Histopathology 1977, I, 319-330

Methyl-methacrylate as an embedding medium in histopathology J . T E VELDE, " R , B U R K H A R D T , * * K A R I N KLEIVERDA," LINEKE LEENHEERS-BINNENDIJK" & WILTRUD SOMMERFELD"" *Pathology Department, University Medical Centre, Leiden, The Netherlands ""Department for Bone and Bone Marrow Investigation, University Hospital, Ziemssenstrasse, and Department f o r Haematomorphology, Institute of Haematology, Ges.f Strahlenund Umweltforschung mBH, associated with EURATOM, Munich, West Germany Accepted for publication

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VELDEJ., BURKHARDT R., KLEIVERDA K., LEENHEERS-BINNENDIJK L. & SOMMERFELD W. (1977) Histopathology I, 3 19-330

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Methyl-methacrylate as an embedding medium in histopathology Methyl-methacrylate embedding makes it possible to obtain semi-thin sections rich in detail and without tissue shrinkage. The procedure requires considerable time and labour, and great care must be taken to prevent heat damage to the tissue during the exothermic polymerization process. The original method given by Burkhardt (1966) and later modifications are described and discussed, with special attention to the practical problems encountered and their solutions. Keywords: methyl-methacrylate embedding, plastic histological techniques

Introduction

Plastics are used as embedding media because of advantages they offer over softer media. Tissue shrinkage is minimal, about 2% as against 10-50% for Paraplast? (Figures I & 2 ) . The resulting semi-thin sections of high histological quality are employed for diagnostic purposes and histopathology research, but for routine diagnostic services the technique has some disadvantages. It is time and labour consuming, and requires the utmost technical care and a thorough knowledge of the difficulties involved. Once improperly embedded in plastic, the specimen usually cannot be retrieved without irreparable damage, whereas material in Paraplast can be

* Address for correspondence: J. te Velde, Pathologisch Anatomisch Laboratorium, University Medical Centre, PO Box 9603, (Wassenaarseweg 62), 2300 RC Leiden, The Netherlands. -1 Paraplast-plus (Paraffin wax-polymer mixture), Sherwood Medical Industries, USA.

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Figure I. Lack of shrinkage. Section through a femoral head embedded in methyl-methacrylate ; 2 pm thick section, Gomori’s reticulin. Block and section photographed at the same magnification.

Figure 2. Differences in section surface. Trephine biopsy specimens of the same length, cut longitudinally and photographed at the same magnification. Lefr: methacrylate, 2 pm thick section, Gomori’s reticulin, mounted and stretched. Middle: same block, approximately 7 pm thick section (Jung-K sledge microtome), stained according to Goldner by floating, stretched by pulling, and then glued to the slide under pressure. Right: routine Paraplast embedding after decalcification in formic acid, 7 pm, H & E.

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re-embedded. Moreover, the risk of tissue damage during plastic embedding is hardly negligible. Polymerization is an exothermic reaction. If not controlled, temperatures reached in the specimen may exceed IOO'C, which causes denaturation of the tissue components. These will then fail to take up stains, and the sections will show pale and swollen, exploded cells (Yaeger 1958, Figure 9). In I 966, Burkhardt tested various methacrylate monomers in different mixtures and after different types of fixation. Methyl-methacrylate gave the best histological detail, and the chosen procedure gave the least chance of explosion artefacts. Since then, the procedure has been further modified and adopted by various laboratories. This article describes the procedure and its modifications since 1966, with special attention to the main problems encountered in its application.

