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Gunnar H. Heine - One of the best experts on this subject based on the ideXlab platform.
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supersage evidence for cd14 cd16 monocytes as a third monocyte subset
Blood, 2011Co-Authors: Adam M. Zawada, Kyrill S. Rogacev, Danilo Fliser, Bjorn Rotter, Peter Winter, Rolfr Marell, Gunnar H. HeineAbstract:Monocytes are a heterogeneous cell population with subset-specific functions and phenotypes. The differential expression of CD14 and CD16 distinguishes classical CD14++CD16−, intermediate CD14++CD16+, and nonclassical CD14+CD16++ monocytes. Current knowledge on human monocyte heterogeneity is still incomplete: while it is increasingly acknowledged that CD14++CD16+ monocytes are of outstanding significance in 2 global health issues, namely HIV-1 infection and atherosclerosis, CD14++CD16+ monocytes remain the most poorly characterized subset so far. We therefore developed a method to purify the 3 monocyte subsets from human blood and analyzed their transcriptomes using SuperSAGE in combination with high-throughput sequencing. Analysis of 5 487 603 tags revealed unique identifiers of CD14++CD16+ monocytes, delineating these cells from the 2 other monocyte subsets. Gene Ontology (GO) enrichment analysis suggests diverse immunologic functions, linking CD14++CD16+ monocytes to Ag processing and presentation (eg, CD74 , HLA-DR , IFI30 , CTSB ), to inflammation and monocyte activation (eg, TGFB1 , AIF1 , PTPN6 ), and to angiogenesis (eg, TIE2 , CD105 ). In conclusion, we provide genetic evidence for a distinct role of CD14++CD16+ monocytes in human immunity. After CD14++CD16+ monocytes have earlier been discussed as a potential therapeutic target in inflammatory diseases, we are hopeful that our data will spur further research in the field of monocyte heterogeneity.
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cd14 cd16 monocytes and cardiovascular outcome in patients with chronic kidney disease
European Heart Journal, 2011Co-Authors: Kyrill S. Rogacev, Adam M. Zawada, Danilo Fliser, C Ulrich, Sarah Seiler, Birgit Reichart, Esther Herath, Daniel Roth, Gunnar H. HeineAbstract:Aims Patients with chronic kidney disease (CKD) pose a worldwide growing burden to health care systems due to accelerated atherosclerosis and subsequent high cardiovascular (CV) morbidity. Atherogenesis is prominently driven by monocytes and monocyte-derived macrophages. The expression of CD14 and CD16 characterizes three monocyte subsets: CD14++CD16−, CD14++CD16+, and CD14(+)CD16+ cells; the latter two are often denoted as ‘proinflammatory’ CD16+ monocytes. Despite an association between CD16+ monocyte counts and higher CV risk in cross-sectional cohorts, the prognostic impact of elevated CD16+ monocyte counts is poorly understood. Methods and results We assessed monocyte heterogeneity using flow cytometry in 119 patients with non-dialysis CKD, who were prospectively followed for a median of 4.9 (inter-quartile range 4.8–5.0) years for the occurrence of CV events. In addition, we assessed expression of chemokine receptors on monocyte subsets. CD14++CD16+ monocyte were independently associated with CV events [hazard ratio (for an increase of 10 cells/μL) 1.26 (confidence interval: 1.04–1.52; P = 0.018)] after adjustment for variables that significantly affected CD14++CD16+ cell counts at baseline. Across the spectrum of CKD, CD14++CD16+ monocytes selectively expressed CCR5. Conclusion We found that CD14++CD16+ monocytes were independently associated with CV events in non-dialysis CKD patients. Our results support the notion that CD16+ monocytes rather than CD16− monocytes are involved in human atherosclerosis.
