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[MT 6321] Histopathology Lecture > [2S] UNIT 6.2 Fixation > Flashcards

[2S] UNIT 6.2 Fixation Flashcards

1
Q

First step in histotechnology

A

Fixation

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2
Q

Chemical process by which biological tissues are preserved from decay

A

Fixation

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3
Q

Process of preserving cells and tissue constituents in a
LIFE-LIKE manner

A

Fixation

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4
Q

Common specimens that are submitted in the laboratory

A

Surgery (Biopsy)
Dissection Room (Autopsy)

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5
Q

Tissue sample thickness should be?

A

5mm

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6
Q

Preserve the morphological and chemical integrity of
the cell

A
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7
Q

Harden and preserve tissue for further handling

A
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8
Q

Prevent or arrest degenerative processes

A
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9
Q

disruption of the cell due to lysosome

A

Autolysis

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10
Q

bacterial decomposition

A

Putrefaction

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11
Q

TYPES OF FIXATION

● Heat fixation
● Microwave fixation
● Cryo-preservation

A

Physical Method

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12
Q

TYPES OF FIXATION

● Immersion Fixation and Perfusion Fixation
● Coagulant fixatives
● Non-coagulant cross-linking fixative

A

Chemical Method

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13
Q

T/F: In immersion & perfusion fixation, the volume of the fixative is 10-20 or 25 times the volume of the specimen

A

T

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14
Q

BASIC MECHANISM IN FIXATION

Fixing agent is not incorporated into the tissue

A

NON-ADDITIVE FIXATION / COAGULANT FIXATIVE

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15
Q

BASIC MECHANISM IN FIXATION

● Non-coagulant cross-linking fixatives
○ Cross-linking of proteins (as in scaffolding of buildings)
■ Stabilizing physical characteristics of tissue

A

ADDITIVE FIXATION/ NON-COAGULANT FIXATIVES

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16
Q

BASIC MECHANISM IN FIXATION

Chemical constituent taken into the cell, forming molecular complexes and stabilizing proteins

A

ADDITIVE FIXATION/ NON-COAGULANT FIXATIVES

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17
Q

BASIC MECHANISM IN FIXATION

Formalin, Mercury and Osmium tetroxide

A

ADDITIVE FIXATION/ NON-COAGULANT FIXATIVES

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18
Q

BASIC MECHANISM IN FIXATION

● Dehydrant coagulant fixatives
○ Capable of dehydration/ removing of water molecules
for hardening or stabilizing tissue

A

NON-ADDITIVE FIXATION / COAGULANT FIXATIVE

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19
Q

BASIC MECHANISM IN FIXATION

● Alteration of tissue composition by removing bound water molecules at Hydrogen bonds within protein molecules
○ Stabilizes proteins by forming crosslinks after water molecule removal

A

NON-ADDITIVE FIXATION / COAGULANT FIXATIVE

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20
Q

BENEFITS OF FIXATION

T/F: Allows sectioning of the tissue by softening tissue

A

F; hardening

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21
Q

BENEFITS OF FIXATION

T/F: Prevents autolysis and inactivates infectious age

A

T

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22
Q

BENEFITS OF FIXATION

T/F: Improves cell avidity for special stains

A

T

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23
Q

MAIN FACTORS AFFECTING FIXATION: HYDROGEN CONC

T/F: High acidity decreases effectiveness

A

T

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23
Q

MAIN FACTORS AFFECTING FIXATION: HYDROGEN CONC

pH of a satisfactory fixation

A

ph 6 & 8 (neutral)

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24
Q

MAIN FACTORS AFFECTING FIXATION: HYDROGEN CONC

Best fixative

A

10% neutral buffered formalin

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24
Q

MAIN FACTORS AFFECTING FIXATION: HYDROGEN CONC

best blood buffer for systemic circulation

A

Bicarbonate

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25
Q

MAIN FACTORS AFFECTING FIXATION: HYDROGEN CONC

Common buffers

A
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25
Q

MAIN FACTORS AFFECTING FIXATION: HYDROGEN CONC

T/F: Outside the pH range, changes may occur that are
detrimental to ultrastructural preservation of the tissue.

A

T

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25
Q

MAIN FACTORS AFFECTING FIXATION: HYDROGEN CONC

T/F: High pH changes:

Precipitation of formalin pigments: Over time, formalin turns into formic acid, which has a lower pH

A

T

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26
Q

MAIN FACTORS AFFECTING FIXATION: TEMP

T/F: Increasing the temperature decreases the
rate of diffusion into the tissue and speeds up the rate of chemical reaction between the fixative and tissue elements

A

F; increases the rate of diffusion

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27
Q

MAIN FACTORS AFFECTING FIXATION: TEMP

Fixation of surgical specimens is done at what temp?

A

room temperature

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28
Q

MAIN FACTORS AFFECTING FIXATION: TEMP

T/F: The higher the temperature, the faster is the rate of fixation and the less time is needed for fixation

A

T

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28
Q

MAIN FACTORS AFFECTING FIXATION: TEMP

Fixation of surgical specimens can be followed by further fixation at temp up to [temp]?

