Histopathologic and Cytologic techniques Lecture

SECOND SEMESTER 4th UNIT EXAM

MOUNTING
Purpose of Mounting:
1. to protect the specimen from physical injury

③
2. to protect the section from bleaching or deterioration due to oxidation
3. to preserve the slides from
ease permanent keeping
4. to facilitate easy handling and storage
⇐
-
5. to prevent damage of sections which may lead to distortion of image during microscopic
- -

examination

Mounting media - usually a syrupy fluid applied between the section and the coverslip after
staining, setting the section firmly, preventing the movement of the coverslip.

Characteristics of a Good Mounting Medium

1. Refractive index: 1.518
Almost similar to the refractive index of glass and tissue
2. Should not dry quickly
Quick drying may break and distort the tissue
3. Should not dissolve out or fade tissues
4. Should not cause shrinkage and distortion of tissues
5. Should set hard, thereby producing permanent mounting of sections
6. Should set hard without granularity or cracking
7. Should be freely miscible with xylene and toluene

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It is necessary for removing the alcohol from the tissue and making the tissue
transparent.
8. Should not be nonreactive or will not change pH or color
9. Should not leach out any stain or affect staining

Classifications of Mounting Media

Temporary or Aqueuos
o Designed to mount water-miscible preparations directly from water in cases where
the stains is decolorized or removed by alcohol and xylene eg. Frozen sections for
fat stains (Sudan methods) or for metachromatic staining of amyloid.
o usually made up of:
gelatin, glycerin jelly or gum arabic (to solidify the medium)
glycerol (to prevent cracking and drying of the preparation)
sugar (to increase the refractive-index)
preservative solution
Permanent or resinous
o are used for preparations that have been dehydrated and cleared in xylene or
toluene, and are recommended for majority of staining methods.
o Natural or synthetic
o The most important synthetic resins are used for embedding undecalcified bones,
a
-

and for electron microscopy.

 Permanent Mounting Media
1. Canada Balsam
Abus balsamea - natural resin extracted fromftp.qq.gs the Canadian tree -

Recommended for whole mounts and for thick sections

= =

Refractive index: 1.524

ADVANTAGES
i. transparent, almost colorless in thin layers
ii. adheres firmly to glass
iii. sets to a hard consistency without granulation
iv. recommended for whole mounts and for thick sections because it does not
shrink much
v. miscible with xylene
vi. refractive index is 1.524
DISADVANTAGES
i. darkens slightly with age and upon exposure to sunlight, thus it should be
kept in a dark glass bottle
ii. slowly becomes acid because it oxidizes xylene to toluic and phthalic acid,
which causes gradual fading of many stains. Calcium carbonate may be
added to maintain its neutral reaction.
iii. (Benzene may be substituted for xylene as solvent.) Toluene or benzene
could be used instead of xylene.
2. DPX - (Dibutyl Phthalate and Xylene) of Kirkpatrick and Lendrum
It is prepared by Refractive Index = 1.532 It tends to set quickly and, in doing so, often retract from the edge of the coverslip. 
dissolving the
common plastic,
is a resinous medium recommended for small tissue sections but not for whole
polystyrene, in a mounts because of shrinkage produced on drying; hence, it should be used in
suitable hydrocarbon
solvent (usually
excessd-amounts.
xylene).  Made up of the resin “Distrene 80” plasticized by the addition of tricresylphosphate
It has a greater advantage over Canada balsam in that slides can be cleaned of excess
or dibutylphthalate mountant simply by stripping it off after cutting around the edge of coverslip.
It is available in neutral colorless solution which dries rapidly
3. Clarite (Clarite X)
Refractive Index = 1.544
One of the most widely used mounting media in North America
is a synthetic resin which is soluble in xylene (it is used as a 60% solution in xylene)
is generally preferred over D.P.X.
4. XAM
Refractive Index = 1.52
synthetic resin mixture in xylene, available in a pale yellow or colorless solution
dries quickly without retraction
preserves stains well
Sections are quickly mounted from xylene.
Semipermanent/Temporary Mounting Media
1. Water
Used in Clinical Microscopy
Least permanent
Dry off quickly
vantage
Not
• for oil immersion lens
Has a low refractive index, is moderately transparent and evaporates easily, hence
-

is good only for temporary mounting.

2. Glycerin
may also be used as a preservative
with a refractive index of 1.46, sets quite hard
will keep sections mounted for months, if care is taken in handling and especially
if sealed on the edges with paraffin wax.
provides greater visibility if slightly diluted with water (for moist sections)
It is miscible with water, is inexpensive, and is non-poisonous. It is also not

⇐
-

necessary to treat the specimens with alcohol or organic solvents, which may
introduce artifacts and remove pigments. This is usually regarded as the standard
mountant for fat stains.
3. GLYCERIN JELLY
Refractive Index = 1.4-1.47
the standard mounting medium used when dehydration and clearing with xylene
•
cannot be made (as in fat stains)
stains may fade
Tgi or sealing (The mountant is not set in the desired amount of
requires ringing
hardness and therefore requires "ringing”)

e-
Gelatin is added to distilled water and incubated in a water bath at 60°C until
-

?
dissolved. Glycerol and then phenol crystals are added and mixed. The solution is
-

labeled, and stored in a refrigerator at 40°C. For use, the gelatin must be heated
. Too much gelatin makes the jelly
-

