Biomedical subjects

L W Terstappen

Publications and source records attributed to L W Terstappen.

14 recordsLinked to original sources

PubMed1992

Flow cytometric assessment of human T-cell differentiation in thymus and bone marrow.

Using multidimensional flow cytometry we have defined and quantified the human T-cell differentiation pathway, focusing on those events occurring among the most immature thymocytes and putative bone marrow (BM) T-precursors. Early thymocytes were found to express the CD34 antigen and consisted of a mean 1.2% of cells within human pediatric (n = 9) and 2.0% in fetal thymi (n = 4). All CD34+ thymocytes were atypical blast by morphology, expressed intracytoplasmatic, but not cell surface, CD3, and were cell surface CD2+, CD5+, CD7+, CD38+, CD45+, CD45RA+, CD49d+, and LECAM-1(Leu8)high. CD34high thymocytes lacked surface expression of CD4 and CD8, but as CD34 expression diminished there was a coordinate increase in CD4 levels, followed by the appearance of CD8. The expression of CD1 and CD10 also increased concomitant with the loss of CD34, whereas expression of LECAM-1 diminished with CD34 downregulation. The differential expression of these antigens on early thymocytes (as well as the number of thymocytes displaying these patterns) was highly reproducible among the nine pediatric and four fetal specimens examined, suggesting a precise, stereotyped regulation of early differentiation events. Cell populations with antigen expression patterns suggestive of pluripotent stem cell (CD34high, CD38-), or non-T-lineage committed stem cells (CD34+, CD33+ or CD34+, CD19+) were not identified in either fetal or pediatric thymi (sensitivity = 1/10(4)). The presence of cells with the antigenic profile of the earliest CD34+ thymocytes was explored in human BM. Putative BM T-cell precursors with the appropriate phenotype (CD34+, CD7+, CD5+, CD2+, LECAM-1high) were readily identified in fetal specimens (constituting +/- 2% of the CD34+ population), but could not be reliably detected in adults. In contrast with thymi, only 13% of these cells expressed cytoplasmatic CD3, suggesting the presence of the immediate precursor of the putative prothymocyte population. This was further supported by the detection of CD34bright, CD7+, CD2-, CD5-, LECAM-1moderate cells in fetal specimens. Our results document the flow of cell surface differentiation during T-lymphopoiesis and suggest that T-lineage features are first acquired in the BM. The ability to reproducibly identify and isolate T-cell precursor populations of precisely defined maturational stage in marrow and thymus by multiparameter flow cytometry will facilitate characterization of the molecular events controlling T-lineage differentiation.

Antibodies, Monoclonal↗

PubMed1992

Expression of the DAF (CD55) and CD59 antigens during normal hematopoietic cell differentiation.

Expression of decay-accelerating factor (DAF or CD55) and of CD59 during hematopoietic cell development in normal bone marrow and on peripheral blood leukocytes were characterized by three-color immunofluorescence experiments. With this technique cell subsets were identified by forward light scatter, orthogonal light scatter, and two cell-surface antigens. For each cell lineage, specific combinations of two monoclonal antibodies labeled with different fluorochromes were used. DAF or CD59 were then quantitated on the defined cell subsets from the fluorescence signal of the respective antibody conjugated with a third fluorochrome. Early uncommitted hematopoietic progenitor cells (CD34+, CD38-) all expressed both proteins homogeneously. Initial commitment to the erythroid (CD71+, CD45dim), myeloid (CD33+), or B lymphocyte (CD10+) lineages was not associated with changes in DAF or CD59 levels. With erythroid development, i.e., after loss of CD45 and decrease of CD71, expression of both proteins decreased. With myeloid maturation, expression of CD59 remained constant and expression of DAF varied. During neutrophil maturation, DAF decreased initially and then reemerged on maturing neutrophils concurrently with the appearance of CD16 (Fc gamma RIII), whereas during monocyte maturation, DAF increased concurrently with up-regulation of CD14. With B cell development, expression of DAF increased concurrently with down-regulation of CD10 and up-regulation of CD20, whereas expression of CD59 diminished slightly late in B cell maturation. Analysis of peripheral blood elements showed that monocytes, neutrophils, and B lymphocytes expressed both proteins homogeneously, but that in contrast to other cell subsets, which all expressed CD59, only a subset of (CD3+) T lymphocytes and (CD16+) Natural killer cells expressed DAF. The absence of DAF was not related to CD4 or CD8 expression or to the presence of activation markers (CD25+, CD38+), memory cell markers (CD58+, CD45RO+), or virgin T cell markers (CD45RA+), but was correlated with expression of CD11b (CR3) and CD11c (gp150/95). Although CD21+ (CR2) and CD35+ (CR1) cells all expressed DAF, CD11a (LFA-1) levels correlated inversely with those of DAF. Although the presence of CD55 and CD59 on early progenitor cells and throughout hematopoietic cell development is consistent with the requirements for both proteins in protection of host cells from complement-mediated injury, the physiological relevance of the unique patterns of variation for each cell lineage is unclear. Nevertheless, the availability of a detailed DAF and CD59 expression map in normal marrow will facilitate analyses of alterations during hematopoietic development that may occur in hematological disorders including paroxysmal nocturnal hemoglobinuria (PNH).

