• Open Access

Fibrin Deposits and Organ Failure in Newborn Foals with Severe Septicemia

Authors


  • This manuscript represents part of a thesis submitted by the first author at the Universitat Autònoma de Barcelona as partial fulfillment of the requirements for a PhD program supervised by the corresponding author. This study was partially presented at the 2nd meeting of the European College of Equine Internal Medicine in Naas (Ireland), February 2007. The abstract was published in the J Vet Intern Med 2007; 21:886.

Corresponding author: Luis Monreal, Servei de Medicina Interna Equina, Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de Barcelona, 08193-Bellaterra, Barcelona, Spain; e-mail: lluis.monreal@uab.es.

Abstract

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.

Alterations in the coagulation and fibrinolytic systems secondary to sepsis have been documented in several studies in humans, dogs, and horses.1–3 These hemostatic changes are consistent with hypercoagulation, and subsequently a consumption coagulopathy and disseminated intravascular coagulation (DIC). Septicemia has been implicated as the major cause of morbidity and mortality in newborn foals.4,5 It also has been reported that septicemia and endotoxemia are predisposing factors for the development of hypercoagulation and possible production and accumulation of fibrin.6–8 In septic shock, DIC is an important mediator in the development of multiple organ dysfunction syndrome (MODS),9,10 which is associated with increased mortality rates in humans.9

A severe systemic activation of coagulation can occur with subsequent deposition of fibrin in small and midsize vessels, which ultimately may lead to microvascular occlusion, hypoxia, and organ dysfunction.11,12 Previous studies in experimental animal models with lipopolysaccharide-induced systemic activation of coagulation reported fibrin deposition in tissues with secondary organ injury.13 In some reports in human patients with DIC,12,14 sepsis-related organ failure correlated significantly with mortality. Moreover, in a recent study of septic patients,15 42% had DIC and MODS, and the number of dysfunctioning organs in these patients was statistically higher than in non-DIC patients; although, 54% of non-DIC septic patients also had MODS. In fact, the definition of DIC by the Scientific Subcommittee on Disseminated Intravascular Coagulation of the International Society on Thrombosis and Haemostasis considers “DIC is an acquired syndrome characterized by the intravascular activation of coagulation with loss of localization arising from different causes. It can originate from and cause damage to the microvasculature, which if sufficiently severe, can produce organ dysfunction.”16

In human medicine, detection of microvascular fibrin deposition in 1 or more organs is considered essential for histopathologic diagnosis of DIC,17–19 and the same is suggested in animals.20 To our knowledge, only 2 previous studies21,22 reported the occurrence of fibrin deposits in equine tissues, consistent with the diagnosis of DIC. A recent study of our group23 demonstrated that 50% of hospitalized septic foals had clinicopathologic evidence of DIC, and this percentage increased to 59% in those septic foals that were euthanized (Monreal, personal communication). In addition, some studies suggested that foals <1-day old were unable or had some delay in their ability to form fibrin.23,24 However, no previous study has reported the frequency of fibrin deposition in septic newborn foals and its relation to organ failure. Considering that some septic foals may suffer from a hypercoagulable state and DIC, the hypotheses of the present study were that (1) nonsurviving newborn foals with severe septicemia have fibrin deposits in their tissues unrelated to age, and (2) the fibrin deposits are correlated with organ failure. Thus, the primary purposes of the study presented here were to assess fibrin deposition in tissues of nonsurviving septic newborn foals, and to evaluate the incidence of clinical signs consistent with DIC and MODS, and their relationship to fibrin deposits. Secondary purposes of the study were to determine if fibrin formation and deposition is age dependent in the 1st weeks of life in nonsurviving septic foals, and to evaluate the most commonly affected organ in these foals.

