Histological Technique PDF

Histological Technique Histopathological Technique is the branch of Biology concerned with the demonstration of tissue structure in various diseases. The specimens submitted to the Histopathology Lab come mostly from the operation theatre, and morgue in the form of small tis...

Histological Technique Histopathological Technique is the branch of Biology concerned with the demonstration of tissue structure in various diseases. The specimens submitted to the Histopathology Lab come mostly from the operation theatre, and morgue in the form of small tissue, biopsy, whole organ, autopsy etc. The preparation of tissue for Microscopy involves the following steps; Receiving of Specimen Grossing Decalcification (If needed) Fixation Dehydration Clearing Impregnation Blocking Section Cutting Staining Mounting The basic purpose of the Specimen receiving room is to receive samples safely and securely. Specimens for Laboratory Examination should be received in separated zone away from the working Laboratory.  Steps of Receiving Biopsy Specimens at Laboratory ⚫ Receiving When a Biopsy or Autopsy material is sent to a Laboratory, the Technologist must check the following; 1) Whether it is in a Fixative or not. 2) Presence of the tissue is there or not. 3) Clinical short notes of the patient. 4) Whether it is referred by a Medical practitioner or Surgeon. 5) Where from the tissue is taken. 6) Physical Examination of big tissue/organ (Length, breadth, weight, colour etc.) ⚫ Registration Every Laboratory has a Clinical report book. After receiving the specimen the Technologist must note in the book which contains Name, Age, Sex, Ward (IPD), diagnosis, tissue and description. It is useful for loss or confusion of labels and also for correct reporting. Details of Clinical biopsies are entered in the appropriate book, including a record of the number of pieces taken and each specimen is numbered. ⚫ Labelling of Specimen Correct labelling and identification of Specimens are the first essentials of any technique. If any mistake is made, it may lead to an incorrect diagnosis and also cause the death of the patient. Labelling should be done with a Graphite pencil. Different Criteria to Reject a Specimen  The patient’s details on the requisition form and specimen label are different.  Details of site/nature of Specimen not mentioned.  The specimen is not received in specific containers.  The specimen is not in proper Fixative.  Patient history not found.  In case of slide/blocks – Broken slides, improperly processed blocks or insufficient contents in Paraffin blocks. Different types of specimens may be received in the Histopathology Laboratory:  Surgical Biopsy  Needle Biopsy  Endoscopy Biopsy  Incision Biopsy  Excision Biopsy  Punch Biopsy  Cone Biopsy Grossing - Gross Examination of Tissue The term Grossing means inspecting the specimen or investigating the Specimen describing, measuring the tissue and sectioning the tissue to a suitable size for further processing to diagnose. The ideal size for a tissue after grossing should be 20mm x 15mm x 5mm. Steps of Grossing Check fixation status Prepare thin slices Avoid specimen trauma and cross-contamination Choose an appropriate tissue cassette for specimen processing. Labelling of cassette. Basic requirements for Grossing Tissue Section / Organ Scissors (Various sizes) Forceps Scale / Metal ruler Scalpel Scalpel handles Surgical blades Metal probe Sharp Knife Pins Tray Weighing scale Cassettes Filter paper Disinfectants etc. Decalcification It is the process of complete removal of Calcium ions from tissue like bone, teeth and other Calcified tissue. Aim of the Decalcification: The presence of Ca++ salts in tissue prevents the preparation of good sections by routine methods. The incomplete removal of these salts results in torn and ragged sections and damage in the cutting edge of the microtome knife. Done to ensure that the specimen is enough to allow grossing and cutting with of the Microtome. Criteria of good decalcifying agents: ❖ Complete removal of Calcium  ❖ Absence of damage to tissue cells and fibers. ❖ Non-impairment of subsequent staining technique. ❖ Reasonable speed of decalcification. Procedure: The techniques of Decalcification follow; 1. Selection of Tissues 2. Fixation 3. Decalcification 4. Neutralization of acid 5. Washing 1. Selection of Tissues - Bone, Calcified tissue 2. Fixation - 10% Formalin (Preferred) - Zenker’s formal (best fixed) 3. Decalcification A. Diluted or Weak Mineral Acid - Nitric acid: most common, so fast, produces minimal distortion. - HCl, Formic acid, Trichloroacetic acid, Picric acid, Acetic acid etc. B. Chelating Reagents - Organic compounds which have power to bind certain Metals. EDTA has the power to capture Metal ions. C. Ion - Exchange resins - Decalcifying fluids are used to remove Ca ions from tissue.  