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HISTOPATHOLOGY
HISTOPATHOLOGY
LABORATORY SAFETY
• Risk management pertains to the process FIRST STEP!
of ensuring and maintaining personal as • Identify all electrical, mechanical and
well as environmental health and safety in biological hazards that can potentially
cause harm in the laboratory
the laboratory.
• The employer and the employee share
safety responsibility.
o Individual employee has an obligation to follow safe work practices and be attentive
to potential hazards
o Employer has the ultimate responsibility for safety and delegates authority for safe
operations to laboratory managers and supervisors.
FIRE HAZARD
CLASSIFICATION OF FIRES
Class of Fire Type of Hazards Type of Extinguisher
Ordinary combustibles: cloth, paper, rubbish, Water, dry, chemical and loaded
Type A
plastics, and wood steam
Flammable liquids: grease, gasoline, paints, and oil Dry chemical, carbon dioxide and
Type B
halon foam
Electrical equipment & motor switches Carbon dioxide, dry chemical, and
Type C
halon
Flammable metals: mercury, magnesium, sodium Metal X, sand or dry powder, should
Type D
and lithium be fought by firefighters only
Detonation (arsenal fire) Usually allowed to burn out and
Type E
nearby materials are protected
Grease, oils, fats Liquid designed to prevent splashing
Type K
and cool the fire

REMEMBER: Water (A), CO2 (BC), Dry Chemicals DEGREE OF HAZARDS: No SMS ex
(ABC) • 0 – No or minimal Hazard
• 1 – Slight Hazard
• Most common type of extinguisher: • 2 – Moderate Hazard
multipurpose Type ABC • 3 – Serious Hazard
• Flash Point – lowest temperature that • 4 – Extreme Hazard
produces ignitable vapor
WHAT TO DO WHEN FIRE IS DISCOVERED
o Flammable liquid: flash point below ✓ Rescue: rescue anyone in immediate
37.8°C danger
o Combustible liquid: flash point at or above ✓ Alarm: activate the institutional fire alarm
system
37.8°C ✓ Contain: close all doors to potentially
affected areas
CHEMICAL HAZARDS ✓ Extinguish/Evacuate: attempt to
extinguish the fire, if possible or
• If chemical spill occurs, first step is to evacuate, closing the door
assist/evacuate personnel
• When skin contact occurs, the best first aid is HOW TO OPERATE FIRE EXTINGUISHER
to flush the area with large amounts of ✓ Pull the pin
water for at least 15 minutes, then seek ✓ Aim at base of fire
✓ Squeeze handle/trigger
medical attention ✓ Sweep nozzle side to side

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HISTOPATHOLOGY
• Always add acid to water → to
avoid splashing or explosions to occur
• Permissible Exposure Limits
(PELs), Threshold Limit Values
(TVLs), or Occupational Exposure
Limits (OELs) – are some of the terms
used to define the maximum allowable
airborne concentration of a chemical
(vapor, fume, or dust) to which a worker
may be exposed
MSDS (Material Safety Data Sheet) – should
always be available that contains a chemical’s
information

• Storage of chemicals above eye level must

be avoided.
o Dangerous liquids are best stored below countertop height to minimize the risk of
bodily exposure in case a bottle is dropped and broken
• Separate acids and bases
CLASSFICATION EXAMPLE EFFECT
Visible destruction of human tissue on
Glacial acetic acid, Hydrochloric
Corrosives contact; can cause injury on inhalation
Acid, Sodium Hydroxide
or contact
Interfere with metabolic processes
Toxic Substances Cyanides, sulfides when ingested, inhales or absorbed
through skin
Benzidine, formaldehyde dyes
such as auramine, basic fuchsin,
Carcinogens and any dye derived from Capable of causing cancer
benzidine (including Congo Red
& Diaminobenzidine)
Benzene, lead, mercury, Mutagens – induce genetic mutations;
Mutagens &Teratogens
radioactive material, toluene Teratogens – cause defects in embryo
Ignitable Acetone, alcohols, ether, xylene May cause fire
May cause explosions:
• Ether forms explosive on exposure to
air or light; store in explosion-proof
refrigerator,
• Perchloric acid may react explosively
Ether, perchloric acid, picric acid with organic compounds; separate
Reactive
(with preservative), sodium azide from other acids.
• Picric acid is shock sensitive when
dehydrated; more powerful than TNT
• Sodium azide solutions can form
explosive lead or copper azides in
drains
Cause allergic reactions in some
Sensitizers Chlorine, alkalis, some solvents. exposed workers, not just in
hypersensitive individuals

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HISTOPATHOLOGY

BIOLOGICAL HAZARDS – Prevention:

A. Personal Protective Equipment
a. The order or wearing PPE is: Gown → Mask → Gloves
b. PPE is removed in the order of most contaminated first: Gloves → Gown → Mask
B. Proper Handwashing
Wash with detergent soap or
Alcohol-based hand rubs:
antimicrobial soap:
1. When hands are visibly soiled 1. Before having direct contact
2. Before eating 2. After contact with patient’s intact skin, fomites,
3. After using restroom samples (if not visibly soiled)
3. Before wearing and after removing gloves
4. In hands are not visibly soiled
HANDWASHING PROCEDURE:
1. Wet hands with warm water
2. Apply antimicrobial soap

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HISTOPATHOLOGY
3. Rub to form a lather, create friction and loosen debris
4. Thoroughly clean all parts of hands up to wrist for at least 15 seconds
5. Rinse in a downward position
6. Dry with a paper towel
7. Turn off faucets with clean paper towel
C. Disinfection
a. Use 1:5 or 1:10 dilution of sodium hypochlorite with contact time of at least 20 minutes
i. Hepatitis B Virus: inactive at 10-minutes
ii. Human Immunodeficiency Virus: inactivated at 2-minutes
ELECTRICAL HAZARDS
• Before attempting to repair any instrument → Turn the instrument off and unplug it
• Grounding
o Connecting of electrical equipment and wiring systems to the earth by a wire or other
conductor
o Purpose: reduce the risk of serious electric shock from current leaking into uninsulated
metal parts of electrical equipment
o Grounding is also done by using: TREE PRONGED PLUGS
• If electrical shock occurs → NEVER TOUCH THE PERSON OR EQUIPMENT
INVOLVED instead:
o Turn off circuit breaker or unplug equipment
o Move the equipment using non-conductive glass or wood object
ERGONOMIC/PHYSICAL HAZARDS
GARBAGE BAG COLOR
• Laboratory-associated ergonomic risks • Black – non-infectious dry waste
• Green – non-infectious wet waste
are the same as those found in the office (kitchen, dietary etc.); biodegradable
and general industry. These risk factors • Yellow – infectious and pathological
include awkward or sustained postures, waste
• Yellow with Black Band – chemical waste
highly repetitive movements, excessive including those with heavy metals
force or strain, contact stresses, and • Orange – radioactive waste (PNRI –
vibration Philippine Nuclear Research Institute)
• Red – Sharps and pressurized containers

EMBRYOLOGY & HISTOLOGY

• Fertilized egg divides into three germ layers: Ectoderm, Mesoderm, Endoderm
• Histos – Greek for web or tissue; Logia – Greek for branch of learning
• Cells → Tissues → Organs → Organ System → Organism

Epithelial Tissues – continuous sheets of cells (one or more layers thick); can be covering
or glandular epithelia.

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HISTOPATHOLOGY
Number of Layers:
1. Simple – single layer of cells
2. Stratified – multiple layers
3. Pseudostratified – falsely stratified,
rests on same basement membrane
Cell Shape:
1. Squamous – thin cells
2. Cuboidal – cells width and thickness
roughly similar
3. Columnar – taller than they are wide
4. Transitional – dome-shaped if
contracted flattened when stretched
also called urothelium
SUMMARY OF EPITHELIAL TISSUE TYPES
Simple
Squamous Stratified
Non-Keratinized
Stratified Keratinized
Simple
Cuboidal
Stratified
Simple
Pseudo-stratified, ciliated Respiratory tract including nose and sinuses
Columnar
Simple, ciliated
Transitional/urothelial
Glandular Epithelia
• Organized collection of secretory
epithelial cells
o Exocrine – has ducts to discharge
secretions into epithelial surfaces
(e.g. sweat, prostate, gastric
glands, salivary glands)
Covering Epithelia
• Classified also based on: Number of
Layers, Cell Shape, and Apical/Free Surface
Specialization
Apical/Free Surface Specialization:
a. Microvilli – increase surface area for absorption
b. Cilia – brush out dust in lungs
c. Keratin – prevention of destruction and drying
d. Stereocilia – smaller and longer version of
microvilli for absorption (epididymis)
Endothelium, mesothelium, lung alveoli, Bowman’s
capsule, thin loop og Henle
Lining oral cavity, epiglottis, esophagus, anus, cervix,
vagina, vulva, glans penis, cornea
Epidermis of skin
Collecting tubule of kidney, rete testis, small ducts or
exocrine glands, surface of ovary
Larger ducts of exocrine glands
Gallbladder, collecting ducts of kidney, endocervix
Fallopian tubes/oviducts/uterine tubes – pushes the egg
cells
Lower urinary tracts (renal pelvis, ureters, bladder and
urethra)
Exocrine Method of Secretion:
• Merocrine – via exocytosis, no part of cells is lost;
most common (sweat, salivary glands)
• Apocrine – apical part of cell is lost (mammary
glands)
• Holocrine – breakdown of entire cell during
secretions (sebaceous glands)
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HISTOPATHOLOGY
Endocrine Exocrine o Endocrine – ductless, highly
Pancreas Insulin, Glucagon Lipase, Amylase vascular – secretions directly into
Kidney Erythropoietin Urine
Liver IGF, THPO Enzymes
bloodstream (e.g. thyroid, adrenals)
Gonads Testosterone, Estrogen Sperm, Egg Cell

CONNECTIVE TISSUES – space-filling, supports SPECIALIZED CONNECTIVE TISSUE:

and forms body’s framework • Regular Connective Tissue
• Adipose Tissue
• Collagen – most abundant (Van Gieson’s – • Cartilage
pink deep red; Masson Trichrome; Mallory’s a. Hyaline
Aniline Blue; Azocarmine) b. Elastic
c. Fibrocartilage
• Reticular – contains mostly type III collagen • Bone
(Argyrophilic – silver impregnation) • Blood
• Elastic – composed of elastin, allows
stretching (Weigert’s Elastic Tissue stain, Taenzer-Unna Orcein Method; Verhoeff’s stain;
Gomori’s Aldehyde-Fuchsin stain; Krajian’s method)

Pathologic Changes & Deposits Found in Connective Tissues

Mallory’s PTAH
Insoluble fibrillar protein resulting from polymerization or
Fibrin Lendrum’s MSB: Early fibrin
enzymatic coagulation of plasma globulin and fibrinogen
(yellow); Late fibrin (blue)
Identical in staining to fibrin but is a mixture of exudate
and altered cytoplasmic constituents; seen in collagen
Fibrinoid Same with Fibrin
diseases, hypersensitivity, SLE, Rheumatic Heart
Disease
Seen id degenerated collagen, hypertension. Atheroma,
Hyaline Period Acid Schiff
and diabetic kidney
Seen in connective tissue, cells, kidney, spleen, Gram’s Iodine; Congo Red –
adrenals, lymph nodes. Pancreas during chronic GOLD STANDARD;
Amyloid
suppurative and inflammatory conditions such as Metachromatic staining; induced
Tuberculosis, Leprosy, and Osteomyelitis fluorescence with thioflavin

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HISTOPATHOLOGY
MUSCLE TISSUE – specialized for producing movement of body/organs
• Skeletal – straited, voluntary, found attached to bones
• Smooth – involuntary, found in visceral organs, GI tract, blood vessels and exocrine
glands
• Cardiac – straited, involuntary, found in the heart

