P007 Purity and yield optimization of EasySep human whole blood CD34 + cell isolation kit

Abstract

Aim Relapse of the underlying hematological malignant disease after stem cell transplantation remains a major cause of mortality. Decline of CD34 + cell subset donor chimerism is an early marker of relapse before it can be detected in unfractionated peripheral blood or other cell subsets. One of the main challenges in performing CD34 + cell donor chimerism is obtaining pure CD34 + cell due to very low frequency of this cell population in peripheral blood. In this report, we describe our experience in optimizing the EasySep Human Total CD34 + isolation protocol to maximize the yield without compromising the purity of this rare cell subpopulation. Methods CD34 subset is isolated from 6 ml of buffy coat using EasySep Human Total CD34 + Kit, StemCell Technologies. CD15+, CD3+, CD19+, and CD34 + subsets are isolated by immunomagnetic positive selection in series from buffy coat. First, CD15 + subset is isolated using 1 ml buffy coat. The remaining 5 ml sample is used to obtain pure lymphocyte using Lymphocyte Enrichment Kit. CD3 + subset is isolated and followed by CD19 + subset isolation from supernatant of CD3 + subset isolation. Supernatant of CD19 + subset isolation was treated with RosetteSep cocktail provided in the kit and 10% whole blood mixture. CD34 + subset was then isolated from RosetteSep cocktail treated supernatant. Lineage-specific subsets were measured in flow cytometry. Results CD34 + purity was confirmed via flow cytometry. Conclusions The optimized method improved the efficiency of vendor recommended protocols by using less starting blood sample and producing higher yield and purity. Download high-res image (307KB) Download full-size image