Functional magnetic resonance imaging (fMRI) of the human spinal cord (SC) is a unique non-invasive method for characterizing neurovascular responses to stimuli. Group-analysis of SC fMRI data involves co-registration of subject-level data to standard space, which requires manual masking of the cord and may result in bias of group-level SC fMRI results. To test this, we examined variability in SC masks drawn in fMRI data from 21 healthy participants from a completed study mapping responses to sensory stimuli of the C7 dermatome. Masks were drawn on temporal mean functional image by eight raters with varying levels of neuroimaging experience, and the rater from the original study acted as a reference. Spatial agreement between rater and reference masks was measured using the Dice Similarity Coefficient, and the influence of rater and dataset was examined using ANOVA. Each rater's masks were used to register functional data to the PAM50 template. Gray matter-white matter signal contrast of registered functional data was used to evaluate the spatial normalization accuracy across raters. Subject- and group-level analyses of activation during left- and right-sided sensory stimuli were performed for each rater's co-registered data. Agreement with the reference SC mask was associated with both rater (F_{(7, 140)} = 32.12, P < 2 × 10^-16, η² = 0.29) and dataset (F_{(20, 140)} = 20.58, P < 2 × 10^-16, η² = 0.53). Dataset variations may reflect image quality metrics: the ratio between the signal intensity of spinal cord voxels and surrounding cerebrospinal fluid was correlated with DSC results (p < 0.001). As predicted, variability in the manually-drawn masks influenced spatial normalization, and GM:WM contrast in the registered data showed significant effects of rater and dataset (rater: F_{(8, 160)} = 23.57, P < 2 × 10^-16, η² = 0.24; dataset: F_{(20, 160)} = 22.00, P < 2 × 10^-16, η² = 0.56). Registration differences propagated into subject-level activation maps which showed rater-dependent agreement with the reference. Although group-level activation maps differed between raters, no systematic bias was identified. Increasing consistency in manual contouring of spinal cord fMRI data improved co-registration and inter-rater agreement in activation mapping, however our results suggest that improvements in image acquisition and post-processing are also critical to address.

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