LESSON 1-2: Histopathologic and Cytologic Techniques LABORATORY

Histology (microanatomy) – the Pathology

science concerned with the Anatomic Pathology Specialty Clinical Pathology Specialty
microscopic structure of tissues and Microscopic analysis of tissue • Hematology
organs in relation to their functions. changes. Pathologist plays a • Microbiology
central role in the diagnosis of • Immunology
surgically removed tissues. • Clinical Chemistry
• Blood Bank (Transfusion Medicine)
• Laboratory Data Management
• Molecular Pathology
Histopathology
HISTOPATHOLOGY
• The branch of pathology which
SECTION
concerns with the demonstration
- A basic component
of minute structural alterations in
of a tertiary hospital
tissues as a result of disease.
laboratory where
• Minute histological changes can
human tissues and
provide maximum information
body fluids are
• Permanent sections as a most
processed into slides
convenient way to study for
for microscopic
morbid tissues
examination by the
HISTOLOGY PROCEDURE
anatomic
1. Specimen Accessioning
pathologist.
2. Gross Examination
3. Tissue Fixation
Challenges of Histopathology in the Safety in the Histopathology
4. Tissue Processing
Philippine Setting Laboratory
a. Dehydration
• Histopathology remains a neglected • Histopathology section is
b. Clearing
branch of medical technology in the one of the most dangerous
c. Impregnation
Philippines sections in the clinical
5. Tissue Embedding
• Majority of hospital laboratories do not laboratory
6. Tissue Sectioning
have basic equipment (such as pH • The histotechnologist should
7. Slide Staining
meter) always have:
• Most laboratories are neglected with • Presence of mind for
old, obsolete, defective, and missing potential hazards in the
apparatuses. laboratory
• Inadequately – trained medical • Ensure vigilance on safety
technicians and non – paramedical (head of the laboratory)
personnel • Follow regulations and laws
• Production of low quality slides given to on proper waste disposal
the pathologists • Trainings done on security
• Basic Histopathologic Techniques by and protective gear for the
Alo, et.al (2015) personnel
• Xylene, alcohol and formalin may be re – used by investing in distillation or
filtration systems
First Step in Specimen Processing How do pathologists examine tissue?
Identification of the specimen and all • Gross examination - "grossing" is the process by which pathology specimens
of its components are inspected with the bare eye to obtain diagnostic information, while
being processed for further microscopic examination.
• Microscopic examination
• Risk management - Pertains to
personal as well as environmental
health
• What is the first step in risk
management? - IDENTIFY ALL
HAZARDS IN AND EMANATING
FROM THE LABORATORY!
S.O.P.!
✓ Control of hazardous substances
✓ Risk assessments
✓ Health and safety information
INSTRUMENTATION IN
HISTOTECHNOLOGY
• Microscope
• Microtome
• Cryostat
• Autotechnicon
• Automated coverslipper
• Automated H & E stainer

It is imperative that the laboratory maintain a current file for every piece of equipment in the laboratory.
This file should contain the following information:
1. Name, Manufacturer, Model Number and Serial Number
2. Record of preventive maintenance performed, as prescribed by the manufacturer
3. Record of service calls and repairs performed
4. Copy of operating manual
 What is the 1st and most What is the • FIXED SPECIMENS have a much less risk because of
important step in the recommended step for nearly all infectious agents are deactivated by
operation of every new employee? histological fixation (THOROUGLY FIXED)
equipment? presentation of checklist!
- Read the manual that
accompanies the
equipment! Prions
MANUAL CHECKLIST • Infectious agents that cause spongiform
provides information to provides a step-by-step encephalopathies such as creutzfeld – jakob disease
the machines operation approach to the and mad cow disease.
machine’s operation • Specimens of patients with suspected cjd can be
decontaminated by immersing the spx in formalin (48
hrs) – treatment in concentrated formic acid (1 hour)
How to handle spills?
– additional formalin fixation (48 hours)
• Small spills – defined as those that can be safely
• Complete penetration by alcohol will destroy all
handled by the immediate staff
infectious agents except PRIONS.
• Spill neutralizing and containment kits
• If the amount of spilled material is limited to a few
grams or mL, it can be simply wiped off with towel or Quality Control
sponge, while protecting the hands with suitable • Quality of sections and quality of staining produced
gloves. It must be appropriately disposed (not in the
by the histopathology laboratory must be checked.
general trash) with sealed impermeable plastic bag
• Special stains must be accompanied by a control
or container.
section.
• For other spills of dangerous materials, all personnel
should evacuate the room.
• If the spill is large, the area must be sealed off and an
Health hazards Physical hazards
emergency team must be called. • Biohazards •Combustibles
• Corrosive chemicals •Flammables
• Sensitizers •Explosive chemicals
First Aid • Carcinogens •Oxidizers
• The most common accidents requiring first aid are • Toxic materials
ingestion, eye contact and extensive skin contact
and splashing of dangerous chemicals into the eyes. • PELs-Permissible Exposure Limits
• All laboratories should be equipped with emergency • TLVs-Threshold Limit Values
eye wash stations either as free standing devices or • OELs-Occupational Exposure Limits
small appliances affixed to sink faucets. (devices no
more than 100 feet from hazardous work areas; water • Terms used to define the maximum allowable
temperature be controlled to 15 – 35C) airborne concentration of a chemical (vapor,
• Accidental splashing of the eyes – eyes should be fume or dust) to which a worker may be
rinsed for 15 – 30 minutes exposed.
• Accidental skin contact with hazardous chemicals –
wash with water for 15 – 30 minutes
Storage of Hazardous Chemicals
• Dangerous liquids
Handling of potentially infectious specimens o Stored below countertop height
• Pathologists, histotechnologists and technicians may • Dangerous reagents
be exposed to a certain level of risk when handling o Stored in plastic or plastic – coated glass
and processing potentially infectious specimen bottles
through inhalation of aerosols, contact with non – • Flammable liquids
intact skin and contact with mucous membranes. o Never store in refrigerator or freezer
• FRESH TISSUE and BODY FLUIDS must always be o If usage cannot be avoided, small amount
considered potentially infectious and GROSSING of must be available and used up asap
specimen has the highest risk of all histological
activities.
 Methods of fresh tissue examination
• Fine needle aspiration • Core needle biopsy • Punch biopsy