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haemodialysis induced transient cd16 monocytopenia and cardiovascular outcome
Nephrology Dialysis Transplantation, 2009Co-Authors: Kyrill S. Rogacev, Danilo Fliser, Maren Ziegelin, C Ulrich, Sarah Seiler, Matthias Girndt, Gunnar H. HeineAbstract:Background. Haemodialysis with bioincompatible membranes led to transient leukocyte activation and intra-dialytic leukopenia due to endothelial adherence. After the introduction ofbiocompatible membranes, only CD16 + (i.e. CD14 ++ CD16 + and CD]4 (+) CD16 + ) monocytes showed an impressive transient intra-dialytic decrease. Presently, it is unclear whether this CD16 + monocyte drop is detrimental. We investigated whether a prominent intra-dialytic decrease of CD16 + monocytes predicts future cardiovascular (CV) events. Methods. We measured leukocyte and monocyte subpopulations in 70 patients before and 10 min after haemodialysis initiation. Patients were stratified by their intra-dialytic CD14 ++ CD16 + monocyte drop (pre-defined major drop: decline of cell counts at 10 min to 5 0% of pre-dialytic counts). Patients were followed up for 42 ± 2 months; endpoints were CV events and death. Results. Patients with a minor CD14 ++ CD16 + monocyte drop had more CV events than patients with a major drop. In multivariate analysis, a minor CD 14 ++ CD16 + monocyte drop was the strongest independent predictor of future CV events [hazard ratio 2.405 (95% CI 1.192―4.854)]. Conclusions. These data refute the assumption that a prominent intra-dialytic decrease of CD14 ++ CD16 + monocytes is detrimental. Instead, a minor cell drop could mirror CD14 ++ CD16 + monocyte dysfunction, with inadequate migratory reaction towards an immunologic stimulus posed by membrane and tubing contact.
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cd14 cd16 monocytes in coronary artery disease and their relationship to serum tnf α levels
Thrombosis and Haemostasis, 2004Co-Authors: Axel Schlitt, Gunnar H. Heine, Stefan Blankenberg, Christine Espinolaklein, Joern F Dopheide, Christoph Bickel, Karl J Lackner, Juergen Meyer, Harald Darius, Hans J RupprechtAbstract:Monocytes play a central role in the inflammatory disease atherosclerosis. CD14+CD16+ monocytes are considered proinflammatory monocytes, as they have an increased capacity to produce proinflammatory cytokines, such as TNF-α, and are elevated in various inflammatory diseases.We hypothesized that patients with coronary artery disease (CAD) have increased levels of CD14+CD16+ monocytes, and that CD14+CD16+ monocytes are associated with inflammation markers. We investigated CD14+CD16+ monocytes in 247 patients with CAD and 61 control subjects using flow cytometry. In addition serum concentrations of TNF-α, IL-6, and Hs-CRP were assessed. Patients with CAD had higher levels of CD14+CD16+ monocytes than controls (13.6% versus 11.4%; p
Kyrill S. Rogacev - One of the best experts on this subject based on the ideXlab platform.
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cd14 cd16 monocytes independently predict cardiovascular events a cohort study of 951 patients referred for elective coronary angiography
Journal of the American College of Cardiology, 2012Co-Authors: Kyrill S. Rogacev, Adam M. Zawada, Sarah Seiler, Bodo Cremers, Nadine Binder, Philipp Ege, Gunnar Grosedunker, Isabel Heisel, Florian Hornof, Jana JekenAbstract:Objectives The aim of this study was to analyze the yet ill-defined relationship of distinct human monocyte subsets with cardiovascular outcomes in a broad patient population at cardiovascular risk. Background Monocytes, the most abundant immune cell type found in atherosclerotic plaques, are crucial promoters of atherogenesis. Three distinct human monocyte subsets exist: classical CD14++CD16−, intermediate CD14++CD16+, and nonclassical CD14+CD16++ monocytes. Immunomodulation of distinct monocyte subsets has recently been discussed as a new therapeutic avenue in atherosclerosis. Methods Cardiovascular events in 951 subjects referred for elective coronary angiography were prospectively analyzed. Monocyte subset analysis was performed using flow cytometry, blinded to patients’ clinical characteristics, and patients were categorized according to quartiles of total monocyte and monocyte subset counts. The primary endpoint was defined a priori as the first occurrence of cardiovascular death, acute myocardial infarction, or nonhemorrhagic stroke. Endpoint adjudication was done blinded to monocyte subset distribution. Results During a mean follow-up period of 2.6 ± 1.0 years, 93 patients experienced the primary endpoint. In univariate Kaplan-Meier analysis, counts of total (p = 0.010), classical CD14++CD16− (p = 0.024), and intermediate CD14++CD16+ (p Conclusions CD14++CD16+ monocytes independently predicted cardiovascular events in subjects referred for elective coronary angiography. Future studies will be needed to elucidate whether CD14++CD16+ monocytes may become a target cell population for new therapeutic strategies in atherosclerosis.
