Lymph node biopsy specimens

Transcription

1 CE Update Anatomic Pathology Achieving Technical Excellence in Lymph Node Specimens: An Update Carolyn Beard, HTL(ASCP); Kay Nabers, HTL(ASCP); Mary C. Bowling, MT(ASCP); and Costan W. Berard, MD Lymph node biopsy specimens present unique problems to the pathologist and the histotechnologist. Because lymph nodes are composed of millions of tiny cells with little connective tissue to hold them in place and, furthermore, are surrounded by a capsule that inhibits penetration of chemicals, the utmost care is required to transform them into superior histologic preparations. In no other area of pathology is the diagnosis predicated to such a degree solely on the microscopic evaluation of a tissue section, without the benefit of definitive clinical symptoms or laboratory tests. Not only is the histologic pattern (the relation of cells to each other) important, but each cell must be studied to categorize the malignancy (if one is indeed present) and indicate the optimal treatment for the patient. Technically excellent histologic preparations from lymph node biopsies result when a complete understanding of all factors involved exists on the part of the surgeon, the pathologist, and the histotechnologist. 1 " 5 Each makes a crucial contribution to the end product. Properly blocked and fixed lymph nodes are difficult to diagnose if the tissue is inadequately processed or poorly sectioned and stained. Conversely, the best histotechnologist cannot prepare Mary C. Bowling, the original author of the 1979 article in Laboratory Medicine on which this article is based, is now retired from her position at the National Institutes of Health in Bethesda. This classic CE Update article has been revised and updated. Kay Nabers is affiliated with Baptist Memorial Hospital in Memphis. Carolyn Beard and Dr Berard are affiliated with St. Jude Children's Research Hospital, Memphis, TN excellent slides from thickly blocked and poorly fixed tissue. This article reviews all of the steps necessary for the preparation of lymph node slides, the rationale behind each procedure, the pitfalls encountered, and the corrective action to be taken. The histotechnologist when sectioning the paraffin block, and both the pathologist and the technologist when examining the slide microscopically, should be able to recognize which step (or steps) in the preparation is responsible for less than excellent results. Comprehensive Preparation Lymph node tissue should arrive from surgery fresh and in a quantity of saline or tissue culture medium sufficient to ensure that the tissue does not dry out. The authors have found that transporting the node wrapped in a dry gauze pad can produce drying artifact (Fig 1) even more quickly than does exposure to air. Such transport may also cause crush artifact. Prompt blocking of the node is essential. If gross abnormalities are observed, one should make sure that portions submitted for special studies are representative of the specimen. Touch preparations may be of diagnostic value and should be made from the cut surface of the fresh node. Sterile conditions are necessary for certain special studies: (1) microbiology for culture, (2) immunology for surface markers, and (3) cytogenetics. Prompt blocking should precede fixation for electron microscopy and light microscopy because the capsule around 468 LABORATORY MEDICINE VOL. 16, NO. 8, AUGUST 1985 the node retards penetration of the fixative. Lymph nodes fixed whole will show a fixed outer rim and a central area of autolysis (Fig 2). Thin blocks are essential. The extra effort expended to cut 3-mm thin blocks will ensure adequate and uniform fixation (Figs 3 and 4). Thick blocks, even if completely fixed, will not dehydrate, clear, and infiltrate on an overnight tissue processor schedule. Fixation Fixation is the most important step in the preparation of histologic sections: inadequately fixed tissue is beyond repair (Fig 5). Tissue blocks can be reinfiltrated, recleared, and redehydrated, but nothing can restore them if the initial fixation was inadequate. Fixation is the point of no return. Poor fixation is responsible for a large proportion of those lymph node slides that are found to be too technically poor to be adequate for diagnosis. Lack of proper fixation is costly: to the patient whose biopsy has been rendered useless; to the histotechnologist who has spent an inordinate amount of time preparing inadequate slides from blocks that will not section properly; to the pathologist who wastes valuable time searching for definitive microscopic details that have been obliterated; and finally to the consultant who finds that most of the problem cases he receives are technically inadequate for diagnosis. Technically excellent slides can be prepared from lymph nodes using any fixative, if the solution is of proper

