​HISTOPATHOLOGY: FIXATION ​

A. Histotechnology-the art and science performed by the medical technologist to

provide the pathologist a good quality tissue sections on which to base patient’s
diagnosis.
B. 2 Types of Specimen Submitted:
1. Autopsy-from dead source
2. Biopsy/Surgical-from living source
Specimens Excluded from Mandatory Submission to the Laboratory:
1. Bone donated to the bone bank
2. Bone segments removed as part of corrective or reconstructive orthopedic
procedures
3. Cataracts removed by phacoemulsification
4. Dental appliances and teeth with no attached soft tissue
5. Fat removed by liposuction
6. Foreign bodies such as bullets or other medicolegal evidence given directly to law
enforcement personnel
7. Foreskin from circumcision
8. Intrauterine contraceptive devices without attached soft tissue
9. Medical devices (catheters, gastrostomy tubes, stents, and sutures) that have not
contributed to the patient’s illness, injury, or death
10. Middle ear ossicles
11. Orthopedic hardware and other radiopaque medical devices, provided that there is
an alternative policy for documentation of their surgical removal
12. Placentas that do not meet institutionally specified criteria for examination
13. Rib segments or other tissues removed only for purposes of gaining surgical
access, provided that the patient does not have a malignancy
14. Saphenous vein segments harvested for coronary artery bypass
15. Skin or other tissues removed during a cosmetic or reconstructive procedure,
provided it is not contiguous with a lesion or the patient does not have a history of
malignancy.
16. Therapeutic radioactive devices
17. Normal toenails and fingernails that are incidentally removed
C. Pre-Analytic Factors
1. Warm Ischemia-initial insult a tissue suffers when blood supply is interrupted
2. Cold Ischemia-lack of oxygen when the tissue is removed from the patient’s body

D. Purpose of Tissue Processing

1. A tissue specimen is processed into slides for microscopic examination by the
pathologist for diagnosis.
2. Usually, to rule out cancer.
3. It is important that the pathologist is provided with good quality specimen.
4. To ensure that the pathologist receives a good quality tissue section, the morphology
and chemical integrity is maintained from the time it is removed from the body until it is
processed.

E. Specimens for Gross Description Only

1. Accessory digits
2. Bunions and hammer toes
3. Extraocular muscle from corrective surgical procedures
4. Inguinal hernia sacs in adults
5. Nasal bone and cartilage from rhinoplasty
6. Prosthetic breast implants

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 7. Prosthetic cardiac valves without attached tissue
8. Tonsils and adenoids from children
9. Torn meniscus
10. Umbilical hernia sacs in children
11. Varicose veins

F. Fixation
1. Definition
-Traditionally, the killing, penetration, and hardening of tissues
-process of preserving the morphology and chemical integrity of cells and tissues as
close to the original as possible
-it involves the immersion of tissue specimen in a fixing agent
Primary Goal: Preserve the tissue, prevent autolysis due to bacterial attack and
putrefaction due to release of enzymes
Secondary Goal: Harden the tissue and protect it from trauma of further handling.

2. Basic Mechanisms Involved in Fixation

2.1. Additive Fixation-the fixative is taken in and becomes part of the tissue by forming
cross-links or molecular complexes and giving stability to the protein.
(e.g. Formalin, Mercury, Osmium tetroxide)
2.2. Non-Additive Fixation-the fixing agent does not combine with the tissue. The
tissues are preserved when the fixative alters the tissue structure.
(e.g. Alcoholic fixatives)

