• Exosomes in blood are proinflammatory and may contribute to transfusionrelated immune modulation.• Exosomes act via antigenpresenting cells to potentiate T-cell survival and mitogeninduced proliferation.Extracellular vesicles (EVs) are small, double membrane vesicles derived from leukocytes, platelets, and cells of other tissues under physiological or pathological conditions. Generation of EVs in stored blood is thought to be associated with adverse effects and potentially immunosuppression in blood transfusion recipients. We measured the quantity and cells of origin for EVs isolated from stored red blood cell (RBC) units and tested whether they had any effects on T-cell-mediated immune responses. Mixing peripheral blood mononuclear cells (PBMCs) with EVs resulted in secretion of proinflammatory cytokines and chemokines and increased survival of unstimulated PBMCs. EVs augmented mitogen-induced CD4 1 and CD8 1 T-cell proliferation in an antigenpresenting cell (APC)-dependent manner. We demonstrated that EVs interacted primarily with monocytes and induced proinflammatory cytokine secretion. We also showed that the exosome fraction of EVs and not larger microvesicles was responsible for induction of TNF-a production by monocytes. Furthermore, blockade of CD40 or CD40L accessory molecules largely neutralized the EV augmentation of T-cell responses, implying a role for cell-cell interaction between T cells and EV-activated monocytes. Contrary to our hypothesis, the data demonstrate that EVs isolated from RBC units increase the potency of APCs and boost mitogen-driven T-cell proliferative responses. (Blood. 2014;123(5):687-696)

UVB plus riboflavin treatment of WBC-enriched PRP effectively blocks alloimmunization and modulates immune responses to subsequent exposures.

BACKGROUND-Donor white blood cells (WBCs) present in transfusion products can lead to immune sequelae such as production of anti-HLA antibodies or GVHD in susceptible transfusion recipients. Eliminating the immunogenicity of blood products may prove to be of clinical benefit, particularly in patients requiring multiple transfusions in whom allosensitization is common. This study examines a method of pathogen reduction based on UV light illumination in the presence of riboflavin. In addition to pathogens, WBCs treated with this system are also affected and fail to stimulate proliferation of allogeneic PBMCs in vitro.

• High, but not low to moderate, HLA antibody levels are associated with platelet refractoriness.In the Trial to Reduce Alloimmunization to Platelets (TRAP) study, 101 of 530 participants became refractory to platelet transfusions without evidence of HLA or human platelet antigen (HPA) antibodies. We used a more sensitive bead-based assay to detect and quantify HLA antibodies and a qualitative solid-phase enzyme-linked immunosorbet assay for HPA to determine whether low-level antibodies could predict refractoriness in longitudinal panels from 170 lymphocytotoxicity assay (LCA) 2 and 20 LCA Continuing Medical Education online This activity has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education through the joint sponsorship of Medscape, LLC and the American Society of Hematology. Medscape, LLC is accredited by the ACCME to provide continuing medical education for physicians. Medscape, LLC designates this Journal-based CME activity for a maximum of 1.0 AMA PRA Category 1 Credit(s)™. Physicians should claim only the credit commensurate with the extent of their participation in the activity. All other clinicians completing this activity will be issued a certificate of participation. To participate in this journal CME activity: (1) review the learning objectives and author disclosures; (2) study the education content; (3) take the post-test with a 70% minimum passing score and complete the evaluation at http://www.medscape.org/journal/blood; and (4) view/print certificate. For CME questions, see page 3299. Disclosures The authors, Associate Editor Mortimer Poncz, and CME questions author Charles P. Vega, Associate Professor and Residency Director, Department of Family Medicine, University of California-Irvine, declare no competing financial interests. Learning objectives Upon completion of this activity, participants will be able to:1. Describe alloimmunization due to HLA after platelet transfusion. 2. Analyze the significance of human platelet antigen (HPA) antibodies (Abs) in cases of alloimmunization after transfusion. 3. Evaluate the performance of newer tests for HLA Ab and HPA Ab. 4. Assess the role of HLA Ab and HPA Ab among patients refractory to treatment with platelet transfusions.

