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.     

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.     

Co-localization of multiple antigens and specific DNA. A novel method using methyl methacrylate-embedded semithin serial sections and catalyzed reporter deposition

Co-localization of proteins and nucleic acid sequences by in situ hybridization and immunohistochemistry is frequently difficult as the process necessary to detect the target structure of one technique may negatively affect the target of the other. Morphological impairment may also limit the application of the two techniques on sensitive tissue. To overcome these problems we developed a method to perform in situ hybridization and immunohistochemistry on semithin sections of methyl methacrylate-embedded tissue. Microwave-stimulated antigen retrieval, signal amplification by catalyzed reporter deposition, and fluorescent dyes were used for both techniques, yielding high sensitivity and excellent morphological preservation compared to conventional paraffin sections. Co-localization of in situ hybridization and immunohistochemistry signals with high morphological resolution was achieved on single sections as well as on adjacent multiple serial sections, using computerized image processing. The latter allowed for the co-localization of multiple antigens and a specific DNA sequence at the same tissue level. The method was successfully applied to radiation bone marrow chimeric rats created by transplanting wild-type Lewis rat bone marrow into TK-tsa transgenic Lewis rats, in an attempt to trace and characterize TK-tsa transgenic cells. It also proved useful in the co-localization of multiple antigens in peripheral nerve biopsies.     

Immunohistochemical staining of plastic embedded bone marrow trephine biopsy specimens after microwave heating.

AIMS--To investigate (1) whether adequate immunohistochemical staining can be achieved on sections cut from plastic embedded bone marrow trephine biopsy specimens after microwave heating in citrate buffer; and (2) whether this immunohistochemical staining is comparable with that achieved on routine sections cut from paraffin wax embedded trephine biopsy specimens after decalcification procedures. METHODS--Sixty five consecutive bone marrow trephine biopsy specimens of more than 1 cm in length were divided transversely into two equal parts. One part was processed in paraffin wax followed by decalcification. The other part was embedded in the epoxyresin Polarbed 812 followed by the cutting of 1 micron sections. Both parts underwent immunohistochemical staining by an identical panel of antibodies. With Polarbed 812 plastic embedded sections, microwave heating in citrate buffer was undertaken before the application of antisera. RESULTS--On sections cut from plastic embedded material, immunohistochemical staining was generally satisfactory, easy to interpret and comparable with that achieved with paraffin wax embedded material. Exceptions were antibodies to neutrophil elastase and CD61 where immunostaining was consistently negative on plastic embedded sections. Immunohistochemical staining for CD20 was consistently more reliable on plastic embedded sections. CONCLUSIONS--The results provide evidence that, with few exceptions, satisfactory immunohistochemical staining is possible on plastic embedded bone marrow trephine biopsy specimens after microwave heating in citrate buffer. This, combined with the advantage of superior cellular morphology with semi-thin (1 micron) sections of plastic embedded material, make such embedding procedures the preferred method for the processing of bone marrow trephine biopsy specimens.     

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.     

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.     

Diagnosis and quantification of bone marrow fibrosis are significantly biased by the pre-staining processing of bone marrow biopsies

AIMS: Marrow fibrosis (MF) is an unfavourable, often lethal complication of haematological neoplasms. Although biopsy technique and staining procedure are standardized, the prestaining processing of bone marrow biopsies (BMBs) varies markedly without any existing data on its significance for the diagnosis of MF. METHODS AND RESULTS: In this study on 712 BMBs from 296 patients with chronic idiopathic myelofibrosis (CIMF), chronic myeloid leukaemia (CML), or healthy bone marrow, MF was a characteristic complication of CML and CIMF. However, diagnosis and quantification of MF and detection of its prognostic significance were significantly biased by fixation, decalcification, embedding, marrow tissue shrinkage during biopsy processing and the thickness of marrow sections (P < 0.000005). The relevance of these influences was explained by their effect on the marrow volume to which the fibre content was related, whereas the stainability of fibres was not affected. Semiquantitative grading of fibrosis and measurements of fibre density could not be adjusted to various methods of processing of bone marrow biopsies (P < 0.003). CONCLUSIONS: Evaluations of MF and its prognostic significance should consider the bias due to the prestaining processing of BMBs and the necessity of an adjustment to the thickness of tissue sections and the degree of marrow tissue shrinkage.     

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.     

