A method for preserving monoclonal marker expression in bone marrow biopsies from haematological disorders using a glycol methacrylate cold-embedding technique

We report a simple glycol methacrylate (GMA) cold embedding technique that can be used on bone marrow biopsy core. The biopsies were fixed in cold Bouins solution, dehydrated in cold methanol followed by infiltration and embedding in cold GMA then polymerised at 4°C under vacuum. This provided an effective cooling system, which preserved a wide range of the cell antigens by preventing heat damage due to excessive rise in temperature. This might be a useful diagnostic tool for the analyses of bone morphology and immuno-histochemistry on the same section. We have described a method of bone marrow biopsy which allows us to achieve excellent antigenic preservation and high-quality observation of morphological details in relation to expression with immuno-histochemical markers.An erratum to this article can be found at     

Glycol methacrylate embedding for light microscopy. I. enzyme histochemistry on semithin sections of undecalcified marrow cores.

A simple, routine procedure for water miscible glycol methacrylate (GMA) embedding of undecalcified bone marrow cores, which preserves the activity of enzymes useful in diagnosing various haematopoietic disorders, is described. The GMA used in this study has a low acid content that eliminates background staining, and the modified May-Grünwald-Giemsa stain provides good definition and excellent colour differentiation of various haematopoietic cells in the bone marrow, thereby providing optimal conditions for the study of the morphology and enzyme activity of bone marrow cells in the same preparation. The method is simple, reproducible, requires no expensive equipment, and is suitable for routine processing of small bone marrow cores in any histopathology or haematology laboratory.     

Biopsy specimen identification by detection of sex chromosomes: application of in situ hybridisation.

AIMS: To investigate the feasibility of non-radioactive in situ hybridisation (ISH) for the identification of sex-mismatched plastic embedded bone marrow biopsy specimens. METHODS: After a suspected accidental transposition of two glycol-methacrylate embedded bone marrow specimens, in situ hybridisation with sex chromosome specific probes was performed. RESULTS: Quantitative analysis of the hybridisation signals established unequivocably the origin of the specimens. CONCLUSIONS: ISH is feasible on GMA embedded bone marrow specimens, and can be used for the identification of accidentally transposed specimens provided that they are of sex-matched origin.     

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.     

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.     

Glycol Methacrylate for Routine,Special Stains,Histochemistry, Enzyme Histochemistry and Immunohistochemistry: A Simplified Cold Method for Surgical Biopsy Tissue

Abstract

A method of preparing surgical pathology bone marrow, lymph node, and kidney biopsies for glycol methacrylate embedding, while preserving enzymes and antigenicity in each tissue biopsy block, is described. Biopsy tissues are fixed in cold phosphate buffered formalin at pH 7.3, rinsed in cold phosphate buffer, dehydrated, infiltrated and embedded in cold glycol methacrylate, then polymerized at 4°C. Sections are cut at 1–2 pm in thickness with disposable blades or glass knives. Room temperature drying of sections and slides preserves enzymes. Antigenicity is preserved when drying slides at room temperature but, due to section removal during fluorescence staining, it is necessary to incubate slides at 37°C. This technic has proved to be a valuable tool in diagnostic biopsy work.     

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.     

Antigen localization in immunoperoxidase-stained plastic-embedded soft tissues

Immunoperoxidase stains were performed on normal and neoplastic tissue from prostate, colon, thyroid, lung, nerve, uterus, and placenta embedded in both plastic (glycolmethacrylate [GMA]) and paraffin. Positive results in plastic section were obtained for carcinoembryonic antigen (CEA), keratin, epithelial membrane antigen (EMA), thyroglobulins, S-100, prostate-specific antigen, human chorionic gonadotrophin (HCG), and beta-HCG. More delicate staining with more precise localization of antigens is noted. Superior (paraformaldehyde) fixation and cold processing followed by GMA polymerization (4 degrees C) allow for optimum antigen survival. After fixation, tissue processing involves a series of 0.1 mol/L phosphate buffer rinses with sucrose and ammonium chloride in a conventional dip-and-dunk processor placed in a 4 degrees C cold room. Acetone dehydrations are used before GMA infiltration, cold polymerization, and sectioning. Before immunoperoxidase staining, the plastic section is digested in .25% bovine trypsin for ten minutes. The immunoperoxidase methods described can be useful when small biopsies are routinely embedded in plastic to obtain improved histologic (hematoxylin-eosin) sections. There may also be research applications in quantifying antigen expression in benign, dysplastic, and neoplastic tissues by examining the stains under high power.     