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supersage evidence for cd14 cd16 monocytes as a third monocyte subset
Blood, 2011Co-Authors: Adam M. Zawada, Kyrill S. Rogacev, Danilo Fliser, Bjorn Rotter, Peter Winter, Rolfr Marell, Gunnar H. HeineAbstract:Monocytes are a heterogeneous cell population with subset-specific functions and phenotypes. The differential expression of CD14 and CD16 distinguishes classical CD14++CD16−, intermediate CD14++CD16+, and nonclassical CD14+CD16++ monocytes. Current knowledge on human monocyte heterogeneity is still incomplete: while it is increasingly acknowledged that CD14++CD16+ monocytes are of outstanding significance in 2 global health issues, namely HIV-1 infection and atherosclerosis, CD14++CD16+ monocytes remain the most poorly characterized subset so far. We therefore developed a method to purify the 3 monocyte subsets from human blood and analyzed their transcriptomes using SuperSAGE in combination with high-throughput sequencing. Analysis of 5 487 603 tags revealed unique identifiers of CD14++CD16+ monocytes, delineating these cells from the 2 other monocyte subsets. Gene Ontology (GO) enrichment analysis suggests diverse immunologic functions, linking CD14++CD16+ monocytes to Ag processing and presentation (eg, CD74 , HLA-DR , IFI30 , CTSB ), to inflammation and monocyte activation (eg, TGFB1 , AIF1 , PTPN6 ), and to angiogenesis (eg, TIE2 , CD105 ). In conclusion, we provide genetic evidence for a distinct role of CD14++CD16+ monocytes in human immunity. After CD14++CD16+ monocytes have earlier been discussed as a potential therapeutic target in inflammatory diseases, we are hopeful that our data will spur further research in the field of monocyte heterogeneity.
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cd14 cd16 monocytes and cardiovascular outcome in patients with chronic kidney disease
European Heart Journal, 2011Co-Authors: Kyrill S. Rogacev, Adam M. Zawada, Danilo Fliser, C Ulrich, Sarah Seiler, Birgit Reichart, Esther Herath, Daniel Roth, Gunnar H. HeineAbstract:Aims Patients with chronic kidney disease (CKD) pose a worldwide growing burden to health care systems due to accelerated atherosclerosis and subsequent high cardiovascular (CV) morbidity. Atherogenesis is prominently driven by monocytes and monocyte-derived macrophages. The expression of CD14 and CD16 characterizes three monocyte subsets: CD14++CD16−, CD14++CD16+, and CD14(+)CD16+ cells; the latter two are often denoted as ‘proinflammatory’ CD16+ monocytes. Despite an association between CD16+ monocyte counts and higher CV risk in cross-sectional cohorts, the prognostic impact of elevated CD16+ monocyte counts is poorly understood. Methods and results We assessed monocyte heterogeneity using flow cytometry in 119 patients with non-dialysis CKD, who were prospectively followed for a median of 4.9 (inter-quartile range 4.8–5.0) years for the occurrence of CV events. In addition, we assessed expression of chemokine receptors on monocyte subsets. CD14++CD16+ monocyte were independently associated with CV events [hazard ratio (for an increase of 10 cells/μL) 1.26 (confidence interval: 1.04–1.52; P = 0.018)] after adjustment for variables that significantly affected CD14++CD16+ cell counts at baseline. Across the spectrum of CKD, CD14++CD16+ monocytes selectively expressed CCR5. Conclusion We found that CD14++CD16+ monocytes were independently associated with CV events in non-dialysis CKD patients. Our results support the notion that CD16+ monocytes rather than CD16− monocytes are involved in human atherosclerosis.
