Introduction to Pathology

Microscope

The microscope is the most commonly used instrument in diagnostic laboratories. It produces
enlarged images of minute objects. The light microscope is the most widely used, followed
by special types of microscopy.

Light Microscope

A light microscope uses visible light for illumination. It may be simple or compound.

Simple Microscope

A simple hand magnifying lens with magnification ranging from 2× to 200×.

Compound Microscope

Uses a combination of lenses: an objective lens near the specimen and an eyepiece lens near
the observer. It may be monocular (single eyepiece) or binocular (two eyepieces).

Parts of a Compound Microscope

Stand and Body

The stand provides stability. The body or limb arises from the stand and supports the
microscope parts. Modern microscopes are ergonomically designed.

Body Tubes

The external tube carries a revolving nosepiece with slots for objective lenses. The nosepiece
may be triple, quadruple, quintuple, or sextuple. The internal draw tube holds the eyepiece.

Mechanical Stage

A metallic platform that holds the glass slide. It has a central aperture for light passage. Slide
movement is controlled by two knobs allowing forward–backward and side-to-side
movement. Graduated scales permit measurements along X and Y axes.

Substage

Located below the stage and contains the condenser and iris diaphragm. The condenser
focuses light on the specimen. The iris diaphragm controls light intensity, opened for high
power and closed for low power.

Coarse and Fine Adjustment Knobs

Used for focusing. In modern microscopes, the stage moves up and down while the objective
remains fixed. Fine adjustment moves the stage by 0.002 mm per division.
Optical System

Eyepiece

Contains two plano-convex lenses. Magnification may be 5×, 10×, or 15×, with 10×
commonly used.

Objectives

Made of multiple lenses with prisms. Common magnifications are 4×, 10×, 40×, and 100× oil
immersion. Types include achromat, apochromat, and planapochromat.

Condenser

Consists of two simple lenses and concentrates light onto the specimen with the help of the
iris diaphragm.

Illumination System

Older microscopes use a mirror, plane for sunlight and concave for artificial light. Most
modern microscopes have built-in electrical illumination ranging from 20 to 100 watts.

Magnification and Resolving Power

Magnification

Total magnification is obtained by multiplying the magnification of the objective lens by that
of the eyepiece. Standard tube length is 160 mm.

Resolving Power

Resolving power is the ability to distinguish two closely placed objects.

R = 0.61 λ / NA

Where λ is the wavelength of light and NA is the numerical aperture. The resolving power of
a light microscope is approximately 200 nm.

Use and Maintenance of Light Microscope

The microscope should be placed on a stable surface and kept dust free. Separate cleaning
materials should be used for optical and mechanical parts. Proper illumination should be
adjusted. For colourless objects, the condenser should be lowered and the diaphragm partially
closed. The 100× objective should be used with oil. After oil immersion, the objective lens
should be cleaned.

Newer Applications of Light Microscopy

Teaching Microscopes
Modern microscopes can be modified with multi-headed viewing systems or camera
attachments for teaching and group discussions. Images can be displayed on monitors and
stored digitally.

Image Analysis and Morphometry

Microscopes attached to computers convert images into digital form, allowing measurement
of cellular parameters such as nuclear area and cell size.

Telepathology

Slides are examined remotely. Robotic telepathology allows remote control of the
microscope, while static telepathology involves transmission of scanned images. It is used for
consultation and primary diagnosis.

Other Types of Microscopy

Dark Ground Illumination

Used for examination of unstained living microorganisms such as Treponema pallidum.

Oblique light illuminates the organism against a dark background.

Phase Contrast Microscopy

Used for studying unstained living cells. Differences in refractive indices of cell organelles
produce contrast.

Polarising Microscopy

Used to demonstrate birefringent substances such as amyloid, hair, and foreign bodies using
polarised light.

Fluorescent Microscopy

Used to demonstrate fluorescent substances or organisms stained with fluorescent dyes such
as FITC and thioflavin. UV light is used for illumination.

Electron Microscopy

Electron microscopy is used for ultrastructural study of tissues and cells. Tissue is fixed in
glutaraldehyde and ultrathin sections are prepared. Resolution is 0.2 nm or less.

Transmission Electron Microscopy

Used to study internal cell structures and produces two-dimensional images.