3. Main Factors Involved in Fixation

3.1. Hydrogen Ion Concentration-pH of 6-8. The use of unbuffered formalin (acidic,
low pH) can develop dark pigments that can obscure cell details.
3.2. Osmolality-slightly hypertonic (400-450 mOsm), isotonic (340 mOsm). Hypertonic
solutions causes shrinkage while hypotonic solutions causes swelling.
3.3. Temperature-Traditionally, at room temperature only. If autotechnicon is used, 40
degrees Celsius. For electron microscopy and histochemistry, 0-4 degrees Celsius.
Formalin heated @ 60 degree Celsius is used for rapid fixation of urgent biopsies.
Formalin heated @ 100 degree Celsius is used for fixing tissues with tuberculosis.
​*Autotechnicon is an automatic tissue processor that can do fixation,
dehydration, clearing, and infiltration.
3.4. Volume-10-25 times the volume of the specimen. 20 times the volume of the
specimen for maximum effectiveness. For museum preparation, it should not be less
than 50-100 times the volume of the specimen. If osmium tetroxide is used, 5-10 times
the volume of the specimen because it is expensive.
3.5. Thickness of Section-Fixatives penetrate tissues at a rate of 1 mm/hour. It should
not be more than 5 mm. If tissue cassette is used, 3 mm. for lung specimens, 1-2 mm.
3.6. Concentration-Traditionally, formaldehyde is used as 10% solution and
glutaraldehyde is used as 3% solution. 0.25% glutaraldehyde is for immune-electron
microscopy.
3.7. Duration of Fixation-It should be within 20-30 minutes from the time blood supply
is interrupted. Fixation time is 6-48 hours.

4. Factors that RETARD Fixation

4.1. Size and thickness of tissues-larger tissues require longer time
4.2. Cold temperature-inactivate enzymes
4.3. Presence of blood and mucus-wash with NSS

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 *Brain specimens must undergo Intravascular Perfusion which is washing out of blood
using Ringer’s Lactate
4.4. Presence of fat-must be cut thinly

5. Factors that ACCELERATE Fixation

5.1. Size and thickness of tissues-smaller tissues require shorter time
5.2. Heat-37-56 degree Celsius
5.3. Agitation-continuous mixing but not performed in manual fixation

6. Factors to be Considered When Choosing the Right Fixative

6.1. Need for immediate examination
6.2. Type of tissue to be processed
6.3. Tissues structure to be studied
6.4 Staining technique to be applied
6.5 Type of section to be made, whether serial or individual

7. Effects of Fixatives in General

7.1. Harden the tissue
7.2. Inhibit bacterial decomposition
7.3. Reduce the risk of infection
7.4. Promote optical differentiation of cells and tissues
7.5. Act as mordant, thereby facilitating staining
7.6. Make the tissue resistant to damage caused by solutions used during tissue
processing

8. Characteristics of a Good Fixative

8.1. Cheap, stable, safe to handle, easy to prepare
8.2. Fast-acting (Kill the cell quickly thereby producing minimum distortion of cells)
8.3. Autolysis (Inhibit bacterial growth)
8.4. Produce minimum shrinkage of tissue
8.5. Permit rapid and even penetration of tissues
8.6. Isotonic

9. Types of Fixation
9.1. Physical Methods
9.1.1. Heat Fixation-carried out for frozen sections and for fixing bacterial smears. It
involves thermal coagulation of proteins.
9.1.2. Microwave Technique-more rapid than heat fixation. It can accelerate
staining and decalcification. Used in preserving neurochemical substances in brain like
acetylcholine. It can penetrate tissues to a thickness of only 10-15 mm.
9.1.3. Freeze-drying/Freeze Substitution-also known as quenching. It is carried out
at -160 to -180 degree Celsius. It can freeze tissues in 2-3 seconds. A tissue that is 2
mm thick is immersed in liquid nitrogen, isopentane, or propane-isopentane. Then,
dehydration, it is placed in a vacuum drying chamber (-40 degree Celsius). Freeze
substitution does not involve use of vacuum drying chamber. The tissue is immersed in
Rossman’s solution or 1% acetone.

9.2 Chemical Methods

9.2.1. Coagulant Fixatives-capable of forming networks to allow rapid penetration.
Maintains cellular architecture, tissue morphology at light microscopic level. Results in
poor preservation of mitochondria and secretory granules.
(e.g. Ethanol, Methanol, Acetone, Trichloroacetic acid, Picric acid)

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 9.2.2. Non-Coagulant Fixatives-capable of forming gel instead of networks thus
making the process slower.
(e.g. Formaldehyde, Glutaraldehyde, Aldehyde-chloral hydrate, glyoxal)
9.2.3. Compound Fixatives-usually formaldehyde-based. It is useful for specific
tissues.
(e.g. Alcoholic Formalin-fatty tissues like breast)

