MLS Review - Histopathologic Techniques

MEDICAL LABORATORY SCIENCE REVIEW • HISTOPATHOLOGIC TECHNIQUES OUTLINE • Histopathology o Fixation o Decalcification o Dehydration o Clearing/Dealcoholization o Infiltration/Embedding • HISTOPATHOLOGY • • • • • Once specimen is received, first thing to do is to assign specimen number and enter patient details in the logbook o Check type of specimen if it is same with the request o Details in the request form: px name, px age, doctor, initial diagnosis, type of specimen to be submitted Specimen is given to pathologist for gross examination o Pathologist to get dimensions, color, consistency, gross weight, etc. The pathologist will cut/dissect the specimen for processing by medical technologist The pathologist will then place the cut specimens on tissue cassettes that is ready for processing Most commonly used reagents o Fixative: 10% buffered neutral formalin o Decalcifying agent: 5-10% nitric acid o Dehydrant: ethyl alcohol/ethanol o Clearing agent: xylene o Infiltrating & embedding agent: paraffin • FACTORS TO CONSIDER WHEN CHOOSING THE RIGHT FIXATIVE • • • FIXATION • • • • Preservation of tissues as close to the original as possible Primary goal: to preserve tissues Secondary goals o To harden the specimen to facilitate easy cutting of tissues into thin slices o To protect the tissue from trauma of further handling ▪ In tissue processing, the specimen is exposed to various reagents that may destroy the tissue Fixation occurs once the fixative penetrates the tissue FACTORS THAT INFLUENCE FIXATION • • • • Size and thickness o Recommended size: 3x2 cm o Recommended thickness: 3-5 mm ▪ Lung specimens: 1-2 cm (thicker) • Required longer fixation time o Increasing thickness lengthens fixation time ▪ Thickness = fixation time pH - between 6.0 to 8.0 Osmolality o Slightly hypertonic is used (by book) o In practice, isotonic solutions are commonly used o Hypotonic - cell swelling o Hypertonic - cell shrinkage Concentration o Concentrated fixative can damage/destroy tissues ▪ Concentrated solutions are not used o Formalin is diluted to produce 10% solution Volume o For maximum effectiveness, the amount of fixative must be 20x the volume of the specimen ▪ Fixative to tissue ratio = 15-20:1 o Penetration rate of formalin: 1mm per hour o Osmium tetroxide - 5-10x the volume of specimen ▪ For election microscopy (EM) o 20x the volume of the specimen - Fixative volume for maximum effective fixation Time and duration of fixation o Specimens are subjected for fixation for 24-48 hours o Some factors can prolong or accelerate fixation time Temperature o Traditionally - room temperature o Fixation temp for EM & histochemistry: 0-4 degC o Fixation temp using autotechnicon: 40 degC o Temperature for manual fixation: room temp o Heating hastens fixation • • Consider urgency of case, need for immediate exam o Urgent biopsy - requires fast-acting fixative o Formalin - rapid in action, most used fixative Consider type of tissue to be processed o Carnoy’s & Acetone - used for preserving brain tissues for diagnosis of rabies o Some fixatives may be contraindicated for certain txs ▪ Bouin’s - not used for kidneys Consider the tissue structure to be studied o Brasil’s fluid - effectively preserves glycogen o Acetone ▪ Not used for lipids as it dissolves fats ▪ Preserves enzymes, lipases, phosphatases Consider the staining technique to be applied o H&E staining - routine staining as it is compatible with many fixatives o Osmium tetroxide - can inhibit hematoxylin; not recommended for H&E Staining Consider type of section to be made (serial or individual) FACTORS THAT WILL RETARD/PROLONG FIXATION PROCESS • • • • Cold temperature (prolongs fixation time) Presence of blood/mucus (challenges penetration) o Remedy: remove blood/mucus with NSS (wash) Size and thickness (larger and thicker specimen) Presence of fats (challenges penetration) o Remedy: cut the specimen thinly to ensure rapid entry of fixative to tissues FACTORS THAT WILL ACCELERATE/SHORTEN FIXATION PROCESS • • • Size and thickness (smaller and thinner specimen) Application of heat o Increasing