introduction and fixation Flashcards
why is specimen receiving important?
cells and tissues begin to die/degrade immediately after removal from the body
how are specimens transported?
tissue is placed in a fixative solution
T or F: fresh tissue to the lab is not a urgent priority
F: it is urgent
what are the two sources of samples for the specimens
- surgical
- post mortem
what is grossing
describing the tissue macroscopically
what are some parameters of block selection
- 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”
what is fixation
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
what is the function of fixatives
- prevent putrefaction and autolysis
- help maintain proper relationships between cells and extracellular substances
- bring out differences in refractive indexes and increase the visibility of the contrast between different tissue elements
- secondary functions: enhacing staining, limiting osmotic effects, preventing dessication
what is autolysis
self destruction after cell death via intracellular enzymes
- highly specialized cells are more rapidly and seriously affected
- affected by temperature
nuclear changes of autolysis
pyknosis, karyorrhexis, karyolysis
how does the cytoplasm appear in autolysis
increasingly granular and swollen
delayed fixation…
makes it difficult to differentiate
putrefaction
decomposition by microorganisms
what are the three modes of action
- stabilize
- kill
- make
stabilize
render enzymes inactive by stabilizing proteins
kill
kill bacteria and molds
make
make tissue more receptive to dyes
common impacts on tissue
- changes size - often shrinks
- changes texture - becomes more brittle and hard
- material can be lose - can dissolve away
- chemical alterations - charges on various components may change, molecules of fixative may attach to tissue and change properties
- fixation artifacts - deposits on and around tissues that impact microscopic image
what are some things to consider when fixing tissue
- penetration rate and density of tissue
- volume ration
- time
- ensuring quality of fixative
what are the 4 major classifications of fixatives
- 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
coagulant chemical action of proteins
- tertiary structure
- many organelles - destroyed or distorted
- mesh
non coagulant chemical action on proteins
- cross-linkages
- insoluble gel
additive chemical action on proteins
combines with protein
non additive chemical action on proteins
fixes proteins by changing nature, structure configuration or activity
microanatomical fixatives
used to preserve the microarchitecture
cytological fixative
preserves intracellular structure or inclusions
histochemical fixatives
produces minimal changes in an element to be demonstrated
simple fixative solution
has one fixing agents
compound fixative solution
more than one fixative agents
tolerant tissue fixation
remain for a long time
intolerant time of fixation
size, math and penetration rate affect the amount of time tissue can be left in fixative
what are a list of simple fixatives
- formaldehyde and formalin based fixative
- glutaraldehyde
- osmium tetroxide
- potassium dichromate
- mercuric chloride
- picric acid
- ethanol
- acetic acid
what is the universal fixative
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
cautions of formaldehyde
toxic (30mL), probable carcinogen
paraformaldehyde may form
formic acid may form
formalin based fixatives
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
glutaraldehyde
- 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
osmium tetroxide
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
drawbacks of osmium tetroxide
- very expensive and toxic
- used in a fumehood
potassium dichromate
- 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
mercuric chloride
microanatomical, additive, coagulant, intolerant
- excellent nuclear and cytoplasmic preservation
- always forms a pigment
- enhances staining
- very poisonous
- phased out of many institutions
picric acid
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
ethanol
cytological, nonadditive, coagulant
coagulates protein by dehydration
not recommended for tissue fixation
precipitates glycogen
dissolved lipids
denatured
acetic acid
does not fix proteins
precipitates nucleic acids: increased basophilia, excellent nuclear morphology
added to compound fixative – never used alone
components of compound fixatives
- 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
B-Plus fixative
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
Bouin’s
- 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
secondary fixation
post fixation or mordanting
done to increase staining reaction
how is fixative of choice determined for secondary fixation
tissue under study and technique to be applied
fixation artefact
produced during processing
- usually lie on top of the tissue and not within the cells
1. formalin pigment
2. mercury pigment
3. chrome pigment
formalin pigment
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
formalin pigment removal
treat unstained tissues with
- saturated alcoholic picric acid
- alcoholic ammonium hydroxide
- alcoholic solutions of sodium or potassium hydroxide
mercury pigment
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
chrome pigment
- 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
