KLUBSYBEAR MTLE

TUTORIALS
BASIC HISTOLOGY, HISTOPATHOLOGY, CYTOLOGY, AND
MEDICAL TECHNOLOGY LAWS AND ETHICS

______________________________, RMT
“Don't count the days, make the days count.”

Princess Erica B. Garcia, RMT

[email protected]
HISTOLOGY:
The study of tissues of the body and of how these tissues are arranged to constitute organs.
HISTOPATHOLOGY:
Microscopic study of tissues affected by diseases.
4 FUNDAMENTAL TISSUES are recognized:
a) Epithelial tissue
➢ These cells have strong adhesion and form cellular sheets that cover surface of the body and line its
cavities.
➢ The principal functions of epithelial tissues are the covering and lining of surfaces (ex. Skin), absorption
(ex. Intestines), secretions (ex. Epithelial cells of glands), sensation (ex. Neuroepithelium), and
contractility (ex. Myoepithelial cells).
➢ Epithelia are divided into 2 main groups according to their structure and function: COVERING EPITHELIA
and GLANDULAR EPITHELIA.
A. COVERING EPITHELIA:
(According to number of cell layers)
• Simple Epithelium – contains only 1 layer of cells
• Stratified Epithelium – contains more than 1 layer of cells
(According to cell shape)
• Squamous – flat
• Cuboidal – cube like
• Columnar – tall and thin

Number of Cell Layers Cell Form Examples of Distribution Main Function

SIMPLE Squamous Lining of blood vessels Facilitates the movement
(one layer) (endothelium). Serous of the viscera
lining of cavities; (mesothelium), active
pericardium, pleura, transport by pinocytosis
peritoneum (mesothelium and
(mesothelium), alveoli of endothelium), secretion of
the lungs, portions of the biologically active
kidney tubules molecules (mesothelium)
Cuboidal Covering of the ovary, Covering and secretion
thyroid, kidney tubules,
glands and their ducts,
choroid plexuses of the
brain, lung the terminal
bronchioles of the lungs
Columnar Lining of the intestine, Protection, lubrication,
gallbladder, bronchioles of absorption, secretion
the lungs, auditory tubes,
uterus, uterine tubes,
stomach, bile ducts,
ventricles of the brain,
glands and some ducts
PSEUDOSTRATIFIED *The cells are almost Lining of the trachea, Protection, secretion; cilia-
(layers of cells with nuclei always ciliated and are bronchi, and nasal cavity mediated transport of
at different levels, not all associated with goblet particles trapped in mucus
cells reach surface but all cells that secrete mucus out of the air passages
adhere to the basal lamina) onto the free surface.
STRATIFIED Squamous keratinized Epidermis Protection against
(two or more layers) (dry) abrasion; prevents water
loss
Squamous nonkeratinized Mouth, esophagus, larynx, Protection, secretion,
(moist) vagina, anal canal prevents water loss
Cuboidal Sweat glands, developing Protection and secretion
ovarian follicles
Columnar Conjunctiva Protection
Transitional Lining of the Urinary Protection and
Bladder, ureters, and renal distensibility
calyces
ADDITIONAL NOTES:
✓ Neuroepithelial cells – are cells of epithelial origin with specialized sensory functions (ex. Cells of taste buds)
✓ Myoepithelial cells – branched cells that contain myosin and a large number of actin filaments. They are
specialized for contraction, mainly of the acini of the mammary, sweat, and salivary glands.
B. GLANDULAR EPITHELIA:
➢ Tissues formed by cells specialized to form secretions. The molecules to be secreted are
generally stored in the cells in small membrane-bound vesicles called secretory granules.
➢ May synthesize, store and secrete proteins (ex. Pancreas), lipids (ex. Adrenal and Sebaceous
glands, or complexes of carbohydrates and proteins (ex. Salivary glands).
MUST KNOW:
✓ EXOCRINE – Glands that retain their connection with the surface epithelium from which they originated. This
connection takes the form of tubular ducts lined with epithelial cells through which glandular secretions pass
to reach the surface.
Additional Information:
➢ Classification of Exocrine Glands (according to the way the secretory products leave the cell)
a) MEROCRINE: The secretory granules leave the cell by exocytosis with no loss of other cellular
material. (ex. Pancreas)
b) HOLOCRINE: The product of secretion is shed with the whole cell – a process that involves
destruction of the secretion-filled cells. (ex. Sebaceous glands)
c) APOCRINE: The secretory product is discharged together with the parts of the apical cytoplasm.
(ex. Mammary glands)
✓ ENDOCRINE – Glands are those whose connection with the surface from which originated was obliterated
during development. These glands are therefore ductless, and their secretions are picked up and transported
to their site of action by the bloodstream rather than by a duct system.

b) Connective tissue – Functions in a mechanical role, they provide a matrix that connects and binds the cells and
organs and ultimately gives support to the body.
The connective tissues are divided into the following groups: (Bancroft’s)
• Connective tissue proper – includes loose or areolar, dense, regular and adipose irregular, reticular
• Cartilage – hyaline elastic and fibrocartilage
• Bone – spongy or cancellous and dense or cortical
• Blood – erythrocytes, leukocytes, thrombocytes
• Blood-forming – hematopoietic
➢ Unlike other tissues, CTs major constituent is the extracellular matrix.
➢ CTs are formed by 3 classes of components:
i. Cells
ii. Protein Fibers – Collagen, Reticular, and Elastic
iii. Ground substance – highly hydrophilic, viscous complex of anionic macromolecules
(glycosaminoglycans and proteoglycans) and multiadhesive glycoproteins (laminin, fibronectin,
and others) that imparts strength and rigidity to the matrix by binding to the receptor proteins
(integrins) on the surface of the cells and to the other matrix components.
➢ Connective tissue matrix also serves as the medium through which nutrients and metabolic wastes are
exchanged between cells and their body supply.
➢ CT originates from the mesenchyme, an embryonic tissue formed by elongated cells, the mesenchymal
cells. The mesenchyme develops mainly from the middle layer of the embryo, called the mesoderm.

FIBROBLAST: cells that synthesize collagen, elastin, glycosaminoglycans, proteoglycans and multiadhesive proteins.
They are the most common cells in connective tissues and are responsible for the synthesis of the extracellular matrix
components. Fibroblasts are also involved in the production of growth factors that influence cell growth and
differentiation. TAKE NOTE! Fibroblast play an important role in wound healing.

CLASSIFICATION OF CONNECTIVE TISSUES:

Connective Tissue Proper Loose (fewer fibers, more ground substance)
• Areolar
• Adipose
• Reticular
Dense (more fibers, less ground substance)
• Dense, regular collagenous
• Dense, regular elastic
• Dense, irregular collagenous
• Dense, irregular elastic
Supporting Connective Tissues Cartilage (semisolid matrix)
• Hyaline
• Fibrocartilage
• Elastic
Bone (solid matrix)
• Spongy
• Compact
 Fluid Connective Tissue Blood
• Red blood cells
• White blood cells
• Platelets
Hematopoietic Tissue
• Red marrow
• Yellow marrow

Three Main types of Connective Tissue Fibers:

A.COLLAGEN – The most abundant protein in the human body. Collagen contains 2 amino acids that are characteristic
of this protein: hydroxyproline and hydroxylysine.
• Vitamin C (Ascorbic acid) – Cofactor for proline hydroxylase, which is essential for normal synthesis of
collagen. Without this vitamin, fibroblast synthesize defective collagen, and the defective fibers are not
replaced. This process leads to general degeneration of connective tissue that becomes more
pronounced in areas where collagen renewal takes place at a faster rate. [Deficiency in Vitamin C =
SCURVY]
• Keloid: A local swelling caused by abnormal amounts of collagen that form in scars in the skin.

Different Types on Collagen:

✓ Type I – Type 1 is the most common in humans. This type accounts for most of the organic matrix of bases, but
is also a major structural protein in the lung. This collagen forms the thick collagenous fibers that have been
demonstrated histologically and form the bulk of the body’s collagen.
▪ Type 1 collagen is found in fibrous supporting tissue, the dermis of skin, tendons, ligaments, and bone.
It is very eosinophilic, readily visible with light microscopy birefringent upon polarization, and reveals a
characteristic pattern of cross-striations with the electron microscope.
✓ Type II - This collagen is found in hyaline and elastic cartilage and is produced by chondroblast activity. Type II
collagen is usually not readily visible by light microscopic methods because the fibers are thin and composed of
fibrils arranged in a meshwork with copious amounts of proteoglycans. The cross-banding of Type II collagen is
less evident due to the masking effect of the abundant interfibrillar material. Treatment with hyaluronidase
may unmask Type II fibers to render them accessible for immunohistochemical evaluation.
✓ Type III - Fibers classically known as ‘reticular fibers’ contain Type III collagen. Ultrastructural studies of reticular
fibers reveal loosely packed fibrils surrounded by abundant carbohydrate-rich interfibrillar material.
✓ Type IV - This collagen has been characterized in structures identified morphologically as basement
membranes. Type IV collagen is closely associated with significant amounts of carbohydrate complexes, which
explains the strong reaction of basement membranes to the Periodic Acid-Schiff (PAS) method.
✓ Type V & Type VI

COLLAGEN STAINS:
Collagen may be differentially stained by any of the following techniques:
1. Van Gieson's stain
2. Masson's Trichrome stain
3. Mallory's Aniline Blue stain
4. Azocarmine stain
5. Krajian's Aniline Blue stain

ADDITIONAL NOTES: (Junqueira’s)

• Collagen fibers are acidophilic; they stain PINK with eosin, BLUE with Mallory’s Trichrome stain, GREEN with
Masson’s Trichrome stain, and RED with Sirius red.

B.RETICULAR FIBERS – Consists mainly of collagen type III. They are not visible in hematoxylin-and-eosin (H&E)
preparations but can be easily stained BLACK by impregnation with silver salts. Because of their affinity for silver salts,
these fibers are called ARGYROPHILIC (Gr. argyros, silver, + philein, to love)

• Reticular fibers are also Periodic Acid-Schiff (PAS)-positive. PAS also stains reticulin purplish red. Both PAS
positivity and argyrophilia are considered to be due to the high content of sugar chains associated with these
fibers.

• NOTE: The argyrophilia (affinity for silver) of reticular fibers is due to the proteoglycan content of the fibers and
is not dependent upon the proteins of the fibrils themselves.

• They provide the bulk of the supporting framework of the more cellular organs (e.g. spleen, liver, lymph nodes,
etc.), where they are arranged in a three-dimensional network to provide a system of individual cell support.

• Reticular fibers may be demonstrated distinctly, in paraffin sections, using one of the many argyrophil-type
silver impregnation techniques available or, in frozen section, by the periodic acid-Schiff technique.

RETICULIN STAINS:
Reticular fibers may be differentially stained by the following:
1. Gomori's Silver Impregnation Stain for Reticulin – Reticulin: BLACK
2. Reticulin Stain -Gordon and Sweets’ Method – Reticulin fibers: BLACK, Nuclei: BLACK, Background: RED

C. ELASTIC FIBERS – composed of 3 types of fibers- oxytalan, elaunin, and elastic. Elastic fibers are rich in aromatic
desmosine and isodesmosine residues.
Fibrillin: a family of proteins related to the scaffolding necessary to the deposition of elastin.
NOTE:
✓ Mutations in the fibrillin gene result in Marfan syndrome, a disease characterized by a lack of resistance in
the tissues rich in elastic fibers. Because the large arteries are rich in components of the elastic system and
because the blood pressure is high in the aorta, patients with this disease often experience aortic rupture, a
life-threatening condition.
ELASTIC FIBERS STAINS:
Elastic fibers may be differentially stained by any of the following techniques:
1. Van Gieson
2. Orcein
3. Weigert’s
4. Verhoeff’s hematoxylin
AMYLOID
Amyloid – meaning starch-like and was first used botany to describe the starchy cellulose material found in plants.
Starch gives a deep blue color when iodine is applied whereas cellulose gives a violet color.
Amyloidosis – a disorder of protein folding, in which normally soluble proteins accumulate in the tissues as abnormal
insoluble fibrils or filaments.
STAINS FOR AMYLOID
1. Gram’s Iodine Stain
2. Congo Red method
3. Krajian’s Amyloid Stain (Modified Bennhold Method) – amyloid: red on clear background; nuclei: blue
4. High pH Congo Red Technique
5. Metachromatic staining
6. Induced Fluorescent Staining with Thioflavine
7. Methyl violet – crystal violet method – amyloid: purplish red; nuclei, cytoplasm, CT: shades of violet
Supporting Connective Tissue:
CARTILAGE: Characterized by an extracellular matrix enriched with glycosaminoglycans and proteoglycans,
macromolecules that interact with collagen and elastic fibers. Cartilage consists of cells called Chondrocytes (Gr.
chondros, cartilage, +kytos, cell) and an extensive extracellular matrix composed of fibers and ground substance.
3 Forms of Cartilage:
1. Hyaline cartilage – the most common form, type II collagen is the principal collagen type. Fresh hyaline cartilage
is bluish-white and translucent. In the embryo, it serves as a temporary skeleton until it is gradually replaced by
bone.
Example: Walls of the larger respiratory passages (nose, larynx, trachea, bronchi), ventral ends of ribs, & epiphyseal
plate
2. Elastic cartilage – essentially identical to hyaline cartilage except that it contains an abundant network of fine
elastic fibers in addition to collagen type II fibrils. Fresh elastic cartilage has a yellowish color owing to the
presence of elastin in the elastic fibers.
Example: auricle of the ear, walls of the external auditory canals, auditory (eustachian) tubes, epiglottis, & cuneiform
cartilage of the larynx
c) Fibrocartilage – it resists pulling and tearing forces
Example: Intervertebral disks

Muscular tissue – Divided into 3 types – Skeletal, Cardiac, and Smooth Muscle.
SKELETAL MUSCLE CARDIAC MUSCLE SMOOTH MUSCLE
Very long, cylindrical, Elongated, BRANCHED, Fusiform cells, SPINDLE-SHAPED,
MULTINUCLEATED (peripheral), that UNINUCLEATED (central) cells, with UNINUCLEATED (central), DO NOT
show CROSS-STRIATIONS CROSS-STRIATIONS SHOW CROSS-STRIATIONS in the light
microscope (No Striations)
VOLUNTARY CONTROL INVOLUNTARY CONTROL INVOLUNTARY CONTROL
Has INTERCALATED DISK, a special
structure found only in Cardiac
muscles

d) Nervous tissue -The nervous system can be subdivided into the central, peripheral and autonomic nervous
system. The principal components of the of the CNS are:
a) Neurons - an excitable cell that is responsible for processing and transmitting information. Most
neurons consist of 3 parts namely:
Neurons or Nerve Cells:
➢ Responsible for the reception, transmission, and processing of stimuli; the triggering of certain cell activities;
and the release of neurotransmitters and other informational molecules.
• Dendrites – (Greek; Dendron, tree) usually short and divide like a tree; receive many synapses
and are the principal signal reception and processing sites on neurons
 • Cell body or Perikaryon – (Greek: peri, around, +karyon, nucleus) part of the neuron that
contains the nucleus and surrounding cytoplasm, exclusive of the cell processes. It is primarily
a trophic center, although has receptive capabilities. It contains highly developed endoplasmic
reticulum organized into aggregates of parallel cisternae.
✓ Nissl Bodies/Nissl substance/Tigroid substance/Chromatoidal substance:
Refers to the basophilic material in the cytoplasm of the neuron. Ultrastructurally, this
material can be identified as large aggregates of rough endoplasmic reticulum (rER),
with the RNA content providing the basis for demonstration by special light microscopic
techniques.
✓ Nissl substance is sharply stained with Basic Aniline Dyes such as:
o Thionin
o Cresyl Echt Violet
✓ Injury to a neuron may cause the Nissl substance to disappear, first from around the
nucleus and then entirely, this loss is referred to as Chromatolysis and demonstrating
this loss is useful in assessing neuronal damage.
• Axon or Nerve Fibers – Neuronal processes that carry nerve impulse over long distances. Each
neuron has a single axon that originates from a cone-shaped elevation, the Axon hillock of the
cell body and terminates on the dendrites or the cell body of other neurons (a synapse), or in
a specialized ending associated with an effector organ such as muscle.
b) Neuroglia (Nerve Glue) – These are the “supporting elements” of the CNS. Neuroglia may be thought
of as Neural Connective Tissue because connective tissue proper is NOT found in the CNS except in the
meninges covering the brain and the spinal cord.