Adult↗

PubMed1992

Flow cytometric characterization of acute myeloid leukemia: IV. Comparison to the differentiation pathway of normal hematopoietic progenitor cells.

Gradual increase of CD38 on cells expressing CD34 characterizes the early cell differentiation pathway of normal human hematopoietic progenitors. In this study the coordinated expression pattern of CD34 and CD38 was assessed on leukemic blasts from bone marrow aspirates of 95 patients with newly diagnosed acute myeloid leukemia (AML). Expression was divided into six categories analogous to the differentiation pathway of normal bone marrow. The CD38 antigen was expressed on the leukemic cells of all patients and CD34+ leukemic cells were found in 79 patients (83%). In 93 patients, the leukemic cells were found along the differentiation pathway defined by CD34 and CD38. In 33 of the 93 patients, a part of the CD34+ cells did not express the CD38 antigen (categories 1 and 2). In another 33 patients, all CD34+ cells expressed CD38 (categories 3 and 4). In the remaining 27 patients, only cells were found which dimly expressed CD34 or did not express CD34 (categories 5 and 6). Of the 93 patients, 88 were treated with intensive chemotherapy according to the protocol of the German AML Cooperative Group. Of these, 21 died early and were not evaluable for treatment response. Complete remission was achieved in 14 of 22 patients (64%) in categories 1 and 2, in 19 of 26 patients (73%) in categories 3 and 4, and in 18 of 19 patients (95%) in categories 5 and 6. The event-free survival was significantly longer in patients of categories 5 and 6 compared to patients in categories 1 and 2 (p less than 0.01) and categories 3 and 4 (p less than 0.05), respectively. We conclude that in the majority of AML patients the immunophenotype of leukemic cells follows the early cell differentiation pathways defined by coordinated expression of CD34 and CD38 similar to that of normal hematopoietic progenitors. The presence of cells in the late cell differentiation stages (CD34+/-, CD38 /+) identifies patients with a higher complete remission rate and longer complete remission duration.

ADP-ribosyl Cyclase↗

PubMed1992

Differentiation and maturation of growth factor expanded human hematopoietic progenitors assessed by multidimensional flow cytometry.

Non-adherent cord blood and bone marrow mononuclear cells were analyzed by multiparameter flow cytometry before and at day 2, 4, 7, and 11 of culture in recombinant interleukin 3 (IL-3) and granulocyte colony-stimulating factor (G-CSF, cord blood) or stem cell factor (SCF), IL3 and granulocyte-macrophage colony-stimulating factor (GM-CSF, BM) to assess the differentiation and maturational pathway of myeloid cells. Before cell culture cord blood contained progenitor cells (CD34+) in various differentiation stages (CD38(-)----CD38bright), mature lymphocytes, monocytes, and neutrophils, but no immature neutrophils and immature monocytes. During cell culture, all CD34+ cells acquired the CD38 antigen between day 2 and 5 of cell culture, the CD34 antigen was lost between day 5 and 11 of cell culture. Differentiation of cells into the myeloid cell lineage was characterized by the acquisition of both CD33 and CD71. The latter is indicative for the active proliferation of these cells. Maturation of the cells into the neutrophilic pathway was indicated by the acquisition of first the CD15 antigen followed by CD11b and CD16 respectively. Whereas maturation of the cells into the monocytic pathway was indicated by the acquisition of first CD11b followed by CD14 and a dim expression of both CD15 and CD16. In normal bone marrow, cells of various maturational stages are already present before cell culture. During cell culture differentiation of cells into the myeloid lineage and maturation of the cells along the monocyte and neutrophilic lineage followed identical pathways as was observed before cell culture. Differentiation and maturational pathways of cord blood and adult bone marrow were identical. The results confirm the surface-antigen-defined pathways of myeloid cell differentiation described previously for non-cultured normal bone marrow aspirates. The detailed assessment of cell maturation and differentiation of cultured cells by multidimensional flow cytometry permits the determination of the specific effects of various recombinant human growth factors on myeloid cells.