Materials and Methods

Animals

Foals <21 days old admitted to the Equine Teaching Hospital of Barcelona between January 2004 and June 2007 with a clinical diagnosis of septicemia that died during admission or hospitalization or were euthanized due to poor prognosis were selected. Foals were considered septic if they had a positive blood culture or sepsis score >11.25,26 All of the foals also were presented with 2 or more clinical signs of systemic inflammatory response syndrome: hyper- or hypothermia (rectal temperature >39.2 or <37.2 °C), tachycardia (heart rate >120 beats/min), tachypnea (respiratory rate >30 breaths/min), leukocytosis or leukopenia (peripheral white cell count >12.5 × 109/L or <4 × 109/L) or >10% immature (“band”) neutrophils.25–27 When blood culture was negative or not performed, the diagnosis of septicemia was confirmed at postmortem examination by the observation of pathologic findings consistent with polyarthritis, omphalophlebitis or omphaloarteritis, enteritis or peritonitis or meningoencephalitis. The septic foals also were classified into 3 subgroups according to the age of the foals at the time of death or euthanasia as previously cited1,24: <24 hours, 2–7 and 8–21 days. Foals were kept alive as long as possible and reasonable, considering the foal's suffering. Septic foals were euthanized for humane reasons when the senior clinician's experience and the clinicopathologic data indicate a poor prognosis.

Sick newborn foals that were hospitalized due to gastrointestinal adhesions (n = 2), nonseptic enteritis (n = 1), and proximal phalanx fracture (n = 1) also were included in this study as the nonseptic control group. These foals died or were euthanized due to their poor prognosis or the owners's economic limitations and their diagnoses were confirmed on postmortem examination. Other foals that died or were euthanized and had nonseptic clinical conditions associated with hypercoagulation and DIC,23 such as severe hypoxic-ischemic encephalopathy, ischemic gastrointestinal disorders, prematurity or immaturity, and massive muscle destruction, were not included.

Sampling and Processing of Tissues

On postmortem examination, tissue samples from kidney, lung, and liver were collected and fixed in 10% neutral buffered formalin for further histopathologic study. Renal tissue specimens included cortex and medulla. Pulmonary and hepatic samples were taken randomly from these organs. Formalin-fixed samples were processed in an automated tissue processora and embedded in paraffin wax before triplicate sections were prepared at 3 μm.

Histologic and Immunohistochemical Procedures

Sections were stained using both hematoxylin and eosin (HE) and phosphotungstic acid hematoxylin (PTAH), by standard procedures.28 For immunohistochemistry (IHC), 3-μm sections were mounted on silane-coated slides and dried. Immunolabeling with polyclonal rabbit anti-human fibrinogen/fibrin antibodyb was performed with a streptavidin biotin complex method as described previously.22

Examination of Sections

Microscopic evaluation was performed in parallel by 2 different researchers (MC and JP) without knowing the origin of the samples and an average value was determined. Presence of histopathologic lesions was evaluated on the HE-stained sections. Presence of microvascular fibrin deposits in glomerular and alveolar capillaries and in hepatic sinusoids was recorded separately for each sample stained with PTAH and IHC, and extravascular fibrin deposits were not considered to avoid possible misclassification of fibrin of inflammatory origin.

Regarding the PTAH-stained slides, the amount of fibrin present in kidney sections was graded from 0 to 4, as described previously.21,22,29 Briefly: 0 = absence of fibrin; 1 = presence of partial fibrin deposits in some glomerular capillaries; 2 = presence of partial deposits in all glomerular capillaries; 3 = presence of large quantities of fibrin in all glomeruli; and 4 = presence of fibrin thrombi in glomerular capillaries and in noncapillary vessels. A 0–4 scoring system also was applied for lung and liver sections as described previously.21,22,30 Sections in which 20 of 20 fields were positive received a score of 4; sections in which none of the fields were positive received a 0 score.

For grading the amount of fibrin deposits in the kidney when using the IHC method, slides were assigned a fibrin score as previously described,22 where 0 = absent staining or positive in ≤ 10% of the glomeruli; 1 = staining in glomerular capillaries in 11–25% of glomeruli in nonconsecutive fields; 2 = staining in glomerular capillaries in 26–50% of glomeruli; 3 = staining in glomerular capillaries in 51–75% of glomeruli; and 4 = staining in glomerular capillaries in ≥ 76% of glomeruli. For grading the amount of fibrin deposits in the lung and liver when using IHC, a fibrin score previously described22 was applied, where 0 = absent staining or ≤10% positive fields; 1 = 11–25% positive fields with staining in alveolar capillaries or hepatic sinusoids in nonconsecutive fields; 2 = 26–50% positive fields with staining in alveolar capillaries or hepatic sinusoids in half of the fields in an almost continuous staining pattern; 3 = 51–75% positive fields with staining in alveolar capillaries or hepatic sinusoids in >50% of the fields in a continuous staining pattern; and 4 =≥ 76% positive fields with staining in alveolar capillaries or hepatic sinusoids in a continuous staining pattern with a fibrillar meshwork.

