Histopathology - May 2024 PDF

HISTOPATHOLOGY PROF. HENRICK AARON LO MAY 2024 HISTOPATHOLOGY Harden soft and friable tissue...

HISTOPATHOLOGY PROF. HENRICK AARON LO MAY 2024 HISTOPATHOLOGY Harden soft and friable tissue TISSUE PROCESSING Make tissue resistant to damage and distortion Effects of Table of Contents: FDCIETS SML Inhibit bacterial decomposition A. Numbering/Logging/Receiving Fixatives in Increase optical differentiation of cells B. Fixation General Act as mordants or accentuators C. Decalcification (not routine) (enhancers) a. For hard tissues (e.g. bone) b. If bone is not decalcified, sectioning will Reduced the risk of infection be difficult Cheap D. Dehydration Stable E. Clearing/Dealcoholization Safe to handle F. Infiltration/Impregnation Prevent distortion G. Embedding Characteristics Inhibit bacterial decomposition H. Trimming of a Good I. Section/Microtomy Produce minimal shrinkage Fixative J. Staining Rapid penetration of tissue K. Mounting Harden tissue L. Labeling Isotonic with minimal effect on tissue Permit staining A. NUMBERING/LOGGING/RECEIVING Additive Becomes part of the tissue by Essentially the first step Fixation formation of cross links or Description Accessioning complexes (ex. S-2024-0072) Mechanism of E.g., Formalin, Hg, S (surgical) Osmium tetroxide Action of A (autopsy) Non-Additive Not incorporated in the tissue, Fixative C (cytology) Fixation stabilizes tissue by removing P (Pap smear)* of the bound water Label E.g., Alcoholic *Separated so that there won’t be fixatives cross-contamination with other cytological samples when staining 1. pH: 6 - 8 *Pap smears are unsterile 2. Temperature Instrument for Use PENCIL in writing the description Autotech 40°C labeling Ink-based labels are erasable Traditionally RT Specimen size for 3X2 cm and 3-5mm THICK EM/Histochem 0 - 4°C processing Rapid examination 60°C Storage of cell Indefinitely For tissue with TB 100°C blocks and slides 3. Thickness B. FIXATION/PRESERVATION EM 1 - 2𝑚𝑚 2 Factors LM 2 2𝑐𝑚 (no more than 0.4 ❖ 1st and most critical step ❖ Process of preserving cells and tissue constituents involved in cm/4mm thick) ❖ The tissue is preserved by preventing degeneration, Fixation Brain Tissue Suspended whole 2 - 3 putrefaction, decomposition and distortion weeks (circle of willis) Ideally done Immediately after collecting of specimen Large Solid Open or sliced thinly To preserve the morphologic and chemical Tissue Primary Aim integrity of the cell in as life-like manner as possible 4. Osmolality: slightly hypertonic solution around 400 - 450 mOsm To harden and protect the tissue from the Secondary Aim 5. Concentration trauma of further handling a. 10% Formalin (100% is toxic to Most important reaction for maintaining Stabilization of tissue) tissue morphology Proteins b. 3% Glutaraldehyde Formation of cross-linkage of proteins c. 0.25% Glutaraldehyde (IEM) 1 HISTOPATHOLOGY Transcribed by: AVENDAÑO, BAUTISTA, BORILLA, BRUNO, CASTOR, CLEMOR, DE LEON, DEL ROSARIO, DOMINGO, ESGUERRA, FERMO, GONZALEZ, LAGYAP, LAW, MARASIGAN, MOLINA, ROPEREZ, VICENCIO 6. Time Duration Brasil’s Solution - Too short: feathery or soft Preserve specific parts and particular - Too long: shrinkage microscopic elements of the cell itself Primary 2 - 6 hours Nuclear Preserve the nuclear Fixation (generally, may be 8 hours) Fixative structures (e.g., EM Fixation of 3 hours (BFNCH) chromosomes) Contains glacial acetic 1. Speed - placement of specimen in acid Example: fixative as soon as it was removed ○ Bouin’s Fluid from the body ○ Fleming’s Fluid 2. Rate of penetration: Formalin ○ Newcomer’s Fluid (1mm/hour) ○ Carnoy’s Fluid - Hollow organs are cut so fixative Cytological ○ Heidenhain’s SuSa can go inside as it tends to float Fixative Cytoplasmic No acetic acid to avoid - Must penetrate the core of the Fixative swelling tissue (HORFF) Preserve cytoplasmic structures 3. Volume: 10 - 25 times that of the Example: Practical tissue ○ Helly’s Fluid Considerations 20:1 Maximum effectiveness ○ Orth’s Fluid (same ratio in ○ Regaud’s Fluid decalcification) ○ Fleming Fluid 5 - 10:1 Osmium tetroxide without acetic acid 50 - 100:1 Prolonged fixation Resin?? ○ Formalin with post (museum preparation) chroming 4. Duration - depends on the structure Preserve the chemical constituents of the tissue; can be cut down by of cells and tissues using heat, vacuum, agitation or Example: microwave ○ 10% Formol saline ○ Absolute ethyl alcohol ○ Newcomer’s fluid CLASSIFICATION OF FIXATIVES ○ Acetone ACCORDING TO COMPOSITION Histochemical Lipid Fixation Fixatives containing Made up of only one component Fixative mercuric chloride and substance (FANA) potassium dichromate Simple Fixative E.g., Formaldehyde, glutaraldehyde, in cryostat section chromate fixatives, lead