(E-Notes) Histopath Lec and Lab

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Histopathologic
Techniques &
Cytology
Review Lec & Lab
Notes
Page 1
TABLE OF CONTENTS
Chapter Topic Pages
1 Review: Cells sturctures & Fundamentals of Histology 3 - 78
2 Introduction to Pathology 79 - 117
3 Cell Adaptation 118 - 123
4 Safety in the Histopathology laboratory 124 - 131
5 Quality Management System 132 - 141
6 Instrumentation 142 - 147
7 Examination of Tissues 148 - 151
8 Fixation 152 - 184
9 Decalcification 185 - 212
10 Gross Room or Surgical Cut-up 213 - 218
11 Tissue Processing (Dehydration, Clearing, Impregnation) 219 - 269
12 Microtomy 270 - 297
13 Staining 298 - 320
14 Mounting Media 321 - 325
15 Frozen & Related Sections 326 - 337
16 Basic Diagnostic Cytology 338 - 347
17 Special Stains 348 - 413
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01
Review: Cells sturctures
& Fundamentals of
Histology
Page 3
Topic 1: Normal Cell Structure • Cells are the smallest working unit of all
living things.
Cells have distinctive shapes and functions which • All cells come from preexisting cells
depend on how they have differentiated, the through cells division.
proteins that they express, and their interactions
with other cell types. Some organisms consist of a single cells =
unicellular organism, others are multicellular
It is difficult to be precise, but there are probably at aggregates of specialized cells.
least 200 different types of cells within the body.
This topic aims to help you remember the things
you learned about normal cell structures and
functions.
The Cell Structure and Function

• The cell is the lowest level of structure capable
of performing all the activities of life.
• The first cells were observed and named by
Robert Hooke in 1665 from slice of cork.
The Cell Thoery

• Proposed by Matthais Schleiden and Theodor
Schwann in 1839:
• All living things are made up of cells.
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Whether multicellular or unicellular, all organisms • A major difference between prokaryotic and
must accomplish the same functions: eukaryotic cells is the location of chromosomes.
• Uptake and processing of nutrients • Eukaryotic cell, chromosomes are contained in a
• Excretion of wastes membrane-enclosed organelle, the nucleus.
• Response to environmental stimuli • Prokaryotic cell, the DNA is concentrated in the
• Reproduction among others. nucleoid without a membrane separating it from
the rest of the cell.
Cells
• Most cells are between 1-100 μm in diameter Plasma Membrane
which can be visualized by light microscope. • Double layer of phospholipids
• Various proteins are attached to it
A Panoramic View of the Cell • Carbohydrate side chains are found only on the
Basic features of cells: outer surface of plasma membrane
• All cells are bounded by a plasma membrane.
• The semifluid substance within the membrane
is the cytosol, containing the organelles.
• All cells contain chromosomes which carry
genes in the form of DNA.
• All cells also have ribosomes, tiny organelles
that make proteins using the instructions
contained in genes.
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• Function of plasma membrane = selective

barrier that allows passage of oxygen, nutrients,
and wastes for the whole volume of the cell.
Nucleus and Its Envelope

• The nucleus contains most of the genes in a
eukaryotic cell.
• Some genes are located in mitochondria and
chloroplast
• The nucleus averages about 5 microns in
diameter.
• The nucleus is enclosed by a nuclear envelope
which is a double membrane of 20 -40nm apart.
• Where the double membranes are fused, a
nuclear pore complex allows large
macromolecules and particles to pass through.
• The nuclear side of the envelope is lined by the
nuclear lamina, a network of intermediate
filaments that maintain the shape of the nucleus.
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Level of Chromatin Packing

• In the nucleus, the DNA and the associated
proteins are organised into firous material called
chromatin.
• In a normal cell they appear as diffuse mass.
• However, when the cell prepares to divide, the
chromatin fibers coil up to be seen as separate
structures, chromosomes.
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Nucleolus • Eukaryotes (80s ribosome):

• A region (in the nucleus) of densely stained • Large subunit = 60S
fibers and granules adjoining chromatin. • 45 proteins
• In the nucleolus, rRNA is synthesized and • rRNA = 28S, 8S and 5S
assembled with proteins from the cytoplasm to • Small subunit = 40S
form ribosomal subunits. • 30 proteins
• The subunits pass from the nuclear pores to the • rRNA = 18S
cytoplasm where they combine to form
ribosomes.
Ribosomes
• Particles consisted of proteins and ribosomal
RNA (rRNA)
• Function = protein synthesis
• Prokaryotes = 70S
• Eukaryotes = 80S
• Free ribosomes in cytosol synthesize proteins

that function within cytosol
• Bound ribosomes are attached to the outside of
the endoplasmic reticulum or nuclear envelope:
membrane protein, organelle proteins or
secretory protein
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• Bound and free ribosomes are structurally Endoplasmic reticulum (ER)

identical and can alternate between the two • ER consists of a network of membranous tubules
roles. and sacs called cisternae (cisterna = a reservoir
for a liquid)
Endomembrane System • The network are interconnected
• The collection of membranes inside and around • The ER membrane is continuous with the nuclear
a eukaryotic cell. envelope and the cisternal space of the ER is
• These membranes are related either through continuous with the space between the two
direct physical contact or by transfer of vesicles membranes of the nuclear envelope.
(sac of membrane). • Smooth and rough ER (with & without ribosomes)
• In spite of these links, these membranes have
diverse functions and structures: Smooth ER:
• Nuclear envelope • Synthesis of lipid (oils, phospholipids, and
• Endoplasmic reticulum steroids)
• Golgi apparatus • Glycogen metabolism in the liver cells
• Lysosomes • Detoxification of drugs and poisons
• Vacuoles • Store calcium for muscle contraction
• Plasma membrane
Rough ER:
• Ribosomes are attached to the outside
• Is abundant in cells that secrete protein
• Synthesis secretory proteins, cell membrane
protein and organelle protein
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• Proteins are targeted to determined • Unlike ER cisternae, the Golgi cisternae are not
location according to the sorting signals. physically connected.
• Sorting signals are encoded in a.a
sequences or in the attached
oligosaccharides)
• Synthesis of phospholipids and ER associated
protein
• Proteins are synthesized from the bound
ribosomes
• Threaded into the cisternal space through a
pore formed by a protein in the ER membrane
• Protein folds into its native conformation
• An oligosaccharide is attached to the protein =
glycosylation
• Those proteins are wrapped in the transport • Golgi stacks has polarity:
vesicles that bud from the ER • cis face (the receiving side) and trans face
(the shipping side)
Golgi apparatus • Transport vesicle from ER fuses to the cis face to
• Major sites for carbohydrate symthesis transfer the material to the Golgi
• Sorting and dispatching station for the products • Proteins and lipids are altered; glycosylation and
of the ER phosphorylation (tagging the sorting signal)
• Consists of flatten membranous sacs, cisternae • Oligosaccharides portion of the glycoproteins are
modified:
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• Golgi removes some sugar monomers

and substitutes others
• Some polysaccharides are synthesized in the
Golgi e.g pectin and cellulose of the plant cell
wall and most of the glycosaminoglycans of
animal extracellular matrix
• Golgi products (that will be secreted) depart
from the trans face of the Golgi by transport
vesicle for the correct docking.
Relationships among Organelles of the

Endomembrane System
• Secretory proteins, lysosomes, vacuoles and
membrane are synthesized by the
• rough ER, then transported through the Golgi
as a vesicle. Lysosomes
• During this process, their molecular • Principal sites of intracellular digestion
compositions and metabolic functions are • Contain hydrolytic enzymes (required acidic pH)
modified. to digest proteins, polysaccharides, fats and
nucleic acids (if those hydrolases leak out of the
lysosmes, they are not likely to do damage
unless the cells become acidic)
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• After digestion, the organic monomers are

returned to the cytosol for reuse
• Lysosomal enzymes and membranes are made

by rough ER and transferred to the Golgi for
processing
• Lysosomal membranes are highly glycosylated
to protect them from lysosomal proteases
• Food particles engulfed as a food vacuole
(phagocytosis) or an endosome (product of
receptor-mediated endocytosis) is fused with
the lysosome.
• The digestion products are passed to cytosol
and become nutrient for the cell.
• Autophagy = process which cells recycle its
own organic material
• The organelles are fused with a lysosome
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Vacuoles Mitochondria and Chloroplast

• Membrane-bound sacs diverse functions in cell • Both are energy transformers of cells
maintenance food vacuoles formed by • mitochondria = cellular respiration
phagocytosis and digested by lysosomes • chloroplast = photosynthesis
• Contractile vacuoles (in protists) pump excess • Both are not part of the endomembrane system
water out of the cells most of their proteins are synthesized by the free
• Central vacuole (a versatile compartment in ribosomes in the cytosol
plants) stores protein and metabolic by- • A few of the proteins are synthesized from their
products, reservoir of inorganic ions, pigments own ribosomes
• Both organelles contain small quantity of DNA
that direct the synthesis of polypeptides produced
by the internal ribosomes
• Both organelles grow and reproduce as
semiautonomous organelles
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Mitochondria Chloroplast
• 1-10 μm long • Is one of the generalized plant structure called
• Some cells contain a single large mitochondrion plastids
but most cells contain several mitochondria • Found in mesophyll cells of the leaves and in
• Enclosed by two membrane: outer and inner algae
membrane with different permeability • 2-4 μm wide and 5-10 μm long
• Cristae = fold innermembrane to increase the • 2 membranes: inner and outer membrane
surface area • Stroma ~ matrix of mitochondria
• Matrix and intermembrane space • Ds circular DNA
• Matrix contains: • 70S ribosomes
• Ds circular DNA • Enzyme for carbohydrate biosynthesis
• Prokaryote like ribosome (70S) • Thylakoids contain photosynthetic machinery of
• Enzymes in TCA cycle the chloroplast:
• Enzymes for β-oxidation of fatty acid • Lght absorbing pigment
• Glucose and fatty acids are catabolized in the • Electron carriers
matrix of mitochondria. • ATP synthesizing apparatus
• Inner membrane of mitochondria contains:
• Electron transport chain
• ATP synthase
• The energy from catabolism in the matrix is
• converted into ATP.
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Microbodies Cytoskeleton
• Electron-dense cytoplasmic particles bound by • A network of fibers extending throughout the
a single membrane cytoplasm
• Contain oxidative enzymes: D-amino acid • Function:
oxidase, ureate oxidase and catalase • Provide mechanical strength to the cell
• They are not formed in the Golgi complex • Establish cell shape
• They are self replicating, like the mitochondria • Locomotion (several types of cell motility)
• e.g. peroxisomes, glyoxisomes • Intracellular transport of organelles
• 3 main types of fiber:
Peroxisomes • Microtubules: determine the positions of
• Single membrane membraneenclosed organelles and
• Contain enzymes that transfer hydrogen from intracellular transport
various substrate to oxygen and produce H2O2 • Microfilament: determine the shape of
as intermediate product the cell and necessary for the whole cell
• But peroxisomes contain another enzyme that locomotion
convert H2O2 to H2O • Intermediate filament: provide
• Some peroxisomes break down fatty acids to mechanical strength and resistance to
smaller molecules that are transported to shear stress
mitochondria for fuel
• Glyoxysomes = specialized peroxisomes (found
in fat storing tissues of plant seeds) convert
fatty acid to sugar which can be used as energy
for seedling.
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Cytoskeleton and Cell Motility

• Cell motility requires interaction of cytoskeleton
with proteins called motor molecules in ATP
dependent manner.
• Sliding of neighboring microtubules moves cilia
and flagella.
• In muscle contraction, motor molecules slide
microfimaments.
Microtubules
• Are straight, hollow cylinders
• Have a diameter of about 25 nm
• Are variable in length but can grow 1000 times as
long as they are thick
• Are built by the assembly of dimers of alpha
tubulin and beta tubulin.
Walking of organelles along microtubules • Are found in both animal and plant cells
• Motor molecule attached to receptor on • Intermediate filaments strengthen the long
organelles can “walk” the organelles along extension (axon) of nerve cells that transmit
microtubules or microfilaments. impulse
• e.g. migration of neurotransmitters containing
vesicles to the tips of nerve cell axon.
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Topic 2: Basic Types of Tissues • There are four basic tissue types defined by their
• Histology is the study of tissues. morphology and function: epithelial tissue,
• A tissue is a group of cells with similar structure connective tissue, muscle tissue, and nervous
and function plus the extracellular substances tissue.
located between the cells.
• The extracellular matrix is nonliving chemical Four basic tissue types:
substances located between cells. • Epithelial tissue
• Although there are many types of cells in the • Connective tissue
human body, they are organized into four broad • Muscular tissue
categories of tissues: epithelial, connective, • Nervous tissue
muscle, and nervous.
• Each of these categories is characterized by Epithelial tissue
specific functions that contribute to the overall • Epithelial tissue covers surfaces, usually has a
health and maintenance of the body. basement membrane, has little extracellular
• A disruption of the structure is a sign of injury or material, and has no blood vessels.
disease. • A basement membrane attaches the epithelial
• Such changes can be detected through cells to underlying tissues.
histology, the microscopic study of tissue • Most epithelia have a free surface, which is not in
appearance, organization, and function. contact with other cells.
• A tissue is a group of cells, in close proximity, • Epithelia are classified according to the number
organized to perform one or more specific of cell layers and the shape of the cells.
functions. • Covers the body surface and forms the lining for
most internal cavities.
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• The major function of epithelial tissue includes • Transitional (changes shape when relaxed or
protection, secretion, absorption, and filtration. distented by fluid)
• The skin is an organ made up of epithelial
tissue that protects the body from dirt, dust, Functions of Epithelium:
bacteria, and other microbes that may be • Simple epithelium involved with diffusion, filtration,
harmful. secretion, or absorption
• Cells of the epithelial tissue have different • Stratified epithelium protects from abrasion
shapes. • Squamous cells function in diffusion or filtration
• Cells can be thin, flat to cubic to elongate. • Cuboidal or columnar cells secrete or absorb
Categories of epithelium based on cell layers: • A gland is a single cell or a multicellular structure
• Simple epithelium has one layer of cells. that secretes substances onto a surface, into a
• Stratified epithelium has more than one layer of cavity, or into the blood.
cells. • Most glands are composed primarily of
• Pseudostratified epithelium consists of a single epithelium.
layer of cells, in which some cells are tall and • Exocrine glands have ducts.
thin and reach the free surface and others do • A duct is a tube or vessel which carries a
not. secretion from a gland.
• Ducts can be simple or compound.
Categories of epithelium based on cell shape: • The end of a duct can be tubular or expanded
• Squamous (flat) into a sac-Iike structure called an acinus or
• Cuboidal (cubelike) alveolus.
• Columnar (tall and thin)
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Types of exocrine glands: Connective tissue

• Unicellular (e.g., goblet cells in mucous • Is the most abundant and the most widely
membranes) distributed of the tissues.
• Simple tubular (e.g., sweat glands and stomach • Connective tissues perform a variety of functions
glands) including support and protection.
• Simple acinar or alveolar (e.g., sebaceous • The following tissues are found in the human
glands) body, ordinary loose connective tissue, fat tissue,
• Compound tubular (e.g., duodenal glands) dense fibrous tissue, cartilage, bone, blood, and
• Compound acinar or alveolar (e.g., pancreas) lymph, which are all considered connective tissue.
• Connective tissue is usually characterized by
Endocrine glands- have no ducts and empty their large amounts of extracellular materials that
secretions (hormones) directly into the blood. separate cells from each other, whereas
epithelial tissue is mostly cells with very little
extracellular material.
• The extracellular substance of connective tissue
consists of protein fibers which are embedded in
ground substance containing tissue fluid.
Functions of Connective tissue:

• Joins together other tissues
• Supporting framework for the body (bone)
• Fat stores energy
• Blood transports substances
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Fibers in connective tissue can be divided into Specific Types of connective tissue cells:
three types: • Fibroblasts
• Collagen fibers are the most abundant protein • Fibrocytes
fibers in the body. • Chondroblasts
• Elastic fibers are made of elastin and have the • Chondrocytes
ability to recoil to original shape. • Osteoblasts
• Reticular fibers are very fine collagen fibers that • Osteocytes
join connective tissues to other tissues. • Osteoclasts
Connective tissue cells are named according Notes:

to their functions: • Mast cells release chemicals promoting
• Blast cells produce the matrix of connective inflammation.
tissues • Macrophages are large cells, capable of moving
• Cyte cells maintains the matrix of connective about and ingesting foreign substances, including
tissues microorganisms that are found in connective
• Clast cells breaks down the matrix for tissue.
remodeling (found in bone) • Adipose- very little extracellular matrix; large cells
filled with lipids
Word parts:
• Fibro (fibrous connective tissue- tendons and
ligaments)
• Chondro (cartilage)
• Osteo (bone)
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• Bone- hard tissue with mineralized matrix; • Hyaline cartilage- most abundant type of
strong and rigid (two types- compact and cartilage, very smooth, and can withstand
cancellous) compression (makes up skeleton of embryos,
• Compact bone - found in the shaft of a found many other places such as covering the
long bone ends of long bones at joints).
• Cancellous bone - found in the ends of • Elastic cartilage- recoils to original shape when
long bones and in flat bones (contains bent (found in the external ear)
red marrow). • Fibrocartilage- compressible, but resists tearing
• Loose or areolar connective tissue- found or pulling forces (found in intervertebral disks).
covering muscles, glands, and nerves (has
widely separated collagen fibers running in
random directions)
• Dense connective tissue- found in the dermis,
also in tendons and ligaments (has an
extracellular matrix consisting mostly of
collagen fibers)
• Blood-fluid matrix allows cells to travel freely
Cartilage-3 Types:
• Has cells located in spaces in the matrix
(lacunae), has a small quantity of collagen
fibers, no elastic fibers, and no blood vessels in
the matrix.
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Muscle tissue Three types of muscle tissue:

• Contracts to initiate movement in the body. • Skeletal muscle
• There are three types of muscle tissue: skeletal, • Has cylindrical shaped cells with striations,
smooth, and cardiac. Skeletal muscle is a cells have many nuclei, is under voluntary
voluntary type of muscle tissue that is used in control, comprises about 40% of a
the contraction of skeletal parts. person's body weight, and connects to the
• Smooth muscle is found in the walls of internal skeleton.
organs and blood vessels. • Cardiac muscle
• It is an involuntary type. • Has cylindrical shaped cells that branch,
• The cardiac muscle is found only in the walls of has striations, has intercalated disks
the heart and is involuntary in nature. connecting the cells together, has one
• The main characteristic of muscle tissue is the nucleus per cell, is under involuntary
ability to contract (shorten). control, and is found only in the walls of
• Muscle contraction results from contractile the heart.
proteins found inside muscle cells. Muscle cells • Smooth muscle
are called muscle fibers because they often • Has spindle shaped cells with one nucleus
resemble tiny threads. per cell, does not have striations, is under
involuntary control, and is the most varied
in location and function of the muscle
tissue types.
• Found inside the eye, inside hollow tubes
such as blood vessels, respiratory tract,
digestive tract, and inside hair follicles.
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Components of a nerve cell:

• The cell body is the part of the nerve cell in which
the nucleus is located.
• Axons are nerve cell processes that conduct
action potentials away from the cell body.
• Dendrites are nerve cell processes that receive
action potentials and conduct them toward the
nerve cell body.
Nervous tissue
• Nervous tissue is specialized to conduct action
potentials (electrical signals).
• Neurons are cells that conduct action potentials.
• Neuroglia are cells which support the neurons.
• Is composed of specialized cells that not only
receive stimuli but also conduct impulses to and
from all parts of the body.
• Nerve cells or neurons are long and string-like
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Membranes
• Mucous membranes line cavities that open to
the outside of the body (digestive, respiratory,
excretory, and reproductive tracts).
• They contain glands and secrete mucus.
• Serous membranes line trunk cavities that do
not open to the outside of the body (pleural,
pericardial and peritoneal cavities).
• They do not contain glands, but do
secrete serous fluid.
• Other membranes include cutaneous
membranes (skin), synovial membranes (line
joint cavities), and periosteum (surrounds bone).
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The differentiation of the four basic tissue types based on cell morphology, extracellular
matrix and main functions.
Tissues Cells Extracellular Matrix Main Functions

Intertwining elongated Transmission of nervous
Nervous None
processes impulses
Lining of surface or body
Aggregated polyhedral
Epithelial Small amount cavities, glandular
cells
secretion
Muscle Elongated contractile cells Moderate Movement
Several types of fixed and
Connective Abundant amount Support and protection
wandering cells
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Topic 3: Cellular Response to Injury • Most of these adaptations take place at a

biochemical level and represent fine regulation of
• Inflammation is the standard, initial response of metabolic function, known as homeostasis.
the body to injury. • However, some adaptations are also
• Whether biological, chemical, physical, or accompanied by structural changes that are
radiation burns, all injuries lead to the same visible microscopically.
sequence of physiological events. • Many such structural changes fall within the
• Inflammation limits the extent of injury, partially normal pattern of growth of a tissue; for example,
or fully eliminates the cause of injury, and the thyroid gland enlarges in pregnancy owing to
initiates repair and regeneration of damaged the action of increased thyroid stimulating
tissue. hormone on thyroid epithelial cells.
• After containment of an injury, the tissue repair • This is an example of physiologic cellular
phase starts with removal of toxins and waste adaptation to a stimulus within the normal range.
products. • In contrast, certain environmental changes lie
• Normal cells exhibit a considerable degree of outside the normal physiological range and result
cellular adaptability in response to the in cellular malfunction, damage or even cell
constantly changing environment. death: these stimuli are termed pathological.
• For instance, over the course of a day most • Pathological stimuli may simply be a more
normal bodies are exposed to varying levels of extreme form of normal physiological event, e.g.
hydration, nutrition, temperature, and energy exposure to extremes of temperatures compared
expenditure, to name but a few variables. to normal day to day variations or maybe
intrinsically pathological agents such as poisons,
toxins or infective organism.
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Causative agents and processes

• A wide range of possible agents or
circumstances result in cellular injury
• Could be categorized according to the nature of
the injurious agent, the cellular target, the
pattern of cellular reaction or mode of cell death.
• The sequence of agent, target and mode will be
uniform, but some injurious agents have
variable effects depending on concentration,
duration or other contributory influences such
as co-existent disease.
Major types of cellular injury:

• Trauma
• Thermal injury, hot or cold
• Poisons
• Drugs Mechanisms of cellular injury. Different agents can
• Infectious organisms injure the various structural and functional
• Ischaemia and reperfusion components of the cell. Some cells with specific
• Plasma membrane failure function are selectively prone to certain types of
• DNA damage injury.
• Loss of growth factors
• Ionizing radiation
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Major Types of Cellular Injury

Example Agent Mode of Action
Trauma (e.g. road traffic accident) Mechanical disruption of tissue
Carbon monoxide inhalation Prevents oxygen transport
Contact with strong acid Coagulates tissue proteins
Paracetamol overdose Metabolites bind to liver cell proteins and lipoproteins
Bacterial infections Toxins and enzymes
Ionizing radiation (e.g. X-rays) Damage to DNA
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Physical agents • Some are highly toxic systemic

• Most physical agents cause passive cell metabolic poisons, while others
destruction by gross membrane disruption or exert their damage locally; the latter
catastrophic functional impairment. includes caustic liquids applied to
• Trauma and thermal injury cause cell death by skin or mucous membranes, or
disrupting cells and denaturing proteins, and gases that injure the lung.
also cause local vascular thrombosis with • Furthermore, some substances
consequent tissue ischemia or infarction. produce one effect locally and
another systemically.
Chemical and biological agents • Infectious organisms
• Cells may be injured by contact with drugs and • The mechanisms of tissue damage
other chemicals; the latter may include produced by infectious organisms
enzymes and toxins secreted by micro- are varied, but with many bacteria it
organisms. is their metabolic products or
• This category of agents can give rise to the full secretions that are harmful.
range of modes of death. • Thus, the host cells receive a
• Drugs and poisons chemical insult that may be toxic to
• Many naturally occurring and their metabolism or membrane
synthetic chemicals cause cellular integrity.
injury; the effect is usually dose-
related, but in a few instances the
effect is exacerbated by
constitutional factors.
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• The mode of cell death generally • Blockage of metabolic pathways

induces an acute inflammatory • Cell injury may result from specific
response, which may be interference with intracellular metabolism,
damaging to adjacent cells; affected usually by relative or total
organisms that do this are called blockage of one or more pathways.
pyogenic. • Ischaemia and reperfusion injury
• In contrast, bacterial endotoxin • Impaired blood flow causes
(lipopolysaccharide) induces inadequate oxygen delivery
apoptosis with different to cells.
pathological consequences. • Mitochondrial production of
• Intracellular agents such as ATP will cease, and
viruses often result in the physical anaerobic glycolysis will
rupture of infected cells, but with result in acidosis due to the
some viruses such as hepatitis B accumulation of lactate.
local tissue damage may result • The acidosis promotes
from host immune reactions. calcium influx.
• Therefore, the cellular response to • Cells in different organs vary
injury caused by infections will widely in their vulnerability to
depend on a combination of the oxygen deprivation; those
damage inflicted directly by the with high metabolic activity
agent and indirectly as a result of such as cortical neurons and
the host response to the agent. cardiac myocytes will be
most affected.
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• When the blood supply is • Failure of membrane integrity

restored, the oxygen • Cell membrane damage is one of the
results in a burst of consequences of complement activation;
mitochondrial activity and some of the end products of the
excessive release of complement cascade have cytolytic
reactive oxygen species activity.
(free radicals). • Another effector of cytolysis is perforin, a
• Free radicals mediator of lymphocyte cytotoxicity that
• Free radicals are atoms or causes damage to the cell membrane of
groups of atoms with an the target cells such as those infected by
unpaired electron viruses.
(symbolized by a • Incidental membrane tears or perforations
superscript dot); they avidly can be repaired very quickly, so do not
form chemical bonds. necessarily result in cell death.
• They are highly reactive, • DNA damage or loss
chemically unstable, • Damage to DNA results primarily from
generally present only at reactive oxygen species attack, for
low concentrations, and example following ionizing radiation.
tend to participate in or Damage may not be evident immediately;
initiate chain reactions. dividing cells are more susceptible.
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• Cell populations that are constantly • While consequence of unsuccessful adaptation to

dividing (i.e. labile cells such as intestinal an environmental change leads to cell damage
epithelium and hemopoietic cells) are and/or cell death.
soon affected by a dose of radiation • Further consequences of unsuccessful
sufficient to alter their DNA. adaptation to an environmental change may lead
• Other cell populations may require a to development of neoplasia.
growth or metabolic stimulus before the
DNA damage is revealed. Inflammation (Acute and Chronic)
• Since non-lethal DNA damage may be • Inflammation is the reaction of vascularized living
inherited by daughter cells, a clone of tissue to local injury evoked by microbial
transformed cells with abnormal growth infections, physical agents, chemicals, necrotic
characteristics may be formed; this is the tissue, and immunologic reactions.
process of neoplastic transformation that • Its roles include the following:
results in tumors. • To contain and isolate injury
• To destroy the invading microorganisms
Cellular Adaptation and inactivate toxins
• If a cell makes a successful response to an • To prepare the tissue or organ for healing
environment change, then it will either return to and repair.
normal or it may make an adaptive change,
adaptive changes, such as hyperplasia and
atrophy.
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Acute inflammation 2) Extravasation of polymorphonuclears from the

• Acute inflammation can result from exposure to microcirculation and their accumulation in the
a substance, such as a bee sting or dust an focus of injury.
injury, an infection. • White blood cells migration in interstitial tissues
• When the body detects damage or pathogens, toward a chemotactic stimulus by margination,
the immune system triggers a number of rolling, adhesion in the lumen of the blood
reactions. vessels.
• Transmigration across the endothelium,
Major events in acute inflammation include the diapedesis
following: • Migration in interstitial tissues toward a
chemotactic stimulus
1) Changes in vascular caliber that lead to an
increase blood flow. 3) Elimination of the inciting microorganisms or
• Transient vasoconstriction will occur stimulus through phagocytosis .
• Vasodilation, accounting for the heat and • This involve three basic steps:
redness. • Recognition and attachment of the particle
• Increased in vascular permeability in the to be ingested by the WBC
microvasculature leads to the escape of plasma • Engulfment by pseudopods encircling the
proteins and white blood cells into the phagocytosed particle forming a
interstitium, causing edema. phagosome which fuses with lysosome
forming a phagolysosome
• Killing and degradation of bacteria which
involves two mechanisms:
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• Oxygen-dependent mechanism
(H2O2-MPO-halide system)
• Oxygen-independent mechanisms
(lysozyme, lactoferrin, MBP of
eosinophils, and defensins)
The killed organisms are then degraded by
hydrolases and other enzymes in lysosomes.
Sequence of events in an inflammatory reaction.

Sentinel cells in tissues (macrophages, dendritic
cells, and other cell types) recognize microbes and
damaged cells and liberate mediators, which trigger
the vascular and cellular reactions of inflammation.
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The vascular phenomena in acute • Phagocytosis

inflammation are characterized by: • Of the offending agent follows, which may
• Increased blood flow to the injured area lead to the death of the microorganism.
• Resulting mainly from arteriolar dilation • During chemotaxis and phagocytosis
and opening of capillary beds. • Activated leukocytes may release toxic
• Increased vascular permeability metabolites and proteases extracellularly,
• Results in the accumulation of protein- potentially causing tissue damage.
rich extravascular fluid, which forms the • Exudate
exudates. • Is an inflammatory extravascular fluid that
• Plasma proteins has a high protein concentration, much
• Leave the vessels most commonly cellular debris, and a specific gravity
through widened interendothelial cell above 1.020.
junctions of the venules or by direct • Transudate
endothelial cell injury. • Another extravascular fluid has lower
• Leukocytes protein content and a specific gravity less
• Initially predominantly neutrophils, than 1.012.
adhere to the endothelium via adhesion • Is essentially an ultrafiltrate of blood
molecules, transmigrate across the plasma and results from hydrostatic
endothelium, and migrate to the site of imbalance across the vascular
injury under the influence of chemotactic endothelium.
agents. • Pus
• A purulent inflammatory exudate rich in
WBCs and parenchymal cell debris
Page 37
Signs of acute inflammation can appear within Some factors and infections that can lead to
hours or days, depending on the cause. In some acute inflammation include:
cases, they can rapidly become severe. How they • Acute bronchitis, appendicitis, etc.
develop and how long they last will depend on the • An ingrown toenail
cause, which part of the body they affect, and • A sore throat from a cold or flu
individual factors. • Physical trauma or wound
There are five key signs of acute inflammation Outcomes of acute inflammation
these include the following: • Complete resolution — regeneration of native
cells and restoration of the tissue to normal
• Pain (dolor): This may occur continuously or • Abscess formation — infections with pyogenic
only when a person touches the affected area. organisms
• Redness (rubor): This happens because of an • Healing by connective tissue replacement
increase in the blood supply to the capillaries in (fibrosis) and scarring
the area. • Progression to chronic inflammation
• Loss of function (function laesa): There may
be difficulty moving a joint, breathing, sensing Chronic inflammation
smell, and so on. • Is also referred to as slow, long-term
• Swelling (tumor): A condition call edema can inflammation lasting for prolonged periods of
develop if fluid builds up. several months to years. (weeks or months)
• Heat (calor): Increased blood flow may leave
the affected area warm to the touch.
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• It is characterized by: • Prolonged exposure to potentially toxic

• Infiltration with mononuclear cells, which exogenous (e.g., silica and silicosis of the
include macrophages, lymphocytes, and lung) or endogenous (e.g., plasma lipid
plasma cells components and atherosclerosis)
• Tissue destruction induced by the substances
inflammatory cells • Immune reactions, particularly
• Attempts to repair damaged tissue is by autoimmune diseases (e.g., SLE, RA,
connective tissue replacement which Sjogren’s Syndrome, Systemic Sclerosis)
involves angiogenesis and fibrosis. • Generally, the extent and effects of chronic
• May follow acute inflammation inflammation vary with the cause of the injury and
• Persistence of the inciting the ability of the body to repair and overcome the
stimulus damage.
• Interference in the normal process
of healing
• May result from repeated bouts of acute
inflammation
• May begin as a low-grade response
• Persistent infection by intracellular
microbes (e.g., tb bacilli, viral infection)
which are of low toxicity but evoke an
immunologic reaction
Page 39
Outcomes of acute inflammation: resolution, healing by fibrosis, or

chronic inflammation. The components of the various reactions
and their functional outcomes are listed.
Page 40
Chronic inflammation may begin as a low- • Persistent acute inflammation

grade response caused by the following • In some cases, a person may not fully
factors: recover from acute inflammation.
• Sensitivity • Sometimes, this can lead to chronic
• Inflammation happens when the body inflammation.
senses something that should not be • Persistent infection by intracellular microbes
there. • (e.g., tb bacilli, viral infection) which are of
• Hypersensitivity to an external trigger low toxicity but evoke an immunologic
can result in an allergy. reaction.
• Exposure
• Sometimes, long-term, low-level Factors that may increase the risk of chronic
exposure to potentially toxic exogenous inflammation include:
(e.g., silica and silicosis of the lung) or • Older age
endogenous (e.g., plasma lipid • Obesity
components and atherosclerosis) • A diet that is rich in unhealthful fats and added
substances sugar
• Autoimmune disorders • Smoking
• The immune system mistakenly attacks • Low sex hormones
normal healthy tissue, as in psoriasis. • Stress
• Autoinflammatory diseases • Sleep problems
• A genetic factor affects the way the
immune system works.
Page 41
Long-term diseases that doctors associate A test for Inflammation:

with inflammation include: • How do you know if you have chronic
• Asthma inflammation?
• Chronic peptic ulcer • A blood test measures a protein produced
• Tuberculosis by the liver, C-reactive protein (CRP),
• Rheumatoid arthritis which rises in response to inflammation.
• Periodontitis • A CRP level between 1 and 3 milligrams
• Ulcerative colitis and Crohn’s disease per liter of blood often signals a low, yet
• Sinusitis chronic, level of inflammation.
• Active hepatitis • The erythrocyte sedimentation rate is
another blood test for inflammation.
Inflammation plays a vital role in healing, but • It is used for people with inflammatory
chronic inflammation may increase the risk of conditions, like rheumatoid arthritis.
various diseases, including some cancers,
rheumatoid arthritis, atherosclerosis, periodontitis, Morphologic Patterns in Acute and Chronic
and hay fever. Inflammation
Granulomatous inflammation
• Is a chronic inflammatory reaction in which the
predominant cell type is an activated
macrophage with a modified epithelial-like
(epithelioid) appearance.
Page 42
• It is characterized by the presence of

granulomas—focal collections of epithelioid
macrophages that are surrounded by
mononuclear leukocytes (lymphocytes and
plasma cells); epithelioid cells may coalesce to
form mononuclear giant cells.
2 types of granulomas
• Foreign body granulomas
• Incited by relatively inert foreign bodies
• Immune granulomas
• Formed by immune T-cell mediated
reactions to poorly degradable antigens.
Page 43
Disease Cause Tissue

• Non-caseating tubercle (granuloma
prototype):
• A focus of epithelioid cells, rimmed by
Mycobacterium fibroblasts, lymphocytes, histiocytes,
Tuberculosis
tuberculosis occasional Langhans giant cells
• Caseating tubercle:
• Central amorphous granular debris,
loss of all cellular detailacid-fast bacilli
• Acid-fast bacilli in macrophages
Leprosy Mycobacterium leprae
• Granulomas and epithelioid cells
• Gumma:
• Microscopic to grossly visible lesion,
Syphilis Treponema pallidum enclosing wall of histiocytesplasma cell
infiltrate center cells are necrotic
without loss of cellular outline
• Rounded or stellate granuloma containing
Cat-scratch Gram-negative
central granular debris and recognizable
disease bacillus
neutrophils, giant cells uncommon
Page 44
Serous inflammation Fibrinous inflammation

• Is marked by the outpouring of a thin fluid that, • A fibrinous exudate develops when the vascular
depending on the size of injury, is derived from leaks are large enough or there is a procoagulant
either the plasma or the secretions of stimulus in the interstitium (e.g., cancer cells).
mesothelial cells lining the peritoneal, pleural, • This exudate contains large amounts of
and pericardial cavities (called effusion). fibrinogen, which is converted to fibrin.
• The skin blister resulting from a burn or viral
infection represents a large accumulation of Fibrinous exudate
serous fluid, either within or immediately • Is characteristic of inflammation in the lining of
beneath the epidermis of the skin. body cavities, such as the meninges (fibrinous
meningitis), pericardium (fibrinous pericarditis),
and pleura (fibrinous pleuritis).
• Fibrinous exudates may be removed by
fibrinolysis and clearing of other debris by
macrophages.
• The process of resolution may restore normal
Biologic purpose: Immediate dilution of the tissue structure, but when the fibrin is not
harmful agent at the site of inflammation. removed, it may stimulate the ingrowth of
fibroblasts and blood vessels and thus lead to
Etiologic factors: scarring.
• Hypersensitivity reactions
• Bacterial and viral tissue injury
• Physical and chemical tissue injury
Page 45
Biologic purpose: Immediate temporary barrier Fibrinous Serosal Inflammation

against additional effects of inflammation. • Exudative fibrinous inflammations of the serous
membranes may occur as a reaction of the
Etiologic factors: serosa to other underlying disorders (serositis) or
• Infectious toxic tissue injury in the presence of tissue injury occurring in the
• Tissue injury from physical trauma serosa (such as infarction).
• Chemical and toxic tissue injury
• Excretion of toxic metabolites (uremic toxins) Fibrinous Mucosal Inflammation
• Ischemic tissue injury • Fibrinous inflammations in the mucosa, the
fibrinous exudation process is usually preceded
Types of fibrinous inflammation: by superficial necrosis
Fibrinous Parenchymal Inflammation Croupous

• Exudative inflammation with exudation of fibrin • A wide area of fibrinous exudate forms an easily
on the inner surfaces of the PULMONARY removable pseudomembrane covering the
parenchyma (pulmonary alveoli). necrosis, which is limited to the mucosal
• Example: epithelium
• Lobar pneumonia in the gray • Example:
hepatization stage • Diphtheric laryngotracheitis
Page 46
Suppurative or purulent inflammation

• Characterized by the production of purulent
Diphtheria exudates or pus consisting primarily of died
• Necrosis extending into the submucosa is neutrophils and cellular debris (detritus).
covered by a wide area of fibrinous exudate in
the form of an adhesive pseudomembrane that Biologic purpose: Damaged tissue is dissolved
can only be forcibly removed along with the pathogen.
• Example:
• Diphtheric tonsillitis and pharyngitis, Etiologic factors – pyogenic bacteria:
dysentery • Staphylococc
• Antibiotic associated colitis and • Streptococci
pseudomembranous colitis
Page 47
Types of suppurative inflammation: Phlegmon

• Diffuse suppurative inflammation that spreads
Empyema primarily in loose fibrous connective tissue
• Suppurative inflammation in a body cavity. without sharp demarcation
• Pathogenesis: an empyema usually occurs • Example:
when a suppurative inflammation of an organ • Muscular phlegmon, and erysipelas- an
breaks through into an adjacent cavity inflammation in the connective tissue of
• Example: the skin usually caused by beta hemolytic
• Pericardial, peritoneal, and pleural streptococci involving a map like pattern of
empyema erythema and swelling
• Gallbladder and appendiceal empyema
• Middle ear and nasal sinus empyema
Page 48
Abscess Tissue Repair

• An accumulation of pus with tissue destruction
and a cavity formation Repair
• Example: • Also called healing
• Pulmonary abscesses, liver abscesses, • Refers to the restoration of tissue architecture
and furuncle- forming inflammations of and function after an injury.
the hair follicle usually following • By convention, the term repair is used for
staphylococcal infection parenchymal and connective tissues and healing
for surface epithelia, but these distinctions are
not based on biology, and we use the terms
interchangeably.
• Critical to the survival of an organism is the ability
to repair the damage caused by toxic insults and
inflammation.
• Hence the inflammatory response to microbes
and injured tissues not only serves to eliminate
these dangers but also sets into motion the
process of repair.
Ulcers
• Local defects, or excavations, of the surface of
an organ or tissue (skin, epithelium or mucous
membrane) that are produced by shedding of
inflammatory necrotic tissue.
Page 49
• Repair of damaged tissues occurs by two However, mammals have a limited capacity to
processes: regenerate most damaged tissues and organs, and
• Regeneration - replacement of dead cells only some components of these tissues are able to
by proliferation of cells of the same type fully restore themselves.
• Replacement by connective tissue or
fibroplasia If the injured tissues are incapable of regeneration,
or if the supporting structures of the tissue are too
Regeneration severely damaged to support regeneration of the
• Some tissues are able to replace the damaged tissue cells, repair occurs by the laying down of
components and essentially return to a normal connective (fibrous) tissue, a process that may
state result in scar formation.
• Regeneration may occur by proliferation of
differentiated cells that survive the injury and Although the fibrous scar is not normal, it usually
retain the capacity to proliferate, notably provides enough structural stability that the injured
hepatocytes in the liver. tissue is able to function.
In other tissues, particularly the epithelia of the The term fibrosis is often used to describe the
skin and intestines, tissue stem cells and their deposition of collagen that occurs in the lungs, liver,
progenitors contribute to the restoration of kidney, and other organs as a consequence of
damaged tissues. chronic inflammation or in the myocardium after
extensive ischemic necrosis (infarction).
Page 50
If fibrosis develops in a tissue space occupied by Granulation Tissue

an inflammatory exudate, it is called organization • Richly vascular connective tissue, containing new
(as in organizing pneumonia affecting the lung). capillaries, proliferating fibroblast, and variable
numbers of inflammatory cells
4 components
• Angiogenesis
• Migration and proliferation of fibroblast
• Deposition of extracellular matrix
• Remodeling — maturation and reorganization of
the fibrous tissue into a scar
Notes to Remember!!!
• Tissue repair is the substitution of viable cells for
dead cells, and it can occur by regeneration or
replacement. In regeneration, the new cells are
the same type as those that were destroyed, and
normal function is usually restored. In
replacement, a new type of tissue develops that
eventually causes scar production and the loss of
some tissue function.
Page 51
• When the edges of a wound are close together, Mechanisms of Tissue Regeneration
the wound fills with blood, and a clot forms. The • We will consider liver regeneration as a model of
clot contains a threadlike protein, fibrin, which tissue regeneration, because it has been studied
binds the edges of the wound together and extensively and illustrates the mechanisms that
stops the bleeding. The clot is replaced by underlie this process.
granulation tissue, a delicate connective tissue • The human liver has a remarkable capacity to
which consists of fibroblasts, collagen, and regenerate, as demonstrated by its growth after
capillaries. partial hepatectomy.
Cell and Tissue Regeneration Regeneration of the liver occurs by two major
• The regeneration of injured cells and tissues mechanisms
involves cell proliferation, which is driven by
growth factors and is critically dependent on the Proliferation
integrity of the ECM, and by the development of • Proliferation of remaining hepatocytes
mature cells from tissue stem cells. • In humans, resection of up to 90% of the liver can
• The regeneration of injured cells and tissues be corrected by proliferation of the residual
involves cell proliferation, which is driven by hepatocytes.
growth factors and is critically dependent on the • This classic model of tissue regeneration has
integrity of the ECM, and by the development of been used experimentally to study the initiation
mature cells from tissue stem cells. and control of the process.
Page 52
• The process occurs in stages, including Restoration of normal tissue structure can occur
priming-where cytokines such as IL-6 produced only if the residual tissue is structurally intact, as
mainly by Kupffer cells act on hepatocytes to after partial surgical resection.
make the parenchymal cells competent to
receive and respond to growth factor signals, By contrast, if the tissue is damaged by infection or
followed by growth factor–induced proliferation. inflammation, regeneration is incomplete and is
accompanied by scarring.
Repopulation
• From progenitor cells- in situations where the For example, extensive destruction of the liver with
proliferative capacity of hepatocytes is impaired, collapse of the reticulin framework, as occurs in a
such as after chronic liver injury or inflammation, liver abscess, leads to scar formation even though
progenitor cells in the liver contribute to the remaining liver cells have the capacity to
repopulation. regenerate.
Repair by Connective Tissue Deposition
If repair cannot be accomplished by regeneration

alone, it occurs by replacement of the injured cells
with connective tissue, leading to the formation of a
scar, or by a combination of regeneration of some
residual cells and scar formation.
Page 53
In contrast to regeneration, which involves the 2) Cell proliferation

restitution of tissue components, scar formation is • In the next stage, which takes up to 10 days,
a response that “patches” rather than restores the several cell types, including epithelial cells,
tissue. endothelial and other vascular cells, and
fibroblasts, proliferate and migrate to close the
The term scar is most often used in connection to now clean wound.
wound healing in the skin, but may also be used to • Each cell type serves unique functions.
describe the replacement of parenchymal cells in
any tissue by collagen, as in the heart after Epithelial cells
myocardial infarction. • Respond to locally produced growth factors and
migrate over the wound to cover it up.
Steps in Scar Formation
Endothelial cells and pericytes
1) Inflammation • Proliferate to form new blood vessels, a process
• Breakdown products of complement activation, known as angiogenesis.
chemokines released from activated platelets,
and other mediators produced at the site of Fibroblasts
injury function as chemotactic agents to recruit • Proliferate and migrate into the site of injury and
neutrophils and then monocytes over the next lay down collagen fibers that form the scar.
6 to 48 hours.
Page 54
3) Formation of granulation tissue Factors That Influence Tissue Repair

• Migration and proliferation of fibroblasts and • Tissue repair may be altered by several factors,
deposition of loose connective tissue, together which impact the quality or adequacy of the
with the vessels and interspersed mononuclear reparative process. Variables that modify healing
leukocytes, form granulation tissue. may be extrinsic (e.g., infection) or intrinsic to the
injured tissue and systemic or local:
4) Deposition of connective tissue • Infection
• Granulation tissue is progressively replaced by • Diabetes
deposition of collagen. • Nutritional status
• The amount of connective tissue increases in • Glucocorticoids (steroids)
the granulation tissue, eventually resulting in • Mechanical factors such as increased
the formation of a stable fibrous scar. local pressure or torsion may cause
wounds to pull apart, or dehisce.
• Poor perfusion, due to peripheral vascular
disease, arteriosclerosis, and diabetes or
due to obstructed venous drainage (e.g.,
in varicose veins), also impairs healing.
• Foreign bodies such as fragments of steel,
glass, or even bone impede healing by
perpetuating chronic inflammation.
• Type and extent of tissue injury and the
character of the tissue in which the injury
occurs affect the subsequent repair.
Page 55
• Location of the injury is also important. • The repair consists of the same three connected
For example, inflammation arising in processes that we have described previously:
tissue spaces (e.g., pleural, peritoneal, • Inflammation, proliferation of epithelial and
synovial cavities) develops extensive other cells, and maturation of the
exudates. connective tissue scar.
Examples of Tissue Repair and Fibrosis Healing by second intention

• In this section we describe two clinically • Occurs when there is more extensive loss of
significant types of repair—the healing of skin tissue (e.g., infarction, ulceration, abscess
wounds (cutaneous wound healing) and fibrosis formation, and large wound).
in injured parenchymal organs.
Wound Healing
Healing by first intention • Complex but orderly phenomenon involving:
• When the injury involves only the epithelial layer, • Induction of an acute inflammation by the
the principal mechanism of repair is epithelial initial injury
regeneration, also called primary union or • Regeneration of parenchymal cells
healing by first intention. • Migration and proliferation of parenchymal
• One of the simplest examples of this type of and connective tissue cells
wound repair is the healing of a clean, • Synthesis of extracellular matrix
uninfected surgical incision approximated by • Remodeling of connective tissue and
surgical sutures. parenchymal components
• Collagenization and acquisition of wound
strength
Page 56
Healing By First Intention • Wound strength reaches approximately 70 % to

• Healing of a clean surgical approximated 80 % of normal by 3 months but usually does not
incision substantially improve beyond that point.
Healing By Second Intention

• Occurs when there is more extensive loss of
tissue (e.g., infarction, ulceration, abscess
formation, and large wound)
Wound Strength
• At the end of the first week is approximately
10% of normal
Wound Strength
• Carefully sutured wounds have approximately
70% of the strength of normal skin, largely
because of the placement of sutures.
• The recovery of tensile strength results from the
excess of collagen synthesis over collagen
degradation during the first 2 months of healing
by crosslinking of collagen fibers and
increased fiber size.
Page 57
Topic 4: Abnormalities in Cell Growth Aplasia

• Incomplete or defective development of a tissue
• Cancer affects everyone – the young and old, or organ, represented only by a mass of fatty or
the rich and poor, men, women and children – fibrous tissue, bearing no resemblance to adult
and represents a tremendous burden on structure Common to paired organs
patients, families and societies.
• Cancer is one of the leading causes of death in Hypoplasia
the world. • Failure to reach full mature or adult size due to
• In this topic we will be discussing about the incomplete development
different changes in tissues and factors that
may lead to the formation of cancer cells, the Atresia
difference between a benign and malignant • Failure of organ to form an opening
tumor and the different diagnostic techniques
for cancer. Atrophy
• Acquired decrease in size of a normally
1. Retrogressive Changes developed or mature tissue or organ, resulting
• Organs or tissues smaller than normal from reduction in cell size or decrease in total
number of cells or both.
Developmental defects
Agenesia Pathogenesis of Atrophy
• Complete non-appearance of organ • Due to increased proteolytic activities associated
with deficient blood and lymphatic circulation
Page 58
• Increase metabolic activity leading to • Exhaustion atrophy

accumulation of CO2 an organic acids in the • Endocrine atrophy
cell, causing loss of cell substances
Pathologic Changes Seen in Atrophy
Types of Atrophy • Atrophic organs are smaller and firmer due to
• Physiologic atrophy increase amount of connective tissue
• Occurs as natural consequence of • Vessels are prominent and increased in number
maturation, as in atrophy of thymus and due to proximity of vessels formerly separated by
lymphoid tissues larger masses of tissues
• Senile atrophy • Microscopically, cells are smaller than normal,
• Occurs in old age characterized hence, tissue may appear relatively cellular
by dry, lusterless, wrinkled skin • Atrophic parenchymal cells may contain yellow
due to atrophy of sweat and granular lipid-containing pigments, imparting
sebaceous glands and other “brown atrophy”
changes • Parenchymal cells may be replaced by
• Pathologic atrophy connective tissue
• Decrease in size of tissues and organs
as a consequence of disease 2. Progressive Changes
• Vascular atrophy • Organs or tissues larger than normal
• Pressure atrophy
• Starvation atrophy
• Atrophy of disuse
Page 59
Hypertrophy Hyperplasia
• An increase in the size of cells resulting to • Increase in the number of cells resulting to
increase in the size of the tissue or organ. increase in the size of the tissue or organ.
True hypertrophy Physiologic hyperplasia

