Cell Pathology Flashcards
Intro
Our bodies react to physiological stress or pathological stimuli
Cells constantly adjust their structure and function to accommodate extracellular stresses
Maintain normal homeostasis
Adaptive changes lead to a new steady state to preserve viability and function
Cell death can occur as a normal or abnormal process
Causes of cell injury
Infectious agents range from viruses, bacteria and fungi to tapeworms
Immune reactions can cause cell and tissue injury e.g. autoimmune reactions or allergic reactions
Genetic defects may cause injury through deficiency of functional proteins, accumulation of DNA damage or misfolded proteins. Sickle cell anaemia a.a. substitution in hemoglobin S
Adaptive responses
Reversible changes in:
Number
Size
Phenotype
Metabolic Activity
Function
2 Adaptation examples
Physiological Adaptations
e.g. hormone-induced enlargement of breast during pregnancy
Pathological Adaptations
Pathological adaptations are cellular responses to adverse stimuli or diseases that, while attempts to survive, result in changes that impair normal function or lead to disease.
What are the principal adaptive responses?
Principal adaptive responses are:
Hypertrophy
Hyperplasia
Atrophy
Metaplasia
Hypertrophy
- Increased size of cells
- Increased size of organ
Cells enlarged due to increased amount of structural proteins and organelles
Occurs largely in cells that do not divide
Can be physiological or pathological
Can occur simultaneously with hyperplasia
Requires mechanical signals –stretch and trophic triggers eg alpha adrenergic stimulation
Which turn on genes that increase protein synthesis
Hyperplasia
- Increased number of cells
- Increased organ size
Takes place in cells capable of replication
Can occur together with hypertrophy
Can be physiological eg hormonal; compensatory hyperplasia
or pathological – excessive hormone or growth factor secretion
Gingival hyperplasia from phenytoin.
Examples e.g. receding gum, enlarging prostate
Atrophy
- Reduced size of Cells
- Reduced size of Organ
Diminished function but not dead, caused by decreased work load through: Loss of innervation; Diminished blood supply; Inadequate nutrition; Loss of endocrine stimulation; Ageing; Reduced protein synthesis (due to reduced metabolic activity), increased protein degradation
Metaplasia
- Reversible change in phenotype
- Replaced by a cell type which can better withstand the adverse environment
Cells are replaced with a different cell type.
This is thought to arise from genetic reprogramming of stem cells
. An example is epithelial metaplasia in smokers where specialised ciliated columnar epithelial cells are replaced with hardy squamous epithelial cells. This adaptation helps survival but protective mechanisms are lost eg cilia clearance
It is often caused by chronic irritation and persistence can lead to the a transformation to malignant cells.
Cellular changes in injury
Reversible
- Cell swelling
- Swelling of organelles
- Lipid deposition
- Detachment of ribosomes
- Loss of microvilli
- Clumping of chromatin
- Surface blebs
Irreversible
- lysosomal enzyme release
- Protein digestions
- Severe cell swelling
- Membrane damage
- Mitochondrial vacuolation
- Nuclear changes
Adaptive Responses FLOWCHAT
MEMORISE
Cellular changes in injury
What can the microscopes see
Light microscope
- fatty change
- vacuoles
- cloudy swelling
- Membrane blebbing
- Necrosis
- Nuclear changes
Electron microscope
- ER swelling
- loss of ribosomes
- loss of specialised membrane structures
- myelin bodies
- condensation of mitochondria
- vacuolisation of mitochondria
Summary
Our bodies react to any form of injury or assault
All adaptive responses are reversible
If stimulus is not removed, cells die or undergo neoplastic change
Cellular pathological investigations allow us to see the changes
What is apoptosis
Apoptosis is the process of programmed cell death. It is used during early development to eliminate unwanted cells; for example, those between the fingers of a developing hand. In adults, apoptosis is used to rid the body of cells that have been damaged beyond repair.