fixatives, Carbohydrate Alcoholic fixative for picric acid, acetic acid Fixation glycogen (Rossman’s fluid or cold absolute Made up of two or more fixatives alcohol) which have been added together to Compound Protein Neutral buffered obtain the optimal combined effect of Fixation formalin Fixative their individual actions upon the cells and tissue constituents ACCORDING TO ACTION TYPES OF FIXATIVES Permit the general microscopic study of tissue structures without altering the ALDEHYDE FIXATIVES structural pattern and normal - Chemical formula: CHO (simplest carbohydrate) Gas produced by the oxidation of intercellular relationship of the tissues methyl alcohol in question Concentrated solutions should not Microanatomical 10% Formol Saline Formaldehyde be neutralized (explosion) Fixative 10% Neutral Buffered Formalin (NBF) (Formalin) Stock solutions: 36 - 40% Heidenhain’s SuSa Working solutions: 10% (no buffer Formol sublimate will result to it being unstable, will Zenker’s Solution lose its concentration) Zenker’s Formol Bouin’s Solution 2 HISTOPATHOLOGY Transcribed by: AVENDAÑO, BAUTISTA, BORILLA, BRUNO, CASTOR, CLEMOR, DE LEON, DEL ROSARIO, DOMINGO, ESGUERRA, FERMO, GONZALEZ, LAGYAP, LAW, MARASIGAN, MOLINA, ROPEREZ, VICENCIO ○ Difference between NBF and Fixing small pieces of liver, 10% formalin: presence of f. Zenker spleen, CT fibers, and nuclei phosphate buffer in NBF for FT: 12-24 hourse stability g. Zenker’s formol Fixative for pituitary gland, BM, Formalin pigments: (Helly’s solution) and blood-containing organs White crystalline 2. CHROMATE FIXATIVES (CROP) precipitates Has potassium dichromate Paraformaldeh Due to prolonged To demonstrate chromatin, yde standing a. Regaud’s mitochondria, mitotic figures, Removed by: 10% (Mollers / Molliflex) golgi bodies, RBC METOH or filtration Acid Brown/black granu To study early degenerative Formaldehyde deposits that may b. Orth’s fluid processes and tissue necrosis, Hematin obscure microscopic preserves myelin better details c. Chromic acid Preserves CHO d. Potassium Diluted in 10% NaCl Preserves lipids and 10% Formol Saline dichromate CNS tissue mitochondria (pH 4.5-5.2) (K2CrO4) 10% Neutral 3. LEAD FIXATIVES Best general tissue fixative Buffered Formalin Fixes CT mucin and is Best fixative for tissue containing (NBF) Description recommended for acid iron granules or mucopolysaccharides With double phosphate buffer PO4 buffered 4. PICRIC ACID FIXATIVES (BB) Rate of tissue penetration: 1 mm/hr Formalin ❖ Yellow staining For routine post-mortem tissues ❖ Removal of picrates Formol corrosive Picrates → Protein → Precipitate (water soluble) → 70% With mercuric chloride (HgCl2) (formol sublimate) alcohol → insoluble ○ Also a compound fixative Protein: renders picrates water-soluble Glutaraldehyde Electron microscopy (EM) HIGHLY EXPLOSIVE when dry Karnovsky’s EM: electron histochemistry and Excessive YELLOW STAINING paraformaldehyde- electron immunocytochemistry of tissues glutaraldehyde Best aldehyde fixative (expensive) Description NEVER WASH IN WATER before Mixture with dehydration Acrolein formaldehyde/formaldehyde For glycogen (excellent) Formol calcium Lipids (frozen section) Recommended for fixation of a. Bouin’s embryos and pituitary biopsies TYPES OF FIXATIVES b. Brasil’s METALLIC FIXATIVES Excellent fixative for glycogen alcoholic 1. MERCURIC CHLORIDE (BOSCHZZ) and less messy then Bouin’s picroformal ❖ Black deposits solution (excellent) fixative ❖ Dezenkerization / Removal of black deposits Water → Iodine → Water → Sodium Thiosulfate → Water 5. GLACIAL ACETIC ACID Iodine: condensation of black deposits It is normally used in conjunction Sodium thiosulfate: renders black deposits with other fixatives to form a water-soluble compound solution For BM biopsies Solidifies at 17°C a. B5 fixative Description FT: 1 ½ - 2 hours Fixes & precipitates b. Ohlmacher’s nucleoproteins, chromosomes, & c. Schaudinn’s chromatin material d. Carnoy-Lebrun Excellent nuclear fixative For tumor biopsies especially of the skin TYPES OF FIXATIVES e. Heidenhain Su: sublimat = mercuric chloride ALCOHOL FIXATIVES Susa Sa: saure = acetic acid (removes water) ○ Nuclear fixative Methyl For fixing dry and wet smears (PBS and FT: 3 - 12 hours Alcohol BM tissues) 3 HISTOPATHOLOGY Transcribed by: AVENDAÑO, BAUTISTA, BORILLA, BRUNO, CASTOR, CLEMOR, DE LEON, DEL ROSARIO, DOMINGO, ESGUERRA, FERMO, GONZALEZ, LAGYAP, LAW, MARASIGAN, MOLINA, ROPEREZ, VICENCIO For fixing dry and wet smears (PBS and Ethanol BM tissues) TYPES OF FIXATIVES Isopropyl For fixing dry and wet smears (PBS and ACETONE Alcohol BM tissues) Used at ice cold temperature from -5°C to 4°C ★ For fixing chromosomes, lymph glands For diffusible enzymes such as phosphatases and Carnoy’s Fluid and urgent biopsies lipases ★ MOST RAPID (1-3 