Histotechnique Review Lecture PDF

REVIEW LECTURE IN HISTOTECHNIQUE DOC J.E.M. HISTOLOGICAL TECHNIQUE 3. SMEAR PREPARATION Useful in cytological examina...

REVIEW LECTURE IN HISTOTECHNIQUE DOC J.E.M. HISTOLOGICAL TECHNIQUE 3. SMEAR PREPARATION Useful in cytological examinations – particularly in cancer A. FIXATION diagnosis B. DEHYDRATION C. CLEARING METHODS OF SMEAR PREPARATION D. EMBEDDING a) STREAKING – applied in a direct or zigzag line E. CUTTING throughout the slide, attempting to obtain a F. STAINING relatively uniform distribution of secretion. SPECIMEN HANDLING AND IDENTIFICATION b) SPREADING – a moderately thick film by teasing a mucous strands apart with an applicator stick. The specimen container label and accompanying request from should include: - Little more tedious than streaking, but has o Patient’s name advantage in maintaining the intercellular o Age of Birth relationship. o A medical record number Label should be firmly attached to the body of the Recommended for smear preparation of: container --- not the lid of the container Fresh Sputum Bronchial Aspirates METHODS OF FRESH TISSUE EXAMINATION Thick Mucoid Secretions 1. TEASING OR DISSOCIATION c) PULL-APART – useful for preparing a smears of A process whereby a selected tissue specimen is thick secretions such as: immersed in a watched glass containing isotonic salt Serous fluids solution (NSS or Ringer’s Lacate), carefully dissected or Concentrated sputum separated, and examined under the microscope. Enzymatic Lavage Samples from GIT Blood Smear d) TOUCH PREPARATION – IMPRESSION SMEAR Advantage: Cells may be examined without destroying their actual intercellular relationship 4. FROZEN SECTION Normally utilized when a rapid diagnosis of the tissue in question is required 2. SQUASH PREPARATION Recommended when LIPIDS and NERVOUS TISSUE AKA: CRUSHING elements are being demonstrated A process whereby small pieces of tissue not more than 1mm in diameter are placed in a microscopic slied and Very thin slices (10-15u in thickness) forcibly compressed with another slide or with a cover Cryostat – temperature of -10C to 20C glass Applications: Rapid pathologic diagnosis during surgery Diagnostic and research enzyme histochemistry Diagnostic and research demonstration of soluble substances such as lipids and carbohydrates Immunofluorescent and immunohistochemical staining Some specialized silver stains – particularly in neuropathology MARK LOUIE B. DELA PEÑA 1 Tissue for freezing should be fresh, and freezing should be done as quickly as possible (not used if may fixative na) Slow freezing can cause distortion of tissue due to ice crystal artifacts. - since 70% of tissue are made with water - will make “Swiss cheese” appearance More commonly used methods of freezing include: “LICA” a) LIQUID NITROGEN – generally used in histochemistry and during operative procedures. - Most rapid of the commonly available freezing agent b) ISOPENTANE COOLED BY LIQUID NITROGEN c) CARBON DIOXIDE d) AEROSOL SPRAY PROCESSING OF TISSUE Solid structures and tissues must be preserved and carefully processed in the following order: “FD-CIETS-SML” 1. Fixation 2. Dehydration 3. Clearing 4. Infiltration 5. Embedding 6. Trimming 7. Section-Cutting 8. Staining 9. Mounting 10. Labelling HISTOTECHNOLOGY To produce microscopic preparations of tissue that represents as closely as possible their structure in life – AIM OF GOOD To preserve the tissue – AIM OF IDEAL FIXATION FIXATION – first and most critical step in histotechnology - Fixing or preserving fresh tissue for examination. - The quality of the section on the slide is only as good as the quality of the fixed tissue specimen. Underfixed -> soft -> mabubulok / difficult to cut Overfixation -> harden -> difficult to cut MARK LOUIE B. DELA PEÑA 2  FIXATION MAIN FACTORS INVOLVED IN FIXATION - Tissues are fixed in a physical, and partly in a chemical state, so that they will withstand subsequent treatment with a various reagents with a minimum loss, significant distortion, or decomposition. - Fixation prevents degeneration, decomposition, putrefaction, and distortion of tissues after removal from the body. - To preserve the tissue – by stopping all cellular activities - To prevent breakdown of cellular elements - To coagulate or precipitate protoplasmic substances (proteins) ------------------------------------------------------------------------------------ Primary aim of fixation - Preserve the morphologic and chemical integrity of the cell in as life-like as possible Secondary goal of fixation - To harden and protect the tissue from trauma of further handling - For easy cutting during gross examination --------------------------------------------------------------------------- The most important reaction in fixation: Stabilization of proteins - This can be achieved by: Forming cross-links between proteins Leaving a tissue specimen in air cause it to: dry-out (distortion of morphologic appearance) Leaving the tissue in water (hypotonic solution) will cause the cell to: swell - If the environment is hypotonic, more water is outside the cell and the solute concentration inside the cell is high, the water would enter the cell and swells, bursts, or lyses (cytolysis) PRACTICAL CONSIDERATION OF FIXATION 1. SPEED Leaving the tissue in strong salt (hypertonic solution) will - The specimen should be placed in fixative as soon as cause the cell to: shrink it is removed from the body. - More solutes are outside the cell (strong salt solution) - This is done to prevent autolysis and and water is less, so water will move outside the cell putrefaction/decomposition. and the cell shrinks - If bacteriologic and toxicologic studies should be encouraged, therefore, fixation is not required. 2 BASIC MECHANISM INVOLVED IN FIXATION o Fixation can kill microorganisms and prevent growth in culture o Fixatives can neutralize drugs and toxins. 2. PENETRATION - Time of fixation varies with different types of tissue - Formalin diffuses into the tissues at a rate of 1mm/hour. 3. VOLUME/AMOUNT OF FIXATIVE - 10-25x the volume of the tissues to be fixed - 20x – maximum effectiveness of fixation - OSMIUM TETROXIDE: 5-10x MARK LOUIE B. DELA PEÑA 3 4. DURATION “Heiden Bought a New Car from Flemming” - Fibrous organs such as uterus and intestinal track a. Heidenhain’s susa take longer. b. Bouin’s fluid - Fixation can be cut down using HEAT, VACUUM, c. Newcomer’s fluid AGITATION OR MICROWAVE d. Carnoy’s Fluid e. Flemming’s fluid IDEAL SIZE OF THE TISSUE TO BE FIXED - Not more than 2cm2 in diameter 2. CYTOPLASMIC FIXATIVE - Not more than 4mm2 thick o Must NEVER CONTAIN GLACIAL - Ideal number of hours for fixation: 4-6 hours (New ACETIC ACID (it destroy cytoplasmic Book: 6-18 hours) structures: Mitochondria and Golgi Bodies) TYPES OF FIXATIVE o Have pH of more than 4.6 ACCORDING TO COMPOSITION “HORFF” a. Helly’s fluid A. SIMPLE FIXATIVE b. Orth’s fluid Made up of only one component substance c. Regaud’s Fluid 1. Aldehydes (Formaldehydes, Glutaraldehydes) d. Flemming’s fluid without acetic acid 2. Metallic Fixatives e. Formalin with post-chroming - Mercuric Chloride - Chromate Fixative 3. HISTOCHEMICAL FIXATIVE Potassium dichromate o Preserves the chemical constituents of Chromic Acid cell and tissues. - Lead Fixative Picric Acid a. 10% Formol Saline b. Absolute Ethyl Alcohol Acetic Acid c. Newcomer’s Fluid Alcohol d. Acetone Osmium Tetroxide - Heat Fixation of tissues can be accomplished by: B. COMPOUND FIXATIVE a. Physical Method Two or more fixatives o Heating o Freezing-drying ACCORDING TO ACTION o Mircrowaving b. Chemical Method A. MICROANATOMICAL FIXATIVES o Coagulant Fixatives Tissues structures without altering the structural pattern o Compound Fixatives and normal intercellular relationship of tissue o Cross-linking Fixatives 1. 