Application of the FICTION technique for the simultaneous detection of immunophenotype and chromosomal abnormalities in routinely fixed, paraffin wax embedded bone marrow trephines

The use of interphase fluorescence in situ hybridisation (FISH) to study cytogenetic abnormalities in routinely fixed paraffin wax embedded tissue has become commonplace over the past decade. However, very few studies have applied FISH to routinely fixed bone marrow trephines (BMTs). This may be because of the acid based decalcification methods that are commonly used during the processing of BMTs, which may adversely affect the suitability of the sample for FISH analysis. For the first time, this report describes the simultaneous application of FISH and immunofluorescent staining (the FICTION technique) to formalin fixed, EDTA decalcified and paraffin wax embedded BMTs. This technique allows the direct correlation of genetic abnormalities to immunophenotype, and therefore will be particularly useful for the identification of genetic abnormalities in specific tumour cells present in BMTs. The application of this to routine clinical practice will assist diagnosis and the detection of minimal residual disease.     

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.     

Amplification of PCR products in excess of 600 base pairs using DNA extracted from decalcified, paraffin wax embedded bone marrow trephine biopsies.

AIMS: To establish a robust method of extracting DNA from paraffin wax embedded bone marrow trephine (PBMT) biopsies for the amplification of relatively long polymerase chain reaction (PCR) products. METHOD: Xylene and ethanol were used to remove paraffin wax from eight formalin fixed, EDTA decalcified PBMT biopsies and DNA extraction was performed using a Qiagen QIAamp tissue kit. The DNA samples were amplified using nine different PCR primers sets, including those used to detect chromosomal translocations (t(11;14) and t(14;18), and clonal B cell populations. A t(11;14) PCR product of approximately 600 base pairs (bp) was sequenced using dye terminator cycle sequencing. RESULTS: All eight DNA samples extracted from PBMT biopsies were amplified successfully to generate DNA fragments up to 643 bp in length. Chromosomal translocations and immunoglobulin gene rearrangements were detected by PCR in some of the samples. Sequencing of the t(11;14) PCR product demonstrated the presence of chimaeric sequences, which included both bcl-1 and immunoglobulin heavy chain (IgH) gene sequences, consistent with the presence of this translocation. CONCLUSIONS: This method enables PCR analyses of PBMT biopsies that were not previously possible, offering the prospect of improved accuracy of diagnosis and the monitoring of patients with bone marrow disease.     

Knochenmarkbiopsie

The rapid technological development in diagnostic pathology, especially of immunohistochemical and molecular techniques, also has a significant impact on diagnostic procedures for the evaluation of bone marrow trephine biopsies. The necessity for optimal morphology, combined with preservation of tissue antigens and nucleic acids on one hand and the wish for short turnaround times on the other hand require careful planning of the workflow for fixation, decalcification and embedding of trephines. Although any kind of bone marrow processing has its advantages and disadvantages, formalin fixation followed by EDTA decalcification can be considered a good compromise, which does not restrict the use of molecular techniques. Although the majority of molecular studies in haematological neoplasms are routinely performed on bone marrow aspirates or peripheral blood cells, there are certain indications, in which molecular studies such as clonality determination or detection of specific mutations need to be performed on the trephine biopsy. Especially, the determination of B- or T-cell clonality for the diagnosis of lymphoid malignancies requires stringent quality controls and knowledge of technical pitfalls. In this review, we discuss technical aspects of bone marrow biopsy processing and the application of diagnostic molecular techniques.     

Intracellular hemoglobin-a specific marker for erythroid cells in paraffin sections. An immunoperoxidase study of normal, megaloblastic, and dysplastic erythropoiesis, including erythroleukemia and other myeloproliferative disorders.

Intracellular hemoglobin represents an excellent marker for specific characterization of normal, megaloblastic, or dysplastic erythroid cells in paraffin sections. Using an immunoperoxidase indirect sandwich technique for detection of intracellular hemoglobin, erythroid cells at all stages of maturation were readily identified in bone marrow biopsies (58 specimens total) with a) normal erythropoiesis, b) megalobastic erythropoiesis, and c) various myeloproliferative disorders, including erythroleukemia. In other tissues (6 spleens, 2 lymph nodes, 1 liver) with extramedullary hematopoiesis, erythroid cells were similarly defined on the basis of this immunohistochemical method. Initial fixation in Zenker's-acetic acid solution (employed for bone marrow biopsies), B5 solution, or formalin, appeared equally effective in preserving the antigenicity of intracellular human hemoglobin. This sensitive and specific immunoperoxidase technique for erythroid cell characterization is particularly applicable to tissues with abnormal erythropoiesis, in which precise cell identification generally presents a diagnostic problem.     

