Tissues

 Cells work together in functionally

related groups called tissues
o How is this done?
▪ Attachments
▪ communication
 Types of tissues:
▪ Epithelial – lining and
covering
▪ Connective – support Classifications of Epithelia
▪ Muscle – movement  First name of tissue indicates number of
▪ Nervous – control layers
Epithelial Tissue o Simple – one layer of cells
 Covers a body surface or lines a body
cavity
 Forms most glands
 Functions of epithelium
o Protection o Stratified – more than one layer of
o Absorption, secretion, and cells
diffusion
o Filtration
o Forms slippery surfaces (mucus
secretion)
Special Characteristics:  Last name of tissue describes shape of cells
o Squamous – cells wider than
 Cellularity
tall (plate or “scale” like)
o cells are in close contact with
each other with little or no
intercellular space between them
 Specialized contacts
o Cuboidal – cells are as wide as
o may have junctions for both
tall, as in cubes
attachment and communication
 Polarity
o epithelial tissues always have an
apical and basal surface
o Columnar – cells are taller than
 Support by connective tissue
they are wide, like columns
o at the basal surface, both the
epithelial tissue and the
connective tissue contribute to
the basement membrane
 Avascular
o nutrients must diffuse from basal Naming Epithelia
layer  Naming the epithelia includes both the
 Innervated layers (first) and the shape of the cells
 Regenerative (second)
o epithelial tissues are highly o i.e. stratified cuboidal epithelium
mitotic  The name may also include any accessory
structures
o Goblet cells
o Cilia
o Keratin
 Special epithelial tissues (don’t follow
naming convention)
o Psuedostratified
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 o Transitional
Simple Squamous Epithelium Simple Cuboidal Epithelium
 Description  Description
o single layer of flat cells with disc- o single layer of cube-like cells
shaped nuclei with large, spherical central
 Special types nuclei
o Endothelium (inner covering)  Function
▪ slick lining of hollow o secretion and absorption
organs  Location
o Mesothelium (middle covering) o kidney tubules, secretory
▪ Lines peritoneal, pleural, portions of small glands, ovary
and pericardial cavities surface
▪ Covers visceral organs of
those cavities
 Function
o Passage of materials by passive
diffusion and filtration
o Secretes lubricating substances in
serous membranes
 Location
o Renal corpuscles (kidneys)
o Alveoli of lungs
o Lining of heart, blood and
lymphatic vessels
o Lining of ventral body cavity
(serosae/serous memb)

If it’s from a
mesothelial lining

Simple Columnar Epithelium

 Description
o single layer of column-shaped
Simple (rectangular) cells with oval
squamous lining nuclei
the walls of the ▪ Some bear cilia at their
capillary apical surface
▪ May contain goblet cells
 Function
o Absorption; secretion of mucus,
enzymes, and other substances
o Ciliated type propels mucus or
reproductive cells by ciliary
action
 Location
o Non-ciliated form
▪ Lines digestive tract,
gallbladder, ducts of
some glands
o Ciliated form
▪ Lines small bronchi,
uterine tubes, and uterus
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 Stratified Epithelia
 Contain two or more layers of cells
 Regenerate from below
 Major role is protection
 Are named according to the shape of
cells at apical layer

Stratified Squamous Epithelium

 Description
Pseudostratified Columnar o Many layers of cells – squamous
Epithelium in shape
 Description o Deeper layers of cells appear
o All cells originate at basement cuboidal or columnar
membrane o Thickest epithelial tissue –
o Only tall cells reach the apical adapted for protection
surface  Specific types
o May contain goblet cells and o Keratinized – contain the
bear cilia protective protein keratin
o Nuclei lie at varying heights ▪ Surface cells are dead
within cells and full of keratin
▪ Gives false impression of o Non-keratinized – forms moist
stratification lining of body openings
 Function  Function
o secretion of mucus; propulsion o Protects underlying tissues in
of mucus by cilia areas subject to abrasion
 Locations  Location
o Non-ciliated type o Keratinized – forms epidermis
▪ Ducts of male o Non-keratinized – forms lining
reproductive tubes of esophagus, mouth, and vagina
▪ Ducts of large glands  Non- Keratinized
o Ciliated variety
▪ Lines trachea and most
of upper respiratory tract

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 Keratinized Epithelial Surface Features –
Apical Surface Features
Microvilli
 finger-like extensions of plasma
membrane
o Abundant in epithelia of small
intestine and kidney
o Maximize surface area across
which small molecules enter or
leave
o Act as stiff knobs that resist
abrasion
Transitional Epithelium Cilia
 Description  whip-like, highly motile extensions of
o Basal cells usually cuboidal or apical surface membranes
columnar o Contains a core of nine pairs of
o Superficial cells dome-shaped or microtubules encircling one
squamous middle pair
 Function o Axoneme – a set of microtubules
o stretches and permits distension o Each pair of microtubules –
of urinary bladder arranged in a doublet
 Location o Microtubules in cilia – arranged
o Lines ureters, urinary bladder similarly to cytoplasmic
and part of urethra organelles called centrioles
o Movement of cilia – in
coordinated waves

Relaxed state

Stretched state

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 Connective Tissue
 Most diverse and abundant tissue  Structures within areolar connective
 Main classes tissue allow:
o Connective tissue proper o Support and binding of other
o Cartilage tissues
o Bone tissue o Holding body fluids
o Blood o Defending body against
 Components of connective tissue: infection
o Cells (varies according to tissue) o Storing nutrients as fat
o Matrix  Loose Connective Tissue
▪ Fibers (varies according o Areolar
to tissue) o Reticular
▪ Ground substance o Adipose
(varies according to  Dense Connective Tissue
tissue) o Regular
o dermatin sulfate, o Irregular
hyaluronic acid, o Elastic
keratin sulfate,
chondroitin
sulfate…
 Common embryonic origin –
mesenchyme

 Areolar connective tissue

o Underlies epithelial tissue Areolar Connective Tissue
o Surrounds small nerves and  Description
blood vessels o Gel-like matrix with:
o Has structures and functions ▪ all three fiber types
shared by other connective (collagen, reticular,
tissues elastic) for support
o Borders all other tissues in the ▪ Ground substance is
body made up by

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 glycoproteins also made Reticular Connective Tissue
and screed by the  Description
fibroblasts. o network of reticular fibers in
o Cells – fibroblasts, macrophages, loose ground substance
mast cells, white blood cells  Function
 Function o form a soft, internal skeleton
o Wraps and cushions organs (stroma)
o Holds and conveys tissue fluid o supports other cell types
o Important role in inflammation  Location
Main battlefield in fight against o lymphoid organs
infection o Lymph nodes, bone marrow,
 Defenders gather at infection sites and spleen
o Macrophages
o Plasma cells
o Mast cells
o Neutrophils, lymphocytes, and
eosinophils
 Location
o Widely distributed under
epithelia
o Packages organs
o Surrounds capillaries

Dense Regular Connective Tissue

 Description
o Primarily parallel collagen fibers
o Fibroblasts and some elastic
fibers
o Poorly vascularized
 Function
Adipose Tissue o Attaches muscle to bone
 Description o Attaches bone to bone
o Closely packed adipocytes o Withstands great stress in
o Have nucleus pushed to one side one direction
by fat droplet Function  Location
o Provides reserve food fuel o Tendons and ligaments
o Insulates against heat loss o Aponeuroses
o Supports and protects organs o Fascia around muscles
 Location
o Under skin
o Around kidneys
o Behind eyeballs, within
abdomen and in breasts

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 Cartilage
 Characteristics:
o Firm, flexible tissue
o Contains no blood vessels or
nerves
o Matrix contains up to 80% water
o Cell type – chondrocyte
 Types:
o Hyaline
o Elastic
o Fibrocartilage

Hyaline Cartilage
 Description
o Imperceptible collagen fibers
Fibrocartilage
(hyaline = glassy)
o Chodroblasts produce matrix  Description
o Chondrocytes lie in lacunae o Matrix similar, but less firm than
 Function hyaline cartilage
o Supports and reinforces o Thick collagen fibers
o Resilient cushion predominate
o Resists repetitive stress  Function
 Location o Tensile strength and ability
o Fetal skeleton to absorb compressive
o Ends of long bones shock
o Costal cartilage of ribs  Location
o Cartilages of nose, o Intervertebral discs
trachea, and larynx o Pubic symphysis
o Discs of knee joint

Bone Tissue
Elastic Cartilage  Function
 Description o Supports and protects organs
o Similar to hyaline cartilage o Provides levers and attachment
o More elastic fibers in matrix site for muscles
 Function o Stores calcium and other
o Maintains shape of structure minerals
o Allows great flexibility o Stores fat
 Location o Marrow is site for blood cell
o Supports external ear formation
o Epiglottis  Location
o Bones

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 o Voluntary movement
o Manipulation of
environment
o Facial expression
 Location
o Skeletal muscles attached to
bones (occasionally to skin)

Blood Tissue
 Description
o red and white blood cells
in a fluid matrix
 Function
o transport of respiratory
gases, nutrients, and wastes
 Location
o within blood vessels Cardiac Muscle Tissue
 Characteristics  Function
o An atypical connective tissue o Contracts to propel blood into
o Develops from mesenchyme circulatory system
o Consists of cells surrounded by  Characteristics
nonliving matrix o Branching cells
o Uninucleate
o Striations
o Intercalated discs
 Location
o Occurs in walls of heart

Muscle Tissue
 Types
o Skeletal muscle tissue
o Cardiac muscle tissue
Smooth Muscle Tissue
o Smooth muscle tissue  Characteristics
o Spindle-shaped cells with
central nuclei
Skeletal Muscle Tissue o Arranged closely to form
 Characteristics sheets
o Long, cylindrical cells o No striations
o Multinucleate  Function
o Obvious striations o Propels substances along
 Function internal passageways
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 o Involuntary control o Destroys particular
 Location microorganisms at site of
o Mostly walls of hollow organs infection

The Tissues Throughout Life

 At the end of second month of
development:
o Primary tissue types have
appeared
o Major organs are in place
 Adulthood
o Only a few tissues regenerate
o Many tissues still retain
populations of stem cells
 With increasing age:
o Epithelia thin
o Collagen decreases
o Bones, muscles, and nervous
tissue begin to atrophy
Nervous Tissue o Poor nutrition and poor
 Function circulation – poor health of
o Transmit electrical signals tissues
from sensory receptors to
effectors
 Location
o Brain, spinal cord, and nerves
 Description
o Main components are brain, Blood: Human blood, human blood
spinal cord, and nerves components, and products made from human
o Contains two types of cells blood
▪ Neurons – excitatory Bloodborne pathogen: Any pathogenic
cells microorganism that is present in human blood
▪ Supporting cells and can cause disease in humans; eg. HBV &
(neuroglial cells) HIV
Clinical laboratory: workplace where diagnostic
or other screening procedures are performed on
blood or other potentially infectious materials
Contaminated: anticipated presence of blood or
other potentially infectious materials on an item
or surface
Contaminated sharps: Any contaminated object
that can penetrate the skin eg. needles, scalpels,
broken glass, broken capillary tubes, and exposed
ends of dental wires
Decontamination: use of physical or chemical
means to remove, inactivate, or destroy
bloodborne pathogens on a surface or item where
they are no longer capable of transmitting
infectious particles and the surface or item is
Tissue Response to Injury rendered safe for handling, use, or disposal
 Inflammatory response – non-specific,
local response Other potentially infectious materials:
o Limits damage to injury site  Anybody fluid that is visibly
 Immune response – takes longer to contaminated with blood, and all body
develop and very specific fluids in situations where it is difficult or
impossible to differentiate between body
fluids (semen, vaginal secretions,
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 cerebrospinal fluid, synovial fluid, sifting through waste dumps could lead
pleural fluid, pericardial fluid, peritoneal to the spread of hepatitis B, HIV-AIDS
fluid, amniotic fluid, saliva in dental and other blood-borne diseases.
procedures)
 Any unfixed tissue or organ (other than
intact skin) from a human (living or
dead)
 HIV-containing cell or tissue cultures,
organ cultures, and HIV or HBV-
containing culture medium or other
solutions and blood, organs, or other
tissues from experimental animals
infected with HIV or HBV

Work practice controls: Controls that reduce the

exposure by altering the manner in which a task
is performed, eg. prohibiting recapping of needles
by a two-handed technique

Chain of Infection
 a model used to understand the infection Method of Compliance
process.  Universal precautions
 a circle of links, each representing a o An approach to infection control
component in the cycle. in which all human blood and
o Each link must be present and in certain human body fluids are
sequential order for an infection treated as if known to be
to occur. infectious for HIV, HBV, and
o The links are: infectious agent, other bloodborne pathogens
reservoir, portal of exit from the
reservoir, mode of transmission, Exposure determination
and portal of entry into a High risk
susceptible host.  handling leaking or contaminated
 Modes of transmission- involve contact specimen containers
(e.g., contaminated needles or blood  working with frozen sections that involve
splatter), vehicle-borne (e.g., handling fresh unfixed human tissue
contaminated wastewater), air-borne including
(e.g., aerosolized pathogens from broken o freezing specimens
culture dishes or the rapture of yellow o sectioning in the cryostat
bags), and vector borne (e.g., rodents in o washing off freezing compound
an HCW storage area) transmission. prior to fixation
 Portals of entry include breaks in the  grossing specimens that involve handling
skin and mucous membranes (e.g., unfixed and fixed human tissue and
needlestick injuries or blood splashes into organs
the mucous membranes), the respiratory o examining and handling
tract (inhalation of pathogenic aerosols), specimens as when searching for
etc. lymph nodes
 Potential susceptible host include HC o sectioning to obtain the proper
workers, waste handlers, patients and size for processing
visitors in the HCF, landfill operators, o wrapping small specimens in
scavengers and the general public. paper prior to cassetting
 The consequences of improper o sawing bone or calcified
handling and disposal of HCW are specimens with the band saw or
serious. For example, the reuse of Beuhler bone saw
improperly discarded needles by o using disposable needles—
intravenous (IV) drug users or accidental disposable needles should never
needle stick injuries suffered by recyclers be recapped; the syringe and

