*Purpose: The intracellular tacrolimus concentration in peripheral blood mononuclear cells (PBMCs) (TAC_[PBMC]) was proposed to better represent active concentration than pre-dose whole blood concentration (C₀). However, in previous studies, the TAC_[PBMC] did not correlate well with acute rejection. Since tacrolimus acts within T lymphocytes and other white blood cells such as monocytes, we investigated the association between the tacrolimus concentration in CD3⁺ T lymphocytes and CD14⁺ monocytes, and acute rejection after kidney transplantation.

*Methods: In this case-control study, blood samples of 37 kidney transplant recipients who underwent a for-cause biopsy were studied. Sixteen out of 37 had a biopsy-proven acute rejection (rejection group) and 21 patients had no rejection (control group). PBMCs were collected from both cryopreserved samples (retrospectively) and freshly obtained samples (prospectively). T lymphocytes and monocytes were isolated from PBMCs and their intracellular tacrolimus concentration was measured by liquid chromatography-tandem mass spectrometry.

*Results: The correlation between the tacrolimus whole blood C₀ and intracellular concentrations was poor, with a correlation coefficient of r=0.33 for PBMCs, r=0.54 for T lymphocytes, and r=0.67 for monocytes. Remarkably, TAC_[CD3] was significantly lower than TAC_[CD14]:13.3(9.9-19.1) vs 72.8(57.3-101.7) pg/million cells (pgm);p<0.001). Patients with rejection had a comparable whole blood C₀ to those without rejection (mean 9.8±2.9 vs 8.8±3.3 ng/mL;p=0.32). Also, no difference was found between the rejection group and control group regarding the TAC_[PBMC] (36.0(22.8-48.5) vs 34.0(32.0-45.5) pgm;p=0.82), TAC_[CD3] (13.1(9.9-15.7) vs 11.4(8.3-13.3) pgm;p=0.19), and TAC_[CD14] (74.8(58.6-105.1) vs 62.3(51.0-78.8) pgm; p=0.19). However, PBMCs that were freshly isolated showed significantly higher TAC_[PBMC] compared with PBMCs from cryopreserved samples (91.5(60.5-113.75) vs 34.5(28.1-47.0) pgm;p<0.001). Subgroup analysis of the intracellular tacrolimus concentration from freshly isolated cells again did not show difference between the rejectors and non-rejectors.

*Conclusions: The correlation between whole blood and intracellular tacrolimus concentrations is poor. No difference in TAC_[CD3] nor TAC_[CD14] concentrations between patients with and without acute rejection could be demonstrated. However, further optimization of the cell isolation process is required because a difference in TAC_[PBMC] between fresh and cryopreserved cells was demonstrated.

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