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There have been great advances over the last decades in haematopoietic stem cell (HSC) transplantation, using either bone marrow, peripheral blood or cord blood-derived stem cells. The coming into force of the European legislation on tissues and cells and the consequent transposition of Directives into national laws have required the health authorities in the Member States (MS) and the scientific societies to review the transplantation activities to ensure the circulation of safe HSC products. Here, the regulatory inspection process performed by the Competent Authorities and the professional voluntary accreditation process of the Transplant Programmes active in Italy is compared.

This issue of the Stem Cell International journal contains papers from many of the leading scientists in the emerging field: ex vivo expansion of hematopoietic progenitor cells into erythrocytes for transfusion. Blood transfusion, the first form of successful cell therapy and, at least to some, “transplantation”, was inspired by the discovery of the circulation by Richard Harvey in the 1600s [1] and begun in earnest later in that century. The development of this clinical practice into the safe and routine therapy we all know today has been both an exciting scientific adventure and the foundation for a number of other scientific disciplines. More specifically, the immunology of transfusion and transplantation began with the discovery of the heterogeneity of human blood group antigens by Dr. Karl Landsteiner in 1901 (recognized with a Nobel Prize in 1930). The discovery of clinically relevant infectious diseases transmitted by transfusion played an important role in the development and advancement of virology. The inheritance of certain form of anemias was discovered during blood transfusion practice and led to development of the genetics of human red cell disorders. In the 1940–1950s, the establishment of blood banks followed by the development of rigorous donation criteria and standardization of blood manufacturing processes has made transfusion safe and widely available and has provided a paradigm for the development of emerging therapies using ex vivo expansion and differentiation of many cell types. An example of one such therapy is represented by the tumor immunotherapy described by Lapteva and Vera. The blood supply of industrialized countries is adequate overall. Nearly one hundred million donations are made every year worldwide (http://www.who.int/mediacentre/factsheets/fs279/en/index.html). The availability of blood and blood products in these nations has permitted the development and implementation of numerous life-saving surgical procedures (open heart surgery, organ transplantation, damage control resuscitation for trauma, and others) and cancer treatments which were not even imaginable without assurance that blood for transfusion would be readily available and safe. However, blood is not an unlimited resource and its potential need as the world rapidly develops requires a significant increase in blood donation. By some estimates (CDH), given the world's population and given the per capita transfusion of Canada as a utilization benchmark, nearly 250 million whole blood donations would be needed. Furthermore, and despite its high level of safety, human donated, unit-by-unit-derived blood donation/transfusion (i.e., without batched blood manufacturing into an aliquoted and homogenized pharmaceutical product), still leads to morbidity and mortality of its own accord and has significant variation from product to product based on the nature of the collection, manufacturing and storage processes, and the antigenic variation of any given donor, amongst others. Finally, it is not known what effect the aging of the world's population will have both on per capita utilization and on the ability of the smaller, younger populations to donate [2]. These issues, and the nearly 20-year-old search for alternative products to meet the transfusion need are discussed in the paper by Whitsett et al. Scientific research is inspired by the prospect of a clinical goal. In recent years, a revolution in stem cell biology has occurred that has far reaching implications, specifically, the discovery that it is possible to generate a potentially unlimited supply of stem cells by epigenetic/genetic treatments of somatic cells (T cells, fibroblasts, others) from any individual (see Pourcher et al., Hyroyama et al., and Chang et al.). In addition, techniques have been discovered to reprogram any cell into another cell type avoiding the induction of pluripotency. These techniques are fascinating though there are numerous scientific, safety, and scaling-up issues to be resolved before cells which have been genetically “altered” in the laboratory may be considered ready for widespread clinical use. As red blood cells do not have a nucleus, it is possible that they will be accepted as genetically safe. Indeed, it is this notion that supports that red blood cells from Hematopoietic stem/progenitor cell expansion or redifferentiation may represent the first therapeutic product to be generated by genomic reprogramming technology. Reprogramming technology is still under development. Therefore, red blood cells expanded ex vivo from primary stem cell sources currently discarded (buffy coats produced during the blood manufacturing processes and low-volume umbilical cord blood) are being considered for first-in-man studies. Tirelli et al. identify the cell populations present in adult blood which are responsible for massive production of red blood cells ex vivo. The first-in-man proof-of-principle study for the use of in vitro expanded red blood cells for transfusion was reported on September 1st 2011, by Luc Douay and colleagues [3], who have also coauthored Pourcher et al. This paper reported that red blood cells generated in vitro from mobilized CD34pos cells collected by apheresis have normal survival (determined by 51Cr labeling) when transfused into an autologous recipient [3]. This first-in-man autologous transfusion described also what would be the most likely safety data necessary for a larger clinical study with such products [in vitro characterization (blood group antigen expression profiling, deformability, hemoglobin content and O2 dissociation curves) and in vivo functional studies in animal models (survival and morphology); http://www.clinicaltrials.gov/ct2/show/NCT00929266]. In vivo functional studies of human red blood cells in animal models will likely allow more complete characterization in many ways [4]. Ghinassi et al. describe an improved animal model which allows in vivo imaging and cell fate determination of human erythroid cells by labeling the cells before transfusion with a fluorescent reporter gene by retroviral technology. Although red blood cells do not have nuclei, their immediate precursors the erythroblasts do. The terminal maturation of erythroblasts into functional red cells requires a complex remodeling process which ends with extrusion of the nucleus and the formation of an enucleated red blood cell [5]. These late stages of maturation are intrinsically controlled by epigenetic/genetic expression programs of the erythroblast itself. Cell reprogramming methodologies may (and at present appear to) disrupt these programs, leading to inefficient enucleation. Keerthivasan et al. discuss novel insights into the critical mechanisms of terminal maturation of a red blood cell and strategies to improve the efficiency of these processes. As represented by all the information, data, and in fact vision contained in this issue, we are clearly at the beginning of a rapidly expanding field. The papers herein provide a broad and comprehensive overview of the most relevant areas of research which have been pursued and are needed to advance the field. Still, as state of the art as this issue is presently, the field is moving so rapidly that one may predict that new knowledge will rapidly follow. Anna Rita Migliaccio Giuliano Grazzini Christopher D. Hillyer

