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Histotechnology 1 > Fixatives and Decalcification > Flashcards

Fixatives and Decalcification Flashcards

1
Q

Which is the best primary fixative for electron microscopy?

A

Glutaraldehyde (but for a short time 2hrs)

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2
Q

Upon microscopic examination, an H+E stained sections of routinely processed spleen shows small brown to black granules evenly distributed throughout the tissue. How can the granules be removed from the tissue?

A

By treating the tissue sections with alcoholic picric acid or alkaline alcohol.

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3
Q

What is an advantage of using 5% hydrochloric or nitric acid for decalcifying tissues?

A

Decalcifies rapidly.

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4
Q

The 3 ingredients in B5 fixative are:

A

HgCl2 (Mercuric chloride), concentrated Formaldehyde, Sodium Acetate

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5
Q

Which fixative makes lipids insoluble?

A

Osmium Tetroxide (OsO4)

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6
Q

True or False: 40% formaldehyde can also be called 100% formalin.

A

True

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7
Q

Which fixative enhances trichrome staining and can be used either as a primary fixative or a secondary for this purpose?

A

Bouin’s.

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8
Q

After fixing tissue in Bouin’s solution the excess picric acid is removed by washing in:

A

50% to 70% alcohol (Post fixation – 70% Ethanol, ETOH).

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9
Q

What fixative is commonly used in cytology?

A

Fixative for all either ethanol or methanol (either 95% or 100%)

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10
Q

Formalin binds with amino groups on tissue proteins and form ____________ bridges. These stabilize the protein structure.

A

methylene bridges

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11
Q

These characteristics belong to which fixative: Hemolyzes RBC’s, dissolves Iron, and increases acidophilia in staining.

A

Bouin’s

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12
Q

What is acidophilia?

A

Acidophile (or acidophil, or, as an adjectival form, acidophilic) is a term used by histologists to describe a particular staining pattern of cells and tissues when using haematoxylin and eosin stains. Specifically, the name refers to structures which “love” acid, and take it up readily.

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13
Q

A patient, Mr. Davidson, is suspected to have a glycogen storage disease. A liver biopsy is done to confirm this. Which fixative is best in providing maximum preservation of glycogen in the tissue?

A

Ethanol, Methanol, Picric Acid, Bouin’s, Zinc Formalin will preserve glycogen.

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14
Q

A lymph node biopsy is received in the lab. The pathologist request immunological studies to be carried out on the lymph node biopsy. Describe the fixative of choice for this scenario.

A

Zinc Formalin

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15
Q

Describe formaldehyde.

A

Formaldehyde (CH2O)

  1. Colorless gas, often dissolved in water at a maximum concentration of 37% to 40%.
  2. This is also known as 100% Formalin
  3. If we dilute this down 1 part Concentrated Formalin to 9 parts water we have a 10% Formalin.
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16
Q

Describe characteristics of 10% Formalin

A

10% Formalin

  1. Additive, Non-coagulant
  2. Reacts with the amino group (NH2) on proteins (side chain of amino acids) and forms methylene bridges that link protein chains together; Can remove some of the methylene bridges by washing in water
  3. Penetrates tissues very quickly but takes a while to form the methylene bridges
  4. Best if fixed 24 to 48 hours to complete cross-linkages
  5. Often tissues stored in it indefinitely
  6. It negatively affects (hinders) eosin dye from binding to the amino groups
  7. Enhances staining with basic or cationic dyes
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17
Q

What effects of 10% formalin are reversible?

A
  1. Formalin pigment AFH - If the pH of formalin is below 6.1 (check textbook) it can break down to formic acid which reacts with heme to form a brown/black pigment called acid formaldehyde hematin or AFH, in particular with blood rich tissue; Remove with alcoholic picric acid or alkaline alcohol (check textbook is 10% formalin different for 10%NBF?).
  2. Paraformaldehyde; Remove by filtering or methanol
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18
Q

Describe characteristics of acetic acid.

A
  1. 5% acetic acid is a common preservative for pickles
  2. Glacial acetic acid is concentrated acetic acid
    It is never used alone as a fixative as it does not fix carbohydrates or lipids. It lyses red blood cells.
    It is used as an ingredient in compound fixatives as it penetrates quickly and does not harden tissues
  3. It is an excellent fixative for nucleoproteins
  4. Causes swelling (may counteract shrinkage from other fixatives).
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19
Q

Describe 10% formalin effects on tissues.

A
  1. It traps glycogen in the tissues
  2. Fixes lipids but does not make them insoluble and they can still be lost during processing
  3. Can use formalin fixed tissues on the cryostat to observe lipids using special stains
  4. Preserves some antigens and enzymes
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20
Q

Describe 10% Neutral Buffered Formalin (NBF).

A

10% Neutral Buffered Formalin (NBF)
Formalin works best if at a pH of 7.0
Requires buffers to stabilize the pH
Uses sodium phosphate, monobasic and sodium phosphate, dibasic
Most common fixative and usually purchased that way
If the pH of formalin is below 6.1 it can break down to formic acid which reacts with heme to form a brown/black pigment called acid formaldehyde hematin or AFH.
Pigment can be removed by treating tissue with alcoholic picric acid or alkaline alcohol.

