BACKGROUND: Trypan blue exclusion staining is probably the most frequently applied method (Method I) for assessment of viability in peripheral blood progenitor cell grafts after cryopreservation. Alternatively, a flow cytometry-based method (Method II) was established and optimized. STUDY DESIGN AND METHODS: In a first series of 22 autologous apheresis products, the influence of duration of antibody staining and red cell (RBC) lysis on viability was investigated. In a second series of 21 autologous and 1 allogeneic apheresis products, the effect of omitting the RBC lysis was evaluated. On the basis of the results of the first two series, 155 autologous and 57 allogeneic apheresis products were analyzed with Method I and the now optimized Method II. RESULTS: Halving the incubation times did not influence the viability of CD45+ or CD34+ cells. Omission of RBC lysis resulted in a significantly (p = 0.022) increased median viability of CD45+ cells (75.8% vs. 71.0%) without any influence on CD34+ cells. In the third series, the median viability of CD34+ cells (96.9%) was significantly (p < 0.0001) higher compared with the viability of CD45+ cells (76.2%) and the viability determined by Method I (86.5%). CONCLUSION: The viability of CD34+ cells was significantly higher compared with the viability of all white blood cells. The presented cytometry-based method is superior to the standard trypan blue method regarding the number of analyzable cells and documentation, regarding observer independence and standardization; it allows the analysis of the cells of interest for transplantation after minimal sample manipulation.
BACKGROUND:Trypan blue exclusion staining is probably the most frequently applied method (Method I) for assessment of viability in peripheral blood progenitor cell grafts after cryopreservation. Alternatively, a flow cytometry-based method (Method II) was established and optimized. STUDY DESIGN AND METHODS: In a first series of 22 autologous apheresis products, the influence of duration of antibody staining and red cell (RBC) lysis on viability was investigated. In a second series of 21 autologous and 1 allogeneic apheresis products, the effect of omitting the RBC lysis was evaluated. On the basis of the results of the first two series, 155 autologous and 57 allogeneic apheresis products were analyzed with Method I and the now optimized Method II. RESULTS: Halving the incubation times did not influence the viability of CD45+ or CD34+ cells. Omission of RBC lysis resulted in a significantly (p = 0.022) increased median viability of CD45+ cells (75.8% vs. 71.0%) without any influence on CD34+ cells. In the third series, the median viability of CD34+ cells (96.9%) was significantly (p < 0.0001) higher compared with the viability of CD45+ cells (76.2%) and the viability determined by Method I (86.5%). CONCLUSION: The viability of CD34+ cells was significantly higher compared with the viability of all white blood cells. The presented cytometry-based method is superior to the standard trypan blue method regarding the number of analyzable cells and documentation, regarding observer independence and standardization; it allows the analysis of the cells of interest for transplantation after minimal sample manipulation.
Authors: Andreas Humpe; Ute Buwitt-Beckmann; Natalie Schub; Martin Gramatzki; Andreas Günther Journal: Transfus Med Hemother Date: 2013-07-25 Impact factor: 3.747
Authors: Yong-Gang Yao; Sachiko Kajigaya; Leigh Samsel; J Philip McCoy; Giuseppe Torelli; Neal S Young Journal: Mutat Res Date: 2013-09-14 Impact factor: 2.433
Authors: Carla Cristina Pedrosa de Lira de Morais; Juliana Dias Alves Pinto; Karen Wagner de Souza; Marina Izu; Luis Fernando da Silva Bouzas; Flávio Henrique Paraguassú-Braga Journal: Hematol Transfus Cell Ther Date: 2020-12-04