Tissue Processing PDF

Tissue Processin g Dr SKB Bani 1 …Introduction Tissues from the body taken for diagnosis of disease processes are processed in the histology laboratory to produce microscope slides The techniques for processing the tissues, whether Biopsies Larger specimens removed at surgery,...

Tissue Processin g Dr SKB Bani 1 …Introduction Tissues from the body taken for diagnosis of disease processes are processed in the histology laboratory to produce microscope slides The techniques for processing the tissues, whether Biopsies Larger specimens removed at surgery, Or Tissues from autopsy 2 Introduction There are 3 main techniques which are used in preparing microscopical sections from tissues: The paraffin technique (most common method) The celloidin technique (most perfect method) The freezing technique (most rapid method) 3 Specimen Assessment Tissue specimens sent to the histopathology laboratory are accompanied with request form that lists the patient information and history along with a description of the site of origin The specimens are accessioned by giving them a number that will 4 identify each specimen for each patient. 5 Tissue Processing The technique of getting tissue fixed into paraffin is called tissue processing. The main steps in this process are dehydration and clearing. 6 Fixation is to preserve tissues permanently in as life- like state as possible. should be carried out as soon as possible after removal of the tissues to prevent autolysis. There is no perfect fixative, though formaldehyde comes the closest. Therefore, a variety of fixatives are available for use, depending on the type of tissue present and features to be demonstrated. 7 Types of fixatives five major groups of fixatives, classified according to mechanism of action: Aldehydes Mercurials Alcohols Oxidizing agents Picrates 8  ALDEHYDES: Include formaldehyde (formalin) and glutaraldehyde. It is good for immuno-histochemistry techniques. Formalin penetrates tissue well, but is relatively slow. The standard solution is 10% neutrally buffered formalin. 9  Mercurials fix tissue by an unknown mechanism. They contain mercuric chloride and include such well-known fixatives as Zenker’s. These fixatives penetrate relatively poorly and cause some tissue hardness, but are fast and give excellent nuclear10  Alcohols: including methyl alcohol (methanol) and ethyl alcohol (ethanol). They are very good for cytologic smears because they act quickly and give good nuclear detail. 11  Oxidizing Agents: Include permanganate fixatives (potassium permanganate), dichromate fixatives (potassium dichromate), and osmium tetroxide. Picrates: include fixatives with picric acid. Foremost among these is Bouin's solution. 12 fixation There are a number of factors that will affect the fixation process: Buffering: Fixation is best carried out close to neutral pH, in the range of 6- 8. Penetration of tissues depends upon the diffusability of each individual fixative. 13  The volume of fixative is important. There should be a 10:1 ratio of fixative to tissue. Increasing the temperature, as with all chemical reactions, will increase the speed of fixation. Concentration of fixative should be adjusted down to the lowest level possible. Time interval: Also very important is time interval from removal of the tissues to the fixation. 14 Gross examination Tissues removed from the body for diagnosis 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 holds the tissue while it is being processed to a paraffin block. Initially, the cassettes are placed 15 into a fixative. 16 17 18 …Gross examination Note: When a malignancy is suspected, then the specimen is often covered with ink in order to mark the margins of the specimen. Different colored inks can be used to identify different areas if needed. When sections are made and processed, the ink will mark the actual margin on the slide. 19 Tissue Processing steps  Biological tissues are generally rather soft, making it quite difficult to cut acceptably thin sections directly from the fresh or fixed tissues.  Methods must be used to hold the tissues firm, which facilitates cutting thin sections with a sharp knife.  Firmness can be achieved either by embedding the tissues in a suitable embedment or by freezing the tissue.  Once the tissue has been fixed, it must be processed into a form in which it can be made into thin microscopic sections. 20 steps  The usual way this is done is with paraffin.  