What is necrosis
Necrosis is the death of the cells in your body tissues. Necrosis can occur due to injuries, infections or diseases
Cell death
Main targets for cell injury are cell membranes, mitochondria, cytoskeleton and DNA
Loss of ability to divide
Loss of normal synthetic functions
Biochemical changes, followed by structural changes
Cell death via necrosis or apoptosis
How can cell death occur
1) Small blebs form; the structure of the nucleus changes
2) The blebs fuse and become larger; no organelles are located in the blebs
3) The cell membrane ruptures and releases the cell’s content; the organelles are not functional
Nuclear Changes in Cell Death
- Pyknosis
- Karyolysis
- Karyorrhexis
What is Pyknosis
Nuclear shrinkage
What is Karyolysis
Nuclear fading
What is Karyorrhexis
Nuclear fragmentation
What is necrosis + cause
Necrosis is caused by factors external to the cell or tissue e.g. infections, toxins or trauma
It results in unregulated digestion of the cell components
Loss of cell membrane integrity and uncontrolled release of products of cell death brought about by autolysis
Inflammatory response in the surrounding tissue
Tissue Response to Necrosis
Haemorrhage
Resolution
- Site, type and extent of injury
- If cannot resolve, body attempts to repair
Repair
- Removal of dead tissue
- Action of neutrophils and macrophages
- Granulation tissue
Necrosis can be divided into 5 distinct types based on the morphological changes that occur:
Necrosis can be divided into 5 distinct types based on the morphological changes that occur:
- Coagulative necrosis
- Liquefaction (colliquative necrosis)
- Caseating necrosis
- Fat necrosis
- Gangrenous necrosis
What is Coagulative Necrosis
Coagulative Necrosis
Most common
Affects solid organs
Artery occlusion - ischaemia is most common cause
Firm and pale
Microscopic view of coagulative necrosis
- Cellular outline and tissue architecture maintained
- Lack of lysosomes/ injury destroys lysosomal enzymes
What is Colliquative or Liquefactive Necrosis
- Semi-liquid
- Degradation of cellular and extracellular components by hydrolytic enzymes
- Cystic cavities containing fluid and cell debris
- Main causes include cerebral infarction or bacterial infection
What happens in Liquefactive Necrosis in the Brain
- Loss of neurones and neuroglia cells
- Many macrophages clearing cell debris
What is Caseating Necrosis
Soft and white – resembling cream cheese
- Unstructured protein mass
- Associated by tuberculosis
What is Fat Necrosis
- Hard yellow material seen in dead adipose tissue
- Pancreatitis - release of proteolytic & lypolytic enzymes
- Breast trauma - release of fatty acids - inflammatory response
What is Calcification of Necrotic Tissue
Dystrophic calcification
- Large amounts of calcium enter the cell
- Combine with phosphate to form crystals
- Extracellular calcification can also occur
What is Gangrene
Not a distinctive type of necrosis
Describes black dead tissue
Most common in lower limb of patients with severe atherosclerosis
Irreversible ischaemic damage
Types of Gangrene
1) Dry gangrene
- Coagulative pattern of necrosis
2) Wet gangrene
- Gram-negative bacteria converts it to liquefactive necrosis
3) Gas gangrene
- Follows infection with gram-positive organism Clostridium welchii (found in the soil)
- Releases toxins which produce gas
What is Dry Gangrene
- Coagulative necrosis from ischaemic injury
- Commonly caused by poor circulation or obstruction
- Slow, gradual process
- Usually seen in smokers, diabetics, atherosclerosis
What is Wet Gangrene
- Largely affects moist tissues and organs
- Characterized by liquefactive necrosis
- Untreated wound infections.
2) Wet Gangrene
- Swelling and inflammation
- Blockage of blood supply
oedematous, decayed, reddish to dark brown - Promotes further bacterial growth necrosis and gangrene
Gas Gangrene
Clostridia are the main etiologic agent
Invade wounds and injuries with diminished blood supply
Release gas-producing toxins.
