Pathology is the study (logos) of structural and functional abnormalities (pathos).

The knowledge of the pathologic

changes is important to explain the different clinical features (signs and symptoms), course, and prognosis of the
disease and also for the proper clinical care and therapy of the patient.

[More specifically, pathology may be defined as the - scientific study of the molecular, cellular, tissue, or organ
system response to injurious agents or adverse influences].

Pathology has its roots deeply implanted in medical history.

The earlier observers, from Celsus (about 30 BC - AD 38) to Morgagni in the eighteenth century, based their work
upon the naked-eye appearances of the diseased individuals and organs.

Only as the technique of microscopy improved was the Germanic School of Pathology, headed by Virchow (1821-
1905), able to investigate changes at a cellular level.

He proposed that the basis of all disease is injury to the smallest living unit of the body, namely the cell.

More than a century later, clinical and experimental pathology was added to the Virchow's cellular pathology.

At the start of the nineteenth century, doctors and scientists had no idea that bacteria caused illness. It was Louis
Pasteur, who first made this link. In France, Pasteur, using the microscope, laid the foundation of the science of
bacteriology.

Later on the German dye industry enabled Robert Koch, Paul Ehrlich, and  Gerhard Domagk to extend this
knowledge and open the era of chemotherapy.

German chemical industry was manufacturing many synthetic coal tar dyes. Robert Koch was able to use these dyes
to stain and to see invisible microbes. He identified the germs that caused anthrax, tuberculosis and cholera.

Advances in pathology thus have been closely related to advances in technology. This in no way belittles the effort
of inspired experimenters like Edward Jenner, who in England pioneered the way to active immunization.

Nowadays technological advances are occurring so rapidly that it has become difficult to have a working knowledge
of all the methods that are available. Utilizing all these complex techniques and along with investigators of different
disciplines, the knowledge of pathology advances rapidly.

In 21st century, pathology has undergone changes from a visual, descriptive morphology to one defined in terms and
interpreted in molecular basis.

Pathology is considered in four headings:

Etiology or cause of the disease:

In 2500 BC, illness was thought to be due to patient’s own fault (for having sinned) or the making of outside agents,
such as bad smell, cold, evil spirits or gods

In modern terms two major etiologic factors are intrinsic (genetic) or acquired (infective, nutritional, chemical and
physical).

Pathogenesis:

Pathogenesis is the sequence of changes from the initial change after injury to the ultimate expression of the disease.
Morphological changes:

It refers to gross and microscopic changes of the tissues or organs in thedisease. It often signifies the etiology of the
disease.

Pathophysiology:

This refers to functional changes due to morphological alteration in the disease and is dependent on the nature and
distribution of lesions in different organs.

Microscopy:

- Light microscopy:

Usually three objectives are used, low power with magnification of 10 times, high power with 45 times and oil
immersion 100 times).

The shorter the wavelength of the light used the better is the resolution, and with ultraviolet light it can be improved
two to three times.

Examination of living tissue:

Living tissue is transparent and the homogeneity in optical  density of its component hides its detailed structures.
Staining techniques must therefore be used to see cellular details, but these must be performed on dead fixed tissues.

In supravital techniques, e.g. mitochondria of living leukocytes can be stained by Janus green but even this damages
them rapidly so that the cells soon lose their motility and begin to die.

Three techniques have been developed to overcome these difficulties in examining living cells.  

-  Dark-ground illumination:

Dark-ground illumination relies upon the fact that objects placed in a beam of light may be seen by the rays which
they reflect.  This method demonstrates the organisms which cannot be readily stained, e.g. Treponema
pallidum.       

-  Phase contrast Microscopy:

Different parts of a cell have different refractive index. These differences are converted into differences in optical
density. Living cells are examined by this method. This is commonly used in virology

-  Interference Microscopy:

The light that passes through the microscope is split into two beams. The light passing through the specimen is
retarded, and interferes with the light of the other beam when the two are recombined in an image plane. The
method finds little application in routine pathology.

Examination of fixed tissue:

Paraffin section: It is the most commonly used routine method of examination.

The tissue is fixed, dehydrated in graded  alcohols, cleared in xylol, chloroform or other solvent which is miscible
with both alcohol and wax, and finally embedded in paraffin wax.

