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Cytometry Part B (Clinical Cytometry) 74B:182–188 (2008)

Electronic Volume of CD34 Positive Cells from Peripheral Blood Apheresis Samples Sherry Shariatmadar, Siddharth Sharma, Raquel Cabana, Scott Powell, Phillip Ruiz, and Awtar Krishan* Department of Pathology, Jackson Memorial Medical Center and Sylvester Cancer Center, University of Miami Miller School of Medicine, Miami, Florida

Background: The authors have used a flow analyzer to measure electronic cellular volume of peripheral blood hematopoietic stem/progenitor cells obtained by granulocyte-colony stimulating factor (G-CSF) mobilization and apheresis (HPC-A) of patients with hematological malignancies. Methods: Fifty three apheresis samples stained with CD45-fluorescein isothiocyanate (FITC) and CD34R-phycoerythrin (PE)-labeled antibodies after erythrocyte lysis with BD FACSTM Lysing Solution were analyzed for electronic cell volume and two-color FITC and PE fluorescence. Results: Lymphocytes, monocytes and granulocytes in the HPC-A samples had a mean electronic volume of 414, 797, and 670 lm3, respectively corresponding to cell diameter of 9.25, 11.5, and 10.85 lm. In 53 HPC-A samples analyzed, the mean electronic volume of the CD34 positive mononuclear cells was 407 lm3 while the CD45 positive cells had mean volume of 453 lm3. Conclusions: CD34 positive stem/progenitor cells have a smaller volume and diameter than CD45 positive mononuclear cells in HPC-A samples. In the present study the CD34 stem/progenitor cells had a considerably larger diameter than that of stem cells previously reported in the literature. With the availability of electronic cell volume as a parameter in flow cytometric analysis, further studies can be carried out to correlate stem cell volume with specific phenotypic marker expression. q 2008 Clinical Cytometry Society

Key terms: stem cells; hematopoietic progenitors; CD34 antigen; electronic cell volume

How to cite this article: Shariatmadar S, Sharma S, Cabana R, Powell S, Ruiz P, Krishan A. Electronic volume of CD34 positive cells from peripheral blood apheresis samples. Cytometry Part B 2008; 74B: 182–188.

Peripheral blood hematopoietic progenitor cells obtained by granulocyte-colony stimulating factor (GCSF) mobilization and apheresis (HPC-A) contain both the committed progenitors capable of producing shortterm hematopoietic engraftment as well as primitive stem cells essential for long-term bone marrow reconstitution (1,2). Most human hematopoietic stem cells and early committed hematopoietic progenitors bear the surface glycoprotein CD34 (3), which has long been recognized as a marker for cells able to provide short- and long-term hematopoietic reconstitution following high dose chemotherapy and transplantation (4). Several studies have described early stem cells from bone marrow and other tissues to be homogeneous with small size (3–7 lm), large nucleus, and scant cytoplasm (5,6). Fluorescence-activated cell sorters have been used to report size and morphology of cells with stem cell

q 2008 Clinical Cytometry Society

phenotype (7). Radley et al. (5) collected drug effluxing stem cells from mouse bone marrow with very high clonogenecity and described them to be 4.6 6 0.2 lm in size. Ratajczak et al. described human bone marrow stem cells with CD34 and CD117 positive expression and low rhodamine 123 retention to be the size of ‘‘quiescent lymphocytes’’ (7). Berardi et al. (8) described

*Correspondence to: Awtar Krishan, Department of Pathology, University of Miami Miller School of Medicine, Mail locator R-71, P.O. Box 016960, Miami, FL 33101, USA. E-mail: [email protected] Received 2 July 2007; Revision 30 August 2007; Accepted 19 November 2007 Published online 27 March 2008 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/cyto.b.20399

