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PART -1
Introduction
Tissue specimens received in the surgical pathology laboratory have a request form that lists the patient information and history along with a description of the site of origin. The specimens are given a number that will identify each specimen for each patient.
Tissues arrive in the Pathology Department and are examined. Gross examination consists of describing the specimen and placing all or parts of it into a small plastic cassette which after fixation will be processed to a paraffin block.
INTRODUCTION
Histo techniques deals with the preparation of tissues for microscopic examination. Submission of total or selective part of the tissue for examination into a series of processes, 1.Fixation 2.Dehydration tissue 3.Clearing processing 4.Embedding 5.Cutting 6.Staining.
Fixation : It is a process to preserve tissues permanently in as life-like state as possible. Dehydration: Gradual removal of water from the tissue using ascending grades of ethyl alcohol to prevent tissue shrinking. Clearing: Replacement of alcohol in tissue by clearing fluid like xylene, benzene, or acetone Embedding: Tissues are impregnated in paraffin with in a capsule. Cutting: Paraffin blocks are cut by microtome using metal knife, into thin sections ~ 3-4. Staining : Sections are spread on the glass slide coated with egg-albumin and taken for staining. Mounting : Stained slides are dried and mounted with a cover slip via mounting media.
Other reasons
Cells contain colloidal solution of salts, proteins, CHO, lipids, enzymes and organic acids. Were the cells not fixed many of them would be lost. Autolysed tissues loose their staining affinities. Fine intracellular organelles are lost.
Definition
Fixation is a process by which the constituents of the cells and therefore of the tissues, are fixed in a physical and partly also in a chemical state, so that they withstand subsequent treatment with various reagents with a minimum loss, less significant distortion or decomposition.
Fixatives act by denaturing / precipitating the proteins. This forms a meshwork which holds the other cell constituents together. Produce some tissue hardening which helps in cutting. But this hardening is reinforced by the alcohols used in dehydration. Micro-organisms are also fixed and killed prevents putrefaction. Mordant to enhance the staining process.
Ideal fixative
Cheap, stable, safe to handle. Penetrate tissues easily ,rapidly, isotonic Cause minimum loss of properties Preserve tissues in their natural state Avoid excessive hardness of tissues Non toxic and non allergic. There is no perfect fixative, though formaldehyde comes the closest.
Types of fixatives
There are five major groups of fixatives, classified according to mechanism of action:
Aldehydes Mercurials Alcohols Oxidizing agents Picrates
1.Aldehydes
Formaldehyde (formalin) Glutaraldehyde Tissue is fixed by cross-linkages/polymerization formed in the proteins forming methylene bridges, particularly between lysine residues. This cross-linkage does not harm the structure of proteins greatly, so that antigenicity is not lost. Therefore, formaldehyde is good for immunohistochemical techniques. However, they cause some alteration which may interfere with the staining, which can be reversed by washing in water.
Formalin
Commercial formaldehyde (formalin) saturated solution of formaldehyde gas in water (40%gas by weight) regarded as 100% formalin. A mixture of 10ml formalin with 90ml water or saline is known as 10%formalin. If stored in cold environment formalin becomes turbid due to the formation of paraformaldehyde. This can be removed by filtration or by adding 11-16% ethanol. Formation of formic acid due to oxidation causes interference in staining techniques. This is prevented by using formal saline with calcium carbonate.
Amount of fixative should be approx 10-20 times the volume of the specimen. 4mm thick sections of the tissues will be adequately fixed with the above amount in 8hrs at room temp & 4hrs with agitation. Complete fixation requires 12-24 hrs at room temperature.
Advantages
Cheap, easy to prepare, stable. Penetrates tissues easily. No excessive hardening/brittleness. Tissue enzyme studies can be done.
Disadvantages
Irritant dermatitis. If exposure is >1ppm conc for 30min reduction in ventilating capacity, asthma in allergic individuals. Forms artifactual pigment acid formaldehyde hematin.