hours) For fixing BRAIN TISSUE (Rabies Diagnosis) Alcoholic Formalin To preserve sputum Note: (Gendre’s Center for Rabies is San Lazaro Hospital Fixative) Brain of dogs can be preserved using acetone For fixing mucopolysaccharides and What is found? Negri bodies nuclear proteins. Newcomer’s Give better reaction in Fuelgen stain TYPES OF FIXATIVES than Carnoys HEAT FIXATION Thermal coagulation of tissue proteins Note: For bacteriologic smears Alcohol fixatives are non-additive fixative ○ Non-additive fixative - remove water from Microwave: 44-55°C tissues Underheating: poor sectioning Carnoy’s - one of the rapid fixative Overheating (>65°C): vacuolation, overstained cytoplasm ○ Fixation time is 1 to 3 hours only (some would take 6 to 9 hours, depending on the Note: thickness of tissue) Heat fixation is not generally good if tissues are big Tissue on slide (cells) - it can condense protein TYPES OF FIXATIVES ○ Heat → protein coagulates (nagdidikit) → specimen is fixed OSMIUM TETROXIDE ○ Is it long term? No. Pale yellow powder which dissolves in water (up to 6% at 20°C) to form a strong oxidizing solution OTHER FIXATIVES Inhibits hematoxylin 4% formalin or formol saline, Produce black precipitate crystals Fixatives for enzyme acetone or formalin for (osmium oxide) histochemistry cryostat section For lipids Glutaraldehyde, P+Cl3, Used for electron microscopy P+Cl3-formalin (Zamboni’s), Fleming’s Most common Fixatives for EM AuCl, Osmium tetroxide, 10% Formaldehyde Solution chrome-osmium acetic NBF = acceptable but not acid fixative used (Formalin) recommended (FIXATIVE & DECALCIFYING Fixative for electron KARNOVSKY’S AGENT) histochemistry and PARAFORMALDEHYDE Permanently fixes fat electron For nuclear GLUTARALDEHYDE immunocytochemistry structures (excellent) Fleming’s Improve cytoplasmic Solution details FIXATION TERMINOLOGIES without Acetic Recommended for SECONDARY Process of placing an already fixed Acid mitochondria FIXATION tissue in a second fixative Fixation whereby a primarily fixed POST- tissue is placed in Aqueous solution TYPES OF FIXATIVES CHROMATIZATION of 2.5-3% potassium dichromate TRICHLOROACETIC ACID Removing excess fixative Precipitates proteins Tap water Helly’s, Zenker’s, MAIN USE FOR HISTOPATH: Swelling effect → WASHING OUT (chromates) Flemming’s counteract shrinkage by other fixatives 50-70% Picric acid Weak decalcifying agent (softening effect) alcohol (Bouin’s solution) 4 HISTOPATHOLOGY Transcribed by: AVENDAÑO, BAUTISTA, BORILLA, BRUNO, CASTOR, CLEMOR, DE LEON, DEL ROSARIO, DOMINGO, ESGUERRA, FERMO, GONZALEZ, LAGYAP, LAW, MARASIGAN, MOLINA, ROPEREZ, VICENCIO Alcoholic Mercuric fixatives 15. When handling MUSCLE TISSUES, it should be iodine stretched for 30 min. to avoid rigor 16. WATER SHOULD NOT BE USED FOR GLYCOGEN Note: containing tissue Tissue is fixed already → still incompletely fixed after 17. HARD TISSUES may be washed out overnight with → can immerse this on another type of fixative = running water, and immersed in 4% aqueous phenol secondary fixation for 1-3 days (LENDRUMS METHOD) Post-chromatization - type of secondary fixation in which, the tissue is submerged on chromates DIFFICULTIES ENCOUNTERED BECAUSE OF IMPROPER FIXATION Failure to arrest early Cause: failure to fix FACTORS AFFECTING FIXATION OF TISSUES autolysis of cells immediately Size and thickness of the tissue Removal of substances Cause: wrong choice of specimen soluble in fixing agent fixative Presence of mucus Presence of artifact Cause: incomplete fixation Retarded by Presence of fat pigments Presence of blood Tissues are soft and Cause: incomplete fixation Cold temperature feather-like Loss or inactivation of Cause: wrong choice of Size and thickness of tissue enzymes fixative Enhanced by Agitation Shrinkage and swelling of Cause: overfixation Moderate heat (37 - 56°) cells Tissue blocks are brittle and Cause: prolonged fixation PRINCIPLES AND PRECAUTIONS IN HANDLING hard FIXATIVES 1. Autopsy/surgical materials should be fixed as soon as Pigment Color Removed by: after death Brown/black Saturated picric 2. All tissue must be properly labelled granules acid 3. If tissue are refrigerated, avoid 0°C Acid formaldehyde Alcoholic KOH a. Repeated freezing and thawing distorts hematin Kardasewitsch method tissues Lillie’s method 4. Tissue should not be more than 5mm thick, except in Mercuric chloride Black granules Alcoholic iodine lung edema (1-2 cm thick) pigment 5. Purulent, exudates or transudates should be kept for Chromate pigment Fine, yellow Acid-alcohol bacteriologic culture brown 6. Amount of fixative must be adequate 20:1 Osmium tetroxide Black precipitate Cold H2O 7. CONTAMINATION of tissue should be avoided pigment crystals 8. WASH the tissue before staining Intense eosinophilic staining at the Crush