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:

A

i. Preserve the morphological and chemical integrity of the cell
ii. Harden and preserve tissue for further handling

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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

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
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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?

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?

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?

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?

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