NERVOUS TISSUE – neurons form the parenchyma, ad diverse glial cells form the stroma
• Anatomically organized into: Central Nervous System and Peripheral Nervous System
• Functionally organized into: Somatic
NERVOUS TISSUE STAINS
Nervous System and Anatomical Central Nervous System Tissue
Nervous System ➢ Bielschowsky’s Technique – neurons,
axons, neurofibrils
➢ Bodian’s Stain – nerve fibers and nerve
endings
HISTOPATHOLOGIC TECHNIQUES
➢ Siever-Munger Technique – neural tissue
• Histopathology – study of abnormal or ➢ Cresyl Fast Violet – Nissl Bodies
Myelin Sheath
diseased tissue states; the microscopic ➢ Weigert-Pal Technique
examination of biological tissues to ➢ Kluver and Barrera Luxol Fast Blue Stain
observe the appearance of abnormal ➢ Weil’s Method
cells and tissues in very fine detail Astrocytes
➢ Cajal’s Gold Sublimate
• Pathology – study of disease, of Greek ➢ Modified PTAH
origin: pathos = disease/suffering & ➢ Modified Holzer’s Method
logos = study
QUALITY ASSURANCE IN HISTOPATHOLOGY
PRE-ANALYSIS ANALYSIS POST-ANALYSIS
• Specimen fixation • Block labeling • Turn-around time
• Specimen delivery • Slide labeling o Surgical pathology and
• Adequacy of clinical history • Loss of specimen cytopathology: within 24-
• Specimen rejection • Specimens that require hours
grossing only o Autopsy: 7days
o Prepuce/foreskin

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HISTOPATHOLOGY
o Vaginal mucosa; o Frozen section: 5 to 15-
Scars/cicatrix minutes
o Foreign bodies (e.g. o Written reports
bullets, ortho/medical • Signatories
devices, silicone o Request form - physician
implants) o Result form - pathologist
o Hair, fingernails and • Storage
toenails removed from o Specimen: 6-months to
cosmetic reasons 1-year
o Lens cataracts; Nasal o Tissue Blocks: 3-years to
septum (rhinoplasty) 10-years
o Calculi, stones o Slides: indefinite
o Eyelid; Placenta from o Autopsy report
NSD; Teeth; Fetuses o Surgical pathology or
cytopathology report
• Pap’s smear – screening test for cervical
cancer PERSONS OF HISTORICAL SIGNIFICANCE
➢ Hippocrates – father of medicine
• Uses of Histopathology: ➢ Rudolf Virchow – father of modern
o Biopsy – most common; tissue from pathology
living person ➢ Giovanni Battista Morgagni – father of
modern anatomical pathology
o Forensic Pathology – autopsy;
➢ George Papanicolau – inventor of the
tissue from dead person "Pap smear"
o Histopalaeopathology – ancient ➢ William Osier – father of modern
diseases medicine
➢ Paracelsus – father of toxicology
General Workflow of Histopathology ➢ Cornelius Celsus – De Medicina
Section ➢ Julius Cohnheim – founder of ex-
perimental pathology
➢ Receiving of specimen → Accessioning ➢ Andreus Vesalius – founder of modern
human anatomy
of specimen → Gross examination →
➢ Marcello Malphigi – father of
Fixation → Dehydration → Clearing → microscopical anatomy, histology,
Impregnation → Embedding → physiology and embryology
Trimming → Section Cutting/Microtomy
→ Staining → Mounting & Labeling

ADDITIONAL NOTES:
➢ Accessioning – giving a pathologic code
or a unique code for sample
➢ Gross Examination – measures the
shape, weight, and size.
➢ Labeling: Procedure; Year; #
➢ Autotechnicon – tissue processor; fix,
dehydrate, clear, and impregnate
➢ Tissuetech System – embedding
machine
➢ Course Trimming – knife; Fine
Trimming – microtomy
➢ Tissue Block Shape – Truncated Pyramid
MNEMONICS: R-A-G-F-D-C-I-E-T-S-S-M

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HISTOPATHOLOGY
Surgical Procedures for Obtaining Histopathology Specimens
• Fine Needle Aspiration – simplest, least invasive, simply removes cells in area of
abnormality; not always adequate to obtain diagnosis, depending on area of biopsy.
• Core Needle Biopsy – removes not only cells, but also a small amount of the surrounding
tissue
• Incisional Biopsy – Takes out even more surrounding tissue; takes out some of the
abnormality, but not all
• Excisional/Surgical Biopsy – removes entire area in question
• Punch Biopsy – considered the primary technique for obtaining diagnostic full-thickness
skin specimens; circular blade is rotated from epidermis to subcutaneous fat (3-4 mm
cylindrical core of tissue).
• Shave Biopsy – small fragments of tissue are “shaved” from a surface (usually skin).
• Curettings – tissue is scooped or spooned to remove tissue or growths from body cavity
(e.g. endometrium or cervical canal).
FRESH TISSUE EXAMINATION
METHODS
Teasing/Dissociation Fresh tissue → immersed in isotonic saline/ringer’s solution → dissected or separated
→ examined either stained (differential dyes) or unstained (microscope: phase
contrast/brightfield microscope)
Crushing/Squash <1 mm in diameter → placed in a slide and forcibly compressed using another slide or
Preparation cover glass → may be stained by vital stains
Smear Preparation Cell materials are spread lightly over a slide by means of a wire loop/applicator
stick/another slide
a. Streaking Material is gently applied in a direct or zigzag line throughout slide using applicator stick
or platinum loop
b. Spreading Gentle spreading of material into moderately thick film by teasing mucous strands apart
using applicator stick; for fresh sputum, bronchial aspirates and thick mucoid secretions
c. Pull-apart For thick secretions such as serous fluids, concentrated sputum, enzymatic lavage
samples from GI tract and blood smears
Touch Preparation or Surface of a freshly cut piece of tissue is brought into contact and pressed on the surface
Impression Smear of a clean glass slide; has an advantage of cells may be examined without destroying
their intercellular relationship
Frozen Section – normally used when a rapid diagnosis of tissue is required
Applications: Methods of Freezing:
1. Rapid pathologic diagnosis during surgery • Liquid nitrogen – most rapid
(intraoperative) • Isopentane cooled by liquid nitrogen
2. Enzyme histochemistry • CO2 gay
3. Demonstration of soluble substances such as lipid • Aerosol sprays
and carbohydrates Methods of Sectioning: 10 to 15 µm sections
4. Immunofluorescence and immunohistochemistry
staining
5. Some specialized silver stains, particularly in
neuropathology
Useful for lipid and nervous tissues
Can be fixed or unfixed
Cold Knife Procedure Cryostat Procedure (Cold Microtome)
• CO2 gas for freezing • Cryostat – refrigerated chamber housing a
• Almost any microtome can be used modified microtome, usually a rotary microtome
• Optimum sectioning conditions: • Optimum working temp. = -18 to -20°C
o Knife: -40 to -60°C o May also vary to type of tissue being cut

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HISTOPATHOLOGY
o Tissue = 5 to 10°C o E.g. breast, skin, and fatty tissues must be
o Environment = 0 to -10°C kept in -20 to -25°C or they will be too soft to
cut.
o Lymph nodes, spleen, brain, liver, and
uterine curettings cut better if the
temperature in higher (-10°C)

Special Frozen Tissue Techniques for MOUNTING MEDIA

Histochemical Studies ➢ Water
➢ 20 to 30% Bovine Albumin
• Freeze-Drying – rapid freezing or quenching of ➢ Von Apathy’s Gum Syrup
fresh tissue at -160°C then removing ice water ➢ O.C.T – best (synthetic water-
molecules (desiccation) by physical transfer of soluble glycols and resins)
STAINING FROZEN SECTIONS
frozen tissue block in a vacuum of higher
➢ H&E – most common
temperature, -40°C (sublimation) without use of ➢ Thionine
any chemical fixative. ➢ Polychrome Methylene Blue
• Freeze Substitution – same as freeze-drying in ➢ Alcoholic Pinacyanol Method
NOTE: Progressive H&E – frozen
rapid freezing step but does not use vacuum in sections; Regressive H&E – routine
desiccation. Instead, fixes tissue in Rossman’s paraffin sections
formula or 1% acetone and dehydrated in absolute
alcohol.
GROSS EXAMINATION
• Also called as Macroscopic Examination
GROSS EXAMINATION
• The process by which pathology specimens are PRODUCTS:
inspected with the bare eye to obtain diagnostic ✓ Gross description
✓ Tissue block
information. This is typically performed by a
Pathologist/Histopathologist
MATERIALS FOR GROSS EXAM
Criteria for Rejection ✓ Cutting tools: surgical blades,
knife
a. Discrepancies between the requisition and ✓ Tissue Cassettes
o 3 x 2.5 x 0.4 cm
specimen labels
✓ Weighing Scale
b. No label; mislabeled ✓ Multicolor Ink Set
c. Leaking specimen containers o inking of the margins
✓ Forceps, etc
d. No clinical data or history
e. Inappropriately identified specimens
NOTE:
CAN WE USE CAMERA IN ✓ Measurements are usually given in centimeters
GROSS EXAMINATION? YES unless the specimen is very small in which millimeters
ca be used.
✓ Endometrial and prostatic tissue should be measured by aggregate pieces in volume
✓ Most specimens from solid tissues are cut in the
form of pieces measuring 10 to 15 mm on the slides BREAD LOAFING – knife
and 2 to 3 mm in thickness technique for a liver specimen
✓ Discrete areas of calcification or ossification should
be taken out and should be decalcified in Nitric Acid
✓ Small fragments of tissue must be wrapped in thin paper

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HISTOPATHOLOGY
FIXATION
PRINCIPLE OF FIXATION: The • First and most critical step in tissue processing
fixative brings about cross linking of • Ideal time to perfume fixation – within 20 to 30-
protein which produces denaturation minutes after loss of blood supply
or coagulation of proteins so that the
semifluid state is converted into • It is a MAIN FACTORS INVOVLED IN
semisolid state; so that it maintains complex series FIXATION
everything in vivo in relation to each 1. pH – 6 to 8 (acidic + basic
of chemical
other. Thus, semisolid state facilitates [phosphate and carbonate])
easy manipulation of tissue. events which 2. Temperature
brings about a. Surgical specimens –
changes in the various chemical constituents of room temperature
b. Electron microscopy &
cell like hardening, however the cell morphology some histochemistry – 0
of cell like hardening, however the cell to 4°C
morphology and structural detail is preserved. 3. Thickness of Section – no
thicker than 3 to 5 mm
• Primary aim: preserve morphologic and 4. Osmolality – slightly
chemical integrity of cell in as life-like manner as hypertonic solutions
5. Concentration
possible
6. Duration of fixation
• Secondary aim: harden and protect tissue from
trauma of further handling
• Most important reaction in fixation: denaturation/coagulation of protein (fixative
have property of forming cross-links between proteins)
Benefits of Fixation
• Allows thin sectioning of tissue by hardening PRACTICAL CONSIDERATIONS
tissue OF FIXATION
• Prevents autolysis and inactivates infectious • Speed
• Penetration – formalin
agents (except prion diseases) diffuses at ~1 mm/hr
• Improves cell avidity for special stains • Volume
o Fixative should be 10 to
Mechanisms Involved in Fixation: 20x the tissue volume
o Most common error in
• Additive Fixation – chemical constituent of histotechnology:
fixative is taken in and becomes part of the tissue insufficient ratio of tissue
volume to fixative volume
• Non-additive Fixation – agent is NOT taken in • Duration of fixation
but fixes tissue by
RATIO OF TISSUE & FIXATIVE
▪ Routine – 1:20 removing bound water attached to hydrogen bonds of
▪ Museum – 1:50 certain groups within protein molecule
• Coagulant fixation – creating a network that
allows solutions to readily penetrate the interior of the tissue
• Non-coagulant Fixation – creating a gel that makes it difficult to penetrate by
subsequent solution
ADDITIONAL NOTES: Aims of Fixation
• Prevent autolysis (lysosomes release lysoenzyme) and putrefaction (bacterial
decomposition)

