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Histopathology > Histopathologic techniques > Flashcards

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

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2
Q

Two important goals of fixation: i. Preserve the morphological and chemical integrity of the cell; ii. Harden and preserve tissue for further handling

A

Goals of Fixation

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3
Q

Heat fixation; usually for microbiology

A

Heat Fixation

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4
Q

Fixation via blood flow

A

Perfusion

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5
Q

Most common fixation method in the laboratory; immersion of tissue in fixative solution

A

Immersion

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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

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7
Q

Fixative does not become part of the tissue; stabilizes tissue by removing water; examples include alcoholic fixatives

A

Non-Additive Fixation

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8
Q

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

A

General Effects of Fixatives

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9
Q

pH range for effective fixation

A

pH 6-8

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10
Q

Traditional temperature for fixation

A

Room Temperature

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11
Q

Temperature for auto-tech fixation

A

40°C

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12
Q

Temperature for electron microscopy and histochemical fixation

A

0-4°C

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13
Q

Rapid fixation temperature

A

60°C

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14
Q

Temperature for tissues with TB

A

100°C

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15
Q

What is the recommended tissue thickness for electron microscopy (EM)?

A

1-2 mm^2 or 1 mm^3

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16
Q

What is the recommended tissue thickness for light microscopy (LM)?

A

2x3 cm or 2 cm^2

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17
Q

What is the maximum tissue thickness recommended, except for edematous lung tissue?

A

4mm/5mm

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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

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19
Q

What should be done with large solid tissues (e.g., uterus) before fixation?

A

They should be opened or sliced thinly

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20
Q

What is the typical osmolality for fixatives?

A

Slightly hypertonic solution around 400-450 mOsm

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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

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22
Q

How long should primary fixation in buffered formalin last?

A

2-6 hours for initial fixation

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23
Q

How long should EM fixation last before being placed in a holding buffer?

A

3 hours for EM fixation

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24
Q

What is the penetration rate of formalin?

A

1 mm per hour

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25
Q

What is the recommended volume of fixative for tissue fixation?

A

10-20 times the tissue volume, or 5-10x for expensive fixatives like osmium oxide

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26
Q

For museum preparations, what should the volume of fixative be?

A

At least 50x the tissue volume

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27
Q

What factors affect fixation duration?

A

Tissue structure: fibrous tissues need longer fixation, while small or loosely textured tissues need shorter fixation

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28
Q

How can fixation be hastened?

A

By using heat, agitation, vacuum, or microwave

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29
Q

What is the recommended temperature for mortuary refrigeration when autopsy materials cannot be fixed immediately?

A

4°C

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30
Q

What method is used when arterial embalming is required for autopsy materials?

A

Arterial embalming

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31
Q

Which tissues should be fixed before grossing during an autopsy?

A

Brain and eyes (use formol alcohol)

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32
Q

What is the problem with hollow organs like intestines and stomach during autopsy?

A

They tend to float

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33
Q

What is the solution for preventing hollow organs like intestines and stomach from floating?

A

Use moistened cotton

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34
Q

What is the issue with air-filled lungs during autopsy?

A

They tend to float

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35
Q

What can be done to prevent air-filled lungs from floating?

A

Wrap them with gauze (lungs from pneumonia will tend to sink)

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36
Q

What should be done with hard tissues such as cervix, fibroids, hyperkeratotic skin, and nails?

A

Wash them in running water and immerse in tissue softeners (e.g., Perenyi’s or Rum’s)

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37
Q

What are simple fixatives made up of?

A

1 component (aldehydes, metallic fixatives, heat)

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38
Q

What are compound fixatives made up of?

A

2 or more fixatives

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39
Q

What is the function of microanatomical fixatives?

A

Permits the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of tissues

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40
Q

What is the purpose of cytological fixatives?

A

Preservation of specific parts or elements of cells

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41
Q

What is the role of nuclear fixatives?

A

Preserves the nucleus and chromatic material with glacial acetic acid

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42
Q

What do cytoplasmic fixatives preserve?

A

Organelles and cytoplasm, with no glacial acetic acid to prevent swelling

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43
Q

What do histochemical fixatives preserve?

A

Chemical components of the cell

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44
Q

Which fixatives are classified as microanatomical?

