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Three-dimensional reconstruction of non-Hodgkin's lymphoma in bone marrow trephines.

The objective of this study was to analyse the location in the bone marrow of deposits of low-grade non-Hodgkin's lymphoma. This was achieved using computer-generated three-dimensional reconstruction techniques applied to serial tissue sections of five bone marrow trephines involved by lymphoma. For comparative purposes, previously published three-dimensional models of benign lymphoid aggregates in the bone marrow were need. The images generated by this new study showed that deposits of low-grade non-Hodgkin's lymphoma involving the bone marrow always assumed a paratrabecular pattern of infiltration at some point. This is in direct contrast to the pattern of bone marrow infiltration shown by benign lymphoid aggregates. It is concluded that location within the marrow space is a crucial factor in distinguishing between benign and low-grade malignant lymphoid infiltrates in the bone marrow.

Biopsy↗

Detection of the bcr/abl gene in bone marrow macrophages in CML and alterations during interferon therapy--a fluorescence in situ hybridization study on trephine biopsies.

A fluorescence in situ hybridization (FISH) study was performed on trephine biopsies of the bone marrow in chronic myelogenous leukaemia (CML) to evaluate the bcr/abl translocation in macrophages before and during interferon (IFN) therapy. Mature macrophages and osteoclasts were identified by the monoclonal antibody PG-M1, which recognizes a fixative-resistant epitope of the CD68 molecule. In contrast to a control group, in 145 of 479 (30 per cent) macrophages of the CML bone marrow, a positive fusion signal was found, together with corresponding translocation sites in the surrounding myeloid cells. In patients following IFN treatment for approximately 12 months and with haematological/cytogenetic remission by clinical standards, this number was reduced to 10 of 136 (9 per cent) macrophages. In addition, the few multinucleated osteoclasts of the untreated CML bone marrow displayed positive translocation spots. This finding supports the hypothesis that stromal macrophages and osteoclasts are of haematogenic stem cell origin. Moreover, clinical remission and reduction of bcr/abl-positive macrophages under IFN therapy lend support to the hypothesis that the presence of malignant stromal macrophages may contribute to the selective expansion of leukaemic precursors and the suppression of normal haematopoiesis in CML.

Antigens, CD↗

Bone marrow examination in small cell carcinoma of the lung. Comparison of trephine biopsy with aspiration.

Bone marrow examinations in 146 patients with small cell carcinoma of the lung were reviewed. The results of trephine biopsy and aspiration were compared in 128 patients in whom both techniques were performed. Twenty-seven of the 128 examinations (21.2%) showed positive aspirates and 30 biopsies (23.4%) revealed metastatic disease. The overall agreement between the aspirates and biopsies was 96.1%. Thirty-one patients (24.2%) had metastatic tumor in either aspirate or biopsy: 4 of these (12.9%) had a positive biopsy with negative aspirate, and 1 (3.2%) showed a positive aspirate and negative biopsy. The Jamshidi needle was used, yielding an average of 4.1 cm of core material per biopsy. The authors conclude that bone marrow biopsy using the Jamshidi needle is equivalent to aspiration. Although both procedures are complementary, a satisfactory specimen obtained with the Jamshidi needle alone may yield nearly equal detection to the combined procedures. Some guidelines for obtaining an optimal biopsy specimen are suggested.

Biopsy↗

Trephine needle bone marrow biopsy in the initial staging of Hodgkin disease: sensitivity and specificity of the Ann Arbor staging procedure criteria.

The purpose of this study was to test the value of the Ann Arbor staging procedures committee criteria in defining a group of newly diagnosed patients with Hodgkin disease who do not have involvement of the bone marrow and do not need this procedure performed. One hundred sixty-six bilateral and 16 unilateral trephine bone marrow biopsies were performed in a consecutive series of 182 patients undergoing initial staging for Hodgkin disease. Bone marrow involvement was found in 13 patients. Advanced stage, defined as stage III or IV, occurring in 92%, anemia occurring in 100%, and "B" symptoms present in 100% were found to be the most sensitive indicators for bone marrow involvement. Osseous disease (99%), platelet count less than 150,000/mm3 (98%), and WBC less than 4,800/mm3 (94%) were the most specific parameters. The known association of bone marrow involvement with older patients, lymphocyte depleted histology, lower blood cell counts, anemia, advanced stage, and poorer survival is verified. The Ann Arbor staging procedures committee criteria for performing a bone marrow biopsy were shown to be 100% sensitive and 40% specific. Use of these criteria would have found all 13 patients with bone marrow involvement. Of the 73 patients who did not meet the criteria, no patient had bone marrow involvement. Use of the Ann Arbor staging procedures committee criteria is recommended.