Scanning Electron Microscopy

Used to study surface details and produces three-dimensional images.

Histology is the study of normal tissues at the microscopic level, whereas histopathology
involves the microscopic examination of tissues to detect structural changes caused by
disease. Both are performed on thin tissue sections stained with specific dyes to highlight
cellular details.

For examination, a representative tissue sample not exceeding 4 mm thickness is placed in a

cassette and subjected to sequential tissue processing steps: fixation, dehydration, clearing,
impregnation, embedding and blocking, section cutting (microtomy), and routine staining
with haematoxylin and eosin (H & E).

Fixation prevents tissue decomposition that begins immediately after removal from the body
due to autolysis, bacterial action, and metabolic waste accumulation. It preserves tissue in a
life-like state by denaturing or precipitating proteins, rendering cellular components
insoluble. Fixation prevents autolysis and putrefaction, hardens tissue for sectioning,
stabilises cells against osmotic changes, enhances staining, and improves morphological
clarity.

An ideal fixative should be inexpensive, stable, safe, fast-acting, provide uniform penetration,
preserve tissue colour, avoid tissue loss, and not interfere with staining reactions.

Fixatives are classified as simple (single agent, e.g. formalin) or compound (multiple agents,
e.g. Bouin’s fluid), and functionally as microanatomical, cytological, or histochemical
fixatives.

Formalin is the most commonly used routine fixative. Commercial formalin is a 40%
formaldehyde solution, considered 100% formalin. Routine fixation uses 10% formalin,
prepared by diluting it with water. Fixation usually takes 6–8 hours for tissue 4 mm thick,
using a fixative volume 15–20 times that of the specimen. Formalin fixes tissue by forming
methylene bridges between proteins. Its advantages include rapid penetration, preservation of
tissue colour, low cost, and suitability for nervous tissue. Disadvantages include excessive
tissue hardening, irritation, and formation of formalin pigment in blood-rich tissues.

Glutaraldehyde is mainly used for electron microscopy and for renal and testicular biopsies. It
is effective but expensive and penetrates tissues slowly.

Bouin’s fluid, containing picric acid, formaldehyde, and acetic acid, stains tissues yellow and
is useful for glycogen preservation. Its disadvantages include tissue brittleness and red cell
lysis.

Alcohol fixatives, such as Carnoy’s fixative, are mainly used for cytological smears and
glycogen preservation. They act by protein denaturation and dissolve fat.

Osmium tetroxide is used for CNS tissue and electron microscopy and is the best fixative for
lipids, imparting a black colour to tissues.

Dehydration removes water from tissues using ascending grades of alcohol to allow wax
infiltration. Clearing replaces alcohol with a wax-soluble agent such as xylene, making
tissues transparent. Impregnation fills tissue spaces with molten paraffin wax, which hardens
the tissue for sectioning.
These steps are performed using automated tissue processors, which may be open (hydraulic)
or closed (vacuum). Closed processors reduce toxic exposure and allow faster processing
using heat and vacuum.

Embedding involves placing tissue in molten wax to form blocks, which are then sectioned
using a microtome. The rotary microtome is most commonly used. Sections of 3–4 µm
thickness are floated on a warm water bath to remove folds, mounted on glass slides, dried,
and prepared for staining.

Routine staining uses haematoxylin and eosin. Haematoxylin stains nuclei blue after
oxidation to haematein and binding with a mordant, while eosin stains cytoplasm and
extracellular components pink. After staining, sections are dehydrated, cleared, and mounted.

Frozen section technique involves rapid freezing of fresh tissue, allowing quick intraoperative
diagnosis. It is useful for margin assessment and demonstration of lipids and enzymes.
Although rapid, frozen sections are thicker, less detailed, and unsuitable for long-term
storage. They are obtained using either a freezing microtome with CO₂ or a cryostat.

Special stains are used to identify specific tissue components, such as fats (Sudan black, Oil
Red O), collagen (Van Gieson), muscle (Masson’s trichrome), reticulin (silver impregnation),
amyloid (Congo red, methyl violet), glycogen (PAS), and iron (Prussian blue).

