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HISTOPATH MTAP > DECALCIFICATION - DEHYDRATION - CLEARING > Flashcards

DECALCIFICATION - DEHYDRATION - CLEARING Flashcards

1
Q

● Not commonly done in the Histopath Laboratory
● Only certain hard human tissues need decalcification
(bone, teeth, nails)
● Same ratio in fixation and decalcification

A

DECALCIFICATION

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2
Q

● High concentrations and greater amounts of fluid will increase the speed of the process
● More concentrated acid solutions decalcify bone more rapidly but may cause more harm to the tissue.

A

DECALCIFICATION

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3
Q

FD CIETS SML

A

Fixation → Dehydration → Clearing → Impregnation, → Embedding → Trimming → Sectioning → Staining → Mounting → Labeling

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4
Q

● Heat can hasten _____________
but can also damage the tissue

A

decalcification

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5
Q

Recommended ratio of fluid to tissue volume for decalcification

A

20:1

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6
Q

Ideal time required for Decalcification

A

24-48 hours

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7
Q

● Dense bone tissues usually
require up to ___ days or longer
in order to complete the
process.

A

14 days

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8
Q

At ___C impaired nuclear staining of Van Gieson’s stain for collagen fibers.

A

At 37 C

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8
Q

Optimum temperature = Decalcification

A

Room temp (18-30°C)

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8
Q

At __ C = tissue will undergo
complete digestion within
24-48 hours.

A

55 C

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9
Q

DECALCIFYING AGENTS (4)

A

● Acids
● Chelating Agents
● Ion Exchange Resins
● Electrical Ionization

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10
Q

● Most commonly used, but not the best decalcifying agent

A

Nitric Acid

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11
Q

○ EDTA (Ethylene Diamine Tetraacetic Acid)
○ Used as an anticoagulant to bind calcium

A

Chelating Agents

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12
Q

2 examples of Nitric Acid

A

○ Perenyi’s Fluid
○ Phloroglucin-Nitric Acid

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13
Q
  • functions as both decalcifying agent and tissue softener
A

Perenyi’s Fluid

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14
Q

most rapid decalcifying agent

A

Phloroglucin-Nitric Acid

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15
Q
  • best general decalcifying agent
A

5% Formic Acid

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15
Q

● Both a fixative and decalcifying agent
● Recommended for small pieces of bones and teeth

A

Formic Acid

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16
Q

Example of Hydrochloric Acid

A

Von Ebner’s Fluid

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17
Q

What is the best general decalcifying agent?

A

5% Formic acid

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17
Q

recommended for teeth and small pieces of bones

A

Von Ebner’s Fluid

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18
Q

What kind of acid makes up Von Ebner’s?

A

Hydrochloric acid

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18
Q

What is the most commonly used decalcifying agent?

A

Nitric Acid

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19
Q

3 WAYS TO MEASURE DECALCIFICATION

A
  1. Physical/Mechanical Test
  2. X-ray/Radiological Method
  3. Chemical Method
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20
Q

● Inaccurate since it is very subjective
● Commonly used in schools since it is cheap.

A

Physical/Mechanical Test

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21
Q

● Done by touching or bending tissue with the fingers
● Alternate method: pricking the
tissue with a fine needle or a probe

A

Physical/Mechanical Test

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22
Q

● For unduly hard tissues that may damage the microtome knives
● Utilized to soften tough tissue

A

TISSUE SOFTENERS

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22
Q

● Most ideal, Most Sensitive, Most
reliable method.
● The best way to measure the extent of decalcification

A

X-ray/Radiological Method

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23
Q

● Very Expensive; not commonly done
● Uses X-ray to measure the extent of decalcification

A

X-ray/Radiological Method

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24
Q

● Calcium Oxalate test
● Simple, reliable, recommended for routine purposes

A

Chemical Method

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25
Q

● Must not be skipped so that the next step, Impregnation (Paraffin Wax), to completely fill the spaces in between the specimen.

  1. Water and Paraffin Wax does not mix
A

DEHYDRATION

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26
Q

(4) Examples of TISSUE SOFTENERS

A

● 4% aq. Phenol.
● Molliflex
● 2% HCl
● 1% HCl in 70% alcohol

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27
Q

● We want the impregnating medium to be able to penetrate
the spaces between the organ and tissue
● ___________ fluids are generally used in increasing strengths (all the aqueous tissue fluids are removed but with little disruption to the tissue due to diffusion currents)

A

DEHYDRATION

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27
Q

● Aims to remove fixative and water from the tissue and replacing them with dehydrating fruit in preparation for impregnation

A

DEHYDRATION

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28
Q

● Used in increasing strengths or
ascending grades
○ In order for the tissue to not be
distorted
○ 70% Alcohol → 80% Alcohol →
90% Alcohol → 100% Alcohol (2x)

