DAVAO DOCTORS COLLEGE

MEDICAL LABORATORY SCIENCE DEPARTMENT

STUDENT NOTES: GPHCT

FIXATION Factors Affecting Fixation

- First and most critical step in tissue processing because if fixation 1. Fixative of  10% Neutral Buffered Formalin (NBF)
is inadequate, the succeeding tissue processing steps will also Choice  Morphologic criteria for diagnosis have
be inadequate been established based on Formalin-
- Primary purpose: Fixed Paraffin Embedded Specimen
 Preserve morphological & chemical integrity of cell in a life- (FFPES)
like manner as possible by stopping all cellular activities
2. Time  Fixation must be done 20-30mins after
- Performed as soon as tissue is removed from the body
cutting off blood supply (to shorten
- If tissues/cells are exposed to: warm ischemia time)
a. Air  drying of tissue  Prolonged fixation shrinkage
b. Water  swelling of cells
c. Saline  shrinkage of cells 3. Tissue-to-  1:10 or 1:20 (tissue to fixative ratio)
Fixative Ratio  1:20 is more common
 Osmic acid fixatives: 1:5
Effects of Fixatives
■ Hardens soft tissues in preparation for further tissue processing
■ Render cells resistant to damage caused by chemicals used in 4. Penetration Rate  Formalin: 1 mm/hr (but slows down as
further processing it goes deeper into the tissue)
■ Inhibit decomposition caused by bacteria and fungi 5. Thickness of  Larger  Longer fixation time, more
■ Minimize the risk of occupational infection Section fixative
■ Act as mordant for certain stains, thus promoting or hastening  Light Microscopy: 2cm2 x 0.4cm
staining, or inhibit certain dyes  Electron Microscopy: 1-2 mm2

Characteristics of Ideal Fixative 6. Tissue  Longer fixation time:

1. Cheap Components Fibrous tissues
2. Stable Presence of Mucus (wash with NSS)
3. Safe to handle Fat (cut into thin slices fixed longer)
Blood (flushed out with saline)
4. Kill cells quickly to minimize cell distortion
5. Inhibit bacterial decomposition and autolysis  Shorter fixation time:
6. Permit rapid and even penetration of tissues Small or loosely textured tissues
7. Must harden tissues thus easier cutting of tissues
8. Must make cellular components insoluble to hypotonic 7. Hydrogen Ion  Optimal pH: 6 to 8
solutions, and insensitive to subsequent processing Concentration  If outside this pH, ultrastuctural
9. Permit application of staining procedures (pH) changes may occur
 May require the use of buffers
NOTE: No single fixative has all the mentioned characteristics. Each  For Electron microscopy: pH should
of them has own advantages and disadvantages. match physiologic pH
8. Temperature - Higher temp  faster fixation rate and
Mechanism of Fixation autolysis
1. Additive Fixative will forms cross-links between - Cold temp  enzyme inactivation
Fixation soluble molecules, thus gluing them Room temp to 45OC - Optimal Temperature (routine)
together into an insoluble meshwork 40 OC - Tissue processors
Up to 65OC - Microwave processing
2. Non-additive Fixative will not chemically bind with tissue 0-4OC - Electron microscopy
Fixation but removes water from tissue protein 100OC - Tuberculosis
groups thus causing denaturation of cell 60 OC - Rapid biopsy
proteins
9. Osmolality  Hypertonicitycell shrinkage
 Isotonicity and hypotonicity cell
swelling
 Thus, maintain tissues at slightly
hypertonic solution (400-450 mOsm)

