Histopathology Lecture Notes PDF

HSTCYT1 - LECTURE LICAO, MARY LAVIENNE OVER...

HSTCYT1 - LECTURE LICAO, MARY LAVIENNE OVERVIEW OF THE HISTOPATHOLOGY SECTION METHODS OF TISSUE SAMPLE COLLECTION deceased live / What is Histopathology? -Tissue samples are acquired through biopsies, autopsies, - Diagnosis and study of disease using tissue secretions or during surgeries - Examination of tissues with potential course of a disease - Fragments, entire tissues, organs, even amputated limbs under the microscope may vary in sizes SMAK NEEDLE ! ! ! ruse Importance 1. Fine Needle Aspiration (smallest) - Diagnosis of diseases can be made using a small tissue - simple and least invasive sample (biopsies) - insert needle with large gauge/small - Histopathologists can perform several diagnoses using size to a certain body part in order to small, well-prepared slides extract cell - Histopathological slides are GOLD standard when it comes - guided by ultrasound ) to assessing certain kinds of diseases. 2. Core Needle Biopsy (same WIFNAB : eg - Bone aspirate needle Courses of disease can be observed 3. Incisional Biopsy - Macroscopically - use of needle larger from fine - Microscopically collection) usin9e needle aspiration (more scalp - Collect small amount of cell / HISTOPATHOLOGISTS surrounding tissue - perform tissue samples - Incise or cut-off certain part - if quality of slides are affected, the diagnosis would also be affected ¥:*. of organ - Can remove abnormal tissue QUALITY is important in histopathology (but not all) unless lesion is cancerous STEP-BY-STEP PROCESS OF HISTOPATHOLOGY 4. Excisional Biopsy even surrounding tissues - ③ ÷: - For cancerous tisssues 1. Biopsy * - Excise / remove completely S--EI☒t - done in surgical ward u the entire lesion - tissue or organ sample 5. Punch Biopsy (FULL LAYERS OF SKIN ! ) G describe 2. Gross Sectioning - Use of circular blade to 1) - Biopsy samples are going to be describe, cut remove cylindrical piece of tissue sample (3-4 mm) 2) measure size down to smaller sizes to process different tissue usually done on skin. samples 6. Shave Biopsy fungal elements - shave off fragment of tissue scraping from SURFACE ! infected F 3. Fixation of - heat - using - preservation of sample in fixative / preservative 7. Curettage chemical agent or - scraping out body tissue, done in exfoliative tissue 4. Decalcification (optional) cases help D in - soften very hard tissues that can damage - using spat # BODY CAVITIES ! ! ! PAP SMEAR ! - preserving microtome Cuse chemicals ) WHAT COLLECTION METHOD TO USE 5. Dehydration 1) help preservation - bring out water molecules and replace with. - Depending on these factors, you can perform biopsy samples 2) to ready for clearing something that would replace water in its place according to: * Cyst sac 6. Clearing The structural and chemical components to be Benign - improve opacity of tissue / optical clarity studied WHOLE LESION 7. Impregnation Nature and amount of sample for evaluation (MALIGNANT ) § diff waxes - removal of clearing agent and replacing it with Need for immediate diagnosis entry of not compress tissue something that could support the structure of the tissue A. TISSUE ACQUISITION 8. Embedding (Blocking) - Tissues come in the laboratory already preserved / Ex section encast - creating tissue block for microtomy : Frozen - Samples can also come in fresh (example: Pap smear) 9. Microtomy - Fresh samples are prone to autolysis thus it must be sam%nmiʰ%*[qfabC# * m.name - tissues are cut down to ribbon section, only done , ,, a , processed ASAP an section singular when microtomy is done well slice into thin sections breakdown - of tissue use adhesive , *AUTOLYSIS - enzymatic breakdown of cells 10. Routine Staining LESA) * in order to avoid breakdown of cells, samples should be - usage of different dyes/stains that brings contrast to preserved with PRESERVATIVE (best option) or COOL different parts of the