Fixation

Question 1

10% aqueous formalin

Answer 1

○ Formaldehyde, 37-40%
○ DI water

Hypotonic fixative that may produce formalin pigment (acidic)

Question 2

10% formalin saline

Answer 2

○ Formaldehyde, 37-40%
○ Sodium chloride
○ DI water

Isotonic fixative that may produce formalin pigment

Question 3

Calcium formalin

Answer 3

○ Formaldehyde, 37-40%
○ Calcium chloride
○ DI water

Recommended for the fixation and preservation of phospholipids. The Ca ions prevent the phospholipids from taking up water and distorting.

Question 4

Formalin ammonium bromide

Answer 4

○ Formaldehyde, 37-40%
○ Ammonium bromide
○ DI water

Recommended for central nervous system, especially in conjunction with the Cajal astrocyte procedure.

It is acidic, lyses RBC, and cause nuclei to give a direct positive Feulgen reaction)

Question 5

Acetate formalin

Answer 5

○ Formaldehyde 37-40%
○ Sodium acetate
○ DI water

Question 6

10% neutralized formalin

Answer 6

○ Formaldehyde, 37-40%
○ DI water
○ Calcium or magnesium carbonate

Not recommended as a fixative because the solution becomes acidic after withdrawal from the storage bottle

Question 7

10% neutral-buffered formalin (NBF)

Answer 7

○ Formaldehyde, 37-40%
○ DI water
○ Sodium phosphate, monobasic
○ Sodium phosphate, dibasic

Most widely used for routine formalin fixation. pH of 6.8-7.2 and is hypotonic.

Question 8

Modified Millonig formalin

Answer 8

○ Formaldehyde, 37-40%
○ DI water
○ Sodium phosphate, monobasic
- Sodium hydroxide

Isotonic with a pH of 7.2-7.4. Allows electron microscopy on stored tissue

Question 9

Define the purpose of fixation.

Answer 9

○ Prevent autolysis and putrefaction from occurring so cells and tissue can be viewed as they appeared in their living state.
○ Insolubilize cellular components and
○ Bring out differences in refractive indexes and to increase the visibility of, or the contrast between, different tissue element

Question 10

Define: Autolysis

Answer 10

Destruction or digestion of tissues and cells by the enzymes normally present in cells

Question 11

Define: Fixation

Answer 11

The stabilization of protein

Question 12

Define: Artifact

Answer 12

A structure or substance not normally present but produced by some external force or action (e.g. mercury pigment, tissue floaters, knife lines, air bubbles)

Question 13

Define: Pigment

Answer 13

A heterogenous group of substances that contain enough natural color to be visible without any further staining (e.g. melanin - a brownish-black pigment found normally in skin, hair and eyes / hematin - brownish black pigment found in blood rich tissues fixed in acidic formalin solution)

Question 14

Define: Denaturation

Answer 14

To change the nature of.
○ Fixatives denature proteins by changing the soluble (liquid) contents of the cell into insoluble substances so that those substances are not lost during the subsequent processing steps.
○ This change can be caused by either chemical (fixative solutions) or physical (heat, desiccation) means.
○ Denaturation causes the protein molecule to unfold and the internal bonds to become disrupted

Question 15

Define: Nonaqueous fixative

Answer 15

Solutions that do not use water

Question 16

Define: Coagulating fixative

Answer 16

This fixative transforms the protein of the cytoplasm into fine mesh which does not interfere with light microscopy but not used in electron microscopy. They separate proteins from water as coagulum
e.g. zinc salts, mercuric chloride, curric sulfate, ethyl alcohol, methyl alcohol, acetone, and picric acid.

Question 17

Define: Additive fixative

Answer 17

Chemically link, or add themselves, to the tissue and change it with this action.
e.g. mercuric chloride, chromium trioxide, picric acid, formaldehyde, glutaraldehyde, glyoxal, osmium tetroxide, and zinc sulfate or chloride

Question 18

Define: Non additive fixative

Answer 18

Acts on tissue without chemically combining with it. Dissociates bound water molecules from tissue protein groups, eliminating the solubility of the protein and causing it to precipitate. Excessive removal of bound water results in shrinkage and hardening. Predominately organic compounds like acetone and the alcohols.

Question 19

Define: Hypertonic

Answer 19

A solution that has a higher concentration of solutes (particles) compared to the inside of a cell. This causes water molecules to move out of the cell, leading to cell shrinkage.

Question 20

Define: Isotonic

Answer 20

Fluids into which normal animal cells can be placed without causing either swelling or shrinkage of the cells. A 0.9% solution of sodium chloride (saline) or a 5% glucose solution is approximately isotonic

Question 21

Describe the effect of temperature on tissue fixation.

Answer 21

○ Can affect tissue morphology
○ An increase in temperature increases the rate of fixation but also the rate of autolysis and diffusion of cellular elements (up to 45C has little effect on morphology)
○ 0-4C was considered ideal for electron microscopy

Question 22

Describe the effect of size on tissue fixation.

