HPCT WEEK 1 Flashcards by Potato Patata

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

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Most diverse and abundant tissue
Main classes are:
____ tissue proper
Blood-Fluid ____ tissue (blood)
Cartilage and Bone tissue - Supporting ____ tissues

Connective tissues

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Components of connective tissue

Cells
Matrix (Protein fibers, Ground substances)

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Common embryonic origin

Mesenchyme

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Cells found in connective tissue proper

Fibroblasts, Macrophages, Lymphocytes, Adipocytes, Mast cells, Stem cells

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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)

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Ground susbtances:

Along with fibers, fills the extracellular space

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The origin of a disease

Refers to why a disease arises

Etiology

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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

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Refers to the structural alterations in cells or tissue

Either Gross morphologic changes (Anatomic/Macroscopic)
or
Microscopic Changes

Morphologic changes

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Referring to the clinical features (acute or malignant), course and prognosis of the disease

Functional derangements and Clinical Significance

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Indication of a disease perceived BY THE PATIENT

Symptoms

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Objective findings noticed BY THE DOCTOR on examination of the patient

Signs

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Start of the disease

Onset

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PREDICTION of the outcome of the disease

Prognosis

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Outcome of the disease

Fate

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New disease conditions that may occur during or after the usual course of the original disease

Complications

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Undergoes replication all throughout life

Labile cells

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Does not undergo replication unless injury

Stable cells

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Does not undergo divisions following maturation

Permanent cells

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Incomplete or defective development of tissue/organs
Affected organs shows no resemblance to normal mature form

Aplasia

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Complete NON-APPEARANCE of organ

Agenesia

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Failure of organ to form an opening

Atresia

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Failure of organ to reach normal mature adult size

Hypoplasia

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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

1. Abnormal metabolism - Inadequate removal of a normal substance secondary to defects 2. Defect in protein folding, transport - Accumulation of an abnormal endogenous substance 3. Failure to degrade a metabolite 4. 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)

1. Recognition of the injurious agent 2. Recruitment of leukocytes 3. Removal of the agent 4. Regulation of the response 5. 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

1. Phase inflammation 2. Phase of demolition 3. 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

1. Aldehydes (Formaldehyde, Glutaraldehyde) 2. Metallic Fixatives Mercuric chloride Chromate fixatives (Potassium dichromate, Chromic acid) Lead fixatives (Acetone, Alcohol, Picric acid, Acetic Acid, Osmium tetroxide (Osmic acid) ) 3. 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

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