Histopathology Brief Tutorial

in the Preparation of Exit Exam

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Presentation Outline
1. Introduction
2. Histopathology specimen collection and handling.
3. Fixation and fixatives
4. Tissue processing
5. Tissue Sectioning
6. Staining
7. Special staining methods

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 1. Introduction
Definition of Terminologies:

• Histology: The Study Of Tissues

• Pathology: is the study of disease

• Histopathology:

- The scientific study of diseased tissue

- Microscopic study of architectural alteration of tissue

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 Causes of Disease
• Environmental factors
➢Physical factors
➢Chemicals
➢Infections and infestations
➢Immunologic factors
➢Nutritional deficiencies and excesses
• Genetic factors
➢Hereditary factors inherited from parents.
• Multifactorial - both are implicated

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Clinical significance of histopathology
• To diagnose cancer (Benign or Malignant)
• To differentiate different types of tumor cells (Tumor Markers)
• To identify the degree of malignancy (tumor grade)
• To confirm the complete removal of the cancerous cells after
surgical removal

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 2. Histopathology specimen collection and handling.
• Biopsy – Specimen from patients taken by clinician – surgeons or
gynecologist
➢Excisional biopsy
➢Incisional biopsy
➢Endoscopic biopsy - inaccessible
• Necropsy –lesion representation from postmortem examination
• Scrapes sampling easily from approachable body surface by spatula uterine
cervix
• Aspirations (FNAC)- sampling from solid organ using needle.(BM)
• Body fluids- body space eg. Pleural, CSF, Peritoneal fluids

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 3. Fixation and fixatives
• Fixatives are used to preserve cells and tissue constituents in as
close a life-like state as possible
• Allow tissue to undergo further preparative procedures without
change.
• Arrests autolysis and bacterial decomposition
• Produce coagulation of tissue proteins and constituents
• Prevent their loss or diffusion during tissue processing

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Functions of Fixation:
• Kill tissue
• Inhibition of autolysis and putrefaction
• Maintain relationship of cellular elements
• Enhance staining
• Make cellular elements insoluble

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 Fixatives
• An agent employed in the preparation of histologic or pathologic specimens
for the purpose of maintaining the existing form and structure of all their
constituent elements
• Fixatives have the property of forming cross-links b/n proteins there by
forming a gel
➢Ideally keeping them in their in vivo relation to each other
• Soluble proteins are fixed to structural proteins and made insoluble
➢The tissue gets mechanical strengths that permits subsequent preparative procedures
to take place

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 Types of chemical fixatives
• Formaldehyde - containing
• Mercury - or zinc-containing
• Alcoholic Based Fixation
• Combinations

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 Formaldehyde - Containing Fixatives

Formaldehyde • Disadvantages of Formalin

➢Irritant vapor
• Reacts with several parts of proteins ➢Allergic reaction
• It is used as 10 % solution ➢Inferiors' alcohol in fixing
• Don’t over hardened tissue Fe and pigments
➢Formation of formalin
• Cheap pigments
• Permits use of variety of stains
• Preserve colour of specimen
• Useful for frozen section and lipid
demonstration
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 Mercury Fixatives
Zenker and Helly fluid B-5 fixative
Stock formulation (both use the same) • Stock formulation
• Mercuric Chloride ➢Mercuric chloride
• Potassium dichromate ➢Sodium acetate
• Sodium sulfate ➢Distilled water
• Distilled water • Working solution: add
Working formulations: formaldehyde just before use
• Zenker's: add acetic acid before using
• Helly's: add formaldehyde before using

• Disadvantages: It is Toxic
• Substitutes for mercury by Zinc
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 Alcoholic Fixatives
• Alcoholic fixatives are good in fixing Fe and pigments
• Examples: Ethanol, methanol
Compound fixatives
➢Mixture of fixatives are developed to minimize deleterious
effect of single fixative
• Formol saline (10% formalin in normal saline)
• Alcoholic formaldehyde
• Zenker’s fluid
• Carry’s fixatives
• Bouin’s fluid 13
 Factors affecting fixation
• PH & Buffers
• Size of specimens & Penetration of fixative
• Volume
• Temperature
• Concentration of the fixative
• duration of fixation &
• Osmolality (refers to the number of particles in solution.
• If the tissue osmolality varies greatly from the solution that it is immersed
in, either shrinkage or swelling of the tissue cells will occur.)

