ALBERT

Description: Key Points TRALI may be induced by antibodies to HLA or HNA antigens or lipids, which accumulate during storage. Prestorage experimental filtration of RBCs removes HLA and HNA antibodies, decreases lipid priming activity, and mitigates TRALI in an animal model.

Description: Abstract 41 Transfusion-related acute lung injury (TRALI) is a leading cause of transfusion-related death with a majority of the reported cases secondary to the infusion of antibodies (Abs) contained within the plasma/blood component. An experimental filter that removes IgG was developed. We hypothesize that filtration of plasma with antibodies to leukocyte antigens will decrease both antibody-mediated priming of PMNs and antibody-mediated TRALI in a two-event in vivo model. Methods: Human plasma was drawn from healthy volunteers and IgG concentrations were measured before and after filtration. Plasma was obtained from two multiparous female donors: one with antibodies to HLA-A2 and to DR7 and the other with antibodies against HNA-3a. These plasma samples were filtered (F-Plas) or left as an unmodified control (Plas) and the anti-leukocyte antibodies were measured in a blinded fashion in referral labs using flow cytometry and Luminex™ beads or standard granulocyte antibody detection assays. These plasma samples were then used to prime the fMLP-activated respiratory burst, measured as the SOD-inhibitable reduction of cytochrome c (nmol O2−/min), of PMNs from HNA-3a+ donors or donors homozygous donors for HLA-A2, respectively. For the two-event in vivo modeling rats were incubated with 2 μg/ml endotoxin (LPS, S. enteritides) or saline (NS) for 2 hours (first event) and then were transfused with heat-treated human plasma that contained 25 μg/ml of an antibody against the MHC class I antigen OX27 that was either filtered (or left unmodified) prior to infusion (second event) followed by Evans Blue dye (EBD). ALI was measured as %EBD leak from the plasma into the bronchoalveolar lavage fluid. Statistical differences were measured via paired (PMN priming) or independent (in vivo TRALI) ANOVA, and data are reported as the mean ± the standard error of the mean. *=p

Description: Transfusion-related acute lung injury (TRALI) is a life-threatening complication of hemotherapy. We report a series of 90 TRALI reactions in 81 patients secondary to transfusion with whole blood platelets (72 reactions), apheresis platelets (2), packed red cells (15), and plasma (1). The overall prevalence was 1 in 1120 cellular components. To examine the epidemiology of TRALI, we completed a nested case-control study of the first 46 patients with TRALI compared with 225 controls who had received transfusions. We then completed a prospective analysis of possible biologic response modifiers responsible for 51 of the TRALI cases, including human leukocyte antigen (HLA) class I, class II, and granulocyte antibodies in donors and neutrophil (PMN) priming activity in the plasma of the implicated units and recipients. Two groups were at risk: patients with hematologic malignancies (P 

