Effect of red blood cell transfusion on inflammation, endothelial cell activation and coagulation in the critically ill.

BACKGROUND AND OBJECTIVES: Red blood cell (RBC) transfusion is a frequently applied intervention in an intensive care unit. However, transfusion is associated with adverse outcomes including organ failure and thrombo-embolic events. Mechanisms of these effects are not known but may be related to activation of the endothelium or of the coagulation or inflammatory system. We hypothesized that a RBC transfusion in the critically ill would result in further activation of these systems. MATERIALS AND METHODS: In 74 non-bleeding critically ill patients receiving one RBC unit, markers of inflammation, endothelial cell activation and coagulation were measured before transfusion, at 1 h after transfusion and 24 h after transfusion. The impact of disease severity of the recipient on these changes was assessed by comparing septic and non-septic patients (according to sepsis-3 definition) and by correlation of biomarkers with the sequential organ failure assessment (SOFA) score. RESULTS: Levels of von Willebrand Factor (vWF), soluble ICAM-1, soluble thrombomodulin, fibrinogen and d-dimer were already high at baseline, whereas ADAMTS13 levels were low. VWF levels increased significantly 24 h after RBC transfusion (median 478% (338-597) vs. 526% (395-623), p = 0.009). The other biomarkers did not change significantly. Post transfusion change was not dependent on the presence of sepsis and was not correlated with SOFA score. CONCLUSION: RBC transfusion in critically ill patients was associated with an increase in circulating vWF levels, suggesting a further increase in activation of the endothelium, a finding that was independent of the presence of sepsis or organ injury level.

Red blood cell transfusion results in adhesion of neutrophils in human endotoxemia and in critically ill patients with sepsis.

BACKGROUND: Red blood cell (RBC) transfusion is associated with adverse effects, which may involve activation of the host immune response. The effect of RBC transfusion on neutrophil Reactive Oxygen Species (ROS) production and adhesion ex vivo was investigated in endotoxemic volunteers and in critically ill patients that received a RBC transfusion. We hypothesized that RBC transfusion would cause neutrophil activation, the extent of which depends on the storage time and the inflammatory status of the recipient. STUDY DESIGN AND METHODS: Volunteers were injected with lipopolysaccharide (LPS) and transfused with either saline, fresh, or stored autologous RBCs. In addition, 47 critically ill patients with and without sepsis receiving either fresh (<8 days) or standard stored RBC (2-35 days) were included. Neutrophils from healthy volunteers were incubated with the plasma samples from the endotoxemic volunteers and from the critically ill patients, after which priming of neutrophil ROS production and adhesion were assessed. RESULTS: In the endotoxemia model, ex vivo neutrophil adhesion, but not ROS production, was increased after transfusion, which was not affected by RBC storage duration. In the critically ill, ex vivo neutrophil ROS production was already increased prior to transfusion and was not increased following transfusion. Neutrophil adhesion was increased following transfusion, which was more notable in the septic patients than in non-septic patients. Transfusion of fresh RBCs, but not standard issued RBCs, resulted in enhanced ROS production in neutrophils. CONCLUSION: RBC transfusion was associated with increased neutrophil adhesion in a model of human endotoxemia as well as in critically ill patients with sepsis.

The effect of red blood cell transfusion on platelet function in critically ill patients.

INTRODUCTION: Red blood cell (RBC) transfusion is associated with an increased risk of pro-thrombotic events, but the underlying mechanism is poorly understood. We hypothesized that RBC transfusion modulates platelet activity in critically ill patients with and without sepsis. METHODS: In a prospective cohort study, 37 critically ill patients receiving a single RBC unit to correct for anemia were sampled prior to and 1 h after transfusion. Platelet exposure of P-selectin, CD63 and binding of PAC-1 as well as formation of platelet-leukocyte complexes were measured by flow cytometry. The ability of plasma from critically ill patients to induce ex vivo platelet aggregation was assessed by flow cytometry after incubation with platelets from a healthy donor. RESULTS: RBC transfusion neither triggered the expression of platelet activation markers nor the formation of platelet-leukocyte complexes. Plasma from critically ill patients induced more spontaneous platelet aggregation prior to RBC transfusion compared to healthy controls, which was further augmented following RBC transfusion. Also collagen-induced platelet aggregation was already increased prior to RBC transfusion compared to healthy controls, and this response was unaffected by RBC transfusion. In contrast, ristocetin-induced platelet agglutination was decreased when compared to controls, suggesting impaired vWF-dependent platelet agglutination, even in the presence of high vWF levels. Following RBC transfusion, ristocetin-induced platelet agglutination further decreased. There were no differences between septic and non-septic recipients in all assays. CONCLUSION: Ex vivo platelet aggregation is disturbed in the critically ill. Transfusion of a RBC unit may further increase the spontaneous platelet aggregatory response.

