Mod 5: HISTOPATHOLOGIC TECHNIQUES Flashcards
This is prepared by cutting a thin slice from a small piece of fixed tissue
Section
What stain is used for viral inclusions?
H&E Staining
What stain is used for Negri Bodies (Rabies)?
Seller’s stain
A machine that fixes, dehydrates, clears, and infiltrates the tissue
Automatic processors
How many steps are in histopathologic techniques?
15 steps
These are samples for the purpose of cytological studies (FNA)?
Fine Needle Aspirations
Primary technique used for diagnosis of “Skin Specimens”
Punch biopsy
This biopsy is you took a portion of the cell and the surrounding tissue.
Used for bone marrow
Core Needle Biopsy
Histological Preparation made from blood, bone marrow, or any fluid such as pleural or ascitic fluid
Smears
This involves swabbing, brushing, lavage, washing, scraping, collection of secretions, shavings, and curettings (layman term of raspa)
Foliative cytology
Usually applied for small samples such as samples that are easily macerated
Whole mount
Whole Mount specimen thickness
No more than 0.2-0.5 mm in thickness
Majority of the preparation in histopathology. It is the cutting of the tissue
Sections
Sections specimen thickness
3-5 mm thick pieces
5 microns thick sections are cut on a microtome
Blocks of tissues taken for processing should be left in __ formalin at __C till processing
This would be fixed in __ hours
10% formalin
60C
2 hours
What is the size of the specimen piece to achieve better penetration of fixative
1cm
Tissues should be fixed in less than __ hour to avoid biochemical changes
less than “1 hour”
These are fixatives that are made up of only one component substance
Simple Fixatives
In Fresh Samples, what type of fixation is required for “electron microscopy”?
Glutaraldehyde fixation
It is the process of the spooning or scooping the tissue out of the endometrial or cervical canal
Curettage
The specimen is washed with __________ to achieve maximum penetration of fixative
normal saline
This is the ability to remove the adipose substrate from any specimen and it is important in the study of lymph nodes
Fat Clearance
2 groups of Chemical Fixatives, classified according to their mechanism of action
Crosslinking Fixative
Precipitating (or denaturing) Fixatives
These are fixatives that are made up of two or more fixatives which been added together to obtain the optimal combined effect
Compound Fixative
Formaldehyde/Formalin Derivatives
“Most widely used fixative for routine histology”
Buffer: pH = 7
Best fixative for “Iron pigments and elastic fibers”
10% Neutral Buffered Formalin
Examples of Simple Fixatives (AAAAMOP)
Aldehydes
Acetic Acid
Acetone
Alcohol
Metallic Fixatives
Osmium
Picric Acid
2 Types of Aldehydes (Cross-Linking Fixative)
Formaldehyde
Glutaraldehyde
Most commonly used fixative in histology, which fixes the tissue by forming cross-linkages in the proteins
Formaldehyde
Difference between “Formaldehyde” and “Formalin”
Formaldehyde: gas produced by oxidation of methyl alcohol
Formalin: made with formaldehyde but the percentage denotes a different formaldehyde concentration
Best fixative for iron pigments and elastic fibers
10% Neutral Buffered Formalin
Formaldehyde/Formalin Derivatives
Simple microanatomical fixative made up of “saturated formaldehyde diluted to 10% with sodium chloride”
Fixation Time: 12-24 hours
Preservation of “lipids”, especially phospholipids
Tissues tend to shrink during alcohol dehydration
10% Formal-Saline
Identify the Formaldehyde/Formalin Derivative based on their application
- Immunohistochemistry and FISH
- Fixation of CNS Tissue and post-mortem tissues
- Immunohistochemistry only
- Routine post-mortem tissues
- 10% Neutral Buffered Formalin
- 10% Formal-Saline
- Zinc-Formalin (unbuffered)
- Formol-Corrosive (Formol-Sublimate)
Formaldehyde/Formalin derivative best for the preservation of “lipids”, especially phospholipids
10% Formal-Saline
Formaldehyde/Formalin Derivatives
Was devised as alternatives to “mercuric chloride” formulations
Zinc Formalin (unbuffered)
Identify the Metallic Fixative
Fixation Time: 4-24 hours
“Brown Pigments” produced due to lysis of RBC (if prolonged fixation)
Zenker-Formol (Helly’s Solution)
Made up of 2 formaldehyde residues, linked by a three-carbon chain.