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HISTOPATHOLOGY
• Solidification – converts the normal semifluid TYPES OF FIXATIVE:
consistency of cells (gel) to an irreversible According to Composition
semisolid consistency (solid). o Simple Fixative – made up
only one component (picrate)
• Optical Differentiation – it alters to varying o Compound Fixative – two or
degrees the refractive indices of the various more fixative
According to Action
components of cells and tissues so that
o Microanatomical – for
unstained components are more easily general microscopic study of
visualized that when unfixed. tissue structure
o Cytological – specific part
• Effects in staining – certain fixative like and particular microscopic
formaldehyde intensifies the staining character of element of the cell itself
tissue especially with hematoxylin o Histochemical

TYPES OF FIXATIVES PURPOSE/USAGE

10% Formalin Routinely used
CNS tissue; port-mortem; histochem
10% Formol Saline
exam
Surgical; post-mortem and research
10 NBF
specimen
Formalin
Formol
Routine post-mortem tissue
Corrosive/Sublimate
Alcoholic
Sputum
Formalin/Gendre’s
Microanatomical Glutaraldehyde Enzyme histochem; electron microscopy
Small pieces of liver, spleen, connective
Zenker’s Solution
tissue and nuclei
Mercuric
Pituitary gland; bone marrow;
(Mercuric Zenker’s Formol
cytoplasmic granules
Chloride)
B5 Bone marrow
Heidenhain’s Susa Tumor biopsy of skin
Bouin’s Solution Embryos and pituitary
Picric (fix,
Brasil’s Picroformol Glycogen (excellent)
decal, stain)
Hollande’s Solution Primary fixative
Bouins Embryos and pituitary
Heidenhain’s Susa Tumor biopsy of skin
Chromosomes; lymph glands; urgent
Carnoy’s
Nuclear biopsies; brain tissue – rabies
Mucopolysaccharides; nuclear proteins;
Newcomer’s Fluid
Fuelgen stain
Flemming’s Nuclear structure
Cytological Flemming’s Most common
Kelly’s Pituitary gland
Formalin with Post
Routinely used
Chroming
Cytoplasmic
Regaurd’s Organelles
Degenerative process and tissue
Orth’s necrosis; Rickettsia and other bacteria;
myelin
CNS tissue; port-mortem; histochem
10% Formol Saline
exam
Absolute Ethanol Blood, tissue, nucleic acids
Histochemical Histochemical Enzymes; phosphatase, lipases brain
Acetone
specimen – rabies; Freezeq substitution
Mucopolysaccharides; nuclear proteins;
Newcomer’s Fluid
Fuelgen stain

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HISTOPATHOLOGY
OTHER FIXATIVES
FIXATIVE SPECIFIC TYPE NOTES
Chromic Acid Carbohydrates
Regaurd’s Organelles
Chromate Degenerative process and tissue necrosis Rickettsia
Orth’s
and other bacter; myelin
Potassium Dichromate Lipid, mitochondria
Lead 4% Lead Mucin and mucopolysaccharides
Glacial Acetic Acid Solidifies at 16°C; swells tissues, preserves nucleus, destroys cytoplasmic organelles
Methanol 100% Blood and Bone Marrow Smears (cause blindness)
Isopropanol 95% Touch preparation specimen
Ethanol Blood, tissue, and nucleic acids (Pap’s Smear)
Alcohol
Chromosomes, Lymph glands, Urgent biopsies, brain
Carnoy’s
tissue – rabies virus; most rapid fixative
Newcomer’s Mucopolysaccharides, nucleir proteins, Fuelgen stain
Osmic Tetroxide Flemming’s Nuclear structure, most common
(Osmic-Acid) Flemming without acetate Mitochondria; both cause conjunctivitis and blindness
Trochloroacetic Both acts as a fixative and decalcifying agent
Ice cold Acetone (– 5 to Enzymes: phosphatases, lipids, brain tissue – rabies;
Acetone
4°C) Freeze substitution
Bacteriologic smear (direct flaming)
Heat
Microwave Fixation 45 to 55°C

ADDITIONAL NOTES IN FIXATION:

• Secondary Fixation – process of placing an already fixed tissue in a second fixative in
order:
o To facilitate and improve the demonstration of particular substances.
o To make special staining techniques possible (with secondary fixative acting as a
mordant)
o To ensure further and complete hardening and preservation of tissues
• Post-chromatization – a form of secondary fixation in 2.5 to 3% potassium dichromate
acting as mordant for staining
• Wash out – process of removing excess fixative and artifacts
o Tap water – chromates from tissues fixed in Kelly’s, Zenker’s and Flemming’s
Solutions, Formalin and Osmic Acid
o 50 to 70% Alcohol – Picric Acid
o Alcoholic Iodine – Mercuric fixatives
• Crush Artifact – found in liver biopsies, associated with an intense eosinophilic staining
at the center of the tissue in H&E stained sections; due to partial coagulation of partially
fixed protein by ethanol or by incomplete wax impregnation.
• Hollow organs should be packed with cotton soaked in fixative or completely opened
before being immersed in adequate fixing solution
• Human brains may be suspended by a cord tied under the Circle of Willis to prevent
flattening
• Lendrum’s Method: to soften hard tissues (e.g. cervix, uterine, fibroids, hyperkeratotic
skin, fingernails, etc.) may be washed out in running water overnight and immersed in
4% aqueous phenol solution for 1 to 3 days.

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HISTOPATHOLOGY
DECALCIFICATION
• The process of removal of calcium from the tissues like bone and teeth and other
calcified tissues following fixation that makes them flexible and easier to cut.
RATIO OF TISSUE &
• More concentrated acid solutions decalcify bone more
DECALCIFYING AGENT rapidly but more harmful to the tissue
▪ Routine – 1:20 • Highly concentrated and greater amount of fluid will
increase the speed of the process.
• Heat will serve to hasten decalcification, but it also increases the damaging effects on
tissues
• The ideal time required for decalcifying tissue is 24 REQUIRED TEMPERATURES
to 48 hours ▪ 18 to 30°C – optimum
• Dense bone tissues usually require up to 14 days temperature (RT)
or longer to complete the process ▪ 37°C – impaired nuclear
staining of Van Gieson’s Stain
DECALCIFYING AGENTS: ACIEM Methods for collagen fibers
▪ 55°C – tissue will undergo
1. Dissolution of calcium by a dilute mineral acid complete digestion within 24
(most widely used decalcifying agent) to 28 hours
2. Using chelating agents – EDTA (most common
Grating Effect – unwanted
chelating agent; pH 7) to chelates calcium to form calcium deposits; resolve by
soluble salts performing surface decalcification
a. Forms (1) Versene – solid form and that uses 10% HCl for 1 hour.

Sequestrene – liquid form

EXTENT OF DECALCIFICATION – three
ways to measure extent of decalcification
• Physical/Mechanical – inaccurate,
unreliable; Flexibility Method
• X-Ray/Radiologic Method – very
expensive; most reliable, most ideal (e.g. tissue by use of electric current –
Kodak X-Omat or Faxitron – x-paper)
• Chemical Methods/Calcium Oxalate
Test – simple, reliable, recommended for
routine purpose
ACID DECALCIFYING AGENTS
STRONG ACIDS
Nitric Acid – most common, most rapid; over
exposure impairs nuclear staining
Perenyi’s Fluid – both tissue softener and
decalcifying agent
Phloroglucin Nitric Acid – most rapid
decalcifying agent
Hydrochloric Acid – surface decalcification;
recommended in small pieces of bones and teeth
3. Removal of calcium by used of dilute
mineral and along with ion exchange resin to
keep the decalcifying fluid free of calcium
4. Electrolytic removal of calcium ions from
electrophoresis
5. Microwave oven decalcification – faster
than the routine procedure
WEAK ACIDS
Formic Acids – both fixative and decalcifying
agent; best general decalcifying agent because of
less tissue distortion that can do to the tissue; from
ants
Trichloroacetic Acid – uses Heidenhain’s Susa
for tumor biopsy; good nuclear staining
Sulfurous Acid – very weak
Chromic Acid/Flemming’s Fluid – both fixative
and decalcifying agent; environmental toxin
Citrate Buffer Solution – pH 4.5; no tissue
distortion

14
HISTOPATHOLOGY
DEHYDRATION
• Aim of Dehydration: to remove fixative and water from TISSUE SOFTENER – for
the tissue and replacing them with dehydrating fluid unduly hard tissue that may
in preparation form impregnation. damage the microtome knives
• 4% Aqueous Phenol
• Definition: the process of removing intracellular and • Molliflex – foamy tissue
extracellular water from the tissue following fixation • 2% Hydrochloric Acid
• 1% Hydrochloric Acid in 70%
and prior to wax impregnation. Solution
• Drying vs. Dehydration: Drying – removal of water by • Perenyi’s Fluid
evaporation; Dehydration – removal of water by
RATIO OF TISSUE &
replacing by dehydrants DEHYDRATING AGENT
• Grades of Alcohol: Dehydration – increasing grades; ▪ Routine – 1:10
Sections to Alcohol – decreasing grades (rehydrate)
• Increasing strength – all the aqueous tissue fluid is GENERAL RULE: amount in
each stage should not be less
removed but with little disruption to the tissue due to than 10x the volume of the tissue
diffusion currents
o Routine – starts with 70% usually ethyl alcohol
o Embryonic Tissues – starts with 30% of ethyl alcohol
• 30% (delicate specimen) < 65% < 70% < 75% < 85% < 95% < 100% Alcohol
COMMONLY USED DEHYDRATING AGENTS

INDICATORS OF INCOMPLETE 1. Alcohol – most common used dehydrating agents

DEHYDRATIONS a. Ethanol – routine dehydration of tissues; ethyl
1. Anhydrous Copper Sulfate –
alcohol; Grain’s Alcohol – routinely used
white to blue if preserve of water
2. Xylene – turn milky b. Methanol – for blood and tissue films – cause
3. 4% Phenol – softener for hard
tissues
blindness; use as fixative and dehydrant; employed for
blood and tissue films; Wood’s Alcohol
c. Butanol – for plants and animals’ micro techniques
d. Industrial Methylated Spirit/Denaturated Alcohol – combination of ethanol and
small amount of methanol
e. Isopropyl Alcohol – 70% disinfectant and fresh tissue examination
2. Acetone – both fixative and dehydrating agent; utilized for urgent biopsies
3. Diethylene Dioxide (Dioxane) – both dehydrating and clearing agent
4. Cellosolve (Ethylene Glycol Monoethyl Ether/EGME) – BP: 156, 4°C

CLEARING/DEALCOHOLIZATION
• Definition: process of replacing the dehydrating
RATIO OF TISSUE &
fluid with a fluid that is miscible with both the CLEARANT
dehydrating fluid and the ▪ Routine – 1:20
impregnating/embedding medium.
OTHER CLEARING AGENTS:
• End Product: Translucent Tissue ▪ Oil of Bergamot
• All clearing agents can de-alcoholise but not all ▪ Oil of Wintergreen
▪ Trepenol
de-alcoholising agents can clear tissues ▪ Histoclear
▪ Coconut Oil
▪ Beach Palm Oil

15
HISTOPATHOLOGY
CLEARING AGENTS FOR ROUTINE USE
CLEARANT NOTES
Most common, most rapid; it turns milky when dehydration is incomplete
Xylene (Xylol)
(15 to 30/60-min)
Toluene Usually more expensive than xylene; alternative for xylene
Recommended for urgent biopsies; carcinogenic or may damage bone
Benzene
marrow resulting to Aplastic Anemia
Recommended for tough tissues (skin, decalcified tissues), nervous
Chloroform tissues, lymph nodes, embryos; toxic to liver after prolonged inhalation –
liver disease
Used for both paraffin and celloidin sections; recommended for central
Cedarwood Oil & Clove Oil
nervous system and cytological studies; expensive
Recommended for clearing embryos and very delicate specimens –
Aniline Oil
embryos and insects
Methyl (Benzoate/Salicylate) Double embedding technique
Citrus Fruits Oil Limonene
Trichloretane Petrol Substitute for xylene
Carbon Tetrachloride Tissue hardening
Gum Syrup and Glycerine Alternative