BACKGROUND Trauma and transfusion can both alter immunity, and while transfusions are common among traumatically injured patients, few studies have examined their combined effects on immunity. STUDY DESIGN AND METHODS We tracked the plasma levels of 41 immunomodulatory proteins in 56 trauma patients from time of injury up to 1 year later. In addition, a murine model was developed to distinguish between the effects of transfusion and underlying injury and blood loss. RESULTS Thirty-one of the proteins had a statistically significant change over time after traumatic injury, with a mixed early response that was predominantly anti-inflammatory followed by a later increase in proteins involved in wound healing and homeostasis. Results from the murine model revealed similar cytokine responses to humans. In mice, trauma/hemorrhage caused early perturbations in a number of the pro- and anti-inflammatory mediators measured, and transfusion blunted early elevations in IL-6, IL-10, MMP-9, and IFN-γ. Transfusion caused or exacerbated changes in MCP-1, IL-1α, IL-5, IL-15, and soluble E-selectin. Finally, trauma/hemorrhage alone increased KC and IL-13. CONCLUSIONS This work provides a detailed characterization of the major shift in the immunological environment in response to trauma and transfusion and clarifies which immune mediators are affected by trauma/hemorrhage and which by transfusion.

Pathogen reduction significantly reduces alloimmunization in repeatedly transfused mice and combined with leukoreduction provides a high level of protection from alloimmunization.

Measurement of peripheral blood cytokines and other immunomodulatory proteins is a useful and popular tool for assessing human immune responses to a wide range of assaults. A common challenge in this work is obtaining fresh, high-quality samples and limiting the time between blood collection and the separation of plasma or serum from cells. In this study we sought to determine the effect of sample age at the time of processing on the measured levels of 41 soluble immune mediators. Two cohorts were examined: healthy lab donors and trauma patients, who have significant immune perturbation. Whole-blood samples were aliquoted, and plasma was isolated, at days 0, 1, 2, and 3 after collection. Multiplexing techniques were used to measure protein concentrations, and general estimating equations were used to determine if there was a significant change over time. Over the 3-day period examined, only 15 of the 41 proteins showed no significant change in either cohort. Among the remaining proteins both increases and decreases were observed, with changes ranging from 2.4% per day to 325% per day. Proteins with significant changes in one cohort did not always show significant changes in the other group. These results support the need to separate plasma or serum from whole blood as quickly as possible and/or to standardize the length of time to processing within a given study of peripheral blood protein concentrations. When this is not possible, care should be taken to account for differences due to sample age.

BACKGROUND Both leukoreduction and UV light treatment of blood products have been shown to reduce the incidence of HLA antibody development in recipients, but the impact of these treatments on the magnitude and persistence of the antibody response is less clear. STUDY DESIGN AND METHODS Longitudinal samples from 319 subjects taken from 4 different study cohorts were evaluated for HLA antibodies to determine the effects of leukoreduction and UV treatment on HLA antibody generation and persistence. RESULTS Subjects receiving leukoreduced or UV treated blood products were less likely to generate class I HLA antibodies, and those receiving leukoreduced blood were also less likely to generate class II HLA antibodies. Among those receiving non-leukoreduced blood, 55% developed class I HLA antibodies and 51% developed class II HLA antibodies compared with 28% (class I) and 15% (class II) for those receiving leukoreduced blood and 36% (class I) and 54% (class II) for those receiving UV treated blood. Among alloimmunized subjects, leukoreduction resulted in a significant 2-fold reduction in the magnitude of class I HLA antibodies, and UV treatment resulted in a significant 3-fold reduction in the magnitude of class II HLA antibodies. Both treatments resulted in shorter persistence of class I HLA antibodies. CONCLUSIONS These data demonstrate that leukoreduction and UV treatment of blood products results not only in a reduction in the incidence of HLA antibody production, but also in lower and more transient HLA antibody levels among sensitized transfusion recipients.