Description: Abstract SCI-48 Stored packed red blood cells (PRBCs) have been implicated in increased morbidity and mortality in critically ill patients, patients requiring cardiothoracic surgery, and injured patients following blunt and/or penetrating trauma. Lipids are generated during the routine storage of cellular blood components and have been implicated in transfusion-related acute lung injury (TRALI) and postinjury multiple organ failure (MOF). These lipids are comprised of two classes, as denoted by their retention times on normal-phase HPLC: 1) nonpolar lipids, including arachidonic acid, 5-hydroxy-eicosotetranoic acid (HETE), 12-HETE, 15-HETE, and 5-oxo-eicosotetranoic acid (5-oxo-ETE); and 2) a mixture of lysophosphatidylcholines (lyso-PCs), including stearoyl-, oleoyl-, or palmitoyl-lyso-PCs as well as C16 or C18 lyso-platelet activating factor. Importantly, all of these lipids were identified by quadrupole LC/MS/MS. The former are derived from red blood cells, because they are generated during the routine storage of prestorage leuko- and platelet-reduced PRBCs, while the latter are platelet-derived, and are generated during storage of platelet concentrates (PC) and unmodified PRBCs but not leukocyte-reduced PRBCs (LR-PRBCs). Generation of the nonpolar lipids requires an active phospholipase, such as peroxiredoxin-6, which accumulates during PRBC and LR-PRBC storage and appears to be active and T-phosphorylated. The nonpolar lipids do not accumulate during PC storage. These lipids are released into the plasma fraction of the stored component and have been shown to: 1) prime the polymorphonuclear leukocytes (PMN) oxidase; 2) activate primed PMNs; and 3) serve as the second event in a two-event model of TRALI in both rodents and sheep. This lipid priming of PMNs occurs through activation of specific receptors, for example G2A, for lyso-PCs, and causes activation of G-protein-linked cellular receptor, G-protein release, and stimulation of kinase cascades. This results in translocation of the cytosolic oxidase components and a change from a resting PMN phenotype to a hyperactive, adherent phenotype. Conversely, stored PRBCs have also been implicated in MOF. However, acute lung injury does not occur until 72 hours postinjury, implicating PRBCs as a first event(s). The nonpolar lipids that accumulate during routine storage activate primary human pulmonary microvascular endothelial cells (HMVECs) and primary human liver sinusoidal endothelial cells (LSECs), as quantified by increased surface expression of intercellular adhesion molecule-1 (ICAM-1) and the synthesis and release of chemokines, for example IL-8. This proinflammatory activation results in PMN adherence, and such HMVEC activation occurs through activation of the BLT2 receptor and activation of a PKC-dependent kinase cascade. Stored PRBCs (day 42) may serve as the first event in a two-event rodent model of acute lung injury. We conclude that lipids generated during the routine storage of cellular blood components have proinflammatory effects in vitro and in vivo, which may explain some of the adverse events of transfusion, including TRALI and postinjury MOF, and that inhibition and avoidance of these compounds may make transfusions safer. Further research is already under way in several prospective trials, including: 1) Age of Blood Components transfused in the PICU (ABC-PICU); 2) red cell storage duration and outcomes in cardiac surgery at the Cleveland Clinic; 3) Red Cell Storage duration Study (RECESS); and 4) the Age of Blood Evaluation trial (ABLE). Disclosures: Silliman: Pall Corporation: Honoraria, Research Funding.

Description: Abstract 1178 Microparticles (MP) are submicron size fragments produced from most cells. Although their role in disease is still not known, concern has been raised over their role in RBCs and the adverse events of RBC transfusion, particularly their pro-inflammatory effects on the host. We hypothesize that MP's increase during routine RBC storage, contribute to the priming activity that accumulates in the supernatant of RBCs, and that pre-storage leukoreduction affects these processes. Methods: RBC's were separated from the whole blood of 8 healthy donors and stored in AS-5 via current U.S. industry standards. The first 5 units were split into two equal aliquots (weight) with 50% undergoing standard leukoreuction (LR) and the remaining was left as an unmodified control (NLR). The other three units were processed as standard LR-RBCs. RBC samples were taken at days (D) 1 and 42 and cell-free supernatants were separated by centrifugation and stored (-80°C). The supernatant was centrifuged at 100,000g – 120 minutes into MP and MP free (MPF) portions and MP's resuspended in equal volume of 1.25 % HSA (MP-HSA) or cell-free plasma (MP-FFP). MP fractions were incubated with specific antibodies to RBCs (CD235), WBCs (CD45), and PLTs (CD41a) and analyzed by flow cytometry employing commercially obtained counting beads. Isolated neutrophils (PMNs) were incubated with the MP and MPF fractions [10%] FINAL for 5 min at 37°C followed by activation of the NADPH oxidase with fMLF. The maximal rate of O2− production was measured as the SOD-inhibitable reduction of cytochrome c. Results were analyzed as an ANOVA with a post-hoc Newman Keuls test for multiple comparisons. Results: The total number of MP's increased during storage in all units irrespective of LR. The predominant MPs came from RBCs with PLTs representing the least (Table 1). Although the total number of MPs increased in all units, the number of MPs that specifically marked for precursor cell types decreased over storage (Table 1). The MPF fractions from stored (D.42) NLR-RBCs and LR-RBCs caused significant priming of the PMN oxidase vs. both the MPF from D.1 and the fMLF controls (Table 1). The MP fraction from NLR-RBCs did not evidence priming activity (data not shown); however, the LR-MP-HSA fractions from both D.1 and D.42 caused significant priming of the oxidase. Resuspension in plasma (LR-MP-FFP) inhibited this priming activity (Table 1). We conclude that during storage of NLR- and LR-RBCs, MPs increase; however the number of MPs associated with a specific cell type decrease but not significantly. This loss of cellular markers may be due to non-specific loss, antigen capping, internalization or proteolysis because proteases are released and increase during RBC storage. The presence of MPs from leukocytes and platelets are likely due to filtration, which is known to cause release of WBC- and PLT-derived proteins. While the majority of the PMN priming activity is in the acellular MPF supernatant in all NLR- and LR-RBC units, there is a modest amount of priming activity in the MP fraction following LR (LR-MP-HSA), which is likely a function of LR and may be inhibited by resuspension of the MPs in plasma. Disclosures: Silliman: Pall Corporation: Honoraria, Research Funding.

