A Double Histochemical/Immunohistochemical Staining for the Identification of Canine Mast Cells in Light Microscopy.

Francesca Gobbo,Giuseppe Sarli,Margherita De Silva,Roberto Chiocchetti,Giorgia Galiazzo,Maria Morini

doi:10.3390/vetsci8100229

Abstract

Immunohistochemistry (IHC) is a widely used technique in diagnostic pathology, but the simultaneous analysis of more than one antibody at a time with different chromogens is rather complex, time-consuming, and quite expensive. In order to facilitate the identification of mast cells (MCs) during immunohistochemical analysis of membrane and/or nuclear markers, we propose a new staining method that includes the association of IHC and toluidine blue as a counterstain. To achieve this goal, we tested c-kit, Ki67, and cannabinoid receptor 2 on several cases of cutaneous canine mast cell tumors (MCTs), cutaneous mastocytosis, and atopic dermatitis. The results obtained show how this double staining technique, although limited to non-cytoplasmic markers and of little use in poorly differentiated MCTs in which MC metachromasia is hard to see, can be used during the evaluation of nuclear and/or membranous immunohistochemical markers in all canine cutaneous disorders, especially if characterized by the presence of a low number of MCs. It can help to evaluate those MCTs in which neoplastic MCs must be clearly distinguished from inflammatory cells that can infiltrate the tumor itself, in facilitating the calculation of the Ki67 index. Moreover, it can be used to study the expression of new markers in both animal and human tissues containing MCs and in MC disorders.

Highlights

In diagnostic pathology, immunohistochemistry (IHC) is a technique used for the identification of cellular markers in tissue samples that could influence disease diagnosis and, patient management [1]
C-kit immunohistochemical analysis of canine mast cell tumors (MCTs) showed the three different patterns, and we found that the metachromatic reaction was clearly detectable in all cases of membranous and Golgi-like c-kit expression (Figure 1B,C and Figure 2C); by contrast, the metachromatic reaction can be hidden from the immunohistochemical reaction of c-kit in the cytoplasm (Figure 1A)—in our cases observed in poorly differentiated MCTs (Patnaik grade III, Kiupel high grade) (Figure 3F)
A4 of 9 comparison between the Harris hematoxylin (HH) and FTB counterstains revealed the clear detection of Mast cells (MCs) within the tissue if counterstaining was performed by toluidine blue (TB) (Figure 3G–L)

Summary

Introduction

Immunohistochemistry (IHC) is a technique used for the identification of cellular markers in tissue samples that could influence disease diagnosis and, patient management [1]. A hallmark of their identification is the metachromatic red/violet color of the cytoplasmic granules [4] that follows some staining, such as toluidine blue, May-Grünwald Giemsa, and Leishman. This staining allows the detection of MCs in tissues and is strongly recommended as a routine stain for this purpose [4]. Tryptase and chymase are both used in the detection of normal [8,9] or neoplastic [10,11,12] conditions, and chymase activity is closely correlated with inflammatory diseases progression [8]

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