Histopathologic Techniques - 04 Sectioning

HISTOPATHOLOGY AND CYTOLOGIC TECHNIQUES TYPES OF TISSUE SECTIONS SECTIONING • • OUTLINE • • • Introduction Microtomy - Sectioning o Types of Tissue Sections o Types of Microtome o Honing & Stropping o Floatation Water Bath o Deparaffinization o Adhesives o Frozen Sections Faults o Faults Observed During Section Cutting • • Paraffin sections - 4-6 um o Uses Rotary microtome Celloidin sections - 10-15 um o Uses Sliding microtome Frozen sections(cryostat) - 4 um o For rapid diagnosis (patient is in OR) o Uses Cold Knife & Cold Microtome INTRODUCTION • • • Staining is the process of applying dyes on the sections to see and study the architectural pattern of the tissue and physical characteristics of the cells. This is made possible because different tissues and cells display varying affinities for most dyes and stains during the process, so that they become more visible, morphologic changes are more easily identified, and the presence or absence of disease process can be established. Physical characteristics and structural relationships of tissues and their cells are thereby studied and evaluated. In order to create permanent slides for histoparhology, mounting of stained tissue section must be done. Mounting media holds the specimen in place, in between the slide and coverslip, and allow the specimen to be stored for years. Mounting media may also provide the maximum degree of transparency to stained tissue section. Fixation → Dehydration → Clearing → Infiltration → Embedding → Sectioning o Fixation - formalin o Dehydration - descending grades of alcohol o Clearing - xylene o Infiltration - paraffin wax o Embedding - embedding mold & media (paraffin) o Sectioning - use microtome TYPES OF MICROTOME • • Microtome - cutting engine in histopathology; used to cut tissue into thin slices Rocking microtome/ Cambridge o Invented by Trefall o Most simple o Thickness of sections: 10-12 micra (u) o Purpose: prepare serial section of large paraffin blocks o Disadvantage: ▪ Difficulty in re-orienting the block ▪ Restriction in the size of block that can be cut • Tissue block should be the same size as the block holder MICROTOMY – SECTIONING • • • • Tissue block - product of embedding Sectioning - cutting tissues into thin slices to facilitate their study under microscope Microtome - used to cut tissue into thin slices; cutting engine Microtome and its parts o Block holder/chuck - holds tissue block during section cutting o Knife carrier & knife and disposable blades - most important parts of microtome; used for actual cutting of tissues into thin slices ▪ Knife - traditionally used ▪ Disposable blades - commonly used nowadays o Rotating wheel - mechanically moved or manipulated to start the cutting process o Pawl, ratchet feed wheel & adjustment screw manipulated to line up the tissue block in proper position with the knife o Micrometer gauge - determines thickness of sections • Rotary Microtome o Most common type o Thickness of sections: 4-6 micra o Invented by Minot o Purpose: to cut paraffin embedded tissues • Sliding Microtome o Invented by Adams, most dangerous due to exposed knife o Thickness of sections: 7-9 micra o Purpose: to cut celloidin embedded tissues & to cut extremely hard/tough tissues o Base sledge - less dangerous because the moveable part is the block holder and the stationary is the knife o Standard Sliding - more dangerous, the movable part is the knife and the stationary part is the block holder • In the laboratory, we usually prepare 2 types of sections o Paraffin section - use rotary microtome o Frozen section - tissue section usually requested for rapid diagnosis (while patient is in OR) ▪ Do not undergo fixation METHODS OF PREPARING FROZEN SECTIONS • • • Frozen section - for rapid diagnosis Cold Knife Procedure - uses freezing microtome Cold Microtome Procedure/Cryostat method - uses Cryostat MICROTOME KNIVES • • • • Ultrathin Microtome o Purpose: for EM specimens o Thickness of Sections: 0.5 micra o Tissues are usually embedded in plastic Freezing Microtome o Invented by Queckette o Used to prepare frozen section o Can immediately harden tissues without prior fixation o Uses intermittent burst of carbon dioxide to freeze the block holder and tissue ▪ CO2 - freezing agent to immediately harden fresh soft tissue in order to facilitate immediate cutting o Also with a second cooling device for lowering temperature of knife o Uses CO2 + device tha can lower knife temp o Thickness of sections: 10-15 micra o Purpose: need to demonstrate fats o For demonstration of fats and other neurological structures o To cut tissues with heat-sensitive structures • • • • • Cryostat / Cold Microtome o More commonly used nowadays o Cryostat - a refrigerated apparatus ▪ Rotary microtome enclosed in a cold chamber o For fresh tissue microtomy, refrigerated (-5 to -30 degC) average is -18 to -20C o The temperature inside the chamber will immediately harden tissues to facilitate immediate cutting o For preparing thin sections of fresh frozen tissues for fluorescent antibody staining or histochemical enzyme studies • • • To cut tissues, traditionally we use microtome knives but now we use disposable blades Plane concave – 25mm long, o One side of knife is flat and the other side is concave o Concave side - for cutting paraffin embedded tissues o Flat side - cutting celloidin embedded tissues Biconcave – 120mm long, o Both sides are concave o Used to cut paraffin embedded tissue Plane wedge – Usually 100mm long recommended for frozen sections and extremely hard or tough tissues o Both sides of the knife are flat If we use knife: o Blunt/dull = pumurol o Nicks = may bungi Important angles considered in sectioning o Bevel angle - set at 27-32 degrees; angle formed between the cutting edges o Clearance angle/tilt angle/inclination angle - set at 0-15 degrees; but during sectioning it is set at 5-10 degrees; angle formed between the surface of the block and the cutting edge of the knife ▪ <5 degrees - increased thickness ▪ >10 degress - decreased thickness ▪ Observed to prevent uneven section ▪ Sections that are alternately thin/thick o Wedge angle - set at 14-15 degrews; angle formed by the sides of the wedge knife Disposable Blades - 2-4 u Glass Knives - for EM Diamond Knives - for EM HONING & STROPPING • HONING • • • • • Process of grinding the cutting edge to have an even edge Purpose: to remove gross nicks in microtome knives Uses hones/oil stones (rough stones) o Belgium yellow - gives the best result o Arkansas - gives a more polishing effect than Belgium yellow o Fine Carborundum - recommended for badly nicked knives; type with a much coarser bite Procedure o Heel to toe movement, edge first o No of strokes required: 20-30 strokes (30+ strokes depending on condition of the knife) Lubricants: soapy water. Mineral oil, clove oil, xylene or liquid paraffin • Fishing out o Removal of ribbons from the float out bath once the ribbons have flattened out o Orientation - process of placing the tissue in a precise position in the center of slide ▪ Orientation must be observed Alternative to float out bath - manual stretching using applicator sticks STROPPING ADDITIONAL NOTES • • • • • • • • • • • • • • Process of polishing and sharpening the cutting edge Purpose: to remove burrs and other irregularities formed during honing Use leather paddle strop made of horse leather o Provides smoother polishing Strops are usually treated with oil Do not use mineral oil which can blister and destroy the leather Procedure: With a toe-to-heel movement edge last Uses 40-120 double strokes o May go beyond 120 depending on knife condition Do not require lubricant Must be oiled prior to use Stropping may be carried out without prior honing Stropping must be done after the process of honing Disposable blades will not require honing and stropping NOTES • • • • • • • ADHESIVES • • • • • • • • • • • Glass plates with finely powdered aluminum oxide made into paste with water = abrasive Diamantine to be used for final polishing FLOTATION WATER BATH Product of sectioning - thin slices/tissue sections/ribbons Ribbons - thin slices of tissue Removes the wrinkles with tissue ribbons Float out bath - thermostatically controlled bath used to remove wrinkles and folds; flatten out the ribbons o Temperature: 5-10/6-10C lower than the wax melting point (45-50C) Drying of slides o Leaving slides in 37 degC incubator overnight o Placing an oven 50-60 degC 2 hours o Drying using a hot plate 45-55 deg C 30-45 minutes Paraffin oven in use - 55-60C or 2-5C higher than wax MP Float out bath - 45-50C or 6-10C lower than wax MP Purpose: to promote attachment of tissue sections to slides to prevent detachment from slide Placed before fishing out to promote attachment Mayer’s egg albumin - routine/common tissue adhesive o Combination of equal amount of egg white and glycerine with thymol crystals to prevent mold growth Dried Albumin 1% Gelatin Gelatin- Formaldehyde mixture Starch Paste Plasma Poly-L-Lysine - recommended for immunohistochemistry APES (3-aminopropylthriethoxysilane) - for cytology Sodium silicate – commercial syrup 1:10 and dilution with strong adhesive property DEPARAFFINIZATION • • Process of removing excess paraffin wax from tissues once properly fixed on the slide before staining Methods of Deparaffinization o Passing the