Bone marrow cell differential counts obtained by multidimensional flow cytometry.

Five-dimensional flow cytometric analysis of normal bone marrow aspirates was utilized to determine the frequency of neutrophils, eosinophils, monocytes, lymphocytes, nucleated erythrocytes, reticulocytes, platelets, and a cell population that included blasts of each of the cell lineages, megakaryocytes, plasma cells, and basophils. Each of these bone marrow cell populations had unique features with respect to forward light scatter, orthogonal light scatter, and staining with Thiazole-Orange, LDS-751, and CD45 labeled with Phycoerythrin (PE). The identity of the cell populations was verified by sorting each of the cell populations and subsequent light microscopic examination of the cells. The frequencies of the nucleated bone marrow cell subpopulations of 50 normal donors were for neutrophils, mean 72.3%; SD +/- 5.1; 95% limits, 70.9-73.8%; eosinophils, mean 1.8%; SD +/- 1.3; 95% limits, 1.4-2.1%; monocytes, mean, 2.8%; SD +/- 1.2; 95% limits, 2.5-3.1%; lymphocytes, mean 12.1%; SD +/- 3.6; 95% limits 11.1-13.2%; nucleated erythrocytes, mean 8.9%; SD +/- 3.9; 95% limits, 7.8-10.1%; and the cell population that included blasts of each of the cell lineages, megakaryocytes, plasma cells, and basophils, mean 1.6%; SD +/- 1.2; 95% limits, 1.3-1.9%. The percentage of reticulocytes in bone marrow aspirates from 50 normal donors correlated with the reticulocyte frequency in the peripheral blood of these donors. However, the mean frequency of reticulocytes was significantly greater (p < 0.0001) in bone marrow (mean 2.19%; SD +/- 0.88) than in peripheral blood (mean 1.71%; SD +/- 0.88). The technique could discriminate between immature and mature reticulocytes based on the brighter staining with both Thiazole-Orange and LDS-751 of the immature reticulocytes. This was confirmed by cell sorting of both reticulocyte populations, which revealed larger clumps of New Methylene Blue staining material in the brighter Thiazole-Orange and LDS-751 stained reticulocytes. The immature reticulocytes were present in normal bone marrow, but not in normal peripheral blood. As expected, a significantly greater frequency of nucleated cells was found in bone marrow aspirates (mean 0.85%; SD +/- 0.59) than in peripheral blood (mean 0.20%; SD +/- 0.11). The frequency of platelets was significantly lower in bone marrow (mean 1.24%; SD +/- 0.69) than in peripheral blood (mean 2.94%, SD +/- 1.14). Flow cytometric bone marrow analysis can provide clinical laboratories with a technique that generates quantitative bone marrow cell differentials and potentially can reduce the need for light microscopic examination of bone marrow smears.     

Bone marrow stem cell transplantation for cardiac repair

Cardiomyocytes respond to physiological or pathological stress only by hypertrophy and not by an increase in the number of functioning cardiomyocytes. However, recent evidence suggests that adult cardiomyocytes have the ability, albeit limited, to divide to compensate for the cardiomyocyte loss in the event of myocardial injury. Similarly, the presence of stem cells in the myocardium is a good omen. Their activation to participate in the repair process is, however, hindered by some as-yet-undetermined biological impediments. The rationale behind the use of adult stem cell transplantation is to supplement the inadequacies of the intrinsic repair mechanism of the heart and compensate for the cardiomyocyte loss in the event of injury. Various cell types including embryonic, fetal, and adult cardiomyocytes, smooth muscle cells, and stable cell lines have been used to augment the declining cardiomyocyte number and cardiac function. More recently, the focus has been shifted to the use of autologous skeletal myoblasts and bone marrow-derived stem cells. This review is a synopsis of some interesting aspects of the fast-emerging field of bone marrow-derived stem cell therapy for cardiac repair.     

Bone marrow transplantation. Part I--Allogeneic.

Major progress in experimental and clinical research has made allogeneic bone marrow transplantation a highly effective therapy for a variety of malignant and nonmalignant diseases. Allogeneic bone marrow transplantation from histocompatible donors is now the therapy of choice for some of these disorders. We review in part I the history, technical approach, complications, and the results achievable with this therapeutic approach. Further experimentation and future goals are also discussed.     

Bone marrow transplantation. Part II--autologous.

Autologous bone marrow transplantation provides an effective form of "rescue" following high-dose therapy used for treating certain malignant diseases. The high doses of radiotherapy or chemotherapy, or both, should allow for greater tumor cell kill if dose-response to therapy exists for that tumor. The use of autologous bone marrow obviates the need for an HLA-identical donor, and the need for pretransplant immunosuppression; no graft-versus-host disease would ensue. We review in part II the history and background, methods of obtaining autologous stem cells, and details of the results achievable with this type of therapy. We discuss potential difficulties with autologous transplantation, as well as possible future areas of research.     