Tissues embedded in paraffin, which is similar in density to tissue, can be sectioned at anywhere from 3 to 10 microns, usually 5-8µ routinely.  The technique of getting fixed tissue into paraffin is called tissue processing.  The main steps in this process are dehydration and clearing. 21 Technique 1. DEHYDRATION is the gradual removal of water from the tissue using ascending grades of ethyl alcohol to prevent tissue shrinking. Wet fixed tissues (in aqueous solutions) cannot be directly infiltrated with paraffin. First, the water from the tissues must be removed by dehydration. This is usually done with a series of alcohols; say 70% to 95% to 100%. The organic solvent must replace the water gradually to prevent turbulence at the interface between water and pure ethanol. Turbulence could cause damage or distortion to cellular components. 22 Technique The duration for which tissues are kept in each strength of alcohol depends upon the size of tissue, fixative used and type of tissue. The volume of alcohol should be 50- 100 times that of tissue. 23 Technique DEHYDRATION The number of steps or the gradient differences should be determined by The degree of fixation The delicacy of the tissue The degree of cellular detail to be preserved Sometimes the first step is a mixture of formalin and alcohol. Other dehydrants can be used, but have major disadvantages. Acetone is very fast, but a fire hazard, so is safe only for small, hand-processed sets of tissues. Dioxane can be used without clearing, but has 24 toxic fumes. 2. Clearing The next step alcohol should be replaced by paraffin wax. As paraffin wax is not alcohol soluble, we replace alcohol with a substance in which wax is soluble. This step is call clearing. Xylene is commonly used. Small piece of tissue are cleared in 0.5 – 1 hour whereas larger (5cm or more thick) are cleaned in 2-4 hours. 25 CLEARING: is the gradual replacement of alcohol in tissue by clearing fluid like xylene, benzene, acetone, chloroform, carbon tetrachloride, toluene etc. Clearing is removal of the dehydrant with a substance that will be miscible with the embedding medium (paraffin). The commonest clearing agent is xylene. Toluene works well, and is more tolerant of small amounts of water left in the tissues, but is 3 times more expensive 26 than xylene. CLEARING Chloroform was used, but it’s a health hazard, and is slow. Methyl salicylate is rarely used because it is expensive, but it smells nice (it is oil of wintergreen). Excessive exposure to clearing reagents may cause excessive hardness or shrinkage. 27 IMPREGNATION IN PARAFFIN The tissues is put in hot soft paraffin at 50°C for 6-24hrs, then in hot hard paraffin at 55 °C in the oven. The paraffin penetrates the tissues. This process of paraffin infiltration is a necessary step to harden the tissues before embedding. 28 Wax: Volume of wax should be about 25-30 times the volume of tissues.  The duration of impregnation depends on size and types of tissues and the clearing agents employed.  Total duration of 4 hours is sufficient for routine impregnation.  Paraffin wax is used routinely. It has hard consistency, so section of 3-4 micron thickness can be cut. 29 3. EMBEDDING Finally, the tissue is infiltrated with the embedding agent, almost always paraffin. In early days of microscopy histologists tried to harden tissues artificially with fixatives, in order to be able to cut suitably thin sections for microscopy. Nearly 100 years ago, the method of embedding tissues in paraffin was developed 30 EMBEDDING Paraffin is a derivative of crude petroleum. It is a group of variable length, long- chain hydrocarbons of the methane series Most paraffins suitable as embedding media melt between 52° and 58°C, and infiltrates the cells while it is hot. 31 Infiltration must be carried out at only a few degrees above the melting point of paraffin. Firmness was achieved with a supporting medium (an embedment), rather than by hardening the tissue itself. Most of our knowledge from microscopy has been gained from sections cut from paraffin-embedded tissues. 32 Paraffin differ in melting point, for various hardness, depending upon the way the histotechnologist likes them and upon the climate (warm vs. cold). Recently a product called Paraplast Plus was introduced into the market. It contains added plasticizers that make the paraffin blocks easier for some technicians to cut. 33 A vacuum can be applied inside the tissue processor to assist penetration of the embedding agent. The time required for embedding tissues using ethanol dehydration and xylene clearing usually exceeds eight hours. Normally, the tissue is processed overnight with an automatic tissue processing machine. 