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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
Urgent biopsies
Formol-Nitric Acid
26
Has chromic acid and ROH (thus, no tissue breakup)
Perenyi's fluid
27
Also a tissue softener
Perenyi's fluid
28
Most rapid decalcifying agent
Phloroglucin Nitric acid
29
Complete decalcification cannot be determined through chemical means
Phloroglucin Nitric acid
30
Removed with 3 changes of 70-90% ETOH
Phloroglucin Nitric acid
31
Slower and causes more distortion compared to HNO3
Hydrochloric acid
32
Provides good nuclear staining
Hydrochloric acid
32
Recommended for surface decalcification if used in 1% with 70% ROH
Hydrochloric acid
33
HCl + 36% NaCl
Von Ebner's
34
1 TYPE OF Hydrochloric acid:
Von Ebner's
35
For teeth and small bones
Von Ebner's
36
Only weak acid used as a primary decalcifying agent
Formic Acid
36
Complete decalcification cannot be determined through chemical means
Von Ebner's
37
For routine decalcification of post-mortem research tissues, small pieces of bone and teeth, immunohistochemical staining
Formic Acid
38
# ``` Better nuclear staining, less tissue distortion than HNO3
Formic Acid
39
Addition of citrate  faster decalcifying rate
Formic Acid
40
P: autopsy, BM, cartilage, research tissues
Formic acid-Sodium citrate solution
41
Small bone spicules
Trichloroacetic acid
42
Good nuclear staining, does not require washing out
Trichloroacetic acid
43
Weak decalcifying agent and very slow
Trichloroacetic acid
44
Very weak agent thus for minute pieces of bone pieces only
Sulfurous acid
45
Both a fixative and decalcifying agent
Chromic acid (Flemming's Fluid)
46
Minute bone spicules
Chromic acid (Flemming's Fluid)
47
DADV: highly corrosive to skin, carcinogenic, and environmental toxin
Chromic acid (Flemming's Fluid)
48
Excellent nuclear and cytoplasmic staining but too slow
Citric acid-citrate buffer solution
49
pH 4.5
Citric acid-citrate buffer solution
50
For immunohistochemistry, enzyme staining, and electron microscopy
CHELATING AGENTS
51
Principle: Use of other salts to form weakly dissociated complexes with calcium salts for ease of removal
CHELATING AGENTS
52
CHELATING AGENTS Duration:
1-3 weeks for small specimens; 6-8 weeks for longer & dense bones
53
CHELATING AGENTS Optimum pH:
7-7.4
54
CHELATING AGENTS Examples:
Cal-Ex & Versene (EDTA)
55
Hastens decalcification by removing calcium ions from formic acid-containing decalcifying solutions, thereby increasing solubility from the tissue
ION EXCHANGE RESINS
56
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)
ION EXCHANGE RESINS
57
Not recommended for fluids with mineral acids such as HNO3 and HCl
ION EXCHANGE RESINS
58
ION EXCHANGE RESINS Duration:
1 to 14 days
59
Calcium ions (positively charged) moves to cathode (negative electrode)
ELECTROPHORESIS
60
Uses 88% formic acid
ELECTROPHORESIS
60
For small bone fragments
ELECTROPHORESIS
60
Shorter time for calcium removal because of heat and electrolytic reaction involved
ELECTROPHORESIS
61
Decalcification must be frequently monitored to avoid maceration of tissue. The methods of measuring extent of decalcification are as follows:
1. Physical/Mechanical Test 2. Radiologic MethodChemical or X-Ray 3. Chemical Method
62
# MEASURING EXTENT OF DECALCIFICATION Touching and bending tissue using fingers; and poking using fine needle or a probe
Physical/Mechanical Test
63
# MEASURING EXTENT OF DECALCIFICATION Rubbery consistency, and soft = Tissue is decalcified
Physical/Mechanical Test
64
# MEASURING EXTENT OF DECALCIFICATION DADV: vague and inaccurate artifact production, destruction of cellular details, small calcified foci may not detected
Physical/Mechanical Test
65
# MEASURING EXTENT OF DECALCIFICATION ADV: most ideal, most sensitive, and most reliable method; can detect smallest focus of calcium
Radiologic Method or X-Ray
66
# 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
Radiologic Method or X-Ray
67
# MEASURING EXTENT OF DECALCIFICATION Opaque result = Incomplete decalcification
Radiologic Method or X-Ray
68
# MEASURING EXTENT OF DECALCIFICATION DADV: very expensive, never used with HgCl2-fixed tissues (radio-opacity, = xrays do not pass through)
Radiologic Method or X-Ray
69
# MEASURING EXTENT OF DECALCIFICATION Simple, reliable, and convenient
Chemical Method
70
# MEASURING EXTENT OF DECALCIFICATION PCPL: precipitation of calcium hydroxide or calcium oxalate
Chemical Method
71
# 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
Chemical Method
72
# MEASURING EXTENT OF DECALCIFICATION If no cloudiness, add saturated aqueous solution of ammonium oxalate, and let it stand for 30 minutes  Cloudiness = incomplete decalcification
Chemical Method
73
# POST-DECALCIFICATION Removal of decalcifying agent through:
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
# TISSUE SOFTENERS Performed prior to dehydration or sectioning; for unduly hard tissues that may damage microtome knives. The agents are as follows
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 (13 decks)

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