µ
in a water bath or incubator at 60°C to melt.
difficult to melt and included bubbles found on the slide will not burst. The melted
medium should not be shaken or stirred before use, if formation of air bubbles is
to be avoided.
The disadvantage is that it should be melted before use (due to the presence of
gelatin). Stains mounted on glycerin jelly tend to fade.
FORMULA:
i. Gelatin 10 gm.
ii. Glycerol 70 ml.
iii. Distilled water 60 ml.
iv. Phenol crystals (preservative) 0.25 gm.
i
4. FARRANT'S MEDIUM require ringing
Refractive Index = 1.43
FORMULA:
i. Gum Arabic 50 gm.
ii. Distilled water 50 ml.
iii. Glycerol 50 ml.
iv. Sodium merthiolate 0.025 gm.
Dissolve gum arabic in distilled water with gentle heating and add glycerol and
sodium merthiolate. Mix well and label.
This gum arabic medium does not solidify upon storage and therefore does not
need to be heated before use. However, it takes a longer time to harden and may
therefore require
. ringing.
Arsenic trioxide may be used as a substitute of sodium merthiolate for preservation
of the medium. Addition of 50 gm. potassium acetate will produce a neutral (pH
7.2) instead of an acid (pH 4.4) medium, and therefore, will raise the refractive
index to 1.44.

÷
5. VON APATHY'S GUM SYRUP MEDIUM does not require ringing
-

Refractive Index = 1.52

FORMULA:
i. Pure gum arabic (crystals not powder) 50 gm.
ii. Pure cane sugar or sucrose 50 gm.
iii. Distilled water 50 ml.
iv. Thymol crystals 0.05 gm.
This medium is used for methylene blue-stained nerve preparations and as a
general purpose aqueous mountant.
It is one of the most useful aqueous mountants for fluorescent microscopy, being
-2
virtually non-fluorescent.
Von Apathy s medium is not compatible with normal histological stains.
The pH of the medium is near 4.0 (highly acidic) so stains fade or bleed into the
medium.
Addition of 50 grams potassium acetate, 20 grams of calcium chloride or 10 grams
pg

sodium chloride can raise the pH to near 7.0 and will prevent "bleeding" of
pg

metachromatic stains for amyloid.

The medium sets quite hard, has a higher refractive index, and does not require
ringing.
6. BRUN'S FLUID
FORMULA:
i. Glucose 24 gm.
ii. Glycerine 6 ml.
iii. Spirits of camphor 6 ml.
iv. Distilled water 84 ml.
is recommended for mounting frozen sections from water
#

Frozen sections that are mounted directly from water or paraffin sections which
require dehydration and clearing, usually should be mounted on glycerin, gum
syrup or Brun's fluid.
 7. Levulose (fructose) syrup
Refractive index = 1.5
Does not leach metachromatic stains
To prepare, dissolve 30 g levulose in 200 ml distilled water, by placing in a
stoppered bottle in the incubator (37O) for 24 hours, with occasional shaking.
8. Mineral Oil (Liquid Paraffin)

@
For Romanowsky-stained smears, which should be well dried before mounting -

Techniques of Permanent Mounting

Since all resins used in mounting are soluble in xylene, it follows that the stained sections
must be brought up to xylene, that is, dehydrated and cleared. When the sections are
placed in the final bath of xylene, a series of suitable-sized coverglass are selected,
cleaned with a fine cloth (if necessary, after washing in alcohol), and placed on a sheet
of white paper or large sheet of white blotting paper. Coverglasses may be round, square
or rectangular; 22 x 22, 22 x 30, 22 x 40, and 22 x 50 mm are popular sizes.
The slide carrying the section to be mounted is taken from the last xylene bath with the
forceps; the ends are first cleaned so that the diamond-inscribed number is seen and the
front of the slide can be identified. The back of the slide is then wiped dry with a clean,
dry, fine cloth and the excess xylene is wiped off from the front to within 2 or 3 mm of
the margin of the section. With a small glass rod, a streak of mounting medium is placed
down the center of suitably sized coverglass. The slide is placed lengthwise on its edge,
touching the edge of the coverglass and gradually inclined downward, onto the coverglass
until the section touches the streak of the mounting medium; the slide is then inverted.
As this happens the mounting medium quickly spreads through the whole area of the
section, which is still moist with xylene, and to the edges of the coverglass. Once the
sections are mounted they are ready for microscopic examination but must be handled
with care so that the coverglass is not moved. It time allows setting of the mounting
medium may be hastened by placing the slides on the hot plate (50oC) or in the wax over
for up to 2 hours. Before filing, the mounting medium must be thoroughly dried and
hardened by placing the slides in a 37 to 60°C oven for 24 hours or leaving them at room
temperature for 2 to 3 days.

