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HISTOPATHOLOGY > MIDTERM LEC: DECALCIFICATION > Flashcards

MIDTERM LEC: DECALCIFICATION Flashcards

1
Q

Follows fixation

A

DECALCIFICATION

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2
Q

Removal of calcium or lime salts from calcified tissues

A

DECALCIFICATION

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3
Q

Inadequate decalcification  poor cutting of hard tissues an damage to the knife edge during sectioning

A

DECALCIFICATION

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4
Q

Hastened by heat and agitation

A

DECALCIFICATION

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5
Q

With a common 2-day duration

A

DECALCIFICATION

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6
Q

Calcifications cause a grating sensation during sectioning
Remedy:

A

Remove block from the chuck, and place face down on cotton or gauze with 10% HCl

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7
Q

Hematoxylin-stained microcalcifications =

A

dark purple granular
masses with light purple halos

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8
Q

DECALCIFICATION IS DONE:

A

 Bone
 Teeth
 Teratoma (means monster)
 Calcified tissues: tuberculous organs, arteriosclerotic vessels

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9
Q

DECALCIFICATION GROSSING:

A

 Done before fixation
 Double gloves, eye protection glasses, face mask
 Specialized table for bone processing
 Bone specimens are grossed in fresh state
 Bone dust particles can be loaded with blood or infectious malts (osteomyelitis, gangrene)
 Fine fret-saw saw  hand razor

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10
Q

Factors Affecting Decalcification:

A
  1. Concentration
  2. Tissue-to-volume ratio
  3. Temperature
  4. Mechanical agitation
  5. Size & consistency of tissue sample
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11
Q

HIGH=faster, but may be more harmful to tissue

A

Concentration

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12
Q

Tissue-to-volume ratio

A

optimum: 1:20

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13
Q

HIGH=faster, but may be more harmful to tissue

A

Temperature

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14
Q

TEMPERATURE OPTIMUM:

A

18 to 30 degrees Celsius

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15
Q

Hastens decalcification

A

Mechanical agitation

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16
Q

Larger specimens will slow the rate of decalcification

A

Size & consistency of tissue sample

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17
Q

Methods of Decalcification:

A
  1. Acids
  2. Chelating Agents
  3. Ion Exchange Resin
  4. Electrical ionization (electrophoresis)
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18
Q

Most widely used, stable, easily available, relatively inexpensive:

A
  1. Nitric acid
  2. Hydrochloric acid
  3. Formic acid
  4. Trichloroacetic acid
  5. Sulfurous acid
  6. Chromic acid
  7. Citric acid
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19
Q

Most common and fastest agents

A

Nitric acid

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20
Q

Removed by 70% ROH during dehydration

A

Nitric acid

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21
Q

Imparts yellow coloration d/t nitrous acid formation
[Remedy: add Urea or Sodium thiosulfate/sulfate]

A

Nitric acid

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22
Q

NITRIC ACID TYPES:

A
  1. 10% Aqueous Nitric Acid
  2. Formol-Nitric Acid
  3. Perenyi’s fluid
  4. Phloroglucin Nitric acid
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23
Q

Urgent, needle and small biopsies

A

10% Aqueous Nitric Acid

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24
Q

Has formalin (allows less destruction)

A

Formol-Nitric Acid

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25
Q

Urgent biopsies

A

Formol-Nitric Acid

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26
Q

Has chromic acid and ROH (thus, no tissue breakup)

A

Perenyi’s fluid

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27
Q

Also a tissue softener

A

Perenyi’s fluid

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28
Q

Most rapid decalcifying agent

A

Phloroglucin Nitric acid

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29
Q

Complete decalcification cannot be determined through chemical means

A

Phloroglucin Nitric acid

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30
Q

Removed with 3 changes of 70-90% ETOH

A

Phloroglucin Nitric acid

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31
Q

Slower and causes more distortion compared to HNO3

A

Hydrochloric acid

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32
Q

Provides good nuclear staining

A

Hydrochloric acid

32
Q

Recommended for surface decalcification if used in 1%
with 70% ROH

A

Hydrochloric acid

33
Q

HCl + 36% NaCl

A

Von Ebner’s

34
Q

1 TYPE OF Hydrochloric acid:

A

Von Ebner’s

35
Q

For teeth and small bones

A

Von Ebner’s

36
Q

Only weak acid used as a primary decalcifying agent

A

Formic Acid

36
Q

Complete decalcification cannot be determined through chemical means

A

Von Ebner’s

37
Q

For routine decalcification of post-mortem research tissues, small pieces of bone and teeth, immunohistochemical staining