2 . V Fig 1. Section from air-dried lymph node displays drying around edge. The rim is darkly stained and smudgy. artifact Fig 2. Section from lymph node fixed whole displays intact periphery and pale shredded center. strength and volume, and if sufficient time is allotted for fixation (Fig 6). A 10% buffered neutral formalin solution is used most widely for fixation and is satisfactory. Most histopathology texts recommend overnight or 24-hour processing to ensure adequate fixation. Although fixatives containing mercury are rapid and enhance the sectioning quality and cytologic detail, they penetrate poorly and are unsatisfactory if the blocks are not thin. Overfixation in these solutions will make the tissue brittle, cause fragmentation during sectioning, and result in poor nuclear staining. The authors use B-5 fixative, a formalinmercuric chloride mixture made nearly neutral with sodium acetate. As with the mercury-chromate mixtures, tissues do not require washing after fixation and excellent fixation of 3-mm blocks is achieved in four hours. Tissue should not remain in B-5 longer than 24 hours, and is best stored in 10% neutral buffered formalin. Some fresh lymph node specimens are difficult to section at intervals of 3 to 4 mm upon gross dissection. The node should be sectioned as thinly as possible, placed in B-5 fixative, and checked after 10 to 30 minutes. Sections may be removed and cut ideally at this time because the fixative has firmed the tissue. B-5 can also be used as a post-fixative after fixation in 10% neutral buffered formalin. Such postfixation can yield almost the same crisp cytologic detail as initial fixation in B-5. Given the solution's near neutrality, sections fixed in B-5 react with most special staining procedures in the same way as formalin-fixed tissue. As with any solution containing mercury, B-5 fixative should be han- died and disposed of properly (Appendix). Glutaraldehyde at 2% to 3% is the fixative of choice for electron microscopic preparations. The fixative must be fresh, the ph being checked for acidity, and it must be kept refrigerated. Glutaraldehyde penetrates slowly but has excellent properties of preservation. In the authors' experience, the fixation time has ranged from a minimum of two hours to a maximum of two to three days. After three days in fixative, there is a loss of cellular contrast. Place a large drop of fixative on a piece of dental wax. Mince a piece of tissue to no greater than 0.1 cm in size, making sure that the tissue stays flooded with the fixative. The volume of any fixative should be at least 20 times that of the tissue, allowing ample room for the fixative to permeate the tissue. *' \»-> Fig 3. Thin (3-mm) section of lymph node. Note the white collagenous bands and pale-gray cellular areas. Fig 4. Section from thin, well-fixed section (shown in Fig 3) allows the direct comparison of gross and microscopic features. LABORATORY MEDICINE VOL. 16, NO. 8, AUGUST

3 Fig 5. Section from poorly fixed node. Linear tears ("Venetian blind" effect), which are often misinterpreted as sectioning artifact, are caused by the friable nature of unfixed tissue. Fig 6. Thin serial sections free of artifact prepared from properly fixed lymph node. Tissue Processing Thinly blocked and properly fixed lymph node tissue should present no problem to the histotechnologist. The responsibility for preparing technically excellent slides from these blocks (barring unrealistic budgetary restrictions on chemicals and equipment) belongs entirely to the technologist. If an automatic tissue processor is used for fixation, one must ensure that (1) the fixative be changed often, preferably rotated every other day, because one full basket of tissue can dilute and deplete the solution; and (2) the time scheduled for fixation should not "short-change" the remaining processing steps. The