ASCP Histology Prep - Fixation & Lab Operation
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| Fixation is the... | stabilization of proteins in tissue
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| Stabilization of proteins is done in order to prevent | tissue destruction during subsequent steps in processing
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| stabilization is accomplished by | denaturing of proteins
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| The most important step in the histology laboratory | fixation
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| 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
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| a good fixative will... | penetrate tissue quickly + harden the tissue retain micro anatomical structure; maintain histological relationships so the cells can be studied
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| Autolysis | dissolving of cells by enzyme action
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| Putrefaction | breakdown of tissue by bacterial action from within. also called postmortem decomposition
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| T or F: Fixation brings out the differences in refractive indexes of tissues. | True
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| The cell is composed of | proteins, carbohydrates and lipids held loosely together by hydrogen bonds
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| Fixation changes the weak hydrogen bonds into | stable bonded complexes
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| Fixation causes ________ linkage of protein molecules. | cross linkage
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| Cross links makes tissue suitable for microtomy by... | strengthening and hardening the tissue
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| RNA and DNA are entrapped in the nucleus by the process of ... | fixation
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| Coagulation/Precipitation render the ________ more resilient. | nucleus.Giving it a sharper more intact appearance.
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| T or F: Lipids in the cell are easily fixed. | False
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| Only 2 chemicals can fix lipids so they are not lost in subsequent processing. | (1) Osmium tetroxide (2) Chromic Acid
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| Some _________ are usually LOST during fixation. | carbohydrates
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| 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.
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| Nine factors which influence tissue fixation: | Temperature, Size, Tissue/Fixative Volume ratio, Time, Choice of Fixative, Penetration Capability, Tissue storage, pH, Osmolality
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| Osmolality is defined as... | the # of particles in a solution
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| normal osmolality | isotonic
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| hypotonic solution will | cause tissue to swell (ie. acetic acid)
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| hypertonic solution will | cause tissue to shrink (ie. picric acid)
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| Fixatives in order of decreasing speed of penetration: | formaldehyde-acetic acid-mercuric chloride-methy alcohol-osmium tetroxide-picric acid
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| The rate of penetration is affected by _______. | heat
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| The rate of penetration is NOT affected by _______. | the concentration of the fixative
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| NON-coagulant fixatives produce a gel that makes penetration difficult.Examples are... | formaldehyde, glutaraldehyde, acetic acid, potassium dichromate, osmium tetroxide
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| 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
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| 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.
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| Kidney biopsy specimens for immunofluorescence frequently are held, or even mailed in ________. | Michel transport solution.
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| Acetic Acid is a non-coagulant and... | Does NOT fix or destroy carbohydrates. Does NOT fix lipids. YES penetrates slowly. YES preserves nucleoproteins.
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| Acetic acid lyses _________, and thus their preservation is poor in in any fixative containing it. | red blood cells
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| Acetic acid warnings: | add TO water; use under a hood; causes severe burns; exposure limit of 10ppm
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| Formaldehyde is colorless ________. | gas
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| Formaldehyde (manufacturer label) = Formalin (37%-40% in water) | 37%-40% formalin
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| Let's look at Stains...momentarily |
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| 2 categories of dyes... | Natural & Artificial
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| Natural dyes are | from natural resources (ie. HEMATOXYLIN)
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| Artificial dyes are | from chemical reactions (greatly outnumber natural dyes)
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| 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
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| Biological stain commission DOES NOT | manufacture biological stains
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| Dyes are substances... | capable of imparting color chemically or physically binding with materials
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| Dyes impart color due to | the presence of color bearing chemical groups called CHROMOGENS
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| ABsorption | a PHYSICAL process by which the dye DISSOLVES DIRECTLY INTO elements int he sample.
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| ADsorption | which the dye BINDS TO ELEMENTS IN THE SAMPLE that have an affinity for the dye.
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| CONCENTRATION of the dye | the greater the concentration, the more the dye is bound to tissue components
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| TEMPERATURE of the dye | increased temperature = increased rate of diffusion throughout the tissue
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| pH of the staining sollution | cells have specific affinity for stains/dyes with specific pH ranges
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| 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.
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| T or F: Unfixed tissue elements have LIMITED binding sites for dyes. | TRUE
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| pH scale: 1 - 14 | 1-6.9 ACIDIC.
7-7.4 NEUTRAL.
7.5-14 BASIC.
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| BUFFERS | prevent fluctuations in pH
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| BASIC stains love ACID tissue elements (nuclei & other basophilics) | ACID stains love BASIC tissue elements (cytoplasm & other acidophilic)
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| OXIDATION = removal of electrons from molecules | REDUCTION = adding electrons
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| 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.
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| show positive PAS (Rose Red) staining | Glycogen.
Neutral mucins.
Some epithelial mucins.
Basement membranes.
Fungal walls.
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| 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.
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| PAS counterstain with _______ hematoxylin | HARRIS hematoxylin with Acetic Acid
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| Mucicarmine | rose red dye in the mucicarmine staining solution gets its color from the aluminum-carminic acid complex carmine
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| 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.
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| Mucicarmine is also used to identify | ADENOCARCINOMAS & Cryptococcus neoformans
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| Alcian Blue | has an affinity for acidic tissue elements like mucin. The dye gets color from the copper in the molecule
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| 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.
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| Alcian Blue solution with a pH of 2.5 | stains CARBOLYLATED mucins in connective tissues and cartilage.
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| 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.
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| 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).
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| Glutaraldehyde fixes ____ and binds ________. | fixes slowly and binds slowly
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