10% Formol Saline PHYSICAL METHODS 2. 10 % Neutral Buffered Formaline HEAT FIXATION 3. Heidenhain’s Susa - Simplest form of fixation 4. Formol Sublimate (Corrsive) - Primarily used to accelerate other forms of fixation as 5. Zenker’s Solution well as the steps of tissue processing. 6. Zenker Formol (Helly’s) 7. Bouin’s Solution MICROWAVE HEATING 8. Brasil’s Solution - From more than 12 hours to less than 20 minutes. - Commercial glyoxal-based fixatives B. CYTOLOGICAL FIXATIVE Particular microscopic elements of the cell FREEZE-DRYING AND FREEZE SUBSTITUTION - Preserve specific parts and particular microscopic elements of the cell itself CHEMICAL METHODS COAGULANT FIXATIVES TYPES OF CYTOLOGICAL FIXATIVES: 1. NUCLEAR FIXATIVES - Coagulate proteins making them insoluble. o Preserves nuclear structure - Cytoplasmic flocculation o CONTAINS GLACIAL ACETIC ACID (since DNA is acid) o have pH of 4.6 or less MARK LOUIE B. DELA PEÑA 4  TYPES OF COGULANT FIXATIVES Reagent-grade formaldehyde contains 10% methanol as a preservative to retard decomposition to formic acid. Prevents the formation of 1. DEHYDRANT COGUALANT FIXATIVE Paraformaldehyde. (Both Dehydrant and Fixative) - Formalin pigments – are also formed due to overfixation. - Fixation Time: 24 hours - METHANOL – 80% OR MORE Removal of formalin pigments: - ETHANOL – 50 TO 60% 1. Lillies Method – involves placing formaldehyde fixed specimens in acetone, 28% ammonia water and hydrogen ALCOHOL AND ACETONE – can react to polar and peroxide. It uses 70% alcohol as a rinsing agent. non-polar material - Alcohol denatures proteins differently depending on: 2. Kardasewitch’s Method – a method of formaldehyde o Concentration of Alcohol clearance involving 70% ethanol and 28% ammonia water. o Presence of organic(non-polar) and non- organic(polar) substances 3. Saturated Alcoholic Picric Acid o pH ------------------------------------------------------------------------------------ o Temperature ROUTINE FORMALIN FIXATIVE 2. ACIDIC COAGULANTS 1. 10% FORMOL-SALINE FIXATION TIME: 24 HOURS AT 35C - ACETIC ACID 48 HOURS AT 20-25C - PICRIC ACID OR TRINITROPHENOL o Produces brighter staining - A simple microanatomical fixative saturated o May cause hydrolysis and loss of nucleic formaldehyde (40%) -> 10% + sodium chloride. acids – due to low pH - Fixation of CENTRAL NERVOUS TISSUES and o Can cause yellow discoloration of tissue GENERAL POST-MORTEM TISSUE for histochemical examination. ROUTINE FIXATIVES - It is the most commonly used fixative in pathology I. ALDEHYDES FIXATIVES FORMULA: FORMALDEHYDE, 40% NaCl A. FORMALDEHYDE DISTILLED WATER FIXATION TIME: 24 HOURS 2. CALCIUM ACETATE FORMALIN (LILLIES - AKA: FORMALIN / METHANAL FIXATIVE) - Most widely used fixative - A saturated solution of formaldehyde: 35% - 40% gas - Used to preserved PHOSPHOLIPIDS by weight (Gregorios: 37% - 40%) - Replaced Formol-saline as the most commonly used o 10% Formalin – mixture of 10mL of fixative in pathology because: Formalin with 90mL of water o Simple to Prepare - It is usually buffered to pH 7 with a phosphate buffer o Buffered with pH 7 by acetate - The best fixative for NERVOUS TISSUE --------------------------------------------------------------------------- 3. 10% NEUTRAL BUFFERED FORMALIN/ PRECAUTIONS: PHOSPHATE - BUFFERED FORMALIN - Concentrated solutions of formaldehyde must never FIXATION TIME: 4-24 HOURS be neutralized – voilent explosion - Recommended for preservation of and storage of (do not add water; you add formalin to water) SURGICAL, POST-MORTEM AND RESEARCH - At low temperature, Formalin becomes turbid – due specimen to the formation of PARAFORMALDEHYDE (whitish - Best general tissue fixative precipitate) - BEST FIXATIVE FOR: REMEDY FOR PARAFORMALDEHYDE: o Frozen sections - Filtration o Iron containing pigments and elastic - Addition of 10% methanol fibers --------------------------------------------------------------------------- - Prevents precipitation of acid formalin pigments Disadvantages: - In tissues containing much blood (spleen), unbuffered formalin FORMULA: leads to the formation of dark brown artifact pigment granules SODIUM DIHYDROGEN PHOSPHATE → these granules consist of acid formaldehyde hematin and DISODIUM HYDROGEN PHOSPPHATE are doubly refractile. FORMALDEHYDE - Prolonged fixation may cause bleaching, fat dispersal and DISTILLED WATER dissolution or loss of glycogen, biurates of sodium crystal and uric acid. --------------------------------------------------------------------------- MARK LOUIE B. DELA PEÑA 5 4. FORMOL CORROSIVE/ FORMOL-SUBLIMATE/ - Tissues fixed with mercury chloride containing FORMOL-MERCURIC CHLORIDE compounds produce black precipitates except FIXATION TIME: 3-24 HOURS Heidenhain’s SuSa - Recommended for ROUTINE POST-MORTEM - Satisfactory for immunoperoxidase techniques but TISSUES. ultrastructural preservation is poor. - Excellent for silver reticulum methods. - Fixes lipids, especially neutral fats and Disadvantages: phospholipids 1. It causes marked shrinkage of cells. - Forms BLACK DEPOSITS Metal = Mercury - No frozen sections are made How can one counteract this? o By addition of an acidic solution such as 5. ALCOHOLIC FORMALIN/ GENDRE’S FIXATIVE glacial acetic acid - It contains ethyl alcohol saturated with picric acid o Through secondary fixation - Enhances immunoperoxidase studies for EM if post- 2. Has black granular deposits and is extremely corrosive to metals. fixed with phenol formalin for 6 hours or more. - It fixes and dehydrates at the same time and fixes sputum since it coagulates mucus Removal of black deposits caused by Mercuric Chloride: 6. SPECIAL FORMALIN FIXATIVES: a. 0.5% iodine in 70% ethanol (alcoholic iodine) for 5-10 minutes + water + 5% sodium thiosulfate CAJOL’S FORMOL AMMONIUM BROMIDE (decolorized-5mins)-wash in running water. - good fixative for nervous tissue (Astrocytes) o Alcoholic iodine is used to remove excess - Fixatives for acid mucopolysaccharide mercury o 5% sodium thiosulfate is used to remove BAKER’S FORMOL CALCIUM excess iodine - Used for the preservation of lipids since most formalin fixatives are inert to lipids. b. Addition of saturated iodine solution in 96% alcohol --------------------------------------------------------------------------- (alcoholic iodine) and iodine decolorized with B. GLUTARALDEHYDE absolute alcohol in the subsequent stages of FIXATION TIME: ½ - 2 HOURS dehydration. - Used form electron microscopy – buffered glutaraldehyde then osmium tetroxide c. Dezenkerization - Made up of two formaldehyde residues, linked by o Bring