An Attempt to Find Alternative Stains for Routine Hematoxylin and Eosin

Introduction

Paraffin embedded tissue blocks are treasures of any pathology department. As soon as the tissue is removed from the body, it is processed through a series of steps to prepare a microscopic slide of diagnostic value. After the tissue processing, staining of the microscopic slides is carried out to study the histopathology of the given tissue sample.^1-3 If the tissue processing or staining has not been properly done, it is often difficult for oral pathologists and general pathologists to provide an accurate diagnosis of the given tissue sample. Encouraging the innovations or studies like this would help government doctors, dental hospitals and general hospitals practice pathology with ease.

Materials and Methods

Mehendi stain, Washing dye, Disclosing agent, Sarasaparilla root extract, Beta vulgaris, Genus Vitis extract, Xerox tonor, Whitener, Ayurvedic Neeli, Harda and pure extracts of Vitis vinifera were chosen for the study.

Extraction of Mehendi stain

The leaves of Lawsonia inermis (natural henna) plant were taken. They were properly washed and allowed to dry. The dried leaves were grinded into a fine powder. The extracted powder was again sieved. The fine powder was mixed in water at a powder-to-water ratio of 1:5. The obtained paste was compressed in a thin cloth and the dye filtrate thus obtained was stored in an air tight container.

Extraction of Washing dye stain

Two kilograms of washing dye was completely allowed to dissolve in 7.5 L of absolute ethanol. It was boiled until maceration and the extracted dye was preserved in an air tight container.

Disclosing agent

It is the dye which on application to the teeth will specifically bind to plaque and allows easy visibility of plaque deposits. The dye is readily available commercially and was used easily.

Extraction of Sarasaparilla root extract dye stain

The roots of the plant Sarasaparilla were obtained and were cleaned thoroughly after immersing in water for one day. The roots were immersed in distilled water and allowed to boil until the color of the distilled water changed to amber color or dark red. The water was then filtered using filter paper and filtrate was stored in an air tight container.

Similarly other dye stains were obtained.

Paraffin embedded well differentiated squamous cell carcinoma tissue block, only to focus on squamous epithelium, was freshly cut using semi-automated tissue processing unit. The samples were taken from the ribbon sections and fixed on to the microscopic slides. Squamous cell carcinoma blocks were chosen because they are the most commonly reported malignant lesions in dentistry. The microscopic slides with the tissue samples were kept on the slide warmer to remove the impregnated wax and kept in xylene for clearing. After air drying, simple staining of the microscopic slides was carried out with the individual stains chosen for the study. Direct staining was also done among few stains followed by mounting in DPX and visualization under BX40 binoculwasar research microscope.

Results

Histomorphological criteria examined are elaborated below.

a) Cellular outline

b) Nuclear detail

c) Overall morphology

d) Staining quality

e) Epithelium and connective tissue differentiation.

Each histomorphologic criteria was rated on a following scale of 1-4.

1. Poor

2. Satisfactory

3. Good

4. Excellent⁴

Scoring Criteria (Table 1)

High diagnostic value or Excellent = 24-20

Moderate diagnostic value or satisfactory = 19-12

Low diagnostic value or poor = 11-0

Among the stains used for the study, mehendi and food coloring agent stain showed excellent scoring criteria. The hair dye stain and tobacco stain showed satisfactory scoring criteria. Further studies are required to assess the clear scoring criteria for hair dye stain. Remaining stains like washing dye stain, disclosing agent stain, Sarasaparilla root extract stain and textile dye stain showed poor scoring criteria. Other stains such as Beta vulgaris extract, xerox toner, whitener, pure extracts of Vitis vinifera were null or negative and their scoring criteria was negative (zero). Interestingly, on staining the malignant lymphoma slide with textile dye, the surrounding tissue around the lymphoid follicle was darkly stained in pink color and the prominent lymphoid follicle was light pink stained.

 To determine the direct staining among these stains, the washing dye stain and disclosing agent stain were used. After the initial staining with washing dye stain, one dip in 1% acid alcohol was carried out followed by staining with disclosing agent. With this, the staining of the localized nucleus was good and prominent. This suggests that further research on excellently stained, satisfactorily stained and poorly stained dyes are equally important to know their affinity towards various tissue architecture. Further, addition of salts and separation of salts in these staining dyes would definitely lead to standardization or standard dye or stain.

Mehendi stain

Epithelium and connective tissue could be differentiated. Epithelium was dark brown in color while the connective tissue was light brown in color. The dark band of cellular proliferation and cellular outline could be noticed from the basal layer. Individual cellular architecture and cellular outline could be visualized. Demonstrated high diagnostic value. On 4X microscopic view, epithelium and connective tissue regions could be clearly differentiated using Mehendi stain (Figure 1a). On 10X microscopic view, epithelium and connective tissue could be clearly differentiated along with dark band of cellular proliferation and cellular outline could be noticed from the basal layer (Figure 1b). On 20X microscopic view, individual cellular architecture and cellular outline could be visualized (Figure 1c). On 40X microscopic view, localized dark nucleus of individual cells and intercellular attachment could be noticed (Figure 1d). Mehendi stain, thus can be classified as an epithelial stain.

