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Histology Spring 2020 > Chapter 7: Carbohydrates and Amyloid > Flashcards

Chapter 7: Carbohydrates and Amyloid Flashcards

1
Q

What is a carbohydrate?

A

hydrated carbons which are organic compounds such as sugars, starch, cellulose, and polymers linked to protein

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2
Q

What are the 3 types of Carbohydrates?

A

Monosaccharides: one sugar unit
Oligosaccharides: few, 2-10 sugar units
Polysaccharides: many sugar units

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3
Q

What is glycogen?

A

the polymer form of glucose used to store carbohydrates/energy

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4
Q

2 places where glycogen is located

A

Liver and skeletal muscles

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5
Q

Why is fixation important when staining for glycogen?

A

Because glycogen is quickly broken down to glucose after death

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6
Q

What is a polysaccharide?

A

a carbohydrate containing many sugar units, such as glycogen

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7
Q

What are acid mucosubstances?

A

Refers to Group 2 acid mucopolysaccharides and Group 3 glycoproteins
Both stain with alcian blue, but not all stain with PAS
epithelial or connective tissue mucins

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8
Q

What are the 4 groups of natural polysaccharides?

A

Group 1: Neutral Polysaccharides (nonionic homoglycans)
Group 2: acid mucopolysaccharides (anionic heteroglycans)
Group 3: Glycoproteins (mucins, mucoid, mucoprotein, mucosubstances)
Group 4: Glycolipids

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9
Q

What is glucose and its characteristics

A

free floating monosaccharide abundant in the body

Because glucose is soluble in aqueous solutions and small it cannot be demonstrated in tissue sections

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10
Q

What is mucin, and what are its properties?

A

Large, heavily glycosylated proteins secreted by epithelial and connective tissues in most humans
PAS positive
most are metachromatic and basophilic
precipitated by acetic acid
soluble in alkaline solutions (only use tap water for bluing)

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11
Q

Birefringence

A

Light is split into 2 waves refracted in different directions
Especially relevant for visualizing amyloid deposits in the Congo red stain under polarized light

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12
Q

Metachromasia

A

Change of color
Some tissue elements will stain a different color from the dye, and the background will stain the expected color
Ex: Toluidine blue on mast cell granules stains red to purple against a blue background

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13
Q

Polychromasia

A

A single solution that stains various tissue elements different colors based on which elements of the dye they interact with. This is not metachromasia

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14
Q

What is amyloid?

A

“Starchlike”

A fibrillar protein that deposits in tissue under certain pathogenic conditions and contains 1-2% carbohydrate

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15
Q

Where is amyloid typically found in the body?

A

Extracellular space of organs

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16
Q

What is amyloidosis?

A

Deposition of amyloid in extracellular space that gradually replaces cellular elements of organs eventually leading to death

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17
Q

What are the four old groups of amyloid?

A

Primary: spontaneous without prior disease; muscle, heart, skin, tongue
Secondary: usually associated with inflammatory disease; kidney, liver, spleen, and adrenal glands
Myeloma: associated with diseases of the immune system; muscle, heart, skin, tongue
Tumor associated: esp with tumors of the amine precursor uptake and decarboxylation system

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18
Q

What is the current number of amyloid types?

A

20

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19
Q

How are amyloids currently classified?

A

Abbreviation of originating protein, preceded by an A

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20
Q

How to test the quality of Schiff Reagent?

A

10mL of 37-40% formaldehyde in a beaker
Add a few drops of Schiff’s reagent
rapid reddish purple is good
slow bluish purple is bad

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21
Q

PAS detects which type of carbohydrate?

A

glycogen

polysaccharides such as glycogen, mucosubstances such as glycoproteins, glycolipids, and mucins in tissues

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22
Q

PAS requires which fixative?