automatic tissue processor only moves the tissues from one container to another. The technologist selects the combination of chemicals or solutions to be used and ensures the purity of each of the solutions used, changing them frequently to avoid dilution and contamination. Dehydrating agents must be watermiscible, and it is generally believed that this process should be done in graded strengths of ethanol to replace the water in the tissue with ethanol. The authors find isopropyl alcohol to be a good substitute for ethanol if ethanol is unavailable. Each basket of tissue carries with it some of the solution from the previous beaker or container into the next. If the last dehydrant has any trace of water in it, the tissues are incompletely dehydrated and the clearing agent (the authors use xylene), which Fig 7. Section from inadequately dehydrated block displays irregular stellate cracks referred to as "dry earth" effect. will not mix with water, cannot remove this moisture. One should avoid xylene contamination by alcohol carryover. The first container should be changed frequently or alcohol may be further carried over to the subsequent xylene beaker. Infiltration with paraffin occurs only where the paraffin solvent (ie, the clearing agent) is replaced by paraffin itself. Any residual moisture or dehydrant will prevent the infiltration of the paraffin. Under any of these conditions, paraffin blocks will be difficult or impossible to cut. Sectioning time will be prolonged. Tissues not completely dehydrated will not be adequately cleared or infiltrated, and will crumble when sectioned and fragment or explode on the water bath (Fig 7). Tissues that are properly dehydrated but 470 LABORATORY MEDICINE VOL. 16, NO. 8, AUGUST 1985 only partially cleared and therefore only partially infiltrated with paraffin will section unevenly (thick and thin), compress, wrinkle on the water bath, and often will not ribbon (Fig 8). Properly dehydrated and cleared tissue that is not completely infiltrated with paraffin will contain some of the clearing agent, which can be detected by smelling the cut surface of the block. As the residual clearing agent evaporates, the tissue will shrink away from the paraffin (retract into the block) and fall out during sectioning, or else will not section at all (Fig 9). The infiltration of tissues with paraffin is greatly improved when a vacuum is used. The vacuum helps to remove trapped gases and residual clearing agent and pro-

4 Fig 8. Section from a block that was not properly cleared. As a result, the tissue becomes wrinkled and compressed during sectioning. Fig 9. Paraffin block containing incompletely infiltrated lymph node. The shrinkage of tissue away from the paraffin in the block causes the mottled appearance. duces more uniform infiltration of paraffin into the tissue. Ideally, none of these problems should occur. If they do, the tissue can be "backed up" through the process to the point of error and then reprocessed. The quality of the sections will not, however, be as good as if they were properly processed in the first place. One should check the temperature of the paraffin baths often. Overheating affects the quality of all tissues, but overheating will destroy lymph nodes. "Cooking" makes them hard and brittle: the tissue when sectioned often falls out of the ribbon like sawdust. Microscopic detail is lost because the cells are coagulated (Figs 10 and 11). Lymph node biopsies can be embedded in the paraffin routinely used in any laboratory. Most commercial preparations contain a small percentage of plastic and are excellent. The authors add piccolyte 3 to the embedding paraffin. Piccolyte is of great benefit in obtaining thin sections. Hammond and Beckman" describe a method for adding piccolyte to paraffin. The authors, however, use the Nabers modification of this procedure. Following the Nabers modification, a 5% to 7% volume of ground piccolyte is added to melted paraffin (peel-a-way paraffin pellets b ). The piccolyte resin is first ground to a fine powder in a coffee grinder. Melted paraffin is placed in a steel beaker and the ground piccolyte is slowly added while the mixture is continuously stirred with a Teflon rod. The beaker is then placed overnight in a 60 C oven to facilitate dissolving the piccolyte. One should always stir the piccolyte-paraffin mixture before using it for embedding. Several blocks of lymph node, each containing one piece, can be sectioned easier and faster than one block containing many pieces. If this is not feasible in your laboratory, the proper embedding of multiple pieces is depicted in Fig 12. If the paraffin block is chilled too rapidly after embedding or during sectioning, this will cause cracking, and very often the lymph node will crack along with the paraffin (Figs 13 and 14). This cracking can occur when Freon sprays are used to cool the block. The authors recommend that one use.