• Excisional biopsy and Incisional biopsy • Shave biopsy

• Curettings The methods of tissue examination

may vary according to:
• Structural and chemical
components of the cells
• Nature and amount of the tissue
• Need for an immediate
examination of a tissue structure
o Examination may be
done on fresh or
preserved tissues,
depending upon
necessity. Fresh tissues
have the advantage of
being examined in the
living state, thereby
allowing protoplasmic
activities to be observed.
Teasing or Dissociation Smear Preparation
• Immersed in a watch glass • Process of examination wherein cellular materials are spread lightly over a
containing isotonic salt solution, slide (wire loop, wire applicator, or apposition smear with another slide.)
carefully dissected or separated, • Useful in cytological examinations particularly for cancer diagnosis
and examined under the
microscope A. Streaking
• Unstained – phase contrast or • With an applicator stick or a platinum loop, the material is rapidly
bright field microscopy and gently applied in a zigzag line throughout the slide, attempting
• Stained – differential dyes to obtain a relatively uniform distribution of secretion.
B. Spreading
• A selected portion of the material is transferred to a clean slide and
gently spread into a moderately thick film by teasing the mucous
strands apart with an applicator stick.
• Has the advantage of maintaining cellular interrelationships of the
material to be examined.
• Recommended for smear preparations of fresh sputum and
bronchial aspirates and thick mucoid secretions.
C. Pull – Apart
• Done by placing a drop of secretion or sediment upon one slide
and facing it to another clean slide.
• The material disperses evenly over the surface of the two slides.
Squash Preparation (Crushing) • The two slides are then PULLED APART with a single UNINTERRUPTED
Small pieces of tissue not more than motion and the specimen is placed under the microscope for
1mm in diameter are placed in a immediate examination or applied with vital stains.
microscopic slide and forcibly o Useful for preparing smears of thick secretions such as
compressed with another slide or with serous fluids, concentrated sputum, enzymatic lavage
a cover slip. samples from the gastrointestinal tract and blood smears.

Touch Preparation (Impression Smear)

• A special method of smear preparation whereby the surface of a
freshly cut piece of a clean slide, allowing the cells to be transferred
directly to the slide for examination by Phase Contrast microscopy or
stained for light microscopic study.

