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Bone marrow biopsy instead of 'marrow juice' for cell kinetic analysis. Comparison of bone marrow biopsy and aspiration material.

Since cell kinetic bone marrow studies have so far exclusively been carried out on aspiration material and have yielded inconsistent or even contradictory results, we investigated the adequacy and reliability of aspirates for cell kinetic analyses in comparison to biopsies. Paired samples of bone marrow (133) were taken simultaneously by aspiration and Jamshidi biopsy from 48 patients with acute leukemias and 67 patients with non-leukemic disorders. Cell kinetic analysis by (1) flow cytometry (FCM) of cellular DNA and RNA content, (2) autoradiography for [3H]TdR pulse labelling indices and (3) liquid scintillation counting of [3H]TdR uptake revealed significantly higher values in biopsies (p less than 0.001) exceeding the corresponding results from aspirates on average by factors of 1.65 for FCM S-phase index, 1.90 for G0/1 cells with high RNA content, 1.82 for [3H]TdR LI and 1.90 for [3H]TdR uptake. In more than 70% of all samples results from biopsies were 1.1-11.4 times higher, indicating that aspirates were equivalent to biopsies in fewer tan 30% of cases. Cell kinetic analysis in vitro blood/biopsy mixtures and measurements of DNA synthesis rate in corresponding aspirates and biopsies revealed that these discrepancies are due to the contamination of aspirates with non-proliferating nucleated blood cells. Biopsy, however, was found to provide representative and reproducible sampling of marrow for cell kinetic studies and should replace the presently used aspirate already characterized as "unreliable marrow juice" by Dameshek et al. in 1937 [18].

Acute Disease↗

[Effect of marrow cavity curettage on bone marrow stromal cell precursors].

Changes in the number of bone marrow stromal precursor cells of guinea pigs after the bone marrow curettage was studied by cloning in monolayer cultures in vitro. About half of the colony-forming units (CEU-F) is extracted during curettage. Later the number of CFU-F in the curetted bone marrow continues to fall, reaching its minimum in 12 hours. Regeneration begins after 24 hours, and on the 7th--12th day the CFU-F content in the curetted bone marrow reacts the normal level; by the 20th day the number of CFU-F exceeded the normal 2.5 times. The CFU-F content in the contralateral bones of donors exceeded their normal content (at periods from 6 hours to 20 days) 2--2 1/2-fold.

Animals↗

Bone marrow 'boosts' following T cell-depleted haploidentical bone marrow transplantation.

Nine patients transplanted for non-malignant conditions (eight severe combined immunodeficiency, one aplastic anemia) received lectin-treated T cell-depleted BMTs from haploidentical donors. Each patient subsequently received a second T cell-depleted transplant (¿boost') from the same donor, without conditioning, because of a delay in the recovery of T cell immunity associated with evidence of engraftment. The median time to boost after initial BMT was 0.5 years (range 0.2-4.6 years). No conditioning therapy was used prior to the boost (except one patient who received ATG) and no GVHD prophylaxis was used during either the initial or subsequent BMTs. Eight of the nine patients are surviving at a median follow-up of 2.9 years (range 0.3-6.2 years). Following BMT boost, T cell function improved with the lymphocyte proliferative responses to PHA, PWM and alloantigen all increasing at least eight-fold. The mean percentage of CD3+ lymphocytes increased from 20 +/- 7% of total lymphocytes following the first BMT to 66 +/- 7% following the marrow boost (P < 0.001). This increase was generated primarily by an increase in the CD4+ lymphocyte subset which increased from 13 +/- 3% post-transplant to 44 +/- 6% after the BMT boost (P < 0.005), and was reflected in both the CD4+/Leu8+ and CD4+/Leu8- lymphocyte populations. Measurements of B cell immunity (immunoglobulins, isohemagglutinins and B cells) showed no significant effect of the marrow boosts. These results suggest that bone marrow 'boosts' are an effective means for improving T cell immunity in patients who fail to recover adequate immune function after T cell-depleted bone marrow transplantation and may be applicable as immunotherapy following allogeneic BMT undertaken to treat malignancy.

Anemia, Aplastic↗

Late taste disorders in bone marrow transplantation: clinical evaluation with taste solutions in autologous and allogeneic bone marrow recipients.

