Stem Cells, Vol. 16, No. 4, 294-300,
July 1998
© 1998 AlphaMed Press
Direct Measurement of CD34+ Blood Stem Cell Absolute Counts by Flow Cytometry
Junko Fukuda,
Takako Kaneko,
Motoki Egashira,
Kazuo Oshimi
Department of Medicine, Division of Hematology, Juntendo University School of Medicine, Tokyo, Japan
Key Words. CD34+ cells • Hematopoietic stem cells • Flow cytometer • Direct measurement • Absolute number • Gating on mononuclear cells
Dr. Junko Fukuda, Department of Medicine, Division of Hematology, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113, Japan.
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Abstract
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For the collection of adequate numbers of peripheral blood stem cells (PBSC) for PBSC transplantation, an accurate quantification of circulating CD34+ stem cells is required for deciding the optimal time of the collection. To enumerate peripheral blood (PB) CD34+ stem cells, the percentage of CD34+ cells in the gated PB mononuclear cells should be multiplied by the percentage of the gated mononuclear cells among white blood cells (WBC) and by the total WBC count. Accordingly, a minor difference in the measured percentage of the CD34+ cells can lead to a major difference in the PB CD34+ cell concentration. In the present study, we measured the concentration of PB CD34+ stem cells with a flow cytometer designed to provide direct absolute counts of cell subsets from a single instrument. Whole blood was stained with a phycoerythrin-conjugated anti-CD34 monoclonal antibody, and, after the lysis of red blood cells, CD34+ cells were counted in a fraction of the lymphocyte and monocyte gate. The accuracy of our method was demonstrated in an experiment in which various dilutions of known numbers of CD34+ leukemic cells were mixed with normal blood; the predicted value of the CD34+ cell count was observed. The concentration of CD34+ cells in leukapheresis products was measured both by our direct assay and an indirect assay that calculates the number from the percentage of CD34+ cells in mononuclear cells, and our assay was shown to produce less variation. Further, our assay showed a significant correlation between the concentration of mobilized CD34+ cells in the PB and the number of harvested CD34+ cells in leukapheresis. These findings indicate that the monitoring of the concentration of PB CD34+ cells by the present method can be used to predict the number of stem cells collected in leukapheresis. This procedure is easy to perform and can be applied to daily monitoring to decide the appropriate timing for harvest of mobilized stem cells.
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Introduction
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Peripheral blood stem cell transplantation (PBSCT) is being increasingly used for the treatment of patients with hematological malignancies or solid tumors as a replacement for conventional bone marrow transplantation [1-3]. In PBSCT, early hematopoietic recovery after myeloablative therapy seems to depend mainly on the number of peripheral blood stem cells (PBSC) reinfused [4, 5].
To decide the optimal time of collection and to collect adequate numbers of PBSC, the quantification of self-replicating hematopoietic stem cells in the peripheral blood (PB) and leukapheresis products is necessary. The best-established determinant for granulocyte recovery after PBSCT appears to be the number of reinfused granulocyte-macrophage colony-forming units (CFU-GM) [6], but the correlation of this number with platelet recovery is less evident [7]. Due to the CFU-GM assay requirements of specific equipment for culture, refined techniques, and the time to complete the assay, its use to guide stem cell harvests is prohibitive. Another common measure used to guide stem cell harvests is the number of harvested PB mononuclear cells (PBMC), which contain the progenitor cell population. However, although this is the easiest method, the wide variation in the percentage of progenitor cells within this population makes this an unreliable determinant [8].
Previous studies have documented a correlation between the number of reinfused CD34+ cells and the time to hematopoietic recovery [3, 4, 9, 10]. Thus, the measurement of PB CD34+ cells is used as a practical laboratory marker of hematopoietic progenitor cells [4]. Successful engraftment requires 1.5 - 2 x 106 CD34+ cells/kg [9, 11-13]. To obtain the concentration of blood CD34+ cells, the percentage of CD34+ cells measured in the PBMC or lymphocyte fraction of the flow cytogram is multiplied by the percentage of gated PBMC or lymphocyte fraction in white blood cells (WBC) and then by the total WBC count [7, 11, 14]. In these indirect assays, a minor difference in the measured percentage of the CD34+ cells can lead to a major difference in the PB CD34+ cell concentration.
