Histology: More Art than Science

      Histology is an interesting department; it is certainly part of the lab, but it is very different from the more normally thought of sections such as chemistry and hematology.  Histology is concerned with the pathology of tissues, and the bulk of the work involves the preparation, sectioning, and staining of specimens for examination by the pathologist.  Medical technologists, histotechnologists (who have gone through histotechnology school) and pathologist's assistants work under the supervision of pathologists (or a pathologist in smaller hospitals).  Examples of specimens that would be processed by histology include biopsies, removed organs such as appendixes and tonsils, and tumors that have been surgically removed.  Autopsy specimens are also handled by histology.  The main goal is to preserve the specimen as a whole and then to remove small sections from the specimen for staining and subsequent examination by a pathologist to see if there are any ongoing pathological processes.  While it sounds and is very scientific, much of it - especially when it comes to microtomy, which will be discussed more later - is also an art form, including the final product.  Below are a few images of stained tissues and after those I will discuss histology in more detail.

Thyroid gland stained with a Hematoxylin and Eosin stain at 100X magnification

Skin, also stained with Hematoxylin and Eosin at 100X.  The purple layer is the epidermis.

Lung, yet again with Hematoxylin and Eosin at 100X.  Open spaces are the alveoli or air sacs.

Colon, Alcian Blue PAS at pH 2.5 and at 400x magnification. The flower-like shapes are various carbohydrates and other macromolecules found in the cells of the lining of the colon that are involved in absorption.

      The histological process starts when the tissue arrives in the lab.  Samples can come from many different sources and methods of collection, and while the exact details are different for each, the general theory and procedure remains the same.  First, the sample is "grossed".  That means that the macroscopic appearance and features are observed and recorded.  For larger specimens, the next step is sectioning of areas of interest into smaller, more user-friendly pieces.  Where in the tissue the sections are from is recorded so that the pathologists can get a picture of what the whole specimen looks like.  Smaller specimens, such as needle biopsies, do not need sectioning.  Next, all specimens are fixed in one of a variety of fixatives.  The choice is based on a number of factors, many of which I don't know, but suffice it to say that fixation is done to preserve the tissue in a state as close to how it was in the body while allowing for subsequent stains to be performed; formalin is the most common fixative.
      Following fixation, samples are embedded into blocks to allow for cutting.  There are various embedding media available, but by and large the most common one is paraffin.  While the exact formulation varies from company to company and many companies offer multiple formulations, it is essentially a waxy compound that is solid at room temperature (roughly 24 degrees Celsius) but melts at a readily attainable temperature (roughly 58 degrees Celsius for the most commonly used versions).  The tissue arrives from the grossing room in plastic blocks and is then placed in a metal tray that is slightly larger than the tissue section.  A little melted paraffin is placed in the bottom of the tray, and then the tissue is pressed firmly into the bottom of the tray, orienting it according to any included instructions.  The tray is then filled with paraffin and allowed to harden.  These blocks are then sent to be cut.
      Cutting the blocks is accomplished by the use of a microtome.  Essentially, the microtome holds a block and moves it up and down while a blade is advanced towards the block.  It is calibrated so that it moves a set amount each time the block moves up and down, producing sections of a consistent thickness, usually around 4 micrometers.  Due to the waxy nature of the paraffin, these sections will form a ribbon when microtomy is done properly.  These sections are then placed on a hot water bath to allow them to smooth out and then they are picked up on microscope slides.  After drying, they are fixed to the slide, and then they are stained according to one of the myriad of staining procedures and techniques.  These stains are performed in order to demonstrate features of interest to the pathologist, such as connective tissue, muscle, carbohydrate stores, overall morphology, and deposits of various substances, just to name a few.  The pathologist then examines the slides microscopically to make a diagnosis.
      The above is the standard procedure, but as you can tell, this is a lengthy process.  In some instances, a diagnosis is needed much more quickly.  The primary case when a rapid diagnosis is needed is when a surgeon is removing a tumor and needs to know if it was entirely removed or if more tissue needs to be taken out.  The patient is in surgery, and waiting a few hours for a diagnosis is unacceptable.  When this scenario comes about, frozen sectioning is done in place of the fixation and embedding process.  By freezing and sectioning the tissue at temperatures well below 0, they can be made ready for routine examination rapidly.  In fact, the average time from when the sample enters the laboratory to when the pathologist is hanging up after calling the surgeon is 20 minutes whereas normal processing can take over day.  Frozen sections are not as good for many stains and must be processed additionally after initial examination to preserve them long-term, which is why that frozen processing is not the norm.  It should be noted that certain stains and procedures require a frozen section.
      While interesting, I am not sure that histology is where I will end up working, although it certainly is a unique section of the lab.