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Histology > Fixation > Flashcards

Fixation Flashcards

1
Q

what is fixation

A

stabilization ( denaturation ) of proteins

stops autolysis : enzymatic breakdown of tissue (tissue is killed)
stops putrefaction : bacterial breakdown of tissue (bacteria is killed )

coagulation of protoplasmic substances or denaturation through addition renders cells resistant to further changes

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2
Q

physical fixation

A

desiccation- touching tissue to slide making touch prep

heat- such as passing through a flame- causing extreme distortion

  • unsuitable for tissue but used in micro for smears
  • controlled microwave fixation ( is without distortion)

freezing ( temporary)

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3
Q

aim of fixation( 4 )

A
  1. autolyse tissue
  2. provide support
  3. remove water
  4. stop enzymatic action
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4
Q

chemical fixation

A

when we speak of histological fixation, we mean chemical fixation

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5
Q

additive fixatives

A

link themselves to tissue. in doing so the tissues electrical charge & structure can be changed

examples: mercuric chloride, chromium trioxide, picric acid, formaldehyde, gluteraldehyde, osmium trioxide, zinc sulfate or chloride.

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6
Q

side effects of fixatives

A

adding itself to important side chain groupings affect staining ( amino NH2 or carboxyl COOH are important side groupings along protein molecules

formaldehyde adds itself to amino group and changes overall charge to negative

heavy metals (chromium, mercury & osmium) add to -SH -COOH & PO4 changing the overall charge to positive

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7
Q

non-additive fixatives

A

act on tissue without chemically combining with it and stabilize through the removal of water
examples : acetone and alcohol

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8
Q

coagulant fixatives

A

produce a mesh or lattice type arrangement allowing good solution penetration
examples: alcohol, zinc salts, mercuric chloride, chromium trioxide, picric acid

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9
Q

non-coagulant fixatives

A

produce a gel which makes penetration more difficult
examples : formaldehyde, gluteraldehyde, osmium tetroxide, potassium dichromate, acetic acid

embossing media other than paraffin work best with non- coagulant fixatives

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10
Q

factors influencing fixation

A

temperature
size
volume
time

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11
Q

temperature & fixation

A

increase temp

  • increases rate of fixation
  • increases rate of autolysis
  • increases rate of diffusion
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12
Q

the compromise

A

EM fixation in the cold; flaws are far more obvious under extreme magnification

formalin at RT is general practice ( perhaps 37 degrees on processor)

microwave or heat to 45 degrees and no higher (saline)

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13
Q

size

A

whole organs and large tissue samples should be opened on day one

sections should be no more than 3mm thick

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14
Q

volume ratio

A

15-20x the volume of tissue should be the volume of fixative
or fixative may be depleted, salt conc increases, staining may be adversely affected

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15
Q

time

A

place tissue in fixative immediately

perform autopsy immediately after death( ideal but unlikely)

allow a total of 24-48 hrs of formalin fixation

at least 24 hrs is crucial for immunohistochemistry

alcohol fixation is much faster but may not be as complete
- however the microscope appearance & some physical properties are altered in comparison to neutral buffered formalin

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16
Q

choosing a fixative

A

fixatives are chosen according to the testing that will follow
some specimens , however, will not be fixed at all
- enzymes
- B& T studies
- immunohistochemistry of immunization process
- others

frozen sections & sometimes quick acetone fixation are used as the substances being examined are adversely affected by other types of fixation

usually fixatives are chosen according to the specific tissue elements to be demonstrated. some tissue elements can be destroyed by the use of an incorrect fixative

INCORRECT FIXATIVE USE OR DELAY CANT BE CORRECTED

17
Q

penetration

A

fixative solutions penetrate at variable rates
penetration starts at the periphery and works inward
some fixatives work on contact
some penetrate quickly but cross links are not completed for some time

18
Q

tissue storage

A

an entire tissue specimen may not be required for initial study. wet storage of the remainder for possible testing is therefore necessary . neutral buffered formalin is suitable for both fixation and storage. other fixatives man need a change in solution

19
Q

pH

A

although pH will affect tissue elements, light microscopy will generally appear unaffected except formalin reacts with blood in tissue to firm an artifact pigment at an acid pH

electron microscopy (EM) requires pH of 7.2-7.4 againgthe effects are visible at this level of magnification if pH is outside of range

20
Q

osmolality

A

isotonic : a solution with the same salt concentration as the living cell also called normal or physiological saline

hypotonic : solution with less salt than the cell so cell swells

hypertonic : solution with more salt than cell so cell shrinks

fixatives should be isotonic but effects of non-isotonic fixatives are not as prominent for light microscopy as for EM ( similar to pH)

specimens being held or transported unfixed are placed in saline or michels medium

21
Q

reactions within cell

A

the nucleus RNA& DNA ( nucleic acid) and attached protein are found here
most fixatives react with the attached protein and hold the nucleic acids intact ; however for specific assay of DNA & RNA the preferred fixatives are Acetic alcohol* or Carnoys solution* . formaldehyde works for this purpose but only at 45 degrees

22
Q

outside the nucleus

A

staining will be effected by the specific group oma protein that a fixative pacts with

additive fixatives alter the 3-D shape

non additive fixatives cause insolubility or precipitation of proteins

23
Q

lipids

A

usually destroyed by either routine fixation or the subsequent processing

frozen sections are generally used

osmium tetroxide or chromic acid prevent loss but alter chemical activity

24
Q

carbohydrates

A

glycogen is thought to be trapped by fixed proteins, others may be lost

fixation with aqueous fixatives may allow movement of carbohydrates from its usual post into tissue this is referred to as glycogen streaming
*** alcohol based fixatives are generally considered best for preservation of carbohydrates

25
Q

why do we fix

A

maintains life like appearance

preserve inclusion bodies

stop autolysis& putrefaction

enhance difference in refractive index

enhance staining

prepare the tissue to hold up during processing

26
Q

ideally a fixative must

A 
not shrink or swell
not history or dissolve 
render enzymes inactive 
kill bacteria and molds 
modify tissue to retain form through processing
27
Q

what is staining dependent on

A

fixatation ; incorrect fixation may lead to false pos or false neg

28
Q

considerations of fixative

A 
speed and completeness 
penetration 
cost effective 
stability 
effect of prolonged use 
safety
29
Q

which volume ration of fixative is recommended

A

15-20x

30
Q

what occurs with fixation occurs when temp is increase

A

fixation speed increased

31
Q

formaldehyde cross links with which of the following

A

NH2

32
Q

which fixatives best preservative of carbohydrates

A

alcohol

33
Q

which fixative is best for [reserving lipids

A

osmium tetroxide

34
Q

what is the maximum tissue thickness recommended for fiction penetration

A

3-4mm

35
Q

putrefaction is

A

breakdown cause by bacteria

36
Q

autolysis is

A

breakdown caused by enzymes

37
Q

which type of fixative allows for best penetration of fluids

A

coagulant

38
Q

microwave and fixation

A

form of heat fixation

optimum temperature 45-55 degrees C

use of formalin at this temperature is not compatible with human life

EM requires fixation with osmium after microwave

advantages

  • speed
  • non chemical

disadvantage
- spores and pathogens serve not standardized for immunoassays

39
Q

transportation of specimen

A

very short time
saline moistened gauze

longer term 
michels media - usually purchased
- 6 month shelf life
- transport renal, skin biopsies, flow cytometry or immunology 
-tissue must be washed

Histology (15 decks)

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