HISTOPATHOLOGY

ANGELIKA B. RAMOS, RMT

 Histology
- Is the study of tissues, their functions and their arrangement to
constitute an organ.
What is a tissue?

- Group of cells with interrelated functions

- Example: Nervous Tissue
- In the nervous tissue, these cells are categorized into neurons
and neuroglia.
- Neurons = cell that you can find in the center, which creates
nerve impulse.
- Microglia = is a type of neuroglia that is a phagocytic cell. It eats
up bacteria that tries to kill the neurons.
- Function: to support and keep the neurons.
- Astrocyte = Star shape.
- Function: Protect the neurons from the invading microorganism.
 Why study cells in a subject
that is supposed to study
Tissues?

You can never understand the characteristic of a tissue

without understanding the characteristics of its individual
components, which are the cells.
ORGANIZATION IN
THE HUMAN BODY

Cell – the smallest/basic

structural functional unit of the
human body.
Tissue – Group of cells with
interrelated functions
Organs – Group of tissues with
interrelated functions
System – An organ will always
belong to a certain human body
system.
 URINARY BLADDER (Urinary System)
- First X = is positioned in one of the
tissues that you can find in the
urinary bladder. It is composed of
cells because tissues are actually
group of cells.
- Second X = Appears on another
are where you can find another
tissue and that area is also made
up of group of cells.

Therefore, this proves that the urinary

bladder since its is an organ, it is made
up of different types of tissues.
- This proves that tissues are made
up of group of cells with
interrelated functions.
 HISTOLOGY
- Branch of anatomy = also known as Microscopic anatomy
- Is the study of tissues, their functions and their
arrangement to constitute an organ.

4 Basic types of Tissue

1. Epithelial tissue
2. Connective tissue
3. Muscle tissue
4. Nervous tissue
 All of these types of issue
have group of cells
If these tissues are
combined; you can form
an organ.

- The 4 tissues are grouped together to

form an organ.
Why is the lining of epithelium of the
stomach is situated nearest to its lumen?
- Stomach functions to produce
hydrochloric acid and when you say acid
it has something to do with being
destructive.
LINING
EPITHELIUM OF
THE STOMACH

- Cells are tightly packed

together
- Avascular = doesn’t have
its own blood supply
- Since they are not
getting oxygen, then
most likely these cells
would die.
How do the organs in the body
maintain the cell of the lining
epithelium?

- Below the epithelium is the connective

tissue, because it is highly vascularized.
Meaning the connective tissue has its own
blood supply. Thus, the epithelium is above
the connective tissue to protect the
connective tissue from the acid.
- And what will keep the cells of the epithelium
alive is because they will be getting blood
supply from the connective tissue.
- Epithelium is avascular
(no blood supply)
- Black arrow = lining of
the epithelium (simple
columnar epithelium)
- Yellow star = connective
tissue
- There is a presence of a
blood vessel above the
yellow star.
Connective Tissue
- Provides blood supply to the
epithelium and links to other
tissue
- Links/connects the
epithelium tissue to the
muscle tissue
Muscle Tissue
- Provide movement of tissue
Nervous Tissue
- Provide sensation, control
and information
processing
 - Not all spaces in the
tissue are occupied by
cells
o Fibers = hair-like structure
o Ground substance =
empty spaces
composed of chemicals
- Tissue is a group of cells
with interrelated functions
+ fibers and ground
substance
Group of cells specialized to
carry on an interrelated
function and their associated
extracellular matrix

Blue arrow = Cell

Yellow arrow = Fiber
Red arrow = empty space
Since the fiber and ground
substance are outside the cell, they
are referred are extracellular matrix
Tissue = Cells + extracellular matrix
HISTOLOGY
LABORATORY
- We prepare the organ
and we make a thin cut
section of the organ so
that the light coming from
the light source of the
microscope can actually
traverse through the slide
and the specimen so that
the light can reach the
eye of the observer.
Cross section of the appendix
PREPARATION OF TISSUES FOR STUDY

To study tissues, one must prepare thin

and translucent histological sections or
tissue slices that can be studied with the
aid of a microscope
POST MORTEM CHANGES
Autolysis The destruction of the tissues (breaking down of the protein of
the cell) by enzymes which are produced by the tissues and
eventually liquefy it. It is the first to occur among all post-mortem
changes
Putrefaction or The decomposition of organic matter under the influence
Decomposition of microorganisms accompanied by the development of
disagreeable odors.
Degeneration A retrogressive pathologic process in cells in which the
cytoplasm undergoes deterioration while the nucleus is
preserved.
How long
would it take
for the tissue
to be ready?
At least 48-72 hours tissue processing time
- Patient: Colon cancer (transverse
colon)
- Surgical plan: remove that portion of
the transverse colon with the cancer
and anastomose (connect/ link the
remaining colon)
- Before you anastomose the remaining
portion of the transverse colon you
have to be sure that the remaining
portions should no longer contain
cancer cells or else you should expect
the cancer to re- occur.

