Histological Staining In Pathology: Techniques And Applications

Applications of Histological Staining in Pathology

Histological staining is a key feature of tissue preparation. It is done after the tissue biopsy has been fixed, cleared, embedded and sectioned (Alturkistani et al 2016, p.72.). It is done to enhance the visual contrast between different cellular components and to allow for distinction based on the different properties that each cellular component holds.

It is widely used in pathology as a means of diagnosis. Pathologies often result in disruption of the normal architecture of the cell. Histological staining highlights disruptions in cellular or tissue architecture. (Ventura et al 2000, p.550).

Hematoxylin and eosin stain is one of the stains used principally in histology. It is the most widely used stain by pathologists. It’s a combination of two stains, hematoxylin and eosin. Hematoxylin is basophilic and stains the nucleus of the cell purple while eosin is acidophilic and stains the other cellular components pink or varying combinations of purple and pink(Fischer, A.H et al, 2008). These colours create patterns that can be used to elaborate tissue architecture.

The Hematoxylin and Eosin stain is used due to its inexpensiveness ease of application and the rapidity with which it can be applied to yield observable samples. Its method of preparation gives room for human error. It is not overly dependent on the type of fixative used. On account of its merits it is used in the diagnosis of many histopathologies (Bancroft et al 2012 pp.173-186).

Each slide containing the samples from the biopsied tissue was labeled with a proper medium such as graphite, as required, this is to enable correct placement of slides and prevent any confusion or misplacement occurring. Caution was taken not to touch or scrape the tissue sample as this may result in destruction of the tissue sample hence may affect the quality of the final product of tissue processing.

The tissue sample was dewaxed by placing it in xylene for five minutes. It was done in order to allow for penetration of aqueous solutions; in light of the rehydration and staining procedures.The slide was immersed into a xylene container for 5 minutes to allow the penetration of aqueous solutions to be used in future procedures.

The excess xylene was drained and the slide dipped in decreasing grades of alcohol as follows; 100% concentration of alcohol for 3 minutes, 90% concentration for 3 minutes and 70% concentration for three minutes. This rehydrated the tissue gradually allowing for staining to occur. The slide was dipped in a tap water bath to remove the excess ethanol, care was taken not to damage it by exposing it to a running water stream.

The slide was immersed in hematoxylin for five minutes. Hematoxylin is basophilic and stains the nucleus purple enabling it to be appreciated. The slide was dipped in a tap water bath for 5 minutes to wash off the excess hematoxylin stain. The slide was dipped in acid alcohol in order to decolourise (1% HCL in 70% Alcohol solution) for 3-5 seconds in order to define nuclei and remove excess stain.The excess acid alcohol was washed off by dipping in a tap water bath for 5 minutes.

Hematoxylin and Eosin Stain: Usage and Preparation

The slide was dipped in a 1% eosin solution in order to decolourise. Eosin stains the extracellular matrix and cytoplasm pink. The slide was placed in a tap water bath to wash off the excess eosin stain. The tissue was dehydrated by placing it in increasing grades of alcohol; 70% alcohol, 90% alcohol and 100% alcohol for 3 minutes each in order to remove all water molecules from the sample and allow for complete clearing by xylene. The slide was placed in a xylene bath in order to clear off the excess alcohol. This serves as a clearing agent and ensures alcohol removal from the sample.

The sample was immersed in another xylene bath for an additional 5 minutes to ensure that complete clearance has been done. The excess xylene was removed onto separate tissue and a drop of mountant added onto the tissue sample and covered with a coverslip. Draining ensures visualization without blurriness, addition of a mountant enhances clarity. Finally labeling and identification of each slide was confirmed to be readable to minimize any confusion. (Feldman et al 2014 pp. 31-43).

The results display both normal lung tissue and pathological lung tissue, specifically lung adenocarcinoma. The results show the slide biopsy samples at high and low magnification powers respectively, magnification 40 and 1000.

The normal lung sample shows intact alveoli with continuous walls. Blood vessels were observed as well as red blood cells surrounding the alveolar spaces. The alveolar walls were continuous. Shown below are a portion of the microscopic image and drawing of the normal lung tissue observed.

The pathological lung tissue showed bits of tar as darkened patches, the alveolar wall is discontinuous and there is destruction of alveoli leading to loss of tissue architecture. Tumour cells can be appreciated in the sample as they stand out with their nuclei staining a deep purple colour. The microscopic and hand drawn images of the pathologic tissue are displayed below.

The specimen under review,TCGA-55-8203 displayed below, is a pathological sample showing lung adenocarcinoma, three predominant patterns observed ,the acinar, micropapillary and lepidic patterns. The sample appears to be moderately differentiated and  its spread is relatively moderate as well. Grading and staging of the specimen would place it at quite advanced, possibly stage iii disease. 