Antigens, CD↗

PubMed1992

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.

Benzothiazoles↗

PubMed1992

Flow cytometric characterization of acute myeloid leukemia. Part II. Phenotypic heterogeneity at diagnosis.

The frequency and distribution of aberrant antigen expression are analyzed on bone marrow aspirates from 80 patients with newly diagnosed acute myeloid leukemia (AML) by multidimensional flow cytometry. Parameters examined are the light scatter profile of the leukemic cells and the correlative expression of different combinations of the CD2, 4, 5, 7, 11b, 11c, 13, 14, 15, 16, 33, 34, 38, and HLA-DR antigens. Antigen expression on leukemic cells in bone marrow is described by characteristic antigen expression patterns describing: (i) the percentage of cells expressing the antigen; (ii) the antigen density; and (iii) the distribution of the antigen on the leukemic cells. Typically the non-myeloid antigens are homogeneously expressed by the leukemic cells, whereas the myeloid associated antigen CD11b, CD11c, CD14, and CD15 are heterogeneously expressed. Comparison of the antigenic profiles of 80 bone marrow aspirates revealed an extreme interclonal heterogeneity. Comparison of the antigen expression patterns found in AML patients with the antigen expression in normal bone marrow revealed four patterns of aberrant antigen expression in AML: (i) expression of nonmyeloid antigens (i.e. CD2, CD5, and CD7 were present in 57, 60, and 37% of the patients, respectively); (ii) asynchronous expression of myeloid associated antigens (i.e. co-expression of CD34 and CD15 in 25% of the patients and expression of CD16 on immature myeloid cells in 15% of the cases); (iii) over-expression of myeloid associated antigens (e.g. CD34 in 16% of the cases and CD14 on neutrophilic cells in 19% of all patients); and (iv) absence of expression of myeloid associated antigens (e.g. lack of CD33 in 21% of the cases and lack of both CD11b and CD15 in 6% of all patients. Multidimensional flow cytometric analysis of bone marrow aspirates of AML patients disclosed that the leukemic cells of each AML patient had a unique antigenic profile and could be discriminated from their normal counterparts based on aberrant antigen expression and typical light scatter profiles. The ability to distinguish leukemic cells from normal cells allows the detection of residual leukemic cells during and after chemotherapy.

Acute Disease↗

PubMed1991

Sequential generations of hematopoietic colonies derived from single nonlineage-committed CD34+CD38- progenitor cells.

Multiparameter flow cytometry was applied on normal human bone marrow (BM) cells to study the lineage commitment of progenitor cells ie, CD34+ cells. Lineage commitment of the CD34+ cells into the erythroid lineage was assessed by the coexpression of high levels of the CD71 antigen, the myeloid lineage by coexpression of the CD33 antigen and the B-lymphoid lineage by the CD10 antigen. Three color immunofluorescence experiments showed that all CD34+ BM cells that expressed the CD71, CD33, and CD10 antigens, concurrently stained brightly with anti-CD38 monoclonal antibodies (MoAbs). In addition, the CD38 antigen was brightly expressed on early T lymphocytes in human thymus, characterized by CD34, CD5, and CD7 expression. Only 1% of the CD34+ cells, 0.01% of nucleated cells in normal BM, did not express the CD38 antigen. The CD34+, CD38- cell population lacked differentiation markers and were homogeneous primitive blast cells by morphology. In contrast the CD34+, CD38 bright cell populations were heterogeneous in morphology and contained myeloblasts and erythroblasts, as well as lymphoblasts. These features are in agreement with properties expected from putative pluripotent hematopoietic stem cells; indeed, the CD34 antigen density decreased concurrently with increasing CD38 antigen density suggesting an upregulation of the CD38 antigen on differentiation of the CD34+ cells. Further evidence for a strong enrichment of early hematopoietic precursors in the CD34+, CD38- cell fraction was obtained from culture experiments in which CD34+ cell fractions with increasing density of the CD38 antigen were sorted singularly and assayed for blast colony formation. On day 14 of incubation, interleukin-3 (IL-3), IL-6, and GM-CSF, G-CSF, and erythropoietin (Epo) were added in each well. Twenty-five percent of the single sorted cells that expressed CD34 but lacked CD38 antigen gave rise to primitive colonies 28 to 34 days after cell sorting. The ability to form primitive colonies decreased rapidly with increasing density of the CD38 antigen. During 120 days of culture, up to five sequential generations of colonies were obtained after replating of the first-generation primitive colonies. This study provides direct evidence for the existence of a single class of progenitors with extensive proliferative capacity in human BM and provides an experimental approach for their purification, manipulation, and further characterization.

