MT TERM
HISTOPATHOLOGY TECHNIQUES
LECTURE / LIWANAG
GENERAL PATHOLOGY AND EXFOLIATIVE CYTOLOGY
PATHOLOGY
• Area of science that deals with the study of diseases
o Involves study of all changes that underlie a disease
▪ Like functional, structural, or biochemical
changes
▪ Starting point of any disease process is the cell
and ends up to a disease
▪ During cell growth, abnormalities may set in
• Cells have limited lifespan, that’s why it undergoes cell
division
ABNORMALITIES IN CELL GROWTH
• APLASIA – incomplete or defective development of tissue
or organ
o Usually happens in paired organs like kidneys, and
gonads
o Tissue or the affected organ shows no resemblance to
the normal matured adult structure
• AGENESIA – complete non-appearance of organ
o Example: during birth, infant has one kidney only
• ATRESIA – failure of organ to form an opening
o Passage in the body is either absent or blocked
o Example: Microtia (absence of ear canal), imperforate
anus
• HYPOPLASIA – failure of organ to reach its normal mature
size
o Example: an organ should be 5x5, but on maturation,
it only grew in a 2x3 size
CELLULAR ADAPTATIONS
• Cells in the body may be exposed to a lot of stress or
injurious agents
o Physical agents: e.g., UV
o Biological agents: e.g., parasites, viruses, or bacteria
o Mechanical agents: e.g., trauma
• Cells can either acquire adaptation or nonadaptation
during an injury
o Nonadaptation can lead to cell death
• Two types of cell injury:
o REVERSIBLE – affected tissue or organ can revert
morphologically into its normal state using several
adaptation mechanisms
o IRREVERSIBLE – the point of no return; affect is cell
death
ADAPTATION MECHANISMS
• ATROPHY – acquired decrease in tissue or organ size
o Two types:
▪ Physiologic atrophy – if it’s a normal
consequence of maturation
➢ Example:
Atrophy of thymus during puberty: as a person
grows, thymus normally decrease in size
Atrophy of uterus size after childbirth
▪ Pathologic atrophy – due to a disease
➢ Example:
Endocrine atrophy: can be attributed to lack of
hormones needed to maintain normal size
Hunger atrophy: affected organ decreased in
size due to lack of nutritional supply
Vascular atrophy – decrease in size is due to
sudden cut off blood supply
4B 01
Exhaustion atrophy – frequent use (over
usage) of tissue or organ resulted into its
decrease in size
• HYPERTROPHY – increase in tissue or organ size due to
an increase in the size of individual cells that comprise that
organ
o No new cells are produced
o Three types:
▪ Physiologic – a normal process
➢ Example: increase in the size of the skeletal
muscles following exercise
▪ Pathologic – due to a disease
➢ Example: hypertrophy of myocardium due to
hypertension or aortic valve disease
▪ Compensatory – one of the paired organs is
removed
➢ Renal hypertrophy: one of the kidneys was
removed, the remaining kidney will
compensate for the other one, as a result, it
will increase in size
• HYPERPLASIA – increase in tissue or organ size due to an
increase in the number of cells that comprise that organ
o New cells are produced
o Three types:
▪ Physiologic
➢ Example: increase in breast and uterus size
during pregnancy (a normal process)
▪ Pathologic
➢ Example: increase in the number of lymph
nodes in TB of cervical lymph nodes
▪ Compensatory
➢ Adrenal hyperplasia
• METAPLASIA – transformation of adult cell into another
adult cell type
o Classified as a reversible change
o Two types:
▪ Epithelial metaplasia – cells involved are
epithelial cells
➢ Example:
Ciliated columnar cells – lining the surface of the
bronchi, due to cigarette smoking, these cells can
transform to squamous epithelial cells
Squamous epithelial cells can return to ciliated
columnar cells by eliminating the stimulus,
cigarette smoking
▪ Mesenchymal metaplasia
➢ Cells involved are connective tissue cells
• ANAPLASIA – transformation of adult cell into primitive cell
type (embryonic)
o Classified as an irreversible change
▪ A.k.a. De-differentiation
• DYSPLASIA – characterized by change in cell size, shape,
and orientation
o Also known as Atypical Metaplasia or Preneoplastic
lesion
o No transformation, only change
o A reversible process
• NEOPLASIA – process of tumor formation, characterized
by abnormal proliferation of cells
o If the tumor is malignant → Cancer
o Specimen from an alive individual:
▪ Excisional biopsy – removing the entire mass or
organ
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 Histopathology Techniques

