title: Comparison of steady-state trough sirolimus samples by HPLC and a radioreceptor assay.
abstract: OBJECTIVE
  We have previously identified a minor immunophilin of 52 kDa molecular weight capable of binding tacrolimus and sirolimus. Because immunophilins are capable of binding both parent drug and metabolites and HPLC assays are typically used to assess parent drug in clinical situations, we used this immunophilin in a radioreceptor assay (RRA) to determine if any metabolites not included in the HPLC measurement would bind to the immunophilin and be associated with thrombocytopenia in patients receiving sirolimus.
  
  
  METHODS
  We tested 51 steady-state trough whole blood samples from non-thrombocytopenic patients and 51 steady-state trough samples from thrombocytopenic patients and compared them to HPLC measurements of parent drug in the same samples. We also tested whole blood samples spiked with authentic sirolimus metabolites using RRA to ascertain the effect these metabolites have on the technique.
  
  
  RESULTS
  We found minimal cross-reactivity in this assay for sirolimus metabolites (binding ranged from <10% to 26%), and good correlation of the radioreceptor 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-using the paired t-test (p<0.005) and Bland-Altman analysis.
  
  
  CONCLUSIONS
  These findings indicate that although the RRA could be substituted for HPLC in therapeutic drug monitoring, the 52 kDa immunophilin does not offer an advantage in terms of detecting metabolites associated with thrombocytopenia. However, the RRA offers the advantages of shorter turnaround time, smaller sample volume and potential for automation.
authors.affiliation: Department of Laboratory Medicine, Children's National Medical Center, Washington, DC 20010, USA.
authors.forename: D L
authors.initials: DL
authors.lastname: Davis
authors.forename: J N
authors.initials: JN
authors.lastname: Murthy
authors.forename: K L
authors.initials: KL
authors.lastname: Napoli
authors.forename: B D
authors.initials: BD
authors.lastname: Kahan
authors.forename: H
authors.initials: H
authors.lastname: Gallant-Haidner
authors.forename: R W
authors.initials: RW
authors.lastname: Yatscoff
authors.forename: S J
authors.initials: SJ
authors.lastname: Soldin
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000592:standards*; D003429:Cross Reactions; D016572:Cyclosporine / Q000627:therapeutic use; D020104:Immunophilins / Q000302:isolation & purification / Q000378:metabolism; D007166:Immunosuppressive Agents / Q000097:blood*; D016030:Kidney Transplantation; D007959:Lymphocyte Culture Test, Mixed; D011241:Prednisone / Q000627:therapeutic use; D011485:Protein Binding; D011869:Radioligand Assay / Q000592:standards*; D020123:Sirolimus / Q000031:analogs & derivatives / Q000097:blood*; D013921:Thrombocytopenia / Q000097:blood / Q000628:therapy
chemical_list: D007166:Immunosuppressive Agents; D016572:Cyclosporine; D020104:Immunophilins; D011241:Prednisone; D020123:Sirolimus