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HISTOPATH LEC - TRANS > 3-DECALCIFICATION > Flashcards

3-DECALCIFICATION Flashcards

(74 cards)

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1
Q

What is the removal of calcium ions from bones or calcified tissues through a histological process?

A

Decalcification

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2
Q

What tissues require calcium removal during histological processing?

A

Bones+teeth+calcified tumors+calcified heart valves

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3
Q

What are the three main types of agents used to remove calcium from tissues?

A

Strong mineral acids+weaker organic acids+chelating agents

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4
Q

What method uses ion exchange to form soluble calcium salts for calcium removal?

A

Strong mineral acids

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5
Q

What risks occur if strong mineral acids are used excessively?

A

Loss of nuclear staining+maceration of tissues

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6
Q

What strong acid is most common for urgent biopsies due to rapid action?

A

Nitric acid

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7
Q

What concentration of nitric acid is typically used for rapid decalcification?

A

5-10% aqueous solution

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8
Q

What are the advantages of 10% aqueous nitric acid for large mineralized bones?

A

Rapid action+minimal distortion+good nuclear staining+easy removal with 70% alcohol

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9
Q

What are the disadvantages of 10% aqueous nitric acid?

A

Tissue distortion+yellow color+damage to tissue antigens

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10
Q

What decalcifying agent combines formaldehyde and nitric acid to reduce tissue damage?

A

Formol-Nitric Acid

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11
Q

What steps neutralize Formol-Nitric Acid after decalcification?

A

Neutralizing with 5% sodium sulfate or adding 0.1% urea to nitric acid

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12
Q

What slow decalcifying agent avoids maceration and softens tissues?

A

Perenyi’s Fluid

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13
Q

What rapid decalcifier uses phloroglucin but causes extreme tissue distortion?

A

Phloroglucin-Nitric Acid

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14
Q

What are the disadvantages of Phloroglucin-Nitric Acid?

A

Poor nuclear staining+yellow color+inability to chemically confirm complete decalcification

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15
Q

What acid is recommended for surface decalcification with good nuclear staining?

A

Hydrochloric acid

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16
Q

What are the drawbacks of hydrochloric acid?

A

Slower action+greater tissue distortion

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17
Q

What decalcifier is used for teeth and small bones without requiring post-washing?

A

Von Ebner’s Fluid

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18
Q

What limitation does Von Ebner’s Fluid have?

A

Inability to chemically measure decalcification completion

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19
Q

What resin-embedded bone section method demonstrates calcium (black) and osteoid (blue)?

A

Von Kossa staining

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20
Q

What artifact occurs in un-decalcified bone fragments forced into marrow spaces during preparation?

A

Tissue compression artifacts (arrows in trephine specimens)

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21
Q

What staining method visualizes calcium in un-decalcified resin sections?

A

Von Kossa’s method

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22
Q

What type of decalcifying agents are slower but gentler in action and less likely to interfere with nuclear staining?

A

Weak acids

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23
Q

What weak acid is moderate-acting and provides better nuclear staining with less tissue distortion?

A

Formic acid

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24
Q

What concentration of formic acid is considered the best all-around decalcifier for routine use?