Immunohistochemistry (IHC) applies immunological techniques to detect specific antigens in

tissues using labelled antibodies visualised by chromogens like DAB. IHC aids in tumour
classification, prognostication, therapy prediction, and infection identification but does not
distinguish benign from malignant lesions.
PA2.8 Identify and describe various forms of cell injuries with their manifestations and
consequences in gross and microscopic specimens

Degeneration

Degeneration refers to the morphologic changes seen in reversible cell injury. It includes
early alterations such as hydropic swelling, intracellular or extracellular deposition of pink
proteinaceous hyaline material, and accumulation of mucoid or myxoid substances in
epithelial or connective tissues.

Vacuolar Nephropathy

Vacuolar nephropathy is an early, reversible renal injury characterized grossly by cloudy

swelling and microscopically by hydropic vacuoles in tubular epithelial cells. It results from
failure of sodium–potassium membrane pumps, leading to intracellular sodium and water
accumulation with potassium loss. Causes include bacterial toxins, chemicals, poisoning,
burns, high fever, and intravenous hypertonic glucose or saline.

Gross Appearance
Kidneys are mildly enlarged, pale, and show a cloudy, opaque, bulging cut surface.

Microscopic Examination:
i. Proximal convoluted tubular cells contain clear, well-defined watery vacuoles representing
distended endoplasmic reticulum; these do not stain for fat or glycogen.
ii. Interstitial microvasculature is compressed due to tubular epithelial swelling.

Vacuolar Nephropathy
Hyaline Change in Leiomyoma

Hyaline change (hyalinisation) denotes deposition of glassy, pink, homogeneous material on

haematoxylin and eosin staining and represents an end-stage change in many lesions. In
uterine leiomyoma, it is an example of extracellular hyaline deposition.

Gross Appearance:
Leiomyomas are well-circumscribed, firm tumours with a whorled cut surface; areas of
hyaline change appear glassy and homogeneous.

Microscopic Examination:
i. Interlacing smooth muscle fibres and fibrous tissue arranged in whorls.
ii. Smooth muscle cells show eosinophilic cytoplasm with short, plump nuclei, while
fibroblasts have paler cytoplasm and elongated nuclei.
iii. Hyaline areas appear as pink, homogeneous, acellular material within or between the
whorls.

Hyaline Change in Leiomyoma

Intracellular Accumulations
Abnormal buildup of substances in cells occurs due to cell injury. These include excess
normal metabolites (fatty change), excess endogenous pigments (melanin), abnormal
endogenous pigments (lipofuscin), and exogenous pigments (carbon).

Fatty Change (Steatosis) of Liver

Commonest site is the liver. Causes include alcohol abuse (most common), malnutrition,
obesity, diabetes mellitus, anoxia, and toxins.
Gross: Enlarged, yellow, greasy liver with rounded margins.
Microscopy: Clear fat vacuoles in hepatocytes, either microvesicular or macrovesicular with
peripheral nuclei. Initially centrilobular, later diffuse. Fatty cysts, lipogranulomas, and fat
positivity with Sudan and Oil Red O stains may be seen.
 Fatty Change (Steatosis) of Liver

Anthracotic Pigment in Lung

Benign deposition of carbon in lungs due to pollution or smoking; severe exposure in coal
miners causes coal-workers’ pneumoconiosis.
Gross: Black lungs and hilar lymph nodes with mottling.
Microscopy: Carbon-laden macrophages around bronchioles, beneath pleura, and in lymph
nodes; usually no functional impairment except in pneumoconiosis.

Anthracosis Lung

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NECROSIS
Morphologic expression of irreversible cell death.
Types
 Coagulative – ischaemia (kidney, heart, spleen)
 Liquefactive – enzymatic digestion (brain)
 Caseous – tuberculosis
 Fat necrosis – pancreatic lipase (acute pancreatitis)

COAGULATIVE NECROSIS (RENAL INFARCT)

Cause: Sudden ischaemia
GROSS APPEARANCE
 Pale, wedge-shaped infarct
 Base under capsule, apex toward medulla
 Subcapsular rim spared
 Initially red → pale yellow (day 4) → depressed (1 week)
MICROSCOPIC EXAMINATION
 Architecture preserved, cellular details lost
 Marginal inflammation: neutrophils → macrophages, lymphocytes, fibrosis

COAGULATIVE NECROSIS (RENAL

INFARCT)

LIQUEFACTIVE NECROSIS (BRAIN INFARCT)