slides to water. three carbon chains. o Immerse in Lugol’s Iodine (5 minutes) - Recommended for histochemistry and electron o Wash in running water (5 minutes) microscopy o Immerse in 5% Na thiosulfate (5 minutes) - Most effective aldehyde for protein cross-linking o Wash in running water (5 minutes) - 2.5% solution – for small tissue fragments and o Proceed with required water soluble stain. needle biopsies fixed in 2-4 hours at room temperature. MERCURIC CHLORIDE FIXATIVES - 4% solution – recommended for larger tissued less 1. ZENKER’S FLUID than 4mm thick fixed in 6-8 hours to 24 hours FIXATION TIME: 12-24 HOURS - Mercuric Chloride Stock Solution + GLACIAL ACETIC Disadvantages: 1. It is more expensive. ACID – added before its use - Good general fixative for adequate preservation of all 2. It reduces PAS positivity of reactive mucin. kinds of tissues and give excellent staining results * How can this be prevented? - Solutions must always be freshly prepared → Immersion of Glutaraldehyde fixed tissues in a mixture - Recommended for fixing small pieces of liver, spleen, of Concentrated glacial acetic acid and aniline oil. connective tissue fibers and nuclei. - Produces Mercuric deposits II. METALLIC FIXATIVE o Remedy: Alcoholic Iodine Dezenkerization A. MERCURIC CHLORIDE - The most common metallic fixative 2. ZENKER-FORMOL / HELLY’S SOLUTION - Frequently used in aqueous saturated solutions of FIXATION TIME: 12-24 HOURS 5-7%. - Mercuric Chloride Stock Solution + 40% - Permits brilliant metachromatic staining FORMALDEHYDE – added before its use - The routine fixative of choice for preservation of cell - Excellent microanatomic fixative for pituitary gland, detail in tissue photography bone marrow, and blood containing organs – spleen and liver. “BLOODY HELLY” MARK LOUIE B. DELA PEÑA 6 - Produces brown pigments FORMULA: o Remedy: immerse the tissue in o 3% Potassium dichromate ALCOHOLIC PICRIC ACID or SODIUM o 40% Strong Formaldehyde HYDROXIDE 3. ORTH’S FLUID (REGAUD’S + SODIUM SULFATE) 3. HEIDENHAIN’S SUSA FIXATION TIME: 36-72 HOURS FIXATION TIME: 3-12 HOURS - For study of early degenerative processes and - For TUMOR BIOPSIES ESPECIALLY OF THE SKIN tissue necrosis - An excellent cytologic fixative - Myelin and Rickettsia FORMULA: FORMULA: o Mercuric Chloride (shrinkage) o 2.5% Potassium dichromate o Sodium Chloride o Sodium sulfate (optional) o Trichloroacetic acid (swelling) o 40% Strong Formaldehyde o Glacial Acetic Acid o Formaldehyde, 40% --------------------------------------------------------------------------- o Distilled Water C. LEAD FIXATIVES - Used in 4% aqueous solution of basic lead acetate - Counterbalance effect - Recommended for acid mucopolysaccharides o Su (Sublimat) = metal (mercury) = shrink o Sa (Saure) = acid (tichloroacetic acid) = Disadvantage: swell - It takes up CO2 to form insoluble lead carbonate especially on prolonged standing. 4. B-5 FIXATIVE o Remedy: Filtration or by adding acetic FIXATION TIME: 1 ½ - 2 HOURS acid drop by drop to lower the pH and - For BONE MARROW BIOPSY dissolve the residue. FORMULA: III. PICRIC ACID FIXATIVES o Distilled Water - Excellent for glycogen demonstration o Mercuric chloride - Normally used in strong saturated aqueous solution o Sodium Acetate - It dyes the tissues yellow, thus preventing the tissue o Just prior to use, add 1cc of 40% fragments from being overlooked. On the other hand, Formaldehyde for 10cc of B-5 fixative this hinders proper staining. --------------------------------------------------------------------------- o REMEDY: o Add 50-70% ethanol B. CHROMATE o Acid dye/ lithium carbonate - a class of fixatives which are strong oxidizing agents o 70% ethanol → 5% sodium thiosulfate → used for precipitating proteins and preserving Wash in running tap water carbohydrates. - recommended for Chromaffin tissue, Adrenal - Can be used for fragmentary biopsies Medulla, Mitochondria - The chemical name for the general picric acid fixatives: 2,4, 6 - trinitrophenol CHROMATE FIXATIVES - Picric acid is highly explosive when dry, and “CROP” therefore must be kept moist with distilled water or saturated alcohol at 0.5 to 1% concentration 1. CHROMIC ACID during storage. - Used in 1-2%, used as a constituent of compound fixative. A. BOUIN’S SOLUTION - It precipitates all proteins and adequately preserves FIXATION TIME: 6-24 HOURS carbohydrates. - Embryos and pituitary biopsies - Formaldehyde must be added to chrome- - Preferred fixative for Masson’s trichrome staining containing tissues before use to prevent for collagen, elastic or connective tissue. counteracting effects and consequent decomposition of solution upon prolonged standing. FORMULA: o Saturated solution of Picric Acid 2. REGAUD’S FLUID/ MOELLER’S FLUID o Strong formaldehyde 40% FIXATION TIME: 12-48 HOURS o Glacial acetic acid - For Chromatin, Mitochondria, Mitotic Figures, Golgi Bodies, RBC and Colloid-containing tissues. MARK LOUIE B. DELA PEÑA 7 B. BRASIL’S ALCOHOL/ PICROFROMOL FIXATIVE D. CARNOY’S FLUID - Best routine fixative for glycogen FIXATION TIME: 1-3 HOURS - Most rapid tissue fixative FORMULA: - Fixing chromosomes, lymph glands and urgent o Formaldehyde 37% biopsies. o Picric Acid - used to fix brain tissue for diagnosis of RABIES o Ethanol or Isopropyl Alcohol o Trichloroacetic acid FORMULA: o Chloroform IV. GLACIAL ACETIC ACID o Absolute Alcohol - Solidifies at 17C o Glacial Acetic acid - Fixes and precipitates nucleoproteins - It precipitates chromosomes and chromatin materials. E. NEWCOMER’S FLUID - Causes tissues to swell specially those containing - Mucopolysaccharide and nuclear proteins collagen. - Both a nuclear and a histochemical fixative Disadvantage: It is contraindicated for cytoplasmic FORMULA: fixation because it destroys mitochondria and golgi o Isopropyl alcohol elements. o Propionic Acid o Petroleum ether o Acetone V. ALCOHOL FIXATIVES o Dioxane - Denatures and precipitates proteins - 70-100% concentrations are used; Less VI. OSMIUM TETROXIDE/ OSMIC ACID concentrations results to red blood cell lysis/ - A poor penetrating agent ad very expensive hemolysis due to hypotonicity - Should be kept in cool place or refrigerated to prevent deterioration Advantages: - Extremely volatile - May be used both as a fixative and dehydrating agent - Excellent for glycogen preservation - It is a pale yellow powder which dissolves in water (up to 6% at 20°C) to form strong oxidizing solution. Disadvantages: - Adequately fixes materials for ultrathin sectioning - Causes RBC hemolysis and dissolves fats and lipids in electron microscopy, since it rapidly fixes small - Tissue shrinks on long usage pieces of tissues and aids in their staining - Polarization (major disadvantage); glycogenic granules moves to towards the ends or poles of cells Disadvantage: - Inhibits Hematoxylin and makes counterstaining General Rule: difficult - Alcohol-containing fixatives are contraindicated when - Very expensive (less amount is required for fixation; lipids are to be studied. 