2.Mercurials
Mercurials fix tissue by an unknown mechanism. and they contain mercuric chloride Zenker's fixative. These fixatives penetrate relatively poorly and cause some tissue hardness, but are fast and give excellent nuclear detail. Mercury deposition on the tissues must be removed. Best application is for fixation of hematopoietic and reticuloendothelial tissues. Since they contain mercury, they must be disposed of carefully.
3.Alcohols
Alcohols, including methyl alcohol (methanol) and ethyl alcohol (ethanol ), are protein denaturants and are not used routinely for tissues because they cause too much brittleness and hardness. However, they are very good for cytological smears because they act quickly and give good nuclear detail. Contraindicated when lipids are to be demonstrated. Carnoys fixative for glycogen.
4.Oxidizing agents
Oxidizing agents include permanganate fixatives (potassium permanganate), dichromate fixatives (potassium dichromate), and osmium tetroxide. They cross-link proteins, but cause extensive denaturation.
Formalin is used for all routine surgical pathology and autopsy tissues when an H and E slide is to be produced.
Summary
Bouin's (Picric acid) solution is recommended for fixation of testis, GI tract, and endocrine tissue
Alcohols, specifically ethanol, are used primarily for cytologic smears. Ethanol (95%) is fast and cheap.
Tissue processing
The technique of getting fixed tissue into paraffin is called tissue processing
Dehydration
Wet fixed tissues (in aqueous solutions) cannot be directly infiltrated with paraffin. First, the water from the tissues must be removed by dehydration. A reagent which can easily react with water so that it can easily penetrate into the tissues. Ethyl alcohol graded, beginning with 70%. Other option isopropyl alcohol. Amount at each stage should not be <10 times the volume of tissue to be dehydrated.
Clearing (dealcoholization)
The next step is called "clearing" and consists of removal of the dehydrant with a substance that will be miscible with the embedding medium (paraffin). The commonest clearing agent is xylene. Others :Toluene - expensive than xylene. Chloroform - health hazard & slow. Methyl salicylate - expensive.
Refractive index of the clearing agents is equal to that of the tissues so that they are rendered transparent. Amount not <10 times the volume of the tissue. Applications: before wax impregnation. before mounting the slides. for making tissues transparent.
Graded alcohols
In our lab
Microm automated tissue processor Contains 12 bottles Each adjusted for one hour. 0ne 10% formalin 6 isopropyl alcohol in graded percentages. 3 xylene 2 -paraffin
Two types of embedding : paraffin wax embedding celloidin embedding Paraffin is routinely used. Tissues
dehydrated and cleared
impregnated with paraffin wax by immersion in successive molten wax baths. embedded in wax block For firmness of the medium many combinations were in use Introduction of paraplast.
Advantages
It is a mixture of highly purified paraffin wax and synthetic plastic polymers. Maintains the melting point. It gives uniform blocks, regardless of the rate of setting. Sections may be cut without cooling on the block face. Does not crack.
Carbowax
Does not need dehydration and clearing soluble in water lipids/neutral fats are not removed and processing time is reduced. It is highly hygroscopic blocks must not come in contact with water. Water soluble sections cannot be floated on water needs other solutions. Tendency to crumble while cutting. However, can be used for enzyme histochemical studies.
Procedure
Wax used is kept molten at 2 deg.cel higher than its MP. The capsules are opened and the number noted.
Suitable mold is chosen and is filled with wax within a few mm of its top.
Tissue is picked out of its cassette by a blunt forceps that was preheated.
Tissue is placed on the molten wax with the cutting surface facing down and kept pressed for some time.
When the wax becomes partly solid, they removed from the steel base and are cooled in the refrigerator prevents crystallization of the wax.
At least 2mm of wax should surround the tissue. A small paper tag bearing the tissue no. must be attached. Dimensions 5mm depth 2mm - surrounding tissue 4mm - surface
Leuckharts moulds : L-molds L- shaped pieces of brass base 1/8th inch 3/2 inches square good initial setting of blocks but too slow for busy labs.
In our lab
Embedding unit contains 2 parts EC 350-1 paraffin in molten state (67 deg.cel) EC 350-2 cooling chamber Microm company Uses tissue tek system of embedding.
to be continued in PART-II