▪ Incisional biopsy – removing only a part of the SECONDARY CHANGES

mass (the new growth) • ALGOR MORTIS – cooling of the body, establishes time of
o Biopsy is carried out to detect malignant conditions death
o Fine Needle Aspiration Biopsy (FNAB) – method o Happens at a rate of 7°F per hour
practiced nowadays
• RIGOR MORTIS – stiffening, rigidity of muscles
▪ For small tumor masses only
• LIVOR MORTIS – postmortem hemolysis, purplish
▪ Disadvantage: specimen is collected using needle
discoloration of skin
or syringe, therefore, only few cells are collected,
o Also known as Lividity
cancer cells might be missed
AUTOPSY
PATTERNS OF CELL DEATH
• Examination of dead body
• APOPTOSIS – programmed cell death; death of a single
o Complete autopsy
cell in a cluster of cells
o Partial autopsy – only few regions of the body are
o Physiologic cell death
examined
o Cell shrinkage, therefore, outline of the cell is still
▪ e.g., Head area only
intact
• Carried out for the purpose of determining the cause of
o No leaking out of cellular contents, so, there is no
death
inflammation
o Also known as Necropsy
• NECROSIS – pathologic cell death
o Pathologist carries out autopsy
o Cell swelling
o Most important requirement to perform autopsy is
o Cell outline is ruptured
consent from the nearest kin
o Leaking out of cellular contents, so, there is
• Two types of autopsies:
inflammation
o Y-shaped incision – for adult cadavers
TYPES OF NECROSIS o Straight cut incision – for infants or children cadavers
1. Coagulative Necrosis – happens due to sudden cut off of AUTOPSY TECHNIQUES
blood supply • Rudolph Virchow – organs are removed from the body one
o Cell death is due to ischemia by one
o Happens in solid organs like kidneys, spleen, or • Carl Rokitansky – in situ dissection
lungs, but not the brain
o Organs are examined or dissected in their original
o In necrosis, changes can be seen grossly or
place
microscopically
o Not removed
o Affected organ appears like boiled material (and color
• Anton Ghon – en-bloc removal of organs
gray) on gross inspection
o All organs belonging in the same system are removed
o The action of hydrolytic enzymes is blocked
together as a group
2. Liquefactive Necrosis – type characterized by complete
• M. Letulle – en-masse removal
digestion of cells due to action of hydrolytic enzyme
o Affected organ appears liquid-like and creamy yellow o All organs are removed at the same time
due to pus SIDE NOTES
o Action of hydrolytic enzymes are not blocked,
• In histopathology, there are three specimens:
compared to coagulative necrosis
o Biopsy specimen – to rule out cancer
3. Caseous Necrosis – combination of coagulative and
o Autopsy specimen – to determine cause of death
liquefactive
o Specimens for cytology – e.g., Pap smear
o Seen in TB
o Affected organ appears like “cottage cheese” • Medical technologist → processes the specimen
4. Fat Necrosis – seen in acute pancreatitis, necrotic material • Pathologist → performs the microscopy
appears like chalky white precipitates Table No. 1 Record and specimen retention
o Cells involved are fat cells SPECIMEN RETENTION
5. Fibrinoid Necrosis – type of necrosis seen in immune CLINICAL PATHOLOGY
reactions involving blood vessels REPORT
o Changes are too small to be seen on gross PATHOLOGY SLIDES
inspection PATHOLOGY BLOCKS
SURGICAL 10 years
INFLAMMATION AND CHANGES IN SOMATIC
PATHOLOGY AND
DEATH
BONE MARROW
• Tissue reaction to energy REPORTS
• Five cardinal signs of inflammation:
AUTOPSY REPORTS Indefinitely
o RUBOR – redness caused by increased rate of blood
flow SECTIONING
o DOLOR – pain • Numbering → fixation to preserve and harden
o TUMOR – swelling • Performed once tissue block is obtained
o CALOR – heat Table No. 2 Microtome
o Functio laesa – loss of functioning units of the cell MICROTOME PURPOSE
• Somatic death – death of the entire body
ROTARY MICROTOME For paraffin embedded tissues
o Primary changes: changes that can be noted
Invented by Minot
immediately after death
o Secondary changes: changes few hours after death SLIDING MICROTOME For cutting celloidin embedded
Developed by Adams tissues
ULTRATHIN For Electron Microscopy
MICROTOME → Thickness of section: 0.5 𝜇
For preparing frozen sections