• Usually observed in the skeletal muscle, heart, • Example hyperplasia of uterus during pregnancy
kidneys, and smooth muscles of the hollow
viscera due to increased workload and Pathological hyperplasia
endocrine stimulation • Brought by a disease
• Example hyperplasia of lymphoid follicles and
False hypertrophy Payer’s patches in typhoid fever
• Due to edema fluid and connective tissue
proliferation 3. Degenerative Changes Due to Aberrations of
Cellular Growth Patterns
Compensatory hypertrophy
• Involve one pair of organ when another is Metaplasia
removed or suffered from functional • Reversible transformation of adult cell from one
insufficiency type to another
Page 60
Dysplasia • As they grow, neoplasms can impinge upon and

• Regressive alteration in adult cells manifested damage adjacent structures.
by variation in size, shape and orientation • The term neoplasm can refer to benign (usually
associated with chronic inflammation and curable) or malignant (cancerous) growths.
protracted irritation
1. Benign Tumors
Anaplasia • In general, the names of benign tumors end with
• Marked regressive change in adult cells toward the suffix – oma
primitive or embryonic cell types, usually utilized • Benign tumors are non-malignant/non-cancerous
as criterion toward malignancy tumor.
• A benign tumor is usually localized, and does not
Neoplasia spread to other parts of the body.
• Continuous abnormal proliferation of cells • Most benign tumors respond well to treatment.
without control accompanied by increase in size • However, if left untreated, some benign tumors
and pigmentation, mitosis, number, metaplastic can grow large and lead to serious disease
and anaplastic changes because of their size.
• Benign tumors can also mimic malignant tumors,
Neoplasia/Tumor and so for this reason are sometimes treated.
• Tumor or neoplasm is an abnormal mass of • In general, the names of benign tumors end with
tissue, the growth of which is virtually the suffix –oma.
autonomous and exceeds that of normal tissues
• Growth persists after cessation of the stimuli
that initiated the change.
Page 61
Benign mesenchymal tumors

• (e.g., lipoma, fibroma, angioma, osteoma,
leiomyoma)
Benign epithelial tumors

• (e.g., adenomas, papillomas, polyp)
2. Malignant Tumors
• Often called cancers
• Malignant tumors are cancerous growths.
• They are often resistant to treatment, may
spread to other parts of the body and they
sometimes recur after they were removed.
• Malignant tumors are divided into two broad
categories:
Carcinomas
• Arising from epithelial cells
• (e.g., adenocarcinomas)
Sarcomas
• Arising from mesenchymal tissues
• (e.g., leiomyosarcomas)
Page 62
Nomenclature of Tumors
Connective Tissue
Tissue Benign Tumors Malignant Tumors
Adult fibrous tissue Fibroma Fibrosarcoma
Embryonic (myxomatous) Myxoma Myxosarcoma
fibrous tissue
Fat Lipoma Liposarcoma
Cartilage Chondroma Chondrosarcoma
Bone Osteoma Osteosarcoma
Notochord - Chordona
Connective tissue, probably Fibrous histiocytoma Malignant fibrous histiocytoma
fibrous
Endothelium and Mesothelium

Blood vessels Hemangloma • Hemanglosarcoma
• Angiosarcoma
Lymph vessels Hemanglopericytoma Lymphanglosarcoma
Mesothelium - Mesothelioma
Page 63
Blood and Lymphoid Cells

Hematopoietic cells “Preleukemia” • Leukemia, of various types
“Myeloproliferative disorders” • Aleukemic leukemia
Lymphoid tissue Plasmacytosis • Plasmacytoma
• Multiple myeloma
• Hodgkin lymphoma
• Non-Hodgkin lymphoma
Muscles

Smooth muscle Leiomyoma Leiomyosarcoma
Striated muscle Rhabdomyoma Rhabdomyosarcoma
Page 64
Epithelial Cells
Stratified squamous • Pailoma • Squamous carcinoma
• Seborrheic erstosis and • Epidemoid carcinoma and some
some skin adnexal tumors malignant skin adnexal tumors
Glandular epithelium Adenoma Adenocarcinoma
• Liver • Hepatic adenoma • Hepatoma: Hepatocellular
• Kidney • Renal tubular adenoma carcinoma
• Bile duct • Bile duct adenoma • Renal Carcinoma
• Hypernephroma
• Cholangiocarcinoma
Transitional epithelium Transitional cell papilloma • Transitional cell carcinoma
Placenta Hydatidiform mole • Choriocarcinoma
Testis - • Seminoma
• Embryonal cell carcinoma
Page 65
Neural
Glial cells (of several - • Glioma
types) • Grades I-III
• Anaplastic
• Glioblastoma multiforme (Grade (IV)
Nerve cells - • Neuroblastoma
- • Medulloblastoma
Ganglioneuroma • -
Meninges Meningioma • Malignant meningioma
Nerve sheath Schwannoma, • Malignant meningioma
Neurilemomma • Malignant schwannoma
Neurofibroma • Neurofibrosarcoma
More than One Neoplastic Cell Type—Mixed Tumors, Usually Derived From One Germ
Layer
Salivary glands Pleomorphic adenoma • Malignant mixed tumor
(mixed tumor)
Breast Fibroadenoma • Malignant cystosarcoma phyllodes
Renal anlage - • Wilm’stumor
Page 66
More Than One Neoplastic Cell Type Derived From More Than One Germ Layer—
Teratogenous
Totipotential cells in • Mature teratoma • Immature teratoma
gonads or inembryonic • Dermoidcyst • Teratocarcinoma
rests
Page 67
Characteristics of Benign and Malignant Anaplasia

Neoplasms • Lack of differentiation
• Distinction between benign and malignant • The hallmark of malignant cells
neoplasm is usually based on appearance • Cytologic features of anaplastic cells:
(morphology) and on behavior (clinical course), • Nuclear and cellular pleomorphism
using the following four criteria: • Hyperchromatism
• Differentiation and anaplasia • N:C ratio —approaches 1:1 instead of 1:4
• Rate of growth or 1:6
• Local invasion • Abundant mitoses
• Metastases • Tumor giant cells
1. Differentiation and Anaplasia 2. Rate of Growth

• Most malignant tumors grow more rapidly than
Differentiation benign tumors
• Extent to which tumor cells resemble normal • Some cancers grow slowly for years, then enter a
cells phase of rapid growth
• Cells within most benign tumors closely mimic • Growth of cancers arising from hormone-
corresponding normal cells sensitive tissues (uterus, breast, prostate gland)
• Cells in lipomas look like those in normal may be affected by the variations in hormone
adipose tissue levels (pregnancy, menopause)
• Malignant neoplasms are in general less
differentiated than their benign counterparts
Page 68
3. Local Invasion
• Most benign tumors develop a capsule at the
periphery
• Do not penetrate the capsule or the surrounding
normal tissues
• Malignant neoplasms are invasive, infiltrating
and destroying normal tissues surrounding
them making surgical excision difficult or
impossible
4. Metastasis
• Single most important feature distinguishing
benign from malignant tumors
• Involves invasion of the lymphatics, blood
vessels, and body cavities by the tumor,
followed by transport and growth of secondary
tumor cells to a distant site
• Example:
• Carcinoma of the breast spread to
axillary lymph nodes
• Almost all malignant tumors have the capacity
to metastasize
Page 69
Characteristics Benign Malignant

Differentiation/ • Well differentiated • Some lack of differentiation with
Anaplasia structure may be typical of anaplasia structure is often
tissue of origin atypical
Rate of growth • Usually progressive and • Erratic and may be slow to
slowmay come to a rapidmitotic figures may be
standstill or regressmitotic numerous and abnormal
figures are rare and normal
Local invasion • Usually cohesive and • Locally invasive, infiltrating the
expansile well demarcated surrounding normal tissues
masses that do not invade sometimes may be seemingly
or infiltrate surrounding cohesive and expansile
normal tissues
Metastasis • Absent • Frequently presentthe larger and
more undifferentiated the primary,
the more likely are metastases
Page 70
Predisposing Factors to Cancer Development Acquired Preneoplastic Disorders

• A variety of factors predispose an individual or a • Clinical conditions associated with an increased
population to the development of cancer. risk of developing cancers
• Cirrhosis of the liver
Environmental factors • Hepatocelullar carcinoma
• Influence the occurrence of specific forms of • Chronic ulcerative colitis
cancer • Carcinoma of the colon
• Association of carcinomas of the oropharynx,
larynx, and lung with cigarette smoking Certain benign tumors are also associated with
the subsequent development of cancer
Age • Villous adenomas of the colon
• Cancer is most common in those older than 55 • Colonic cancers
years old
• Certain cancers are particularly common in Molecular Basis of Cancer
children • Cancer is a genetic disease - genetic injury may
• Leukemias and lymphomas be acquired by exposure to environmental agents
• Neuroblastomas (chemicals, radiation, or viruses) or inherited in
• Wilm's tumor the germ line
• Retinoblastomas • Tumors develop from the clonal expansion of a
single genetically damaged progenitor cell (i.e.,
Heredity tumors are monoclonal).
• Plays a role in the development of cancer
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• Four classes of normal regulatory genes that 1. Chemical Carcinogenesis

are targets of genetic damage. • DNA is the primary and most important target of
• These include the following: chemical carcinogens
• Growth-promoting proto-oncogenes • Chemical carcinogens are mutagens that induce
• Growth-inhibiting tumor suppressor mutations in proto-oncogenes, cancer-
genes (anti-oncogenes) suppressor genes, apoptotic genes, and DNA
• Genes that regulate apoptosis repair genes
• Genes that regulate DNA repair
2. Radiation Carcinogenesis
Carcinogenic Agents
Ultraviolet Rays
Carcinogen • Natural UV radiation, especially UVB, derived
• Is something that can cause you to have cancer. from the sun can cause skin cancer
• It may be a substance in the air, a product you
use, or a chemical in foods and drinks. Ionizing Radiation
• Particulate radiations (α-particles, neutrons) are
Just because you had contact with a carcinogen more carcinogenic than electromagnetic
doesn't mean that you'll get cancer. Your chance radiations (x-rays, gamma rays)
of getting sick depends on many things. How • Most common radiation-induced neoplasms are
much you've been exposed to it is part of it. myeloid leukemia, followed by thyroid cancer in
Your genes also play a role. children
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3. Viral and Microbial Carcinogenesis 2. Fine-Needle Aspiration Biopsy

• HPV types 1, 2, 4, and 7 and benign squamous • Aspiration of cells and fluid from tumors that
papillomas (warts) occur in readily palpable sites
• HPV types 16 or 18 and SCCA of the uterine • Example:
cervix • Breast
• HBV infection and liver cancer • Thyroid
• Helicobacter pylori and gastric lymphoma, • Lymph nodes
gastric carcinoma • Aspirated cells are smeared, stained, and
examined
Laboratory Diagnosis of Cancer
3. Cytologic (Papanicolau) Smears
1. Histologic and Cytologic Methods • Exfoliative cytology—examination of cancer cells
• Most important method of diagnosis that are readily shed
• Routine fixed and paraffin-embedded sections, • Most commonly used in the diagnosis of cancers
quick-frozen sections (employed to obtain a of the uterine cervix, and tumors of the stomach,
rapid diagnosis while the patient is still under bronchus, and urinary bladder
anesthesia)
4. Immunocytochemistry/immunohistochemistry
• Useful in the following settings:
• Diagnosis of undifferentiated tumors
• Categorization of leukemia/lymphomas
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• Determination of the site of origin of 7. Tumor Markers

metastases • Tumor-derived or tumor-associated molecules
• Example: that can be detected in blood or body fluid
• PSA for prostate cancer • May be of value in determining recurrence and
• Detection of molecules that have response to therapy
prognostic or therapeutic significance
• Example:
• ERPR
• C-erb B2 on breast cancers
5. DNA Probe Analysis

• Involves PCR
• Involves amplification of DNA, using DNA
polymerases and subsequent identification of
DNA mutations
• Widely used in the molecular diagnosis of
human disease
• cCurrently used in the diagnosis of lymphomas
6. Flow Cytometry
• Measurement of the DNA content of tumor cells
useful in the diagnosis of leukemias/lymphomas
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Tumor Markers Associated Cancers

Hormones
• Human chorionic gonadotropin • Trophoblastic tumors, Nonseminomatous testicular tumors
• Calcitonin • Medullary carcinoma of thyroid
• Pheochromocytoma and related tumors
• Catecholamine and metabolites • See Paraneoplastic Syndromes
• Ectopic hormone
Oncofetal Antigens
• α-fetoprotein • Liver cell cancer, Nonseminomatous germ cell tumors of testis
• Carcinomas of the colon, pancreas, lung, stomach, and breast
• Carcinoembryonic antigen
Isoenzymes
• Prostatic acid phosphatase • Prostate cancer
• Neuron-specific enolase • Small cell cancer of lung
• Neuroblastoma
Specific Proteins
• Immunoglobulins • Multiple myeloma and other gammopathies
• Prostate-specific antigen • Prostate cancer
Mucins and Other Glycoproteins
• CA-125 • Ovarian cancer
• CA-19-9 • Colon cancer
• Pancreatic cancer
• CA-15-3 • Breast cancer
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Differentiation of Tumor base on Histologic Broder’s Classification

Characteristics Differentiated Undifferentiated
Cells Cells
Medullary
• More cells than supporting tissue GRADE I 100% - 75% 05 - 25%
GRADE II 75% - 50% 25% - 50%
Scirrhous carcinoma GRADE III 50% - 25% 50% - 75%
• More connective tissue than cells, cells are GRADE IV 25% - 0% 75% - 100%
apparently trapped within fibrous tissue
If a grading system for a tumor type is not specified,
Grading of Tumors the following system is generally used (1):
• The grade of a cancer depends on what the
cells look like under a microscope. • GX: Grade cannot be assessed (undetermined
• In general, a lower grade indicates a slower- grade)
growing cancer and a higher grade indicates a • G1: Well differentiated (low grade)
faster- growing one. The grading system that's • G2: Moderately differentiated (intermediate
usually used is as follows: grade)
• G3: Poorly differentiated (high grade)
• G4: Undifferentiated (high grade)
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Value of Grading Tumors Degeneration and Infiltration

• Doctors use tumor grade and other factors,
such as cancer stage and a patient's age and Degeneration
general health, to develop a treatment plan and • Injury to cell precedes and result in an
to determine a patient's prognosis accumulation of metabolites
• Regressive change in the cells which cytoplasm
Limitations of Grading takes on homogenous, glassy, appearance.
• Histologic grading may vary from section to • A form of protein coagulation, usually indicating
section severe cell damage
• higher grades of tumor are generally regarded • Substances responsible for producing hyaline
as having more tendecy to metastasize, but degeneration:
more benign tumors may have metastasis • Mucin
• Sometimes metastasis has lower grade than • Colloid
primary tumor; sometimes higher grade than • Amyloid
the primary tumor • Glycogen
• Some tumors canot be graded, as most of the
sarcomas Infiltration
• Injury is caused by overloading of metabolites
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Degeneration and Infiltration Involving Lipids Fatty metamorphosis

Fatty degeneration • Accumulation of fat within hepatic and
• Abnormal accumulation of fat within previously parenchymal cells
injured parenchymal cells • Include both changes seen in fatty infiltration and
• Phase or stages of cell degeneration: fatty degeneration
• Cloudy swellin Fatty phanerosis
• Fatty degenration • Presumed unmasking of previously invisible fat in
• Cell death or necrosis the cytoplasm of cells
• Calcification • Marked fatty metamorphosis is associated with
• Common in the liver, kidney, heart brought by an absolute increase in the fat content of cells, so
the any of the following: that the occurrence of phanerosis is doubted.
• Anoxia Hyaline degeneration
• Infections • Regressive change in the cells which cytoplasm
• Intoxication takes on homogenous, glassy, appearance.
• Biologic agents • A from of protein coagulation, usually indicating
• Endogenous toxins severe cell damage
Fatty infiltration • Substances responsible for producing hyaline
• Abnormal accumulation of fats within healthy degeneration:
cells as result of systemic metabolic • Mucin
derangement, affecting connective tissue • Colloid
stroma or parenchyma • Amyloid
• Example is parenchymal fatty infiltration in liver • Glycogen
Page 78
02
Introduction to
Pathology
Page 79
Introduction Moreover, pathology is a branch of medical science

that involves the study and diagnosis of disease
Pathology literally translates as the study of through the examination of surgically removed
suffering (Greek pathos = suffering, logos = organs, tissues (biopsy samples), bodily fluids, and
study); more prosaically, and as applied to modern in some cases the whole body (autopsy).
medicine, it is the study of disease.
Aspects of a bodily specimen that may be
Virchow was prescient in asserting that disease considered include its gross anatomical make up,
originates at the cellular level, but we now appearance of the cells using immunological
appreciate that cellular pathologies arise from markers and chemical signatures in the cells.
perturbations in molecules (genes, proteins, and
metabolites) that influence cell survival and
behaviors. Thus the foundation of modern
pathology understands the cellular and molecular
aberrations that give rise to diseases.
Areas of study include cellular adaptation to injury,

necrosis (death of living cells or tissues),
inflammation, wound healing, and neoplasia
(abnormal new growth of cells).
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Topic 1: Divisions of Pathology Pathology

• Pathology is the study of the molecular basis of
Although medical pathologists do not treat patients disease
of their own, they diagnose disease in the patients • Pathology is a branch of medical science that
of other physicians through laboratory tests involves the study and diagnosis of disease
performed by the medical technologists. through the examination of surgically removed
organs, tissues (biopsy samples), bodily fluids,
They examine body tissues, secretions, and other and in some cases the whole body (autopsy).
specimens to see whether a disease is present • Aspects of a bodily specimen that may be
and to determine its stage. They evaluate the considered include its gross anatomical make up,
extent of the disease, estimate the course it is appearance of the cells using immunological
likely to take, and suggest ways to treat the markers and chemical signatures in the cells.
disease. • Pathology also includes the related scientific
study of disease processes where by the causes,
In this topic, we will learn about the divisions of mechanisms and extent of disease are examined.
pathology and the techniques used in the study of • Areas of study include cellular adaptation to
patient’s specimen as wells as roles of the injury, necrosis (death of living cells or tissues),
pathologists and medical technologists both in inflammation, wound healing, and neoplasia
anatomic and clinical pathology laboratory (abnormal new growth of cells).
sections.
Page 81
Extensive Defiinition of Pathology Pathophysiology

• Study of the nature, causes, different processes, • Pathology describes the abnormal condition,
development, consequences of disease and the whereas pathophysiology seeks to explain the
modification in the cellular function and changes physiological processes because of which such
in cellular structure produced in any cell, organ condition develops and progresses.
or part of the body by disease i.e. disease • In other words, pathophysiology defines the
originates at the cellular level (Virchow) functional changes associated resulting from
• Cellular disturbance arises from alterations in disease or injury
molecules (genes, proteins, etc.). that Divisions of Pathology
influences the survival and behavior of cells. • Gross and microscopic pathology
Thus, the foundation of modern pathology is • Anatomic pathology
understanding the cellular and molecular • Clinical pathology
abnormalities that give rise to disease.
Anatomic Pathology
Pathogenesis • Is the branch of medicine that studies the effect
• Greek pathos (“suffering”, “disease”) and of disease on the structure of body organs, both
genesis (“creation”) as a whole (grossly) and microscopically.
• The development of a disease and the chain of • The primary role of anatomic pathology is
events leading to that disease to identify abnormalities that can help to
• A study of the biological mechanism (or diagnose disease and manage treatment.
mechanisms) that leads to a diseased state
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• Other than its purpose to help identify • Surgical pathology involves macroscopic (gross)
and manage various types of tumors or and microscopic (histologic/histopathologic)
cancers, it is also valuable in evaluating tissue analysis where the molecular properties of
other conditions, including: tissue samples are assessed by
• Kidney and liver diseases immunohistochemistry or other laboratory tests.
• Autoimmune disorders
• Infections
• Branches of anatomic pathology include
the following:
• Surgical pathology
• Autopsy
• Cytopathology molecular
pathology
Histopathology: Biopsies and Examination of
Surgical Pathology Tissues
• Is the most significant and time consuming • Histopathology involves the examination of
branch of pathology with a primary focus on sampled tissues under the microscope.
examining tissues with the naked eye or under • These may be small pieces of tissue obtained
a microscope for definitive diagnosis of disease from a part of the body using: biopsy or samples
• Surgically removed specimens are received taken from whole organs or parts of organs
from sources such as small biopsies of skin, removed during surgery
core biopsies for the diagnosis of cancer, and
the operating room where tumors are removed.
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Biopsy Excision Biopsy

• A biopsy is a procedure to remove a piece of • Also called a wide local incision, involves surgical
tissue or a sample of cells from your body so removal of a tumor and some normal tissue
that it can be analyzed in a laboratory. around it.
• Examination of cells or tissues from a living • The amount of normal tissue taken (also called
organism and studied in order to diagnose the surgical margin) depends on the size,
disease or to confirm findings of normality. histologic type and thickness of the tumor.
• Tumors are routinely biopsied in order to
determine whether they are benign or malignant. Types of biopsy procedures used to make a
cancer diagnosis:
Incision Biopsy 1. Bone marrow biopsy
• Partial removal of a small portion of tissues in • Bone marrow biopsy is commonly used to
the form of wedges, cylindrical pieces, cores, diagnose a variety of blood problems — both
punch, or scrapings of the suspected tissue or noncancerous and cancerous — including blood
organ. cancers, such as leukemia, lymphoma and
• The intention is to sample only a representation multiple myeloma.
portion of the lesion of interest.
• The surgeon when performing an incision 2. Endoscopic Biopsy
biopsy does not intend on removing the entire • Tubes used in an endoscopic biopsy can be
tumor inserted through your mouth, rectum, urinary tract
or a small incision in your skin.
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• Examples of endoscopic biopsy procedures 4. Skin biopsy

include: • A skin (cutaneous) biopsy removes cells from the
• Cystoscopy to collect tissue from inside surface of your body to diagnose skin conditions,
your bladder including melanoma and other cancers.
• Bronchoscopy to get tissue from inside • What type of skin biopsy you undergo will depend
your lung on the type of cancer suspected and the extent of
• Colonoscopy to collect tissue from inside the suspicious cells
your colon • Proceudres:
• Shave biopsy
3. Needle Biopsy • Punch biopsy
• A needle biopsy is often used on tumors that • Incisional biopsy
your doctor can feel through your skin, such as • Excisional biopsy
suspicious breast lumps and enlarged lymph
nodes. 5. Surgical Biopsy
• When combined with an imaging procedure, • These procedures can be used to remove part of
such as X-ray, needle biopsy can be used to an abnormal area of cells (incisional biopsy) or
collect cells from a suspicious area that can't be surgical biopsy may be used to remove an entire
felt through the skin. area of abnormal cells (excisional biopsy)
• Procedures:
• Fine-needle aspiration
• Core needle biopsy
• Vacuum-assisted biopsy
• Image-guided biopsy
Page 85
Histopathology Techniques Frozen section

• Histopathology Techniques refers to techniques • When time is crucial (for instance, when a
in the preparation of tissue for histopathologic surgeon needs an answer while performing
(histologic) studies surgery), the pathologist will bypass the fixation,
processing, and embedding in paraffin steps and
1. Tissue preparation perform a frozen section.
• Freezing of the tissue can result in some
Formalin-fixed, paraffin-embedded (FFPE) distortion of cells and some staining artifact.
• Biopsies and samples of tissue removed from • Sometimes making imprint smears (“touch
organs are usually placed in formalin (diluted preps”) by pressing cut tissue onto a glass slide
formaldehyde), which "fixes" the tissue by can help because this avoids freezing artifacts
cross-linking proteins and reduces the time needed to make a
• The tissue goes through various chemical steps diagnosis.
(dehydration and dissolving of fat) in
preparation for embedding into a paraffin (wax) 2. Special Techniques
block and blocks are placed on a special
machine that uses an extremely sharp knife (a Special staining
microtome) to shave very thin pieces of tissue • Pathologists use different special stains which
of about 5 µm may highlight fat, different tissue fibers, mucus,
• The thin pieces are placed on a glass slide and microbes such as bacteria or fungi, proteins, or
stained with special reagents such as other biochemical substances that might be
hematoxylin and eosin useful in identifying key elements that are
characteristic of certain diseases.
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Immunohistochemistry Genetic testing and other novel techniques

• Immunohistochemical stains are more specific • Chromosomal translocations and deletions can
in what they stain be detected using fluorescent in situ hybridization
• It takes advantage of the unique properties of technique (FISH).
antibodies that have been developed to • DNA and RNA isolated from FFPE tissue can
recognize specific components on or within also be used to identify specific mutations or
cells. screened for abnormalities.
• The antibodies are bound to certain markers • Most of these molecular techniques are used to
that allows a pathologist to identify key cellular identify mutations that help to guide therapy of
elements or tissue types that have been malignant tumors
associated with certain diseases and assist in
obtaining a final diagnosis. Autopsy
• Greek autopsia = "to see for oneself", derived
Electron microscopy from autos = "oneself" and opsis = "sight, view",
• A higher level of microscopy than that provided a seeing for oneself:
by standard light microscopy which can magnify • Auto + opsis = sight
up to two million times • Also called post-mortem examination, obduction,
necropsy, or autopsia cadaverum
Page 87
• Surgical procedure that consists of a thorough • The state of health of the person before
examination of a corpse by dissection to they died
determine the cause, mode, and manner of • Any medical diagnosis and treatment
death or to evaluate any disease or injury that before death was appropriate
may be present for research or educational
purposes Cytopathology
• Is another branch of anatomic pathology and is • Is a branch of anatomic pathology that studies
a highly specialized surgical procedure that is and diagnoses diseases on the cellular level.
performed by a pathologist and consists of a • It is usually used to aid in the diagnosis of cancer,
thorough examination of a corpse to determine but also helps in the diagnosis of certain
the cause and manner of death and to evaluate infectious diseases and other inflammatory
any disease or injury that may be present. conditions.
• Systematic examination of a cadaver for study • It is generally used on samples of free cells or
or for determining the cause of death. tissue fragments that spontaneously exfoliate or
• Uses many methodical procedures to determine are removed from tissues by abrasion or fine
the etiology and pathogenesis of diseases, for needle aspiration
epidemiologic purposes, for establishment of
genetic causes, for family counsel, and for
improvement of safety standards for the living.
• The principal aim of an autopsy or post-mortem
examination is to determine:
• The cause of death
Page 88
Cytopathology Techniques 2. Special Techniques

• Cytometry
1. Routine Techniques • Immunocytochemistry
Exfoliative cytology Molecular Pathology

• Is the analysis of cells that are shed from body • Is a relatively recent discipline that has achieved
surfaces. remarkable progress over the past decade.
• The cervical (Pap) smear is the most common • It emphasizes the study and diagnosis of disease
example, but samples from the urinary bladder, through the examination of molecules within
abdominal cavity, chest cavity, cerebrospinal organs, tissues or bodily fluids.
fluid, and washings from the lung are also • Molecular analysis is leading the way towards
frequently examined. personalized medicine by allowing us to predict a
patient’s response to certain anti-cancer therapy
Fine needle aspiration based on their own genetic make-up by
• Is performed using a thinner needle and is identifying specific hallmark mutations.
particularly useful in evaluating the presence of • Molecular Pathology includes the development of
normal or abnormal cell types molecular and genetic approaches to the
• The type of material is usually liquid or loosely diagnosis and classification of human tumors and
packed cell mass rather than solid tissue also to design and validate predictive biomarkers
• It generally involves a visible or palpable lump for prognosis of the disease, and susceptibility of
or cyst, complex FNAs involve sampling of developing certain cancers in individuals.
tissues located internally.
Page 89
• The high levels of sensitivity provided by • A physician who studies all aspects of disease
molecular assays allows for the detection of with an emphasis on the nature, causes, and
very small tumors that are otherwise development of abnormal conditions, as well as
undetectable by other means the structural and functional changes that result
from disease processes
Clinical Pathology • A physician specialized in the interpretation and
• Clinical pathology (or laboratory medicine), diagnoses of the gross, microscopic, and
which deals with the measurement of chemical molecular cause by disease in the body.
constituents of blood and other body fluids • The laboratory specialist behind the front-line
(clinical chemistry), analysis of blood cells clinical team.
(hematology), and identification of microbes • Pathologists specialize in a wide range of
(microbiology), to name a few examples. diseases including cancer and the vast majority
• While most of the tests described on this site of cancer diagnoses are made by pathologists
would be categorized as clinical pathology, • Pathologists also employ genetic studies and
many are used in conjunction with anatomic gene markers in the assessment of various
pathology procedures diseases
• There are varying amounts of laboratory work
Pathologist involved in pathology, depending on the specialty
• A pathologist is a doctor who interprets and and the role itself.
diagnoses the changes caused by disease in • Some pathologists don't tend to have any patient
the body's cells and tissues. contact, whereas others combine lab work with
clinical, direct patient care.
Page 90
• It's a myth that pathologists only deal with dead • Their main function is to prepare tissue samples
bodies - this is only the case with forensic for analysis.
histopathology, a sub-specialty of • Histotechnologist: MT specialized in
histopathology. histopathologic techniques. HT(ASCP)
• Pathology is involved in over 70% of all
diagnoses in 'live' patients.
Medical Technologists
• A medical technologist provides information for
diagnosis, treatment, and prevention of disease
by conducting medical laboratory tests,
procedures, experiments, and analyses
• Laboratory professional who performs
diagnostic analysis on human blood, urine, and
body fluids such as cerebral spinal fluid,
peritoneal, pericardial, and synovial, as well as
other specimens such as stool, sputum, etc.
Histotechnologist
• A histotechnologist is someone that is part of a
medical laboratory team that works with human
specimens to diagnosis disease and
abnormalities.
Page 91
Topic 2: Necrosis and Autopsy Pathology Necrosis

• Necrosis is the death of group of cells or part of a
Although medical pathologists do not treat patients tissue or organ due to disease or injury. On the
of their own, they diagnose disease in the patients other hand, a slower process of physiologic death
of other physicians through laboratory tests of cells with immediate regeneration is termed
performed by the medical technologists. They necrobiosis.
examine body tissues, secretions, and other • The main causes of necrosis include:
specimens to see whether a disease is present • Loss of oxygen supply (ischemia)
and to determine its stage. They evaluate the • Exposure to microbial toxins
extent of the disease, estimate the course it is • Burns and other forms of chemical and
likely to take, and suggest ways to treat the physical injury
disease. • Unusual situations in which active
proteases leak out of cells and damage
In this topic, we will learn about the divisions of surrounding tissues (as in pancreatitis).
pathology and the techniques used in the study of
patient’s specimen as wells as roles of the All of these initiating triggers lead to irreparable
pathologists and medical technologists both in damage of numerous cellular components.
anatomic and clinical pathology laboratory
sections.
Page 92
Necrosis is characterized by: • Numerous other molecules that are

• Denaturation of cellular proteins normally confined within healthy cells and
• Leakage of cellular contents through whose release is an indicator of severe
damaged membranes cell injury.
• Local inflammation • These molecules are recognized by receptors
• Enzymatic digestion of the lethally present in macrophages and most other cell
injured cell. types, and trigger phagocytosis of the debris as
• When damage to membranes is severe, well as the production of cytokines that induce
lysosomal enzymes enter the cytoplasm and inflammation.
digest the cell.
• Cellular contents also leak through the Causes of Necrosis
damaged plasma membrane into the • Ischemia or anoxia
extracellular space, where they elicit a host • Cell death due to ischemia is known as
reaction (inflammation). infarction, manifested by histologic
• Some specific substances released from injured appearance called “coagulation necrosis’
cells have been called damage-associated • Physical agents
molecular patterns (DAMPs). • Trauma
• These include: • Extreme heat or cold
• ATP (released from damaged • Radiant energy
mitochondria) • Electrical energy
• Uric acid (a breakdown product of DNA) • Chemical agents
• Biological products
Page 93
Microscopic Changes during Necrosis Types of Necrosis according to Basic

Morphologic Changes
1. Nuclear Changes
Coagulation necrosis
Pyknosis • Consists of more or less rapid coagulation of
• Reduction in size and condensation of the cytoplasm brought by leaking enzymes
nuclear material
Karyorrhexis Liquefaction necrosis
• Segmentation and fragmentation of the nucleus, • Refers to fairly rapid enzymatic dissolution of
where nuclear contents are broken up and cells with complete destruction of cells
released into the cytoplasm
Karyolysis Fat Necrosis
• Dissolution of the nucleus where all basophilism • Peculiar destruction of adipose tissue, particularly
is lost and nucleus disappears found in pancreatic degenerations
2. Cytoplasmic Changes Caseous necrosis

• Initially cells appear larger and granular (‘cloudy • Form of cell death produced by MTB. Necrotic
swelling’). tissue have appearance of soft, friable cheese.
• Then later, become more acidophilic, dense
and opaque. Gangrenous necrosis
• Cell boundary is lost • Massive death or necrosis of tissue brought by
• Nuclear death ultimately causes combination of ischemia and superimposed
cytoplasmic death bacterial infection
Page 94
Dry gangrene Massive Necrosis

• Arterial occlusion, producing ischemic necrosis, • Involves whole or greater part of organ,
and desiccation/mummification exemplified by gangrene
Wet gangrene Autopsy Pathology

• Venous occlusion, causing putrefactive • Autopsy pathology refers to postmortem
changes, with collection of offensive and foul- examination of a body to determine the cause of
odor fluid death or the nature of pathological changes.
• Autopsy has served medicine in numerous ways
Types of Necrosis according to Location or and continues to play evolving roles in a time
Extent when technologies have dramatically improved
and when new diseases, naturally occurring or
Focal Necrosis iatrogenic, continue to arise on the medical
• Confined to specific organs or particular horizon.
structures, usually found as minute, • An autopsy is usually carried out within 48 hours
circumscribed lesions after the death of a person since autolytic change
• Found in liver, spleen, bone marrow, lymph starts to happen
nodes • An autopsy can be hospital-based (non-coronial)
• Usually seen in infectious disease such as or coronial (medicolegal case)
typhoid and diphtheria • Coronial autopsies are ordered by the state
coroner, whereas hospital based autopsies may
be performed at the request of the family of the
deceased
Page 95
Brief History and Perspective of Autopsy • Over the last 2,500 years, as medicine moved
from mystic art to proper science, so did the field
460 B.C. of pathology and the quest to figure out what a
• Even in ancient Greece, they realized the truth person's body can tell us about how they died.
or cause of death can be deduced from • Over these centuries, medicine was
examining direct observations of the body considered as a vague and mystical belief,
• Was considered the ultimate medical audit which evolved to become a modern day
• Necessary to ensure the quality of the science during the early 17th century
services rendered by a medical service during the time of Virchow
• Autopsy was categorized by five different • Autopsies could provide answers - but
rulings for manner of death: even now, sometimes they find nothing but
• Natural a cloud of uncertainty (cannot provide
• Accident evidence)
• Homicide
• Suicide 44 B.C.
• Undetermined • Time of Roman Empire
• Not everyone received an autopsy upon death • First recorded autopsy: Antistius examining Julius
• In cases where suspicious circumstances Caesar’s body after his assassination
surround the death, a medical examiner or • Wished to determine which of the 23 stab
coroner can order an autopsy without consent wounds proved fatal
from next of kin • It was one wound to the chest that
ruptured Caesar’s aorta
Page 96
• The assassination of Julius Caesar was the • La mort de Cèsar

result of a conspiracy by approximately 60 • The Death of Julius Caesar
Roman senators who called themselves • An 1806 painting by Vincenzo
Liberators Camuccini
• Led by: • Originally commissioned in 1793
• Gaius Cassius Longinus • Frederick Hervey, 4th Earl of
• Marcus Junius Brutus Bristol
• Julius Caesar was stabbed to death in a
location adjacent to the Theatre of 1247
Pompey • “Hsi Yüan Lu, or The Washing Away of Wrongs,”
• Happened on the Ides of March • Written by Song Ci during the Song
(March 15), 44 BC Dynasty
• Caesar was the dictator of the Roman • An instruction manual on how to conduct
Republic at the time, having recently medico-legal investigations, examine
been declared dictator perpetuo by the corpses, and determine the time and
Senate cause of death
• This declaration made several • Advanced writing at the time
senators fear that Caesar wanted • Other forward-thinking forensic issues were
to overthrow the Senate in favour illustrated, such as poisoning, decomposition,
of tyranny wounds from various weapons, strangulation,
and fake wounds
Page 97
1302 • Leonardo da Vinci and Michelangelo

• The first-known legal autopsy where the death performed several “autopsies,” dissecting
was investigated explicitly to determine if there corpses and observing (and recorded by
was fault drawings) the anatomy unseen by the
• Performed by Bartolomeo da Varignana naked eye
• Was influenced by The Washing
Away of Wrongs 1870s
• What is referred to as a medico-legal • Rudolf Ludwig Karl Virchow
autopsy • Established “cellular pathology’ Due to
• The investigation was requested by a importance of microscopic examination
magistrate in Bologna when conducting research
• Before the advent of the microscope, his • Uncovered minute details not seen
observations were limited by the power by naked Eye
of the human eye and his tools • There is correlation between gross
• Throughout the Renaissance, anatomy findings and microscopic findings
teachers and students in medical schools did • The father of Modern Pathology
not perform dissections themselves. • Realized the importance of the microscope
• They would congregate in an operating when conducting pathological research to
theater and watch as a cadaver was uncover minute details
opened by a “lay dissector” • Through his examinations, he
characterized a case of leukemia, and his
resulting report is one of the earliest formal
reports on this cancer
Page 98
Why is an autopsy done? • Obtain and review clinical records

• Autopsies may be done for several reasons, • Contact clinical team and staff pathologist
including the following: • In this step, we review and collate all clinical
• When a suspicious or unexpected death records and data that will help us investigate
occurs further the correlation as well as assemble the
• When there's a public health concern, clinical and pathology staff
such as an outbreak with an
undetermined cause Preparation of the autopsy room
• When no doctor knows the deceased • Set up of dissection instruments and tools
well enough to state a cause of death • Lay out swabs, media, etc. for any ancillary
and to sign the death certificate studies to be performed
• When the doctor, the family or legally • Prepare photographic equipment
responsible designee of the deceased • Assemble PPE
person requests an autopsy
Confirmation of decedent identity
Preparations Before Postmortem Examination • This is the most important step in the autopsy
procedure we want to make of the identity of
Administrative preparations decedent
• Obtain and confirm consent for autopsy • Identifiers on the body must be confirmed and
• From the next of kin matched with the autopsy consent form
• No need for consent if this is for medico • Also confirm any limitations or restrictions to the
legal cases or death under suspicious autopsy by this time by the next of kin
circumstances
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Consent for Autopsy When it is necessary to complete the death

Next of kin consist of blood relatives in the 1st-3rd certificate
degree of: • For conscientious physician, he/ she may request
• Spouse for an autopsy and physicians enjoy this privilege
• Adult children
• Adult grandchildren When the deceased himself has given consent
• Parent before he died (advanced directive)
• Brother/sister • Very popular in the west but not that acceptable
• Nephew in our country
• Grandparent • Many individuals upon entry to a hospital may
• Uncle/Aunt give their advance directive in case something
• Cousin happens in their stay in the hospitals.
• Stepchildren • They would gladly contribute to medical science
by consenting to their own postmortem
Post Mortem Examination is permitted w/o examination, research for a rare unusual disease
consent in the following circumstances: or contribute an organ for transplantation
purposes
When it is ordered by the police or coroner
• Can be ordered by a judge or higher official in Deceased military personnel who dies in active
senate hearings duty/training in the military service
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3 Types/levels of Autopsy Notes to Remember!!!

Autopsy
Complete autopsy • Extent – Selective, partial, complete
• Requires consent • Retention of organs
• Complete examination of all organs, including • For organs that may be vital for the next of
the brain kin such as the brain and heart
• All body cavities are examined (including the • Must be explained to next of kin and kin
head/brain) will give consent to making it property of
• Must be clearly understood and agreed upon by the hospital
the next of kin as there may be individual’s • Medico-legal – forensic
sensitive regarding examining organs for • Under jurisdiction of medicolegal officer/
reasons such as religion, etc. coroner
• In PH = NBI office
Limited (Partial) autopsy • Death certificate
• Part of the anatomy • Grows findings (provisional anatomic
• Which may exclude the head/brain diagnosis)
• You have permission to examine all other • Data such as time, nature of death and
organs except brain witness attesting to the death of the
individual
Selective autopsy • Pathologist signs the certificate if
• Restricted to at least a single organ (Ex. MI – postmortem exam is performed and this
heart) appears at the back portion of certificate
• Specific organs only are examined
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• There is a provision there for Criteria for Autopsies

anatomic diagnosis which
includes the gross findings College of American Pathologists Criteria
observed by the pathologist used 1) Deaths in which autopsy may help to explain
to give possible explanation as unknown and unanticipated medical
evidence to cause of individuals complications to the attending physician.
death 2) All deaths in which the cause of death or a major
• Final cause of death is only diagnosis is not known with reasonable certainty
plausible after all things have on clinical grounds.
been examined including all slides 3) Cases in which an autopsy may help to allay
and tests, microbiological tests, concerns of the family and/or the public
angular tests, etc. regarding the death, and to provide reassurance
• Final diagnosis to them regarding the same.
• Gross and microscopic findings (final 4) Unexpected or unexplained deaths occurring
anatomic diagnosis) during or following any dental, medical or
• Final report released by pathologist surgical diagnostic procedures and/or therapies.
explaining circumstances, pathogenesis 5) Deaths of patients who have participated in
and pathophysiology of the cause of clinical trials (protocols) approved by institutional
death of individual review boards.
6) Unexpected or unexplained deaths which are
apparently natural and not subject to a forensic
medical jurisdiction.
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7) Natural deaths which are subject to, but Jurisdiction of Medico-Legal Autopsy
waived by, a forensic medical jurisdiction such
as: The medicolegal examiner or the coroner has
• Persons dead on arrival at hospitals jurisdiction in medicolegal cases and may authorize
• Deaths occurring in hospitals within 24 the pathologist to proceed with an autopsy.
hours of admission, and
• Deaths in which the patient sustained or • Coroner also known as the forensicpathologist
apparently sustained an injury while • A forensic pathologist must be qualified, certified,
hospitalized. and authorized to perform such forensic
8) Deaths resulting from high-risk infectious and autopsies
contagious diseases. • As you have seen in the list many of the cases
9) All obstetric deaths. are medicolegal in nature
10)All perinatal and pediatric deaths. • The medical legal are under the jurisdiction of the
11)Deaths at any age in which it is believed that coroner
autopsy would disclose a known or suspected
illness which also may have a bearing on The coroner has authority in the following cases:
survivors or recipients of transplant organs. 1) All-natural deaths occurring in the hospital within
12)Deaths known or suspected to have resulted 24 hours of admission, unless the case was
from environmental or occupational hazards. attended by a private physician within 36 hours
of death
• This is very important: Any death happening to
any patient within the first 24 hours is a
suspicious case, unless
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• The patient has been seen by an • Medicolegal examination of the mother who
inhouse physician sustained the trauma and complications of an
• A known case within that institute illegal abortion
2) Newborns in the first 24 hours of life • Abortion is illegal in the PH, no such thing as
• It is imperative that examination of the therapeutic abortion
possibility of why the newborn died is of great • Person behind the management of this illegal
concern medical procedure is investigated
• All personnel involved in the care, delivery, and • Investigation of the mother’s death points out to
management of the newborn infant becomes the possibility of illegal activity
part of the investigation 7) All violent deaths
• Anesthesiologist or Obstetrician gynecologist 8) All accidental deaths
are first examined 9) All sudden deaths
3) All injury cases, old or recent 10)All cases without medical attendance within 36
• Abnormal and extensive scar hours prior to the hour of death
• Presence of such abnormal scar would indicate 11)All deaths due to drowning, hanging or
that the individual sustained a recent or recent strangulation
physical injury because of a penetrating wound • A thorough investigation is undertaken if the case
• The wound is suspicious is natural or accidental
4) All deaths due to unknown cases • In extreme cases, the suspicious death would be
• Indeterminate/indeterminate causes of death even considered homicide in nature
5) All deaths due to suspicious cases
6) All abortion cases, whether self-induced or
otherwise
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12)All deaths due to shooting, stab wounds, burns, Technique of Ghon

electricity, lightning, tetanus, etc. • En bloc technique
13)All homicides
14)All suicides Technique of Letulle
15)All cases in which there is suspicion of • En masse technique
poisoning • En masse: all organs of thoracic, abdominal, &
16)Stillborns pelvic are removed at the same time
17)Prematures • Sweeping of all organs
Techniques of Autopsy Somatic Death

Technique of Virchow • Somatic death refers to death or complete
• Organs removed & dissected individually in the cessation of metabolic and functional activities of
body the organism or body as a whole.
• Most widely used method • Death of an organism (Bodily death)
• Cessation of circulation and respiration (1960s)
Technique of Rokitansky • Criteria for the pronouncement of death
• In-situ dissection in part combined with en bloc • American Bar Association and the National
technique Conference of Commissioners of Uniform State
• En bloc: by cavity Laws legislative definition of death (1980)
• Interrelated to each other • Further expanded definition of somatic
• Systemic dissection death
• Ex. thoracic cavity (lungs, heart, diaphragm), • Irreversible cessation of circulatory
respiratory system and respiratory functions or
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• Irreversible cessation of all Criteria for Death

functions of the entire brain,
including the brainstem is dead 1) Advances in resuscitation techniques that are
• “Brain dead” legally defined as capable of reviving effectively cases of clinical
Somatic death death.
• Individual who is knowledgeable in resuscitation
Three Primary Signs of Somatic Death is regarded as an individual who can save an
otherwise near death experience for a patient
Circulatory failure 2) Advanced life- sustaining equipment capable of
• Cessation of cardiac function, evidenced by maintaining cardiovascular and respiratory
absence of pulse and heart beat functions despite severe brain injury.
3) Redefinition from cessation to irreversible
Respiratory failure cessation of cardiorespiratory functions after
• Absence of oxygen and accumulation of carbon resuscitation attempts
dioxide, loss of oxidative processes necessary • Even after exhaustive resuscitation attempts
for life have been rendered
4) Brain death
CNS failure • National Institute of Neurological Diseases and
• Loss of coordination of various body functions, Stroke in the United States (1977)
loss of reflexes
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Criteria for Brain Death • EEG emits a pattern, “delta wave” pattern that is
1. Coma and Cerebral unresponsiveness indicative of coma
• When we talk of a state of coma: the individual • Criteria should be present for 30 minutes at least
is unable to be aroused by physical stimulation 6 hours after onset of coma and apnea.
2. Apnea American Academy of Neurology

• Absence of spontaneous respiration 1. Coma most obvious clinical manifestation
2. Absence of the ff:
3. Absent cephalic (brainstem) reflexes • Motor response: contraction of the skeletal
• Reflexes of the central nervous system: this can muscle when physical, chemical, electrical, and
be tested by examination of neurologic test thermal stimulation is applied
• Often a test for the 12 cranial nerves • Pupillary response to light and pupils at mid-
position: patient who is declared brain dead, the
4. Electrocerebral silence pupils will not constrict:
• The brain physiologically works in the same • Often the test done immediately after
manner as the heart pronouncing the death of the patient
• They release electrical activity in the living state • Corneal reflexes: elicited when there is a blinking
• Neurologists haver a way of determining this response when conjunctiva is lightly touched
electrophysiologic activity: brain wave test or • Caloric response: even application of extreme
EEG (electroencephalogram) thermal stimulation to the skin, there is no
skeletal muscle reflex at all
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• Gag reflex: elicited when there is any form of Medical Certification of Death
manipulation in the throat or touching the • Filling out a death certificate properly, in the
posterior portion of the tongue by any foreign proper context
material • Includes the causes of death
• Coughing in response to tracheal suctioning: • Type of category of death
deep stimulation by a foreign object to the • Many physicians don’t know how to fill out a
trachea does not elicit a response - violent death certificate
cough
• Sucking and Rooting reflexes: for infants Completing a Death Certificate
Immediate Cause of Death
Republic Act 7170 or Organ Donation Act of • The immediate cause of death is the final disease,
1991 injury, or complication directly causing death.
• As amended by Republic Act No. 7885, organ • It precedes death as a consequence of an
and tissue donations from donors who have underlying cause or causes.
been declared brain dead has been allowed. • In the case of sudden or traumatic death, the
• To donate their organ or tissues for violent act or accident is the antecedent to an
transplantation purposes with the permission for injury entered, although these two events are
the next of kin often almost simultaneous.
• Somatically dead: • Example: Congestive Heart Failure
• Legally, medically, ethically defined as
dead
• Identify potential patients who may be potential
donors
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Antecedent Cause of Death • It is the most important entry in the certificate

• The condition(s) that led to or precipitated the since mortality statistics is based on this
immediate cause of death, as recorded on a underlying cause. All certification of death must
death certificate. include an underlying cause.
• This antecedent cause, is the direct result for • Example: coronary arterial atherosclerosis
the immediate cause
• Other intervening cause (or causes) of death 1. Immediate cause of Death:
occurring between the underlying and • Congestive heart failure
immediate causes is called the antecedent • Patient died directly because of this
cause.
• Example: Myocardial Ischemia caused by 2. Antecedent cause of Death:
coronary artery disease • Myocardial Ischaemia caused by coronary artery
disease
Underlying (proximate) Cause of Death • Caused the immediate cause of death
• Defined for public health and legal purposes as
“the disease or injury that initiated the train of 3. Underlying cause of Death:
events leading to death.” • Coronary arterial atherosclerosis
• Or the circumstances of the accident or • Collectively what happened
violence which produced the fatal injury.
• Without an underlying cause, the death would
not have happened.
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Descriptions of Various Manners of Death 5) Indeterminate or Undetermined: Unknown cause?