34 4. Cutting:- Paraffin block are cut by microtome using metal knife, into thin sections ~ 6µ 6. Mounting:- Sections spread on the hot plate or water bath and mounted on glass slides. 7. Staining:- Variable stains are used for specific tissues. 35 Automated tissue processor 36 Sectioning Once the tissues have been embedded, they must be cut into very thin sections (4 to 6 microns) that can be placed on a slide. This is done with a microtome. The important thing for proper sectioning is a very sharp knife. 37 38  Frozen sections are performed Frozen Sections with an instrument called a cryostat. The cryostat is just a refrigerated box containing a microtome. The temperature inside the cryostat is about -20 to -30 C. The tissue sections are cut and picked up on a glass slide. The sections are dried and then stained. 39 Staining 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. Therefore, before any staining can be done, the slides are "deparaffinized" by running them through xylene then, to alcohols and lastly to water. There are no stains that can be done on tissues containing paraffin. 40 Automated stainer Frozen sections are stained by hand, because this is faster for one or a few individual sections. 41 Coverslipping The stained section on the slide is 1. covered with a thin piece glass to protect the tissue from being scratched, and to 2. preserve the tissue section for years to come. 42 Decalcification Bone specimens as well as calcified tissues are the most common type here. The calcium must be removed before embedding to allow sectioning. A variety of reagents have been used to decalcify tissue such as mineral acids, organic acids, EDTA, and electrolysis. 43  Strong mineral acids such as nitric and hydrochloric acids  Strong acids will remove large quantities of calcium at a rapid rate, but they will cause damage of cellular morphology. 44  Organic acids such as acetic and formic acid. However, they act more slowly on dense cortical bone. EDTA can remove calcium safely, it works slowly, it penetrates tissue poorly, but it is expensive in large amounts. 45 Using a completely different rationale for dehydration, Prento (1978) was able to reduce the time required for embedding fixed tissues to less than 3 hours, using far few steps. He used Dimethoxypropane (DMP), which served as both the dehydrating and clearing agent. Acidified DMP does not simply replace the water but chemically reacts with water to form methanol and acetone which both act as dehydrants. After the block of tissue has been completely infiltrated with paraffin, it is placed in a mold 46 containing hot paraffin and oriented in the desire manner. The paraffin is then allowed to solidify. The most objectionable aspects of paraffin as an embedding medium: The heat required for melting- the critical shrinkage point of collagen is approximately 65°C. Exposure of collagenous tissues to this temperature must be carefully guarded against to avoid excessive shrinkage. Upon solidifying, paraffin shrinks 16.5% in volume. Paraplast plus shrinks 14% by volume. Despite these problems, paraffin has been far the most widely used embedding medium for many years, and it will probably not be readily replaced by another medium. 47  The above processes are almost always automated for the large volumes of routine tissues processed. Automation consists of an instrument that moves the tissues around through the various agents on a preset time scale. Newer processors have computers to control them and have sealed reagent wells to which a vacuum and/or heat can be applied. 48 The paraffin technique Tissues that come off the tissue processor are still in the cassettes and must be manually put into the blocks by picking the tissues out of the cassette and pour molten paraffin over them. This "embedding" process is very important, because the tissues must be aligned, or oriented, 49 properly in the block of Some General Rules for the biopsy Procedure: 1. The larger the lesion, the numerous the biopsies that should be taken from it because of the fact that the diagnostic areas may be present only focally. 2. In ulcerated tumour, biopsies should be taken from the periphery that includes normal and diseased tissue. 50 Some General Rules for the biopsy Procedure: 3. Crushing or squeezing of the tissue with forceps should be carefully avoided. 