Aldehyde Dehydrogenase (ALDH) Activity in Fresh (Pre-Freezing) and Post-Thawing Leukapheresis and Cord Blood Collections

Introduction: Primitive hematopoietic progenitor cells may be characterized by the detection of the intracellular enzymatic activity of aldehyde dehydrogenase (ALDH ). The use of a fluorescent substrate for ALDH (Aldefluor) permits the cytofluorimetric study of different stem cell populations in the functional way rather then as markers of cell surface. Aim of this study was to evaluate the ALDH activity of the stem cells in pre-freezing (fresh) leukapheresis (LKF) colllections and in cord blood (CB) units, to verify the feasibility of detecting the ALDH activity in the post-thawing units and finally to compare the results between the fresh and post thawed stem cells to be transplanted. Materials and methods: Samples containing 2 x106 total nucleated cells obtained from 5 LKF and 5 CB units immediately after collection and from the same units after thawing, were incubated with Aldeflour (StemCo Biomedical, Durham, NC, USA) at 37°C for 45 min. and successively for 15 min. with anti CD34 PE, (Beckman Coulter, Fullerton, CA, USA) and anti CD45 PerCP (BD Biosciences, San Jose, CA, USA). Viability of CD34+ cells was evaluated using 7AAD, (Beckman Coulter, Fullerton, CA, USA). The setting of thawed samples was carried out immediately after thawing, taking care to make the sample dilution very quickly, in order to avoid the detrimental action on the cells exerted by DMSO at 37°C and at room temperature. All samples were analized using a BD Biosciences FACSCalibur flow cytometer device (San Jose, CA, USA). Results: The results of the ALDH and CD34+ cell analysis and stem cell viability on fresh and thawed samples are detailed in table 1. Conclusions: Our results demonstrate the feasibility of ALDH determination even in post thawed samples on condition that the test is conducted with accuracy with particular attention at the dilution step that must be completed in a very short time. The ALDH activity tends to decline in thawed stem cells demonstrating the detrimental action on stem cell functionality of the entire cryopreservation and thawing processes. In particular the contact of the stem cell with cryoprotectant mixture (DMSO) at room temperature has a negative, time dependent, impact on stem cell quality, confirming that the reinfusion phase of the graft must be carried out as soon as possible. The introduction of a functional test to evaluate the graft quality may be helpful in transplant clinical practice. Table 1: Results of ALDH assayes on pre-freezing and post-thawing leukapheresis and cord blood collections. CD34+ (%) ALDH+ (%) CD34+/ALDH+ (%) ALDH+/CD34+ (%) Viability (%) Fresh LKF n=5 0.56 0.12 64.6 97.8 99.2 Fresh CB n=5 0.28 0.23 76.7 99.8 98.1 Thawed LKF n=5 0.48 0.09 65.5 94.4 81.2 Thawed CB n=5 0.24 0.19 71.5 96.8 77.3