[PMC free article] [PubMed] [Google Scholar] 8

[PMC free article] [PubMed] [Google Scholar] 8. these lipids as arachidonic acid and 5-, LDN-27219 12-, and 15-hydroxyeicsotetranoic acid. At concentrations from Day 42, but not Day 1, three of four of these lipids individually, and the mixture, primed PMNs. The mixture also caused ALI as the second event in a two-event model of TRALI. CONCLUSION We conclude that the nonpolar lipids that accumulate during LR-RBC storage may represent the agents responsible for antibody-negative TRALI. Transfusion-related acute lung injury (TRALI) remains the most common cause of transfusion-related mortality.1,2 TRALI has been linked to a number of mediators including antibodies against white blood cell (WBC) antigens, biologically active lipids that accumulate during routine storage of cellular blood components, and soluble CD40 ligand.3C6 During routine storage of red blood cells (RBCs), lipids accumulate in the plasma fraction, supernatant.7 These lipids consist of nonpolar lipids and a mixture of lysophosphatidylcholines (lyso-PCs), as defined by their retention times on normal-phase high-pressure liquid chromatography (HPLC).7 Lyso-PCs at concentrations that accumulate during RBC and platelet (PLT) concentrate storage precipitate polymorphonuclear neutrophil (PMN)-mediated endothelial damage and acute lung injury (ALI) in a two-event in vivo model of TRALI.8 Universal prestorage leukoreduction has been instituted in a number of countries, and despite decreasing the numbers of LDN-27219 febrile transfusion reactions, outside of a single institution in the United States, there has been no mention of its effects on the rates of TRALI.9,10 In addition, prestorage leukoreduction by filtration is known to decrease WBC contamination by greater than 3 logs and decrease PLT contamination by 4 to 5 logs, as evidenced by eradication of soluble CD40 ligand accumulation during routine storage because it is PLT derived.3 It is hypothesized, therefore, that prestorage leukoreduction changes the lipids that accumulate during routine storage but does not affect their ability to rapidly prime the PMN oxidase or to serve as the second event to precipitate ALI in an in vivo model. MATERIALS AND METHODS Materials All chemicals were purchased from Sigma Chemical Co. (St Louis, MO) unless otherwise stated. Solutions were made from sterile water for injection, USP (Baxter Healthcare Corp., Deerfield, IL). All buffers Rabbit Polyclonal to ZNF287 were made from stock USP solutions as previously described and sterile-filtered with disposable sterilization filter units (Nalgene, MF75 series, Fisher Scientific Corp., Pittsburgh, PA).7 RBC collection Whole blood (450 mL) was collected from 10 healthy volunteer donors after obtaining informed consent under a protocol approved by the Colorado Multiple Internal Review Board at the University of Colorado Denver.8 Each unit was equally divided (weight) with 50% being leukoreduced (LR-RBCs) via filtration (Pall BPF4 filter, Pall Corp., Westbury, NY) and stored at 4C per industry standards.11 Sterile couplers were employed to obtain samples on Day 1 and Day 42, and the supernatant was isolated and stored at ?80C.11 HPLC separation of lipids and analysis by mass spectrometry Plasma lipids were solubilized and separated by normal-phase HPLC and these lipid fractions were solubilized LDN-27219 in albumin for use in PMN-priming assays and for the identification of the active lipids.7 To identify the nonpolar lipids in the supernatant ice cold methanol was added (50% vol/vol) to the acellular supernatant, the proteins were precipitated, a stable internal standard was added (2 ng 2H8-5- hydroxyeicosatetranoic acid [HETE]), and the nonpolar lipids were extracted and analyzed as reported using an HPLC system directly interfaced into LDN-27219 the electrospray source of a triple quadrupole mass spectrometer (liquid chromatography coupled to electrospray ionization mass spectrometry [LC/MS/MS]).12,13 An estimation of lipid concentration was completed using ratios to an internal standard (1.0 = 2ng of lipid).12C14 PMN isolation and.