The impact of iron supplementation efficiency in female blood donors with a decreased ferritin level and no anaemia. Rationale and design of a randomised controlled trial: a study protocol.

ABSTRACT: BACKGROUND: There is no recommendation to screen ferritin level in blood donors, even though several studies have noted the high prevalence of iron deficiency after blood donation, particularly among menstruating females. Furthermore, some clinical trials have shown that non-anaemic women with unexplained fatigue may benefit from iron supplementation. Our objective is to determine the clinical effect of iron supplementation on fatigue in female blood donors without anaemia, but with a mean serum ferritin </= 30 ng/ml. METHODS/DESIGN: In a double blind randomised controlled trial, we will measure blood count and ferritin level of women under age 50 yr, who donate blood to the University Hospital of Lausanne Blood Transfusion Department, at the time of the donation and after 1 week. One hundred and forty donors with a ferritin level </= 30 ng/ml and haemoglobin level >/= 120 g/l (non-anaemic) a week after the donation will be included in the study and randomised. A one-month course of oral ferrous sulphate (80 mg/day of elemental iron) will be introduced vs. placebo. Self-reported fatigue will be measured using a visual analogue scale. Secondary outcomes are: score of fatigue (Fatigue Severity Scale), maximal aerobic power (Chester Step Test), quality of life (SF-12), and mood disorders (Prime-MD). Haemoglobin and ferritin concentration will be monitored before and after the intervention. DISCUSSION: Iron deficiency is a potential problem for all blood donors, especially menstruating women. To our knowledge, no other intervention study has yet evaluated the impact of iron supplementation on subjective symptoms after a blood donation. TRIAL REGISTRATION: NCT00689793.

Erythropoietin and blood doping.

OBJECTIVE AND METHOD: To outline the direct and indirect approaches in the fight against blood doping in sports, the different strategies that have been used and are currently being used to fight efficiently against blood doping are presented and discussed. RESULTS AND CONCLUSIONS: The paper outlines the different approaches and diagnostic tools that some federations have to identify and target sports people demonstrating abnormal blood profiles. Originally blood tests were introduced for medical reasons and for limiting misuse of recombinant human erythropoietin (rHuEPO). In this way it became possible to prevent athletes with haematocrit levels well above normal, and potentially dangerous for their health, competing in sport. Today, with nearly a decade of blood testing experience, sports authorities should be familiar with some of the limitations and specially the ability of blood tests performed prior to competitions to fight efficiently against the misuse of rHuEPO, blood transfusion, and artificial haemoglobin.

Hepatitis E Virus Seroprevalence among Blood Donors in Southwest Switzerland.

Aim: The aim of this study was to determine the seroprevalence of Hepatitis E virus (HEV) among blood donors in southwest Switzerland.Background: HEV is recognized as a food-borne disease in industrialized countries, transmitted mainly through pork meat. Cases of transmission through blood transfusion have also been reported. Recent studies have revealed seroprevalence rates of 13.5%, 16.6% and 20.6% among blood donors in England, France and Denmark, respectively.Methods: We analyzed 550 consecutive blood donor samples collected in the region of Lausanne, canton of Vaud, Switzerland, for the presence of anti-HEV IgG, using the MP Diagnostics HEV ELISA kit. For each donor, we documented age, sex and alanine aminotransferase (ALT) value.Results: The study panel was composed of 332 men (60.4%) and 218 women (39.6%). Overall, anti-HEV IgG was found in 27 of 550 samples (4.9%). The seroprevalence was 5.4% (18/332) in men and 4.1% (9/218) in women. The presence of anti-HEV IgG was not correlated with age, gender or ALT values. However, we observed a peak in seroprevalence of 5.3% in individuals aged 51 to 70 years old.Conclusions: Compared with other European countries, HEV seroprevalence among blood donors in southwest Switzerland is low. The low seroprevalence may be explained by the sensitivity of commercial tests used and/or the strict regulation of animal and meat imports. Data regarding HEV prevalence in Swiss livestock are lacking and merit exploration.

Inactivation des pathogènes des produits sanguins labiles: entre enjeux financiers et conséquences possibles à long terme [Pathogen reduction of blood components: from financial issues to possible long-term consequences].