artifact center of the tissue (H&E) 9. SOLID ORGANS must be injected with fixatives Due to partial coagulation of partially a. Ex. Liver fixed protein 10. HOLLOW ORGANS should be packed with cotton soaked in fixative/ or opened a. Ex. intestine and stomach B. DECALCIFICATION 11. AIR-FILLED LUNGS may float on fixative, to avoid Removal of calcium or lime salts from this cover the lungs in several layers of gauze bones and calcified tissue soaked with fixative Description More concentrated acid solutions a. Death by Drowning: lung will sink in water decalcify bone more rapidly but are 12. HUMAN BRAIN should undergo intravascular more harmful to the tissue. perfusion (avoid washing out of blood with Ringers High concentrations and greater lactate). Suspended in a cord tied in the circle of Willis amounts of fluid will increase the speed of the process. (Fixation time: 2 weeks) The recommended ratio of fluid to 13. EYES should not be dissected before they are tissue volume for decalcification is fixed. 20:1 14. Frozen section may lead to formation of ICE 1-2 days CRYSTALS Heat will serve to hasten decalcification BUT it also increases the damaging effects on tissues. At 37°C Impaired nuclear staining of Van 5 HISTOPATHOLOGY Transcribed by: AVENDAÑO, BAUTISTA, BORILLA, BRUNO, CASTOR, CLEMOR, DE LEON, DEL ROSARIO, DOMINGO, ESGUERRA, FERMO, GONZALEZ, LAGYAP, LAW, MARASIGAN, MOLINA, ROPEREZ, VICENCIO Gieson’s stain for collagen fibers. MEASURING THE EXTENT OF DECALCIFICATION At 55°C Tissue will undergo complete digestion within 24-48 hours. 1. Physical or Poking the tissue with a stick Optimum ROOM TEMP (18-30 C) Mechanical temperature most ideal, most sensitive, Ideal time for 24-48 hours most reliable but very decalcification expensive Dense bone tissues 2. X-ray Method When bones are no longer 14 days or longer in order to complete the process. visible in X-ray, calcium content has been removed 3. Chemical Method Calcium oxalate TYPES OF DECALCIFYING AGENTS ACIDS 1. NITRIC ACID (5-10%) POST DECALCIFICATION Description Most common Immersing in saturated Li2CO3 or 5-10% NaHCO3 a. Formol nitric Rinsing in running water acid Storing in formol saline containing 15% sucrose or b. Perenyi’s Tissue softener & decalcifying agent PBS with 15-20% sucrose at 4°C fluid Done to neutralize the acids used in the c. Phloroglucin Most rapid decalcification process nitric acid 2. HYDROCHLORIC ACID (HCl) a. Von Small pieces, surface decalcification TISSUE SOFTENERS Ebner’s - Softens tissue slightly without putting back to its 3. 5% FORMIC ACID primary decalcifying agent Description Fixative and decalcifying agent 1. Lendrum’s (4% phenol) Best general decalcifying agent 2. Molliflex 4. TRICHLOROACETIC ACID (TCA) - tissue appear swollen and soapy 5. SULFUROUS ACID 3. 2% HCL 6. CHROMIC ACID / FLEMMING’S FLUID 4. 1% HCL in 70% alcohol TYPES OF DECALCIFYING AGENTS CHELATING AGENTS C. DEHYDRATION Sequestration of calcium AIM: to remove fixative and water from the tissue Commonly used is EDTA (versene) and replacing them with dehydrating fluid in ○ Versene = powdered form of EDTA preparation for impregnation. Excellent for EM, immunohistochemical or enzyme ○ Dehydrating fluids are generally used in staining increasing strengths. Rehydration = decreasing strengths TYPES OF DECALCIFYING AGENTS ○ INCREASING STRENGTHS = all the ION EXCHANGE RESINS aqueous tissue fluids are removed but with Hastens decalcification by using formic acid little disruption to the tissue due to diffusion containing decalcifying solutions currents. NOT recommended for fluids that contain mineral ○ For delicate tissues, particularly embryonic acids such as nitric acid or HCL and animal tissues, it is recommended to Accompanied with other decalcifying agents to hasten start processing with 30% ethyl alcohol the decalcification process (embryonic & animal tissues) TYPES OF DECALCIFYING AGENTS CHARACTERISTICS OF AN IDEAL ELECTROPHORESIS DEHYDRATING SOLUTION: (+) charged calcium ions move towards the negative 1. It should dehydrate rapidly without producing electrode using an electric charge considerable shrinkage or distortion of tissues Shorter time of calcium removal 2. It should NOT evaporate very fast One of the most expensive decalcification process 3. It should be able to dehydrate even fatty tissues Calcium will be attracted to the cathode 4. It should NOT harden tissues excessively (negatively-charged) 5. It should NOT remove stains 6. It should NOT be toxic to the body 6 HISTOPATHOLOGY Transcribed by: AVENDAÑO, BAUTISTA, BORILLA, BRUNO, CASTOR, CLEMOR, DE LEON, DEL ROSARIO, DOMINGO, ESGUERRA, FERMO, GONZALEZ, LAGYAP, LAW, MARASIGAN, MOLINA, ROPEREZ, VICENCIO 7. It should NOT be a fire hazard D. CLEARING COMMONLY USED DEHYDRATING AGENT 1. Alcohol- MOST COMMON