Haem is associated with thrombosis in neonates and infants undergoing cardiac surgery for congenital heart disease.

BACKGROUND: Haem levels are associated with thrombosis in a variety of diseases, as well as being a contributing cause of thrombotic events in animal models. MATERIALS AND METHODS: We retrospectively analyzed samples from 39 children who underwent cardiac surgery with cardiopulmonary bypass, including 15 children who developed a postoperative thrombosis and 24 controls. RESULTS: Patients who developed thrombosis postoperatively had statistically significant higher average haem levels over time (presurgery to 12 h postsurgery) compared to patients who did not develop thrombosis. CONCLUSION: Higher cell-free total haem levels are associated with a higher risk of thrombosis in a paediatric cardiac surgical cohort.

Maresin 1 induces a novel pro-resolving phenotype in human platelets.

Essentials Specialized proresolving mediators (SPMs) promote the resolution of inflammation. This study sought to investigate the effects of SPMs on human platelet function. The SPM, Maresin 1, enhanced hemostatic, but suppressed inflammatory functions of platelets. SPMs uniquely regulate platelet function and may represent a new class of antiplatelet agents. SUMMARY: Background Antiplatelet therapy is a cornerstone of modern medical practice and is routinely employed to reduce the likelihood of myocardial infarction, thrombosis and stroke. However, current antiplatelet therapies, such as aspirin, often have adverse side-effects, including increased risk of bleeding, and some patients are relatively 'aspirin-resistant'. Platelets are intimately involved in hemostasis and inflammation, and clinical consequences are associated with excessive or insufficient platelet activation. Objectives A major unmet need in the field of hematology is the development of new agents that safely prevent unwanted platelet activation in patients with underlying cardiovascular disease, while minimizing the risk of bleeding. Here, we investigate the potential of endogenously produced, specialized pro-resolving mediators (SPMs) as novel antiplatelet agents. SPMs are a recently discovered class of lipid-derived molecules that drive the resolution of inflammation without being overtly immunosuppressive. Methods Human platelets were treated with lipoxin A4, resolvin D1, resolvin D2, 17-HDHA or maresin 1 for 15 min, then were subjected to platelet function tests, including spreading, aggregation and inflammatory mediator release. Results We show for the first time that human platelets express the SPM receptors, GPR32 and ALX. Furthermore, our data demonstrate that maresin 1 differentially regulates platelet hemostatic function by enhancing platelet aggregation and spreading, while suppressing release of proinflammatory and prothrombotic mediators. Conclusions These data support the concept that SPMs differentially regulate platelet function and may represent a novel class of antiplatelet agents. SPMs also may play an important role in the resolution of inflammation in cardiovascular diseases.

ABO-immune complex formation and impact on platelet function, red cell structural integrity and haemostasis: an in vitro model of ABO non-identical transfusion.

BACKGROUND: Transfusion of ABO non-identical platelets has been associated with fatal haemolytic reactions, increased red cell transfusion needs and other adverse effects, but the practice of ABO matching in platelet transfusion is controversial. Immune complexes can be formed from the anti-A and/or anti-B antibodies and ABO soluble antigen(s) present in donor and recipient plasma after ABO non-identical transfusions. We hypothesized that these immune complexes affect recipient red cell structural integrity, platelet function and haemostasis. STUDY DESIGN AND METHODS: Haemolysis, platelet function and haemostatic function were assessed before and after incubation of recipient red cells, platelets and whole blood with normal saline controls, ABO-identical plasma controls or in vitro-generated ABO-immune complexes. RESULTS: ABO-immune complexes caused significantly increased haemolysis (P < 0·001), inhibition of platelet function (P = 0·001) and disruption of clot formation kinetics (P < 0·005) in both group A and O recipient samples. CONCLUSIONS: Substantial changes in platelet function, red cell integrity and haemostasis occur after in vitro exposure to immune complexes. These in vitro findings may explain, in part, previously observed associations of ABO non-identical platelet transfusions with adverse effects including increased red cell transfusion needs, organ failure and mortality.

Haemovigilance of reactions associated with red blood cell transfusion: comparison across 17 Countries.

