1. Surgical Removal of Tissue- In the laboratory, tissue is usually obtained from patients undergoing surgery. The tissue is analysed to make diagnoses as to the cause or histologic manifestion of the patient's problem. In each case, particular questions may be asked such as "Is it cancer?". Tissues for research are obtained in a variety of ways with approval from the Institution Review Board (IRB) and patient's consent if needed. In any case the preparation of tissue from the operating room is critical. For most histologic work, tissue will require preservation which may call for freezing or chemical "fixation".2. Fixation- Fixation is the interruption of dynamic cell processes such as self destruction from enzymes. There are many methods of fixation but the common thread is to render the structural elements insoluble via chemicals that precipitate proteins and other compounds. Various fixatives yield different visual and biochemical results and sometimes special fixatives are desired. The vast majority of tissue will be received in some chemical fixative from the operating room. If consulted ahead of time the pathology laboratory selects the type of fixative. Here are the most commonly used fixatives in eye pathology:
- Formalin- Neutral buffered formalin (10%) produces very little nuclear clumping and works by crosslinking proteins and DNA. Formalin penetrates tissue at a rate of about 1 mm /hour. Proteins are crosslinked via methylene bridges between amino groups. DNA can be crosslinked with proteins. Specifically Lys, Cys, His, and Trp cross link with dG, dA, and dC. Similarly DNA is crosslinked and denatured with interruption of interchain hydrogen bonds in AT-rich regions. DNA isolated in fomalin fixed tissue has variable sequence alterations that hinder its use for accurate sequencing. In general formalin is excellent for preserving most antigens for antibody reactions (used in immunohistochemical reactions).
- Ethanol- 50-80% acidified ethanol is an excellent fixative that appears to precipitate proteins and nuclei acids with little cross linking. This leaves DNA and even RNA intact for analysis. DNA and RNA are often stored for long periods in ethanol. Acidified ethanol is much better for preservation of DNA and RNA than formalin. For genetic studies ethanol is an excellent fixative to prepare the samples when DNA sequencing may be needed, such as in a tumor of children called retinoblastoma. Immunohistochemistry (intact antigens) may work very well in alcohol fixation but this is not guaranteed because as with formalin, epitopes may be altered by fixation. However, the disadvantage of alcohol is that it causes clumping of nuclear chromatin and shrinkage of cells that gives the appearance of nuclear atypia under the microscope. Actually the normal cells may appear atypical to the untrained eye because the nuclear clumping and shrinkage mimics the appearance of cancer cells. However, there are numerous advantages. The acidified ethanol mixture may be just a combination of vinegar and ethanol and can be relatively non-toxic, an asset for specimens referred from a far distance and transported through the a mail service. Formalin is poisonous. Also, alcohol and vinegar are household substances that are readily accessible to clients that don't keep formalin in stock.
- Paraformaldehyde- A polymer of 8-100 formaldehyde units, paraformaldehyde has the advantage of adequate fixation for electron microscopy and presumably somewhat reversible. Although expensive to maintain in the laboratory (out of light and refrigerated) paraformaldehyde is an excellent fixative when one is uncertain of the pathology. DNA extraction from paraformadehyde is somewhat similar to formalin and not ideal. Although presumably reversible, cross linking of DNA does seem to occur that limits the yield.
- Freezing- Freezing is an excellent preservative that keep antigens, DNA, and RNA relatively intact. For this purpose, rapid freezing is a preferred method of preservation for diseases in which the antibody epitopes are destroyed by cross linking. Rapid freezing also is excellent (like ethanol) when genetic studies are needed, particularly if RNA is required. Although acidified ethanol works, dry ice or liquid nitrogen may be the method of choice if readily available. The disadvantage is that the tissue must be kept frozen and shipped and stored at low temperature. This is expensive.