The effect of red blood cell transfusion on iron metabolism in critically ill patients.

BACKGROUND: Anemia of inflammation (AI) has a high prevalence in critically ill patients. In AI, iron metabolism is altered, as high levels of inflammation-induced hepcidin reduce the amount of iron available for erythropoiesis. AI is treated with red blood cell (RBC) transfusions. The effect of RBC transfusion on iron metabolism during inflammatory processes in adults is unknown. We investigated the effect of RBC transfusion on iron metabolism in critically ill patients. METHODS: In a prospective cohort study in 61 critically ill patients who received 1 RBC unit, levels of iron variables were determined before, directly after, and 24 hours after transfusion in septic and nonseptic patients. RESULTS: Serum iron levels were low and increased after transfusion (p = 0.02). However, RBC transfusion had no effect on transferrin saturation (p = 0.14) and ferritin levels (p = 0.74). Hepcidin levels increased after RBC transfusion (p = 0.01), while interleukin-6 levels decreased (p = 0.03). In septic patients, RBC transfusion induced a decrease in haptoglobin levels compared to baseline, which did not occur in nonseptic patients (p = 0.01). The effect of RBC transfusion on other iron variables did not differ between septic and nonseptic patients. CONCLUSION: Transfusion of a RBC unit transiently increases serum iron levels in intensive care unit patients. The increase in hepcidin levels after transfusion can further decrease iron release from intracellular storage making it available for erythropoiesis. RBC transfusion is associated with a decrease in haptoglobin levels in septic compared to nonseptic patients, but did not affect other markers of hemolysis.

Transfusion of 35-day-stored red blood cells does not alter lipopolysaccharide tolerance during human endotoxemia.

BACKGROUND: Transfusion-related immunomodulation (TRIM) encompasses immunosuppressive and proinflammatory effects induced by red blood cell (RBC) transfusion. Changes that occur during storage in the RBC product have been hypothesized to underlie TRIM, mediated by tolerance of toll-like receptors (TLR). We investigated whether transfusion of 35-day-stored autologous RBCs alters cytokine production in response to stimulation with lipopolysaccharide (LPS) or lipotheic acid (LTA), in a clinically relevant model of endotoxemia. STUDY DESIGN AND METHODS: Eighteen volunteers received 2 ng/kg LPS intravenously, followed by normal saline or 2- or 35-day-stored autologous RBC transfusion. Before LPS, before transfusion, and 6 hours after transfusion blood was collected to measure cytokine gene expression. Whole blood was used for ex vivo stimulation with LPS and LTA, after which cytokine levels were measured with enzyme-linked immunosorbent assay. RESULTS: In vivo LPS induced a biphasic response in cytokine mRNA with peak values 2 hours after LPS infusion. Storage time of RBC transfusion did not influence cytokine mRNA levels. In vivo infusion of LPS resulted in tolerance for ex vivo stimulation with LPS and LTA. However, transfusion of either fresh or stored RBCs did not further affect the capacity to produce cytokines after ex vivo stimulation. CONCLUSION: In a clinically relevant model of human endotoxemia, autologous transfusion of 35-day-stored RBCs does not influence cytokine mRNA levels nor does it change the capacity of white blood cells in whole blood to produce cytokines after ex vivo stimulation with LPS or LTA.

Monocyte-mediated activation of endothelial cells occurs only after binding to extracellular vesicles from red blood cell products, a process mediated by ß-integrin.