Cell suspensions from collagenase digestion of bone marrow trephine biopsy specimens.

A technique for the extraction of cells from bone marrow trephine core biopsy specimens using collagenase digestion was assessed in 39 cases (33 diagnostic and six normal). Diagnostically useful numbers of cells were extracted from all marrows. Morphological assessment of cytocentrifuge preparations of these cells gave a correct diagnosis in 23 (60%) of cases compared with 27 (70%) for the corresponding aspirated marrow smears. Phenotypic analysis using flow cytometry showed persistence of a range of surface membrane antigens following collagenase digestion. Increased autofluorescence was a problem in some cases. Cytochemistry, bone marrow culture, and cytogenetic analysis could also be carried out on these cells. It is concluded that this technique has useful diagnostic applications in cases of dry taps.     

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.     

Bone marrow biopsy in clinical medicine: an overview

Bone marrow biopsies are now employed in the investigation of many disorders in haematology, oncology and internal medicine. This review provides a survey of the recent literature and a summary of observations made on undecalcified bone marrow biopsies embedded in plastic. The conditions investigated include osteopathies, myelopathies, haematologic and non-haematologic malignancies in the bone marrow. The interrelationship and interdependence of bone and bone marrow have been emphasized, and examples of the effects of diseases of bone on marrow, and of disturbancies of marrow function on bone, have been given. In the myelo- and lympho-proliferative disorders bone marrow biopsies contribute to diagnostic evaluation and classification, as well as to provide factors of prognostic significance. In the investigation of patients with solid tumours bone marrow biopsy may detect metastases in 20 per cent (bronchus), 35 per cent (prostate), 40 per cent (breast), to 80 per cent (unknown primaries) of the patients. Bone marrow biopsy constitutes an additional investigative parameter capable of providing valuable information in many different clinical situations.     

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.     

Plastic embedded core biopsy: a complementary approach to bone marrow aspiration for diagnosing acute myeloid leukaemia.

Bone marrow aspirates and biopsy specimens were taken at diagnosis from 51 patients with acute myeloid leukaemia (AML). The diagnosis was based on morphological and cytochemical analyses, and the leukaemias were classified by FAB criteria. A considerable difference was observed between the results of bone marrow aspirates and the findings of plastic-embedded bone marrow biopsy specimens, particularly in marrow cellularity, extent of blast cell infiltration, and cell type involved in the leukaemic process. The myelomonocytic cell type seemed to predominate in the sections. In four cases there was considerable marrow infiltration with maturing, but dysplastic, granulocytic cells in the sections, but not in the aspirate smears. Features of potential prognostic importance, such as bone marrow infiltration with inflammatory cells, were easily recognised and quantified in the sections. These results indicate that plastic embedded bone marrow biopsy sections complement the findings of bone marrow aspiration in the diagnosis of AML and may also provide information of independent prognostic importance that cannot be obtained by other means.     

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.     

Comparative evaluation of bone marrow aspirate particle smears, imprints and biopsy sections

Comparative evaluation of bone marrow aspirate particle smears, imprints and biopsy sections was done on 30 haematological problems. Core needle biopsy of the bone marrow is a safe and useful procedure. It is a valuable diagnostic aid for measurement of marrow cellularity, metastatic tumours and fibrosis. It should not be taken as a substitute for examination of the marrow by aspiration smear but is a complementary procedure which affords several advantages. Bone marrow biopsy was of maximum utility in myelofibrosis which was diagnosed on biopsy alone. There were three additional cases with normal bone marrow aspiration in which specific diagnosis could only be made from bone marrow biopsy sections. New methodologies i.e. plastic embedding and semi thin sections of undecalcified bone marrow, can be expected to improve the cytological details of tissue obtained by biopsy. Imprint preparations obtained from biopsy can be useful in patients of malignancy but we have found them to be of limited value except in cases of dry tap.     

Adequacy of bone marrow trephine biopsy specimens in children.