Filed by an attending physician • Very difficult to identify
• Ex: trauma, perinatal death, drug related death
1) Natural: Death resulting from disease
• Known medical disease Poarmortem Changes
• Ex: Dying from Lobar pneumonia situation as a • Refers to a continuum of changes that occur in a
consequence of a long standing hematologic dead body following death
pathology called thalassemia • These changes include livor mortis, rigor mortis,
decomposition and taphonomy
2) Accidental: Death as a result of environmental • Taphonomy - study on how organisms
influence decay and become fossilized in the
• Ex: Pedestrian being hit, dies from massive archaeological record.
internal bleeding called hypovolemic shock
7 Secondary Signs of Somatic Death (follow after
3) Suicide: Death intentionally self-inflicted death, observed on post mortem examination)
• Challenging because it is hard to distinguish
from homicide Algor Mortis
• First demonstrable change observed,
4) Homicide: Death resulting from the deliberate characterized by cooling of the body to equalize
action of another person that of the surrounding environment
• Distinguished from murder (legally: action is • Occurring at a rate of about 7°F per hour
planned or pre-meditated or planned) • Important in establishing the approximate time of
death
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• Accelerated during cold weather, in lean, Rigor Mortis

malnourished, dehydrated individuals, after long • Rigidity or stiffening of the muscles, occurring
wasting diseases, and severe hemorrhage about 6 to 12 hours after death, and persisting for
• Slowed down in certain infectious diseases, 3 to 4 days
when death is followed by increase in • First seen in muscles of the head and neck,
temperature spreading towards the lower extremities, and
• Algor - means temperature subsequently disappearing in the same sequence
• First demonstrable change after death is • The position usually affected by the muscular
cooling of the body activity at the time of death
• Constant drop of body temperature • Rigidity of the body due to hardening of the
• At room temp: skeletal muscles caused by a series of
• 2 to 2.5 deg F/hr - 1st hour physiochemical events after death
• 1.5 to 2 deg F/hr - next 12hrs • Lack of ATP regeneration and increased acidity
• 1 deg F /hr - next 12 - 18hrs (hypoxia) result in the formation of locking
• As a rule, the body cools at 1.5°F/hr (50% of chemical bridges between actin & myosin
cases) • Due to depletion of ATP and accumulation of
• Not a reliable indicator as to the time of death lactic acid; in a dead body, the glycogen stores
• As many factors may affect the are rapidly depleted, preventing the energy
temperature of the body examples are a dependent breakage of sarcomere contraction
corpse being found on a pool, pyogenic • This interlocking is fixed and produces rigor
infection or a body of an obese person. mortis without shortening of the muscle
• Muscle interaction is left in a suspended state of
animation.
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• Sets within 2 hrs after death (head & neck) • It is important for forensic investigators to know
initially notable in the small muscles (i.e. jaw) what positioning at which the individual died.
followed by larger muscle groups (i.e. legs)
• The warmer the temperature the faster Sliding filament mechanism of the sarcomeric unit:
rigor mortis becomes fully fixed. 1) Action potential sets off a biochemical reaction
• The colder the temperature the slower that sets the myosin head to bend/ flex which
rigor mortis becomes fixed. causes a cross reaction with the actin
• Bodies must be transferred immediately myofilament. Which causes the actin to slide on
to hospital refrigerator morgue. the stationary myofilament.
• To prevent unnecessary rigor mortis. 2) Then it pulls thick and thin filament towards each
• Complete and fully fixed after approx. 6-12 hrs other. Actin myofilament moves together toward
• Dissipates after approximately 36 - 48 hours the center at the level of the H band. Once there
• Reversal of the rigidity phenomenon. is the narrowing of the so called, I band you
• Muscles become softer because of onset know have the phenomenon seen if full muscle
of the putrefaction of the muscles and contraction.
soft tissue. 3) Relaxation, ATP is needed to break the
contraction and release myosin head from actin
Antigravitational rigor mortis filaments. ATP attaches to the myosin and
• Fixed rigor mortis of the upper extremities forces it to release its crosslink with actin. Here
wherein the arms are suspended against gravity is where rigor mortis occurs because of the
indicating they were previously held in that absence of the ATP unit.
position while rigor was fixing.
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Livor Mortis • Livor mortis is spared in these areas due

• Purplish discoloration or lividity of the skin in the to localized pressure preventing blood
dependent portions of the body, due to stasis from entering the skin
and settling of blood into the dependent vessels • Showed darkened discoloration and
which usually dilate due to loss in muscle tone blanching (light areas)
• Discoloration disappears on pressure, and • Becomes evident as early as 20 min after death
reappears when pressure is released, and on • Fully evident within 4 hrs and fixed in approx. 8 -
incision, oozing of the blood is observed 12 hrs
• Aka Postmortem (Lividity) Hypostasis • Tardien spots aka:
• Aka Dependent lividity • Tardieu petechiae
• Blood supply gravitates to the skin vessels • Tardieu spots
which become toneless and dilate after
circulation ceases. Shifting the body position will not alter the
• Evident as deep purple-red discoloration in the geographic distribution of rigor mortis.
skin and internal organs This becomes an indication of the position of the
• Blood pools in capillary bed body, the time the individual died.
• Blanching occurs in gravity dependent areas of
the body that come into contact with firm
surfaces (i.e. floor, tight clothing)
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Tardieu spots Desiccation

• Tardieu's ecchymoses, subpleural spots of • Drying and wrinkling of the cornea and anterior
ecchymosis that follow the death of a newborn chamber of the eye due to the absorption of the
child by strangulation or suffocation, were first aqueous humour
described by Tardieu in 1859, and were so • After death, the skin and mucosal membranes
named in his honor. may desiccate (dry) resulting in a change in color
• Lividity is pink to purple discoloration of the skin and character of these tissues
from blood pooling in dependent areas of the • Usually a blackened or dark mummified
body. looking tissue.
• Tardieu spots are purple to black spots on the • Tache noir de la sclérotique (black spot of the
skin that can develop along with lividity, from sclera): horizontal linear scleral blackening along
the rupture of capillaries (because of increased the equator of the globe of the eye
pressure). • The sclera is exposed to drying when the
eyelids are incompletely shut
Postmortem Clotting • Tache noir is often initially red in
• Occurs slowly, immediately after death in appearance and over time becomes black
contrast to ante-mortem thrombi, postmortem • Lips, tip of tongue and scrotum darken with
thrombi show definite settling and separation of postmortem drying
the red cells from the plasma • Mummification/ drying of the skin and mucous
• Portions of the blood clot show a yellow membrane
"chicken fat" appearance, while other parts
assume the shape of the vessel where they are
found, and are called "currant jelly" clots
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Putrefaction Autolysis
• Usually noticeable after 3 days • Self-digestion of the cells, by their own ferments,
• Production of foul-smelling gases, due to the eventually undergone by all the tissues of the
invasion of the tissue by multiplying saprophytic body
organisms, associated with the following • Putrefactive bacteria which diffuse from their
changes: intestinal location into the surrounding tissues,
• Greenish blue discoloration in the belly, enhance the destruction of cells
due to the formation of iron sulfide • Post-mortem autolysis evokes no inflammatory or
• Softening of the muscles, due to auto- cellular response so characteristic of ante-
digestion mortem necrosis of cells
• Retraction of the cornea, due to • Progressive desiccation, putrefaction and
absorption of aqueous humour autolysis will eventually produce total digestion of
• Loss of rigor mortis, due to liquefaction of the soft tissues
coagulated myosin
• Peeling off of the skin, with crepitation in Techniques of Autopsy
the subcutaneous tissue and swelling of
the face Primary Autopsy Incisions (On Skin)
1. For scalp:
• Mastoid-to-Mastoid incision (Coronal scalp
incision and Sawing the skull)
Page 115
2. For Trunk: Conventional Techniques of Autopsy

• I shaped
• Y shaped • Technique of Virchow (organs removed and
• Modified Y shaped dissected individually)
• Used for medico-legal and forensic
Secondary Autopsy Incisions examinations
• The cutting of bones exposes the cavities • Painstaking, and takes several hours to
• Sawing of the skull complete the autopsy
• Cutting of the sternal plate • Technique of Rokitansky (in- situ dissection in
part combined with en bloc- technique)
Quaternary Autopsy Incisions (For Viscerae) • Organs that belong to the same cavity
• To expose the chambers of the Heart were examined in a systematic faction
• To expose the inner lungs • Organs were dissected while inside the
• To expose the inner liver cavity
• To open the urinary bladder cavity • Technique of Ghon (en bloc Technique)
• To expose the GIT lumen • Organs were removed and then dissected
• Technique of Letulle (en masse Technique)
• Removal of all organs from the thoracic,
abdominal, and pelvic cavities simply by
cutting the mesenteric attachment of these
organs.
Page 116
• All the organs are easily removed, and is Suggested Autopsy Material Retention
popular especially when the
prosector/physician is in a hurry. Material/Record (Non- Forensic Autopsy
• Organs that need not further be Records)
examined are returned judiciously into • Wet Tissue: 3 months after final report
the body. • Up to 6 months after the final report has
• Minimally invasive been released
• Needle autopsy • Paraffin blocks: 10 years
• Aspiration of blood, urine, cytology, etc., • Slides: 10 years
for analysis • Reports: 10 years
• Multiple percutaneous needle biopsies
after death (“blind biopsies”) Material/Record (Forensic Autopsy Records)
• Laparoscopic and thoracoscopic • Wet Tissue: 3 years
investigation with tissue sampling • Paraffin blocks: Indefinitely
• Mini-autopsy” - extensive organ sampling • Slides: Indefinitely
or removal via a limited incision (e.g., a • Reports: Indefinitely
15-cm upper abdominal wall incision) • Gross Photographs / Negatives: Indefinitely
• Imaging “autopsies” • Body Fluids and Tissues for Toxicology: 1 year
• MRI autopsy • Can be kept for more than a year using
• CT autopsy ultra low freezing temperatures
• Radiology-Virtopsy (“Virtual autopsy”) • Dried Blood stain or frozen tissue for DNA:
indefinitely
Page 117
03
Cell Adaptation
Page 118
Adaptation • Reversible
Physiologic • The most important aspect of cellular
• Represents cells to normal stimulation by adaptation: capacity for reversing back to
hormones/endogenous chemical substances. normal once the offending injury is
• Just the cells responding to normal stimulations removed
• Usually by hormones or endogenous • Injury is often referred to as stimuli
chemicals • Cells may undergo various adaptation in
• Example: physiological and pathological conditions
• Breast feeding • These are controlled by complex molecular
• Pregnancy mechanisms
Pathologic • Reverses back to normal once the offending
• The cells have the ability to modulate their stimulus is removed
environment.
• The environment becomes a hostile Types of Cellular Adaptations
environment, cell has the ability to respond to • Hyperplasia
adapt to that particular change to the • Hypertrophy
environment • Atrophy
• Metaplasia
Cellular Adaptation • Dysplasia
• Cellular responses to persistent sublethal injury,
physical, biological, radiation, or chemical.
Page 119
• Cells are able to adapt to changes in work

demands or threats to survival by changing their
size (atrophy and hypertrophy), number
(hyperplasia), and form (metaplasia).
• Normal cellular adaptation occurs in response to
an appropriate stimulus and ceases once the
need for adaptation has ceased.
Cell Cycle
• The life of a cell is regulated by numerous
checkpoints and proteins
• A mutation in a protein that is meant to either
slow or stop the cell cycle can cause a cell to
lose control (point when dysplastic and
metaplastic changes happen)
• Normally the number of cells produced = the
number of cells that die (unlike in cancer where
cell production overtakes cell death)
• The total number of cells in the body remains
constant
Page 120
Mitosis There are some cells that end up in the G0 phase

• Cell divides to produce 2 new daughter cells forever, some from G0 can reenter the cell cycle
• The process in which a eukaryotic cell nucleus once more
splits in two, followed by division of the parent
cell into two daughter cells. G1 phase
• The word "mitosis" means "threads," and it • The cell contents increase in number and
refers to the threadlike appearance of duplicated as the cell functions and matures
chromosomes as the cell prepares to divide. (excluding the chromosome)
• The cell grows physically and increases the
Interphase volume of both protein and organelles.
• Cell is growing, maturing and differentiating until
it reaches a point and enters a mitotic phase S phase / Synthesis phase
again • Each of the 46 chromosomes is duplicated
• Interphase is the portion of the cell cycle that is • Copying of the entire set of the 46 chromosomes
not accompanied by gross changes under the • The cell copies its DNA to produce two sister
microscope, and includes the G1, S (synthesis chromatids and replicates its nucleosomes
phase), and G2 phases.
Gap 2 phase
After mitosis, the cell either proceeds to g1 or G0 • Brief period
(meaning in the g) phase, the cell cycle simply • Involves further cell growth and organisation of
arrests cellular contents.
• Never reenters the cell cycle to divide • Cell double checks the duplicated chromosome
again for any errors
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• Making any needed repairs of necessary Cells Capable of Proliferation

• Double checks the integrity of the duplication of
your chromosomes just before the cell enters Labile cells
mitosis • Continuously dividing cells
• You know they should proliferate
• Continuously enter and reenter the cell cycle
• They have a fast and short recycling time
• These cells continue to divide and proliferate
• Seen in cells with a short life span
• Cycling is very frequent
• Example:
• Epithelial cells lining the skin
• Mucosa
• Mouth
• Excretory area
Stable cell or quiescent levels

• Low level of replication
• When stimulated they can divide
• In the gap 0 phase just the same as the
permanent cells
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• They can be stimulated to reenter the cell cycle • When damage occurs in permanent cells, healing
at the G1 phase once there is a need for the is carried out by repair (scarring)
body to replenish what has been destroyed • There is an expected decrease in function
• Cells with the capacity for regeneration • After they are produced or developed during
• Example: embryonic development, It enters the G0 phase
• Parenchymal cells of the kidney • Left the cell cycle after being produced in the
• Cells of the liver embryonic period and remains in your gap 0
• Kidney and hepatocytes are • The lost cells are replaced by healing or
stable: the number of cells reparative connective tissue
produced = number of cells that • Example:
die • Cardiac cells
• Total number of cells in the body • Neurons
remains constant
Regeneration occurs in labile and stable cells
Permanent cells
• Remain there forever
• Never reenter the cell cycle
• Remain permanent from birth to death
• No capacity to proliferate anymore
• Unable to proliferate
• Left the cell cycle
Page 123
04
Safety in the
Histopathology
laboratory
Page 124
Types of Hazards • Carcinogens

• Health hazards • Are substances that may induce tumors
• Physical risks during exposure in unrealistically higher
dosages
Health Hazards
• Biohazards Toxic Materials
• Can be infectious agents themselves or • Substances or materials capable of causing
items (solutions, specimens, or objects) death by ingestion, skin contact or inhalation at
contaminated with them certain specified concentrations
• Irritants • Sometimes termed as poisons
• Are chemicals that cause irreversible • Pose an immediate risk greater than the hazards
inflammatory effects at the site of contact • Examples:
with living tissue • Methanol
• Corrosive chemicals • Osmium tetroxide
• When exposed to living tissue, • Uranyl nitrate
destruction or irreversible alteration
occurs Target Organ Effects
• Sensitizers • Characteristic of chemicals that cause specific
• Cause allergic reactions in substantial harm to select anatomical or physiological
proportion of exposed subjects systems
• Effects are not immediately evident but are
cumulative and frequently irreversible
Page 125
• Examples: Control of Chemicals Hazardous to Health &

• Xylene Environment
• Toluene • Personal hygiene practices
• Benzene • Labeling
• Chloroform • Warning signs
• Methanol • Protective equipment
• Mercuric chloride • Ventilation
• First aid
Physical Risks • Radiation
• Combustibles have flash points at or above a • Storage of hazardous chemicals
specified temperature • Spills and containment
• Flammable have flash points below the • Recycling
specified temperature • Hazardous chemical waste disposal
• Explosive –examples picric acid
• Oxidizers initiate or promote combustion in Control of Biological Substances Hazardous to
other materials Health & Environment
• Organic peroxides are particularly dangerous • Preparation
oxidizers sometimes used to polymerized • Handling
plastic resins • Disposal of biohazardous waste
Page 126
Hazards & Handling of Common Histological Aniline

Chemicals • A very dangerous reagent, which should not be
used if possible
Acetic acid • Moderate skin and eye irritant
• Irritating to respiratory system (target organ • Carcinogen
effects) • Excessive exposure cause blue discoloration of
• Sever skin and eye irritants extremities
• Corrosive to most metals and combustible
• Use a chemical fume hood Celloidin
• Do not use latex gloves • Harmless as a health hazard but dangerously
• Always add acid to water flammable
• Do not mix concentrated acetic acid to chromic • Usually contain highly flammable ether and
acid alcohol
Acetone Chloroform
• Highly flammable and very volatile • Target organ affects liver, reproductive, fetal,
• Great risk of fir from heavy vapors central nervous, blood and GIT systems
• Not as a serious health hazard undermost • Carcinogenic
condition o fuse • Do not burn
• Can be narcotic in high concentration • Do not evaporate solvent to the atmosphere
• Skin contact can cause excessive drying and
dermatitis
Page 127
Chromic acid Picric acid

• Highly toxic with target organ effects on kidney • Toxic by skin absorption
• Corrosive to skin and mucuos membranes • Explosive when dry or complexed with metallic
• Carcinogenic strong oxidizers salts
• Do not move bottles containing dry picric acid
Ether
• Skin and eye irritant Hazard Prevention Strategies in the Laboratory
• Over-exposure to vapors can produce
disorientation, unconsciousness or death Universal Precautions
• Target organ effect on nervous system • All blood and body fluids are considered
potentially infectious
Formaldehyde
• Severe eye and skin irritant Work Practice Controls
• Sensitizer by skin and respiratory contact • Good housekeeping should include regular area
• Considered to be the most serious hazards for cleaning and daily trash emptying
most laboratory workers • Ergonomically-designed chairs and computer
• Target organ effect on respiratory system stations and facilitate safe but effective human-
• Carcinogen machine interaction
• Mouth-pipetting is prohibited.
Glutaraldehyde • Centrifuge must be kept closed and well-
• Severe eye and skin irritant balanced when used
• Toxic by ingestion • Tubes of specimens or flammable liquids must be
capped
Page 128
• Laboratory work surfaces should be Labeling and Warning Signage

decontaminated with sodium hypochlorite • All primary and secondary containers of
(bleach) or the approved work placed is infect hazardous materials must have appropriate
after a spill of blood or other body fluids and warning labels with the substance name, its
when work activities are completed manufacture’s name and address, its physical
and health hazards, and its target organs
Personal Hygiene • Signs should be posted on entrances of
• Proper hand washing before and after work laboratories to indicate the hazardous biological,
should be practiced radioactive, or carcinogenic materials used inside
• Personal hygienic habits, like covering the these areas
mouse or nose
• Smoking, eating, and drinking are prohibited in Exits and Aisles
technical work • Exits and aisles must be conspicuous and not be
• Laboratory coats must be worn while working blocked, bolted, or obstructed in any way to
• Shoes-comfortable, rubber-soled, and flat- prevent egress
should cover the entire foot
• Hair must be secured back and off the Personal Protective Equipment, Emergency
shoulders Fixtures, Barriers, Spill kits, and First Aid Kits
• Gloves must be worn when handling liquids that
can cause harm or be absorbed through the skin
Page 129
• Eye washing stations, safety showers, fire Documentation and Record-Keeping

extinguishers, smoke detectors, and alarm • All maintenance and troubleshooting activities
levers must be installed in strategic places and should be recorded in a dedicated logbook
checked regularly • Meetings, incident reports, injuries, and sentinel
• Physical barriers must be present events should be filed.
• Spill kits for biological or chemical hazards must • Temperatures of refrigerators and other
be stored in designated areas machines should be monitored and recorded
• First aid kits should be available • MSDS of all reagents should be compiled and
made accessible
Vaccination
• Vaccines must be completed Mechanical Safety
Staff Training and Awareness Sharps and Needles

• Seminars and hazard drills must be conducted • Cuts are the second most common incident in
regularly histopathology next to accident falls
• Safety manuals and pamphlets must be • Never leave blades uncapped or unguarded in
distributed the microtome
• Big laboratories may organize a committee that • When changing specimens without removing the
would meet periodically to discuss safety issues blade, cover it with a knife guard and then lock
related to the laboratory the hand wheel
• Remove and replace microtome blades with
rubber-tipped forceps or small pencil magnets
Page 130
Machines Disposal of Glass Slides and Paraffin Blocks

• Documented periodic maintenance and • Slides with unfixed tissues should be disposed of
calibration extend the life of the equipment and as biohazard waste or fixed first then discarded in
prevent mechanical malfunctions which can sharps container
cause problems • Paraffin blocks, except those exposed to CJD,
should not be considered infectious
Infection Control
• Reported agents that infect laboratory staff Classification of Proper Waste Disposal (Color
include: Coding)
• M. tuberculosis
• Hepatitis B and C Black:
• HIV • General waste
• Creutzfeldt-Jakob or prion disease • Non-infectious and dry
Green:
Disposal • General waste
• Non-infectious and wet
Strategies for Waste Handling Yellow:
• Recycling • Infectious
• Scaling down • Pathological
• Re-use or Treat-and-release • Pharmaceutical wastes
• Storing for transportation and disposal by third Orange:
party waste management companies • Radioactive wastes kept in leaded containers
Page 131
05
Quality Management
System
Page 132
Management is an important aspect of the day-to- Quality Control (QC)

day life of the histopathology laboratory • Quality control is a set of procedures or technical
particularly since the emergence of accreditation. activities on fulfilling quality requirements.
The accreditation standards include management • It is the system that checks that the work process
as part of the evaluation and it is necessary that is functioning properly and include processes in
the laboratory worker is familiar with the laboratory the laboratory to recognize and eliminate errors.
processes involved. • QC ensure that the quality of work produced by
the laboratory conforms to specified requirements
This unit discusses about quality control, quality prior to its release for diagnoses.
assurance and continual quality activities in the • Errors/ problems reported by pathologists and
laboratory as part of its quality management others should be included as part of the lab QC
system. data collection.
• QC evaluation will include, but is not limited to:
In conjunction with “quality assurance” and • Fixation
“continuing quality improvement”, “quality control” • Adequate processing
is an integral component of a required quality • Appropriate embedding techniques
system. A good QC system provides information • Acceptable microtomy
for quality assurance activities. • Unacceptable artefacts
• Inspection of controls to determine
correctness of staining and
immunohistochemistry methods
Page 133
Quality Assurance (QA) • Signatories

• Quality assurance aims to generate the • Request forms are signed by attending
confidence of the patient in the final report a physician who is requesting for the test or
shift from focus on the end product or service to examination done to the patient.
a focus on the process. • Result forms are signed by the pathologist.
• Reviewing data allows identification of declining
quality in specific areas and should trigger In specimen handling the following should be
appropriate corrective action. remembered:
• The participation in external programs/ • FIX first
schemes also contributes valuable information • LABEL
for quality assurance program • Routine Turn-over of results
• Quality assurance is “a means of getting the • Surgical pathology and cytology = 24
right test at the right time on the right specimen hours
from the right patient with the right diagnosis • Frozen section = 5-15 minutes
and at the right price”. • Autopsy report
• 1 week (autopsy procedure: 24 hours)
Quality Assurance and Documentation • Storage of specimen, tissue blocks and slides
• Histopath Reports • Specimen (tissue) = 1 month to 1 year
• Surgical pathology • Tissue blocks = 3 to 10 years
• Cytopathology report • Slides = indefinite
• Autopsy report
Page 134
Suggested Guidelines for Record and Specimen Retention

Record/ Specimen Type Retention
Records
Requisitions 2 years
Quality Control 2 years
Instrument Maintenance 2 years
Blood bank donor/recipient records Indefinitely
Blood bank employee signatures/initials 10 years
Blood bank quality control 5 years
Reports
Clinical pathology lab reports 2 years
Autopsy forensic reports Indefinitely
Surgical pathology (and BM) reports 10 years
Cytogenetics reports 20 years
Specimens
Serum/other body fluids 48 hours
Blood smears (routine) 7 days
Pathology/BM slides 10 years
Pathology blocks 10 years
Microbiology smears 7 days
BB donor/recipient specimens 7 days (post transfusion)
Cytogenetics slides 3 years
Cytogenetic diagnostic images 20 years
Page 135
Quality Management System (QMS) • Preventive maintenance of equipment

• Quality management system is a set of • Continuous professional education of staff
coordinated activities to regulate laboratory in • Documentation and control
order to continually improve the efficiency of its • Proper coordination
performance. • Timely customer’s feedback
• QMS is concerned with the following:
• Good sampling Factors Affecting the Three Phases of
• Tissue processing with quality reagents Examination
• Providing supplies and equipment • Reliability of histopathology results depends on 3
• Receiving, documenting and validating phases of examination which include pre-
results examination, examination, and post-examination
• Releasing of reports procedures.
• QMS requires good management, and • Each phase is affected by different factors:
responsibility shared from top to bottom. Its
principle goal is to guarantee cost-effectiveness Pre- Examination (Pre-Analytical Factors)
in aid of increased productivity. • Pre-examination factors include the:
• To ensure an effective QMS, histopathology lab • Collection of the right specimen
should have the following: • Proper fixation of the specimen
• Skilled histotechnologists/technicians • Correct identification of the specimen
• Proper specimen collection • Timely transportation of the specimen
• Proper processing of specimen
• Efficient processing of results
• High quality of reagents and equipment
Page 136
Examination (Analytical) Factors • Ensure that the report reaches the appropriate
• Grossing of tissues clinicians/surgeons
• Processing • Several copies may have to be made to the
• Procedure reliability using technical manuals surgeon, the attending physician, or the medical
• Reagent integrity and efficiency records keeper
• Cutting of paraffin sections • Filing of paraffin blocks must be done in a cool
• Staining area to prevent them from melting together and
• Slide labeling being consumed by vermin. Blocks maybe stored
• Equipment reliability for at least 10 years
• Adequate calibration • The following guidelines should be kept in mind:
• Proficiency of personnel and continuous • Slides are stored for 10 years, while
updating of their knowledge - Good internal reports can be stored for even longer in
quality control safe or humidity-free locations
• Any possible remarks on the diagnosis
Post-Examination (Post-Analytical Factors) obtained should also be indicated
• Pathologist records the findings and diagnosis • Frequent dialogues between the
on the correct requisition slip clearly pathologist and the surgeons for the right
• The following guidelines should be kept in mind: diagnosis of results obtained in the
• Render histopathologic diagnosis (hard copy or laboratory should also be done
electronic) free of clerical errors by checking for
laterality, the correct patient’s name, and other
demographics
Page 137
Factors Influencing the Quality of Reports in

the Different Phases of Examination
Page 138
Quality Management System • Create an orientation program for a new

• QMS also consists of management personnel
responsibility and human resource training. • Finalize and maintain job description for all
personnel
Management Responsibility • Organize a training program for new
• QMS will not be successfully implemented personnel and ongoing training for all
without the support of the managers of the other personnel as needed
organization • Periodically assess individual competence
• Ensure laboratory complies with legal and • Establish to maintain and control records
regulatory requirements of qualification, experience, training and
• Quality manual is the most important document competence assessment
in lab that describes in detail the policies and
procedures to implement the QMS Physical Facilities
Human Resource Management Reagents and Supplies

• Every laboratory shall:
• Have an adequate number of qualified or Delivery of Service
trained, and competent staff to ensure
efficient and effective delivery of quality
services
• Establish policies and procedures on
recruitment and appointment
Page 139
Equipment • Provides the pathologist with properly processed

• Acquisition or replacement of equipment and stained slides which are labeled and
• Installation of equipment presented in the right order and free of dirt,
• Identification of equipment fingerprints, paraffin, or excess mounting medium
• Operation of equipment
• Calibration and validation Continual Quality Improvement (CQI) Activities
• Preventive maintenance • Continual quality improvement is a system used
• Inspection trouble shooting and repair proactively to approach, evaluate, and identify
opportunities to improve quality before problems
Responsibility of the histotechnologist / occur through evaluation of all systems /
histotechnician processes in the laboratory.
• Ensures that formalin solutions are fresh and of • Its goal is to improve patient care and safety
the appropriate pH through recognition of potential problems/errors
• Regularly filters reagents, especially before they can occur.
hematoxylin • The lab should establish, implement and maintain
• Maintains equipment in high quality condition a system for quality improvement and must also
and conducts preventive maintenance do the following:
• Ensure tissue processors with tissue sections • Prepare policies and procedure on QMS
are not overloaded and CQI
• Work systematically to minimize errors • Draft a written quality plan that must be
• Analyze the problems as they occur and implemented.
corrects them • The plan should be monitored and
reviewed regularly
Page 140
• Develop a system to improve customer

service
• Measure customer satisfaction by
surveying, collating, analyzing, and
resolving all complaints
• File all records of complaints from the
customers
• Participate in EQAS/NEQAS
Document Control Management

• Document everything in the lab. No document
means it did not happen.
• List of records in the lab:
• Request forma
• Patient’s report
• Incidence report
• Safety records
• Communication with patients
• Telephone reports
• Preliminary reports
• Final reports
• Corrected reports
Page 141
06
Instrumentation
Page 142
In the preparation of tissue blocks and slides for Microtomes

histopathological study, several materials or • Microtome is a tool used to cut extremely thin
equipment are needed. slices of material, known as sections.
• The cut tissue is floated over a water bath, in
These are very essential in the handling, grossing order to eliminate wrinkles and distortion in the
and surgical cutting, and processing of tissue tissue, and picked up on a slide.
specimens to come up with tissue blocks and
slides. Thus, this unit introduces the material and
equipment used in the histopathology laboratory
Histology is the study of tissues, especially their

microscopic anatomy, and pathology is the study
and diagnosis of disease. Histology and pathology
are sciences that are often used together in
biology and medical laboratories.
Histology and pathology laboratory equipment are

tools that help to prepare and examine tissues.
Page 143
Types of microtome: Sliding Microtome

• Holds the block in a stationary position, and the
Rotary microtome knife is moved along a horizontal plane past the
• Paraffin blocks move up and down and either block face
the blade holder or he block advances by preset • Not used for routine histopathology
number of micrometers
• Commonly used for sectioning glycol
methacrylate and paraffin-embedded material
• Found on most cryostat machines
Page 144
Clinical Freezing Microtome Microtome blades

• A portable microtome clumped into a tabletop • Replace microtome steel knives
• Carbon dioxide supplied to a chuck freezes the • Have edges superior to knives
tissue placed on a horizontal plane • Classified as either high-profile (for tough
• Disadvantages: not suitable for friable tissues, materials or for frozen sectioning) or low-profile
airborne disease transmission s more likely to blades (sections < 3 um), distinction dictated by
occur, section thickness is not exact the type of blade holder used
• Replace by cryostat in most laboratories • Angle used is approximately is 3-8 degrees
Tissue Processing Equipment

• Fluid transfer (closed system) type of processor:
most commonly used
• Tissue is stationary and fluids are pumped in and
out of the pressurized chamber holding the tissue
• Advantages of close systems: reduction of
exposure to toxic vapors, and specimens not
drying out in the tissue chamber in case of
malfunction
• Some routine processors use heat and vacuum
with agitated and spinning movements
Page 145
Embedding Center
• Typically composed of a paraffin reservoir, a
work stage for orientation of specimens, cold
plates/chilling plates, and a warm storage for
embedding molds
Microwave Processing Equipment

• Results to better antigen preservation
compared with formalin
• Can be applied for histohemical staining for light Glasswares
and electron microscope, preservation of • Include staining racks, Coplin jars, and staining
cryostat sections, and immunohistology dishes that are resistant to most common
staining reagents
• Graduated cylinders (50 ml to 1000 ml)
Page 146
Accessory Equipment
• pH meters
• Weighing scales
• Freezers and refrigerators
• Flotation baths
• Gross lab/ gross table
• Basic features of the gross lab: sink,
stable table top, water supply, irrigation
Paraffin Dryers and Ovens system, fume extraction system/
• Convection ovens ventilation system, waste disposal unit
• Maintained at temperature just above the
melting point of paraffin (60C)
• Complete drying of slide: 1 hour in conventional
oven
• 15 minutes in forced-air slide dryers
Incubator ovens
• Used for many enzyme reactions, in situ
hybridization techniques and some special
stains
• Maintained at 37℃ Once samples have been processed and are ready
for analysis, microscopes are used to analyze the
tissues.
Page 147
07
Examination of
Tissues
Page 148
The methods of tissue examination may vary 3. Smear Preparation

according to the structural and chemical • Cellular materials are spread lightly over a slide
components of the cells to be studied, the nature by means of a wire loop or applicator, or by
and amount of the tissue to be evaluated and the making apposition smear with another slide. It is
need for an immediate examination of a tissue useful in cytologic investigations.
structure. • Streaking - material is applied gently in a zigzag
line on a slide using a applicator stick or platinum
This unit discusses the different methods of tissue loop
examination using preserved or fresh specimens. • Spreading - a selected portion of material is
gently spread into moderately thick film by
Methods of Fresh Tissue Examination teasing the mucous strands apart with an
applicator stick
1. Teasing or Dissociation • Recommended for thick mucoid secretions
• Selected tissue specimen is immersed in a • Pull-apart - dispersion of secretion or sediment
watch glass containing isotonic salt solution, placed upon one slide facing another slide
carefully dissected or separated, and examined through pulling apart the slides in opposite
under the microscope directions
• Touch preparation - done by allowing cells of
2. Squash Preparation freshly cut tissues to be transferred directly to the
• Tissues not more than 1 mm are placed in a slide through contact
microscopic slide and compressed with another
slide or cover slip
Page 149
4. Frozen Section 3. Clearing

• Is use for rapid diagnosis of tissue and for lipid • Alcohol is removed in the tissue by immersing in
and nervous tissue demonstration. In this a clearing agent.
technique the tissue (10-15u) is frozen on a
microtome with carbon dioxide or a cryostat 4. Infiltration
• Tissue is then placed in melted paraffin until it
Processing of Preserved Tissue becomes completely infiltrated with the
substance.
1. Fixation
• Small pieces of tissues are immersed in a 5. Embedding
solution called fixative after removal from the • The paraffin- infiltrated tissue is placed in a mold
body. with melted paraffin and allowed to harden
• To avoid tissue digestion by enzymes present • Note: The medium used in infiltration of tissue is
within the cells or bacteria the same medium used for embedding.
• To preserve cell and tissue structure
6. Trimming
2. Dehydration • The resulting paraffin block is trimmed to expose
• Tissue is transferred through a series of the tissue for sectioning (slicing) on a microtome.
increasingly concentrated alcohol solutions
called as dehydrating agents, ending in 100%, 7. Section-Cutting
which removes all water. • Tissue block is sliced into thin films using a
microtome which are placed on glass slides and
allowed to adhere, deparaffinized, and stained.
Page 150
8. Staining
• Methods of staining have been devised that not
only make the various components conspicuous
but also permit distinctions to be made between
them.
• Cell components with a net negative charge
(anionic) stain more readily with basic dyes and
are termed basophilic
• Cationic components have affinity for acidic
dyes and are termed as acidophilic
9. Mounting
• Stained tissue slides are mounted with a cover
slip using a mounting media.
10. Labeling
• Tissue slides are labeled on the frosted areas
with assigned tissue numbers or codes
• Tissue slides should be properly labeled
Page 151
08
Fixation
Page 152
In the fields of histology, pathology, and cell Fixation

biology, fixation is the preservation of biological • First and most critical step in histotechnology
tissues from decay due to autolysis or putrefaction. • Chemical process by which biological tissues are
It terminates any ongoing biochemical reactions preserved from decay
and may also increase the treated tissues' • Process of preserving cells and tissue
mechanical strength or stability. constituents in a LIFE-LIKE manner
In this topic, we will be discussing everything • Chemical process by which the tissues are
about tissue preservation, types of fixative, factors preserved from decay
affecting fixation and the commonly used agents • Decay is either by putrefaction or autolysis
for fixation of tissues.
Principle of Fixation
Fixation is the first and most critical step in tissue • The fixative brings about crosslinking of proteins
processing. It is a complex series of chemical which stabilizes protein to maintain tissue
events which brings about changes in the various morphology
chemical constituents of cell like hardening,
however the cell morphology and structural detail The Aims and Effect of Fixation
is preserved. 1° aim
• To preserve the morphologic and chemical
Unless a tissue is fixed soon after the removal integrity of the cell in as life-like manner as
from the body it will undergo degenerative possible, stopping all cellular activities
changes due to autolysis and putrefaction so that • Prevents degradation, decomposition,
the morphology of the individual cell will be lost. putrefaction
Page 153
2° aim • Putrefaction
• To harden and protect tissue from the trauma of • The breakdown of tissue by
further handling so that it is easier to cut during bacterial action often with formation
gross examination of gas.
• Hardening a tissue increases the • Further decomposition after death
mechanical strength and stability of the due to bacterial or fungal
tissues, for easier cutting and sectioning overgrowth
• Bacterial decomposition brought
3° aim about by microorganisms which
• To prevent postmortem changes like autolysis may already be present in the
and putrefaction. specimen.
• Autolysis
• Is the lysis or dissolution of cells • Preservation of chemical compounds and
by enzymatic action probably as a microanatomic constituents so that further
result of rupture of lysosomes. histochemistry is possible- to coagulate or
• Results from tissue digestion by precipitate protoplasmic substances
intracellular enzymes that are
released when organelle Solidification
membranes rupture • Converts the normal semifluid consistency of
• Postmortem decomposition cells (gel) to an irreversible semisolid consistency
• Due to action of hydrolytic (solid).
enzymes
Page 154
Optical differentiation • Make cellular components insoluble to hypotonic

• It alters to varying degrees the refractive indices solutions and render them insensitive to
of the various components of cells and tissues subsequent processing
so that unstained components are more easily • Permit the subsequent application of many
visualized than when unfixed. staining procedures to facilitate easier and more
profitable examination
Effects of staining
• Certain fixatives like formaldehyde intensifies 2 Basic Mechanisms in Tissue Fixation
the staining character of tissue especially with
haematoxylin. Additive Fixation
• Chemical constituent of the fixative is taken in
Characteristics of a Good Fixative and becomes a part of the tissue
• Cheap • Forms crosslinks and gives stability to proteins
• Stable • Non-coagulant cross linking fixatives
• Safe to handle • Chemical constituent taken into the cell, forming
• Kill the cell quickly molecular complexes and stabilizing proteins
• Inhibit bacterial decomposition and autolysis • Ex:
• Produce minimum shrinkage of tissue • Formalin
• Permit rapid and even penetration of tissues • Mercury
• Harden tissue • Osmium tetroxide
• Isotonic
Page 155
Non-additive Fixation • Microwave fixation

• The fixing agent is NOT taken in, but changes • Widely used
the tissue composition and stabilizes the tissue • Glyoxal based fixatives
by removing the bound water attached to • Relatively high temperatures up to 56C but
hydrogen bonds of certain groups within the for relatively short periods
protein molecule • Cryo-preservation
• Fixing agent is not incorporated into the tissue • Freeze drying & freeze substitution
• Dehydrant coagulant fixatives
• Alteration of tissue composition by removing Chemical methods of fixation
bound water molecule at Hydrogen bonds • Immersion fixation and perfusion fixation
within protein molecules • Coagulant fixatives
• Stabilizes proteins by forming cross links • Non-coagulant cross linking fixative
after water molecule removal
• Ex: Benefits of Fixation
• Alcoholic fixatives • Allows sectioning of tissue by hardening tissue
• Prevents autolysis and inactivates infectious
Types of Fixation agents
• Improves cell avidity for special stains
Physical methods of fixation
• Heat fixation
• Rarely used on tissues
• Mainly smears
Page 156
Factors Affecting the Quality of Fixation • Buffering capacity in the fixative will
prevent the excessive acidity
Buffers and Hydrogen ion concentration (pH) • Acidity favors formation of formalin-heme
• Satisfactory fixation occurs at pH 6 – 8. pigment that appears black, polarizable deposit in
• Outside this range changes may occur tissues
detrimental to ultrastructural preservation of
tissues Temperature
• Precipitation of formalin pigments • Fixation of surgical specimens are done at room
• Common buffers include phosphate, temperature
bicarbonate, cacodylate, and veronal • This can be followed by further fixation at
• Hypoxia of the tissues lowers the temp up to 40 to 45°C
pH so there must be a buffering • Electron microscopy (EM) & some histochem: 0 –
capacity in the fixative to prevent 4°C
excessive acidity • Except for mast cells: room temperature
• pH <5.7 brown-black insoluble crystalline • General rule:
birefringent pigment forms • Increasing the temperature, increases the
• pH 3-5 increase pigment formation rate of diffusion into the tissue and speeds
• pH 7- optimal pH for buffering formalin up the rate of chemical reaction between
• High acidity decreases effectiveness the fixative and tissue elements
• More hydrogen concentration is equal to lower • Nucleic acids do not react with fixatives at room
pH or higher acidity temperature
• If there is tissue hypoxia, this lowers the pH • Chemical reactions are rapid at high temperature
• You need a buffer (60 to 65°C)
Page 157
Thickness of Section Osmolality and Ionic composition

• Generally, the thicker the tissue the loner the • Hypertonic or hypotonic solutions lead to
fixation time, and the thinner the tissue the shrinkage and swelling respectively
faster it is. • Isotonic and Hypotonic fixatives cause swelling
• Formalin penetrates 3-4 mm /hr and poor fixation
• NBF penetrates 1 mm/hr • Best result are obtained at slightly hypertonic
• Desired thickness of tissue for electron (400-450 mOsm)
microscope: 1-2 sq. mm • 10% NBF- 1500 mOsm
• Desired thickness of tissue for light microscope: • Ionic composition of fluids should as isotonic as
2 sq. cm or not more than 0.4cm possible
• It is done to abstain full penetration and
satisfactory fixation Concentration of fixative
• Sections should be no more than 3 mm • Formaldehyde: 10%
thick • Glutaraldehyde: 3%
• Sections should not touch the top and • Presence of buffer causes polymerization of
bottom of tissue processing cassette aldehyde, with consequent decrease in its
• Brain is usually suspended whole in 10% effective concentration
buffered formalin for 2 to 3 weeks to ensure • 0.25% Glutaraldehyde:
fixation and some hardening prior to sectioning • For immune electrochemistry
Page 158
Duration of Fixation Fixation is enhanced by:

• Ideal time to perform fixation after interruption of • Size and thickness of tissue specimen- the
blood supply: 20 to 30 minutes smaller and thinner the tissue the faster the
• Usual fixation time: 24 hours fixation time.
• Primary fixation in buffered formalin is usually • Agitation
carried out for 2 to 6 hours during the day the • Moderate heat (37-56 C)
specimen is obtained
• When surgeons remove an organ, they must Practical Considerations of Fixation
put it in formalin right away
• Prolonged fixation may cause shrinkage and Speed
hardening of the tissue and may severely inhibit • Specimen should be placed in a fixative= solution
enzyme activity and immunological reaction as soon as it is removed from the body to prevent
autolysis and putrefaction
Fixation is retarded by the following: • Putrefaction:
• Presence of mucus • Process of decay or rotting in a body
• Presence of fat
• Presence of blood Penetration
• Cold temperature • Most fixatives diffuse into the tissue at the rate of
1 mm per hour and slows down as it goes deeper
in the tissue
Page 159
Volume Factors to Consider in Selecting Fixatives

• This tells you how much of a tissue should be • Need for immediate examination
submerged into how much of a fixative • Tissue structure or component to be studied
• 10 to 25 times the volume of the tissue to be • Type of tissue to be processed
fixed • Staining technique to be applied
• The maximum effectiveness of fixation is noted • Type of section to be made
to be 20 times the tissue volume
• Correct Fixative to Tissue Ratio Difficulties Encountered Because of Improper
• Ideal ratio is at 20:1 Fixation
• Failure to arrest early autolysis
Duration of Fixation • Removal of substances soluble in fixing agent
• Fibrous organs take longer than small or loosely • Presence of artefact pigments on tissue sections
textured tissues such as biopsies or scrapings • Tissues are soft and feather-like in consistency
• Fixation tie can be cut down by using heat, • Loss or inactivation of enzymes needed for study
vacuum, agitation or microwave • Shrinkage and swelling of cells and tissue
structure
Consequences of Delayed, Incomplete, or Poor • Tissue blocks are brittle and hard
Fixation
• Loss or total disappearance of nuclear
chromatin
• Disappearance of some cells
• Cell shrinkage with artifactual space around
cells
Page 160
Troubleshooting Fixation Problems • Change the formalin solution frequently to

prevent depletion
Prevention of autolysis • Do not pack tissue section tightly in the cassettes
• Minimize cold ischemia time • Use agitation of cassettes in formalin holding
• Ensure adequate ratio of fixative to specimen solutions during or after grossing
• Uterus must be opened by the bivalve
technique to expose endoterium Reactions of Cellular Components to Fixation
• GIT specimens must be opened and pinned to
corkboard or Styrofoam sheets to expose Nucleus
mucosa • Formalin does not react well with either DNA or
• Lymph nodes for possible lipoma must be RNA unless fixation temperature is elevated at
bisected, sectioned at 3 mm intervals 65C for DNA and 45C for RNA
• Formalin fixation of the nucleus results in the
Recommendation for incomplete fixation formation of nuclear bubbling
• Increase time in fixative solution
• Change to another fixative (zinc formalin, Proteins
gloyoxal) • Fixation disrupts tertiary configuration of protein
• Place formalin-alcohol in the first three stages by changes that occur with protein bonds
of the processing cycle • Reactions of Cellular Components to Fixation
• Ensure that grossed sections are < 3mm for
good reagent penetration
• Amount of fixative is 15-20 times that of the
tissue
Page 161
Lipids • Formalin is widely used because of number of

• Generally lost during the procedure factors:
• Fixative of choice: osmium tetroxide and • High speed of diffusion in tissue
chromic acid • The capability to preserve the shape and
structure of samples
Carbohydrates • Provide long term fixation
• Some carbohydrates are lost during fixation • The mechanism of fixation is based on the
• Glycogen is preserved due to entrapment in interaction of formalin with tissue proteins
fixed proteins • It reacts with the hydrogen in amino acids
and forms methylene bridges between
Steps of Histologic Technique: Fixation them
• Fixation is necessary to preserve the native • It’s important to use 10% formalin, since this
structure of the tissue concentration ensures the good quality of fixation
• If the tissue is not fixed, the autolysis will start, • If the concentration is too low, then the tissue will
and the structure will change because of not be fixed
enzymatic decomposition • If it is too high, then the outer later of the samples
• There are many different fixative solutions, but will be fixed too quickly and will become too
the golden standard in histology is 10% neutral dense so the formalin molecules will have no
buffered formalin opportunity to penetrate the tissue
• Most methods of histochemistry are validated • As a result, the inner later will stay raw and the
for formalin-fixed tissues autolysis will start
• Formalin is a 40% aqueous solution of
formaldehyde stabilized with methanol
Page 162
• Importance of using neutral buffered formalin • 2 Steps of Fixation

• If the concentrated formalin is just diluted • When the whole organ is fixes in formalin
with water. during tis transportation to the laboratory
• This solution will be acidic. • When the samples are fixed after grossing
• Excision of tissue with such solution will • If the material was not fixed
lead to formation of formalin pigments properly in the 1st step, it is very
(black or brown spots) on samples which important to comply all the
will obstruct diagnostics conditions on the 2nd step
• To make the solution neutral, it is • Besides the use of 10% neutral buffered formalin
necessary to add some salt for the buffer. of good quality, there are several other conditions
• Example: which are important for proper fixation
• Sodium hydrophosphate • Thickness of the samples after grossing
• Dihydrophosphate should be about 3 to 4mm
• The white sediment can be formed in • It will provide the good penetration of the
formalin solution because of the storage formalin in the tissue and all parts of the
and the low temperature. materials will be fixed
• This is formalin polymer- • The volume rate of the material and the
paraformaldehyde fixative solution should be 1 to 20
• It is not recommended to use • Example:
formalin with sediment for • If your sample has a volume of 675
histological study uL then you need to add a certain
and a half mL of fixative solution
Page 163
• If you have 20 samples then you Classification of Fixatives

need to use 270mL of formalin
• Another important factor is the duration A. According to Composition
of fixation Simple Fixatives
• 24 hours is enough for good • Made up only of one substance (One component
quality only)
• Some methods have restrictions • Glacial Acetic Acid - it solidifies at 17°C
on formalin fixation • It fixes nucleoprotein, destroys
• Example: Special stains for lipids mitochondria and Golgi bodies
can be done only in frozen • Causes tissue to swell
sections • Aldehydes
• Always pay attention on what is • Formaldehyde
written in the instructions • Glutaraldehyde
• Fixation is the first step in histological study, • Metallic fixatives
that’s why all the mistakes will lead to the loss • Mercuric Chloride
of diagnostic value of the sample • Chromate Fixatives
• Lead Fixatives
• Picric Acid
• Acetic Acid
• Acetone
• Alcohol
• Osmium tetroxide
• Heat
Page 164
Compound Fixatives • 2007 = American Society of Clinical Oncology

• 2 or more simple fixatives to obtain optimal (ASCO) & the College of American Pathologists
combined effect (CAP) released guidelines to improve accuracy of
• Combination of simple fixatives Her-2 testing for invasive breast cancer which
• Zenker’s fluid - composed of glacial acetic acid recommend that tissue for Her-2 testing be fixed
and mercuric chloride in 10% neutral buffered formalin for a minimum of
• Glacial acetic acid causes tissue to swell 6 hours and a maximum of 48 hours.
• Mercuric chloride causes tissue to shrink
Cytologic fixatives
B. According to Action • Chemical preserve specific parts of the
Microanatomical cytoplasm specifically microscopic elements
• General microscopic study of tissue structures • Example:
• Examples: • When you want to see the mitochondria
• 10% Formol Saline clearly, then there are specific fixatives for
• 10% Neutral Buffered Formalin it.
• Heidenhain’s Sus • Preserve specific parts and particular
• Formol Sublimate (Formol Corrosive) microscopic elements
• Zenker’s Solution
• Zenker-Formol (Helly’s Solution) Cytoplasmic Fixatives
• Bouin’s Solution • Preserves cytoplasmic structures in particular
• Brasil’s Solution • Does NOT contain Glacial HAc
• pH > 4.6
Page 165
• Examples: Histochemical fixatives

• Flemming’s fluid without Glacial Acetic • Preserves chemical constituents of cells and
Acid tissues
• Helly’s fluid • Example:
• Formalin with post-chroming • 10% formol-saline
• Regaud’s fluid (Moller’s Fluid) • Absolute ethyl alcohol
• Orth’s fluid • Acetone
• RNA: • Newcomer’s fluid
• Ethanol
• Acetone Lipid Fixation
• Used to preserve fat droplets in the cell
Nuclear fixatives • Largely removed during processing
• Preserve nuclear structures • Use frozen sections and special stains
• pH ≤4.6 • Aldehydes can be used in general
• Glacial acetic acid as primary component
• Flemming’s fluid Carbohydrate Fixation
• Carnoy’s fluid • Alcoholic fixatives for better retention
• Bouin’s fluid
• Newcomer’s fluid
• Heidenhain’s susa
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Fixatives in Order of Decreasing Speed of Formaldehyde

