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NOVEL HISTOPATHOLOGICAL PATTERNS IN CORTICAL TUBERS IN TSC
size of 50MB. Files without visible tissue were discarded. Qualitative and semi-quantita- tive image analysis was performed with the Image-Pro Premiere software package v. 9.1 (Media Cybernatics, USA). The mean and standard deviation was calculated for every parameter and used for statistical analysis.
Calcifications
The presence or absence of calcifications was scored based on the H&E staining. This was converted into a nominal variable of 0 and 1.
Giant cells
The numbers of giant cells per mm2 were calculated from 10 representative fields (each representing 1.081mm2) of an anti-vimentin stained section.
Microglial activation, gliosis, mTORC1 activation and myelin content
Quantification of all available tissue was taken into account. In a first step the total tissue surface area was calculated for each case utilizing the smart segmentation tool pro- vided by the ImagePro software package. Second, the DAB positive area (anti-Cr3/43, anti-GFAP, anti-pS6-Ser235/236 and anti-MBP) was separated from background using an adjusted protocol for segmentation (S1 Fig). All stacks of images were submitted to a batch processing algorithm that was kept consistent throughout the whole analysis. In a final step, the overall percentage of positivity was assessed for each case and used for statistical analysis. In the myelin stainings we further addressed the optical density (OD) of the MBP positive area. The integrated OD of MBP was also used. For simplicity and due to the linear correlation (p < 0.001), the product of intensity x frequency (percent- age x OD) was calculated. This product is further referred to as overall myelin content (OMC).
Cellular densities
For the calculation of cellular densities the available images were split into RGB chan- nels. To establish the positive count a brightness threshold in the blue channel was determined for each staining (NeuN: 120; CD3, SMI32 and Olig2: 100; CD34: 150) and size ranges were defined to allow more accurate counts (pixel areas: NeuN: 120-2500; CD3: 20-155; Olig2: 20-155 in surgical tissue and 5-120 in autopsy material; CD34: 100-unlimited; SMI32: 750-5000). These parameters were kept the same throughout the analysis and were also implemented in the batch processing algorithm. All cellular densities are rep- resented as total number/mm2.
Clinical data
All available MRIs were retrospectively reviewed and the resected tubers were scored according to the classification system proposed by Gallagher et al. 11. The presence of “focal cortical dysplasia (FCD)-like” features (thickened cortex and blurred grey-white matter border in areas surrounding one or several tubers and presence of transmantle sign) were assessed as well as the presence of calcifications. In addition, the following clinical data was obtained: TSC1/TSC2 mutation status, gender, age at seizure onset, local- ization of the resected area, age at epilepsy onset, mean seizure frequency (daily-week-
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