10
 needle combined should be Ingestion: Encourage the victim to drink large
disposed of in the sharps amounts of water. Refer to the MSDS for special
container precautions and additional information.
o cleaning up and disinfecting
counter tops, cryostats, and Skin contact: Promptly flush the affected area
refrigerators. with water and remove any contaminated
clothing. If symptoms persist after washing, seek
Low risk medical attention.
 performed by the histology technicians,
histology supervisor, and laboratory Cleanup: Promptly contain chemical spills and
aides: alert people in all parts of the facility including
 picking up, grossing and cassetting isolation areas. If the spill is small, clean it up
kidney biopsies using appropriate protective apparel and
 fixing, decalcifying, and cassetting bone equipment. Remember to dispose of
marrow biopsies contaminated items as hazardous waste. For
 loading tissue processors and handling large spills or those containing extremely toxic or
contaminated specimen containers such hazardous materials contact safety services
as with autopsy cases personnel for assistance.
 staining frozen sections and touch prep
slides (smears) Eating, smoking, drinking, gum chewing, or
 handling processed tissue sections (there application of cosmetics are prohibited in areas
is a slight risk in CJD infected brain where laboratory chemicals are present. Wash
cases) hands before conducting activities. Avoid storing
 cleaning counters or handling food or beverages in storage areas,
refrigerators, glassware, or utensils which are
used for laboratory operations.
Chemical hazards
 Exposure to toxic chemicals poses a real Equipment and glassware Handle and store
threat to the health and safety of
laboratory glassware with care to avoid damage.
laboratory staff.
3 main routes by which chemicals enter the Exiting: Wash hands prior to leaving the
body. laboratory
 Inhalation—major route of entry when
working with solvents; great rapidity of Horseplay: Avoid practical jokes or other
absorption when fumes are inhaled. behavior that might confuse, startle or distract
 Absorption through skin—this may another worker.
produce systemic poisoning; the
condition of the skin determines the rate Mouth suction Do not use mouth suction for
of absorption. pipetting or starting a siphon. use pipet pumps.
o Examples of chemicals with these
risks are organic lead, solvents Personal protective equipment The MSDS will
such as xylene and methylene specify which protective equipment are to be
chloride, organophosphate, used
pesticides and cyanides.
 Ingestion —accidental ingestion is Personal housekeeping Keep the work area clean
generally due to poor hygiene practices, and uncluttered. Chemicals and equipment
such as eating or smoking in the should be properly labeled and stored. Clean up
laboratory. the work area at the completion of each operation
and at the end of each day. Replace microtome
Working with chemicals blades in the original box.
Accidents and spills
Eye contact: Promptly flush eyes with water for Activities which require protection or PPE
at least 15 minutes and seek medical attention. o working with formaldehyde
o hazardous waste and associated
contaminants
o working with acids, bases, or organic
material changing processors
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 o grossing and cassetting specimens 2. Benzoyl peroxide
o making reagents and solutions  easily ignited and sensitive to shock,
 decomposes spontaneously at
Chemical Storage and Handling temperatures above 50°C,
 Good laboratory practice should be  can be desensitized by the addition of
observed even when handling substances 20% water.
of no known significant hazard. 3. Anhydrous aluminum chloride
Minimize exposure by working in an  completely enclose the bottle in a heavy
exhaust hood, wearing eye and hand towel and open slowly,
protection, and a laboratory coat or an  add water slowly.
apron. 4. Ammonia
 Corrosives are strong acids or bases – it  becomes explosive when mixed with
may be liquid or solid. iodine,
o Inhalation of the vapors or mists  reacts with hypochlorite to become
of these substances can cause chlorine,
severe bronchial irritation;  may become explosive with silver nitrate
o can erode the skin and harmful and should be disposed of after use.
to the eyes;
o also damaging to steel. 5. Chromic acid (chromium trioxide) will react
1. Wear protective clothing when working with with alcohol and other flammables.
corrosives. 6. Dry ice and liquid nitrogen should not keep
2. Acids should be stored separate from other in sealed containers; CO2- build-up will
chemicals cause the container to burst. Containers
3. When mixing solutions, always add the acid specifically designed for such storage should
slowly to the water. Never pour water into be used.
concentrated acids or bases.
4. Do not mix incompatible chemicals. If in Known Carcinogens
doubt check the MSDS  aniline
 chloroform
Flammables: any liquid having a flashpoint  formaldehyde
below 100°F.  chromium compounds chromic acid
 Must be stored in a safety can with a potassium dichromate
spring closing lid and spout cover.
 Do not store flammables in areas exposed Carcinogenic stains
to direct sunlight.  Auramine o
 Do not store combustibles (e.g.,  Basic fuchsin (pararosaniline
cardboard boxes) with flammables. hydrochloride)
 Tissue processors and similar automatic  Chlorazol black e
equipment using flammable or  Ponceau de xylidine (ponceau 2R)
combustible reagents should be operated  Substances that cause cancer in animals
at least 5 feet away from heat sources (no human data)
such as embedding centers. o acridine orange
 Label all flammables with a o basic fuchsin (rosaniline
“Flammables” label. hydrochloride)
o Congo red
“Routine chemical reaction" is used, refers to a o crystal violet (gentian violet),
relatively slow or easily controllable reaction. o Eosin Y,
o fast green FSF
Highly reactive chemicals and explosives o hematoxylin
When working with the following highly reactive o light green SF yellowish
chemicals, special procedures must be taken: o orange g,
1. Picric acid crystal o rhodamine B,
 always have 10% water added to the o Sudan III
container (it is explosive when dry) o Sudan IV
 be kept out of contact with metal,
 never be used near an open flame.
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 Safety in the Lab Specific hazards that YOU
 Should be well-ventilated. SHOULD KNOW
 Regulations governing formalin and  Bouin's solution- is made with picric
hydrocarbons (xylene and toluene) acid.
should not be exceeded the limits set by  acid is only sold in the aqueous state.
the Occupational Safety and Health When it dries out, it becomes explosive.
Administration (OSHA)  Na azide - as a preservative.
 MSDS-should specify the Nature  Flush solutions containing Na azide
(chemical & physical), Nomenclature, down the drain with lots of water, (there
Common Name, known health effects, is a tendency for the azide to form metal
routes of entry, Toxicity & safety azides in the plumbing -explosive.)
precautions in handling
 Benzidine, benzene, anthracene, and
 Must have a method for disposal of
naphthol containing compounds are
hazardous wastes.
carcinogens and should not be used.
 Proper Waste Mgt- Health care facilities
 Mercury-containing solutions (Zenker's
processing tissues often contract this to a
or B-5) should always be discarded into
waste management company.
proper containers. Mercury, if poured
o Tissues that are collected should
down a drain, will form amalgams with
be stored in formalin and may be
the metal that build up and cannot be
disposed by incineration or by
removed
putting them through a "tissue
grinder" attached to a large sink
(similar to a large garbage Labels And Warning Signs
disposal unit).  Chemical containers MUST BE checked
 Do not discharge concentrated acids or for warning labels & good condition.
bases, flammables, highly toxic  Any time a chemical is transferred into a
substances, or heavy metals such as different container, the new container
mercury (B-5 fixative) into the sewer- MUST BE appropriately labelled.
must be recycled or reduced when o The name of the chemical as
possible, or placed in appropriate found on the MSDS.
containers to be picked up by safety o Any hazards associated with the
services. chemical or mixture.
 A Hazardous Waste Manifest must o Name and address of the
accompany waste material. chemical manufacturer or
 Every instrument used in the laboratory importer.
should meet electrical safety  All solutions MUST BE labelled w/the
specifications and have written chemical ingredients, preparation date,
instructions regarding its use. initials identifying the technician who
 Flammable materials should be in prepared solution, and expiration date.
properly stored in approved rooms and Known chemical hazards must be on the
only in storage cabinets that are designed label.
for its purpose.  Label - any written, printed, or graphic
 Fire safety procedures are to be posted. material displayed on or affixed to
Safety equipment such as fire containers of hazardous chemicals
extinguishers, fire blankets, and fire  NFPA Label - National Fire Protection
alarms should be within easy access. Association
 A shower and eyewash should be readily  Code for hazards of materials—
available. information is obtained from the MSDS
 Laboratory accidents must be o Red fire hazard
documented and investigated with o Blue health hazard
incident reports and industrial accident o Yellow reactivity
reports. o White specific hazard
 Caustic or corrosive - Acids and alkalis
cause burns of skin, eyes and may
damage to equipment and storage areas.

13
  Poisons - may cause acute or chronic o Loss of endocrine stimulation
effects if inhaled, ingested, or placed in (uterine, breast)
contact with skin.
 Carcinogens - chemicals designated by Brain atrophy
the Occupational Safety and Health  Research has shown we are less likely to
Administration as cancer causing lose brain function if we continue to
 Flammables -materials that easily ignite, engage all parts of the brain
burn, or serve as fuel for a fire  (Brain atrophy with Alzheimer's) -
 Explosives - materials which may Atrophic cells have lost endoplasmic
explode under special circumstances reticulum, have fewer mitochondria, and
myofilaments.
Chemical Waste
 Toxic: chemicals that have the capacity
to harm biological tissue
 Corrosive: chemicals that can cause
severe burns to skin and other biological
tissues including eyes and lungs (e.g.
acids of pH<2 and bases of pH>12)
 Flammable: chemicals that ignite/burn
easily in normal working temperatures
(e.g. chemicals with flashpoint below
37.8oC or 100oF)
 Reactive: chemicals that can react by
themselves when exposed to heat,
pressure, shock, friction, catalyst
presence or by contact with air or water
Hypertrophy ( size of cells)
 D/T  workload requirement of an
organ part
 Causes an increase in cell size & cell
function
Cellular Adaptation o *See  protein in cellular
 The cell is the fundamental unit of life components with no  in cellular
 Permits survival and maintenance of cell fluid
function  Results in an increase in tissue mass
 May alter differentiation of genes o Seen in cardiac, skeletal, and
enabling a cell to change size or form. muscle tissue
 Is a normal adaptive response to o These cells are not capable of
an appropriate stimulus mitosis (so, no  number or
 Abnormal cellular changes may also hyperplasia)
occur  May be a normal physiologic response
o as seen in an increase in muscle
Atrophy (cells shrink) size with exercise
 D/t  workload or adverse  May be a pathological response as in
environmental conditions myocardial hypertrophy from HTN or
o Is adaptive and reversible valve disease
o Example: Left ventricular
o results in a decrease in cell size
hypertrophy (LVH) –
 Types
▪ A Pathological Hypertrophy
o Disuse atrophy (paralysis)
resulting in  size of
o Degeneration atrophy (MS)
heart d/t  workload
o Ischemic atrophy (kidney, heart)
caused by HTN.
o Malnutrition atrophy
o There is an increase in size but
(starvation)
function is compromised

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 o However, there is a LIMIT to the
amount the tissue can enlarge
( DNA synthesis
and mitotic
division)

Verrucae vulgaris Plantar warts -  in #

– common warts of epidermal cells

• Left Ventricular Hypertrophy LVH) – seen with Metaplasia

poorly controlled HTN ▪ One cell type is replaced by another
• Mmyocardial cells enlarge d/t  workload of
o May predispose to cancer
pumping
o Involves reprogramming of
Hyperplasia ( # of cells) undifferentiated stem cells
 Occurs d/t to a response from ▪ Allows to cells to better survive in a
appropriate stimulus and ceases when hostile environment
stimulus is removed o Is Reversible
 An increase in NUMBER of cells o Is a response to chronic irritation
 Restricted to cells capable of mitosis and inflammation
 epidermis, intestinal epithelium, ▪ Cells that are normally columnar or
and glandular tissue. stratified may change to squamous.
 Physiological hyperplasia ▪ Examples:
 uterus and breast enlarge in o With continued smoke exposure,
pregnancy ciliated columnar cells are
 May be a Physiological response and changed to stratified squamous
occur with hypertrophy ( in both cell cells
size & number) o Cervical cells change when
 Hyperplasia is important in wound exposed to STDs or HPV
healing ▪ Think metamorphosis or change from
 May also be a Non-Physiologic one form to another
Hyperplasia o Continued exposure may
 Seen in prostatic hypertrophy (BPH), predispose to cancerous
endometrial hyperplasia d/t increased transformations.
hormone stimulation, or thyroid
enlargement
Benign Prostatic
Hypertrophy/Hyperplasia
(BPH) – can cause difficulty
with urination

Dysplasia (Atypical Hyperplasia)

 Deranged cell growth resulting in cells of
varying size, shape, and appearance
Goiter – thyroid o May be associated with chronic
hyperplasia irritation or inflammation
o May be reversible if offending
agent is removed
 Dysplasia is considered A Strong
 Hyperplasia seen in finger warts: Precursor of Cancer
epidermal hyperplasia d/t viral o Example: Cervical dysplasia
stimulation o However, dysplasia is an
adaptive process – may or may
not lead to cancer
15
 ▪ Decrease risk if irritation  Example: elevated triglyceride
is removed or levels can result in a “Fatty”
inflammation treated. liver.
 This may reverse when
Anaplasia triglyceride level is
 Cells differentiate to a more lowered with medication
IMMATURE or embryonic form.
 Malignant tumors are characterized by Lipids and Edema
anaplastic cell growth.  If the offending cause is not or cannot be
corrected
Summary  CELL DEATH may occur
 Example:
 Abnormal lipids in the brain of
child with Tay-Sachs disease (a
genetic disorder) ultimately
results in cell death, and a
shortened life span.
 Cellular Swelling –
extracellular water shifts
into cells

 Chemical agents
 Hypoxia
 Free radicals
 Infectious agents (bacteria, viruses, fungi)
 Physical and mechanical agents
 Genetic factors
 Nutritional imbalances
o **Individuals have different
abilities to adapt to these stressors
Cellular Accumulations
(Cellular Infiltrations) What are Free Radicals?
 Is a buildup of substances the cells are  atoms or groups of atoms with an odd
unable to dispose of (unpaired) number of electrons
 Is a result of cell injuries  Free radical theory of aging states that
 Examples organisms age because cells accumulate
 Carbon/coal dust free radical damage over time
 Lipids  Some free radicals are formed during
 Fluid/edema normal metabolism
 Antioxidants are believed to protect
against free radical damage
Cellular Infiltrations  Free radicals are line robbers which are
(carbon, triglycerides) deficient in energy
 Carbon can accumulate via inhaled coal  Free radicals attack and snatch energy
dust. Can cause black lung disease and snatch energy from other cells to
(pneumonconiosis) satisfy themselves
 Organs enlarged, i.e., “Megaly”
 Ex. spleenomegaly,
hepatomegaly
 Diseases d/t celular accumulations
MAY BE REVERSIBLE if d/t a
correctable systemic disorder

16
 Cellular Injury o Average 20 years from exposure to
Mechanical Forces development of skin cancer
 Cellular injury may be reversible or
irreversible determined by: the severity of Radiation Injury: Ionizing Radiation
the insult, blood supply, nutritional status, • Direct injury causes immediate cell death or
and regeneration capability deranged replication.
o Physical Agents – trauma
o The endothelial cells in blood vessels
o Electrical
o Temperature extremes are very sensitive.
o Radiation • Examples:
o Radiation dermatitis
Physical Agents o Stomatitis
 Mechanical forces such as physical • Ionizing radiation
trauma resulting in lacerations, fractures, o Chronic effect causes scarring or
burns, injury to blood vessels, and
fibrosis
disruption in blood flow.
o Most radiation injury is d/t localized
Electrical irradiation used to treat cancer.
• Can cause extensive tissue injury and • Effects DNA synthesis and interferes with
disruption of neural and cardiac impulses. mitosis
o Vessels degenerate and create thrombi o Bone marrow and intestines are very
or clots. vulnerable to radiation exposure.
• The body acts as a conductor of electrical
current. Can see seizures or cardiac
Chemical Injury
arrhythmias.  Cells may be damaged by a vast array of
o Extent of injury determined by the chemicals
amount of voltage and duration of  A biochemical interaction between a toxic
exposure. substance and the cell’s plasma membrane
occurs and leads to  cell permeability
• Lightning and high voltage wires produce
 Examples:
most severe damage.
o air and water pollution
Temperature o tobacco smoke
o some processed or preserved foods
• Heat stroke or partial-thickness burns cause
o carbon monoxide
cellular injury.
o insecticides
o With an increase in intensity of heat,
o trace metals such as lead, and
blood vessels coagulate and tissue
DRUGS
protein loss occurs Fried egg effect.
Chemicals In Tobacco Smoke
• Cold exposure increases blood viscosity and
induces vasoconstriction which may cause
tissue hypoxia.
o Disrupts the cell membrane.

