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53 Cerebral Blood Flow | Trigonocephaly Arterial Spin Labeling Data Analysis The arterial spin labeling technique uses water molecules in the blood to measure perfusion. This imaging technique magnetically labels arterial blood water when it flows through the neck, and after a postlabeling delay during which the blood is allowed to flow to distal brain tissue an image of the brain is able to depict this change in magnetization in the blood. Therefore, an arterial spin labeling image is weighted for the cerebral blood flow (i.e., the volume of blood that perfuses 100 g of tissue per minute). Data processing and evaluation were performed with the ExploreASL pipeline and included the basic processing of arterial spin labeling and M0 calibration images as described in the ExploreASL review article.17 T1-weighted images were excluded from the complete analysis due to insufficient differentiation between white and gray matter. This is common for the age group we studied because of incomplete myelination and precluded successful segmentation and spatial normalization in some of the subjects. To be able to use the structural atlas of brain regions for the evaluation, the arterial spin labeling images were directly aligned with the Montreal Neurological Institute template. In this age group, the brain is of considerably different shape and size than the adult brain. Therefore, a dedicated template, the University of North Carolina 0-1-2 infant atlases for 1-year-olds, was used to replace the adult template.18 The control images were not of high enough contrast to allow alignment with the mean Montreal Neurological Institute T1-weightedimage; therefore, registration of the individual cerebral blood flow to a standard-space pseudo cerebral blood flow, based on the gray matter and white matter maps of the template, was performed.19 Because of the relatively high amount of skull deformations in the patient group, rigid or affine registration was not considered sufficient for the alignment of the individual data to the template. Therefore, an affine registration, followed by nonlinear deformations using a linear combination of three-dimensional discrete cosine transform basis functions, was used.20 The cerebral blood flow was quantified according to the consensus article by Alsop et al.11 Regional cerebral blood flow was evaluated in both hemispheres taken together in the superior, middle, and inferior gyri of the frontal lobe. The anatomical regions of interest were taken from Hammer’s atlas in the Montreal Neurological Institute space and transformed to the subject’s native space using the previously obtained transformation for the spatial normalization.21 Mean cerebral blood flow values and the frontal lobe, occipital lobe, parietal lobe, temporal lobe, insula, cerebellum, caudate, putamen, and thalamus brain regions were investigated. Statistical Analyses Statistical analyses were performed using R version 4.0.3 (R Foundation for Statistical Computing, Vienna, Austria).22 The descriptive statistics of the patient demographics are presented as mean and SD or as median and interquartile range, depending on if the data are normally distributed or not. Categorical data are presented as counts. 3

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