Microscopy and Related Techniques

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800px-kidney_cd10_ihcSince microscopes offer the ability to see tiny things in an incredible amount of detail, it’s not so surprising that they are used by medical professionals to diagnose and treat diseases on a daily basis. The two main categories of microscope used in clinical settings is the basic light microscope and the more advanced electron microscope.

 

The smallest visible object that can be seen by the human eye is less than 100 micrometres, and since animal cells range from 10 to 100 micrometres, the microscope is an essential tool in cellular pathology. The most commonly used being the compound microscope.

Biopsy Analysis

When a disease is suspected a tissue specimen is regularly required in the process of deriving a diagnosis and confirming the presence of a disease state. This process begins with a biopsy, which can include a wide range of different techniques, common biopsies include core biopsies from breast, prostate and renal tissue, as well as excision biopsies from the skin.

labpict11Once the biopsy has been removed, it is immediately placed into a specimen pot with a fixative, such as 100% formalin. The purpose of this measure is to preserve the cells in a life-like state, it achieves this in a variety of ways including prevention of autolysis, putrification as well as maintaining antigenicity. It is then transported to the pathology laboratory for processing.

Prior to the specimen being placed into the cassette(s), the specimen dissection takes place. Typically, the areas of interest are sampled, and if applicable, the lymph nodes and tumour margins are also sampled as these can help guide treatment options and prognosis. For example, if cancer cells are still remaining outside of the margin, further surgery may be required; additionally if cancer cells are discovered in the lymph nodes, this can indicate metastasis.

cassetteAlthough, many samples warrant just a single cassette, there are often many cassettes per patient sample. Once labelled, cut and placed into their cassettes, they can be processed, which today is typically a heavily automated process. In short, this involves a series of steps including tissue sample dehydration, clearing, embedding in paraffin wax and staining with a histological stain. Dehydration is an important step since the beginning stage i.e. formalin is not miscible with the end stage i.e. paraffin wax. This involves the tissue being submerged in a series of graded alcohol’s to remove water e.g. 70%, 90% and 100% ethanol. Once the dehydration process is complete, clearing is the next step, which removes the dehydrating agent e.g. ethanol and replaces it with a solvent miscible with wax e.g. xylene.

The paraffin wax is crucial to the embedding process, which involves placing the tissue into a mould, and hot paraffin wax is then added and left until cool and solid. The specimen now embedded in wax can be efficiently cut into sections using an apparatus referred to as a microtome, and 4µm slices are the typical thickness for histological samples. The resultant and extremely delicate sections are immediately placed onto a water bath and then onto glass slides ready to be stained.

Haematoxylin and eosin (H&E) is the principal and routine stain used on all received samples, and the diagnosis of malignancies is based largely upon this procedure. Additionally, there are many other stains, termed “special stains” that are utilized to identify other structures not possible with H&E, for example silver stains, which are commonly employed as a connective tissue stain to identify and observe disturbed patterns of reticulin fibres in cirrhosis and some tumours.

Looking through the Microscope

compoundpartsOnce the patient sample has reached the final stage of tissue preparation i.e. been microscopically prepared and stained, it is ready to be observed by a pathologist and the results reported. There are 3 important questions asked of every specimen, including what’s the diagnosis? As well as the prognosis? And the resulting treatment? In short, the diagnosis involves the clinician looking for any structural and morphological changes in the tissue, and checking if they fit with the data set. Furthermore, the pathologist will need to request further special staining if they can’t be sure with an H&E stain alone. In addition to a diagnosis, the prognosis and treatment are also points of interest, for example, if neoplasia was observed, the pathologist would need to answer whether it was malignant or benign, as well as the grade, whether it was metastatic and provide potential treatment options.

Immunohistochemistry

300px-immunohistochemicalstaining2At approximately day 4 immunohistochemistry may also be employed for Identification of a certain antigen in tissue by an antibody specific to that antigen. The site of antibody/antigen binding must then be labelled for microscopic visualisation, and this technique is particularly useful for tumour typing, prognosis and therapy.

Immunohistochemistry (IHC) is a technique which detects antigens in cells or tissue by utilising the ability of antibodies to bind to specific antigens in biological tissues. IHC staining is commonly utilised in the diagnosis of neoplasia, as for example, specific molecular markers can be detected, which are characteristic of a particular cellular event e.g. cellular proliferation or apoptosis or a particular tissue, helping to differentiate between a primary of secondary tumour. Thus, common immunohistochemistry investigations include primary and secondary tumour typing, and the confirmation of metastasis.

The visualisation of the antibody-antigen interaction can be achieved in numerous ways, for example, the antibody can be conjugated to an enzyme such as peroxidase, which catalyses a colour-producing reaction. Additionally, there are other labels that can be conjugated to the antibody including enzyme-Horse radish peroxidase + Chromogen-Diaminobenzidine tetrahydrochloride (DAB), as well as fluorescent labels.

A good example of IHC utility in the diagnosis of neoplasia is the identification of specific markers for diagnosis, tumour typing, and confirmation of metastasis, which often involves the use of particular antibody panels. For example, a CK7, CK20, and TTF-1 antibody panel, which is useful in the diagnosis of lung tumours and for the differential diagnosis of primary pulmonary adenocarcinomas from extrapulmonary adenocarcinomas metastatic to the lung.

 

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