ASH 2010 meeting report—Top 10 clinically oriented abstracts in transfusion medicine
Authors: Janice McFarland, Ravi Sarode
Journal: American Journal of Hematology (2011)

At the 2010 American Society of Hematology annual meeting held December 4–7, in Orlando, FL, USA. Fifty-five articles in the field of Transfusion Medicine comprised two oral and two poster sessions. In addition, one educational session and one scientific committee session in Transfusion Medicine were presented. As is the case for all abstracts submitted to the Society in the months preceding the meeting, the Transfusion Medicine articles were read and evaluated by a committee of experts in the field and ranked according to scientific merit and impact on practice. The articles scoring the highest in this evaluation were selected for oral and poster presentation. The following 10 articles were selected for summarization because they either represent potentially important breakthroughs in the evolution of a new direction in transfusion medicine (ex vivo production of blood cells for cellular and transfusion therapy) and/or contain information that is immediately relevant to clinical practice. Space constraints force the omission of many other impactful articles in the field, all of which, including those summarized here, are published in abstract form in the Society's journal Blood, Vol. 116 (21) Nov 2010. Articles included in this summary are identified by abstract number. MSC are capable of differentiating into different tissues and also manifest immunosuppressive properties [1]. These features make them prime targets for use in cellular therapy, a relatively new field being emphasized within Transfusion Medicine [2]. The quantity of MSC needed for therapeutic use is thought to require large scale ex vivo expansion, and a number of protocols to achieve this have been published. In this study one- (1 passage) and two- (2 passages) step procedures were compared using either fetal calf serum (fcs) or human platelet lysate (hpl) to potentiate growth of MCS derived from normal adult bone marrow. The investigators demonstrated that the two-step protocol resulted in increased numbers of MCS (over 1.5 billion cells per μL of initial bone marrow seeded) and that hpl was superior to fcs in effecting MSC expansion. Evaluation of the cytokines/chemokines in the hpl found large amounts of PDGF-AB/BB, PDGF-AA, RANTES, sCD40L, GRO, sVCAM, and sICAM. The concentration of PDGF-AB/BB declined progressively in culture inversely proportional to the increase in the number of MSC while the remaining cytokines/chemokines were stable. Using an automated cell expansion system (CES) (Caridian BCT) they were able to further increase the yield of MCS while improving control of lactate, PDGF and glucose in the system. Eventual yields of >2.0 billion MCS/μL of initial bone marrow in under 3 weeks were achieved using animal free reagents. While the MSC remained phenotypically stable over one or two passages, experiments with prolonged culture of MSC with multiple passages resulted in phenotypic changes in the cells, sounding a note of caution and the need for standardization of ex vivo cell expansion techniques before their wide scale use in clinical medicine. Blood cells transfused today are for the most part mature cells with finite lifespan. In a new twist to the cellular therapy aspect of TM, these researchers sought to produce erythrocyte progenitors ex vivo that could be infused and produce mature red cells in the recipient. Reprogramming of CD34+ cells from cord blood to produce erythroid precursors in vitro was achieved using histone deacetylase inhibitors (HDACI), in particular, valproic acid (VPA). Commitment of progenitor cells to the erythroid lineage as measured by phenotypic markers was increased significantly in the VPA treated cells versus those exposed to cytokines alone. This was further supported by showing higher acetylation levels of VPA treated cells versus controls. The behavior of ex vivo expanded cell products were then tested in sublethally irradiated NOD/SCID mice, and both FACS and RT-PCR analyses showed evidence of circulating nucleated erythroid and enucleated red cells, providing proof of the concept that progenitor cells differentiated and expanded ex vivo could be used as a source of mature blood cells after transfusion into the recipient. One of the main goals in the pursuit of ex vivo generation of RBCs for transfusion is to produce a sufficient quantity of cells to be clinically useful. Previous work has shown that large numbers of erythroid cells (EBs) can be produced from adult buffy coat in culture stimulated with stem cell factor, IL-3, erythropoietin, dexamethasone, and estrogens (human erythroid massive amplification, HEMA) [3]. However, the production of EBs plateaus after about 2 weeks in culture and transfusion quantities of cells are not produced. Experience with HEMA has shown that two types of EBs can be