Dr.

Shampa Rani
Lecturer
Department of Pathology and Parasitology
(FANSVM, PSTU)

CHAPTER-I
Introduction, death of cells and tissues

Pathology: Greek word, Pathos means disease, Logos means science.

Disease- Dis – negative sense, ease- to maintain easy.
Health is the state of an individual living in complete harmony with the environment.
A disease is a condition in which an individual shows an anatomical, chemical, or physiological
deviation from the normal.
Definition: Pathology studies the derangement of molecules, cells, tissues, and functions in
living organisms in response to injurious agents or deprivations.
Key word:
• Response
• Injurious agents- Like mechanical, chemicals, biological, radiation, and different forms of
energy.
• Deprivation- Example, oxygen and various nutrients.
Pathology is the main gate of entrance to medical science. Without the study of Pathology, no
entry into the field of Pharmacology, Medicine, Surgery and Obstetrics.
Purposes of studying:
1.To know the pathogenesis and pathology of a disease or disease condition.
Pathogenesis: is the progressive development of a disease process from the time it is initiated to
its conclusion in recovery or death.
Pathology: Gross and microscopic lesions.
2.To diagnose the disease.
3.To apply the knowledge of pathology in medico-legal practice.
First, changes occur at the molecular, cellular, and tissue levels, and finally, signs appear.

Branches of Pathology:
1.General Pathology: is the branch of Pathology that deals with the fundamental alterations in
tissues that occur in living organisms in response to injurious agents or deprivation.
2. Special Pathology: is the branch of Pathology which deals with systemic disorders and
specific diseases by applying knowledge of general pathology. It includes both systemic
pathology and pathology of infectious diseases.

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3.Clinical Pathology: includes some laboratory tests used by clinicians to aid them in arriving
at a diagnosis.
4.Necropsy/Autopsy/ Post mortem Pathology: Examination of an individual after death by
systematic dissection.
5.Poultry Pathology
6.Ruminant Pathology
7.Experimental Pathology
8.Microscopic Pathology or Histopathology
9.Gross Pathology
10.Humoral Pathology
11.Chemical Pathology
12.Physiological Pathology
13.Pathology of infectious diseases
14.Pathology of non-infectious diseases
15.Oncology.
Terminology
Cell injury is any biochemical and structural alterations that impair a cell's ability to function normally.
Injury may be mild, transient, reversible, and irreversible.
Degeneration is the physico- chemical changes of a cell that may lead to death.
Cell death is a process during which the cell losses its integrity as a structural and functional unit of life
at a point of no return. Cell deaths are, biochemically and morphologically, of two types- i) Programmed
cell death/apoptosis, ii) Accidental cell death.
Necrosis: The term necrosis refers to the characteristics of light microscopic changes resulting from
enzymatic degradation of the nucleus and cytoplasm that define cell death.
Necrosis occurs 12 to 18 hours after the death of the cell.
Normal cell (injurious agent) cell injury cell death Necrosis Autolysis.
Autolysis is the digestion or dissolution of the cell by its enzyme. Autolysis may occur either
ante-mortem or post-mortem. The acid hydrolysis enzymes carry out this process in lysosomes
(especially cathepsin enzyme).
Necrobiosis: It is the physiologic or normal death of cells or tissues as a result of changes associated
with development, aging or use (wear and tear). e.g. the maturation and shedding of epidermal cells,
replacement of chondrocytes as bones grow, the phagocytosis of aged erythrocytes and so on.

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 Causes of cell death:
Cell death occurs in two biochemically and morphologically distinct ways:
(I) Accidental cell death (II) Apoptosis or Programmed cell death.
(I) Causes of cell injury and accidental cell death:
1. Forms of kinetic injury- Example, mechanical, thermal, radiation.
2. Exposure to reactive forms of exogenous and endogenous chemicals.
Exogenous- Example, (a) Various chemicals, e.g., acid, (b) plant poisons, (c) Toxins of
microbial origin.
Endogenous- Example, (a) Toxic products of metabolism (b) Free radicals.
3. Deprivation of essential nutrients – Example, Oxygen, water, food stuffs.
4. Immunologic reactions and genetic disorders.
Biochemical and ultrastructural changes of cell death:
Accidental cell death: causes many, but basic mechanisms and alterations are the same. In all
cases, there is a loss of cell membrane integrity and acute cellular swelling.
(A) Hypoxic cell injury leads to Acute cellular swelling:
Acute cellular swelling results from failure of the energy-dependent active process (Na –
K ion pump). Typically cells are not at equilibrium with their environment. The ion
pump maintains the nonequilibrium condition.
Typical cell Failure of ion pump Acute cellular swelling
Pathogenesis (Schematic):
Hypoxia
Decrease oxidative phosphorylation

Decrease ATP production

Failure of ion pump Increase glycoslysis that causes Detachment Membrane injury*
decrease
(a) decrease
cellcell
glycogen
glycogen
and of ribosome
Increase influx of Na & Ca (b)
decrease
decreasePH PH - ribosomes Increase Ca influx
Increase efflux of K & Mg Clumping
1.Clumping
of nuclear
of nuclear
chromatin. Decrease
Intracellular
chromatin.
release of protein
Decrease Loss of cell membrane
Increase osmotic pressure in lysosomal
2.Intracellular
enzyme.release of synthesis
protein phospholipid
cell lysosomal enzyme- synthesis
Increase influx of water (i)Decrease basophilia Fatty change Death of cell
(ii)Nuclear changes Fatty change
•Cellular swelling (iii) Protein digestion3 Incrase exit of enzyme
•Loss of microvilli e.g., SGOT, SGPT
•Bleb formation
•ER swelling
 * Membrane injury release of cytokines leucotaxis infiltration of leucocytes

(B) Cell injury induced by free radicals: Free radicals have a single unpaired electron in their outer
orbit. These radicals are extremely reactive to lipids, proteins, and DNA which initiate autocatalytic
processes. These are produced in the body due to the action of chemical and radiation injury,
ageing processes, and phagocytosis. Free radicals cause loss of cell membrane integrity and acute
cellular swelling.

(C) Cell injury induced by chemicals: Injury occurs in two ways- (i) binds directly to critical structures,
less ATP production, and finally, acute cellular swelling (ii) transforms into more reactive
metabolites that generate free radicals.

(D) Cell injury induced by viruses:

(i) Interfere in protein and macromolecules synthesis
(ii) Redirecting organelles of a cell to synthesize viral RNA or DNA
(iii) Mechanical pressure
(iv) By stimulating the host immune system

(II) Apoptosis or programmed cell death.

It results from the activation of “suicidal genes” that are found in all cells. Activation of these
genes occurs programme-wise, e.g., digits of limbuds and involution of the thymus.
Activation may occur under particular pathological conditions-
(a) Thymic atrophy- induced by corticosteroid or radiation.
(b) Cachexia induced by TNF-α
(c) Lymphoid depletion in AIDS
(d) Elimination of foreign or virally infected cells by cytotoxic T-Lymphocytes

Apoptotic protein is present in all cells in the inactive stage due to the synthesis of antiapoptotic
protein by the same cells. Antiapoptotic proteins are synthesized in response to survival signals.
Survival signals receive from neighboring cells and extracellular substrates or fluids (survival
signals include growth factors, extracellular matrix and a variety of hormones.)

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I) HYPOXIC CELL DEATH: In hypoxic cell death, there is acute cellular swelling. It occurs
early stages of cell death. When it is severe, the change can be seen by light microscope and is
hydropic or vascular degeneration.

Mechanism/Pathogenesis
a) Normally, the cells are not in equilibrium with the environment. The energy dependent active
ionic process maintains this imbalance so that Na+ and Ca++ cannot enter the cell and K+ and
Mg++ con not leak out the cell. In hypoxia, there is disturbances in mitochondria and no ATP is
produced and as a result the active ionic process is disturbed. So, influx of Na+ and Ca++ in the
cell and leak out of K+ and Mg++ from the cell. So, the selective permeability of the cell is lost
and osmotic pressure of the cell is increased due to Na+ and hence influx of water into the cell
and there is swelling of cell and organelles.
b) For transient period ATP is produced by anaerobic glycolytic pathway and ultimately there is
formation of lactic acid. This lactic acid decreases the pH of the cell.
i) Due to decrease in pH of the cell, the phosphofructokinase is inhibited and so glycolytic
pathway is ceased.
ii) Decreased pH of the cell causes clumping of the nuclear chromatin materials and inhibits the
synthesis of RNA.
c) When there is influx of Ca++ in the cell this Ca++ react with phosphate and initiate
phospholipase activity. So, there is loss of phospholipid from the cell membrane. So, there is
loss of cell membrane integrity and there is cell death.
d) When there is swelling of the organelles of the cell, there is leak out of the enzymes present in
the organelles. These enzymes will ultimately come to the circulation and give the clue of
diagnosis of the disease.
E.g. GPT is present only in hepatocyte, where there is damage of the hepatocyte, the GPT will
be liberated and ultimately will come to the circulation and gives increased SGPT. So increased
SGPT indicates liver damage.
SGOT is found in → Heart, Liver and Bone.
Changes in the organelles after death of the cell.
i) Ribosome will be detached from RER
ii) Mitochondria and other organelles swell
iii) Large fluid blebs protrude from the plasma membrane
iv) Rupture of the plasma and nuclear membrane
v) Nuclear chromatin losses its density
II) Cell death induced by free radicals:
Most of the free radicals that cause cell death are the toxic metabolites derived from oxygen.
The free radicals are-
i) Superoxide (O2−)
ii) Peroxide (H2O2)
iii) Hydroxyl ion (OH−)

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All these compounds inactivate cellular enzyme and damage DNA of the cell. So, there is
ultimately acute cellular swelling.
III) Cell death by exogenous chemicals
Exogenous chemicals induce cell death by one of the following two methods:
a) Binding directly to critical structure or reactive chemicals of cellular organelles and thereby
damaging cell membrane or ability of the cell to generate ATP.
b) By being chemically transformed to more reactive metabolites that are capable of generating
free radicals.
IV) Cell death induced by viruses:
Two types of viruses cause cell death and cell injury
a) Cytolytic viruses by
1. Interfering with the ability of a cell to synthesize protein and other macromolecules essential
for maintenance of cell life.
2. Redirecting organelles of the cell to preferentially synthesize viral RNA/DNA and structural
protein needed for replication and assembly of the virus.
3. Mechanically damaging cellular organelles and disrupting cytoskeleton with accumulation
of viral nuclei acid and proteins (viral inclusion bodies) some of which are cytotoxic.
4. Inserting viral encoded protein in cell membrane causing them to malfunction and leak.
b) Non-cytolytic viruses:
The non-cytolytic viruses cause cell death in vivo by stimulating the host’s immune response to
viral antigens expressed on the surface of the infected cell. The cell killing can be mediated by
1. In humoral immune response, antibody and complement generate a membrane attack
complex that cause infected cell to undergo damage of the membrane and swelling of the
cell as in accidental cell death. This mechanism is known as complement dependent
cytolysis.
2. The Fc portion of the antibody bound to infected cell interact with leukocytes such as
macrophages, neutrophils, eosinophils and natural killer cells (NK cells) to cause death of
the virally infected cell. This mechanism is known as antibody dependent cell mediated
cytotoxicity.
3. Virus infected cell may be killed by cell mediated cytotoxicity. In this case sensitized
cytotoxic T-lymphocytes or NK cells bind to the cell expressing specific viral antigens on
the membrane.
4. Cell mediated cytotoxicity mechanism may cause apoptosis.

