Background Platelet transfusions (PTxs) are often given to septic preterm neonates at high platelet count thresholds in an attempt to reduce bleeding risk. However, the largest randomized controlled trial (RCT) of neonatal transfusion thresholds found higher mortality and/or major bleeding in infants transfused at higher thresholds. Using a murine model, we investigated the effects of adult PTx on neonatal sepsis‐induced mortality, systemic inflammation, and platelet consumption. Study design and methods Polymicrobial sepsis was induced via intraperitoneal injection of cecal slurry preparations (CS1, 2, 3) into P10 pups. Two hours after infection, pups were transfused with washed adult Green Flourescent Protein (GFP+) platelets or control. Weights, platelet counts, and GFP% were measured before 4 and 24 h post‐infection. At 24 h, blood was collected for quantification of plasma cytokines. Results The CS batches varied in 24 h mortality (11%, 73%, and 30% in CS1, 2, and 3, respectively), due to differences in bacterial composition. PTx had differential effects on sepsis‐induced mortality and systemic inflammatory cytokines, increasing both in mice infected with CS1 (low mortality) and decreasing both in mice infected with CS2 and 3. In a mathematical model of platelet kinetics, the consumption of transfused adult platelets was higher than that of endogenous neonatal platelets, regardless of CS batch. Discussion Our findings support the hypothesis that transfused adult platelets are consumed faster than endogenous neonatal platelets in sepsis and demonstrate that PTx can enhance or attenuate neonatal inflammation and mortality in a model of murine polymicrobial sepsis, depending on the composition of the inoculum and/or the severity of sepsis.

Platelet transfusions (PTx) are frequently given to thrombocytopenic preterm neonates at higher platelet count (PC) thresholds than those used in adults, in an attempt to reduce their bleeding risk. However, in the largest randomized trial of neonatal PTx thresholds, infants transfused at a higher PC threshold had a significantly higher mortality and/or major bleeding compared to infants transfused at a lower threshold. Since platelets carry multiple cytokines and chemokines, and since activated adult platelets may have a higher ability to interact with immune cells than neonatal platelets (due to their higher P-selectin expression levels), we hypothesized that the deleterious effects of PTx in neonates would be related to pro-inflammatory effects. We further hypothesized that the effects of PTx on the systemic inflammatory response would be different in thrombocytopenic neonates with non-inflammatory conditions (e.g., intrauterine growth restriction, drugs, genetic syndromes) compared to neonates with underlying inflammation (e.g., sepsis, necrotizing enterocolitis). To test the effects of PTx in the absence of inflammation, we transfused healthy post-natal day 10 (P10) C57BL/6 pups with washed platelets (5x10 7/g, isolated from adult C57BL/6J mice or eGFP+ mice) or with Tyrode's buffer (TY control). Blood was collected via terminal bleed 2h, 4h, and 6h after transfusion, and plasma was separated for quantification of 31 pro- and anti-inflammatory cytokines by multiplex (n=5-10 mice per group/timepoint). Two hours after PTx, the transfused mice exhibited significantly higher levels of G-CSF, IL-1, IL-1, IL-6, IL-17, KC (CXCL1) and MCP-1 compared to controls, with the most striking increases observed in IL-6 (928±19 vs. 135±36 pg/dL, p<0.001) and KC (1201±239 vs 371±77 pg/dL, p=0.001). At 4h post-transfusion, the levels of most cytokines were decreasing, with the exception of G-CSF (1940±276 vs. 825±126 pg/dL, p=0.003), MCP-1 (185±39 vs. 58±14 pg/dL, p=0.003), and IL-17 (2.12±1 vs. 0.66±0.3 pg/dL, p=0.002), which peaked at four hours. All cytokines were decreasing by 6h. Next, to model neonates with inflammatory conditions, we injected P10 pups with lipopolysaccharide (LPS) IP at a sub-lethal dose (1µg/g), which induced mild weight loss, thrombocytopenia (~ 50% drop in PC), and leukopenia followed by leukocytosis. Two hours after LPS injection, pups were transfused with washed platelets from adult C57BL/6 mice or TY (as above). Blood was obtained by terminal phlebotomy 4h, 8h or 18h post LPS injection and plasma was separated and stored for cytokine quantification by multiplex. 4h after LPS, PTx pups had significantly higher levels of leukemia inhibitory factor (LIF, a member of the IL-6 family) compared to TY controls (35±6 pg/mL vs. 17±3.9 pg/mL, p<0.01). At both 4 and 8h, IL-6 and G-CSF levels were extremely high and at or above the upper limit of the standard curve in both groups. By 18h post-LPS, the majority of cytokines had decreased to near-normal levels in TY control pups, while IL-6, IL-5, KC (CXCL1) and IL-10 remained significantly elevated in PTx mice (IL-6: 601±114 vs. 187±38 pg/mL, p=0.0007; IL-5: 659±257 vs. 486±191 pg/mL, p=0.01; KC: 4569±1370 vs. 2686±827 pg/mL, p=0.04; IL-10: 729±283 vs. 330±131 pg/mL, p=0.009). Since IL-10 is an anti-inflammatory cytokine, we also evaluated the relation of IL-6 to IL-10 in PTx vs. TY control mice. This analysis showed that IL-6 levels were 2.3 times higher for any given IL-10 level in pups who received PTx compared to controls. In conclusion, our findings suggest that platelet transfusions induce an inflammatory response in newborn mice without underlying inflammation, characterized mostly by elevations in IL-6, G-CSF and KC. In newborn pups with underlying sub-lethal inflammation, platelet transfusions seem to prolong the inflammatory response. These observations may provide an explanation for the increased morbidity and mortality in human neonates receiving liberal PTx. Studies to identify the mechanisms through which platelets induce these responses are ongoing. Disclosures No relevant conflicts of interest to declare.