To summarize all of the information, all animals were later categorized for each stain from grades 0–4 according to the amount of fibrin and thrombi observed in their tissue samples, as previously described.21,22 Briefly, a foal was classified as grade 4 when it had at least 1 tissue sample with a 4 score; grade 3 foals were those with at least 1 tissue sample scored 3 and no samples with higher scores; foals classified as grade 2 were those that had at least 1 tissue sample scored 2 and no tissue samples with higher scores, and grade 1 foals were those that had at least 2 tissue samples scored 1 and no tissue samples of higher score. The remaining animals were classified as grade 0.

Clinical Data

Medical records from all neonates were reviewed to assess clinicopathologic evidence of organ dysfunction and spontaneous bleeding during hospitalization and before death. Renal failure was diagnosed whenever plasma creatinine concentrations remained above the reference range (>2 mg/dL) in foals with normal hydration status, excluding prerenal azotemia, receiving fluid therapy at maintenance rate, with an estimated reduction of urine production, and without a diagnosis of a previous renal dysfunction.31 In foals <2 days old, only large increments in creatinine concentration were considered abnormal, because normal foals can have increased plasma creatinine concentration (above 2 mg/dL) for 24–48 hours after birth without having renal failure.32,33 Respiratory failure was diagnosed whenever arterial blood analysis indicated a decreased PaO2 considering the age of the foal, with or without an increased PaCO2, with the foal in standing position or in lateral recumbency and normal mean arterial pressure.34–36 Foals with a diagnosis of renal failure, respiratory failure or both were included in the organ failure group and the remaining foals were included in the group without organ failure.

Statistical Analysis

Data were analyzed using a computerized programc and a significance level of P<.05 was used for all statistical tests used throughout the study. For each method (PTAH and IHC), median (25th–75th percentiles) fibrin deposition scores were calculated for the septic and nonseptic groups and for the septic subgroups (regarding age, type of death, and postmortem findings). Differences regarding the number of animals positive for fibrin deposits among the septic subgroups were evaluated using the χ2-test, with additional Fisher's exact test for small groups. The Mann-Whitney U-test was used to compare medians between 2 subgroups, and the Kruskal-Wallis test was used to compare medians when there were 3 subgroups.

Differences regarding the presence and the amount of fibrin deposits among organ groups from septic newborn foals were evaluated using the Friedman test followed by the Wilcoxon paired test with Bonferroni's correction for multiplicity of contrasts.

After the observation of the slides but before evaluating the association with organ failure, and considering that a mild fibrin deposition (grade 1) may not be sufficient to cause organ dysfunction, a minimum cut-point of 2 for fibrin deposits was established to study the association between fibrin deposition and organ failure. Differences regarding the number of septic foals with a fibrin score ≥2 between the group of foals with organ failure and the group of foals without organ failure were studied using the χ2-test, with additional Fisher's exact test for small groups.

