Histology Past Paper PDF - MTAP 2 - Infiltration to Embedding

MTAP 2 - INFILTRATION to EMBEDDING ○​ DISADVANTAGE: Not suitable for fatty tissues or specimens with RECAP fats as the fats will be me...

MTAP 2 - INFILTRATION to EMBEDDING ○​ DISADVANTAGE: Not suitable for fatty tissues or specimens with RECAP fats as the fats will be melted due to the presence of heat. TI ​ In histopath, we process different type of specimens in the same manner ○​ THREE METHODS OF PARAFFIN WAX INFILTRATION ○​ Autopsy ​ Manual = use of oven ○​ Biopsy / Surgical Specimens ​ A dissecting pan is needed along with a melted M ○​ Cytology specimens paraffin wax; the small pieces of tissues is placed in ​ Ex. Pap Smear the pan with the wax ​ Initially we do fixation to preserve the tissues and to somehow harden the ​ To be placed inside the oven = the oven temp tissue to facilitate cutting. should be 55-60 degC/ 2-5 deg higher than the S. ​ Next to fixation, we do dehydration, most of the time using ascending wax’s melting point, with 4 changes of paraffin concentrations of ethyl alcohol, the routine dehydrating agent is ethanol wax at 15 minutes interval for 1 hour. S. ​ After dehydration we perform dealcoholization or clearing, most commonly used clearing agent is xylene ​ After clearing we do infiltration/ impregnation P. INFILTRATION ​ Also known as IMPREGNATION ​ Automatic = Autotechnicon ​ PURPOSE: ​ Autotechnicon is an automatic tissue processor S, ○​ To remove clearing agent from tissues that can do the first 4 steps in tissue processing ○​ To fill up cavities and tissue spaces (fixation, dehydration, clearing, and infiltration) ​ In order to give the specimens a firm consistency. ​ Heat generated from electricity TO ​ A firm consistency is a must to facilitate easy ​ 2-3 changes of wax; more rapid cutting. ​ Constant agitation will ensure rapid entry of paraffin ​ Infiltration is a step in tissue processing that involves use of a medium that wax into the tissue will fill all cavities and tissue spaces ​ Vacuum N THREE TRADITIONAL METHODS TO DO INFILTRATION ​ Also requires use of oven ​ PARAFFIN WAX INFILTRATION ​ Paraffin wax infiltration method under negative SA ○​ Medium: PARAFFIN WAX atmospheric pressure inside the oven ○​ Melting point: 56 degrees C ​ Most rapid method ○​ Most common method of infiltration is paraffin wax infiltration, because the process is rapid due to the application of heat.  MTAP 2 - INFILTRATION to EMBEDDING ​ The negative atmospheric pressure ensures however, a much longer time is needed for dense rapid removal of bubbles and also the clearing specimens TI agent. ○​ SUBSTITUTES FOR PARAFFIN WAX — synthetic wax or artificial wax M ​ Paraplast – recommended for bones and brain specimens ​ Bioloid; Embeddol –recommended for eye specimens ​ Carbowax – water-soluble (advantage), eliminates clearing and dehydration; therefore, when carbowax is S. ​ Embedding produces tissue block used for used, both clearing and dehydration is optional; cutting/sectioning recommended for enzyme studies. ​ Embedding product: Tissue blocks S. ​ Ester wax - compared with paraffin wax it has a lower ○​ TWO METHODS OF CELLOIDIN INFILTRATION; SAME melting point but harder than paraffin wax, specimens PROCEDURE WITH DIFFERENT REAGENT USED could be harder, hence a heavy duty type of microtome is ​ Wet celloidin P. needed ​ Use of 70% alcohol ​ CELLOIDIN INFILTRATION ○​ To store the block prior to sectioning ○​ Also known as COLLODION ​ Used for bones, brains, teeth specimens, and S, ○​ No heat; slow method (disadvantage) ○​ For specimens with large cavities/spaces that tends to collapse like bones, brain, and whole organs