4 Types of Glial Cells:

1) Oligodendroglia - small cells that function in the CNS to produce, and probably maintain, the myelin sheath
surrounding many axons.
2) Astroglia - Astrocytes are stellate cells of 2 types: protoplasmic, which occur in the gray matter, and fibrous, which
occur in the white matter. Following injury or trauma of the CNS, astrocytes function in scar formation by
proliferation of cell processes and the formation of an area of gliosis.
3) Microglia - Microglia are fixed phagocytic cells found throughout the brain and spinal cord; stains for the
demonstration of microglia rarely are needed except for research purposes.
4) Ependymal cells - Ependymal cells are true epithelial cells that line the ventricles and spinal canal. They form a
selective barrier between the cerebrospinal fluid and nervous tissue.
c) Meninges
d) Blood vessels

STAINS FOR CENTRAL NERVOUS TISSUE

1. Bielschowsky’s Technique – neurons, axons, & neurofibrils (Black on grayish background)
2. Bodian’s stain – nerve fibers & nerve endings
3. Sevier-Munger Technique – neural tissues
4. Cresyl Fast Violet – Nissl stain
5. Kluver & Barrera Luxol Fast Blue – myelin with Nissl counterstain
6. Weil’s method – myelin sheaths
7. Modified Palmgren’s method – nerve fibers

ASTROCYTES STAINS
✓ Astrocytes – represent the major supporting cells in the brain. They respond to injury by producing a dense
network of processes, somewhat analogous to the fibrous scar that occurs elsewhere in the body. However, in
contrast to the fibroblast, astrocytes do not produce collagen. Instead, the glial “scar” is made up predominantly
of cytoplasmic processes, with little or no extracellular protein.
✓ Astrocyte swelling is often associated with increased synthesis of glial fibrillary protein (GFAP), the astrocyte’s
major cytoskeletal protein.
1. Cajal’s Gold Sublimate Method
2. Modified PTAH stain – for reactive astrocytes
3. Modified Holzer’s Method
TISSUE CELLS EXTRACELLULAR MAIN FUNCTIONS
MATRIX
Nervous Intertwining elongated None Transmission of nervous impulses
processes
Epithelial Aggregated polyhedral cells Only small amount Lining of surface or body cavities,
glandular secretions
Muscle Elongated contractile cells Moderate amount Movement
Connective Several types of fixed and Abundant amount Support and protection
wandering cells
PATHOLOGY: Pathology is devoted to the study of the structural, biochemical, and functional changes in cells,
tissues, and organs that underlie disease. It literally translates to study of suffering. (Greek: pathos = suffering; logos =
study). The term pathology is invoked to represent the study of disease
➢ Father of Cellular Pathology – Rudolf Ludwig Carl Virchow
➢ Father of Modern Pathology – Rudolf Ludwig Carl Virchow

Four Aspects of a disease process that form the core of Pathology:

A. Etiology – the cause
B. Pathogenesis – the biochemical and molecular mechanisms of its development
C. Morphologic changes – the structural alterations induced in the cell and organ of the body
D. Clinical manifestation – the functional consequences of these changes

1. Etiology or Cause - grouped into two classes: genetic (e.g., inherited mutations and disease-associated gene
variants, or polymorphisms) and acquired (e.g., infectious, nutritional, chemical, physical).
2. Pathogenesis - Pathogenesis refers to the sequence of cellular, biochemical, and molecular events that follow
the exposure of cells or tissues to an injurious agent.
3. Morphologic changes - Morphologic changes refer to the structural alterations in cells or tissues that are either
characteristic of a disease or diagnostic of an etiologic process.
4. Functional Derangements and Clinical Manifestations - The end results of genetic, biochemical, and structural
changes in cells and tissues are functional abnormalities, which lead to the clinical manifestations (symptoms
and signs) of disease, as well as its progress (clinical course and outcome). Hence, clinicopathologic correlations
are very important in the study of disease.

Cellular Responses to Stress and Toxic Insults: (Robbin’s and Cotran 9th)
• 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 are able to divide or contain abundant tissue stem cells.
• Atrophy: decreased cell and organ size, as a result 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. (Ex. Smoking)

INFLAMMATION
➢ It is a response of vascularized tissues to infections and damaged tissues that brings cells and molecules of host
defense from the circulation to the sites where they are needed, in order to eliminate the offending agents.
➢ A protective response involving host cells, blood vessels, and proteins and other mediators that is intended to
eliminate the initial cause of cell injury, as well as the necrotic cells and tissues resulting from the original insult,
and to initiate the process of repair.

5 Cardinal Signs of Inflammation:

1) Rubor (redness) – the result of increased blood flow at the site of inflammation
2) Calor (heat) – Due to transfer of internal heat to the site of injury
3) Tumor (swelling) – caused by fluid exudation and hyperemia
4) Dolor (pain) – resulting from release of bradykinin and PGE2; due to pressure upon the sensory nerve or it
can also be due to direct damage to the nerve
5) Functio laesa (loss of function) – caused by interference with nerve supply; can be an adaptive mechanism
by the body to protect the injured area from further damage

Classification of Inflammation:
o According to DURATION:
a) Acute Inflammation
✓ Rapid onset; Short duration
✓ Lasting from a few minutes to as long as few days
✓ Characterized by fluid and plasma protein EXUDATION
✓ Predominantly NEUTROPHILIC leukocyte accumulation
b) Subchronic Inflammation
✓ Intergrade between acute and chronic inflammation
c) Chronic Inflammation
✓ Insidious; Longer duration
✓ Lasts from days to years
✓ Associated with more tissue destruction
✓ Presence of lymphocyte and macrophages, the proliferation of blood vessels, and the
deposition of connective tissues.
 Features of Acute and Chronic Inflammation
FEATURE ACUTE CHRONIC
Onset Fast: minutes or hours Slow: days
Cellular infiltrate Mainly neutrophils Monocytes/Macrophages and
lymphocytes
Tissue injury, fibrosis Usually mild and self-limited Often severe and progressive
Local and systemic signs Prominent Less

ACUTE INFLAMMATION
Acute inflammation has three major components:
1. Dilation of small vessels leading to an increase in blood flow
2. Increased permeability of the microvasculature enabling plasma proteins and leukocytes to leave the circulation
3. Emigration of the leukocytes from the microcirculation, their accumulation in the focus of injury, and their
activation to eliminate the offending agent
IMPORTANT TERMS:
• EXUDATION: The escape of fluid, proteins, and blood cells from the vascular system into the interstitial tissue
or body cavities.
• EXUDATE: An extravascular fluid that has a high protein concentration and contains cellular debris.
• TRANSUDATE: a fluid with low protein content (most of which is albumin), little or no cellular material, and low
specific gravity.
EXUDATE TRANSUDATE
High protein concentration Low protein concentration
Contains cellular debris Little or No cellular material
Its presence indicates increased vascular permeability An ultrafiltrate of blood plasma that is produced as a
triggered by some sort of tissue injury and an ongoing result of osmotic or hydrostatic imbalance across the
inflammatory reaction. vessel wall without an increase in vascular permeability.
• EDEMA: denotes an excess of fluid in the interstitial tissue or serous cavities; it can be either an exudate or a
transudate.
• PUS, a PURULENT exudate, is an inflammatory exudate rich in leukocytes (mostly neutrophils), the debris of
dead cells and, in many cases, microbes.

Granulomatous Inflammation:
➢ A form of chronic inflammation characterized by collections of activated macrophages, often with T
lymphocytes, and sometimes associated with central necrosis.
➢ The activated macrophages may develop abundant cytoplasm and begin to resemble epithelial cells, and are
called epithelioid cells. Some activated macrophages may fuse, forming multinucleate giant cells.

Examples of Diseases with Granulomatous Inflammation:

Disease Cause Tissue Reaction
Tuberculosis Mycobacterium tuberculosis Caseating granuloma (tubercle):
focus of activated macrophages
Langhans giant cells; central necrosis
with amorphous granular debris;
acid-fast bacilli
Leprosy Mycobacterium leprae Acid-fast bacilli in macrophages;
noncaseating granulomas
Syphilis Treponema pallidum Gumma: microscopic to grossly
visible lesion, enclosing wall of
histiocytes; plasma cell infiltrate;
central cells are necrotic without
loss of cellular outline
Cat-scratch disease Gram negative bacillus Rounded or stellate granuloma
containing central granular
debris and recognizable
neutrophils; giant cells
uncommon
Sarcoidosis Unknown etiology Noncaseating granulomas with
abundant activated macrophages
Crohn disease (inflammatory bowel Immune reaction against intestinal Occasional noncaseating
disease) bacteria, possibly self-antigens granulomas in the wall of the
intestine, with dense chronic
inflammatory infiltrate

Systemic Effects of Inflammation:

• Fever: cytokines (TNF, IL-1) stimulate production of prostaglandins in hypothalamus.
• Production of acute-phase proteins: C-reactive protein, others; synthesis stimulated by cytokines (IL-6, others)
acting on liver cells
 • Leukocytosis: cytokines (colony-stimulating factors) stimulate production of leukocytes from precursors in the
bone marrow
• In some severe infections, septic shock: fall in blood pressure, disseminated intravascular coagulation,
metabolic abnormalities; induced by high levels of TNF and other cytokines.
NOTE:
✓ Defective inflammation is responsible to increased susceptibility to infections.
✓ The most common cause of defective inflammation is leukocyte deficiency resulting from replacement of the
bone marrow by leukemias and metastatic tumors, and suppression of the marrow by therapies for cancer and
graft rejection.
✓ Inherited genetic abnormalities of leukocyte adhesion and microbicidal function are rare.

Morphologic Patterns of Acute Inflammation:

The morphologic hallmarks of acute inflammatory reactions are:
1. ________________________________________
2. ________________________________________

Special Morphologic Patterns of Acute Inflammation:

Marked by the exudation of cell-poor fluid into spaces created by cell injury
or into body cavities lined by the peritoneum, pleura, or pericardium.
Example: Typically, the fluid in serous inflammation is not infected by destructive
Skin blister resulting from a burn or organisms and does not contain large numbers of leukocytes (which tend to
viral infection produce purulent inflammation).
With greater increase in vascular permeability, large molecules such as
fibrinogen pass out of the blood, and fibrin is formed and deposited in the
Example: extracellular space. A fibrinous exudate develops when the vascular leaks are
Fibrinous pericarditis large or there is a local procoagulant stimulus (e.g., cancer cells).
Characterized by the production of pus, an exudate consisting of neutrophils,
the liquefied debris of necrotic cells, and edema fluid. The most frequent
Example: cause of purulent (also called suppurative) inflammation is infection with
Abscesses bacteria that cause liquefactive tissue necrosis, such as staphylococci; these
pathogens are referred to as pyogenic (pus-producing) bacteria.
A local defect, or excavation, of the surface of an organ or tissue that is
Example: produced by the sloughing (shedding) of inflamed necrotic tissue.
Peptic ulcer

Overview of Tissue Repair:

Repair (Healing) – refers to restoration of tissue architecture and function after an injury.
2 Types of Reaction:
1. _________________– occurs by proliferation of residual (uninjured) cells that retain the capacity to divide, and
by replacement from tissue stem cells.
2. ________________________ - If the injured tissues are incapable of regeneration, or if the supporting
structures of the tissue are severely damaged, repair occurs by the laying down of connective tissue, a process
that results in scar formation.
FIBROSIS – is the most often used to describe the extensive deposition of collagen that occurs in the lungs, liver, kidneys,
and other organs as a consequence of chronic inflammation.
Morphologic Alterations in Cell Injury:
o REVERSIBLE INJURY - Two features of reversible cell injury can be recognized under the light microscope:
cellular swelling and fatty change.
• Cellular swelling - Cellular swelling appears whenever cells are incapable of maintaining ionic and fluid
homeostasis and is the result of failure of energy-dependent ion pumps in the plasma membrane.
Cellular swelling is the first manifestation of almost all forms of injury to cells. It is a difficult
morphologic change to appreciate with the light microscope; it may be more apparent at the level of
the whole organ.
NOTE: When it affects many cells, it causes some pallor, increased turgor, and increase in weight of the
organ. On microscopic examination, small clear vacuoles may be seen within the cytoplasm; these
represent distended and pinched-off segments of the ER. This pattern of nonlethal injury is sometimes
called hydropic change or vacuolar degeneration. Swelling of cells is reversible. Cells may also show
increased eosinophilic staining, which becomes much more pronounced with progression to necrosis.
The ultrastructural changes of reversible cell injury include:
1. Plasma membrane alterations, such as blebbing, blunting, and loss of microvilli
2. Mitochondrial changes, including swelling and the appearance of small amorphous densities
3. Dilation of the ER, with detachment of polysomes; intracytoplasmic myelin figures may be present
4. Nuclear alterations, with disaggregation of granular and fibrillar elements
• Fatty change - Fatty change occurs in hypoxic injury and various forms of toxic or metabolic injury. It is
manifested by the appearance of lipid vacuoles in the cytoplasm. It is seen mainly in cells involved in
and dependent on fat metabolism, such as hepatocytes and myocardial cells.
NOTES:
o Restoration of normal tissue structure can occur only if the residual tissue is structurally intact, as after partial
surgical resection.
o By contrast, if the entire tissue is damaged by infection or inflammation, regeneration is incomplete and is
accompanied by scarring.
o For example, extensive destruction of the liver with collapse of the reticulin framework, as occurs in a liver
abscess, leads to scar formation even though the remaining liver cells have the capacity to regenerate.
o LIVER REGENERATION: The human liver has a remarkable capacity to regenerate, as demonstrated by its
growth after partial hepatectomy, which may be performed for tumor resection or for living donor hepatic
transplantation.
o Regeneration of the liver occurs by two major mechanisms: proliferation of remaining hepatocytes and
repopulation from progenitor cells.

ABNORMALITIES IN CELL GROWTH:

1. Retrogressive changes
A. Developmental defects
• _____________– defective or incomplete development of a tissue or an organ
• _____________– absence of an organ
• _____________– failure of an organ to reach its mature or adult size
• _____________ – failure of an organ to form an opening
B. Atrophy - decreased cell and organ size, as a result of decreased nutrient supply or disuse; associated with
decreased synthesis of cellular building blocks and increased breakdown of cellular organelles. Atrophy can
be physiologic or pathologic.
o Physiologic Atrophy - The decrease in the size of the uterus that occurs shortly after parturition is
a form of physiologic atrophy.
o Pathologic Atrophy - Has several causes and it can be local or generalized.
The common causes of atrophy are the following:
Decreased workload When a fractured bone is immobilized in a plaster cast or when a patient is restricted
(atrophy of disuse) to complete bed rest, skeletal muscle atrophy rapidly ensues. The initial decrease in
cell size is reversible once activity is resumed. With more prolonged disuse, skeletal
muscle fibers decrease in number (due to apoptosis) as well as in size; muscle atrophy
can be accompanied by increased bone resorption, leading to osteoporosis of disuse.
Loss of innervation The normal metabolism and function of skeletal muscle are dependent on its nerve
(denervation atrophy) supply. Damage to the nerves leads to atrophy of the muscle fibers supplied by those
nerves.
Diminished blood supply A gradual decrease in blood supply (ischemia) to a tissue as a result of slowly
developing arterial occlusive disease results in atrophy of the tissue. In late adult life,
the brain may undergo progressive atrophy, mainly because of reduced blood supply
as a result of atherosclerosis. This is called senile atrophy, which also affects the heart.
Inadequate nutrition Profound protein-calorie malnutrition (marasmus) is associated with the utilization of
skeletal muscle proteins as a source of energy after other reserves such as adipose
stores have been depleted. This results in marked muscle wasting - cachexia. Cachexia
is also seen in patients with chronic inflammatory diseases and cancer. In the former,
chronic overproduction of the inflammatory cytokine tumor necrosis factor (TNF) is
thought to be responsible for appetite suppression and lipid depletion, culminating in
muscle atrophy.
Loss of endocrine Many hormone-responsive tissues, such as the breast and reproductive organs, are
stimulation dependent on endocrine stimulation for normal metabolism and function. The loss of
estrogen stimulation after menopause results in physiologic atrophy of the
endometrium, vaginal epithelium, and breast.
Pressure Tissue compression for any length of time can cause atrophy. An enlarging benign
tumor can cause atrophy in the surrounding uninvolved tissues. Atrophy in this setting
is probably the result of ischemic changes caused by compromise of the blood supply
by the pressure exerted by the expanding mass.

2. Progressive Changes
• _____________– increase in the tissue/organ size due to increase in the cell size
• Physiologic – due to a natural process
• Pathologic – due to a removal of a paired organ
• _____________ – increase in the tissue/organ size due to increase in the number of cells
3. Degenerative Changes
• ____________– reversible transformation of one adult cell type to another cell type
• ____________ – when one mature cell changes in structural components of one cell type
• ____________ – when one mature cell transforms to more primitive form
• ____________ – the continuous abnormal proliferation of cells
NEOPLASIA:
→ Literally means “new growth.” Neoplastic cells are said to be transformed because they continue to replicate,
apparently oblivious to the regulatory influences that control normal cell growth.
→ In common medical usage, a neoplasm often is referred to as a tumor, and the study of tumors is called oncology
(from oncos, “tumor,” and logos, “study of”).

Basic Components of a Tumor:

1. _________________ - made up of transformed or neoplastic cells
2. _________________ - the supporting, host-derived, non-neoplastic cells, made up of connective tissue, blood
vessels, and host-derived inflammatory cells.

