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.     

Diagnosis of nasopharyngeal tuberculosis by detection of tuberculostearic acid in formalin fixed, paraffin wax embedded tissue biopsy specimens.

The use of gas chromatography and mass spectrometry with selected ion monitoring detected tuberculostearic acid (TBSA) in 10 of 12 formalin fixed, paraffin wax embedded nasopharyngeal and head and neck biopsy specimens from patients with confirmed tuberculosis and carcinoma, and in one of 50 control specimens (giving a sensitivity of 83% and a specificity of 98%). The two false negative cases had very small tissue fragments and the patient with a false positive result may have had pulmonary tuberculosis. Tuberculostearic acid (TBSA) was also detected in nine of 16 specimens from the head and neck region with non-caseating granulomas suspected, but not confirmed, to be tuberculosis. It is concluded that nasopharyngeal tuberculosis is relatively common in Hong Kong and should be considered when biopsy specimens show granulomas. The detection of TBSA in tissue biopsy specimens is a useful, rapid method for the diagnosis of tuberculosis and other mycobacterial infections, and can be conveniently performed within two days on formalin fixed and paraffin wax embedded material.     

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 monoclonal antibodies to recognise osteoclasts in routinely processed bone biopsy specimens.

In decalcified (5% nitric acid) and undecalcified (glycol-methacrylate or resin embedded) routinely processed bone specimens osteoclasts against resorbing surfaces were identified with monoclonal antibodies directed against leucocyte common antigen (LCA) (PD7/26, 2B11), CD68 (KP1), and gpIIIa (Y2/51) but not against HLA-DR (CR3/43 and Ta11B5). Mononuclear cells on resorbing surfaces and occasional mononuclear cells against or near resting surfaces showed a similar pattern of reactivity. This study shows that immunohistochemistry is a sensitive and useful technique for identifying osteoclasts in routinely processed bone specimens. It also suggests a role for mononuclear cells (possibly pre-osteoclasts) in bone resorption.     

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.     

A method of processing small biopsy specimens embedded in paraffin using xylene

The method proposed for the processing of gastrobiopsies is as follows: 30-40 min in solution I (seventy per cent 96% ethyl alcohol and 30% of turpentine), then 30-40 min in solution II (100 parts of turpentine, 5 parts of balzam and 8 parts of castor oil), 4 portions of melted paraffin for 7.5-10 min in each portion. Small biopsies (up to 5 mm in diameter) are treated in the same way but till 3 hrs in solution I, 6-8 hrs in solution II and 30 min in each portion of paraffin. For an instant processing of biopsies of operation and autopsy material the basic solution is prepared: 1000 ml of xylene, 100-120 g of balzam, 150 ml of ol. ricini. The material is treated in two portions of ethyl alcohol (30-150 min in each portion); two portions of mixture A; 80% of acetone and 20% of the basic solution), 3 hrs in each portion; two portions of mixture B (20% of acetone and 80% of basic solution), 2 hrs in each portion; and in 6 portions of melted paraffin, 30 min in each portion. The technique provides good results for the processing of small biopsies from other organs.     

Production of a monoclonal antibody as immunohistochemical marker on paraffin embedded tissues using a new immunization method

This report describes a method for the production of murine monoclonal antibodies (MAbs) against cellular antigens preserved during formol fixation and paraffin embedding of human tissues in an attempt to select markers that would be useful in immunopathology. Hybridomas were prepared using spleen cells from mice immunized with cell suspensions obtained from formalin-fixed paraffin block sections of a human breast carcinoma. A monoclonal antibody 83 D4 was selected, which was reactive with paraffin embedded breast carcinoma tissues, but not with normal breast. The reactive antigen has a high molecular weight (400-1000 kD) and was detected on the cell surface of live human breast cancer cell lines and on frozen tissues sections. These results demonstrate that the MAb 83 D4 identifies a native breast tumor associated epitope conserved during tissue fixation and embedding and could be used as an immunohistochemical marker.     

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.     

