HPCT WEEK 1 Flashcards
Covers a body surface or lines a body cavity
Forms most glands
Functions are:
Protection
Absorption, secretion, and ion
Filtration
Forms slippery surfaces
Epithelial Tissue
Most diverse and abundant tissue
Main classes are:
____ tissue proper
Blood-Fluid ____ tissue (blood)
Cartilage and Bone tissue - Supporting ____ tissues
Connective tissues
Components of connective tissue
Cells
Matrix (Protein fibers, Ground substances)
Common embryonic origin
Mesenchyme
Cells found in connective tissue proper
Fibroblasts, Macrophages, Lymphocytes, Adipocytes, Mast cells, Stem cells
Fibers:
Collagen - Very strong and abundant, long and, straight
Elastic - Branching fibers with a wavy appearance when relaxed
Reticular - Form a network of fibers that form a supportive frameworks in soft organs (i.e Spleen and Liver)
Ground susbtances:
Along with fibers, fills the extracellular space
The origin of a disease
Refers to why a disease arises
Etiology
Refers to the steps in the development of disease.
It describes how etiologic factors trigger cellular and molecular changes
Describes how a disease develops
Pathogenesis
Refers to the structural alterations in cells or tissue
Either Gross morphologic changes (Anatomic/Macroscopic)
or
Microscopic Changes
Morphologic changes
Referring to the clinical features (acute or malignant), course and prognosis of the disease
Functional derangements and Clinical Significance
Indication of a disease perceived BY THE PATIENT
Symptoms
Objective findings noticed BY THE DOCTOR on examination of the patient
Signs
Start of the disease
Onset
PREDICTION of the outcome of the disease
Prognosis
Outcome of the disease
Fate
New disease conditions that may occur during or after the usual course of the original disease
Complications
Undergoes replication all throughout life
Labile cells
Does not undergo replication unless injury
Stable cells
Does not undergo divisions following maturation
Permanent cells
Incomplete or defective development of tissue/organs
Affected organs shows no resemblance to normal mature form
Aplasia
Complete NON-APPEARANCE of organ
Agenesia
Failure of organ to form an opening
Atresia
Failure of organ to reach normal mature adult size
Hypoplasia
A state that lies intermediate between normal cell and injured cell
Cellular adaptation
Acquired decrease in tissue/organ size
Atrophy
The decrease in size happens as a consequence of maturation
Physiologic atrophy
Occurs if blood supply to an organ or tissue may directly injure the cell or may secondarily promote diminution of blood supply
Vascular Atrophy
Persistent PRESSURE on the organ or tissue may directly injure the cell or may secondarily promote diminution of blood suppliy
Pressure Atrophy
Due to lack of nutritional supply to sustain normal growth
Hunger atrophy
Due to lack of HORMONES needed to maintain normal size and structure
Endocrine Atrophy
Cardinal sign:pain
Dolor
Cardinal sign:Redness
Rubor
Cardinal sign:heat
Calor
Cardinal sign:swelling
Tumor
Cardinal sign: Destruction of functioning units of the cell
Function laesa
Watery, low protein fluid (Inflammation)
Serous inflammation
Fibrinogen is present in exudate (Inflammation)
Fibrinous inflammation
Pus / Purulent exudates (Inflammation)
Purulent / Suppurative inflammation
Blood and elements of exudates are present (Inflammation)
Hemorrhagic inflammation
Mucus is the main component (Inflammation)
Catarrhal Inflammation
Form of chronic inflammation characterized by collection of activated macrophages, T Lymphocytes, and sometimes associated with central necrosis
Granulomatous Inflammation
Pertains to the process of ensuring and maintaining personal as well as environmental health and safety in the laboratory
Risk management
Used to define the maximum allowable airborne concentration of a chemical
Permissible Exposure limits, Threshold Limit values, Occupational exposure Limits
Every Chemical should be labeled with
Chemical name and all ingredients (if mixture)
Manufacturer’s name and address or name of the person making the reagent
Date Purchased or made
Expiration date
Hazard warnings and safety procedures
Chemicals that cause reversible inflammatory effects
Irritants
Chemicals that cause destruction or irreversible alterations when exposed to living tissue
Corrosive chemicals
Cause allergic reactions in some exposed workers, not just in hypersensitive individuals
Sensitizers
Substances that can induce tumors, not only in experimental animals but also in humans
Carcinogens
Chemicals capable of causing death by ingestion, skin contact or inhalation at certain specified concentrations
Toxic materials
Intracellular changes associated with reversible injury (Plasma membrane)
Blebbing, Blunting, or distortion of microvilli, and loosening of intracellular attachments
Intracellular changes associated with reversible injury (Mitochondrial changes)
Swelling and the appearance of phospholipid-rich amorphous densities
Intracellular changes associated with reversible injury
Dilation of the endoplasmic reticulum with detachment of ribosomes and dissociation of polysomes
Intracellular changes associated with reversible injury (Nuclear alteration)
With clumping of chromatin (Pyknosis)
Intracellular changes associated with reversible injury (Myelin figure)
Phospholipid masses, derived from damage cellular membranes
Cell injury results from
Functional and biochemical abnormalities
Phospholipases increase will cause
membrane damage
Proteases increase will cause
break down of both membrane and cytoskeletal proteins
Endonucleases are responsible for
DNA and chromatin fragmentation
Mitochondrial damage
Decrease ATP, Increase ROS (reactive oxygen species)
Entry of Calcium
Increase mitochondrial permeability and will cause activation of multiple cellular enzymes
Membrane damage (Plasma membrane)
Loss of cellular components (cellular swelling)
Membrane damage (Lysosomal membrane)
Enzymatic digestion of cellular components (Necrotic to the cells)
Protein misfolding, DNA damage
Activation of pro-apoptotic proteins (apoptosis)
Cell size: Enlarge (Swelling)
Nucleus: Pyknosis > Karyorrhexis > Karyolysis
Plasma membrane: Disrupted
Cellular contents: Enzymatic digestion; may leak out of the cell
Adjacent inflammation: Frequent
Physiologic or pathologic role: Invariably pathologic
Necrosis
Cell size: Reduce (Shrinkage)
Nucleus: Fragmentation into nucleosome size fragments
Plasma membrane: Intact; altered structure
Cellular contents: Intact
Adjacent inflammation: No
Physiologic or pathologic role: Often physiologic. Means of eliminating unwanted cells, may be pathologic after some forms of cell injury, especially DNA and protein damage
Apoptosis
Types of cell death that is associated with loss of membrane integrity and leakage of cellular content
Necrosis
Individual organ removal
Virchow
Organ dissection in-situ
Rokitansky
En-masse dissection and organ separation
Letulle
Separate block dissection and organ separation
Ghon
Organ remove one by one from the cranial cavity down to the abdominal organs
For high-risk autopsies where permission is LIMITED TO ONE ORGAN
Good for DEMONSTRATING PATHOLOGICAL CHANGES in individual organs but the relationship between various organs may be hard to interpret
Virchow
En masse removal then subsequently dissected into organ blocks
Best technique for PRESERVING THE VASCULAR SUPPLY and relationships between organs, and for routine inspection because it is fast and the body can be made available to the undertaker
Letulle
Thoracic and cervical organs, abdominal organs, and the urogenital system are removed in functionally related blocks preserving ANATOMICAL RELATIONSHIPS
Simple to execute and appears as a compromise between Virchow and Letulle techniques
Gohn
In situ dissection (In local) combined with en bloc removal
Rokitansky
Computation for time of death
37c - Current body temp / 0.78 (C)
98.6F - Current body temp / 1.4 (F)
Rigor mortis occurs in ___ after death
2-3 hours
Starts in small muscle group (Head and Neck)
Fixed in 6-8 hours completed in 8-12 hours. Post mortem staining
Livor mortis
Takes out even more surrounding tissue.
Takes out some of the abnormality but not all
Incision biopsy
Removes the entire area in question
Excision biopsy
Simplest, least invasive test and uses the smallest needle to simply remove cells from the are of abnormality.
Not always adequate to obtain a diagnosis
FNAB
Considered as the primary technique for obtaining diagnostic full-thickness skin specimens
Use of circular blade that is rotated down through the epidermis and dermis, and into the subcutaneous fat, yielding a 3-4mm cylindrical core of tissue sample
Punch Biopsy
Removes not only cells, but also a small amount of the surrounding tissue
Provides additional information to assist in the examination of lesion
Core needle biopsy
Yield pleural fluid, directly aspirated from lungs
Thoracentesis / Chest tube thoracostomy
Samples are acquired though this procedure is received by hematology section or histopathology section
Similar to CSF collection
Bone marrow biopsy
Represent responses of cells to normal stimulation by hormones or endogenous chemical mediators.
