Midterm Histopath (2) PDF

GENERAL PATHOLOGY AND HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES INTRODUCTION TO TISSUE PROCESSING EXAMINATION OF FRESH TISSUE PUNCH BIOPSY BIOPSY...

GENERAL PATHOLOGY AND HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES INTRODUCTION TO TISSUE PROCESSING EXAMINATION OF FRESH TISSUE PUNCH BIOPSY BIOPSY  Uses circular blade to  Gold standard for obtain deeper skin Tumor Diagnosis sample  Excision and  Full-thickness skin examination of tissue sample coming from a living person.  Bios = life, Opsy = look SHAVE BIOPSY  Small fragments of SPECIMEN COLLECTION METHOD tissue are “shaved” 1. Core Needle Biopsy from surface / top 2. Incisional Biopsy layers of the skin 3. Excisional Biopsy 4. Punch Biopsy CURRETINGS 5. Shave Biopsy 6. Curettings  Tissue is scooped or 7. Fine Needle Aspiration spooned to removed tissue or growths CORE NEEDLE BIOPSY from body cavity  Remove cells + small amount of surrounding tissue FINE NEEDLE BIOPSY  Large (4mm)  Small (2mm)  Aspiration of fluid for Cytological examination  Simplest,Inexpensive FINE NEEDLE ASPIRATION INCISIONAL BIOPSY  Aspiration of fluid for  Remove cells + large Cytological amount of examination surrounding tissue  Simplest,Inexpensive EXCISIONAL BIOPSY  Removes the entire area in a tissue SCM ADD ONS.. 1. BITE BIOPSY 2. CUTAENOUS BIOPSY 3. WEDGE BIOPSY METHODS OF FRESH TISSUE EXAMINATION 1. TEASING / DISSOCIATION 2. SQUASH PREPARATION / CRUSHING 3. TOUCH PREPATION / IMPRESSION SMEAR 4. SMEAR PREPARATION  Streaking  Spreading  Pull-Apart MT304 LECTURE / MIDTERM DOLORSKIEEE  FROZEN SECTION TEASING / DISSOCIATION 1. Rapid Diagnosis 2. Enzyme Immunohistochemistry  selected tissue 3. Demonstration of soluble substances (lipids & specimen is immersed carbohydrates) in a watch glass 4. Immunofluorescent/chemical staining containing isotonic 5. Specialized silver stains (esp. Neuropathology) salt solution, & ADVANTAGE: Rapid TAT carefully dissected or DISADVANTAGE: Poor Quality of Slide separated and METHODS OF PREPARING FROZEN SECTION examined under microscope 1. COLD KNIFE 2. CRYOSTAT SQUASH PREPARATION / CRUSHING COLD KNIFE  Different temperature is employed between the tissue  Small pieces of tissue and the knife (not more than 1mm  Carbon dioxide is the most common freezing agents in diameter) are  Depends on the Ambient Temp. & Humidity placed in microscopic  Dewline: slides & forcibly the point at which sections may be ut at 10 micra compressed with thickness another slide or cover slip Optimum Condition For Cold Knife Sectioning TOUCH PREPARATION / IMPRESSION  special method of smear preparation whereby the surface CRYOSTAT of the freshly cut tissue is brought into  Consist of isolated rotary microtome housed in a contact and pressed electrically driven refrigerated chamber and on the surface of a maintained at near -20 oC where microtome , knife, clean glass slide and specimen atmosphere are kept at the same temperature SMEAR PREPARATION  Techniques useful in Cytological examinations particularly for cancer diagnosis ‘’SMEAR PREPARATION’’ STREAKING  Use of an applicator stick or platinum loop, and the specimen is rapidly & gently applied in a direct or zigzag line throughout the slide SPREADING  Selected portion of the specimen is transferred to a clean slide and gently spread into a moderately thick film by teasing using an applicator stick. METHODS OF FREEZING PULL-APART 1. LIQUID NITROGEN  Two slides are pulled apart in a single uninterrupted 2. ISOPENTANE motion 3. CARBON DIOXIDE 4. AEROSOL SPRAY 5. DRY ICE MT304 LECTURE / MIDTERM DOLORSKIEEE  LIQUID NITROGEN  Most rapid freezing agent  USAGE: Histochemistry & Intra-operative  procedures  ADVANTAGE: Fast TAT  DISADVANTAGE: Tissue prone to crack & Uneven cooling ISOPENTANE  Liquid at room temperature  Cooled by immersing it in liquid nitrogen at -170 oC CARBON DIOXIDE  Commonly used in cold knife procedure  Quick freezing spray cans with fluorinated hydrocarbon  Cryokwik DRY ICE  Not routinely used SPECIAL PROCESSING TECHNIQUES 1. FREEZE-DRYING 2. FREEZE-SUBSTITUTION FREEZE DRYING  Rapid freezing of tissue & subsequently removing ice water molecules (desiccation) by physical process of transferring the still frozen tissue block in a vacuum at higher temperature QUENCHING : -160 oC to -180 oC SPECIMEN IDENTIFICATION SUBLIMATION: -30 oC to -40 oc 1. Patient Information 2. History USAGE: 3. Description of Site of Origin  Demonstration of Hydrolytic Enzymes LABELLING  Mucous Substance  Give each specimen a unique identifier  Glycogen  Proteins Example: S-13-001 ADVANTAGE: S = Surgical, 13 = Year, 001 – Accession Number  Minimum tissue shrinkage GROSSING  Allows tissue processing in fresh state  Inspecting the specimens, describing and measuring DISADVATAGE: the tissue, inking if needed, and sectioning the tissue  Time Consuming Expensive to be processed for diagnosis.  Skin sample provides the most diagnostically valuable parts of a specimen for the pathologist's FREEZE SUBSTITUTION review.  Useful to select relevant portions of tissue for  Similar to freeze drying, the only variation is that the microscopic examination frozen tissue –instead of being subjected to dehydration in an expensive vacuum during MANDATORY PREREQUISITES: apparatus – is fixed in ROSSMAN’S FLUID or in 1%  Knowledge of the Clinical History ACETONE and DEHYDRATE ABSOLUTE ALCOHOL  Thorough knowledge of the anatomy of organ GROSS EXAMINATION MACROSCOPIC ASSESSMENT 1. Location of the lesion 2. Size, Weight, Length 3. Shape and Architecture 4. Color and Consistency 5. Description of pathologic changes MT304 LECTURE / MIDTERM DOLORSKIEEE  SELECTION, CUTTING, AND COLLECTION  Selected tissue should contain the portion of the lesion  Take the youngest lesion from the periphery  Take specimens of from more than one area of affected organs  Cut must be made quickly, sharply, and accurately ( 5-6 mm thick) INSTRUMENT USED IN GROSSING HANDLING OF SPECIMEN  Properly labeled  Wide-mouthed screw-capped, leaked-proof container MT304 LECTURE / MIDTERM DOLORSKIEEE GENERAL PATHOLOGY AND HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES FIXATION AND DECALCIFICATION TISSUE PROCESSING CHARACTERISTIC OF A GOOD FIXATIVE  Steps required to take an animal 1. It must be cheap or human tissue from fixation to 2. It must be stable the state where it is completely 3. It must be safe to handle infiltrated with a suitable 4. It must kill the cell quickly thereby producing minimum histological wax and can be distortion of cell constituents embedded ready for section 5. It must inhibit bacterial decomposition and autolysis cutting on the microtome. 