slide over a flame using alcohol lamp o Immersion of slides in xylene o Placing the slides inside the oven (55-60C) FROZEN SECTIONS • • • • Rapid diagnosis; For enzyme histochemistry Demonstration of soluble substance Immunofluorescent & immunocytochemical staining ; Specialized silver stains Two methods: o Cold knife ▪ Requires freezing microtome ▪ Uses freezing agents to facilitate immediate hardening of fresh tissues for immediate cutting ▪ Tissue block – 3-5 mm ▪ Dew Line – point in which section may be cut at 10 um o Cryostat - requires cold microtome • Methods of freezing o Liquid nitrogen - generally used in histochemistry; most rapid freezing agent o Isopentane - liquid at room temp o Aerosol sprays (cryokwik) o Carbon dioxide - often used when using freezing microtome o Freon 2.2 – high thermal conductivity FAULTS FAULTS OBSERVED DURING SECTION CUTTING FAULTS REASON Sections expand or • Poor impregnation disintegrate on water of tissue bath • Water temperature too high in flotation bath Sections roll into a • Blade dull coil instead of • Rake angle too remaining flat on small knife edge • Section too thick Grooved, Scored, smeared and deformed sections • Dull knife /blade edge • Too warm room Sections fail to form ribbons Sections roll up on cutting so that they adhere and get broken against the knife edge Ribbon is curved, crooked or uneven instead of straight FAULTS Sections are • Knife is blunt or compressed wrinkled dull or jammed • Paraffin block is warm and soft • Knife edge is coated with paraffin • Sections are too thin • Microtome set screw is loose • Tilt of knife is too vertical • Too rapid cutting REMEDY • Re-process tissue • Turn down the temperature of the flotation bath • Use a new blade • Reduce blade tilt if clearance angle is excessive • Reduce section thickness • Re-sharpen the knife/blade or move the existing blade to an unused surface or replace it with a new one • Cool/change the temperature of the room or cool the block in ice water • Re-trim the block • Surfaces and edges of the block are not parallel • Horizontal surface • Re-adjust and reof the block is not orient the block parallel to the knife • Paraffin wax is too • Coat horizontal hard edges of the block with wax of lower melting point • Knife is tilted too • Reduce the tilt much • Sections are too • Re-adjust thick thickness of the section • Knife is dull • Hone and strop • Knife is blunt • Sharpen the knife • Tilt of knife is too • Reduce the tilt great • Knife edge is dirty • Clean the knife edge • Blunt of dull spot • Adjust the knife so on the knife, that knife edge will producing an present a irregular knife uniformly sharp edge edge to the block or sharpen • Edges of the block • Re-trim the block are not parallel but round or wedge shape • Knife is not parallel • Readjust knife and to the block block REASON • Paraffin is impure • Too warm room • Unparallel block edges Sections are squashed (width of each section is less than that of block A hole is formed in the section Sections of unequal thickness are produced Sections adhere to the knife or other parts of the microtome REMEDY • Repeat impregnation using pure wax • Re-sharpen the knife • Cool the block on ice water until firm • Clean the knife edge • Re-adjust thickness of section • Tighten screw • Reduce the tilt • Decrease cutting speed for every thin section • Cool/change the room temperature or cool the block in ice water • Trim the block edges until they are parallel • Re-sharpen using a knife back or automatic knife sharpener • Re-embed in freshly filtered wax if necessary • Bevel of knife is lost due to incorrect sharpening • Bubble or dirt formed in the embedding medium • Hard spot in tissue • Once embedded in due to calcium paraffin wax, decalcification is impractical; use a base sledge microtome with a wedge knife • Excessive • Expose the tissue, dehydration or then soak the improperly block in ice water processed tissue or with a piece of cotton before sectioning • Tilt of knife is too • Reduce the tilt great or bevel is not cleared hence object is compressed against the knife edge • Clamp set screw • Tighten the screw on knife or blockholder is loose • Blocks are too • Cut blocks into large smaller fragments • Blocks are too • Soften the blocks hard in detergent or phenol • Static electricity • Breathe out or due to low blow gently on the atmospheric block and knife to humidity breakup static electricity, or boil water in the room FAULTS Ribbon is split or lengthwise vertical scratches are seen on sections REASON • Knife edge is dirty • Knife edge is dull • Tilt of knife is too great • Nicks or damage on the knife edge • Dirty embedding • Knife edge is