A

10% NBF (or Bouin’s ONLY IF NOT DIGESTING)

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23
Q

PAS Purpose

A

demonstrate polysaccharides, neutral mucosubstances, and basement membranes

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24
Q

PAS Principle

A

Periodic acid oxidizes reactive groups to form aldehydes (-CHO)
Basic fuschin + sulfurous acid = leucofuschin/Schiff’s reagent which binds to exposed aldehyde groups
Water washes away the sulfur resulting in the rose chromophore

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25
Q

PAS Basic Procedure

A
  1. Deparaffinize and hydrate
  2. 0.5% Periodic acid for 5 minutes
  3. Wash with distilled water 3X
  4. Stain with Schiff reagent for 15 minutes
  5. Wash in 0.55% potassium metabisulphate to remove nonspecific Schiff background staining
  6. Wash in running tap water for 10 min to develop full color
  7. Counterstain 30 seconds in Harris Hematoxylin with acetic acid
  8. Wash sections well to blue the Hematoxylin
  9. Dehydrate, clear, coverslip
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26
Q

PAS Results

A

Glycogen, neutral mucosubstances, some epithelial sulfomucins and sialomucins, thyroid colloid, basement membranes, and fungal walls stain Bright Rose

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27
Q

PAS Technical Notes

A

Make sure to test Schiff: fast red purple is good, slow blue purple is bad
Fast green is an alternative counterstain to Hematoxylin
Don’t fix with glutaraldehyde because it causes non-specific binding of Schiff’s
Liver with abundant glycogen is a bad positive control because of abundance makes stain visualization difficult

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28
Q

PAS with Diastase digestion detects which type of carbohydrate?

A

Specifically glycogen, more specific than PAS alone

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29
Q

PAS-D requires which fixative?

A

10% NBF, formalin alcohol, or 100% alcohol

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30
Q

PAS-D Purpose

A

demonstrate glycogen in tissue sections (by comparing to PAS without digestion)

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31
Q

PAS-D Principle

A

This is a very sensitive reaction
Diastase and a-amylase depolymerize glycogen into glucose and maltose subunits that wash out of the section during the reaction, while mucin in glands remains polymerized
Schiff reaction is the same as in PAS

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32
Q

PAS-D Basic Procedure

A
  1. Deparaffinize, hydrate
  2. digest slide in diastase or saliva (a-amylase) for 20 minutes at room temp
  3. Wash in running water for 5 minutes
  4. 0.5% periodic acid for 5 minutes
  5. Wash in distilled water 3X
  6. Stain with Schiff for 15 minutes
  7. Wash for 1 minute in potassium metabisulfate to reduce background and non-specific staining
  8. Wash in running water for 10 minutes
  9. Counterstain for 30 seconds in Harris Hematoxylin
  10. Wash in running water
  11. Dehydrate, clear, coverslip
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33
Q

PAS-D Results

A

Positive rose stain for Glycogen is pale compared to undigested PAS stained slides which are Bright Rose

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34
Q

PAS-D Technical Notes

A

A-amylase is preferred because it doesn’t lift tissue off slides like diastase
Picric acid fixative (ex Bouin’s) can cause glycogen to be resistant to digestion and give a false result

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35
Q

Best Carmine detects which type of carbohydrate?

A

Glycogen (Group 1)

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36
Q

Best Carmine requires which fixative?

A

Absolute alcohol (Carnoy and Bouin’s are also acceptable)

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37
Q

Best Carmine Purpose

A

Demonstrate glycogen (inferior to PAS)

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38
Q

Best Carmine Principle

A

High pH of the staining solution (9-11) allows the dye to hydrogen bond to glycogen

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39
Q

Best Carmine Basic Procedure

A
  1. deparaffinize, hydrate
  2. Stain with Mayer or Harris Hematoxylin
  3. Wash in running water
  4. Stain in carmine solution (contains ammonium hydroxide, hazardous, use in hood)
  5. Differentiating solution
  6. 80% alcohol rinse
  7. Dehydrate, clear, coverslip
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40
Q

Best Carmine Results

A

Glycogen: Pink to red
Nuclei: Blue

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41
Q

Best Carmine Technical Notes

A

PAS is better because its more specific for glycogen, and the ammonia solutions in Best carmine are hazardous, and evaporate easily

Important to obtain fresh liver for controls because glycogen quickly breaks down after death

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42
Q

Mayer Mucicarmine detects which type of carbohydrate?

A

“epithelial” mucins and Cryptococcus neoformans fungus (Group 3)

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43
Q

Mayer Mucicarmine requires which fixative?

