Journal of Orofacial Sciences

: 2022  |  Volume : 14  |  Issue : 1  |  Page : 71--79

Microinvasive Oral Squamous Cell Carcinoma Redefined by Using Modified Cajal Trichrome Differential Stain − A Histochemical Study

Vaishnavi Srinivasan, Gundamaraju Kiran Kumar, Naragani Durga Venkata Naga Shyam, Vaishali Narayen, Paremala Konda, Korra Swetha Rani 
 Department of Oral Pathology and Microbiology, Government Dental College and Hospital, Hyderabad, Telangana, India

Correspondence Address:
Dr. Korra Swetha Rani
Department of Oral Pathology and Microbiology, Government Dental College and Hospital, Hyderabad, Telangana, 500012


Introduction: In histopathologic laboratories, hematoxylin and eosin (H and E) staining is the routine and gold standard technique employed for biopsy tissues. But, few differential stains are being used in questionable diagnostic cases of oral squamous cell carcinoma (OSCC) to confirm its final diagnosis. Modified Cajal trichrome stain (CTS) is one such stain which may be employed as an adjunctive supporting aid for arriving at a conclusive diagnosis. To assess epithelial and connective tissue components in normal oral mucosa, oral epithelial dysplasia (OED), and OSCC cases stained with routine H and E and modified CTS. And also to compare and evaluate the efficacy of Modified CTS with that of H and E stain. Materials and Methods: A total of 40 tissue samples, 10 each of normal oral mucosa and OED, 20 cases of OSCC (10 cases each of microinvasive and frank OSCC) were stained with standard H and E and modified CTS simultaneously. Results: Modified CTS showed statistically significant and better results in differentiated terms of parameters such as depth of tumor invasion and differentiation between epithelial and connective tissue components in OSCC cases. Epithelial basement membrane interface and microinvasion are clearly appreciated in challenging cases of early epithelial malignancies such as microinvasive OSCC using this stain. Conclusion: As this stain can easily delineate epithelial structures from various connective tissue components, it may be utilized as an auxiliary diagnostic support along with routine H and E stain for confirmatory diagnosis.

How to cite this article:
Srinivasan V, Kumar GK, Shyam ND, Narayen V, Konda P, Rani KS. Microinvasive Oral Squamous Cell Carcinoma Redefined by Using Modified Cajal Trichrome Differential Stain − A Histochemical Study.J Orofac Sci 2022;14:71-79

How to cite this URL:
Srinivasan V, Kumar GK, Shyam ND, Narayen V, Konda P, Rani KS. Microinvasive Oral Squamous Cell Carcinoma Redefined by Using Modified Cajal Trichrome Differential Stain − A Histochemical Study. J Orofac Sci [serial online] 2022 [cited 2022 Sep 30 ];14:71-79
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Early detection and accurate diagnosis of epithelial neoplasms is essential for proper treatment intervention and also determining prognosis and survival of the patients.[1] Histopathologic assessment of surgically removed formalin-fixed tissues has remained the essential tool for surgical pathologist in everyday clinical practice.[2]

Biopsy tissue samples stained with hematoxylin and eosin (H and E) are regarded as the root basis for its diagnosis and histopathologic staging.[3] It is considered as a gold standard stain for routine histopathologic examination of tissues.[4] H and E stain is a permanent histologic stain which imparts blue color to the nuclei of the cells and stains the cytoplasm as pink.[5]

The final diagnosis of some initial epithelial neoplasms may be challenging and difficult even with routine H and E staining, especially in cases of microinvasive oral squamous cell carcinoma (OSCC) and some atypical epithelial neoplasms.[6] In few cases, H and E stain may not provide the accurate identification or recognition of some abnormal components which the pathologists or researchers need.[7] Hence, diagnosing such pathologies can be challenging using H and E stain only. In such cases, special or differential staining procedure is very much helpful, and facilitating an easy and accurate diagnosis of some challenging cases. They are simple to interpret for diagnosis and remain as an essential auxiliary tool for routine histopathologic diagnosis.[5] One such differential stain is modified Cajal trichrome stain (CTS).

