María C López1, David A Lawrence. 1. The Wadsworth Center, New York State Department of Health, Albany, NY 12201-0509, USA.
Abstract
BACKGROUND: Successful hematopoietic engraftment depends on the number of viable CD34+ stem cells. Therefore, accurate quantification of viable CD34+ stem cells is required. STUDY DESIGN AND METHODS: To evaluate clinical laboratory performance, the New York State Department of Health initiated proficiency testing (PT) for viable CD34+ stem cells. Preserved adult peripheral blood was spiked with preserved cord blood CD34+ stem cells and was shipped to the participating laboratories. Three educational and two graded PTs were performed by participating laboratories, and their results were analyzed for consistency. Comparative analysis of viability with 7-aminoactinomycin D (7-AAD) and ToPro-3 dyes also was performed. RESULTS: Laboratories had to adapt their standard operating procedures to include a viability dye to quantify the number of viable CD34+ stem cells. The majority of laboratories chose 7-AAD as their preferred viability dye, but propidium iodide (PI) and ToPro-3 were used by two laboratories. Once all laboratories started to simultaneously analyze viability and staining for CD34, graded PTs started. Lower numbers of viable CD34+ stem cells were obtained for ToPro-3 when the dye was compared with 7-AAD. CONCLUSION: It is concluded that ToPro-3 stains more cells than 7-AAD and likely includes compromised cells. The use of new vital dyes, like ToPro-3, that may stain preapoptotic cells could represent an important advance to improve the quantification of viable CD34+ stem cells, for engraftment purposes. Further studies are needed to document the benefits of switching to a method that excludes not only dead cells, but apoptotic cells as well.
BACKGROUND: Successful hematopoietic engraftment depends on the number of viable CD34+ stem cells. Therefore, accurate quantification of viable CD34+ stem cells is required. STUDY DESIGN AND METHODS: To evaluate clinical laboratory performance, the New York State Department of Health initiated proficiency testing (PT) for viable CD34+ stem cells. Preserved adult peripheral blood was spiked with preserved cord blood CD34+ stem cells and was shipped to the participating laboratories. Three educational and two graded PTs were performed by participating laboratories, and their results were analyzed for consistency. Comparative analysis of viability with 7-aminoactinomycin D (7-AAD) and ToPro-3 dyes also was performed. RESULTS: Laboratories had to adapt their standard operating procedures to include a viability dye to quantify the number of viable CD34+ stem cells. The majority of laboratories chose 7-AAD as their preferred viability dye, but propidium iodide (PI) and ToPro-3 were used by two laboratories. Once all laboratories started to simultaneously analyze viability and staining for CD34, graded PTs started. Lower numbers of viable CD34+ stem cells were obtained for ToPro-3 when the dye was compared with 7-AAD. CONCLUSION: It is concluded that ToPro-3 stains more cells than 7-AAD and likely includes compromised cells. The use of new vital dyes, like ToPro-3, that may stain preapoptotic cells could represent an important advance to improve the quantification of viable CD34+ stem cells, for engraftment purposes. Further studies are needed to document the benefits of switching to a method that excludes not only dead cells, but apoptotic cells as well.