Affected skin may appear greyish to purplish red
Bubbly skin which makes a crackling sound whenever pressed
Apoptosis
- Programmed cell death
- Normal process
- Energy-dependent
- Orderly packaging in membrane bound vesicles
- Vesicles phagocytosed – no inflammatory response
Apoptosis why is it important
Important for:
- Controlling cell number
- Tissue remodelling
- Removing damaged cells
Necrosis + Apoptosis
Necrosis
Group of cells affected
Caused by injurious agent
Changes due to energy deprivation
Cells swell
Ruptured lysosome & cell leakage
Inflammatory response
Apoptosis
Single/few cells affected
Programmed cell death
Energy-driven
Cells shrink
Organelles & nuclear fragments packaged in vesicles
No inflammatory response
Summary
Cells and tissues react in different ways to injury and assault, attempting to limit damage, thus avoid cell death
Cell death occurs as necrosis or apoptosis
Different tissues undergo necrosis in different ways
Apoptosis is largely a physiological process
INFLAMMATION FLOWCHART
Inflammation causes
Causes:
Mechanical injury
Bacteria, viruses, parasites
Ischaemia
Chemical injury
Temperature
Radiation
Immune mechanism
Foreign body
Symptoms of inflammation
1) Redness
2) Swelling
3) Heat
4) Pain
5) Loss of function
Inflammatory Process
Microvasculature changes
- Vasodilation - Redness & heat
⬆️ Permeability - Swelling
Inflammatory exudate
- Fluid
Cells - Chemical mediators
What is the immediate transient response
Immediate-transient response:
Peaking at 5-10 minutes
Lasts 15-30 minutes
Produced by chemical mediators
Nettle stings or insect bits
What is immediate persistent response
Immediate-persistent response
Direct endothelial damage
Peaking at around 1h
Lasts until thrombus plugs vessel
Severe direct injury e.g. burns
What is the Delayed-persistent response and its causes
Delayed-persistent response
Subtle endothelial damage
Leakage starts 18-24hr later
Last 36h or more
Causes include:
Sunburn
Radiotherapy
Bacterial toxins
Cellular Events to inflammation
Margination and Adhesion
-Changes in Blood flow
-White cells fall to edge of vessels
-Adhesion molecules allow attachments between endothelial cell and leukocyte
Emigration
- Pseudopodia form
- Push between endothelial cells
- Proteases released
- Digest basement membrane
Chemotaxis What is it
Leukocyte migrates between endothelial cells
Basement membrane seals behind them
Cells move towards chemical signal
Towards inflammatory mediator
Phagocytosis
1) Bind Foreign Material (opsonin)
2) Engulf foreign material (Phagosome)
3) Kill foreign material (Phagolysosome)
4) Damage to surrounding tissue
What is opsonin
Opsonins are molecules, like antibodies or complement proteins, that act as “tags” to mark foreign substances or cells, making them more easily recognized and ingested by phagocytes
What are granulocytes
Granulocytes are a type of white blood cell characterized by the presence of granules in their cytoplasm. The primary examples of granulocytes are neutrophils, eosinophils, and basophils
Leukocyte order in blood
Neutrophils > lymphocytes>monocytes>eosinophils >basophils
What are Neutrophils
Neutrophils
- Mediates majority of acute inflammatory effects
- Granule contents increase vascular permeability
- Migrate to damaged area by chemotaxis
- Phagocytic
What is Eosinophils
(Granulocyte)
Eosinophils
- Allergic/parasitic conditions
- Granules include major basic protein
What is Basophils
(Granulocyte)
Basophils
- Give rise to mast cells
- Granules include histamine
What are Macrophage
Macrophage
- Derived from monocytes
- Migrate to damaged area (later than neutrophils)
- Low numbers which increase with time
- Survive much longer than neutrophils
What are lymphocytes + Examples
Lymphocytes
T Cells:
- Antigen recognition and presentation
- Cell killing
B cells
- Give rise to plasma cells
- Synthesize immunoglobulin’s
Cell-derived mediators:
- Vasoactive amines (histamine and serotonin)
- Arachidonic acid derivatives (prostaglandin and leukotrienes)
- Cytokines (interleukins, interferons, chemokines and GFs)
- Platelet activating factor
- Nitric oxide
- Lysosomal content
Plasma-derived mediators:
- Kinin system (bradykinin)
- Coagulation and fibrinolytic system (plasmin/fibrin degradation products)
- Complement system (C3a and C5a)
Systemic effects of inflammation
Systemic effects: ALL ARE ACUTE PHASE REACTIONS
- Leukocyte production
- Fever
- Tachycardia
- ↓ Blood pressure
- loss of appetite
- vomiting
- skeletal weakness
- aching
What is Acute Inflammation