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 Fixation of specific substances

• Even though we usually use the same fixative on most tissues that come to
the histology lab, some fixatives are preferred to be used over others for
specific tissue.

Choice of Fixative:
• Immunofluorescence
• Immunohistochemistry
• Enzymes

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 Choice of Fixative…
Immunofluorescence:
• Immunofluorescence (IF) staining is generally done on unfixed (fresh) frozen
sections
Enzymes:
• Enzymes are generally not preserved well when tissue is fixed, so most stains are
done on fresh frozen tissue
• However, some enzyme procedures such as the Leder stain can be done on FFPE
tissue
Immunohistochemistry:
• Identifies tissue constituents by detecting Ag using Ab( Ag-Ab) rxn
• Immunohistochemistry (IHC) is generally done on formalin-fixed paraffin
embedded (FFPE) tissue sections.
• Some IHC procedures require fresh frozen tissue, but those are rare
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 Other Choices of Fixative
• Chromaffin cells: are best preserved with a chromate fixative such as Orth.
• Uric acid crystals: in gout are best preserved in a non-aqueous fixative such
as absolute alcohol.
• Bone marrow and blood cells are best preserved using a fixative that does
not lyse the erythrocytes. E.g. B-5 fixative
• Fat: is adequately fixed in formalin-based fixatives, but it is dissolved in the
processing alcohols and xylenes.

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 4. Tissue processing

➢Tissue processing refers to any treatment of tissue necessary to

impregnate them with a solid medium to facilitate the production of
sections for microscopy.

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 Purpose of tissue processing:
To move tissue from its natural aqueous state to a solid supportive
medium(paraffin wax)
✓Give it sufficient rigidity to enable thin sections to be cut.
✓Easy to store tissue specimen for a later day
• Paraffin wax is not water miscible but most fixatives are water
based
• Tissue should be treated to allow paraffin wax embedding
• paraffin processing, involves steps of dehydration, clearing , and
infiltration with wax

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• There are two types of tissue processing methods:
➢ Conventional and
➢ Frozen.
• Conventional method of tissue processing is preferred than frozen method
of producing tissue sections because of the following advantages.
✓ Helps to section large number of tissues routinely;
✓ Suitable for impregnating tissues with a solid medium easy to store;
✓ Further section can be readily produced at a later date;
✓ Requires paraffin wax, which is the most suitable for routine microtomy.

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Three Steps of Tissue Processing
• Dehydration
• Clearing
• Infiltration (impregnation)

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 4.1 Dehydration
• Ultimate purpose of tissue processing to embed the tissue with an impregnating
media
• since water and paraffin wax are not miscible, the sample must be dehydrated after
the fixation step
• Remove aqueous fixatives from the tissue
• Mechanisms of action:
➢Hydrophilic attracts water from tissue
➢Repeated dilution of aqueous tissue fluids (repeatedly diluting the waters found in the tissue
with alcohols until alcohol is the only fluid in the tissues.)

• Dehydration is the first step in tissue processing and is done by alcohol of various
types to remove fixative and water from tissue and replace them with dehydrating
fluids.
• Dehydration is achieved by immersing tissues in increasing strength of alcohol
(usually starting with 70% alcohol).
• Generally, dehydration is done sequentially; first in 70% alcohol for three changes
then 95% alcohol for three changes and then absolute alcohol for three changes.
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 Dehydrating fluids
• Water is present in tissues in free and bound (molecular) forms.
• Tissues are processed to the embedding medium by removing some or all of
the free water from the tissues
• The commonly used dehydrating fluids include
➢Alcohols
➢Acetone
➢Universal Solvents

Alcohols
• Carried out by increasing strength 50%, 70%, 95%, 100%
• For delicate/ embryonic tissue start with 30% ethanol
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 Remove the water & replace with wax-solvent
Imbed the oriented specimen in molten wax