the temperature may also increase chances of destroying the tissues o Only apply heat up to 56 degC (beyond 56 degC is damaging to the tissues) Agitation o Use of autotechnicon provides continuous agitation that ensures constant entry of fixative 1 o PROBLEMS ENCOUNTERED IN FIXATION • • • • • Loss of substances soluble in fixing agents o Example: loss of glycogen o Probable cause: wrong choice of fixative Too soft tissues o Probable cause: incomplete/inadequate fixation Too hard and brittle tissues o Probable cause: overfixation Presence of artefacts o Probable cause: inadequate washing Note: Failure to follow protocols will affect specimen quality • TYPES OF FIXATIVES BASED ON MECHANISM OF ACTION • • Additive fixative - when used, binds with the tissue and becomes part of the tissue o All fixatives are additives exc. acetone & alcohol Non-additive fixatives - do not bind with the tissue and do not become part of the tissues o Ex.: Acetone & alcohol FIXATIVES CLASSIFIED ACCORDING TO ACTION • • • Microanatomical Cytological - to preserve parts of cell (nucleus, etc.) o Nuclear Fixatives ▪ Used to preserve the nucleus and its components ▪ Carnoy’s; Bouin’s; Newcomer’s Heidenhain’s Susa; Flemming’s with HAc o Cytoplasmic fixatives ▪ Use to preserve cytoplasmic components ▪ Regaud’s/Moller’s; Orth’s; Formalin with post chroming Flemming’s without HAc Histochemical - preserve chemical components of tissues o Acetone - preserves enzymes • TYPES OF FIXATIVES BASED ON ACTIVE COMPONENT SUBSTANCE • Aldehyde Fixatives o Formaldehyde/Formalin Fixatives ▪ Formalin - highly concentrated, diluted for use ▪ 10% Formalin • Most commonly used fixative • To prepare a 1L 10% formalin solution: combine 100 mL 37-40% formalin + 900 mL distilled water • Disadvantage: prolonged storage may cause precipitation to white paraformaldehyde o 10% methanol - Added to prevent precipitation of formaldehyde ▪ 10% Formol Saline • Uses NSS as diluent • Used for CNS tissues, post-mortem tissues ▪ 10% Neutral Buffered Formalin • 10% formalin + sodium phosphate (as buffer) • For tissues with iron pigments ▪ Formol corrosive/formol sublimate • Mercuric chloride + formaldehyde/formalin • Best for lipids and neutral phospholipids ▪ Alcoholic formalin/Gendres • Ethanol + Glacial acetic acid + formaldehyde • For preserving sputum specimens & microincineration techniques • • Glutaraldehyde - for Electron Microscopy (EM) ▪ 2.5% solution - for small tissue fragments/needle biopsies ▪ 4% solution - larger tissues less than 4mm thick o Glyoxal - smallest and rapid aldehyde Metallic Fixatives o Mercuric Chloride Fixatives ▪ Most common & excellent metallic fixative ▪ Excellent for trichrome staining & tissue photography ▪ Disadvantage: produce black mercuric deposits/precipitates • Alcoholic iodine - used to wash out excess mercuric fixatives (black precipitates) ▪ Mercuric Chloride - base ingredient ▪ Zenker’s fluid - liver, spleen, CT, & nuclei ▪ B5 - bone marrow ▪ Heidenhain’s Susa - tumor skin biopsies ▪ Helly’s fluid - pituitary gland, BM, spleen, liver • Aka Zenker formol/Formol Zenker ▪ Zinc sulfate - proposed as mercuric chloride substitute • Mercuric chloride is not recommended as it can be toxic to man • Zinc sulfate may be toxic as well o Chromate Fixatives ▪ Contains potassium dichromate ▪ 1-2% Chromic acid - for carbohydrates ▪ 3% Potassium dichromate - for lipids, mitochondria ▪ Regaud’s fluid/Moller’s fluid - mitochondria, RBCs & colloid containing tissues ▪ Orth’s fluid - Rickettsiae & other bacteria, tissue necrosis o Lead Fixatives ▪ For acid mucopolysaccharide Picric Acid fixatives - Bouin’s, Brasil’s, Hollande’s o Excellent for glycogen demonstration o Picric acid - a versatile reagent used as fixative, decalcifying agent, stain ▪ Disadvantage: can impart a yellow coloration to specimen when used as fixative as it is a stain also o Bouin’s fluid ▪ Contains picric acid as main ingredient ▪ For embryos; pituitary biopsies & endometrial curetting ▪ Not for kidney structures ▪ Not compatible with Feulgen’s stain o Brasil’s o Hollande’s o 50-70% alcohol - used to wash out excess amount of