IMPREGNATION/INFILTRATION
• After clearing, tissue is submerged in 2 or more changes
RATIO OF TISSUE &
IMPREGNATING MEDIA of melted paraffin wax
▪ Routine – 1:25 • Process of replacing the
clearing agent with FOUR TYPES OF MEDIUM:
infiltrating or impregnating medium ▪ Paraffin
▪ Celloidin
• The medium used to infiltrate the tissue is usually the ▪ Gelatin
same medium used for embedding – purpose in filling ▪ Plastic
in tissue cavities/hole/space by using melted wax
PARAFFIN
TEMPERATURES FOR
PARAFFIN WAX
• Butchlii: first used the paraffin wax; simple, most
▪ 55 to 60°C – paraffin
common and best infiltrating/embedding medium oven temperature; 2 to 5°C
• Can be used twice and recycle once above the melting point of
the paraffin wax
• Not recommended for fatty tissue – frozen section
▪ 100 to 105°C –
• Methods of paraffin wax temperature to remove
impregnation/embedding excess water in the wax
o Manual processing – four changes of wax
at 15 minutes interval each
o Automatic processing – 2 to 3 changes using Autotechnicon
o Vacuum processing – fastest
• Factors Affecting Paraffin Wax Impregnation
o Nature if size of tissue
o Type of clearing agent used: benzene and xylene (easily removed) or
Chloroform and cedarwood oil (difficult to remove)
SUBSTITUTES FOR PARAFFIN WAX
Melting Point: 56 to 57°C; more elastic and resilient than paraffin; for large dense
Paraplast
tissue blocks such as bones and brain

16
HISTOPATHOLOGY
Embeddol Melting Point: 56 to 58°C; less brittle and less compressible than paraplast
Bioloid Melting Point: 56 to 58°C; recommended for embedding eyes
Tissue Mat A product of paraffin, containing rubber with the same property as paraplast
Melting Point: 46 to 48°C; harder than paraffin; not soluble in water; solublr in
Ester Wax 95% ethanol and other clearing agents; can be used for impregnation without prior
clearing of tissue (can omit clearing)
Melting Point: 38 to 42°C or 45 to 56°C; mostly Polyethylglycol (PEG); most
commonly used – Carbowax; soluble and miscible with water (hence does not
Water Soluble Waxes
require dehydration and clearing of the tissue); soluble for many enzyme
histochemical studies

CELLOIDIN – purified form of nitrocellulose METHODS FOR CELLOIDIN

▪ Wet – recommended for bones,
• Suitable for specimens with large hallow teeth, large brains and whole organ
cavities and dense tissues (bone and teeth), ▪ Dry – preferred for processing whole
eye sections
large tissue section of the whole embryo
• Low Viscosity Nitrocellulose (LVN) – another
form of celloidin; soluble in equal concentration of ether and alcohol with lower viscosity,
allowing it to be used in higher concentrations and still penetrate tissues rapidly.
GELATINE
• Rarely used when dehydration is to be avoided
• Usage: histochemistry and enzyme studies
• Embedding medium for delicate specimens and frozen sections because it prevents
fragmentation of tough and friable tissues when
frozen sections are cut THREE TYPES OF EPOXY
BRAND
• Water soluble and does not require dehydration and ▪ Bisphenol A (Alardite) –
clearing slowest
▪ Glycerol (Epon) – lower
PLASTIC/RESIN viscosity
▪ Cyclohexene Dioxide
• Classified into epoxy, polyester, acrylic (Spur) – very low viscosity;
infiltrate fastest
• Used in electron microscope

EMBEDDING/CASTING/BLOCKING
• the process by which the impregnated tissue is TYPES OF MOLDS
placed into a precisely arranged position in a mold ▪ Leuckhart’s L Pieces
containing a medium which is then allowed to ▪ Compound Embedding Units
▪ Plastic Embedding Blocks
solidify. ▪ Disposable Embedding Molds
• End Product: Tissue Block
• Orientation, process by which tissue is arrange in precise positions in the mold during
embedding on the microtome before cutting and on the slide before staining
• Temperature of melted paraffin used for embedding: 5 to 10°C above its melting point
• Solidify by using refrigerator or cold plate: -5°C
• The surface of the section to be cut should be placed parallel to the bottom of the mold
in which it is oriented.

17
HISTOPATHOLOGY
• Double Embedding – first embedded or fully infiltrated with a supporting medium such
as agar or nitrocellulose (celloidin) then infiltrated a second time with paraffin wax
o Used to facilitate cutting a large block of dense firm tissues like brain
TYPES OF MOLDS
1. Leuckhart’s L Pieces – these are two “L”
which are resting metal usually bras, which
are resting on a flat metal or glass plate.
2. Compound Embedding Units – consists of
square shaped brass or metal plates in a
series of interlocking plates
3. Plastic Embedding Blocks/Tissue Tek System – consist of a stainless-steel base
mold fitted with plastic embedding ring, which is later serves as the block holder during
cutting
4. Disposable Embedding Molds – Types
TISSUE CASSETTE DIMENSION:
a. Peel-away ▪ 3 x 2.5 x 0.4 cm
b. Plastic Ice Trays
c. Paper Boats
TRIMMING
• Removal of excess wax after embedding by using a knife or blade to expose tissue
surface in preparation for actual sectioning
• Ideal block shape: Truncated Pyramid or Cube-like
SECTION CUTTING/MICROTOMY
• From the Greek words: “mikos” meaning small MICROTOMY DISTANCE
and “temnein” meaning to cut ▪ 4 to 6 µm – Routine histologic
• The process by which a processed tissue is cut into procedure (rotary microtome
with biconcave knife)
uniformly thin slices (sections) to facilitate studies ▪ 10 to 15 µm – Frozen sections
under the microscope (cryostat); unfixed tissues
▪ 0.5 to 1 µm – electron
• Machine: Microtome microscope
• Father of Microtomy: Wilheim His Sr.
• Principle: Spring-balanced teeth or Pawl is brought into contact with and turns a ratchet
feed wheel connected to a micrometer screw, which in turn rotated, moving the tissue
block at a predetermined distance towards the knife for cutting sections at uniform
thickness.
• Three essential parts: Block holder, knife carrier and knife, Pawl, ratchet feed wheel
and adjust
MICROTOME
TYPE INVENTOR YEAR TISSUE SECTION NOTES
Rocking Simplest; disadvantages: restriction in
Padwell Trefall
Microtome In large blocks paraffin sizes; difficulty of reorienting the block (10
(1881)
(Cambrige) to 12 µm)
Rotary Routinely use and most common;
Charles Minot Paraffin embedded
Microtome mechanically or electrically driven; has
(1885 -86) section
(Minot) flywheel

18
HISTOPATHOLOGY
Most dangerous due to movable expose
Sliding
Celloidin embedded knife; Two Types (1) Base-Sledge –
Microtome Adams (1789)
tissue moving: block holder; (2) Standard –
(Adams)
moving: knife; most dangerous
Freezing John Thomas Provided with carbon dioxide supply for
Frozen section
Microtome Queckett (1848) freezing; needs table top
Temperature: -5 to -30°C (-18 to -20°C); a
refrigerated cabinet in which a modified
James Dewar
Cryostat (Cold) Frozen section microtome (rotary) is housed; all the
(1897)
controls to the microtome are operated
from the outside the cabinet
Section for electron Cut tissue section as thin as 0.5 to 1 µm;
Ultrathin
microscopy (gelatin) the knife used consists mainly of broken
Microtome
embedded tissue plate glass fragments
Disadvantage: sections are liable to
Vibrotome Unfixed, unfrozen
disintegrate
MICROTOME KNIVES NOTES
Sharpest but delicate; one side flat, other concave; Less concave –
Plane Concave (Celloidin; 25µm)
celloidin (sliding); More concave – paraffin (rotary/rocking)
Biconcave (Paraffin; 120µm) Prone to vibration; Both sides concave
Standard knife; most commonly used in all types of microtome; both
Plane Concave (Frozen; 100µm)
sides straight/flat
HONING – hard sharpening STROPPING – polishing
➢ Removal of gross nicks ➢ Removal of burr
➢ Direction: Edges first, heel to hoe ➢ Direction: Edges last, toe to heel
➢ 10 to 20 strokes ➢ 40 to 120 strokes
➢ Purpose: to remove irregularities from the knife ➢ Purpose: to polish and sharpen the cutting edge
➢ Instrument: paddle strop made of horse leather

MAINTANACE OF MICROTOME
• Three types of Honing Stone (8x3.5/3in):
▪ Accumulated paraffin and small (1) Belgium Yellow – best result, (2) Arkansas –
pieces of tissues must be brushed
more polishing effect, and (3) Fine carborundum
away by soft brush (Camel
Hair/Squirrel Hair)
– for badly nicked
▪ After drying, wiped with xylene • Clearance Angle – angle formed between
▪ Movable parts should be oiled the cutting facet presenting to the block and the
▪ Covered when not in use
▪ Inspected at least once a year surface of the block, normally 0 to 15°
• Bevel Angle/Cutting Facet – angle
formed between the cutting edges, normally 27 to 37°
• Wedge Angle – angle formed by the sides of the wedge knife, normally 15°
• Other Cutting Tools: (1) disposable blade, (2), glass knives - 40x2.5 cm (3) diamond
knives – good cutting edge [2 to 3µm]
OTHER EQUIPTMENT NOTES
Water/Floatout bath Purpose to flatten out the ribbon; temperature of the water: 5 to 10°C
below melting point of paraffin; reduces surface tension: detergent
alcohol; less than 30 seconds
Drying Oven Hot Plate Drying tissue section on slides and for heat fixation
Forceps & Squirrel Hair Brush Use for handling tissue sections; remove folds and creases on sections
Clean Slides Dimension: 76 x 25 x 1to1.2 mm
Slide Rack

19
HISTOPATHOLOGY
• Adhesives – a substance which can be
ADVANTAGE OF ADHESIVES
smeared on the slides so that tissue sections ▪ Prone to bacterial or fungal growth
adhere well in the slides o Thymol – prevents fungal
o Mayer’s egg Albumin – best growth
o Sucrose – prevent cracking
o Plasma and higher refractive index
o Dried Albumin
o 1% Gelatin HEAT FIXATION
o Starch Paste • Oven: 56 to 60°C
• Hot Plate: 45 to 55°C
o Poly-L-lysine – immunochemistry • Blower: 50 to 55°C
o APES (3-Aminopropyl) • Direct Heating: emergency
triethoxysilane • Incubator: 37°C Overnight for
Nerve Tissue