6. It must produce minimum shrinkage of tissue PREANALYTICAL STEP 7. It must permit rapid and even penetration of tissues NUMBERING 8. It must harden tissues thereby making the cutting of  first and most important step before performing sections easier tissue processing FACTORS AFFECTING FIXATION PROCESS  it will serve as an identifier of the patient instead of  Volume its full name  pH / Hydrogen Ion Concentration  use PENCIL (DIAMOND PENCIL)  Temperature  Thickness of Section EXAMPLE: S-24-001  Osmolality SURGICAL; DATE ; ACCESSION NUMBER  Concentration of Fixative S- SURGICAL;  Duration of Fixation C-CYTOLOGICAL;  Time Interval A-AUTOPSY CASSETE  Rate of Penetration OVERVIEW OF CONVENTIONAL TISSUE FIXATIVES VOLUME: PROCESSING 10-25:1 1. Fixation FORMAT: 2. Decalcification WHAT IS TISSUE TO FIXATIVES ON FORMALIN? 3. Dehydration  1:20 4. Clearing WHAT IS FIXATIVES TO TISSUE ON FORMALIN? 5. Impregnation/Infiltration  20:1 6. Embedding/Casting Volume 7. Trimming CHEMICAL TO TISSUE RATIO 8. Sectioning/Cutting Traditional 10-25:1 Dehydration 10:1 9. Staining Fixation 10.Mounting Routine Fixation 20:1 Acetone 20:1 11.Ringing 12.Labeling Dehydration Museum Fixation 50- Clearing 10:1 FIXATION 100:1  Alteration of the tissue by stabilizing protein so that the Osmium 5-10:1 Infiltration 25:1 tissue becomes resistant to further changes Tertoxide  Preserving cells and tissue constituents in a condition Decalcification 20:1 Gelatin 25:1 identical to that existing during life Infiltration FUNCTION: pH/ Temperarure Primary: To preserve morphological and characteristics of FIXATION METHOD TEMPERATURE tissue Traditional Formalin / Mast Cell Room Room Secondary: To harden and protect tissue Temperature temperature Electron Microscopy / Histochemistry 0-4°C *WHAT FIXATIVES FOR mucopolysaccharides? LEAD* Automatic Processor 40°C ‘’FRESH TISSUE IS MORE PRONE TO DECOMPOSITION AND BACTERIA’’ Microwave 45-55°C BENEFITS/ EFFECTS OF FIXATION 1. Allow thin sectioning of tissue by hardening tissue Rapid Formalin 60°C 2. Prevents autolysis and inactivates infectious agents MTB (Tuberculosis) 100°C (except prion diseases) 3. Improved cell avidity for special stains NOTE: Thickness *MAS MAKAPAL MAS MATAGAL Light Microscopy 2cm2 dimension to 0.4 cm thick MAS MANIPIS MAS MABILIS Electron Microscopy 1-2 mm2 MT304 LECTURE / MIDTERM DOLORSKIEEE Osmolality ACCORDING TO ACTION Slightly Hypertonic or Isotonic  MICROANATOMICAL 400 to 450 mOsm/kg - Fixative that permits general microscopic study of tissue * Hypertonic = Shrink; Hypotonic =Swell* _addition structures without altering the structural pattern and normal intercellular relationship of tissues. Concentrations  NUCLEAR Routine Fixation 10% Formalin - preserve the specific elements of the cell  CYTOLOGICAL - preserve the specific elements of the cell Duration of Fixation  HISTOCHEMICAL Routine 2-6 hours - preserve the chemical constituent of cell and tissues Electron Microscopy 3 hours NUCLEAR CYTOPLASMIC PRESERVES Preserves Nuclear Preserves Cytoplasmic Time Interval Structures Elements Immediately to prevent autolysis of cell in tissues GLACIAL Contains Glacial Acetic NEVER contain Glacial ACETIC Acid Acetic Acid Rate of Penetration ACID (YES) (NO; because it Routine 1 mm per hour destroys mitochondria and FACTORS AFFECTING FIXATION PROCESS Golgi bodies) IMPROVES FIXATION: pH ≤ 4.6 >4.6 Mema mnemonics 1. Size & Thickness 2. Agitation TYPES OF FIXATIVE ACCORDING TO ACTION 3. Moderate MICROANATOMICAL “AMP****” Aldehydes, Metallic RETARDS FIXATION: (delay or hold back) (Mercuric, Chromate), Picric 1. Larger Tissues NUCLEAR (w/ Glacial A.A.) “BFNCH” 2. Presence of Mucus Bouin’s. Flemming’s, 3. Presence of Blood Newcomer, Carnoy’s, 4. Presence of Fat Heidenhain's Susa 5. Cold Temperature CYTOPLASMIC (w/o Glacial “FFORK” TYPES OF FIXATION A.A.) Flemming's w/o G.A.A.,  PHYSICAL METHOD Formalin w/ Post-Chroming,  CHEMICAL METHOD Orth 's, Regaud 's fluid MAJOR GROUP OF FIXATIVES (Muller 's fluid), Kelly's  ALDEHYDES HISTOCHEMICAL “FANA”  OXIDIZING AGENTS Formal Saline, Absolute Ethyl  ALCOHOL Alcohol, Newcomer's Fluid,  METALLIC Acetone FIXATIVE MAJOR CLASSIFICATION MICROANATOMICAL + Nuclear Heidenhain's  ACCORDING TO COMPOSITION Susa  ACCORDING TO ACTION Cytoplasmic Helly’s (Zenker- ACCORDING TO COMPOSITION Formol)  SIMPLE FIXATIVES Histochemical Formol - made of only one component (pure) Saline Examples: OLD VERSION: ACCORDING TO ACTION Aldehydes, Acetone, Metallic, Alcohol, Picric acid,  CROSSLINKING FIXATIVE Osmium tetroxide, and Acetic acid - act by creating covalent chemical bonds between proteins  COMPOUND FIXATIVES in tissue. - made of two or more components (mixed)  PRECIPITATING/DENATURING FIXATIVE - act by reducing the solubility of protein molecules and disrupting the hydrophobic interaction in proteins MT304 LECTURE / MIDTERM DOLORSKIEEE  FIXATION MECHANIMS II. PARAFORMALDEHYDE  ADDITIVE  Polymerized form of formaldehyde - chemical constituents of the fixative are taken in and become part of the tissue  Obtained as fie white powder which depolymerize back to formalin when heated  NON-ADDITIVE - fixing agent is not taken into the tissue but rather alters III. GLUTARALDEHYDE the composition of tissue for stability  Made up of 2 formaldehyde residue linked by 3 SECONDARY FIXATION carbon chain Process of placing an already fixed tissue in a second fixative in  Provide better cellular structure order: CONCENTRATION: 1. To facilitate and improve the demonstration of a particular  2.5 % -small tissue fragments substance.  