whole organs ​ Dry celloidin ​ Use of Gilson’s mixture (chloroform + cedarwood TO ○​ PROCEDURE: oil) ​ Prepare 3 container ​ Recommended for eye specimens ​ Thin celloidin (2-4% celloidin solution; immerse ​ GELATIN INFILTRATION specimen for 5-7 days) ○​ Recommended for enzyme studies N ​ Medium celloidin ( 4-6% celloidin solution; ○​ PROCEDURE: immerse specimen for 5-7 days) ​ Immerse the specimen under 10% Gelatin + 1 % phenol for ​ Thick celloidin (8-12% celloidin solution; immerse SA 24 hours specimen for 3-5 days) ​ After 24 hours, immerse the specimen under 20% gelatin + ​ This method takes up to approximately 3 weeks of 1% phenol for 12 hours process before embedding for small specimens;  MTAP 2 - INFILTRATION to EMBEDDING ​ After 12 hours, immerse the specimen under 20% gelatin + ​ Placing the infiltrated tissue at the precise position is called 1% phenol allowed to cool inside the refrigerator for at least orientation; the surface of the section to be cut should be TI 30 minutes to 1 hour placed parallel to the bottom of the mold in which the ​ Then embed to produce a tissue block. The tissue block sample is oriented. produced should be immersed in 10% formalin for 12-24 ​ Embedding medium = paraffin wax; plastic resin M hours ​ Embedding mold (disposable and non-disposable) = ​ The purpose of formalin in gelatin infiltration is hulmahan not as a fixative, but rather used to harden the ​ Non-disposable embedding mold tissue prior to cutting/sectioning. ○​ Leuchhart’s embedding molds / “L” S. ○​ Phenol pieces ​ Remember that phenol is also used as a tissue softener in ​ Used for embedding one tissue S. decalcification sample ​ But in gelatin infiltration, it is used to prevent the growth ​ Two L-shaped molds made with of molds heavy brass or metal P. ○​ 10% gelatin used to fill up the cavities ​ Not commonly used as it is too slow or cumbersome in busy EMBEDDING laboratories as it requires S, ​ Also known as CASTING / BLOCKING assembling. ○​ A step in tissue processing that involves placing the tissue sample ​ An advantage of this you can into a mold containing an embedding medium allowed to solidify. produce tissue blocks of TO ○​ Produces tissue blocks used for cutting/sectioning. different sizes ○​ Usually in the embedding process, the infiltrating agent used is ○​ Compound embedding unit the one we also use as medium of embedding. ​ For embedding more than one ​ Paraffin wax is the most commonly used medium in specimen N embedding ​ A type of mold consist of a series ○​ Procedure: of interlocking plates resting on a ​ Manual method: Place the infiltrated tissue in the mold flat metal piece forming several SA specifically at the bottom of the center of the mold. We then compartments pour the melted paraffin and allow it to solidify or cool rapidly ○​ Plastic embedding rings (Tissue-Tek) in the refrigerator at -5 degC or we immerse it in cold water and Bass molds  MTAP 2 - INFILTRATION to EMBEDDING ​ Advantage: requires less paraffin wax TI ​ Rapid embedding ​ Orientation of specimen is easy ​ Disposable M ○​ Paper Boats / Paper molds ​ Used for embedding paraffin wax and celloidin blocks. ○​ Plastic Ice Trays S. ​ The mold should be smeared with glycerin for easy removal of block S. upon removal once it solidifies. ○​ Peel Away Mold ​ Can produce perfect tissue P. blocks even without trimming ​ Trimming is not required as it LECTURE NOTES FROM MA’AM CRISTINA LIWANAG produces perfect square molds INFILTRATION / EMBEDDING S, ​ Advantage: the tissue can be placed directly on the block holder DRY CELLOIDIN Celloidin infiltration method recommended for whole eye specimens TO ​ Infiltrated