10. Types of Fixatives

10.1 According to Composition
10.1.1. Simple-only one component substance
10.1.2. Compound-2 or more fixatives combines

10.2 According to Action

10.2.1. Histochemical-preserve chemical components of tissues, phosphatases and
lipases
(e.g. 10% formol saline, Absolute ethyl alcohol, Acetone, Newcomer’s fluid)
10.2.2. Cytological-Cytoplasmic fixatives are those with pH greater than 4.6 usually
with no glacial acetic acid (e.g. Flemming’s fluid without glacial acetic acid, Kelly’s fluid,
Formalin with “post-chroming”, Regaud’s fluid, Orth’s fluid). Nuclear fixatives are those
with pH leser than 4.6 usually with glacial acetic acid (e.g. Flemming’s fluid, Carnoy’s
fluid, Bouin’s solution, Newcomer’s fluid, Heidenhain’s Susa)
10.2.3. Microanatomical-allows the study of the general microscopic structures of
tissues and cells
(e.g. 10% formol saline, 10% neutral buffered formalin, Heidenhain’s susa, Formol
sublimate, Zenker’s solution, Helly’s solution, Bouin’s solution, Brasil;s solution)

10.3 According to Active Component Substance

10.3.1 Trichloroacetic Acid
Fixative Definition Advantage & Disadvantage
Trichloroacetic Acid -both a fixative and a -precipitates proteins.
decalcifying agent. -suitable only for small
-used in conjunction with pieces of tissues or bones.
other fixatives to form
compound fixatives.

10.3.2 Glacial Acetic Acid

Fixative Definition Advantage & Disadvantage
Glacial Acetic Acid -for nuclear fixation -preserve nucleoproteins,
-solidifies @ 17 degree chromosomes, chromosome
Celsius materials
-used in conjunction with -destroys mitochondria and
other fixatives to form golgi apparatus
compound fixatives.

10.3.3 Acetone
Fixative Definition Advantage & Disadvantage
Acetone -used att ice cold -fix phosphatases and
temperature (-5 to -4 degree lipases
Celsius) -dissolves fat, volatile,
-for fixing brain tissues for evaporates rapidly
the diagnosis of rabies
-used as both fixative and
dehydrating agent

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 10.3.4 Picric Acid Fixatives
-for glycogen determination
-it can also dye tissues
-to remove: treat with another acid such as lithium carbonate, immersion
in 70% alcohol and 5% sodium thiosulfate then wash with water

Fixative Definition Advantage & Disadvantage

Bouin’s Solution -for pituitary, embryos, and
endometrial curettings
Brasil’s Solution -less messy than Bouin’s
Hollande’s solution -for GIT biopsies and
endocrine tissues

10.3.5 Alcohol Fixatives

-they cause glycogen polarization
-can be used as dehydrating agent
Fixative Definition Advantage & Disadvantage
100% Methyl Alcohol -for wet and dry smears and
blood smears
Ethyl Alcohol -for blood, tissue films, and
smears
95% Isopropyl Alcohol -for touch preparations
Carnoy’s Fluid -for fixing chromosomes, -MOST RAPID FIXATIVE
lymph glands, and urgent -produces RBC hemolysis
biopsies
-fix brain tissue for diagnosis
of rabies
New Comer’s Fluid -for mucopolysaccharides -compatible with Feulgen
and nucleoproteins
Clarke’s solution -produce good results with
H&E
-preserves nucleic acid
Methacarn -causes less hardening and
shrinkage than Carnoy’s
Rossman’s Solution -good for CT mucins and
umbilical cord

10.3.6 Metallic Fixatives

10.3.6.1 Lead Fixatives
Fixative Definition Advantage & Disadvantage
Lead -for acid -formation of insoluble lead
mucopolysaccharides and carbonate
connective tissue mucin

10.3.6.2 Chromate Fixatives

Fixative Definition Advantage & Disadvantage
Chromic Acid -preserves carbohydrates
Potassium Dichromate -preserves lipids and
mitochondria
Regaud’s (Muller’s) Fluid -for demonstration of
chromatin, mitochondira,
mitotic figures, Golgi bodies,