Pathogen inactivation of blood products represents a global and major paradigm shift in transfusion medicine. In the next near future, it is likely that most blood products will be inactivated by various physicochemical approaches. The concept of blood safety will be challenged as well as transfusion medicine practice, notably for donor selection or biological qualification. In this context, it seems mandatory to develop analytical economic approaches by assessing costs-benefits ratio of blood transfusion as well as to set up cohorts of patients based on hemovigilance networks allowing rigorous scientific analysis of the benefits and the risks of blood transfusion at short- and long-term.

Iron and transfusion medicine.

Blood bankers have focused their energy to secure blood transfusion, and only recently have studies been published on the effect of blood donation on iron metabolism. In many facilities, hemoglobin measurement is only performed just before or even during blood donation, but the determination of iron stores is largely ignored. The 2013 paradox of transfusion medicine is due to the fact that blood donation may be harmful and leads to iron deficiency with or without anemia, but for other individuals, it may be a healthy measure preventing type 2 diabetes. The purpose of this review is to discuss iron metabolism in the perspective of blood donation, notably regarding their possible genetic profiles that eventually will discriminate "good" iron absorbers from "bad" iron responders.

Red blood cell microparticles and blood group antigens: an analysis by flow cytometry.

BACKGROUND: The storage of blood induces the formation of erythrocytes-derived microparticles. Their pathogenic role in blood transfusion is not known so far, especially the risk to trigger alloantibody production in the recipient. This work aims to study the expression of clinically significant blood group antigens on the surface of red blood cells microparticles. MATERIAL AND METHODS: Red blood cells contained in erythrocyte concentrates were stained with specific antibodies directed against blood group antigens and routinely used in immunohematology practice. After inducing erythrocytes vesiculation with calcium ionophore, the presence of blood group antigens was analysed by flow cytometry. RESULTS: The expression of several blood group antigens from the RH, KEL, JK, FY, MNS, LE and LU systems was detected on erythrocyte microparticles. The presence of M (MNS1), N (MNS2) and s (MNS4) antigens could not be demonstrated by flow cytometry, despite that glycophorin A and B were identified on microparticles using anti-CD235a and anti-MNS3. DISCUSSION: We conclude that blood group antigens are localized on erythrocytes-derived microparticles and probably keep their immunogenicity because of their capacity to bind specific antibody. Selective segregation process during vesiculation or their ability to elicit an immune response in vivo has to be tested by further studies.

A cell therapy approach for erythropoietin delivery using encapsulated human primary fibroblasts