FROZEN SECTION
• Normally utilized when a rapid diagnosis of the tissue under question is required.
• Recommended when lipids and nervous tissue elements are to be demonstrated.
• Very thin slices, around 10 – 15μ in thickness are cut from a fresh tissue frozen on a microtome with CO2 OR on a
cryostat (cold chamber kept at an atmospheric temperature of -10°to -20°C).
• Frozen sections are then transferred to a slide and processed for a light microscopic study.
Frozen sections, both fixed and unfixed, have many The tissue for freezing should be FRESH! Done as quickly as
applications in histotechnology and commonly used for: possible!
• Rapid pathologic diagnosis during surgery. Slow freezing?
• Diagnostic and research enzyme histochemistry. - Distortion of tissue due to ice crystal artifacts!
• Diagnostic and research demonstration of soluble Commonly used methods of freezing:
substances such as lipids and carbohydrates. *Liquid nitrogen
• Immunofluorescent and immunohistochemical *Isopentane cooled by liquid nitrogen
staining.
*Carbon dioxide gas
• Some specialized silver stains, particularly in
*Aerosol sprays
neuropathy.
 LESSON 3: From the operating room to histopathology laboratory
Operating Room • Cold Ischemia Time
1. The tissue is removed from the patient. o Interval between the interruption of blood supply
2. The OR staff (circulating nurse) immediately and the time the tissue is immersed in the fixative
transports the fresh tissue to the laboratory. *for o Controlled by the surgeon. The longer the tissue
frozen sections, IHC, IF, cytogenetic studies, etc.* sits in the operating room without fixative, the
a. Large tissues can be sectioned serially more autolytic changes are seen in the sections
b. Uteri and intestines can be opened to taken from the tissue
expose mucosal linings • Fixation Time
3. The tissues will be immersed in adequate fixatives. o Period the tissue is exposed to formalin
4. A properly filled – up surgical pathology request Histopathology Department
form must accompany the specimen to the 1. Specimens submitted must be entered into the surgical
histopathology laboratory done by the clinician. pathology database via the accessioning process.
a. a. Patient’s history a. Unlabeled specimens are unacceptable. For
b. b. Physical and laboratory c. Imaging inconsistencies, return the specimen to the OR for
findings correction.
c. d. Pre – operative diagnosis b. Incomplete specimens in which the request form
specifies a number of samples which do not
correspond to the number submitted are
unacceptable.

Fixation

Functions of Fixatives
• Classically defined as the killing, penetration, and hardening of • They change the soluble contents of cells
tissues. into insoluble structures that can
• Currently defined as the alteration of tissues by stabilizing protein so otherwise be lost during subsequent
that the tissues become resistance to further changes. processing.
• Primary aim: Preserve the morphologic and chemical integrity of the o Subjecting the tissue by heat or
cell in as life – like a manner as possible dessication (denaturation)
• Secondary goal: Harden and protect the tissue from trauma of • Stabilize structures to maintain the proper
further handling relationship of cells and their stroma
• Involves fixing or preserving fresh tissue for examination (collagen / reticulin / elastin / amorphous
• Good quality of fixed tissue specimen = good quality of section on substances)
slide o Prevent distortion of cellular
• Cells must be viewed under the microscope as if they are still in their elements which may affect a
original living state. pathologist’s interpretation
• Specimen exposed to air for a long time = dry out resulting to • Enhance staining
distortion of its morphological appearance • Hardening of tissues for proper grossing
• Tissue in hypotonic solution = cell will swell and cutting thin sections for processing
• Tissue in hypertonic solution = cell will shrink
o Autolysis is more commonly known as self- How Fixatives Work
digestion, refers to the destruction of a cell • Additive fixation –
through the action of its own enzymes. the chemical
o Denaturation is the alteration of a protein constituent of the
shape through some form of external stress (for fixative is taken
example, by applying heat, acid or alkali), in and becomes
such a way that it will no longer be able to part of the tissue
carry out its cellular function. by forming cross –
o Degeneration is deterioration in the medical links or molecular
sense. complexes and
o Decomposition is the natural process of dead giving stability to
animal or plant tissue being rotted or broken the protein.
down. • Non – additive –
o Putrefaction process references the breaking fixing agent
down of a body of a human or animal post (predominantly
mortem (meaning after death).
organic
o Distortion is the alteration of the original shape
compounds) is not incorporated into the tissue but alters the
(or other characteristic) of something.
tissue composition and stabilizes the tissue by removing bound
water attached to H – bonds of certain groups within the
protein molecule.

