Fixation Flashcards
what is fixation
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
physical fixation
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)
aim of fixation( 4 )
- autolyse tissue
- provide support
- remove water
- stop enzymatic action
chemical fixation
when we speak of histological fixation, we mean chemical fixation
additive fixatives
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.
side effects of fixatives
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
non-additive fixatives
act on tissue without chemically combining with it and stabilize through the removal of water
examples : acetone and alcohol
coagulant fixatives
produce a mesh or lattice type arrangement allowing good solution penetration
examples: alcohol, zinc salts, mercuric chloride, chromium trioxide, picric acid
non-coagulant fixatives
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
factors influencing fixation
temperature
size
volume
time
temperature & fixation
increase temp
- increases rate of fixation
- increases rate of autolysis
- increases rate of diffusion
the compromise
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)
size
whole organs and large tissue samples should be opened on day one
sections should be no more than 3mm thick
volume ratio
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
time
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
choosing a fixative
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
penetration
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
tissue storage
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
pH
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
osmolality
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
reactions within cell
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
outside the nucleus
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
lipids
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
carbohydrates
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
why do we fix
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
ideally a fixative must
not shrink or swell not history or dissolve render enzymes inactive kill bacteria and molds modify tissue to retain form through processing
what is staining dependent on
fixatation ; incorrect fixation may lead to false pos or false neg
considerations of fixative
speed and completeness penetration cost effective stability effect of prolonged use safety
which volume ration of fixative is recommended
15-20x
what occurs with fixation occurs when temp is increase
fixation speed increased
formaldehyde cross links with which of the following
NH2
which fixatives best preservative of carbohydrates
alcohol
which fixative is best for [reserving lipids
osmium tetroxide
what is the maximum tissue thickness recommended for fiction penetration
3-4mm
putrefaction is
breakdown cause by bacteria
autolysis is
breakdown caused by enzymes
which type of fixative allows for best penetration of fluids
coagulant
microwave and fixation
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
transportation of specimen
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
