Hematopoietic stem cell (HSC) transplantation (HSCT) is most often performed in patients with hematologic disorders such as severe aplastic anemia, leukemia, and multiple myeloma [1^,2]. The transplanted HSCs can be derived from bone marrow (BM), peripheral blood (PB), or cord blood. One of the most important factors affecting the outcome of HSCT is the quantity of progenitor cells infused [3^,4]. Higher cell doses have been associated with lower transplant-related mortality rates, faster engraftment, and better survival [5^,6]. The progenitor cell dose is generally measured in terms of the number of viable CD34-positive (CD34+) cells, because CD34 is the most commonly used marker for HSCs/progenitor cells in clinical hematology [5^,6^,7].

The use of peripheral blood stem cells (PBSCs) in HSCT has increased because of an advantage in the hematopoietic function recovery rate [8]. PBSCs can be collected from allogeneic or autologous donor and are usually harvested from PB using leukapheresis. Historically, flow cytometric enumeration of CD34+ cells is the most widely used tool for optimizing harvest timing and the harvest of HSCs for both BM transplantation and PBSC transplantation [9^,10^,11^,12]. Ensuring a relatively acceptable turnaround time for flow cytometry is also important to assess the optimal timing and duration of the leukapheresis procedure and processing volume. However, the existing flow cytometric methods require large, complex equipment and skilled operators, and they cannot be performed as urgent or point-of-care tests (POCTs).

A previous study evaluated image based-immunofluorescence cell counter ADAMII (NanoEntek, Seoul, Korea) and showed acceptable analytical performance for enumerating CD34+ cells among total nucleated cells (TNCs); however, its enumeration of TNC was not very accurate because of the presence of dead cells and debris [13]. We evaluated the ability of an improved version of ADAMII (NanoEntek) to enumerate CD34+ cells, which simultaneously analyzed CD45 expression from PB and leukapheresis samples. We specifically considered analytical performance and efficacy as a POCT for determining mobilized stem cell collection time and predicting an adequate apheresis stem cell product.

CVs of vCD34 ranged from 3.49% to 9.51% and those of CD34/45 ranged from 2.48% to 10.07% (Table 1). The CVs were within acceptable levels at ≤10%. R² was 0.99 for vCD34 when analyzed with the intended count and flow cytometry data (Fig. 2). No significant changes were observed in vCD45 and vCD34 after sample storage at 4℃ for 48 hours prior to the experiment and storage on ice for one hour before acquisition (Fig. 3).

Regression analysis revealed a strong relationship between the ADAMII and FACSCanto II (Fig. 4). Spearman's rho for vCD45, vCD34, and CD34/CD45 between ADAMII and FACSCanto II were 0.993, 0.996, and 0.989, respectively (P<0.0001), and those between ADAMII and FACSAria II were 0.986, 0.979, and 0.972 (P<0.0001). The slope and intercept of the regression line did not significantly differ between the two methods. The Bland–Altman plot showed no significant bias between vCD45, vCD34, and CD34/CD45 of ADAMII and FACSCanto II (data are presented as mean [95% CI]: 2,303.92 [−8.09 to 4,615.92], −7.32 [−15.62 to 0.98], and −0.01 [−0.02 to 0.002], respectively) or between ADAMII and FACSAria II (−6,043.04 [−10,760.06 to −1,296.02], −38.15 [−61.29 to −15.01], and −0.03 [−0.05 to −0.01], respectively).

It is well-established that the absolute number of CD34+ cells correlates with in vitro colony-forming unit assay activity and with clinical engraftment of hematopoietic grafts prepared from PB, BM, and cord blood sources [2^,15]. Enumeration of CD34+ cells is based on the staining of cells with a labeled monoclonal antibody against CD34, as well as flow cytometry. Commercial stem cell enumeration kits have been used for simultaneous enumeration of viable CD34+ and CD45+ cells in clinical samples [16]. In addition, a non-flow cytometry-based method, the microvolume fluorimetry-based STELLer CD34 assay, has demonstrated excellent analytical performance [15^,17]. ADAMII-CD34, an image-based fluorescence cell counter, is another alternative technology that can be used in conjunction with simpler instruments. We evaluated the improved version of the ADAMII, which includes enumerate viable CD45+ cells using a PerCP channel instead of bright channel. This change reduced false detection of CD34+ cells by simultaneously considering the CD45 expression of each cell (Supplemental Data Fig. S1). The vCD45 results obtained using the PerCP channel were slightly lower than TNC from bright channel and similar to vCD45 obtained using FACSCanto II.

The improved ADAMII-CD34 enumeration system showed excellent analytical performance including precision and linearity. A reproducibility of 10% of the mean between replicates is acceptable for routine clinical application of CD34+ cell enumeration [11]; our CVs of vCD34 were within the acceptable range. Comparison of the data showed no significant differences between the various instruments, and the results of ADAMII was strongly related to the results of both FACSCanto II and FACSAria II. We also found that the samples were stable in 4℃ for 48 hours and could be stored on ice for one hour after adding the RBC lysis solution before capturing images. Our results demonstrated that the ADAMII and two flow cytometry-based assays generated very similar data for a range of PB and leukapheresis samples.

A routine clinical CD34+ cell enumeration assay should possess simplicity, high sensitivity, accuracy, reproducibility, and speed [11]. ADAMII is not only satisfactory in terms of analytical performance but is also advantageous because it is a simple and cost-effective instrument. Additionally, this system is technically simple to use and can be used by less experienced laboratory personnel because of its built-in optics and analysis software. All procedures were finished within 40 minutes; hence, use of this system could reduce the waiting time prior to PBSC collection [18]. Intra-procedural CD34+ cell enumeration is also available to estimate the final stem cell yield and to set fixed-volume apheresis [19^,20^,21]. Furthermore, CD34+ cell enumeration can be repeated on the same day if the precollection PB vCD34 is not sufficient to initiate collection.

In conclusion, ADAMII demonstrated excellent performance as a routine clinical assay with respect to CD34+ cell enumeration obtained from PB and leukapheresis samples. Moreover, it can be used as a POCT for determining optimal timing for mobilized PBSC collection and predicting an adequate apheresis stem cell yield. Our results should be further validated in terms of clinical utility, using a larger number of serial samples from each donor and the relationship of PB vCD34 with vCD34 in leukapheresis sample.

ADAMII-CD34 assay procedure.

Linearity of viable CD34-positive cell count (vCD34) using two samples. vCD34 counts compared with intended values (A, C) and FACSCanto II values (B, D).

Stability results of ADAMII for viable CD45-positive cell count (vCD45) (A) and viable CD34-positive cell count (vCD34) (B), along with box plots showing percentage of recovery (C, D).

Correlations between vCD45, vCD34, and CD34/CD45 in ADAMII and flow cytometry, shown through Passing–Bablok regression analyses (A–F) and Bland–Altman plots (G–L). Data from flow cytometry including FACSCanto II and FACSAria II on the horizontal (x) axis. Data from ADAMII and their differences from flow cytometry on the vertical (y) axis.

Abbreviations: vCD45, viable CD45-positive cell count; vCD34, viable CD34-positive cell count; CD34/CD45, percentage of viable CD34-positive cells among viable CD45-positive cells.