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Histopathology (Lec & Lab) > FIXATION > Flashcards

FIXATION Flashcards

1
Q

The first and most critical step in histotechnology

A

Fixation

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2
Q

PRIMARY AIM OF FIXATION:

A

to preserve the morphologic and chemical integrity of the cell in as lifelike manner as possible

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3
Q

SECONDARY GOAL OF FIXATION:

A

to harden and protect the tissue from the trauma of further handling, so that it is easier to cut during gross examination

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4
Q

Satisfactory fixation occurs between the pH of

A

pH 6 and 8

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5
Q

Fixation of surgical specimens is traditionally carried out at

A

room temperature

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6
Q

Many laboratories use tissue processors that work at

A

40°C

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7
Q

Formalin heated to ______ is sometimes used for the rapid fixation of very urgent biopsy specimens, although the risk of tissue distortion is increased

A

60°C

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8
Q

Can be used to fix tissues with tuberculosis

A

Formalin at 100°C

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9
Q

Brain is usually suspended whole in ____________ for 2-3 weeks to ensure fixation and some hardening prior to sectioning

A

10% buffered formalin

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10
Q

For fixation, the best results are usually obtained using what type of solution?

A

slightly hypertonic solutions (400-450 mOsm)

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11
Q

Causes polymerizationof the aldehyde, with consequent decrease in its effective concentration

A

presence of a buffer

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12
Q

This have been found to be an ideal concentration for immuno-electron microscopy

A

Low concentrations of glutaraldehyde (0.25%)

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13
Q

Primary fixation using buffered formalin should be for how hours?

A

2-6 hours

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14
Q

The maximum effectiveness of fixation is noted to be _____ the tissue volume

A

20 times

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15
Q

Traditionally, the amount of fixative used has been ________the volume of tissue to be fixed.

A

10-25 times

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16
Q

What is the adequate fixation time?

A

4 - 6 hours

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17
Q

What is the recommended size of the tissue?

A

2 cm2 and no more than 4 mm thick

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18
Q

Used to slow down decomposition if the tissue needs to be photographed and cannot be fixed immediately

A

Refrigeration

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19
Q

Continues to undergo mitosis (growth) up to 30 minutes after death when refrigerated

A

bone marrow

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20
Q

This deteriorate very quickly

A

Brain cells

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21
Q

Are made up of only one
component substance

A

Simple Fixatives

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22
Q

Are those that are made up of two or more fixatives which have been added together to obtain the optimal combined effect of their individual actions upon the cells and tissue constituents.

A

Compound Fixatives

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23
Q

Are those that permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues in question

A

Microanatomical Fixatives

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24
Q

Enumerate the fixatives under the aldehydes of Simple fixatives

A
  • Formaldehyde
  • Glutaraldehyde
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25
Q

Enumerate the fixatives under metallic fixatives of simple fixatives

A
  • Mercuric chloride
  • Chromate fixatives ( potassium dichromate, and chromic acid)
  • Lead fixatives (Picric acid, acetic acid, acetone alcohol, and osmium teroxide or osmic acid)
  • Heat
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26
Q

Enumerate the fixatives under microanatomical fixatives

A
  • 10% Formol saline
  • 10% Neutral buffered formalin
  • Heidenhain’s Susa
  • Formol sublimate (formol corrosive)
  • Zenker’s solution
  • Zenker-formol (Helly’s solution)
  • Bouin’s solution
    -Brasil’s solution
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27
Q

Enumerate the fixatives that have a ph of 4.6 or less ( hint: Nuclear fixatives)

A
  • Flemming’s fluid
  • Carnoy’s fluid
  • Bouin’s fluid
  • Newcomer’s fluid
  • Heidenhain’s fluid
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28
Q

Enumerate the fixatives that have a pH of more than 4.6 (hint: Cytoplasmic fixatives)

A
  • Flemming’s fluid without acetic acid
  • Kelly’s fluid
  • Formalin with “post-chroming”
  • Regaud’s fluid (Muller’s fluid)
  • Orth’s fluid
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29
Q