It delineates various components in the tissue visually with different hues of color. This differential stain helps in the detection of tumors where connective tissue stromal components are associated with epithelial cells, for example, OSCC.[1] Microinvasive OSCC is an early stage epithelial malignancy which shows the invasion of atypical epithelial cells confining to the superficial lamina propria without invading into the deeper structures. In some cases, identification of invasive components is difficult using H and E stain due to its smaller amount or masking of basement membrane integrity by inflammatory cells.[8] In such cases, modified CTS helps in distinguishing and identification of atypical epithelial cells within the connective tissue stroma.

In our study, we attempt to explore the usage of modified CTS in making diagnosis much easier rather than using expensive diagnostic aid such as immunohistochemistry. Even though immunohistochemistry is well known for its specificity in confirming the diagnosis, it is not given much preference as it will be costly and time consuming.[9] Hence, prior to opting for any immunohistochemical or advanced molecular markers, differential stains can acts as a simple, beneficial, economic way out to detect and diagnose various pathologies of oral cavity.[10]

 Materials and Methods

This retrospective study was carried out after obtaining ethical approval (ethical clearance number: GDCH-IEC/PG/1922) from the Institutional Ethical Committee, Osmania Medical College, Hyderabad on November 13, 2019. Total sample size includes 40 cases of formalin-fixed, paraffin wax-embedded tissue blocks, which were selected and taken from the archives of our department. The group I category included 10 samples of normal oral mucosa obtained during disimpaction of third molars in patients with no tobacco or alcohol habits. The group II category comprised of 10 samples of oral epithelial dysplasia (OED) and group III category included 20 tissue samples of OSCC (10 cases each of microinvasive and frank OSCC). Selected cases were diagnostically challenging using H and E staining.

Inclusion criteria

Histopathologically diagnosed cases ofOED,microinvasive OSCC, andFrank OSCC.

Exclusion criteria:

Patients undergoing treatment for OED and OSCC (surgery, chemotherapy, and radiotherapy).Patients with metastatic tumors in jaws from systemic malignancies.

Pair of sections of 3 μ thickness was taken from the selected tissue blocks and these were stained with H and E and modified CTS simultaneously.

Staining procedure of modified Cajal trichrome stain

Three-micron thickness sections were taken from the selected tissue blocks. Deparaffinization was carried out by heating sections on a slide warmer for 1 hour at 70°C and treated with xylene, followed by rehydration in decreasing grades of isopropyl alcohol and then washed in distilled water. Then, sections were stained with Ziehl acetic fuchsin for 10 minutes (Ziehl fuchsin − 5 mL, acetic acid − 0.5 mL, distilled water − 100 mL) followed by rinsing with distilled water. Vitrofixation has been carried out using formalin acetic acid mixture for 15 minutes (glacial acetic acid − 0.5 mL, formalin − 10 m, distilled water − 10 mL) and subsequently washed with distilled water. Sections were treated with picroindigocarmine solution for 30 minutes (Indigo carmine − 0.5 mg and picric acid solution − 100 mL, and distilled water − 15 mL). Then sections were dehydrated using alcohol, cleared, dried, and mounted.


Using modified CTS, normal epithelial cell cytoplasm shows pale pink color, whereas the dysplastic epithelial cell cytoplasm appears green and nucleus shows magenta-red color. Keratin and RBCs show grass-green, collagen shows blue, and muscle appears as olive-green color. A comparison of these interpretations with H and E staining is shown in [Table 1].{Table 1}

The normal mucosal tissue stained with modified CTS showed epithelial cells with pale pink color cytoplasm and magenta-red nuclei [Figure 1]. In OED, dysplastic epithelial cells appeared with green color cytoplasm with hyperchromatic magenta-red nuclei [Figure 2]. In one case of oral submucous fibrosis transformed into OSCC, the orientation of collagen fibers (appearing as blue) was clearly appreciated in modified CTS stain than H and E staining [Figure 3].{Figure 1}{Figure 2}{Figure 3}

