Beyond the Ribbons: A Look at Fixing Agents Used in Blood Cancer Biopsy

Beyond the Ribbons: A Look at Fixing Agents Used in Blood Cancer Biopsy

September is blood cancer awareness month. We appreciate and encourage the sharing of potentially life-saving information about leukemia, lymphoma, and myeloma as well as the rarer forms of blood cancer throughout the month of September and beyond.

While we’re on the subject, we also want to give a shout out to the dedicated hard-working professionals responsible for the behind-the-scenes action… the work that takes place in the lab after the surgery or procedure. The work that involves the preparation and examination of the tissues of more than 16,000 newly diagnosed patients every month of the year (based on 2018 US average estimates).

One of the many steps that take place in the lab is fixation of the tissue for biopsy. The choice of which method to use out of the dozen or more possible options requires careful consideration of the specific tissue and circumstances.

Done as soon as possible after removal, the point of fixation is to preserve the structure and composition of the tissue in as close to its original state as possible. The fixative needs to penetrate the sample, diffusing into the inner layers and hardening the tissue in order to prevent decomposition, putrefaction, and autolysis. The fixation method chosen also affects the morphology, cytological detail, and immunoreactivity of the tissue.

What goes in will always be reflected in the quality of what comes out. Achieving high-quality biopsy results means starting with the correct materials and conditions. According to the University of Utah, getting these six things right is crucial to the process:

  1. Fresh tissue – fixation should be done as soon as possible after collection.
  2. Appropriate fixative with
    • Stable buffering 
    • Ample penetration capability
  1. Correct temperature – increased temperature speeds up fixation, enhances penetration, and affects the sample’s morphology.
  2. Optimal time – depends on the fixative, temperature, and thickness of the sample.
  3. Sufficient volume of fixative – recommendations range from 10 to 20 times the tissue’s volume.
  4. Proper fixative concentration – too high of a concentration can have adverse effects on the tissue (such as over-hardening), while too low may be less effective or increase the time needed for fixation.

Chemical Fixative Categories

Chemical fixatives can be grouped into four separate categories based on their chemical properties, according to a study published by the National Institute of Health. Here’s a quick summary.


This group works by cross-linking proteins, forming covalent bonds. They cause extensive denaturation of the proteins, however, which limits their use for immunohistochemical biopsy.

  • Potassium permanganate
  • Osmium tetraoxide


Alcohols also denature proteins and potentially result in brittle and over-hardened samples. They do act quickly and give good, clear nuclear detail, making them a viable option for cytological use.

  • Methyl alcohol
  • Ethyl alcohol
  • Acetic acid


Those in this group form insoluble metallic precipitates. They tend to show good nuclear details and can be excellent options for bone marrow biopsy.

  • Zinc (zinc chloride)
  • Mercury (mercuric chloride)


The most frequently used overall are the aldehydes, which work by cross-linking proteins.

  • Formaldehyde (formalin)
  • Glutaraldehyde

Formaldehyde does little damage to the structure, making it a top choice for immunohistochemical applications.  Glutaraldehyde is more commonly used for electron microscopy as it penetrates more slowly, preserving the cell structure. The cross-linkages formed by glutaraldehyde are considered to be irreversible.

Of the fixatives named above, many are used in combinations as solutions. A few of the most notable are below.

Mercury-Based Fixatives


Toxic but effective, mercuric chloride-based fixatives like B-5 yield high-quality results for hematopoietic tissue. B-5 produces ample cytologic detail but isn’t the best for DNA extraction and some immunostains. Close attention needs to be paid to the fixation time of between 2 and 4 hours… going over 4 hours will result in brittle tissue.

Hazardous materials handling and disposal requirements make it a more time-consuming and expensive option.

Zenker’s fixative

A mixture that includes mercuric chloride, potassium dichromate, and acetic acid, Zenker’s provides superb morphological detail and is fast-acting. Again, the handling and disposal of this mercury-containing solution involves being extra cautious and following hazardous materials disposal procedures. Fixation time is 4 to 24 hours.

Mercury-Free Fixatives

Favored for its superior morphological results, B-5 was once the gold standard. However, the hazards associated with the handling of mercuric chloride and disposal of the waste products, in addition to the fact that it is no longer allowed in many places, have pushed us to explore safer alternatives to mercury-containing fixatives.

AZF (acetic acid-zinc-formalin)

AZF is a less toxic alternative to B-5. A study published in the American Journal of Clinical Pathology concluded that it results in equally detailed morphology and possibly better antigen preservation for immunohistochemical studies, and it’s useful for DNA amplification. Fixation times are not nearly as stringent as B-5’s at 2 to 72 hours and as an added bonus, decalcification time is shorter.

A study published in 2005 by the American Society for Clinical Pathology found AZF to be the preferred fixative to replace mercury-containing formulas.

 Bouin’s solution

There aren’t many compounds that can be used as an antiseptic, yellow dye, and an explosive but picric acid, a component of Bouin’s solution, is all that and more. With a fixation time of between 4 and 18 hours, it provides good nuclear and chromosomal preservation and is a viable, mercury-free alternative to Zenker’s solution.

B-5 Modified with Zinc Chloride

Replacement of mercuric chloride with zinc chloride in this formulation yields clear nuclear detail. It works well for bone marrow biopsy with a reasonable fixation time of 3 to 72 hours.

Zenker’s Modified with Zinc Chloride

Zenker’s fixative can also be modified, using zinc chloride in place of the mercuric chloride. Another viable alternative, its fixation time is 2 hours to a maximum of 24 hours.


According to theCollegeofAmerican Pathologists, the most commonly used fixative is a buffered 10 percent formalin solution (3.7 to 4.0 percent formaldehyde and 1 percent methanol in water). The addition of methanol prevents the precipitate paraformaldehyde from forming in the concentrated solution. Buffering to a pH of 6.8 to 7.2 keeps the solution from oxidizing and becoming formic acid, which would react with hemoglobin. Another benefit to using buffered formalin is that it works better at a neutral pH.

The use of formalin as a fixative comes with several advantages:

  • It yields accurate results,
  • it doesn’t cause a lot of tissue shrinkage or distort cellular morphology,
  • it’s relatively inexpensive,
  • and clean-up is easy by comparison.

Fixation time is a minimum of 12 to 24 hours with the upper limit varying from days to months depending on multiple factors such as antigens and antibodies.

Neutral buffered formalin is referred to as “a standard fixative” by The International Council for Standardization in Hematology (ICSH) in their ICSH guidelines for standardization of bone marrow specimens reports adding that “It gives adequate morphology, preserves antigen for IHC and nucleic acid for molecular studies.”


While there are many almost-perfect options for fixation, formalin… 10 percent neutral buffered formalin to be exact… is the most universal and widely used of them. The final choice of which chemical fixative to use remains a careful balance of need, accessibility, and personal preference. 

We’re here to help those who help patients. Check out PathSUPPLY for your anatomical pathology or other laboratory supply needs or give us a call at 1-800-631-3556.