«. t I f % 1f» m %jf «\ *w*ikj * 4 % pom m & w *^* Fig 10. Section from lymph node infiltrated in overheated paraffin Fig 11. Higher magnification of section in Fig 10 shows waxy bath. Connective tissue (center) appears pale and homogeneous. collagen and smudged nuclei in connective tissue. v < LABORATORY MEDICINE VOL. 16, NO. 8, AUGUST

5 Incorrect Correct 0000 Fig 12. Illustration of incorrect embedding (left) and correct embedding (right) of multiple pieces of tissue. for the diagnosis of these diseases (Fig 17). Sections 5 to 6 i.m thick are acceptable but even better detail is afforded if sections are cut at 3 to 4 ^.m (Fig 18). This takes a skilled technologist, properly processed tissue, and a correctly sharpened microtome knife that is properly used. These tissues should be segregated from the routine materials and assigned to the best technologist in the laboratory. Improper sectioning can introduce a wide variety of artifacts. a piece of ice to chill the block just before cutting after facing. Allow a margin of paraffin on all sides of the tissue to facilitate ribboning during sectioning. Even if the microtome knife edge looks fine, do not assume that the knife is really sharp. Compare the section retrieved from the water bath with the paraffin block. The section should be nearly the same size as the tissue in the block. If it is smaller, and if the knife angle is correct, the microtome knife is dull (Fig 15). With a sharp knife and a bit of patience, it is possible to produce technically excellent sections.... with just a few more things to remember. The sections must be thin, preferably one-cell thick. (Since lymphocytes are quite small, this means 3 to 4 xm.) Do not be lulled into a false sense of security by the microtome setting. If the microtome wheel is turned rapidly, the heat of friction between the block and the knife will make the sections 2 or 3 n-m thicker than the indicated setting. Blocks should be cut with a slow and even rotation of the wheel (Fig 16). Disposable knives are helpful in sectioning, but their proper use requires patience and good technique. A disposable blade enables one to change knives to ensure a sharp edge without time-consuming mechanical sharpening. Even properly processed lymph node tissue is delicate. If an attempt is made to stretch the tissue to original size on the water bath (because it was compressed by a dull knife during sectioning), it will crack and pull apart, destroying the pattern the pathologist seeks to examine. Any method that distorts the sections when separating them on the water bath should be avoided. Thin sections with crisp cytologic detail are essential for the diagnosis of hematopoietic and lymphoreticular tissues. Thick sections obscure cellular morphology and lead to impressions based on "pattern," which provides a notoriously risky criterion Staining Any routine hematoxylin and eosin procedure, properly controlled, is satisfactory for staining lymph nodes. Difficulty arises when slides are overstained in a regressive type of hematoxylin (such as Harris' hematoxylin) and subsequently not properly differentiated. This problem can be avoided only by microscopic monitoring of differentiation to the proper degree. Nevertheless, Harris' hematoxylin in the hands of a skilled technologist yields excellent nuclear detail. In a routine histology laboratory, consistently excellent hematoxylin staining can be achieved easily by using a progressive type of hematoxylin (such as Mayer's or Gill's hematoxylin) that does not overstain and, therefore, does not require differentiation in acid alcohol. Nuclear details essential for diagnosis, such as the chromatin pattern, nucleoli, and clefts or lobulations of the nuclear membrane, can only be Fig 13. Paraffin block chilled too rapidly. Note the cracks in both the lymph node and the paraffin. Fig 14. Section from paraffin block chilled too rapidly. 472 LABORATORY MEDICINE VOL. 16, NO. 8, AUGUST 1985

