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MTAP - HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES MODULE > LESSON 2: FIXATION > Flashcards

LESSON 2: FIXATION Flashcards

1
Q

 first and most critical step in tissue processing

A

FIXATION

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2
Q

 fixing or preserving fresh tissue for examination

A

FIXATION

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3
Q

 should be done immediately to preserve cellular and tissue morphology

A

FIXATION

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4
Q

Two Purposes of Fixation

A
  1. Preservation: primary purpose
  2. Protection: secondary purpose
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5
Q

 capable of forming cross-links between proteins

A

Fixative agents

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6
Q

 stabilizes tissue components making them insoluble to lysosomal enzymes

A

Fixative agents

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7
Q

Types of Fixative:

A
  1. Additive: becomes part of the cross-link itself
  2. Non-Additive: facilitates the removal of water in order for cross-links to form
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8
Q

: becomes part of the cross-link itself

A
  1. Additive
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9
Q

: facilitates the removal of water in order for cross-links to form

A
  1. Non-Additive
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10
Q

 capable of inactivating lysosomes

A

Fixative agents

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11
Q

RETARDED BY:

A

 Increase in size and thickness
 Presence of mucus
 Presence of fats
 Presence of blood
 Decrease in temperature

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12
Q

ENHANCED BY:

A

 Decrease in size and thickness
 Presence of agitation
 Presence of heat

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13
Q

 pH:

A

6 to 8

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14
Q

 Routine Manual:

A

Room Temp (20 to 22oC)

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15
Q

 Routine Automated:

A

40 oC

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16
Q

 Electron Microscopy:

A

0 to 4oC

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17
Q

 Formalin at 60 oC:

A

very urgent biopsies

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18
Q

 Light Microscopy:

A

2cm2 by 0.4cm thick

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19
Q

 Electron Microscopy:

A

1 to 2 mm2

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20
Q

 Lung Edema:

A

1 to 2 cm thick

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21
Q

 Light microscopy:

A

slightly hypertonic (400-450 mOsm)

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22
Q

 Electron Microscopy:

A

more or less isotonic (340 mOsm)

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23
Q

 Formaldehyde:
 Glutaraldehyde:

A

10%

3%

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24
Q

 Routine:

A

10 to 25 times the volume of the specimen

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25
Q

 Museum:

A

50 to 100 times the volume of the specimen

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26
Q

 Cover with several layers of gauze

A
  1. Air-filled Lungs
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27
Q

 Dilate with cotton soaked in fixative

A
  1. Hollow Organs
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28
Q

 Completely open specimen

A
  1. Hollow Organs
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29
Q

 Fix first before sampling

A
  1. Brain
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30
Q

 Suspended by a cord tied under the Circle of Willis

A
  1. Brain
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31
Q

 Intravascular perfusion using Ringer’s lactate

A
  1. Brain
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32
Q

 Fixed whole

A
  1. Eyes
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33
Q

 Inject formol-alcohol

A
  1. Eyes
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34
Q

 Lendrum’s Method: washed out with running water overnight and immersed in 4% aq. phenol solution for 1-3 days

A
  1. Hard Tissues
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35
Q

 Stretched with sutures on each end

A
  1. Muscles
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36
Q

 Laid flat in a moist filter paper

A
  1. Muscles
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37
Q

CHARACTERISTICS OF A GOOD FIXATIVE

A
  1. Cheap & economical
  2. Stable and safe to handle
  3. Fast acting, permits rapid an even penetration
  4. Inhibits bacterial decomposition & autolysis
  5. Must harden tissues
  6. At least isotonic
  7. Must render tissues insensitive to subsequent processing
  8. Must be compatible with many staining procedures
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38
Q

: made up of only one component substance

A
  1. Simple Fixatives
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39
Q

: made up of two or more fixatives

A
  1. Compound Fixatives
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40
Q

:  permit general microscopic study of tissue structures and normal intercellular relationship of tissues

A
  1. Microanatomical Fixatives
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41
Q

