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Vascular architecture and collagen type IV in primary myelofibrosis and polycythaemia vera: an immunomorphometric study on trephine biopsies of the bone marrow.

Using a monoclonal antibody against collagen type IV, an immunohistochemical and morphometric study was performed on trephine biopsies of the bone marrow to determine the vascular architecture of the myeloid stroma as well as the occurrence of subendothelial collagen type IV deposits. In comparison with a control group (20 patients), we investigated specimens from 20 patients with polycythaemia vera (P. vera) and 40 patients showing primary (idiopathic) osteomyelofibrosis/-sclerosis (OMF). Morphometric analysis disclosed a significant increase in the number of sinusoids and also in the extent of collagen type IV deposits in the latter disorder. Sinusoids of P. vera showed only a dilatation of their lumina corresponding with enlarged areas of sectioned vascular profiles. Contrary to this finding, in OMF sinusoidal distension was accompanied by an excessive deviation from a circular profile and prominent intravascular haematopoiesis. Furthermore, there was a significant formation of basement membrane collagen (collagen type IV). All these features were far less pronounced in early hypercellular stages of OMF than in the late fibro-osteosclerotic subtypes. Our morphometric findings confirm haemodynamic studies on advanced stages of OMF which revealed an increased vascularity. Moreover, these results are in keeping with the assumption that angiogenesis in the evolution of the fibroosteosclerotic marrow process is mediated by certain growth factors. The increase in collagen type IV formation is thought to be generated by the pronounced endothelial cell proliferation in the course of neovascularization.

Aged↗

The airway resistance of the naso-frontal duct during and after acute frontal sinusitis. A long term follow-up of 49 trephined frontal sinuses.

A new pressure flow method for function tests and calculation of the airway resistance in the naso-frontal duct is presented. With this pressure flow technique the function and airway resistance of the naso-frontal duct was tested in 49 patients who were trephined for acute frontal sinusitis. The tests were performed during the primary healing period (0-16 days) and in a long term study 2, 6 and 12 months after operation. The patients were also checked radiologically and clinically. A prolonged healing period (more than 6 days) corresponded significantly to a high incidence of recurrent disease. With this method it is possible to define groups where recurrences might be expected because of impaired function of the naso-frontal duct.

Adolescent↗

Bone marrow trephine biopsy in infants. European Neuroblastoma Study Group.

Central review of bone marrow trephine biopsies obtained between January 1990 and July 1996 from 282 children with neuroblastoma showed that 18% of cores from older children and an unacceptably high 36% from infants were inadequate (p = 0.0002). Centres should choose their operators for this invasive investigation of infants with more care in order to reduce the failure rate.

Biopsy↗

Optimal processing of bone marrow trephine biopsy: the Hammersmith Protocol.

Specimens of bone marrow trephine biopsy (BMT) are transported and fixed in acetic acid-zinc-formalin fixative, decalcified in 10% formic acid-5% formaldehyde and processed with other specimens to paraffin-wax embedding. Sections, 1-microm-thick, are cut by experienced histotechnologists and used for haematoxylin and eosin, Giemsa, reticulin silver and other histological stains. Further, all immunohistochemical procedures used in the laboratory, including double immunostaining, can be used on these sections with no or minimal modifications. About 10,000 BMT specimens have been analysed using this procedure since 1997 and diseases involving the bone marrow have been classified successfully. More recently, standardised polymerase chain reaction-based analysis and mRNA in situ hybridisation studies have been conducted. Excellent morphology with good antigen, DNA and RNA preservation is offered by the Hammersmith Protocol.

Biopsy↗

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.

Antigens, CD20↗

Quantitative study of the immunoglobulin-containing cells in trephine samples of bone marrow.

Immunoglobulin (Ig) was demonstrated in paraffin sections of 12 trephine bone marrow biopsies by means of the unlabelled antibody peroxidase-antiperoxidase (PAP) method. The Ig-containing cells, which were counted with the Reichert-Jung (Kontron) MOP-AMO(3) user-controlled image-analyser, were found to constitute approximately 4.2% of all the nucleated cells in the marrow, a figure significantly higher than those reported by previous workers.

Bone Marrow↗

Comparison of two bone trephine instruments used for quantitative histomorphometry.

Transilial bone samples were obtained at necropsy using trephine instruments with internal diameters of 2 mm and 7 mm. Undecalcified histological sections were prepared and two structural histomorphometric variables (percentage of trabecular bone volume and surface density of bone) were estimated by a Quantimet 720 image analyser. The differences between the variables measured from the two adjacent cores were due to differences in biopsy site rather than the size of the sample. The smaller diameter needle is therefore recommended for use as the biopsy procedure is simpler and tissue sections can be prepared sooner than is the case with the larger needle.

Adult↗

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.

Adult↗

Immunohistochemical detection of neuroblastoma in frozen sections of bone marrow trephine biopsy specimens.