- So, before the surgeon will

anastomose the remaining portions,
the surgeon will have to send the
cancer tissue to the laboratory.
- Resected colon mass will be sent to
the laboratory for the pathologist to do
a biopsy of the edges of the resected
colon to look for the presence of
cancer cells.
- If both edges of the colon do not
contain cancer cells, therefore the
surgeon can procced to the
anastomosin.
 Histopathologic
Techniques
- Deals with the preparation for microscopic examination
- It is accomplished by submitting the total or a selected part
of the tissue presented for examination to a series of
processes:
- Examination may be done on:
1. Fresh Tissues
o usually examined when there is an immediate need

for evaluation
2. Preserved Tissues
1. Routinely done in the histopathology section
 Advantage: Examined in the living state, thereby
allowing protoplasmic activities such as:

• Motion (Cellular Movement)

FRESH TISSUE • Mitosis (Cellular Reproduction)
EXAMINATION •
•
Phagocytosis (Cell-eating activity)
Pinocytosis (Cell-drinking activity)

Disadvantage: Its use has been limited, however,

because of the fact that tissues examined in the
fresh state are not permanent and therefore, are
liable to develop the changes that have usually
been observed after death.
METHODS OF FRESH TISSUE
EXAMINATION
TEASING OR - Process wherein selected tissue specimen is
DISSOCIATION immersed in a watch glass containing isotonic
salt solution (NSS or ringer’s lactate),
carefully dissected or separated and
examined under the microscope, either
unstained by phase contrast microscope or
bright-field microscope, or stained with
differential dyes.
 - Process where small pieces of tissue not more
than 1mm in diameter are placed in a
SQUASH PREPARATION/ microscopic slide and forcibly compressed with
CRUSHING another slide or with coverglass.
- Vital dyes are placed at the slide and coverslip
junction and absorbed through capillary action.
- Useful in cytological examinations, particularly for
Smearing
cancer diagnosis.
Smearing Technique Material Process/Important Notes
a. Streaking Applicator stick or - Rapid and gentle direct or zigzag application to
platinum loop. obtain uniform distribution.
- Too thick or too thin smears are unsuitable for
examination.
b. Spreading technique Applicator stick to tease - Little more tedious than streaking, but has advantage
the mucous strands to in maintaining the intercellular relationship.
make a moderately - Especially recommended for fresh sputum, bronchial
thick film. aspirates and thick mucoid secretions.
c. Pull-apart technique Slides facing each other - The material disperses evenly over the surface of 2
as a drop of slides.
secretion is sandwiched - A single uninterrupted motion of pulling apart is
in- between. applied.
- It is useful for serous fluids, concentrated sputum,
enzymatic GIT lavage and blood smears
d. Touch preparation or One slide. - Special method where slide surface is in contact and
Impression smear pressed on the site.
- Cells may be examined without destroying their actual
intercellular relationship and without separating them
from their normal surroundings.
FROZEN SECTION

Fixation is done rapid freezing

Compressed carbon dioxide

Sectioning is done thru cryostat, a refrigerated compartment containing microtome

Method is rapid

Routinely done in hospital to study specimens during surgery

Lipids and enzymes are best preserved in this method

CRYOSTAT
Compressed carbon dioxide
needs to be maintained at a
cold temperature, the entire
procedure should be done
within a cryostat.

Comes with a built-in

microtome within the cryostat
so that after freezing the
tissue u can immediately
proceed to the sectioning
- Before we can prepare a thin histological section
of the organ, the organ has to undergo different
tissue processing steps.
- Always start with fixation
1.
FIXATION

- 1st step
- To preserve the
organ and prevent
changes in the
organ
- Tissues must be
fixed to prevent
alterations in their
structure through
decomposition.
FIXATION

1. Avoid tissue destruction

by digestive enzymes
(autolysis) or through
bacterial degradation.
2. Terminate cell metabolism
a. Glycolysis without

oxygen = lactic acid

LEFT: Inflamed appendix
with fixation
RIGHT: No fixation
so the organ
undergone
decomposition/decaying
1. Hardens the tissue by cross-linking or denaturing
proteins (So it would be easy to do the thin
sectioning)
2. Kill pathogenic microorganisms such as bacteria,
fungi and viruses
3. Commonly used fixative is formalin.
STEPS IN PROCESSING PRESERVED TISSUES

1. Fixation
• AKA: Preservation
• The first and most critical (important) step in histotechnology
• Why is it the most critical? Because it is the first procedure and is able to affect the
subsequent procedures
• Primary aim of fixation
• Preserve the morphologic and chemical integrity of the cell in as life- like as possible
• Secondary goal of fixation
• To harden and protect the tissue from trauma of further handling
• For easy cutting during gross examination
- The most important reaction in fixation: Stabilization of proteins
▪ This can be achieved by: Forming cross-links between proteins
 Two Basic Mechanisms in Fixation