5-10x volume of fixative used) - Formation of artifact pigments/black precipitate A. METHYL ALCOHOL 100% - Prolonged exposure to acid vapors causes eye - Fixing dry and wet smears, blood smears and bone irritation(conjunctivitis) or black osmic oxide marrow tissues. deposition in the cornea (blindness) - Fixes and dehydrates at the same time - Not to fix for more than 48 hours – tissues may be 1. FLEMMING’S SOLUTION overharden and difficult to cut FIXATION TIME: 24-48 HOURS - Used in WRIGHT’S STAIN as diluent - The most commonly used Chrome-Osmium - acetic acid, recommended for nuclear preparation B. ISOPROPYL ALCOHOL 95% of such sections. - Used for fixing touch preparation (impression smear) - Excellent for nuclear structures such as - WRIGHT-GIEMSA STAIN chromosomes - Permanently fixes fats/lipids C. ETHYL ALCHOL FIXATION TIME: 18-24 HOURS FORMULA: - Used concentrations of 70% to 100% o Aqueous chromic acid 1% - Lower concentrations --- RBC’s become hemolyzed o Aqueous osmium tetroxide and WBC’s are inadequately preserved. o Glacial Acetic Acid - Used as simple fixative MARK LOUIE B. DELA PEÑA 8 2. FLEMMING’S SOLUTION WITHOUT ACETIC ACID FIXATION TIME: 24-48 HOURS - Chromic and osmic acid - For cytoplasmic structures- mitochondria --------------------------------------------------------------------------- VII. TRICHLOROACETIC ACID (TCA) - Sometimes incorporated into compound fixatives (Ex. Heidenhain’s SuSa) - It precipitates proteins - Causes marked swelling effect on tissues - Can be used as a weak decalcifying agent. - Has softening effect on dense fibrous tissue - Suitable only for small pieces of tissues or bone because of its poor penetration VIII. ACETONE - It is used at ice cold temperature from -5C to 4C (New book: 0-4C) - Recommended for the study of water diffusible enzymes especially lipases and phosphatases - Used in fixing brain tissues for diagnosis of rabies (Negri bodies). - Used in freeze substitution techniques as a solvent for certain metallic salts - Evaporates rapidly MARK LOUIE B. DELA PEÑA 9  DECALCIFICATION - Procedure whereby calcium or lime salts are removed from tissues (most especially bones and teeth) - It is a form of ionization - Done after fixation and before impregnation (before dehydration) - Calcified tissues are harder to cut - Tuberculous organs, arteriosclerotic vessels, bones, and teeth Recommended temperature: Room temperature (18-30C) Ideal time required for decalcifying tissue: 24-48 hours Volume: 20x the volume of the tissue Factors Influencing Rate of Decalcification a. Structure b. Temperature c. Concentration and Volume *too rapid removal of CA+2 salts may produce complete digestion of the tissue specimen and poor staining capacity of the cell Decalcifying Agents: 1. Acids 2. Chelating Agents 3. Ion Exchange Resins 4. Electrical Ionization / Electrophoresis For routine purposes, only acids are recommended: - Nitric acid - almost 2X as fast as formic acid - Formic acid - provides better tissue preservation and staining I. ACID DECALCIFYING AGENTS II. CHELATING AGENTS - Substances which combine with calcium ions and other salts (iron and magnesium) to form weakly dissociated complexes and facilitate removal of Calcium salt - The most common chelating agent: EDTA (Used as an anticoagulant and water softener) o Commercial name: Versene, Sequestrene III. ION EXCHANGE RESIN - Ion exchange resin (ammonium form of polystyrene resin) hastens decalcification - Removes calcium ions from formic acid, thereby increasing solubility from the tissue. - Not recommended for fluids containing mineral acids such as nitric or hydrochloric acids. MARK LOUIE B. DELA PEÑA 10  IV. ELECTRICAL IONIZATION (ELECTROPHORESIS) - Process whereby positively charged Calcium ions are attracted to a negative electrode (cathode) and subsequently removed from the decalcifying solution. - The time required for decalcification is thereby shortened due to the heat and electrolytic reaction produced in this process. - The principle applied is similar to that of chelating agents; with the main difference that this process utilizes electricity and is dependent upon a supply of direct current to remove calcium deposits. Solutions used: 1. 88% Formic acid 2. Concentrated H 3. Distilled Water 3 WAYS TO MEASURE THE EXTENT OF DECALCIFICATION TISSUE SOFTENERS - Unduly hard tissues which are reliable to damage the microtome knives may require tissue softener, aside from decalcification. 1. Perenyi’s fluid – act both as a decalcifying agent and tissue softener 2. 4% phenol (1-3 days) 3. Molliflex (tissues may appear swollen and soapy) 4. 2% Hydrochloric acid 5. 1% HCl in 70% alcohol MARK LOUIE B. DELA PEÑA 11  DEHYDRATION PROBLEM: Concentrated alcohols (95% or absolute) - Process of removing intercellular and extracellular resulting in a relatively unequal impregnation of tissue water from tissue following fixation and prior to wax o REMEDY: 70% or lower concentration of impregnation alcohol, gradually increased to 95% is used. - Many of these dehydrating agent are ALCOHOLS of (IBABALIK MO LANG ULIT SA LOWER increasing strengths to remove aqueous tissue fluids CONCENTRATION) with little disruptions to the tissue. - Removal of water through osmosis. II. ACETONE - A cheap, rapid dehydrating agent utilized for most urgent biopsies - DEHYDRATION TIME: ½ to 2 hours - With water, ethanol, and most organic solvents III. DIOXANE/ DIETHYLENE DIOXIDE - Excellent DEHYDRATING AND CLEARING AGENT miscible to water, melted paraffin, alcohol and xylol/xylene - 70% ethyl alcohol in water, 95% ethyl alcohol to 100% - Expensive and toxic and extremely dangerous ethyl alcohol IV. CELLOSOLVE/ETHYLENE GLYCOL MONOETHYL GENERAL RULE: whatever dehydrating agent is ETHER used, the amount in each stage should not be less - Dehydrates rapidly and not harmful to tissues than 10 times - Toxic by inhalation, skin contact and ingestion (use propylene-based glycol esters) 1. Should dehydrate rapidly without producing V. TRIETHYL PHOSPHATE considerable shrinkage or distortion of tissues. - Tissues can be transferred directly after fixation & 2. Should NOT evaporate very fast. washing 3. Should be able to dehydrate even fatty tissue (Kasi - Produces very little distortion and hardening of tissue. ang alcohol ay may polar part) - Soluble in alcohol, water ether benzene, chloroform, 4. Should NOT harden tissues excessively acetone and xylene. 