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HISTOPATH MTAP > Part 1 (FIXATION) > Flashcards

Part 1 (FIXATION) Flashcards

1
Q

First and most critical step in histotechnology

A

FIXATION

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2
Q

Stabilization of proteins is the most important reaction for maintaining tissue morphology

A

FIXATION

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3
Q

Process of preserving the tissue for future examination

A

FIXATION

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4
Q

have the property of forming cross-links between protein

A

FIXATION

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5
Q

Primary aim: preserve the morphological and chemical integrity of the cell in life-like manner

A

FIXATION

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5
Q

Secondary aim: to harden and protect tissue from trauma of further handling

A

FIXATION

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6
Q

First or Secondary Aim?

preserve the morphological and
chemical integrity of the cell in life-like manner

A

First Aim

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7
Q

First or Secondary Aim?

to harden and protect tissue from
trauma of further handling

A

Secondary Aim

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8
Q

In the process, it is possible that the organ or tissue will be exposed to different forces and
pressure.

A

Secondary Aim of Fixation

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9
Q

Ideal time for fixation

A

Within 20-30 minutes after
interruption of blood supply.

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10
Q

Once you have already cut off or interrupted the blood supply or remove the organ, it is already the start of death of the cells.

A

Ideal time for fixation (Within 20-30 minutes)

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11
Q

Usual fixation temperature for
surgical specimen

A

Room Temperature (22-24 deg)

● Temperature may go higher if
you want to enhance the fixation process.

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12
Q

Fixative-to-Tissue Ratio (F:T Ratio)

A

F:T Ratio = 20:1
● 20 = Fixative
● 1 = Tissue

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13
Q

Tissue-to-Fixative Ratio (T:F Ratio)

A

T:F Ratio = 1:20
● 1 = Tissue
● 20 = Fixative

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14
Q

● 20x the tissue volume

A

Volume

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15
Q

Specimen must be placed in fixative as soon as it is removed from the body (20-30 mins after
interruption of blood supply)

A

Speed

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16
Q

● Rate of formalin penetration = 1mm per hour
● Formalin was previously a brand name of formaldehyde, but now synonymous with it

A

Penetration

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17
Q

(4) PRACTICAL CONSIDERATIONS OF FIXATION

A

1.) Speed
2.) Penetration
3.) Volume
4.) Duration of fixation

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18
Q

Rate of formalin penetration =

A

= 1mm per hour

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19
Q

is previously the brand name of
acetylsalicylic acid

A

Aspirin

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20
Q

(2) MECHANISMS INVOLVED IN FIXATION

A

1) Additive Fixation
2.) Non- Additive Fixation

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21
Q

(3) Examples of Additive Fixation

A

formalin, mercury, and osmium tetroxide

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22
Q

Example of Non- Additive Fixation

A

alcoholic fixatives

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23
Q

● Whereby the fixing agent is not taken in, but changes the tissue composition and stabilizes the tissue by removing bound water attached to hydrogen bonds of
certain groups within the protein molecule
● Example: alcoholic fixatives

A

Non- Additive Fixation

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24
Q

● Whereby the chemical constituent of the fixative is taken in and becomes part of the tissue
● Example: formalin, mercury, and osmium tetroxide

A

Additive Fixation

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25
Q

Hydrogen Ion Concentration (pH)

Satisfactory fixation:

A

pH 6-8

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26
Q

(6) MAIN FACTORS INVOLVED IN FIXATION

A
  1. Hydrogen Ion
    Concentration (pH)
  2. Temperature
  3. Thickness of
    section
  4. Osmolality
  5. Concentration
  6. Duration of fixation
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27
Q

Surgical specimen: TEMPERATURE

A

room temperature (22-24°C)

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28
Q

Electron microscopy and some
histochem: TEMPERATURE

A

Refrigerator temperature (0-4°C)

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29
Q

Tissue blocks should be either small, thin, or as prescribed by tissue processor manufacturer

A

Thickness of section

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30
Q

● Formalin aims to harden the
specimen; the longer the specimen stays in the fixative,
the harder it gets

A

Brain

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31
Q

● Suspended whole in 10%
buffered formalin for 2-3
weeks

A

Brain

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32
Q

Brain
● Suspended whole in __%
buffered formalin for ___
weeks

A

Brain
● Suspended whole in 10%
buffered formalin for 2-3
weeks

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33
Q

(2) TYPE OF FIXATIVES ACCORDING TO…

A
  1. ACCORDING TO COMPOSITION
  2. ACCORDING TO ACTION
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34
Q

● One component

A

Simple Fixatives

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35
Q

● Glacial acetic acid
- Considered as a fixative in itself
- Solidifies at ___°C
- Causes tissues to swell
- Preserve _________

