CHAPTER 4

Sectioning
Microtomy Sectioningditficulties Frozen sections Tissue-handling chart for sectioning

MICROTOMY Microtomy is an art. All the pages printed cannot teach this very important a-rt, since it must be learned by experience in the histopathology laboratory. It is here that the welltrained experienced histotechnologist stands out in sharp contrast against those with poor training, for she recognizesthe difficulty, what caused it, and how it can be remedied. Successful sectioning of tissue has four requirements: 1. A shilled technologist. Most of the problems of sectioning should be eliminated in the future when the histotechnologists, who are part of the laboratoryteam, have been trained in CAHEA approvedschools. When on-the-job training is no longer the norrn, students will be taught the why, when, where, and how of tissue technology and the importance of their work in the total care of the patient. 2. A sharp microtome knife. The microtome knife is an extremely important instrument in the histopathologylaboratory. A well-sharpened, good-quality microtome knife may, at times, section poorly prepared material. A poor knife never pro* duces good sections of perfectly prepared tissue. 3. A proper microtome. Different kinds of microtomesare availablefor different uses (see Chapter 1). These are precision instruments and require good care to give many years of service. Unless the microtome is abused, mistreated, rusty, caked with paraffin and dirt, and left unoiled, it is rarely, if ever, the cause of poor sections. 4. Properly prepared material Specimens from surgery, for rapid diagnosis,section

better if frozen in OCT rather than in water. Other specimensrequire extensive fixation, dehydration, clearing, and embedding in a supporting medium that must match the physical characteristics of the specimen and have properties suitable for the cutting procedure to be used. SECTIONING DIFFICULTIES Tables 4-l to 4-3 should help the technologist overcome many of the common difficulties. Since the microtome'is rarely the cause of poor sections unless it is old, damaged,or obviouslyworn out, they should aid the technologist in overcoming difficulty in sectioning. The processof sectioning paraffin-embedded material leads to distortion so that the sections are a little bit thicker and a little shorter than the block itself. The distortion results from the following: 1. Nature of the paraffin 2. Nature of the tissue 3. Action of the microtome knife Different paraffins have different plastic points. The plastic point is the lowest temperature at which permanent deformafion may be made without fracture. A paraffin with a low plastic point appears more translucent, is less brittle, but compresses more in sectioning than does one with a higher point. The hardness of the paraffin depends on its plastic point, which lies a few but variable number of degrees below the melting point. Large crystals are sectionedor pushed apart by the wedging of the knife, especially when the paraffin is brittle and has a high plastic point. This upsetting of crystalsgives the smooth and velvety appearance to the topside of the sectionsas the ribbon is laid on the water. When the crystals are not of proper size to fit closelyto the tissue, they cannot support it adequatelyand a section will be cut that cannot be completely flattened out. A fine crystalline paraffin gives adequate support, whereas a paraffin with large crystals produces artifacts in sections. Warm paraffin

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ANDPRACTICE OF HISTOTECHNOLOGY THEoRY

Table 4-1. Difficulties common to all methods

Difficulty lrregular sections, skipped sections, or thick and thin sections Scored, grooved, smeared, and deformed sections Regular, lengthwise scratches and splits in sections Sections fall out of matrix or show an amount of compression different from the embedding medium frame Mushy-appearing sections indicate insufficient dehvdration or clearing Corection These are usually the result of insufficient tilt of the knife, which compresses the block on the return stroke, or of too much tilt, which scrapes off the section instead of cutting it. Turn the knife holder to give the proper clearance angle between the cutting facet of the knife and the specimen. These are caused by a dull knife. Resharpen. These are caused by a defect in the knife edge or by dirt or hard material in the specimen. Moving the knife to an unused region, or replacing with a sharper knife, may restore good sectioning. Embedding medium is inade{uate. Try different medium for embedding.

Sections of tissue that have not been dehydrated properly can rarely be salvaged. If the clearing agent has not been removed completely, retum the tissue to several changes of paraffin and reembed. If sections are mushy in the celloidin technic, they can rarely, if ever, be salvaged, and new tissue must be processed.