5. Should NOT remove stain 6. Should NOT be toxic to the body VI. TETRAHYDROFURAN (THF) 7. Should NOT be a fire hazard. - Both DEHYDRATES AND CLEARS tissue – miscible in both water and paraffin. I. ALCOHOL - It does not dissolve out aniline dyes A. ETHANOL/ ETHYL ALCOHOL - Less shrinkage and easier cutting of sections with - For routine dehydration fewer artifacts. - Best dehydrating agent - Toxic if ingested or inhales - A clear, colorless, flammable liquid - An eye and skin irritant and prolonged exposure may o Fast-acting conjunctivitis irritation o Mixes with water and many organic - Vapor cause nausea, dizziness, headache and solvents anesthesia o Penetrates tissue easily o Not poisonous and not very expensive - Should be at least 99.7% pure B. METHANOL/METHYL ALCOHOL - Toxic dehydrating agent - For blood and tissue films and for smear preparation 3. BUTANOL/ BUTYL ALCOHOL - Slow dehydrating agent - Producing less shrinkage and hardening - Utilized for plant and animal microtechnique 4. ISOPROPANOL/ ISOPROPYL ALCOHOL - Should be used id good-grade absolute ethyl alcohol is not easily available MARK LOUIE B. DELA PEÑA 12  CLEARING III. BENZENE AKA: DE-ALCOHOLIZATION CLEARING TIME: 15 – 60 MINUTES - Dehydrating agent (alcohol) is removed from the - Rapid acting - recommended for urgent biopsies (15 tissue - 60 minutes) and routine purposes. - Replaced with a substance that will dissolve the wax - Volatilizes rapidly in paraffin oven (De-paraffin) - Miscible with absolute alcohol - The tissue has translucent appearance – “Clearing - Makes tissue transparent Agent” - Does NOT make tissue hard and brittle - Must be miscible with paraffin and alcohol - Highly flammable - Microscopic tissue preparations TRANSPRENT due - APLASTIC ANEMIA to their high index of refraction. - Not all clearing agents exhibit property of making IV. CHLOROFORM tissues transparent CLEARING TIME: 6 – 24 HOURS - Miscible with Canada balsam and other resins that - Slower action and causes less brittleness are used for mounting sections - XYLENE most - Thicker blocks (even up to 1 cm in thickness) can commonly used be processed. - In frozen sections, GLYCERIN and GUM SYRUP are - Do NOT become transparent. (Translucent only) used when tissue is to be cleared directly from water- - Recommended for routine work (6 - 24 hours) -- no de-alcoholization process involved. - Miscible with absolute alcohol - Tough tissues, nervous tissues, lymph nodes and CHARACTERISTICS OF A GOOD CLEARING AGENT: embryos. Miscible with alcohol to promote rapid removal of the - Large tissue specimens dehydrating agent from the tissue. - Toxic to the liver Should be miscible with, and easily removed by - Does not make the tissues transparent melted paraffin wax and/or mounting medium to - ­SG = Tissue floats facilitate impregnation and mounting of sections. - Not very volatile in paraffin Should NOT produce excessive shrinkage, hardening or damage of tissue V. CEDARWOOD OIL Should NOT dissolve out aniline dyes CLEARING TIME: 2-3 DAYS Should NOT evaporate quickly in a water bath CELLOIDIN: 5-6 DAYS Should make tissue transparent - clear both paraffin and celloidin sections during the embedding process - central nervous system tissues and cytological I. XYLENE/XYLOL studies - smooth muscles and skin. CLEARING TIME: 30 MINS TO 1 HOUR - Least distortion - Most commonly used - Clearing with this agent often improves cutting of the - Used for clearing and mounting procedures sections. - Suitable for most routine histologic processing - Very expensive and it requires 2 changes in clearing - Rapid clearing agent and cheap solution - Miscible with absolute alcohol and paraffin - Quality is not always uniform and good and is - For mounting procedures ---it does NOT dissolve extremely slow celloidin and can be used for celloidin sections - It becomes milky on prolonged storage - Highly inflammable - Should not exceed 3 hours ---makes tissues VI. ANILINE OIL excessively hard and brittle - Recommended for clearing embryos, insects, and - NOT suitable for nervous tissues and lymph nodes very delicate specimens -- due to its ability to clear - MILKY when an incompletely dehydrated tissue. 70% alcohol without excessive tissue shrinkage and hardening. II. TOLUENE CLEARING TIME: 1-2 HOURS VII. CARBON TETRACHLORIDE - Substitute for xylene or benzene - Used in clearing tissues for embedding - Acts fairly rapid and is recommended for routine - Produces considerable tissue hardening purposes - Dangerous to inhale on prolonged exposure due to its - Miscible with both absolute alcohol and paraffin. highly toxic effects. - Does not excessively make tissue brittle. - Properties are very similar to chloroform but it is - NOT carcinogenic cheaper - More expensive and relatively slower than benzene - Emit fumes -> toxic upon prolonged exposure. VIII. CLOVE OIL - It removes aniline dyes and dissolves Celloidin; - Tissues become brittle MARK LOUIE B. DELA PEÑA 13 - Its quality is not guaranteed due to its tendency to be adulterated - Not suitable for routine purposes because it is expensive IX. TETRAHYDROFURAN (THF) - Dehydrates and clears at the same time since it is miscible in both water and paraffin X. METHYL BENZOATE/ MEHYL SALICYLATE - These are slow-acting clearing agents that can be used when double embedding techniques are required. MARK LOUIE B. DELA PEÑA 14  IMPREGNATION - Disadvantage: AKA: INFILTRATION 1. Slow and tedious - The process whereby the clearing agent is completely 2. Serial sections are difficult to prepare removed from the will completely fill all the tissue 3. Very thin sections (EMBED) processing of tissues, resulting in a more rapid diagnosis with less technicality - Autotechnicon/Elliot-Bench Type Tissue Processor: Fixation, Dehydration, Clearing, Infiltration 3. VACUUM EMBEDDING - Impregnation under negative atmospheric pressure - Recommended for urgent biopsies and delicate tissues such as lung, brain, connective tissues, decalcified bones, eyes spleen and CNS. - Reduces 25-75% of impregnation time - Most rapid NOTE: Infiltration in overheated paraffin (above 60C) produces shrinkage and hardening of tissues. Paraffin oven must be maintained at a temperature 2-5°C above the melting point of the wax. II. CELLOIDIN IMPREGNATION - Celloidin - purified form of nitrocellulose soluble in many solvents - suitable for specimens containing large cavities or hollow spaces which tend to collapse, for hard and dense tissues (bones and teeth) and for large tissue sections of whole embryos. - Advantage: Causes much less shrinkage and distortion than paraffin wax MARK LOUIE B. DELA PEÑA 15  EMBEDDING 2. COMPOUND EMBEDDING UNITS AKA: CASTING OR BLOCKING - Made up of a series of Interlocking plates resting on - Precisely positioned in a mold flat metal base, forming several compartments - Embedding medium same as the medium used in Impregnation ORIENTATION - process by which a tissue is arranged in precise positions in the mold during embedding, on the microtome before cutting, and on the slide before staining - 3. PLASTIC EMBEDDING RING AND BASE MOLD 4 WAYS OF EMBEDDING - Consist of special stainless steel and mold fitted with 1. PARAFFIN a plastic embedded ring 2. CELLOIDIN - TISSUE TEK 3. GELATIN o Consist of white plastic cassette mold 4. PARAFFIN SUBSTITUTE with detachable, perforated stainless steel hinge and snap-on lid. I. PARAFFIN WAX - Simplest, most common and best embedding 4. DISPOSABLE EMBEDDING MOLDS medium used for routine tissue processing - PEEL-AWAY - MELTING POINT: 55-60 C - PLASTIC ICE TRAYS o Used for routine work - 56°C - PAPER BOATS o If laboratory temperature is from 20 - 24°C - paraffin wax with a melting point OTHER EMBEDDING METHODS of 54 - 58°C is indicated. PRECAUTIONS: 1. CELLOIDIN/NITROCELLULOSE METHOD - Above 60°C → shrinkage and hardening of tissues - Recommended for embedding hard tissues such as and destroy lymphoid tissues completely. bones and teeth and for large sections of whole - To avoid this, parafin oven must be maintained at a organs like eyes. temperature 2 to 5°C above the melting point of - Low Viscosity Nitrocellulose (L.V.N.) is another form paraffin