tissue Will require use of Gilson’s mixture to store blocks ○​ In many laboratories, embedding is used automatically due to the 56 DEGREES CELSIUS Melting point of paraffin wax commonly used invention of embedding units. PARAPLAST Substitute for paraffin wax recommended for bones and brain specimens CARBOWAX Substitute for paraffin wax that is water soluble & is N recommended for enzyme histochemical studies 55-60 DEGREES CELSIUS Temperature range of paraffin oven when in used SA 2-5 DEGREES HIGHER Approximate temperature of paraffin oven when in THAN THE WAX MELTING used POINT VACUUM METHOD Infiltration under negative atmospheric pressure inside the oven  MTAP 2 - INFILTRATION to EMBEDDING PARAFFIN WAX Infiltration method not suited for fatty tissues LEUCHHART’S Embedding mold consisting of L shaped strips made of INFILTRATION METHOD EMBEDDING MOLD heavy brass TI BIOLOID Substitute for paraffin wax used for eye specimens ESTER WAX Substitute for paraffin wax that will require heavy duty *Advantage can produce tissue blocks of various sizes type of microtome AUTOMATIC METHOD Method of paraffin infiltration that require 2-3 changes 56-57 DEGREES CELSIUS Melting point of paraplast (AUTOTECHNICON) of wax M 46-48 DEGREES CELSIUS Melting point of ester wax Use of Phenol in gelatin To prevent growth of molds GELATIN INFILTRATION Infiltration method for enzyme & histochemical studies infiltration CELLOIDIN Thin - 2 - 4% of solution * Gelatin is also used as an embedding medium for INFILTRATION Medium - 4 - 6% solution S. delicate specimens and for frozen tissue sections Thick - 8 - 12% solution WET CELLOIDIN Celloidin infiltration method for bones, brain & teeth **SPUR Fastest epoxy resin specimens **POLYESTER Plastic resin originally introduced for EM S. **POLYGLYCL Extremely hydrophilic Will require 70-80% alcohol to store blocks METHACRYLATE/GMA GILSON’S MIXTURE Combination of chloroform & cedarwood oil **METHYL Plastic resin considered ideal for undecalcified bones P. (DRY CELLOIDIN) METHACRYLATE/MMA & hard tissues CELLOIDIN INFILTRATION Infiltration method recommended for specimens with * - Added notes METHOD large and hollow cavities that tends to collapse ** - For resin or for plastic embedding S, OLEUM RECINI & Plasticizers to prevent cracking tissues when infiltrated Plastic embedding - is used for hard tissues like undecalcified bones, renal CASTOR OIL using LVN (Low Viscosity Nitrocellulose) biopsies, and bone marrow biopsies; however, it is not often used since the GELATIN INFILTRATION Infiltration method that will require tissues not to be components are carcinogenic; can provide high resolution for light microscopic exam TO more than 2-3 mm thick PLASTIC ICE TRAYS A disposable embedding mold that will require smearing of inner mold with glycerine COMPOUND EMBEDDING An embedding mold consisting of a series of UNIT interlocking plates resting on a flat metal base N PEEL AWAY MOLD Disposable mold that gives perfect blocks without trimming IMPREGNATION Other term for infiltration SA Other term for embedding 1.​ CASTING 2.​ BLOCKING DOUBLE EMBEDDING Process of infiltrating tissue with celloidin and embedding with paraffin  HISTOPATHOLOGY | MICROTOMY TO DEPARAFFINIZATION MTAP 2ND SEM | A.Y. 2024-2025 ​ TISSUE BLOCK - the product of embedding, ​ Standard sliding — to be used in cutting. And cutting tissues in to MOVABLE PART (Knife); thin slices is what we call SECTIONING. STATIONARY (Block Holder); ​ SECTIONING — involves cutting tissue into requires a slow but very thin slices. And in order to cut tissue sections, steady motion during microtomes are used. manipulation ​ Freezing microtome — Invented