A

10% NBF

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44
Q

Mayer Mucicarmine Purpose

A

Stain epithelial mucins, esp goblet cells

carboxylated and sulfonated (acidics) nut not neutrals

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45
Q

Mayer Mucicarmine Principle

A

Stains carboxylated and sulfonated mucins by attaching to the acid groups of mucins through an aluminum chelation complex, thus does not stain neutral mucins

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46
Q

Mayer Mucicarmine Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Stain in Weigert (Iron) Hematoxylin (resists destaining in subsequent acidic solutions)
  3. Wash in running water (helps with bluing)
  4. Stain in mucicarmine solution
  5. Rinse and remove excess water
  6. Counterstain in metanil yellow (careful not to overstain)
  7. Dehydrate, clear, coverslip
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47
Q

Mayer Mucicarmine Results

A

Mucin: Deep rose to red
Cryptococcus capsule: deep rose to red
Nuclei: black/grey
Other tissue elements: blue (Gill Hematoxylin) or yellow

48
Q

Mayer Mucicarmine Technical Notes

A

Only rinse in tap water because mucins are soluble in alkaline solutions

don’t overstain with metanil yellow or you may mask the mucicarmine

mucicarmine stock deteriorates over time, monitor your control slides for gradual fading as your stock ages

prepare stock under hood because it can release HCl vapors

49
Q

Alcian Blue pH 2.5 detects which type of carbohydrate?

A

Group 2 Acid mucoploysaccharides, also known as connective tissue mucins
carboxylated only
carboxylated and sulfonated
sulfonated only

50
Q

Alcian Blue pH 2.5 requires which fixative?

A

10% NBF or Bouin’s

51
Q

Alcian Blue pH 2.5 Purpose

A

demonstrate acid mucopolysaccharides

52
Q

Alcian Blue pH 2.5 Principle

A

Alcian blue is a water soluble copper dye
In a 3% acetic solution Alcian blue stains both sulfated and/or carboxylated acid mucopolysaccharides and sulfated and/or carboxylated glycoproteins
Alcian blue forms salt linkages with the acidic groups of acid mucopolysaccharides

53
Q

Alcian Blue pH 2.5 Basic Procedure

A
  1. Deparaffinize, hydrate
  2. 3% acetic acid (condition the tissue)
  3. Alcian blue
  4. 3% Acetic acid rinse (prevents nonspecific staining)
  5. Wash in running tap water, warm preferred
  6. Rinse in distilled water
  7. Counterstain in Nuclear Fast Red
  8. Wash in running tap water to prevent cloudiness caused by NFR mixing with alcohol
  9. Dehydrate, clear, coverslip
54
Q

Alcian Blue pH 2.5 Results

A

Weakly acidic sulfated mucosubstances: dark blue
Hyaluronic acid: dark blue
Sialomucins: dark blue
Background: pink to red (may have some light blue areas)
Nuclei: Red

55
Q

Alcian Blue pH 2.5 Technical Notes

A

3% Acetic acid before Alcian blue stain conditions tissue to be more dye receptive
Incomplete dehydration prior to staining leads to weak staining
Overstaining with Alcian blue leads to blue nuclei (which should be red)
Sections that are not washed well after Nuclear Fast Red counterstain will become cloudy because it reacts with the alcohol dehydrant prior to coverslipping

56
Q

Alcian Blue pH 1.0 detects which type of carbohydrate?

A

Sulfated mucosubstances including:

Group 2 acid mucopolysaccharides and Group 3 glycoproteins

57
Q

Alcian Blue pH 1.0 requires which fixative?