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 Differences between apoptosis and accidental cell death:

Apoptosis Accidental cell death

1. Deletion of a single cell or small group of cells
1. 1. 1. Death of cell groups, usually many types.
of the same type.
2. No loss of cell membrane integrity 2. Loss of cell membrane integrity
3. Cell shrinkage occur 3. Cell swells, and then lysis occurs.
4. No inflammatory response 4. Significant inflammatory response.
5. Phagocytized by adjacent normal cells and some 5. Phagocytized by macrophages.
macrophages.
6. Lysosomes remain intact. 6. Lysosomal leakage occurs.
7. Compaction of chromatin in uniformly dense 7. Clumpy, ill-defined aggregation of
masses (usually semilunar shape) chromatin
8. Budding of the cell membrane occurs. 8. Blebing of the cell membrane occurs.
9. Generally induced by physiologic stimuli. 9. Evoked by nonphysiologic/pathologic
stimuli.
10. Regular fashion nucleosomal cleavage (Ladder 10. Random nucleosomal cleavage
formation). (Smear formation)

Apoptotic DNA fragmentation is a crucial feature of apoptosis, a type of programmed cell

death. Apoptosis is characterized by activating endogenous endonucleases with subsequent
cleavage of chromatin DNA into internucleosomal fragments of roughly 180 base pairs (bp) and
multiples thereof (e.g., 360bp and 540bp). This effect can be used to detect apoptosis, for
example, via the DNA laddering assay.

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Characteristics of necrotic cells and tissues:
The microscopic changes that occur in dead tissue as a consequence of ante mortem autolysis
are collectively known as necrosis. Cells & tissues exhibiting these changes are referred to as
necrotic.
A) Microscopic features of necrosis:
1) Changes in the nucleus:
a. Pyknosis:
Pyknosis is the irreversible condensation of chromatin in the nucleus of
a cell undergoing necrosis. Pyknosis (from Greek pyknono meaning "to thicken up, to close or
to condense"). More common nuclear alteration is seen in dead cells but not in all dead cells.
The pyknotic nucleus is characterized by-
❖ The nucleus is decreased in size (i.e. smaller than usual)
❖ Homogenous due to breakdown of chromatin.
❖ Hyperchromatic (Dark blue to black color)
❖ Relatively round in appearance.
Occurrence: Pyknosis is best seen in dead epithelial cells, mononuclear inflammatory cells (e.g.
lymphocytes or monocytes) & nerve cells. Pyknosis is seen in coagulation necrosis.
b. Karyorrhexis:
Karyo→Nucleus, rrhexis→Broken down into tiny pieces. The term karyorrhexis is used to
describe the fragmentation of chromatin into tiny basophilic granules as a consequence of rupture
of the nuclear membrane. The fragmented bits of chromatin may remain in the original position
of the nucleus or be scattered throughout the cytoplasm of the necrotic cell & adjacent areas. It is
a prominent finding in abscess, purulent exudates & early stage of caseation necrosis.
c. Karyolysis:
Karyolysis is the complete dissolution of the chromatin matter of a dying cell due to the activity
of DNase. The whole cell will eventually stain uniformly with eosin after Karyolysis.
d.Loss of nucleus: No nucleus means dead cell. Exception: erythrocytes, thrombocytes
(or platelet)

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 2) Changes in the cytoplasm:
a. Depletion of cytoplasmic glycogen:
It is one of the earliest events in hypoxic cell injury. Glycogen depletion occurs in anaerobic
glycolysis. Here, cytoplasm appears homogenous rather than irregular vacuolation.
b. Increased eosinophilia of cytoplasm:
Cytoplasms appear more red than usual. Due to- (i) Cytoplasmic RNA gives a degree of
basophilia which reduces by enzymatic degradation. (ii) Denaturation of cytoplasmic proteins
gives rise to polypeptide chains, increasing eosin's reaction sites.
Occurrence: Usually in epithelial cells of renal tubules, hepatocytes, coagulation necrosis and
dead neutrophil.
c. Cytoplasmolysis:
This change progresses with the progress of necrosis. The cytoplasm becomes less & less dense
& ultimately disappears completely.

3) Changes in the whole cell- indicate cell death

a.Loss of cell outline:
Completely loss of cell outline is seen in caseous necrosis. e.g. Tuberculosis
Normally→ not visible early in stratified squamous epithelial & smooth muscle.
b. Loss of differential staining:
In this situation, tissue can still be seen; the nuclear and cytoplasmic colour cannot be
distinguished. Chromatolysis is responsible for this.
Occurrence:
Found in caseous & also in post-mortem autolysis.
c. Loss of cells:
In three ways- i. Autolysis ii. Heterolysis iii. Ingested by macrophages or other cells.

Gross characters of dead tissue:

i. Loss of colour: Dead tissue is typically paler than healthy tissue, except if it is filled with
increased blood, it may be black.
ii. Loss of strength: Necrotic tissue has less tensile strength than non-necrotic tissue. It occurs
due to enzymatic digestion of cytoskeleton, cell membrane & intercellular connection. A finger
can easily be thrust into a liver or lung.

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iii. Odors: Odors of putrefaction may emit from dead tissue produced by saprophytic bacteria after
gangrene or post-mortem autolysis. It is because foul-smelling compounds, such as hydrogen
sulfide, ammonia and mercaptans (all are by-products of bacterial fermentation of tissues,
especially proteins) have formed.
iv. Necrosis: Specific gross lesions of various types of necrosis.

Difference between Necrosis & Postmortem Autolysis:

Microscopic lesions:
Necrosis Postmortem Autolysis
i. Presence of alive, sick, and dead cells in a Presence of uniformly dead cells in a
microscopic focus. microscopic focus.
ii. Erythrocytes within the blood vessels are normal in Presence of hemolyzed blood within the
colour with a sharp outline. blood vessels
• Formalin-fixed tissue- H & E→Bright copper red
• Mercuric chloride fixed- H & E→Rose red
• Alcohol fixed- any stain- empty circles
iii. Presence of inflammatory cells & inflammatory zone No inflammatory cells or zone are present.
at the junction between living and necrotic tissue.
iv. No definite sequence of necrosis. Any tissue can There is a definite sequence of post-
undergo necrosis at any time. mortem autolysis.
e.g. Adrenal medulla→Gall bladder→ CNS
→ GIT →→→Enamel of teeth.

Gross Lesions:
Necrosis Postmortem Autolysis
i. Presence of normal blood in the left Presence of hemolyzed blood in the left
ventricle. ventricle of the heart.
ii. No imbibition of blood & bile Imbibition of blood & bile
iii. Usually, no desquamation of Desquamation of epithelium of GIT
epithelium of GIT
iv. Focal discolouration. Uniform discolouration
v. No pseudomelanosis Pseudomelanosis occur.
vi. No putrefactive odor except Putrefactive odor.
gangrene.
vii. Patchy paleness Paleness of whole organ or tissue.
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 Forms / Types of Necrosis:
Named based on gross appearance & tissue involvement.
1. Coagulation or coagulative necrosis.
2. Liquefaction or liquefactive necrosis.
3. Caseation or caseative necrosis.
4. Necrosis of fat or fat necrosis.
5. Zenker’s necrosis.
6. Gangrenous necrosis
7. Fibrinoid necrosis

Brief description of necrosis:

1.Coagulation necrosis:
It is the most common type of necrosis and occurs in almost all organs. There is the persistence of
architectural details but the loss of cellular details for some days. Any injury or the increasing
intracellular acidosis is probably denaturing the structural and enzymatic proteins.
Microscopic appearance:
• Preservation of cellular outline but the loss of cellular details
• The cytoplasm is more acidophilic.
• Nuclei are pyknotic, karyolytic or karyorrhectic
• Comparative less reactive cells.
Gross appearance:
• The necrotic tissue is gray or white, firm, dense
• Necrotic tissue is often depressed compared with surrounding living tissue.

Causes:
i. Local ischemia. e.g. Infract
ii. Toxic products of some bacteria. e.g.Human diphtheria→ Carynobacterium diphtheriae,
Calf diphtheria→ Fusobacterium necrophorum
iii. Certain locally acting poisons. e.g. Mercuric chloride.
iv. Physical injury (heat, cold, electricity, ionizing radiation)
v. Zenker’s necrosis of muscle.

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2. Liquefactive Necrosis:
It is known that liquefactive necrotic tissue liquefies slowly & disappears slowly. However, if the
liquefaction rate is higher than removal, then a measurable amount of fluid will be accumulated
in the necrotic tissue without the noticeable precursory changes in the surrounding cells. This
process is called liquefaction necrosis. Liquefactive necrosis involves the complete breakdown
and digestion of the dead cells, leaving the dead tissue as a viscous liquefied material.
Occurrence:
i. CNS
ii. Abscesses
iii. Sometimes in the tuberculous lung.

Microscopic characteristics:
I. Empty spaces without any definitive lining but have frayed and irregular edges.
II. Cells of edges show some features of necrosis.
III. Pink staining proteinaceous precipitate may remain in empty spaces.
IV. In an abscess, the liquid contains large numbers of neutrophils.
V. In chronic cases, dystrophic calcification and fibrous connective tissue encapsulation are
found.

Why pink colour ?

Ans: Tissue→ Processing→ Dehydration with alcohol→ Fluid remove→ Proteinoceous
precipitate→ H & E stain→ Pink colour
Composition of abscess:
a. Dead or alive neutrophils are found
b. Pyogenic bacteria ( pus-forming bacteria)
c. Pink-coloured proteinaceous precipitate
d. Cellular debris.

Gross appearance:
i. The cavity contains yellowish-white fluid, which is opaque.
ii. In an abscess, semi-solid yellowish pus is present.

[NB: It is confused with the cyst. In cyst, the fluid-filled spaces are lined by the usual
epithelium & the fluid is usually a secretion. In general, the fluid is clean]
Causes: liquefaction necrosis occurs either through heterolysis/or by autolysis, heterolysis and
bacterial toxin-lysins.
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 In CNS- liquefaction occurs by autolysis, high lipid content; less coagulable protein content of
CNS enhance the process.
Abscess: In this case, liquefaction occurs due to autolysis, heterolysis & bacterial enzymes
➢ Toxins ( lysins) from bacteria
➢ If neutrophils come, autolysis & heterolysis occurs & ultimately liquefy.
Pulmonary tubercle: In secondary pyogenic infection, the caseous mass undergoes liquefaction
rapidly.
3. Caseous necrosis:
Caseous necrosis can be considered a combination of coagulative and liquefactive necroses. There
is a loss of both architectural & cellular details. It forms a fine granular mass forming a mixing of
coagulated protein & lipid. The toxins of various organisms cause it.

Occurrence:
i. Typical lesions of tuberculosis ( Mycobacterium tuberculosis)
ii. Human syphilis (Treponema pallidum)
iii. Ovine caseous lymphadenitis ( Corynebacterium pseudotuberculosis)
Another granulomatous disease (primary reactive cells are macrophages with
lymphocytes, giant cells and fibroblasts)
a. Aspergillosis- Aspergillus spp.
b. Glanders- Pseudomonas mallei
iv. Caseous material slowly liquefies and removes.

Microscopic appearance:
i. Loss of cell outline & differential staining.
ii. Tissue disintegrates to form a purplish-coloured fine granular mass ( mixture of
cytoplasm and nuclear materials).
iii. In chronic cases-
a. Fibrous capsule formation
b. Dystrophic calcification.

Gross appearance:
i. The caseous appearance is characterized by white, grayish or yellowish color. The color
of the affected area is similar to milk card or cottage cheese.
ii. The necrotic tissue is dry, slightly greasy & firm.
iii. Loss of cohesive strength, so easily separable with a blunt instrument.
iv. Easily separate into granular fragments with a blunt instrument.
In chronic cases:
a. Fibrous capsule→hard to cut
b. Dystrophic calcification→ gritty sound & sensation

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4. Fat necrosis:
It occurs only in adipose tissue. When adipose tissue (fat) undergoes necrosis, the fat is
decomposed into fatty acid & glycerin. The fatty acid combines with metallic ions (chiefly Na,
K, & Ca) to form soap. These soaps are not dissolved by the solvents used to remove fat from
tissue during processing. Hence the compounds persist.
Microscopic appearance:
i. Sheets of lipocytes present with colored, homogenous substances instead of empty spaces in
adipose tissue. Na- soap gives bluish colour, K- soap gives pinkish colour & Ca- soap gives
purple colour.
ii. Sometimes tiny clear slits are present in lipocytes indicating dissolved fatty acid crystals.
iii. Usually, the nuclei of lipocytes are pyknotic.