A

40-45C

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29
Q

MAIN FACTORS AFFECTING FIXATION: TEMP

Fixative used for EM

A

Glutaraldehyde

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29
Q

MAIN FACTORS AFFECTING FIXATION: TEMP

Recommended temperature

A

body temperature

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29
Q

MAIN FACTORS AFFECTING FIXATION: TEMP

Temp for EM

A

0-4C

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30
Q

MAIN FACTORS AFFECTING FIXATION: TEMP

T/F:
Mast cells - Room temp
Nucleic acids - Not room temp

A

T

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31
Q

Light-colored areas = ___ areas
Dark colored areas = ____ areas

A

fixed
unfixed

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32
Q

THICKNESS OF SECTION

for EM

A

1 to 2 mm square

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33
Q

THICKNESS OF SECTION

for light microscopy

A

2 cm

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34
Q

T/F: Brain is usually suspended whole in 10% buffered formalin for 2-3 weeks to ensure fixation and some hardening prior to sectioning

A

T

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35
Q

OSMOLALITY

give rise to cell shrinkage

A

hypertonic

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36
Q

OSMOLALITY

cause swelling and poor fixation

A

Isotonic and Hypotonic fixatives

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37
Q

OSMOLALITY

The best result is usually obtained using ____ _______ solutions (400-450 mOsm)

A

slightly hypertonic

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38
Q

Concentration of formaldehyde and glutaraldehyde

A
  • Formaldehyde – 10%
  • Glutaraldehyde – 3%
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39
Q

Concentration of glutaraldehyde for immunoelectrochemistry

A

0.25%

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40
Q

CONCENTRATION

T/F: Presence of buffer causes polymerization of
aldehyde, with consequent increase in its effective concentration

A

F; decrease

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41
Q

DURATION OF FIXATION

________ in buffered formalin is usually carried out for 2-6
hours during the day the specimen is obtained

A

Primary Fixation

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42
Q

DURATION OF FIXATION

T/F: Prolonged fixation may cause shrinkage and hardening of the tissue
and may severely inhibit enzyme activity and immunological reaction

A

T

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43
Q

PRACTICAL CONSIDERATIONS OF FIXATION

The specimen should be placed in a fixative solution as soon as it is removed from the body to prevent autolysis and putrefaction

A

Speed

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44
Q

PRACTICAL CONSIDERATIONS OF FIXATION: PENETRATION

T/F: A commonly quoted rate of penetration for aldehyde
fixative is two to three millimeters per hour and slows down as it goes deeper into the tissue.

A

T

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45
Q

PRACTICAL CONSIDERATIONS OF FIXATION: PENETRATION

____ per hour fixative penetration

A

2-3 mm

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46
Q

PRACTICAL CONSIDERATIONS OF FIXATION: VOLUME

T/F: 10-25 times the volume of the tissue to be fixed

A

T

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47
Q

PRACTICAL CONSIDERATIONS OF FIXATION: VOLUME

T/F: The maximum effectiveness of fixation is
noted to be ____ the tissue volume.

A

20 times

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48
Q

PRACTICAL CONSIDERATIONS OF FIXATION: DURATION OF FIXATION

T/F: Small or loosely textured tissues such as biopsies / scrapings take longer than fibrous organs

A

F; Fibrous organs take longer than small or loosely textured tissues such as biopsies or scrapings

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49
Q

PRACTICAL CONSIDERATIONS OF FIXATION: DURATION OF FIXATION

T/F: Fixation time can be cut down by using heat, vacuum, agitation or microwave

A

T

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49
Q

PRACTICAL CONSIDERATIONS OF FIXATION: DURATION OF FIXATION

Negative pressure in a container that helps
fixative penetration to reach up to 5mm per hour

A

Vacuum

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50
Q

PRACTICAL CONSIDERATIONS OF FIXATION: DURATION OF FIXATION

The fixative container is slightly shaken so that the ions would not be relatively at rest but are excited and evenly distributed in the entire solution

A

Agitation

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51
Q

PRACTICAL CONSIDERATIONS OF FIXATION: MISCELLANEOUS CONSIDERATIONS

T/F: To maintain an adequate fixation time of 4-6 hours, the tissue size must be 2cm square

A
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52
Q

PRACTICAL CONSIDERATIONS OF FIXATION: MISCELLANEOUS CONSIDERATIONS

T/F: The tissue selected for sectioning should be thin enough to allow penetration by fixative within a reasonable amount of time

A

T

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53
Q

PRACTICAL CONSIDERATIONS OF FIXATION: MISCELLANEOUS CONSIDERATIONS

T/F: Refrigeration is used to fasten decomposition if the
tissue that needs to be photographed and cannot be fixed immediately

A

F; slow down decomposition

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54
Q

EFFECTS OF FIXATIVES

T/F: It hardens soft tissues and make the handling and cutting of section easier

A

T

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55
Q

EFFECTS OF FIXATIVES

T/F: It inhibits bacterial decomposition and reduces risks of infections during handling

A

T

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56
Q

EFFECTS OF FIXATIVES

T/F: Decreases optical differentiation of cells

A

F; Increases

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57
Q

EFFECTS OF FIXATIVES

T/F: Fixatives diluted and/or contaminated by bodily fluids (e.g. bile, blood, feces) will be increased in concentration and must be replaced regularly to ensure effectiveness

A

F; reduced in conc

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58
Q

EFFECTS OF FIXATIVES

T/F: Pinning specimens to a corkboard or inserting a paper or gauze “wick” into tubular structures can improve fixation and reduce tissue distortion

A

T

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59
Q

EFFECTS OF FIXATIVES

T/F: Prolonged fixation may be more difficult to reverse and may also result in loss of immunohistochemical
antigenicity

A

T

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60
Q

FIXATIVES ACCORDING TO COMPOSITION

made up only of one substance

A

Simple Fixatives

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61
Q

FIXATIVES ACCORDING TO COMPOSITION

○ Two or more fixatives to obtain optimal combined
effect
○ A combination of different simple fixatives