Common fixative is formaldehyde (10% aqueous solution of formalin).

Staining:

Hematoxylin and Eosin staining (with its limitation) is preferred for paraffin sections because it is relatively quick,
inexpensive and allows accurate diagnosis of the vast majority of cases received in a laboratory. Nucleus takes the
colour of hematoxylin (blue) and cytoplasm eosin (pink).

In paraffin section certain substances are removed (e.g. fat )and alters other substances (e.g. enzymes and some
antigens).

Hence frozen sections are used.

Frozen section: 

Frozen section is done in cryostat (microtome in which sections are cut at - 30 C.) This method is used for
preservation of fat, enzymes and some antigens (lost in paraffin method).  Commonly used stain for neutral fat is
Sudan III or oil red O; for other tissues Toluidine blue is used.

Histochemistry:

For different types of tissues, enzymes and antigens different stains are used.

Example:

- Van Gieson’s stain is used to differentiate muscle and connective tissue).

- Hale's colloidal iron stain (Prussian blue) is used to stain iron.

- Periodic acid-Schiff (PAS) is used to stain glycogen, the glycoproteins of ground substance and basement
membrane, and epithelial mucin.

- Fuelgen stain is used to show nuclei and chromosomes  (magenta coloured).

- Perl's Prussian-blue reaction is used to stain hemosiderin.

- Reduction of silver nitrate to metallic silver to show argentaffin cells (black).

Metachromasia means the change of colour of the stain in the coloured tissue or cell (Eg. Toluidine blue stains
the granules of mast cells and some acid mucopolysaccharides red ).  

Immunohistochemistry:

The antibody can be labelled with fluorescein and is examined under ultraviolet light.

Alternative method:
i) Sandwich technique using commercial labelled Coomb’s reagent.

ii) Radioactive isotopes in conjunction with autoradiography

iii) Immunoperoxide technique.

iv) Double staining by using two separate antigen in one section. This is done by using horseradish peroxidase with
different substrate chromogen system.

Electron microscopy:

The tissue must be fixed immediately after removal from the body, commonly in osmium tetroxide. Other fixative is
glutaraldehyde. Formaldehyde fixes slowly.

It is useful in the morphological diagnosis of poorly differentiated tumours and to differentiate various types of
glomerulonephritis and epidermolysis bullosa.

Ultracentrifuge:

The ultracentrifuge is used to separate particles of different sizes from a mixture. The technique is used mainly to
separate macroglobulins.

Electrophoresis:

If a mixture of proteins is placed in an electric field at a known pH, individual proteins move at particular rates
dependent mainly to their size and charge.

Chromatography:

Chromatography is an important technique for separating pure substances from mixtures. The separation depends on
the fact that different substances follow the moving solvent at different rates. Large molecules cannot enter the
beads and so pass rapidly through the column. Small particles enter the beads and pass slowly through the column.

Chromatography is of great use in the separation and purification of proteins. It is also used for separation of
aminoacids or sugars in solution such as urine.

Radioactive isotopes:

Radioactive isotopes are treated by living cells in the same way as the normal elements.

Their radiation can be detected by suitable counter e.g. investigation of thyroid function by radioactive iodine.

The isotopes can also be used at a microscopic level.

A tissue section or peritoneal spread is placed on a photographic film subsequent photographic development will
reveal the site of isotope localization as black grain.

An example is seen in mast cell granules in peritoneal spread  [International J Appl Radiation & Isotopes (London &
New York), 1959].
Tissue culture:

Tissue culture is an extremely important technique to study the behavior of cancer cell and normal cell. The
technique is used to cultivate viruses and to study the tissue metabolism.

Microdissection:

[ Microdissection is a technique that is very useful both in the research setting and for clinical molecular testing in
paraffin-embedded tissue samples. The available techniques range from simple and inexpensive (manual
microdissection) to complex and expensive (laser-capture microdissection). All of the techniques, however, require
the user to be familiar with microscopy and histology.] (Arch Pathol Lab Med. 2004;128:1372-1378)

A cell can be dissected and its nucleus can be removed. Micro-beams of ultraviolet light, or Laser rays (Light
Amplification by stimulated Emission of Radiation), have been used to produce damage in a particular part of a cell.

