Robust Multi-Parameter Single-Platform Quantification of Myeloid and B-Lymphoid CD34 Progenitor Cells in All Clinical CD34 Cell Sources and in Thawed PBSC

Abstract

As B-lymphoid progenitor cells do not give rise to in vitro colony formation and are unlikely to support myeloid engraftment, we validated a five-color extension of the single platform Stem Cell Enumeration (SCE) kit, to routinely quantify myeloid and B-lymphoid progenitor cells. Fresh samples (n > 20 each) of granulocyte colony stimulating factor mobilized blood (peripheral blood (PB)), cord blood (CB), bone marrow (BM), and apheresis products (APs) were stained in TruCOUNT™ tubes and the results were compared with those from the two-color CD45/CD34 reagent combination and the three-color SCE kit. To address repeatability, 10 samples from one AP were prepared by four technicians. Aliquots (n = 15) of four frozen AP were analyzed after thawing. Excellent correlations were observed between the three kits (R² > 0.99), for the quantification of white blood cells and total CD34. The extended kit showed considerable amounts of B-lymphoid progenitors in all CD34 sources (0–20% of all CD34 in PB, AP, and CB; 3–90% in BM). Very similar results were obtained when the same sample was prepared by different technicians. After thawing of frozen AP, the recovery of viable cells varied depending on the freezing medium employed, but the results from the different quantification methods were identical. Most non-viable cells were clearly identified with 7 Aminoactinomycin D (7AAD) but an additional gate in the forward scatter/side scatter was necessary to address dead cells negative for 7AAD. The extended SCE kit allows rapid and exact quantification of viable B-lymphoid and myeloid CD34⁺ cells in all cell sources and in thawed stem cell harvests, and may thus improve the correlation between CD34 number and engraftment kinetics.

Keywords :

• 7AAD
• CD34 enumeration
• CD34 subpopulations
• multicolor analysis
• single-platform analysis