The Importance of Fixation

Proper fixation is the most important step in Histotechnology, and the quality of fixation will affect every subsequent step.  Fixation stabilizes the proteins throughout the specimen and prevents changes typically caused by postmortem activities such as mold or bacterial attack (putrefaction) and the continuation of enzyme metabolic processes (autolysis). Fixatives help maintain the relationship between cells and extracellular substances and change soluble substances within the cell to insoluble substances. They also provide contrast among different tissue constituents by enhancing the differences in their refractive indexes.  One of the most important actions of a fixative is to protect the specimen from the denaturing effects of the dehydrating agents and the ravages of subsequent processing steps. 

 

Actions of Fixatives

The primary method of fixation in the histopathology laboratory is by using one or more chemical reagents.  The chemicals alter the protein structure by either combining with the molecules or, most commonly, by disrupting the molecular structure. With the advances in histochemistry, immunohistochemistry and molecular biology procedures in recent years, some of the previous concepts of good fixation are no longer valid.  Fixatives and fixation techniques that offer good preservation of morphologic features may interfere with histochemical stains or antigen-antibody interactions by modifying or masking the reactive sites.  Selection of the proper fixative and determining the optimum exposure time requires careful evaluation of the desired structures and entities to be demonstrated and the effect of the fixative on the procedures that will follow.

Formalin Fixatives

Formaldehyde (CAT# 28530) is found in the majority of fixatives used in histopathology.  Concentrated formaldehyde is a solution of approximately 37% to 40% formaldehyde gas dissolved in water.  It normally contains methanol to inhibit the polymerization of paraformaldehyde, maintaining the proper concentration.  The concentrated solution is available in 1 gallon bottles or 5 gallon cubitainers. 

 

10% Neutral Buffered Formalin (CAT# 28600) is the most common routine fixative.  It is a 1:9 dilution of formaldehyde in water so, although it is referred to as a 10% solution, it is actually only 3.7% to 4.0% formaldehyde.  It is buffered with phosphate salts to achieve a pH of 7.0 which prevents the formation of formic acid.  Subsequent dehydration of tissues fixed in this solution should begin with no greater than 70% alcohol or the salts could precipitate in the alcohol and within the specimen.  The ready to use solution is available in 1 gallon bottles or 2.5 gallon and 5 gallon cubitainers.

 

10% Modified Carson/Millonig Formalin (CAT# 28610) is a dual purpose fixative that is excellent for routine histology or electron microscopy (even sections taken later from the periphery of stored specimens).  The 1:9 aqueous dilution of formaldehyde is buffered with sodium phosphate, monobasic and sodium hydroxide to a pH of 7.4.  Because the milliosmolality of the solution is very close to that of plasma, there is little extraction of cellular elements and ultrastructure is well preserved.  The ready to use solution is available in 1 gallon bottles or 2.5 gallon and 5 gallon cubitainers.

 

Zinc Formalin (CAT# BFZ6400) is gaining popularity as a routine fixative because it has the benefits of NBF but it does not crosslink.  This results in greatly enhanced immunopreservation and reduces the need for epitope retrieval.  The quality of the nuclear morphology makes it an excellent replacement for B-5, which contains mercury.  The buffered solution is readily soluble in alcohol and is no more corrosive than NBF.  The concentrated solution is available in 1 gallon bottles.  Each gallon of concentrate, when mixed with 4 gallons of water, will make 5 gallons of ready-to-use fixative solution.

 

Stat-Fix (CAT#SF-1) is a buffered alcoholic formalin fixative that was designed to enhance fixation of fatty or thick tissue sections.  It penetrates three times faster than NBF and has the added benefit of simultaneous fixation and dehydration making it an excellent choice as a secondary fixative on an automatic tissue processor.  It also performs well as a frozen section fixative.  Stat-Fix is available in 1 gallon containers.

 

Formalin Substitutes

GTF (CAT# FSXXXX) can be used just like 10% NBF, either as a primary or secondary fixative.  The active component is glyoxal, a two-carbon di-aldehyde, in a non-toxic, buffered solvent system containing ethanol and water.  It produces morphological patterns similar to aldehyde fixation without the disadvantages of cross-linking.  It is ideal for individuals with formaldehyde sensitivity.  It is non-carcinogenic and, at room temperature, has almost no vapor pressure; therefore, it does not evaporate and poses no inhalation hazard.  It has proven to be an excellent choice for microwave fixation.  Drain disposal of non-contaminated product is usually granted by wastewater treatment authorities and it is not considered a hazardous material when shipped in the U.S. by ground transportation.   GTF is available in 20ml, 40ml and 60ml prefilled containers.

