Lower cell viability determined by 7-AAD flow cytometry in HPC-A that have been stored overnight at 2-8 degrees prior to cryopreservation

Abstract

Background & Aim Yearly retrospective evaluations of year-cohorts of autologous stem cell transplants are performed at the Stem Cell Laboratory of the Acadamic Hospital in Maastricht, The Netherlands. In this evaluation cell viability by trypan blue and 7-AAD, CD34+ cell recovery and engraftment time is analysed for all patients that have had their autologous stem cell transplantation. Cell viability by 7-AAD and CD34+ cell recovery is analysed by flow cytometry. Trypan blue staining is used as another method to determine cell viability. Engraftment time is defined as the number of days between stem cell transplantation and the the first of the days in which the 3 consecutive neutrophil counts above 0.5 × 10E9/L and platelet counts above 20 × 10E9/L were recorded. Data are grouped in tranplants that have been stored overnight at 2-8 degrees Celsius prior to cryopreservation (n = 24) and those that are cryopreserved directly at the day of harvest (n = 40). Comparison of data is done by a a 2-sided Student T-test with two populations with equal variances. Methods, Results & Conclusion Retrospective evaluation of CD34+ cell recovery and cell viability after cryopreservation and thawing revealed a significant difference in cell viabilitiy (42% versus 60%) determined by 7-AAD flow cytometry of HPC-A that were stored overnight before being cryopreserverd versus HPC-A that were cryopreserved on the day of harvest. There was no significant difference in cell viability determined by trypan blue staining (70% versus 79%), CD34+ cell recovery (48% versus 56%), nor in the length of hematologic recovery (both 12 days for neutrophils and 14 days for platelets) in the patients transplanted with HPC-A that were stored overnight versus cryopreserved directly. HPC-A that have a WBC concentration > 275 × 10E6/mL are diluted with autologous plasma or 4% human serum albumin prior to cryopreservation, but not prior to overnight storage at 2-8 degrees Celsius. Further investigation into the cause for this difference in viability between HPC-A that were stored overnight versus cryopreserved directly did not show high cell concentration at overnight storage to be a factor. The diagnosis of multiple myeloma was more present in the overnight stored (n=14) stem cell products than in the cryopreserved directly group (n= 17). Conclusion: No definite cause for the difference in viability determined by 7-AAD between could be found in this evaluation of overnight stored and directly cryopreserved stem cell products could be found.