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Histotechnology > Staining of Pigments > Flashcards

Staining of Pigments Flashcards

1
Q

What are pigments?

A

Any of the various colouring agents deposited as cytoplasmic inclusions or granules within tissues and cells

Pigments can be coloured, thus not always requiring staining with biological dyes

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2
Q

How are pigments classified?

A

Three Groups:
* Artifact
* Endogenous
* Exogenous

Each group has distinct origins and characteristics

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3
Q

What are artifact pigments?

A

Produced during tissue processing, commonly from fixation

Usually lie on top of tissue, with exceptions like formalin pigment

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4
Q

Name examples of artifact pigments.

A
  • Formalin pigment
  • Mercury pigment
  • Chrome pigment

Formalin pigment is an acid hematin and birefringent

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5
Q

What is formalin pigment?

A

An acid hematin, birefringent, microcrystalline, dark-brown pigment formed from acidic formaldehyde solutions

Resistant to strong acids and can be dissolved by alcoholic picric acid

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6
Q

What are endogenous pigments?

A

Pigments formed within the body

Subdivided into hematogenous and non-hematogenous categories

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7
Q

What are hematogenous pigments?

A

Derived from blood, includes:
* Hemoglobin
* Hemosiderin
* Bile pigments

Hemoglobin breaks down into heme and globin

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8
Q

What is hemosiderin?

A

The storage form of ferric iron, a yellow-brown intracellular granular pigment

Found in liver, bone marrow, and spleen; increased in pathologic conditions

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9
Q

What is the Prussian Blue reaction used for?

A

Detection of ferric (Fe3+) iron in tissues

Forms a bright blue pigment called Prussian Blue when Fe3+ reacts with ferrocyanide

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10
Q

What are exogenous pigments?

A

Pigments formed externally and taken into the body

Examples include carbon, asbestos fibers, and tattoo pigments

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11
Q

What is lipofuchsin?

A

Known as the ‘wear and tear’ pigment, a yellow-brown pigment that collects in permanent cells

Formed by slow progressive oxidation of lipids and lipoproteins

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12
Q

What distinguishes ceroid from lipofuchsin?

A

Ceroid stains positive in acid-fast staining techniques, while lipofuchsin does not

Both stain with oil red O and sudan black B

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13
Q

What is the significance of melanin?

A

A brown-black pigment derived from tyrosine, present in skin, hair, and certain parts of the CNS

Variations in melanin amount contribute to differences in color among individuals

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14
Q

What are urates?

A

Deposits found in tissues or joints associated with gout, appearing as sodium urate crystals

Chronic gout can lead to accumulation of ‘tophi’ in soft tissues

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15
Q

Which minerals can be demonstrated with special stains?

A
  • Calcium (Ca2+)
  • Ferrous/Ferric (Fe2+/Fe3+)
  • Cupric (Cu2+)
  • Phosphate (PO4^3-)
  • Carbonate (CO3^2-)

Some metallic elements like silver, lead, and copper can be deposited pathologically

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16
Q

What are neuroendocrine cells?

A

Cells involved in hormone synthesis, including adrenal chromaffin cells and enterochromaffin cells

They have high uptake of amine precursors and the ability to decarboxylate

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17
Q

What is the role of fixation in demonstrating cytoplasmic granules?

A

Fixation is critical to preserving and demonstrating cytoplasmic granules

Different fixatives preserve different types of granules

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18
Q

What is the appearance of carbon as an exogenous pigment?

A

A black pigment that resists bleaching and extraction procedures

Insoluble in concentrated sulfuric acid

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19
Q

True or False: Formalin pigment can be removed using saturated alcoholic picric acid.

A

True

This is done before staining to prevent interference

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20
Q

Fill in the blank: Hemosiderin is a _______ form of ferric iron.

A

storage

It is usually found in small amounts in specific organs

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21
Q

What is the purpose of Perl’s Prussian Blue Technique?

A

Used for the demonstration of ferric (Fe3+) iron in tissues

Ferric iron is normally found in small amounts in liver, bone marrow, and spleen

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22
Q

What condition is characterized by excess iron storage due to defects in iron absorption?

A

Hemochromatosis

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23
Q

What can lead to excess storage of iron in the body?

A
  • Increased dietary consumption of iron
  • Hemolysis
  • Multiple transfusions
  • Severe congestion
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24
Q

What is hemosiderin?

A

A storage form of iron that accumulates when iron storage is beyond normal limits

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25
Q

What is the endpoint of the reaction in Perl’s Prussian Blue Technique?

A

An insoluble, highly visible pigment (ferric iron) that remains in situ

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26
Q

What type of fixative should be used when demonstrating hemosiderin?

A

Buffered neutral formalin

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27
Q

What reagent is used in Perl’s Prussian Blue Technique?

A

A solution of equal parts HCl and potassium ferrocyanide

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28
Q

What is the result of the reaction between ferric iron and potassium ferrocyanide?

A

Ferric-ferrocyanide; a bright blue coloured insoluble substance

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29
Q

What is the quality control requirement for Perl’s Prussian Blue Technique?