ADP-ribosyl Cyclase↗

PubMed1991

Single laser three color immunofluorescence staining procedures based on energy transfer between phycoerythrin and cyanine 5.

Monoclonal antibodies specific for phycoerythrin (PE) were covalently labeled with the fluorescent dye cyanine 5 (Cy5). Excitation at 488 nm of immune complexes obtained by mixing Cy5-anti-PE with PE resulted in a 4-fold reduction of PE fluorescence measured at 565 nm and an increase of fluorescence measured at 655 nm. The observed energy transfer between PE and Cy5-anti-PE was used to develop three color immunofluorescence staining procedures for flow cytometers equipped with an Argon laser tuned at 488 nm. Mouse IgG1 monoclonal antibodies specific for cell surface antigens were cross-linked with either unlabeled or Cy5 labeled mouse IgG1 anti-PE using F(ab')2 fragments of monoclonal rat anti-mouse IgG1. PE was added to these immune complexes in sufficient amounts to saturate all PE binding sites. Cells were incubated with PE-labeled and PE/Cy5-labeled tetrameric antibody complexes together with FITC labeled antibodies and analyzed by flow cytometry. The emission from FITC, PE and PE/Cy5 could be readily separated and bright three color immunofluorescence staining of mononuclear cells from human peripheral blood and bone marrow was observed. The results of these experiments demonstrate that useful probes for single laser three color staining of cell surface antigens can be readily obtained by mixing of selected reagents. Compared to standard procedures for the covalent labeling of PE (tandem) molecules to antibodies, the non-covalent procedures described in this report provide significant advantages in terms of the amount of reagents, time and equipment required to obtain suitable reagents for three color immunofluorescence staining.

Animals↗

PubMed1991

Multidimensional flow cytometric blood cell differentiation without erythrocyte lysis.

Forward light scattering, orthogonal light scattering, and the fluorescence intensities of unlysed peripheral blood cells, labeled with CD45-phycoerythrin and the nucleic acid dyes LDS-751 and thiazole orange, were measured simultaneously, utilizing a flow cytometer. Erythrocytes, reticulocytes, platelets, neutrophils, eosinophils, basophils, monocytes, lymphocytes, nucleated erythrocytes, and immature nucleated cells occupied unique positions in the five-dimensional space created by the listmode storage of the five independent parameters. A software program was developed which identified and enumerated each of these cell populations. Platelets in this study were identified by LDS-751 staining, in addition to their forward and orthogonal light-scattering characteristics. Validation of this approach was obtained by demonstrating that all CD41- or CD42-expressing platelets also stained with LDS-751. Furthermore, the staining by LDS-751 did not change following platelet activation with ADP. The quantification of erythrocytes, platelets, neutrophils, eosinophils, monocytes, and lymphocytes correlated well with data obtained with a commercial hematology whole blood analyzer (H-1). Reproducibility of the identification of these populations was shown by repeated measurement of the same sample and by staining and analysis of multiple aliquots of identical blood samples. Stability studies demonstrated that 8 hours after blood collection, the number of damaged cells increased. This could be measured by a greater thiazole orange uptake by the damaged cells. This investigation demonstrates the feasibility of multidimensional flow cytometric blood cell differentiation for an automated whole blood cell analysis without the necessity of erythrocyte lysis. The ability to simultaneously identify reticulocytes, nucleated erythrocytes, and immature nucleated cells in one measurement is unique and promises to be a powerful tool for the assessment of abnormal blood samples.

