The subject of pathology. Methods in the general and clinical pathology. Basic notions – disease/death. Classif ication of the pathogenic factors.

Radina Ivanova, MD, PhDAssociate Professor of PathologyAssociate Professor of Pathology

Medical University of SofiaMedical University of Sofia

Synopsis Subject of pathological anatomy. General and

clinical pathology. Main notions in pathology. Biopsy and

necropsy methods. Biopsy examination in different ways of

obtaining material and tissue processing. Cellular injuries – etiology. General mechanisms of cellular injury –

ischemia and hypoxia. General mechanisms of cellular injury - free

radicals injury.

PATHOLOGY

Literally translated, pathology is the study (logos) of suffering (pathos).

Pathos (suffering)

Logos (study)

PATHOLOGY Hartman (19th century)

theory of the diseases It involves the investigation of the causes

(etiology) of disease as well as the underlying mechanisms (pathogenesis) that result in the presenting signs and symptoms of the patient to understand structural and functional changes

in cells, tissues and organs

PathologyClinical practice

Bridging Subject

Basic science

It is a discipline that bridges clinical practice and basic science

PATHOLOGY

Rudolph Virchow

1821-1902

The Father of Modern Pathology• “All diseases are the results of visible cell abnormalities”, i.e., abnormal histology, i.e., histopathology.

History of pathology• Morgagni, 1761- founder of pathological anatomy

• Bishat, 1800 – founder of pathological histology

PATHOLOGY

GENERAL fundamental cellular and tissue responses

to pathologic stimuli. E.g.. Inflammation, cancer, ageing.

SYSTEMIC particular responses of specialized organs

E.g.. Goiter, pneumonia, breast cancer.

Basic notions in pathology

Disease

Death

What is Disease?

dis + ease (not at ease…) Disease

“Expression of discomfort due to structural or functional abnormality”

Health Complete physical, mental and social well being,

not merely absence of disease…

What is Disease?

Disease A condition of impaired dynamic equilibrium of the

organism with the environment Biologic processes in which the function and structure of the

organs are abnormal Health

A condition of dynamic equilibrium of the organism with the environment

Homeostasis – internal equilibrium of different organs and systems

Disease Types:

Inflammatory / Neoplastic / Degenerative Acute / Chronic

Acute – short days to weeks. Chronic – long, months to years.

Congenital / Familial / Acquired Genetic / Environmental Mild / Moderate / Severe

DISEASE

Each disease - 4 aspects Etiology (“cause”) Pathogenesis (“insidious development”) Morphology (abnormal anatomy) Clinical expression

Etiology: What is the cause?

Environmental agents Physical Chemical Nutritional Infections Immunological Psychological

Genetic Factors

Multifactorial:Multifactorial:Diabetes,Diabetes,HypertensionHypertensionCancerCancer

One agent One agent One disease One disease - Malaria- Malaria

Several agents Several agents One disease One disease - - DiabetesDiabetes

One agent One agent Several diseases Several diseases - Smoking- Smoking

Disease

Disease

Disease

DiseaseDisease

Pathogenesis Development (underlying mechanisms)

“Sequence of events in the cells and tissues to a stimulus/pathogen” starting from the initial stimulus to the ultimate expression of disease.”

Morphology To render diagnoses, pathologists identify:

changes in the gross or microscopic appearance (morphology) of cells and tissues

Other changes (molecular)

Clinical Significance

Patient Signs and symptoms are related to underlying pathology…

Prognosis of disease depends on inside pathology Is he going to recover or die soon?

Guided therapy What treatment is suitable for this patient?

Outcome of disease

Restitutio ad integrum (recovery) Substitutio (not fully recovery,

chronic disease) Death

What is death?