Factors affecting fixation

Practical Considerations in Fixation
Hydrogen Ion Concentration Temperature Duration and Penetration of Fixation
• pH between 6 and 8 • Increased fixation temperature • 1mm / hour – formalin diffusion
• Outside this range, changes may (60oC) accelerates the fixation of • Primary fixation in NBF: 2 -6 hours
occur that are detrimental to tissues but likewise increases the • Most formalin can be washed out
ultrastructural preservation of the rate of autolysis. (rapid fixation of after the fixation for 24 hours.
tissue. urgent spx) • Some tissues take longer
• Increased fixation temperature depending on their structure.
(100oC) for tissues with • Can be cut down by using HAMV
tuberculosis
Thickness of section • Traditionally carried out at RT. Osmolality
• Small tissues: 2 mm2 for EM and 2 • Tissue processors: 40oC • Refers to the number of
cm2 for light microscopy • For electron microscope: 0o – molecules of a particular
• Thin: no more than 0.4 cm2 for light 4oC substance in solution.
microscopy o For light microscopy initial • Isotonic/hypotonic – cause cell
• Large solid tissue should be opened fixation is usually carried swelling and poor fixation
or sliced thinly. out at room temperature • Hypertonic – contain more
• Brain – suspended whole in 10% NBF and this may be followed dissolved substances (cells to
for 2 – 3 weeks. by further fixation at shrink)
• Fixation of 4 – 6 hours = size is 2 cm2, temperatures up to 45°C • Hypotonic – contain less
not more than 4 mm thick. during tissue processing. dissolved substances (cells swell)
o The thickness of any Concentration • 400 – 450 Osm = best results using
specimen or tissue slice • Formaldehyde – 10% slightly hypertonic solutions
should not exceed 4 mm if • Glutaraldehyde – 3%; 0.25% (ideal
optimal fixation is to be for immuno – electron
achieved. microscopy)
Volume
• Maximum effectiveness of fixation is 20 times the tissue volume.
• The ideal ratio of fixative volume to specimen volume is 15 – 20 is to 1.
• Current recommendations allow a ratio of 10 : 1 (Lo, et.al.)
• Position or shape desired for sectioning should be maintained before fixation
• Refrigeration is used to slow down decomposition if tissue needs to be photographed and cannot be fixed
immediately.
• Cells from different parts of the body die at different time intervals.
• So far, no single fixative has yet been known to possess all the qualities of an ideal solution. Many fixatives and each
has its own advantages and disadvantages.
 Types of fixative according to Action and Composition
According to Composition
Simple Fixative Compound Fixatives
• Aldehydes • Those that are made up of two or more fixatives which
• Metallic Fixatives have been added together to obtain the optimal
combined effect of their individual actions upon the
cells and tissue constituent
According to Action
Microanatomical Fixatives Cytological Fixatives Histochemical Fixatives
• permit the microscopic study of - preserve specific parts and • Preserve the chemical
tissue structures without altering the particular microscopic elements constituents of cells and tissues
structural pattern and normal of the cell itself. • F, A, A, N
intercellular relationship of the • Nuclear Fixatives - preserve the
tissues in question. nuclear structures in particular.
• FSA, NBF, HS, FSU, ZS, ZF, B, B Contain glacial acetic acid for its
affinity to chromatin. pH of 4.6 or
less.
• B , C, F, N, H
• Cytoplasmic Fixatives – preserve
cytoplasmic structures in
particular. Never contain glacial
acetic acid. pH of more than 4.6
• F, H, FPC, R, O

Fixatives for nucleic acids Lipid fixation Carbohydrate Fixation

• Mercuric chloride - can react with • Lipids are largely removed during • Alcoholic fixatives: generally
viruses and cause the loss of their preparation of tissues. (cryostat or recommended for glycogen
infective power frozen sections should be used) fixation
• Native DNA and RNA do not react • Mercuric chloride & potassium • Alcoholic formaldehyde is better
with formalin at RT. • 45C for RNA dichromate – effective for fixative in human skin compared
• 65C for DNA preservation of lipids in cryostat with NBF
• Ethanol, methanol, Carnoy’s sections • Rossman’s fluid or cold abs. ROH:
solution – used for nucleic acids • Baker’s formol – calcium: used to most useful esp. tissues from
• Ethanol – for PCR preserve phospholipids patients with glycogen storage
• Imidazole osmium tetroxide: Post disease; better retention of
– fixation for improved glycogen if section is coated with
Protein Fixation
ultrastructural demonstration of celloidin
• Neutral Buffered Formol Saline or
lipids
formaldehyde vapor: most • Digitonin: can fix cholesterol for
commonly used fixatives for amino
ultrastructural demonstration
acid histochemistry

Fixatives for Electron Cytochemistry

• Karnovsky’s paraformaldehyde – glutaraldehyde solution
• Acrolein mixed with formaldehyde or glutaraldehyde

Aldehydes
Formaldehyde (formalin)
• Formaldehyde – colorless gas produced by the oxidation of methyl alcohol and soluble in water to the extent of 37 –
40% weight in volume.
• Formaldehyde is commonly used as 4% solution, giving 10% formalin for tissue fixation.
• Diluted at 1:10 or 1:20 to make a 10% or 5% solution.
• 10% formalin is usually buffered to a pH of 7
• If unbuffered:
o Reduces quality of routine cytology staining
o Oxidizes to formic acid with blood forming brown or black crystalline precipitate
• Prolonged storage at low temperature may produce white paraformaldehyde deposits (turbidity)
Figure 2: A formalin-fixed paraffin Figure 3: An electron micrograph Figure 4: The Schmidt-Lanterman
section of kidney showing the typical showing a Schmidt-Lanterman incisure in a myelinated nerve fibre
deposition of acid formaldehyde incisure in a myelinated nerve fibre. shown at a higher magnification than
hematin (formalin pigment) This specimen was fixed in buffered in Figure 2. The multiple layers of
associated with red blood cells. The glutaraldehyde, washed in buffer phospholipid membrane forming the
pigment is brown to black in color then given secondary fixation in myelin sheath are well-preserved by
and is birefringent under polarized buffered osmium tetroxide, a the glutaraldehyde/osmium tetroxide
light. In this case the specimen standard procedure when preparing fixation.
remained in fixative for an extended specimens for transmission electron
period before processing. microscopy. The multiple layers of
phospholipid membrane forming the
myelin sheath are well-preserved by
this procedure.

