of collaborative studies. The values assigned to the two ERMs carry a 95% uncertainty interval of +/- 1-4% of the mean.

Improved preparation of cholesterol calibration and control sera.

We prepared several serum batches with various cholesterol concentrations, to be used as calibrators and controls in a proficiency testing program of an organization in The Netherlands that is in charge of the standardization of cholesterol determinations for epidemiological purposes. The sera were of human origin, to avoid abnormal matrix effects. To decrease the cholesterol content in some samples, we adsorbed them onto colloidal silicic acid. To increase it, we added lipoproteins that had been precipitated from human serum with heparin and calcium ions. The precipitation method we used allowed us to dilute the serum as little as possible and to keep the final concentrations of calcium and heparin as low as possible, while maintaining a high cholesterol content. By mixing these sera having high and low cholesterol concentrations, we could prepare batches with any desired concentration. The stabilities of these sera were excellent. We used the sera to calibrate enzymic determinations of cholesterol.