Clinical death-minutes medical term for cessation of blood

circulation and breathing cardiopulmonary resuscutation

Biological death irreversible cessation of circulatory and

respiratory functions and of all functions of the entire brain (areflexia)

Late signs of death Algor mortis -cooling of the body after death

Depends on body temperature at the time of death and environmental temperature

Livor mortis –discoloration the red-purple discoloration caused by the settling of the

blood in the dependent portions of the body due to gravity Rigor mortis

the stiffening of the muscles after death due to chemical changes in the muscle fibers (depletion of ATP, lowered pH)

The decomposition of a body – several stages 2-3 days -green staining on the right side of the abdomen,

smell

Methods in the general and clinical pathology

Autopsy hospital (pathologo-anathomic)

performed by pathologists in hospitalized patients who died of natural causes

forensic investigate the death in cases of violence or presumed

violence Biopsy

histological cytological

Autopsy

Aims to identify the cause of death to clarify cases without clinical diagnosis or in those in

which the patient death was unexpected recognition of the effect of the treatment in the evolution

of the disease. recognition of new diseases and of new lesion patterns source of information, allowing the making of precise

statistics on the most frequent diseases material for the residents, students and staff learning material for scientific research.

Autopsy-performance

Stages External examination Dissection -instrumentarium

internal examination (necropsies) Pathology protocol

Macroscopic diagnosis Final pathologcal diagnosis

Biopsy

Histological investigation-pieces of tissue Surgery

Frozen sections (gefrieren) Endoscopic

Bronchoscopy, gasto-, colonoscopy, etc Large needle biopsy

Liver, ren, pulmo, prostate Cytological investigation – isolated cells, smears

Body fluids – ascites, urine, pleural effusions Fine needle biopsy of thyroid, prostate, lymph nodes Vaginal smears (PAP)

Processing of biopsies Histological tissue processing

Formalin-10% Paraffin embedding-block Microtome cutting – 5 µ Staining of sections

HE Special (histochemical) stainings

Van Gieson, Gomori, Sudan, PAS, Perls, congo-rot

Cytological smears Drying on air/alcohol fixation Staining

MGG, Papanicolau

Other methods in pathology Electron microscopy Immunofluoresce Immunohistochemistry Morphometry/flow

cytometry DNA

Molecular pathological methods In situ hybridization PCR

Pathogenic factorsClassification

1. Oxygen deprivation (hypoxia, ischemia)2. Chemical agents and drugs 3. Physical agents4. Infectious agents5. Immunologic reactions 6. Nutritional imbalances7. Genetic derangements8. Aging

1. Hypoxia

= or oxygen deficiency an extremely important and common cause of cell

injury and death. should be distinguished from ischemia ( a loss of

blood supply in a tissue due to impeded arterial flow or reduced venous drainage).

Reasons for oxygen deprivation Ischemia inadequate oxygenation of the blood (pneumonia) reduction in the oxygen-carrying capacity of the

blood (anemia) carbon monoxide (CO) poisoning (CO forms a stable

complex with hemoglobin that prevents oxygen binding.)

2. Chemical Agents

An enormous number of chemical substances can injure cells Concentrated glucose or salt Oxygen at sufficiently high partial pressures Poisons and potentially toxic agents (air pollutants,

insecticides, CO, ethanol) Therapeutic drugs

if used excessively or inappropriately

Pathogenic factors

3. Physical Agents Trauma, extremes of temperatures, radiation, electric

shock, sudden changes in atmospheric pressure

4. Infectious Agents Viruses, tapeworms, rickettsiaes, bacteria, fungi,

protozoas.

5. Immunologic Reactions autoimmune reactions against one's own tissues allergic reactions against environmental substances in

genetically susceptible individuals

Pathogenic factors6. Nutritional Imbalances

Nutritional deficiencies - protein-calorie insufficiency, specific vitamin deficiencies

Excesses of nutrition obesity –DMdiets rich in animal fat - atherosclerosis

7. Genetic Defects congenital malformations associated with Down

syndrome, sickle cell anemia deficiency of functional proteins -enzymes in inborn

errors of metabolism8. Aging

alterations in replicative and repair abilities of individual cells and tissues

Cellular responses to stress and pathogenic factors

Normal cells have a fairly narrow range of function or steady state

Homeostasis Excess physiologic or

pathologic stress may force the cell to a new steady state

Adaptation Too much stress

exceeds the cell’s adaptive capacity

Injury

Cell injury Reversible/Irreversible

Reversibility depends on the type, severity and duration of injury

Cell death is the result of irreversible injury

Responses of the heart to different types of stress:• Hypertension – hypertrophy (adaptation)• Ischemia