Materials and methods FIXATION AND DEHYDRATION

For the preparation of each biopsy specimen, mix before use: 12.5 cm3 filtered formaldehyde 37% with CaCO,; 24 cm3 methanol absolute; I cm3 of a solution composed of 5 g glucose jn IOO cm3 Sorensen phosphate buffer (pH 7.4). This mixture may be stored but the pH must be checked after storage as it may become more acid. The glucose-buffer mixture can be safely stored in a refrigerator for one week. Tissue no thicker than 5 mm, is fixed for 16-24 h. Thicker specimens require longer fixation, and the fixative must be refreshed. Dehydrate in absolute methanol for at least 12 h and refresh the methanol several times, especially during the first few hours. Specimens in methanol can be stored for several weeks or sent by mail if the jars are completely filled and tightly sealed. Cork stoppers should be avoided. INFILTRATION

The mixture used for infiltration consists of pure, destabilized methyl-methacrylate monomer, with nonylphenol-polyglycoletheracetate(Plastoid N, Rohm Pharma) as softener, and benzoylperoxide as initiater. Destabilize the monomer by passing it over two or three columns (diameter 2 cm, height 25 cm) of alkaline A1203.Refresh the Al,03 after passage of 800 cm3monomer, and keep the last column free of colour changes. The monomer should be stored in a refrigerator, but not for long periods or dubious results may be obtained. Although the monomer is delivered as pure, there can be considerable differences in polymerization rate between batches. Plastoid N should be used within 6 months of manufacture and within 6 weeks after opening the bottle. Store in a cool place. Benzoylperoxide must be dried thoroughly before use, but since it is highly explosive in this state, it is preferable to dry it in small quantities, on paper, in an

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uncovered petri dish in a dessicator or in an oven at 37°C. Avoid storage of dried material in closed bottles. For each specimen measuring not more than 2 x 2 x 0.8 cm, mix 20 cm3 monomer with 5 cm3 Plastoid N and 700 mg benzoylperoxide. This mixture can be stored for one day in the dark at room temperature which prevents prepolymerization by light or heat. Use only clean and completely dry glassware. Remove the specimen from the methanol and bring into 3 cm3 of the mixture in a glass jar (diameter 2.5 cm) and allow infiltration to occur under vacuum conditions for 30 min before transfer to another completely clean and dry jar containing 3 cm3 of fresh mixture. Then replace under the vacuum. Repeat this procedure four times, infiltrating for +,$, I, and I f h, decreasing the vacuum each time, commencing with 5-8 kPa and ending at 10.5-13.0 kPa. Small gas bubbles may escape, but prevent overt boiling, which leads to selective evaporation of the monomer and an increase in the relative contents of the softener and the oxidant. After the Iast infiItration, transfer the specimen into a glass jar containing 10 cm3 of the mixture and upon which a plastic lid can be tightly screwed. Apply a vacuum of 13.5 kPa for about 30 min, then release the vacuum and leave the open jars in the vacuum container overnight. On the following morning reapply a vacuum of 13.5 kPa for 2-3 h, subsequently closing the jars very tightly. Avoid any unnecessary stirring, and initiate the polymerization at once. POLYMERIZATION

Initiate polymerization in a waterbath in an oven at 47°C for no longer than 45 min. This step should be omitted for specimens larger than 2 cm, and must be modified

Figure 3. Bone marrow, methacrylate,

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,urn, Gallamine-Giemsa. x 900.

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Figure 4. Lymphnode (left) and bone marrow (right) in a patient with Sezary’s syndrome. Arrow: a cerebriform SBzary cell in the bone marrow. Methacrylate, 2 ,urn, PAS. x 500 and x 600.

Figure 5- Bone marrow, methacrylate.

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Gomori’s reticulin. x 400.