4% larger tissues 2. To make special staining techniques possible (secondary IV. KARNOVSKY’S FIXATIVE fixative acting as a mordant)  4% Paraformaldehyde + 1% Glutaraldehyde in 0.1 3. To ensure complete and further hardening and Preservation  M Phosphate buffer POST-CHROMATIZATION  Used in Electron Microscopy Form of secondary fixation whereby a primarily fixed tissue is METALLIC FIXATIVES placed in aqueous solution of 2.5 to 3% potassium dichromate METALLIC FIXATIVES - MERCURIC CHLORIDE for 24 hours to act as mordant for better staining effects and to  Most common metallic fixative aid in cytologic preservation of tissues.  Use in tissue photography WASHING OUT  Tissue fixed with mixtures containing mercury Process of removing excess fixative from the tissue after fixation chloride contain black precipitates of mercury in order to improve staining and remove artifacts from the  Corrosive to metal tissue.  Must not be drained in sink COMMONLY USED: Tap Water, Alcohol, Iodine MAJOR GROUP OF FIXATIVE I. ZENKER FLUID  ALDEHYDES  Recommended for fixing a small piece of liver, spleen,  OXIDIZING AGENTS connective tissue fibers, and nuclei  ALCOHOL  Before using, add glacial acetic acid first  METALLIC ALDEHYDE FIXATIVES II. ZENKER-FORMOL I. FORMALDEHYDE / FORMALIN  Excellent for bone marrow, extramedullary  FORMALDEHYDE- Precursor of formalin hematopoiesis and intercalated discs of the cardiac  Produced from methanol oxidation muscle  10% Formalin  Excellent microanatomic fixative for pituitary gland,  Buffered at pH=7 using Phosphate Buffer bone marrow and blood-containing organs such as STOCK SOLUTION: 37-40% (*40% CONSIDERED EQUAL TO 100%) spleen and liver WORKING SOLUTION: 10% III. HEIDENHAIN’S SUSA DILUTION: 1:10 (1 part stock, 10 parts water)  Excellent cytologic fixative recommended for tumor FORMALDEHYDE / FORMALIN VARIANTS: biopsies, especially of the skin  10% Formol Saline  Produces minimum shrinkage and hardening of  10% Neutral Buffered Formalin tissues due to the counter-balance of the swelling  Formol Sublimate / Corrosive effects of the acids and the shrinkage effect of  Alcoholic Formalin / Gendre’s Fluid mercury  Zinc Formalin IV. LILLIE’S B-5 FIXATIVE  Enhances nuclear detail, which is important for identifying normal and abnormal cell types in (hematopoietic tissue) bone marrow  Used for cytology of bone marrow biopsies MT304 LECTURE / MIDTERM DOLORSKIEEE MEMAMNEMONICS 3. Will produce excessive HEIDENHAIN’S SUSA staining of tissues What fixative for tumor biopsies? 4. highly explosive when dry - si susa may tumor II. BOUIN’S SOLUTION LILLIE’S B-5 FIXATIVE  Recommended for fixation of embryos and pituitary - si lili ay buntis biopsies METALIC FIXATIVES- CHROMATE  Excellent in preserving soft and delicate structures I. CHROMIC ACID (endometrial curetting)  It precipitates all proteins and adequately preserves  Yellow stain is useful in fragmentary biopsies carbohydrates.  Preserved glycogen  It is a strong oxidizing agent; hence a strong reducing  Not suitable for fixing kidney structures, lipid and agent (e.g. formaldehyde) must be added to chrome- mucus containing fixatives before use to prevent What fixatives for embryos? counteracting effects and consequent decomposition - bouin’s solution of solution upon prolonged standing. II. POTASSIUM DICHROMATE III. BRASIL’S ALCOHOLIC PICROFORMOL  Used in a 3% aqueous solution  Better and less messy than Bouin’s  Preserves mitochondria (if used in pH 4.5 to 5.2,  Excellent fixative for glycogen mitochondria is fixed; If the solution becomes COMPOSITION: acidified, cytoplasm, chromatin bodies and  37% Formaldehyde chromosomes are fixed but the mitochondria are  Picric Acid destroyed)  Ethanol or Isopropyl III. REGAUD’S / MULLER’S FLUID IV. HOLLANDE’S SOLUTION  Recommended for demonstration of chromatin, Golgi  Recommended for gastrointestinal tract specimens bodies, mitochondria, mitotic figures, RBC and and fixation of endocrine tissue colloid-containing tissues  Has decalcifying properties METALIC FIXATIVES- LEAD COMPOSITION: I. LEAD FIXATIVE  Copper acetate Acetic Acid  Lead oxaloacetate, a primary reaction product  Picric Acid Distilled Water precipitate for the visualization of the activity of  40% Formaldehyde glutamic oxaloacetic transaminase in tissue sections GLACIAL ACETIC ACID  Recommended for Acid mucopolysaccharides  An essential constituent of most compound nuclear  Fixes connective tissue mucin fixatives PICRIC ACID FIXATIVES  An anhydrous (water-free) acetic acid and it solidifies I. PICRIC ACID FIXATIVE at 17°C  It dyes the tissue, but the yellow color can be removed  Fixes and precipitates nucleoproteins by treatment with another acid dye or lithium  Precipitates chromosomes and chromatin materials; carbonate hence very useful in the study of nuclear components PICRIC ACID FIXATIVE of the cell ADVANTAGES DISADVANTAGES  Causes tissues (esp. those containing collagen) to 1. Excellent fixative for 1. Causes RBC hemolysis and swell which counteracts the shrinkage produced by glycogen demonstration reduces the demonstrable other compound fixative components (e.g. mercury) amount of ferric iron in 2. Yellow stain prevents ALCOHOLIC FIXATIVES tissue small fragments to be ALCOHOL overlooked 2. Not suitable for frozen  Denatures and precipitates proton by destroying section, because it causes hydrogen and