Micromanipulation of embryos can produce allophonic mouse. Genetic alteration can produce clones of different
genetic constitution.

Cells encounter many stresses as a result of changes in their internal and external environments.

The patterns of response to this stress constitute the cellular basis of disease.

If an injury exceeds the adaptive capacity of the cell, it dies.

Pathology is the study of cell injury and the expression of a pre-existing capacity to adapt to such injury, on the part
of either injured or intact cells.

Causes of Cellular Injury:

Diagram showing Structural Changes in Reversible and Irreversible Cell Injury: click here

1. Lack of O2 supply (Hypoxia)- e.g. Ischemia, Cardio-respiratory failure.

2. Loss of O2 carrying capacity of blood as in Anemia, CO poisoning

3. Physical agents - heat, cold, radiation etc. Hypothermia ; Hyperthermia.

4. Chemical agents including - acids, alkali, drugs, insecticides etc.

5. Infections - bacteria, virus, fungus, parasites etc. Infectious Disease Online

6. Immunologic reactions such as autoimmune diseases

7. Genetic disorders: chromosomal alteration or mutation - e.g. Sickle cell disease

8. Nutritional injury [e.g. Protein Calorie Malnutrition (Kwashiorkor), Vitamin A Deficiency causing
Xerophthalmia, Keratomalacia and Night blindness].Nutritional Pathology Online

Normal cell contains higher K+ and low Na+ than extra-cellular fluid maintained by Sodium Pump (ATP dependent
Cell membrane Transport system).
A cell needs ATP to maintain its normal metabolic functions:

 i) Membrane transport system (Failure of Na pump causes swelling of cell).

 ii) Protein synthesis (Reduced protein synthesis causes lipid deposition).

 iii) Phospholipid turnover (lack of peroxidation causes damage of cell membrane).

Lesions:

Gross features:

The organ involved is pale and increased in weight.

Microscopic features: 

Injured cells show cellular swelling due to increased volume of water, sodium and potassium and are characterized
by a large, pale cytoplasm and a normally located nucleus called hydropic or vacuolar degeneration. Lipid vacuoles
appear later mainly in cells participating in metabolism (e.g. hepatocytes, myocardial cells).

Ultrastructural Structures:

i) Plasma membrane shows alteration, blebbing, blunting or distortion of microvilli and loosening of intercellular
attachment.

ii) Mitochondria show swelling and there is appearance of phospholipid-rich amorphous densities.

Reversible cell injury:

These are the pathologic changes that can be reversed when  the stimulus is removed or if the cause of injury is
mild. 

Reversible cell injury is characterized by the cellular swelling with accumulation of fat, protein and other substances
e.g. steatosis, cholesterosis, glycogen, and others. If the injury persists the changes become irreversible.

Irreversible cell injury (Cell Death):

Injury to cell causes:

1. Change in mitochondria : Swelling and abnormal cristae.

2. Damage of the plasma membrane causing leakage of soluble enzymes (detected in serum in myocardial injury).

3. Fragmentation of the nuclear membrane.

Cell death:  This means the series of morphological changes which occur in relation to a cell or group of cells
following lethal injury. It is the element of time and the action of enzymatic degradation and protein denaturation
which determine the differences between functional cell death and cell death as morphologically defined.

Two main types:

1. Necrosis

2. Apoptosis

Necrosis:
Necrosis is the cellular death in living cell or tissue and is characterized by swelling, denaturation and coagulation of
proteins, breakdown of cellular organelles and cell rupture.

Necrosis is mainly caused by extra-cellular enzymes, liberated from inflammatory cells.

Presence of inflammatory cells around the necrotic area differentiates infarct from autolysis.

Cytoplasmic changes :

The cytoplasm shows a decrease in basophilia ( indicating a loss of RNA protein) and an increased infinity for acid
dyes such as eosin. This increased eosinophilia is due to denaturation of some of the cytoplasmic proteins with
exposure of basic groups which bind the eosin. When appropriate special stains such as the periodic acid-Sciff
method are used, loss of glycogen is noted and there may be some fragmentation and clumping of the cytoplasmic
contents.

Nuclear changes :

- Karyolysis - There is a gradual fading away of the basophilic nuclear material, presumably due to action of
DNAses.

