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Processing of MRI and PET data The PET images were corrected for head movement using a frame-by-frame realignment algorithm, in which all frames were individually realigned to the first minute of acquisition using SPM5 (Wellcome Department of Cognitive Neurology, University College London). For comparison, a parallel data set was created for which no realignment was performed. The T1-weighted MR images were reoriented according to the line defined by the anterior and posterior commissures (AC-PC line) being parallel to the horizontal plane and the inter-hemispheric plane parallel to the sagittal plane. The MR images were then co-registered to the summation PET image (9–51 min) using SPM5 (Wellcome Department of Imaging Neuroscience, London, UK) using the normalized mutual information algorithm and the default 7 × 7 FWHM smoothing of the 256 × 256 joint histogram. The MR images were used to delineate anatomical regions of interest (ROI) for the caudate nucleus (CAU), the putamen (PUT), the dorsolateral prefrontal cortex (DLPFC), and the insular cortex (INS). Each region was defined by a manual and an automated method, respectively.

The regions were chosen to represent the regions of central interest in schizophrenia research and include high- and low D 1-DR density regions with different degrees of proximity to CSF. Regions with more CSF borders may be particularly vulnerable to errors in ROI definition or motion artifacts. The caudate and putamen are high-D 1-DR density regions bordering and not bordering to CSF, respectively. The DLPFC and INS are low-density D 1-DR regions bordering more and less to CSF, respectively. Manual ROIs For the manual method, an in-house software, HBA , was used where the reoriented MP-RAGE volume was loaded for manual delineation of the ROIs on any of the three orthogonal projections.

The manual segmentation was performed by one investigator (PS) who has more than 20 years of experience in manual ROI delineation. The CAU and PUT were delineated as described by Mawlawi et al. , with the modification that the sagittal planes were used instead of the coronal. The DLPFC was traced on all the coronal planes anterior to the genu of the corpus callosum. The INS was delineated on all of its transaxial planes. The cerebellum was drawn on the central six transaxial images of the cerebellum separately on each hemisphere and about 1 cm distant from the subarachnoidal space. The manual ROIs were not masked by the GM map in order to have a complete manual method of gray-white matter segmentation for comparison with the automated ROIs that were masked by the GM map.

The ROIs were translated into each PET study space using the inter-modality co-registration matrices. Automated ROIs The automated definition of target ROIs was performed using FreeSurfer (FS, version 5.0.0, ) to obtain subject-specific anatomical delineation by reconstruction of the cortex and segmentation of subcortical structures as described elsewhere ,. The FreeSurfer morphometric procedures have been shown to exhibit good reproducibility across scanner manufacturers and across different field strengths and have been validated against histological and manual measurements. In addition, the cortical structures are divided based on individual cortical folding patterns to match the cortical geometry across subjects.

The ROI for the dorsolateral prefrontal cortex used here comprises the ctx-rostralmiddlefrontal and ctx-parstriangularis regions of the Desikan-Killiany Atlas in FreeSurfer. Finally, an automated ROI for the cerebellum was defined using FSL (the FMRIB Software Library), a library of analysis tools for brain imaging data. This region was defined using the maximum probability FSL MNIfnirt atlas segmentation with 25% probability threshold , from which a mask was defined containing cerebellar regions VI, crus I, and crus II (indices , ).

This mask was then registered to the space of each individual’s MR using the inverse FNIRT warp parameters. This mask was subsequently then trimmed as follows: 8 mm from the cortex, 8 mm from the vermis (defined using the same atlas), 4 mm from the edge of the brain mask (FSL brain mask). In addition, voxels belonging to the two most inferior planes of the PET image were excluded from the ROI. The resulting ROI was multiplied by the FreeSurfer gray matter segmentation mask to obtain a ROI consisting of only those voxels identified as belonging to gray matter.

For more information, see Matheson et al. The purpose was to compare the different impact on the BP ND value using a standardized automatically delineated cerebellum compared to using the manual cerebellum as reference region. Statistical analysis—general In the following, the terminology related to the statistical analysis follows the recommendations of the Terminology Working Group of the Quantitative Imaging Biomarker Alliance. The inclusion of a test-retest analysis serves two purposes. First, it allows for the examination of the repeatability of PET measurements with 11C. Second, it can be viewed as a duplicate measure of the same variable, i.e., in the same way as the mean of triplicate measurements commonly are used in biochemistry and bioanalyses. 4 where MS B is the between-subject mean sum of squares, MS W is the within-subject mean sum of squares, and k represents the number of observations (in this case 2).