ELECTRONIC VOLUME OF CD34 POSITIVE CELLS FROM PERIPHERAL BLOOD APHERESIS SAMPLES

CD34 and C-kit positive, lineage negative cells in human bone marrow to be small in size (6 lm). As phenotypic analysis in a laser flow cytometer provides a rapid, quantitative, and reproducible method for identification and enumeration of CD34 positive cells, forward angle light scatter has been used as an approximate measure of stem cell size (9). However, light scatter is influenced by a number of factors and may not be an accurate measure of cell size (10). We have described the development and applications of the QuantaTM SC MPL system (henceforth referred to as Quanta analyzer), which can simultaneously measure electronic cell volume and fluorescence of phenotypic markers (11,12). In the present study, we used Quanta analyzer to determine electronic cell volume of CD34 positive cells with CD45dim expression from peripheral blood HPC-A samples of patients mobilized with G-CSF. MATERIALS AND METHODS Subjects The University of Miami Medical School Institutional Review Board approved the study protocol, and informed consent was obtained from patients with hematologic malignancies referred for autologous hematopoietic progenitor cell apheresis (HPC-A). Large volume leukapheresis was performed using a continuous flow blood cell separator (COBE Spectra, Gambro, Englewood, CO) following 5 days of mobilization by subcutaneous administration of 5–10 lg/kg/day of G-CSF. At the completion of leukapheresis, 1 ml of the final HPC-A product was used for CD34 enumeration. After analysis on a FACSCaliburTM flow cytometer (BD Biosciences, San Jose, CA) in the clinical laboratory at the Jackson Memorial Medical Center (henceforth referred to as the clinical laboratory), samples were analyzed on a Quanta analyzer in our laboratory. A total of 53 HPC-A samples were analyzed in the present study. The patient diagnosis included multiple myeloma (n 5 27), non-Hodgkin’s lymphoma (n 5 14), Hodgkin’s lymphoma (n 5 10) and acute myelogenous leukemia (n 5 2). Peripheral blood samples from volunteers were used for monitoring the effect of erythrocyte lysis on electronic volume of the mononuclear cells. Flow Cytometric Analysis The percentage of CD34 positive cells was determined using immunofluorescence staining with FITC-labeled CD45 antibody (clone 2D1, catalog number 340664) and PE-labeled CD34 antibody (clone 8G12, catalog number 340669) obtained from BD Biosciences, San Jose, CA. IgG1 fluorochrome-matched control isotype antibodies were used for correction of background staining. Samples were analyzed on a Becton Dickinson FACSCalibur flow cytometer using excitation wavelength of 488. Emission of FITC and PE fluorescence was collected by the use of appropriate filters. In the clinical laboratory, the percentage of CD34 positive cells amongst the gated

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mononuclear cells was determined by the Milan-Mulhouse protocol (13,14). Aliquots were incubated with CD45-FITC, CD34-PE antibodies or the appropriate isotype control for 15 min at room temperature. Erythrocytes were lysed by incubation for 15 min with BD FACSTM Lysing Solution (BD Biosciences). Samples were centrifuged for 5 min and cell pellets resuspended in 1 ml phosphate buffered saline (PBS). In both the laboratories, the percentage of CD34 positive cells per 100,000 mononuclear cells was recorded. The Quanta analyzer uses a triangular flow cell (transducer) through which cells suspended in a weak electrolyte flow between two electrodes. The displacement of the electrolyte by a cell increases the impedance which in turn is used by the Coulter Principle (15) for measurement of volume. Electronic volume, fluorescence and side scatter signals were amplified, digitized, and then analyzed for pulse height and generation of dot plots and histograms. The Quanta analyzer data collection software was used for control of the analyzer and the multiplate loader, sample introduction and fluidics and for data acquisition and real-time analysis. Data acquired was saved in a Microsoft SQL Server database and retrieved by the analysis software for further processing and export to a list mode file. The analysis software provides multiple capabilities including protocol and region creation, statistical analysis, gating, compensation, electronic volume calibration and for viewing and reporting of results. This software can create up to 64 regions and logical regions for Boolean AND and OR gating. Electronic cell volume measurements were calibrated using National Institute of Standards and Technology (NIST) traceable beads obtained from Beckman Coulter, Miami, FL (Standard L5, 5.15 lm, Standard L10, 10.08 lm) and 8.18 lm beads from Polysciences (Warrington, PA). Bead diameter was confirmed under a microscope using a stage micrometer slide. Calibration data could be expressed either as electronic volume (lm3) or calculated as diameter of a spherical cell (lm). To monitor the effect of sample preparation on electronic volume, peripheral blood samples from a male and female volunteer were incubated with phosphate buffered saline (PBS), BD FACSTM Lysing Solution (BD Biosciences) or ImmunoPrepTM (Beckman Coulter) and analyzed on the Quanta for cell volume. Single color compensation was performed using Cytotrol1 (Beckman Coulter) stained with single antibodies (CD45-FITC and CD45-PE). The stained samples were analyzed for side scatter, electronic cell volume and two-color fluorescence emission of CD45-FITC and CD34-PE on the Quanta analyzer using default filter configuration of 525 BP (FL1), 575 BP (FL2), and 670 LP (FL3) with excitation from a 488-nm solid-state laser. Electronic gates were used in dot plots of side scatter versus electronic volume to exclude debris and platelets and select the mononuclear cells or lymphocytes for further analysis.