Ultraviolet Radiation
• Sunburns increase the risk of skin cancers.
o Damage to melanin-producing
processes in skin and damage to
DNA occurs.
• Risk of skin cancers  with number and
severity of sunburns.

17
 Chemical Injury: Drugs
• May damage cells directly or indirectly.
• Both prescription (RX) and over-the-counter Viruses, Bacteria, And Parasites
(OTC) medication can cause cell damage. • Biological agents are able to replicate and
o Tylenol can damage liver cells thus can continue to cause injury to cells
o Ibuprofen can injure kidney cells
• ETOH (ethyl) can harm gastric mucosa Viruses
(gastritis) and liver cells and harm the • Viruses incorporate themselves into the
developing fetus. DNA of the cell.
o Fetal Alcohol Syndrome
• Anti-cancer drugs can directly injure normal Bacteria
cells • Bacteria secrete exotoxins or endotoxins that
o Some drugs produce metabolic end- interfere with cellular reproduction of ATP,
products that are toxic to the cells or release antitoxins that cause injury
• Example:
o Tylenol, which is detoxified in the Parasites
liver, can cause massive liver necrosis • Parasites can cause direct injury to the cell.
when taken in large amounts.

Chemical Injury: Lead Toxicity

• Lead is very toxic to the cells.
• Sources include: flaking paint, lead- • Nutritional excesses such as obesity and diets
contaminated dust and soil, lead- high in fats can predispose to atherosclerosis
contaminated root vegetables, lead water leading to ischemia.
pipes, and newsprint. o The body needs minerals, vitamins,
o Inhaled or ingested lead accumulates certain fatty acids, and specific amino
over time. acids.
• Lead is absorbed via the GI tract or via the • Nutritional and vitamin deficiencies interfere
lungs into the blood. with cell metabolism and delay wound
• Lead is toxic d/t multiple biochemical healing.
effects:
o RBC’s become small and pale Hypoxic (Ischemia) Cell Injury
(anemia). • The most common cell injury.
o Since lead is excreted by the kidneys,
• Generalized
kidney damage leading to renal
o Is an inadequate supply of O2 in
failure can occur.
BLOOD affecting the entire body.
o Sx include change in LOC,
Lead confusion, combativeness.
• Lead causes demyelination of NERVE o Low pulse oximetry (less than 90%)
CELLS in the cerebral and cerebellar matter. • Localized
o causing death of neural and cortical o Causes ischemia of TISSUES
cells o If not corrected, can see infarction or
• Lead toxicity can cause death of tissues (heart, renal).
o a decrease in IQ • Ischemia affects local tissue and is reversible
o impaired neurobehavioral o Sx include pain, cyanosis,weak or
development in children. absent pulses
• In adults see peripheral neuropathy. • Infarction is tissue death and is not reversible.

18
• The cell reverts to anaerobic metabolism Three Types of Gangrene:
causing a fall in PH Dry Gangrene
o lactic acid accumulates in the cell • Part of the tissue becomes dry and shrinks,
lactic acidosis the skin wrinkles and color changes to brown
• Causes of hypoxia or black
o Decrease of oxygen in the air o Shriveled/mummified
o Respiratory disease o Spread is slow
o Ischemia o Results from chronic ischemia of
o Anemia d/t  hemoglobin tissue
o Edema o Symptoms not as marked as with wet
gangrene.
• Line of inflammatory reaction occurs
between dead tissue and health tissue
Apoptosis o Usually due to interference IN
• Is essentially “cell suicide” or programmed ARTERIAL supply to a part without
cell death. interfering with venous return so no
• Apoptosis is the process that eliminates: swelling
o Worn out cells (RBCs) ▪ No bacterial infection
o Cells which have been produced in o Mostly confined to extremities
excess
▪ WBCs with infectious Moist Or Wet Gangrene
response/hepatocytes with • Frequently occurs in internal organs
hepatitis). (appendix).
o Cells which have developed o If on the skin, surrounding area is
improperly. cold, swollen, and pulseless.
▪ Example: spontaneous abortion ▪ Skin in moist, black, and
o Cells which have genetic damage. under tension.
▪ Example cancer. o Blebs form on the skin, liquefaction
Necrosis occurs, and foul odor develops from
• Interferes with cell replacement and tissue bacteria
regeneration. • No line of demarcation
• Necrotic cells or tissue can: o Spread is rapid
o Liquefication • Severe systemic symptoms may develop
▪ cells becomes liquefied o Changes in VS, LOC, kidney
▪ Ex: abcess, brain tissue function
▪ May result in wet gangrene o Death may occur
o INFARCTION o Venous return is impaired so see
▪ cells die and become BLACK swelling
AND SHRIVELED • Bacterial invasion is present
o May affect extremities or internal
Gangrene organs
• Gangrene is necrosis of a mass of tissue o Dry gangrene may become wet
gangrene if a bacterial invasion
• Death of many cells:
occurs
 Results from severe hypoxia and
subsequent ischemia Gas Gangrene
• Often seen with impaired circulation to lower • Destroys connective tissue and cellular
extremities (PVD, Diabetes) membrane.
• Is a specialized type of wet gangrene.
19
 o Results from infection of devitalized Classification of Diseases:
with clostridium bacteria. • Developmental – genetic, congenital.
• Anaerobic and spore forming, clostridium • Acquired:
are often found in the soil. o Inflammatory – Trauma,
• Gas gangrene is more prone to occur when infections, immune, etc.
there is trauma with a compound fracture. o Neoplastic – tumors cancers
• Bacteria produce toxins which dissolve cell o Degenerative – ageing.
membranes causing: o Metabolic- biochemical process.
o death of muscle cells o Iatrogenic: Drug induced;
o massive spreading edema caused by med tx.
o hemolysis of RBCs
o renal toxicity
Branches of Pathology
• Characteristic bubbles of hydrogen (sulfide
• General Pathology
gas) form in the muscle.
• Systemic Pathology
o Can be fatal
• Gross Pathology
• Cellular Pathology
• Surgical Pathology
• It is the “Scientific study of disease". • Clinical Pathology
• "scientific study of the molecular, • Immunopathology
cellular, tissue, or organ system response
to injurious agents." General Pathology
• Pathology serves as a "bridge" or "link" • Common changes in all tissues. e.g.
between the preclinical sciences Inflammation, cancer, ageing, edema,
(anatomy, physiology, ……etc.) and the hemorrhage …. etc.
courses in clinical medicine.
• Disease Systemic Pathology
o It is the “State in which an • Discussing the pathologic mechanisms in
individual exhibits an relation to various organ systems e.g.,
anatomical, physiological, or CVS, CNS, GIT…. etc.
biochemical deviation from the
normal” Pathology focuses on 4 aspects of
o an abnormal alteration of disease
structure or function in any part
• Etiology: Cause of disease.
of the body.
• Pathogenesis: Mechanisms of
Basic Language of Pathology
development of disease.
• Pathology is the study of suffering (Latin
• Morphology: The structural alterations
word)
induced in cell and tissues.
– Logos = study
• Functional Consequences: Functional
– Pathos= suffering
results of the morphologic changes, as
• Etiology = Cause
observed clinically.
• Pathogenesis = sequence of events
• Morphology = structural alterations in
cells and tissues
Etiology
• – Gross = Changes in the tissue or organ • Study of the cause of a diseas
• – Microscopy = Changes noted under a • Knowledge of etiology remains the
light microscope backbone of:
o Disease diagnosis

20
 o Understanding the nature of Morphology: Structural Changes
diseases
• Structural changes in disease.
o Treatment of diseases.
o Tumor in a cancer.
• An etiologic agent : o Ulcer in an infection.
o is the factor (bacterium, virus, o Atrophy in dementia.
etc.) responsible for lesions or a o Gross & Microscopic.
disease state.
o One etiologic agent
Technique of Morphology
- one disease, as Malaria.
• Gross appearance:
o Several etiologic agents one
o size, shape
disease, as diabetes.
o weight
o One etiologic agent several
o color
diseases, as smoking.
o consistency
o surface
Predisposing Causes of Disease:
o edge, section
• Factors which make an individual more • Histologic and cytologic observation:
susceptible to a disease (damp weather, o most common and basic formalin
poor ventilation, smoking , etc.) fixed → HE (hematoxylin and
eosin) stained
Exciting Causes of Disease:
• Factors which are directly responsible for
a disease (hypoxia, chemical agents….
etc.).

Etiology: What is the cause Hemangioma of

• Environmental agents: ventrical wall
o Physical
o Chemical Immunohistochemistry
o Nutritional • Ag-Ab specific reaction
o Infections • Applications
o Immunological o Location analysis: cytokeratin →
o Psychological cell membrane
• Genetic Factors: o Clinical diagnosis and
o Age distinguishing diagnosis of tumor
o Genes histogenesis

Pathogenesis Flow cytometry (FCM)

• The sequence events in the response of • One kind of cells → quantitative
the cells or tissues to the etiologic agent, • DNA ploidy analysis
from the initial stimulus to the ultimate • Protein nucleus acid→ quantitative
expression of the disease,” from the time analysis
it is initiated to its final conclusion in • Selection of collection of cells
recovery or death”
• The core of the science of pathology —
the study the pathogenesis of the disease.

21
 Molecular biology technique Early Signs: Immediate Signs
• Polymerase chain reaction (PCR) • Somatic death
• DNA sequencing • Cessation of circulation:
• Biochip technique o no pulse
o Gene chip (DNA chip) o pallor mortis
o Protein chip (protein microarray) • Cessation of respiration
o Tissue chip (tissue microarray) • Cessation of nervous activity:
o no reflexes
Prognosis o no sensibility
o muscular relaxation: primary flaccidity
• Expected outcome of the disease, It is the
clinician's estimate of the severity and
Algor Mortis
possible result of a disease.
• Cooling of the body: “algor” = coolness
o Body temperature: decreases after 2/3
hours
• Variables: ambient temperature, clothing,
Physiology of Death body fat,
• Timing: mathematical estimation
• Complete, permanent and irreversible
cessation of the vital functions.
Livor Mortis
• Somatic or Clinical death
• Permanent and irreversible damage to; • Discoloration of the body:
o Brain activity stops o “lividus” = bluish
o Heart
o Lungs

Molecular Death
• Death of individuals tissues and cells
Rigor Mortis
• Process competes by two to three hours after
somatic death • Hardening of the body: “rigor” = stiffening
o Chemical changes
• Changes in the eye, skin muscles etc.
o Smaller muscles of the jaw -> arms ->
legs
Time Of Death
• Changes in the eye
• Can estimate time of death from
• Cooling of the body
o Body temperature
• Cadaveric spam
o Insect action
o Stomach contents
o Last known
o Normal postmortem changes Late Signs: Decomposition and
Autolysis
Normal Postmortem Changes Decomposition (Putrefaction)
• Early signs: • Invasion of microorganism, production of
o Immediate signs noxious ODOR
▪ Algor mortis o destruction of soft tissues
▪ Livor mortis o GI microorganisms
▪ Rigor mortis
• Late signs:
o Decomposition and autolysis
22
Autolysis Types Of Wounds (Trauma)
• self-destruction by internal enzymes Lacerations

Adipocere Formation
• a crumbly, waxy, water-insoluble material
consisting mostly of saturated fatty acids.
o Grayish white or tan in color Incised Wounds
• Corpses - firm cast of adipocere allows come • Puncture – penetrating injury due to an object
estimation of body shape and facial feature with no blade.
and injuries are often well-preserved.

Normal Postmortem Changes Abrasions

• Rigor mortis
• Livor mortis
• Desiccation
• Putrefaction (days 4-10)
• Cell autolysis (days 10-20)
• Dry decay (days 20-50)

• “CAUSE of DEATH”
o Petechial hemorrhage as a result of
strangulation.
Role Of the Pathologist
• Determine type of wound
• Measure the dimensions (length, width,
depth)
• Position relative to anatomical Contusions
landmarks
• Color changes a bruise goes through can give
• Determine initial location if wound
rough estimate of time of injury
involves cutting,
o Dark blue/purple (1-18 hours)
• slashing, etc. o Blue/brown (~1 to 2days)
• Determine height from heel o Green (~ 2 to 3 days)
o Yellow (~3 to 7 days)

23
 Gunshot Wounds Purpose
• Things for pathologist to learn: Saving Lives:
o Type of firearm • Enhance the understanding of diseases
o Distance of gun to victim and how a person die, and contribute
o Entrance vs exit wounds critical medical knowledge.
o Track of projectile • Forensic pathologists have identified
public health emergencies, such as the
anthrax terrorist attacks or other lethal
infection diseases, as well as public
health hazards, such defective cribs
that kill babies
• Stippling – powder burns on the skin when Discovering Hereditary Illness:
the gun is inches to a few feet from the victim • Can help family members learn
whether a relative died from an
undiagnosed or misdiagnosed illness or
disease that may be hereditary
Providing Legal Evidence:
• Determines a death to be the result of a
• Starring of a contact wound – barrel work or environmental hazard, it may
touching the skin. lead to compensation for family.
• If an autopsy reveals evidence of
medical malpractice, it may be the
grounds for a lawsuit
Easing the Stress of the Unknown:
• A way for families and loved ones to
seek reassurance or peace of mind after
death.

Two types of Autopsies

Forensic Clinical
Autopsy • Examines • preformed in
external and hospitals by
Necropsy, Postmortem or Postmortem
internal surface pathologists or
Examination for the evidence the attending
• Dissection and examination of a dead body, • Part of an physician to
its organs and structures. overall police determine the
• “To see for oneself” and has been in use in investigation. cause of death
reference to determining cause of death by for research and
examining a body since the 17th century. study purposes.
• Ancient Egyptians were one of the first
civilizations to practice the removal and
examination of the internal organs of humans
2 Types of Mortuary Cold
in the religious practice of mummification. Chambers:

• Morgue +: (35.6 -
Morgue - 39.2 °F)
Positive • most usual for
temperature keeping bodies for a
few days or weeks.

