Enumeration of CD34-positive Stem Cells Using the ADAMII Image-based Fluorescence Cell Counter

BACKGROUND: It is very important to accurately enumerate CD34-positive (CD34+) cells for successful hematopoietic stem cell transplantation (HSCT). We evaluated the ability of the newly developed image based-immunofluorescence cell counter ADAMII (NanoEntek, Seoul, Korea) to enumerate CD34+ cells, which was improved through simultaneous CD45 analysis. METHODS: We enumerated CD34+ cells with ADAMII using 19 peripheral blood (PB) and 91 leukapheresis samples from HSCT donors. Analytical performance, including precision and linearity, was analyzed, and sample stability during storage was evaluated. Viable CD34+ cell count (vCD34) and viable CD45+ cell count (vCD45) and the percentage of viable CD34+ cells among viable CD45+ cells (CD34/CD45) as measured by ADAMII were compared with the corresponding values from two flow cytometry assays, using regression analysis. RESULTS: ADAMII demonstrated acceptable precision, as CV values of vCD34 from six samples with different counts were all < 10% (range: 3.49–9.51%). CV values of the vCD45 and CD34/45 ranged from 4.03% to 9.67% and from 2.48% to 10.07%, respectively. The linearity of vCD34 showed an excellent R 2 value (0.99) when analyzed using the intended count and flow cytometry data. The ADAMII and two flow cytometry-based assays generated very similar data for the PB and leukapheresis samples. CONCLUSIONS: ADAMII demonstrated excellent performance for use as a routine clinical assay in terms of CD34+ cell enumeration from PB and leukapheresis samples. Moreover, it could be used as a point-of-care-test for determining mobilization time and predicting an adequate apheresis stem cell product.

Clinical Applicability of Newly Developed Image-based Cell Counter for Counting CD34+ Cells: Comparison with Flow Cytometric Analysis / 임상소아혈액종양

BACKGROUND: Flow cytometric analysis is the standard method for enumerating CD34+ stem cells in hematopoietic stem cell transplantation. However, it has some limitations such as expensive instrumentation, high reagent costs, and discrepancies between technicians and laboratories. We compared counts of total nucleated cells (TNCs) and CD34+ cells counts obtained from a flow cytometer with a newly-developed image-based microscopic cell counter (ADAM II) to evaluate the possibility of clinical application of the ADAM II.METHODS: We used 18 samples of circulating peripheral blood (PB) and waste tube fractions of peripheral blood stem cells (PBSCs) harvested by apheresis after G-CSF mobilization from adult volunteer donors. We assessed the reproducibility and linearity of the new procedure and compared the numbers of TNCs and viable CD34+ cells determined with the ADAM II and two different flow cytometers (FACSCalibur, FACSCanto II).RESULTS: Numbers of viable CD34+ cells determined with the ADAM II were accurate over the expected range; the intra-assay coefficient of variation was ≤19.8%. Linearity was also satisfactory (R²=0.99). TNC counts obtained with the ADAM II were highly correlated with those obtained with the FACSCalibur (R²>0.9841, P<0.0001) and FACSCanto II (R²>0.9620, P<0.0001), as were the numbers of viable CD34+ cells obtained with the ADAM II and the FACSCalibur and FACSCanto II (R²>0.9911, P<0.0001 and R²>0.9791, P<0.0001), respectively.CONCLUSION: The newly developed image-based microscopic cell counter (ADAM II) appears to be suitable for enumerating TNCs and viable CD34+ cells.

Clinical Applicability of Newly Developed Image-based Cell Counter for Counting CD34+ Cells: Comparison with Flow Cytometric Analysis / 임상소아혈액종양

BACKGROUND: Flow cytometric analysis is the standard method for enumerating CD34+ stem cells in hematopoietic stem cell transplantation. However, it has some limitations such as expensive instrumentation, high reagent costs, and discrepancies between technicians and laboratories. We compared counts of total nucleated cells (TNCs) and CD34+ cells counts obtained from a flow cytometer with a newly-developed image-based microscopic cell counter (ADAM II) to evaluate the possibility of clinical application of the ADAM II. METHODS: We used 18 samples of circulating peripheral blood (PB) and waste tube fractions of peripheral blood stem cells (PBSCs) harvested by apheresis after G-CSF mobilization from adult volunteer donors. We assessed the reproducibility and linearity of the new procedure and compared the numbers of TNCs and viable CD34+ cells determined with the ADAM II and two different flow cytometers (FACSCalibur, FACSCanto II). RESULTS: Numbers of viable CD34+ cells determined with the ADAM II were accurate over the expected range; the intra-assay coefficient of variation was ≤19.8%. Linearity was also satisfactory (R²=0.99). TNC counts obtained with the ADAM II were highly correlated with those obtained with the FACSCalibur (R²>0.9841, P0.9620, P0.9911, P0.9791, P<0.0001), respectively. CONCLUSION: The newly developed image-based microscopic cell counter (ADAM II) appears to be suitable for enumerating TNCs and viable CD34+ cells.