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haemodialysis induced transient cd16 monocytopenia and cardiovascular outcome
Nephrology Dialysis Transplantation, 2009Co-Authors: Kyrill S. Rogacev, Danilo Fliser, Maren Ziegelin, C Ulrich, Sarah Seiler, Matthias Girndt, Gunnar H. HeineAbstract:Background. Haemodialysis with bioincompatible membranes led to transient leukocyte activation and intra-dialytic leukopenia due to endothelial adherence. After the introduction ofbiocompatible membranes, only CD16 + (i.e. CD14 ++ CD16 + and CD]4 (+) CD16 + ) monocytes showed an impressive transient intra-dialytic decrease. Presently, it is unclear whether this CD16 + monocyte drop is detrimental. We investigated whether a prominent intra-dialytic decrease of CD16 + monocytes predicts future cardiovascular (CV) events. Methods. We measured leukocyte and monocyte subpopulations in 70 patients before and 10 min after haemodialysis initiation. Patients were stratified by their intra-dialytic CD14 ++ CD16 + monocyte drop (pre-defined major drop: decline of cell counts at 10 min to 5 0% of pre-dialytic counts). Patients were followed up for 42 ± 2 months; endpoints were CV events and death. Results. Patients with a minor CD14 ++ CD16 + monocyte drop had more CV events than patients with a major drop. In multivariate analysis, a minor CD 14 ++ CD16 + monocyte drop was the strongest independent predictor of future CV events [hazard ratio 2.405 (95% CI 1.192―4.854)]. Conclusions. These data refute the assumption that a prominent intra-dialytic decrease of CD14 ++ CD16 + monocytes is detrimental. Instead, a minor cell drop could mirror CD14 ++ CD16 + monocyte dysfunction, with inadequate migratory reaction towards an immunologic stimulus posed by membrane and tubing contact.
Dennis E. Van Epps - One of the best experts on this subject based on the ideXlab platform.
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phenotypic analysis and characterization of cd34 cells from normal human bone marrow cord blood peripheral blood and mobilized peripheral blood from patients undergoing autologous stem cell transplantation
Clinical Immunology and Immunopathology, 1994Co-Authors: James G. Bender, S F Williams, Kristen L Unverzagt, D E Walker, Stephen D Smith, Dennis E. Van EppsAbstract:Abstract Single- and multicolor flow cytometry were used to define progenitor subsets in normal human bone marrow and peripheral blood, cord blood, and blood following mobilization of CD34+ progenitor cells by cyclophosphamide or cyclophosphamide/etoposide/G-CSF treatment. CD34 cells were quantitated and subsets of CD34+ cells were defined by coexpression of CD33, CD13, CD10, CD19, CD45RA, and CD71. Myeloid and erythroid progenitors were quantitated by sorting single CD34+ cells into individual wells of 96-well plates containing methylcellulose, IL-3, GM-CSF, G-CSF, IL-6, and erythropoietin. Comparative studies of CD34 cells showed that the percentage of CD34+ mononuclear cells was greatest in blood samples from patients following mobilization treatment with cyclophosphamide/etoposide/G-CSF averaging 2%. By comparison, the remaining sample groups ranged from 1.68 to 0.15% CD34 cells in this order, bone marrow > cord blood > cyclophosphamide mobilized blood > peripheral blood. Comparison of CD34 cells per milliliter of bone marrow or blood showed a range of 22.4 × 104 to 0.65 × 104/ml in the following order, bone marrow > chemotherapy/etoposide/G-CSF > cord blood > cyclophosphamidemobilized blood. Comparative analysis of CD34 subsets from different sources showed significant differences, particularly bone marrow and blood samples. A distinct population of CD34+ CD19+ (Leu 12) CD10+ (CALLA) pre-B lymphocyte cells was defined in bone marrow with lower side and forward light scatter characteristics and was variable between donors (29.8 ± 16.9%, mean ± 1 SD; range, 3-54%; n = 8). This population was not found to a significant degree in blood and also expressed CD45RA (Leu 18). Coexpression studies of CD45RA and CD71 (transferrin receptor) expression on CD34+ cells defined a CD45RA-CD71+ population containing 89 ± 6.3% (n = 4) BFU-E and a CD45RA+CD71+ population that contained all CFU-GM (n = 4). LeuM7 (CD13) stained a larger percentage to a greater intensity than MY7 (CD13). Coexpression of CD45RA (Leu 18) and CD13 (LeuM7) defined a subset of CD13+ CD45RA+ cells enriched for CFU-GM and CFU-M with a cloning efficiency of 31%. Coexpression of CD33 (MY9) and CD13 (MY7) defined a population that was predominantly CFU-GM with a cloning efficiency of 38%. These studies define CD34+ phenotypes containing pure populations of B lymphocyte, granulocyte-macrophage, or erythroid progenitors and demonstrate the utility of multiparameter flow cytometry to define lineage-committed CD34+ cells.