Difficulties in Mounting:
1. Excessive blotting dries up the section, causing shrinkage and checking of the specimen
2. Too much mountant = ooze out at the sides of the coverglass, makes cleaning of the slide
difficult, should be carefully wiped away with cotton swab dipped in xylol
3. Too little mountant = too diluted, will not cover the entire area of the coverslip and may
form bubbles around the edges of the coverslip; may require repeat mounting removing
the coverglass by soaking in xylene. Only experience will teach one how much
mounting medium to place on the coverglass.
4. Cloudy areas = moisture caused by incomplete dehydration (requires repeat dehydration)
a. Too rapid passage through the alcohols
b. Water contamination of the alcohols
c. Splashing
d. Breathing on the slide
e. A very moist atmosphere
 5. Air bubbles = interfere in microscopy; can be removed by gently pressing on the coverslip
a. Should not happen:
i. if proper amount of xylene is on the section
ii. If streak of mountant on the coverglass is in line with the axis in which the
slide is inclined down onto it
iii. If the slide is lowered gently and evenly onto the coverglass
b. Too many bubbles= remount the section
c. 1 or 2 bubbles= tease out by gentle pressure on the coverglass with the point of a
mounting needle

Ringing/Sealing
the process of sealing the margins of the cover-slip to prevent the escape of fluid or
semi-fluid mounts and evaporation of mountant, to fix the coverslip in place, and to
prevent sticking of the slides upon storage.
The term “ringing” originated because round coverslips were initially used and the coating
applied in the form of a circle or “ring.”
The ringing media include:
o paraffin wax,
o Kronig mixture (Du Noyer s) made up of two parts paraffin wax mixed with 4-9
parts powdered colophonium resin, heated and filtered
o Nail polish
o Household plastic cements
o Also available are cellulose adhesives such as Durofix.

Restaining Sections
REASONS:
1. Old, bleached or faded sections
2. For using a different or additional stain (superimposed staining)
3. For staining 2 or 3 pieces of tissues with different stains or techniques

PROCEDURE:
1. Remove coverslip by soaking in xylene for 24 hours, or by warming gently over a Bunsen
burner and gently pushing off the coverglasses with the point of a mounting needle.
2. Immerse in 75% alcohol
3. Transfer to 1% acid alcohol until all color has been removed (decolorize)
4. Wash thoroughly in running tap water (if water is acid, place in a weakliy alkaline
solution e.g. Scott’s tap water substitute, and then rinse in distilled water.)
5. Proceed to staining.
Alternatively, after hydration the sections are placed in 0.25% KMnO 4 for 5
minutes, then washed in water and placed in 1% oxalic acid until white, followed
by 5 minutes washing in tap water and restaining.
Broken Slides
If the slide has not been fragmented and the section is not too important
o Pieces may be reassembled from xylene atop a clean xylene-moist slide on which a
streak of Clarite (or any permanent mounting media) has been placed. This will
usually suffice for immediate examination, but a new section should be cut and
stained.
If the section or vital part thereof, and a replacement is not available, it may be
transferred to another slide as follows:
1. Remove coverslip by soaking in xylene. (This may be expedited by placing in the
incubator at 37°C) Leave until all the mountant has been removed.
2. The whole slide is then covered with a mixture of 6 parts butyl acetate and 1 part
durofix.
3. Leave in the incubator for 30 minutes until the mixture hardens into a film.
4. Using a sharp scalpel blade, the hardened film is cut around the section, and the
slide is placed in cold water until the film and section float off.
5. The film containing the section is mounted on a clean slide, placed in the 37°C
incubator until dry, washed gently with butyl acetate, then washed well with
xylene, and mounted in Clarite or Permount.
SPECIAL PROCESSING TECHNIQUE
when the important chemical constituents of the tissue eg. Enzymes should not have
been removed, altered or displaced, such as in Histochemistry
Employed when chemical constituents of the tissue will be preserved and/or examined

Frozen section

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- The most ideal and preferred means of preserving tissues to avoid complete or partial
loss of enzymes.
- Difficulties, however, arise in obtaining thin and serial sections of uniform thickness;
o_0 be easily handled without
since cut sections of tissue tend to disintegrate and cannot
prior fixation. These disadvantages will have to be considered in determining the
necessity and advisability of such sections.

Methods to employ if Chemical Fixation of Tissue Blocks are to be Avoided:

1. Freeze-drying
2. Freeze-substitute
3. Fresh frozen section

General Principle
- rapidly preserving the tissue block by freezing (quenching) to produce instant cessation of
cellular activity thereby preventing chemical alteration of tissue and displacement of
cellular tissue components. Freezing must be rapid, accomplished within seconds to
prevent the formation of ice crystal artefacts in tissue blocks and produce optimum tissue
preservation.
- The freezing agent commonly employed is liquid nitrogen, and the tissue is sectioned
into thin slices using a cryostat machine under very low temperature. The use of
isopentane, pentane and propane and most recently of dichloro-difluoromethane, which
can be cooled to very low temperature in order to retain the fluidity of the freezing
agents, have contributed much in giving higher conductivity to this liquefied gas.

Freeze-Drying
special way of preserving tissues by rapid freezing (quenching) of fresh tissue at -160°C

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and subsequently removing ice water molecules (dessication) by transferring the still
frozen tissue block into a vacuum chamber at a higher temperature, e.g. -40°C
(sublimation) without the use of any chemical fixative.