Immunohistochemical identification of erythroid precursors in paraffin embedded bone marrow sections: spectrin is a superior marker to glycophorin

AIM: To investigate whether spectrin can be used as an immunohistochemical marker for erythroid precursors in routinely processed paraffin embedded bone marrow sections. METHODS: Bone marrow biopsies and clot sections were stained with rabbit antihuman erythrocyte spectrin antibodies, specific for erythroid cells as shown by western blotting and bone marrow smears, and compared to sections stained with antiglycophorin monoclonal antibodies (JC159 and Ret49f). RESULTS: Antispectrin antibodies resulted in diffuse cytoplasmic staining of early erythroblasts and membranous staining of late erythroblasts as well as erythrocytes. In haematopathological samples, immature erythroid cell clusters were clearly identified. In contrast, antiglycophorin monoclonal antibodies resulted in only membranous staining of late erythroblasts, and faint staining of early erythroblasts. CONCLUSIONS: Spectrin may be a superior marker to glycophorin for the identification of erythroid precursors in paraffin embedded sections.     

The Mi15 monoclonal antibody (anti-syndecan-1) is a reliable marker for quantifying plasma cells in paraffin-embedded bone marrow biopsy specimens

Plasmocyte selective monoclonal antibodies (MAb) recognizing syndecan-1 have recently been described. They belong to a new cluster, CD138. Using the MAb MI15, we investigated the expression of syndecan-1 in routinely paraffin-embedded tissues. Nontumoral lymph nodes (25 cases) and bone marrow biopsy specimens (63 cases) showed strong membrane staining of plasma cells only, allowing accurate analysis of the nuclear structure. The MI15 positivity correlated with kappa and lambda light chain expression in the cytoplasm. The percentages of plasma cells calculated in bone marrow biopsy specimens after MI15 staining were, respectively, 2.1% (range, 1% to 4%) in normal bone marrows, 8.5% (range, 5 to 17) in reactive plasmocytosis, and 4.66% in monoclonal gammapathy of undetermined significance (MGUS) patients (range, 1 to 13), in the same range but slightly higher than those obtained on smears or on hematoxylin and eosin (H&E)-stained sections. In multiple myeloma (40 cases), all plasma cell types were marked, and Mi15 MAb gave additional information in 8 of 40 (20%) patients. In lymph nodes, Mi15 MAb reacted with Reed-Sternberg cells of classical Hodgkin's disease in 23 of 31 cases (74%) with variable intensity. In contrast, nodular lymphocyte predominance Hodgkin's disease (10 cases), most B cell lymphomas (88 of 107 cases) and all T cell lymphomas (30 cases) were negative. In B cell lymphomas, plasmocytomas (8 cases), plasmocytic lymphomas (2 cases), and 5 of 13 cases of immunoblastic lymphoma with plasmocytoid differentiation were stained. In lymphoplasmocytoid lymphomas (4 lymph nodes and 20 bone marrow biopsy specimens), only mature plasma cells were positive. Moreover, a wide distribution of syndecan-1 was observed in normal and tumoral epithelial tissues. Finally, Mi15 MAb appears to be a reliable marker for identifying and quantifying normal and tumoral plasma cells in paraffin-embedded bone marrow and lymph node samples.     

Value of bilateral bone marrow biopsy specimens in non-Hodgkin''s lymphoma.

A study of 260 patients with non-Hodgkin's lymphoma (NHL) who underwent bilateral bone marrow biopsy at initial diagnosis showed marrow disease in 99 (38%) cases. The highest incidence of disease (83%) was seen in small lymphocytic lymphoma (SLL) and the lowest (19%) in diffuse large cell lymphoma (DLCL). Among cases with positive marrows, disease was bilateral in all 15 cases of SLL but in only 10 of 20 (50%) of the DLCL cases. In 30 of 99 (30%) positive marrows disease was unilateral. Follicular lymphomas were strongly associated with a paratrabecular pattern, with 40 of 45 positive cases showing this. Discordant histology was seen in six of 20 positive cases of DLCL and two of 37 positive cases of follicular small cleaved cell lymphomas (FSCCL). A bone marrow aspirate was positive in only 56 of the 99 (57%) cases. Peripheral blood disease was present in 15% of the bone marrow positive cases and in 6% of the cases overall. The incidence of marrow disease varies with the histological subtype of lymphoma. The paratrabecular pattern is associated with follicular lymphoma, and bilateral biopsy specimens increase the positivity rate in most subtypes of NHL.     