other bonds 3. Suitable for Aniline stains frozen  Used in concentrations ranging from 70 to 100% (less sections to crumble when cut concentrated solutions will produce cell lysis)  Fix and preserve glycogen, pigments, blood tissue films and smears MT304 LECTURE / MIDTERM DOLORSKIEEE   Both fixative and a dehydrating agent  Used for the precipitation of proteins and nucleic I. METHYL ALCOHOL (100%) acids  Fixing dry and wet smears, blood smears and bone  Also a weak decalcifying agent marrow tissues ACETONE II. ETHYL ALCOHOL  Fixes by dehydration and precipitation  Used for histochemistry and enzyme studies  Used at ice cold temperature ranging (-5 to 40c)  Most usable DNA fragments for PCR  Recommended for water diffusible enzymes  If lower concentrations are used, RBCs become  Used in fixing brain tissue for the diagnosis of rabies hemolyzed and WBCs are Inadequately preserved TYPES OF FIXATION III. CARNOY’S  PHYSICAL METHOD  Most rapid fixative  CHEMICAL METHOD  Recommended for chromosomes, lymph glands and PHYSICAL METHODS OF FIXATION urgent biopsies  HEAT FIXATION  Fix brain tissues for the diagnosis of rabies  MICROWAVE FIXATION IV. ALCOHOLIC FORMALIN (GENDRES)  FREEZE-DRYING & FREEZE-SUBSTITUTION  Used for fixation or post-fixation of large fatty specimens RABIES FIXATIVES  (breasts), since it allows lymph nodes to be more 2 FIXATIVES FOR RABIES DIAGNOSIS easily 1. Carnoy’s 2. Acetone  detected as it clears and extracts lipids V. NEWCOMER’S Example for brain tissue  Recommended for fixing mucopolysaccharides and nuclear proteins MEMANEMONICS: CAR (Carnoys & Acetone for Rabies)  FIXING TIME: 12-18 hours psychopathic effects- VI. CLARKE’S nangyayari sa brain due to infection (negri bodies)  Recommended for frozen sections and smears negri bodies are eosinophilic, sharply outlined inclusion bod *GENDRE’S ies found in the cytoplasm of certain nerve cells infected wit  Good preservation of glycogen and for microincineration h the rabies virus technique  Used to fix sputum, since it coagulates mucus Carnoy's solution is a mixture of 60% ethanol, 30% chloroform, and FORMOL-ACETIC ALCOHOL 10% glacial acetic acid. It is primarily used for fixing tissues in histol  Faster acting agent compared to alcoholic formalin ogical studies and enhancing lymph node detection during dissectio  It may be used to fix diagnostic cryostat sections* n.It helps preserve tissue structure and is often used in immunohist OSMIC ACID FIXATIVES ochemical detection OSMIUM TEROXIDE  Pale yellow powder which dissolves in water to form Acetone is an organic solvent used as a fixative for smears of brain t issue in the fluorescent antibody test (FAT) for rabies, a strong oxidizing solution It is preferred because it preserves antigenicity and maintains the s  Used traditionally in electron microscopy as a fixative tructure of the tissue  Excellent lipid stain in membranous structures and vesicles *note natatanong sa board exam  Fixes fats and lipids  Preserves cytoplasmic structures FLEMMING’S  Most common Chromic-Osmium Acetic Acid fixative  Recommended for nuclear preparation FLEMMING’S without GLACIAL ACETIC ACID  Recommended for cytoplasmic structures particularly the Mitochondria OTHER FIXATIVES TRICHLOROACETIC ACID MT304 LECTURE / MIDTERM DOLORSKIEEE  OVERVIEW OF CONVENTIONAL TISSUE PROCESSING 1. Fixation COMMON FORMULATIONS: 2. Decalcification  10% Aqueous Nitric Acid 3. Dehydration  Formol Nitric Acid: Nitric acid + formaldehyde + water 4. Clearing  Pereyeni's Fluid: Nitric acid + formic acid + ethyl 5. Impregnation/Infiltration alcohol (softens tissue) 6. Embedding/Casting 7. Trimming PHLOROGLUCIN-NITRIC ACID: Fastest agent 8. Sectioning/Cutting STRONG ACIDS -HYDROCHLORIC ACID 9. Staining HYDROCHLORIC ACID 10.Mounting  Used for surface decalcification (e.g., Von Ebner 11.Ringing solution) 12.Labeling WEAK ACIDS (WEAKER ORGANIC ACIDS) DECALCIFICATION FORMIC ACID:  Process of completely removing calcium or lime salts from  Safer than strong acids; used for post-mortem tissues tissues (bones, teeth, calcified tissues) following fixation. COMPOSITION: Formic acid + 10% Formol saline TIMING: after fixation and before impregnation TRICHLOROACETIC ACID:  A weak decalcifying agent. *Which is conventional is optional? COMPOSITION: Trichloroacetic acid +10% Formol saline - Decalcification SULFUROUS ACID: - Matagal ang process ng decalcification –umaabot ito  Very weak decalcifying agent. ng 1-2days (24-48 hrs) CHROMIC ACID (Flemming's Fluid): - FACTORS INFLUENCING DECALCIFICATION RATE  Used as both fixative and decalcifying agent  Concentration of Decalcifying Agent CARCINOGENIC AND CORROSIVE; composition:  Fluid to Tissue Ratio COMPOSITION: Chromic acid + osmium tetroxide + glacial acetic  Temperature acid  Mechanical Agitation CHELATING AGENTS  Ideal Time Required  Substances that bind with calcium ions and other salts (e.g., iron, magnesium) to form weakly dissociated CONCENTRATION OF DECALCIFYING AGENT: complexes, facilitating the removal of calcium  Higher concentrations lead to faster decalcification EDTA (Versene) but may damage tissues. COMMON USE: Recommended for detailed microscopic studies FLUID TO TISSUE RATIO: MECHANISM: Binds with metallic ions (calcium and magnesium)  RECOMMENDED RATIO: 20:1 (fluid volume). to form insoluble, non-ionized complexes TEMPERATURE: ADDITIONAL USES: Anticoagulant and water softener  IDEAL: 18-20°C  37°C: Impairs nuclear staining (e.g., Van Gieson stain) DECALCIFICATION RATE:  55°C: Tissues may undergo complete digestion within  Small specimens: 1-3 weeks 24-48 hours  Dense cortical bone: 6-8 weeks or longer ION EXCHANGE RESINS MECHANICAL AGITATION  Ammonium form of polystyrene resin used to hasten  Enhances the process decalcification IDEAL TIME REQUIRED MECHANISM: Removes calcium ions from formic acid-  Generally 1-2 days (24-48 hours) containing decalcifying solutions VOLUME RATIO: 20-30 times the volume of the tissue METHODS OF DECALCIFICATION ELECTROLYTIC DECALCIFICATION CHEMICAL METHODS PROCESS: Utilizes electricity to attract positively charged  Most common approach using acids. calcium ions to a negative electrode STRONG ACIDS APPLICATION: Effective for small bone fragments STRONG ACIDS- NITRIC ACID (5-10%)  Fastest decalcifying agent. SOLUTION COMPOSITION: Usually involves a specific  Inhibits nuclear stains and can damage tissue; formulation (not detailed) mitigated by combining with formaldehyde or alcohol MT304 LECTURE / MIDTERM DOLORSKIEEE  MICROWAVE DECALCIFICATION Fastest is OVERVIEW: A novel technique compared to traditional methods Phoglucin- nictric acid PROCEDURE: Gold standard decalcification to check bono have tissue?  