The aim of this work was to determine the type and the significance of taste disorders in allogeneic bone marrow transplanted patients. In a retrospective study the taste threshold of a cohort of 15 allogeneic bone marrow transplanted patients, 4-51 months after transplantation (mean: 30.6 +/- 15.8), was compared to the taste threshold of 8 autologous bone marrow recipients, 4-48 months after transplantation (mean: 24.12 +/- 12.18), and to the taste threshold of a group of 20 consecutive normal subjects. Allogeneic bone marrow transplanted patients showed a significant hypogeusia for salt (Pearson's chi square p = 0.0002; Yates' correction p = 0.0007) and sour (Pearson's chi square p = 0.001; Yates' correction p = 0.008). No significant variations were observed for sweet and bitter. Autologous bone marrow recipients did not show any significant variation of taste acuity for sweet, salt or sour; a constant reduction of the taste threshold for bitter was observed, but the values were not significantly different from normal (Pearson's chi square p = 0.47; Yates' correction p = 0.83). So, late and selective taste disorders are observed in allogeneic bone marrow transplanted patients. Since the severity of the disorders is not strictly related to the severity of chronic oral G.V.H.D., taste analysis could discover the slightest, clinically undetectable cases of chronic oral G.V.H.D. The mechanism of immune aggression on the sensorial taste cells is poorly understood. Further trials are needed to define variations of taste acuity not only after allogeneic bone marrow transplantation, but also in systemic immune diseases.

Adolescent↗

Long duration of erythrocyte hypoplasia after bone marrow transplantation.

Bone marrow transplantation was performed on a 15 year old girl with chronic myelogenous leukemia. The bone marrow was obtained from her younger sister, who was human leukocyte antigen haplo-identical but major ABO incompatible. As a result, the condition of pure red cell aplasia (PRCA) persisted over a long period of time. In order to overcome major ABO incompatibility, erythrocytes were eliminated from the bone marrow graft before transplantation, and methotrexate and cyclosporine (CsA) were used to prevent graft-versus-host disease (GVHD). Administration of erythropoietin proved ineffective. B19 parvovirus infection could not be detected during that time. Agglutinin titers decreased to less than fourfold in parallel with the recovery of erythrocytes. Reports on similar PRCA have been limited to cases of transplantation with ABO incompatibility and cases where CsA was administered to prevent GVHD. This suggests that ABO incompatibility and CsA might be related to the development of PRCA.

ABO Blood-Group System↗

Philadelphia chromosome positive acute lymphoblastic leukemia masquerading as persistent asymptomatic bone marrow necrosis.

Bone marrow necrosis is an uncommon histologic finding of malignant diseases at presentation. Clinically it is characterized by pyrexia and severe bone pain, and heralds a poor prognosis if untreated. We report an unusual patient with asymptomatic bone marrow necrosis persisting for 10 mths, a hitherto undescribed length of time for this condition. The diagnosis of Philadelphia chromosome positive acute lymphoblastic leukemia was finally made after multiple bone marrow biopsies.

Aged↗

Bone marrow transplantation with major ABO incompatibility. Experience of the Bone Marrow Transplantation Unit--Francisco Gentil Portuguese Institute of Oncology, Lisbon Centre.

Thirteen patients submitted to bone marrow transplantation (BMT), HLA A and B identical, with mixed negative lymphocyte culture and major ABO incompatibility were retrospectively evaluated. Gravity sedimentation by hydroxyethyl starch was used in almost all cases to deplete erythrocytes from bone marrow (12/13): a removal rate of 90%--corresponding to an erythrocyte residual volume of 27,4 rate ml--and a nucleated cell recovery of 76% (mean values) were obtained. All patients underwent a hyperhydration regimen and received corticosteroid prophylaxis before bone marrow infusion. Considering the most important immunohematological problems associated to this type of transplant, we verified that seven patients developed minor complications and among them only one had long term consequences. In comparison with the control group, ABO identical and with minor incompatibility, the study group had delayed onset of erythropoiesis and needed greater erythrocyte transfusion support. We concluded that major ABO incompatibility does not constitute a drawback to BMT success in most patients.

ABO Blood-Group System↗

Frequency and indication of unrelated bone marrow transplants in Europe. European Group of Bone Marrow Transplantation (EBMT).

In 1991, 4,961 bone marrow transplants (BMT) were performed in Europe by 171 teams in 21 countries. 217 were from an unrelated donor. Two years ago, this number was 99, five years ago less than ten. Main indications for unrelated BMT were CML (115), AML (32), ALL (24), SAA (19), MDS (10). Major differences exist in the European countries with regard to the number of BMT performed per number of inhabitants and with regard to indications. The present study documents the marked increased in the use of unrelated BMT in Europe. It illustrates the need for health care strategies and for clearer definitions of indications.

