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HSTCYT > 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
✓ this process is also known as demineralization

A

DECALCIFICATION

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2
Q

✓ Poor cutting of hard tissues
✓ Damage to the knife edge during sectioning
✓ Bone dust and other cellular debris obscures microanatomic details

A

Consequences of NOT Performing Decalcification

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3
Q

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

A

Consequences of Performing Decalcification

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4
Q

hematoxylin is inhibited

A

Basic dyes

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5
Q

eosin produces a deep brick red color without differential staining

A

Acid dyes

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6
Q

➢ Failure of sections to stain properly is compounded by: (1) overtreatment in acid & (2) insufficient washing out of the acid
➢ Basic dyes: hematoxylin is inhibited
➢ Acid dyes: eosin produces a deep brick red color without differential staining

A

Consequences of Performing Decalcification 2. Affects staining

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7
Q

➢ Observed after immersion in acid alcohol

➢ Collodionize prior to staining

A

Consequences of Performing Decalcification

3. Sections Float-off During Staining

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8
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)

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9
Q

Fixative of choice

A

10% neutral buffered formalin

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10
Q

partial or complete decalcification is required before cutting samples

A

Teeth

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11
Q

➢ detected during sectioning or examination
➢ REMEDY IF DETECTED DURING SECTIONING: surface decalcification using a pad of cotton/gauze soaked with 10% HCl for 1 hour
➢ APPEARANCE UNDER THE MICROSCOPE: dark purple granular masses with lighter purple halos
➢ commonly found in malignancy

A

Microcalcified samples

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12
Q

➢ Dense/Hard Bone: 2-5 mm thick

➢ Softer Tissue: 4-6 mm thick

A

Thickness of the Specimen

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13
Q

➢ Ideal: 24-48 hours

➢ Dense Cortical Bone: 14 days

A

Duration

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14
Q

✓ Required temperature: 18-30 degrees Celsius (ROOM TEMP)
✓ Heat enhances destructive action of acids on matrices
✓ 37 degrees Celsius: impairs nuclear staining with Van Gieson’s
→ reduced effectiveness of Trichrome and PAS
✓ 55 degrees Celsius: tissues will undergo complete digestion within 24-48 hrs

A

Temperature and Heat

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15
Q

Required temperature

A

18-30 degrees Celsius (ROOM TEMP)

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16
Q

Temp that impairs nuclear staining with Van Gieson’s

A

37 degrees Celsius

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17
Q

temp at w/c tissues will undergo complete digestion within 24-48 hrs

A

55 degrees Celsius

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18
Q

stains for muscles, for blood samples, and bone marrow samples

A

Van Gieson’s, Trichrome and PAS

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19
Q

❖ Concentration of Solutions
➢ Directly proportional to the rate of decalcification
❖ Strong Acids
➢ Affects the antigenicity of cells and tissue components

A

Solution Used

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20
Q

antigens which are tumor markers that can be used to detect malignancy or can detect cancer found in the surface of cells or tissue cells

A

antigenicity of cells

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21
Q

bone sample can be destroyed is used acid is too strong; must be diluted so that reagent won’t be too concentrated in the sample

A

Strong Acids

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22
Q

the more concentrated the reagent is, the faster the decalcification

A

Directly proportional to the rate of decalcification

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23
Q

❖ Protect tissues but slows down decalcification

A

Presence of Additives

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24
Q

❖ Tissues are to be suspended in the upper portion of the jar/container

A

Fluid Access

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25
Q

❖ Once or twice a day – depends on the type of reagent (usually once or twice a day) because it causes turbidity

A

Changing of the Solution

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26
Q

Optimum: 20 times the volume of the tissue

A

Volume

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27
Q

❖ Mechanical agitation or moving of tissue in the solution which influences fluid exchange
❖ Gentle fluid agitation: low speed rotation, rocking, mechanical stirrer, bubbling air into the solution
❖ Vigorous agitation: sonication

A

Agitation

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28
Q

remove acid in the tissue so that it won’t affect staining soon

A

Removal of Decalcifying Solution

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29
Q

– use of heat and electricity for faster decalcification

A

Microwave and Electrolytic Methods

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30
Q

acids or chelating agents

A

DECALCIFYING AGENTS

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31
Q
  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
A

Characteristics of a Good Decalcifying Agent:

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32
Q

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

A

DECALCIFYING METHODS

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33
Q

Injurious to the organic ground substance of tissues

A

USE OF ACID SOLUTIONS

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34
Q

✓ most common and fastest
✓ 5 to 10% is the recommended concentration when used as a simple solution
✓ rapid decalcifying agent: may inhibit nuclear stains and damage tissues
✓ formaldehyde or alcohol and chromic acid may be added as additives
✓ Washing of tissue: acid removed by 3 changes of 70-90% ethanol
✓ Washing slide: brought to water and placed in 1% aq. lithium carbonate for 1
hour→ wash for 15 min
✓ causes spontaneous yellow discoloration
➢ impairs staining reaction of the tissue
➢ IF PRESENT IN TISSUES: neutralize with 5% NaSO4 → wash in running tap
water (at least 12 hours)
➢ IF PRESENT IN SOLUTION: add 0.1% urea to pure conc. nitric acid