Hard tissues are placed in a decalcifying agent inside X ray or radiologic method a microwave oven  The process involves intermittent periods of Most ginagamit is CHEMICAL method microwaving, with regular changes of the solution until the endpoint is reached MEASURING DECALCIFICATION PHYSICAL OR MECHANICAL TEST  Involves touching, bending, or applying pressure with a fingernail or fine needle to assess pliability LIMITATIONS:  Vague and Inaccurate: May yield unreliable results  False Positives: Can indicate micro-fractures that may not be present  Tumor Disruption: Risks damaging tumors during testing  Detection Issues: Small calcified foci may go unnoticed. X-RAY OR RADIOLOGICAL METHOD  Most sensitive and reliable method for measuring decalcification  LIMITATIONS:  Cost: Expensive to implement  Incompatibility: Not suitable for tissues fixed with mercuric chloride CHEMICAL METHOD (CALCIUM OXALATE TEST)  Simple, reliable, and convenient for routine decalcification assessment SOLUTIONS USED:  Ammonium Hydroxide  Saturated Ammonium Oxalate PROCEDURE:  Conducted on the discarded decalcifying fluid  Detection involves the precipitation of insoluble calcium hydroxide or calcium oxalate TISSUE SOFTENERS TISSUE SOFTENERS  are used to facilitate better processing of calcified tissues  PERENYI'S FLUID: A common tissue softener  4% AQUEOUS PHENOL SOLUTION: Used to soften tissues effectively  MOLLIFEX: Another option for tissue softening  2% HCL OR 1% HCL IN 70% ALCOHOL: Used for softening tissues MT304 LECTURE / MIDTERM DOLORSKIEEE GENERAL PATHOLOGY AND HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES DEHYDRATION AND CLEARING OVERVIEW OF CONVENTIONAL TISSUE PROCESSING 4 BUTYL 1. Fixation ✓ Excellent substitute for ethanol 2. Decalcification ✓ Suitable for microwave processing 3. Dehydration 4. Clearing 5 PENTANOL 5. Impregnation/Infiltration ✓ Less common but used similarly 6. Embedding/Casting 6 DENATURED ALCOHOL 7. Trimming ✓ Industrial methylated spirit 8. Sectioning/Cutting ✓ Contains toxic additives 9. Staining 10.Mounting ACETONE  Clear, colorless, miscible with water, ethanol, and 11.Ringing many solvents 12.Labeling  Rapid dehydrator; useful for urgent biopsies DEHYDRATION (dehydrates in 17 to 2 hours)  Process of removing water from the tissue following  Requires volume of at least 20x that of tissue fixation in preparation for wax impregnation.  Placing fixed tissues in ascending grades of a LIMITATIONS: dehydrating agent.  Not recommended for routine use (removes  RATIO (Dehydrating agent to Tissue): 10:1 lipids, causes shrinkage).  Controlled substance in large quantities due to WHY WE NEED TO DEHYDRATE TISSUE? use in methamphetamine production. - To remove excess water DIOXANE  Excellent dehydrating and clearing agent If incomplete dehydration  Miscible with various solvents - used xylene  Cumulative toxicity and carcinogenic  Risk of explosive peroxides TISSUE PLACE DEHYDRATION TURN INTO MILKY? - because of incomple process of dehydration DIOXANE DEHYDRATION METHODS DEHYDRATING AGENTS 1. GRAUPNER'S METHOD: 1. Alcohol  Uses pure dioxane and paraffin. 2. Acetone 3. Dioxane 2. WEISHBERGER'S METHOD: 4. Other dehydrating agents  Tissues wrapped in gauze and submerged in I. cellosolve dioxane with calcium oxide. II. tetrahydrofuran OTHER DEHYDRATING AGENTS III. triethyl phosphate CELLOSOLVE  Α.Κ.Α. ETHYLENE GLYCOL MONOETHYLETHER ALCOHOL  Combustible and toxic by inhalation and skin contact  Most commonly used dehydrating agent.  Best at 37°C for quicker dehydration. TETRAHYDROFURAN (THF)  Use anhydrous copper sulfate (1½ inch layer) to  Excellent dehydrating agent absorb moisture; blue discoloration indicates  Toxic if ingested or inhaled saturation.  Irritant to eyes and skin; prolonged exposure risks conjunctival irritation. TYPES OF ALCOHOL 1 ETHYL TRIETHYL PHOSPHATE ✓ Clear, colorless, flammable  Least commonly used dehydrating agent ✓ Fast-acting, non-toxic, and inexpensive. ✓ Ideal for routine dehydration 2 METHYL ✓ Toxic ✓ used mainly for blood and tissue smears 3 ISOPROPYL ✓ Excellent substitute for ethanol ✓ Suitable for microwave processing MT304 LECTURE / MIDTERM DOLORSKIEEE  OVERVIEW OF CONVENTIONAL TISSUE PROCESSING TOLUENE 1. Fixation  Clearing time of 1 to 2 hours 2. Decalcification 3. Dehydration USES: 4. Clearing  Can substitute for xylene or benzene in both 5. Impregnation/Infiltration embedding and mounting procedures 6. Embedding/Casting 7. Trimming BENZENE 8. Sectioning/Cutting  Rapid acting (15 to 60 minutes) 9. Staining  Highly flammable 10.Mounting 11.Ringing RISKS: 12.Labeling  Extremely toxic and carcinogenic with excessive exposure CLEARING  Potential to damage bone marrow and cause aplastic  A.K.A DECOLORIZATION anemia  Process of removing alcohol/dehydrating fluid from tissue and replacing it with an intermediate solvent that CHLOROFORM is miscible with both ethanol and paraffin wax  Suitable for tough tissues (skin, decalcified tissues)  RATIO (Clearing agent to tissue): 10:1  Causes minimal shrinkage and hardening APPLICATIONS OF CLEARING TRANSPARENCY: LIMITATIONS:  Makes tissues, embryos, and parasites transparent.  