Secondary Fixation
• The process of placing an already fixed tissue in a second fixative to improve demonstration of a specific
substance, as a mordant for special stains, ensure complete hardening and preservation of tissues
• Usually done on 10% NBF or 10% formol saline as primary fixative

Post – Chromatization
• A form of secondary fixation whereby a primarily fixed tissue is placed in aqueous solution of 2.5 – 3% potassium
dichromate for 24 hours to act as mordant for better staining effects and cytologic preservation of tissues

Washing Out
• The process of removing excess fixative from the tissue after fixation to improve staining and remove artifacts from
the tissues

Fixation is retarded by:

o Size and thickness of tissue specimen
o Presence of mucus
o Presence of fats
o Presence of blood
o Cold temperature

Fixation is enhanced by:

o Size and thickness of tissues
o Smaller ones require less fixatives and shorter fixation time
o Agitation
 LESSON 4-6: Decalcification Dehydration Clearing
DECALCIFICATION - A special step but not part of the whole tissue processing
Figure 10: A section from a Figure 3: A transverse section from a Figure 4: A decalcified section of
granuloma specimen in which long bone optimally decalcified cancellous bone (pink) and hyaline
unanticipated calcium deposits using formic acid (H&E). Numerous cartilage (blue) from the epiphysis of a
were encountered (blue). Sections of osteons with peripheral cement lines long bone (H&E). The delicate trabeculae
better quality could have been are shown. Well-stained osteocyte of the bone are well preserved as is the
prepared if surface decalcification nuclei are present indicating that fine structure of the bone marrow and
had been applied to this block. the decalcification endpoint was associated adipocytes. The
not exceeded. basophilic/acidophilic balance of the
stain is well maintained indicating that the
formic acid decalcification was optimal.

Decalcification Calcium may be removed by any one of

• Specimens such as BONES, TEETH, OTHER CALCIFIED TISSUES the following agents:
(tuberculous lungs) which contain some amount of calcium that is • Acids
apt to interfere with the accurate evaluation and examination of • Chelating agents
histological sections. • Ion exchange resin
• The purpose of bone decalcification is to make the bone flexible • Electrical ionization
and easy for pathological investigation. (electrophoresis)
• Necessary to obtain soft sections of the bone using the microtome.
• Bones, calcified tissues – cut into
• A procedure whereby calcium or lime salts are removed from small pieces with a fine fret – saw;
tissues following fixation trimmed with a hand razor to
• Should be done after fixation and before impregnation permit complete penetration of
• Inadequate process may result in poor cutting of hard tissues and the decalcifying solution with
damage to the knife edge during sectioning minimal distortion
• Small foci of microcalcification –
can be sectioned without
noticeable damage to the knife
or disruption of surrounding tissues

• After trimming, the tissue surface may reveal small foci of calcification
causing resistance and „grating‟ sensation when sectioned/cut in the
microtome.
• Remove block – place face down on a pad of cotton or gauze
saturated with 10% HCl for 1 hour – reset to microtome – cut. (SURFACE
DECALCIFICATION)
o Grating - scraping, scratching, grinding, rasping, jarring

WHAT IS THE QUALITY OF A GOOD DECALCIFYING AGENT?

• Capable of removing calcium salts and/or lime salts from tissues
COMPLETELY and with no destruction of cells and its staining