In microinvasive OSCC, the epithelial connective tissue interface was more distinctly visible in modified CTS when compared with H and E staining. In addition, the invaded atypical epithelial cells were evident within the connective tissue stroma using this stain [Figure 4]. Modified CTS stain helped in distinguishing keratin pearls very clearly and distinctly from some other connective tissue stromal elements, which appeared as grass-green in color. Another important histomorphologic parameter is the depth of tumor invasion, which was more precisely determined using this stain, as the correct extent of the neoplasm was also accurately analogized [Figure 5]. Another case of OSCC with questionable muscle invasion of neoplastic cells with H and E staining was taken. It was more readily appreciated in modified CTS as the muscle appears in olive-green color invaded by atypical epithelial cells within it [Figure 6].{Figure 4}{Figure 5}{Figure 6}

After staining with H and E and modified CTS, the slides were analyzed by two observers independently using following criteria such as (1) cytoplasmic intensity, (2) nuclear intensity, (3) grading of OED, (4) depth of tumor invasion, and (5) differentiation between epithelial and connective tissue components.

The assessment and comparison of the two stains were carried out with a four-degree scale in which each parameter was analyzed by two observers independently and a numerical representation of qualitative score was given as poor (1), average (2), good (3), and excellent (4).

The data were collected and tabulated, and Pearson Chi-square test was performed using SPSS Version 20.0 (IBM Corp. Released 2011, IBM SPSS Statistics for Windows, Version 20.0; IBM Corp, Armonk, NY, USA). To analyze interobserver agreement, kappa statistical analysis was performed.

The cytoplasmic intensity of cells stained with H and E, and modified CTS was compared and it was more distinct in modified CTS with high statistical significance (P = 0.000) [Table 2]. Similarly, nuclear intensity of cells was compared, which was more clear in H and E staining with statistical significance (P = 0.000) [Table 3]. There was also a statistically significant difference (P = 0.003) in the grading of OED, which was better in H and E stain than modified CTS [Table 4]. When the depth of tumor invasion in OSCC cases was compared, there was a high statistically significant difference between H and E and modified CTS stained sections (P = 0.001) [Table 5]. The differentiation between epithelial and connective tissue components in OSCC was compared and it showed statistically significant difference with P-value of 0.001 among H and E and modified CTS-stained sections [Table 6]. A kappa value of 0.94 was obtained which represents almost perfect agreement between the two independent observers [Table 7].{Table 2}{Table 3}{Table 4}{Table 5}{Table 6}{Table 7}


Since 1850s, H and E staining remains a traditional method and cornerstone for diagnosis among pathologists for routine histopathologic diagnosis. This is regularly used as a histologic stain for light microscopy. It works excellently with various fixative agents and also shows an ample range of nuclear and cytoplasmic findings.[1] But structures such as collagen, muscle, keratin, and other extracellular matrix components will stain as eosinophilic; hence, it does not differentiate these various eosinophilic structures observed in the connective tissue components distinctly.[9],[11] Hence, the routine H and E stain helps in the identification and diagnosis of conventional OSCC, in which the invasive structural elements are more and clearly appreciable within the connective tissue stroma. Besides this conventional OSCC, different subtypes present with various diagnostic challenges to pathologists have been identified.[1]

In such OSCC cases, there may be smaller amount of invasive epithelial components or sometimes these components may remain obscure or masked by the inflammatory elements leads to difficulty in its accurate diagnosis.[8] Hence, there is a need for appropriate special or differential stain which helps in distinguishing epithelial components within the stromal elements of connective tissue, which may acts as an additional diagnostic aid. For epithelial pathologies, various special stains have been identified and that are useful in differentiation and diagnosis.[4] One such differential or special stain is modified CTS.

