Use of paraffin wax embedded bone marrow trephine biopsy specimens as a source of archival DNA.

AIMS: To evaluate the use of DNA extracted from paraffin wax embedded trephine biopsy specimens as a source of archival nucleic acid for Southern hybridisation studies and polymerase chain reaction (PCR) amplification. METHODS: DNA was extracted simultaneously from paraffin wax embedded bone marrow trephine and lymph node biopsy specimens after incubation of tissue sections for one to five days in lysis mix and proteinase K with periodic sampling. DNA from 10 trephine biopsy specimens was subjected to PCR amplification using HLA-DPB primers to determine whether the extracted nucleic acid was of sufficient quality to permit amplification. RESULTS: For most specimens the greatest yield of high molecular weight DNA was seen after five days' incubation. Unlike lymph node material the quality of extracted nucleic acid and the quantity obtained from trephines was insufficient for Southern blot analysis. PCR amplification using HLA-DPB primers yielded positive results in six out of 10 trephine biopsy specimens. CONCLUSIONS: DNA extracted from paraffin wax embedded trephine biopsy specimens is largely degraded and unsuitable for Southern analysis but serves as a useful source of archival nucleic acid for PCR amplification.     

Use of APAAP technique on paraffin wax embedded bone marrow trephines.

The immunoalkaline phosphatase (APAAP) technique was applied to the labelling of decalcified sections of formalin fixed, paraffin wax embedded bone marrow trephine biopsy specimens. A panel of monoclonal antibodies reactive with haemopoietic and epithelial antigens, which survive routine formalin fixation, was assessed on 72 cases of haematological malignancy (including acute and chronic leukaemias and lymphomas) showing bone marrow infiltration. The APAAP method showed clear distinct labelling of antigen positive cells without loss of antigens due to decalcification. Both normal or reactive single cells present in the sample and neoplastic cell populations could be identified morphologically and their antigenic phenotype and cellular origin, whether lymphoid or myeloid, established. The application of the APAAP method to routinely prepared paraffin wax embedded trephines has many advantages over the assessment of specially prepared cryostat sections of bone marrow.     

Plastic embedding of bone marrow trephine biopsies for routine immunohistochemistry and diagnosis: our developments,updates and experiences over 20 years

Bone marrow trephine specimens are routinely examined for the histological investigation and diagnosis of lymphoma and other disorders. To achieve this, biopsies are usually fixed in formalin and embedded in paraffin wax for subsequent tinctorial and, often, immunohistochemical staining. However, in this review the authors report the historical developments of immunohistochemical staining on plastic sections, and, in particular, our own developments and updates during the last 20 years of using a plastic embedding procedure for the routine reporting of over 50,000 bone marrow trephines. Many evolutionary changes during this period have occurred to provide a simple technique for the successful and excellent demonstration of numerous cellular antigens. While the volume of work and experience relating to immunohistochemistry on plastic-embedded tissue is, the authors we believe, unique, the review also present why the current procedure may revert to use of paraffin wax in the future.     

Immunohistochemical analysis of decalcified paraffin-embedded human bone marrow biopsies with emphasis on MHC class I and CD34 expression

 

Aims:

To identify the stromal structures and haematopoietic cell lineages in normal bone marrow. The optimal conditions were studied for the reactivity of a panel of antibodies, applicable to paraffin sections of decalcified trephine biopsies using antigen retrieval methods.  

Methods and results:

Two methods of antigen retrieval (pepsin and acid citrate buffer) were tested. For the demonstration of most antigens and for reduction of background staining, heating in acid citrate buffer was preferred. In the case of elastase and von Willebrand Factor (factor VIIIrAg) pepsin pre-treatment was optimal, whereas Ulex europaeus agglutinin (UEA-1) and α-smooth muscle actin (α-SMA) required no pre-treatment. Staining patterns obtained after 48 h EDTA decalcification and short electrolytic decalcification were identical. Both methods allowed recognition of HLA-A and HLA-B antigens, isolated CD34+ cells, mono-histiocytic cells (CD68+), myeloid cells (elastase and myeloperoxidase), erythroid cells (glycophorin C) and of megakaryocytic cells (Factor VIIIrAg). A relative simple lymphocyte-subset analysis was possible in decalcified paraffin sections allowing recognition of B-cells (CD20+) and T-cells (CD3+ and CD45RO+) in frequencies comparable to frozen sections. Suitable stromal cell staining was achieved by vimentin and desmin antibodies, whereas the bone marrow capillary network was visualized by CD34, factor VIIIrAg and UEA-1.  

