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Updated: Mar 14, 2026
Quantification of the Immunosuppressant Tacrolimus on Dried Blood Spots Using LC-MS/MS
Published on: November 8, 2015
Analytical Validation and Cross-Validation of an NFAT-Regulated Gene Expression Assay for Pharmacodynamic Monitoring
Emaad Abdel-Kahaar1, Thomas Giese, Claudia Sommerer
1*Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Stuttgart, Germany; and Departments of †Immunology and ‡Nephrology, University Hospital Heidelberg, Heidelberg, Germany.
Background:
Analysis of residual gene expression of the nuclear factor of activated T cell (NFAT)-regulated genes has been developed as a pharmacodynamic biomarker to monitor therapy with calcineurin inhibitors. The availability of commercial primer sets (Search-LC) and the well-established assay protocol makes this biomarker a promising candidate to be used clinically in different laboratories. However, implementation of the method in routine practice requires analytical robustness and comparable results across laboratories. Therefore, a protocol originally established at the Institute of Immunology, Heidelberg was verified at the Institute of Laboratory Medicine, Klinikum Stuttgart, and a comparison study was conducted between the 2 laboratories.
Methods:
For the analytical verification, whole blood samples of healthy individuals were incubated with tacrolimus in vitro. Linearity, imprecision, and limit of quantification, as well as sample stability, were investigated. For interlaboratory comparison, samples of patients under cyclosporine A therapy were analyzed in Heidelberg and then reanalyzed in Stuttgart within 24 hours.
Results:
Tacrolimus (6.25-50 mcg/L) decreased the expression of NFAT-regulated genes in vitro dose dependently (15%-89%). Within- and between-assay coefficient of variations (n = 6 each) were <17%. The limit of quantification was <200 cDNA copies for each of the interleukin-2, interferon-γ, and granulocyte-macrophage colony-stimulating factor genes. Samples were stable for 24 hours. Interlaboratory comparison using patient samples correlated well (r = 0.951) but showed an inconsistent bias depending on the magnitude of residual gene expression.
Conclusions:
The assay can be set up with a satisfactory analytical performance in a routine molecular biological laboratory and shows comparable results between laboratories. The reproducibility of the NFAT-regulated gene expression assay across laboratories can facilitate the implementation of this assay for pharmacodynamic routine monitoring of calcineurin inhibitors in different centers.
Insights
This study validates a gene expression assay for monitoring calcineurin inhibitor therapy. The NFAT-regulated gene expression assay demonstrates analytical robustness and reproducibility for clinical use.
Area of Science:
- Molecular Biology
- Pharmacology
- Clinical Diagnostics
Background:
- Nuclear factor of activated T cell (NFAT)-regulated gene expression serves as a pharmacodynamic biomarker for calcineurin inhibitor therapy.
- Commercial availability and established protocols make this assay promising for clinical application.
- Analytical robustness and inter-laboratory comparability are crucial for routine clinical implementation.
Purpose of the Study:
- To verify the analytical performance of the NFAT-regulated gene expression assay.
- To compare results between two independent laboratories for routine clinical use.
- To assess the assay's suitability for pharmacodynamic monitoring of calcineurin inhibitors.
Main Methods:
- Analytical verification involved in vitro incubation of whole blood with tacrolimus to assess linearity, imprecision, limit of quantification, and sample stability.
- Inter-laboratory comparison utilized patient samples from cyclosporine A therapy, analyzed in two different institutes.
- Standard molecular biological techniques were employed for gene expression analysis.
Main Results:
- Tacrolimus demonstrated a dose-dependent decrease in NFAT-regulated gene expression in vitro.
- The assay exhibited good precision (CV <17%) and a low limit of quantification (<200 cDNA copies).
- Inter-laboratory comparison showed a high correlation (r = 0.951) with minor bias, indicating good reproducibility.
Conclusions:
- The NFAT-regulated gene expression assay possesses satisfactory analytical performance for routine molecular diagnostic laboratories.
- Comparable results between laboratories facilitate the assay's implementation for pharmacodynamic monitoring.
- This validated assay supports consistent, multi-center clinical application of calcineurin inhibitor therapy monitoring.