Answer 22

○ Sections should be less than or equal to 3mm thick, even thinner for short protocols (at no time should a section be so thick that is touches the both the top and bottom of the cassette)
○ Large specimens like segments of the colon or small intestine should be surgically opened before being placed in fixative to reduce autolysis of the epithelium
○ Solid organs like spleen or kidney should be “bread-loafed” as soon as possible and covered with adequate fixative

Question 23

Describe the effect of volume ration on tissue fixation.

Answer 23

○ Fixative volume should be at least 15-20x greater than the tissue volume
○ 2-way exchange: The diffusion of the fixative into the tissue displaces the free water and serum proteins of the tissue
○ A tissue volume that is too large will compromise the fixative composition and therefore alter the characteristics

Question 24

Describe the effect of Ischemic time on tissue fixation.

Answer 24

○ Ideally the tissue should be placed in fixative immediately, the more time that elapses between interruption of the blood supply and fixation, the more postmortem changes that can be demonstrated microscopically (enzyme rich tissue like liver, pancreas, and brain are more subject to rapid autolysis
○ Often seen as surface autolysis (degradation or loss of epithelium)

Question 25

Describe the effect of duration on tissue fixation.

Answer 25

○ Adequate fixation is needed so that the morphology of the tissue is preserved and tissue will not be distorted by the subsequent processing steps ○ Tissue must be removed from certain fixatives, washed if indicated, and then stored in an appropriate storage solution - if allowed to remain in these fixatives for too long, the tissue becomes over hardened and staining may be impaired

Question 26

Describe the effect of fixative choice on tissue fixation.

Answer 26

○ Specimens for immunofluorescence study or enzyme profile must be frozen without fixation ○ Rarely a particular fixative must be chosen to ensure optimal demonstration of a particular tissue element ○ Some tissue elements cannot be demonstrated if the wrong fixative is chosen

Question 27

Describe the effect of penetration rate on tissue fixation.

Answer 27

depth (d) to which the fixative penetrates is proportional to the square root of time (t) hours the tissue was in fixative. K being the coefficient of diffusibility d=k√t ○ The Medawar constant (k) value increases as the rate of penetration increases ○ Increased pressure improves formalin penetration ○ The presence of muscle fibers and capillaries as well as increased surface area with crypts can increase penetration rate ○ Order of decreasing penetration rates: formaldehyde > acetic acid > mercuric chloride > methyl alcohol > osmium tetroxide > picric acid ○ Rate of penetration also impacted by heat

Question 28

Describe the effect of tissue storage on tissue fixation.

Answer 28

Wet tissue storage is important because the wet tissue often will be needed for additional studies

Question 29

Describe the effect of pH on tissue fixation.

Answer 29

Influences the reactivity of the fixative and ultrastructure preservation

Question 30

Describe the effect of Osmolality on tissue fixation.

Question 31

Describe the properties, functions, and actions of Acetone.

Question 32

Describe the fixation properties, functions, and actions of Alcohols.

Question 33

Describe the fixation properties, functions, and actions of B-5 solution.

Question 34

Describe the fixation properties, functions, and actions of Bouin solution.

Question 35

Describe the fixation properties, functions, and actions of Carnoy & methacarn solution.

Question 36

Describe the fixation properties, functions, and actions of Davidson solution.

Question 37

Describe the fixation properties, functions, and actions of Formalin.

Question 38

Describe the fixation properties, functions, and actions of Aqueous Formalin.

Question 39

Describe the fixation properties, functions, and actions of Buffered Formalin.

Question 40

Describe the fixation properties, functions, and actions of Neutralized Formalin.

Question 41

Describe the fixation properties, functions, and actions of Acetate Formalin.

Question 42

Describe the fixation properties, functions, and actions of Formalin alcohol.

Question 43

Describe the fixation properties, functions, and actions of Calcium Formalin.

Question 44

Describe the fixation properties, functions, and actions of Formalin ammonium bromide.

Question 45

Describe the fixation properties, functions, and actions of Gendre solution.

Question 46

Describe the fixation properties, functions, and actions of Glutaraldehyde.

Question 47

Describe the fixation properties, functions, and actions of Glyoxal.

Question 48

Describe the fixation properties, functions, and actions of Helly solution.

Question 49

Describe the fixation properties, functions, and actions of Hollande solution.

Question 50

Describe the fixation properties, functions, and actions of Mercuric chloride.

Question 51

Describe the fixation properties, functions, and actions of Orth solution.

Question 52

Describe the fixation properties, functions, and actions of Osmium tetroxide.

Question 53

Describe the fixation properties, functions, and actions of Paraformaldehyde.

Question 54

Describe the fixation properties, functions, and actions of Potassium dichromate.

Question 55

Describe the fixation properties, functions, and actions of Zamboni solution.

Question 56

Describe the fixation properties, functions, and actions of Zenker solution.

