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Keywords:

  • colic;
  • D-dimer;
  • subclinical coagulopathies;
  • TAT levels

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Statistical Methods
  6. Results
  7. Discussion
  8. Acknowledgment
  9. Footnotes
  10. References

Objective: The objective of this study was to evaluate coagulation profiles in horses with surgical treatment of large colon volvulus (LCV), and determine if an association exists between hemostatic dysfunction and outcome.

Design: Prospective clinical investigation from February to December 2000.

Setting: Large animal intensive care unit in a veterinary teaching hospital.

Interventions: Blood was collected from horses intra-operatively, 24, and 48 hours following surgical treatment for LCV.

Measurements: Coagulation profiles, thrombin–antithrombin (TAT) levels, and D-dimer concentrations were determined for each time point. The number of tests abnormal in the standard coagulation profile, defined as the degree of hemostatic dysfunction, was determined for each horse for the duration of the study period. The association between each test and outcome, as well as the degree of hemostatic dysfunction for each horse and outcome, was determined using univariate analysis and logistic regression. TAT levels and D-dimer concentrations were compared to the results of the standard coagulation profile and to patient outcome using univariate analysis and logistic regression.

Main results: Seventy percent of horses evaluated with surgical treatment of LCV had evidence of hemostatic dysfunction (3/6 tests abnormal). Only 18% of those patients had clinical signs recognized by the attending clinician as a coagulopathy. There was an association between the development of a coagulopathy and outcome, with horses with 4/6 tests abnormal being more likely to be euthanized, and those with 3/6 tests abnormal having a prolonged hospital stay. Platelet count, prothrombin time, and TAT levels may be helpful in predicting outcome in horses with LCV.

Conclusions: Hemostatic function should be evaluated in horses with surgical treatment of LCV to detect subclinical coagulopathies and direct subsequent intervention.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Statistical Methods
  6. Results
  7. Discussion
  8. Acknowledgment
  9. Footnotes
  10. References

The survival rate reported in the literature for horses with surgical treatment of large colon volvulus (LCV) is variable1,2, ranging from 36 to 71%,3 depending on the degree of rotation, duration of the lesion, and severity of vascular compromise. For the purposes of this study, LCV is defined as a 360° twist of the large colon at the cecocolic junction. Annually, approximately 40 horses are surgically treated for LCV in our clinic. A review of medical records in the year prior to the initiation of this study revealed a survival rate of 49%. This is slightly higher compared to a study conducted at this facility in the early 1980s, which reported a survival rate of 34.7%.4

Horses presenting with LCV demonstrate various degrees of abdominal discomfort and clinical signs consistent with hypovolemic shock, based on the severity and duration of the lesion. Regional variation of clinical signs on presentation may exist, and reflect specific characteristics of the referral population that impact duration of the lesion and corresponding vascular compromise. Failure to correct the lesion in a timely fashion results in further exacerbation of hypovolemic shock and, in cases of severe vascular compromise over a long duration, the development of endotoxemia. Currently, large colon resection is not typically performed in our hospital in cases of 360° volvulus at the cecocolic junction. Clinical post-operative observations in these horses include tachycardia, tachypnea, hyperemic mucous membranes, hypothermia or hyperthermia, leukopenia, hypoproteinemia, respiratory compromise, and death. The presumed mechanism of this clinical picture is massive absorption of endotoxin from a vascularly compromised large colon, reperfusion injury, and severe tissue trauma. Many of these horses progress from endotoxemia to systemic inflammatory response syndrome (SIRS); coagulopathies and organ dysfunction have been observed clinically. Colonic thrombosis has been observed at post-mortem examination in horses with surgical treatment for LCV as long as 8 days post-operatively, raising the concern that hypercoagulability and/or fibrinolytic dysfunction may be playing a role in patient morbidity and mortality.

Analysis of hemostatic parameters has been reported in horses with colic,5–7 revealing that horses with the most severe intestinal ischemia had decreased survival and coagulation profiles interpreted as hypercoagulable.8 Coagulation profiles associated with a specific surgical lesion such as LCV have not been reported to our knowledge, although the vascular injury associated with this diagnosis is well documented.9

Currently in our hospital, coagulation profiles are not commonly performed unless the animal has overt clinical signs of disseminated intravascular coagulation (DIC). Recent literature in the human critical care realm has introduced the concept of subclinical or compensated DIC, in which early identification of hypercoagulability or hemostatic dysfunction, followed by aggressive intervention resulted in improved outcome.10 The purpose of this prospective clinical trial was to determine the incidence of hemostatic dysfunction in equine patients with surgical treatment of LCV, and investigate if an association exists between abnormal coagulation profiles and outcome. Thrombin–antithrombin (TAT) levels are not currently a part of our clinical hemostatic evaluation. An ELISA test measuring TAT complexes has been evaluated both in normal horses11 and horses with acute gastrointestinal disorders, and was deemed to be a good assay to diagnose early hypercoagulable states in horses with the most severe forms of colic.12 D-dimer concentrations are also not routinely performed at our hospital, although one report suggests that knowledge of D-dimer concentrations may predict non-survival and DIC in horses with colic.13 TAT and D-dimer concentrations were determined to be useful in the diagnosis of pre-DIC in human studies evaluating laboratory tests predicting the development of a fulminant coagulopathy.14 TAT and D-dimer concentrations have also been useful in characterizing a relationship between the degree of hemostatic dysfunction and SIRS in human patients.15 Based on this information, we chose to evaluate TAT and D-dimer concentrations in addition to standard coagulation profiles in horses with surgical treatment of LCV. The goal of this study was to determine the predictive value of abnormalities of these coagulation tests with respect to patient outcome.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Statistical Methods
  6. Results
  7. Discussion
  8. Acknowledgment
  9. Footnotes
  10. References