To evaluate the disease progression after human immunodeficiency virus infection, monitoring the absolute number of blood CD4+ cells is important, and a flow cytometer we used has made this monitoring more accurate [15]. We therefore used this flow cytometer to directly measure the precise concentration of CD34+ cells in the PB and leukapheresis products.
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Materials and Methods
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Blood Samples
PB from normal donors and patients with hematological malignancies was directly assayed for the concentration of CD34+ cells. Twenty-two patients with hematological malignancies underwent leukapheresis using a COBE Spectra Apheresis System (Cobe Laboratories; Lakewood, CO). At each apheresis, 0.17 l/kg (mean; range: 0.08-0.28 l/kg) of blood was processed in three to four hours and 49 leukapheresis products were obtained. An aliquot from each apheresis product was tested for CD34+ cell concentration.
Staining with Antibodies
Ten µl phycoerythrin (PE)-conjugated anti-CD34 monoclonal antibody ([mAb] Q BEND 10, Ortho Diagnostic Systems; Tokyo, Japan) were added to 100 µl blood samples. As a negative control, a PE-conjugated mouse isotype-matched IgG1 antibody (DAKO; Glostrup, Denmark) was used. The samples were incubated for 30 min at 4°C, and then 2 ml of erythrocyte-lysing solution (Ortho-Mune Lysing Reagent; Ortho Diagnostic Systems) were added. They were kept at room temperature for 10 min and then at 4°C until use. The leukapheresis products were diluted 10 times with phosphate-buffered saline and processed for the flow cytometric assay.
Flow Cytometry and Data Analyses
After staining, the cells were analyzed by a flow cytometer, CytoronAbsolute (Ortho Clinical Diagnostic, Inc.; Tokyo, Japan), that operates on the basic principle of flow cytometry and analyzes cells according to their size, density, and surface marker, with a function of directly measuring absolute counts. This flow cytometric system represents a single platform from which absolute subset determinations may be made without the need for a separate hematological measurement of the mononuclear cell count.
Calibration of the CytoronAbsolutes was accomplished by running the calibration program provided by Ortho Clinical Diagnostic, Inc. This software requires that the operator enter a target value (such as known absolute lymphocyte count), which is determined on a sample of whole blood by using a hematological instrument or other counting device. The CytoronAbsolute uses syringes, which are driven by precision stepper motors, to deliver sample at a constant and precise flow rate. At a constant flow rate, the volume of sample analyzed is directly proportional to the run time. To calibrate the CytoronAbsolute, a suspension is run that has a known number of events in a specified volume. The time required to reach the expected number of events is measured and stored. Calibration, therefore, is simply measuring the time required to deliver a specific volume of sample [15].
By forward-side scatter, the detection gain parameter was adjusted to make lymphocytes and monocytes occupy one-half to two-thirds of the cytogram area ( Fig. 1A, left), and an electronic gate "a" was set as broadly as possible around the mononuclear cell population in order not to exclude lymphocytes or monocytes. Using a fluorescence analysis with negative control immunoglobulin, the positive rectangle area of gate "b" was set as broadly as possible to not include three or more cells in that gate ( Fig. 1A, middle). We then directly measured the absolute number of PE-conjugated CD34+ cells in the positive area of gate "b" ( Fig. 1B, middle). When PB CD34+ cells harvested from a patient are shown in a forward-side scatter, CD34+ cells are found to be located from an area of medium- to large-sized lymphocytes to an area between those of lymphocytes and monocytes ( Fig. 1B, right).
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Figure 1. Gating and analysis of apheresis products by the CytoronAbsolute flow cytometer. A) (Left panel): By forward-side scatter (FW-Sc) and right-side scatter (RT-Sc), the detection gain parameter was adjusted to make lymphocytes and monocytes occupy one-half to two-thirds of the cytogram area, and the electronic gate "a" was set as broadly as possible around the mononuclear cell population in order not to exclude lymphocytes or monocytes. (Middle panel): Using a fluorescence analysis with negative control immunoglobulin (IgG-PE), positive rectangle area of gate "b" was set as broadly as possible to not include three or more cells in that gate. B) (Middle panel): The absolute number of CD34+ cells reactive to the PE-conjugated anti-CD34 mAb (CD34-PE) was directly measured in the positive area of gate "b." (Right panel): In the FW-Sc and RT-Sc, CD34+ cells were found to be located from an area of medium- to large-sized lymphocytes to an area between lymphocytes and monocytes.