Penetration • Formed from oxidation of methanol
1. Formaldehyde • High concentration (37-40%)
2. Acetic Acid • Tend to over harden the outer layer of the tissue
3. Mercuric chloride and affect staining adversely
4. Ethyl/Methyl Alcohol • Should be diluted 1:10 or 1:20 to make 10% or
5. Osmium tetroxide 5% solution or combined with another
6. Picric Acid • Fixation time: 24 hrs
I. Aldehyde Fixatives 10% Formalin

Mechanism
• The aldehydes form cross-links between Advantages
proteins, creating a gel, thus retaining cellular • Cheap, readily available, easy to prepare
constituents in their in vivo relationships to each • Relatively Stable
other • Compatible with many stains
• When you put tissue in a formalin • For Routine Paraffin Sections, Electron
(formaldehyde) it forms crosslinks on Microscopy, Histochemistry, and Enzyme Studies
how it interacts with hydrogen ions on • Pigments removed by treatment with alcoholic
the amino acid on tissue and then form picric acid
crosslinks known as methylene bridges • It preserves fat and mucin, making them resistant
• Give some mechanical strength to the entire to subsequent treatment with fat solvents, and
structure which enables it to withstand allowing them to be stained for demonstration
subsequent processing • It preserves glycogen
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• It preserves but does not precipitate proteins, 10% Formol Saline

thereby allowing tissue enzymes to be studied • Made from saturated formaldehyde diluted to
• It does not make tissues brittle and is therefore 10% with NaCl
recommended for nervous tissue preservation. • For CNS tissues and general post mortem
• It allows natural tissue colors to be restored tissues for histochemical exam
after fixation by immersing formalin-fixed • 24 hours at 35°C or 48 hours at 20-25°C
tissues in 70% alcohol for one hour, and is • 10% NaCl solution
therefore recommended for colored tissue • The solution should have a pH of 6.8
photography
• It allows frozen tissue sections to be prepared Advantages
easily. • Preserves microanatomic and cytologic details
• It does not require washing out, unless tissues with minimum shrinkage and distortion
have stayed in formalin • Preserves enzymes and nucleoproteins
• For excessively long periods of time • Demonstrates fats and mucin
• Does not over-harden tissues; facilitating
Disadvantages dissection of the specimen
• Fumes irritating to nose and eyes
• Solution is irritating to skin Disadvantages
• May produce considerable shrinkage of tissues • Like formaldehyde with the following additions
• It is a soft fixative and does not harden some • Slow fixative
cytoplasmic structures adequately enough for • Fixed tissues tend to shrink during alcohol
paraffin embedding dehydration
• May be reduced by secondary fixation
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• Metachromatic reaction of amyloid is reduced Advantages

• Acid dye stains less brightly than when fixed • Fast penetration
with mercuric chloride • Non-coagulant and additive
• Notes: • Prevents alterations during processing
• Formaldehyde 40% = 100 mL • Less shrinkage than other fixatives
• NaCl = 9 gm • Not osmotically active
• Distilled water = 900 mL • Hardens tissue better (except alcohol and
• Sodium dihydrogen phosphate acetone)
monohydrate = 4 gm • Tissue can be stored in formalin indefinitely
• Disodium hydrogen phosphate (except for IHC)
anhydrous = 6.5 gm • Fixative of choice for immunohistochemistry and
molecular tests
10% Neutral buffered formalin • Cheap and stable
• Most commonly used and BEST general tissue
fixative Disadvantages
• Preservation and storage of surgical, • Takes longer to prepare
postmortem and research specimens • Positivity of mucin to PAS is reduced
• Usually buffered with phosphate buffer (pH=7.2- • May produce gradual loss I basophilic staining of
7.4) cells
• Contains iron pigments • Reactivity with myelin to Weigert’s iron
• Fixation time: 4 to 24 hours hematoxylin stain is reduced
• 10% NBF is the optimal choice of fixative • Inert towards lipids, especially neutral fats and
phospholipids
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• Notes: • Fixes lipids, especially neutral fats and

• Sodium dihydrogen phosphate = 3.5 gm phospholipids
• Disodium hydrogen phosphate = 6.5 gm • Brightens cytoplasmic and metachromatic stains
• Formaldehyde 40% = 100 mL
• Distilled water = 900 mL Disadvantages
• Slow penetration
Formol-Corrosive (Formal-Sublimate) • Tissue sections should not be more than
• Fixes fats and lipids 1cm thick
• Saturated aqueous Mercuric Chloride as diluent • Forms mercuric chloride deposits
• Fixation time: 3 to 4 hours • Does not allow frozen tissue sections to be
• Recommended for routine postmortem tissues made
• Inhibits the determination of the extent of
Advantages tissue decalcification
• Penetrates small pieces of tissues rapidly • Notes:
• Produces minimum shrinkage and hardening • Sat. aq. Mercuric Chloride = 90 mL
• Excellent for many staining procedures • Formaldehyde = 10 mL
including silver reticulum methods
• Cytological structures and blood cells are well Glutaraldehyde
preserved. • For enzyme histochemistry and EM studies
• There is no need for “washing-out”. • Should be stored at 4C and pH 5
• Tissues can e transferred directly from • 2 concentrations
fixative to alcohol
Page 170
• 2.5% for small tissue fragments and needle • This may be prevented by immersing
biopsies/aspirates glutaraldehyde-fixed tissues in a mixture of
• Fixation time: 2 to 4 hours concentrated glacial acetic acid and aniline oil
• Room temperature
• 4% solution for large specimens (<4mm thick) Formol Calcium
• Fixation time: 6 to 8 hours up to 24 hours • For preservation of fats and lipids
Advantages Karnovsky’s paraformaldehyde-glutaraldehyde

• For routine light microscopy and EM solution and Acrolein
• Preserves cellular structures better • For electron cytochemistry
• Recommended for electron microscopy
• Less irritating to nose and skin II. Metallic Fixatives
• Does not cause dermatitis Mechanism
• Produces less tissue shrinkage • Mercuric Chloride and Lead Fixative’s Mercuric
Salts bind with sulfhydryl groups in acidic
Disadvantages solutions
• More expensive • Chromate fixatives in water form Cr-O-Cr
• Less stable complexes that have an affinity for -COOH and -
• Slow penetration OH groups of proteins, so that complexes
• Tends to make tissue (renal biopsy) more brittle between adjacent molecules are formed
• Reduces PAS positivity of reactive mucin. • This leads to free amino group for eosin binding
Page 171
Mercuric Chloride • Notes:

• Mercuric Chloride = 5 gm
5-7% Mercuric chloride • Potassium Dichromate = 2.5 gm
• Most commonly used METALLIC fixative • Sodium Sulfate = 1 gm
• Included in most compound fixatives • Distilled Water = 100 mL
• Nuclear components are show in fine detail
• Excellent Trichrome staining Zenker’s fluid (with Glacial HAc)
• Pigments removed by treatment with iodine • Good fixative for blood (congested) sepcimens
solution in 95% alcohol • For trichrome staining
• For fixing small pieces of liver, spleen, CT fibers
Advantages and nuclei
• Permits brilliant metachromatic staining of cells • With Glacial Acetic Acid for affinity to nuclear
• Routine fixative of choice for tissue photography chromatin
• Mercuric chloride with Glacial acetic acid
Disadvantages • Permits brilliant staining of nuclear and
• Produce black granular deposit (except for connective tissue fibers
Heidenhain Susa w/c is removed by De- • Fixation time: 12 to 24 hours
Zenkerization) • Lyses RBC and can make tissues brittle
• Corrode metal (except nickel alloy) • Stock Solution = 95 mL
• Decrease the amount of demonstrable glycogen • Glacial Acetic Acid (Before Use) = 5 mL
• Causes considerable lysis of cell
• Causes tissue shrinkage
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Zenker-formol (Helly’s solution) B-5 Fixative

• For pituitary gland, BM extramedullary • For bone marrow, lymph nodes, spleen and
hematopoiesis, blood containing organs and hematopoietic tissues
intercalated discs • Commonly used for bone marrow biopsies
• Example: • Fixation time: 1 to 2 hours
• Spleen • Unstable
• Liver • Can make tissues brittle
• With 40% formaldehyde (5mL) • Good clear nuclear details
• Preserves cytoplasmic granules well • Notes:
• Distilled Water = 90 mL
Heidenhain’s Susa Solution • Mercuric Chloride = 6 gms
• Recommended for Tumor biopsies of skin • Sodium Acetate Anhydrous = 1.25 gms
• Reason why it is recommended for tumor • Add 1cc Formaldehyde for 10cc of above
biopsies:
• Excellent cytologic fixative or fixation Chromate Fixatives
structures • Appear as yellow brown deposits
• Penetrates and fixes tissue rapidly and evenly
• With: 1-2% Chromic Acid
• Trichloroacetic Acid • Strong oxidizing agent hence strong reducing
• Glacial Acetic Acid agent must be added (formaldehyde)
• 40% Aldehyde • Usually constituent of a compound fixative
• Fixation time: 3 to 12 hours • Compound fixative utilizes more than one type of
your simple fixative
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• Preserves carbohydrates & precipitates all Orth’s fluid

proteins • 2.5% Potassium Dichromate
• May produce sub oxide precipitates • 40% Formaldehyde
• Fixation time: 36 to 72 hours
3% Potassium Dichromate • Recommended for study of Early Degenerative
• Preserves lipids processes and Tissue Necrosis
• Preserves mitochondria • For Rickettsia and other bacteria
• Fixes mitochondria at pH 4.5 – 5.2
Lead fixatives
Regaud’s fluid (Muller’s fluid) • Generally for Acid MPS
• Recommended for demonstration of chromaffin • For CT mucin
tissues (phaeochromocytoma), mitochondria,
mitotic figures, Golgi bodies, RBC, and colloid - 4% Lead Acetate
containing tissues • Recommended for acid mucopolysaccharides
• Fixation time: 12 to 48 hours • Fixes connective tissue mucin
• Penetrates well • Takes up CO2 to form insoluble lead carbonates
• Unstable on standing
• Poor nuclear staining
• Notes:
• 3% Potassium Dichromate = 80 mL
• 40% Formaldehyde = 20 mL
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III. Picrate Fixatives Bouin’s solution

• For glycogen demonstration • Yellow stain is useful for fragmented biopsies
• Dyes tissues, but the yellow color can be • Minimal distortion of microanatomical structures
removed by treatment with another acid or • Excellent general fixative for connective tissue
lithium carbonate or washing with 50-70% stains
ethanol • For embryos and pituitary biopsies
• Highly explosive when dry • For fatty tissue:
• Must NEVER be washed in water before • Bouin’s solution (75 ml) plus 95% ethanol
dehydration (25ml)
• Mechanism is still unknown • Require 48 hours for good sections of lipomas or
• Picrates are soluble in water liposarcomas
1% Picric acid solution Disadvantage

• Penetrates and fixes small tissue rapidly • Destroys membranes, intact nuclei cannot be
• Excellent for Glycogen Demonstration recovered
• Suitable for aniline stains • Notes:
• Causes RBC hemolysis • Picric Acid = 75 mL
• Not suitable for frozen sections: • Formaldehyde = 25 mL
• Causes it to crumble when cut • Glacial Acetic Acid = 5 mL
• Prolonged fixation makes tissue hard
Brasil’s Alcoholic Picroformol Fixative
• For glycogen
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IV. Glacial Acetic Acid • Alcohol fixative destroy hydrogen bonds in amino
• Used in conjunction with other fixatives with acids.
other fixatives to form a compound solution • To stabilize tertiary structure of proteins.
• Solidifies at 17°C
• Fixes and precipitates nucleoproteins, 100% Methanol
chromosomes, and chromatin materials • Recommended for fixing dry and wet smears,
• For Nuclear Component studies blood smears and bone marrow tissues
V. Alcohol Fixatives Advantages

Mechanism • Preserves but does not fix glycogen
• Rapidly denatures and precipitates proteins by • Fixes blood, tissue films and smears
destroying hydrogen bonds to stabilize the • Preserves nucleoproteins and nucleic acids,
tertiary structure of proteins hence, is used for histochemistry, especially for
• Must be used in concentrations (70 to 100% ) enzyme studies
• Lesser concentrations produce cell lysis • Fixes tissue pigments fairly well
• Used both as fixative and dehydrating agent • It is ideal for small tissue fragments
• POLARIZATION is the general disadvantage • May be used both as a fixative and dehydration
movement of glycogen granules towards the agent
ends/poles of cells
• Aldehyde fixatives form cross-link or bridges Disadvantages
methylene bridges by interacting with the • Hemosiderin preservation is less than in buffered
hydrogen of the amino acids. formaldehyde
Page 176
• A strong reducing agent 70-100% Ethanol

• Should not be mixed with chromic acid, • Most commonly used smear fixative for
potassium dichromate and osmium exfoliative cytology
tetroxide which are strong oxidizing • May be used as simple fixative
agents • Usually incorporated into compound fixatives for
• Lower concentrations (70 to 80%) will cause better results
RBC hemolysis and inadequately preserve • Fixes blood, tissue films, and smears
leukocytes • Preserves nucleoproteins and nucleic acids for
• Dissolves fats and lipids, as a general rule. histochemistry and enzyme studies
• Alcohol-containing fixatives are • Dissolves fats and lipids
contraindicated when lipids are to be • May shrink tissues
studied • Fixation time: 18-24 hours
• Causes glycogen granules to move towards the
poles or ends of the cells (polarization) Isopropanol 95%
• Tissue left in alcohol too long will shrink, making • For touch preparations
it difficult or impossible to cute
• Causes polarization of glycogen granules Carnoy’s fluid
• Produces considerable hardening and • Most RAPID fixative (1 – 3 hours)
shrinkage of tissues • For chromosomes, lymph glands and urgent
biopsies
• Also for brain tissue and diagnosis of rabies
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Advantages • Slow penetration

• The most rapid fixative • Dissolves acid-soluble cell granules and
• Fixation time: 1 to 3 hours only pigments
• Fixes and dehydrates at the same time
• Used to fix brain for diagnosis of rabies • Notes:
• Permits good nuclear staining and • Absolute Alcohol = 60 mL
differentiation • Chloroform = 30 mL
• Preserves Nissl granules and cytoplasmic • Glacial Acetic Acid = 10 mL
granules well
• Preserves nucleoproteins and nucleic acids Newcomer’s Fluid
• Excellent fixative for glycogen since aqueous • For MPS and nuclear proteins
solutions are avoided • Both nuclear and histochem fixative
• Very suitable for small tissue fragments such as • Fixation time: 12-18 hours at 3C
curetting’s and biopsy materials • Isopropanol, propionic acid, petroleum ether,
• Following fixation for one hour, tissues may be acetone, dioxane
transferred directly to absolute alcohol- • Produces better reaction in Feulgen stain
chloroform mixture, thereby shortening • Recommended for fixing mucopolysaccharides
processing time
Alcoholic formalin (Gendre’s fixative)
Disadvantages • Also called alcoholic Bouin’s
• May cause RBC lysis • 95% ethanol sat. with picric acid
• Shrinks and may harden tissues excessively • 40% formaldehyde
• Glacial Acetic Acid
Page 178
• Useful for sputum: it coagulates mucus • Prevention:

• Cause better retention of carbohydrate • Addition of saturated aq. Mercuric chloride
(glycogen) in tissues • Remedy:
• Fixation time: between 4 hours and overnight • Black osmic oxide crystals may be
followed by washing in 70% ethanol, followed dissolved in cold water
by 95% ethanol • PRECAUTION:
• May cause conjunctivitis or blindness
VI. Osmium Tetroxide Fixatives
Mechanism Flemming’s solution
• Various hypotheses of lipid stabilization have • Most common chrome-osmium acetic acid
been postulated fixative
• Oxidation of double bonds between adjacent • For nuclear preparation of sections
carbon atoms to form monoesters and diesters • With Glacial Acetic Acid for Nuclear affinity
• Binding of lipid to protein • With 1% Chromic Acid as diluent
• Conversion of unsaturated fatty acids to stable • Fixation time: 24 to 48 hours
glycol osmates • Recommended for nuclear preparation of such
• Should be kept in DARK-COLORED, chemically sections
clean bottle to prevent evaporation and • Permanently fixes fat
reduction by sunlight and organic matter • Excellent fixative for nuclear structures
• Inhibits hematoxylin and makes counterstaining • Example: Chromosomes
difficult
• Produces BLACK PRECIPITATE (Osmium
oxide)
Page 179
• Expensive VII. Trichloroacetic Acid

• Has a tendency to form artifact pigments; these • Incorporated into compound fixatives
may be removed by washing the fixed tissue in • Poor penetrating agent, suitable for small pieces
running tap water for 24 hours before of tissues or bones only
dehydration • It Precipitates proteins
• Softening Effect on Dense Fibrous Tissues
Flemming’s Solution without Acetic acid • May be used as a weak decalcifying agent
• Made up only of chromic and osmic acid
• For cytoplasmic structures (mitochondria) VIII. Acetone
• The removal of acetic acid improves the • Used at ice cold temperature ( -5°C to 4 °C)
cytoplasmic detail of the cell • For the study of water diffusible enzymes
(phospatases and lipases)
6% Osmium tetroxide • Used in Fixing Brain Tissues for diagnosis of
• Fixes conjugated fats and lipids permanently by Rabies
making them insoluble to alcohol and xylene • Volatile
during dehydration and clearing • Dissolves fat
• Preserves cytoplasmic structures well • Shrinks tissues excessively
• Fixes materials for Ultrathin Sectioning in
Electron Microscopy
• Very Expensive
• Penetrates poorly
• Forms black precipitates upon exposure to
sunlight
Page 180
IX. Physical Methods of Fixation • Tissue are cut into thin sections, immersed in
liquid nitrogen, and water is removed in a
Heat Fixation vacuum chamber at -40 C
• Involves Thermal Coagulation of tissue protein • Tissue can be post-fixed with formaldehyde
• For Rapid diagnosis vapor
• Employed for Frozen Tissue Sections
and Bacteriologic Smears Freeze Substitution
• Primarily used to accelerate other forms of • Specimen are immersed in cold fixatives such as
fixation as well as the steps of tissue processing acetone or alcohol, then temperature will brought
in histopathology gradually to 4 C to complete fixation
Microwave Fixation X. Chemical Fixation

• Speed fixation and can reduce times for fixation
from more than 12 hours to < 20 minutes Coagulant Fixatives
• Microwaving tissue in formalin results to large • Organic and non-organic solutions that coagulate
amount of dangerous vapors proteins making them insoluble, thus, maintaining
• Efficient method of microwave fixation: cellular architecture
• Glyoxal-based fixatives heated at 55 C • Disadvantage:
• Result in cytoplasmic flocculation as well
Freeze Drying as poor preservation of mitochondria and
• Useful technique in studying soluble materials secretory granules
and small molecules
Page 181
Dehydrant Coagulant Fixatives Compound fixative

• Most commonly used coagulating fixatives: • Useful for specific tissues
alcohols and acetone • Unreacted aldehyde groups in glutaraldehyde-
• Disrupts tertiary structure of protein causing formaldehyde fixation
insolubility and loss of function • Increase background staining
• Examples: • Alcoholic formalin for fatty tissues such as breast
• Ethanol coagulation fixation: 50-60% • Also for lymphnodes embedded in fats
conc. • Good in preserving antigen
• Methanol coagulation fixation: 80% conc. immunorecognition
• Acetone - up to 13% of protein may be • Cause non-specific staining of myelinated
lost nerves
• Other examples include picric acid and
TCA Secondary Fixation
• Secondary fixation is the process of placing an
Non-coagulant Cross linking Fixatives already fixed tissue in a second fixative to
• Form crosslinks within and between proteins facilitate and improve the demonstration of
and nucleic acids as well as between nucleic particular substances, to act as mordant and to
acids and proteins ensure further and complete hardening and
• Sometimes called covalent additive fixatives preservation of tissues.
• It is done before dehydration and on
deparaffinized sections before staining.
• The pimary fixative used is 10% formalin or 10%
formol saline
Page 182
Post-Chromatization Fixation for Selected Individual Tissues

• Post-chromatization is a form of secondary
fixation where primarily fixed tissue is placed in Eyes
aq. solution of 2.5 – 3% potassium dichromate • May be fixed in NBF for 48 hours
for 24 hours. • For 24 hour, cut the globe in one or two small
• The post-chroming agent acts as mordant and windows avoiding the retina and iris
is also used for the purpose of cytologic • After gross description, components of the globe
preservation. are fixed for an additional 48 hours or more in
buffered formaldehyde
Washing Out • Embedded in celloidin or paraffin
• Washing-out is used in removing excess fixative
from tissue after fixation to improve staining and Brain
to remove artifacts from tissues. • Conventional fixation: 2 weeks
• Examples of agents used for wahing-out are the • Fixative may be enhance by using microwave
following: • Alcoholic method should not be used if biotin-
• Tap water - remove excess chromates, avidin immunohistochemistry is to be performed
excess formalin, excess osmic acid
• 50-70% alcohol - remove excess amount Testis
of picric acid (Bouin’s Solution) • Routinely fixed with NBF
• Alcoholic iodine - remove excess • Lymphoid tissue
mercuric fixatives • Special care should be taken as many organisms
may sequester themselves in the reticular system
• Fixed with NBF, or B5 or zinc
Page 183
Lungs Principle and Precautions in Handling and

• Typically fixed in NBF Fixation of Specimens
• Gross sections are fixed overnight • Autopsy material should be fixed as soon after
• Fixation for Selected Individual Tissues death as possible. If not possible, should be
placed in mortuary refrigerator or undergo arterial
Muscle Biopsies embalming.
• Portion is separated for enzyme histochemistry • Surgical specimens should be fixed as soon as
• For routine histology, fixed with NBF and possible or refrigerated
embedded • All tissue specimens must be properly labelled
• Stained with H&E and identified
• Congo red if amyloid is suspected • If tissue specimens are refrigerated, slow
freezing of unfixed tissue near 0C must be
Renal Biopsies avoided.
• Routine histology: Neutral Buffered Formalin • Avoid repeated freezing and thawing.
(NBF) or Carson’s modified Millonig’s • Tissue slices must be taken right angles to the
• Ultrastructural analysis: Carson’s modified surface of the organ
Millonig’s fixative or 2% buffered glutaraldehyde • Tissue should not be >5mm thick except in lung
• Immunofluorescence examination: commercial edema
transfer solution. • Purulent materials, exudates or transudates
should be marked and kept for possible
bacteriologic examinations
• Amount of fixative must be adequate
Page 184
09
Decalcification
Page 185
Decalcification describes the techniques used for • Unless the tissues in completely decalcified the
removing mineral from bone, or other calcified sections will be torn and ragged and may
tissue, so that high-quality paraffin sections can be damage the cutting edge of microtome knife.
prepared. • Adjust the hard substance of bones to the
softness of paraffin embedding medium
In this topic, we will be learning about the • Bones
principles of decalcification, different methods and • The main object of decalcification in a
agents used, and methods in measuring the surgical pathology laboratory
extent of decalcification. • But other specimens, such as teeth,
calcified tumors
Decalcification • Enables the histotechnologist to cut soft sections
• Is a process of complete removal of calcium salt of the bone using the microtome, so that they can
from the tissues like bone and teeth and other be processed like any other soft tissue of the
calcified tissues following fixation. body
• Removal of calcium ions or lime salts from the • Fine detail radiographs: often used to assist in
organic extracellular matrix, calcified collagen, the selection of appropriate bone specimens for
and surrounding tissues of the bone. processing
• It is done after fixation and before impregnation • If the calcified areas in tissue specimens are
• Decalcification is done to assure that the substantial, it may be impossible to obtain decent
specimen is soft enough to allow cutting with sections without first decalcifying the entire
the microtome knife. specimen
Page 186
• One alternative is to apply “surface

decalcification” to a paraffin block, allowing
sections to be obtained where the presence of
calcium was not anticipated when the specimen
was processed
• Lengthy procedure: as bone pieces have to be
left in the decalcifying agent for several days or
even weeks, depending on the size of the tissue
• Many of the grossing and cutting techniques for
bone require the use of a high-speed saw An unstained ground section of compact bone. A
and/or long periods in a decalcifying solution, number of osteons (Haversian systems) are cut
prior to reducing the specimen to a size that can transversely. The osteons consist of concentric
be easily processed, embedded, and sectioned layers of bone (lamelle) surrounding a central
• The poor quality of thin sections obtained from Haversian canal containing blood vessels. Within
these methods contribute to the already difficult the lamelle are lacunae (spaces) normally
task of evaluating the pathology and making a containing osteocytes. In dried ground sections
correct diagnosis such as this they appear as black structures due to
• A low speed saw may be sufficient to routinely particles of abrasive and air contained within them.
and rapidly reduce undecalcified surgical
specimens of hard tissue, to a thickness of 2 to • As mineralized bone is such a hard material,
3mm, without compromising the integrity of the there is a limited range of techniques available to
tissue produce sections from it
Page 187
• The same final effect makes 14% ethylene • Hence, one must see to it that all such
diamino tetracetic acid (EDTA) an ideal extraneous materials have been removed
chelating agent that sequestes metallic ions, before proceeding to the next step in the
including calcium, in aqueous solutions tissue processing
• It is also possible to prepare bone specimens
by infiltrating them with acrylic or epoxy resins General Rule
which when polymerized, have a hardness • Specimens should be FIXED first before being
equivalent to that of mineralized bone decalcified
• Do not require decalcification at all • Fixation before decalcification
• Decalcification should be done after fixation and • Because unfixed specimens submerged in
before impregnation, to ensure and facilitate the • Buffered formalin is a satisfactory fixative for
normal cutting of sections and to prevent bone but where the preservation of bone marrow
obscuring the microatomic detail of such is important, some laboratories use alternatives
sections by bone dust and other cellular debris. such as:
• Inadequate decalcification may result in • Zinc formalin mixtures
poor cutting of hard tissues and damage • B-5
to the knife edge during sectioning • Formol-acetic alcohol (Davidson’s fixative)
• There are certain specimens e.g., bones, teeth, • Bouin’s solution
and other calcified tissues like tuberculous • In order to protect the cellular and fibrous
lungs, which contain some amount of calcium elements of bone from damage caused by the
that is apt to interfere with the accurate acids used as decalcifying agents, it is
evaluation and examination of histologic particularly important to thoroughly fix these
sections. specimens prior to decalcification
Page 188
• Unfixed specimens that are decalcified tend to • Coarse saws can cause considerable
be macerated (masisira) mechanical damage and force bone
• Poorly-fixed specimens become fragments into the soft tissues present in
macerated during decalcification and the specimen
stain poorly afterwards
• This is very noticeable in areas containing bone Tissues that are Decalcified:
marrow. • Bone specimens (e.g. femur)
• It is therefore common practice for • Calcified tissues
laboratories to extend fixation times for
bone specimens before commencing Not decalcified:
decalcification. • Naturally hard tissues like toenails are not
• It is to penetrate the bone, so skin and decalcified
soft tissue should be removed from large • Cartilage generally is not decalcified
specimens if practicable. • There are cartilaginous organs that are hard, we
• Bone specimens should be sawn into only decalcify them if really needed
thin slices as soon as possible to
enhance fixation and an adequate Steps of Decalcification
volume of fixative provided 1) To ensure adequate fixation and complete
• High-quality fine tooth saws should be used to removal of the calcium it is important that the
prepare bone slices. slices are 4-5 mm thick. Calcified tissue needs
2-3 hours only, for complete decalcification to be
achieved so it in necessary to check the
decalcification after 2-3 hours.
Page 189
1) Fixative of choice for bone or bone marrow is Factors Affecting Rate of Decalcification
Zenker formal or Bouin's fluid. Unfixed tissue
tends be damaged 4 times greater during Concentration and Volume of the agent
decalcification than a properly fixed tissue. • Higher concentrations of active agent decalcify
bone more rapidly but are more harmful to tissue
Notes to Remember!!! • General rule: the more concentrated the agent,
• Ideal bone thickness: 1-3 mm the faster it will decalcify the tissue
• Ideal length of time for decalcification: 24-48 • Disadvantage:
hours • The more rapid the decalcification the
• Recommended fluid to tissue volume for tissue is more prone to shrinkage
decalcification: 20:1 • Preferred: combination of fixative and acid
• Optimum temperature: RT (18° to 30 °C) decalcifier
• Volume = 20:1(like fixative)
Types of Specimens
• Amputated limbs Fluid access
• Resected specimens • The tissue should be surrounded by the agent in
all surfaces to ensure adequate entry
• Surrounded on all sides
• If the tissue is in contact to the bottom of the
contained, then that portion is not being
penetrated by the agent, thus the rate of the
decalcifying agent is affected as well
Page 190
Temperature Suspension
• Increase temperature will hasten decalcification, • Decalcification may be hastened by suspending
but it will laso increase the damaging effects if the tissue in decalcifying solution for greater fluid
acids on tissue. access.
• At 37° C
• Impaired nuclear staining of Van Tissue size
Gieson’s statin (collagen fibers) • Generally, larger tissues take longer to be
• Penetration becomes faster but can decalcified
cause tissue damage • The amount would determine the rate if
• The temperature should be controlled decalcification, including the concentration
• At 55 ° C • The more concentration, the faster the
• Tissue undergo complete digestion decalcification
within 24 – 48 hours
• Optimum temperature: 18 to 30C Decalcification Process
Acid Decalcification
Agitation • Acid decalcifying agents are the most widely
• Accelerates the rate of diffusion and speeds up used agents for routine decalcification of large
decalcification process amounts of bony tissues because they are stable,
• Gentle agitation may hasten the penetration of readily available, and relatively inexpensive as
the agent into the tissues compared to other decalcifying agents.
• Disadvantage:
• Agitation can also cause tissue damage
if done improperly
Page 191
• As soon as fixation is complete, the selected Strong Acid

pieces of tissues are usually placed in a gauze • Hydrochloric or nitric acid at concentrations up to
bag and suspended in liberal amounts of 10% are the most rapid in action
decalcifying solution by means of a thread to • But if used longer than necessary will
ensure complete decalcification and to protect rapidly cause a loss of nuclear staining
the tissue from any precipitate that might be and can macrate tissues
settled at the bottom of the container. • It is important that an appropriate end-point test
• Due to the corrosive action of the acid, it is used to minimize exposure of the specimens to
is recommended that the thread be these agents
dipped in melted paraffin wax and that • Generally, proprietary decalcifiers that are
use of metal cap containers be avoided. claimed to be rapid in action are based on strong
acids, most commonly hydrochloric acid, and
Methods of Decalcification should be used conservatively with attention to
• Acids the provided instructions if good results are to be
• Chelating method obtained
• Ion exchange resin method • Rapid decalcifying agents are more likely to
• Electrolysis adversely affect any subsequent staining
• Decalcifying Agents • Especially noticeable in cell nuclei (acidic
solutions make it hard for nuclear
Types of Decalcifying Agents chromatin to take up hematoxylin and
• Strong acids other basic dyes)
• Weak Acids
• Chelating agents
Page 192
• Acidic dyes aren’t as affected as basic Nitric Acid

dyes (although eosin can produce a • Most common and fastest decalcifying agent
deep, brick red stain without differential • Recommended concentration = 5 to 10%
shading) • For routine purposes
• Theses effects on H&E can be corrected • Combined with formaldehyde or alcohol
with post-decalcification and removal,
and by appropriately adjusting the Advantages
staining procedure • Rapid: Good for urgent biopsy
• Produces minimal tissue distortion
• Recommended for routine processing
• You can meet the 3 to 4 day turnaround time
using nitric acid
Disadvantage
• Inhibits nuclear stains and destroy tissues
(concentrated sol’s)
A section of decalcified cancellous bone (H&E).
This specimen was decalcifie with a hydrochloric
acid decalcifier for an excessive time without using
an appropriate endpoint test. Although the tissue
elements are cohesive the staining is very poor
showing a complete absence of nuclear staining
together with strong, poorly-differentiated eosin
Page 193
• Causes problems for staining the cell nuclei due • Acid is easily removed by 70% alcohol
to its acidic nature • Recommended for urgent biopsies
• The more acidic, the milieu of your tissue
section is, the hematoxylin will have Disadvantages
trouble entering the nuclei • Prolonged immersion can cause tissue distortion
• This is why formalin is neutral: to not • Seriously damage tissue sustainability (because
make the nucleus more acidic than it of the acidic nature)
already is (acidic cause of DNA: Nucleic • Imparts a yellow color
ACID) • Might destroy antigens in the cell.
• Usually the routine hematoxylin we use • Important in immunohistochemistry
is the Harris Hematoxylin: enters your purposes
nuclei the best when it is not acidic
Notes:
Aqueous Nitric Acid Base Solution • Concentrated nitric acid = 10mL
• Decalcification time: 12 to 24 hours • Distilled water = 100mL
• Aq. Nitric Acid 10%
• For urgent biopsies Formol Nitric Acid
• Decalcification time: 1 to 3 days
Advantages
• Rapid Advantages
• Causes minimal distortion of tissues • Rapid: recommended for urgent biopsies
• Produces good nuclear staining as long as the • Relatively good nuclear staining
procedure is properly monitored
Page 194
• Produces less tissue destruction compared to Advantages

aqueous nitric acid • Recommended for routine purposes
• Decalcifies and softens tissues at the same time
Disadvantages • Nuclear and cytoplasmic staining is good
• Imparts a yellow color which can interfere with • Maceration is avoided due to the presence of the
the staining procedure ethanol and chromic acid
• This is prevented by neutralizing the tissue with
5% sodium sulfate Disadvantages
• Addition of 0.1% urea solution in the nitric acid • Decalcification is slow for dense bones therefore
can lessen this yellow discoloration it is not recommended for urgent biopsies
• Irritant: should be used using a fume hood • Complete decalcification cannot be determined
by a chemical test
Notes:
• Concentrated nitric acid = 10mL Notes:
• Strong formaldehyde (40%) = 5mL • 10% nitric acid = 40mL
• Distilled water = 85mL • Chromic acid (0.5%) = 30mL
• Absolute ethanol = 30mL
Perenyi’s fluid
• Decalcification time: 2-7 days
• Acts as both tissue softener and decalciying
agent
Page 195
Phloroglucin-Nitric Acid • However, it produces better nuclear staining

• Decalcification time: 12 to 24 hours • For surface decalcification of tissue blocks (1%
sol’n with 70% OH)
Advantages
• Most rapid decalcifying agent: Von Ebner Fluid
• Good for urgent biopsies • For teeth and small pieces of bones
Disadvantages Advantages
• Poor nuclear staining • Permits relatively good cytologic staining
• Tissue distortion if not monitored properly • Moderately rapid
• Impart yellowish color • Does not require washing out prior to dehydration
• Completeness of decalcification cannot be • Recommended for teeth and small pieces of
determined by chemical means bone
Notes: Disadvantage
• Concentrated nitric acid = 10mL • Extent of decalcification cannot be determined by
• Phloroglucin = 1g a chemical test
• 10% nitric acid = 30mL • Saturated aqueous solution of NaCl 50mL
Hydrochloric acid Notes:

• Inferior to nitric acid due to its slower action and • 36% hydrochloric acid 8mL
greater tissue distortion • Distilled water 50mL
Page 196
Weak Acids • Recommended for routine decalcification and

• Weak acids, such as formic acid, are popular postmortem specimens
and are widely used for decalcification. • Both fixative and decalcifying agent
• Organic acids such as acetic and formic acid • For routine decalcification of postmortem
are better suited to bone marrow, since they are research tissues
not as harsh. • Most suitable for decalcification of long bone
• However, they act more slowly on dense • Safer to handle
cortical bone. • Only weak acid used as primary decalcifying
• Other acids such as trichloracetic acid (TCA) agent
have also been used. • Addition of sodium citrate or sodium
• Picric acid and acetic acid are not used alone formate accelerates decalcification by
as decalcifying agents but are found as chelating the calcium
components of Carnoy's and Bouin's fixatives.
• These fixatives may act as incidental, albeit, Advantages
weak decalcifiers, and can be used in urgent • Can be used both as a fixative and decalcifying
cases when there is only minimal calcification. agent
• It permits excellent nuclear and cytoplasmic
Formic acid staining
• Decalcification time: 2 to 7 days • Recommended for small bones and teeth
• Moderate acting decalcifying agent • Suitable for routine use especially if
• Easier to handle compared to nitric and immunohistochemistry is contemplated
hydrochloric acid
Page 197
• It is slower than the strong acid agents, but it is • 10% formalin-formic Acid
much gentler in action and less likely to • For very small bones
interfere with nuclear staining • Fixative and decalcifier
• Formic acid in a 10% concentration is the best • 5% Formic Acid
all-around decalcifier • Best GENERAL decalcifying agent
• Addition of citrate probably accelerates
decalcification by chelating the calcium as it is Advantages
liberated from the bone. • Permits better staining
• Better than without citrate no sht
Disadvantages • Recommended for autopsy specimens, bone
• Relatively slow in action marrow, cartilage, and tissues studied for
• Requires neutralization of 5% sodium sulfate research purposes
and washing out to remove acid from the tissue
Disadvantages
Notes: • Relatively slow
• Formic acid (sp.gr 1.20) = 10mL • Turnaround time 14 days (2 weeks)
• Normal saline = 90mL • Requires neutralization by 5% sodium sulfate
Formic Acid + Sodium Citrate Solution Notes:

• For autopsy materials, BM, cartilage and • Aqueous sodium citrate 20% 50mL
tissues for research purposes • Formic acid 50mL
• Decalcification time: 3 to 14 days
Page 198
Trichloroacetic Acid and Picric acid Chromic acid (Flemming fluid)

• Weak decalcifying agents • Both fixative and decalcifying agent
• Needs stronger concentrations • PRECAUTION:
• Decalcification is carried out with large • Environmental toxin
quantities (10% Aq. Solution) • Highly corrosive
• For good nuclear staining • Carcinogenic
• Components in Carnoy’s and Bouin’s fixatives
Advantages
Advantages • Can be used both as a fixative and decalcifying
• Permits good nuclear staining agent
• Does not require washing out. • Good for small bone fragments
• Since it has osmium tetroxide and is used
Disadvantages for small bone fragments then it may be
• Weak decalcifying agent. used for electron microscopy
• Not recommended for dense tissues.
• Recommended for small pieces of bone tissue. Disadvantages
• Not for urgent biopsies. • Nuclear staining with hematoxylin is inhibited.
• Tends to undergo reduction and forms
precipitates.
• Insoluble pigments are formed when decalcified
tissue is dehydrated with alcohol.
• Thus tissues should be washed out first
before dehydration.
Page 199
• Degree of decalcification cannot be determined Citric Acid – Citrate Buffer Solution

by chemical testing • Decalcification Time: around 6 days
• About 5 to 6
Chromic Acid Warning • pH = 4.5
• Environmental Toxin
• Carcinogen Advantages
• Technician/Technologist should have proper • Permits excellent nuclear and cytoplasmic
PPE when handling chromic acid. staining
• Has a special way of disposal. • Does not produce tissue or cell distortion
• This chemical is not simply thrown down
the drain of a sink. Disadvantages
• Special methods of disposal should be • Too slow for routine purposes.
employed.
Chelating Agents
Notes: • Substances that combine with calcium ions and
• Chromic acid = 15mL other salts to form weakly dissociated complexes
• Osmium tetroxide = 4mL and facilitate removal of calcium salts.
• 2% glacial acetic acid = 1.0mL • If preservation of nuclear DNA is important, or if
histochemical methods for nucleic acids or
enzyme activities are intended, a chelating agent
is preferred to an acid.
• Usually the disodium salt of EDTA is used, with
the pH adjusted to a level between 7 and 8.
Page 200
Ethylenediamine tetraacetic acid (EDTA) • Work by capturing the calcium ions from the
• EDTA – most common chelating agent surface of the apatite crystal, slowly reducing its
• MOA: Combines with calcium forming an size.
insoluble non-ionized complex. • Because the process is very slow but very
• Excellent bone decalcifier for immunohistochem gentle (weeks may be required depending
and EM on the size of the specimen), this reagent
• Not recommended for urgent and routine is not suitable for urgent specimens but
purposes more appropriate for research applications
• Slow decalcifying agent where very high quality morphology is
• Same as the ones we used in lavender top tube required or particular molecular elements
but this time it is used for decalcification must be preserved for techniques such as
• Chelating agents are the preferred agent if: IHC, FISH or PCR.
• Preservation of nuclear DNA is the goal • It is used at a concentration of
• Enzyme activity is going to be assessed approximately 14% as a neutralized
• Tissue is going to be processed for solution.
research/academic purposes • The rate at which EDTA will decalcify is
• Since we don't want it to be distorted pH dependent.
• Main disadvantage: Slow decalcifier • It is generally used at pH7.0.
• Not suitable for urgent work • It works more rapidly at pH 10 but some
• Requires monitoring tissue elements can be damaged at
• Equipment used needs to be cleaned alkaline pH. 10
and monitored
Page 201
Neutral EDTA • Preferred decalcifier if the tissue will undergo

• Rate of decalcification depends on the pH tests such as immunohistochemistry, enzyme
• Optimal pH: 7 to 7.6 studies or electron microscopy.
• Works more rapidly at pH10 but some • Usually for research and academic use
tissue elements can be damaged at
alkaline pH Disadvantages
• Does not work in formic acid with pH 3 • Very slow decalcifier (takes weeks)
as a decalcifier yie jianna usto mo yan • Causes slight tissue hardening
• Inactivates alkaline phosphatase activity which
Advantages can be restored using magnesium chloride
• Permits excellent staining results
• Minimal tissue distortion Notes:
• Minimal artifact formation • EDTA disodium salt = 250g
• Extent of decalcification can be determined by • Distilled water = 1750mL
chemical means • Bring to pH 7.0 by adding sodium hydroxide =
• So we can easily see the end point 25g
• pH of the medium can be adjusted
• EDTA works too slowly under pH 5,
owing to insolubility, but over pH 8,
tissue maceration starts due to alkaline
sensitive protein bonds.
Page 202
Other Techniques to Increaase Efficiency of • The resin is used up over time.

Decalcification • Once there are no more H+ or OH-, then
the resin has been exhausted
Ion Exchange Resin (IER) • The resin must be regenerated
• Ammonium form of polysterene • HCl solution is used to remove the cations
• Hastens decalcification by removing Ca ions bounded in a cation resin
from formic acid-containing decalcifying • Cation resin
solutions. • The cations are replaced by H+
• Increasing solubility from the tissue over time
• In the ion exchange process, the resin is ready • NaOH solution is used to remove the anions
to exchange ions from the liquid bound in an anion resin
• The ions (cations or anions) are attracted by the • Anion resin
resin • The anions are replaced by OH-
• Cation resin over time
• Cations such as Na, K, Ca, & Mg • Once all the cations (Na, K) or anions (Cl, PO4)
are replaced by H+ ions, reducing are removed from the resin, the resin has been
the pH of the liquid whey regenerated
• Anion resin • The resin can now be used again
• Anions such as Cl & PO4 are • IER hastens decalcification by removing calcium
replaced by OH- ions, increasing ions from formic acid-containing solutions thereby
the pH increasing solubility from the tissue
Page 203
• It is not recommended for fluids containing Disadvantages

mineral acids such as: • Degree of decalcification cannot be measured
• Nitric acid using chemical means
• Hydrochloric acid • The endpoint is also hard to know since it cannot
• A layer of the resin about ½ inch thick is spread be measured by chemical means
over the bottom of the container to be used and • Very slow.
the specimen is placed on top of it • Not recommended for routine use.
• The decalcifying agent is then added. • Slow decalcification
• The amount of the agent is 20-30 times • Causes slight tissue hardening
the volume of the tissue.
STEP ACTION
• The tissue is immersed for 1-14 days.
1 • Spread ½ inch-thick ion exchange resin
• Degree of decalcification can be assessed
over the bottom of container
using xray.
2 • Place the specimen on top of it
• No bones should be seen in the x-ray
3 • Add decalcifying agent (20 –30 times the
volume of tissue)
Advantages
4 • Allow tissue to stay in the solution for 1
• Cellular detail is preserved
–14 days
• Daily washing of solutions is eliminated
5 • Measure the decalcification using the
• Permits excellent staining results
physical or X-ray method
• Hastens decalcification
• Produces minimal tissue distortion
• Forms minimal artifacts
Page 204
Electrophoresis • It shows that this method somehow hastens the

• Positively charged Ca ions are attracted to a decalcification process.
negative electrode and subsequently removed • Temperature should be monitored
from the decalcifying solution • This method does not adversely affect cell
• The principle is similar to chelating agents. morphology under the microscope
• Histologic details are sometimes not preserved.
• Utilizes electricity Measuring Extent of Decalcification
• For small bone fragments • Frequent monitoring is required to insure that
bone tissue is taken from the acid solution as
Notes: soon as all calcium is removed from the
• Formic acid 88% = 100mL specimen, and before the tissue becomes
• Concentrated HCl = 80mL completely macerated.
• Distilled water = 1000mL • Tissue decalcified for long time periods or in high
acid concentration are more likely to show the
Microwave Over Decalcification effects of over-decalcification.
• Uses heat • Prolonged decalcification of tissue is liable to
• A tissue immersed in a decalcifying agent (e.g. prevent hydrolysis and lead to maceration and
nitric acid) and then put inside a microwave destruction of tissue components which are
oven. poorly stained.
• The solution is heated intermittently.
Page 205
• Over- decalcification, particularly with the strong Physical/Mechanical Test

acid decalcifiers, spoils the staining of • Inaccurate, done by touching or bending the
basophilic elements such as cell nuclei and in tissue with fingers.
certain circumstances can cause maceration of • Decalcified tissue are usually softer to touch.
the softer tissue elements • Require manipulation, bending, probing or
• On the other hand, when the tissue is allowed trimming of the specimen to “feel” for remaining
to stay in the decalcifying agent for a very short calcified areas
period of time, decalcification may be • Mechanical damage can occur during bending or
incomplete thereby interfering with the normal probing, and small deposits of calcium can easily
cutting of sections and staining of specimens be missed
• If high-quality results are to be obtained from • Carefully weighing the specimen after rinsing and
decalcified tissue, it is important to determine blotting has also been described and may be an
the point at which all the calcium has been • effective method for large specimens
removed because, from this point on, tissue • An alternate ethod of evaluating tissues
damage seems to occur at an increasing rate mechanically is by pricking the tissue with a fine
• There are three ways by which the extent of needle or a probe
decalcification may be measured, these are the • This method is apt to produce needle tract
following: artifacts and destroy important cellular
• Physical details.
• Chemical • Pricking, slicing, bending or squeezing tissue can
• Radiologic (X ray) disrupt soft tumors from the bone or cause false
positive microfractures of fine trabeculae, leading
to a potential misdiagnosis
Page 206
• Small calcified foci may not even be • An alternate method of evaluating tissues
detected mechanically is by pricking the tissue with a fine
• For probing and bending, the one that does this needle or a probe.
are very experienced technologists because if • This method is apt to produce needle tract
you have too much fun in performing either of artifacts and destroy important cellular
the two then the tissues can become rubbery details.
specially the bones. • Pricking, slicing, bending or squeezing
• While this method may be successful in tissue can disrupt soft tumor from the bone
experienced hands it is generally considered to or cause false positive microfractures of
be unreliable fine trabeculae, leading to a potential
• Generally not recommended because it can misdiagnosis.
cause further tissue damage. • Aside from this disadvantage, small calcified foci
• Mechanical damage can occur during bending may not even be detected.
or probing and small deposits of calcium can
easily be missed Chemical Method (CaOx Test)
• A method of determining the endpoint by • Simple, reliable, convenient method
carefully weighing the specimen after rinsing recommended for routine purposes.
and blotting has also been described, and may • It involves the detection of calcium in acid
be an effective method for large specimens. solutions by precipitation of insoluble calcium
hydroxide or calcium oxalate.
• Applied when some acid decalcifiers are used
(particularly formic acid)
Page 207
• Decalcifying fluid is usually changed every 24 - Disadvantage