Antibodies, Monoclonal↗

PubMed1991

Flow cytometric characterization of acute myeloid leukemia. Part 1. Significance of light scattering properties.

Acute leukemias are classified using the morphological and cytochemical criteria set forward by the French, American and British (FAB) group. Immunophenotyping is helpful for the differential diagnosis but is secondary to the morphological criteria. Immunophenotyping performed by flow cytometry, however, can yield valuable information on cell morphology in addition to cell surface antigen expression. To provide a basis of a combined evaluation of both morphology, i.e. light scattering, and immunophenotype by flow cytometry we have compared the light scattering profiles of 70 patients newly diagnosed with acute leukemia with normal bone marrow and related the findings to the FAB classification. Three main light scattering profiles were observed in the bone marrow aspirates of the 70 patients (A1,2; B1,2,3; C1,2,3,4). A1,2, characterized by a predominant cell cluster with low forward and orthogonal light scattering, contained only and all patients diagnosed as acute lymphoblastic leukemia, acute undifferentiated leukemia, and acute non-lymphocytic leukemia M6 and M1. B1,2,3 is characterized by a predominant cell cluster with large forward and low to high orthogonal light scattering. Category B1 contained the majority of patients classified as M5; the M3 leukemias were categorized as B2. C1,2,3,4 is characterized by a predominant cell cluster with low forward and orthogonal light scattering that branches towards regions with larger light scattering. Categories C1 and C2 contained the majority of the patients classified as M2. Category C3 was specific for M4 and M4eo leukemias. The patients diagnosed as M4 were heterogeneous and equally distributed over the B and C categories. The clear relationship found between the FAB classification and classification by the light scattering profile of the acute leukemias enhances the importance of the flow cytometric classification of leukemias. In contrast with light microscopy, flow cytometry can now provide the hematologist with an objective technique to classify leukemias by the simultaneous assessment of cell surface antigen expression and cell morphology, i.e. light scattering.

Acute Disease↗

PubMed1990

Differential expression of homing-associated adhesion molecules by T cell subsets in man.

The ability of lymphocyte populations to recognize and bind high endothelial venules during homing into lymphoid tissues and sites of chronic inflammation is critically dependent on their expression of certain homing-associated adhesion molecules known as homing receptors (HR). In animal models, certain lymphocyte populations, particularly subsets of memory or previously activated lymphocytes, demonstrate tissue-selective homing behavior, and it has been hypothesized that differential expression of HR accounts for this selective migration. In this study, we analyzed expression of human HR--the Dreg 56/Leu 8-defined peripheral lymph node (PLN) HR (also known as LECAM-1), H-CAM (CD44), and alpha 4-integrins (CD49d; VLA-4)--among subsets of thymocytes and peripheral blood T cells to identify populations with differential homing potential. In the thymus, these three HR classes are differentially regulated relative to phenotypically defined maturational stages, but are all expressed on the mature, surface CD3high subset. In the peripheral blood, virgin T cells (LFA-3/CD58low) show uniform high expression of the PLN HR, uniform relatively low expression of H-CAM and alpha 4-integrin, and lack markers of tissue association--the mucosal and cutaneous lymphocyte associated Ag (MLA and CLA Ags) defined by mAb Ber ACT8 and HECA-452, respectively. In contrast, circulating memory T cells (LFA-3/CD58high) are bimodal with respect to PLN HR expression, show uniform high expression of H-CAM and alpha 4-integrin, and contain essentially all the CLA and MLA Ag-bearing T cells. The circulating skin-associated T cell subset (CLA Ag+; 10 to 15% of total T cells) is predominantly PLN HR+, and shows high levels of both the alpha 4- and beta 1-integrin chains. The distinct mucosa-associated T cell subset (MLA Ag+; 1 to 3% of peripheral blood T cells) is predominantly PLN HR-; and is alpha 4high, but beta 1low. These findings indicate the independent regulation of homing-associated adhesion molecules among populations of memory/previously activated T cells, and suggest that the expression patterns of these molecules contribute to, or perhaps determine, the tissue distribution of these subsets.

Adult↗

PubMed1990

Cytotoxic lymphocytes in B-cell chronic lymphocytic leukemia. A flow cytometric study of peripheral blood, lymph nodes and bone marrow.