BENIGN TUMORS MALIGNANT TUMORS

Remains localized at its site of origin Locally invasive and metastasize to distant sites
Most develop an enclosing fibrous capsule that Poorly circumscribed and invade the surrounding normal
separates them from the host tissue tissues
NOTE: NOT all benign neoplasms are encapsulated
(Ex. leiomyoma of the uterus)
Usually slow-growing Generally, grow faster
Usually well-differentiated Malignant tumors are poorly or completely
undifferentiated (anaplastic)

Summary of Histological Features of Neoplasm:

BENIGN MALIGNANT
Behavior Expansile growth only; grows locally Expansile and invasive growth; may metastasize;
Often encapsulated Not encapsulated
Histology Resembles cell of origin (well-differentiated) May show failure of cellular differentiation
Few mitoses Many mitoses, some of which are abnormal form
Normal or slight increase in ratio of nucleus to High nuclear to cytoplasmic ratio
cytoplasm
Cells are uniform through the tumor Cells vary in shape and size (cellular
pleomorphism) and/or nuclei vary in shape and
size (nuclear pleomorphism)
Modes of Spread of Malignant Neoplasms:
1. ___________________ - Invasive tumors tend to spread into surrounding tissues by the most direct route.
Examples include carcinoma of the breast invading into overlying skin or deeply into underlying muscle, and
carcinoma of the cervix invading into the rectum or bladder.
2. _______________________ - Tumors may spread via lymphatic vessels draining the site of the primary tumor;
neoplastic cells are conducted to local lymph nodes where they become trapped and set up secondary tumors,
e.g. breast cancer spreading to lymph nodes in the axilla, or carcinoma of the tongue spreading to lymph nodes
in the neck.
3. _______________________ - Tumors can spread via the venules veins draining the primary site; gut tumors
tend to spread via the portal vein to the liver where secondary tumors are very common. In the systemic
circulation, neoplastic cells may be trapped in the capillaries of the lungs to form pulmonary metastases.
4. _______________________ - Certain tumors can spread directly across coelomic spaces, for example across
the peritoneal or pleural cavities. Carcinoma of the ovary may spread transcoelomically to produce large
numbers of metastatic deposits on the peritoneal surfaces.
Differentiation of Tumor depending on histological characteristics:
a. _________________ - Where there are more cells than supporting tissue; It is soft and very malignant.
b. _________________ - (stony/hard): Where there is more connective tissue than cells, where the cells are
apparently trapped within the fibrous tissue.

Grading of Tumors (Broder’s Classification):

The grading of tumors based on cellular differentiation.
_____________________: resemble normal cells
_______________________: resemble younger forms (AKA: ___________ in form)

Broder’s Classification
Grade Differentiated Cells Undifferentiated Cells
Grade I 75-100% 0-25%
Grade II 50-75% 25-50%
Grade III 25-50% 50-75%
Grade IV 0-25% 75-100%
TNM System of Cancer Staging:
It is the most widely used method and it involves scoring the extent of local Tumor spread, regional lymph Node
involvement and the presence of distant Metastases.
‘T’ score – size of the tumor
‘N’ score – spread to the lymph nodes
‘M’ score – presence of metastasis

NOMENCLATURE OF NEOPLASMS:
i. BENIGN – suffix used: ________oma_
ii. MALIGNANT:
A. Mesenchymal/Connective Tissue – suffix used: _____________
B. Epithelial Tissue – suffix used: _____________

NECROSIS:
→ Necrosis is a pathologic process caused by direct external injury to cells such as, from burns, radiation, or toxins.
→ The morphologic appearance of necrosis as well as necroptosis is the result of denaturation of intracellular
proteins and enzymatic digestion of the lethally injured cell.
→ Necrotic cells show increased eosinophilia in hematoxylin and eosin (H & E) stains, attributable in part to the
loss of cytoplasmic RNA (which binds the blue dye, hematoxylin) and in part to denatured cytoplasmic proteins
(which bind the red dye, eosin).

Patterns of Tissue Necrosis:

COAGULATIVE NECROSIS A form of necrosis in which the architecture of dead tissues is preserved for a span of
at least some days. Ischemia caused by obstruction in a vessel may lead to a
coagulative necrosis of the supplied tissue in all organs except the brain.
NOTE: Infarct – a localized area of coagulative necrosis
LIQUEFACTIVE NECROSIS Characterized by digestion of dead cells, resulting in transformation of the tissue into
a liquid viscous mass.
NOTE: The necrotic material is frequently creamy yellow because of the presence of
dead leukocytes and is called pus.
GANGRENOUS NECROSIS Not a specific pattern of cell death; usually applied too limb, generally the lower leg,
that has lost its blood supply and has undergone coagulative necrosis.
CASEOUS NECROSIS Refers to the cheese-like friable white appearance of the area of necrosis; Often seen
in tuberculosis
NOTE: Granuloma – Necrotic area appears as structureless collection of fragmented
or lysed cells and amorphous granular debris enclosed within a distinctive
inflammatory border on microscopic examination
FAT NECROSIS FAT NECROSIS – It refers to focal areas of fat destruction, typically resulting from
release of activated pancreatic lipases into the substance of the pancreas and
peritoneal cavity.
NOTE: On histologic examination, the necrosis takes the form of foci of shadowy
outlines of necrotic fat cells, with basophilic calcium deposits, surrounded by an
inflammatory reaction.
FIBRINOID NECROSIS FIBRINOID NECROSIS – A special form of necrosis usually seen in immune reactions
involving blood vessels. This pattern of necrosis typically occurs when complexes of
antigens and antibodies are deposited in the walls of the arteries. Deposits of these
“immune complexes,” together with fibrin that has leaked of vessels, result in a
bright pink and amorphous appearance in H&E stains, called “fibrinoid” (fibrin-like)
by pathologists.

Nuclear Changes in Necrosis:

Nuclear changes appear in one of three patterns, all due to nonspecific breakdown of DNA.
PYKNOSIS Reduction and Characterized by nuclear shrinkage and increased basophilia.
condensation of nucleus The chromatin condenses into a solid, shrunken basophilic
mass.
KARYORRHEXIS Fragmentation of The pyknotic nucleus undergoes fragmentation. With the
nucleus passage of time (a day or two), the nucleus in the necrotic cell
totally disappears.
KARYOLYSIS Dissolution of nucleus The basophilia of the chromatin may fade (karyolysis), a change
that presumably reflects loss of DNA because of enzymatic
degradation by endonucleases.

Apoptosis:
• “Programmed cell death”
• A form of cell death that is characterized by nuclear dissolution, fragmentation of the cell without complete
loss of membrane integrity, and rapid removal of the cellular debris.
 FEATURES OF NECROSIS AND APOPTOSIS
NECROSIS APOPTOSIS
Cell size Enlarged (swelling) Reduced (shrinkage)
Nucleus Pyknosis →karyorrhexis →karyolysis Fragmentation into nucleosome size
fragments
Plasma membrane Disrupted Intact; altered structure, especially
orientation of lipids
Cellular contents Enzymatic digestion; may leak out of Intact; may be released in apoptotic
cell bodies
Adjacent inflammation Frequent No
Physiologic or pathologic role Invariably pathologic (culmination of Often physiologic, means of
irreversible cell injury) eliminating unwanted cells; may be
pathologic after some forms of cell
injury, especially DNA damage

The following morphologic features of cells undergoing APOPTOSIS, some best seen with the electron microscope:
1. Cell shrinkage. The cell is smaller in size, the cytoplasm is dense, and the organelles, although relatively normal,
are more tightly packed. (Recall that in other forms of cell injury, an early feature is cell swelling, not shrinkage.)
2. Chromatin condensation. This is the most characteristic feature of apoptosis. The chromatin aggregates
peripherally, under the nuclear membrane, into dense masses of various shapes and sizes. The nucleus itself
may break up, producing two or more fragments.
3. Formation of cytoplasmic blebs and apoptotic bodies. The apoptotic cell first shows extensive surface
blebbing, then undergoes fragmentation into membrane-bound apoptotic bodies composed of cytoplasm and
tightly packed organelles, with or without nuclear fragments.
4. Phagocytosis of apoptotic cells or cell bodies, usually by macrophages. The apoptotic bodies are rapidly
ingested by phagocytes and degraded by the phagocyte’s lysosomal enzymes.

Primary Signs of Death:

1. Circulatory failure – cardiac function ceases; absence of pulse and heartbeat
2. Respiratory failure – absence of oxygen; accumulation of carbon dioxide
3. Nervous failure – loss of coordination of various body function; chiefly loss of reflex

Secondary Signs of Death:

Algor mortis o First change to be observed
o “Cooling of the body”
o Used to determine time of death
Livor mortis o Purplish discoloration of the skin
o Lividity of the skin
Rigor mortis o Rigidity or stiffening of muscle occurring 6 to 12 hours after death
o Persists for 3 to 4 days
Post-mortem o Settling or separation of red cells from the fluid phase of blood
clotting
Desiccation o Drying of wrinkling of the cornea and the anterior chamber of the eye
Putrefaction o Production of foul-smelling gases due to saprophytic microbe invasion (accumulation of
saprophytic microbes)
Autolysis o Self-digestion of cells
HISTOTECHNOLOGY:
Microscopy
1. Bright-Field Microscopy – widely used by students of histology; used by histotechnologist and pathologist in
viewing results of enzyme immunohistochemistry slide.
Different parts:
• Condenser – collects and focuses a cone of light that illuminates the object to be observed.
• Objective lens – enlarges and projects the image of the object in the direction of the eyepiece.
• Eyepiece or Ocular lens – further magnifies this image (secondary magnification) and projects it onto
the viewer’s retina or a charge-coupled device (CCD)
2. Fluorescence Microscopy - tissue sections are usually irradiated with ultraviolet (UV) light and the emission is
in the visible portion of the spectrum. Fluorescence is the light emitted with characteristic low energy and
longer wavelength. The fluorescent substances appear brilliant on a dark background.
3. Phase-Contrast Microscopy – a modified light microscope used to view unstained cells and tissue sections; uses
a lens system that produces visible images from transparent objects, and it can be used for living or culture
cells.
NOTE: A modification of phase-contrast microscopy is differential interference microscopy with Nomarski optics,
which produces an image of living cells with a more apparent three-dimensional (3D) aspect.
4. Confocal Microscopy – limits the problem with stray (excess) light; achieves high resolution and sharp focus by
using (1) a small point of high-intensity light, often from a laser, and (2) a plate with a pinhole aperture in front
of the image detector.
5. Polarizing Microscopy - allows the recognition of stained or unstained structures made of highly organized
subunits. When normal light passes through a polarizing filter, it exits vibrating in only one direction.
NOTE: The ability to rotate the direction of vibration of polarized light is called birefringence and is a feature of
crystalline substances or substances containing highly oriented molecules, such as cellulose, collagen, microtubules,
and actin filaments.
6. Electron Microscopy - based on the interaction of tissue components with beams of electrons. The wavelength
in the electron beam is much shorter than that of light, allowing a 1000-fold increase in resolution.
NOTE: Transmission Electron Microscope (TEM) is an imaging system that permits resolution around 3 nm.

Microtome:
Three essential parts:
1. Block Holder – Holds the tissue block in proper position
2. Knife and Knife Carrier – used for the actual cutting of tissue sections
3. Paul, Rachet Feed, and Adjustment Screws – It lines up the tissue block in proper position with knife, adjusting
thickness of tissue for successive sections.
Different Types of Microtome:
1. Rocking Microtome or Cambridge Microtome (Inventor: Paldwell Trefall)
→ for cutting serial sections of large blocks of paraffin embedded tissue
→ Simplest type
2. Rotary Microtome (Inventor: Charles Minot)
→ for cutting paraffin embedded sections
→ The most common type of microtome used for both routine and research laboratories
3. Sliding Microtome (Inventor: Adams)
→ for cutting celloidin embedded sections
→ The most dangerous type of microtome due to its movable knife (Standard Sliding Microtome)
→ 2 Types: Standard Sliding (movable knife) and Base Sledge Microtome
→ Base Sledge Microtome: Ideal for resin-embedded decalcified bone (heavy-duty electrically driven). It
is used for all forms of media especially for hard tissue or large blocks.
NOTE: Block holder – moving; Knife – stationary
→ Standard Sliding Microtome: developed for celloidin-embedded tissue blocks; most dangerous due to
its movable knife
NOTE: Block holder – stationary; Knife – moving (forward and backward motion)
4. Freezing Microtome (Inventor: Queckett)
→ Used to cut un-dehydrated tissues in frozen state (for unembedded frozen section)
→ Most common freezing agent used is Carbon dioxide (CO2)
5. Cryostat or Cold Microtome
→ It is the most commonly used for rapid preparations
→ It is a refrigerated apparatus used in fresh tissue microtomy (intraoperative procedures)
→ Temperature is maintained by an adjustable thermostat at -5 to -30°C (Average is -20°C)
→ The usual type of microtome enclosed inside is the Rotary Microtome.
6. Ultra-thin Microtome
→ Used to produce sections for Electron microscopy
→ Tissues are embedded in Plastic resin
→ Uses special knives: Glass knives & Diamond knives
Section thickness:
A. Semi-Thin: 0.5 to 1 micron (Glass knives)
B. Ultra-Thin: 500 to 1200 angstrom (50 to 120 nm) (Diamond knives)
 CRYOSTAT or COLD MICROTOME:
• It is most commonly used for rapid preparation of urgent tissue biopsies for intraoperative diagnosis.
• The cryostat provides a means of preparing thin sections of fresh frozen tissues especially for fluorescent
antibody staining techniques or histochemical enzyme studies.

➢ FROZEN SECTION - Most ideal and preferred means of preserving tissues for histochemical evaluation involving
enzyme studies.
➢ CARBON DIOXIDE - The freezing agent commonly employed for frozen sections.

METHOD QUENCHING DESSICATION

Freeze-drying -160°C to -180°C -30°C to -40°C
Freeze-substitution -160°C -70°C

Difference between Freeze-Drying and Freeze-Substitution:

The only variation is that in FREEZE-SUBSTITUTION, the frozen tissue instead of being subjected to dehydration in an
expensive vacuum drying apparatus, is fixed in Rossman's formula or in 1% Acetone and dehydrated in absolute alcohol.

FIXATIVES:
• The first and most critical step in histotechnology is ____________.
• Inhibits decay, autolysis, and putrefaction of specimen.
• Fixation should be carried out as soon as possible after removal of the tissues (in the case of surgical pathology)
or soon after death (in the case of autopsy) to prevent autolysis.
Objectives of Fixation:
1. ________________________________________________________________
2. ________________________________________________________________

Different types of Fixatives according to Composition:

1. Simple Fixative – it is made up of only one component substance.
2. Compound Fixative – made up of two or more fixatives.
Different types of Fixatives according to their function:
1. MICROANATOMICAL FIXATIVES
• Fixatives that permit the general microscopic study of histological tissue structures without altering its
structural pattern and normal intercellular relationship
• REMEMBER: Microanatomical fixatives should NOT contain ___________________ because it is known
to inhibit Hematoxylin (nuclear stain)
2. CYTOLOGICAL FIXATIVES
• Those that preserve specific parts and particular microscopic elements of the cell.
A. Nuclear Fixatives
• Fixes nuclear structure.
• Must contain _______________ as their primary component due to its affinity for nuclear chromatin.
• pH ______ or less
B. Cytoplasmic Fixatives
• Preserve the cytoplasmic structures.
• It must NEVER contain ________________ because it destroys the mitochondria and Golgi bodies of
the cell.
• pH of more than ______
3. HISTOCHEMICAL FIXATIVES
• Preserve the chemical constituents of cells and tissues.

Histochemical Fixatives Microanatomical Fixatives Cytological Fixatives

Acetone 10% Formol Saline Nuclear Fixatives Cytoplasmic Fixatives
Absolute Ethyl Alcohol 10% Neutral Buffered Bouin’s fluid Helly’s
10% Formol Saline Formalin Flemming’s Orth’s fluid
Newcomer’s fluid Bouin’s solution Newcomer’s Regaud’s
Brasil’s solution Carnoy’s fluid Flemming’s fluid WITHOUT
Heidenhain’s Susa Heidenhain’s Susa Acetic acid
Formol Sublimate (Formol Formalin with Post
Corrosive) Chroming
Zenker’s Solution
Zenker-Formol (Helly’s)

Secondary Fixation: A process wherein an already fixed tissue specimen is placed in a second fixative.

1. To facilitate and improve the demonstration of a particular substance.

2. To make special staining techniques possible (secondary fixative acting as mordant).
3. To ensure complete and further hardening and preservation of tissues.
Post chromatization is a form of secondary fixation whereby a primarily fixed tissue is in a 2.5 to 3% Potassium
Dichromate solution for 24 hours to act as mordant for better staining effects and to aid in cytologic preservation of
tissues.

Washing out: Process of removing excess fixative from tissue after fixation to improve staining, and to remove artifacts
from the tissues.

1. Tap water
2. 50 to 70% Alcohol
3. Alcoholic Iodine
Fixatives for Smears: Fixatives that are Volatile (Explosive):
• Schaudinn’s fixative • Formalin – when neutralized
• Ether alcohol • Dioxane – should not be recycled; creates explosive peroxides
• Methanol • Picric acid – explosive when dry
• Low Viscosity Nitrocellulose (LVN) – explosive when dry
• Ammoniacal Silver Solution – when dry
• Other Silver solutions – when aging

Chemical Fixatives:
• Stabilize the proteins, nucleic acids and internal components of the tissue by making them insoluble
There are two major groups of chemical fixatives, classified according to their mechanism of action:

• Crosslinking Fixatives (e.g., Aldehydes) that act by creating covalent chemical bonds between proteins in tissue.
This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to the tissue.
• Precipitating (or denaturing) fixatives (e.g., alcoholic fixatives) that act by reducing the solubility of protein
molecules and (often) by disrupting the hydrophobic interactions that give many proteins their tertiary
structure. The precipitation and aggregation of proteins is a very different process from the crosslinking that
occurs with the aldehyde fixatives.
ALDEHYDE FIXATIVES
1. Formaldehyde – The most commonly used fixative in histology. It fixes the tissues by forming cross-linkages in the
proteins, particularly between lysine residues.
• A good fixative for immunohistochemical techniques.
• The standard solution is 10% neutral buffered formalin or approximately 3.7%-4.0% formaldehyde in
phosphate buffered saline.
• Phosphate Buffer - prevents acidity that would promote autolysis and cause precipitation of formol-heme
pigment in the tissues.
• Other benefits of formaldehyde include long term storage and good tissue penetration.
• A good fixative for immunohistochemistry techniques, and formaldehyde vapor can be used as a fixative
for cell smears.
• Formaldehyde is a gas produced by the oxidation of methyl alcohol. It is soluble in water to the extent of
37 to 40% weight in volume.
• The commercially available solution of formaldehyde contains 35-40% gas by weight (equivalent to 40%
stock or concentrated formalin).
• Formalin is prepared from commercial-grade 37 to 40% formaldehyde, by diluting the concentrated
formalin 1:10 in phosphate buffer.
• The most widely used fixative for routine histology is 10% neutral buffered formalin (NBF, approximately
4% formaldehyde), buffered to pH 7 with phosphate buffer.
• It is considered the fixative of choice for many other procedures that require paraffin embedding,
including immunohistochemistry and interphase Fluorescent In-Situ Hybridization (FISH).