Summary Cell therapy has emerged as a strategy for the treatment of various human diseases. Cells can be transplanted considering their morphological and functional properties to restore a tissue damage, as represented by blood transfusion, bone marrow or pancreatic islet cells transplantation. With the advent of the gene therapy, cells also were used as biological supports for the production of therapeutic molecules that can act either locally or at distance. This strategy represents the basis of ex vivo gene therapy characterized by the removal of cells from an organism, their genetic modification and their implantation into the same or another individual in a physiologically suitable location. The tissue or biological function damage dictates the type of cells chosen for implantation and the required function of the implanted cells. The general aim of this work was to develop an ex vivo gene therapy approach for the secretion of erythropoietin (Epo) in patients suffering from Epo-responsive anemia, thus extending to humans, studies previously performed with mouse cells transplanted in mice and rats. Considering the potential clinical application, allogeneic primary human cells were chosen for practical and safety reasons. In contrast to autologous cells, the use of allogeneic cells allows to characterize a cell lineage that can be further transplanted in many individuals. Furthermore allogeneic cells avoid the potential risk of zoonosis encountered with xenogeneic cells. Accordingly, the immune reaction against this allogeneic source was prevented by cell macro- encapsulation that prevents cell-to-cell contact with the host immune system and allows to easy retrieve the implanted device. The first step consisted in testing the survival of various human primary cells that were encapsulated and implanted for one month in the subcutaneous tissue of immunocompetent and naturally or therapeutically immunodepressed mice, assuming that xenogeneic applications constitute a stringent and representative screening before human transplantation. A fibroblast lineage from the foreskin of a young donor, DARC 3.1 cells, showed the highest mean survival score. We have then performed studies to optimize the manufacturing procedures of the encapsulation device for successful engraftment. The development of calcifications on the polyvinyl alcohol (PVA) matrix serving as a scaffold for enclosed cells into the hollow fiber devices was reported after one month in vivo. Various parameters, including matrix rinsing solutions, batches of PVA and cell lineages were assessed for their respective role in the development of the phenomenon. We observed that the calcifications could be totally prevented by using ultra-pure sterile water instead of phosphate buffer saline solution in the rinsing procedure of the PVA matrix. Moreover, a higher lactate dehydrogenase activity of the cells was found to decrease calcium depositions due to more acidic microenvironment, inhibiting the calcium precipitation. After the selection of the appropriate cell lineage and the optimization of encapsulation conditions, a retroviral-based approach was applied to DARC 3.1 fibroblasts for the transduction of the human Epo cDNA. Various modifications of the retroviral vector and the infection conditions were performed to obtain clinically relevant levels of human Epo. The insertion of a post-transcriptional regulatory element from the woodchuck hepatitis virus as well as of a Kozak consensus sequence led to a 7.5-fold increase in transgene expression. Human Epo production was further optimized by increasing the multiplicity of infection and by selecting high producer cells allowing to reach 200 IU hEpo/10E6 cells /day. These modified cells were encapsulated and implanted in vivo in the same conditions as previously described. All the mouse strains showed a sustained increase in their hematocrit and a high proportion of viable cells were observed after retrieval of the capsules. Finally, in the perspective of human application, a syngeneic model using encapsulated murine myoblasts transplanted in mice was realized to investigate the roles of both the host immune response and the cells metabolic requirements. Various loading densities and anti-inflammatory as well as immunosuppressive drugs were studied. The results showed that an immune process is responsible of cell death in capsules loaded at high cell density. A supporting matrix of PVA was shown to limit the cell density and to avoid early metabolic cell death, preventing therefore the immune reaction. This study has led to the development of encapsulated cells of human origin producing clinically relevant amounts of human EPO. This work resulted also to the optimization of cell encapsulation technical parameters allowing to begin a clinical application in end-stage renal failure patients. Résumé La thérapie cellulaire s'est imposée comme une stratégie de traitement potentiel pour diverses maladies. Si l'on considère leur morphologie et leur fonction, les cellules peuvent être transplantées dans le but de remplacer une perte tissulaire comme c'est le cas pour les transfusions sanguines ou les greffes de moelle osseuse ou de cellules pancréatiques. Avec le développement de la thérapie génique, les cellules sont également devenues des supports biologiques pour la production de molécules thérapeutiques. Cette stratégie représente le fondement de