BACKGROUND AND OBJECTIVES: The recent establishment of the National Healthcare Safety Network Hemovigilance Module in the United States affords an opportunity to compare results with those of other developed nations. MATERIALS AND METHODS: Using data from national haemovigilance systems, reactions associated with red blood cell (RBC) transfusion and residual risks of transfusion-transmitted infectious diseases were assembled from 17 nations. Country-specific rates of adverse events were pooled using random-effects Poisson regression. RESULTS: Febrile non-haemolytic and delayed serologic transfusion reactions were the most frequent adverse events reported after RBC transfusion, occurring in 26 patients per 100 000 RBC units and 25 patients per 100 000 RBC units administered, respectively. Rates of allergic, febrile non-haemolytic and delayed haemolytic transfusion reactions in the United States were significantly greater than the pooled rates from other countries. Frequencies of adverse events generated from the national haemovigilance programme in the United States were considerably lower than when obtained through active surveillance. CONCLUSION: Haemovigilance reports of adverse events in the United States are comparable to, or greater than, reports from other developed countries. Rates generated from haemovigilance programmes are lower than those obtained through active surveillance. The lack of universal leucoreduction of RBC units may be a contributing factor to the higher rate of some adverse events in the United States.

Skeletal injuries sustained during the Haiti earthquake of 2010: a radiographic analysis of the casualties admitted to the Israel Defense Forces field hospital.

PURPOSE: To report the distribution and types of skeletal injuries demonstrated on the images taken at the field hospital following the Haiti 2010 earthquake. METHODS: Following the January 12, 2010, earthquake, the State of Israel dispatched a field hospital to Haiti, managing 1,111 patients from January 17, 2010, to January 26, 2010. Four hundred and seven patients (37 %) had 684 radiographic images, most of them (87 %) due to presumed skeletal injuries. RESULTS: There were 224 limb fractures (excluding the hands and feet), with 77 % of them in the lower limbs (30 % femur, 17 % tibial shaft, 16 % ankle). Out of 37 axial skeleton fractures, 30 involved the pelvis (21 anterior posterior, three vertical shear, three lateral compression, three combined). Nine traumatic dislocations (five hips, three shoulders, one knee) were reduced. After reviewing all the digital radiographs, on a PACS-compatible radiography screen, few false diagnoses (2 %) were encountered, with none of them affecting the management of these injuries. CONCLUSIONS: To the best of our knowledge, this is the first report of the radiological results emerging from a field hospital following a mass casualty event. Laptop personal computer-based workstations provide an adequate solution for radiographic image viewing in a field hospital setting. Recognition of the prevalence and distribution of skeletal injuries can improve the preparedness of such delegations before departure in the future.

A novel method for overexpression of peroxisome proliferator-activated receptor-γ in megakaryocyte and platelet microparticles achieves transcellular signaling.

BACKGROUND: Microparticles are submicrometer vesicles that contain RNA and protein derived from their parent cells. Platelet and megakaryocyte microparticles represent 80% of circulating microparticles, and their numbers are elevated in diseases such as cancer and type 2 diabetes. The ability of microparticles to transport protein, lipid and RNA to target cells, as a means of transcellular communication, remains poorly understood. Determining the influence that microparticles have on circulating cells is essential for understanding their role in health and in disease. OBJECTIVES: To develop a novel approach to modify the composition of platelet microparticles, and understand how such changes impact their transcellular communication. METHODS: This novel model utilizes a lentiviral technology to alter the transcription factor peroxisome proliferator-activated receptor-γ (PPARγ) content of megakaryoblastic cell lines and primary megakaryocytes, and also the protein composition of generated platelets and microparticles. The subsequent microparticles were isolated and added to target cells for assessment of uptake and resultant signaling events. RESULTS: We successfully engineered microparticles to contain green fluorescent protein and elevated levels of PPARγ. We found that these altered microparticles could be internalized by the monocytic cell line THP-1 and primary human microvascular endothelial cells. Importantly, microparticle-delivered PPARγ was shown to increase the expression of fatty acid-binding protein 4 (FABP4), which is a known PPARγ target gene in THP-1 cells. CONCLUSION: This proof-of-concept modification of megakaryocyte, platelet and microparticle composition and subsequent change in target cell physiology is an important new tool to address transcellular communication of microparticles.

