Histopathologic techniques Flashcards
1
Q
Most crucial step for preservation; preserves cells and tissue constituents in a condition identical to that existing during life; prevents autolysis
A
Fixation
2
Q
Two important goals of fixation:
A
i. Preserve the morphological and chemical integrity of the cell
ii. Harden and preserve tissue for further handling
3
Q
Heat fixation; usually for microbiology
A
Heat Fixation
4
Q
Fixation via blood flow
A
Perfusion
5
Q
Most common fixation method in the laboratory; immersion of tissue in fixative solution
A
Immersion
6
Q
Fixative becomes part of the tissue by formation of cross-links or complexes; stabilizes tissue proteins; examples include formalin, Hg, osmium tetroxide
A
Additive Fixation
7
Q
Fixative does not become part of the tissue; stabilizes tissue by removing water; examples include alcoholic fixatives
A
Non-Additive Fixation
8
Q
General Effects of Fixatives
A
- 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
9
Q
pH range for effective fixation
A
pH 6-8
10
Q
Traditional temperature for fixation
A
Room Temperature
11
Q
Temperature for auto-tech fixation
A
40°C
12
Q
Temperature for electron microscopy and histochemical fixation
A
0-4°C
13
Q
Rapid fixation temperature
A
60°C
14
Q
Temperature for tissues with TB
A
100°C
15
Q
What is the recommended tissue thickness for electron microscopy (EM)?
A
1-2 mm^2 or 1 mm^3
16
Q
What is the recommended tissue thickness for light microscopy (LM)?
A
2x3 cm or 2 cm^2
17
Q
What is the maximum tissue thickness recommended, except for edematous lung tissue?
A
4mm/5mm
18
Q
What fixation method should be used for brain tissue and where should it be suspended?
A
Suspended in pole tie at the circle of Willi’s in 10% buffered formalin
19
Q
What should be done with large solid tissues (e.g., uterus) before fixation?
A
They should be opened or sliced thinly
20
Q
What is the typical osmolality for fixatives?
A
Slightly hypertonic solution around 400-450 mOsm
21
Q
What is the concentration of fixatives for formalin, glutaraldehyde, and immunoelectron microscopy?
A
10% Formalin, 3% Glutaraldehyde, 0.25% Glutaraldehyde for immunoelectron microscopy
22
Q
How long should primary fixation in buffered formalin last?
A
2-6 hours for initial fixation
23
Q
How long should EM fixation last before being placed in a holding buffer?
A
3 hours for EM fixation
24
Q
What is the penetration rate of formalin?
A
1 mm per hour
25
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
26
For museum preparations, what should the volume of fixative be?
At least 50x the tissue volume
27
What factors affect fixation duration?
Tissue structure: fibrous tissues need longer fixation, while small or loosely textured tissues need shorter fixation
28
How can fixation be hastened?
By using heat, agitation, vacuum, or microwave
29
What is the recommended temperature for mortuary refrigeration when autopsy materials cannot be fixed immediately?
4°C
30
What method is used when arterial embalming is required for autopsy materials?
Arterial embalming
31
Which tissues should be fixed before grossing during an autopsy?
Brain and eyes (use formol alcohol)
32
What is the problem with hollow organs like intestines and stomach during autopsy?
They tend to float
33
What is the solution for preventing hollow organs like intestines and stomach from floating?
Use moistened cotton
34
What is the issue with air-filled lungs during autopsy?
They tend to float
35
What can be done to prevent air-filled lungs from floating?
Wrap them with gauze (lungs from pneumonia will tend to sink)
36
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)
37
What are simple fixatives made up of?
1 component (aldehydes, metallic fixatives, heat)
38
What are compound fixatives made up of?
2 or more fixatives
39
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
40
What is the purpose of cytological fixatives?
Preservation of specific parts or elements of cells
41
What is the role of nuclear fixatives?
Preserves the nucleus and chromatic material with glacial acetic acid
42
What do cytoplasmic fixatives preserve?
Organelles and cytoplasm, with no glacial acetic acid to prevent swelling
43
What do histochemical fixatives preserve?
Chemical components of the cell
44
Which fixatives are classified as microanatomical?
10% formol saline, 10% neutral buffered formalin, Heidenhain's Susa, formol sublimate, Zenker's, Bouin's, Brasil's
45
Which fixatives are classified as nuclear fixatives?
Flemming's, Carnoy's, Bouin's, Newcomer's, Heidenhain's Susa
46
Which fixatives are classified as cytoplasmic fixatives?
Flemming's without HOAC, Kelly's, formalin with post chroming, Regaud's, Orth's
47
Which fixatives are classified as histochemical?
10% formol saline, absolute ethanol, acetone, Newcomer's
48
What is formalin?