la thérapie génique ex vivo, caractérisée par le prélèvement de cellules d'un organisme, leur modification génétique et leur implantation dans le même individu ou dans un autre organisme. Le choix du type de cellule et la fonction qu'elle doit remplir pour un traitement spécifique dépend du tissu ou de la fonction biologique atteintes. Le but général de ce travail est de développer .une approche par thérapie génique ex vivo de sécrétion d'érythropoïétine (Epo) chez des patients souffrant d'anémie, prolongeant ainsi des travaux réalisés avec des cellules murines implantées chez des souris et des rats. Dans cette perpective, notre choix s'est porté sur des cellules humaines primaires allogéniques. En effet, contrairement aux cellules autologues, une caractérisation unique de cellules allogéniques peut déboucher sur de nombreuses applications. Par ailleurs, l'emploi de cellules allogéniques permet d'éviter les riques de zoonose que l'on peut rencontrer avec des cellules xénogéniques. Afin de protéger les cellules allogéniques soumises à une réaction immunitaire, leur confinement dans des macro-capsules cylindriques avant leur implantation permet d'éviter leur contact avec les cellules immunitaires de l'hôte, et de les retrouver sans difficulté en cas d'intolérance ou d'effet secondaire. Dans un premier temps, nous avons évalué la survie de différentes lignées cellulaires humaines primaires, une fois encapsulées et implantées dans le tissu sous-cutané de souris, soit immunocompétentes, soit immunodéprimées naturellement ou par l'intermédiaire d'un immunosuppresseur. Ce modèle in vivo correspond à des conditions xénogéniques et représente par conséquent un environnement de loin plus hostile pour les cellules qu'une transplantation allogénique. Une lignée fibroblastique issue du prépuce d'un jeune enfant, nommée DARC 3 .1, a montré une remarquable résistance avec un score de survie moyen le plus élevé parmi les lignées testées. Par la suite, nous nous sommes intéressés aux paramètres intervenant dans la réalisation du système d'implantation afin d'optimaliser les conditions pour une meilleure adaptation des cellules à ce nouvel environnement. En effet, en raison de l'apparition, après un mois in vivo, de calcifications au niveau de la matrice de polyvinyl alcohol (PVA) servant de support aux cellules encapsulées, différents paramètres ont été étudiés, tels que les procédures de fabrication, les lots de PVA ou encore les lignées cellulaires encapsulées, afin de mettre en évidence leur rôle respectif dans la survenue de ce processus. Nous avons montré que l'apparition des calcifications peut être totalement prévenue par l'utilisation d'eau pure au lieu de tampon phosphaté lors du rinçage des matrices de PVA. De plus, nous avons observe qu'un taux de lactate déshydrogénase cellulaire élevé était corrélé avec une diminution des dépôts de calcium au sein de la matrice en raison d'un micro-environnement plus acide inhibant la précipitation du calcium. Après sélection de la lignée cellulaire appropriée et de l'optimisation des conditions d'encapsulation, une modification génétique des fibroblastes DARC 3.1 a été réalisée par une approche rétrovirale, permettant l'insertion de l'ADN du gène de l'Epo dans le génome cellulaire. Diverses modifications, tant au niveau génétique qu'au niveau des conditions d'infection, ont été entreprises afin d'obtenir des taux de sécrétion d'Epo cliniquement appropriés. L'insertion dans la séquence d'ADN d'un élément de régulation post¬transcriptionnelle dérivé du virus de l'hépatite du rongeur (« woodchuck ») ainsi que d'une séquence consensus appelée « Kozak » ont abouti à une augmentation de sécrétion d'Epo 7.5 fois plus importante. De même, l'optimisation de la multiplicité d'infection et la sélection plus drastique des cellules hautement productrices ont permis finalement d'obtenir une sécrétion correspondant à 200 IU d'Epo/10E6 cells/jour. Ces cellules génétiquement modifiées ont été encapsulées et implantées in vivo dans les mêmes conditions que celles décrites plus haut. Toutes les souris transplantées ont montré une augmentation significative de leur hématocrite et une proportion importante de cellules présentait une survie conservée au moment de l'explantation des capsules. Finalement, dans la perspective d'une application humaine, un modèle syngénique a été proposé, basé sur l'implantation de myoblastes murins encapsulés dans des souris, afin d'investiguer les rôles respectifs de la réponse immunitaire du receveur et des besoins métaboliques cellulaires sur leur survie à long terme. Les cellules ont été encapsulées à différentes densités et les animaux transplantés se sont vus administrer des injections de molécules anti-inflammatoires ou immunosuppressives. Les résultats ont démontré qu'une réaction immunologique péri-capsulaire était à la base du rejet cellulaire dans le cas de capsules à haute densité cellulaire. Une matrice de PVA peut limiter cette densité et éviter une mort cellulaire précoce due à une insuffisance métabolique et par conséquent prévenir la réaction immunitaire. Ce travail a permis le développement de cellules encapsulées d'origine humaine sécrétant des taux d'Epo humaine adaptés à des traitements cliniques. De pair avec l'optimalisation des paramètres d'encapsulation, ces résultats ont abouti à l'initiation d'une application clinique destinée à des patients en insuffisance rénale terminale.