A

Alcohol

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29
Q

● MOST COMMONLY USED DEHYDRATING AGENT
● Also used for fixation

A

Alcohol

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30
Q

For routine dehydration of
tissues. BEST DEHYDRATING
AGENT

A

Ethyl Alcohol

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31
Q

● Used for blood and tissue films

A

Methyl Alcohol

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32
Q

● Used for plant and animal microtechniques

A

Butyl Alcohol

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33
Q

● Less shrinkage and hardening than ethanol

A

Isopropyl Alcohol

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33
Q

● Denatured Alcohol
● Ethanol + Small amount of Methanol

A

Industrial Methylated Spirit

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34
Q

● Both Fixative and Dehydrating Agent
● Also used for diffusible enzymes, brain (for rabies diagnosis)

A

Acetone

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35
Q

● Sometimes, instead of using 100% again at the end of dehydration, ___________ can be used.

A

Acetone

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36
Q

● Both dehydrating and clearing agent at the same time

A

● Diethylene Dioxide and
Tetrahydrofuran

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37
Q

(6) COMMONLY USED DEHYDRATING AGENTS

A
  1. Alcohol
  2. Acetone
  3. Dioxane
  4. Tetrahydrofuran (THF)
  5. Cellosolve (Ethylene Glycol Monoethyl Ether)
  6. Triethyl Phosphate
38
Q

● Acts as a tissue softener when added with 95% ethanol
● If the tissue is already soft, no need to add this

A

4% Phenol

39
Q

ADDITIVES TO DEHYDRATING AGENTS (2)

A
  1. 4% Phenol
  2. Anhydrous Copper Sulfate
40
Q

● BOTH dehydrating and indicator of H2O (water) content (100% ETOH)

A

Anhydrous Copper Sulfate

41
Q

Anhydrous Copper Sulfate

● If copper sulfate is dry = ______ in color
● Has water/Hydrated = ________ in color (incomplete dehydration)

A

White
Blue

42
Q

A typical dehydration sequence for specimens NOT more than 4 mm thick would be:

A
  1. 70% ethanol - 15 min
  2. 90% ethanol - 15 min
  3. 100% ethanol - 15 min
  4. 100% ethanol - 15 min
  5. 100% ethanol - 30 min
  6. 100% ethanol - 45 min
43
Q

NOTE AND RECALL:

A

Higher concentration of alcohol = water concentration will decrease (slowly removing the water from interstitial spaces so tissues will not wrinkle)

After you remove the water, alcohol is now inside the interstitial spaces after the dehydration process

44
Q

What is the Best General Decalcifying Agent?

A

5% Formic Acid

45
Q

What is considered to be the Best General Tissue Fixative?

A

10% Buffered Neutral Formalin

46
Q

What is the Best Way to Measure the Extent of Decalcification?

A

X-Ray or Radiological Method

47
Q

What is the Best Dehydrating Agent?

A

Alcohol

48
Q

Most commonly used Impregnating Medium?

A

Paraffin Wax

49
Q

■ Not soluble to water
■ Not soluble to alcohol
■ Needs to be the one to fill the spaces in the specimens so that when it is cut in the microtome, there will be no empty holes.

A

Paraffin Wax

50
Q

Aka: DEALCOHOLIZATION

A

CLEARING

51
Q

● Making the tissues transparent
● Process of replacing the dehydrating fluid with a fluid that
is miscible with BOTH the dehydrating fluid and the
impregnating/embedding medium

A

CLEARING

52
Q

CLEARING AGENTS SUITABLE FOR ROUTINE USE:

● Most commonly used
● Most rapid

A

Xylene/Xylol

53
Q

● Some of the other uses:

○ Removes alcohol from tissue
section, fat, and paraffin
○ For cleaning tissue processors

A

Xylene/Xylol

54
Q

If xylene turns MILKY =
___________ DEHYDRATION

A

INCOMPLETE DEHYDRATION

55
Q

● Toxic to the liver after prolonged inhalation and it does not make the tissues transparent

A

Chloroform

56
Q

● Used for TOUGH tissue
specimens

A

Chloroform

57
Q

especially recommended for CNS tissues and cytological studies,
particularly of smooth muscles and skin

A

Cedarwood oil and clove oil

58
Q

Can be utilized to replace Xylene
because it is not as toxic and is
cheaper

A

Citrus Fruits Oils

59
Q
A
59
Q

Recommended for clearing
embryos, insects, and very
delicate specimens

A

Aniline Oil

60
Q

Why do we need to remove the alcohol?