Table 4-2. Dtfficulties common to paraffin-embedded material

Difficulty Ribbon fails to form Cause Room too cold or paraffin too hard. Remedy Use lower melting point paraffin. Breath on knife and warm it slightly. Place a desk lamp so that light and heat fall on the knife and block. Tilt the knife less. Cut thinner sections. Resharpen. Unroll a section and hold it lightly against the knife with a camel's hair brush; if the first few sections can be held down, the ribbon will often form and follow. Trim blocks parallel or reembed in molds so that the edge of block is parallel to knife edge. Try another part of knife edge. Reembed the tissue and stir the melted paraffin. Relocate microtome for uniform temperature. Cool block in ice water. Retilt knife either more or less.

Knife tilt too gleat. Sections too thick. Knife is dull.

Crooked ribbons

Blocks not trimmed parallel.

Irregularity of knife edge. Paraffin may be softer on one side of block. One side of block may be warmer than the other from a radiator, lamp, or draft. Sections vary in thickness or are skipped Knife not tilted enough to clear facet or bevel, or tilted too much, and tissue is compresseduntil the inevitable expansion gives a thick section. Clamping set screws are loose. Knife block holder is loose. Microtome worn through lack of lubrication, or not in adjustment.

Retighten. Clamp firmly. Have microtome checked by manufacturer.

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material-cont'd Table 4-2. Difficulties common to paraffin-embedded

Difficulty
Cause

Remedy Soak in water or a solution of g parts of glycerin to I part of aniline oil for 30 minutes; if specimen is embedded in a plastic mold, be careful to keep the mold out of the solution. Resharpen. Cool block in ice water. Increase tilt. Remove with facial tissue soaked with xylene and remove xylene with another facial tissue soaked with absolute alcohol. Very thin sections should be cut slowly and evenly. Salvagerarely possibleif material was incompletely dehydrated. Reinfiltrate with paraffin and reembed. Check thermostatically controlled paraffin baths daily and record temperatures. Try chloroform or toluene instead of xylene for clearing, or a mixture of toluene and cedar oil. Use a harder wax or wax mixture. Resharpen. Use less tilt of knife so that it will cut rather than scrape. Clean as previously recommended. Use celloidin embedding. Dirt in paraffin; refi.lter. Washing was insufficient. Decalciff or desilicify. Change knife Ult. Soak in water to soften. Try celloidin. Increase knife tilt. Try harder paraffin, cooler room, or cool block. Resharpen. Clean with xylene and alcohol as previously described. Increase tilt. Resharpen. Tighten all screws. Clamp tighdy. Decreaseknife tilt. Try celloidin embedding. Boil water in open pan. Burn a Bunsen bumer. Ground microtome to a water pipe with wire or chain. Ionize the air by an electrical method.

Very large blocks, or blocks with hard regions, may spring knife edge while sectioning.

Sections compressed, wrinkled, or jammed together

Knife too dull. Room too warm. Knife tilt too slight. Knife edge gummed with paraffin.

Cutting too rapidly. Sectionscrumble and specimen may tear out Material incompletely dehydrated or not completely cleared. Soft and mushy material incompletely infiltrated with embedding medium. Object too long in paraffin bath or bath too hot. Subject hard and brittle becauseof clearing fluid. When the specimen shatters and falls out of the wax, it is too hard for paraffin. A split ribbon or lengthwise scratches in ribbon Nicks in knife. Too much tilt. Knife edge dirty. Object too hard for paraffin. Hard particles in block may cause scratching. Crystals from mercuric chloride fixation. Calcareous or silicious particles. Knife tilt too great or too little. Material is too hard. Material is too tough for paraffin method. Not enough knife tilt. Room too warm or paraffin too soft. Knife dull. Sections stick to knife Knife edge dirty. Knife tilt too little. Knife dull. Undulations in the surface of the section Scratching noise during cutting Sections fly and stick to parts of microtome or other nearby objects Set screwsloose. Knife holder loose. Excessive knife tilt. Material may be too hard. Static electricity,

Knife rings on up stroke and sections are scratched Sections lifted from knife on upstroke

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ANDPRACTICE OF HISTOTEcHNoLoGY THEoRY

