Whether removed surgically, traumatically, or via autopsy; tissue samples to be reviewed by pathology will be processed to facilitate creation of microscopic slides. Slides are sheets of glass with mounted slices of tissue cut so thin that light shines through. Through staining, the pathologist can use a microscope to identify characteristics of the cells based on their shape and color. The formation of the cells and relationships with adjacent cells is how pathologists make such a precise diagnosis. How the large pieces of tissue are processed to create diagnostic slides is an interesting process involving many steps and various laboratory professionals.
The first step to creating high quality diagnostic slides is appropriate sampling of the tissue during the gross examination and dissection. Pathologists or Pathologists’ Assistants (PA, ASCP) use their knowledge of anatomy to remove sections of tissues that are about the dimension of a quarter, or smaller. The small sections of tissue are oriented such that the area of pathologic interest and the surrounding structures can be seen in the same plane when the sample is embedded in paraffin. For example, when sampling a colon tumor the pathologist will need to see the tumor’s relation to the anatomic structures of the colon; primarily the muscularis, serosa, and pericolonic fat. The size of the tumor and involvement of adjacent structures are used to assign the Tumor portion of TNM staging. A great diagnostic slide will show multiple features needed to determine an accurate diagnosis, this is accomplished through skillful gross dissection.
The tissue samples are placed into special holders called cassettes then placed into a fixative. The fixative used is determined by the type of studies that will be performed. The most common fixative used in the creation of diagnostic tissue slides is 10% neutral buffered formalin. Other common fixatives include Alcohol, B-5, Bouin’s, Carnoy’s, and Zenker’s solution. Regardless of the agent, the purpose of fixation is to rapidly stop the metabolic processes of cells and preserve them so that they can be observed in as natural appearing state as possible. Formalin only penetrates tissues approximately 1 mm/hour so tissues are allowed to bathe in the fixative for an appropriate period prior to processing. Fatty tissues tissues such as breast or brain are often soaked for longer periods to ensure adequate fixation. Samples containing bone go through an additional process called decalcification.
The procedure used to process tissues to make slides is not much different than it was 100 years ago; formalin fixed tissues are dehydrated, cleared, and infiltrated prior to embedding. Dehydration is accomplished by bathing the tissues in high concentrations of alcohol. As water moves through membranes from areas of greater concentration to lesser, the alcohol bath draws about 99% of water from the cells.
Unfortunately, paraffin and alcohol do not mix so the alcohol must be removed from the tissue prior to infiltration. The removal of alcohol is accomplished by bathing the tissue in an organic solvent and the process is called clearing. Many organic solvents used in tissue processing such as xylene, benzine, acetone, and chloroform are hazardous to workers health so some facilities use a synthetic xylene substitute.
The tissues, now free of water and alcohol, are soaked in a warm paraffin bath. The paraffin is absorbed into the tissues providing support for cellular structures. This processing step is called infiltration.
To complete all the steps needed to process tissue prior to embedding can take twelve hours, or longer. Some hospitals or research labs still process histology samples by hand but most use an automated processor. Automated processors allow for uniformity in samples and work while unattended. The processor works by either moving baskets containing samples to different baths or by pumping chemicals into and out of a common bath.
A histotechnologist (HT or HTL) will open the cassette used to hold the tissue during processing and embed the tissue in a paraffin block. The histotech removes the sample from the cassette and aligns it in a metal mold that matches the size of the cassette. The histotech may orient the tissue as it was found in the cassette or, if instructed by the person that grossed the sample, embed the tissue on-edge. The cassette is placed over the oriented tissue sample then melted paraffin is poured through the cassette to fill the mold. When the paraffin cools it is removed from the mold. Using the cassette as a base for the mold allows for thorough tracking of samples and many of today’s microtomes are designed to use cassettes as a chuck.
The paraffin block is placed on a special machine designed to cut very thin slices called a microtome. Slices 3-15 microns (0.00035 inches) thick are cut from the block as it moves across a very sharp blade. The paraffin holds the adjoining slices together to form a ribbon of sequential slices that can be floated onto a bath of warm water. The paraffin floats on the water and can be manipulated to remove folds created during cutting. A glass slide is inserted beneath the best area and lifted, allowing the tissue to rest on the glass. The glass slide is placed into an oven and baked. Baking allows excess paraffin to melt away and causes the tissue to adhere to the glass. Some slides contain a protein such as albumin or are statically charged to facilitate tissue adherence. Ensuring the tissue does not fall off the slide is important as they are about to go through another series of baths.
The the process of dehydration and clearing, some color components of tissue is removed, leaving only a faint outline of cell walls on the slide. Staining the tissue is necessary for the pathologist to observe the cells shape and structures. The most common stains used for anatomic pathology is hematoxylin and eosin (H&E). Hematoxylin stains the nuclear material blue while eosin counter-stains the cellular cytoplasm pink. A multitude of special stains and antibodies may be used to to further classify tissue types. Once stained, the glass slide has a thin, clear cover slip attached as a final step.
From grossing to cover-slipping, the steps to process a specimen into permanent samples on a glass slide can take 12-24 hours; or longer if decalcification is required. While the pathologist reading the slide may only take a matter of minutes, it is the work that goes into the sampling, processing, embedding, and staining that make the sample suitable for making a diagnosis.