Does not render tissue transparent PREPARATION FOR WAX IMPREGNATION:  Toxic to the liver with prolonged inhalation  Prepares tissues for embedding in paraffin. DE-ALCOHOLIZATION:  Prepares stained sections prior to mounting. CEDARWOOD OIL CLEARING AGENTS  Used for clearing both paraffin and celloidin sections 1. Xylene  Recommended for CNS tissues and cytological studies 2. Toluene 3. Benzene LIMITATIONS:  Extremely slow clearing; becomes milky upon 4. Chloroform prolonged storage 5. Cedarwood Oil 6. Aniline Oil ANILINE OIL 7. Clove Oil  Effective for clearing embryos and delicate specimens 8. Methyl benzoate/salicylate  Clears 70% alcohol without excessive shrinkage or 9. OTHER CLEARING AGENTS hardening I. Terpenes (Plant Oils) II. Limonene CLOVE OIL III. N-Butyl Acetate  Quality is variable IV. OTHER AGENTS  Often adulterated a. Carbon Tetrachloride b. Tetrahydrofuran METHYL BENZOATE/SALICYLATE c. Dioxane  Slow-acting clearing agents d. Orange Oil Clearing Agents  Useful for double embedding techniques e. Chlorinated Hydrocarbons f. Bleached Palm Oil OTHER CLEARING AGENTS XYLNE / XYLOL Terpenes (Plant Oils)  Colorless  Historical solvents  Most commonly used clearing agent  Examples include turpentine, bergamot oil  Rapid clearing (15 to 30 minutes) Limonene USES:  Citrus-derived xylene substitute  Suitable for embedding and mounting procedures  Acts as a de-waxing agent during staining N-Butyl Acetate LIMITATIONS:  Xylene substitute and nitrocellulose solvent  Causes hardening and shrinkage  Unsuitable for nervous tissues and lymph nodes  May turn milky with incompletely dehydrated tissues MT304 LECTURE / MIDTERM DOLORSKIEEE  Other Agents:  Carbon Tetrachloride  Tetrahydrofuran  Dioxane  Orange Oil Clearing Agents  Chlorinated Hydrocarbons  Bleached Palm Oil SUMMARY Aspect DEHYDRATION CLEARING DEFINITION Removal of water Removal of from tissue dehydrating fluid and replacement with a solvent PURPOSE Prepares a tissue Prepares tissue for for embedding in wax impregnation paraffin and transparency VOLUME Sufficient to 10 times the REQUIREMENT replace moisture volume of the tissue COMMON  Ethyl  Xylene AGENTS  Alcohol  Toluene  Acetone  Chloroform  Dioxane SPEED Varies; some Generally Rapid agents are fast- (e.g., XYLENE clears acting in 15-30 min.) EFFECT ON Can cause Varies; some cause TISSUE shrinkage and minimal shrinkage brittleness USE CASES Routine Making tissues processing and transparent for fixation microscopy LIMITATIONS May remove Some agents may lipids be toxic (e.g., BENZENE) TRANSPARENCY No transparency Enhances transparency for better visualization MT304 LECTURE / MIDTERM DOLORSKIEEE GENERAL PATHOLOGY AND HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES IMPREGNATION AND EMBEDDING OVERVIEW OF CONVENTIONAL TISSUE TYPES OF IMPREGNATING & EMBEDDING MEDIA PROCESSING 1. Fixation 1. PARAFFIN 2. Decalcification 2. CELLOIDIN 3. Dehydration 3. GELATIN 4. Clearing 4. PLASTIC 5. Impregnation/Infiltration PARAFFIN WAX 6. Embedding/Casting 7. Trimming  Simplest and most common embedding medium. 8. Sectioning/Cutting 9. Staining TEMPERATURE CONTROL: 10.Mounting  OPTIMAL INFILTRATION at 2-5°C above the melting 11.Ringing point (MP). 12.Labeling  ROUTINE MELTING POINT: 66°C. IMPREGNATION/INFILTRATION  FOR 20-24°C LAB TEMPERATURES: Use wax with MP IMPREGNATION of 34-58°C.  A.K.A. INFILTRATION  Process of removing clearing agents from tissue and  FOR 15-18°C LAB TEMPERATURES: Use wax with MP replacing them with a medium that fills all tissue cavities of 50-54°C.  Ensures that the tissue is adequately filled and PARAFFIN WAX SPECIFICATIONS supported for subsequent slicing and staining  PURITY REQUIREMENTS: RATIO (Impregnation agent to Tissue): 25:1  Must be free from dust, water, and foreign EMBEDDING matter  -A.K.A. CASTING, BLOCKING, MOLDING  Fresh wax should be filtered before use at 2°C  -Paraffin-impregnated tissues are placed into a mold above Melting point with the embedding medium. METHODS OF PARAFFIN IMPREGNATION & EMBEDDING PROCESS: 1. Manual Processing (Hand Processing)  Each tissue is labeled appropriately 2. Automatic Processing (Autotechnicon)  Mold is immersed in melted paraffin at 3. Vacuum Embedding temperatures 6 to 10°C above the melting point (MP) of paraffin. 1. MANUAL PROCESSING (HAND PROCESSING)  Rapid cooling occurs at -5°C or through immersion  Requires 4 changes of wax at 15-minute intervals in cold water to solidify the medium.  Final immersion in fresh melted paraffin for 3 Hours ORIENTATION IN EMBEDDING  Precise arrangement of tissue in the mold during 2. AUTOMATIC PROCESSING (AUTOTECHNICON) embedding, on the microtome before cutting, and on  Involves 12 processing steps the slide before staining  Temperature maintained at 3°C above wax MP  Most crucial step in embedding → ensuring that  Advantages include constant agitation tissue sections are cut accurately and can be stained 3. VACUUM EMBEDDING effectively for analysis  Conducted under negative pressure  Recommended for urgent biopsies and delicate ASPECT IMPREGNATION/ EMBEDDING tissues INFILTRATION  Fastest method, temperature at 2-4°C above MP Definition Process of Process of placing replacing clearing paraffin- PARAFFIN WAX SUBSTITUTE agents in tissue impregnated 1. PARAPLAST with a filing tissue into a mold 2. EMBEDDOL medium for sildification 3. BIOLOID Purpose Ensures comple Prepares tissue for filing of tissue sectioning and 4. TISSUE MAT cavities for support staining 5. ESTER WAX 6. WATER-SOLUBLE WAX MT304 LECTURE / MIDTERM DOLORSKIEEE  WATER- 38-42°C Plastic Excellent Enzyme 1.PARAPLAST = Melting Point: 56-57°C SOLUBLE 45-56°C polymers preservation of Histo- WAX (e.g., cytological chemical  A mixture of purified paraffin and synthetic polyethylene details studies polymer glycol) >18 Polyethylene Water-soluble Cytology  Suitable for large, dense tissue blocks CARBOWAX Carbon glycol And atoms Requires Nato- 2. EMBEDDOL = Melting Point: 56-58°C special chemistry sectioning  Less brittle and compressible than paraplast techniques 3.BIOLOID  Semi-synthetic wax CELLOIDIN  Ideal for embedding eyes METHOD OF CELLOIDINN EMBEDDING 4.TISSUE MAT 1. DRY CELLOIDIN 2. WET CELLOIDIN  Paraffin-based product containing rubber 1. DRY CELLOIDIN 5.ESTER WAX = Melting Point: 46-48°C APPLICATION: Preferred method for processing whole eye  Soluble in 95% ethanol sections.  