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tion were larger with a mean volume of 639 lm3 (diameter 10.7 lm), as shown in Figure 2E. Gating Strategy Used in the Quanta Analyzer

FIG. 1. Bland-Altman plot of mean and standard deviation of the percent of CD34 positive cells detected on the FACSCalibur and the Quanta analyzer. The mean difference value was 0.06 and most of the values were within the confidence range of þ0.29 and 20.17, except for three outliers. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

RESULTS Comparison of Data from FACSCalibur and Quanta Analyzer Figure 1 shows a Bland-Altman plot of data on the percent of CD34-PE positive cells analyzed on the FACSCalibur and the Quanta analyzer. This plot shows the mean and standard deviation of the percentage of CD34 positive cells reported on the two platforms. The mean difference value of 0.06 indicates that the percentage of CD34 positive cells measured by the two instruments was approximately similar. Most of the values were within the confidence interval of þ0.29 and 20.17, except for three outliers. Linearity of Electronic Volume Measurement and the Effect of Sample Preparation Plots in Figure 2 show electronic volume (Fig. 2A) and mean diameter (Fig. 2B) of NIST beads with diameters of 5.15, 8.18, and 10.08 lm analyzed on the Quanta analyzer. Correlation coefficient (r2) of mean channel value with mean volume was 0.998 indicating that linearity of electronic volume measurement in the Quanta analyzer was excellent. Dot plots in Figures 2C–2E show the effect of incubation with phosphate buffered saline (PBS) or the erythrocyte lysis solutions (BD FACSTM Lysing Solution from BD Biosciences and ImmunoPrepTM reagent from Beckman Coulter) on electronic volume and side scatter of mononuclear cells in a peripheral blood sample. In general, lymphocytes incubated with PBS (Fig. 2C) had mean volume of 204 lm3 (diameter 7.3 lm) as compared with lymphocytes treated with the ImmunoPrepTM (Fig. 2D), which had mean volume of 196 lm3 (diameter 7.2 lm). In contrast, cells incubated with BD FACSTM Lysing Solu-

As shown in Figure 3A, electronic gates were used to exclude debris and platelets with subsequent selection of mononuclear cells for further analysis. Plots in Figure 3B show that the lymphocytes, monocytes, and granulocytes in the HPC-A samples prepared after erythrocyte lysis with BD FACSTM Lysing Solution had a mean electronic volume of 414, 797, and 670 lm3, respectively corresponding to cell diameters of 9.25, 11.5, and 10.85 lm. Dot plot in Figure 3C of CD45 vs. CD34 expression shows that CD34 positive cells (0.17% of the total mononuclear cells) had CD45dim expression. In dot plot in Figure 3D, CD34 positive expression is seen in 0.24% of the gated lymphocyte population. CD34 Versus Side Scatter, Electronic Volume, and CD45 Expression In samples evaluated on the Quanta analyzer (Figs. 4A and 4B), CD45-FITC fluorescence was characterized as very dim (recorded in first log on x-axis), dim (second log), or bright (third log). Vertical line of the quadrants was set to distinguish CD45very dim cells from those with dim and bright expression. The horizontal line of the quadrant was set to exclude 95% of CD34 positive cells from the isotype control. Dot plot in Figure 4A is of cells stained with CD45-FITC antibody and the isotype for CD34-PE, while dot plots in Figure 4B is of cells stained with CD45-FITC and CD34-PE antibody. In dot plot Figure 4B, most of the CD34-PE positive cells (0.35% of the total mononuclear cells) had CD45dim expression while only a few CD34-PE positive cells (0.02%) had CD45very dim expression. In dot plots in Figures 4C and 4D, we have plotted cell diameter (lm) vs. CD34 and CD45 expression of the mononuclear cells from a HPC-A sample. In Table 1, we have plotted data on electronic volume of CD34/ CD45dim cells and the CD45 positive mononuclear cells from the 53 apheresis samples. The mean volume of the CD34 cells was 407 lm3 with standard deviation of 69.6. In contrast the CD45 positive cells had mean volume of 453 lm3 with a standard deviation of 75.4. DISCUSSION CD34 antigen identifies cells in the early stages of hematopoietic differentiation. This population contains progenitors committed to myeloid, erythroid, megakaryoid, and lymphoid lineages, as well as primitive stem cells capable of long term reconstitution (1,2,16). The length of time to neutrophil and platelet engraftment in the recipient following high dose chemotherapy and transplantation in general correlates well with the number of CD34 positive cells in the transplanted product. CD34 dim and negative primitive stem cells with selfrenewal capability have also been reported, suggesting



