I Clin Pathol 1997;50:45-49

Use of methyl methacrylate resin for embeddingbone marrow trephine biopsy specimens

D Blythe, N M Hand, P Jackson, S L Barrans, R D Bradbury, A S Jack

AbstractAims-To evaluate the use of methylmethacrylate resin as an embedding me-dium for undecalcified bone marrow tre-phine biopsy specimens.Methods-About 2500 undecalcified bonemarrow trephine biopsy specimens wereprocessed, and embedded in methyl meth-acrylate resin. Semithin sections (2-3 gm)were stained by routine tinctorial andimmunocytochemical staining methodswith a wide range of antibodies using astandard streptavidin biotin horseradishperoxidase technique. Different antigenretrieval pretreatments were evaluated.Results-Bone marrow trephine biopsyspecimens are embedded routinely inmethyl methacrylate at the Haematologi-cal Malignancy Diagnostic Service at TheLeeds General Infirmary. Over 50 differ-ent primary antibodies are in current use;for the majority of these, microwave anti-gen retrieval or trypsin digestion, or both,is either essential or greatly enhances theresults.Conclusions-Embedding bone marrowtrephine biopsy specimens in methylmethacrylate resin retains morphologyand permits reliable, high quality immu-nocytochemistry. This is particularly de-sirable for the demonstration ofneoplasticcells in regenerative marrow after chemo-therapy, and in the detection of residualdisease after treatment. The use ofmethylmethacrylate for routine use on bonemarrow trephine biopsy specimens isadvocated.(i Clin Pathol 1997;50:45-49)

Keywords: methyl methacrylate resin; bone marrow tre-phine biopsy; morphology; immunocytochemistry.

The investigation of neoplastic and otherdisorders of the bone marrow requires mor-phological examination of a cellular aspirateand a trephine biopsy specimen in conjunctionwith immunophenotypic and molecular bio-logical techniques. Two- or three-colour flowcytometry is currently the method of choice forthe immunophenotypic characterisation of cellpopulations in bone marrow aspirate samples.However, immunohistological studies on bonemarrow trephine biopsy specimens also have animportant and complementary role. The tre-phine biopsy specimen is particularly impor-tant in cases where the aspirate sample is inad-equate for flow cytometric investigation orwhere a population of tumour cells is poorly

represented in the aspirate. This is mostcommon where marrow infiltration is focal, orwhere fibrosis is associated with the tumour.

Decalcified paraffin wax embedded biopsyspecimens can be studied using immunohisto-chemistry, but this technique has the disadvan-tage that morphology and antigenicity may bepoorly preserved. Resin section histology offersthe prospect of excellent high resolution lightmicroscopy, and both acrylic and epoxy resinshave been advocated as being suitable for thispurpose.' The most popular resin is thatbased on the monomer glycol methacrylate(GMA). Various GMA mixtures have beenused and most of these will give high qualitymorphological preservation, but the results ofimmunohistological studies have been disap-pointing. Although there are reports thatimmunohistochemistry is possible on GMAembedded tissue,58 we agree with others thatthe techniques are complicated, troublesomeand impractical in the routine laboratory.9 Forthis reason, decalcification and paraffin waxembedding continues to be widely used in theimmunohistological investigation of bone mar-row trephine biopsy specimens.""-3

In an attempt to overcome the problemsassociated with the GMA resin, a methodbased on a methyl methacrylate (MMA) resinhas been reported'4 '5 which retains good mor-phological detail and permits the use of stand-ard immunohistological methods. This paperdescribes the application of this method toroutine diagnostic practice, based on theexperience of processing around 2500 bonemarrow trephine biopsy specimens.

MethodsSPECIMEN PROCESSINGSpecimens were fixed for a minimum of 18hours in 10% formalin. Other formalin basedfixatives such as formol saline, neutral bufferedformalin and formol calcium have also beenevaluated and produce similar results. Bonemarrow trephine biopsy specimens were notdecalcified prior to processing. The tissue wasprocessed manually in small glass vials on aroller mixer (automated tissue processors maybe used for the dehydration stages). The tissuewas dehydrated through 50%, 70% and 95%ethanol for a minimum of one hour in each,and 100% ethanol for two hours minimum.