It has a larger molecule than formaldehyde, thus the rate of diffusion is “SLOW”
Glutaraldehyde
Formaldehyde/Formalin Derivatives
Fixation Time: 3-24 hours
Used in “silver reticulum methods”
Brightens cytoplasmic and metachromatic stains
No need for washing-out
Formol-Corrosive (Formol-Sublimate)
2 Types of Metallic Fixatives
Mercuric Chloride
Chromate Fixatives
This fixative leads to formation of “black granular” deposits in the tissues
Mercuric Chloride
Identify the Metallic Fixative
Fixation Time: 12-24 hours
May act as “mordant”
Stable, but will not be stable when added with acetic acid
Zenker’s Solution
A metallic fixative used for PTAH Staining, congested specimens, and Trichome Staining
PTAH = Phosphotungstic acid-haematoxylin
Zenker’s Solution
Most common metallic fixative
Frequently used in saturated aqueous solutions of 5-7%
Penetrates poorly and produces shrinkage of tissues, so it is usually combined with other fixative agents
Mercuric Chloride
A metallic fixative used for “bone marrow, extramedullary hematopoiesis, and intercalated discs of cardiac muscles”
Zenker-Formol (Helly’s Solution)
Zenker-Formol is also known as?
Helly’s Solution
Identify the Metallic Fixative
4% aqueous formaldehyde with 0.22 M mercuric chloride and 0.22 M acetic acid
Enhances nuclear detail
Lillie’s B-5 Fixative
A metallic fixative used for identifying “normal and abnormal cell types” in bone marrow specimens
Lillie’s B-5 Fixative
Identify the Metallic Fixative
Fixation Time: 3-12 hours
Excellent cytologic fixative
Penetrates and fixes tissues rapidly and evenly
Tissue should be transferred directly to a high-grade alcohol, to avoid undue swelling of tissues
Heidenhains Susa Solution
A metallic fixative used for “tumor biopsies” especially of the skin
Heidenhain’s Susa Solution
A chromate fixative used in 1-2% aqueous solution, usually as a constituent of a compound fixative
precipitates “all proteins” preserves “carbohydrates”
Strong oxidizing agent, hence, a strong reducing agent
Chromic Acid
A chromate fixative used in a 3% aqueous solution
preserves “lipids” and “mitochondria”
Potassium Dichromate
Regaud’s Fluid is also known as?
Muller’s Fluid
Identify the Chromate Fixative
Fixation Time: 12-48 hours
Hardens tissues better and more rapidly than Orth’s Fluid
Demonstration of:
- chromatin
- mitochondria
- mitotic figures
- Golgi bodies
- Rbc
- Colloid-containing tissues
Regaud’s Fluid (Muller’s)
Identify the Chromate Fixative
Fixation Time: 36-72 hours
Demonstrates “Rickettsiae and other bacteria”
Preserves “myelin” better than buffered formalin
Applied in the study of “early degenerative processes” and “tissue necrosis”
Orth’s Fluid
An explosive hazard in dry form and normally used in strong saturated aqueous solution
Dyes the tissues “yellow”
Picric Acid
Penetrates tissue well to react with “histones” and “basic proteins”
Preserves: glycogen
Extracts: lipids
Causes a loss of basophilia unless specimen is thoroughly washed following fixation
Picric Acid
3 Types of Acetic Acid
Glacial Acetic Acid
Lead Fixatives
Trichloroacetic Acid
Identify the Picrate
Fixation Time: 4-18 hours
Gives very good results with tissue that is subsequently stained with “trichome”
Preserves: Glycogen
Lyses: RBC
Not used: Frozen sections
Bouin’s Solution
A picrate applied in fixation of:
Embryos
Pituitary biopsies
GIT biopsies
Animal embryos
Endocrine gland tissues
Bouin’s Solution
Identify the Picrate
Fixation Time: 4-18 hours
Produces “less lysis” than Bouin’s Solution
Has “decalcifying properties”
For GIT and Endocrine Tissues
Hollande’s Solution
Identify the Picrate
Fixation Time: 4-18 hours
An alcoholic Bouin’s solution that appears to improve upon aging
Yellow-stain is useful when handling fragmentary biopsies
Not suitable for fixing kidney structures, lipid, and mucus
Produces RBC hemolysis
Preserve: Glycogen and Carbohydrates
Gendres Solution
Identify the Picrate
Better and less messy than Bouin’s Solution
Fixative for Glycogen
Brasil’s Alcoholic Picroformol Fixative
Identify the Acetic Acid:
Recommended for “acid mucopolysaccharides”
Fixes: Connective tissue mucin
Used in 4% aqueous solution of basic lead acetate
Lead Fixatives
Identify the Acetic Acid:
Fixes and precipitates nucleoproteins
Precipitates: Chromosomes and chromatin materials
Causes tissues to swell
Destroys: Mitochondria and Golgi elements
Corrosive to skin
Solidifies at 16C when undiluted
Glacial Acetic Acid
Identify the Acetic Acid:
Precipitates: proteins and nucleic acids
Marked swelling effect on tissues and used as a weak decalcifying agent
Suitable for small pieces of tissues or bones
Trichloroacetic Acid
Used to fix specimens at cold temperatures (0-4C = book) (-5 - 4C = ppt)
