Development Of Transfusion-related Acute Lung Injury Research Articles

Adverse effects of microparticles on transfusion of stored red blood cell concentrates

BackgroundSystemic and pulmonary coagulopathy and inflammation are important characteristics of transfusion-related acute lung injury (TRALI). Whether microparticles that accumulate in transfused red blood cell concentrates (RBCs) have proinflammatory and procoagulant potential and contribute to adverse reactions of RBC transfusions is unclear. AimTo investigate the ability of microparticles in stored RBCs to promote thrombin generation and induce human pulmonary microvascular endothelial cell (HMVEC) activation and damage. MethodsThe number and size of microparticles were determined by flow cytometric and nanoparticle tracking analyses, respectively. Thrombin generation and the intrinsic coagulation pathway were assayed by a calibrated automated thrombogram and by measuring activated partial thromboplastin time (aPTT), respectively. The expression of ICAM-1 and the release of cytokines by endothelial cells were detected by flow cytometric analyses. HMVEC damage was assessed by incubating lipopolysaccharide-activated endothelial cells with MP-primed polymorphonuclear neutrophils (PMNs). ResultsThe size of the microparticles in the RBC supernatant was approximately 100–300 nm. Microparticles promoted thrombin generation in a dose-dependent manner and the aPTT was shortened. Depleting microparticles from the supernatant of RBCs stored for 35 days by either filtration or centrifugation significantly decreased the promotion of thrombin generation. The expression of ICAM-1 on HMVECs was increased significantly by incubation with isolated microparticles. Furthermore, microparticles induced the release of interleukin-6 (IL-6) and interleukin-8 (IL-8) from HMVECs. Microparticles induced lipopolysaccharide-activated HMVEC damage by priming PMNs, but this effect was prevented by inhibiting the PMNs respiratory burst with apocynin. ConclusionMicroparticles in stored RBCs promote thrombin generation, HMVEC activation and damage which may be involved in TRALI development.

Transfusion-Related Acute Lung Injury (TRALI): A Narrative Literature Review

Transfusion-related acute lung injury (TRALI) is defined as the onset of respiratory distress in a patient after receiving a blood component transfusion. So far, TRALI is considered a rare complication in the field of blood transfusion. However, in the last decade, there has been a shift in perspective. Currently, the US Food and Drug Administration recognizes the syndrome as the leading cause of transfusion-related death. This literature review aims to describe TRALI and its prevention strategies. Understanding the pathogenesis of TRALI drives prevention strategies from a blood bank perspective. A major breakthrough in efforts to reduce the incidence of TRALI was excluding female donors from the high plasma volume product, which resulted in an approximately two-thirds reduction in incidence. However, this strategy has not completely eliminated complications of transfusion. In recent years, research has identified patient-associated risk factors for the development of TRALI and empowered clinicians to take an individualized approach to patients requiring transfusion.

Fc-Mediated Complement Activation Is Associated with Macrophage Trafficking and Induction of Neutrophil Extracellular Traps in Transfusion-Related Acute Lung Injury

Transfusion-related acute lung injury (TRALI) is a leading cause of blood transfusion-related fatalities without available therapies. The pulmonary endothelium is damaged in TRALI, through incompletely understood pathogenic mechanisms, resulting in pulmonary edema. Generally, anti-leukocyte antibodies or biological response modifiers in the transfusion product, in combination with predisposing risk factors in the transfused recipient (e.g. inflammation), are responsible for initiation of TRALI. Remarkably, not all anti-leukocyte antibodies cause TRALI. In a previous in vitro study, we identified increased Fc-mediated complement activation to be a key feature of murine TRALI-inducing antibody 34-1-2S (anti-H-2K d) compared to TRALI-resistant antibody SF1.1.10 (anti-H-2K d) (Zeeuw van der Laan et al, Blood Adv 2020).In the current study, we further explored antibody-mediated TRALI mechanisms in vivo using our previously established TRALI mouse model in which mice are pre-depleted of protective CD4+ T cells and primed with LPS, followed by infusion of antibody 34-1-2S (Kapur et al, Blood 2017, Blood Adv 2018, Blood 2019). A key read-out for TRALI was the lung wet/dry weight ratio (Lung W/D, measure for pulmonary edema). We found that in vitro antibody-mediated complement activation was associated with in vivo antibody-mediated TRALI. 34-1-2S caused severe TRALI (complement activation: +++, Lung W/D: 7.4 ± 0.21), while SF1.1.10 did not cause TRALI (complement activation: +, Lung W/D: 4.68 ± 0.16). Furthermore, Fc-deglycosylated 34-1-2S still caused significant TRALI (complement activation: ++, in vivo Lung W/D: 5.16 ± 0.52). TRALI development was fully Fc-dependent as 34-1-2S-Fab did not cause any TRALI (no complement activation, Lung W/D: 4.35 ± 0.18). Importantly, we found significantly increased levels of complement C1q-C4 complexes in plasma samples of TRALI patients (n=46) compared to healthy controls (n=25): 6.49 ± 5.27 vs 3.84 ± 2.27 AU/ml, respectively, P: 0.0005,***. Similarly, C5a levels were significantly elevated in plasma samples of TRALI patients (n=53) compared to healthy controls (n=30): 2.52 ± 2.17 vs 1.37 ± 1.08 ng/ml, respectively, P: 0.0006,***. To further dissect the effect of the Fc-part of 34-1-2S in TRALI-induction, we aim to investigate the contribution of Fc-mediated complement activation vs Fc-receptor interaction. We therefore successfully generated chimeric variants of 34-1-2S with a humanized IgG1 Fc-domain containing mutations making them functionally complement dead (K322A), Fc-receptor dead (dG236) or both complement and Fc-receptor dead (PG LALA). We are currently investigating the effects of these variants on in vivo TRALI induction.We next investigated how the in vivo murine TRALI reaction was related to numbers of macrophages, monocytes and neutrophils in blood and lungs. We found that 34-1-2S-TRALI was associated with significantly decreased levels of macrophages in the lungs and increased levels in blood, compared to mice infused with PBS, SF1.1.10 or 34-1-2S-Fab, suggesting that Fc-mediated complement activation and TRALI induction is related to macrophage trafficking from lungs to blood.We did not observe any significant differences between blood and lung neutrophil levels of 34-1-2S-TRALI mice compared to SF1.1.10 TRALI-resistant mice. We hypothesized that in TRALI neutrophils undergo formation of neutrophil extracellular traps (NETs) induced by complement. We observed that C5a enabled potent neutrophil-chemotaxis in vitro (P: 0.0048,**). In addition, using direct immunofluorescence staining of extracellular DNA and Citrullinated histone H3, we observed that both LPS and C5a on their own could induce NET formation in vitro, which was synergistically increased with a combination of both LPS and C5a (P: 0.0417,*), as occurring during TRALI. Furthermore, we found increased levels of NETs to be present in plasma samples of TRALI patients (n=53) compared to healthy controls (n=30): 1.64 ± 0.97 vs 0.80 ± 0.34 MPO-DNA OD, respectively, P: 0.0002,***.Finally, we targeted the C5a-receptor (C5aR) using a C5aR-antagonist in our TRALI mouse model. Surprisingly, this did not prevent but even worsened TRALI (P: 0.0398,*), with elevated levels of blood monocytes and macrophages. This suggests that an approach directly targeting complement components may be a more promising therapeutic strategy to explore in combatting TRALI. DisclosuresNo relevant conflicts of interest to declare.