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This technique is generally not used in routine surgical laboratories, and is restricted to
specialized or research laboratories.
Very important for enzyme studies
Tissues are difficult to section and are brittle
1. A tissue around 2 mm. thick is plunged into isopentane or propaneisopentane
mixture which has been chilled to -160° to -180°C with liquid nitrogen.
2. This will effectively solidify the tissue in 2-3 seconds, thus preventing the
formation of large ice crystals, autolysis and putrefaction.
3. The frozen tissue is then transferred into a high vacuum drying chamber
maintained at a temperature of -30° to -40°C depending upon the size of the
tissue. Water is sublimated and dehydrated from the tissue, thereby completing
the dessication process within 24-48 hours.
 Freeze Dry
-

4. Once drying is completed, the tissue is removed, fixed and embedded, either in
molten paraffin wax, water soluble waxes or celloidin.
②
5. Infiltration and impregnation are usually performed in a vacuum embedding oven.
6. The tissue is then sectioned in the usual routine manner.
7. Specific staining is applied, depending upon individual necessity.
ADVANTAGES:
o produces minimum tissue shrinkage

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o allows tissues to be processed in a fresh •state
o causes minimal chemical change on the cells, most especially on the protein
components
o less displacement of tissue and cellular constituent
o very important for enzyme studies
DISADVANTAGES:
o time-consuming
•
o expensive
o freeze-dried materials are generally more difficult to section than ordinary
paraffin blocks
o tissue is brittle and inadequately supported due to the relatively short period for
wax impregnation

a
o it is not advisable as a routine procedure.

Freeze-substitution

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It is similar to freeze-drying in preparing and preserving tissue blocks for subsequent
sectioning because both involve the rapid freezing of tissues and the subsequent
infiltration and embedding of the frozen tissue block in paraffin or celloidin.
The only variation is that the frozen tissue, instead of being subjected to dehydration in
an expensive vacuum drying apparatus, is fixed in Rossman's formula or in 1% Acetone and
dehydrated in absolute alcohol for 1-6 days at -60 to -70 degree Celcius, respectively
Infiltration and embedding is then carried out in the same way as in paraffin section.
more economical than freeze drying
-

may be used for routine purposes

Fresh Frozen Tissue Sectioning

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requires that the tissue be maintained in the frozen solid state during cutting of section,
thereby supporting and protecting the tissue from damage and distortion by the knife
during the process of cutting.
The tissue must be sufficiently cold to prevent compression
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and displacement of cell and
-
tissue structures
=
as the knife passes thru it.
DECALCIFICATION
the removal of calcium ions from a bone or calcified tissue through a histological process
so that softening will take place to facilitate embedding and sectioning.
Done after fixation and before dehydration ( not impregnation )
- e-

Inadequate decalcification may result in poor cutting of hard tissues and damage to the
knife edge during sectioning
dd-
Decalcification adjusts the hard substance of bones to the softness of paraffin embedding
medium.
Tissues subjected to decalcification:
o Bones
@

•
o Teeth
o Tuberculous organs
o Arteriosclerostic vessels
Before decalcification, tissues are cut into small pieces, approximately 5 mm, with a fine
fret-saw, hackooo o
saw, or jig saw, and trimmed with a hand razor to permit complete
penetration of the decalcifying solution with minimal surface and tissue distortion. Since
O which are injurious
all decalcifying agents contain acids @ to the organic substance of the
↳ ----
bone or other tissue, they must be protected by adequate fixation before decalcification

TEXT
tissue
is begun. mass

Fixation is best accomplished by placing cut tissues in buffered neutral formalin for 2 to 4
days, or in Helly's fluid or Zenker for 15 to 24 and longer for formalin so that the nucleic
acids become resistant hydrolytic enzyme used in decalcification.
The choice of a decalcifying agent depends on the length of time which can allotted for

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the purpose.
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For paraffin-embedded tissues with small foci, the block can be placed face down on a
fsnomtetdodgalusffffehbhtanoedecal-I.fm
②gauze or cotton
pad of @ saturated with 10% HCl.
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A good decalcifying agent must be capable of removing calcium salts from tissues
completely without producing considerable destruction of cells and tissue components
and without adversely affecting the staining (hematoxylin) capacity of the cell,
particularly the nucleus.
Eshleman
staining
:
Different Types of Decalcifying Agents:
1. Acids
[ 2. Chelating agents
3. Ion exchange resins
4. Electrical ionization

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Acid decalcifying agents
Stable t.hmodhaua.ae be minimized can

Easily available
Relatively inexpensive
After fixation, tissue blocks are placed in a gauze bound by a thread, and suspended in a
liberal amount of decalcifying agent. This ensures complete decalcification and protects
the tissue from any precipitate which may form at the bottom of the tube. Since the
solution is characterized by the corrosive action of its acids, it is recommended that the
thread be dipped in melted paraffin wax before use and metal cap containers be avoided.
-
dissolve Calcium out of the tissue
-
acids makes calcium salts soluble to mix w/ acid
Rate of decalcification can be affected by:
1. Structure of the tissue
a. Longer decalcification for larger and denser tissues

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b. Ideal time: 24-48 hours
c. Dense tissues require up to 14 days or longer
d. Solution should be changed everyday and the degree of decalcification tested
-2ft TAT

2. Temperature
÷
a. Higher temperature accelerates decalcification but it will also increase the
if

damaging effects of acids on tissue.

b. Optimum temp is 18-30oC (room temp)
3. Volume and concentration of solutions
÷ volume and concentration accelerate decalcification
a. Higher

Tie
4. External factors: agitation and moving
a. Gentle fluid agitation accelerated the rate of diffusion and decalcification
-

Precautions: Too rapid decalcification produces complete digestion of tissue specimens with
a (marked swelling), hydrolysis
accompanying swelling e of body matrix and poor e staining capacity
of the wall. arm deem
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Acid decalcifying agents p

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staining
1. Nitric acid
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-

Most common and fastest decalcifying agent

utilized both as a simple solution or combined with other reagents.
Inhibits nuclear stains and tissue destruction especially if prolonged
i. May be prevented by combining nitric acid with formaldehyde or alcohol
a very rapid decalcifying agent, producing minimal distortion and is, therefore,
.

recommended for routine purposes.