Cause: Hydrolytic enzymes
GROSS APPEARANCE
 Soft, liquefied area → cyst formation
MICROSCOPIC EXAMINATION
 Cyst with necrotic debris and macrophages
 Wall shows capillaries, inflammatory cells, gliosis
 LIQUEFACTIVE NECROSIS (BRAIN INFARCT)

CASEOUS NECROSIS (TB LYMPH NODE)

Cause: Tuberculosis
GROSS APPEARANCE
 Matted nodes
 Yellow, granular, cheese-like necrosis
MICROSCOPIC EXAMINATION
 Structureless eosinophilic granular debris ± calcification
 Surrounding granuloma: epithelioid cells, Langhans giant cells, lymphocytes

CASEOUS NECROSIS (TB LYMPH NODE)

ENZYMATIC FAT NECROSIS (PANCREAS)
Cause: Acute pancreatitis
GROSS APPEARANCE
 Yellow-white, firm, chalky deposits
MICROSCOPIC EXAMINATION
 Cloudy necrotic fat cells
 Basophilic calcium soaps
 Surrounding inflammation

GANGRENE
Ischaemic necrosis with bacterial infection
Types
 Dry – arterial obstruction (foot)
 Wet – venous obstruction or arterial block (bowel)

DRY GANGRENE (HAND)

GROSS APPEARANCE
 Dry, shrunken, black tissue
 Clear line of demarcation
MICROSCOPIC EXAMINATION
 Coagulative necrosis
 Tissue breakdown
 Granulation tissue at margin

MONCKEBERG’S ARTERIOSCLEROSIS
Type: Dystrophic calcification
GROSS APPEARANCE
 Pipestem rigidity
 No significant luminal narrowing
MICROSCOPIC EXAMINATION
 Media replaced by hyalinised tissue
 Basophilic calcific granules
 No inflammation

MONCKEBERG’S ARTERIOSCLEROSIS
Cellular Adaptations
Cellular adaptations are structural and functional changes that cells develop in response to
stress. These include decrease or increase in cell size (atrophy and hypertrophy), increase in
cell number (hyperplasia), and change in differentiation (metaplasia and dysplasia). Dysplasia
is discussed separately in Exercise 40.

Testicular Atrophy
Atrophy is reduction in size and number of previously normal cells, commonly seen with
senility.
Gross Appearance: Testis is small, firm, and fibrotic.
Microscopic Examination: Seminiferous tubules show progressive loss of germ cells,
thickened basement membrane, peritubular fibrosis, and occasional hyalinisation. Interstitial
stroma is increased with prominent fibrovascular tissue and conspicuous Leydig cells.

Cardiac Hypertrophy
Hypertrophy is enlargement of an organ due to increased size of parenchymal cells,
commonly caused by aortic stenosis and systemic hypertension.
Gross Appearance: Heart is enlarged and heavy, weighing up to seven hundred to eight
hundred grams. Left ventricular wall thickness exceeds two centimetres. Hypertrophy without
chamber dilatation is concentric, while that with dilatation is eccentric.
Microscopic Examination: Myocardial fibres are enlarged with prominent nuclei, and focal
degeneration or necrosis may be present.

Reactive Hyperplasia of Lymph Node

Hyperplasia is an increase in cell number, commonly occurring in lymphoid tissue due to
local irritation.
Gross Appearance: Lymph nodes are enlarged, firm, and non-tender.
Microscopic Examination: Patterns include follicular hyperplasia with enlarged germinal
centres, paracortical hyperplasia with effacement of follicles, and sinus histiocytosis with
distended sinusoids filled with histiocytes and endothelial cells.

Squamous Metaplasia of Cervix

Squamous metaplasia is a reversible replacement of specialised epithelium by squamous
epithelium.
Gross Appearance: Common in prolapsed uterus, the cervix appears pearly white with
elongated isthmus.
Microscopic Examination: Columnar epithelium and glands show squamous change,
surface keratinisation or hyperkeratosis, and variable subepithelial chronic inflammatory
infiltrate.
Squamous Metaplasia of Cervix
PA3.4 Identify and describe acute and chronic inflammation in gross and microscopic
specimens

Inflammation
Inflammation is the local response of the body to injury from various agents. Depending on
the host defense and duration, it may be acute, such as in abscesses, or chronic. Chronic
inflammation is divided into chronic nonspecific inflammatory reactions and specific or
chronic granulomatous reactions, as seen in tuberculosis, leprosy, and sarcoidosis.