Allows direct Impregnation TECHNIQUE: Utilizes Gilson's mixture (a blend of chloroform 6.WATER-SOLUBLE WAX = Melting Point: vary 38-42°C, 45- and cedarwood oil) to render the tissue transparent 56°C COMPOSITION: Primarily polyethylene glycols 2. WET CELLOIDIN EXAMPLE: Carbowax APPLICATION: Suitable for embedding bones, teeth, large FLOATING SECTIONS: brain sections, and whole organs  Cannot float on water TECHNIQUE Tissue blocks are stored in 70%-80% alcohol to  use alternative solutions: prevent dehydration and shrinkage Pearse Solution: Diethylene glycol, distilled water, strong formaldehyde. CELLOIDIN ALTERNATIVE METHODS Blank and McCarty Solution: Gelatin and 1. LOW VISCOSITY NITROCELLULOSE (LVN) potassium dichromate. 2. DOUBLE EMBEDDING METHOD SUMMARY in paraffin wax 1. LOW VISCOSITY NITROCELLULOSE (LVN) TYPES MELTING POINT (°C) COMPOSITION CHARACTERISTICS USES  A variant of celloidin, soluble in equal parts ether Common and alcohol PARRAFIN 66°C Pure Standard SAFETY NOTE: WAX paraffin Routine used embedding  Highly explosive when dry; care must be taken to for tissue Prone to processing prevent striking or dropping the container shrinkage of  Typically sold while wet with alcohol overhead PARAPLAST 56-57°C Mixture of Less brittle Embedding STORAGE PRECAUTIONS: purified bones and  Containers must be tightly sealed and kept out of paraffin and Ideal for large brain tissue synthetic dense blocks sunlight to minimize alcohol evaporation polymer EMBEDDOL 56-58°C Synthetic Less brittle General 2. DOUBLE EMBEDDING METHOD wax embedding TECHNIQUE OVERVIEW: Compressible than paraplast BIOLOID  Tissue is first infiltrated with celloidin, then N/A Semi- Recommend Embedding synthetic for dedicate eyes embedded in paraffin wax wax tissues TISSUE N/A Paraffin with Similar Embedding BENEFITS: MAT rubber properties to various  Facilitates the cutting of large blocks of dense and paraplast tissues firm tissues ESTER WAX 46-48°C Ester-based Soluble in 95% Tissue ethanol embedding  Recommended for making small sections from without celloidin blocks Allows direct prior impregnation clearing MT304 LECTURE / MIDTERM DOLORSKIEEE  SUMMARY in celloidin PLASTIC CELLOIDIN  provides superior results for light microscopy ASPECT DETAILS  For hard tissues such as undecalcified bone Definition Purified form of nitrocellulose, soluble in many  used in electron microscopy, enhancing the detail and solvent clarity of tissue structures Primary use Store tissue blocks in 70%-80% alcohol to prevent dehydration RESOLUTION: CELLOIDIN METHODS  This method allows for high-resolution light METHODS APPLICATION TECHNIQUES microscopy of tissue sections that are thinner than Dry Celloidin Whole eye sections Use Gilson’s mixture the standard 4-6 μm, making it ideal for renal and (chloroform and bone marrow biopsies cedarwood oil) for transparency TYPES OF PLASTIC EMBEDDING MEDIA Wet Celloidin Bones, teeth, large Store tissue blocks in 1. EPOXY brain section, whole 70%-80% alcohol to 2. POLYESTER organs prevent dehydration 3. ACRYLIC Nitrocellulose Low viscosity Explosive when dry; 1. EPOXY PLASTICS Method Nitrocellulose keep containers sealed (LIV.N.), soluble in and away from COMPOSITION: ether and alcohol sunlight  Made from a carefully balanced mixture of epoxy Double First infiltrate with Facillitates cutting of plastic, catalysts, and accelerators Embedding celloidin, then large, dense tissue Method embed in paraffin blocks CARCINOGENIC COMPONENT: wax  Vinyl cyclohexane dioxide (VCD) is a known, ‘’carcinogen’’ GELATIN  Gelatin method is rarely used TYPES OF EPOXY PLASTICS  Primarily when dehydration of tissues needs to be 1. BISPHENOL A avoided 2. GLYEROL  Suitable for histochemical and enzyme studies 3. CYCLOHEXENE DIOXIDE  For delicate specimens and frozen sections 4. POLYESTER PLASTICS VOLUME REQUIREMENT: 5. ACRYLIC PLASTICS  should be at least 25 times the volume of the tissue 1. BISPHENOL A to ensure proper embedding  Also known as ARALDITE TISSUE THICKNESS: INFILTRATION: Slow due to the large size of its molecules  tissue does not exceed a thickness of 2-3 mm to 2. GLYCEROL achieve optimal results  Known as EPON. MOLD PREVENTION: VISCOSITY: Has a lower viscosity than some other epoxy  To prevent mold growth during the embedding plastics process, 1% phenol is added to the gelatin FORM: Sold as a mixture of isomers SUMMARY in gelatin GELATIN 3. CYCLOHEXENE DIOXIDE  Known as SPURR ASPECT DETAILS CHARACTERISTICS: Pure with very low viscosity Usage Rarely used INFILTRATION SPEED: Infiltrates the fastest among the Primarily for avoiding dehydration epoxy plastics Applications Suitable for histochemical and enyme studies 4. POLYESTER PLASTICS Delicate specimens and frozen sections  Originally introduced for electron microscopy, Volume At least 25 times the volume of the tissue they are now seldom(rarely) used Requirement Tissue Should not exceed 2-3 mm thickness 5. ACRYLIC PLASTICS Mold Addition of 1% phenol prevents mold growth  Extensively used for light microscopy, they include preservation polyglycol methacrylate (GMA) and methyl methacrylate (MMA) MT304 LECTURE / MIDTERM DOLORSKIEEE  SUMMARY in plastic 3.PLASTIC EMBEDDING RINGS AND BASE MOLDS PLASTIC  Made of stainless steel ASPECT