A

0.1

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25
What are the advantages of formic acid as a decalcifying agent?
Acts as both fixative and decalcifier+excellent nuclear and cytoplasmic staining+suitable for routine surgical and IHC applications
26
What are the disadvantages of formic acid?
Relatively slow+requires neutralization with 5% sodium sulfate and washing out
27
What weak acid solution provides better nuclear staining for autopsy materials
bone marrow
28
What are the disadvantages of formic acid-sodium nitrate solution?
Requires neutralization with 5% sodium sulfate+not recommended for routine or dense tissues
29
What weak acid provides good nuclear staining without requiring washing out but acts very slowly?
Trichloroacetic acid
30
What are the disadvantages of trichloroacetic acid?
Weak decalcifying agent+very slow-acting
31
What fixative and decalcifying agent inhibits nuclear staining with hematoxylin and forms insoluble pigments?
Chromic acid (Flemming’s fluid)
32
Why is chromic acid rarely used nowadays?
Health hazards+nuclear staining inhibition with hematoxylin+formation of insoluble pigments
33
What buffer solution provides excellent nuclear and cytoplasmic staining but acts too slowly?
Citric acid-citrate buffer solution (pH 4.5)
34
What type of decalcifying agents combine with calcium ions to form weakly dissociated complexes
preserving nuclear DNA?
35
Why are chelating agents preferred for histochemical methods or enzyme studies?
Acids destroy nucleic acids
36
For what applications are chelating agents recommended?
Research+IHC+FISH+PCR+molecular studies requiring intact nucleic acids or enzymes
37
What chelating agent provides excellent soft-tissue integrity and quality staining but requires weeks to decalcify dense cortical bone?
Ethylenediaminetetraacetic acid (EDTA)
38
What concentration and pH is neutral EDTA typically used at for decalcification?
14% neutralized solution+pH 7.0 (7-7.6)
39
How long does EDTA take to decalcify small specimens vs. dense cortical bone?
1-3 weeks for small specimens+6-8 weeks for dense cortical bone
40
What are the advantages of neutral EDTA as a decalcifier?
Excellent staining results+minimal cell/tissue distortion+minimal histological artifacts+suitable for enzyme or immunohistochemical testing and electron microscopy (EM)
41
What are the disadvantages of neutral EDTA?
Very slow turnaround time+slight tissue hardening+inactivates alkaline phosphatase activity
42
What technique involves the use of EDTA with ultrasound to accelerate decalcification for molecular analysis?
Sonication
43
What are the advantages of sonication in decalcification?
Speeds up decalcification+preserves tissue morphology and antigenicity
44
What resin-based method removes calcium ions from formic acid-containing solutions to hasten decalcification?
Ion exchange resin
45
What are the advantages of ion exchange resin?
Well-preserved cellular detail+excellent staining results+minimal cell/tissue distortion+eliminates daily washing of solution
46
What are the disadvantages of ion exchange resin?
Very slow+causes slight tissue hardening
47
What method uses electrical ionization to attract positively charged calcium ions to a negative electrode?
Electrophoresis
48
What are the advantages of electrophoresis for decalcification?
Shortens decalcification time+satisfactory for small bone fragments
49
What are the disadvantages of electrophoresis?
Good cytologic and histologic details are not always preserved+heat may damage tissues
50
What technique uses microwave energy to accelerate decalcification by intermittently exposing tissues to heat while changing solutions?
Microwave-assisted decalcification
51
What is the primary advantage of microwave-assisted decalcification?
Reduces decalcification time significantly (from days to hours)
52
How does concentration affect the rate of decalcification?
Higher concentrations and greater amounts increase speed (recommended ratio: 20:1)
53
Why is fluid access important in decalcification?
Ensures ready access to all tissue surfaces+enhances diffusion and penetration+facilitates calcium removal
54
How does tissue size and consistency affect decalcification time?
Smaller/softer tissues decalcify faster while dense bones may require up to 14 days
55
How does agitation improve decalcification efficiency?
Mechanical agitation or sonication facilitates solution penetration and calcium removal
56
What temperature range is ideal for decalcification?
18-30°C
57
What physical methods are used to check decalcification completion?
Bending+probing+trimming+weighing (if tissue becomes soft or cuts easily with a blade)
58
What are the risks of physical end-point tests?
Tissue damage/loss from repeated probing or cutting
59
What chemical method detects calcium ions in the decalcifying solution?
Calcium oxalate test (ammonium hydroxide+ammonium oxalate)
60
What result in the calcium oxalate test indicates incomplete decalcification?
Cloudy solution (calcium oxalate precipitate)
61
What result indicates complete decalcification in the calcium oxalate test?
Clear solution (no precipitate)
62
Why are chemical tests rarely used despite accuracy?
Time-consuming+requires multiple reagents+each tissue must be tested separately
63
What is the gold standard method for end-point determination?
X-ray (calcified areas appear white; decalcified areas black)
64
What are the disadvantages of X-ray for end-point testing?
High cost+limited availability in labs
65
What is required after decalcification and before processing?
Extensive tap water washing to remove residual acid/EDTA
66
What alkaline solutions neutralize acid-decalfied tissues?
Saturated lithium carbonate or 5-10% sodium bicarbonate
67
How should EDTA-decalfied tissues be treated before alcohol exposure?
Avoid direct 70% alcohol immersion; wash thoroughly first
68
What solution preserves acid-decalfied tissues for frozen sections?
Formol-saline with 15% sucrose or PBS with 15-20% sucrose
69
What method removes unexpected calcium deposits in paraffin blocks?
Surface decalcification with 10% hydrochloric acid on gauze for 1 hour
70
When is surface decalcification performed?
After processing when calcified areas disrupt microtomy (e.g.
71
What artifact occurs if calcium is not removed before sectioning?
Holes or tears in tissue sections (e.g.
72
What agents soften paraffin-embedded tissues for microtomy?
Perenyi’s fluid+4% phenol solution+Molliflex+1-2% hydrochloric acid in alcohol
73
What preliminary steps ensure proper decalcification?
Specimen assessment+thorough fixation+thin tissue slices (2-4 mm)
74
What factors optimize decalcifier efficiency?
Adequate volume (20:1 ratio)+regular solution changes+proper end-point testing

HISTOPATH LEC - TRANS (29 decks)

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