Results

During the study period, 104 newborn foals were admitted in the hospital with a diagnosis of septicemia based on the stated criteria, and 38 of them died or were euthanized, of which 32 were included in the study. During the study period, 6 foals died, but a postmortem examination was not performed or samples were not in adequate condition to be studied. From the 32 nonsurviving septic foals included in the study, 20 were males and 12 were females. Breed representation was as follows: Andalusian (27), crossbred (3), Arabian (1), and Menorquin (1). Regarding the 20 foals in which blood culture was carried out, 11 had positive results, with E. coli being the main bacteria isolated (n = 8) followed by Klebsiella pneumoniae (n = 2) and Pasteurella spp (n = 1). The main reason for not performing a blood culture was previous antibiotic administration by the referring veterinarian. The diagnosis of sepsis in the other 21 foals was based on sepsis score and confirmation on postmortem examination. Of these, the main diagnoses were enteritis (n = 9), omphalophlebitis or omphaloarteritis (n = 3), meningoencephalitis (n = 2), polyarthritis (n = 2), peritonitis (n = 1), and systemic septicemia (n = 4). Eighteen foals died or were euthanized within 48 hours after admission, and the remaining 14 foals were hospitalized for more than 2 days (mean, 9.4 days; range, 4–19 days). Regarding cause of death, 23/32 foals were euthanized, and the remaining 9 foals died naturally. Regarding the age of the foals at the time of death,1,24 the 1st group included foals that died or were euthanized after <24 hours of life (n = 9); the 2nd group included foals that died or were euthanized when they were 2–7 days old (n = 11), and the 3rd group included foals that died or were euthanized when they were 8–21 days old (n = 12).

Histochemical and Immunohistochemical Results in Sick Neonates and Controls

Evaluation of HE-stained tissue sections showed that some kidney samples from septic foals were congested (12/32; 37.5%) and had evidence of renal cortical necrosis with hemorrhage (5/32; 16%). The lungs of some neonates had thickened alveolar septae (9/32; 28%), patchy alveolar edema (6/32; 19%), and hemorrhage (9/32; 28%), and intra-alveolar inflammatory cell infiltration (3/32; 9%).

Antifibrin staining (IHC) in septic foals showed extensive diffuse fibrin deposition along the septae in 5/32 (16%), on alveolar capillaries in 19/32 (60%) (Fig 1) and in alveolar fluid in 5/32 (16%). Fibrin deposition also was observed in glomeruli of septic foals with obliteration of capillary architecture in 5/32 (16%) (Fig 2). Vessels occluded by fibrin clots were readily identified.

Figure 1.

 Lung: Numerous fibrin deposits (brown) in alveolar capillaries in a foal with severe septicemia, classified as grade 3; extensive diffuse fibrin deposition occludes alveolar capillaries. Immunohistochemistry, magnification × 400.

Figure 2.

 Kidney: Marked positive staining of fibrin deposits (brown) by immunohistochemistry in glomerular capillaries from a foal with severe septicemia, classified as grade 4; fibrin deposits obliterate the capillaries. Immunohistochemistry, magnification × 400.

Fibrin deposits were clearly observed in 28/32 septic newborn foals (87.5%) (median, 2; 25th–75th percentiles, 1–2.75) when using the IHC technique, from which 23/32 (72%) animals were classified grades 2–4. When using the PTAH stain, fibrin deposits were observed in 21/32 (66%) (median, 2; 25th–75th percentiles, 0–3) all of which were classified grades 2–4 (Table 1). No fibrin deposition was observed in nonseptic neonates using both techniques.

Table 1.   Number of animals classified as positive and negative for presence of fibrin deposits with phosphotungstic acid hematoxylin (PTAH) and immunohistochemistry (IHC).
 Septic Foals (n = 32)Nonseptic Foals (n = 4)
  1. The nonseptic group includes 4 sick neonate foals without septicemia.

PTAH
 Positive210
 Negative114
IHC
 Positive280
 Negative44

Fibrin Deposits in Septic Neonates Subgroups

On postmortem examination, 8/32 (25%) septic neonates had gross evidence of epicardial or subserosal pulmonary petechiations, which was considered consistent with DIC. The median (25th–75th percentiles) score for fibrin deposition was similar between the foals with multiple subserosal petechiations on postmortem examination and the foals that did not have any evidence of subserosal petechiations (Table 2), and between the foals that died naturally or were euthanized. When the amount of fibrin deposits was compared among age subgroups, no significant differences were observed.

Table 2.   Fibrin score (median, 25th–75th percentiles) in septic foals subgroups classified with phosphotungstic acid hematoxylin (PTAH) and immunohistochemistry (IHC).
 PTAHIHC
  • a

    Age of foals was recorded at the moment of death.

  • No statistical differences were observed among subgroups for both techniques.

