In 1957, Edward Donnall Thomas reported the first successful bone marrow transplant on a patient (Thomas et al., 1957). Over the years, the quality and safety in hematopoietic stem cell transplants have increased (D'Souza et al., 2020). Different factors need to be considered to make a transplantation successful. One of the critical steps is the precise enumeration of viable CD34+ hematopoietic stem and progenitor cells.

The ISHAGE protocol was first described in 1996 (Sutherland et al., 1996). The single platform approach is currently used for progenitor cell counting by combining CD34, CD45, 7-AAD staining and beads (Keeney et al., 1998). General recommendations includes the acquisition of a minimum of 75,000 CD45+ events with a minimum of 100 CD34+ cells, using a flow cytometer equipped with at least one blue laser and collecting the fluorescence in the green (FITC-CD45), orange (PE-CD34), and red (7-AAD) channels.

Fluid stability is critical for positioning the sample stream for accurate counting of thousands of cells, especially for high throughput applications and reliable rare cell counting. Sheath fluid is delivered to provide constant velocity. The fluidic sensors maintain a constant pressure (Shapiro, 2003). One of the problems with the use of reference microbeads is the phenomenon of vanishing of the counting beads (Brando et al., 2001). This phenomenon occurs when leukapheresis or blood samples containing microspheres are vortexed and diluted in phosphate buffered saline (PBS), resulting in a decrease in the concentration of microspheres and consequently reducing the total and relative number of microspheres in the calibration procedures. In addition, changes in the pressure of the sheath fluid delivered to the nozzle will result in a non-linear or continuous flow rate.

In the last years, flow cytometers including a volumetric system to quantify cells have been developed and may represent a promising alternative to enumerate CD34+ cells avoiding the use of beads (Gisselø et al., 2002; Gutensohn et al., 2012; Mortazavi et al., 2012; Mariani et al., 2014; Saraiva et al., 2019). In this study we have used a direct true volumetric counting of CD34+ cells under continuous flow pump to overcome potential drawbacks with impact in rare cell analysis. To confirm this hypothesis, we have compared the results of CD34+ cell enumeration using non-volumetric vs. volumetric systems with FC500 (Beckman Coulter) and Attune NxT (ThermoFisher) flow cytometers, respectively, in mobilized peripheral blood samples.