Principle/ Notable Characteristics Example

1. Additive - The chemical constituent of the fixative is Formaldehyde

taken in and becomes part of the tissue Osmium tetroxide/Osmic acid
through cross-link formation or molecular fixative
complexes. Mercuric chloride

2. Non- - The fixing agent is not incorporated into the Alcoholic

additive tissue but alters the tissue composition and
stabilizes it through water removal.
- New cross-links are formed preventing
autolysis and bacterial decomposition.
 Main Factors Involved in Fixation
1. Hydrogen Ion - pH: 6.0-8.0
Concentration - Average: 7.0 (neutral pH)
-
Traditional/usual: Room temperature (18-30oC)
2. Temperature Tissue processors: Autotechnicon (40-42 oC)
Electron Microscopy and Histochemistry: 0-4 oC
Small
Electron Microscopy: 1-2 mm2
3. Thickness of Light Microscopy: 2 cm2
sections Thin
Light Microscopy: ≤0.4 cm or as prescribed by tissue
processor manufacturer
Best results are obtained using slightly hypertonic
solutions (400-450 mOsm)
4. Osmolality Hypertonic solutions = Shrinkage
Isotonic (340 mOsm)/Hypotonic solutions = swelling and
poor fixation
Formaldehyde: 10%
5. Concentration Glutaraldehyde: 3%
Glutaraldehyde for Immunoelectron microscopy: 0.25%
Most formalin fixatives: 24 hours (washed out)
Buffered formalin: 2-6 hours up to 1 week
6. Duration
EM: 3 hours (New books: 0-4 hours; Average: 2 hours)
of fixation
Prolonged fixation may cause shrinkage and hardening
of tissue
 1) Speed
- The specimen should be placed in
fixative as soon as it is removed from
the body.
- This is done to prevent autolysis and
Practical putrefaction/decomposition.
Considerations - If bacteriologic and toxicologic studies
of Fixation should be encouraged, therefore,
fixation is not required.
o Fixation can kill microorganisms and
prevent growth in culture
o Fixatives can neutralize drugs and toxins
 Formalin diffuses into
the tissue at the rate of
approximately 1mm/hr.
2. Penetration
Time of fixation varies
with different types of
tissue
 3. Volume/Amount of
Fixative
- Traditionally, the amount of fixative
used has been 10-25x the volume of
the tissue to be fixed.
- Recently, 20x is known as the maximum
effective concentration for fixation
- Except when osmium tetroxide (5-10x)
is used.
4. Duration
Some tissues take longer to fix than others,
depending on their structures.

Fibrous organs such as uterus and intestinal tract

take longer.

Fixation can be cut down by using

Heat, Vaccum, Agitation or Microwave.
IDEAL SIZE OF
TISSUE TO BE
FIXED:
Not more than 2 cm2 in
diameter
Not more than 4 mm
thick.
Ideal number of hours for
fixation: 4-6 hours (new
books: 6-18 hours)
TYPES OF FIXATIVES
According to Composition
• SIMPLE FIXATIVES (made up of only 1 component substance)
• Aldehydes (Formaldehyde, Glutaraldehyde)
• Metallic Fixatives (Mercuric chloride, Chromic acid, Lead)
• Picric acid
• Acetic acid
• Acetone
• Alcohol
• Osmium tetroxide
• Heat
• COMPOUND FIXATIVES (Made of up of 2 or more fixatives to obtain optimal results)
TYPES OF
FIXATIVES
According to Action
• Microanatomical Fixatives: Permit the general microscopy
study of tissue structures

1.10% Formol saline

2.10% NBF
3.Heidenhain’s susa
4.Formol sublimate (corrosive)
5.Zenker’s solution
6.Zenker-formol (Helly’s)
7.Bouin’s solution
8.Brasil’s solution
TYPES OF
FIXATIVES
According to Action
• Cytological Fixatives: Preserve specific parts and particular microscopic
elements of the cell itself

1.Nuclear Fixatives
- Preserve nuclear structures (ex. chromosomes).

- They usually contain Glacial acetic acid as their primary component due to
its affinity for nuclear chromatin.
- The pH is <4.6.

a. Bouin’s fluid
b. Flemming’s fluid
c. Newcomer’s fluid
d. Carnoy’s fluid

e. Heidenhain’s Susa
TYPES OF
FIXATIVES
According to Action
• Cytological Fixatives: Preserve specific parts and particular microscopic elements
of the cell itself

1.Cytoplasmic Fixatives
- Preserve cytoplasmic structures.
- They must never contain glacial acetic acid because it
• destroys the mitochondria and golgi bodies of the cytoplasm.
- The pH >4.6

a. Helly’s
b. Orth’s
c. Regaud’s
d. Flemming’s fluid w/o HAc
e. Formalin w/ post-chroming
TYPES OF
FIXATIVES
According to Action
• Cytological Fixatives: Preserve specific parts and particular
microscopic elements of the cell itself