Agea
 < 24 hours (n = 9)0 (0–2.5)2 (0.5–3.5)
 2–7 days (n = 11)2 (0–3)2 (2–3)
 8–21 days (n = 12)2 (0.25–3)2 (1–2)
Type of death
 Euthanized (n = 23)2 (0–3)2 (1–2)
 Natural death (n = 9)2 (1.5–2.5)2 (1.5–3.5)
Postmortem findings
 With serosal petechiae (n = 8)2 (0–2.75)2 (0.25–2)
 Without serosal petechiae (n = 24)2 (0–3)2 (1.25–3)

Fibrin Deposits by Organ in Septic Neonates

When analyzing the samples by organ, the kidney was the most frequently affected organ (44%) when using the PTAH stain, although the observed differences were not statistically significant. When the IHC method was used, the lung was the most frequently affected organ (97%) with significant statistical differences (P<.001) compared with kidney and liver. The median (25th–75th percentiles) score of fibrin deposition in the lung with IHC technique was 2 (1–2), which was significantly higher (P<.001) than the score of kidney (0, 0–1) and liver (0, 0–1).

Fibrin Deposits and Organ Failure in Septic Neonates

None of the septic foals showed signs of hemorrhagic diathesis (bleeding form of DIC), but 18/32 septic neonates (56%) had clinical signs consistent with organ failure (8 foals had a diagnosis of respiratory failure, 14 foals had a diagnosis of renal failure, and 4 foals had a diagnosis of both).

Regarding the 14 foals with histologic evidence of renal lesions on HE-stained sections, 6 foals were included in the renal failure group and the remaining 8 had normal renal function. Of the 21 foals that had histologic pulmonary lesions on HE-stained sections, 8 foals belonged to the respiratory failure group (with at least 1 pulmonary lesion), and in the remaining 13 foals with pulmonary lesions respiratory failure was not diagnosed.

In the septic group, 23/32 foals had a fibrin score ≥2 by the IHC method; 21/32 foals had a fibrin score ≥2 with PTAH staining, and 18/32 foals had clinical signs consistent with organ failure. Of the 21 foals with fibrin score ≥2 using PTAH, 9/21 (43%) had organ failure. When the number of foals with a fibrin score ≥2 on PTAH staining was compared between animals with organ failure and animals without organ failure, statistical differences were not observed (P= .06). Of the 23 foals with a fibrin score ≥2 using IHC, 14/23 (60%) had clinical signs consistent with organ failure. When the number of foals with a fibrin score ≥2 on IHC was compared between animals with organ failure and animals without organ failure, no difference was identified (P= .6).

Discussion

In the present report, 2 different methods to identify fibrin deposits in tissues were used and both techniques allowed clear observation of fibrin deposits in several tissues from nonsurviving septic neonates. In contrast, no fibrin deposits were observed in tissues from nonseptic newborn foals. Several authors reported the use of PTAH stain17,21 and IHC methods10,22,37 to identify fibrin deposits in humans, horses and laboratory animals with satisfactory results. Regarding the nonsurviving septic foals that were classified as positive or negative for the presence of fibrin deposits, comparison of PTAH with IHC showed an agreement of 72%, which is very similar to the value obtained in a previous study,22 in which both methods were compared in horses with colic of poor prognosis. Both the histologic and immunohistochemical methods presented in this paper had similar effectiveness in demonstrating fibrin deposits in the capillary meshwork of nonsurviving septic foals, which is similar to previous studies in adult horses with severe colic.21,22

