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Histopathology Lec&Lab > MODULE 2 - Decalcification & Dehydration/Clearing > Flashcards

MODULE 2 - Decalcification & Dehydration/Clearing Flashcards

1
Q 
Removal of calcium ions or lime salts from the organic extracellular matrix, calcified collagen and surrounding tissues of bones
A. Decalcification
B. Fixation
C. Dehydration/Clearing
D. Embedding
A

A

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2
Q

Both decalcification and processing depend on

A

Bone thickness

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3
Q

Ideal thickness of bone tissues

A

1-3 mm

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4
Q

Decalcification must be done following

A

Fixation

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5
Q

Ratio of fluid to tissue in decalcification

A

20:1 or 10-20x the volume of tissue

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6
Q

Factors that can speed up decalcification

A

Heat and agitation

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7
Q

This can decalcify more rapidly but may destroy tissue.

A

Concentrated acid solutions

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8
Q

Optimal temperature in decalcification

A

Room temperature

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9
Q

Decalcification can be done on autotechnicon

True or false

A

False, it must be done MANUALLY

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10
Q

It is not usually done and not suited on urgent biopsies
A. Electrolytic method and Ion exchange resin
B. Use of acid and use of chelating agent
C. Ion exchange resin and use of chelating agent

A

C

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11
Q

It Inactivates alkaline phosphatase activity

A

Use of chelating agent: EDTA

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12
Q

A combination of nitric acid and formaldehyde

A

Formol nitric acid

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13
Q

It requires fumehood.

A

Formol nitric acid

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14
Q

Duration of ion exchange resin

A

1-14 days

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15
Q

Use of chelating agent: EDTA is best for

A

EM and immunohistochemistry

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16
Q 
Calcium is rapidly removed by the decalcifying solution containing formic acid, thereby increasingly solubility from tissues
A. Ion exchange resin
B. Electrolytic method
C. Use of Acid
D. Use of chelating agent: EDTA
A

A

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17
Q 
The positively charged calcium ions are attracted to negative electrodes from the decalcifying solution
A. Ion exchange resin
B. Electrolytic method
C. Use of Acid
D. Use of chelating agent: EDTA
A

B

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18
Q

Duration for small specimen in using chelating agent: EDTA

A

1-3 weeks

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19
Q

Duration for dense tissue in using chelating agent: EDTA

A

6-8 weeks

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20
Q

Duration in using Ion Exchange resin.

A

1-14 days

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21
Q

Use of Chelating agent: EDTA inactivates alkaline phosphatase activity, what is the remedy?

A

Add magnesium chloride

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22
Q

In ion exchange resin, tissue must be placed at the bottom of container with what to increase solubility from tissues

A

ammonium form of polystyrene resin

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23
Q 
The most rapid method of decalcification
A. Ion exchange resin
B. Electrolytic method
C. Use of Acid
D. Use of chelating agent: EDTA
A

B

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24
Q 
It is not routinely used because it is weak and slow. 
A. Nitric acid and formol nitric acid
B. Von ebner's
C. HCl and chromic acid w/ flemming's
D. HCl and TCA and sulfurous acid
A

D

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25
Q

4 solutions under Nitric acid

A

10% aqueous nitric acid
Formol nitric acid
Perenyi, chromic acid and ethyl alcohol
Phloroglucin nitric

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26
Q 
Most rapid type of nitric acid
A. 10% aqueous nitric acid
B. Formol nitric acid
C. Perenyi, chromic acid and ethyl alcohol
D. Phloroglucin nitric
A

D

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27
Q

It is commonly used but should not be used in concentrated solutions

A

Nitric acid

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28
Q

It contains chloroform as preservative

A

Citric acid citrate buffer

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29
Q

It is highly corrosive on skin and is considered as an environmental toxin

A

chromic acid and flemming’s w/ HAc

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30
Q

Used for cartilage, research, autopsy and BM specimen

A

Formic acid sodium citrate

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31
Q

Duration when using 10% aqueous nitric acid

A

24 hrs

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32
Q

Duration of using Formol nitric acid

A

1-3 days

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33
Q

Duration of using Perenyi’s nitric acid, chromic acid and ethnyl alcohol

A

2-7 days

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34
Q

Perenyi’s nitric acid, chromic acid and ethnyl alcohol all are:

A

2-in-1 decalcifying and softener

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35
Q

HCl is used for

Von Ebner’s is used for:

A

minute pcs of bones

teeth and small pcs of bones, surface decalcification of blocks

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36
Q

Physical / mechanical method is done by

A

bending, probing and pricking tissue with needle

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37
Q

Physical/mechanical method is reliable

True or false

A

False

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38
Q 
The easiest METHOD OF TESTING FOR THE COMPLETENESS OF DECALCIFICATION
A. Physical/Mechanical
B. X-ray/Radiological
C. Bubble Test
D. Chemical/Calcium oxalate
A