picric acid fixatives Glacial acetic acid - solidifies at 17 degC o A nuclear fixative o Contraindicated for cytoplasmic fixation o May be used by itself o Most of the time is used in combination with other fixatives to produce compound fixatives Alcohol Fixatives o Ethanol o Methanol o 95% isopropyl alcohol (IPA) - for touch preparations o Carnoy’s - Most rapid tissue fixative for fixing chromosomes & lymph glands o Newcomer’s - for mucopolysaccharides & nuclear protein 2 • Acetone o Fixative & dehydrating agent o For phosphatases & lipases o Must be used at ice cold temperature o Disadvantages: ▪ Evaporates easily ▪ Dissolves fats TWO GENERAL METHODS OF FIXATION • • Physical fixation: o Heat fixation - most common ▪ Fixation method that involves thermal coagulation of proteins ▪ Used in microbiology/bacteriology to prepare bacterial smears ▪ Eg. microwave technique Chemical fixatives o Most commonly used o Uses chemical fixatives • 4 METHODS FOR DECALCIFICATION • • ADDITIONAL NOTES ON FIXATION • • • • • • • • Presence of mucus, blood, fatty tissues, cold temperature retard fixation 37-56 degC - temperature range that will accelerate fixation Fixative volume for museum preparations should not be less than 50-100x the volume of the specimen Fixatives for Enzyme Histochemistry: 4% formaldehyde & formol saline EM fixatives: Osmium Tetroxide, Glutaraldehyde, paraformaldehyde Osmium tetroxide - can be used for nervous tissues (myelin & peripheral nerves) but can inhibit hematoxylin o Used for electron microscopy Carnoy’s fluid & Acetone - For brain tissues for the diagnosis of rabies Trichloroacetic acid (TCA) - often used in conjunction with other fixatives to produce a compound fixative o Also a weak and slow decalcifying agent DECALFICATION • • • • • Performed immediately after fixation Involves removal of calcium and other salts from bones, teeth, etc. Not all specimens will undergo decalcification To soften hard tissues to facilitate easy cutting Factors affecting Decalcification o Degree of mineralization ▪ Higher degree of mineralization requires longer time of decalcification ▪ Eg. osteoporotic px = deficient calcium = short decalcification time o Concentration/Volume ▪ Increasing concentration and volume will shorten decalcification time ▪ Required is 20x volume of the specimen • Decalcifying agent to tissue ratio = 20:1 o Temperature ▪ Heating can accelerate/hasten decalcification ▪ Room temp (18-30 degC) - optimum temperature range for decalcification o Agitation ▪ Constant agitation accelerates decalcification Decalcification is not performed in autotechnicon o Performed separately and manually o There is no special machine to perform decalcification o Autotechnicon: ▪ Fixation → Dehydration → Clearing → Infiltration • • Use of acid o Hydrochloric acid (HCl) - decalcifying acid used for surface decalcification of block o 5-10% nitric acid - most commonly used decalcifying agent especially recommended for urgent biopsies, for needle & small biopsies ▪ Routine decalcifying acid o Phloroglucin - most rapid nitric containing decalcifying agent ▪ Phloroglucin nitric = phloroglucin + nitric acid Use of chelating agent (EDTA) o EDTA ▪ Potent anticoagulant but weak decalcifying agent ▪ Binds with calcium to form weakly dissociated complexes ▪ An excellent bone decalcifier for EM and enzyme histochemistry ▪ Disadvantage: inactivation of alkaline phosphatase • Magnesium chloride - remedy for inactivation of alkaline phosphatase by EDTA Ion-exchange resin o 1-14 days - duration of decalcification thru ion exchange resin o Larger & heavily mineralized = longer time Electrolytic method o Decalcification method that involves attraction of positively charged calcium ions with negatively charged electrodes o Most rapid method of decalcification ▪ Remove calcium through electric current METHODS TO MEASURE EXTENT OF DECALCIFICATION • • • • • Mechanical/physical method o Method of testing for the completeness of decalcification done by probing tissue with a needle o Bending, probing, or pricking the tissue Radiologic method (X-ray) o Most reliable method of testing for