Follicular disorganization of the spleen in sickle cell disease identified by a convolutional neural network model

Abstract The spleen is the largest immune organ and is among the first to be functionally impaired in sickle cell disease (SCD). Histopathologic studies have consistently documented marginal zone depletion in the lymphoid follicles of SCD spleens. Our group and others have reported alterations in B, T, and dendritic cell populations in the peripheral blood of persons with SCD, paralleling changes observed in splenic tissue. We aim to directly compare immune cell phenotypes in peripheral blood with spatial patterns in spleen tissue from patients with SCD. Here, we describe our novel analysis of human spleen tissue. Fresh spleen tissue was collected from 12 patients undergoing total splenectomy (10 SCD, 2 hereditary spherocytosis [HS] controls). Formalin-fixed, paraffin-embedded sections were stained with hematoxylin & eosin (H&E) and immunohistochemical markers: IgD (unswitched memory B cells), CD21 (naïve follicular B cells and dendritic cells), CD3 (T cells), and Pax5 (B cell nuclei). Whole-slide images were acquired using a high-resolution scanner and analyzed using a convolutional neural network (cNN) model adapted from Cellpose (Stringer et al., 2021) to identify and segment follicles from the H&E images. Pathologist-in-the-loop training of the model improved follicle identification precision from 75% to 90%. Follicle metrics were derived, and distances from follicle centers to immunostained cells were calculated. Group comparisons were performed using one-way ANOVA with Tukey's post hoc test. Across all samples, a median of 33 follicles per subject were analyzed (IQR: 8–57). Compared with HS controls, SCD spleens showed similar mean follicle count (33.0 vs. 30.6, n.s.) and follicle density (1.48 vs. 1.76 follicles/1M pixels, n.s.). For all stains, mean intensity per follicle and the percent of positive cells were significantly higher (27-57% and 86-145%, p < 0.001) in subjects with SCD compared with controls. Spatial intensity profiles of immunohistochemical stains in controls displayed the expected pattern of highest signal at the follicle center with progressive decline outward, whereas SCD samples demonstrated a shifted distribution of staining, indicating follicular disorganization of CD3+ cells and potential loss of IgD+ and Pax5+ cells in adjacent tissue. These findings reveal structural disorganization and altered immune cell localization within SCD spleens beyond what is seen by H&E alone, consistent with functional impairment. While interpretation is limited by the small control group, ongoing recruitment and integration with peripheral blood phenotyping aim to elucidate mechanisms driving altered B cell differentiation and immune dysregulation in SCD.