A

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39
Q

METHOD OF TESTING FOR THE COMPLETENESS OF DECALCIFICATION

It is done by adding calcium carbonate

A

Bubble test

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40
Q

Can detect even the smallest amount of calcium

A

X-ray/radiologic

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41
Q

Disadvantage of using X-ray / Radiologic Method

A

cannot be used on mercuric chloride fixed tissues

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42
Q 
The most reliable and accurate method
A. Physical/Mechanical
B. X-ray/Radiological
C. Bubble Test
D. Chemical/Calcium oxalate
A

B

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43
Q

In using x-ray method, appearance of this will indicate incomplete decalcification

A

opaqueness in x-ray films

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44
Q

In using chemical/calcium oxalate test, what will you add to make it alkaline?

A

Strong ammonia

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45
Q

You can check the solution if its alkaline by using

A

litmus paper

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46
Q

In using chemical/calcium oxalate test, what will be added after it becomes alkaline?

A

ammonium oxalate

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47
Q

In using chemical/calcium oxalate test, this result will indicate incomplete decalcification

A

Cloudiness

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48
Q

In using chemical/calcium oxalate test, for how long will you allow the solution to stand?

A

30 mins

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49
Q

In using chemical/calcium oxalate test, how many mL of discarded fluid will you put in the tube?

A

5 mL

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50
Q

Decalcified tissues are neutralized by

A

(1) Immersing in saturated lithium carbonate or 5-10% sodium bicarbonate
(2) Rinsing in tap water
(3) Storing in formol saline with 15% sucrose or PBS with 15-20% sucrose at 4 degC

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51
Q

Enumerate the tissue softeners used for unduly hard tissues

A

(1) 2% HCl or 1% HCl in 70% alcohol
(2) Perenyi’s fluid
(3) Molliflex fluid

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52
Q

What is lendrum’s method?

A

It is used for hard tissues like cervix and it is done by immersing tissues in 4% aqueous phenol for 1-3 days

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53
Q

Decalcification is mandatory

True or False

A

False

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54
Q

Dehydration is mandatory

True or False

A

True

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55
Q

In dehydration, we may use autotechnicon

True or False

A

True

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56
Q

It pertains to the removal of intercellular and extracellular water from tissues after _____ and prior to_____.

A

Fixation and wax impregnation

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57
Q

In dehydration, concentration used in delicate tissues like embryo may be

A

30% concentration

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58
Q

Dehydration uses _______ of alcohol.

A

Ascending/increasing concentration

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59
Q

In dehydration, the amount of dehydrating agent must be

A

not less than 10x the volume of specimen

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60
Q

In dehydration, the initial concentration for routine is

A

between 70-80%

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61
Q

In dehydration, low concentration tends to

A

macerate the tissues

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62
Q

In dehydration, higher/95% concentration will

A

harden only the surface, deeper parts will not be penetrated

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63
Q 
This is used as routine dehydrating agent, FAST and NON-TOXIC
A. Ethanol
B. Methyl alcohol
C. Butyl alcohol
D. Pentanol
A

A

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64
Q 
It can be used as ethanol and xylene substitute, usually used for microwave techniques
A. Ethanol
B. Methyl alcohol
C. Butyl alcohol
D. Isopropanol/IPA
A

D

65
Q 
It is not commonly used because it is toxic.
A. Ethanol
B. Methyl alcohol
C. Butyl alcohol
D. Pentanol
A

B

66
Q

Methyl alcohol as dehydrating agent can only be used for

A

blood and tissue films

67
Q 
It is used for plant and animal microtechniques
A. Ethanol
B. Methyl alcohol
C. Butyl alcohol
D. Pentanol
A

C

68
Q 
It dissolve paraffin.
A. Ethanol
B. Methyl alcohol
C. Butyl alcohol
D. Pentanol
A

D

69
Q

Enumerate dehydrating agents under alcohol

A
  1. Ethanol
  2. Methyl alcohol
  3. Butyl alcohol/butanol
  4. Isopropanol/IPA/isopropyl alcohol
  5. Tertiary butanol
  6. Pentanol
70
Q

In dehydration, prolonged storage in alcohol will

A

affect staining

71
Q

Dehydration can be hasten when you apply ___ @____

A

heat @37 degrees

72
Q

It can be used too accelerate dehydration process and acts as an indicator for water saturation

A

Anhydrous copper sulfate

73
Q

Where will you put anhydrous copper sulfate to accelerate dehydration process?

A

at bottom of the container

74
Q

In using anhydrous copper sulfate, this will indicate full saturation of dehydrating fluids with water.