A

Compound Fixatives

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62
Q

FIXATIVES ACCORDING TO ACTION

General microscopic study of tissue structures

A

MICROANATOMICAL

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63
Q

FIXATIVES ACCORDING TO ACTION

Well preservation to the totality of physical characteristic or morphology of tissue
○ Not only used for the tissue but also the cytoplasm
○ Can preserve all tissue structures

A

MICROANATOMICAL

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64
Q

FIXATIVES ACCORDING TO ACTION

Preserve specific parts and particular microscopic
elements

A

CYTOLOGIC FIXATIVES

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65
Q

CYTOLOGIC: NUCLEAR OR CYTOPLASMIC

Preserves cytoplasmic structures in particular

A

Cytoplasmic

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66
Q

CYTOLOGIC: NUCLEAR OR CYTOPLASMIC

Preserve cytoplasmic
organelles better than
nucleus

A

Cytoplasmic

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67
Q

CYTOLOGIC: NUCLEAR OR CYTOPLASMIC

> 4.6 pH

A

Cytoplasmic

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68
Q

CYTOLOGIC: NUCLEAR OR CYTOPLASMIC

Flemming’s Fluid without
Acetic Acid
Helly’s Fluid
Formalin with
Post-Chroming
Regaud’s Fluid (Moller’s
Fluid)
Orth’s Fluid

A

Cytoplasmic

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69
Q

CYTOLOGIC: NUCLEAR OR CYTOPLASMIC

RNA preservation: Ethanol
(best) and Acetone
(alternative)

A

Cytoplasmic

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69
Q

CYTOLOGIC: NUCLEAR OR CYTOPLASMIC

Preserve nuclear structures

A

Nuclear

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70
Q

CYTOLOGIC: NUCLEAR OR CYTOPLASMIC

Preserve nucleus better
than cytoplasm

A

Nuclear

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71
Q

CYTOLOGIC: NUCLEAR OR CYTOPLASMIC

Glacial acetic acid as
primary component

A

Nuclear

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72
Q

CYTOLOGIC: NUCLEAR OR CYTOPLASMIC

<4.6 pH

A

Nuclear

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73
Q

CYTOLOGIC: NUCLEAR OR CYTOPLASMIC

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

A

Nuclear

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74
Q

FIXATIVES ACCORDING TO ACTION

Preserves chemical constituents of cells and tissues

A

HISTOCHEMICAL FIXATIVES

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75
Q

FIXATIVES ACCORDING TO ACTION

May preserve tissues, nucleus, and cytoplasm but aside from that, it can preserve the chemical component

A

HISTOCHEMICAL FIXATIVES

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76
Q

FIXATIVES ACCORDING TO ACTION

ODD ONE OUT: Histochemical Fixatives

○ 10% Formol Saline
○ Absolute Ethyl Alcohol
○ Acetone
○ Newcomer’s
○ Heidenhain’s Susa

A

Heidenhain’s Susa - Microanatomical

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77
Q

FIXATIVES ACCORDING TO ACTION

ODD ONE OUT: Cytoplasmic Fixatives

  • Flemming’s Fluid without Acetic Acid
  • Helly’s Fluid
  • Bouin’s Fluid
  • Formalin with Post-Chroming
  • Regaud’s Fluid (Moller’s Fluid)
  • Orth’s Fluid
A

Bouin’s Fluid - Nuclear

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78
Q

FIXATIVES ACCORDING TO ACTION

ODD ONE OUT: Nuclear Fixatives

  • Flemming’s Fluid
  • Formalin with Post-Chroming
  • Carnoy’s Fluid
  • Bouin’s Fluid
  • Newcomer’s Fluid
  • Heidenhain’s Susa
  • RNA: ethanol and acetone
A

Formalin with Post-Chroming - Cytoplasmic

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79
Q

FIXATION FOR IMMUNOHISTOCHEMISTRY

Fixation methods generally fall into two classes

A

organic solvents and cross-linking reagents

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80
Q

FIXATION FOR IMMUNOHISTOCHEMISTRY

Use frozen sections and special stains (fixative: mercuric chloride and potassium dichromate)

A

Lipid Fixation

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80
Q

FIXATION FOR IMMUNOHISTOCHEMISTRY

such as alcohols and acetone remove lipids and dehydrate the cells while precipitating the proteins on the cellular architecture

A

Organic solvents

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81
Q

FIXATION FOR IMMUNOHISTOCHEMISTRY

(such as paraformaldehyde) form intermolecular bridges, normally through free amino
groups, thus creating a network of linked antigens

A

Cross-linking reagents

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81
Q

FIXATION FOR IMMUNOHISTOCHEMISTRY

T/F: Cross-linkers preserve cell structure better than organic solvents but may reduce the antigenicity of some cell components and require the addition of a permeabilization step to allow access of the antibody to the specimen

A

T

82
Q

FIXATION FOR IMMUNOHISTOCHEMISTRY

Baker’s formal-calcium may be used to preserve phospholipids

A

Lipid Fixation

82
Q

FIXATION FOR IMMUNOHISTOCHEMISTRY

T/F: 10% neutral buffer formalin would be effective

A

T

82
Q

FIXATION FOR IMMUNOHISTOCHEMISTRY

T/F: Antigen retrieval phase is not necessary

A

F; is necessary

83
Q

FIXATION FOR IMMUNOHISTOCHEMISTRY

Largely removed during processing

A

Lipid Fixation

84
Q

FIXATION FOR IMMUNOHISTOCHEMISTRY

Alcoholic fixatives for better retention

A

Carbohydrate Fixation

84
Q

FIXATION FOR IMMUNOHISTOCHEMISTRY

Aldehyde fixatives can be used in general

A

Lipid Fixation

85
Q

FIXATION FOR IMMUNOHISTOCHEMISTRY

Glycogen can be demonstrated satisfactorily enough for diagnosis, although losses of
glycogen can be high (60-80%) in aqueous solution