cell in order to observe structural ENVIRONMENT (freezer) µ using mounting agent act as glue covslip stays , components of tissues 11. Mounting /cover slipping - mount cover slip is placed in slides - It must come with a request form, containing: a. Patient’s info (name, age, sex) protect tissue from trauma b. Clinical history (diagnosis of patient) 12. Labelling ✗ - putting number specific label depending on aqvisition c. Description of site of origin avoid - Specimens are given a unique number [ The - entire “processing” procedure of tissue processing - Minimizes risks of specimen mix up by giving a unique includes: number to a certain patient (unique ACESSION o Fixation b) microscopy NUMBER/IDENTIFICATION NUMBER) µ o Dehydration done by hist pathologist ◦ o Clearing B. TISSUE EXAMINATION o Impregnation - Tissues can be examined in either a fresh or preserved state o Decalcification - A greater section can be acquired from preserved stained L sections - Sections can be kept permanent by mounting a coverslip ↓ INDEFINITE  HSTCYT1 - LECTURE LICAO, MARY LAVIENNE 1. FRESH TISSUE EXAMINATION Disadvantages: Advantage: - Workers are more prone to toxic substance (ex. a. Able to examine cells in its living state Formalin, xylene) b. Immediate examination - Time-consuming c. Simple, easy to perform Disadvantage: 3. FROZEN SECTION EXAMINATION SURGERY a. Not permanent - Allows for an immediate diagnosis b. Prone to changes - Aids during surgery, as freezing quickly hardens tissue - subjected to autolysis and putrefaction - Recommended for lipid and nervous tissue elements *AUTOLYSIS - Uses: " - can be observed after hours of tissue excision and a. Rapid diagnosis during surgery ;÷¥¥¥ has not been preserved b. Enzyme histochemistry *PUTREFACTION c. Demonstration of soluble substances (lipids, CHO) ÷ - degredation of cells because of bacterial enzymes acting on tissues d. IF and IHC staining e. Neuropathology (Ag staining) - produce pungent smell - Methods: - produce macroscopic and microscopic changes to 1. Cold Knife Procedure the tissues - use freezing microtome c. Limited use 2. Cryostat (Cold Microtome) Advantages: Methods of Fresh Tissue Examination or - Rapid Processing 1. Teasing/Dissociation ( NSS ) < - Less equipment requirement - cut out fresh tissue while submerged in Bringer- - Less need for ventilation mounted OF lactose solution or normal saline sol then placing Disadvantages: WET on slide for examination - Inferior slide quality 2. Squash/Crush - place tissue in between 2 glass slide then a) CRYOSTAT squish for distribution - Consists of an insulated microtome in a refrigerated 3. Smear Preparation chamber - smearing on glass slide - Creates an ambient temperature near -20°C a. Spreading application stick - - Tissues are frozen depending on the type of tissue - done with pap smear wherein swab is before cutting placed in the cavity of patient then directly Advantage: spread on slide (Tuber c.) - Simple, quick, least labor-intensive process for APPOSITION b. Pull-apart putting 1- drop suspension of producing frozen sections - like blood smear, pull apart 2 slides - Helpful in intraoperative diagnosis containing tissue sample ( Bone Ma Aspi ). Disadvantage: c. Streaking apply zigzag pattern - on - Creates single sections 4. Touch Preparation / IMPRESSION SMEAR Storage in Histopathology - simply put slide on top of tissue to get an impression - As with any section in the clinical laboratory, the of tissue sample histopathology laboratory has protocols when it comes to - some cells might be present storage of specimens 2. CONVENTIONAL TISSUE PROCESSING IN HOLE PROCESS) - Processing of preserved specimens Specimens: 1 month - 1 year : ADV - Involves the entire process 1¥ Tissue blocks 3-10 years Better - GOAL: provide a tissue hard enough to section thinly enough for proper staining and microscopic examination section quality Slides Indefinite (Quality) * Is for removing