These fixatives preserve the chemical constituents of cells and tissues

A

Histochemical fixatives

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30
Q

Enumerate the fixatives under histochemical fixatives

A
  • Formol saline 10% or 10% Formol Saline
  • Absolute Ethyl alcohol
  • Acetone alcohol
  • Newcomer’s fluid
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31
Q

For lipid fixation, fixatives that contain ___________ and _________ can be effective for preservation of lipids in cyrostat sections

A

mercuric chloride and potassium dichromate, respectively

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32
Q

These fixatives are generally recommended for glycogen fixation

A

Alcoholic fixatives

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33
Q

These are the most commonly used fixatives for amino acid histochemistry

A

Neutral buffered formol saline or formaldehyde vapor

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34
Q

The most useful fixatives for preserving glycogen are alcohol-based such as

A

Rossman’s fluid or cold absolute alcohol

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35
Q

One of the most widely used fixatives is________ which is made from formaldehyde, a gas produced by the oxidation of methyl alcohol, and is soluble in water to the extent of 37-40% weight in volume

A

10% formalin

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36
Q

A simple microanatomical fixative made up of saturated formaldehyde (40% by weight volume) diluted to 10% with sodium chloride.

A

10% Formol- Saline

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37
Q

It is recommended for fixation of central nervous tissues and general post-mortem tissues for histochemical examination.

A

10% Formol-Saline

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38
Q

Recommended for preservation and storage of surgical, post-mortem and research specimens.

A

10% Neutral Buffered Formalin or Phosphate-Buffered Formalin (pH 7)

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39
Q

This aldehyde fixative is recommended for routine post-mortem tissues.

A

Formol-Corrosive (Formol-Sublimate)

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40
Q

Post-fixation with ________ for 6 hours or more can enhance immunoperoxidase studies on the tissues, and in some cases, electron microscopy, if it is necessary at a later time to establish a diagnosis.

A

phenol-formalin (Alcoholic Formalin/ Gendre’s Fixative)

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41
Q

It acts in a manner similar to formaldehyde and is sometimes utilized for routine light microscopic work.

A

Glutaraldehyde

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42
Q

Enumerate the six fixatives under Aldehyde Fixative

A
  • Formaldehyde (Formalin)
  • 10% Formol-Saline
  • 10% Neutral Buffered Formalin or Phosphate-Buffered Formalin (pH 7)
  • Formol-Corrosive (Formol-Sublimate)
  • Alcoholic Formalin (Gendre’s) Fixative
  • Glutaraldehyde
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43
Q

Enumerate the fixatives under Mercuric Chloride (Metallic fixative)

A
  • Zenker’s fluid
  • Zenker-formol (Helly’s solution)
  • Heidenhain’s Susa solution
  • B-5 Fixative
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44
Q

Buffered glutaraldehyde, followed by secondary fixation in _________ is satisfactory for electron microscopy.

A

osmium tetroxide

45
Q

The most common metallic fixative, frequently used in saturated aqueous solutions of 5-7%; it is included in many compound fixatives

A

Mercuric Chloride

46
Q

Widely used as a secondary fixative reacting with a number of amino acid residues and accompanied by spectroscopic changes, probably due to reaction with histidine residues.

A

Mercuric Chloride

47
Q

Made up of mercuric chloride stock solution to which glacial acetic acid has been added just before its use to prevent turbidity and formation of a dark precipitate.

A

Zenker’s fluid

48
Q

It is recommended for fixing small pieces of liver, spleen, connective tissue fibers and nuclei.

A

Zenker’s fluid

49
Q

It is an excellent microanatomic fixative for pituitary gland, bone marrow and blood containing organs such as spleen and liver.

A

Zenker-formol (Helly’s solution)

50
Q

Recommended mainly for tumor biopsies especially of the skin; it is an excellent cytologic fixative.

A

Heidenhain’s Susa Solution

51
Q

Commonly used for bone marrow biopsies.

A

B-5 fixative

52
Q

Used in 1-2% aqueous solution, usually as a constituent of a compound fixative. It is a strong oxidizing agent; hence, a strong reducing agent (e.g. formaldehyde) must be added to chrome-containing fixatives before use in order to prevent counteracting effects and consequent decomposition of solution upon prolonged standing.

