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Histology Spring 2020 > Chapter 11: Pigments, Minerals, and Cytoplasmic Granules > Flashcards

Chapter 11: Pigments, Minerals, and Cytoplasmic Granules Flashcards

1
Q

Endogenous Pigment

A

A pigment formed within the body

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2
Q

Exogenous Pigment

A

A pigment formed externally which is then taken into the body
Carbon: black, often in lung and associated lymph nodes
Asbestos fibers: birefringent in lungs
Tattoo ink: skin and associated lymph nodes
Metals

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3
Q

Hematogenous Pigment

A

Pigments derived from the blood

Hemoglobin, Iron, Hemosiderin, Biliverdin (bile)

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4
Q

Anthracotic Pigment

A

black discoloration of bronchi from carbon pigment, usually due to smoke or coal inhalation

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5
Q

Endogenous, Nonhematogenous Pigment

A

Not derived from blood

Lipidic (lipofucsin and ceroid) and non-lipidic pigments (melanin)

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6
Q

Mineral

A

Metallic and non-metallic ions necessary for growth and other bodily functions

Calcium, ferric/cupric phosphate/carbonate
Silver, lead, copper, gold

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7
Q

Artifact

A

Deposited on tissue as a result of chemical action
Usually occur during processing due to certain fixatives
Mercury, Chrome, Formalin, Malarial

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8
Q

Hemoglobin

A

Protein in red blood cells for transporting oxygen
Stains with acid dyes such as eosin
May be pathologically found in recent hemorrhages or renal tubules after hemolysis

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9
Q

Hemosiderin

A

Storage form of iron in bone marrow and spleen, recycled for production of hemoglobin
Yellow to brown pigment
Large deposits are typically pathological since a majority of the iron is in use as RBCs

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10
Q

Bile (biliverdin)

A

Greenish bile pigment, component of Heme in RBCs
Reduced to bilirubin in the liver and is secreted as a component of bile
Demonstrated by oxidizing bilirubin (yellow-brown) to biliverdin (green)
Abnormal accumulations can result in jaundice (yellowing of the skin), and deposition in the Kupffer cells and hepatocytes

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11
Q

Hematoidin

A

Formed in tissues as a result of hemorrhage and reduced oxygen tension
A pigment similar to bilirubin that is also oxidized to biliverdin by bile-demonstrating techniques

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12
Q

Lifespan and degradation of erythrocytes

A

Lifespan of 120 days
Then split open by hemolysis or phagocytosed by macrophages in the spleen
Break down into Heme and Globin
Heme breaks down into iron and biliverdin (greenish bile pigment)

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13
Q

Function of Iron, where is it found?

A

Component of Red blood cells, also found in liver and bone marrow as hemosiderin

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14
Q

Hemachromatosis

A

Disease caused by excessive absorption of dietary iron characterized by excessive hemosiderin deposits
Can be differentiated from other yellow-brown pigments by the Prussian-Blue reaction

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15
Q

Bilirubin function and characteristics

A

(Yellow-brown) Reduced form of biliverdin (green bile pigment )

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16
Q

Melanocytes

A

Cells responsible for demonstration of melanin, contain dopa oxidase
Demonstrated by exposure of frozen sections to a buffered solution of dopa, melanin, or melanin-like pigment

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17
Q

Melanin

A

Derived from tyrosine and is characteristically a brown-black pigment

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18
Q

Summary of melanin production

A

tyrosine is oxidized to dopa which is oxidized to melanin

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19
Q

What is lipofucsin, where is it found?

A

A lipidic pigment indicating “wear and tear” which collects in more permanent tissues such as the heart, liver, and neurons

Yellow-brown and stains with Oil Red O, Sudan Black B, and PAS

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20
Q

What is ceroid, where is it found?

A

A lipid pigment seen in hepatocytes and macrophages with liver cirrhosis

Yellow-brown, stains with Oil Red O, Sudan Black B, and PAS.
Can be differentiated from lipofucsin using acid fast stain (ceroid is acid-fast positive)

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21
Q

Where are three places melanin is found in the body?

A

eyes, hair, skin

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22
Q

Method for bleaching melanin pigment

A

Expose to an oxidizing agent, such as 10% hydrogen peroxide or 0.25% potassium permanganate followed by oxalic acid (to clear sections of color)

This is to help retain cell detail when melanin pigment is present in large quantities

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23
Q

Preferred fixative for minerals

A

Formalin because metallic fixatives (mercury, chrome) would leave artifacts in the tissue

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24
Q

What are urate crystals, where are they found?