:  preserve specific cellular components

A
  1. Cytological Fixatives
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42
Q

:  preserve nuclear structures

A

A. Nuclear Fixatives

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43
Q

 contain glacial HAc

A

A. Nuclear Fixatives

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44
Q

 high affinity to nuclear chromatin

A

glacial HAc

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45
Q

 destroys mitochondria and Golgi bodies

A

glacial HAc

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46
Q

 pH 4.6 or less

A

A. Nuclear Fixatives

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47
Q

:  preserve cytoplasmic structures

A

Cytoplasmic Fixatives

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48
Q

 do not contain glacial HAc

A

Cytoplasmic Fixatives

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49
Q

 pH of more than 4.6

A

Cytoplasmic Fixatives

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50
Q

:  preserve chemical constituents

A

Histochemical Fixatives

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51
Q

a. Lipids:

A

mercuric chloride or potassium dichromate

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52
Q

b. Phospholipids:

A

Baker’s formol-calcium

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53
Q

c. Cholesterol:

A

digitonin

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54
Q

d. Carbohydrates:

A

alcoholic fixatives

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55
Q

e. Glycogen:

A

Rossman’s fluid or cold absolute alcohol

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56
Q

f. Proteins:

A

neutral buffered formol saline or formaldehyde vapor

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57
Q

g. Electron microscopy:

A

double fixation

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58
Q
  1. Electron Cytochemistry:
A

Karnovsky’s paraformaldehydeglutaraldehyde solution

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59
Q
  1. Acrolein:
A

mixture of glutaraldehyde or fomaldehyde

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60
Q

 rapid penetration, preserves morphology and enzyme activity at low concentrations

A

Acrolein

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61
Q

 immersion fixation of surgical biopsies

A

Acrolein

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62
Q

:  for routine paraffin sections, electron microscopy, histochemistry and enzyme studies

A

ALDEHYDE FIXATIVES

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63
Q

 gas produced by the oxidation of methyl alcohol

A

Formaldehyde (Formalin)

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64
Q

 buffered at ph 7 to 8

A

Formaldehyde (Formalin)

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65
Q

 hypoxia in tissues leads to acidity which favors the formation of Formalin heme pigments (black, polarizable deposits)

A

Formaldehyde (Formalin)

66
Q

 Formaldehyde (Formalin) Pure Stock:

A

40% formalin

67
Q

 Formaldehyde (Formalin) Dilution:

A

1:10 (10% solution); 1:20 (5% solution)

68
Q

 Formaldehyde (Formalin) Concentration for fixation:

A

10% formalin

69
Q

: white precipitate due to prolonged storage

A

 Paraformaldehyde

70
Q

 may be removed by filtration or addition of 10% methanol

A

Paraformaldehyde

71
Q

 central nervous tissues and general post-mortem tissues for histochemical examination

A

10% Formol-Saline

72
Q

 ideal with most stains including silver impregnation

A

10% Formol-Saline

73
Q

 duration of fixation: more than 24 hours (slow fixative)

A

10% Formol-Saline

74
Q

 preservation and storage of surgical, post-mortem and research specimens

A

10% Neutral Buffered Formalin

75
Q

 best fixative for frozen sections

A

10% Neutral Buffered Formalin

76
Q

 best fixative for iron pigments and elastic fibers

A

10% Neutral Buffered Formalin

77
Q

 routine post-mortem tissues

A

Formol-Corrosive

78
Q

 for lipids, especially neutral fats and phospholipids

A

Formol-Corrosive

79
Q

 no need for washing-out

A

Formol-Corrosive

80
Q

 used to fix sputum

A

Alcoholic Formalin

81
Q

 for the demonstration of immunoperoxidase activity

A

Alcoholic Formalin

82
Q

 made up of two formaldehyde residues linked by three carbon chains

A

Glutaraldehyde

83
Q

 used in conjunction with osmium tetroxide

A

Glutaraldehyde

84
Q

 Glutaraldehyde Fixation time:

A

½ hour to 2 hours

85
Q

 most common metallic fixative

A

Mercuric Chloride

86
Q

 tissue photography

A

Mercuric Chloride

87
Q

 tissues contain black precipitates of mercury (except Susa)

A

Mercuric Chloride

88
Q

 fixing small pieces of liver, spleen, connective tissues fibers and nuclei

A

Zenker’s Fluid

89
Q

: removal of mercuric deposits in tissues

A

De-zenkerization

90
Q

 pituitary gland, bone marrow and blood containing organs (spleen and liver)

A

B. Zenker-formol

91
Q

 for tumor biopsies

A

C. Heidenhain’s Susa Solution

92
Q

 for bone marrow biopsies

A

D. B-5 Fixative

93
Q

 preserves carbohydrates

A

A. Chromic Acid

94
Q

 preserves lipids and mitochondria

A

B. Potassium Dichromate

95
Q

 demonstration of chromatin, mitochondria, mitotic figures, Golgi bodies, RBC and colloid-containing tissues

A

C. Regaud’s Fluid

96
Q

 study of early degenerative processes and tissue necrosis

A

D. Orth’s Fluid

97
Q

 demonstrates Rickettsia and other bacteria

A

D. Orth’s Fluid

98
Q

 demonstration of acid mucopolysaccharides

A

Lead Fixatives

99
Q

are soluble in water, therefore, tissues should not be washed in running tap water (use 70% alcohol instead)

A

Picrates

100
Q

 fixation of embryos and pituitary biopsies

A
  1. Bouin’s Solution
101
Q

 fixative for glycogen

A
  1. Brasil’s Alcoholic Picroformol Fixative
102
Q

 fixation of nucleoproteins

A

GLACIAL ACETIC ACID

103
Q

 Preserves but does not “fix” glycogen granules

A
  1. 95 % Ethanol
104
Q

 For dry and wet smears, blood and bone marrow samples

A
  1. Methanol
105
Q

 for touch preparations

A
  1. Isopropyl alcohol (95%)
106
Q

 most rapid fixative

A
  1. Carnoy’s fluid
107
Q

 for chromosomes, lymph glands, urgent biopsies and brain tissue for the diagnosis of rabies

A
  1. Carnoy’s fluid
108
Q

 demonstration of mucopolysaccharides and nucleoproteins

A
  1. Newcomer’s
109
Q

 Permanently fixes fats and recommended for fixing nuclear structures

A
  1. Flemming’s (chrome-osmium HAc)
110
Q

 For cytoplasmic structures

A
  1. Flemming’s w/o acetic acid
111
Q

 may also be used as a weak decalcifying agent

A

TRICHLOROACETIC ACID

112
Q

 use at cold temperature (-5 to 4oC)

A

ACETONE

113
Q

 fixation of brain tissues for rabies diagnosis

A

ACETONE

114
Q

Routinely used fixative

A

Formaldehyde

115
Q

Used for electron microscopy

A

Glutaraldehyde

116
Q

For enzymes, nucleoproteins, fats and mucins

A

10% Formol-Saline

117
Q

Best for frozen sections, iron pigments and elastic fibers

A

10% Neutral Buffered Formalin

118
Q

For lipids, especially neutral fats and phospholipids

A

Formol Corrosive/Sublimate

119
Q

For immunoperoxidase activity, glycogen, microincineration & sputum

A

Gendre’s

120
Q

For small pieces of liver, spleen, connective tissue fibers & nuclei

A

Zenker’s

121
Q

For pituitary, bone marrow and blood-containing organs

A

Zenker-Formol

122
Q

For tumor biopsies of the skin

A

Heidenhain’s SuSa

123
Q

For bone marrow biopsies

A

B-5

124
Q

For carbohydrates

A

Chromic Acid

125
Q

For lipids and mitochondria

A

Potassium dichromate

126
Q

For chromatin, mitochondria, mitotic figures, Golgi bodies, RBC and colloid-containing tissues