Several studies in humans and experimental animals with DIC have demonstrated that fibrin is formed in the systemic circulation due to a hypercoagulable state, and may accumulate in peripheral capillaries of several organs, forming fibrin deposits.17,37,38 The most relevant finding of the present study is that most nonsurviving septic foals had fibrin deposits in their tissues, specially demonstrated by IHC, and systemic fibrin deposition is consistent with the postmortem diagnosis of DIC. Even considering only the nonsurviving septic foals, this finding is consistent with our clinical impression and the results of other previous in vivo studies1,24 that nonsurviving septic foals have some hemostatic abnormalities despite the paucity of reported cases in the veterinary literature. In the present study, fibrin deposits were observed in 87.5% of the nonsurviving septic foals when using the IHC technique. This percentage is slightly higher than those reported in some in vivo studies1,23 that reported a diagnosis of DIC in 50% of septic foals during hospitalization. However, a higher percentage of foals that did not survive were diagnosed with DIC. The different percentage of septic foals with DIC between the in vivo study and the present postmortem study may be explained because our septic group was composed by foals with poor prognosis that did not survive or were euthanized. Additionally, clinicopathologic criteria for the diagnosis of DIC may have a lower sensitivity than the histologic observation of fibrin deposits or the presence of fibrin deposits may be too sensitive and not very specific. Furthermore, these fibrin deposits have only been evaluated in nonsurviving foals.

The physiologic response to inflammation and sepsis includes activation of the coagulation system and inhibition of fibrinolysis. This may initiate a hypercoagulable state responsible for acute thrombosis in horses,1,39 as well as MODS and death.39,40 In this study, nonsurviving septic neonates had fibrin deposits in several tissues, which is consistent with presence of a hypercoagulable state. Although not conclusive, the fibrin deposits observed in the different organs of nonsurviving foals with septicemia may have resulted from DIC. In some reports of experimental DIC in animals, fibrin deposits have been histologically observed in the capillaries of different organs, demonstrating that fibrin deposition is one of the consequences of DIC.10,38

Several studies in humans and experimental animal models of DIC demonstrate that fibrin accumulated in peripheral capillaries produces a disturbance of microcirculation and ischemia which may cause necrosis and multiple organ failure syndrome (MOFS).10,41 MOFS is the leading cause of morbidity and mortality in patients admitted to the intensive care unit, and can be characterized by different degrees and combinations of organ dysfunction or failure.42,43 In the study reported here, the occurrence of organ failure in nonsurviving septic newborn foals was not statistically associated with the large amounts (grade ≥ 2) of fibrin deposits observed in tissues from those animals. The analysis of the data could indicate an association between organ failure and fibrin deposition (from the 18 foals with organ failure, 14 had a score value of fibrin deposition ≥2 with IHC technique), but possibly the small number of animals in each group influenced the lack of statistical association. Even without statistical differences, our results suggest a possible association between fibrin deposits and organ failure and this would be consistent with a study of human neonatal sepsis, in which coexisting shock and DIC were significantly associated with acute renal failure.44 However, in the present study, there were some foals with fibrin deposition in their organs that did not have signs of organ dysfunction. Possibly, these foals died or were euthanized after the occurrence of fibrin deposition, but before tissue ischemia or necrosis and subsequent organ dysfunction could be identified by clinicopathologic examination. During MOFS, different organs are affected in a complex pathophysiologic process at different time points45 and a given evaluation can miss the full extent of organ dysfunction sustained by the patient, leading to an underestimation of the cumulative insult suffered. Mortality due to MOFS depends on the number of organs failing,46,47 on the severity of the dysfunction or failure, on treatments received, on the particular combination of failing organs,46,48 and on the duration.46,47 On the other hand, there were some septic nonsurviving foals that had signs of organ dysfunction without a large amount of fibrin deposition. The organ dysfunction observed in these animals may have been caused by other factors associated with sepsis. A recent study of septic patients reported the occurrence of MODS without DIC in 54% of the patients.15 In the present study, the diagnosis of organ failure was limited by available information, because it was based on retrospective clinical data. Also, the small number of animals included in this study made it difficult to draw conclusions about organ failure. Thus, additional prospective studies should be performed to assess the relationship between MODS and DIC.

