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mlsci 250 > introduction and fixation > Flashcards

introduction and fixation Flashcards

1
Q

why is specimen receiving important?

A

cells and tissues begin to die/degrade immediately after removal from the body

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2
Q

how are specimens transported?

A

tissue is placed in a fixative solution

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3
Q

T or F: fresh tissue to the lab is not a urgent priority

A

F: it is urgent

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4
Q

what are the two sources of samples for the specimens

A
  • surgical
  • post mortem
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5
Q

what is grossing

A

describing the tissue macroscopically

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6
Q

what are some parameters of block selection

A
  • size: “1cm x 1cm round mass”
  • texture: “soft fragments of tissue”
  • number/proportion “specimen submitted entirely”
  • markings “medial edge with india ink”
  • locations “resection margins”
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7
Q

what is fixation

A

preservation of cells and tissues in as life-like a manner as possible by stabilizing the protein so that it is resistant to further changes

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8
Q

what is the function of fixatives

A
  1. prevent putrefaction and autolysis
  2. help maintain proper relationships between cells and extracellular substances
  3. bring out differences in refractive indexes and increase the visibility of the contrast between different tissue elements
  4. secondary functions: enhacing staining, limiting osmotic effects, preventing dessication
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9
Q

what is autolysis

A

self destruction after cell death via intracellular enzymes
- highly specialized cells are more rapidly and seriously affected
- affected by temperature

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10
Q

nuclear changes of autolysis

A

pyknosis, karyorrhexis, karyolysis

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11
Q

how does the cytoplasm appear in autolysis

A

increasingly granular and swollen

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12
Q

delayed fixation…

A

makes it difficult to differentiate

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13
Q

putrefaction

A

decomposition by microorganisms

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14
Q

what are the three modes of action

A
  1. stabilize
  2. kill
  3. make
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15
Q

stabilize

A

render enzymes inactive by stabilizing proteins

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16
Q

kill

A

kill bacteria and molds

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17
Q

make

A

make tissue more receptive to dyes

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18
Q

common impacts on tissue

A
  1. changes size - often shrinks
  2. changes texture - becomes more brittle and hard
  3. material can be lose - can dissolve away
  4. chemical alterations - charges on various components may change, molecules of fixative may attach to tissue and change properties
  5. fixation artifacts - deposits on and around tissues that impact microscopic image
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19
Q

what are some things to consider when fixing tissue

A
  • penetration rate and density of tissue
  • volume ration
  • time
  • ensuring quality of fixative
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20
Q

what are the 4 major classifications of fixatives

A
  • chemical action on proteins
  • effect on the microscopic appearance of the tissue
  • number of fixing reagents in the fixative solution
  • amount of time tissue can remain in fixative
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21
Q

coagulant chemical action of proteins

A
  • tertiary structure
  • many organelles - destroyed or distorted
  • mesh
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22
Q

non coagulant chemical action on proteins

A
  • cross-linkages
  • insoluble gel
23
Q

additive chemical action on proteins

A

combines with protein

24
Q

non additive chemical action on proteins

A

fixes proteins by changing nature, structure configuration or activity

25
Q

microanatomical fixatives

A

used to preserve the microarchitecture

26
Q

cytological fixative

A

preserves intracellular structure or inclusions

27
Q

histochemical fixatives

A

produces minimal changes in an element to be demonstrated

28
Q

simple fixative solution

A

has one fixing agents

29
Q

compound fixative solution

A

more than one fixative agents

30
Q

tolerant tissue fixation

A

remain for a long time

31
Q

intolerant time of fixation

A

size, math and penetration rate affect the amount of time tissue can be left in fixative

32
Q

what are a list of simple fixatives

A
  • formaldehyde and formalin based fixative
  • glutaraldehyde
  • osmium tetroxide
  • potassium dichromate
  • mercuric chloride
  • picric acid
  • ethanol
  • acetic acid
33
Q

what is the universal fixative

A

formaldehyde
- clear colourless gas, soluble to 37-40% inw ater
- microanatomical, additive, noncoagulant
- penetrate tissue very quick but fixes slowly
- preserves lipids
- CHO: trapped
- histones: fixed
- methylene bridges : crosslinking

