Obtaining Specimens

There are three main ways of obtaining specimens for pathological examination: 1) biopsies, where small amounts of tissue are removed, often using a wide bore needle; 2) surgical resection, where parts or whole organs are removed during surgery; and 3) autopsy, where specimens are obtained after a person has died to try and determine the cause of death.

Preservation

When cells, tissues, and organs are removed from the body and deprived of their normal environment and blood supply, they start to die, and will release enzymes that start to break down the cellular and tissue structures. Therefore, in order to distinguish any pathological changes to the organ, these artificially induced changes must be prevented from occurring. The main method of preserving the tissue and preventing these changes is using a chemical fixative.

Chemical Fixatives

The most common chemical fixative is formalin, which is a buffered solution of formaldehyde. There are other chemical fixatives that can be used that are better at preserving specific components of the tissue, but their use is specialized. An important limitation of a fixative is the slow penetration rate, which requires the specimens to be thinly sliced to make sure they get complete penetration before degradation occurs. When a specimen is cut up for fixation, care must be taken to retain the features that you want to look at (e.g. perpendicular to the surface if you want to look at the layers).

Preparing for Viewing

Specimens intended to be examined under the microscope need more extensive preparation than those to be viewed grossly, or macroscopically.

Specimens for Gross Examination

Pathologists will examine specimens before fixation to mark any changes on the outside of the specimen before dissecting and viewing the inside of the specimen. They will usually then select areas of the organ that they want to view microscopically to see the changes occurring at that level. They will then remove a block of tissue, which will be processed for microscopic viewing.

Specimens for Viewing in the Museum

Specimens that show interesting features of a disease, with consent obtained from the individual or family for educational use, may be processed for viewing in the museum. The specimens are already sliced and fixed in formalin. The specimens are soaked in a series of solutions to try to remove excess red blood cells, which tend to cloud the solutions, restore colour as much as possible, and to replace the water-based solutions with a glycerol-based solution to provide the right refractive index so that there is no distortion when viewing the specimen through plexiglass or glass. This final solution, Kaiserling III, is non-toxic.

The specimens are then mounted in a plexiglass case with a stopper that allows the fluid to be topped up and replaced when it gets depleted, cloudy or discoloured.

Specimens for Viewing Under the Light Microscope

In order to view tissue under the light microscope, the tissue must be thin enough to: a) allow light to pass through, and b) thin enough to distinguish features (i.e. not have too many cells piled up on one another so you don’t know which features belong to which cell). They also need to be coloured, or stained, in some way. Otherwise, you are looking at a clear specimen, making the details hard to distinguish. They are then covered with a coverslip to preserve and allow viewing at high magnification with oil immersion.

Paraffin Embedding

If you have ever tried to cut into fresh chicken or a steak, you will notice how ‘wobbly’ the tissue is and how the skin has a different consistency from the muscle below. These characteristics make it difficult to section tissue thinly and consistently without embedding in a supportive medium. Paraffin wax is the most commonly used medium to provide this support. However, if we just placed a tissue directly into paraffin, it would pop out of the paraffin during sectioning as paraffin cannot penetrate into the water-based tissue (paraffin is immiscible with water). Therefore, we need to put the tissue through a series of solutions where we replace water with alcohol, then alcohol with xylene and finally xylene with melted paraffin wax. This process is known as tissue processing.

Fortunately, processing has been automated, or you would be there all night.

Once tissue is completely infiltrated with paraffin wax, it can be brought to the tri-console embedding machine, where it is placed into the molten wax in the cassette holding tank. The technician then removes the tissue from the cassette and embeds the specimen completely into molten wax. Placing the final product on the cold plate solidifies the wax in preparation for cutting (sectioning).

Paraffin Sectioning

There are several types of microtomes, depending on what it is that you need to cut. Smaller rotary microtomes are used to cut paraffin embedded specimens into thin sheets (typically 5 – 8 µm thick) that can be placed in a warm water bath and picked up on glass slides. So why are we doing all of this work and completing all of these steps? We need to cut VERY THIN pieces of tissue in order to see them with a microscope.

Staining

Unstained tissues cannot be viewed using a standard light microscope so we must use different dyes to stain the tissue and distinguish components. Unfortunately, these dyes cannot bind to the tissue when it is full of paraffin, so we must remove the paraffin completely. Basically, what happens is we reverse the process we used to infiltrate the tissues with wax using xylene to remove the wax, alcohol to remove the xylene, and if it is a water-based stain, we use water to remove the alcohol.

Using multiple dyes makes it easier to distinguish between components. The most commonly used stain combination is Hematoxylin and Eosin (H&E).

Coverslipping

We are now almost ready to view the tissue under the microscope, but first we need to put on a coverslip, which is a thin piece of glass or plastic that is 0.17mm thick to maximize resolution. This won’t stick by itself, so we need to use an adhesive to attach it. This adhesive cannot interfere with the dye or the viewing of the specimen. They are usually dissolved in toluene or xylene. So, guess what? We get to replace the water from the dye baths with alcohol and then the alcohol with xylene.

Section Review

Pathology specimens are obtained from patients (given with consent and permission) during their health journey. The specimens can be a block of tissue, which is preserved in chemicals (to prevent tissue degradation) before mounting in a plexiglass case, for viewing the gross anatomy. Tissue specimens, which are to be viewed microscopically, have the additional step of being solidified (paraffin wax embedding) and sliced thinly before staining and covering with a coverslip.