Physiological Adaptation
Responses to stress that allow cells to modulate their structure and function
Pathologic adaptations
An increase in the size of cells resulting in increase in the size of the organ
No new cells are made
Occurs in tissues incapable of cell division
Hypertrophy
Enzyme substrate that colors viable myocardium magenta. Failure to stain is due to enzyme loss after cell death
Triphenyltetrazolium chloride
Takes place if the tissue contains cell populations capable of replication. It may occur concurrently with hypertrophy and often in response to the same stimuli
New cells are produced
Hyperplasia
Residual tissue grows after removal or loss of part of an organ
Compensatory hyperplasia
Shrinkage in the size of the cell by the loss of the cell substance
Decreased cell and organ size as a result of decreased nutrient supply or disuse
Associated with decreased synthesis and increased proteolytic breakdown of cellular organelles
Atrophy
Decreased workload
Loss of innervation
Inadequate Nutrition
Loss of endocrine stimulation
Aging
Causes of Atrophy
A reversible change in which one adult cell type is replaced by another adult cell type
Response to chronic irritation that makes cells better able to withstand the stress
Usually induced by altered differentiation pathway of tissue stem cells
May result in reduced functions or increased propensity for malignant transformation
Metaplasia
Occurs in epithelium exposed to mechanical trauma or chronic irritation of prolonged inflammation
Prolonged Vitamin A deficiency
Most commonly leading to replacement of columnar cells by stratified squamous epithelium
Epithelial Metaplasia
Occurring in connective tissues whereby fibroblasts are transformed into more highly differentiated forms such as osteoblasts, fat cells or tissue macrophages
Mesenchymal metaplasia
Cells may accumulate abnormal amounts of various substances. It may be harmless or associated with varying degrees of injury
The substance may be located at:
Cytoplasm
Within organelles (Lysosomes)
In the nucleus,
May be synthesized by the affected cells or may be produced elsewhere
Intracellular accumulations
Mechanism / Pathways of abnormal intracellular Accumulations
- Abnormal metabolism - Inadequate removal of a normal substance secondary to defects
- Defect in protein folding, transport - Accumulation of an abnormal endogenous substance
- Failure to degrade a metabolite
- Deposition and accumulation of an abnormal exogenous substance
Refers to any abnormal accumulation of triglycerides within parenchymal cells
Mostly seen in the liver
AKA steatosis
Fatty Change
Causes of steatosis
Toxins, protein malnutrition, diabetes mellitus, obesity, anoxia
Common causes of fatty change in the liver
Alcohol abuse and diabetes
Alter mitochondrial and the Smooth Endoplasmic Reticulum function
Inhibits fatty acid oxidation
Hepatotoxins (Alcohol)
Decrease the synthesis of apoproteins
CCI4 and Protein Malnutrition
Inhibits fatty acid oxidation
Anoxia
Increases fatty acid mobilization
Starvation
Immunoglobulins that may occur in the Rough Endoplasmic Reticulum of some plasma cells
Found in the peripheral areas of tumors
Eosinophilic Russel bodies
Is an eosinophilic cytoplasmic inclusion in liver cells that is highly characteristic of alcoholic liver disease
Damaged intermediate filaments within the hepatocytes
Mallory Body, or Alcoholic Hyalin
Are aggregates of hyperphosphorylated tau protein that are most commonly known as primary marker of Alzheimer’s disease
Found in the brain in alzheimer disease aggregated protein inclusion
Neurofibrillary tangle
____ Accumulates in renal tubular epithelium, cardiac myocytes, and B cells of the islet of langerhans
Glycogen
Most common exogenous pigment
A ubiquitous air pollutant. When inhaled, it is phagocytosed by alveolar macrophages and transported through lymphatic channels to the regional tracheobronchial lymph nodes
Aggregates of the pigment blacken the draining lymph nodes and pulmonary parenchyma
Carbon
Heavy accumulations may induce emphysema or a fibroblastic reaction that can result in a serious lung disease called coal worker’s pneumoconiosis
Exogenous carbon
An endogenous, brown-black pigment that is synthesized by melanocytes located in the epidermis and acts as a screen against harmful ultraviolet radiation
Melanocytes are the only source
Melanin
A hemoglobin-derived granular pigment that is golden yellow to brown and accumulates in tissues when there is a local or systemic excess of iron
Identified by its staining reaction (blue color) with the prussian blue dye
Hemosiderin
Hemosiderin types
1. Accumulation is primarily within tissue macrophages and is NOT ASSOCIATED WITH TISSUE DAMAGE
Hemosiderosis
Hemosiderin types
2. Extensive accumulation within parenchymal cells, WHICH LEADS TO TISSUE DAMAGE, SCARRING, and ORGAN DYSFUNCTION
Hereditary Hemochromatosis
An insoluble brownish-yellow granular intracellular material that accumulates in a variety of tissues particularly the heart, liver, and brain as a function of age or atrophy
Represents complexes of lipid and protein that derive from the free radical-catalyzed peroxidation of polyunsaturated lipids of subcellular membranes
Not injurious to the cell but is a marker of a past free radical injury
Brown pigment when present in large amounts imparts an appearance to the tissue that is called brown atrophy
Lipofuscin or wear-and-tear pigment
Abnormal deposition of calcium salts, together with smaller amount of iron, magnesium, and other minerals.
Pathologic Calcification
When the deposition occurs in dead or dying tissues, it occurs in the absence of calcium metabolic derangements in calcium metabolism
(Normal serum level of calcium)
Dystrophic
Deposition of calcium salts in normal tissues
Always reflects some derangement in calcium metabolism
Increase calcium in serum (Hypercalcemia)
Metastatic
Encountered in areas of necrosis of any type
Virtually inevitable in the atheroma of advance atherosclerosis
Dystrophic calcification
Initiation in ______ sites occurs in membrane bound
Extracellular sites
Initiation of ______ calcification occurs in the mitochondria of dead or dying cells that have lost their ability to regulate intracellular calcium
Intracellular calcification
Definition: Deposits of calcium salts in dead and degenerated tissue
Calcium metabolism: Normal
Serum Calcium level: Normal
Reversibility: Irreversible
Causes: aging or damaged heart valves
Dystrophic
Definition: Deposits calcium salts in normal tissues
Calcium metabolism: Deranged
Serum Calcium level: Hypercalcemia
Reversibility: Reversible upon correction of metabolic disorders
Causes: Hypercalcemia
Metastatic
A protective response involving
host cells
blood vessels
proteins and other mediators
Inflammation
It’s main goal is to eliminate the initial cause of cell injury, its protective mission by
diluting
Destroying
Neutralizing
Inflammation
Exogenous cases of inflammation
Physical agents -
a. Mechanic agents such as fractures, foreign, sand
b. Thermal agents: burns, Freezing
Chemical agents - Toxic gases, acids, bases
Biological agents - Bacteria, viruses, parasites
Endogenous cases of inflammation
Circulation disorders - Thrombosis, infarction, hemorrhage
Enzyme activation - acute pancreatitis
Metabolic products deposals - uric acid, urea
Changes in inflammation
Tissue damage
Cellular-vascular response
Metabolic changes
Tissue repairs
Onset: Fast
Cellular infiltrate: PMN (Mainly neutrophils)
Tissue injury, fibrosis - Mild and self-limited
Local and systemic signs - Prominent
Acute
Onset: Slow
Cellular Infiltrate: monocytes/macrophages and lymphocytes
Tissue injury, fibrosis: Often sever and progressive
Local and systemic signs: Less prominent, may be subtle
Chronic
An immediate and early response to an injurious agent
Short duration
Acute inflammation
Acute inflammation is characterized by
exudation of fluids and plasma proteins
Emigration of neutrophilic leukocytes to the site of injury
Cardinal signs of acute inflammation:
Due to dilation of small blood vessels within damage tissue (Cellulitis)
Rubor (Redness)
Cardinal signs of acute inflammation:
Results from increased blood flow (hyperemia ) due to regional vascular dilation
Calor (Heat)
Cardinal signs of acute inflammation:
Due to accumulation of fluid in the extravascular space
Tumor (Swelling)
Cardinal signs of acute inflammation:
Results from the stretching and destruction of tissues due to inflammatory edema
Dolor (Pain)
Cardinal signs of acute inflammation:
Inflamed area is inhibited by pain
Sever swelling may physically immobilize the tissue
Function laesa (Loss of function)
Chemicals of acute inflammation
Bradykinins
Prostaglandins
Serotonin
High protein content, High RBC, Pus present
Exudates
Low pus cells, Low protein content
Transudates
A peripheral position of white cells along the endothelial cells
Margination
Rows of leukocytes tumble slowly along the endothelium
Rolling
Endothelium can be lined by white cells. The binding of leukocytes with endothelial cells is facilitated by cell adhesion molecules
- Selectins, immunoglobulins, integrins
Pavementing
The process of movement of leukocytes by extending pseudopodia through the vascular wall
Diapedesis
Unidirectional attraction of leukocytes from vascular channels towards the site of inflammation within the tissue space guided by chemical gradients
Chemotaxis
The important chemotactic factors of neutrophils are:
C5a - Complement system, bacteria, and mitochondrial products of arachidonic acid metabolism
Leukotriene B4
Cytokine (IL-8)
Process of engulfment and internalization by specialized cells of particulate material
Phagocytosis
Leukocyte molecule: Sialyl-Lewis X
modified proteins
Major role: Rolling
P-selectin
Leukocyte molecule: Sialyl-Lewis X
modified proteins
Major role: Rolling and adhesion
E-selectin
Leukocyte molecule: L-selectin
Major role: Rolling Neutrophils, monocytes)
GlyCam-1, CD34