Adult↗

A study of cell proliferation in formalin-fixed, wax-embedded bone marrow trephine biopsies using the monoclonal antibody PC10, reactive with proliferating cell nuclear antigen (PCNA).

We have investigated proliferation in bone marrow trephine biopsies from 32 patients with normal or abnormal haemopoiesis, using the monoclonal antibody PC10, which detects proliferating cell nuclear antigen (PCNA), together with immunohistochemical markers of haemopoietic cell lineage. PCNA immunostaining revealed the pattern of proliferation within individual haemopoietic lineages in normal marrow. Two unexpected observations were made: of erythroid cells, only pro-erythroblasts and occasional early normoblasts reacted, and positivity of megakaryocytes was unrelated to nuclear lobulation or CD61 expression. The pathological cases represented conditions in which haemopoiesis is increased (reactive hyperplasia, chronic granulocytic leukaemia, myeloproliferative and myelodysplastic syndromes, megaloblastic anaemia). Increases in the number, and disturbances of the spatial organization, of PCNA-expressing cells were present to a variable extent in all cases. Sheets of PCNA-positive megaloblastoid erythrocytes were frequently found in myelodysplastic and myeloproliferative tissue, associated with marked disturbances in the spatial organization of all haemopoietic lineages. Cases of megaloblastic anaemia due to vitamin B12/folate deficiency also demonstrated greatly increased erythroid PCNA expression, with positivity in some giant metamyelocytes. In addition to reflecting increased proliferation, elevated PCNA expression in some bone marrow pathologies may be due to altered kinetics of the protein induced by disturbances in growth factor production.

Autoantigens↗

Microwave histoprocessing of bone marrow trephine biopsies.

In recent years, the microwave oven has been increasingly used in the pathology laboratory for processing of tissue for diagnostic purposes with a remarkable reduction in processing time and also reports of excellent morphology and immunohistochemistry. We evaluated some of these processes on post mortem bone marrow trephine biopsies and describe a novel way of processing these biopsies in the microwave oven.

Biopsy↗

Lectin histochemistry of human bone marrow: investigation of trephine biopsy specimens in normal and reactive states and neoplastic disorders.

The lectin binding pattern of bone marrow cells in normal and reactive states and in various neoplastic disorders was investigated using trephine biopsy specimens taken from the iliac crest. The tissue samples were routinely processed (fixed in formalin and embedded in paraffin wax) and subjected to mild decalcification with EDTA. The following results were obtained. (1) More than half of the 23 fluoresceinated lectins used reacted with normal blood cells and/or their neoplastic derivatives. Inhibition tests with the appropriate sugars confirmed the specificity of binding for the majority, but not all, of the lectins. (2) WGA, Con A, PSA, STA and RCA60 and RCA120 produced a particularly intense reaction with normal, reactive and neoplastic myeloid cells. Erythroblasts exhibited weak staining in a few cases by a few lectins (WGA producing the strongest staining), while megakaryocytes nearly always remained unstained. Neoplastic lymphoid cells in various lymphoproliferative disorders and plasmacytoma cells generally reacted with the same lectins as the myeloid cells. (3) Since neoplastic myeloid cells in various myelodysplastic and myeloproliferative disorders exhibited a lectin binding pattern similar to that of myeloid cells in normal and reactive bone marrow, it is unlikely that lectin histochemistry of the bone marrow will prove of great value in the diagnosis of myelodysplastic-myeloproliferative disorders.

Blood Cells↗

Macrophages in normal human bone marrow and in chronic myeloproliferative disorders: an immunohistochemical and morphometric study by a new monoclonal antibody (PG-M1) on trephine biopsies.

An immunohistochemical and morphometric study was performed on routinely processed trephine biopsies of the bone marrow in 30 normal individuals and in 90 patients with various subtypes of chronic myeloproliferative disorder. Using a new monoclonal antibody (PG-M1) directed against a formalin-resistant epitope on macrophages and by employment of the Prussian blue reaction, quantitation of this cell population was feasible. Morphometric analysis revealed that the number of iron-laden macrophages represented only a fraction of the total number of histiocytic reticular cells. As could be expected, in polycythaemia rubra vera, no haemosiderin deposits were detectable, but the content of macrophages slightly exceeded that of the normal bone marrow. In chronic myeloid leukaemia 9 of 30 patients showed a significant increase in PG-M1-positive reticular cell elements. These were consistent with pseudo-Gaucher cells, sea-blue histiocytes and intermediate cell types. Primary (idiopathic) myelofibrosis-osteomyelosclerosis was characterized by a significant increase in macrophages (25 of 30 patients). Involvement of macrophages in the complex mechanisms generating bone marrow fibrosis and angiogenesis and in bone remodelling (osteosclerosis) may be responsible for this finding.