STAINING
• Application of dyes on sections to
DYE-TO-TISSUE MECHANISMS SUMMARY
visualize the architectural pattern of 1. Coulombic Interactions – acid and
tissues and cells. basic dyes and other ionic reagents
including inorganic salts
DYE-TO-TISSUE MECHANISM – tissue will 2. Van der Waals’ Forces – elastic fiber
bind dyes by one of the following mechanisms: stains, and final reactions products in
enzyme histochemistry
1. Coulomb Force/Electrostatic Force 3. Hydrogen Bonding – staining of
glycogen by carmines and collagen by
– majority of tissue-dye reactions Sirius Red (Congo Red, Weigert-Type
a. e.g. Neutral red, light green Resorcinol Dye)
b. Opposite charges attract: 4. Covalent Bonding – Feulgen, PAS,
Mercury Orange for thiols
i. Nucleus (acidic) – (eosin) – 5. Electrostatic – majority of tissue-dye
cytoplasm; cationic; positive reaction, include Neutral Red and Light
charge Green
6. Physical Staining – Sudan dyes
ii. Cytoplasm (basic) – 7. Natural Affinity – Janus green
hematoxylin; anionic dye;
negative charge. Oil Red O – a lysochrome dye that is not a
2. Van der Waals – the partially negative true stain
portion of one of the polar molecules is
attracted to the partially positive portion of the second polar molecule; WEAKEST; e.g.
Orcein
3. Hydrogen Bonding – dye-tissue attraction arising when a hydrogen atom lies
between two electronegative atoms; e.g. Congo Red, Carmine (glycogen).
4. Covalent Bonding – polar covalent bonds between metal ions and mordant dyes; e.g.
Period Acid Schiff; Feulgen;
a. Covalent bond = nonmetal + nonmetal
b. Ionic bond = nonmetal + metal
5. Hydrophobic Effect/Sudanophilia: property of tissue to be stained with oil-soluble
dyes, regardless of the type of the dye used, due to the essential lipid nature; e.g.
Sudan Dyes – lysochromes
a. Sudan Black B – most sensitive; phospholipids, Neutral fats – black
b. Sudan IV – a.k.a. Scharlach R; Triglycerides – intense red
c. Sudan III – 1st dye introduced in histochemistry

20
HISTOPATHOLOGY
METHODS OF STAINIG
According to Presence of Mordant
1. Direct – staining processed by which sections are stained with simple aqueous
alcoholic solutions dye; without mordant, only one dye is used; use of cytoplasmic
stains; e.g. Methylene Blue, Eosin
2. Indirect – action of the dye is intensified by a mordant; Mordant (link/bridge between
tissue and dye) versus Accentuator (hasten the staining process)
According to the Presence of Differentiator/Decolorizers
1. Progressive – frozen section; without
decolorizer or washing step NOTE: Counterstaining – application of
different color or stain to prove counterstain or
2. Regressive – biopsy; with decolorizer background.
or with washing step
According to Resultant Color
1. Orthochromatic – color of dye is same with the tissue
2. Metachromatic – color of dyes is
PRINCIPLE: Metallic Ions (Silver Nitrate/Gold
different with the tissue Chlorite) is reduced to black precipitate on the
3. Metallic Impregnation – specific surface of the tissue.
tissue elements are demonstrated by
colorless solutions of metallic salts; never exposed to sunlight to avoid EXPLOSION
According to Method Application to the Living Cell/Vital Cell
1. Intravital – injections of dye to the animal body; e.g. Lithium, India Ink, Carmine
2. Supravital – the stain applied onto cells after removal from the animal body
a. NJTNTT – Neutral Red (best vital dye), Janus Green (mitochondria), Trypan
Blue, Nile Blue, Thionine, Toluidine Blue.
NATURAL VERSUS SYNTHETIC DYES
• Natural Dyes – obtained from plants and animals
1. Hematoxylin – derived from Mexican tree heartwood (Hematoxylin
campechianum).
2. Cochineal dyes – dye extracted from the female cochineal bug (Coccus cati);
treated with aluminum to produce dye = Carmine
3. Orcein – vegetable dye extracted from lichens which are normally colorless –
treated with ammonia and exposed to air to produce blue or violet color; excellent
stain for elastic fibers
4. Saffron – rusty/orange red
• Synthetic Dyes – coal tar dyes; derived
from hydro-carbon benzene, collectively
as Aniline Dyes
Three Components of Dye
1. Chromophore – color bearer
2. Chromogens – carrier; colorless in nature;
oxidant to produce

21
HISTOPATHOLOGY
3. Auxo chrome – anchor color; increaser that
form salt linkages between the tissue and dye;
used for stabilizer
Hematoxylin & Eosin Staining:
I: Hematoxylin
• Hematin is oxidized to Hematoxylin – a
natural dye derived from extraction from the
heartwood of the Mexican Tree known as
Hematoxylin campeche from Campeche,
Mexico.
• Jamaica Wateyer: first person to use
Hematoxylin in Histology
Ripening/Oxidation
• May be done by exposing the substrate to air or
sunlight (slow).
• May be done by adding oxidizing agents such
as: Hydrogen Peroxide, HgCl2 (ripening agent of
Harris Hematoxylin), KMnO4, Sodium Perborate,
and Sodium Iodate (ripening agent of Ehrlich’s
Hematoxylin)
A. Aluminum Hematoxylin – routinely used in H &
E Staining; mordant: potash aluminum sulfate;
produce good nuclear staining color: Red and
treated with Alkaline Solution turns into Blue THREE FORMS OF EOSIN:
B. Iron Hematoxylin – iron salts are used as
oxidants and mordants
C. Tungsten Hematoxylin – mordant: tungsten
D. Copper Hematoxylin – used for study of
spermatogenesis: spermatogonia – spermatid –
sperm cell
E. Molybdenum Hematoxylin – Thomas Hematoxylin: used for collagen and endocrine
cell granules
F. Lead Hematoxylin – Solicia Hematoxylin: used for endocrine cell granules
ALUMINUM HEMATOXYLIN:
• Ehrlich’s – slowly ripened, natural or sodium
iodate; glycine
• Delafield’s – slowly ripened
• Harris – mercuric oxide, sex chromosome
• Gill’s – sodium iodate, mucins
• Mayer’s – sodium iodate, chloral hydrate;
preservative
• Coles – alcoholic iodine
• Carrazis – potassium iodate
IRON HEMATOXYLIN:
• Weigert’s – feris chloride; combination with
Van Gieson’s stain, can demonstrate
connective tissue elements and Entamoeba
histolytica in sections
• Standard Iron Hematoxylin – for
nucleus/connective tissue fibers; Van
Gieson’s Stain – good for demonstrating
collagen
• Heidenhain’s – ferric ammonium sulfate; for
mitochondria, muscle striations, chromatin,
and myelin
• Verhoeffs – ferric chloride, photomicrography
• Lozey – ferric ammonium sulphate, myelin
TUNGSTENHEMATOXYLIN:
• Mallory PTAH (Phosphotungstic Acid
Hematoxylin) – to ripen: stand in the light for
several weeks or use potassium for
immediate ripening; for staining muscle
striations, fibrin and glial fibers
• Eosin Y (Yellowish) – most commonly used
• Eosin/Erythrosin B (Bluish) – deeper red
color
• Ethyl Eosin – eosin S – stock alcoholic
solution

HEAT FIXATION:
• Oven – 50 to 60°C for 2-Hours
• Hot Plate – 45 to 55°C for 30 to 45-
Minutes
• Blower – 50 to 55°C for 20 to 30-Minutes
• Direct Heating – emergency
• Incubator – 37°C Overnight for Nervous
Tissues
II: Eosin
• Routinely used dye in histopathology as a
counterstain after hematoxylin; with thymol,
to prevent fungal growth.
• Red acid dye used as counter stain after
hematoxylin and before methylene blue

22
HISTOPATHOLOGY
ROUTINE H&E STAINING TECHNIQUE
STEPS NOTES H&E RESULTS:
1. Xylol (2 Changes) Deparaffinization • Nuclei – blue to blue
2. Descending grade of Alcohol Rehydration black
3. Water • Karyosome – dark
4. Stain with Harris/Ehrlich’s/Delafield’s Nuclear Staining blue
5. Rinse slides in tap water • Calcium & Calcified
6. Acid Alcohol Differentiation Bone – purplish blue
7. Ammonia Water Bluing • Muscle Fibers – deep
8. Wash well in running tap water pink
9. Stain with Eosin Y Cytoplasmic Staining • Cytoplasm, CHON in
10. Ascending grade of alcohol Dehydration edema fluid – pale
11. Xylol/Xylene Clearing pink
12. Mount and label
PAP’S SMEAR STAINING
STEPS NOTES
1. Fix with 95% ethanol 15-seconds
2. Stain with Harris Hematoxylin 6-minutes
3. Acid Alcohol 0.5%HCl for 1 to 2 quick dips
4. Bluing step 0.1%Ammoniated Water
5. Stain with OG-6 Orange green; 2 minutes
6. 70 to 95% ethanol 10 dips
7. Stain with EA 36/50 Yellowish; 3 minutes
8. Dehydrate 10 dips
9. Xylol 10 dips
10. Mount and label DPX using coverslip

OTHER STAINS
1. Acid Fucshin-Picric Acid (Van Gieson’s Stain) – demo of connective tissue
Acid Fucshin 2. Acid Fucshin (Masson) – stain collagen, smooth muscle, or mitochondria
3. Picrofucshin
1. Carbolfuschin
2. Coleman’s Feulgen Reagent
Basic Fucshin 3. Schiff’s Reagent
4. Mallory’s Fuchsin
5. Aldehyde Fuchsin (Gomori’s)
Fluorescence Method for Nucleic Acid: DNA – green fluorescence, RNA – red
Acridine Orange
fluorescence
Feulgen Demonstrate sugar for Nucleic Acid: DNA – red purple; Cytoplasm - green
Methyl Green- Demonstrate phosphate for Nucleic Acid: DNA (chromatin) green or blue-green;
Pyronin RNA (nucleoli) rose-red
Acridine Red 3B Calcium salts and demo of phosphatase activities
Alcian Blue Stains mucopolysaccharides (Wharton’s Jelly)
Aniline Blue Cytoplasmic stain; counterstaining of epithelial tissues
Night Blue Substitute for carbolfuchsin in Acid-Fast Staining
Victoria Blue For demonstration of neuroglia in frozen sections
Celestine Alternative to iron hematoxylin nuclear stain
Contrast stain for Gram’s technique, in acid fast and Pap’s method, and for staining
Bismarck Brown
diphtheria.
Benzidine Stain hemoglobin
1. Picrocarmine = Carmine + Picric Acid (for neuropathological studies)
2. Best Carmine = Carmine + aluminum chloride (for glycogen demonstration)
Carmine
3. Mucicarmine = Carmine + aluminum hydroxide (for mucin and Cryptococcus
neoformans)
Crystal Violet For amyloid in frozen sections and platelets in blood

23
HISTOPATHOLOGY
Cresyl Violet Stains nervous tissues
Gentian Violet Crystal violet + methyl violet + dextrin
Stain for axis cylinders in embryos; used as a 4% aqueous solution in Krajian’s
Congo Red
Method of staining elastic tissues, amyloid and myelin
Oldest stain; stains amyloid, cellulose, starch, carotenes, and glycogen –
Iodine
dezenkerization,
Malachite Green Contrast stain for Ascaris eggs and RBCs; used as bacterial spore stain
Janus Green B Used for demonstration of mitochondria in supravital staining
NOT real dyes do not have auxochrome groups
Provide colors in lipids due to solubility in tissues that their medium of 70% alcohol
Lysochromes (Oil
• Sudan Black B – black (only stain for phospholipids)
soluble dyes)
• Sudan III – orange
• Sudan IV (Scharlach R) – Red
Stains glycogen mucin, mucoprotein, glycoprotein, basement membranes, capsules,
Periodic Acid Schiff and blood vessels as well as fungi and intracellular carbohydrates such as glycogen
Reaction in hepatocytes. (Mucoproteins – most common PAS positive substance); PAS
Positive Color – Red/Magenta Red
STAINS FOR MICROORGANISMS
• Gram’s Method
• Brown and Brenn – for Nocardia and Actinomyces
• Toluidine Blue and Cresyl Violet Acetate – Helicobacter pylori
Bacteria • Dieterle Method – Legionella pneumophilia
• Levaditi’s – Spirochete
• Modefied Steiner and Steiner – Spirochete, Helicobacter and Legionella
• Warthin-Starry – Spirochete
• Ziehl neelsen – ‘hot’ staining method for Mycobacterium
Acid-Fast • Kinyoun Method – ‘cold’ staining method for Mycobacterium
Organisms • Auramine-Rhodamine – fluorescent staining method for Mycobacteria;
• Wide-Fite Technique – Mycobacterium leprae, Nocardia
• Grocott Methanamine Silver – demonstrates all fungi, including Pneumocystis
(black)
▪ Also stains Actinomyces, Nocardia, and some encapsulated bacteria
Fungi
• Mucicarmine (A & B) – capsule of Cryptococcus (red and blue)
• Fontana-Masson – melanin pigment in Cryptococcus and pigment filamentous
fungi (black)
• Lendrum’s Phloxine-Tartrazine Method – viral inclusion
Viruses
• Orcein Method -HBsAg/Australian Antigen
• Giemsa – blood parasites
Protozoans
• PAS – Entamoeba
PIGMENTS & MINERALS
A. Endogenous – can be physiologic or pathologic when in excessive quantities
Contain iron in the form of ferric hydroxide which is bound to a
Hemosiderin
protein framework and is unmasked by various chemicals
Free iron in the form of Fe(OH)3 bound to protein complexes and
Hemoglobin can be identified in organs such as the liver, spleen, and bone
marrow.
Hematogenous Iron-free pigment of hemoglobin, found un places where there is
Hematoidin poor oxygenation, participating in the formation of bile pigment;
chemically similar with bilirubin – stained by Hall’s Method
Hematin Hemoglobin minus the globin molecule, found in old blood clots
Black granule formed by malarial parasites living in red blood cells,
Hemozoin
and may be removed by alcoholic picric acid method

