A Novel Enumeration Method for Hematopoietic Progenitor Cells in Collecting Peripheral Blood Stem Cell for Transplantation
Authors: Tanosaki Ryuji , Kumazawa Tomoko , Yoshida Shigehisa , Nakano Atsuya , Yamagata Shizuka , Takahashi Noriko , Kurosawa Saiko , Mori Shin-Ichiro , Heike Yuji , Fukuda Takahiro , Furuta Ko , Tsuda Hitoshi 
Published: October 2019

Abstract
Abstract 1922
The number of infused CD34+ cells is crucial to the success of peripheral blood stem cell transplantation (PBSCT). Although counting CD34+ cells currently depends solely on flow cytometry technology, the complexity of the procedure and the high cost of reagents (including monoclonal antibodies) are the main disadvantages. The SYSMEX SE-9000 (SE) and XE-2100 (XE) automated hematology analyzers quickly estimate the number of immature cells, referred to as hematopoietic progenitor cells (HPCs), at very low cost. The number of peripheral blood SE/XE-determined HPC (SE/XE-HPC) is used to determine the optimal timing of peripheral blood stem cell (PBSC) collection. However, SE/XE-HPCs are limited as a substitute for CD34+ cells because they are likely to be affected by co-existing immature cells (e.g. immature granulocytes), resulting in overestimation of the HPC count. Therefore, we developed a new technology for counting HPCs. The assay's mechanism is based on finely-tuned hemolysis reactions and chemical staining with a specific dye, and does not require monoclonal antibodies. The assay is followed by a flow cytometry-based optical detection technique that differs from the SE or XE former types, which use the electrical radiofrequency/direct currency impedance detection method. This modified program has been installed into an revised model of an automated hematology analyzer, the XN Prototype (SYSMEX corporation, Kobe, Japan), which enables us to cost-effectively obtain the number of new, marked HPCs, designated as 'XN-determined HPC (XN-HPC)', within 4 minutes using small (200 μL) samples. The purpose of this study is to evaluate the XN-HPC in comparison with CD34+ cells, and this is the first report of the results.
Between 2008 and 2011, a total of 87 blood or G-CSF-mobilized apheresis samples were taken from healthy donors (n=20) or patients undergoing autologous PBSCT (n=5) at the National Cancer Center Hospital, Japan. Next, CD34+ cells and XN-HPCs were analyzed in the same samples. XN-HPCs were counted using the XN Prototype, and CD34+ cells were quantified using a flow cytometer (FACSCalibur, BD, New Jersey, USA) using the dual platform method according to the International Society of Hematology and Graft Engineering protocol. This study was approved by Institutional Review Board, and informed consent was obtained from all patients.
There was a very good correlation between the numbers of XN-HPCs and CD34+ cells (R2=0.952) in all samples, at a wide range of CD34+ cell concentrations (range; 0.3–12830.5 cells /μL) (Fig. 1). The correlation was unaffected by WBC counts, use of EDTA as an anticoagulant, sample type, or timing of collections. The XN-HPC concentration in the 3L-apheresis products (3L-HPCs) correlated well with CD34+ cell concentration in the final products (R2=0.948). The estimated total number of XN-HPCs in the final products, which was calculated from the 3L-HPC concentration or pre-apheresis HPC concentration in the peripheral blood (PB-HPCs), also correlated well with the total number of CD34+ cells in the final products (R2=0.918 or 0.950, respectively), suggesting that the final amount of collected CD34+ cells could be predicted from the total number of HPCs in the final products, as well as from pre-apheresis PB-HPCs and from the intermediate products during apheresis (3L-HPCs). The change in PB-HPCs closely resembled that of CD34+ cells during the bone marrow recovery phase after chemotherapy (Fig. 2), also suggesting that XN-HPC might be a good indicator for the optimal timing of PBSC collection. In conclusion, XN-HPC could be a surrogate for CD34+ cells in PBSCT, and further investigation of their usefulness and clinical applications are warranted.

Disclosures:
Tanosaki: Sysmex Corporation: They provided hematology analyzers, a flow cytometer and reagents.