IJD FOCUS: LICHEN PLANUS
|Year : 2015 | Volume
| Issue : 5 | Page : 450-456
|Comparing the efficacy of hematoxylin and eosin, periodic acid schiff and fluorescent periodic acid schiff-acriflavine techniques for demonstration of basement membrane in oral lichen planus: A histochemical study
Ashwini Pujar, Treville Pereira, Avinash Tamgadge, Sudhir Bhalerao, Sandhya Tamgadge
Department of Oral and Maxillofacial Pathology and Microbiology, Dr. D. Y. Patil Dental College and Hospital, Sector 7, Nerul, Navi Mumbai, Maharashtra, India
|Date of Web Publication||4-Sep-2015|
Department of Oral and Maxillofacial Pathology and Microbiology, Dr. D. Y. Patil Dental College and Hospital, Sector 7, Nerul, Navi Mumbai - 400 706, Maharashtra
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Basement membrane (BM) is a thick sheet of extracellular matrix molecules, upon which epithelial cells attach. Various immunohistochemical studies in the past have been carried out but these advanced staining techniques are expensive and not feasible in routine laboratories. Although hematoxylin and eosin (H-E) is very popular among pathologists for looking at biopsies, the method has some limitations. This is where special stains come handy. Aims and Objectives: The aim of the present study was to demonstrate and compare the efficacy of H-E, periodic acid Schiff (PAS) and fluorescent periodic acid-acriflavine staining techniques for the basement membrane and to establish a histochemical stain which could be cost effective, less time consuming, and unambiguous for observation of the basement membrane zone. Materials and Methods: A total number of 40 paraffin-embedded tissue sections of known basement membrane containing tissues including 10 - Normal oral mucosa (NOM) and 30 - oral lichen planus (OLP) were considered in the study. Four-micron-thick sections of each block were cut and stained with H-E stain, PAS and fluorescent periodic acid-acriflavine stain. Sections were evaluated by three oral pathologists independently for continuity, contrast and pattern. Results: Though all the three stains showed favorable features at different levels, acriflavine stain was better than the other stains in demonstrating BM continuity, contrast and also the pattern followed by PAS stain. Acriflavine stain was the better in demonstrating a fibrillar pattern of a BM. Acriflavine stains a BM distinctly and is less time consuming and easy to carry out using readily available dyes as compared to other stains. Conclusion: The continuity and contrast along with the homogenous pattern and the afibrillar pattern of the BM was better demonstrated by acriflavine followed by the PAS stain.
Keywords: Basement membrane, fluorescent microscope, fluorescent periodic acid-acriflavine, oral lichen planus, periodic acid Schiff
|How to cite this article:|
Pujar A, Pereira T, Tamgadge A, Bhalerao S, Tamgadge S. Comparing the efficacy of hematoxylin and eosin, periodic acid schiff and fluorescent periodic acid schiff-acriflavine techniques for demonstration of basement membrane in oral lichen planus: A histochemical study. Indian J Dermatol 2015;60:450-6
|How to cite this URL:|
Pujar A, Pereira T, Tamgadge A, Bhalerao S, Tamgadge S. Comparing the efficacy of hematoxylin and eosin, periodic acid schiff and fluorescent periodic acid schiff-acriflavine techniques for demonstration of basement membrane in oral lichen planus: A histochemical study. Indian J Dermatol [serial online] 2015 [cited 2019 Oct 20];60:450-6. Available from: http://www.e-ijd.org/text.asp?2015/60/5/450/159626
What was known?
Basement membrane is known to play a role in the pathogenesis of inflammatory and autoimmune diseases.
| Introduction|| |
A basement membrane (BM) is a thick sheet of extracellular matrix molecules, upon which epithelial cells attach and form a boundary between epithelium and the connective tissue.  It also provides structural support for epithelial, mesothelial, and endothelial cells and surrounds adipocytes, muscles and Schwann cells.  Various immunohistochemical studies have proved the role of BM components in inflammatory and autoimmune diseases like Lichen planus, pemphigus, pemphigoid, where the BM and basal cells are the primary targets. To understand its exact role in the pathogenesis of inflammatory and autoimmune diseases, various immunohistochemical studies in the past have been carried out but these advanced staining techniques are expensive and not feasible in routine laboratories.