Gross characteristics:
Adipose tissue becomes dull instead of shiny, opaque, whitish, solid or slightly granular.

Causes:
Depending on the causative mechanism, fat necrosis are two types-
i) Pancreatic necrosis of fat: Occurs in acute pancreatitis. The pancreatic lipase enzyme escapes
the acinus and ducts and is released into the pancreatic tissue. This act on fat cell membranes
and liquefy them, and the lipases split the triglyceride esters and release FA. The released FA
combines with calcium to produce a chalky white appearance (fat saponification). This enables
the surgeon to identify the lesion quickly.

It is caused by direct mechanical injury and inflammation of the pancreas (acute pancreatitis).

ii) Traumatic necrosis of fat:

Pressure & trauma can cause necrosis of subcutaneous fat in any region of the body. e.g.
due to sternal recumbency, fat necrosis is found in the sternal region in a cow.

[NB: In cattle- lipomatosis may be found in retro peritoneal tissue. Sometimes large masses or
nodules of necrotic adipose tissue are found in the abdominal cavity or retroperitoneal tissue in
the absence of pancreatic disease. It may lead to intestinal stenosis. It occurs due to the
crystallization of fatty acids when lipocytes exceed the rate of fatty acid transport.]

Significance:
• Pancreatic necrosis of fat indicates pancreatic disease and has significance to meat
inspection.
• Traumatic necrosis of fat has significance in medico-legal practice.

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5. Zenker’s necrosis/ Zenker’s degeneration:
Zenker’s necrosis (a particular type of coagulation necrosis) is a severe glassy or
waxy hyaline degeneration or necrosis of skeletal muscles in acute infectious diseases. The
condition was named by Friedrich Albert von Zenker. It occurs only in striated muscles.

Microscopic appearance:
i. Muscle fibres are swollen and have a homogenous hyaline or glassy appearance.
ii. Sarcoplasm is more eosinophilic than a typical cell.
iii. Nucleus is pyknotic
iv. Loss of cross- striations of myofibrils.

Gross appearance:
I. The affected muscle is pale tan to white but sometimes black due to pseudomelanosis (black
leg).
II. Muscle fibres are swollen and somewhat shiny.
III. It looks like a cooked meat appearance.

Causes:
I. Pathogenic microorganisms produce toxins. e.g., Black leg.
II. Vitamin E and selenium deficiency e.g., white muscle disease.

6. Gangrenous necrosis
Gangrenous necrosis is usually associated with the lower limb, where the blood supply to tissue
has been severely affected. It causes coagulative necrosis with a distinct black colour due to the
deposition of iron sulphide from the breakdown of haemoglobin. When infection of the
gangrenous area occurs, liquefactive necrosis occurs (formation of pus) - it is known as “wet
gangrene” and presents a very foul odour.
Caused by ischaemia (coagulative necrosis), and bacterial infections (particularly Chlostrida
spp).

7. Fibrinoid necrosis
Fibrinoid necrosis is associated with smooth muscle necrosis surrounding arterioles and other
vessels. It causes fibrin deposits and other plasma proteins to be deposited. Fibrin is easily
identified in light microscopy due to its eosinophilic appearance.
It is caused by injuries to blood vessels.

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Disposition of necrotic tissue
Necrotic tissue may persist in the body for some time, but ultimately its disposal occurs by one
of the following mechanisms:

1. Liquefaction by autolysis and heterolysis and removal of the fluid by way of the blood
and lymph. It is the typical process and route in CNS.
2. Liquefaction and cyst formation occurs when fluid accumulates faster than it is drained
away.
3. Liquefaction and abscess formation: There is purulent exudation in pyogenic bacterial
infection. Abscesses tend to rupture and ultimately develop necrosis in the overlying
tissue.
4. Encapsulation without liquefaction: Occurs when there is less moisture in dead tissue
(coagulation and caseation necrosis). Dead tissue acts as an irritant and incites
inflammatory reactions and fibrous tissue encapsulation.
5. Desquamation and sloughing
6. Replacement by scar tissue.
7. Dystrophic calcification: Ca-salt deposits in dead tissue and may remain harmlessly
though out life. May mechanically interfere with function as in joint.
8. Gangrene: Necrotic tissue is invaded by saprophytic, specially putrefactive bacteria. Fate
is dangerous. It occurs when necrotic tissue is directly or indirectly exposed to air.
9. Numerical atrophy: The organ or tissue becomes smaller than usual due to the loss of
cells.
10. Regeneration: Replacement of necrotic tissue by the same type of cells. It is a fortunate
outcome. Best seen in lining epithelia, alveoli of lung and bronchia and parenchymal
cells of liver and kidneys.

Cholesterol cleft: Occurs as by-products of necrosis and appears as empty spaces. Grossly –
shiny, yellowish, granular material. Found in old haemorrhages, old abscesses, and atheromas.

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 Gangrene
Gangrene is a condition in which necrotic tissue is invaded by saprophytic and, usually,
putrefactive bacteria. Saprophytes are usually air-borne bacteria. So, gangrene occurs in those
organs or tissues with direct or indirect access to air.
Saprophytic bacteria-- The bacteria (air-borne) which multiply in the dead decaying organic
material. Saprophytes are air borne bacteria. So, gangrene occurs in that organ which is
excessively in contact directly or indirectly with air. Hence gangrene does not occur in the liver
or brain.
Types of gangrene: Mainly gangrene is two types: 1. Moist gangrene, and 2. Dry gangrene and
other one is Gas gangrene.
1) Moist gangrene-- is a more frequent type of gangrene that occurs in tissues that are well-
filled with blood at the time of necrosis begins. It is the common type of gangrene. This type of
gangrene is usually found in visceral organ.
Microscopic appearance:
• Large bacilli are found in a mixture of coagulation and liquefaction necrosis. Bacilli need not
be numerous. It may be 2 or 3 in focus.
• In H&E stain, bacteria are bluish but less blue than the nucleus and hazy in outline.
• Gas bubbles may be found as variable-sized empty spaces with no wall of their own but are
usually spherical.
• There is a zone of inflammation and reactive cells between normal and gangrenous tissue.
Grossly:
• The affected part is swollen, soft, pulpy, and usually dark or black.
• Areas have a foul or putrefactive odour.
• The affected part is cold and insensitive to touch or pain.

2) Dry gangrene-- occurs in tissues with a limited content of blood and fluid or in tissues
where necrosis has developed slowly with retardation of natural circulation. This type of
gangrene is found in extremities. Multiplication and spread of bacteria is slow.
Microscopic character-same as moist gangrene.
Grossly:
•In tissues that are poor in moisture and from those in which evaporation is possible (e.g.,
extremities and surface of the body)
•The affected area becomes cool, shrivelled, leathery and discolored. Dry gangrene spread
slowly.

17
 •Areas of gangrene (in both types) are separated from the adjacent living tissue by a sharp
line of demarcation, which is grossly swollen, reddish or bluish due to hyperemia and
inflammation.
Causes of gangrene:
Causes of gangrene are the causes of necrosis plus exposure to saprophytic bacteria.
• In the intestine, interference with blood supply is the leading cause. Thrombus, embolus,
twisting, intussusception, volvulus, parasite, etc., may interfere with blood supply.
• In the lungs and udder, toxic products of highly lethal bacteria are the usual causes.
• Irritant medicines inadvertently inhaled gangrenous cause pneumonia in the lung.
• In extremities, low temperature (freezing, frostbite) causes thrombosis of the vessel, so
necrosis of tips of ears, snout, feet, and tail causes infection by putrefactive bacteria
resulting in gangrene. Ergot causes constriction of the smooth muscle of arterioles of the
limb, causing gangrene. Senile gangrene occurs in men in old age due to arteriosclerosis,
narrowing the arteries and causing ischemia. Diabetic gangrene occurs due to the
narrowing of arteries. Bacteria grow well in tissues with high sugar content.
• In mastitis, caused by staphylococcus, necrosis occurs either due to toxins or due to
thrombosis of the mammary vessels. Saprophytes results in gangrene subsequently
invading it.
Significance and effect:
The principal target is to stop the gangrenous process. Highly toxic substances are produced
during the bacterial decomposition of proteins in gangrenous tissue.
Gangrene is dangerous because toxemia, sapremia, and septicemia produce toxins that lead to
death. So, amputation is necessary to preserve patients' life. In the intestine, surgical removal of
gangrenous parts and anastomosis of a healthy segment is the only hope for survival. If the
patient has good resistance capacity, the gangrenous extremity may drop off, and the stump
slowly heals. This is the usual outcome of dry gangrene.
[NB: Sapremia: a toxic state resulting from the presence in the blood of toxic products of
putrefactive bacteria and often accompanying gangrene of a part of the body; Septicemia:
Septicemia is bacteria in the blood that often occurs with severe infections; Toxaemia: Presence
of toxin in the blood.]

18
 3) Gas gangrene:
In this type of gangrene there is accumulation of gas. Some anaerobic spore-forming bacteria of
the genus Clostridium are both pathogens and saprophytes. They can kill animal tissue and
continue to multiply in it as saprophytes. They produce excess gas during the time of
proliferation in the necrotic tissues, known as gas gangrene. E.g., Name of some bacteria
responsible for production of gas gangrene--
i) Clostridium chauvoei − Black quarter or black leg
ii) Clostridium septicum − Malignant edema
iii) Clostridium novyi − Black Disease
iv) Clostridium sordelli − Wound infection
v) Clostridium canaris − wound infection, etc.
The group of diseases produced by these organisms is known as gas gangrene.

19
 Infarct
An infarct is a localized area of tissue necrosis (coagulative type) resulting from sudden
obstruction of the blood supply to the affected area. Infarct is derived from the Latin word
‘infarcire’ to plug or cram. The term used in ancient pathology originally referred to a supposed
consolidation of humors of affected parts. Area-infarct; process- infarction.
Ordinarily, the area is supplied by a single end artery, having no collateral blood supply, and the
boundaries are sharply delimited.
Types of infarct (Based on how much blood is present in the necrotic tissue): Infarcts are two
types- (i) hemorrhagic or red infarcts and (ii) anemic or pale infarcts.
Mechanism of infraction:
Blood vessel obstruction
↓
No blood supply to the tissues
↓
Results hypoxia (deficiency of O2 in the tissues)
↓
Coagulative necrosis in the affected tissue & necrosis of blood capillaries occur
↓
Dead tissue without normal strength & resistance
↓
A certain amount of blood diffuses back from the living into the dead capillaries
↓
Blood enters into necrotic tissue
↓
As a result, recent infarcts in specific organs are filled with blood
↓
Red or hemorrhage infract

The area is filled with blood in a hemorrhagic infarct, but the area is paler than normal in a pale
infarct. Congestion and hemorrhage were seen at the peripheral rim of the area. The type of
infarct depends upon the time of observation or the type of tissue involved.
Red or hemorrhagic infarct occurs in some organs (soft and porous). e.g. spleen, lung.
Infracts in solid tissues such as the kidney appear paler than usual (anemic or pale infarct).
Pale infarct looks pale because, in long-standing cases, RBC is breakdown and hemoglobin is
released to the surrounding area, which is engulfed by inflammatory cells.

20
Microscopic appearance:
i. Coagulative necrosis filled with RBC in red infract or without filled with RBC in pale infarct.
ii.Necrotic area is triangular or wedge-shaped, while the apex is near the point of obstruction &
its base at or just beneath the organ's wall or capsule.
iii. Zone of inflammation present around the infarct, but cell no. are less.
iv. Old infarcts are chiefly fibrous.
v.Small amounts of blood continue to flow. Structures with previous access may survive and
remain as living oases in the necrotic desert.

