Calretinin Staining Pattern is Variable in Hirschsprung Disease Occurring in Patients with Down Syndrome

Abstract Introduction/Objective Hirschsprung disease (HD), a congenital absence of enteric ganglion cells, affects 2-10% of patients with Down syndrome (DS). Submucosal nerve hypertrophy (SNH) and abnormal calretinin immunohistochemistry (IHC) support a diagnosis of HD, but the concordance of these findings can be variable in DS. Recognizing differences in HD occuring in DS can provide insight into the disease and help to improve outcomes and reduce delays in diagnosis for these patients. We sought to describe the histologic and calretinin staining patterns in HD occurring in patients with DS. Methods/Case Report From 2017-2023, 22 rectal biopsies from 20 patients with DS were retrospectively collected and reviewed. Subsequent pull-through (PT) resections were evaluated from 8 patients. Biopsies were evaluated for ganglion cells and SNH. Calretinin IHC was qualitatively evaluated for intensity (strong, weak) and density (many, partial, rare). Quantitative analysis used a calculation of the percent area above threshold (%AAT) function of the NIH ImageJ software version 2.1.0. Representative areas of the PT resections were also evaluated by ImageJ %AAT analysis. Results (if a Case Study enter NA) HD biopsies (12/22, 54.5%) were from male patients (age 3 days-6 weeks) and showed SNH in 11/12 (91.7%) with a mean nerve diameter of 57.3 microns (range 25-89 microns). Ganglionated biopsies (10/22, 45.5%) were collected from patients 3 days to 12 years old (M:F ratio 1:1.5) and showed SNH in 6/10 (60%) with a mean nerve diameter of 44.4 microns (range 10-70 microns). Strong normal calretinin staining was only seen in ganglionated biopsies (7/10 non-HD versus 0/12 HD). Negative calretinin staining was only seen in HD (0/10 non-HD vs 7/12 HD). Abnormal staining was seen in both non-HD (3/10, 30%) and HD (5/12, 41.7%) as partial strong positivity and rare weak positivity, respectively. ImageJ %AAT quantitative analysis revealed an average of 1.71 in biopsies with strong calretinin expression, 0.130 with weak staining, and 0.033 with negative staining. Evaluation of 8 PT resections (mean length of aganglionosis 7.1 cm) showed a mean nerve diameter in the aganglionic segment of 88.3 microns, 49.6 microns in the TZ, and 32.7 microns in the ganglionated segment. ImageJ %AAT quantitative analysis of calretinin revealed a mean of 0.044 in the aganglionic area, 0.66 in the TZ, and 1.56 in the ganglionated segment. Conclusion Recognition of the variable calretinin patterns seen in rectal biopsies from patients with DS can assist in accurate and timely diagnosis of HD.