the completeness of decalcification o Detects even the smallest amount of calcium Chemical method Bubble test o Method of testing for the completeness of decalcification that uses calcium carbonate If the tissues are still hard after decalcification, they are placed in tissue softeners o Eg. Molliflex-Perenyi’s o Perenyi’s - act as decalcifying agent & tissue softener 3 DEHYDRATION • • • • • • • • • • • Removal of intra- and extracellular water in tissue Includes submerging in increasing/ascending concentrations - important requirement when using alcohol as dehydrating agent 37 degC - temperature that will hasten dehydration time Ethyl alcohol/Ethanol o Fast-acting and non-toxic o Most commonly used alcohol for dehydration process Triethyl phosphate - for dehydrating sections and smears Cellosolve/Ethylene glycol monoethyl ether o Toxic by ingestion, inhalation, & by skin contact o Highly corrosive on skin o Toxic to reproductive, fetal, urinary system o Decomposes when exposed to sunlight Dioxane/Diethylene dioxide o Can dehydrate and clear tissues o Dehydrating agent whose main disadvantage is its being toxic o Not commonly used due to its being toxic to man Tetrahydrofuran (THF) o Dehydrating agent that is both a skin & eye irritant o Has an offensive odor o Must be used in a well-ventilated room Acetone - rapid acting dehydrant suited for urgent biopsies but not for routine tissue processing o Can be used as fixative or dehydrant Butyl alcohol/Butanol - alcohol for plant & animal microtechniques Purpose of phenol in alcohol bath - acts as tissue softener o Recommended for quite hard tissues without calcium (nails, etc.) CLEARING / DEALCOHOLIZATION • • • • • • • • • • Removes excess alcohol Most agents can make tissues transparent as the reagents used make tissue transparent o Aka clearing Benzene - clearing agent that can damage the bone marrow & may cause aplastic anemia on prolonged use Chloroform o Toxic to liver; recommended for skin, fibroid & decalcified tissues o For tough tissues, nervous tissues, lymph nodes, embryos o Clearing agent that will not make tissues transparent o Clearing agent that can attack rubber seal used in vacuum impregnation Xylene o Routinely used clearing agent but not for nervous tissues & lymph nodes o Can become milky after placing an incompletely dehydrated tissue in it Cedarwood oil o Clearing agent for CNS tissues & cytological studies o An extremely slow clearing agent o Can become milky because of prolonged storage Aniline oil - for clearing insects, embryos, & other delicate specimens Clove oil - when used, tissues become adulterated Carbon tetrachloride - same property as chloroform Terpineol - recommended for eye specimens • • Limonene o Clearing agent obtain from citrus fruit o With faint pleasant odor Toluene - can be used as xylene & benzene substitute ADDITIONAL NOTES IN CLEARING • • • • Chloroform & cedarwood oil o Clearing agents that can be used for dense tissues like uterus o Clearing agents difficult to remove from tissues ▪ Make infiltration time longer Xylene & benzene - clearing agent easily removed from tissues Clearing agents for double embedding which are slow acting agents o Methyl salicylate o Methyl benzoate o Amyl acetate Steps in tissue processing that will require complete clearing of tissues o Infiltration o Mounting INFILTRATION • • Aka impregnation Carried out for two reasons: o Remove clearing agents from tissues o To fill up cavities and tissue spaces to give the tissue a firm consistency which is required to facilitate easy cutting of tissues into very thin slices THREE GENERAL METHODS OF INFILTRATION • Paraffin o Most common method of infiltration o Paraffin wax requires heat o Application of heat hastens the process o 56 degC - melting point of paraffin wax commonly used o Heat is applied to paraffin through oven o 55-60 degC or - temperature range of paraffin oven when in use o Approx. 