Melissa A. Claus

Red blood cell (RBC) transfusion is associated with recipient inflammation and infection, which may be triggered by excessive circulating iron. Iron chelation following transfusion may reduce these risks. The aim of this study was to evaluate the effect of deferoxamine on circulating iron and inflammation biomarkers over time and in vitro growth of Escherichia coli (E. coli) following RBC transfusion in dogs with atraumatic hemorrhage. Anesthetized dogs were subject to atraumatic hemorrhage and transfusion of RBCs, then randomized to receive either deferoxamine or saline placebo of equivalent volume (n = 10 per group) in a blinded fashion. Blood was sampled before hemorrhage and then 2, 4, and 6 h later. Following hemorrhage and RBC transfusion, free iron increased in all dogs over time (both p < 0.001). Inflammation biomarkers interleukin-6 (IL6), CXC motif chemokine-8 (CXCL8), interleukin-10 (IL10), and keratinocyte-derived chemokine (KC) increased in all dogs over time (all p < 0.001). Logarithmic growth of E. coli clones within blood collected 6 h post-transfusion was not different between groups. Only total iron-binding capacity was different between groups over time, being significantly increased in the deferoxamine group at 2 and 4 h post-transfusion (both p < 0.001). In summary, while free iron and inflammation biomarkers increased post-RBC transfusion, deferoxamine administration did not impact circulating free iron, inflammation biomarkers, or in vitro growth of E. coli when compared with placebo.

Background: Prestorage leukoreduction of red blood cell (RBC) bags prevents accumulation of pro-inflammatory mediators and experimentally attenuates post-transfusion inflammation in healthy dogs. However, the effect of leukoreduction on post-transfusion inflammation in critically ill dogs is unclear. Hypothesis: Dogs transfused with leukoreduced (LR) RBC will have lower concentrations of leukocytes, interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), and C-reactive protein (CRP) within 24 hours of post-transfusion compared to dogs transfused with nonleukoreduced (NLR) RBC. Animals: Sixty-one RBC-transfused dogs (LR = 34, NLR = 27). Methods: Randomized, blinded, controlled preliminary clinical trial. Blood bag processing was randomized to create identically appearing LR and NLR bags. Group allocation occurred with transfusion of the oldest compatible RBC bag. Blood samples were collected pretransfusion and at 8 and 24 hours post-transfusion for leukocyte count, IL-6, IL-8, MCP-1, and CRP. Data were analyzed on an intention-to-treat basis using linear mixed effects models. Significance was set at P < .05. Results: No significant differences were found between groups in concentrations of leukocytes (P = .93), IL-6 (P = .99), IL-8 (P = .75), MCP-1 (P = .69), or CRP (P = .18) over time. Eleven LR dogs (32%) and 4 NLR dogs (15%) were euthanized in the hospital (P = .14). No natural deaths occurred. Conclusions and Clinical Importance: No differences in inflammation biomarker concentrations were detected over time between dogs transfused with LR or NLR RBC, but heterogeneity likely hampered the ability to detect a difference with this sample size. The novel randomization and enrollment protocol was successfully implemented across 2 participating institutions and will be easily scaled up for a future multicenter clinical trial.

Objective The primary objective of this study was to document coagulation factor activity in canine “NEVER-FROZEN” and “THAWED” refrigerated plasma for the purposes of defining recommended expiration dates. We hypothesized that NEVER-FROZEN and THAWED refrigerated plasma would maintain >50% activity of coagulation factors V (FV), VII (FVII), VIII (FVIII), IX (FIX), X (FX), and von Willebrand factor antigen (vWF) and a concentration of fibrinogen above the lower bound of the reference interval (>0.982 g/L) for greater than 14 days but less than 42 days. Design Prospective laboratory-based study. Setting University teaching hospital blood bank. Animals Ten canine plasma units derived from healthy client-owned blood donors. Interventions Serial sampling (days 0, 1, 3, 5, 7, 10, 14, 17, 21, 24, 28, 32, 35, 39, 42) from NEVER-FROZEN and THAWED refrigerated canine plasma units was conducted for measurement of activities of FV, FVII, FVIII, FIX, FX, vWF, and fibrinogen concentrations using the ACL TOP 300. Plasma was defined as “suitable for transfusion” at a given time point if the entire 95% confidence interval for each factor was above 50% activity and above a fibrinogen concentration of 0.982 g/L. Measurements and main results The lower bounds of the FVIII and vWF confidence intervals were above 50% up to and including day 32 for NEVER-FROZEN refrigerated plasma and day 28 for THAWED refrigerated plasma. Confidence intervals for FV, FVII, FIX, and FX remained above 50% activity at all time points. The lower bound of the fibrinogen concentration was <0.982 g/L on day 39 for NEVER-FROZEN refrigerated plasma and on day 35 for THAWED refrigerated plasma. Conclusions Refrigerated canine plasma from these 10 dogs retained coagulation factor activity above the limit that we defined as suitable for transfusion for up to 32 days when NEVER-FROZEN and 28 days when THAWED. Further studies should evaluate the clinical outcomes and effects on coagulation factor activity of dogs receiving refrigerated plasma transfusions.