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characterization of chemotherapy mobilized peripheral blood progenitor cells for use in autologous stem cell transplantation
Bone Marrow Transplantation, 1992Co-Authors: James G. Bender, S F Williams, Sage R Myers, D Nottleman, Kristen L Unverzagt, D E Walker, L B To, Dennis E. Van EppsAbstract:: Twenty patients were treated with chemotherapy to mobilize progenitors into the blood. Peripheral blood stem cells were quantitated in peripheral blood or leukapheresis products using colony assays and flow cytometric measurement of CD34+ cells. In four patients where complete sets of serial samples were obtained, the appearance of CD34+ cells preceded the increase in CFU-GM by 24-48 h. Peak levels of CD34+ cells ranged from 0.6-5% and coincided with the peak increase in CFU-GM. Mobilized CD34+ cells contained subsets expressing CD33, CD13, CD45RA, CD38, HLA-DR, CD61 and CD41. Subsets of CD34+ cells expressing CD33, CD13, or CD45RA represent committed myeloid progenitors. In contrast to bone marrow CD34+ cells, few mobilized CD34+ cells expressed CD71, CD7, CD19 or CD10. Prompt engraftment of granulocytes greater than 500 x 10(6)/l at a median of 13 days and platelets greater than 50 x 10(9)/l at a median of 15 days was observed in patients reconstituted with mobilized cells. These data indicate that CD34+ cells mobilized during recovery from chemotherapy are predominantly myeloid in phenotype and contain few actively proliferating cells or cells with lymphoid phenotypes.
E G Van Lochem - One of the best experts on this subject based on the ideXlab platform.
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immunophenotypic differentiation patterns of normal hematopoiesis in human bone marrow reference patterns for age related changes and disease induced shifts
Cytometry Part B-clinical Cytometry, 2004Co-Authors: E G Van Lochem, V H J Van Der Velden, Henk Wind, J Te G Marvelde, N A C Westerdaal, J J M Van DongenAbstract:Background The abundance of monoclonal antibodies (mAb) and the routine use of quadruple stainings in flow cytometry allow stepwise analysis of bone marrow (BM) samples that are suspected for abnormal hematopoiesis. A screening phase that precedes lineage-specific classification phases should be sufficient to assess whether the BM has a normal or abnormal composition, as well as to identify the abnormal differentiation lineage. Methods For a quick and easy flow cytometric screening of BM samples, we selected six quadruple immunostainings that cover multiple differentiation stages of the B-cell, monocytic, granulocytic, and erythroid lineages: TdT/CD20/CD19/CD10 and CD45/CD34/CD19/CD22 for B cells, CD34/CD117/CD45/CD13.33 for precursor granulocytic and precursor monocytic cells (myelo/monoblasts), CD14/CD33/CD45/CD34 for monocytic cells, CD16/CD13/CD45/CD11b for granulocytic cells, and CD71/CD235a/CD45/CD117 for erythroid cells. Results The six quadruple immunostainings reveal specific staining patterns in normal BM, which allow the recognition of various subpopulations of the respective lineages. These staining patterns can be used as a frame of reference for recognition of normal and abnormal BM development. Examples of normal (age-related) variations in these otherwise stable staining patterns are presented together with several abnormal differentiation patterns. Conclusions Although alternative immunostainings can be used (e.g., including NK- and T-cell markers), we feel that the selected six stainings represent a comprehensive and easy screening phase for quick identification of shifts in the composition of the studied differentiation lineages, reflecting age-related changes or disease-induced BM abnormalities. © 2004 Wiley-Liss, Inc.
