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83 Cards in this Set

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