24
HISTOPATHOLOGY
Stains for Iron Stains for Bile Pigment
B. Ferric Iron – Perl’s Prussian Blue A. Modified Fouchet’s Technique
C. Ferrous Iron – Turnbull’s Blue B. Gmelin’s Technique
C. Stain’s Iodine
Stained by DOPA-oxidase method, Mason-Fontana and other silver
Melanin
staining methods
• Yellow-brown to reddish-brown pigment that is due to a slow
• Wear and tear pigment or brown atrophy – associated with
Non - Lipofuscin aging
Hematogenous • Stained by lipid stains and Schmorl’s Ferric-Ferricyanide
reduction technique, Gomori’s Aldehyde Fuchsin
Found in the cells of the adrenal medulla as dark brown, granular
Chromaffin material. It may occur in tumors of the adrenal medulla
(pheochromocytomas).
Iron Found in liver in cases of iron overload
Endogenous
Calcium Stained by Von Kossa’s Silver-Nitrate Method
Minerals
Copper Lindquist Modified Rhodamine Technique
B. Exogenous
Most common; jet black pigments in lung sections and bronchial
Carbon
glands of chronic smokers
Iron From shrapnel wounds
Others Tattoos, asbestos, silver, talcum, artifacts of fixation

MOUNTING
• Added to slid before the application of coverslip for protection
• Refractive index – ratio of speed of light in air and
speed of light in a specific medium
Resinous Media (Refractive Index ≥ 1.518)
▪ Eukitt – 5% Acrylic Resin, 55% Xylene (1.510)
▪ Entallan
▪ DPX (1.532)
▪ Histmount
▪ XAM (1.52)
▪ Paramount
▪ Canada Balsam – Abus balsamea (1.524)
▪ Clarite (1.544)
• Ringing – the process of sealing the
margins of the cover-slip to prevent the
escape of fluid or semi-fluid mounts
and evaporation of mountant, to fix the
coverslip in place, and to prevent
sticking of the slides upon storage.
o Uses: Kronig Cement (made up
of two parts paraffin wax mixed
with 4-9 parts powdered
colophonium resin, heated and
filtered) or cellulose adhesives
such as Durofix
Refractive Index of glass – 1.518
Aqueous Media (for lipids because resinous
media contain xylene which may dissolve fat)
▪ Glycine (1.47)
▪ Gum Arabic (Farrant’s Medium) (1.43)
▪ Karo Corn Syrup
▪ Apathy’s Medium (1.52)
▪ Brun’s Fluid – recommended for mounting
frozen sections from water
▪ Water – poor, evaporates easily
Removal of Coverslips
a. Place slides in xylene until coverslip
detaches on its own accord
b. Leave slides in xylene until all the mounting
media is dissolved
c. Dip in 100% Ethyl alcohol, 95% ethyl alcohol,
then water (10 dips each)
Removal of Stain (Restaining Method)
a. Place slide in 1% Acid-Alcohol for 30-minutes
or longer (until stain is completely removed)
b. Wash in running tap water for 5-minutes
c. Stain as desired

25
HISTOPATHOLOGY
RESTAINING OF MOUNTED SLIDES – Employed to all sub optimally stained slides, usually
from other laboratories
IMMUNOHISTOCHEMISTRY (IHC)
• Use of antibodies as histological tools for POLYCLONAL ANTIBODIES – produced
identifying patterns of antigen distribution by immunizing an animal with an
within a tissue or an organism immunogen (antigen of interest)
• IgG – most commonly used antibody for IHC - Most frequently used animal:
Rabbit > Goat > Pig > Sheep >
Horse > Guinea Pig > Others
Tissue Preparation for Immunohistochemistry MONOCLONAL ANTIBODIES – produced
• Some tissue must be prepared as a cryostat by an individual clone of plasma cells from
animal immunized with specific
section and fixed for a few seconds in absolute
immunogens
methanol or acetone.
o To preserve immunological activity
and prevent destruction of some of the labile antigenic sites
1. Proteolytic Enzyme Digestion
a. Useful in demonstration of
i. FORMALIN FIXED PARAFFIN SECTION – heavy chain
immunoglobulin, complement, specific antigen (such as cytokeratin).
b. Most commonly used enzymes: TRYPSINE – 0.1% in 0.1% Ca2+ Ch NaOH
pH:7.8 & PROTEASE (may also use ficin, pepsin, pronase)
2. Microwave Antigen Retrieval
a. Boiling of formalin-fixed deparaffinized sections in certain solutions such as:
i. 0.01M Citrate Buffer (pH 6)
ii. EDTA (pH 8)
iii. Tris-EDTA (pH 9.9 or 10)
3. Microwave and Trypsin Antigen Retrieval
4. Pressure Cooker Antigen Retrieval – less time consuming but allows for more
constituent recovery of many antigens.
I: EPITHELIAL TUMOR MARKERS
• Highly sensitive marker for epithelial cells, present in epithelial tumors
(carcinoma)
• Also positive: mesotheliomas as non-seminomatous germ cell tumors
➢ CK 7 (Cytokeratin 7) – CA of lungs. Breast, uterus, and ovaries
(typically negative for CK 20)
➢ CK 20 (Cytokeratin 20) – CA of colon and stomach (negative for CK
Keratin
7)
➢ CK 7 and CK 20 (+) = transitional cell carcinomas of bladder and
mucinous ovarian tumors
➢ CK 7 and CK 20 (-) = renal cell carcinomas, hepatocellular
carcinomas, prostatic adenocarcinomas, thyroid carcinomas, and
squamous cell carcinomas (skin, lung and esophagus)
• Positive for adenocarcinoma of the breast, lungs, and kidneys
EMA (Epithelial • Non-reactive for hepatocellular carcinomas, adrenal carcinomas and
Membrane Antigen) embryonal carcinomas
• Negative for non-epithelial tumors (sarcomas, lymphomas, melanomas)
• Oncofetal antigen; present un carcinomas of GI tract, pancreas, lung,
CEA (Carcinoembryonic
breast, ovary, uterus, and cervix
Antigen)
• Useful in differentiate between adenocarcinoma (+) and mesothelioma (-)

26
HISTOPATHOLOGY
TTF -1 (Thyroid • Useful in differentiating lung adenocarcinoma from mesothelioma
Transcription Factor - 1) • Positive in thyroid, lung, and neuroendocrine tumors
PSA (Prostate Specific • Useful in diagnosis of prostatic adenocarcinoma
Antigen) • Also positive in certain pancreatic and salivary gland tumors
II: INTERMIATE FILAMENT MARKERS
Actin • Used to identify tumors derived from smooth, skeletal, and cardiac muscle
Vimentin • Melanomas and schwannomas – always stain positive for vimentin
• Highly specific for myogenic tumors, including leiomyoma (smooth muscle
Desmin
tumor) and rhabdomyosarcoma (skeletal muscle tumor)
Glial Fibrillary Acidic • Expressed in CNS glial cells, particularly astrocytes
Protein/GFAP • Most widely used to conform the disease of astrocytoma
• Tumors that show neuronal or neuroendocrine differentiation will stain (+)
Neurofilament for neurofilament (neuroblastomas, ganglioneuromas, neuromas,
chemodectomas, pheochromocytoma)
• Low molecular weight calcium-binding protein that is expressed in CNS
glial cells, Schwann cells, melanocytes, histiocytes, chondrocytes, skeletal
S100 Protein
and cardiac muscle, myoepithelial cells and some epithelial cells of breast,
salivary, and sweat glands epithelium
III: NEUROENDOCRINE MARKERS
Neuron -Specific • Provides strong evidence of neural or neuroendocrine differentiation
Enolase (NSE)
Chromogranin • Recognized as a marker for neuroendocrine differentiation
Synaptophysin • Marker protein for neuroendocrine cells
IV: GERM CELL TUMOR MARKERS
HCG (Human Chorionic • Synthesized by placental syncytiotrophoblast
Gonadotropin) • Marker for choriocarcinomas
• Used as a marker for endodermal sinus tumors showing yolk sac
AFP (α-fetoprotein)
differentiation
PLAP (Placenta-like • Used as a marker for germ cell tumors, particularly germinomas
Alkaline Phosphatase)
V: MESENCHYMAL TUMOR MARKERS
• Use actin and desmin and/or other muscle markers such as myo-D1,
Myogenic tumors
myoglobin and myogenin
• Used for showing fibroblastic, myofibroblastic and histiocytic
Fibriohistiocytic Tumor
(macrophage-like) differentiation
Vascular Tumors • Factor VII-related antigen =, CD31 and Ulex europaeus 1 (UEA)
• Melanocytes are derived from neural crest and will be reactive for S100
protein
Melanomas
• Intensity of staining for S100 is usually inversely proportional to the
melanin content of the tumor
• Best screening marker for lymphoma is LCA (Leukocyte Common
Lymphomas
Antigen) a.k.a. CD45
VI: CELL PROFERATION MARKERS
Ki67 • Reference monoclonal antibody for demo of Ki67
PCNA • Proliferating cell nuclear antigen
VII: CONTROLS
• Uses section known and proven to contain antigen in question because
Positive Control absence of staining in test section does not mean that antigen is absent
in tissue being studied
• Uses parallel section from tissue; omitting primary antibody from staining
Negative Control schedule or replacing the specific primary antibody by immunoglobulin
directed to an unrelated antigen
Internal Tissue Control • A.k.a. built-in control; eliminates variability of tissue fixation and controls

BASIC PATHOLOGY
INFLAMMATION – protective response of the tissues of the body to irritation or injury

27
 HISTOPATHOLOGY
Five Cardinal Signs of Inflammation
1. Rubor – redness; due to the arteriolar and capillary dilatation with increased rate of
blood flow towards the site of injury
2. Tumor – swelling; due to increased capillary permeability and extravasation of blood
fluid
3. Calor – heat; due to transfer of internal heat to the surface or site of injury, brought
about by increased blood content
4. Dolor – pain; due to pressure upon sensory nerve by exudate/tumor
5. Functio Lasia – loss of function; due to tissue damage
Classification of Inflammation – Duration

ACUTE INFLAMMATION –
vascular and exudative; innate
immunity; hours to weeks;
predominantly PMNs
CHRONIC INFLAMMATION
SUBCHRONIC
– weeks to month to years;
INFLAMMATION –
vascular and fibroblastic cell
transition between
mediated; mononuclears but
acute and chronic
PMNs may also be present

