Yellow–green laser‐based flow cytometry for CD34+ progenitor cell counting

THE CD34 molecule is a cell surface marker widely used to both identify and isolate hematopoietic progenitor cells. Although this marker was initially identified as an antigen expressed in progenitors cells, it has also been used to detect CD341 leukemic cells, vascular endothelial cells, muscle satellite cells, and epidermal precursors (1–3). Flow cytometry is a rapid and reproducible method to quantify the numbers of circulating CD341 cells following stem cell mobilization with cytokines, as well as to predict the total number of CD341 harvested cells in leukapheresis products. Nowadays, CD341 cell enumeration is routinely used in clinical transplantation centers to optimize stem cell collections for reconstituting the hematopoietic system following myeloablative therapies (2,3). First flow cytometry assays for CD341 cell enumeration used indirect immunofluorescence techniques and red-cell lysing procedures with centrifugation and washing steps. CD341 counts were initially obtained using a dual-platform technique, also known as the Milan protocol. Since then, different methodologies have been described for improved detection of CD341 cells. Using mutiparametric flow cytometry and, in agreement with the International Society of Hematotherapy and Graft Engineering (ISHAGE), CD341 cells are counted in combination with CD45 staining to eliminate nonleukocytes and debris (4). Current flow cytometry-based methods for human hematopoietic progenitor cell counting consist of a series of consensus steps. Most accepted and standardized protocols include a single-platform strategy, absolute counting beads, lyse no-wash procedures and single laser excitation at 488 nm for PE-CD341/FITC-CD45dim/7-AAD negative cell counting. The single-platform ISHAGE protocol is the most widely accepted method for CD341 progenitor cell counting. This methodology follows the more recent recommendations for different monoclonal antibody clones and fluorescent conjugates or different viability dyes for excluding dead cells practice. As an example, the UK NEQAS quality control program has determined that this methodology and minor variations of this assay is used by 95% of participating centers in the United Kingdom, 81% of international participants use ISHAGE in the proficiency testing programme (5). Moreover, the Iberian Society for Cytometry Working Group for counting CD341 cells has highlighted that there is significant subjectivity related to variability in gating, as demonstrated using CD34 in silico studies. Participating laboratories (n 5 50) analyzed four representative FCS data files, showing unexpectedly high coefficients of variation, from 12.62% to 58.72% for CD341 events (6).