Oral lichen planus (OLP) is an inflammatory autoimmune, mucocutaneous disease, the etiology of which is unknown. It affects the skin, genital mucosa, scalp, nails, and areas in the oral cavity, the clinical diagnosis of which can be confirmed by the classical histopathological findings.  OLP was first described by a British physician Erasmus Wilson in 1869.  Chronic inflammation may lead to malignant transformation; hence, early diagnosis is important as many studies have supported malignant transformation of OLP. , Lichen planus has a varied prevalence based on different geographic regions. OLP affects people of all ethnic groups. Many studies have proved that lichen planus is a disease of middle age  and only 4% of which occurs in children. ,
For nearly 100 years, pathologists have utilized special histochemical stains to assist in tissue-based diagnosis. Although hematoxylin and eosin (H-E) stains are very popular stains among histotechnologists and pathologists for looking at biopsies and surgical and post mortem specimens, this method has some limitations. This is where special stains come in handy: For identifying microorganisms or specific structures or molecules in cells or in tissue sections. The "special stain" terminology is most commonly used in the clinical environment, and simply means any technique other than the H-E method that is used to impart colors to a specimen. Although the presence of the BM can be detected very specifically by immunohistochemical methods on paraffin-embedded tissue sections, the antikeratin-antisera are not economical and are time consuming. 
Hence, this study was designed to demonstrate the efficacy of acriflavine stain in comparison with H-E and periodic acid Schiff (PAS) stain. The present study was done to evaluate the BM changes of lichen planus under light microscope and fluorescent microscope using a histochemical stain which could be cost effective, less time consuming, and unambiguous for the observation of the BM zone.
| Materials and Methods|| |
Since the study was planned to study the BM in tissue sections, H-E-stained sections were reviewed for the presence of BM in tissue sections and the slides were accordingly selected.
The study group included:
- 10 cases of normal oral mucosa (NOM)
- 30 cases of oral lichen planus (OLP)
A total number of 40 paraffin-embedded tissue blocks were retrieved from the archives for the present study. Institutional ethical clearance was obtained prior to beginning of the study.
Three sections of 4-μm-thick paraffin-embedded sections were taken and stained with (1) hematoxylin and eosin,  (2) periodic acid Schiff (McManus, 1946)  and (3) fluorescent periodic acid-acriflavine,  respectively.
The staining procedure for fluorescent periodic acid-acriflavine method
- Solution a: Periodic acid 0.5%
- Periodic acid - 1 g
- Distilled water - 200 ml
- Solution b: Fluorescent Schiff reagent
- Acriflavine - 1 gm
- Distilled water - 200 ml
- Potassium metabisulfite - 2 gm
- Hydrochloric acid, 1N - 20 ml.
Dissolve the dye in water and mix well. Add potassium metabisulfite and hydrochloric acid and mix well. Close the stopper tightly and leave for 48 hours in the dark and then filter it.
- Dewax and hydrate the paraffin sections and place in periodic acid solution for 10 minutes
- Wash well with water, and place in fluorescent Schiff reagent for 20 minutes
- Wash well with tap water and place in acid alcohol for two changes of 5 minutes each
- Wash well with tap water dehydrate, clear and mount in a non-fluorescent resinous medium.
| Results|| |
Basement membrane: Golden yellow.
- The use of fluorescent microscopy to identify the BM in fluorescent periodic acid -acriflavine -stained sections is not only responsible for its specificity, but also increases the method's sensitivity and resolution. This last property is due to the fact that the basement membrane, undetectable in normal microscopy, becomes visible when a source of light is used against a dark background.
All the slides were scanned under 40X for BM analysis. Stained sections were evaluated by three oral pathologists independently and consensus was taken when required.