CLEARING AGENT ○ Ethanol 1. It should be miscible with alcohol to remove For routine dehydration of tissues dehydrating agent from the tissues BEST DEHYDRATING AGENT 2. Generally clearing agent should be miscible with ○ Methyl alcohol paraffin wax to facilitate embedding. Employed for blood and tissue films 3. Should be miscible with Canada balsam and other ○ Butyl alcohol resins for during mounting. Utilized in plant and animal micro 4. Should make tissue transparent after staining due to techniques their high refractive index ○ Industrial methylated spirit (denatured 5. Most clearing agents are flammable liquids that alcohol) requires proper handling Ethanol + small amount of 6. Viscosity affects speed of pentration of the clearing methanol; used in the same way as agent ethanol 7. Prolonged exposure to most clearing agents causes ○ Isopropyl alcohol thetissue to become brittle and more difficult to cut Many of the processing methods for use in a microwave oven CLEARING AGENT recommend this agent 2. Acetone Colorless clearing agent ○ BOTH fixative and dehydrating agent MOST COMMONLY USED in 3. Dioxane (Diethylene dioxide) routine procedures ○ BOTH dehydrating and clearing agent XYLENE/XYLOL Clearing time: 30-60 mins ○ Saves time because 2-in-1 and will skip a Xylene turns MILKY when step in the tissue process tissues are incompletely 4. Cellosolve (Ethylene glycol monoethyl ether) dehydrated ○ BOTH dehydrating and clearing agent Substitute for xylene or 5. THF (Tetrahydrofuran) benzene ○ BOTH dehydrating agent and clearing agent TOLUENE Time for clearing: 1-2 hours 6. Triethyl phosphate Also used both for embedding and mounting process Note: Recommended for URGENT When using dehydrating agents that also clear the BIOPSIES tissue like dioxane and cellosolve, there is no need to BENZENE CARCINOGENIC undergo clearing. Can cause damage to BM & Aplastic anemia ADDITIVES TO DEHYDRATING AGENT Slower in action 1. 4% phenol + each 95% ethanol baths May cause HEPATOTOXICITY ○ Acts as a tissue softener for hard tissues CHLOROFORM Does not make tissue such as tendons, nails or dense fibrous translucent but removes tissues alcohol 2. Anhydrous copper sulfate Recommended for CNS ○ Can act as BOTH dehydrating agent and an TISSUE AND CYTOLOGY indicator of water content of the last bath No tissue distortion even if left (100% ETOH) CEDARWOOD OIL in oil Very expensive Notes to remember when using alcohol: Clearing agent for paraffin and Strength of alcohol depends upon the size, and the celloidin sections nature of the tissue and fixative used Length of storage Not for routine purposes Temperature Recommended for embryos, ANILINE OIL Anhydrous copper sulfate serves as an indicator insects, and VERY DELICATE that dehydration is complete (white -> blue) TISSUES Xylene - Turns milky 7 HISTOPATHOLOGY Transcribed by: AVENDAÑO, BAUTISTA, BORILLA, BRUNO, CASTOR, CLEMOR, DE LEON, DEL ROSARIO, DOMINGO, ESGUERRA, FERMO, GONZALEZ, LAGYAP, LAW, MARASIGAN, MOLINA, ROPEREZ, VICENCIO Similar to chloroform but Wax with melting point = 56 C CARBON cheaper is normally used for routine TETRACHLORIDE Highly toxic upon prolonged work. exposure MANUAL If the lab temperature = 20-24 METHYL BENZOATE Slow acting PROCESSING C → paraffin wax MP to use: AND METHYL For double embedding (54-58 C) SALICYLATE techniques If the lab temp. = 15-18 C → No dealcoholization but paraffin wax MP to use: 50-54 GLYCERINE/ GUM make the tissues clearer This method makes use of an SYRUP Used to increase refractive automatic tissue processing index machine (Autotechnicon) 1. Oil of bergamot Usually only 2-3 changes of 2. Oil of origanum wax are required to remove 3. Oil of wintergreen the clearing agent 4. Carbon disulfide Parts: 12 individual processing OTHER CLEARING steps 5. Carbol xylene AGENTS ○ Transfer arm 6. Terpineol 7. Phenol AUTOMATIC ○ Electrical clock 8. High test aviation lead free PROCESSING gasoline E. WAX IMPREGNATION AND EMBEDDING TYPES OF TISSUE IMPREGNATING MEDIUM Paraffin wax - most commonly used Celloidin (collodion) Gelatin and plastic - ideal and accurate but has consequences Vacuum embedding involves PARAFFIN WAX IMPREGNATION the wax impregnation under Simplest, most common and best embedding medium negative atmospheric pressure Very rapid inside an embedding oven Melting point of 56°C Provides more rapid wax If the lab temp is between 20-24: 54- 58C penetration of tissue If the lab temp is between 15-18: 50-54 C The time required for complete impregnation is reduced from ADVANTAGES 25-75% of the normal time Thin sections maybe cut without undue distortion required for tissue processing VACUUM Rapid, and can be prepared within 24 hrs EMBEDDING Tissue blocks may be stored in paraffin for an indefinite period of time after impregnation Permits