We hypothesized that blood transfusion could induce platelet-mediated innate immune reactions caused by the interaction between TLR4 and damage-associated molecular patterns (DAMPs). To test this hypothesis, we assessed the levels of TLR4, lipopolysaccharide (LPS)-binding protein, and certain DAMPs including high mobility group box 1 (HMGB1), S100 calcium-binding protein A8 (S100A8, MRP8, and calgranulin A), S100 calcium-binding protein A9 (S100A9, MRP14, and calgranulin B), and serum amyloid A (SAA) in a simulated cross-reaction ex vivo. Furthermore, pro-inflammatory cytokines downstream of TLR4 signaling, including TNF-α, IL-1β, and IL-6, were evaluated. The excel sheet include demographic variables of the four groups (control, M, S, and I). The sheet named Fig 3 was the raw data of Effect of Toll-like receptor 4 (TLR4) expression after 0.9% saline was mixed with washed platelets . The sheet named Fig 4 was the raw data of effect of Toll-like receptor 4 (TLR4) expression on matched blood type mixing (Group M), uncross-matched ABO type-specific mixing (Group S), and ABO incompatible blood mixing (Group I) after mixing donor red blood cells and washed platelets.The sheet named Fig 5 was the raw data of Effect of DAMPs (HMGB1, S100A8, S100A9, and SAA) in the plasma prepared from mixing donor red blood cells and recipient whole blood on total blood mixing, matched blood type mixing (Group M), uncross-matched ABO type-specific mixing (Group S), and ABO incompatible blood mixing (Group I). The sheet named Fig 6 was the raw data of Effect of LPS-binding protein and pro-inflammatory cytokines in the plasma, prepared from mixing donor red blood cells and recipient whole blood, on matched blood type mixing (Group M), uncross-matched ABO type-specific mixing (Group S), and ABO incompatible blood mixing (Group I).
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Flow cytometry analysis of TLR4 expression Recipient whole blood was centrifuged at 37°C for 10 min at 200 × g. The upper phase was carefully collected, whereas the lower phase and interphase were discarded. The upper phase was then centrifuged at 2,000 × g for 10 min and the remaining pellet was gently resuspended in platelet wash buffer. The suspension was centrifuged at 2,000 × g for 10 min to prepare washed platelets, which were resuspended in HEPES-buffered Tyrode’s solution; the suspension was adjusted to a final platelet count of 150,000–450,000 platelets/μL. Thereafter, recipient washed platelets without or with 0.9% saline added at 1%, 5%, or 10% (v/v) were used as controls, respectively. Consequently, for blood mixing reactions, donor red blood cells were immediately mixed with recipient washed platelets at 1%, 5%, or 10% (v/v) and incubated at 37°C for 5 min. Quantification of TLR4 expression in washed platelets We investigated platelet TLR4 expression in mixed samples in the presence of thrombin (final concentration: 0.2 U/mL) by incubating the samples at an ambient temperature of 23–26 °C for 5 min. To quantify TLR4 expression, the samples were stained with a saturating concentration of the anti-CD41a-FITC and anti-TLR4-PE monoclonal antibodies and incubated at 22–26 °C in the dark for 20 min. The samples were then fixed with 1% paraformaldehyde at 4 °C for 30 min and analyzed using flow cytometry. FITC-labeled IgG1κ and PE-labeled IgG1κ served as background controls. Individual platelets were identified through side scatter (granularity characteristics) and anti-CD41a-FITC immunofluorescence in a logarithmic-scaled dot plot. The results are expressed as the mean fluorescence intensity (MFI) of TLR4-PE expression, and reads from 10,000 platelets were collected for each sample. ELISA analysis Donor red blood cells were immediately added to recipient whole blood at 1%, 5%, or 10% (v/v) and incubated at 37°C for 30 min. The mixed blood was then centrifuged at 37°C for 10 min at 100 × g. The upper phase was carefully collected, whereas the lower phase and interphase were discarded. The plasma was analyzed to determine the concentrations of HMGB1, S100A8, S100A9, SAA, and LPS-binding protein using ELISA kits according to the manufacturer’s protocols. Multiplex immunoassays The plasma levels of pro-inflammatory cytokines, i.e., TNF-α, IL-1β, and IL-6, were assessed using multiplex immunoassay following the manufacturer’s instructions.