AN APPRAISAL OF FIXATIVES AND THEIR FUNCTIONS IN

HISTOPATHOLOGY

BY

OGBENI, COMFORT
(INTERN OF MEDICAL LABORATORY SCIENCE)
A SEMINAR PRESENTED TO ANATOMIC PATHOLOGY IRRUA SPECIALIST TEACHING HOSPITAL
EDO-STATE

SUPERVISOR: MR. OSIGBEMHE VICTOR

NOVEMBER, 2023
 ABSTRACT
Fixation is a process involving a series of complex chemical modifications of
macromolecules present in tissues and cells to preserve the structural and
functional components as closely as possible to the living state. By definition,
fixatives change the original chemical and physical compositions of tissues. In
addition to altering the chemical nature of the cells and tissues to which they are
applied, fixatives also cause physical changes to cellular and extracellular
components. Fixation of tissues can be achieved by chemical or physical means.
Physical methods include heating, micro-waving and cryo-preservation (freeze
drying). Classification of chemical fixative includes simple fixative and Compound
Fixatives. There are two major mechanisms which are important in fixation of
proteins and protein complexes: denaturation, and addition and cross-link
formation. The common functions of fixatives in the pathology laboratory based
upon the nature of the fixatives, the type of tissue, and the histologic details to be
demonstrated. Factors affecting fixation includes buffering, penetration, volume,
temperature, concentration, time interval. Faults in fixation are permanent and
cannot be corrected at any later stage whereas any other step after proper fixation
can be reversed to refine a problem
 OUTLINE
1. ABSTRACT
2. OUTLINE
3. INTRODUCTION
4. TYPES OF FIXATIVE
5. CLASSIFICATION OF CHEMICAL FIXATIVE
6. MECHANISM OF ACTION OF FIXATIVE
7. FUNCTIONS OF FXATIVES
8. QUALITIES OF A GOOD FIXATIVE
9. FACTORS AFFECTING FIXATION
10. CONCLUSION
11. REFERENCE
 INTRODUCTION
• Fixation is a process involving a series of complex chemical
modifications of macromolecules present in tissues and cells to
preserve the structural and functional components as closely as
possible to the living state.

• By definition, fixatives change the original chemical and physical

compositions of tissues.

• In addition to altering the chemical nature of the cells and tissues

to which they are applied, fixatives also cause physical changes to
cellular and extracellular components

(Lykidis, Van Noorden and Armstrong, 2007; Puchtler and Meloan,

2015)
 TYPES OF FIXATION
Fixation of tissues can be achieved by two
methods:

Physical methods:
• Heating, micro-waving and cryo-preservation
(freeze drying).

Chemical methods
• Immersion fixation
• Perfusion fixation
 CLASSIFICATION OF CHEMICAL FIXATIVE
Simple Fixative
– Formaline
– Mercuric chloride
– Osmic acid
– Picric acid
– Acetone
– Ethyl alcohol
– Osmium tetroxide

(Srinivasan et al., 2002).

 CLASSIFICATION OF CHEMICAL FIXATIVE
Compound Fixatives Microanatomical
– Formal Saline
– Neutral buffer Formaline
– Zenker’s fluid
– Bouins fluid
•
Cytological
– Nuclear: carnoy’s fluid
– Cytoplamsic: champy’s fluid
•
Histochemical
– Cold acetone
– Ethanol

(Srinivasan et al., 2002).

 Figure 1: A paraffin section of kidney that has been fixed using neutral
buffered formalin. This is an example of well-fixed tissue showing good
nuclear and cytoplasmic morphology with minimal shrinkage showing clearly
defined basement membranes and cell margins (WHO, 2006).
 CLASSIFICATION OF FIXING AGENTS AND THE
MECHANISMS OF FIXATION
• Denaturation: Most commonly this effect is induced
by dehydrants such as the alcohols or acetone.