Conclusions:

This immunohistochemical study indicates that all cellular components of the haematopoietic microenvironment of the bone marrow can be identified in decalcified paraffin sections using antigen retrieval methods and that the time of decalcification can be reduced to 1–1.5 h.     

Microwave antigen retrieval in immunocytochemistry: a study of 80 antibodies.

AIMS--To evaluate the effect of microwave irradiation on the staining quality of a range of commonly used primary antibodies in archival, formalin fixed, paraffin wax embedded material, with emphasis on antibodies that have previously worked successfully only on frozen tissue. METHODS--Immunocytochemistry (streptavidin-biotin complex technique) was performed on histological sections of a range of normal and pathological tissues, after varying treatment with microwave irradiation. The staining quality of each antibody was compared with that achieved without prior treatment of the sections or after enzyme predigestion. RESULTS--Microwave irradiation permitted successful immunostaining with 20 antibodies that stained only frozen tissues before. The staining characteristics of 21 antibodies that were already known to stain formalin fixed, paraffin wax embedded material were improved. Another 39 antibodies did not show enhanced staining with microwave irradiation. The method preserves tissue morphology and produces more consistent staining than that achieved by enzyme predigestion with many antibodies. Microwave irradiation may also allow some primary antibodies to be used at higher working dilutions. The citrate buffer used in this study avoids the necessity of exposure to heavy metal salts. CONCLUSIONS--Microwave antigen retrieval represents an important technical advance within immunocytochemistry that will greatly increase the range of antibodies which can be used to study formalin fixed, paraffin wax embedded tissues.     

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

AIMS: To evaluate the use of methyl methacrylate resin as an embedding medium for undecalcified bone marrow trephine biopsy specimens. METHODS: About 2500 undecalcified bone marrow trephine biopsy specimens were processed, and embedded in methyl methacrylate resin. Semithin sections (2-3 microns) were stained by routine tinctorial and immunocytochemical staining methods with a wide range of antibodies using a standard streptavidin biotin horseradish peroxidase technique. Different antigen retrieval pretreatments were evaluated. RESULTS: Bone marrow trephine biopsy specimens are embedded routinely in methyl methacrylate at the Haematological Malignancy Diagnostic Service at The Leeds General Infirmary. Over 50 different primary antibodies are in current use; for the majority of these, microwave antigen retrieval or trypsin digestion, or both, is either essential or greatly enhances the results. CONCLUSIONS: Embedding bone marrow trephine biopsy specimens in methyl methacrylate resin retains morphology and permits reliable, high quality immunocytochemistry. This is particularly desirable for the demonstration of neoplastic cells in regenerative marrow after chemotherapy, and in the detection of residual disease after treatment. The use of methyl methacrylate for routine use on bone marrow trephine biopsy specimens is advocated.     

How we process trephine biopsy specimens: epoxy resin embedded bone marrow biopsies

Improved cytomorphology of semithin resin sections over paraffin wax embedded sections may be important in diagnostic haematopathology. However, resin embedding can make immunohistochemical antigen detection or DNA isolation for clonal gene rearrangement assays difficult. This review describes the processing of bone marrow biopsies using buffered formaldehyde based fixation and epoxy resin embedding, with or without EDTA decalcification. Traditional semithin resin sections are completely rehydrated after etching in home made sodium methoxide solution. Resin elimination allows high resolution staining of tissue components with common histological stains. Efficient antigen retrieval and the Envision-HRP system permit the immunohistological detection of many antigens of diagnostic relevance, with retention of high quality cytomorphology. Furthermore, DNA can be extracted for clonality analysis. The technique can be completed within a similar time period to that of paraffin wax processing with only approximately 30% increase in cost. This technique has been used for diagnosis in over 4000 bone marrow biopsies over the past 14 years. By meeting traditional and contemporary demands on the haematopathologist, it offers a powerful alternative to paraffin wax processing for diagnosis and research.     

Glycol methacrylate (GMA) embedding for light microscopy. II. Immunohistochemical analysis of semithin sections of undecalcified marrow cores.