50 %
ethanol
70 %
ethanol
95 %
ethanol label
100 %
Fresh tissue ethanol
Benzene
/Xylene
10% Formalin Paraffin wax
fixative Miscible with ethanol;
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dissolves wax
Ethanol
• Ethanol is a clear, colorless, flammable liquid. It is probably the most commonly
used dehydrant in histology.
• It is supplied as 99.85% ethanol and as special Methylated Spirits (99.85%
ethanol denatured with 2% methanol).
Methanol
• Methanol is a clear, colorless, flammable fluid, miscible with water, ethanol and
most organic solvents.
- but poisonous, so rarely used.
• It is a poor lipid solvent and will not dissolve nitrocellulose unless mixed with
acetone.
Isopropyl alcohol
• Isopropyl alcohol is miscible with water, ethanol and most organic solvent.
• Isopropanol shrinks and hardens tissues less than ethanol and is used to dehydrate
hard, dense tissues, which can remain in the solvent for extended periods without
harm.
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Acetone
• Acetone is a colorless flammable liquid with sharp characteristic kenotic odor,
low toxicity and is freely miscible with water and organic solvents.

• It is a fast, effective Dehydrant though it may cause tissue shrinkage; it may

also act as a coagulant secondary fixative.

• Acetone is the best Dehydrant for processing fatty specimens.

• makes tissue brittle and is flammable so that not widely used

• Tissues are dehydrated through four changes of acetone, the last of which
should always be fresh.
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 Universal Solvents
• Can be used in place of dehydrating and clearing step
(consolidates the two steps)

• Miscible with water and paraffin

• Not suitable for delicate (embryonic) tissues

• Common universal solvents are tetrahydrofuran (THF), dioxane

(diethylene dioxide), and butanol (most are highly odorous)

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 4.2 Clearing
• After dehydration the tissue sample may still not be capable of accepting
paraffin wax

• A chemical solution, selected to be miscible with both alcohol and paraffin,

may be used to clear the alcohol from the sample

• Substitution of alcohols with a wax miscible solvent

• Clearing agents are miscible with both dehydrating agent and the embedding
media

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Selection of the clearing agent must be based on

• Speedy removal of dehydrating agent

• Ease of removal by molten paraffin wax
• Minimal tissue damage
• Flammability Clearing agents suitable for routine use
• Toxicity • Xylene
• Cost • Benzene
• Toluene
• chloroform
• carbon tetrachloride

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• Xylene • Toluene
➢Similar property with xylene
➢Best routinely used
➢Less damaging on prolonged immersion
➢Reasonably Cost effective ➢Better in preserving tissue structure
➢Must not prolonged ➢More expensive than xylene and more toxic
➢It is flammable • Chloroform
• Benzene ➢Slower in action than xylene/toluene
➢ similar with xylene ➢Cause less brittleness
➢Carcinogenic ➢Not flammable but release toxic gas with heat
➢High rate of evaporation ➢More tolerant toward tissue.

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 4.3 Impregnation (infiltration)
• Is the uniform penetration of tissue with a suitable embedding medium to
support cellular components adequaetly during microtomy
• Replacing of the clearing agents with the embedding medium
• Must have complete removal of clearing agents
• Impregnation with paraffin wax was done in an oven heated at 58⁰c
• The volume of paraffin wax should be 25-50x the bulk of tissue
• Time consumption depends on:
➢Size of tissue
➢Type of tissue
➢The clearing agent used
➢Use of vaccum embedding method(Speeds up wax impregnation and removes any
residual air bubbles)
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 Paraffin wax
• Popular medium for a number of reasons:
✓ Cheap
✓ Easily handled
✓ Has wide range of melting point (55 - 60 °c)
• Lower melting point wax (soft paraffin 38° to 40° C) is softer, so use with soft
tissues
• Higher melting point wax (hard paraffin 50°c to 58°C) is harder, so use with denser
tissues
• Heating paraffins 5-10 °c above its melting point causes crystallization of paraffin
and difficulty in cutting