10. Agitation,  Hastens fixation

Vacuum
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 TYPES OF FIXATIVES I. ALDEHYDES
According to Composition 1. Formaldehyde AKA Formalin
A. Simple made of one component  For routine HP techniques
 Produced from oxidation of methanol
i. Aldehyde  Usually buffered to pH 7 with phosphate buffer
a. Formaldehyde
b. Glutaraldehyde Concentrations:
ii. Metallic Fixatives o 100% - gas form
a. Mercuric chloride o 37-40% - stock concentration (causes overhardening
b. Chromate of the external surfaces of tissues)
c. Lead o 10% - working solution; most commonly used
iii. Picric acid
iv. Glacial acetic acid  ADV: cheap, readily available, easy to prepare, stable,
v. Alcohol compatible with most stains
vi. Osmium Tetroxide
 DADV: nose and eye-irritant, may cause allergic dermatitis
vii. Trichloroacetic acid
viii. Acetone
ix. Heat Effect Cause Remedy
White Prolonged  Filter o
B. Compound 2 or more components or fixatives paraformaldehyde storage  Add 10% methanol
precipitatesturbidity (but denatures
proteins thus
unsuitable for EM)
According to Action
A. Microanatomical (10,10,HFZZBB)
Formation of formic Unbuffered  Buffer
permits general study of tissues without altering the
acid  reduced  Methanol
structure of the subjects of interest
staining quality, and
1. 10% NBF
formation of formalin  10% formol saline +
2. 10% Formol-Saline
pigment Mg++/Ca++ carbonate
3. Heidenhain’s Susa
in jar with marbles
4. Formol-Sublimate/Corrosive
5. Zenker’s
Brown/black Action of  Saturated alcoholic
6. Zenker-formol (Helly’s)
precipitates formic acid picric acid
7. Bouin’s
with excess  1% KOH in 80%
8. Brasil’s
blood ROH
B. Cytological
 Kardasewitsch’s
preserve specific parts of the cell
Method
1. Nuclear Fixatives 2. Cytoplasmic Fixatives
(70% ETOH & 28%
o Preserve nucleus o Other organelles aside
ammonia H20)
from nucleus
o pH ≤ 4-6 o pH > 4-6
o HAc destroys
 Lillie’s MTD
o Glacial acetic acid
mitochondria and Golgi (Acetone, H2O2
has affinity to
bodies 70% ETOH & 28%
nuclear chromatin
ammonia water)
a. Flemming’s with a. Helly’s
glacial acetic acid b. Orth’s
c. Regaud’s/Moller’s 2. 10% Formol-Saline
b. Carnoy’s
d. Formalin with Post-chroming  Formalin diluted with 10% NaCl
c. Bouin’s
e. Flemming’s without glacial  Traditionally, the most common fixative
d. Newcomer’s
e. Heidenhain’s acetic acid  Recommended for CNS tissue and general post-mortem
tissues for histochemical examination
(FCBNH) (HORFF)  ADV: ideal for Silver impregnation staining technique
C. Histochemical  DADV: tissue shrinks during alcohol dehydration [Remedy:
Preserves chemical constituents of cells & tissues Secondary fixation]
1. 10% Formol Saline
2. Absolute ethanol 3. 10% Neutral Buffered Formalin (NBF) or Phosphate
3. Newcomer’s Buffered Formalin
4. Acetone  pH 7
(10FANA)  Best general tissue fixative

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  Best for iron-containing pigments and elastic fibers which II. METALLIC FIXATIVES
do not stain well after Susa, Zenker or Chromate fixation, Mercuric chloride Zenker
 DADV: longer to prepare, inert to phospholipids and neutral (ZZCHBS) Zenker-Formol (Helly’s)
fats Carnoy-Lebron
Heidenhain’s Susa
4. Formol-Sublimate/Corrosive B5
 Has HgCl2 Schaudinn’s
 ADV: Excellent for silver reticulum staining method, does
not need washing, fixes lipids Chromates Chromic acid
 DADV: forms mercuric chloride deposits (CROP) Regaud’s/Muller’s
Orth’s
5. Gendre’s (Alcoholic Formalin) Potassium dichromate
 Has 95% ETOH, Picric acid, and GHAc
 ADV: good for microincineration techniques, fixes sputum Lead

6. Hollande’s
 For gastrointestinal (GI) tissues, prostate biopsies, and 1. Mercuric Chloride (HgCl2)
bone marrow (BM)  Most common metallic fixative
 A: penetrates and hardens tissue rapidly
7. Glutaraldehyde  Routine fixative of choice for preservation of cell detail in
 Made up of 2 formaldehyde resides linked by three carbon tissue photography
chains  Conc. 5-7%
 For enzyme histochemistry and electron microscopy  Mostly incorporated in compound fixatives
 ADV: more pleasant and less irritating compared to  DADV: Banned worldwide d/t extreme toxicity, marked cell
formalin shrinkage [Remedy: add acid]
 DADV: less stable and more expensive than formalin  May produce black granular deposits except in
 Container must be refrigerated Heidenhain’s Susa