Autolysis and Putrefaction Dls : Records Permanent Initial Steps in Tissue Processing workers prone to toxic substances 1. Specimen Association/Identification : formalin (carcinogen ) - Specimen is received or collected eg xylene less toxic ) - Request must accompany sample ( narcotic) - Request must contain diagnosis and brief clinical details time consuming regarding px and sample - Assigned an accession/ID number 2. Gross Examination and Sampling - Specimen is described by pathologist - Specimen is cut up to pieces to allow processing and examination afterwards 3. Tissue Processing - Specimen is processed according to the protocols of the laboratory Advantages: - Better section quality - Slides can be stored PERMANENTLY  HSTCYT1 - LECTURE LICAO, MARY LAVIENNE TECHNICAL PROCESSES METHODS OF FIXATION FIXATION 1. PHYSICAL a. Heat fixation - use of flame to preserve - Process of preserving tissues - can destroy tissue sample - Prevents decay b.Microwave contamination putrefaction - Retains the state of the tissue as to how it was removed c. Cryo-preservation = from the body - freeze-drying; freeze-substitution - Eliminate reactivating of enzymes - ideal for examining lipids - Stabilizes cellular structures by making macromolecules resistant to water 2. CHEMICAL a. Immersion fixation (IF) FIXATIVE - just submerge the tissue - inactivates enzymes inside the cell, preventing autolysis and i. Coagulation fixation putrefaction ii. Dehydrant coagulation fixation - for bacterial protection interact w/ proteins iii. Formaldehyde fixation stabilise cellular structures by making macromolecules resistant to water - most common IF - DENATURATION - to inactivate proteins and enzymes b. Perfusion fixation c. Vapor-fix fixation GOALS OF FIXATION FIXATION MECHANISM 1.*Reserves the chemical and morphological integrity of the ' cell making it “life-like” 1. ADDITIVE FIXATION harder when < 2. Harden and protect the tissue - The fixative is incorporated inside the cell more exposed 3. Support high quality staining properties with Hematoxylin & - It forms cross-links or complexes, providing stability Eosin staining procedure 2. NON-ADDITIVE FIXATION ·Alters the tissue composition FACTS ABOUT TISSUE PROCESSING -Removes water from tissues by competing with the H-bonds formed by water with molecules. 1. Fixation makes cells slightly swell then shrink - tissues lose 20% - 30% of their total volume TYPES OF FIXATION 2. It affects the degree of staining 3. It stops the activity of enzymes FOUR MAJOR GROUPS: Denaturation < A. Aldehydes D 4. It prevents the breakdown of cellular components * inactivate enzymes 5. It allows retention of certain tissue components - Forms Cross-links proteins the entry OF water Fixative - Denature protein but will deposit it on cellular cytoskeleton 2) Prevent BIOPSY FACTORS AFFECTING FIXATION ↓ providing them stability Fixative but not to the point that it will swell) ration 20 :| B. Oxidizing Agents 1. VOLUME ( DILUTE : ↓ - Cross-links proteins - there should be more fixative than sample < 2. HYDROGEN ION CONCENTRATION (PH) - Composed of salts of Acids GOLDILOCKS Tissue 3. TEMPERATURE sample - Denature protein but will deposit it on cellular cytoskeleton *. - the hotter the chemical the faster the movement providing them stability - Hot = faster penetration C. Alcohol-based Fixatives - Colder = slower penetration for fixatives -Protein-denaturing agents 4. THICKNESS OF SECTION I / mm / hour) - Denatured in such a way that it is removed or precipitated - the thicker the longer it gets preserved and longer time for outside the cell the fixative to penetrate D. Metallic Fixatives 5. OSMOLALITY - Forms insoluble metallic precipitates 6. s CONCENTRATION - mechanism is somewhat misunderstood until today but their - not too strong not too weak fixatives job as fixative is good 7. DURATION OF FIXATION ( 3,4 -55) , - precipitate can cause confusion in reading so ppt must be 8. TIME