After dehydration, the tissue was incubatedovernight at room temperature in the resinmixture, consisting of a 3:1 solution of methylmethacrylate and dibutyl phthalate, on a rollermixer in a fume cupboard. The tissue was thenembedded in heavy duty polyethylene

HaematologicalMalignancy DiagnosticService, Institute ofPathology, TheGeneral Infirmary atLeeds,Leeds LS1 3EXD BlytheA S JackS L BarransR D Bradbury

HistopathologyDepartmentP Jackson

Division of Pathology,Clinical LaboratorySciences, UniversityHospital, Queen'sMedical Centre,Nottingham NG7 2UHN M Hand

Correspondence to:Dr A S Jack.

Accepted for publication25 October 1996

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Figure 1 Early infiltration by myeloma (haematoxylin and eosin). This specimelow level interstitial infiltrate ofplasma cells. The improved morphology associatecuse ofMMA is clearly evident.

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Figure 2 HIV infection (Ziehl-Neelsen). This HIVpositive patient developedpancytopenia. The bone marrow biopsy specimen showed a granulomatous infiltrcontaining very large numbers of mycobacteria.

moulding cup trays with a block stub, and anidentification label attached. The embeddingformulation consisted of a fresh 3:1 methylmethacrylate/dibutyl phthalate mixture with5% pre-dried benzoyl peroxide and 1% N,Ndimethylalinine. Benzoyl peroxide is poten-tially explosive in the dry state and is supplieddampened; aliquots are dried overnight at37°C. MMA acts as the monomer, dibutylphthalate as the plastisicing/softening agent,benzoyl peroxide as the catalyst, and N,Ndimethylaniline as the accelerator. The embed-ding mixture was allowed to polymerise for aminimum of two hours at room temperature ina desiccator until the resin solidified. Becauseoxygen inhibits polymerisation of acrylic res-ins, including MMA, some laboratories preferto polymerise in an anaerobic environment,either by using a vacuum or by displacing the

en shows air with oxygen-free nitrogen. We have foundi with the that these methods are not necessary, as the

resin stubs seal the resin mixture into themould sufficiently to prevent oxygen contami-nation. All infiltration and embedding proce-dures should be done in a fume cupboard.

Following polymerisation, the sections werecut at 2 ptm using glass Ralph knives on aReichert-Jung 1140 Autocut. Sections werefloated out on a 56°C distilled water bath,picked up on clean slides, drained, and placedon a hot plate for a minimum of 10 minutes.Sections for reticulin demonstration were cutat 4 ,um, and those for immunocytochemistry at3 jim. These were picked up on slides coatedwith 2% aminopropyltriethoxysilane in acetone(APES), and either placed on a hot plate for 20minutes, or dried overnight at 60°C.

TINCTORIAL STAININGPrior to staining, MMA resin was removedwith xylene at 37°C for 20 minutes. Sectionswere then rinsed in alcohol and washed inwater prior to conventional histological stain-ing. Routinely, the following tinctorial stains

,ate are applied to all bone marrow trephine biopsyspecimens: haematoxylin and eosin (fig 1);May-Grunwald Giemsa (MGG); Gordon andSweet's reticulin ammoniacal silver stain; andPerls Prussian blue. A wide range of tinctorialstaining techniques may be applied to MMAsections (fig 2).

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IMMUNOHISTOLOGICAL METHODSThe MMA resin was removed from thesections as described earlier. Endogenous per-oxidase activity was blocked using 0.5%hydrogen peroxide in methanol for 30 minutes.The optimal antigen retrieval pretreatmentprocedures will depend upon the antibodiesunder investigation, and are shown in tables1-4.