by T TYPES OF MICROTOME QUECKETTE; uses (1) intermittent burst of ​ Ultrathin Microtome — intended for carbon dioxide (freezing agent) to freeze the M ELECTRON MICROSCOPY; thickness of block holder and the tissue (2) it has cooling sections produced is 0.5 micra; tissues are device that can lower the knife temp; usually embedded in plastic Thickness of sections 10-15 u; can ,R ​ Rotary microtome — was developed by immediately harden tissues without prior MINOT and it is used to cut paraffin fixation embedded tissue; the MOST COMMON and ○​ PURPOSE: commonly used for research purposes; ​ Demonstration of fats and thickness of sections 4-6 microns other neurological ES ​ Rocking microtome — invented by structures TREFALL; thickness of sections 10-12 micra; ​ Used to cut tissues with heat used to prepare serial sections of large sensitive structures. paraffin block; MOST SIMPLE ○​ FREEZING AGENTS: ○​ DISADVANTAGES: ​ Liquid nitrogen EL ​ Difficulty in re-orienting the ​ Isopentane cooled by liquid block nitrogen ​ Restriction in the size of block ​ Dry ice that can be cut ​ Carbon dioxide gas ​ Aerosol sprays G ​ Sliding microtome — invented by ADAMS; thickness of the section 7-9 micra; MOST ​ Cryostat / Cold microtome — the DANGEROUS because of the exposed knife temperature inside the chamber will AN ○​ PURPOSE: immediately harden tissues to facilitate ​ For celloidin embedded immediate cutting; For fresh tissue microtomy, tissue or ester infiltrated refrigerated (-5 to -30 degC) average is -18 to tissue -20 degC; uses cryostat – a refrigerated ​ To cut extremely hard or tough apparatus (a rotary microtome) enclosed in a tissues cold chamber ○​ TYPES: ○​ PURPOSE: ​ Base sledge — MOVABLE ​ For preparing thin sections of PART (Block Holder); fresh frozen tissues for STATIONARY (Knife); less fluorescent antibody dangerous 1  staining or histochemical concave (paraffin embedded); flat enzyme studies celloidin embedded) ​ Freezing microtome uses freezing agent to ○​ Plane wedge – for frozen sections; immediately harden the blocks, while cryostat extremely hard or tough tissues; uses the temp inside the chamber to harden both sides are flat the blocks ○​ Disposable blades – commonly used today; size of sections 2-4 u FROZEN SECTIONS ○​ Diamond knife and glass knives – ​ In the laboratory, we usually prepare 2 types for electron microscopy of sections: ​ Diamond knives — are brittle T ○​ Paraffin section — we use ROTARY and quite expensive; cuts MICROTOME resin blocks for EM M ○​ Frozen section – type of tissue ​ Glass knives — used for section usually requested for RAPID trimming and semi-thin DIAGNOSIS; we prepare frozen sectioning of tissue blocks for ,R section to come up with diagnosis electron microscopy while the patient is in the OPERATING ROOM processing fresh specimen HONING & STROPPING ​ 2 METHODS OF PREPARING FROZEN ​ Honing — process of grinding the cutting SECTIONS: edge to have an even edge ES ○​ Cold knife procedure – requires ○​ Purpose: To remove gross nicks freezing microtome; use of freezing ○​ Material for honing: Oil stones and agent to immediately harden fresh hones tissues for immediate cutting; tissue ​ Belgium yellow — type that blocks should be 3-5 mm; obsolete gives the best result EL ○​ Cold microtome / Cryostat method – ​ Arkansas — gives a more uses cryostat; preferred nowadays polishing effect than belgium ​ Cryostat and freezing microtome are used for yellow FRESH TISSUES ​ Fine carborundum — G recommended for badly MICROTOME BLADES nicked knives; with a much ​ Honing and stropping are not done if we coarser bite AN utilized disposable blades ○​ Procedure: Heel to toe movement, ​ To cut tissues TRADITIONALLY, we use edge first microtome knives, but now we commonly use ○​ Number of strokes required: 20-30 DISPOSABLE BLADES strokes ​ MICROTOME BLADES: ○​ During honing, we need to use ○​ Biconcave – for cutting paraffin lubricants like soapy water, xylene, embedded tissues; both sides of the etc. knives are concave ​ Stropping — process of polishing and ○​ Plane concave – one side of the knife sharpening cutting edge is concave, while the other one is flat; ○​ Purpose: to remove burrs and other irregularities formed during honing 2  ○​ Material: Leather strop or horse float out bath in order to remove the wrinkles leather and fold. ○​ Procedure: Toe to heel movement, ○​ The main purpose of float out bath is edge last to FLATTEN THE RIBBONS ○​ Number of strokes: 40-120 double ​ Temperature of the float out bath: 5-10 deg strokes lower than the melting point of the wax, ○​ Do not require lubricant specifically 45-50 deg ○​ Must be OILED prior to use ​ Fishing out — process of removing ribbons ○​ Commonly used oils: Vegetable oils from float out bath like castor oil ​ Orientation — process of placing the tissue T ​ MINERAL OIL should not be in a precise position (middle of the slide) used because it tends to M blister or destroy the leather. ADHESIVES ​ Stropping may be carried out without prior ​ Before fishing out, a slide must be prepared honing and it should be coated with adhesives ,R ​ Stropping must be done after the process of ​ Process: Placing adhesives onto the slides > honing fishing out > orienting ​ Disposable blades will not require honing and ​ Adhesives — promotes adhesion of the tissue stropping; Microtome knives will require honing sections into the slides; to promote attachment; and stropping to prevent detachment of ribbons from slide ES ○​ MAYER’S EGG ALBUMIN — most IMPORTANT ANGLES commonly used adhesive; a ​ Bevel angle — set at 27-37 deg; the angle combination of egg white and formed between cutting edges of the knife glycerine ​ Wedge angle — set at 14-15 deg; the angle ​ equal amount of egg white EL formed by the sides of the wedge knife and glycerine PLUS thymol ​ Clearance angle / tilt angle / inclination crystals in order to prevent angle — is the most important angle the growth of molds because in sectioning we need to follow the ○​ Poly-l-lysine — widely used as a G correct clearance angle to prevent uneven section adhesive in sections; the angle betwen the cutting edge of immunohistochemistry; its the knife and the surface of the tissue effectiveness as an adhesive slowly AN block; set at 0-15 deg, but during sectioning decreases as the time goes by we set it at 5-10 deg to prevent uneven ○​ Apes — very useful for cytology; sections/sections that are alternately thin or recommended for specimen that is thick bloody and has high protein content ○​ Starch paste RIBBONING, FISHING OUT & ORIENTING ○​ Dried albumin ​ Product of sectioning: Thin slices/tissue sections/ribbons DEPARAFFINIZATION ​ The ribbons tend to be wrinkled after ​ Deparaffinization — the process of removing sectioning, and therefore should be placed in a paraffin wax from the tissue once properly fixed on a slide; process before staining 3  ○​ Methods: ​ BASE SLEDGE MICROTOME: Specific type ​ Immersion of slides in xylene of siding microtome in which the movable part ​ Place slides inside the oven — is the block holder oven temp is 55 to 60 degC ​ CRYOSTAT: A refrigerated apparatus used in ​ Pass the slide over a flame fluorescent antibody staining techniques or using alcohol lamp histochemical enzyme studies ​ ULTRATHIN MICROTOME: Tissues SUMMARY QUESTIONS: embedded using plastic