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MTAP - HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES MODULE > LESSON 3: DECALCIFICATION > Flashcards

LESSON 3: DECALCIFICATION Flashcards

1
Q

 process that entails the removal of calcium or lime salts from tissue samples after fixation

A

DECALCIFICATION

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2
Q

 this process is also known as demineralization

A

DECALCIFICATION

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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

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4
Q

Consequences of Performing Decalcification

A

 Distortion or damage to tissues
 Affects staining
 Sections Float-off During Staining

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5
Q

 Failure of sections to stain properly is compounded by:

A

(1) overtreatment in acid & (2) insufficient washing out of the acid

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6
Q

: hematoxylin is inhibited

A

 Basic dyes

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7
Q

: eosin produces a deep brick red color without differential staining

A

 Acid dyes

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8
Q

SAMPLES THAT REQUIRE DECALCIFICATION

A
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9
Q

 cut into small pieces using fret-saw, trimmed with a hand razor and fixed with 10% neutral buffered formalin

A
  1. Bones and other calcified samples (tuberculous organs, atherosclerotic vessels, teratomas)
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10
Q

 partial or complete decalcification is required before cutting samples

A
  1. Teeth
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11
Q

 detected during sectioning or examination

A
  1. Microcalcified samples
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12
Q
  1. Microcalcified samples
     REMEDY IF DETECTED DURING SECTIONING: surface decalcification using a pad of cotton/gauze soaked with
A

10% HCl for 1 hour

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13
Q
  1. Microcalcified samples  APPEARANCE UNDER THE MICROSCOPE:
A

dark purple granular masses with lighter purple halos

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14
Q

 commonly found in malignancy

A
  1. Microcalcified samples
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15
Q

 Dense/Hard Bone:

A

2-5 mm thick

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16
Q

 Softer Tissue:

A

4-6 mm thick

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17
Q

 Ideal:
 Dense Cortical Bone:

A

24-48 hours

14 days

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18
Q

 Required temperature:

A

18-30 degrees Celsius

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19
Q

 enhances destructive action of acids on matrices

A

Heat

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20
Q

: impairs nuclear staining with Van Gieson’s > reduced effectiveness of Trichrome and PAS

A

 37 degrees Celsius

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21
Q

: tissues will undergo complete digestion within 24-48 hrs

A

 55 degrees Celsius

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22
Q

 Concentration of Solutions = Directly proportional to the

A

rate of decalcification

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23
Q

 Affects the antigenicity of cells and tissue components

A

 Strong Acids

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24
Q

 Protect tissues but slows down decalcification

A

Additives

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25
Q

Tissues are to be suspended in the [?] of the jar/container

A

upper portion

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26
Q

Changing of the Solution

A

Once or twice a day

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27
Q

Volume  Optimum: [?] the volume of the tissue

A

20 times

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28
Q

influences fluid exchange

A

 Mechanical agitation or moving of tissue

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29
Q

: low speed rotation, rocking, mechanical stirrer, bubbling air into the solution

A

 Gentle fluid agitation

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30
Q

: sonication

A

 Vigorous agitation

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31
Q

after decalcification and/or prior to staining

A

 Washing-out or neutralization

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32
Q

DECALCIFYING AGENTS:

A

acids or chelating agents

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33
Q

Characteristics of a Good Decalcifying Agent:

A
  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
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34
Q

use of acids, use of chelating agents, ion exchange resins and electrical ionization

A

DECALCIFYING METHODS

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35
Q

:  Injurious to the organic ground substance of tissues

A

ACID SOLUTIONS

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36
Q

 most common and fastest

A

NITRIC ACID

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37
Q

 5 to 10% is the recommended concentration when used as a simple solution

A

NITRIC ACID

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38
Q

 rapid decalcifying agent: may inhibit nuclear stains and damage tissues

A

NITRIC ACID

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39
Q

 formaldehyde or alcohol and chromic acid may be added as additives

A

NITRIC ACID

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40
Q

 Washing slide: brought to water and placed in 1% aq. lithium carbonate for 1 hour > wash for 15 min

A

NITRIC ACID

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41
Q

 Washing of tissue: acid removed by 3 changes of 70-90% ethanol

A

NITRIC ACID

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42
Q

 causes spontaneous yellow discoloration that impairs staining reaction of the tissue