Classification of Inflammation – CELLULAR ADAPTATIONS TO STRESS

Character of Exudate
• Serous Inflammation – characterized
by secretions of mesothelial cells; seen
in some cases of Pulmonary
Tuberculosis
• Fibrinous Inflammation – Large
amount of fibrinogen in inflamed tissue;
seen in diphtheria, rheumatic
pericarditis, pneumonia
• Catarrhal Inflammation
hypersecretion of mucosa
• Hemorrhagic Inflammation
admixture of blood and other elements
of the exudate; seen in bacterial
infections
• Suppurative/Purulent Inflammation
– large amounts of pus (dead tissue
cells and necrotic PMNs)
❖ Hypertrophy: increased cell and organ size,
often in response to increased workload;
induced by growth factors produced in
response to mechanical stress or other stimuli;
occurs in tissues incapable of cell division
❖ Hyperplasia: increased cell numbers in
response to hormones and other growth
factors; occurs in tissues whose cells can
divide or contain abundant tissue stem cells
❖ Atrophy: decreased cell and organ size,
because of decreased nutrient supply or
disuse; associated with decreased synthesis
– of cellular building blocks and increased
breakdown of cellular organelles
– ❖ Metaplasia: change in phenotype of
differentiated cells, often in response to
chronic irritation, that makes cells better able
to withstand the stress; usually induced by
altered differentiation pathway of tissue stem
cells; may result in reduced functions or
increased propensity for malignant
transformation

PHYSIOLOGIC & PATHOLOGIC

CELLULAR CHANGES
Retrogressive Changes
• Developmental Defects:
o Aplasia – incomplete/defective development of tissue/organ; most commonly seen in
paired organs (kidneys/gonads)
o Agenesia – non-appearance of an organ
o Hypoplasia – failure of an organ to reach its full, mature size

28
HISTOPATHOLOGY
o Atresia – failure of an organ to form an opening (e.g. biliary atresia)
• Atrophy – decrease in size of a normally mature tissue/organ due to reduced cell size or
cell number
o Physiologic – occurs as natural consequence of maturation (e.g. atrophy of
thymus during puberty or atrophy of brain and sexual organs – senile atrophy)
o Pathologic – as a consequence of disease (e.g. vascular atrophy; pressure
atrophy; starvation atrophy; atrophy of disuse; exhaustion atrophy; endocrine
atrophy)
Progressive Changes
TYPES OF TUMORS
• Hypertrophy – increase in size of individual According to Capacity to Cause Death
cells • Benign – microscopic and gross
characteristics are relatively innocent;
o Physiologic – e.g. hormone-induced
localized and amendable to surgical
enlargement of the breast and uterus removal; good prognosis. “-oma” suffix
during pregnancy • Malignant – lesion can invade and destroy
o Pathologic – e.g. cardiac enlargement adjacent structures and spread to distant
that occurs when hypertension sites (metastasize) to case death.
• Hyperplasia – increase in number of cells “sarcoma” – mesenchymal origin;
“carcinoma “– epithelial cells
making up the tissue
According to Histologic Changes
o Physiologic – e.g. (1) hormonal
• Medullary Tumor – if cell > connective
hyperplasia, by the proliferation of the tissue framework
glandular epithelium of the female breast • Scirrhous Tumor – if connective tissue
at puberty and during pregnancy, and (2) framework > cells
compensatory hyperplasia, in which
residual tissue grows after removal or loss of part of an organ as in liver resection
o Pathologic – e.g. hyperplasia of
Neoplasm – new and abnormal growth of tissue
lymphoid follicle and Peyer’s patches in in some parts of the body
typhoid fever
Degenerative Changes
• Metaplasia – reversible change from one type of differentiated tissue to another,
usually an adaptive response to an irritating stimulus
o E.g. change from mucus-secreting epithelium to stratified squamous epithelium
in the bronchial irritation associated with cigarette smoking
• Dysplasia – disorder development of cells resulting in an alteration n in their size,
shape, and organization
o Also known as: Atypical Hyperplasia
o Reversible but is known to precede neoplasia
• Anaplasia – a criterion toward malignancy; also known as Differentiation; lack of
cellular differentiation
• Neoplasia – continuous abnormal proliferation of cells without control (no purpose or
function)
o Parts of a tumor:
▪ Parenchyma – transformed or neoplastic cells
▪ Stroma – connective tissue and vascular framework

29
HISTOPATHOLOGY
TUMOR NOMENCLATURE
Histological Type Benign Malignant
Epithelial Tissue
Glandular Adenoma Adenocarcinoma
Non-Glandular Papilloma Carcinoma
Connective Tissue
Adipose Lipoma Liposarcoma
Cartilage Chroma Chondrosarcoma
Bone Osteoma Osteosarcoma
Smooth Muscle Leiomyoma Leiomyosarcoma
Voluntary Muscle Rhabdomyoma Rhabdomyosarcoma
Blood Vessels Angioma/Hemangioma Angiosarcoma
Nerve Neurofibroma Neurofibrosarcoma
Nerve Sheath Neurilemmoma Neurilemmosarcoma
Glial Cells Glioma Malignant glioma
Other
Hematopoietic Leukemia
Lymphoreticular Lymphoma
Melanocytes Malignant melanoma
Germinal Cell Benign teratoma Malignant teratoma

Tumor Grading
• Done to estimate aggressiveness or level of malignancy for appropriate modality
• Based on cytologic differentiation of tumor cells and number of mitosis within the tumor
• Well differentiated = less malignant than undifferentiated tumors
BRODER’S CLASSIFICATION
Differentiated Cells Undifferentiated Cells Modality
Grade I 75 – 100% 0 – 25%
Lower Grade = Radiation
Grade II 50 – 75% 25 – 50%
Grade III 25 – 50% 50 – 75%
Higher Grades = Surgery
Grade IV 0 – 25% 75 – 10%
Tumor Staging TNM System of Cancer Staging
• Based on size of primary lesion, extent • T – size of primary tumor (T1, T2, T3, T4 with
of spread to regional lymph nodes and increase size of primary lesion)
presence or absence of metastases. • N – spread to lymph node (N0, N1, N2, N3)
• M – presence or absence of metastases
Follows criteria from the following (M0/M1)
agency:
o UICC – Union for International Cancer Control
o AJCS – American Join Committee on Cancer Staging
• TNM System of Cancer Staging – developed by UICC
• Teratomas (Gk. “monstrous tumors”) – a type of neoplasm or tumor characterized by
normal tissue or organ components that are inappropriate to the surrounding tissue (e.g.
presence of hair, teeth, bones, and very rarely eyeballs, torso, and hands)
DEATH
A. Cell Death
• Apoptosis – pathological or physiological/programmed cell death
o Nucleus: fragmentation into nucleosome size fragments
o Plasma membrane: membrane remains intact
o Cell morphology: cell shrinkage and fragmentation, formation of characteristic
apoptotic bodies

30
HISTOPATHOLOGY
o Inflammation: no inflammatory
TYPES OF NECROSIS
response • Coagulation Necrosis – most common
o Fate of Cells: phagocytosed by type; caused by ischemia/infraction; cells
neighboring cells lose organelles, but cellular outline
remains; may be found in myocardium,
• Necrobiosis – physiologic cell death lungs, kidneys, and spleen
• Necrosis – pathological • Liquefaction/Colliquative Necrosis –
characterized by pus formation; seen in
o Nucleus: Pyknosis →
brain and spinal cord
Karyorrhexis → Karyolysis • Caseous Necrosis – characterized by
o Plasma membrane: loss of formation of yellow, cheesy masses,
crumby materials; seen in MTB infraction,
membrane integrity
tularemia, syphilis, LGV
o Cell morphology: Cell swelling • Gangrenous Necrosis – histologic patter
and lysis, incorporated with = coagulation + liquefaction necrosis.
eosinophilia Types of Gangrene (1) Dry – arterial
occlusion; (2) Wet – venous occlusion
o Inflammation: with • Fat Necrosis – chalky white precipitate
inflammatory response from degraded fats; seen in pancreatitis
o Fate of Cells: phagocytosed by
neutrophils and macrophages
B. Somatic Death
• Primary changes: (1) circulatory (2) respiratory (3) central nervous system
• Secondary changes:
1. Algor mortis – first demonstratable change observed; cooling of the body at a rate
of 7°C/hour
2. Rigor mortis – stiffening of skeletal muscles after death; first occurs in the muscles
of head and neck
3. Livor mortis – postmortem lividity/postmortem suggillations; purplish
discoloration/lividity of the skin; due to the pull of gravity on RBCs
4. Postmortem clot – chicken fat
LIVER MORTIS
• Disappears on pressure but reappears appearance; currant jelly appearance;
when relieved; on inclusion: blood flows assumes shape of blood vessel; rubbery
from the cut vessel consistency
ECCHYMOSES
• Does not disappear on pressure; on 5. Antemortem clot – granular
incision: blood coagulate in tissue friable; not easily detached from blood
vessel wall; seldom assumes shapes of
the blood vessels
6. Desiccation – drying and wrinkling of anterior chamber of the eye and cornea
7. Putrefaction – manifested as greenish discoloration of the abdomen caused by
invasion of intestinal microorganism
8. Autolysis – self-destruction of cells due to organelles; lysosomes

AUTOPSY
• Greek word “autopsia” which means eyewitness
• Also known as post mortem or necropsy

31
HISTOPATHOLOGY
Types of Autopsies
Processor
• Forensic (medico-legal autopsy or • Individual in-charge of actual dissection of
the body (Pathologist or Pathology-
coroner’s autopsies) – with legal Resident)
implications Coroner
• Clinical/Pathological – to diagnose a • One who confirms and certify the death of
individual (can be a pathologist depending
particular disease or for research on request of the state)
purposes Diner
• Anatomical/Academic – for • On assisting the autopsy procedure
anatomical study purpose; possible
only when a person has given permission ahead of death
Autopsy Techniques
ALL AUTOPSIES REQUIRE
AUTHORIZATION FROM NEXT-
• Technique of Virchow – involves Head-Throrax-
OF-KIN OF THE DECEASED IN
Abdomen removal; THE FOLLOWING ORDER:
• Technique or Rokitansky – an in-situ dissection • Spouse
examination of viscera with removal of notable • Children
• Parents
organs; • Siblings
• Technique of Ghon – an en block removal of • Guardians
viscera into thoracic, intestinal, upper and lower • Other Affairs
abdominal, brain & neck;
Prohibited Autopsy – mutilation
• Technique of M. Letulle – the cervical-thoracic- and conspiracy
abdominal-pelvic organs-en masse removal of all
the viscera
Normal Weight of Organs:
➢ Right Lung – 300 to 400 g Restricted Autopsy – may or may not be done
➢ Left Lung – 250 to 350 g
• Autopsy of anus and vagina
➢ Heart – 250 to 300 g
➢ Liver – 1100 – 1600 g • Autopsy of surgical wounds
➢ Adrenals – 4 gm or so each • Needle autopsy
➢ Thyroid – 10 to 50 g
➢ Spleen – 60 to 300 g
➢ Brain – 1150 to 1450 g

BASIC DIAGNOSTIC CYTOLOGY

• Microscopic examination of cells from different body sites; two divisions: EXFOLIATIVE
& FINE NEEDLE ASPIRATION
Exfoliative Cytology
EXFOLIATIVE SPECIMEN:
• Microscopic exam of desquamated cells from
➢ Vaginal smears
epithelial surfaces ➢ Cervicovaginal smears
• Purpose: assessing malignant or cancerous ➢ Endometrial & endocervical smears
conditions; assessment of female hormonal activity ➢ Nipple discharge
(sterility and endocrine disorders); detection of ➢ Smears of urine
➢ Prostatic and breast secretion
asymptomatic cancers in women; determination of ➢ Pleural and Peritoneal
genetic sex; detection of precancerous cervical ➢ Gastric and Bronchial
lesions in women (Pap’s smear); detection of ➢ Sputum
presence of possible infection. ➢ CSF