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.     

骨髓活检塑料包埋切片免疫双标法的建立

双重免疫染色用于恶性血液病骨髓活检的研究国内外报道甚少,我们借鉴了非洗脱法中的间接法一异种动物抗体法,对多发性骨髓瘤患者选用了浆细胞抗体CD38和增殖细胞抗体Ki-67抗体的配对,建立了重复性和稳定性好的双标记法。     

Early reconstitution of haematopoiesis after allogeneic bone marrow transplantation: a prospective histopathological study of bone marrow biopsy specimens.

To study early haematopoietic reconstitution after bone marrow transplantation bone marrow biopsy specimens taken in the third week after transplantation were evaluated. Cellularity was highly variable; localisation of the various cell lineages and the ratios of myeloid cells to erythroid cells were abnormal. Clustering of cells of the same lineage in the same stage of maturation was prominent. The bone marrow stroma showed many anomalies, including increased fibre content, periodic acid Schiff positivity of fat cells, oedema, sinus ectasia and granulomas. A comparison of biopsy findings with clinical and laboratory data showed a correlation between the amount of erythroid cells and the day of appearance of reticulocytes, as well as the number of reticulocytes. Absence of clustering of haematopoietic cells in four of five patients was associated with either failure of engraftment or early leukaemic relapse. Variables such as infections and administration of possibly myelosuppressive drugs did not influence bone marrow biopsy findings.     

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.     

Central review of bone marrow biopsy specimens from patients with neuroblastoma.

In order to achieve some uniformity in histological detection of bone marrow infiltration by neuroblastoma and to provide a measure of variation in histological opinions, sections from 712 evaluable trephine biopsy cores from children in a European Neuroblastoma Study Group (ENSG) study were reviewed centrally. Biopsy specimens were graded as tumour positive or negative. Discordance between local and central review opinions was found in 5% of specimens. Only five of 165 children at presentation and nine of 256 re-staging procedures in 126 children, affecting one child each, had their diagnosis upgraded to positive. In six re-staging procedures, affecting one child each, the diagnosis was downgraded. The low discordance rate is encouraging and substantially less important than previously documented difficulties in obtaining adequate specimens.     

Orange-red birefringence of gold particles in paraffin wax embedded sections

Chrysiasis, the systemic deposition of gold pigment in patients on long term chrysotherapy, is identified histologically as small black granules within macrophages. Histological sections from 12 confirmed cases of chrysiasis were examined under crossed polarized light. This revealed a striking orange–red birefringence of the pigment not detected in other histologically similar deposits. This technique provides a valuable adjunct to the histological identification of gold without the need to resort to ultrastructural and analytical procedures.     

Evolution of myelofibrosis in chronic idiopathic myelofibrosis as evidenced in sequential bone marrow biopsy specimens

Although the new World Health Organization (WHO) classification acknowledges "prefibrotic" phases, progression of myelofibrosis in chronic idiopathic myelofibrosis (cIMF) is controversial because there are only a few studies about sequential biopsy specimens, and they yield conflicting results. The conflicting results might be due to a mixture of different degrees of myelofibrosis and therapy regimens within the respective groups studied. To prove this hypothesis, we studied sequential bone marrow biopsy specimens from patients with cIMF and compared 3 groups with different degrees of myelofibrosis at initial diagnosis with a group of patients with primarily unfibrosed disease who met the WHO criteria for prefibrotic cIMF. Patients receiving chemotherapy were considered separately from patients without treatment. Our results favor a steady progression of myelofibrosis unrelated to therapy modalities, whereas confusing literature data can be explained: fibrosis may remain static or lessen, especially in more advanced stages of cIMF.