Elastic Staining on Paraffin-embedded Slides of pT3N0M0 Gastric Cancer Tissue

Summary

Here, we present a protocol of elastic staining to identify elastic fibers in pT3N0M0 gastric cancer tissues on formalin-fixed, paraffin-embedded sections. Subsequently, we describe a method to determine whether tumor cells invade beyond the elastic lamina.

Abstract

The elastic lamina, which is usually located in the sub-mesothelial layer, adjacent to the peritoneum mesothelial cells, is amphiphilic on hematoxylin and eosin (H&E) staining but can be visualized through elastic staining. One of the important benefits of elastic staining is that it makes it easy to determine whether tumor cells have invaded beyond the elastic lamina. This helps in examining the extent of peritoneal surface invasion, thereby distinguishing the pT3 and pT4 stages in gastrointestinal cancer. In this study, we present a protocol to identify elastic fibers in the formalin-fixed, paraffin-embedded pT3N0M0 gastric cancer tissue sections. We prepare 5-µm paraffin sections fixed on slides, and then all the slides are deparaffinated and rehydrated during the staining procedure. Subsequently, all the sections are oxidized by potassium permanganate, bleached by oxalic acid, stained by elastic staining, and counterstained by Van Gieson's. Finally, we examine the effect of staining; elastic lamina is stained blue-black and collagen fibers are stained red, while the cancer cells are stained in varying shades of yellow. We also describe in detail the methods used for determining the positional relationship between cancer cells and elastic lamina. This method is simple, low-cost, and widely applicable in the identification of peritoneal surface invasion in gastrointestinal cancer because of its outstanding selectivity for elastic fibers and authentic results.

Introduction

As per the Tumor, Node, and Metastasis (TNM) cancer staging system, the prognosis of gastric cancer at pathologic stage pT4 is significantly worse than that at pT3 (8th UICC/AJCC)¹. With gastric cancer, classification of the primary tumor (pT) usually depends on the depth of tumor invasion². Pathologic stage pT3 gastric cancer is defined as cancer invading through the muscularis propria into subserosal tissues, whereas pT4 gastric cancer is defined as cancer penetrating to the surface of the visceral peritoneum. The presence of the peritoneal surface invasiveness is the key to distinguish the two stages

Protocol

Between 1994 and 2005, patient samples were selected by two experienced gastrointestinal pathologists in the Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, from patients who suffered gastrectomy of stomach neoplasms, with a postoperative pathological diagnosis of gastric cancer at the pT3N0M0 stage (according to the seventh TNM staging). This study was approved by the ethics review committee.

1. Sectioning

1. Place the paraffin block into a fixed holder of the sledge microtome, which can move backward and forward across the blade. Adjust the optimal angle between the block and the microtome knif....

Results

A successful elastic staining clearly reveals elastic lamina and cancer cells in pT3 gastric cancer. Figure 1 - low-power field, microscopically checked after elastin solution staining, prior to Van Gieson's counterstain as per the protocol mentioned - shows the elastic lamina is close to the serosal mesothelial cells and is stained blue-black in the form of filaments, and there is still a certain distance between the tumor cells and the elastic lamina. <.......

Discussion

This method proposed here provides an accurate approach for identifying the subserosal elastic lamina. The method can be used to evaluate whether tumor cells have invaded the elastic lamina in pT3N0M0 gastric cancer¹². Elastic lamina is reddish in H&E-stained sections, which could not be easily distinguished from collagen fibers, and other existing auxiliary diagnosis methods, such as cytology and immunohistochemistry, also exerted limited diagnostic value. The finding of tumor cells in the pe.......

Materials

Name	Company	Catalog Number	Comments
Elastin-Van Gieson staining kit	Baso	BA4083B	Used for staining of elastic fibers, collagen fibers and myofibers
Permount TM Mounting Medium	MXB Biotechnologies	DAB-0033	Used for mounting
Ethanol	LMAI Bio	LM64-17-5	100%
Xylene	LMAI Bio	LX820585