differentiated using CD36 (thrombospondin receptor) and CD235a (glycophorin A) expression: immature EBs (iEB) and mature EBs (mEB). This study looked at the contribution of these two populations relative to the growth of total EBs in HEMA culture. Via mixing studies the investigators confirmed that mEBs had an inhibitory affect on the increase of cell numbers that were achieved using iEB alone. The loss of cells did not appear to be due to apoptosis, but rather to autophagy, a process that is activated in the natural maturation pathway of erythroid cells. By further co-culture studies of iEBs and mEBs, and staining with Acridine Orange (AO), a marker for activated autophagy machinery within cells, they were able to show that in the presence of mEBs, iEBs were induced to undergo autophagy. This then is the likely explanation for the limitation of EB production in the HEMA culture systems. The findings suggest that selective inhibition of this process may lead to improvement in yields of RBCs in ex vivo culture systems. Development of large scale quantities of red blood cells ex vivo systems will require efficient devices that will allow access to differentiating and maturing cells for analysis as well as production high yields of mature blood cells. In this study, the traditional two-dimensional tissue culture flasks, which require high concentrations of cytokines in order to sustain cultures of hematopoietic cells in vitro, were compared with a three-dimensional synthetic scaffold device that more closely mimics bone marrow architecture. The three-dimensional device had previously been shown to provide expansion of CBMNC in a cytokine-free environment for at least 28 days ex vivo [4]. In this study, two near-physiological concentrations of exogenous erythropoietin (EPO) were added to established cultures of CBMNC in the three-dimensional device without the use of serum or cytokines. Cultured cells were assessed at 14, 21, and 28 days both by extracting cells from the scaffold and testing for cell differentiation markers by flow cytometry, and by examining them in situ via light and electron microscopy. In the three-dimensional system, erythrocyte maturation was seen through the enucleation stage and viable cultures were maintained over 28 days. The higher dose of EPO (1.8 U/ml vs. 0.2 U/ml) resulted in greater production of RBCs. In comparison, cultures in two-dimensional tissue flasks supported with high concentrations of cytokines collapsed within 5 days. The three-dimensional scaffold structures provided a sound model for studying erythropoiesis ex vivo with physiological concentrations of EPO. Recent retrospective trials have shown poorer outcomes in patients transfused with “older” versus “younger” blood. These adverse effects are thought to be related to a “storage lesion” suffered by red blood cells. The fall in pH, 2,3-DPG, ATP, glucose, and RBC deformability, and rise in lactate and spicule formation are but some of the changes seen with increasing duration of RBC storage. This presentation focused on the accumulation of microparticles (MPs) of RBCs (RMP), WBC (LMP) and platelets (PMP) which also occurs with RBC storage. Depending on the source of MPs (RBC, WBC, platelets) the kinetics of accumulation differed. RMPs were stable until day 10 of storage, and then rose rapidly to 450% of baseline by day 30. LMPs were stable until day 20 of storage, and then increased to 160% by day 30. PMP rose steadily from day 0 to day 20 where they peaked at 220% of baseline. MP-mediated thrombin generation mimicked the RMP profile with stable levels increasing after day 10. Proinflammatory activity (Leukocyte CD11b expression) increased linearly from day 0 to 30, mimicking the kinetics of PMP. Two of the 12 experimental blood bags had high initial concentrations of platelets and these contained exceptionally high levels of PMP and RMP with high inflammatory and coagulant activities. The investigators concluded that RMPs are the predominate MP species in stored RBC increasing dramatically after10 days of storage. PMP were also significant and the two types of MPs appeared to be related to prothrombotic and/or proinflammatory markers. Interestingly, initial levels of platelets in the blood bags were correlated with both PMP and RMP. These studies suggest that MPs, particularly in relation to their proinflammatory and procoagulant effects, may be related to adverse reactions to transfusion of longer storage-age blood. Anemia is recognized as a predictor of poor outcomes in acute myocardial infarction (MI), possibly due to increase in ischemia and greater tissue damage. Acutely, the only way to correct anemia is via transfusion, although its role in supporting anemic patients with acute MI remains unclear. These researchers had shown in rats that transfusion of fresh blood reduces infarct size in a model of MI, improving both cardiac function and survival [5]. In this study, they sought to test whether