Notes to Remember:

• Recommended for colored tissue photography

• Allows frozen sections to be prepared easily
• A ‘tolerant fixative’ (for long term storage), used for mailing the specimen.
• Disadvantages: fumes are irritating to the nose and eyes and may cause sinusitis, allergic rhinitis or excessive
lacrimation. Solution is irritating to the skin and may cause allergic dermatitis on prolonged contact.
• 10% Methanol can be added as a preservative to formaldehyde to prevent precipitation of white
paraformaldehyde thus making it unsuitable for electron microscopy.
• Concentrated solutions should never be neutralized since it might precipitate violent explosions.
• Bleaching may be prevented by changing the solution every 3 months.
• It is a known carcinogen because the metabolite of Formaldehyde is methanol which can cause cancer and toxic
levels can lead to blindness.
• Cadmium or Cobalt salts are added to prevent dispersion of fat into the fluid.
 • Acid reaction due to formic acid formation can be neutralized by adding magnesium carbonate or calcium
carbonate to 10 to 15% Formalin.
• Calcium acetate can also be used to buffer formalin but it leaves calcium deposits.

Formaldehyde disposal:

✓ Can be recycled by distillation

✓ Can be detoxified by a commercial product
✓ Can be disposed by a licensed waste hauler

3) Automatic Tissue Processor

▪ Fixation Stations 1-2 10% formalin

▪ Dehydration Stations 3-6 Ascending grads of ethyl alcohol
▪ Clearing 70% to 95%
▪ Infiltration (AKA impregnation) Stations 7-8 2 changes of Acetone
Stations 9-10 2 changes of Chloroform or Xylol
4) Vacuum Embedding Apparatus
Stations 11-12 2 changes of liquid Paraffin
▪ Wax impregnation under negative atmospheric pressure inside an embedding oven.
▪ Vacuum impregnation gives the fastest result.

5) Flotation Water Bath

▪ Used to float out and flatten paraffin ribbons. 7° Fx. Of Flotation water bath
▪ Set at a temperature of 45°C to 50°C.
▪ Approximately 6°C to 10°C above than the melting point of the wax used.
▪ Small amount of detergent may be added to the water to reduce surface tension.

6) Oven (Paraffin Oven/Drying Oven)

▪ Infiltration process: regulated at 55°C to 60°C.

▪ Drying process: done at 56-60°C for 2 hours.

7) Hot Plate

▪ Hot plate may be used instead of drying oven, although it is not recommended since they can cause overheating
and there is a risk of dust falling onto the section during the drying period.

8) Forceps (fine pointed or curved) and Squirrel Hair Brush

▪ Used in handling sections during cutting, and for removing folds and creases on the sections during “floating
out” in water bath.

9) Clean slides

a.) Coplin Jars: Slotted jar holding from 5 to 9 slides.

b.) Slotted staining dishes: Holding from 5 to 19 slides, over which different solutions are poured. Slides are placed
on end singly or in staggered fashion, in the arm.

c.) Metal or glass staining racks or carriers: Holding from 10 to 30 slides upright. Slides are transferred to
appropriately sized glass dishes containing the staining solutions.

Types of Biopsy Specimen

Shave Biopsy Small fragments of tissue are “shaved” from a surface.
Incisional Biopsy Takes out even more surrounding tissue. It takes out some of the abnormalities, but
not all. The doctor will slice into the lesion and remove only a portion of it. If the
lesion is found to be cancerous, further surgery may be needed to remove or excise
the entire lesion.
Core Needle Biopsy Removes not only cells, but also a small amount of surrounding tissue. This provides
additional information to assist in the examination of the lesion.
Curettings Tissue is scooped or spooned to remove the tissue or growth from the body cavity
such as endometrium or cervical canal.
Fine Needle Aspiration Simplest, least invasive test and uses the smallest needle to remove cells from the
area of abnormality.
Punch Biopsy Considered the primary technique for obtaining diagnostic full-thickness skin
specimen. This requires surgical and suture-tying skills. This technique involves the
use of a circular blade that is rotated down through the epidermis and dermis, and
into the subcutaneous fat, yielding a 3 to 4 mm cylindrical core of tissue sample.
Excisional Biopsy Removes the entire area in question.
 Methods of Fresh Tissue Examination
Teasing/Dissociation Selected tissue specimen is immersed in a watch glass containing isotonic salt solution,
carefully dissected or separated and examined under the microscope.
Squash Preparation Small pieces of tissue not more than 1 mm, in diameter placed in a microscopic slide and
(Crushing) forcibly compressed with another slide or with a cover glass; Vital stain may be placed at
the junction of the slide and cover glass and allowed to absorb through capillary
attraction
Smear Preparation Techniques useful in cytological examinations particularly for cancer diagnosis.

Streaking Use of an applicator stick or platinum loop, material is

rapidly and gently applied in a direct or zigzag line
throughout the slide.

Spreading Material is transferred in a clean slide and gently spread

into a moderately thick film by teasing using an applicator
stick; More tedious than steaking and it maintains cellular
interrelationships of the material. Recommended for
preparations of fresh sputum, bronchial aspirates, and
thick mucoid secretions.
Pull-Apart A drop of secretion or sediment upon 1 slide and facing it
to another clean slide. 2 slides are pulled apart with a
single uninterrupted motion. Useful in preparation of thick
secretions.
(Serous fluids, concentrated sputum, enzymatic lavage
samples from gastrointestinal tract and blood smear).
Touch Preparation Special method of smear preparation whereby the surface
(Impression smear) of the freshly cut tissue is brought into contact and pressed
on the surface of a clean glass slide.
Frozen sectioning For rapid diagnosis during surgery

Pre-Analytical Factors in Tissue Processing

1. Warm Ischemia: Term used for the initial anoxic insult a tissue suffers when blood supply is cut off.
2. Cold Ischemia: Lack of oxygen, once the tissue sample is removed from the patient’s body and before all
metabolic processes are stopped.
3. Fixation: The earlier the tissue is fixed, the better its preservation. Most critical step in MANUAL
Histotechniques
4. Properly Filled-Surgical Pathology Request
5. Accessioning Procedure
HISTOTECHNIQUES
1. Fixation
2. Decalcification (*special histotechnique; only for Calcified spx.)
3. Dehydration
4. Clearing
5. Infiltration / Impregnation
6. Embedding / Casting / Molding / Blocking
7. Trimming
8. Sectioning/ Cutting (Microtomy)
9. Staining
10. Mounting
11. Ringing
12. Labelling

NUMBERING
S SURGICAL
A AUTOPSY
C CYTOLOGY
Year is indicated usually in 2 digits
Accession Number (unique identifier for each patient)
PENCIL is utilized
 1. FIXATION:
• Classically defined as killing, penetration and hardening of tissues.
• Currently defined as the alteration of tissues by stabilizing protein so that the tissues become resistant to further
changes (i.e., degeneration, decomposition, putrefaction, and tissue distortion).
▪ _______________________ in histotechnology.
No process of histotechnology is more critical to slide preparation than fixation.

▪ _________________: Preserve the morphologic and chemical integrity of the cell in as life-like manner as possible.

▪ _________________: Harden and protect tissue from trauma of further handling, so that it is easier to cut during
gross examination.
▪ Most important reaction in maintaining the morphology of the tissue in fixation: Stabilization of proteins.

Actions of Fixative:

❖ Preserve the tissue.

❖ Prevent breakdown of cellular elements.
❖ Coagulate or precipitate protoplasmic substances.

Main Factors Involved in Fixation (2nd Ed.):

1. pH (Hydrogen Ion Concentration):

2. Temperature:

3. Thickness of section:

4. Osmolality:

5. Concentration:

6. Duration of fixation:

Practical Considerations of Fixation (2nd Ed.):

1. Speed:

2. Rate of Penetration:

3. Volume:

4. Duration of Fixation:

Effects of Fixatives in General:

▪ Harden soft friable tissues and make the handling and cutting of sections easier.
▪ They make cells resistant to damage and distortion by hypotonic and hypertonic solutions used.
▪ Inhibit bacterial decomposition.
▪ Increase optical differentiation of cell and tissue components.
▪ They act as mordants or accentuators to promote and hasten staining.
▪ Reduce risk of infections during handling and actual processing of tissues.

Characteristics of Good Fixative:

▪ It must be cheap, stable, and safe to handle.

▪ It must kill cells quickly; It must inhibit bacterial decomposition and autolysis.
▪ It must produce minimum shrinkage of tissues.
▪ It must permit rapid and even penetration of tissues; It must harden tissues.
▪ It must make cellular components insoluble by hypotonic solution and render them insensitive to subsequent
processing.
▪ It must permit application of many staining procedures.
▪ It must be isotonic causing minimal physical and chemical alteration of the cells and their constituents. This is not
however, a strict rule since there are some hypotonic solutions (i.e., glacial acetic acid) produce tissue swelling which
 are being used in conjunction with hypertonic solutions (e.g., picric acid) causing shrinkage of cells, to produce a
compound which would give an optimal effect on the tissues structure.)

Miscellaneous Consideration:

▪ Tissue selected for sectioning should be thin enough to allow penetration by fixative within a reasonable amount
of time.
❖ The recommend size of the tissue is 2cm², and no more than 4mm thick.
▪ Most tissue can be out and trimmed without prior fixation, EXCEPT for the BRAIN which is generally soft when
unfixed.
❖ The brain must be fixed “grossing” or sectioning.
▪ Refrigerator is used to slow down decomposition if the tissue needs to be photographed and cannot be fixed
immediately.
❖ (Body cells die at different time intervals. Brain cells, in particular, deteriorate very quickly. On the other
hand, bone marrow continues to undergo mitosis up to 30 minutes after death when refrigerated.)

Factors that affect Fixation of Tissues:

Factors that ENHANCE (ACCELERATES) the fixation Factors that RETARD (SLOWS) the fixation
▪ _____________ (Smaller and thinner tissues) ▪ ________________ (Larger tissues)
▪ _____________ ▪ Presence of ____________
▪ _____________ (37°C) ▪ Presence of ____________
▪ Presence of ____________
▪ _____________

Major Groups of Chemical Fixatives:

1. Aldehydes
2. Oxidizing Agents
3. Alcohol based Fixatives
4. Metallic Group of Fixatives

Two basic Mechanisms Involved in Fixation

1. _________________: Chemical constituent of the fixative is taken in and becomes a part of the tissue.
2. _________________: Fixing agent is not taken into the tissue. But it alters the tissue composition thus,
stabilizing the tissue which makes them unsuitable for bacterial decomposition.

Types of Fixatives According to Composition:

1. ________________: Made up of only one component substance.

2. ________________: Made up of two or more fixatives.

Types of Fixatives According to Action:

1. Microanatomical Fixatives:
• Are those that permit the general microscopic study of tissue structures without altering the structural
pattern and normal intercellular relationship of the tissue in question.
• It must never contain __________________________ because it inhibits hematoxylin.
2. Cytological Fixatives:
✓ Are those that preserve specific parts and particular microscopic elements of the cell
a.) Nuclear Fixatives
✓ Preserve the nuclear structures
✓ Contain ______________ as their primary component due to its affinity for nuclear
chromatin
✓ pH 4.6 or less

b.) Cytoplasmic Fixatives

✓ Preserve the cytoplasmic structures
✓ Must NEVER contain _________________ which destroys the mitochondria and golgi bodies
✓ pH of more than 4.6
3. Histochemical Fixatives:
✓ Are those that preserve the chemical constituents of cells and tissues.
 Histochemical Fixatives Microanatomical Fixatives Cytological Fixatives
Acetone 10% Formol Saline Nuclear Fixatives Cytoplasmic Fixatives
Absolute Ethyl Alcohol 10% Neutral Buffered Bouin’s fluid Helly’s
10% Formol Saline Formalin Flemming’s Orth’s fluid
Newcomer’s fluid Blouin’s solution Newcomer’s Regaud’s
Brasil’s solution Carnoy’s fluid Flemming’s fluid WITHOUT
Heidenhain’s Susa Heidenhain’s Susa Acetic acid
Formol Sublimate (Formol Formalin with Post
Corrosive) Chroming
Zenker’s Solution
Zenker-Formol (Helly’s)

Secondary Fixation: A process wherein an already fixed tissue specimen is placed in a second fixative.

1. To facilitate and improve the demonstration of a particular substance.

2. To make special staining techniques possible (secondary fixative acting as mordant).
3. To ensure complete and further hardening and preservation of tissues.

Post chromatization is a form of secondary fixation whereby a primarily fixed tissue is in a 2.5 to 3% Potassium
Dichromate solution for 24 hours to act as mordant for better staining effects and to aid in cytologic preservation of
tissues.

Washing out: Process of removing excess fixative from tissue after fixation to improve staining, and to remove artifacts
from the tissues.

1. Tap water
2. 50 to 70% Alcohol
3. Alcoholic Iodine

CHEMICAL FIXATIVES

ALDEHYDE FIXATIVES
1. Formaldehyde (Formalin)
✓ 10% formalin is one of the most commonly used fixatives.
✓ Formaldehyde is usually buffered at pH7 with phosphate buffer.
Advantages:
✓ Preserves fats and mucin, allowing them to be stained for demonstration.
✓ Fixative of choice for immunohistochemistry and interphase Fluorescent In-Situ Hybridization.
✓ It does not make tissues brittle, and is therefore, recommended for nervous tissue demonstration.
✓ Allows natural tissue colors to be restored after fixation by immersing formalin-fixed tissues in 70% alcohol for
1 hour, therefore recommended for colored tissue photography.
✓ Allows frozen sections to be prepared easily.
✓ A ‘tolerant fixative’ (for long term storage), used for mailing the specimen.
Disadvantages:
✓ Fumes are irritating to the nose and eyes and may cause sinusitis, allergic rhinitis, or excessive lacrimation.
✓ Solution is irritating to the skin and may cause allergic dermatitis on prolonged contact.
Precautions:
✓ Prolonged storage of formaldehyde, especially at very low temperature, may induce precipitation of white
paraformaldehyde deposits (which can be removed via filtration or by adding 10% methanol)
✓ Methanol added as a preservative to formaldehyde.
✓ Concentrated solutions should never be neutralized since it might precipitate violent explosions.
✓ Bleaching may be prevented by changing the fluid every three months.
✓ Brown or black crystalline precipitate formed by the action of formic acid with blood can be removed from the
sections prior. Saturated alcoholic picric acid or a 1% solution of potassium hydroxide in 80% alcohol. The use
of 10% neutral (phosphate) buffered formalin will prevent pigmentation.
✓ Cadmium/cobalt salts are added to prevent dispersion of fat.
✓ Acid reaction due to formic acid formation can be buffered or neutralized by adding magnesium carbonate or
calcium carbonate to 10 to 15% formalin. Calcium acetate can also be used to buffer formalin, but it leaves
calcium deposits.
Formaldehyde disposal:

✓ Can be recycled by distillation.

✓ Can be detoxified by a commercial product.
✓ Can be disposed by a licensed waste hauler.
10% Formol Saline
✓ Recommended for fixation of central nervous tissues and general post-mortem tissues.
Composition:
✓ 40% Formaldehyde
✓ Sodium Chloride
✓ Distilled water
Advantages:
✓ Preserves microanatomic and cytologic details with minimum shrinkage and distortion.
✓ Preserves enzymes and nucleoproteins.
✓ Ideal for most staining techniques. Including silver impregnation.

10% Neutral Buffered Formalin/Phosphate Buffered Formalin (pH7)

✓ Recommended for preservation and storage of surgical, post mortem, and research specimens
Advantages:
✓ Prevents precipitation of acid formalin pigments.
✓ Best fixative for tissues containing iron pigments.
Disadvantage:
✓ Positivity of mucin to PAS is reduced

Formal-Corrosive (Formal-Sublimate)
✓ Formol-Mercuric Chloride solution is recommended for routine post-mortem tissues.
Advantage:
✓ Excellent for many staining procedures including silver reticulum methods.
Disadvantages:
✓ Forms mercuric chloride deposits.
✓ Does not allow frozen tissue sections to be made.

Alcoholic Formalin (Gendre’s Fluid) Fixative

Advantages:
✓ Good preservation of glycogen and for micro-incineration technique.
✓ Used to fix sputum, since it coagulates mucus.

Paraformaldehyde
✓ Polymerized form of formaldehyde, usually obtained as a fine white powder, which depolymerizes back to formalin
when heated. Can be used in EM formation.

Glutaraldehyde
✓ Made up of 2 formaldehyde residues, linked by a three-carbon chain.
✓ Satisfactory for electron microscopy

Advantage of glutaraldehyde over formaldehyde:

✓ Preserves cellular structures better; hence recommended for enzyme histochemistry and electron microscopy (fixes
well at 4C)

Disadvantage:
✓ Reduces PAS positivity of reactive mucin.