Compound Fixatives

Bouin’s Solution (CAT# 11750) contains formaldehyde, an aqueous saturated picric acid solution and glacial acetic acid.  It is recommended for fixation of tissue containing soft, delicate structures such as testis, GI biopsies, and endocrine tissue and for any tissues that will be trichrome stained.  The acetic acid in the solution lyses red blood cells.  It can be used as a routine fixative for zoology, histology, embryology and parasitology.  Fixed tissues should be washed in 50% to 70% alcohol to remove the yellow color of the picric acid.  Fixation time should be limited to less than 24 hours.  The tissue can then be stored indefinitely in 70% alcohol.  It is available in 32 oz. bottles or 5 gallon cubitainers.

 

Hollande’s Solution (CAT# HFXXXX) is a stable modification of Bouins.  It is used routinely for the fixation of gastrointestinal biopsies.  The cupric acetate present in the solution preserves red blood cell membranes, the granules of eosinophils and endocrine cells.  It provides excellent preservation of protozoa, flagellates and ciliates to be demonstrated with silver stains.  It is capable of decalcifying small pieces of bone.  Like Bouins, fixation time must be limited and the fixed specimens must be washed in 50% to 70% alcohol before placing on the processor to prevent the formation of an insoluble phosphate precipitate.  The ready-to-use solution is available in ½ filled 20 oz and 60 oz plastic, screw top containers.

 

Factors to Consider When Using a Formalin Fixative

 

Three factors to consider when using formalin fixatives are ratio, size and time.  Maintaining an appropriate fixative concentration is critical for proper fixation.  Fixative concentration is affected during its penetration into the specimen because it’s diluted as it replaces the water in the specimen and further affected as its molecules attach to the tissue.  The ratio of fresh fixative to specimen should be at least 15:1, preferably 20:1.  Ratios of 10:1 have been shown to have an adverse affect on the overall quality of fixation. 

 

Specimen size is also a critical factor in fixation.  Formalin penetrates rather slowly and specimens greater than 4 mm thick may show signs of degradation in the center.  Large specimens should be “bread loafed” and specimens with capsular surfaces should be bisected when placed in fixative for grossing at a later time. 

 

Recommendations for proper formalin fixation range from 1 to 7 days, with a minimum of 24 hours.  This time frame is impractical in most labs and is consistently ignored, usually at the expense of specimen quality.  Formalin actually fixes in two steps.  The first step, which occurs as the fixative penetrates the cells and stops the metabolic processes, involves one end of the formaldehyde molecule reacting with and loosely attaching to a select tissue protein.  At this point, the attachment is easily reversible, histochemical reactivity is rarely impaired and the macromolecules within the specimen are not rigid.  The second step is much slower and can continue for a period of years.  It consists of the formation of a more stable bond, or “crosslinking”, between the remaining reactive end of the formaldehyde molecule onto the same macromolecule with the loose bond or with one that is nearby.  This causes firming of the macromolecules and masking of biologically active sites.  Different tissue types have different rates of reactivity during fixation.  Reactive sites respond differently too.  Some are masked within minutes while others can take days or longer to show signs of weakening.  The conformational changes caused by formalin fixation create the familiar microscopic morphologic images that have been the basis for pathologic diagnosis for many years.

 

It’s important to note that microwave accelerated fixation techniques offer quality fixation in a fraction of the time with no adverse affect on morphology or immunoreactivity.

 

Specimen Receipt

A specimen can be severely compromised before it ever reaches the laboratory.  Other than being cut off from its blood supply for quite some time before it’s removed, there are issues such as chemical or thermal treatment of the surgical site, physical trauma during removal, freezing and thawing of fresh tissue during cryotomy, fixation exposure delays and drying out during transport.  Needless to say, after removal from the body it is extremely important either to place the tissue into an appropriate fixative or freeze it as soon as possible.  Tissue can be held for short periods of time without extensive damage on saline-moistened gauze at refrigerator temperatures.  Large specimens should be “bread loafed” and capsular specimens should be bisected for adequate fixative penetration.

 

Gross Sectioning

A little time spent to correctly gross section tissue specimens will save a lot of time and energy later.  If the time isn’t taken to submit thin, well trimmed, homogeneous specimens, it will be difficult, if not impossible, to obtain good sections during microtomy.  Thick gross sections and overfilled cassettes do not permit adequate fixation or processing.  When specimens are too thick to process adequately and have to be “reverse-processed” before they can be sectioned, it compromises specimen quality and wastes time. 

 

Margin Identification

It is sometimes necessary to mark margins for orientation or to distinguish one specimen from another.  India ink was used routinely for many years but new substances have now been developed that are easier to use and don’t interfere with microscopic examination.

 

Margin Markers (CAT# SL62XX) are easy to apply and fast drying.  They are available in six colors; black, blue, green, orange, red and yellow, making them ideal for Moh’s surgery specimens.  The colors won’t fade and, since they are not absorbed by the cells, they are easily distinguishable from the staining used for microscopic evaluation.