Gross appearance:
i. Red infarct is red, protruded, a sharp line of demarcation, triangular or conical shaped,
and softer than usual.
ii. Pale infarct is white, slightly depressed, sharply demarcated, slightly denser, and more
rigid than usual.
iii. Old infarcts are sunken on the surface of the affected organ.
Causes :
Obstruction of a single-end artery is the main cause. e.g. Coronary arteries, Renal arteries,
certain arteries of the brain etc. The common cause of arterial occlusion is (i) thrombus (ii)
embolus (iii) arteriosclerosis (iv) hypotension (Severe hypotension that may occur in shock or
anesthesia may cause ischemia of the brain resulting in necrosis). Mechanical obstruction – (i)
twisting (ii) volvulus (iii) intussusceptions (iv) parasitic larvae.
Infract can also result secondarily from occlusion of the venous drainage of portions of an organ.
Pressure on vein
↓
Prevent inflow of blood
↓
Prevent outflow of blood through arteries
↓
Accumulated blood becomes stagnant
↓
Hypoxia
↓
Necrosis
↓
In addition to necrosis tissues engorged with static blood
↓
Not true infract.

21
[ N:B: Thrombus→ Blood clot found in the blood vessels of living organisms.
Embolus → Foreign bodies in circulation bifurcated artery.
Atherosclerosis→ Harden & thickening of the blood vessel due to accumulation of lipid or
cholesterol & proliferation of connective tissue.

Some particular types & diagnoses (infract)

1. Kidney: It is common in cows and pigs. Infarct is typically conical, apex near the cortico-
medullary junction. Found frequently anemic type, maybe multiple sometimes healed up with
a fibrous scar. In dogs and pigs, the infarct is accompanied by chronic valvular endocarditis.
2. Spleen: Always hemorrhagic, usually sub capsular infarct. This is common in Hog cholera.
3. Heart: Red or grey, common in humans but rare in animals. The common cause is
arteriosclerosis.
4. Brain: Usually anemic, liquefies quickly, leaving a hole in the brain.
5. Intestine: always haemorrhagic
6. Lung: Infarct may occur due to embolism in the pulmonary artery. Always hemorrhagic.
Difficult to differentiate from simple haemorrhage. Usually red due to double blood supply
(pulmonary and bronchial).
7. Liver: Usually red due to double blood supply (arterial and portal).
8. Intestine: these are common in the horse due to thrombosis of an anterior mesenteric artery by
the migrating Strongyle larvae.

Sequel of infarcts:
1. The tissue may be organized, and a scar may form
2. Gangrene is common in infarcts of the intestine or extremities due to
saprophytes' invasion of necrotic tissue.
3. Death: If infarction occurs in the brain, heart, or intestines, death occurs due to
shock. Death may also be due to toxemia or septicemia.

22
 Chapter-II
Intracellular and extracellular depositions; degeneration:
Cells may undergo reversible or irreversible cell injury due to metabolic disturbances resulting
accumulation of abnormal amounts of endogenous substances within the cytoplasm, nucleus or
surrounding extracellular spaces.
Deposits may be in a disturbed metabolic cell or a distant cell.

A. Fatty change
Fatty change is the term used to indicate the accumulation of discrete droplets of neutral lipids
(triglycerides) within the cytoplasm of parenchymal cells. Fatty change occurs most often in the
liver, kidney and heart.
Causes:
If the transport of lipids to an affected cell is more than its capacity to metabolize it, if the cell’s
ability to synthesize proteins and lipoproteins is impaired, fatty change occurs.
Fat metabolism:

23
Some of the basic mechanisms involved in the pathogenesis of fatty change follow:
1. Excessive release of free fatty acids: Excess FA release; utilization is limited, so excess fatty
acids are stored as triglycerides. Fatty change by this mechanism is found during starvation
or severe caloric restriction. Idiopathic hepatic lipidosis occurs in anorexic obese cats. Fatty
change in the liver of a patient with diabetes mellitus occurs by this mechanism.
2. Decreased utilization or oxidation of fatty acids: Fatty change occurs due to interference
with co-factors such as carnitine which is essential for the oxidation of long-chain fatty
acids. The bacterial toxin is responsible for interference with co-factors.
3. Lipotrope deficiency: Deficiency of methionine and choline (lipotrope) decreases
phospholipid synthesis and leads to esterification of diglycerides to triglycerides.
4. Fatty acids preferentially esterified to triglycerides: may result from increased levels of
glycerophosphate. It is one of the mechanisms of fatty change that occurs with excessive
alcohol consumption.
5. Impairment of apoprotein synthesis: RER synthesizes apoprotein. In some poisoning like
ethionine, carbon tetrachloride, puromycin and yellow phosphorus, RER was destroyed. So,
interfere with apoprotein synthesis, thus no lipoprotein synthesis.
The deficiency of dietary protein causes poor amino acid substrate resulting in less protein
synthesis and fatty change.
6. Impairment of lipoprotein secretion: Found in experimental orotic acid intoxication.

Gross appearance:
In case of less fatty change- no gross leions
In more fatty change- organ larger than usual, the color of the organ changes to yellow tan or
paler than usual.
In liver:
i. Liver somewhat enlarged with rounded edges.
ii. Paler in color, may have a yellow reticular pattern of discoloration.
iii.The liver tends to float in fixative due to low specific gravity.
iv. Glistening knife.
v. Fat droplets floats on the surface of fixative.
vi. Organs soft to cut.
In heart:
• Yellow tan streaking of the myocardium. In severe cases, flabby consistency.

24
In kidney:
• Radial, yellow-tan streaking of the cortex and medulla (PCT or medullary rays)
Microscopic appearance:
Liver: With H & E, the cytoplasm contains empty spherical space, a single large pushing the
nucleus to the periphery or multiple small empty vacuoles with the nucleus in normal position.
Heart: numerous fine vacuoles.
Kidney: Multiple variables- sized vacuoles in the epithelial cells of PCT or ascending loops of
Henle. Cat’s epithelia of PCT usually contain fat droplets.
Special stain:
No fat solvent used, frozen section or cryostat section. Colored spherical substances are present
in cytoplasm.
Oil red O- Red.
Sudan III or Sudan IV- Scarlet red.
Osmic acid- Black
Nile blue sulphate- blue.
Excess fatty change in hepatocytes- rupture of hepatocytes- release of neutral lipids in
intercellular space- phagocytize by macrophages- formation of widely scattered aggregates of
lipid-laden macrophages – a condition known as lipogranuloma.
Fatty change may be reversible or irreversible. In heart it is usually irreversible.

Extracellular accumulation of lipids:

1. Excess fatty change- cell rupture- accumulates extracelllarly- lipogranuloma.
2. Cholesterol cleft.
3. Fat emboli
4. Fatty casts.
Fatty infiltration:
Presence of normal-appearing lipocytes in interstitial connective tissues of organs that do not
usually contain adipose tissue. It is associated with obesity and found in the heart and pancreas.

25
 B. Glycogen deposition
Usually, considerable amounts of glycogen are stored in the liver and skeletal muscle. Excessive
accumulation of glycogen may occur pathologically in the cytoplasm of epithelial cells of the
liver and kidneys, leukocytes, and cardiac muscle; and less often in smooth muscle, spleen,
lymph nodes and brain.
Gross appearance: Not grossly visible
Microscopic appearance:
• Presence of irregular-shaped clear spaces with indistinct outline in the cytoplasm of cells.
• The nucleus remains in normal position except in steroid induced hepatopathy in dogs where
glycogen deposits in mid-zonal hepatocytes and the nucleus pushed to periphery.
• Hepatocytes of well-nourished animals usually contain considerable glycogen and give finely
foamy appearance of cytoplasm. The deposition depends on the (a) amount of
carbohydrate consumed and (b) the interval between fixation of the liver and
carbohydrate consumption.
Special stain- tissue alcohol fixed and use of alcoholic based stains and no contact with water.
• Best’s carmine- Bright pink
• PAS- red
Causes:
(i) The frequent cause of excessive glycogen deposition is hyperglycemia, as in diabetes
mellitus.
(ii) In dogs- hyperadrenocorticism or dogs receiving cortico-steroid therapy (steroid-induced
hepatopathy) is the cause.
(iii) Other causes include glycogen storage diseases. These are autosomal recessive inherited
disorders characterized by the deficiency of various enzymes involved in the synthesis or
degeneration of glycogen.
Eight distinct types of glycogen storage diseases have been described in humans, designated as
types I through VIII; four of these have been reported in animals.
In animals:
1. Type II glycogenosis (Pompe’s disease)
2. Type III glycogenosis (Cori’s disease)
3. Type IV glycogenosis

26
 4. Type VII glycogenosis

C. Hydropic change/ hydropic degeneration

This is the advanced stage of acute cellular swelling. The causes and mechanisms are the same
as acute cellular swelling. Here empty round spaces without distinct outlines are found in the
cytoplasm of cells. The nuclei remain in a normal position.
[NB: Empty spaces in the cytoplasm are found in (a) Fatty change, (b) Glycogen deposition (c)
Hydropic degeneration]

D. Intracellular accumulation of proteins

The protein accumulates in the form of discrete eosinophilic droplets in the cytoplasm of cells-
termed hyaline droplet formation. It indicates increased absorption of protein by particular cells.
Most often seen in the cytoplasm of epithelial cells of proximal convoluted kidney tubules.
• Slight glomerular damage – no protein in the cytoplasm of PCT, because digested.
• Mild glomerular damage- highly eosinophilic droplets found, because resorption
exceeds the rate of digestion.
• Severe glomerular damage- albuminous or hyaline casts are present in the tubular
lumen.
Lysosomal storge dieases
The lysosome is the primary disposal and recycling center of cells. Two types of lysosomes-
primary and secondary. About 72 hydrolytic enzymes are produced, they mostly work at
acid PH , so-called acid hydrolases.
About three dozen of genetically determined, heterogenous, metabolic and acquired
lysosomal disorders are classified as lysosomal storage diseases. Thre are eight mechanisms
are involved, but in most cases, it results from deficient activity of lysosomal enzymes
resulting from mutation of the gene coding for specific hydrolases.
Lysosomal storage diseases are classified according to their major stored material eg,.
•Mucopolysaccharidoses (Mucopolysaccharide)
•Sphingolipidosis (Glycolipid)
•Glycogen storage diseases, etc.
Classification of lysosomal storage diseases:
A. Mucopolysaccharidoses (MPS): (i) MPS-I (ii) MPS-II (iii) MPS-VI (iv) MPS-VII
27
 B. Sphingolipidoses (Lipid storage disease): (i) GM1gangliosidosis (ii) GM2gangliosidosis
C. Galactosialidosis
D. Sphingomyelin- cholesterol lipidoses
E. Glycoprotein storage diseases
F. Neuronal glycoproteinosis
G. Glycogen storage diseases
H. Neuronal ceroid- lipofucinosis
I. Drug-induced lysosomal diseases.

Extracellular accumulation of proteins

A.Amyloidosis
B.Albumins and albuminous fluids
C.Fibrin
A) Amyloidosis
Amyloidosis is not a single disease but a diverse group of diseases. The extracellular deposition
of ultrastructurally similar but biochemically distinct proteins (amyloid) in various organs and
tissues.
Amyloid means starch-like. However, amyloids are proteins. It is composed principally of
fibrillar protein and a small amount of glycoprotein.
The name is derived from early observation because amyloid gives a similar colour when
painting iodine solution as starch.
[Cut surface of organ- painted with iodine solution – rinsed with dilute sulfuric acid – blue-
violet color in starch and amyloid deposition.]
Gross appearance: Small amount – not visible. Large amounts- appear as firm, white, opaque,
lard-like deposits. Mainly deposits in the spleen, kidney, and liver.
In spleen-
(a) Sago spleen- When deposits around the central artery grossly appear as discrete nodules
of firm, white material resembling tapioca grains (sago), or sago spleen.
(b) Bacon spleen- When deposits along with the reticular pulp, they grossly appear as
bacon, brown alternately with white streaks, so called bacon spleen.
The liver appears larger, paler and firmer than usual.