INTERVAL - tissue removed from body to tissue to fixation removed after fixation process or before staining process CONSIDERATIONS ON FIXING TISSUES I. ACCORDING TO COMPOSITION than 1. Tissues must be fixed WITHIN 1 HOUR not longer 2. Tissue-to-fixative ratio must be at least 1:10 or in A. Simple Fixatives general 20:1 - Contains one component I hour 3. Anatomical barriers must be incised or removed 1. Aldehydes 4. Large specimens must be section or inflated 2. Metallic Fixatives 5. Pin tissues on a cork-board or insert a wick (cotton 3. Picric Acid balls or gauze) into tubular structures to maintain shape 4. Acetic Acid 6. Duration and the rate of penetration must be considered 5. Acetone 7. Prolong fixation can result in the loss of 6. Alcohol IMMUNOHISTOCHEMICAL antigenecity 7. Osmium Tetroxide 8. Tissues harden during fixation B. Compound Fixatives - Combination of two or more fixatives. - Combines effects of theindividual action of fixatives. HSTCYT1 - LECTURE LICAO, MARY LAVIENNE II. ACCORDING TO ACTION FIXATION FOR EM ENZYME HC, IF, and IHC A. Microanatomical Fixatives - Permits microscopic study A. ELECTRON MICROSCOPY - Doesn't distort the structuralpatterns of the cells - Done at 4°C B. Cytological Fixatives - Fixatives containing glutaraldehyde - Preserves SPECIFIC parts - Followed by secondary fixation using osmium tetroxide a. NUCLEAR (≤ pH 4.6) B. ENZYME HISTOCHEMISTRY - preserve the nucleus only - Maximal preservation of enzyme activity to where it b. CYTOPLASMIC (> pH 4.6) originates, while also preserving structural integrity. - preserve only the contents of cytoplasm - Overnight fixation using 4% formaldehyde or formal- c. HISTOCHEMICAL saline - preserve the biochemical molecule in the tissues - Fixed with acetone or formaldehyde for frozen sections. - Ex. Lipids C. IMMUNOFLUORESCENCE and SECONDARY FIXATION IMMUNOHISTOCHEMISTRY - Fixing an ALREADY PRESERVED tissue in a different - Epitopes of antigens must be preserved for antibodies to fixative. bind on antigens to be detected. - Contribution - Antigen retrieval can be done to bring back antigenicity a. Demonstration of particular substances. using Heat-induced Epitope Retrieval (HIER). b. Makes special staining possible (MORDANT). c. Ensures further and complete hardening and *EPITOPES - sites where antibodies will attached to. preservation of tissues. D. MICROWAVE FIXATION - Done before dehydration and on deparaffinized tissues - Employing the advantage of heat to fix tissue before staining. * Microwave fixation - Microwaves aid in the fixation process by either allowing heat *Post-Chromatization to permeate and fix the tissue itself - process where tissues have enhanced staining quality * Microwave- assisted fixation because of the use of the fixative potassium dichromate - heat using microwave assists the permeation of a chemical WASHING OUT fixative - ADVANTAGES: - Process of removing excess fixative. Allows examination of rapid cellular processes. - Improves staining; removes artifacts. Non-chemical; allows preservation of - Solutions used: neurochemical substances. a. Tap Water Rapid fixation of tissues of routine surgical b. 50%-70% Alcohol specimens. - true to tissues fixed with picric fixatives Significantly reduces time for IHC and in-situ c. Alcoholic lodine hybridization. - helpful in removing deposits formed by metallic Allows the satisfactory production of tissues for EM. fixatives specially mercuric fixatives - DISADVANTAGES: May only penetrate tissues with 10-15 mm BIOCHEMICAL FIXATION / HISTOCHEMICAL FIXATION thickness. No significant cross-linking of proteins. A. LIPID FIXATION Spores and other pathogens may remain in the - Hydrophobic tissues. - Must be fixed with a solution that mustn't affect lipid composition. E. CHEMICAL FIXATION - Frozen tissues stained with a general lipid stain. Use of chemical reagents to preserve tissues. - Use fixatives containing HgCl2 or potassium dichromate. Chemical