Horses

This prospective study was designed to include approximately 30 horses with surgical treatment of LCV presenting to the George D. Widener Large Animal Hospital from February to December 2000. Diagnosis of LCV was made at the time of surgery, and defined as a 360° rotation of the large colon at the junction of the cecum and colon. Thirty-four horses presented to the hospital for surgical treatment of LCV during the defined study period. Horses with less than a 360° rotation, or failure to collect a sample at the appropriate time during the horse's hospitalization were criteria for exclusion from the study, resulting in the evaluation of coagulation profiles of 27 horses surgically treated for LCV. All horses in the non-survivor group were euthanized due to poor prognosis (complete colon devitalization) or a dramatic decline in clinical progression. No horses died naturally, or were euthanized due to financial reasons in circumstances in which the attending clinician predicted a fair or reasonable prognosis. Horses that were euthanized prior to the 48-hour point were included in the study, provided they had complete coagulation profiles, TAT and D-dimer concentrations collected at the appropriate times, allowing temporal comparisons to be made.

Sample collection

Samples were collected intra-operatively prior to surgical correction of the volvulus, 24 hours post-operatively, and 48 hours post-operatively. A previous study indicated that the most dramatic changes in hemostatic parameters were observed in this time period.2 Blood was collected from a single venipuncture site and placed in respective tubes for sample analysis. Standard coagulation profiles consisted of platelet count, fibrinogen, fibrin-degradation products (FDPs), antithrombin activity (AT), prothrombin time (PT), and activated partial thromboplastin time (PTT). TAT and D-dimer concentrations were also measured to further characterize hemostatic dysfunction, evaluate the degree of correlation with the standard coagulation profile, and determine the predictive value with respect to outcome.

Data collection

Data collected from medical records included signalment, temperature, pulse, respiration on initial presentation, duration of clinical signs, time to surgery, and specific descriptions of surgical and post-operative management. No horse received heparin therapy as part of its peri-operative care, and horses receiving greater than 10 mL/kg of hydroxyethyl starcha would have been excluded from the study (no horse met this criterion for exclusion).

Initial laboratory findings reported were taken on admission to the hospital and included blood pH, blood lactate concentration, plasma creatinine concentration, packed cell volume (PCV), and plasma total solids concentration (TS). Blood lactate concentration and blood pH were determined with a blood gas analyzer;b both parameters were measured directly via electrodes following standard protocol according to the manufacturer's recommendations. Blood gas analysis (whole blood lactate and pH) was performed only at the time of admission and immediately post-operatively. Post-operative PCV and TS were also obtained from the blood gas sample obtained immediately post-operatively. Plasma creatinine concentration was determined by the use of a colorimetric assay on a chemistry analyzer,c and was evaluated only at the time of admission. The heart rate was averaged over a 24-hour period post-operatively, and the mean duration of tachycardia (defined as greater than 48 beats per minute (bpm) was determined. Complications encountered during the surgery and post-operative period were also recorded, and a successful outcome was defined as discharge from the hospital as opposed to death or euthanasia.

Sample analysis

Blood for platelet counts was placed in a K3 EDTAd tube and analyzed immediately on an automated analyzer.e Samples with low platelet counts or a questionable distribution curve were rechecked manually. Blood for fibrinogen, PT, PTT, and AT was placed in a buffered citrate sodium tube (3.8%)d and processed immediately by centrifugation at 1,240×g followed by plasma aspiration. All samples were placed in appropriate tubes, stored at −20°C, and evaluated within 2 weeks of sample collection. Plasma fibrinogen and PT levels were simultaneously determined on an automated analyzerf using lyophilized rabbit brain calcium thromboplastin as an activator of the extrinsic pathway of coagulation. Activated partial thromboplastin time was determined by incubation of recalcified sample plasma with rabbit brain phospholipid and a particulate activator to activate the intrinsic coagulation pathway. Antithrombin activity was measured by chromogenic assay on an automated analyzer,f in which plasma is incubated in the presence of factor Xa and excess heparin, followed by quantification of residual factor Xa. All samples were collected and handled according to published laboratory recommendations16 and the manufacturer's instructions.17

Blood for FDP analysis was placed in tubes containing Bothrops atrox venom with soybean trypsin inhibitor.g Serum FDPs were measured using a commercially available slide latex-agglutination testh containing antibodies produced in sheep against the human D and E fragments. Slide evaluation resulted in a score of <10, 10–20, or >40 μg/dL, and for ease of statistical analysis, these scores were converted to 0, 1, or 2, respectively.