Acute Inflammation
- Short duration
- Minutes to days
- Sequential process
- Neutrophil and macrophage driven
- Protective and reparative mechanism
- Sometimes inflammatory response more harmful than initial tissue damage/infection
What is Chronic Inflammation
- Persistent (months or years)
- Defined by type of cell present (Lymphocyte, macrophage and plasma cell)
- Necrotic cell debris
- Tissue granulation
- Fibrosis
- Balance between repair and continues tissue damage
What are the main effector cells in chronic inflammation
Macrophage are the main effector cells in chronic inflammation:
- Antigen presentation (to lymphocytes)
- Scavenging
- Phagocytosis
- Giant cell formation
- Harbouring of organisms
- Secretion
- Reactive oxygen metabolites
- Proteases
- Arachidonic acid metabolites
- Cytokines
- Growth factors
What is Granulomatous Inflammation
Special type of chronic inflammation, characterized by the presence of a granulomata
Can occur through foreign material or immune response – microbe digested and antigens presented to TH cells – release INFy, which activated the macrophage to transform into epithelioid cell
- Collection of macrophages frequently surrounded by lymphocytes
- Macrophage fuse forming multi-nucleated giant cell or epitheliod cell
- Micro-organisms which excite an immune response eg mycobacteria
- Non-living foreign material
Chronic inflammation Types
Nonspecific
Occurs following infection
Lymphocyte and plasma cells
Chronic suppurative
Continued neutrophil production and recruitment
Surrounded by fibrous tissue & poorly vascularized
Eosinophil-rich
Granulomatous
Agents difficult to destroy using lysosomal enzymes
Autoimmune
What is Tuberculosis
- World wide issue
Each accounted for between 1.1 million and 1.2 million deaths in 2014 – world most deadly infectious disease together with HIV
There are two strains of TB which can affect humans. Bovine TB can pass to humans through milk – enters the GI tract causing abdominal TB.
Infection with Mycobaterium Hominis is the common form and it causes pulmonary TB with symptoms including cough, blood stained sputum, night sweats, weight loss.
The incidence of TB was relatively low at the beginning of the twentieth century, however it has increased over recent decades
Tuberculosis primary
Primary
Never affected before
Produce lesions
Neutrophil inflammatory response - macrophage influx
Macrophage ingest bacilli - present antigen to lymphocytes
Proliferation of specific T lymphocytes - attract macrophage - Granuloma
Caseating necrosis
Tuberculosis secondary
Secondary
Patients previously sensitized
Largely due to reactivation of latent mycobacteria
More common in immune compromised e.g. steroid treatment, HIV/AIDS
Tuberculosis Miliary
Miliary
Extensive caseous necrosis
Tissue destruction
Bacilli may enter bloodstream
Granulomas appear in other
organs
What is Crohn’s Disease
Chronic inflammation
Affects all layers of the bowel wall
Unknown aetiology
Non-caseating granulomas
Four stages in wound healing
Four stages in process
1) Haemostasis
2) Inflammation (removal)
3) Cell proliferation (regeneration)
4) Remodelling (replacement)
What is primary intention wound healing
Primary Intention
-Cleanest type of wound
-Aseptic
-Minimal tissue trauma
- Two sides need to be held together (sutures)
What is Secondary intention wound healing
Secondary Intention
- Fibrin clot
- Cell infiltration
Temporary matrix
Wound contraction
Epithelial migration
Clot dissolution - Surface intact
New basement membrane
Definitive matrix - Scar formation
Factors which affect wound healing can be local or systemic:
What is systemic factors
Systemic:
Nutrition (vitamin C and zinc)
Therapy (steroids, chemotherapy and radiotherapy)
Disease (metabolic, connective tissue disorders, diabetes)
Factors which affect wound healing can be local or systemic:
What is Local factors
Local:
Foreign bodies and/or Infection
Blood supply
Mechanical (direction of wound and movement)
Size of wound
Capacity of cell for regeneration
Role of Cellular Pathology
Involves investigation of the aetiology and pathogenesis that result in the signs and symptoms of the patient
Cell pathology looks at cells in tissue or fluid
Cell arrangement, development and function is investigated to determine if a patient has a disease, inflammation, or cancer
Multi-disciplinary teams are involved to decide an appropriate treatment plan or review how a treatment is progressing.
Can be used to diagnose patients or determine cause of death
When are autopsies undertaken?
