CHAPTER 8 DECALCIFICATION Flashcards
1
Q
Strong Mineral Acids:
A
I. NITRIC ACID
II. HYDROCHLORIC ACID
III. FORMIC ACID
IV. TRICHLOROACETIC ACID
V. CHROMIC ACID (FLEMMING’S FLUID)
VI. CITRIC ACID-CITRATE BUFFER SOLUTION (pH 4.5)
2
Q
I. NITRIC ACID:
A
3
Q
II. HYDROCHLORIC ACID:
A
4
Q
III. FORMIC ACID:
A
5
Q
IV. TRICHLOROACETIC ACID:
A
6
Q
is the most common and the fastest decalcifying agent used so
far, utilized both as a simple solution or combined with other reagents.
A
NITRIC ACID
7
Q
This may be used as simple aqueous solutions with recommended concentrations of 5- 10%.
A
NITRIC ACID
8
Q
It is a very rapid decalcifying agent, producing minimal distortion and is, therefore, recommended for routine purposes.
A
NITRIC ACID
9
Q
It has, however, the disadvantage of inhibiting nuclear stains and destroying tissues, especially in concentrated solutions.
A
NITRIC ACID
10
Q
The endpoint of decalcification must be carefully watched for, to prevent progressive tissue damage and impaired staining. This may be prevented by combining nitric acid with formaldehyde or alcohol.
A
NITRIC ACID
11
Q
DECALCIFICATION TIME: 12-24 hours
A
Aqueous Nitric Acid Solution 10%
12
Q
- It is rapid in action.
A
Aqueous Nitric Acid Solution 10%
13
Q
- It produces minimum distortion of tissues.
A
Aqueous Nitric Acid Solution 10%
14
Q
- It produces good nuclear staining (although less than in slower acting agents).
A
Aqueous Nitric Acid Solution 10%
15
Q
- The acid may be easily removed by 70% alcohol.
A
Aqueous Nitric Acid Solution 10%
16
Q
- It is recommended for urgent biopsy, and for needle and small biopsy specimens to permit rapid diagnosis within 24 hours or less.
A
Aqueous Nitric Acid Solution 10%
17
Q
- It can be used for large or heavily mineralized cortical bone specimen if decalcification progress is carefully monitored by a decalcification endpoint test.
A
Aqueous Nitric Acid Solution 10%
18
Q
1 Prolonged decalcification may lead to tissue distortion.
A
Aqueous Nitric Acid Solution 10%
19
Q
- It can seriously damage tissue stainability.
A
Aqueous Nitric Acid Solution 10%
20
Q
- It imparts a yellow color with nitrous acid, thereby impairing the staining reaction of the tissue.
A
Aqueous Nitric Acid Solution 10%
21
Q
- Old nitric acid solution is particularly damaging and should be replaced with fresh stock solution.
A
Aqueous Nitric Acid Solution 10%
22
Q
- Strong acids tend to be more damaging to tissue antigens for immunohistochemical staining, and enzymes may be totally lost.
A
Aqueous Nitric Acid Solution 10%
23
Q
DECALCIFICATION TIME: 1-3 days
A
24
Q
- It is rapid-acting; hence, is recommended for urgent biopsies.
A
Formol-Nitric Acid
25
2. Nuclear staining is relatively good.
Formol-Nitric Acid
26
3. It produces less tissue destruction than 10% aqueous nitric acid.
Formol-Nitric Acid
27
1. The yellow color imparted by nitrous acid formation will impair staining reaction of the cell. This may be prevented by neutralizing the tissue with 5% sodium sulfate and washing in running tap water for at least 12 hours. Addition of 0.1% urea to pure concentrated nitric acid will also make discoloration disappear without considerably affecting the efficiency of the decalcifying solution.
