HISTOPATHOLOGY - MIDTERM o Failure to observe these minimum and maximum fixative

FIXATION times may show false negative results for HER-2.

Classically defined as the killing, penetration, and hardening of Choice of fixative
tissue. 10% Neutral Buffered Formalin
Currently defined as the alteration of tissue by stabilizing protein so  Fixative of choice
that the tissues become resistant to further changes.  It is the fixative of choice is that morphologic criteria used for
Functions of Fixative diagnosis throughout the years have been established based on
 Change the soluble contents of cells into insoluble structures. formalin-fixed tissues.
o Occur through denaturation. Penetration
 Avert autolysis  Formalin is the fastest penetrating fixative.
 Stabilize structures to maintain the proper relationship of cells o 3.6 mm per hour and 7.22mm in 4hrs at a temp. of
and their stroma. 25C and a pH of 7
 Affects refraction index – the ratio of velocity of light in air and pH
velocity of light in liquid.  Does not affect light microscopy
 Enhance staining  Not buffered formalin is acidic (pH4) necessary to buffer
How fixatives work formalin at pH 7.
Fixative classified as either additive/non-additives, and either Osmolality
coagulant/non-coagulant.  Refers to the number of molecules of molecules of a particular
Additive fixatives – chemically alter the tissue by bonding with it substance in solution.
and adding themselves to the tissue.  Isotonic
 Cause a change in electrical charge at the site of attachment o Osmolality solution is the same as that body fluid
Non-additive fixatives – act on a tissue without chemically o Used as holding solutions for tissues to be transported to
combining with it. frozen sections or kidney biopsies for special processing.
 E.g., Acetone, and alcohol  Hypertonic
Coagulant fixatives – act by creating a network that allows o Contain more dissolved substance
solutions to readily penetrate the interior of the tissue. o Cause cell to shrink
 E.g., zinc salt, mercuric chloride, picric acid, ethyl alcohol,  Hypotonic
methyl alcohol, and acetone. o Contains less dissolved substance
Non-coagulant fixatives – creates a gel that makes it difficult to o Cause cell to swell.
penetrate by subsequent solutions.
Consequences of delayed, Incomplete, or poor fixation
Factors Affecting Fixation
1. Loss or total disappearance of nuclear chromatin
Temperature
2. Disappearance of some cells
 Affects tissue morphology 3. Cell shrinkage with artifactual space around the cells
 ↑ fixation temp. ↑ rate of autolysis Cellular components and their reactions to fixation
 The consensus is that fixation temp. up to 45C Nucleus
 Room temp. to have satisfactory results.  Contains DNA and RNA aside from protein.
Specimen size  45C for RNA and 65C for DNA
 Pre analytical issue  Nuclear bubbling – formation of chromatin strands with
 Tissue thickness (important consideration) intervening clear areas due to formalin fixation of the nucleus.
 Large tissue such as colon resections should be opened and Proteins
sectioned.  Have a primary, secondary, and tertiary structure
 3mm sectioning is required  Primary – covalent, secondary – hydrogen, tertiary – hydrogen
Volume ratio bond, electrostatic bonds, hydrophobic bond, and disulfide
 The ideal ratio of fixative volume to specimen volume is 15-20 bonds
is to 1. Additive Fixative
 Current recommendations allow a ratio of 10:1  Alter the tertiary structure of protein by changing the electrical
Time charges at the site of attachment.