4. Once the biopsy is obtained, it should be placed immediately into container with adequate volume of fixative. 51 Specimen Specimen should be transported in glass, plastic or metal container or in a plastic bag in 10% formalin. If formalin is not available at hand, place the specimen in refrigerator at 4°C to slow down autolysis. The container should have an opening larger enough so that the tissue can be removed easily after 52 fixation. Examination: 1. Proper identification and orientation of the specimen. 2. Unlabeled specimen should never be processed. 3. A properly completed histopathology requisition form containing patient’s name, age, sex/gender, relevant clinical data, surgical findings, nature of operation 53 and name of tissue submitted. Examination: 4. In an orderly manner, carefully search and examination of all the tissue submitted. 5. Place the specimen on cutting board in an anatomic position and record the following information: a.Type of specimen b.Structure of specimen c. Dimensions d. Weight e. Shape f. Colour g. Consistency 54 h. Surgical margin Examination 6. Measurements are usually given in centimeter unless the specimen is very small in which mm can be used. 7. Endometrial and prostatic tissue should be measured by aggregate pieces in volume. 55 Sampling for Histopathological Examination Tissue submitted for histopathology laboratory must not be more than 3 mm thick and not larger than the diameter of slides used. Most specimens from solid tissues are cut in the form of pieces measuring 10 to 15 mm on the slides and 2 to 3 mm in thickness. Discrete areas of calcification or ossification (or osteogenesis) should be taken out and should be decalcified in nitric acid. Small fragments of tissue must be wrapped in thin paper or gauze. 56 Technique: Histological technique deals with the preparation of tissue for microscopic examination. The aim of good histological techniques is to preserve microscopic anatomy of tissue. This is achieved by passing the tissue through 1. Fixation 2. Dehydration 3. Clearing 4. Embedding 57 5. Cutting 6. Staining Fixation This is the process by which the constituents of cells and tissue are fixed in a physical and a chemical state so that they will withstand subsequent treatment with various reagents with minimum loss of architecture. This is achieved by exposing the tissue to chemical compounds, called fixatives. 58 Fixation Mechanism of action of fixatives: Most fixatives act by precipitating proteins No fixative will penetrate a piece of tissue thicker than 1 cm. For dealing with specimen thicker than 1 cm, following methods are recommended: 1. Solid organ: Cut slices as necessary as but not thicker than 5mm. 2. Hollow organ: Either open or fill with fixative or pack lightly with wool soaked in fixative. 3. Large specimen, Inject fixative along the vessels or bronchi as in case of lung so that 59 it reaches all parts of the organ Fixative: 1. Prevents autolysis and bacterial decomposition. 2. Preserves tissue in their natural state and fix all components. 3. Make the cellular components insoluble to reagent used in tissue processing. 4. Preserves tissue volume. 5. Avoid excessive hardness of tissue. 6. Allows enhanced staining of tissue. 7. Should be non-toxic and non-allergic for user. 8. Should not be very expensive. 60 fluid: should be approximately 10-20 times the volume of the specimen. Fixative should surround the specimen on all sides. 61 Fixatives A. Tissue fixatives a. Buffered formalin b. Buffered gluteraldehyde c. Zenker’s formal saline d. Bowen’s fluid B. Cytological fixatives a.Ethanol b.Methanol c.Ether C. Histochemical fixatives a. Formal saline b. Cold acetone 62 c. Absolute alcohol Tissue Processing Tissue processing is a long procedure and required 24 hours. Tissue processing can be done by manually or mechanically. It is important that all specimens are properly labeled before processing is started. For labeling, pen containing ordinary ink should not be used. Printed, or graphite pencil written, are 63 satisfactory. Blocking: Impregnated tissues are placed in a mould with their labels and then fresh melted wax is poured in it and allowed to settle and solidify. Once the block has cooled sufficiently to form a surface skin it should be immersed in cold water to cool it rapidly. After the block has completely cooled it is cut into individual blocks and each is trimmed. Labels are made to adhere on the surface of the block. 64