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for hot climatic conditions, where polymerization starts earlier. After polymerization has been initiated, bring the jars into a water bath in an oven set at 34-37°C (in this temperature range the polymerization can proceed slowly). Do not stir or open the jars unnecessarily. The blocks will harden within 24-38 h, although the top layer may remain sticky for longer. Finally, break the glass with a hammer after submerging the jar in a bucket of water (to avoid flying splinters). SECTIONING, STRETCHING AND MOUNTING

Blocks with adiameter up to 6 cm arecut at 0.1-3opm on a Zeiss heavy-duty microtome. Routinely, a thickness of 2 or 3 pm is used for the study of cellular detail and 0.9 and 7.5 pm for the structure of basement membranes. During sectioning, wet the block with 30% ethanol, which will soften the cutting surface. Lift the section from the knife by gently pulling with a pair of foreceps and transfer into distilled water. Sections to be stained for calcium or osteoid are mounted immediately, but for other bone staining procedures it is advisable to decalcify sections by floating them in 3% acetic acid for not more than 3 min. Microscope slides are pretreated by thorough cleaning in 4% NaOH, rinsing, neutralizing in 2% HCI, rinsing again, and drying. Before use, they are dipped in a warmed solution of 2.5 g gelatin powder with 19.5 cm3 4% chromalum in 500 cm3 distilled water, and dried. Even spreading of the gelatin coating can be obtained by placing the glasses in a rack mounted on an old gramophone turntable and spinning until dry. Transfer the sections onto the slides and drip some 97% ethanol on the surface after which the section will soften for a few minutes during which it can be stretched

Figure 6. Spleen, methacrylate, methenamine-silver Hi& E; left: 7 ,urn, x 200; right:

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by gentle pulling. With a thick, very soft brush stretch it further and brush till dry Then cover the section with a PVC coverslip and allow to dry further in an oven at 46°C for 1-2 h under some pressure, eg. by weighting a pile of 15 or so sections with lead. After this treatment the section will adhere to the glass without folds and will not float during staining. The methacrylate can be dissolved from the sections by immersion in two changes of benzene for 30 min each. Wash in 10o0/, ethanol and bring to water via 70% ethanol with ammonia. STAINING

All routine histopathology staining methods can be used, but some require slight modification of the staining times depending on the thinness of the sections. Our standard set of staining methods includes the following: Giemsa after gallamine etching; PAS with and without diastase; Gomori’s reticulin (Figures 3 , 4 & 5 ) ; iron-staining by Perl’s or Turnbull’s methods ; Trevan’s methylgreen pyronin can be

Figure 7. Polymerized blocks with maximal thermometers inserted. Lefi: routine procedure; maximal temperature reached 44°C. Right: procedure with omission of waterbath during polymerization; maximal temperature reached 92°C. Note the gasbubbles trapped in the overheated block.

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used as a counterstain instead of nuclear fast red. For connective tissue and osteoid, trichrome staining according to Ladewich (1938), Masson, and Goldner; and Romeis (1968), elastic van Gieson. For basement membranes, methenamine silver HE (Figure 6). Alcian blue in varying MgClz solutions according to Scott & Dorling (1965) gives results comparable to those obtained in Paraplast. For qualitative and quantitative determinations we use Feulgen’s DNA stain or naphthol yellow S, alkaline fast green or, alternatively gallocyanin and chromalum. Satisfactory results for enzyme histochemistry are achieved for alkaline phosphatase and naphthol-ASD-chloroacetate-esterase, but other enzymes have not been detected. To the present, reproducible results have not been achieved for the demonstration of the different immunoglobulins in plasma cells. Gomori’s stain for reticulin requires special precautions. The silver solution should be titrated twice, the washes doubled or tripled, and water and formalin solutions refreshed very often. Impregnation must be restricted to a few seconds and repeated when necessary under microscopical control.

Discussion Since Burkhardt’s original description in 1966, this embedding procedure has been modified in details, but the main objective has always been the production of reliable semi-thin sections of high histological quality, avoiding not only tissue shrinkage and decalcification but also heat damage. Therefore fixation can be kept to a minimum, the fixative proposed here being chosen for its gentleness. Stronger fixatives may give greater contrast, but at the cost of cytoplasmic detail. It should be remembered that methanol can displace, or later abolish, PAS-positivity due to glycogen. no waterbath during- polymerization -____

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-Hours Figure 8. Peak temperatures measured in polymerizing blocks, prepared according to the routine pracedure or with omission of one detail in the procedure.

