Histotechnology topic

1. Decalcification of Bony and Hard Tissue
for Histopathology Processing
• The presence of calcium salt in the tissues
makes them very firm to hard and this may
damage the knife. Therefore, it is often
necessary to remove calcium salt from the
tissue and to make it soft for cutting in a
microtome. The process of removal calcium
salt from the tissue is known as
decalcification.

2. • The basic aims of decalcification are:
1. Removal of calcium salt from tissue
2. No damage to tissue morphology
3. No significant effect in staining

3. Calcium-containing tissue
• The tissue containing heavy amount of calcium salts
are (1) the bone, (2) tooth, (3) pathological tissue such
as in tuberculous lymph node, dystrophic
calcification, and certain tumours such as teratomas,
etc. Requisites for successful decalcification: The
following measures are helpful for successful
decalcification:
• Consistency: Exact assessment of the consistency of the
tissue is required for successful decalcification.
• Small pieces: The tissue should be cut in 2–6 mm thick
sections because thicker tissue may take longer time to
be decalcified.
• Fixation: Adequate fixation of the tissue is necessary for
proper decalcification.

4. • Washing: The fixed tissue should be washed
thoroughly before decalcification.
• Choice of decalcifying agent: Suitable choice
of the decalcifying agent is required.
• Volume: Optimum volume of the decalcifying
agent is a prerequisite for proper
decalcification.
• End point detection: The end point of the
decalcification should be determined

5. Factors Controlling the Rate of Decalcification
• Concentration: The increased concentration of
the decalcifying agent increases the rate of
decalcification.
• Temperature: Increased temperature fastens
the decalcification rate.
• Density of bone: Hard bone takes longer time
to be decalcified.
• Agitation: Mild agitation of the decalcifying
solution increases the rate.
• Thickness of tissue: Thinner tissue is quickly
decalcified.

6. The Methods of Decalcification
1. Acid decalcification
2. Ion-exchange resin
3. Electrical ionization
4. Chelating solution
5. Surface decalcification

7. • Acid Decalcification; This is the commonest
method of decalcification in routine
laboratory process. Acid makes the soluble
calcium salt, and thereby calcium is removed
from the tissue.
The strong acids:
• Hydrochloric acid
• Nitric acid
Weak acids:
• Formic acid
• Trichloroacetic acid

8. • Strong Acid The strong acids are used in 5–
10% concentration.
• They are rapid in action. However, careful
attention is needed to prevent tissue damage.
Neutralizer is also used to prevent any tissue
distortion.
• Aqueous Nitric Acid; This is rapid in action. It
does not impair staining if the end point is
not crossed.

15. Chelating Agents
• Chelating agents are organic substances that
adsorb metals. Ethylenediaminetetraacetic
acid (EDTA): EDTA is the most commonly
chelating agent.
• It binds with calcium of the hydroxy-apatite
crystals and forms a non-ionized soluble
complex.

16. • The action of EDTA is slow and gentle, and it
may take several weeks to remove calcium
from the tissue. Therefore EDTA is not a
suitable decalcification agent for dense bone
or urgent removal of calcium. The main
advantage of EDTA is the preservation of
morphology and to maintain the tissue for
various other techniques for research
purpose. The action of EDTA is pH dependant
and it works best in pH 7–7.6.

18. Advantages:
• It gives best morphological preservation of
tissue.
• Various other laboratory tests can be done on
the tissue such as immunohistochemistry,
fluorescent in situ hybridization technique
Disadvantages;
1. Very slow process.
2. Maintenance of pH around 7 is necessary.
3. Thin tissue is needed.

19. • Ion-exchange resin method:
• Ion-exchange resins involve the use of formic
acid over a layer of an ammoniated salt of a
sulfonated resin. Ammonium ions from the
resin are exchanged for calcium ions; this
keeps the solution free of calcium ions and
speeds up the reaction. Staining is excellent
after application of this method, and the time
tissue remains in the decalcifying reagent is
not critical. Because the solution remains
relatively free of calcium ions, it does not
need to be changed frequently. This is one of
the best, if not the best, methods of
decalcification.

20. • Electrolysis method:
• In this process electrolysis of the tissue is done
in a solution of hydrochloric acid and formic
acid. Calcium from the tissue moves to the
cathode plate. This is a very rapid method of
decalcification and takes only a few hours to
decalcify the bone. However there is a risk of
tissue damage in this technique.