PUNDAVELA, N. 2
 Histopathology Techniques

CRYOSTAT → To cut fresh tissues TYPE DEFINITION

MICROTOME (common microtomy BELGIUM YELLOW Gives the best result
nowadays), AND Purpose of frozen sections: for ARKANSAS With more polishing effect than
FREEZING rapid diagnosis; requested while Belgium yellow
MICROTOME the patient is inside the OR FINE CARBORUNDUM For badly nicked knives
Table No. 3 Important LUBRICANTS: Mineral oil, clove oil, xylene, liquid paraffin,
Thickness of sections 4 to 6 𝜇 and soapy water
using → While honing, lubricants should be used
Rotary Microtome STROPPING
Cryostat microtome
Refrigerated apparatus → Microtome enclosed in a • Process of polishing and sharpening the cutting edge
for preparing frozen chamber o Purpose: to remove the “BURRS” and other
sections → For fresh tissues, consistency irregularities formed during honing
is soft • Material used: Horse leather
-5 to -30C • Must be oiled prior to use
Temperature of → Facilitates immediate Table No. 6 Angles
Cryostat hardening of tissues, thus, TYPE ANGLE
making the specimen easier to CLEARANCE ANGLE/ Angle formed between the
cut TILT ANGLE/ cutting facet of the knife and the
Freezing agents Liquid nitrogen, isopentane INCLINATION ANGLE surface of the block
used cooled by liquid nitrogen, CO2 Set at 5 to 10°
gas, and aerosol sprays WEDGED ANGLE Angle formed by the sides of the
Most dangerous type Sliding microtome knife
of microtome → Knife is exposed Set at 14 to 15°
Mayer’s Egg Albumin BEVEL ANGLE Angle formed between cutting
(To promote attachment of edges
Commonly used as a ribbons to the slide) Set at 27 to 32°
tissue adhesive Prepared by equal amounts of • Float-out Bath
egg white + glycerin o Thermostatically controlled bath
→ Add thymol crystals to o Temperature: 45 to 50C or 5 to 10 degrees lower
prevent molds than the wax melting point
▪ Melting point of paraffin wax commonly used: 56C
SIDE NOTES o Purpose: To flatten the ribbons, and to remove
• Ribbons – tissue sections wrinkles and folds
o Usually wrinkled • DEPARAFFINIZATION
• Ribbons in thin slices are placed on a float out bag, and o Removal of paraffin wax from tissues once properly
when it hardens, adhesive is applied, then it is fished out fixed on the slide
• Place on a slide, with paraffin wax surrounding the tissue o Three methods:
• Disposable blades are used nowadays, instead of knives 1. Place the slides in the oven (55 to 60C)
2. Use of alcohol lamp
TYPES OF MICROTOME KNIVES 3. Immerse the slides in xylene
• Biconcave
• Plane concave STAINING
• Plane wedge • Step in tissue processing that involves application of dyes
Table No. 4 Knives • Nucleus – acid; cytoplasm – alkaline
TYPE DEFINITION • NUCLEUS
o Part of the cell with greater affinity for basic dyes
BICONCANVE KNIFE Both sides of the knife are
concaved • CYTOPLASM
→ Used for cutting paraffin o Part of the cell with greater affinity for acid dyes
embedded tissues THREE STAINING CATEGORIES
PLANE WEDGE KNIFE Both sides of the knife are flat o Histological
→ Used for frozen sections, and o Histochemical
extremely hard and tough o Immunohistochemical
tissues Table No. 7 Staining categories
PLANE CONCAVE One side is concave, the other TYPE DEFINITION
is flat Tissue components are
Concave part – cutting paraffin demonstrated through direct
embedded tissues interaction with a dye or a
Flat part – for celloidin HISTOLOGICAL staining solution
embedded tissues STAINING/ → Stains both nucleus and
HONING MICROANATOMICAL cytoplasm (e.g., H&E, Gram
STAINS stain, AF stain)
• Grinding the cutting edge to form an even edge To demonstrate the general
o Purpose: to remove gross nicks relationship of tissues and
• Microtome knives are prone to nicks, dullness, and cells with differentiation of
bluntness, remedy can be honing nucleus and cytoplasm
→ Examples: Methylene blue
Table No. 5 Types of Hones or oil stones
PUNDAVELA, N. 3
 Histopathology Techniques
Saining category in which
tissue components
demonstrated through
HISTOCHEMICAL chemical reactions
STAINING → Examples: Periodic-Acid
Schiff (to demonstrate
carbohydrates),
Pearl’s Prussian Blue (to
demonstrate hemosiderin)
Staining technique which is a
combination of immunologic
and histochemical techniques
IMMUNOHISTOCHEMICAL For the detection
STAINING phenotypic markers
tissue antigens
→ Dyes are not used;
antibodies are used instead
(monoclonal or polyclonal)
STAINING METHODS
Table No. 8 Staining methods
METHOD DEFINITON
Uses aqueous or alcoholic
DIRECT STAINING solutions of dye
→ Tissue + dye = color
Staining technique that employs
INDIRECT STAINING use of mordant and accentuator
→ Tissue + dye with
mordant/accentuator
Substances that serve as link or
MORDANT bridge between the dye and the
tissue
Examples: Iron and K alum
Increases or heightens the
ACCENTUATOR staining power of the dye (color
intensity)
Examples: Phenol and KOH
HEMATOXYLIN WITH K Mayer’s hematoxylin, Ehrlich’s
ALUM AS A MORDANT hematoxylin, Harris hematoxylin
HEMATOXYLIN WITH Weigert’s hematoxylin,
IRON AS A MORDANT Heidenhain’s hematoxylin
Involves gradual application of
PROGRESSIVE dye
STAINING Follows definite sequence
NO excess dye
No decolorization needed
REGRESSIVE Tissue is overstained
STAINING Excess dye
Decolorization is needed
DECOLORIZATION/ Selective removal of excess dye
DIFFERENTIATION Commonly used is acid alcohol
Staining technique that involves
use of dye/stain that gives
different color, different from the
color of the dye itself
METACHROMATIC → Mainly used for
STAINING demonstration of cartilage, CT,
epithelial mucins, mast cell
granules, amyloid
Examples: Methyl violet crystal
violet/safranin,
Bismarck brown/basic fuchsin,
Methylene blue,
Thionine/Toluidine blue,
Azure A, B, C, Cresyl blue
Staining technique in which
ORTHOCHROMASIA tissue components are stained
with the same shade or hue as
that of the dye
→ Same shade, but not the
are same color as that of the dye
(blue → light blue)
Staining method that involves
COUNTERSTAINING use of another dye to provide
contrast and background
Commonly used eosin
Tissue components are
METALLIC demonstrated not by stains but
IMPREGNATION by colorless solutions of metallic
salts that produces black
deposits on the surface of
of tissues
and EXAMPLES OF Gold chloride, silver nitrate, or
AGENTS USED IN ammoniacal silver
METALLIC
IMPREGNATION
Staining of living cell
components
VITAL STAINING Involves cytoplasmic
phagocytosis
Methods: Intravital and
Supravital staining
Dye is injected to any part of the
INTRAVITAL STAINING living body
Examples: India ink, lithium, and
carmine
Involves immediate application
of stain to living cells following
SUPRAVITAL STAINING removal from living body
Examples: Neutral red, janus
green, trypan blue, nile blue,
thionine, toluidine blue
THE BEST VITAL DYE Neutral red
SUPRAVITAL STAIN Janus green
RECOMMENDED FOR
MITOCHONDRIA
HEMATOXYLIN AND EOSIN STAINING
• H&E staining
o Staining category → histological staining
o Staining method → regressive staining
• Hematoxylin – primary dye
o Basic dye
o Nuclear stain
o Nucleus → blue
• Eosin – counterstain
o Acid dye
o Cytoplasmic stain
o Cytoplasm → pink
PROCEDURE OF H&E STAINING
1. 2 changes of xylene
o For further deparaffinization
2. Descending grades of alcohol
o 90 – 80 – 70
o For hydration
3. Hematoxylin
o Primary/nuclear stain
4. Acid alcohol
o Decolorizer
5. Ammonia water
o Bluing agent
6. Eosin
o Counterstain/Cytoplasmic stain
7. Ascending concentrations of water
o For dehydration process
8. 2 changes of xylene
o For clearing prior to mounting
PUNDAVELA, N. 4
 Histopathology Techniques