Not recommended as morphological fixative for tissue blocks
Acetone
Rapidly denatures and precipitates proteins by destroying hydrogen and other bonds
Uses: 70-100% concentration
Alcohol
Identify the Alcohol
Fixation Time: 12-18 hours at 3C
Produces better reaction in Feulgen stain than Carnoy’s fluid
Acts both as a “nuclear and histochemical fixative”
Fixes: Mucopolysaccharides and nuclear proteins
Newcomer’s Fluid
Recommended for the study of “water diffusible enzymes” especially “phosphatases and lipases”
Fixes: brain tissue for diagnosis of rabies
Solvent: For metallic salts to be used in freeze substitution techniques for tissue blocks
Dissolves: Fat
Preserves: Glycogen (poorly)
Acetone
Identify the Alcohol
Fixes and dehydrates at the same time
Slow penetration
Fixes: Dry and wet smears, blood smears, and BM tissues
Methyl Alcohol (100%)
Pale-yellow powder which dissolves in water (up to 6% at 20C) to form strong oxidizing solution
Causes “complete denaturation” of protein
1% OsO4 buffered at pH 7.3-7.5 with acetate-veronal buffer is recommended as an appropriate fixative for electron microscopy
Osmium
Identify the Alcohol
Used for fixing “touch preparations”
Used for special staining procedures such as “Wright-Giemsa”
Isopropyl Alcohol (95%)
Identify the Alcohol
Fixation Time: 18-24 hours
Preserves but does not fix “glycogen”
Preserves: glycogen, nucleoprotein, nucleic acids
Strong reducing agent
Ethyl Alcohol (70-100%)
Identify the Alcohol
Fixation Time: 1-3 hours
“Most rapid fixative”
May be used for urgent biopsies
Produces RBC hemolysis, dissolves lipids, and can produce excessive hardening and shrinkage
Fixes: brain tissues for diagnosis of rabies
Used for “curettings”
Carnoy’s Fixative
Identify the Alcohol
Fixation Time: 12-24 hours
Used during processing to complete fixation
Fixation or post-fixation of “large-fatty” specimens
Alcoholic Formalin
Identify the Alcohol
Fixation is faster
Used for rapid diagnosis because it fixes and dehydrates at the same time
Fixes: sputum
Frozen section room
Gendre’s Fixative
Identify the Alcohol
Fixation Time: 3-4 hours
Produces fair results after conventional processing if fixation time is kept very short
Preserves: Nucleic acids
Extracts: Lipids
For frozen sections and smears
Clarke’s Solution
Identify the Osmium Fixative
“Most common” chrome-osmium acetic acid fixative used
Fixation Time: 24-48 hours
“permanently fixes FAT”
Poor penetrating agent
Excellent fixative for “nuclear structures” (chromosomes)
Flemming’s Solution
Identify the Alcohol
Fixation Time: 1-6 hours
“Faster acting agent” than alcoholic formalin
Produce “formalin pigment”
Formol-Acetic Alcohol
Identify the Osmium Fixative
Made up of only “Chromic and Osmic Acid”
Fixation Time: 24-48 hours
“permanently fixes FAT”
Poor penetrating agent
Fixative for “cytoplasmic structures” particularly the “mitochondria”
Flemming’s Solution without Acetic Acid
Involves thermal coagulation of tissue proteins for rapid diagnosis, usually employed for frozen sections and precipitation of bacteriologic smears.
Heat Fixation
Type of Fixative that permit the general microscopic study of tissue structures without altering the structural pattern and normal intracellular relationship of the tissues in question
Microanatomical Fixatives
Microanatomical Fixatives (10-10HFZZBB)
● 10% formal saline
● 10% neutral buffered formalin
● Heidenhain ‘s Susa
● Formal sublimate (formal corrosive)
● Zenker ‘s solution
● Zenker-formal (Kelly ‘s solution)
● Bouin’s solution
● Brasil’s solution
A type of fixative that preserve specific parts and particular microscopic elements of the cell itself
Cytological Fixatives
A cytological fixative that preserve the “nuclear structures”
Contain “glacial acetic acid” due to its affinity for nuclear chromatin
pH = 4.6 or less
Nuclear Fixative
5 Nuclear Fixatives (FCBNH)
Flemming’s Fluid
Carnoy’s Fluid
Bouin’s Fluid
Newcomer’s Fluid
Heidenhain’s Susa
A cytological fixative that preserve “cytoplasmic structures”
Does NOT contain GAA = destroys the structures
pH = >4.6
Cytoplasmic Fixatives
5 Cytoplasmic Fixatives (FFORK)
Flemming’s without acetic acid
Formalin with “post-chroming”
Orth’s Fluid
Regaud’s Fluid (Muller’s)
Kelly’s Fluid
A cytological fixative that preserve the “chemical constituents” of cells and tissues
Histochemical Fixatives
4 Histochemical Fixatives (FANA)
Formal Saline 10%
Absolute Ethyl
Newcomer’s Fluid
Alcohol Acetone
To make special staining techniques possible, adding the secondary fixative makes it act as a?
Mordant
Form of secondary fixation whereby a primarily fixed tissue is placed in aqueous solution of 2.5-3% potassium dichromate for 24 hours to act as a mordant for better staining effects
Post- Chromatization