Table 4-3. Difficulties common to celloidin-embedded material

DifficuIty Improperly prepared material Correction Inadequate impregnation or inadequate dehydration prior to impregnation, as well as improperly hardened blocks, make it impossible to get good sections, start over with new tissue. Nicks in the knife. Use a different portion of the knife or resharpen it. These may be caused by dirt in the stock solution; filter it. Or if too much calcium in the tissue, decalcify it. Dehydration was incomplete Infiltration was incomplete. Hardening was incomplete. Tissue may be reinfiltrated and rehardened but never redehydrated. Loose set screws or block holder on microtome; tighten all screws. Do not use pressure on the knife holder since this may depressthe section and raise it while sectioning. Knife is not tilted enough to clear the facet of the cutting bevel. Knife is dull. Microtome is worn and out of adjustment. Material is not properly hardened; reharden. Do not let block dry out during sectioning. Slight dryrng will give this variation. A wooden cradle, available from American Optical Company, may be used for this, or the knife may be carefully laid on a piece of smooth white paper on the stage of the microscope. The only effective test is whether the knife cuts the tissue properly. Testing the knife by any other means merely dulls the edge in the process of testing. March (1878) observed: "Of not less importance than the microtome is the section tnife, to be used in conjunction with it. How perfect soever the former, and whatever the dexterity of the operator, unless he is provided with a suitable, well made knife, he will never succeed in obtaining satisfactory results." His statement is still true. and written direction cannot take the place of experience in learning to sharpen a knife. It is an art that must be learned if the knifes must be sharpened in the laboratory. With the high cost of technical time and the use of this time to its greatest advantage in the many technical procedures in the modern histopathology laboratory, we recommend commercial sharpening of microtome knives. (See section on microtome knives, p. 21.) Routine paraffin sections are cut at 4 to 6 /im. A micrometer (formerly micron) is 1/1000 of a millimeter or 1125.4A0 of an inch. Tissue to be stained for amyloid is cut at l0 pcm, since small deposits of amyloid are demonstrated at this thickness. Tissues for the demonstration of myelin sheath are cut at l0 to 15 p,m. Renal biopsies are cut at 2 to 4 pm.

Lengthwise scratches or splits in the section Specimen falls out of the section

Variation in thickness of section

shrinks as it cools and compressesthe tissue in the block. Tissue that is harder than the paraffin withstands this pressure, but soft or spongy tissuemay be under considerable strain. When sectioned,the tissue tends to expand to the shape and size it had before compression, and if the tissue is confined by the paraffin around it, pleating or wrinkling results. The melting point of paraffin therefore has to be adaptedto the chmate in which you live or the temperatureat which sectioningis done. Additionatly,it must be matched to the hardness of the tissue,which, for the most part, depends on the fixation, dehydration, and clearing of the tissue, as well as the temperature at which it is impregnated with the paraffin. Unless the hardnessof the paraffin is adapted to the temperature at which the cutting is done and to the nature of the tissue, good sections cannot be made, regardlessof the excellence of the microtome and the microtome knife. If the tissue has been properly dehydrated and cleared, it is possible to reembed it in another paraffin, after several changes to wash out the old paraffin. If it has not been properly fixed, dehydrated,and cleared,salvage is rarely possible. The microtome knife should be set and tilted to as little clearance angle as possible to give good sections. The clearance angle should be within 3 to 8 degrees. The knife edge should not show any definite serrations when examined with the microscopeat 100 diameters.

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SECTIONING 83

FROZEN SECTIONS Frozen sectioning of either fixed or fresh tissue may be accomplished by the use of either the clinical (standard) freezing microtome or the cryostat. The various planes for sectioning are shown in Fig. 4-1. Refer to pp. 14 and 15 for sectioning and instrument information.

sagittal-section,frontal-secFig. a-1. Cross-section, tion embeddingand sectioning.