48 hours, and the chemical test is performed on • Cumbersome and useless in the everyday
the discarded fluid practice of surgical pathology when many
• Piece of blue litmus paper is added to a test samples are placed in the decalcification solution
tube containing 5mL of the discarded simultaneously.
decalcifying agent (litmus paper will turn red • Definitely more objective than the bubble method
due to the acidity of the fluid) which includes the observation of carbon- dioxide
• The presence of cloudiness indicates that there bubbles at the surface of the bone during acid
is still calcium found in the solution decalcification.
• The tissue is then immersed in a new
solution of decalcifying agent • If a chemical method of determination is to be
• If the solution remains clear after neutralization done, the decalcifying agent should be prepared
with concentrated ammonia, 0.5 mL of with distilled water, since false positive readings
saturated aqueous solution of ammonium may be produced by the calcium ions present in
oxalate is added and the solution is allowed to tap water.
stand for 30 minutes. • It is unsuitable for solutions containing
• Cloudiness will signify incomplete over 10% acid, although these could be
calcium removal diluted and result in a less sensitive test.
• Need for further decalcification. • Can be applied when some acid decalcifiers are
• If the solution remains clear after 30 used (particularly formic acid)
minutes, decalcification is considered to • The decalcifying fluid is usually changed every 24
be complete. to 48 hours, and the chemical test is performed
on the discarded fluid
Page 208
• Decalcifying fluid is immersed in fluid and let’s 6) If the solution remains clear, decalcification is
say after a day or so, we will see how many deemed complete.
calcium is present in that fluid
• If there are many calcium present = Warnings:
Decalcification is not complete • This test is cumbersome and useless in the
everyday practice of surgical pathology when
The steps below are how we determine the many samples are placed in the decalcification
endpoint of decalcification: solution simultaneously.
• It is definitely more objective than the bubble
1) The decalcifying fluid is transferred to a method which includes the observation of
different container. carbon-dioxide bubbles at the surface of the bone
2) A blue litmus paper is added into a test tube during acid decalcification
containing ~ 5 mL of the used decalcifying fluid.
(the litmus paper will turn red due to the acidity X-ray/Radiological Method
of the fluid). • Very expensive, most ideal, most sensitive, and
3) Strong ammonia is added until the solution is most reliable method.
neutralized. • However, is not recommended for mercuric
4) If there is a precipitate (“cloudiness”) then that chloride-fixed tissues due to the ability of
means the solution still contains calcium. mercuric chloride to produce radio-opacity which
5) If the solution remains clear after ammonia will interfere with the correct interpretation of the
treatment 0.5 mL of saturated aqueous plate.
solution of ammonium oxalate is added and let
the solution stand for 30 minutes.
Page 209
• Most sensitive and most reliable method of Post Decalcification

determining extent of decalcification due to its • After decalcification is complete, the acid can be
ability to detect even the smallest focus of removed from tissues or neutralized chemically
calcium which appears opaque in an X-ray plate by immersing the decalcified bone in either
• A good-quality X-ray will clearly reveal tiny saturated lithium carbonate solution or 5-10%
residual calcium deposits and allow further sodium bicarbonate for several hours.
treatment if required • Other laboratories simply rinse the decalcified
• Good for larger bones such as femoral heads specimen with running tap water.
• The best method, particularly with large • Saturated lithium carbonate solution
specimens such as femoral heads, is to X-ray • 5-10% aq. Sodium bicarbonate
the specimen. • Tap water
• This is a very expensive although the most ideal,
most sensitive and most reliable method of
determining extent of decalcification Treatment Following Decalcification &Prior to
• Ability to detect even the smallest focus of Testing
calcium which appears opaque in an X-ray plate • Usual method:
• A good-quality X-ray will clearly reveal • Extensive washing by tap water or alkaline
tiny residual calcium deposits and allow solutions
further treatment if required • Main principle:
• Removal of acid in the tissues
• Tissues are immersed in lithium carbonate
solution or 5 to 10% aqueous sodium bicarbonate
for several hours
Page 210
• Adequate water rinsing can usually be • Tissue decalcified using EDTA solutions should
accomplished in 30 minutes for small samples not be placed directly into 70% alcohol because
and 1 to 4 hours for larger specimens. this will cause residual EDTA to precipitate in
• Samples that need to be immediately alcohol and within the tissue.
processed, such as small needle biopsies, can • Rinsing the decalcified tissue with water or
be blotted or quickly rinsed with water to storing overnight in formol-saline or phosphate
remove acid from the surface, before buffer saline (PBS) will prevent formation of the
transferring the specimen to a dehydrating fluid. crystalline precipitate.
• It is important to remove the bulk of the • Soak first before proceeding to
decalcifier to avoid contaminating the dehydration
processing reagents and the processor with
acid. Surface Decalcification
• Application of vacuum during wax infiltration • Method of dealing with small unexpected
should improve the quality of the finished blocks. deposits of calcium that may be encountered in
• Acid Decalcified tissues for frozen sections: paraffin blocks.
• Thoroughly washed with water or stored • Done if there is a “grating” sensation during
in formol saline containing 15% sucrose cutting the paraffin block.
or phosphate buffered saline with 15 to • The tissue block is placed down in a gauze or
20% sucrose at 4 degrees centigrade cotton saturated with 10% hydrochloric acid for
before freezing. approximately 1 hour.
• This surface treatment will allow the decalcifier to
penetrate a small distance into the block and
dissolve the calcium.
Page 211
• The block is then rinsed then cutting may • The tissue block may be submerged in the fluid
proceed. for 1 to 2 hours before sectioning.
• Careful realignment of the block will be • Washing out an immersion of fixed tissues in 4%
required because the decalcifier will aqueous phenol solution for 1 to 3 days may
penetrate a very small distance into the cause considerable tissue softening thus making
block allowing only a couple of sections tissues easier to section using the microtome.
to be taken.
• This method might cause staining problems. Other softeners:
• Sections from blocks treated this way • Molliflex
should have an adjusted staining • Commercially prepared tissue softener
procedure. • Molliflex may appear swollen and soapy.
• This does affect the normalizing and
Tissue Softeners subsequent staining of tissue sections.
• For unduly hard tissues that may damage • 70% alcohol
microtome knives may require tissue softeners, • 4% Aq. Phenol
aside from decalcification. • 2% HCl (Hydrochloric Acid)
• Perenyi Fluid • 1% HCl (Hydrochloric Acid) in 70% OH
• Both a decalcifier and a tissue softener
Procedure
• Selected portions are left in the fluid for 12 to 24
hours
• Dehydrate in the usual manner
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10
Gross Room or
Surgical Cut-up
Page 213
The term "grossing" means inspecting the Gross Examination of Specimens

specimens, describing and measuring the tissue, • The first step in specimen processing is the
inking if needed, and sectioning the tissue to be identification of the specimen and all its
processed for diagnosis. The skin sample components.
provides the most diagnostically valuable parts of
a specimen for the pathologist's review. Notes to Remember!!
• The specimen container must bear the same
In this topic, we will be learning about the name and accession number as those with the
specimen reception laboratory, the criteria for requisition form.
accepting or rejecting the specimens, and the • All other components of the specimen sample
grossing lab. container must be present: patient’s surname,
name, birth date, and hospital number
Gross room or Specimen Reception
Laboratory Criteria for Gross Specimen Rejection
• Is where tissue specimens from the operating • Discrepancies between the requisition and
theaters and clinics are received. specimen labels
• An accurate diagnosis from this tissue is • Specimens with no labels, or mislabeled
dependent upon the correct identification, specimens
handling, processing in this busy area. • Leaking specimen containers
• Absent clinical data or history
• Inappropriately identified specimens
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“Incorrect identification of any specimen General Considerations during Gross

results in the wrong diagnoses and incorrect Examination
treatment to potentially two patients” • All anatomic structures presented must be
identified and verified
Materials used for gross examination • Some surgeons include diagram in the requisition
• Cutting Tools for easier identification of the specimen’s parts
• Gross Laboratory or Grossing Workstations • If the purpose of the surgical procedure is not
• Standard dissecting kits/sets containing: clear, then it should always be confirmed with
scissors, forceps, blade holders surgeon
• Weight of intact specimen is taken and rounded
Grossing to the nearest 0.1 g
• A pathologist, resident, physician assistant, • Dimensions are taken and rounded to the nearest
histotechnologist, or biomedical scientist can 1.0 cm.
gross specimen. • Color and consistency and the presence of blood
• This is dependent upon the nature of the clot and foreign material are likewise reported
specimen and the local and national regulations
in place in the laboratory.
• The term "grossing" means inspecting the
specimens, describing and measuring the
tissue, inking if needed, and sectioning the
tissue to be processed for diagnosis.
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Seven major components in grossing a • Nasal bone and cartilage from rhinoplasty
specimen • Prosthetic breast implants
1) Reliable and rapid transfer of the specimen • Prosthetic cardiac valves without attached tissue
from surgery to pathology • Tonsils and adenoids from children
2) Accurate identification of the specimen • Torn meniscus
3) Description of additional specimens received • Umbilical hernia sacs in children
from the same patient • Varicose veins
4) Gross description of the specimen’s normal
and abnormal features Limbs
5) Recording the sites from which tissue blocks of • A specimen release forms should accompany the
tissue are taken limbs when these are returned to the patient
6) Recording markers (e.g. sutures) that help with
the correct orientation Specimens that Maybe Excluded from
7) Identifying special studies requested and/or Mandatory Submission to the Histopathology
needed Laboratory
8) Other specimens • Bone donated to the bone bank
• Bone segments removed as part of corrective or
Specimens For Gross Description Only reconstructive orthopedic procedures
• Accessory digits • Cataracts removed by phacoemulsification
• bunions and hammer toes • Dental appliances and teeth with no attached soft
• Extraocular muscle from corrective surgical tissue
procedures • Fat removed by liposuction
• Inguinal hernia sacs in adults
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• Foreign bodies such as bullets or medicolegal • Normal toe nails and fingernails that are
evidence accidentally removed
• Foreskin from circumcision
• Intrauterine contraceptive devices without Sectioning
attached soft tissue • After grossing, specimens are dissected in 1.0
• Medical devices (catheters, gastrostomy tubes, cm thick sections (bread loafing) ensures that
stents and sutures) pathologic areas or tumoral areas are identified
• Middle ear ossicles • All hollow structures are opened as part of the
• Orthopedic hardware and other radiopaque initial examination suspected disease processes
medical devices are identified
• Placentas that do not meet institutionally • If a lesion that has been previously reported is
specified criteria for examination not found in the submitted specimen, the surgeon
• Rib segments or other tissues removed only for must be informed accordingly
purpose of gaining surgical access, provided • Specimen are squared when possible before
that the patient does not have a malignancy placing in the cassette
• Saphenous vein segments harvested for • Specimen should NOT be >0.3 cm thick
coronary artery bypass • Wrap minute and multiple tissue fragments in
• Skin or other normal tissue removed during a filter paper or similar material to prevent them
cosmetic or reconstructive procedure, provided from getting lost during the processing
it is not contiguous with a lesion or the patient • Number of sections and blocks are then indicated
does not have a history of malignancy on the specimen description and on the
• Therapeutic radioactive sources specimen worksheet
• Each organ is sectioned differently
Page 217
Things to Identify for Specimens with Tumor

• Site and size of the tumor
• Location and structures invaded by the tumor
• Vascular invasion
• Distance from resection margins
• Presence of lymph nodes
Specimen worksheet
• The specimen worksheet or gross worksheet
contains the accession number, number of
sections, and blocks taken per case.
• Worksheet primarily guides the histotechnician
in assuring that all blocks are processed.
• Care must be taken to ensure that the
specimen worksheets are properly filled up and
also filled for future reference.
Page 218
11
Tissue Processing
(Dehydration, Clearing,
Impregnation)
Page 219
The tissue processing is the heart of any tissue Completion of Fixation before Processing
section which will be cut adequately only if the • The tissues should be dissected 3-4 mm in
tissue is properly preserved and processed. thickness
• A rule of thumb for most specimen is the size of
The study of this topic is to understand the coarse small coin
and fine details of tissue processing so that
excellent sections are obtained. Post Fixation Treatment
• The longest phase in routine histopathology • Special fixation techniques may require additional
• Tissue must be adequately prepared and set in steps before processing is initiated
a paraffin block after fixation and before staining • Picric acid fixative should be placed directly into
70% alcohol for processing
Pre-eminent type of tissue processing treatment • Carnoy’s fluid should be placed directly into
considered to be the most suitable for routine 100% alcohol
preparation, sectioning, staining, and subsequent
storage of large tissue samples. It utilizes a series
of alcohol as dehydrating fluid
Labelling of Tissues
• A unique identification number or code is
assigned to the tissue sample accessioned in
the laboratory
Page 220
Post Fixation Treatment of Common Fixative used in Histopathology

Fixative Post fixation treatment Comment
• Zenker’s • Thorough rinsing in water • To remove chromic acid
• Helly’s that may form insoluble
• Reagents containing chromate precipitate
• To remove mercury
pigments
• Bouin’s • Washing in ethanol until • To remove picric acid
• Gendre’s supernatant is faintly which may prevent wax
• Reagents containing picrate yellow or clear infiltration and to get good
ribbons on sectioning
• Hollande’s • Brief washing in water • To remove phosphate salt
• Reagents containing which may precipitate in
phosphate tissues and cause
sectioning problems
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Principles of Tissue Processing • Processing reagents having miscibility with water,

Stages of tissue processing: solvent and embedding media should still be
• Dehydration used in graded series
• Clearing • Polarity of the reagents must be change
• Infiltrating gradually, from highly polar aqueous fixative and
• Embedding dehydrant to the non-polar embedding medium to
lessen undue tissue effect
Factors Influencing the Rate of Processing • High viscosity of the clearing and infiltrating
• Tissue density agents results in slow diffusion through tissues
• Agitation
• Heat
• Viscosity
• Vacuum
• Fixation
• Solvent effect
• Tissue modifiers
• Tissue porosity
Properties of the Processing Reagents

• Concentration of the reagents must be
increased in graded increments
• Infiltrating and embedding media must fill all
spaces with the tissue to support section cutting
Page 222
Topic 1: Dehydration • This process is commonly carried out by

• Removal of unbound water and aqueous immersing specimens in a series of
fixative from the tissue components ethanol (alcohol) solution with increasing
• It is the step in tissue processing by which the concentration to avoid excessive distortion
intercellular and extracellular water from the of tissue until a water-free tissue in alcohol
tissue are removed after fixation and prior to is reached.
wax infiltration • Water soluble proteins are removed at
• Specimens are processed through series of lower concentrations of ethanol
graded reagents of increasing concentration • When ethanol concentration is increased
• Amount of each dehydrating agent should not to 100%, certain lipids may be dissolved
be less than 10 times the volume of the tissue • Fatty tissues such as breast or lipoma may be
• Removal of intercellular and extracellular inadequately processed in what is normally a
water from the tissue successful schedule for other tissues
• Increasing concentration of alcohol • Ethanol is a poor fat solvent
• Routine – starts with 70% usually ethyl • To ensure complete dehydration, a
alcohol superior fat solvent such as acetone or
• Embryonic tissues – starts with 30% of isopropanol should be added before the
ethyl alcohol final absolute ethanol, and chloroform or
• Dehydrating fluid to tissue ratio: 10:1 trichloroethane used as the transition
• Melted paraffin wax is hydrophobic solvent
• Most of the water in a specimen must be
removed before it can be infiltrated with
wax
Page 223
Characteristics of an Ideal Dehydrating Commonly Used Dehydrating Agents

Solution • Alcohol (most common)
• Dehydrate rapidly • Acetone
• Do not evaporate very fast • Dioxane 4-cellosolve
• Do not harden tissues excessively • Triethyl phosphate
• Do not remove stains • Tetrahydrofuran
• Not toxic to the body • Cellosolve
• Not a fire hazard
A. Alcohol
Notes to Remember!!! • Alcohols are most commonly used in the
• As a general rule, whatever dehydrating agent laboratory for tissue dehydration, since they are
is used, the amount in each step should not be miscible with aqueous fixatives like 10% formalin
less than 10 times the volume of the tissue in • Alcohol penetrates tissue quickly and the water is
order to ensure complete penetration of the replaced with alcohol
tissue by the dehydrating solution • Performed at room temperature
• Keep the dehydration times as brief as possible
to minimize the risk of extracting cellular Ethyl alcohol (Ethanol)
constituents • Most common
• Almost any water miscible, anhydrous fluid can • Used most often
be used as a dehydrating agent provided that it • Best dehydrating agent
does not damage the tissue proteins and is also • For routine dehydration of tissues
miscible with the fluids to be used subsequently
• Cost may also be a factor
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• Characteristics: • Long periods in absolute ethanol will cause

• Clear excessive shrinkage and hardening
• Colorless • May be difficult to obtain
• Flammable fluid • May have prohibitive taxes that necessitate
• Boiling point: 78.3C troublesome book-keeping
• Extracts methylene blue and other thiazine dyes
Advantages from sections
• Nontoxic • Extracts more lipids than acetone
• Miscible in all proportions with water • May cause more shrinkage of specimen
• Little shrinkage if graded alcohols are used • May react with an unreduced Osmium tetroxide
• Can be used on eyes and embryos, if graded (OsO4) remaining in the specimen
alcohols are used • Only slightly miscible with most resins
• Can be used on eyes and embryos, if graded
alcohols are used fast acting Methyl alcohol (Methanol)
• Still considered best dehydrating solution • Toxic to the body
• Reliable • Employed for blood and tissue films
• Appears to cause less extraction of cellular
components in general than other agents Butyl alcohol (Butanol)
• Inexpensive and easily obtained • Utilized for plants and micro-techniques
• Boiling point: 117.7C
Disadvantages
• Expensive
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Advantage • Mixes with water, ethanol, xylene, and paraffin in

• Slow dehydrating agent producing less all
shrinkage and hardening than ethanol
• Less shrinkage and hardening than with ethyl Disadvantages
• Excellent for slow processing • Odorous
• Miscible with paraffin • More expensive than butanol
• Primary infiltration must be done in half tertiary
Disadvantages butanol and half paraffin, prior to paraffin
• Odorous impregnation
• Slow acting dehydrating agent • Reagent tends to solidify at room temperature or
• Long periods of infiltration necessary below 25C
• Low dehydrating power
• Not suitable for rapid tissue processing Isopropyl alcohol (Isopropanol)
• Boiling point: 82.3C
Tertiary butanol (butyl alcohol)
• Boiling point: 82.8C Advantages
• Excellent substitute for ethanol
Advantages • Less shrinkage and hardening than ethanol
• Universal solvent: acts as dehydrating and • No government restrictions on its use
clearing agent • Sufficiently water-free to use in place of absolute
• May be used in staining series as a dehydrating ethanol
agent • Less expensive than tax-free alcohol
Page 226
Disadvantages Notes to Rememeber!!!

• o Cannot be used in the celloidin technique • Delicate tissues (i.e. embryonic tissues):
• since nitrocellulose is insoluble in it • Dehydration starting with 30% ethanol is
• o Cannot be used for preparing staining recommended
• solutions, since dyes are not soluble in it • It is not advisable to transfer fixed tissues directly
from water or aqueous fixative directly into
Pentanol (amyl alcohol) absolute ethanol as this causes a rapid removal
• Boiling point: 128C of water which can distort the appearance of
more delicate cells and structures.
Advantages • Formalin fixed tissue should never be transferred
• Miscible with 90% alcohol, toluene and xylene directly to higher grades of alcohol
• Dissolves paraffin wax • Example: 85 to 95% alcohol
• Can produce considerable shrinkage and
Disadvantages tissue hardening leading to distortion
• Toxic • Will result in a relatively unequal
• Cannot be used in poorly ventilated rooms impregnation of tissue with consequently
• Not miscible with water poor cutting of sections
• To avoid this, 70% or lower concentrations
Industrial methylated spirit (denatured OH) of alcohol, gradually increased to 95% are
• Ethanol + small amount of methanol used
• It is advisable to remove water gently and allow
the tissue to slowly adjust to its removal.
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• The more delicate the tissue, the more gently • More miscible when epoxy resins (and most
this should be done but there is no hard and embedding resins) than alcohol
fast rule. • Does not extract methylene blue and other dyes
• In most instances, dehydration starts by placing from stained sections
the fixed specimen in 70% ethanol in water, • Not reactive with OsO4 remaining in specimens
progressing through 95% ethanol to 100%
ethanol Disadvantages
• Needs good ventilation
B. Acetone • Evaporates rapidly
• Not recommended for routine dehydration • Penetrates tissues poorly
purposes due to its considerable tissue • Highly flammable
shrinkage production • Requires clearing agent
• Both fixative and dehydrating agent • Most lipids are removed from tissues
• Utilized for urgent biopsies • Volume must be 20 times that of tissue
• Boiling point: 56C • Not recommended for routine dehydration
• Characteristics purposes
• Clear colorless fluid that mixes with • Causes brittleness (tissues placed for prolonged
water, ethanol, and most organic solvent period of time)
• Absolute acetone is easily contaminated with
Advantages water, resulting in incomplete dehydration
• Cheap (less expensive than ethanol), rapid- • Uranyl acetate and phosphotungstic acid are only
acting dehydrating agent which it dehydrates in soluble in dilute solutions of acetone
½ to 2 hours
Page 228
C. Dioxane (Diethylene Dioxide) Disadvantages

• Phase out – toxic, carcinogenic • Expensive
• Refractive index: 1.42 • Extremely dangerous
• Boiling point: 101.5C • Its vapor can accumulate and cause cumulative
• Characteristics: toxicity to the body
• Excellent dehydrating and clearing agent • Tissue sections tend to ribbon poorly
• Readily miscible in water, melted paraffin, • Its vapor produces a cumulative and highly toxic
alcohol, and xylol action in man
• Should not be used routinely
• Laboratory room should be properly Weisberg’s method
ventilated, and all residues should be • The tissue is wrapped in a gauze bag and
washed down in the sink suspended in a bottle containing dioxane and a
• Should not be recycled as the risk of little anhydrous calcium oxide.
creating explosive peroxides increases • Water displaced from the tissues by dioxane and
greatly in turn absorbed by calcium oxide or quicklime.
• Dehydration period range from 3-24 hours
Advantages
• Produces less tissue shrinkage Advantages
• Tissues can be left in this reagent for long • Universal solvent—it dehydrates and clears
period of time w/o affecting consistency or • Miscible with water, alcohol, xylene, and paraffin
staining properties of the specimen • Does not harm tissue over long time periods
• Tissues may be placed directly into the solution • Faster dehydrant than ethanol
after washing out
Page 229
Disadvantages • The tissue may be transferred from water or

• Needs large volume for dehydration normal saline directly to cellosolve and stored in
• Costs about for times more than does absolute it for months without producing hardening or
alcohol distortion.
• Must be used in well-ventilated rooms
• Cumulatively toxic Advantages
• Odorous • Avoids distortion and does not require graded
• Distorts tissue-containing cavities dilutions
• Rapid dehydrating agent
D. Cellosolve (Ethylene Glycol Monoethyl • Tissue may remain in it for months without injury
Ether)
• Combustible at 110° - 112oF Disadvantages
• Toxic by inhalation, skin contact and ingestion • Expensive
• Following exposure, the reproductive, fetal, • Rapidly absorbs water from the air
urinary and blood systems are particularly • Requires clearing agent
vulnerable to their toxic side effects.
• If it cannot be avoided, propylenebased glycol E. Triethyl Phosphate
ethers should be used instead of ethylene- • Boiling point: 215C
based glycol ethers • Removes water very readily and produces very
• Dissolves nitrocellulose but tends to little distortion and hardening
decompose when exposed to sunlight • Used to dehydrate section and smears following
• Boiling point: 156.4C certain stains
• Produce minimum shrinkage
Page 230
Advantages Advantages
• May be used in routine paraffin technique • Miscible with both water, paraffin, lower alcohols,
• Displaces water readily with slight distortion ether, chloroforms, acetone, and benzene
• Does not harden tissue excessively • Rapid without excessive shrinkage and
• May be used as a dehydrating solution in the hardening
staining sequence • Low toxicity; low fire and explosion hazard
• Soluble in alcohols, benzene, toluene, xylene, • Not toxic
ether, and chloroform • Better results than most universal solvents
• Solvents of mounting media
F. Tetrahydrofuran (THF)
• Both dehydrates and clears tissue Disadvantages
• Dehydrating and clearing agent • Odorous: should be used in well-ventilated room
• Toxic if ingested or inhaled • Evaporates rapidly
• Best universal solvent for its fast action and • Dyes are not soluble in tetrahydrofuran
minimal tissue shrinkage or hardening • Should be avoided if possible as there is no
• Dissolve many substances including fats practical way to absolutely protect skin against
• May be used for demixing, clearing, and contact
dehydrating paraffin section • Toxic if ingested or inhaled
• Most staining procedures give improved results • It is an eye and skin irritant, and prolonged
with tetrahydrofuran exposure (up to 6 months) may cause
• Should be in a well ventilated room conjunctival irritation
Page 231
• Because of this and its rather offensive • May combine with reactive groups
odor, processing with THF should be in cells
done in a wellventilated room • May cause certain cytochemical
and staining reactions
G. Additives to Dehydrating Agents • Traces may be retained in
• 4% Phenol – softener for hard tissues polymerized resin
• Copper sulfate • It may react with epoxy groups and
partially inhibit polymerization which
Dehydrating Agents for Electron Microscopy adversely affects hardness and
• Tissue processing for transmission electron cutting properties of blocks
microscopy (TEM) is commonly accomplished
using ethanol as a dehydrating solvent and Acetonitrile
propylene oxide as a transition fluid • Good substitute for propylene oxide
• Both solvents have some undesirable • Reported to be non-carcinogenic, less toxic and
properties: not as flammable as propylene oxide
• Ethanol solubilizes lipids • Freely miscible with water, alcohols, acetone,
• Propylene oxide and epoxy resins
• Highly flammable • Does not interfere with epoxy polymerization
• Volatile • The resulting cured resins have excellent cutting
• Toxic quality and beam stability
• Potentially carcinogenic • Excellent dehydrating agent whose use does not
• Very reactive even at low necessitate modification of current techniques
temperatures
Page 232
• Low solubility of phospholipids in acetonitrile

limits the loss of membrane lipids and, hence,
leads to a better preservation of tissue features
• Used as a dehydrating agent for cells prepared
for Scanning Electron Microscopy (SEM)
Indicators of Incomplete Dehydration

• When copper sulfate CuSO4 changes in color
(from white to blue)
• When xylene turns milky
Page 233
Topic 2: Clearing Clearing Agent

• The choice of a clearing agent depends upon the
Clearing (DEALCOHOLIZATION) following:
• Removal of alcohol from interstitial spaces and • Processor system to be used
replace with a substance that dissolves the wax • Intended processing conditions such as
with which tissue is to be impregnated or the temperature, vacuum, and pressure
mounting medium • Safety factors
• After dehydrating the tissues, the alcohol • Cost and convenience
present in them must be removed before they • Speedy removal of dehydrating agent
are impregnated and embedded with paraffin • Ease of removal by molten paraffin wax
• Remove a substantial amount of fat from the • Minimal tissue damage
tissue which otherwise presents a barrier to wax
infiltration Characteristics of a Good Clearing Agent
• All but a tiny residue of tightly bound • Miscible with OH for rapid removal of dehydrating
(molecular) water should have been agent from tissue
removed from the specimen • Miscible with, and easily remove by melted
• Miscible with both dehydrating fluid and paraffin wax mounting medium to facilitate
impregnating or embedding medium impregnation and mounting of sections
• Paraffin and mounting medium • Do not produce excessive shrinkage, hardening
• It will make tissue transparent due to their high and damage of tissues
index of refraction • Do not dissolve aniline dyes
• Viscosity, temperature will affect the procedure • Do not evaporate quickly in water bath
• Make tissues transparent
Page 234
Commonly Used Clearing Agents • Used in cover slipping, in cleaning tissue

• Xylene processors, as solvent to remove synthetic
• Toluene immersion oil from the microscope objective and
• Benzene in recycling of used slides
• Chloroform • Several toxicities believed to be caused by
• Cedarwood oil intermediate products of xylene metabolism such
• Aniline oil as methyl benzaldehyde have been reported
• Clove oil • Includes:
• Carbon tetrachloride • Central nervous system disorders
• Respiratory depression
A. Xylene • Abdominal pain
• Most commonly used • Dryness and redness of skin
• Most rapid • Dermatitis
• Turns milky when dehydration is incomplete • Liver disease
• Clearing time: 15-30 minutes (½ to 1 hour) • Nephrotoxicity
• Colorless • Conjunctivitis
• Used for clearing, both for embedding and • Teratogenic and fetotoxic effects
mounting procedures
• Generally suitable for routine histologic Advantages
processing schedules of less than 24 hours and • Cheap
when tissue block is less than 5mm in thickness • For urgent biopsies
• Makes tissues transparent
• Miscible with absolute alcohol and paraffin
Page 235
• Does not extract out aniline dyes Advantages

• For mounting procedures: does not dissolve • Miscible with both absolute alcohol and paraffin
celloidin • Acts fairly rapidly: recommended for routine
• Embedding and impregnation purposes
• Evaporates quickly in paraffin oven: can • Does not make tissues excessively hard and
be readily replaced by wax brittle if left for 24 hours
Disadvantages Disadvantages
• Highly inflammable • Slower than xylene and benzene
• Makes tissue excessively hard if used over 3 • Acidifies in a partially filled vessel
hours: • Highly concentrated solutions will emit fumes that
• Causes considerable hardening and are toxic upon prolonged exposure
shrinkage • More expensive
B. Toluene C. Benzene
• May be used as substitute for xylene or • Recommended for urgent biopsies
benzene for clearing both during embedding • Preferred by some in the embedding process
and mounting processes because it penetrates and clears tissues rapidly
• Clearing time: 1-2 hours • Toxic and carcinogenic or may damage bone
marrow resulting to aplastic anemia
• Clearing time: 15- 60 minutes
Page 236
Advantages • Tissues do not become translucent

• Rapid acting: for urgent biopsies and routine • When used for clearing in embedding process,
purposes slower than xylene
• Volatizes rapidly in paraffin oven: easily • Causes less brittleness
eliminated from the tissue • Can be used for thicker tissue blocks (up to 1cm)
• Does not make tissues hard and brittle • Clearing time: 6- 24 hours
• Cause minimum shrinkage
• Makes tissues transparent Advantages
• For routine work
Disadvantages • For large tissue specimens
• Highly inflammable • Not inflammable
• Causes considerable tissue shrinkage if left in
benzene for a long time Disadvantages
• Excessive exposure may be extremely toxic to • Toxic to the liver after prolonged inhalation
man, may damage bone marrow: aplastic • Wax impregnation after chloroform clearing
anemia relatively slow
• Does not make tissues transparent
D. Chloroform • Not very volatile in paraffin oven
• Recommended for tough tissues specimen • Vapor may attack rubber seal used in vacuum
(skin, decalcified tissues), nervous tissues, impregnation bath
lymph nodes, embryos • Complete clearing is difficult to evaluate
• Toxic to liver after prolonged inhalation • Tissues tend to float in chloroform
• Does not make tissue transparent • Evaporates quickly from a water bath
Page 237
E. Cedarwood Oil • Cedarwood oil previously used to clear

• Use for both paraffin and celloidin sections aceticalcohol fixed tissues may produce crystals
during the embedding process • Extremely slow: not for routine purposes
• Recommended for CNS tissue and cytological • Hard to be eliminated from tissues in paraffin
studies (smooth muscles and skin) bath
• Requires two changes in clearing solution • Quality not always uniform and good
• Clearing time: 2 to 3 days • Tissues cleared in cedarwood oil initially float
before gradually staying to the bottom as clearing
Advantages proceeds
• Very penetrating • Hence, the tissue may dry out before it is
• Miscible with 96% alcohol which it removes completely cleared
readily
• Clears celloidin in 5 to 6 days F. Aniline Oil
• Causes minimal shrinkage and hardening • Recommended for clearing very delicate
• Tissues may be left indefinitely without causing specimen (embryo and insects)
considerable damage and distortion • Ability to clear 70% alcohol w/o excessive
• Makes tissues transparent shrinkage and hardening
• Improves cutting of sections • Not normally utilized as a routine clearing agent
Disadvantages
• Very expensive
• Cedarwood oil becomes milky upon prolonged
storage, should be filtered before use
Page 238
G. Carbon Tetrachloride J. Citrus fruits oils (Lemon Oil)

• Similar to chloroform • Essential oil “Terpene containing” (oxygenated
• Used in clearing tissues for embedding derivatives of terpenes)
• Produces considerable tissue hardening • Derived from Citrus limonum or lemon tree
• Highly toxic: dangerous to inhale on prolonged • Clears slower than Xylene
exposure • Volatile oil found in citrus peels which goes by
several trade names.
H. Clove oil • Natural component found in the skins of citrus
• Expensive fruits, such as lemons or oranges, and in cooking
• Unsuitable for routine clearing purposes is usually referred to as lemon or orange zest
• Causes minimum shrinkage • Clear, colorless, and with distinct citrus aroma
• Quality not guaranteed: tendency to become • Available in the Philippines under the brand
adulterated name “Limonene
• Wax impregnation slow and difficult • Limonene is often sold as a xylene
• Tissues become brittle, aniline dyes removed, replacement and some technologists
celloidin is dissolved substitute it for xylene in other uses, but
this is not universally successful
I. Methyl benzoate and methyl salicylate
• Can be used when double embedding Disadvantages
techniques are required • Allergies: rashes
• Slow acting clearing agents
Page 239
K. Terpenes M. Coconut Oil

• Terpenes are moderately effective solvents, but • Can be used as a dealcoholization agent in the
they too are considered toxic histopathological laboratory, without losing the
• They are the earliest transition solvents to be quality of the histological details
used in histology and include turpentine and oils • An efficient substitute for xylene, as it is
of bergamot, cedarwood, clove, lemon, nonhazardous, less expensive and causes less
oreganum and sandalwood shrinkage of the tissue
• Many terpenes clear tissues and celloidin
sections from 80%-95% alcohol, render tissues N. Histoclear
transparent and have a slow gentle non- • Compatible with most popular mounting media
hardening action • Rapid clearing agent without the toxic hazards of
xylene
L. Orange Oil Based Clearing Agents
• Orange oil based clearing agents offer the Advantages
clearing action with the lowest hazard rating of • Enhances the clarity and vibrance of acidophilic
all xylene alternatives. stains and improves staining
• Excellent for preserving fine tissue structure • Leaves tissues less hard and brittle than xylene,
and can often be used in place of xylene with facilitating the cutting of thin sections
no alteration of protocol • Nuclear morphology is rendered in fine detail
Page 240
O. Ultraclear R. Tetrahydrofuran
• Less toxic • Tetrahydrofuran is superior to ordinary
• Less flammable dehydrating and clearing agents dur to its ability
• Friendlier to the environment and easy to to perform two processes at the same time,
handle, but it is two times more expensive than thereby shortening the total processing time and
xylene allowing more time for fixation
• It is not toxic but has an offensive odor and
P. Beach Palm Oil should be used in a well-ventilated room
• Gives good tissues, sections, and histological
slides S. Trichloroethane and petrol
• Nontoxic • Is a clear liquid that was considered to be the
• Nonhazardous least toxic haloalkane solvent.
• Nonflammable
• Biodegradable
• Economic
• Easy to handle
• Readily available
Q. Chlorinated Hydrocarbons
• Can be effective solvents, but they are
considered toxic chemicals, posing serious
health risks
Page 241
Topic 3: Impregnation Paraffin Wax Impregnation

• Inexpensive
Impregnation • Provides quality sections
• Impregnation (infiltration) – is the process of • Easily adaptable
complete removal of the clearing agent and is • Simplest, most common, most popular best
replaced by a medium that will completely fill all impregnating/infiltration and embedding medium
the tissue cavities and give a firm consistency • Forms a matrix preventing tissue distortion during
to the specimen. microtomy
• This allows easier handling and cutting of • Paraffin wax is a polycrystalline mixture of solid
suitably thin sections without any damage or hydrocarbons produced during the refining of
distortion to the tissue and its cellular coal and mineral oils.
components. • Not recommended for fatty tissues
• General types of tissue impregnation: • It is solid at room temperature but melts at
• Paraffin wax impregnation temperatures up to about 65 celsius or 70 celsius
• Celloidin impregnation • Paraffin wax can be purchased with melting
• Gelatin impregnation points at different temperatures, the most
• The medium is for impregnation is the same common for histological use being about 56
medium used for embedding celsius to 58 celsius.
• At its melting point, it tends to be slightly viscous,
but this decreases as the temperature is
increased.
Page 242
• Wax hardness (viscosity) depends upon the • Manual Processing - at least 4 changes of wax at
molecular weight of the components and the 15 minutes interval each
ambient temperature. • To decrease viscosity and improve infiltration of
• Paraffin oven temperature: 2-5C above the the tissue, technologists often increase the
melting point of wax (55-60’C) temperature to above 60 celsius or 65 celsius.
• Embedding center: 2-4C above the melting • High molecular weight mixtures melt at higher
point of wax temperatures than waxes comprised of lower
• Temperature of melted paraffin wax for molecular weight fractions.
embedding: 5-10’C above the MP of wax • Paraffin wax is traditionally marketed by its
• Common waxes have melting points of 45 melting points which range from 39 celsius to 68
celsius, 52 celsius, 56 celsius and 58 celsius celsius.
• The 56 celsius wax is normally used for routine • Tissue-wax adhesion depends upon the crystal
work morphology of the embedding medium.
• In a laboratory with temperature ranging from • Small, uniform sized crystals provide better
20- 24 celsius, paraffin wax with a melting point physical support for specimens through close
of 54-58 celsius is indicated packing.
• If the laboratory temperature is between 15-18 • Crystalline morphology of paraffin wax can be
celsius, the melting point of wax to be used altered by incorporating additives which result in
should be between 50 and 54 celsius a less brittle, more homogeneous wax with good
• Hard tissues require wax with a higher melting cutting characteristics
point than soft tissues • There is consequently less deformation during
thin sectioning
Page 243
• Setting temperature does not appreciably affect Factors Affecting Paraffin Wax Impregnation
crystal size • Nature and size of tissue to be processed
• The duration and number of changes required • Type of clearing agent used
for thorough impregnation of tissue depends on: • Benzene and xylene – easily removed
• Size and type of tissues: Longer time is • Chloroform and cedarwood oil – difficult to
required for thicker tissues remove
• Use of vacuum imbedding: Vacuum • Use of vacuum embedding oven
reduces the time required for complete
impregnation Important points in Paraffin Wax
• Clearing agent employed • Paraffin oven must be maintain a temperature 2
• Automatic Processing - 2-3 changes of wax, to 5 degrees above the melting point of paraffin
decreased processing time because of constant used in impregnation
agitation (e.g. Autotechnicon) • Paraffin wax must be pure, free from dust, water
• Vacuum Embedding droplets and other foreign materials
• Vacuum impregnation under negative • Paraffin wax can only be used twice
atmospheric pressure inside the • T-shirt should not be left in the medium for longer
embedding oven periods of time
• Hastens removal of air bubbles • Water in recycled paraffin wax must be removed
and clearing agent
• Promotes more rapid penetration
of wax
• Recommended for urgent biopsies
• Gives the fastest result
Page 244
Substitute For / Type of Paraffin Wax • Serial sections may be cut with ease, without
Paraplast cooling the tissue block, thereby preventing the
• Mixture of highly purified paraffin and synthetic formation of ice crystal artefacts.
plastic polymers • Soluble in common clearing agents and follows
• Melting point: 56-57°C the same time schedule for paraffin impregnation,
• Paraplast with a melting point of 56 to 58 and does not tend to crack like other paraffin wax
celsius is recommended substitutes.
• Winter: 54 to 56 celsius Paraplast may be used
if the tissue is cut in a cool room. Bioloid
• Summer: Necessary to use 60 to 63 celsius, • A semisynthetic wax recommended for
although this is to be avoided if possible, in embedding eyes
order to not to "cook" the tissue.
• "Cooked" tissue does not section well or, Tissue Mat
if it does, it does not stain well and most • Product of paraffin, containing rubber, with the
details are destroyed. same property as Paraplast.
• More elastic and resilient than paraffin wax
thereby permitting large dense tissue blocks Embeddol
such as bones and brain to be cut easily with • Melting point: 56-58°C
the same result as in double embedding. • Synthetic wax substitute similar to Paraplast
• Blocks obtained are more uniform than with any • Less brittle and less compressible than Paraplast
other medium, with better ribboning of sections.
Page 245
Ester Wax • Sectioning of ester wax-impregnated tissues

• Lower Melting point: 46-48 °C should be done on a heavy duty microtome (e.g.
• It is harder than paraffin. sliding or sledge type microtome) due to the
• Not soluble in water but is soluble in 95% relative hardness of the wax.
Ethyl Alcohol and other clearing agents • Soluble in 95% ethyl alcohol and other clearing
• It can be used for impregnation • Insoluble in H2O
without prior clearing of the tissue.
• Cellosolve (ethylene glycol monoethyl ether) or Water Soluble Wax
xylene may be used as clearing agents, if • Plastic polymers, mostly polyethylene glycols
indicated • Melting point: 38-42 °C or 45-56 °C
• In such instances, removal of the • Tissue can be embedded directly from water
clearing agent must be gradual • Allow demonstration of neutral fats and lipids
• That is, the tissue must be placed • For enzyme histochemical and cytologic studies
in a solution containing equal • Incorporated in the majority of proprietary
proportion of clearing agent and histological paraffin wax blends presently
ester wax for 3- 6 hrs before available to improve adhesion, hardness and
finally transferring it to pure wax. plasticity.
• Three to four changes of wax are • Carbowax
required to ensure complete tissue • Most commonly used water soluble wax
impregnation. • It is a polyethylene glycol containing 18 or
more carbon atoms, which appears solid
at room temperature.
Page 246
• Soluble in and miscible with water; hence Dimethyl sulphoxide (DMSO)

does not require dehydration and • When added to proprietary blends of plastic
clearing of the tissue polymer paraffin waxes reduces infiltration times
• The tissues are fixed, washed out and and facilitates thin sectioning.
transferred directly into the melted • It scavenges residual transition solvent and
Carbowax. probably alters tissue permeability by substituting
• Processing time is reduced with the for or removing bound water thus improving
special advantage that harmful effects infiltration.
produced by ordinary dehydrating agents • Some individuals who handle DMSOparaffin wax
are consequently avoided. may experience an unpleasant and annoying
• It does not remove neutral fats and lipids oyster or garlic taste probably caused by DMSO
which are soluble in reagents used for metabolites.
routine processing with paraffin, hence, • Possible health risks associated with the use of
allowing these substances to be DMSO-paraffin wax are minimal if correct
demonstrated in thin sections. laboratory hygiene is observed.
• Due to its hygroscopic nature, Carbowax
is very easily dissolved in water. Advantages of Paraffin
• Care must be taken to avoid • Thin individual serial sections may be cut with
contact of the block with water or ease from the majority of tissues without
ice distortion.
• The process is very rapid, allowing sections to be
prepared within 24 hours.
Page 247
• Tissue blocks and unstained mounted sections • Tissues that are difficult to infiltrate, e.g. bones,
may be stored in paraffin for an indefinite period teeth, brains and eyes, need long immersion for
of time after impregnation without considerable proper support; otherwise, they will crumble on
tissue destruction. sectioning.
• Because formalin-fixed, paraffin-embedded • Prolonged immersion in paraffin, on the
tissues may be stored indefinitely at room other hand, is not advisable.
temperature, and nucleic acids (both DNA and • Paraffin processing is not recommended for fatty
RNA) may be recovered from them decades tissues.
after fixation, they are an important resource for • The dehydrating and clearing agents used
historical studies in medicine. in the process dissolve and remove fat
• Many staining procedures are permitted with from the tissues.
good results.
Celloidin Impregnation
Disadvantages of Paraffin • Suitable for specimen with large hollow cavities
• Overheated paraffin makes the specimen brittle. which tends to collapse
• Prolonged impregnation will cause excessive • For large dense tissues and large tissue sections
tissue shrinkage and hardening, making the of the whole embryo (For hard tissue specimens)
cutting of sections difficult. • Supplied in thin, medium or thick medium of
• Inadequate impregnation will promote retention cellulose dissolved in equal parts of ether and
of the clearing agent. alcohol
• Tissues become soft & shrunken, & • Does not require heat during processing
tissue blocks crumble when sectioned & • Amorphous, slightly yellowish substance
break up when floated out in a H2O bath. • Purified form of collodion or nitro-cellulose
Page 248
Wet Celloidin Method • The cedarwood oil used in the dry celloidin
• Recommended for bones, teeth, large brain and technique helps to soften the brittle layers.
embryo sections and whole organs
Disadvantages of Celloidin
Dry Celloidin Method • Vapor of the ether solvent is very flammable;
• Preferred for whole eye section hence, it should never be used near an open
flame.
Advantages of Celloidin • Photomicrographs are difficult to obtain.
• Permits cutting of tissue sections which are • It is very volatile and therefore must be kept in
thicker than in paraffin wax, and is bottles with ground glass stoppers to prevent
recommended for processing of neurological evaporation.
tissues.
• Dense tissues which are hard to infiltrate (e.g. Gelatin Impregnation
bones and brain) and specimens which tend to • Rarely used except when dehydration and
collapse easily due to air spaces (e.g. eyes) are clearing are to be avoided
supported better, thereby avoiding the • For tissues subjected to histochemical and
crumbling of tissues during sectioning. enzyme studies
• When eye sections are embedded by the • Embedding medium for delicate specimen and
paraffin method, the retina may be frozen sections because it prevents
detached from the harder tissues (e.g. fragmentation of tough and friable tissues when
sclera and choroid) that encircle it. frozen sections are cut.
Page 249
Topic 4: Embedding • Homogeneous

• Capable of flattening after ribboning
Embedding nontoxic
• Tissues are placed in a mold filled with molten • Odorless
embedding medium then allowed to harden • Easy to handle
• Require an/a: • Inexpensive
• Ample supply of clean, filtered, paraffin • The medium used to infiltrate the tissue is usually
wax held at 2C-4C above melting point the same medium utilized for impregnation, and
• Cold plate for general purposes is known as an Embedding
• Supply molds Medium.
• Embedding (Casting or Blocking) is the process • There are generally four types of impregnation
by which the impregnated tissue is placed into and embedding medium, namely:
aprecisely arranged position in a mold • Paraffin wax
containing a medium which is then allowed to • Celloidin (collodion)
solidify. • Gelatin
• Ideally, an infiltrating and embedding medium • Plastic
should be:
• Soluble in processing fluids Orientation of Tissues
• Suitable for sectioning and ribboning • Tubular structures:
molten between 30 celsius and 60 • Cut in cross sections of the lumen
celsius translucent or transparent; • Skin, intestines, gallbladder, and other epithelial
colorless stable biopsies:
• Cutting plane at right angles to the surface
Page 250
• Muscle biopsies: Agar

• Sections containing both transverse and • Main use is in double embedding technique with
longitudinal planes ester wax or paraffin wax
• Multiple pieces of tissues: • Cohesive agent for multiple fragments or friable
• Oriented side-by-side with the epithelial tissue
surface facing in the same direction
Gelatin
Other Embedding Methods • Has a lower melting point than agar
• Main use in the production of whole organ
Celloidin or nitrocellulose method sections
• Recommended for embedding hard tissues • For friable tissues
(such as bones and teeth) and large sections of • Rarely used except when dehydration is to be
whole organs (such as the eye) avoided and when tissues are to be subjected to
• Bell jars can be used to control the rate or histochemical and enzyme studies.
evaporation of the solvent. • It is used as an embedding medium for delicate
• The use of celloidin is discouraged now specimens and frozen tissue sections because it
because of the special requirements needed for prevents fragmentation of tough and friable
processing and the limited use of these types of tissues when frozen sections are cut.
sections in neuropathology.
Page 251
Double embedding method Epoxy embedding plastics

• Tissue are 1st infiltrated with celloidin and • 3 Types Used in Microscopy:
subsequently embedded in paraffin mass • Bisphenol A (Araldite)
• Used in dealing with hard tissues or making • Glycerol (Epon)
small sections of celloidin blocks • Cyclohexene dioxide (Spurr)
• For maintenance of the morphological • Reduces antigenicity, hydrophobic, toxic
appearance of the tissue [example being vinyl cyclohexane dioxide (VCD)
• Serial sections are easily prepared is known to be carcinogenic], and damages
• Extra degree of resilience is given when cutting tissue as it oxidizes peroxide
hard tissues • Carefully balanced mixture of epoxy plastic,
• Availability of paraffin waxes containing different catalysts and accelerators
types of resins has made this technique • Bisphenol A (araldite) infiltration is slow because
obsolete the epoxy plastic is a large molecule
• Glycerol-based epoxy plastics (Epon) have a
Plastic or Resin Embedding Medium lower viscosity but are often sold as mixtures of
• Provides superior results for light microscopic isomers
studies, particularly in hard tissues such as and • Cyclohexene dioxide-based plastics (Spurr) can
for high resolution light microscopy of tissue be obtained pure, have very low viscosity,
sections thinner than the usual 4-6 μm biopsies. compatible with ethanol and infiltrate fastest
• Classified into epoxy, polyester or acrylic based
on their chemical composition Polyester plastics
• Are used in the mid-1950s for tissue examined
under electron microscopy
Page 252
Acrylic plastics • Thin sections of polymerized GMA can be cut

• Are used for tissue examined under light with glass or diamond knives
microscopy • Low acid GMA can resist the uptake of stain,
greatly reducing nonspecific background staining
Polyglycol methacrylate / 2-hydroxyethyl • Enzyme digestion, a variety of stains and
methacrylate (GMA) immunological localizations may be performed on
• Popular embedding medium for light thick sections of GMA without removal of the
microscopy, being very hydrophilic but also plastic
being tough enough when dehydrated to
section well on most microtomes Benzoyl peroxide
• Also being used for transmission electron • Added to the plastic as a catalyst that
microscopy for the preservation and decomposes to form phenyl radicals acting as an
observation of fine structure not previously active site for the polymerization of acrylics
subjected to solvent dehydration
• Does not need a hardener Methyl methacrylate (MMA)
• Can dehydrate tissues directly in aqueous • Its hardness is an ideal embedding medium for
dilutions of GMA or organic solvents undecalcified bone
• Can infiltrate tissue with monomer at room • Used for bone histomorphometry and bone
temperature or lower marrow hematopathology
• Polymerization of GMA can be performed at • Penetrates better than GMA
ambient temperature of 0°C with UV radiation to • Produces high quality bone sections
60°C in an oven
Page 253
• Can easily be completely removed from tissue • It is important that no water is present before
sections dissolving the catalyst (2 minutes) in the
• Conventional MMA embedding causes almost infiltrating solution.
complete loss of enzyme activity and protein • It must be completely dissolved in the infiltrating
antigenicity in the tissues solution, and this may take up to 30 minutes.
• All acrylic hydrophilic media are insoluble • The acrylic plastic mixes are best prepared only
so staining has to occur in situ in the quantity required, preferably using a large
• Results in embedding medium becoming glass vial.
stained and matrix being a physical • It is advisable to measure the quantities
barrier for some molecules volume by weight.
• Methyl methacrylate is a hydrophobic • Any waste solutions containing plastic
alternative that may be used if this components must be handled and discarded in
problem occurs accordance with local and legal requirements.
Practical Considerations for Plastic Mediums ORIENTATION – tissue is arranged in precise

• Specimens should only be processed under an position in the mold during embedding, on the
operational fume hood. microtome before cutting, and on the slide before
• Processing is best achieved if the specimen is staining
agitated continuously on a roller mixer.
• Small aliquots of benzoyl peroxide should be Methods for solidifying tissue block: placing in the
dried carefully away from direct heat and refrigerator or in cold water at 5C
sunlight as it is potentially explosive.
Page 254
Molds for Embedding
Leuckhart’s Embedding Mold

• Molds for routine work and are widely used
• Consist of 2 L-shaped pieces of metal
• Aarranged on a flat metal plate which can be
moved to adjust the size of the mold to the size
of the specimen
Plastic Embedding Ring & Base Mold

• Consist of a special stainless steel base mold
fitted with a plastic embedding ring, which later
serves as the block holder during cutting.
• Used in positioning histological tissues accurately
in base molds.
• Compatible with most commonly-used
processing and storage systems.
Compound Embedding Unit • Rings are precision-molded from premium-grade,
• Made up of series of interlocking plates resting chemically-inert, high impact polystyrene for
on a metal bras, forming several compartments dimensional rigidity and sturdiness.
• Embeds more specimen at a time
• Has the advantage of embedding more
specimens at a time
Page 255
Disposable Embedding Mold
Peel Away
• They can be placed directly in the chuck of the
microtome.
• No need for blocking and trimming disposable
thin plastic embedding molds, available in 3
different sizes, are simply peeled off one at a
time, as soon as the wax has solidified, giving
Plastic Embedding Ring perfect even block without trimming.
• It may be placed directly in the chuck or block
holder of the microtome.
Base Molds
Page 256
Plastic Ice Tray Test Tubes

