Histopathology & MedTech Laws PDF

lOMoARcPSD|129 359 57 MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 2 HISTOPATHOLOGY & MEDTECH LAWS Lecturer: Prof. Edmund Francisco, RMT, MLS (ASCPi)...

lOMoARcPSD|129 359 57 MEDICAL TECHNOLOGY ASSESSMENT PROGRAM 2 HISTOPATHOLOGY & MEDTECH LAWS Lecturer: Prof. Edmund Francisco, RMT, MLS (ASCPi) By: Xiao - The Conqueror of Demons, The Vigilant Yaksha, & Alatus, the Golden-Winged King HISTOPATHOLOGIC TECHNIQUES Squash - Process where small pieces of tissue not more than - It deals with the preparation for microscopic examination preparation/ 1mm in diameter are placed in a microscopic slide and - It is accomplished by submitting the total or a selected part of the tissue Crushing forcibly compressed with another slide or with presented for examination to a series of processes: coverglass. “FDD-CIET-SSML” - Vital dyes are placed at the slide and coverslip Fixation - Preservation junction and absorbed through capillary action. Decalcification - Calcium or lime salts are removed from the tissues Frozen section - Normally utilized when a rapid diagnosis of the tissue - Optional process: for calcified tissues only such as in question is required, and especially recommended bones and teeth when lipids and nervous tissue elements are to be demonstrated. Dehydration - Desiccation - Removing intracellular and extracellular fluid/water Smearing - Useful in cytological examinations, particularly for Clearing cancer diagnosis. - De-alcoholization - Removing alcohol used in dehydration Infiltration - Impregnation Smearing Material Process/Important Notes Embedding - Casting or Blocking Technique Trimming - Removing excess wax from the tissue block a. Streaking Applicator stick - Rapid and gentle direct or zigzag - Optional process: not all tissue blocks have excess wax or platinum application to obtain uniform Sectioning - Section cutting loop. distribution. - Cutting tissue blocks into uniformly thin slices - Too thick or too thin smears are Staining - Dyeing unsuitable for examination. Mounting b. Spreading Applicator stick - Little more tedious than streaking, Labelling - Proper labelling technique to tease the but has advantage in maintaining - Examination may be done on: mucous strands the intercellular relationship. 1. Fresh Tissues to make a - Especially recommended for O usually examined when there is an immediate need for evaluation moderately fresh sputum, bronchial aspirates 2. Preserved Tissues thick film. and thick mucoid secretions. O routinely done in the histopathology section. FRESH TISSUE EXAMINATION c. Pull-apart Slides facing - The material disperses evenly technique each other as a over the surface of 2 slides. Advantage: Examined in the living state, thereby allowing protoplasmic activities drop of - A single uninterrupted motion of such as: secretion is pulling apart is applied. a. Motion (Cellular Movement) b. Mitosis (Cellular Reproduction) sandwiched in- - It is useful for serous fluids, c. Phagocytosis (Cell-eating activity) between. concentrated sputum, enzymatic d. Pinocytosis (Cell-drinking activity) GIT lavage and blood smears d. Touch One slide. - Special method where slide Disadvantage: Its use has been limited, however, because of the fact that preparation surface is in contact and pressed tissues examined in the fresh state are not permanent and therefore, are liable to develop the changes that have usually been observed after death. or on the site. Impression - Cells may be examined without POST MORTEM CHANGES smear destroying their actual Autolysis - the destruction of the tissues (breaking down of the intercellular relationship and without separating them from their protein of the cell) by enzymes which are produced by the tissues and eventually liquefy it. normal surroundings. - It is the first to occur among all post-mortem changes STEPS IN PROCESSING PRESERVED TISSUES Putrefaction or - the decomposition of organic matter under the influence Decompositionof microorganisms accompanied by the development of FIXATION disagreeable odors. - AKA: Preservation Degeneration - a retrogressive pathologic process in cells in which the cytoplasm undergoes deterioration while the nucleus is - The process by which the constituents of the cells, and therefore of the preserved. tissues are fixed in a physical, and partly also in a chemical state so that they will withstand subsequent treatment with various reagents with minimum loss or significant distortion or decomposition. METHODS OF FRESH TISSUE EXAMINATION - The first and most critical (important) step in histotechnology Teasing or Process wherein selected tissue specimen is - Why is it the most critical? Because it is the first procedure and is able to - affect the subsequent procedures Dissociation immersed in a watch glass containing isotonic salt - Primary aim of fixation solution (NSS or Ringer’s lactate), carefully dissected or separated and examined under the microscope,  Preserve the morphologic and chemical integrity of the cell in as life-  like as possible either unstained by Phase Contrast microscope or - Secondary goal of fixation Bright-field microscope, or stained with differential  To harden and protect the tissue from trauma of further handling dyes.  