Histopathologic techniques Flashcards
Most crucial step for preservation; preserves cells and tissue constituents in a condition identical to that existing during life; prevents autolysis
Fixation
Two important goals of fixation: i. Preserve the morphological and chemical integrity of the cell; ii. Harden and preserve tissue for further handling
Goals of Fixation
Heat fixation; usually for microbiology
Heat Fixation
Fixation via blood flow
Perfusion
Most common fixation method in the laboratory; immersion of tissue in fixative solution
Immersion
Fixative becomes part of the tissue by formation of cross-links or complexes; stabilizes tissue proteins; examples include formalin, Hg, osmium tetroxide
Additive Fixation
Fixative does not become part of the tissue; stabilizes tissue by removing water; examples include alcoholic fixatives
Non-Additive Fixation
Effects of fixatives: hardens soft and friable tissues for easy handling, makes cells resistant to damage and distortion, inhibits bacterial decomposition, increases optical differentiation of cells, acts as mordants or accentuators, reduces the risk of infection
General Effects of Fixatives
pH range for effective fixation
pH 6-8
Traditional temperature for fixation
Room Temperature
Temperature for auto-tech fixation
40°C
Temperature for electron microscopy and histochemical fixation
0-4°C
Rapid fixation temperature
60°C
Temperature for tissues with TB
100°C
What is the recommended tissue thickness for electron microscopy (EM)?
1-2 mm^2 or 1 mm^3
What is the recommended tissue thickness for light microscopy (LM)?
2x3 cm or 2 cm^2
What is the maximum tissue thickness recommended, except for edematous lung tissue?
4mm/5mm
What fixation method should be used for brain tissue and where should it be suspended?
Suspended in pole tie at the circle of Willi’s in 10% buffered formalin
What should be done with large solid tissues (e.g., uterus) before fixation?
They should be opened or sliced thinly
What is the typical osmolality for fixatives?
Slightly hypertonic solution around 400-450 mOsm
What is the concentration of fixatives for formalin, glutaraldehyde, and immunoelectron microscopy?
10% Formalin, 3% Glutaraldehyde, 0.25% Glutaraldehyde for immunoelectron microscopy
How long should primary fixation in buffered formalin last?
2-6 hours for initial fixation
How long should EM fixation last before being placed in a holding buffer?
3 hours for EM fixation
What is the penetration rate of formalin?
1 mm per hour
What is the recommended volume of fixative for tissue fixation?
10-20 times the tissue volume, or 5-10x for expensive fixatives like osmium oxide
For museum preparations, what should the volume of fixative be?
At least 50x the tissue volume
What factors affect fixation duration?
Tissue structure: fibrous tissues need longer fixation, while small or loosely textured tissues need shorter fixation
How can fixation be hastened?
By using heat, agitation, vacuum, or microwave
What is the recommended temperature for mortuary refrigeration when autopsy materials cannot be fixed immediately?
4°C
What method is used when arterial embalming is required for autopsy materials?
Arterial embalming
Which tissues should be fixed before grossing during an autopsy?
Brain and eyes (use formol alcohol)
What is the problem with hollow organs like intestines and stomach during autopsy?
They tend to float
What is the solution for preventing hollow organs like intestines and stomach from floating?
Use moistened cotton
What is the issue with air-filled lungs during autopsy?
They tend to float
What can be done to prevent air-filled lungs from floating?
Wrap them with gauze (lungs from pneumonia will tend to sink)
What should be done with hard tissues such as cervix, fibroids, hyperkeratotic skin, and nails?
Wash them in running water and immerse in tissue softeners (e.g., Perenyi’s or Rum’s)
What are simple fixatives made up of?
1 component (aldehydes, metallic fixatives, heat)
What are compound fixatives made up of?
2 or more fixatives
What is the function of microanatomical fixatives?
Permits the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of tissues
What is the purpose of cytological fixatives?
Preservation of specific parts or elements of cells
What is the role of nuclear fixatives?
Preserves the nucleus and chromatic material with glacial acetic acid
What do cytoplasmic fixatives preserve?
