In vitro pentamer formation as a biomarker of tacrolimus-related immunosuppressive activity after liver transplantation

Therapeutic drug monitoring of tacrolimus (FK) is widely performed to assis t adjustments of drug dosage but may be an inadequate surrogate of the immu nosuppression induced. The aim of this investigation was to develop an alte rnative method for measuring FK-related immunosuppressive activity in blood samples from liver transplant recipients. A pentamer formation assay (PFA) was devised based on the attachment of the 12 kDa FK-binding protein (FKBP 12) to microtitre plates in the presence of calcineurin, calmodulin. Ca++ a nd FK. Pentamer formation could be detected at FK concentrations greater th an or equal to 0.2 mug/l by optimising assay conditions, particularly by in cluding Ca++ (0.5 mM) only during the formation of the pentameric complex. Three methods (blood lysis, proteolytic digestion and use of commercial sol utions used in a microparticle enzyme immunoassay (MEIA) technique) were in compatible with PFA measurements after extracting immunosuppressive FK-rela ted material from patients' blood samples. However, therapeutic amounts of FK-related material could be quantified by the PFA assay after extraction o f blood samples with methanol. There was a moderate correlation (r = 0.689) of FK equivalents assayed by PFA with results using MEIA in 56 blood sampl es from 14 liver graft recipients, but no obvious relationship of results t o variables reflecting their clinical status.