For easy cutting during gross examination  lOMoARcPSD|129 359 57 - The most important reaction in fixation: Stabilization of proteins Practical Considerations of Fixation   This can be achieved by: Forming cross-links between proteins 1. Speed - Leaving a tissue specimen in air cause it to: dry-out (distortion of - The specimen should be placed in fixative as soon as it is removed from the morphologic appearance) body. - Leaving the tissue in water (hypotonic solution) will cause the cell to: swell - This is done to prevent autolysis and putrefaction/decomposition.  If the environment is hypotonic, more water is outside the cell and the - If bacteriologic and toxicologic studies should be encouraged, therefore, solute concentration inside the cell is high, the water would enter the fixation is not required.  cell and swells, bursts, or lyses (cytolysis) O Fixation can kill microorganisms and prevent growth in culture O - Leaving the tissue in strong salt (hypertonic solution) will cause the cell to: Fixatives can neutralize drugs and toxins shrink  More solutes are outside the cell (strong salt solution) and water is 2. Penetration less, so water will move outside the cell and the cell shrinks - Formalin diffuses into the tissue at the rate of approximately 1mm/hr. - Time of fixation varies with different types of tissue Two Basic Mechanisms in Fixation Principle/ Notable Characteristics Example 3. Volume/Amount of Fixative 1. Additive- The chemical constituent of the Formaldehyde - Traditionally, the amount of fixative used has been 10-25x the volume of the fixative is taken in and becomes Osmium tissue to be fixed. part of the tissue through cross-link tetroxide/Osmic - Recently, 20x is known as the maximum effective concentration for fixation formation or molecular complexes. acid fixative - Except when osmium tetroxide (5-10x) is used. Mercuric chloride O Because Osmium tetroxide is very expensive 2. Non- - The fixing agent is not incorporated Alcoholic additive into the tissue but alters the tissue 4. Duration composition and stabilizes it - Some tissues take longer to fix than others, depending on their structures. through water removal. - Fibrous organs such as uterus and intestinal tract take longer. - New cross-links are formed - Fixation can be cut down by using Heat, Vaccum, Agitation or Microwave. preventing autolysis and bacterial decomposition. IDEAL SIZE OF TISSUE TO BE FIXED: Main Factors Involved in Fixation - not more than 2cm2 in diameter 1. Hydrogen Ion - pH: 6.0-8.0 - not more than 4mm thick Concentration - Average: 7.0 (neutral pH) - Ideal number of hours for fixation: 4-6 hours (New books: 6-18 hours) 2. Temperature - Traditional/usual: Room temperature (18-30oC) - Tissue processors: Autotechnicon (40-42 oC) TYPES OF FIXATIVES - Electron Microscopy and Histochemistry: 0-4 oC O Mast cells for EM: Room temperature - Nucleic acids fixation: Rapid at higher temperature I. According to Composition - Formalin heated to 60°C → rapid fixation of very urgent biopsy specimens A. SIMPLE FIXATIVES (made up of only 1 component substance) - Formalin heated to 100°C → to fix tissues with tuberculosis 1. Aldehydes (Formaldehyde, Glutaraldehyde) 3. Thickness of- Small 2. Metallic Fixatives (Mercuric chloride, Chromic acid, Lead) sections O Electron Microscopy: 1-2 mm2 3. Picric acid O Light Microscopy: 2 cm2 4. Acetic acid - Thin 5. Acetone O Light Microscopy: ≤0.4 cm or as prescribed by 6. Alcohol tissue processor manufacturer 7. Osmium tetroxide O Large solid tissue, such as uterus, should be 8. Heat opened or sliced thinly O Brain is usually suspended whole in 10% Neutral B. COMPOUND FIXATIVES (Made of up of 2 or more fixatives to obtain Buffered Formalin for 2-3 weeks. optimal results) 4. Osmolality- Best results are obtained using slightly hypertonic solutions (400-450 mOsm) II. According to Action O Hypertonic solutions = Shrinkage O Isotonic (340 mOsm)/Hypotonic solutions = A. Microanatomical Fixatives: Permit the general microscopy study of swelling and poor fixation tissue structures - Added to Osmium tetroxide fixatives for EM: Sucrose 5. Concentration- Formaldehyde: 10% 1. 10% Formol saline - Glutaraldehyde: 3% 2. 10% NBF - Glutaraldehyde for Immunoelectron microscopy: 3. Heidenhain’s susa 0.25% 4. Formol sublimate (corrosive) 6. Duration of- Most formalin fixatives: 24 hours (washed out) 5. Zenker’s solution fixation - Buffered formalin: 2-6 hours up to 1 week 6. Zenker-formol (Helly’s) - EM: 3 hours (New books: 0-4 hours; Average: 2 hours) 7. Bouin’s solution - Prolonged fixation may cause shrinkage and 8. Brasil’s solution hardening of tissue  lOMoARcPSD|129 359 57 B. Cytological Fixatives: Preserve specific parts and particular Removal of formalin pigments: microscopic elements of the cell itself 1. Lillies Method – involves placing formaldehyde fixed specimens in 1. Nuclear Fixatives acetone, 28% ammonia water and hydrogen peroxide. It uses 70% alcohol - Preserve nuclear structures (ex. chromosomes). as a rinsing agent. - They usually contain Glacial acetic acid as their primary 2. Kardasewitch’s Method – a method of formaldehyde clearance involving component due to its affinity for nuclear chromatin. 70% ethanol and 28% ammonia water. - The pH is 4.6 O Simple to prepare O Buffered to pH 7 by acetate a. Helly’s 3. 