A

● Glacial acetic acid
- Considered as a fixative in itself
- Solidifies at 17°C
- Causes tissues to swell
- Preserve nucleoprotein

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35
Q

● Glacial acetic acid
- Considered as a fixative in itself
- Solidifies at 17°C
- Causes tissues to swell
- Preserve nucleoprotein

A

Simple Fixatives

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36
Q

● Made up of 2 or more fixatives

A

Compound Fixatives

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37
Q

Compound Fixatives example

A

Zenker’s Fluid

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38
Q

(2) Zenker’s Fluid is made up of:

A
  1. Glacial acetic acid
  2. Mercuric chloride
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39
Q
  • Causes the tissues to swell
A

Glacial acetic acid

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40
Q
  • Minimizes the shrinkage effect of the Mercuric chloride
A

Glacial acetic acid

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41
Q
  • Causes the tissues to
    shrink
A

Mercuric chloride

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42
Q
  • Minimizes the swelling effect
    of the Glacial acetic acid
A

Mercuric chloride

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43
Q

Glacial acetic acid and Mercuric chloride

● These two _________ each other that minimizes the adverse effect

A

● These two neutralizes each other that minimizes the adverse effect

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44
Q

For GENERAL microscopic study of tissue structures

A

MICROANATOMICAL FIXATIVES

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44
Q

(2) According to Action

A

A. MICROANATOMICAL FIXATIVES
B. CYTOLOGICAL FIXATIVES

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45
Q

Examples
- 10% Formol Saline
- 10% BNF (Buffered neutral formalin)/Neutral buffered
formalin
- Heidenhain’s SuSa
- Bouin’s
- Formol sublimate
- Zenker’s solution
- Zenker-Formol
- Brasil’s

A

MICROANATOMICAL FIXATIVES

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46
Q

● Preserves SPECIFIC parts and particular microscopic elements of the cell itself

A

CYTOLOGICAL FIXATIVES

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47
Q

(3) Types of CYTOLOGICAL FIXATIVES

A

○ Nuclear fixatives
○ Cytoplasmic fixatives
○ Histochemical fixatives

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48
Q

● Preserves the NUCLEAR STRUCTURES of the cells

A

Nuclear fixatives

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49
Q

● WITH glacial acetic acid
○ Preserves the nucleoproteins
which preserves the nuclear
structures

A

Nuclear fixatives

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50
Q

Fixatives:
● Flemmings
● Carnoy’s
● Bouin’s
● Newcomer’s
● Heidenhain’s SuSa

A

Nuclear fixatives

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51
Q

● Preserve the CYTOPLASMIC STRUCTURES

A

Cytoplasmic fixatives

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52
Q

General pH of Nuclear fixatives

A

General pH: 4.6 or less (acidic)

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53
Q

Preserve CHEMICAL CONSTITUENTS of cells

A

Histochemical fixatives

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53
Q

General pH of Cytoplasmic fixatives

A

General pH: greater than 4.6

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53
Q

● WITHOUT glacial acetic acid
○ Destroys the mitochondria and
golgi bodies (organelles in the
cytoplasm)

A

Cytoplasmic fixatives

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54
Q

Fixatives:
● Flemming’s fluid w/o acetic acid
● Helly’s fluid
● Formalin with post-chroming
● Regaud’s (Moller’s)
● Orth’s

A

Cytoplasmic fixatives

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55
Q

Fixatives:
● 10% Formol Saline
● Absolute ETOH (Ethanol)
● Acetone
● Newcomer’s Fluid

A

Histochemical fixatives

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56
Q

Functions as both a nuclear and
histochemical fixative

A

Newcomer’s Fluid

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56
Q

All of the following fixatives contain Glacial Acetic Acid except

What is the question looking for? Nuclear or Cytoplasmic fixatives?