1.Histochemical Fixatives
- Preserve the chemical constituents of cells and tissues

a. 10% Formol saline

b. Absolute Ethyl alcohol
c. Newcomer’s fluid (Both Nuclear and Histochemical fixative)
d. Acetone
Routine Fixatives
1. Aldehyde Fixatives
A. Formaldehyde:Formalin
- Commercial formaldehyde/formalin
- A saturated solution of formaldehyde gas in water, approximately 35-40%
gas by weight (Gregorios: 37-40% weight in volume).
- A mixture of 10 mL formalin with 90 mL of
water/saline is known as 10% formalin (most
widely used fixative of all)
- It is usually buffered to pH 7 with a phosphate buffer.
- Formalin is the best fixative for the Nervous tissue
Disadvantages:

In tissues containing much blood (spleen), unbuffered formalin

Prolonged fixation may cause bleaching, fat dispersal and dissolution or loss of
leads to the formation of dark brown artifact pigment granules glycogen, biurates of sodium crystal and uric acid.
→ these granules consist of acid formaldehyde hematin and
are doubly refractile.

Reagent-grade formaldehyde contains 10% methanol as a preservative to retard decomposition to formic acid. Prevents the
formation of Paraformaldehyde.

Formalin pigments – are also formed due to overfixation.

Fixation Time: 24 hours

 ROUTINE FORMALIN FIXATIVES
1. 10% Formol-Saline Traditionally, it is the most commonly used fixative in pathology
Fixation time:
Best fixative for central nervous tissues and general post-mortem tissues
24 hours at 35ºC
for histochemical examination.
48 hours at 20-25ºC
2. Calcium formalin Fixative) acetate (Lillies It is used to preserve phospholipids

3. 10% Neutral Buffered Formalin/Phosphate buffered Best general tissue fixative

Formalin (pH 7) Best Fixative for frozen sections
Recommended for surgical, post mortem and research specimens.
Fixation time:
Prevents precipitation of acid formalin pigments.
4-24 hours
Best fixative for iron-containing pigments and elastic fibers.
4. Formol corrosive/ Formol-sublimate/ Formol- Recommended for routine post-mortem tissues.
Mercuric Chloride It is excellent for silver reticulum methods.
It fixes lipids, especially neutral fats and phospholipids.
Fixation time:
3-24 hours
Alcoholic Formalin/ It fixes and dehydrates at the same time and fixes sputum since it coagulates
Gendre’s fixative mucus

Cajol’s formol ammonium bromide - good fixative for nervous tissue

SPECIAL FORMALIN FIXATIVES: (astrocytes).

Fixatives for acid mucopolysaccharides.

Baker’s formol calcium - used for the preservation of lipids since most
formalin fixatives are inert to lipids.
 B. Glutaraldehyde
- It is made up of two fomaldehyde residues, linked by three carbon chains.
- It is utilized for Light microscopy.
- Buffered Formaldehyde with secondary fixation in osmium tetroxide is
satisfactory for Electron Microscopy.
o 2.5% - for small tissue fragments and needle biopsies for 2-4 hours at
room temperature.
o 4% - large tissue fragments less than 4 mm thick for 6-8 hours up to 24
hours
o Fixation time: ½-2 hours
- Recommended for histochemistry and electron microscopy.
- The most effective aldehyde for protein cross-linking
 A. Mercuric Chloride
- The most common metallic fixative

- The routine fixative of choice for preservation of

cell detail in tissue photography
II. Metallic
Fixatives 1.
Disadvantages:
It causes marked shrinkage of cells.
o Metal = Mercury
2. Has black granular deposits and is extremely
corrosive to metals.
 Mercuric Chloride Fixatives
1. Zenker’s fluid
Fixation time: 12-24 hours
2. Zenker- formol/ Helly’s
Solution
Fixation time: 12-24 hours
“Bloody Helly”
3. Heidenhain’s
Susa solution
Fixation time: 3-12 hours
4. Schaudinn’s Solution/
Sublimated alcohol
5. B-5 Fixative
Fixation time:
1 ½ to 2 hours (Rapid fixation)
6. Carnoy- Lebrun
7. Ohlmacher
Mercuric chloride + glacial acetic acid just before its use.
Good general fixative for all kinds of tissue
Recommended for fixing small pieces of liver, spleen,
connective tissue fibers and nuclei
Mercuric chloride + 40% formaldehyde just before its
use.
It is an excellent microanatomic fixative for pituitary gland,
bone marrow and blood- containing organs such as
the liver and spleen.
Recommended for tumor biopsies especially of the skin
It is an excellent Cytologic fixative
Counterbalance effect
o Su (sublimat) = metal (mercury) → cell
shrinkage
o Sa (saure) = acid (trichloroacetic acid) →
cell swelling
Used on wet smears for cytologic examinations.
Composed of mercuric chloride and sodium acetate.
Commonly used for bone marrow biopsies.
Just prior to use, add 1 mL of 40% formaldehyde
to 10 mL of B5
 A class of fixatives which are strong
oxidizing agents used for
precipitating proteins and preserving
carbohydrates.

