1. THE UTILITY OF AZAN TRICHROME STAINING IN AMELOBLASTOMA
Akinyele Adisa1, Samuel Udeabor2, Alicia Kubesch3,4, Mike Barbeck3,4, Shahram Ghanaati3,4
1Department of Oral Pathology, College of Medicine, University of Ibadan, Nigeria
2Department of Oral and Maxillofacial Surgery, College of Health Sciences, University of Port Harcourt, Nigeria
3 Departments for Oral, Cranio-Maxillofacial and Facial Plastic Surgery,
Medical Center of the Goethe University Frankfurt, Frankfurt am Main, Germany
4 REPAIR-Lab, Institute of Pathology, University Medical Center, Johannes Gutenberg University, Mainz, Germany
Introduction
Results
Discussion
It is occasionally difficult to distinguish the stellate reticulum-like region of ameloblastoma from the fibrous connective tissue stroma area. This difficulty is further pronounced in the plexiform variant of ameloblastoma that has intricate interlacing islands, strands and cords of tumor and connective tissue, such that in some cases it is virtually impossible to distinguish the fibrous connective tissue from the stellate reticulum-like region. It has been shown that the estimated monthly growth rate of desmoplastic ameloblastoma, which has abundant dense fibrous connective tissue stroma, is significantly lower than other conventional variants of ameloblastoma1. Hence it can be projected that the less the fibrous connective tissue stroma, the faster the rate of monthly growth of ameloblastoma. Compared to other conventional variants, the fibrous connective tissue stoma of plexiform ameloblastoma is very sparse indeed. It may thus be useful to be able to estimate the area covered by connective tissue in this variant as a possible predictor of growth rate.
Trichrome staining is a method in which three anionic dyes are used to mark a varied number of structures histologically2. It is commonly used to distinguish cells from extracellular components and stains muscle fibers red, cartilage and bone matrix blue2.
Our study tested the utility of Azan trichrome stain in marking tumor regions and the peri-tumor environment of ameloblastoma, especially the distinguishing of the stellate reticulum-like region from the fibrous connective tissue stroma area.
Eighteen cases of ameloblastoma comprising 7 plexiform, 3 follicular, 6 cystic and 2 hemangiomatous variants were selected for inclusion in the study. There was no correlation between histological variant and azan staining coloration/pattern. The tumor areas were stained mostly brown; with the ameloblasts mainly marked as deep brown while the stellate reticulum-like region was light brown (Table1). The structures in the peri-tumor region were marked with different shades of blue (Table1)
In this report we have demonstrated the staining characteristics of Azan in ameloblastoma. We did not find any other such study in the English literature either describing this pattern in ameloblastoma or correlating any deductions with biologic behavior or outcome after management. This was a retrospective study and since all the cases were treated by surgical resection with an apparently normal margin of at least 1.5 cm, follow-up and outcome were not factored into the analysis. The clinical-pathological relevance, or lack thereof, of our description may be clarified by future longitudinal studies.
Conclusion
Azan trichrome staining was mostly able to clearly distinguish between the fibrous connective tissue and the stellate reticulum-like areas
References
Materials and methods
Effiom OA, Odukoya O. Desmoplastic ameloblastoma: analysis of 17 Nigerian cases. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;111(1):27-31.
2. Kiernan JA (2008) Histological and Histochemical Methods. Theory and Practice. 4th
ed. Bloxham, UK: Scion. ISBN
Eighteen FFPE ameloblastoma cases from the Oral Pathology Department of the University College Hospital and University of Ibadan, Nigeria were sectioned and stained with hematoxylin and eosin for re-evaluation and inclusion. At the REPAIR laboratory, Institute of Pathology, School of Medicine, University of Mainz Germany, sections were prepared for Azan trichrome staining using the manufacturer’s specification. The sections were de-paraffinized using xylene and hydrated with alcohol. Thereafter, they were rinsed in distilled water for 3 minutes and then placed in nuclear fast red solution for 30 minutes, rinsed 2 times under running water and once in distilled water. The sections were differentiated by placement in 0.1% aniline alcohol solution for 3 minutes. Differentiation was stopped by placement in 1 change of acetic alcohol solution. They were placed in 5% aqueous phosphotungstic acid for 10 minutes and rinsed in distilled water. After which they were then placed for 5 minutes in 1part Aniline Blue-Orange mixture to 3 parts distilled water and briefly rinsed again in distilled water. Absolute alcohol (100% Isopropanol) was then used to differentiate and dehydrate the sections through 2 changes for 2 minutes each. Xylene was finally used 3 times for 3 minutes each to clear the slides and cover slips placed.