Reducing the cost of cyclosporin assays: modification of the EMIT 2000 method.

Cyclosporin is the leading immunosuppressant agent in organ transplantation, and therapeutic drug monitoring forms an integral part of patient management in most institutions. In the authors' laboratory, the cost of cyclosporin assays represents a major fraction of total consumable expenditure. At present, an average of 4,300 patient cyclosporin assays are performed annually using the EMIT 2000 method (Behring-Syva) on the Cobas Mira analyser (Roche), at a cost of AUD$50,000 in kits alone. As a means of reducing laboratory costs, the manufacturer's recommended method was modified by decreasing all of the reagent and sample volumes in the "Analytical" section of the Cobas Mira cyclosporin programme by 33%. Assay performance was monitored over a 10-month period and compared to that of the unmodified method. Calibration curves were stable, requiring a one-point correction on average of once every 12 days, and a full calibration once ever 1.7 months. Interassay variability was not different to that previously reported for the unchanged method, with mean (SD, CV) concentrations for trilevel quality control specimens of 86.5 micrograms/L (10.2, 11.9%), 185.9 micrograms/L (11.4, 6.2%) and 408.5 micrograms/l (28.9, 7.1%). From 24 specimens assayed in an international quality assurance programme, the results of 23 were within 1.2 SD of the group mean for the EMIT method, with an average bias of 0.8%. With the current modifications, we were able to perform an average of 105 patient assays per kit compared to the previous 71, equating to an annual saving the AUD$16,600.

High-performance liquid chromatography determination of clonazepam in plasma using solid-phase extraction.

The use of high-performance liquid chromatography for therapeutic drug monitoring of clonazepam has previously been limited by low sensitivity and labor-intensive liquid-liquid extractions. The present method was developed employing a rapid solid-phase extraction, thus minimising sample workup and providing analytical sensitivity down to 2 micrograms/L using 1 ml of plasma. Plasma samples were loaded onto C18 solid-phase extraction columns, and clonazepam and its internal standard (methyl-clonazepam) were eluted with methanol, dried, and reconstituted in 130 microliters of mobile phase. Chromatographic separation was achieved using a 3-microns RP18 column at 40 degrees C and a mobile phase of 32% acetonitrile and 0.5% glacial acetic acid in distilled water at 0.5 ml/min. Detection was carried out using ultraviolet absorbance at 306 nm. Retention times for clonazepam and methyl-clonazepam were approximately 7 and 12 min respectively. Standard curves were linear over a range of 5-200 micrograms/L with intraassay coefficients of variation of 1.2 and 4.8% at 200 and 5 micrograms/L, respectively. Plasma concentrations measured in patient samples were not statistically different from those obtained using an established gas chromatographic method, and quality control specimens from the Heathcontrol EQA Scheme were consistently within +/- 1.2 SD of the group means. There was no chromatographic interference from other benzodiazepines or other drugs used for the treatment of epilepsy.

Experiences with the enzyme-multiplied immunoassay cyclosporine specific assay in a therapeutic drug monitoring laboratory.

Cyclosporin-A (CsA) monitoring is well established in the management of most organ transplant patients. The present communication reviews the performance of the recently introduced specific enzyme-multiplied immunoassay (EMIT) CsA method during the first 7 months of its operation and compares costs of providing this service with those of the specific 125I radioimmunoassay (RIA) method previously employed in this clinical laboratory. Results suggest that the EMIT method performed well, giving long calibration curve stability (up to 12 weeks), and only 4.4% of the 31 kits through this period were consumed in assaying calibration standards compared with 20.8% with RIA. However, more quality control assays were performed, with the net result that only a slight improvement in the percentage of kit consumed in patient assays was noted (74.0% compared with 70.3%) with the EMIT method. This method appears to have been well accepted clinically as the CsA assay request rate over this period increased by 23% and, since it is both specific and rapid, is, therefore, recommended as the best CsA method currently available.