2 aspects: Carbohydrates
 Cold ischemia time  Lost during fixation
o interval between the interruption of blood supply and the time Types of fixatives
the tissue is immersed in the fixative. Classification of fixatives:
o Under the control of the surgeon and his team. 1. Simple aqueous fixatives
o The ideal time to perform fixation is within 20-30min after the 2. Compound aqueous fixatives
interruption of the blood supply. 3. Non-aqueous fixatives
 The tissue must be immersed in the fixative for no longer than Simple aqueous fixatives:
60mins. 1. Acetic acid
 Fixation time 2. Formaldehyde
o Time period the tissue is exposed to formalin. 3. Glutaraldehyde
o Ideally fixed for a minimum of 6hours and a maximum of 48 4. Glyoxal
hours in 10% neutral buffered formalin 5. Mercuric chloride
o Importance of fixation time is particularly emphasized by the 6. Osmium tetroxide
guidelines of the ASCO-CAP 7. Picric acid
8. Potassium dichromate
 9. Zinc sulfate Recommendations to prevent autolysis:
10. Others 1. Minimize cold ischemia time
Acetic acid 2. Ensure adequate ratio of fixative to specimen
 Common household product (vinegar) 3. In cutting room this should be done:
 Glacial acetic acid – concentrated form a. Uterus specimens – opened by the bivalve technique
o Stored at room temperature and kept away from strong b. Gastrointestinal tract specimen – opened and pinned to
acids corkboards or Styrofoam sheets to allow contact
 Penetrates rapidly, thus acetic acid-fixed tissues are soft c. Lymph nodes – bisected, and if large sectioned at 3mm
 Main use is for the preservation of nucleic acids by intervals
precipitation. Incomplete Fixation
 Using fume hood is required.  Separation of tissue components during microtomy and poor
Formaldehyde/Formalin tissue morphology.
 Colorless gas commonly available at 37%-40% solution in  Nucleic may be smudgy or may exhibit bubbling.
water. Recommendations:
 Commonly referred as 100% formalin 1. Increase time in fixative solution
10% formalin 2. Change another fixative
3. Place formalin-alcohol in the first 3 stages
 Most widely used fixative in histopath today.
4. Ensure that grossed sections are 3mm or less
 Fast penetrating
5. Change the formalin solution frequently
 Non-coagulant and additive
6. Do not pack tissue tightly in the cassettes
 Buffered to a pH of 7 to prevent formation of black acid hematin 7. Use agitation of cassettes in formalin
pigment.
PROCESSING OF TISSUES
 Penetration rate: 3.6mm per hour
 After proper fixation and before the staining process, the tissue
 Cross linking complete 24-48hours
must be adequately prepared and set in a paraffin block, ready to
 At a pH below 6, black acid hematin pigment be cut in ribbons.
 Black acid hematin – obscures cellular components and masks  Longest phase in routine histopathology.
or stimulates microorganisms. o Dehydration
Glutaraldehyde
o Clearing
 Dialdehyde
o Infiltration
 Mainly used for specimen fixation for electron microscopy since
o Impregnation
it preserves ultrastructure best.
Post-fixation Treatment
 Best limited to 2hrs.
 Buffered formalin forgoes any post-fixation treatment.
Glyoxal
 Complete fixation prevents secondary post-fixative changes
 40% aqueous solution
 Rapidly penetrating
 Surgical specimens are fixed within 4-6 hrs and small biopsy
specimens within 45 mins.
 Has the ability to cross-link which can be prevented during
normal fixation.
Mercuric Chloride
 Extremely toxic compound that has been banned from use
worldwide.
Osmium Tetroxide
 Very expensive and hazardous agent that can readily fix nasal
mucosa and conjunctiva of the eye.
 Post-fixative for electron microscopy
Picric Acid
 A fixative and a stain
 Strong coagulant and nucleoprotein.
 Avoided if DNA or RNA staining is desired
 Explosive compound, used by the military until displaced by
TNT.
Potassium Dichromate (K2Cr2O7)
 Carcinogenic and corrosive to skin and mucous membrane
Zinc Sulfate (ZnSO4)
 Replacement for mercuric chloride
 A respiratory, skin, and eye irritant.
 Ingestion of 10grams of zinc can cause death.
Troubleshooting Fixation Problems
Autolysis
 Once the tissue is autolyzed, nothing can be done to improve its
appearance.
 The best remedy is prevention