The ICC is an estimation of reliability, which is the “true” variance/total variance. Thus, a value of 1 means that there is no measurement error at all, while a value of 0 means that the obtained measurements are entirely comprised of measurement error.

Zero is therefore technically the lowest value for the ICC, and a negative value can be approximated by 0. Comparisons of ROI volumes and BP ND values between the methods for image analyses The advantages of interval estimate compared to point estimate as the output of statistical analysis have been demonstrated for medical research in general and specifically for the comparison of quantitative biomarkers. Hypothesis testing for a point estimate using a p value is a binary (yes or no) decision even for minor differences that may not be of practical importance. It may thus be more useful to evaluate the data with an interval estimate that in addition to a p value provides a plausible range for the true value, which can be judged subjectively on an agreement between and precision of the methods. Computation of median, interquartile range (25th to 75th), and coefficient of variation (CoV; standard deviation mean) was applied to evaluate the distribution and variability of the BP ND values.

Computation of a 95% confidence interval was applied for the evaluation of the agreement and precision of the two ROI volumes (manual and automated), and the two BP ND values from the three comparisons in the image analysis (ROI definition, realignment and definition of reference region). The two PET measurements and the two methods in each of the three comparisons of the analysis amounted to 2 × 2 × 2 × 2 equals 16 sets or 8 pairs of BP ND values for each ROI as illustrated in Additional file: Table S1 for the test-retest combinations. The balanced data set made it possible to test for agreement between the pairs of BP ND values only once instead of for each of the eight combinations of the test-retest and three pairs of methods of analysis (manual target ROI/automated target ROI, no realignment/realignment, manual reference ROI/automated cerebellum ROI). Hence, the statistical computations were applied once for each pair of BP ND values using the mean BP ND value of the other eight combinations of data (2 × 2 × 2). This averaging was justified after testing by ANOVA for the absence of two- and three-way interactions among the pairs of variables.

The two absolute variability (AV) values and ICC values were also evaluated for agreement and precision by the mean differences and confidence intervals in the same way as for the BP ND values with the difference that there were 2 × 2 × 2 (i.e., eight sets or four pairs) AV and ICC values for each of the three parts of the image analysis. The comparison of ICC values between the methods was assessed by using the 95% confidence intervals to examine whether there was an overlap between the confidence intervals between the pairs of methods compared. Results The subjects completed the study according to the protocol. Each subject underwent one T1 MRI scan on 1 day and two PET measurements in the morning and afternoon of another day after a negative urine drug screen on the same day. The time gap between PET and MRI studies was 1 to 96 days, median 6 days.

The spatial agreement of the manual and FreeSurfer ROIs estimated by the Jaccard coefficient varied between 32 and 69% (Table ). The highest values were obtained for the caudate (59%), putamen (57%), and insula (69%), indicating that the ROIs overlapped to a major extent. The percentage of the manual ROI covered by the FreeSurfer ROI varied between 51.3 and 92.5%. The ROI volumes were calculated by the number of voxels multiplied by the voxel volume for each paired ROI (both hemispheres) extracted from the ROI files. Worth noting is that the positioning of the head in the gantry differed slightly between the two PET measurements, and the subsequent two sets of re-sliced ROIs in PET space differed in volumes by 1.7 ± 3.0% (mean ± SD). Each of the FreeSurfer ROIs and the modified FSL cerebellum ROI were significantly larger compared to the corresponding manual ROIs (Figs.

And, Table ). The 95% CI for the differences was relatively narrow indicating a high precision for both methods (Fig. a, Table ). The CoV (%) for the manual ROI volume was generally higher than for the automatically generated ROIs. The values were obtained from four brain regions in 15 subjects, each value represents the mean of the eight values for that measurement or part of the analysis.

Interquartile range = 25th to 75th percent CoV coefficient of variation, CI confidence interval aThe confidence interval does not contain zero As can be seen from the TACs, there was a rapid initial increase of brain radioactivity that peaked after 5–10 min followed by a rapid decline (Fig. ). The AUC of the TACs for the realigned PET images was 2–18% higher for the manually generated target ROIs when compared to that of the FreeSurfer ( p. Time-activity curves from a healthy volunteer after i.v. Injection of 11C in the morning (left) and afternoon (right).