Formol-Nitric Acid
28
2. The solution should be used inside a fume hood.
Formol-Nitric Acid
29
DECALCIFICATION TIME: 2 - 7 days
Perenyi’s Fluid
FORMIC ACID
30
1. It is recommended for routine purposes.
Perenyi’s Fluid
31
2. It decalcifies and softens tissues at the same time.
Perenyi’s Fluid
32
3. Nuclear and cytoplasmic staining is good.
Perenyi’s Fluid
33
4. Maceration is avoided due to the presence of chromic acid and alcohol.
Perenyi’s Fluid
34
1. It is a slow decalcifying agent for dense bones; hence, is not recommended for urgent diagnosis.
Perenyi’s Fluid
35
2. Complete decalcification cannot be determined by chemical test because a precipitate is formed upon the addition of ammonia to Perenyi's fluid even in the absence of calcium ion.
Perenyi’s Fluid
36
This may be dissolved by adding glacial acetic acid drop by drop. About 0.5 ml. of saturated aqueous ammonium oxalate is then added to the solution. Reappearance of a white precipitate within 30 minutes will reaffirm the presence of calcium in the agent, signifying that decalcification is still incomplete.
Perenyi’s Fluid
37
DECALCIFICATION TIME: 12-24 hours
Phloroglucin-Nitric Acid
38
It is the most rapid decalcifying agent so far, recommended for urgent cases.
Phloroglucin-Nitric Acid
39
1. Nuclear staining is poor.
Phloroglucin-Nitric Acid
40
2. Prolonged decalcification produces extreme tissue distortion.
Phloroglucin-Nitric Acid
41
3. Yellow color must be neutralized with 5% sodium sulfate and thoroughly washed with running tap water for at least 24 hours.
Phloroglucin-Nitric Acid
42
4. Complete decalcification cannot be determined by chemical means.
Phloroglucin-Nitric Acid
43
When decalcification is complete, the acid must be removed by three changes of 70% to 90% ethanol, since washing in watery solutions will lead to excessive swelling and deterioration of tissue. When the sections are cut, the slides are brought to water and placed in 1% aqueous lithium carbonate for I hour, washed in later for 15 minutes, and then stained.
Phloroglucin-Nitric Acid
44
is inferior compared to nitric acid in its role as a
decalcifying agent because of its slower action and greater distortion of tissue produced on the decalcified section.
HYDROCHLORIC ACID
45
However, it produces good nuclear staining and if used in 1% solution with 70% alcohol, may be recommended for surface decalcification of the tissue blocks.
HYDROCHLORIC ACID
46
Rapid proprietary solutions usually contain hydrochloric acid, whereas slow proprietary mixtures contain buffered formic acid or formalin/formic acid.
HYDROCHLORIC ACID
47
Dilution of a proprietary [?] is not deleterious for effective decalcification or staining, and this is an option if a strong mixture is considered too concentrated.
HYDROCHLORIC ACID
48
1. It permits relatively good cytologic staining.
Von Ebner's Fluid
49
2. It is a moderately rapid decalcifying agent.
Von Ebner's Fluid
50
3. It does not require washing out before dehydration.
Von Ebner's Fluid
51
4. It is recommended for teeth and small pieces of bone.
Von Ebner's Fluid
52
The extent of decalcification cannot be measured by a chemical test.
Von Ebner's Fluid
53
is a moderate-acting decalcifying agent which produces better nuclear staining with less tissue distortion, and is safer to handle than nitric acid or hydrochloric acid.
FORMIC ACID
54
It is recommended for routine decalcification of postmortem research tissues, although not suitable for urgent examinations.
FORMIC ACID
55
can be used as a simple 10% aqueous solution or combined with formalin or with a buffer.
FORMIC ACID
56
It is slower than the strong acid agents, but it is much gentler in action and less likely to interfere with nuclear staining.
FORMIC ACID
57
[?] in a 10% concentration is the best all-around decalcifier.
FORMIC ACID
58
Some commercial solutions are available that combine formic acid with formalin to fix and decalcify tissues at the same time.
