Histopathology

The following material is intended to familiarize the student with techniques of tissue preparation and steps required to achieve histopathologic evaluation and diagnosis. The case presented here is that of a middle aged man with cancer of the colon first noted by an endoscopic procedure performed by a gastroenterologist. Subsequently the patient was scheduled and prepared for resection of a part of the colon which contains the tumor.

A partial colectomy was performed, removing the colonic segment which contains the tumor. In the operating room, the tubular shaped colon was opened in order for the surgeon to visualize the tumor. The specimen was then sent to the pathology department for gross and microscopic examination to determine the type of tumor and related prognostic factors.

Here the pathologist first verifies patient identifiers and necessary paperwork. Each patient will receive a surgical accession number by which her or his tissue can be identified. The tissue is then removed from the container and inspected in the fresh state. It is then placed in a solution of 10% formalin to fix overnight. The formalin stops any further decay in the tissue and renders it amenable to subsequent treatments needed for histopathologic processing.

The following day the formalin fixed tissue is further examined. The ulcerated tumor is visible at one end of the resected colon. Remember this is a view of the inside of an opened tube. The pathologist identifies the closest surgical margin and measures its distance from the tumor. Additional observations and measurements of the tumor are recorded. The tumor is sectioned revealing a firm white/tan tissue occupying the mucosa, or the lining of the bowel. The tumor thickness and its depth of invasion into the wall of the colon are measured. A slice of the tumor and adjacent uninvolved colonic mucosa is cut, and the more external uninvolved adipose tissue lying outside of the colonic wall is trimmed away. Selected tissue samples are then placed in individual cassettes each numbered by the patient’s surgical accession number. The cassettes are then transferred into a metal basket for further tissue processing.

In the histology lab, the metal baskets which are loaded with tissue cassettes are placed in a tissue processor to be treated overnight. Under a vacuum system, the formalin-fixed tissues will get exposed to graded higher concentrations of alcohol until dehydrated. Then the alcohol in the tissue is replaced by xylene which is a solvent capable of dissolving paraffin. Progressively the xylene is replaced by xylene/paraffin solutions as higher concentrations of paraffin are introduced. Ultimately the tissue is infiltrated by paraffin which renders it amenable to very thin slicing. The usual tissue thickness used for histopathology is between 4 and 8 micrometers.

The paraffin-infiltrated tissue is removed from the processor and taken out of its cassette. It is embedded in paraffin using a rectangular mold and the numbered cassette the tissue came with, to maintain tissue identification. Once cooled, the paraffin block containing the tissue will be cut on a microtome, with a sharp blade, at 5 micrometer thickness. Thin paraffin slices of tissue are then placed on a warm water bath and picked up on glass slides. Here the outline of tissue section is evident on the glass slide numbered according to the cassette number.

In preparation for staining, the tissue section must first be de-paraffinized and rehydrated so that aqueous stains can react with it. First the paraffin is removed by passing the glass slides through xylene, and then through graded concentrations of alcohol starting with 100% and ending with a 70% solution. For routine staining, hematoxylin and eosin, also known as H&E, are used. Being a basic dye, the dark blue hematoxylin has affinity for acidic molecules in the cells, such as deoxyribonucleic acid, or DNA, and ribonucleic acid, or RNA. For this reason the cell nucleus and the cytoplasmic RNA stain dark blue to purple. The red eosin, which is an acidic dye, has affinity for basic molecules, including the majority of proteins of the cytoplasm. For this reason, the red blood cells, which contain abundant cytoplasmic protein, but which lack a nucleus, stain bright red.

Following staining, the tissue must be protected for subsequent handling and archival filing. It will once again undergo dehydration through increasing concentrations of alcohol and then clearing with xylene which in turn allows for introduction of a form of glue called Permount. Permount will glue another piece of glass on top of the tissue, thus sandwiching it and protecting it against physical contact. Another property of the Permount is to unify the optical density of the entire slide allowing pathologist to look through the tissue sample. Note our section is in profile, similar to the piece of tissue which was initially sampled and placed in the cassette for processing.

Lastly the pathologist must review the tissue sections and reach a diagnostic conclusion based on the microscopic examination, as well as on the gross and clinical findings. In academic institutions as is the case at University of Wisconsin Hospital, residents and faculty work together to reach a diagnosis. The pathologist carefully examines all tissue sections using a light microscope which provides tissue magnifications which usually range between 20 and 1000.

Here at low magnification the lining of the colon also known as the mucosa is seen. On the right the mucosa is normal, containing organized columns of glands appearing as light staining tube-like structures. To the left half of the field the organization is disrupted due to crowded abnormal aggregates of dark staining glands which are haphazardly arranged. As you will see in a few minutes the dark staining is due to presence of increases number of cells in the tumor. These cells have abnormally enlarged nuclei as well. The size of nuclei, their increased number and their affinity for Hematoxylin causes the tumor to stain darker.

At a slightly higher magnification, the junction between normal mucosa on the right and the abnormal mucosa is observed. Amongst the abnormal glands necrotic tissue is seen. This is a reflection of abnormal growth of this tumor where at times due to rapid cellular proliferation the tumor cells become deprived of oxygen and nourishment and die.

At yet a higher magnification at the junction of tumor and normal mucosa, the difference between malignant cells and normal colonic cells is evident. On the right, the normal gland is lined by a single layer of columnar cells that have basally positioned nuclei and contain clear staining secretory material on the luminal side. On the left an abnormal gland is lined by multiple layers of haphazardly arranged cells. These cells contain relatively small amount of cytoplasm in relation to their enlarged nuclei. Several mitoses are also seen. This is a malignant gland of colonic mucosa and is also referred to as adenocarcinoma.

Finally back to lower magnification, it becomes evident that this adenocarcinoma is invading the wall of the colon and is about to enter its muscle layer on the bottom of the field. As of this point the histopathologic diagnosis would be: Invasive colonic adenocarcinoma.