The occurrence of cytotoxic lymphocyte subpopulations (i.e., CD 16+, CD 57+ and cytotoxic CD 8+) wa studied in the peripheral blood of 18 B-cell chronic lymphocytic leukemia (B-CLL) patients. The absolute numbers of CD 57+, CD 16+ and cytotoxic CD 8+ lymphocytes were increased in the peripheral blood of untreated patients as compared with healthy donors, suggesting a causal relation with the accumulation of malignant B-cells. For 5 B-CLL patients and 5 hematological normal donors, the lymphocyte subpopulations in peripheral blood, lymph nodes and bone marrow were determined. A significant immune response was observed in the lymph nodes of the patients, as reflected by the CD 3+ lymphocytes, which were 1.7-27 times larger in the patients lymph nodes than in their peripheral blood and bone marrow. In contrast, with peripheral blood this was mainly caused by an increase in CD 4+ lymphocytes. The CD 57 lymphocytes in the lymph nodes of the patients had abnormal orthogonal light-scattering signals and an abnormal density of CD 57+ receptors in comparison with their peripheral blood CD 57+ lymphocytes or the CD 57+ lymphocytes in the peripheral blood, bone marrow and tonsils of the hematological normal donors. This study shows that although a significant increase of cytotoxic lymphocytes in the peripheral blood of B-CLL patients is observed, the actual distributions of the non-malignant lymphocytes can be quite different at the actual tumor sites, i.e., bone marrow and lymph nodes.

Adult↗

PubMed1990

Myeloid cell differentiation in normal bone marrow and acute myeloid leukemia assessed by multi-dimensional flow cytometry.

A detailed analysis of normal myeloid differentiation was performed using mutlidimensional flow cytometry. Based on two light scattering and three color immunofluorescence signals, the normal maturation pathways of both the monocyte and neutrophil lineages could be elucidated. Gradual changes of light scattering properties and cell surface antigen expression defined the pathways of each of the lineages. The consistency of the location of these lineage specific pathways found in normal individuals provided the basis for the discrimination between normal and leukemic cells in acute myeloid leukemia (ANLL). The position of leukemic cells in patients with ANLL in a five-dimensional space was compared with the position of the maturation tracks in normal individuals. The expression of normal antigens on leukemic cells provided the tools to: (1) distinguish normal from clonal populations of leukemic cells in all 15 patients; (2) detect a lineage predominance, either monocytic or neutrophilic, in all 15 patients; (3) detect maturation heterogeneity in all 15 patients. Although maturation pathways of the monocytic and the neutrophilic lineages were analogous to the normal patterns they were distinct in several ways. The expression of normal antigens on leukemic cells may provide the tools to: (1) obtain a new frame-work for classification of leukemia based on the ability to quantify the aberrant antigen expression and to define a 'distance from normal' based on the characteristics studied (the maturation heterogeneity of the leukemic cells also can be correlated with the clinical outcome of the patients); (2) detect minimal residual disease using the difference in locations of the leukemic cells in the multidimensional space from the normal maturation pathways (3) monitor relapse and changes in phenotypes which may accompany chemotherapy, suggesting the appearance of variant or new clones.

Antibodies, Monoclonal↗

PubMedDate not supplied

Formation of haematopoietic microenvironment and haematopoietic stem cells from single human bone marrow stem cells.

Haematopoietic stem cells are a population of cells capable both of self renewal and of differentiation into a variety of haematopoietic lineages. Enrichment techniques of human haematopoietic stem cells have used the expression of CD34, present on bone marrow progenitor cells. But most CD34+ bone marrow cells are committed to their lineage, and more recent efforts have focused on the precise characterization of the pluripotent subset of CD34+ cells. Here we report the characterization of two distinct subsets of pluripotent stem cells from human fetal bone marrow, a CD34+, HLA-DR+, CD38- subset that can differentiate into all haematopoietic lineages, and a distinct more primitive subset, that is CD34+, HLA-DR-, CD38-, that can differentiate into haematopoietic precursors and stromal cells capable of supporting the differentiation of these precursors. These data represent, to our knowledge, the first identification of a single cell capable of reconstituting the haematopoietic cells and their associated bone marrow microenvironment.

ADP-ribosyl Cyclase↗