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 Alternate Processing Procedures
• Plastics
➢ Epoxy resins
➢ Glycol methacrylate
➢ Methyl methacrylate
➢ Used for very thin sectioning
• Nitrocellulose (celloidin)
➢ Uses no heat in processing
➢ LVN (low viscosity celloidin)

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Ideally, an impregnating and embedding medium should be:
✓Soluble in processing fluids;
✓ Suitable for sectioning and ribboning;
✓ Molten between 30°C and 60°C;
✓ Translucent or transparent and colorless;
✓ Stable;
✓Homogeneous;
✓ Capable of flattening after ribboning;
✓ Non-toxic;
✓ Odorless;
✓ Easy to handle;
✓ Inexpensive
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Processing methods and routine schedules
Large /fatty tissues:
• penetrated more slowly and requires Longer processing times,
possibly 1 hr in each solution.
‘Routine’ tissues:
• Specimens taken by surgical procedures such as gallbladders,
appendices, uterus.
• Penetrated more rapidly and requires 30-45 minutes per processing
fluid.
Biopsies or small tissues:
• require less time for penetration, displacement and replacement of
processing fluids.
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• Tissues are most conveniently processed through dehydration,
clearing and impregnation stages automatically by machine.

There are two types of automatic tissue processors;

A. Tissue-transfer processors
• Characterized by transfer of tissue contained with a basket, through a
series of stationary reagents
• Allow maximum flexibility in the choice of reagents
B. Fluid-transfer processors.
• Fluids are pumped to and from a retort in which the tissue remain
stationary
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 Embedding
• Is surrounding of tissue by a medium such as Agar, Wax or Gelatin.
• Provide sufficient external support during sectioning.
• The tissue are embedded by placing them in a mold filled with Paraffin wax
• The [Link] used for embedding should be the same with the impregnation
type.

Precaution while embedding in wax

• The wax should be clear of clearing agent
• No dust particles must be present
• Immediately after tissue embedding, the wax must be rapidly cooled.

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 Decalcification
• Removal of inorganic calcium from collagen matrix, calcified cartilage and
bone tissue
Reason for decalcification:
• Calcium deposits cause torn and ragged sections
• Calcified tissue damage microtome knifes/ blades
• To make bone soften
Decalcifiers
• Good decalcifying agent removes all Ca without adverse effect on the cell
Two types of decalcifying agents
• Acid decalcifiers
• Chelating agents

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 Acid decalcifiers
• Calcium salts dissolves and then ionize when exposed to acids
• Two types of acid decalcifiers
➢ Strong/ inorganic acid
➢ Weak/ organic acid

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Strong inorganic acids
• HNO3 and HCl
• Decalcify rapidly
• Swelling and can seriously damage tissue stain ability
• For rapid diagnosis
• Larger or heavily mineralized cortical bone specimen
Weak organic acids
• Formic, acetic & picric acids
• Formic acid used as 10 decalcifiers
• Acetic acid & picric acid – cause tissue swelling
➢ Are not used alone as a decalcifier - fixatives
• Decalcification completed within 1-10 days
• Used both as fixative and decalcifying agent.

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 Chelating agents
• EDTA- binds Ca & Mg
• PH dependent
• Excellent bone decalcifier for enzyme staining
• Require long time for complete decalcification

• The choice of decalcification is influenced by three independent factors

➢Urgency of the case
➢The degree of mineralization
➢The scope of investigation and subsequent staining technique required
Factors influencing the rate of decalcification
➢Concentration of decalcifying agents
➢Temperature
➢Agitation 41
Factors influencing tissue processing

• Size of tissue
• Penetrability of tissue
• Viscosity of reagents
• Temperature
• Agitation
• Vacuum

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• Tissue should be in small enough pieces that the processing fluids can easily
penetrate the tissue in the time allotted for that step of the procedure.
• Some tissues are more dense (like a uterus) and
• Some are less dense (like a lung), and that will impact the speed at which
processing fluids penetrate the tissue.
• Reagents used in tissue processing must have low viscosity, which means that
it will penetrate more rapidly.
• Examples of high viscosity fluids are honey or motor oil, and some low
viscosity fluids are water and alcohol.