Abscess Lung
An abscess is a cavity formed by extensive tissue necrosis following pyogenic bacterial
infection, accompanied by intense neutrophilic infiltration. Lung abscesses may result from
inhalation, emboli, or pneumonia, most commonly affecting the right lung in the upper or
lower lobe. Cavities vary in size; walls are initially ragged and necrotic but may become
smooth and fibrous in advanced stages, sometimes communicating with bronchi.

Microscopic and Experimental Features

 Abscess walls show dense infiltration by polymorphonuclear leucocytes and

macrophages.

 Chronic cases show peripheral fibroblasts.

 Alveolar walls in affected areas are destroyed.

 Abscess lumina contain pus, red blood cells, tissue debris, and fibrin.

Chronic Inflammatory Granulation Tissue

Granulation tissue appears granular and pink in healing ulcers and secondary wound unions.

Microscopic and Experimental Features

 Ulcer surface contains blood, fibrin, and inflammatory exudate.

 Underlying granulation tissue consists of proliferating fibroblasts, new blood vessels,

and inflammatory cells—initially polymorphonuclear cells, later macrophages and
lymphocytes predominate.

 Epithelium grows from wound edges as spurs.

 Mature granulation tissue shows dense collagen, reduced vascularity, and fewer
inflammatory cells.

Tuberculous Lymphadenitis
Tuberculosis of lymph nodes is always secondary to infection elsewhere.
Gross Appearance
Lymph nodes are enlarged, matted together due to periadenitis. Tuberculous areas are yellow,
cheesy, opaque, and caseous; other areas are grey-brown.

Microscopic and Experimental Features

 Caseous areas contain nuclear debris from fragmented cells.

 Granulomatous inflammation surrounds caseous foci, consisting of epithelioid cells

with lymphocytes and some plasma cells; epithelioid cells may fuse into giant cells,
such as Langhans’ or foreign body type.

 Fibroblasts may surround older granulomas.

 Dystrophic calcification may appear as bluish granules in caseous areas.

 Ziehl-Neelsen staining can demonstrate acid-fast bacilli.

 Normal nodal architecture may remain at the periphery.

Granulomatous Inflammation—Tuberculosis
Granulomatous inflammation is a specific chronic response to poorly digestible agents,
exemplified by Mycobacterium tuberculosis infection, producing caseating granulomas in
lungs, lymph nodes, and other organs.

Fibrocaseous Tuberculosis Lung

Chronic breakdown of caseous lung tissue leads to cavitary fibrocaseous tuberculosis, usually
at the apex. Cavities are large, may communicate with bronchi, and have smooth fibrous
walls traversed by bronchi and vessels.

Microscopic and Experimental Features

 Basic lesion is the tubercle: epithelioid cells, lymphocytes, giant cells, and central
caseation necrosis. Tubercle coalescence forms confluent areas.

 Peripheral fibroblasts and fibrosis develop.

 Arterioles may show endarteritis obliterans.

 Surrounding alveoli may contain cellular exudate.

Tuberculosis Intestine
Occurs as primary, secondary, or hyperplastic; secondary tuberculosis of the small intestine is
most common.
Gross Appearance

 Large ulcers transverse to bowel axis, sometimes coated with caseous material.

 Advanced cases show transverse fibrous strictures and obstruction.

Microscopic and Experimental Features

  Caseating tubercles present in all intestinal layers.

 Mucosal ulceration with slough.

 Muscular layer fibrosis varies.

Miliary Tuberculosis Lung

Discharge of caseous pulmonary lesions into blood or lymph leads to disseminated
tuberculosis affecting lungs or other organs.

Gross Appearance

 Lungs show intense congestion and multiple millet seed-sized yellowish-white

lesions.

Microscopic and Experimental Features

 Tubercles with minute central caseation scattered in fibrous lung stroma.

 Intervening alveoli are empty or contain cellular exudate.

Microsvopy

Miliary Tuberculosis Spleen

Hematogenous spread of pulmonary tuberculosis may produce acute miliary lesions in the
spleen.
Gross Appearance

 Scattered yellowish-white firm lesions a few millimeters in diameter.