The primary focus of national blood programs is the provision of a safe and adequate blood supply. This goal is dependent on regular voluntary donations and a regulatory infrastructure that establishes and enforces standards for blood safety. Progress in ex vivo expansion of blood cells from cell sources including peripheral blood, cord blood, induced pluripotent stem cells, and human embryonic stem cell lines will likely make alternative transfusion products available for clinical use in the near future. Initially, alloimmunized patients and individuals with rare blood types are most likely to benefit from alternative products. However, in developed nations voluntary blood donations are projected to be inadequate in the future as blood usage by individuals 60 years and older increases. In developing nations economic and political challenges may impede progress in attaining self-sufficiency. Under these circumstances, ex vivo generated red cells may be needed to supplement the general blood supply.

In Italy, the law does not permit the setting up of private banks to preserve cord blood (CB) stem cells for personal use. However, since 2007 the right to export and preserve them in private laboratories located outside Italy has existed, and an increasing number of women are requesting this collection of umbilical CB at delivery to enable storage of stem cells for autologous use.Since private banks recruit clients mainly via the Internet, we examined the content of 24 Italian-language websites that offer stem cells storage (from CB or amniotic fluid), to assess what information is available.We found that the majority of private banks give no clear information about the procedures of collection, processing, and banking of CB units and that the standards offered by private CB banks strongly differ in terms of exclusion or acceptance criteria from the public banks. These factors may well influence the overall quality of the CB units stored in private CB banks. Of note, during the period 2007 to 2009, the number collected for autologous use did not create a downward trend on the number of units stored in public CB banks for allogeneic use.CB is a valuable community resource but expectant parents should be better informed as to the quality variables necessary for its storage, both by institutions and by professionals. Currently, most of the advertising is insufficient to justify the expense and the hopes pinned on autologous use of CB stem cells.