A

10% NBF or Bouin’s

58
Q

Alcian Blue pH 1.0 Purpose

A

Demonstrate sulfated mucosubstances, rarely used, image is crisper than pH2.5

59
Q

Alcian Blue pH 1.0 Principle

A

At pH1 Alcian blue only stain sulfated acid mucopolysaccharides and sulfated glycoproteins, not unsulfated ones
carboxylated groups aren’t stained because they can’t ionize at pH1

60
Q

Alcian Blue pH 1.0 Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Rinse in 0.1N HCl (instead of Acetic acid) conditioning
  3. Stain in 1% Alcian blue in 0.1N HCl for 30 minutes
  4. Rinse in 0.1N HCl (reduces nonspecific staining)
  5. Blot sections dry (reduces nonspecific staining)
  6. 5 minute counterstain in Nuclear Fast Red
  7. Wash in distilled water (to prevent cloudy reaction)
  8. Dehydrate, clear, coverslip
61
Q

Alcian Blue pH 1.0 Results

A

Sulfated mucosubstances: pale blue
Background: pink to red
Nuclei: red

62
Q

Alcian Blue pH 1.0 Technical Notes

A

Same as pH2.5

Section is blotted dry after HCl rinse because washing it in water could cause a pH change and nonspecific staining

63
Q

Alcian Blue with Hyaluronidase digestion detects which type of carbohydrate?

A

Group 4 Glycoproteins, also known as epithelial mucins

64
Q

Alcian Blue with Hyaluronidase digestion requires which fixative?

A

10% NBF

65
Q

Alcian Blue with Hyaluronidase digestion Purpose

A

Differentiates epithelial mucins from connective tissue mucins

66
Q

Alcian Blue with Hyaluronidase digestion Principle

A

Alcian blue reaction is the same as previous protocols
Blue staining is dramatically reduced for connective tissue mucins such as: hyaluronic acid, chondroitin sulfate A, and chondroitin sulfate C
While stain remains strong for epithelial mucins such as Group 4 glycoproteins

67
Q

Alcian Blue with Hyaluronidase digestion Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Digest slides in hyaluronidase 37C for 2 hours
  3. 5 minute wash in running water
  4. 3% acetic acid (condition the tissue)
  5. Alcian blue
  6. 3% Acetic acid rinse (prevents nonspecific staining)
  7. Wash in running tap water, warm preferred
  8. Rinse in distilled water
  9. Counterstain in Nuclear Fast Red
  10. Wash in running tap water to prevent cloudiness caused by NFR mixing with alcohol
  11. Dehydrate, clear, coverslip
68
Q

Alcian Blue with Hyaluronidase digestion Results

A

Without digestion: deep blue acid mucopolysaccharides and sialomucins
with digestion: pale blue mucosubstances that contain hyaluronic acid and chondroitin sulfates A and C
Nuclei: reddish pink

69
Q

Alcian Blue with Hyaluronidase digestion Technical Notes

A

3% Acetic acid before Alcian blue stain conditions tissue to be more dye receptive
Incomplete dehydration prior to staining leads to weak staining
Overstaining with Alcian blue leads to blue nuclei (which should be red)
Sections that are not washed well after Nuclear Fast Red counterstain will become cloudy because it reacts with the alcohol dehydrant prior to coverslipping

70
Q

Alcian Blue-PAS-Hematoxylin detects which type of carbohydrate?

A

Acidic and neutral mucosubstances

sometimes abbreviated PAB or AB-PAS

71
Q

Alcian Blue-PAS-Hematoxylin requires which fixative?

A

10% NBF or Zenker

72
Q

Alcian Blue-PAS-Hematoxylin Purpose

A

Differentiates between neutral and acidic mucosubstances

Used to detect intestinal metaplasia

73
Q

Alcian Blue-PAS-Hematoxylin Principle

A

Alcian blue stains the acidic mucosubstances

PAS stains the neutral mucosubstances

74
Q

Alcian Blue-PAS-Hematoxylin Basic Procedure

A
  1. Deparaffinize, hydrate
  2. 3% acetic acid (conditioning)
  3. Stain with alcian blue
  4. Wash in tap water, rinse in distilled water
  5. Incubate in periodic acid (to form aldehyde groups)
  6. Wash in tap water, rinse in distilled water
  7. Stain with Schiff reagent
  8. Reducing rinse of sodium metabisulfate (to prevent nonspecific PAS staining)
  9. Wash in running tap water
  10. Stain with Harris Hematoxylin
  11. Dehydrate, clear, coverslip
75
Q

Alcian Blue-PAS-Hematoxylin Results

A

Exclusively acid mucosubstances: blue
Neutral polysaccharides: magenta
some substances will stain purple because they react to both PAS and alcian blue

76
Q

Alcian Blue-PAS-Hematoxylin Technical Notes

A

Same as PAS and Alcian blue pH2.5

77
Q

Colloidal Iron detects which type of carbohydrate?