Leukocyte molecule:C11/CD18, Integrins (LFA-1, Mac-1)
Major role: Firm adhesion, transmigration
ICAM-1 (immunoglobulin Family)
Leukocyte molecule: VLA-4 Integrin
Major role: Adhesion
VCAM-1 (Immunoglobulin family)
Leukocyte molecule: CD31 (homotypic interaction)
Major role: Transmigration of leukocytes through endothelium
CD31
What is the defect of the ff:
Bone marrow suppression: Tumors (including leukemias, radiation, and chemotherapy)
Production of leukocytes
What is the defect of the ff:
Diabetes, malignancy, sepsis, chronic dialysis
Adhesion and Chemotaxis
What is the defect of the ff:
Anemia, sepsis, diabetes, malnutrition
Phagocytosis and microbicidal activity
What is the defect of the ff:
Leukocyte adhesion deficiency 1
Defective leukocyte adhesion because of oil mutations in Beta chain of CD11/CD18 integrins
What is the defect of the ff:
Leukocyte adhesion deficiency 2
Defective leukocyte adhesion because of mutations in fucosyl transferase required for synthesis of sialylated oligosaccharide (receptor for selectins)
What is the defect of the ff:
Chronic granulomatous disease
Decreased oxidative burst
What is the defect of the ff:
X-Linked
Phagocyte oxidase (membrane component)
What is the defect of the ff:
Autosomal recessive
Phagocyte oxidase (cytoplasmic component)
What is the defect of the ff:
Myeloperoxidase deficiency
Decreased microbial killing because of defective MPO-H202 system
What is the defect of the ff:
Chediak-lligashi syndrome
Decreased leukocyte functions because of mutations affective protein, involved in lysosomal membrane traffic
Steps of the inflammatory response (5Rs)
- Recognition of the injurious agent
- Recruitment of leukocytes
- Removal of the agent
- Regulation of the response
- Resolution (Repair)
Characterized by the outpouring of a watery, relatively protein-poor fluid that, depending on the site of injury
Fluid in a serous cavity is called an effusion
Serous inflammation
Resulting in greater vascular permeability allows large molecules to pass the endothelial barrier
A fibrinous exudate is characteristic of inflammation in the lining of body cavities such as the meninges, pericardium, and pleura
Fibrinous inflammation
Manifested by the presence of large amount of purulent exudate
Consisting of neutrophils, necrotic cells, and edema fluid (Staphylococci)
Suppurative inflammation
Focal collections of pus that may be caused by seeding of pyogenic organisms into a tissue
Secondary infections of necrotic foci
Abscesses
A local defect, or excavation, of the surface of an organ or tissue that is produced by necrosis of cells and sloughing/shedding of inflammatory necrotic tissue
Ulcer
Source: Mast cells, basophils, platelets
Actions: Vasodilation, increased vascular permeability, ENDOTHELIAL ACTIVATION
Histamine
Source: Platelets
Actions: VASOCONSTRICTION
Serotonin
Source: Mast cells, leukocytes
Actions: Vasodilation, PAIN, FEVER
Prostaglandins
Source: Mast cells, Leukocytes
Actions: Increased vascular permeability, chemotaxis, leukocyte adhesion and activation
Leukotrienes
Source: Leukocytes, mast cells
Actions: Vasodilation, increased vascular permeability, leukocyte adhesion, chemotaxis, DRGRANULATION, OXIDATIVE BURST
Platelet-activating factors
Source: Leukocytes, mast cells
Actions: KILLING OF MICROBES, TISSUE DAMAGES
Reactive-activating factor
Source: Endothelium, Macrophages
Actions: VASCULAR SMOOTH MUSCLE RELXATION; killing of microbes
Nitric Acid
Source: Macrophages, Endothelial cells, mast cells
Actions: Local: Endothelial activation
Systemic: Fever, metabolic abnormalities, hypotension
Cytokines (TNF, IL-1, IL-6)
Source: Leukocytes, activated macrophages
Actions: Chemotaxis, Leukocyte activation
Chemokines
Source: Plasma (produced in liver)
Actions: Increased vascular permeability, smooth muscle CONTRACTION, VASODILATION, Pain
Kinins
Source: Plasma (Produced in liver)
Actions: Endothelial activation, leukocyte recruitment
Proteases activated during coagulation
Inflammatory component:
Vasodilation
Mediators:
Prostaglandins
Nitric oxide
Histamine
Inflammatory component:
Increased vascular permeability
Mediators:
Histamine and Serotonin
C3a and C5a (by liberating vasoactive amines from mast cells, other cells)
Bradykinin
Leukotrienes (C4,D4,E4)
PAF
Substance P
Inflammatory component:
Chemotaxis, Leukocyte recruitment and activation
Mediators:
TNF, IL-1
Chemokines
C3a,C5a
Leukotriene B4
Bacterial products (N-formyl methyl peptides)
Inflammatory component:
Fever
Mediators:
IL-1
TNF
Prostaglandins
Inflammatory component:
Pain
Mediators:
Prostaglandins
Bradykinin
Inflammatory component:
Tissue Damage
Mediators:
Lysosomal enzymes of leukocytes
Reactive oxygen species
Nitric oxide
The dominant cells of chronic inflammation
Macrophages
Macrophages in liver
Kupffer cells
Macrophages in Spleen and lymph nodes
Sinus histiocytes
Macrophages in CNS
Microglial cells
Macrophages in lungs
Alveolar macrophages
Develop from activated B lymphocytes, produce antibodies
Plasma Cells
Characterized found in inflammatory sites around parasitic infections or as part of immune reactions mediated by IgE, typically associated with allergies
Eosinophils
Distributed in connective tissues throughout the body, and they can participate in both acute and chronic inflammatory response
Mast cells
This involves a diffuse accumulation of macrophages and lymphocytes at site of injury that is usually productive with new fibrous tissue formations
Nonspecific Chronic Inflammation
- Characterized by the presence of granuloma
- Granuloma: is a microscopic aggregate of
epithelioid cells - Epithelioid: cells is an activated macrophage, with a modified epithelial cell-like appearance. The epithelioid cells can fuse with each other & form multinucleated giant cells
Specific Inflammation
- Is a distinctive pattern of chronic inflammation characterized by aggregates of activated macrophages that assume an epithelioid appearance
- A typical granuloma resulting
from infection with Mycobacterium tuberculosis showing central caseous necrosis,
activated and epithelioid macrophages, many giant cells, and a peripheral accumulation of lymphocytes
Granulomatous Inflammation
Irregularly scattered nuclei in presence of indigestible materials
Foreign body-types giants cells
Nuclei are arranged peripherally in a horse-shoe pattern which is seen typically in tuberculosis, and sarcoidosis
Langhans Giant cells
Granulomas are initiated by inter foreign
Foreign body granuloma
Antigen presenting cells engulf a poorly soluble inciting agent
Macrophages inhibitory factor helps to localize activated macrophages and epithelioid cells
Immune granulomas
Major cause of Granulomatous inflammation:
Tuberculosis, Leprosy, Syphilis, Cat scratch disease, Yersiniosis
Bacterial
Major cause of Granulomatous inflammation:
Histoplasmosis, Cryptococcosis, Coccidioidomycosis, Blastomycosis
Fungal
Major cause of Granulomatous inflammation:
Schistosomiasis
Helminthic
Major cause of Granulomatous inflammation:
Leishmaniasis, Toxoplasmosis
Protozoal
Major cause of Granulomatous inflammation:
Lymphogranuloma venerum
Chlamydia
Mechanisms regulating cell populations
Cellular proliferation
Cell numbers can be altered by
Increased or decreased rates of stem cell input
Process in the proliferation of cells
DNA replication and Mitosis
Continuously dividing tissues
Cells are continuously proliferating
Can easily regenerate after injury
Contains a pool of stem cells
E.G - Bone marrow, Skin, Epithelium
Stable tissues
Cells have limited ability to proliferate
Limited ability to regenerate
Con proliferate if injured
E.G - Liver, Kidney, Pancreas
Permanent tissue
Cells can’t proliferate
Can’t regenerate (Injured always lead to scar)
E.G - Neurons, Cardiac muscle
Three phases in granulation-tissue
- Phase inflammation
- Phase of demolition
- Ingrowth of granulation tissue
Inflammatory exudate, platelet aggregation, and fibrin deposition
Phase Inflammation
The dead cells liberate their autolytic enzymes
There is an associated macrophage infiltration
Phase of demolition
Proliferation of fibroblasts, and an ingrowth of new blood vessels, and an ingrowth of new blood vessels (angiogenesis) into the area of injury, with a variable number of inflammatory cells
Ingrowth of granulation tissue
A mechanical reduction in the size of the defect
Contraction: results in much faster healing
If contraction is prevented, healing is slow and a large scar is formed
Wound contraction
Have the capacity to stimulate cell division and proliferation (Promote cell survival)
Growth factors
Sources of growth factors
Platelets, activate (TGF - Transforming growth factor)
Damaged epithelial cells (EGF - Epidermal growth factors)
Macrophages (Angiogenic factor)
Lymphocytes recruited to the area of injury
Network that surrounds scells
Extracellular Matrix
Provides mechanical supports to tissues
Collagens and elastin
Defined as the process of preparing the tissue
Tissue processing
Is the microscopic study of the normal tissues of the body
Histology
Microscopic tissue affected by DISEASE
Histopathology
Fresh tissue examination advantages
Protoplasmic activities
Mitosis
Phagocytosis
Pinocytosis - (an active, energy consuming process where extracellular fluid and solutes are taken up into a cell via small vesicles)
Methods of fresh tissue examination
Teasing or dissociation
Squash preparation (Crushing)
Smear preparation: Streaking, Spreading, Pull-apart, Touch preparation (Impression smear)
Frozen section
A process whereby a selected tissue specimen is immersed in a watch glass containing ISOTONIC SALT SOLUTION (NSS OR RINGER’S SOLUTION)
Unstained by Phase contrast or Bright Field microscopy, or stained with differential dyes
Teasing or Dissociation
A process whereby small pieces of tissue not more than 1mm in diameter are placed in a microscopic slide and forcible compressed with another slide or with a cover glass
Squash preparation (Crushing)
More than 1 mm in diameter in squash preparation what will happen?