Organelles and cytoplasm, with no glacial acetic acid to prevent swelling
What do histochemical fixatives preserve?
Chemical components of the cell
Which fixatives are classified as microanatomical?
10% formol saline, 10% neutral buffered formalin, Heidenhain’s Susa, formol sublimate, Zenker’s, Bouin’s, Brasil’s
Which fixatives are classified as nuclear fixatives?
Flemming’s, Carnoy’s, Bouin’s, Newcomer’s, Heidenhain’s Susa
Which fixatives are classified as cytoplasmic fixatives?
Flemming’s without HOAC, Kelly’s, formalin with post chroming, Regaud’s, Orth’s
Which fixatives are classified as histochemical?
10% formol saline, absolute ethanol, acetone, Newcomer’s
What is formalin?
Formaldehyde, a gas produced from the oxidation of methanol
What is the concentration of formalin for routine use?
10% formalin (1:9 dilution of stock solution)
How is formalin usually buffered?
Buffered with PO4 buffer to pH 7 to minimize artifacts (formalin pigments)
What is added to formalin to retard decomposition?
10% methanol
How long is the usual fixation time with formalin?
12-24 hours
What are the advantages of formalin?
Cheap, readily available, easy to prepare, relatively stable, compatible with many stains
What are the disadvantages of formalin?
Fumes irritating to the nose (rhinitis) and eyes (lacrimation); solution is irritating to skin (allergic dermatitis)
What is the remedy for skin irritation caused by formalin?
Use rubber gloves
What happens if 10% formalin is stored for prolonged periods?
Formation of paraformaldehyde (white precipitate)
What is the remedy for paraformaldehyde formation?
Add 10% methanol or filtration
What causes brown or black crystalline precipitates on blood-containing tissues (e.g., spleen) in formalin?
Action of formic acid with blood
What is the remedy for removing formalin pigments?
Kardasewitsch’s Method (70% ethanol & 28% ammonia water), Lilli’s Method (Hydrogen Peroxide & 28% ammonia water), Picric Acid Method (Saturated Alcoholic Picric Acid), 1% KOH in 80% Alcohol
What is 10% Formol Saline used for?
Microanatomical fixative; ideal for silver impregnation techniques, fixation of CNS tissues, and general postmortem tissues for histochemical examination
What is the best fixative for iron-containing pigments and elastic fibers?
10% Neutral Buffered Formalin or PO4 Buffered Formalin (pH 7)
What is the best fixative for routine histopathology?
10% Neutral Buffered Formalin
What is the disadvantage of 10% Neutral Buffered Formalin?
It is inert to lipids and takes longer to prepare
What is Formol Sublimate composed of?
Contains mercuric chloride, excellent for stains like silver reticulin methods, no washing out, fixes lipids
What is the advantage of Formol Sublimate?
Fixes lipids and does not require washing out
What is the composition of Gendre’s (alcoholic formalin)?
95% ethanol with picric acid and glacial acetic acid
What is Gendre’s fixative used for?
Good for glycogen preservation, sputum, and microincineration technique
What does Gendre’s fixative preserve?
Glycogen and sputum
What is glutaraldehyde’s chemical composition?
Two formalin residues linked by three carbon chains
What are the concentrations of glutaraldehyde used for fixation?
2.5% for small tissue fragments, 4% for larger tissues
What is glutaraldehyde recommended for?
Enzyme histochemistry and electron microscopy
What is the advantage of glutaraldehyde over formalin?
Better preservation of cellular and fluid proteins, more pleasant and less irritating
How should glutaraldehyde specimens be stored?
Specimen vials should be refrigerated
What is paraformaldehyde?
Polymer of formalin in white powder form
What is paraformaldehyde used for?
Used for thin and ultrathin sections for plastic embedding (electron microscopy)
What is the most common metallic fixative?
Mercuric Chloride
What is the concentration of mercuric chloride used in fixatives?
5-7%
Is mercuric chloride used in compound fixatives?