Acute Disease↗

Evidence for the persistence of paramyxoviruses in human bone marrows.

Human bone marrow cells derived from multiple sclerosis (MS) and control patients were screened for a number of virus antigens by the fluorescent antibody technique using monoclonal antibodies. The results showed that antigens of the paramyxovirus, simian virus 5, were present in about 60% of MS and 25% of control bone marrows. About 25% of the MS and 50% of control bone marrows were found to contain nucleoprotein antigen of the human parainfluenza types 1 and 3. These experiments demonstrated that paramyxoviruses can persist in human tissues possibly in a defective or repressed state.

Animals↗

Use of DNA polymorphisms to monitor engraftment after allogeneic bone marrow transplantation.

Bone marrow transplantation (BMT) has been established as a life-saving procedure in hematologic malignancies and bone marrow failure syndromes, and it may be valuable in other types of neoplastic disease. DNA polymorphisms are used to monitor engraftment after transplantation from a related or unrelated donor. DNA polymorphisms are not useful after autologous BMT or if the donor is an identical twin. The most valuable polymorphism for this purpose is caused by variation in certain repeated sequences that are known as variable number of tandem repeats (VNTR). The VNTRs are valuable because they each have several alleles increasing the probability of finding one that is useful in a given case. This method can be used to sensitively detect small amounts of residual recipient hematopoiesis. To accomplish this the laboratory must first find a polymorphic allele that is unique in the recipient. Detection of the unique allele in peripheral blood or bone marrow after BMT is tantamount to finding recipient hematopoiesis. The presence of both donor and recipient hematopoiesis can result in a state of stable mixed chimerism and not necessarily presage a relapse after BMT for leukemia; however, the presence of residual recipient cells in some cases may indicate an increased probability of relapse, particularly in chronic myelogenous leukemia.

Bone Marrow Transplantation↗

[Hepatic complications of bone marrow transplantation].

Bone marrow transplantation has become an accepted procedure for the treatment of severe aplastic anemia, hematologic malignancies (particularly lymphoma and leukemia) and certain inborn errors of metabolism and immunity. However, numerous complications follow bone marrow transplantation. Liver disease is a very common and complex complication after human transplantation. The major complications are: veno-occlusive disease, graft-versus-host disease acute and/or chronic. In this decade, the results of bone marrow transplantation will enhance.

Bone Marrow Transplantation↗

Haemopoiesis in mammalian bone marrow.

The bone marrow supports haemopoiesis of all blood cell types and delivers mature cells to the blood. Haemopoiesis is characterized not only by the differentiation and proliferation of haemopoietic stem cells but by a number of physically associated cell types. These include macrophages, lymphocytes and, when haemopoiesis is intense, a multinucleate branched stromal cell. The venous vasculature of the bone marrow is associated with both haemopoiesis and the delivery of blood cells to the circulation. The wall of the vascular sinus consists of an endothelium lying upon a basement membrane. On the outside surface of the basement membrane lie adventitial cells or pericytes which branch out into the haemopoietic space forming a scaffolding upon which haemopoietic clusters are arranged. These cells move away from the wall of the vascular sinus to permit maturing blood cells to penetrate the endothelium and enter the circulation. Under other circumstances, adventitial cells accumulate fat, becoming the adipocytes of marrow.

Animals↗

The morphology of hematopoietic layers in long-term cultures of mouse bone marrow.

Mouse bone marrow cells in long-term culture were examined with scanning electron microscopy during the first 10 days of growth and with phase contrast microscopy during the first 4 weeks. The development of stroma and hematopoiesis was studied, and phase microscopic observation was used in order to achieve positive cell identifications with scanning EM. We analyzed those cell populations that could not be washed away from the adherent culture layer. These adherent cells in 24-hour cultures contain the full potential of hematopoietic long-term production. Stromal cells started to spread almost immediately and by 5 days had established several layers. Although in early cultures hematopoietic cells were found resting on the surfaces of stromal cells, they were later packed between stromal layers. The blast cells, especially, were usually buried under and between thin sheets of reticulum cells. The study confirms the three-dimensional nature of bone marrow in culture and points to close correspondence with bone marrow structure as studied by others in vivo.

Animals↗

Selective secretion of chemoattractants for haemopoietic progenitor cells by bone marrow endothelial cells: a possible role in homing of haemopoietic progenitor cells to bone marrow.