28
 Microscopic appearance: In H & E stain, amyloid appears as irregular, globular, or linear masses
of an eosinophilic hyaline material. Sometimes may appear faintly fibrillar.
As it accumulates extracellularly, so can compress the adjacent parenchymal cells causing
atrophy or death of cells.
Other eosinophilic extracellular deposits are collagen, fibrin and immune complexes.
Special stain:
• Congo red stain: orange-red under ordinary light and apple green birefringence under
polarized light.
• Tissue section- treatment with potassium permanganate – stain with congo red stain (No
stain- AA protein ie. Secondary amyloidosis; red stain- AL protein i.e. primary
amyloidosis)
• Thioflavin T or S, and Toluidine blue are also special stains. An immunohistochemical
stain can be used.
Physical properties:
a) Major/ fibrillar protein; masses of non-branching filaments.
b) Minor protein or P components.
Chemical properties:
The principal component is a fibrillar protein (90%), and the minor component (10%) is a
glycoprotein or P component. P component is similar in all types of amyloidosis but the fibrillar
component varies.
Fibrillar proteins:
• Amyloid light chain protein (AL protein)- is primary derived from plasma cells and
contained immunoglobulin light chain.
• Amyloid-associated protein (AA protein) – seconday amyloidosis
* Tissue damage- stimulate macrophages- secrete cytokines (interleukin-I, IL-6 & TNF)-
simulate hepatocyte- synthesize serum amyloid associated protein (SAA) (acute phase protein)-
SAA + HDL-3 –AA protein.
P component has structural homology with C –reactive protein (another acute phase protein)

Classification: 2 basic types-

A. Primary amyloidosis- occurs as a consequence of various forms of plasma cell dyscrasias,
including multiple myeloma and other monoclonal proliferation of B lymphocytes.

29
B. Secondary amyloidosis- occurs in association with chronic inflammatory diseases. E.g.,
tuberculosis and long-standing suppurative processes.
Repeated injection of tetanus antigen, casein, or certain durgs also produces secondary
amyloidosis.
• Senile amyloidosis- related to the aging process
•Familial amyloidosis- family trends.

Pathogenesis:
Pathogenesis is not fully understood. It is thought that inadequate or defective degradation of
soluble precursors by the mononuclear phagocytic system leads to extracellular deposition of
insoluble forms of the protein. Insoluble forms of proteins are relatively resistant to proteolysis.
Amyloidosis associated with plasma cell dyscrasias (Primary amyloidosis):
It is the standard form in man, rare in animals. It is often associated with multiple myeloma and
other plasma cell abnormalities. Major protein is AL type. Here plasma cells secrete large
quantities of whole Ig molecules of a single idiotype. The cells may secrete only light chains
also. The circulating Ig light chains are known as Bence-Jones protein. This is excreted through
urine of patients with multiple myeloma.

Amyloidosis associated with chronic inflammatory processes (secondary or reactive

amyloidosis): It is characterized by deposition of AA protein, an insoluble derivative of SAA.

Organs affected:
May affect only a single organ or involve multiple organs. Kidneys, liver and spleen are
common sites of amyloid deposition, but other organs like lymphnodes, adrenals, pancreatic
islets and GIT may also be affected.
It is a slowly progressive condition, usually, diagnose with necropsy. If deposits are extensive
the affected organs cease to function normally.
In the kidney, amyloid first appears in the glomeruli, then peritubular connective tissue.
Affected glomeruli become converted into solid, globular mass.
Amyloid usually deposits around the small blood vessels.
In spleen- mainly deposits in the germinal center.
In the liver- deposits begin in the space of Disse.
Significance: It’s a progressive disorder. If cause is withdrawn, amyloid does not regress.

30
B) Albumins and albuminous fluids:

Serum albumin, often referred to simply as albumin is a globular protein that in humans is
encoded by the ALB gene. Serum albumin is the most abundant plasma protein in mammals.
Albumin is essential for maintaining the oncotic pressure needed to properly distribute body
fluids between intravascular compartments and body tissues.
The albumin or albuminous fluids are found in following situations-
• Lumen of blood vessel may be filled with pink staining homogenous material which is
dehydrated plasma with albumin.
• In albuminuria or proteinuria, dense and deep-staining albumin found in the renal tubule.
• Most cysts are filled with albumin-containing fluid. Similar fluids were found
physiologically also, such as liquor folliculi, colloid of thyroid acini and salivary gland.
• Serous inflammatory exudates or transudate of edema. Best found in alveoli of lungs.
Microscopic appearance: Bright clear pink colored smoothly homogenous mass, at higher
magnification may appear as uniformly fine granules. Amyloid and fibrin have an opaque pink
appearance.
Gross appearance: look like watery fluid unless the fluid is mixed with other substances, such as
blood or pus. Foamy appearance after shaking.

C) Fibrin
Fibrin is another proteinaceous substance. It may be physiologic or pathologic.
Occurrence: Fibrin is seen in –
• Whenever blood clots either antemortem or post mortem
•Rarely, in the clotting of lymph
•In fibrinous inflammatory exudates
Gross appearance:
After washing the blood cells from the blood clots, fibrin appears as dull white stringy material,
the strands of which form a tangled mass. In fibrinous exudation, the form and color varies
according to the admixed substances like dead tissue, inflammatory cells (purulent), fecal
materials, etc.
Microscopic appearance:
Fibrin stains “dirty or smudgy pink”. Ordinarily, it is in the form of minute, tangled fibrils, but
in some cases, it forms a solid, homogenous mass. Fibrin has sponge-like absorption capacity, so
31
if Karyolysis occurs in the vicinity, the chromatin is absorbed by fibrin and takes blue stain.
Fibrinous exudation best seen in the alveoli of lungs.
Causes:
• Causes of clotting of blood and causes of formation of fibrinous exudation.
Significance:
Fibrin is formed from the polymerization of fibrinogen, so the source of fibrin is blood.
Fibrin may be beneficial-
• Form protective coating an serous and mucous surfaces & prevent irritation.
•Close the opened blood vessel and save the life.
Fibrin may be harmful-
• Within the blood vessel may form emboli
•If in pulmonary alveoli, prevent gaseous exchange
•If fibrin deposits on serous or mucous surface, it interfere with normal functioning of
surface cells.

Fate of fibrin:
• Completely liquefied by plasmin or hydrolytic enzymes of leukocytes.
•Fibrin stimulates the proliferation of fibroblasts, the process is known as organization.
Organization in the alveoli of lungs is known as carnification. Organization between two body
cavities surfaces can permanently bind two surfaces, known as adhesion.

Serous atrophy of fat/ myxomatous degeneration/ Mucoid atrophy of fat:

In this condition, the animal becomes cachectic, and the fat becomes gelatinous, flaccid, more
translucent and less yellowish.
It occurs when the animal suffers from malnutrition or chronic wasting diseases like TB, para
TB, etc.

32
 Chapter-III
Mineral deposition
Includes pathological deposition of minerals encountered in tissues on gross and microscopic
examination.
Calcification:
Deposition of calcium salts in tissues. 2 types:
(a) Physiologic calcification: deposition of calcium salts in bone and teeth.
(b) Pathologic calcification: deposition of calcium salts in tissues other than bone and teeth.
Calcium salt usually deposits as calcium phosphate and calcium carbonate or rarely in the form
hydroxy appatite. Often the salts are not chemically pure, accompanied with other ions (iron),
then the term mineralization is used. 2 major types of pathologic calcification:
1. Dystrophic calcification
2. Metastatic calcification
1. Dystrophic calcification:
Dystrophic calcification is the deposition of calcium salts in dead or degenerating tissues.
[Normal blood calcium level: 10mg/dl, Normal blood phosphorus level: 3.5 mg/dl]
Dystrophic calcification is not related to the calcium content of the blood.

Microscopic appearance:
In H & E stain, purplish colored irregular granules. Color varies with minor variations in
technique and thickness of the particles.

Special stain: Von kossa stain- black, Alizarin red S- red, Calcium oxalate- not stained.

Gross appearance:
Some lesions may only be microscopic and so not visible to the naked eye. Calcium particles are
white or gray, irregularly rounded which is often honey-combed. It is less dense than ordinary
limestone. A gritty sound is heard when cut, and the characteristic gritty feeling is felt.
Commonly found in chronic tuberculosis.

Causes:
Presence of dead or dying animal tissue is the fundamental cause of calcium deposition, not
related to increased blood calcium level. Local alkalinity in dead tissue favours the precipitation
of calcium. Another suggestion is that fatty acids formed in necrotic tissue combine with
calcium, forming soap that is replaced by phosphates and carbonates.
Calcification is prone to develop in –
• Caseous center of tuberculous nodules
•Actinomycotic and staphylococcal granulomas
•Old thrombus
•Degenerating thrombus
•Old scarrings, etc.

33
 Significance:
• The deposition is relatively permanent.
•Usually harmless unless it interferes mechanically with function or reduces tissue strength.
•One of the outcomes of necrosis.
•May undergo pathologic ossification, etc.

2. Metastatic calcification
Metastatic calcification is the precipitation of calcium salts in tissues as the result of a
persistently high concentration of calcium in the blood.
Microscopic appearance: As dystrophic calcification but no surrounding dead tissue.
Gross appearance: Same as dystrophic calcification.
Causes:
a. Hyperparathyroidism – an excess function of the parathyroid gland, more parathormone
secretion, more resorption of calcium from bone, hypercalcemia, leading to metastatic
calcification,
• Primary hyperparathyroidism: rare in animals. A tumor of parathyroid gland is responsible.
Due o excess parath hormone, increase resorption of calcium.
• Secondary hyperparathyroidism: these are of two types-
▪ Renal secondary hyperparathyroidism- In kidney damage (especially in dog, due to
leptospirosis)- less excretion of phosphorus-increase blood P level – ratio imbalance-
hyperparathyroidism- excess hormone, so calcium resorption increase- increased blood
calcium level- Metastatic calcification (Renal rickets; bone is soft)
▪ Nutritional secondary hyperparathyroidism- common in horse. Excess wheat bran
consumption continuously (rich of P)- excess blood P level- ratio imbalance-
hyperparathyroidism (Bran disease/ Big head disease)
b. Excess vit D in diet: Excess absorption of Ca, so, hypercalcemia.
c. Consumption of plants containing active principle of vitamin D (25, HCC). e.g., Solanum
malacoxylon.
Manchester wasting disease/ enzootic calcinosis.
d. Hyperadrenocorticism in dogs: Calcification occurs in skin, lungs and skeletal muscle.

The tissues affected are:

(i) Kidney: The tubular epithelial cells are calcified, and casts are formed, which plug the
tubules.
(ii) Lungs: The alveoli of lungs are the places where calcification occurs.
(iii) Stomach: The mucous membrane is calcified.

Calcification of kidneys, lungs, and stomach can be explained by their acid excreting properties
(kidney- hippuric acid and uric acid; lungs- carbondioxide; stomach-HCl). During this process, a
local increment of the tissue occurs, which lowers the solubility product of calcium and
phosphate; hence, these minerals are deposited. The formation of kidney stones is a frequent
feature noticed in hypercalcemia. Metastatic calcification also occurs in the elastica of heart,
aorta & muscular arteries. Also involves elastic fibers of pulmonary parenchyma, trachea,
bronchial cartilage, heart valves, tendons of forelimbs etc. Reduce the strength of the affected
part and may rupture. The mechanical problem may occur depending on the site of Ca
deposition. Calcification in the septa of the lungs may lead to pathologic ossification.