B. CHROMATE Recommended for

Chromaffin tissues,
Adrenal medulla,
Mitochondria
 CROP
CHROMATE FIXATIVES
1. Chromic acid- Used in 1-2%, used as a constituent of a compound fixative.
- It precipitates all proteins and adequately preserves carbohdyrates.
- Formaldehyde must be added to chrome
2. Regaud’s Fluid/ Moeller’s fluid - Recommended for demonstration of Chromatin, Mitochondria, Mitotic figures,
Golgi bodies, RBC’s and colloid-containing tissues
3. Orth’s Fluid- Recommended for study of early degenerative processes and tissue necrosis
- Demonstrates Rickettsia and other bacteria
Fixation time: 36-72 hours
4. Potassium dichromate - Preserves mitochondria (pH 4.5-5.2)
- Fixes lipids
- Used in 3% aqueous solution
- It fixes but does not precipitate cytoplasmic structures.
 Lillie’s alcoholic lead nitrate formalin

Lead subacetate Advantages:

C. LEAD It is recommended for acid

FIXATIVES mucopolysaccharides

It fixes connective tissue mucin

lower the pH and dissolve the residue.

 1. Excellent for glycogen demonstration
2. It dyes the tissues yellow.

III. PICRIC 3. The chemical name for the general picric

acid fixatives: 2,4, 6 - trinitrophenol
ACID - Picric acid is highly explosive when dry,

FIXATIVES and therefore must be kept moist with

distilled water or saturated alcohol at 0.5 to
1% concentration during storage.
 PBB
- Fixation of embryos and pituitary biopsies
- It is an excellent fixative for preserving soft and
Bouin’s solution delicate structures (endometrial curettings)
- Yellow stain is useful in fragmentary biopsies
Fixation time: - Preferred fixative for Masson’s trichrome
6-24 hours staining for collagen, elastic or connective tissue
Brasil’s Alcoholic - Best routine fixative for glycogen
Picroformol Fixative - It is better and less messy than Bouin’s solution
 It solidifies at 17ºC (New book: 16oC)

Fixes and precipitates nucleoproteins

IV. GLACIAL It precipitates chromosomes and chromatin

materials.
ACETIC ACID
Causes tissues to swell specially those containing
collagen.

Disadvantage: It is contraindicated for cytoplasmic

fixation because it destroys mitochondria and golgi
elements.
 Denatures and precipitates proteins

70-100% concentrations are used; Less

concentrations results to red blood
• cell lysis/hemolysis due to hypotonicity

V. ALCOHOL Advantages:

FIXATIVES • May be used both as a fixative and dehydrating agent

• Excellent for glycogen preservation

Disadvantages:

• Causes RBC hemolysis and dissolves fats and lipids

• Tissue shrinks on long usage
• Polarization (major disadvantage); glycogenic granules
moves to towards the ends or poles of cells
1. Methyl Alcohol
100% (CH3OH/
Methanol)
2. Isopropyl Alcohol
95% (Isopropanol)
3. Ethyl Alcohol
(C2H5OH/ Ethanol)
Fixation time: 18-24 hours
4. Carnoy’s Fluid
Fixation time: 1-3 hrs.
5. Alcoholic Formalin/ Gendre’s
6. Newcomer’s Fluid
Fixation time:
12-18 hours @ 3ºC
- Used in Wright’s stain as a diluent
- Excellent for fixing wet and dry smears, blood smears and bone marrow tissues
- It is used for fixing touch preparations (impression smears), although some are air
dried and not fixed, for certain procedures such as Wright-Giemsa staining.
- Is used in 70-100% concentrations.
- Lower concentrations (70-80%) will cause RBC lysis and inadequate WBC
preservation
- It fixes blood, tissue films and smears
- Used for histochemistry especially for enzyme studies.
- The most rapid tissue fixative
- Recommended for fixing chromosome, lymph glands and urgent biopsies
- It is used to fix brain tissue for rabies diagnosis.
- It preserves Nissl granules and very suitable for Curettings (small tissue
fragments).
- Better preserves glycogen
- Capable of coagulating mucus and is used as a fixative for sputum cytology
- For fixing mucopolysaccharides and nuclear proteins
- It is both a nuclear and histochemical fixative.
 VI. OSMIUM
TETROXI/OSMIC ACID
- It is a pale yellow powder which
dissolves in water (up to 6% at
20°C) to form strong oxidizing
solution.