Blood donor motivation: what is ethical? What works?

The retention of previous donors and the recruitment of new donors is a serious challenge for many blood donation services in their effort to prevent blood shortages. More and more services make use of some sort of donation incentives. However, the use of (material) incentives to motivate blood donors is fiercely controversial and there is a longstanding (ethical) debate about whether it should be allowed that donors receive material rewards. Interestingly, this debate is dealt with in almost complete absence of systematic empirical evidence on the effectiveness of material incentives in encouraging people to donate. In this paper, we argue that the discussion on what is ethical in motivating blood donors should be enriched with empirical evidence based on field experiments. We confront the Titmuss controversy with recent results from an experiment administering lottery tickets as a motivation device. Moreover, we take up a neglected phenomenon in the study of blood donors: many non-donors are not principally against donating blood they have just never made up their mind about becoming active blood donors. We propose active decisions as a mechanism to transform latent prosocial preferences into actual prosocial behavior.

Low seroprevalence of hepatitis E virus among blood donors in Switzerland

Introduction: Hepatitis E virus (HEV) emerged as an autochthonous food-borne disease in developed countries, transmitted mainly through pork meat. Cases of transmission through blood transfusion have been reported. Recent studies revealed sero-prevalence rates of 13.5%, 16.6% and 20.6% among blood donors in England, France and Denmark, respectively. The aim of this study was to determine the sero-prevalence of HEV among Swiss blood donors. Method: We screened 550 consecutive blood donations from the Service Régional Vaudois de Transfusion Sanguine of Epalinges, for the presence of anti-HEV IgG (MP Diagnostics HEV ELISA). The sample size was based on the Lorentz formula considering an expected prevalence of 3% with a precision of 1.5%. For each donor, we studied the following variables: age, sex and alanine aminotransferase (ALT) value. Results: All blood donors were Caucasian, and included 332 men (60.4%) and 218 women (39.6%). The median age was 55 years (IQR 46-63 years). Overall, anti-HEV IgG were found in 27 of 550 samples (4.9%). The sero-prevalence was 5.4% (18/314) in men and 4.1% (9/209) in women. The presence of anti-HEV IgG was not correlated to age, gender or ALT values. Conclusion: Compared to other European countries, the HEV sero-prevalence among blood donors in Switzerland is surprisingly low. Possible explanations include the strict regulation of animals and meat import. However, to confirm this hypothesis, further studies assessing the prevalence of HEV in Swiss swine will be necessary.

Microparticles from stored red blood cells induce thrombin generation

Background: Microparticles are small phospholipid vesicles of <1 lm shed in blood flow by various cell types including red blood cells. Erythrocyte-derived microparticles (EMPs) accumulate in erythrocyte concentrates (ECs) during their storage time. EMPs are considered as part of storage lesion and as their exact role is not elucidated, they could be involved in these clinical outcomes. Aims: The aim of this study is to evaluate the impact and implication of EMPs isolate from ECs on coagulation. Methods: EMPs were first isolated from erythrocyte concentrates by centrifugation and counted by flow cytometry. Using a calibrated automated thrombogram, EMPs were then added to different type of plasmas in order to evaluate the potential of thrombin generation. Results: We demonstrate that EMPs isolated from ECs are capable to accelerate and amplify thrombin generation in presence of a low exogenous tissue factor concentration, thanks to their negatively charged membrane necessary for the assembly of coagulation complexes. Interestingly, in the absence of exogenous tissue factor, EMPs are also able to trigger thrombin generation. In addition, thrombin generation induced by EMPs is not affected by the presence of anti-TF antibodies. Finally, thrombin generation induced by EMPs is not affected by using plasma samples deficient in factor VII, XI or XII. However, thrombin generation is reduced in plasma deficient in factor VIII or IX and is completely abolished in plasma deficient in factor X, V or II. No thrombin generation was observed in plasma samples without EMPs. Summary/conclusion: Several studies have shown a link between storage time of blood products and post transfusion complications. We provide evidence that EMPs accumulated during storage of erythrocyte concentrates were not only able to accelerate and support thrombin generation in plasma in presence of a low exogenous tissue-factor concentration, but also to trigger thrombin generation in absence of exogenous TF. The impact of those transfused EMs is unknown on recipients, nevertheless it could be hypothesized that under certain circumstances, transfused EMPs could be involved in thrombin generation and could be linked to adverse clinical outcome. Further work is needed to determine whether procoagulant EMPs transfused with erythrocyte concentrate may account for some of the complications occurring after red blood cell transfusion, and more particularly after transfusion of ''older''stored blood, rich in EMPs.

Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008.