Pulmonary disease as a risk factor for transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is a severe condition characterised by noncardiogenic pulmonary oedema that develops within 6 h of blood transfusion. Patient factors and blood products have both been implicated in the development of TRALI; however, the role of pulmonary disease has not been investigated. We aimed to determine whether pulmonary disease is a risk factor for TRALI.We conducted a nested case-control study using data from the Diagnosis Procedure Combination database, a nationwide inpatient database in Japan, between July 2010 and March 2015. Case patients who developed TRALI were 1:4-matched with control patients for sex, age and same hospital for receipt of blood transfusion. We conducted a multivariable conditional logistic regression analysis to evaluate the associations of TRALI with various factors including comorbidities, body mass index (BMI) and plasma-containing blood products.We identified 2 019 501 hospitalised patients who received a blood transfusion. Among these patients, 72 developed TRALI. The 72 case patients had higher proportions of haematological malignancy, trauma and interstitial lung disease (ILD) than the 288 matched control patients. The multivariable conditional logistic regression analysis showed that occurrence of TRALI was associated with ILD (odds ratio, 3.88; 95% confidence interval, 1.11–13.6), BMI ≥25.0 kg·m−2 (2.10; 1.05–4.24) and plasma-containing blood products (1.94; 1.10–3.42), but not with infectious lung disease or obstructive airway disease.In conclusion, ILD was an independent risk factor for the development of TRALI. Physicians should be aware of the increased risk of TRALI in patients with ILD.

Risk Factors for Transfusion-Related Acute Lung Injury.

Until now, transfusion-related acute lung injury (TRALI) has been considered the leading cause of blood transfusion-related diseases and death. In addition, there is no clinically effective treatment plan for TRALI. The aim of this study was to systematically summarize the literature on risk factors for TRALI in critical patients. Electronic searches (up to March 2020) were performed in the Cochrane Library, Web of Knowledge, Embase, and PubMed databases. We included studies reporting on the risk factors of TRALI for critical patients and extracted risk factors. A total of 13 studies met the inclusion criteria. We summarized and analyzed the potential risk factors of TRALI for critical patients in 13 existing studies. Host-related factors were age (odds ratio [OR] 1.16 [95% CI 1.08-1.24]), female sex (OR 1.26 [95% CI 1.16-1.38]), tobacco use status (OR 3.82 [95% CI 1.91-7.65]), chronic alcohol abuse (OR 3.82 [95% CI 2.97-26.83]), positive fluid balance (OR 1.24 [95% CI 1.08-1.42]), shock before transfusion (OR 4.41 [95% CI 2.38-8.20]), and American Society of Anesthesiologists (ASA) score of the recipients (OR 2.72 [95% CI 1.43-5.16]). The transfusion-related factors were the number of transfusions (OR 1.40 [95% CI 1.14-1.72]) and units of fresh frozen plasma (OR 1.21 [95% CI 1.01-1.46]). The device-related factor was mechanical ventilation (OR 4.13 [95% CI 2.20-7.76]). The risk factors that were positively correlated with TRALI in this study included number of transfusions and units of fresh frozen plasma. Age, female sex, tobacco use, chronic alcohol abuse, positive fluid balance, shock before transfusion, ASA score, and mechanical ventilation may be potential risk factors for TRALI. Our results suggest that host-related risk factors may play a more important role in the occurrence and development of TRALI than risk factors related to blood transfusions.