• May be recommended for busy routine • Used for small fragments which have been
laboratories. processed (e.g. Bone marrow) which
• Each compartment may be utilized for concentrates them without the damage caused
embedding one tissue block, which may then be by orientation with forceps
removed by bending the plastic tray once the
wax has solidified or by smearing the inner General Process & Tips of Using An Embedding
mold with glycerin or liquid paraffin before Mold
embedding. • Hot paraffin is added to the mold from the
paraffin dispenser.
Paper Boats • Be sure there is enough paraffin to cover
• Made from thick paper or (or cartolina) the face of the plastic cassette.
cardboard paper • Cool the top surface of the Paraplast by blowing
• Cheap to make and allow blocks to be stored gently on it.
without being removed • Tissues at this stage are very brittle and
• One of the readily used embedding should be handled with care.
• If necessary, fill cassette with paraffin
Watch Glasses while cooling, keeping the mold full until
• Ideal for embedding fragmentary biopsies solid.
• Not essential to smear them with glycerin • Cool thoroughly in cold running tap water.
• Blocks are hard to remove • If you use ice water for the final cooling,
you may split the block owing to too rapid
shrinkage.
Page 257
• Paraplast naturally splits in the line of • Orientation of tissues in the Paralast block is
least resistance-right through the tissue important for tissues so that they can be properly
• Paraffin should solidify in 30 minutes. placed in a predetermined plane
• When the wax is completely cooled and • Trimming is excessively difficult in a block
hardened (30 minutes) the paraffin block embedded with two or more tissues if they are
can be easily popped out of the mold not carefully lined up before the Paraplast is
• The wax blocks should not stick. cooled
• If the wax cracks or the tissues are not
aligned well, simply melt them again and Methods of Tissue Processing
start over.
• If you use plastic cups, the Paraplast block can Manual Tissue Processing
be removed as soon as it is cooled. • Rarely used, circumstances requiring manual
• The stainless steel mold should slip off tissue processing:
easily when cool and can be used again. • Power failure or equipment malfunction
• Large tissue sample requiring more time
Other important tips: than can be allocated on an automated
• Work quickly while transferring specimens or processor
wax from oven because wax hardens quickly • Small biopsies kneading rapid diagnosis
• Always remember to put wax container back • Can be accelerated using laboratory grade
into oven immediately and close oven door microwave ovens and ultrasonics
between transfers
Page 258
• At least four changes of wax are required at 15 • Impregnation:

minutes intervals in order to insure complete • Paraffin wax = 15 minutes
removal of the clearing agent from the tissue • Paraffin wax = 15 minutes
• The specimen is then immersed in another • Paraffin wax = 15 minutes
fresh solution of melted paraffin for • Paraffin wax = 15 minutes
approximately 3 hours to insure complete • Embedding:
embedding or casting of tissue • Paraffin wax = 3 hours
• The following is an example of a time schedule
for manual processing of tissues about 3 mm.
thick:
• Fixation:
• 0% Buffered Formalin = 24 hours
• Dehydration:
• 70% Alcohol 6 hours 95% Alcohol
= 12 hours
• 100% Alcohol = 2 hours
• 100% Alcohol = 1 hour
• 100% Alcohol = 1 hour
• Clearing:
• Xylene or Toluene = 1 hour
• Xylene or Toluene = 1 hour
Page 259
Sample manual time schedule for 3mm (Thick Specimen)

Process Time
Fixation 10% Buffered Formalin 24 hours
Dehydration 70% alcohol 6 hours
95% alcohol 12 hours
100% alcohol 2 hours
100% alcohol 1 hour
100% alcohol 1 hour
Clearing Xylene or toulene 1 hour
Xylene ortoulene 1 hour
Impregnation Paraffin wax 15 minutes
Paraffin wax 15 minutes
Embedding Paraffin wax 3 hours
Manual Tissue Processing for Small Biopsies

Process Time
10% formalin 10 minutes
95% alcohol 10 minutes
100% alcohol 10 minutes
Xylene 10 minutes
Paraffin 10 minutes
Page 260
Automatic Tissue Processing • It makes use of 12 individual processing steps,

• This method makes use of an automatic tissue with ten 1-liter capacity glass beakers and two
processing machine (i.e., Autotechnicon) which thermostatically controlled wax baths with a
fixes, dehydrates, clears and infiltrates tissues, safety device cut-out switch to protect the wax
thereby decreasing the time and labor needed against overheating
during the processing of tissues • A transfer arm controlled by electrical current
• This results in a more rapid diagnosis with less moves the tissues from one processing reagent
technicality to another (by clock schedules)
• Usually, only 2- 3 changes of wax are required • It can be removed by raising a spring-loaded
to remove the clearing agent and properly plunger in the center of the cover plate, thereby
impregnate the specimen allowing the tissue to be arranged manually
• This is made possible due to constant tissue anytime during the processing
agitation which accelerates and improves tissue • Agitation of fluid is accompanied by a continuous
penetration giving rise to more consistent vertical movement or rotation of the specimen
results carrier by a mechanism connected to the transfer
• One example of an automatic tissue processing arm
machine is the Elliott Bench-Type Processor • An electrical clock connected to a metal disc
• The machine is mounted on rollers to permit the notched in positions of 15 minutes or more,
turning of platforms and easy access to beakers serves to control the time needed for each
and wax baths processing step
Page 261
• The clock rotates and sets the transfer arm and Fluid-Transfer (Enclosed)
mechanism into motion, moving the tissue to • Enclosed, self-contained vacuum tissue
the next position processor
• A delay mechanism is provided in instance • Reagents and melted paraffin are moved
where processing time may exceed 24 hours sequentially into and out of the retort chamber
using vacuum pressure
Tissue-Transfer (Dip and Dunk) • Advantages:
• Uses carousel type processors which fixes, • Vacuum and heat can be applied at any
dehydrates, clears and infiltrate tissues, stage
decreasing time and labor needed • Customized schedules
• Length of time the specimens were submerged • Fluid spillage contained
in each reagent was electronically programmed • Fumes eliminated
Page 262
Microwave Ovens
• Shortens processing time
• Stimulate diffusion of the solutions into the
tissue
• Processing is dependent on the thickness and
density of the specimen
• Reagents:
• Ethanol
• Isopropanol
• Paraffin
• Graded concentrations are not
require
• Clearing agents not necessary
• No formalin and xylene
• Advantages:
• Provide uncompromised morphology and
antigenicity of specimens
• Increased efficiency
• Environmentally friendly reagents
• Disadvantages:
• Process is labor intensive
• Lab-grade microwave are costly
• Requires calibration and monitoring
Page 263
Continuous Input, Rapid Tissue Processor

• Uses microwave technology, vacuum infiltration,
and proprietary reagents
• Reagents:
• Acetone
• Isopropanol
• Polyethylene glycol
• Mineral oil
• Paraffin
• Advantages:
• Toxic vapors eliminated
• Environmentally safe reagents
• Disadvantages: Precautions with Automatic Processing
• Costly processor • The frequency with which fluids are changed
• Grossing of specimen requires depends on the number and sizes of the tissues
standardized dissection processed
• The presence of any odor in the clearing agent
during final paraffin wax bath indicates that the
paraffin wax needs to be changed
• Dehydrating fluids should be changed frequently
since dehydration is the most critical stage of
tissue processing and inadequate dehydration is
difficult to correct once the tissue is in paraffin
Page 264
• The first 100% ethanol bath should be • Reduces the time when tissues are subjected to
discarded, and the others moved down, so that high temperatures thus minimizing heat-induced
the final bath has fresh 100% ethanol after two tissue hardening.
complete processing runs of loads of at least • Facilitates complete removal of transition
three-quarters capacity solvents, and prolongs the life of wax by reducing
• The clearing agent and the dilute ethanols solvent contamination.
should be changed at least once a week • Vacuum infiltration requires a vacuum infiltrator
• To avoid spillage, fluid and wax containers must or embedding oven, consisting of wax baths, fluid
be filled to the appropriate level and correctly trap and vacuum gauge, to which a vacuum of up
located in the machine. to 760 mm Hg is applied using a water or
mechanical pump.
Vacuum Embedding • With vacuum embedding, the time required for
• Involves wax impregnation under negative complete impregnation is reduced by 25% -75%
atmospheric pressure inside embedding oven of the normal time required for tissue processing
• Hasten removal of air bubbles and clearing • The tissue is not over-exposed to heat
agent from tissue block, thereby promoting a • Brittleness, shrinkage and hardening of
more rapid wax penetration of the tissue. tissues consequent to overheating is
• Recommended for urgent biopsies, for delicate therefore prevented.
tissues (lungs, brain, CT, decalcified bones, • Tissue can also be transferred after clearing to a
eyes, spleen, and CNS) heated bath of paraffin wax from which air can be
• Give the fastest result evacuated
Page 265
• The vacuum embedding oven consists of a Processor Maintenance Tips

flatbottomed heavy brass chamber covered with • Any spillage or overflow should be wiped away
a heavy glass lid resting on a wide and thick immediately
rubber valve which produces an airtight seal • Accumulation of wax on any surface should be
when the chamber is being used. removed
• Temperature of paraffin bath should be set at 3C
Vacuum embedding oven above melting point of the paraffin
• Consists of a flat-bottomed heavy brass • Timing should be checked when placing tissue
chamber covered with a heavy glass lid resting cassettes in the processors, especially when
on a wide and thick rubber valve which delayed schedules are selected
produces an airtight seal when the chamber is
being used. Advantages of Newer Technology in Processing
• The vacuum chamber is enclosed in a • Custom program specific to tissue being
thermostatically controlled water-jacket, usually • Rapid schedules
maintained at a temperature of 2- 4°C above • Fluid and fume containment
the melting point of the wax. • Environmentally-friendly reagents
• The degree of the vacuum should not exceed • Delay schedules
500 mmHg.
• A stopcock is provided to prevent water from Automated Processing Schedules
being sucked back into the trap bottle and • Overnight processing
vacuum chamber when the water or suction • Schedule for processing eyes
pump is closed. • Rapid processing schedule for small biopsies
Page 266
Sample Schedule for Overnight Processing

Pressure/Vacu
Sections Reagents Time Temperature
um
1 10% formalin 1 hr On 38 C
2 10% formalin 1 hr On 38 C
3 50% alcohol/formalin 1 hr On 38 C
4 70% alcohol 30 min On 38 C
9 Xylene 40 min On 38 C
11 Paraffin 30 min On 60 C
Page 267
Schedule for Processing Eyes

Station Reagents Time
1 10% formalin 0 hr
2 4% phenol/ 70% alcohol 1 hr
3 4% phenol/ 70% alcohol 1 hr
4 95% alcohol 1 hr
5 95% alcohol 1 hr
6 100% alcohol 1.5 hr
7 100% alcohol 1.5 hr
8 100% alcohol/ chloroform 2 hr
9 Chloroform 2 hr
10 Chloroform 2 hr
11 Paraffin 2 hr
12 Paraffin 3 hr
Page 268
Schedule for Processing Eyes

Station Reagents Time Vacuum Temperature
1 10% formalin 10 min On 38 C
2 10% formalin 10 min On 38 C
Page 269
12
Microtomy
Page 270
Definition of Terms: Block Trimming

• The sides, top and bottom of the tissue block are
Microtome trimmed until perfectly level and all sides are
• Sectioning instrument that allows for the cutting parallel, almost to the edge of the tissue.
of extremely thin slices of material called • An old knife or blade may be used for this
sections. procedure, but it must still be relatively sharp to
• A specialized precision cutting instrument, avoid damage to the tissue
which accurately and repeatedly slices sections • Samples of small biopsy tissue may be trimmed
from a block of embedded tissue. only to the depth of the first representation of
several levels that will be collected
Micron (μ)
• 1/1000th of a millimeter / (1/25000th of an inch) Coarse Trimming/Facing
• Unit for measurement for the thickness of • Since tissue is completely surrounded by paraffin,
sections it is useful to uncover the surface of the block to
reveal the tissue
Trimming • When using the coarse feed, avoid cutting
• Cutting of the excess wax from the block to unintentional thick sections as this will damage
expose the tissue surface in preparation for the knife and possibly the block face.
actual microtomy or section-cutting • Care should be taken to avoid removing too
• Sides, top and bottom of blocks are trimmed much tissue in this step
until perfectly level and all sides parallel
Page 271
• Tissue that was embedded improperly may not • Re-chilling of the block may be required if
reveal the entire tissue surface and will have to the block face becomes warm or if deeper
be reembedded levels are required
• After coarse trimming, a heated spatula is held • The block is then placed in the microtome
between the tissue block and the block holder for fine trimming and cutting
until the wax begins to melt
• The spatula is then withdrawn, and the Fine Trimming
block is gently pressed into position • The knife is usually tilted at 0-15 degrees
• The block is allowed to harden for cutting angulation on a microtome to allow a clearance
proper by facing them down in ice cold angle between the cutting facet and the tissue
water or refrigerator for 5-10 mins block
• Placing blocks in a freezer can cause • Biconcave knives require smaller
surface cracking, where the friable tissue clearance angles than wedge-shaped
separates from the surrounding wax knives
cohesive sections become difficult to
obtain Trimming Process
• Cooling both the tissue and the wax will • The block that is clamped on the chuck must be
give them a similar consistency, and retracted enough to ensure that the knife does
make sectioning easier not touch the chuck or block on initial down
stroke
• The surface block is then trimmed away
until the entire tissue surface has been
partly exposed
Page 272
• The block is advanced into the knife and • Using the microtome handle, try to cut in a slow
cutting is continued until complete and consistent manner - don’t start and stop
sections come out of the block and a while the blade is cutting a block as this may
regular cutting rhythm is maintained produce horizontal lines across the block and the
• The cutting rate depends upon the type sections (and very slight changes in thickness)
of the tissue, the size of the block, and • Sectioning is generally improved when the
the model or type of the microtome that specimen and the wax are well matched in
is used hardness.
• Sections usually form ribbons due to slight heat • It is for this reason that most paraffin
generated between the block and the knife blocks must be cold when sections are cut.
edge during the process of cutting • The actual method used to chill the block
• Sections are cut between 4-6μ in thickness for is important
routine histologic procedures, after the block • Cold wax provides better support for the harder
has been fixed and secured to the block holder elements in a specimen allowing thinner sections
• The micrometer gauge is set to the required to be obtained
thickness and the knife is positioned in such a • Place the blocks on a cold plate or a cold
way that the center of the blade is in line with wet surface for a few minutes (such as the
the block and the knife has been securely surface of melting ice)
clamped in place • Water penetrates a small distance into the block
• The actual thickness of the first couple of face, swelling tissues and making them more
sections in a ribbon may be thicker than amenable to cutting
indicated because of thermal expansion when • This is particularly important to
cutting a cold paraffin block overdehydrated, dry or crumbly tissues
Page 273
• Incomplete sections are discarded • The application of warm, moist breath

• Complete ribbons are picked up at once with a tends to make sections more cohesive, but
camel hair brush, or a pair of forceps it also causes thermal expansion thus
• Tissues which tend to crumble (e.g. blood clots, making the section thicker
bone marrow) or do not form a smooth flat • Debris adhering to upper or lower edges of the
surface can be sectioned with ease, by exhaling block, or the back of the blade, can make it
gently into the block surface while the section is difficult to obtain cohesive ribbons and cause the
being cut slowly, to reduce the effects of static ribbon to lift off the blade on the upstroke
electricity • If debris is present clear it away, re-chill
• Successive sections will usually stick edge-to- the block and start again
edge due to local pressure with each cutting • Sections are removed in ribbons of ten to allow
stroke, thereby forming a ribbon easy location of serial sections
• Generally a slow, uniform cutting stroke
produces the best results and the least
compression
• Do not stop and restart during a cutting stroke
as this will produce bands of different thickness
across the section
• The practice of gently breathing on the face of a
chilled block immediately before cutting each
section, is common in some laboratories
Page 274
Microtome knives • Use steel, glass, or diamond blades depending

• Used for trimming and section-cutting upon the specimen being sliced and the desired
• Cutting edges has angle between about 27 to thickness of the section being cut:
32 degrees • Standard Steel blades / Disposable
• A good cutting edge must be able to cut steel blades
sections about 2-3 micra thick, without any • Used for routine microtomy and
serration noted in examination cryotomy
• Basic types: • Produce flawless to 2-4 mm section
• Plane-concave knife • Coated with PTFE allowing ribbons
• Less concave side for celloidin to cut with ease
sections cut on a sliding • Glass knife
microtome • Generally, used for trimming and
• More concave sides for paraffin semi-thin sectioning of tissue block
sections cut on base-sledge, for EM
rotary or rocking microtome • Industrial grade diamond knife
• Biconcave knife • To cut any type of resin block for
• For paraffin sections cut on rotary EM
microtome
• Plane-wedge knife Honing
• For frozen sections and hard • Purpose:
specimens in paraffin blocks, • To remove gross nicks and grinding the
using a base-sledge or sliding cutting edge of the knife to acquire an
microtome even edge
Page 275
• “HEEL to TOE” Microtomy

• Uses a hone, a natural sharpening stone or • Tissues are cut into uniformly thin slices or
hard grinding surface “sections” with the aid of a machine to facilitate
• Belgium yellow or Belgian black vein the studies under the microscope
• Usually gives the best result • The means by which tissue can be sectioned and
• Arkansas attached to a surface for further microscopic
• Gives more polishing effect examination.
• Fine carborandum
• Used for badly nicked knives
• Lubricants for hone:
• Soap water
• Liquid paraffin
• Castor oil
• Clove oil
Stropping
• Purpose:
• To polish and sharpen the cutting edge
• Fine quality horseleather is used as paddle
strops which may be either flexible / hanging or
rigid.
• Firm surface is preferred
• Action is reverse of honing-toe to heel
Page 276
Microtome Rocking Microtome

• Essential Parts: • Invented by Paldwell Trefall
• Block holder • Simplest
• Where tissue is held in position • For cutting serial sections of large blocks of
• Knife Carrier and Knife paraffin embedded tissues
• For actual cutting of sections • Cambridge Rocking Microtome
• Pawl, Ratchet Feed Wheel, and • Common in early cryostats
Adjustment Screws
• To line up the tissue block in
proper position with the knife,
adjusting the proper thickness of
the tissue for successive section
• Kinds:
• Rocking Microtome
• Rotary Microtome
• Sliding Microtome
• Freezing Microtome
• Ultrathin Microtome
Page 277
Rotary Microtome Sliding Microtome

• Invented by Minot • Invented by Adams
• Most common type used for routine and • Most dangerous type
research laboratories • Movable and exposed knife
• For cutting paraffin embedded tissues • For cutting celloidin embedded sections
• Manual, semi-automated, or fully-automated • Two Types:
• Advantage: • Base Sledge Microtome
• Ability to cut thin 2-3 mm sections • Useful in neuropathology and
• Easy adaptation to all types of tissue opthalmic pathology
sectioning • Standard Sliding Microtome
Page 278
Freezing Microtome Ultrathin Microtome

• Invented by Queckett • For cutting tissue sections at 0. 5 micra for EM
• For cutting un embedded Frozen sections • Specimen is small, fixed in OSMIUM
TETROXIDE, and embedded in resins (epoxy,
acrylic resins)
Page 279
Microtome Knives Biconcave Knife

• *NOTE: • It is the classical knife shape with concavity on
• Bevel Angle – angle formed between the both sides
cutting edges (27° to 32°) • Introduced by Heiffor
• Clearance Angle – angle formed • Rocking microtome and sledge microtome
between knife and cutting plane (5°to • Length 100-250mm
10°) • Less rigid
• Prone to vibration
Plane Concave Knife
• One side of cutting surface is flat and the other
is concave with different degrees of concavity.
• Extremely sharp, but delicate.
• Used for cutting soft samples like nitrocellulose
embedded tissues.
• The plane surface is closest to tissue block.
• Sledge and rotary microtome.
• Used in sliding, rotary, and rocking microtomes
• Lesser concavity: for celloidin sections
• More concave: for paraffin sections
Page 280
Plane-Wedge knife Other Equipments

• Use in all type of microtome to cut all type of • Water bath
materials • Drying Oven or Hot Plate
• Both cutting surface are plane • Fine pointed or curved forceps
• Known as standard profile • Squirrel or camel hair brush
• Size 100-350mm • Scalpel
• Clean slides
• Ice tray
• Chemical resistant pencil or pen
• Slide rack
Notes to Remember!!!
• Position the microtome on a stable bench, away
from air drafts, doorways and passing staff. Any
air movement from air conditioners or other
causes can make section handling very difficult.
• It is very important that staff are not distracted
when using the microtome because of the risks of
injury from extremely sharp blades.
• The potential for interaction with other staff
members should be considered when
positioning microtomes in a laboratory.
Page 281
• Microtome knives and disposable blades are

extremely sharp and can inflict serious injuries
unless appropriate care is taken when working
with them.
• Accidents occur when a microtomist is
distracted and not concentrating fully.
• Use forceps or brush instead of your fingers to
pick up sections or wax fragments from blade or
block face.
• The knife or blade should be removed from the
microtome when the instrument is left
unattended or when cleaning the instrument.
• The orientation of the specimen to the blade
during the cutting stroke can affect the ease
with which a ribbon can be obtained and directly
influence section quality.
Page 282
Orientation of Tissue Blocks
A. Intestine B. Cervix C. Skin
Page 283
• Blade clearance angle is adjustable and MUST Types of Tissue Sections

BE set for optimum performance. • Paraffin Section
• The facet angle is the angle between the • Rocking and rotary microtome
two facets that form the cutting edge. • Thickness: 4-6 u
• Celloidin Section
• Sliding microtome
• Thickness: 10-15 u
• Frozen Section
• Cryostat and freezing microtome
• Thickness: 4 u
This ribbon has been cut with a low and steady

stroke from a well, processed, thoroughly chilled
block.
This section show very little compression even
before floatation.
Page 284
Floating Out of Sections

• Expand the section to its original dimensions
and ensure it is completely flat .
• Folds and creases may be removed by
stretching the sections gently with a pair o
dissecting needles or forceps
• Sections should not be left on the water bath for
a long time (30 seconds enough) to avoid
distortion of the tissue
• The sections are floated out on a water bath set
at 45- 50°C, approximately 6-10°C lower than
the melting point of the wax used for embedding
the tissue
• This is to flatten the sections and prepare
them for mounting onto the slides
• Bubbles may be teased out from beneath the
sections by means of the same needle
Page 285
Fishing Out Sections Drying of Slides

• A section is selected for staining and picked up • Proper drying ensures that sections are
onto a clean slide in a vertical position completely dehydrated, free of heat damage, flat
• The slide is immersed in the water bath in a and unlikely to lift during staining.
near vertical position as close as possible to the • Besides the paraffin oven which is maintained at
section a temperature of 2-5°C above the melting point of
• When the slide touches the section, it is lifted the paraffin used, small thermostatically
vertically out of water and drained controlled incubators may be used, regulated at
• Sections may also be flattened out by placing 37°C, and at 45-55°C, for enzyme digestion,
them on a slide which has been flooded with chemical extraction, metallic impregnation and
20% alcohol, producing convection currents enzyme localization techniques
which will serve to remove the creases in the • Hot plates are not recommended because they
tissue within a few seconds can cause overheating and there is a risk of dust
• Sections are very easily damaged when falling onto the section during the drying period
dislodging wrinkles or bubbles with brush or • Excessive heat can cause droplets of
forceps water underneath a section to boil and this
• Examine each section as it floats on the water will cause damage
surface as imperfections can be readily seen • Proper drying ensures that sections are
• Leave the section on the water surface just long completely dehydrated, free of heat damage, flat
enough for it to flatten and unlikely to lift during staining
• Drain excess water from beneath the section
before drying
Page 286
• Dry sections for between 5 and 30 minutes • If staining is to include antigen retrieval (IHC),
• Some delicate specimens will produce bes enzyme pretreatment (ISH), or prolonged
results when dried at 37 celsius for a longer incubation steps, charged slides or an adhesive
time (several hours to overnight) must be used
• Metal racks with 25-slide divisions are used to • Some special stains, particularly those that
store the mounted sections during the drying employ alkaline reagents, can also cause
process which usually takes about 5 minutes in sections to lift
the heated oven • Extended storage (usually more than 3 days) of
• Once dry, the whole rack of slides can be unstained formalin-fixed paraffin embedded
taken for manual batch staining or placed slides should be avoided as this may result in the
on an automated staining machine loss of antigens
• Staining of serial sections should never • While not established, vacuum sealing and
be attempted unless they are completely refrigeration may help preserve some unstable
dried antigens
• Overheating should be avoided because • For nucleic acid extraction sections, allow the
it will distort the tissue and melt some of individual sections to roll up naturally and place
the structures like collagen them directly into sterile microfuge tubes ready
for nucleic acid extraction
Practical considerations when staining is • The extraction buffer can be added directly to the
involved microfuge tube in order to preserve the molecular
• Slides must always be grease- and dust-free integrity of the sample
and stored and handled correctly
Page 287
• When cutting sections for DNA or RNA • The resulting restrictions on the type of
extraction, all instruments and equipment must staining methods that may be used.
be pre-cleaned and wiped down with RNAse- • Celloidin may be purchased either as a solution
away before and between each specimen or as a solid, damped with a liquid (usually
• Gloves must be worn ethanol) to reduce flammability.
• Molecular grade water must be used for floating • Celloidin is used in the form of solution, usually in
sections for RNA extraction. a 1:1 mixture of ethanol-ether at concentrations
of 2%, 4% and 8%.
Celloidin Sections • The fastest way to dissolve celloidin is to soak it
• The advantage of celloidin embedding is that it first in half the final volume of anhydrous ethanol
completely avoids the use of heat at any stage. to soften it (50 mL for each 8 grams celloidin)
• As a consequence, heat produced artifacts are with intermittent mixing in a tightly stoppered
avoided. container.
• Shrinkage is minimal
• Disadvantages:
• Longer time to cut the thickness of the
sections
• The necessity for staining to be done on
free floating section
• The inconvenience of having to store the
blocks in sealed jars with tight lids to
prevent complete evaporation of 70%
ethanol
Page 288
Summary Table on Troubleshooting

PROBLEM REASON REMEDY
Surfaces and edges of the block are not Re-Trim the block
parallel
Horizontal surface of the block is not
Re-adjust and re-orient the block
parallel to the knife
Sections fail to form
Coat horizontal edges of the block with
ribbons Paraffin wax is too hard
wax of lower melting point
Knife is tilted too much Reduce the tilt
Sections are too thick Readjust the thickness of the sections
Knife is dull Hone and strop
Sections roll up on Knife is blunt Sharpen the knife
cutting so that they Tilt of knife is too great Reduce the tilt
adhere and get broken
Knife edge is dirty Clean the knife edge
against the knife edge
Adjust the knife so that knife edge will
Blunt or dull spot on the knife, producing
present a uniformly sharp edge to the
Ribbon is curved, crooked an irregular knife edge
block, or sharpen
or uneven
Edges of the block are not parallel but
instead of straight Re-trim the block
round or wedge shaped
Knife is not parallel to the block Readjust the knife and block
Page 289

Knife is blunt or dull Re-sharpen the knife
Paraffin block is warm and soft Cool the block on ice water until firm
Sections are compressed, Knife edge is coated with paraffin Clean the knife edge
wrinkled or jammed Sections are too thin Readjust the thickness of the section
Microtome set screw is loose Tighten the screw
Tilt of knife is too vertical Reduce the tilt
Sections are squashed
Bevel of knife is lost due to incorrect Re-sharpen, using a knife back or
(width of each section is
sharpening automatic knife sharpener
less than that of the block)
Bubble or dirt formed in the Re-embed in freshly filtered wax if
embedding medium necessary
Tissue is not processed properly and
will not form a section (especially if Re-process tissue
center is raw)
A hole is formed in the
Under-processed portion of tissue
section Re-process tissue
bursts on contact with warm water
Once embedded in paraffin wax,
decalcification is impractical
Hard spot in tissue due to calcium
Use a basesledge microtome with a
wedge knife
Page 290

Tilt of knife is too great, or bevel is
not cleared, hence object is Reduce the tilt
compressed against the knife edge
Sections of unequal
Clamp set screw on knife or block
thickness are produced Tighten the screw
holder is loose
Blocks are too large Cut blocks into smaller fragments
Blocks are too hard Soften the blocks in detergent or phenol
Breathe out or blow gently on the block
Static electricity due to low and knife to break up static electricity,
Sections adhere to the knife atmospheric humidity or boil water in the room to increase
or other parts of the humidity
microtome Knife edge is dirty Clean the knife edge
Knife edge is dull Sharpen the knife
Knife tilt is too great Reduce the tilt
Nicks or damage on the knife edge Sharpen the knife
Ribbon is split or lengthwise
Dirty embedding Re-embed in freshly filtered wax
vertical scratches are see on
Knife edge is dirty Clean knife edge with xylene
sections
Tilt of knife is too great Reduce the tilt
Page 291

Knife tilt is too great Reduce the tilt
Sections are lifted from the Knife is dull Sharpen the knife
knife on upstrokes Paraffin is too soft or room
Cool paraffin wax in ice water
temperature is warm
Resistance is felt on the Tilt of knife is too small, paraffin block
lower part of the section is therefore compressed against the
Increase the tilt
during cutting base of the knife towards the end of
stroke
Horizontal or parallel lines Knife edge vibrates due to hardness Treat with phenol during processing or
or furrows across the of tissue collodionize
section (“chatters”) are seen Tilt of knife is too great Reduce the tilt
Knife is blunt Sharpen the knife
Adjust the knife so that knife edge will
Knife is not clamped properly present a uniformly sharp edge to the
Section cut is sometimes block or sharpen
thin, sometimes thick Knife or block holder is loose Tighten adjusting and locking screws
Knife tilt is too small that block is
compressed by bevel and section is Increase the tilt
not cut
Page 292

Knife makes a hard metallic Tilt of knife is too slanted or too big Readjust the tilt
scraping or ringing sound on Take fresh block treated with phenol
Tissue is too hard
backstroke, when section is during processing
cut Knife blade is too thin Change the knife
Frozen tissue crumbles and
comes off the block holder Freezing is not adequate Refreeze the tissue block
when cut
Frozen tissue chips into Adjust the block holder to make the
Tissue is frozen too much
fragments when cut block edges parallel to the knife
Sections are too thick Wrong micrometer setting Microtome needs recalibration
Block is trimmed down nearest to the
Clearing agent not completely tissue. Remaining wax is melted on
On trimming, tissue smells of
removed due to insufficient embedding oven and paraffin
clearing agent
impregnation impregnation is repeated, changing the
paraffin at least once before embedding
Tissue is opaque, section Repeat clearing; if object has already
cutting is difficult due to Insufficient clearing been embedded, prolong clearing up to
presence of alcohol 12 hours, then reembed
Insufficient dehydration,
Tissue shrinks away from wax
therefore incomplete clearing and Repeat the whole procedure
when trimmed
impregnation
Page 293

Contaminated wax Re-embed in freshly filtered wax
On trimming, wax appears
Re-embed in freshly
crystalline Block not cooled rapidly enough
filtered wax
Paraffin block, after cooling, is Repeat paraffin impregnation, then re-
Insufficient paraffin
moist and crumbles embed
Page 294
Adhesives Dried Albumin

• Alternative to drying • Crystals of thymol is added
• Essential for methods that require exposure to • Components:
acids and alkalis (ammoniacal silver solutions) • Dried albumin
• Smeared on to the slides so that the sections • NaCl
stick well to the slides
1% Gelatin
Common Adhesives • Added and mixed to the water bath
• Most convenient alternative to direct coating of
Mayer’s Egg Albumin slides
• Most commonly used • Provides firmer attachment then albumin has to
• Easy to make, convenient, inexpensive be gently heated before use to melt the gelatin.
• Equal parts of glycerin, distilled water, and egg • Shouldn’t be kept molten for long periods as in
white are mixed filtered through coarse filter will lose its ability to solidify
paper • Components:
• Thymol crystal is added to inhibit the growth of • Gelatin
molds, solution kept in refrigerator • dH2O
• Small quantity of the solution is smeared over • glycerol
the surface of the slide immediately before • phenol crystals
mounting sections from the water bath
• Components:
• Egg white
• Glycerin (same volume)
Page 295
Gelatin-formaldehyde mixture Poly-L-Lysine

• Coated slides are dried at 37°C for 1 hour or • Widely used section adhesive in
overnight before use immunohistochemistry
• Components: • Use as a general-purpose section adhesive.
• 1% gelatin • No production of background staining.
• 2% formaldehyde
APES (3-aminopropylthriethoxysilane)
Starch Paste • Useful in cytologymicrons
• Thymol crystals are added to prevent mold • Produces improved section bonding over other
formation commonly used adhesives such as poly-L-lysine,
• Greater adhesion than gelatin glycerin , albumin, and gelatin and withstands
• Disadvantage: protease digestion.
• Stains with many dyes • Withstand repeated washings with a variety of
• Components: inorganic solvents.
• Powdered starch • There is no background staining
• dH2O • Does not interfere with the routine histological or
• HCl immunostaining methods
Plasma Charge or plus slides

• Readily available from outdated blood • Proven resistance to cell and tissue loss
• Dispensed into sterile tubes (0.5 mL)
Page 296
Cellulose Resins
• In the form of 1% Methyl cellulose • Greatest adhesion
• Advantage: • Diluted 1:10 with acetone
• Not staining to any appreciable extent • Little affect by most fluids in any treatment of
with commonly used in stains of sections
histochemical reagents.
Sodium Silicate
• Has strong adhesive properties
• Commercial syrup = 1:10 dilution
• Advantages:
• Little tendency to staining with most dyes
• Not affected by the use of mild alkaline
solutions
• Disadvantages:
• Blackening in some silver impregnation
techniques, in some reticulin methods,
and red staining in methyl green pyronin
technique
Page 297
13
Staining
Page 298
Staining • A histologic stain is the purified form of a coloring

• Process of applying dyes on the sections agent or crude dye that is generally applied in an
• To see and study architectural pattern of the aqueous solution.
tissue and the physical characteristics of the • The actual staining process may involve
cells immersing the sample (before or after fixation
• To make the tissues and cells become more and mounting) in dye solution.
visible
• To easily identify morphologic changes in the Staining of Paraffin Section
tissues/cells • After staining, the section is again dehydrated
• To establish presence or absence of a disease with increasing grades of alcohol and cleared
process with two changes of xylene to prepare the section
• The main reason why cells are stained is to for mounting, since most mountants are miscible
enhance contrast and visualization of the cell or in xylene.
certain cellular components under a microscope. • The second change of xylene will also raise the
• Cells may be stained to highlight metabolic refractive index of the glass slide, thereby
processes, to differentiate between live and reducing light refraction during microscopic
dead cells in a specimen, to demonstrate the examination.
relationship between internal and external • The stained section may be left in xylene for an
structures of the cells, and to identify different indefinite period of time until it is finally mounted
types of cells. on the slide.
Page 299
• The section should not be allowed to stay in Three Major Groups of Staining
alcohol for a long time because many stains are
usually removed by prolonged immersion in Histological Staining
alcohol. • Tissue constituents are demonstrated in sections
• After the section is cut and mounted on the by direct interaction with a dye or staining
slide, it is drained and dried thoroughly to solution
ensure that all moisture between the section • Micro-anatomic stains
and slide has evaporated, and that the section • Bacterial stains
is firmly attached to the slide. • Specific tissue stains
• If drying is not complete, the section (or part of • Used to demonstrate the general relationship of
it), especially from bone and nervous tissue, tissues and cells with differentiation of nucleus
may become detached from the slide during the and cytoplasm
process of staining, usually after adding the • Histologists have developed many stains which
acid differentiator. are suited to particular purposes, allowing cell
• Sections may float off the slide during staining if structures to be differentiated.
the slides are dirty or greasy, or if the sections • It is important to remember that the colors of
have not been left in the paraffin oven long stains are not the real color of a particular tissue,
enough to dry and be fixed in the slide. and that a structure that appears as one color
• Sections must be left in the oven for a minimum using one stain, may be a quite different color
of 30 minutes before they are finally stained to using another stain
avoid such problems.
Page 300
Histochemical Staining/Histochemistry Immunohistochemical Staining

• Tissues are studied thru chemical reactions that • Combination of immunologic and histochemical
will permit microscopic localization of specific techniques that allow phenotypic markers to be
tissue substance detected and demonstrated under the
• Perl’s Prussian Blue – Hemoglobin microscope, using a wide range of polyclonal or
• Periodic Acid Schiff – Carbohydrates monoclonal, fluorescent labeled or enzyme
• Enzyme histochemistry labeled antibodies.
• The active reagent serves as a substrate • Immunohistochemical staining is widely used in
where enzymes act. the diagnosis of abnormal cells such as those
• The final color produced is from the found in cancerous tumors, in the localization of
substrate not on the tissue. biomarkers and differentially expressed proteins
• In many instances, histochemical methods used in different parts of a biological tissue, and in the
to stain several chemical constituents will also detection of specific molecular markers that are
ultimately stain the tissue itself, thereby characteristic of particular cellular events such as
producing an overlapping of techniques. proliferation or cell death (apoptosis)
• The staining techniques employed for • Immunohistochemical staining techniques are
histochemistry are also usually applied for used to label defined antigens with monoclonal
staining of histologic structures. and polyclonal antibodies.
• Commercially produced antibodies most
frequently originate from mice, and less
frequently from rabbits
Page 301
• Unlike conventional histological staining Hematoxylin

methods, immunohistochemical techniques are • Hematoxylin campechianum
based on antigen–antibody bindings, which can • Not a stain
be affected by inappropriate fixative selection • HEMATIN – active coloring agent
and duration • Most frequently used with mordant
• Alum
Two Categories • Iron
• Natural Dyes • Copper
• Synthetic Dyes
Cochineal Dyes (Carmine)
Natural Dyes • Scarlet dye made from the ground bodies of
• Obtained from plants and animals female cochineal bug (Coccus cacti)
• Staining of basophilic cell structures such as • A powerful chromatin and nuclear stain for fresh
nuclei and some cytoplasmic substances material and smear preparations
(cationic) • Picrocarmine
• Derived from plants and animals, previously • Used in neuropathological studies
used for dyeing wool and cotton • Best Carmine stain
• Hematoxylin • Used for glycogen demonstration
• Cochineal dyes
• Orcein
• Saffron
Page 302
Orcein • Quinone-amine group (OXAZIN and

• Vegetable dye extracted from certain lichens THIAZINS)
which are normally colorless, but when treated
with ammonia and exposed to air, produce Auxochromes
blue/violet colors. • Substance which imparts to the compound the
• Used for staining elastic fibers property of electrolytic dissociation to retain the
color of the tissue
Saffron • CATIONIC (Amino group)
• ANIONIC (Hydroxyl and Carboxyl group)
Synthetic Dyes (Coal Tar Dyes)
• Derived from hydrocarbon benzene Dye modifiers (attached on benzene rings)
• Collectively known as aniline dyes • Ethyl groups
• Consist of a chromophore and auxochrome • Methyl groups
group attached to a hydrocarbon benzene ring • Sulphonic Acid
• They were originally manufactured from
substances that have been taken from coal tar. Dye-to-Tissue Mechanisms
• Electrostatic
Chromophores • Majority of tissue-dye reactions
• Substance with definite atomic groupings and • Neutral red and Light green
are capable of producing colors
• Quinoid rings • Hydrogen bonding
• Azo groups • Congo Red, Carmie, Weigert-type
• Xanthene resorcinol dye
Page 303
• Van der Waals forces Basic Dyes

• Alum hematoxylin solutions • The active coloring substance is found in the
basic component
• Physical staining • Cationic dyes and stain nuclei, basophilic
• Sudan dyes granules or bacteria
• SUDANOPHILIA – property of tissues to
be stained with fat soluble dyes Neutral Dyes
regardless of the type of dye used due to • Formed by combining aq. solutions of acid and
their essential lipid nature basic dyes
• Stains cytoplasm and nucleus simultaneously
• Natural affinity and differentially
• Janus Green
Methods of Staining
Classification of dyes depending on where the According to the presence of mordant:
chromophore is found
Direct Staining
Acid Dyes • Process of giving color to the sections by using
• The active coloring substance is found in the aqueous or alcoholic dye solutions:
acid component • Methylene Blue
• Anionic dyes and stain mainly cytoplasm, • Eosin
eosinophilic granules • No mordant is used
Page 304
Indirect Staining According to the presence of a differentiator /

• Process whereby the action of the dye is decolorizer:
intensified by adding another agent
• Uses mordant Progressive Staining
• Process whereby tissue elements are stained in
Mordant a definite sequence and the staining solution is
• Serves as a link or bridge between the tissue applied for specific periods of time or until the
and the dye desired intensity of coloring of the different tissue
• Potassium alum with hematoxylin (Ehrlich’s elements is attained.
hematoxylin) • No differentiator
• Iron (Weigert’s hematoxylin) • H/E for frozen sections
Accentuator Regressive Staining

• Accelerates or hastens the speed of the • Tissue is first over-stained to obliterate the
staining reaction by increasing the staining cellular details and the excess stain is removed
power and selectivity of the dye or decolorized from unwanted parts of the tissue,
• Potassium hydroxide (Loeffler’s methylene until the desired intensity of color is obtained.
blue) • With differentiator
• Phenol (Carbol thionine and Carbol fuchsin) • H/E for routine histological staining
Page 305
Differentiation/Decolorization • Epithelial mucins

• Selective removal of excess stain from the • Mast cell granules
tissue during regressive staining • Amyloid
• Done by washing the section in simple solution
(water/alcohol) or by the use of acids and Metachromatic Dyes
oxidizing agents. • Basic dyes belonging to thizine AND
• Alcohol acts as a differentiator for both basic triphenylmethane groups
and acidic dyes • Methyl violet or crystal violet
• Cresyl blue (reticulocytes)
According to resultant color: • Safranin
Ortochromatic Staining • Bismarck brown
oSubstances are stained with a color that is the • Basic fuchsin
same from that of the dye used. • Methylene blue
• Thionine
Metachromatic Staining • Toluidine blue
• Entails the use of specific dyes which • Azure A, B, C
differentiate particular substances by staining
them with a color that is different from that of
the stain itself
• Employed for staining:
• Cartilage
• CT
Page 306
Vital Staining Counterstaining

• Selective staining of living cell constituents, • Application of a different color or stain to provide
demonstrating cytoplasmic structure. contrast and background to the staining of the
structural components to be demonstrated.
Intravital Staining
• Injecting the dye into any part of the animal Metallic Impregnation
body producing specific coloration of certain • Process where specific tissue elements are
cells particularly those of the reticulo-endothelial demonstrated, not by stains, but by colorless
system solutions of metallic salts.
• Lithium, Carmine, India Ink • Characteristics:
• Structures demonstrated are opaque and
Supravital Staining black
• Stain living cell after removal from the • The colouring matter is particulate
immediately after removal from the living body. • The deposit is on or around but not in the
• Neutral red (best vital dye) element so demonstrated
• Janus green (mitochondria)
• Trypan blue Factors Influencing Dye Binding
• Nile blue • pH of the solution
• Thionine • Increase in temperature
• Toluidine blue • Increase concentration of dye molecules
• Presence of other salts
Page 307
H & E Staining • Example:

• Most widely-used histological stain • Erlich’s
• Hematoxylin – blue black nuclei • Deladfield’s
• Eosin – pink, ornage, red cytopasm and C.T.
fibers Ripening/Oxidation
• Done by:
Hematoxylin • Exposing the substance to air and sunlight
• Extracted from H. campechianum (slow)
• A natural dye derived from the extraction from • Adding oxidizing agents (H2O2, Mercuric
the heartwood Mexican tree oxide, Potassium permanganate, Sodium
perborate, Soidium iodate)
Hematein
• Responsible for the color properties Mordants for Hematein
• Anionic and inadequate as a nuclear stain • Aluminum, Iron, Tungsten, Lead
• Are cations binds the dye to tissue
Chemical oxidation
• Using sodium iodate, mercury oxide Alum Hematoxylin
• The same mordant, different ripening agent
Natural oxidation • Mordant: Potassium aluminum sulfate / “alum”
• Exposure to light and air • “Potash alum”
• Takes as long as 3-4 months • Can be used progressively and progressively
Page 308
• Produce good nuclear stain Cole’s 20 – 40 min

• Nuclear is red, then turns blue-black Delafield’s 15 – 20 min
using the procedure of “bluing” Ehrlich’s (progressive) 20 – 45 min
• Appears RED upon contact with acid Mayer’s (progressive) 10 – 20 min
• Stain times with alum hematoxylins Mayer’s (regressive) 5 – 10 min
• Factors affecting staining time with alum Haris’s (progressive in 4 – 30 sec
hematoxylins: cytology)
• Type of hematoxylin used Haris’s (regressive) 5 – 15 min
• Age of stain Carazzi’s (progressive) 1 – 2 min
• Intensity of use of stain Carazzi’s (regressive) 45 sec
• Whether stain is used Carazzi’s (frozen 1 min
progressively or reggressively section)
• Pre-treatment of tissues or Gill’s I (regressve’s) 5 – 15 min
sections
• Post-treatment of sections Disadvanatges of Alum Hematoxylins
• Personal preference • Nuclear Stain are sensitive to any subsequently
applied acidic stains
• Satisfactory nuclear staining can be done in
combination with celestine blue staining solution
Page 309
• Stained sections fade much slowly than other

Celestine Blue Solution
hematoxylins
Celestine blue B 2.5 g
• Suitable for t-shirt that have been exposed to
Ferric Ammonium 25 g acids
Sulfate • Not ideal for frozen sections
Glycerin 70 ml
Preparation of Solution
Distilled water 500 ml
Hematoxylin 2g
Gill’s Hematoxylin Absolute alcohol 100 ml
• Double or triple hematoxylin concentrations Glycerin 100 ml
• More frequently used for routine H and E than Distilled water 100 ml
Mayer’s Glacial acetic acid 10 ml
• More stable than Haris’s Potassium alum 15 g approx.
• Disadvantage:
• Staining of adhesive and the glass Delafield’s Hmeatoxylin
• Stained mucus darkly • Naturally ripened with similar longevity with
Ehrlich’s
Ehrlich’s Hmeatoxylin Preparation of Solution
• Takes 2 months to ripen Hematoxylin 4g
• An excellent nuclear stain 95 % alcohol 120 ml
• Also stains mucins and recommended for Sat. aq. Ammonium 400 ml
staining of bone and cartilage alum (15 g/100ml)
• Can be chemically oxidized using sodium iodate Glycerin 100 ml
• Stains nuclei intensity and crisply
Page 310
Harris’s Hematoxylin Preparation of Solution

• Traditionally ripened using mercuric oxide Hematoxylin 1.5 g
• Useful as general purpose hematoxylin- Sat. aq. Ammonium 700 ml
diagnostic stain for exfoliative cytology alum (15 g/100ml)
• Most often used for progressive staining with 1% iodine in 95% 50 ml
acetic acid-alcohol rinse alcohol
Preparation of Solution
Hematoxylin 2.5 g Carazzi’s Hematoxylin
Absolute alcohol 25 ml • Chemical ripened with potassium iodate
Potassium alum 50 g • Suitable as progressive nuclear counterstain
Distilled water 500 ml since it is a pale and precise nuclear stain
Mercuric oxide 1.25 g • Used for frozen sections from urgent surgical
Sodium iodate 0.5 g biopsy
Glacial acetic acid 20 ml • Excellent and clear nuclear staining when used in
double and triple strength
Cole’s Hematoxylin • Carazzi’s Hematoxylin and Eosin for FROZEN
• Artificially ripened with an alcoholic iodine SECTIONS
solution • Freeze suitable tissue block on to a chuck
• Cut cryostat section at 3-6 mm thickness
• Fix section in 10% NBF at RT for 20
seconds
• Rinse in tap water
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• Stain in double strength Carazzi’s • Use in Celestine blue hemalum method of

hematoxylin for 1 minute nuclear staining
• Wash well in tap water wash well in tap • Deparaffinize sections, rehydrate
water for 10-20 seconds • Stain in celestine’s blue for 5 minutes
• Stain in 1% aqueous Eosin for 10 • Rinsed in distilled water
seconds • Stain in Mayer's hematoxylin for 5 minutes
• Rinse in tap water • Wash in water until blue
• Dehydrate, clear and mount • Proceed with required staining technique
Preparation of Solution Preparation of Solution

Hematoxylin 5g Hematoxylin 1g
Glycerol 100 ml Distilled water 1000 ml
Potassium alum 25 g Potassium or 50 g
Distilled water 400 ml ammonium alum
Potassium iodate 0.1 g Sodium iodate 0.2 g
Citric acid 1g
Chloral hydrate SLR 50 g
Mayer’s Hematoxylin
Chloral hydrate AR 30 g
• Chemical ripened with sodium iodate
• Useful as progressive stain
• Used as nuclear counterstain in demonstration
of glycogen
• Applied for 5-10 minutes
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Iron Hematoxylin Heidenhain’s Hematoxylin

• Iron salts are used as oxidizing agents and • Mordant/oxidant/differentiating fluid: ferric
mordants ammonium sulfate
• Standard iron hematoxylin • Used to demonstrate structures according to
• For muscles/CT fibers degree of differentiation
• *Van Gieson’s stain – good for • Results: all components are black or dark gray
demonstrating collagen black
• Can be all demonstrated four:
Weigert’s Hematoxylin • Mitochondria
• In combination with van Gieson’s stain, can • Muscle striations
demonstrate CT elements and Entamoeba • Chromatin
hystolytica sections • Myelin
• Uses ferric chloride as a mordant/ oxidant • Staning time for tissue fixed in Susa’s, Bouin’s,
• Iron and hematoxylin solutions are prepared Carnoy’s: 1 hour
separately and are mixed immediately before • Staining time for tissue fixed in Helly’s or Zenker:
use 3 hours
• Working solution remains active for 1 – 2 days • Staining time for tissue fixed in Osmium
• Mixture is violet-black tetraoxide and Flemming’s fluid: 24 hour
• Use as a nuclear stain where acidic stains are • Cytoplasmic counterstain: aq. Eosin or Orange G
to be applied to tissue subsequently • Sections should be wash well after differentiation
• Useful stain for CNS tissues stage to resist fading of sections
• Staining time: 15 – 30 minutes
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Loyez Hematoxylin Tungsten Hematoxylin