- Preservative Buffer- Tissue may be kept in some sterile neutral buffer (such as phosphate buffered saline) for several hours especially if kept cold. Specimens in the buffer can be transferred to the desired fixative or frozen once they reach the laboratory. This is a preferred way to handle ocular cicatricial pemphigoid cases that are sent locally where shipping can be occur in the same day. Buffers are used for specimens for ocular cicatricial pemphigoid, a disease where fluorescent antibodies are directly applied to sections in order to visualize basement membrane antigens. These antigens are not stable with fixatives such as formalin, paraformaldehyde and perhaps ethanol. So called Zeus Fixative provides stability for 1 day and is composed of potassium citrate, n-ethyl maleimide, magnesium sulfate and ammonium sulfate. Zeus Media is used for transport for outside specimens to a referral laboratory. It is not ideal.
- Osmication- Lipids are incidentally extracted from tissue by organic solvents used in histologic processing for paraffin embedding. Lipid removal can be vitiated by fixation with osmium tetroxide. Osmication is usually done in the electron microscopy laboratory. Osmium tetroxide is used to fix the lipids of a cancer of the eyelids known as sebaceous cell carcinoma. The lipids can also be preserved by freezing, because no organic solvents are used in processing this tissue prior to microtomy. Specimens fixed in this manner are generally embedded in a plastic resin and very fine 1 micron "thick" sections are made with a special microtome. The plastic sections (called "thick" sections) can be stained with toluidine blue, a dye that stains lipid green and viewed with a light microscope. If needed ultrathin sections (nm thick) can be made for electron microscopy.
- Decalcification- Bone fragments are very hard due to the presence of calcified osteoid and can ruin the microtome blade if not softened prior to sectioning. Calcium is removed by a process called decalcification, which uses acid and/or EDTA for removal. It is important that the specimen be well fixed prior to decalcification. Many of the decalcification solutions contain formic acid, hydrochloric acid and acetic acid. These are compatible with different fixatives. Formic acid works well with formalin. It is important to wash the specimen after decalcification with water and then to place it in ethanol. The acidic solutions are also inherently fixative although when used alone in high concentration can deleteriously affect the morphologic appearance.
3. Histologic Processing, Sectioning and Staining.- Tissues that have been fixed need to be infiltrated with some solution of paraffin, gelatin, celloidin, or plastic that later hardens. Tissue can then be cut into very thin sections. Paraffin is the most common infiltration and embedding compound and requires that the water be removed (dehydration). This is accomplished by using progressively more concentrated alcohols, e.g 50, 70, 80, 95, 100% with several changes at 95, and 100% and then an organic solvent like xylene for several changes with final immersion into a hot paraffin bath. The tissue can then be carefully mounted in a slab of paraffin (called a paraffin block) for fine cutting (microtomy). Histologic sections cut from a paraffin block with a rotary microtome are generally 4-6 microns in thickness in experienced hands. This is a very important skill that some lab members will need to know well. Histologic stains are varied and can be used for visualization of specific features. In general, the laboratory uses hematoxylin and eosin stains (H&E) for almost every tissue as a start. Nuclei are stained blue and cytoplasm pink with H&E. H&E is a work horse stain that is good for a general view. Periodic acid-Schiff (PAS) is an excellent stain for carbohydrates. For example mucins in the conjunctiva and cornea stain bright pink with this stain. There are a litany of other special histologic stains used by the laboratory and varied antibodies available for immunohistochemistry.
4. Microscopic Analysis- The most common microscope used in the laboratory is the compound light microscope. Visible light shines through the stained tissue section and is collected by objectives and condensing lenses. The resolution of the microscope is limited by the numerical aperature of the lenses and approaches about .25 microns. For reference a red blood cell is about 7-10 microns in diameter and bacteria are about .1-10 microns depending on the species. Epithelial cells may measure 20-50 microns in diameter depending on the location.
For gross dissection a stereomicroscope is used to carefully identify and ink margins. The total magnification here is equivalent to the low mag of a compound microscope.
Also used in the laboratory are the fluorescent microscope, electron microscope, and laser scanning confocal microscope depending on the needs (usually research and rarely a clinical specimen). The best resolution is obtained with an electron microscope in tissue sections.