34
Pathologic ossification:

Definition: Pathological ossification is a form of metaplasia of non osseous tissue to form

osseous tissue. Example:
(i) Ossification of tendons of leg muscles in adult turkey.
(ii) Ossification of lateral cartilage of horse leading to lameness. It is found in old heavy
breed horses working on pavements.
(iii) In old man, there is partial ossification of cartilages of trachea and larynx.
(iv)Bony specules may replace Intra alveolar septa of the lung.
(v) Myocytic ossification is found in chronic cases.

Microscopic character: Formation of bony tissue with osteocyte and lacunae. Sometimes
presence of functional bone marrow.

Causes:
(i)Prolonged irritation
(ii)Hereditary tendency
(iii) Causes of dystrophic and metastatic calcification, etc.

Significance: It interferes in movements and gaseous exchange if it occurs in the lungs.

Gout:

Gout is a condition in which crystals of uric acid or urates are deposited in tissues.
Gout usually occurs in humans, reptiles and bird. In humans, it occurs mainly in articular form.
In the case of birds and reptiles, gout mainly occurs in visceral form not in articular form.

Microscopic character:
Articular form: In articular gout, the crystals deposits in articular surface, joint capsule and
adjacent tissues. An accumulation of neutrophil, macrophages, and foreign body giant cells is
associated with clusters of sharp acicular birefringent crystals or space left after removal by
washing.
Visceral form: Renal gout is characterized by the presence of urate deposits and inflammatory
reactions in renal tubules and ureter. In visceral form various serous surfaces are covered with
finely crystalline or granular material that does not stain.

Gross character:
In articular gout, presence of white chalky nodules known as tophi (A tophus (Latin: "stone",
plural tophi) is a deposition of monosodium urate crystals in people with long-standing high
levels of uric acid in the blood. Tophi are pathognomonic for the disease gout.) It may deposit in
subcutaneous tissue and may ulcerate without pain.
In visceral gout, the visceral organ, especially the parietal surface of the pericardium, is covered
by a thin layer of grayish, granular material with a metallic sheen.

Causes:
Uric acid and urates are the end product of purine metabolism. So, disturbances of purine
metabolism lead to gout. The causes are-
35
 (a) Deficiency or increase activities of the specific enzymes necessary for purine
metabolism.
(b) Impaired excretion of urates through kidney due to kidney damage.
(c)Genetic factors.
(d)Environmental factors.
In case of bird, the causes are-
(a) Impaired renal function
(b) Excess dietary intake of protein and calcium
(c) Deficiency of vit A
(d) Dehydration
Significance: Incase of bird, visceral gout is seen only at necropsy.

Pseudogout/Chondrocalcinosis:
The lesions clinical and radiological features are similar to articular gout but caused by
deposition of Ca-pyrophosphate dehydrate or hydroxyappatite.
It can found in dog and monkey.

Incase of dog, there is slowly enlargement of the joint with deposition of chalky mass in the
joint but not in joint capsule. Pathogenesis is not known.

36
 Chapter-IV

Pigments

A large number of pigments can be found in animal tissues-

a) Some are normal e.g., Melanin
b) Some are important components of specific diseases.

Classification of pigments:
1. Exogenous pigments (formed outside the body)
A. Carbon
B. Dusts (i) Silicosis (ii) Siderosis (iii) Asbestosis.
C. Metals (i) Silver (ii) Lead (iii) Bismuth
D. Tattoos
E. Kaolin
F. Carotenoids
2. Endogenous pigments (formed inside the body)
A. Phenolic pigments, autogenous pigment- Melanin
B. Haematogenous pigments
I. Haemoglobins II. Haematins III. Parasitic pigments
IV. Haemosiderosis V. Bile pigments VI. Porphyrins
C. Lipogenic pigments-
(i) Tissue lipofuscins (ii) Ceroid (iii) Vitamin E deficiency pigment
D. Miscellaneous pigments-
(i) Ochronosis pigment (ii) Dubin- Johnson pigment (iii) Cloisonne kidney.

A . Carbon (Anthracosis)
[New Latin : Greek anthrax, anthrak-, charcoal + -osis]. Anthracosis is the condition in which
carbon particles are found as a black pigment in the tissues. Carbon is the most common
exogenous pigment.
Occurrence: Carbon appears in the lungs, the lymphnodes that drain them, and in other organs if
carried by macrophages. Anthracosis is common in humans and animals that live in smoky cities
and in persons that work in coal mines.
Microscopic appearance:
Minute black granules, either between cells or in their cytoplasm.
In lungs- in alveolar walls, CT septa, usually within macrophages.
In lymphnodes- chiefly found between the lymphoid cells, often phagocytized by large
mononuclear cells and carried to other organs. It is always black colour, resistant to all solvents
and bleaching agents.
Gross appearance:
Ventral portions of the lung lobes are affected more than dorsal. Moderate or large amounts of
carbon give mottling black or grey. Lymphnodes are blackened. Bovine lymph nodes appear
black or brown in the medullary region (due to recently hemolyzed blood)- may be confused
with anthracosis.

37
Causes and significance:
Occurs due to repeated and continued inhalation of coal dust or smoke as a consequence of
living in cities. The heavily blacked lung is called “miner’s lung” or “black lung disease”. Small
amount of deposition- does little harm and causes no symptoms but remain in the tissues for life.
Excessive amounts may cause slight pulmonary fibrosis and predispose to lung infections.

Pneumoconiasis- dust in the lungs. Anthracosis is a specialized form of pneumoconiosis. Some

mineral dust deposition may induce life-threatening pulmonary disease.
(i) Silicosis: silicon dioxide is inhaled in rock quarries and mines where rock is being cut or
sandblasted.
Microscopically- fine, anisotropic crystals visualized only with polarized light. Crystals
incite collagenous CT proliferation in the form of dense sclerotic nodules. It’s visual cycle.
Grossly- Lungs are nodular and firm, may be pigmented if associated with anthracosis.
(ii) Siderosis- iron inhaled from mines as hematite or iron oxide. Iron does not incite fibrosis
or an inflammatory reaction. So, little significance. Microscopically- red crystals of
varying size.
(iii) Asbestosis: Asbestos is used in the manufacture of fire-retardant insulating materials.
Asbestos incites fibrous scarring of the pleura, around bronchioles and with in
alveolar septa. No nodular scarring. Foreign body giant cells form around the
asbestos particles. In human, asbestosis is associated with bronchogenic carcinoma
and mesothelioma.
Metals:
(i) Silver: Argyria. Used as medicine. Silver not injurious to cells, does not incite cellular
reaction.
(ii) Lead: Chronic poisoning caused by prolonged ingestion or assimilation of small amounts
of lead compounds; a blue- black discoloration is imparted to the gums adjacent to
the teeth, know as lead line.
(iii) Bismuth: Fistulous tract or sinuses are treated with bismuth-containing paste or powder,
colouring the wall of tracts.
Tattos: Indian ink, China ink, Bismark brown, cinnabar and kurkuma are used as tattos.
These pigments are inert and produce no tissue reaction.
Kaolin: Also known as china clay or Fuller’s earth. Kaolin cause pneumoconiosis which
leads to dense pulmonary scarring in man and non-human primates. Also, produce extensive
subcutaneous granulomas in the pharyngeal and neck tissues.

38
Carotenoid pigments:
Carotenoids are organic pigments that are found in the chloroplasts and chromoplasts of plants
and some other photosynthetic organisms like algae, some bacteria, and some fungi. Carotenoids
can be produced from fats and other basic organic metabolic building blocks by all these
organisms. Carotenoids generally cannot be manufactured by species in the animal kingdom so
animals obtain carotenoids in their diets, and may employ them in various ways in metabolism.
These are fat soluble pigments of plant origin. They include α carotene, β carotene and
xanthophyll. Also known as lipochrome pigments.
Occurrence: Usually occurs-
• Epithelia of adrenal gland.
•Lutein cells of corpus luteum
•Epithelia of testis and seminal vesicles
•Kupffer cells of the liver
•Ganglion cells
•Yolks of egg
•Butter fat
•Adipose cells of animal like, horse, jersey cattle.
The amount of carotenoid pigments in tissues varies between species-
➢ Relative efficiency in converting β carotene to vitamin A.
➢ Rejecting carotenoids in the ingesta that are not required for vit A synthesis.
These two functions are low in foul, horses, cattle, humans, and non human primates.
Occurs pathologically – in xanthomas. These are not true neoplasm but disorders of
mononuclear phagocytic system.
Lipochrome pigments are only seen grossly, because these are soluble in organic solvents used
in tissue preparation.
Hepatic carotenosis:
Bovine livers sometimes appear brilliant yellow or perhaps slightly reddish. The pigment in this
case is carotene, which itself is harmless, but affected liver show focal or centrilobular necrosis
and limited fatty change, may advance to portal cirrhosis or almost replacement of liver tissues
by fibrous tissue with mild lymphocytic infiltration. The meat inspectors suspected some
unidentified toxic plant as the cause.
Yellow liver found in 3 condition; Jaundice, hepatic carotenosis and in fatty change.
[Jaundice- Color is greenish yellow, Hepatic carotenosis- color brilliant yellow, Fatty change-
Color lighter yellow.]

39
Phenolic pigment/Autogenous pigment/Melanin:
Melanin is the pigment that gives color to the skin, hair and iris and provides the black reflexion
to the choroid of the eye. Chemistry of the melanin is not fully understood but it is supposed to
form from amino acid tyrosine.

Phenyl alanine OH hydroxyl phenyl alanine which is oxidized to tyrosine; by the action
of the enzyme tyrosinase or dopa oxydase melanin is formed.

Tyrosinase is copper containing enzyme. So, deficiency of cu no melanin is produced and the
condition is sometimes known as Achromotrichia (focal depigmentaiton).

[*Phenyl alanine → Tyrosine (-OH phenyl alanine) → dihydroxy phenyl alanine →dioxyphenyl
alanine (DOPA)→Dopamine →Norepinephrine→
Epinephrine
Dopachrome

Indole 5, 6 quinone (melanin)

Melanin is produced by the action of hormone
a) MSH-a
b) MSH-b]

Occurrence:
•Melanin is present in the cytoplasm of melanocyte or epithelial cells of stratum
germinativum.
• Melanin pigment is present as brown to black color granules.
•In addition to skin and hair pigment is found in the mucosa of mouth in the breed of cattle
specially Jersey breed.
•Melanin pigment also present in hoofs, iris and anterior portion of brain.

In pathological condition we get melanin pigment in –

• Melanomas
• Melanosis- is the deposition of excess melanin in tissues specially in lungs and aorta.
• Acanthosis nigricans- a pathological condition in which thickening of epidermis with
proliferation of basal cell layer. It usually occurs in anal region of dog.
• Hyper pigmentation of the skin due to hyper adrenalism. It is also known as Crushing
syndrome.
• In inherited disease of man- Dubin –Johnson syndrome.
• Freckles in human- Brown to black pigmentation in skin.
• Mutant corriedale sheep- melanin pigment may deposit in hepatocyte.
• Albinism- Pathologic absence of melanin pigment. In this condition melanocytes are present
but they have no capacity to produce melanin due to absence of tyrosinase enzyme.

40
Microscopic character:
1.Melanin is present as minute brown to black granules. In melanomas there is excess
production of melanin and this melanin may present extracellularly and it excrete
through urine (Bronze diabetes)
2.Melanin may be engulfed by macrophages. These melanin containing macrophages are
known as melanophores or melanophage. These melanophores are present in the
epidermis or dermis or in the regional lymphnode.

Melanophores can be differentiate from melanocyte by using “dopa reaction” .