A 
Urate crystals (sodium/uric acid deposits) are found in tissue and joints of people suffering from gout
They are an endogenous deposit, originating inside the body
They must be fixed in methanol (non-aqueous) because they are water soluble; are birefringent (polarizing microscopy), and can be demonstrated with argentaffin methenamine silver techniques
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25
Q

Argentaffin vs Argyrophilic silver reactions

A

Argyrophilic cells can be impregnated with silver, but require an external reducer (chemical or light) to demonstrate metallic silver. Can only be demonstrated with Argyrophil techniques.

Argentaffin cells can be impregnated with silver and reduce those ions to their visible metallic form. Can be demonstrated wit both argentaffin and argyrophil techniques

Silver stains are used to demonstrate cytoplasmic granules

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26
Q

Prussian Blue Purpose

A

Detection of ferric iron in tissues
Typically found in small amounts in the bone marrow and spleen
Excess hemosiderin (idiopathic hemochromatosis can lead to organ failure and death

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27
Q

Prussian Blue Principle

A

Detects the ferric iron (Fe3+) in loosely bound protein complexes (such as hemosiderin)

Strongly bound iron in hemoglobin will not react with Prussian Blue

Iron ions in the tissue react with acidic potassium ferrocyanide to form Prussian-Blue

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28
Q

Prussian Blue Preferred Fixative

A

Alcohol or 10% NBF

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29
Q

Prussian Blue Basic Procedure

A
  1. Deparaffinize, hydrate (use non-metallic forceps)
  2. Place slides in potassium-ferrocyanide and hydrochloric acid solution for 20 minutes
  3. Wash in distilled water
  4. Counterstain in nuclear fast red
  5. Rinse thoroughly in tap water to prevent cloudiness
  6. Dehydrate, clear, coverslip
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30
Q

Prussian Blue Results

A

Nuclei and hemofuchsin: bright red
Hemosiderin (iron): blue
Background: pink

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31
Q

Prussian Blue Technical Notes

A

Staining containers must be clean or residual iron will cause diffuse background staining

Acid decalcifiers for bone can dissolve iron; use 3% or 5% acetic acid or formic acid to preserve iron

Excess nuclear fast red must be thoroughly washed out to prevent cloudiness when placed in alcohol dehydrant

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32
Q

Turnbull Blue Purpose

A

Detection of Ferrous iron Fe2+ (very toxic) in tissue, rapidly converted to ferric form

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33
Q

Turnbull Blue Principle

A

Ferrous iron in the tissue reacts with acidic potassium-ferricyanide to form Turnbull blue

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34
Q

Turnbull Blue Preferred Fixative

A

alcohol or 10% NBF

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35
Q

Turnbull Blue Basic Procedure

A
  1. Deparaffinize, hydrate (use non-metallic forceps)
  2. Place slides in freshly prepared ferricyanide staining solution
  3. Wash in acetic acid
  4. Counterstain in nuclear fast red
  5. Rinse thoroughly in distilled water to prevent cloudiness
  6. Dehydrate, clear, coverslip
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36
Q

Turnbull Blue Results

A

Ferrous iron: blue

Background: pink-red

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37
Q

Turnbull Blue Technical Notes

A

Staining containers must be clean or residual iron will cause diffuse background staining

Acid decalcifiers for bone can dissolve iron; use 3% or 5% acetic acid or formic acid to preserve iron

Excess nuclear fast red must be thoroughly washed out to prevent cloudiness when placed in alcohol dehydrant

38
Q

Schmorl Technique Purpose

A

Demonstrates reducing substances present in tissue including melanin, argentaffin granules, and formalin pigment

39
Q

Schmorl Technique Principle

A

Ferric iron Fe3+ in the staining solution reacts with reducing substances in teh tissue to become ferrous iron Fe2+. The ferrous ions Fe2+ combine with ferricyanide to form insoluble Turnbull blue (ferrous ferricyanide)

40
Q

Schmorl Technique Preferred Fixative

41
Q

Schmorl Technique Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Place in ferric chloride-potassium ferricyanide
  3. Rinse in distilled water
  4. Stain in Mayer mucicarmine
  5. Rinse in distilled water
  6. Counterstain in Metanil yellow
  7. Rinse in distilled water
  8. Dehydrate, clear, coverslip
42
Q

Schmorl Technique Results

A

Reducing substances: blue-green
Goblet cells, mucin: rose
Background: yellow-green

43
Q

Schmorl Technique Technical Notes

A

Works well on GI tract because of mucin demonstration, nuclear fast red is an acceptable alternative to Mayer mucicarmine

Glassware must be clean and solutions must be fresh to avoid non-specific iron background staining

44
Q

Fontana Masson Purpose

A

Demonstrate argentaffin substances such as: melanin, argentaffin granules of carcinoid tumors, and some neurosecretory granules