Bone marrow transplantation in Canada.

Bone marrow transplantation is an established form of therapy for aplastic anemia and severe combined immunodeficiency. It is also a therapeutic option for acute leukemia in remission. Unfortunately, compatible donors are not available for most patients who could benefit from it. Further refinement of the techniques involved may make it suitable for more patients. Graft rejection, recurrent leukemia, graft-versus-host disease and interstitial pneumonia continue to be the main unsolved complications of bone marrow transplantation, but recent advances have decreased their frequency and severity. Most of the complications of allogeneic bone marrow transplantation may be eliminated with the use of autologous stem cells. For further refinement bone marrow transplantation should continue to be performed in large centres that combine treatment with research.     

Bone marrow biopsy (BMB). II. Bone marrow biopsy in myeloproliferative disorders

The myeloproliferative disorders (MPD) are a domain in which the bone marrow biopsy (BMB) greatly proved its utility. We have studied the histology of the bone marrow (BM) in all the four entities of MPD: chronic myeloid leukemia (CML) with its subtype, chronic megakaryocytic granulocytic myelosis (CMGM), polycythemia vera (PV), hemorrhagic thrombocythemia (HT) and myeloid metaplasia with myelofibrosis (MMM). The work presents in short some of the clinical and hematologic characters of MPD with special stress upon the histologic modifications of BM, either specific or common to all MPD entities, underlying also the criteria for differential diagnosis.     

Identification of young megakaryocytes by immunofluorescence and cytophotometry.

The DNA-content of fluoresceine-labeled platelet antigen containing cells of mouse bone marrow was measured. For immunofluorescence highly specific anti-mouse-platelet-serum and fluoresceine-conjugated antigammaglobuline was used, applying the "sandwich" technique. Three hundred panoptically identifable megakaryocytes served as control group. The DNA-polyploidization pattern of megakaryocytes and immunofluorescence positive cells was almost identical. However, among the immunofluorescence positive cells a considerable amount of cells showed DNA-values lower than 4c, whereas the megakaryocytes of the Pappenheim stained smears revealed no DNA-values lower than 4c. The percentages of diploid and tetraploid cells, respectively, was 6 and 7% compared with 0 and 1% of panoptically identifiable megakaryoctyes. The results suggest that young megakaryocytic cells with diploid and tetraploid DNA-values can be detected by immunofluorescence technique, indicating that the flow from the uncommited to the committed megakaryocytic precursor cell appears at this early stage of megakaryocyte production.     

Escherichia coli growth studied by dual-parameter flow cytophotometry.

The growth of Escherichia coli cells has been analyzed for the first time by dual-parameter flow cytophotometry, in which the deoxyribonucleic acid and protein contents of single bacteria have been measured simultaneously with an accuracy of a few percent and at a rate of 3,000 cells/s.     

Bone marrow osteoblast damage by chemotherapeutic agents

Hematopoietic reconstitution, following bone marrow or stem cell transplantation, requires a microenvironment niche capable of supporting both immature progenitors and stem cells with the capacity to differentiate and expand. Osteoblasts comprise one important component of this niche. We determined that treatment of human primary osteoblasts (HOB) with melphalan or VP-16 resulted in increased phospho-Smad2, consistent with increased TGF-β1 activity. This increase was coincident with reduced HOB capacity to support immature B lineage cell chemotaxis and adherence. The supportive deficit was not limited to committed progenitor cells, as human embryonic stem cells (hESC) or human CD34+ bone marrow cells co-cultured with HOB pre-exposed to melphalan, VP-16 or rTGF-β1 had profiles distinct from the same populations co-cultured with untreated HOB. Functional support deficits were downstream of changes in HOB gene expression profiles following chemotherapy exposure. Melphalan and VP-16 induced damage of HOB suggests vulnerability of this critical niche to therapeutic agents frequently utilized in pre-transplant regimens and suggests that dose escalated chemotherapy may contribute to post-transplantation hematopoietic deficits by damaging structural components of this supportive niche.     

Assessment of monocyte esterase activity by flow cytophotometry.

An azo dye supravital method has been devised for selectively staining human monocytes in suspension for nonspecific esterase activity. Stained cells can be identified and rapidly enumerated by presenting the suspension of stained cells to the Cytograf, a flow-through cell discriminating cytophotometer. The intensity of stain is proportional to the intracellular esterase activity. By analysis of the oscilloscope display, it has been possible to obtain relative data concerning the degree of activity of monocyte nonspecific esterase activity. These observations suggest a unique approach to the measurement of intracellular enzyme activity in selected cells in a mixed population.