DECALCIFYING AGENTS
Acid Decalcifying Agents Examples:
✓ Most widely used • Nitric acid • Sulfurous acid
✓ Stable • Hydrochloric acid • Chromic acid
✓ Easily available • Formic acid • Citric acid
✓ Relatively inexpensive • Trichloroacetic acid
Nitric Acid
• Most common and fastest decalcifying agent
• 5-10% may be used as a simple aqueous solution
• Rapid; minimal distortion
• Recommended for routine process
Disadvantage:
• Inhibits nuclear stains and destroys tissue in concentrated solutions
• Combine nitric acid with formaldehyde or alcohol to prevent tissue damage
A. AQUEOUS NITRIC ACID SOLUTION 10% Maceration
• Recommended for urgent biopsy, for needle and small • In histology, the softening of a tissue by soaking,
biopsy specimens especially in acids, until the connective tissue fibers
• Can be used for large or heavily mineralized cortical are dissolved so that the tissue components can be
bone specimen teased apart.
• Rapid in action
• Minimum distortion of tissue ENDPOINT TEST
• Produces good nuclear staining • Do not use chemical test
• Can be easily removed by 70% alcohol • Dissolve precipitates by adding glacial acetic acid
Disadvantages: drop by drop
• Imparts yellow color with nitrous acid • Then add 0.5 mL of saturated aqueous ammonium
oxalate to the solution
B. FORMOL-NITRIC ACID • Reappearance of white precipitates within 30 minutes
• For urgent biopsies = presence calcium (incomplete decalcification)
• Nuclear staining is relatively good
• Produces less tissue destruction than 10% aqueous nitric D. PHLOROGLUCIN-NITRIC ACID
acid • Most rapid, recommended for urgent works
Disadvantages: Disadvantages:
• Impair staining reaction (yellow color – nitrous acid) • Nuclear staining is poor
• Prevent: neutralize tissue with 5% sodium sulfate – wash • Yellow discoloration
in running tap water for at least 12hrs o Neutralize with 5% sodium sulfate – wash in
o Addition of 0.1% urea to pure concentrated running tap water (24h)
nitric acid
COMPLETE DECALCIFICATION IS DONE
C. PERENYI’S FLUID • Three changes of 70% to 90% ethanol (washing out)
• Decalcifies and softens tissues (tissue softener) at the • NO TO WATER because watery solutions will lead to
same time excessive swelling and deteriorating of tissue
• Maceration is avoided due to chromic acid and • When sections are cut, slides are brought to water –
alcohol placed in 1% aqueous lithium carbonate (1h) –
• For routine processes washed in later (15m) - STAINED
Disadvantages:
• Slow decalcifying agent

2. HYDROCHLORIC ACID A. Von Ebner’s Fluid

• Inferior compared to nitric acid • Permits good cytological staining
• Slower action and imparts greater distortion of the tissue • Does not require washing out before dehydration
• Produce good nuclear staining if used in 1% solution • Recommended for teeth and other small pieces of
with 70% alcohol bone
• Recommended for surface decalcificatio Disadvantage
• Endpoint test cannot be measured by chemical test

3. FORMIC ACID Disadvantages: A. FORMIC ACID – SODIUM

• Most commonly used • The only weak acid used as a CITRATE SOLUTION
• Moderate-acting decalcifying agent primary decalcifying agent • Permits better nuclear
• Safer to handle • Addition of citrate accelerates staining than nitric acid
• Produce better nuclear staining and less decalcification • Recommended for autopsy
tissue distortion • May be hastened by increasing materials, bone marrow,
• Recommended for routine the proportion to 25ml but yields cartilage and tissues for
decalcification of postmortem research solution opaque thereby interferes research studies
tissues staining result Disadvantages:
• Not suitable for urgent examinations • Requires neutralization with 5% • Slow
• Can be both fixative and decalcifying sodium sulfate and washing out to • Requires neutralization with
agent remove the acid from the tissue 5% sodium sulfate
• Requires neutralization and washing out
 4. TRICHLOROACETIC ACID 6. CHROMIC ACID (FLEMMING’S FLUID) 7. CITRIC ACID-CITRATE BUFFER
• Permits good nuclear staining • Both fixative and decalcifying SOL’N
• Does not require washing out (excess agent • Permits excellent nuclear
acid may be removed by several • Used to decalcify minute bone and cytoplasmic staining
changes of 90% alcohol) spicules • Does not produce cell or
Disadvantages: Disadvantages: tissue distortion
• Weak; not for dense tissues and suitable • Nuclear staining with hematoxylin Disadvantage:
for small spicule of bone is inhibited • Too slow for routine
• Very slow acting and not • Forms precipitates purposes
recommended for urgent examinations • Forms insoluble pigments if
undergo dehydration with alcohol
5. SULFUROUS ACID • Environmental toxin
• Very weak decalcifying agent suitable
only for minute pieces of bones

Chelating agents
• combine with calcium ions
and other salts to form
weakly dissociated
complexes and facilitate
removal of calcium salt.

Ethylenediaminetetraacetic Acid
• Versene (commercial name)
• For detailed microscopic studies
• Binds metallic ions (calcium and magnesium)
• Tissue is placed in EDTA from 1-3 weeks – small spx
• 6-8 weeks or longer – dense cortical bone
• Solution should be changed every 3 days
• Below ph 3 – not bind to calcium
• Ph 7-7.4 - faster
• Ph 8 and above – optimal binding but may damage alkaline – sensitive protein linkages