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biomed i concerted action report flow cytometric immunophenotyping of precursor b all with standardized triple stainings biomed 1 concerted action investigation of minimal residual disease in acute leukemia international standardization and clinical evaluation
Leukemia, 2001Co-Authors: P Lucio, E G Van Lochem, M W M Van Den Beemd, E R Van Wering, Anna Porwitmacdonald, Giuseppe Gaipa, Andrea Biondi, T Faria, E Bjorklund, E BaarsAbstract:The flow cytometric detection of minimal residual disease (MRD) in precursor-B-acute lymphoblastic leukemias (precursor-B-ALL) mainly relies on the identification of minor leukemic cell populations that can be discriminated from their normal counterparts on the basis of phenotypic aberrancies observed at diagnosis. This technique is not very complex, but discordancies are frequently observed between laboratories, due to the lack of standardized methodological procedures and technical conditions. To develop standardized flow cytometric techniques for MRD detection, a European BIOMED-1 Concerted Action was initiated with the participation of laboratories from six different countries. The goal of this concerted action was to define aberrant phenotypic profiles in a series of 264 consecutive de novo precursor-B-ALL cases, systematically studied with one to five triple-labelings (TdT/CD10/CD19, CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19 and CD19/CD34/CD45) using common flow cytometric protocols in all participating laboratories. The use of four or five triple-stainings allowed the identification of aberrant phenotypes in virtually all cases tested (127 out of 130, 98%). These phenotypic aberrancies could be identified in at least two and often three triple-labelings per case. When the analysis was based on two or three triple-stainings, lower incidences of aberrancies were identified (75% and 81% of cases, respectively) that could be detected in one and sometimes two triple-stainings per case. The most informative triple staining was the TdT/CD10/CD19 combination, which enabled the identification of aberrancies in 78% of cases. The frequencies of phenotypic aberrations detected with the other four triple-stainings were 64% for CD10/CD20/CD19, 56% for CD34/CD38/CD19, 46% for CD34/CD22/CD19, and 22% for CD19/CD34/CD45. In addition, cross-lineage antigen expression was detected in 45% of cases, mainly coexpression of the myeloid antigens CD13 and/or CD33 (40%). Parallel flow cytometric studies in different laboratories finally resulted in highly concordant results (>90%) for all five antibody combinations, indicating the high reproducibility of our approach. In conclusion, the technique presented here with triple-labelings forms an excellent basis for standardized flow cytometric MRD studies in multicenter international treatment protocols for precursor-B-ALL patients.
