Cellular Pathology

Histopathology

Histopathology (or histology) involves the examination of sampled whole tissues under the microscope. Three main types of specimen are received by the pathology laboratory.

1. Larger specimens include whole organs or parts thereof, which are removed during surgical operations. Examples include a uterus after a hysterectomy, the large bowel after a colectomy or tonsils after a tonsillectomy.
2. Pieces of tissue rather than whole organs are removed as biopsies, which often require smaller surgical procedures that can be performed whilst the patient is still awake but sedated. Biopsies include excision biopsies, in which tissue is removed with a scalpel (e.g. a skin excision for a suspicious mole) or a core biopsy, in which a needle is inserted into a suspicious mass to remove a slither or core of tissue that can be examined under the microscope (e.g. to investigate a breast lump).
3. Fluid and very small pieces of tissue (individual cells rather than groups of cells, e.g. within fluid from around a lung) can be obtained via a fine needle aspiration (FNA). This is performed using a thinner needle than that used in a core biopsy, but with a similar technique. This type of material is usually liquid rather than solid, and is submitted for cytology rather than histology (see Cytopathology).

Specimens received by the pathology laboratory require tissue preparation then are treated and analysed using techniques appropriate to the type of tissue and the investigation required. For immediate diagnosis during a surgical procedure a frozen section is performed.

Tissue preparation

When these organs/tissues/fluids are received by the pathology laboratory, they are first placed into formalin to stop them from deteriorating (called fixation). They must then be examined by a medical scientist or anatomical pathologist. The pathologist or scientist will describe any abnormalities in the tissue and then carefully dissect it to aid in diagnosing what disease is present, and to select the areas of the specimen that should be put onto glass slides (processed) for microscopic examination. This initial process of tissue examination is called macroscopic pathology.

The areas of interest within the tissues are cut into small pieces, numbered and labelled and put through a series of procedures to end up with a prepared slide. These steps include dehydrating the tissue, placing it into a wax block to harden it, slicing extremely thin layers off of the block (less than half a millimetre thick), mounting these on a glass slide, staining them so that the tissue will be visible under the microscope and covering them with a cover slip so that the tissue on the slide will be preserved for many years. This process may take one to two days.

Special techniques in histopathology

Many additional methods of tissue analysis are available to assist in diagnosis if routine processing and microscopy do not give a definitive answer.

• Staining
• Immunohistochemistry
• Electron microscopy
• Flow cytometry
• Cytogenetics

Special stains

Pathologists use the chemical properties of components of the tissues being studied in their choice of different stains. The stain(s) are applied to the thin sections on glass slides to allow the pathologist to see the cells under the microscope. The most widely used stain is haematoxylin and eosin. This stain is a combination of a basic stain (haematoxylin) and an acidic stain (eosin), which react with acidic and basic cell components on the slide to give purple and pink colours to the tissues. Other stains available highlight fats, different tissue fibres, different types of mucus, microorganisms, proteins etc.

Immunohistochemistry

A major change in histopathology in recent times has been the development of immunohistochemistry. Where special stains are a relatively crude and, in most cases, relatively non-specific way of staining tissue components, immunohistochemical stains are by comparison far more specific in what they stain.

This technique involves attaching a dye to an antibody that will only bind to a certain protein type on or within a cell. Antibodies are like keys that can only open a certain lock (cell protein or antigen). Hundreds of antibodies are available which allow labelling of hundreds or even thousands of different protein types. Where a special stain may allow the pathologist to identify, for example, a cell as being cancerous, immunohistochemistry can identify which organ in the body that cancerous cell came from and how aggressively it may behave.

The dyes that attach to the labelling antibody can be also altered, including using different coloured dyes or even fluorescent dyes that are easier to see on microscopy. Some laboratories can use fluorescence-labelled antibodies to allow for computerised slide analysis, reducing the time taken to examine large numbers of slides and identifying which slides need to be reviewed by a pathologist and which are within the normal range.

Electron microscopy

The usual microscopes used by pathologists are not powerful enough to see the smallest parts that make up a cell. This is not usually a problem, but some diseases can only be diagnosed at this subcellular level. Examples include types of kidney disease (glomerulonephritis) or aggressive cancers which lose their normal proteins, making immunohistochemistry less useful in their identification.

In these cases a very powerful type of microscope is used called the electron microscope. This utilises beams of electrons rather than visible light to magnify the cells in a tissue sample. It can magnify up to 2 million times, whereas the maximum power of a conventional light microscope is only 1 to 2 thousand times.

