Cost-Effective Staining Alternatives to Immunohistochemistry for Keratin Demonstration in Oral Epithelial Pathologies

ABSTRACT

Background:

Demonstrating keratin is crucial in diagnosing various epithelial pathologies and is typically done through histological and immunohistochemical (IHC) methods. While IHC staining for keratin is highly specific, it can be costly and time-consuming. Therefore, common histological staining methods for keratin, suitable for routine histotechniques labs, are gaining importance.

Methodology:

Twenty formalin-fixed, paraffin-embedded tissue blocks, each representing histologically confirmed normal oral mucosa (NOM), hyperorthokeratosis (HOK), and well-differentiated squamous cell carcinoma (WDSCC), were retrieved. Five histological sections of 4-μ thickness from each block were stained using routine hematoxylin and eosin (H and E), modified pap stain (mPap), Ayoub-Shklar (AS), Dane-Herman (DH), and Alcian blue-PAS stains (AB-PAS). Two independent observers evaluated the stained sections and scored staining specificity and intensity. Statistical comparisons were made.

Results:

All sections of NOM, HOK, and WDSCC showed positive staining for keratin with each of the five stains used. The staining specificity and intensity scores were highest with the AS stain and lowest with the AB-PAS stain.

Conclusion:

Routine H and E, mPap, AS, and DH stains effectively stained keratin with adequate intensity in NOM, HOK, and WDSCC. However, while AB-PAS stain also positively stained keratin, its staining intensity was poor across all three tissue types.

INTRODUCTION

Keratins, a diverse group of proteins, are responsible for the form and structural integrity of keratinocytes. Keratin filaments, being crucial stabilizers of the structure of epithelial cells, draw inexorable interest in keratins in embryology, biology, dermatology, and oral epithelial pathologies.[1,2] The expression of these proteins is guided by the type of epithelium and the stage of cellular differentiation.[1,3]

Keratins and the associated proteins have been exploited as diagnostic markers owing to their characteristic of region- and differentiation-specific expression.[1,2,3] Epithelial tumors, including metastases, retain the patterns of keratin expression of the epithelial origin, which opens another exploitable characteristic of enabling cell and tumor typing by the determination of keratin patterns of tumors.[4,5,6] Since the routine hematoxylin and eosin (H and E) stain does not differentiate the eosinophilic components of the connective tissue and the keratin,[7] antibodies specific against several keratins are widely used for the immunohistochemical diagnosis of epithelial premalignant and malignant and many hereditary mucocutaneous lesions.[8] However, immunohistochemistry being time-consuming and expensive, the use of special stains is luring diagnosticians.[9]

We attempted to observe and compare the effectiveness of the stains shown to have a possible affinity for keratin protein, namely the modified Papanicolaou (mPap), Ayoub-Shklar (AS), Dane-Herman (DH), and Alcian blue periodic acid Schiff (AB-PAS) stains in demonstrating keratin in epithelial pathologies.

MATERIALS AND METHODS

Redo materials and method as the oral pathology department provided 60 formalin-fixed, paraffin-embedded tissue blocks for sample collection.

The microbiology study included 20 blocks of NOM, HOK, and WDSCC.

Staining procedures:

• H&E Staining: Deparaffinization, dehydration, Harris’ hematoxylin, differentiation, bluing, eosin, clearing, and mounting.

Deparaffinization, dehydration, Harris’ hematoxylin, acid alcohol differentiation, orange G and eosin azure staining, dehydration, and mounting for mPAP staining.

• AS staining: Deparaffinization, dehydration, acid fuschin, aniline blue OG, clearing, and mounting.

• DH Staining: Deparaffinization, dehydration, Mayer’s hematoxylin, phloxine B, AB, orange G, clearing, and mounting.

• AB-PAS Staining: Hydration, AB staining, acidified permanganate oxidation, oxalic acid rinsing, Schiff’s reagent, Mayer’s hematoxylin staining, clearing, and mounting

• Evaluation: Two independent observers rated keratin staining specificity and intensity from 1 to 4 (1 = poor, 2 = satisfactory, 3 = good, 4 = excellent).

Kruskal-Wallis, Mann-Whitney Each stain’s specificity and intensity were compared using U tests.

RESULTS

All sections of NOM, HOK, and WDSCC stained positive for keratin with each of the five stains used in the study [Figure 1]. The staining specificity and intensity scores for each of the stains are tabulated in Tables 1 and 2. The staining specificity was found to be higher by the AS stain, followed by mPap, H and E, DH, and AB-PAS, and the score was maximum in NOM samples stained with the AS stain. Likewise, the staining intensity was maximum in the tissues stained with the AS stain, followed by mPap, DH, H and E, and AB-PAS stains, with the score obtained being maximum for the NOM samples stained with the AS stain. There was a statistically significant difference in the staining specificity and intensity in scores among the NOM samples stained by the different stains. However, there was no statistically significant difference in the scores obtained by the different stains among the HOK and WDSCC samples.

Figure 1.

Sections of NOM, HOK, and WDSCC (right to left in every row) stained using PAS-Alcian Blue stain, modified PAP stain, Ayoub-Shklar Stain, Dane-Herman stain, and routine H and E stain (top to bottom)

Table 1.

Scores of staining specificity for H and E, mPap, AS, DH, and AB-PAS stains in the three groups of sections

Stains	NOM	HOK	WDSCC
H and E	1.80±0.79	2.00±1.05	1.30±1.34
mPap	2.00±0.82	2.00±0.94	1.40±1.35
AS	2.20±0.79	2.00±1.33	1.30±1.25
DH	1.70±0.68	1.60±1.08	1.10±1.10
AB-PAS	1.00±0.67	1.20±0.79	1.00±0.94
P	<0.05	>0.05	>0.05

Table 2.

Scores of staining intensity for H and E, mPap, AS, DH, and AB-PAS stains in the three groups of sections

Stains	NOM	HOK	WDSCC
H and E	1.75±0.69	1.80±1.08	1.20±1.40
mPap	2.02±0.72	2.02±0.84	1.30±1.32
AS	2.10±0.59	2.02±1.23	1.20±1.45
DH	1.80±0.48	1.80±1.04	1.00±0.90
AB-PAS	1.10±0.47	1.10±0.79	1.20±0.84
P	<0.05	>0.05	>0.05

DISCUSSION

Diagnostic and surgical pathology heavily rely on visual inspection and differentiation of tissue components stained on microscopic slides. While H and E stain remains the gold standard for diagnostic purposes, pathologies involving abnormal keratinization necessitate more specific contrasts. Special stains, cost-effective alternatives to immunohistochemistry, serve as adjuncts to emphasize normal or abnormal keratinization.[10]

The AS stain, initially introduced by Ayoub P and Shklar G, stains keratin in a brilliant orange color. Similarly, the DH stain, introduced by Dane ET and Herman LH, has shown remarkable staining specificity and intensity for keratin in various studies, including ours.

mPap, a modification of the Papanicolaou stain, has been less utilized on paraffin-embedded tissue sections.

AB-PAS stain, utilizing acidified permanganate solution, has shown effective staining of keratin in previous studies. However, in our study, AB-PAS demonstrated lower staining specificity and intensity compared to routine H and E stains.

CONCLUSION

Keratinization, whether abnormal, excessive, or unintended, serves as a crucial diagnostic indicator for pathologists. While routine H and E stain effectively stain keratin, it may not clearly distinguish small foci of abnormal keratinization.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.