Blood for TAT and D-dimer analysis was also placed in a buffered citrate sodium tube (3.8%)d upon collection, centrifuged at 1380×g, and plasma aspirated and placed in respective tubes for storage at −70°C until tested within 6 months from collection as a group. TAT levels were determined using a sandwich-type ELISA test. An optical density vs. TAT concentration (μg/L) standard curve was generated using the standards provided in the ELISA kit, and human normals (provided) and 10 normal horse samples were also run simultaneously. All samples, including normal horses and standards, were run in duplicate. TAT levels for normal horses were determined to be 1.54±0.054 μg/L.

D-dimer concentration was determined via an automated latex enhanced turbidimetric immunoassay on an automated analyzer.f Latex particles coated with a monoclonal antibody highly specific for the D-dimer domain were mixed with sample plasma in the presence of a reaction buffer, resulting in particle agglutination. The degree of agglutination was determined by measuring the decrease in transmitted light at 405 nm. Thirty normal horses were used as controls. The mean D-dimer concentration in this normal population was determined to be 224±185 ng/mL. This is consistent with published normals6 for D-dimer concentrations in the horse using a semi-quantitative turbidimetric assay.j

Test results for the standard coagulation profile were identified as abnormal if the following criteria were met: platelet count <100,000/μL (reference range 100,000–300,000 cells/μL), PT≥12.5 seconds (reference range 8–10 seconds), PTT ≥50 seconds (reference range 29–40 seconds), AT <110% (reference range 150–200%), fibrinogen <150 mg/dL (reference range 200–375 mg/dL), FDPs >10 μg/dL (reference range <10 μg/dL).

These values were determined to be abnormal based on the evaluation of coagulation profiles routinely performed on normal horses in our clinical laboratory. The PT, PTT, and AT had to vary minimally by 20% from standards determined in the hospital clinical laboratory to be considered abnormal. In order for horses to be defined as having hemostatic dysfunction (subclinical DIC), at least 3 tests had to be abnormal at a single sample time. Single tests abnormal at different sample times did not result in a ‘cumulative’ degree of hemostatic dysfunction, and horses demonstrating such laboratory results were not defined as having abnormal hemostasis.

Statistical Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Statistical Methods
  6. Results
  7. Discussion
  8. Acknowledgment
  9. Footnotes
  10. References

Analysis of individual coagulation profile parameters and outcome

All statistical analyses were performed using a statistical software program. Prior to the analysis, coagulation profile variables were dichotomized into normal and abnormal ranges according to previously described parameters defined by our clinical laboratory. χ2 analysis was used to determine any preliminary associations between individual coagulation profile parameters (PT, PTT, AT, platelet count, fibrinogen, FDPs) and outcome. In cases in which numbers were small (cells <5), the Fisher's exact test was substituted. To quantify established associations, univariate logistic regression was applied and odds ratios are reported with their respective P values. A significance level of P≤0.05 was used for all statistical tests used throughout the study, including further analysis to quantify associations with univariate logistic regression.

Analysis of degree of hemostatic dysfunction and outcome

Prior to the analysis, the coagulation profile was assessed to determine the number of abnormal tests, resulting in dichotomization of data into normal and abnormal ranges. Horses having 3/6, and 4/6 abnormal tests were evaluated with respect to outcome. χ2 analysis was used to determine any preliminary association between the number of abnormal tests (degree of hemostatic dysfunction) and patient outcome, again relying on Fisher's exact test in situations where small sample size was a feature. Univariate logistic regression was used to quantify the above-determined associations, providing odds ratios and associated P values. Evaluation of associations between the development of a coagulopathy (3/6 abnormal tests) in the post-operative period (at sample times 24 or 48 hours), or having an abnormal coagulation profile at more than one time point, and duration of hospitalization were performed using the Kruskal–Wallis test. This non-parametric test was chosen because of small sample size.

Evaluation of lactate as a predictor of outcome

Univariate logistic regression was used to evaluate the association between blood lactate and outcome. Odds ratios are reported with their P values to provide quantitative information on the degree of the association. Sensitivity and specificity were generated using Receiver Operator Characteristics (LROC functionk). Specificity was plotted against sensitivity, and from this plot the lactate level associated with the highest specificity/sensitivity with respect to outcome was determined. Sensitivity, specificity, positive, and negative predictive values are reported.

Evaluation of TAT and D-dimer concentrations: associations with abnormal coagulation profiles and outcome

Determination of D-dimer concentrations in 30 normal horses was performed in our clinical laboratory as previously described. Examination of the data distribution of the study population indicated that a normalizing transformation for the data was the 1/√D-dimer. Summarizing this transformation for D-dimer concentrations in normal horses resulted in a 95% range of 0.077±0.042. Transformation and examination of the study population D-dimer concentrations indicated that all horses in the study had abnormal D-dimer concentrations. Univariate logistic regression was used to determine any association between transformed D-dimer concentration and outcome. Robust regression was used where indicated by small sample size. The Kruskal–Wallis test was also used to determine if there was any difference between D-dimer concentrations in survivor vs. non-survivor groups. This approach was also used to determine if an association existed between increased D-dimer and the development of a coagulopathy. Again, a significance level of P≤0.05 was used for all statistical analyses.