Concentrations: Remedy: Dezenkerization

o 0.25% - for immunoEM 0.5% Iodine + 70% ETOH  H20  5% Na thiosulfate  H20
o 2.5% - for small TSE fragments
o 3% - most common a. Zenker’s
o 4% - for large TSE  HgCl2 + potassium dichromate + glacial acetic acid
 Good general fixative for adequate preservation of all
8. Paraformaldehyde kinds of tissues
 Polymer of formalin  Good for Trichrome staining
 Powder in form, used in 4%
 Plastic embedding b. Zenker-formol/Helly’s
 For ultrathin and electron microscopy - HgCl2 + potassium dichromate + strong formalin (40%)
 For piituitary gland, BM, blood-containing organs,
9. Karnovsky’s Paraformaldehyde/Glutaraldehyde preserves cytoplasmic granules
 Acrolein in glutaraldehyde or formalin  Brown pigments are removed with saturated alcoholic
 P: for Electron Histochemistry and Electron picric acid or NaOH
Immunocytochemistry
c. Heidenhain Susa
10. 40% Aqueous Glyoxal  Susa: Su = sublimate ; Sa = saure (acid)
 ADV: no smudging of nuclei and distortion of staining  HgCl2 + NaCl + TCA + glacial acetic acid + formalin
compared with formalin  Skin tumor biopsy
 D: reduced staining capacity  ADV: minimum cell shrinkage and tissue hardening
[Remedy: increase staining time] due to counter-balance effect of acids and mercury:
Acids : swelling
Mercury: shrinkage
 Does not produce black pigments
 DADV: Weigert’s staining of elastic fibers not possible

d. B5 Fixative
 HgCl2 + Anhydrous Na acetate
 BM biopsies

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2. Chromate Fixatives IV. GLACIAL ACETIC ACID
 Incorporated in compound fixatives
a. Chromic acid  Solidifies at 17OCI
 Conc.: 1-2% aqueous solutions  Important for nuclear fixatives (precipitates nucleoproteins,
 Precipitates all proteins, and preserves carbohydrates chromatins)
 Destroys mitochondria and Golgi elements, thus not for
b. Regaud’s/Muller’s cytoplasmic fixation
 P: chromatin, mitochondria, mitotic figures, golgi
bodies, RBC and colloid-containing TSEs
 DADV: prolonged fixation may lead to blackening of V. ALCOHOL FIXATIVES
tissue pigment  ADV: good for glycogen
[Remedy: Wash in running tap water before  DADV: never for FATs and LIPOPROTEINS (dissolves); causes
dehydration] polarization of glycogen (granules will move towards the poles
or ends of the cells)
c. Orth’s  Effect: rapidly denatures and precips CHONs, preserves nuclear
 P: early degenerative processes and necrosis, stains
demonstration of Rickettsia and other bacteria  (CEMING)
 E: preserves myelin
1. Carnoy’s Fixative
d. 3% Potassium dichromate  Most rapid tissue fixative
 E: preserves lipids, mitochondria, at pH4.5-5.2,  Fixing brain tissues for rabies diagnosis
cytoplasm, chromatin and chromosome are fixed  E: fixes Nissl granules (Tigroid substance) and cytoplasmic
 Corrosive, thus avoid skin contact granules
3. 4% Aqueous Lead 2. 70-100% Ethanol
 P: for acid mucopolysaccharides and mucin  Enzyme studies
 DADV: Prolonged standing  formation of insoluble lead  Does not fix but preserves glycogen
carbonate
[Remedy: add drops of acetic acid to dissolve residue] 3. 100% Methanol/Wood alcohol
 Dry and wet smears, BM smears, bacterial smears
III. PICRIC ACID
4. 95% Isopropyl Alcohol/Rubbing Alcohol
 Used in strong saturated aqueous solution (1%)  Touch prep smears to be Wright-stained
 For Glycogen preservation
 ADV: may be used as a stain as yellowing of tissue will prevent 5. Newcomer’s
small fragments from being overlooked; suitable also with  Mucopolysaccharides and nuclear CHONs
Aniline stains  Better reaction in Feulgen stain than Carnoy’s
 DADV:
1. Explosive when dry 6. Gendre’s (Alcoholic Formalin)
[Remedy: add distilled H2O or 0.5-1% saturated alcohol]
2. Yellowing of tissues  excessive staining
[Remedy: immerse in Li2CO3 with 70%ROH  water  VI. OSMIUM TETROXIDE / OSMIC ACID
70% ethanol  5% Na thiosulfate  water]  Pale yellow powder in water (6% in 20OC)
3. RBC hemolysis
 Ultrathin sections in Electron Microscopy
 (PBB)
 E: Fixes and stains conjugated fats and lipids black
 DADV: very expensive, very volatile, inhibits hematoxylin
1. Bouin’s
 Tissue-to-fixative ratio: 1:5
 P: for embryo and pituitary biopsies, and tissues to be
stained with Masson’s Trichrome  (OFF)
 ADV: minimum cell shrinkage and tissue hardening due to
1. Flemming’s
counter-balance effect of glacial acetic acid (swelling) and
picric acid (shrinking)  Most common osmic acid fixative
 DADV: poorly penetrates large tissue, thus limited to small  P: nuclear structures
fragments of tissues  Effect: permanently fixes fat
 ADV: needs less amount of fixative
2. Brasil’s
 C: TCA 2. Flemming’s w/o acetic acid
 ADV: Better and less messy than Bouin’s  Cytoplasmic structures