21. • Surface Decalcification
• the surface layer of paraffin blocks is inverted
in 1% hydrochloric acid (HCl) for 1 h. The
exposed top 30 μm tissue of the paraffin
block is decalcified. The block should be
washed thoroughly before cutting. Only the
first few paraffin sections are expected to be
free from calcium.

22. • End Point Determination of Decalcification
1. Radiographic examination
2. Chemical test
3. Physical test
• Radiographic examination: X-ray
examination of the tissue is the most
accurate technique to detect the end point
of decalcification. However, this is a costly
procedure, and the pre-decalcification
radiograph is also needed to assess the
extent of decalcification.
• the small X-ray units available in many
histology laboratories are easy to use.

23. • This method yields visual evidence that
demineralization is complete. Radiographic
methods cannot be used with metallic-fixed
tissue, such as zinc formalin, Zenker, or B-5
fixed specimens because the metal will
render the specimen radiopaque.

24. • Chemical methods
• This test is done to assess the presence of calcium in
the decalcifying solution in two successive times. The
chemical test is applied when weak acid solution (e.g.
formic acid) is used.
• Now for the chemical test, 5 ml of the decalcifying
agent from the container containing the tissue is
withdrawn.
• Mix the decalcifying agent with 5 ml of ammonium
hydroxide and ammonium oxalate mixture solution.
• The mixture is kept overnight.
• Any precipitation is noted.
• The presence of precipitation (calcium oxalate)
indicates that the decalcifying agent contains calcium
and decalcification is not completed.

26. Physical test:
• This is a crude test and it does not accurately
detect the end point of decalcification. The
tissue is bent or a pin is introduced within the
tissue. In case of adequate decalcification, it
is expected that the tissue will be soft and
could be bent easily. Pin also should
penetrate easily within the tissue. The major
disadvantage of physical test is the tissue
damage by making a hole or by bending it.

27. • The embedding medium most frequently used
for undecalcified bone is GMA.

28. • Microscopes
• A microscope is a critical tool for quality control
in the histology laboratory and should be readily
available for monitoring quality of both routine
and special stains.
• the slides should be examined for the quality of
processing, sectioning, and staining.
• LIGHT MICROSCOPE
A magnifying glass (1 lens) is a simple microscope.
The light microscope used for the examination of
tissue sections combines 2 simple microscopes, or
magnifying lens systems; therefore, the light
microscope is called a compound microscope.

29. • POLARIZING MICROSCOPE
The polarizing microscope is finding increased
use as a diagnostic tool in histopathology
primarily for the identification of crystals such
as talc, silica, or urate. It is also used to make
the identification of amyloid stained with Congo
red more specific.
• PHASE-CONTRAST MICROSCOPE
is used for the examination of unstained
specimens, especially unstained living cells

30. • DARKFIELD MICROSCOPE
• FLUORESCENCE MICROSCOPE
• ELECTRON MICROSCOPE

31. • FIXATION
• A fixative alters tissue by stabilizing the
protein so that it is resistant to further
changes.
• Enzymes, which are proteins, are rendered
inactive as a result of the protein-stabilizing
action of fixatives.

32. • Simple Aqueous Fixatives or fixative
Ingredients
I. Acetic acid
2. Formaldehyde (formalin alcohol is included)
3. Glutaraldehyde
4. Glyoxal
5. ilercuric chloride
6. Osmium tetroxidc
1. Picric acid
8. Potassium di ch romatc
9. Zinc salts

36. • Compound or Combined Fixatives
• fixative solutions are combined in such a way that the
disadvantage of one component will be
counterbalanced by an advantage (or even a
disadvantage) of another. For example, the swelling
caused by acetic acid is a disadvantage that can be
counteracted by the shrinking effect of picric acid, a
disadvantage if picric acid is used alone
1. B-5
2. Bouin
3. Gendre
4. Hollan de
5. Zenker and Helly
6. Orth
7. Zamboni
8. Zinc formalin

41. • Mechanism of Fixation
Dehydration and coagulation of protein:
Methanol and ethanol are commonly used
coagulative fixatives. These two alcohols remove
water from the tissue and causing
destabilization of the hydrogen bonds and
thereby disruption of the tertiary structure of
protein.
• Cross-linking fixatives
Formaldehyde
Glutaraldehyde
Osmium tetroxide

42. • In cross linking there is connecting of protein
group and change their active structure or
denaturation of protein occurs
• Glutaraldehyde is used as a fixative for
electron microscopy because it fixes and
preserves the ultrastructure.
• Osmium tetroxide is used for fixation in
electron microscopy.
• Methyl alcohol (methanol) and ethyl alcohol
(ethanol) are used as dehydrating agent, and
these two alcohols are used mainly as
fixatives of cytology smears.