24
 • Morgue –: (5 / -13 I Incision
F) • A straight-line incision extending from the
Morgue -
• used for keeping chin to the symphysis pubis.
Negative
unidentified bodies.
temperature Modified Y
• Body is completely
frozen. • made from suprasternal notch over the
clavicle to its center on both sides and passes
upwards over the neck, behind the ear to
symphysis pubis

• Waist high and is

plumbed for running
water and has
several faucets for
washing away blood
that is released
Cadaver Table during the
procedure. Raised
edges keep blood
• All the organs are removed and weighed
and fluids from
o (usually removed in one unit or in
running into the
sequence depending on the trauma to the
floor.
body).
• Placed under the • Slices of each organ are taken and tested
back of the body
• Depending on type of death, stomach
causing arms and contents are removed, examined and
neck to fall back recorded
while pushing the
• If gun shot was involved, then any bullets
chest upward to would be removed and documented and
Body Block make it easier to cut saved for evidence
open.
Removal of Organs
• Organs are removed one by
one from the body to be
examined.
Virchow • This is done in the
following order: expose the
Technique
cranial cavity, the spinal
cord, followed by the
thoracic, cervical, and
Procedure abdominal organs.
Y Incision • This method is
• Starts near the acromion process and characterized by in situ
progresses downwards towards the xiphoid dissection.
process. Extended till the symphysis pubis. • The dissection begins at the
neck and slowly works its
• used by the pathologist or examiner to open
way down the body and the
up the breastplate of the deceased and gain Rokitansky
organ is removed as a bloc.
access to the body's major organs; heart, Technique
• A cut is first made at the
lungs, liver, stomach, spleen etc. larynx to separate the
esophagus from the
pharynx. Larynx and
trachea are pulled
downward, and using a
25
 scalpel, the chest organs After Completing the Internal
are cut. The diaphragm is Examination
cut to expose the • Body cavities should be cleaned and made
abdominal organs. Other free from blood, fluid, etc
organs like the bladder and
• Organs are packed back in and excess space
rectum are separated from
the body by using a scalpel is packed with cotton/cloth (pelvis and neck
to cut the pelvic ligaments. region)
• Like the Rokitansky • Dissection flaps are closed and sutured with
method, the Ghon method thin twine
also employs the bloc • Skull is filled with cotton and absorbent
method of removing material and the skull cap is placed back in
Ghon
thoracic, cervical, and scalp is stitched
Technique
abdominal organs. • Body is washed with water, dried, covered
• However, the organs are with clothes and handed over to the police
removed "en bloc," rather officials
than with in situ dissection.
• Thoracic, cervical, Rigor Mortis
abdominal, and pelvic
Body warm Not stiff < 3 hours
organs are removed "en
masse," meaning as one big Body warm stiff 3-8 hours
organ block. Body cool stiff 8-36 hours
• While, his organ mass is Body cool Not stiff >36 hours
Letulle
awkward to handle, it
Method
allows the body to be made
available for examination
in less than 30 minutes
because the process of
removing organs is much
quicker.

Procedure: FACTS:
1. The body block is moved to underneath the • the 17th century, lacking chemical tests
head (and knowledge of disease
2. Deep incision begins behind one ear, travels transmission mechanisms), Italian
over the top of the head and behind the physicians and autopsist ANTONIO
opposite ear. VALSALVA sometimes tasted the
3. Scalp is pulled away from the skull in two fluids he encountered in cadavers in an
flaps; front going over the face and the rear effort to better characterize them.
going over the back of the neck so the skull is • In 1828 Irish immigrants WILLIAM
fully exposed. BURKE and WILLIAM HARE
4. Electric saw known as the “Stryker saw” is partnered to murder 16 people in
used to cut and remove a wedge shape
Scotland for cadaver bounties paid for
portion of the skull which exposes the brain.
by a doctor who didn’t ask questions.
5. Brain is removed, weighed and examined.
6. Any findings noted
• Hare testified against Burke who was
7. Once everything has been examined, all the hung in 1829.
internal organs are returned to the body • Burkes cadaver was publicly dissected
cavities or incinerated. and his skeleton remains on display at
8. The body is sewn back together the University of Edinburg.
• Wallets made from his skin which was
stolen during the autopsy were offered
for sale on the streets.

26
 • In 1912, Boston physician RICHARD Expanding Cells not
CABOT analyzed autopsies and mass adhesive,
claimed that some diseases were being Frequently infiltrate
misdiagnosed at an alarming rate of encapsulate tissue
80%. A 2005 study in Histopathology d No capsule
suggests that doctors still misdiagnose
fatal Spread Remains Invades
diseases about a third of the time. localized nearby tissues
or
metastasizes
to distant sites
Neoplasia through blood
and lymph
• “New growth” abnormal mass of tissue, the vessels
growth of which exceeds and is Systemic Rare Often present
uncoordinated with that of the normal Effects
tissues and persists in the same excessive Life- Only in Tissue
manner after cessation of the stimuli which Threateni certain destruction
evoked the change” ng locations and spread of
• - Willis (e.g., brain tumors

Two Basic Components

Parenchyma
• Component from which the tumor derives its
name.
• Made up of transformed or neoplastic cells.

Stroma
• provides support for the growth of
parenchymal cells
• Host-derived, non-neoplastic made up of Nomenclature
connective tissue, blood vessels, and host-
derived inflammatory cells
Benign Tumors
• Designated by attaching the suffix -oma to
Characteristics of Benign and the cell type from which the tumor arises.
• -OMA = benign neoplasm
Malignant Tumors
Benign Malignant Mesenchymal Epithelial Tumors
Tumors Tumors Tumors
Cells Similar to Varied in size • Chrondroma • Adenoma
normal cells and shape • Fibroma • Papilloma
Differentiat with large • Osteoma • Papillary
ed nuclei Many cystadenoma
Mitosis undifferentiat • Polyp: a “tumor”
fairly ed that projects
normal. Mitosis above a mucosal
increased and surface
atypical
Growth Relatively Rapid growth
slow
27
 Malignant Tumors are produce to the site
• Referred to as cancers-derived from the Latin of the lesion, e.g., lung
word for “crab” can invade and destroy • congenital anomaly
adjacent structures and spread to distant sites • ectopic focus of
to cause death, arising in “solid” normal tissue
mesenchymal tissues or its derivatives are Choristoma
(heterotopia), e.g.,
called sarcomas. pancreas,
endometriosis
Sarcomas: Carcinomas:
• Hepatoma: malignant
Mesenchymal Epithelial Tumors
liver tumor
Tumor
• Melanoma: malignant
• Chondrosarcoma • adenocarcinoma:
skin tumor
• Fibrosarcoma gland forming
• Seminoma: malignant
• Osteosarcoma tumor
testicular tumor
• Leukemias or • squamous cell
carcinoma: • Lymphoma: malignant
lymphomas- Misnomers
squamous tumor of lymphocytes
arise from
differentiation • Hodgkin’s Disease: -
mesenchymal
Malignant tumors of
cells of the blood • undifferentiated
carcinoma: no the lymphoid tissues.
differentiation • Multiple Myeloma: -
• carcinomas can malignant tumors
arise from from the plasma
ectoderm,
endoderm, or less
likely, mesoderm.

Mixed Tumors
• Tumors with mixed differentiation
o Mixed Tumors: e.g. pleomorphic adenoma
of salivary gland
o Carcinosarcoma

Teratoma
• Gr teraton-monster like.
• Tumor comprised of cells from more than
one germ layer.
• Arise from totipotent cells (usually gonads-
ovary & testis).
• Benign cystic teratoma of ovary is the most
common teratoma.

ABBERANT DIFFERENTIATION
• Not true neoplasms.

• developmental
malformations
HAMARTOMA
• disorganized mass of
tissue whose cell types

28
 Tissue Origin Benign Malignant
Components of One Parenchymal Cell Type
Connective tissue and Fibroma Fibrosarcoma
derivatives Lipoma Liposarcoma
Chondroma Chondrosarcoma
Osteoma Osteogenic sarcoma
Endothelial and Related Tissues
Blood vessels Hemangioma Angiosarcoma
Lymph vessels Lymphangioma Lymphangiosarcoma
Mesothelium
Brain coverings Meningioma Mesothelioma
Invasive meningioma
Blood Cells and Related Cells
Hematopoietic cells Leukemias
Lymphoid tissue Lymphomas
Muscle
Smooth Leiomyoma Leiomyosarcoma

Striated Rhabdomyoma Rhabdomyosarcoma

Tumors Of Epithelial Origin
Stratified squamous Squamous cell papilloma Squamous cell or epidermal
carcinoma
Basal cells of skin or adnexa Basal cell carcinoma

Epithelial lining of glands or Adenoma Adenocarcinoma

ducts Papilloma Papillary carcinomas
Cystadenoma Cystadenocarcinoma
Respiratory passages Bronchial adenoma Bronchogenic carcinoma
Renal epithelium Renal tubular adenoma Renal cell carcinoma
Liver cells Liver cell adenoma Hepatocellular carcinoma
Urinary tract epithelium Urothelial papilloma Urothelial carcinoma
(transitional)
Placental epithelium Hydatidiform mole Choriocarcinoma
Testicular epithelium (germ Seminoma
cell) Embryonal carcinoma
Tumors of melanocytes Nevus Malignant melanoma

More Than One Neoplastic Cell Type (mixed tumors, usually derived from one germ cell
layer)
Salivary glands Pleomorphic adenoma Malignant mixed tumor of salivary
(mixed tumor of salivary gland
gland)
Renal glands Wilms tumor

29
 More Than One Neoplastic Cell Type Composed of primitive cells with little
o
Derived From More Than One Germ Cell differentiation
• Correlation with biologic behavior
Layer - Teratogenous
o Poorly differentiated malignant tumors
Totipotential Mature Immature usually have worse prognosis than well
cells in teratoma, teratoma, differentiated malignant tumors.
gonads or in dermoid teratocarcinoma • Anaplasia literally means “backward formation”
embryonic cyst o Implies dedifferentiation, or loss of
rests structural and functional differentiation of
normal cells

Characteristics of Benign and Features of Anaplasia

Malignant Neoplasms • Marked pleomorphism
• Nuclei are extremely hyperchromatic (dark-
• 4 fundamental features by which benign and
staining) and large resulting in an increased
malignant tumors can be distinguished:
1. differentiation and anaplasia nuclear-to-cytoplasmic ratio
2. rate of growth • 1: 1 instead of the normal 1:4 or 1:6.
3. local invasion
4. metastasis Giant Cells
• Larger than the neighboring cells;
• May formed and possess either one enormous
nucleus or several nuclei.

Anaplastic Nuclei
• Varies in shape and size.
o Chromatin is coarse and clumped, and
nucleoli may be of great in size.
• Mitoses often are numerous and distinctly
atypical
• Anaplastic cells usually lose normal polarity:
fail to develop recognizable patterns of
orientation to one another.

4 Fundamental Features
Differentiation And Anaplasia
• Well differentiated neoplasm
o Resembles mature cells of tissue of origin
• Benign tumors are well differentiated closely
resemble their normal counterparts.
• LIPOMA: mature fat cells laden with
cytoplasmic lipid vacuoles A. Nests of anaplastic tumor cells are seen with
• CHONDROMA: mature cartilage cells that round to oval eccentric nuclei, coarse chromatin,
synthesize their usual cartilaginous matrix. prominent nucleoli and moderate homogeneous
cytoplasm (×1000).
• Mitoses are usually rare and are of normal
configuration. B. Single giant malignant cells with similar
morphology are also seen.
Malignant Neoplasm
• Wide range of parenchymal cell differentiation
• Undifferentiated or “anaplastic” tumor
• Poorly differentiated neoplasm

30
 Local Invasion
Benign Neoplasm
• Remains localized at its site of origin.
• It does not infiltrate, invade, or metastasize to
distant sites.
o Encapsulated (separated from the host tissue
by a fibrous capsule).
o Capsule derives from stroma of the host tissue
and stroma of the tumor itself.
o Not all benign neoplasms are encapsulated:
Leiomyoma of the uterus. This benign, well- o Leiomyoma of the uterus is discretely
differentiated tumor contains interlacing bundles of separated from the surrounding smooth
neoplastic smooth muscle cells that are virtually
muscle by a zone of compressed normal
identical in appearance to normal smooth muscle
myometrium.
cells in the myometrium

Dysplasia Cancers
• Literally means abnormal growth • Grow by progressive infiltration, invasion,
• Disorder but non-neoplastic proliferation. destruction, and penetration of the surrounding
• Encountered principally in epithelial lesions. tissue
• Loss in the uniformity of individual cells and in • Do not develop well-defined capsules
their architectural orientation cells exhibit • Occasionally, a slowly growing malignant
• Pleomorphic and hyperchromatic nuclei that tumor appears to be encased by the stroma of
are abnormally large for the size of the cell the surrounding host tissue
• Dysplasia may develop into malignancy: • MICROSCOPIC EXAMINATION- reveals
1. Uterine cervix tiny crablike feet penetrating the margin and
2. Colon polyps infiltrating adjacent structures
• Graded as low-grade or high-grade, prompting • It is necessary to remove a wide margin of
different clinical decisions surrounding normal tissue when surgical
excision of a malignant tumor is attempted
• HIGH grade dysplasia often classified with CIS
(clean margins).
- carcinoma in situ, a preinvasive stage of
cancer
Metastasis
• Secondary implants of a tumor that are
Rate of Growth discontinuous with the primary tumor and
• Most benign tumors grow slowly, with some located in remote tissues.
exceptions:
• Property of metastasis identifies a neoplasm as
o LEIOMYOMAS OF THE UTERUS are
malignant.
influenced by circulating estrogen level:
• Not all cancers have equivalent ability to
increase in size during pregnancy and cease
metastasize.
growing after menopause
• Basal cell carcinomas of the skin and most
• Most cancers grow much faster, spreading locally
primary tumors of the cns are highly invasive
and to distant sites and causing death, with
locally but rarely metastasize.
some exceptions:
• Osteogenic sarcomas, usually have
o Some grow slowly for years and then enter a
metastasized to the lungs at the time of initial
phase of rapid growth (aggressive subclone of
discovery.
transformed cells)
o Others grow relatively slowly and steadily;
o In exceptional instances, growth may come
almost to a standstill