At request of a coroner or hospital doctor
Coroner-
if death is sudden, suspicious, unexplained, violent, occurs soon after a hospital stay or occurs from accident or injury
Doctor –
To gain a better understanding of the illness
To further research
What Happens in an Autopsy?
First, the height, weight, age, and gender of the body should be noted and recorded.
Distinguishing characteristics like birthmarks, scars, or tattoos should be noted as well.
An external examination is performed.
- Performed ASAP (usually within 3 days following death)
- Performed in examining room licensed and inspected by HTA
- Prior to an autopsy information about the subject and the events leading to his or her demise is gathered, consulting medical records, doctors and family members and examining the location and circumstances of death.
What happens in an autopsy 2
- A blood sample for toxicology and biochemical analysis
Autopsy internal examination
Internal examination:
A large “Y” shaped incision is made from each shoulder across the chest to the brisket, then down to the belly button,
The skin is then spread open to check to see if any ribs are broken.
The ribcage is split open, and the internal organs examined
Each organ is examined individually
Organs are weighed and tissue samples taken and fixed for further examination
What is Histology/Histopathology
- The microscopic examination of healthy or diseased tissue
What is Cytology/Cytopathology
-The microscopic examination of healthy or diseased cells
-Usually split into gynaecological and non- gynaecological cytology
Body fluids naturally contain cells
Cells can also be scraped from the surface of tissues.
These cells can be collected and transferred onto glass slides where they are dyed and examined under the microscope for abnormalities
Specimen Collection-Biopsy
Cells can be taken from tissue removed:
During surgical procedures in theatres
At outpatient clinics
At GP clinics
During post-mortem examination
Or can be collected from fluid
Blood
Urine
CSF
Sputum
What is Endoscopic biopsy
Endoscope is used to remove tissue, such as from the stomach during agastroscopy
What is Punch Biopsy
The lines of least skin tension are determined.
Skin is stretched 90 degrees perpendicular to the lines of least skin tension
The punch biopsy is performed.
Following relaxation the wound can be closed with sutures parallel to the lines of least skin tension.
When is a punch biopsy used
A punch biopsy– is for diagnosing skin conditions using a special instrument to punch a small hole in the skin to obtain a skin sample
Punch biopsy 2
The instrument is pressed down and rotated clockwise and anti-clockwise, cutting down into the subcutaneous fat.
The punch biopsy instrument is removed.
The biopsy specimen is gently lifted with a needle to avoid crush artifact.
Scissors are used to cut the specimen free at a level below the dermis.
What is Needle Biopsy
Fine-needle aspirate
- Superficial lumps or masses
- Usually palpable
- A thin hollow needle is used
- Cells aspirated
a needle biopsy– a special hollow needle, guided byultrasound, is used to obtain tissue from an organ or from tissue beneath the skin
What is a core needle biopsy
A Core needle biopsy
- A larger hollow needle is used
- Solid sample of tissue is removed
- Assisted by ultrasound, PET or CT
What happens in the needle biopsy
A needle is used to draw sample fluid and tissue from a lump to be studied
What is Surgical Biopsy 2 types
- An excisional biopsy
- Perioperative biopsy
What is an excisional biopsy
An excisional biopsy– where surgery is used to remove a larger section of tissue
What is a Perioperative biopsy
Perioperative biopsy– if consenthas been given, a perioperative biopsy can be carried out during surgery; the sample will be tested straight away so that the surgeon can be given the diagnosis and provide appropriate treatment
Why is fixing crucial immediately
It is crucial that tissue is fixed immediately to prevent degradation/ loss of integrity
What is Fixing and whys it necessary
1) Fixation is an attempt to preserve tissue as close to its natural state as possible
- Fixatives kill tissue/disable biomolecules (eg proteolytic enzymes) preventing autolysis and putrefaction
2) Protects sample from extrinsic damage
- Fixatives are toxic to most common organisms preventing infection
3) Preserve morphology
- Fixatives increase the strength and stability of the tissue
Properties of Fixatives
- Penetrate tissue quickly and evenly
- Kill cells quickly and evenly
- Prevent autolysis
- Prevent putrifaction
- Not add extraneous material
- Not swell or shrink tissue
- Be compatible with later processing
- Prevent tissue from drying out
- Safe
- Cheap
- Convenient
No fixative has all of these properties, compromises must be made.