OBJECTIVE: To provide an update to the original Surviving Sepsis Campaign clinical management guidelines, "Surviving Sepsis Campaign Guidelines for Management of Severe Sepsis and Septic Shock," published in 2004. DESIGN: Modified Delphi method with a consensus conference of 55 international experts, several subsequent meetings of subgroups and key individuals, teleconferences, and electronic-based discussion among subgroups and among the entire committee. This process was conducted independently of any industry funding. METHODS: We used the Grades of Recommendation, Assessment, Development and Evaluation (GRADE) system to guide assessment of quality of evidence from high (A) to very low (D) and to determine the strength of recommendations. A strong recommendation (1) indicates that an intervention's desirable effects clearly outweigh its undesirable effects (risk, burden, cost) or clearly do not. Weak recommendations (2) indicate that the tradeoff between desirable and undesirable effects is less clear. The grade of strong or weak is considered of greater clinical importance than a difference in letter level of quality of evidence. In areas without complete agreement, a formal process of resolution was developed and applied. Recommendations are grouped into those directly targeting severe sepsis, recommendations targeting general care of the critically ill patient that are considered high priority in severe sepsis, and pediatric considerations. RESULTS: Key recommendations, listed by category, include early goal-directed resuscitation of the septic patient during the first 6 hrs after recognition (1C); blood cultures before antibiotic therapy (1C); imaging studies performed promptly to confirm potential source of infection (1C); administration of broad-spectrum antibiotic therapy within 1 hr of diagnosis of septic shock (1B) and severe sepsis without septic shock (1D); reassessment of antibiotic therapy with microbiology and clinical data to narrow coverage, when appropriate (1C); a usual 7-10 days of antibiotic therapy guided by clinical response (1D); source control with attention to the balance of risks and benefits of the chosen method (1C); administration of either crystalloid or colloid fluid resuscitation (1B); fluid challenge to restore mean circulating filling pressure (1C); reduction in rate of fluid administration with rising filing pressures and no improvement in tissue perfusion (1D); vasopressor preference for norepinephrine or dopamine to maintain an initial target of mean arterial pressure > or = 65 mm Hg (1C); dobutamine inotropic therapy when cardiac output remains low despite fluid resuscitation and combined inotropic/vasopressor therapy (1C); stress-dose steroid therapy given only in septic shock after blood pressure is identified to be poorly responsive to fluid and vasopressor therapy (2C); recombinant activated protein C in patients with severe sepsis and clinical assessment of high risk for death (2B except 2C for postoperative patients). In the absence of tissue hypoperfusion, coronary artery disease, or acute hemorrhage, target a hemoglobin of 7-9 g/dL (1B); a low tidal volume (1B) and limitation of inspiratory plateau pressure strategy (1C) for acute lung injury (ALI)/acute respiratory distress syndrome (ARDS); application of at least a minimal amount of positive end-expiratory pressure in acute lung injury (1C); head of bed elevation in mechanically ventilated patients unless contraindicated (1B); avoiding routine use of pulmonary artery catheters in ALI/ARDS (1A); to decrease days of mechanical ventilation and ICU length of stay, a conservative fluid strategy for patients with established ALI/ARDS who are not in shock (1C); protocols for weaning and sedation/analgesia (1B); using either intermittent bolus sedation or continuous infusion sedation with daily interruptions or lightening (1B); avoidance of neuromuscular blockers, if at all possible (1B); institution of glycemic control (1B), targeting a blood glucose < 150 mg/dL after initial stabilization (2C); equivalency of continuous veno-veno hemofiltration or intermittent hemodialysis (2B); prophylaxis for deep vein thrombosis (1A); use of stress ulcer prophylaxis to prevent upper gastrointestinal bleeding using H2 blockers (1A) or proton pump inhibitors (1B); and consideration of limitation of support where appropriate (1D). Recommendations specific to pediatric severe sepsis include greater use of physical examination therapeutic end points (2C); dopamine as the first drug of choice for hypotension (2C); steroids only in children with suspected or proven adrenal insufficiency (2C); and a recommendation against the use of recombinant activated protein C in children (1B). CONCLUSIONS: There was strong agreement among a large cohort of international experts regarding many level 1 recommendations for the best current care of patients with severe sepsis. Evidenced-based recommendations regarding the acute management of sepsis and septic shock are the first step toward improved outcomes for this important group of critically ill patients.