TISSUE.HANDLING CHART FOR SECTIONING lmportant points in cutting paraffin-embedded material l. The block must be trimmed so that edgesparallel to the knife are straight and parallel to each other. tf this is not done, the ribbon will not be straight and distortion will be increased. 2. A camel'shair brush, a fine-eye forceps, or a dissecting needle, may be used to handle the ribbon as it comes off the microtome knife. We have found a fine-eye forceps excellent for this step, since it may also be used to remove bubbles when placed underneath the ribbon on the water bath to break the bubble. It is also excellent for removing fine wrinkles from the tissue on the water bath. 3. An intermediate step between the cutting of the ribbon and the water bath prevents wrinkling on the water bath. FiU a staining dish with distilled water and 95Voalcohol (5 ml of gSVo alcohol to 95 ml of distilled water). Float the ribbon on this solution prior to floating it on the warrn water bath. 4. The temperatureof the water bath should be 5 to 10 degreesbelow the melting point of the embeddingmedium, dependingon the rapidity with which the technologistremovesthe tissue from the water bath. The bath is usually kept at 47" to 51' C, and a thermometer should be kept in the water bath while cutting is being done. 5. A thin coating of egg albumen glycerin is rubbed on the slide before the tissue is picked up. When the sldes are dried, prior to staining, the albumen coagulatesand sets the tissue firmly against the slide. Alternately, another method is to add 7a teaspoonof U.S.P. gelatin to the warrn water bath before the sections are floated on the water. 6. Avoid too much egg albumen. Thick albumen stains and makes a messyhistologicpreparation. 7. Tissues that cut well rarely come loosefrom the slides during staining. The exception is the tissue from the central nervous system and those for silver impregnation technics. The high alkalinity of the silver solutions will, at times, float the tissue off the slide. These may be coated with celloidin prior to staining. a. Remove the paraffin with xylene and place the slide in absolute alcohol for 2 minutes. b. Placethe slide in O.SVo celloidin in ether alcohol 1 :1 for 2 minutes. c. Drain off excess celloidin and place the slide in 80Voalcohol for 5 minutes to harden the celloidin, prior to staining. 8. Be sure to tighten all set screws on the microtome to prevent undulations and thick and thin secfions. 9. The water bath must be kept scrupulously clean and should be cleaned thoroughly after each block is cleaned. Kimwipes are excellent for this purpose. Floatersare dangerous-the result of carelessness.

Continued.

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OF HISTOTECHNoLoGY THEoRY ANDPRAcTICE

TISSUE-HANDLING CHART FOR SECTTONTNG-cont'd 10. Sections should be cut at a slow or moderately even pace. The rapid turning of the wheel cuts a ribbon of thick sections. A student may make a test by cutting a ribbon rapidly and laying it on the water bath with one cut at a slow even pace. The rapidly cut ribbon appears thick to the naked eye. 11. The technologist is frequently requested to cut a cross section, a sagittal section, or a frontal section of tissue. The embedding and cutting of cross, sagittal, and frontal sections is demonstratedin Fig. 4-1. 12. When ribbons fail to form, it may be the result of too slow a cutting speed, but too fast a cutting speedmay cause the same defect or excessiverwinkling of the sections. 13. The crystalline structure of the wax will exert an influence on the quality of sections. Do not keep wax for prolonged periods in an oven or paraffin vat since it can lose its low melting point fractions and present difficulties in ribboning. 14. The quality of the sections you obtain is influenced by the degree of crystallinity of the wax in which you embed the tissue. A fine crystalline wax is preferred. Large and nonhornogeneous crystals in wax are detrimental to good sectioning. Embeddedblocks must be cooled rapidly to prevent the formation of large crystals in the wax, and any race of the clearing agent will result in large crystal formation. 15. When cutting bone sections, do not have the microtome knife enter the bone on the long axis. Embed the bone in the manner shown in Fig. 3-9, B, so that you enter the block at a small corner to cut better sections. 16. Be sure to remove the oil from your knife with xylene and remove the xylene with absolute alcohol before you start to cut sections. 17. If the problem is too hard a block, and softening is required to permit sectioning, then one of the following ways may be tried: a. The block may be faced off (paraffin removed) and placed in water for several hours to several days. b. Place the block in a solution of 9 parts of 6OVoethartol and I part of glycerol for a few hours . or days. c. Place the block in a solution of 9 pa';ts of glycerol and 1 part of aniline. Be careful of using glycerol, since it could prevent the tissue from sticking to the slide flatly. d. The detergent "Joy" may be used in a lVo aqueous solution for a few hours or overnight. 18. To facilitate sectioning after the block has been faced off to the tissue level, hold a piece of cotton or facial tissue paper soaked in ice water against the face of the block, or directly apply an ice cube to the block surface. Caution: Crank the block back slightly or the first section will be too thick. 19. When cutting tissue like thyroid, use the alternate heat and cool method. Cool the block with the ice-cooled tissue paper and alternately heat it by dipping your thumb in warm water and and holding it against the block. Repeat several times until you get a good ribbon. 2O. Section leuels It is important when cutting small biopsies to cut them at several levels. Thefrst leuel should be taken when you first reach the tissue. Do not shaae off the tissue. Carefully cut into the block and take a ribbon of first-level sections.Ribbon off 10 sections and take the second level ribbon. Carefully cut into the block (using the adjustment screws) until the ribbon marches the tissue in the block. When this happens, you have a full level for your third section. 21. Serial sections When preparing serial sections, slides are numbered, consecutively, and section ribbons are flattened and mounted on the slides from left to right. If the block is small enough, two ribbons are mounted on each slide from left to right. All sections are used after they are cut serially; so it is important to avoid wrinkles, if possible, since these cannot be eliminated from a serial sectionset. Rememberto move the block babk betweenribbons to avoid losing any of the tissue. 22. Affixing sections to slidesuith Mayer's albumen fixatiue Preparedsolution is economical to buy but may be made in the laboratory. a. Beat egg white until broken up but not stiff. Let stand overnight. b. Measure liquid from bottom of cylinder and add an equal volume of glycerin. c. Add a grain of thymol and filter through glass wool.