Objective: To evaluate the effect of 6% hydroxyethyl starch (HES) 130/0.4, compared with a Hartmann's solution control (CRYST), on urine biomarkers of acute kidney injury (AKI) in dogs prescribed a fluid bolus.Design: Randomized, controlled, blinded clinical trial January 2018 to February 2019.Setting: University teaching hospital.Animals: Forty client‐owned dogs.Interventions: Dogs prescribed a fluid bolus were randomized to receive at least 10 mL/kg of HES or CRYST with clinicians and investigators blinded to fluid type. Study fluid was used for further boluses as required in the following 24 hours, to a limit of 40 mL/kg total, after which fluid administration was open‐label.Measurements and Main Results: Urine was collected prior to and 6, 12, and 24 hours after the first study fluid bolus. Urine concentrations of AKI biomarkers: neutrophil gelatinase‐associated lipocalin (NGAL), cystatin C, kidney injury molecule‐1 (KIM), clusterin, and osteopontin were measured using a magnetic bead multiplexed assay. Osmolality‐indexed biomarker concentrations were compared between groups over time with linear mixed‐effects models, with P < 0.05 considered significant. The mean volume of study fluid administered was not significantly different between groups (HES: 23.1 mL/kg, CRYST: 25.9 mL/kg; P = 0.47, t‐test). There were no significant differences between groups in change over time of osmolality‐indexed urine concentrations of NGAL (P = 0.91), cystatin C (P = 0.95), KIM (P = 0.77), clusterin (P = 0.63), or osteopontin (P = 0.91). The maximum Veterinary Acute Kidney Injury (VAKI) score up to 7 days during hospitalization (P = 1.0) and in‐hospital mortality (P = 0.49) were not significantly different between groups, as compared by Fisher's exact test.Conclusions: There were no differences in change over time of urine AKI biomarkers in dogs treated with 10 – 40 mL/kg HES or CRYST over 24 hours. Larger clinical trials with patient‐centered outcomes are required to investigate the safety of HES in dogs.

Hepcidins are an evolutionarily conserved class of liver-expressed peptide, from which the twenty-five amino acid hormone, hepcidin-25 (herein hepcidin), has gained significant notoriety as the master regulator of iron homeostasis in mammals. Hepcidin maintains iron homeostasis by controlling the dietary absorption of iron and the mechanisms of recycling cellular iron stores. With the physiological significance of this hormone well established, it has emerged as an informative biomarker. In a comparison of the genome, transcriptome and peptidome of Canis lupis familiaris, we reveal the size of the hepcidin peptide in the canine, previous reports of which were contradictory to the evolutionary conservation predicted by genome annotation. Here, measurement of the peptide by mass spectrometry, following isolation from greyhound blood serum, revealed an amino acid sequence and peptide mass, differing from all accounts to date, yet demonstrating perfect sequence identity to that of the greater Canidae lineage of the Carnivora. Importantly, in the greyhound, the measured hepcidin peptide showed a similar temporal pattern to total serum iron, consistent with our understanding of hepcidin regulating iron homeostasis, in agreement with human diagnostics, and providing added translational evidence of the measured peptide being the iron regulatory hormone of the Canidae.

The peripheral nervous system communicates specifically with the immune system via local interactions. These interactions include the “hardwiring” of sympathetic/parasympathetic (efferent) and sensory nerves (afferent) to primary (e.g., thymus and bone marrow) and secondary (e.g., lymph node, spleen, and gut-associated lymphoid tissue) lymphoid tissue/organs. To gain a better understanding of this bidirectional interaction/crosstalk between the two systems, we have investigated the distribution of nerve fibres and PNS-immune cell associations in situ in the mouse lymph node by using immunofluorescent staining and confocal microscopy/ three-dimensional reconstruction. Our results demonstrate i) the presence of extensive nerve fibres in all compartments (including B cell follicles) in the mouse lymph node; ii) close contacts/associations of nerve fibres with blood vessels (including high endothelial venules) and lymphatic vessels/sinuses; iii) close contacts/associations of nerve fibres with various subsets of dendritic cells (e.g., B220+CD11c+, CD4+CD11c+, CD8a+CD11c+, and Mac1+CD11c+), Mac1+ macrophages, and B/T lymphocytes. Our novel findings concerning the innervation and nerve-immune cell interactions inside the mouse lymph node should greatly facilitate our understanding of the effects that the peripheral nervous system has on cellular- and humoral-mediated immune responses or vice versa in health and disease.

