Between June 2000 and December 2002, 378 renal transplant recipients were enrolled into this observational prospective study. Participants were recruited from the renal transplant clinics at Belfast City Hospital and Antrim Area Hospital in Northern Ireland, United Kingdom.
The study was approved by the Research Ethics Committee Queen's University Belfast and fully informed written consent was obtained from each participant prior to enrolment. Patients were eligible for entry if they had a functioning renal transplant present. No formal exclusion criteria were imposed. However, patients who were clinically unwell or had signs of sepsis at initial assessment were deferred until a subsequent clinic re-assessment.
All the renal transplant patients recruited to this study were greater than 2 months post-renal transplant and 94% were recruited more than 12 months after transplant surgery. All participants had stable graft function and were on standard immunosuppression regimens.
The 378 renal transplant recipients enrolled in this study represented 71.7% of all patients with a functioning renal transplant in Northern Ireland at the end of 2002 and 98% of all renal transplant recipients attending the transplant clinics at Belfast City Hospital and Antrim Area Hospital. The patients not enrolled either did not consent to participate in the study or, more commonly, were attending renal transplant clinics at other geographically distant hospitals in the Northern Ireland region.
At enrolment, with the assistance of a research nurse, each participant completed a cardiovascular risk assessment questionnaire. This recorded drug history, the presence of traditional cardiovascular risk factors (age, gender, diabetes and smoking history) and history of vascular disease. Prior vascular disease was defined as history of stroke, myocardial infarction, coronary artery bypass grafting, angioplasty, amputation for peripheral vascular disease or angiographic evidence of atherosclerotic vascular disease.
Each participant also had a measurement of blood pressure. This was recorded as the average of the last three blood pressure measurements (measured using Disytest sphygmomanometer Welch-Allyn, Buckinghamshire, UK) assessed at the renal transplant clinic.
A fasting blood sample was obtained from each participant and stored at -70°C until biochemical analysis.
VCAM and ICAM were measured in plasma samples using a commercially available solid phase sandwich ELISA technique (Diaclone; available from IDS, Tyne and Wear, UK). The within run coefficient of variation for VCAM was 3% and the between run coefficient of variation was 10%. The within run coefficient of variation for ICAM was 1.2% and the between run coefficient of variation was 7.1%.
Serum total cholesterol and high density lipoprotein (HDL) cholesterol were measured using VITROS slides and analysed using a VITROS 700 System (Ortho Clinical Diagnostics, Rochester, NY, USA).
Serum creatinine was measured using the VITROS Slide System and the VITROS 950 analyser system (Ortho Clinical Diagnostics, Rochester, NY, USA). Detection range for creatinine was 4 - 1238 μmol/l and within lab coefficient of variation was 1.1%. Estimated glomerular filtration rate (eGFR) was calculated for all patients using the 4-variable MDRD equation :
High sensitivity C reactive protein (hsCRP) was measured using a high sensitivity immunoturbidimetric assay (Randox, Crumlin, UK). Samples were analysed using a Roche Cobas Fara (Roche, Basel, Switzerland).
All 378 participants had measurements for VCAM, ICAM, total cholesterol, HDL cholesterol and creatinine. 375 participants had a measurement for hsCRP.
Prospective Data Collection
The collection of prospective follow-up data was completed in April 2008 at a mean of 2243 days and a median of 2441 days after enrolment. The longest period of follow-up after recruitment to the study was 2844 days. Mortality data, including date of death, where applicable, was available for all participants. This information was obtained from the mortality data recorded on the Regional Nephrology Database at Belfast City Hospital and via letter and direct telephone contacts with the Primary Care Physicians of the renal transplant recipients enrolled in this study. No patients were lost to follow up.
Data analysis was performed using SPSS (version 11.0 Chicago, Illinois, USA). Kolmogorov-Smirnoff analysis was used to test if variables were normally distributed. Logarithmic transformation was performed for variables that did not conform to a normal distribution. For normally distributed variables data is expressed as arithmetic mean +/- standard deviation (SD). For those variables that were not normally distributed data is expressed as median with the interquartile range in brackets. The significance of differences between two groups was assessed using independent samples t-test for normally distributed variables. A two-tailed p value <0.05 was considered to be statistically significant.
Kaplan-Meier analysis with log rank test was used for univariate survival analysis. As there are no established reference values for either VCAM or ICAM, these variables were banded into thirds prior to inclusion in survival analysis.
A Cox Regression model was used for multivariate survival analysis. As cardiovascular disease is the leading cause of death in patients with a renal transplant , multivariate survival analysis was performed including traditional cardiovascular risk factors as co-variates. Given the size of the study population, traditional cardiovascular risk factors were banded into thirds prior to inclusion in the Cox Regression model. Since the risk of cardiovascular disease is also associated with hsCRP and eGFR, these variables were also included as co-variates, banded into thirds, in the Cox Regression analysis.