A routine method allows bone marrow biopsy specimens to be embedded in glycol methacrylate (GMA), a water miscible plastic, and to benefit from the advantages of good morphology with immunoperoxidase detection of a wide range of cellular antigens useful in diagnosing and classifying various haematopoietic disorders. Marrow cores were fixed in cold Bouin's solution, rinsed in cold phosphate buffer, dehydrated in cold methanol, infiltrated and embedded in cold GMA, then polymerised at 4 degrees C. Sections were cut at 2 micron thickness with a Tungsten carbide knife in a Jung's high performance microtome (Autocut). Antigenecity was preserved when drying slides at room temperature but pronase digestion was necessary to re-expose the antigens in bone marrow biopsy sections embedded in GMA. Histostik, a new adhesive, was used to coat the glass slides to prevent section loss during enzyme digestion and immunostaining procedures. This method of adapting plastic embedding to undecalcified marrow cores preserves marrow architecture and cellular details and it can serve as a useful adjunct to analyse the bone marrow from patients with myeloproliferative and lymphoproliferative disorders. This technique may also be applicable in non-haematological malignant conditions which affect the marrow.     

Acute lymphoblastic leukaemia: correlation between morphological/immunohistochemical and molecular biological findings in bone marrow biopsy specimens.

BACKGROUND: Although numerous antibodies suitable for use on paraffin wax embedded sections are available for the subtyping of acute leukaemia (acute myelogenous leukaemia (AML) and acute lymphoblastic leukaemia (ALL)) in bone marrow biopsy sections, unequivocal identification of the cell line involved is sometimes impossible. METHODS: Forty eight formalin fixed, paraffin wax embedded bone marrow biopsy specimens that had been decalcified in EDTA were investigated, including 42 thought to exhibit ALL on the basis of bone marrow smears. Five specimens exhibited AML and one biphenotypic leukaemia, as diagnosed immunohistochemically in bone marrow biopsies. Immunostaining was performed with antibodies against relatively specific B and T cell antigens. The blasts were investigated for rearrangements of the immunoglobulin heavy chain (IgH) and the T cell antigen receptor (TCR) genes. RESULTS: Amplifiable DNA was obtained from all 48 specimens. An IgH gene rearrangement was detected in 20 of 23 c-ALL specimens. Four of seven T cell ALL (T-ALL) specimens had a TCR-gamma gene rearrangement, and the one B cell ALL (B-ALL) specimen exhibited a clonal IgH gene. Three of four cases of unclassifiable ALL could be assigned to the B cell lineage on the basis of gene rearrangement analysis. Seven cases originally diagnosed in smears as ALL were rediagnosed as AML (n = 5) or biphenotypic leukaemia (n = 2) because of immunohistochemical reactivity for myeloperoxidase or lysozyme. Two of these AML cases and two of three cases of biphenotypic leukaemia exhibited a monoclonal IgH gene rearrangement. CONCLUSIONS: Acute leukaemia can be subtyped in bone marrow sections with a limited panel of antibodies suitable for use on paraffin wax embedded sections (against CD3, CD10, CD20, CD79a, myeloperoxidase, and lysozyme). In patients with ALL and a diagnostically equivocal immunophenotype, gene rearrangement analysis might indicate whether the B or T cell lineage is involved.     

Immunohistochemical analysis of Hodgkin's disease using microwave heating.

AIMS--To assess the effect of microwave heating on immunohistochemical staining of CD15 and CD30 antigens in Hodgkin''s disease tissue samples. METHODS--Formalin fixed, paraffin wax embedded sections from 20 cases of Hodgkin''s disease (six mixed cellularity, 14 nodular sclerosis) were immunostained for CD15, using two antibodies (DAKO-M1 and Leu-M1) and for CD30 using the antibody Ber-H2. The staining was carried out by conventional techniques which included pretreatment of sections with trypsin and on untreated sections following heating with microwaves. With antibody Leu-M1 an additional method, using a specific antimouse IgM bridge both with and without microwave heating, was also included. The results for each method were compared by counting positively stained Reed-Sternberg cells and estimating the staining intensity. RESULTS--Microwave heating resulted in a substantial increase in the number of cells stained with antibodies to CD15 and also in the staining intensity. The best results were obtained using Leu-M1 with specific rabbit anti-mouse IgM bridge and microwave heating. Dramatic enhancement of the staining of Reed-Sternberg cells for CD30 was achieved following microwave heating, together with disappearance of the non-specific staining of plasma cells. CONCLUSION--Microwave heating is strongly recommended for the immunohistochemical staining of CD15 and CD30 expressed by Reed-Sternberg cells in Hodgkin''s disease.     

