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Fixation
Lab
| Question | Answer |
|---|---|
| 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 |
| Best fixative(s) for rbc? | Methanol |
| Best fixative(s) for fat | Formalin |
| Best fixative(s) for collagen & muscle | Bouin |
| Best fixative(s) for immunoglobin in lymphoid tissue? | B-5 |
| Best fixative(s) for spirochetes? | NBF |
| Best fixative(s) for uric acid? | Absolute alcohol |
Created by:
CCF
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