The research will be conducted under the rules of Good Clinical Practice, Good Laboratory Practice of the International Conference on Harmonisation (ICH)and the Principles of Declaration of Helsinki.
Groups of study are included once inclusion/exclusion criteria were verified and after written informed consent was given: healthy volunteers, patients of the waiting list for liver transplantation, liver transplant recipients on tacrolimus and cyclosporine and a longitudinal cohort of patients enrolled since the waiting list for transplantation.
From this instance proceed the sampling plan. Considering potential dropouts or withdrawals during the study, the intention is to recruit an additional 10% to compensate for the cohorts, as they allow.
The study design does not suppose applying masking methods, since it is an open study.
All data required in the registration forms will be recorded, however in case of persistent failure, will be properly documented the reasons for the absence. Each instance will merit a particular analysis, dated and signed.
Models could be used to estimate the impact of bias due to potential missing data, and if applicable will be complemented with a sensibility analysis.
Loading data will be conducted electronically. Data will be validated according to the data management plan, jointly defined by the principal investigator and the biostatistician, including freezing and thawing processes.
Pharmacokinetic, pharmacogenetic and pharmacodynamic modeling will be done using R software.
Data back ups will be run everyday, and will be archived on tape and a USB storage drive.
Besides self monitoring procedures, the study may be audited by the health authority (during the course of the study or even when it is completed), to assess compliance with the standards of Good Clinical Practice.
Atypical results if any, will be handled according to the criteria of results outside of specification. If re-analysis of samples will occur, they will be properly documented.
Sample size calculation includes the percentage of occurrence of acute cellular rejection and adverse events, as reported by the National Liver Transplant Program and the possible rates of abandonment or premature retirement of the in study subjects.
Under national rates, will be studied at least 30 healthy volunteers, 50 patients and 12 patients with end stage liver disease enlisted for liver transplantation, with serial follow-up during the first year.
Statistical analysis will be performed according to the principle of "intention to treat", and will be the responsible for at least one specialist in biostatistics.
It will be developed a descriptive analysis of all variables collected. Categorical variables will be expressed in percentages and number of observations. Continuous variables will be expressed as mean and coefficient of variation or median and 25th and 75th percentiles, minimum and maximum. Lof transformations will be held whenever needed.
The variables will be compared between groups according to the patient's original listing (intention to treat). They will be calculated and presented the estimated relative risks and their effect with 95% confidence intervals.
For comparison of categorical variables, it will be used the Chi-square test or Fisher's exact test as appropriate.
For comparison of continuous variables, it will be used the test "t" of Student.
For variables that develop with time, they will be represented by Kaplan-Meier curves and compared using the "log-rank" test. Their relative risk with 95% confidence interval will be calculated.
To identify variables associated with different responses multivariate linear analysis, logistic or Cox proportional will be used.
All analyzes will be performed with hypothesis testing and a 2-tailed significance level of 5%.
The program used will be R. Multivariate analysis will be held corrected according Bonferroni's criteria.
We have established standard operating procedures to describe blood collection instances, biological fluids sampling circuit, evaluation of the candidates to include in the study, verification of the inclusion criteria, monitoring of patients during the study, record of undesirable events and report of adverse drug reactions, terminating tracking.