Adipose Tissue↗

The impact of interferon versus busulfan therapy on the reticulin stain-measured fibrosis in CML--a comparative morphometric study on sequential trephine biopsies.

To evaluate treatment-related changes of the reticulin stain-measured fibrosis in Ph(1+)-CML, a clinicopathological study was performed on sequential trephine biopsies of the bone marrow following either interferon (IFN) or busulfan (BU) monotherapy. Using the monoclonal antibody CD61 for the identification of megakaryopoiesis and Gomori's silver impregnation method, number of megakaryocytes and density of argyrophilic (reticulin and collagen) fibers were determined by morphometry. We studied specimens from 26 patients with IFN-alpha 2b (including nine patients with additional IFN gamma) therapy and from 23 patients who had received BU. In both groups, repeated bone marrow biopsies (total 125) revealed a significant increase in the fiber content, as well as in the number of megakaryocytes during treatment. To assess the dynamics of myelofibrosis more precisely, computation of differences in the degree of fiber density between the first and last examination was carried out. Regarding the considerable variations in the biopsy intervals, a so-called myelofibrosis progression index (MPI) was calculated. Following this rationale, we were able to demonstrate that, in comparison to the BU-group, speed of progression of bone marrow fibrosis was significantly increased in CML patients treated with IFN. Preliminary statistical analysis indicated a relationship between myelofibrosis on admission, which was always associated with increased growth of megakaryocytes, and the MPI with survival. Even when these parameters were regarded, prognosis was significantly more favorable in the IFN-treated patients. The failure of IFN and BU to inhibit the evolution of myelofibrosis may be related to several conversely acting pathomechanisms. Among others, the inability of both therapeutic agents to reduce the number of megakaryocytes more effectively should be taken into consideration.

Adolescent↗

Histological features of prognostic significance in CML--an immunohistochemical and morphometric study (multivariate regression analysis) on trephine biopsies of the bone marrow.

To evaluate the prognostic significance of clinical as well as histological disease features at the time of diagnosis, an immunohistochemical and morphometric study was performed on bone marrow trephine biopsies in 130 patients with Ph(1+)-CML. For identification of all cell elements of the megakaryocytopoiesis we used the monoclonal antibody CD61 (Y2/51) and for the macrophages, the recently characterized antibody PG-M1. Density of argyrophilic fibers was determined per fat cell-free marrow area. Based on a multivariate analysis-derived risk model, the reproducibility of the prognostic score described by Sokal and co-workers was tested, particularly with regard to histological variables. Additionally, we calculated the disease-specific loss in life expectancy. Our prognostic model (Cox model) consisted of the variables: age, spleen size, peripheral erythro-normoblasts, pseudo-Gaucher cells, and fiber density. To assess the validity of this new CML score, a receiver-operating curve (ROC) of sensitivity and specificity was constructed. The improved prognostic efficiency of this newly developed risk model in predicting death within 3 years after diagnosis of CML was demonstrated in comparison with generally accepted staging systems. Immunohistochemistry revealed that not the total number of macrophages, but only the subfraction of pseudo-Gaucher cells exerted a significant impact on survival. Furthermore, it was feasible to calculate the number of atypical micromegakaryocytes and pro- and megakaryoblasts. This abnormal and immature cell population showed a significant correlation with fiber density and prognosis. Finally, the practical value of the Hannover classification was tested. This histological classification enabled a discrimination between two groups with different survival patterns, i.e., granulocyte and/or megakaryocyte-rich subtypes versus subtypes with increase in reticulin and collagen fibers.

Adolescent↗

Atypical micromegakaryocytes, promegakaryoblasts and megakaryoblasts: a critical evaluation by immunohistochemistry, cytochemistry and morphometry of bone marrow trephines in chronic myeloid leukemia and myelodysplastic syndromes.