10% Neutral - Best general tissue fixative b. Orth’s Buffered - Best Fixative for frozen sections c. Regaud’s Formalin/Phosphate - Recommended for surgical, post mortem d. Flemming’s fluid w/o HAc buffered Formalin and research specimens. e. Formalin w/ post-chroming (pH 7) - Prevents precipitation of acid formalin pigments. 3. Histochemical Fixatives Fixation time: - Best fixative for iron-containing pigments - Preserve the chemical constituents of cells and tissues 4-24 hours and elastic fibers. 4. Formol corrosive/ - Recommended for routine post-mortem a. 10% Formol saline Formol-sublimate/ tissues. b. Absolute Ethyl alcohol c. Newcomer’s fluid (Both Nuclear Formol-Mercuric - It is excellent for silver reticulum methods. Chloride - It fixes lipids, especially neutral fats and and Histochemical fixative) phospholipids. d. Acetone Fixation time: - Forms black deposits. 3-24 hours - No frozen sections are made ROUTINE FIXATIVES Alcoholic Formalin/ - It contains ethyl alcohol saturated with picric Gendre’s fixative acid. I. ALDEHYDE FIXATIVES - It enhances immunoperoxidase studies for EM if post-fixed with phenol formalin for 6 A. FORMALDEHYDE: FORMALIN hours or more. - It fixes and dehydrates at the same time and - Commercial formaldehyde/Formalin fixes sputum since it coagulates mucus - A saturated solution of formaldehyde gas in water, approximately 35-40% - Cajol’s formol ammonium bromide - good gas by weight (Gregorios: 37-40% weight in volume). SPECIAL FORMALIN fixative for nervous tissue (astrocytes). - A mixture of 10 mL formalin with 90 mL of water/saline is known as 10% FIXATIVES: - Fixatives for acid mucopolysaccharides. formalin (most widely used fixative of all) - Baker’s formol calcium - used for the - It is usually buffered to pH 7 with a phosphate buffer. preservation of lipids since most formalin - Commercial formalin often becomes turbid, especially if stored in a very fixatives are inert to lipids. cold place, because of the formation of paraformaldehyde (whitish precipitate) → may be removed by filtration, but almost all commercial B. GLUTARALDEHYDE formaldehyde contains 10% methanol, which tends to inhibit the formation of paraformaldehyde. - It is made up of two fomaldehyde residues, linked by three carbon chains. - It is utilized for Light microscopy. - Formalin is the best fixative for the Nervous tissue - Buffered Formaldehyde with secondary fixation in osmium tetroxide is satisfactory for Electron Microscopy. Disadvantages: O 2.5% - for small tissue fragments and needle biopsies for 2-4 hours at room temperature. - In tissues containing much blood (spleen), unbuffered formalin leads to the O 4% - large tissue fragments less than 4 mm thick for 6-8 hours up to 24 formation of dark brown artifact pigment granules → these granules consist hours. of acid formaldehyde hematin and are doubly refractile. - Fixation time: ½-2 hours - Prolonged fixation may cause bleaching, fat dispersal and dissolution or - Recommended for histochemistry and electron microscopy. loss of glycogen, biurates of sodium crystal and uric acid. - The most effective aldehyde for protein cross-linking Disadvantages: Reagent-grade formaldehyde contains 10% methanol as a preservative to retard decomposition to formic acid. Prevents the formation of Paraformaldehyde. 1. It is more expensive. - Formalin pigments – are also formed due to overfixation. 2. It reduces PAS positivity of reactive mucin. * How can this be prevented? - Fixation Time: 24 hours → Immersion of Glutaraldehyde fixed tissues in a mixture of Concentrated glacial acetic acid and aniline oil.  lOMoARcPSD|129 359 57 II. METALLIC FIXATIVES - Counterbalance effect O Su (sublimat) = metal (mercury) → cell A. MERCURIC CHLORIDE shrinkage - The most common metallic fixative O Sa (saure) = acid (trichloroacetic acid) → - Frequently used in aqueous saturated solutions of 5-7%. cell swelling - Permits brilliant metachromatic staining 4. Schaudinn’s - A solution of mercuric chloride, sodium - The routine fixative of choice for preservation of cell detail in tissue Solution/ chloride, alcohol, and glacial acetic acid photography Sublimated - Used on wet smears for cytologic examinations. - Tissues fixed with mercury chloride containing compounds produce alcohol black precipitates except Heidenhain’s SuSa 5. B-5 Fixative - Composed of mercuric chloride and sodium - Satisfactory for immunoperoxidase techniques but ultrastructural acetate. preservation is poor. Fixation time: - Commonly used for bone marrow biopsies. Disadvantages: 1 ½ to 2 hours - Just prior to use, add 1 mL of 40% formaldehyde 1. It causes marked shrinkage of (Rapid fixation) to 10 mL of B5 cells. O Metal = Mercury 6. Carnoy- O How can one counteract this? Lebrun  By addition of an acidic solution such as glacial acetic acid 7. Ohlmacher  Through secondary fixation 2. Has black granular deposits and is extremely corrosive to metals. B. CHROMATE Removal of black deposits caused by Mercuric Chloride: - A class of fixatives which are strong oxidizing agents used for precipitating proteins and preserving carbohydrates. a. 0.5% iodine in 70% ethanol (alcoholic iodine) for 5-10 minutes + water + - Recommended for Chromaffin tissues, Adrenal medulla, Mitochondria. 