Aqueous Nitric Acid Solution 10%

FORMULA:
Concentrated nitric Acid 10 ml.
Distilled water added up to 100 ml.
DECALCIFICATION TIME: 12-24 hours
Advantages:
1. It is rapid in action.
2. It produces minimum distortion of tissues.
3. It produces good nuclear staining (although less than in slower acting agents).
4. The acid may be easily removed by 70% alcohol.
5. It is recommended for urgent biopsy, and for needle and small biopsy specimens to
permit rapid diagnosis within 24 hours or less.
6. It can be used for large or heavily mineralized cortical bone specimen if
decalcification progress is carefully monitored by a decalcification endpoint test.
Disadvantages:
1. Prolonged decalcification may lead to tissue distortion.
2. It can seriously damage tissue stainability.
3. It imparts a yellow color with nitrous acid, thereby impairing the staining reaction
 of the tissue.
4. Old nitric acid solution is particularly damaging and should be replaced with fresh
stock solution.
e
5. Strong acids tend to be more damaging to tissue antigens for immunohistochemical
staining,
- and enzymes
-
may be -
totally lost.
Procedure:
1. Fixation for 2-3 days in buffered neutral formalin.
2. Place in 10% Nitric Acid
3. Change solution daily until bubbles stop evolving from the tissue (usually 1-3 days,
depending on tissue size and consistency)
4. Wash in 3 changes of 90% alcohol (washing in watery solutions may lead to
excessive swelling and deterioration of tissue)
5. Dehydrate, clear in xylene or benzene, and embed in paraffin. It is important that
the time in the nitric acid be kept to the absolute minimum; otherwise staining
will suffer severely.

Formol-nitric acid -

weak acid
FORMULA:
Concentrated nitric Acid 10 ml.
Strong formaldehyde, 40% 5 ml.
Distilled water 85 ml.
DECALCIFICATION TIME: 1-3 days
Advantages:
1. It is rapid-acting; hence, is recommended for urgent biopsies.
2. Nuclear staining is relatively good.
3. It produces less tissue destruction than 10% aqueous nitric acid.
Disadvantages:
1. The yellow color imparted by nitrous acid formation will impair staining reaction
of the cell. This may be prevented by neutralizing the tissue with 5% sodium
sulfate and washing in running tap water for at least 12 hours. Addition of 0.1%
urea to pure concentrated nitric acid will also make discoloration disappear
without considerably affecting the efficiency of the decalcifying solution.
2. The solution should be used inside a fume hood.

Perenyi’s fluid
FORMULA:
Nitric acid 10% 40 ml.
Chromic acid 0.5% 30 ml.
Absolute ethyl alcohol 30 ml.
Mix shortly before use. Chromic acid must be collected for proper disposal.
DECALCIFICATION TIME: 2-7 days
Advantages:
1. It is recommended for routine purposes.
2. It decalcifies and softens tissues at the same time.
3. Nuclear and cytoplasmic staining is good.
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4. Maceration is avoided due to the presence of chromic acid and alcohol.
 Disadvantages:
1. It is a slow decalcifying agent for dense bones; hence, is not recommended for
urgent diagnosis.
2. Complete decalcification cannot •
• be determined by chemical test because a
precipitate is formed upon the addition of ammonia to Perenyi's fluid even in the
absence of calcium ion. This may be dissolved by adding glacial acetic acid drop by
drop. About 0.5 ml. of saturated aqueous ammonium oxalate is then added to the
solution. Reappearance of a white precipitate within 30 minutes will reaffirm the
presence of calcium in the agent, signifying that decalcification is still
incomplete.

Phloroglucin-nitric acid
FORMULA:
Concentrated nitric acid 10 ml.
Phloroglucin 1 gm.
Nitric acid 10% 100 ml.
(To be added after disappearance of dense white fumes formed by combining the
first two ingredients.)
DECALCIFICATION TIME: 12-24 hours
Advantages:
1. It is the most rapid decalcifying agent so far, recommended for urgent cases.
Disadvantages:
1. Nuclear staining is poor.
2. Prolonged decalcification produces extreme tissue distortion.
3. Yellow color must be neutralized with 5% sodium sulfate and thoroughly washed
with running tap water for at least 24 hours.
• be determined by chemical
4. Complete decalcification cannot • means.
When decalcification is complete, the acid must be removed by three
changes of 70% to 90% ethanol, since washing in watery solutions will lead to
excessive swelling and deterioration of tissue. When the sections are cut, the
slides are brought to water and placed in 1% aqueous lithium carbonate for I hour,
washed in later for15 minutes, and then stained.