AIMS: To evaluate success in obtaining adequate bone marrow trephine biopsy cores from children. METHODS: Sections of trephine biopsy cores submitted by 25 centres from children with neuroblastoma over a five year period were reviewed centrally. In cores containing no tumour adequacy was defined as 0.5 cm of well preserved bone marrow after processing. Occasional smaller cores containing obvious tumour were also considered adequate. RESULTS: Of 822 biopsy specimens, 139 (17%) were inadequate. In 13 centres submitting at least 20 cores failure rates ranged from 2.6 to 50%. There was no improvement over the five years of the study. There was no practically important correlation between the numbers of cores submitted and success in obtaining adequate specimens. Although a lower rate of inadequate biopsy specimens was found when haematologists rather than paediatricians (13 v 29%) were the predominant operators this should not be overinterpreted, not least because of the potentially confounding association between haematologist operators and larger numbers of biopsy specimens, and because the arbitrary subdivision of centres according to operator specialty was crude. The skill of individual operators could not be assessed. CONCLUSIONS: Many operators do not obtain adequate bone marrow biopsy specimens from children. Improvement is necessary because this is an invasive investigation, often performed under general anaesthesia. Reporting pathologists are well placed to influence practice by pointing out inadequacies in the specimen and suggesting retraining or even a change in operator. Improvement would almost certainly occur if this investigation was restricted to locally recognised successful operators, whatever their specialty. Most centres should review their practice and devise strategies to improve their ability to obtain adequate cores.     

Techniques for obtaining differential cell counts from bone marrow trephine biopsy specimens.

Differential cell counts were performed on 200 paired bone marrow aspirates and trephine biopsy specimens to compare the distribution of cell types. Relatively more immature myeloid cells were found in the trephine biopsy specimens and relatively more polymorphs and lymphocytes in the aspirates. Two methods for sampling areas of the trephine biopsy specimens for counting were assessed, and the differences between aspirates and trephine specimens were found to be more consistent when the second, more extensive, sampling method was used. This method also permitted quantitation of some features of bone marrow topography and provided information that would not normally be obtainable from aspirated material. The techniques were easy to apply and took relatively little time to perform. They could offer useful information in the study of bone marrow disorders, particularly those such as myelodysplastic syndromes in which disturbances of marrow architecture are prominent.     

Preparation and light-microscopic examination of fixed hematopoietic cells in soft agar.

Hematopoietic cells from the blood or bone marrow (of leukemic and nonleukemic patients) grown in vitro using soft agar tissue-culture technics may be fixed in formalin, embedded in paraffin, sectioned, and mounted on glass slides. Light microscopic examination of these sections stained with hematoxylin and eosin and with other histologic stains provides information useful in investigative and diagnostic hematology. Morphologic interpretation of the characteristics of cultured cells is within the capability of pathologists and clinical hematologists. The slides provide a permanent record of growth in vitro.     

Histopathologic diagnosis of bone marrow in leukemia and related disorders]

Histopathologic diagnosis of the bone marrow in leukemia is usually a supplementary method to the cytological in acute and chronic leukemia. However, for patients with MDS and MPD and with dry tap bone marrow biopsy is very important. Important morphological findings and useful immunohistochemical methods for differentiation and characterization of leukemia are reported and the usefulness of sequential examination of bone marrow in leukemia during and after chemotherapy is emphasized. In addition to leukemia, histological features and differential points of myelodysplastic syndrome (MDS) and myeloproliferative disorders (MPD) are mentioned. The proliferating megakaryocytes differed in size and shape between MDS and MPD. The difference in proliferating rate of the cells examined by PCNA was also useful to differentiate the two disorders histologically.     

Methodological aspects and application of the immunoperoxidase staining technique in diagnostic fine-needle aspiration cytology

The immunoperoxidase method was modified and adapted for use on cells obtained by fine-needle aspiration biopsy for routine diagnostic cytology. Combinations of different modes of fixation and graded trypsinization were tested. Best results were obtained with fixation in formol-acetone followed by enzyme digestion for 3-6 min; exact times were adjusted for the individual antigen. With optimal conditions as to fixation and proteolytic digestion, the method was found to be sensitive and reproducible and without artifactual background staining. Various intracytoplasmic antigens of diagnostic importance such as immunoglobulins, prostate-specific antigen, keratin, thyroglobulin, S-100, alpha-1-antitrypsin, and lysozyme in lymphoid cells, bone marrow cells, and tumor cells of epithelial and mesenchymal origin were detected. Staining of newly prepared or up to 2-yr-old specimens gave equally good results. Both cellular morphology and the results of immunoperoxidase staining can be studied simultaneously. The method is considered valuable for increasing accuracy of diagnostic cytology.