5% sodium thiosulfate (decolorized-5mins)-wash in running water. “CROP” O Alcoholic iodine is used to remove excess mercury CHROMATE FIXATIVES O 5% sodium thiosulfate is used to remove excess iodine 1. Chromic acid - Used in 1-2%, used as a constituent of a b. Addition of saturated iodine solution in 96% alcohol (alcoholic iodine) and compound fixative. iodine decolorized with absolute alcohol in the subsequent stages of - It precipitates all proteins and adequately dehydration. preserves carbohdyrates. c. Dezenkerization - Formaldehyde must be added to chrome- - Bring slides to water. containing tissues before use to prevent - Immerse in Lugol’s Iodine (5 minutes) counteracting effects andconsequent - Wash in running water (5 minutes) decomposition of solution upon prolonged - Immerse in 5% Na thiosulfate (5 minutes) standing. - Wash in running water (5 minutes) 2. Regaud’s - Recommended for demonstration of Chromatin, - Proceed with required water soluble stain. Fluid/ Mitochondria, Mitotic figures, Golgi bodies, Moeller’s fluid RBC’s and colloid-containing tissues Mercuric Chloride Fixatives 3. Orth’s Fluid - Recommended for study of early degenerative 1. Zenker’s fluid - Mercuric chloride + glacial acetic acid just before processes and tissue necrosis its use. Fixation time: - Demonstrates Rickettsia and other bacteria Fixation time: O To prevent turbidity and formation of dark 36-72 hours 12-24 hours precipitates 4. Potassium - Preserves mitochondria (pH 4.5-5.2) - Good general fixative for all kinds of tissue dichromate - Fixes lipids - Recommended for fixing small pieces of liver, - Used in 3% aqueous solution spleen, connective tissue fibers and nuclei - It fixes but does not precipitate cytoplasmic - May act as mordant to make certain special structures. staining possible - Contains glacial acetic acid which makes the C. LEAD FIXATIVES solution unstable 1. Lillie’s alcoholic lead nitrate formalin 2. Zenker- - Mercuric chloride + 40% formaldehyde just 2. Lead subacetate formol/ Helly’s before its use. Advantages: Solution - It is an excellent microanatomic fixative for 1. It is recommended for acid mucopolysaccharides pituitary gland, bone marrow and blood- 2. It fixes connective tissue mucin Fixation time: containing organs such as the liver and spleen. Disadvantage: 12-24 hours - Better nuclear fixations and staining than Zenker’s - It takes up CO2 to form insoluble lead carbonate especially on prolonged - It presevres cytoplasmic granules better than standing. “Bloody Helly” Zenker’s - This may be removed by filtration or by adding acetic acid drop by drop to - Disadvantage: Brown pigments are produced if lower the pH and dissolve the residue. tissues are allowed to stay in the fixative for more III. Picric Acid Fixatives than 24 hours due to RBC lysis. 1. Excellent for glycogen demonstration - Remedy: immerse the tissue in alcoholic picric 2. Normally used in strong saturated aqueous solution acid or sodium hydroxide 3. It dyes the tissues yellow, thus preventing the tissue fragments from being 3. Heidenhain’s - Recommended for tumor biopsies especially of the overlooked. On the other hand, this hinders proper staining. Susa solution skin - Can be used for fragmentary biopsies - It is an excellent Cytologic fixative - The chemical name for the general picric acid fixatives: 2,4, 6 - trinitrophenol Fixation time: - Produces minimum shrinkage and hardening of - Picric acid is highly explosive when dry, and therefore must be kept moist 3-12 hours tissues due to the counter-balance of the swelling with distilled water or saturated alcohol at 0.5 to 1% concentration during effects of acid (trichloroacetic acid) and the storage. shrinkage effect of a metal (mercury).  lOMoARcPSD|129 359 57 - What must be done to remove this yellow color? VI. Osmium Tetroxide/Osmic Acid O Add 50-70% ethanol - It is a pale yellow powder which dissolves in water (up to 6% at 20°C) to O Acid dye/lithium carbonate form strong oxidizing solution. O 70% ethanol → 5% sodium thiosulfate → Wash in running tap water - Adequately fixes materials for ultrathin sectioning in electron microscopy, “PBB” since it rapidly fixes small pieces of tissues and aids in their staining A. Bouin’s solution - Fixation of embryos and pituitary biopsies - It is an excellent fixative for preserving soft and Disadvantage: Fixation time: delicate structures (endometrial curettings) - Inhibits Hematoxylin and makes counterstaining difficult 6-24 hours - Yellow stain is useful in fragmentary biopsies - Very expensive (less amount is required for fixation; 5-10x volume of - Preferred fixative for Masson’s trichrome fixative used) staining for collagen, elastic or connective tissue - Formation of artifact pigments/black precipitate B. Brasil’s Alcoholic - Best routine fixative for glycogen - Prolonged exposure to acid vapors causes eye irritation(conjunctivitis) or Picroformol Fixative - It is better and less messy than Bouin’s solution black osmic oxide deposition in the cornea (blindness) IV. Glacial Acetic Acid “OFF” 1. Flemming’s Solution - The most commonly used Chrome-Osmium 1. It solidifies at 17ºC (New book: 16oC) acetic acid, recommended for nuclear 2. Fixes and precipitates nucleoproteins Fixation time: preparation of such sections. 