Description: Introduction: TRALI is acute lung injury occurring during or within hours of a blood transfusion. The etiology is thought to be infusion of leukocyte antibodies or neutrophil priming and activation caused by biologically active lipids in blood components. We report a TRALI reaction associated with fresh frozen plasma (FFP) and activation of complement in both the unit of FFP and the patient at the time of the reaction. Case History: A 59 year old male with factor XI was admitted to the hospital with hematochezia and given 3 units of FFP. During infusion of the third unit, he developed dyspnea and cyanosis requiring ventilator and O2 support. A chest x-ray showed bilateral diffuse pulmonary infiltrates, CVP was 3 mm Hg, and an echocardiogram was normal. The symptoms resolved in 3 days. Methods: Samples from donors and/or units were screened for the presence of HLA antibodies by ELISA and lymphocytotoxicity and antibodies detected were typed for HLA specificity and antibody class. Reactivity was determined by flow crossmatch. Serologic and molecular HLA typing was completed on donor and patient samples. Priming activity of the implicated FFP, fresh plasma from donor and recipient, and plasma from controls was completed against freshly isolated neutrophils from the three sources. Significant activity was defined as 〉1.5 times the fMLP stimulated superoxide anion (O2−) production. C3aLE, C4aLE, SC5b-9, and Bb were determined by standard techniques. Results: HLA antibodies were only detected in the third unit of FFP. Samples from this unit and the donor exhibited HLA Class I and II reactivity by ELISA but not lymphocytotoxicity. Flow crossmatch cells demonstrated Class II, IgG reactivity of donor serum against recipient DR11, 13. No autologous reactivity was demonstrated. The FFP unit primed the fMLP response in donor, recipient and control neutrophils 2.6, 3.1, and 3.4 fold above baseline. Testing of donor, recipient and control plasma obtained 3 months after the reaction showed no priming against the same battery of cells (priming ratio 0.8–1.3). C4aLE (105%, control range 24–176%); C3aLE (476%, control range 21–180%); and Bb (351%, control range 31–169%) were elevated in recipient samples obtained during the TRALI reaction and SC5b-9 was at the high end of normal (164%, control 0–200%). These returned to normal after the reaction. Strikingly, evidence of complement activation was seen in the FFP unit (C4aLE 214%, C3aLE 402%, C5b-9 213%) but not in subsequent samples from the donor. Conclusion: These studies document a TRALI reaction with symptoms expressed during the administration of FFP. One unit exhibited HLA Class I and II antibodies, the latter of which bound to the recipient’s cells. Priming activity was seen with plasma from the implicated unit, not in subsequent samples from the donor. Laboratory studies document activation of complement in the FFP infused but not donor samples. Plasma from the recipient at the time of the reaction also exhibit activation of complement which became normal after the TRALI resolved. Infusion of the FFP with activated complement capable of priming neutrophils may have induced pulmonary leukostasis and TRALI quite distinct from any subsequent effect of antibodies. Although the cause of FFP complement activation is not defined, these results suggest alternative mechanisms involving complement may be responsible for HLA antibody-associated TRALI.

Description: Transfusion-related acute lung injury (TRALI) is a life-threatening adverse effect of transfusion that is occurring at increasing incidence in the United States and that, in the past 2 reporting years, has been the leading cause of transfusion-related death. TRALI and acute lung injury (ALI) share a common clinical definition except that TRALI is temporally and mechanistically related to the transfusion of blood/blood components. In prospective studies, 2 patient groups, 1 requiring cardiac surgery and 1 with hematologic malignancies and undergoing induction chemotherapy, were predisposed. Two different etiologies have been proposed. The first is a single antibody-mediated event involving the transfusion of anti-HLA class I and class II or antigranulocyte antibodies into patients whose leukocytes express the cognate antigens. The second is a 2-event model: the first event is the clinical condition of the patient resulting in pulmonary endothelial activation and neutrophil sequestration, and the second event is the transfusion of a biologic response modifier (including lipids or antibodies) that activates these adherent polymorphonuclear leukocytes (PMNs), resulting in endothelial damage, capillary leak, and TRALI. These hypotheses are discussed, as are the animal models and human studies that provide the experimental and clinical relevance. Prevention, treatment, and a proposed definition of TRALI, especially in the context of ALI, are also examined.