It will interfere with the objective of the microscope
Cellular materials are spread lightly over a slide by means of a wire loop or applicator
Useful in CYTOLOGIC EXAMINATIONS
Used for CANCER DIAGNOSIS
Smear preparation
Smear preparation methods
Streaking, Pull-apart, Touch preparation, and Spreading
Used in rapid diagnosis of the tissue
Recommended for lipids and nervous tissue
10-15u in thickness
Frozen section
A cold chamber kept at an atmospheric temp of -10 to -20C
Cryostat
Utilized for RAPID pathologic diagnosis during surgery
Diagnostic and research enzyme histochemistry
Diagnostic and research demonstration of soluble such as lipids and carbs
Immunofluorescent and immunohistochemical staining
Some specialized silver stains, particularly in NEUROPATHOLOGY
Frozen Section
Advantages: Used in IHC
Most Rapid of the commonly available freezing agents
Disadvantages: Soft tissue is liable to crack producing ice crystals or freeze artifacts
Causes a vapor phase
Liquid nitrogen
Advantages: Excellent method for freezing muscle tissue
Disadvantages:
Isopentane cooled by liquid nitrogen
Advantages: Adapting a conventional freezing microtome
Disadvantages:
Carbon dioxide gas
Advantages: Adequate for freezing small pieces
Rapidly freezing Blocks of any type of tissue
Disadvantages:
Aerosol sprays
Most common method of freezing
Liquid nitrogen
Tissue blocks can be frozen by adapting a conventional freezing microtome gas supply of carbon dioxide gas from a CO2 cylinder, or by using a specially made piece of equipment known as cryostat
Cold knife procedure
This method makes use of the cryostat, an apparatus used in fresh tissue microtomy
The cryostat consists of an insulated microtome housed in an electrically driven refrigerated chamber and maintained at temperatures near -20C, where microtome, knife, specimen, and atmosphere are kept at the same temp
Cryostat procedure
Optimum working temp of cryostat is
-19 to -20C
Most common
Pathologist diagnose the presence or absence of disease
Histotechnologist needs to produce a tissue section of good quality that allows for adequate interpretation of microscopic cellular changes
Solid tissue needs to be fixed and processed to preserve their structures
Conventional tissue processing
Most critical step in tissue processing, depends on the type, ratio, concentration of the solution
Fixative
Not all specimens are subjected to this
Decalcification
Used of alcohol
Dehydration
Dealcoholization step, commonly used is xylene
Clearing
Steps in tissue processing
Fixation
Decalcification (Optional)
Dehydration
Clearing (Dealcoholization)
Infiltration or impregnation
Embedding
Trimming
Section-cutting
Staining
Mounting
Labelling
Step that removes excess wax in preparation of section-cutting
Trimming
The FIRST and MOST CRITICAL STEP IN HISTOTECHNOLOGY
Fixation
Primary aim of fixation
To preserve the morphologic and chemical integrity of the cell in as like-like manner as possible
Secondary goal of fixation
To harden and protect the tissue from the trauma of further handling
So that it is easier to cut during gross examination
It prevents degeneration, decomposition, putrefaction, and distortion of tissues after removal from the body
Fixation
How does fixation prevents breakdown of cellular elements
Fixation prevents autolysis by inactivating the lysosomal enzymes or by chemically altering, stabilizing, and making the tissue components insoluble
It also protects the tissue from further decomposition after death due to bacterial or fungal colonization
How does fixation coagulate or precipitate protosplasmic substances?
Fixation renders insoluble certain tissue components that may otherwise leak out during subsequent histologic handling
Chemical constituent of the fixative is taken in and becomes part of the tissue by FORMING cross-links or molecular complexes and giving stability to the protein
E.g - Formalin, mercury, Osmium, tetroxide
Additive fixation
Fixative is not incorporated into the tissue, but ALTERS the tissue composition and stabilizes the tissue by REMOVING the bound water within the protein molecule
E.g - Alcoholic fixative
Non-additive fixation
Effects of fixative
Preserve the morphologic and chemical integrity of the cell
Harden soft and friable tissues
Inhibits bacterial decomposition
Act as mordants or accentuators
Osmolality
Slightly hypertonic solutions - ______
Isotonic solutions - _____
Slightly hypertonic - 400-450 mOsm (Shrinkage of tissue)
Isotonic - 340 mOsm
Concentration:
Formaldehyde - ____
Glutaraldehyde - ____
___ is the ideal concentration in immuno Electro microscopy
Formaldehyde - 10%
Glutaraldehyde - 3%
0.25 % is the ideal concentration in immuno electro microscopy
Duration of fixation
2-6 hours
Formalin can be washed after fixation for 24 hours
Hydrogen Ion concentration in fixation
6 and 8 pH satisfactory
Temperature in fixation
Room temp
Tissue processors - 40C
EM and Histochemistry - 0 to 4C
Formalin Heated to 60: Rapid fixation
Formalin at 100C - fix tissues with tuberculosis
Thickness of section
1-2 mm2 for electron microscopy
2cm2 for light microscopy
Large solid tissue (Uterus) (Brain (suspended whole in 10% NBF for 2-3 weeks)
Practical considerations of fixation
Prevent autolysis and putrefaction
Speed
Practical considerations of fixation
Formalin diffuses at 1mm/hr (depends on the concentration of formalin) recommended is 10% NBF
Penetration
Practical considerations of fixation
Amount of fixative - 20X the tissue volume = max effectiveness
10-25x (before)
Volume
Practical considerations of fixation
Fibrous organs take longer to fix than biopsies or scrapings
Can be cut down using heat, vacuum, agitation, or microwave
Duration of fixation
Characteristics of fixative
Cheap, stable, safe to handle
Must be isotonic
Inhibits bacterial decomposition
Must permit rapid and even penetration of tissues
Must make cellular components insoluble to hypotonic solutions
One component fixative
Simple fixatives
Compound fixatives
2 or more components
Type of fixative according to action
Microanatomical fixative
Cytological fixatives
Histochemical fixatives
Simple fixatives examples
- Aldehydes (Formaldehyde, Glutaraldehyde)
- Metallic Fixatives
Mercuric chloride
Chromate fixatives (Potassium dichromate, Chromic acid)
Lead fixatives (Acetone, Alcohol, Picric acid, Acetic Acid, Osmium tetroxide (Osmic acid) ) - Heat
Made up of two or more fixatives which have been added
Compound fixatives
Fixatives according to action that permits general MICROSCOPIC STUDY of tissue structures
Microanatomical fixatives
Fixatives according to action that preserve specific parts NUCLEAR or CYTOPLASMIC
Cytological fixative
Fixatives according to action that preserve the CHEMICAL constituents of cells and tissues
Histochemical fixatives
Microanatomical fixatives example
10% Formol Saline
10% Neutral buffer formalin
Heidenhein’s susa
Zenker’s solution
Zenker’s Formol (Helly’s solution)
Bouin’s solution
Brasil Solution
Preserves NUCLEAR structure (Chromosomes).