- Adequately fixes materials for

ultrathin sectioning in electron
microscopy, since it rapidly fixes
small pieces of tissues and aids in
their staining.
1. Flemming’s Solution The most commonly used Chrome-
Osmium acetic acid, recommended
Fixation time: for nuclear preparation of sections
24-48 hours such as chromosomes

Permanently fixes fats/lipids

2. Flemming’s Solution -
Recommended for cytoplasmic OFF
without Acetic Acid structures such as mitochondria
(The removal of acetic acid from the
Fixation time: formula serves to improve the
24-48 hours cytoplasmic detail of the cell).
 It is used at ice cold Recommended for the study Used in fixing brain tissues for
temperature from -50C to of water diffusible enzymes diagnosis of rabies (Negri
40C (New book: 0-4oC) especially lipase and bodies).
phosphatases

VIII.ACETONE

Used in freeze substitution Evaporates rapidly

techniques as a solvent for
certain metallic salts
 SECONDARY
FIXATION
- The process of placing an already fixed
tissue in a second fixative in order to:
a. To facilitate and improve the demonstration
of particular substances
b. To make special staining techniques
possible (secondary fixative acting as
mordant)
c. To ensure further and complete hardening
and preservation of tissues
POST-
CHROMATIZATION
Post-chroming or Post-mordanting
- A secondary fixation whereby a primary
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 to aid in cytologic preservation of
tissues.
WASHING-OUT
The process of removing excess fixative from the tissue after fixation in order to improve
staining and remove artifacts from the tissue.

Solutions that may be used:

• Tap water – used to remove:
• Excess Chromates from tissues fixed (Helly’s, Zenker’s and Flemming’s solutions)
• Excess Formalin
• 10% formalin is extracted more rapidly in 70% alcohol than in water
• Excess Osmic acid/Osmium tetroxide
• 50-70 % alcohol – used to wash out excess amount of picric acid

3. Alcoholic Iodine – used to remove excessive mercury

 Important Notes on Improper Fixation
1. Failure to arrest early cellular Due to the failure to fix immediately by which one first
autolysis allowed the tissue to dry
before fixing or insufficient fixative
2. Too brittle and too hard blocks Due to prolonged fixation
3. Soft and feather-like tissues Due to incomplete fixation
4. Removal of fixative soluble Wrong choice of fixative
substances
5. Presence of artefact pigments on Incomplete washing of fixative
sections
6. Shrinkage and swelling of cells in Due to overfixation
tissue blocks
7. Enzyme inactivation and loss Wrong choice of fixative
Incomplete fixation → Improper and incomplete clearing and impregnation →
incorrect sectioning and staining of pathologic slides
 2. DECALCIFICATION

- 2nd step
- Only done in specimens such as
bones, teeth and calcified tissues
- To soften and easily cut them
- Reagent: Nitric acid
- Not only limited to bones and teeth but
can also be done to arteries especially
arteries with atherosclerotic plaque
formation = caused by the deposition
of the cholesterol on the walls/ lumen
of the artery.
 - Done by successively bathing the specimen in mixture of ethanol and
water from 70% to 100%. (increasing concentration of alcohol)
- Alcohol removes water out of tissue.

3. DEHYDRATION -
How do we do dehydration?
By successively dipping/bathing the tissue in a mixture of alcohol from
a lower concentration to a higher concentration until pure/100%
alcohol
- Commonly used: Ethanol
Why do we have to start with lower
concentration to a higher concentration?

RIGHT = Immediately immersed to

100% alcohol
LEFT = Immersed to a lower
concentration and then to a higher
concentration
 Remove the alcohol and replace it with the
clearing agent

Removal of the dehydrating agent by

immersing the specimen in the solvent that the
4. CLEARING alcohol and embedding medium is miscible

Clearing agent are highly volatile (becomes

vapor once exposed to heat)

Reagents: Xylene and Toluene

4. Impregnation/Infiltration
- Remove the clearing agent and replace it with paraffin
- After the clearing procedure, the tissue is placed in a melted paraffin in an
oven set at 52-60 degree Celsius.
- The heat causes the clearing agent to evaporate so that the tissue will be
filled up with the paraffin.
- After dipping, the melted paraffin will cool down forming a solid plaque
containing the tissue in the center.
- Using melted paraffin, medtechs are prone to having burned injuries.
That’s why in some lab, paraffin is replaced with plastic resins
- The tissue and paraffin will harden after removal from oven
- Plastic resins – makes use of plastic solution which hardens tissue by
cross-linking polymers
- Eliminates the need to use oven and paraffin; little tissue distortion
- Plastic resins are fluid at room temperature but once you try to place
something on it, it will solidify immediately.

- PLASTIC RESINS ARE TOO EXPENSIVE

Plastic resin
Melted
paraffin
The paraffin oven is set at
a.58-60C c. 45-50C
b.65-70C d. 50C
58-60 C
6. Trimming
A trimmed tissue block is similar in
appearance to
a.Truncated pyramid c. Either
b.Four sided prism d. Neither
c. Either
 7. SECTIONING
- Uses knife to remove excess
paraffin
- The block is then mounted in
microtome and cut with a steel knife.
o Sectioning is done with the
aid of microtome
- Instrument: Microtome
- Steel knife
- Drive wheel
o Production of the thin
sections
- Allow the thin section to
float on the water and try to
scoop them out using the
slide
- Glass slide used should be
covered with adhesives so
that once we scoop them
out, the tissue will be
adhere to the slide
8.
STAINING
Since paraffin is colorless,
staining is a must.

