Bioanalytical procedures and genotyping
Whole blood cyclosporine and tacrolimus were determined by a validated affinity chrome-mediated immunoassay (ACMIA, Siemens, Germany). Total plasma MPA was determined by high pressure liquid chromatography (HPLC) with UV/VIS spectrophotometric detection (at 215 nm, 25°C, workflow 1 mL/min) using a commercially available HPLC kit for MPA in plasma (Chromsystems, Germany). All analytes were included in the external proficiency testing schemes (RfB and Instand).
Creatinine clearance was estimated (Cockroft-Gault) based on serum creatinine quantified by an enzymatic assay on an automated analyzer (Cobas c 501; Roche, Germany) validated by isotope dilution mass spectrometry.
Genomic DNA was isolated from whole blood using BioSprint 15 DNA Blood Kit (Qiagen, Hilden, Germany) on KingFisher mL System (Thermo Labsystems, Vantaa, Finland). Genotyping was performed on an Applied Biosystems 7500 Real Time PCR System, according to manufacturer’s instructions (Applied Biosystems, CA, USA by) using a validated TaqMan® Drug Metabolism Genotyping Assays (Life Technologies, Carlsbad, CA, USA) for the following polymorphisms: ABCG2 c.421C > A (rs2231142, ID C_15854163_70); ABCC2-24C>T (rs717620, ID C_2814642_10) and1249G>A (rs2273697; ID C_22272980_20);SLCO1B1 c.521T > C (rs4149065, ID C_30633906_10); UGT2B7161C>T (rs7668258, ID C_27827970_40);UGT1A9-275 T>A (rs6714486, ID C__27843087_10) and-2152C>T (rs17868320, ID C__34418857_10);ABCB1 3435C>T (rs1045642, ID C___7586657_20) and1236C>T (rs1128503, ID C___7586662_10); CYP3A4*22 (rs35599367, ID C__59013445_10 ) and CYP3A5*3(rs776746, ID C__26201809_30). Genotyping of ABCB1 c.2677G> T/A (rs2032582) was performed by real-time PCR genotyping on the LightCycler® instrument (Roche Diagnostics, Mannheim, Germany).