Question 57

Describe the fixation properties, functions, and actions of Zinc Formalin.

Question 58

Identify the chemicals in and any special indication for B-5 fixative.

Question 59

Identify the chemicals in and any special indication for Bouin solution.

Question 60

Identify the chemicals in and any special indication for Carnoy & methacarn solution.

Question 61

Identify the chemicals in and any special indication for Davidson solution.

Question 62

Identify the chemicals in and any special indication for Gendre solution.

Question 63

Identify the chemicals in and any special indication for Helly solution.

Question 64

Identify the chemicals in and any special indication for Hollande solution.

Question 65

Identify the chemicals in and any special indication for Orth solution.

Question 66

Identify the chemicals in and any special indication for Zamboni solution.

Question 67

Identify the chemicals in and any special indication for Zenker solution.

Question 68

Identify which fixatives require post-fixation washing & identify the preferred washing agent.

Question 69

Identify the fixation pigments and the conditions under which the pigment may be formed.

Question 70

Identify which fixation pigments can be prevented and which of the fixation pigments can be removed, and how.

Question 71

Explain the difference between buffered and neutralized formalin.

Question 72

State how paraformaldehyde differs from formaldehyde.

Question 73

Describe the difference between formalin & formaldehyde.

Question 74

Identify the percentage and volume of formaldehyde in 1L of a 10% formalin solution.

Question 75

List 2 methods of fixation other than using chemical reagents.

Question 76

Identify the preferred method of fixation (or lack of fixation) for Enzyme histochemistry.

Question 77

Identify the preferred method of fixation (or lack of fixation) for Immunofluorescence.

Question 78

Identify the preferred method of fixation (or lack of fixation) for Skeletal muscle cross-striation (nonimmunohistochemical staining).

Question 79

Identify the preferred method of fixation (or lack of fixation) for Pheochromocytomas.

Question 80

Identify the preferred method of fixation (or lack of fixation) for Urates.

Question 81

Identify the preferred method of fixation (or lack of fixation) for Immunohistochemical methods.

Question 82

Identify the preferred method of fixation (or lack of fixation) for trichrome stains.

Question 83

Identify which fixative reagents are protein coagulants.

Question 84

Identify which fixative reagents are protein noncoagulants.

Question 85

Identify which fixative reagents are additive fixatives.

Question 86

Identify which fixative reagents are nonadditive fixatives

Question 87

Identify the site or group with which the additive reagent reacts.

Question 88

Describe the effect of acetic acid on erythrocytes & collagen.

Question 89

Describe the action of zinc on fixation.

Question 90

State 2 major problems associated with fixation.

Answer 90

Autolysis & incomplete fixation.

Question 91

Identify 3 corrective actions for autolysis.

Answer 91

○ Place specimens in fixative solution as soon as possible, ensuring the volume is 15-20x that of the tissue ○ Open uterus specimens upon receipt so the fixative can immediately come into contact with the endometrium ○ Open and pin open gastrointestinal tract specimens upon receipt so that the fixative can immediately come into contact with the mucosal surface ○ Slice any organ resection, breast, or large tissue specimens into thin slices & placing in a large volume of fixative ○ Bisect lymph nodes if appropriate & place in fixative

Question 92

Identify 3 corrective actions for incomplete fixation.

Answer 92

○ Increase the time allowed in fixative solution. Some problems may be noted even after 24 hours of fixation in 10% NBF ○ Change to another fixative such as zinc formalin, which still requires several hours for complete fixation. Glyoxal should also be considered because it is an extremely rapid fixative ○ Place formalin alcohol in the first 3 stages of the processing cycle. This will decrease fixation time, and also begin dehydration ○ Ensure that the gross sections are thin enough for good reagent penetration, and that the amount of fixative is 15-20x that of the tissue ○ Ensure the formalin solution is not depleted because of overuse, change the solution frequently ○ Do not pack cassettes tightly in the processing holders ○ Use agitation of cassettes in fixative holding solutions during or after gross examination

Question 93

List 3 criteria for determining the quality of fixation for ultrastructural studies.

Question 94

Give 2 advantages & 2 disadvantages of fixation for ultrastructural studies with Osmium tetroxide.

Question 95

Give 2 advantages & 2 disadvantages of fixation for ultrastructural studies with Aldehydes.

Question 96

Give 2 advantages & 2 disadvantages of fixation for ultrastructural studies with Zamboni solution.

Question 97

Identify the hallmarks of good fixation.

Answer 97

○ Nuclei with various crisp chromatin patterns & a crisp blue nuclear membrane; nuclei should not show smudginess, bubbling, or fading ○ There should not be any cell shrinkage or artifactual spaces between individual cells ○ Cell cytoplasm should be well preserved & should stain well with eosin

Question 98

What are the primary fixatives for ultrastructural studies (electron microscopy)?

Answer 98

Osmium tetroxide Aldehydes (formaldehyde & glutaraldehyde) Buffered PAF (Zamboni)