A morphometric analysis of bone marrow trephine biopsies has been performed to study the frequency and planimetric characteristics of so-called atypical micromegakaryocytes in chronic myeloid leukemia (CML) and myelodysplastic syndromes (MDS). In addition, an attempt was made to discriminate this particular cell population from small immature elements of megakaryocytopoiesis, such as promegakaryoblasts and megakaryoblasts. The staining reactions employed included periodic acid-Schiff (PAS), alpha-naphthyl acetate esterase (ANAE) and immunohistochemistry with a monoclonal antibody against platelet glycoprotein IIIa (Y2/51-CD61). Comparison of the various staining reactions applied to the different megakaryocytic elements together with morphometric measurements resulted in a clearcut identification of promegakaryoblasts. These were defined as the earliest immature and exclusively CD61-positive precursors. Atypical micromegakaryocytes were characterized by their dysplastic features and strong ANAE reactivity in addition to their positive CD61 staining. When stringent diagnostic criteria (diameter ranging between 10 to 15 microns, mean size about 12 microns) were applied, this abnormal cell population comprised less than 10% of total megakaryocytopoiesis in CML and MDS. It may be assumed that dysmegakaryocytic features in the latter disorders are partially generated by small to medium-sized megakaryocytes (diameter less than 30 microns). In conclusion, the relative frequency of promegakaryoblasts in the normal bone marrow (range 6-8%) is confirmed by evaluation of the immunohistochemical and cytochemical staining methods (CD61 and ANAE). Furthermore, the ANAE reaction facilitates the recognition of atypical micromegakaryocytes as well as small megakaryocytes. Thus cytochemistry provides a better insight into alterations of these cell lineages in various pathological conditions.

Adult↗

Increased cell apoptosis in bone marrow trephine biopsies and immunomagnetically isolated myeloid progenitor cells in patients with chronic idiopathic neutropenia.

The frequency of apoptotic cells in bone marrow trephine biopsies and cytospins of immunomagnetically isolated myeloid progenitor cells was determined in 39 patients with chronic idiopathic neutropenia (CIN) and 12 hematologically normal individuals using the in situ end-labeling (ISEL) apoptosis detection method. We found that 66.7% of the patients but none of the normal controls displayed apoptotic cells equal to or higher than 5% of the total mononuclear cells in bone marrow biopsies (p<0.01). In the double stain, we also found that the proportion of apoptotic CD15(+) myeloid precursor cells did not differ significantly between patients and control subjects, while the proportion of apoptotic CD34(+) hemopoietic cells could not be estimated with accuracy because of the presence of CD34(+) endothelial cells. Significantly increased apoptosis was noted in cytospins of immunomagnetically isolated patient CD34(+) and CD34(+)/CD33(+) cells but not CD34(-)/CD33(+) cells, compared to the controls ( p<0.001, p<0.02 and p>0.05, respectively). These findings confirm and extend our previous observations in flow-cytometric studies of apoptosis in CIN, indicating that increased apoptosis in CIN bone marrow concerns mainly the CD34(+) and CD34(+)/CD33(+) progenitor cell compartments. We conclude that the accelerated apoptosis in these compartments may account for the impaired neutrophil production in CIN patients.

Adult↗

Syndrome of the trephined: theory and facts.

The pathophysiology behind "the syndrome of the trephined" has been under investigation over the past 50 years. Research related to barometric pressure, cellular metabolism, cerebrospinal fluid (CSF) dynamics, and the vasculature have attempted to decipher the mechanism of disease. These subjects are discussed in five papers along with specific topics related to the syndrome. The symptoms experienced after craniectomy, the resolution of symptoms with cranioplasty as well as CSF, cerebral blood flow, and metabolic studies are presented, respectively, with a review of the theories.

Journal Article↗

Controlled nail trephination for subungual hematoma.

An injury to a finger or toe can result in a collection of blood under the nail plate that if unrelieved can cause extreme discomfort due to pressure. In this case, a 47-year-old man developed a subungual hematoma of the right thumb due to a crush injury. Controlled nail trephination was performed using a uniquely designed drill that penetrated the nail plate without breaching the nail bed. The subungual hematoma was successfully drained, and there was a substantial relief in pain over the subsequent 8 hours. This technique appears to be a quick and convenient method of evacuating subungual hematomas with minimal discomfort and minimal risk.

Drainage↗

Overexpression of delta-like (Dlk) in a subset of myelodysplastic syndrome bone marrow trephines.

The mRNA expression level of the gene delta-like (Dlk), coding for a signal transducer related to the Delta-Notch family, was measured using real-time PCR methodology in a large series of control biopsies (n = 61) and bone marrow trephines from patients with myelodysplastic syndrome (MDS) (n = 93) and related myeloid disorders (AML, n = 16 and chronic myeloproliferative disease (CMPD), n = 38). It turned out that dlk is strongly overexpressed in a subset of MDS (16%) cases, however with higher frequency in blast rich MDS cases. Therefore, the quantitative detection of dlk mRNA can support the morphological diagnosis of MDS in selected cases.