RIL-35 prevents murine transfusion-related acute lung injury by inhibiting the activation of endothelial cells.

Transfusion-related acute lung injury (TRALI) is an important cause of death associated with transfusion, and no specific clinical treatments are available. Endothelial cells are believed to play an important role in the development of TRALI. This study investigated whether IL-35, an endothelial stabilizing cytokine could regulate the severity of antibody-mediated TRALI in vivo. Human microvascular endothelial cells (HMVECs) were cultured in vitro, rIL-35(2 μg/mL) was added before HMVECs activation, and HMVECs were fully activated by LPS (0.5 μg/mL). Then cells were collected for flow cytometry analysis. We used a previously established "two-event" mouse model of TRALI with naive and lipopolysaccharide (LPS)-injected mice as controls. rIL-35(100 μg/kg) was injected into the tail vein for 3 consecutive days before the induction of the TRALI model. Samples were collected 2 hours after TRALI induction and tested for lung tissue myeloperoxidase activity, total protein levels, lung tissue histology, endothelial cell activation assay, and cytokine assay. In vitro culture of HMVECs with rIL-35 verified that rIL-35 inhibited endothelial cells. In a mouse model, prophylactic administration of rIL-35 prevented pulmonary edema, increased lung protein levels, and reduced polymorphonuclear neutrophil accumulation in the lung. This work suggests that antibody-mediated murine TRALI can be prevented by rIL-35, and that rIL-35 appears to work by inhibiting the activation of lung endothelial cells.

Platelet depletion limits the severity but does not prevent the occurrence of experimental transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is a severe pulmonary reaction due to blood transfusions. The pathophysiology of this complication is still not widely elucidated by the scientific community, especially regarding the direct role of blood platelets within the cellular mechanism responsible for the development of TRALI. In this study, a mouse model was used to induce the development of antibody-mediated acute lung injury through injections of lipopolysaccharide and an anti-major histocompatibility complex Class I antibody. BALB/c mice were pretreated with an anti-GPIbα antibody, which induces platelet depletion, or ML354, a protease receptor 4 pathway inhibitor, 30 minutes before TRALI induction. Depletion of platelets before TRALI induction appeared to reduce the severity of TRALI without completely inhibiting its development. Also, inhibition of platelet activation by ML354 did not prevent the onset of TRALI. Finally, the stimuli used for TRALI induction also triggered specific platelet activation upon ex vivo stimulation. This study suggests that blood platelets are not critically required for TRALI induction, although they are to some extent involved in its pathophysiology.

An animal model of transfusion-related acute lung injury and the role of soluble CD40 ligand.

Transfusion-related acute lung injury (TRALI) is a life-threatening complication of transfusion and is one of leading causes of transfusion-associated fatalities. However, the pathogenesis of TRALI is still unclear. Soluble CD40 ligand (sCD40L) is a proinflammatory cytokine that accumulates during blood component storage and is involved in transfusion reactions. The objective of this study was to establish a clinically relevant TRALI animal model and to evaluate the role of sCD40L in TRALI. Rats' red-blood-cell (RBC) suspensions were prepared, and the quality of RBC was evaluated. A trauma-haemorrhage-transfusion strategy was applied to build the animal model. Lung oedema was evaluated by histopathology examination, total bronchoalveolar lavage fluid (BALF) protein concentration, Evans blue dye (EBD) leakage and inflammatory cytokines. The sCD40L concentrations were measured. Storage lesions of RBCs gradually increased over time. Obvious histological evidence of lung injury of rats transfused with a 35-day RBC was observed. The total BALF protein concentration, EBD leakage, inflammatory cytokines concentration were increased significantly in the Day 35 group. The sCD40L concentration increased significantly in the storage RBC suspension over time but was slightly elevated in rat plasma. These findings indicated successful establishment of a TRALI animal model with trauma-haemorrhage-transfusion, in which sCD40L may play a minor role in the development of TRALI.

Osteopontin Mediates Murine Transfusion-Related Acute Lung Injury through Stimulation of Pulmonary Neutrophil Accumulation

