D. Segura

Fibrin Deposits and Organ Failure in Newborn Foals with Severe Septicemia

BACKGROUND Septicemia in human neonates frequently is complicated by activation of the coagulation system, disseminated intravascular coagulation (DIC) and multiple organ failure syndrome, which may contribute to high mortality. In adult horses with DIC, the lung has been the organ most frequently affected by fibrin deposits. In addition, in vivo studies suggest that hemostatic mechanisms may be immature in foals < 1-day old. HYPOTHESIS Newborn foals with severe septicemia have fibrin deposits in their tissues independently of their age, and these fibrin deposits are associated with organ failure. ANIMALS Thirty-two septic and 4 nonseptic newborn foals euthanized for poor prognosis. METHODS Tissue samples (kidney, lung, and liver) collected on postmortem examination were stained with phosphotungstic acid hematoxylin (PTAH) and immunohistochemistry (IHC) for blind histologic examination. A fibrin score (grades 0-4) was established for each tissue sample and for each foal. Medical records were reviewed for assessing clinical evidence of organ failure during hospitalization. RESULTS Fibrin deposits were found in most septic foals (28/32 when using IHC and 21/32 when using PTAH), independently of the age of the foal. The lung was the most affected tissue (97% of the septic foals). Additionally, organ failure was diagnosed in 18/32 septic foals (8 with respiratory failure, 14 with renal failure), although a statistical association with severe fibrin deposition was not identified. CONCLUSIONS AND CLINICAL IMPORTANCE Nonsurviving septic foals have fibrin deposits in their tissues, a finding consistent with capillary microthrombosis and DIC.

Peritoneal d-Dimer Concentration for Assessing Peritoneal Fibrinolytic Activity in Horses with Colic

BACKGROUND Plasma D-dimer concentration is a useful marker to assess systemic coagulation and fibrinolytic activities in humans, dogs, and horses. Peritoneal fibrinolytic activity increases in horses with colic, especially in horses with endotoxin in the peritoneal fluid. HYPOTHESIS/OBJECTIVES Peritoneal D-dimer concentration can be used to assess peritoneal fibrinolytic activity in horses with severe gastrointestinal (GI) disorders and altered peritoneal fluid. ANIMALS Two hundred and twenty-one colic horses and 15 control horses. METHODS Prospective observational clinical study. Blood and peritoneal fluid were collected on admission. Horses were grouped according to diagnosis, peritoneal fluid analysis, and outcome. Peritoneal D-dimer concentration was determined, together with peritoneal tissue-plasminogen activator (t-PA) and plasminogen activator inhibitor (PAI-1) activities. Plasma D-dimer concentration also was measured. RESULTS Peritoneal D-dimer concentration was significantly higher in all colic groups compared with controls, and in horses with enteritis, peritonitis, and ischemic disorders compared with horses with large intestinal obstructions. Peritoneal D-dimer concentration was significantly higher in horses with altered peritoneal fluid (modified transudate and exudate) compared with horses with normal peritoneal fluid analysis. Plasma D-dimer concentration also was significantly higher in the peritonitis group, and in horses with altered peritoneal fluid analysis. Peritoneal and plasma D-dimer concentrations also were significantly higher in nonsurvivors. Peritoneal d-dimer concentration was significantly correlated with decreased peritoneal t-PA activity and increased peritoneal PAI-1 activity. CONCLUSIONS AND CLINICAL IMPORTANCE Peritoneal D-dimer concentration is markedly higher in severe GI disorders, and it can be used to assess peritoneal fibrinolytic activity in horses with colic.

Plasma d-Dimer Concentration in Sick Newborn Foals

BACKGROUND Septicemia is associated with a systemic inflammatory response, hemostatic activation, and disseminated intravascular coagulopathy (DIC). HYPOTHESIS Increased plasma d-dimer concentration occurs in septic neonates and can reliably detect sepsis or DIC, and predict death in ill neonatal foals. ANIMALS 40 septic, 41 nonseptic hospitalized foals, and 22 healthy neonates. METHODS Prospective observational clinical study. Blood samples were collected on admission, at 24-48 hours after admission, and at the time of discharge or euthanasia. Plasma d-dimer concentration, clotting times, antithrombin activity, and fibrinogen concentration were determined. RESULTS On admission, d-dimer concentration values were significantly higher in septic foals (median, 25-75th percentiles; 568, 245-2013 ng/mL) compared with the nonseptic and healthy groups (386, 175-559 and 313, 152-495 ng/mL, respectively), and in septic foals at the age of 2-7 days compared with similar-age nonseptic foals. By means of samples taken at 24-48 hours of hospitalization and a cut-off value of > 2000 ng/mL, D dimer concentration was significantly associated with the diagnosis of septicemia (odds ratio [OR] = 19.6, 95% confidence interval [95% CI] 1.9-203) and death (OR = 8.7, 95% CI 1.8-43). Owing to a high false-positive prediction rate (71%), a normal d-dimer concentration is better at eliminating the diagnosis of sepsis than an increased d-dimer concentration at predicting sepsis. Fifty percent of septic foals had a diagnosis of DIC, but d-dimer concentration was not significantly associated with the diagnosis of DIC. CONCLUSIONS AND CLINICAL IMPORTANCE Septic foals showed a marked activation of coagulation and fibrinolytic systems and a high prevalence of DIC. Increased plasma d-dimer concentration is significantly associated with the diagnosis of sepsis.