A

The paraffin wax that will be utilized later on is not soluble to alcohol

60
Q

Aka: INFILTRATION

A

IMPREGNATION

61
Q

● Process of replacing the clearing agent with the infiltrating medium
● The medium used to infiltrate the tissue is usually the same medium used for embedding

A

IMPREGNATION

62
Q

FOUR TYPES OF TISSUE IMPREGNATION AND EMBEDDING MEDIA

A

1 Paraffin
2 Celloidin (Collodion)
3 Gelatin
4 Plastic

63
Q

● The man who introduced paraffin wax embedding:

A

Butschlii

64
Q

● Simplest, most common and the BEST infiltrating/embedding medium
● NOT recommended for fatty tissues

A

PARAFFIN

65
Q

Temperature of paraffin oven =

A

55-60 C

66
Q

Paraffin oven must be maintained at a temperature

A

2-5 C above the MP (Melting Point) of the paraffin wax

67
Q

● MP: 56-57 C
● Mixture of highly purified paraffin and synthetic plastic polymers
● More elastic and resilient than
paraffin
● SPECIMEN: For large dense tissue blocks such as bones and brain

A

Paraplast

68
Q

● MP: 56-58 C
● Less brittle and less compressible than paraplast

A

Embeddol

69
Q

● Recommended for embedding
eyes

A

Bioloid

70
Q

A product of paraffin, containing
rubber, with the same property as paraplast

A

Tissue Mat

71
Q

● MP: 46-48 C
● Harder than paraffin
● NOT soluble in water
● Soluble in 95% ETOH (ethyl alcohol) and other clearing agents
● Can be used for impregnation
without prior clearing of the tissue
○ as it is soluble in ETOH

A

Ester Wax

72
Q

● MP: 38-42 C or 45-56 C
● Mostly polyethylene glycols
○ No need to perform dehydration and clearing

A

Water-soluble waxes

72
Q
A
73
Q

Suitable for many enzyme
histochemical studies

A

Carbowax

73
Q

● Purified form of nitrocellulose

A

CELLOIDIN (COLLODION)

74
Q

● Suitable for specimens:
○ With large hollow cavities
○ Hard and dense tissues (bones and teeth)
○ Large tissue sections of the whole embryo

A

CELLOIDIN (COLLODION)

75
Q

2 METHODS FOR CELLOIDIN IMPREGNATION

A
  1. Wet celloidin
  2. Dry celloidin
76
Q

Recommended for bones, teeth, large brain sections, and whole organs/organs with large cavities

A

Wet celloidin

77
Q

Preferred for processing whole eye sections

A

Dry celloidin

78
Q

Name of the other impregnating medium that may also be for the eye section?

A

Bioloid

79
Q

Fixative For Embryos

A

Bouin’s solution

80
Q

Impregnation Medium
For Embryos

A

Celloidin

81
Q

Impregnating Medium
For Eyes

A

Bioloid or Celloidin

82
Q

● Share the same characteristic as the carbowax
● Rarely used except when dehydration is to be avoided
● Used when tissues are for histochemical and enzyme
studies

A

GELATIN

83
Q

● Embedding medium for delicate specimens and frozen
tissue sections
○ When we are using frozen tissues usually it is fresh meaning there is still a water inside
■ Even though there is still water inside the gelatin can still pass through

A

GELATIN

84
Q

● Water-soluble impregnating medium
○ Not a wax
○ No need for dehydration

A

Gelatin

85
Q

Why are we allowed not to do dehydration if we intend to use gelatin as our impregnating
medium?

A

Because gelatin is a water soluble
impregnating medium

Gelatin → Water-Soluble Impregnating Medium

86
Q

Why do we remove water?

A

○ To prepare the tissue into the paraffin wax impregnation
○ Paraffin wax is not soluble to water

87
Q

What is the impregnating medium that we can use for impregnating frozen sections?

A

Gelatin

87
Q

Used for Electron Microscopic Studies

A

PLASTIC / RESIN

88
Q

PLASTIC / RESIN are CLASSIFIED INTO 3 categories

A

1 Epoxy
2 Polyester
3 Acrylic

89
Q

Brand name: Bisphenol A

A

Araldite

90
Q

Brand name: Glycerol

A

Epon

90
Q

Brand name: Cyclohexene dioxide

A

Spurr

91
Q

Most commonly used routine tissue impregnation medium

A

Paraffin wax

92
Q

Impregnation medium for large dense tissues like bones, brain and whole organs (embryo

A

Celloidin

92
Q

Impregnation medium for
frozen sections

A

Gelatin

93
Q

Impregnation medium for
electron microscopic studies

A

Plastic / Resin

94
Q

Since Epon is a glycerol, and a
glycerol is a form of epoxy, Epon
therefore is used for _______
microscopic studies

A

Electron

94
Q

What is the use/purpose of EPON?

A

Utilize for electron microscopic studies

■ One of the impregnating medium to utilize for that purpose
■ A kind of glycerol epoxy plastic
impregnating medium
■ A brand name of glycerol epoxy
plastic impregnating medium

HISTOPATH MTAP (5 decks)

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