2-5 deg higher than wax melting point approximate temperature of paraffin oven when in use o Three methods for paraffin wax infiltration ▪ Manual - requires oven (55-60 degC) ▪ Automatic - requires autotechnicon • Would require 3-4 changes of wax ▪ Vacuum Infiltration - infiltration under negative atmospheric pressure inside the oven • Tissue processing time is reduced because tissues are subjected to very high temperature • Facilitates removal of transition solvents • Prolongs the life of wax by reducing solvent contamination o Paraffin wax method is not suited for fatty tissues ▪ Due to application of heat o Paraplast - substitute for paraffin wax recommended for bones and brain specimens ▪ 56-57 degC - melting point of paraplast 4 o • • Carbowax - substitute for paraffin wax that is water soluble & is recommended for enzyme histochemical studies ▪ Polyethylene glycol containing 18 or more carbon atoms ▪ Appears solid at room temperature o Bioloid - substitute for paraffin wax used for eye specimens o Ester wax - substitute for paraffin wax that will require heavy duty type of microtome ▪ Harder than paraffin ▪ 46-48 deg C - melting point of Ester wax Celloidin o Recommended for enzyme and histochemical studies o Infiltration method recommended for specimens with large and hollow cavities that tends to collapse Disadvantage: the process is slow o Two methods: Wet & Dry o Wet Celloidin Method ▪ Celloidin infiltration method for bones, large brain sections, whole organs, & teeth specimens ▪ Will require 70-80% alcohol to store blocks o Dry Celloidin Method ▪ Celloidin infiltration method recommended for whole eye specimens ▪ Will require use of Gilson’s mixture to store blocks o Gilson’s mixture ▪ combination of chloroform & cedarwood oil ▪ Added to celloidin blocks before hardening to make tissues transparent ▪ Used in dry method of celloidin o Low Viscosity Nitrocellulose (LVN) ▪ Another form of celloidin ▪ Soluble in ether and alcohol ▪ Lower viscosity that can be used at high concentrations and still penetrate rapidly ▪ More preferred than ordinary celloidin for impregnation and embedding methods ▪ Produces harder tissue blocks and allow cutting of thinner sections ▪ Disadvantage: infiltrated have tendency to crack • Plasticizers are added to prevent cracking tissues when infiltrated using (LVN) o Plasticizers such as oleum ricini or Castor oil o Procedure: Thin → Medium → Thick 2-4% 4-6% 8-12% 5-7 days 5-7 days 3-5 days Gelatin - not usually carried out o Infiltration method for enzyme & histochemical studies o Performed when there is a need to avoid the dehydration process o Infiltration method that will require tissues not to be more than 2-3 mm thick ▪ Allow rapid entry of infiltrating agents in tissue o Use of phenol in gelatin infiltration - to prevent molds EMBEDDING • • • • • • • • • Aka Casting/ Blocking Performed after infiltration Involves placing infiltrated tissue in a mold containing an embedding medium which is allowed to solidify Tissue block - product of embedding Requires embedding medium o Usually paraffin wax o Paraffin wax - most common embedding medium Infiltrating agents can also be used as embedding medium Embedding molds o Plastic ice trays - a disposable embedding mold that will require smearing of inner mold with glycerine o Compound embedding unit - an embedding mold consisting of a series of interlocking plates resting on a flat metal base ▪ Advantage: can embed many tissues at a time o Peel away mold - disposable mold that gives perfect blocks without trimming o Leuckhart’s Embedding mold - embedding mold consisting of L shaped strips made of heavy brass ▪ Not usually used in the lab as it required assembly prior to use ▪ Advantage: size of the block can be adjustable depending on the required size Double embedding - process of infiltrating tissue with celloidin and embedding with paraffin o Uses two different medium Plastic embedding o Use of plastic resin to embed has provided superior results for light microscopic studies especially when processing hard tissues like undecalcified bones o Spur - fastest epoxy resin o Polyester - plastic resin originally introduced for EM o Polyglycol methacrylate/GMA - extremely hydrophilic o Methylmethacrylate/MMM - plastic resin considered ideal for undecalcified bones & hard tissues 5