BackgroundThe various properties of Positron Emission Tomography (PET) ligands often place limitations on the identification of specific binding to a target. Consequently, analysis techniques employed form an important step in the quantification of the specific binding potential. [11C](R)-PK11195 is a ligand for the translocator protein (TSPO), a marker of microglial activation. [11C]-DASB is a ligand for the serotonin transporter (SERT). Methods[11C](R)-PK11195: The study group comprised forty patients undergoing brain PET scans recruited as healthy controls across sites in Manchester (N = 22) and Hammersmith (N = 18). Parametric images of binding potential (BPND) were generated with the simplified reference tissue model (SRTM) using grey matter of cerebellum and supervised cluster analysis (SVCA) as reference input functions. [11C]-DASB: Twelve healthy volunteers (Age 30-51; mean 40.5; SD 7.4; 12 males) have also been previously recruited and had [11C]-DASB PET brain scans at The University of Manchester. Paired PET scans were undertaken before and after dosing with oral sibutramine. Parametric images of binding potential using SRTM with basis function implementation were generated and the parameters used in this method were optimised. Results[11C](R)-PK11195: The pattern of binding potential values produced is broadly similar between the two reference tissue input functions, however there are important differences. Particular cases in which the accuracy of the SVCA method is reduced have been identified. Using the cerebellar reference region significant changes in binding potential with age were noted using multiple measures ANOVA. These changes were not statistically significant using the SVCA input function. [11C]-DASB: BPND values read from the parametric maps showed a high degree of consistency with those generated using time-activity curves in regions of the brain with high SERT density.Conclusions[11C](R)-PK11195: SVCA requires further validation with arterial plasma input and development to reduce potential sources of error and ideally permit multicentre trials. Future WMIC studies seeking to detect regional variation in BPND and directly compare values with other PET centres should utilise the cerebellar reference region method until further advances in the SVCA method are made. There is evidence that the variation in [11C](R)-PK11195 BPND with age is dependent upon the region of the brain however a consistent relationship between BPND in individual regions of interest and age was not demonstrated between two PET centres. [11C]-DASB: The method of generation of parametric images of BPND developed for the [11C]-DASB ligand is suitable for use in future studies investigating differences between controls and subjects with hypothesised alterations of the SERT.