Yes, it is included in compound fixatives
What does mercuric chloride produce during fixation?
Black granular deposits
Which fixative does not produce black granular deposits?
Heidenhain Susa
How can black granular deposits from mercuric chloride be removed?
By washing out or dezenkerization
What solution is added to remove black granular deposits?
Saturated iodine solution of 96% alcohol & 5% sodium thiosulfate
What is the effect of the iodine solution on tissue?
It penetrates and hardens tissue rapidly
What is the routine fixative of choice for preservation of cell detail in tissue photography?
Zenker’s fluid with glacial acetic acid
What tissues is Zenker’s fluid recommended for?
Small pieces of liver, spleen, connective tissue fibers, and nuclei
What type of staining is Zenker’s fluid recommended for?
Trichrome staining
What does Zenker Formol (Helly’s/Kelly’s solution) contain?
Potassium dichromate and formalin
What is Zenker Formol excellent for fixing?
Pituitary gland, bone marrow, and blood-containing organs
What cytoplasmic feature does Zenker Formol preserve?
Cytoplasmic granules
What pigment does Zenker Formol produce, and how can it be removed?
Brown pigments; removed using picric acid or NaOH
What components are present in Heidenhain Susa, and what is it recommended for?
TCA, glacial acetic acid, formalin; recommended for skin tumor biopsies
What fixative is used for bone marrow biopsies and what does it contain?
B5 fixative; contains anhydrous sodium acetate
What are chromate fixatives, and what do they preserve?
1-2% aqueous solutions of strong oxidizing agents; preserve carbohydrates
What does potassium dichromate preserve, and at what pH?
Lipids and mitochondria; at pH 4.5-5.2
What is another name for Regaud’s fixative, and what does it preserve?
Muller’s fixative; preserves chromatin, mitochondria, mitotic figures, Golgi bodies, RBCs, and colloid-containing tissues
What is Orth’s Fluid used for?
Early degenerative processes, tissue necrosis, and demonstration of Rickettsia
What does Orth’s Fluid preserve?
Myelin
What concentration is used for lead fixatives, and what do they preserve?
4% aqueous solution; preserves acid mucopolysaccharides and fixes mucin
What are picric acid fixatives commonly used for, and what is their chemical composition?
Used in strong or saturated solutions (aqueous, alcoholic); chemically 2,4,6-trinitrophenol
What is the major disadvantage of picric acid fixatives, and how is it remedied?
Yellow staining of tissue; remedied with a saturated solution of lithium carbonate in 70% alcohol, followed by washing with water, 70% ethanol, 5% sodium thiosulfate, and water
What tissue component is excellently preserved by picric acid fixatives?
Glycogen
What is Bouin’s fixative recommended for?
Embryo and pituitary biopsies; soft and delicate structures; tissues to be stained by Masson’s trichrome stain
What tissue is Bouin’s NOT recommended for?
Kidney fixation
What is the composition and use of Brasil’s Alcoholic Picroformol Fixative?
Contains TCA; better and less messy than Bouin’s; fixes glycogen
What is Hollande’s solution used for, and what are its advantages?
Used for GI tract samples and endocrine tissues; causes less lysis than Bouin’s and has decalcifying properties
What does glacial acetic acid fixatives precipitate, and what are they not suitable for?
Precipitates nucleoproteins and chromatin materials; not suitable for cytoplasmic fixation
At what temperature does glacial acetic acid solidify?
17 degrees Celsius
What is the function of alcohol fixatives, and what is their required concentration?
Rapidly denature and precipitate proteins; act as fixatives and dehydrating agents; at least 70% concentration
What tissue component is alcohol fixative excellent for preserving?
Glycogen
What components are preserved and dissolved by alcohol fixatives?
Preserves nuclear stains but dissolves fats and lipids
What is the concentration and use of methanol (wood alcohol)?
100%; used for dry and wet smears, blood smears, and bone marrow
What is a major disadvantage of methanol as a fixative?
Toxic when ingested; can cause blindness and death
What is the concentration and use of isopropyl alcohol (rubbing alcohol)?