staining DISADVANTAGES Overheated paraffin can make the specimen brittle Prolonged impregnation will cause excessive shrinkage and hardening Inadequate impregnation will cause retention of the clearing agent Paraffin processing is not recommended for fatty PRECAUTIONS IN WAX IMPREGNATION tissues Tissue should not be left in paraffin wax for longer THREE WAYS WHICH PARAFFIN WAX MAY BE periods of time than is necessary. PERFORMED 8 HISTOPATHOLOGY Transcribed by: AVENDAÑO, BAUTISTA, BORILLA, BRUNO, CASTOR, CLEMOR, DE LEON, DEL ROSARIO, DOMINGO, ESGUERRA, FERMO, GONZALEZ, LAGYAP, LAW, MARASIGAN, MOLINA, ROPEREZ, VICENCIO Over heated paraffin wax will also produce shrinkage and hardening of tissues and destroy lymphoid tissues completely. Paraffin wax must be from free dust water droplets and other foreign matters. PARAFFIN WAX SUBSTITUTES MP: 56-57°C Mixture of highly purified Rarely used paraffin and synthetic plastic Water soluble polymers Add 1% phenol to prevent Paraplast For large dense tissue blocks molds such as bones and brain Plastic/Resin - Classified into: Combination of paraffin and Gelatin ○ Epoxy plastic ○ Polyester MP: 56-58°C ○ Acrylic Embeddol Less brittle and less Disadvantage: Temporary compressible than paraplast Gelatin is a agar - susceptible Synthetic wax to have growth (organism) Bioloid Used for eye specimens A product of paraffin, containing rubber, with the F. EMBEDDING/CASTING/BLOCKING Tissue Mat same property as paraplast Done after wax impregnation Rubber containing paraffin Placing the tissue in a mold containing the medium MP: 46-48°C Orientation - Process by which the tissue is arranged Harder than paraffin in a precise positions (middle) in the mold Ester Wax Not soluble in water Soluble in 95% ETOH and other clearing agents Soluble and miscible with water (hence does not require dehydration and clearing of the tissue). Tissues are fixed, washed out and transferred directly into melted carbowax. Carbowax Suitable for many enzyme histochemical studies. AKA colloidin Very slow (days to weeks) Methods: ○ Wet celloidin Celloidin ○ Dry celloidin ○ Nitrocellulose method (Low viscosity nitrocellulose) 9 HISTOPATHOLOGY Transcribed by: AVENDAÑO, BAUTISTA, BORILLA, BRUNO, CASTOR, CLEMOR, DE LEON, DEL ROSARIO, DOMINGO, ESGUERRA, FERMO, GONZALEZ, LAGYAP, LAW, MARASIGAN, MOLINA, ROPEREZ, VICENCIO C. Paper boats TYPES OF MOLDS THAT IS USED Advantage: big tissues can be embedded (can be resize) Double embedding method Plastic (resin) embedding Leuckhart’s classified into: Embedding Molds ○ Epoxy - reduces antigenicity, toxic and damage tissue Bisphenol A (araldite) Compound Many tissue can be embedded Other embedding Glycerol Embedding Unit methods Cyclohexene Tissue tek dioxide (spurr) ○ Polyester - not often Plastic Embedding used Rings and Base ○ Acrylic - used Molds extensively for light microscopy Other types: maraglass 665 TRIMMING AND SECTIONING A. Peel away TRIMMING Disposable Process of removing excess wax after embedding Excess wax is cut off from the block to expose the tissue surface in preparation for actual cutting Disposable Knife/blade may be used Embedding Molds Ideal: four-sided prism/truncated pyramid SECTIONING (CUTTING OR MICROTOMY) The process by which a processed tissue is cut into B. Plastic ice trays uniformly thin slices (sections) to facilitate studies under the microscope 4-6 um in thickness for routine histologic procedure 10-15 um for frozen section 0.5 um for electron microscopy MICROTOME KNIVES 10 HISTOPATHOLOGY Transcribed by: AVENDAÑO, BAUTISTA, BORILLA, BRUNO, CASTOR, CLEMOR, DE LEON, DEL ROSARIO, DOMINGO, ESGUERRA, FERMO, GONZALEZ, LAGYAP, LAW, MARASIGAN, MOLINA, ROPEREZ, VICENCIO LESS concave sides are recommended for cutting 30 double strokes are given each side of the knife to celloidin-embedded tissue blocks on a sliding which very gentle pressure is applied microtome MORE concave sides are used to cut paraffin sections on base sledge, rotary or rocking microtome BEVEL - cutting facet, found on the tapered edge of all knives, the sides of which are more acutely inclined towards each other BEVEL ANGLE - the angle formed between the cutting edges, about 27°-32° CUTTING ANGLE - sides of the knife are inclined at 15° angle CLEARANCE ANGLE - the knife should be inclined with a 0°-15° angle HONING PROPER – removal of blemishes and ○ Will prevent uneven sections and will prevent the grinding the cutting edge of the knife on a stone to cutting facet to compress the tissue block during acquire an even edge. the cutting process The surface of the hone is wiped clean with a soft 25 mm in length cloth moistened with XYLENE in order to remove the One side of the knife is PLANE-CONCAVE scattered small particles of stones and metal. flat while the other is concave Then it is covered with a film of mineral and clove oil, 120 mm in length xylene, liquid paraffin or soapy water for With both sides concave LUBRICATION. Recommended for The knife is placed on one end of the hone and with BICONCAVE cutting paraffin the cutting knife edge first the “heel” end of the handle embedded sections on drawn diagonally towards the operator on the stone a rotary microtome until the “toe” head portion is reached. 