Transfusion-related acute lung injury (TRALI) is a syndrome of respiratory distress which develops within 6 hours of blood transfusion. It is the leading cause of transfusion-related deaths and the pathogenesis is complex and incompletely understood. In the majority of the cases, anti-leukocyte antibodies present in the transfused blood product, in combination with recipient predisposing risk-factors such as inflammation, are implicated to be responsible for the onset of TRALI. Unfortunately, no therapies are available for TRALI.Osteopontin (OPN) is an extracellular matrix protein with multiple biological functions. OPN is involved in normal physiological processes, such as cell migration and adhesion, but has also been implicated in a wide range of disease states, including cancer, atherosclerosis, glomerulonephritis, and several chronic inflammatory diseases. Interestingly, OPN is upregulated at sites of inflammation and tissue remodeling. As inflammation is an important risk factor for TRALI development, and as neutrophils (PMNs) are known effector cells in the pathogenesis of TRALI which migrate and accumulate in the lungs during TRALI development, we investigated the potential contribution of OPN in the onset of antibody-mediated TRALI.We utilized a previously established murine TRALI model (Kapur et al, Blood 2017, Blood Advances 2018) in which C57BL/6 mice were first primed with a low dose of lipopolysaccharide (LPS) and depleted of their CD4+ T cells in vivo followed by injection of anti-major histocompatibility complex (MHC) class I antibodies (clones 34-1-2s and AF6-88.5.5.3). The TRALI response was analyzed after 90 minutes by analysis of pulmonary edema (lung wet-to-dry weight ratios, W/Ds) and the levels of pulmonary neutrophils. Wildtype (WT) mice suffered from antibody-mediated TRALI compared to untreated naïve mice, as was shown by their significantly increased lung W/Ds (4.72 vs 4.50, respectively, P<0.0001). This also corresponded to significantly increased levels of pulmonary PMNs compared to untreated naïve mice (34% vs 5%, P<0.0001). In contrast, C57BL/6 OPN knock-out mice were resistant to antibody-mediated TRALI induction as they did not display any significant increase in lung W/Ds levels or pulmonary PMNs compared to untreated naïve OPN knock-out mice (lung W/Ds 4.83 vs 4.75, and pulmonary PMNs 18% vs 16%, respectively). Strikingly, administration of purified recombinant OPN during TRALI induction in C57BL/6 OPN knock-out mice, significantly induced a TRALI reaction (lung W/Ds 5.12 vs 4.75 as compared to untreated naïve OPN knock-out mice, P<0.05). Mechanistically, this TRALI inducing effect of OPN administration to OPN knock-out mice was associated with increased levels of pulmonary PMNs (38% vs 16%, as compared to untreated naïve OPN knock-out mice, P<0.0001). In vivo blocking of OPN in WT mice with an anti-OPN antibody demonstrated decreased lung W/Ds as compared to treatment with an isotype antibody (4.48 vs 4.69, respectively, P<0.05). The OPN blocking response during TRALI was associated with a decreased level of pulmonary PMN accumulation as compared to treatment with an isotype antibody (14% vs 34%, respectively, P<0.0001). As the PMN-chemoattractant macrophage inflammatory protein (MIP)-2 has previously been described to be upregulated in murine antibody-mediated TRALI, we investigated if the OPN-associated pulmonary PMN accumulation and TRALI induction could be related to the levels of MIP-2. We found that plasma MIP-2 levels were increased in mice that were infused with anti-MHC class I antibodies as compared to naïve controls, but that addition or blocking of OPN did not affect these MIP-2 levels. This indicates that the OPN-related PMN responses in TRALI are independent of plasma MIP-2 levels.Collectively, these data indicate OPN as a novel pathogenic factor which enhances antibody-mediated murine TRALI through stimulation of PMN migration towards the lungs, independent of MIP-2. This may suggest that blocking OPN (using an anti-OPN antibody) may prevent TRALI by impairing pulmonary PMN accumulation and could be a therapeutic avenue to explore in combatting this serious adverse complication of blood transfusion. DisclosuresNo relevant conflicts of interest to declare.

Establishment of An Animal Model of Transfusion-related Acute Lung Injury and the Role of Soluble CD40 Ligand in the Development of Transfusion-related Acute Lung Injury

Objective To establish an animal model of transfusion-related acute lung injury (TRALI) and investigate the role of soluble CD40 ligand (sCD40L) in the development of TRALI. Methods The TRALI animal model established by trauma-hemorrhage-transfusion. Lung edema was evaluated by histopathological examination and the protein and Evans blue dye accumulation in bronchoalveolar lavage fluid. The concentration of sCD40L in storage packed red blood cell (PRBC) and rat's plasma was measured by enzyme-linked immunosorbent assay (ELISA). Results There were obvious epithelial hyperplasia and inflammatory cell infiltration in the lung tissue of 7 d-PRBC-treated group. The accumulation of protein in bronchoalveolar lavage fluid of 7 d-PRBC-treated group [(13.17±5.76)mg] was significantly higher than that in normal controls [(1.21±0.66)mg] and normal saline (NS)-treated group [(4.94±2.15) mg] (F=17.605,P<0.001). The leakage amount of Evans blue dye in 7 d-PRBC-treated group [(0.0109±0.0067)%/min] was significantly higher than that in NS-treated group [(0.0026±0.0006) %/min] (t=2.998,P=0.03). The concentration of sCD40L of the 7 d PRBC [(451.58±73.28) pg/ml] was significantly higher than 0 d PRBC [(277.94±98.18)pg/ml] (t=2.834,P=0.03). The concentration of sCD40L in the plasma of 7 d-PRBC-treated group [(878.21±125.30)pg/ml] was significantly higher than those in normal controls [(289.78±62.60)pg/ml] and NS-treated group [(418.07±47.68)pg/ml] (F=78.715,P<0.001). Conclusion The TRALI animal model was successfully established with trauma-hemorrhage-transfusion. The concentration of sCD40L in plasma of rats with massive transfusion is remarkably increased,suggesting sCD40L may play a role in the development of TRALI.