Formaldehyde, a gas produced from the oxidation of methanol
49
What is the concentration of formalin for routine use?
10% formalin (1:9 dilution of stock solution)
50
How is formalin usually buffered?
Buffered with PO4 buffer to pH 7 to minimize artifacts (formalin pigments)
51
What is added to formalin to retard decomposition?
10% methanol
52
How long is the usual fixation time with formalin?
12-24 hours
53
What are the advantages of formalin?
Cheap, readily available, easy to prepare, relatively stable, compatible with many stains
54
What are the disadvantages of formalin?
Fumes irritating to the nose (rhinitis) and eyes (lacrimation); solution is irritating to skin (allergic dermatitis)
55
What is the remedy for skin irritation caused by formalin?
Use rubber gloves
56
What happens if 10% formalin is stored for prolonged periods?
Formation of paraformaldehyde (white precipitate)
57
What is the remedy for paraformaldehyde formation?
Add 10% methanol or filtration
58
What causes brown or black crystalline precipitates on blood-containing tissues (e.g., spleen) in formalin?
Action of formic acid with blood
59
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
60
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
61
What is the best fixative for iron-containing pigments and elastic fibers?
10% Neutral Buffered Formalin or PO4 Buffered Formalin (pH 7)
62
What is the best fixative for routine histopathology?
10% Neutral Buffered Formalin
63
What is the disadvantage of 10% Neutral Buffered Formalin?
It is inert to lipids and takes longer to prepare
64
What is Formol Sublimate composed of?
Contains mercuric chloride, excellent for stains like silver reticulin methods, no washing out, fixes lipids
65
What is the advantage of Formol Sublimate?
Fixes lipids and does not require washing out
66
What is the composition of Gendre's (alcoholic formalin)?
95% ethanol with picric acid and glacial acetic acid
67
What is Gendre's fixative used for?
Good for glycogen preservation, sputum, and microincineration technique
68
What does Gendre's fixative preserve?
Glycogen and sputum
69
What is glutaraldehyde's chemical composition?
Two formalin residues linked by three carbon chains
70
What are the concentrations of glutaraldehyde used for fixation?
2.5% for small tissue fragments, 4% for larger tissues
71
What is glutaraldehyde recommended for?
Enzyme histochemistry and electron microscopy
72
What is the advantage of glutaraldehyde over formalin?
Better preservation of cellular and fluid proteins, more pleasant and less irritating
73
How should glutaraldehyde specimens be stored?
Specimen vials should be refrigerated
74
What is paraformaldehyde?
Polymer of formalin in white powder form
75
What is paraformaldehyde used for?
Used for thin and ultrathin sections for plastic embedding (electron microscopy)
76
What is the most common metallic fixative?
Mercuric Chloride
77
What is the concentration of mercuric chloride used in fixatives?
5-7%
78
Is mercuric chloride used in compound fixatives?
Yes, it is included in compound fixatives
79
What does mercuric chloride produce during fixation?
Black granular deposits
80
Which fixative does not produce black granular deposits?
Heidenhain Susa
81
How can black granular deposits from mercuric chloride be removed?
By washing out or dezenkerization
82
What solution is added to remove black granular deposits?
Saturated iodine solution of 96% alcohol & 5% sodium thiosulfate
83
What is the effect of the iodine solution on tissue?
It penetrates and hardens tissue rapidly
84
What is the routine fixative of choice for preservation of cell detail in tissue photography?
Zenker's fluid with glacial acetic acid
85
What tissues is Zenker's fluid recommended for?
Small pieces of liver, spleen, connective tissue fibers, and nuclei
86
What type of staining is Zenker's fluid recommended for?
Trichrome staining
87
What does Zenker Formol (Helly's/Kelly's solution) contain?
Potassium dichromate and formalin
88
What is Zenker Formol excellent for fixing?
Pituitary gland, bone marrow, and blood-containing organs
89
What cytoplasmic feature does Zenker Formol preserve?
Cytoplasmic granules
90
What pigment does Zenker Formol produce, and how can it be removed?
Brown pigments; removed using picric acid or NaOH
91
What components are present in Heidenhain Susa, and what is it recommended for?
TCA, glacial acetic acid, formalin; recommended for skin tumor biopsies
92
What fixative is used for bone marrow biopsies and what does it contain?
B5 fixative; contains anhydrous sodium acetate
93
What are chromate fixatives, and what do they preserve?
1-2% aqueous solutions of strong oxidizing agents; preserve carbohydrates
94
What does potassium dichromate preserve, and at what pH?
Lipids and mitochondria; at pH 4.5-5.2
95
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
96
What is Orth’s Fluid used for?