DETAILS with a plastic embedding Advantage Superior results for light microscopy ring, which serves as a block holder during cutting Ideal for hard tissues like decalcification 4. DISPOSABLES Resolution High-resolution for tissue sections 1. PEEL-AWAY MOLDS thinner than 4-6um (e.g., renal and bone marrow 2. PAPER BOATS biopsies) Applications Suitable for embedding in electron 3. PLASTIC ICE TRAYS microscopy A. PEEL-AWAY MOLDS TYPES OF PLASTIC EMBEDDING -These provide perfect, 1. EPOXY Mixture of epoxy plastic, catalyst, even blocks without the PLASTICS and accelerators need for trimming Biphenol A Known as ARALDITE Slow infiltration due to large molecules B. PAPER BOATS Glycerol Known as EPON -Typically used for Lower viscosity celloidin but can also be Sold as mixture of isomers adapted for paraffin Cyclohexene Dioxide Known as SPURR Low viscosity Fastest infiltration C. PLASTIC ICE TRAYS 2. POLYESTERS Introduced for electron microscopy -Recommended for busy PLASTICS laboratories for efficient Now rarely (seldom) used embedding. 3. ACRYLIC Extensively used for light microscopy PLASTICS (e.g. GMA and MMA) MOLDS FOR EMBEDDING 1. LEUKHART'S EMBEDDING MOLD 2. COMPOUND EMBEDDING UNITS 3. PLASTIC EMBEDDING RINGS AND BASE MOLDS 4. DISPOSABLES 1.LEUKHART'S EMBEDDING MOLD  Consists of L-shaped strips with adjustable heavy brass sizes  Recommended for routine used  However, it may be too slow and Cumbersome for busy Laboratories. 2.COMPOUND EMBEDDING UNITS  Consist of interlocking plates resting on a flat metal base  Allowing multiple specimens to be embedded simultaneously MT304 LECTURE / MIDTERM DOLORSKIEEE  SUMMARY for molds embedding MOLDS FOR EMBEDDING MOLD TYPE DESCRIPTION ADVANTAGES/NOTES Leukhart's L-shaped strips Recommended for Embedding Mold with routine use adjustable size Cumbersome for busy labs Compound Interlocking Allows simultaneous Embedding Units plates for embedding embedding multiple specimens Plastic Embedding Stainless steel Serves as a block Rings And Base base mold holder during cutting Molds with a plastic ring Disposables Options like peel-away molds, paper boats, and a plastic ice trays or covenience CONSIDERATIONS FOR EMBEDDING MEDIA COMPATIBILITY:  It is crucial that the embedding medium matches the tissue type in terms of strength and hardness CONSEQUENCES OF MISMATCH:  If the embedding medium is too soft, sections may tear or shred  If too hard, sections may become brittle and shatter MT304 LECTURE / MIDTERM DOLORSKIEEE GENERAL PATHOLOGY AND HISTOPATHOLOGIC AND CYTOLOGIC TECHNIQUES TRIMMING AND SECTIONING /CUTTING OVERVIEW OF CONVENTIONAL TISSUE PROCESSING 1. COARSE TRIMMING (DULL KNIFE/DULL BLADE) 1. Fixation  Trims the sides, tips, and bottom of the tissue block 2. Decalcification using a knife/blade 3. Dehydration  Forms a truncated pyramid or 4-sided prism shape 4. Clearing 5. Impregnation/Infiltration 2. FINE TRIMMING 6. Embedding/Casting  Block is placed in a microtome 7. Trimming  Surface is trimmed until the tissue is fully exposed 8. Sectioning/Cutting by adjusting the microtome or advancing the block 9. Staining MICROTOMY 10.Mounting MICROTOMY / SECTIONING / CUTTING 11.Ringing  Process of cutting a processed tissue into thin, uniform 12.Labeling slices (sections) using a microtome, allowing for microscopic examination MICROTOMY & TRIMMING *Note: Tissue blocks should be cold before sectioning PARTS OF MICROTOME  BLOCK HOLDER/CHUCK  KNIFE CARRIER AND KNIFE  PAWL, RATCHET FEED WHEEL  ADJUSTMENT SCREWS: Used to align the tissue block and control section thickness CASSETTE KNIFE HOLDER BASE  A part that anchors the knife holder to the microtome stage  The knife holder base can be moved toward or away from the block, but MUST be TRIMMING stationary and locked during microtome KNIFE HOLDER This part is comprised of several components includes:  BLADE CLAMP- holds the blade  KNIFE TILT- for adjusting the knife angle  FACE PLATE- guides that ribbons away from the blade and toward the operator COARSE HANDWHEEL  Moves the block holder either toward the knife or away from the knife TRIMMING MICRON ADJUSTMENT  Process of removing excess wax after embedding  Micros settings for section thickness the tissue in a paraffin block can range from 1-60 microns on most microtomes  Tissue must be surrounded by at least 2mm of wax PURPOSE: To facilitate easy sectioning *WHAT IS MOSTLY USED/TRADITIONAL KNIFE? DULL KNIFE TYPES OF TRIMMING 1. COARSE TRIMMING 2. FINE TRIMMING MT304 LECTURE / MIDTERM DOLORSKIEEE note: what is end product of microtome? RIBBON ADVANCEMENT HANDWHEEL FREEZING MICROTOME (1848)  Turns in one direction and  For cutting frozen tissue advances the block toward sections  Invented by Queckett the knife at the specified  USES CO2 as a freezing agent microns for frozen sections SAFETY LOCK Cryostat: Modern variant used for  Most handwheels are equipped with a safety lock to rush frozen sections, typically cuts prevent the wheels from 4-10 μm thick sections releasing and having the block holder come down towards the blade while a block is inserted or removed  It should be used anytime the microtomist is not actively sectioning paraffin blocks ULTRATHIN MICROTOME  Primarily for electron microscopy  Cuts sections as thin as 0.5 μm using a diamond knife (durable but expensive) SUMMARY of microtome MICROTOME INVENTOR DESCRIPTION PURPOSE ROCKING Paldwell Simplest For larged Trefall/ paraffin TYPES OF MICROTOME Caldwell embedded ROCKING MICROTOME (1881) (1881) sections(cut A.K.A Cambridge Microtome 10-12 um)  Simplest type ROTARY Charles Most Paraffin  Invented by Caldwell / Sedgwick common embedded Paldwell Trefall Minot sections  Cuts 10-12 μm tissue (1885) sections SLIDING George Most Celloidin and  Used for large paraffin- Adams dangerous hard embedded blocks but not suitable for serial sections or (1789) embedded tissue ribbons