Gastrointestinal Flora Dictates the Biological Response in Murine Transfusion Related Acute Lung Injury (TRALI)

Transfusion related acute lung injury (TRALI) is a syndrome of respiratory distress which occurs within 6 hours of blood transfusion. It is the leading cause of transfusion-related fatalities and the pathogenesis is incompletely understood. In the majority of the cases, anti-leukocyte antibodies present in the transfused blood product, in combination with recipient predisposing risk-factors such as inflammation, are implicated to be responsible for the onset of TRALI. Unfortunately, no therapies are available for TRALI. Recently, using novel murine models of TRALI, CD4+T cells were found to be important protector cells against antibody-mediated TRALI and administration of interleukin (IL)-10 was demonstrated to be a successful treatment strategy for TRALI, rescuing mice therapeutically from pulmonary edema, the hallmark of acute lung injury (Kapur et al, Blood 2017, 129(18):2557-2569). Whether the gut microbiome plays any role in the development of TRALI is currently unknown. For that purpose, we compared the biological TRALI response in mice housed in a barrier-free (BF) setting versus mice housed in a specific pathogen-free (SPF) environment. We utilized our TRALI model in which C57BL/6 mice were first depleted of CD4+ T cells in vivo followed by injection of anti-major histocompatibility complex class I antibodies (clones 34-1-2s and AF6-88.5.5.3). The TRALI response was analyzed after 90 minutes for several parameters including pulmonary edema (lung wet-to-dry weight ratios, W/Ds), rectal temperatures (indicative of systemic shock), plasma levels of macrophage inflammatory protein (MIP)-2 (murine homologue of IL-8, a neutrophil chemoattractant), levels of pulmonary neutrophils (major effector cells in TRALI) and lung histology. We observed that after one week of housing, baseline rectal temperatures of BF mice significantly increased from 38.7o C to 39.5° C (p<0.001) while SPF mice remained stable around 38.3 °C. When comparing BF versus SPF mice, the baseline temperature of BF mice was significantly higher than SPF mice after one week of housing (P<0.0001). The gut flora was subsequently depleted in BF mice via administration of broad-spectrum antibiotics through the drinking water. When both aerobic and anaerobic gut microbes were efficiently depleted after a week of antibiotic treatment, rectal temperatures of the treated BF mice significantly dropped again to 38.7 °C (P<0.05). When inducing TRALI by depleting CD4+ T cells and injecting TRALI-antibodies, the rectal temperatures of the untreated BF mice remained low while the rectal temperatures of SPF mice and gut flora depleted-BF mice normalized again from 60 minutes onwards (34.9 versus 36.8 versus 37.8 °C, respectively). Moreover, the untreated BF mice suffered from TRALI while the SPF mice and the gut flora depleted-BF mice were protected from TRALI development (lung W/Ds 5.77 versus 4.63 versus 4.49, respectively) which was also evident from lung histology-analyses. The prevention of TRALI in SPF mice and gut flora depleted BF mice was paralleled by decreased plasma MIP-2 levels compared to the untreated BF mice which suffered from TRALI (MIP-2: 0.09 versus 0.08 versus 82.94 pg/ml, respectively) which also corresponded to pulmonary neutrophil numbers. As previously shown, low IL-10 levels were associated with TRALI development and IL-10 KO mice were found to be susceptible to antibody-mediated murine TRALI (without prior in vivo cell-depletion). However, when IL-10 KO mice were housed under SPF conditions and injected with TRALI-inducing antibodies, they did not display increased lung W/Ds levels compared to naïve SPF mice indicating that the gut flora precedes and dictates the biological TRALI response. These data collectively link the gut flora to the development of antibody-mediated TRALI in mice and antibiotic treatment prevents TRALI and may perhaps prevent the onset of human TRALI which may help in TRALI risk-assessment prior to transfusion. DisclosuresSemple:Rigel: Consultancy; Novartis: Consultancy; Amgen: Consultancy; UCB: Consultancy.

Reporting transfusion-related acute lung injury by clinical and preclinical disciplines.

Disciplines involved in diagnosing transfusion-related acute lung injury (TRALI) report according to a "one-hit" theory. However, studies showed that patients with an underlying condition are at increased risk of the development of TRALI. We investigated whether accumulating evidence on the "two-hit" theory has changed the practice of reporting TRALI. Departments of haematology, haemovigilance, transfusion medicine, intensive care and anaesthesiology from all Dutch hospitals with at least five beds equipped for mechanical ventilation were invited to participate in an online survey. Using clinical vignettes with conjoint analysis we investigated the effect of patients' age, admission diagnosis, type and number of transfusions and presence of risk factors for acute lung injury on TRALI reporting. A positive β-coefficient indicated a higher likelihood of reporting TRALI. We received 129 questionnaires (response rate 74%). Respondents were more likely to report TRALI in younger patients, if symptoms developed within 2 hours of transfusion and if patients had received multiple transfusions. Sepsis and the presence of a risk factor for acute lung injury reduced the inclination to report. Transfusion medicine physicians and haemovigilance staff no longer took the age of transfusion products into account in their diagnostic considerations on TRALI. We conclude that the multidisciplinary team involved in TRALI reporting, still considers TRALI a "one-hit" event, despite accumulating evidence that supports the "two-hit" theory. These results suggest that the patients most at risk of developing TRALI are not reported to the blood bank.