Changes in the hemostatic profile of septic foals within the 1st weeks of life have been documented in vivo,1,23,24 showing alterations consistent with a hypercoagulable state at the age of 2–7 and 8–21 days. Therefore, the effect of age in the population studied here also was considered. When using the IHC method, the most affected subgroups were foals <24 hours old and those 2–7 days old (median fibrin deposition 2 [range, 0.5–3.5] and 2 [range, 2–3], respectively). Previous studies reported that during the 1st day of life, equine neonates have some hemostatic immaturity which might contribute to a delay in fibrin formation.49 However, the results reported here seem to demonstrate that fibrin formation and deposition in different tissues of septic foals at age <24 hours was similar to that observed in older septic foals, revealing the capability of foals <24 hours of age to form fibrin. Findings observed in previous studies1,24 could be explained by a dysfunction or inhibition of the fibrinolytic system in septic foals <24 hours of age. To avoid misclassification in age categories, the subgroups were created before looking at the descriptions in previous studies.23,24 However, bias may have been introduced by the influence of humane reasons in the choice of time of euthanasia. There likely were some foals for which certain treatments (eg, plasma transfusion, parenteral nutrition) were withheld due to financial considerations, although most owners were advised of treatment costs before or at the time of admission.

In a previous report on neonatal septic foals,24 hemorrhage was considered more apparent than thrombosis at postmortem examination. However, in our study, microthrombosis was observed in more animals than was hemorrhage and petechiation. This observation is not consistent with the previous study24 although 8 animals had petechiations on epicardial or pulmonary surfaces. The foals with evidence of subserosal petechiation on postmortem examination had a median score of fibrin deposition lower than that recorded in the other animals. This observation may mean that the higher amount of fibrin deposition does not correspond necessarily to larger macroscopic evidence of hemostatic dysfunction. Despite the derangements of fibrin deposition in the study reported here, bleeding or thrombus formation was not observed in the foals by the senior clinician during their hospitalization.

In bacterial sepsis, the lung is the organ that usually is affected first and most severely.50 When the type of organ affected was analyzed in the samples from the septic neonates processed by IHC technique, the lung was the most frequently affected organ. This result may indicate that sepsis can cause a generalized systemic hemostatic dysfunction, with subsequent lesions in several organs related to the fibrin deposition in capillaries. The higher incidence of fibrin deposition in the lung could be caused by the fact that the lung may act as a filter for fibrin microthrombi formed in the venous vasculature,19,37 but the more extensive fibrin deposition in the lung also may be related to the percentage of cardiac output passing through the lungs or other local or systemic factors.

This study had some limitations. The study design was based on foals with severe septicemia that died naturally or were euthanized, which may have introduced some bias by the specific characteristics of this population. This limitation does not allow us to draw conclusions about the surviving foals, because they were not studied. Nevertheless, previous in vivo studies23,24 showed activation of coagulation and fibrinolysis in similar populations that survived, which potentially indicates fibrin formation. On the other hand, some nonseptic diseases also could be associated with DIC, which necessitates strict selection of septic cases. In addition, the study was dependent on a small number of nonseptic neonatal foals that did not survive and that could be included as controls, which represented a substantial limitation. To control for the dynamic changes that occur in the hemostatic and fibrinolytic system during the 1st month of life, few nonseptic foals were used as internal controls. Because this nonseptic control group was small, it was impossible to statistically compare data in the septic group with the nonseptic group, as previously described in vivo.24 Moreover, in the animals used in this study, clinicopathologic data concerning the diagnosis of DIC in vivo were not available and it was impossible to assess the correlation between DIC in vivo and postmortem fibrin deposition.

In conclusion, our results demonstrate that by using specific techniques (ie, PTAH or IHC) fibrin deposition can be easily observed in tissues of almost all nonsurviving foals with sepsis. This fibrin deposition observed in septic foals is supportive of the presence of DIC, and may be related to organ dysfunction and failure. The fibrin deposition that occurs in the organs of some foals with septicemia may contribute to the high mortality rates in these animals, and might be decreased by the administration of antithrombotics at prophylactic dosages. Prospective studies should be performed to demonstrate the association among DIC, fibrin deposits, and MODS and the effect of anticoagulant therapy in these animals.

Footnotes

aSHANDON – Hipercenter XP, Thermo Electron Corporation, Waltham, MA

bPolyclonal Rabbit Anti-Human Fibrinogen, Dakocytomation, Denmark

cSPSS version 11.5 for Windows software, SPSS Inc, Chicago, IL

Acknowledgment

We thank the personnel of the Equine Teaching Hospital of Barcelona for their support and sample collection.

Ancillary