- Karyorrhexis - There is fragmentation of nucleus and the debris is either phagocytosed by other cells or just
disappears.

- Pyknosis - There is condensation of nucleus into a deep basophilic mass. This stage is often followed by
karyorrhexis.

Coagulation (Coagulative) necrosis:

It is the most common pattern of necrosis. It is characterized by denaturation of cytoplasmic proteins with
preservation of the framework of the coagulated cell.  Necrosed area becomes dry, homogeneous and opaque.
Example: Infarct kidney, heart. Normal architectural pattern is preserved but the cellular detail is lost.

2. Caseative (Caseous) necrosis:

Cellular death with complete loss of architectural pattern. Necrotic area is dry, cheesy and friable.  Example:
Tuberculosis.

3. Liquefaction necrosis:

Occurs when autolysis and heterolysis prevail over protein denaturation. The necrotic area is soft and filled with
fluid with obliteration of normal architecture. This type of necrosis is most frequently seen in localized bacterial
infection (abscesses) and in the brain.
4. Fat necrosis:

  - Traumatic fat necrosis - mostly seen in female breast. Rupture of adipocytes causes release of fat with
lipolysis releasing fatty acids and glycerol.                                                                                                                   
Microscopically, there is fatty acid crystals, lipid filled macrophages and foreign-body giant cells.

Fat necrosis is also seen in subcutaneous fat - Example: thigh, abdomen etc.

  - Pancreatic fat necrosis - In acute hemorrhagic pancreatitis ( Pancreatitis ; Acute Pancreatitis ) there is
liberation of pancreatic lipase, which acts on the adipose tissue liberating glycerol and fatty acids. Glycerol
combines with calcium forming soap, which appear as small, opaque and intensely white patches on adipose tissues
of pancreas, omentum and other areas of peritoneum.

5. Fibrinoid necrosis:

Necrotic area becomes homogeneous and eosinophilic and involves mainly the small blood vessels in Immune-
complex-vasculitis and  Malignant hypertension.

Gangrene:
Gangrene is characterized by extensive necrosis superadded with putrifaction.   Visit: Necrosis

Dry gangrene (mummification):

This is due to arterial obstruction, commonly in old age with atherosclerosis followed by thrombosis of the large and
medium size arteries of the limbs, slowly over a long period. Venous drainage is normal.    Visit: Hemostasis
and Thrombosis

Dry gangrene usually starts in the most distal part of the extremities.

Skin of the affected areas is dry, shrivelled and black like that of mummy due to diffusion of hemoglobin from small
vessels into the extra-vascular space (Example:  Senile gangrene, diabetic gangrene).

Gangrene extends upwards till it reaches a point where circulation is sufficient to keep the part alive.

This junction of living and dead tissue is called line of  demarcation and consists of granulation tissue formed
due to irritation of dead tissue.

Granulation tissue erodes the dead tissue finally causing complete detachment (spontaneous amputation).

In younger age, it is seen in thrombo-angiitis-obliterans (Buerger’s disease)- due to inflammatory occlusion of both
artery and vein.

Wet Gangrene:

This is due to obstruction of vein with intact arterial supply (Example: strangulated loop of gut in hernia, prolonged
application of tourniquet etc).

Gangrene is distal to the site of obstruction.

Marked edema allows rapid growth of bacteria often with gas formation.

Absorption toxin causes profound toxemia.

Gas Gangrene: Gas Gangrene:click here

This is rapidly spreading tissue necrosis, often involving muscle as in crush injury in road accident.

This is due to infection by saccharolytic and proteolytic Clostridia along with other pyogenic bacteria.

This may be rarely seen in suppurative appendicitis, strangulated loop of intestine, puerperium. 

Affected muscles and soft tissues are edematous, crepitant on palpation due to gas bubbles in the tissue and
commonly develops profound toxemia and septicemia.

    Microscopic features of Gangrene:

i) Absence of inflammation.

ii) Cell shrinkage.   

iii) Chromatin -condensation and fragmentation.

iv) Cellular blebbing and fragmentation are followed by removal by shedding or phagocytosis by macrophage.