TAT levels for the 10 normal control horses were consistent with previously reported values.11 Logistic regression was used to evaluate associations between mean TAT and the degree of hemostatic dysfunction and outcome, and odds ratios and associated P values are reported, with robust measures employed in most cases due to small sample size. Sensitivity and specificity were generated using Receiver Operator Characteristics (LROC functionk), and from this plot the TAT level associated with the highest specificity/sensitivity with respect to outcome was determined. Sensitivity, specificity, and positive and negative predictive values are reported.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Statistical Methods
  6. Results
  7. Discussion
  8. Acknowledgment
  9. Footnotes
  10. References

From February to December 2000, 32 horses presented to the George D. Widener Large Animal Hospital for surgical treatment of LCV. Coagulation profiles were successfully collected at the appropriate time intervals in 27 of these horses, excluding those horses that had <360° rotation of the large colon at the cecocolic junction. The mean age was 8.5±4.9 years, with a breed distribution reflective of the hospital population, consisting predominantly of thoroughbreds and standardbreds. Females were over-represented, with 19/27 or 70.4% of horses presenting for surgical treatment of LCV. Six mares were peri-parturient, defined as being either within 2 weeks pre- or post-partum. Initial physical examination and laboratory findings of note included tachycardia, hemoconcentration, azotemia, and metabolic acidosis (Table 1). As best as could be determined from medical history, horses had clinical signs of abdominal discomfort for an average of 11.2±9.5 hours, with a median of 8.5 hours (range 2–48 hours). The average time to surgery was 2.25±2.0 hours, with a median time of 1 hour (range 0.5–16 hours).

Table 1.    Initial physical examination and laboratory parameters of horses presenting to the George D. Widener Large Animal Hospital for surgical treatment of large colon volvulus. All parameters were collected within 15 minutes of arrival.
ParameterSurvivor (n=12)Non-Survivor (n=15)
MeanMedian (range)MeanMedian (range)
  • *

    16/27 horses had creatinine levels >1.8 mg/dL (normal range 1.0–1.8).

Heart rate57±20 bpm51 bpm (44–100)84±33bpm84 bpm (36–160)
PCV44±6%42% (39–53)54±10%54% (37–72)
Total solids concentration5.9±0.8 g/dL6.2 g/dL (4.1–6.8)5.9±0.6 g/dL6.0 g/dL (4.6–7.2)
Creatinine*2.2±1.0 mg/dL1.63±mg/dL (1.2–2.9)2.6±1.2 mg/dL2.4±mg/dL (1.5–5.9)
pH7.40±0.067.40 (7.30–7.47)7.37±0.087.40 (7.13–7.44)
Lactate5.1±3.4 mmol/L5.2 mmol/L (0.7–10)6.8±2.2 mmol/L6.4 mmol/L (3.2–11.8)
Duration of clinical signs7.5±3.1 hours7.5 hours (2–12)13.3±12 hours10 hours (3–48)
Time to surgery3.3±4.5 hours1.4 hours (0.4–16)1.12±0.5 hours1 hour (0.3–1.75)

In all cases, horses received intravenous fluid therapy, intravenous pre-operative anti-microbial therapy, and analgesics as indicated, and were placed under general anesthesia. Anesthesia was maintained in a semi-closed circle system with oxygen and inhalant anesthetic, with mechanical ventilation used as indicated. The mean surgery time in horses that were recovered was 1.9±0.6 hours. An enterotomy was performed in 6/27 horses treated surgically. No large colon resections were performed. Fifteen horses received an average of 4 L of colloid therapy consisting of synthetic colloidsa (n=5), plasma (n=8), or a combination of both (n=2). Twenty horses were placed in the recovery stall, and 19 recovered successfully from general anesthesia and were transferred to the intensive care unit for post-operative management. Post-operative heart rate, duration of tachycardia, PCV, total solids concentration, pH, and blood lactate are reported in Table 2. One horse was euthanized in the recovery stall following rapid cardiovascular collapse secondary to endotoxemia and shock. One horse was euthanized in the ICU prior to the 24-hour sample due to rapid clinical decline characterized by severe tachycardia, tachypnea, metabolic and respiratory acidosis, and severe endotoxemia. Five horses were euthanized prior to the 48-hour sample (n=14 surviving at the 48-hour sample), demonstrating similar clinical progressions, and 2 horses were euthanized after 48 hours. One of these 2 horses had repeated episodes of colic upon reintroduction of feed, and the other had a second surgery and a large colon resection, but was euthanized shortly after recovery due to failure to improve.

Table 2.    Post-operative physical examination and laboratory parameters of horses surgically treated for large colon volvulus. Blood pH and lactate were sampled immediately post-operatively, and PCV, TS, and heart rate were evaluated over the first 24 hours post-operatively.
ParameterSurvivor (n=12)Non-Survivor (n=15)
MeanMedian (range)MeanMedian (range)
Heart rate56±7.8 bpm56 bpm (48–68)83±15 bpm83 bpm (62–110)
Duration of tachycardia18±17 hours12 hours (12–48)39.4±22 hours30 hours (12–72)
PCV39±7%39% (28–55)45±9%47% (30–57)
Total solids concentration4.5±1 g/dL4.3 g/dL (3.9–7.2)3.9±0.6 g/dL4 g/dL (2.5–4.7)
pH7.38±0.307.40 (7.13–7.45)7.28±0.057.29 (7.18–7.41)
Lactate2.5±1.2 mmol/L2.6 mmol/L (0.6–4)5.7±2.1 mmol/L5.6 mmol/L (4–9.9)

After a median hospital stay of 9 days (range 4–13, 25%=0.5; 75%=8), 12 horses were successfully discharged. This results in a survival rate of 44% for horses surgically treated for LCV. Post-mortem results were available for 8 horses that were euthanized. Relevant post-mortem findings included pathologic evidence of severe enterocolitis, devitalized colon, possible thrombosis of colonic vasculature, severe fibrinous peritonitis, and gross evidence suggestive of DIC in 2 horses. Complications associated with the post-operative period included thrombophlebitis (4/19), incisional infection (3/19), and moderate abdominal discomfort (8/19). Several horses had increased digital pulses, but there were no cases in which a radiographic diagnosis of laminitis was confirmed.