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Polymerization of the monomer to long interconnecting chains within the tissue is a process which requires heat for its initiation, but later produces warmth which, in turn, gives rise to new bonds which generate heat. This process, if uncontrolled, Every effort should be made can very quickly generate temperatures in excess of 100°C. to infiltrate the tissue with a mixture than can polymerize slowly and evenly at the same speed throughout the block. This means that it is imperative to avoid all potentially interfering substances, such as water, alcohol, or the stabilizing agent in commercial monomers, If these precautions are taken, the tissuecomponents interfering with polymerization, eg. fat need not be removed by pretreatments with acetone or other substances, and pre-polymerization (Conkie 1965) can be ommitted. Plastoid N not only produces softer and less brittle blocks, but also has a favourable effect on the polymerization itself (Figure 8). Once started, polymerization progresses adequately at temperatures between 34 and 37°C. Excess heat can be efficiently removed by placing the jar in a waterbath (Figure 7 & 8). The start-up must be short, and this stage can safely be omitted in hot weather. To demonstrate the importance of each detail in the procedure, we measured the temperatures attained in the polymerized blocks by omitting individual details, eg. we continued polymerization at the start-up temperature of 47"C, omitted the use of vacuum to remove residual methanol from the tissue, etc. Temperatures were measured with a maximal thermometer inserted into the block (Figure 7). The curve (Figure S), clearly demonstrates the very rapid rise of temperature. Once the peak temperature is reached the block is hard. Since evaporated monomer gas bubbles may be trapped within the hardening plastic (Figure 7), the presence of even a single

Figure 9. Explosion artefact: erythroid cells without (right) and with (left) heat damage during polymerization, causing swelling and loss of stainability. Gallamine-Giernsa, x 600.

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Figure 10.Schematic drawing of the excursion of the knife in conventional, unadapted microtomes. Figure 11. Schematic drawing of knife and block holder in the Zeiss heavy duty microtome.

gas bubble in the block must be taken as a sign that temperatures have been above the boiling temperature of the monomer and tissue damage from overheating can be expected. Some cells, eg. erythroid precursors, are particularly susceptible to explosion artefact (Figure 9); other tissue components, eg. bone, are much less affected. Heat damage can be masked by previous overfixation, as by formaldehyde; the nuclei appearing more resistant, but having already lost the fine detail of chromatin distribution during fixation. A further difficulty is encountered in sectioning the hard, but elastic blocks. With an ordinary microtome, due to the bevel angle, the knife tends to fret its way into the block and, as the tension accumulates, tears towards the surface, producing some shattering of the material. Thicker sections will show alternating thick and thin bands (Figure 10). Consequently, the microtome must be adapted in several respects. It should be motor-driven to produce a slow, steady cutting speed and both the knife and the block must be fixed so that bending cannot occur. The construction of the moving parts of the instrument must be so stable that cutting deviations are not possible (Figure 11). The Zeiss heavy duty microtome was specially designed for Burkhardt to meet these requisites. Using this instrument, sections as large as 6 cm diameter can be cut at 0.05-30 pin. Tests have shown that variations between sections and across random sections cut at 0.9 and 2 pm thick, are less than 10% (measurements made with a Leitz interference microscope by Dr J.Josselin de Jong, Erasmus University, Rotterdam). The use of alternative types of microtomes demands not only considerable skill but several modifications of techniques, eg. reduction of the width of the cutting area by trimming the block to a narrow strip of tissue: variation of the bevel angle, cutting speed, or the amount of softner applied may also improve results. The occurrence of thick and thin bands in a section impedes mounting and stretching and likewise leads to uneven staining. Unstretched sections with folds are very difficult to examine at high magnifications. The stretching procedure described here does, however, lead to artefacts on bone tissue which seems partially unavoidable. During cutting, the section curves over the knife (Figure 11) and under this stress, fully mineralized bone breaks even though the fragments are held together by the polymer chains. Some compression also occurs