FORMIC ACID
59
1. It may be used both as a fixative and decalcifying agent.
FORMIC ACID
60
2. It permits excellent nuclear and cytoplasmic staining.
FORMIC ACID
61
3. It is recommended for small pieces of bones and teeth.
FORMIC ACID
62
4. It is suitable for most routine surgical specimens, particularly when immunohistochemical staining is needed.
FORMIC ACID
63
1. It is relatively slow; hence, is not suitable for urgent specimens. Decalcification may be hastened by increasing the proportion of formic acid to 25 ml. However, such concentration may make the solution opaque, thereby interfering with the staining results.
FORMIC ACID
64
2. It requires neutralization with 5% sodium sulfate, and washing out to remove the acid from the tissue.
FORMIC ACID
65
DECALCIFICATlON TIME: 3 -14 days
Formic Acid-Sodium Citrate Solution
66
1. It permits better nuclear staining than nitric acid method.
Formic Acid-Sodium Citrate Solution
67
2. It is recommended for autopsy materials, bone marrow, cartilage and tissues studied for research purposes.
Formic Acid-Sodium Citrate Solution
68
1. It is relatively slow; hence, is not recommended for routine purposes and for dense tissues.
Formic Acid-Sodium Citrate Solution
69
2. It requires neutralization with 5% sodium sulfate.
Formic Acid-Sodium Citrate Solution
70
DECALCIFICATION TIME: 4- 8 days
TRICHLOROACETIC ACID
71
1. It permits good nuclear staining.
TRICHLOROACETIC ACID
72
2. It does not require washing out; the excess acid may be removed by several changes of 90% alcohol, thus improving tissue dehydration.
TRICHLOROACETIC ACID
73
1. It is a weak decalcifying agent, not used for dense tissues, and is suitable only for small spicules of bone.
TRICHLOROACETIC ACID
74
2. It is very slow-acting; hence, is not recommended for urgent examinations.
TRICHLOROACETIC ACID
75
-is a very weak decalcifying solution suitable only for minute pieces of bone.
SULFUROUS ACID
76
1. It may be used both as a fixative and decalcifying agent.
CHROMIC ACID (FLEMMING'S FLUID)
77
2. It may be used for decalcifying minute bone spicules.
CHROMIC ACID (FLEMMING'S FLUID)
78
1. Nuclear staining with hematoxylin is inhibited.
CHROMIC ACID (FLEMMING'S FLUID)
79
2. It tends to undergo reduction and forms precipitates at the bottom of the container thus requiring frequent changes of solution.
CHROMIC ACID (FLEMMING'S FLUID)
80
3. Insoluble pigments are formed when decalcified tissue is dehydrated with alcohol; hence, tissues must be washed out prior to dehydration.
CHROMIC ACID (FLEMMING'S FLUID)
81
1. Chromic acid is highly corrosive to skin and mucous membranes.
CHROMIC ACID (FLEMMING'S FLUID)
82
2. It is carcinogenic.
CHROMIC ACID (FLEMMING'S FLUID)
83
3. Suitable protective material is not readily available or practical for laboratory use.
CHROMIC ACID (FLEMMING'S FLUID)
84
4. Drain disposal is not a legitimate option for any solution containing chromium, including subsequent processing of fluids following fixation or rinses following staining procedures involving chromium.
CHROMIC ACID (FLEMMING'S FLUID)
85
DECALCIFICATION TIME: 6 days
CITRIC ACID-CITRATE BUFFER SOLUTION (pH 4.5)
86
1 It permits excellent nuclear and cytoplasmic staining.
CITRIC ACID-CITRATE BUFFER SOLUTION (pH 4.5)
87
2. It does not produce cell or tissue distortion.
CITRIC ACID-CITRATE BUFFER SOLUTION (pH 4.5)
88
Its action is too slow for routine purposes.
CITRIC ACID-CITRATE BUFFER SOLUTION (pH 4.5)
89
acts slowly but causes little tissue damage. Conventional stains are largely unaffected.