2. Hydrochloric acid (HCI) -

strong
Inferior to nitric acid; slower and greater distortion
produces good nuclear staining
. solution with 70% alcohol, may be recommended for surface
if used in 1%
decalcification of the tissue blocks.
Von Ebner’s Fluid
FORMULA:
Saturated aqueous solution of NaCl 50 ml.
36% concentrated hydrochloric acid 8 ml.
Advantages:
1. permits relatively good cytologic staining; moderately rapid decalcifying agent.
2. Does not require washing out before dehydration
 3. Recommended for teeth and small pieces of bone
Disadvantage: The extent of decalcification cannot be measured by a chemical test.

3. Formic acid (CH2O2) weak acid

Moderate-acting decalcifying agent
Better nuclear staining with less tissue distortion
Safer to handle than nitric acid and hydrochloric acid
It is recommended for routine decalcification of postmortem research tissues,
although not suitable for urgent examinations

Ee
Formic acid in a 10% concentration is the best all-around decalcifier.
Formic acid is the only weak acid used extensively as a primary decalcifying agent.
e
Addition of citrate probably accelerates decalcification by chelating the calcium as
it is liberated from the bone.
Formic acid-formol saline
FORMULA:
Formic acid (Sp. grav. 1.20) 10 ml.
Normal saline 10% 90 ml.
DECALCIFICATION TIME: 2-7 days
Advantages:
1. Fix and decalcify tissues
2. Suitable for most routine surgical specimens, particularly when
immunohistochemical staining is needed
3. recommended for small pieces of bones and teeth.
4. permits excellent nuclear and cytoplasmic staining.
Formic acid-sodium citrate solution
FORMULA:
Aqueous sodium citrate 20% 50 ml.
Formic acid 45% 50 ml.
DECALCIFICATlON TIME: 3 -14 days
Advantages:
1. It permits better nuclear staining than nitric acid method.
2. It is recommended for autopsy materials, bone marrow, cartilage and tissues
studied for research purposes.
Disadvantages:
1. It is relatively slow; hence, is not recommended for routine purposes and for dense
tissues
2. It requires neutralization with 5% sodium sulfate.

4. Trichloroacetic acid Weak acid

FORMULA:
Trichloroacetic acid 5 gm
Formol saline 10% 95 ml.
DECALCIFICATlON TIME: 4-8 days
Advantages:
1. Good nuclear staining
2. Does not require washing out; excess acid may be removed by several changes of
 90% alcohol
Disadvantages:
1. It is a weak decalcifying agent, not used for dense tissues, and is suitable only for
small spicules of bone.
2. It is very slow-acting; hence, is not recommended for urgent examinations.

5. Sulfurous acid strong

Very weak decalcifying solution suitable only for minute pieces of bone.

6. Chromic acid Strong

FORMULA:
Chromic acid % 15 ml.
Osmium tetroxide 4 ml.
2% Glacial acetic acid 1 ml.
Advantages:
1. It may be used both as a fixative and decalcifying agent.
2. It may be used for decalcifying minute bone spicules.
Disadvantages:
1. Nuclear staining with hematoxylin is inhibited.
2. It tends to undergo reduction and forms precipitates at the bottom of the
container thus requiring frequent changes of solution.
3. Insoluble pigments are formed when decalcified tissue is dehydrated with alcohol;
hence, tissues must be washed out prior to dehydration.
4. Degree of decalcification cannot be measured by the routine chemical test.
Caution: Chromic acid is an environmental toxin.
1. Chromic acid is highly corrosive to skin and mucous membranes.
2. It is carcinogenic.
3. Suitable protective material is not readily available or practical for laboratory use.
4. Drain disposal is not a legitimate option for any solution containing chromium,
including subsequent processing of fluids following fixation or rinses following
staining procedures involving chromium.

7. Citric acid-citrate buffer solution Weak acid

FORMULA:
Citric acid (monohydrate) aqueous solution 7% 5.0 ml.
Ammonium citrate (anhydrous) aqueous solution 7.4% 95.0 ml.
Zinc sulfate aqueous solution. 1% 0.2 ml.
Chloroform (as preservative) - a few drops
DECALCIFICATION TIME: 6 days
Excellent nuclear and cytoplasmic staining
Does not produce or tissue distortion
action is too slow for routine purposes.
Chelating agents
Combine with calcium ions and other salts to form weakly dissociated complexes and to
facilitate removal of calcium salt
Most common chelating agent: EDTA (ethylene diamine tetra acetic acid)
Optimum pH 7-7.4; EDTA works too slowly I under pH 5, owing to insolubility, but over@
as
pH
8, tissue maceration starts due to alkaline sensitive protein bonds.
#

②bind calcium at pH 3
do not
The tissue is usually placed in EDTA Atf
from 1-3 weeks for small specimens, but it may take
egg ÷

6-8 @weeks or longer

@ to totally decalcify dense cortical bone. The solution should be
÷

changed every 3I days, and in the final stage, everyI day, to facilitate decalcification.
•

It permits excellent #staining results.

