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Histopathology (Lecture) Pathology & Fixative > Decalcification/Dehydration/Clearing > Flashcards

Decalcification/Dehydration/Clearing Flashcards

1
Q

Removal of calcium ions or lime salts from the organic extracellullar matrix, calcified collagen and surrounding tissues of bones.

A

Decalcification

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2
Q

Ideal bone thickness?

A

1-3mm

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3
Q

Decalcifying agent fluid to tissue ratio?

A

20:1

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4
Q

What speeds up decalcification?

A

Heat and Agitation

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5
Q

True or False:
More concentration acid solutions decalcify more rapidly but may destroy tissue

A

True

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6
Q

Optimal temperature for decalcification?

A

Room temp

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7
Q

The positively charged calcium ions are attracted to negative electrode from the decalcifying solution.

A

Electrolytic method

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8
Q

Most rapid method. Physically dependent upon the supply of electric current for calcium removal

A

Electrolytic method

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9
Q

Calcium is rapidly removed by the decalcifying soln contaning formic acid, increasing solubility from tissues

A

Ion Exchange Resin

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10
Q

This increases solubility of tissues to facilitate removal of calcium.

A

Ammonium form of Polyklear resin

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11
Q

What is the duration of Ion Exchange resin?

A

1-14 days

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12
Q

This is a potent anticoagulant but a weak decalcifying agent.

A

EDTA

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13
Q

Best decalcifier in EM and Immunohistochemistry

A

EDTA

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14
Q

Inactivates alkaline phosphatase activity remedy?

A

Magnesium chloride

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15
Q

EDTA specimen durations: small specimens, dense tissues

A

Small 1-3 weeks; Dense 6-8 weeks

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16
Q

Most common method of decalcification

A

Use of Acid

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17
Q

Most commonly used acid?

A

Nitric acid

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18
Q

Acid used Routinely with a 24hr duration of process

A

10% Aqueous Nitric acid

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19
Q

Nitric acid + Formaldehyde

What is its name and duration?

A

Formol Nitric acid; 1-3 days

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20
Q

Chromic acid + ethyl alcohol.

What is its duration?

A

Perenyi’s Nitric acid; 2-7 days

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21
Q

Double purpose; used as a decelcifying acid and tissue softener

A

Perenyi’s nitric acid

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22
Q

Phloroglucin + nitric acid
Considered the most rapid

A

Phloroglucin Nitric

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23
Q

Not routinely used; it is a weak and slow acid.

Used only for minute pieces of bone

A

Hydrochloric acid

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24
Q

HCl +. NaCl

For teeth and small pieces of bone.

Surface decalcification of plaques

A

Von Ebner’s

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25
Q

Recommended for decalcifying cartilage, respiratory spx, autopsy spx, bone marrow

A

Formic acid sodium citrate

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26
Q

These are weak and slow decalcifying agents

A

TCA, Sulfurous acid, (Hydrochloric acid)

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27
Q

Not commonly used acid because it is an environmental toxin and is highly corrosive on the skin

A

Chromic acid (Flemmings with HAC)

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28
Q

This acid contains chloroform as a preservative

A

Citric acid citrate buffer

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29
Q

What are the Methods of Decalcification?

A
  • Electrolytic method
  • Ion Exchange method
  • Use of Chelating agents: EDTA
  • Use of Acid
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30
Q

Not a reliable method of testing completeness of decal.

Done by bending, probing, pricking the tissue with needle

A

Physical/ Mechanical method

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31
Q

Most reliable mtd to test the completeness of decal.

Can detect even the smallest amount of calcium.

A

X-ray/ Radiologic method

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32
Q

In this method, opaqueness = incomplete decalcification

A

X-ray/ Radiologic method

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33
Q

Disadvantage of Radiologic method?

A

Not suitable for Mercuric chloride fixed tissues

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34
Q

Cloudiness = to be decalcified further

Clear = complete decalcification

A

Chemical method/ Calcium Oxalate test

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35
Q

State the process of Chemical method/ Calcium Oxalate test

A

5ml discarded fluid + strong ammonia —> check for alkalinity using litmus paper—> add ammonium oxalate —> stand for 30 mins

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36
Q

This test is carried out by adding calcium carbonate.

Bubbles = incomplete decalcification

A

Bubble test

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37
Q

Decalcified tissues are neutralised by immersing in what?

A

Immersing in saturated lithium carbonate or 5-10% sodium bicarbonate

38
Q

Most common method in neutralizing decalcified tissues

A

Rinsing in tap water

39
Q

What is the third method for post decalcification?

A

Store in formol saline with 15% sucrose OR PBS with 15-20% sucrose at 4C

40
Q

Tissue softeners for unduly hard tissues

A
  • 1-2% HCl in 70% alcohol
  • Serenyi’s fluid
  • Molliflex
  • Lendrum’s method
41
Q

Tissue softener, Lendrum’s method contain what

A

Spx in 4% phenol for 1-3 days

42
Q

This uses increasing concentration of alcohol

A

Dehydration

43
Q

Amount of dehydrating agent to tissue ratio

A

Should not be less than 10x the volume of the specimen

44
Q

Initial concentration for Routine and Delicate tissues?