Other Granulomatous Inflammations

Granulomas may also occur in leprosy and sarcoidosis.

Lepromatous Leprosy
Caused by Mycobacterium leprae, affects skin and peripheral nerves; polar form shows poor
cell-mediated immunity.

Gross Appearance
 Nodules or masses on skin, especially face, hands, and feet.

Microscopic and Experimental Features

 Dermis contains lipid-laden foamy macrophages (lepra cells).

 Clear subepidermal zone; epidermis thin and flat.

 Fite-Faraco stain shows abundant acid-fast lepra bacilli.

Tuberculoid Leprosy
Represents high resistance and good cell-mediated immunity.

Gross Appearance

 Skin lesions with hyperpigmented margins and pale center; neuronal involvement
predominates.

Tuberculoid leprosy

Microscopic and Experimental Features

 Dermal granulomas of epithelioid cells, Langhans’ giant cells, few lymphocytes; little
or no caseation.

 Granulomas erode basal epidermis; dermal nerves affected.

 Fite-Faraco stain shows almost no lepra bacilli.

Fungal Infections
Many fungal infections in humans are opportunistic, occurring when host immunity is
impaired. Key examples include oral candidiasis, Madura foot, pulmonary aspergillosis, and
nasal rhinosporidiosis.

Oral Candidiasis
Caused mainly by Candida albicans, a normal commensal that becomes pathogenic in
immunocompromised individuals. Clinically, it produces creamy white pseudomembranes on
the tongue, palate, and buccal mucosa (oral thrush).

 Microscopy/Pathology: Ulcers may form; organisms appear as budding spores or

non-branching pseudohyphae; positive with PAS stain.

Madura Foot (Eumycetoma)

Chronic suppurative infection caused by Madurella mycetomatis or Madurella grisea, usually
affecting the foot.

 Gross Features: Swelling with black granules discharging from sinus tracts.

 Microscopy/Pathology: Purulent sinuses contain fungal grains; surrounding tissue

shows mononuclear inflammation and fibrosis.

Pulmonary Aspergillosis
Most common opportunistic fungal lung infection, caused by Aspergillus fumigatus,
presenting as allergic bronchopulmonary aspergillosis, aspergilloma, or invasive
aspergillosis.

 Gross Features: Aspergilloma forms a fungal ball in pulmonary cavities or

bronchiectasis.

 Microscopy/Pathology: Abscess cavity with chronic inflammatory cells; mass of

septate hyphae (2–7 μm) with acute-angle dichotomous branching, highlighted by
GMS stain.

Rhinosporidiosis (Nose)
Caused by Rhinosporidium seeberi, forming gelatinous nasal polyps.

 Microscopy/Pathology: Polyp has edematous connective tissue with mucous glands

and inflammatory cells; numerous thick-walled sporangia contain spores on mucosal
surface and submucosa.

Actinomycosis and Parasitic Infections

Includes actinomycosis (bacterial) and tissue parasites such as cysticercosis and hydatid
cysts.
Actinomycosis (Skin/Soft Tissue)
Chronic suppurative infection caused by Actinomyces israelii, commonly in head and neck.

 Gross Features: Firm swelling in lower jaw, later forming sinuses with yellow
“sulfur” granules.

 Microscopy/Pathology: Granulomatous inflammation with central abscess; colonies

show basophilic filaments with hyaline club-shaped ends.

Actinomycosis of skin
Cysticercosis (Soft Tissue/Brain/Muscle)
Larval infection by Taenia solium. Lesions are solitary or multiple 1 cm white cysts with
milky fluid.

• Microscopy/Pathology: Parasite within cyst may degenerate or calcify, triggering

intense tissue reaction; cyst wall lined by histiocyte palisades and mixed inflammatory
infiltrate with eosinophils.

Hydatid Cyst (Liver)

Caused by Echinococcus granulosus larvae.

 Gross Features: Cysts can exceed 10 cm; cyst wall has laminated structure, lumen
contains clear fluid.

 Microscopy/Pathology:

o Pericyst: Outer host inflammatory response with mononuclear cells,

eosinophils, giant cells, fibroblasts.

o Ectocyst: Acellular, chitinous laminated hyaline intermediate layer.

o Endocyst: Inner germinal layer with daughter cysts and scolices.