A

Carbohydrate Fixation

86
Q

FIXATION FOR IMMUNOHISTOCHEMISTRY

There is better retention of glycogen if the section is coated with celloidin

A

Carbohydrate Fixation

87
Q

FIXATION FOR IMMUNOHISTOCHEMISTRY

Alcoholic formaldehyde is a better fixative in human skin compared with neutral
buffered formaldehyde.

A

Carbohydrate Fixation

87
Q

FIXATION FOR IMMUNOHISTOCHEMISTRY

Neutral buffered formal saline

A

Protein fixation

88
Q

FIXATION FOR IMMUNOHISTOCHEMISTRY

Routinely osmium tetroxide or glutaraldehyde is used with the whole procedure performed at 4°C.

A

Fixation for Electron Microscopy

88
Q

FIXATION FOR IMMUNOHISTOCHEMISTRY

is the process of placing an already fixed tissue in a second fixative

A

Secondary Fixation

89
Q

To make special staining techniques possible (with secondary fixative acting as
a mordant)

A

Secondary Fixation

89
Q

To facilitate and improve the demonstration of particular substances

A

Secondary Fixation

90
Q

To ensure further and complete hardening and preservation of tissues

A

Secondary Fixation

90
Q

CHARACTERISTICS OF A GOOD FIXATIVES

T/F:

● It must be cheap.
● It must be stable.
● It must be safe to handle

A

T

90
Q

CHARACTERISTICS OF A GOOD FIXATIVES

T/F: It must kill the cell slowly, thereby producing a minimum distortion of cell constituents

A

F; quickly

91
Q

CHARACTERISTICS OF A GOOD FIXATIVES

T/F: It must be hypotonic, causing minimal physical and chemical alteration of the cells and their constituents (not strictly implemented).

A

F; isotonic

91
Q

CHARACTERISTICS OF A GOOD FIXATIVES

T/F: It must inhibit bacterial decomposition and autolysis. It must produce minimum shrinkage of tissue

A

T

92
Q

CHARACTERISTICS OF A GOOD FIXATIVES

T/F: It must permit rapid and even penetration of tissues

A

T

93
Q

CHARACTERISTICS OF A GOOD FIXATIVES

T/F: It must harden tissues, thereby making the cutting of sections easier

A

T

93
Q

CHARACTERISTICS OF A GOOD FIXATIVES

T/F: A good fixative can be slightly hypertonic to slightly
harden tissues

A

T

94
Q

CHARACTERISTICS OF A GOOD FIXATIVES

T/F: It must make cellular components insoluble to hypotonic solutions and render them insensitive to subsequent processing.

A

T

94
Q

CHARACTERISTICS OF A GOOD FIXATIVES

T/F: It must permit the subsequent application of many staining procedures to facilitate easier and more profitable examination

A

T

95
Q

6 Fixatives in order of decreasing speed of
penetration

A

FAMOP

Formaldehyde > Acetic acid > Mercuric chloride > Ethyl/Methyl alcohol > Osmium tetroxide > Picric acid

96
Q

The aldehydes form cross-links between proteins, creating a gel, thus retaining cellular constituents in their in vivo relationships to each other.

A

ALDEHYDE FIXATIVES

96
Q

Gives some mechanical strength to the entire structure which enables it to withstand subsequent
processing.

A

ALDEHYDE FIXATIVES

97
Q

T/F: Aldehyde fixatives are non-coagulant

A

T

98
Q

ALDEHYDE FIXATIVES

Produced from oxidation of methyl alcohol/ methanol.

A

Formaldehyde

99
Q

ALDEHYDE FIXATIVES

● Fixation time 24 hrs
● Irritating especially to mucosa (nose, throat) because of its fumes

A

Formaldehyde

100
Q

ALDEHYDE FIXATIVES

If formaldehyde is not used immediately, it becomes acidic over time (formic acid), which forms a white precipitate called _________

A

paraformaldehyde

101
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

● Cheap, Readily Available, Easy to Prepare
● Relatively Stable
● Compatible with many Stains
● Fumes Irritating to Nose and Eyes
● Solution is Irritating to Skin

A

10% FORMALIN

102
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

For Routine Paraffin Sections, Electron Microscopy, Histochemistry & Enzyme Studies

A

10% FORMALIN

103
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

Pigments removed by treatment with alcoholic picric acid

A

10% FORMALIN

104
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

10% NaCl Solution as diluent

A

10% FORMOL-SALINE

105
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

Fixation of central nervous tissues

A

10% FORMOL-SALINE

106
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

Post-mortem tissues for histochemical examinations

A

10% FORMOL-SALINE

107
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

● Fixation time: 24 hrs at 35 degrees/ 48 hrs at RT
● 10% formalin with NSS

A

10% FORMOL-SALINE

108
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

A
109
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

Components of 10% Formalin

A

900 mL Distilled H2O
100 mL Formaldehyde 40%
9 gm NaCl

110
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

○ Fast penetration
○ Non-coagulant and additive
○ Prevents alterations during processing