TYPES OF HEMATOXYLIN
• HARRIS HEMATOXYLIN
o Form of hematoxylin used in exfoliative cytology and
for staining of sex chromosomes
o Pap’s
• MAYER’S HEMATOXYLIN
o Form of hematoxylin used for immunohistochemistry
• COPPER HEMATOXYLIN
o Form of hematoxylin used to demonstrate
spermatogenesis
• Component of dye responsible for coloring property:
o CHROMOPHORE
• Component of dye responsible for dyeing property:
(retaining the imparted color)
o AUXOCHROME
• Lysochrome dyes
o Dyes without auxochrome component
o Also called Oil Soluble dyes
o Used as Fat stains
o Examples: Sudan dyes
MOUNTING / COVERSLIPPING
• Prevents bleaching of sections
• Protect tissue from damage
• CANADA BALSAM
o Routinely used mordant
o Refractive index: 1.524
o Natural mountant
• BRUN’S FLUID
o Recommended mountant for mounting frozen sections
directly from water
• APATHY’S
o Mountant used for methylene blue stained nerve
preparations
• Refractive indices
o Glycerin jelly/Kaiser’s: 1.47
o Farrant’s/Gum Arabic: 1.43
o Apathy’s: 1.52
o DPX: 1.532
o XAM: 1.52
o Clarite: 1.544
• RINGING
o Process of sealing margins of coverslip to immobilize
coverslip, to prevent escape of mountant and to
prevent sticking of slides upon storage
o Used Durofix or Kronig cement
o Not a routine step, only optional

PUNDAVELA, N. 5