Correspondence: The Influence of Flow Cytometric Gating Strategy and Cell Preparation Procedures on CD34+ Cell Quantification

Abstract

11 BECAU SE THE SM ALL PO PU LATION of cells that express the CD34 antigen is known to correlate with multilineage engraftment, CD34 1 cell quantification by flow cytometric analysis is of great importance in determining the adequate number of hematopoietic stem cells (HSC) for both autologous and allogeneic peripheral stem cell transplantation (1). Flow cytometric enumeration of CD34 1 stem cells varies according to the differences in the method of cell preparation, the reagents used, and the flow cytometric analysis (2). The proposed Milano and ISHAGE guidelines define different gating strategies for CD34 1 cell analysis by flow cytometry (3,4). We analyzed 11 bone marrow (BM) and 11 peripheral blood (PB) samples obtained from healthy donors and 11 leukapheresed stem cell (LSC) harvest samples to compare the influence of cell preparation procedures on the gating strategies. Briefly, 100 m l of sample (LSC and BM diluted to 10 3 103 cells/ m l) was incubated with 10 m l of anti-CD34-PE (BIRMA-K3, Dako, Carpenteria, CA) and 10 m l of anti-CD45-FITC (T29-33, Dako), followed by isotypic controls for 30 min at 4°C in the dark. Red cells were lysed with lysing solution (FACSLyse, Becton Dickinson, Mountain View, CA), and specimens were analyzed without washing or after one wash with PBS or one wash with PBS 1 2% human albumin or two washes with PBS. Following the ISHAGE guidelines and the Milano protocol, listmode data of each sample were analyzed by two different gating strategies. In the Milano protocol, forward scatter (FSC) and side scatter (SSC) were used to gate the nucleated cells and to exclude the RBC, debris, and cell aggregates (live gate) (3). The gated nucleated cell population was plotted on CD34 versus SSC. Only CD34 1 events with low SSC were used to calculate the number of CD34 1 cells, expressed as a percentage of the gated nucleated cells. In the second gating strategy, proposed by ISHAGE, the first gate selected the CD45 1 events, and a second gate chose the CD34 1 events. CD34 1 cells were selected by their CD34 expression, their characteristic low to in-