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83 Cards in this Set
- Front
- Back
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Why must fixation be carried out ASAP?
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To prevent autolysis- the longer you wait, the more cellular organelles will be lost and the more nuclear shrinkage and artefactual clumping will occur.
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Name two aldehydes
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Formaldehyde and Glutaraldehyde.
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How does Formaldehyde fixate?
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Cross-linkages, particularly between lysine residues.
They do not harm the structure of proteins greatly, so antigenicity is not lost. |
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What is Formaldehyde good for, and why?
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Immunoperoxidase techniques, because it does not harm the structure of proteins greatly, so antigenicity is not lost.
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Name two mercurials
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B-5, Zenker's
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Properties of formalin?
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Penetrates well, but slowly.
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Why is Glutaraldehyde not good for immunoperoxidase techniques?
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Because it causes deformation of alpha-helix structure in proteins.
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What is Glutaraldehyde good for, and why?
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EM, because it fixed very quickly
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What are mercurials good for?
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hematopoietic and reticuloendothelial tissues.
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What must you do before staining after fixating with mercurials?
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Remove the mercury deposits (dezenkerize) or black deposits will result in the sections.
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What do alcohols (methanol, ethanol) do to tissues?
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Denature proteins.
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Name a picrate
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Bouin's solution
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What pH is fixation best carried out at?
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Close to normal: 6-8.
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What does acidity cause when fixating with formalin?
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Formation of formalin-heme pigment that appears as black deposits in tissue.
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Who penetrates the best? Who the worst?
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Formalin and alcohol the best.
Glutaraldehyde the worst. |
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What effect does increasing the temp has on fixation?
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Speeds things up.
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What can you use for fixation of frozen tissues?
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Just about anything, but alcohols are the best.
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What are the two steps in tissue processing?
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1. Dehydration.
2. Clearing (of dehydrant). Xylene. |
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Can you stain on tissues that contain paraffin?
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NO.
(thus the embedding process must be reversed in order to get the paraffin wax out of the tissue and allow water soluble dyes to penetrate the sections) |
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H&E- who is acidic and who is basic?
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Hematoxylin is basic- likes acids (the acids are basophiles).
Eosin is acidic- likes bases (such as cytoplasmatic components). |
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What problems do you encounter with Eosin?
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Overstaining, especially with decalcified tissues.
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Fine black precipitate on the slides, with no relationship to the tissue?
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Formalin-heme pigment.
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Large irregular clumps of black precipitate in tissues fixed in a mercurial?
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The tissues were not "dezenkerized" prior to staining
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What are mucins?
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Family of high molecular weight, heavily glycosylated proteins produced by many epithelial tissues.
Most mucins are secreted onto mucosal surfaces or secreted to become a component of saliva. |
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What is AMP (colloidal iron)?
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Mucin stain.
Requires formalin fixation. Phospholipids and free nucleic acids may also stain. |
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What is Alcian Blue?
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Mucin stain.
The pH of this stain can be adjusted to give more specificity. |
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What is PAS?
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Period acid-Schiff.
Stains glycogen as well as mucins, but tissue can be pre-digested with amylase to remove glycogen. |
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What is Mucicarmine?
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Very specific for epithelial mucins.
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Where can you find neutral mucins, and what stains them?
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Glands of the GI tract and in prostate.
Stain with PAS alone. |
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Where can you find acid (simple- non sulfated, epithelial) mucins, and what stains them?
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Epithelial cells containing sialic acid.
stain with PAS, Alcian blue at pH 2.5, colloidal iron, and metachromatic dyes. |
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Where can you find acid (simple, mesenchymal) mucins, and what stains them?
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Tissue stroma, contain hyaluronic acid.
Do NOT stain with PAS, but do stain with Alcian blue at pH 2.5, colloidal iron, and metachromatic dyes. |
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Where can you find acid (complex- or sulfated, epithelial) mucins, and what stains them?
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Adenocarcinomas.
PAS is usually positive. Alcian blue is positive at pH 1, and colloidal iron, mucicarmine, and metachromatic stains are also positive. |
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Where can you find acid (complex, connective tissue) mucins, and what stains them?
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Tissue stroma, cartilage, and bone.
Stain selectively with Alcian blue at pH 0.5. |
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What are the 3 stains for biogenic amines (hormones, NT)?
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1. "Chromaffin"- cells have cytoplasmic granules that appear brown when fixed with a dichromate solution.
2. "Argentaffin" cells reduce a silver solution to metallic silver after formalin fixation. 3. "Argyrophil" - after using a pre-reduction step that may get more cells to stain. |
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Types of stains for Argentaffin?
(4) |
1. Diazo.
2. Fontana-Mason (also for melanin). 3. Schmorl's. 4. Autofluorescence. |
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Types of stains for Chromaffin?
(3) |
1. Modified Giemsa.
2. Schmorl's. 3. Wiesel's. |
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Types of stains for Argyrophil?
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1. Grimelius (Bouin's preferred).
2. Pascual's. |
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What is the malanin stain?
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Fontana-Mason.
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Describe a histochemical method to identify melanocytes.
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DOPA-oxidase: the DOPA substrate is acted upon by DOPA-oxidase in the melanin-producing cells to produce a brownish black deposit.