A

NITRIC ACID

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35
Q

not too concentrated because too concentrated samples destroy the antigenic properties of tissue

A

5 to 10%

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36
Q

acid removed by 3 changes of 70-90% ethanol

A

Washing of tissue

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37
Q

used in dehydration which is the next step after decalcification

A

Ethanol

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38
Q

alkaline solution for neutralization; not too acidic slide

A

1% aq. lithium carbonate

39
Q

silver impregnation of nerve fibers

A

De Castro’s Fluid

40
Q

nitric acid, 40% formaldehyde

A

Formol-Nitric Acid:

41
Q

nitric acid, 0.5% chromic acid, absolute alcohol

A

Perenyi’s Fluid

42
Q
  • slow-acting

- complete decalcification cannot be determined by chemical testing

A

DISADVANTAGES of Perenyi’s Fluid

43
Q

conc. nitric acid, phloroglucin, 10% nitric acid
✓ most rapid decalcifying agent
✓ recommended for urgent work

A

Phloroglucin-Nitric Acid

44
Q

changes of 70 to 90% ethanol

A

WASHING OF TISSUES using Phloroglucin-Nitric Acid

45
Q

o bring slides to water
o place in 1% aqueous lithium carbonate for 1 hour
o wash for 15 minutes

A

WASHING OF SECTIONS using Phloroglucin-Nitric Acid

46
Q

✓ fixative and decalcifying agent
✓ for small and large pieces of bones, and teeth
✓ gentler on tissues
How will you know if your tissue is completely decalcified?
1&2. PHYSICAL AND CHEMICAL TESTING – bend or prick and touch the bone
3. RADIOLOGICAL: x-ray bone sample then the radtech will interpret if there is a calcified portions or completely decalcified sample or will be interpreted by the use of chemicals
✓ post-mortem research tissues
✓ concentrated reagent: 90%
➢ Aqueous Formic Acid: formic acid, formalin
➢ Formic Acid-Sodium Citrate
❖ WASHING: neutralize with 5% sodium sulfate

A

FORMIC ACID

47
Q

concentrated reagent of FORMIC ACID

A

90%

48
Q

formic acid, formalin

A

Aqueous Formic Acid

49
Q

✓ slower action, with greater distortion
✓ provides good nuclear staining
✓ surface decalcification: 1% HCl with 70% alcohol
✓ cannot be measured by chemical testing

A

HYDROCHLORIC ACID

50
Q

36% saturated aqueous NaCl, concentrated HCl

✓ for teeth and small pieces of bones

A

Von Ebner’s Fluid

51
Q

✓ for minute samples
✓ provides good nuclear staining
✓ does not require washing-out
✓ very slow and weak decalcifying acid

A

Trichloroacetic Acid (TCA)

52
Q

✓ also for minute samples

✓ fixative and decalcifying agent

A

Flemming’s Fluid

53
Q

✓ provides excellent nuclear and cytoplasmic staining

✓ does not produce distortion

A

Citric Acid-Citrate Buffer

54
Q

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

A

Sulfurous Acid

55
Q
  • H and E
  • Masson’s Hematoxylin-Phloxine-Safran
  • Giemsa Stain
A

STAINS USED AFTER ACID DECALCIFICATION

56
Q

NITRIC ACID DECALCIFYING AGENTS -

Most commonly used decalcifying agent

A

5 to 10% Nitric acid

57
Q

NITRIC ACID DECALCIFYING AGENTS

-For silver impregnation of nerve fibers

A

De Castro’s fluid

58
Q

NITRIC ACID DECALCIFYING AGENTS

Nitric acid + 40% formaldehyde

A

Formol-Nitric acid

59
Q

NITRIC ACID DECALCIFYING AGENTS

Slow-acting decalcifying agent

A

Perenyi’s fluid

60
Q

NITRIC ACID DECALCIFYING AGENTS

Most rapid decalcifying agent

A

Phloroglucin-Nitric acid

61
Q

NITRIC ACID DECALCIFYING AGENTS

For small and large pieces of bones and teeth

A

FORMIC ACID

62
Q

HYDROCHLORIC ACID DECALCIFYING AGENTS

For surface decalcification

A
  • 1% HCl with 70% alcohol

- 10% HCl

63
Q

HYDROCHLORIC ACID DECALCIFYING AGENTS For teeth and small pieces of bones

A

Von Ebner’s fluid

64
Q

OTHER ACIDS

For minute samples

A
  • Trichloroacetic acid

- Flemming’s fluid

65
Q

OTHER ACIDS

Excellent nuclear and cytoplasmic staining

A

Citric acid-Citrate buffer

66
Q

OTHER ACIDS

For very minute samples

A

Sulfurous acid

67
Q

✓ excellent bone decalcifying agent for immunohistochemistry and enzyme studies
✓ acts as both decalcifying agent and water softener
✓ does not interfere with staining
✓ does not distort tissues and enzymes
✓ available in 2 formulations