45
Q

Fontana Masson Principle

A

Some tissue components are argentaffin and can bind metallic silver without the need of an external reducer

46
Q

Fontana Masson Preferred Fixative

A

10% NBF

Skin as melanin control, small appendix as argentaffin granule control

47
Q

Fontana Masson Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Immerse slides in silver nitrate solution. The reaction should b stopped when granules are dark brown and the background is colorless.
  3. Rinse in distilled water
  4. Immerse in gold chloride (toner)
  5. Rinse in distilled water
  6. Place in sodium thiosulfate (remove excess unreduced silver)
  7. Rinse in distilled water
  8. Counterstain in nuclear fast red
  9. (wash thoroughly in running water to prevent cloudiness)
  10. Dehydrate, clear, coverslip
48
Q

Fontana Masson Results

A

Melanin: black
Argentaffin granules: black
Nuclei: pink

49
Q

Fontana Masson Technical Notes

A

This technique is not specific for melanin and argentaffin granules. Other reducing substances, such as formalin will also give a positive reaction

Overstaining will result in a dirty grey background and loss of contrast

Silver solutions should be discarded on the day of use because they can form explosive compounds after standing for several days

For melanin, duplicate sections should be treated with the melanin bleach procedure. if the pigment is melanin it will disappear with this treatment

50
Q

Grimelius Purpose

A

Demonstrate argyrophil granules in neurosecretory tumors

51
Q

Grimelius Principle

A

Tissue binds silver ions, but requires an external reducer to reduce silver to its visible metallic form

52
Q

Grimelius Preferred Fixative

53
Q

Grimelius Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Place in working silver solution
  3. Drain slides
  4. Place in reducing solution (hydroquinone)
  5. Rinse in distilled water
  6. Repeat step 2
  7. Drain and place slides in reducing solution again
  8. Rinse in distilled water
  9. Counterstain with nuclear fast red
  10. Rinse thoroughly in distilled water to prevent cloudiness
  11. Dehydrate, clear, coverslip
54
Q

Grimelius Results

A

Argentaffin granules: dark brown to black
Argyrophil granules: dark brown to black
Nuclei: red
Background: pale yellow brown

55
Q

Grimelius Technical Notes

A

Argentaffin substances will also stain with this method because they will bind and reduce the silver on their own. To differentiate between argentaffin and argyrophil both Grimelius and Fontana Masson techniques must be performed and compared

56
Q

Churukian-Schenk Purpose

A

Demonstrate argyrophil granules in neurosecretory tumors

57
Q

Churukian-Schenk Principle

A

Argyrophil stains/substances require an external reducer to demonstrate visible metallic silver

58
Q

Churukian-Schenk Preferred Fixative

A

10% NBF

argyrophil-positive carcinoid tumor, or section of small intestine (containing argentaffin granules)

59
Q

Churukian-Schenk Basic Procedure

A
  1. Deparaffinize, hydrate to acidified water, pH 4.0-4.2
  2. Place slides in silver-nitrate
  3. Rinse in distilled water
  4. Transfer to reducing solution (hydroquinone)
  5. Rinse in distilled water
  6. Return slides to the same silver stain
  7. Rinse in distilled water
  8. Place in reducing solution
  9. Rinse in distilled water
  10. Counterstain in nuclear fast red
  11. Rinse thoroughly in distilled water to prevent cloudiness
  12. Dehydrate, clear, coverslip
60
Q

Churukian-Schenk Results

A

Argyrophil granules: black
Argentaffin substances: black
Nuclei: red
Background: yellow-brown

61
Q

Churukian-Schenk Technical Notes

A

Shelf-life of silver-nitrate and hydroquinone can be greatly increased by storing these chemicals at 4C

62
Q

Gomori Methenamine Silver Purpose

A

Demonstrate urates in tissue

Gout is the deposition of urate crystals, especially around joints and in soft tissues

63
Q

Gomori Methenamine Silver Principle

A

Urates are stained black by reacting with silver (argentaffin/ no external reducer)

64
Q

Gomori Methenamine Silver Preferred Fixative

A

absolute alcohol must be used because urates are water soluble

65
Q

Gomori Methenamine Silver Basic Procedure

A
  1. Deparaffinize and rinse with absolute alcohol. DO NOT HYDRATE (urate is water soluble)
  2. Place sections in working methenamine silver solution that has been preheated to 60C
  3. Incubate for 30 minutes. Urate crystals should be black
  4. Rinse in distilled water
  5. Place sections in sodium thiosulfate (removes unreduced silver)
  6. Wash in running water GENTLY to prevent tissue from washing off
  7. Rinse in distilled water
  8. Counterstain with light green
  9. Dehydrate, clear, coverslip
66
Q