A

Bluish discoloration

75
Q

Disadvantage of using acetone

A

evaporates easily and highly flammable

76
Q

It is fast-acting and is used for urgent biopsies, but NOT ROUTINELY USED.

A

Acetone

77
Q

Cellosolve is also known as

A

ETHYLENE GLYCOL MONOETHYL ETHER

78
Q

Main disadvantage of using Diethyl dioxide/dioxane

A

extremely dangerous and toxic to man

79
Q

Dioxane is also known as _____ and its 2nd disadvantage is

A

Diethylene dioxide, it ribbons poorly

80
Q

Dioxane can act both as ___ and ____

A

dehydrating and clearing agent

81
Q

2 Methods of Dioxane dehydration

A

Graupner’s and Weisenberger’s

82
Q

Cellosolve is combustible @ what temperature

A

110-120 degrees fahrenheit

83
Q

In Weisenberger’s – tissue wrap in gauze bag and placed in a bottle with _______ and ______________.

A

Dioxane and calcium oxide/quicklime

84
Q

Cellosolve decomposes upon exposure to _______.

A

sunlight

85
Q

ETHYLENE GLYCOL MONOETHYL ETHER is toxic to:

A

inhalation, ingestion, skin contact (prolonged exposure)

fetal, urinary, reproductive and blood systems

86
Q

It is used for sections and smears and it causes minimum shrinkage

A

Triethyl phosphate

87
Q

Triethyl phosphate causes _________

A

minimum shrinkage

88
Q

Both dehydrating ang clearing agents
A. Diethylene dioxide and Tetrahydrofuran
B. Ethylene glycol monoethyl ether and Triethyl phosphate
C. Diethylene dioxide and Triethyl phosphate

A

A

89
Q

It has offensive odor and may cause conjunctival irritation

A

Tetrahydrofuran (THF)

90
Q

Tetrahydrofuran is _______ and can cause

A

nausea, dizziness, headache and conjunctival irritation

91
Q

A fast acting and a routine clearing agent

A

xylene

92
Q

Can be a substitute for xylene and benzene

A

Toluene

93
Q

Xylene becomes _______ when an incompletely dehydrated tissue is immersed in it

A

milky

94
Q

rapid clearing time of xylene is

A

30 mins - hrs

95
Q

clearing time of toluene

A

1-2 hrs

96
Q

Xylene is not suitable for

A

lymph nodes and nervous tissue

97
Q

disadvantage of using toluene

A

acidifies in partially filled vessel

98
Q

Benzene is fast acting and is suited for

A

urgent biopsies

99
Q

Disadvantages of using benzene

A

highly flammable, carcinogenic and can damage BM leading to aplastic anemia

100
Q

Chloroform is used for

A 
rough tissues (skin, fibroid, decalcified tissues) 
lymph nodes, nervous tissues and embryos
101
Q

Chloroform is toxic to

A

liver

102
Q

Disadvantage of using chloroform and its remedy

A

tissue will float , wrap in gauze

103
Q

In using chloroform tissues do not become _______ and can lead to _____

A

translucent , wax deterioration

104
Q

may be used for dense tissues like uterus

A

chloroform and cedarwood oil

105
Q

Clearing time of cedarwood oil

A

2-3 days

106
Q

Cedarwood oil can be used for

A

cytological studies like smooth muscles of skin and CNS tissues

107
Q 
It is recommended for embryos, insects and delicate specimens
A. Chloroform
B. Benzene
C. Xylene
D. Aniline oil
A

D

108
Q 
Tissues tend to become adulterated
A. Chloroform
B. Clove oil
C. Xylene
D. Aniline oil
A

B

109
Q 
Similar to chloroform but cheaper
A. Chloroform
B. Clove oil
C. Carbon tetrachloride
D. Aniline oil
A

C

110
Q

extremely slow clearing agents and used only in double-embedding processes

A

Methyl benzoate, amyl acetate and methyl salicylate

111
Q

can be used as xylene substitute

A

N-butyl acetate

112
Q

obtained from citrus fruit

A

Limonene

113
Q

Oil of bergamot and Oil of origanum is used to clear

A

smooth muscles of the skin

114
Q

Oil of wintergreen is used for

A

delicate tissues

115
Q

Carbon disulfide is used for

A

for smooth muscles; with foul odor

116
Q

Carbon xylene is for

A

friable tissues

117
Q

Terpineol is used for and has _______________ and __________.

A

eyes, has faint odor, low evaporation rate

good substitute for cedarwood oil

118
Q

excellent clearing agent

A

High Test Aviation Lead Free gasoline-

119
Q 
Removal of dehydrating agent from tissues to replace it with a medium that will dissolve the wax with which the tissues to be impregnated
A. Decalcification
B. Fixation
C. Dealcoholization/Clearing
D. Embedding
A

C

120
Q

Prolonged clearing time will cause

A

tissues to be brittle

121
Q

The use of ____ will make tissues transparent and __________.