A

EDTA: ethylenediaminetetraacetic acid

68
Q
  • Binds to calcium and removes it in bones

- Used for non-urgent processing and for research purposes only

A

EDTA: ethylenediaminetetraacetic acid

69
Q

❖ 5-10% EDTA Disodium

❖ EDTA Tetrasodium

A

2 formulations of EDTA

70
Q

pH adjusted to 7.4 using concentrated Hac

A

EDTA Tetrasodium

71
Q

➢ 1 to 3 weeks: small specimens

➢ 6 to 8 weeks: dense cortical bone

A

DURATION

72
Q

➢ pH 3: inhibits calcium binding – cannot remove calcium
➢ pH 8: optimum binding
➢ pH 7.0 to 7.4: allows binding and does not destroy tissue components

A

pH

73
Q

DURATION of chelating small specimens

A

1 to 3 weeks

74
Q

DURATION of chelating dense cortical bone

A

6 to 8 weeks

75
Q

pH w/c inhibits calcium binding – cannot remove calcium

A

pH 3

76
Q

pH for optimum binding

A

pH 8

77
Q

pH w/c allows binding and does not destroy tissue components

A

pH 7.0 to 7.4

78
Q

✓ uses ammonium-sulfonated polysterene + formic acid
✓ volume of the acid solution: 20-30 times the volume of the sample
✓ Resin and Formic Acid (RAF)
➢ Cellular detail is well preserved
➢ Decalcification is faster
➢ Daily changing of solution is eliminated

A

ION EXCHANGE RESIN

79
Q

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

➢ 5-6 mm thick (4-8 hours)

A

10 % and 20 % RAF (Resin and Formic Acid)

80
Q

pieces of dense bone (24 hrs)→trimmed to 3 mm thick→40 % RAF

24 hrs

A

40 % RAF

81
Q

→ 2 washings with N/10 HCl

→ final washing with distilled water

A

REACTIVATION OF USED RESIN:

82
Q

fastest way to decalcify hard tissue and bone samples bec it adds electricity and heat
✓ positively charged calcium ions are attracted to a negative electrode
✓ uses heat and electrolytic reaction
✓ dependent on electricity for the removal of calcium
✓ temperature: 30 to 45 degrees Celsius
✓ uses 90%/88% formic acid and concentrated HCl as the acid solution

A

ELECTROPHORESIS

83
Q

temperature for ELECTROPHORESIS

A

30 to 45 degrees Celsius

84
Q

not recommended bec it can damage bone
✓ done by touching or bending to determine consistency or pricking with fine needle or probe - can destroy sample and may have a needle crash artifacts
✓ vague and not a reliable method – only surfaces only you will feel decalcified and not the inner portions of the bone

A

PHYSICAL TEST / MECHANICAL WAY

85
Q

✓ calcium and mineral salts produce opaque areas (interpreted by trained radtechs)
✓ very expensive, most ideal method, most sensitive and most reliable method
✓ DO NOT use for mercuric chloride fixed tissues
mercuric chloride – produce opaque appearance in

A

RADIOLOGIC TEST / X-RAY METHOD

86
Q

✓ Simple, reliable and convenient method for routine purposes
Why is it called calcium oxalate test? Bec the one causing turbidity and precipitation is the formation of calcium and oxalate → formation of calcium oxalate
REAGENTS USED: ammonia water or ammonium hydroxide, and ammonium oxalate – calcium oxalate is the one formed when calcium is present upon addition of ammonium or sodium oxalate
❖ 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
❖ COMPLETE DECALCIFICATION – clear solution after 15-30 mins

A

CHEMICAL TEST or CALCIUM OXALATE TEST

87
Q

done to remove acid for succeeding procedure which is staining

A

POST-DECALCIFICATION TREATMENT

88
Q 
POST-DECALCIFICATION TREATMENT 
Water Rinsing
➢ 30 minutes for small samples
➢ 1 to 4 hours for larger samples
➢ quick rinsing and blotting for small needle biopsies
A

WASHING-OUT

89
Q

POST-DECALCIFICATION TREATMENT
❖ 2% Lithium Carbonate
❖ 5 to 10% Aqueous Sodium Bicarbonate

A

NEUTRALIZATION

90
Q 
POST-DECALCIFICATION TREATMENT
❖ 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

FROZEN SECTIONING

91
Q

❖ wash in water NEVER IN ALCOHOL
❖ storage in formol-saline or PBS overnight
PBS (Phosphate Buffered Saline)

A

EDTA

92
Q

✓ surface blocks submerged for 1 to 2 hours

✓ tissues immersed for 12 to 24 hours

A

Perenyi’s Fluid

93
Q

✓ may cause swelling or make tissues soapy

A

Molliflex

94
Q 
Perenyi’s Fluid
4% Aqueous Phenol
Molliflex
2% HCl
1% HCl in 70% alcohol
A

TISSUE SOFTENERS

HSTCYT (6 decks)

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