ALBERT

Description: Introduction Clinical studies suggest packed red blood cell (PRBC) transfusion modulates recipients' immune responses, resulting in increased rates of infection, morbidity and mortality. In addition, there is evidence that increased length of storage of PRBC before transfusion is associated with worse outcomes. The mechanisms driving immunosuppression and transfusion-related poor patient outcomes remain largely undefined. As dendritic cells (DC) play a critical role in initiation and regulation of the immune response, this study investigated transfusion-mediated modulation of expression of DC genes involved in inflammation, antigen presentation and signal transduction. Methods Monocyte-derived DC (MoDC) were differentiated following exposure to PRBC (at either D2, D14, D28 or D42 of routine storage) in an in vitro transfusion model. Gene expression profiling of MoDC generated “post-transfusion” was assessed at each time point using quantitative real time PCR using ΔΔCT method (relative to GAPDH and matched “no transfusion control”) and data analysed using ANOVA (95% CI). Expression array (Applied Biosystems) consisted of 28 genes broadly categorised into 1) chemokines, cytokines and receptors, 2) antigen presentation, 3) signal transduction. Results PRBC transfusion significantly modulated expression of genes associated with signal transduction (CD86, CD44, TAP2, all p

Description: Background Transfusion-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. Methodology Standard 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). Results 5-, 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. Conclusions These 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. Disclosures No relevant conflicts of interest to declare.

Description: Storage lesion is a critical issue facing transfusion treatments, and it adversely affects the quality and viability of stored red blood cells (RBCs). RBC deformability is a key indicator of cell health. Deformability measurements of each RBC unit are a key challenge in transfusion medicine research and clinical haematology. In this paper, a numerical study, inspired from the previous research for RBC deformability and morphology predictions, is conducted for the first time, to investigate the deformability and morphology characteristics of RBCs undergoing storage lesion. This study investigates the evolution of the cell shape factor, elongation index and membrane spicule details, where applicable, of discocyte, echinocyte I, echinocyte II, echinocyte III and sphero-echinocyte morphologies during 42 days of in-vitro storage at 4 °C in saline-adenine-glucose-mannitol (SAGM). Computer simulations were performed to investigate the influence of storage lesion-induced membrane structural defects on cell deformability and its recoverability during optical tweezers stretching deformations. The predicted morphology and deformability indicate decreasing quality and viability of stored RBCs undergoing storage lesion. The loss of membrane structural integrity due to the storage lesion further degrades the cell deformability and recoverability during mechanical deformations. This numerical approach provides a potential framework to study the RBC deformation characteristics under varying pathophysiological conditions for better diagnostics and treatments.

Description: Introduction During routine storage, packed red blood cells (PRBC) undergo numerous biochemical and biophysical changes collectively referred to as the “RBC storage lesion”. A number of factors reported to accumulate during the routine storage of PRBCs are hypothesized to mediate inflammatory cell responses and contribute to poor patient outcomes following transfusion. In addition, donor variability in red blood cell (RBC) characteristics and onset of the storage lesion has been reported. We investigated changes in levels of potential biological response modifies in the supernatant (SN) of PRBC relevant to storage, and, variance between donations. Methods Cytometric bead array was utilised to quantify a panel of 32 potential biological response modifiers (BRMs) in the SN of PRBC during storage. Potential BRMS were analysed in the SN of 8 leukodepleted PRBC units at weekly intervals (D2, D7, D14, D21, D28, D35, D42). The CBA panel was comprised of soluble(s) CD40 Ligand, sCD62E, sCD62L, sCD14, sCD54 (ICAM-1), sCD106 (VCAM-1), CXCL9, VEGF, Fractalkine (CX3CL1), IL-1β, IL-6, IL-8, IL-10, IL-12p70, TNF-α, MIP-1α, MIP-1β, IP-10, RANTES, sCD62P, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-7, IL-9, IL-13, IFN-α, IFN-γ, angiogenin, MCP-1. Storage related changes were analysed using ANOVA (95% CI). Donor variance was indicated by fold difference and range. “High” sub population of donations compared to remaining donations at each time point using Mann-Whitney (95% CI). Results Of the 32 potential BRMs studied, angiogenin, sCD14, sCD106 (VCAM-1), sCD62L, sCD62P, ICAM-1, IL-1α, IP-10, RANTES and IL-9 were consistently detected in all units throughout the time course. There was no evidence of a storage related increase in these biological mediators during storage of the PRBC, although angiogenin levels significantly declined during storage (P

Description: Introduction During routine storage packed red blood cells (PRBC) undergo biochemical and biophysical changes collectively referred to as the “RBC storage lesion”. Donor-to-donor variability in the severity of the storage lesion has been reported. The extent to which donor-associated differences in blood component storage affect blood product quality and post-transfusion outcome remains unknown. Murine models with single nucleotide variants (SNV) in gene encoding spectrin-1β were used to investigate the impact of mutations on the RBC storage lesion. Methods Two murine lineages with N-ethyl-N-nitrosourea (ENU) generated single SNV in Spnb1, encoding spectrin-1β (Table 1), were selected from the Australian Phenomics Facility library (http://databases.apf.edu.au/mutations). Using genetic selection, homozygous (HOM), heterozygous (HET) and unaffected (WT) mice from each strain were generated (C57BL/6 background strain). Murine blood was leucoreduced, prepared in SAGM (0.4 HCT) and stored at 4°C for time course assessment of RBC characteristics. At day (D), D2, D7, D14 and D21 of storage, RBC integrity and evidence of storage-related changes were investigated using RBC osmotic fragility and flow cytometric analysis of CD44, CD47, TER119 and phosphatidylserine (PS). Data were generated from analysis of blood from Spnb1 (pedigree spectrin-1β a) homozygous (HOM, n=3), heterozygous (HET, n=3) and unaffected (WT, n=2 ); Spnb1 (pedigree spectrin-1β b) HOM (n=4), HET( n=4); C57BL/6 (n=4). The Mann-Whitney Test and ANOVA were utilised for statistical analyses (95% CI). Results At D2 of storage SNV in Spnb1 did not alter RBC characteristics, with all mice studied demonstrating a similar resistance to osmotic lysis and levels of CD44, CD47, TER119 and PS. By D7 of storage, clear pedigree-related differences in RBC characteristics were evident. At D7, RBC from spectrin-1β(a) HOM mice had significantly increased osmotic fragility and exposure of PS as well as significantly reduced CD44 and TER119 expression compared to unaffected siblings and background strain. Of note, these changes were not evident in the spectrin-1β(b) HOM mice at D7. For both strains at D7, heterozygous SNV did not exhibit altered storage parameters. By D14 both HOM and HET spectrin-1β(a) mice demonstrated a phenotype consistent with an exacerbated RBC storage lesion, characterised by significantly increased osmotic fragility and exposure of PS, and reduced CD44 and CD47 compared to background strain. At D14 there was also evidence of exacerbation of the storage lesion in stored RBC from HOM spectrin-1β(b) mice (significantly increased PS), though this was not to the extent observed in the spectrin-1β(a) mice. By D21 all murine RBC were substantially degraded under these storage conditions. Conclusions SNV in Spnb1,encoding RBC structural protein spectrin-1β, resulted in both early onset and exacerbation of the RBC storage lesion. Further, the degree of storage lesion and the point at which RBC degradation was observed was not only dependent on the homozygous or heterozygous status, but the mutation itself. These data demonstrate that minor genetic variation in genes encoding important RBC proteins contribute to donor related differences in PRBC storage. Disclosures: No relevant conflicts of interest to declare.