Application of color to the

tissue to highlight structures

Most commonly used stain:

• Hematoxylin and Eosin
CLASSIFICATIONS OF
STAINING
CLASSIFICATIONS
OF STAINING
CLASSIFICATIONS
OF STAINING
Common Staining Solutions Used
 HEMATOXYLIN EOSIN

- Imparts blue or purple color - Imparts red or pink color

- Basic dye thus, it will stain - Acidic dye, thus it will stain the
acidic portions of the cell basic portion of the cell
- Blue arrow = Nucleus
- Nucleus is in blue, thus it imparts
hematoxylin
- Since the nucleus contains our
DNA, our nucleus is acidic
therefore it will be attracted to an
alkaline/basic dye that’s why it
retained the hematoxylin
- Cytoplasm is red in color, meaning
the cytoplasm retained the eosin
dye
Counterstains
Cytoplasmic Counterstains
RED Yellow Green

Eosin Y Picric acid Light green SF

Eosin B Orange G Lissamine green

Phloxine B

Rose Bengal
Counterstains
Nuclear Counterstains
Red Blue

Neutral red Methylene blue

Safranin Toluidine Blue

Carmine Celestine Blue Hematoxylin

Red Blue
 LYSOCHROMES (Oil Soluble Dyes)
-
1. Sudan Black B Greatest affinity for phospholipids or neutral fats (triglycerides)
(SBB) A more sensitive coloring agent than other lysochromes
Should be discarded if the brownish black color appeared.
Demonstrates lipids that are resistant to paraffin embedding
-
2. Sudan IV or Recommended for neutral fats (triglycerides)
Sharlach R Do not color phospholipids and fine lipid droplets
-
3. Sudan III First Sudan dye introduced into Histochemistry
Fat soluble
A good stain for the CNS
 8. MOUNTING
- Ensure the section will adhere to the slide
- Placing cut sections on a slide with adhesives
such as pinene or acrylic resins
- Done after staining & involves putting cover
slip on top of the stained tissue section

TAKE NOTE: Sections are transparent

- You won’t see anything because the tissue is
transparent. That’s why STAINING is always
done before MOUNTING.
 COMMONLY USED ADHESIVES
1. Mayer’s Egg - Most commonly used because it is very
Albumin
easy to make and relatively inexpensive
Formula: Egg white (50 mL)
Glycerin (50 mL)

Adhesives
Thymol crystals (100 mg)
2. Dried Albumin Formula: Dried albumin (5 g)
Sodium chloride (5 g) Distilled
water (100 mL) Thymol crystals
3. 1% Gelatin Formula: Gelatin (1 g)
Distilled water (100 mL) Glycerol
(15 mL) Phenol crystals (2 g)
4. Gelatin Formula: 1% Gelatin (5 mL)
2% Formaldehyde (5 mL) - Used in order to promote adhesion of
5. Starch Paste Formula: Powdered starch (1 g)
Distilled water (30 mL = 10 mL cold, 20
sections to slides.
mL boiling)
Hydrochloric acid (2 drops) Thymol
- Spread thinly and evenly on a clean
crystals
6. Plasma - Readily available from outdated blood
grease-free glass slide which is then
stored in blood banks, dispensed into
sterile tubes of 0.5 mL each. gently approximated to the end of the
7. Poly-L-Lysine - 0.1% aqueous detergent solution which
is further diluted 1:10 with distilled water
ribbon and drawn upwards in a near
Used
prior to use (Final dilution: 0.01%).
as a section adhesive in
vertical motion
immunohistochemistry.
8. 3-APES (3- - Very useful in cytology particularly for
aminopropyltrietho cytospin preparations of proteinaceous or
xysilane) bloody material.
Mounting
Mounting medium
AKA: Mountant
- Necessary to protect the
stained section from
physical injury and from
bleaching or
deterioration of the stain
as a result of oxidation.
- RI (Refractive Index)
close to that of glass
(1.518).
 Two Main Groups
a. Water
- Low refractive index; good only
for temporary mounting of Mounting Media
b. Glycerin (RI:1.47) - Also used as preservative, has
high index of refraction
1. Aqueous/Water Soluble
c. Farrant’s Medium - Does not solidify upon storage Mountants
(Gum syrup) (RI:1.43)