A Preliminary Analysis of the Incidence of Transfusion-Related Acute Lung Injury (TRALI) in a Sheep Model Following Transfusion with the Supernatant from Leukodepleted Red Cell Units

IntroductionDespite the introduction of risk reduction strategies, transfusion-related acute lung injury (TRALI) continues to be a significant cause of morbidity and mortality. TRALI development may be associated with the transfusion of anti-leucocyte antibodies or biological response modifiers (BRMs). Our group has previously developed a model in which the development of TRALI in lipopolysaccharide (LPS) treated sheep was precipitated by the transfusion of pooled supernatant from date-of-expiry (day (d) 42) packed red blood cell (PRBC) units. This work pre-dated the introduction of pre-storage leukodepletion of PRBCs in Australia, therefore we investigated whether TRALI would still develop in LPS-treated sheep transfused with supernatant from d42 leukodepleted PRBCs.MethodsOn either d2 (n=75 units) or d42 (n=113 units) of storage, leukodepleted PRBCs underwent dual centrifugation to obtain acellular supernatants. Two supernatant pools (d2 and d42) were then prepared by pooling and heat-treating (56°C for 30min) the supernatants. Levels of potential BRMs in the PRBC supernatant pools were characterised using cytometric bead array and ELISA. Instrumented sheep (n=14) were infused with LPS then transfused (10% v/v) with either d2 (n=7) or d42 (n=7) pooled PRBC supernatant. Two hours later sheep were euthanized and post-mortem lung samples were collected. Physiological data were recorded continuously and then averaged in 30 minute blocks for these preliminary analyses. Blood and broncoalveolar lavage (BAL) fluid samples were collected at specific intervals. Blood samples were used for arterial blood gas analyses, coagulation tests (ROTEM), platelet function tests (MultiPlate) and ELISAs. TRALI was defined by hypoxemia (PaO2/FiO2 < 300 on arterial blood gas) and histological evidence of pulmonary edema (by two blinded histopathological assessments of hematoxylin and eosin stained lung sections). Data were compared by group (sheep transfused with d2 PRBC supernatant vs. d42 PRBC supernatant) and by outcome (sheep who developed TRALI vs. those who did not) with either t-tests or 2-way ANOVAs as appropriate.Results and DiscussionStorage duration of leukodepleted PRBC was associated with increased levels of the potential BRMs 5-HETE, 12-HETE, 15-HETE and IL-8, but not soluble CD40 ligand. Only 3 sheep developed TRALI: one transfused with d2 leukodepleted PRBC supernatant and two transfused with d42 leukodepleted PRBC supernatant corresponding to an incidence of 14% and 29% respectively. This indicated a reduced incidence of TRALI in the sheep model compared to previous data using supernatant from d42 non-leukodepleted PRBC which resulted in an incidence of 75% (Tung et al. Vox Sanguinis. 2011). Preliminary analyses of the physiological data revealed a number of differences. Heart rate (P<0.001), blood pressure (P=0.003), pulmonary artery pressure (P<0.001), cardiac output (P<0.001) and PaO2/FiO2 (P<0.001) were different between sheep transfused with either d2 or d42 PRBC supernatant. Heart rate (P<0.001), pulmonary artery pressure (P<0.001), pulse oximeter oxygen saturation (P=0.003), cardiac output (P<0.001) and PaO2/FiO2 (P<0.001) were different between sheep that developed TRALI and sheep that did not. However, given the complexity of these data, further analyses using mixed effects modelling are required to better understand these differences. Sheep transfused with d42 PRBC supernatant had reduced IL-1β lung gene expression and reduced ADP-induced platelet aggregation (both total and area under curve) compared to sheep transfused with d2 PRBC supernatant (P=0.047, 0.012 and 0.028 respectively). In addition, sheep that developed TRALI had worse lung histology scores as well as reduced collagen-induced platelet aggregation (both total and area under curve) compared to sheep who did not develop TRALI (P=0.024, 0.049 and 0.018 respectively).ConclusionsComparison of these results to previously published data from the sheep model is suggestive that pre-storage leukodepletion of PRBCs is associated with a reduced incidence of TRALI. Unsurprisingly, the development of TRALI was associated with worsened respiratory function (oxygen saturation and PaO2/FiO2) and lung histology scores, while changes in hemodynamics and platelet function were also observed. DisclosuresFraser:Fisher and Paykel Healthcare: Consultancy, Other: Provision of equipment for research use, Research Funding; De Motu Cordis: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Platelets are dispensable for antibody‐mediated transfusion‐related acute lung injury in the mouse

Essentials Role of platelets in immunological transfusion-related acute lung injury (TRALI) is debated. Immunological TRALI was tested in mice exhibiting severe thrombocytopenia or platelet dysfunction. Platelets are required to prevent lung hemorrhage but not edema formation and respiratory distress. Platelets are dispensable for the initiation and development of TRALI. Background Transfusion-related acute lung injury (TRALI) is a serious transfusion-related complication. Previous conflicting studies have indicated that platelets are either crucial or dispensable for TRALI. Objectives To evaluate the role of platelets in major histocompatibility complex (MHC) I-induced-TRALI. Methods Antibody-mediated TRALI was experimentally induced in mice by lipopolysaccharide priming followed by the administration of an anti-MHC I mAb. Results TRALI was tested in the context of severe thrombocytopenia provoked by the administration of diphtheria toxin (DT) in transgenic iDTR mice selectively expressing DT receptor in megakaryocytes. The pathologic responses occurring within the first 10 min following the injection of the anti-MHC I mAb, i.e. the severity of lung edema and the drop in aortic blood oxygenation, were similar in severely thrombocytopenic DT-iDTR and control mice. At later times, mortality was nevertheless increased in DT-iDTR mice, owing to lung hemorrhages. When less severe thrombocytopenia was induced with an antiplatelet mAb, TRALI started and developed similarly as in control mice, but hemorrhages were absent. Furthermore, when platelet functions were defective because of administration of aspirin or clopidogrel, or because of glycoprotein (GP)IIbIIIa deficiency, TRALI still developed but no lung hemorrhages were observed. In contrast, when GPVI was immunodepleted, TRALI still occurred, but was occasionally accompanied by hemorrhages. Conclusions Platelets are dispensable for the initiation and development of MHC I-induced TRALI. Although they do not protect against the disruption of the vascular endothelial cell barrier and the subsequent plasma leakage and edema formation, platelets are essential to prevent more serious damage resulting in hemorrhages in alveoli.