fixatives have their own different - Phospholipids are fixed with aldehydes (Baker's formol- actions. citrate). *Cross-links with proteins, providing - Digitonin for cholesterol demonstration. additional rigidity to the cells. Denatures proteins. B. CARBOHYDRATE FIXATION - Hydrophilic I. FORMALDEHYDE and FORMALIN - DO NOT USE FIXATIVES WITH HIGH WATER CONTENT - Sold as 35% - 40% w/v formaldehyde solution. - Alcoholic fixatives (Rossman's fluid) - Diluted to a 10% formalin solution in phosphate-buffered saline (diluent) to meet C. PROTEIN FIXATION adequate fixative properties. - For amino acid histochemistry, NB Formol-Saline or - Advantage: Cheap, easy to prepare, penetrates Formaldehyde vapor. tissues well, and can be stored for a long time. D. NUCLEIC ACIDS - Stabilize using 15% methanol -Disadvantage: Irritating, Carcinogen - Product of the reduction of methanol HSTCYT1 - LECTURE LICAO, MARY LAVIENNE II. PARAFORMALDEHYDE 9. Newcomer’s Fluid - White powder; formaldehyde polymers. - for fixing mucopolysaccharides and nuclear - Allows paraffin embedding and sectioning, and proteins IHC. - Effects are reversible by excess water. VI. METALLIC FIXATIVES - Avoids pigmentation produced by formalin. - Actions of these fixatives are still not - In high blood content tissues, formalin may act understood. up in the heme molecule of hemoglobin of RBC, - Allows a good to excellent staining quality. thus producing a formalin heme pigment (brown - Poor penetration; produces shrinkage. color) - Ideal for hematopoietic and reticuloendothelial - Good tissue penetration. Long term storage. tissue processing. III. GLUTARALDEHYDE MERCURIC CHLORIDE - 2%, 2.5% or 4% - Most common metallic fixative - Standard fixative for Electron Microscopy - Used as a 5-7% aqueous solution - Pleasant and less irritating on the nose. - Works by additive and coagulative means. - Gives excellent nuclear detail. 2% Electron Microscopy - Allows excellent trichrome staining. - Poor penetration (due to its large mol.structure) Small tissue biopsies and and allows significant cell shrinkage (due to its 2.5% needle biopsies concentrated nature). - Routine fixative of choice for tissue photography. 4% for larger tissues less than 4 - Mercury deposits may form. mm thick OTHER NOTABLE METALLIC FIXATIVES (MERCURIC BASED FIXATIVES) IV. KARNOVSKY'S FIXATIVE 1. Zenker's Solution - 4% Paraformaldehyde - 1% Glutaraldehyde in - recommended for congested specimens, and for 0.1 M Phosphate Buffer trichrome staining and Phosphotungstic acid- - Suitable to use when tissues are to be embedded haematoxylin (PTAH) staining in resin. - Suitable fixative for electron microscopy. 2. Zenker-Formol (Helly's) Solution - excellent fixative for bone marrow, liver (to V. ALCOHOLIC FIXATIVES demonstrate extramedullary hematopoiesis), spleen, - Denatures proteins pituitary glands, and cardiac muscle. - Not routinely used for tissues 3. Lillie's B-5 Fixative or B-5 - Rarely used alone for fixing blocks, unless - for fixing hematopoietic BM biopsies, and lymphoid studying nucleic acid. tissues. - Good for cytologic smear fixation 4. Heidenhain's Susa - Used as 70% to 100% concentration. - for tumor biopsies of the skin; permits easier - Mixed with acetic acid, water, and/or chloroform to sectioning of large blocks of fibrous connective tissue reduce distortions. *DE-ZENKERIZATION NOTABLE ALCOHOLIC FIXATIVES - Removal of mercury deposit from tissues fixed 1. Methanol with mercury or mercury-based fixatives - general fixative for Peripheral Blood Smear (PBS), - Lugol’s iodine is used or an alcoholic solution dry and wet smears, and Bone Marrow Aspirate complexes with precipitate (BMA) smears - Sodium thiosulfate (5%) solution is used to rinse 2. 