A

Cytoplasmic fixatives

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57
Q

Most commonly used aldehyde fixative

A

FORMALDEHYDE

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57
Q

Aka Formalin

A

FORMALDEHYDE

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58
Q

is only a brand name. But due to its popularity, formaldehyde can be called as

A

“Formalin”

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59
Q

A gas produced from the oxidation of methanol

A

FORMALDEHYDE

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60
Q

Rate of fixation: FORMALDEHYDE

A

1mm per hour

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61
Q

Usual fixation Time of FORMALDEHYDE

A

● 24 hours

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62
Q

2 types of concentration

A

● 40% Formaldehyde (Stock Solution)
● 10% Formaldehyde (Working
Solution)

63
Q
  • Concentration is too strong resulting to outer part of the tissue tends to overharden
  • Not used as routine concentration aldehyde
A

40% Formaldehyde (Stock
Solution)

64
Q
  • Used to fixed organs and
    tissues
  • An unstable solution and may
    produce artifacts that can affect
    in the observation of cells in the
    microscope
A

10% Formaldehyde (Working
Solution)

65
Q
  • Needed to be further diluted
    with water before using → 10%
    formaldehyde
A

40% Formaldehyde (Stock
Solution)

66
Q

40% Formaldehyde aka

A

Stock Solution

67
Q

10% Formaldehyde aka

A

Working Solution

68
Q

Artifacts That May Be Produced By The 10% Formaldehyde (2)

A

Paraformaldehyde

Acid Formaldehyde Hematin

69
Q

● White precipitates which may
be found on the solution itself
● Affects microscopic details

A

Paraformaldehyde

69
Q

Paraformaldehyde

Removed by: (2)

A
  • Addition of 10% methanol
  • Filtration
70
Q

● Brown or black granules that
may obscure microscopic details

A

Acid Formaldehyde Hematin

71
Q

● If present, they can block the
microscopic view of the cells that are underneath them
● Can be a problem because you
won’t be able to see the abnormalities that are on the cells

A

Acid Formaldehyde Hematin

72
Q

Acid Formaldehyde Hematin

Removed By: (4)

A

● Saturated alcoholic picric acid
● Alcoholic Potassium Hydroxide
(KOH)
● Kardasewitsch method
● Lillie’s method

73
Q

A. ALDEHYDE FIXATIVES (8)

A
  1. FORMALDEHYDE
  2. 10% Formol saline
  3. 10% Buffered Neutral
    Formalin (BNF)
  4. Formol-corrosive
  5. Glutaraldehyde
  6. Formol-calcium
  7. Acrolein
  8. Karnopsy’s ParaFormaldehyde
    Glutaraldehyde Solution (KPG)
74
Q

● Best fixative for tissue containing iron pigments and for elastic fibers
● Best general tissue fixative

A

10% Buffered Neutral Formalin (BNF)

74
Q

2 concentrations:

A

● 2.5% Solution
● 4% Solution

75
Q

AKA: Formol-Sublimate

A

Formol-corrosive

76
Q

Used for enzyme histochemistry and electron microscopy

A

Glutaraldehyde

77
Q

Used for small tissue fragments and needle biopsy

A

2.5% Solution

78
Q

Used for the preservation of lipids

A

Formol-calcium

78
Q

Used for larger tissue specimens

A

4% Solution

79
Q

(2) Used for electron cytochemistry

A

Acrolein

Karnopsy’s ParaFormaldehyde
Glutaraldehyde Solution (KPG)

80
Q

40% Formaldehyde can be also called as?

A

Stock Solution

80
Q

What is the best fixative for tissue containing pigments and the best general tissue fixative?

A

10% BNF

81
Q

What is the next fixative for tissue containing pigments?

A

10% BNF

82
Q

What solution can function both as a nuclear and histochemical fixative?

A

Newcomer’s Fluid

83
Q

What fixative is used for electron microscopy?

A

Glutaraldehyde

84
Q

Most common metallic fixative

A

MERCURIC CHLORIDE (HgCl2)

84
Q

● Routine fixative of choice for
tissue photography
● Permits brilliant metachromatic
staining of cells

A

MERCURIC CHLORIDE (HgCl2)

85
Q

● Decreases amount of
demonstrable glycogen
● Do not use if you wish to
demonstrate glycogen on tissues

A

MERCURIC CHLORIDE (HgCl2)

85
Q

● Causes tissue shrinkage
- MgCl2 is a compound fixative
that causes the tissue to shrink
- To neutralize shrinking, mix with
glacial acetic acid

A

MERCURIC CHLORIDE (HgCl2)

86
Q

● Corrodes all metals, except for
the nickel alloy (Monel)
- If you were to fix a tissue in
MgCl2, the material you should
use to get that organ/tissue should be made of Monel nickel
alloy

A

MERCURIC CHLORIDE (HgCl2)

87
Q

● Produces black granular deposits
- removed by: De-Zenkerization
Technique
- Reagent: Alcohol Iodine

A

MERCURIC CHLORIDE (HgCl2)

88
Q

MERCURIC CHLORIDE (HgCl2)

● Produces black granular deposits
- removed by:
- Reagent:

A
  • removed by: De-Zenkerization
    Technique
  • Reagent: Alcohol Iodine
89
Q

● Fixation of tumor biopsies (esp. of the skin)
● Came from two German wor

A

Heidenhain’s SuSa

90
Q
  • Sublimat -
  • Säure -
A
  • Sublimat - Mercuric chloride
  • Säure - Acid
91
Q

What is glycogen?