A

Formic Acid

38
Q

```

Better nuclear staining, less tissue distortion than HNO3

A

Formic Acid

39
Q

Addition of citrate  faster decalcifying rate

A

Formic Acid

40
Q

P: autopsy, BM, cartilage, research tissues

A

Formic acid-Sodium citrate solution

41
Q

Small bone spicules

A

Trichloroacetic acid

42
Q

Good nuclear staining, does not require washing out

A

Trichloroacetic acid

43
Q

Weak decalcifying agent and very slow

A

Trichloroacetic acid

44
Q

Very weak agent thus for minute pieces of bone pieces only

A

Sulfurous acid

45
Q

Both a fixative and decalcifying agent

A

Chromic acid (Flemming’s Fluid)

46
Q

Minute bone spicules

A

Chromic acid (Flemming’s Fluid)

47
Q

DADV: highly corrosive to skin, carcinogenic, and environmental toxin

A

Chromic acid (Flemming’s Fluid)

48
Q

Excellent nuclear and cytoplasmic staining but too slow

A

Citric acid-citrate buffer solution

49
Q

pH 4.5

A

Citric acid-citrate buffer solution

50
Q

For immunohistochemistry, enzyme staining, and electron microscopy

A

CHELATING AGENTS

51
Q

Principle: Use of other salts to form weakly dissociated complexes with calcium salts for ease of removal

A

CHELATING AGENTS

52
Q

CHELATING AGENTS Duration:

A

1-3 weeks for small specimens; 6-8 weeks for longer & dense bones

53
Q

CHELATING AGENTS Optimum pH:

A

7-7.4

54
Q

CHELATING AGENTS Examples:

A

Cal-Ex & Versene (EDTA)

55
Q

Hastens decalcification by removing calcium ions from formic acid-containing decalcifying solutions, thereby increasing solubility from the tissue

A

ION EXCHANGE RESINS

56
Q

Ion exchange resin (ammonium form of polystyrene resin) is spread over the bottom of container  put tissue on top  add
decalcifying agent (20 to 30 times the volume of the tissue)

A

ION EXCHANGE RESINS

57
Q

Not recommended for fluids with mineral acids such as HNO3
and HCl

A

ION EXCHANGE RESINS

58
Q

ION EXCHANGE RESINS Duration:

A

1 to 14 days

59
Q

Calcium ions (positively charged) moves to cathode (negative electrode)

A

ELECTROPHORESIS

60
Q

Uses 88% formic acid

A

ELECTROPHORESIS

60
Q

For small bone fragments

A

ELECTROPHORESIS

60
Q

Shorter time for calcium removal because of heat and electrolytic reaction involved

A

ELECTROPHORESIS

61
Q

Decalcification must be frequently monitored to avoid maceration of tissue. The methods of measuring extent of decalcification are as
follows:

A
  1. Physical/Mechanical Test
  2. Radiologic MethodChemical or X-Ray
  3. Chemical Method
62
Q

MEASURING EXTENT OF DECALCIFICATION

Touching and bending tissue using fingers; and poking using fine needle or a probe

A

Physical/Mechanical Test

63
Q

MEASURING EXTENT OF DECALCIFICATION

Rubbery consistency, and soft = Tissue is decalcified

A

Physical/Mechanical Test

64
Q

MEASURING EXTENT OF DECALCIFICATION

DADV: vague and inaccurate artifact production, destruction of cellular details, small calcified foci may not detected

A

Physical/Mechanical Test

65
Q

MEASURING EXTENT OF DECALCIFICATION

ADV: most ideal, most sensitive, and most reliable method; can detect smallest focus of calcium

A

Radiologic Method or X-Ray

66
Q

MEASURING EXTENT OF DECALCIFICATION

Rinse decal agent from tissue  Put tissue on waterproof polyethylene sheet on top of X-ray film  Expose to X-ray for 1 minute at 30kV  Leave until film is developed

A

Radiologic Method or X-Ray

67
Q

MEASURING EXTENT OF DECALCIFICATION

Opaque result = Incomplete decalcification

A

Radiologic Method or X-Ray

68
Q

MEASURING EXTENT OF DECALCIFICATION

DADV: very expensive, never used with HgCl2-fixed tissues (radio-opacity, = xrays do not pass through)

A

Radiologic Method or X-Ray

69
Q

MEASURING EXTENT OF DECALCIFICATION

Simple, reliable, and convenient

A

Chemical Method

70
Q

MEASURING EXTENT OF DECALCIFICATION

PCPL: precipitation of calcium hydroxide or calcium oxalate

A

Chemical Method

71
Q

MEASURING EXTENT OF DECALCIFICATION

Addition of strong ammonia to the discarded decalcifying fluid (until solution becomes ALK).
 Cloudiness = presence of Ca in the discarded fluid

A

Chemical Method

72
Q

MEASURING EXTENT OF DECALCIFICATION

If no cloudiness, add saturated aqueous solution of ammonium oxalate, and let it stand for 30 minutes
 Cloudiness = incomplete decalcification

A

Chemical Method

73
Q

POST-DECALCIFICATION

Removal of decalcifying agent through:

A
  1. Immersion in saturated lithium carbonate or 5-10% aqueous sodium bicarbonate solution for several hours;
  2. Rinsing in tap water (small spx=30 mins; large spx=1-4 hours);
  3. For frozen section, acid decalcified tissue is stored in formol saline with 15% sucrose or PO4-buffered saline (PBS) with 15- 20% sucrose at 4 OC before freezing
74
Q

TISSUE SOFTENERS

Performed prior to dehydration or sectioning; for unduly hard tissues that may damage microtome knives. The agents are as follows

A
  1. Perenyi’s
  2. 4% Aqueous Phenol
  3. Molliflex (Effect: tissues appear swollen/soapy [not a negative effect])
  4. 2% HCl
  5. 1% NaCl in 70% ROH

HISTOPATHOLOGY (12 decks)

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