A

NITRIC ACID

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43
Q

 IF PRESENT IN TISSUES: neutralize with 5% NaSO4 > wash in running tap water (at least 12 hours)

A

NITRIC ACID

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44
Q

 IF PRESENT IN SOLUTION: add 0.1% urea to pure conc. nitric acid

A

NITRIC ACID

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45
Q

: silver impregnation of nerve fibers

A
  1. De Castro’s Fluid
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46
Q

De Castro’s Fluid:

A

chloral hydrate, distilled water, alcohol, nitric acid

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47
Q
  1. Formol-Nitric Acid:
A

nitric acid, 40% formaldehyde

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48
Q
  1. Perenyi’s Fluid:
A

nitric acid, 0.5% chromic acid, absolute alcohol

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49
Q

o slow-acting

A

Perenyi’s Fluid

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50
Q

o complete decalcification cannot be determined by chemical testing

A

Perenyi’s Fluid

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51
Q

 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

A

Perenyi’s Fluid

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52
Q

Phloroglucin-Nitric Acid: conc. nitric acid, phloroglucin, 10% nitric acid

A
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53
Q

 most rapid decalcifying agent

A

Phloroglucin-Nitric Acid

54
Q

 recommended for urgent work

A

Phloroglucin-Nitric Acid

55
Q

 WASHING OF TISSUES:
o 3 changes of 70 to 90% ethanol

A

Phloroglucin-Nitric Acid

56
Q

 WASHING OF SECTIONS:
o bring slides to water
o place in 1% aqueous lithium carbonate for 1 hour
o wash for 15 minutes

A

Phloroglucin-Nitric Acid

57
Q

 fixative and decalcifying agent

A

FORMIC ACID

58
Q

 for small and large pieces of bones, and teeth

A

FORMIC ACID

59
Q

 gentler on tissues

A

FORMIC ACID

60
Q

 post-mortem research tissues

A

FORMIC ACID

61
Q

 concentrated reagent: 90%
—Aqueous Formic Acid: formic acid, formalin
—Formic Acid-Sodium Citrate

A

FORMIC ACID

62
Q

 WASHING: neutralize with 5% sodium sulfate

A

FORMIC ACID

63
Q

 slower action, with greater distortion

A

HYDROCHLORIC ACID

64
Q

 provides good nuclear staining

A

HYDROCHLORIC ACID

65
Q

 surface decalcification: 1% HCl with 70% alcohol

A

HYDROCHLORIC ACID

66
Q

 cannot be measured by chemical testing

A

HYDROCHLORIC ACID

67
Q

: 36% saturated aqueous NaCl, concentrated HCl

A
  1. Von Ebner’s Fluid
68
Q

 for teeth and small pieces of bones

A
  1. Von Ebner’s Fluid
69
Q

 for minute samples

A
  1. Trichloroacetic Acid (TCA)
70
Q

 provides good nuclear staining

A
  1. Trichloroacetic Acid (TCA)
71
Q

 does not require washing-out

A
  1. Trichloroacetic Acid (TCA)
72
Q

 very slow and weak decalcifying acid

A
  1. Trichloroacetic Acid (TCA)
73
Q

 also for minute samples

A
  1. Flemming’s Fluid
74
Q

 fixative and decalcifying agent

A
  1. Flemming’s Fluid
75
Q

 provides excellent nuclear and cytoplasmic staining

A
  1. Citric Acid-Citrate Buffer
76
Q

 does not produce distortion

A
  1. Citric Acid-Citrate Buffer
77
Q

 very weak decalcifying agent, thus it is recommended for very minute samples only