Frozen sections from bone marrow trephine biopsy specimens from children with disseminated neuroblastoma were stained using the monoclonal antibody UJ13A. An immunoalkaline phosphatase technique was the preferred staining method. Acceptable histological detail was obtained from this material and deposits of tumour cells detected. Some apparently fibrous tissue was also stained by this antibody. The results show that this immunohistological approach is feasible and provide encouragement for its addition to the range of investigations currently available for assessing the marrows of children with this disease.

Antibodies, Monoclonal↗

Dysmegakaryopoiesis in myelodysplastic syndromes (MDS): an immunomorphometric study of bone marrow trephine biopsy specimens.

An immunohistochemical and morphometric analysis was performed on trephine biopsy specimens of the bone marrow in 40 patients (23 men and 17 women, mean age 62 years) with different subtypes of myelodysplastic syndromes (MDS) to determine dysmegakaryopoiesis, but particularly precursor cells--that is, pro- and megakaryoblasts. In 31 of the 40 patients the numbers of megakaryocytes were increased which was associated with a predominance of smaller cell forms (micromegakaryocytes). Compared with periodic acid Schiff, immunostaining with a formalin resistant monoclonal antibody against glycoprotein IIIa (Y2/51(CD61) showed a clinically important proportion of immature elements. These could be designated pro- and megakaryoblasts by taking morphometric measurements on smears and bone marrow sections. There was a relevant increase in the number of promegakaryoblasts in 32 patients, consistent with uncontrolled expansion of the precursor pool. Seventeen repeated bone marrow biopsy specimens taken after chemotherapy largely showed a decrease in the numbers of megakaryocytes including the precursor cell population. Moreover, morphometric evaluation disclosed that micromegakaryocytes in MDS differ significantly from those in chronic myeloid leukaemia (CML) due to distinctive nuclear features and a disturbed nuclear:cytoplasmic ratio. These changes generate a more pleomorphic or atypical appearance of this cell population in MDS, compared with micromegakaryocytes in CML. It is concluded that the disproportionate increase in megakaryocyte precursors and the grossly abnormal aspects of micromegakaryocytes in MDS are characteristics of the severe defect involving haematopoiesis in this disorder.

Adult↗

Effects of prolonged processing in standard and isotonic trichloroacetic acid (TCA) on cellular preservation in bone marrow trephines.

The effects of a combined fixing and decalcifying solution, trichloroacetic acid, on prepared bone marrow specimens were evaluated to try to improve the speed of trephine biopsy processing in our laboratory. The morphological preservation obtained immediately after decalcification was complete was assessed, and also after prolonged immersion in each of two solutions. Trichloracetic acid was made up in the standard manner using distilled water and in an isotonic form by the addition of sodium chloride. Prolonged immersion was performed to assess any possible adverse effects of this acidic solution. By the time decalcification was complete, each solution gave morphological preservation sufficient for diagnostic purposes. However, after prolonged immersion, only the isotonic solution performed adequately.

Biopsy↗

Alveolar rhabdomyosarcoma infiltrating bone marrow at presentation: the value to diagnosis of bone marrow trephine biopsy specimens.

AIMS: To describe the histological appearances of bone marrow infiltrated with rhabdomyosarcoma at presentation and to determine their value in establishing the diagnosis. METHODS: Patients presenting over seven years in the northern health region of England with rhabdomyosarcoma were studied. Bone marrow aspirates and trephine biopsy specimens taken at presentation were examined. RESULTS: Seven of 32 patients with rhabdomyosarcoma had bone marrow infiltration, resulting in marrow failure in all cases, at diagnosis. The diagnosis was established in these seven by the typical cytological appearances and immunophenotype of the infiltrating cells (all seven patients) and cytogenetic abnormalities (three patients). Histological examination of the bone marrow showed a pseudoalveolar pattern with fibrous septal bands, enlarged vascular channels, and lack of cohesion of the tumour cells within the subdivided aggregates in all seven. In four cases multinucleate giant cells, often with peripherally sited nuclei, were found. CONCLUSIONS: These histological features of infiltrated marrow are so characteristic that the diagnosis of alveolar rhabdomyosarcoma can be made, or at least suspected, in many cases even without recourse to technically difficult and expensive further investigations. Bone marrow biopsy should be a routine part of the investigation of patients with bone marrow failure and will be of particular value in the diagnosis of those with disseminated alveolar rhabdomyosarcoma.

Adolescent↗

An enhanced immunocytochemical method for staining bone marrow trephine sections.