Two-event Transfusion-related Acute Lung Injury Mouse Model

Transfusion-related acute lung injury (TRALI) is defined as acute lung injury that occurs within 6 hours of a blood product transfusion. TRALI continues to be a leading cause of transfusion-related mortality and we have developed a mouse model of TRALI to better understand the mechanisms by which injury occurs and to test therapeutic approaches. Our model is a two-event model based on immune priming and the challenge of BALB/c wild-type mice with cognate MHC Class I monoclonal antibody (MHC I mAb). Immune priming with LPS mimics the primed state of recipients (first event) that is important for the development of TRALI. Donor HLA antibodies are frequently implicated in TRALI reactions, and cognate MHC Class I antibody (second event) produces acute lung injury in primed animals. Here, we describe a detailed protocol with high reproducibility within animals.

Potential Role of Bioactive Lipids in the Development of Non-Antibody Mediated Transfusion-Related Acute Lung Injury (TRALI)

BackgroundTransfusion-related acute lung injury (TRALI) has been the leading cause of transfusion-related morbidity and mortality in the UK and the USA in recent years. A threshold mechanism of TRALI has been proposed in which both patient factors (type and/or severity of clinical insult) and blood product factors (strength and/or concentration of antibodies or biological response modifiers) interact to surpass a threshold for TRALI development (Bux et al. Br J Haematol; 2007; 136: 788-99). The risk of developing antibody-mediated TRALI has been minimised by the introduction of risk-reduction strategies such as limiting the use of plasma from female donors. In contrast, there are no strategies currently in place to mitigate the development of non-antibody mediated TRALI as the mechanisms remain largely undefined. Previous studies have implicated non-polar lipids such as arachidonic acid and various species of hydroxyeicosatetranoic acid (HETE) in the development of non-antibody mediated TRALI (Silliman et al. Transfusion; 2011; 51: 2549-54), however the contribution of these lipids to the development of an inflammatory response in TRALI is poorly understood.MethodologyStandard leucodepleted packed red blood cell (PRBC) units were sampled at either day (D) 2 (n=75) or at D42 (n=113). PRBC-supernatants were obtained via centrifugation, pooled (D2, D42) and levels of arachidonic acid, 5-, 12- and 15-HETE determined using commercial ELISA kits. In an in vitro transfusion model, fresh human whole blood (“recipient”; n=8 for each lipid) was mixed with combinations of culture media (control) or lipopolysaccharide (LPS, 0.23 µg/mL) as a first hit. A range of concentrations of either 5-HETE (200; 1,000; 2,500; 10,000; 250,000 pg/mL), 12-HETE (1,500; 5,000; 62,500; 250,000 pg/mL) or 15-HETE (150; 1,000; 2,000; 8,000 pg/mL) were added as the second hit, and incubated for 6 hours with the addition of a protein transport inhibitor. Neutrophil- and monocyte-specific inflammatory response was assessed using multi-colour flow cytometry (panel: IL-6, IL-8, IL-10, IL-12, IL-1α, IL-1β, TNF-α, MCP-1, IP-10, MIP-1α, MIP-1β). Significance was determined as P < 0.05 by one-way ANOVA with Bonferonni’s correction used to determine dose response (indicated by asterisks).Results5-, 12- and 15-HETE were all detectable in both of the PRBC supernatant pools, with levels increased in D42 compared to D2 (5-HETE: 20,347 vs. 3,449; 12-HETE: 240,967 vs. 1,572; 15-HETE: 7,900 vs. 934; all levels in pg/mL). Arachidonic acid was not detectable in either of the PRBC supernatant pools. In the absence of LPS as a first hit, the addition of non-polar lipids had a predominantly immunosuppressive effect in the transfusion model. 12-HETE suppressed monocyte production of MIP-1α* and neutrophil production of IL-6, IL-8 and IL-12. Also, 15-HETE modulated monocyte IL-8 production and reduced neutrophil production of IL-8, IL-12, IP-10, MIP-1α, MIP-1β and TNF-α. In contrast, in the presence of LPS as a first hit, a predominantly pro-inflammatory response was evident to these lipids. 12-HETE increased monocyte production of IL-1α, IL-8* and MIP-1β* as well as neutrophil production of IL-1α*, IP-10*, MCP-1, MIP-1α* and MIP-1β. In addition, 15-HETE increased neutrophil expression of IL-1α and IL-6, and 5-HETE modulated monocyte production of MIP-1β.ConclusionsThese data suggest that the non-polar lipid mediators investigated here, in particular 12-HETE, may contribute to TRALI pathogenesis. A storage related accumulation of 5-, 12- and 15-HETE was evident in leucodepleted PRBC units. The in vitro model indicated that exposure to these lipid mediators supressed the recipient inflammatory responses in the absence of LPS, but contributed to a pro-inflammatory profile in the presence of LPS as a first hit. Together these data provide further evidence of the importance of both patient (first hit) and blood component (second hit) factors in the development of TRALI. Furthermore, the dose-associated response observed for a number of inflammatory markers is consistent with the threshold hypothesis of TRALI pathogenesis. DisclosuresNo relevant conflicts of interest to declare.