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• Increased temperature speeds up processing steps, however, high
temperatures can be deleterious to the tissue.
• Heat in the alcohol and xylene steps will make tissues more brittle and
dry.
• Heat is applied only to the paraffin wax step to alleviate the brittleness
problem.
• Vacuum and pressure are available on most modern tissue processors, to
improve the removal of air from the tissue spaces and to ensure the quick
penetration of solutions into the tissue.

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 5. Tissue Sectioning
➢ Is cutting transparent thin slices from embedded tissue specimen
• Generally, 5-15 mm sections
• Thickness depends upon tissue, subsequent procedures
➢Equipment required for tissue sectioning – Microtome
• Mechanical instrument used to cut biological specimens into very thin segments
• Microtomy is the means by which tissue can be sectioned and attached to a surface
➢Main parts of a microtome
• base
• knife holder
• materials/tissue holder

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 Types of microtome
I. Cambridge rocking microtome:
• it is cheap machine operated by a simple lever action
• Used in paraffin wax-embedded tissue and cryostat
II. Rotary microtome
• Also called Minot microtome
• most popular for routine paraffin wax embedded tissues
• It has the ability to cope with very hard tissues and cuts good accurate sections
III. Base sledge microtome
• for cutting very hard tissue or large blocks
• neuropathology and ophthalmic pathology
IV. Sliding microtome
• it is used for Cutting celloidin embedded tissue
V. Ultra microtome
• Ultra microtome it is used exclusively for electron microscopy
VI. Cryostat
• is a microtome mounted within a freezer box
• A piece of tissue can be quickly frozen to about -15 to -20⁰c. 46
 Why sectioning?
1. To see internal structure of tissue
2. Allows stains or specific markers to more easily infiltrate tissue
3. Allows light to pass through tissue making structure visible
NB. in some cases tissues are stained and examined without
sectioning

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 Frozen sections
• Produce sections with out the use of dehydrating solutions, clearing agents
and embedding media.
• The applications of frozen sections are for
➢Rapid production of sections for urgent diagnosis
➢histological demonstration of fats, enzymes, and neurological structure when they
are labile
➢Immunocytochemistry method
➢Immunoflouresence method
The principle of cutting frozen section
➢When tissue is frozen -<the water - turns to ice
➢ The ice acting as the embedding medium
➢ Varying the temperature of the tissue can alters the consistency of the frozen block.

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 6. Staining
• The purpose of staining is to differentiate the tissue elements.
• The differential staining is due to the fact that certain parts of the tissue take
up more stain than others.
Factors contributing to dye-tissue affinity
• different tissues and cells display varying affinities for most dyes and stains
used during the process
➢Solvent-solvent interaction
➢Reagent-Reagent interaction
➢Reagent-tissue interaction
Factors controlling selectivity of stain፡
➢Number and affinity of binding sites
➢Rate of reagent uptake
➢Rate of reaction
➢Rate of reagent loss 51
 General method of staining
A. Based on use of mordant
• Direct staining;
➢ Sections are stained with simple aqueous or alcoholic solution of the dye
➢Methylene blue and eosin
• Indirect staining:
➢process which needs mordant
➢It combines with a dye to form a colored “lake”,
➢Tissue- mordant -dye complex

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 General method of staining …
B. Based on endpoint attainment
Progressive staining:
• the tissue is left in the stain just long enough to reach the proper endpoint
Regressive staining;
• the tissue is deliberately over stained and then destained (differentiated) until the
proper endpoint is reached
C. Based on the component stained
• Vital staining; selective staining of living cells
• Negative staining
• staining of a contrasting color used to color the components