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FIG. 2. Mean cell volume (A) and mean diameter (B) of NIST beads analyzed on the Quanta analyzer. Correlation of mean channel value with mean cellular volume had a coefficient (r2) of 0.998. Dot plots in (C–E) show the effect of incubation with PBS or the erythrocyte lysis solutions on electronic volume and side scatter of mononuclear cells. Lymphocytes incubated with PBS or ImmunoPrepTM (C, D) had approximately similar mean volume while cells incubated with FACSTM Lysing Solution were significantly larger (E). [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

that more than one phenotypically identifiable population can produce long-term hematopoietic reconstitution (17). As suggested by Gratama et al. (13), CD34 dim cells may include precursors committed to erythroid, myeloid and lymphoid lineages and thus may be involved in short term engraftment. Current methods for identification and enumeration of CD34 positive cells cannot easily separate the more primitive and committed cell populations and clearly remain a limiting factor in determining the true number and type of stem/progenitor cells required for successful hematopoietic reconstitution (18). Significant site-to-site variability in quantitation of CD34 positive cells attributed to different sample preparation techniques and reagents has been previously reported. Gratama et al. (19), suggest that the use of single-platform ISHAGE pro-


tocol can reduce the variation in enumeration of absolute CD34 positive numbers between different laboratories. In a recent comparison of the various methods for CD34 enumeration in leukapheresis/bone marrow products, Gajkowska et al. (14) reported a high correlation among the three methods of single- and double-platform ISHAGE and Milan-Mulhouse protocols for determination of CD34 cells in leukapheresis samples. Several studies have reported on the size of stem cells in bone marrow and in different tissues. Matsuoka and Tavassoli described small cells measuring 4–5 lm with high nuclear cytoplasmic ratio characteristics of hematopoietic progenitor cells (20). In mouse bone marrow, Radley et al. (5) described the stem cells with a mean diameter of 4.6 6 0.2 lm. Vacanti et al. (6) identified adult stem cells ranging in size from 3 to 7 lm. Berardi et al.

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FIG. 3. (A) Gates used in Quanta analyzer to exclude debris and platelets and select mononuclear cells for further analysis. Histograms in (B) show that the lymphocytes, monocytes, and granulocytes in this HPC-A sample prepared after erythrocyte lysis with BD FACSTM Lysing Solution had a mean electronic volume of 414, 797, 670 lm3, respectively corresponding to cell diameters of 9.25, 11.5, and 10.85 lm. Dot plot in (C) of CD45 vs. CD34 expression in the mononuclear cells of a HPC-A sample show that most of the CD34 positive mononuclear cells (0.17 % of the total) had CD45dim expression. Dot plot in (D) of gated lymphocytes shows that in this sample, 0.24% of the lymphocytes had CD34 positive expression. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

(8) described CD34, c-Kit positive and lineage marker negative human stem cells to be small in size (6 lm). A recent report has suggested the presence of a heterogeneous CD45 negative nonhematopoietic tissue committed stem cells in bone marrow (21). These cells measured 5–7 lm in size with nuclear DNA resembling that of embryonic stem cells. The cells were CXCR4 negative, CD34 positive, AC133 positive, lineage negative, and CD45 negative in humans. The authors hypothesized that these cells could also be present in the so-called side population of marrow mononuclear cells and have low retention of Hoechst 33342, pyronin, and rhodamine 123. Because of their small size, the cells would easily be lost during isolation procedures, especially those employing density gradient or velocity centrifugation. To our knowledge, the present study is the first accurate measurement of electronic cell volume in hemato-

poietic stem/progenitor cells in a flow cytometer. In our study, the CD34 positive/CD45 dim cells had mean electronic volume of 407 lm3. As shown in comparison of the peripheral blood lymphocytes incubated with the two erythrocyte lysing solutions, in general HPC-A samples prepared with the BD FACSTM Lysing Solution tend to be larger than those treated with the ImmunoPrepTM Reagent or PBS. The CD34 positive cells in our samples were obviously much larger than the stem cells reported by other workers using nonflow cytometric methods such as light or electron microscopy. It is possible that the methods used for collection of the HPC-A samples do not favor collection of the small CD34 cells and only cells with large volume are preferentially collected. The CD34 positive stem/progenitor cells in the HPC-A samples showed heterogeneity in their volume. It is likely that these cells represent various committed progenitors at different stages of hematopoietic differentia-