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After counting the cells, Immunocount 2 software (Ortho Clinical Diagnostic, Inc.) was used to acquire, analyze, and process data from the CytoronAbsolute.
Comparison of the Direct and Indirect Assasy
The concentration of CD34+ cells in the leukapheresis products was enumerated by our direct assay and an indirect assay. To enumerate the concentration of CD34+ cells by the indirect assay, the percentage of CD34+ cells measured in the mononuclear cells of apheresis products was multiplied by the percentage of gated mononuclear cells in WBC and then by the total WBC count. We prepared five tubes from each of 10 leukapheresis samples, and the CD34+ cell concentration of each tube was determined with the above two assays.
Statistical Analysis
The numbers of CD34+ cells measured by the two (direct and indirect) methods were compared using the computer software program DAStat. Coefficients of variation (CV) were calculated according to the following formula: standard deviation (SD)/mean x 100 (%).
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Results
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First, a known number of CD34+ cells obtained from a leukemic patient was mixed with the normal PB of a healthy donor at various dilutions, and the actual measured number of CD34+ cells was compared with the predicted number of CD34+ cells in three repeated experiments. The PB of the leukemic patient contained 98% CD34+ leukemic cells, and the PB of a healthy donor contained three CD34+ cells/µl. The comparison of the actual measured value and predicted value, and the CV of measured values are shown in Figure 2. The actual measured number of CD34+ cells was equal to the predicted number of CD34+ cells when the concentration of the CD34+ cells was five cells or higher per µl. However, when the concentration was four cells or lower per µl, the actual measured number tended to be slightly lower than the predicted number, indicating that the use of the flow cytometer in measuring a low concentration of blood CD34+ cells is unreliable. The CV of the actual measured number tended to be larger when concentrations of blood CD34+ cells became lower, as was reported by others [16].
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Figure 2. Measurement of CD34+ cells after dilution. A known number of CD34+ leukemic cells was mixed with normal peripheral blood of a single donor at various dilutions (1,000:0-0:1,000), and the concentration of CD34+ cells was assayed. This value was then compared with the predicted concentration of CD34+ cells. The results of mean values ± SD and CV from three experiments are shown.
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Next, we measured PB CD34+ cells in 15 normal donors, five times for each individual (n = 75), and found that 1.9 ± 1.4 CD34+ cells were present per µl. However, these values were too low to be interpreted, as is shown in Figure 2 and elsewhere [17, 18], and were not different from those obtained in negative controls (data not shown).
The concentration of CD34+ cells in the patient leukapheresis products was enumerated with our direct assay and an indirect assay. We prepared five tubes from each of 10 leukapheresis samples, and the CD34+ cell concentration of each tube was examined by the different methods. The results are shown in Table 1. The mean CV was 3.4% in the direct assay and 9.0% in the indirect assay. In the direct assay, CV values of less than 5% were obtained in eight samples, and in the indirect assay, such values were obtained in two samples, indicating that the direct assay is more reproducible than the indirect assay, and gives minor differences.
As shown in Figure 3, the concentration of patient PB CD34+ cells was assayed immediately prior to the leukapheresis procedure, and the value was compared with that of CD34+ cells in harvested leukapheresis products. A significant correlation was observed (r = 0.87, p < 0.0001), indicating that the enumeration of the mobilized PB CD34+ stem cells can predict the concentration of the harvested CD34+ stem cells in apheresis products and can, therefore, be used to predict the optimal time of PBSC harvest.
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Figure 3. Correlation of the CD34+ cell concentration between the patient PB and the harvested apheresis products. The concentration of PB CD34+ cells assayed immediately prior to the leukapheresis procedure was compared with that of CD34+ cells in the harvested leukapheresis products (n = 49). A significant correlation was observed (r = 0.87, p < 0.0001).