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 Classifications of common stains
• Acid dyes:
➢anionic(negatively charged)
➢has affinity for cytoplasm which has +ve charge
➢Stains acidophilic structures
➢e.g Eosin
• Basic dyes:
➢cationic (positively charged)
➢has affinity for nuclei & ribosome which have –ve charge
➢Stains basophilic structures
➢Hematoxylin
• Neutral dye:
➢combining aqueous solutions of acid and basic dyes
➢capable of staining cytoplasm and nucleus simultaneously and differentially
➢E.g. giemsa’s stain and leishman’s stain.
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Basic reaction of stains = attraction of opposites
a) Tissues that stain with a basic stain = Basophilic (stain acid component - ie.
Nuclei or
b) Tissues that stain with an acidic stain = Acidophilic (stain basic component
- i.e. cytoplasm)
• Hematoxylin & Eosin (H & E) - most common stain - good for general
structure
a) Nuclei = blue (basophilic) → Hematoxylin
b) Cytoplasm = pink (acidophilic) → Eosin

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Common stains in histopathology
Hematoxylin and Eosin
• The most commonly used histological stain
• Routinely performed on all specimens and is the core stain for all
microscopic diagnoses.
• The pathologist uses the H & E
➢ Diagnose disease
➢ Identify cancer
➢ Confirm a metabolic disorder or identify tissue type.

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 Hematoxylin stain

• Natural dye
• coloring agent is hematein- oxidation of hematoxylin
-< ripening
• Ripened hematoxylin has low affinity for the tissue itself
• Require combination with mordant(form links b/n the
hematine stain and the tissue)

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 Hematoxylin stain…
➢ Common mordants are salts of alum, iron, tungsten,
molybdenum and lead:-
• Alum hematoxyline
• Iron hematoxylin
• Tungsten hematoxylin
• Molybdenum hematoxylin
• Lead hematoxylin

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 Eosin
• The counterpart to hematoxylin in the H & E stain
• Eosin Y is the most widely used
• The complementary pairing of H&E demonstrates tissue structure
and cellular architecture
• Stains both cytoplasm and connective tissue fibers differentially

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 Staining procedure for H & E stains
• Dewax sections; hydrate through graded alcohols to water.
• Remove fixation pigments if necessary.
• Stain in an alum hematoxylin of choice for a sutialble time.
• Wash well in running tap water until sections ‘blue’ for 5 minutes or less.
• Differentiate in 1 percent acid alcohol for 5-10 seconds.
• Wash well in tap water until sections are again ‘blue’ (10-15 minutes), or
• Stain in 1 percent eosin Y for 10minutes.
• Wash in running tap water for 1-5 minutes.
• Dehydrate through alcohols, clear and mount.

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Results
• Nuclei ---------------------blue/black
• Cytoplasm ---------------varying shades of pink
• Muscle fibers -----------deep pink/red
• Red blood cells ---------orange/red
• Fibrin ---------------------deep pink

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 Epoxy resin section (1 μm)
Paraffin section (8-10 μm)
Toluidine blue
Hematoxylin & Eosin

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 7. Special staining methods
Staining Nucleic Acids (DNA and RNA)
1. Feulgen stain:
• Used for staining DNA
• Results * DNA - red to purple
* Background - Light green
2. Methyl Green- Pyronin Y Stain:
• Methyl green preferentially binds to DNA and pyronin Y to RNA
Results
• RNA - Red
• DNA - Green or blue-green
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 7. Special staining methods …
• Reticular fibers - silver solutions
• staining of Lipids - Sudan black B or oil red O dyes
• Staining of proteins (Amyloid) - Congo red
• Glycogen - Periodic Acid-Schiff (PAS)
• Hemosiderin and iron - Perl’s Prusian blue

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Staining a frozen section

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Coverslipping

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 Mounting of sections
• After staining the section is mounted under a cover slip using a syrup fluid,
• Why mounting?
➢to protect the specimen from physical injury
➢to protect the section from deterioration
➢to facilitate easy handling.
➢preserving the slides for permanent storage

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 Mounting medium
• a syrup fluid applied between the section and the cover slip
Characteristic of a good mounting medium
– It should not dry quickly
– It should not dissolve out or fade tissue sections
– It should not cause shrinkage and distortion of tissues
– It should set hard thereby producing permanent mounting of sections

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Thank You!
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