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Cancer Research

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

Published: May 1st, 2019



1Department of Medical Oncology, Lung Cancer and Gastrointestinal Unit, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University, 2Hunan ClinicaI Research Center in Gynecologic Cancer, Hunan Cancer Hospital and The Affiliated Cancer Hospital of Xiangya School of Medicine, Central South University
* These authors contributed equally

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.

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.

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

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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 h.......

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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. <.......

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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.......

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The authors thank the support in part from the National Natural Science Foundation (NO.81401902 and NO.81501992) and the Hunan Natural Science Foundation (NO.2017SK2134, NO.2018JJ2238). The manuscript is written by Guang Lei. The whole experiment is performed by Guang Lei and Haiyan Yang. The authors thank the assistance of pathologists and of fellows who follow up patients' data.


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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

  1. Jiang, C. G., et al. Clinicopathologic characteristics and prognosis of gastric cancer invading the subserosa. Journal of Surgical Oncology. 102 (7), 737-741 (2010).
  2. Amin, M. B., et al. . AJCC Cancer Staging Manual. 8th ed. , (2017).
  3. Kojima, M., et al. Elastic laminal invasion in colon cancer: diagnostic utility and histological features. Frontiers in Oncology. 2, 179 (2012).
  4. Ludeman, L., Shepherd, N. A. Serosal involvement7 in gastrointestinal cancer: its assessment and significance. Histopathology. 47 (2), 123-131 (2005).
  5. Shepherd, N. A., Baxter, K. J., Love, S. B. The prognostic importance of peritoneal involvement in colonic cancer: a prospective evaluation. Gastroenterology. 112 (4), 1096-1102 (1997).
  6. Liang, W. Y. Retrospective evaluation of elastic stain in the assessment of serosal invasion of pT3N0 colorectal cancers. American Journal of Surgical Pathology. 37 (10), 1565-1570 (2013).
  7. Carter, D., True, L., Otis, C. N., Mills, S. E. Serosal membranes. Histology for Pathologists. 3rd ed. , 547-562 (2007).
  8. Knudsen, P. J. The peritoneal elastic lamina. Journal of Anatomy. 177, 41-46 (1991).
  9. Shepherd, N. A., Baxter, K. J., Love, S. B. Influence of local peritoneal involvement on pelvic recurrence and prognosis in rectal cancer. Journal of Clinical Pathology. 48 (9), 849-855 (1995).
  10. Uitto, J., Li, Q., Urban, Z. The complexity of elastic fibre biogenesis in the skin--a perspective to the clinical heterogeneity of cutis laxa. Experimental Dermatology. 22 (2), 88-92 (2013).
  11. Kazlouskaya, V., et al. The utility of elastic Verhoeff-Van Gieson staining in dermatopathology. Journal of Cutaneous Pathology. 40 (2), 211-225 (2013).
  12. Lei, G., et al. Elastic staining-a rejuvenated method to reassess prognosis and serosal invasion in patients with pT3N0M0 gastric cancer. Human Pathology. 65, 79-84 (2017).
  13. Hernández-Morera, P., Travieso-González, C. M., Castaño-González, I., Mompeó-Corredera, B., Ortega-Santana, F. Segmentation of elastic fibres in images of vessel wall sections stained with Weigert's resorcin-fuchsin. Computer Methods and Programs in Biomedicine. 142, 43-54 (2017).
  14. Kojima, M., Nakajima, K., Ishii, G., Saito, N., Ochiai, A. Peritoneal elastic laminal invasion of colorectal cancer: the diagnostic utility and clinicopathologic relationship. American Journal of Surgical Pathology. 34 (9), 1351-1360 (2010).
  15. Nakanishi, Y., et al. Reappraisal of Serosal Invasion in Patients With T3 Colorectal Cancer by Elastic Stain. ARCHIVES OF PATHOLOGY & LABORATORY MEDICINE. 140 (1), 81-85 (2016).
  16. Grin, A., et al. Peritoneal elastic lamina invasion: limitations in its use as a prognostic marker in stage II colorectal cancer. Human Pathology. 44 (12), 2696-2705 (2013).
  17. Stewart, C. J., Brennan, B. A., Crook, M. L., Russell, P. Value of elastin staining in the assessment of peritoneal implants associated with ovarian serous borderline tumours. Histopathology. 51 (3), 313-321 (2007).
  18. Kojima, M., et al. Pathological diagnostic criterion of blood and lymphatic vessel invasion in colorectal cancer: a framework for developing an objective pathological diagnostic system using the Delphi method, from the Pathology Working Group of the Japanese Society for Cancer of the Colon and Rectum. Journal of Clinical Pathology. 66 (7), 551-558 (2013).
  19. Elston, C. A., Kazlouskaya, V., Elston, D. M. Elastic staining versus. fluorescent and polarized microscopy in the diagnosis of alopecia. Journal of the American Academy of Dermatology. 69 (2), 288-293 (2013).
  20. Vos, A., et al. Predominance of Nonatherosclerotic Internal Elastic Lamina Calcification in the Intracranial Internal Carotid Artery. Stroke. 47 (1), 221-223 (2016).

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