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21
Q

What safety hazards are associated with 10% Neutral Buffered Formalin (NBF)?

A

Hazards
1. Many studies consider it a carcinogen
2. Skin and eye irritant, proper PPE is required
Levels must be monitored
3. Can only be used in a well ventilated area
4. Must be treated with formalex to be discarded
5. On standing, formaldehyde may polymerize to a white precipitate called paraformaldehyde, it can be filtered out or prevented by adding methanol.

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22
Q

Describe acetone as a fixative.

A

Coagulant, intolerant, non-additive
Penetrate tissues rapidly and dehydrates tissues at the same time
Dissolve lipids
Overhardens and shrinks tissues, not routinely used except for special circumstances
Allows good demonstration of enzymes
Frozen sections to be stained for cell surface antigens in histochemical techniques
Used as a fixative of brain tissue if known or suspected cases of rabies
Flammable, poisonous

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23
Q

Describe ethanol, methanol as a fixative

A

Coagulant, intolerant, non-additive
Penetrate tissues rapidly
Dissolve lipids
Not often used alone for tissues as it overhardens and shrinks tissues, little effect on staining
Methanol used to fix touch preps and blood smears
Ethanol preserves glycogen, urate crystals, pigments (things that are soluble in water)
Used alone for a cytology fixative or slide fixative
Flammable, poisonous, government regulated

24
Q

Glutaraldehyde

A

Another aldehyde, similar to formaldehyde in that it causes methylene bridges but it has an extra aldehyde group at the other end of the molecule that is free
Cannot be used in staining techniques that bind with aldehydes (Periodic Acid Schiff’s)
Fixes at the rate it penetrates (slow)
Additive and non-coagulant, causes tissue to harden
Used mostly for a primary fixative for electron microscopy, but keep the time short (2 hours)

25
Q

Mercuric Chloride (HgCl2)

A

Was widely used but not common any more due to its extreme toxicity – affects central nervous system and acute nephritis– requires special disposal and special tracking
Very corrosive
Never used alone but in compound fixatives, or as a secondary fixative as it causes swelling and penetrates slowly
Coagulant and additive (binds to sulfhydryl groups)
Enhances staining as it leaves tissues very receptive to dyes (both cytoplasm and nuclei)
Leaves a black pigment- can’t prevent, but you can remove with alcoholic iodine, followed by sodium thiosulfate (steps called dezenkerization)

26
Q

What effects of mercury are reversible?

A

Leaves a black pigment.

Removed by Dezenkerization: A mixture of 0.5% iodine in 70% alcohol, then 2.5% sodium thiosulphate, and water

27
Q

Osmium Tetroxide (OsO4)

A

Osmium Tetroxide (OsO4)
Used as a secondary fixative for electron microscopy
Preserves lipids
Chemically combines with lipids and phospholipids of cell membranes
Makes them insoluble
Makes them black and is electron dense
Non-coagulant, additive
Poor penetration, tissues must be thin
Must be used in a fume hood (can fix your cornea and nasal mucosa)
Expensive

28
Q

Picric Acid (C6H3N3O7)

A

Used as a fixative ingredient and as a stain
Optimal fixation time – less than 24 hours
Strong coagulant of nuclear proteins, but leaves DNA soluble, additive, keeps tissues soft, but shrinks them
Not used alone but as a component of compound fixative
Excellent nuclear and cytoplasmic staining
Good for demonstration of glycogen
Enhances trichrome stains
Lyzes red blood cells, can remove ferric iron from tissues
Can colour tissues yellow – remove with 70% alcohol

29
Q

Zinc Salts (ZnSO4)

A

Used as an acceptable replacement for mercury
Zinc chloride can be used but it is very corrosive
Zinc sulphate is more commonly used
It is not used alone but usually with formaldehyde
It enhances nuclear detail
Often used for fixing lymph nodes and bone marrow biopsies
It is good for immunohistochemistry (preserves enzymes)

30
Q

Why are compound fixatives used?

A

Often fixatives are combined so that one disadvantage counteracts another
If one fixative causes shrinkage of tissue it can be combined with another one that causes swelling to limit the adverse affects on the cells

31
Q

Describe B5 (FORMOL SUBLIMATE).

A

HgCl2, concentrated Formaldehyde, Sodium Acetate
Formalin & Buffer minimize the bad effects of Mercury
Time: (4-6hrs)
3 hours min.
18 hours max.
Layered fixation
PROTEINS –
COAGULANT - Hg, NON-COAGULANT – Formalin, INTOLERANT (LESS)
ADDITIVE
CHO – no rxn
LIPIDS – no rxn

32
Q

What mineral does bone, dentine, arteries, cartilage and certain other abnormal tissues contain will cause cutting difficulties during microtomy due to its extreme hardness?

A

Hydroxyapatite, a complex molecule composed of calcium and phosphate.

33
Q

What is the risk in using decalcifying agents? When do you decalcify?

A

Decalcifying agents are very harmful to the tissue.