to be used for impregnation. of celloidin soluble in equal concentration of ether and alcohol II. PARAFFIN SUBSTITUTE 1. PARAPLAST 2. DOUBLE EMBEDDING METHOD MELTING POINT: 56-57 C - The process in which tissues are first infiltrated with - Highly purified paraffin + synthetic plastic polymers celloidin and subsequently embedded in paraffin - Elastic and resilient mass - Used to facilitate cutting of large blocks of dense firm 2. ESTER WAX tissues (brain) and small sections of celloidin blocks MELTING POINT: 46-48 C - Insoluble in water but soluble in 95% ethyl alcohol 3. PLASTIC OR RESIN METHOD - It has superior results for light microscopic 3. WATER SOLUBLE WAXES studies, particularly in hard tissues (undecalcified MELTING POINT: 38-42 C OR 45-56 C bone) and for high resolution light microscopy of - Mostly polyethylene glycols tissue sections (renal and bone marrow biopsies). - CARBOWAX - most commonly used o Epoxy plastics (recommended for o Does NOT require dehydration and electron microscopy) clearing of tissue o Polyester plastics o Acrylic plastics TYPES OF BLOCKING-OUT MOLDS 1. LEUCKHART’S EMBEDDING MOLD - Two 'L'-shaped pieces of brass which, together with a base plate, can be arranged to form a square or rectangular mold for molten paraffin wax during histological embedding. MARK LOUIE B. DELA PEÑA 16  SECTIONING - Not be left on the water bath for a long time to avoid - Process whereby tissues are cut into uniformly thin expansion and distortion of tissue (30 seconds will be slices or "sections" w/ the aid of a machine to facilitate enough) the studies under the microscope. - Section is selected and picked up by onto a clean 3 GENERAL TYPES OF TISSUE SECTION slide in a vertical position = fishing – out 1. PARAFFIN SECTIONS - rocking and rotary microtomes - Sections can also be flattened out by placing them on 2. CELLOIDIN SECTIONS - usually by means a slide which has been flooded w/ 20% alcohol of a sliding microtome - Mounted section is placed in a paraffin oven to dry 3. FROZEN SECTIONS - cut from tissues that whose temperature is maintained 2 - 5°C above the have been fixed and frozen w/ CO2 , or for melting point of the paraffin fresh or fixed tissues frozen w/ the cryostat - Small thermostatically controlled incubators may be used regulated at 37°C and at 45 - 55°C for I. PARAFFIN SECTIONS enzyme digestion, chemical extraction, metallic - Excess wax is cut off from the block to expose the impregnation, enzyme localization techniques tissue surface in preparation for actual cutting – TRIMMING - Mounted sections are placed in metal racks for the drying process usually lasting 5 minutes in the - Sides, top and bottom of the tissue block are trimmed heated oven until perfectly level and all sides are parallel almost to the edge of the tissue; old knife or blade may be used II. CELLOIDIN SECTIONS for this procedure - Usually cut between 10 – 15u thickness - FINE TRIMMING - set thickness adjuster at 15mm or - Do not requires hardening by chilling before cutting advancing the block using the coarse feed mechanism; surface block is trimmed away until the - To avoid dehydration and shrinkage, sections are entire tissue surface has been partly exposed usually cut by the Wet method - both sections and block are kept moist w/ 70% alcohol during cutting - Cutting is continued until complete sections come out of the block and a regular cutting rhythm is maintained - Celloidin sections do not come off in ribbons and have - Thickness of tissue for routine histologic procedures to be collected into 70% alcohol immediately = 4-6 u - Stored in jars w/ 70% alcohol in tightly fitting lids and - Knife is usually tilted at 0 - 15° angulation = finally mounted on to slides after they have stained clearance angle (Average = 5 – 10°) - Biconcave knives needs smaller clearance angles than wedge-shaped knives - Incomplete sections are discarded and complete ribbons are picked up at once with a camel hair brush, a pair of forceps or the fingers - Sections are removed in ribbons of ten to allow easy location of serial sections - Sections are floated out on a water bath set at 45 - 50°C, 5- 10°C lower than the melting point of the wax used for embedding the tissue o Flatten the sections and prepare them for mounting into the slider - Fold and creases may be removed by stretching the sections gently w/ a pair of dissecting needles or forceps - Bubbles may be teased by means of needle MARK LOUIE B. DELA PEÑA 17  STAINING - Uses a wide range of monoclonal or polyclonal, - The process of applying dyes on the sections to see flourescent labeled or enzyme-labeled antibodies. and study the architectural pattern of the tissue and physical characteristics of the cells. METHODS OF STAINING - Different tissues and cells display varying affinities for 1. DIRECT STAINING most dyes and stains --- become more visible, - The process of giving color to the sections by using morphologic changes are more easily identified, and aqueous or alcoholic dye solutions. the presence or absence of disease process can be - Examples: Methylene blue and Eosin established. - Certain parts of cells and tissues that are: 2. INDIRECT STAINING o Acidic - greater affinity for basic dyes - The process whereby the action of the dye is o Basic - take more of the acid dyes. intensified by adding another agent or MORDANT - In general, microscopic examination is facilitated if to make staining reaction possible. two contrasting stains are used. --------------------------------------------------------------------------- E.g. Hematoxylin (Basic) -nuclear detail (Acid) MORDANT Eosin (Acid) – cytoplasmic detail (Basic) - Serves as a link or bridge between the tissue and - IMPREGNATION - is a related procedure that makes the dye. use of heavy metal salts which are selectively - Combines with a dye to form a colored "lake" --- in precipitated on certain cellular and tissue turn combines with the tissue to form --- a "TISSUE- components. MORDANT-DYE COMPLEX" - Utilized for silver staining of the nervous system - may be applied to the tissue before the stain, may be - Used to demonstrate reticulin included as part of the staining technique, or may be - SILVER NITRATE - most commonly used agent for added to the dye solution itself. impregnation and as staining agent. Examples: - Potassium Alum with Hematoxylin in Ehrlich’s THREE MAJOR GROUP OF STAINING hematoxylin 1. HISTOLOGICAL STAINING - Iron in Weigert’s hematoxylin - Process whereby the tissue constituents are demonstrated in section by direct interaction with dye ACCENTUATOR or staining solution --- producing coloration of the - Is not essential to the chemical union of the tissue and active tissue component the dye. - Used to demonstrate the general relationship of - Does not participate in the staining reaction tissues and cells with differentiation of nucleus and accelerates or hastens the speed of the staining cytoplasm. reaction by increasing the staining power and o Micro-anatomic stains selectivity of the dye. o Bacterial stains Examples: o Specific tissue stains - Potassium hydroxide in Loeffler's methylene blue - Phenol in Carbol thionine and carbol fuchsin 