A

10% Neutral Buffered Formalin

110
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

Most recommended fixative

A

10% NEUTRAL BUFFERED FORMALIN

111
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

● Buffered to a pH level near 7 so that there will be no
acidity and no precipitation
○ pH level makes the fixative usable for 1 month

A

10% NEUTRAL BUFFERED FORMALIN

111
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

● Preservation and routine storage of surgical, post-mortem and research specimens
● Fixation time: 4-24 Hrs

A

10% NEUTRAL BUFFERED FORMALIN

112
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

900 mL D H2O
100 mL Formaldehyde 40%
6.5 gm Disodium hydrogen
phosphate
3.5 gm Sodium dihydrogen
phosphate

A

10% Neutral Buffered Formalin

113
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

○ Less shrinkage than other fixatives
○ Not osmotically active
○ Hardens tissue better (except alcohol and acetone)

A

10% Neutral Buffered Formalin

114
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

○ Tissue can be stored in formalin indefinitely (except
for IHC)
○ Cheap and stable

A

10% Neutral Buffered Formalin

115
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

Fixative of choice for immunohistochemistry and
molecular studies

A

10% Neutral Buffered Formalin

116
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

10% Neutral Buffered Formalin Disadvantage

T/F: It is longer to prepare; hence, is time-consuming

A

T

117
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

10% Neutral Buffered Formalin Disadvantage

T/F: Positivity of mucin to the periodic acid shift (PAS) is
increased

A

F; reduced

118
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

10% Neutral Buffered Formalin Disadvantage

T/F: It may produce gradual loss in basophilic staining of
cells

A

T

119
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

10% Neutral Buffered Formalin Disadvantage

T/F: Reactivity of myelin to Weigert’s iron hematoxylin
stain is reduced

A

T

120
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

10% Neutral Buffered Formalin Disadvantage

T/F: It is inert towards lipids, especially neutral fats and
phospholipids

A

T

121
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

● Brightens cytoplasmic and metachromatic stains
● Slow penetration
● After fixation, we can do the dehydration step immediately using absolute (100%) alcohol

A

FORMOL-CORROSIVE (FORMOL-SUBLIMATE)

121
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

Sat. Aq. Mercuric chloride as diluent

A
122
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

● Fixation Time: 3-24 Hrs
● Recommended for routine post-mortem tissues

A

FORMOL-CORROSIVE (FORMOL-SUBLIMATE)

123
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

90 mL Sat aq Mercuric Chloride
10 mL Formaldehyde

A

FORMOL-CORROSIVE (FORMOL-SUBLIMATE)

124
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

Polymerized form of formaldehyde usually obtained as a fine white powder (precipitate), which depolymerizes back to formalin when heated

A

PARAFORMALDEHYDE

125
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

It is suitable for paraffin embedding and sectioning and also for immunocytochemical analysis

A

PARAFORMALDEHYDE

126
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

also be stained for general histology, but the degree of fixation is less vigorous than Bouin’s, so the quality of the morphology obtained will be less

A

PARAFORMALDEHYDE

127
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

a mixture of paraformaldehyde and glutaraldehyde.

A

Karnovsky’s Fixative

128
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

It is suitable for use when preparing samples for light
microscopy in resin embedding and sectioning and for electron microscopy

A

Karnovsky’s Fixative

129
Q

ALDEHYDE FIXATIVES

● 2.5% SOLUTION – small tissue fragments and needle
aspirates
● 4% SOLUTION – larger tissues

A

Glutaraldehyde

130
Q

ALDEHYDE FIXATIVES: FORMALDEHYDE

T/F: Karnovsky should always be prepared fresh

A

T

131
Q

ALDEHYDE FIXATIVES

● Less Irritating to nose and skin
● More Expensive
● Less stable
● Slow penetration

A

Glutaraldehyde

132
Q

ALDEHYDE FIXATIVES

● For Routine Light Microscopy, also for EM
● Preserves cellular structures better

A

Glutaraldehyde

133
Q

MECHANISM: Mercuric Chloride and Lead Fixatives’ Mercuric salts bind with sulfhydryl groups in acidic solutions

A

METALLIC FIXATIVE

134
Q

Chromate Fixatives in water form Cr-O-Cr complexes that
have an affinity for –COOH and –OH groups of proteins,
so that complexes between adjacent molecules are
formed.

A

METALLIC FIXATIVE

135
Q

METALLIC FIXATIVE: MERCURIC CHLORIDE

  • Included in most compound Fixatives
  • Nuclear components are
    shown in fine detail
A

5-7% MERCURIC CHLORIDE

136
Q

METALLIC FIXATIVE: MERCURIC CHLORIDE

  • Excellent Trichome Staining
  • Preservation of cell detail in
    Tissue Photography
A