H. W. Reesink, S. Panzer, C. A. Gonzalez, N. Lena, P. Muntaabski, S. Gimbatti, E. Wood, M. Lambermont, V. Deneys, D. Sondag, T. Alport, D. Towns, D. Devine, P. Turek, M.-K. Auvinen, T. Koski, C. K. Lin, C. K. Lee, W. C. Tsoi, E. Lawlor, G. Grazzini, V. Piccinini, L. Catalano, S. Pupella, H. Kato, S. Takamoto, H. Okazaki, I. Hamaguchi, J. C. Wiersum-Osselton, A. J. W. van Tilborgh, P. Y. Zijlker-Jansen, K. M. Mangundap, M. R. Schipperus, D. Dinesh, P. Flanagan, O. Flesland, C. T. Steinsvag, A. Espinosa, M. Letowska, A. Rosiek, J. Antoniewicz-Papis, E. Lachert, M. B. C. Koh, R. Alcantara, M. Corral Alonso & E. Muniz-Diaz

Factor VIII is an important glycoprotein involved in hemostasis. Insertion of expression vectors containing either the full-length cDNA sequence of human factor VIII (FLrFVIII) or B-domain deleted (BDDrFVIII) into mammalian cell lines results in the production of recombinant factor VIII (rFVIII) for therapeutic usage. Three commercially available rFVIII concentrates (Advates, Helixate NexGens and Refactos), either FLrFVIII or BDDrFVIII, were investigated by 1- and 2-DE and MS. The objective of this study was to compare the heterogeneity and the high purity of both rFVIII preparations before and after thrombin digestion. In particular, the 2-D gel was optimized to better highlight the presence of contaminants and many unexpected proteins. Recombinant strategies consisting of insertion of expression vectors containing BDDrFVIII and FLrFVIII resulted in homogeneous and heterogeneous protein products, respectively, the latter consisting in a heterogeneous mixture of various B-domain-truncated forms of the molecule. Thrombin digestion of all the three rFVIII gave similar final products, plus one unexpected fragment of A2 domain missing 11 amino acids. Regarding the contaminants, Helixate NexGens showed the presence of impurities, such as Hsp70 kDa, haptoglobin and proapolipoprotein; Refactos showed glutathione S-transferase and b-lactamase, whereas Advates apparently did not contain any contaminants. The proteomic approach will contribute to improving the quality assurance and manufacturing processes of rFVIII concentrates. In this view, the 2-DE is mandatory for revealing the presence of contaminants. L'artcoo è disponibile sul sito dell'editore http://onlinelibrary.wiley.com/

Umbilical-cord blood (UCB) has growingly become an accepted alternative source of hematopoietic stem cells for transplantation purposes. However, the low cell dose limit within a single unit is still an obstacle hindering the way of a broader diffusion. The real deal is the lack of knowledge about the molecular processes governing the events of expansion and differentiation of these cells. In order to fill this void, several studies were focused on the identification of the peculiar whole protein profile of UCB-derived hematopoietic stem cells. In this review article we provide a referenced list of overall proteins from UCB-derived hematopoietic stem and progenitor cells. This list has been elaborated for pathway and network analyses, along with GO term enrichment for biological and molecular functions, in order to individuate main classes of proteins governing functioning of these cells. From these analyses it seems to emerge a central role for heat shock proteins in immature hematopoietic stem cells. Their role might be relevant in protecting crucial transcription factors which drive proliferation and differentiation towards a specific lineage (e.g. erythroid, myeloid). Hereby we also stress the helpfulness of interactomics elaboration in providing a unified overview of independent proteomics data. It appears that maturation, other than representing a bottleneck to protein expression, could sculpt interaction maps via reducing complexity of immature interactomics profiles.

The first umbilical cord blood (UCB) transplant to a sibling with Fanconi's anaemia in 1988 represented a breakthrough in the field of transplantation. Thereon, several transplants have been performed with UCB-derived hematopoietic stem cells (HSCs) and a plethora of studies have investigated the plasticity of UCB-derived stem and progenitor cells. However, these studies have not been hitherto translated into clinical trials and, although UCB is routinely used as an alternative source of HSCs, no substantial advances have been made in the field of clinical regenerative medicine. The real deal is the lack of knowledge about the molecular processes governing the events of differentiation which transform immature UCB stem cells into terminally-committed hematopoietic, muscle, bone and nervous cells. In order to fill this void, several studies have been recently focused on the identification of the peculiar proteomic profile of UCB-derived stem cells. Hereby, we concisely review recent proteomic surveys addressing UCB-derived stem and progenitor cells. Notably, comparative studies detected a wider spectrum of proteins in immature cells rather than in more differentiated populations, as if maturation events could represent a bottleneck to protein expression. Future research projects should try to shed light on these processes and their completion could pave the way for unprecedented treatments.