What are common fixatives
- Formaldehyde
- Glutaraldehyde
- Alcohol
What is formaldehyde
Formaldehyde
Most commonly used fixative
40% solution in H20 called formalin
Commonly used diluted 1:10 in PBS buffer called 10% neutral buffered formalin (10% NBF)
Same as 4% Formaldehyde
Best for good architecture
What is Glutaraldehyde
Glutaraldehyde
Reacts more quickly than formaldehyde
Difficult to remove from tissue
Inactivates all enzymes preventing enzyme histochemistry
Binds and masks many antigens
Gives excellent morphology (widely used in EM)
Alcohol
Alcohol
Non-additive fixative
Preserves chemical reactivity
Tissue shrinks and becomes brittle
More common for fixing smears or fresh sections
FIXATIVES ADV AND DISADV
What is Sectioning
Fixed tissue is still quite soft and needs extra support to allow thin sections to be cut (usually 3-6µM)
Hard embedding medium such as paraffin wax is therefore used to support the cells and keep the tissue firm
Processing is the method by which tissues are converted into a block for microtomy
How does Processing work
1) Dehydration
(Ethanol or Methanol)
2) Clearing
(Toluene or Xylene)
3) Impregnation
(Molten wax)
4) Removal of clearing agent
5) Blocking out tissue
6) Microtomy
What is the rule on Paraffin wax impregnation in processing phase
Paraffin wax impregnation
Specimen cannot simply be immersed in wax, it must be impregnated
Water must be replaced with wax
What do all microtomes have
Various different types of microtome but all have:
Mechanisms for holding block firmly in place
A knife holder
Mechanism to move blade across block
A feed mechanism to advance the block so that subsequent sections can be cut
Frozen Tissue pros and cons and use
- Tissue is frozen quickly to reduce damage by ice crystals
Pros:
- Freezing H20 is quicker than replacing it
- Doesn’t involve treating with chemicals
- Better survival of many antigens
Cons:
- Poor morphology
- Poor resolution at higher magnification
- Special storage
- Difficulties cutting
What is used to freeze tissue
Chlorofluorocarbons (commercial spray)
Liquid CO2
Liquid N2
Sectioning how is it done + prepared
Using a cryostat
Deep freeze cabinet with microtome inside and controls outside
Sections are cut from frozen tissue
Sections are then picked up onto warm (room temp) slides
Adhesion good due to sudden change in temperature
Why is staining necessary and most common methods?
- Staining is required to provide contrast between different cell types and cell components
- Haematoxylin and eosin (H&E) is the most common staining method
What is Haemotoxylin
Haemotoxylin (blue) is a basic dye and therefore stains acidic components eg DNA & RNA
What is eosin
- Eosin (Pink) is an acidic dye and therefore binds and stains basic components eg. Proteins in the cytoplasm, cytoplasmic filaments and extracellular fibres
What is Periodic acid-Schiff (PAS)
- PAS is a staining method used to detect polysaccharides such as glycogen, glycoproteins and glycolipids
Often used to stain kidney biopsies, liver biopsies, certain glycogen storage diseases in striated muscles and suspected fungal infections.
What is Masson’s trichrome
Masson’s Trichrome (skin).
Rat airway & skin sections
This stain is intended for use in histological observation of collagenous connective tissue fibres in tissue specimens. It is used to assist in differentiating collagen and smooth muscle in tumours and assists in the detection of diseases or changes in connective/muscle tissue
What is Alcian blue
- A mucin stain that stains certain types of mucin blue. Cartilage is also stained blue. It can be used with H&E, and with van Gieson stains.
What is Van Gieson
- Stains collagen red, nuclei blue, and erythrocytes and cytoplasm yellow. It can be combined with an elastic stain that stains elastin blue/black. It is often used for blood vessels and skin.
What is Luxol Fast Blue
Attracted to bases found in lipoproteins of the myelin sheath.
Commonly used to detect demyelination in the CNS but cannot discern myelination in the PNS.
What is Silver Impregnation
Variety of silver stains available to stain neurons
Bielschowsky silver stain highlights tangles and plaques in Alzheimer’s disease
What is Sudan Black
Stains triglycerides, lipids and lipoproteins
Stains myeloblasts but not lymphoblasts
What is Immunohistochemistry
Antibodies used to detect specific molecules (antigens)