A

10% formol saline, 10% neutral buffered formalin, Heidenhain’s Susa, formol sublimate, Zenker’s, Bouin’s, Brasil’s

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45
Q

Which fixatives are classified as nuclear fixatives?

A

Flemming’s, Carnoy’s, Bouin’s, Newcomer’s, Heidenhain’s Susa

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46
Q

Which fixatives are classified as cytoplasmic fixatives?

A

Flemming’s without HOAC, Kelly’s, formalin with post chroming, Regaud’s, Orth’s

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47
Q

Which fixatives are classified as histochemical?

A

10% formol saline, absolute ethanol, acetone, Newcomer’s

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48
Q

What is formalin?

A

Formaldehyde, a gas produced from the oxidation of methanol

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49
Q

What is the concentration of formalin for routine use?

A

10% formalin (1:9 dilution of stock solution)

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50
Q

How is formalin usually buffered?

A

Buffered with PO4 buffer to pH 7 to minimize artifacts (formalin pigments)

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51
Q

What is added to formalin to retard decomposition?

A

10% methanol

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52
Q

How long is the usual fixation time with formalin?

A

12-24 hours

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53
Q

What are the advantages of formalin?

A

Cheap, readily available, easy to prepare, relatively stable, compatible with many stains

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54
Q

What are the disadvantages of formalin?

A

Fumes irritating to the nose (rhinitis) and eyes (lacrimation); solution is irritating to skin (allergic dermatitis)

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55
Q

What is the remedy for skin irritation caused by formalin?

A

Use rubber gloves

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56
Q

What happens if 10% formalin is stored for prolonged periods?

A

Formation of paraformaldehyde (white precipitate)

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57
Q

What is the remedy for paraformaldehyde formation?

A

Add 10% methanol or filtration

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58
Q

What causes brown or black crystalline precipitates on blood-containing tissues (e.g., spleen) in formalin?

A

Action of formic acid with blood

59
Q

What is the remedy for removing formalin pigments?

A

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
Q

What is 10% Formol Saline used for?

A

Microanatomical fixative; ideal for silver impregnation techniques, fixation of CNS tissues, and general postmortem tissues for histochemical examination

61
Q

What is the best fixative for iron-containing pigments and elastic fibers?

A

10% Neutral Buffered Formalin or PO4 Buffered Formalin (pH 7)

62
Q

What is the best fixative for routine histopathology?

A

10% Neutral Buffered Formalin

63
Q

What is the disadvantage of 10% Neutral Buffered Formalin?

A

It is inert to lipids and takes longer to prepare

64
Q

What is Formol Sublimate composed of?

A

Contains mercuric chloride, excellent for stains like silver reticulin methods, no washing out, fixes lipids

65
Q

What is the advantage of Formol Sublimate?

A

Fixes lipids and does not require washing out

66
Q

What is the composition of Gendre’s (alcoholic formalin)?

A

95% ethanol with picric acid and glacial acetic acid

67
Q

What is Gendre’s fixative used for?

A

Good for glycogen preservation, sputum, and microincineration technique

68
Q

What does Gendre’s fixative preserve?

A

Glycogen and sputum

69
Q

What is glutaraldehyde’s chemical composition?

A

Two formalin residues linked by three carbon chains

70
Q

What are the concentrations of glutaraldehyde used for fixation?

A

2.5% for small tissue fragments, 4% for larger tissues

71
Q

What is glutaraldehyde recommended for?

A

Enzyme histochemistry and electron microscopy

72
Q

What is the advantage of glutaraldehyde over formalin?

A

Better preservation of cellular and fluid proteins, more pleasant and less irritating

73
Q

How should glutaraldehyde specimens be stored?

A

Specimen vials should be refrigerated

74
Q

What is paraformaldehyde?

A

Polymer of formalin in white powder form

75
Q

What is paraformaldehyde used for?

A

Used for thin and ultrathin sections for plastic embedding (electron microscopy)

76
Q

What is the most common metallic fixative?

A

Mercuric Chloride

77
Q

What is the concentration of mercuric chloride used in fixatives?

A

5-7%

78
Q

Is mercuric chloride used in compound fixatives?

A

Yes, it is included in compound fixatives

79
Q

What does mercuric chloride produce during fixation?