It produces minimal cell and tissue distortion.
#
It forms minimal histological artifacts, usually caused by production of-2-0
CO2 bubbles.
Extent of decalcification can be measured by routine chemical test.
EDTA is an excellent bone decalcifier for enzyme or immuno-histochemical staining, and
for electron microscopy
Enzymes require specific pH conditions in order to maintain activity, and EDTA solutions
can be adjusted to a specific pH for enzyme staining.
Very slow and is therefore not recommended for urgent and routine purposes.
It causes slight tissue hardening.
EDTA inactivates alkaline phosphatase activity, which can be restored by addition of
magnesium chloride. ↳ too much alkalinity damage the tissue =

alkali pH =

Ion exchange resins -

do not remove calcium

iohsenhanee acid
Ammonium form of polystyrene resin , hasten action of Formic
Hastens decalcification by removing calcium ions from formic acid-containing decalcifying
solutions, thereby increasing solubility from the tissue
Not recommended for nitric acid and HCl, which contain minerals
Placed over the bottom of the container to be used
May be reactivated by immersing it in N/10 HCl and washing it with distilled water thrice
Advantages:

Nutty
HA cannot be used w/ acid w/ minerals
-

1. Cellular detail is well-preserved. ca ngtaumnpisygm

in

2. Daily washing of solutions is eliminated.

3. It permits excellent staining results.
4. Decalcification is hastened.
5. It produces minimal cell and tissue distortion.
6. It forms minimal histological artifacts, usually caused by production of CO 2 bubbles.
7. Extent of decalcification can be measured by routine chemical test.
Disadvantages: p
determined by amount of Calcium ions

The degree of decalcification cannot be measured by chemical means.

It is very slow, and is therefore not recommended for urgent and routine purposes.
It causes slight tissue hardening. has its own end Time
Each decal agent duration
recommended or

" "
end time tissue will
beyond
=

•
go
be damaged or
macerated
difficulty in sectioning
or

.
shorten decal =

monotony
Electrophoresis hasten dual
@
-

Positively charged ions are removed by using negative electrode

Faster decalcification due to the heat and electrolytic reaction produced in the process.

€-0
Recommended for small bone fragments
Cytologic and histologic details are not well-preserved
processing only a limited number of specimens at a time

Degree of Decalcification
Prolonged decalcification of tissue is liable to prevent hydrolysis and lead to
maceration and destruction of tissue components which are poorly stained. Over-
decalcification, particularly with the strong acid decalcifiers, spoils the staining of
basophilic elements such as cell nuclei and in certain circumstances can cause maceration
of the softer tissue elements. On the other hand, when the tissue is allowed to stay in the
decalcifying agent for a very short period of time, decalcification may be incomplete
thereby interfering with the normal cutting of sections and staining of specimens. If high-
quality results are to be obtained from decalcified tissue, it is important to determine the
point at which all the calcium has been removed because, from this point on, tissue damage
seems to occur at an increasing rate. There are several methods to check if the end point
of decalcification has been reached:
Physical or mechanical test
o Done by touching or bending the tissue or by pricking with a needle or a probe
o Vague and inaccurate
o manipulation, bending, probing or trimming of the specimen to “feel” for remaining
calcified areas.
o Mechanical damage can occur during bending or probing and small deposits of
calcium can easily be missed.
o A method of determining the endpoint by carefully weighing the specimen after
rinsing and blotting has also been described, and may be an effective method for
large specimens.
o An alternate method of evaluating tissues mechanically is by pricking the tissue
with a fine needle or a probe. This method is apt to produce needle tract artifacts
and destroy important cellular details. Pricking, slicing, bending or squeezing
tissue can disrupt soft tumor from the bone or cause false positive microfractures
of fine trabeculae, leading to a potential misdiagnosis. Aside from this
disadvantage, small calcified foci may not even be detected.
X-ray or radiological method
o Can detect the smallest focus of calcium
o Very expensive and not for mercuric chloride-fixed tissues due to the latter's
characteristic radio-opacity which will interfere with the correct interpretation of
the plate.
o best method, particularly with large specimens such as femoral heads
most
Chemical method - common

o Uses the discarded decalcifying solution, litmus paper, concentrated ammonia, and
sat. aq. Ammonium ocalate
o Cloudiness indicates incomplete calcium removal
1. The decalcifying fluid is usually changed every 24 -48 hours and the
Familiaris
 chemical test is performed on the discarded fluid.
2. A piece of blue litmus paper is added to a test tube containing 5 ml. of the
discarded decalcifying agent (the litmus paper will turn red due to the
acidity of the fluid).
3. Strong ammonia is then added drop by drop until the fluid is neutralized
(this can be detected by the change in color of the litmus paper from red to
blue, indicating alkalinity). The presence of cloudiness indicates that there
is still calcium found in the solution.
4. The tissue is then immersed in a new solution of decalcifying agent. If the
solution remains clear after neutralization with concentrated ammonia, 0.5
ml. of saturated aqueous solution of ammonium oxalate is added and the
•
solution is allowed to stand for 30 minutes. Cloudiness will signify
incomplete calcium removal; hence, the need for further decalcification. If
=

the solution remains clear after 30 minutes, decalcification is considered to

@
be complete. #

used few acid deal agents

chemical - can be in only

by transparency of solution after addition

D of D determined

of
calcium resin >

complete = Clear

cloudiness
incomplete
=

postal
✓ - after decal the decal agent esp
.