34
Q

cautions of formaldehyde

A

toxic (30mL), probable carcinogen
paraformaldehyde may form
formic acid may form

35
Q

formalin based fixatives

A

10% neutral buffered formalin
- widely used
10% aqueous formalin
- very hypotonic, may produce pigment
10% formal saline
- isotonic (due to NaCl) buy may get pigment
zinc formalin
- zinc is a protein coagulant superior nuclear detail better paraffin infiltration
fewer crosslinks

36
Q

glutaraldehyde

A
  • forms many cross links, some aldehydes may be left free
  • may cause flase positives in certain stains
  • microanatomical, additive, noncoagulant
  • used as the primary fixative in electron microscopy
  • penetrates very slowly; tissue blocks must be very small about 1mm
37
Q

osmium tetroxide

A

additive, noncoagulant, microanatomical, intolerant
- excellent preservation of ultrastructural detail
- fixes lipids but renders them black and insoluble
- penetrates slowly
- main use is secondary fixative after glutaraldehyde

38
Q

drawbacks of osmium tetroxide

A
  • very expensive and toxic
  • used in a fumehood
39
Q

potassium dichromate

A
  • reaction with protein depends on pH
    >3.5 microanatomical , additive, non coagulant
    <3.5 microanatomical, additive, coagulant
  • used in compound fixative
  • disadvantage: tissues washed 24-48 before processing
  • can fix lipids at a higher pH, bu takes many weeks
  • chromium dermatitis, carcinogen, corrosive
40
Q

mercuric chloride

A

microanatomical, additive, coagulant, intolerant
- excellent nuclear and cytoplasmic preservation
- always forms a pigment
- enhances staining
- very poisonous
- phased out of many institutions

41
Q

picric acid

A

trinitrophenol
microanatomical, coagulant, additive
enhances acid dyes
is a yellow dye; tissues appear yellow
component of bouins fixative
it is a dye and a differentiator
removes formalin pigment

42
Q

ethanol

A

cytological, nonadditive, coagulant
coagulates protein by dehydration
not recommended for tissue fixation
precipitates glycogen
dissolved lipids
denatured

43
Q

acetic acid

A

does not fix proteins
precipitates nucleic acids: increased basophilia, excellent nuclear morphology
added to compound fixative – never used alone

44
Q

components of compound fixatives

A
  • contains one or more coagulating agents
  • may contain a non coagulating agent
  • are always in solution
  • may contain an indifferent salt to overcome osmotic effects, or a buffer to maintain pH
  • may contain acetic acid to counteract shrinking effects and heighten nuclear morphology and staining
45
Q

B-Plus fixative

A

advantages:
- cytoplasm well fixed
- acid dyes and metachromatic stains enhanced
- used a secondary fixative following formalin
- useful for many immunoperoxidase techniques, especially lymphoid markers
- good nuclear detail seen
ingredients: water, formalin, zinc chloride, buffering agent

46
Q

Bouin’s

A
  • rapid and even penetration
  • give brilliant staining with Trichrome
  • solution keeps well
  • glycogen is well preserved
  • very small pieces of tissue stain yellow so easier to see
    ingredients: formalin, picric acid
47
Q

secondary fixation

A

post fixation or mordanting
done to increase staining reaction

48
Q

how is fixative of choice determined for secondary fixation

A

tissue under study and technique to be applied

49
Q

fixation artefact

A

produced during processing
- usually lie on top of the tissue and not within the cells
1. formalin pigment
2. mercury pigment
3. chrome pigment

50
Q

formalin pigment

A

tissue that is fixed in acidic formalin solutions
found in blood rich tissues
can be avoided by buffering
- will show up as a dark brown pigment, usually birefringent

51
Q

formalin pigment removal

A

treat unstained tissues with
- saturated alcoholic picric acid
- alcoholic ammonium hydroxide
- alcoholic solutions of sodium or potassium hydroxide

52
Q

mercury pigment

A

cannot be prevented but can be removed
appears as an amorphous brown to black precipitate on top of stained section
remove by iodine followed by sodium thiosulfate

53
Q

chrome pigment

A
  • may form if tissues are fixed in solutions of dichromate and not washed in running water before dehydration
  • brownish green to black pigment
  • once formed, cannot remove

mlsci 250 (11 decks)

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