Contain Glacial acetic acid
pH is 4.6 or less
Nuclear fixatives
Nuclear fixatives example
Flemming’s fluid
Carnoy’s Fluid
Bouin’s Fluid
Newcomer’s Fluid
Heidenhain susa
Preserver CYTOPLASMIC structure
No glacial acetic acid
pH is more than 4.6
Cytoplasmic fixatives
Cytoplasmic fixatives example
Flemming’s fluid without acetic acid
Helly’s Fluid
Regaud’s Fluid (Muller’s fluid)
Orth’s Fluid
Histochemical Fixatives example
Formol saline 10%
Absolute Ethyl Alcohol
Acetone
Newcomer’s Fluid
Fixatives for satisfactory for routine paraffin sections
For electron microscopy
For Histochemical and enzyme studies
Aldehyde Fixatives
Most widely used concentration for this fixative is 10%
A gas produced by the oxidation of METHYL ALCOHOL
Pure stock solution of this fixative is 40% which is unsatisfactory for routine fixation
Dilution is 1:10 or 1:20
usual fixation time of this fixative is 24 hours
Buffered to pH 7 with PHOSPHATE BUFFER
Formaldehyde
Cheap, Readily available, easy to prepare, Relatively stable
Compatible with most stain
Preservers fats, glycogen, and mucin
Allows tissue enzymes to be studied because it does not precipitate proteins
Recommended for nervous tissue preservation
Allows natural tissue colors to be restored; recommended for colored tissue photography
Tolerant fixative used for mailing specimen
Advantages of formaldehyde
Disadvantages of formaldehyde
May cause sinusitis, allergic rhinitis, excessive lacrimation or allergic dermatitis
May produce considerable shrinkage of tissues
A soft fixative and does not harden some cytoplasmic structures adequately enough for paraffin embedding
Advantages of formalin
Cheap, Readily available, easy to prepare, Relatively stable
Compatible with most stain
Preservers fats, glycogen, and mucin
Allows tissue enzymes to be studied because it does not precipitate proteins
Recommended for nervous tissue preservation
Allows natural tissue colors to be restored; recommended for colored tissue photography
Tolerant fixative used for mailing specimen
May cause sinusitis, allergic rhinitis, excessive lacrimation or allergic dermatitis
May produce considerable shrinkage of tissues
A soft fixative and does not harden some cytoplasmic structures adequately enough for paraffin embedding
Disadvantages of formalin
Microanatomical fixative
Recommended for fixation of CNS and general postmortem tissues for histochemical explanation
Preserves enzymes and nucleoproteins
Demonstrates fats and mucin
10% Formol saline
Recommended for preservation and storage of SURGICAL, POST-MORTEM, and RESEARCH specimen
Fixation time is 4-24 hours
Best fixative for tissues containing iron pigments and for elastic fibers
10% Neutral buffered formalin or Phosphate-buffer formalin
Recommended for routine POST-MORTEM TISSUES
Fixation time of this fixative is 3-24 hours
Penetrates SMALL PIECES of TISSUES RAPIDLY
Excellent for many staining procedures including SILVER RETICULUM METHODS
Formol-Corrosive or Formol-Sublimate
Fixation of this fixative is FASTER
for RAPID DIAGNOSIS because it FIXES AND DEHYDRATES at the same time
God for preservation of GLYCOGEN and for MICRO-INCINERATION technique
Used to fix SPUTUM since it COAGULATES mucus
Produces GROSS HARDENING of TISSUES
Causes partial LYSIS of RBCs
Preservation of iron-containing pigments is POOR
Alcoholic formalin or Gendre’s fixative
Made up of 2 formaldehyde residues, linked by 3 carbon chains
For ROUTINE LIGHT MISCROCOPIC WORK
Buffered glutaraldehyde, followed by secondary fixation in osmium tetroxide is satisfactory for ELECTRON MICROSCOPY
Fixation time of this fixative is 1/2 hour to 2 hours
Preserves PLASMA PROTEINS
Produces LESS TISSUE SHRINKAGE
EXPENSIVE
LESS STABLE
Penetrates tissue SLOWLY
Tends to make tissue more BRITTLE
Reduces PAS (Periodic acid–Schiff) positivity of reactive mucin
Glutaraldehyde
List of aldehyde fixatives
Formaldehyde (Formalin)
10% Formol Saline
10% NBF or Phosphate-buffered formalin
Formol- corrosive / Formol sublimate
Alcoholic formalin / Gendre’s Fixative
Glutaraldehyde
Most common metallic fixative; used in 5-7%
Penetrates poorly and produces shrinkage of tissues
May form BLACK PRECIPITATES of MERCURY
Precipitates ALL PROTEIN
Recommended for RENAL TISSUES, FIBRIN, CONNECTIVE TISSUES, and MUSCLES
Rapidly HARDENS the OUTER LAYER of the TISSUE with incomplete fixation of the center
Trichrome staining is excellent. Permits brilliant metachromic staining of cells
Mercuric Chloride
Mercuric chloride stock solution + GLACIAL ACETIC ACID
Recommended for fixing small pieces of LIVER, SPLEEN, CONNECTIVE TISSUE FIBERS, and NUCLEI
Fixation time is 12 - 24 hours
RECOMMENDED FOR TRICHROME STAINING
Permits BRILLIANT STAINING of NUCLEAR and CONNECTIVE TISSUE FIBERS
COMPATIBLE with MOST stains
May ACT as a MORDANT
PENETRATION is POOR
Zenker’s Fluid
Fixation time of this fixative is 12-24 hours
EXCELLENT MICROANATOMIC FIXATIVE for PITUITARY GLAND, BONE MARROW, and BLOOD containing organs such as SPLEEN, and LIVER
PRESERVES CYTOPLASMIC GRANULES well
Zenker-Formol / Helly’s solution
Recommended mainly for TUMOR BIOPSIES especially of the skin
Excellent CYTOLOGIC FIXATIVE
Fixation time : 3-12 hrs
Produces brilliant results with SHARP NUCLEAR and CYTOPLASMIC details
Permits EASIER sectioning of large blocks
of FIBROUS CONNECTIVE TISSUES
RBC preservation is POOR
Some CYTOPLASMIC granules are DISSOLVED
Weigert’s method of staining elastic fibers is not possible in Susa-fixed tissues
Heidenhain’s Susa Solution
commonly used for BONE MARROW BIOPSIES
Rapid fixation can be achiever in 1 1/2 - 2 hours
B-5 Fixative
List of metallic fixatives
Mercuric Chloride
Zenker’s Fluid
Zenker-Formol or Helly’s Solution
Heidenhain’s Susa Solution
B-5 Fixative
Use in 1-2% aqueous solution
Precipitates ALL PROTEINS and ADEQUATELY PRESERVES CARBOHYDRATES
A STRONG OXIDIZING AGENT
Not used because IT IS HAZARDOUS
Chromic Acid
Used in 3% Aqueous solution
PRESERVES LIPIDS AND MITOCHONDRIA
Potassium Dichromate
Fixation time of this fixative is 12-48 hours
HARDENS TISSUES BETTER and MORE RAPIDLY than Orth’s Fluid
Recommended for DEMONSTRATION OF CHROMATIN, MITOCHONDRIA, MITOTIC FIGURES, GOLGI BODIES, RBC, AND COLLOID-CONTAINING TISSUES
Must always be FRESHLY PREPARED
GLYCOGEN penetration is POOR
NUCLEAR STAINING is POOR
DOES NOT preserve FATS
Intensity of PAS reaction is REDUCED
Regaud’s Fluid/ Muller’s Fluid
Fixation time is 36-72 hours
RECOMMENDED for STUDY of EARLY DEGENERATIVE PROCESSES AND TISSUE NECROSIS
Demonstrates RICKETTSIAE and OTHER BACTERIA
Preserves MYELIN better than BUFFERED FORMALIN
Orth’s Fluid
Used in 4% aqueous solution of basic lead acetate
Recommended for ACID MUCOPLYSACCHARIDES
Fixes CONNECTIVE TISSUE MUCIN
Takes up CARBON DIOXIDE to FORM INSOLUBLE CARBONATE especially on PROLONGED STANDING
Lead Fixatives
Normally used in strong saturated aqueous solution (1%)
Excellent Fixative for GLYCOGEN DEMONSTRATION
Also DYES the tissue. ALLOWS Brilliant staining with the TRICHROME method
Precipitates ALL proteins
STABLE
causes RBC HEMOLYSIS and REDUCES the amount of DEMONSTRABLE FERRIC IRON in TISSUES
Must NEVER be washed in water before dehydration
HIGHLY EXPLOSIVE when DRY
ALTERS AND DISSOLVES LIPIDS
SUITABLE for ANILINE Stains
Causes shrinkage of tissue (Slightly Hypertonic)
Picric Acid
recommended for FIXATION of EMBRYOS and PITUITARY BIOPSIES
Excellent Fixative for preserving SOFT and DELICATE structures
Fixation time of this fixative is 6-24 hours
PRESERVES Glycogen
Does NOT need washing out
Bouin’s Solution
BETTER and LESS MESSY than Bouin’s Solution
EXCELLENT FIXATIVE for GLYCOGEN
Brasil’s Alcoholic Picroformol Fixative
Solidifies at 17C
FIXES and PRECIPITATES NUCLEOPROTEINS