Objective: To investigate the association between synthetic colloids and biomarkers of acute kidney injury (AKI) in dogs with hemorrhagic shock.Design: Experimental interventional study.Setting: University.Animals: Twenty‐four healthy ex‐racing Greyhounds.Interventions: Anesthetized Greyhounds subjected to hemorrhage for 60 min were resuscitated with 20 mL/kg of fresh whole blood (FWB), 6% hydroxyethyl starch (HES) 130/0.4, 4% succinylated gelatin (GELO), or 80 mL/kg of isotonic crystalloid (CRYST) over 20 min (n = 6 per treatment). Concentrations of biomarkers of AKI were measured at baseline, end of hemorrhage, and at 40 (T60), 100 (T120), and 160 (T180) min after fluid bolus. Biomarkers included neutrophil gelatinase‐associated lipocalin in urine and serum (uNGAL; sNGAL), and urine cystatin C (uCYSC), kidney injury molecule‐1 (uKIM), clusterin (uCLUST), osteopontin, gamma‐glutamyl transferase, monocyte chemoattractant protein‐1 (uMCP), interleukin‐6, interleukin‐8, protein (uPROT), hyaluronan, and F₂‐isoprostanes. Renal histology was scored for tubular injury and microvesiculation. Biomarker fold‐change from baseline was compared between groups using mixed effects models (Bonferroni–Holm corrected PMeasurements and main results: In dogs treated with GELO, uNGAL fold‐change was markedly greater compared with all other groups at T60, T120, and T180 (all PPPP = 0.015) although tubular injury scores were comparable.Conclusion: In dogs with hemorrhagic shock, GELO was associated with greater magnitude increases in urine biomarkers of AKI and more frequent marked tubular microvesiculation, compared with FWB, CRYST, and HES.

Background: Hemorrhagic shock and volume replacement can alter coagulation. Synthetic colloids, hydroxyethyl starch (HES) and gelatin, may enhance hypocoagulability. Our primary objective was to describe the effect of four fluid products on coagulation in canine hemorrhagic shock. Our secondary objective was to compare measurements of coagulation during shock to baseline in all dogs. Methods: Anesthetized greyhounds subjected to atraumatic hemorrhage for 60 minutes were administered 20 mL kg-1 of either fresh whole blood (FWB), 6% HES 130/0.4, 4% succinylated gelatin (GELO), or 80 mL kg-1 of isotonic crystalloid over 20 minutes (n=6 per group). Platelet closure time (PCT), rotational thromboelastometry (ROTEM) and plasma coagulation assays were measured at baseline, end of hemorrhage (shock), and 40 (T60) and 160 (T180) minutes after study fluid. ROTEM parameters included clotting time (CT), clot formation time (CFT), alpha angle, maximum clot firmness (MCF), lysis index at 60 minutes (LI60) and thrombodynamic potential index (TPI) for INTEM, EXTEM, FIBTEM (MCF only) and APTEM (LI60 only) profiles. Plasma coagulation assays included prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen concentration and activities of factor VII (FVII), factor VIII (FVIII) and von Willebrand Factor antigen (vWF). Between-group differences were tested using linear mixed models with post hoc between-group comparisons (Bonferroni-Holm corrected). Differences between baseline and shock were tested using paired t-tests. Significance was set at P<0.05. Results: GELO showed longer PCT at T60, compared with FWB and CRYST, and at T180, compared with all other groups. HES showed longer EXTEM CT at T60, compared with all other groups. HES showed lower INTEM and EXTEM MCF at T60 and lower INTEM MCF at T180, compared with FWB. Some plasma coagulation assays showed greater hypocoagulability with HES. Comparing shock to baseline, EXTEM CT, INTEM CFT, EXTEM CFT, PT and FVIII significantly increased and PCT, INTEM CT, INTEM MCF, EXTEM MCF, EXTEM LI60, EXTEM TPI, FIBTEM MCF, APTT, fibrinogen, FVII and vWF significantly decreased. Conclusions: In dogs with hemorrhagic shock, volume replacement with GELO caused mild platelet dysfunction and HES was associated with coagulation changes consistent with hypocoagulability, beyond effects of hemodilution. Shock alone produced some evidence of hypocoagulability.