Flow cytometric-based isolation of nucleated erythroid cells during maturation: an approach to cell surface antigen studies.

Nucleated red blood cells (NRBCs) are involved in normal physiologic processes, as well as in several malignancies. They are usually counted manually under the microscope. However, blood sample manipulation may be a source of variability and manual counting is imprecise, time-consuming, and subjective. To improve identification of CD45-negative cells, we used a flow cytometry technique that avoids the addition of lysing reagents and stains viable cell nuclei. We applied this method for counting and isolating NRBC subpopulations in whole blood samples, using DNA/RNA viable staining to discriminate nonnucleated erythroid cells and debris. NRBC counts gave 197.95 cells per mm(3) in mobilized peripheral blood samples (1.00%, n = 20), 3897.59 cells per mm(3) in leukapheresis products (3.08%, n = 20), and 765.21 cells per mm(3) in cord blood samples (6.09%, n = 20). Normal bone marrow counts were 5449.42 cells per mm(3) (11.76%, n = 20). Scatter profiles showed three distinct populations, from early to late-stage erythroblasts, consisting of erythroblasts, orthochromatic erythroblasts, and ejected nuclei, as confirmed by Wright-Giemsa staining. In addition, flow cytometry immunophenotyping showed that glycophorin A was expressed dimly on NRBCs during maturation. These findings point to the feasibility of live NRBCs studies, which offer great potential for a wide range of disciplines.

[1]  H. Tanke,et al.  Detection of fetal erythroblasts in maternal blood by one-step gradient enrichment and immunocytochemical recognition. , 1996, Early human development.

[2]  R. Degowin,et al.  Suppressive effects of an extramedullary tumor on bone marrow erythropoiesis and stroma. , 1978, Experimental hematology.

[3]  I. Syllm-Rapoport,et al.  Separation and characterization of red blood cells from newborns and infants during the first trimenon of life by means of a dextran density gradient. Density distribution curves of erythrocytes and reticulocytes. , 1977, Biology of the neonate.

[4]  A. Seaman,et al.  THE CELLULAR COMPOSITION OF NORMAL BONE MARROW AS OBTAINED BY STERNAL PUNCTURE , 1944 .

[5]  N. Tatsumi,et al.  Counting and differential of bone marrow cells by an electronic method. , 1986, American journal of clinical pathology.

[6]  J. Petriz,et al.  Flow cytometry counting of CD34+ cells in whole blood , 2000, Nature Medicine.

[7]  C. Rogers,et al.  Flow cytometric analysis of human bone marrow perfusion cultures: erythroid development and relationship with burst-forming units-erythroid. , 1996, Experimental hematology.

[8]  R. Gelman,et al.  Analyses of quality assessment studies using CD45 for gating lymphocytes for CD3(+)4(+)%. , 2000, Cytometry.

[9]  W. Nijhof,et al.  Biogenesis of the red cell membrane and cytoskeletal proteins during erythropoiesis in vitro. , 1988, Experimental cell research.

[10]  I. Weissman,et al.  The monoclonal antibody TER‐119 recognizes a molecule associated with glycophorin A and specifically marks the late stages of murine erythroid lineage , 2000, British journal of haematology.

[11]  D Barnett,et al.  Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis. , 2000, Cytometry.

[12]  M. von Lindern,et al.  The use of in vitro expanded erythroid cells in a model system for the isolation of fetal cells from maternal blood , 1999, Prenatal diagnosis.

[13]  D. Danon,et al.  Comparative study of nuclear expulsion from the late erythroblast and cytokinesis. , 1970, Experimental cell research.

[14]  H. Tanke,et al.  Fetal cell detection in maternal blood: a study in 236 samples using erythroblast morphology, DAB and HbF staining, and FISH analysis. , 1998, Cytometry.

[15]  B. Houwen,et al.  New rapid flow cytometric method for the enumeration of nucleated red blood cells. , 1999, Cytometry.

[16]  S. Wickramasinghe,et al.  Observations on the Ultrastructure of Erythropoietic Cells and Reticulum Cells in the Bone Marrow of Patients with Homozygous β‐Thalassaemia , 1975, British journal of haematology.

[17]  H. Rinneberg,et al.  Flow cytometric differentiation of erythrocytes and leukocytes in dilute whole blood by light scattering. , 1998, Cytometry.

[18]  P. Edwards,et al.  Expression of cell-surface HLA-DR, HLA-ABC and glycophorin during erythroid differentiation , 1981, Nature.

[19]  L. Terstappen,et al.  Multidimensional flow cytometric blood cell differentiation without erythrocyte lysis. , 1991, Blood cells.

[20]  B. Pace,et al.  Globin Gene Silencing in Primary Erythroid Cultures , 1997, The Journal of Biological Chemistry.

[21]  R. Neiman.  Erythroblastic transformation in myeloproliferative disorders. Confirmation by an immunohistologic technique , 1980, Cancer.

[22]  M R Loken,et al.  Five-dimensional flow cytometry as a new approach for blood and bone marrow differentials. , 1988, Cytometry.

[23]  P. Secchiero,et al.  Ionizing radiation sensitizes erythroleukemic cells but not normal erythroblasts to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)--mediated cytotoxicity by selective up-regulation of TRAIL-R1. , 2001, Blood.

[24]  Naiman Jl,et al.  Hematologic problems in the newborn. Third edition. , 1982 .

[25]  D. Sutherland,et al.  Single platform flow cytometric absolute CD34+ cell counts based on the ISHAGE guidelines. International Society of Hematotherapy and Graft Engineering. , 1998, Cytometry.

[26]  L. Glasser,et al.  The effect of various cell separation procedures on assays of neutrophil function. A critical appraisal. , 1990, American journal of clinical pathology.