Evaluation of coagulation profiles

Of the 27 horses that met criteria for inclusion in the study, 19 (70% of horses evaluated) had a coagulation profile consisting of 3/6 tests abnormal at a minimum of one sample time. Nine out of the 27 (33% of the study population) horses had a coagulation profile consisting of 4/6 tests abnormal at least one sample time.

An association was identified between the development of an abnormal coagulation profile and clinical outcome. Horses with a post-operative coagulation profile consisting of 4/6 tests abnormal were much more likely (OR, 47:1) to be euthanized in the clinic (P=0.004). Horses which were euthanized intra-operatively due to severe colon devitalization (n=7) were 18 times more likely to have 4/6 coagulation tests abnormal at the time of lesion identification and evaluation (P=0.02). An association between persistent, less severe hemostatic dysfunction and duration of hospital stay was also observed, with horses with 3/6 tests abnormal at more than one sample time having a significantly longer hospital stay (P=0.03). There was no association between an abnormal coagulation profile and other complications observed (P=0.40).

Evaluation of individual laboratory tests as predictors of outcome

Coagulation profile results in survivor and non-survivor groups are presented in Table 3, and individual laboratory tests were evaluated as predictors of outcome. There was no association between hypofibrinogenemia and patient outcome (P=0.26). Non-survivors did not exhibit a fibrinogen increase in response to surgical intervention. FDPs were increased in both survivor and non-survivor groups, and no significant association was observed between an abnormal level (out of relative range defined by the clinical laboratory) and patient outcome (P=0.43). The mean antithrombin activity decreased over time in patients surgically treated for LCV, but there was no statistically significant distinction between abnormally low antithrombin activity and horses that did not survive (P=0.32).

Table 3.    Coagulation profile results for survivors and non-survivors surgically treated for large colon volvulus (means±SEM). Reported P values are for all sample times, *P=0.01, †P=0.04, ‡P=0.06, demonstrating an association between abnormal individual coagulation tests and non-survival.
Test (normal range)Survivor (n=12)Non-survivor (n=15)
Sample timeSample time
Intra-op24 hours48 hoursIntra-op24 hours48 hours
  • *

    P =0.01.

  • P =0.04.

  • P =0.06.

Platelet count (×1000 cells/μL)* (100–300)128.5±4.7130±5.3131.7±9.1101.1±7.884.3±10.692.4±16.8
Prothrombin time (s) (8–10)10.1±0.411.4±0.411.2±0.314.0±2.214.1±1.413.7±.9
Partial thromboplastin time (s) (29–40)50.9±3.560.5±5.153.6±4.358.5±5.780.1±7.589.4±19.2
Antithrombin (% activity) (150–220)135±11134±9120±11122±8109±4108±6
Fibrin degradation products (score 0–2) (0)0.5±0.11.1±0.21.4±0.20.7±0.21.2±0.31.5±0.7
Fibrinogen (mg/dL) (175–350)242±24415±41568±46179±152.31±213.05±98

Activated partial thromboplastin time was increased over our normal reference range for most horses treated surgically for LCV, with no horse euthanized post-operatively having a PTT <50 seconds, and only 4 surviving horses having a normal PTT at all 3 sample times. However, data were only suggestive of an association between prolonged PTT and non-survival, with P=0.06 (OR, 9:1; CI, 0.86–93.6).

Horses that developed prolonged PT in the post-operative period were significantly more likely to be euthanized (P=0.04), with mean PT remaining prolonged 48 hours post-operatively in non-survivors compared to survivors (Figure 1). The development of thrombocytopenia post-operatively was also significantly associated with poor outcome (P=0.01), with mean platelet count declining steadily over time in the non-survivor group (Figure 2).

image

Figure 1.   Prothrombin time (secs) intra-operatively, 24 hours, and 48 hours following surgical treatment for large colon volvulus.

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image

Figure 2.   Platelet count (cells/ul) intra-operatively, 24 hours, and 48 hours following surgical treatment for large colon volvulus.

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Blood lactate levels were consistently measured on the horse's arrival and immediately post-operatively, prior to the entrance to the recovery stall. There was no association between pre-operative lactate levels and outcome (P=0.40). Increased lactate levels post-operatively were associated with non-survival (P=0.01), with horses with lactate levels greater than 3.7 mmol/L being more likely to be euthanized (sensitivity 92.8%, specificity 80%, positive predictive value (PPV) 86.7%, negative predictive value (NPV) 88.9%).