44. • Acetone; enzyme study and
immunocytochemistry.
• Bouin’s solution contains picric acid. This is an
excellent fixative for glycogen.
• Zenker’s Fluid; It is a good fixative for nuclear
chromatin and collagen.
• Helly’s Fluid; This is a good cytoplasmic
fixative.

78. • Staining of Different Carbohydrates
• Glycogen
• Glycogen, the polysaccharide, is
demonstrated by periodic acid-Schiff’s (PAS)
reaction.
• To demonstrate polysaccharides: PAS helps to
demonstrate glycogen, cellulose and starch.
• demonstrates glycogen in glycogen storage
disorders. Basement membrane of the
glands, glomeruli, etc. can also be
demonstrated by PAS stain.

79. • Alcian Blue Alcian blue stains acid mucin (in
acidic pH 2.5), such as sialomucin and
sulphomucin. It stains mucin of the salivary
glands, prostate and large intestine. Alcian
blue also stains proteoglycans of cartilaginous
material.
• Combined PAS-Alcian Blue Staining
Indications Combined use of Alcian blue and PAS
in a same section helps to demonstrate both
acidic and neutral mucin in the same section

80. • Oil Red O
Indications Oil red O stain is used for
demonstration of lipid material. The lipid
material takes a deep red colour in this stain.
• Other special stains for lipids includes; Sudan
Black B, Ferric Haematoxylin for Phospholipid

82. • Special stains for pigments;
• Prussian Blue Reaction (Perls’ Reaction)
for Ferric Iron. A useful control would be
postmortem lung tissue that contains a
reasonable number of iron-positive
macrophages (heart failure cells).
• Masson-Fontana stain demonstrates melanin
and argentaffin granules.

83. • Formalin Pigment
Formalin pigment is brownish black in colour. Acidic
formalin fixation commonly produces formalin
pigment (Fig. 9.13). The pigment can be removed
by alcoholic picric acid. Using buffered formalin
helps to reduce formalin pigment.
• Malarial Pigment
Malarial pigment is similar to formalin pigment.
This is also brownish black in colour. Unlike formalin
pigment, malarial pigment is intracellular
in location. RBCs are usually loaded with malarial
pigment. This pigment can be removed by treating
the section with alcoholic picric acid.

84. • TURNBULL BLUE STAIN FOR FERROUS
• Detection of ferrous (Fe2+) iron in tissues.
• SCHMORL TECHNIQUE FOR REDUCING
SUBSTANCES
• To indicate reducing substances present in
tissue. Melanin, argentaffin granules, and
even formalin pigment will be stained

85. • Components of connective tissues
• Three major components:
Cells
Fibers
Ground substances
• Cells are the living part of connective tissue
responsible for continuation of life
• Fibers and ground substances are also called
non-living or intercellular elements

86. • Fibers of connective tissues
• Three major types of fibers:
Collagen fibers
Elastic fibers
Reticular fibers
• Fibers are formed by cells of the individual
connective tissues
• The ratio of cells to intercellular substances
depend on the type of CT

87. • Collagen fibers
Van Gieson
Masson trichrome and Gomori 1 step
• Reticula fibers
Gordon and Sweet
Gomori method
The reticular fibers techniques are also called
silver technique

88. • Elastic fibers
Verhoff techniques
Aldehyde-Fuchsin method
Orcein
Resorcin-Fuchsin method
• In the demonstration of any connective tissue
fibers, it is important to note that the dyes are
acidic

89. • Amyloid Staining
• The deposition of this extracellular amyloid
protein in various organs and tissues is known
as amyloidosis.
• Alkaline Congo Red Stain
• Stains for the Microbial Organisms
• Gram’s Stain
• Ziehl-Neelsen Stain
Ziehl-Neelsen stain demonstrates acid-fast
tubercular bacilli.
• Fite Acid-Fast Stain for Leprosy
Fite acid-fast stain helps to demonstrate
mycobacterium leprosy.

90. • Grocott’s Methenamine Silver
Grocott’s methenamine silver stains the cell wall
of the fungi.