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Six Steps of Processing a Biopsy in the Histology Lab

Updated on February 3, 2017
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Chris has spent 26 years in laboratory work and has had personal experience with cancer, alcoholism and Willis-Eckbom Disease (RLS).

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

Pathologist looks at a slide prepared in a histology laboratory.
Pathologist looks at a slide prepared in a histology laboratory. | Source

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.

tissue in 10% formalin
tissue in 10% formalin

Step One-Fixation

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.

Step Two-Grossing

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:

  • Formalin
  • 70% alcohol
  • 80% alcohol
  • 95% alcohol
  • 100% alcohol
  • Xylene
  • 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.

Tissue Processors

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

hot paraffin pours from the embedding station into the mold.  Tissue is then placed in the paraffin mold and allowed to cool
hot paraffin pours from the embedding station into the mold. Tissue is then placed in the paraffin mold and allowed to cool | Source
a paraffin block with embedded tissue
a paraffin block with embedded tissue | Source

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.

Tissue Stainer

a robotic arm moves slides through the various reagents on this automatic tissue stainer
a robotic arm moves slides through the various reagents on this automatic tissue stainer | Source

The H&E Stain

Dark blue-cell nucleus; light pink-cell cytoplasm; red-erythrocytes (red blood cells)
Dark blue-cell nucleus; light pink-cell cytoplasm; red-erythrocytes (red blood cells) | Source

The Pathologist

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

one days worth of paraffin blocks in the histology laboratory
one days worth of paraffin blocks in the histology laboratory | Source

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.


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    • cam8510 profile image

      Chris Mills 2 months ago from Maple City, Michigan

      I am not a physician, so I won't comment on possible complications regarding your surgery. But I can comment on the process of getting the results to you. Depending on the facility where the surgery was done and where the lab work was to be done, it could be that the results are simply slow in coming. Since the surgery was on Friday, it is possible the slides were not ready for the pathologist until Monday. Results should have been to your doctor by Tuesday. But if your Doctor is busy and has other patients in more demand of immediate treatment, your case might be put on the bottom of a pile of charts. At this point, I would consider a long wait to be due to your case not needing immediate attention. I don't say that to minimize your situation, but if I am right, the wait is worth the good news.

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      Afi 2 months ago

      Hi .thanks for you nice information. I had tonsils remnant out in last Friday and the surgen sent that to the lab . I'm really worried because now it's six days and still no result came out . Pls tell my is that period time means there is something wrong ?

    • cam8510 profile image

      Chris Mills 13 months ago from Maple City, Michigan

      Thank you, Faith. It looks like this one could use some attention to rough edges and layout. I'll take advantage of the renewed activity and give it a facelift. Maybe it will get moved to a niche sight in time.

    • Faith Reaper profile image

      Faith Reaper 13 months ago from southern USA

      I pinned it to two of my Boards on Pinterest, "HubPages Writers' Work I Love" and "Health".

    • Faith Reaper profile image

      Faith Reaper 13 months ago from southern USA

      Oh, my pleasure, Chris.

      I came back to share on Pinterest, Twitter and G+ because (for some reason) I am unable to share to those sites from my tablet. The buttons are there, but the sites do not come up as normal.

      I for one, know firsthand about just how important it is to have the biopsy done as soon as possible with accurate results to determine further treatment.

      It seems our most important hubs were written when we first joined HubPages and, therefore, did not get the deserved attention they should have, being we were new and no one knew us or followed us at that time. I've tried to highlight the most important ones to me on my wheel on my profile page, and it seems to have worked, plus with the help of my followers ...once they knew of those hubs.

      Bless you for the important work you do,


    • cam8510 profile image

      Chris Mills 13 months ago from Maple City, Michigan

      RTalloni, I appreciate you visiting this hub and your comment. Thanks for helping keep this hub alive for people to read.

    • cam8510 profile image

      Chris Mills 13 months ago from Maple City, Michigan

      Paula, Thanks for visiting this hub. Yes, it is packed with information most people are not aware of. I am always excited to help others understand what happens when the biopsy is taken.

    • cam8510 profile image

      Chris Mills 13 months ago from Maple City, Michigan

      Faith, thanks for reading this hub and most of all, thanks for commenting. I wrote and published this article over four years ago. It gets about a thousand page views each year, which is fairly low, and you have the honor of posting the first comment ever.

      Yes, Histopathtology is important work, critical to the diagnosis and subsequent treatment of many diseases, most notably, many cancers. Thank you for helping to show people the process. Where there is knowledge and understanding, better choices are made.

    • RTalloni profile image

      RTalloni 13 months ago from the short journey

      Thanks for this practical look at what it takes for pathology to process a biopsy. It will benefit the average person wanting to know more than what docs usually say after, "You need a biopsy." Going to check out your other work.

    • fpherj48 profile image

      Paula 13 months ago from Beautiful Upstate New York

      Chris, I admit I find pathology a very fascinating study. You have presented an impeccable step by step explanation of tissue biopsy. Millions of people have discovered a biopsy has been necessary to diagnose any number of issues involving having a snip of tissue put through this process. These people know the anxiety of the dreaded wait for results .

      It is so important for patients to understand the reasons biopsies are done and especially how the process works & steps taken upon results.

      Thanks for this educational piece, Chris. Paula

    • Faith Reaper profile image

      Faith Reaper 13 months ago from southern USA

      Chris, you do important work! I found this all fascinating and was clueless what goes on behind the scenes after a biopsy is taken. I wish I knew of this when it was discovered I had DCIS breast cancer after they did a biopsy. You're so right about needing results as qickly as possible! I know once I had the mammogram and it showed large calcification, they did the biopsy within a matter of days.

      Seeing the photos there of all that was done in one day is sad knowing so many people had to have a biopsy, but that is what can help to save lives!

      Thank you for the important work you do, Chris.

      Wonderful comprehensive hub.