95% Isopropanol out the complexes formed by mercury and iodine - Touch preps or Imprint Slides VII. OXIDIZING AGENTS 3. Ethanol (70-100%) - Permanganates, Dichromates and Chromatic - used as a fixative and a dehydrating agent acid, Osmium Tetroxide, Picric Acids 4. Carnoy's Fluid (Absolute alcohol in - Reacts with various side chains of proteins Chloroform) and other biomolecules, forming cross-links, - preserves Nissl bodies, cytoplasmic granues, stabilizing tissue structures nucleoproteins, nucleic acids - May extensively denature biochemical - for fixing brain tissues for rabies dx. components despite preserving fine structures 5. Alcoholic Formalin - Mainly used as secondary fixative. - fixation or post-fixation of large fatty specimens 6. Clarke's Solution VIII. OSMIUM TETROXIDE; OSMIC ACID - can be used on frozen sections - Causes complete denaturation of proteins 7. Formol-Acetic Alcohol - Traditionally used in EM (secondary fixative & - Sometimes used for fixing tissues for diagnostic stain) cryostat - Good fixative and stain (black) for lipids. 8. Gendre's Fluid - May act as stain because of its black color - enhances immunoperoxidase studies - Preserves cytoplasmic structures. - for glycogen and micro-incineration - Penetrates at a slower rate vs. metallic fixatives. HSTCYT1 - LECTURE LICAO, MARY LAVIENNE - May give off a black stain XIII. TRICHLOROACETIC ACID (TCA) - Extremely volatile; prolonged exposure to vapors - Fixative and decalcifying agent may cause conjunctivitis. (Must be done in a fume- - Precipitates proteins and nucleic acids. hood) - Used only on small pieces; penetrates poorly. OSMIUM-BASED FIXATIVES XIV. ACETONE 1. Flemming's Fluid - Not recommended as a morphological fixative - most common; permanent fixative for fat (can somehow distort tissues) 2.Flemming's without acetic acid - Used in fixing cryostat sections and for tissues - recommended for demonstrating mitochondria requiring enzyme preservation. - Used to fix tissues at cold temperatures (0-4°C) IX. CHROMATE FIXATIVES - Recommended for fixing brain tissues to diagnose 1. Chromic acid (1-2%) rabies - good in preserving mitochondria at pH 4.5- 5.2 - Preserves glycogen poorly; dissolves fats. 2. Regaud's Fluid (because of its organic nature) - recommended for demonstrating mitochondria, mitotic figures, Golgi bodies, RBC and colloid- *MICHEL'S SOLUTION containing tissues - Use when transporting tissues 3. Orth's Fluid - Not a fixative - recommended for studying early degenerative - Stable medium for transporting fresh, unfixed tissues that will processes and tissue necrosis; undergo frozen sectioning or IF studies. - also advisable for demonstrating rickettsiae and - Specimens can be kept in the solution for 5 days until it other bacteria can be delivered to a reference laboratory. - Not suitable for keeping tissues for Fluorescence in situ X. PICRATE FIXATIVES hybridization (FISH). - Performs almost as well as mercuric fixative; - Tissues in such solution must be kept at an ambient doesn't harden as much. temperature of 4-22°C. - Reacts with histones and basic proteins; precipitates all proteins. - Good fixative for connective tissues, preserves DECALCIFICATION glycogen well, extracts lipids. - May impart a yellow pigment. - Removed using lithium carbonate or wash in - remove calcified structures (calcium ions or magnesium alcohol in changes of 50-70% ions) inside tissue samples ; Ex: bones - the section must be as soft as the embedding medium 1. Bouin's Fluid - facilitates an easier cutting for histotechnologist during - recommended for fixing embryos and pituitary microtomy biopsies, also glycogen 2. Hollande's Solution -recommended for gastro-intestinal tract specimen calcified and endocrine tissues; has some decalcifying properties 3. Gendre's Fluid - highly recommended for glycogen and decalcified carbohydrate preservation; - produces minimal distortion of micro-anatomical structures. 