100 mm in length Have both sides straight ○ For plane wedge knife: The knife is turned Recommended for over to sharpen the other surface every frozen sections or for 10-20 strokes. cutting extremely hard ○ For plane concave knives: Only the PLANE-WEDGE and tough specimens surface should be rubbed on the hone embedded in paraffin ADDITIONAL: use a circular glass plate usually ¼ to blocks, using a base 3/8 inch thick, about 14 inches long and 1-2 inches sledge type or sliding microtome wider than the length of the knife with finely powdered aluminum oxide made into paste with water is used as an abrasive (polishing) TYPES OF HONES Belgium yellow – for manual sharpening when cutting edge has been rendered blunt or nicked. This type usually gives the best result KNIFE MATERIALS Arkansas – Gives more polishing effect than the Disposable knives belgium yellow Stainless steel ○ Tungsten carbide Fine carborundum – much coarser than the first two Glass knives = “ralph knives” for ultramicrotomy typed and used only for badly nicked knives. Followed Diamond knives - cutting resin sections by either one of the first 2 knife sharpeners Steel knives STROPPING To polish and sharpen the cutting edge HONING AND STROPPING Removal of burrs HONING (HARD SHARPENING) From toe to heel direction From HEEL TO TOE 20-30 TIMES Around 40-120 double strokes are usually required Removal of nicks (irregularities) Material: PADDLE STROP made up of horse leather Hones (8 inch x 3 inch) firmly attached to a solid back to prevent sagging Mechanical honing – makes use of a vibrating frosted plate or wheel driven by an electrical motor. 11 HISTOPATHOLOGY Transcribed by: AVENDAÑO, BAUTISTA, BORILLA, BRUNO, CASTOR, CLEMOR, DE LEON, DEL ROSARIO, DOMINGO, ESGUERRA, FERMO, GONZALEZ, LAGYAP, LAW, MARASIGAN, MOLINA, ROPEREZ, VICENCIO G. SECTIONING TYPES OF SECTION Paraffin sections – rocking and rotary microtome Celloidin sections – sliding microtome Frozen section (Cryostat) - Microtome inside freezer ○ Cold knife ○ Cryostat METHODS OF FREEZING Liquid nitrogen Isopentane Aerosol sprays - Ideally if a frozen section is used, aerosol spray is used to freeze. Carbon dioxide MICROTOMY PRINCIPLE: a spring balanced pawl is brought into contact with a ratchet feed wheel connected to a micrometer screw, which is rotated, moving the tissue block at a known distance towards the knife for cutting Rotary/Minot Microtome sections at uniform thickness *inventor: Minot in 1885-1886 3 essential parts *MOST COMMON type used ○ Block holder – the tissue is held in position. today especially for ○ Knife carrier and knife – actual cutting of paraffin-embedded tissues. tissue sections. Has little movement because the cutting is set at 4 to 6 ○ Pawl, ratchet feed wheel and adjustment Rotary Microtome screws – to line up the tissue block in the proper position with the knife. Knife holder moves forward, block holder moves up and down when spinning ratched feet Sliding Microtome = MOST DANGEROUS TYPE DUE TO MOVABLE EXPOSED KNIFE! *inventor/developer: Adams in 1789 *There are 2 types: ○ Base-Sledge Sliding Microtome for all forms of media block holder: moving knife: stationary ○ Standard Sliding Microtome Dangerous type - knife is moving not the holder KINDS OF MICROTOME block: stationary Rocking Microtome (Cambridge knife: moving Rocking Microtome) *inventor: Paldwell Trefall in 1881 *simplest among the microtomes Rocking Microtome *disadvantage: difficulty in reorienting the block Like a rocking chair, stationary movement 12 HISTOPATHOLOGY Transcribed by: AVENDAÑO, BAUTISTA, BORILLA, BRUNO, CASTOR, CLEMOR, DE LEON, DEL ROSARIO, DOMINGO, ESGUERRA, FERMO, GONZALEZ, LAGYAP, LAW, MARASIGAN, MOLINA, ROPEREZ, VICENCIO Tissue adhesive - pag minelt yung paraffin, pwedeng sumama yung na-cut na sections if walang tissue adhesives ○ Disadvantage: background staining - staining the slide will also stain the adhesive ALBUMIN (MAYER’S EGG Invented by Queckett in 1848 ALBUMIN) Used in the frozen section. Disadvantage of use: background staining Components: ○ Egg white: adhesive ○ Glycerol: to increase viscosity and prevent drying Freezing Microtome ○ Thymol: prevent growth of molds 1% GELATIN Disadvantage: stains with different dyes Provides firmer attachment than albumin Ultrathin Microtome –for cutting Gently heated to melt sections for Electron Added