LECTURE - Histotechnique Flashcards
Fixation
preservation of cells and tissues in as a life-like a manner as possible by stabilizing the protein so that it is resistant to further changes
functions of fixatives: (4)
- prevent putrefaction and autolysis
- help maintain proper relationships between cells and extracellular substances
- bring out differences in refractive indexes and increase the visibility of or the contrast between different tissue elements
- secondary functions = enhancing staining, limiting osmotic effects, preventing desiccation
self-destruction after cell death via intracellular enzymes
autolysis
- affected by temp (faster if warmer)
- highly specialized cells are more rapidly and seriously affected
- nuclear changes = pyknosis => karyorrhexis => karyolysis
- cytoplasm appears increasingly granular and swollen
putrefaction
decomposition by microorganisms
modes of actions of fixatives
- render enzymes inactive by stabilizing proteins
- kill bacteria and molds
- make tissue more receptive to dyes
fixation impacts on tissue
- often shrinks tissue
- changes texture = becomes more brittle and hard
- material can be lost = 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
important considerations in fixation step
- penetration rate and density of tissue
- volume ratio = should be at least 20/1
- time = before and during
- ensuring quality of fixative (Storage, etc.)
most often, tissue is placed in a ______ solution for transport to the lab
fixative
sources of samples
surgical
post-mortem
block selection parameters (5)
- size
- texture
- number/proportion
- markings
- locations
Grossing
Gross processing or “grossing” is the process by which pathology specimens undergo examination with the bare eye to obtain diagnostic information, as well as cutting and tissue sampling in order to prepare material for subsequent microscopic examination.
4 major classifications of fixatives
- by their chemical action on proteins
- by their effect on the microscopic appearance of the tissue
- by the number of fixing reagents in the fixative solution
- by the amount of time tissue can remain in fixative
fixative classification: by their chemical action on proteins (4)
- coagulant = many organelles destroyed or distorted; tertiary???; mesh
- non-coag = cross-linkages (harder for stain to penetrate tissue + interact with it); insoluble gel
- additive = combines with protein
- non-additive = fixes proteins by changing nature, structure configuration or activity
fixative classification: by their effect on microscopic appearance of tissue
- microanatomical fixatives: to preserve microarchitecture
- cytological fixative: preserves intracellular structures or inclusions; EM
- histochemical fixative: produces minimal changes in an element to be demonstrated
fixative classification: by the # of fixing agents in solution
- simple: just one; formalin
- compound: mix of fixatives
fixative classification: by the amount of time tissue can remain in fixative
- tolerant
- intolerant
simple fixatives (8)
- formaldehyde and formalin-based fixatives
- glutaraldehyde
- osmium tetroxide
- potassium dichromate
- mercuric chloride
- picric cid
- ethanol
- acetic acid
the universal fixative
- formaldehyde
- penetrates tissue very quickly but fixes slowly
- preserves lipids
- CHOs: trapped
- histones: fixed (traps NAs)
- methylene bridged = cross-linking; lysine and glutamine
formaldehyde cautions
- toxic; carcinogen possibly
- paraformaldehyde may form (does not fix tissues; from spontaneous polymerization)
- formic acid may form (if you don’t buffer; annoying deposits when interacts with RBCs)
formalin-based fixatives
- 10% neutral buffered formalin (NBF): widely used
- 10% aqueous formalin: very hypotonic, may produce pigment
- 10% formal saline: isotonic (due to NaCl) but may get pigment
- Zinc formalin: protein coagulant (Zinc); superior nuclear detail, better paraffin infiltration, fewer crosslinks
Glutaraldehyde
- forms many cross-links, some aldehydes may be left free
> may cause false positives in certain stains - microanatomical, additive, noncoag
- primary fixative in EM
- penetrates very slowly; tissue blocks must be very small
osmium tetroxide
- additive, non-coagulant, microanatomical, intolerant
- excellent preservation of ultrastructural detail
- fixes lipids (black + insoluble)
- penetrate slowly
- 2ry fixative after glutaraldehyde
- drawbacks: very expensive and toxic(corneas); used in a fumehood
potassium dichromate
- rxn with protein depends on pH
> 3.5 = microanatomical, additive, non-coag
<3.5 “ “ coag - used in compound fixatives
- tissue washed 24-48 hrs before processing (disadvantage)
- can fix lipids at a higher pH bt takes many weeks
- chromium dermatitis, carcinogen, corrosive
mercuric chloride
- actively getting rid of this
- microanatomical, additive, coag, intolerant
- excellent nuclear and cytoplasmic preservation
- always forms a pigment
- enhances staining
- very posionous; acute and cumulative poison
picric acid
- trinitrophenol (TNP; explosie like TNT)
- microanatomical, coagulant ,additive
- enhances acid dyes
- is a yellow dye; tisues = yellow
- componenet of Bouin’s fixative
- van Giesen stain
- differentiator
- removes formalin pigment
- *SAFE if in solution; dry = explosion!**
ethanol
- cytological, nonadditive, coagulant
- coagulates protein by dehydration
- NOT recommended for tissue fixation
- orecipitates glycogen
- dissolves lipids
- denatured form is always used
acetic acid
- does not fix proteins
- precipitates nucleic acids; increased basophilia; excellent nuclear morphology
- added to compound fixative and never used alone bc it counteracts tissue shrinage but does not fix proteins
general formula for compound fixatives
- usually 1 or more coagulating agents
- may contain a non-coagulating agent
- are always in solution
- may have indifferent salt to overcome osmotic effects; or a buffer to maintain pH
- may contain acetic acid to counteract shrinking effects and heighten nuclear morphology + staining
B-Plus fixative
- example of a compound fixative
- advantages:
> well-fixed cytoplasm
> acid dyes and metachromaticstains enhanced
> used as a 2ry fixative following formalin
> immunoperoxidase; especially lymphoid markers
> good nuclear detail seen - water, formalin, zinc chloride, buffering agent
Bouin’s
- compound fixative
- rapid and even penetration
- gives brilliant staining with Trichrome
- solution keeps well
- glycogen is well preserved
- very small pieces of tissue stain yellow so easier to see
- formalin, picric acid
what is secondary fixation?
- AKA post-dfixation or mordanting
- to increase staniing rxn
- fixative of choice determined by:
> tissue under study
> technique to be applied
what are fixation artefacts?
- produced during processing
- usually lie on top of the tissue and not within cells
- types:
> formalin pigment
> mercury pigment
> chrome pigment
formalin pigment (acid formaldehyde hematin)
- tissue fixed in acidic formalin solutions
- usually found in blood-rich tissues
- can avoid by buffering
- appearance: dark-brown, birefriengent
formalin pigment removal
treat unsained tissue with:
- saturated alcoholic picric acid
- alcoholic amonium hydroxide
- alcoholic solutions of sodium or potassium hydroxide
mercury pigment
- cannot be prevented, but can be removed
- appears as an amorphous brown-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