A

Chromic Acid

53
Q

Preserves lipids and mitochondria

A

Potassium Dichromate

54
Q

It is recommended for demonstration of chromatin, mitochondria, mitotic figures, Golgi bodies, RBC and colloid-containing tissues.

A

Regaud’s (Muller’s) Fluid

55
Q

Recommended for study of early degenerative processes and tissue necrosis. It demonstrates rickettsiae and other bacteria.

A

Orth’s fluid

56
Q

Recommended for acid mucopolysaccharides. It fixes connective tissue mucin.

A

Lead fixatives

57
Q

Enumerate the fixatives under Chromate fixatives

A
  • Chromic acid
  • Potassium Dichromate
  • Ragaud’s /Ragard’s (huhuhu) (Muller’s fluid)
  • Orth’s fluid
58
Q

Enumerate the fixatives under Picric acid fixatives

A
  • Bouin’s solution
  • Brasil’s Alcoholic Picroformol Fixative
59
Q

Normally used in conjunction with other fixatives to form a compound solution.

A

Acetic acid

60
Q

It fixes and precipitates nucleoproteins. It precipitates chromosomes and chromatin materials.

A

Glacial Acetic acid

61
Q

Rapidly denatures and precipitates proteins by destroying hydrogen and other bonds

A

Alcohol (Alc. fixatives)

62
Q

Can be used to fix and preserve glycogen, pigments, blood, tissue films and smears.

A

Absolute alcohol

63
Q

It is excellent for fixing dry and wet smears, blood smears and bone marrow tissues. It fixes and dehydrates at the same time.

A

Methyl Alcohol 100%

64
Q

Used for fixing touch preparations, although some touch preparations are air dried and not fixed, for certain special staining procedures such as Wright-Giemsa.

A

Isopropyl Alcohol 95%

65
Q

Used at concentrations of 70-100%. If lower concentrations are used, the RBC’s become hemolyzed and WBC’s are inadequately preserved. It may be used as a simple fixative.

A

Ethyl alcohol

66
Q

Recommended for fixing chromosomes, lymph glands and urgent biopsies.

A

Carnoy’s Fluid

67
Q

Rapid in action and may be used for urgent biopsy specimens for paraffin processing within 5 hours

A

Carnoy’s Fluid

68
Q

Enumerate the fixatives under Alcohol fixative

A
  • Methyl Alcohol 100%
  • Isopropyl Alcohol 95%
  • Ethyl alcohol
  • Carnoy’s fluid
  • Newcomer’s fluid
69
Q

This is a pale yellow powder which dissolves in water (up to 6% at 20°C) to form a strong oxidizing solution.

A

Osmium tetroxide (Osmic acid)

70
Q

Most common chrome-osmium acetic acid fixative used, recommended for nuclear preparation of such sections

A

Flemming’s solution

71
Q

Made up only of chromic and osmic acid, recommended for cytoplasmic structures particularly the mitochondria

A

Flemming’s solution without acetic acid

72
Q

IDENTIFY:
- It precipitates proteins.
- Its marked swelling effect on tissues serves to counteract shrinkage produced by other fixatives.
- It may be used as a weak decalcifying agent.
- Its softening effect on dense fibrous tissues facilitates preparation of such sections.

A

Trichloroacetic acid

73
Q

Used at ice cold temperature ranging from -5°C to 4°C

74
Q

Enumerate the fixatives under Osmium Tetroxide (Osmic acid)

A
  • Flemming’s solution
  • Flemming’s solution without acetic acid
75
Q

This procedure involves thermal coagulation of tissue proteins for rapid diagnosis, usually employed for frozen tissue sections and preparation of bacteriologic smears.

A

Heat fixation

76
Q

This may be done before DEHYDRATION and on DEPARAFFINIZED sections before staining

A

Secondary fixation

77
Q

A form of secondary fixation whereby a primarily fixed tissue is placed in aqueous solution of 2.5 - 3% potassium dichromate for 24 hours to act as mordant for better staining effects and to aid in cytologic preservation of tissues.

A

Post- Chromatization

78
Q

The process of removing excess fixative from the tissue after fixation in order to improve staining and remove artefacts from the tissues.

