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Fixation

General

QuestionAnswer
Functions of fixation? Stop putrefaction, autolysis; stabilize relationships of cells & extracellular materials; emphasize differences in refractive indices of tissue elements for greater contrast
2 general methods of protein stabilization? Physical & chemical
Heat fixation Stabilize & denatures protein, one- or two-stage microwave fixation
2-step microwave fixation 1) tissue immersed in saline to make firm; 2) 2 mm thick block in saline heated to 50°C to 68°C or 45°C to 55°C
Microwave fixation temp should not exceed what and why? 68°C, causes overstained nuclei, loss enzyme activity & antigenicity, lysis rbc
Fixation by desiccation Only used for air-drying of touch preps for Wright stain
2 physical methods fixation? Heat & desiccation
Additive fixatives work by__ Chemically adding to tissue
Common additive fixatives Formaldehyde, glutaraldehyde, OsO4, HgCl, picric acid, chromium trioxide, Zn/Cl sulfate
Non-additive fixatives work by__ Dissociating bound water, non-additive are coagulating
Common non-additive fixatives Ethanol, methanol, acetone
Coagulant fixatives form__ Network in tissue, better penetration
Common coagulant fixatives Alcohols, acetone, HgCl, picric acid, chromium trioxide, Zn salts
Noncoagulant fixatives form__ Gel, worse penetration, better for non-paraffin embedding media
Common noncoagulant fixatives Formaldehyde, glutaraldehyde, OsO4, potassium dichromate, acetic acid
Light microscopy fixation temperature Higher temp, faster fix, more autolysis; max temp 45°C
Fixation tissue size Open large structures, 3mm thick routine processing, smaller shorter schedule
Fixation volume ratio Fixative 15x to 20x > tissue; Additive fixatives depleted as combine w/ tissue
Fixation timing Tissue into fixative asap so fewer post-mortem changes; tissue in fixative long enough; some fixatives be careful not to leave in too long
Formalin fixation time? 6 to 8 hours
Light microscopy fixative pH pH 4 to 9, less important than w/ EM
Fixation osmolality important b/c__ Aq. fixatives may swell (damage) cells, add unreactive ionizing salts
Osmolality of body fluids? 340 mOsm or 0.3 Osm
Fixation & the nucleus Most fixatives react @ 65°C for DNA, 45°C for RNA, both fixed by stabilization of nucleic proteins
Fixation & proteins Additive fixatives alter protein shape, change binding site charges Nonadditive make proteins insoluble
Fixation & lipids OsO4 & chromic acid fix lipids, otherwise lose in processing; both change chemical reactivity of lipids
Fixation & carbs Most lost, glycogen entrapped by fixed proteins
EM fixation temperature 0 to 4 °C
EM fixation pH Important for EM, ultrastructural preservation, pH 7.2 to 7.4
Commercial formaldehyde contains __ to __ Methanol to prevent polymerization of paraformaldehyde
Cytology smears Fix w/i 1-2 seconds to prevent air-drying artifact
Best fixative for preservation of stainability in long-term storage? 70%, good for H&E & IHC staining
What fixatives should be avoided for silver stains? Mercury & chromate-containing fixatives
What fixative should be used for Warthin-Starry staining? Formalin only
Best antibody to determine if tissue is over-fixed? Vimentin
Best fixative for HER2 antibody testing? Formalin
How does choice/change of fixative affect staining? Different fixatives give different stain binding sites, especially effects eosin uptake, may need different staining procedure depending on fixative
What does S-100 antibody stain? Schwannomas, ependymomas, astrogliomas, & most melanomas
What does CD20 antibody stain? B cells, to ID in tissue
What does Cam 5.2 antibody stain? Cytokeratins, can ID neoplasms of epithelial differentiation
Which fixatives react specifically w/ DNA & RNA Acetic acid, Carnoys
Hypertonic solution is: High salt, low water, water drawn out of cell, cell shrinks
Hypotonic solution is: Low salt, high water, water sucked into cell, cell swells/ruptures
Isotonic solution is: Physiological saline, cells stable
Ultrastructural (EM) good characteristics of good fixation are: Complete plasmalemma, uniform nuclear envelope, no swelling of mitochondria, finely precipitated cytoplasm, well-preserved ER & nucleus
Signs of poor paraffin fixation include: Tissue separating on water bath, poor morphology, smudgy nuclei w/ no chromatin pattern, nuclear bubbling, center of tissue has more intense eosin staining
Best fixative(s) for nuclear material? Acetic acid, Carnoy
Best fixative(s) for hematopoietic & lymphoreticular tissue? B-5
Created by: CCF