AUTOLOGOUS BONE MARROW TRANSPLANTATION - RELATION BETWEEN CD34+ CELLS AND COLONY FORMING UNITS FOR QUALITY ANALYSIS IN CRYOPRESERVED APHERESIS PRODUCTS

INTRODUCTION AND OBJECTIVE: Bone marrow transplantation (BMT) is one of the indications for the treatment of hematological diseases. The quantification of hematopoietic progenitor cells (PHC) by phenotype (CD34+/CD45+) or by cell culture (Clonogenic Assay evaluates Colony Formation Units-CFU) is used to determine the quality of leukapheresis products applied to BMT. The ratio between CD34+ and CFUs of granulocytes and macrophages (CFU-GM) shows great variations, but may express damage resulting from cryopreservation. The objective was to evaluate the loss of quality of these post cryo products using pre cryo RCD34/CFU as references. MATERIAL AND METHODS: In this study we included 399 clonogenic assay of 120 patients transplanted from 2013 to 2019 at the HUCFF Bone Marrow Transplantation Unit. Of these, 69 had multiple myeloma, 35 had Hodgkin's lymphoma, and 16 had non-Hodgkin's lymphoma. Therefore, 170 fresh samples and 170 thawed samples and 38 thawed samples were submitted to a new culture to confirm the result. To assess the amount of MHC by flow cytometry, samples were incubated with anti-CD45 and anti-CD34 antibodies and analyzed as indicated by ISHAGE/ISCT. The clonogenic assay was performed by culturing nucleated cells of the leukapheresis product in semi-solid culture containing Iscove's Medium, agar, fetal bovine serum and conditioned medium of the 5637 strain, as a source of GM-CSF. The plates were incubated for 12-14 days. Afterwards, the samples were fixed with formalin vapor until the colonies were counted. RCD34/CFU was determined by the ratio between the number of CD34+ cells and the number of CFU from apheresis. The cryopreservation method was automated and the products were kept in liquid N2. Statistical analysis of values was performed using the Mann-Whitney and t-paired tests. RESULTS AND CONCLUSION: The median of RCD34/CFU in fresh samples was 35 (5-2143) and in thawed samples it was 102 (2-19171). The loss of post-cryo proliferative capacity was significant p<0.0001 (Main-Whitney U). Post cryo samples that had losses outside of tolerance levels were repeated and showed similar results (RCD34/CFU with median 113 and post repeat 472). The loss of post-cryo proliferative capacity is very variable between patients. It is necessary to identify the associated risk factors to predict satisfactory collection for patients with greater risk of loss of post-cryo graft quality, a study of patients' clinical characteristics or cytological properties in these products may help in the interpretation of these results. The clonogenic assay in cryopreserved products reveals the loss of proliferative capacity of post-thawed progenitors and can add quality values, along with cell viability. We observed that a rate of about 25% of samples show significant hematopoietic loss in vitro. The results were confirmed by repetition and showed reproducibility.