To elucidate the mechanisms by which haemopoietic progenitor cells lodge in the bone marrow, we examined the secretion of chemoattractants for haemopoietic progenitor cells by bone marrow and lung endothelial cells. The bone marrow endothelial cells, but not lung endothelial cells, secreted chemoattractants for the haemopoietic progenitor cell line, FDCP-2, and normal haemopoietic progenitor cells. Checkerboard analysis demonstrated that the conditioned medium of the bone marrow endothelial cells had chemotactic activity and random motility-stimulating activity. The bone marrow endothelial cells expressed stromal-cell-derived factor-1 (SDF-1) mRNA and produced SDF-1 protein, whereas the lung endothelial cells did not. Adhesion of FDCP-2 cells to the bone marrow endothelial cells was partially inhibited by anti-SDF-1 antibody. These findings suggest that the chemoattractants for haemopoietic progenitor cells including SDF-1 and random motility-stimulating factor(s) selectively secreted by the bone marrow endothelial cells may contribute to the homing of haemopoietic progenitor cells to bone marrow.

Animals↗

IL-3-dependent growth of basophil-like cells and mastlike cells from human bone marrow.

Human bone marrow cultured in the presence of human rIL-3 has been reported to give rise to basophils. In contrast, mouse bone marrow, cultured in the presence of mouse IL-3, leads to the growth of mast cells. To determine if human rIL-3 might also stimulate the growth of human mast cells, we cultured human bone marrow in the presence of human rIL-3 in suspension cultures, methylcellulose, and in "interphase" cultures where cells are layered over agar. The presence of mast cells was determined using a variety of histochemical techniques. In agreement with previous reports, basophil-like cells were identified in all culture systems. Mastlike cells were identified only in interphase cultures. By 3 wk, such cultures consisted of basophil-like cells (20 to 50%) and mastlike cells (1 to 5%). Cultures supplemented with rIL-4 showed no additional increase in basophil-like and mastlike cells. Both basophil-like and mastlike cells fluoresced with o-phthaldialdehyde and exhibited IgE receptors. Unlike basophil-like cells, mastlike cells were chloroacetate esterase, amidase, and human mast cell tryptase positive. We conclude that human rIL-3 can support the growth of human mastlike cells under selected culture conditions.

Basophils↗

Production and characterization of monoclonal antibodies to mesenchymal stem cells derived from human bone marrow.

The bone marrow (BM) serves as a reservoir for different classes of stem cells. In addition to haematopoietic stem cells, bone marrow contains a population of mesenchymal stem cells (MSCs). These cells have a multilineage differentiation capacity and are able to generate progenitors with restricted developmental potential, which include fibroblasts, osteoblasts, adipocytes, and chondrocytes. Characteristic markers have been reported for expanded MSCs, but none of these markers are specific for MSCs. Thus, the objective of this study was to produce monoclonal antibodies against MSCs. MSCs derived from human bone marrow were cultured, expanded, and immunized into mice, and spleen cells subsequently harvested were used to generate hybridoma cell lines secreting antibodies against MSCs. Hybridoma culture supernatants were screened for antibodies against MSCs by enzyme-linked immunosorbent assay (ELISA), and 33 positive clones were then screened against cell suspensions of MSCs by immunofluorescence staining and flow cytometry. Ten clones were positive in immunofluorescence staining. Among these, three hybridoma cell lines, namely, YS08, YS14, and YS18 were found to be reactive with MSC by flow cytometry, but non-reactive with human tumor cell lines and hematopoietic stem cells. YS08 and YS14 showed specific bands in Western blotting. In conclusion, we developed three monoclonal antibodies, YS08, YS14, and YS18, that recognize human MSC cell surface antigen.

Antibodies, Monoclonal↗

The use of soybean agglutinin (SBA) for bone marrow (BM) purging and hematopoietic progenitor cell enrichment in clinical bone-marrow transplantation.

Soybean agglutin (SBA) is a plant lectin that has been used to fractionate bone marrow cells. It binds bone marrow mononuclear cells, including mature myeloid, erythroid, and lymphoid cells, but has very low binding affinity and no toxic effect to the human hematopoietic cells. In this article we describe the possibilities of enriching bone-marrow-derived CD34+ hematopoietic progenitor cells using SBA. As the method is simple and elegant. SBA is of vast importance to the field of clinical bone marrow transplantation.

Adult↗