d. Apathy’s Medium - Used for Methylene

Designed to mount water-miscible
preparations directly from water in
e. Brun’s Fluid - Recommended for mounting
frozen sections from water or cases where the stain is removed or
paraffin sections which require
dehydration and clearing
decolorized with alcohol or xylene.
 Two Main Groups
a. Canada
Balsam
-
Natural resin extracted from the
Canadian Tree, Abus balsamea
of Mounting Media
(RI: 1.524) Recommended for whole mounts and for
thick sections because it does not shrink
much 1. Resinous Mountants
Miscible with xylene and is quite expensive
Used for preparations that have
b. DPX - Recommended for small tissue sections been dehydrated and cleared in
(RI:1.532)
c. XAM - Synthetic resin mixture in xylene in pale xylene or toluene and are
(RI:1.52) yellow or colorless solution recommended for majority of
d. Clarite - Synthetic resin which is soluble in xylene
(RI:1.544) (usually diluted to 60% with xylene) staining methods.
Ringing
- Process of sealing the margins of
the coverslip to prevent escape of
fluid or semi-fluid mounts and
evaporation of mountant, to
immobilize the coverslip, and to
prevent sticking of slides upon
storage.

Kronig Cement
- Made up of 2 parts paraffin wax
mixed with 4-9 parts powdered
colophonium resin, heated and
filtered
 Epithelial Tumor Markers
1. Keratin - Highly sensitive marker for epithelial cells (Epithelial
tumors)
a. Cytokeratin 7 (CK7) - Carcinomas of the lung, breast, uterus and ovaries
(serous tumors)
b. Cytokeratin 20 (CK20) - Carcinomas of colon and stomach
c. CK 7 and CK20 - Transitional carcinomas of the bladder and mucinous
ovarian tumor
2. Epithelial membrane antigen (EMA) -
Determining site tumor, Breast, lung and kidney
adenocarcinomas
3. Carcinoembryonic membrane antigen - Differentiating adenocarcinoma and mesothelioma
(CEA) GIT, pancreas, lung, breast, ovary, uterus and cervix
Thyroid, lung and neuroendocrine tumors

4. Thyroid - Distinguishing lung adenocarcinoma from mesotheliomas

transcription factor- 1 (TTF-1)
5. Prostate specific antigen (PSA) - Prostatic carcinoma; pancreatic and
salivary gland tumor.
Quiz time! ☺
 Which of the following fixatives can be
used to demonstrate rickettsia?
a.Orth’s
b.Helly’s
c.Zenker’s
d.B5 fixative
A. Orth’s
The optimal fixative to tissue ratio:
a.1:20
b.20:1
c.1:30
d.1:10
B. 20:1
Commonly used decalcifying agent
a.5-10% Nitric Acid
b.Formic Acid
c.TCA
d.Versene
A. 5-10% Nitric Acid
Usual optimum temperature for the
decalcification process
a.1-6C
b.22-25C
c.37C
d.55C
B. 22-25C
Process in which excess water is removed from
a preserved tissue
a.Dehydration
b.Clearing
c.Decalcification
d.Fixation
A. Dehydration
Simplest, most common and best embedding
medium
a.Paraffin c. Gelatin
b.Plastic Resins d. Celloidin
A. Paraffin

Paraffin wax Impregnation

o the simplest, most common,
and by far the best for routine
use
o melting point is 54-58 0C
It is a process by which a tissue is arranged
in precise positions in the mold during
embedding, on the microtome before cutting
and on the slide before staining
a.Waxing c. Paraplasting
b.Blocking d. Orientation
d. Orientation
Considered as the hallmark of acute inflammation
a.Monouclear infiltrates c. Swelling
b.Increased vascular permeability d. Edema
B. Increased
vascular
permeability
Pathology tissue blocks should
be kept for a period of
2 years
A.

10 years
B.

Indefinite
C.

5 years
D.
B. 10 years
Adjustable type of embedding mold
a.Peel-away c. Leuckhart’s
b.Paper boat d. Tissue-tek
c.
Leuckhart’
s
Considered to be the MOST RAPID fixative/ recommended
for fixing chromosomes, lymph glands and urgent
biopsies:
A. Gendre's fixative
B. Carnoys fluid
C. Newcomers fluid
D. Flemmings solution
B. Carnoys fluid
 The last container through which tissue
pass through in an automatic tissue
processor contains:
A. Paraffin C. Formalin
B. Xylol D. Alcohol
A. Paraffin
To avoid distortion of the image,
the refractive index of the
mountant should be near as
possible to that of the glass which
is:
A. 1.581
B. 1.185
C. 1.518
D. 1.155
C. 1.518
__________ provides the direction of where an
organization is going whereas ____ provides the
“road” to get there.
a. leadership; management
b. management; leadership
c. either
d. neither
a. leadership;
management
How do you prepare a 1L working solution of
formalin?
a.900mL stock formalin soln + 100mL dH2O
b.100mL stock formalin soln + 900mL dH2O
c.500mL stock formalin soln + 500mL dH2O
d.400mL stock formalin soln + 600mL dH2O
B. 100mL stock formalin soln +
900mL dH2O