A new method to assess the viability of collected CD34+ cells before reinfusion: a prospective study in 50 autotransplanted patients.

Introduction: In the setting of peripheral blood stem cell transplantation (PBSCT),the strongest predictive factor for successful engraftment is the dose of reinfused CD34+ cells (per kg of body weight, not corrected for viability). Currently the amount of harvested CD34+ cells is assessed after completing the aphereses, before cryopreservation. However, such measurement does not account for the variable loss of viable CD34+ cells which occurs during freezing or thawing processes. Here we propose a novel method to measure the CD34+ cell viable content after cryopreservation, before reinfusion, through the use of a small bag (mini-bag, mB) which replicates the conditions to which CD34+ cells are subjected in the mother-bag (MB) to be reinfused. Material (or patients) and methods: We analyzed 93 samples from 63 aphereses collected from 50 patients undergoing PBSCT for hematological malignancies (22 multiple myeloma, 24 lymphoma, 4 acute leukemia). Total and viable CD34+ cells were quantified by flow cytometry; analyses were performed according to ISHAGE method with 7-AAD exclusion, before controlled freezing (ICE-CUBE14M system) from an aliquot of the final apheresis volume (FAV); after 1 week of storage for the mB; just prior to reinfusion from an aliquot of the MB. The mB contained 5 ml from FAV, were structurally similar to the MB and were cryopreserved together. Results were statistically compared by Student’s t test and Pearson correlation. Univariate and multivariate linear regression analyses were used to identify variables influencing the viability of CD34+ cells. Results: Mean viability before cryopreservation was 99.7% (range 94-100%); median cell concentration in the FAV was 220x106/ml for leukocytes (range 10-376)and 3x106/ml for CD34+ cells (range 0.3-27).After diluting and splitting the FAV, the median amount of CD34+ in each MB was 2.2x106cells (range 0.2-25) and the median concentration of neutrophils was 19x106/ml (range1-37). After thawing the mean viability of CD34+ cells was 76% (range 23-97%) for mB and 71% (range 28-99%) for MB (P=NS). The two viability values had good linear correlation with high statistical significance (Pearson’s rho 0.59, p40.0001). We investigated factors affecting the viability of CD34+ cells according to the two methods: atunivariate analyses, FAV and the bag leukocyte content were inversely correlated with viability both for mB and MB. In a multivariate model including all covariates with significance P40.2, FAV remained as the only significant factor for viability (both for mB and MB). FAV was highly correlated with both the leukocyte and neutrophil content of the apheresis, MB and mB. Conclusion: We showed that viability check of CD34+ cells after cryopreservation can be done with comparable results in thawed MB or mB. However, results of mB are available before reinfusion, allowing for a correction of the planned CD34+ dose to be reinfused, thus making it preferable as quality control of the freezing/thawing process. Leukocyte/neutrophil contamination of the collection (reflected by the size of FAV) significantly impacts CD34+ cells viability and should be minimized.