Gomori Methenamine Silver Results

A

Urates: black, also birefringent
background: blue-green

67
Q

Hall Purpose

A

Demonstrate presence of bilirubin and distinguish bile pigment from other pigments

68
Q

Hall Principle

A

Easily identifiable green color develops when bilirubin is oxidized to biliverdin in an acid medium

ferric chloride in trichloroacetic acid facilitates the oxidation (fouchet reagent)

69
Q

Hall Preferred Fixative

70
Q

Hall Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Wash in distilled water
  3. Stain in Fouchet reagent (trichloroacetic acid and ferric chloride)
  4. Wash in tap water, rinse in distilled water
  5. Stain in van Gieson
  6. Place in 95% alcohol and rinse well
  7. Dehydrate, clear, coverslip
71
Q

Hall Results

A

Bile or bilirubin: emerald green to olive drab

Background: yellow

72
Q

Von Kossa Purpose

A

Identify the presence of calcium

73
Q

Von Kossa Principle

A

Silver reacts with carbonate and phosphate anions of the calcium salts, not calcium directly.

Bright light reduces the silver salt to metallic silver and unreduced silver is removed by sodium thiosulfate (argyrophil)

74
Q

Von Kossa Preferred Fixative

A

Alcohol or 10% NBF

Avoid acidic fixatives because they break down calcium deposits into ions

75
Q

Von Kossa Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Place in silver nitrate, and expose to bright sunlight. Stop the reaction when the calcium salts are brown-black
  3. Rinse in distilled water
  4. Place in sodium thiosulfate
  5. Wash in distilled water
  6. Counterstain in nuclear fast red
  7. Wash thoroughly in water to prevent cloudiness
  8. Dehydrate, clear, coverslip
76
Q

Von Kossa Results

A

Calcium salts: black

Background: red

77
Q

Von Kossa Technical Notes

A

Alcoholic iodine for removing mercury may also remove calcium salts

Unbuffered formalin may reduce silver
Artificial light produces a brown rather than black pigment

78
Q

Alizarin Red S Purpose

A

Identify the presence of calcium

79
Q

Alizarin Red S Principle

A

Alizarin Red S will actually react with all of the following cations: Calcium ,magnesium, manganese, barium, and strontium.
However, only Calcium is typically present in large enough quantities ot be demonstrated

80
Q

Alizarin Red S Preferred Fixative

A

Alcoholic formalin or 10% NBF

81
Q

Alizarin Red S Basic Procedure

A
  1. Deparaffinize and hydrate to 50% alcohol
  2. Rinse in distilled water
  3. Place slides in Alizarin Red S, remove slides when an orange-red lake forms
  4. Shake off excess dye and blot
  5. Dehydrate in acetone and in acetone-xylene
  6. Clear and coverslip
82
Q

Alizarin Red S Results

A

Calcium deposits: orange-red (also birefringent)

83
Q

Rhodanine Copper Purpose

A

Detection of copper in tissue, especially when looking for Wilson disease in the liver

84
Q

Rhodanine Copper Principle

A

Demonstrates the protein that copper bonds to rather than the copper itself, so there is a chance for false positives

85
Q

Rhodanine Copper Preferred Fixative

A

10% NBF

Control must contain copper and be cut at -10uM

86
Q

Rhodanine Copper Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Place slides in Rhodanine solution for 18 hours at 37C
  3. Rinse in distilled water
  4. Stain in Mayer hematoxylin
  5. Rinse with distilled water
  6. Rinse in sodium borate
  7. Rinse in distilled water
  8. Dehydrate, clear, coverslip
87
Q

Rhodanine Copper Results

A

Copper: bright red to red-yellow
Nuclei: light blue

88
Q

Rhodanine Copper Technical Notes

A

If the concentration of copper is too low, fading may occur and make it difficult to distinguish between copper and lipofucsin

Fetal liver is a good control for copper

Do not overstain with hematoxylin or the copper may be masked

89
Q

How to remove Chrome pigment

A

Wash tissue in running water before dehydration

90
Q

How to remove Mercury pigment

A

Iodine, then sodium thiosulfate

91
Q

How to remove Formalin pigment

A

Absolute alcohol saturated with picric acid, or 70% alcohol containing ammonium hydroxide

92
Q

Melanin Bleaching Procedure

A
  1. Deparaffinize, hydrate
  2. Treat with potassium permanganate
  3. Wash well in tap water
  4. Place slides in oxalic acid
  5. Wash in running tap water
  6. Rinse in distilled water
  7. Stain in nuclear fast red
  8. Rinse thoroughly with water to prevent cloudiness
  9. Dehydrate, clear, coverslip

Histology Spring 2020 (14 decks)

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