4. Brasil's Alcoholic Picroformol Fixative - better and less messy than Bouin's; excellent for preserving glycogen. XI. GLACIAL ACETIC ACID - Anhydrous - Freezes at 16°C - Enables cell swelling - Precipitates and fixes nucleoproteins PRINCIPLES OF DECALCIFICATION - Compounded with other fixatives to counteract the swelling effect. - Sections are immersed or exposed to methods that would facilitate the exchange of ions to form calcium salts. XII. LEAD FIXATIVES - Chelating agents applied to calcified structures "bind" - Recommended for fixing acid calcium and other metallic ions to soften tissues. mucopolysaccharides, mucin. - Acrylic or epoxy resins can also be used to eliminate the - May produce lead carbonate upon prolong need of decalcification. standing or exposure to Carbon dioxide. (White ppt on the bottom of the sol) HSTCYT1 - LECTURE LICAO, MARY LAVIENNE FACTORS INFLUENCING DECALCIFICATION B. NITRIC ACID - MOST COMMON; FASTEST Volume - Used as a 5% - 10% solution - Recommended ratio of decalcifying agent to tissue: 20:1 - Used on its own or may be mixed with other decalcifying - Lower amount will retard the decalcifying process agents, enhancing the property. 2. Fluid access - Can impart a yellow color (nitrous acid) - All the sides of the tissue must be exposed (to avoid uneven decalcification process) C. NITRIC ACID-BASED DECALCIFIERS - Suspended using gauze (will hold the tissue) and thread i. Formol-Nitric Acid (will suspend the tissue). ii. Perenyi’s Fluid 3. Size and Consistency iii. Phloroglucin-Nitric Acid - Dense tissues must be soaked for > 14 days. - Changed daily to ensure proper penetration. D. HYDROCHLORIC ACID 4. Agitation - Inferior compared to HNO3 - Swirling of the decalcifying agent in order to facilitate the - Slow action and produces great distortion circulation of agent around the container - Produces good nuclear staining - No agitation means uneven decalcifying process - May be used as a surface decalcifier or tissue softener 5. Temperature (1% HCI in 70% alcohol) - Increased temperature increases perfusion, but also E. HYDROCHLORIC-ACID BASED DECALCIFIERS have damaging effects. - Von Ebner's Fluid - Low temperatures retard the process. F. WEAK ACIDS METHODS OF DECALCIFICATION - Mostly organic acids, but some are inorganic. A. Acid decalcification - Formic acid (HCOOH) and Trichloroacetic acid (TCA) - use of various acids to form soluble salts. - Better suited for urgent biopsy samples. B. Chelation G. FORMIC ACID (HCOOH) - binds with calcium to soften tissue - Moderate-acting C. lon exchange resin - Recommended for post- mortem research tissue studies. - removes calcium from decalcifying agents that may - Used as a 10% aqueous solution or combined with contain formic acid formalin or a buffer. D. Electrophoresis - Only weak acid extensively used as a primary decalcifier - use of electrodes (anodes and cathodes) to remove because it will not distort much the cell calcium ions based on their charges E. Microwave decalcification H. FORMIC ACID-BASED DECALCIFIER - will only enhance the decalcification process by the - Formic Acid - Sodium Citrate application of microwaves to hasten the process. F. Tissue Softening I. TRICHLOROACETIC ACID (TCA) - application of a decalcifying agent to soften unexpected - Slow; not recommended for urgent examinations. foci of calcium during microtomy. - Does not require washing out because of its organic nature (TCA is normally found inside tissues thus there is no need to NOTES ON DECALCIFICATION wash out but there is no harm to wash excess TCA) - Excess may be washed out using several changes of 90% - Decalcifying agents are basically composed of strong acids, alcohol. weak acids or chelating agents. - Dilute mineral acids can be used only if the endpoint is J. SULFUROUS ACID monitored carefully. - Very weak; for minute bone pieces. - Prolonged decalcification can macerate the tissues, and cellular details can be compromised. K. CHROMIC ACID (Flemming's) - Fixative and decalcifier *PROCESS OF MACERATION - May form insoluble pigments - their will be excessive softening of tissues that will result - Highly