resins (for EM) i.e. ​ ROCKING MICROTOME: Type of microtome epoxy must be cut using what type of used to prepare serial sections microtome T ​ FREEZING MICROTOME: Microtome type ​ FREEZING MICRTOME: Type of microtome used to cut tissues to demonstrate neurological invented by Queckette M structures ​ ROTARY MICROTOME: Type of microtome ​ 4-6 micra: thickness of sections produced operated by the rotation of the flywheel, using rotary microtome (range in micra) causing reciprocal motion of knife over the ,R ​ STROPPING: Process of sharpening and block polishing the cutting edge ​ DIAMOND KNIFE: Type of microtome knife ​ BICONCAVE: Type of microtome knife used to used to cut any type of resin block for EM cut paraffin embedded tissues ​ WEDGE ANGLE: It is defined as the angle ​ BEVEL ANGLE: Defined as the angle formed formed by the sides of the knife ES between cutting edges ​ SLIDING MICROTOME: Type of microtome ​ 5-10 deg: Actual angulation in degrees of developed by Adam clearance angle to prevent uneven sections ​ 10-12 micra: Thickness of sections produced ​ GLYCERINE: Mayer’s egg albumin is prepared using rocking microtome by adding equal amounts of egg white and ​ 10-15 micra: Celloidin sections are usually cut EL _______ between _________ thickness ​ CHUCK: Block holder is also called ______ ​ FINE CARBORUNDUM: Type of hone used for ​ CRYOSTAT and FREEZING MICROTOME: badly nicked knives Types of microtome which can be used to ​ 10-20 STROKES: In honing, when using plane G prepare frozen sections wedge knife, the knife is turned over as to ​ GLASS KNIFE: Type of microtome knife used sharpen the other surface every _____ strokes for trimming and semi-thin sectioning of tx ​ FREEZING MICROTOME: Type of microtome AN blocks for EM used to cut tx with heat sensitive structures ​ ROCKING MICROTOME: Type of microtome ​ 0.5 micra: Thickness of sections produced invented by Trefall using ultrathin microtome ​ BELIGIUM YELLOW: Type of hone that gives ​ ROCKING MICROTOME: Type of microtome the best result that has restriction as to the size of block that ​ 40 to 120 double strokes: Number of strokes can be cut required when doing stropping ​ TO PREVENT GROWTH OF MOLDS: ​ ROCKING MICROTOME: The type of Purpose of adding thymol crystals in mayer’s microtome that when used will make block egg albumin re-orientation difficult 4  ​ PLANE WEDGE: Type of microtome knife used to cut tough specimens embedded in paraffin blocks ​ ULTRATHIN MICROTOME: Type of microtome required for tissues fixed using osmium tetroxide (for EM) ​ 27-32 deg: Bevel angle is set at what angulation ​ OILING USING VEGETABLE OIL: Must be done to leather strop prior to use T ​ 45-50 degC: Actual temperature of float out bath M ​ STANDARD SLIDING MICROTOME: Specific type of Sliding microtome regarded as the most dangerous ,R ​ LUBRICANTS FOR HONING: Mineral oil clove oil, xylene, liquid paraffin & soapy water ​ STROPPING: To remove burrs and other irregularities ​ METHODS FOR PREPARING FROZEN ES SECTIONS: Cold knife procedure & Cold Microtome procedure/Cryostat procedure ​ FREEZING AGENTS FOR FREEZING MICROTOME: Liquid nitrogen, isopentane cooled by liquid nitrogen, CO2 gas & aerosol EL sprays ​ FREEZE DRYING: Special way of preserving tissues by rapid freezing (quenching) of fresh tissues at -160 degC G CHAPTER READINGS: INFILTRATION AND SECTIONING AN 5 FAULTS DURING SECTION CUTTING COMPILED BY – MA. CRISTINA SJ LIWANAG pg. 1 FAULTS DURING SECTION CUTTING COMPILED BY – MA. CRISTINA SJ LIWANAG pg. 2 FAULTS DURING SECTION CUTTING COMPILED BY – MA. CRISTINA SJ LIWANAG pg. 3 FAULTS DURING SECTION CUTTING COMPILED BY – MA. CRISTINA SJ LIWANAG pg. 4