Novel swine model of transfusion-related acute lung injury.

Transfusion-related acute lung injury (TRALI) is a life-threatening complication of blood transfusion. Antibodies against human leukocyte antigens in donors' plasma are the major causes of TRALI. Several animal models of TRALI have been developed, and the mechanism underlying TRALI development has been extensively investigated using rodent models. Although sheep models of nonimmune TRALI have been developed, large-animal models of antibody-mediated TRALI are not yet available. To develop a swine model of TRALI, male Clawn strain miniature pigs were used. A monoclonal antibody (MoAb) against swine leukocyte antigens (SLAs) Class I (4G8, 0.3 or 1.0 mg/kg body weight [BW]) and a control antibody (1.0 mg/kg BW) were injected into the peripheral vein after priming with or without 1 μg/kg BW lipopolysaccharide (LPS; n = 3 each). Lung injury was assessed using PaO2 /FiO2 (P/F) ratio and by chest X-ray imaging. Histopathologic analysis was also conducted. Lung injury could be induced by injecting 4G8 at an amount of 1.0 mg/kg BW, after LPS. The P/F ratio 90 minutes after the administration of 4G8 significantly decreased (p < 0.05). Bilateral infiltration was shown in chest X-ray imaging. Lung injury was confirmed by histopathologic analysis. Lung injury in pigs was successfully induced by anti-SLA MoAb. Priming with LPS is a prerequisite for inducing lung injury and the amount of the antibody is a critical condition.

Remote ischemic preconditioning as prevention of transfusion-related acute lung injury

Transfusion-related acute lung injury (TRALI) is a serious complication of transfusion medicine, considered now as the leading cause of transfusion-related mortality. It may occur in up to 1 in 5000 transfusions and carries an elevated morbidity and mortality. Clinically it presents as hypoxia and non-cardiogenic pulmonary edema, usually within 6h of transfusion. It consists of an immunological phenomenon involving the activation of neutrophils and endothelial injury, leading to capillary leak and pulmonary edema, mechanisms shared with lung ischemia–reperfusion (IR) injury. Brief and repetitive periods of ischemia in an organ or limb have been shown to protect against subsequent major IR injury in distant organs, a phenomenon called remote ischemic preconditioning (RIPC). Limb RIP has been shown to protect the lung against IR injury trough modulation of endothelial function as well as neutrophil activation and infiltration. The protective effects of RIPC on the lung have been confirmed in clinical trials of orthopedic and cardiothoracic surgery. RIPC is a safe, tolerable and cheap procedure. I propose that limb RIPC could be used as a preventive strategy against the development of TRALI.

Storage of Red Blood Cells and Transfusion-Related Acute Lung Injury.

Transfusion-related acute lung injury (TRALI) is a major complication post-transfusion. A consensus definition of TRALI has been recently established to improve diagnosis but the pathogenesis of TRALI is yet to be understood. Although the antibody-mediated two-hit model of TRALI is the classical narrative, increasing evidence of the probable implications of prolonged storage of blood provides novel mechanisms towards storage lesion- the potentially injurious cellular and biochemical changes that occur in stored red blood cells. Red blood cell-derived lipids and micro vesicles may have been playing an important role in the development of TRALI. This article will provide a brief overview of the current understanding of TRALI and then discuss the implications and the potential mechanisms by which stored red blood cells may lead to TRALI.

Transfusion‐related acute lung injury and potential risk factors among the inpatient US elderly as recorded in Medicare claims data, during 2007 through 2011

Transfusion-related acute lung injury (TRALI) is a serious complication leading to pulmonary edema and respiratory failure. This study's objective was to assess TRALI occurrence and potential risk factors among inpatient US elderly Medicare beneficiaries, ages 65 and older, during 2007 through 2011. This retrospective claims-based study utilized large Medicare administrative databases. Transfusions were identified by recorded procedure and revenue center codes. TRALI was ascertained via ICD-9-CM diagnosis code. The study evaluated TRALI rates among the inpatient elderly overall and by calendar year, age, sex, race, blood components, and units transfused. Logistic regression analyses were used to assess potential risk factors. Of 11,378,264 inpatient transfusion stays for elderly Medicare beneficiaries, 2556 had a recorded TRALI diagnosis code, an overall rate of 22.46 per 100,000 stays. TRALI rates were higher for platelet (PLT)- and plasma-containing transfusions and increased by year and number of units transfused (p < 0.0001). Significantly higher odds of TRALI were also found for persons ages 65 to 79 years versus more than 79 years (OR, 1.19; 95% confidence interval CI, 1.09-1.29), females versus males (OR, 1.26; 95% CI, 1.16-1.38), white versus nonwhite (OR, 1.43; 95% CI, 1.27-1.66), and with 6-month histories of postinflammatory pulmonary fibrosis (OR, 1.89; 95% CI, 1.52-2.20), tobacco use (OR, 1.16; 95% CI, 1.00-1.26), and other diseases. Our study among the elderly suggests TRALI to be a severe event and identifies a substantially increased TRALI occurrence with greater number of units and with PLT- or plasma-containing transfusions. The study also suggests importance of underlying health conditions, prior recipient alloimmunization, and nonimmune mechanism in TRALI development among the elderly.