3. It precipitates chromosomes and chromatin materials. 24-48 hours - Excellent for nuclear structures such as 4. Causes tissues to swell specially those containing collagen. chromosomes - Permanently fixes fats/lipids - Disadvantage: It is contraindicated for cytoplasmic fixation because it 2. Flemming’s Solution - Recommended for cytoplasmic structures destroys mitochondria and golgi elements. without Acetic Acid such as mitochondria - The removal of acetic acid from the formula V. Alcohol Fixatives Fixation time: serves to improve the cytoplasmic detail of the - Denatures and precipitates proteins 24-48 hours cell. - 70-100% concentrations are used; Less concentrations results to red blood cell lysis/hemolysis due to hypotonicity VII. Trichloroacetic acid (TCA) Advantages: 1. May be used both as a fixative and dehydrating agent 1. Sometimes incorporated into compound fixatives (Ex. Heidenhain’s SuSa) 2. Excellent for glycogen preservation 2. It precipitates proteins Disadvantages: 3. Causes marked swelling effect on tissues 1. Causes RBC hemolysis and dissolves fats and lipids 4. Can be used as a weak decalcifying agent. 2. Tissue shrinks on long usage 5. Has softening effect on dense fibrous tissue 3. Polarization (major disadvantage); glycogenic granules moves to towards 6. Suitable only for small pieces of tissues or bone because of its poor the ends or poles of cells penetration General Rule: - Alcohol-containing fixatives are contraindicated when lipids are to be VIII. Acetone studied. 1. Methyl Alcohol - Used in Wright’s stain as a diluent 1. It is used at ice cold temperature from -50C to 40C (New book: 0-4oC) 100% (CH3OH/ - Excellent for fixing wet and dry smears, blood 2. Recommended for the study of water diffusible enzymes especially lipases Methanol) smears and bone marrow tissues and phosphatases 2. Isopropyl Alcohol - It is used for fixing touch preparations 3. Used in fixing brain tissues for diagnosis of rabies (Negri bodies). 95% (Isopropanol) (impression smears), although some are air 4. Used in freeze substitution techniques as a solvent for certain metallic salts dried and not fixed, for certain procedures 5. Evaporates rapidly such as Wright-Giemsa staining. 3. Ethyl Alcohol - Is used in 70-100% concentrations. IX. Heat Fixation (C2H5OH/ Ethanol) - Lower concentrations (70-80%) will cause - This procedure involves thermal coagulation of tissue proteins for rapid RBC lysis and inadequate WBC preservation diagnosis, usually employed for frozen tissue sections and preparations of Fixation time: - It fixes blood, tissue films and smears bacteriologic smears. 18-24 hours - Used for histochemistry especially for enzyme studies MIXTURE OF FIXATIVES - Can be both used as a simple and compound Useful for Electron Microscopy fixative. O Karnovsky’s paraformaldehyde-glutaraldehyde solution 4. Carnoy’s Fluid - The most rapid tissue fixative O Acrolein – mixture with glutaraldehyde or formaldehyde - Recommended for fixing chromosome, lymph O Other fixatives for Electron Microscopy: Fixation time: 1-3 hrs. glands and urgent biopsies Glutaraldehye - It is used to fix brain tissue for rabies Osmium tetroxide diagnosis. - It preserves Nissl granules and very suitable Secondary Fixation for Curettings (small tissue fragments). - The process of placing an already fixed tissue in a second fixative in order 5. Alcoholic - Better preserves glycogen to: Formalin/ - Capable of coagulating mucus and is used as a. To facilitate and improve the demonstration of particular substances Gendre’s a fixative for sputum cytology b. To make special staining techniques possible (secondary fixative 6. Newcomer’s Fluid - For fixing mucopolysaccharides and nuclear acting as mordant) proteins c. To ensure further and complete hardening and preservation of tissues Fixation time: - It is both a nuclear and histochemical fixative. 12-18 hours @ 3ºC  lOMoARcPSD|129 359 57 Post-chromatization DECALCIFICATION AKA: Post-chroming or Post-mordanting - Procedure whereby calcium or lime salts are removed from tissues (most - A secondary fixation whereby a primary fixed tissue is placed in aqueous especially bones and teeth) solution of 2.5 – 3% potassium dichromate for 24 hours, to act as mordant - It is a form of ionization for better staining effects and to aid in cytologic preservation of tissues. - Done after fixation and before impregnation (before dehydration) - Calcified tissues are harder to cut Washing Out - Tuberculous organs, arteriosclerotic vessels, bones, and teeth - The process of removing excess fixative from the tissue after fixation in Recommended temperature: Room temperature (18-30oC) order to improve staining and remove artifacts from the tissue. Ideal time required for decalcifying tissue: 24-48 hours Volume: 20x the volume of the tissue Solutions that may be used: Factors Influencing Rate of Decalcification 1. Tap water – used to remove: a. Structure a. Excess Chromates from tissues fixed (Helly’s, Zenker’s and b. Temperature Flemming’s solutions) c. Concentration and Volume b. Excess Formalin O 10% formalin is extracted more rapidly in 70% alcohol than in *too rapid removal of CA+2 salts may produce complete digestion of the tissue water specimen and poor staining capacity of the cell c. Excess Osmic acid/Osmium tetroxide 2. 