•incomplete occluded coronary artery – cell injury•complete or prolonged occlusion – cell death

Mechanisms of cell injury 1. Depletion of ATP2. Damage to Mitochondria 3. Influx of Calcium4. Accumulation of Oxygen-Derived Free Radicals (Oxidative Stress)5. Defects in Membrane Permeability5. Damage to DNA and Proteins

1. Depletion of ATP

ATP - the energy store of cells required for almost all

synthetic and degradative processes within the cell

membrane transport, protein synthesis, lipogenesis

The major causes of ATP depletion

reduced supply of oxygen and nutrients,

mitochondrial damage, toxins (e.g., cyanide).

2. Damage to Mitochondria

Mitochondria -critical players in cell injury and death.

Mitochondria can be damaged by ↑ cytosolic Ca2+, reactive oxygen species (ROS) oxygen deprivation (hypoxia, toxins).

Two major consequences of mitochondrial damage:

loss of mitochondrial membrane potential and pH changes, resulting in failure of oxidative phosphorylation and progressive depletion of ATP

leakage of cytochrome c and other proteins into the cytosol and death by apoptosis.

3. Influx of Calcium

Increased cytosolic Ca++ activates a number of enzymes,

with potentially deleterious cellular effects

induction of apoptosis by direct activation of caspases and by increasing mitochondrial permeability

Increase in cytosolic Ca++ by: increased influx across the

plasma membrane ischemia and certain toxins release of Ca2+ from the

intracellular stores

4. Accumulation of Oxygen-Derived Free Radicals (Oxidative Stress)

Free radicals have an unpaired electron in their outer orbit cause autocatalytic reactions (molecules that react with free radicals

are in turn converted into free radicals, thus propagating the chain of damage)

Oxidative stress – a condition called in ↑ production of ROS or ↓ degradation - an excess of free radicals

Generated by: Absorption of radiant energy Oxidation of endogenous and exogenous compounds Oxidation of exogenous compounds

4. Accumulation of Oxygen-Derived Free Radicals (Oxidative Stress)

Reactions relevant for cell injury by ROS

Lipid peroxidation damage to cellular and organellar membranes

Protein cross-linking and fragmentation due to oxidative modification of amino acids and proteins

DNA damage due to reactions of free radicals with thymine

5. Defects in Membrane Permeability

The plasma membrane can be damaged by:

ischemia, various microbial toxins, lytic complement components, physical and chemical agents.

The most important sites of membrane damage during cell injury

are: Mitochondrial membrane damage Plasma membrane damage

leads to loss of osmotic balance and influx of fluids and ions, as well as loss of cellular contents.

Injury to lysosomal membranes leads to leakage of their enzymes into the

cytoplasm and enzymatic digestion of cell components, necrosis.

6. Damage to DNA and Proteins

Damage to DNA and proteins in:

radiation injury oxidative stress, inherited mutations

Cells have mechanisms that repair damage to DNA

if this damage is too severe to be corrected the cell initiates its suicide program and dies by apoptosis.

INJURY MECHANISMS IN REVERSIBLE CHANGES

Decreased ATP Mitochondrial damage Increased intracellular calcium Increased free radicals Increased cell membrane permeability

(cellular swelling)

INJURY MECHANISMS IN IRREVERSIBLE CHANGES

Mitochondrial irreversibility Irreversible membrane defects Lysosomal digestion

General mechanism of cellular injury in hypoxia and ischemia

↓ intracellular generation of ATP failure of many energy-

dependent cellular systems ion pumps depletion of glycogen stores,

with accumulation of lactic acid and lowering the intracellular pH

reduction in protein synthesis If hypoxia continues, worsening

ATP depletion causes further deterioration

If oxygen is restored, all of these disturbances are reversible.

If ischemia persists, irreversible injury and necrosis ensue.