95%; fixes touch preparations and Wright-Giemsa-stained samples
What is the concentration and specific use of ethanol (grain alcohol)?
70-100%; does not fix glycogen, useful for PCR
What are the components and special properties of Carnoy’s fixative?
Contains absolute alcohol, glacial acetic acid, and chloroform; fixes and dehydrates simultaneously; fixes Nissl granules and cytoplasmic granules; most rapid fixative with fixation time of 1-3 hours
What is Newcomer’s fixative recommended for, and what dual role does it play?
Recommended for mucopolysaccharides and nuclear proteins; acts as both nuclear and biochemical fixative
What are the uses of osmium tetroxide (osmic acid)?
Fixative and stain for uncojugated fats; secondary fixative for electron microscopy; preserves mitochondria and Golgi bodies
What are the disadvantages of osmium tetroxide?
Expensive, inhibits hematoxylin, and leads to corneal blindness
What is Flemming’s fixative used for?
Most common chrome-osmium-acetic acid fixative; excellent for nuclear structures; requires lesser amount of fixative
What is Flemming’s fixative without acetic acid used for?
Fixation of cytoplasmic structures, especially mitochondria”Function and concentration of alcohol fixatives?
Preserves and dissolves?
Preserves glycogen, nuclear stains; dissolves fats, lipids
Methanol use and issue?
Dry/wet smears, blood, marrow; toxic—blindness, death
Isopropyl alcohol use?
Touch prep, Wright-Giemsa; 95%
Ethanol use?
PCR; does not fix glycogen; 70-100%
Carnoy’s fixative components?
Alcohol, acetic acid, chloroform; fixes/dehydrates, 1-3 hrs
Newcomer’s fixative?
For mucopolysaccharides, nuclear proteins; dual nuclear/biochemical role
Osmium tetroxide uses?
Fixative/stain for fats; EM secondary fixative; preserves mitochondria, Golgi
Osmium tetroxide issues?
Expensive, inhibits hematoxylin; corneal blindness
Flemming’s fixative?
Chrome-osmium-acetic; nuclear structures
Flemming’s w/o acetic acid?
For cytoplasmic structures, esp. mitochondria
Precipitates proteins; weak decalcifying agent; softens dense tissues
TCA Fixatives; Trichloroacetic acid
Ice cold (-5°C to 4°C); for diffusible enzymes (phosphatases, lipases); fixes brain tissues; Dx of rabies (Negri bodies)
Acetone Fixatives
Thermal coagulation of proteins; for frozen tissue sections; bacteriologic smears
Heat Fixation
Physical agent; accelerates fixation, staining, decalcification, EM, IHC; 45-55°C; penetrates 10-15 mm thick tissue
Microwave Technique
4% formalin, formol saline; acetone/formalin for cryostat sections
Fixatives for Enzyme Histochemistry
Fixatives for EM
Osmium tetroxide, Pallade’s, Millonig’s, glutaraldehyde, paraformaldehyde; optimum temp: 4°C
Fixative for Electron Histochemistry and Electron Immunocytochemistry
“Best: Karnovsky’s paraformaldehyde; paraformaldehyde with acrolein and glutaraldehyde or formaldehyde”
UMFIX (Rapid Microwave Techniques)
“Mixture of methanol and PEG; cost-effective formalin alternative; recovers RNA
Factors Affecting Fixation
“Retarded by: size
Placing an already fixed tissue into another fixative; facilitates and improves the demonstration of substances, special staining, and ensures further and complete hardening and preservation
Secondary Fixation
Secondary fixation using 2.5-3% potassium dichromate for 1 day to act as a mordant; also known as post-mordanting
Post Chromatization
Removal of excess fixative in order to improve staining and remove artefacts; tap water for chromates, formalin, osmic acid; 50-70% alcohol removes excess picric acid; alcoholic iodine removes excess mercury fixation
Washing Out
Mighel’s Solution — for unfixed tissues (renal, skin, oral mucosa biopsies); refrigerated; not a fixative
Transport Medium