31
• Metastasis occur by one of Three Pathways: o Leukoplakia (thick white patches of
1. Seeding within body cavities epithelium) of mucous membranes
• Cancers of the ovary often cover
the peritoneal surfaces widely Staging Of Cancer
• Tumor cells break away and travel • Classification process applied to a specific
easily with the movement of fluid malignant tumor at the time of diagnosis
and tissue • Describes the extent of the disease at the time
2. Lymphatic spread and provides a basis for treatment and
• More typical of carcinomas prognosis.
3. Hematogenous spread • Staging systems are based on the:
• Favored by sarcomas, most frequently liver o Size of the primary tumor (T)
and lungs are common secondary sites for o Extent of involvement of regional lymph
many tumors. nodes (N)
• Venous invasion, the blood-borne cells follow o Spread (invasion or metastasis) of the
the venous flow draining the site of the tumor (M)
neoplasm • Tumor stage: scale I to IV or A to D
• Spread to distant sites by blood or lymphatic • Grading of Tumors:
channels. o Grade I tumors are well-differentiated
• Tumor cells erode into a vein or lymphatic o Grade II lesions are well-differentiated
vessel, travel through the body, and eventually o Grade III lesions are undifferentiated
lodge in a hospitable environment to reproduce o Grade IV tumors are well-advanced,
and create one or more secondary tumors. difficult to treat at multiple sites, and
have a poorer prognosis.
Predisposing Factors for Cancer
Geographic And Environmental
Age • Sun exposure
• Most cancers occur in persons ≥ 55 years o Melanomas
• Childhood cancers • Smoking and alcohol abuse
o Leukemias & CNS neoplasms • Can be found particularly in urban settings
o Bone tumors (e.g., asbestos in building and construction
• Major lethal cancers in children are: industries)
Leukemias, tumors of CNS, lymphomas, and • Body mass
soft tissue and bone sarcomas. o Overweight = 50% increase in cancer
• Viral exposure
Genetic Predisposition o Human papilloma virus (HPV) and
• Familial cancer syndromes cervical cancer
o Early age at onset o Hepatitis B virus (HBV) and liver cancer
o Two or more primary relatives with the (Africa, Asia)
cancer (“soil” theory) o Epstein-Barr Virus (EBV) and
o Multiple or bilateral tumors lymphoma
• Polymorphisms that metabolize
procarcinogens, e.g., nitrites
Diagnostic Tests
• Early detection of cancer and in long-term
Nonhereditary Predisposing Conditions
monitoring.
• Precancerous conditions
o Chronic ulcerative colitis
Blood Tests
o Atrophic gastritis of pernicious anemia
• As an indicator of a problem and in monitoring
the effects of chemotherapy and radiation.
32
 o Eg: Leukemia- cell characteristics are Diagnostic Techniques Used in
diagnostic when confirmed by a BM
exam Pathology
Histopathological Techniques
Chromosome Markers • Studies abnormal tissue structure under the
• Philadelphia chromosome microscope.
o Chronic myelocytic leukemia • Tissues are obtained by biopsy.
• Biopsy is a tissue sample from a living person
BRAC-1 to identify the disease.
• Specific genes associated with higher ▪ Can be either incisional or
probability of breast or ovarian cancer. excisional.
• Once the tissue is removed from the patient, it
X-Rau, UTZ Magnetic Resonance has to be immediately fixed by placing it into
Imaging and Computer Tomography adequate amount of 10% Formaldehyde (10%
(CT) Scans formalin) before sending it to the pathologist.
• Methods of examining changes in tissues or
organs. Purpose of Fixation
• To prevent autolysis and bacterial
Cytologic Tests (FNAC) decomposition and putrefaction.
• Used to screen high-risk individuals. • To coagulate the tissue to prevent loss of easily
• Confirm a diagnosis, or follow a clinical course
diffusible substances.
and monitor change.
• Histologic and cytologic exams • To strengthen the tissue against the harmful
• Used to evaluate biopsies of suspicious masses effects of the various stages in the preparation
and check sloughed cells in specific tissues of sections and tissue processing.
(exfoliative cytology). • To leave the tissues in a condition which
• Dependable confirmation of malignancy. facilitates differential staining with dyes and
other reagents.
Tumor Markers
• Tumor markers
o CEA Histopathological Techniques
▪ Colon cancer • Gross anatomy
o hCG • Tissue processed
▪ Testicular cancer, • Purpose of tissue processing:
teratocarcinoma,
choriocarcinoma, molar o To prepare a very thin tissue (i.e., 4 to
pregnancy. 6 μm/micron) or one cell thick tissue)
o AFP which can be clearly seen under the
▪ Hepatocellular cancer microscope.
o CA125 • Tissue is processed into different chemicals.
▪ Ovarian cancer
• Stains can be Hematoxylin/Eosin stain or
o CA 19-9
▪ Pancreatic colorectal cancer special stains such as PAS, IHC, etc.
o CA 15-3 o H&E stain is routinely used.
▪ Breast ca after mastectomy o It gives the nucleus a blue color
o CA 27.229 o Cytoplasm & extracellular matrix a
▪ Early recurrence of breast CA pinkish color.
o PSA
• Microscopic examination by Pathologist.
▪ Prostate cancer
• Presence of abnormal morphology is the basis
for the diagnosis.
• Histopathology is usually the gold standard for
pathologic diagnosis.

33
 retroperitoneum are aspirated with guidance
by fluoroscopy, ultrasound or CT scan.
• Study of cells from various body sites to
determine the cause or nature of disease Advantages
Applications: • Cheap, fast
1. Screening for early detection of • Accurate in diagnosing many diseases.
asymptomatic cancer
▪ Ex: Scrapings from cervix for early Abrasive Cytology
detection and prevention of • Cells are dislodged by various tools from body
cervical cancer. surfaces (skin, mucous membranes, and serous
2. Diagnosis of symptomatic cancer. membranes).
▪ Cytopathology may be used alone o E.g. preparation of cervical smears
or in conjunction with other with a spatula or a small brush - to
modalities to diagnose tumors detect cancer of the uterine cervix at
revealed by physical or radiological early stages.
examinations. • Cervical smears, also called Pap smears, can
▪ It can be used in the diagnosis of significantly reduce the mortality from cervical
cysts, inflammatory conditions and cancer.
infections of various organs.
3. Surveillance of patients treated for cancer Exfoliative Cytology
▪ Most feasible method of • Microscopy study of cells that have been shed,
surveillance to detect recurrence. desquamated/ scrapped off from lining of
▪ Ex: Periodic urine cytology to epithelium and mucosal surfaces.
monitor the recurrence of cancer of o Eg: Sputum, CSF, Urine, etc
the urinary tract. Purposes
• Assess malignant/cancerous condition
Advantages Of Cytologic • Detect asymptomatic cancer in women
Examination • Assess female hormonal activity (sterility,
• Cheap endocrine disorder)
• Takes less time • Determine genetic sex
• No anesthesia to take specimens. • Determine presence of infection
• Appropriate for developing countries with
limited resources. Interpretation Of Cellular Materials Depens On
• Complementary to histopathological • Method of SPN collection
examination. • Fixation, fixatives
Methods • PRSV of fluid SPN prior to processing
• Prep of materials for microscopic exam
Fine-Needle Aspiration Cytology (FNAC)
• Cells are obtained by aspirating the diseased • Staining and mounting of cell sample
organ using a very thin needle under negative
pressure. Collection Of SPN
• Any organ or tissue can be sampled by FNAC. • Vaginal scraping
• Aspirated cells are then stained & are studied o Scraping/swab
under the microscope. • Endocervical & endometrial scrapings -swab,
• Superficial organs (e.g., thyroid, breast, lymph curettage
nodes, skin and soft tissues) can be easily • Prostatic secretion
aspirated. o Prostatic massage
• Deep organs, such as the lung, mediastinum, • Bronchial aspirate/ sputum
liver, pancreas, kidney, adrenal gland, and o Deep cough/ bronchoscopy.
34
• Serous fluid • Culture and serological techniques is used to
o Pleural(thoracentesis) identify microorganisms responsible for many
o Pericardial (pericardiocentesis) diseases.
o Peritoneal (paracentesis) Biochemical Examination
• Gastric & duodenal • Metabolic disturbances of disease are
o Nasogastric tube aspiration investigated by assay of various normal and
• Nipple discharge abnormal compounds in the blood, urine, etc.
• CSF fluid
o Lumbar puncture
Clinical Genetics (Cytogenetics)
• BM aspirate
• Method for inherited chromosomal
o BM aspirate
abnormalities in the germ cells or acquired
• Urinary sediment
chromosomal abnormalities in somatic cells
o Urine collection, catheterization
are investigated using the techniques of
molecular biology.
Commonly Used Staining
Techniques
• Pap smear (Papanicolaou)
• H&E Fluorescent In Situ Hybridization
• Modified staining (Wright’s) (FISH) and Southern Blot
• PAP’S STAIN: Hematoxylin, • Can be used to detect genetic diseases
Orange G6, Eosin 36
Selection Preparation
Advantages
• Transparent blue staining of cytoplasm Two Strategies
• Excellent nuclear staining Frozen Sections
• Color range is predictable and great value in • Rapid process
cells ID. • Uses cryostat microtome
0 • Used for intra-operative diagnosis to guide a
Disadvantages surgical procedure or type of interference with
• Lengthy procedure the chemical makeup of the cells (as in some
• Complicated procedure histochemical investigations)
• Doesn’t give accurate acidophile index Paraffin Sections
• Specimens can be infiltrated with a liquid agent
that can subsequently be converted into a solid.
Hematological Examination
• Thin sections are cut from it by using rotary
• Abnormalities of the cells of the blood and microtome.
precursors in the bone marrow to diagnose the
• “Rotary” describes the cutting action of the
different kinds of anemia & leukemia
instrument

Immunohistochemistry
• Used to detect a specific antigen in the tissue in
order to identify the type of disease.

Microbiological Examination
• Body fluids, excised tissue, etc. are examined
microscopically.

35
 Biopsy Target Tissue Advantages Disadvantages Equipment
Surgical removal of May require major
Combines Dx &
Excision entire lesion for many surgery under Scalpel
Tx of lesion
tissue anesthetic
Disturbs cell
Flexible or
architecture,
Fine aspiration
Permits study of
Avoids surgery; needle,
Aspiration Bone marrow or breast individual cells but not
For breast cyst needle guide,
intercellular structure.
aspiration
May cause seeding of
syringe
malignant cells
May cause seeding of
malignant cells when
Punch
Core of lesion in skin or part of mass is
Punch Avoids surgery (Tischler
cervix removed. May require
forceps)
excision/ TX based on
histologic results
Generally safe,
May require excision or
combines (DX
Tissue from raised TX if lesion is
+TX of benign
Shaving surface lesion on the malignant. May cause Scalpel
lesion), yields
skin seeding of malignant
good cosmetic
cells
result
Core of tissue from BM, May require
Avoids surgery,
bone, breast, lung, excision/TX, may be Cutting
furnishes a
pleura, lymph node, traumatic to needle (cope
Needle representative
liver, kidney, prostate, surrounding tissue. orvimsilverman
spn, preserves
synovial membrane, May cause seeding of cutting needle
cell architecture
thyroid malignant cells

Definition Of Terms
• New abnormal growth because of abnormal cellular reproduction.
Neoplasm
• It is synonymously used with tumor.
Aberrant Cellular • An alteration in normal cell growth
Growth
• A growth of Neoplastic cells clustered together to form a mass.
Tumor
• It can be benign or malignant.
• Is characterized by abnormal cell division but no metastasis or invasion of the
Benign Tumor
surrounding tissues
• Abnormal cell division characterized by ability to invade locally, metastasize
Malignant Tumor and reoccur.
• It is cancer cells
Carcinogenesis • production or origination of cancer cells.

36
 • Immigration of leukocytes: the migration of
WBC from within the blood vessel towards
the inflammation site
• It is the response of the living tissue to mild
to moderate irritant • Diapedesis: the passage and movement of
RBC from within the blood vessel towards
• The response is directed to defend the tissue
the inflamed area
for foreign irritants and to prevent further
damage Cardinal Signs of Inflammation
• It is denoted by the suffix “itis”
• The aim is to bring more blood to the • Redness (RUBOR)
damaged area by acceleration of the blood • Hotness (CALOR)
stream • Swelling (edema) due to inflammatory
exudate (TUMOR)
• Pain: due to pressure of edema on nerves and
irritation of nerve ends by metabolites
(DOLOR)
• Loss of function: this is to make the inflamed
part of tissue rest and heal. (FUNCTIO
LAESA)
Effects Of Inflammation
• Vascular phenomena
o Transient vasoconstriction rapidly
followed
o Vasodilatation
o Stasis
o Migration of leucocytes
• Exudative stress
o Immigration of leukocytes
o Inflammation fluid exudate

Inflammation Fluid Exudates Types Of Inflammation

• Fluid exudates Acute Inflammation
o Dilution of bacterial toxins
o Also contain antibodies • Acute non-suppurative inflammation: acute
o fibrin threads: help the movement of without the formation of pus
leucocytes and limit the spread of • Acute suppurative inflammation: with pus
infection o Localized: Abscess, Furuncle, Carbuncle
• Cellular part ▪ A carbuncle is a red, swollen, and
o Phagocytosis: engulfing of and painful cluster of boils that are
destruction of bacteria and necrotic tissue connected to each other under the
▪ Engulfing of and destruction of skin. A boil (or furuncle) is an
bacteria and necrotic tissue by infection of a hair follicle that has a
phagocytes and PMNL small collection of pus (called an
POLYMORPHONUCLEAR abscess) under the skin
CELLS o Diffused: cellulitis, septic meningitis

Mechanism Of Inflammation Chronic Inflammation

• Chemotaxis: the movement of WBC in the • Chronic specific: TB

area of inflammation towards the irritant • Chronic non-specific: follows acute or
chronic from the beginning

37
 Cells Of Inflammation Types Of Healing
• Fate of acute inflammation • Regeneration:
o Regression: by resolution for example o Healing by the same type of tissue cells
when the body (immune system) from surrounding healthy living cells,
overcomes the bacterial infection this occurs within small damages of labile
o Progression which can lead to chronic cells and stable cells for examples liver
inflammation and spread: the bacteria cirrhosis and bone fractures
overcome the immune system and can • Fibrous (scar tissue):
spread by: o Healing by granulation tissue (fibroblast
▪ Blood: septecemia, bacterimea, with new capillaries formed) which
toximia pyaemia mature a vascular fibrous tissue (scar),
▪ Lymphatic: lyphangitis, lyphadenites this occurs in the healing process of
permanent cells and stable cells with high
damage. for example, myocardial
Type Of Cells infraction and wounds
• Continuously dividing cells or labile Healing Process
o Epithelium
o Hematopoietic (blood) • Four distinct phases
• Quiescent or stable o The homeostasis phase
o Hepatic o The inflammatory phase
o Kidney o The proliferative phase
o Pancreas o The remodeling phase
• Non-dividing or permanent
o Nerve cells Sequence Of Events in Healing
o Skeletal muscle cells
Initial Phase - Hemostasis
Cell Development
• Following vasoconstriction, platelets adhere
• Proliferation: increased number to damaged endothelium
• Differentiation: development through stages o Following vasoconstriction, platelets
adhere to damaged endothelium and
discharge adenosine diphosphate (ADP),
promoting thrombocyte clumping, which
• Tissue repair involves replacement of dams the Wound
damaged tissue with new healthy living tissue • The inflammatory phase is initiated by the
when resolution cannot occur release of numerous cytokines by platelets.
• Healing is a complex and dynamic process of • Fibrinogen is cleaved into fibrin and the
restoring cellular structures and tissue layers framework for completion of the coagulation
process is formed.
o Fibrin provides the structural support for
cellular constituents of inflammation.
• This process starts immediately after the
insult and may continue for a few days