6 fcffetfii \ Fig 15. Lymph node sectioned with a dull knife. Although wrinkles and folds resemble "compression" effect, they are caused by the dull knife. Fig 16. Same lymph node as seen in Fig 15, sectioned with a sharp knife. visualized in material that is properly fixed, processed, sectioned, and stained. These nuclear structures are poorly delineated in understained sections and obscured in overstained sections. The depth of eosin counterstaining has traditionally been based on the personal preference of the pathologist. Taste and color perception do vary and, unless the nuclear stain is obliterated by the eosin, individual preference should prevail. The most common error in eosin staining involves a passage through the subsequent alcohol steps that is too rapid. The alcohols are used primarily to dehydrate the sections, but they also serve to remove excess eosin. If slides are rushed through the alcohol, the sections may appear muddy and the excess eosin may obscure pigments, inclusion bodies, or other easily overlooked, but nonetheless important features. Special staining procedures are too numerous to include in this article, and descriptions of most such procedures are easily found in the literature. The Appendix includes two procedures that can be troublesome: an esterase reaction for neutrophilic myeloid and tissue mast cells, and a methyl green-pyronine (MGP) stain for RNA and DNA. Plastic Sections The authors have used plastic embedding from time to time when it has been specially required. Plastic embedding provides very thin sections (2 (j.m or less) with greater clarity, contrast, and higher resolution. When plastic is used, shrinkage is not as severe and embedding damage is reduced. The authors have used Sorvall and Polyscience plastic embedding kits and have found that both work successfully. The plastic sections can also be stained with the MGP and esterase reactions with little or no modification in the staining techniques used on paraffin (Appendix). Common Errors and Their Causes Common errors introduced in the processing of lymph node specimens occur in each of the steps previously described in this article. A summary of these common errors and their causes is listed in the Table. Fig 17. Lymph node section that is several cells thick. Large cell (center) is suggestive of the Reed-Sternberg cell of Hodgkin's disease, but the cytologic detail is obscured. Fig 18. Lymph node section that is one cell thin. Diagnostic binucleated Reed-Sternberg cell and a similar neoplastic mononuclear cell are evident within the monolayer of small lymphocytes. LABORATORY MEDICINE VOL. 16, NO. 8, AUGUST

7 Common Errors and Their Results Step Error Result Fixation Delay in fixation; tissue open to air Block too thick or encapsulated Periphery air-dried; central autolysis. Periphery well-fixed and stained; center shows cellular ballooning, distortion, and pale staining Dehydration Clearing Infiltration Embedding Overfixation (eg, blocks left too long in Zenker's or B-5 fixative) Acid formalin Failure to process tissue from mercurycontaining fixatives through iodine and sodium thiosulfate (hypo). Acetone used as dehydrant Aqueous contamination of absolute alcohol (or other dehydrant) leading to incomplete dehydration Excessively prolonged clearing time in xylene. Chloroform used as clearing agent on tissue processor Contamination of xylene by alcohol leading to poor infiltration with paraffin Paraffin too hot Delay in embedding after removal from paraffin bath. Tissue brittle and shatters. Sections thick and thin. With Zenker's, affinity for nuclear stains may be diminished or lost. Poor hematoxylin affinity for nuclei gives inadequate differential staining, with a predominantly eosinophilic stain. Formalin pigment (acid formaldehyde hematin). Randomly distributed brown to black crystalline deposits (mercuric chloride crystals, "Zenker'' crystals). Tissue shrunken, dry. and hard; shatters during sectioning and sections may be thick and thin. Sections crumbled, torn, or "exploded." May exhibit "dry earth effect" (ie, small irregular cracks throughout sections). Stained sections appear muddy. Tissue hard and brittle. Sections shatter and may be thick