Precaution:
✓ Specimen vial must be kept refrigerated during the fixation process.
 METALLIC FIXATIVES
1. Mercury Chloride Fixatives
Mercuric Chloride
✓ Tissues fixed with mixtures containing mercury chloride contain black Mercuric deposits may be
precipitates of mercury except Susa removed by immersing
tissues in alcoholic iodine
Advantages: solution prior to staining
✓ Routine fixative of choice for preservation of cell detail in tissue photography through a process known as
✓ Permits brilliant metachromatic staining of cells dezenkerization. Chemically,
✓ Recommended for renal tissues, fibrin, connective tissues and this is done by the oxidation
✓ muscles with sodium to mercuric
iodide, which can be
Disadvantages:
subsequently removed by
✓ Causes marked shrinkage of cells
treatment with sodium
✓ Leads to the formation of black granular deposits
✓ Extremely corrosive to metals thiosulfate.

Precaution: Black deposits may be removed by adding saturated iodine solution

in 96% alcohol

1.A. Zenker’s Fluid Mercurial fixatives and reagents used

✓ Recommended for fixing small pieces of liver, spleen, connective in dezenkerization MUST NOT go
tissue fibers and nuclei
through drain disposal.
Advantages:
✓ Recommended for trichrome staining
✓ May act as a mordant to make certain special staining reactions possible

1.B. Zenker-Formol (Helly’s Solution)

✓ Excellent for bone marrow, extramedullary hematopoiesis and intercalated discs of the cardiac muscle.
✓ Excellent microanatomic fixative for pituitary gland, bone marrow and blood-containing organs such as a
spleen and liver.

1.C. Heidenhain’s Susa Solution

✓ Excellent cytologic fixative recommended for tumor biopsies esp. of the skin.
Advantage:
✓ Produces minimum shrinkage and hardening of tissues due to the counter-balance of the swelling effects of
the acids and the shrinkage effect of mercury.

1.D. B-5 Fixative (Lillie’s B-5 Fixative)

Precaution:
✓ Over fixation hardens the tissue and makes cutting difficult.
✓ Mercury pigment can be removed via dezenkerization.

2. Chromate Fixatives
2.A. Chromic Acid
✓ It precipitates all proteins and adequately preserves carbohydrates.
✓ It is a strong oxidizing agent.

2.B. Potassium Dichromate

✓ Fixes but does not precipitate cytoplasmic structures
✓ Preserves lipids
✓ Preserves mitochondria (if used in pH 4.5 to 5.2, mitochondria is fixed; If the solution becomes acidified,
cytoplasm, chromatin bodies and chromosomes are fixed but the mitichondria are destryoed).

2.C. Regaud’s (Muller’s Fluid)/Mollers

✓ Recommended for demonstration of chromatin, golgi bodies, mitochondria, mitotic figures, RBC and
colloidcontaining tissues.

Composition: 3% Potassium Dichromate and 40% Formaldehyde added just before use.
Disadvantage:
✓ Prolonged fixation blackens the tissue pigment.

2.D. Orth’s Fluid

✓ Recommended for the study of early degenerative processes and tissue necrosis.
✓ Demonstration of Rickettsia and other bacteria.
✓ Preserves myelin than buffered formalin
 3. Lead Fixative
Lead Fixative (4% Aqueous Solution of Basic Lead Acetate)
✓ Recommended for Acid mucopolysaccharides.
✓ Fixes connective tissue mucin.

PICRIC ACID FIXATIVES

✓ Picric Acid is normally used in strong saturated aqueous solution (approx. 1%). It dyes the tissue, but the
yellow color can be removed by treatment with another acid dye or lithium carbonate.

Advantage:
✓ Excellent fixative for glycogen demonstration.
✓ Yellow stain prevents small fragments to be overlooked.
✓ Suitable for Aniline stains (Mallory’s Heidenhain’s or Masson’s Trichrome Methods)

Disadvantages:
✓ Causes RBC hemolysis and reduces the demonstrable amount of ferric iron in tissue.
✓ Not suitable for frozen section, because it causes frozen sections to crumble when cut.
✓ Picric acid fixed tissues must never be washed in water before hydration.
✓ Picric acid will produce excessive staining of tissues; to remove the yellow color, tissues may be placed in
70%/50% ethyl alcohol followed by 5% Sodium Thiosulfate and then washed in running water.
✓ Picric acid is highly explosive when dry.

1. Bouin’s Solution
✓ Recommended for fixation of embryos and pituitary biopsies

Advantages:
✓ Excellent in preserving soft and delicate structures (e.g. endometrial curettings)
✓ Yellow stain is useful in fragmentary biopsies.
✓ Preferred fixative for tissues to be stained by Masson’s Trichrome for collagen, elastic or connective
tissues.
✓ Preserves glycogen.

Disadvantages:
✓ Not suitable for fixing kidney structures, lipid and mucus.
✓ Destroys cytoplasmic structures (e.g. mitochondria)

2. Brasil’s Alcoholic Picroformol

Advantage:
✓ Better and less messy than Bouin’s Solution.
GLACIAL ACETIC ACID
✓ It solidifies at 17°C.
Advantages:
✓ Fixes and precipitates nucleoproteins.
✓ Precipitates chromosomes and chromatin materials; hence very useful in study of nuclear components of the
cell.
✓ Causes tissues (esp. those containing collagen) to swell.

PRECIPITATING (ALCOHOL) FIXATIVES

✓ Alcohol rapidly denatures and precipitates protein by destroying hydrogen and other bonds. It must be used in
concentrations ranging from 70 to 100%, because less concentrated solutions will produce lysis of the cells.
✓ Alcohol can be used to fix and preserve glycogen, pigments, blood tissue films and smears.
✓ Color of the specimen can be preserved for photographic work using 80% alcohol. Sometimes the specimen is
taken out from fixative (formaldehyde) and placed in 80% alcohol.

Advantages:
✓ Used as a both fixative and a dehydrating agent.
✓ Excellent for glycogen preservation.
Disadvantage:
Lower concentrations (70 to 80%) will cause RBC hemolysis and inadequately preserve leukocytes.

1. 100% Methyl Alcohol

Advantages:
✓ Excellent ion fixing dry and wet smears, blood smears and bone marrow tissues.
✓ Fixes and dehydrates at the same time.

3.70 to 100% Ethyl Alcohol

✓ If lower concentrations are used, RBCs becomes hemolyzed and WBCs are inadequately preserved.
Advantages:
✓ Preserves nucleoproteins and nucleic acids, hence, is used for histochemistry esp. for enzymes studies.
✓ Give the most usable DNA fragments to PCR.

4. Carnoy’s Fixative
✓ Recommended for chromosomes, lymph glands and urgent biopsies.
✓ Used to fix brain tissues for the diagnosis of rabies.

Advantages:
✓ Fixes and dehydrates at the same time.
✓ Preserves nissl granules and cytoplasmic granules well.
✓ Preserves nucleoproteins and nucleic acids.

6. Newcomer’s Fixatives
Advantage:
✓ Acts as both nuclear and histochemical fixative.

OSMIUM TETROXIDE (OSMIC ACID) FIXATIVES

Advantage:
✓ Fats from hydrated osmium dioxide, are stained black.
✓ Preserves cytoplasmic structures well.
✓ Brilliant nuclear staining with safranin.

Disadvantage:
✓ Expensive.
✓ Poor penetrating agent.
✓ Prolonged exposure to acid vapor can irritate the eyes, producing conjunctivitis, or cause the deposition of
black osmic oxide in the cornea, producing blindness.
✓ It inhibits hematoxylin and makes counterstaining difficult.
✓ Extremely volatile.

Precautions:
✓ Wear protective plastic mask of gloves while using Osmium tetroxide.
✓ It should be kept in a dark-colored, chemically clean bottle.
✓ Black osmic oxide crystals may be dissolved in cold water.
✓ Osmic acid fixed tissues must be washed in running water for at least 24 hours to prevent formation of
artifacts.

1. Flemming’s Solution
✓ Most common Chrome-Osmium Acetic Acid Fixative.
✓ Recommended for nuclear preparation.

Advantage:
✓ Excellent fixative for nuclear structures.
✓ Permanently fixes fats.

2.A Flemming’s Solution without Acetic Acid

✓ Recommended for cytoplasmic structures particularly the mitochondria.

TRICHLOROACETIC ACID
✓ Used for the precipitation of proteins and nucleic acids.
✓ Weak decalcifying agent.
ACETONE
✓ Fixes by dehydration and precipitation.
✓ This is not recommended as a morphological fixative.
✓ Used at ice cold temperature ranging from -5 to 4°C.

Advantages:
✓ Recommended for water diffusible enzymes esp. phosphatases and lipases.
✓ Used in fixing brain tissue for the diagnosis of rabies.
✓ Used as a solvent for certain metallic salts to be used in freeze substitution techniques for the tissue blocks.
PHYSICAL METHODS OF FIXATION:

1. Heat Fixation:
✓ Accelerate other forms of fixation as well as the steps in tissue processing.
✓ Usually employed for frozen tissues sections and preparation of bacteriologic smears.
2. Microwave Fixation:
✓ Works as a physical agent. Agitation to increase the movement of molecules and accelerate fixation.
✓ Allows light microscopic techniques.
✓ Microwave treated tissue (at 50%), post fixed in osmium tetroxide, gives satisfactory results for electron
microscopy.

Two Ways for Microwave Tissue Fixation:

1. Microwave fixation or Microwave stabilization: No chemical fixative is used at this stage.

2. Microwave assisted fixation: Microwave is carried out while tissues is in fixative. Microwave is used to
assist the action of the fixing agent. (**450 watts, at 55°C for 1.5 to 4 minutes)
Advantage:
✓ Tissue is heated right through the block in a very short time, allowing the study of cellular
processes that proceed very rapidly.
Disadvantage:
✓ Only penetrate tissue to thickness of 10 to 15mm.

3. Freeze-Drying and Freeze Substitution:

✓ Freeze drying is a special way of preserving tissues by rapid freezing (quenching _________________). Higher
temperature (sublimation __________________).
✓ Freeze substitution is similar to freeze drying, the only variation is that the frozen tissues-instead of being
subjected to dehydration in an expensive vacuum drying apparatus – is fixed in Rossman’s formula or in 1%
acetone and dehydrated in absolute alcohol.

2. DECALCIFICATION:
✓ Process of complete removal of calcium or lime salts from tissues (bones, teeth, calcified tissues) after fixation.
✓ Decalcification should be done after Fixation and before Impregnation to ensure and facilitate normal cutting of
sections.

FACTORS INFLUENCING THE RATE OF DECALCIFICATION:

1. Concentration of decalcifying agent. 5-10% Nitric Acid

2. Ratio of decalcifying fluid to tissue. 20:1 or 20x the Volume of the tissue
3. Temperature 18°C to 30°C (room temperature) @37°C w/ causion impaired nuclear staining
4. Mechanical agitation enhances diffusion of w/ Von Gieson fluid
5. Ideal time required 1-2 days (24 to 48 hrs)

TYPES OF DECALCIFYING AGENTS USED

1. Acid Decalcifying Agents: The most widely used agents for routine decalcification of large amounts of bony tissues
because they’re stable, easily available and relatively inexpensive.
A. 5 to 10% Nitric Acid:
• Most common and fastest decalcifying agent
• It has a disadvantage of inhibiting nuclear stains and destroying tissues.
a.1) 10% Aqueous Nitric Acid Solution
• Composition: Concentrated Nitric Acid solution and Distilled water.
• Decalcification time: 12 to 24 hours
a.2) Formol Nitric Acid
• Composition: Concentrated Nitric Acid, 40% Formaldehyde and Distilled Water.
• Decalcification time: 1 to 3 days
a.3) Perenyi’s Fluid: Decalcifying agent & a tissue softener at the same time.
• Composition: 10% Nitric Acid, 0.5% Chromic Acid, Abs. Ethyl Alcohol
• Decalcification time: 2 to 7 days
a.4) Phloroglucin-Nitric Acid: most rapid decalcifying agent
• Composition: Concentrated Nitric acid, Phloroglucin, 10% nitric acid
• Decalcifying time: 12 to 24 hours
 B. Hydrochloric Acid: recommended for surface decalcification of tissue blocks
• Von Ebner’s
• Composition: Distilled water. HCI and NaCl

C. Formic Acid
• Safer to handle than nitric acid or hydrochloric acid
• Recommended for routine decalcification of post-mortem research tissues.
c.1) Formic Acid-Sodium Citrate Solution: recommended for autopsy materials, bone marrow cartilage and
tissues studied for research purposes.
• Composition: Aq. Sodium Citrate and formic Saline
• Decalcification time: 3 to 14 days

D. Trichloroacetic Acid: weak decalcifying agent

• Composition: Trichloroacetic Acid and 10% Formol Saline
• Decalcifying time: 4 to 8 days

E. Sulfurous Acid: very weak decalcifying agent

F. Chromic Acid (Flemming’s Fluid)

• Used as a fixative and a decalcifying agent - chromic acid
• Carcinogenic
• Environmental toxin
• Corrosive to skin and mucus membranes

G. Citric Acid-Citrate Buffer Solution

Composition: Citric acid, Ammonium Citrate, Zinc Sulfate and Chloroform - acts as a preservative

2. Chelating Agent: substances that combine with calcium ions and other salts (e.g. iron and magnesium deposits)
to form weakly dissociated complexes and facilitate the removal of calcium salt.
a. EDTA (Versene) -pH dependent
• Most common chelating agent
• Also used as an anticoagulant and water softener
➢ Slow decalcifying agent:
• Small specimens may take 1 to 3 weeks.
b. Cal-Ex

3. Ion Exchange Resins

- Ammonium form of polystyrene resin that hastens decalcification by removing calcium ions from formic
acid containing decalcifying solutions.
- Decalcifying agent is usually 20 to 30 times the volume of the tissue.

4. Electrophoresis (Electrical Ionization)

- This process uses electricity.
- Solution used for electrolytic decalcification: 88% Formic Acid, concentrated HCl, and Distilled Water.

MEASURING THE EXTENT OF DECALCIFICATION

1. Physical or Mechanical Test

❖ This is done by touching or bending (check for pliability).

2. X-ray or Radiological Method

❖ Most sensitive; most ideal and most reliable method.
❖ Expensive

3. Chemical Method (Calcium Oxalate Test) -uses blue pH paper

❖ Solutions used: Sat. Aqueous Ammonium Oxalate and Ammonium hydroxide.
❖ Simple, reliable, convenient, recommended for routine decalcification.
2.3.DECALCIFICATION
DEHYDRATION:
• Process of removing water from the tissue following fixation in preparation for wax impregnation. Dehydration
usually occurs by placing fixed tissues in ascending grades of dehydrating fluids. As a general rule, whatever the
dehydrating agent is used, the amount should not be less than 10x the volume of the tissue.

Incomplete Dehydration is the most common processing problem.

Incomplete Dehydration prevents the ingress of the clearing agents to the tissue, making tissue soft and non-receptive to
wax infiltration.

Dehydrating Agents

1. Alcohol

✓ A temperature of 37°C will hasten the dehydration time.

✓ A layer of anhydrous Copper Sulfate (about ¼ inch thick) placed in the bottom of the container and covered with
a. Ethyl Alcohol
filter paper. This will accelerate dehydration by removing water from dehydrating fluid. A BLUE discoloration of
✓ Clear, colorless, flammable liquid
copper sulfate crystal will indicate full indicate full saturation of dehydrating fluid-alcohol should be charged with
✓ Most commonly dehydrating agent
a fresh solution.
✓ Best dehydrating agent
b. Methyl Alcohol
✓ Toxic dehydrating agent, primarily employed for blood and tissue films and for smear
preparation.
c. Isopropyl Alcohol (IPA)
✓ Versatile ethanol substitute (Best all-around-ethanol substitute)
✓ Used in microwave processing schedules
d. Butyl Alcohol
✓ For plant and animal micro techniques
✓ Slow dehydrating agent
2. Acetone
✓ More miscible with epoxides than alcohol
✓ Rapid acting and very cheap utilized for urgent biopsies (dehydrates in ½ to 2 hours)
✓ Volume: At least 20x that of the tissue because it is highly volatile
Raw material in making methamphetamine (“shabu”)
3. Dioxane (Diethylene Dioxide)
✓ Excellent dehydrating and clearing agent.
a. Graupner’s – uses pure dioxane and paraffin.
b. Welseberger’s – tissues are wrapped in a gauze and submerged in dioxane with Calcium oxide or
quicklime.
4. Cellosolve (Ethylene Glycol Monomethylether)
Combustible (110-120F)
Toxic by inhalation, skin contact and ingestion
5. Triethyl phosphate
6. Tetrahydrofuran (THF)
✓ Dehydrates and clear tissue at the same time.
✓ Best universal solvent for its fast action and less shrinkage or hardening.

4. CLEARING (DEALCOHOLIZATION):
Process wherein alcohol/dehydrating fluid is removed from the tissue and replaced with an intermediate solvent that is
fully miscible with both ethanol and paraffin wax.