• For myelin • Most widely used tungsten hematoxylin: Mallory’s
• Mordant: Ferric ammonium sulfate PTAH
• Differentiator: Weigert’s differentiator (borax • To ripen, stand in the light for several weeks or
and potassium ferricyanide) use potassium for immediate ripening
• Used to demonstrate myelin, and can be • For staining muscle striations
applied to paraffin, frozen or nitrocellulose • Mallory PTAH
sections • Mallory combine hematoxylin with 1% aq.
• 2 methods: Phosphotungstic acid (mordant)
• Heidenhain myelin stain • Oxidant: Potassium permanganate (usable
• Weil technique for 24 hours only)
• Ripening: usable for years
Verhoeff’s Hematoxylin • Applicable for CNS tissue and general
• Used to demonstrate elastic fibers tissue structure
• Contains ferric chloride, Lugol’s Iodine, 2% aq.
Ferric Chloride (differentiator) Molybdenum Hematoxylins
• Intense black staining produced is thee most • Rare
ideal for photomicrography • Most accepted technique: Thomas technique
• Recommended for demonstration of collagen and
coarse reticulin, and argentaffin cell granules
• Differentiator: Picro-acetic alcohol
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Lead Hematoxylins • Three forms:

• Used in demonstration of granules and tumors • Eosin Y (Yellowish) – most commonly
in the endocrine cells of doubtful origin used
• Also used in localization of gastrin secreting • Bluish – gives deeper red color
cells in stomach • Ethyl eosin/Eosin S
• Xanthine dyes:
Copper Hematoxylin • Eosin Y
• Used for spermatogenesis studies • Most widely used
• Water and alcohol soluble
Eosin • Usually used as 0.5% - 1.0%
• More suitable stain to combine with an alum solution in distilled water with
hematoxylin crystal thymol
• Has the ability of differentiating cytoplasm of • Ethyl eosin
different types of cells, CT fibers and matrices • Eosin B
• Differentiation occurs in subsequent tap water
wash and during dehydration with alcohol Routine H and E Staining
• Secondary stain, cytoplasmic stain, acidic stain Steps:
• Used as a counterstain after hematoxylin and 1. 2 changes of xylol (for deparaffinization)
before methylene blue 2. DESCENDING grade of OH (for rehydration)
3. WATER
4. Hematoxylin
5. Rinse with tap water
6. Acid OH (differentiator)
Page 315
7. Ammonia water (bluing) 5. Differentiate in 1% acid alcohol for 5-10 seconds

8. Rinse with tap water 6. Wash well in tap water until sections again blue
9. Eosin 10-15 minutes
10. ASCENDING grade of OH 7. Blue by dipping in alkaline solution
11. Xylol 8. Dip in tap water for 5 minutes
12. Mount and label 9. Stain in 1% eosin Y for 10 minutes
10. Wash in running water for 1-5 minutes
Result: 11. Dehydrate through alcohol, clear and mount
• Nuclei – blue to blue black
• Karyosome – dark blue Papanicalaou stain for Cytological Preparations
• Cytoplasm, proteins in edema fluid – pale pink Papanicolaou Formula
• Calcium and calcified bone – purplish blue Harris’s Hematoxylin
• Muscle fibers – deep pink/red Orange G 6
• Fibrin – deep pink 10 % aq. Orange G 50 ml
Alcohol 950 ml
Routine Staining Procedure using Alum Phosphotungstic acid 0-15 g
Hematoxylin: EA 50
1. Deparaffinize section, rehydrate 0.04 M light green Sf 10 ml
0.3 M eosin Y 20 ml
2. Remove fixation pigments
Phosphotungstic acid 2g
3. Stain in alum hematoxylin of choice for suitable Alcohol 750 ml
time Methanol 250 ml
4. Wash with running water until sections ‘blue’ Glacoal acetic acid 20 ml
for 5 minutes or less
Page 316
Papanicolaou Staining Method: Papanicolaou Staining Results:

1. Remove PEG fixative in 50% alcohol, 2 MIN • Nuclei – blue/black
2. Hydrate in 95% alcohol, 2 min, and 70% • Cytoplasm (non-keratinizing squamous cells) –
alcohol, 2 min blue/green
3. Rinse in water, 1 min • Kerainizing cells – pink/orange
4. Stain in Haris’s hematoxylin, 5 min
5. Rinse in tap water, 2 min Other Stains Used
6. Differentiate in 0.5% aq. HCl, 10 sec Benzidine
7. Rinse in tap water, 2 min • For staining hemoglobin
8. Blue’ in Scotts tap water substitute, 2 min
9. Rings in water, 2 min Acridine orange
10. Dehydrate in 70%, 95%, 95% alcohol for 2 min • DNA (green fluorescence)
in each solution • RNA (red fluorescence)
11. Stain in OG 6
12. Rinse in two changes of 85% alcohol for 2 min Crystal violet
each • For staining amyloid in frozen sections and
13. Stain in EA 50, 3 min platelets in blood
14. Rinse in 95% alcohol
Gentian violet
• Formed by the mixture of:
• CV
• Methyl violet
• Dexterin
Page 317
Congo red Janus green

• Stain for axis cylinders in embryos • Used to demonstrate mitochondria during
• Used as 4% aq soln in Kraijan’s mtd (elastic intravital staining
tissues, amyloid and myelin)
Night blue
Iodine • A substitute for carbol fuchsin in AFS
• Oldest stain
• For: Van Gieson’s stain
• Amyloid • Mixture of picric acid and acid fuchsin used to
• Cellulose demonstrate CT
• Starch
• Carotenes Acridine Red 3B
• Glycogen • Used to demonstrate calcium salt deposits and
• Widely used for removal of mercuric fixative possible sites of phosphatase activities
pimets
Alcian blue
Malachite green • Stain acid MPS
• Contrast stain for staining Ascaris eggs and • More specific for CT and epithelial mucin
RBCs
• Used also as a bacterial spore stain Bismarck brown
• For diphtheria organisms
Page 318
Giemsa Victoria blue

• For staining blood to differentiate leukocytes • Used for the demonstration of neuroglia in frozen
tissues
Gold sublimate
• Used for metallic impregnation Lysochromes (Oil soluble dyes)
• Not real dyes
Orcein • Do not have auxochrome groups
• Excellent stain for elastic fibers • Used for demonstration of intracellular fats
• Recommended in dermatological studies • Sudan Black B (black)
• Most sensitive
Osmium tetroxide • Sudan III (orange)
• Stains fat • First sudan dye to be introduced in
histochem
Rhodamine B • For CNS tissues
• Stain blood and glandular tissues • Sudan IV / Scharlach R (red)
• Addition of benzoic acid –
Silver nitrate intensifies fat and prevent rapid
• Used in identification of spirochetes, reticulum deterioration of solutions
and other fiber stains • Recommended for staining
triglycerides
Toluidine blue
• Recommended for staining Nissl granules
Page 319
Causes of poor quality of staining

• Poor or inadequate fixation of tissue.
• Over or under-ripened Haematoxylin.
• Overused or worked out Haematoxylin.
• Over or under differentiation of haematoxylin
• Insufficient blueing following differentiation.
• Failure to wash blueing agent out of section
before counter staining with eosin (especially
when ammonia is used).
• Insufficient differentiation of eosin during
washing or dehydration.
• Insufficient dehydration and clearing of sections.
• Contamination of stains.
Page 320
14
Mounting
Media
Page 321
Mounting media • Two Groups

• Done AFTER staining • Aqueous Mounting Media
• Preventing the movement of the cover slip • Resinous Mounting Media
• Prevent distortion of image during microscopy
Aqueous mounting media
• Designed to mount water-miscible preparations
directly from water
• Water
• Low refractive index, moderately
transparent and evaporates easily
• Good only for temporary mounting
• Glycerin
• Also used as a preservative
Characteristics of a good mounting medium • High refractive index, lasts for a few
• The refractive index of the mountant must be minutes
close to the glass slide*** • Suitable for semi-permeable
• Freely miscible with xylene or toluene*** mounting medium (RI = 1.46)
• Do not dry quickly*** • Glycerin Jelly (Kaiser’s 1880)
• Do not crack or produce artefactual granularity • Farrant’s Medium
on the slide upon drying • RI = 1.43
• Do not dissolve out or fade tissue sections • Sodium Merthiolate/Arsenic trioxide: used
• Do not cause shrinkage and distortion of tissue for preservation of the medium
• Do not affect staining*** • Require ringing
Page 322
• Apathy’s Medium • Canada Balsam

• RI= 1.52 • RI= 1.524
• For methylene blue stained nerve • Natural resin extracted from for a
preparations Canadian tree (Abus balsamea)
• Potassium acetate/NaCl: prevent • For whole mounts and thick sections
“bleeding” of metachromatic stains for • Quite expensive
amyloid • DPX
• Does not require ringing • RI=1.532
• Brun’s Fluid • For small tissue sections
• For frozen sections from water • XAM
• RI=1.52
Resinous mounting media • Synthetic resin
• For preparations that have been dehydrated • CLARITE/CLARITE X
and cleared in xylene or toluene • RI=1.544
• For majority of staining methods • Synthetic resin
• Divided into: • Permount
• Natural resins • Clearmount
• Synthetic resins: for undecalcified bones, • Harleco Synthetic Resin (HSR)
EM, LM
Page 323
Ringing Example of Coated Slides

• Sealing the margins of the cover slip to prevent
the escape of fluid or semi-fluid mounts and Silanized slides
evaporation of mountant • Are prepared by cleaning the slides by washing,
• To immobilize the coverslip followed with a rinse in 95% ethanol.
• To prevent sticking of the slides upon • 4 ml of 3-aminopropyltriethoxy silane is added to
storage 200 ml of acetone and slides are dip for 30-60
• Kronig cement seconds, followed by 60 seconds in agitated
• Duroflix (cellulose adhesive) distilled water.
• Then, slides are dried for 1 hour
Coating of Slides
• To act as an adhesive to keep difficulty tissue Polylysine slide
specimens attached • Are prepared similarly to silanized slides.
• To aid to aid in cellular attachment when cells • Slides are washed followed by 95% ethanol.
are to be grown on slides or coverslips • Immerse non-frosted portion in a 5% polylysine
• To make staining more consistent when for more than 1 minute.
staining small structures spread across • Dry for an hour.
microscope slides
Albumen- coated slides
• Slides are washed followed by 95% ethanol.
• Immersed non- frosted portion of slide in 5%
crude egg albumin for 1 minute.
Page 324
• Dry for an hour and then fix in 10% NBF for

over 1 hour
Chrome alum gelatin slides or ‘subbed slides’

• Slides are washed followed by 95% ethanol.
• Spread a working solution of mixed gelatin and
chromic potassium sulfate over slides and dry
for at least 1 hour.
Page 325
15
Frozen &
Related Sections
Page 326
Uses For Frozen Sections

• Rapid production of sections for intra-operative
diagnosis
• Diagnostic and research enzyme histochemistry
• Immunofluorescent methods
• Immunohistochemistry methods
• Diagnostic and research non-enzyme
immunohistochemistry
Cryostat
• A refrigerator cabinet in which specialty
microtome is housed
• To produce thin and high-quality frozen sections Freezing of Fresh Unfixed Tissue
• Maintained at temperature between -5C to -30C • BEST frozen sections are obtained when the
• Freezes tissue within 2-3 minutes tissue is frozen quickly
• Cut sections 4u in thickness • Techniques:
• Liquid nitrogen (-190C)
• Isopentane cooled by liquid nitrogen (-150
C)
• Dry ice (-70 C)
• Carbon dioxide gas (-70 C)
• Aerosol sprays (-50 C)
Page 327
Fixed Tissue and Cryostat • Temperature control of ultra-cryotomes is set

• For most diagnostic procedure in the laboratory between -20C and -212C
• Fixed: to localize hydrolytic enzymes and other • -180C, suitable for most tissues
antigens • Useful for localization of enzymes for
• Tissue must be fresh and place in formal ultrastructural level
calcium at 4 C for 18 hours
Freeze-Drying
Cryostat Sectioning • Rapid freezing of fresh tissue at -160C and
• Most unfixed material will section well between - subsequent removal of water molecules by
15C to -23 C sublimation in a vacuum at higher temperature (-
• Warmer temp: for tissues containing large 40C)
amount of water • Technique minimizes:
• Colder temp: for harder tissues and tissues • Loss of soluble substance
containing fats • Displacement of cell constituents
• Fixed material section well between -7C to -12C • Chemical alteration of reactive groups
• Denaturation of proteins
Ultracryotomy • Destruction or inactivation of enzymes
• Used primarily in research laboratories
• Involves rapid freezing of fixed or unfixed tissue Four Stages to Freeze Drying
by using isopentane • Quenching
• Sections cut into 50-150 nm • Drying
• Vapor Fixation
• Embedding
Page 328
Applications and Uses of Freeze-dried • Advantage:

Materials • Easy
• Immunohistochemical methods • Convenient
• Demonstration of hydrolytic enzymes • Labor- saving
• Fluorescent antibody studies
• Autoradiography Additional Notes:
• SEM
Definition / general
Freeze Substitution • A frozen section (cryosection) is a pathological
• Similar with freeze-drying the only difference is laboratory technique used for rapid microscopic
the fixing of tissue in Rossman’s fluid or in analysis / diagnosis of a specimen / disease
Osmium tetroxide in 1% acetone for 1-6 days at • Usually used with oncologic surgery
-60C to -70C • Rapid diagnosis can guide intra-operative patient
• Then dehydrated in Abs. alcohol or acetone management
Frozen Section Substitution Indications

• Rapid freezing of tissue in isopentane super • Use frozen section to
cooled in liquid nitrogen • Provide rapid gross or microscopic
• Cryostat sections are cut at 8-10um diagnosis to identify an unknown
• And transferred to acetone and cooled at -70C pathologic process, identify extent of
for 12 hours disease / evaluate margins, identify
metastases or simply identify a tissue
Page 329
• Process tissue to provide appropriate Tissue type

and accurate diagnosis, prognosis and to • Tissue should be received fresh, otherwise it will
adhere to research and special study not stay on slide
protocols • At time of receipt of tissue, decide whether to
• Confirm that pathological tissue is obtain smears or touch preps and whether to
present for diagnosis on permanent freeze all or part of it
sections • Touch preps and smears are often
• Do not use if performed on lymph nodes suspicious for
• Frozen section diagnosis has no lymphoma
immediate implications for decision • Some primary small lesions should not be
making entirely submitted for frozen section
• Tissue is needed for permanent • There is debate on whether sentinel nodes
processing (is unique or small or requires should be entirely or representatively
extensive study for diagnosis) submitted for frozen section
• Consider not freezing tissue if • Fixed tissue:
• Frozen section is known to produce • There are special slides to keep tissue
severe artifacts that hinder proper affixed to slide
interpretation • To freeze fixed tissue, make sure it has
• Tissue is heavily ossified / calcified been preserved in formalin and not
• Risk of serious infection (HIV, TB, alcoholic fixatives like Carnoy's, because
hepatitis B or C) tissue fixed in alcohol is harder to freeze
• Tissue is fatty
Page 330
• Avoid freezing tissue fixed with heavy • Place the chuck into a -20 to -15 degree (optimal)
metal salts such as B5 and Helly's cryostat
(Zenker’s formal solution), which can • Note that the OCT media should not be
denature proteins and shrink the tissue frozen completely
• Avoid hard tissues like bone and • It is better to have a semisolid consistency
cartilage that require decalcification • This will alleviate tissue artifact
• Avoid tissues with a lot of fat • Tissue size should be no greater than 3mm -
• Avoid tissues from patients with known 5mm in greatest dimension (thinner specimens
TB or other infection (if absolutely have shorter freezing time and minimal ice crystal
necessary, wear appropriate protection) artifact formation)
• Avoid freezing tissue that will be needed • The smaller the tissue, the more even and
to make a permanent diagnosis thorough the freeze
• Place the tissue on the semisolid chuck and add
Freezing tissue more media rapidly over the tissue, covering it
• OCT (optimal cutting temperature) or similar entirely but avoiding overflow
embedding media like TBS or Cryogel should • Place chuck quickly back into the cryostat
be placed on an appropriately sized chuck that • Apply heat sink or CO2 aerosol (optional) to
has been precooled in a cryostat rapidly freeze or use "quick freeze" option on
• The chuck should be clean cryostat
• A toothbrush is useful to remove tissue and
OCT
• Dipping the chuck in methanol removes ice
crystals
Page 331
• Histobath: being phased out 1) Once the chuck is in position, there should be a
• Cryowells: useful in keeping all tissue on manual or an automatic advance option to move
an even plane; also helpful in eliminating the block close to the cutting blade
loss of smaller tissues that are frozen 2) Fully face the tissue by using a trim setting on
with larger ones, although recommended your cryostat
to not freeze different sizes together • If you do not have this setting, then an
• Aerosol sprays: often canned CO2 (but advance button should be available, which
may aerosolize infectious diseases) should be pressed each time before one
• Liquid nitrogen full revolution of the instrument's wheel
• Isopentane based workflow (Virchows 3) If wells are used to freeze the blocks, then the
Arch 2008;452:305) tissue should be on an even plane and the
tissue will be faced faster
Cryosectioning 4) To polish the tissue, avoid advancing the
cryostat or deselect the trim setting on the
cryostat and turn 10 - 15 times
5) As you cut the tissue, anchor the tissue to
prevent folding or curling
• This can be done with an anti-roll bar (a
plastic plate attached to cryostat) or by
using a precooled paintbrush with stiff
bristles and a wide gripping surface
Tissue embedded within OCT 6) The brush should be held like a pen with your
left hand at an angle
Page 332
7) You can rest your fifth finger on the stage for 11)Move the brush as the chuck moves towards the
stabilization blade
8) Cutting the brushes' bristles at an angle can • Your brush should move down in pace
aid in the brush meeting the tissue flat over its with the chuck
length because you will hold it at an angle
Left: brush with angled tip; right: holding the brush Riding the block: as the block descends toward the
brush, the brush keeps pace
9) Turn the wheel with your right hand in a with the block by gently resting on the bottom 2 - 3
continuous motion without stopping; avoid mm of the block
speeding up or slowing down
10)Avoid stopping the wheel at the beginning of 12)You can rest your brush softly on the very
the section, slowly grabbing the tissue and bottom of your chuck avoiding tissue contact
then resuming wheel revolutions; this can 13)Pull the brush away easily as the chuck meets
cause artifacts such as variation in section the blade
thickness and tissue folding
Page 333
Catching the curl: as the block meets the blade Pull over the blanket: the brush holding the curl
and the section begins its curl, the brush leaves pulls the section horizontally over the stage, like
the block while catching the curling edge of the pulling the blanket over yourself, without pressing
section; then the brush jumps off the block with the tissue to the stage
the curl
15)A glass slide is gently laid upon the tissue
14)The downward motion of the brush allows you section
to keep a continuous motion as you take your
section
Page 334
Staining slides
• Keep all stains and solutions fresh and well
maintained
• Dip slide in reagents in this order for H&E
staining:
• After obtaining frozen section,
IMMEDIATELY fix in 95% ethanol (even
Gently touch the section to the slide; avoid 15 seconds of delay can cause significant
stretching or folding the section by keeping a artifact)
steady hand, and keep the transverse axis of the • Formal alcohol, formalin or 95% alcohol:
slide parallel to the section 45 - 60 second
• Water: 5 - 7 seconds
16)The tissue section should melt onto the slide • Hematoxylin: 60 seconds
17)Prepared slides should immediately go into • Lithium carbonate or 0.2 % aqueous
formal alcohol, 95% alcohol (methanol/ethanol) ammonia (Bluing): 15 - 20 seconds
or formalin while awaiting the stain line • Eosin: 20 - 60 seconds
• If you delay this step, drying artifact will • 95% alcohol: 10 seconds
occur • 100% alcohol: 10 seconds
18)You can take a deeper level after • Xylene, toluene, limonene derivatives and
approximately 20 turns (multiple levels may be Clearite: 10 seconds
needed for breast or prostate biopsies) • Then add mounting media for cover
19)Optimal cutting thickness is 4 - 7 microns for slipping
sectioning and 20 - 40 microns for trimming
Page 335
Troubleshooting • Solution:
• Ice crystal artifacts • Change your blade every few cases
• Due to slow freezing of tissue • Some institutions use a new blade
• Solution: for each case
• Freeze fast (flash / snap) • Over freezing
• The faster the freeze, the smaller • Can cause section to have holes
the ice crystals, the less tissue • Solution:
damage (best freezing method is • Polish block with a couple extra
arguably liquid nitrogen) turns of the blade to create friction
• Smaller tissues yield less artifact - and warm up block by pressing on it
optimally tissue should be 0.5 x 0.5 x 0.3 with your finger (5 - 10 seconds)
cm or less • Under freezing
• Never freeze fragments larger than the • Under freezing can be troublesome for
diameter of the chuck fatty tissue
• Avoid freezing fat around tissue • Solution:
• Blot the outer surface of the tissue dry • Add heat sink to block or select
with gauze before making your block rapid freeze setting on your cryostat
• Knife artifact (if available)
• A nicked cutting blade will produce a split • Staining issues
/ tear in your section • Dirty "stain line" can cause floaters
(extraneous foreign tissue) to adhere to
slides
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• Overly diluted stains and alcohols can • Solution:

diminish slide quality • Maintain an extremely cold cutting
• Poor staining hinders frozen section temperature (-20C)
diagnoses, as nuclear detail is • Firm lymph nodes, spleen, brain and liver
compromised cut better at -10C
• Solutions: • Rissue may shatter if sectioning is
• (a) maintain a clean stain line by performed at lower temps
frequent solution changes • Air bubbles
• (b) follow recommended staining • May be trapped under cover slips, which
times can cause the underlying tissue to dry out
• (c) don't rush • Solution:
• Note: brain tissue may stain best in eosin • Make sure an appropriate amount
for 60+ seconds of resin (2 drops) is applied
• Water: should be changed after each • Gently move air bubbles off the
frozen section slide with finger or tweezers
• Alcohols and stains: change at least • Do not press on the slide too hard
weekly, alcohols may need to be or it will break
changed more frequently depending on • Overly thick sections
work load • May cause tissue to fall off slide
• Fatty tissue • Solution:
• Includes lymph nodes, breast, skin • Reduce the cryostat's sectioning
• May be too soft to cut thickness
Page 337
16
Basic Diagnostic
Cytology
Page 338
Basic Diagnostic Cytology Specimen for Examination

• Microscopic examination of cells from different • Vaginal smears
body sites • Endometrial and endocervical smears
• Two divisions: • Prostatic and breast secretions
• Exfoliative Cytology • Gastric or bronchial secretions
• Fine Needle Aspiration (FNA) • Pleural and peritoneal fluids
• Sputum
Exfoliative Cytology • Smears of urine sediment
• Microscopic study of cells that have been • Cerebrospinal fluid
desquamated from epithelial surfaces.
Adhesion
Importance • Some specimens require addition of adhesive
• For assessing malignant or cancerous agent to provide adequate adhesion of smeared
conditions material throughout fixation and staining.
• For detection of asymptomatic cancer in women • Urinary sediment
• For assessment of female hormonal activity in • Bronchial lavage specimen
case of sterility and endocrine disorders • Specimen that utilizes proteolytic enzymes
• Determination of sex during processing
• Determination of the presence of possible • Trypsin, concentrated sputum and
infection enzymatic lavage specimen from
GIT
Page 339
Characteristics of Adhesive Agents Fixatives for Smears

• It must be permeable to both fixative and stain • Equal parts of 95% Ethyl alcohol and Ether
• It must not retain the stain • 95% Ethyl alcohol
• Egg albumin is NOT recommended as an • Carnoy’s fluid
adhesive agent
• Intensely stained by the basic light green Fluid Specimen
counterstain of Pap’s method. • Fixatives:
• 50% alcohol (all types of effusion)
Adhesive Agents Used in Cytology • Saccomano’s fixative (50% ethanol and
• Pooled human serum/plasma 2% carbowax)
• Celloidin ether alcohol • Centrifugation: 2000 rpm for 2 minutes
• Leuconostoc culture
Precautions during Fixations
Fixation • Identify the slides before preparing smears.
• Some can be fixed in 15 minutes • Use paper clips to the identified end of the slide
• Recommended to be left for a minimum of 1 before preparing smears.
hour before staining • Smears should be placed into the fixative
• Assure optimum dehydration and adhesion container immediately after preparations.
permitting more complete penetration of cells • Place each smear in fixative by single
uninterrupted motion to avoid rippling of smeared
material.
• Avoid striking the bottom of the fixative container
forcefully to prevent dislodging of the cells
Page 340
Non-Gynecologic Specimens GIT Specimens

• Types of specimen:
Respiratory Tract Specimens • Gastric lavage
• Sputum • Gastric brush
• Obtain at least 3 consecutive morning • FNA (for submucosal lesions)
sputum specimens (through deep cough)
• Use wide-mouthed jar with Saccomano’s Urine
fixative • At least 50 mL is needed
• Alveolar macrophages – sputum from • Second urine is preferred
deep cough • Use of preservatives is NOT recommended
• Absence of alveolar macrophages – • Use for the diagnosis of urothelial malignancy
specimen is not sputum but SALIVA • Types of specimens:
• Bronchoalveolar lavage/bronchial washing • Males: Voided urine
• Performed in patients with AIDS to rule • Females: Catheterized specimen (to
out Pneumocystis carinii prevent contamination of vulvar cells)
• Bronchial brushings • Washings from bladder or renal pelvis
Peritoneal, pleural and pericardial fluids

• To prevent Jelly-like clots, add 300 u of heparin
per 100 mL of aspirate
Page 341
Gynecologic Specimen Vaginal Hormonal Cytology

• Relatively inexpensive
Staining of Smear Preparations • May be performed regularly even in pregnant
• Papanicolaou Method (Paps Smears) women without undue risk
• Staining method of choice for exfoliative
cytology Papanicolaou Staining Method
• Remove PEG fixative in 50% alcohol, 2 min
Advantages of Paps Smear • Hydrate in 95% alcohol, 2 min, and 70% alcohol,
• Transparent blue staining of cytoplasm is 2 min
obtained due to the action of high alcoholic • Rinse in water, 1 min
content of the cytoplasmic counterstain, • Stain in Harris’s hematoxylin, 5 min
allowing overlapping cells to be seen. • Rinse in tap water, 2 min
• Excellent nuclear detail is produced. • Differentiate in 0.5% aq. HCl, 10 s
• Color range is predictable and of great value in • Rinse in tap water, 2 min
the identification and classification of cells • ‘Blue’ in Scotts tap water substitute, 2 min
producing a good differential coloring of • Rinse in water, 2 min
basophilic and acidophilic cells. • Dehydrate in 70%, 95%, 95% alcohol for 2 min in
• It is valuable in comparing cellular appearances each solution
in smears with their counterpart in similarly • Stain in OG 6
stained sections. • Rinse in two changes of 95% alcohol for 2 min
each
• Stain in EA 50, 3 min
• Rinse in 95% alcohol
Page 342
Sites for the Detection of Genital Cancer Intermediate cells

• Upper lateral third of vaginal wall • Medium-sized polyhedral or elongated cells with
• For vaginal hormonal cytology basophilic cytoplasm showing vacuoles
• More accessible and less likely to be • Navicular Cells
contaminated by cellular debris or • Boat-shaped cells with strong tendency to
vaginal discharges. fold or curl on edges (presence of
• Ectocervix progesterone)
• Transformation Zone • Pregnancy cells
• Endocervix • Round, oval, boat-shaped cells with
translucent basophilic cytoplasm
Cells found in cervico-vaginal smears (glycogen accumulation)
Mature superficial cells/superficial cell • Nucleus pushed aside or towards the cell
• 45-50 um membrane
• With dark pyknotic nuclei • With double walled boundary appearance
• With true acidophilia (under estrogen influence) (deeper blue stain of cytoplasm at the
• Pseudoacidophilia periphery)
• Drying of smears especially before
fixation Parabasal cells
• Prolapsed and drying of vaginal • 15-30 um
epithelium • Thick, round to oval
• Infection • Smaller than intermediate cells
• Chemicals • Similar to fried fresh eggs with sunny side up
(presence of large nucleus)
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• Normally found: Basal cells

• From 2 weeks of age to puberty • Small, round to slightly oval cells with relatively
• After childbirth large nuclei
• Abortions • Strongly basophilic cytoplasm
• After menopause • Found before puberty and after menopause
Other cells Doderlein bacillus

Endometrial cells • Presence means healthy vagina
• Similar in appearance to parabasal cells • Most common organism of the normal vaginal
• Slightly cylindrical with less basophilic flora
cytoplasm • Found due to:
• Occurring in groups of 3 or more • Last trimester of pregnancy
• Found during and 1-10 days after menstruation • DM
• Infection
Endocervical glandular cells • Estrogen deficiency
• Cytoplasm is usually stained pale blue/gray, • Paps: Lavender/blue
finely vacuolated
• Nuclei with finely granular chromatin Candida albicans
• Having a honeycomb appearance when viewed • Commonly seen in:
on end • Patients with DM
• Patients taking oral contraceptives
• Immunocompromised states
• Leukemia and lymphoma
Page 344
Trichomonas vaginalis Quantitative Evaluation of Vaginal Cytology

• Pear-shaped
• Causes Strawberry cervix Maturation Index (MI)
• Paps: Blue green to blue gray • Percentage of cells from the main layers of
vaginal epithelium (superficial, intermediate, deep
Gardnerella vaginalis (parabasal/basal) cells)
• Clue cells – squamous epithelial cells which • Pyknosis – used as a criterion for mature
contains coccobacilli in the cytoplasm superficial cells
Koilocytes Acidophilic Index (AI)

• Squamous epithelial cells that show the • Percentage of cells staining pink-orange to red
cytopathic effects of HPV with Paps smear
• Cells with atypical nucleus surrounded by • Not a reliable index due to possible
perinuclear halo Pseudoacidophilia
Ferning Pyknotic Index (PI)

• Cervical mucus exhibiting palm leaf pattern • Percentage of cells with shrunken, dark, small
• Due to formation of salt crystals structureless nuclei.
• Signifies a high, persistent estrogen effect
• One of the basis of the diagnosis of early
pregnancy
Page 345
Report for Cytologic Smear

• For the diagnosis of cancer
CLASS DESCRIPTION
Class I Absence of atypical or abnormal cells
Atypical cytologic picture but no evidence of
Class II
malignancy
Cytologic picture suggestive but not conclusive of
Class III
malignancy
Class IV Cytologic picture strongly suggestive of malignancy
Class V Cytologic picture conclusive of malignancy
Page 346
Other Cytological Staining Methods Phase Contrast Microscopy

• Polychrome staining method – rapid and to • Second best choice for routine cytologic
some extent, differential examination
• Cresyl violet method – simple and rapid • Used for hormonal evaluation of gynecologic
technique with resulting clarity of morphologic specimen and for cancer detection
cellular details
• Supravital staining by Wet Film method Interference Microscopy
• PAS staining • Determines the dry weight of individual cells or
• Giemsa procedures cellular constituents, cancer cells, nucleus and
• Feulgen reaction cytoplasmic dry wet content being less than that
of normal.
Additional Microscopic Exam for Cytologic • Very expensive and complex.
Smears
Acridine Orange Fluorescence Technique

• RNA in cytoplasm and nucleus stains brick to
orange red
• DNA stains green and yellow
Page 347
17
Special Stains
Page 348
Special Stain 1: Carbohydrate Stains Basic Classification of Carbohydrates

Carbohydrate Stains • Monosaccharides
• Glycobiology • Glucose
• A complex discipline that permeates • Mannose
diverse fields as: • Galactose
• Cell biology • Oligosaccharides
• Microbiology • Sucrose
• Enzymology • Maltose
• Molecular biology • Polysaccharides
• Histochemical techniques • Glycogen
• Are used for the detection and • Starch
characterization of carbohydrates and
carbohydrate containing macromolecules Basic Classification of Glycoconjugates
(glycoconjugates) • Connective tissue glycoconjugates
• Provide invaluable information • Proteoglycans
which may aid the pathologist in • Hyaluronic acid
diagnosing and characterizing • Mucins
various pathological conditions • Neutral mucins
including: • Sialomucins
• Neoplasia • Sulfomucins
• Inflammation
• Autoimmune disorders
• Infectious diseases
Page 349
• Other glycoproteins
• Membrane proteins (receptors, cell
adhesion molecules)
• Blood group antigens
• Glycolipids
• Cerebrosides
• Ganglioside
Page 350
Summary of Different Types of Carbohydrates & Glycoconjugates

ASSOCIATED
TYPE LOCATION FUNCTION PATHOLOGICAL
CONDITION
Glycogen • Liver • Storage form of Found in wide range of
• Skeletal muscle carbohydrate malignancies:
• Cardiac muscle • Ewing’s sarcoma
• Hair follicles • Rhabdomyosarcoma
• Cervical epithelium • Seminoma
• Glycogen storage
disease
Proteoglycans • Cartilage • Support Found in:
andhyaluronic • Heart valves • Lubrication • Myxoid
acid • Blood vessels • Cell adhesion chondrosarcomas
• Tendons • etc. • Myxoid liposarcomas
• Ligaments • Myxoid fibrous
• ECM histiocytomas
• Membranes of many • Patients with
cell types mucopolysaccharidoses
Page 351

ASSOCIATED
TYPE LOCATION FUNCTION PATHOLOGICAL
CONDITION
Mucins • Epithelia of the GIT • Secreted • Frequently found
• Respiratory tract mucinslubrication inadenocarcinomas of
• Reproductive tract and protection the GIT
• Membrane-bound • Aberrant or
mucinscell adhesion inappropriate expression
and regulation of of specific mucin types
proliferation occurs frequently in
neoplastic process
Glycoproteins • Cell membrane- Multiple and diverse • Aberrant expression of
Blood group antigens functions such as: blood group antigens in
• Secreted products • Cell adhesion, various malignancies
(peptide hormones immune recognition
and immunoglobulins) • Regulation of
receptor ligand
binding etc.
Page 352
Proteoglycans
• Referred to as connective tissue mucins or
mucopolysaccharides
• Found in the connective tissues ECM and act in
stabilizing and supporting fibrous elements of
connective tissue
• Highly glycosylated (90-95% is carbohydrate)
• Carbohydrated component: GAGs
• Composed of repeating disaccharides
• Ionized and carry a negative charge
• Most abundant type: chondroitin sulfates
Page 353

Glycosaminoglycans Disaccharide repeat Location
Chondroitin sulfate • Glucoronic acid and N- • Cartilage
acetylglucosamine • Tendons, ligaments, aorta, cell
membranes
Dermatan sulfate • Iduronic acid and N- • Skin
acetylglucosamine • Blood vessels
• Heart valves
Keratan sulfate • Galactose and N- • Cartilage
acetylglucosamine • Cornea
Heparin sulfate • Glucoronic acid and N- • Blood vessels
acetylglucosamine or N- • Aorta
sulfate lglucosamine • Cell membranes
Heparin • Glucoronic acid and N- • Mast cell granules
acetylglucosamine or N-
sulfate lglucosamine
Hyaluronic acid • Glucoronic acid and N- • Synovial fluid
acetylglucosamine • Humor
• Loose connective tissues
• Small amount in cartilage
Page 354
Mucins • Mucopolysacharidoses
• Consist of polysaccharide chains covalently • Fresh or frozen sections are most
linked to a protein core recommended although formalin is also
• Defining structure of epithelial mucins is the satisfactory
presence of tandemly repeated amino acid • Typical Mucins & Proteoglycans
sequences within the protein core • Recommended fixatives: formalin or
• Sialic acids: alcoholic formalin
• Sialisidase resistant – not detected by
PAS Techniques for Demonstration of Carbohydrates
• Sialisidase labile – clearly visible with • Periodic Acid Schiff (PAS) technique
PAS • Mild PAS technique
• Alcian Blue techniques
Fixation • Combine Alcian blue-PAS technique
• Glycogen • Gomori’s aldehyde fuschin stain
• Recommended fixative: alcoholic • Combined- Aldehyde fuschin-Alcian blue
formalin • Mucicarmine technique
• Others: NBF, Rossman’s fluid, alcoholic • Colloidal Iron technique
formalin with picric acid • Metachromatic methods (ex. Azure A)
• Zenker’s-acetic acid and Susa’s are not • Fluorescent Acridine Orange Technique
recommended
• Fixation should be carried out in 4 C to
minimize artifacts
Page 355
Periodic Acid-Schiff (PAS) Technique • Reticulin

• Most versatile and widely used technique for • Fungi (capsules)
demonstration of carbohydrates and • Pancreatic zymogen granules
glycoconjugates • Thyroid colloid
• First histochemical use was by McManus for • Corpora amylacea
mucin • Russel bodies
• Also used for glycogen and certain
glycoproteins PAS Technique (Modified McManus)
• Aid in differential diagnosis of tumors through Components
detection of glycogen or mucins • Periodic Acid solution
• Periodic acid (1g)
Mechanism of Pas Technique • Deionized or distilled water (100ml)
• PAS is based upon the reactivity of free
aldehyde groups within carbohydrates with • Schiff reagent preparation
Schiff reagent to form a bright red magenta end • Dissolve 1g basic fuchsin and 1.9 g of
product sodium metabisulfite in 100 ml of 0.15 N
HCl.
PAS-Reactive Cells & Tissue Components • Shake solution at intervals or with
• Glycogen mechanical rotator for 2 hours.
• Starch • Solution should be clear and yellow to light
• Mucin (Sialomucins, neutral mucin) brown.
• Basement membranes
Page 356
• Add 500 mg of activated charcoal and PAS Technique (Modified McManus) Results
shake for 1-2 minutes • Glycogen, neutral/Sialomucins – magenta
• Filter through Whatman filter paper (No. • Various glycoproteins – magenta
1). • Nuclei – blue
• Filter should be clear and
colorless. PAS Technique (Modified McManus) Notes
• Store at 4C • For basement membranes a longer time in
Periodic acid (10 min) AND Schiff reagent (20
PAS Technique (Modified McManus) Method min) may give better result
• Deparaffinize in Xylene and rehydrate through • Post-Schiff bisulfite rinses are used for reduction
graded ethanols of background
• Oxidize with Periodic acid for 5 minutes • Glutaraldehyde fixatives should be avoided when
• Rinse in several changes of deionized water using PAS
• Cover sections with Schiff reagent for 15 • Glycolipids staining may be detected using frozen
minutes sections
• Rinse in running tap water for 5-1 minutes • Glycolipids and unsaturated lipid are significantly
• Stain the nuclei with Harris’s or Mayer’s loss from paraffin-embedded sections
hematoxylin.
• Differentiate and blue the sections
• Dehydrate, clear and mount
Page 357
Best Carmine Method Langhan’s Iodine Method for Glycogen

• Highly specific for glycogen • Oldest stain
• Best Carmine is brought about due to the • Not specific for glycogen
affinity of alkaline carminic acid for glycogen, • Rapid but not permanent
producing a bright red color • Fixation:
• Counterstain: Ehrlich’s hematoxylin • 10% formol alcohol
• Potassium carbonate and potassium chloride • Formol-saline
salts are added to the stock solution to inhibit • Sections: Paraffin sections, floating in 70%
non-specific background carmine staining alcohol
• Fixation: • Result:
• 10% NBF • Tissue constituents – yellow
• Abs. alcohol • Glycogen – mahogany brown
• Formol-saline
• Carnoy’s fluid Mild PAS technique
• Sections: celloidin are the best • Utilizes a weak periodic acid oxidation step to
• Paraffin sections should be covered with thin demonstrate N-acetyl sialic acid – containing
layer of celloidin mucins
• Result:
• Nuclei – blue or grayish blue
• Glycogen – bright red granules
• Mucin, fibrin – weak red
Page 358
Standard Alcian Blue Technique Combined Alcian Blue-PAS Results

• Most popular method for demonstration of acid • Glycogen, neutral mucins, various glycoproteins
mucins – magenta
• Initially used for dyeing textile fibers • Acid mucins (Sulfomucins and Sialomucins) –
• Alcian blue 8GX - recommended dye for blue
histological techniques • Proteoglycans & hyaluronic acid – blue
• Demonstrates and precipitate hyaluronic acid,
chondroitin sulfate and heparin from an Gomori’s Aldehyde Fuchsin Stain
aqueous solution (pH 2.5) • First introduced by Gomori as an elastic tissue
• Also demonstrates acidic epithelial mucins stain
(Sialomucins & Sulfomucins) of large intestine • Other tissues equally stained:
• Neutral mucins of gastric mucosa and Brunner’s • Acid mucopolysaccharides
gland are not reactive with alcian blue • Sulfated mucosubstances
• Pancreatic islets of Langerhans
Combined Alcian Blue-PAS • Thyrotropic hormones
• Use to differentiate neutral mucins from acidic • Secretory substances
mucins within a tissue section • Results:
• Sections are stained with standard alcian blue • Sulfated mucin – purple
(pH 2.5) method followed by PAS • Carboxylated mucins (Sialomucins) – blue
Page 359
Mucicarmine Technique Colloidal Iron Technique

• Oldest Histochemical methods for the • Initially described by Halen for detection of acid
visualization of mucins in specimens mucopolysaccharides
• Remains valuable means for the demonstration • Based upon the attraction of ferric cations for the
of acidic mucins negatively charged carboxyl and sulfate groups
• Dye molecule used: aluminum-carminic acid of acid mucins and proteoglycans
complex (carmine) • Tissue-bound ferric ions are visualized by
• Useful for identification of adenocarcinomas treatment with potassium ferrocyanide to form
• Also, for demonstration of C. neoformans bright blue or Prussian blue color
capsule • Can be used in combination with PAS reaction
for mucin differentiation
Mucicarmine Results
• Acidic epithelial mucins – deep rose to red Colloidal Iron Technique Results
• Nuclei – black • Proteoglycans, hyaluronic acid and acidic mucins
• Other tissue elements – light yellow – bright blue
• Note: • Collagen – red
• Ehrlich’s hematoxylin should not be used • Nuclei – red
as counterstain because mucins will not • Muscle and cytoplasm – yellow
be stained
Page 360
Colloidal Iron Technique Notes Uranyl Acetate-Azure A technique Results

• pH of colloidal iron is critical • Acid mucins and proteoglycans – crimson or red
• pH 2.0 or higher non-specific staining of violet
structures other than acidic carbohydrate • Tissue background – blue
will occur
• Nuclear fast red can be used as a counterstain Azure A technique Results
• Stock colloidal iron should be dialyzed to • Most useful metachromatic dye for acid mucin
remove free acid and unhydrolyzed iron salts • Acid mucins and proteoglycans – purple to red
• Tissue background – blue
Metachromatic Methods
• Oldest histochemical technique for identification Fluorescent Acridine Orange Technique
of charged mucins and proteoglycans • Mucin on section stained with iron hematoxylin
• Methylene blue, Azure A, and toluidine blue and acridine orange gives a selective brilliant
stain tissue blue but under conditions of orange fluorescence
metachromasia they stain tissue components • Disadvantage: temporary
purple-red • Fixatives: formalin and other fixatives except
• The more acidic or highly sulfated heavy metals
proteoglycans the stronger and more stable the • Section: paraffin and frozen sections
metachromasia
Fluorescent Acridine Orange Technique Results
• Acid mucopolysaccharides – Black
• Fungi – greenish red fluorescence
• Background – reddish orange fluorescence
Page 361
Pathologic Changes & Deposits Found In Fibrinoid

Connective Tissue • A mixture of exudate and altered cytoplasmic
• Fibrin constituents forming a homogenous eosinophilic
• Fibrinoid material
• Hyalin • Gives the same staining reaction as fibrin
• Amyloid • Found in:
• Collagen disease
Fibrin • Hypersensitivity
• Material resulting from enzymatic coagulation of • SLE
plasma globulin and fibrinogen, forming bundles • Rheumatic heart disease
which contract into dense homogenous masses
• Substances in the blood such as hemoglobin Hyalin
and glycoproteins become entangled with the • Refers to a wide variety of pathologic exudates
fibrin, giving a weak reaction when tested for and deposits appearing in the form of a smooth
hemoglobin and glycoprotein homogenous eosinophilic material in routine
stains
Stains for demonstration of FIBRIN • Seen in:
• Hematoxylin and eosin stain • Degenerated collagen
• Mallory’s PTAH • Hypertension
• Gram Staining • Atheroma
• PAS • Diabetic kidney
• No specific stain
Page 362
Amyloid Congo red method

• A semi-translucent, ground-glass or hyalin • Amyloid – deep pink to red color
eosinophilic substance made up of chondroitin
sulfuric acid-protein complex Metachromatic Staining
• Deposited in kidneys, spleen, lymph nodes, • Amyloid – purplish red or red
pancreas, during chronic suppurative conditions
and inflammatory conditions Induced Fluorescence staining with thioflavin
• TB, leprosy, osteomyelitis • Amyloid – yellow fluorescence in olive-green
• Also found in Idiopathic or primary amyloidosis background
in:
• Heart
• Tongue
• Larynx
• Intestine
• Skeletal muscle
• Blood vessels
Stains for demonstration of AMYLOID

Gram’s iodine stain
• Amyloid – brown to blue
Krajians amyloid stain

• Amyloid – red in clear background
Page 363
Special Stain 2: Connective Tissue & Stains Formed or Fibrous Intercellular Substances
• Develops from the mesenchyme Collagenic fibers

• Consists of cellular portion and a surrounding • Most frequently encountered
network of non-cellular substances • Appear as individual fibers or large bundles of
• Cells include: fibers
• Fibroblasts • Strongly birefringent in polarized microscope
• Mast cells
• Histiocytes Collagen Type I
• Adipose cells • Forms thick collagenous fibers that form the bulk
• Osteoblast of collagen in the body
• Chondrocytes • Major structural protein in the lung
• Blood cells
• Intercellular substance contains amorphous and Collagen Type II
formed elements • Found in hyaline and elastic cartilage produced
• Formed or fibrous types by chondroblast activity
• Amorphous or gel types • Fibers are thin with proteoglycans
• Not readily visible by light microscope
• Those that are found in articular cartilage are
thicker and resembles ultrastracturally type 1
collagen
• Hyaluronidase is used to unmask type 2 to
render immunohistochemical evaluation
Page 364
Collagen Type III • May be demonstrated in paraffin sections using

• Found only on tissues with type I collagen argyrophilic-type silver impregnation techniques
• Examples: or frozen section by PAS
• Liver
• Spleen Elastic Fibers
• Lung • Found throughout the body especially associated
• Kidney with the:
• Referred to as fetal collagen (fetal skin contains • Respiratory system
60% of collagen type III compared to adults • Circulatory system
which is only 20%) • Integumentary system
• Appears as fine single fibers (upper dermis) or
Staining reaction of Collagen membrane-like structures (large arteries)
• Type I collagen strongly stains with acidic dyes
due to cationic group of proteins Basement Membrane
• Resilient matrix that separates connective tissue
Reticular Fibers from:
• Fine and delicate fibers connected to collagen • Epithelial cells
fibers • Endothelial cells
• Provide bulk of supporting framework of more • Mesothelial cells
cellular organs such as spleen, liver, lymph • Muscle cells
nodes, where they arranged in 3D network • Fat cell
• Shows weak birefringence under light • Nervous cells
microscopy
Page 365
• Support epithelial surfaces, glands and other Factors Affecting Trichome Staining
structures like: • Tissue permeability and dye molecular size
• Renal tubules • General rule in trichrome staining:
• Endothelial cell linings of blood vessels • Smaller dye molecule penetrates and stain
• Difficult to distinguish using H&E technique a tissue element but whenever larger dye
• Techniques used for critical examinations: molecule can penetrate the same element,
• PAS reaction the smaller molecule will be replaced by it
• Oxidation-aldehyde demonstration • Heat – increase rate of staining and penetration
techniques of dye molecule)
• pH – 1.5-3.0
Connective Tissue Stains
Nuclear Stains for Trichrome
Trichrome Stains • Iron hematoxylins are more preferred than alum
• A general name for number of techniques for hematoxylins to prevent decolorization in the
the selective demonstration of: subsequent staining with acid containing dyes
• Muscle • Improved and resistant stain can also be done
• Collagen fibers using Celestine blue followed by conventional
• Fibrin alum hematoxylin
• Erythrocytes
• Example: Effects of Fixation
• Van Gieson stain • Satisfactory fixatives for Trichrome staining:
• Masson Trichrome stain • Zenker’s
• Formal mercury
Page 366
• Bouin’s fixative Van Gieson’s Stain Results

• Picro-mercuric alcohol • Nuclei – black
• Tissues fixed in formaldehyde will not produce • Collagen – red
optimum result with Trichome stains • Muscle, cytoplasm, RBC and fibrin – yellow
• Thus, should be treated with picric acid
and/or mercuric chloride solution to Van Gieson’s Stain Notes
enhance intensity and brilliance • Fixation is not critical – NBF is satisfactory
• Washing in water after Van Gieson’s should be
Van Gieson’s Stain avoided
• Simplest method of differential staining of • Nuclear staining should be intense – picric acid in
collagen using a mixture of picric acid and acid Van Gieson’s will act as differentiator
fuchsin • For celloidin-embedded tissues, Weigert’s is
• Fixation: recommended than celestine blue
• Mercuric chloride fixative
Masson’s Trichrome Stain
SOLUTION
• Fixation:
Sat. aq. Picric acid 50 ml
• Zenker
solution
• Helly’s
1% aq. Acid fuchsin 9.0 ml
• Bouin’s
solution
• Formal sublimate
• Formal saline
• Sections: All types
Page 367
Masson’s Trichrome Stain Results Mallory’s Aniline Blue

• Nuclei – blue/black • Not differential because other than staining
• Cytoplasm, muscle and erythrocytes – red collagen fibers and reticulum it also stains:
• Collagen – blue • Hyalin fibrils
• Fibro glia fibrils
Masson’s Trichrome Stain Procedure • Smooth and striated muscle fibers
• Deparaffinize and bring to water • Amyloid
• Remove mercuric pigment by iodine, sodium
thiosulfate sequence Mallory’s Aniline Blue Results
• Wash in tap water • Collagen fibrils, cytoplasm, fibro glia fibrils, axon
• Stain nuclei by celestine blue or Wiegert’s cylinders and neuroglia – Red
• Differentiate with 1% acid alcohol (when using • Elastic fibers – pale pink or yellow
celestine blue) • RBC and myelin sheath – yellow
• Wash well in tap water
• Stain in acid fuchsin for 5 minutes Heidenhain’s Azan Technique
• Rinse in distilled water • Also known as Azocarmine stain – a modification
• Treat with PMA solution for 5 minutes of Mallory’s Aniline blue
• Drain • Not recommended as general connective tissue
• Stain with methylene blue (or light green) stain
• Treat with 1% acetic acid, 2 minutes • Useful in the demonstration of “wire loop lesions”
• Dehydrate, clear and mount in the diagnosis of lupus nephritis in renal
biopsies
Page 368
Heidenhain’s Azan Technique Results • Soluble blue