A

clearing, improve refractive index

122
Q

Incomplete clearing will cause

A

uneven H&E staining

poor nuclear chromatin patterns

123
Q

often used to assist in the selection of appropriate bone

specimens for processing.

A

Fine detail radiographs

124
Q

should be used to prepare bone slices.

A

High-quality fine tooth saws

125
Q

Bone specimens should be sawn into

A

thin slices

126
Q

can cause considerable mechanical damage and force

bone fragments into the soft tissues present in the specimen.

A

Coarse saws

127
Q

After fixation, depending on the amount of adjusted soft tissue, the toenail should be

A

rinsed off with soapy water once it becomes pliable

128
Q

does not require any softening

A

Cartilage

129
Q

three main types of decalcifying agents:

A

Those based on strong mineral acids
Those based on weaker organic acids
Those composed of chelating agents.

130
Q

what happens if specimens are exposed for too long to acidic decalcifying agents

A

Nuclear and cytoplasmic detail are compromised

it will extract RNA and remove the purine and pyrimidine bases from DNA

131
Q

effects of decalcifying agents on H&E staining can be reduced by

A

post-decalcification and removal, and by

appropriately adjusting the staining procedure.

132
Q

In Formol-Nitric Acid, the yellow color imparted by nitrous acid formation may be prevented by

A

neutralizing the tissue with 5% sodium sulfate and washing in running tap water for at least 12 hours.
Addition of 0.1% urea to pure concentrated nitric acid will
also make discoloration disappear without considerably affecting the
efficiency of the decalcifying solution

133
Q

In Phloroglucin-Nitric Acid, yellow color must be neutralized with

A

5% sodium sulfate and thoroughly washed with running tap water for at least 24 hours.

134
Q

It does not require washing out before dehydration

A

Von Ebner’s Fluid

135
Q

the only weak acid used extensively as a primary decalcifying
agent

A

Formic acid

136
Q

-is a very weak decalcifying solution suitable only for

minute pieces of bone.

A

SULFUROUS ACID

137
Q

pH of CITRIC ACID-CITRATE BUFFER SOLUTION

A

4.5

138
Q

The rate at which EDTA will decalcify is pH

A

7.0

139
Q

gives excellent results for soft-tissue

integrity, and best quality of both soft-tissue and hard-tissue staining

A

Neutral EDTA

140
Q

EDTA works more rapidly at pH

A

10

141
Q

The optimal pH when using EDTA

A

7-7.6

142
Q

EDTA when used under pH 5 will result in

A

insolubility

143
Q

EDTA when used over pH 8 will result in

A

tissue maceration

144
Q

used to accelerate decalcification of trephine specimens for subsequent molecular analysis.

A

Sonication with EDTA

145
Q

Due to the corrosive action of the acid, it is recommended that the thread be

A

be dipped in melted paraffin wax

146
Q

In decalcification, what happens at 37°C

A

there will be impaired nuclear staining of Van Gieson’s stain for collagen fibers.

147
Q

In decalcification, what happens at 55°C

A

the tissue will undergo complete digestion within 24-48 hours.

148
Q

Acid decalcified tissues for frozen sections must be

A

washed in water or stored in formol-saline containing 15% sucrose or phosphate-buffered saline (PBS) with 15-20% sucrose at 4°C before freezing.

149
Q

involves slow substitution of the water in the tissue with an organic solvent.

A

Dehydration

150
Q

In dehydration, 85-95% alcohol will cause

A

considerable shrinkage and hardening of tissues leading to distortion

151
Q

Another important role of the clearing agent is to remove a substantial amount of fat from the tissue which otherwise presents a barrier to wax infiltration.
True or False

A

True

152
Q

used when the tissue is to be cleared directly from water, as in
a frozen section.

A

Glycerin and gum syrup

153
Q

affects the speed of penetration of the clearing agent.

A

Viscosity

154
Q

It is used for clearing, both for embedding and mounting procedures.

A

Xylene (Xylol)

155
Q

Cedarwood oil becomes _______ upon prolonged storage and should be filtered before use.

A

milky

156
Q

offer the clearing action with the lowest hazard rating of all xylene alternatives.

A

Orange oil based clearing agents

157
Q

an efficient substitute for xylene, as it is non-hazardous, less expensive and causes less shrinkage of the tissue.

A

Coconut oil

158
Q

The only drawback associated with coconut oil

A

tendency to get solidified at a lower temperature

Histopathology Lec&Lab (7 decks)

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