TAT and D-dimer analysis

The median D-dimer concentration in horses at the time of diagnosis (intra-operative) was 902 ng/mL (range 308–22,125, 25%=630.5 ng/mL, 75%=1,540 ng/mL). D-dimer concentrations increased 24 hours post-operatively, rising to a median 4,950 ng/mL (range 1,480–24,000, 25%=3,583, 75%=6,025), and continued to increase at the 48-hour sample, with a median of 6,125 ng/mL (range 1,355–23,500, 25%=4,125, 75%=8,025). Based on normal values determined prior to evaluation of the study population, all horses in the study had an abnormal D-dimer concentration. Normalizing transformation resulted in mean±SE values of 0.03±0.01, 0.015±0.004, and 0.014±0.006 ng/mL for the intra-operative, 24-hour, and 48-hour samples, respectively. No association between increased D-dimer concentration and poor outcome (P=0.20), or increased D-dimer and the development of an abnormal coagulation profile (P=0.46) was observed. Comparison of D-dimer concentrations in survivor vs. non-survivor groups revealed no significant differences in the groups (P=0.11); however, a wide range for D-dimer concentration was observed in the non-survivor group, as opposed to the survivor group.

Horses with a TAT level greater than 7.07 μg/L at any sample point were significantly more likely to have 4/6 tests abnormal on the standard coagulation profile (P=0.02). As a predictor of hemostatic dysfunction, TAT levels were 75% sensitive and 65% specific, with a PPV=30%, and an NPV=93%. Horses that were euthanized had mean±SE TAT levels of 8.26±2.3 μg/L intra operatively, 9.90±1.74 μg/L 24 hours post-operatively, and 5.01±1.3 μg/L 48 hours post-operatively. Horses that survived surgical treatment had mean±SE TAT levels of 6.49±1.9 μg/L intra-operatively, 4.24±0.54 μg/L 24 hours post-operatively, and 2.72 ±0.27 μg/L 48 hours post-operatively. Horses with increased TAT levels at the time of diagnosis (intra-operative sample) were significantly more likely to have an abnormal coagulation profile (P=0.001), and be euthanized (P=0.01). There was a significant association between an abnormal TAT level and euthanasia in the post-operative period, at both the 24- and 48-hour sample times (P≤0.01 and ≤0.04).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Statistical Methods
  6. Results
  7. Discussion
  8. Acknowledgment
  9. Footnotes
  10. References

Survival for horses with surgical treatment of LCV in our hospital population has been low, although relatively similar with other university referral centers. Our geographical location in combination with our client profile may result in the rather long duration of clinical signs observed in this study prior to arrival at the hospital, although this time has decreased slightly compared to a previous study evaluating LCV at this facility.4 Initial physical examination and laboratory parameters observed in horses surgically treated for LCV are consistent with previously reported studies.3,4 Post-operatively, these patients undergo a severe inflammatory response and endotoxemia, and often death or euthanasia results despite intensive management following surgical correction. As a result, these horses are among the most challenging critical care cases facing the equine gastrointestinal surgeon. The development of coagulation dysfunction as a result of gastrointestinal strangulation18–20 and severe endotoxemia is well documented,21,22 and DIC as a component of uncontrolled inflammation has received much attention in the human literature.23,24 It has been these concepts, in combination with the focus on early identification of subclinical DIC in the human and veterinary literature,10,25,26 that provided the impetus for this study. It was our goal to evaluate coagulation parameters in a specific patient population, determine what tests were most useful, and explore if coagulation dysfunction had an effect on outcome.

Seventy percent of horses (19/27) surgically treated for LCV over approximately one year exhibited some degree of coagulation dysfunction (3/6 tests abnormal) at least once during the study. Additionally, approximately one-third of these patients (9/27) had 4/6 tests abnormal. In only 2 cases did the attending clinician comment in the medical record that the horse may be exhibiting overt clinical signs that could be associated with a coagulopathy, and neither horse had any tests ordered assessing coagulation parameters. In this patient population, horses with coagulation dysfunction were more likely to have either a prolonged hospital stay or be euthanized, depending on the severity of the coagulopathy. It, therefore, is indicated to evaluate coagulation parameters in horses treated surgically for LCV. Coagulation dysfunction can be as difficult to diagnose as it is to treat, and post-mortem confirmation can also be challenging due to lysis of thrombi prior to pathological evaluation.27 Coagulation testing ante mortem is, therefore, necessary both to characterize the severity of the ongoing inflammatory process further and to direct any additional clinical intervention that might improve outcome.

The degree of inflammatory response and the resulting effect on coagulation may vary with respect to the inciting event. Coagulation tests that may be helpful in characterizing the coagulopathy resulting from a neoplastic process may not necessarily be the same as those characterizing a coagulopathy resulting from trauma or exposure to endotoxin. In this study, levels of FDPs were not useful in predicting outcome. Similarly, D-dimer concentrations were increased in the entire population, and were not correlated with an abnormal coagulation profile or the likelihood of euthanasia. This may relate to the actual inciting cause of the systemic inflammation, which is severe vascular compromise of the large colon due to strangulation. Both FDPs and D-dimer concentrations attempt to characterize the fibrinolytic component of the hemostatic system. It is possible in this patient population that increased levels are reflective of the regional microthrombi in the large colon as a result of strangulation, and the body's attempt to restore blood flow, as opposed to an accelerated state of systemic clot formation and degradation. The previous study evaluating D-dimer as a predictor of outcome in horses with gastrointestinal disease did not characterize lesions as strangulating vs. non-strangulating, but horses successfully treated with medical therapy alone did have significantly lower D-dimer concentrations.13