Superheating Using Pressure Cooking: Its Use and Application in Unmasking Antigens Embedded in Methyl Methacrylate

Abstract

Unmasking of antigens in formalin fixed, paraffin embedded tissue sections for immunohistochemical staining has recently been reported using superheating with the aid of a pressure cooker. We describe the use and application of this technology to semithin sections of tissue that have been embedded in methyl methacrylate, the plastic we routinely employ for high resolution light microscopy immunohistochemical studies. In particular, we have investigated the use of superheating for the detection of bcl-2, CD3, CD79a, and Ki-67, as these antigens had previously proved more difficult to demonstrate following other pretreatment procedures. Using a protocol of superheating in 10 mM citrate buffer (pH 6.0) for 3 min, we easily localized all the antigens, with superior immunocytochemical staining to that achieved with microwave antigen retrieval. However, CD3 was best demonstrated when pretreatment with trypsin at 37°C was used in addition to pressure cooking. We have also shown that the choice and concentration of the accelerators N-N dimethylaniline or N-N tetramethylaniline employed for polymerizing the plastic affected immunocytochemical staining. (The J Histotechnol 21:231–236, 1998)     

CD31 (JC70) expression in plasma cells: an immunohistochemical analysis of reactive and neoplastic plasma cells.

AIMS: To investigate the immunohistochemical expression of CD31 (JC70) in normal and neoplastic plasma cells. METHODS: Plasma cells in bone marrow biopsies and extramedullary locations were examined. All extramedullary biopsies were formalin fixed and paraffin embedded. The bone marrow biopsies were fixed in formal acetic acid and embedded in paraffin wax. Twenty multiple myelomas (12 bone marrow and eight extramedullary deposits), 10 extramedullary plasmacytomas, and 30 biopsies with reactive plasma cells (10 bone marrow, 20 extramedullary biopsies) were stained with anti-CD31 (JC70) using the streptavidin-biotin detection system with diaminobenzidine as a chromogen. Antigen retrieval in bone marrow biopsies was achieved by pressure cooking. In all other biopsies, antigen retrieval was achieved by microwave pretreatment. RESULTS: All 20 extramedullary cases with reactive plasma cells showed intense membrane staining. Focal staining was detected in reactive plasma cells in bone marrow biopsies. Five of 10 plasmacytomas showed membrane staining. None of the cases of multiple myeloma, either medullary or extramedullary, showed any immunoreactivity for CD31. CONCLUSIONS: CD31, a member of the immunoglobulin supergene family of cell adhesion molecules, is strongly expressed in extramedullary reactive plasma cells, focally in bone marrow reactive plasma cells, and occasionally in extramedullary plasmacytomas.     

Bone marrow trephine biopsy

Trephine biopsies of the bone marrow should be carried out, when clinically indicated, by trained individuals following a standard operating procedure. A bone marrow aspiration should be performed as part of the same procedure. For patient safety and convenience, biopsies are usually performed on the posterior iliac crest. The biopsy specimen should measure at least 1.6 cm and, if it does not, consideration should be given to repeating the procedure, possibly on the contralateral iliac crest. If bone marrow aspiration is found to be impossible, imprints from the biopsy specimen should be obtained. Otherwise, the specimen is placed immediately into fixative and after fixation is embedded in a resin or, more usually, decalcified and embedded in paraffin wax. Thin sections are cut and are stained, as a minimum, with haematoxylin and eosin and with a reticulin stain. A Giemsa stain is also desirable. A Perls' stain does not often give useful information and is not essential in every patient. The need for other histochemical or immunohistochemical stains is determined by the clinical circumstances and the preliminary findings. Trephine biopsy sections should be examined and reported in a systematic manner, assessment being made of the bones, the vessels and stroma, and the haemopoietic and any lymphoid or other tissue. Assessment should begin with a very low power objective, the entire section being examined. Further examination is then done with an intermediate and high power objective. Ideally, reporting of trephine biopsy sections should be done by an individual who is competent in both histopathology and haematology, and who is able to make an appropriate assessment of both the bone marrow aspirate and the trephine biopsy sections. When this is not possible, there should be close consultation between a haematologist and a histopathologist. The report should both describe the histological findings and give an interpretation of their importance. A signed or computer authorised report should be issued in a timely manner. If the report is a preliminary, this must be clearly stated.     