A

Storage form of glucose in humans

91
Q

Commonly used for bone marrow biopsies

A

B-5 fixative

92
Q

The mercuric chloride deposits on tissues may be removed using?

A

Alcohol Iodine

93
Q

Conversion of Glycogen to Glucose =

A

Glycogenolysis

94
Q

Conversion of Glucose to Glycogen =

A

Glycogenesis

94
Q

What is the meaning of Y in eosin Y?

A

Y is yellowish

95
Q

● Chromic acid
● Potassium dichromate
● Regaud’s (Moller’s)
● Orth’s Fluid

A

CHROMATE FIXATIVES

95
Q

What is the meaning of LASER?

A

Light amplified by stimulated emission of radiation

96
Q

○ For Rickettsia and other bacteria
○ For study of early degenerative process

A

CHROMATE FIXATIVES

97
Q

● Generally for acid mucopolysaccharides

A

LEAD FIXATIVES

98
Q

● May be used for the fixation of umbilical cord/wharton’s jelly

A

LEAD FIXATIVES

99
Q

● Used as:
○ Fixative
○ Decalcifying agent
○ Stain

A

PICRIC ACID FIXATIVES

100
Q

● Versatile solution
● Highly explosive when dry
○ When transported it is in a liquid form/solution

A

PICRIC ACID FIXATIVES

101
Q

● Produces excessive yellow staining of tissues
○ Can be an advantage/disadvantage
depending on viewpoint

A

PICRIC ACID FIXATIVES

102
Q

● Picrates are formed upon protein; precipitates are soluble in water; hence tissues must be first rendered insoluble by direct immersion in 70% ETOH
● Picrate fixatives must never be washed in water before dehydration

A

PICRIC ACID FIXATIVES

103
Q

Also an example of picric acid
fixative

A

Brasil’s alcoholic picro formol

104
Q

● Recommended for embryos &
pituitary biopsies

A

Bouin’s solution

105
Q

What fixative is recommended for embryos?

A

Bouin’s

106
Q

What is used for tumor biopsy of skin?

A

Heidenhain susa

107
Q

What fixative is used for bone marrow biopsy?

A

B-5

108
Q

● Fixes nucleoprotein
○ Able to preserve nucleoprotein

A

GLACIAL ACETIC ACID

109
Q

● Destroys mitochondria & golgi bodies
○ Not a component of cytoplasmic fixatives
● Solidifies at _____

A

GLACIAL ACETIC ACID

17C

109
Q

● Causes tissues to swell/shrink
○ Can be paired with mercuric chloride because it can shrink which glacial acetic neutralizes the shrinking effect

A

GLACIAL ACETIC ACID

110
Q

ALCOHOLIC FIXATIVES

● Generally recommended for:

A

Glycogen fixation

110
Q

movement of glycogen granules towards the ends or poles of the
cells

A

Polarization

111
Q

One of the disadvantages of these fixatives is

A

Polarization

112
Q

What group of fixative which we cannot use for glycogen fixation?

A

Mercuric chloride

113
Q

What group of fixative that can decrease the amount of demonstrable glycogen?

A

Mercuric chloride

114
Q

● Also known as methyl alcohol
● Used for the fixation of bone marrow smears and blood smears

A

Methanol

115
Q

Is bone marrow smear and bone marrow biopsy different?

A

YES

116
Q

Used for bone marrow biopsy?

A

B-5

117
Q

Used for bone marrow smear?

A

Methanol

118
Q

● Used for fixation of cytological smears

(Ex: Pap Smear - 95% Ethanol)

A

Ethanol

119
Q

● Most rapid fixative
- Fixation Time:

A

Carnoy’s Fluid

1-3 hrs

120
Q

● Uses:
- Preservation of chromosomes**
- Preservation of glycogen
- Fixation of urgent biopsies and
lymph glands
- Preservation of brain tissues for
RABIES diagnosis
- Acetone can also be used

A

Carnoy’s Fluid

121
Q

● Uses:
- Preservation of chromosomes**

A

Carnoy’s Fluid

122
Q

● Also known as “Gendre’s Fixative”
● Useful in preserving sputum

A

Alcohol Formalin

123
Q

● Functions as both nuclear and
histochemical fixative

A

Newcomer’s Fluid

124
Q

● Used for the preservation OR fixation of mucopolysaccharides and nuclear proteins