An Introduction to Pathology

Pathology is the study and diagnosis of disease through examination of organs, tissues, bodily
fluids, and whole bodies (autopsies).   The history of pathology can be traced to the earliest
application of the scientific method to the field of medicine, a development which occurred in
the Middle East during the Islamic Golden Age and in Western Europe during the Italian
Renaissance.

Early systematic human infections were carried out by the Ancient Greek physicians Herophilus
of Chalcedon and Erasistratus of Chios in the early part of the third century BC.   The first
physician known to have made postmortem dissections was the Arabian physician Avenzoar
(1091–1161). Rudolf Virchow (1821–1902) is generally recognized to be the father of
microscopic pathology. Most early pathologists were also practicing physicians or surgeons.

General Pathology

General pathology is a broad and complex scientific field which seeks to understand the
mechanisms of injury to cells and tissues, as well as the body's means of responding to and
repairing injury. Areas of study include cellular adaptation to injury, necrosis, inflammation,
wound healing and neoplasia.   It forms the foundation of pathology, the application of this
knowledge to diagnose diseases in humans and animals.
The term "general pathology" is also used to describe the practice of both anatomical and clinical
pathology.

Anatomic Pathology

Anatomic pathology (U.S.) is a medical specialty that is concerned with the diagnosis of disease
based on the gross, microscopic, chemical, immunologic and molecular examination of organs,
tissues, and whole bodies (autopsy).

Anatomic pathology is itself divided in subspecialties, the main ones being surgical pathology,
cytopathology and forensic pathology. To be licensed to practice pathology, one has to complete
medical school and secure a license to practice medicine. An approved residency program and
certification (in the U.S., the American board of Pathology or the American Osteopathic Board
of Pathology) is usually required to obtain employment or hospital privileges.

Anatomic pathology is one of two branches of pathology, the other being clinical pathology, the
diagnosis of disease through the laboratory analysis of bodily fluids and/or tissues. Often,
pathologists practice both anatomic and clinical pathology, a combination known as general
pathology. The distinction between anatomic and clinical pathology is increasingly blurred by
the introduction of technologies that require new expertise and the need to provide patients and
referring physicians with integrated diagnostic reports. Similar specialties exist in veterinary
pathology.

Clinical Pathology

Clinical pathology or Labortory medicine, is a medical specialty that is concerned with the
diagnosis of disease based on the laboratory analysis of bodily fluids such as blood and urine,
and tissues using the tools of chemistry, microbiology, hematology and molecular pathology.
Clinical pathologists work in close collaboration with medical technologists, hospital
administrations, and referring physicians to ensure the accuracy and optimal utilization of
laboratory testing.

Clinical pathology is one of the two major divisions of pathology, the other being anatomic
pathology. Often, pathologists practice both anatomic and clinical pathology, a combination
sometimes known as general pathology.

Forensic Pathology

Forensic pathology is a branch of pathology concerned with determining the cause of death by
examination of a cadaver. The autopsy is performed by the pathologist at the request of a coroner
usually during the investigation of criminal law cases and civil law cases in some jurisdictions.
Forensic pathologists are also frequently asked to confirm the identity of a cadaver.

Veterinary Pathology
Veterinary pathologists are doctors of veterinary medicine who specialize in the diagnosis of
diseases through the examination of animal tissue and body fluids. Like for medical pathology,
veterinary pathology is divided in two branches, anatomical pathology and clinical pathology.
Veterinary pathologists are critical participants in the drug development process.

Pathology as a Medical Specialty

Pathologists are physicians who diagnose and characterize disease in living patients by
examining biopsies or bodily fluid. The vast majority of cancer diagnoses are made or confirmed
by a pathologist. Pathologists may also conduct autopsies to investigate causes of death. 
Pathology is a core discipline of medical school and many pathologists are also teachers. As
managers of medical laboratories, pathologists play an important role in the development of
Laboratory information systems. Although the medical practice of pathology grew out of the
tradition of investigative pathology, most modern pathologists do not perform original research.

Pathology is a unique medical specialty in that pathologists typically do not see patients directly,
but rather serve as consultants to other physicians (often referred to as "clinicians" within the
pathology community). To be licensed, candidates must complete medical training, an approved
residency program and be certified by an appropriate body. In the US, certification is by the
American Board of Pathology or the American Osteopathic Board of Pathology.