Precipitates CHROMOSOMES and CHROMATIN materials
Causes tissue to SWELL (Hypotonic)
Glacial Acetic Acid
List of Chromate fixatives
Chromic acid
Potassium Dichromate
Regaud’s Fluid or Muller’s Fluid
Orth’s Fluid
Lead Fixatives
Picric Acid
Bouin’s Solution
Brasil’s Alcoholic picroformol fixative
Glacial Acetic Acid
Must be used in concentrations ranging from 70-100% because less concentrated solution will produce lysis of cells
Alcohol fixatives
Used to fix and preserve GLYCOGEN PIGMENTS, BLOOD, TISSUE FILMS, and SMEARS
Ideal for SMALL TISSUE FRAGMENTS
Excellent for GLYCOGEN PRESERVATION
Preserves NUCLEAR STAINS
Lower concentrations will cause RBC HEMOLYSIS and INADEQUATELY preserve leukocytes
DISSOLVES fats and Lipids
Absolute alcohol
Excellent for fixing DRY and WET smears, BLOOD SMEARS, and BONE MARROW TISSUES
FIXES and DEHYDRATES at the same time
Penetration is SLOW
Tissues may be OVERHARDENED and DIFFICULT to cut if left for more than 48 HOURS
Methyl Alcohol
Used for fixing TOUCH PREPARATIONS
95% Isopropyl alcohol
Used at 70-100% concentration
a SIMPLE FIXATIVE
Fixation time is 18-24 hours
Preserves but DOES NOT fix glycogen
Ethyl Alcohol
Used to fix BRAIN TISSUES for the diagnosis of RABIES
Fixation time is 1-3 hours
Considered as the MOST RAPID FIXATIVE
Fixes and dehydrates at the SAME TIME
Preserves NISSL’s granules and Cytoplasmic granules WELL
Preserves NUCLEOPROTEINS and NUCLEIC acids
Excellent fixative for GLYCOGEN
Carnoy’s Fluid
Histochemical fixative and nuclear fixative
Produces BETTER reaction in FEULGEN STAIN than Carnoy’s Fluid
Recommended for fixing MUCOPOLYSACCHARIDES and NUCLEAR PROTEINS
Fixation time is 12-18 hours at 3c
Newcomer’s Fluid
Used in ELECTRON MICROSCOPY
Preserves CYTOPLASMIC STRUCTURES well such as GOLGI BODIES and MITOCHONDRIA
Produces BRILLIANT NUCLEAR STAINING with SAFRANIN
Adequately fixes materials for ULTRATHIN sectioning in EM
VERY EXPENSIVE
POOR penetrating agent, suitably ONLY for SMALL PIECES of tissues
INHIBITS hematoxylin and makes counterstaining DIFFICULT
EXTREMELY VOLATILE
Can IRRITATE the EYES producing conjunctivitis or may cause deposition of BLACK OSMIC OXIDE in the cornea leading to blindness
Stains Fat BLACK
Osmium Tetroxide
Most common chrome-osmium acetic acid fixative
Fixation time is 24- 48 hours
Excellent fixative for NUCLEAR STRUCTURES
PERMANENTLY fixes FATS
Flemming’s Solution
Made up of only chromatic acid and osmic acid
Recommended for cytoplasmic structures particularly the MITOCHONDRIA
Fixation time is 24-48 hours
Flemming’s solution w/o acetic acid
Precipitates proteins
WEAK decalcifying agent
Softening effect on DENSE FIBROUS TISSUES facilitates preparation of such sections
POOR penetrating agent
Suitable only for SMALL PIECES OF TISSUES or BONES
Trichloroacetic acid
Used at ice cold temperature ranging from -5c to 4c
Recommended for study of WATER DIFFUSIBLE ENZYMES especially PHOSPHATES and LIPASES
Used in fixing brain tissues for diagnosis of RABIES
Used as solvent for certain METALLIC SALTS to be used in FREEZE SUBSTITUTION techniques for tissue blocks
Evaporates RAPIDLY
Acetone
Involves thermal coagulation of tissue protein for rapid diagnosis
HEAT FIXATION
A process of placing an already fixed tissue in a second fixative
SECONDARY FIXATION
Form of secondary fixation
2.5-3%K dichromate for 24 hrs to act as mordant for better staining and aid in cytologic preservation of tissues
Post-Chromatization
The process of removing excess fixative from the tissue after fixation
Washing out
Solution used for washing out Helly’s solution, Zenker’s Solution, Flemming’s solution, Formalin, Osmic acid
Tap Water
Solution used for washing Picric’s acid (Bouin’s Solution)
50-70% Alcohol
Solution used for washing out Mercuric fixation
Alcoholic Iodine
Retarded by:
Size and thickness of the tissue specimen
Larger Tissue requires more fixatives and longer Fixed time
Retarded by presence of mucus
Tissue that contain mucus are fixed slowly and poorly
Retarded by presence of fat
Fatty Tissues should be cut in thin sections and fixed longer
Retarded by: Presence of blood
Tissues containing blood, large amt of blood should be flushed out with saline before fixing
Retarded by: Cold temp
Inactivates enzymes
Enhanced by: Size and thickness of tissue
Smaller and thinner Tissues require less fixative and shorter fix times
Enhanced by agitation
Fixation is accelerated when automatic or mechanical tissue processing is used
Enhanced by moderate heat (35-56C)
Accelerates fixation but hastens autolytic changes and enzymes destruction
beyond 35-56 can damage or distortion to the tissues
Known artefact produced under acid conditions
Reduced by fixation in phenol-formalin
Formalin Pigment
Found in surgical spec (Liver biopsies)
Associated with an intense eosinophilic staining
Due to partial coagulation of protein by ethanol
Incomplete wax fixation
Crush artefact
Fixative of choice and Fixative to avoid when your target to study is PROTEIN
Fixative of choice: NBF, Paraformaldehyde
Fixative to avoid: Osmium Tetroxide
Fixative of choice and Fixative to avoid when your target to study is Enzymes
Fixative of choice: Frozen section
Fixative to avoid: Chemical Fixatives
Fixative of choice and Fixative to avoid when your target to study is Lipids
Fixative of choice: Frozen section, Glutaraldehyde, Osmium tetroxide
Fixative to avoid: Alcoholic and NBF
Fixative of choice and Fixative to avoid when your target to study is Nucleic acid
Fixative of choice: alcoholic fixatives
Fixative to avoid: Aldehydes
Fixative of choice and Fixative to avoid when your target to study is Mucopolysaccharides
Fixative of choice: Frozen section
Fixative to avoid: Chemical
Fixative of choice and Fixative to avoid when your target to study is Biogenic amines
Fixative of choice: Bouin’s solution and NBF
Fixative of choice and Fixative to avoid when your target to study is Glycogen
Fixative of choice: Alcoholic fixatives
Fixative to avoid: Osmium tetroxide
What are the cause/s for the ff difficulty:
Failure to arrest early autolysis of cells
Cause: Failure to fix immediately
Insufficient fixative
What are the cause/s for the ff difficulty:
Removal of substances soluble in fixing agent
Loss or inactivation of enzyme needed for study
Cause: Wrong choice of fixative
What are the cause/s for the ff difficulty:
Presence of artefact pigments on tissue sections
Cause: Incomplete washing of fixative
What are the cause/s for the ff difficulty:
Tissues are soft and feather-like in consistency
Cause: Incomplete fixation
What are the cause/s for the ff difficulty:
Tissue blocks are brittle and hard
Cause: Prolonged fixation
What are the cause/s for the ff difficulty:
Shrinkage and swelling of cells and tissue structures
Cause: Over fixation
Works as physical agent to increase the movement of molecules and accelerates fixation
Used to accelerate staining, decalcification, IHC, and Electron Microscopy
Microwave technique
The process whereby calcium or lime salts are removed from tissues following fixation
It should be done AFTER fixation and BEFORE impregnation to ensure and facilitate the normal cutting of sections
Decalcification
Different methods to remove calcium in the tissues
Acid
Chelating Agents
Ion exchange
Electrophoresis
To form soluble calcium to remove the lime salts or calcium
Acid
Binds the calcium ion
Chelating agents
To ensure and facilitate the normal cutting of sections
To prevent obscuring the microanatomical detail of sections
Inadequate decalcification may result in poor cutting of hard tissues and damage to the knife edge during sectioning
Purpose of decalcification
Three main types of decalcifying agents