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flow cytometric analysis of normal b cell differentiation a frame of reference for the detection of minimal residual disease in precursor b all
Leukemia, 1999Co-Authors: P Lucio, E G Van Lochem, Antonio Parreira, M W M Van Den Beemd, E R Van Wering, E Baars, Anna Porwitmacdonald, Elisabet Bjorklund, Giuseppe Gaipa, Andrea BiondiAbstract:During the last two decades, major progress has been made in the technology of flow cytometry and in the availability of a large series of monoclonal antibodies against surface membrane and intracellular antigens. Flow cytometric immunophenotyping has become a diagnostic tool for the analysis of normal and malignant leukocytes and it has proven to be a reliable approach for the investigation of minimal residual disease (MRD) in leukemia patients during and after treatment. In order to standardize the flow cytometric detection of MRD in acute leukemia, a BIOMED-1 Concerted Action was initiated with the participation of six laboratories in five different European countries. This European co-operative study included the immunophenotypic characterization and enumeration of different precursor and mature B cell subpopulations in normal bone marrow (BM). The phenotypic profiles in normal B cell differentiation may form a frame of reference for the identification of aberrant phenotypes of precursor-B cell acute lymphoblastic leukemias (precursor-B-ALL) and may therefore be helpful in MRD detection. Thirty-eight normal BM samples were analyzed with five different pre-selected monoclonal antibody combinations: CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19, CD19/CD34/CD45 and TdT/CD10/CD19. Two CD19- immature subpopulations which coexpressed B cell-associated antigens were identified: CD34+/CD22+/CD19- and TdT+/CD10+/CD19-, which represented 0.11 +/- 0.09% and 0.04 +/- 0.05% of the total BM nucleated cells, respectively. These immunophenotypes may correspond to the earliest stages of B cell differentiation. In addition to these minor subpopulations, three major CD19+ B cell subpopulations were identified, representing three consecutive maturation stages; CD19dim/CD34+/TdT+/CD10bright/CD22dim/CD45dim /CD38bright/CD20- (subpopulation 1), CD19+/CD34-/TdT-/CD10+/CD22dim/CD45+/CD38bright/ CD20dim (subpopulation 2) and CD19+/CD34-/TdT-/CD10-/CD22bright/CD45bright/ CD38dim/CD20bright (subpopulation 3). The relative sizes of subpopulations 1 and 2 were found to be age related: at the age of 15 years, the phenotypic precursor-B cell profile in BM changed from the childhood 'immature' profile (large subpopulations 1 and 2/small subpopulation 3) to the adult 'mature' profile (small subpopulation 1 and 2/large subpopulation 3). When the immunophenotypically defined precursor-B cell subpopulations from normal BM samples are projected in fluorescence dot-plots, templates for the normal B cell differentiation pathways can be defined and so-called 'empty spaces' where no cell populations are located become evident. This allows discrimination between normal and malignant precursor-B cells and can therefore be used for MRD detection.
Andrea Biondi - One of the best experts on this subject based on the ideXlab platform.
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biomed i concerted action report flow cytometric immunophenotyping of precursor b all with standardized triple stainings biomed 1 concerted action investigation of minimal residual disease in acute leukemia international standardization and clinical evaluation
Leukemia, 2001Co-Authors: P Lucio, E G Van Lochem, M W M Van Den Beemd, E R Van Wering, Anna Porwitmacdonald, Giuseppe Gaipa, Andrea Biondi, T Faria, E Bjorklund, E BaarsAbstract:The flow cytometric detection of minimal residual disease (MRD) in precursor-B-acute lymphoblastic leukemias (precursor-B-ALL) mainly relies on the identification of minor leukemic cell populations that can be discriminated from their normal counterparts on the basis of phenotypic aberrancies observed at diagnosis. This technique is not very complex, but discordancies are frequently observed between laboratories, due to the lack of standardized methodological procedures and technical conditions. To develop standardized flow cytometric techniques for MRD detection, a European BIOMED-1 Concerted Action was initiated with the participation of laboratories from six different countries. The goal of this concerted action was to define aberrant phenotypic profiles in a series of 264 consecutive de novo precursor-B-ALL cases, systematically studied with one to five triple-labelings (TdT/CD10/CD19, CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19 and CD19/CD34/CD45) using common flow cytometric protocols in all participating laboratories. The use of four or five triple-stainings allowed the identification of aberrant phenotypes in virtually all cases tested (127 out of 130, 98%). These phenotypic aberrancies could be identified in at least two and often three triple-labelings per case. When the analysis was based on two or three triple-stainings, lower incidences of aberrancies were identified (75% and 81% of cases, respectively) that could be detected in one and sometimes two triple-stainings per case. The most informative triple staining was the TdT/CD10/CD19 combination, which enabled the identification of aberrancies in 78% of cases. The frequencies of phenotypic aberrations detected with the other four triple-stainings were 64% for CD10/CD20/CD19, 56% for CD34/CD38/CD19, 46% for CD34/CD22/CD19, and 22% for CD19/CD34/CD45. In addition, cross-lineage antigen expression was detected in 45% of cases, mainly coexpression of the myeloid antigens CD13 and/or CD33 (40%). Parallel flow cytometric studies in different laboratories finally resulted in highly concordant results (>90%) for all five antibody combinations, indicating the high reproducibility of our approach. In conclusion, the technique presented here with triple-labelings forms an excellent basis for standardized flow cytometric MRD studies in multicenter international treatment protocols for precursor-B-ALL patients.