Food coloring agent stain

Epithelium and connective tissue could be differentiated. Epithelium was dark eosinophilic in color while the connective was light eosinophilic in color. Thick band of cellular proliferation at the basal layer was noted. The intercellular architecture of the individual cells in the epithelial layer could be seen along with localized cellular nucleus. Demonstrated high diagnostic value.

Under 4X, the epithelium and connective tissue regions could be differentiated using food coloring agent (Figure 2a). Under 10X, epithelium and connective tissue could be clearly differentiated along with thick band of cellular proliferation at the basal layer. Localized cellular nucleus was observed in the epithelial region along with light eosinophilic stain in relation to connective tissue (Figure 2b). Under 20X, the intercellular architecture of the individual cells in the epithelial layer could be noticed along with localized cellular nucleus (Figure 2c). Under 40X, the individual cellular architecture could be clearly noticed along with palely stained nucleus (Figure 2d). Food color agent stain can be classified as an epithelial stain.

Hair dye stain

Figure 3 shows predominantly stained keratin pearls in well differentiated squamous cell carcinoma slide. Further studies are required to conclude this. Demonstrated moderate diagnostic value.

Washing dye stain

Figure 4a shows generalized epithelium and connective tissue stained using washing dye. Demonstrated poor diagnostic value. Washing dye showed good diagnostic value in the malignant lymphoma slide. On staining the malignant lymphoma slide with textile dye (Figure 4b), the surrounding tissue around the lymphoid follicle was darkly stained and the prominent lymphoid follicle was lightly stained. Whole tissue sample showed a blend of dark pink color with washing dye stain (Figure 4c). Demonstrated poor diagnostic value.

Disclosing agent stain

Figure 5a shows generalized epithelium and connective tissue stained using disclosing agent as a stain. Epithelium was light pink in color and connective tissue was dark pink in color. Demonstrated low diagnostic value.

Saraparilla root extract and tobacco stain

Generalized epithelium and connective tissue stained with Sarsaparilla stain demonstrated poor diagnostic value [Figure 5b (i)]. Epithelium and connective tissue could be differentiated using tobacco stain [Figure 5b (ii)] demonstrating moderate diagnostic value.

There were distinct localized nuclei in few cells which were light blue in color. Few of the cells did not show any stained nucleus. Standardization and repeated staining of various tissue samples with washing dye stain and disclosing agent was required for the final conclusion. Demonstrated poor diagnostic value (Figure 6a).

In this staining, eosin stain was not used. Hematoxylin was included in the study along with Ayurvedic Neeli and Harda stains. The results were not so fascinating. Harda and Neeli did not show affinity for cellular cytoplasm and connective tissue components. The diagnostic value was poor (Figure 6b).

Discussion

The golden standard stain for histopathological examination is Hematoxylin and Eosin staining. The principle of H&E staining is chemical bonding of the stain or dye to the tissue architecture. The Hematoxylin which is a basophilic stain shows affinity to basophilic components such as nucleus, ribosomes and RNA in cytoplasm and eosin which is an acidophilic stain shows affinity towards cytoplasm, collagen fibers, RBCs, and muscle.5 Similarly, on staining with mehendi stain and food coloring agent stain, the whole epidermal or epithelial cells took up the stain leaving the connective tissue. The standardization of these stains is required as these stains can be applied to tumors of epithelial origin. Repeated studies on these stains gives us clear clarity about the affinity of these stains towards the tissue architecture. Though hair dye stain, disclosing agent stain, tobacco stain, textile dye stain were not under excellent scoring criteria, they are as important as the mehendi and food coloring agent stains. Further research on these stains would give us excellent results and standardization of these stains is 100% possible. This original study was presented at XXIII National conference of Indian Association of Oral and Maxillofacial Pathology at Bangalore, 2014.⁶

Conclusion

Thomas Alva Edison discovered 10,000 ways in which an electric light bulb will not work. But later, he and his team successfully designed electric bulb with tungsten f ilament. Similarly, I made a sincere effort to find alternative stain from the readily available commercial stains and I was half successful as the mehendi stain, food colouring stain were highly positive. Further research on the alternative stains from commercially available stains or natural stains could lead to the development of a successful stain. So, here I would like to conclude that there is a definite possibility that some commercially available stains or naturally occurring stains would replace hematoxylin & eosin for routine staining.

Conflicts of Interest

None