AIMS: The detection of cellular antigens in fixed decalcified bone marrow trephine (BMT) sections depends on the method of processing, the nature of the antigen and antibody, antigen retrieval techniques, and the sensitivity of the immunocytochemical method. This study evaluated a tyramide enhanced avidin-biotin immunostaining method on formalin fixed decalcified BMT sections to determine whether the method could detect previously undetectable antigens. METHODS: Nineteen BMT biopsies from a range of haematological disorders were evaluated with 43 antibodies to haemopoietic antigens using horseradish peroxidase and alkaline phosphatase detection methods, using the tyramide enhanced avidin-biotin immunostaining method. RESULTS: Compared with standard avidin-biotin immunostaining methods the tyramide enhanced immunostaining method showed enhanced signal intensity, gave positive labelling for antigens that require pretreatment by other methods, and previously unreactive antigens were detected. Primary antibodies could be used at up to 200 times higher dilutions. CONCLUSION: The tyramide enhanced immunostaining method, while retaining specificity, is highly sensitive and enables an increased number and range of antigens to be detected than previously possible. The method could be applied to BMT sections for the routine diagnosis and classification of haematological disorders.

Antibodies, Monoclonal↗

A panel of antibodies for the immunostaining of Bouin's fixed bone marrow trephine biopsies.

AIMS: To assess a panel of antibodies on Bouin's fixed bone marrow trephine (BMT) biopsies. These biopsies are widely used in routine diagnosis of various haematological malignancies and may be the sole material available in many centres; however, information regarding the immunostaining of this material is lacking. METHODS: Biopsies were taken from 72 patients presenting with various haematological malignancies (leukaemia, 38; lymphoma, 14; multiple myeloma, 20). A panel of antibodies was assessed on Bouin's fixed BMT biopsies by the alkaline phosphatase-antialkaline phosphatase method. RESULTS: Three B (MB2, LN-2, Ki-B5) and two T cell lineage antibodies (UCHL-1, CD3-r) reliably identified lymphoid cells, while MPO-r, Leu-M1/CD15, and KP-1/CD68 recognised cells from the myeloid or histiocytic/macrophage series. Reed-Sternberg cells were stained by LN-2, Leu-M1, and CD30. Antibodies specific for plasma cells (VS38) and hairy cells (DBA.44) gave a variable pattern of staining. Among the proliferation markers, proliferative cell nuclear antigen but not Ki-67 related antibodies were effective. CONCLUSION: This study presents a panel of antibodies with reactivity not restricted to common fixatives that are also suitable for Bouin's fixed BMT biopsies.

Antibodies↗

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.

Base Pairing↗

Trephine bone biopsy system: a refined needle for radiologists.

A new type of bone biopsy unit that uses standard engineering and machinery principles of self cleaning and clearance allows for greater ease and more accurate performance of biopsy. Its use consistently yields excellent biopsy material. Included is a semi-rigid K wire, which may be used with a drill, for ease of insertion and a stylet in a cannula, which allows for multiple biopsies without reinsertion. The needle is compared with standard saw tips (trephines) and sharpened cannulas.

Biopsy, Needle↗

Bone marrow biopsy for the staging of non-Hodgkin's lymphoma: bilateral or unilateral trephine biopsy?

AIM: The occurrence of unilateral involvement in bilateral bone marrow trephine biopsies in non-Hodgkin's lymphomas (NHL) at disease onset (10-20% of cases) has been reported since the early 70s. Therefore, although these studies were based on small series, the use of bilateral bone marrow biopsies has become the rule. However, the clinical value of this procedure has never been clearly established. The aim of the present study was to ascertain the true value of bilateral bone marrow biopsy in the staging of NHL. STUDY DESIGN: We examined 368 cases of NHL (A-H according to the Working Formulation) (WF), without leukemic involvement of the peripheral blood, in order to evaluate: 1) the incidence of unilateral bone marrow involvement; 2) the percentage of patients who, as a result of unilateral bone marrow involvement, changed from stages I-II to stage IV; 3) assessment of response to therapy for patients with both bilateral or unilateral bone marrow involvement. RESULTS: In the A-C NHL groups of WF there was a unilateral bone marrow involvement of 8.8%. Overall, bone marrow involvement induced a change from clinical stages I-II to stage IV in 5.6% of cases, a figure which would correspond to a false negative rate of 2.8%, if unilateral bone marrow biopsy was performed. In the D-F and G, H groups of WF, unilateral involvement was 10.1% and 8.5% respectively; the change in stage from I-II to IV by unilateral bone marrow involvement respectively amounted to 1.4% and 2.8%, which correspond to respective false negative rates of 0.7% and 1.4%. CONCLUSIONS: On the basis of these results and of the present therapeutic strategies, we propose: bilateral bone marrow biopsy for clinical stages I-II of all NHL; no bone marrow biopsy at disease onset for clinical stages III and IV of A to H histologic subtypes of the WF; unilateral bone marrow biopsy (A-C subtypes of the WF) or bilateral (D-H of the WF), after the regression of extramedullary localizations.

Antineoplastic Combined Chemotherapy Protocols↗

The Rochester bone trephine for small joint arthrodesis in the foot.

The Rochester bone trephine is a useful instrument for performing small joint arthrodesis in the foot. The technique involves harvesting a dowel graft from the iliac crest and then inserting the graft into a recipient bed across the small joints in the foot. Minimal surgical trauma and patient discomfort result from this technique. Three cases are presented to illustrate the usefulness and diversity of this system.

Adult↗