Clearing Agents

1. Xylene (xylol)
✓ Colorless clearing agent that is most commonly used.
✓ Most rapid clearing agent (clears within 15 to 30 minutes)
✓ Used for clearing, both embedding and mounting procedure.
✓ De-waxing agent during staining
✓ Causes considerable hardening and shrinkage of tissues; hence not suitable for nervous tissues and
lymph nodes.
✓ Xylene becomes milky when an incompletely dehydrated tissue is immersed in it.
 2. Chloroform
✓ For tough tissues (skin, fibrinoid, decalcified tissues), nervous tissues lymph nodes and embryos because
it causes minimum shrinkage and hardening of tissues.
Does not make tissue transparent.
Toxic to the liver after prolonged inhalation.
3. Benzene
✓ Rapid acting (15 to 60 minutes)
✓ Highly inflammable
Excessive exposure to benzene may be toxic and may become carcinogenic.
May damage the bone marrow and cause APLASTIC ANEMIA.
4. Toluene
✓ Clearing time 1 to 2 hours
✓ May be used as a substitute for xylene or benzene for clearing both during embedding and mounting
processes.
5. Cedarwood Oil
✓ Used to clear both paraffin and celloidin sections during embedding process.
✓ Recommended for central nervous system tissues and cytological studies, particularly of smooth
muscles and skin.
Becomes milky upon prolonged storage and should be filtered before use.
6. Aniline Oil
✓ Recommended for clearing embryos, insects and very delicate specimens.
7. Clove Oil
✓ Quality is not guaranteed due to its tendency to become adulterated.
8. Carbon tetrachloride
9. Methyl benzoate and Methyl salicylate
✓ Slow-acting clearing agents that can be used when double embedding techniques are required.

Other Clearing Agents:

1. N-Butyl Acetate
✓ Xylene substitute and nitrocellulose solvent.
2. Terpenes (Plant oils)
✓ Earliest transition solvents used in histology and include turpentine and oils of bergamot, clove, lemon,
oreganum and sandalwood.
✓ Limonene: Xylene substitute from the terpene family, derived from oil of citrus peels.
3. Orange oil clearing agents
4. Chlorinated hydrocarbons
5. Coconut oil
6. Bleached palm oil

5. IMPREGNATION (INFILTRATION):
Process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will
completely fill all the tissue cavities.

The impregnating medium should be at least 25x the volume of the tissue.

6. EMBEDDING (CASTING/BLOCKING/MOLDING):
Paraffin impregnated tissues are placed into a mold containing the embedding medium together with their proper labels
and immersed in melted paraffin at a temperature between 5 to 10°C above the MP and then coded rapidly at -5°C or
immersed in cold water to solidify.

ORIENTATION – The MOST CRITICAL STEP in embedding. It is a process by which a tissue is arranged in a precise position
in the mold during embedding, on the microtome, before cutting and on the slide before staining.

3 Types of Impregnation and Embedding Medium:

1. Paraffin Wax
2. Celloidin (collodion)
3. Gelatin
4. Plastic

Paraffin Wax Impregnation

✓ Simplest, most common and best embedding medium used for the routine tissue processing.
✓ Infiltration in overheated paraffin (>60°C) will produce considerable shrinkage and hardening of tissues. To avoid
this, the paraffin over must be maintained at a temperature of 2 to 5°C above the MP of the paraffin used.
✓ Melting point of paraffin wax normally used for routine work: 56°C
 ❖ Laboratory temperature ranging from 20-24°C, paraffin wax with a melting point of 54-58°C is indicated.
❖ Laboratory temperature ranging from ranging from 15-18°C, paraffin wax with a melting point of 50-54°C
is indicated.
✓ Wax that has been trimmed away from the impregnated tissue may be melted and filtered for future use, with
a coarse filter paper (e.g. Green’s No. 904)

3 Ways of Paraffin Impregnation and Embedding

1. Manual Processing (hand processing)

✓ At least 1 changes of wax are required at 15 minutes intervals in order to ensure complete removal of the clearing
agent from the tissue.
✓ The specimen is then immersed in another fresh solution of melted paraffin for approximately 3 hours to ensure
complete embedding or casting of tissue.
2. Automatic processing (e.g. Autotechnicon)
✓ 12 processing steps
✓ Fixation, Dehydration, Clearing and Infiltration
✓ Wax bath temperature: 3°C above the MP of wax
✓ Advantage: Contrast Agitation
3. Vacuum Embedding
✓ Recommended for urgent biopsies, delicate tissues such as lungs, connective tissues, decalcified bones, eyes,
spleen and central nervous system.
✓ Temperature is maintained at 2°C to 4°C above the MP of the wax.

Substitutes for Paraffin Wax

1. Paraplast (MP 56 to 57°C)

✓ Mixture of highly purified paraffin and synthetic plastic polymer.
✓ For large dense tissue blocks (bones and brain).
2. Embeddol (MP 56 to 58°C)
✓ Synthetic wax substitute.
✓ Less brittle and less compressible than paraplast.
3. Ester Wax (MP 46 to 48°C)
✓ Insoluble in water but soluble in 95% ethanol.
✓ Sectioning should be done using a heavy-duty microtome. (Sliding/sledge type)
4. Water Soluble Wax (MP 38 to 42°C or 45 to 56°C)
✓ Suitable for enzyme histochemical studies.
✓ Cytologic details are excellently preserved.

*Because of the water-soluble properties of carbowax, sections cannot be floated out on water, they must be floated
out in one or other of the following solutions:
Pearse solution Diethylene glycol, distilled water, strong formaldehyde
Blank and McCarthy Solution Gelatin and potassium dichromate

5. Dimethyl sulphoxide (DMSO)

✓ Substance added to proprietary blends of plastic polymer paraffin waxes.
✓ Reduces infiltration times and facilitates thin sectioning.

Celloidin (Collodion) Impregnation

• Recommended for large hollow organs, which tend to collapse, for hand and dense tissues such as bones and
teeth, for large tissue sections of the whole embryo.

Nitrocellulose Method

Low Viscosity Nitrocellulose (L.V.N.) is another form of celloidin soluble in equal concentration of ether and alcohol.

Explosive: when dry, striking or dropping the container will cause the substance to explode.

Gelatin Method of Embedding

- Volume should be at least 25 times the volume of the tissue; Tissue should not be more than 2-3mm
thick.
- 1% phenol prevents growth of molds.
Molds for Embedding

1. Leuckhart’s Embedding Mold: 2 ‘L’ shaped strips with heavy brass which the size can be adjusted.
2. Compound Embedding Unit: Made up of a series of interlocking plates forming several compartments.
3. Plastic embedding rings and base mold
4. Disposable embedding molds
a. Paper Boat
b. Peel-a-way
c. Plastic ice trays

Plastic Classification:

1. Epoxy Plastics (mixture of epoxy plastics, catalysts and accelerators)

• Bisphenol A (oraldite) = 3rd
• Glyceral (epon) = 2nd fastest
• Cyclohexane Dioxide (spurr) = 1st fastest
2. Polyester plastics
3. Acrylic plastics
✓ Used extensively for high resolution light microscopy and un-decalcified bone.

7.TRIMMING:
Process of removing excess wax after embedding. Tissue should be surrounded by at least 2mm of wax.

• Coarse Trimming: Sides, tips and bottom of the tissue are trimmed using a knife or a blade to form a truncated
pyramid/4-sided prism.
• Fine Trimming: Block is placed in the microtome – setting the adjuster at 15mm or by advancing the block using
the coarse feed mechanism – where the surface is trimmed away until the entire tissue surface has been exposed.

8. SECTIONING/CUTTING:
Process wherein a processed tissue is cut into uniformly thin slices (sections) using microtome to facilitate studies under
the microscope.

Section cut is picked by:

1. Index finger
2. Camel hairbrush
3. Spatula
4. Flat-bladed forceps

Adhesives

1. Mayer’s Egg Albumin:

✓ Most commonly used adhesive for paraffin sections.
✓ Very easy to make, convenient and relatively inexpensive.
✓ Composition
• Egg white
• Glycerin: increases viscosity and prevents drying.
• Thymol Crystal: preservative; prevent growth of moids.
2. Dried albumin
3. Starch paste
4. Plasma
5. Gelatin (1%)
6. Gelatin-formaldehyde mixture
7. Poly-L-Lysine: widely used as a section adhesive for immunohistochemistry.
8. APES (3-aminopropythriethoxysilane): utilized for cytology, particularly for cytospin preparations of
proteinaceous material.

Methods of Drying Slides

1. Wax oven at 56°- 60°C for two hours

2. Incubator at 37°C overnight
3. Hot plate at 45°- 55°C for 45 minutes
4. Blower type slide dryer at 50°- 55°C for 20-30 minutes
5. In urgency, by carefully holding the slide section upwards, above a Bunsen flame, add mounting medium until
the wax melts
 FAULTS DURING TISSUE PROCESSING:
PROBLEM REASON REMEDY
Sections fail to form Surfaces and edges of the block are not Re-trim the block
ribbons parallel
Horizontal surface of the block is not Re-adjust and re-orient the block
parallel to the knife
Paraffin wax is too hard Coat horizontal edges of the block with wax of
lower melting point
Knife is tilted too much Reduce the tilt
Sections are too thick Readjust the thickness of the sections
Knife is dull Hone and strop
Sections roll up on Knife is blunt Sharpen the knife
cutting so that they Tilt of knife is too great Reduce the tilt
adhere and get broken Knife edge is dirty Clean the knife edge
against the knife edge
Ribbon is curved, Blunt or dull spot on the knife, producing Adjust the knife so that knife edge will present
crooked or uneven an irregular knife edge a uniformly sharp edge to the block, or sharpen
instead of straight Edges of the block are not parallel but Re-trim the block
round or wedge shaped
Knife is not parallel to the block Knife is not parallel to the block
Sections are Knife is blunt or dull Re-sharpen the knife
compressed, wrinkled
or jammed
Paraffin block is warm and soft Cool the block on ice water until firm
Knife edge is coated with paraffin Clean the knife edge
Readjust thickness of the section
Microtome set screw is loose Tighten the screw
Tilt of knife is too vertical Reduce the tilt
Sections are squashed Bevel of knife is lost due to incorrect Re-sharpen, using a knife back or automatic
(width of each section sharpening knife sharpener
is less than that of the
block)
A hole is formed in the Bubble or dirt formed in the embedding Re-embed in freshly filtered wax if necessary
section medium
Tissue is not processed properly and will Re-process tissue
not form a section (especially if center is
raw)
Under-processed portion of tissue bursts Re-process tissue
on contact with warm water
Hard spot in tissue due to calcium Once embedded in paraffin wax, decalcification
is impractical; use a base-sledge microtome
with a wedge knife
Sections of unequal Tilt of knife is too great or bevel is not Reduce the tilt
thickness are produced cleared, hence object is compressed
against the knife edge
Clamp set screw on knife or block holder Tighten the screw
is loose
Blocks are too large Cut blocks into smaller fragments
Blocks are too hard Soften the blocks in detergent or phenol
Sections adhere to the Static electricity due to low atmospheric Breathe out or blow gently on the bock and
knife or other parts of humidity knife to break up static electricity, or boil
the microtome water in the room to increase humidity
Knife edge is dirty Clean the knife edge
Knife edge is dull Sharpen the knife
Knife tilt is too great Reduce the tilt
Ribbon is split or Nicks or damage on the knife edge Sharpen the knife
lengthwise vertical Dirty embedding Re-embed in freshly filtered wax
scratches are seen on Knife edge is dirty Clean knife edge with xylene
sections Tilt of knife is too great Reduce the tilt
Sections are lifted from Knife tilt is too great Reduce the tilt
the knife on upstrokes Knife is dull Sharpen the knife
Paraffin is too soft or room temperature is Cool paraffin wax in ice water
warm
Resistance is felt on the Tilt of knife is too small; paraffin block is Increase the tilt
lower part of the therefore compressed against the base of
section during cutting the knife towards the end of stroke
 Horizontal or parallel Knife edge vibrates due to hardness of Treat with phenol during processing or
lines or furrows across tissue collodionize
the section ("chatters") Tilt of knife is too great Reduce the tilt
are seen
Section cut is Knife is blunt Sharpen the knife
sometimes thin, Knife is not clamped properly Adjust the knife so that knife edge will present
sometimes thick a uniformly sharp edge to the block, or sharpen
Knife or block holder is loose Tighten adjusting and locking screws
Knife tilt is too small that block is Increase the tilt
compressed by bevel and section is not
cut
Knife makes a hard- Tilt of knife is too slanted or too big Readjust the tilt
metallic scraping or Tissue is too hard Take fresh block treated with phenol during
ringing sound on processing
backstroke, when Knife blade is too thin Change the knife
section is cut
Frozen tissue crumbles Freezing is not adequate Refreeze the tissue block
and comes off the
block holder when cut
Frozen tissue chips into Tissue is frozen too much Warm the tissue with the fingers
fragments when cut
Ribbons are crooked Top and bottom edges of block are not Adjust the block holder to make the block
parallel to edge of blade/sides of block are edges parallel to the knife
not perpendicular to the blade
Ribbons are crooked Top and bottom edges of block are not Adjust the block holder to make the block
parallel to edge of blade/sides of block are edges parallel to the knife
not perpendicular to the blade
Sections are too thick Wrong micrometer setting Microtome needs recalibration
On trimming, tissue Clearing agent not completely removed Block is trimmed down nearest to the tissue.
smells of clearing agent due to insufficient impregnation Remaining wax is melted on embedding oven
and paraffin impregnation is repeated,
changing the paraffin at least once before
embedding
Tissue is opaque, Insufficient clearing Repeat clearing; if object has already been
section cutting is embedded, prolong clearing up to 12 hours,
difficult due to then re-embed
presence of alcohol
Tissue shrinks away Insufficient dehydration, therefore Repeat the whole procedure
from wax when incomplete clearing and impregnation
trimmed
On trimming, wax Contaminated wax Re-embed in freshly filtered wax
appears crystalline Block not cooled rapidly enough Re-embed in freshly filtered wax
Paraffin block, after Insufficient paraffin Repeat paraffin impregnation, then re-embed
cooling, is moist and
crumbles
The table below provides some troubleshooting tips for poor processing:

Problem Possible Causes Corrections

Tissue feels soft or mushy during embedding • Tissue may have been grossed • Reprocess tissue on
in too thick proper program
• Tissue may have been • Reprocess tissue on
processed on a program that correct processing
was too short for that tissue protocol
type • Change reagents and
• Processing reagents may be reprocess tissue
saturated with water • Change paraffin and
• Paraffin may be saturated with reprocess tissue
xylene or isopropanol

Tissue bounces out of paraffin block during • Poor dehydration and paraffin • Change reagents and
microtomy or tissue does not adhere to block infiltration due to water left in reprocess tissue on
or slides (Commonly experienced with uterus the tissue proper processing
and prostate tissue, as well as dense organ protocol
core samples)
Tissue looks greasy and "explodes" or • If the temperature of the water • Reprocess tissue on
separates rapidly when ribbon is placed on bath is between 45-50° C, then correct processing
water bath the tissue is under-processed protocol
 • Tissue may have been grossed • Reprocess on proper
in too thick program
• Tissue may have been • Reprocess tissue on
processed on a program that correct processing
was too short for that tissue protocol
type • Change reagents and
• Processing reagents may be reprocess
saturated with water • Change paraffin and
• Paraffin may be saturated with reprocess tissue
xylene or isopropanol

Tissue does not adhere to slide or falls off • If tissue slides are placed in • Reprocess tissue on
easily oven prior to deparaffinization correct processing
in xylene, tissue is under- protocol
processed • Change reagents and
• Reagents saturated with water paraffin and reprocess
or contaminated with the tissue on proper
preceding reagent processing protocol

Hematoxylin and eosin (H&E) stained tissue • If tissue was fixed properly, • Change reagents and
section shows uneven nuclear staining and then sample was improperly reprocess tissue on
"blue blobs" lacking distinct chromatin dehydrated and infiltrated with proper processing
patterns paraffin protocol

9. STAINING:
Histological staining - The process whereby the tissue constituents and general relationship between cell and tissue are
demonstrated in sections by direct interaction with a dye or staining solution, producing coloration of the active tissue
component.

• ____________________ - The process of giving color to the sections by using aqueous or alcoholic dye solutions
and only 1 dye is used.
• ____________________ - The process whereby the action of the dye is intensified by adding another agent or a
MORDANT which serves as a link or bridge between the tissue and the dye, to make the staining reaction
possible.

• ____________________ - The process whereby tissue elements are stained in a definite sequence, and the
staining solution is applied for specific periods of time or until the desired intensity of coloring of the different
tissue elements is attained.
NOTE: Once the dye is taken up by the tissue, it is NOT WASHED OR DECOLORIZED.

• ____________________ - With this technique, the tissue is first overstained to obliterate the cellular details,
and the excess stain is removed or decolorized from unwanted parts of the tissue, until the desired intensity of
color is obtained.
NOTE: With WASHING OR DECOLORIZATION STEP
• ____________________ - The selective removal of excess stain from the tissue during regressive staining in
order that a specific substance may be stained distinctly from the surrounding tissues.
• ____________________ - Uses more than one chemical stain to better differentiate between various
microorganisms or structures/cellular components of a single organism.
• ____________________ - Entails the use of specific dyes which differentiate particular substances by staining
them with a color that is different from that of the stain itself (metachromasia).

Examples of Metachromatic Dyes:

✓ Methyl violet or crystal violet
✓ Cresyl blue (for reticulocytes)
✓ Safranin
✓ Bismarck brown
✓ Methylene blue
✓ Thionine
✓ Toluidine blue – staining of H. pylori
✓ Azure A, B, C
 • ____________________ - A process where specific tissue elements are demonstrated, not by stains, but by
colorless solutions of metallic salts which are thereby reduced by the tissue, producing an opaque, usually black
deposit on the surface of the tissue or bacteria.
• ____________________ - The application of a different color or stain to provide contrast and background to the
staining of the structural components to be demonstrated.

RED YELLOW GREEN

CYTOPLASMIC STAINS Eosin Y Picric acid Light green SF
Eosin B Orange G Lissamine green
Phloxine B Rose Bengal
RED BLUE
NUCLEAR STAINS Neutral Red Methylene blue
Safranin O Toluidine blue
Carmine Celestine blue
Hematoxylin

• ____________________ - The most widely used nuclear stain.

