Summary:wide may reduce the present delay between the initiation of a search for an unrelated donor and actual transplantation that occurs using existing unrelated marrow donor panels. 3 Cord blood transplantation has been proven to be a suitable form of treatment for a variety of diseases in Separation and processing of cord blood samples in large numbers for storage in cord blood banks ideally needs to childhood and more recently in an increasing number of adult patients. Banks of cord blood cryopreserved after be partially automated to allow large numbers of samples to be processed efficiently. A closed system reduces the risk HLA testing are required in order to provide various HLA types for unrelated transplantation. To optimize of bacterial contamination after collection. The processing method must allow adequate recovery of nucleated cells storage space cord blood needs to be stored as a separated product. Several early methods of cord blood sepand progenitors to enable engraftment. Early attempts at separating cord blood by density gradient techniques led to aration resulted in a significant loss of progenitor cells. We used a separation procedure where the donation loss of mononuclear cells and to the suggestion that cord blood should be stored unseparated. 4,5 These donations, was separated by centrifugation into a buffy coat fraction, a red cell fraction, and a plasma fraction. Twentycryopreserved as whole blood, have been transplanted successfully. 1,2,5 However, for cord blood banks to be econfive samples, (mean initial volume 81 ml) were assessed. Nucleated cells were recovered in the buffy coat fracomical and efficient, volumes smaller than that of whole packs need to be stored. Furthermore, clinical problems tion. Recoveries of nucleated cell count, total progenitors and CD34-positive cells in the buffy coat were 90%, could arise with red cell lysis and some form of red cell depletion is necessary to reduce the risk of transfusion reac-88% and 100%, respectively. The buffy fraction was tested for sterility by aerobic and anaerobic culture.tions in cases of ABO incompatibility between donor cord blood and recipient. A study using the density gradient Using this closed bag system, volume reduction was achieved while maintaining sterility and retaining progenimethod to separate cord blood 6 did not find a major loss of progenitor cells, and a later study 7 suggested a modifitor cells in a final mean buffy coat volume of 44 ml. Red cell and plasma fractions were available for ABO cation of the common density gradient separation methods as suitable for processing cord blood before cryopreservgrouping, virology testing and cryopreservation. The results show that cord blood can be effectively volumeation. Falkenburg et al 6 compared cell separation of cord blood and bone marrow by red cell lysis, methylcellulose reduced using simple and readily available blood banking techniques.sedimentation and density gradients, and showed similar recovery of nucleated cells from both cell sources provided

Human fetal cord blood contains subsets of mononuclear cells with the potential to form both hematological and endothelial cells. Vascular progenitor cells, which can produce all three elements of mature blood vessels, including smooth muscle, have been identified in animals. We hypothesized that similar multipotential progenitor cells exist in humans and used the expression of alpha-smooth muscle actin (alpha-SMA) to identify such cells in fetal cord blood. Mononuclear cell preparations were isolated from human umbilical cord blood and CD34(+) and CD133(+) cells obtained by magnetic bead separation. Isolated cells were cultured on fibronectin-coated dishes with medium containing vascular endothelial growth factor, basic fibroblast growth factor, and insulin-like growth factor. mRNA was extracted, and the expression of alpha-SMA and a number of endothelial cell markers (VEGFR-2, vWF, eNOS, VE-Cadhein, PECAM-1 and Tie-2) was determined by reverse transcriptase-PCR techniques. Human umbilical vein endothelial cells (HUVECs) were used as positive controls. Freshly isolated CD34(+) and CD133(+) cells expressed all endothelial cell markers, but did not express alpha-SMA. HUVECs expressed alpha-SMA. Following 4 weeks of culture, CD34(+) isolates produced morphologically endothelial-like cells that expressed both endothelial cell markers and alpha-SMA. CD133(+) cells failed to produce morphological endothelial-like cells but expressed a range of endothelial markers. However, they did not express alpha-SMA. Following culture in an endothelial cell-promoting environment, CD34(+), but not CD133(+), isolates produced endothelial-like cells that expressed alpha-SMA. Human fetal cord blood contains a population of cells that may differentiate toward both an endothelial and a smooth muscle phenotype in culture.

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Summary:the mature T cells from the donated marrow. 2 This approach has subsequently been successful in over half of the transplanted children with SCID. Graft failure is much Bone marrow transplantation is the only curative treatment for children with severe combined immunodefiless common in children than in older leukaemic patients given similarly treated marrow perhaps because children ciency (SCID). In the absence of an HLA-identical sibling, haploidentical parental donor marrow can be used with SCID lack the alloreactive immune responses that mediate graft rejection. 3-5provided it is depleted of T cells to prevent otherwise inevitable GVHD. Campath 1M has been successfully T cell depletion has been accomplished by a variety of methods including physical separation with soya bean lecused for this procedure in several centres. In our centre 17 SCID patients plus one with combined immunodefitin and sheep erythrocyte rosetting, 6 as well as immunochemical methods using monoclonal antibodies to lymphocyte ciency (CID) were transplanted with Campath 1M T cell-depleted bone marrow. Progenitor cell recovery, surface antigens, such as Campath 1M in vitro and Campath 1G added to the bag of donor marrow or infused in before and after T cell depletion, was monitored using granulocyte-macrophage colony-forming cell assays vivo. 7,8 We describe a series of 18 T cell-depleted transplants carried out in our BMT centre over the past 8 years, (GMCFU) and CD34 analysis. The numbers of GMCFU/kg transplanted correlated with engraftment during which time the method of marrow manipulation during T cell depletion was changed in an attempt to conform and survival post-transplant and monitoring CD34+ cell numbers in the T cell-depleted marrow pretransplant with other European centres who mainly used physical methods of T cell depletion. Unfortunately this change may be an additional indicator of successful engraftment. Use of a buffy coat marrow preparation appeared to contribute to several failures of engraftment. We therefore attempted to identify markers that would prewith restriction of the number of T cells to Ͻ5 × 10 5 /kg was associated with graft failure in four and death in dict lymphoid engraftment and our results suggest that the number of both CD34 + cells and GMCFU infused per kg five of eight children, probably because too few stem cells were infused. T cell depletion of a mononuclear cell correlates with sustained engraftment and survival following T cell-depleted donor marrow transplantation in preparation of donor marrow with no arbitrary ceiling of infused T cells is highly effective at preventing clinipatients with SCID. cally important GVHD and cured nine out of 10 children transplanted with such material.

The present study provides the first clear in vitro evidence to suggest that CBMCs are less able than PBMCs to induce skin GVH type alloreactivity in HLA-mismatched pairs. The severity of in vitro GVH type alloreactivity (graded as I-IV) was strongly associated with the levels of alloreactive CTLp frequencies. The low cellular alloreactivity of CBMCs detected in vitro suggests that in a proportion of cases HLA-mismatched unrelated CB may not give rise to severe GVHD in vivo after transplantation.