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Figure 12. Artefacts due to sectioning and mounting. Left: 2 pm thick section, Gomori’s reticulin, mounted. Right: approximately 7 pm thick section (Jung-K sledge microtome), stained by floating according to Goldner, then stretched by pulling, and glued to the slide under pressure. x 300.

which can only partially be avoided by gentle pulling. During stretching and mounting the surface of the sectionis restored to the state occurring in the block (Figure I), but the broken pieces of calcified bone are readily brushed from the section leaving holes within the trabeculae (Figures 2 & 12). Unstretched sections can also show these holes; moreover, the bone and bone marrow are left compressed. The section surface may be reduced as much as 30%. Preliminary investigations have, however, shown that the percentage of bone tissue is the same in both stretched and unstretched sections. Both methods result in artefacts, but in stretched sections bone marrow is available for study at high magnifications without folds, compression or concomitant variations in staining. Sections from well-prepared blocks pose no real problem in routine histopathology staining. Tf pale or unevenly stained sections are obtained the possibilities of heat damage during polymerization or unequal section thickness, should be considered. One should not resort to overfixation to mask this unwanted and avoidable effect, and section thickness should not be blamed until it has been adequately determined, since the setting of the mircotome may not correspond with the actual section thickness. In our laboratories we havenot yet solved the problems of combining high definition histological preparations with reproducible detection of various enzymes or immunoglobulins. This is partially due to the choice of fixative and to some extent to polymerization itself. In these procedures temperature is not an important factor. Monomers with longer side chains, such as butyl-methacrylate, are said to permit enzyme histochemistry, but we have found that they lead to a loss of histological detail and give less reliable results than have been claimed. Therefore, a choice has to be

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made between a loss of cellular detail and the possibility of detecting some enzymes present in large amounts on one hand, or, on the other, a technique giving a high degree of histological detail with which most cells can be differentiated on morphological grounds and which is suitable for routine diagnostic work. The further disadvantage is the amount of time and labour required. Both can be reduced, but the price is heat damage and uneven staining. Again, a choice has to be made between a quick diagnosis or a delayed diagnosis based on highly detailed histological and cellular information (Burkhardt 1971).In some tissues the diagnostic advantage of the methacrylate embedding technique can be minimal when the sections are compared with corresponding Paraplast embedded sections of small biopsies on which an equal amount of care and time have been spent. In calcified, or large biopsy specimens, or tissues which are easily compressed or shrink readily, especially bone marrow and lymphatic tissues, the histological detail of well-prepared plastic sections can be crucial for accurate diagnosis and provide much additional research information.

References BURKHARDT R. (I 966) Technische Verbesserungen und Anwendungsbereich der Histo-biopsie von Knochenmark und Knochen. Klinisches Wochenschrift 4, 326 BURKHARDT R. (1966) Praparative Voraussetzungen zur Klinischen Histologie des menschlichen Knochenmarks. Blut 14,30 BURKHARDT R. (1971) Color atlas of clinical histopathology of bone marrow and bone. SpringerVerlag Berlin, Heidelberg & New York CONKIE D. (1965) Plastic embedding in routine histology. Acta Anatomica 60, 531-538 LADEWICH P. (1938) Ueber eine einfache und vielseitige Bindegewebsfarbung (Modifikation der Mallory-Heidenhainschen Methode). ZeitschriJt f u r Wissenschaftliche Mikroskopie 5 5 2 15-2 17 ROMEIS B. (1968) Mikroskopische Technik, 16th Edition, R. Oldenburg Verlag, Miinchen-Wien SCOTTJ.E. & DORLINGJ. (1965) Differential staining of acid glycosaminoglycans (mucopolysacharides) by Alcian Blue in salt solutions. Histochemie 5, 221-233 YAEGER J.A. (1958) Methacrylate embedding and sectioning of calcified bone. Stain Technology 33, 229-239