Association of post-thaw viable CD34 þ cells and CFU-GM with time to hematopoietic engraftment H Yang 1 , JP Acker 1,2 , M Cabuhat 1 , B Letcher 1 , L Larratt 1,3 and LE McGann 1,2 1 Canadian Blood Services, Edmonton Alberta, Canada; 2 Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton Alberta, Canada; and 3 Division of Clinical Hematology, University of Alberta, Edmonton Alberta, Canada Summary: In all, 78 peripheral hematopoietic progenitor cell collections from 52 patients were evaluated using our previously published validated post-thaw assays at the time of collection and following transplantation by assessment of viable CD34 þ cells, and granulocyte– macrophage colony-forming units (CFU-GM) cryopre- served in quality control vials. The median (range) post-thaw recovery of viable CD34 þ cells and CFU-GM was 66.4% (36.1–93.6%) and 63.0% (28.6–85.7%), respectively, which did not show significant correlation with the engraftment of either neutrophils (P ¼ 0.136 and 0.417, respectively) or platelets (P ¼ 0.88 and 0.126, respectively). However, the reinfused viable CD34 þ cells/ kg of patient weight pre- or post-cryopreservation showed significant correlation to engraftment of neutrophils (P ¼ 0.0001 and 0.001, respectively) and platelets (P ¼ 0.023 and 0.010, respectively), whereas CFU-GM pre- or post-cryopreservation was significantly correlated to neutrophils (P ¼ 0.011 and 0.007, respectively) but not to platelets (P ¼ 0.112 and 0.100, respectively). The results show that post-cryopreservation assessment of viable CD34 þ cells or CFU-GM is as reliable a predictor of rapid engraftment as that of pre-cryopreservation measures. Therefore, the post-cryopreservation number of viable CD34 þ cells or CFU-GM should be used to eliminate the risks of unforeseen cell loss that could occur during cryopreservation or long-term storage. Bone Marrow Transplantation (2005) 35, 881–887. doi:10.1038/sj.bmt.1704926 Published online 21 March 2005 Keywords: cryopreservation; engraftment; viability; CD34 þ ; CFU-GM A minimum number of CD34 þ cells is required for rapid hematologic recovery following myeloablative therapy for hematologic malignancies. 1 Some studies have reported that 2.5–5.0  10 6 CD34 þ cells/kg is associated with rapid engraftment, 1–3 but the minimum CD34 þ cells/kg for autologous peripheral blood progenitor cell transplantation has not been defined or standardized. An obstacle in defining such threshold is that the quantification of CD34 þ cells has been performed pre-cryopreservation. As the extent of loss of viable CD34 þ cells after thawing is not assessed, the actual number of viable CD34 þ cells infused into patients is not known. Some studies have compared CD34 þ cell number and colony growth before and after cryopreserva- tion of peripheral hematopoietic progenitor cells (HPC), 4,5 and concluded that post-thaw CD34 þ cell number may be useful in monitoring cell loss during cryopreservation. 4,6,7 However, the results reported showed that viable CD34 þ cells after cryopreservation did not decrease but instead increased by up to 77%. 5 In addition, the reported viability of CD34 þ cells varied from 0 to 304%. 4,8 This wide variation of the post-cryopreservation viability resulted from the post-thaw assessment assays being based on the number of nucleated cells remaining after cryopreservation. 9,10 However, the number of nucleated cells, particularly neutrophils, is decreased during cryopreservation and during post-thaw incubation due to cell loss and clumping of the damaged cells. 11,12 In dual-platform CD34 þ enumeration assays, this decrease in the number of nucleated cells results in an apparent increase in the percentage of CD34 þ cells when nucleated cell count is used as the denominator to calculate the total post-cryopreservation CD34 þ cells. Even when using a validated single-platform CD34 þ enumeration technique to obtain absolute CD34 þ cells, assays did not account for cells lost during cryopreservation or post-thaw washing, 13 nor were comparisons made with proliferation of progenitor cells (granulocyte–macrophage colony-forming units (CFU-GM)). 7 In previous studies, 9,10 we validated the use of post-cryopreservations assays for assessing post- cryopreservation viability of CD34 þ cells and CFU-GM with high accuracy. 7 It is our hypothesis that post-thaw assessment of CD34 þ cell viability and CFU-GM may be a more accurate method to predict hematopoietic engraftment. In this study, we assessed 78 consecutive collections of autologous HPCs from 52 patients before and after cryopreservation using validated post-cryopreservation assays to investigate the association of viability of CD34 þ cells and CFU-GM with hematopoietic engraftment. Received 10 May 2004; accepted 1 December 2004 Published online 21 March 2005 Correspondence: Dr H Yang, Laboratory and Scientific Director, Progenics Cord Blood Cryobank, 701 Sheppard Ave. East, Suite 310, Toronto, Ontario, M2K 2Z3, Canada; E-mail: ycryoscientific@yahoo.ca Bone Marrow Transplantation (2005) 35, 881–887 & 2005 Nature Publishing Group All rights reserved 0268-3369/05 $30.00 www.nature.com/bmt