Six Steps of Processing a Biopsy in the Histology Lab
This article describes the six steps which biopsy tissue goes through in the pathology lab. In order, they are fixation in formalin, grossing by a Pathologist or Pathology Assistant, processing through four reagents, embedding in paraffin, cutting sections for mounting on slides, staining with Hematoxylin and Eosin stains.
Pathologist Looks at a Thin Cross Section of a Biopsy on a Glass Slide
What is a Biopsy?
A biopsy refers to tissue which has been removed from a living body for examination, in order to discover the presence, cause, or extent of a disease.
The Histology Lab
So, you had a biopsy taken. It may have been a biopsy of your breast, cervix, prostate, lung, liver, kidney or something else. Probably the last thing on your mind while waiting to hear from your doctor, is "I wonder what's happening to my biopsy right now?" No, you want your medical laboratory results, and you want them quickly because life could change dramatically with one call from your physician. I work as a traveling histology technician in labs around the United States. The histology lab is where all biopsies go to be prepared for analysis by a pathologist. I would like to describe this process through which virtually all surgical specimens will pass.
You are probably familiar with how a rope unravels when you cut it. The same thing happens, on a microscopic level, to the small piece of tissue called the biopsy. Chains of protein molecules begin to degenerate almost immediately after the tissue is removed. We call this tissue autolysis. Think again of the cut rope. One thing that can be done to stop it from unraveling is to hold a lit match up to the frayed ends so that the fibers melt together. To stop tissue autolysis, the biopsy is put into a tissue processor which uses 10% neutral buffered formalin to cross link the loose ends of protein molecule chains, thereby preventing further decomposition. This process is known as tissue fixation. After the Doctor removes the piece of tissue, the first thing he does is to place it into a container of 10% neutral buffered formalin.
When the surgical specimen arrives in the Histopathology Lab, it is taken to a grossing station. This is where it is thoroughly inspected for disease, measured and otherwise described. A voice recording, known as gross dictation, is made and subsequently typed into the patient's permanent report. The biopsy is then secured in a plastic tissue cassette which is placed in a bath of formalin.
Grossing of a Biopsy
Step Three-Tissue Processing
A histology technician takes all of the cassettes grossed that day and puts them in a tissue processor. Depending on the size of the individual pieces, tissue processing can last from about four hours to thirteen hours. Tissue processors move the tissue through the following series of chemical reagents:
- 70% alcohol
- 80% alcohol
- 95% alcohol
- 100% alcohol
- Liquid paraffin
Formalin fixes or stops decomposition of the tissue. Three grades or percentages of alcohol slowly dehydrate the tissue. Xylene removes the alcohol from the tissue. Liquid paraffin replaces the xylene and permanently infiltrates the tissue.
Step Four-Embedding Tissue in Paraffin
The plastic cassettes which contain pieces of tissue are then taken to an embedding station. The tissue is removed from the cassette and placed in a mold which is then filled with liquid paraffin. The part of the cassette which has the patient's hospital number printed on it is then placed on top of the mold. After cooling, the cassette top and the paraffin embedded tissue become one unit known as a paraffin block.
Tissue Embedding Station
Step Five-Microtomy: Cutting Thin Sections of Biopsy Tissue to Place on Glass Slides
The paraffin block is then taken to an instrument known as a microtome. The block is placed in the block holder, which then passes across a blade. Each time the block passes the blade, it advances forward by three or four micrometers. This is approximately the thickness of one tissue cell. The result is a section of paraffin and biopsy tissue. As the tissue continues to pass across the blade, several sections are produced in a long ribbon.
This ribbon is picked up by the histology technician and floated on a bath of warm water. Wrinkles are allowed to smooth out, and the technician slips a glass slide under the section and lifts it from the water. The slide, with a very thin section of paraffin and tissue, is the result of microtomy.
Step Six-Staining the Biopsy Tissue
After drying in a sixty degree celsius oven, the slide is passed through another series of chemical reagents. Xylene removes the paraffin and absolute alcohol removes the xylene. The tissue on the slide is then rehydrated to prepare it for staining.
The two staining reagents are hematoxylin and eosin. Hematoxylin stains the nucleus of the tissue cells dark blue to black. Eosin stains the interior of the cell surrounding the nucleus light pink and the tissue outside the cell dark pink to red. These stains enable the pathologist to view the whole cellular makeup of the specimen in order to make a diagnosis.
The stain, using hematoxylin and eosin, is known as an H&E stain. It is the primary stain used in the Histology Laboratory and is performed on virtually every tissue specimen that comes through the lab. Other stains can also be used to emphasize different structures in the tissue. These Special Stains are usually ordered after the pathologist has viewed the initial H&E stain.
The H&E Stain
The Pathologist is a Medical Doctor who specializes in the diagnosis of diseases. He/She, after microscopically examining the tissue, communicates with the patient's physician. The whole histology process is recorded in the pathology report which is, in most laboratories, electronically sent to the physician's office.
All in a Day's Work
The Results of Processing a Biopsy in the Histology Lab
The phone call from the doctor's office comes. The physician communicates to the patient the content of the pathology report. In most cases the patient's doctor hears from the pathologist within twenty-four hours. Of course, the doctor is busy and may not communicate with the patient immediately unless the diagnosis requires immediate attention.
Understanding and Appreciating the Role of the Histology Lab
Hopefully, as a result of this article, you can appreciate all the scientific work that is going on behind the scenes if you ever have a biopsy taken. The scientific process is performed by highly trained individuals who try always to keep in mind that there is a person out there waiting to know whether or not their life is about to drastically change.