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); UGT2B7 –161C>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).