50-70 % alcohol – used to wash out excess amount of picric acid Decalcifying Agents: 1. Acids 3. Alcoholic iodine – used to remove excessive mercury 2. Chelating Agents Special Factors Affecting Fixation 3. Ion Exchange Resins 4. Electrical Ionization / Electrophoresis A. Retarded by: 1. Size and thickness - Larger and thicker tissues require more fixative For routine purposes, only acids are recommended: and longer fixation time - Nitric acid - almost 2X as fast as formic acid 2. Presence of - Prevents complete penetration of fixative, - Formic acid - provides better tissue preservation and staining mucus hence, tissues that contain mucus are fixed slowly and poorly I. Acid Decalcifying Agents - Excess mucus may be washed away with NSS. 1. Nitric acid - The most common and fastest decalcifying agent 3. Presence of fats - Fatty tissues should be cut in thin sections and used so far should be fixed longer. Recommended - Very rapid decalcifying agent, producing minimal 4. Presence of blood - Tissues containing large amount of blood (e.g. concentrations: distortion Blood vessels and spleen) should be flushed 5-10% - Recommended for routine purposes out with saline (arterial cannulization) before - It undergoes spontaneous yellow discoloration owing fixing. to formation of nitrous acid (HNO2) 5. Cold temperature - Inactivates enzymes 6. Hot temperature - Denatures enzymes a.10%aqueous - Recommended for urgent biopsies and for needle Nitric Acid and small biopsy specimen - Used for large or heavily mineralized cortical bone B. Enhanced by: Decalcification time: specimen 12-24 1. Size and thickness - Smaller and thinner tissues require less fixative hours and shorter fixation time b. Formol-Nitric - For urgent biopsies with good nuclear staining. 2. Agitation - Fixation is accelerated when automatic or acid - Yellow color imparted by nitrous acid is removed mechanical tissue processing is used. through neutralization with 5% sodium sulfate and - Autotechnicon Decalcification time: running tap water for 12 hours 3. Moderate heat - Accelerates fixation but hastens autolytic 1-3 days changes and enzyme destruction c. Perenyi’s fluid - It is recommended for routine purposes. - It decalcifies and softens tissues at the same time Decalcification time: - Maceration is avoided due to the presence of Important Notes on Improper Fixation 2-7 days chromic acid and alcohol 1. Failure to arrest early cellular Due to the failure to fix immediately by d. Phloroglucin - Most rapid decalcifying agent, recommended for autolysis which one first allowed the tissue to dry Nitric Acid urgent works before fixing or insufficient fixative - Has poor nuclear staining 2. Too brittle and too hard blocks Due to prolonged fixation Decalcification time: - Yellow color formation 3. Soft and feather-like tissues Due to incomplete fixation 12-24 hours 4. Removal of fixative soluble Wrong choice of fixative substances 2. Hydrochloric - Inferior compared to nitric acid (slower action and 5. Presence of artefact pigments on Incomplete washing of fixative acid/ Muriatic greater distortion of tissue) sections acid - Good nuclear staining 6. Shrinkage and swelling of cells in Due to overfixation - Recommended for surface decalcification of tissue blocks tissue blocks if used in 1% solution with 70% 7. Enzyme inactivation and loss Wrong choice of fixative alcohol Incomplete fixation → Improper and incomplete clearing and impregnation → incorrect sectioning and staining of pathologic slides Von Ebner’s fluid Formula: 36% Sat. Aq. NaCl, Conc. HCl, Distilled water  lOMoARcPSD|129 359 57 - Recommended for teeth and small pieces of 3 Ways to Measure the Extent of Decalcification bones 1. Physical or - By touching or bending the tissue with the fingers to 3. Formic acid - Moderate-acting decalcifying agent Mechanical determine the consistency of tissues - Recommended for routine decalcification of post- Method - By pricking the tissue with a fine needle/probe mortem research tissues 2. X-ray/ - Very expensive, although it is the most ideal, most Decalcification time: Radiological sensitive and most reliable method 2-7 days Disadvantage: Not suitable for urgent examination method - Good but not always convenient - Not recommended for mercuric chloride-fixed tissues a. Aqueous Formic Acid (radio-opacity will interfere with the plate interpretation) (formic acid + 10% formol saline) 3. Chemical - Simple, reliable and convenient method recommended - Recommended for small pieces of bones and method/ for routine purposes (still favoured) teeth; Suitable for most routine surgical specimen Calcium - Decalcifying fluid is changed every 24-48 hrs and immunohistochemical staining. Oxalate test - Solutions used: 1. Concentrated Ammonium Hydroxide b. Formic Acid –Sodium Citrate 2. Saturated Aqueous Ammonium Oxalate (20% Aq. Sodium citrate + 45% Formic acid) - Detection of calcium in acid solutions by precipitation - Recommended for autopsy materials, bone of insoluble calcium hydroxide or calcium oxalate marrow, cartilage and tissues studied for research - Presence of cloudiness indicates the presence of Ca purposes (incomplete decalcification) 4. Trichloroacetic - Does not require washing out acid - Not recommended for urgent examinations (very Tissue Softeners slow acting) - Unduly hard tissues which are reliable to damage the microtome knives 5. Sulfurous acid - Weakest decalcifying agent, suitable only for may require tissue softener, aside from decalcification. minute pieces of bone 1. Perenyi’s fluid – act both as a decalcifying agent and tissue softener 6. Chromic acid - Used as a fixative and decalcifying agent 2. 