Figure 3 Post-treatment multiple myeloma (VS38c). This specimen was taken as part ofroutine follow up. The use of VS38c, which in the haemopoietic system is a specific markerofplasma cells, shows an interstitial infiltrate ofplasma cells corresponding with a low levelof residual disease.

Microwave antigen retrievalSections were placed in 400 ml 0.01 M citratebuffer (pH 6.0) (using plastic staining racksand a microwave resistant plastic container).The sections were then microwaved (PanasonicNN6402) at high power (800 W) for eightminutes, allowed to cool for five minutes, andirradiated again for a further three minutes.

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Use of methyl methacrylate resin for embedding bone marrow trephine biopsy specimens

Table 1 Optimal antigen demonstration after microwave antigen retrieval

Antibodylclone Species Source Dilution

IgA Polyclonal Dako 1 in 800IgG Polyclonal Dako 1 in 400Kappa Polyclonal Dako 1 in 400Lambda Polyclonal Dako 1 in 300CD15 (Leu MI) Monoclonal Becton Dickinson 1 in 10CD20 (L26) Monoclonal Dako 1 in 100CD34 (QBend 10) Monoclonal Oxoid 1 in 100CD43 (L20) Monoclonal Dako 1 in 50CD45 (PD7/26+2B1 1) Monoclonal Dako 1 in 20CD45RO (UCHL1) Monoclonal Dako 1 in 100MB2 Monoclonal Europath 1 in 20CD68 (PG-Mi) Monoclonal Dako 1 in 100CD68 (KP1) Monoclonal Dako 1 in 100BLA.36 (A27-42) Monoclonal Dako 1 in 50DBA.44 Monoclonal Dako 1 in 10Bcl-2 (124) Monoclonal Dako 1 in 50BNH-9 Monoclonal Dako 1 in 25EMA (E29) Monoclonal Dako 1 in 200HLA-DR (TAL. 1B5) Monoclonal Dako 1 in 500LMP-1 (CS 1-4) Monoclonal Dako 1 in 10Glycophorin C (Ret 40f) Monoclonal Dako 1 in 100OPD4 Monoclonal Biogenex 1 in 50PGP 9.5 Polyclonal Ultraclone 1 in 1000P53 (pAB 1801) Monoclonal HMDS 1 in 10S100 Polyclonal Dako 1 in 200Tdt Polyclonal Harland Seralab 1 in 20Vimentin (V9) Monoclonal Dako 1 in 50Desmin (DE-R-1 1) Monoclonal Europath 1 in 10Plasma cell (VS38c) Monoclonal Dako 1 in 100J-Chain Polyclonal Biogenex Neat

Source: Becton Dickinson UK Ltd, Between Towns Road, Cowley OX4 3LY, UK; Biogenex, dis-tributed by Biomen Ltd, Pentos House, Falcon Business Park, Ivanhoe Road, Finchampstead,Berks RGl 1 4QQ; Dako Ltd, 16 Manor Courtyard, Hughenden Avenue, High Wycombe, BucksHP13 5RE; Europath Ltd, Highland Comfort, Union Hill, Stratton, Bude, Cornwall EX23 9BL;Harland Seralab Ltd, Hophurst Lane, Crawley Down, Sussex RH10 4FF; HMDS Laboratory,Algernon Firth Building, Institute of Pathology, The General Infirmary at Leeds, Leeds LS1 3EX;Immunotech, distributed by Coulter Electronics Ltd, Northwell Drive, Luton, Beds LU3 3RH;Novocastra Laboratories Ltd, 24 Claremont Place, Newcastle upon Tyne NE2 4AA; UltracloneLtd, Rossiters Farmhouse, Wellows, Isle of Wight P041 OTE.