The slides were analyzed for:
Basement membrane in NOM and OLP and the following parameters were considered:
- Continuous - BM is seen throughout the tissue section
- Fragmented - BM is seen fragmented in focal areas.
- Distinct - BM is clearly demarcated as a bright line from surrounding tissue
- Indistinct - BM is not clearly demarcated as a bright line from surrounding tissue.
- Fibrillar - BM seen as fine delicate fibers
- Afibrillar - BM seen as homogenous structure.
The results were analyzed and tabulated. Frequency and percentage was calculated. For the efficacy of three staining techniques statistical analysis of the data was performed using Statistical Package for Social Sciences (SPSS version 17) and inferences were drawn. A P ͳ was considered to be statistically significant.
| Results|| |
In the present study all the three staining procedures, i.e. H-E, PAS, acriflavine stains showed a clear, distinct BM as pink, magenta pink and yellow to orange, respectively.
All three stains showed similar results for staining of the BM in normal mucosa [Figure 1]; however, when the BM of cases of OLP was evaluated, using the H-E stain, 10 cases showed a continuous BM while 20 cases showed a fragmented BM. When using the PAS stain, 19 cases showed a continuous and 11 showed a fragmented BM. In acriflavine-stained sections 20 cases showed a continuous and10 showed a fragmented BM [Figure 2] and [Figure 3].
|Figure 1: Photomicrograph showing a continuous and distinct basement membrane in normal buccal mucosa under light microscopy using H and E stain (a), PAS stain (b), and fluorescent microscopy acriflavine stain (c). (×40)|
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|Figure 2: Photomicrograph showing a continuous basement membrane in oral lichen planus under light microscopy using H and E stain (a), PAS stain (b), and fluorescent microscopy acriflavine stain (c). (×40)|
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|Figure 3: Photomicrograph showing a fragmented basement membrane in oral lichen planus under light microscopy using H and E stain (a), PAS stain (b), and fluorescent microscopy acriflavine stain (c). (×40)|
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When an evaluation was done as to which stain provides better contrast for visualization of the BM, 6 cases stained with H-E showed a distinct contrast while 24 cases of the same stain showed an indistinct BM. In the PAS-stained sections, 18 cases showed a distinct while 12 showed an indistinct BM. In acriflavine-stained sections, the BM was distinct in 21 cases and indistinct in 9 cases [Figure 4] and [Figure 5].
|Figure 4: Photomicrograph showing a distinct basement membrane in oral lichen planus under light microscopy using H and E stain (a), PAS stain (b), and fluorescent microscopy acriflavine stain (c). (×40)|
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|Figure 5: Photomicrograph showing an indistinct basement membrane in oral lichen planus under light microscopy using H and E stain (a), PAS stain (b), and fluorescent microscopy acriflavine stain (c). (×40)|
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When an evaluation was done for the pattern of the BM, the H-E-stained sections showed 1 case of a fibrillar type and 29 cases of afibrillar type of BM. The PAS-stained slides showed 5 cases of fibrillar and 25 cases of afibrillar type of BM and acriflavine-stained slides showed 10 cases of fibrillar and 20 cases of afibrillar BM [Figure 6] and [Figure 7] [Table 1], Graph 1 [Additional file 1] ].
|Figure 6: Photomicrograph showing fibrillar pattern of the basement membrane in oral lichen planus under light microscopy using H and E stain (a), PAS stain (b), and fluorescent microscopy acriflavine stain (c). (×40)|
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|Figure 7: Photomicrograph showing afibrillar pattern of the basement membrane in oral lichen planus under light microscopy using H and E stain (a), PAS stain (b), and fluorescent microscopy acriflavine stain (c). (×40)|
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|Table 1: Comparison of staining techniques using frequency and percentage |
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When the three stains were compared in OLP, the continuity of BM showed statistical significant result with a Chi - square value of 8.153 and a P value of 0.017, whereas the contrast showed a Chi-square value of 16.6 and a P value of 0 which was significant. In the pattern group the Chi-square value was 9.274 and P value was 0.01 which is significant. Therefore, all three groups were statistically significant. In the continuity group, the P values among all three stains were statistically significant.