2. HISTOCHEMICAL STAINING --------------------------------------------------------------------------- HISTOCHEMISTRY - The process whereby various constituents of tissues 3. PROGRESSIVE STAINING are studied thru chemical reactions that will permit - The process whereby tissue elements are stained in microscopic localization of a specific tissue a definite sequence, and the staining solution is - substance. applied for specific periods of time or until the desired Examples: intensity of coloring of the different tissue elements is o Perl's Prussian Blue - for hemoglobin attained. (dart but, dark purple); demonstrates - The differentiation or distinction of tissue detail relies ferric iron (Fe3+) in tissues solely on the selective affinity of the dye for different o Periodic Acid Schiff's - for carbohydrates cellular elements. - Less favored due to the difficulty of producing ENZYME HISTOCHEMISTRY sufficiently intense progressive staining of cell - the active reagent serves as a substrate upon which structures without staining other parts -- resulting in the enzymes act -- final opacity or coloration diffused color and obscured details. produced from the substrate. Example: - Wright-stain 3. IMMUNOHISTOCHEMICAL STAINING - Is a combination of immunologic and histochemical techniques that allow phenotypic markers to be detected and demonstrated under the microscope. MARK LOUIE B. DELA PEÑA 18 4. RERGRESSIVE STAINING - The tissue is first overstained to obliterate the cellular details, and the excess stain is removed or decolorized from unwanted parts of the tissue, until the desired intensity of color is obtained. - Usually coupled with counterstain DIFFERENTIATION - Decolorization - Selective removal of excess stain from the tissue during regressive staining in order that a specific substance may be stained distinctly from the surrounding tissues. 7. METALLIC IMPREGNATION - Done by washing the section in simple solution, or by - Is a process where specific tissue elements are the use of acids and oxidizing agents. demonstrated by colorless solutions of metallic - ALCOHOL – act as differentiator for both basic and salts which are thereby reduced by the tissue, acidic dyes – simply dissolving out the excess dye. producing an opaque, usually black deposit on the - A mordant can act as a differentiating agent surface of the tissue or bacteria. - Usually controlled by following the exact times - A metallic impregnating agent is not absorbed by the specified for staining, or by examination under the tissue, but is held physically on the surface as a microscope. precipitate or as a reduction product. - Gold (gold chloride) and silver (silver nitrate) 5. METACHROMATIC STAINING o Allowed to precipitate -> black deposit - Entails the use of specific dyes which differentiate in the surface of the tissue -> seen in particular substances by staining them with a color microscope that is different from that of the stain itself. - All reagents to be used should be chemically pure and (Iba yung color ng stain sa tissue compare sa stain glassware should be clean. mismo) - A formalin-laden atmosphere which is apt to - Particularly employed for staining cartilage, precipitate such pigment deposition should be connective tissues, epithelial mucins, mast cell avoided. granules, and amyloid. - Ammoniacal silver solutions are potentially - Basic dyes belonging to the thizine and explosive --- care should be taken to prepare all triphenylmethane groups, such as: solutions in clean containers just before use, and o Methyl violet or Crystal violet silvered glassware should be avoided. o Cresyl blue - Used in demonstration of tissues in CNS and for o Safranin demonstration of reticulin. o Bismarck brown (Pap’s) (dye=brown, - Solution should never be exposed to sunlight if stained=pink) explosion is to be avoided. o Basic fuchsin - All unused reagents should be immediately o Methylene blue (dye=blue, stained= inactivated by sodium chloride or dilute green) hydrochloric acid solution and discarded. o Thionine - The use of metallic instruments should be avoided o Toluidine blue when handling sections for metallic impregnation. o Azure A, B, C - All metachromatic dyes are cations or basic 8. VITAL STAINING whose peculiar staining property depends upon their - Is the selective staining of living cell constituents, tendency to polymerize. demonstrating cytoplasmic structures by - Major group of metachromatic tissues consists of phagocytosis of the dye particle acidic polysaccharides that occur in ground - Nucleus of the living cell is resistant to vital stains. substance and in connective tissue mucin that bind basic dye. INTRAVITAL STAINING - WATER is necessary for most metachromatic - done by injecting the dye into any part of the animal staining techniques. body - producing specific coloration of certain cells. - Common dyes used are lithium, carmine, and India 6. COUNTERSTAINING ink. - Is the application of a different color or stain to provide contrast and background to the staining of the SUPRAVITAL STAINING structural components to be demonstrated. - is used to stain living cells immediately after removal from the living body. MARK LOUIE B. DELA PEÑA 19 - Thin slices of tissues are placed in small staining STAINING OF PARAFFIN SECTION dishes and enough staining solution is added to cover - Drying of the specimen should be complete. to avoid the tissue. detachment of the section from the slide during the - Commonly dyes used are: process of staining o NEUTRAL RED - best vital dye o JANUS GREEN - recommended for - Paraffin wax is poorly permeable to most staining mitochondria solutions - should be removed from the section prior o TRYPAN BLUE - 1 gram of dye is to staining dissolved in 100 mL of sterile distilled water to be used immediately - Done by immersing the paraffin section in a solvent § Dangerous to allow suspension 2x to stand for more than 1 hour -- likely to become toxic to the cell. - If alcoholic stain is to be used, there is no more need o NILE BLUE to replace the alcohol with water at 1-2 minutes o THIONINE duration each, for sections up to 10 micra thick. o TOLUIDINE BLUE - SECTIONS TO WATER - Paraffin wax immersed in CLASSIFICATION OF DYES XYLENE --- Remove xylene with ABSOLUTE 1. NATURAL DYES ALCOHOL, followed by descending grades of alcohol - Are those obtained from plants and animals, -- then finally replaced with WATER before actual previously utilized for dyeing of wool and cotton. staining of sections is performed. - If alcoholic stain is to be used, there is no more need to replace the alcohol with water. - SECTIONS TO ALCOHOL- deparaffinization with xylene. the section is subjected to grades of alcohol then staining - If tissue has been fixed in mercuric chloride solution, the section shall be taken to water. 2. SYNTHETIC DYES - Sometimes known as “coal tar dyes” since they - After staining, the section is again dehydrated with were originally manufactured from substances that increasing grades of alcohol and cleared with two have been taken from Coal Tar. changes of xylene to prepare the section for - Derived from the hydrocarbon benzene and are mounting. collectively known as aniline dyes. - Avoid prolong immersion in alcohol -- many stains are usually removed. A DYE SHOULD CONSIST OF 2 SUBSTANCES: MATERIALS NEEDED FOR STAINING: 1. CHROMOPHORES 1. COPLIN JAR - slotted jar holding from 5 to 9 - Greek. “color-bearers” slides. - Substances capable of producing visible colors. 