• ____________________ - The most widely used histological stain.

Hematoxylon campechianum - Hematoxylin is extracted from a logwood named.

• The major oxidization product of Hematoxylin is _______, a natural dye that is responsible for the color
properties.
• ____________________ - The most suitable stain to combine with an alum hematoxylin to demonstrate the
general histological architecture of a tissue.

EOSIN Y - Most widely used eosin dye.

• ____________________ - The selective staining of living cell constituents.

Examples of VITAL STAIN:

✓ Janus green – Mitochondria
✓ Trypan blue – Reticuloendothelial system
✓ Propidium iodide – Eukaryotic cells

• ____________________ - Staining of living cells is done by injecting the dye into any part of the animal body.

Examples of INTRAVITAL STAIN:

✓ Lithium
✓ Carmine
✓ India ink

• ___________________ - It is used to stain living cells immediately after removal from the living body.

Examples of SUPRAVITAL STAIN:

✓ Neutral Red – the BEST VITAL DYE
✓ Janus Green – especially recommended for Mitochondria
✓ Trypan Blue
✓ Nile Blue
✓ Thionine
✓ Toluidine Blue -can be used to demonstrate Helicobacter pylori (causative agent of Peptic ulcer)

• Most fixatives can be used for routine H&E staining EXCEPT: _________________ as it inhibits Hematoxylin.

Stains for PROTEINS:

✓ Alkaline Fast Green: For Basic proteins
Histones & Protamines – Green
✓ Peracetic Acid-Alcian Blue: For Cystine & Cysteine
Cystine & Cysteine – BLUE GREEN
✓ Sakaguchi’s Test: For Arginine
✓ Arginine – Orange-red

• ___________________ - Mixture of picric acid and acid fuchsin for the demonstration of connective tissues.
• ___________________ - Used to demonstrate deposits of calcium salts and possible sites of phosphatase
activities.
 • ___________________ - A complex, water-soluble phthalocyanine dye, similar to chlorophyll, which stains acid
mucopolysaccharides by forming salt linkages with them. It produces a striking blue color, and it is resistant to
various counterstaining procedures.
• ___________________ - Basic acridine fluorochrome which permits discrimination between dead and living
cells, giving green fluorescence for DNA and a red fluorescence for RNA.
• ___________________ - A cytoplasmic stain used for counterstaining epithelial sections.
• ___________________ - Plasma stain utilized for deep staining acid-fast organisms, for mitochondria, for
differentiation of smooth muscles with the use picric acid.
• ___________________ - Used for staining hemoglobin.
• ___________________ - Used as a contrast stain for Gram’s technique in acid fast and Papanicolau’s method,
and for staining diphtheria organisms.
• ___________________ - Omitted from the EA-50 formula in the Modified Papanicolau’s Stain.
• ___________________ - Used as a chromatin stain for fresh materials in smear preparations.

Best Carmine solution Carmine + Aluminum Chloride - Stains GLYCOGEN

• ___________________ - Best known as an indicator, but may be utilized as a stain for axis cylinders in embryos.
It is used as a 4% aqueous solution in Krajian’s method of staining elastic tissues, amyloid, and myelin.
• ___________________ - Nuclear or chromatin stain used for staining amyloid in frozen sections and platelets in
blood.
• ___________________ - Used for staining blood to differentiate leukocytes.

GIEMSA STAIN - Most common Romanowsky-type of stain used in histopathology.

Examples of Romanowsky Stain:

✓ Wright's stain
✓ Jenner's stain
✓ Leishman stain
✓ May-Grunwald
✓ Giemsa stain

NOT a Romanowsky stain - SILVER STAIN

• ___________________ - Stain used for metallic impregnation, made up of gold chloride and mercuric chloride.

Astrocytes are selectively stained with the Cajal gold sublimate method on frozen sections.
Results: Astrocytes with perivascular feet – BLACK

• ___________________ - The oldest of all stains, originally used for microscopic study of starch granules. It stains
amyloid, cellulose, starch, carotenes, as well as glycogen.

0.5% Iodine - Widely used for removal of mercuric fixative artefact pigments.

• ___________________ - Used for demonstrating mitochondria during intravital staining.

• ___________________ - Weakly basic dye used as a contrast stain for staining Ascaris eggs and erythrocytes,
and as a bacterial spore stain; it can also be used both as decolorizer and counterstain.
• ___________________ - Stain for bacterial endospore which uses Malachite green as a primary stain and
Safranin as a counterstain.
• ___________________ - Stains chromatin green in the presence of an acid.
• ___________________ - It is a common basic nuclear stain employed with Eosin to provide differentiation. It is a
valuable stain for plasma cells and may also be employed in cytological examinations of fresh sputum for
malignant cells, as a bacterial stain for evaluation and differentiation of bacterial organisms, for diagnosis of
diphtheria, and for vital staining of the nervous tissue.
• ___________________ - A metachromatic dye formed whenever methylene blue is heated in fixed alkali or
alkali carbonate, coloring nuclei of leukocytes reddish-purple in the presence of methylene blue.
• ___________________ - Formed by boiling Nile blue with sulfuric acid. Nile red is a lipophilic stain; it will
accumulate in lipid globules inside cells, staining them red.
• ___________________ - A dye that is more soluble in fat than in water or alcohols, hence it is used as a stain for
neutral lipids.
• ___________________ - A basic dye recommended for observing cell granules and vacuoles of phagocytic cells.
• ___________________ - Used as a substitute for carbol fuchsin is acid-fast staining.
• ___________________ - Excellent stain for Elastic fibers and is especially recommended in dermatological
studies due to its ability to demonstrate the finest and most delicate fibers of the skin.
• ___________________ - A selective stain for unsaturated lipids and for lipoproteins such as myelin, which it
stains BLACK.
• ___________________ - Employed as a contrast stain to acid fuchsin, for demonstration of connective tissue
(Van Gieson’s stain), as a cytoplasmic stain in contrast to basic dyes, as a counterstain to crystal violet, as a
tissue fixative, and as a decalcifying agent.
 • ___________________ - An insoluble colored salt of ferric ferrocyanide (an iron cyanide compound) normally
utilized for the manufacture of paints, but may be used for microanatomical color contrast of specimens and for
demonstration of the blood and lymph vessels by injection (intravital staining).
• ___________________ - Used with osmic acid to fix and stain blood and glandular tissues.
• ___________________ - A nuclear stain. It produces red nuclei, and is used primarily as a counterstain. It gives a
yellow color to collagen.
• ___________________ - Used in 10% aqueous solution to prepare various dilutions to be used in identification
of spirochetes, reticulum and other fiber stains.
• ___________________ - A nuclear stain for fixed tissues, used as a substitute for thionine in fresh frozen tissue
sections. It is recommended for staining of Nissl granules or chromophilic bodies.
• ___________________ - Binds to collagen in the extracellular matrix, staining it pink. Often it is combined with a
stain for elastic fibers (elastic van Gieson) which stain black, allowing the two major elements of connective
tissue to be differentiated.
• ___________________ - Used for demonstration of neuroglia in frozen sections.
• ___________________ - A silver reduction method that demonstrates phosphates and carbonates, but these
are usually present along with calcium. This stain is most useful when large amounts are present, as in bone.
• ___________________ - Causes blood cells to exhibit four major staining properties that allow the cell types to
be distinguished. Basophilia (affinity for methylene blue), azurophilia (affinity for the oxidation products of
methylene blue called azures, which are reddish purple), acidophilia (affinity for eosin), and neutrophilia
(affinity for a complex of dyes in the mixture, which are pale lilac).

OIL SOLUBLE DYES (LYSOCHROMES)

• ___________________ - A stain that colors fat droplets black and is the most sensitive of the oil soluble dyes.
• ___________________ - It is different from Sudan Black because it has no secondary amino group and it does
not color phospholipids or the fine lipid droplets.
• ___________________ - The first Sudan dye to be introduced into histochemistry. It is also fat soluble, and is
good as a fat stain for central nervous system tissues, giving a less deep and lighter orange stain compared to
the darker staining Sudan IV.

Stains for CARBOHYDRATES:

✓ Periodic Acid Schiff (PAS) - a histochemical stain that will demonstrate carbohydrates and other substances in
the tissue. It widely used for demonstration of glycogen.
Results: PAS-positive substances - red or magenta red
Nuclei - blue
✓ Best Carmine Method - is a good staining technique due to the affinity of alkaline carminic acid for glycogen,
producing a bright red color.
✓ Langhan’s Iodine – OLDEST STAIN

Stains for MUCINS:

Mucins are polysaccharides bound to other substances (the nature of which serves as the basis for their classification),
forming the ground substance of connective tissues primarily.
✓ H&E - light pink color
✓ Ehrlich’s hematoxylin (alkaline) - bluish or basophilic
✓ Alcian Blue technique - Acidic mucosubstances are stained by alcian blue.
Results: Acid mucins (sulfomucins and sialomucins) - blue
Proteoglycans and hyaluronic acid - blue Nuclei – red

Stains for ACID MUCOPOLYSACCHARIDES:

✓ Metachromatic staining
1. Toluidine blue and Azure A
Results: Mucopolysaccharide - red purple
Tissue background - blue
2. Uranyl nitrate- Azure method
Results: Acid mucopolysaccharides – Crimson or Red-violet
Other tissue components - blue
✓ Alcian blue technique
Results: Acid mucins - blue
Nuclei - red
✓ Colloidal iron technique
✓ Aldehyde fuchsin stain
✓ Mucicarmine stain
✓ Fluorescent acridine orange technique
Results: Acid mucopolysaccharides - Black
Fungi - Greenish red flourescence
Background - Reddish orange fluorescence

Stains for NUCLEIC ACIDS:

1. Feulgen Technique – for nuclear DNA
• DNA – Red-purple
• Cytoplasm – Green
2. Methyl Green-Pyronin – for DNA & RNA
• DNA (chromatin) – Green or Blue-green
• RNA (nucleoli) – Rose-red
3. Acridine Orange (Fluorescent Staining) – for DNA & RNA
• DNA – Yellow-green fluorescence
• RNA – Brick to Orange-red fluorescence
4. Acriflavine – stains DNA (fluorescent)
• DNA – Fluorescent Yellow

Stains for FATS and LIPIDS:

Sudan Black B – most sensitive lipid stain known
Results: Lipids – blue black
Nuclei – Red
2. Sudan IV (Scharlach R) – most commonly used lipid stain, producing rapid and permanent coloration of lipid
Results: Lipids (mainly triglycerides) – Red
Nuclei – Blue/Black
Oil Red O
Results: Fat – Brilliant red
Nuclei – Blue
4. Osmic Acid Stain – It is NOT a dye but it has an unstable oxide which is reduced to a black substance by unsaturated
fats and fatty acids.
Results: Nuclei – Yellow-orange
Fats – Black
5. Nile Blue Sulfate Method – Dye capable of differentiating 2 lipid classes simultaneously by the action of 2
components: a red oxazone which dissolves neutral lipids, and a blue oxazine which is basic and reacts with
phospholipids and fatty acids.
Results:
Neutral fat – pinkish red
Cholesterin esters and cholesterin fatty acids – light red
Cerebrosides – light blue
Fatty acids and Soap – Deep blue to violet

Stains for MUSCLE AND BONE:

1. Modified Gomori’s Trichrome Stain
Results: Muscle fibers – red
Collagen – green
Nuclei – Blue to black
2. Mallory’s Phosphotungstic Acid Hematoxylin (PTAH)
Results: Fibrin, muscle striations, neuroglia and amoebae - dark blue
Nuclei, cilia, red blood cells - blue
Myelin - lighter blue
Collagen, osteoid cartilage, elastic fibers - deep brownish red
Cytoplasm - pale pinkish brown
3. Lissamine Fast Red – Tartrazine Method
Results: Nuclei – Black
Muscles, RBCs – Red
Collagen – Yellow
4. Heidenhain’s Iron Hematoxylin Method
Results: Muscle striations, mitochondria, myelin, and chromatin are stained grey-black.
5. Schmori’s Picro-Thionin Method – for Bones only
Results: Lacunae and canaliculi – filled with air
Lamellae – brown or brownish black lines

Stains for BM and BLOOD ELEMENTS:

1. Romanowsky Stains – consists of Methylene Blue/Azure B and Eosin dissolved in acetone-free methanol.
• Jenner
• Giemsa – most commonly used in histopathology
• May-Grunwald
• Leishman
2. Wright’s Stain -Methylene blue is polychromed by heating with Sodium bicarbonate
3. Wright-Giemsa or Jenner-Giemsa – Combination of a Romanowsky stain with another stain for improved staining of
cytoplasmic granules.
Results: Nuclei – purple/blue
Cytoplasm – pink/blue
Eosinophils – pink/red
4. Peroxidase reaction – For Myeloid cells
Results: Myeloid cells (except Basophils) – POSITIVE; presence of Green to Dark Blue granules in the cytoplasm
Basophils, Lymphocytes, and Erythroblasts – negative
Monocytes – show slight peroxidase activity.

ESTERASE GRANULOCYTE MONOCYTE

Naphthol AS-D Positive Negative
chloroacetate
α-naphthyl Negative Positive
acetate
α-naphthyl Negative Positive
butyrate

Stains for Tissue Pigments and Deposits:

ENDOGENOUS PIGMENTS:

• ___________________ - The iron-containing pigment of hemoglobin. It is usually seen as yellow to brown

granule and is normally found inside the cell.
• ___________________ - The iron-free pigment of hemoglobin, found in places where there is poor oxygenation,
participating in the formation of bile pigments.
• ___________________ - Hemoglobin minus the globin molecule, found in old blood clots, but may be
encountered in malaria, pernicious anemia, and toxic hemolysis.
• ___________________ - The black granule formed by malarial parasites living in RBCs, and may be removed by
alcoholic picric acid method.
• ___________________ - Iron-free brownish yellow pigment that occurs with hemosiderin in hemochromatosis.
It is a hemoglobin derivative which does not react with ferrocyanide, but stains intensely with basic dyes.
• ___________________ - This histochemical method is based on the unmasking of ferric iron by dilute HCl which
is a components of the acid ferrocyanide solution.

Results of Perl’s Purssian Blue Method:

Hemosiderin and ferric salts stain deep blue.
Other pigments retain their natural color.
Tissues and nuclei stain red (according to counterstain)

Gomori’s Prussian Blue Stain

Results: Iron pigments – bright blue

Nuclei – Red
Cytoplasm – pink to rose

Turnbull’s Blue Reaction – for Ferrous form of Iron

Results: Hemosiderin – Blue

Nuclei – Red

Benzidine-Nitroprusside Stain - for Hemoglobin and Oxidase granules

Results: Hemoglobin and some “oxidase” granules in leukocytes – Dark Blue

Nuclei – Red
Most other tissue components – Faint pink

Staining of Bile Pigments and Hematoidin:

1. Modified Fouchet’s Technique – for liver bile pigments
Results:
• Bile pigments – emerald to blue green
• Muscle – Yellow
• Collagen – Red
2. Gmelin Technique – for Bile and Hematoidin
Results:
• Bilirubin – Blue-green
3. Stein’s Iodine test – for Bile pigments
4. Schmorl’s Ferric Ferricyanide – for Reducing substances
Results:
• Bile, lipofuscins, and melanin – dark blue
• Argentaffin cells, Chromaffin – dark blue
• Thyroid colloid – dark blue
• Nuclei – Red
5. Gomori’s Aldehyde Fuchsin Technique – for lipofuscin
Results:
• Lipofuscin – Purple
• Background – Yellow
6. Mallory’s Fuchsin Stain – for Hemofuscin pigment
Results:
• Nuclei – Blue
• Hemofuscin – Red
• Hemosiderin – Unstained
7. Masson Fontana Technique – for Melanin and Argentaffin granules
Results:
• Melanin – Black
• Argentaffin granules – Black
• Nuclei – Red
8. Linquist’s Modified Rhodamine Technique – for Copper
Results:
• Copper, Copper-associated protein – red to orange-red
• Nuclei – Blue
• Bile – Green

Staining of MICROORGANISMS:
1. Gram’s Method – for demonstration of bacteria; differentiates GP from GN
Results:
• Gram positive – Blue/Black (Gregorio’s)
• Gram negative – Red
• Nuclei – Red
• Fibrin and Some fungi – Blue
2. Gram-Twort Stain – for Bacteria
Results:
• Gram positive – Blue-black
• Gram negative – pink-red
• Nuclei – Red
• RBCs and most cytoplasm – Green
• Elastic fibers – Black
3. Brown and Brenn Method – for Nocardia and Actinomycetes
Results:
• Gram positive – Blue
• Gram negative – Red
• Nuclei – Red
• Other tissues – Yellow
4. Ziehl-Neelsen’s Method – for Acid-Fast Bacteria
• Acid-Fast Bacilli – Red
• Cells and Nuclei – Blue
• RBCs – Pink
5. Fite-Fraco AFB stain – An AFB stain modification that uses Hematoxylin as a counterstain instead of Methylene Blue
6. Wade-Fite Technique – For Leprosy bacilli and Nocardia
Results:
• Leprosy and other Mycobacteria, Nocardia – Red
• Background – Blue
• Nuclei – blue-black (if Hematoxylin is used)
7. Toluidine Blue – for Helicobacter pylori
Results:
• pylori – Dark Blue against a variably blue background
8. Cresyl Violet Acetate Method – Helicobacter
Results:
• Helicobacter and nuclei – blue-violet
• Background – shades of blue-violet
9. Dieterle Method – for Legionella pneumophila
Results:
• L. pneumophila bacillus and Spirochetes – Dark brown to black
• Background - Yellow
10. Levaditi’s Method – for Spirochetes
Results:
• Spirochetes – Black on yellowish brown background
11. Modified Steiner and Steiner Technique – for Spirochetes
Results:
• Spirochetes, Donovan bodies, Fungi and Bacteria – Black
• Background – Yellow to brown
12. Warthin-Starry Method – For Spirochetes
Results:
• Spirochetes – Black
• Background – Golden Yellow
13. Grocott’s Methenamine Silver Stain – For Fungi
Results:
• Fungi – Sharply outlined BLACK
• Mucin and Glycogen – Gray-black
• Inner parts of Mycelia and Hyphae – Old Rose
• Red blood cells – Yellow
• Background – Pale Green
14. Lendrum’s Phloxine-Tartrazine Method – for Viral inclusions
Results:
• Viral inclusions – Bright red
• RBCs – Variable yellow to orange-red
• Nuclei – Blue-gray
• Background – Yellow to pink
15. Orcein Method – For Hepatitis B Surface antigen
Results:
• Hepatitis B antigen, elastic and some mucins – Brown-Black
• Background – Yellow

ADHESIVES AND MOUNTING MEDIA

• ___________________ - Most commonly used adhesive because it is very easy, convenient, and is relatively
inexpensive.
• ___________________ - Adhesive used in cytology, particularly for cytospin preparations of proteinaceous or
bloody material.
• ___________________ - To avoid distortion of the image, the refractive index of the mountant should be as
near as possible to that of the glass which is _______.

