Calretinin immunohistochemical staining in Hirschsprung’s disease: An institutional experience

OBJECTIVE: This study aims to evaluate the results obtained by calretinin staining on tissue samples for diagnosing Hirschsprung’s disease (HD) in a single institution, by single expert.
METHODS: A retrospective evaluation was done for calretinin immunostaining in HD patients for a period of 3 years. Calretinin staining was evaluated in nerve fibers. Calretinin immunohistochemistry was considered positive if any staining was seen in nerve fibers and/or ganglion cells in the lamina propria, muscularis mucosa or submucosa. According to staining intensity, staining was classified as strong, weak or negative. The pathological diagnosis was based on presence or absence of ganglion cells (G0/G1) and nerve hypertrophy (N0/N1). Samples were classified according to the depth (presence of submucosa or intermuscular area), the type (biopsy or resection specimen) and staining intensity of calretinin (strong, weak, or negative staining).
RESULTS: A total of 96 tissue samples from 56 patients were studied. Tissues were from colon (43.8%), rectum (43.8%), stoma (6.2%), ileum (3.1%) and appendix (3.1%). The pathological diagnosis was G0N0 in 14.6%, G1N0 in 54.2%, G0N1 in 25% and G1N1 in 6.2% of cases. Our materials consisted of 92 tissue biopsies and four resection specimens. Intermuscular layer was present in 87.5% of materials and 12.5% of biopsies contained submucosa. Calretinin staining was negative (C0) in 37.5% of cases, strong positive (C1) in 47.9%, and weak positive (C2) in 14.6%. When the C0 category was taken as the reference, the status of calretinin staining as C2 (weak positive) in cases with pathological diagnosis of G1N0 was found to be 37.575 times that of cases with G0N0 (OR [95% CI]: 37.575 [2.928, 482.176], p=0.006) and the status of calretinin staining as C1 (strong positive) in cases with pathologic diagnosis of G1N0 was found to be 131.401 times that of G0N0 (OR [95% CI]: 131.401 [9.263, 1864.082), p<0.001).
CONCLUSION: Calretinin staining is positive whenever ganglion cells are present independent from presence of nerve hypertrophy, the depth and the site of the biopsy or staining intensity. It is negative in all aganglionic samples. Calretinin staining is a reliable ancillary test in HD diagnosis.

Hirschprung Hastalýðý’nda Ýmmunhistokimyasal Kalretinin Boyanmasý: Bir Merkezin Deneyimi

Amaç: Konusunda uzman tek patolog tarafýndan tek bir kurumda Hirschsprung Hastalýðý (HH) tanýsý koymak için alýnmýþ doku örneklerinde Kalretinin boyanmasý ile elde edilen sonuçlarý deðerlendirmek.
Materyal Metod: 3 yýllýk periodda (2013-2016) Hirschprung Hastalýðý tanýsý almýþ dokulara retrospektif olarak Kalretinin boyamasý uygulanarak sonuçlarý deðerlendirildi. H&E boyalý kesitlerin incelenmesinden sonra seçilen paraffin bloklara Kalretinin immunohistokimyasal boyamasý uygulandý. Kalretinin boyanmasý lamina propria, submukoza ve intermusküler alandaki sinir liflerinde deðerlendirildi. Kalretinin boyanmasý sinir liflerinde herhangi bir lineer ya da granüler boyanma varlýðýnda ya da ganglion hücrelerinde sitoplazmik boyanmna varlýðýnda pozitif olarak kabul edildi. Boyanma þiddetine göre negatif, zayýf ya da kuvvetli olarak sýnýflandý. Patolojik tanýda ganglion hücre varlýðý/ yokluðu (G1/G0) ve sinir hipertrofisinin varlýðý/yokluðu ( S1/S0) esas alýndý. Örnekler materyalin derinliði ( submukozanýn ve intermusküler alanýn varlýðý/ yokluðu) ve türü (biyopsi /rezeksiyon) ve Kalretinin boyanma durumuna ( Güçlü, zayýf, negatif) göre sýnýflandýrýldý.
Bulgular: 56 hastadan toplamda 96 biyopsi örneði üzerinde çalýþýldý. Biyopsilerin % 43.8’i kolon, %43.8 ‘i rektum,%6.2 ‘si stoma, %3.1’i ileum ve %3.1 ‘i appendikse aitti. Vakalarýn patolojik tanýlarý þu þekildeydi; %14.6’sý G0S0, %54.2 ‘si G1 S0, %25’i G0S1 ve %6.2’si G1S1. Materyallerin 92’si doku biyopsisi iken 4 tanesi rezeksiyon materyaliydi. Intermüsküler tabaka vakalarýn %87.5’inde mevcut idi; %12.5’i submukoza içermekteydi. Kalretinin boyanmasý vakalarýn %37,5’inde negatif (K0), %47.9’unda kuvvetli pozitif (K1) ve %14.6 ‘sýnda zayýf pozitif olarak izlendi (K2). Anatomik yerleþimin, biyopsi türünün ya da derinliðinin kalretinin boyanmasý üzerine etkisi olmadýðý görüldü (p: 0.931, p: 0.327, p: 0.999). Patolojik taný kalretinin boyanma sonuçlarýyla anlamlý olarak korele olarak izlendi. (F: 5.407, p <0.001). K0 kategorisi referans olarak alýndýðýnda G1S0 tanýlý hastalarda kalretinin zayýf boyanmasý (K2) G0S0 tanýlý hastalarýn 37.575 katýydý. [OR (95% CI): 37.575 (2.928, 482.176), p: 0.006] ve yine bu hastalarda kalretininin güçlü boyanmasý (K1) G0S0 tanýlý hastalarýn 131.401 katýydý. [OR (95% CI): 131.401 (9.263, 1864.082), p <0.001].
Sonuç: Sinir hipertrofisi, biyopsi derinliði ya da biyopsi yerinden baðýmsýz olarak Kalretinin boyanmasý ve boyanmanýn þiddeti ganglion hücrelerinin varlýðýnda pozitif olarak izlenmektedir. Bütün aganglionik segmentlerde negatif olarak izlenmektedir. HH tanýsýnda Kalretinin boyanmasý güvenilir bir yardýmcý testtir. (NCI-2020-0417.R1)