In the contrast group, only the acriflavine stain with P values 0.043 was statistically significant whereas H-E, PAS were statistically non-significant. In the pattern group, only acriflavine stain with P values 0.053 was statistically significant whereas H-E, PAS was statistically non-significant [Table 2] and [Table 3] [Graph 2 [Additional file 2] ] and [Graph 3 [Additional file 3] ].
|Table 2: Comparison of staining technique in the study group using the Chi-square test |
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|Table 3: Comparison in between three groups and three stains using kappa test |
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| Discussion|| |
A BM is a simple, flat junction which enables the forces applied at the surface of the epithelium to be dispersed over a greater area of connective tissue.  Epithelial-mesenchymal interactions are necessary for development of the oral epithelium during embryogenesis, but adult oral epithelium is also under the influence of mesenchymal tissue, both in normal and neoplastic conditions. BM helps in the epithelial-mesenchymal communication in two ways: It is a dynamic sieve that can control the passage of molecules between the two tissues and it provides a spatial configuration to which mesenchymal cells can react.
A BM acts as a selective macromolecular filter and influences the growth morphology and cytodifferentiation. It also functions as a barrier for the passage of malignant tumor cells, scaffold for salivary gland morphogenesis and tissue regeneration. It also plays a role in odontogenesis and odontogenic tumor pathosis. 
In the histopathology laboratory, the H-E stain is considered to be routine and often referred to as the "gold standard." Many special stains are used for identification of BM, among them PAS is most commonly used. Uses of the PAS method in histopathology include detection of glycogen which is stored in increased amounts in some inherited diseases, staining abnormally thick glucose-rich renal glomerular BMs in diabetes mellitus. Very often PAS is considered a standard stain for all mucus producing tissues, but this is not always possible. Glycogen and some types of mucus stain strongly with PAS, whereas, BM and collagen fibers stain less strongly. BM comprises of fine reticulin fibers and thin collagen Types III and IV fibrils embedded in a carbohydrate-rich matrix. Although BM is PAS positive, reticulin can be shown with greater contrast by oxidizing with periodate and detecting the resulting aldehydes with an ammoniacal silver nitrate solution, which deposits black colloidal silver. 
From the literature it is known that in H-E stain, a BM is seen as fine eosinophilic line beneath the epithelium, whereas PAS shows a BM as a fine magenta line. Fluorescent periodic acid-acriflavine shows a BM as golden orange color. The literature search did not reveal any studies that compared all three stains for demonstration of a BM.
Thus, aim of the present study was to stain the basement membrane in tissue sections with H-E, PAS and fluorescent periodic acid-acriflavine and to compare the efficacy of these stains with each other so as to determine the best possible staining technique for the BM.
H-E staining method being the gold standard involves the application of haemalum, which is a complex formed from aluminum ions and oxidized hematoxylin. But some structures do not stain well. For example, basal laminae need to be stained by PAS stain or some silver stains, if they have to be well visible. Reticular fibers also require silver stain.
Our results were in agreement with studies done relating BM and cancer invasion. Wilson et al.  stated that the BM appears to be crucial in tumor invasion and metastasis. Its loss has been associated with many types of carcinomas and with tumor cells in lymph node and organ metastases. The basal membrane is a dynamic, rather than a static structure, being continuously remodeled by glycoprotein rupture and synthesis (such as laminin). These processes are extremely important in inflammation and tissue repair, as the membrane becomes fragmented to allow the entry and exit of inflammatory cells. Such fragmentation needs to be orderly, rapidly and accurately repaired and this process does not take place if tumor cells are present. During neoplastic invasion, glycoprotein synthesis in the basal membrane is slower and disorganized, leading to permanent loss of continuity.  Using electron microscopy and immunohistochemistry, discontinuities, duplication, thickening, and intensive and/or absent staining have been identified. ,
Contrast is the difference in visual properties that makes an object distinguishable from other objects and the background.
