LESSON 3: DECALCIFICATION Flashcards
1
Q
process that entails the removal of calcium or lime salts from tissue samples after fixation
A
DECALCIFICATION
2
Q
this process is also known as demineralization
A
DECALCIFICATION
3
Q
Consequences of NOT Performing Decalcification
A
Poor cutting of hard tissues
Damage to the knife edge during sectioning
Bone dust and other cellular debris obscures microanatomic details
4
Q
Consequences of Performing Decalcification
A
Distortion or damage to tissues
Affects staining
Sections Float-off During Staining
5
Q
Failure of sections to stain properly is compounded by:
A
(1) overtreatment in acid & (2) insufficient washing out of the acid
6
Q
: hematoxylin is inhibited
A
Basic dyes
7
Q
: eosin produces a deep brick red color without differential staining
A
Acid dyes
8
Q
SAMPLES THAT REQUIRE DECALCIFICATION
A
9
Q
cut into small pieces using fret-saw, trimmed with a hand razor and fixed with 10% neutral buffered formalin
A
- Bones and other calcified samples (tuberculous organs, atherosclerotic vessels, teratomas)
10
Q
partial or complete decalcification is required before cutting samples
A
- Teeth
11
Q
detected during sectioning or examination
A
- Microcalcified samples
12
Q
- Microcalcified samples
REMEDY IF DETECTED DURING SECTIONING: surface decalcification using a pad of cotton/gauze soaked with
A
10% HCl for 1 hour
13
Q
- Microcalcified samples APPEARANCE UNDER THE MICROSCOPE:
A
dark purple granular masses with lighter purple halos
14
Q
commonly found in malignancy
A
- Microcalcified samples
15
Q
Dense/Hard Bone:
A
2-5 mm thick
16
Q
Softer Tissue:
A
4-6 mm thick
17
Q
Ideal:
Dense Cortical Bone:
A
24-48 hours
14 days
18
Q
Required temperature:
A
18-30 degrees Celsius
19
Q
enhances destructive action of acids on matrices
A
Heat
20
Q
: impairs nuclear staining with Van Gieson’s > reduced effectiveness of Trichrome and PAS
A
37 degrees Celsius
21
Q
: tissues will undergo complete digestion within 24-48 hrs
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55 degrees Celsius
22
Q
Concentration of Solutions = Directly proportional to the
A
rate of decalcification
23
Q
Affects the antigenicity of cells and tissue components
A
Strong Acids
24
Q
Protect tissues but slows down decalcification
A
Additives
25
Tissues are to be suspended in the [?] of the jar/container
upper portion
26
Changing of the Solution
Once or twice a day
27
Volume Optimum: [?] the volume of the tissue
20 times
28
influences fluid exchange
Mechanical agitation or moving of tissue
29
: low speed rotation, rocking, mechanical stirrer, bubbling air into the solution
Gentle fluid agitation
30
: sonication
Vigorous agitation
31
after decalcification and/or prior to staining
Washing-out or neutralization
32
DECALCIFYING AGENTS:
acids or chelating agents
33
Characteristics of a Good Decalcifying Agent:
1. Remove calcium salts completely.
2. Does not produce considerable destruction of cells and tissue components.
3. Does not adversely affect the staining capacity of the cell
34
use of acids, use of chelating agents, ion exchange resins and electrical ionization
DECALCIFYING METHODS
35
: Injurious to the organic ground substance of tissues
ACID SOLUTIONS
36
most common and fastest
NITRIC ACID
37
5 to 10% is the recommended concentration when used as a simple solution
NITRIC ACID
38
rapid decalcifying agent: may inhibit nuclear stains and damage tissues
NITRIC ACID
39
formaldehyde or alcohol and chromic acid may be added as additives
NITRIC ACID
40
Washing slide: brought to water and placed in 1% aq. lithium carbonate for 1 hour > wash for 15 min
NITRIC ACID
41
Washing of tissue: acid removed by 3 changes of 70-90% ethanol
NITRIC ACID
42
causes spontaneous yellow discoloration that impairs staining reaction of the tissue
NITRIC ACID
43
IF PRESENT IN TISSUES: neutralize with 5% NaSO4 > wash in running tap water (at least 12 hours)
NITRIC ACID
44
IF PRESENT IN SOLUTION: add 0.1% urea to pure conc. nitric acid
NITRIC ACID
45
: silver impregnation of nerve fibers
1. De Castro’s Fluid
46
De Castro’s Fluid:
chloral hydrate, distilled water, alcohol, nitric acid
47
3. Formol-Nitric Acid:
nitric acid, 40% formaldehyde
48
4. Perenyi’s Fluid:
nitric acid, 0.5% chromic acid, absolute alcohol
49
o slow-acting
Perenyi’s Fluid
50
o complete decalcification cannot be determined by chemical testing
Perenyi’s Fluid
51
precipitate forms upon addition of ammonia even in the absence of calcium ion
re-dissolve by adding glacial HAc drop by drop
add 0.5ml saturated aqueous ammonium oxalate
reappearance of white precipitate after 30 minutes
presence of calcium ions
decalcification is not yet complete
Perenyi’s Fluid
52
Phloroglucin-Nitric Acid: conc. nitric acid, phloroglucin, 10% nitric acid
53
most rapid decalcifying agent
Phloroglucin-Nitric Acid
54
recommended for urgent work
Phloroglucin-Nitric Acid
55
WASHING OF TISSUES:
o 3 changes of 70 to 90% ethanol
Phloroglucin-Nitric Acid
56
WASHING OF SECTIONS:
o bring slides to water
o place in 1% aqueous lithium carbonate for 1 hour
o wash for 15 minutes
Phloroglucin-Nitric Acid
57
fixative and decalcifying agent
FORMIC ACID
58
for small and large pieces of bones, and teeth
FORMIC ACID
59
gentler on tissues
FORMIC ACID
60
post-mortem research tissues
FORMIC ACID
61
concentrated reagent: 90%
—Aqueous Formic Acid: formic acid, formalin
—Formic Acid-Sodium Citrate
FORMIC ACID
62
WASHING: neutralize with 5% sodium sulfate
FORMIC ACID
63
slower action, with greater distortion
HYDROCHLORIC ACID
64
provides good nuclear staining
HYDROCHLORIC ACID
65
surface decalcification: 1% HCl with 70% alcohol
HYDROCHLORIC ACID
66
cannot be measured by chemical testing
HYDROCHLORIC ACID
67
: 36% saturated aqueous NaCl, concentrated HCl
1. Von Ebner’s Fluid
68
for teeth and small pieces of bones
1. Von Ebner’s Fluid
69
for minute samples
1. Trichloroacetic Acid (TCA)
70
provides good nuclear staining
1. Trichloroacetic Acid (TCA)
71
does not require washing-out
1. Trichloroacetic Acid (TCA)
72
very slow and weak decalcifying acid
1. Trichloroacetic Acid (TCA)
73
also for minute samples
2. Flemming’s Fluid
74
fixative and decalcifying agent
2. Flemming’s Fluid
75
provides excellent nuclear and cytoplasmic staining
3. Citric Acid-Citrate Buffer
76
does not produce distortion
3. Citric Acid-Citrate Buffer
77
very weak decalcifying agent, thus it is recommended for very minute samples only
4. Sulfurous Acid
78
STAINS USED AFTER ACID DECALCIFICATION
H and E
Masson’s Hematoxylin-Phloxine-Safran
Giemsa Stain
79
Most commonly used decalcifying agent
5 to 10% Nitric acid
80
For silver impregnation of nerve fibers