4% phenol (1-3 days) - Caution: It is an environmental toxin, highly 3. Molliflex (tissues may appear swollen and soapy) corrosive to skin and mucous membrane and 4. 2% Hydrochloric acid carcinogenic 5. 1% HCl in 70% alcohol 7. Citricacid– - Does not produce cell or tissue distortion Citrate buffer DEHYDRATION - The process of removing intracellular and extracellular water from the II. Chelating Agents tissue following fixation (if decalcification is not required) and prior to wax - Substances which combine with calcium ions and other salts (iron and impregnation (clearing). magnesium) to form weakly dissociated complexes and facilitate removal - Naturally carried out by the use of some reagents (dehydrating of Calcium salt agents/dehydrants) that mixes with and has a certain affinity for water so - The most common chelating agent: EDTA (Used as an anticoagulant and that it may penetrate easily between the tissue cells. water softener) - Ideal volume: not less than 10x the volume of tissue to be dehydrated O Commercial name: Versene, Sequestrene EDTA + Ca Insoluble non-ionized complex Commonly Used Dehydrating Agents (Removes Ca) 1. Alcohol - Dehydration is best accomplished by the use of increasing grades of alcohols, beginning with 70% III. Ion-Exchange Resin (70→95→100→100→100) - Ion exchange resin (ammonium form of polystyrene resin) hastens - Transfer of tissue directly from formalin to higher grades decalcification of alcohol (85% -95%) is risky, since it could lead to - Removes calcium ions from formic acid, thereby increasing solubility from distortion of tissues (shrinkage and hardening of tissues). the tissue. - Concentrated solutions (above 80%) makes tissue hard, - Not recommended for fluids containing mineral acids such as nitric or brittle and difficult to cut hydrochloric acids. - Prolonged storage in lower concentrations (below 70%) IV. Electrical ionization (Electrophoresis) tends to macerate the tissue - Tissue stored in 70-80% may interfere with the staining - Process whereby positively charged Calcium ions are attracted to a negative electrode (cathode) and subsequently removed from the properties of the specimen decalcifying solution. - The time required for decalcification is thereby shortened due to the heat a. Ethanol/ Ethyl - Undoubtedly the best dehydrating agent (fast acting, and electrolytic reaction produced in this process. alcohol mixes with water and organic solvents and penetrates tissues easily) - The principle applied is similar to that of chelating agents; with the main difference that this process utilizes electricity and is dependent upon a - Has the advantage of not being poisonous and not supply of direct current to remove calcium deposits. very expensive - Should be at least 99.7% pure Solutions used: b. Isopropanol/ - Should be used if good-grade absolute ethyl alcohol is 1. 88% Formic acid Isopropyl not easily available 2. Concentrated HCl alcohol 3. Distilled water c. Methanol/ - Toxic dehydrating agent Methyl alcohol - For blood and tissue films and for smear preparations d. Butanol/ Butyl - Utilized for plant and animal microtechniques. alcohol  lOMoARcPSD|129 359 57 2. Acetone - A cheap, rapid dehydrating agent utilized for - It does not make tissues transparent and it is (CH3COCH3) most urgent biopsies (1/2 to 2 hours) toxic to the liver (hepatotoxic) after prolonged - Its use is limited only to small pieces of tissues inhalation due to its extreme volatility and flammability 5. Cedarwood oil - Clears both Paraffin and Celloidin sections 3. Dioxane/ - Excellent dehydrating and clearing agent - Recommended for CNS tissues and cytological Diethylene miscible to water, melted paraffin, alcohol and Clearing time: studies (esp. Smooth muscles and skin) Dioxide xylol/xylene 2-3 days - Very expensive and it requires 2 changes in - Expensive and toxic and extremely dangerous clearing solution (main disadvantage) - Quality is not always uniform and good and is 4. Cellosolve/ - Dehydrates rapidly and not harmful to tissues extremely slow Ethylene glycol - Toxic by inhalation, skin contact and ingestion - It becomes milky on prolonged storage monoethyl ether (use propylene-based glycol esters) 6. Aniline oil - Recommended for clearing embryos, insects 5. Triethyl - Tissues can be transferred directly after fixation and very delicate specimens since it clears phosphate & washing 70% alcohol without excessive tissue shrinkage - Used to dehydrate sections and smears and hardening following certain stains 7. Clove oil - It removes aniline dyes and dissolves Celloidin; 6. Tetrahydofuran - Dehydrates and clears tissues since it is Tissues become brittle miscible to water and paraffin - Its quality is not guaranteed due to its tendency - It is toxic if ingested or inhaled. Vapors cause to be adulterated