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Figure 4 shows a correlation between the concentration of PB CD34+ cells and the number of harvested CD34+ cells per kg of body weight in the leukapheresis products. The patients underwent leukapheresis for two to four hours and the processed blood volume ranged from 0.08 to 0.28 l/kg (mean, 0.17 l/kg). In Figure 4A, the concentration of PB CD34+ cells is depicted on the x axis, and the actual measured number of CD34+ cells per kg of body weight in leukapheresis products is depicted on the y axis (r = 0.63, p < 0.0001). Because the harvested number of CD34+ cells is affected by the processed blood volume, and the processed blood volume varied from patient to patient, all of the patients were assumed to have undergone blood processing of an equal volume of 0.2 l/kg, and the number of CD34+ cells harvested was corrected based on this assumption. In Figure 4B, the corrected number of CD34+ cells per kg of body weight is depicted on the y axis, and this showed a more significant correlation with the concentration of PB CD34+ cells (r = 0.88, p < 0.0001) than the actual measured number of CD34+ cells shown in Figure 4A. Further, Figure 4B can predict that, for example, a patient PB CD34+ cell concentration of 17.2/µl and blood processing of 0.2 l/kg will be required on average to obtain 2 x 106/kg CD34+ cells in a single apheresis.
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Figure 4. Correlation between the concentration of PB CD34+ cells and the number of harvested CD34+ cells per kg of body weight in leukapheresis products. The concentration of PB CD34+ cells was assayed immediately prior to the leukapheresis procedure (n = 39). The patients then underwent leukapheresis for two to four hours, and the processed blood volume ranged from 0.08 to 0.28 l/kg (mean, 0.17 l/kg). A) The concentration of PB CD34+ cells is depicted on the x axis, and the actual measured number of CD34+ cells per kg of body weight in leukapheresis is shown on the y axis (r = 0.63, p < 0.0001). B) Because the harvested number of CD34+ cells is affected by the processed blood volume, all of the patients were assumed to have undergone blood processing of an equal volume of 0.2 l/kg. Based on this assumption, the corrected number of CD34+ cells per kg was calculated and is depicted on the y axis (r = 0.88, p < 0.0001).
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Discussion
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The use of autologous PBSC, instead of autologous bone marrow, to provide hematopoietic support following myeloablative therapy has been notably increasing in the last few years. To obtain adequate numbers of PBSC, it is important to optimize the monitoring procedures to precisely detect circulating clonogenic stem cells. Problems remain regarding the assessment of the optimal time to start progenitor cell collections and the number of apheresis procedures providing safe engraftment. Clonogenic assays for CFU-GM have been used for monitoring, but they require two weeks to complete, and require refined techniques and specific equipment for culture. These problems can be overcome by an assessment of CD34+ cells. Siena et al. [4] reported a correlation between CD34+ cells and CFU-GM colonies. The method for measuring the CD34+ cells, however, is not standardized. The concentration of blood CD34+ stem cells is usually calculated indirectly from the percentage of CD34+ cells measured in the PBMC or lymphocyte region; i.e., the percentage of CD34+ cells in the PBMC or lymphocyte region is multiplied by the percentage of PBMC or lymphocyte region in WBC and then by the total WBC count. In 1996, the International Society of Hematotherapy and Graft Engineering [19] published a guideline for CD34+ cell determination, but the guideline was also based on an indirect assay. When measurements from two instruments are combined, the variability in the final absolute CD34+ count is greater than the greatest variability in either the flow cytometric or hematologic measurements. We thus used a flow cytometer designed to provide direct absolute counts of cell subsets from a single instrument. Indeed, our direct assay demonstrated smaller variations in CD34+ cell concentration than the indirect assay, as is shown in Table 1.
In an analysis of CD34+ stem cells with flow cytometry, the gating is identified as another major variable among the methods, and that leads to a great variation in the results [20-22]. This variation may not be acceptable for clinical use. Some strategies set the gate on only the lymphocyte region. Leibundgut et al. [7], in contrast, reported a significant correlation between CFU-GM colonies and CD34+ stem cells in the mononuclear cell fraction. Roscoe et al. [17] also reported that gating on the lymphocyte region detects only a mean of 27% of the total CD34+ cells. Other studies [23-25] confirmed the clonogenicity of the CD34+ cells in the monocyte region. For these reasons, we made the gate wide enough around the mononuclear cell population to count all of the CD34+ cells. Indeed, many of the CD34+ cells in this study were located in the region between lymphocytes and monocytes, and some were in the monocyte region, as shown in Figure 1.
Figure 2 shows that the absolute number of circulating CD34+ stem cells can be accurately measured by this method when the concentration of blood CD34+ stem cells is five cells or more per µl. Measurements of lower concentrations of circulating CD34+ stem cells are inaccurate and uninterpretable. Indeed, in the dilution experiments of Figure 2, lower concentrations of CD34+ cells yield a higher CV percentage. A similar tendency is shown by others [16].