• Addition and cross-link formation: The non-coagulant

fixing agents chemically react with proteins and other
cell and tissue components, becoming bound to them
by addition and forming inter-molecular and intra-
molecular cross-links.
(Gillespie et al., 2002; Delfour et al., 2006)
 FIXATIVES AND FUNCTIONS
• to preserve tissues permanently in as life-like a state as
possible.

• after removal of the tissues (in the case of surgical

pathology) or soon after death (with autopsy) to prevent
autolysis.

• a variety of fixatives are available for use, depending on

the type of tissue present and features to be demonstrated
(Becker et al., 2007; Hopwood,2016).
 GENERAL FUNCTIONS
• Formalin is used for all routine surgical pathology and autopsy
tissues when an H and E slide is to be produced.

• Zenker's fixatives are recommended for reticuloendothelial tissues

including lymph nodes, spleen, thymus, and bone marrow.

• Bouin's solution is sometimes recommended for fixation of testis,

GI tract, and endocrine tissue.

• Glutaraldehyde is recommended for fixation of tissues for electron

microscopy.

• Alcohols, specifically ethanol, are used primarily for cytologic

smears. Ethanol (95%) is fast and cheap.
 FACTORS AFFECTING FIXATION
There are a number of factors that will affect the
fixation process:
• Buffering
• Penetration
• Volume
• Temperature
• Concentration
• Time interval
(Eltoum and Fredenburgh,2001).
 CONCLUSION
Faults in fixation are permanent and cannot be
corrected at any later stage whereas any other step
after proper fixation can be reversed to refine a
problem. No single fixative is ideal. Every fixative has
advantages and disadvantages which include
molecular loss from fixed tissue, swelling and
shrinkage of tissue, quality of histochemical staining.
Long term fixation in absolute alcohol results in
excessive shrinkage and tissue hardening which
causes microscopic distortion.
 REFERENCES
Becker, K.F., Schott, C. and Hipp, S. (2007): Quantitative protein analysis from
formalin-fixed tissues: implications for translational clinical research and
nanoscale molecular diagnosis. Journal of Pathology. 211(3):370–378.
Delfour, C., Roger, P. and Bret, C. (2006): RCL2 a new fixative, preserves
morphology and nucleic acid integrity in paraffin-embedded breast
carcinoma and microdissected breast tumor cells. Journal of Molecular
Diagnosis. 8(2):157–169.
Eltoum I, and Fredenburgh J. (2001). Introduction to the theory and practice of
fixation of tissues. J Histotechnol;24;173 -190.
Gillespie, J.W., Best, C.J. and Bichsel, V.E. (2002): Evaluation of non-formalin
tissue fixation for molecular profiling studies. America Journal
Pathology.160(2):449–457.
Hopwood, D. (2016). Fixation and fixatives. In Bancroft J and Stevens A eds.
Theory and practice of histological techniques. New York: Churchill
Livingstone,Pp.12-24
Leong A.S (2014). Fixation and fixatives. In Woods AE and Ellis RC eds.
Laboratory histopathology. New York: Churchill Livingstone Pp.41-261.
 REFERENCES
Lykidis, D., Van Noorden, S. and Armstrong, A. (2007): Novel zinc-based fixative
for high quality DNA, RNA and protein analysis. Nucleic Acids Research.
35(12):85.
Puchtler, H. and Meloan, S.N. (1985): On the chemistry of formaldehyde
fixation and its effects on immunohistochemical reactions.
Histochemistry. 82(3):201–204.
Srinivasan, M. Sedmak, and D. Jewell, S. (2002): Effect of fixatives and tissue
processing on the content and integrity of nucleic acids. American
Journal of Pathology.161(6):1961–1971.
Williams, J.H and Mepham, B.L (2017). Tissue preparation for
immunocytochemistry. J Clin Pathol;50;422-428.
Winsor, L. (2014). Tissue processing. In Woods A and Ellis R eds. Laboratory
histopathology. New York: Churchill Livingstone, 4: 42-39.
World Health Organization (2006); IARC Working Group. Formaldehyde, 2-
Butoxyethanol and 1-tert-Butoxypropan-2-ol. Lyon, France: IARC Press;
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