The consistent development of a low fibrinogen was also not observed in this patient population, and was not often a characteristic of the coagulopathy observed. It has been stated that hypofibrinogenemia rarely occurs in horses that develop DIC.28 Interestingly, survivors had a relevant fibrinogen increase over time in the post-operative period (567±45.6 mg/dL at 48 hours) compared to non-survivors, who had fibrinogen levels in the normal range (305±98 mg/dL at 48 hours). This ‘lack of fibrinogen response’ has been observed in other studies involving evaluation of coagulation parameters in horses with inflammation of the large colon,26 and may warrant further investigation. It is possible that fibrinogen trends may be more clinically relevant than hypofibrinogenemia in evaluating equine patients at risk for developing a coagulopathy.

Data were suggestive, but not significant, for a prolonged PTT, an indicator of the function of the intrinsic pathway, being associated with a poor outcome. Careful analysis of PTT in this patient population revealed the test to be abnormal in both survivor and non-survivor groups. In previous studies evaluating hemostatic parameters in horses with colic, PTT was one of the most commonly abnormal tests, and not reliable as a predictor of outcome.5,6 One study reported that PTT was likely to be prolonged in horses with strangulating gastrointestinal lesions.8 Results of the study presented here correlate well with those previous findings, and may lead one to conclude that PTT is commonly increased in horses with severe gastrointestinal lesions, but may be too sensitive to predict outcome. Further evaluation of a larger sample size may lead to a critical range for prolonged PTT that could be associated with poor outcome.

Prothrombin time, an indicator of the function of the extrinsic and common pathways, was useful in predicting outcome in horses undergoing surgical treatment of LCV. Prolongation of PT has been observed in horses with acute gastrointestinal lesions in other studies,6 and although prolonged PT has not been as commonly observed as prolonged PTT, it has been a better predictor of outcome.5 PT assessment may be useful in evaluating patients with LCV, providing evidence for intervention aimed at restoring hemostatic function, as well as information regarding patient outcome.

The development of thrombocytopenia in patients with LCV at any time point was associated with poor outcome. These findings correlate well with studies in human ICUs in which thrombocytopenia on admission, or blunted platelet response during hospitalization in critically ill patients, was associated with non-survival.29,30 Certainly, thrombocytopenia can be related to surgical intervention, but in this study population there were no large colon resections and only 6 enterotomies were performed. It would seem that the thrombocytopenia could be related either to the severity of damage to the large colon, or the degree of ensuing inflammatory response. Pseudothrombocytopenia can also be observed when EDTA has been used as the anticoagulant.31 Horses with thrombocytopenia in this study had further confirmation via a manual platelet count. This study supports the evaluation of platelet counts in horses with surgical treatment of LCV. Platelet function has also become an area of interest in the critically ill human patient.32 The evaluation of platelet function in horses with LCV may be helpful in further characterizing the platelet response to strangulating gastrointestinal disease.

Increased TAT levels were significantly correlated with an abnormal coagulation profile and poor outcome in horses in this study population. The generation of thrombin is the final step of the coagulation cascade, and antithrombin is the main inhibitor of thrombin through the formation of inactive TAT complexes. The TAT complex has a relatively short half-life;33 therefore, the level of TAT complexes should be reflective of the degree of recent coagulation activation. TAT levels in this study correlated with previous work reporting an association between increased TAT levels and outcome in horses with acute gastrointestinal disease.12 From a practical standpoint, the test currently available has limited clinical usefulness, as it is quite expensive, requires a plate reader of appropriate wavelength, and is most useful if large numbers of samples are to be analyzed. The development of a ‘stall-side’ ELISA for TAT complexes would be quite useful, but, to the authors' knowledge, does not exist at the time of this writing.

Horses with a lactate level greater than 3.7 mmol/L post-operatively were more likely to be euthanized. It is unclear whether this is reflective of the development of a coagulopathy or merely reflective of the lesion severity and resulting inflammatory response. An increased lactate level may result from hypovolemia, hypotension, endotoxemic shock, or a coagulopathy, and could be indicative of poor end-organ perfusion. Hyperlactatemia has been associated with poor outcome in dogs with surgical treatment of gastric dilatation-volvulus.34 An increased lactate post-operatively in horses with surgical treatment of LCV should alert the clinician to possible poor outcome, and may indicate the need for aggressive intervention.

The identification of horses at risk for the development of coagulopathic dysfunction, particularly in patients with no overt clinical signs of DIC, is important for both an improved ability to manage our most critical cases and for the evolution of large animal critical care in general. In horses presenting with a high suspicion of LCV, treatment should focus on rapid surgical intervention and restoration of physiologic hemostasis, and evaluation of coagulation parameters should be an important component of patient monitoring. Treatment of coagulation dysfunction may include, but are not limited to, heparin, low-molecular-weight heparin, aspirin, or replacement therapy consisting of fresh frozen plasma. Prospective clinical trials evaluating treatment efficacy in this particular patient group are needed, as mortality is high due to the severity of the inciting gastrointestinal lesion, and additional therapeutic intervention in such cases is quite costly. Treatment should also focus on removing the inciting cause, and large colon resection or enterotomy (evacuation of endotoxin) may be indicated in this patient population. Hemostatic evaluation in other critically ill equine patients in the future may help identify those at risk for a coagulopathy, as well as provide additional knowledge as to the usefulness of specific coagulation tests.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Statistical Methods
  6. Results
  7. Discussion
  8. Acknowledgment
  9. Footnotes
  10. References

The Raymond C. Firestone Fund, Kennett Square, PA, provided funding for this study.

