The plasma from stored PRBCs causes lung injury in a 2-event in vivo model of TRALI. (A) Sprague Dawley rats were injected intraperitoneally with 0.9% saline (NS; ■ left or NS middle) or endotoxin (LPS, left, LPS right) 2 hours prior to transfusion (first event) and then infused with NS or 10% FFP (controls), 4.5 μM lysophosphatidylcholines (LPCs, positive control), or the plasma from D1, D28, and D42 PRBCs () or LR-PRBCs () at 5% or 10% of the total blood volume. The percentage of Evans blue dye (EBD) leak from the plasma to the bronchoalveolar lavage (BAL) was measured (% BAL EBD/% plasma EBD, y-axis) and is shown as a function of treatment group (x-axis). No rats treated with NS as the first event demonstrated significant EBD leak (ALI) nor did animals injected with LPS followed by NS, 10% FFP, the plasma (10%) from D1 PRBC or D1 LR-PRBCs, or the plasma (5%) from D28 PRBCs and LR-PRBCs. However in rats treated with LPS and then infused with the plasma from D28 PRBCs (10%) or D28 LR-PRBCs (10%), D42 PRBCs (5%-10%) or D42 LR-PRBCs (5%-10%), or LPCs, the lipids from stored PRBCs, significant EBD leak occurred compared with the controls (*P < .05 from LPS/NS, †P < .05 from LPS/10% FFP, n = 4 for each bar). (B) Rats were pretreated with NS or LPS 2 hours prior to infusion of plasma from D42 PRBCs or D42 LR-PRBCs at 10% of the total blood volume. After 6 hours, the rats were humanely killed, and the lungs were removed, embedded in OCT compound, and snap-frozen. The lungs were sectioned and stained with H&E (left column) or underwent immunohistochemistry to identify rat PMNs (right columns) that used a specific rabbit antirat granulocyte antibody followed by a fluorescently labeled goat antirabbit antibody (red). The sections were visualized at 40×. Rats pretreated with NS and infused with plasma (10% total blood volume) demonstrated normal pulmonary histology (i) with intact endothelium and alveoli with a few scattered PMNs (red) in the vasculature; and the lung parenchyma including the vasculature were stained with WGA (green, membrane stain) and bis-benzamide (blue, nuclear stain) (ii). In rats treated with LPS and then infused with 10% plasma from D42 PRBCs (iii,iv) or D42 LR-PRBCs (v,vi) the histology demonstrates PMN infiltration, pulmonary edema, arcuate inflammation, and hyaline membrane formation. In addition, the PMN infiltration was confirmed by the immunohistochemistry, which demonstrated widespread PMN margination into the lung (red), further demarcated with the white arrows (iv,vi; n = 2).