ION EXCHANGE RESINS ELECTROPHORESIS: ELECTRICAL

• Ammonium form of polystyrene resin IONIZATION
• Hastens decalcification by removing calcium ions from formic acid- • Process whereby positively charged
containing decalcifying solutions calcium ions are attracted to a
• Not recommended for fluids containing mineral acids negative electrode and
• 1⁄2 inch thick of ion exchange resin is spread over the bottom of the subsequently removed from the
container to be used decalcifying solution
• Specimen is placed on top • Time required thereby shortened
• Decalcifying agent is then added, 20-30 times the volume of the tissue due to heat and electrolytic
• Let it stay in the solution for 1-14 days reaction
Use resin for later decalcification • Same with chelating agent the only
It can be reactivated by immersing it in 0.1 N HCL twice – wash it with distilled difference is the use of electricity
water thrice • Satisfactory for small bone
fragments
 FACTORS INFLUENCING RATE OF DECALCIFICATION
CONCENTRATION RATE OF DECALCIFICATION VOLUME RATIO
More concentrated solutions decalcify This will depend upon the Recommended ratio of fluid to tissue
bone more rapidly, but are more harmful structure, temperature and volume for decalcification is 20:1
to the tissue volume of the solution to be used
TEMPERATURE AGITATION TIME
Heat hastens decalcification, also Mechanical agitation and moving • Ideal time required for decalcifying
increases the damaging effects of acids of the tissue in the solution tissue is 24-48 hours
on tissue. accelerates the rate of diffusion • Dense bone tissues require up to 14
• At37C, there will be impaired and speeds up the days or longer in order to complete
nuclear staining of Van Gieson’s stain decalcification process the process
for collagen fibers Gentle fluid agitation is achieved
• At55C,thetissuewillundergocomplete by low-speed rotation, rocking,
digestion within 24-48 hours stirring or bubbling air into the
• Lowertemperaturedecreasesreaction solution
rates
• Optimum temperature for
decalcification- Room temperature
(18-30 C)

MEASURING EXTENT OF DECALCIFICATION

PHYSICAL OR MECHANICAL TEST X-RAY OR RADIOLOGICAL TEST CHEMICAL METHOD
• Touching or bending the tissue • Very expensive although most • Simple, reliable and convenient
with the fingers to determine the ideal method recommended for routine
consistency of tissues/ pricking the • Most sensitive and most reliable purposes
tissue with a fine needle or a method due to its ability to detect • Involves detection of calcium in
probe even the smallest focus of calcium acid solutions by precipitation of
• Decalcified tissues are softer to which appears opaque in an X- insoluble calcium hydroxide or
touch Ray plate calcium oxalate
• Inaccurate

Step Action
1 Insert a pipette into the decalcifying solution containing the specimen
2 Withdraw approximately 5mL of the HCL / formic acid decalcification solution from under the specimen
and place it in a test tube.
3 Add approximately 10 mL of the ammonium hydroxide / ammonium oxalate working solution
4 Mix well
5 Let it sit overnight
Result: Decalcification is complete when NO precipitate or turbidity is observed.
X-ray or Radiological Method

Figure 8: An X-ray series following the process of decalcification of a femoral head with formic acid/citrate decalcifier. The
radiographs were produced using a Hewlett-Packard Faxitron® and allow the process to be accurately followed and the
endpoint to be properly identified.
 POST-DECALCIFICATION TISSUE SOFTENERS
• Saturated lithium carbonate solution or 5-10% aqueous sodium bicarbonate • PERENYI‟S FLUID – decalcifying
solution for several hours agent and tissue softener
• Rinse in tap water for 30 minutes (small samples) and 1-4 hours (larger • Washing out and immersion in
specimens) 4% aqueous phenol solution
• ASAP spxs can be blotted, quickly rinsed with water to remove acid from the • Molliflex
surface • 2% hydrochloric acid
• Spx for frozen sections must be thoroughly washed in water or stored in formol – • 1% hydrochloric acid in 70%
saline (15% sucrose or PBS) at 4C before freezing alcohol
• Tissues decalcified in EDTA solutions should not be placed directly into 70%
alcohol
DEHYDRATION
• The process of removing Characteristics of an Ideal
intercellular and extracellular Dehydrating Solution:
water from the tissue as well • Should dehydrate rapidly
as the fixative. without causing considerable
• Remove fixative and water shrinkage.
and replace it with • Should not evaporate fast.
dehydrating fluid in • Should be able to dehydrate
preparation for impregnation even fatty tissues.
• “Dehydrating agents” • Should not harden tissues
• Must be used in increasing excessively.
strengths to avoid diffusion • Should not remove stains.
currents • Should not be toxic to the
• 1:10–Tissue to fluid ratio body.
• Should not be a fire hazard.

Dehydration
• Under no circumstances should a formalin-fixed tissue be transferred directly to higher grades of alcohol.
o cause distortion, shrinkage and hardening of the tissue.
• A temperature of 370C will hasten dehydration time.

