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Histology > Histo Techniques 3 > Flashcards

Histo Techniques 3 Flashcards

1
Q

What is the stain most used in Histology

A

Haematoxylin and Eosin stain

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2
Q

What does the Haematoxylin part stain?

A

Produces well delineated blue/ purple crisp nuclear staining

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3
Q

What does the Eosin part stain?

A

Stains red blood cells red/pink
Stains collagen, muscle and cytoplasm varying shades of orange/pink

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4
Q

Acidic/Basic properties of H and E stain

A

Hematoxylin stains acidic molecules shades of blue
Eosin stains basic materials shades of red, pink and orange

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5
Q

Why stain tissues?

A

To promote good contrast between different cell and tissue components and to diagnose disease

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6
Q

2 phases of tissues

A

Tissue sections are a solid phase and interact with the applied stains to form the liquid phase

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7
Q

Chemical bonds involved in staining :

A

Hydrophobic bonding, Van der Waals forces, hydrogen bonding, electrostatic attraction, covalent bonding, ionic bonds

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8
Q

What are many synthetic dyes produced from?

A

Benzene

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9
Q

What is a chromophore?

A

Any chemical group which alters the absorption range and makes n organic compound like benzene coloured

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10
Q

What is a chromogen?

A

A substance that will dissolve to give coloured molecules

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11
Q

What is an auxochrome?

A

A substance that causes ionisation of a chromogen e.g NO2 , SO2
Auxochromes turn coloured compounds into dyes and enables it to bind to the tissues

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12
Q

What are modifiers?

A

A group of chemicals that alter the final colour of a dye. Usually use ethyl or methyl groups to deepen the final colour of the dye

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13
Q

Different groups of the staining method

A
  1. Dye methods
  2. Vital staining
  3. Lysochrome methods
  4. Histochemistry
    5.Immunohistochemistry
  5. Metallic impregnantion
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14
Q

Affinity of direct dyes between tissues and examples of direct dyes

A

Have a direct affinity for tissues e.g by an affinity of dissimilar charge
E.g Eosin, acid fuchsin, methylene blue and neutral red
-Don´t need an intermediate layer

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15
Q

Affinity of indirect dyes to tissues

A

Used when there is poor affinity between a dye and tissue components
-Intermediate layer (mordant) required

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16
Q

What are mordants used in histology usually made of?

A

Salts of metals

17
Q

How do mordants work?

A

They have binding sites which attach to tissue and to dyes
Mordant metals have an affinity for tissues rich in glycols, carboxylic acids and certain phenol compunds

18
Q

3 ways mordants can be used

A
  1. Combined in staining solutions
  2. Used prior to staining solutions
  3. Used after staining solutions
19
Q

Examples of mordant dyes

A
  1. Ehrlich’s haematoxylin (potassium)
  2. Best’s Carmine (Aluminium)
20
Q

What are accelerators?

A

Materials added to staining solutions to improve staining reactions.
Used to enhance the affinity between metals and tissue components
Used in metallic impregnation techniques i .e Potassium Hydroxide

21
Q

What are trapping agents?

A

They form large aggregates with the dye and result in the dye precipitating in the tissues. This precipitate is difficult to differentiate.
The trapping reagent increases the hold the tissue component has for the stain.

22
Q

What does differentiation mean with regards to staining?

A

Removing excess stain from a tissue

23
Q

What do metachomasia, chromotrope and metachromic mean?

A

Metachromasia - term used when a dye stains the tissue component a different colour to the dye solutions
The tissue component that causes the change in colour is termed a CHROMOTROPE and the dye is termed METACHROMATIC

24
Q

what is in substances that act as chromotrope?

A

Usually rich in sulphate of carboxyl groups and include Amyloid, mast cell granules and cartilage

25
Q

What are dyes that do not exhibit Metachromasia called?

A

Orchochromatic

26
Q

What is a neutral stain?

A

A compound formed by the addition of an acidic dye to a basic dye e.g Romanowsky stain - acidic component eosin and basic component methylene blue

27
Q

What are vital stains used for and what are the 2 types of vital cells?

A

Used to stain living cells
1. Supra-vital stains
2.Intra-vital stains

28
Q

How do supra vital stains work?

A

Supra-vital staining relies on the active uptake of dye particles by the living entity e.g. methylene blue uptake by reticulocytes (young red cells)

29
Q

How do Intra-vital stains work?

A

Intra-vital staining involves the colouring of enclosed spaces by the injection of vital stain e.g demonstration of mitochondria using Janus Green

30
Q

What are lysochrome methods?

A

Colouring agents which dissolve in tissue.
Use restricted to the colouring of lipids with oil soluble dyes

31
Q

How do lysochrome method dyes work?
What are examples of these dyes?

A

The dyes are more soluble in tissue lipids than in their solvents and so by process of elective solubility pass along a partition gradient and colour the tissue liquid.

E.g Sudan Black and Oil Red O

32
Q

What is histochemistry?

A

The demonstration of tissue constituents by the use of chemical or enzymatic reactions

33
Q

What 2 groups can Histochemistry be divided into?

A

a) Those that produce a coloured end product in the tissue - uses chemical reagents products
b) Those where a colourless dye has colour restored by the tissue component - uses dye products

34
Q

What can reagent products be divided into?

A
  1. Chemical reactions
  2. Enzymatic reactions
35
Q

Give examples of chemical reactions in histochem

A
  1. Perl’s reaction- where ferric ions in the tissue react with potassium ferrocyanide to give a blue reaction product - demonstrates the presence of iron in bone marrow tissue
  2. Fouchet’s test for bilirubin, ferric chloride reacts with bilirubin in the tissue to form cholecyanin (blue colour)
36
Q

Examples of enzymatic reactions in histochem

A

The presence or absence of the enzyme alkaline phosphatase can be demonstrated in neutrophils where the enzyme causes a colour change on the addition of an appropriate substrate - deficiency of this enzyme in neutrophils of Chronic Myeloid Leukaemia

37
Q

What is metal impregnation?

A

The reduction of metal salts to the metallic states by tissue constituents

38
Q

What is the metallic salt used for metallic impregnation in histology and what is it reduced to?

A

Silver nitrate and it is reduced to a black silver deposit on many tissue constituents

39
Q

What happens on each day the sample is in the lab?

A

Day 1: Sample reception
-Sample received and accessioned
Day 2: Cut up
-Macroscopic grossing
-Inking
-Overnight processing
Day 3: Main lab
-Embedding, Mircotomy, Staining, Case assembly
Day 4: Pathologist
-Slide review, further tests ordered
Day 5: IHC/Special stains
-Microtomy, staining, slide review
Day 6: Pathologist
-Reporting

Histology (3 decks)

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