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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
Formalin pigment is brown granular material formed by the action of formalin in excess of blood. It is removed by:
* Picric acid * Kardasewitsch’s method
26
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.
Picric acid
27
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
Kardasewitsch’s method
28
Maintains color - also known as museum fixative
10% formal saline
29
recommended for CNS material
10% formal saline
30
Slow & liable for shrinkage during dehydration
10% formal saline
31
Recommended for routine post-mortem material
Formol Sublimate
32
No hardening/shrinkage and the cytological details and RBCs are well preserved
Formol Sublimate
33
Recommended for lipid fixation
Formol calcium (Lillie; 1965)
34
Have a near neutral pH
Formol calcium (Lillie; 1965)
35
Formalin pigment (acid formaldehyde haematin) is not formed
Formol calcium (Lillie; 1965)
36
Used for glycogen
Gender's fluid
37
First used in 1962 by Sabatini et al
Glutaraldehyde
38
Shown to preserve properties of subcellular structures by EM
Glutaraldehyde
39
Glutaradehyde forms a _____ with amino groups on proteins and polymerizes via this catalyzed reactions
Schiff's base
40
Reacts with the B-amino group of lysine, a-amino group of amino acids - reacts with tyrosine, tryptophan, histidine, phenylalanine and cysteine
Extensive crosslinks
41
Fixes protein rapidly, but has slow penetrate rate
Glutaraldehyde
41
Fixes protein rapidly, but has slow penetrate rate
Glutaraldehyde
42
Can cause cells to form membrane blebs
Glutaraldehyde
43
The simplest aldehyde
Formaldehyde
44
An aldehyde molecule which is sold as "Cidex"
Glutaraldehyde
45
Formaldehyde vs Glutaraldehyde: One functional group per molecule
Formaldehyde
46
Formaldehyde vs Glutaraldehyde: Two functional groups per molecule
Glutaraldehyde
47
Formaldehyde vs Glutaraldehyde: Moderately toxic
Formaldehyde
48
Formaldehyde vs Glutaraldehyde: Highly toxic
Glutaraldehyde
49
Formaldehyde vs Glutaraldehyde: As a precursor for many organic synthesis processes, as a disinfectant, etc
Formaldehyde
50
Formaldehyde vs Glutaraldehyde: As a disinfectant, medication, preservative and as a fixative
Glutaraldehyde
51
Used in electron microscopy and Used in fixing material for ultrathin sections for electron microscopy
OSMIUM TETROXIDE
52
METALLIC FIXATIVES:
* Mercuric fixatives * Chromate fixatives * Lead fixatives
53
Principle– Mercuric ions act by combining with acidic (carboxyl –COOH) groups of proteins & form especially strong combination with the sulfer (thiol) radicals
Mercuric fixatives
54
Recommended for fixing small pieces of liver, spleen, connective tissue fibers and nuclei.
Zenker's fluid
55
Viral inclusions (Negri bodies)
Zenker's fluid
56
Excellent fixative for pituitary gland, bone marrow, spleen and liver
Zenker-formol (Helly's solution)
57
Recommended for tumor biopsies esp. of the skin.
Haidenhain's Susa solution
58
Commonly used for Bone marrow samples.
B-5 Fixative
59
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.
CHROMATE FIXATIVES
60
This leads to disruption of the internal salt linkages of the protein increasing the reactive basic groups & thereby enhancing acidophilic in staining.
CHROMATE FIXATIVES
61
Chromate fixatives:
1. Chromic acid 2. Potasium dichromate 3. Regaud's (Muller's fluid) 4. Orth's Fluid
62
preserves carbohydrates, precipitates proteins
Chromic acid
63
preserves mitochondria
Potassium dichromate`
64
They are used mainly for mucopolysacharides.
Lead Fixatives
65
forms protein picrates, some of which are water soluble until treated with alcohol
Picric acid fixatives
66
For fixation of embryos & pituitary biopsies
Bouin's fluid
67
Rapidly denatures proteins by destroying hydrogen bonds.
Alcoholic fixatives
68
excellent for fixing dry and wet smears, blood smears, and bone marrow
Methyl alcohol
69
Alcohol fixative for touch preparation
Isopropyl alcohol
70
Alcohol fixative used in histochemistry for enzyme studies
Ethyl alcohol
71
Alcohol fixative recommended for fixing chromosomes and for urgent biopsies
Carnoy's fluid
72
Alcohol fixative considered to be the most rapid fixative
Carnoy's fixative
73
Recommended for the study of water diffuible enzymes ( ex. lipases & phoshatases)
ACETONE
74
For fixing brain tissues for rabies cases.
ACETONE
75
Done only if calcium and lime slats are present in the processed tissue.
DECALCIFICATION
76
Removal of calcium and lime salts.
DECALCIFICATION
77
must be capable of removing calcium without producing considerable tissue destruction.
Decalcifying agents
78
Types of Decalcifying agents
1. Acid decalcifying agents 2. Chelating agents ( EDTA) 3. Ion-Exchange resins (Ammonium form of polystyrene resin) 4. Electrical ionization (Electrophoresis)
79
Problems encountered in fixation: Failure to arrest early autolysis
Cause: Failure to fix immediately Insufficient fixative
80
Problems encountered in fixation: Removal of substances soluble in fixing agent
Cause: Wrong choice of fixative
81
Problems encountered in fixation: Presence of artifacts/pigments on tissue sections
Cause: Incomplete washing
82
Problems encountered in fixation: Tissues are soft and feather-like in consistency
Cause: Incomplete fixative
83
Problems encountered in fixation: Loss or inactivation of enzymes needed
Cause: Wrong choice of fixative
84
Problems encountered in fixation: Shrinkage or swelling of cells and tissues
Cause: Over Fixation

Histopathology (22 decks)

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