Strong mineral acids
Weaker organic acids
Chelating agents
Most widely used agents for routine decalcification because it is stable, easily available and relatively inexpensive
E.G - Chromic acid, Nitric acid, Hydrochloric acid, Formic acid, Trichloroacetic acid, Sulfurous acid, Citric acid
Acid decalcifying agents
Acid decalcifying agent that produces minimum distortion of tissues and GOOD nuclear staining
Prolonged decalcification may lead to tissue distortion
Rapid in Action
Decalcification time is 12-24 hours
Seriously damage tissue stainability
Most COMMON and FASTEST decalcifying agent
Easily removed by 70% alcohol
Imparts YELLOW color which will impair staining reaction
10% Aqueous nitric acid solution
An acid decalcifying agent that is recommended for URGEN BIOPSIES
Decalcification time is 1-3 days
Produces less tissue destruction than 10% aqueous nitric acid
Nuclear staining is RELATIVELY GOOD
The solution should be used inside the fume hood
Formol-Nitric acid
Decalcification of this decalcifying agent is 2-7 days
Relative SLOW decalcifying agent for DENSE BONES
RECOMMENDED for routine purposes
Decalcifies and SOFTENS tissue at the same time
Nuclear and cytoplasmic staining is GOOD
MACERATION is AVOIDED due to the presence of chromic acid and alcohol
CANNOT be determined by chemical test
Perenyi’s Fluid
Decalcification time is 12-24 hours
Most rapid decalcifying agent so far
POOR nuclear staining
Recommended for URGENT works
Prolong decalcification produces extreme tissue distortion
Complete decalcification CANNOT be determined by chemical means
Phologlucin-Nitric acid
Greater distortion of tissues
Inferior to slower reaction
Nitric acid in its role as a decalcifying agent
produces GOOD nuclear staining
1% SOLUTION in 70% alcohol - recommend for surface decalcification of the tissue block
Hydrochloric acid
Permits relatively good cytologic staining
DOES NOT require washing out before hydration
Moderately rapid decalcifying agent
Recommended for TEETH and SMALL PIECES OF BONE
Von Ebner’s Fluid
SAFER to handle than nitric acid or Hydrochloric acid
Moderate acting decalcifying agent
Recommended for ROUTINE DECALCIFICATION OF POSTMORTEM RESEARCH TISSUE
SG is 1.20
Decalcification time is 2-7 days
May be used as FIXATIVE and DECALCIFYING AGENT
Relatively SLOW, NOT for urgent works
Recommended for TEETH and SMALL PIECES
Produced better nuclear staining
Permits EXCELLENT NUCLEAR and CYTOPLASMIC STAINING
requires NEUTRALIZATION with 5% sodium sulfate and WASHING OUT
Formic acid
Decalcification time is 4-8 days
Very slow-acting, not recommended for urgent works
Suitable only for SMALL SPICULES of bones
Permits GOOD NUCLEAR STAINING
Weak decalcifying agent, NOT used for dense tissues
Trichloroacetic acid
Decalcification time is 3-14 days
SLOW, NOT for ROUTINE purpose
Requires neutralization with 5% sodium sulfate
Permits better nuclear staining than NITRIC ACID METHOD
Recommended for AUTOPSY MATERIAL, BONE MARROW, CARTILAGE, and TISSUES studies for RESEARCH PURPOSES
Formic acid- Sodium citrate solution
Very WEAK decalcifying agent
Suitable only for minute pieces of bone
Sulfurous acid
May be used as a FIXATIVE and decalcifying agent like formic acid
Used to decalcify MINUTE BONE SPICULES
Nuclear staining with Hematoxylin is INHIBITED
Forms PRECIPITATE AT THE BOTTOM, which requires FREQUENT CHANGES of solution
Degree of decalcification cannot be measured by routine chemical test
Chromic Acid / Flemming’s fluid
Decalcification time is 6 days
Too slow action for routine purposes
Permits EXCELLENT nuclear and cytoplasmic staining
Does NOT produce cell or Tissue distortion
Citric acid- Citrate buffer solution
Chelating agent
Commercial name is Versene/Sequestrene
Very slow decalcifying agent
Permits excellent staining result
Combines with CLCIUM IONS and OTHER SLATS to form weakly dissociated complexes and facilitates removal of CALCIUM SALTS
for small specimen: 1-3 weeks
For Dense cortical bone it will take 6-8 weeks
An excellent bone decalcifier for IHC, Enzyme staining and EM
Inactivates ALKALINE PHOSPHATASE activity, which can be restored by adding MAGNESIUM CHLORIDE
Ethylene Diamine TETRAACETIC ACID (EDTA)
Cellular detail is well-preserved
Daily washing of solution is eliminated
Permits EXCELLENT staining results
The degree of decalcification may be measured by physical or X-RAY method
AMMONIUM FORM of POLYSTYRENE RESIN that hastens decalcification by removing calcium ions from formic acid-containing decalcifying solutions
Ion exchange resin
A layer of ion exchange resin 1/2 thick is spread over the bottom of the container and the specimen is placed on top of it. Then the decalcifying agent is added usually 20-30X the volume of the tissue. Cellular detail is well-preserved
Daily washing of solution is eliminated
Permits EXCELLENT staining reuslts
Tissue may stay for 1-14 days
Ion exchange resin
A process whereby positively charged calcium ions are attracted to a negative electrode and subsequently removed from the decalcifying solution
Decalcification time is short due to the heat and electrolytic reaction
Same principle with chelating agent: This process utilizes electricity and is dependent upon a supply of direct current to remove the calcium deposits
This method is satisfactory for SMALL BONE fragments, processing only a LIMITED number of specimens at a time
GOOD cytologic and histologic details are NOT always preserved
Electrophoresis
Factors influencing rate of decalcification:
High ____ and greater amount of fluid will increase the speed of the process
Concentration
Factors influencing rate of decalcification:
37C impaired nuclear staining of Van Gieson’s stain for collagen fibers
55C tissue will undergo complete digestion within 24-48 hrs
Room temp range of 18-30C
Temperature
Factors influencing rate of decalcification:
Increase in size and consistency of tissue requires longer period
Ratio 20:1
Volume
Factors influencing rate of decalcification:
24-48 hours is the ideal time for decalcification
Dense bone tissue - 14 days longer
Time
Measuring the extend of decalcification:
Done by touching with fingers to determine the consistency of tissue
Bending, needling or by use of scalpel. If it bends easily that means decalcification is complete
Pricking CAUSES DAMAGE and DISTORT of tissue
INACCURATE way
Physical or mechanical test
Measuring the extend of decalcification:
Best method (most ideal, most sensitive, and most reliable) for determining complete decalcification
NOT recommended on tissue fixed in mercuric chloride (Radio opacity)
X-ray or Radiological method
Measuring the extend of decalcification:
Simple, Reliable and convenient method for routine purposes
Calcium oxalate test
Detects calcium in the decalcifying solution by precipitation of insoluble calcium hydroxide or calcium oxalates
Chemical Method (Calcium oxalate test)
The removal of acid from tissue or neutralized chemically by immersing the specimen either saturated with lithium carbonate solution or 5-10% aqueous sodium bicarbonate solution for several hours
or
Simply rinse the decalcified specimens with running tap water
Acid decalcified tissue for frozen sections- washed in water or stored in formol saline containing 15% sucrose or Phosphate buffered saline with 15-20% sucrose at 4c before freezing
Tissue decalcification in EDTA - Wash with water or stored overnight in formol saline or Phosphate buffered saline (Prevents the formation of crystalline precipitate)
Post Decalcification
Tissue softeners:
May act both as a decalcifying agent and tissue softener
Most commonly used tissue softeners
Perenyi’s Fluid
How to soften unduly hard tissues?
