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Histology ASCP

ASCP Histology Prep - Fixation & Lab Operation

Fixation is the... stabilization of proteins in tissue
Stabilization of proteins is done in order to prevent tissue destruction during subsequent steps in processing
stabilization is accomplished by denaturing of proteins
The most important step in the histology laboratory fixation
Intent of fixation is: preserve tissue in life-like state;prevent breakdown of cellular components; coagulate/precipitate proteins (insoluble);keep from losing proteins during processing
a good fixative will... penetrate tissue quickly + harden the tissue retain micro anatomical structure; maintain histological relationships so the cells can be studied
Autolysis dissolving of cells by enzyme action
Putrefaction breakdown of tissue by bacterial action from within. also called postmortem decomposition
T or F: Fixation brings out the differences in refractive indexes of tissues. True
The cell is composed of proteins, carbohydrates and lipids held loosely together by hydrogen bonds
Fixation changes the weak hydrogen bonds into stable bonded complexes
Fixation causes ________ linkage of protein molecules. cross linkage
Cross links makes tissue suitable for microtomy by... strengthening and hardening the tissue
RNA and DNA are entrapped in the nucleus by the process of ... fixation
Coagulation/Precipitation render the ________ more resilient. nucleus.Giving it a sharper more intact appearance.
T or F: Lipids in the cell are easily fixed. False
Only 2 chemicals can fix lipids so they are not lost in subsequent processing. (1) Osmium tetroxide (2) Chromic Acid
Some _________ are usually LOST during fixation. carbohydrates
A ________ - storage form of _________ is retained during fixation. A glycogen-storage form of glucose is retained during fixation. This is due to the entrapment by fixed proteins.
Nine factors which influence tissue fixation: Temperature, Size, Tissue/Fixative Volume ratio, Time, Choice of Fixative, Penetration Capability, Tissue storage, pH, Osmolality
Osmolality is defined as... the # of particles in a solution
normal osmolality isotonic
hypotonic solution will cause tissue to swell (ie. acetic acid)
hypertonic solution will cause tissue to shrink (ie. picric acid)
Fixatives in order of decreasing speed of penetration: formaldehyde-acetic acid-mercuric chloride-methy alcohol-osmium tetroxide-picric acid
The rate of penetration is affected by _______. heat
The rate of penetration is NOT affected by _______. the concentration of the fixative
NON-coagulant fixatives produce a gel that makes penetration difficult.Examples are... formaldehyde, glutaraldehyde, acetic acid, potassium dichromate, osmium tetroxide
Coagulant fixatives establish a network in tissue allowing penetration. Examples are... Mercuric chloride; zinc salts; chromium trioxide; picric acid; and POTASSIUM DICHROMATE AT PH > 3.5
Biopsy specimen which cannot be immediately placed in fixative should be. placed in a saline dampened gauze, put in plastic container and put on ice for the short holding time.
Kidney biopsy specimens for immunofluorescence frequently are held, or even mailed in ________. Michel transport solution.
Acetic Acid is a non-coagulant and... Does NOT fix or destroy carbohydrates. Does NOT fix lipids. YES penetrates slowly. YES preserves nucleoproteins.
Acetic acid lyses _________, and thus their preservation is poor in in any fixative containing it. red blood cells
Acetic acid warnings: add TO water; use under a hood; causes severe burns; exposure limit of 10ppm
Formaldehyde is colorless ________. gas
Formaldehyde (manufacturer label) = Formalin (37%-40% in water) 37%-40% formalin
Let's look at Stains...momentarily
2 categories of dyes... Natural & Artificial
Natural dyes are from natural resources (ie. HEMATOXYLIN)
Artificial dyes are from chemical reactions (greatly outnumber natural dyes)
Biological Stain Commission agency responsible for ensuring the quality of biological stains and -PROMOTE COOPERATION AMONG VENDORS -EDUCATE USERS OF STAINS PUBLISH INFO CONCERNING NEW OR IMPROVED STAINS
Biological stain commission DOES NOT manufacture biological stains
Dyes are substances... capable of imparting color chemically or physically binding with materials
Dyes impart color due to the presence of color bearing chemical groups called CHROMOGENS
ABsorption a PHYSICAL process by which the dye DISSOLVES DIRECTLY INTO elements int he sample.
ADsorption which the dye BINDS TO ELEMENTS IN THE SAMPLE that have an affinity for the dye.
CONCENTRATION of the dye the greater the concentration, the more the dye is bound to tissue components
TEMPERATURE of the dye increased temperature = increased rate of diffusion throughout the tissue
pH of the staining sollution cells have specific affinity for stains/dyes with specific pH ranges
Tissue FIXATION fixation alters and reorganizes certain molecular structures in tissue samples so that they have an increased permeability and are more receptive to staining.
T or F: Unfixed tissue elements have LIMITED binding sites for dyes. TRUE
pH scale: 1 - 14 1-6.9 ACIDIC. 7-7.4 NEUTRAL. 7.5-14 BASIC.
BUFFERS prevent fluctuations in pH
BASIC stains love ACID tissue elements (nuclei & other basophilics) ACID stains love BASIC tissue elements (cytoplasm & other acidophilic)
OXIDATION = removal of electrons from molecules REDUCTION = adding electrons
in the case of staining, FIXATION alters and reorganizes certain molecular structures in tissue samples so that they have an increased permeability and are more receptive to staining.
show positive PAS (Rose Red) staining Glycogen. Neutral mucins. Some epithelial mucins. Basement membranes. Fungal walls.
PAS with Diastase used in conjunction with the PAS staining procedure to specifically identify glycogen granules in tissue samples. Amylase enzymes in a malt extract are used to digest the glycogen, which is then washed out of the tissue sample.
PAS counterstain with _______ hematoxylin HARRIS hematoxylin with Acetic Acid
Mucicarmine rose red dye in the mucicarmine staining solution gets its color from the aluminum-carminic acid complex carmine
Mucicarmine results Nuclei are stained black with Weigert's Iron Hematoxylin and the remaining background tissue elements are stained yellow with Metanil Yellow or Tartrazine.
Mucicarmine is also used to identify ADENOCARCINOMAS & Cryptococcus neoformans
Alcian Blue has an affinity for acidic tissue elements like mucin. The dye gets color from the copper in the molecule
Alcian Blue solution with a pH of 1.0 stains most SULFATED acid mucins. These mucins can be found in cartilage, large intestine goblet cells and bronchial serous glands.
Alcian Blue solution with a pH of 2.5 stains CARBOLYLATED mucins in connective tissues and cartilage.
Alcian Blue used with PAS both acid and neutral mucins can be demonstrated. Alcian Blue will stain acidic mucins blue. PAS will stain neutral mucins rose red. This technique is particularlyuseful in diagnosing diseases of the gastrointestinal (GI) tract.
glutaraldehyde is s dialdehyde and gives false result for PAS becuase its 2nd NH2 group is left free to react w/Schiff (which is used to detect aldehydes).
Glutaraldehyde fixes ____ and binds ________. fixes slowly and binds slowly
Created by: alexinem



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