2. SLOTTED STAINING DISHES - holding from 5 to 19 slides, over which different solutions 2. AUXOCHROME are poured. - Greek. “color increaser” 3. METAL or GLASS STAINING RACKS or - For a chromogen to be a dye, it must be composed CARRIERS - holding from 10 - 30 slides of an acid and a base, and therefore have salt- upright. forming properties, ultimately retaining its color. - The coloring property is attributed to the chromophore and the dyeing property to the salt forming auxochrome. MARK LOUIE B. DELA PEÑA 20  H&E STAINING TECHNIQUE 3. Mallory's Phloxine Methylene Blue Stain - Most common method utilized for microanatomical - Eosin-Methylene Blue (EMB) studies of tissues, using the regressive staining which method consists of overstating the nuclei, removal of - Produces a sharp nuclear stain and superfluous and excessive color of the tissue reveals with marked differentiation constituent by acid differentiation. the various structures in the tissues.. - Four staining methods are commonly employed for should be fixed in Zenker's fluid. frozen sections: o Hematoxylin-eosin method COLLOIDIONIZATION OF SECTIONS o Thionine method - Coating the slide with dilute celloidin solutions -- more o Polychrome Methylene Blue method firmly attached the sections in the slides. o Alcoholic Pinacyanol method - Recommended for sections that will be subjected to - Reagents for rapid H & E stain are generally arranged strong alkaline or acid solutions in sequence using a series of coplin jars -- takes only - For tissues that contain glycogen for demonstration 5-10 minutes and produces well-differentiated - The celloidin will be removed in the final dehydration sections that are semi-permanent and can be stored. with absolute alcohol prior to clearing and mounting. EYE DROPPER METHOD STAINING OF CELLOIDIN SECTIONS - Rapid metachromatic staining of frozen section - CELLULOSE NITRATE - soluble in absolute alcohol - May be stained by picking up sections on albuminized ---should be avoided during dehydration and clearing slides and drying them quickly or by simple direct of stained sections. staining on a wet slide with an eye dropper. PRECAUTIONS IN STAINING: ROUTINE H&E STAINING IN PARAFFIN - Stains on skin should be avoided - because stains are EMBEDDED SECTION (REGRESSIVE STAINING) health hazards per se - FIXATION - most fixatives can be used EXCEPT o REMEDY: Topical application of 0.5% OSMIC ACID solutions which inhibit hematoxylin. acid alcohol, followed by rinsing with tap water. NOTE: Read the procedure and results in the book. - Failure of sections to remain on the slide during staining - due to dirty or oily slide. - Tissues fixed with MERCURIC CHLORIDE.-- staining - Stains that have already been deteriorated should be time with hematoxylin should be increased slightly replaced. while eosin staining should be reduced. - Failure of staining may be due to paraffin, fixative, or - Mercury should be removed using 0.5% solution of decalcifying solution that has not been thoroughly iodine in 80 to 95% alcohol and rinsed in water. washed out and removed. - Removed iodine by placing the slide in 3% SODIUM THIOSULFATE for 1 to 5 minutes and wash well with running water for 3 to 5 minutes. - Staining may be prolonged for: o Chromium and osmium fixed tissues o Tissues subjected to long acid decalcification o After prolonged storage in acid formalin or 70% alcohol. STAINING METHODS 1. Heidenhain's Iron Hematoxylin Method - Cells nuclei, cytoplasmic inclusions and muscle striations stain black. 2. Celestine Blue-Haemalum Sequence Staning - CELESTINE BLUE - an oxazine dye used as an alternative to iron hematoxylin nuclear stain, producing a strong and precise nuclear stain that is resistant to decolorization by succeeding acid stains and solutions. - Forms a strong staining lake with iron alum - acting as a mordant. MARK LOUIE B. DELA PEÑA 21  CYTOLOGY - Study of the microscopic appearance of cells for diagnostic purposes - Can be used as a screening tool for healthy individuals at risk of a particular disease Exfoliative vs FNA cytology - Exfoliative - cells desquamated from epithelial FIXATION OF FLUID SEPCIMEN surfaces 50% - Alcohol is for all types of effusions o Spontaneous shedding 50% - Peritoneal and pleural fluids o Physically removed from epithelial and 70% - Sputum specimen mucous membranes 95% - Urine, gastric and bronchial aspirates. o Vagina, buccal mucosa, from body fluids What fixative is composed of 50% ethanol and 2 % - FNA - fine needle aspiration of palpable and non- carbowax? Saccomano’s Fixative palpable masses APPLICATION OF EXFOLIATIVE CYTOLOGY - Detection of malignant cells in body fluids (for staging cancers) - Detection of precancerous cervical lesions (cervicovaginal or Pap smear). - Assessment of female hormonal status in case of sterility and endocrine disorders o Based on smears taken from vaginal walls o CHMI - cytohormonal maturation index - For determination of genetic sex (Karyotyping) o Demonstration of Barr bodies from buccal or vaginal mucosa - Detection of infectious agents SPECIMEN - Vaginal smears - Endometrial and endocervical smears - Prostatic and breast secretions - Gastric and Bronchial secretions - Pleural and peritoneal fluids GYNECOLOGICAL CYTOLOGY - Sputum - Smears of Urine Sediments - Cerebrospinal Fluid CYTOLOGY: FIXATION - Specimen should be fixed immediately for optimal cell preservation o 95% ethanol o Equal parts of 95% ethanol and ether o 100% methanol - If smears from effusions (liquid prom FNA) cannot be made immediately, place in: o 50% alcohol o Saccomano preservative (50% alcohol and carbowax) - If fluid specimen is enough for cytocentrifugation: o Centrifuge at 2000 rpm for 2 mins., decant supernatant and smear the o Prepare at least 2 cytocentrifuged smears and a cell block (similar to tissue processing) MARK LOUIE B. DELA PEÑA 22 Liquid-based preparations PAPANICOLAU METHOD - alternative to conventional cervicovaginal smears 1. Fix in 95% EtOH - Improved preparation that minimizes cell overlap for 2. Primary stain with hematoxylin (with mordant na) better identification of abnormal cells 3. Differentiate with acid alcohol then wash with water 4. Blue in ammonia water then wash with water ThinPrep technique: membrane filtration 5. Counterstain with OG-6 SurePath technique: density gradient centrifugation 6. Wash with 2 changes of 95% EtOH 7. Counterstain with EA-50 and gravity sedimentation 8. Dehydrate (ascending grades of alcohol) 9. Clear with xylene 10. Mount with resinous media BETHESDA SYSTEM CATEGORIES FOR SPECIMEN ADEQUACY Satisfactory for evaluation - A satisfactory squamous component must be present. - Note the presence/absence of endocervical/transformation zone component. - Obscuring elements (inflammation, blood, drying artifact, other) may be mentioned if 50% to 75% of epithelial cells are obscured Unsatisfactory for evaluation