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Is melanin PAS positive?
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No, but the pseudomelanin of Melanosis Coli is!
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Where is pseudomelanin usually found?
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Macrophages.
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Lipofuscin stains?
(3) |
1. Sudan black.
2. long Ziehl-Neelson acid fast. 3. Schmorl's. |
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Iron stain? Which fixative should be used?
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Perl's iron stain.
Mercurial fixatives seem to do a better job of preserving iron in bone marrow than formalin. |
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Which calcium can you demonstrate?
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Only calcium that is bound to anions.
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What does calcium look like on H&E?
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Forms a blue-black lake with hematoxylin to give a blue color. usually with sharp edges.
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What is the VonKossa stain?
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Silver stain reduction, demonstrates phosphates and carbonates- that usually present along with calcium!
Useful when large amounts of calcium are present, as in bone. |
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Alizarin red S?
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Forms orange-red lake with calcium, pH 4.2.
Works best with small amounts of calcium. |
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Which stain is better for large amounts of calcium (as in bone)? Which for small?
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Large: VonKossa (stains phosphates and carbonates).
Small: Alizarin (pH 4.2). |
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What can you use Azan stains for?
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differentiate osteoid from mineralized bone.
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What are urates?
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Uric acid crystals, can be found in acid urine or as sodium-urate in tissues.
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What do you use to stain urates?
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Methenamine silver, stains urates black.
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Which blood protein is decreased in Wilson's disease?
What do it do? |
Ceruloplasmin.
Transports serum copper. |
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Where does copper accumulate in Wilson's disease?
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Brain, eye, and liver.
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What happens to the liver in Wilson's disease?
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Hepatic copper accumulation results in:
fatty change acute hepatitis chronic hepatitis eventual cirrhosis. |
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Which stains are utilized to detect cytoplasmic accumulation of copper in the liver?
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Rubeanic acid, Rhodanine.
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How can you differentiate carbon from melanin in the lungs?
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Perform a melanin bleach (=removes melanin).
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What happens to asbestos in tissues?
What is it called? |
the fibers become coated with a protein-iron-calcium matrix.
Called "ferruginous bodies" because they are highlighted with an iron stain. |
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Can you see most forms of Silica in stains?
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No.
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How can you demonstate silica?
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White birefringence on polarization.
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Where is Silica most often found?
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Lungs, but it can travel to lymph nodes.
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Where do Silica/Talc from street drugs for injection can usually be found?
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Lymphoreticular tissues
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What techniques are best for demonstrating minerals?
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* Microincineration techniques
* Electron microscopy with energy dispersive analysis (SEM-EDA). |
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What can you use for fat stains?
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ORO (Oil Red O).
Sudan red. |
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What happens to lipids during fixation etc.?
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Fixatives containing alcohols, or tissue processing with clearing, remove lipids.
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What is the Trichrome stain?
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Highlights collagenous stroma. Also aids in identifying normal structures- connective tissue capsules of organs, the lamina propria of gastrointestinal tract, and the bronchovascular structures in lung.
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When do you use reticulin stain?
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Parenchymal organs such as liver and spleen.
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What do you use for elastic tissues?
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van Gieson.
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Unique property of methylene blue and toluidine blue dyes?
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Metachromasia.
A tissue component stains a different color than the dye itself. (For example, mast cell granules, will stain purple and not blue). |
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What is LAP?
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Leukocyte Alkaline Phosphate.
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What is LAP useful for?
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Determining whether a high peripheral blood leukocytosis is a reactive process or a leukemia (CML).
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How is LAP stain read?
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The more differentiated cells in the reactive process will stain more readily with LAP, while leukemic cells will not.
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LAP score?
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A high score generally indicates a "leukemoid reaction" or reactive condition.
A low score suggests CML. |
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What do you use Tartrate-resistant acid phosphatase (TRAP stain) for?
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Help diagnose hairy cell leukemia.
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What is myeloperoxidase (MPO) stain useful for?
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Helps identify cytoplasmic granules characteristic of myeloid cells (identifying myeloid leukemia).
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How do bacteria appear on H&E?
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Blue, regardless of gram reaction.
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How do fungi appear on H&E?
PAS? |
H&E: Blue.
PAS: Red. |
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What is the most sensitive method to identify fungi?
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Methenamine silver.
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What is AFB (Acid Fast Bacilli) stain?
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Uses carbol-fuchsin to stain the lipid walls of acid fast organisms such as M. tuberculosis.
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Name two AFB (Acid Fast Bacilli) stains.
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Auramine- sensitive for mycobacteria, requires a fluorescence microscope.
Ziehl-Neelsen, the most commonly used. |
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What is GMS used for?
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Staining for fungi and Jirovecii- the cell walls are stained, so the organisms are outlined in black-brown.
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What is problematic with GMS?
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There is a tendency to produce a lot of artefact from background staining, so it is essential to be sure of the morphology of the organism being sought.
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What is PAS useful for?
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All-around useful stain for many things. It stains glycogen, mucin, mucoprotein, glycoprotein, as well as fungi.
useful for outlining tissue structures--basement membranes, capsules, blood vessels, etc. |
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What are some problems with PAS?
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It does stain a lot of things and can have a high background.
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