A

Regaud’s

127
Q

For early degenerative processes, tissue necrosis, Rickettsiae and other bacteria

A

Orth’s

128
Q

For acid mucopolysaccharides

A

4% Basic Lead acetate

129
Q

For embryos and pituitary biopsies

A

Bouin’s

130
Q

For glycogen

A

Brasil’s Alcoholic Picroformol

95% Ethanol

131
Q

For dry and wet smears, blood and bone marrow samples

A

100% Methanol

132
Q

For touch preparations

A

95% Isopropyl alcohol

133
Q

For chromosomes, lymph glands, urgent biopsies and brain fixation for rabies diagnosis

A

Carnoy’s

134
Q

For mucopolysaccharides and nuclear proteins

A

Newcomer’s

135
Q

For fats and nuclear structures

A

Flemming’s with HAc

136
Q

For cytoplasmic structures

A

Flemming’s without HAc

137
Q

For nuclear structures

A

Glacial Acetic acid

138
Q

For dense fibrous tissues

A

Trichloroacetic acid

139
Q

For brain fixation for rabies diagnosis and water-diffusible enzymes

A

Acetone

140
Q

 placing a fixed tissue in a second fixative

A

POST MORDANTING/SECONDARY FIXATION

141
Q

 2.5-3% potassium dichromate as the secondary fixative

A

 Post-chromatization

142
Q

 acts as a mordant for better staining effects

A

2.5-3% potassium dichromate

143
Q
  1. Tap Water removes excess
A

C
F
O

144
Q
  1. Tap Water:
A

 Chromates (Helly’s, Zenker’s & Flemming’s)
 Formalin
 Osmic Acid

145
Q
  1. 50 – 70 % Alcohol:
A

 Picric acid (Bouin’s solution)

146
Q
  1. Alcoholic iodide:
A

 Mercuric fixatives

147
Q

: preserve cellular structure & require short fixation time

A

FIXATIVES

148
Q

ALDEHYDE FIXATION

A
  1. Karnovsky’s paraformaldehyde - glutaraldehyde
  2. Acrolein – glutaradehyde / formaldehyde
149
Q

POST FIXATION:

A

Buffered Osmium Tetroxide

150
Q

OSMIC ACID FIXATION

A
  1. Palade’s fixative
  2. Zetterqvist Osmium fixative
  3. Millonig’s Osmium tetroxide
151
Q

Enzyme Histochemistry
1. [?]
2. Frozen sections: [?]

A

4% formaldehyde or formol-saline

acetone or formaldehyde

152
Q

Fixatives for Smears

A
  1. Schaudinn’s
     Mercuric Chloride  Absolute Ethanol  Acetic acid
  2. Ether-Alcohol
  3. Methanol
153
Q

Other Methods of Fixation

A
  1. Heat
  2. Microwave Fixation
154
Q

Microwave Fixation –

A

vacuum, heat & agitation (10 – 15 mm) + formaldehyde fixatives

155
Q

 Chemical fixation is avoided

A

SPECIAL TECHNIQUES

156
Q

 Recommended for histochemical studies

A

SPECIAL TECHNIQUES

157
Q

 Rapid freezing of tissue blocks to allow instant cessation of cellular activities

A

Quenching

158
Q

Freezing Agents

A
  1. Liquid Nitrogen
  2. Isopentane
  3. Pentane
  4. Propane
  5. Dichlorodifluoromethane
159
Q

: tissue plunged in isopentane or propane-isopentane (-160 to -180 OC)

A

 Rapid freezing

160
Q

: removal of tiny ice crystals by sublimation in a vacuum (-30 to -40 OC)

A

 Desiccation

161
Q

 Similar with freeze-drying

A

FREEZE-SUBSTITUTION

162
Q

FREEZE-SUBSTITUTION
 Instead of dehydration in a vacuum, tissue blocks are:

A

 Fixed in Rossman’s fluid or 1% acetone
 Dehydrated with abs. alcohol

MTAP - HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES MODULE (9 decks)

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