stored blood would provide a similar benefit. Anemic and non-anemic rats underwent coronary artery ligation to produce acute MI. The anemic animals were randomly assigned to: (1) no transfusion, (2) transfusion of fresh rat blood, or (3) blood stored for 7 days in CPDA-1, which has been shown to have a storage lesion similar to that seen in human blood stored for 28 days [6]. At 24 hours after MI, measurement of cardiac function, infarct size, and survival was done. In addition, Caspase 3, protein that correlates with myocardial ischemia and apoptosis was also measured. Despite similar degrees of LV myocardial ischemia in all groups, the infarct size to area at risk ratios were significantly increased in anemic versus nonanemic controls. Transfusion of fresh blood decreased infarct size, improved LV function, and survival compared with anemic animals. However, transfusion of stored blood did not have the same salutary effects. The levels of Caspase 3 correlated with these findings. They concluded that fresh, but not stored blood improves infarct size, LV function, and survival in this animal model. Factors in stored but not fresh blood to which these adverse effects were attributed included increased free Hb, and decreased 2,3 DPG and ATP. Extrapolating these animal model results to humans is premature, but these findings are certainly intriguing and suggest that blood storage age ought to be looked at in the setting of AMI in anemic humans. The storage of refrigerated RBC units for up to 42 days has been approved by the FDA, in part, since at least 75% of the initial cells in autologous collections are recovered 24 hours after transfusion in healthy volunteer donors. This leaves the fate of up to 25% of the cells in the bag unclear. It is likely that many of these unaccounted for cells undergo extravascular hemolysis, releasing their hemoglobin and associated iron within the first 24 hours after transfusion. These researchers sought to study the kinetics of iron metabolism after transfusion of blood stored for 40 to 42 days to that of units stored for 3 to 7 days, using timed blood samples in normal volunteers who donated pairs of autologous units; one of which was stored long term (40–42 days) and the other, short term (3–7 days). At four hours after transfusion, older units led to significantly higher levels of both serum iron and bilirubin. No differences were seen in LDH or haptoglobin at any time point. Significant increases in transferrin saturation and non transferrin bound iron (NTBI) after older versus fresher units were not accompanied by differences in inflammatory markers including hepcidin, c-reactive protein, or IL-6. Although no adverse effects were noted in this study of healthy volunteers receiving stored autologous blood, plasma NTBI has been shown to promote bacterial growth and to induce oxidative injury [7, 8], and therefore its impact on ill patients at risk of sepsis, multi-organ failure and death is unclear. Transfusion of allogeneic blood cells is associated with immune-modulatory affects, some of which are beneficial to the patient (e.g. prolonged renal allograft survival after transfusion). Similar to many of the adverse effects that are associated with transfusion, the exact mechanism of this induced immune tolerance is not well understood. Moreover, in animal studies, these researchers have demonstrated that the salutary immunosuppressive effect of fresh blood transfusion in reducing immunogenicity of the blood product is lost when the blood is stored for 2 weeks before transfusion [9]. These observations are consistent with either of the following two hypotheses: (1) older, stored blood is more immunogenic, or (2) younger, fresh blood is more able to induce immune tolerance. In the present study, HOD mice, genetically engineered to express a compound antigen of Hen egg lysozyme, Ovalbumin, and human red cell antigen Duffy-b on their RBCs, were used as blood donors for immunocompetent C57/BL/6 recipients. The latter were given either fresh or 14-day stored blood or a combination of the two. Alloimmunization of recipients was assessed at 2 weeks posttransfusion using flow cytometry to detect antibodies to the HOD protein. The 14-day stored blood reproducibly resulted in higher rates of alloimmunization versus fresh blood. This tended to correlate with levels of IL-6, an inflammatory marker in the recipients posttransfusion. However, the rate of sensitization (and IL-6 generation) in the mixed transfusion (stored plus fresh) group was always less than that of animals receiving the stored blood alone. This effect persisted when volume of sensitizing blood was corrected for. Overall, over 90% of the recipients of stored blood became sensitized; compared with 20% of those receiving fresh and about 50% of those transfused with both types of blood. They concluded that the data supported the hypothesis that fresh blood exerted an immunoregulatory effect and was able to