to floatation bath microscopy Has microscope above CELLULOSE uses DIAMOND KNIVES 1% methyl cellulose specimen is small, fixed in Advantage: non staining to Tissue adhesives osmium tetroxide embedded in commonly used stains plastic POLY-L-LYSINE Ultrathin Microtome General purpose adhesive Widely used in immunohistochemistry Advantage: no background staining SODIUM SALICYLATE Commercial syrup that is diluted 1:10 Floatation water bath - Advantage: less staining with straightens the ribbon cut from most dyes, not affected by mild the microtome. alkaline solutions Drying oven - drying forceps and Disadvantage: Blackening on Other equipments slides silver impregnation and reticulin needed when using ○ 5 deg higher than methods, red staining in methyl microtome melting point of the green pyronin technique wax ○ To melt the paraffin RESIN wax Plastic Greatest adhesion 13 HISTOPATHOLOGY Transcribed by: AVENDAÑO, BAUTISTA, BORILLA, BRUNO, CASTOR, CLEMOR, DE LEON, DEL ROSARIO, DOMINGO, ESGUERRA, FERMO, GONZALEZ, LAGYAP, LAW, MARASIGAN, MOLINA, ROPEREZ, VICENCIO Araldite: made of epoxy resins Hematin Active coloring ○ Diluted 1:10 with substance upon ripening acetone ○ Little affected by EOSIN (EOSIN Y) fluids in sections Most commonly used Cytoplasmic stain, acidic, 2’ stain FAULTS OCCURRING DURING TISSUE Counterstain PROCESSING Brittle or hard tissue EQUIPMENTS Clearing agent turns milky as soon as tissue is placed in COPLIN JAR Holds 5-9 slides it Tissue smells of clearing agent Tissue is opaque due to presence of alcohol Tissue shrinks Tissue is soft airholes On trimming, wax appears crystalline Holds 5-19 slides SLOTTED STAINING Paraffin block after cooling is moist and crumbles DISHES FAULTS OCCURRING DURING SECTION CUTTING Sections fail to form ribbons Sections roll up on cutting METAL/GLASS STAINING Holds 10- 30 slides —Ribbon is curved RACKS/ CARRIERS —Sections are compressed, wrinkled or jammed —Sections are torn and crumble when cut Sections are squashed —A hole is formed Sections of unequal thickness are produced —Sections adhere to the knife —Ribbon is split PROCEDURES (IN ORDER) —Sections are lifted from the knife on upstrokes —Resistance of tissue —Chatters are seen PROCEDURES (IN ORDER) —Section cut is sometimes thin/thick Process Function Additional Knife makes a hard metallic scraping or ringing sound Paraffin is miscible 1 Xylene Deparaffinization —Frozen tissue crumbles with xylene —Frozen tissue chips into fragments Descending Absolute (95%) → 2 Rehydration grades of alcohol 70% 3 Water Rinse H. H&E STAINING Remove fixative 4 Washing out artifact pigments Routine staining Nucleus = light blue HEMATOXYLIN Light blue because of 5 hematoxylin Cytoplasm = light Hematoxylin Nuclear stain acid alcohol blue capechianum / basic Nucleus = light blue Act as a differentiator Hematoxylon 1’ stain 6 Acid alcohol Cytoplasm = (based on affinity) campechianum Extracted from colorless heartwood or log wood Function: mordant Colorless when first Nucleus = blue Blueing agent extracted (intensified = dark (imparts darker blue blue) WALDEYER 1st to use hematoxylin 7 Ammonia Water color; intensifies blue) Cytoplasm = Hematoxylin Ripening Colorless Accentuator or Produces color because NH4OH (ammonium mordant of the addition of hydroxide). LICO3 chemicals 14 HISTOPATHOLOGY Transcribed by: AVENDAÑO, BAUTISTA, BORILLA, BRUNO, CASTOR, CLEMOR, DE LEON, DEL ROSARIO, DOMINGO, ESGUERRA, FERMO, GONZALEZ, LAGYAP, LAW, MARASIGAN, MOLINA, ROPEREZ, VICENCIO (Lithium carbonate), KINDS OF MOUNTING MEDIA Scott’s tap H2O Used to mount water miscible 8 Water wash preparations 9 Eosin Y counterstain Composition: Ascending grades ○ Gelatin, Glycerin jelly, Gum 10 Dehydration of alcohol Arabic → solidify medium Clear the alcohol Aqueous ○ Glycerol = prevent drying and Dealcoholization. 11 Xyelene since you used it Mounting cracking Clearing again medium ○ Sugar = increase refractive index To prolong its shelf life Nucleus = Blue to ○ Preservative = merthiolate (prevent (kept indefinitely blue black 12 Mount & Label fungi) since you can still Cytoplasm = pale restrain it) pink Disadvantage: Not permanent (aqueous meaning its made of water; temporary) Recall! Wet mount (Parasitology) Question: Water Temporary If your fixative is mercuric chloride, what should be your Semi permanent washing out process? Sections are kept for years if sealed Answer: Dezenkerization You need to remove the artifact because if you stain it, it will be with paraffin Glycerin part of the staining itself Standard mounting medium for fat stains I. MOUNTING Refractive index: 1.46 Use of a medium and a coverslip to facilitate the Farrant’s handling storage, protection of the tissue section Medium (Gum Refractive index: 1.44 Mounting medium used Arabic Medium) ○ Syr