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flow cytometric analysis of normal b cell differentiation a frame of reference for the detection of minimal residual disease in precursor b all
Leukemia, 1999Co-Authors: P Lucio, E G Van Lochem, Antonio Parreira, M W M Van Den Beemd, E R Van Wering, E Baars, Anna Porwitmacdonald, Elisabet Bjorklund, Giuseppe Gaipa, Andrea BiondiAbstract:During the last two decades, major progress has been made in the technology of flow cytometry and in the availability of a large series of monoclonal antibodies against surface membrane and intracellular antigens. Flow cytometric immunophenotyping has become a diagnostic tool for the analysis of normal and malignant leukocytes and it has proven to be a reliable approach for the investigation of minimal residual disease (MRD) in leukemia patients during and after treatment. In order to standardize the flow cytometric detection of MRD in acute leukemia, a BIOMED-1 Concerted Action was initiated with the participation of six laboratories in five different European countries. This European co-operative study included the immunophenotypic characterization and enumeration of different precursor and mature B cell subpopulations in normal bone marrow (BM). The phenotypic profiles in normal B cell differentiation may form a frame of reference for the identification of aberrant phenotypes of precursor-B cell acute lymphoblastic leukemias (precursor-B-ALL) and may therefore be helpful in MRD detection. Thirty-eight normal BM samples were analyzed with five different pre-selected monoclonal antibody combinations: CD10/CD20/CD19, CD34/CD38/CD19, CD34/CD22/CD19, CD19/CD34/CD45 and TdT/CD10/CD19. Two CD19- immature subpopulations which coexpressed B cell-associated antigens were identified: CD34+/CD22+/CD19- and TdT+/CD10+/CD19-, which represented 0.11 +/- 0.09% and 0.04 +/- 0.05% of the total BM nucleated cells, respectively. These immunophenotypes may correspond to the earliest stages of B cell differentiation. In addition to these minor subpopulations, three major CD19+ B cell subpopulations were identified, representing three consecutive maturation stages; CD19dim/CD34+/TdT+/CD10bright/CD22dim/CD45dim /CD38bright/CD20- (subpopulation 1), CD19+/CD34-/TdT-/CD10+/CD22dim/CD45+/CD38bright/ CD20dim (subpopulation 2) and CD19+/CD34-/TdT-/CD10-/CD22bright/CD45bright/ CD38dim/CD20bright (subpopulation 3). The relative sizes of subpopulations 1 and 2 were found to be age related: at the age of 15 years, the phenotypic precursor-B cell profile in BM changed from the childhood 'immature' profile (large subpopulations 1 and 2/small subpopulation 3) to the adult 'mature' profile (small subpopulation 1 and 2/large subpopulation 3). When the immunophenotypically defined precursor-B cell subpopulations from normal BM samples are projected in fluorescence dot-plots, templates for the normal B cell differentiation pathways can be defined and so-called 'empty spaces' where no cell populations are located become evident. This allows discrimination between normal and malignant precursor-B cells and can therefore be used for MRD detection.