A

Newcomer’s Fluid

125
Q

Fixatives for biopsy specimens

Tumor biopsies:
Bone Marrow Biopsies:
Pituitary Biopsies:
Urgent Biopsies & Lymph Glands:

A

Tumor biopsies: Heidenhain’s SuSa
Bone Marrow Biopsies: B5 Fixative
Pituitary Biopsies: Bouin’s Solution
Urgent Biopsies & Lymph Glands: Carnoy’s Fluid

126
Q

Precaution: OSMIUM TETROXIDE FIXATIVES

A

Cause Conjunctivitis or Blindness

126
Q

● Inhibits hematoxylin and makes counter staining difficult
- Avoid using osmium tetroxide when performing H & E (hematoxylin and eosin) stain.
● Utilized also for electron microscopy

A

OSMIUM TETROXIDE FIXATIVES

126
Q

● Kept in a dark-colored bottle to prevent evaporation and reduction by sunlight or organic matter

A

OSMIUM TETROXIDE FIXATIVES

127
Q

● It produces black precipitate called
○ It is removed by cold water

A

OSMIUM TETROXIDE FIXATIVES

128
Q

2 KINDS OF OSMIUM TETROXIDE

A

Flemming’s Fluid

Flemming’s Fluid Without Acetic Acid

129
Q

OSMIUM TETROXIDE
● Example of Nuclear fixative

A

Flemming’s Fluid

130
Q

OSMIUM TETROXIDE
● Example of Cytoplasmic fixative

A

Flemming’s Fluid Without Acetic
Acid

130
Q

Function as both a Fixative and Decalcifying agent

A

TRICHLOROACETIC ACID (TCA)

131
Q

Optimum working temperature for cryostat: -18 to -20

A

ACETONE

131
Q

Acetone is used at ice cold temperatures -5 to 4°C

A

ACETONE

132
Q

● It is used for the demonstration of water-diffusible enzymes (like
phosphatases and lipases)

A

ACETONE

133
Q

● Fixation of brain tissue for rabies diagnosis
○ ** Carnoy’s fixative is also
used for rabies

A

ACETONE

134
Q

2 Methods of Fixation

A
  • Direct Method
  • Microwave Fixation
135
Q

It is also applied in bacterial smears
● In gram staining, you put a bacterial colony in the slide then you heat fix it

A

Direct Method

136
Q

● Optimum temperature: Microwave Fixation

A

45 to 55 °C

137
Q

● It is non-chemical technique useful for preservation of neurochemical substances in the brain (such as acetylcholine)

A

Microwave Fixation

138
Q

What is the optimum working temperature for cryostat?

A

-18 to 20°C

139
Q

What is the working temperature for acetone?

A

-4 to 5°C

140
Q

What is the optimum temperature for microwave
fixation?

A

45 to 55°C

141
Q

What is the appropriate temperature for the fixation of
surgical specimens?

A

Room temperature

142
Q

What is the temperature utilized for the fixation of electron microscopy and some histochemistry?

A

0 to 4°C

143
Q

FIXATIVES FOR ELECTRON MICROSCOPY (5)

A

● Glutaraldehyde
○ Used in enzyme chemistry but also used in
microscopy
● Platinic Chloride
● Platonic Chloride-formalin (Zamboni’s Gold Chloride)
● Osmium Tetroxide
● 10% BNF (Buffered Neutral Formalin)

144
Q

FACTORS THAT AFFECT FIXATION

RETARTED BY:

A
  1. Size and thickness of the tissue
  2. Presence of mucus
  3. Presence of blood
  4. Presence of fats
  5. Cold temperature
145
Q

○ Too large or too thick tissues may not be fixed
right away or preserved properly
○ Rate of penetration of formalin: __________
○ Longer fixation time if tissue is thick or big

A

Size and thickness of the tissue

1mm/hr

146
Q

FACTORS THAT AFFECT FIXATION

ENHANCED BY: (3)

A
  1. Size and thickness of the tissue
  2. Agitation
  3. Moderate heat
147
Q

○ Agitating the container will help the fixative to penetrate through the tissues
○ The machines in histopath allow continuous agitation of the tissue cassettes while they are
immersed in a formalin fixative during the course of a 24 hour fixation.

A

Agitation

147
Q

○ Small or thin tissues will be fixed quickly and properly

A

Size and thickness of the tissue

148
Q

○ Specifically 37-56°C
○ Increasing the temperature slightly from room temperature will cause the fixative to better
preserve the tissue.

A

Moderate heat

HISTOPATH MTAP (5 decks)

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