A

Black granular deposits

80
Q

Which fixative does not produce black granular deposits?

A

Heidenhain Susa

81
Q

How can black granular deposits from mercuric chloride be removed?

A

By washing out or dezenkerization

82
Q

What solution is added to remove black granular deposits?

A

Saturated iodine solution of 96% alcohol & 5% sodium thiosulfate

83
Q

What is the effect of the iodine solution on tissue?

A

It penetrates and hardens tissue rapidly

84
Q

What is the routine fixative of choice for preservation of cell detail in tissue photography?

A

Zenker’s fluid with glacial acetic acid

85
Q

What tissues is Zenker’s fluid recommended for?

A

Small pieces of liver, spleen, connective tissue fibers, and nuclei

86
Q

What type of staining is Zenker’s fluid recommended for?

A

Trichrome staining

87
Q

What does Zenker Formol (Helly’s/Kelly’s solution) contain?

A

Potassium dichromate and formalin

88
Q

What is Zenker Formol excellent for fixing?

A

Pituitary gland, bone marrow, and blood-containing organs

89
Q

What cytoplasmic feature does Zenker Formol preserve?

A

Cytoplasmic granules

90
Q

What pigment does Zenker Formol produce, and how can it be removed?

A

Brown pigments; removed using picric acid or NaOH

91
Q

What components are present in Heidenhain Susa, and what is it recommended for?

A

TCA, glacial acetic acid, formalin; recommended for skin tumor biopsies

92
Q

What fixative is used for bone marrow biopsies and what does it contain?

A

B5 fixative; contains anhydrous sodium acetate

93
Q

What are chromate fixatives, and what do they preserve?

A

1-2% aqueous solutions of strong oxidizing agents; preserve carbohydrates

94
Q

What does potassium dichromate preserve, and at what pH?

A

Lipids and mitochondria; at pH 4.5-5.2

95
Q

What is another name for Regaud’s fixative, and what does it preserve?

A

Muller’s fixative; preserves chromatin, mitochondria, mitotic figures, Golgi bodies, RBCs, and colloid-containing tissues

96
Q

What is Orth’s Fluid used for?

A

Early degenerative processes, tissue necrosis, and demonstration of Rickettsia

97
Q

What does Orth’s Fluid preserve?

A

Myelin

98
Q

What concentration is used for lead fixatives, and what do they preserve?

A

4% aqueous solution; preserves acid mucopolysaccharides and fixes mucin

99
Q

What are picric acid fixatives commonly used for, and what is their chemical composition?

A

Used in strong or saturated solutions (aqueous, alcoholic); chemically 2,4,6-trinitrophenol

100
Q

What is the major disadvantage of picric acid fixatives, and how is it remedied?

A

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
Q

What tissue component is excellently preserved by picric acid fixatives?

A

Glycogen

102
Q

What is Bouin’s fixative recommended for?

A

Embryo and pituitary biopsies; soft and delicate structures; tissues to be stained by Masson’s trichrome stain

103
Q

What tissue is Bouin’s NOT recommended for?

A

Kidney fixation

104
Q

What is the composition and use of Brasil’s Alcoholic Picroformol Fixative?

A

Contains TCA; better and less messy than Bouin’s; fixes glycogen

105
Q

What is Hollande’s solution used for, and what are its advantages?

A

Used for GI tract samples and endocrine tissues; causes less lysis than Bouin’s and has decalcifying properties

106
Q

What does glacial acetic acid fixatives precipitate, and what are they not suitable for?

A

Precipitates nucleoproteins and chromatin materials; not suitable for cytoplasmic fixation

107
Q

At what temperature does glacial acetic acid solidify?

A

17 degrees Celsius

108
Q

What is the function of alcohol fixatives, and what is their required concentration?

A

Rapidly denature and precipitate proteins; act as fixatives and dehydrating agents; at least 70% concentration

109
Q

What tissue component is alcohol fixative excellent for preserving?

A

Glycogen

110
Q

What components are preserved and dissolved by alcohol fixatives?

A

Preserves nuclear stains but dissolves fats and lipids

111
Q

What is the concentration and use of methanol (wood alcohol)?

A

100%; used for dry and wet smears, blood smears, and bone marrow

112
Q

What is a major disadvantage of methanol as a fixative?