The curves were derived from realigned dynamic PET images using manually and automatically generated ROIs, respectively. PUT = putamen, DLPFC = dorsolateral prefrontal cortex, CBL = cerebellum. SUV = standardized uptake value = (C ROI/inj dose) × body weight The eight sets of target TACs and two sets of cerebellum TACs amounted to 16 BP ND values for each ROI (Additional file: Table S1). The statistical computations of the regional BP ND values for the three parts of the analysis are listed in Table. The 95% CI for most of the differences was relatively narrow and did not contain zero indicating statistical significance for the difference between methods (Fig. b). The manually generated BP ND values were 3–21% higher with a generally low CoV and interquartile range compared to that of the automatically generated BP ND values. After realignment, the BP ND values became higher for the caudate and DLPFC (Table ).

On the contrary, the BP ND values for putamen and insula did not increase after realignment. The use of the modified FSL cerebellum ROI increased BP ND by 3 to 7% in all regions as compared to that of the manual CBL. The absolute variability values (AV) for each of the four regional BP ND values were calculated for the total of eight pairs of regional BP ND values from the three parts of the analysis (2 × 2 × 2 = 8) (Additional file: Table S1). The AV values among the methods varied between 3.9–11.9% for the caudate and putamen and 8.0–17.9% for the DLPFC and insula. The 95% CI of the mean difference in AV was wide and included zero in most cases indicating no significant differences (Table ).

However, following realignment, the 95% CI of the mean difference in AV did not overlap with zero for caudate, putamen, and DLPFC, suggesting a small but significant improvement. The use of automated cerebellum did not improve the AV values in any region compared to that of the manual.

The ICCs were generally higher using FreeSurfer, but all comparisons fell within the limits of the 95% confidence intervals, meaning that these differences are not significant. Discussion The main objective of the present study was to examine the impact of specific methodological steps used in quantitative image analysis of 11C binding to D1-DR on variability and reliability. The methods were chosen to allow for comparison to historical conditions so that the results would be applicable for interpreting the existing PET literature. For that purpose, data were acquired using the PET system ECAT EXACT HR, which has a resolution comparable to the currently widely used whole-body PET systems. In relation to the major aims of the study, the results show that automated ROIs generally produce lower BP ND values than manual ROIs, whereas absolute variability was similar.

Moreover, the use of realignment improved the absolute variability, and an automated cerebellum ROI yielded slightly increased BP ND values. The volumes were significantly larger for the automated ROIs, with lower interindividual variability except for the cerebellum, which had larger variability for the automated ROI (Table ). The differences in volumes between the methods were largest for the caudate and putamen. Similar results of smaller manual compared to FreeSurfer striatal ROIs, but with greater interindividual variability, have recently been reported when analyzing MRI data for the caudate and putamen ,. Other studies have demonstrated that FS can produce measurements that are comparable to those derived from manual tracing of ROIs ,. However, manual editing of FS ROIs may still be required in order to improve validity. The intersection of the manual and FreeSurfer ROIs was smaller than the manual ROI itself, which indicates that the ROIs did not overlap completely or in a symmetrical fashion (Table ).

Hence, to shrink or erode the FreeSurfer ROI, as shown for partial volume correction , to the same volume as the manual would still result in a placement mismatch. FreeSurfer has become the standard for obtaining cortical metrics with demonstrated high validity and reliability.

For the subcortical regions, manual segmentation still remains the gold standard due to a better specificity of anatomic boundaries compared to FreeSurfer which yields larger caudate and putamen and to FSL-FIRST generating larger caudate ROIs ,. However, manual subcortical ROI volumes do also vary between different raters. Hence, the differences in the size and placement of the ROIs in the present study may be due to both systematic differences in anatomical designation and in software-based segmentation. The higher BP ND for the manual ROIs is consistent with their smaller volumes (Table ).

All regions examined have a higher BP when compared with surrounding tissue or the subarachnoidal space. More spill-out than spill-in of the measured radioactivity will yield a recovery coefficient. Conclusions In summary, the results show that the repeatability of BP ND was similar between the manual and FS ROIs, while it improved after realignment in all regions. The results suggest that the choice of the ROI method is more dependent on questions of validity, such as anatomical precision, which may be particularly important in patients with gross morphological changes or very localized pathology. The importance of the correction of motion artifacts by realignment to obtain higher and more reliable BP ND values may be particularly important in patient populations such as schizophrenia where motion artifacts may be more prevalent.

Ethics approval and consent to participate The study was conducted in compliance with the ethical principles originating in the Declaration of Helsinki and the Good Clinical Practice (GCP) Guidelines of the International Conference on Harmonization (ICH). The study was approved by the Regional Ethical Review Board in Stockholm, Sweden (registration number 2013/1703-31/1) and the Radiation Safety Committee at the Karolinska University Hospital in Solna, Stockholm, Sweden (registration number K1865-2013). Written informed consent to participate was obtained from all individual participants.