A

A section containing ferric iron without excessive amounts to avoid contamination

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30
Q

What is the purpose of the Turnbull Blue Stain?

A

The detection of ferrous (Fe2+) iron in tissues

31
Q

What is the principle behind the Turnbull Blue reaction?

A

Sections are treated with an acid solution of potassium ferricyanide, forming an insoluble bright blue pigment with ferrous iron

32
Q

What is the fixative used for the Turnbull Blue Stain?

A

Alcohol or 10% NBF

33
Q

What color indicates the presence of ferrous iron in Turnbull Blue Stain results?

34
Q

What are melanins classified as?

A

Argentaffin substances

35
Q

What technique is primarily used for the demonstration of melanin?

A

Masson-Fontana technique

36
Q

What is the role of phenolic groups in melanin during the Masson-Fontana technique?

A

Donate an electron to positively charged silver ions, reducing them to visible metallic silver

37
Q

What is the fixative used in the Masson-Fontana technique?

38
Q

What is the result of the Masson-Fontana technique for melanin?

A

Melanin and argentaffin granules appear black, nuclei appear pink

39
Q

What is used to differentiate between melanin and argentaffin granules?

A

Melanin Bleaching

40
Q

What are some strong oxidizing agents that can bleach melanin?

A
  • KMnO4
  • H2O2 (hydrogen peroxide)
41
Q

What is the purpose of the Mallory Bleach Technique?

A

To distinguish between melanin and argentaffin granules

42
Q

What is the first step in the Mallory Bleach Technique?

A

Immerse in 0.25% KMnO4 for 5-20 min

43
Q

What is the result of the Mallory Bleach Technique after washing in running tap water?

A

Removes KMnO4

44
Q

What is the most useful bleaching agent mentioned?

A

KMnO4

Followed by 1% oxalic acid.

45
Q

What is the purpose of the Mallory bleach technique?

A

To distinguish between melanin and argentaffin granules.

46
Q

What is the first step in the Mallory bleach technique?

A

Hydrate tissues.

47
Q

How long should tissues be immersed in 0.25% KMnO4?

48
Q

What does KMnO4 do in the Mallory bleach technique?

A

Oxidizes melanin.

49
Q

What is the purpose of washing in running tap water after using KMnO4?

A

To remove K permanganate.

50
Q

What is the next step after washing with water in the Mallory bleach technique?

A

Immerse in 5% oxalic acid.

51
Q

What is the result of bleaching with oxalic acid?

A

Bleaches melanin.

52
Q

What is the interpretation of a Masson-Fontana stain on a bleached slide?

A

No granules seen for melanin, golden brown granules seen for argentaffin granules.

53
Q

What does the Gomori Methenamine Silver Method for urates demonstrate?

A

The demonstration of urates in tissue.

54
Q

What condition can cause the deposit of urate crystals?

55
Q

What fixative is required for the Gomori Methenamine Silver Method?

A

Absolute alcohol.

56
Q

What is the main principle of the Gomori Methenamine Silver Method?

A

Urates are stained black by reacting with silver, which is then reduced to its metallic form.

57
Q

What are the reagents used in the Gomori Methenamine Silver Method?

A
  • Silver nitrate, 5%
  • Methenamine solution, 3%
  • Sodium borate, 5%
  • Sodium thiosulphate solution, 3%
  • Light green solution.
58
Q

What color do urates stain in the Gomori Methenamine Silver Method?

59
Q

What is the background color in the Gomori Methenamine Silver Method?

60
Q

What is hydroxyapatite?

A

A calcium phosphate salt and the main mineral component of bone and teeth.

61
Q

What are the two main methods used for calcium demonstration?

A
  • Von Kossa
  • Alizarin Red S.
62
Q

What does the Von Kossa Technique demonstrate?

A

Phosphate and carbonate or urate radical.

63
Q

What is the principle behind the Von Kossa Technique?

A

The method depends on the replacement of calcium by silver, which is then reduced to metal form.

64
Q

What type of fixative is preferred for the Von Kossa Technique?

65
Q

What is the result of the Von Kossa stain?

A

Calcium salts appear black/brown.

66
Q

What is the purpose of Alizarin Red S?

A

Used to demonstrate calcium in tissue sections.

67
Q

What is the principle of the Alizarin Red S method?

A

Calcium forms an alizarin red S-calcium complex in a chelation process.

68
Q

What color do calcium deposits stain with Alizarin Red S?

A

Orange-red.

69
Q

What is the background color when using Alizarin Red S?

70
Q

True or False: Alizarin Red S is more sensitive for small amounts of calcium than Von Kossa.

71
Q

What is the common artifact that can affect the Prussian blue reaction?

A

Decalcification with a strong acid.

72
Q

What can prolonged incubation in the silver solution cause in Masson-Fontana staining?

A

Dark black melanosomes and dirty gray staining.

73
Q

What is the likely cause of a marked black precipitate in a section of ileum stained with Masson-Fontana?

A

Using the silver immediately after preparation.

74
Q

What is the significance of calcium in coronary arteries?

A

Marker for coronary arterial disease (CAD).

Histotechnology (17 decks)

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