Comparison of steady-state trough sirolimus samples by HPLC and a radioreceptor assay

Objectives: We have previously identified a minor immunophilin of 52 kDa mo lecular weight capable of binding tacrolimus and sirolimus. Because immunop hilins are capable of binding both parent drug and metabolites and HPLC ass ays are typically used to assess parent drug in clinical situations, we use d this immunophilin in a radioreceptor assay (RRA) to determine if any meta bolites not included in the HPLC measurement would bind to the immunophilin and be associated with thrombocytopenia in patients receiving sirolimus. Design and methods: We tested 51 steady-state trough whole blood samples fr om non-thrombocytopenic patients and 51 steady-state trough samples from th rombocytopenic patients and compared them to HPLC measurements of parent dr ug in the same samples. We also tested whole blood samples spiked with auth entic sirolimus metabolites using RRA to ascertain the effect these metabol ites have on the technique. Results: We found minimal cross-reactivity in this assay for sirolimus meta bolites (binding ranged from <10% to 26%), and good correlation of the radi oreceptor assay with HPLC (linear regression slope 0.92, y-intercept 0.79). There was no statistically significant difference between the RRA and HPLC results' in two patient groups-thrombocytopenic and non-thrombocytopenic-u sing the paired t-test p < 0.005) and Bland-Altman analysis. Conclusions: These findings indicate that although the RRA could be substit uted for HPLC in therapeutic drug monitoring, the 52 kDa immunophilin does not offer an advantage in terms of detecting metabolites associated with th rombocytopenia. However, the RRA offers the advantages of shorter turnaroun d time, smaller sample volume and potential for automation. Copyright (C) 2 000 The Canadian Society of Clinical Chemists.