The pathological lung tissue observed is diagnostic of lung cancer, lung adenocarcinoma. Lung adenocarcinoma manifests many histologic subtypes; lepidic, acinar, papillary, micropapillary, solid and invasive mucinous subtypes (Hutchinson et al 2019 pp. 255-264). Each is characterized by specific features, lepidic pattern demonstrates replacement of pneumocytes in the alveolar wall with the tumour cells. Solid adenocarcinoma demonstrates layers of tumour cells with adequate cytoplasm. Papillary adenocarcinoma is made up of  cuboidal-shaped tumour cells multiplying adjacent to the fibrovascular cores in a finger-like arrangement. Micropapillary pattern shows finger-like clusters in alveoli without the characteristic fibrovascular cores. Acinar and mucinous invasive adenocarcinoma patterns display a malignancy invading the fibrous stroma and columnar shaped cells with mucinous cytoplasm spreading in an acinar-like fashion.

Staining of Normal and Pathological Lung Tissues

Pathologic slides may show more than one histologic subtype. The subtypes observed in this particular slide are micropappilary, lepidic and acinar patterns.

Lung adenocarcinoma falls under non-small cell cancer and is strongly linked to chronic smoking. Other risk factors include exposure to carcinogens such as asbestos and heavy metals, although incidence with these is markedly lower.P53 gene mutations are the tumorigenic cause in more than 50% of cases(Ding, et al 2008 pp.1069-1075).

Symptoms associated with lung adenocarcinoma are linked to staging of the cancer with the earliest stages being asymptomatic and only being detected by chance. Coughing, presence of blood in sputum and noticeable weight loss are attributed to more advanced disease. Pleural effusion with loss of breath sounds may occur as well as chronic obstructive pulmonary disease. Locoregional spread and paraneoplastic syndromes may occur as well, but this is rare and may manifest as phrenic nerve palsy and Cushing syndrome respectively (Myers et al 2021).

Histological samples can be used to assess for progression and prognosis by assessing the degree of spread as limited invasive tumours or limited nodal disease which are associated with stages I,II and IIIa compared to mediastinal, subcarinal and/or contralateral nodes and metastatic disease which are associated with stage IIIb and stage IV(Travis et al 2013 pp.685-705).Surgical resection serves as the treatment modality for stage I to stage IIIa followed by adjuvant chemotherapy because of the risk of relapse. Radiation is the only treatment option for stages IIIb and IV which carry worse prognosis as compared to stages I to IIIa (Travis et al pp.669-692).

Conclusion

In conclusion, histological staining is a key component of tissue processing that is used in the medical field as a means of reaching diagnosis. The hematoxylin and eosin stain particularly is one of the most commonly used stains on account of its numerous merits and ease of preparation. The pathological sample in question is a lung adenocarcinoma which showed three histologic subtypes;the lepidic,acinar and micropapillary subtypes.

References

Alturkistani, H., Tashkandi, F. and Mohammedsaleh, Z., 2015. Histological Stains: A Literature Review and Case Study. Global Journal of Health Science, 8(3), p.72.

Downie, T., 1990. Theory and Practice of Histological Techniques Edited by J.D. Bancroft & A. Stevens, Churchill Livingstone, Edinburgh, 740 pages, £55.00. Histopathology, 17(4), pp.386-386.

Feldman, A.T. and Wolfe, D., 2014. Tissue processing and hematoxylin and eosin staining. In Histopathology (pp. 31-43). Humana Press, New York, NY.

Fischer, A., Jacobson, K., Rose, J. and Zeller, R., 2008. Hematoxylin and Eosin Staining of Tissue and Cell Sections. Cold Spring Harbor Protocols, 2008(5), p.pdb.prot4986.

Hutchinson, B., Shroff, G., Truong, M. and Ko, J., 2019. Spectrum of Lung Adenocarcinoma. Seminars in Ultrasound, CT and MRI, 40(3), pp.255-264.

Myers, D.J. and Wallen, J.M., 2021. Lung adenocarcinoma. StatPearls [Internet].

Tanoue, L., 2010. Somatic mutations affect key pathways in lung adenocarcinoma. Yearbook of Pulmonary Disease, 2010, pp.115-117.

Travis, W., 2011. Pathology of Lung Cancer. Clinics in Chest Medicine, 32(4), pp.669-692.

Travis, W., Brambilla, E., Noguchi, M., Nicholson, A., Geisinger, K., Yatabe, Y., Ishikawa, Y., Wistuba, I., Flieder, D., Franklin, W., Gazdar, A., Hasleton, P., Henderson, D., Kerr, K., Nakatani, Y., Petersen, I., Roggli, V., Thunnissen, E. and Tsao, M., 2013. Diagnosis of Lung Adenocarcinoma in Resected Specimens: Implications of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society Classification. Archives of Pathology & Laboratory Medicine, 137(5), pp.685-705.

Ventura, H., 2000. Rudolph virchow and cellular pathology. Clinical Cardiology, 23(7), pp.550-552.