CD40 ligand (CD154) involvement in platelet transfusion reactions.

Platelet transfusions are commonly used treatments that occasionally lead to adverse reactions. Clinical trials, in vitro and animal studies have been performed to try to understand the causes of such reactions. Multiple studies have shown that the supernatant fraction of platelet concentrates contain prothrombotic and pro-inflammatory mediators. The origin of these mediators was first ascribed to white blood cells contaminating the platelet preparation. However, the accumulation of bioactive mediators after leukoreduction focused attention on platelets themselves during storage. Numerous cytokines, chemokines and prostaglandins are released in stored platelet concentrates. We have focused on a powerful mediator called soluble CD40 ligand (sCD40L, formally known as CD154) as a seminal contributor to adverse reactions. sCD40L can bind and signal the surface receptor, CD40, which is present on various types of human cells including white blood cells, vascular cells and fibroblasts. Downstream results of sCD40L/CD40 signaling include pro-inflammatory cytokine and chemokine production, prothrombotic mediator release, adherence and transmigration of leukocytes to endothelium and other undesirable vascular inflammatory events. Increased plasma levels of sCD40L can be detected in conditions such as myocardial infarction, stroke, unstable angina, high cholesterol, or other cardiovascular conditions. In retrospective studies, correlations were made between increased sCD40L levels of platelet concentrates and adverse transfusion reactions. We hypothesize that transfusion of partially activated, CD40L-expressing platelets along with sCD40L into a recipient with damaged or dysfunctional vascular tissue results in a "double-hit", thus inciting inflammation and vascular damage in the recipient.

ABO-mismatched transfusions are not over-represented in febrile non-haemolytic transfusion reactions to platelets.

ABO-mismatched plasma and platelet (PLT) transfusion have been associated with worse outcomes, including haemolysis and other reactions, compared to recipients of ABO-identical products. The immune complexes that form in a mismatched transfusion have been demonstrated to stimulate pyrogenic cytokine release in vitro. Comparing ABO identical vs. ABO mismatched PLT transfusions, we found no significant difference in the ABO compatibilities between the PLT doses implicated in causing febrile non-haemolytic transfusion reactions (FNHTR) in 162 recipients and both the baseline PLT donor/recipient ABO compatibility (P = 0·67) or the PLTs issued in the 30 days preceding the FNHTR (P = 0·92). ABO-mismatched PLT transfusions do not appear to be aetiological agents of FNHTR in a population routinely receiving both ABO-identical and ABO-mismatched transfusions.

15-deoxy-Delta12,14 prostaglandin J2-induced heme oxygenase-1 in megakaryocytes regulates thrombopoiesis.

BACKGROUND: Platelet production is an intricate process that is poorly understood. Recently, we demonstrated that the natural peroxisome proliferator-activated receptor gamma (PPARgamma) ligand, 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)), augments platelet numbers by increasing platelet release from megakaryocytes through the induction of reactive oxygen species (ROS). 15d-PGJ(2) can exert effects independent of PPARgamma, such as increasing oxidative stress. Heme oxygenase-1 (HO-1) is a potent antioxidant and may influence platelet production. OBJECTIVES: To further investigate the influence of 15d-PGJ(2) on megakaryocytes and to understand whether HO-1 plays a role in platelet production. METHODS: Meg-01 cells (a primary megakaryoblastic cell line) and primary human megakaryocytes derived from cord blood were used to examine the effects of 15d-PGJ(2) on HO-1 expression in megakaryocytes and their daughter platelets. The role of HO-1 activity in thrombopoiesis was studied using established in vitro models of platelet production. RESULTS AND CONCLUSIONS: 15d-PGJ(2) potently induced HO-1 protein expression in Meg-01 cells and primary human megakaryocytes. The platelets produced from these megakaryocytes also expressed elevated levels of HO-1. 15d-PGJ(2)-induced HO-1 was independent of PPARgamma, but could be replicated using other electrophilic prostaglandins, suggesting that the electrophilic properties of 15d-PGJ(2) were important for HO-1 induction. Interestingly, inhibiting HO-1 activity enhanced ROS generation and augmented 15d-PGJ(2)-induced platelet production, which could be attenuated by antioxidants. These new data reveal that HO-1 negatively regulates thrombopoiesis by inhibiting ROS.