The determination of the appropriate timing of the harvest of PBSC is difficult. Some authors [11, 26, 27] suggested that the number of circulating CD34+ cells is more accurate for predicting the CD34+ content of the leukapheretic products than is the peripheral blood WBC count or mononuclear cell count. Figure 3 shows the good correlation observed here between CD34+ cells in the PB and those in the apheresis products. Because circulating CD34+ cells are usually at the level of five cells/µl or more at the time of harvest after intensive chemotherapy and the administration of G-CSF, our present assay can be applied to assess the concentration of PB CD34+ cells, and therefore can be used to assess the optimal time for collecting the circulating hematopoietic progenitor cells. Because it takes less than an hour to enumerate the number of circulating CD34+ cells using our assay, it is possible to quickly determine whether or not leukapheresis should be performed.
Because the number of CD34+ cells harvested in leukapheresis depends on the blood volume processed during leukapheresis, and since the processed blood volume varies among patients, all of the present patients were assumed to have undergone blood processing of an equal volume of 0.2 l/kg, and the number of CD34+ cells harvested was corrected based on this assumption. Figure 4B shows the significant correlation between the concentration of PB CD34+ cells and the corrected number of CD34+ cells per kg of body weight. Thus, when the concentration of CD34+ cells in the peripheral blood is enumerated and the total volume of blood processed is determined, the total number of collected CD34+ cells can be easily estimated.
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Conclusion
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With a flow cytometer designed to provide absolute counts, we directly measured the concentration of mobilized PB CD34+ cells in patients with hematological malignancies. Our direct assay produced less variation in the concentration of CD34+ cells in leukapheresis products than an indirect assay that calculated the number from the percentage of CD34+ cells in mononuclear cells. Further with our direct assay, a significant correlation was found between the number of mobilized PB CD34+ cells and the number of CD34+ cells in harvested apheresis products. Because this procedure can automatically determine the CD34+ stem cell concentration in the PB in a short time, and since the concentration of PB CD34+ cells can be used to predict the number of CD34+ stem cells harvested in leukapheresis, this procedure can be applied to daily monitoring for deciding the optimal time of harvest of the mobilized CD34+ clonogenic stem cells.
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References
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|---|
-
Berenson RJ, Andrews RG, Bensinger WI et al. Antigen CD34+ marrow cells engraft lethally irradiated baboons. J Clin Invest 1988;81:951-955.
-
Berenson RJ, Bensinger WI, Hill RS et al. Engraftment after infusion of CD34+ marrow cells in patients with breast cancer or neuroblastoma. Blood 1991;77:1717-1722.[Abstract/Free Full Text]
-
Langenmayer I, Weaver C, Buckner CD et al. Engraftment of patients with lymphoid malignancies transplanted with autologous bone marrow, peripheral blood stem cells or both. Bone Marrow Transplant 1995;15:241-246.[Medline]
-
Siena S, Bregni M, Brando B et al. Flow cytometry for clinical estimation of circulating hematopoietic progenitors for autologous transplantation in cancer patients. Blood 1991;77:400-409.[Abstract/Free Full Text]
-
Schwartzberg L, Birch R, Blanco R et al. Rapid and sustained hematopoietic reconstitution by peripheral blood stem cell infusion alone following high-dose chemotherapy. Bone Marrow Transplant 1993;11:369-374.[Medline]
-
Douay L, Gorin NC, Mary JY et al. Recovery of CFU-GM from cryopreserved marrow and in vivo evaluation after autologous bone marrow transplantation are predictive of engraftment. Exp Hematol 1986;14:358-365.[Medline]
-
Leibundgut K, von Rohr A, Brulhart K et al. The number of circulating CD34+ blood cells predicts the colony-forming capacity of leukapheresis products in children. Bone Marrow Transplant 1995;15:25-31.[Medline]
-
Brandwein JM, Callum J, Sutchiffe SB et al. Analysis of factors affecting hematopoietic recovery after auto bone marrow transplantation for lymphoma. Bone Marrow Transplant 1990;6:291-294.[Medline]
-
Zimmerman TM, Lee WJ, Bender JG et al. Quantitative CD34 analysis may be used to guide peripheral blood stem cell harvests. Bone Marrow Transplant 1995;9:439-444.