Table 2 Optimal antigen demonstration using trypsinisation followed by microwaveantigen retrieval

Antibodylclone Species Source Dilution

IgD Polyclonal Dako 1 in 50IgM Polyclonal Dako 1 in 50CDla (010) Monoclonal Immunotech NeatCD3 Polyclonal Dako 1 in 100CD8 (C8/144B) Monoclonal Dako 1 in 50CD21 (IF8) Monoclonal Dako 1 in 20CD23 (1B12) Monoclonal Novocastra 1 in 10CD30 (Ber H2) Monoclonal Dako 1 in 15CD45RA (MT2) Monoclonal Biogenex 1 in 10CDw75 (LN-1) Monoclonal Novocastra 1 in 20CD79a (JCB1 17) Monoclonal Dako 1 in 25Ki-67 (MIB-1) Monoclonal Immunotech 1 in 50

Table 3 Optimal antigen demonstration after trypsin or microwave antigen retrieval

Antibodylclone Species Source Dilution

CAM 5.2 Monoclonal Becton Dickinson 1 in 2Factor VIII Polyclonal Dako 1 in 500Poly Keratin Polyclonal Dako 1 in 200

Table 4 Optimal antigen demonstration with no antigen retrieval

Antibodylclone Species Source Dilution

CD57 (HNK-1) Monoclonal HMDS 1 in 20Myeloperoxidase Polyclonal Dako 1 in 4000

After leaving to cool for 20 minutes, thesections were washed in cold water.

Trypsinisation antigen retrievalSections were incubated in 0.025% trypsin(100 mg trypsin, 400 ml distilled water, 8 ml5% calcium chloride, pH to 7.8 with 1%sodium hydroxide) at 37°C for six minutes, and

then washed in cold tap water to preventfurther digestion.

Trypsinisation and microwave antigen retrievalTrypsinisation was done prior to microwaveirradiation, using the methods described ear-lier.

Following antigen retrieval, sections wererinsed in 0.005 M Tris buffered saline (TBS)(pH 7.6), and incubated in 20% normal serumfor 10 minutes. Excess serum was drained fromthe sections, and primary antibody applied foran incubation period of one hour, using 0.01%Tween 20 in TBS as the antibody diluent. Sec-tions were then washed in TBS for fiveminutes, and incubated in biotinylatedsecondary antibody (Dako Duet kit K492) for30 minutes. After washing in TBS, sectionswere incubated in streptavidin/biotin/horse-radish peroxidase complex (Dako Duet kitK492) for 30 minutes. Following washing inTBS, sections were developed in 0.02% diami-nobenzidene in Tris buffer (pH 7.6) containing0.15% hydrogen peroxide. Sections were de-veloped for 10 minutes, then washed in water,and intensified in 0.5% copper sulphate in0.9% sodium chloride for five minutes. Afterwashing in water, the sections were counter-stained in haematoxylin. Sections can beimmunostained either manually or by semi-/automatic methods. Throughout the stainingprocedures, it is important to recognise thatetched MMA sections dry out quicker thanparaffin wax sections when not covered withfluid, and, consequently, this must be avoidedto help prevent background staining.

ResultsThe method described here is routinly used atthe Haematological Malignancy DiagnosticService at Leeds General Infirmary. Approxi-mately 2500 bone marrow biopsy specimenshave been processed in this way. In every casehaematoxylin and eosin, Giemsa, reticulin, andPerls stained sections are examined. Sectionsare studied immunohistologically when neces-sary, and about 3000 resin sections areexamined using immunocytochemistry eachyear. None of the biopsy specimens embeddedin MMA in our department have been difficultto interpret because of technical failure.

Forty six antibodies are currently in routineuse. With the exception of myeloperoxidaseand CD57, microwave antigen retrieval ortrypsin digestion, or both, are either essential orgreatly improve staining intensity andreproducibility."6 The optimal antigen retrievalpretreatment methods and antibody dilutionsare shown in tables 1-4.

Figures 3-6 show representative MMAsections stained immunocytochemically with anumber of routinely used antibodies.