D. Electrolytic method by Electrophoresis - Based on the principle of attracting Ca++ ion to a negative electrode into a decalcifying Solution. 4. Methods of chemical neutralization − It can be done by treatment with a weak alkali by immersing decalcified bone into either saturated lithium carbonate solution or 5-10% aqueous sodium bicarbonate solution for several hours. − Another method is by washing of decalcified tissues in two changes of 70% alcohol for 12-18 hours, which improves staining in most cases. Following this step, dehydration is continued in the usual way. 5. Washing Thorough washing of tissue is essential before processing to remove the acid or alkali which would otherwise interfere with Staining. Determination of End-point of Decalcification: Tissues to be decalcified should not be exposed to decalcifying fluids for longer than needed. Tissue kept for prolonged decalcification adversely affects the tissue. Methods of Determining End-point Accurate determination of End-point of decalcification is necessary to avoid harmful effects of decalcifying fluids. There are various methods used to test the End-point of decalcification. ✓ Radiography of the tissue ✓ Chemical test for Calcium ✓ Physical tests ✓ Bubble test (CO2 method) Radiography of the Tissue: Taking an X-ray picture of the tissue to check for any evidence of Calcium ions in bone or tissue is the most satisfactory, efficient and sensitive method for detecting the decalcification endpoint. Chemical Test for Calcium: Detects the presence of Calcium in the decalcifying solution released from Bone. Calcium oxalate Test - detects Calcium in acid solutions by precipitation of insoluble Calcium hydroxide or Calcium oxalate. Physical tests: Experience hands can tell by feeling the tissue. Physical tests are inaccurate, unreliable and damaging to tissues. Probing, needling (inserting a needle to feel for calcium deposits), slicing, bending, or squeezing tissue should not be used to detect the decalcification end point because they can produce damage to tissue and artefacts, e.g. needle tracks, disrupt soft tumour from bone, or cause false micro- fractures. 'Bubble' test (carbon dioxide method): Acids react with calcium carbonate in bone and form carbon dioxide which produces a layer of bubbles on the bone surface. These bubbles disappear with agitation or shaking but reform. They become smaller as less calcium carbonate is reduced. This as an endpoint test is subjective, unreliable and dependent on worker interpretation. However, it can be used as a guide to check the progress of decalcification, i.e. tiny bubbles indicate less calcium present.  Tissue Fixation Fixation In Histopathology & Cytopathology, tissue fixation is a process by which a tissue specimen is placed in a fixative that preserves the cell most closely to the original form present in the body. The foundation of Histopathological preparation is complete fixation. Fault in fixation cannot be remedied at any later stage. It is essential that tissues are to be fixed as soon as possible after death or removal from the body. For that reason Screw capped jars containing appropriate fixatives should be permanently kept at OT, Post mortem room. The amount of Fixative should be at least 15-20 times the volume of Tissue. Tissue should be thin to be adequately fixed. The length, breadth thickness should be 20 mm x 15 mm x 5 mm. Several hours are needed for Fixation. Time required 12-24 hours. Fixation should be done in Room temp. In case of any emergency, can execute at 60 degrees C for 2-3 hours. Purpose of Fixation To prevent autolysis To prevent Putrefaction To preserve in the museum for future guidance Hardening of Tissue. Solidification of colloidal material. Inactivate infectious agents. Increase refractive index. Better Staining etc. Different kinds of Fixation: Chemical methods of Fixation:  Aldehydes (cross-linking)- Formaldehyde, glutaraldehyde, acrolein, glyoxal  Protein denaturing (coagulants/dehydrating) agents- Acetic acid, methyl alcohol, ethyl alcohol  Oxidizing agents- Osmium tetroxide, potassium permanganate, potassium dichromate  Other cross-linking agents- Carbodiimides Physical methods of Fixation: Heat, microwave, dry Different techniques of Fixation Immersion Fixation Perfusion Fixation Vapour Fixation Spray Fixation Microwave Fixation Freeze Drying Fixation etc. 1. Immersion Fixation The most commonly used technique of Fixation. In this tissue/smear is simply immersed in a Fixative. 