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FIG. 4. Dot plots are of cells from a HPC-A sample analyzed on the Quanta analyzer for CD45 vs. CD34 expression. The quadrant horizontal line was set to exclude 95% of CD34 positive cells from the isotype controls in (A). In dot plot (B), the CD34-PE positive cells (0.35% of the total) had CD45dim expression. In (C,D), electronic volume vs. CD45 and CD34 expression of the mononuclear cells from a HPC-A sample are plotted. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

tion and may include common lymphoid and myeloid progenitors as well as the more mature megakaryocytic and granulocyte/monocyte precursors. Further correlation of cell volume with various cell surface markers specific for early and committed progenitors may lead to

better characterization of these cells in apheresis and bone marrow samples. Since the Quanta analyzer can measure volume of cells as small as 4 lm, it may be valuable to study the presence of these small primitive cells in the HPC-A samples

Table 1 Statistical Comparison of Electronic Volume of the CD34/CD45dim and CD45 Positive Mononuclear Cells from the 53 Apheresis Samples Arithmetic mean (lm) 95% CI for the mean Median 95% CI for the median Variance Standard deviation Relative standard deviation Standard error of the mean Coefficient of Skewness Coefficient of Kurtosis


CD34

CD45

407.1836 385.8025–428.1647 404.6800 380.9608–427.6937 4853.6737 69.6683 0.1712 (17.12%) 10.5029 20.3785 (P 5 0.2743) 0.9640 (P 5 0.1754)

453.3620 430.9625–475.7616 451.9000 420.2312–486.4216 5428.1695 75.4761 0.1625 (16.25%) 11.1071 20.1503 (P 5 0.6587) 20.9209 (P 5 0.1883

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and correlate their presence with the quality of bone marrow reconstitution following transplantation. We expect cells with the smallest cell volume to be primitive, CD45 dim, CD34 positive, without any markers of lineage differentiation and capable of long term marrow reconstitution. In contrast, the heterogeneous population of CD45 and CD34 positive cells with large volume may have marker expression associated with advanced lineage differentiation able to provide short-term engraftment. Further studies are clearly needed to elucidate the role of these CD34 subsets in short and long term hematopoietic reconstitution. The use of electronic cell volume in combination with side scatter and fluorescence may be a simple and rapid method for characterization of stem cell/progenitor cell populations. Future studies will aim to further characterize these cells based on correlation of cell volume with cell surface marker expression. The information obtained may prove to be of prognostic value and impact clinical outcome. ACKNOWLEDGMENTS The authors are thankful to Dr. Azorides Morales, Chairman of the Pathology Department at the University of Miami Miller School of Medicine, for supporting this pilot study. LITERATURE CITED 1. Molineux G, Pojda Z, Hampson IN, Lord I, Dexter TM. Transplantation potential of peripheral blood stem cells induced by granulocyte colony-stimulating factor. Blood 1990;78:2153–2158. 2. Haas R, Witt B, Moble R, Goldschmidt H, Hohaus S, Freuhauf S, Wannenmacher M, Hunstein W. Sustained long-term hematopoiesis after myeloablative therapy with peripheral blood progenitor cell support. Blood 1995;85:3765–3761. 3. Sutherland DR, Keating A. The CD34 antigen: structure, biology, and potential clinical applications. J Hematotherap 1992;1:115– 129. 4. Weaver CH, Hazelton B, Birch R, Palmer P, Allen C, Schwartzberger L, West W. An analysis of engraftment kinetics as a function of the CD34 content of peripheral blood progenitor cell collections in 692 patients after the administration of myeloablative chemotherapy. Blood 1995;86:3961–3969. 5. Radley JM, Ellis S, Palatsides M, Williams B, Bertoncello I. Ultrastructure of primitive hematopoietic stem cells isolated using probes of functional status. Exp Hematol 1999;27:365–369.

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