Early degenerative processes, tissue necrosis, and demonstration of Rickettsia
97
What does Orth's Fluid preserve?
Myelin
98
What concentration is used for lead fixatives, and what do they preserve?
4% aqueous solution; preserves acid mucopolysaccharides and fixes mucin
99
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
100
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
101
What tissue component is excellently preserved by picric acid fixatives?
Glycogen
102
What is Bouin’s fixative recommended for?
Embryo and pituitary biopsies; soft and delicate structures; tissues to be stained by Masson's trichrome stain
103
What tissue is Bouin's NOT recommended for?
Kidney fixation
104
What is the composition and use of Brasil’s Alcoholic Picroformol Fixative?
Contains TCA; better and less messy than Bouin’s; fixes glycogen
105
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
106
What does glacial acetic acid fixatives precipitate, and what are they not suitable for?
Precipitates nucleoproteins and chromatin materials; not suitable for cytoplasmic fixation
107
At what temperature does glacial acetic acid solidify?
17 degrees Celsius
108
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
109
What tissue component is alcohol fixative excellent for preserving?
Glycogen
110
What components are preserved and dissolved by alcohol fixatives?
Preserves nuclear stains but dissolves fats and lipids
111
What is the concentration and use of methanol (wood alcohol)?
100%; used for dry and wet smears, blood smears, and bone marrow
112
What is a major disadvantage of methanol as a fixative?
Toxic when ingested; can cause blindness and death
113
What is the concentration and use of isopropyl alcohol (rubbing alcohol)?
95%; fixes touch preparations and Wright-Giemsa-stained samples
114
What is the concentration and specific use of ethanol (grain alcohol)?
70-100%; does not fix glycogen, useful for PCR
115
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
116
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
117
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
118
What are the disadvantages of osmium tetroxide?
Expensive, inhibits hematoxylin, and leads to corneal blindness
119
What is Flemming’s fixative used for?
Most common chrome-osmium-acetic acid fixative; excellent for nuclear structures; requires lesser amount of fixative
120
What is Flemming’s fixative without acetic acid used for?
Fixation of cytoplasmic structures, especially mitochondria"Function and concentration of alcohol fixatives?
121
Preserves and dissolves?
Preserves glycogen, nuclear stains; dissolves fats, lipids
122
Methanol use and issue?
Dry/wet smears, blood, marrow; toxic—blindness, death
123
Isopropyl alcohol use?
Touch prep, Wright-Giemsa; 95%
124
Ethanol use?
PCR; does not fix glycogen; 70-100%
125
Carnoy's fixative components?
Alcohol, acetic acid, chloroform; fixes/dehydrates, 1-3 hrs
126
Newcomer’s fixative?
For mucopolysaccharides, nuclear proteins; dual nuclear/biochemical role
127
Osmium tetroxide uses?
Fixative/stain for fats; EM secondary fixative; preserves mitochondria, Golgi
128
Osmium tetroxide issues?
Expensive, inhibits hematoxylin; corneal blindness
129
Flemming’s fixative?
Chrome-osmium-acetic; nuclear structures
130
Flemming’s w/o acetic acid?
For cytoplasmic structures, esp. mitochondria
131
Precipitates proteins; weak decalcifying agent; softens dense tissues
TCA Fixatives; Trichloroacetic acid
132
Ice cold (-5°C to 4°C); for diffusible enzymes (phosphatases, lipases); fixes brain tissues; Dx of rabies (Negri bodies)
Acetone Fixatives
133
Thermal coagulation of proteins; for frozen tissue sections; bacteriologic smears
Heat Fixation
134
Physical agent; accelerates fixation, staining, decalcification, EM, IHC; 45-55°C; penetrates 10-15 mm thick tissue
Microwave Technique
135
4% formalin, formol saline; acetone/formalin for cryostat sections
Fixatives for Enzyme Histochemistry
136
Fixatives for EM
Osmium tetroxide, Pallade's, Millonig's, glutaraldehyde, paraformaldehyde; optimum temp: 4°C
137
Fixative for Electron Histochemistry and Electron Immunocytochemistry
"Best: Karnovsky's paraformaldehyde; paraformaldehyde with acrolein and glutaraldehyde or formaldehyde"
138
UMFIX (Rapid Microwave Techniques)
"Mixture of methanol and PEG; cost-effective formalin alternative; recovers RNA
139
Factors Affecting Fixation
"Retarded by: size
140
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
141
Secondary fixation using 2.5-3% potassium dichromate for 1 day to act as a mordant; also known as post-mordanting
Post Chromatization
142
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
143
Mighel’s Solution — for unfixed tissues (renal, skin, oral mucosa biopsies); refrigerated; not a fixative
Transport Medium