Poor Reproducibility of Template Bleeding Time in Horses

BACKGROUND Template bleeding time (TBT) is considered to be a useful test for detecting platelet function disorders and the effect of platelet-activating drugs, but studies in human medicine have concluded that the test has poor reproducibility and sensitivity. HYPOTHESIS TBT has poor reproducibility in horses and has insufficient sensitivity to detect the effect of etamsylate on platelet function. ANIMALS Twenty healthy horses. METHODS TBT was determined and repeated 2 hours and 30 days later. TBT was also performed 2 hours after IV administration of etamsylate. RESULTS Although no statistical differences were seen between the TBT values obtained at different times, the coefficients of variation for TBT replicates ranged from 26.8% to 45.5%. The reference range for TBT was 138.4-860.4 seconds. No statistically significant shortening of the mean TBT value was observed after etamsylate administration. CONCLUSION AND CLINICAL IMPORTANCE TBT has poor reproducibility, and the reference range is too wide to make TBT useful in a clinical setting. Other tests with higher reproducibility should be considered when assessing platelet function disorders in horses.

Neutrophilic myeloperoxidase index and mean light absorbance in neonatal septic and nonseptic foals

BACKGROUND Two neutrophilic indices reported by the ADVIA 120 Hematology Analyzer, neutrophilic myeloperoxidase index (MPXI), and mean light absorbance (neutrophil X mean [NXM]) have been proposed as indicators of systemic inflammatory disease in horses and of neutrophil activation in coronary ischemic syndromes in people. OBJECTIVE The aim of this study was to evaluate NXM and MPXI in healthy, sick nonseptic, and sick septic foals to determine whether conditions likely associated with neutrophil activation result in decreases in these variables. METHODS In this retrospective study, CBC data from 61 neonatal foals presented to the Equine Teaching Hospital of Barcelona were evaluated for correlations between MPXI, NXM, percentage of large unstained cells, neutrophil count, and percentage of band neutrophils. Results obtained in septic (n=32), sick nonseptic (n=22), and healthy foals (n=7) were compared. In addition, results recorded in septic/neutropenic (n=12), septic/non-neutropenic (n=20), nonseptic/neutropenic (n=8), nonseptic/non-neutropenic (n=14), and healthy foals (n=7) were also compared. RESULTS A weak negative correlation was found between MPXI and neutrophil count and between NXM and percentage of band neutrophils. Septic/neutropenic foals had significantly higher MPXI values (median 17.9, minimum-maximum 4.7-42.5) than did septic/non-neutropenic (1.5, -24.4 to 22.3), nonseptic/neutropenic (6.6, 0.6-17.9), and nonseptic/non-neutropenic foals (8.8, -10.1 to 16.8) but did not differ significantly from controls (12.8, -8.5 to 20.4). CONCLUSIONS Significant differences in NXM or MPXI were not found when disease groups were compared with controls; however, septic/neutropenic foals had significantly higher median MPXI than other groups of sick foals. Further prospective studies are needed to clarify if this finding is related to decreased neutrophil function or activation in septic/neutropenic foals.

The secretory mechanisms in equine platelets are independent of cytoskeletal polymerization and occur through membrane fusion.

Studies in animal models are useful to understand the basic mechanisms involved in hemostasis and the functional differences among species. Ultrastructural observations led us to predict differences in the activation and secretion mechanisms between equine and human platelets. The potential mechanisms involved have been comparatively explored in the present study. Equine and human platelets were activated with thrombin (0.5 U/ml) and collagen (20 µg/ml), for 90 seconds, and samples processed to evaluate: i) ultrastructural changes, by electron microscopy, ii) actin polymerization and cytoskeletal assembly, by polyacrylamide gel electrophoresis, and iii) specific molecules involved in activation and secretion, by western blot. In activated human platelets, centralization of granules, cytoskeletal assembly and fusion of granules with the open canalicular system were observed. In activated equine platelets, granules fused together forming an organelle chain that fused with the surface membrane and released its content directly outside the platelets. Human platelets responded to activation with actin polymerization and the assembly of other contractile proteins to the cytoskeleton. These events were almost undetectable in equine platelets. When exploring the involvement of the synaptosomal-associated protein-23 (SNAP-23), a known regulator of secretory granule/plasma membrane fusion events, it was present in both human and equine platelets. SNAP-23 was shown to be more activated in equine platelets than human platelets in response to activation, especially with collagen. Thus, there are significant differences in the secretion mechanisms between human and equine platelets. While in human platelets, activation and secretion of granules depend on mechanisms of internal contraction and membrane fusion, in equine platelets the fusion mechanisms seem to be predominant.