Procedure: Pieces of fresh tissue is placed in dopa solution. After incubation it produce brown to
black precipitation indicates presence of melanocyte/ melanoblast.
These reaction is due to presence of presence of tyrosinase or dopa oxydase enzyme in the
melanin producing cell.
In Fontana silver method (special stain), melanin appears as black pigment.

Significance: Melanin itself is not harmful but melanoma is life threatening. Melanin gives
protection of the body against ultraviolet light. The production of melanin is increased with
exposure of sunlight.

Hematogenous pigment
The pigment originated from the blood. It includes-
1. Hemoglobin (Hb): Hb give the normal color of RBC. It is in colloidal state in erythrocyte.
When Hb is broken down, it forms heme and globin (protein). The globin part is reutilized by
the body. Heme portion is again broken down into iron (which is reutilized) and porphyrin.
Hemoglobin gives straw yellow color in the individual erythrocyte but crimson red in thick
smear.
Hb gives negative test for iron test. The gross ticntorial properties and chemistry of the Hb
slightly alter in some poisoning and after death of individual.
Oxyhemoglobin: It is found in arterial blood. In this case iron is in ferrous state and it is loosely
oxygenated and color is bright red.
Reduced hemoglobin: When Hb gives up its oxygen store. It is found in venous blood and color
is dark red.
Met-hemoglobin: In this condition iron is in ferric state and the color of the blood is chocolate
color. It is found in nitrite and chlorate poisoning.
Carboxyhemoglobin: It results from combination of CO and Hb. In this case cherry red color
blood is found. It results from cyanide and carbon mono oxide poisoning.

41
 Sulf- hemoglobin: It is combination of reduced Hb with an organic sulfide. The color of the
blood is dark brown. This can be caused by taking medications that contain sulfonamides under
certain conditions (i.e. overdosing of sumatriptan).
Sulphur met-hemoglobin: It is formed after death of individual and gives greenish red color of
abdominal cavity. This may be followed by pseudomelanosis.
2. Hematin or acid hematin:
This pigment is formed in the body by the action of acid on Hb. Among these acid hematins
most common is the -
(a) Acid formalin hematin or Formol precipitated hemoglobin.
This acid formalin hematin gives dark brown or black coloration of tissue.
This acid formalin hematin has no pathologic significance but it is an artifact which is annoying
for pathologist. Because this pigment can be confused with melanin or carbon particle.
So, we have to preserve tissue not in 10% formalin but in neutral buffered formalin.
This formed precipitated Hb can be removed from section by use of saturated picric acid
solution.
[Histology artifacts and cytology artifacts are structures that were not originally present in the living tissue. These artifacts can be from a
range of sources including contamination, poor tissue preparation and surgical procedures. Understanding artifacts is important for both
histologists and pathologists as they have the potential to compromise accurate diagnosis]

(b) HCl hematin: it is found at the site of gastric ulcer.

[ Parasitic pigments: hematin like pigment are found in some parasitic diseases which are also
known as parasitic pigment.
• Malaria- In this case, dark pigment found in affected erythrocyte and in macrophages.
• Fascioloides magna: it produce pigment in liver.
• Schistosomiasis: pigment found in macrophages.
• Pneumonyssus simicola: is common in lung mite of monkey. Pigment found in macrophages
of lung.]
3. Hemosiderosis:
It is derived from the iron part of hemoglobin.
It is shiny golden yellow or golden brown pigment. This hemosiderin gives positive test for iron.
Occuernce:
• Principally found in the red pulp of spleen.
• Old hemorrhages
• Regressive stage of corpora hemorrhagica
• Chronically congested lung.

42
Microscopic character:
• Glittering golden colored spherules packed into round to oval macrophages. Presence is
determined by ordinary chemical test (Prussian blue) for its iron component.
Gross character:
• Brownish color tissue in large deposition of pigment.

Causes and special consideration:

• Excessive destruction of erythrocyte. It may be either generalized or localized tissue
destruction.
• Due to excess RBC destruction, hemosiderosis is found in spleen (Deep littoral cell), liver
(Von kupffer cell), tubular epithelial cells, and intersitium of kidney.
• Hemosiderosis also found in other organ due to chronic passive congestion.
• Hemosiderin laden macrophage is called a Heart failure cell which is found in lung in chronic
passive congestion. Due to fibrosis, lung become leathery and firm called Brown induration of
lung.

Significance: Pigment itself is not harmful. Presence in large amount indicates extensive
hemolysis either in general form or local form. Large amount of hemosiderin in spleen indicate
hemolytic anemia.

4.Hemochromatosis:
A rare condition in human. Hemosiderin like pigment deposits in epithelial cells of liver, kidney
and other organs.
Causes: Hemochromatosis is inborn metabolic defect; there is excessive absorption of dietary
iron.
[Bronze diabetes- A condition where 3 conditions occur simultaneously due to
hemochromatosis. Conditions includes- (i) cirrhosis in liver (ii) Diabetes mellitus (iii) Excessive
melanin in skin.

43
Bile Pigment
Bilirubin not found microscopically usually and grossly it gives orange yellow color.
Sometimes, when there is excess deposition then we can get yellowish pigment which is called
hematoidin pigment. Both hematoidin and bilirubin are negative for iron reaction.
Fate of erythrocytes:
Erythrocyte (Hemoglobin)

Heme Globin

Within the RE cell iron Porphyrin (Pyrrole ring open)

Biliverdin (Greenish yellow color)

Bilirubin (Orange yellow pigment)

Bind with α2 albumin

Hemobilirubin/Prehepatic bilirubin/Indirect reacting bilirubin/Unconjugated bilirubin

In liver α2 albumin is separated

In hepatocyte conjugated with mono/diglucuronic acid and formed

Cholebilirubin/Post hepatic bilirubin/Conjugated bilirubin/Direct reacting bilirubin

Come in bile canaliculi bile duct gall bladder Duodenum

Urobilinogen (bacterial reduction)

Through portal Absorbed and goes to Goes lower intestine and

circulation it goes to
liver
Re excreted bilirubin
like compound
general circulation oxidized
Form urobilin and
Pass through urine stercobilin (gives
normal yellow color of
feces)

Hemobilirubin- Large molecule, not pass via urine, water insoluble but soluble in alcohol.
Chole bilirubin- Small molecule, pass via urine, water soluble, normally not found in circulation.

44
 Fate of RBC

Engulfed by RE cells
Old erythrocyte [ Haeme +globin

Recycled
Pyrole ring opens Prophyrin + Iron

Converted to

Biliverdin

Reduced by
biliverdin reductase

Bilirubin] Synonyms:
+ • Haemobilirubin
Albumin • Unconjugated bilirubin
• Prehepatic bilirubin
via circulation
• Indirect reacting bilirubin
albumin

Bilirubin in liver Synonyms:

+ • Cholebilirubin
Glucuronic acid • Conjugated bilirubin
• Post hepatic bilirubin
Uridine diphosphoglucose • Direct reacting bilirubin
glucuronyl transferase

Bilirubin diglucuronide

via bile canalicule

and bile duct

Cholebilirubin in small intestine

Bacterial reduction
Urobilin + stercobilin Come in large intestine
& excreted through feces
& gives the color of faces
Urobilinogen Come to the general circulation and excreted
through urine and gives the color of urine.
Absorbed in intestine

Come to the liver through portal circulation and

form bilirubin-like substance and again come to
the intestine (enterohepatic circulation)
45
Jaundice/Icterus
Definition: is a condition in which levels of bilirubin reach in such a high concentration in blood
that all tissues of the body gives a yellow discoloration.
As yellow is the light color in comparison to red and black so jaundice is seen only in pale or
whitish tissues of the body.
Gross lesions: Sclera of the eye, omentum, mesentery and in some cases mucosa of different
tract gives the indication of jaundice.
Microscopic lesions: Generally absent but incase of severe obstructive jaundice, yellow color is
seen in Van Kupffer cells and epithelia of kidney tubule.
Normal bilirubin level in blood in human is 0.8 to 1.2 mg/dl.
Types of jaundice:
1.Hemolytic jaundice
2.Obstructive jaundice
3.Toxic jaundice
1. Hemolytic jaundice:
Hemolytic jaundice results from excessive destruction of erythrocytes usually within circulation.
Causes:
1.Protozoal: Babesiosis, Anaplasmosis, etc.
2.Viral: Equine infectious anemia
3. Bacterial: Leptospirosis, Bacillary hemoglobinuria, Anthrax, etc.
4. Poisons: Plant (Ricin, Saponin), Chemical (Na &K chlorate, lead), Sanke venom,etc.
5. Massive internal hemorrhage:
6. Congenital hyper bilirubinemia
7. Neonatal jaundice/ Erythroblastosis fetalis/Icterus neonatarum
Pathogenesis of hemolytic jaundice:
Excess destruction of RBC causes excess hemobilirubin production in circulation results excess
urobilinogen production that is pass through urine. Excess stercobilin/urobilin causes excess
yellow color feces.
2. Obstructive jaundice:
It results from obstruction of the biliary system of the liver. It may be either intrahepatic or
extrahepatic (eg., bile duct/gall bladder).
1.Blockage of bile canaliculi be swollen hepatocytes.
2.Obstruction of the bile duct by parasites (Fasciola, ascaris larvae, etc.)
3.Gall stone formation
4.Biliary cirrhosis
5.Cholengitis
6.Pressure on ductal system (by abscess, tumor, granuloma, etc.)
7.Duodenitis
Pathogenesis: Cholebilirubin comes in circulation, no urobilinogen production, no stercobilin/
urobilin formation. So, color of the feces become clay, greasy, foul odor, due to the absence of
yellow pigment.

46
3.Toxic Jaundice/ Hepatocellular jaundice:
It results from the toxic substance which either cause acute cellular swelling/hydropic
change/fatty change/causes of necrosis in liver.
Causes:
1.Infectious disease
2.Extraneous poisons (Lupin, vetches)
3.Chemicals like chronic copper, phosphorus, CCl4 , Phenothiazine poisoning, etc.
4.Hepatotoxic mycotoxins (aflatoxicosis)
5.Some viruses (hepatitis A, B viruses)
Pathogenesis of toxic jaundice:
If there is swelling of hepatocytes, obstruction of bile canaliculi, so cholebilirubin in circulation
resulting changes of obstructive jaundice.
If there is necrosis in hepatocyte, no formation of cholebilirubin, excess hemobilirubin in
circulation resulting changes of hemolytic jaundice.
I.e, toxic jaundice is biphasic. Actually both the process go on simultaneously.

Differences of various types of jaundice:

Events Hemolytic jaundice Toxic jaundice Obstructive jaundice
Van den Bergh test Indirect Biphasic Direct
Urine bililrubin Acholuric Choluric Choluric
Urine urobilinogen Present Present Absent
Color of urine Light yellow Intense yellow Intense yellow
Dye retention Normal Increased Increased
Feces Intense yellow Normal Clay colored, greasy and
foul odor
Liver function test Negative Positive Negative in early stage
Blood prothrombin Normal Prolonged Prolonged
time
Total serum Normal Decreased Increased
cholesterol
Serum albumin Normal Decreased Normal
Blood uric acid and Normal Increased Normal
ammonia
SGOT, SGPT, ICD, Normal Increased Normal
etc

Diagnosis of jaundice:
If jaundice is severe then diagnosis can be done on the gross sign.
1.Icterus index: If jaundice is mild it can be diagnosed by a test known as icterus index.
In icterus index yellow color of the serum is compared with the standard potassium dichromate
solution of definite strength. This test is done to determine the presence or absence of jaundice
but not to detect the specific type of jaundice.