• Amyloid connective tissue and mucous colloid – • Can be substituted with:
deep blue • Durazol blue
• Nuclei – red • Pontamine sky blue
• Fast green FCF
Stains for Demonstration of Fibrin • Naphthalene black 10B
• Gram - Weigert
• Phosphotungstic acid-hematoxylin Stains for Elastic Fibers
• Trichrome methods • Verhoeff’s Stain
• (Martius, Scarlet, Blue technique or MSB • Taenzer-Unna Orcein Method
technique) • Weigert’s Elastic Tissue Stain
• Masson’s Trichrome Stain
MSB technique for Fibrin • Krajian’s Method
• Standard technique employs
• Martius yellow Verhoeff’s Stain
• Can be substituted with: • Classical staining method for elastic fibers and
• Lissamine fast yellow works well with all routine fixatives
• Brilliant crystal scarlet • Satisfactory results may be obtained using
• Can be substituted with: solutions up to 48 hours old
• Ponceau de xylidine
• Azofuchsin
Page 369
Verhoeff’s Stain Results Weigert’s Stain

• Elastic tissue fibers – black • Primary stain: Weigert’s stain (Basic fuchsin,
• Other tissues – depend on the counterstain resorcin and ferric chloride)
used • Differentiator: acid alcohol
• Sharpness and intensity of staining can be • Counterstain:
improved with pre-treatment with 1% potassium • Neutral red
permanganate for 5 minutes • H&E
• Hematoxylin
Orcein Method (Taenzer-Unna Orcein) • Van Gieson’s
• Excellent stain for elastic fibers – stains even • Fixative:
the most delicate and finest fibers of skin • Formalin
• Main advantage: simplicity of preparation • Alcohol (Zenker fixed tissue stains slowly)
• Primary stain: orcein
• Differentiator: acid alcohol Weigert’s Stain Results
• Counterstain: • Elastic fibers – dark blue or blue black
• Methylene blue • Other structures – depend on the counterstain
• Alum hematoxylin used
Orcein Method (Taenzer-Unna Orcein) Results

• Elastic fibers – dark-brown
• Nuclei – blue
Page 370
Krajian Technique Silver Impregnation Techniques

• Rapid method of elastic tissue staining, for fibrin • Employ silver salts which is NOT visible with
and amyloid employing – Congo red routine H&E
• Elastic fibers – bright red • Uses ammoniacal solution of Silver Carbonate
• Fibrin and connective tissues – dark blue reduced by reticulum to dark brown Silver Oxide,
• RBC – orange yellow in turn reduced to black metallic Silver by
formalin
Other Stains For Elastic Fibers • Unreduced Silver is removed by Sodium
• Weigert’s resorcin-fuchsin method Thiosulfate solution
• Elastic fibers – brown-purple • For complete permanent preparation:
• Methyl violet/ethyl violet resorcin method • Silver is partially converted to gold
• Elastic fibers – blue-black impregnation by treatment with gold
• Aldehyde fuchsin method chloride
• Elastic fibers – blue-purple
Sections for Reticular Fiber Silver Impregnation
Stains for Reticular Fibers Techniques
• Metal impregnation techniques • Frozen, cryostat, paraffin, and celloidin sections
• Gordon and Sweet’s method may be employed for reticular fibers
• Gomori’s method demonstration
• Russel Modification of Movat Pentachrome • Fixative containing salts of Mercury or osmium
stain cause little non-specific background precipitation
of silver
Page 371
Gordon and Sweet’s Method Gomori’s Method

• Uses 5 ml of 10% aq. Silver nitrate solution with • Uses 10% potassium hydroxide solution with 40
concentrated ammonia and 3% sodium ml of 10% silver nitrate solution
hydroxide • Results:
• Notes: • Reticular fibers – Black
• Short treatment with iron alum solution, • Nuclei – gray
of < 5 minutes, give less staining of • Other tissues – according to counterstain
nuclei
• Use of Coplin Jar for the silver solution Russell Modification of the Movat Pentachrome
reduces the possibility of precipitation Stain
• Sections can be counterstained with: • Use for tissue fixed in 10% NBF or acetic
• Eosin formalin sublimate with tissue of 5 microns thick
• Nuclear fast red • Employs:
• Tartrazine • Solutions containing:
• Van Gieson • 1% alcian blue solution
• Alkaline alcohol
Gordon and Sweet’s Method Results • Iodine solution
• Reticular fibers – Black • 10% absolute alcohol hematoxylin
• Nuclei – Black or unstained • 10% ferric chloride
• Other elements – according to counterstain • Hematoxylin solution
• Crocein scarlet-acid fuchsin
Page 372
Russell Modification of the Movat Other Stains for Reticular Fibers

Pentachrome Stain Results • Bielchowsky’s method
• Nuclei and elastic fibers – black • Periodic Acid Schiff-Leucofuchsin technique
• Collagen and reticular fibers – yellow • Gold method
• Ground substance, mucin – blue • Laidlaw’s Silver impregnation
• Fibrinoid, fibrin – intense red • Reticulum – violet to blue-black
• Muscle – red • Snook’s reticulum method
• Reticulum – black
Russell Modification of the Movat
Pentachrome Stain Notes
• Differentiation of elastic fibers is usually
complete in 2-3 minutes
• Complete removal of alkaline alcohol using
running water is important, otherwise,
subsequent staining will be inhibited
• The stain may be used to demonstrate C.
neoformans staining brilliant blue
Page 373
Special Stain 3: Proteins & Nucleic Acids • Fixation:

• NBF
• Protein and nuclei are major cell and tissue • Formaldehyde vapor (for freeze-dried
constituents tissue)
• Simple proteins • Section:
• Fibrous proteins and enzymes • Paraffin
• Conjugated proteins • Cryostat
• Nucleic acids • Freeze dried
• Deoxyribonucleic acid (DNA) & • Results:
ribonucleic acid (RNA) • Amino groups – pinkish purple
• Histochemical techniques for
demonstration of nucleic acids are based Hydroxy naphthaldehyde Method of Weiss et al
on all their constituents (1954)
• Another method for demonstration of amino
Amino Acid Histochemical Methods groups
• Non-formalin fixative is preferred
Ninhydrin – Schiff Method for Amino Groups
• Demonstrate protein-bound amino groups Millon Reaction for Tyrosine
• At neutral pH and 37C, ninhydrin reacts with a- • Tyrosine is the only amino acid that contains
amino groups to produce aldehydes which can hydroxyphenyl group which can be demonstrated
be demonstrated using Schiff’s reagent histochemically
• Section is treated with hot mercuric sulfate-
sulfuric acid- sodium nitrite mixture
Page 374
• Results: • Sections:
• Tyrosine-containing proteins – Red or • Paraffin
pink • Cryostat
• Freeze dried
Diazotization – Coupling Method for Tyrosine • Results:
• Less well-known, but more reliable technique • Disulfides – Blue
for tyrosine • Positive control:
• Employs 8-amino-1-naphthol-5-sulfonic acid • Hair – bearing skin
(coupling amine for diazonium nitrites) • Pituitary with basophils
• Carried in dark and at low temperature
• Results: DMAB – Nitrate Method for Tryptophan
• Tyrosine-containing proteins – Purple • Most reliable method
and red • Best results are obtained with freeze-dried
sections
Performic Acid – Alcan Blue Method for • Satisfactory results can be obtained with paraffin
Disulfide sections
• Disulfide & sulfhydryl linkages are found in • Principle:
amino acids cysteine and methionine • Tryptophan reacts with DMAB to produce
• Fixative: B-carboline, then oxidized by nitrite
• NBF solution to produce a deep blue pigment
• Formaldehyde vapor (for freeze-dried
tissue)
Page 375
• Fixative: • Principle:
• NBF • Arginine reacts with a-naphthol and an
• Formaldehyde vapor (for freeze-dried alkaline hypochlorite solution, giving an
tissue) orange-red color
• Sections: • Fixative:
• Paraffin • NBF
• Cryostat • Formaldehyde vapor (for freeze-dried
• Freeze dried tissue)
• Results: • Sections:
• Tryptophan – Dark blue • Paraffin
• Nuclei – Red • Cryostat
• Positive control: • Freeze dried (12-15 mm thick) mounted in
• Pancreas silane-coated slide
• Results:
Modified Sakaguchi Reaction for Arginine • Arginine – Orange red
• Arginine • Positive control:
• Is the only amino acid demonstrated • Testis
histochemically with this reaction
• The only amino acid that contains
guanidyl group
Page 376
Nucleic Acids Feulgen Reaction

• Fixation: • Standard technique for demonstration of
• Best preserved in alcoholic and acidic deoxyribose
fixatives (ex. Carnoy’s fluid) • Developed by Feulgen and Rossenback
• NBF can also be use at 4C to prevent DNA • Fixation:
degradation by cell nucleases • Not critical but not Bouin’s
• Nitric or hydrochloric and Bouin’s fixative should • Principle:
be avoided because they cause nucleic acid • 1 M HCl at 60C is used for mild acid
extraction hydrolysis to break purine-deoxyribose
• Decalcification: bond.
• 5% formic acid and EDTA • And resulting ‘exposed’ aldehydes are
• Basophilia: demonstrated by the Schiff’s reagent.
• DNA and RNA both stains strongly with • Ribose-purine bond are unaffected by the
cationic dye hydrolysis and RNA is not demonstrated
• Methylene blue selectivity is achieved at pH • Results:
3.0-4.0 • DNA – Red-purple
• Cytoplasm – Green
Demonstration of DNA
• Feulgen nuclear reaction for DNA
• Naphthoic acid hydrazine – Feulgen (NAH)
method
• Blue thionine – Feulgen reaction for DNA ploidy
studies
Page 377
Fuelgen Reaction- Hydrolysis Time In Prewarmed 1 M HCl At 60C

Fixative Time (minutes)
Bouin 8
Carnoy 14
Flemming 16
Formalin 8
Helly 8
Newcomer 20
Regaud 8
Susa 18
Zenker 5
Page 378
Naphthoic Acid Hydrazide – Feulgen (NAH) • Fixation:

Method • Formalin
• Can be used as a control method for the • Post – fixation in Boehm-Sprenger fixative
Feulgen reaction • Results:
• Principle: • DNA – Blue
• Sections are hydrolyzed in 1 M HCl as in • Cytoplasm – Unstained
Feulgen reaction and aldehydes
produced are coupled with 2-hydroxy-3- Demonstration of RNA
naphthoic acid, which in turn is coupled • Methyl-green pyronin method
to fast blue B • Methyl-green pyronin Y alternative method
• Fixation:
• Not critical Methyl Green – Pyronin Method by Unna (1902)
• Results: • Demonstrate both DNA and RNA and first
• DNA – Blue to bluish-purple published by Pappenheim (1899)
• Protein material possibly – Purplish-red • Modified by Unna (1902) by converting methyl
green (contains methyl violet removed by
Blue Thionin – Feulgen Reaction for DNA washing with chloroform) to pure methyl green
ploidy studies which appears to be specific for DNA at slightly
• Useful for ploidy studies and in the study of acid pH
cancer cell nuclear morphology • Rationale of technique:
• Employed to show an inverse relationship • When used in combination methyl green
between DNA content of certain lymphoma cells binds specifically to DNA and pyronin
and in tumor prognosis binds to RNA
Page 379
• Acid mucins in epithelium and cartilage will also • At pH 1.64, also stain non-nucleic acid
stain material in the karyoplasm
• Fixation:
• Carnoy Enzyme Digestion Method for RNA & DNA
• Formalin is acceptable • Deoxyribonuclease
• Dehydration in 93% ethanol will give a greener • Ribonuclease
nuclear staining
• Results: Enzyme Extraction of DNA
• DNA – Green-blue • Fixation:
• RNA – Red • Potassium dichromate will inhibit digestion,
and should be avoided
Other Methods – Gallocyanin - Chrome Alum • Reagents:
• Technique relies upon combination of the • Deoxyribonuclease
phosphoric residue of nucleic acids with • 0.2 M Tris buffer
gallocyanin at an acid pH • pH 7.6
• Extraction technique can be used to identify • Distilled water
either DNA or RNA • Results after staining with Feulgen method:
• Results: RNA/DNA - Blue • Test section – DNA negative
• Note: • Control section – DNA red
• Method does not distinguish between
DNA & RNA but appears specific for
nucleic acid if used at pH 1.0
Page 380
Enzyme Extraction of RNA Perchloric acid extraction

• Fixation: • To remove both DNA and RNA, sections are
• Potassium dichromate and mercuric place in 5% perchloric acid solution at 60C for 30
chloride will inhibit digestion, and should minutes
be avoided
• Reagents: Trichloroacetic acid (TCA) acid extraction
• Ribonuclease • Bring sections to water and treat with 4% TCA at
• Distilled water exactly 90C for 15 minutes.
• Results after staining with methyl green - • Wash and stain with toluidine blue.
pyronin method: • Both DNA and RNA are extracted.
• Test section
• RNA negative Hydrochloric acid extraction
• DNA green • Bring sections to water and treat with 1 M HCl for
• Control section 3 hours at 37C
• RNA red • Wash and stain with dilute methylene blue at pH
• DNA green 5.7 for 12-24 hours, or with other suitable basic
stains
Chemical Extraction Method for RNA & DNA • Both nucleic acids are extracted
• Perchloric acid extraction
• Trichloroacetic acid (TCA) acid extraction
• Hydrochloric acid extraction
Page 381
Fluorescent Methods for DNA & RNA

• Acridine orange technique
• Acriflavine
Acridine Orange Technique

• Results:
• DNA – Yellow-green
• RNA – Red
• Results are not permanent and technique
cannot be applied to formalin-fixed tissue
• Has been used in exfoliative cytology and
alcohol-fixed tissues
Acriflavine
• Used either as 0.01% alcoholic solution
precede by acid hydrolysis, or as alternative to
basic fuchsin in Schiff’s reagent, again precede
by acid hydrolysis
• DNA – Fluorescent yellow
Page 382
Special Stain 4: Pigments & Minerals • Entry is gained by inhalation into the lungs or by
implantation into the skin
Endogenous Pigments • Example:
• Substances produced either within tissue and • Minerals which are pigmented
serve a physiological function
• Or are by-products of normal metabolic Consideration before carrying out Various stains
processes and histochemical reactions
• Subdivisions • Pigment’s morphology
• Hematogenous pigments • Tissue site
• Relevant clinical data
Artifact Pigments
• Deposits of artifactually produced material Endogenous Pigments
cause by the interactions between certain tissue • Hematogenous
components and some chemical substances • Non-Hematogenous Pigments
• Example: • Endogenous Minerals
• Malaria
• Formalin Hematogenous Pigments
• Hemosiderins
Exogenous Pigments and Minerals • Hemoglobin
• Gain access to the body accidentally and serve • Bile pigments
no physiologic function • Porphyrins
Page 383
Hemosiderins Perl’s Prussian Blue

• Seen as yellow or brown granules and normally • Considered to be the best first classical
appear intracellularly Histochemical reaction
• Contain iron in the form of ferric hydroxide that • Treatment with acid ferro cyanide solution will
is bound to a protein framework and is result in unmasking of ferric iron in the form
unmasked by various chemicals hydroxide by dilute HCl
• The ferric iron reacts with a dilute potassium
Demonstration of Hemosiderin ferrocyanide solution to produce an insoluble
• Perl’s Prussian Blue compound, ferric ferrocyanide (Prussian blue)
• Lillies Method For Ferric And Ferrous Iron • Fixation:
• Hukill And Putt’S Method • Avoid use of acid fixatives
• Chromates will also interfere with the
Demonstration of Hemosiderin and Iron preservation of iron
• After fixation in formalin, hemosiderin becomes • Sections:
soluble with dilute acids, especially oxalic acids • Works well on all type of section, including
• Fixatives that contain acids can give a negative resin
reaction for iron pigment • Results:
• Can be demonstrated in Perl’s Prussian blue if • Ferric iron – Blue
treated with hydrogen peroxide or if the • Nuclei – Red
ferrocyanide is heated to 60C in water bath,
oven, or microwave oven
• However, use of heat will sometimes cause a
fine blue precipitate
Page 384
Lillie’s Method Hemoglobin

• Fixation: • Leuco Patent Blue V Method
• Avoid the use of acid fixative
• Sections: Demonstration of Hemoglobin
• Paraffin • The need to demonstrate the pigment may arise
• Frozen in certain pathological conditions such as casts in
• Resin the lumen of renal tubules in cases of
• Results: hemoglobinuria or active glomerulonephritis
• Ferric iron – Dark Prussian blue
• Ferrous iron – Dark turnbull’s blue Leuco Patent Blue V Method
• Nuclei – Red • Introduced by Lison (1938) and later was
modified by Dunn and Thompson (1946)
Hukill and Putt’s Method • Fixation:
• Fixation: • Formalin or formal mercury
• Not critical but avoid prolonged exposure • Results:
to acid fixative • Hemoglobin peroxidase (RBCs and
• Sections: neutrophils) – Dark blue
• All types of sections including resin • Nuclei – Red
• Results:
• Ferrous iron – Red
• Nuclei – Blue
Page 385
Bile Pigments & Hematoidin • Pigments is converted to green color of biliverdin

• Modified Fouchet’S Technique For Liver Bile and blue cholecyanin by the oxidative action of
Pigments ferric chloride in the presence of TCA
• Gmelin Technique • Quick and simple to carry out and when
counterstained with Van Gieson’s solution of
Demonstration of Bile Pigments and green color is accentuated
Hematoidin • Fixation:
• The need to identify bile pigments arises mainly • Any fixative appears suitable
in the histological examination of the liver, • Sections:
where lipofuscin may be of significant • Any
importance • Results:
• Both appear yellow-brown in H&E – stained • Bile pigments – Emerald to blue-green
paraffin section • Muscle – Yellow
• Green color of biliverdin is often masked with • Collagen – Red
eosin
• Bile pigments are not auto fluorescent Gmelin Technique
• Lipofuscin is auto fluorescent • The only method that shows an identical result
with liver bile, gall bladder bile, and hematoidin
Modified Fouchet’s technique for Liver Bile tends to be messy, capricious, and gives
Pigments impermanent results
• Commonly used method for demonstration of • Deparaffinized tissue containing bile pigments
bile pigments are treated with nitric acid and a changing color
spectrum is produced
Page 386
• It is unreliable – It is advisable to repeat the test Demonstration of Porphyrin Pigments

for at least three times • In erythropoietic porphyria, porphyrin appears as
• Popular modification is the used of bromine in a dense dark brown pigment and in fresh frozen
carbon tetrachloride as an oxidant sections exhibits a brilliant red fluorescence that
• Sections: rapidly fades with exposure to UV
• Paraffin • In paraffin section viewed in polarized light,
• Results: shows a bright red color with centrally located
• Bile pigments will gradually produce the dark Maltese cross
following spectrum of color change:
yellow-green-blue-purple-red
• The method is impermanent Non-hematogenous Pigments
• The reaction can occur rapidly but can be • Melanins
slowed down using 50-70% solution of nitric • Lipofuscins
acid • Chromaffin
• Pseudo melanosis (melanosis coli)
Porphyrin Pigments • Dubin-Johnson pigment
• These substances normally may occur in the • Ceroid-type lipofuscins
tissues in only small amounts • Hamazaki-Wesenberg bodies
• Considered to be precursors of the heme
portion of hemoglobin
• Porphyria’s are rare pathological conditions that
are disorders of the biosynthesis of porphyrins
and heme
Page 387
Melanins Reducing Melanin methods for Melanin

• Groups of pigments which color varies from Mass on-Fontana method for melanin
light brown to black • Fixation:
• Normally found in the skin, eye, substantia nigra • Formalin is best
of the brain and hair follicles • Chromate and mercuric chloride should be
• These are bound to proteins and localized in avoided
the cytoplasm of cells within “melanin granules” • Sections:
• Common sites where melanin can be found: • Works on all types of section, although
• Skin some adjustment is necessary for resin
• Eye section
• Brain • Results:
• Melanin
Demonstration of Melanin and Melanin- • Argentaffin, Chromaffin and some
producing Cells lipofuscins – Black
• Reducing methods such as the Masson- • Nuclei – Red
Fontana silver Technique and Schmorl’s ferric
Ferricyanide reduction test Microwave Ammoniacal Silver method for
• Enzyme methods argentaffin and melanin
• Solubility and bleaching characteristics • Fixation:
• Fluorescent methods • 10% NBF
• Immunohistochemistry
Page 388
• Results: • The enzyme tyrosinase localized within

• Melanin, Argentaffin, Chromaffin, the cells will oxidize DOPA to form an
lipofuscins and other silver reducing insoluble brown-black pigment
substances – Black • Best results are obtained using post-fixed
• Nuclei – Red cryostat sections
• Note: • DOPA Oxidase Method
• Results obtained with this method are • Method not in use
similar to those obtained with the • Enzyme tyrosinase in cells will oxidized
Masson-Fontana technique DOPA to form an insoluble brown-black
pigment
Schmorl’s Reaction • By Bloch (1917) and Laidlaw and
• Fixation: Blackberg (1932) for tissue sections
• 10% NBF • By Bloch (1917) and Rodriguez and
• Results: McGavran (1969) for tissue blocks
• Melanin, argentaffin cells, chromaffin
some lipofuscins, thyroid colloid – Dark Solubility Methods and Bleaching
blue • Solubility and Bleaching Methods
• Nuclei – Red • Uses strong oxidizing agents such as
• Enzyme methods permanganate, chlorate, chromic acid,
• DOPA Method peroxide, and peracetic acid that will
• Demonstrates cells capable of producing bleach melanin
melanin • Process is slow taking 16 hours
Page 389
• Lipofuscins take longer time to be • Results:

bleached from paraffin sections than • Melanin precursor cells – Weak yellow
melanin fluorescence
• Method of choice: peracetic acid but
treatment with 0.25% potassium Other Methods for Formalin
permanganate followed by 2% oxalic • Ferrous Iron Uptake Reaction for Melanin
acid also works well • Fixation:
• Formalin is best
Formalin-induced fluorescence • Avid all chromate fixatives
• Particularly useful in demonstrating amelanotic • Sections:
melanoma • Paraffin
• Melanin precursors present will form a product • Results:
of isocarboline derivatives that are • Melanin of skin, eye, pin and
dehydrogenated and will show yellow neuromelanin – Dark green
fluorescence • Nuclei – Red
• Best result is seen when using freeze-dried • Notes:
sections and then fixed using paraformaldehyde • According to Lilies this method is
vapor specific for melanin
• Fixation: • Ferric iron and lipofuscin do not
• 10% NBF stain with this method
• Sections:
• Cryostat
• 5-um paraffin sections
Page 390
• Nile blue method for melanin and lipofuscin • HMB-45-demonstrate melanosome

• Fixation: formation and melanocytic
• 10% NBF differentiation
• Sections:
• Paraffin Lipofuscins
• Results: • Yellow-brown to reddish-brown pigments occur
• Melanin – Dark blue widely throughout the body and are thought to be
• Lipofuscin – Dark blue produced by an oxidation process of lipids and
• Nuclei – Blue or unstained LPP
• Notes: • Found in the following sites:
• Using frozen sections, this method • Hepatocytes
will stain neutral lipids red to pink • Cardiac muscle cells
• Acidic lipids stain blue • Adrenal cortex
• Immunohistochemistry • Testis
• Uses antibodies to demonstrate • Ovary
melanocytic lesions • CNS
• More widely use antibodies: • Bone marrow
• S100 • Cervix
• HMB-45 • Kidney
• Melanin A
• To lesser extent PGP9.5
Page 391
Demonstration of Lipofuscins Aldehyde Fuchsin Technique

• Periodic-acid Schiff’s method • Fixation:
• Schmorl’s ferric-ferricyanide reduction test • 10% NBF
• Long-Ziehl-Neelsen method • Sections:
• Sudan Black B method • Paraffin
• Gomori’s aldehyde fuchsin technique • Solutions:
• Masson-Fontana silver method • Acidified potassium permanganate
• Basophilia using methyl green solution
• Churukian’s silver method • Results:
• Lilie’s Nile blue sulfate method • Lipofuscins – Purple
• Elastic – Purple
Long Ziehl-Neelsen method
• Fixation: Chromaffin
• Any • Normally found in the cells of adrenal medulla as
• Sections: dark brown, granular material
• Works well on all types of tissue section • May occur in pheochromocytoma
• Results: • Fixation:
• Lipofuscins – Magenta • Orth’s or dichromate containing fixatives
• Ceroid – Magenta are recommended
• Nuclei – Blue • May be demonstrated by Schmorl’s reaction,
• Background – Pale magenta to pale blue Lilie’s Nile blue A, Masson-Fontana, Churukian’s
microwave ammoniacal silver method and PAS
technique
Page 392
Pseudomelanosis pigment (melanosis cell) Hamazaki-Wesenberg Bodies

• Sometimes seen in macrophages in the lamina • Small, yellow-brown spindle-shaped structures
propria of the large intestine and appendix found mainly in the sinuses of lymph nodes
• Stain in methods that are used to demonstrate • Seen in patients with sarcoidosis and associated
lipofuscin such as Masson-Fontana and with melanosis coli
Schmorl’s • Histochemically similar to lipofuscin
• Ultrastructural level suggest probably are giant
Dubin-Johnson pigment lysosomal residual bodies
• Found in the liver of patients with Dubin-
Johnson syndrome and is due to defective Endogenous Minerals
canalicular transport of bilirubin • Calcium
• Characterized by presence of brown-black, • Copper
granular, intracellular pigment situated in the • Uric acid and urates
centrilobular hepatocytes
• Histochemically similar to lipofuscin Calcium
• Abnormal depositions can be found in necrotic
Ceroid-type lipofuscins areas of tissue associated with:
• Fail to stain with ferric-ferricyanide • Tuberculosis
• A lipofuscin in early stage of oxidation • Infarction (Gandy-Gamma bodies)
• Atheroma in blood vessel
• Malakoplakia of the bladder (Michaelis-
Gutman bodies)
Page 393
• Calcium salts are usually monorefringent Copper

• Calcium oxalate is birefringent • Mallory’s unrepined Hematoxylin – copper forms
• Calcium usually stains purple blue with H&E blue dye lake
• Dyes used for demonstration of calcium: • Other methods include the:
• Alizarin red S • Rubeanic method for copper
• Purpurin • Modified rhodamine technique
• Naphthochrome green B • Accumulation is associated with Wilson’s disease
• Nuclear fast red • Kayser-Fleischer ring – brown ring of deposited
• Von Kossa with silver nitrate – preferred for copper in the cornea
demonstration of calcium in paraffin sections • Deposition is also associated with primary biliary
cirrhosis and certain hepatic disorder
Alizarin red S method for calcium
• Fixation: Rubeanic acid method for Copper
• NBF • Fixative:
• Formal alcohol and alcohol • 10% NBF
• Sections: • Results:
• Paraffin and frozen • Copper – Greenish black
• Results: • Nuclei – Pale red
• Calcium deposits – Orange-red
• Background – Green
Page 394
Modified Rhodamine Technique Artifact Pigments

• Fixative: • Formalin
• 10% NBF • Malaria
• Section: • Schistosome
• Paraffin • Mercury
• Results: • Chromic Oxide
• Copper and copper associated protein – • Starch
Red to orange-red
• Nuclei – Blue Formalin Pigment
• Bile – Green • Seen as brown or brownish-black deposits in
tissues that have been fixed in acidic formalin
Uric Acid and Urates • Usually seen in blood-rich tissues such as:
• Breakdown products of body purine metabolism • Spleen
• Small proportion is obtained in the diet • Hemorrhagic lesions
• High levels result in deposition, which are water • Large blood vessels filled with blood
soluble in tissues causing: • Morphology of pigment is commonly seen as
• Subcutaneous nodular deposits of urate microcrystalline deposit that is anisotropic
crystals (‘tophi’) (birefringent)
• Synovitis and arthritis • One way of removing pigment is by treating
• Renal disease and calculi unstained tissue sections with sat. alcoholic picric
acid 10% ammonium hydroxide in 70% alcohol
for 5-15 minutes
Page 395
Malarial Pigment • Do NOT remove pigment with iodine solutions

• Morphologically similar to formalin pigment which cause CT to take up crystal violet and
• Formed within or in the region of RBCs with the resist acetone decolorization
malarial parasite
• Can be seen over the RBC within tiny blood Chromic Acid
capillaries of the brain in infection with P. • Rarely seen in tissues
Falciparum • Present as fine yellow-brown particulate deposit
• Like formalin pigment, exhibits birefringence in tissue
and can be removed with sat. alcoholic picric • May be reduced using graded alcohol
acid within 12-24 hour • Monorefringent and extracellular
• Can be removed by 1% acid alcohol
Schistosome Pigment
• Occasionally seen in tissue sections where Exogenous Pigments & Mineral
infections with Schistosoma can be seen • Lead
• Chunky and shows similar properties to both • Silver
formalin and malarial pigments
Lead
Mercury Pigment • Can be deposited within many tissues,
• Seen in mercury-containing fixatives particularly bone and kidney tubules
• Usually, monorefringent • Rhodizonate method – most popular method for
• In long storage of tissues, pigment changes demonstration of lead deposits
from crystalline form to globular one (exhibits a
Maltese cross birefringence)
Page 396
• Nonspecific methods: Rhodanine method for silver

• Sulfide-silver • Fixation:
• Unripened hematoxylin technique • Not critical but avoid using Mercury-
containing fixatives
Rhodizonate method for Lead salts • Sections:
• Fixation: • Paraffin
• Avoid use of mercuric-containing • Frozen sections (best results)
fixatives • Results:
• Sections: • Silver deposits – Reddish-brown
• Paraffin
• Results:
• Lead salts – Black
• Background – Green
Silver
• Rarely found in the skin
• More commonly seen as localized change in
the mouth
• Appear fine dark brown or black granules in
unstained and H&E sections, particularly in
basement membrane and sweat glands
Page 397
Special Stain 5: Lipids Stains • Examples:

• Cholesterol
Lipids • Bile acids
• Any group of fats or fat-like substances • Sex and adrenocortical hormones
characterized by their insolubility in water. • Lipids can be demonstrated by standard
• These fats would include: procedures, but exposure to alcohols, acetone,
• True fats – ester of fatty acid and chloroform, xylene and paraffin will destroy them
glycerol • Frozen sections - best to demonstrate simple
• Lipids – phospholipids, cerebrosides and lipids
waxes • Phospholipids, lipofuscin and granules of
• Hydrocarbons – squalene and carotene leukocytes can bind to other tissue elements or
entities and resistant to paraffin processing
Classification • Formal calcium - fixative of choice for
• Simple Lipids – usually found in the body as phospholipids (also demonstrated using oxidation
energy stores in adipose tissue and mordanting solutions or Sudan dyes
• Waxes are usually found in plant and animal
species Fixation
• Compound lipids can be formed in the brain and • Frozen Section – most common method of
central nervous system demonstrating lipids
• Derived lipids can originate from the simple and • Histochemical technique – general method for
compound lipids by means of hydrolysis lipid demonstration
Page 398
• Osmium tetroxide and chromic acid – the only Histophysical Methods

reagents that truly fix lipids, however, cause • Optical properties
great alteration in the chemical reactivity of • Affinity to fat stains and Sudan dyes
lipids • Microscopic discrimination between classes of
• Formal calcium – Choice of fixative for lipid lipids based on their differences in physical
histochemistry and is prepared by adding 2% property
calcium acetate to 10% formalin • Optical properties
• Formal saline should be avoided when dealing • Affinity to fat stains and Sudan dyes
with any material which may require • Example between crystalline and liquid lipids and
examination of lipids hydrophobic and hydrophilic types
Microtomy Birefringence of Lipids

• Frozen or cryostat sections require for lipid • Discrimination between fatty droplets that are
histochemistry stained and are non-birefringent (isotropic) from
• Have morphological appearance that is crystalline lipids which remain unstained but
at least as good as that of a routine appear anisotropic (birefringent)
paraffin section • Polarizing microscopy also distinguish cholesterol
• Mounted directly on slides ester from glycerol esters, where cholesteryl
• Formal-calcium-fixed blocks of tissue are also ester shows ‘Maltese cross’ birefringence
suitable for cryostat sectioning but is advisable
to mount sections in chrome-gelatin- coated
slides or used of ‘charged’ slides
Page 399
Fat Stains & the Sudan Dyes • Organic solvents used to facilitate ease of
• B-naphthol’s such as the original diazo dyes penetration of Sudans to fats
• Sudan III - first Sudan dye introduced • Isopropanol
into histochemistry • Triethyl phosphate
• Sudan IV - preferred by Michaelis (1901) • Propylene glycol
because of its darker staining • 70% ethanol for Oil red O and Sudan
• Basic Aryl amines with very low water solubility black
• Sudan Black B - most sensitive and
versatile and was introduced by Lison Oil Red O in dextrin
and Dagnelie (1935) • Fixation:
• Sudan Red VII B • Fresh frozen or NBF
• Oil red O - more intense in staining and • Rinse
generally more preferred to the other red dyes • Frozen
• Oily and greasy hydrophobic lipids, have an • Sections:
affinity for the Sudan dyes – Sudanophilia • 5 um mounts on Super Frost/ plus slides
• Staining is based on the fact that dyes are more • Air dry
soluble in tissue fats than in their dye solvent • Method:
and by adsorption process • Place slides directly into filtered 0.5% Oil
• Sudan III - first Sudan dye introduced red O in dextrin.
into histochemistry • Stain for 20 minutes, rinse with running
• Sudan IV - preferred by Michaelis (1901) water briefly.
because of its darker staining
Page 400
• Counterstain with Gill II hematoxylin for Bromine Sudan Black method for Lipids
20-30 seconds. • Fixation and sections:
• Rinse with water, blue, cover slip with aq. • Cryostat sections post - fixed for 1 hour in
Mounting medium formal calcium
• Results: • Short-fixed frozen sections
• Fat - Brilliant red • Results:
• Nuclei - Blue • Phospholipids – Gray
• Sphingomyelin - Bronze in polarized light
Standard Sudan black method for fats and
phospholipids Nile Blue Sulfate method for acidic & neutral
• Fixation and Sections: lipids
• Cryostat sections post-fixed in formal • Comprises two components
calcium • Red oxazone - dissolves in neutral lipids
• Short fixed frozen sections • Blue oxazone - reacts with phospholipids
• Unfixed cryostat sections (preferred) and free F.A.
• Results: • pH 9.0 and interference by non-lipid structures is
• Unsaturated esters - & TAG- Blue-black reduced by employing an acid-hydrolyzed dye
• Phospholipids – Gray solution
• Myelin - Bronze dichroism in polarized • Fixation and sections:
light • Cryostat sections post-fixed for 1 hour in
formal calcium
• Short-fixed frozen sections
Page 401
• Results: Osmic Acid Stain for Fats

• Unsaturated Hydrophobic lipids – Pink • Osmic acid is not a dye but is an unstable oxide
• Free fatty acids - Pink to Blue which his reduce to black substance by
• Phospholipids - Blue unsaturated fats and F.A.
• Used as a fixative for EM and histochemistry
Acetone - Nile blue sulfate method for • Fixation: 10% formalin
phospholipids • Section: Cryostat
• A Dunnigan’s modification whereby fatty acids
are first extracted with acetone so the Nile blue Histochemical Methods
sulfate staining will subsequently be confined to • Include methods for each lipid class and other
phospholipids relevant techniques
• Fixation and sections:
• Cryostat sections post-fixed for 1 hour in Histochemical Methods For Free Fatty Acids
formal calcium (converted into a soap • Weigert’s lithium hematoxylin
before fixation) • Developed by Fischler (1904) to stain
• Short-fixed frozen sections copper soaps bound with free F.A.
• Results: • Based on the observation that free fatty
• Phospholipids - Blue acids bind heavy metal ions to form soaps
• Okamoto et. Al. Procedure
• Use dimethylaminobenzylidine rhodanine
for the same purpose
• Neither method is specific as Holczinger’s copper
rubeanic acid method for free F.A.
Page 402
Copper Rubeanic Acid method for Free fatty • Calcium and iron deposits can be verified by
acids extraction with 1% HCl (for calcium) or 5% oxalic
• Visualized copper soaps with rubeanic acid, acid (for iron salts)
using EDTA to remove extraneous copper
• Fixation: Histochemical Methods For Cholesterol
• Cryostat sections post-fixed in formal • Schultz (1924) technique
calcium • Adapted the Liebermann - Burchardt
• Fixed frozen sections reaction to demonstrate cholesterol in
• Results: tissue sections
• Free F.A. - Dark green (rubeanic acid) or • Method: Oxidation in air with iron alum
red (p- dimethylaminobenzylidine followed by treatment with a sulfuric-acetic
rhodanine) acid mixture
• This is absent from acetone- • Results: Free and esterified cholesterol –
extracted control Blue
• If cryostat sections are used, • Rossouw et al
extraction is best performed • Overcame technical problems associated
before post-fixation with Schultz procedure by a using a ‘pre-
mixed’ reagent which is a combination of
Holczinger’s Copper Acetate Procedure ferric chloride and acetic, sulfuric, and
• Provides an equally specific and sensitive phosphoric acids
alternative using Okamoto’s rhodanine reagent • Iodine-sulfuric acid technique
with rather less background staining and is • Described by Okamoto but color is
compatible with hematoxylin nuclear stain transient and masked by iodine
Page 403
Adams’ Perchloric Acid - Naphthoquinone • Used to detect accumulation of free cholesterol in

(PAN) Method cultured fibroblasts from Niemann-Pick disease
• Depends on the different principle for • Fixation and section:
cholesterol detection • Cryostat section, post-fixed
• Sensitive, accurate and has more satisfactory • Frozen sections of fixed tissue
tissue preservation • Results:
• Ineffective unless cholesterol has been oxidized • Free cholesterol - Strong silvery
with ferric salts or exposure to air (same is true fluorescence
with the procedures mentioned beforehand)
• Fixation and Sections: Enzymatic methods for Free cholesterol
• Formal calcium fixed frozen section • Uses enzymes like cholesterol esterase and
• Cryostat sections post-fixed in formal oxidase for cholesterol hydrolysis and oxidation
calcium • Practical value is limited by their cost and
• Results: complexity for routine light microscopy
• Cholesterol and related steroids - Blue • Proved to be useful at the EM level
Filipin Method for Free cholesterol Histochemical Methods for Unsaturated Lipids
• Uses filipin, a compound similar to digitonin • Unsaturated-Schiff Method
• Used in aq. Media to complex with free • Fixation and sections: preferably unfixed
cholesterol to produce a highly fluorescent cryostat sections
compound • Results: Unsaturated lipids – Magenta
Page 404
• Osmium Tetroxide Method Histochemical Methods for Glycerol-Based

• Fixation and sections: Preferably unfixed Phospholipids
cryostat sections • Gold Hydroxamic Acid Method
• Results: Unsaturated lipids - Brown to • Recommended for selective
black demonstration of phosphoglycerides
• Note: Osmium solution must be handled (lecithin & cephalins)
inside a fume hood to avoid effect of its • Ester bonds, converted to hydroxamic
toxic vapor o cornea and mucous acids by alkaline hydroxamine, reduce
membrane silver nitrate to metallic silver and finally
stabilized by gold toning
Histochemical Methods for Triglycerides • Phosphoglycerides - stained purple
• Calcium Lipase Method
• Adams (1966) use pancreatic lipase to Histochemical Methods for Sphingomyelins
hydrolyze triglycerides into their • Dichromate-Acid Hematein (DAH) For
constituent’s fatty acids Choline-Containing Phospholipids
• Highly specific for triglycerides and has • Unfixed cryostat sections are
been adapted for electron histochemistry recommended
• Uses cryostat sections post-fixed in • Lecithin and sphingomyelin - Blue black
formal calcium • Ferric Hematoxylin Method
• Results: Triglycerides - Brown • Simpler, quicker, and more sensitive than
DAH technique
• Method of choice for sphingomyelin
Page 405
• Ideally unfixed cryostat sections are used • Results:

• Phospholipids and nuclei - Blue • Sulfatide deposits - Metachromatic
red-brown or yellow
Histochemical Methods for Sphingomyelins • Modified PAS reaction cerebroside
• Naoh- Ferric Hematoxylin/DAH Method • Fixation:
• Ideally unfixed cryostat sections mounted • Cryostat sections post-fixed with
on chrome - gelatin-coated slides are formal calcium
used • Fixed frozen sections
• Sphingomyelin – Blue • Results:
• Notes: to prevent interference by • Cerebroside – Magenta
plasmalogens, sections are treated with • Borohydride-periodate-Schiff (BHPS) method
1% mercuric chloride in 1% HCl for 10 for gangliosides
minutes • Fixation:
• Cryostat sections post-fixed with
Histochemical Methods for Other Lipids formal calcium
• Toluidine blue-acetone method for sulfatide • Fixed frozen sections
• Fixation: • Results:
• Cryostat sections post-fixed with • Gangliosides – Red
formal calcium • Nuclei – Blue
• Fixed frozen sections
Page 406
• UV method for lipofuscin • Dichromate-acid hematein - Oil red O

• Fixation: combination
• Cryostat sections (5-10 um) air • Recommended for demonstration of
dried normal and degenerating myelin
• Fixed frozen sections • Good color contrast between blue myelin
• Paraffin sections and red-stained esters within lipophages
• Results:
• Lipofuscin - Orange-yellow Methods for Demonstration of Normal &
fluorescence Degenerating Myelin
Application of Lipid Histochemistry in Dichromate-acid hematein - Oil red O

Pathology combination
• Recommended for demonstration of normal and
Myelin & Demyelination degenerating myelin
• Primary demyelination and dysmyelination are • Good color contrast between blue myelin and
demonstrated using paraffin sections red-stained esters within lipophages
• Frozen section
• Preferred for demonstration of active Sudan Black B
demyelination and stain with Sudan dye • Normal myelin – Gray
• Lipophage - convenient hallmark of • Degenerating myelin - Intense blue
ongoing demyelination
Luxol fast blue
• For paraffin sections
Page 407
Osmium tetroxide a-naphthylamine (OTAN)

technique
PAS Sudan red procedure of Hirsch & Peiffer
Parkinson’s disease and the characteristic

Lewy bodies
• Can be demonstrated by:
• NAOH-ACID HEMATEIN PROCEDURE
• SUDAN BLACK B - sphingomyelin is
demonstrated with red birefringence
• Sudan Black B is also use to
demonstrate lipid deposition in
Fabry’s disease
Page 408
Special Stain 6: Microorganisms • Ethyl alcohol-acetone

• Basic fuchsin
• Stains use for the following microorganisms: • Results:
• Viruses • Gram - positive organism, fibrin, some
• Bacteria fungi, Paneth cells granules – Blue
• Fungi • Gram - negative organisms – Red
• Protozoa • Nuclei – Red
• Helminths • Other tissue elements – Yellow
Detection and Identification of Bacteria Gram-Twort Stain

Gram Stain • Results:
• Quick technique such as this enable rapid • Gram- positive organism – Blue – black
identification of the organism causing lung • Gram-negative organisms – Pink – red
abscess, wound infection, septicemic • RBC and cytoplasmic structures – Green
abscesses or meningitis • Elastic fibers – Black
Modified Brow-Brenn Method for Gram -

positive and -negative bacteria in paraffin
sections
• Uses:
• Crystal violet
• Modified Gram’s iodine
Page 409
Techniques for Mycobacteria • Uses:

Ziehl-Neelsen • Carbolfuchsin
• Fixative: • Acid alcohol
• Formalin or fixative other than Carnoy’s • Methylene blue
• Paraffin • Results:
• Results: • AFB including M. leprae – Bright red
• Mycobacteria – Red • Nuclei and other tissue elements – Pale
• Background – Pale blue blue
Fluorescent Method for Mycobacteria Other Methods

• Uses: Cresyl Violet Acetate Method for Helicobacter
• Auramine O sp.
• Rhodamine B • Fixation:
• Results: • Formalin fixed
• Mycobacteria – Golden yellow • Paraffin
• Background – Dark green • Results:
• Helicobacter and nuclei – Blue – violet
Modified Fite Method for M. leprae and • Background – Shades of Blue – violet
Nocardia
• Fixation: NBF
• Sections: 4-5 um
Page 410
Gimenez Method for H. pylori Warthin-Starry Method for Spirochetes

• Fixation: • Fixation:
• Formalin fixed • Formalin fixed
• Paraffin • Paraffin
• Uses: • Employs 1% silver nitrate in pH 3.6 acetate buffer
• Carbolfuchsin • Results:
• Malachite green • Spirochetes – Black
• Results: • Background – Golden – yellow
• Helicobacter – Red magenta
• Background – Blue – green Modified Steiner for Filamentous and Non-
filamentous Bacteria
Toluidine Blue in Sorensen’s Buffer for • Sections:
Helicobacter • Formalin fixed
• Fixation: • Paraffin
• Formalin fixed • Employs 1% uranyl nitrate, 1% silver nitrate
• Paraffin • Results:
• Uses: • Spirochetes, rickettsiaes, Donovan bodies,
• Toluidine blue in pH 6.8 non-filamentous bacteria of L.
• Results: pneumophila – Dark brown-black
• Helicobacter - Dark blue against a • Background – Bright yellow to golden
variably blue background yellow
Page 411
Identification of Fungi Demonstration of Rickettsiae

Grocott Methenamine (hexamine) - Silver for Macchiavello’s Stain for Rickettsiae and Viral
Fungi and Pneumocystis Organisms inclusions, modified
• Sections: • Sections:
• Formalin fixed • Formalin fixed
• Paraffin • Paraffin
• Results: • Results:
• Fungi, pneumocystis, melanin – Black • Rickettsia and some viral inclusions – Red
• Hyphae and yeast – Sharply delineated • Background – Blue
in black
• Mucins and glycogen – Taupe to dark Detection and identification of viruses
gray Phloxine Tartrazine Technique for Viral
• Background – Pale green inclusions
• Sections:
McManus’ PAS Method for Glycogen and • Formalin fixed
Fungal Cell Walls • Paraffin
• Fixation: NBF • Employs tartrazine and phloxine
• Sections: 3-5 um paraffin sections • Results:
• Results: • Viral inclusion – Bright red
• Fungal cell walls and glycogen – • RBCs – Variably orange-red
Magenta to red • Nuclei-blue – Gray
• Background – Pale green • Background – Yellow
Page 412
Shikata’s Orcein Method for Hepatitis B

Surface Antigen
• Sections:
• Formalin fixed
• Paraffin
• Employs acid permanganate, orcein, cellosolve
and tartrazine
• Results:
• HBsAg, elastic and some mucins –
Brown – black
• Background – Yellow
Demonstration of Protozoa and other

organisms
Giemsa Stain for Parasite
• Fixation:
• Not critical
• Preferred is B5 or Zenkers’ is preferred
• Results:
• Protozoa and some other
microorganisms – Dark-blue
• Background – Pink-pale blue
• Nuclei – Blue
Page 413
References: (No copyright infringement

intended)
• Bruce-Gregorios, J., 2006. Histophatologic
Techniques. 2nd ed.
• Rolls, G., 2011. An Introduction to Specimen
Processing. [online] Leicabiosystems.com.
Available at:
<http://www.leicabiosystems.com/pathologyle
aders/an-introduction-to-specimen-
processing/> [Accessed 10 October 2015].
• Credits to the rightful owners of the pictures
that has been used in this Trans/Reviewer
Page 414
Thank you!
Happy Aral
PADAYON!!!
Page 415

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The Cell Structure and Function

The Cell Thoery

• Function of plasma membrane = selective

Nucleus and Its Envelope

Level of Chromatin Packing

Nucleolus • Eukaryotes (80s ribosome):

• Free ribosomes in cytosol synthesize proteins

• Bound and free ribosomes are structurally Endoplasmic reticulum (ER)

• Golgi removes some sugar monomers

Relationships among Organelles of the

• After digestion, the organic monomers are

• Lysosomal enzymes and membranes are made

Vacuoles Mitochondria and Chloroplast

Cytoskeleton and Cell Motility

Types of exocrine glands: Connective tissue

Functions of Connective tissue:

Connective tissue cells are named according Notes:

Muscle tissue Three types of muscle tissue:

Components of a nerve cell:

Tissues Cells Extracellular Matrix Main Functions

Topic 3: Cellular Response to Injury • Most of these adaptations take place at a

Causative agents and processes

Major types of cellular injury:

Major Types of Cellular Injury

Physical agents • Some are highly toxic systemic

• The mode of cell death generally • Blockage of metabolic pathways

• When the blood supply is • Failure of membrane integrity

• Cell populations that are constantly • While consequence of unsuccessful adaptation to

Acute inflammation 2) Extravasation of polymorphonuclears from the

Sequence of events in an inflammatory reaction.

The vascular phenomena in acute • Phagocytosis

• It is characterized by: • Prolonged exposure to potentially toxic

Outcomes of acute inflammation: resolution, healing by fibrosis, or

Chronic inflammation may begin as a low- • Persistent acute inflammation

Long-term diseases that doctors associate A test for Inflammation:

• It is characterized by the presence of

Disease Cause Tissue

Serous inflammation Fibrinous inflammation

Biologic purpose: Immediate temporary barrier Fibrinous Serosal Inflammation

Fibrinous Parenchymal Inflammation Croupous

Suppurative or purulent inflammation

Types of suppurative inflammation: Phlegmon

Abscess Tissue Repair

If fibrosis develops in a tissue space occupied by Granulation Tissue

Repair by Connective Tissue Deposition

If repair cannot be accomplished by regeneration

In contrast to regeneration, which involves the 2) Cell proliferation

3) Formation of granulation tissue Factors That Influence Tissue Repair

Examples of Tissue Repair and Fibrosis Healing by second intention

Healing By First Intention • Wound strength reaches approximately 70 % to

Healing By Second Intention

Topic 4: Abnormalities in Cell Growth Aplasia

• Increase metabolic activity leading to • Exhaustion atrophy

True hypertrophy Physiologic hyperplasia

Dysplasia • As they grow, neoplasms can impinge upon and

Benign mesenchymal tumors

Benign epithelial tumors

Endothelium and Mesothelium

Blood and Lymphoid Cells

Tissue Benign Tumors Malignant Tumors

Striated muscle Rhabdomyoma Rhabdomyosarcoma

Characteristics of Benign and Malignant Anaplasia