During 1897, Ramon Y. Cajal introduced CTS. Later in 1919, it was modified by scientists Gallego and Calleja and called as modified CTS. It is regarded as an excellent stain for analyzing tissues comprised of various structural elements within it. In 1932, the different staining methods and principles of CTS were broadly illustrated by Castroviejo in his review article. Based on his research works, this stain was considered to be excellent for diagnosing corneal pathologies.[12]

The principle of modified CTS is that Ziehl acetic fuchsin, a low molecular weight dye, will impart dark pink to magenta-red color to all the structures. The formol acetic acid is used as a differentiating agent and removes the fuchsin color from most of the tissue elements. But exceptional is acidic tissues such as nuclei which will retain the fuchsin color and hence nuclei appear in magenta-red color. Picroindigocarmine is the counterstain which imparts blue color to the basic tissues (such as collagen) and green color to tissues with high acidic contents (such as dysplastic epithelial cells). Thus, fuchsin color is retained solely in the nucleus due to formol acetic acid mixture by means of vitro-fixation mechanism.[1]

In OED, dysplastic epithelial cells appeared with green color cytoplasm with hyperchromatic magenta-red nuclei [Figure 2]. Thus, modified CTS helped to delineate dysplastic cells from normal epithelial cells more clearly and easily compared to H and E staining. This also assisted in the identification of accurate extend of dysplasia within the layers of epithelium, ‘thereby it helps in minimizing the intraobserver and interobserver variability in the grading of OED.

Modified CTS was incredibly helpful in detecting and redefining the early microinvasion in OSCC cases, which was difficult to identify in routine H and E staining. Keratin pearl formation within the connective tissue components is a characteristic finding in OSCC. Modified CTS stains keratin pearls in grass-green in color. This differentiation also helps in easy grading of OSCC based on degree of keratinization. Thus in OSCC, it helps in easy recognition of epithelial elements and various other components within the connective tissue by giving different hues of colors.[10]

In challenging cases of OSCC stained with modified CTS, invasion of atypical epithelial cells within the connective tissue stroma and existence of keratin pearl formation were remarkably evident. In addition, depth of tumor invasion is precisely recognized using this stain. Thus modified CTS can be useful in diagnosing challenging cases and preferred rather than using pan-cytokeratin immunohistochemical markers in diagnostic dilemmas of microinvasive OSCC cases.[1] The correct diagnosis is essential for successful treatment plan and determining prognosis and thus, this stain helps in easy diagnosis of challenging cases of OSCC.

Our study results were in accordance with the previous study conducted by Sanjai et al.[1] In recent times, increased number of immunohistochemical markers such as pan-cytokeratins and newer molecular techniques are being used for the confirmatory diagnosis of OSCC. But they are tedious, technique sensitive, and expensive. On the other hand, modified CTS is a simple staining method, economical and affirmed to be a time-saving adjunctive diagnostic support to the pathologists for day to day practice. Prompt diagnosis of OSCC is crucial for therapeutic interventions and determining the survival rate of the patients and modified CTS helps in early as well as rapid diagnosis.[1]


Our study included a lesser number of samples. Hence, further studies with larger sample sizes are required to predict the application of this special stain in various lesions of the oral cavity.


Modified CTS is greatly helpful in challenging cases for diagnosis, in which the invasive behavior of the atypical tumor cells is not much evident and in solving the predicament of microinvasion. In addition, for screening of large number of OSCC samples, using modified CTS, various shortcomings in OSCC diagnosis may be overcome. Modified CTS stain was slightly superior compared to H and E stain for assessing depth of tumor invasion and differentiating epithelium and connective tissue components. Thus, it can be a promising differential stain for epithelial pathologies with diagnostic dilemmas. Though, it cannot replace traditional H and E staining in the diagnosis of oral cancer, yet it can certainly be used as an adjunct to it. To the best of our knowledge, this is the first study which was conducted using modified CTS in oral epithelial pathologies within our geographical population.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.


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