Flow cytometry

This technique is used most commonly as an adjunct in the diagnosis of cancers of the blood cells (leukaemias and myelomas). Cells are suspended in a liquid and passed through a laser beam (single wave length light beam). A detector measures how the beam is scattered and if fluorescent light is emitted from excited particles on the cells. This is interpreted by a computer as a number of cells/ particles/ proteins (whatever substance is being examined for) shown on a graph. This can be used to give the quantities and relative proportions of different types of cells in the blood and identify any abnormal cells (e.g. leukaemias).

Cytogenetics

With the explosion in information about cell DNA (the genetic coding material) and genes that has resulted since the completion of the Human Genome Project, increasing numbers of genes are being recognised that, if faulty, may be involved in the development of disease including cancers. This is shaping up to change the way that disease is thought of, diagnosed and treated.

Cytogenetics is an umbrella term for the analysis of the genetic material (chromosomes and their DNA) of cells, and is becoming an increasingly widely requested component of the pathology workup of a submitted tissue. Techniques used include staining chromosomes (the form in which DNA is found in the cell nucleus) to reveal areas where genes may have been deleted, duplicated or broken. Fluorescent labels can also be attached to specific DNA sequences (parts of specific genes) which allow faulty genes to be seen when examining the cells under a special type of microscope. Direct sequencing of cell DNA is not routinely done for medical purposes at the present time.

As an example of the usefulness of cytogenetics one can look at breast cancer. Anatomical pathology can give a diagnosis of what type of breast cancer a patient may have, how far it has spread, whether or not it is likely to be an aggressive tumour and whether it will respond to hormone therapy. Cytogenetics can add to this information by identifying whether the patient has a faulty gene(s) which predisposed them to the development of breast cancer. If present, this would mean that they have an increased chance of developing cancer in the opposite breast and of developing other specific cancer types (e.g. ovarian cancer). It also has implications for the patient’s direct relatives and offspring. Did they inherit the faulty gene(s) and what are the chances that they will develop cancer in the future? There are also treatments being developed which will target the products of specific gene mutations in a patient.

Frozen sections

Frozen section is a technique for histological examination of tissue taken intra-operatively to give the surgeon a preliminary diagnosis whilst the surgery is still in progress. This may tell the surgeon, for example, that a suspicious mass is non-cancerous, allowing for more conservative surgery, or that a lymph node in the axilla (arm pit) contains cancer, requiring all of the axillary fat and lymph nodes to be surgically removed. Time is crucial in performing a frozen section, as the surgical team wants an answer as quickly as possible to reduce the amount of time for the patient to be under the general anaesthetic.

When a frozen section is performed, the histopathology laboratory will initially be notified that the surgical specimen is on its way from theatre, allowing time to prepare to receive the sample. The tissue will be examined on arrival by a pathologist, who describes and measures it before selecting the area of most interest to be processed (put onto a slide). Instead of the routine method of leaving the tissue in formalin to fix for a number of hours before cutting and staining it (see tissue preparation), it will be frozen using a cryostat or liquid nitrogen before sections are cut on a cooled microtome (cutting machine), mounted and stained by the medical scientist or anatomical pathologist. This reduces processing time from a day or two to 10 to 20 minutes. However, freezing results in some distortion of the tissue and a less satisfactory stain, making routine processing of tissue the preferred technique when tissue is submitted following a surgical procedure.

Another method which can be employed instead of freezing the tissue sent by the surgeon intra-operatively is of imprint smears. Here the tissue is cut and pressed or smeared onto a glass slide, then stained and examined under the microscope. This process leaves individual cells from the tissue on the slide and is therefore a cytopathology technique. Imprint smears further reduce the time taken to examine tissue and are most often used to check whether cancer has spread to involve a submitted lymph node (metastasis).

Cytopathology

Cytology is the study of individual cells and cytopathology is the study of individual cells in disease. Sampled fluid/ tissue from a patient is smeared onto a slide and stained (see techniques, below). This is then examined under the microscope by the anatomical pathologist to look at the number of cells on the slide, what types of cells they are, how they are grouped together and what the cell details are (shape, size, nucleus etc). This information is useful in determining whether a disease is present and what is the likely diagnosis.

Cytology is most often used as a screening tool; to look for disease and to decide whether or not more tests need to be performed. An example of screening would be the investigation of a breast lump. In combination with examination by the clinician and imaging tests, a needle aspirate of the lump submitted for cytology will show whether the breast cells are suspicious for cancer or look bland/ benign. If they look suspicious, a core biopsy with a larger needle may be performed which takes more tissue, allowing for a definitive diagnosis to be made before deciding what type of surgery is required (local removal of the lump or removal of the whole breast).