5-7% MERCURIC CHLORIDE

137
Q

METALLIC FIXATIVE: MERCURIC CHLORIDE

Pigments removed by
treatment with Iodine
solution in 95% alcohol

A

5-7% MERCURIC CHLORIDE

138
Q

METALLIC FIXATIVE: MERCURIC CHLORIDE

2.5 g Potassium Dichromate
1 gm Sodium Sulfate
100 mL DH2O

A

5-7% MERCURIC CHLORIDE

138
Q

METALLIC FIXATIVE: MERCURIC CHLORIDE

With Glacial Acetic Acid for affinity to nuclear chromatin

A

ZENKER’S FLUID

138
Q

METALLIC FIXATIVE: MERCURIC CHLORIDE

Permits Brilliant staining of nuclear and connective
tissue fibers

A

ZENKER’S FLUID

139
Q

METALLIC FIXATIVE: MERCURIC CHLORIDE

  • Fixation time: 12-24 hrs
  • Lyses RBC and can make tissues brittle
A

ZENKER’S FLUID

139
Q

METALLIC FIXATIVE: MERCURIC CHLORIDE

For Liver, Spleen, Connective Tissue Fibers, and Nuclei

A

ZENKER’S FLUID

139
Q

METALLIC FIXATIVE: MERCURIC CHLORIDE

  • Mercuric chloride, 5 gm
  • Potassium dichromate, 2.5 gm
  • Distilled water, 100 ml
  • 40% formaldehyde 5 ml (to be added immediately before use)
A

ZENKER-FORMOL (Helly’s solution)

140
Q

METALLIC FIXATIVE: MERCURIC CHLORIDE

  • For Pituitary Gland, Bone Marrow and Blood Containing Organs
    (e.g. Spleen and Liver)
  • Preserves Cytoplasmic Granules well
A

ZENKER-FORMOL (Helly’s solution)

140
Q

METALLIC FIXATIVE: MERCURIC CHLORIDE

Recommended for Tumor biopsies of skin

A

HEIDENHAIN’S SUSA SOLUTION

141
Q

METALLIC FIXATIVE: CHROMATE FIXATIVE

● Usually constituent of a compound fixative
○ NOT a simple fixative
● Preserves Carbohydrates and precipitates all proteins
● May produce sub-oxide precipitates

A

1-2% CHROMIC ACID

141
Q

METALLIC FIXATIVE: CHROMATE FIXATIVE

● Preserves Lipids
● Preserves Mitochondria
● For post-chromatization

A

3% POTASSIUM DICHROMATE

142
Q

METALLIC FIXATIVE: MERCURIC CHLORIDE

  • Excellent Cytologic Fixative
  • Fixation time 3-12 hrs
  • Penetrates and fixes tissue rapidly and evenly
A

HEIDENHAIN’S SUSA SOLUTION

143
Q

METALLIC FIXATIVE: MERCURIC CHLORIDE

● Commonly used for bone marrow biopsies
● Fixation time: 4 – 8 hours
● RAPID FIXATION: 1 1/2-2 hrs
● Unstable
● Can make tissues brittle
● Good and clear nuclear details

A

B-5 FIXATIVE

144
Q

METALLIC FIXATIVE: CHROMATE FIXATIVE

● Fixation time 12-48 hrs
● Penetrates well
● Recommended for demonstration of Chromaffin tissues (phaeochromocytoma), Mitochondria, Mitotic Figures, Golgi Bodies, RBC, and Colloid-containing Tissues
● Unstable
● Poor nuclear staining

A

REGAUD’S FLUID (MULLER’S FLUID)

145
Q

METALLIC FIXATIVE: CHROMATE FIXATIVE

● Recommended for study of Early Degenerative Processes and tissue Necrosis
● 2.5% Potassium Dichromate
● 40% Formaldehyde
● Fixation time: 36-72 hours

A

ORTH’S FLUID

146
Q

METALLIC FIXATIVE: LEAD FIXATIVE

● Recommended for Acid Mucopolysaccharides
● Fixes Connective Tissue Mucin
● Takes up CO2 to form insoluble lead carbonates on standing

A

4% LEAD ACETATE

147
Q

PICRIC ACID FIXATIVES

● Excellent for Glycogen Demonstration
● Penetrates and fixes small tissue rapidly
● Suitable for Aniline Stains

A

1% PICRIC ACID SOLUTION

148
Q

● MECHANISM is still unknown
● Yellow color, which can be removed by lithium carbonate or washing with 50-70% ethanol

A

PICRIC ACID FIXATIVES

149
Q

PICRIC ACID FIXATIVES

● With 37% Formaldehyde, Picric acid, Ethanol or Isopropyl Alcohol, and Trichloroacetic Acid
● Less messy than Bouin’s solution

A

BRASIL’S ALCOHOLIC PICROFORMOL FIXATIVE

149
Q

PICRIC ACID FIXATIVES

● is recommended for fixation of embryos and pituitary biopsies
● Preferred fixative for connective tissue staining

A

BOUIN’S SOLUTION

149
Q

PICRIC ACID FIXATIVES

● Yellow stain is useful for Fragmented Biopsies
● Minimal distortion of microanatomical structures
● Excellent for soft and delicate tissues

A

BOUIN’S SOLUTION

149
Q

PICRIC ACID FIXATIVES

● Causes RBC hemolysis
● Not Suitable for Frozen Sections – causes it to crumble when cut
● Prolonged fixation makes tissue hard

A

1% PICRIC ACID SOLUTION

150
Q

● Normally used in conjunction with other fixatives to form a compound solution
● It causes tissues (especially those containing collagen) to
swell.

A

GLACIAL ACETIC ACID

150
Q

PICRIC ACID FIXATIVES

● Excellent for Glycogen
● Overnight tissue fixation by automatic processing
technique may utilize 3-4 changes of Brasil’s fixative at 1/2 to 2 hours each, succeeded directly by absolute alcohol.