IMMUNOHISTOCHEMISTRY

• A heavily used technique for assisting the pathologist in making a diagnosis.

• Used to determine the origin, prognosis, and treatment of a tumor
• The major role an immunohistochemist plays is troubleshooting for variables introduced by fixation, processing,
and tissue types.
• The immunohistochemist should be familiar with both the controls used and the staining patterns of an
antibody to troubleshoot unexpected results.

• ___________________ - Most commonly used antibody for Immunocytochemistry.

• ___________________ - The structural part of the antigen that reacts with an antibody.
• ___________________ - The structural part of an antibody that reacts with an antigen.
o Recommended thickness of tissue sections for Immunohistochemistry.

METHOD OF VISUALIZATION
Method Label Used Microscope
Immunofluorescence Fluorochrome Fluorescent Microscope
Enzyme Enzyme Light Microscope
immunohistochemistry (Ex. Horseradish peroxidase)

• ___________________ - The enzymes most commonly chosen for antibody visualization in Enzyme
Immunohistochemistry.
A. HORSERADISH PEROXIDASE B. ALKALINE PHOSPHATASE
• ___________________ - The most commonly used fluorochromes in immunofluorescence techniques
A. FLUORESCEIN ISOTHIOCYANATE (FITC) B. RHODAMINE

EPITHELIAL TUMOR MARKERS: o Keratin – Cytokeratin, CK7, CK20

o Epithelial Membrane Antigen (EMA)
o Carcinoembryonic Antigen (CEA)
o Thyroid transcription factor (TTF-1)
o Prostate specific antigen (PSA)
 Intermediate Filament Markers o Actin
o Vimentin
o Desmin
o Glial fibrillary acidic protein (GFAP)
o Neurofilament (NF)
o S100 protein
Neuroendocrine markers o Neuron-specific enolase (NSE)
o Chromogranin
o Synaptophysin
Germ cell tumor markers o Human chorionic gonadotropin (HCG)
o Alpha-fetoprotein (AFP)
o Placenta-like alkaline phosphatase (PLAP)

Mesenchymal Tumor Markers

Myogenic tumors o Muscle-specific actin and desmin
o Myo-D1
o Myoglobin
o Myogenin
Fibrohistiocytic tumors o CD68
o FAM56
o Alpha1-antitrypsin
o Alpha1-antichymotrypsin
o Vimentin
Vascular tumors o Factor VII-related antigen
o CD31
o Ulex europaeus I (UEA)
Melanomas o S100 protein
o Melanosome (HMB-45)
o Melan-A (MART-1)
Lymphomas o Leukocyte Common Antigen (LCA) or CD45
o CD3, CD4, CD8 (T cell markers)
o CD19, CD20, CD23 (B cell markers)
o CD15, CD30 (Reed-Sternberg cells)
o Ig Light chain and Heavy chains
Cell Proliferation Markers:
✓ Ki-67 (MIB-1) and PCNA (Proliferating Cell Nuclear Antigen) are the most common
immunohistochemical markers used to assess proliferation of tumor cells.
✓ Increased expression of these antigens is usually associated with greater aggressiveness and higher
likelihood of recurrence of metastasis.

Cancer-associated Genes
✓ Proto-oncogenes such as p53, c-erbB-2, c-myc, and ras have been found to be activated in cancer,
particularly of the breast.

DIAGNOSTIC CYTOLOGY
• ___________________ - Deals with the microscopic study of cells that have been desquamated from
epithelial surfaces.
• ___________________ - A ____________ appears as a small drumstick-like projection on one of the
lobes of some neutrophils in females.

SPECIMENS FOR CYTOLOGY:

Gynecological Specimen Non-Gynecological Specimens
Cervicovaginal smear (Pap smear) No – Nipple discharge
Gf – Gastric secretions
Bf – Bronchial secretions
Pa – Pleural & Peritoneal fluids
Serious – Sputum
University – Urine sediment
College - CSF
Specimens for Exfoliative Cytology Concentrated sputum
requiring adhesives: Urine sediment
Bronchoalveolar lavage
Enzymatic lavage (GIT)
 • ___________________ - Best Fixative for Cytology:
Disadvantage: Flammability and extreme volatility; NO LONGER USED
• ___________________ - Most commonly used fixative for routine cytologic smears.
• ___________________ - Fixative used for bloody specimens
• ___________________ - Recommended substitute for 95% ethanol
• ___________________ - Commercial aerosol spray
• ___________________ - Father of Exfoliative Cytology
• ___________________ - Adhesives used in Cytology:
NEVER use Mayer’s Egg Albumin as an adhesive for slides to be stained by PAPs. During staining, the excess of
albumin may also take up the stain and interfere with diagnosis.

• ___________________ - Useful adhesive for cytology, especially specimens that may be bloody or
contain proteinaceous material.
• ___________________ - Considered to be the staining method of choice for exfoliative cytology
• ___________________ - Second best choice for examining routine cytologic preparations, after PAP’s.

Immediate fixation is essential for Cytologic smears. The optimal fixation is done by dropping the smears (use of paper
clips on every other slide to avoid slide surface contacts, and proper exposure to the fixative), in 95% ethyl-alcohol
container for 3 to 5 minutes.

• ___________________ - Required volume of urine for Urine cytology.

SMEAR PREPARATION
STREAKING Use of an applicator stick or platinum loop, material is rapidly and
gently applied in a direct or zigzag line throughout a slide
SPREADING Material is transferred in a clean slide and gently spread into a
moderately thick film. Recommended for fresh sputum, bronchial
aspirate, and thick mucoid secretions.
PULL-APART A drop of secretion or sediment upon 1 slide and facing it to
another clean slide. 2 slides are pulled apart with a single
uninterrupted motion. Useful in preparation of thick secretions
(Serous fluid, Concentrated Sputum, Enzymatic lavage from GIT and
blood smear)
TOUCH PREPARATION Special method of smear preparation whereby the surface of the
(IMPRESSION SMEAR) freshly cut tissue is brought into contact and pressed on the surface
of a clean glass slide.
CLASSIC AUTOPSY TECHNIQUES
VIRCHOW Organs are removed one by one
ROKITANSKY “In-situ” dissection; in part combined with the removal of organ blocks
Dissection while organs are in place.
GHON “En Bloc” removal; widely used
LETULLE “En Masse” removal; recommended for pediatric patient
ADDITIONAL PATHOLOGY NOTES:
NECROSIS → is a pathologic process caused by direct external injury to cells such as, from burns,
radiation, or toxins
→ Necrotic cells show increased eosinophilia in hematoxylin and eosin (H & E) stains,
attributable in part to the loss of cytoplasmic RNA (which binds the blue dye, hematoxylin)
and in part to denatured cytoplasmic proteins (which bind the red dye, eosin).

PATTERNS OF TISSUE NECROSIS

1. COAGULATIVE NECROSIS – A form of necrosis in which the architecture of dead tissues is preserved for a span of
at least some days. Ischemia caused by obstruction in a vessel may lead to a coagulative necrosis of the supplied
tissue in all organs except the brain.
NOTE: Infarct – a localized area of coagulative necrosis
2. LIQUEFACTIVE NECROSIS – Characterized by digestion of dead cells, resulting in transformation of the tissue into
a liquid viscous mass.
NOTE: The necrotic material is frequently creamy yellow because of the presence of dead leukocytes and is called
pus.
3. GANGRENOUS NECROSIS – Not a specific pattern of cell death; usually applied too limb, generally the lower leg,
that has lost its blood supply and has undergone coagulative necrosis.
4. CASEOUS NECROSIS – Refers to the cheese-like friable white appearance of the area of necrosis; Often seen in
tuberculosis
NOTE: Granuloma – Necrotic area appears as structureless collection of fragmented or lysed cells and amorphous
granular debris enclosed within a distinctive inflammatory border on microscopic examination.
 5. FAT NECROSIS – It refers to focal areas of fat destruction, typically resulting from release of activated pancreatic
lipases into the substance of the pancreas and peritoneal cavity.

NOTE: On histologic examination, the necrosis takes the form of foci of shadowy outlines of necrotic fat cells, with
basophilic calcium deposits, surrounded by an inflammatory reaction.

6. FIBRINOID NECROSIS – A special form of necrosis usually seen in immune reactions involving blood vessels. This
pattern of necrosis typically occurs when complexes of antigens and antibodies are deposited in the walls of the
arteries. Deposits of these “immune complexes,” together with fibrin that has leaked of vessels, result in a bright
pink and amorphous appearance in H&E stains, called “fibrinoid” (fibrin-like) by pathologists.

Nuclear Changes in Necrosis:

PYKNOSIS Reduction and Characterized by nuclear shrinkage and increased basophilia. The
condensation of chromatin condenses into a solid, shrunken basophilic mass.
nucleus

KARYORRHEXIS Fragmentation of The pyknotic nucleus undergoes fragmentation. With the passage of time
nucleus (a day or two), the nucleus in the necrotic cell totally disappears.
KARYOLYSIS Dissolution of The basophilia of the chromatin may fade (karyolysis), a change that
nucleus presumably reflects loss of DNA because of enzymatic degradation by
endonucleases.

APOPTOSIS • “Programmed cell death”

• A form of cell death that is characterized by nuclear dissolution, fragmentation of the cell
without complete loss of membrane integrity, and rapid removal of the cellular debris.

FEATURES OF NECROSIS AND APOPTOSIS

NECROSIS APOPTOSIS
Cell size Enlarged (swelling) Reduced (shrinkage)
Nucleus Pyknosis →karyorrhexis →karyolysis Fragmentation into nucleosome size
fragments
Plasma membrane Disrupted Intact; altered structure, especially
orientation of lipids
Cellular contents Enzymatic digestion; may leak out of Intact; may be released in apoptotic
cell bodies
Adjacent inflammation Frequent No
Physiologic or pathologic role Invariably pathologic (culmination of Often physiologic, means of eliminating
irreversible cell injury) unwanted cells; may be pathologic after
some forms of cell injury, especially DNA
damage

The following morphologic features of cells undergoing APOPTOSIS, some best seen with the electron microscope:

1. Cell shrinkage. The cell is smaller in size, the cytoplasm is dense, and the organelles, although relatively normal,
are more tightly packed. (Recall that in other forms of cell injury, an early feature is cell swelling, not shrinkage.)
2. Chromatin condensation. This is the most characteristic feature of apoptosis. The chromatin aggregates
peripherally, under the nuclear membrane, into dense masses of various shapes and sizes. The nucleus itself may
break up, producing two or more fragments.
3. Formation of cytoplasmic blebs and apoptotic bodies. The apoptotic cell first shows extensive surface blebbing,
then undergoes fragmentation into membrane-bound apoptotic bodies composed of cytoplasm and tightly
packed organelles, with or without nuclear fragments.

4. Phagocytosis of apoptotic cells or cell bodies, usually by macrophages. The apoptotic bodies are rapidly
ingested by phagocytes and degraded by the phagocyte’s lysosomal enzymes.

MEDICAL TECHNOLOGY LAWS AND ETHICS

Republic Act Title Effectivity Date
RA 1517 Blood Banking Law of 1956 June 16, 1956
RA 4688 Clinical Laboratory Act of 1966 June 18, 1966
RA 5527 Philippine Medical Technology June 21, 1969
Act of 1969
RA 6425 The Dangerous Drugs Act of March 30, 1972
1972
RA 7170 Organ Donation Act of 1991 January 7, 1992
RA 7719 National Blood Services Act of May 5, 1994
1994
RA 8504 Philippine AIDS Prevention and February 13, 1998
Control Act of 1998
 RA 8981 PRC Modernization Act of 2000 December 5, 2000
RA 9165 Comprehensive Dangerous June 7, 2002
Drugs Act of 2002
RA 9288 Newborn Screening Act of 2004 April 7, 2004
RA 10912 Continuing Professional July 21, 2016
Development (CPD) Act of 2016
RA 11166 Philippine HIV and AIDS Policy December 20, 2018
Act

Classification of Clinical Laboratories

(Based on Service Capability)
Primary Secondary Tertiary
1. Routine Hematology 1. Services offered by the 1. Services offered by the secondary category
2. Qualitative Platelet Primary category 2. Special Chemistry
Determination 2. Routine Clinical Chemistry 3. Special Hematology, including coagulation procedures
3. Routine Urinalysis 3. Quantitative Platelet 4. Immunology
4. Routine Fecalysis Determination 5. Microbiology – Culture & Sensitivity
5. Blood Typing – for 4. Cross-matching – for
Hospital-based Hospital-based
5. Gram staining – for Hospital-
based
6. KOH – for Hospital-based
10 sqm 30 sqm 60 sqm

• ___________________ - Head of the laboratory.

• ___________________ - A certified pathologist is authorized to manage or supervise or be an associate in not

more than _______ clinical laboratories.
• ___________________ - Any person who knowingly or negligently causes another to get infected with HIV in the
course of the practice of his/her profession through unsafe and unsanitary practice or procedure is liable to
suffer a penalty of imprisonment for _________.
• ___________________ - Any violation of medical confidentiality shall suffer the penalty of imprisonment for
_________.
• ___________________ - Current PAMET President
• ___________________ - PRC Chairperson
• ___________________ - PRC Commissioners
• ___________________ - Head of BOMT
• ___________________ - What is the REQUIRED age of an applicant for the issuance of certificate of registration
as a registered medical technologist?

MEDICAL TECHNOLOGY CODE OF ETHICS

• As I enter into the practice of Medical Technology, I shall accept the responsibilities inherent to being a
professional; I shall uphold the law and shall not engage in illegal work nor
• cooperate with anyone so engaged; I shall avoid associating or being identified with any enterprise of
questionable character;
• I shall work and act in a strict spirit of fairness to employer, clients, contractors,
employees and in a spirit of personal helpfulness and fraternity toward other members of the profession;
• I shall use only honorable means of competition for professional employment or services and shall refrain from
unfairly injuring, directly or indirectly, the professional reputation, projects or business of a fellow medical
technologist;
• I shall accept employment from more than one employer only when there is no conflict of interest;
• I shall perform professional work in a manner that merits full confidence and trust carried out with absolute
reliability, accuracy, fairness and honesty;
• I shall review the professional work of other medical technologists, when requested, fairly and in confidence
whether they are subordinates or employees, authors of proposals for grants or contracts, authors of technical
papers or other publications or involved in litigation;
• I shall advance the profession by exchanging general information and experience with fellow medical
technologists and other professionals and by contributing to the work of professional organizations;
• I shall restrict my praises, criticisms, views and opinions within constructive limits and shall not use the
knowledge I know for selfish ends;
• I shall treat any information I acquired about individuals in the course of my work as strictly confidential, and
may be divulged only to authorized persons or entities or with consent of the individual when necessary;
• I shall report any infractions of these principles of professional conduct to the authorities responsible of
enforcement of applicable laws or regulations, or to the Ethics Committee of the Philippine Association of
Medical Technologists as may be appropriate.
To these principles, I hereby subscribe and pledge to conduct myself at all times in a
manner befitting the dignity of my profession.
 Suggested Guidelines for Record and Specimen Retention
Requisitions 2 years
Accession logs 2 years
Maintenance/instrument logs 2 years
Quality control records 2 years
Blood bank donor/receipt records 10 years
Blood bank deferred donor records Indefinitely
Blood bank patient records 10 years
Blood bank employee signatures/initials 10 years
Blood bank QC records 5 years
Clinical pathology test records 2 years
Serum/CSF/body fluids 48 hours
Urine 24 hours
Blood/fluid smears 7 days
Microbiology stained slides 7 days
Wet tissue 2 weeks
Surgical pathology (bone marrow) slides 10 years
Paraffin blocks/slides 10 years
Cytology slides 5 years
FNA slides 5 years
Reports (surgical/cytology/nonforensic) 10 years
Cytogenetic slides 3 years
Cytogenetic reports/images 20 years
Flow cytometry plots/histograms 10 years
Retired Laboratory Procedures 2 years

________________________________, RMT