Neutral EDTA
90
1. It permits excellent staining results.
Neutral EDTA
91
2. It produces minimal cell and tissue distortion.
Neutral EDTA
92
3. It forms minimal histological artifacts, usually caused by production of CO2 bubbles.
Neutral EDTA
93
4. Extent of decalcification can be measured by routine chemical test.
Neutral EDTA
94
5. EDTA is an excellent bone decalcifier for enzyme or immuno-histochemical staining, and for electron microscopy.
Neutral EDTA
95
6. Enzymes require specific pH conditions in order to maintain
activity, and EDTA solutions can be adjusted to a specific pH for
enzyme staining.
Neutral EDTA
96
1. It is very slow, and is therefore not recommended for urgent and routine purposes.
Neutral EDTA
97
2. It causes slight tissue hardening.
Neutral EDTA
98
3. EDTA inactivates alkaline phosphatase activity, which can be restored by addition of magnesium chloride.
Neutral EDTA
99
1. Cellular detail is well-preserved.
ION EXCHANGE RESIN
100
2. Daily washing of solutions is eliminated.
ION EXCHANGE RESIN
101
3. It permits excellent staining results.
ION EXCHANGE RESIN
102
4. Decalcification is hastened.
ION EXCHANGE RESIN
103
5. It produces minimal cell and tissue distortion.
ION EXCHANGE RESIN
104
6. It forms minimal histological artifacts, usually caused by production of CO2 bubbles.
ION EXCHANGE RESIN
105
7. Extent of decalcification can be measured by routine chemical test.
ION EXCHANGE RESIN
106
1. The degree of decalcification cannot be measured by chemical means.
ION EXCHANGE RESIN
107
2. It is very slow, and is therefore not recommended for urgent and routine purposes.
ION EXCHANGE RESIN
108
3. It causes slight tissue hardening.
ION EXCHANGE RESIN
109
hastens decalcification by removing calcium ions from formic acid-containing decalcifying solutions, thereby increasing solubility from the tissue.
ION EXCHANGE RESIN
110
It is not recommended for fluids containing mineral acids such as nitric acid or hydrochloric acid.
ION EXCHANGE RESIN
111
is a process whereby positively charged calcium ions are attracted to a negative electrode and subsequently removed from the decalcifying solution.
ELECTROPHORESIS (ELECTRICAL IONIZATION)
112
This method is satisfactory for small bone fragments, processing only alimited number of specimens at a time. Good cytologic and histologic details are, however, not always preserved in tissues that have been electrically decalcified.
ELECTROPHORESIS (ELECTRICAL IONIZATION)
113
is faster than routine decalcification irrespective of the decalcifying agents used
MICROWAVE OVEN DECALCIFICATION
114
has been used quite often for tissue processing, but there are very few studies describing its use in decalcification of bone or teeth
microwave oven
115
Factors influencing the rate of decalcification
Concentration
Fluid access
Size and consistency
Agitation
Temperature
116
The recommended ratio of fluid to tissue volume for decalcification is
20 to 1.
117
Dense bone tissues usually require up to [?] or longer in order to complete the process.
14 days
118
At [?], there will be impaired nuclear staining of Van Gieson's stain for collagen fibers.
37°C
119
At [?], the tissue will undergo complete digestion within 24-48 hours.
55°C
120
The optimum temperature so far recommended is the room temperature range of
18°C -30°C.
121
The decalcifying fluid is usually changed every [?] and the chemical test is performed on the discarded fluid.
24 -48 hours
122
If the solution remains clear after [?], decalcification is considered to be complete.
30 minutes
123
Adequate water rinsing can usually be accomplished in [?] for small samples and [?] for larger specimens.
30 minutes
1-4 hours
124
Acid decalcified tissues for frozen sections must be thoroughly washed in water or stored in formol-saline containing 15% sucrose or phosphate-buffered saline (PBS) with 15-20% sucrose at [?] before freezing.
4°C
125
To soften unduly hard tissues, selected portions are left in the fluid for [?] and dehydrated in the usual manner; or the cut surface of the block may be submerged in the fluid for [?] before sectioning, to facilitate easier cutting of tissues.
12-24 hours
1-2 hours