Selected portions are left in the fluid for 12-24 hours and dehydrated in the same manner
or
Submerge the cut surface of the block in the fluid for 1-2 hours before sectioning to facilitate easier cutting of tissue
Washing out and immersion of fixed tissues + 4% Aqueous phenol solution for 1-3 days
Considerable tissue softening
Easier block sectioning without producing marked delirious effects and tissue distortion
Softening of tissue
Process of removing intercellular and extracellular water from the tissue following fixation and prior to wax impregnation
Dehydration
For routine dehydration of tissues
Best dehydrating agent - fast acting
Not poisonous
Not expensive
Ethyl Alcohol
Toxic dehydrating agent
Primarily employed for blood and tissue films and for smear preparation
Methyl Alcohol
This dehydrating agent is utilized in PLANT and ANIMAL microtechnique
Slow dehydrating agent
Producing less shrinkage and hardening than ethyl alcohol
Recommend for tissue which do not require rapid processing
Butyl Alcohol
Factors to considered in Dehydration
Size and nature of tissue - 30%
Types of Fixative used
Temperature - 37C will hasten dehydration time
Ratio - not be less than 10X
Effects of alcohol concentration
85-95% - Liable to produce considerable shrinkage and hardening of tissues leading to distortion
Above 80% - make tissues hard brittle and difficult to cut
95% or absolute alcohol - tends to harden only the surface of the tissue while the deeper parts are not completely penetrated
Low concentration (Below 70%) - Macerate the tissue and can cause cell lysis
Routine dehydration process
70% alcohol - 6 hours
95% alcohol - 12 hours
100% alcohol - 2 hours
100% alcohol - 1 hour
100% alcohol - 1hour
Dehydration of tissue NOT more than 4mm thick
70% Ethanol -15 min
90% ethanol - 15 mins
100% Ethanol - 15 mins
100% ethanol - 15 mins
100% ethanol - 30 mins
100% Ethanol -45 mins
A cheap, rapid acting dehydrating agent
Dehydrates in 1/2 to 2 hours
More miscible with epoxy resins than alcohol
20:1 ratio (Fixative)
Clear, Colorless highly flammable and extremely volatile fluid
Rapid in action but penetrates tissues poorly and causes brittleness in tissues that are prolonged dehydrated
Most lipids are removed
Produces considerable tissue shrinkage
NOT RECOMMENDED for routine dehydration purposes
Acetone
Excellent dehydrating and CLEARING agent
Produces less tissue shrinkage
Tissues can be left for long periods of time w/o affecting the consistency or staining properties of the specimen
Tissue sections dehydration with this dehydrating agent tends to ribbon POORLY
EXPENSIVE and extremely dangerous (Vapor is toxic)
Formed peroxide may EXPLODE upon air exposure
Dioxane (Diethyl Dioxide)
1st - Pure dioxane solution - 1 hour
2nd - Pure dioxane solution - 1 hour
3rd - Pure dioxane solution - 2 hours
Then proceed to impregnation agad
1st Paraffin wax - 15 mins
2nd Paraffin wax - 45 mins
3rd Paraffin wax - 2 hours
Embed in mold and in cool
Uses pure dioxane and paraffin
Graupner’s method
Tissue is wrapped in gauze bag suspended in dioxane solution and a little anhydrous calcium oxide / Quicklime
Dehydration time - 3-24 hours
Tissue fixed in chromate fixative must be washed in running water
Weiseberger’s method
Dehydrates rapidly
The tissue may be transferred from water or normal saline directly to cellosolve and stored in it for months w/o producing hardening or distortion
Caution: Ethylene glycol ether is combustible at 110F to 120F and is toxic
Propylene based glycol ether should be used instead
Cellosive (Ethylene glycol monoethyl ether)
Removes water
Produces very little distortion and hardening of tissues
Soluble in alcohol, water, ether, benzene, chloroform acetone, and xylene
Used to dehydrate SECTIONS AND SMEARS
Triethyl Phosphate
BOTH DEHYDRATES and CLEARS tissues since it is miscible in water and paraffin
Can be used for demixing, clearing, and dehydration paraffin sections before and after staining
Causes less shrinkage and EASIER cutting of sections with FEWER artefacts
Does NOT dissolve aniline dyes
TOXIC if INGESTED or INHALED
Vapors causes nausea, dizziness, headache, and anesthesia
Tetrahydrofuran
Added to each 95T ethanol bats as part of dehydration process
Acts as a softener for hard tissues
4% Phenol
A glycerol and alcohol mixture
Tissue softener
Molliflex
Dehydrating agent for Electron microscopy
Accompanied by PROPYLENE OXIDE as a transition fluid
Along with propylene oxide, this solvent have some undesirable propery
Ethanol
A good substitute for propylene
Non-carcinogenic, less toxic, and not as flammable as propylene oxide
Excellent dehydrating agent
Acetonitrile
The transition step between dehydration and infiltration with the embedding medium
Clearing
Process whereby alcohol is removed from the tissue and replaced with a substance that will dissolve the wax with which the tissue is impregnated or the medium on which the tissue is to be mounted
De-alcoholization
Characteristic of a good clearing agent
Miscible with alcohol to promote rapid removal for the dehydrating agent
Should be miscible with and easily removed by melted paraffin wax
Should not produce excessive shrinkage, hardening or damage of tissue
Should not dissolve out aniline dyes
Should not evaporate quickly in a water bat
Should make tissue TRANSPARENT
Colorless clearing agent that is MOST COMMONLY used ⭐
Most rapid clearing agent, suitable for urgent biopsies
Clearing time is 1/2 hour to 1 hour
Makes tissue transparent
Does not extract aniline dye
Can be used for Celloidin sections because it does NOT dissolve celloidin
NOT suitable for nervous tissue and lymph nodes
CHEAP
Xylene
May be used as a SUBSTITUTE OR ALTERNATIVE ONLY for xylene or benzene ⭐
Clearing time is 1 - 2 hours
Acts fairly rapidly and is recommended for routine purpose
Tissues do not become excessively hard and brittle even if left for 24 hours
NOT carcinogenic
SLOWER than xylene and benzene
EXPENSIVE
Toluene
Preferred as a clearing agent in the embedding process of tissues because it penetrates and clears tissues rapidly
Clearing time is 15 to 60 minutes
Does not make tissues hard and brittle but it causes MINIMUM SHRINKAGE
Makes tissues transparent
FLAMMABLE
TISSHUE SHRINKAGE may be OBSERVED if left for a long time
Excessive exposure is TOXIC and CARCINOGENIC to human
May damage the bone marrow resulting in APLASTIC ANEMIA ⭐
Benzene
Slower in action than xylene but causes less brittleness
Suitable for LARGE TISSUE SPECIMENS. Thicker blocks can be processed
Clearing time 6-24 hours
Recommended for NERVOUS TISSUES, LYMPH NODES, and EMBRYOS ⭐
NOT FLAMMABLE
Relatively toxic to the LIVER after prolonged inhalataion
Wax impregnation after this clearing agent is relatively slow
DOES NOT make tissue transparent
Difficult to REMOVE from paraffin section because it is NOT very volatile
Complete clearing is difficult to evaluate
Chloroform
Advantages and Disadvantages are the same with chloroform ⭐
Produces CONSIDERABLE tissue HARDENING and DANGEROUS to inhale on prolonged exposure due to its highly toxic effects
Carbon tetrachloride
Used to clear both PARAFFIN and CELLOIDIN sections during embedding process
Recommended for CNS tissues, and Cytological studies
CCC = _____ , CNS, Cytological
Very penetrating agent
Becomes MILKY upon prolonged storage and should be filtered before use ⭐
VERY EXPENSIVE
Extremely slow clearing agent, not for routine purposes
Clearing time is 2-3 days
Celloidin clearing is 5-6 days
Cedarwood oil
Not normally utilized as a clearing agent
Recommended for clearing embryos, INSECTS, and VERY DELICATE SPECIMENS due to its ability to clear 70% ALCOHOL without excessive tissue shrinkage and hardening ⭐
Aniline oil
Causes MINIMUM shrinkage of tissues
Its quality is not guaranteed due to its tendency to become ADULTERATED ⭐
Wax impregnation after clearing with this clearing agent is SLOW and DIFFICULT
Tissues become BRITTLE, aniline dyes are REMOVED and celloidin is DISSOLVED
EXPENSIVE and UNSUITABLE for routine clearing purposes
Clove oil
Slow-acting clearing agents that can be used when DOUBLE EMBEDDING techniques are required ⭐
OIL OF WINTERGREEN
Methyl benzoate and Methyl Salicylate
For frozen section
No de-alcoholization is involved in this process
Glycerin and gum syrup