Paraffin wax embedded muscle is suitable for the diagnosis of muscular dystrophy

AIM: At present, the diagnosis of muscular dystrophy is made by means of immunohistochemistry on frozen sections. The aim of this study was to develop a sensitive and reproducible immunohistochemical method for use on formalin fixed, paraffin wax embedded sections for the demonstration of dystrophin associated proteins and other muscle associated antigens. METHODS: All the cases studied were from the files of the department of histopathology, Great Ormond Street Hospital for Children NHS Trust. Immunohistochemistry was performed on paraffin wax embedded sections with heat mediated antigen retrieval and overnight incubation with the antibodies at room temperature. Four different pretreatment buffers were tested in the attempt to optimise the immunostaining. Frozen sections were run in parallel for direct comparison. RESULTS: All the antibodies except delta sarcoglycan gave strong, consistent immunostaining in paraffin wax embedded sections, comparable with the frozen sections. The most consistent results were obtained using citrate/EDTA as the pretreatment buffer. CONCLUSION: A reliable and reproducible technique has been established, using a heat mediated citrate/EDTA buffer antigen retrieval method, which works well for most of the antibodies needed to make the diagnosis of muscular dystrophy in formalin fixed, paraffin wax embedded sections. This technique overcomes some of the inherent problems encountered using frozen muscle tissue and it could become a valuable tool for the diagnosis of muscular dystrophy.     

Are routine iron stains on bone marrow trephine biopsy specimens necessary?

AIMS: To determine the role of Perls' staining in bone marrow trephine biopsy sections. METHODS: The haemosiderin content of 155 Perls' stained, formic acid decalcified trephine biopsy sections was assessed and compared with Perls' stained aspirate samples in 105 cases and haematoxylin and eosin (H&E) stained biopsy sections in all cases. RESULTS: An evaluable aspirate film with positive iron or at least seven negative particles was available for 105 biopsies. Only 71 of 95 cases with detectable aspirate iron had haemosiderin detectable on a Perls' stained section. None of 10 samples with a negative aspirate had a positive trephine biopsy. Haemosiderin was positive in 101 of the 155 Perls' stained sections, and was detectable on the H&E stained section in 71 of these cases. In five of 54 cases with negative Perls' staining, a small amount of haemosiderin was thought to be present on H&E staining. CONCLUSIONS: Aspirate smears reflect bone marrow iron stores more reliably than formic acid decalcified trephine biopsy sections. The presence of iron in Perls' stained aspirates in 44% of cases with negative Perls' stained sections indicates that iron is often lost from sections during decalcification. However, 61% of cases with unassessable aspirate samples had a positive trephine biopsy Perls' stain, contributing useful clinical information about iron status. Preparation of Perls' stained sections only in cases in which aspirate samples are inadequate for iron assessment and no obvious haemosiderin is present in an H&E stained section could produce savings in staff time and reagent costs.     

Labelling of cells of the mononuclear phagocyte system in routinely processed archival biopsy specimens with monoclonal antibody EBM/11.

When first reported, EBM/11 reacted with mononuclear phagocyte system cells only in fresh frozen sections, but it has been shown to have similar cellular specificity in routine formalin fixed, paraffin wax embedded tissue. This was achieved by limited proteolysis with protease XIV before immunocytochemical staining. In archival biopsy specimens EBM/11 produced granular cytoplasmic staining of alveolar macrophages, Kupffer cells, tingible body macrophages and sinus histiocytes, cells of splenic cords, cortical and medullary macrophages of thymus; blood monocytes, peritoneal and mesothelial macrophages; bone marrow mononuclear cells, megakaryocytes and osteoclasts; lamina propria macrophages in the gastrointestinal tract, and connective tissue cells (presumptive macrophages) of thyroid, gall bladder, skin, pancreas, ovary, myometrium, endometrium, cervix, kidney, prostate, placenta, myocardium and breast. Unlike other anti-macrophage antibodies, EBM/11 did not react with granulocytes, lymphocytes, plasma cells, platelets, endothelial and epithelial cells in paraffin wax sections. It did not label skin Langerhans' cells, microglial cells, and interdigitating reticulum cells (as in frozen sections). This study opens a new area for the specific identification by EBM/11 of mononuclear phagocyte system cells in archival biopsy specimens. It also raises the possibility that some monoclonal antibodies, believed to be reactive only in frozen sections, may react in archival tissue after limited proteolysis with an appropriate enzyme.     