A

BRASIL’S ALCOHOLIC PICROFORMOL FIXATIVE

150
Q

ALCOHOL FIXATIVES

May be used as simple fixative
● Usually incorporated into compound fixatives for better results
● Fixes blood, tissue films, and smears

A

70-100% ETHANOL

150
Q

● Fixes and precipitates nucleoproteins, Chromosomes and
Chromatin Materials
● For Nuclear Component studies

A

GLACIAL ACETIC ACID

151
Q

MECHANISM: Rapidly denatures and precipitates proteins by destroying hydrogen bonds to stabilize the tertiary structure of proteins

Used both as Fixative and Dehydrating Agent

A

ALCOHOL FIXATIVES

151
Q

ALCOHOL FIXATIVES

● The most rapid fixative (1-3 hrs only)
● Fixes and dehydrates at the same time
● Used to fix brain for diagnosis of rabies

A

CARNOY’S FLUID

151
Q

ALCOHOL FIXATIVES

Recommended for fixing dry and wet smears, blood
smears and bone marrow tissues

A

100% METHANOL

152
Q

ALCOHOL FIXATIVES

● Preserves Nucleoproteins and Nucleic Acids for Histochemistry and Enzyme studies
● Dissolves fat and lipids
● May shrink tissues

A

70-100% ETHANOL

153
Q

ALCOHOL FIXATIVES

● May cause RBC lysis
● Shrinks and may harden tissues excessively
● Slow penetration

A

CARNOY’S FLUID

154
Q

ALCOHOL FIXATIVES

FORMULA:
Ethanol (absolute) 75 ml
Acetic acid glacial 25 ml

A

Clarke’s solution

155
Q

ALCOHOL FIXATIVES

  • Fixation time: 1 - 6 hours
  • Recommended Applications
  • It is sometimes used to fix diagnostic cryostat sections. If used for
    primary fixation, the specimens can be placed directly into 95% ethanol for processing.
A

Formol-acetic alcohol

156
Q

ALCOHOL FIXATIVES

FORMULA:
* Ethanol absolute: 85 ml
* 40% formaldehyde: 10 ml
* Acetic acid glacial: 5 ml

A

Formol-acetic alcohol

156
Q

ALCOHOL FIXATIVES
* Fixation time: 3 - 4 hours
* Has been used on frozen sections and smears

A

Clarke’s solution

157
Q

ALCOHOL FIXATIVES

Gendre’s Fixative if with picric acid
○ 95% Ethanol
○ 40% Formaldehyde
○ Glacial Acetic Acid

A

Alcohol Formalin

157
Q

ALCOHOL FIXATIVES

Post-fixation with phenol-formalin for 6 hours or more can enhance immunoperoxidase studies

A

Alcohol Formalin

158
Q

ALCOHOL FIXATIVES

Useful for Sputum as it coagulates mucus

A

Gendre’s

159
Q

ALCOHOL FIXATIVES

New alcohol
○ Isopropanol is used instead of the commonly used ethanol

A

Newcomer’s fluid

160
Q

ALCOHOL FIXATIVES

Acts both as Nuclear and Histochemical Fixative

A

Newcomer’s fluid

161
Q

permanganate fixatives
(potassium permanganate), potassium dichromate,
chromic acid, and osmium tetroxide

A

OXIDIZING AGENTS

162
Q

ALCOHOL FIXATIVES

● Produces better reaction in Feulgen stain
○ Feulgen stain is used for staining DNA or nucleus
● Recommended for fixing mucopolysaccharides
● Fixation time: 12-18 hours at 3°C

A

Newcomer’s fluid

163
Q

react with various side chains of proteins and other biomolecules, allowing the formation of crosslinks that stabilize tissue structure but cause extensive denaturation despite preserving fine cell structure and are used mainly as secondary fixatives

A

OXIDIZING AGENTS

164
Q

OXIDIZING AGENTS

MECHANISM: Various hypotheses of lipid stabilization have been postulated
■ Oxidation of double bonds between adjacent carbon atoms to form monoesters and diesters
■ Binding of lipid to protein
■ Conversion of unsaturated fatty acids to stable glycol osmates.

A

OSMIUM TETROXIDE

165
Q

OXIDIZING AGENTS: OSMIUM TETROXIDE

Fixes conjugated fats and lipids permanently by making them insoluble to alcohol and xylene during dehydration and clearing

A

6% OSMIUM TETROXIDE

166
Q

OXIDIZING AGENTS: OSMIUM TETROXIDE

● Preserves cytoplasmic structures well
● Fixes materials for Ultrathin Sectioning in Electron Microscopy

A

6% OSMIUM TETROXIDE

167
Q

OXIDIZING AGENTS: OSMIUM TETROXIDE

Drawback: forms black precipitates upon exposure to sunlight

A

6% OSMIUM TETROXIDE

168
Q

OXIDIZING AGENTS: OSMIUM TETROXIDE

● With Glacial Acetic Acid for Nuclear affinity
● With 1% Chromic Acid as diluent
● Fixation time: 24-48 hrs (1-2 days)

A

FLEMMING’S SOLUTION

169
Q

OXIDIZING AGENTS: OSMIUM TETROXIDE

● Recommended for nuclear preparation of such
sections
● Permanently fixes fat
● Excellent fixative for nuclear structures (e.g.
Chromosomes)
● Expensive

A

FLEMMING’S SOLUTION

170
Q

OXIDIZING AGENTS: OSMIUM TETROXIDE

Recommended for cytoplasmic structures particularly the mitochondria

A

FLEMMING’S SOLUTION W/O GLACIAL ACETIC ACID

171
Q

OTHERS

● Sometimes incorporated into compound fixatives
● It precipitates proteins
● Softening Effect on Dense Fibrous Tissues
● May be used as a weak decalcifying agent
● Poor penetration