BACKGROUND: Red blood cell (RBC) transfusion is associated with organ failure. The mechanism remains unknown, but may include adherence of blood cells to the microvasculature. We hypothesized that RBC-derived extracellular vesicles (EVs) interact with monocytes to activate endothelial cells. STUDY DESIGN AND METHODS: Human umbilical vein endothelial cells were incubated with supernatant from fresh and stored RBC units either containing EVs or depleted from EVs, with or without the addition of immune cells. We measured expression of adhesion markers by flow cytometry and markers of coagulation and inflammation in the culture medium. We studied phagocytosis of EVs by monocytes by using confocal microscopy and flow cytometry. RESULTS: Incubation of endothelial cells with monocytes alone did not induce up regulation of adhesion markers. The addition of both monocytes and supernatant from RBCs containing EVs resulted in up regulation of endothelial expression of intercellular adhesion molecule 1 and E-selectin when compared to baseline. Up regulation was absent when stimulated with RBC supernatant depleted from EVs. EVs are phagocytosed by monocytes, which was partly abrogated after coincubation with two different complement receptor 3 (CR3)-blocking antibodies. Addition of RBC-derived EVs also increased levels of von Willebrand factor (VWF). There were no differences between groups related to storage time. CONCLUSION: EVs from RBC transfusion bags activate monocytes with subsequent up regulation of endothelial cell adhesion markers. EVs are phagocytosed by monocytes through CR3. Furthermore, these EVs proved to be a source of VWF. These effects are unrelated to storage time. Thereby, EVs from RBC transfusion bags induce a proinflammatory and procoagulant endothelial cell response.

Transfusion of 35-Day Stored RBCs in the Presence of Endotoxemia Does Not Result in Lung Injury in Humans.

OBJECTIVE: Transfusion-related acute lung injury is the leading cause of transfusion-related mortality. Preclinical studies have shown that aged RBCs can induce transfusion-related acute lung injury in the presence of a "first hit" (e.g., sepsis). Clinical studies, however, show conflicting results on this matter. We tested whether maximally stored RBCs are able to induce lung injury in the presence of a "first hit" in humans (Dutch Trial Register: NTR4455). DESIGN: Open-label, randomized controlled trial. PATIENTS: Healthy male volunteers. INTERVENTIONS: Eighteen healthy male volunteers donated one unit of autologous RBCs 2 or 35 days before the experiment. The experiment was started by infusion of 2 ng/kg lipopolysaccharide ("first hit"). After 2 hours, volunteers received normal saline (n = 6), 2-day stored transfusion (n = 6), or 35-day stored transfusion (n = 6) ("second hit"). Blood was sampled hourly. Six hours after transfusion, the diffusion capacity of the lungs for carbon monoxide was tested and volunteers underwent spirometry, chest x-ray study, and a bronchoalveolar lavage. MEASUREMENTS AND MAIN RESULTS: All volunteers fulfilled sepsis criteria after lipopolysaccharide injection. The stored blood transfusion did not result in significant changes in either hemodynamic or respiratory variables compared with the control groups. Furthermore, chest x-rays, lung function, and PaO2/FIO2 ratios did not differ between groups. Transfusion of stored autologous RBCs did not result in an increased level of protein in the lungs or neutrophil influx. CONCLUSIONS: Transfusion of 35-day stored autologous RBCs in the presence of endotoxemia does not result in lung injury in humans.

Pathogenesis of non-antibody mediated transfusion-related acute lung injury from bench to bedside.

Transfusion-related acute lung injury (TRALI) is a major cause of transfusion-related mortality. Causative factors are divided in antibody mediated TRALI and non-antibody mediated TRALI. Antibody mediated TRALI is caused by passive transfusion of cognate antibodies and non-antibody mediated TRALI is caused by transfusion of aged cellular blood products. This review focuses on mechanisms in non-antibody mediated TRALI which includes soluble mediators accumulating during storage of red blood cells (RBCs) and platelets (PLTs), as well as changes in morphology and function of aged PLTs and RBCs. These mediators cause TRALI in two-hit animal models and have been implicated in TRALI onset in clinical studies. Pre-clinical studies show a clear relation between TRALI and increased storage time of cellular blood products. Observational clinical studies however report conflicting data. Knowledge of pathophysiological mechanisms of TRALI is necessary to improve storage conditions of blood products, develop prevention strategies and develop a therapy for TRALI.