A
  1. Sulfurous Acid
78
Q

STAINS USED AFTER ACID DECALCIFICATION

A

H and E
Masson’s Hematoxylin-Phloxine-Safran
Giemsa Stain

79
Q

Most commonly used decalcifying agent

A

5 to 10% Nitric acid

80
Q

For silver impregnation of nerve fibers

A

De Castro’s fluid

81
Q

Nitric acid + 40% formaldehyde

A

Formol-Nitric acid

82
Q

Slow-acting decalcifying agent

A

Perenyi’s fluid

83
Q

Most rapid decalcifying agent

A

Phloroglucin-Nitric acid

84
Q

For small and large pieces of bones and teeth

A

FORMIC ACID

85
Q

For surface decalcification

A

1% HCl with 70% alcohol
10% HCl

86
Q

For teeth and small pieces of bones

A

Von Ebner’s fluid

87
Q

For minute samples

A

Trichloroacetic acid
Flemming’s fluid

88
Q

Excellent nuclear and cytoplasmic staining

A

Citric acid-Citrate buffer

89
Q

For very minute samples

A

Sulfurous acid

90
Q

 excellent bone decalcifying agent for immunohistochemistry and enzyme studies

A

EDTA

91
Q

 acts as both decalcifying agent and water softener

A

EDTA

92
Q

 does not interfere with staining

A

EDTA

93
Q

 does not distort tissues and enzymes

A

EDTA

94
Q

 EDTA available in 2 formulations

A

 5-10% EDTA Disodium
 EDTA Tetrasodium

95
Q

o EDTA pH adjusted to 7.4 using

A

concentrated HAc

96
Q

: small specimens

A

 1 to 3 weeks

97
Q

: dense cortical bone

A

 6 to 8 weeks

98
Q

: very important factor

A

pH

99
Q

: inhibits calcium binding

A

 pH 3

100
Q

: optimum binding

A

 pH 8

101
Q

: allows binding and does not destroy tissue components

A

 pH 7.0 to 7.4

102
Q

 uses ammonium-sulfonated polysterene + formic acid

A

ION EXCHANGE RESIN

103
Q

 volume of the acid solution: 20-30 times the volume of the sample

A

ION EXCHANGE RESIN

104
Q

 Cellular detail is well preserved

A

Resin and Formic Acid (RAF)

105
Q

 Decalcification is faster

A

Resin and Formic Acid (RAF)

106
Q

 Daily changing of solution is eliminated

A

Resin and Formic Acid (RAF)

107
Q

 cancellous bone 2-3 mm thick (2-3 hours)

A

10 % and 20 % RAF

108
Q

 5-6 mm thick (4-8 hours)

A

10 % and 20 % RAF

109
Q

: large pieces of dense bone (24 hrs)>trimmed to 3 mm thick40 % RAF (24 hrs)

A

40 % RAF

110
Q

REACTIVATION OF USED RESIN:
 2 washings with [?]
 final washing with [?]

A

N/10 HCl

distilled water

111
Q

 end-point of decalcification is measured using

A

physical or X-ray methods

112
Q

 positively charged calcium ions are attracted to a negative electrode

A

ELECTROPHORESIS

113
Q

 uses heat and electrolytic reaction

A

ELECTROPHORESIS

114
Q

 dependent on electricity for the removal of calcium

A

ELECTROPHORESIS

115
Q

ELECTROPHORESIS  temperature:

A

30 to 45 degrees Celsius

116
Q

 uses 90%/88% formic acid and concentrated HCl as the acid solution

A

ELECTROPHORESIS

117
Q

 done by touching or bending to determine consistency or pricking with fine needle or probe

A

PHYSICAL TEST

118
Q

 vague and not a reliable method

A

PHYSICAL TEST

119
Q

 calcium and mineral salts produce opaque areas

A

RADIOLOGIC TEST

120
Q

 very expensive, most ideal method, most sensitive and most reliable method

A

RADIOLOGIC TEST

121
Q

 DO NOT use for mercuric chloride fixed tissues

A

RADIOLOGIC TEST

122
Q

RADIOLOGIC TEST  EQUIPMENT:

A

Faxitron and Kodak X-OMAT X-ray film

123
Q

 Simple, reliable and convenient method for routine purposes

A

CHEMICAL TEST

124
Q

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

A
125
Q
  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
A
126
Q
  1. NEUTRALIZATION  2% Lithium Carbonate  5 to 10% Aqueous Sodium Bicarbonate
A
127
Q
  1. 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
A
128
Q
  1. EDTA  wash in water NEVER IN ALCOHOL  storage in formol-saline or PBS overnight
A
129
Q

TISSUE SOFTENERS:

A
130
Q

 surface blocks submerged for 1 to 2 hours

A
  1. Perenyi’s Fluid
131
Q

 tissues immersed for 12 to 24 hours

A
  1. Perenyi’s Fluid

MTAP - HISTOPATHOLOGIC & CYTOLOGIC TECHNIQUES MODULE (9 decks)

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