Second Phase - Inflammation

• Within the first 6-8 hours, the next phase of
the healing process is underway, with
polymorphonuclear leukocytes (PMNs) or
PNLs engorging the wound
• These cells “cleanse” the wound, clearing it
of debris. The PMNs attain their maximal

38
 numbers in 24-48 hours and commence their • In days 5-7, fibroblasts have migrated into the
departure by hour 72 wound, laying down new collagen of the
• As the process continues, monocytes also subtypes I and III
exude from the vessels. These are termed • The wound is suffused with GAGs and
macrophages. The macrophages continue the fibronectin that are bonded covalently to a
cleansing process and manufacture various protein core and contribute to matrix
growth factors during days 3-4. deposition
• Many factors influencing the wound healing
process are secreted by macrophages. Fourth Phase - Remodeling
o Many factors influencing the wound • After the third week, the wound undergoes
healing process are secreted by constant alterations, known as remodeling,
macrophages. These include TGFs,
• This can last for years after the initial injury
cytokines and interleukin-1 (IL-1), tumor
occurred. Collagen is degraded and deposited
necrosis factor (TNF)
in an equilibrium-producing fashion
▪ TGF – transforming growth factor
• The collagen deposition in normal wound
Third Phase - Granulation healing reaches a peak by the third week after
the wound is created.
• This phase consists of different subphases. • Contraction of the wound is an ongoing
These subphases do not happen in discrete process resulting in part from the
time frames but constitute an overall and proliferation of the specialized fibroblasts
ongoing process. The subphases are: termed myofibroblasts, which resemble
o fibroplasia contractile smooth muscle cells.
▪ Fibroplasia - The development of
fibrous tissue, as in wound healing or Complications Of Healing
by other stimulating factors, e.g., as Process
retrolental fibroplasia in the neonate
due to the administration of excessive • This process can go wrong and produce an
oxygen. increase of fibroblastic proliferation with a
o matrix deposition resultant hypertrophic scar.
o angiogenesis • Further exuberance can result in keloid
▪ Angiogenesis is the product of parent formation where scar production extends
vessel offshoots. The formation of beyond the area of the original insult.
new vasculature requires Conversely, insufficient healing can result in
extracellular matrix and basement atrophic scar formation.
membrane degradation followed by • Week scar: this may lead to hernia
migration, mitosis, and maturation of • Cicatrisation: contracture of the size of the
endothelial cells scar
o and re-epithelialization • Implantation epidermoid cyst
▪ Re-epithelization occurs with the
• Stump neuroma: following amputation
migration of cells from the periphery
causing a painful coiled mass of nerves
of the wound and adnexal structures.
• Sinus: is a track of septic granulation tissue
This process commences with the
connecting a cavity to the outside and has
spreading of cells within 24 hours.
one blind end e.g. pilonidal sinus
Division of peripheral cells occurs in
• Fistula: is a tract of septic granulation tissue
hours 48-72, resulting in a thin
connecting 2 epithelial surfaces
epithelial cell layer, which bridges the
wound. • Infection: leading to delayed healing
o This succession of subphases can last up • Rarely scars may develop squamous cell
to 4 weeks in the clean and carcinoma
uncontaminated wound. • Ulcers: discontinuity of cover epithelium or
muscle membrane

Factors Affecting Healing

39
• Systemic & Local factors Smear Preparation
o Immobilization • is the process of examining sections or
o Improper reduction – abnormal position sediments, whereby cellular materials are
o Infection. Debris, dead tissue in wound spread lightly over a slide by means of a wire
o Joint involvement loop or applicator or by making an
opposition smear with another slide. This
technique is especially useful in cytological
examination, particularly for cancer
diagnosis
• Varies according to the following:
o structural and chemical components of Streaking
the cells to be studied • with an applicator stick or platinum loop, the
o the nature and amount of tissue to be material is rapidly and gently applied in a
evaluated direct or zigzag line throughout the slide,
o The need for an immediate examination attempting to obtain a relatively uniform
of a tissue structure distribution of secretion. Too thin or thick
• Examination may be done on fresh or smear have to be avoided since they make the
preserved tissues, depending upon necessity tissues unsuitable for examination
• Fresh tissue haves the advantage of being
examined in the living state, thereby allowing Spreading
protoplasmic activities such as motion, • a selected portion of the material is
mitosis, phagocytosis and pinocytosis to be transferred to a clean slide and gently spread
observed moderately thick film by teasing the mucous
• Its use has been limited, however, because of strands apart with applicator stick.
the fact that tissues examined in the fresh • This method is a little more tedious than
state are not permanent, and therefore, are streaking, but has the advantage of
liable to develop the changes that have maintaining cellular interrelationships of the
usually been observed after death material to be examined. It is especially
recommended for smear preparations of fresh
Methods of Fresh Tissue
sputum and bronchial aspirates and also for
Examination thick, mucoid secretions.

Teasing Or Dissociation
• a process whereby a selected tissue specimen Pull–Apart
is immersed in a watch glass containing • this is done by placing a drop of secretion or
isotonic salt solution, carefully dissected or sediment upon one side and facing it to
separated and examined under the another clean slide. The material disperses
microscope, either unstained, by Phase evenly over the surface of the two slides in the
Contrast or Bright Field Microscopy, or opposite directions may be necessary to
stained with differential dye initiate the flow of materials. The two slides
are then pulled apart with a single
Squash Preparation uninterrupted motion, and the specimen
• a process whereby small pieces of tissues, not placed under the microscope for immediate
more than one mm in diameter, are placed in examination, or applied with vital stain.
a microscopic slide and forcibly compressed • This is useful for preparing smears of thick
with another slide or with a cover glass secretions such as serous fluids, concentrated
• If necessary, a vital stain may be placed at the sputum, enzymatic lavage samples from the
junction of the slide and the cover glass and gastro–intestinal tract and blood smears
allowed to be absorbed by the tissue through
capillary attraction

40
 Touch Preparation o Clearing - Specimen is transparent rather
• impression smears than opaque, so that light can pass
• is a special method of smear preparation through it
whereby the surface of a freshly cut piece of o Section - Specimen is thin and flat so that
tissue is brought into contact and pressed on only a single layer of cells is present
the surface of a clean glass slide allowing the o Staining - Some components can be
cells to be transferred directly to the slide for differentially colored so that they can be
examination by Phase Contrast Microscopy clearly distinguished.
or after vital staining. • Microscopic examination of sections by a
• It has an added advantage in that the cells pathologist forms the corner stone of cancer
may be examined without destroying their diagnosis
actual intercellular relationship, and without
separating them from their normal
Preparation Options
surrounding • Smears and squash preparations provide
detail about individual cells and relative cell
Frozen Sections numbers, but structural relationships are lost.
• This method is normally utilized when a • whole-mounts and sections preserve the
rapid diagnosis of the tissue in question is structural relationships between individual
required and especially recommended when cells and extracellular components
lipids and nervous tissue elements are to be
demonstrated Whole-Mounts
• Very thin slices around 10-15u in thickness • entire organism or small/ thin structure is
are cut from a fresh tissue frozen on a enough to be placed directly onto a
microtome with CO2 or on cryostat, a cold microscope slide
chamber kept at an atmospheric temperature
“Squash” Preparations
of -10oC to -20oC, transferred to a dish
• cells are intentionally squashed or crushed
containing isotonic salt solution and stained
onto a slide to reveal their contents (e.g.
for microscopic examination
botanical specimens where cells are disrupted
to reveal chromosomes)

Smears
• specimen consists of cells suspended in a
fluid (e.g. blood, semen, cerebro-spinal fluid,
• Basis in identifying the changes and links to or a culture of microorganisms)
a particular disease • Cells that have been scraped, brushed or
• Medical and biological research- knowledge aspirated (sucked) from a surface or from
of normal structure and function of cells, within an organ (exfoliative cytology).
tissues and the organs and structures that • Basis of the well-known “Pap test” that is
they make up. used to screen for cervical cancer in women.
• Alterations of structures by means of physical
and chemical influences Sections
• specimens are cut in a very thin slices,
Microscopy mounted on slides, and stained.
• Prepared using an instrument called a
• Brightfield” microscopy-commonly
“microtome”.
employed for specimen viewing or
examination • most technically complicated methods as it
requires specialized equipment and
• Requirements for a specimen to be
considerable expertise.
successfully examined
o Fixation - Cells and other elements are
preserved in a “life-like” state

41
 Specimen Reception “Paraffin Sections”
• Specimens can be infiltrated with a liquid
• Come from a number of different sources. agent that can subsequently be converted
• Range from very large specimens or whole into a solid.
organs to tiny fragments of tissue. • Thin sections are cut from it by using rotary
• Before specimens are accepted by a microtome
laboratory the identification (labelling) and o “Rotary” describes the cutting action of
accompanying documentation will be the instrument
carefully checked, all details recorded and
“specimen tracking” commenced. It is vital
that patient or research specimens are
properly identified and the risk of • A tissue taken from the body for diagnosis
inaccuracies minimized. must be process in histopathology laboratory
• Specimen-types commonly received in a to produce microscopic slides that are viewed
histopathology lab. under the microscope by the pathologists
o Excision specimens (surgical biopsies)-
whole organs or affected areas are Tissue Processing Steps
removed at operation
o Incisional biopsy - tissue is removed for 1. Fixation
diagnosis from within an affected area 2. Dehydration
o Punch biopsies - punches are used to 3. Clearing
remove a small piece of suspicious tissue 4. Infiltration/ Impregnation
for examination (often from the skin) 5. Embedding
o Shave biopsies - small fragments of tissue 6. Trimming
are “shaved” from a surface (usually 7. Cutting/ Sectioning
skin) 8. Staining
o Curettings - tissue is removed in small 9. Mounting
pieces from the lining of the uterus or 10. Labelling
cervix
Role of Histotechnologist
• Core biopsies- small tissue sample is removed
using a special needle sometimes through the • Adhere to policies and procedures.
skin (percutaneously). • Report problems (reagent supply, equipment
malfunction, poor sectioning by residents)
Two Sources of Specimen immediately to pathologist.
• AUTOPSY – from a dead body • Perform PROPER histopathologic
o Ordinary hospital – autopsy is done with techniques.
a consent from the relatives, the person • Submit best quality slides to pathologist with
died of natural causes. attention to PROPER numbering, labeling,
o Medico legal autopsy – done even sequence of slides corresponding to
without the consent Specimen requests and slides free of excess
• BIOPSY – from a living person through adhesive, dirt or paraffin.
surgery • Assist the pathologist in maintaining quality
practices in the histolab.
Section Preparation • Persons who do the tissue processing and
Frozen Sections make the glass microscopic slide are
• Rapid process histotechnologist
• Uses cryostat microtome
• Used for intra-operative diagnosis to guide a
surgical procedure or type of interference
with the chemical makeup of the cells (as in
some histochemical investigations)

42
 • Accelerates staining, decalcification,
immunohistochemistry and electron
• First and most critical step microscopy
• Aim: to prevent decay and preserve cells and
tissues in a “life-like” state. stops enzyme Advantages
activity, killing microorganisms and • Tissue is heated right through the block in a
hardening the specimen while maintaining very short time (main advantage)
sufficient of the molecular structure to enable • Non chemical technique (less interference)
appropriate staining methods to be applied • Rapid
o Inactivation of lysosomal hydrolytic • Lesser time for immunohistochemistry and
enzymes – post mortem decomposition in-situ hybridization
(autolysis); or by chemically altering,
stabilizing, and making tissue Disadvantages
components insoluble • Penetrates 10-15 mm only
o Reducing the risk of infection by • No significant cross linking of protein
preventing putrefaction after death molecules; subsequent chemical fixation may
(bacterial / fungal colonization & be needed
overgrowth) • Viable spores/pathogens (alcohol-based
• Goal: to harden and protect the tissue from fixatives or microwaving alone)
trauma of further handling.
Factors Involved In Fixation
• Ideal volume of the fixative is 10 to 20 times
greater than the size or volume of the • Hydrogen ion concentration
specimen • Temperature
• Promotes staining • Thickness of section
• Concentration
Classification of Fixing Agents • Duration of fixation
• Based on the effect on soluble proteins in
solution Fixatives
• Fixing agents are termed as: Characteristics Of Good Fixative
o “Coagulant” fixatives - result in a • Rapid action and quick penetration
permeable meshwork of protein strands • Cheap and economical
o “Non-coagulant”- additive in nature, • Isotonic to the tissue
produce a less permeable gel • Stable
• Should cause minimal loss in the physical
Mechanism of Fixing Agents and chemical properties of the tissue
• Denaturation: Most common, effect is • Safe to handle
induced by dehydrants such as the alcohols • Kills quickly
or acetone. These reagents remove and • Hardens tissues for easier cutting
replace free water in cells and tissues and
cause a change –irreversible Effects Of Fixatives
• Addition and cross-link formation: non- • Harden soft and friable tissue
coagulant fixing agents (reactive cpds) • Makes cell resistant to damage and distortion
chemically react with proteins and other cell • Inhibit bacterial decomposition
and tissue components, bind to a variety of • Increase optical differentiation of cells and
chemical groups in tissues and effect on tissue components
staining -stable • Acts as mordant or accentuator
Microwave Technique • Reduce risk of infection
• Physical agent like vacuum, oven (heat) and
agitation increase movement of molecules
and accelerate fixation