and thin. Lymphoid tissues brittle. Sections shatter and may be thick and thin. Sections compressed, and wrinkled; will not ribbon. Tissue shrunken and hard. Shatters during sectioning and sections may be thick and thin. Stain "muddy" and homogeneous, with poor nuclear-cytoplasmic differentiation. Air spaces around tissue in paraffin block make sectioning difficult. better than ours," and then check techniques to see where the practice is becoming lax: a sort of a "practice what you preach reaction. With other slides, the first reaction is selfish. The authors are thankful that they are not the patient whose diagnosis and treatment depends on such preparations. This discussion of technical factors, effects, and remedies will help to produce technically excellent lymph node slides in any laboratory. In fact, attention to the details covered will improve the quality of all histologic slides. Once again, technical excellence cannot be achieved alone. In a histopathology laboratory it is essential that the pathologist and the technologist work as a team. Each plays an integral part in ensuring technical excellence. The best histotechnologist cannot produce a good slide from thickly blocked or inadequately fixed tissue. The most skilled and specialized pathologist is severely handicapped by thick sections from partially dehydrated or inadequately infiltrated blocks. Working together, however, technical excellence can be achieved and diagnostic problems can be reduced to a minimum. Rapid Method to Spot Technical Problems Technical trouble can be discovered in just two days by systematically eliminating each major source of difficulty, using the following technique: 1. From a fresh node that has not been allowed to dry, cut a section at the same thickness as usual. Label this section "block 1" and fix and process exactly as usual. 2. Cut a 3-mm thin section from the node. Label this section "block 2." Fix and process exactly as block Cut another 3-mm section, label it "block 3," and fix it in 10% buffered formalin for 24 hours. Place it on the tissue processor with the regular tissues. Skip the fixative beaker if the processor contains fixative. 4. Cut another 3-mm section, label it "block 4," and fix it for 24 hours in 10% buffered formalin (as with block 3). Process this block by hand through unused dehydrant, clearing agent and paraffin, or on the tissue processor after changing all of the solutions and the paraffin. Compare the sections from all four blocks. The section from block 1 will be a positive control: it should show all routine artifacts. In block 2, the problem of blocking too thickly is eliminated. If this is one of the difficulties, the slide from block 2 will be better than the slide from block 1. If it is the same, examine the slide from block 3. In block 3, both blocking and fixation problems are eliminated. If the section is not excellent, contamination is present in the tissue processor or the timing is inadequate. Slides from block 4, if properly sectioned, should be excellent, without artifacts from blocking, fixation, or processing. Summary Working in hospitals in which slides and blocks are received for consultation or referral of patients from all over the country, a little bit of everything is seen, from the ridiculous to the sublime. The authors look at some slides and think, "Gee, theirs look References 1. Bowling MC: Lymph node specimens: achieving technical excellence. Lab Med 1979;10: Berard CW, Bowling MC: Overcoming Technical Errors in the Histologic Preparation of Lymph Nodes. Chicago, American Society of Clinical Pathologists, Enterline HT: Pathologist and histotechnologist: marriage in need of counseling, in Sommers SC led): Pathology Annual. New York, Appleton-Century-Crofts, 1975, pp Bowling MC: Histopathology Laboratory Procedures of the Pathologic Anatomy Branch of the National Cancer Institute. Washington, DC, U.S. Government Printing Office, Sheehan DC, Hrapchak BB: Theory and Practice of Histotechnology. St. Louis, CV Mosby Company, Hammond GF, Beckman B: Piccolyte investments of better sectioned cutting. Stain Technol 1978;53: Lillie RD: Histopathologic Technic and Practical Histochemistry, ed 3. New York, McGraw-Hill, Wasson MT: Histotechnique for mercuric chloride disposal. Microtome Express 1978;1: p Jordan BM, Baker JR: A simple pyronine/methyl green technique. Quart J Micros Science 1955;96: Leder LD: The selective enzymocytochemical demonstration of neutrophil myeloid cells and tissue mast cells in paraffin sections. Klin Wochenschr 1964;42: Suppliers a. C.H.E.M., Maryland Heights, MO b. American Scientific Products, McGaw Park, IL c. Roboz Surgical Instrument Co, Washington, DC d. Sigma Chemical Co, St. Louis, MO LABORATORY MEDICINE VOL. 16, NO. 8, AUGUST 1985

