What Goes on in an Histology Lab?
A Quick Overview
Prior to a historic winter, the Reno winter of 2005, I found myself unemployed. I had finished my Bachelors in Molecular Biology, had worked as a Medical Laboratory Technician in the military, and had worked as a Phlebotomist while at school.
The city was peppered with my resume for multiple positions, from Lab Assistant to Graveyard Phlebotomist. The job market at the time seemed dead and I quickly approached desperation.
It was at this point I found a training program being offered at Washoe Medical Center. Their lab offered a two year training course, with a promise of certification, in the field of Histology.
I had taken one course in Histology at school, that had centered primarily on tissue identification. I was not familiar with the technical skills needed to perform the job when I applied and was taken in as a trainee.
Since then I have finished my training, achieved certification, worked in a few labs, and have been active in my field for a few years now. The problem with my job is no one has ever heard of what Histology is or what it entails, and no one seems to be familiar at all with what the role of Histology is in the diagnoses and treatment of disease.
The best way I could think to describe the process of Histology is to tell the story of a patient and follow this patient from the collection of a biopsy, to the diagnoses, and then to any treatment by the patients physician.
Betty woke up Saturday morning and moved into the restroom to get undressed for her shower. As she undressed something unusual caught her eye, a mole. Had that been there the night before? Where did it come from? Betty decided to call and make an appointment with a dermatologist as soon as she could.
She made the appointment and the day came to see her dermatologist. He told her that it was probably nothing but wanted to get a punch biopsy of the mole and some surrounding skin. He performed the biopsy in the patient room in minutes and sent Betty home with a small bandage.
The specimen, or punch biopsy of mole and surrounding skin, had just started it's journey into the world of Histology.
The first step in specimen preparation is fixation. Fixation is described as a process that stops the tissue from going through autolysis and putrefaction and stabilizes proteins and other cellular components so that they will not change through the Histological process.
Autolysis is the action of enzymes, after the cell has died, that continue to break up cellular components and dissolve waste. Putrefaction is the action of bacteria on dead cells that cause what is called postmortem decomposition.
Fixatives will stop all enzymatic activity in the cell, put to death any bacteria living near the cell, and cause what is called a cross-linking between proteins within the cell that creates a matrix that holds the cellular components in place.
The most common fixative used is 10% formalin, which is 37% formaldehyde (a gas if 100%) diluted in water. Other fixatives are used if there are specific cellular components that need attention, for instance a Carnoy's Fixative is used primarily for RNA, DNA, or fat, for reasons I will not discuss here.
Betty's dermatologist took the fresh punch biopsy and placed it into a small bottle of 10% formalin to be fixed for the Histology lab. The size of the tissue determines how long the fixation, or penetration, takes. Usually only a few hours with small biopsies.
The bottle is labeled and off it went to the next step in the process.
By now Betty's mole has been packaged up and delivered to a local Histology lab for further work.
The mole was checked in at the lab and the patient data along with billing information was logged into a computer.
At this time the mole found itself in a line of fellow tissue also placed in 10% Formalin.
The mole is in a grossing room ready to be grossed by a Pathologist. Grossing is when the Pathologist examined the mole and dictated into a dictation device it's size, color, general appearance, and if there is any abnormalities noticable by the naked eye. Then the Pathologist trimmed down the tissue, or not in the case of the mole, and placed the tissue into a cassette for a procedure called processing.
The end product of Histology is to have a slide made with a thin section of tissue. The tissue, even after fixation, is still not firm enough to obtain a thin section from. The only way to ensure that the tissue can be cut into such a small section is to completely impregnate the tissue with a medium that is harder.
Most tissues are impregnated with Paraffin wax to harden and to hold the cellular components still for microtomy. This is called processing the tissue and is accomplished with three steps; dehydration, clearing, and infiltration.
Dehydration commonly uses alcohol to displace and replace the water in the tissue. Clearing commonly uses a hydrocabon like Xylene to remove the alcohol and to "clear" the tissue, or change the refractive index of the tissue so the cells can be seen on a microscope. Infiltration is the replacement of the xylene with melted paraffin wax that will harden the tissue and make the tissue ready to be cut.
