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Histopathology > PPT: Fixation and Decalcification > Flashcards

PPT: Fixation and Decalcification Flashcards

1
Q

10 STEPS IN TISSUE PROCESSING

A
  1. FIXATION
  2. DECALCIFICATION if necessary; DEHYDRATION.
  3. CLEARING
  4. INFILTRATION/ IMPREGNATION
  5. EMBEDDING
  6. TRIMMING
  7. SECTION CUTTING
  8. STAINING
  9. MOUNTING
  10. LABELLING
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2
Q

a chemical process by which biological tissues are preserved from decay, either through autolysis or putrefaction.

A

Fixation

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3
Q

terminates any ongoing biochemical reactions, and may also increases the mechanical strength or stability of the treated tissues.

A

Fixation

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4
Q

The aims of fixation are:

A
  1. Prevent postmortem (PM) degeneration
  2. Prevent autolysis. It is effective against hydrolytic enzymes
  3. Stop the bacterial effect
  4. Harden the tissues, as fixation causes coagulation of proteins
  5. Fixation has a mordanting effect, facilitating subsequent staining of tissues.
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5
Q

Example of Additive fixation:

A

Mercury and Osmium tetroxide

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6
Q

Example of Non-Additive Fixation

A

Alcoholic fixatives

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7
Q

Duration of fixation can be shortened by:

A
  1. Heat
  2. Vacuum
  3. Agitation
  4. Microwave
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8
Q

Factors that affect fixation:

Retarded by

A
  1. Thick tissue size
  2. Presence of mucus
  3. Presence of fat
  4. Presence of blood
  5. Cold temperature
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9
Q

Factors that affect fixation:

Enhanced by

A
  1. Thin size
  2. Agitation
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10
Q

TYPES OF FIXATIVE:

Based on Composition

A
  1. Simple fixatives
  2. Compound fixatives
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11
Q

TYPES OF FIXATIVE

According to Action

A
  1. Histochemical fixatives
  2. Microanatomical fixatives
  3. Cytologic fixatives
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12
Q

TYPES OF FIXATIVE

Based on Chemical Nature

A
  1. Physical agents
  2. Aldehydes
  3. Coagulants
  4. Oxidizing agents
  5. Miscellaneous
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13
Q

a solution of formaldehyde. It is the most common chemical fixative used in histopathology.

A

Formalin

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14
Q

Formalin itself is a_______ solution of formaldehyde gas in water.

A

37%-40%

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15
Q

10% of formalin used for fixation is prepared by adding 10% of formalin to _________

A

90 ml of saline.

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16
Q

Turbidity in the formalin is due to formation of ________ which is formed due to polymerization of formaldehyde.

A

paraformaldehyde

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17
Q

Usually commercial formaldehydes contain _________ which inhibits the formation of paraformaldehyde.

A

11% to 16% methanol

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18
Q

Formalin acts by ___________, i.e, the formation of complexes by development of links (methylene bridges) between protein molecules.

A

polymerizing action

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19
Q

Usually commercial formaldehydes contain_________which inhibits the formation of paraformaldehyde.

A

11% to 16% methanol

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20
Q

Formalin characteristics:

Hours of fixation time in room temp.

A

8-24 hours

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21
Q

Formalin characteristics:

hours if with agitation

A

4 hours

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22
Q

Formalin characteristics:

hours if temperature is increased up to 45C

A

2-3 hours

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23
Q

Formalin is usually buffered to pH ____ with _____

A

pH 7
phosphate buffer

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24
Q

Use of ____ formalin may cause brown pigments on blood containing tissues.

A

unbuffered

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25
Q

Formalin pigment is brown granular material formed by the action of formalin in excess of blood. It is removed by:

A
  • Picric acid
  • Kardasewitsch’s method
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26
Q

Formalin pigment and removal:

Place the sections in the saturated alcoholic solution of picric acid for 20 min to 2 hrs & then wash under tap water for 10 to 15 min.

A

Picric acid

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27
Q

Formalin pigment and removal:

After washing with
water place the sections in the following mixture for 5 min to 3 hrs then wash with water:
-70% ethyl alcohol – 100 ml
-20% Ammonia – 10-20 ml

A

Kardasewitsch’s method

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28
Q

Maintains color - also known as museum fixative

A

10% formal saline

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29
Q

recommended for CNS material

A

10% formal saline

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30
Q

Slow & liable for shrinkage during dehydration

A

10% formal saline

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31
Q

Recommended for routine post-mortem material

A

Formol Sublimate

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32
Q

No hardening/shrinkage and the cytological details and RBCs are well preserved

A

Formol Sublimate

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33
Q

Recommended for lipid fixation

A

Formol calcium (Lillie; 1965)

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34
Q

Have a near neutral pH

A

Formol calcium (Lillie; 1965)

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35
Q

Formalin pigment (acid formaldehyde haematin) is not formed

A

Formol calcium (Lillie; 1965)

36
Q

Used for glycogen

A

Gender’s fluid

37
Q

First used in 1962 by Sabatini et al

A

Glutaraldehyde

38
Q

Shown to preserve properties of subcellular structures by EM

A

Glutaraldehyde

39
Q

Glutaradehyde forms a _____ with amino groups on proteins and polymerizes via this catalyzed reactions

A

Schiff’s base

40
Q

Reacts with the B-amino group of lysine, a-amino group of amino acids - reacts with tyrosine, tryptophan, histidine, phenylalanine and cysteine