Description: Background: Secretory phospholipase A2 is an enzyme that is elevated in SCD patients with acute chest syndrome (ACS) and vaso-occlusive pain crisis (VOC) and inhibition of its enzymatic activity is in trials for ACS prevention (Styles LA. Blood.1996; 87:2573). This enzyme cleaves arachidonic acid from the phospholipids of red blood cell (RBC) membranes; and arachidonic acid can be converted to other pro-inflammatory lipid compounds (Murakami M. J Biochem.2002; 131:285). sPLA2 cleaves phosphatidylserine (PS) expressing lipid membranes. PS is usually in the inner leaflet (unexposed) in normal red blood cells (RBCs) but becomes externalized in sickle RBCs especially during VOC or ACS. There is modest exposure of PS during in the steady state among certain RBC populations (de Jong K. Br J Haematol.2006; 133:427). PS exposure allows sPLA2 to cleave RBC membranes, producing lipids which prime neutrophils (PMNs) and cause PMN-mediated pulmonary endothelial cell injury as the second event in a two-event model (Ball JB, Blood submitted). Objective: We hypothesize that sickle RBCs treated with hydroxyurea (HU) will be more resistant to digestion by sPLA2, thereby inhibiting the release of bioactive lipids. Design/Methods: Whole blood was collected from children with SCD when healthy or daily during admissions for VOC or ACS. The plasma and RBCs were separated. Plasma and lipids extracted from the plasma were used as priming agents of quiescent PMNs isolated from healthy donors. Additionally, the separated RBCs were treated with exogenous sPLA2, creating sPLA2-liberated lipids, which were also used as priming agents. The plasma sPLA2 levels were measured. Results: There was no difference in the sPLA2 levels of untreated and HU-treated SCD patients in the healthy condition (non-treated SCD: 12.8±3.1 ng/ml, HU: 12.2±4.2 ng/ml, p=0.95) and in VOC (non-treated SCD: 89.2 ± 79.3 ng/ml, HU: 86.5 ± 44.3 ng/ml, p=0.85). The PMN priming of the sPLA2-liberated lipids of untreated SCD patients, both healthy (*, Table 1) and those with VOC (#, Table 1), was elevated compared to the sPLA2-liberated lipids from HU-treated patients. We conclude that hydroxyurea treatment in vivo induces resistance to sPLA2 cleavage of RBC membrane lipids thereby inhibiting priming of PMNs, which has been implicated in PMN-mediated endothelial cell injury (Ball JB, Blood submitted). Table 1: PMN priming activity of plasma lipids and sPLA 2 -liberated lipids from patients with SCD. Healthy VOC pain plasma lipids sPLA2-liberated lipids plasma lipids sPLA2-liberated lipids * - p

Description: Abstract 2109 Poster Board II-86 Introduction: Transfusion of plasma is associated with the highest risk of transfusion related acute lung injury (TRALI)-related death. We hypothesize that the current preparation regimen of plasma derived from whole blood has led to significant platelet contamination of plasma, and these platelets contribute to the high rates of TRALI in recipients of plasma. Materials and Methods: Ten units of whole blood were drawn from volunteer donors (5 females and 5 males). All units were centrifuged at 5,316g for 6 minutes to achieve acellular centrifugal effect (ACE) of 4.61×107 in accordance with the blood center's operating procedure for the primary separation of red cells from plasma. We divided each unit into 4 equal aliquots and compared 4 preparation regimens to evaluate the effect of freezing and cell depletion centrifugation (CDC) on platelets and associated factors: not frozen/no CDC, frozen/no CDC, not frozen/CDC, and frozen/CDC. CDC samples were centrifuged at an additional 12,500g for 7 minutes at 4°C to achieve cell depletion prior to freezing, and frozen samples were prepared according to the American Association of Blood Banks (AABB) standard for the preparation of frozen plasma. For each of the four preparation regimens we measured platelet levels using a Beckman Coulter Counter (model: AcT 5Diff AL). We also measured levels of four biologic mediators associated with platelets using commercially available ELISA kits: adenosine diphosphate (ADP), which is found in dense bodies; soluble CD40 ligand (sCD40L), which is found on the surface of platelets; vascular endothelial growth factor (VEGF) and platelet factor 4 (PF4), both found in the alpha granules. Results: We found significant platelet contamination (p