Kirsty Jones

An immunoassay for the measurement of sirolimus

OBJECTIVE This study assessed the performance characteristics of a new microparticle enzyme immunoassay (MEIA) for the determination of sirolimus in whole blood. BACKGROUND In clinical investigatory studies, dose adjustments of the immunosuppressive drug sirolimus have been carried out using either high-performance liquid chromatography (HPLC) or, more recently, this investigational immunoassay kit based on the MEIA technique. METHODS Calibration was made over the linear range 0 to 30 ng/mL. Inaccuracy and imprecision were assessed by means of 3 control samples supplied with the kit (5, 11, and 22 ng/mL) and dilution of an above-quantitation-limit sample (154 ng/mL). Specificity was determined by the addition of 2 sirolimus metabolites to sirolimus-free human whole blood or to I of the control samples supplied with the kit. In addition, whole-blood samples from patients receiving either cyclosporine or tacrolimus (N = 24) were analyzed for sirolimus. A comparison of the MEIA and a validated HPLC/MS/MS assay analyzed both pooled samples from patients receiving sirolimus and spiked samples (sirolimus 2-60 ng/mL). In a more extensive comparison of patient samples measured by the MEIA assay, a validated HPLC assay with UV detection (HPLC-UV) was used (HPLC-UV sirolimus 7-64 ng/mL). RESULTS Inaccuracy (between-run) was < or =16.2% at all 4 concentrations (N = 5). Within-assay imprecision (repeatability) was <6% (N = 5), and between-assay imprecision (reproducibility) for the same samples was < 11% (N = 5). Recovery, assessed by means of 3 in-house control samples prepared in both fresh and previously frozen sirolimus-free human whole blood, ranged from 93.9% to 109.5%. The limit of detection, determined by dilution of the lowest nonzero calibrator (3 ng/mL), was set at 1 ng/mL, at which repeatability was 20.5% (N = 5). Five ng/mL of hydroxysirolimus cross-reacted with the assay by a mean of between 44% and 50% (N = 4); 5 ng/mL of 41-O-demethylsirolimus cross-reacted with the assay by a mean of between 86% and 127% (N = 4). Assay specificity was further challenged by ethylenediamine-tetraacetic acid (EDTA)-whole-blood samples from transplant patients not receiving sirolimus. These samples had tacrolimus and cyclosporine concentrations of 7.8 to 15.9 ng/mL and 38 to 485 microg/L, respectively. The median result was 0 ng/mL (third quartile, 0.7 ng/mL; maximum, 1.4 ng/mL); no value was above the lowest nonzero calibrator. The results of the comparison between the MEIA and the HPLC/MS/MS assay showed mean positive biases of 21% and 8% for the MEIA in measuring sirolimus in pooled patient samples and spiked samples, respectively. The results of the comparison of the MEIA and HPLC-UV median sirolimus concentrations were 18.2 and 20.1. Whole-blood samples anticoagulated with EDTA and containing sirolimus were stable for analysis by MEIA for 3 freeze-thaw cycles when stored at -20 degrees C and for 10 days when stored at 4 degrees C or at ambient temperature. A decline in sirolimus concentration occurred when samples were stored at 37 degrees C. CONCLUSION The MEIA showed suitable precision across a clinically relevant concentration range. In terms of patient management, the practical significance of cross-reactivity with sirolimus metabolites remains to be assessed.

Quality assessment issues of new immunosuppressive drugs and experimental experience.

There are established quality assessment schemes for the two immunosuppressive drugs that have entered routine clinical use: cyclosporine and tacrolimus. These two drugs, together with sirolimus and mycophenolic acid, have been the subject of recent consensus panel reports that have reached broad agreement on several issues relating to therapeutic monitoring of these agents. While the current quality assessment schemes are not based on validated reference methods, the data they yield on comparative assay performance are of value as a guide to patient management and for clinical studies of drug efficacy.

Proficiency-testing issues relating to sirolimus.

BACKGROUND A need exists to document laboratory proficiency to (1) compare results produced by different analytical techniques and (2) ensure consistency of results from multiple testing sites. OBJECTIVES To enable concentration-controlled studies of sirolimus to proceed with confidence, proficiency-testing schemes were put in place at laboratories selected to act as reference laboratories. The feasibility of establishing an ongoing proficiency-testing scheme was addressed with respect to sample stability. The scheme was then used to test proficiency for the measurement of sirolimus in 3 blinded samples each month. METHODS The method chosen for measurement of sirolimus was a prototype microparticle enzyme immunoassay. Initially, 15 laboratories were asked to analyze a series of 85 blinded samples that tested their inaccuracy, repeatability, and reproducibility for the measurement and their ability to dilute over-range samples competently. Both blood samples spiked with sirolimus and pooled blood samples from patients receiving the drug were circulated to a maximum of 50 laboratories. RESULTS Overall, both inaccuracy and imprecision were acceptable by predefined criteria. Inaccuracy for the immunoassay (percentage difference of the measured value against the nominal value) averaged -5% (95% CI, -9% to -1%). The mean percentage difference between the immunoassay and a high-performance liquid chromatographic assay with mass-spectrometric detection for the measurement of sirolimus in pooled samples (n = 5) from patients receiving the drug was 29% (95% CI, 24% to 34%). CONCLUSION The techniques documented here as part of the International Sirolimus Proficiency Testing Scheme could be applied to other clinical studies requiring protocol-driven dosing adjustments based on sirolimus measurements, irrespective of analytical technique used.

Quality Assurance Programs for Immunosuppressive Drugs: Cyclosporine and Beyond

Monitoring blood cyclosporine concentrations as a guide to therapy has become established as a useful guide for optimal prescription of the drug. Quality assurance programs for external assessment of cyclosporine measurements have helped to clarify many of the methodological problems associated with measurement of the drug. A number of new immunosuppressive agents are in development and, although experience in their routine monitoring in clinical practice is still at an early stage, some observations regarding external assessment of their measurement can be made. This article summarizes the lessons to be drawn from the experience of cyclosporine quality assurance programmes and suggests some areas in which these lessons could be applied in the field of new immunosuppressive drugs.