A

Routine 70-80% ; Delicate 30%

45
Q

Routine dehydrating agent; fast acting and non-toxic

46
Q

Dehydrating agent not commonly used; toxic
Suited only for blood and tissue films

A

Methyl alcohol

47
Q

Dehydrating agent Recommended for plant and animal microtechnique

A

Butyl alcohol

48
Q

Ethanol and Xylene substitute; used in microwave technique

A

Isopropanol

49
Q

This dehydrating agent dissolved paraffin

50
Q

Prolonged storage in low concentration alcohol can _____________ tissues

51
Q

Prolonged storage in 70-80% can affect _______

52
Q

Hasten dehydration by

A

Applying Heat at 37C

53
Q

Accelerates dehydration and used as an Indicator for water saturation

Blue discolouration = full saturation of dehydrating fluids with water

A

Anhydrous copper sulfate

54
Q

Fixative and dehydrate tissues. Not commonly used.

Evaporates early and is highly flammable

55
Q

Used for urgent biopsy and fast-acting

56
Q

Dehydrating and Clearing agent. Tissues tend to ribbon poorly.

A

Diethylene dioxide

57
Q

Diethylene dioxide is also known as _________

Main disadvantage?

A

Dioxane; Extremely dangerous and toxic to man

58
Q

3 changes of pure dioxane —> 3 changes of paraffin wax, embed in cool water

A

Graupner’s method

59
Q

Tissue wrap in gauze bag and placed in a bottle with dioxane anhydrous calcium oxide or quicklime

A

Weisenberger’s method

60
Q

In Weisenberger’s method, Dioxane ___________; anhydrous calcium oxide or quicklime _________________

A

Removes water; absorbs water removed by dioxane

61
Q

Also known as cellosolve

A

Ethylene glycol monoethyl ether

62
Q

Ethylene glycol monoethyl ether combustible at what temp

63
Q

Toxic by ingestion, inhalation, skin contact.

Toxic to reproductive, fetal, urinary, and blood systems

A

Ethylene glycol monoethyl ether

64
Q

Decomposes upon exposure to sunlight

A

Ethylene glycol monoethyl ether

65
Q

Not commonly used; causes minimum shrinkage.

For sections and smears

A

Triethyl phosphate

66
Q

Can dehydrate and clear tissues
Toxic when inhaled and ingested; has an offensive odor

A

Tetrahydrofuran

67
Q

Vapors causes nausea, dizziness, and headache.

May cause conjunctival irritation

A

Tetrahydrofuran

68
Q

Removal of dehydrating agent from tissue to replace it with a medium that will dissolve the wax with wc the tissues is to be impregnated

A

Dealcoholization/Clearing

69
Q

Routine clearing agent; Fast-acting at 30min-hours

70
Q

Clearing agent is not suited for Nervous tissues and lymph nodes.

Incomplete dehydrated tissue turns this _______

A

Xylene; milky

71
Q

Fast acting; suited for urgent biopsies.

Highly flammable and carcinogenic, can damage BM leading to _________

A

Benzene; Aplastic anemia

72
Q

This clearing agent is a substitute for Xylene and Benzene ______

Clearing time ________

A

Toluene

1-2 hours

73
Q

This clearing agent acidifies in a partially filled vessel

74
Q

For rough tissues (/skin, fibroid, and decalcified tissues) for nervous tissues, lymph nodes, and embryos

A

Chloroform

75
Q

Tissues in chloroform tend to float, remedy for this is ________

A

To wrap the tissue in gauze

76
Q

Chloroform is toxic to what organ?

77
Q

This clearing agent is for cytological studies like smooth muscles of skin and CNS tissues

A

Cedarwood oil

78
Q

Cedarwood oil clearing time ___________

A

2-3 days; extremely slow clearing agent

79
Q

Agent for clearing agent for embryos, insects, and delicate spx

A

Aniline oil

80
Q

Agent where tissues tend to become adulterated

81
Q

This clearing agent is similar to chloroform but cheaper

A

Carbon tetrachloride

82
Q

Extremely slow agents used only in double embedding process

A

Methyl benzoate,
Amyl acetate,
Methyl salicylate

83
Q

This clearing agent is a substitute for Xylene

A

N-butyl acetate

84
Q

Clearing agent obtained from a citrus fruit

85
Q

Clearing agent for smooth muscles of the skin

A

Oil of bergamot
Oil of origanum

86
Q

Clearing agent for delicate tissues

A

Oil of wintergreen

87
Q

Clearing agent for smooth muscles with foul odor

A

Carbon disulfide

88
Q

Clearing agent for friable tissues

A

Carbon xylene

89
Q

Clearing agent for eye specimens; with faint odor and low evaporation rate. Good substitute for Cedarwood oil

90
Q

Excellent clearing agent

A

High Test Aviation Lead Free gasoline

91
Q

These clearing agents may be used for dense tissues like the uterus

A

Chloroform and Cedarwood oil

Histopathology (Lecture) Pathology & Fixative (2 decks)

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