Thrombocytopenia affects 18-35% of all neonates in the Neonatal Intensive Care Unit and ~70% of those born extremely prematurely, with sepsis being a frequent cause. Platelet transfusions (PTx) are frequently given to septic preterm neonates at higher platelet count (PC) thresholds than those used in adults, in an attempt to reduce their bleeding risk. However, in the largest randomized trial of neonatal PTx thresholds, infants transfused at a higher PC threshold had a significantly higher mortality and/or major bleeding compared to infants transfused at a lower threshold. We hypothesized that the deleterious effects of PTx would be related to a potential "developmental mismatch" resulting from adult platelets being transfused into a neonate. Among other developmental differences, adult platelets (human and murine) exhibit significantly higher surface P-selectin expression following activation than neonatal platelets. P-selectin is essential for the interaction of platelets with immune cells. Thus, we hypothesized that adult platelets transfused into septic neonates would be consumed faster than endogenous neonatal platelets (due to higher potential for immune interaction), and would increase inflammation and mortality. To test these hypotheses, we used a published murine model of neonatal sepsis, consisting of injecting cecal slurry (CS) into C57BL/6 pups. CS batches were prepared by isolating the cecal content of adult C57BL/6 mice, which was weighted, aliquoted and frozen until use. Three different CS batches were prepared and injected IP into post-natal day 10 pups at a dose of 1.1 (CS1) or 1.0 mg/g (CS2 and 3). Two hours after infection, pups were transfused with washed platelets from adult GFP mice (5x10 7 platelets/g) or Tyrode's buffer (control). Weights, PCs and GFP platelet % were measured before, 4h and 24h post-infection. Blood was collected via terminal bleed at 24h, and plasma separated for quantification of 31 cytokines by multiplex. Despite identical preparation, CS batches varied greatly in their 24h mortality (11% vs 73% vs. 30% for CS1, 2 and 3, respectively). Moreover, PTx had different effects on the mortality of pups infected with different CS batches, increasing the 24h mortality of pups infected with CS1 (30% in transfused vs 11% in non-transfused, RR 2.70, 95% CI 1.02-7.15) but decreasing the mortality of pups infected with CS2 (46% vs. 73%) or CS3 (9% vs. 30%), with a combined RR of 0.52; 95% CI 0.30-0.91. Bacterial counts differed between CS batches, but did not correlate with mortality. Comparison of the microbiome composition using deep sequencing revealed an increased presence of pathogenic bacterial species (Legionella, Sutterella, and Helicobacter species) in CS2 and 3 compared to CS1, and a relative abundance of beneficial bacterial (Actinobacteria and Proteobacteria) in CS1. Different CS batches also elicited different cytokine responses, with significant differences noted in G-CSF, IL-1α, IL-1β, IL-3, IL-7, IL-12p70, and IL-15 levels (p<0.05). For all of these cytokines, except G-CSF, levels were lower in mice infected with CS1 compared to CS2 or 3. Next, we investigated the effects of PTx on the plasma cytokine profile of mice infected with CS1 or CS2/3 (combined), compared to their infected, non-transfused littermates. For nearly all cytokines, PTx increased the response after infection with CS1, but decreased it after infection with CS2/3, with a significant difference in mean global cytokine effect (p<0.0001). For individual cytokines, however, these differences only reached statistical significance for LIX (CXCL5, p=0.04) and approached significance for IL15 and IL17 (p=0.06). Finally, we developed a mathematical model to compare the consumption of endogenous neonatal platelets (GFP-) to that of transfused adult platelets (GFP+) in pups infected with CS1 vs. CS2. In both, the calculated percent consumption was higher for adult platelets than for neonatal platelets (54.8% vs. 32.6% for CS1 and 56.5% vs. 40.4% for CS2). In conclusion, our findings support the hypothesis that adult transfused platelets are consumed faster than endogenous platelets in early neonatal sepsis, and demonstrate that platelet transfusions can either enhance or attenuate the neonatal inflammatory response and the mortality in a model of murine polymicrobial sepsis, depending on the bacterial composition of the inoculum and/or the severity of the sepsis. Disclosures Stowell: Grifols: Speakers Bureau; Argenx: Speakers Bureau; Alexion: Consultancy.

The immature platelet fraction (IPF) is a measure of newly released platelets, which has been used as a marker of platelet production in multiple human studies but is not widely available in multispecies analyzers. We developed gates to measure the IPF in diluted and undiluted murine blood samples on the Sysmex XN-1000V multispecies hematology analyzer. IPF gates were created using undiluted and diluted (1/10) blood samples obtained from adult and newborn (postnatal day 10, P10) C57BL/6J wild-type (WT) mice, and from 3 murine models of thrombocytopenia: c-MPL−/− mice, which lack the thrombopoietin receptor (hyporegenerative); antibody-mediated thrombocytopenia; and acute inflammation-induced thrombocytopenia. P10 mice were chosen because, at their size, we could consistently obtain (by terminal phlebotomy) the blood volume needed to run an undiluted sample. The undiluted blood IPF gate successfully differentiated between mechanisms of thrombocytopenia in both adult and P10 mice. For diluted samples, 2 IPF gates were generated: a thrombocytopenic (T) gate, which performed well in samples with platelet counts (PCs) <800 × 109/L in adult mice and <500 × 109/L in newborn mice, and a non-thrombocytopenic (NT) gate, which performed well in samples with PCs above these thresholds. PCs and IPFs measured in diluted blood using these gates agreed well with those measured in undiluted blood and had good reproducibility. These diluted gates allow for the accurate measurement of PCs and IPFs in small (10 µL) blood volumes, which can be obtained easily from adult and newborn mice as small as P1 to assess platelet production serially.