The results in the present study were in accordance with a study done by David B. Jones, who used MacCallum's crystal violet basement membrane stain, McManus periodic acid-Schiff's reaction, Masson's trichrome stain (Foot's variant), Mallory's azan stain (Heidenhain's variant), Wilder's reticulum stain and a periodic acid silver stain to study glomerular basement membrane. The periodic acid-Schiff's reaction and the periodic acid silver reaction were found to be the most satisfactory compared to others. 
When evaluated for contrast in the different stains, acriflavine showed better contrast of the BM when compared to H-E where it was difficult to identify the BM. It is very important to identify and distinguish the BM as it can be helpful in diagnosing some early invasive carcinomas, which might be missed in H-E stain.
There are many genetic and autoimmune diseases that involve desmosomal proteins including both acquired blistering diseases and rare inherited genetic disorders that result in disruption of desmosome function. These diseases are characterized by reduced cell-cell adhesion and partial or complete loss of attachment of keratin filaments due to altered desmosome function. The pemphigus diseases are autoimmune blistering diseases in which separation of the suprabasal layers is found in conjunction with circulating anti-desmoglein antibodies.  On the other hand, in Pemphigoid, the vesicles and bullae are subepidermal rather than suprabasilar in contrast to pemphigus, thus identification of BM attachment in these types of diseases could be beneficial by special stains.
Linear IgA disease is another subepidermal bullous disease that is characterized by a linear deposition of IgA at the basement membrane zone (BMZ). The loss of adhesion and blistering seen in epidermal and mucosal tissues is probably the result of autoantiantibody-mediated disruption of these proteins that are structural components of the hemidesmosome and basement membrane. 
Pierce, Midgley and Ram (1964), were the first to classify the BM under two structural headings: (a) Homogeneous lamella and (b) fibrillar lamella.  Our result is in accordance with a recent study done by Desai et al. where the fibrillar components were impregnated with salts of silver and the reticulin fibers appeared distinctly black in contrast to the golden brown hue of collagen bundles, providing an interesting avenue of distinguishing the two fibrillar components of the basement membrane. Difference in the staining pattern of the fibrillar components of the basement membrane may be due to their differential argyrophilic nature. This feature permits the delineation of fibrillar components and also elucidates the pattern of their distribution within the amorphous (homogenous) ground substance. An understanding of the complex interplay between the two components of the basement membrane viz., the fibrillar component and the amorphous ground substance and between these and the adjacent epithelial and connective tissue elements, in health and disease, may unravel the nature of derangement seen in a wide spectrum of diseases afflicting the basal lamina scaffold. 
Though all the three stains showed favorable features at different levels, acriflavine stain was better than the other stains in demonstrating BM continuity, contrast and also the pattern followed by PAS stain. Based on these observations from the present study, it was seen that, special stains are an important tool in rapid and easy demonstration of BM and also provide a powerful complement to immunohistochemistry, flow cytometry, in situ hybridization and other diagnostic technologies.
The present study to the best of our knowledge is the first study to document and compare H-E, PAS, and acriflavine stain for a BM. Hence, this study adds to the limited literature on application of acriflavine on paraffin-embedded tissue sections and it is comparable to H-E and PAS stain to stain a BM. From the above-mentioned study and analysis of data, it can be concluded that continuity and contrast, homogenous pattern, fibrillar pattern of a BM were better demonstrated by acriflavine followed by PAS. Acriflavine is an easily available, less time-consuming staining procedure and also distinctly enhanced delineation of structure details and color contrast of a BM. Further studies are required to support the above-mentioned conclusion.
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What is new?
The present study found that the periodic acid-acriflavine stain is better in demonstrating the continuity, contrast, homogenous and fibrillar pattern of the basement membrane in histopathological specimens when compared to the hematoxylin and eosin and the periodic acid Schiff stain.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
[Table 1], [Table 2], [Table 3]
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