corrosive, carcinogenic, and an environmental to “pulp-like” consistency that will facilitate the poor hazard. cutting of tissues because this will lead to compression L. CITRIC ACID - CITRATE BUFFER SOLUTION - expect that tissues will compress during microtomy - pH 4.5 because it cannot withstand the force applied - Very slow tissue decalcification; seldomly used for routine ACID DECALCIFYING AGENTS decalcification. A. STRONG ACIDS M. CHELATING AGENTS - Inorganic acids - Binds calcium and magnesium ions to soften bone and - 10% HCI and HNO3 other calcified tissues - Most rapid technique of decalcification but it can induce * Ethylenediamine Tetra-acetic Acid (EDTA) maceration if prolonged. - More preferred if studying DNA or histochemistry - Will affect staining of nuclear contents, but not of must be preserved for study. acidophilic cell components. - Adjusted to a pH of 7 or 8; pH of 10 may be used, Corrected by post-decalcification, removal and but some tissue elements may be damaged. adjustments on the staining process. - Very slow but very gentle. HSTCYT1 - LECTURE LICAO, MARY LAVIENNE - May take 1-3 weeks to decalcify small bone DEHYDRATION tissues, and 6-8 weeks for dense cortical bone. - Essential removal of water molecules using gradual ENHANCEMENT PROCEDURES USING EDTA concentrations of an anhydrous solution. i. Sonication with EDTA - come before clearing because clearing agents are - use of sound waves to enhance the movement of essentially water immiscible thus it cannot mix or penetrate in molecules of EDTA the presence of water ii. Microwave - allows clearing agents to enter the tissues - same as Sonication - Slowly substitutes water with a strong organic solvent in iii. Electrolytic decalcification gradual increasing series to minimize the effects of shrinkage and extraction of cellular components. CHECKING FOR ENDPOINT OF DECALCIFICATION - Will not completely remove water. 1. PHYSICAL CONSIDERATIONS FOR CHOOSING A DEHYDRATING - use of probe to poke the tissue AGENT - Disadvantage: physical force can damage tissue 2. CHEMICAL - Any water miscible substance can be used as a - not that ideal because of the use of several reagents to dehydrating agent as long as: check the endpoint of decalcification (titration) ✓ Substance is miscible to both water and the *TITRATION following substances used. - done using the neutralization of any acid with ✓Cost may also be a factor. the addition of a strong ammonia sol. - Common dehydrating agents include: - check for cloudness or turbidity (turbid means not ✓ Alcohols completed) ✓ Acetone 3. X-RAY ✓Dioxane - Most ideal, most sensitive and most reliable method ✓Cellosolve - Most expensive method of checking the endpoint ✓Triethyl phosphate ✓Tetrahydrofuran - A complete dehydration can be observed by placing anhydrous copper sulfate crystals in a filter paper at the bottom of the container. ✓Accelerates dehydration by binding water to its structure. ✓Complete dehydration may be observed when a bluish discoloration of the crystals is observed. POST- DECALCIFICATION - Removal of excessive decalcifying agent. Neutralization using: DEHYDRATING AGENT i. Sat'd lithium carbonate ii. 5-10% aq. Sodium bicarbonate 1. ALCOHOLS - Rinsing with tap water. - Most common type of dehydrating agent. - For frozen sections, acid- decalcified tissues must be - Changes of gradual increasing concentrations of alcohols washed with water or stored in formol-saline with 15% sucrose must be used. (to avoid distortion of tissues) or PBS with 15-20% sucrose. - Delicate tissues like embryos must be dehydrated by - Store EDTA-decalcified tissues in formol-saline/PBS before alcohols of lower concentrations first (start with 50%) dehydration. compared to routine dehydration. (start from 70% until you reach 95%) - An immediate use of a high concentration of alcohols will harden tissue surfaces. SURFACE DECALCIFICATION and TISSUE SOFTENERS - Soaking tissues in