2. Perfusion Fixation Basically, it is used in Research work. In this method, tissue is perfused with a fixative. Exmp- Lungs or Brain after removal. 3. Vapor Fixation It is used for imprint or Blood smears. Different types of chemicals used as Vapour are Aldehydes, Osmium tetroxide, Ethanol etc. 4. Spray / Coating Fixatives Basically used in Cytology samples especially when the Slides are to be transported to Laboratory in a distinct place. 5. Microwave Fixation Basically, it can reduce the time required for the Fixation of some gross Specimens and histological sections from more than 12 hours to less than 20 minutes. But it produces a large amount of dangerous vapours. Microwave Fixation preserves tissue Antigen better than routine methods of Fixation. 6. Freeze drying Fixation Freeze drying is an alternative to Fixation that is useful technique for studying soluble materials. In this method, tissue is rapidly frozen at -160 degrees C that forms an ice cube of Tissue. Fixative Definitions A fixative may be defined as a substance that preserves the morphological and chemical characteristics of cells and tissues and prevents autolytic and putrefactive changes. Qualities of an ideal fixative 1) Prevent autolysis and putrefaction 2) Preserve cells, tissue and its constituents in life like manner 3) Make the cellular components insoluble to reagents used in tissue processing 4) Rapid and even penetration and fixation of tissues 5) Mildly hardened tissues 6) Inactivate infectious agents in the tissue (if any) 7) Preserves tissue volume (should be isotonic), maintains shape and prevents structure deformation 8) Support high quality and consistent staining 9) Safe to handle, nontoxic, non-inflammable and nonallergic 10) Economical (cheap), stable, readily disposable or recyclable 11) Should be useful for a wide variety of tissues 12) Prevents fixation artifacts Types of Fixative: By Composition  Simple fixative: Contains single chemical, e.g. formaldehyde (10% formalin), glutaraldehyde, ethyl alcohol, etc.  Compound fixative: A mixture which contains more than one chemical fixative. ▪ Formalin based fixatives: 10% neutral buffered formalin, 10% neutral buffered formal saline, formol calcium ▪ Mercurial fixatives: Zenker's solution, Zenker -formol (Helly's solution), B5 fixatives ▪ Dichromate fixatives: Regaud's solution, Möller's solution and Orth's solution ▪ Picric acid fixatives: Bouin's fluid, Genre's fluid  Alcohol-containing fixatives: Carnoy's fluid, acetic acid formalin (AAF) Fixatives used in Histopathology: 1. Formalin 10% Formal Saline (Formalin – 10 ml, Saline - 90 ml) Duration -12-24 hrs Advantage - Cheap - Preserves most tissues well Disadvantage -Allergenic 2. Zenker’s Fluid Solution of Mercuric Chloride …………………. 5g Potassium dichromate …….…... 2.5g Distilled water …………………….... 100 ml 5 ml Glacial acetic acid is to be added before use. Duration -10-20 hrs. Advantage -Preserves nuclear and connective tissues very well. -Facilitates monochromatic staining. Disadvantage -Can penetrate up to 5 mm. -Expensive -Has to be preserved in Nickel alloy bottles. -Required to be washed for ½ hour in running water -Several changes in 70% alcohol after fixation. 3. Bouin’s Fluid Combination of Picric Acid ………… 75 ml Formalin ……….…. 25 ml Glacial acetic acid….... 5 ml Duration -10-20 hrs Advantages -Rapid penetration -Preserves Glycogen -Improves greatly staining of Nuclei and connective tissue. Disadvantages -Lyses red cell -Makes tissue hardening if kept >24 hrs -Fixed tissues should be transferred to 70% alcohol 4. Helly’s Fluid - Stock Solution of Zenker’s Fluid - Add 5-10% Formalin just before use. Duration -10-20 hrs Advantage -Preserves Nucleus and connective tissues well. -Facilitates metachromatic staining. Disadvantage -Penetrates up to 5 mm -Expensive -A nickel alloy jar is needed to preserve. Other Primary Fixatives are - Baker’s Solution (Formal calcium) Orth’s fluid Carnoy’s fluid. Dehydration The process of removal water and fixative from the tissue is termed as Dehydration. Most of the fixatives used in aqueous solution. The fixative solution as well as tissues contain water which does not mix with Paraffin wax. Hence, first this water must be removed from the tissue by hydrophilic solution before Embedding. Dehydration is a most essential step and must be carried out in a thorough manners. Water is generally removed by immersing the tissue in increasing grades of Ethyl alcohol. Total time required for Dehydration depends on the volume and type of tissue and Dehydrating agents. Amount of Dehydrating agents should not be less than 10 times the volume of tissue. Some dehydrating agents: Ethyl Alcohol Methyl Alcohol Isopropyl Alcohol Butyl Alcohol Tetrahydrofuran Acetone Steps of Dehydration: Now a days Histopathology Laboratory dehydrates tissue like; 70% alcohol ……………………….. 