A

Toxic when ingested; can cause blindness and death

113
Q

What is the concentration and use of isopropyl alcohol (rubbing alcohol)?

A

95%; fixes touch preparations and Wright-Giemsa-stained samples

114
Q

What is the concentration and specific use of ethanol (grain alcohol)?

A

70-100%; does not fix glycogen, useful for PCR

115
Q

What are the components and special properties of Carnoy’s fixative?

A

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
Q

What is Newcomer’s fixative recommended for, and what dual role does it play?

A

Recommended for mucopolysaccharides and nuclear proteins; acts as both nuclear and biochemical fixative

117
Q

What are the uses of osmium tetroxide (osmic acid)?

A

Fixative and stain for uncojugated fats; secondary fixative for electron microscopy; preserves mitochondria and Golgi bodies

118
Q

What are the disadvantages of osmium tetroxide?

A

Expensive, inhibits hematoxylin, and leads to corneal blindness

119
Q

What is Flemming’s fixative used for?

A

Most common chrome-osmium-acetic acid fixative; excellent for nuclear structures; requires lesser amount of fixative

120
Q

What is Flemming’s fixative without acetic acid used for?

A

Fixation of cytoplasmic structures, especially mitochondria”Function and concentration of alcohol fixatives?

121
Q

Preserves and dissolves?

A

Preserves glycogen, nuclear stains; dissolves fats, lipids

122
Q

Methanol use and issue?

A

Dry/wet smears, blood, marrow; toxic—blindness, death

123
Q

Isopropyl alcohol use?

A

Touch prep, Wright-Giemsa; 95%

124
Q

Ethanol use?

A

PCR; does not fix glycogen; 70-100%

125
Q

Carnoy’s fixative components?

A

Alcohol, acetic acid, chloroform; fixes/dehydrates, 1-3 hrs

126
Q

Newcomer’s fixative?

A

For mucopolysaccharides, nuclear proteins; dual nuclear/biochemical role

127
Q

Osmium tetroxide uses?

A

Fixative/stain for fats; EM secondary fixative; preserves mitochondria, Golgi

128
Q

Osmium tetroxide issues?

A

Expensive, inhibits hematoxylin; corneal blindness

129
Q

Flemming’s fixative?

A

Chrome-osmium-acetic; nuclear structures

130
Q

Flemming’s w/o acetic acid?

A

For cytoplasmic structures, esp. mitochondria

131
Q

Precipitates proteins; weak decalcifying agent; softens dense tissues

A

TCA Fixatives; Trichloroacetic acid

132
Q

Ice cold (-5°C to 4°C); for diffusible enzymes (phosphatases, lipases); fixes brain tissues; Dx of rabies (Negri bodies)

A

Acetone Fixatives

133
Q

Thermal coagulation of proteins; for frozen tissue sections; bacteriologic smears

A

Heat Fixation

134
Q

Physical agent; accelerates fixation, staining, decalcification, EM, IHC; 45-55°C; penetrates 10-15 mm thick tissue

A

Microwave Technique

135
Q

4% formalin, formol saline; acetone/formalin for cryostat sections

A

Fixatives for Enzyme Histochemistry

136
Q

Fixatives for EM

A

Osmium tetroxide, Pallade’s, Millonig’s, glutaraldehyde, paraformaldehyde; optimum temp: 4°C

137
Q

Fixative for Electron Histochemistry and Electron Immunocytochemistry

A

“Best: Karnovsky’s paraformaldehyde; paraformaldehyde with acrolein and glutaraldehyde or formaldehyde”

138
Q

UMFIX (Rapid Microwave Techniques)

A

“Mixture of methanol and PEG; cost-effective formalin alternative; recovers RNA

139
Q

Factors Affecting Fixation

A

“Retarded by: size

140
Q

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

A

Secondary Fixation

141
Q

Secondary fixation using 2.5-3% potassium dichromate for 1 day to act as a mordant; also known as post-mordanting

A

Post Chromatization

142
Q

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

A

Washing Out

143
Q

Mighel’s Solution — for unfixed tissues (renal, skin, oral mucosa biopsies); refrigerated; not a fixative

A

Transport Medium

Histopathology (30 decks)

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