• A layer of anhydrous copper sulfate- 1⁄4 inch deep, placed in the bottom of the container and covered with filter
paper to insure complete dehydration
• Blue discoloration of copper sulfate crystal- full saturation of dehydrating fluids with water
1. Alcohol
• Ethyl alcohol – recommended for • Methyl Alcohol – dehydrating • Butyl Alcohol – utilized in plant
routine dehydration of tissues; BEST agent for blood and tissue films and animal micro techniques
DEHYDRATING AGENT and for smear preparation. o slow dehydrating agent
o Most commonly used o toxic o produces less shrinkage
dehydrant in histology and hardening than ethyl
o Fast acting alcohol
o Miscible with water and o recommended for tissues
many organic solvents – which do not require
Not poisonous rapid processing
o Not very expensive
II. Acetone III. Dioxane (diethylene IV. Cellosolve ( ethylene V. Tetrahydrofuran
• Cheap and rapid dioxide) glycol monoethyl ether) • Both dehydrates and
acting dehydrating • Excellent clearing and • Dehydrates rapidly. clears tissues at the
agent utilized for most dehydrating agent. • Combustible at 110 to same time because it is
urgent biopsies which it • Should not be used 120of miscible with both
dehydrates in 1⁄2 to routinely because of its • Toxic by inhalation, skin water and melted
2hours. toxic effects. contact and ingestion. paraffin.
• Causes brittleness • Miscible with water, • Also miscible with
• Flammable melted paraffin, alcohol alcohols, ether,
• Its use has been limited and xylol. chloroform, acetone,
only to small pieces of • Extremely dangerous benzene and xylene.
tissues due to its (main disadvantage) • It can dissolve many
extreme volatility and substances including
inflammability fats
• Not recommended for
routine dehydration
purposes
ADDITIVES TO DEHYDRATING AGENTS
• When phenol (4%) is added to each of the 95% ethanol baths as part of dehydration process, it acts as a softener for
hard tissues such as tendon, nail or dense fibrous tissue.
• In addition, hard tissues can be immersed in a glycerol/ alcohol mixture or in “Molliflex”.

CLEARING
• Also called as de-alcoholization. Characteristics of a good clearing agent
• Removal of the dehydrating agent and replacing • Miscible and can remove the dehydrating fluid
it with a substance that will dissolve the wax with • Miscible, easily removed by paraffin wax and or mounting
which the tissue is to be impregnated or the medium
medium on which the tissue is to be mounted. • NO to distortion, tissue hardening and damages
• Increases the refractive index of the tissue making • Not dissolve out aniline dyes
it translucent, thus the term clearing agent. • Not evaporate quickly
• Make tissues transparent

XYLENE (Xylol) Other clearing agents

- MOST COMMONLY USED CLEARING AGENT! • Toluene - substitute for xylene and benzene.
ADVANTAGES DISADVANTAGES Benzene(carcinogenic) – rapid acting clearing agent, thus
o Rapid o Highly flammable utilized for urgent biopsies.
o Makes tissues o Makes tissue • Chloroform (hepatotoxic) – slow in action than xylene but does
transparent excessively hard not cause shrinkage
o Miscible with and brittle (use o does not make the tissue transparent
absolute alcohol more than • Cedarwood oil – recommended for central nervous system tissues
and paraffin 3hours) and cytological studies particularly of smooth muscle and skin.
o Does not extract o Causes • Aniline oil – utilized for clearing embryos, insects and very delicate
out aniline dyes considerable specimens.
o Does not dissolve hardening and • Methyl benzoate and methyl salicylate – slow-acting clearing
celloidin shrinkage of agents used when ,double embedding techniques are required.
o Evaporates quickly tissues.
in paraffin oven o Becomes milky in • Clove oil • Terpenes
o It is cheap the presence of • Carbon tetrachloride • Limonene
water. • Esters • Terpinol
• N- butyl acetate

Clearing
• Clearing agents (low boiling point) – more readily
replaced by melted paraffin
• Viscosity affects the speed of penetration of the
clearing agent.
• Prolonged exposure to clearing agents – brittle tissues
and difficult to cut
• Benzene and xylene are easily removed from the
tissues while chloroform and cedarwood oil are more
difficult to remove.
• Glycerin and gum syrup are used when the tissue is to
be cleared directly from water, as in a frozen section.

RECAP

Tissue processing was done manually in the 18th and 19th century. A cruel long
process that took days and sleepless nights to achieve this feat. This discomfort
forced scientists into looking for a better and a more efficient way to process
tissues.

The first automatic tissue processors were introduced during the first half of the
20th century. In the USA, they were produced under the name of Auto-
Technicon and in the UK under the name of Histokine, and later by other
companies.
These devices have slowly evolved to be safer to use, handle larger specimen
numbers, process more quickly and to produce better quality outcomes.