43
 Types of Fixatives • Adv vs. HCHO: more stable effect, less tissue
• Aldehydes shrinkage, less irritating
o Formaldehyde • Disadv: more expensive, slow penetration
o Glutaraldehyde 10% Formaldehyde Or 10% Formalin
• Metallic Fixatives 10% Formol Saline Solution
o Mercuric Chloride
• USED TO PRESERVE CADAVERS,
o Chromate Fixatives
Recommended for fixation of central nervous
o Lead Fixatives
tissues and post mortem tissues
• Picric Acid
• Acetic Acid Kaisserling’s Animal Cells
• Acetone • contains potassium salts and formalin
• Alcohol
• Osmium Tetroxide / Osmic Acid Formaldehyde Precautions
• Heat • Formation Paraformaldehyde
• Well ventilated room
Microanatomical Fixatives • Not neutralized if concentrated – explosion
• 10 % Formol Saline • Buffered or neutralized by adding
• 10 % Neutral Buffered Formalin magnesium carbonate/CaCO3 – wide mouth
• Heidenhain’s Susa bottle
• Formol Sublimate (Formol Corrosive) • Changing formalin can prevent Bleaching
• Zenker’s
10% Neutral Buffered Formalin/Phosphate
• Zenker – Formol (Helly’s)
Buffered Formalin
• Bouin’s
• Brasil’s 10% Neutral-Buffered Formalin (1000mL)
Formaldehyde 100mL
Histochemical Fixatives
Distilled water 900mL
• Formol Saline 10% Sodium phosphate, monobasic 4g
• Absolute Ethyl Alcohol Sodium phosphate, dibasic 6.5g
• Acetone • Optimal choice for many reasons.
• Newcomer’s Fluid o fast penetration
o prevents alterations during processing
Cytological Fixatives
o less shrinkage than other fixatives
• Nuclear o hardens tissue better
o Flemming’s
o Tissue can be stored in formalin
o Carnoy’s
indefinitely however, prolonged fixation
o Bouin’s
may bleach tissues
o Newcomer’s
o Fixative of choice:
o Heidenhain’s
Immunohistochemistry & molecular
• Cytoplasmic tests
o Flemming’s w/o acetic acid
o Readily available, time cosuming, stable,
o Helly’s
compatible w/ stains, penetrates tissues
o Formalin w/ post chroming
well, preserves fat, mucin, glycogen, for
o Regaud’s (Moller’s)
tissue photography
o Orth’s
• Na dihydrogen PO4, Disodium H PO4
Common Fixatives Used o For preservation and storage of surgical,
post mortem and research specimens
Formalin/Aldehyde - Containing
o Best fixative for Fe pigments, elastic
Fixatives fibers
Glutaraldehyde
• used for electron microscopy and light
microscopy
44
10% Formol Saline • For tissue photography, recommended for
renal tissues, fibrin, CT, muscles
• Penetrates and fixes tissues well, minimum • Disadv: hardens outer layers only, black
shrinkage & distortion, does not overharden granular deposits formed (removed by adding
tissues iodine), corrosive to metals
• Slow (>24 h)
Zenker’s Fluid (HgCl2 + Glacial HAc)
Undifferentiated Tumor • Recommended for fixing small pieces of
liver, spleen, connective tissue fibers and
Incomplete Fixation
nuclei.
• Good general fixative for adequate
preservation of all kinds of tissue and gives
excellent staining result
• Also recommended for Trichrome staining
Zenker Formol( Helly’s Solution)
• An excellent microanatomic fixative for
pituitary gland, BM & blood containing
tissue such as the liver and spleen.
• Made up of HgCl2 and formaldehyde.
• Produced Brown/ Black pigment

• Proper relationship of tissue structure has not Heidenhain’s Susa Solution

been maintained. • Excellent cytology fixative
• Poor staining. • Recommended for tumor biopsy esp. of the
• Lack of contrast between cell nucleus and skin
cytoplasm • Made up of MgCl2, glacial acetic acid and
formalin.
Well Fixed In 10% Neutral Buffered Formalin
B-5 Fixative
• Commonly used for BM biopsy.
• HgCl2, anhydrous Na acetate
Schaudinn’s Fixative
• Alcohol-containing mercury fixative,
• used for wet smear preparation and
connective tissues.
Dezenkerization

• HgCl2 deposits are removed by alcoholic

iodine solution prior to staining
• B Nucleus has “bubbling artifact” • Oxidation w/ Na to mercuric iodide,
• Better contrast between cell nucleus and removed by treatment with Na thiosulfate:
cytoplasm. o Bring slides to water. Immerse in Lugol’s
• Crisp nuclear membranes. iodine (5mins), running water (5mins),
5% Na thiosulfate (5mins), running water
Metallic Fixatives (5mins), proceed with required water
soluble stain
Mercury Containing
Chromate Fixatives
• Most common metallic fixative
• Routine fixative of choice for preservation of Chromic Acid
cell detail in tissue photography. • Precipitates all CHON and adequately fixes
carbohydrates

45
Potassium Dichromate(K2cr2o7) Methyl Alcohol 100%
• Preserves lipid and mitochondria
• Excellent for fixing dry and wet smears,
Regaud’s (Muller’s) Fluid (3%) K2cr2o7 blood smears and bone marrow tissues.
• Recommended for demonstration of
chromatin, mitochondria, mitotic figures, Ethyl Alcohol
golgi bodies, RBC and colloid containing
tissue • Used at concentrations of 70-100%
• Lower concentration causes RBC’s to be
Orth’s Fluid (2.5% K2cr2o7) hemolyzed and WBC’s are inadequately
• Recommended for study of early preserved.
degenerative processes and tissue necrosis
• Demonstrates Myelin, Rickettsiae and other Gendre’s Fixative
bacteria • Sputum
Lead Containing • Made up of alcoholic formalin

Lillie’s Fixative
• used for preservation of glycogen Other Fixatives
mucopolysaccharide, and amyloid.
Osmium Tetroxide (Osmic Acid)
• Fixes connective tissue mucin
• Forms insoluble lead carbonate – remove by • Preserves cytoplasmic structures well
filtering or adding Hac • Used extensively for neurological tissues
Picric Acid Fixatives • Fixes fats, for EM
• Expensive, poor penetration, reduced w/
• Remove yellow color by 70% ethanol sunlight causes black deposit; dark bottle
followed by 5% sodium thiosulfate & running • Acid vapor cause conjunctivitis, osmic oxide
water in cornea leads to blindness
• Highly explosive when dry • Inhibits hematoxylin
• Extremely volatile
Picric Acid
Flemming’s Solution
• Preserves glycogen well but causes
considerable tissue shrinkage • most common chrome-osmium acetic acid
• Allows brilliant staining with the Trichrome fixative
method • excellent fixative for nuclear structure
Bouin’s Solution Flemming’s Solution W/O Acetic Acid
• Recommended for fixation of embryos and • recommended for cytoplasmic structures.
pituitary biopsy
• for embyros, glycogen, does not need Acetone
washing out; poor penetration, not good for
• it is used in fixing brain tissues for the
kidneys, mitochondria, hemolyzes RBC
diagnosis of rabies
Brasil’s Alcoholic Picroformol Fixative • Dissolves fat, evaporates rapidly, preserves
glycogen poorly
• Excellent fixative for glycogen
Heat Fixation
Alcohol Fixatives
• involves thermal coagulation of tissue
• Denatures/ppt CHONs (destroys H bonds) proteins
• usually employed for frozen tissue sections
and preparation of bacteriologic smear.

46
Carnoy’s Fluid • Tissue that is not fixed well does not process
well, and subsequently will not stain well.
• Recommended for fixing chromosomes,
lymph glands and urgent biopsy. Secondary Fixation
• Also used to fix brain tissue for the diagnosis
of rabies. • Process of placing the previously fixed tissue
• Made up of ethyl alcohol, glacial acetic acid in a second fixative.
and chloroform • Purposes:
o To improve the demonstration of a
Newcomer’s Fluid particular substance
o For special staining methods
• Recommended for fixing o To ensure further complete hardening
mucopolysaccharides and nuclear protein and preservation of tissues
• Acts both as nuclear and histochemical
fixative Post Chromatization
• Made up of isopropyl alcohol
• Process wherein a fixed tissue is placed in an
Factors Affecting Fixation aqueous solution of 2.5 to 3% potassium
dichromate for 24 hrs
• Retarded By:
• Potassium dichromate acts as mordant for
o Size and thickness
better staining effect and preservation of
o Presence of mucus
tissues
o Presence of fats
o Presence of blood Washing Out
o Cold temperature
• Enhanced By: • Done to remove excess formalin or fixative
o Size and thickness from the tissue after fixation for better
o Agitation staining effect and to remove the artifacts
o Moderate heat (37 to 56 degrees C) from the tissue using:
o Tap water – excellent for removal of
Incomplete Fixation chromates, formalin and osmic acid
o Alcoholic iodine – excellent for removal
• Results in: of mercury containing fixatives
o Separation of tissue components on the
o 50 – 70 % alcohol
flotation bath during microtomy
o Poor tissue morphology Grossing Of Specimen
o Smudgy nuclei with no chromatin
pattern defined • Tissue thickness
o Nuclear bubbling o <3mm, especially fatty tissues
o Center of tissue more eosinophilic than o Tissue cassette must not bulge when
periphery closed (allow space)
• Paper tags:
Incomplete Fixation Troubleshooting o Use pencil or computer print (prefer dot
• REMEMBER: The most important pre- matrix), not pen or marker
analytical variable • Dissect fat away from lymph nodes before
• Amount of time the tissue is not immersed in loading
the right solution of formalin (10% neutral Labelling
buffered formalin),
• Right volume (10-20 x the specimen) and • Labeling of sections and blocks
exposed properly to formalin by opening, o Indicate number of sections and
cutting or bisecting specimens before blocks(cassettes) taken
immersing in formalin. o Document samples taken systematically
o Document color code of inked margins

47
• Minute and multiple fragments
o wrap tissues in filter paper or similar
material to prevent loss in processing

• Process of removing calcium from the tissues

following fixation- bones, teeth, calcified
tissues – tuberculous lungs, arteriosclerotic
vessels
• Done to facilitate cutting of tissues
• Done after fixation and before impregnation
• Change decalcifying agent regularly Acids: Nitric Acid

Decalcifying Agents 5%- 10% Aqueous Nitric Acid

• Acids - HNO3, HCl, formic, TCA, sulfurous, • most common acid used, considered as the
chromic, citric best acid for decalcification because of its
• Chelating agents - EDTA – slow rapid action. Bone is immersed for 24 hrs
o EDTA (versene)- Chelating Agents • Disadvantage: inhibits nuclear stain –
o Excellent staining combine with alcohol or formaldehyde
o Very slow
Formol – Nitric Acid
o appropriate for research applications
(high quality morphology is required) or • Rapid acting
particular molecular elements must be
• Good nuclear staining
preserved for techniques such as IHC,
• Less tissue destruction than 10% aqeuous
FISH or PCR
nitric acid
o Slight tissue hardening produced
• Use fume hood
• Ion exchange resin - Ammonium form of
• Lessen yellow tissue discoloration by 5% Na
polystyrene resin) – 1 to 14 days – spread on
sulfate or 0.1 % urea
bottom of container
o Produce artifacts usually caused by CO2 Formic Acid
bubbles
• Electrophoresis (electrical ionization) - • Better nuclear staining with less tissue
attraction of Ca to negative electrode distortion & * safer to handle than nitric and
o Combined with formic acid, conc HCL, HCl
Dist H2O • 2-7 days - slow
• Fixative & decalcifying agent
Mineral Acid Decalcifiers
• Excellent nuclear & cytoplasmic staining

Phloroglucin
.
• 12 –24 hours
• Conc nitric + phloroglucin = dense white
fumes, then add 10% nitric acid
• Most rapid
• Disadv: poor nuclear staining
• when decalcification is complete, acid must
be removed by 3 changes of 70 to 90%
ethanol

48
 Measuring Extent of Tissue Softeners
Decalcification • Perenyi’s – 12 –24 hours
• 4% aqueous phenol – 1 – 3 days
Physical/Mechanical Test • 2 % HCl
• 1 % HCl in 70 % alcohol
• Decalcified tissue are softer to touch
• Mechanically checked by pricking the tissue
with a needle or probe, manipulation,
bending probing or trimming of the specimen • most critical stage of processing – difficult to
correct
Chemical Method Purposes of Dehydration
• Simple, reliable and convenient method for • Process of removing intercellular and
routine purposes. extracellular water from the tissue following
• Involves detection of calcium in acid solution fixation and prior to impregnation.
by precipitation of insoluble hydroxide or • To remove the excess water from the tissue
calcium oxalate. • To prevent the excessive hardening of the
• Litmus paper -> red, add NH3 drop by drop tissue
-> blue; • To facilitate better wax impregnation
• If cloudy – still w/ calcium; if clear: add
ammonium oxalate, 30 mins -> if cloudy, Characteristics of an Ideal Dehydrating
incomplete Agent
• Must dehydrate rapidly without shrinkage of
Xray/Radiologic Method tissues
• Very expensive but the most ideal and • Should not evaporate very fast
reliable method of determining the extent of • Be able to dehydrate even fatty tissues
decalcification. • Should not overharden tissue
• Should not be toxic to the body
Factors Influencing the Rate Of • Should not be a fire hazard
Decalcification • Should not remove the stain

Concentration Commonly Used Dehydrating

• affect the rate at which calcium is removed Agents
• Conc decreases as it combines w/Ca Alcohols
Temperature Ethyl Alcohol
• Increased temperature will speed up the • Best dehydrating agent, fast acting, mixes
decalcification rate but will also increase the w/water and many organic solvents,
rate of tissue damage so must be employed penetrates tissue easily.
with great care • Not poisonous and not very expensive
• Refrigerate At 4oc-To Slow the Process
Methyl Alcohol
Agitation • Best dehydrating agent for blood smear
• Gentle agitation may increase the rate
Butyl
slightly
• slow, for Plant and Animal microtechnique
Fluid Access • Disadvantages:
• access to all surfaces of the specimen o Prolonged storage in 70 % alcohol –
• will enhance diffusion and penetration into macerates tissues
the specimen and facilitate solution, o Directly placed in high grade alcohol –
ionization and removal of calcium shrinkage & hardening of tissues

49
 Acetone • To remove the dehydrating agent and to
make tissue transparent.
• Cheap, rapid acting, used as substitute for
alcohol Characteristics of a Good Clearing
• Rapid in action but penetrates tissues poorly Agent
and causes brittleness • Should be miscible with alcohol to promote
• Not recommended for routine dehydration, rapid removal of dehydrating agent
for small pieces of tissues causes tissue • Should be miscible with, and easily removed
shrinkage by melted paraffin wax and/or by mounting
medium to facilitate impregnation and
Dioxane (Diethylene Dioxide)
mounting
• Excellent dehydrating and clearing agent • Should not produce excessive hardening,
• Produces less tissue shrinkage shrinkage, or damage of the tissue
• Tissues can be stored for a long time • Should not dissolve out aniline dyes
• DISADVANTAGE: • Should not evaporate quickly
• expensive and extremely dangerous and not • Should make tissues transparent
recommended for routine dehydration
Common Clearing Agent Used
Cellusolve (Ethylene Glycol Monoethyl
Xylene
Ether)
• Most commonly used clearing agent
• Dehydrate rapidly, non toxic, has no harmful
effect • Becomes milky when an incompletely
dehydrated tissue is immersed in it
• Tissues can be stored in it for months without
producing hardening or distortion • Best clearing agent
• DISADVANTAGEs: Hardens/shrinks
Triethyl Phosphate tissues – not for nervous system & lymph
nodes; hard/brittle tissues if > 3 hours
• Minimum shrinkage
• Minimum distortion and hardening of tissue Toluene