Processing takes around twelve hours, unless a rapid process is used, and is commonly done overnight for the technician/technologist to work on the following morning.
Betty's mole was placed into a cassette by a pathologist, her information dictated, and the cassette placed into a processor to process overnight.
Histology begins early in the morning. Histotechnicians/Histotechnologist must be present when the process is finished to move the tissue from the processor to what is called an embedding center.
Also the pathologist like to have their slides made when they come in to work around 8:00am.
Anyway, back to the embedding center. An embedding center is a work area that includes a hot area to keep melted parraffin, and stainless steel molds.
The molds are filled with melted wax and the tissue is placed properly in the mold. After the tissue is placed in the mold, the mold is placed onto a cold plate to solidify. Once the wax in the mold is solidified the molded wax is removed from the molds and taken to a microtomy area to be cut.
The block, or the molded wax, is then faced. Facing is removing excess wax from the block and cutting into the block until a nice smooth area of tissue is available for microtomy. The faced blocks are then placed onto a ice bath. The ice bath lowers the temperature of the wax and makes it easier to obtain a thin section. An example would be trying to cut a block of warm cheese into thin slices compared to a block of cold cheese into thin slices.
Microtomy is performed on a microtome. A 3-5 micron section is collected from the microtome in the form of a ribbon. To help the reader understand how thin the section is, 3 microns is also the same thickness as a red blood cell.
A ribbon of sections is removed carefully from the microtome and placed on a waterbath set to 38 C. The waterbath removes any wrinkles from the sections and also spreads out the tissue for the next step.
Next, a section from the ribbon is picked up on a labeled slide and placed into a rack to dry. The slide with Betty's mole is now ready to be stained.
After the slide has dried, either on the countertop for awhile or in an oven, it is placed within an automated stainer that will stain the slide with an Hematoxylin and Eosin stain.
The first step in the automated stainer is to place the slide through xylene and a series of alchohols to remove paraffin from the tissue. Then the slide is placed in water to remove the alcohol. At this time the tissue on the slide is ready to meet a stain.
Hematoxylin and Eosin are the most common stain and counterstain used in modern histology labs.
Hematoxylin is extracted from logwood, or Haematoxylon Campechianum, as Hematein and is oxidized into a dye. Hematein has been extracted from logwood in Jamaica since 1715. Hematoxylin was introduced as a dye in England during Queen Elizabeth I reign, yet was not accepted as a dye until the 1840s.
When a metallic mordant is added to Hematoxylin the dye begins to have a stong affinity for nuclei and nuclear material, making the dye a nuclear stain. Nuclear stains are positively charged and cationic, which accounts for the affinity to amino acids. Hematoxylin stains blue.
Eosin is the common counterstain to Hematoxylin, and stains cell plasma components. Eosin is negatively charged and anionic which accounts for its affinity for positively charged cell plasma proteins. Eosin stains red or pink.
Once the slide is stained it is covered with a glass coverslip to keep the stained area safe. The slide is then ready to be looked under a microscope and examined by a pathologist.
The labs work with Betty's mole is over, the slide with the mole is now sent to the Pathologist to examine.
The End Product
The slide made it onto a Pathologists microscope and was examined promptly. He examined all the cells to ensure that there were no abnormal cells present. He finished up his report and sent it to the Dermatologist that had ordered the Histology work.
Later that week the Dermatologist office got the results and called Betty to let her now that not only was she able to get a mole removed but that the mole was benign, or not cancerous.
Betty was relieved and was able to sleep well since she had the mole removed two days earlier.
I still work in the field of Histology as an Histotechnologist. I love my work and I am proud to be a part of the medical community. There is currently a shortage in the field of Histology and I hope that this article will maybe excite someone to consider it as an employment possibilty.
The current certification agency is ASCP, American Society of Clinical Pathologists, and the requirement for a Histotechnician is an Associate Degree in Science and for an Histotechnologist is a Bachelors Degree in Science. Both require the candidate to sit down for a certification exam.
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