A

carboxylated and sulfated mucopolysaccharides (group 2) and glycoproteins (Group 3)

78
Q

Colloidal Iron requires which fixative?

A

10% NBF (Avoid chromate fixatives B-5, Zenker, Helly)

79
Q

Colloidal Iron Purpose

A

Demonstrate carboxylated and sulfated mucopolysaccharides (group 2) and glycoproteins (Group 3)

80
Q

Colloidal Iron Principle

A

At low pH carboxylated and sulfated mucosubstances absorb Colloidal iron ions. Then Prussian blue reaction detects those bound iron ions to indirectly demonstrate the presence of acid mucosubstances

81
Q

Colloidal Iron Basic Procedure

A
  1. Deparaffinize, hydrate
  2. 12% acetic acid rinse (prevents water dilution of colloidal iron solution)
  3. Stain (primary) in colloidal iron
  4. 12% acetic acid rinse (for specificity)
  5. Ferrocyanide-HCl solution (secondary) (also used in Gomorri’s Iron stain)
  6. wash in running tap water (for specificity)
  7. Counterstain in Nuclear Fast Red
  8. Wash in running water (to prevent cloudiness)
  9. Dehydrate, clear, coverslip
82
Q

Colloidal Iron Results

A

Acid mucopolysaccharides and Glycoproteins (sialomucins): deep Prussian Blue
nuclei: pink-red
Cytoplasm: pink
Cryptococcus neoformans fungus: deep blue

83
Q

Colloidal Iron Technical Notes

A

A control should be run without the initial colloidal iron solution to ensure that Prussian Blue staining is not sue to hemosiderin (excess iron deposits in the tissue)

Less specific than alcian blue for acid mucopolysaccharides

May have some background due to connective tissue mucins

Strongly acid mucins that do not stain with Alcian also won’t stain with colloidal iron

84
Q

Crystal Violet detects which type of carbohydrate?

A

Amyloid

85
Q

Crystal Violet requires which fixative?

A

10% NBF or alcohol

86
Q

Crystal Violet Purpose

A

Faster but less accurate way to detect amyloid, good for rapid screening

87
Q

Crystal Violet Principle

A

Polychromatic stain, amyloid stains rose due to reacting with one of the components of crystal violet

88
Q

Crystal Violet Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Stain in working crystal violet
  3. Rinse in tap water
  4. Mount in Apathy’s or air dry and mount with synthetic resin
89
Q

Crystal Violet Results

A

Amyloid: purplish violet

Other tissue elements: Blue

90
Q

Crystal Violet Technical Notes

A

Bleeding into the mounting media can be prevented by adding sugar, this is why Apathy’s is preferred for aqueous mounting

staining in crystal violet for 5 hours can increase intensity and sensitivity of the stain because it self differentiates

some procedures use a 1% acetic acid wash to differentiate

91
Q

Congo Red detects which type of carbohydrate?

A

Amyloid

92
Q

Congo Red requires which fixative?

A

Alcohol or Carnoy preferred (10% NBF acceptable)

93
Q

Congo Red Purpose

A

Best stain to demonstrate amyloid

94
Q

Congo Red Principle

A

Benzidine derivative that reacts with cellulose (amyloid is similar to cellulose). Alkali acids separate protein chains exposing hydroxyls on the amyloid, which hydrogen bond to azo and amide groups in the Congo Red dye. The dye binds with the polysaccharide or protein component of amyloid. Results in highly specific apple green birefringence.

95
Q

Congo Red Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Stain in Harris Hematoxylin with acetic acid
  3. Wash in running tap water
  4. Alkaline salt solution (makes hydroxyls of amyloid available)
  5. Stain in working Congo Red solution
  6. Dehydrate, clear, coverslip
96
Q

Congo Red Results

A

Amyloid: deep pink to red, apple green birefringence under polarized light
Elastic tissue: pink
Nuclei: blue

97
Q

Congo Red Technical Notes

A

must cut sections 8-10uM for birefringence, too thin results in red, too thick results in yellow

98
Q

Thioflavine T Fluorescent Method detects which type of carbohydrate?

