Variation in Leukocyte Subset Concentrations Affects Calcineurin Activity Measurement: Implications for Pharmacodynamic Monitoring Strategies Huub H. van Rossum, 1* Fred P.H.T.M. Romijn, 1 Kathryn J. Sellar, 1 Nico P.M. Smit, 1 Paul J.M. van der Boog, 2 Johan W. de Fijter, 2 and Johannes van Pelt 1 BACKGROUND: In renal transplantation patients, thera- peutic drug monitoring of the calcineurin (CN) inhib- itor cyclosporin A (CsA) is mandatory because of the drug’s narrow therapeutic index. Pharmacodynamic monitoring of CN inhibition therapy could provide a tool to define and maintain the therapeutic efficacy of CsA therapy. We investigated the effect of variation in cell counts of leukocyte subsets on leukocyte CN activ- ity measurement in renal transplant recipients. METHODS: We measured leukocyte CN activity, whole blood CsA concentrations, and leukocyte subset cell counts in 25 renal transplant recipients. Blood was col- lected before graft implantation and CsA therapy, 1 day before transplantation when CsA therapy was already started, and 5 days after transplantation. Monocyte, granulocyte, CD4 T-cell, CD8 T-cell, B-cell, and natural killer– cell CN activities and CsA inhibition sensitivities were determined in vitro by a spectropho- tometric CN assay. RESULTS: Leukocyte CN activity was inhibited after drug intake. Inter- and intrapatient variation in leuko- cyte subset cell counts resulted in variation of sample composition. The mean (SD) CN activity varied among leukocyte cell subsets, ranging from 650 (230) to 166 (26) pmol/min/10 6 cells for monocytes and CD4 T cells, respectively. CsA half maximal inhibi- tory concentration (IC 50 ) values ranged from 15 to 78 g/L for monocytes and B cells, respectively. CONCLUSION: Inter- and intraindividual leukocyte sub- set cell count variation can affect measured CN activity independent of CsA concentration. Cell-specific activ- ity and drug sensitivity should be considered for sam- ple validation to optimize method specificity when pharmacodynamic monitoring strategies are applied in a clinical setting. © 2008 American Association for Clinical Chemistry Immunosuppression by calcineurin (CN) 3 inhibition therapy is essential for prevention of graft rejection early after renal transplantation. The 2 CN inhibitors (CNI) used in transplantation medicine, cyclosporin A (CsA) and tacrolimus, inhibit CN after formation of a complex with their corresponding binding proteins, and result in suppression of lymphocyte activation (1, 2 ). Unfortunately, side effects, such as nephropa- thy, neuropathy, diabetogenesis, malignancies, and cardiovascular disease occur in CNI-treated patients (3–7 ). Because of the unpredictable kinetic profiles of CNIs and potential drug-drug interactions, patients re- ceiving CNI therapy require intensive drug monitoring (8). Pharmacokinetic monitoring is performed by measuring blood drug concentrations at different time points after drug intake (9 –11 ). However, more accu- rate drug monitoring strategies are desired to maxi- mize efficacy of these drugs (12, 13 ). Pharmacodynamic monitoring of CNI therapy is a new approach for the optimization of CNI monitoring (8), and several methods are available for measure- ment of CN enzyme activity as a pharmacodynamic marker. All methods use the same substrate, a 19- amino acid peptide (RII-peptide), which corresponds to a part of the regulatory subunit of bovine cAMP- dependent protein kinase (14 ), but methods differ in the detection techniques used, such as HPLC-ultravio- let measurement of dephosphorylated peptide (15, 16 ), radioactive measurement of 32 P-labeled phosphate (1, 17, 18 ), and absorbance measurement of malachite green phosphate reagent (19 ). In addition, various Departments of 1 Clinical Chemistry and 2 Nephrology, Leiden University Medical Center, Leiden, the Netherlands. * Address correspondence to this author at: Department of Clinical Chemistry, Leiden University Medical Center, Leiden 2333 ZA, The Netherlands. Fax: 31 71 5266753; e-mail H.Rossum@lumc.nl. Received September 3, 2007; accepted December 21, 2007. Previously published online at DOI: 10.1373/clinchem.2007.097253 3 Nonstandard abbreviations: CN, calcineurin; CNI, CN inhibitor; CsA, cyclosporin A; PBMCs, peripheral blood mononuclear cells; NK cell, natural killer cell; FITC, fluorescein isothiocyanate; PE, phycoerythrin; APC, allophycocyanin; HBS, Hepes buffered saline. Clinical Chemistry 54:3 517–524 (2008) Drug Monitoring and Toxicology 517