12 hours 90% alcohol ……………………….. 12 hours Absolute alcohol …………………... 1 hour Absolute alcohol …………………... 1 hour Absolute alcohol …………………... 1 hour Clearing (Dealcoholization) Usually a dehydrating agent (e.g. alcohol) is not miscible with an infiltrating solution (e.g. wax). Clearing agents act as an intermediary between the dehydration and infiltration solutions. The clearing agent should be soluble (miscible) in both the dehydrating agent as well as in the infiltrating solutions. When the dehydrating agent has been completely replaced by clearing agent, the tissues appear clear and translucent/ transparent. Hence this step is known as clearing and the agent that produces clearing is known as clearing agent. Thus, clearing is the process of removal of dehydrating solutions and making the tissue components receptive to the infiltrating medium. Properties of a good clearing agent: Rapid or quick removal of dehydrating agent Rapid penetration of tissues Clear the tissues quickly without hardening or tissue damage Easily removed by melted paraffin wax Low flammability, toxicity and cost Not evaporate too quickly in the wax baths Time required: 1-3 hours Some clearing agents: Xylene (Most Commonly used) Toluene (Next commonly used) Benzene Cedar wood oil Aniline Oil Citrus fruit oils Chloroform Clove Oil Carbon Di-Sulphide Carbon tetrachloride Techniques of Clearing: Remove the tissue from the last bath/beaker of Absolute alcohol Place in 2 changes of Xylene for 1 hour duration each. The volume of the clearing agent should be 50-100 times that of the tissue. Impregnation It is the permeation of tissue with a support medium. This method is also called as Infiltration. The most common method used for microscopic examination of tissues is by sectioning (with the exception of frozen sections of tissue of normal consistency) by using microtomes. This requires the infiltration and embedding of the tissues in a medium that will permit thin sections to be cut easily. Infiltration and impregnation/embedding of the tissue is the process of replacement of clearing agent by an embedding medium.  After clearing the tissue is transferred to Molten wax in a Paraffin embedding oven (58- 60 degree C) to impregnate the tissue.  Tissue should be left on melted Paraffin wax for 2-10 hours.  Good quality paraffin wax free from dust and other foreign matter should be used.  10-20% bees wax is added to improve the texture and makes it harden.  Volume of the wax should be 20-50 times of the tissue.  The paraffin wax should be replaced as it becomes saturated with the clearing agents. Different embedding reagents: ▪ Paraffin wax ▪ Gelatin ▪ Agar ▪ Water soluble wax ▪ Paraplast ▪ Ester wax ▪ Polyester wax ▪ Carbowax ▪ Celloidin  EMBEDDING (CASTING OR 'BLOCKING') The next step after tissue processing is wax embedding and preparing tissue blocks. Embedding means casting or blocking. Embedding is the process in which the tissue/ the specimen are enclosed in an embedding medium using a mould. In this process, the tissue transferred from the final wax bath (in tissue processor) and is placed in a mould filled with warm liquid/molten paraffin (impregnated with wax). On cooling this forms a solid paraffin block. Embedding enables the tissue to be cut into very thin sections on a microtome. The tissue requires supporting media to cut thin sections and this supporting media is called embedding media. There are different methods of embedding tissue and preparing paraffin blocks. Requirements  L Shape Mould  Melted Paraffin wax  Flat base  Forcep Types of Moulds  Leukhart’s L pieces  Glass Petri dishes  Metal Petri dishes  Paper boats  Watch glass  Test tube  Plastic embedding ring  Porcelin embedding ring Steps of Block making  Fresh Paraffin wax used for Blocking is kept in a Beaker in molten state in Wax oven.  Adjust Mould to suitable size and then fill with melted Paraffin wax.  Take the container with tissues out of the Oven.  Hold it with a pair of forceps and warm over the Bunsen burner for few Seconds.  Warm the lips of the forceps and remove the tissue.  Place the surface to be cut facing downwards in the molten wax-filled mould.  Gently press the tissue down as far as it will go in the wax.  With the help of forceps take out the registered number of tissues and place it on the side of block and allow to harden in Room temp.