Plastic-embedded human marrow biopsy specimens: improved histochemical methods

Improved methods for processing, sectioning, and staining plastic (glycol methacrylate)-embedded human marrow biopsy specimens were studied. Special stains, including naphthol AS-D-chloro-acetate esterase, PAS, reticulin, and iron, have been modified so that they are suitable for undecalcified, 2-microns-thick, plastic-embedded human marrow biopsy specimens. These adaptations permit plastic-embedded marrow specimens to be used for clinical diagnosis. Marrow biopsy specimens embedded in plastic were compared with biopsy specimens preserved by the conventional paraffin method. The plastic-embedded marrows provide better results from morphologic examination (enhancing diagnostic accuracy), permit assessment of bone as well as of marrow, and allow histochemical analysis to be performed.     

Immunohistochemical demonstration of c-erbB-2 oncoprotein in gastric adenocarcinoma: comparison of cryostat and paraffin wax sections and effect of fixation.

AIMS--To investigate the effects of fixation on the immunohistochemical demonstration of c-erbB-2 oncoprotein using paraffin wax and cryostat sections; to compare c-erbB-2 expression in non-neoplastic and neoplastic gastric tissues. METHODS--Adjacent blocks of tumour and non-neoplastic tissue from four gastrectomy specimens were put into a panel of 10 fixatives including acetone, B5, Bouin's fluid, Carnoy's fluid, buffered formalin, formol dichromate, zinc formalin, 4% paraformaldehyde, periodate-lysine-paraformaldehyde (PLP) and periodate-lysine-paraformaldehyde-dichromate (PLPD) before embedding in paraffin wax for sectioning. Similar tissue blocks were snap frozen and cryostat sections were postfixed in these fixatives, either alone or in combination, before immunostaining. RESULTS--In paraffin wax embedded sections the best fixative was PLP, and in frozen tissues the best results were obtained after fixation of cryostat sections in buffered formalin followed by cold methanol and acetone. Applying these fixatives to samples from a further 16 gastrectomy specimens, strong membrane staining of c-erbB-2 protein was found in the tumour in eight of 16 cases (50%) using paraffin wax sections, and staining was stronger in the better differentiated carcinomas. For frozen tissues, positive membrane staining was found in all gastric adenocarcinomas, but differential staining intensity associated with tumour differentiation could not be detected. CONCLUSIONS--These results indicate that fixation and paraffin wax embedding affect the results of immunohistochemical demonstration of c-erbB-2 in gastric cancer. The choice of fixative is critical in the demonstration and evaluation of c-erbB-2 protein expression by immunohistochemistry in gastric carcinomas. Staining results also vary depending on whether frozen or paraffin wax embedded tissues are studied.     

Direct immunofluorescence of skin using formalin-fixed paraffin-embedded sections.

The technique of direct immunofluorescence has been applied to skin biopsy specimens fixed in formalin and embedded in paraffin wax. The results have been compared with those obtained by using snap-frozen biopsy specimens from the same patients. Trypsinisation of the dewaxed material allowed subsequent detection of immunoglobulins, complement, and fibrinogen. When compared to the fluorescence in the snap-frozen specimens, the staining in the paraffin sections was less bright and there was a higher rate of negatives. Even so, it was possible to establish the diagnosis in most cases of pemphigus, pemphigoid, and lupus erythematosus.     

Is it necessary to embed bone marrow biopsies in plastic for haematological diagnosis?

With the increase in the use of bone marrow trephines for diagnosis have come numerous reports that traditional methods of preparation (by decalcification and embedding in paraffin wax) should be replaced by plastic embedding to avoid decalcification. It has been argued that only by this means can the high quality preparations needed for accurate haematopathological diagnosis be achieved. The present study challenges this viewpoint and argues that with a little extra care and attention conventional paraffin embedding techniques can give equally high quality preparations. Sections prepared in this way meet the diagnostic needs of the haematologist, without requiring a separate technique to be established in the pathology laboratory solely for bone marrow trephines.