The PPAR-Platelet Connection: Modulators of Inflammation and Potential Cardiovascular Effects.

Historically, platelets were viewed as simple anucleate cells responsible for initiating thrombosis and maintaining hemostasis, but clearly they are also key mediators of inflammation and immune cell activation. An emerging body of evidence links platelet function and thrombosis to vascular inflammation. peroxisome proliferator-activated receptors (PPARs) play a major role in modulating inflammation and, interestingly, PPARs (PPARbeta/delta and PPARgamma) were recently identified in platelets. Additionally, PPAR agonists attenuate platelet activation; an important discovery for two reasons. First, activated platelets are formidable antagonists that initiate and prolong a cascade of events that contribute to cardiovascular disease (CVD) progression. Dampening platelet release of proinflammatory mediators, including CD40 ligand (CD40L, CD154), is essential to hinder this cascade. Second, understanding the biologic importance of platelet PPARs and the mechanism(s) by which PPARs regulate platelet activation will be imperative in designing therapeutic strategies lacking the deleterious or unwanted side effects of current treatment options.

Release of biologically active CD154 during collection and storage of platelet concentrates prepared for transfusion.

BACKGROUND: Millions of platelet transfusions are given each year. Transfusion reactions occur in as many as 30% of patients receiving unmodified platelet transfusions. The cause of some transfusion reactions remains unclear. The current paradigm suggests that platelet concentrates (PC) contain proinflammatory mediators that are released by white blood cells during collection, processing and storage. CD154 (CD40 ligand, CD40L) is a potent inflammatory mediator, normally sequestered inside the resting platelet, that is known to translocate to the platelet membrane and be shed into plasma in response to agonist activation. We hypothesized that platelet-soluble CD154 (sCD154) is 'spontaneously' released by transfused platelets and plays a major role in transfusion reactions. OBJECTIVES: To determine the time course and biological properties of CD154 translocation and release during collection and storage of platelets for transfusion. METHODS: We measured surface and sCD154 in platelets prepared by the platelet-rich plasma method or apheresis by fluorescence-activated cell sorting and enzyme-linked immunosorbent assay, respectively. The specific biological activity of platelet sCD154 was assayed by stimulation of the CD154/CD40 pathway in known CD40-positive cells with PC-derived supernatants. RESULTS AND CONCLUSIONS: We demonstrate that PCs prepared for transfusion have high levels of membrane-bound CD154 and sCD154, with maximum levels being seen 72 h after platelet collection. Importantly, we show that platelet-derived sCD154 potently stimulates CD40-positive cells. We propose that platelet-derived CD154 is a key 'cytokine' responsible for adverse reactions associated with platelet transfusions. Improved methods of platelet collection and/or storage, which limit CD154 expression, could reduce the risks of transfusion reaction.

What would Karl Landsteiner do? The ABO blood group and stem cell transplantation.

ABO blood group antigens, of great importance in transplantation and transfusion, are present on virtually all cells, as well as in soluble form in plasma and body fluids. Naturally occurring plasma IgM and IgG antibodies against these antigens are ubiquitous. Nonetheless, the ABO blood group system is widely ignored by many transfusion services, except for purposes of red cell transfusion. We implemented a policy of transfusing only ABO identical platelets and red cells in patients undergoing stem cell transplantation or treatment for hematologic malignancies. Major bleeding episodes have occurred in about 5% of patients undergoing induction therapy for acute leukemia as compared with 15-20% in the literature. Overall survival times appear to be superior to that in historical cohorts. In 2002-2004, treatment-related mortality at 100 days in our Blood and Marrow Transplant Unit was 0.7% for autologous transplants (n=148), 13% for sibling allogeneic transplants (n=110), and 24% (n=62) for matched unrelated allogeneic transplants, suggesting that our approach is safe. We speculate that more rigorous efforts on the part of transfusion services to provide ABO identical blood components, and to remove incompatible supernatant plasma, when necessary, might yield reduced morbidity and mortality in patients undergoing stem cell transplantation.

Hematopoietic stem cell transplantation for multiply transfused patients with sickle cell disease and thalassemia after low-dose total body irradiation, fludarabine, and rabbit anti-thymocyte globulin.

