[Improved method for delivery room collection and storage of human cord blood cells for grafting].

Haematopoietic stem cell transplantation is indicated in several haematologic and genetic diseases, the most notable being aplastic anemia and leukemias. Bone marrow has been the traditional source of these cells. Human umbilical cord blood (UCB) has recently become an alternative source of haematopoietic stem cells for transplants. The advantages of cord blood include noninvasive collection without risk to mother and neonate, low risk of viral infection, and immunologic immaturity of cord cells. Single umbilical cord blood donation is usually sufficient for transplantation to adult recipients. Additionally, banking of HLA-typed UCB appears valuable in patients lacking a family donor. This study has focused on basic "perinatological" parameters of umbilical cord blood: average volume of single donation UCB and initial storage conditions before isolation of haematopoietic stem cells. Additionally, the mean content of CD34+ haematopoietic stem cells in leukocyte, lymphocyte and mononuclear cell fractions was established. Correlations between levels of so-called pro-inflammatory cytokines (present in cord blood serum) and number, viability and clonogenicity of cord blood mononuclear cells were checked. UCB samples were obtained by "open" collection during vaginal deliveries and cesarean sections. The collected blood was stored in solutions of anticoagulants (ACD, CPDA-1, heparin) and culture media (PBS, Iscove medium, RPMI), during several time intervals (0-1 h, 1-6 h, 6-12 h, 12-24 h) and at two temperatures (+4 degrees C, ambient). UCB volumes, as well as MNC counts, correlated with delivery type, placental weight, neonatal body weight and duration of pregnancy. The concentration, viability and clonogenicity of MNCs were assessed after collection and storage. The subpopulation of CD34+ haematopoietic stem cells was isolated from MNCs using monoclonal antibodies and magnetic-based separation. The number, viability and clonogenicity of CD34+ cells were evaluated. Subsequently in some samples, the concentration of proinflammatory cytokines (IL-1 alpha, IL-1 beta, IL-6, IL-8, and TNF-alpha), number of mononuclear cells and in vitro clonogenicity of myeloid progenitors (CFU-GM) were determined. It was found that the collected blood volume depended on neonatal body weight (Fig. 1). Umbilical blood could be stored either at ambient temperature (Fig. 4) or +4 degrees C (recommended because of reduced risk of infection) for up to 24 hours in RPMI solution (Fig. 5) with heparin (Fig. 2, 3). CD34+ cell count correlated with mononuclear cell count only (Fig. 6). A negative correlation between the number of mononuclear cells and concentration of TNF-alpha was revealed (Fig. 7), as well as between the number of detectable CFU-GM and concentration of IL-1 beta (Fig. 8). In conclusion, UCB collection and short-term storage is a safe and simple method for graftable haematopoietic stem cell recovery. Save for IL-1 beta and TNF-alpha, cytokine levels did not correlate with the studied parameters of umbilical cord blood.