A

Amyloid

99
Q

Thioflavine T requires which fixative?

A

10% NBF

100
Q

Thioflavine T Purpose

A

Demonstrate amyloid, not as specific as Congo Red

101
Q

Thioflavine T Principle

A

Fluorescent dye that attaches to amyloid. Background nuclear fluorescence is quenched by staining with aluminum Hematoxylin

102
Q

Thioflavine T Basic Procedure

A
  1. Deparaffinize, hydrate
  2. Stain in Mayer hematoxylin to quench nuclear fluorescence
  3. Wash in water
    4 Stain in filtered Thioflavin T
  4. Rinse in distilled water
  5. Differentiate in 1% acetic acid
  6. Wash in running water and blot dry
  7. Mount with a non-fluorescent mountant
103
Q

Thioflavine T Results

A

Amyloid: fluoresces yellow to yellow green

104
Q

Group 1: Neutral Polysaccharides (nonionic homoglycans)

A

Glucose containing: glycogen, starch, cellulose
N-acetyl-glucosamine-containing: chitin
Very positive PAS stain
Negative for alcian blue, colloidal iron, and mucicarmine

105
Q

Group 2: acid mucopolysaccharides (anionic heteroglycans)

A

They are all acidic (anionic) polysaccharides attached to protein
Connective tissue mucins
PAS stains negative
Stains positive for alcian blue, colloidal iron, and mucicarmine

106
Q

Group 3: Glycoproteins (mucins, mucoid, mucoprotein, mucosubstances)

A

Mostly epithelial mucins, but some may occur in connective tissue
Potentially PAS positive, but not always

107
Q

Group 4: Glycolipids

A

Cerebrocides: fatty residue on a carbohydrate structure
phosphatides: PAS positive lipids that don’t contain carbohydrates
PAS positive, may react to other stains

108
Q

PAS (incorrectly) stains acid mucopolysaccharides

A

skipped the metabisulfate wash and then rinsed in over-chlorinated tap water causing non-specific off-target staining

109
Q

PAS/D (incorrectly) shows glycogen staining

A

glycogen wasn’t properly digested by diastase

Probably due to fixation in bouins/picric acid which inhibits diastase

110
Q

Background on mucicarmine is nearly completely yellow

A

overstained with metanil yellow

111
Q

Alcian blue shows non-specific staining

A

the stain is underdifferentiated, pretreat and rinse longer in acetic acid if staining with pH2.5, 1NHCL if using pH1.0

112
Q

Amyloid shows faint red birefringence

A

sections are too thin, make sure they are 8-10uM for Congo Red stain

113
Q

Congo red shows unnecessary background staining

A

make sure that the alkaline salt solution component is properly and fully saturated to prevent retention of excess Congo red dye in the tissue

114
Q

PAS control slide for fungi is very pale

A

make sure to counter stain with fast green

make sure not to use chromate fixatives (like B5, Zenker, or Helly) which may overoxidize reactive groups and lead to a weakened schiff reaction

115
Q

A section mounted with synthetic resin appears cloudy

A

If nuclear Fast Red counterstain was used, the slide probably wasn’t properly rinsed in water before dehydrating

remove the coverslip, back up to the NFR step, then make sure to thoroughly rinse in running tap water before dehydrating and coverslipping

116
Q

Excessive hydrolysis seen in a Feulgen Reaction

A

Probably used a picric acid fixative (like bouins) which overhydrolyzed the DNA resulting in a poor Feulgen stain

117
Q

PAS -
Alcian pH2.5 +
Colloidal Iron +

A

not neutral, not glycogen, not sialomucin
acid mucopolysaccharide “connective tissue mucin” carboxylated or sulfonated, or glycoprotein (sialomucin) “epithelial mucin”
acid mucopolysaccharide “connective tissue mucin”, carboxylated or sulfonated, or glycoprotein (sialomucin) “epithelial mucin”

These results suggest the presence of an acid mucosubstance (Probably Group 2), carboxylated or carboxylated and sulfated since pH1.0 wasn’t performed to demonstrate whether it is sulfonated only

Histology Spring 2020 (14 decks)

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