DiscussionBased on our experience described here, weconclude that MMA embedding is the methodof choice for processing bone marrow trephinebiopsy specimens. The technique has severaladvantages over other previously reported resinembedding methods..The most important of

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Figure 4 Myelodysplastic syndrome (CD34). This patient presented with pancyto)Examination of the bone marrow showed abnormal architecture and trilineage dyspThe number of blast cells was shown using anti-CD34.

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Figure 5 Metastatic carcinoma (cytokeratin). This patient presented withleuco-lerythroblastic changes in the peripheral blood. The bone marrow was replaceddense fibrosis. Using an antibody directed against cytokeratin (Cam 5.2), a numbersmall clusters of metastatic carcinoma were identified.

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Figure 6 Post-transplantation lymphoproliferative disorder (LMP-1). This patientpresented with pancytopenia several years after a kidney transplant. The marrow shnodular infiltrate which included large atypical cells, some of which resembledReed-Sternberg cells. These cells expressed the Epstein-Barr virus protein LMP-1.

these is that the resin is soluble in itspolymerised state in xylene, and unlike otherresins MMA does not undergosuperpolymerisation.'7 Therefore, sections canbe cut and immunostained long after the biopsyspecimen was originally embedded. After re-moval of the resin, the section can be hydratedand handled as a paraffin wax section using

;.t., * optimal antigen retrieval pretreatments asrequired. Almost all of the antibodies that canbe used on paraffin wax sections also work wellon MMA sections (CD40 being an exception).However, pretreatment schedules may need tobe prolonged for MMA sections. Our process-ing method is done entirely at room tempera-ture after conventional histological fixation in10% formalin. The standard of morphologicalpreservation is consistently better than that ondecalcified paraffin wax sections (fig 1).Variables which may be encountered with par-

penia. affin wax sections, such as the length of time inlasia. decalcification fluid and the microtomy of thin

sections, are not a problem with MMAsections. A further important advantage of thismethod is that routinely stained sections areavailable for examination within 48 hours ofthe specimen being taken. This comparesfavourably with the use of decalcificationfollowed by paraffin wax processing andembedding.The diagnostic application in which this

technique has been of most value is the identi-fication of small populations of neoplastic cells,particularly in regenerative marrow followingchemotherapy. Suspicious cells are more read-ily identified in semi-thin MMA sections, andthese can then be identified positively usingappropriate markers. This can be of value indetecting residual disease after treatment ofmyeloma, acute lymphoblastic leukaemia, or

7 hairy cell leukaemia. Low levels of marrow}- 'infiltration by follicle centre lymphoma can

also be detected. In all of these applications, itis essential to correlate closely the cytological

i by appearance of the cell with the antibodyof reactivity. This is much easier to do on MMA

sections than on decalcified paraffin waxsections.Another important area of application is in

the assessment of the marrow in cases ofsuspected myelodysplasia and chronic myelo-proliferative disorders. The combination ofgood morphological preservation and smallpanels of antibodies (for example, myeloper-oxidase, glycophorin C, Factor VIII RAG, andCD34) permits accurate and detailed assess-ment ofbone marrow microarchitecture, whichis one of the key features in the diagnosis ofmyelodyplastic syndrome.'8 '9 Myeloblasts canbe positively discriminated from early meg-akaryocytes and erythroid precursors, andtheir number and distribution assessed. Theprognostic importance of determining thenumbers of CD34 positive cells in myeloprolif-erative disorders has been reported.20 21 Thepresence of ring sideroblasts can also be readilydetected on Perls-stained, MMA-embedded

t sections.owed a In conclusion, we suggest that the use of

MMA has many advantages over other meth-

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Use of methyl methacrylate resin for embedding bone marrow trephine biopsy specimens

ods of processing bone marrow trephine biopsyspecimens, and we would strongly recommendit for routine use.

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21 Orazi A, Neiman RS, Cualing H, Heerema NA, John K.CD34 immunostaining of bone marrow biopsy specimensis a reliable way to classify the phases of chronic myeloidleukemia. Am J Clin Pathol 1994;1O1 :426-8.

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