32
HISTOPATHOLOGY
• Fixation:
PRECAUTIONS IN FIXATION
o Fluid Specimen 1. Identify slides before
▪ Fixative: 50% alcohol (all types of effusion) preparing smears
– Saccomano’s Fixative: 50% Ethanol and 2. Use paper clips to the
identified end of the slide
2% Carbowax before preparing smears
▪ Centrifuge 3. Smears should be placed into
the fixative container
• 2000 rpm for 2-minutes with
immediately after
supernatant discarded preparations’
• Sediment is smeared directly to glass 4. Place each smear in fixative
by a single uninterrupted
slide or cytospin on slides with egg motion to avoid rippling of
albumin (to reduce cellular distortion) smeared material
• Extra sediment → prepare cell block 5. Avoid striking the bottom of
the fixative container
(filtration method, plasma-thrombin forcefully to prevent
method, carbowax method) dislodging of cells
o Smears
▪ Fixatives: equal parts of 95% ethanol and ether – BEST! But is highly volatile
and flammable; 95% ethanol – MOST COMMONLY USED TODAY; Spray
fixatives – slide should be kept 1-feet away from spray.
• Adhesion
o Specimen that require adhesive
▪ Urinary sediment
▪ Bronchoalveolar lavage (BAL)
▪ Specimens that use proteolytic enzymes (trypsin, concentrated sputum,
enzymatic lavage specimen from GIT)
MAILING OF SPECIMENS
• Air drying of specimens after Characteristics of Adhesives
fixation for 2-hours and placed in ▪ Permeable to both fixative and stain
wooden, cardboard or plastic slide ▪ Must not retain the stain
containers
• Glycerin technique – smears are ▪ Do not use: MAYER’s EGG ALBUMIN
fixed for at least 30-minutes and Good Adhesive Agents
covered with glycerin. A clean ▪ Pooled Human Serum/Plasma
glass slide will serve as a
temporary coverslip for the smear ▪ Celloidin Ether Alcohol
▪ Leuconostoc Culture
NON-GYNECOLOGIC SPECIMENS
RESPIRATORY TRACT SPECIMENS
• Sputum
o Obtain at least 3 consecutive morning sputum specimens (deep cough)
o Use wide-mouthed jar with Saccomano’s Fluid
o Sputum induction – inhalation of aerosol solution for 20 minutes
o Presence of alveolar macrophage = sputum from deep cough
o Absence of alveolar macrophage = saliva
• Bronchoalveolar Lavage/Bronchial Washings – performed in patients with AIDS to
rule out P. carinii
• Bronchial Brushing
o Specimen is directly smeared onto 2 labeled slides by pull technique
o Fix slides immediately, if delayed: air drying artifacts will form

33
HISTOPATHOLOGY
PERITONEAL, PLEURAL, AND PERICARDIAL FLUIDS
TYPES OF GI SPECIMENS
• Add 300 units of Heparin/100 mL of aspirate: to prevent • Gastric Lavage
• Gastric Brush
formation of jelly-like clots • FNA (Submucosal
Lesions)
GASTROINTESTINAL SPECIMENS
• Collection is done to exclude the possibility of malignant tumors
• Patient NPO; specimen collected via irrigation or aspiration

TYPES OF URINE SPECIMENS

URINE
• Voided Urine (Males)
• Diagnosis of urothelial malignancy
• Catheterized Urine (Females) – prevent
contamination with vulvar cells • Prostatic CA – rarely found in urinary
• Washings from bladder or renal pelvis specimens
• At least 50 mL is needed
• First voided urine should be discarded: second urine is preferred, due to overnight
degeneration of cells
• Use of preservatives is not recommended
BREAST SECRETIONS
• Collection is from spontaneous nipple discharge via cotton swab or tough prep
• Observe abnormal cells, NOT crystals nor bacteria
GYNECOLOGIC SPECIMENS
• Pap Smears – developed by Dr. George Papanicolau
• Instruments for Cervicovaginal Collection:
MAILING OF SPECIMENS
• Upper Lateral 3rd of the Vaginal
o Glass Pipette and Rubber depressor
Wall – for vaginal hormonal o Sterile Tongue Depressor
cytology o Ayre’s Spatula
• Ectocervix
o Laryngeal Cannula with Syringe
• Endocervix
o Cervical Broom, Cervical brush
• If derived from all three (3) anatomic sites → detection of female genital cancer
o Endocervical Brush – samples of endocervical canal
o Vaginal Scrape – patients with hysterectomy
o Lateral Vaginal Scrape – hormonal evaluation
o Four Quadrant Vaginal Scrape – localization of vaginal adenosis
o Vulvar Scrape – detection of herpetic lesions or carcinoma
VAGINAL HORMONAL CYTOLOGY
• Relatively inexpensive
• May be performed regularly even in pregnant women (taken from at the upper
lateral third of vaginal wall) without undue risk
VAGINAL SMEAR PREPARATION PRECAUTIONS
1. The patient should not have been douched or undergone vaginal examination for at least 24 to 48
hours before smears are prepared
2. The glass pipette used should be absolutely dry since presence of water will distort cellular details
3. No lubricant or powder should be used on the examiner’s gloves
4. Smears should be spread thinly in a rotary motion instead by pull-apart method
5. All materials should have been ready before smear collection is done’

34
HISTOPATHOLOGY
6. Scrapping from the lateral vaginal wall with wooden spatula is recommended only for hormonal studies
7. For detection of female genital cancer, combines vaginal and cervical smear is method of choice.

CELLS FOUND IN CERVICOVAGINAL SMEARS

1. Mature Superficial/Superficial Cells
• Characterized by dark pyknotic nuclei and true acidophilia (estrogen effect)
• “Pseudophilia” – observed in the following instances: (1) drying of smears before fixation (2)
prolapse and drying of vaginal epithelia (3) infection and (4) chemicals
2. Intermediate Cells
• Medium sized, polyhedral or elongated cells
• Basophilic cytoplasm showing vacuoles
Navicular Cells Pregnancy Cells
- Boat-shaped intermediate cells with strong - Round, oval or boat-shaped cells
tendency to fold or curl on edges - With translucent basophilic cytoplasm
- Presence suggests a combine estrogen- - Nucleus is pushed aside or towards the cell
progesterone effect membrane
- Found in the latter half of the menstrual - With double-walled boundary appearance
cycle, during pregnancy and menopause (deeper blue stain of the cytoplasm at the
periphery)
.
3. Parabasal Cells
• Thick, round to oval
• Smaller than intermediate cells
• Fried “sunny side-up” egg appearance
• Normally found from two (2) weeks of age to puberty, after childbirth, abortions, port-menopause
Maturation Index
- Assessment of hormonal status based on proportion of parabasal, intermediate, and superficial
cells in 100 cells counted
- Examples:
✓ Shift to the left (95/5/0) increase parabasal – seen in post menopause
✓ Shift to the middle (5/90/5) increase intermediate cells – seen in pregnancy
✓ Shift to the right (0/0/100) increase mature superficial cells
4. Endometrial Cells
• Similar in appearance to parabasal cells
• Slightly cylindrical with less basophilic cytoplasm
• Occurring in groups of 3 or more cells
• Found during 1 to 10 days after menstruation
5. Endocervical Glandular Cells
• Cytoplasm is usually stained pale blue/gray, finely vacuolated
• Nuclei with finely granular chromatin
• Honeycomb appearance when viewed on end
6. Basal Cells
• Small round slightly oval cells
• With relatively large nuclei (more than half of the cell)
• Strongly basophilic cytoplasm
• Found before puberty and after menopause
7. Doderlein’s bacillus
• Gram-positive, slender rod-shape
• Pap’s stain: lavender to blue in color
• Most common organism in normal vaginal flora and in gastrointestinal tract called as Boa’s Oppler
Bacilli
• Numerous naked nuclei with many Doderlein’s bacilli seen in:

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HISTOPATHOLOGY
o Last Trimester of pregnancy
o Diabetes mellitus
o Infection
o Estrogen deficiency
8. Candida albicans – pathologic yeast seen in diabetic patients, patients on contraceptives,
immunocompromised, leukemia, and lymphoma
9. Trichomonas vaginalis – pear-shaped, blue-green to blue0gray on Pap’s smear
10. Gardnerella vaginalis – coccobacilli, associated with clue cell; bacterial vaginosis
11. Koilocytes
• Squamous epithelial cells that show cytopathic effects of HPV
• Atypical wrinkled prune nucleus surrounded by perinuclear halo
• Presence = low grade squamous epithelial lesion (Bethesda System)

FERNINING
• Fern/palm leaf pattern of salt crystals in dried cervical mucus
• Influenced by estrogen, but inhibited by progesterone
• Signifies a high, persistent estrogen effect
• One of the basis of diagnosis of early pregnancy

Manner of Reporting
A. Class System – obsolete, often used in non-gynecologic specimens also
Class I Absence of atypical cytologic picture
Class II Atypical cytologic picture but no evidence of malignancy
Class III Cytologic picture suggestive but not conclusive of malignancy
Class IV Cytologic picture strongly suggestive for malignancy
Class V Cytologic picture conclusive for malignancy

B. Bethesda System (2014) – used in Pap’s smear reporting and formatted as:
i. Specimen type – indicate conventional smear (Pap smear) vs. liquid-based
preparation vs. other
ii. Specimen adequacy – satisfactory or not satisfactory
iii. General categorization
iv. Interpretation/Result
a. Negative for intraepithelial lesion or malignancy
✓ Squamous metaplasia ✓ Atrophy
✓ Keratotic changes ✓ Pregnancy-associated
✓ Tubal metaplasia changes
b. Organisms
✓ Bacteria morphologically consistent with Actinomyces species
✓ Fungi morphologically consistent with Candida species
✓ Shift in flora suggestive of bacterial vaginosis – Gardnerella vaginalis
✓ Trichomonas vaginalis
✓ Cellular changes consistent with HSV – Human Simplex Virus
✓ Cellular changes consistent with CMV – Cytomegalovirus
c. Other
d. Epithelial cells abnormalities

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HISTOPATHOLOGY
SQUAMOUS CELL
o Atypical squamous cells (of undetermined
significance [ASC-US]/cannot exclude HSIL [ASC-
H])
o Low-grade squamous intraepithelial lesion (LSIL)
[encompassing: HPV/mild dysplasia/CIN 1]
o High-grade squamous intraepithelial lesion (HSIL)
[encompassing: moderate and severe dysplasia,
CIS; CIN 2 and CIN 3] – with features suspicious
for invasion (if invasion is suspected)
o Squamous cell carcinoma
GLANDULAR CELL
o Atypical – endocervical cells, endometrial cells,
glandular cell (NOS or specify in comments)
o Atypical – endocervical cells, glandular cells (favor
neoplastic)
o Endocervical adenocarcinoma in situ
o Adenocarcinoma (endocervical, endometrial,
extrauterine, not otherwise specified [NOS])

REFERENCES

Bancroft, J. D., Layton, C., & Suvarna, S. K. (2013). Bancroft’s Theory and Practice of
Histological Techniques.
Bruce-Gregorios, J. (2017) Histopathologic Techniques (3rd ed.), Katha Publishing Co., Inc.,
Makati City
Dinglasan, R. J. A. R. (n.d.) Histopathologic Techniques Board Review (10th ed)
Kumar, V., Abbas, A, K & Aster, J. C. (2015). Robbins and Cotran Pathologic Basis of Disease
(9th ed). Philadelphia, PA: Elsevier/Saunders
McPherson, R.A., Pincus, M. R. (2017). Henry’s Clinical Diagnosis and Management by
Laboratory Methods (23rd ed.). Maryland Heights, MO: Saunders Elsevier.
Mescher, A. L., & Junqueira, L. C. U. (2013). Junqueira’s Basic Histology: Text and Atlas (13th
ed.). New York, McGraw Hill Medical
Polansky. (2014). Quick Review Cards for Medical Laboratory Science (2nd ed.).
Philadelphia, PA: F.A. Davis Company.
University of the Immaculate Conception – Davao City. Medical Laboratory Science Lecture
Notes
University of the Immaculate Conception – Davao City. Medical Laboratory Science
PowerPoint Presentation

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