-
Urashima M, Ohkawara J, Hoshi Y et al. Peripheral blood progenitor cell transplantation estimated by three-color (CD34, HLA-DR, CD33) flow cytometry. Acta Haematol 1994;92:23-28.[Medline]
-
Elliott C, Samson DM, Armitage S et al. When to harvest peripheral-blood stem cells after mobilization therapy: prediction of CD34-positive cell yield by preceding day CD34-positive concentration in peripheral blood. J Clin Oncol 1996;14:970-973.[Abstract/Free Full Text]
-
Bensinger WI, Longin K, Applebaum F et al. Peripheral blood stem cells collected after recombinant granulocyte colony stimulating factor: an analysis of factors correlating with the tempo of engraftment after transplantation. Br J Haematol 1994;87:825-831.[Medline]
-
Bender JG, To LB, Williams S et al. Defining a therapeutic dose of peripheral blood stem cells. J Hematother 1992;1:329-341.[Medline]
-
Legros M, Fleury J, Cure H et al. New method for stem cell quantification: applications to the management of peripheral blood stem cell transplantation. Bone Marrow Transplant 1995;15:1-8.[Medline]
-
Connelly MC, Knight M, Giorgi JV et al. Standardization of absolute CD4+ lymphocyte counts across laboratories: an evaluation of the Ortho CytoronAbsolute flow cytometry system on normal donors. Cytometry 1995;22:200-210.[Medline]
-
Sauberlich S, Kirsch A, Serke S. Determination of CD34+ hematopoietic cells by multiparameter flow cytometry: technical remarks. In: Wunder E Sovalat H, Hénon PR et al., eds. Hematopoietic Stem Cells: The Mulhouse Manual. Dayton: AlphaMed Press, 1994:45-60.
-
Roscoe RA, Rybka WB, Winkelstein A et al. Enumeration of CD34+ hematopoietic stem cells for reconstitution following myeloablative therapy. Cytometry 1994;16:74-79.[Medline]
-
To LB, Haylock DN, Dowse T et al. A comparative study of phenotype and proliferative capacity of peripheral blood (PB) CD34+ cells mobilized by four different protocols and those of steady-phase PB and bone marrow CD34+ cells. Blood 1994;84:2930-2939.[Abstract/Free Full Text]
-
Sutherland DR, Anderson L, Keeney M et al. The ISHAGE guideline for CD34+ cell determination by flow cytometry. J Hematother 1996;5:213-226.[Medline]
-
Brecher ME, Sims L, Schmitz J et al. North American multicenter study on flow cytometric enumeration of CD34+ hematopoietic stem cells. J Hematother 1996;5:227-236.[Medline]
-
Johnson HE, Knudsen LM. Nordic flow cytometry standards for CD34+ cell enumeration in blood and leukapheresis products: report from the second Nordic workshop. J Hematother 1996;5:237-245.[Medline]
-
Chang A, Ma DDF. The influence of flow cytometric gating strategy on the standardization of CD34+ cell quantitation: an Australian multicenter study. J Hematother 1996;5:605-616.[Medline]
-
Fritsch G, Stimpfl M, Kurz M et al. The composition of CD34 subpopulations differs between bone marrow, blood and cord blood. Bone Marrow Transplant 1996;17:169-179.[Medline]
-
Andrews RG, Singer JW, Bernstein ID. Precursors of colony-forming cells in humans can be distinguished from colony-forming cells by expression of the CD33 and CD34 antigens and light scatter properties. J Exp Med 1989;169:1721-1731.[Abstract/Free Full Text]
-
Teofili L, Rutella S, Pierelli L et al. Separation of chemotherapy plus G-CSF-mobilized peripheral blood mononuclear cells by counterflow centrifugal elutriation: in vitro characterization of two different CD34+ cell populations. Bone Marrow Transplant 1996;18:421-425.[Medline]
-
Knudsen LM, Gaarsdal E, Jensen L et al. Improved priming for mobilization of and optimal timing for harvest of peripheral blood stem cells. J Hematother 1996;5:399-406.[Medline]
-
Armitage S, Hargreaves R, Samson D et al. CD34 counts to predict the adequate collection of peripheral blood progenitor cells. Bone Marrow Transplant 1997;20:587-591.[Medline]
accepted for publication May 29, 1998.
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Abstract
Introduction