Blood-related proteomics is an emerging field, recently gaining momentum. Indeed, a wealth of data is now available and a plethora of groups has contributed to add pieces to the jigsaw puzzle of protein complexity within plasma and blood cells. In this review article we purported to sail across the mare magnum of the actual knowledge in this research endeavour. The main strides in proteomic investigations on red blood cells, platelets, plasma and white blood cells are hereby presented in a chronological order. Moreover, a glance is given at prospective studies which promise to shift the focus of attention from the end product to its provider, the donor, in a sort of Kantian "Copernican revolution". A well-rounded portrait of the usefulness of proteomics in blood-related research is accurately given. In particular, proteomic tools could be adopted to follow the main steps of the blood-banking production processes (a comparison of collection methods, pathogen inactivation techniques, storage protocols). Thus proteomics has been recently transformed from a mere basic-research extremely-expensive toy into a dramatically-sensitive and efficient eye-lens to either delve into the depths of the molecular mechanisms of blood and blood components or to establish quality parameters in the blood-banking production chain totally anew.

Abstract 4195 Directed family cord blood (DCB) storage provides hemopoietic stem cell source for transplantation (HSCT) for families with an existing or a potentially future recipient with HSCT curable disease (D). The National CB Banks Network in Italy (ITCBN) has a leading role in providing public DCB service for high-risk families, in compliance to GITMO directives for eligibility criteria (HSCT curable D: malignant MD, non MD, inherited ID). To provide best cost-effective practices recommendations it is important to report on DCB procedures and HSCT rate (HSCT-R) among public Banks. By 12.12.2008 almost 1800 DCB units were stored in 18 Italian Banks and 104 (9%) issued for HSCT. The present survey aims at summarizing the over 15 yrs DCB experience among 5 ITCBN Banks active since 1997 (range 1990-1997), and including 670 DCB units. Results Preliminary analysis reports a 94% overall compliance to eligibility criteria directives, and overall HSCT- R for an alive sibling of 12% (63/522); the 63 HSCT were 97% matched, for curing ID in 84% and with 72 % overall survival outcome. Different policies among Banks were compared (Bank vs others: 1) eligibility criteria distribution : Bank PV06 DCB for MD Conclusion. Advice for public long-term DCB storage should depend on HLA compatibility, potential recipient, disease progression, and likelihood/ timing of using DCB; Italian CB Banks are actively cohoperating to find best public banking practices to ensure a CDB storage system that is ethical, cost effective and responsive to patient needs. Disclosures: No relevant conflicts of interest to declare.

The overall use of allogeneic blood transfusions in clinical practice remains relatively high and still varies widely among centres and practitioners. Moreover, allogeneic blood transfusions have historically been linked with risks and complications: some of them (e.g. transfusion reactions and transmission of pathogens) have been largely mitigated through advancements in blood banking whereas some others (e.g. immunomodulation and transfusion-related acute lung injury) appear to have more subtle etiologies and are more difficult to tackle. Furthermore, blood transfusions are costly and the supply of blood is limited. Finally, evidence indicates that a great number of the critically ill patients who are being transfused today may not be having tangible benefits from the transfusion. Patient blood management is an evidence-based, multidisciplinary, multimodal, and patient-tailored approach aimed at reducing or eliminating the need for allogeneic transfusion by managing anaemia, perioperative blood conservation, surgical haemostasis, and blood as well as plasma-derivative drug use. From this point of view, the reduction of allogeneic blood usage is not an end in itself but a tool to achieve better patient clinical outcome. This article focuses on the three-pillar matrix of patient blood management where the understanding of basic physiology and pathophysiology is at the core of evidence-based approaches to optimizing erythropoiesis, minimising bleeding and tolerating anemia. Anesthesiologists and critical care physicians clearly have a key role in patient blood management programmes are and should incorporate its principles into clinical practice-based initiatives that improve patient safety and clinical outcomes.