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SECTIONING 85

TISSUE-HANDLING CHART FOR SECTIONTNG-confd When using Mayer's albumen fixative, you should rub a small amount over the surface of the slide. To avoid dead epithelial cells from the finger, use a small piece of plastic foam or a Lipshaw tissue capsule pad no. 84bA. 23. Gelatin in utater both a. Drop Y+teaspoonof U.S.P. gelatin into a clean hot water bath 47' C. The gelatin will dissolve in the water within a few minutes. Do not buy gelahn in large packets; buy it in small packets. b. Float sections on the water and pick them up on clean glass slides. Drain off water and dry.
CAUTION:

(1) Gelatin will pick up dyes if an excess of gelatin is used. (2) Gelatin may produce artifacts in some special stains. (3) When gelatin is used, the water bath must be kept scrupulously clean to avoid bacteria on tissue sections. 24. How to section undecalcified bone a. Use a Sorvall JB-4 Microtome with MicroMacro Stage 45166 assembly and glass/diamond knife holder no. 45211, b. Insert the slide adaptor (45364) to hold the plastic block holder (aSaS8). c. Put the glass knife in the knife holder and the block in the plastic block holder. d. Set the instrument to cut at 4 p.m. e. Position magnifier-illuminator (No. 45314, DuPont Instruments-Sorvall, Newtown, Conn.) to view the cutting process. f. After cutting to desired level, take one section at a time using cuned microdisseetingforceps (Clay Adams, no. 41909; 6439). Hold the very tip of the plastic surrounding the section. Blow lightly and let the section fall to distilled water. Pick up on clean dry slide and place slide on hot surface at 50o C and let it dry. Use canned "Air It" (No. 11555-10, Matheson, Coleman & Belle, East Rutherford, N.J.) to clean away the excess sections and the debris on the knife. Be careful not to get forceps wet and do not touch the glass knife with the fingers since any moisture on plastic will cause it to stick. g. Place slides in 60" C oven overnight. h. Proceed with staining (p.96). Eliminate xylene and hydration. Place slides directly into stains. Note: Glass knives may be made by using LKB knife maker style 7800 (LKB Instrumenrs, Inc., Rockville, Md.) and following their instructions. Store glass knives in a dust-free container.

REFERENCE Richards, O. W.: The effective use and proper care of the microtome, Buffalo, N.Y. 14215, 1959, American Opticd Corporation, Scientific Instrument Division.

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