dirty • Tilt of knife is too great Sections are lifted • Knife tilt is too from the knife on the great upstrokes • Knife is dull • Paraffin is too soft or room temperature is warm Resistance is felt on • Tilt of knife is too the lower part of the small, paraffin section during block is therefore cutting compressed against the base of the knife towards the end of stroke Horizontal or parallel • Knife edge lines or furrows vibrates due to across the section hardness of tissue (chatters) are seen, • Tilt of knife is too forming thin and great thick zones • Overhydration or lack of moisture in tissue • Use of dull knife/blade • Cutting too quickly Section cut is sometimes thin sometimes thick Knife makes a hard metallic scrapping or ringing sound on backstroke, when section is cut Frozen tissue crumbles and comes off the block holder when cut • Knife is blunt • Knife is not clamped properly • Tilt of knife is too great • Knife or block holder is loose • Knife tilt is too small that block is compressed by bevel and section is not cut • Tilt of knife is too slanted or too big • Tissue is too hard • Knife blade is too thin • Freezing is not adequate REMEDY to increase humidity • Clean knife edge • Sharpen the knife • Reduce tilt • Sharpen the knife FAULTS Frozen tissue chips into fragments when cut Pleated, folded and crooked ribbons • Re-embed in filtered wax • Clean knife edge with xylene • Reduce the tilt • Reduce the tilt Brittle or Hard tissues • Sharpen the knife • Cool paraffin wax in ice water • Increase the tilt Clearing agent turns milky as soon as tissue is placed in it • Treat with phenol during processing or collodionize • Reduce tilt • Restore moisture of tissue by letting the block stand and soaking it briefly in ice water or by placing it face down on an ice tray • Resharpen the knife/blade • Decrease cutting speed • Sharpen the knife • Adjust the knife • Reduce tilt • Tighten adjusting and locking screw • Increase the tilt • Readjust the angulation of the knife • Take fresh block treated with phenol during processing • Change the knife • Refreeze the tissue block On trimming, tissue smells of clearing agent Tissue is opaque, section cutting is difficult due to presence of alcohol Tissue shrinks away from wax when trimmed Tissue is soft when block is trimmed Airholes are found on tissue during trimming On trimming wax appears crystalline Paraffin block, after cooling is moist and crumbles Inadequate processing of fatty tissues REASON • Tissue is frozen too hard REMEDY • Warm the tissue with the fingers • Imperfections in the blade edge, i.e. the horizontal edges are not parallel or the lower block edge is not parallel to the knife edge when sectioning • Trim the block parallel to the other edge or reembed them in molds so that the edge of blocks is parallel to the knife edge • Try another part of the knife edge • Prolonged fixation • Tissue may be softened by • Prolonged soaking in a small dehydration dish or bowl • Prolonged clearing containing water • Prolonged paraffin with detergent, infiltration phenol or molliflex • Overheated paraffin oven • Drying out of tissue before actual fixation • Water not • Repeat completely dehydration with removed due to absolute alcohol incomplete then clear again dehydration • Clearing agent not • Block is trimmed completely down nearest to removed due to the tissue. insufficient Remaining wax is impregnation melted on embedding oven and paraffin impregnation is repeated, hanging the paraffin at least once before blocking • Insufficient • Repeat clearing; if clearing object has already been embedded, prolong clearing up to 12 hours, then re-embed • Insufficient • Repeat the whole dehydration procedure therefore incomplete clearing and impregnation • Incomplete fixation • Repeat the whole procedure • Incomplete • Repeat impregnation impregnation • Contaminated wax • Re-embed in freshly filtered wax • Block not cooled rapidly enough • Insufficient paraffin • Repeat paraffin impregnation impregnation, • then re-embed • Routine schedule • Fatty tissues and reagents may should be sampled and cut thinly for • not process fat easy reagent adequately action. Ethanol is a poor fat solvent. FAULTS Hard, dense tissues On embedding, tissue is soft and mushy in the center • REASON • Tough keratin component of certain • tissues • Inadequate processing due to very thick or fatty sections REMEDY To ensure complete dehydration, a far better lipid solvent (acetone or isopropanol) should be inserted before using the final absolute ethanol • Treat fixed tissues using 4% aqueous phenol for 24-72 hrs to soften • At gross, cut thin sections especially fatty tissues, ensure • complete fixation