A

Washing-out

79
Q

Used to remove:
a. excess chromates from tissues fixed in Kelly’s, Zenker’s, and Flemming’s solutions
b. excess formalin
c. excess osmic acid

80
Q

Used to wash out excess amount of **picric acid **(Bouin’s solution).

A

50-70% alcohol

81
Q

Used to remove excessive mercuric fixatives.

A

Alcoholic iodine

82
Q

Prevents complete penetration of fixative; hence, tissues that contain this are fixed slowly and poorly

A

Presence of mucus

83
Q

This inactivates enzymes

A

Cold temperature

84
Q

Tissues containing large amount of blood (e.g. blood vessels and spleen) should be flushed out with?

85
Q

This require less fixative and shorter fixation time.

A

Smaller and thinner tissues

85
Q

Accelerates fixation but hastens autolytic changes and enzyme destruction

A

Moderate heat (37-56°C)

86
Q

A well-known artefact that may be produced under acid conditions

A

Formalin pigment

87
Q

May be found in surgical specimens particularly in liver biopsies, associated with an intense eosinophilic staining at the center of the tissue in H &E stained sections.

A

Crush artefact

88
Q

Works as a physical agent similar in mechanism to vacuum, oven (heat) and agitation to increase the movement of molecules and accelerate fixation

A

Microwave technique

89
Q

Enumerate the factors that affect fixation of tissues

A

Retarded by:
- Size and thickness of the tissue specimen
- Presence of mucus
- Presence of fat
- Presence of blood

Enhanced by:
- Size and thickness of tissue
- Agitation

90
Q

Thorough rinsing out of formalin for tissue less than 1 mm thick: Isopropanol

A

1 change, 3 minutes

90
Q

Thorough rinsing out of formalin for tissue less than 1 mm thick: Ethanol

A

1 change, 5 minutes

91
Q

Thorough rinsing out of formalin for tissue less than 1 mm thick: Paraffin

A

2 changes; 1 at 67°C for 2 minutes; second change at 82°C for 5 minutes

91
Q

Thorough rinsing out of formalin for tissue 1-2 mm thick: Ethanol

A

15 minutes

92
Q

Thorough rinsing out of formalin for tissue 1-2 mm thick: Isopropanol

A

15 minutes

93
Q

Thorough rinsing out of formalin for tissue 1-2 mm thick: Paraffin

A
  • 1 change — 67°C for 10 minutes
  • 1 change — 82°C for 12 minutes
94
Q

Thorough rinsing out of formalin for tissue 2-5 mm thick: Paraffin

A

30 minutes at 67°C; 60 minutes at 82°C

95
Q

Thorough rinsing out of formalin for tissue 2-5 mm thick: Ethanol

A

60 minutes

95
Q

Thorough rinsing out of formalin for tissue 2-5 mm thick: Isopropanol

A

45 minutes

96
Q

Its gneral aim is to preserve the maximum enzyme activity at its original localization, while also preserving structural integrity.

A

Enzyme histochemistry

97
Q

What are the most useful primary fixatives for electron microscopy?

A
  • osmium tetroxide
  • glutaraldehyde
  • paraformaldehyde,

with the whole procedure performed at 4°C.

98
Q

Microwave energy interacts with dipolar molecules, causing oscillation at a frequency of

98
Q

For routine studies in electron microscopy the fixatives used are

A

Glutaraldehyde or osmium
tetroxide

99
Q

For electron histochemistry and electron immunocytochemistry in electron microscopy, the fixative used is?

A

Karmnovsky’s paraformaldehyde-glutaraldehyde

100
Q

What are the usual primary fixative used in secondary fixation

A

10% formalin or 10% formol saline

101
Q

When tissue is fixed in 10% buffered neutral formalin, it may require secondary fixation with ___________ which acts as a mordant

A

Zenker’s solution

102
Q

IDENTIFY the specific use/target of the following stains:
a. Masson’s trichome
b. Mallory’s aniline blue
c. Phosphotungstic acidhematoxylin (PTAH) stain

A

a. connective tissue
b. collagen
c. striated muscle

103
Q

Fixative used for post- fixation in electron microscopy

A

Osmium tetroxide

Histopathology (Lec & Lab) (8 decks)

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