The Journey Towards Histopathology

Today I was fortunate enough to attend a lecture by Carol Stoddard revealing the operation of Derriford Hospital’s pathology department. I was particularly looking forward to this lecture due to my keen interest in learning about histology. Due to the brevity of the lecture relative to the extensiveness of the subject field, I was empowered to conduct further research into the techniques used to diagnose diseased tissues that were mentioned in the lecture.

Histopathology is the branch of pathology which deals with the tissue diagnosis of disease. A tissue diagnosis can be made on the basis of biopsy material taken from the patient on the ward or in the operating theatre, or from autopsy material. The latter is a small but important component of the work, establishing the cause in cases of sudden or unexpected death, monitoring disease progression or the response to treatment, and in criminal cases helping police in their investigations. The former is by far the larger component of a histopathologist’s workload. In any patient subjected to biopsy the final diagnosis is made by the histopathologist and this in most cases determines the clinical management of a patient.

Made very apparent to me today was the breadth of knowledge required by histopathologists; needing a broad-based knowledge and understanding of the pathological and clinical aspects of disease. With the help of the interventional radiologist and sophisticated imaging techniques, biopsy tissue can now be obtained from previously inaccessible sites such as the pancreas. This may involve direct contact with the patient, with the pathologist undertaking fine needle aspiration (biopsy procedure whereby a fine needle is inserted into a prospective tumour) to obtain material for cytological examination for a woman with a lump in her breast or a man with an enlarged prostate. Special staining techniques allow you to examine the specimen immediately and in 90% of cases the diagnosis can be made before the patient leaves the clinic. Carol Stoddard mentioned a biopsy sample could be prepared and sent to a pathologist within just  20 minutes of being taken, however standard protocol uses paraffin wax which requires several hours to set.

In order to examine biopsies the following procedures occur within a hospital. After the doctor removes the biopsy specimen, it’s placed in a container with formalin (a mixture of water and formaldehyde) or some other fluid to preserve it. This specimen is subsequently sent to the pathology lab where a pathologist will perform a gross examination of the tissue (without a microscope). The gross examination includes the tissue sample’s size, colour, consistency, and other characteristics.The gross examination is important since the pathologist often sees features that suggest cancer. It also helps the pathologist decide which parts of a large biopsy are the most critical to study under a microscope for further analysis.

For small biopsies, for example, a punch biopsy or a core needle biopsy, the entire specimen is usually looked at under a microscope. The tissue to be looked at under the microscope is placed into small containers called cassettes. Having seen such a cassette today, the appearance resembles a cassette only in the sense of size. Cassettes are small plastic trays with numerous small holes. The cassettes hold the tissue securely while it’s processed. After processing, which may take a few hours but is usually done overnight, the tissue sample is placed into a mould with hot paraffin wax. The wax cools to form a solid block that protects the tissue.

This paraffin wax block with the embedded tissue is placed on an instrument called a microtome, which cuts very thin slices of the tissue. This was aptly described as a glorifies ham slicer. These thin slices of the specimen are placed on glass slides, and dipped into a series of stains or dyes to change the colour of the tissue. The colour makes cells easier to see under a microscope. For most biopsy specimens, routine processing as just described is all that’s needed. At this point (usually the day after the biopsy was done), the pathologist looks at the tissue under a microscope.

Sometimes, however, a surgeon needs information about a tissue sample during surgery to make immediate surgical decisions. If the surgeon cannot wait the day or more that it will take for routine processing and histology, he or she will request an intra-operative pathology consultation. This is often called a frozen section exam.

When a frozen section exam is done, fresh tissue is sent from the operating room right to the pathologist. Because the patient is often under general anesthesia (kept asleep with drugs) it’s important that the tissue be looked at quickly. It usually takes 10 to 20 minutes. The fresh tissue is grossly examined by the pathologist to decide which part of it should be looked at under the microscope. Instead of processing the tissue in wax blocks, the tissue is quickly frozen in a special solution that forms what looks like an ice cube around the tissue sample. It’s then thinly sectioned (sliced) on a special machine, quickly stained (dipped in a series of dyes), and looked at under the microscope. The frozen sections usually do not show features of the tissue as clearly as sections of tissue embedded in wax, but they are good enough to help the surgeon make decisions during the operation.

How cytology specimens are processed depends on the type of specimen. Some specimens are smeared on glass microscope slides by the doctor who gets the sample. The slides, which are called smears, are then sent to the cytology lab where they are dipped into a series of stains (coloured dyes), much like those used for biopsy samples. Other specimens, such as body fluids, cannot easily be placed on a glass microscope slide because they are too diluted (there are too few cells in a large volume of fluid). Cytology labs have ways to concentrate these cells on a glass slide before staining. A centrifuge is first used to isolate the desired cells, then further concentration precesses (that weren’t made explicit in the lecture) are used to make the sample viably observable.

 

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