Patients with sickle cell disease (N = 3) and thalassemia (N = 1) with high-risk features received hematopoietic stem cell transplantations (HCT) to induce stable (full or partial) donor engraftment. Patients were 9-30 years of age. Fludarabine, rabbit anti-thymocyte globulin (ATG), and 200 cGy total body irradiation were administered pre-transplant. Patients received bone marrow (N = 3) or peripheral blood stem cells (N = 1) from HLA-identical siblings, followed by mycophenolate mofetil and cyclosporine for post-grafting immunosuppression. Significant lymphopenia, but only moderate neutropenia and thrombocytopenia developed post transplant. No grade IV nonhematological toxicities or acute graft-versus-host disease (GVHD) were observed. At 3 months after transplantation, three of four patients had evidence of donor myeloid chimerism (range, 15-100%). However, after post transplant immunosuppression was discontinued, graft rejection occurred in all but one patient. This patient is now doing well 27 months post transplant with full donor engraftment. One patient died after a second transplant, and another patient experienced a stroke as her graft was being rejected. These results suggest that stable donor engraftment after nonmyeloablative HCT is difficult to achieve among immunocompetent patients with hemoglobinopathies and that new approaches will need to be developed before wider application of this transplantation method for hemoglobinopathies.

WBC-reduced blood transfusions and clinical outcome in children with acute lymphoid leukemia.

BACKGROUND: WBC reduction offers a variety of benefits to patients requiring multiple transfusions during induction therapy for childhood acute lymphoid leukemia (ALL), including reductions in febrile transfusion reactions, HLA alloimmunization, and CMV transmission. One potential benefit is a reduction in the deleterious effects of transfusion immunomodulation. In the surgical setting, transfusion immunomodulation has been linked to increases in postoperative infections and decreases in host cellular immunity that are mitigated by WBC reduction of transfused blood. STUDY DESIGN AND METHODS: A retrospective review was conducted of the medical records of 68 consecutive children undergoing induction therapy for newly diagnosed ALL from 1988 through 1995, a period whose midpoint is 1991, the year WBC reduction was introduced in this hospital. RESULTS: WBC reduction of platelet and RBC transfusions was associated with fewer days with fever (mean, 5.7 days [no WBC reduction] and 2.1 days [WBC reduction]; p = 0.012) and days with positive microbial cultures (mean, 1.5 [no WBC reduction] and 0.71 [WBC reduction]; p = 0.0055). There were more high-risk ALL patients in the group receiving WBC-reduced transfusions. CONCLUSION: Allogeneic WBCs in transfused blood may cause impairment of host defenses against microbial infection during induction therapy for childhood ALL.

Association of ABO-mismatched platelet transfusions with morbidity and mortality in cardiac surgery.

BACKGROUND: The transfusion of ABO-mismatched platelets has been associated with increased morbidity and mortality during induction therapy for acute leukemia and allogeneic progenitor cell transplantation. STUDY DESIGN AND METHODS: Reported here is a cohort study of 153 patients undergoing primary coronary artery bypass graft or coronary valve replacement surgery by two surgeons in one institution during 1997 and 1998. All statistics employed nonparametric two-sided tests (Mann-Whitney; Fisher's exact test). RESULTS: Patients receiving at least one ABO-mismatched pool of platelets had a significantly longer hospital stay, more days of fever, greater total hospital charges, and more RBC transfusions. Mortality, hours in the intensive care unit, days on antibiotics, and numbers of platelet transfusions were also greater in recipients of ABO-mismatched platelets, but these differences were of less statistical significance. When the analysis was restricted to the 139 patients who received no more than two pools of platelets, the trends for increased morbidity and mortality (8.6% vs. 1.9%; p = 0.10) in recipients of ABO-mismatched platelets persisted. The number of RBC transfusions required in this latter cohort was 50 percent greater (mean, 6.1 vs. 9.2; p = 0.02), despite the fact that the number of platelet transfusions given was similar (mean, 1.2 vs. 1.3 pools; p = 0.22). CONCLUSIONS: ABO-mismatched platelet transfusions are associated with unfavorable outcomes in cardiac surgery, a relationship that remains unexplained. As this association has been found in three cohort studies in various clinical settings, further investigation of this association is warranted.