Tetrahydrofuran -THF • May be used to replace xylene or benzene for

clearing; Rapid acting
• dehydrates and clears tissue • Tissues do not become excessively hard and
• Causes less shrinkage and easier cutting of brittle even if left here for 24 hours
sections • DISADVAN: toxic upon prolonged
• DISADVANTAGE: it is toxic if inhaled and exposure; expensive
ingested
o Carbon tetrachloride Benzene
o has rapid action but toxic.
• Penetrates and clear tissue rapidly,
Additives To Dehydrating Agents Volatilizes rapidly in paraffin oven
• DISADVAN: carcinogenic; aplastic anemia
• 4 % phenol added to 95 % ethanol – softens
hard tissues Chloroform
• Hard tissues – immerse in glycerol/alcohol
mixture • slower in action than xylene but causes less
brittleness
Clearing Or Dealcoholization • suitable for large tissue specimen
• it is recommended for tough tissues,
• Process whereby alcohol or the dehydrating • DISADVAN: highly toxic to liver and has
agent is removed from the tissue and replaced sleeping effect.; Does not make tissues
with substance that will dissolve the wax with transparent; Tissues tend to float
which the tissue is to be impregnated

50
 Cedarwood Oil o Dry – whole eye sections – 70% ROH not
used
• used to clear specimens to be embedded in • Gelatin
both paraffin and celloidin. • Plastic
• recommended for central nervous system
tissues, and cytological Studies, particularly Automated Tissue Processor
of smooth muscle and skin
• DISADVAN: expensive; extremely slow

Aniline Oil
• not normally used as routine clearing agent
• recommended for clearing embryos and
delicate specimen Conventional Tissue Processor
Clove Oil • Primary processor in use today is the closed
system (fluid transfer), in which tissue is
• causes minimum shrinkage and expensive
stationery and fluids are pumped in and out
• not suitable for routine clearing purposes
of the pressurized chamber holding the
Carbon Tetrachloride tissue.
• Closed systems are computerized, provides
• cheaper than chloroform digital read out on instruments status and has
• DISADVANTAGE: produces considerable alarm to alert personnel to any mechanical
tissue hardening and dangerous to inhale on problems.
prolonged exposure due to highly toxic • Closed systems can assist with keeping
effects. exposure to toxic vapor to a minimum.
• A major advantage is that specimens cannot
dry out in tissue chamber in the event of
malfunction.
• Process whereby clearing agent is completely
removed from the tissue and replaced by a Autotechnicon
medium that will completely fill all the tissue
cavities.
• To completely fill the intracellular and
extracellular spaces
• To set a firm consistency of tissues and to
allow cutting of thinner sections without
distortion, tissues are immersed in
impregnating medium like molten paraffin
wax to penetrate tissue spaces.

Embedding & Impregnation Paraffin Wax Impregnation

Media Paraffin Agent
• Paraffin wax • the most common, simplest and best
o Manual – 4 changes of wax 15 mins embedding medium used for routine tissue
interval processing.
o Automatic – Autotechnicon, Elliott
• Good staining results
Bench type
• Sections cut will stick together edge-to-edge,
o Vacuum – fastest, negative atmospheric
forming a “ribbon” of sections
pressure
• Not recommended for fatty tissues
• Celloidin
• Overheated paraffin -> brittle specimen
o Wet – bones, teeth, large brain sections
51
• Inadequate impregnation -> clearing agent Ways Of Paraffin Impregnation
retained
• Melting point – 56°C – 58°C normally used • Manual
for routine work. • Automatic
• Paraplast: Melting pt is 56°-57°C; more • Vacuum – gives the fastest result
elastic/resilient eg. embeddol.
• Ester wax: harder than paraffin wax; H2O
insoluble; melting pt is lower, 46°-48°C. • Also known as Blocking or Casting.
• Water-soluble waxes: also called as
• Process by which the impregnated tissue is
polyethylene glycol; melting pt is 38°-42°C; placed into a precisely arranged positioning a
Eg. Carbowax.
mold containing a medium which is then
Celloidin Impregnation allowed to solidify.
• Blocking – formation of tissue block
• A purified form of nitrocellulose soluble in • Molding – process of placing the tissue in a
many solvents molder
• Permits cutting of thicker tissue sections
• Crumbling of tissues avoided Orientation
• Suitable for specimens with large cavities • it is the process by which a tissue is arranged
• For hard and dense tissues in a precise position in the mold during
• For large tissue section of the whole embryo embedding, on the microtome, before cutting
• Also recommended for processing of and on the slide before staining.
neurological tissues • will determine the plane through which the
• Disadvantages: section will be cut and ultimately may decide
• Very slow whether an abnormal area will be visible
• Thin sections difficult to cut under the microscope
• Serial sections difficult to prepare • Tissues with a wall, such as cysts, gallbladder
and gastrointestinal tract, must be embedded
• Photomicrographs difficult to obtain
on edge so that all layers are visible.
• Very volatile
• Tubular structures, such as fallopian tubes or
Methods Of Celloidin Impregnation the appendix, are embedded in cross-section
• Wet Celloidin Method so that the lumen and all layers of the
o Recommended for bones, teeth, large mucosa, submucosa, and external muscle
brain sections and whole organ layers are obvious microscopically.
• Dry Celloidin Method
o Preferred for processing of the whole eye Double Embedding
section • 2 % Celloidin for 3 days and subsequent
paraffin
Gelatin Impregnation
• For large blocks of dense firm tissues – brain
• Rarely used except when dehydration is to be • Obsolete – due to paraffin waxes with
avoided and when tissues are subjected to different types of resins
histochemical and enzyme studies
• Used as an embedding medium for delicate Plastic (Resin) Embedding
specimen and frozen section specimen • Epoxy, polyester, acrylic
• Does not require dehydration & clearing • With VCD – vinylcyclohexane dioxide-
• Low melting point carcinogenic
• For hard tissues, renal and bone marrow
Plastic biopsies
• For Tiny tissues Electron microscopy
• Used for automation

52
 Epoxy Resins
• Require dehydration through a graded series
• Microtomy
of ethyl alcohol, and unless miscible with
o means by which tissues can be sectioned
ethanol
and attached to the surface so that
• Requires the use of a transitional fluid (i.e.
examination by microscopy can take
clearing agent) such as propylene oxide.
place.
• Most commonly used epoxy resins are
• Microtome
Araldite, Epon and Spurr.
o basic instrument used in microtomy into
• Polymerization is commonly done at 60°C which cutting tool is clamped.
resulting in a hard block.
o used to cut thin section of tissue.
• Required as embedding media for EM- for o are machines that will advance an object
good resolution very thin sections w/c can be for a predetermined distance then slide
obtained only from epoxy resin-embedded the object to the cutting tool.
material.
• Sections 60 to 90 nm thick can be cut with a Different Microtome Knives
diamond knife. For light microscopic
examination and orientation, 0.5 um thick • Diamond edged knife – designed for
sections are cut with a glass knife ultrathin section microtome, specimen is
examined under electron microscope.
Tissue Block • Biconcave knife – designed for rotary
microtome (most commonly used
• After embedding, the blocks should be cooled microtome).
rapidly to obtain the smallest paraffin crystal
• Plane concave knife – for sliding and
size possible, because the crystal size affects
rocking, celloidin embedded microtome, one
sectioning quality.
side is flat the other side is concave
• Celloidin blocks – do not require chilling or
• Plane wedge knife – used for frozen sections,
refrigeration
or hard specimens in paraffin blocks with
Types Of Blocking-Out Molds base sledge/sliding has both sides straight

• Leuckhart’s embedding mold Sharpening of the Knives

• Compound embedding unit • Honing:
• Plastic embedding rings and base mold o removal of gross nicks, blemishes, and
o Tissue Tek – machine w/ warm plate grinding cutting edge of the knife to
• Disposable embedding mold stone.
o Peel away, plastic ice trays, paper boats o Lubrication: xylene, mineral oil, clove
oil, paraffin and soapy water.
Trimming and Sectioning o “Edge first” heel to toe, 20-30 times,
• TRIMMING abrasives (powdered aluminium oxide).
o excess wax is cut off from the block to o Belgium yellow- Best manual
expose the tissue surface in preparation sharpening,
for actual section cutting. o Arkansas- more polishing effect than
Belgium Yellow
• SECTIONING
o Fine carborundum- much coarser for
o Process by which tissue are cut uniformly
badly nicked knives.
thin slices or sections on a precision
o Plate glass hone - use diamanthine for
instrument called a “microtome” using
final polishing
extremely fine steel blades;
o Machine hone: glass disc or wheel driven
• Microtome – is the cutting engine of the
by electric motor
histopath lab.
• Stropping:
o removal of the burr,

53
 o “Edge last” Toe to heel, 40-120 double • Dry the machine carefully especially the
strokes. knife holder
o Horse leather (use castor oil to prevent • keep the machine well oiled to prevent rust
rusting) formation
• Angle forms of the knife • keep the moving parts of microtome oiled
o Bevel angle – angel formed by cutting • cover the microtome when not in use
edges of the knife, 27 to 32 degrees (prevent accumulation of dust and dirt)(may
o Clearance angle – formed by the knife interfere the sectioning)
and the tissue block, 5 to 15 degrees
Rotary Microtome
Types Of Microtomes
• most commonly used microtome for routines
• Each designed for a specific purpose and research purpose
although many have a functional role. • rotary action of the hand wheel actuates.
• Excluding ultra-microtome, there are 5 basic These machines sometimes called minor
types. named according to the mechanism. microtome after their inventor prof. Minot
• Rocking microtome – for cutting serial (1885-86)
sections of large blocks of paraffin embedded • the knife is fixed & the tissue blocks moves
tissues, 10-12microns; simplest – Caldwell & up & down vertically in front of the knife.
Trefall 1881 • Plane wedge knife is used.
• Rotary microtome – for cutting of paraffin • Thickness is adjusted by micrometer screws.
embedded tissues, 4 to 6 ums; Minot 1885-86 • The block moves forward to the knife at
• Sliding microtome – for cutting celloidin presented thickness during the rotation of fly
embedded tissues, 7 to 9 um; Adams 1798 –wheel handle.
• Base Sledge • It has adjusting screws to make the tissue
o Standard Sliding- block is stationary; block parallel to the knife.
knife is moving; Most dangerous type of
microtome – movable exposed knife Parts Of Rotary Microtome
• Freezing microtome – for cutting frozen
• Block holder or chuck
sections, 10 to 15 microns; Queckett 1881;
o part where the tissue block is being held
bursts of CO2 gas vs. cryostat
in position.
• Ultrathin microtome – used for electron
• Knife clamps screws
microscopy, 0.5 microns
• Knife clamps
Major Parts of a Microtome • Block adjustment
• Thickness gauge
• Block Holder/ chuck - Holds the tissue block • Angle of tilt adjustment
in place
• Operating handle
• Knife carrier and knife - Actual cutting of
• Internal and external lock
tissues
• Micrometer adjustment
• Pawl, ratchet feedwheel, adjs.screw Line
o used to line up the tissue block in proper
up the tissue in proper position to the knife,
position with the knife.
Adjust proper thickness of the tissue
for successive sections Advantages
Care of the Rotary Microtome • It is heavier than rocking microtome.
• No vibration while cutting.
• after cutting, brush away with soft brush: all
• It can be used for cutting hard tissues.
accumulated paraffin and tissue
• The cutting angle of the knife is adjustable.
• wipe clean all metal parts with xylol
• The knife holder is movable.
• avoid continuous application of xylol to the
rest of the machine (can remove the painted • The ability to cope with very hard tissues,
finish) together with cutting good accurate section at
3mm gives the flexibility that is now required.
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• Electrically driven microtome can be used because tissues are sliced larger leading to
when the necessary to produce ribbons for inaccuracy.
serial reactions required.
• Manual or electrically driven rotary Vibrating Microtome
microtome are successfully used in cryostats.
• Designed to cut tissues which has not been
Rocking Microtome fixed , processed or frozen.
• ADVANTAGES
• It is the oldest type of microtome. o Greatest application in enzyme
• Name comes under the rocking action of the histochemistry & ultra structure
handle. histochemistry.
• Cambridge rocking microtome was the most o Tissues are cut at very slow speed to
popular microtome. avoid disintegration.
• Knife is fixed &the block of the tissue moves
through an arc to strike knife.
Adhesives & Mounting Media
• DISADVANTAGES • for entailing significant exposure of section to
o Size of the block that can be cut is limited. acids and alkalis during staining, but cannot
o It is a righter microtome, so it vibrates be used for protein histochemical
while cutting. investigation
o Cutting angle of the knife cannot be • Mayer s albumin: Egg white + glycerin
adjusted. • A drop is smeared into a clean slide before
o Sections cut curved when the block sections are oriented
moves through an arc.
• Dried albumin: Dried albumin + NaCl +
o No serial section is possible.
thymol crystal
Sledge/Sliding Microtome • Gelatin: Gelatin + water + glycerol +phenol
crystal
• Starch paste: Powdered starch +d. water+
HCl + thymol crystal (prevent formation of
molds)
• Plasma: Outdated blood stored in blood
banks

Mounting Of Sections Purpose

• protect specimen from physical injury
• protect section from bleaching
or deterioration
Ultra-Microtome • preserve slide for permanent keeping
• Used almost exclusively for electron Characteristics Of Good Mounting
microscopy Medium
Freezing Microtome • Ref.Index: near to the glass 1.518
• shouldn’t dry quickly
• Although other microtome can be modify for
• shouldn’t dissolve out or fade tissue section
cutting frozen section, this type will give the
best results & is used almost universally. • should not cause shrinkage and distortion
• should set hard
Frozen Section • shouldn’t have granularity/ cracking
• should be miscible with xylene or toluene
• Specimens are frozen using nitrogen gas
• should be nonreactive, no pH or color change
• Used if the patient requires immediate
histopath analysis, not routinely used

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 Classifications of Mounting Medium

Sealing
• RINGING - process of sealing the margins
of coverslip to prevent escape of fluid
• Ringing media:
o paraffin wax, kronig’s/Dunoyer’s
mixture, nail polish ,
Durofix (cellulose adhesive)

Processing Bone Marrow Biopsies

• The fixative used is very important.
• Submit entire needle biopsy after
• fixation in Bouin’s fluid overnight, which is
mildly acidic and removes calcium.
• Serially number eight slides and cut sections
at 4 microns.
• Stain slides 1 & 5 with H&E; slides 2 &6 with
reticulin stain, and slides 3 & 7with iron.

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