A

TRICHLOROACETIC ACID

172
Q

OTHERS

● This could be a backup alternative solution for
glutaraldehyde intended for electron microscopy; hence, it is used in cold temperatures.
● Dissolves fat
● Shrinks tissues excessively
● Used as a fixative and dehydrating agent

A

ACETONE

172
Q

OTHERS

● Used at ice cold temperature (-5 to 4 degrees Celcius)
● Recommended for the study of water-diffusible enzymes (i.e., Phosphatases and Lipases)
● Used in Fixing Brain Tissues for diagnosis of Rabies

A

ACETONE

173
Q

OTHERS

Stable medium for transport of fresh unfixed tissues, such
as renal, skin, and oral mucosa biopsies, which will
undergo subsequent frozen section and
immunofluorescence studies

A

MICHEL’S SOLUTION

174
Q

OTHERS

Not suitable for transporting cells for flow cytometry or for tissues used for fluorescent in-situ hybridization (FISH)

A

MICHEL’S SOLUTION

175
Q

OTHERS

● Specimens may be kept in it at room temperature for 5
days while in transport
● Not a fixative per se but a transport media

A

MICHEL’S SOLUTION

176
Q

MECHANISM:
○ Involves Thermal Coagulation of tissue protein
○ For Rapid diagnosis
○ Employed for Frozen Tissue Sections and Bacteriologic Smears
○ Physical fixation

A

HEAT FIXATION

177
Q

DIFFICULTIES CAUSED BY IMPROPER FIXATION

CAUSE: Failure to fix immediately (the tissue was
probably allowed to dry before fixing); Insufficient
fixative

a. Tissue blocks are brittle and hard
b. Failure to arrest early autolysis of cells
c. Presence of artifact pigments on tissue sections
d. Removal of substances soluble in fixing agent
e. Tissues are soft and feather-like in consistency
f. Loss or inactivation of enzymes needed for study
g. Shrinkage and swelling of cells and tissue structure

A

b. Failure to arrest early autolysis of cells

178
Q

DIFFICULTIES CAUSED BY IMPROPER FIXATION

CAUSE: Wrong choice of fixative

a. Tissue blocks are brittle and hard
b. Failure to arrest early autolysis of cells
c. Presence of artifact pigments on tissue sections
d. Removal of substances soluble in fixing agent
e. Tissues are soft and feather-like in consistency
f. Loss or inactivation of enzymes needed for study
g. Shrinkage and swelling of cells and tissue structure

A

d. Removal of substances soluble in fixing agent

f. Loss or inactivation of enzymes needed for study

179
Q

DIFFICULTIES CAUSED BY IMPROPER FIXATION

CAUSE: Overfixation

a. Tissue blocks are brittle and hard
b. Failure to arrest early autolysis of cells
c. Presence of artifact pigments on tissue sections
d. Removal of substances soluble in fixing agent
e. Tissues are soft and feather-like in consistency
f. Loss or inactivation of enzymes needed for study
g. Shrinkage and swelling of cells and tissue structure

A

g. Shrinkage and swelling of cells and tissue structure

179
Q

DIFFICULTIES CAUSED BY IMPROPER FIXATION

CAUSE: Incomplete washing of fixative

a. Tissue blocks are brittle and hard
b. Failure to arrest early autolysis of cells
c. Presence of artifact pigments on tissue sections
d. Removal of substances soluble in fixing agent
e. Tissues are soft and feather-like in consistency
f. Loss or inactivation of enzymes needed for study
g. Shrinkage and swelling of cells and tissue structure

A

c. Presence of artifact pigments on tissue sections

180
Q

DIFFICULTIES CAUSED BY IMPROPER FIXATION

CAUSE: Incomplete fixation

a. Tissue blocks are brittle and hard
b. Failure to arrest early autolysis of cells
c. Presence of artifact pigments on tissue sections
d. Removal of substances soluble in fixing agent
e. Tissues are soft and feather-like in consistency
f. Loss or inactivation of enzymes needed for study
g. Shrinkage and swelling of cells and tissue structure

A

e. Tissues are soft and feather-like in consistency

181
Q

DIFFICULTIES CAUSED BY IMPROPER FIXATION

CAUSE: Prolonged fixation

a. Tissue blocks are brittle and hard
b. Failure to arrest early autolysis of cells
c. Presence of artifact pigments on tissue sections
d. Removal of substances soluble in fixing agent
e. Tissues are soft and feather-like in consistency
f. Loss or inactivation of enzymes needed for study
g. Shrinkage and swelling of cells and tissue structure

A

a. Tissue blocks are brittle and hard

182
Q

Formalin pigment is a well-known artifact that may be
produced under acid conditions. The pigment may be eliminated or reduced by fixation in phenol-formalin.

A

FIXATION ARTIFACTS

183
Q

FIXATION ARTIFACTS

is a well-known artifact that may be produced under acid conditions. The pigment may be eliminated or reduced by fixation in phenol-formalin

A

Formalin pigment

184
Q

The use of neutral buffered formalin and phenol-formalin almost completely stops the formation of formalin pigment, which occurs with non-buffered formaldehyde solutions and also fixes tissues more rapidly.

A

FIXATION ARTIFACTS

185
Q

FIXATION ARTIFACTS

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. This may be due to partial coagulation of partially fixed protein by ethanol or by incomplete wax impregnation during subsequent histological processing.

A

“Crush artifact”

[MT 6321] Histopathology Lecture (10 decks)

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