47
 2.Van den Bergh reaction:
This reaction is done to determine the type of bilirubin in the serum i.e., to determine the type of
jaundice.
A solution known as the Ehrlich diazo reagent (Sulfanilic acid and sodium nitrite) is used.
(Procedure: At first 0.5 ml reagent is taken in a test tube then add 1ml of serum)
Result:
1.If the pink colored ring is present within 30 second it is called immediate direct reaction. It
indicates there is presence of excess cholebilirubin in circulation. i.e, indicates
obstructive jaundice.
2.If there is delayed direct reaction it indicates there is small amount of cholebilirubin- it
may be obstructive jaundice.
Adding of 0.3ml of 95% alcohol and mix well –
3.If the color (pink) develop it indicates presence of hemobilirubin that means hemolytic
jaundice.
4.If first test give delayed reaction but color of second reaction is deep – indicates toxic
jaundice.
Significance of jaundice:
Jaundice indicates there is some abnormality in the body and it indicates the type of
abnormalities. To understand the type of jaundice, the pathogenesis and interpretation is
essential. Bilirubin is not seriously harmful except it gives some itching sensation at the time of
urination. Sometimes this bile pigment may deposit in liver & kidney giving some degenerative
changes in the organ and the condition is known as hepato renal syndrome.
The most important significance of jaundice is the kern icterus- which occur in young.
[Kern icterus- is the gross deposition of yellow color bile pigment in the neurons of children and
young animal. The deposition mainly occur in the hippocampus, basal ganglia, mid brain,
medulla of the brain, floor of the 4th ventricle causing neuronal degeneration in child and young
animal, become mentally retarded.]

Porphyrin, Photosensitizing pigment/ Photosensitizaiton/ Photosensitizaional dermatitis

Occur in cattle, sheep, goat, pigs and sometimes in horses. For photosensitizational dermatitis
there should be-
•Presence of photodynamic pigment in the peripheral circulation
•Animals should be in exposure to direct sunlight.
•The unpigmentd and uncovered area of the body are usually affected. In cattle and
goat- teat , udder& perineum; in sheep- muzzle, head & ears are affected.
Photosensitizaion dermatitis occur after few hours of exposure to sunlight.
Signs:
There is burning / itching sensation followed by erythema and inflammation of the skin,
ultimately necrosis and sloughing off the epidermis.
There is no death directly from photosensitizaional dermatitis but death may occur if there is
secondary bacterial infection or gangrene occurs.

48
Types of Photosensitization:
1.Type-I /Primary photosensitizaion: In this case preformed photodynamic pigment is
present in some plants or in drugs.
2.Type-II / Congenital erythropoietic porphyria/ Protoporphyria: In this case there is
inherited metabolic defect in the synthesis of the heme resulting excess porphyrin in
circulation.
3.Type-III/ Secondary photosensitization/Hepato toxic photosensitizaion: In this case there is
disturbances in chlorophyll metabolism in the body due to liver disease.
Microscopically this pigment are not seen but grossly, it is visible – some rodents excrete the
pigment through harderian gland as red tears.
1.Type-I /Primary photosensitizaion:
Results from ingestion of preformed pigments present in some plants. There are 2 groups of
plants-
(a)Helianthrons- Plant includes-
Buck wheat (Fagopyrum esculentum), pigment is Fagopyrin and the disease is fagopyrism.
St. John’s wort (Hypericum perforatum), pigment is hyperin, disease is hyperincism
(b)Furocoumarins- pigment is psoralens. Produced by some plants infected with some
fungus.
[Anthelmintic- Phenothiazine cause photosensitization. Phenothiazine ingested by the animal
goes to intestine that is converted to phenothiazine sulfoxide (Photodynamic pigment); come to
the liver and converted to phenothiazine sulfate which is excreted through bile. But incase of
liver disease, less efficiency to convert phenothiazine sulfoxide to sulfate then phenothiazine
sulfoxide come to circulation causing photosensitization.]
2.Type-II / Congenital erythropoietic porphyria/ Protoporphyria:
In this pigment is ferro proto porphyrin. This pigment is accumulate in dentin and bone giving to
red color which is known as osteohemochromatosis/pink tooth. The color of the urine is brown
and anemia is often seen.
3.Type-III/ Secondary photosensitization/Hepato toxic photosensitizaion:
In this case the pigment is phylloerythrin. It occurs due to acute toxic hepatitis. The animals
must eat diet containing liberal amounts of chlorophyll. Plant includes-
Lantana camera and Nolina texana.
Animal affected – if there is hepatic damage by liver fluke infestation and consumption of green
grass along with exposure to direct sunlight.

Lipogenic pigments:
Definition: A group of colored pigments which are derived mainly from lipids is called
lipogenic pigments. These are not similar to the lipochrome (α & β carotene and xanthophil).
Types of lipogenic pigments: 1) Lipofuscin 2) Ceroid
1) Lipofuscin: Lipofuscin is the name given to finely granular yellow
brown pigment granules composed of lipid-containing residues of lysosomaldigestion. It
is considered to be one of the aging or "wear-and-tear" pigments, found in
49
 the liver, kidney, heart muscle, retina, adrenals, nerve cells, and ganglion cells. It is
specifically arranged around the nucleus, and is a type of lipochrome. It appears to be the
product of the oxidation of unsaturated fatty acids, and may be symptomatic of
membrane damage, or damage to mitochondria and lysosomes. Aside from a large lipid
content, lipofuscin is known to contain sugars and metals,
including mercury, aluminum, iron, copper and zinc.
Microscopic appearance: The pigment appear as yellow or dark brown granules. In
cardiac muscle it is found as mainly in the pole of nucleus. In case of skeletal muscle
pigments are found in the cytoplasm. It is mainly found in the cytoplasm near the
nucleus.
Gross appearance: It is not detected in small amount deposition. If there is large amount
deposition, it may give brownish tinge to cardiac and skeletal muscle.
Significance: In some cases, it may be an indication of wasting disease, senility or
emaciation. Due to this pigment carcass may be condemned by meat inspector.
In human, Lipofuscin accumulation is a major risk factor implicated in macular degeneration, a
degenerative disease of the eye. Abnormal accumulation of lipofuscin is associated with a group
of diseases of neurodegenerative disorders. Pathological accumulation of lipofuscin is
implicated in Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,
lipid myopathy, chronic obstructive pulmonary disease, etc.
2) Ceroid --- Histochemically it is similar to lipofuscin. In Choline deficiency and Vit E
deficiency ceroid deposition is observed.
Ceroid deposition is found in the hepatocytes and macrophages. In addition it may also
found in fat cells, cardiac muscle, smooth muscle of intestine and ganglion cells.
Microscopically, ceroid pigment appears as granular to homogenous depending on the
pigment deposition yellow to brown in color.
Grossly, no change is found in small amount deposition. But in large deposition, it
causes brown discoloration of tissue. If deposit in fatty tissue it causes Yellow fat disease
and it deposit in the smooth muscle of intestine it cause Brown dog gut.
Significance: Like lipofuscinosis.

50
Miscellaneous pigment:
1. Ocronosis pigment: Melanin like pigment is found in the wall of blood vessel, cartilage,
tendon, ligament and other dense connective tissue, endocrine glands, kidney, lungs, etc.
This pigment is associated with a hereditary disease called Alkaptonuria.
Alkaptonuria (black urine disease or alcaptonuria) is a rare inherited genetic disorder
of phenylalanine and tyrosine metabolism. This is an autosomal recessive condition that is due
to a defect in the enzyme homogentisate 1,2-dioxygenase (EC 1.13.11.5), which participates
in the degradation of tyrosine. As a result, homogentisic acid and its oxide, called alkapton,
accumulate in the blood and are excreted in urine in large amounts (hence -uria). Excessive
homogentisic acid causes damage to cartilage (ochronosis, leading to osteoarthritis) and heart
valves as well as precipitating as kidney stones.
2. Dubin Johnson pigment: a melanin like pigment, it is found in association with Dubin
Johnson syndrome.
Dubin Johnson syndrome is characterized by – i) Chronic idiopathic jaundice ii) Pigmentation in
hepatocytes iii) Photosensitizaiton
3.Cloisonne kidney: This condition is found in castrated Angora goat. In this case, dark brown,
iron negative pigment is found in the basement membrane of proximal convoluted tubule of
kidney. Histologically, appearance is sometimes Enameled Jewellary (Cloisonne kidney).

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 Some notes
• Desquamation: Separation of the basement membrane only in epithelial cells (surface
epithelium).
• Sloughing off: Separation with underlying tissue.
• Hematoidin pigment: A pigment derived from hemoglobin that contains no iron but is closely
related to or identical to bilirubin. Hematoidin is formed intracellularly, presumably within
reticuloendothelial cells, but is often found extracellularly after 5–7 days in foci of previous
hemorrhage

Role of orotic acid in impairment of lipoprotein secretion in fatty change:

NB: Orotic acid is a heterocyclic compound and an acid; it is also known
as pyrimidinecarboxylic acid. Historically it was believed to be part of theVitamin B complex
and was called vitamin B13, but it is now known that it is not a vitamin. The compound is
manufactured in the body by intestinal flora. It is sometimes used as a mineral carrier in
some dietary supplements (to increase their bioavailability), most commonly for lithium orotate.
Ultrastructural studies of the hepatic changes produced by a variety of agents and methods, all of
which cause some increase in liver lipid with varying degree in alterations in the endoplasmic
reticulum which have included dilatation and degranulation of the rough reticulum and, in some
cases, proliferation of the smooth reticulum.

[>>How does virus cause cell injury ?

Ans : Two types of viruses cause cell injury/ death:
1) Cytotytic viruses cause cell death by:
i. Interfering with the ability of a cell to synthesize proteins & other macromolecules essential to
maintaining cell life.
ii.Redirecting organelles of the cell to preferentially synthesize viral RNA, DNA & structural
proteins needed for replication & assembly of the virus.
iii.Mechanically damaging cellular organelles & disrupting the cytoskeleton with accumulation
of large amounts of viral nucleic acids & proteins, some of which are cytotoxic.
iv.Inserting viral encoded proteins in cell membranes, causing them to mal function.
2) Non cytolytic viruses in cell cultures may indirectly cause cell death in vivo by stimulating
host’s immune response to viral antigens expressed on the surface of infected cells.
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Prussian Blue Staining Protocol for Iron
Description: Small amounts of ferric iron are found normally in the spleen and bone marrow.
Excessive amounts are present in hemochromatosis and hemosiderosis. Prussian blue reaction
involves the treatment of sections with acid solutions of ferrocyanides. Any ferric ion (+3)
present in the tissue combines with the ferrocyanide and results in the formation of a bright blue
pigment called Prussian blue, or ferric ferrocyanide. This is one of the most sensitive
histochemical tests and will demonstrate even single granules of iron in blood cells.
Fixation: 10% Formalin.
Sections: Paraffin sections at 5 um.
Solutions and Reagents:
20% Aqueous Solution of Hydrochloric Acid:
Hydrochloric acid, concentrated ------------ 20 ml
Distilled water -------------------------------- 80 ml
Mix well.

10% Aqueous Solution of Potassium Ferrocyanide:

Potassium ferrocyanide, Trihydrate
(K4Fe(CN)6.3H2O, FW 422.4, Sigma, Cat# P-3289) -----10 g
Distilled water ----------------------------------------------- 100 ml
Mix to dissolve
Working Solution:
Mix equal parts of 20% hydrochloric acid and 10% potassium ferrocyanide solution JUST
before use.
Nuclear Fast Red Solution
Procedure:
1. Deparaffinize and hydrate sections to distilled water.
2. Mix equal parts of hydrochloric acid and potassium ferrocyanide prepared immediately
before use. Immerse slides in this solution for 20 minutes.
3. Wash in distilled water, 3 changes.
4. Counterstain with nuclear fast red for 5 minutes.
5. Rinse twice in distilled water.
6. Dehydrate through 95% and 2 changes of 100% alcohol.
7. Clear in xylene, 2 changes, 3 minutes each.
8. Coverslip with resinous mounting medium.
Results:
Iron (ferric form) ------------------------ bright blue
Nuclei ------------------------------------- red
Cytoplasm -------------------------------- pink

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