nausea, dizziness and headache. - Not suitable for routine purposes because it is - It is an eye and skin irritant and prolonged expensive exposure (up to 6 months) may cause 8. CCl4 (Carbon - Properties are very similar to chloroform but it is conjunctival irritation. Tetrachloride) cheaper - Toxic on prolonged exposure CLEARING 9. Tetrahydrofuran - Dehydrates and clears at the same time since it AKA: Dealcoholization is miscible in both water and paraffin - The process whereby alcohol or a dehydrating agent is removed from the 10. Methyl - These are slow-acting clearing agents that can tissue and replaced by a fluid (clearing agent) that will dissolve the wax benzoate/ be used when double embedding techniques with which the tissue must be impregnated. Methyl are required. - Clearing agent must be miscible with the dehydrating agent, paraffin wax, Salicylate and mounting medium. IMPREGNATION - The word clearing is used because, in addition to removing alcohol, many AKA: Infiltration of these substances have the property of making tissues transparent/translucent. - The process whereby the clearing agent is completely removed from the - Ideal amount: not less than 10x the volume of the tissue tissue and replaced by a medium that will fill all natural cavities, spaces, and interstices of the tissues, even the spaces within the constituent cells, Commonly Used Clearing Agents and that will set to a sufficiently firm consistency to allow the cutting of suitably thin sections without undue distortion and without alteration of the 1. Xylene - An excellent and true clearing agent spatial relationships of the tissue and cellular elements. Advantages: O Without firm consistency: tissue will collapse during cutting Clearing time: 1. The most rapid clearing agent - Ideal volume: at least 25x the volume of the tissue ½ to 2 hours (usual) 2. Cheap and does not extract out aniline dyes 3. It is miscible with absolute alcohol and paraffin 3 Methods of Impregnation Used in Histology 15-30 Minutes 4. Can be used with celloidin sections 1. Paraffin wax Impregnation (urgent biopsies) Disadvantages: O the simplest, most common, and by far the best for routine use 1. It is highly flammable O melting point is 54-58 0C 2. When dehydration is not complete, the xylene 2. Celloidin wax/Collodion Impregnation becomes milky when the tissue or section is 3. Gelatin Impregnation added to it 3 Ways of Paraffin Wax Impregnation (and Embedding) 2. Toluene - Substitute to xylene or benzene 1. Manual - Requires at least 4 changes of wax with 15 minutes’ - It is miscible with absolute alcohol and paraffin processing interval Clearing time: - It is not carcinogenic but highly concentrated - Total: 1 hour 1-2 hours emit fumes that are toxic upon prolonged 2. Automatic - Makes use of an automatic tissue processing machine exposure Processing (e.g. Autotechnicon) which fixes, dehydrates, clears - It acts slower than benzene and is expensive and infiltrates tissues, thereby decreasing the time 3. Benzene - Rapid acting, recommended for urgent biopsies and labor needed during the processing of tissues, and routine purposes resulting in a more rapid diagnosis with less Clearing time: - It is miscible with absolute alcohol and paraffin technicality 15-60 minutes - It is highly flammable - Autotechnicon/Elliot-Bench Type Tissue Processor: - Carcinogenic or may damage bone marrow Fixation, Dehydration, Clearing, Infiltration (Aplastic anemia; If antibiotic: Chloramphenicol) 3. Vacuum - Impregnation under negative atmospheric pressure 4. Chloroform - It is the best of the traditional clearing agents for Embedding - Recommended for urgent biopsies and delicate routine use tissues such as lung, brain, connective tissues, Clearing time: - Gives the widest latitude decalcified bones, eyes spleen and CNS. 6-24 hours - It is recommended for tough (skin, fibroid and - Reduces 25-75% of impregnation time decalcified tissues) and large tissue specimens - Most rapid - Also best for nervous tissue, lymph nodes, NOTE: Infiltration in overheated paraffin (above 600C) produces shrinkage and granulation tissue, and fetal and other delicate, hardening of tissues. Paraffin oven must be maintained at a temperature 2-5°C highly cellular specimens above the melting point of the wax  lOMoARcPSD|129 359 57 Celloidin Impregnation Several Types of Blocking-out Molds - Celloidin - purified form of nitrocellulose soluble in many solvents 1. Leukhart’s - Consist of 2 L-shaped pieces of heavy brass or metal, a - suitable for specimens containing large cavities or hollow spaces which tend to embedding base being formed by a piece of 1/8 inch thick copper collapse, for hard and dense tissues (bones and teeth) and for large mold brass, about 3X2 inches square, or a piece of plate glass. tissue sections of whole embryos. 2. Compound - Made up of a series of interlocking plates resting on a flat Advantage: Causes much less shrinkage and distortion than paraffin wax embedding metal base, forming several compartments. Disadvantage: mold 1. Slow and tedious 3. Plastic - Consist of a special stainless steel base mold fitted with a 2. Serial sections are difficult to prepare embedding plastic embedding ring which later serves as the block 3. Very thin sections (