Plastic embedding in routine histology I: Preparation of semi-thin sections of undecalcified marrow cores

The preparation of sections of bone marrow cores in a routine histology laboratory requires decalcification and paraffin embedding, which produces shrinkage and considerable loss of cellular detail. This may be avoided by using plastic embedding procedures. This report describes a simplified routine procedure for using methylmethacrylate as a plastic embedding medium for the preparation of semi-thin sections of undecalcified bone marrow cores. A modification of the May-Grunwald-Giemsa stain is also given which provides good colour differentiation of various haematopoietic cells in the marrow. The method is simple, reproducible, requires no expensive equipment, and is suitable for routine processing of bone marrow biopsy cores in any histopathology laboratory.     

Rapid detection of hepatitis B virus DNA in liver tissue by in situ hybridisation and its combination with immunohistochemistry for simultaneous detection of HBV antigens.

A rapid technique using a non-radioactive receptor molecule (digoxigenin) for intrahepatic hepatitis B virus (HBV) DNA detection using in situ hybridisation was developed. It can be adapted for use in combination with standard immunohistochemistry for simultaneous detection of both HBV DNA and HBV antigens. The total time required for dual detection of HBV antigens and HBV DNA starting from paraffin wax liver sections was two working days. A good signal to background ratio for the detection of HBV DNA was always obtained using this labelling. This technique is cheap, safe, and relatively simple which makes it an ideal tool for the detection of intrahepatic HBV DNA for both routine diagnostic purposes and in research.     

Bone marrow biopsy: interpretive guidelines for the surgical pathologist

Ideally, the bone marrow core biopsy should be reviewed with knowledge of the clinical history, complete blood count, and findings in the peripheral blood and bone marrow aspirate smears. However, for a variety of reasons, the pathologist may receive the core biopsy and aspirate clot section without all of this information. Although this approach is not optimal, a great deal of valuable information can be generated from these specimens. Over the past 20 years, there has been considerable progress in the fields of flow cytometric analysis, immunohistochemistry, and molecular diagnostic studies that can be performed on smears or extracted DNA from paraffin embedded tissue. These modalities have augmented and refined diagnostic criteria formerly ascertained by light microscopy, cytochemistry, and cytogenetics. This is particularly true of some myeloid and lymphoreticular neoplasms where a collaborative and multidisciplinary approach to the diagnosis has become necessary. Despite this growing complexity and dependence on newer methodologies, the traditional role of histopathology in evaluating the bone marrow biopsy remains as important as it has been in the past. In this review, we focus on contemporary practices and expectations for interpreting bone marrow biopsies and clot sections.     

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.     

Use of glass knives for preparing histological sections from wax-embedded blocks

A technique using glass knives for making histological sections without application of epoxy resin is suggested. The method of embedding of the material fixed in formalin is described, according to which instead of paraffin dental wax is used. Sections were made on a rotation microtome "Leitz" modified for the fixation of a glass knife.     

LN-3: a diagnostic adjunct in cutaneous graft-versus-host disease.

Expression of HLA Class II antigen on keratinocytes has been advocated as a diagnostic marker of acute graft-versus-host disease (GVHD). LN-3 is a murine monoclonal antibody marking an HLA Class II antigen that survives formalin fixation and paraffin embedding. We analyzed the LN-3 staining pattern on 56 skin biopsies from patients treated with bone marrow or peripheral stem cell transplantation, non-transplant patients receiving conventional chemotherapy and/or radiation therapy and controls. Keratinocyte staining, endothelial staining, and the number of epidermal and dermal Langerhans cells were evaluated and analyzed with respect to the histologic and clinical diagnosis in a blind and retrospective fashion. The most predictive parameter for GVHD was marking of endothelial cells by LN-3 which was present in 12 of 16 (75%) biopsies from patients with GVHD. Endothelial staining was found in a total of 19 biopsies of which 12 (63%) had GVHD. These data suggest that LN-3 immunohistochemistry may help identify cutaneous GVHD and discriminate it from conditions that are histologically similar.     

Use of monoclonal anti-actin as a megakaryocyte marker in paraffin wax embedded bone marrow biopsy specimens.

Monoclonal anti-actin was used as a marker of megakaryocytes in Zenker's fixed, paraffin wax embedded bone marrow tissue, using an immunoperoxidase staining method. Twenty bone marrow samples were studied, including controls, and different myeloproliferative and myelodysplastic syndromes. The results were compared with those obtained using factor VIII related antigen (F VIII RAg) immunolabelling. Anti-actin is as good a marker for megakaryocytes as anti-FVIIIRAg and is potentially clinically useful when morphological identification is difficult, when bone marrow aspiration is unsuccessful, or when quantitative evaluation of tissue sections is required.