A

Extensive crosslinks

41
Q

Fixes protein rapidly, but has slow penetrate rate

A

Glutaraldehyde

41
Q

Fixes protein rapidly, but has slow penetrate rate

A

Glutaraldehyde

42
Q

Can cause cells to form membrane blebs

A

Glutaraldehyde

43
Q

The simplest aldehyde

A

Formaldehyde

44
Q

An aldehyde molecule which is sold as “Cidex”

A

Glutaraldehyde

45
Q

Formaldehyde vs Glutaraldehyde:

One functional group per molecule

A

Formaldehyde

46
Q

Formaldehyde vs Glutaraldehyde:

Two functional groups per molecule

A

Glutaraldehyde

47
Q

Formaldehyde vs Glutaraldehyde:

Moderately toxic

A

Formaldehyde

48
Q

Formaldehyde vs Glutaraldehyde:

Highly toxic

A

Glutaraldehyde

49
Q

Formaldehyde vs Glutaraldehyde:

As a precursor for many organic synthesis processes, as a disinfectant, etc

A

Formaldehyde

50
Q

Formaldehyde vs Glutaraldehyde:

As a disinfectant, medication, preservative and as a fixative

A

Glutaraldehyde

51
Q

Used in electron microscopy and Used in fixing material for ultrathin sections for electron microscopy

A

OSMIUM TETROXIDE

52
Q

METALLIC FIXATIVES:

A
  • Mercuric fixatives
  • Chromate fixatives
  • Lead fixatives
53
Q

Principle– Mercuric ions act by combining with acidic (carboxyl –COOH) groups of proteins & form especially strong combination with the sulfer (thiol) radicals

A

Mercuric fixatives

54
Q

Recommended for fixing small pieces of liver, spleen, connective tissue fibers and nuclei.

A

Zenker’s fluid

55
Q

Viral inclusions (Negri bodies)

A

Zenker’s fluid

56
Q

Excellent fixative for pituitary gland, bone marrow, spleen and liver

A

Zenker-formol (Helly’s solution)

57
Q

Recommended for tumor biopsies esp. of the skin.

A

Haidenhain’s Susa solution

58
Q

Commonly used for Bone marrow samples.

A

B-5 Fixative

59
Q

Principle – Chromium salts in H2O form Cr-O- Cr complexes which have an affinity for the COOH & - OH groups of proteins so that complexes between adjacent protein molecular are formed.

A

CHROMATE FIXATIVES

60
Q

This leads to disruption of the internal salt linkages of the protein increasing the reactive basic groups & thereby enhancing acidophilic in staining.

A

CHROMATE FIXATIVES

61
Q

Chromate fixatives:

A
  1. Chromic acid
  2. Potasium dichromate
  3. Regaud’s (Muller’s fluid)
  4. Orth’s Fluid
62
Q

preserves carbohydrates, precipitates proteins

A

Chromic acid

63
Q

preserves mitochondria

A

Potassium dichromate`

64
Q

They are used mainly for mucopolysacharides.

A

Lead Fixatives

65
Q

forms protein picrates, some of which are water soluble until treated with alcohol

A

Picric acid fixatives

66
Q

For fixation of embryos & pituitary biopsies

A

Bouin’s fluid

67
Q

Rapidly denatures proteins by destroying hydrogen
bonds.

A

Alcoholic fixatives

68
Q

excellent for fixing dry and wet smears, blood smears, and bone marrow

A

Methyl alcohol

69
Q

Alcohol fixative for touch preparation

A

Isopropyl alcohol

70
Q

Alcohol fixative used in histochemistry for enzyme studies

A

Ethyl alcohol

71
Q

Alcohol fixative recommended for fixing chromosomes and for urgent biopsies

A

Carnoy’s fluid

72
Q

Alcohol fixative considered to be the most rapid fixative

A

Carnoy’s fixative

73
Q

Recommended for the study of water diffuible enzymes ( ex. lipases & phoshatases)

A

ACETONE

74
Q

For fixing brain tissues for rabies cases.

A

ACETONE

75
Q

Done only if calcium and lime slats are present in the processed tissue.

A

DECALCIFICATION

76
Q

Removal of calcium and lime salts.

A

DECALCIFICATION

77
Q

must be capable of removing calcium without producing considerable tissue destruction.

A

Decalcifying agents

78
Q

Types of Decalcifying agents

A
  1. Acid decalcifying agents
  2. Chelating agents ( EDTA)
  3. Ion-Exchange resins (Ammonium form of
    polystyrene resin)
  4. Electrical ionization (Electrophoresis)
79
Q

Problems encountered in fixation:

Failure to arrest early autolysis

A

Cause: Failure to fix immediately Insufficient fixative

80
Q

Problems encountered in fixation:

Removal of substances soluble in fixing agent

A

Cause:

Wrong choice of fixative

81
Q

Problems encountered in fixation:

Presence of artifacts/pigments on tissue sections

A

Cause: Incomplete washing

82
Q

Problems encountered in fixation:

Tissues are soft and feather-like in consistency

A

Cause: Incomplete fixative

83
Q

Problems encountered in fixation:

Loss or inactivation of enzymes needed

A

Cause: Wrong choice of fixative

84
Q

Problems encountered in fixation:

Shrinkage or swelling of cells and tissues

A

Cause: Over Fixation

Histopathology (22 decks)

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