,
removed
the acid decal must be

from the tissue to minimize damage

&
Pleven t contamination

neutralize acid
-

-
rinse tissue w/ running water
 EXFOLIATIVE CYTOLOGY
Microscopic study of cells that have been shed, desquamanted, or scraped off
Purposes:
1. Assess malignant conditions
2. Detection of asymptomatic cancers
3. Assessment of female hormonal activity
4. Determination of genetic sex
5. Detection of possible infections Refer to
Common specimens:

Moduli
- Vaginal scrapings/swabs
- Endocervical and endometrial scrapings
- Prostatic secretions
- Bronchial aspirate or sputum
- Serous fluids 147 -

160
- Gastric and duodenal fluids pp
-

- CSF
- Bone marrow aspirate
- Urine
Fixation and Fixatives
Alcohol-Ether
o Best for cytology
95% Ethanol
o Most widely used
o “universal fixative”
Carnoy s fluid
o Useful for bloody specimens
o 3-5 minutes only
Coating fixatives
o Spray fixative
o Hair sprays with no lanolin or oil are also effective due to the alcohol content
o Not recommended for routine laboratory use
Precautions:
- Care must be taken to prevent dislodging the smear from the slide during fixing
- Make sure slides do not stick together
- Refrigerate a fluid specimen if it cannot be fixed immediately
- Gastric material must be kept in ice and prepared within minutes
- High mucus content: 12-24 hours if refrigerated
- High protein content: 24-48 hours if refrigerated
- Low mucus content: 1-2 hours delay only
- Low pH specimen: collected on ice
Transport:
- Slides may be air-dried after fixation
- Fixed smears may also be transported in glycerin
Smear Preparation
Methods:
o Streaking
o Spreading
o Pull-apart
o Touch/imprint/impression
Guidelines:
o Slide must be clean and smear spread evenly (no lumps)
o Labels must be indicated legibly on the slide (preferably using a diamond pen)
o Prepare fixative before making the smear
o Angle of spatula or second glass slide: 15o

Concentrating Cells from Fluids

A cell pellet/button may be prepared using the centrifuge
Cells can be directly concentrated onto the slide using a cytocentrifuge
Fluids may be filtered and an imprint smear made from the filter paper

Adhesives
Fluids with low protein content require the use of adhesives
o Urine
o Bronchial lavage
o Specimens that have been treated with proteolytic enzymes or mucolytics

Staining
Common stains for exfoliative cytology
o Papanicolau stain
o H&E
o Modified staining (Wright s)
Papanicolau Stain
Advantage:
o Transparent blue staining of cytoplasm is observed
o Excellent nuclear staining
o Color range is predictable and of great value in identification of cells
Disadvantage:
o Procedure is lengthy and complicated
o Does not give accurate acidophilic index

Hormonal Cytology
- The vaginal epithelium responds to stimulation by steroid hormones, mainly estrogen and
progesterone
- The cell population reflects the biological effects of the current hormonal status
- NORMAL CELLS
o Epithelial cells
Superficial cells Endocervical cells
Intermediate cells Endometrial cells
Parabasal cells Basal cells
 o Non-epithelial cells
Erythrocytes
PMNs
Histiocytes
Spermatozoa
- Acidophilic Index – percentage of cells that stain pink-orange to red with Pap s and red
in Shorr method
- Pyknotic Index – “karyo-pyknotic index”; percentage of cells having shrunken, dark, small
(less than 6µ) structures nuclei
- Maturation index – percentage proportion of cells from the three layers of the vaginal
epithelium

Maturation index
Relation of parabasal cells to intermediate cells to superficial cells
o Estrogen effect leads to increased superficial cells
o Progesterone effect leads to increased intermediate cells

Other indicators
Lactobacillus acidophilus
o Doderlein s bacillus
o Increased numbers indicate corpus luteum phase (pregnancy)
Ferning
o Due to drying of mucus in high NaCl concentration
o Due to influence of estrogen

Sexual Determination
Barr Bodies
o Small, plan-convex mass or a dot of chromatin about 1µm in diameter on the inner
nuclear membrane

o
Reporting
Class I
o Negative for Malignant cells
Class II
o Atypical cells present, but negative for malignancy
Class III
o Suspicious for malignant cells
Class IV
o Strongly Suggestive for Malignant cells
Class V
o Conclusive for Malignant cells

Bethesda System
A standardized system for the reporting of cervical and vaginal cytologic diagnoses
Developed in 1988
Uniform format
Standard terminology

Specimen Adequacy
Satisfactory/Limited/Unsatisfactory
General Categorization:
Negative for Intraepithelial lesion or malignant cell
Epithelial cell abnormality
Benign Cellular Changes
Descriptive Diagnosis:
Infections / Radiation effects
Atypical squamous cells of unknown significance
Low grade squamous intraepithelial lesion
High grade squamous intraepithelial lesion
Squamous Cell Carcinoma
Glandular cell abnormality
Atypical glandular cells
Adenocarcinoma
Others

Pap smear specimens are considered satisfactory for interpretation if there are:
• Adequate numbers of well visualized squamous cells present
• Adequate numbers of well visualized endocervical cells or squamous metaplastic cells
(from the transformation zone).
• Less than 50% of the cells obscured by blood or inflammation
• Properly labeled specimens
Specimens to which the following conditions apply will be rejected:
1. Specimen is submitted without a requisition.
2. Specimen is not labeled with the patient name.
3. The patient name (or other identifying information) on the specimen and requisition do
not correspond.
5. The specimen is labeled appropriately but the requisition is not labeled.
6. The specimen slide(s) is (are) irreparably broken.
7. Specimen is submitted from an unauthorized source.

Specimen collection:
Routine Cervical/Endocervical (Pap Smear)