Bone Marrow Examination↗

Changes of axial length and keratometry after keratoplasty for keratoconus using the guided trephine system.

PURPOSE: The purpose of this study was to examine the cause of increasing myopia after penetrating keratoplasty for keratoconus with the guided trephine system. DESIGN: Prospective interventional study cohort. METHODS: Thirty eyes (28 patients) after keratoplasty for keratoconus were examined. Preoperatively and 1, 3, 6, 12, and 24 months postoperatively subjective refraction was evaluated. Keratometry was calculated with a computerized videokeratoscope (TMS-1). Axial length was measured using applanation ultrasonography before surgery and 2 years after surgery. Anterior chamber depth, lens thickness, and vitreous length were taken into consideration. RESULTS: The mean spherical equivalent was + 2.22 +/- 3.47 diopters 1 month postoperatively and had a continuous myopic shift to -1.02 +/- 2.65 diopters 2 years postoperatively. This was associated with a significant increase in mean keratometric levels from + 41.72 +/- 2.96 diopters 1 month postoperatively to + 43.77 +/- 2.29 diopters 2 years postoperatively (r(s) = -0.36, P =.05). Overall, no significant changes in axial length were observed. However, vitreous length showed a small but statistically significant increase. As expected, mean anterior chamber depth decreased significantly postoperatively (P </=.05). In eyes with a decrease in keratometric values (n = 8) a myopic shift was also observed. In this group a significant elongation of the axial length and the vitreous length was found. CONCLUSIONS: A study of eyes after keratoplasty for keratoconus demonstrated continuous myopic shift over time. Increasing myopia was associated with increasing keratometric values. Thus, the major cause of increasing myopia after keratoplasty in keratoconus eyes seems to be continuing keratectasia. However, ongoing bulbus growth may contribute to continuing myopia in some eyes.

Adult↗

Internal sclerectomy with an automated trephine for advanced glaucoma.

An automated trephine (trabecuphine) was used to perform an internal sclerectomy in seven glaucoma patients who were aphakic or had undergone previous filtering surgery that had failed or both. A patent fistula was achieved intraoperatively in all seven eyes. Postoperatively, six patients received subconjunctival injection of 5-fluorouracil (5-FI) once daily for an average of 8 days. Five of seven patients have retained a functional bleb and a controlled intraocular pressure (IOP) after surgery (follow-up, 4-24 months). The only intraoperative complication was hemorrhage from the sclerectomy site in a patient with aniridia that resulted in a 20% hyphema. The hyphema cleared quickly, and the bleb has remained functional with a pressure of 12 mmHg for 9 months. The trabecuphine makes it possible to perform a glaucoma filtering operation safely from within the anterior chamber. This technique minimizes conjunctival trauma in the filtration area. The absence of a conjunctival incision overlying the fistula simplifies the adjunctive use of antimetabolites such as 5-FU.

Choroid↗

Osteoclasts and bone remodeling in chronic myeloproliferative disorders. A histochemical and morphometric study on trephine biopsies in 165 patients.

In 165 patients with chronic myeloproliferative disorders (CMPD) a morphometric and histochemical study was performed on trephine biopsies of the bone marrow to elucidate osseous remodeling by assessment of trabecular bone area (planimetry) and number of osteoclasts. Osteoclastic elements were identified by the tartrate-resistant acid phosphatase method. In addition to control specimens (n = 20) subtypes of CMPD included chronic myeloid leukemia (CML, n = 65), primary (essential) thrombocythemia (PTH, n = 25), polycythemia vera rubra (P. vera, n = 25) and agnogenic myeloid metaplasia (AMM, n = 50). AMM was discriminated into a so-called early hyperplastic stage without gross myelofibrosis (n = 19) and an overt or advanced stage showing fibro-osteosclerotic changes (n = 31). Total area of trabecular bone and counts for osteoclasts (uni- and multi-nucleated cells as well as a-nuclear cytoplasmic fragments) were not significantly increased in CML, PTH, P. vera and in the initial hypercellular stages of AMM. In contrast to these results, in advanced stages of AMM there was a significant increase in total bone area associated with a high count for all osteoclastic elements and apparently also an increased number of osteoblasts. It is speculated that the marked increase in osteoclastic-osteoblastic elements in late stages of AMM possibly reflects an imbalance of calcitriol (1.25-dihydroxyvitamin D 3) on skeletal homeostasis. This abnormal osseous remodeling may be mediated by the atypical megakaryocytic proliferation in this disorder, which is always a conspicuous feature of bone marrow biopsies.

Biopsy↗