8 APPENDIX B-5 Fixative Formula: Distilled water (hot) 90 ml Mercuric chloride 6.0 g Sodium acetate 1.25 g Add 10 ml concentrated formaldehyde immediately before use. For a reference in the literature, see the article by Lillie. 7 Notes: Always wear gloves when dealing with mercuric fixative. If overheated, a black precipitate will form on the bottom of the beaker and/or the solution will turn brown. If this occurs, start over. In view of the present and continuing problem of environmental pollution, OSHA regulations stipulate that mercury cannot be discarded into drains or wastebaskets, but instead requires special handling. 8 For every 100 ml of fixative to be discarded, add 10.0 ml of ammonium hydroxide. A reaction occurs immediately: HgCI 2 + 2NH 3^ HgNH 2 + NH 4 + Cl" The metallic-colored liquid is then filtered through coarse filter paper. The supernate is discarded and the filtrate is saved. This is allowed to drain for approximately 12 hours, so that most of the liquid is gone. The filter paper containing the filtrate is then wrapped in plastic and sealed with masking tape, collected, and given to a commercial company for disposal. Jordan & Baker's Methyl Green Pyronine (modified) This stain is recommended for mercuric-fixed tissues. 9 Paraffin sections are cut at 3 to 5 p.m and plastic sections are cut at 2 to 2.5 jim. (For plastic sections, start with step 2 below.) Tonsil or a Hodgkin's diseased lymph node is recommended as a control tissue. Method: 1. Section hydrated to distilled water. 2. Methyl-green pyronine stain for 15 to 30 minutes. 3. Rinse quickly in distilled ice water. 4. Blot to almost dry. 5. Rinse slide rapidly with two changes of acetone. 6. Rinse quickly in acetone-xylene mixture. 7. Clear in fresh xylene and mount. Solutions Solution A: 0.5% pyronine Y c (CI 45005), aqueous 37 ml 0.5% methyl green purified 0 (CI 42585), aqueous 13mL Acetate buffer, ph ml Solution B: 0.1 N acetic acid 80 ml 0.1 N sodium acetate 120 ml Results: DNA green RNA rosy red Leder-Esterase For Paraffin Sections For a reference, see the article by Leder. 10 Solution A: 1.6 ml concentrated HCI 8.4 ml distilled water 0.4 g p-rosaniline, acridinfrei = 4% p-rosaniline in 2N HCI Solution B: 0.4 g sodium nitrite 10.0 ml distilled water = 4% sodium nitrite Solution C: 35 ml Sorensen M/10 phosphate buffer, ph 6.5 (Store A and B stock solutions in refrigerator.) A. Sodium phosphate diabasic g per 1 L distilled water B. Potassium phosphate monobasic 13.1 g per 1 L distilled water A = 17.5 ml; B = 32.5 ml, ph 6.5. Mix together and use only 35 ml of the mixture. Procedure Cut paraffin sections at 3 to 5 ^.m; plastic sections at 2 to 2.5 ixm. (For plastic sections, start with step 5 below.) The recommended control is bone marrow smears or skin. 1. Deparaffinize and hydrate sections. Do not remove mercury with iodine. For smears, fix for two minutes in absolute methanol containing 10% formaldehyde by volume, and wash. 2. Mix one drop of Solution A and one drop of Solution B in a small beaker and let stand for one minute. 3. Add Solution C and mix well. 4. Dissolve 0.01 g napthol AS-D chloroacetate d in 1 ml of N,Ndimethylformamide. 5. Add the solution from step 3 to this last beaker, mix well, and filter. Immediately place slides in this solution at room temperature for 30 minutes. 6. If the reaction is not complete after 30 minutes, refilter the solution and replace slides for an additional 15 to 30 minutes. 7. Wash slides in running water for five minutes. 8. Counterstain for three to five minutes in Mayer's hematoxylin, rinse in water, and blue in diluted ammonia water. 9. Wash in running water for five minutes. 10. Air dry, clear in xylene, and mount in Permount. Results Neutrophilic granules Mast cell granules Nuclei scarlet scarlet blue This is the final article in the current Anatomic Pathology CE Update series. An examination for CME credit appears on page 498. LABORATORY MEDICINE VOL. 16, NO. 8, AUGUST