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 RBC and colloid-containing
tissues
Orth’s Fluid -for early degenerative
processes, tissue necrosis,
rickettsia and other bacteria

10.3.6.2 Mercuric Chloride

-compatible with Trichrome stain
-for tissue photography
-can leave black mercury deposits
Fixative Definition Advantage &
Disadvantage
Zenker’s Fluid -for fixing small pieces of -not stable after addition of
liver, spleen, connective Acetic acid
tissue fibers and nuclei.
Zenker-Formol (Helly’s) -for pituitary gland, bone
marrow, and blood-
containing organs such as
liver and spleen
Heidenhain’s Susa -for tumor skin biopsies
B-5 -for bone marrow biopsies
Schaudinn’s Ohlmacher;s -rapid or fast-acting
and Carnoy-Lebrun
Solution

10.3.7 Zinc Sulfate

Fixative Definition Advantage & Disadvantage
Zink Sulfate -replacement for mercuric -respiratory, skin and eye
chloride irritant
-preserves tissue antigenicity -ingestion of 10 grams can
cause DEATH

10.3.8 Osmium Tetroxide/Osmic Acid

Fixative Definition Advantage & Disadvantage
Osmium Tetroxide -pale yellow powder which -VERY EXPENSIVE
dissolves in water to form a -extremely volatile
strong oxidizing solution
-fixes conjugated fats and
lipids
-preserves cytoplasmic
structures
-fixes myelin and peripheral
nerves well
-produces brilliant nuclear
staining with safranin
-it precipitates and gels
proteins

10.3.9 Aldehyde Fixatives

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 Fixative Definition Advantage &
Disadvantage
Formaldehyde -gas produced by oxidation -irritating to nose, eyes and
of methyl alcohol skin and may cause
-commonly used as 4% dermatitis
solution -reduces basophilic and
-cheap, readily available eosinophilic staining of cells
-preserves fat and mucin,
glycogen
-“tolerant fixative”
10% Formol Saline -saturated formaldehyde + -slow fixative
10% sodium chloride -metachromatic reaction of
-for fixation of central amyloid is reduced
nervous tissues and general
post-mortem tissues for
histochemical examination
10% Neutral Buffered -for preservation and -inert towards lipids
formalin or Phosphate storage of surgical, post-
Buffered Formalin mortem, and research
specimens
-best fixative for tissues
containing iron pigments
Formal-Corrosive/Formal -for routine post-mortem
Sublimate tissues
-no need for “washing out”
Alcoholic formalin/ -fix sputum
Gendre’s fixative -preserves glycogen and or
micro-incineration technique
Glutaraldehyde -made up of 2 formaldehyde -MORE EXPENSIVE
residues linked by 3 carbon -less stable
chains
-preserves plasma proteins
better
-it does not cause dermatitis
Glyoxal -THE SMALLEST
ALDEHYDE

*Methods of Removing Formalin Pigmenet

1. Kardasewitch-28% ammonia water and 70% alcohol
2. Lillie’s method-acetone, hydrogen peroxide and ammonia water
3. Picric acid method-saturated picric acid

11. Fixatives for Enzyme Histochemistry: 4% Formaldehyde or Formol Saline

12. Fixatives for Electron Microscopy: Osmium Tetroxide, Glutaraldehyde and
Paraformaldehyde
13. For Electron Histochemistry and Electron Immunocytochemistry: Karnovsky’s
paraformaldehyde-glutaraldehyde and acrolein
14. Secondary Fixation and Post Chromatization
14.1 Secondary Fixation-is the process of placing an already fixed tissue in a second
fixative to (1) facilitate and improve the demonstration of particular substances, (2) to
make special staining techniques possible, and (3) to ensure further and complete
hardening and preservation of tissues
14.2 Pot-Chromatization-is a form of secondary fixation whereby a primarily fixed
tissue is placed in aqueous solution og 2.5-3% potassium dichromate for 24 hours to act
as a mordant.

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 15. Washing Out
-is the pocess of removing excess fixative from the tissue
15.1 Tap water-is used to remove excess chromates, formalin, and osmic acid
15.2 50-70% alcohol-is used to wash out excess picric acid
15.3 Alcoholic iodine-is used to remove excessive mercuric fixatives

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