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 VII. TRICHLOROACETIC FIXATIVES IMPROPER FIXATION
 Incorporated also in compound fixatives Effect Reason
 Marked swelling effect on tissues 1. Failure to arrest early Failure of fixing immediately
 Poor penetrating agent thus for small pieces of tissues or bones cellular autolysis or insufficient fixative
 Weak decalcifying agent, thus has softening effect on dense
fibrous tissues

VIII. ACETONE 2. Too brittle and too hard Prolonged fixation

blocks or tissue
 Used at cold temp -54OC
 For water-diffusible enzymes (Phosphatase, Lipase)
3. Shrinkage and swellings
 For brain tissues (such as in Rabies) of cells in tissue blocks
 DADV: Dissolves fat, evaporates rapidly
4. Soft and feather-like Insufficient or incomplete
tissues fixation
IX. HEAT
5. Presence of artefact Insufficient washing of
 Principle: Thermal coagulation of tissue proteins pigments on sections fixative
 For rapid diagnosis: frozen tissue sections and Bacterial smear 6. Enzyme inactivation and Wrong choice of fixative
prep loss
7. Removal of fixative
Microwave Technique soluble substances
 PCPL: Increases movement of molecules to accelerate
fixation, staining, decalcification
 Electron Microscopy and immunohistochemistry
 ADV: Tissue is heated right through the block in a very
short time; preserves neurochemical substances
(acetylcholine)
 DADV: Penetrates at 10-15mm thickness; spores and References:
pathogen may remain in tissues 1. Bruce-Gregorios, J. H. (2016). Histopathologic techniques.
 Optimum Temp: 45-55OC (2nd Revised Edition). Makati, PH: Katha Publishing
(611.0182/B83)
2. Lo, R., Orillaza, M., Madrid, M., Santiago, F., & Aguilar, P.
SECONDARY FIXATION (2015). Basic histopathologic techniques. Metro Manila, PH:
C & E Publishing
 “Refixation” with another fixative
o Done before dehydration or restaining of deparaffinized
TSEs
o Improve demonstration of substance
o Make special staining techniques possible (with the next
fixative as mordant)
o Ensure further and complete handling
 Post-Chromatization
o Use of 2.5-3% aqueous K2Cr2O7 that will act as mordant

WASHING OUT

 Removal of excess fixative to improve staining and remove

artifacts

1. Tap Water for excess formalin, osmic acid, and

chromates
2. 50-70% ROH for excess picric acid fixatives and
Gendre’s

3. Alcoholic for excess mercuric chloride

iodine

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