Therefore, decalcification is always carried out AFTER appropriate fixation has occurred.

34
Q

Why should decalcification solution be changed frequently?

A

Calcium ions migrate out of the tissue into the surrounding solution and the solution may become saturated with calcium ions and hence no calcium ions come out of solution.

35
Q

What is the purpose of decalcification?

A

Purpose – remove calcium salts from tissue (bone, arteries) to allow routine sectioning with a microtome. If they were left in the sample the sections would just shred making it impossible to view under a microscope.

Note: tissues must be thoroughly fixed prior to this process

36
Q

What are the two main methods of decalcification?

A

There are two main methods of decalcification:

  1. Acid Methods
  2. Chelating Agents
37
Q

What is the method and/or acid selected based on?

A

METHOD and ACID SELECTED BASED ON:

  1. urgency of case
  2. degree of mineralization
  3. scope of investigation
38
Q

What does the rate of decalcification depend on?

A

RATE OF DECAL DEPENDS UPON:

  1. size of tissue
  2. density of tissue
  3. type of decal agent
  4. volume of decal agent
39
Q

What should be done after fixation prior to the decalcification procedure?

A

Wash tissues well with water post fixation and prior to decalcification procedure

40
Q

What affect do fixatives still remaining in the tissue have on decalcification?

A
  • Phosphate buffers in 10%NBF will also buffer the decalcification reagent
  • If using HCl, it can combine with formalin to form a toxic bis-chloromethyl-ether
  • Zinc in fixatives will form a chelate with EDTA
41
Q

What negative effects will acid have on the tissue when used for decalcification?

A
  • Acids will effect the stainability of tissues
  • The stronger the acid = more damage to the tissue and poorer quality stain
  • The longer it is an acid = more damage to the tissue and poorer quality stain
  • Acids affect the nuclear staining the most
42
Q

How do you mitigate the effect of acids?

A
  1. Fixing the tissue first helps minimize the effects on cells and collagen fibres
  2. Must monitor the end point – just leave in as long as required.
43
Q

How do acids act on calcium?

A

Acids act on the calcium - hydroxyapatite to release a calcium salt

44
Q

How is the pH to be adjusted for the decalcification process?

A

Calcium salts are soluble at a pH of 4.5
Acids used to remove calcium will have a pH below that (0.5 – 3.0)
If we buffer the acid it will be closer to pH 4.5 not as harmful to the tissue or staining

45
Q

Describe use of NITRIC ACID and HYDROCHLORIC ACID for decalcification.

A 
NITRIC ACID and HYDROCHLORIC ACID
strong acids
5% - 10% used
rapid – urgent, biopsy
1-2 days
may damage tissue
46
Q

Describe use of FORMIC ACID for decalcification.

A 
FORMIC ACID
Weak acid
Slow - routine, non urgent
Up to 10 days
Very little damage & staining maintained

Can be mixed with 10%NBF to fix and decalcify in one step

47
Q

Describe use of commercial acid decalcifiers for decalcification. Name some examples.

A

COMMERCIAL ACID DECALCIFIERS
CAL-EX, RDO
Contents are a secret
Usually strong acids, buffered to have pH of 4.5
More gentle on tissues and allow better staining
Used frequently in the hospital labs
Are more expensive than other simple acids

48
Q

What are commercial acid decalcifiers really good for?

A

Great for urgent specimens and bone marrow biopsies

49
Q

Why does the acid solution need to be changed frequently?

A

As the acid solution allows the Calcium ions to be removed from the tissue they are brought into the solution.
The acid solution can become saturated with the ions and actually prevents further decalcification
The acid solution needs to be changed frequently

50
Q

What aids can be used to speed up decalcification?

A

Aids to help speed up the decalcification:
Agitation
Vacuum
Suspension in a bag – exposes all surfaces to the action of the decalcification solution, salts go to the bottom of the bag

51
Q

What should one never use to decalcify?

A

NEVER USE HEAT – causes severe damage to other components of the tissue

52
Q

Describe ion exchange resins use for decalcification (Acid type)?

A

Ion Exchange Resins
Uses formic acid over a layer of an ammonical salt of a sulfonated resin.
Calcium ions go out of tissue and ammonium ions go in

53
Q

Describe electrolytic method use for decalcification (Acid type)?

A

Electrolytic Method
Specimen is attached to the anode (positive pole) and when a current is passed through an acid solution, the calcium is drawn toward the cathode (negative pole)
Very quick but may generate heat

54
Q

Describe the chelating agent (EDTA) method of decalcification. Rate, adverse effects, etc.

A 
CHELATING AGENT – EDTA
Uses organic compounds that bind to certain ions such as calcium (also iron, magnesium)
pH more neutral or slightly alkaline
very slow – 6-8 weeks
very  tolerant
Has no adverse effects on staining
55
Q

What is the END POINT DETERMINATION?

A

Decalcification methods can damage the tissues and negatively affect its staining. We must remove the tissue from the solution as soon as the calcium has been removed. We have to be able to tell when the all of the calcium has been removed.

Histotechnology 1 (10 decks)

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