inhibit sensitization to stored blood. The implications for human transfusions are important, particularly for the potential impact on ongoing clinical trials of “younger” versus “older” blood where the storage times of transfused blood in the trials may overlap. Moreover, the finding of increased IL-6 in the recipients of older blood in this animal study is in contrast to the finding of no increase in this inflammatory marker in the human study (abstract 662 above), raising the question about to what extent we can extrapolate results from mice to humans in these transfusion studies. Transfusion related acute lung injury (TRALI) remains a “hot topic” in transfusion medicine. Blood products that contain greater volumes of plasma (FFP, thawed plasma, platelets) carry the greatest risk. There are two main pathogenic mechanisms thought to be responsible for TRALI: (1) HLA or neutrophil-specific WBC antibodies in donor plasma react with recipient neutrophils, causing them to adhere and activate in the pulmonary circulation, and (2) Nonimmune substances accumulate in stored blood and serve as one of two processes or “hits” necessary to cause neutrophils to become activated in the lung. Major efforts in the past decade have reduced patient exposure to WBC antibodies in donor plasma by limiting the source of plasma to male donors and by screening female platelet donors for HLA antibodies [10]. In contrast, no strategies are in place to address the incompletely characterized nonimmune substances related to TRALI in stored blood. This article sheds light on one of these factors: soluble CD40 ligand (sCD40L). This protein is a participant in the inflammatory cascade and is known to accumulate during the storage of platelets [11]. Moreover, it has been has been linked with both TRALI and febrile reactions after platelet transfusion [12, 13]. In the study, two methods were used to reduce the platelets that are known to contaminate licensed plasma: cell depleting centrifugation of plasma before freezing and leukoreduction by filtering of whole blood units before plasma production. The control (standard) FFP units contained about 4.0 × 103 platelets whereas the treated units had significantly fewer. Frozen control and experimental plasma units were thawed and held at 4°C and sampled over 5 days. Both methods of reducing contaminating platelets resulted in significant reduction in sCD40L in the plasma over the 5-day period while control units had elevated levels of the marker that increased significantly over the sampling period. Testing of the plasma samples using a previously published method to detect WBC priming activity known to be essential in inducing TRALI in both an animal model and an ex vivo system using human vascular endothelial cells [13-16] showed a high correlation of the priming activity with accumulation of sCD40L. The implications of this study are significant because this may explain an important nonimmune mechanism of TRALI in some patients who receive stored blood products that contain contaminating platelets (e.g. thawed plasma, RBCs). Moreover, since thawed plasma, i.e. FFP that is thawed and held at 4°C for 5 days, is being used more often by hospital blood banks to prevent wastage; the accumulation of sCD40L from the breakdown of contaminating platelets in this product may represent an increasing risk of TRALI to transfusion recipients. Malaria remains a major threat to world-wide health and continues to exert selective pressure on human populations in underdeveloped countries. Malaria's pathogenicity relies on cytoadhesion via a parasite-derived surface protein on infected RBCs, PfEMP1. In turn, this protein binds to ligands on platelets, endothelium, and other red cells contributing to vascular obstruction common in severe malarial syndromes such as cerebral malaria. Moreover, PfEMP1 exhibits preferential binding to blood Group A through a lectin-like domain, suggesting that individuals with this blood group may be at an increased risk of severe malarial disease compared with those with nongroup A blood types. Retrospective studies support this association [17]. This article described an analysis of a prospective observational study of children with acute malaria in Uganda. Blood groups of those children with uncomplicated malaria (UM) were compared with those with severe or fatal malaria (SM). The proportion of children with Group A or AB increased with categories of increasing disease severity (UM = 28%, SM = 37%, fatal = 48%) while those with blood Group O decreased. This difference was highly statistically significant. According to the investigators, “this represents the largest prospective study on the relationship between ABO blood groups and P. falciparum malaria and the first report sufficiently powered to demonstrate the effect of ABO on survival in P. falciparum malaria.” The study confirms malaria's continuing selection pressure on the developing world's populations.