De Castro’s fluid
81
Nitric acid + 40% formaldehyde
Formol-Nitric acid
82
Slow-acting decalcifying agent
Perenyi’s fluid
83
Most rapid decalcifying agent
Phloroglucin-Nitric acid
84
For small and large pieces of bones and teeth
FORMIC ACID
85
For surface decalcification
1% HCl with 70% alcohol
10% HCl
86
For teeth and small pieces of bones
Von Ebner’s fluid
87
For minute samples
Trichloroacetic acid
Flemming’s fluid
88
Excellent nuclear and cytoplasmic staining
Citric acid-Citrate buffer
89
For very minute samples
Sulfurous acid
90
excellent bone decalcifying agent for immunohistochemistry and enzyme studies
EDTA
91
acts as both decalcifying agent and water softener
EDTA
92
does not interfere with staining
EDTA
93
does not distort tissues and enzymes
EDTA
94
EDTA available in 2 formulations
5-10% EDTA Disodium
EDTA Tetrasodium
95
o EDTA pH adjusted to 7.4 using
concentrated HAc
96
: small specimens
1 to 3 weeks
97
: dense cortical bone
6 to 8 weeks
98
: very important factor
pH
99
: inhibits calcium binding
pH 3
100
: optimum binding
pH 8
101
: allows binding and does not destroy tissue components
pH 7.0 to 7.4
102
uses ammonium-sulfonated polysterene + formic acid
ION EXCHANGE RESIN
103
volume of the acid solution: 20-30 times the volume of the sample
ION EXCHANGE RESIN
104
Cellular detail is well preserved
Resin and Formic Acid (RAF)
105
Decalcification is faster
Resin and Formic Acid (RAF)
106
Daily changing of solution is eliminated
Resin and Formic Acid (RAF)
107
cancellous bone 2-3 mm thick (2-3 hours)
10 % and 20 % RAF
108
5-6 mm thick (4-8 hours)
10 % and 20 % RAF
109
: large pieces of dense bone (24 hrs)>trimmed to 3 mm thick40 % RAF (24 hrs)
40 % RAF
110
REACTIVATION OF USED RESIN:
2 washings with [?]
final washing with [?]
N/10 HCl
distilled water
111
end-point of decalcification is measured using
physical or X-ray methods
112
positively charged calcium ions are attracted to a negative electrode
ELECTROPHORESIS
113
uses heat and electrolytic reaction
ELECTROPHORESIS
114
dependent on electricity for the removal of calcium
ELECTROPHORESIS
115
ELECTROPHORESIS temperature:
30 to 45 degrees Celsius
116
uses 90%/88% formic acid and concentrated HCl as the acid solution
ELECTROPHORESIS
117
done by touching or bending to determine consistency or pricking with fine needle or probe
PHYSICAL TEST
118
vague and not a reliable method
PHYSICAL TEST
119
calcium and mineral salts produce opaque areas
RADIOLOGIC TEST
120
very expensive, most ideal method, most sensitive and most reliable method
RADIOLOGIC TEST
121
DO NOT use for mercuric chloride fixed tissues
RADIOLOGIC TEST
122
RADIOLOGIC TEST EQUIPMENT:
Faxitron and Kodak X-OMAT X-ray film
123
Simple, reliable and convenient method for routine purposes
CHEMICAL TEST
124
CHEMICAL TEST PROCEDURE:
Aliquot 5 ml of used reagent.
Alkalinize with ammonia water
(+) precipitate = (+) for calcium = INCOMPLETE DECALCIFICATION
if clear, proceed to the next step
Add 0.5 ml ammonium oxalate or 1% sodium oxalate
Stand for 15 to 30 minutes
(+) cloudiness or precipitate = (+) for calcium = INCOMPLETE DECALCIFICATION
125
1. WASHING-OUT Water Rinsing 30 minutes for small samples 1 to 4 hours for larger samples quick rinsing and blotting for small needle biopsies
126
2. NEUTRALIZATION 2% Lithium Carbonate 5 to 10% Aqueous Sodium Bicarbonate
127
3. FROZEN SECTIONING thorough washing in water storage in any of the following: formol saline with 15% sucrose phosphate-buffered saline (PBS) with 15 to 20% sucrose at 4 degrees Celsius
128
4. EDTA wash in water NEVER IN ALCOHOL storage in formol-saline or PBS overnight
129
TISSUE SOFTENERS:
130
surface blocks submerged for 1 to 2 hours
1. Perenyi’s Fluid
131
tissues immersed for 12 to 24 hours
1. Perenyi’s Fluid
