Molar absorptivities of bilirubin (NIST SRM 916a) and its neutral and alkaline azopigments.

Three laboratories in the U.S. and two in the Netherlands determined molar absorptivities (epsilon) of Standard Reference Material (SRM) 916a Bilirubin from the National Institute of Standards and Technology. In caffeine reagent the average epsilon values were 50,060 and 48, 980 L.mol-1.cm-1 at 432 and 457 nm, respectively. The epsilon value of the blue azopigment, obtained with the Reference Method for total serum bilirubin, was 76,490 L.mol-1.cm-1 at 598 nm. When the addition of alkaline tartrate was omitted, the molar absorptivity of the red azopigment was 56,600 L.mol-1.cm-1 at 530 nm.

Production and certification of secondary enzyme reference materials (ERMs). Part 1: Preparation of the sera and some of their properties.

We produced three batches of a human-serum-based enzyme reference material (ERM) enriched with human aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2), creatine kinase (EC 2.7.3.2), and lactate dehydrogenase (EC 1.1.1.27). The added enzymes were not exhaustively purified; thus the final ERMs contained some enzymes as contaminants, of which only glutamate dehydrogenase activity might interfere. The stability during storage and after reconstitution was good. The commutability of the four enzymes in the three ERM batches was also good, except when German or Scandinavian methods for aminotransferases were involved. The temperature-conversion factors for the ERMs were equivalent to those for patients' sera. Reactivation after reconstitution was complete within 5 min and was independent of the temperature of the reconstitution fluid. We believe that these secondary ERMs will aid in the transfer of accuracy between well-defined reference methods and daily working methods so that clinical enzymology results will become more comparable from laboratory to laboratory.

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.

International Federation of Clinical Chemistry (IFCC). Scientific Committee, Analytical Section. IFCC/WHO principles and recommendations on evaluation of diagnostic reagent sets used in health laboratories with limited resources. Part 3. Selection and evaluation using reference materials. General considerations.

The purpose of this document is to provide general considerations for the selection and evaluation of clinical chemistry kits in laboratories with limited resources. Separate documents have been developed to provide guidance on experimental procedure, the statistical treatment and interpretation of the data and criteria for acceptable performance of diagnostic kits designed to measure specific analytes.

The influence of sodium bromide in man: a study in human volunteers with special emphasis on the endocrine and the central nervous system.

Sodium bromide was administered orally in capsules to healthy volunteers in doses of 0, 4 or 9 mg Br-/kg/day using a double-blind design. Each treatment was given to seven males for 12 weeks and to seven non-pregnant females (not using oral contraceptives) over three full cycles. Special attention was paid to possible effects on the endocrine and central nervous systems. At the start and end of the study, a full medical history, the results of a physical examination, haematological studies and standard clinical chemistry and urine analyses were recorded for each subject. These showed no changes for individuals following treatment, except for some incidence of nausea associated with bromide-capsule ingestion. Mean plasma-bromide concentrations at the end of treatment were 0.08, 2.14 and 4.30 mmol/litre for males and 0.07, 3.05 and 4.93 mmol/litre for females of the 0-, 4- and 9-mg Br-/kg/day groups, respectively. Plasma half-life was about 10 days. In the females taking 9 mg Br-/kg/day (but in no other group) there was a significant (P less than 0.01) increase in serum thyroxine and triiodothyronine between the start and end of the study but all concentrations remained within normal limits. No changes were observed in serum concentrations of free thyroxine, thyroxine-binding globulin, cortisol, oestradiol, progesterone or testosterone, or of thyrotropin, prolactin, luteinizing hormone (LH) and follicle-stimulating hormone before or after the administration of thyrotropin-releasing hormone and LH-releasing hormone. Analysis of neurophysiological data (EEG and visual evoked response) showed a decrease in delta 1- and delta 2-activities and increases in beta-activities and in mean frequency (Mobility parameter) in the groups on 9 mg Br-/kg/day, but all the findings were within normal limits.

A column chromatographic method for the removal from 2-amino-2-methyl-1-propanol of impurities that inhibit alkaline phosphatase activity.

The selective removal from 2-amino-2-methyl-1-propanol solutions of impurities that inhibit alkaline phosphatase activity by means of Amberlite chromatography is described. The effects of buffer concentration, column dimensions, adsorbent type, and zinc sulphate addition were investigated. The method was found to improve greatly the quality of commercial 2-amino-2-methyl-1-propanol solutions and to have several advantages over alternative purification methods.

Elimination of inhibitors of alkaline phosphatase from 2-amino-2-methyl-1-propanol.

When 2-amino-2-methyl-1-propanol buffer solutions are extracted with chloroform, impurities inhibiting alkaline phosphatase are selectively removed and this source of variation is thereby greatly decreased. We also studied the effect of adding zinc sulfate to this buffer. A combination of chloroform extraction and zinc sulfate, added to give concentrations from 5 to 10 mumol/L, results in buffer of high and consistent quality. The alkaline phosphatase activities of 12 reference sera of different origin were determined with use of the purified buffers. Those sera, which closely resembled human serum in terms of alkaline phosphatase isoenzyme composition, behaved similarly to human serum. Use of such control sera is strongly recommended.