Footnotes

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Statistical Methods
  6. Results
  7. Discussion
  8. Acknowledgment
  9. Footnotes
  10. References

a6% Hetastarch, Abbott Laboratories, North Chicago, IL.

bNOVA Stat Profile M, Nova Biomedical Corp, Waltham, MA.

cKodak Eckachem 250 Analyzer, Kodak Co., Rochester, NY.

dVacutainer, BD Vacutainer, Preanalytical Solns., Franklin Lakes, NJ.

eAbbott Celldyne 3500, Abbott Laboratories, Abbott Park, IL.

fACL 6000 Analyzer, Instrumentation Laboratories, Lexington, MA.

gFDP tubes, BD Vacutainer Systems, Belliver Ind. Est., Plymouth, UK.

hThrombo-Wellcotest, Murex Laboratories, Norcross, GA.

jNycoCard D-dimer, Nycomed Pharma AS, Oslo, Norway.

kSTATA version 7.0, Stata Corporation, College Station, TX.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Statistical Methods
  6. Results
  7. Discussion
  8. Acknowledgment
  9. Footnotes
  10. References
  • 1
    Huskamp B. The diagnosis and treatment of acute abdominal conditions in the horse: the various types and frequency as seen at the Animal Hospital in Hoochmoor. Proc Equine Colic Res Symp 1982;261272.
  • 2
    Fischer AT, Meagher DM. Strangulating torsions of the equine large colon. Compend Contin Educ Pract Vet 1986; 8 (1): S25S30.
  • 3
    Snyder JR, Pascoe JR, Olander HJ, et al. Strangulating volvulus of the ascending colon in horses. J Am Vet Med Assoc 1989; 195 (6): 757764.
  • 4
    Harrison IA Equine large intestinal volvulus, a review of 124 cases. Vet Surg 1988; 17 (2): 7781.
  • 5
    Johnstone IB, Crane S. Haemostatic abnormalities in horses with colic – their prognostic value. Eq Vet Journal 1986; 18 (4): 271274.
  • 6
    Welch RD, Watkins JP, Taylor TS, et al. Disseminated intravascular coagulation associated with colic in 23 horses (1984–1989). J Vet Int Med 1992; 6 (1): 2935.
  • 7
    Collatos C, Barton MH, Prasse KW, et al. Intravascular and peritoneal coagulation and fibrinolysis in horses with acute gastrointestinal tract diseases. J Am Vet Med Assoc 1995; 207 (4): 465470.
  • 8
    Prasse KW, Topper MJ, Moore JN, et al. Analysis of hemostasis in horses with colic. J Am Vet Med Assoc 1993; 203 (5): 685693.
  • 9
    Snyder JR, Pascoe JR, Olander HJ, et al. Vascular injury associated with naturally occurring strangulating obstructions of the equine large colon. Vet Surg 1990; 19 (6): 446455.
  • 10
    Wada H, Wakita Y, Nakase T, et al. Outcome of disseminated intravascular coagulation in relation to the score when treatment was begun. Thromb Haemost 1995; 74 (3): 848852.
  • 11
    Topper MJ, Prasse KW, Morris MJ, et al. Enzyme-linked immunosorbent assay for thrombin–antithrombin III complexes in horses. Am J Vet Res 1996; 57 (4): 427431.
  • 12
    Topper MJ, Prasse KW. Use of enzyme-linked immunosorbent assay to measure thrombin–antithrombin III complexes in horses with colic. Am J Vet Res 1996; 57 (4): 456462.
  • 13
    Sandholm M, Vidovic A, Puotunen-Reinert A, et al. D-dimer improves the prognostic value of combined clinical and laboratory data in equine gastrointestinal colic. Acta Vet Scand 1995; 36 (2): 255272.
  • 14
    Wada H, Sakuragawa N, Mori Y, et al. Hemostatic molecular markers before the onset of disseminated intravascular coagulation. Am J Hematol 1999; 60: 273278.
  • 15
    Garcia-Fernandez N, Montes R, Purroy A, et al. Hemostatic disturbances in patients with systemic inflammatory response syndrome (SIRS) and associated acute renal failure (ARF). Thromb Res 2000; 100: 1925.
  • 16
    National Committee for Clinical Laboratory Standards. Collection, Transport, and Processing of Blood Specimens for Coagulation Testing and Performance of Coagulation Assays, 3rd edn. Villanova, PA: NCCLS; 1991.
  • 17
    AOL 7000 Operator Manual. Lexington, MA: Instrumentation Laboratory. Rev. 3, pp 8.1–8.3, Jan 2001.
  • 18
    Pablo LS, Purohit RC, Teer PA, et al. Disseminated intravascular coagulation in experimental intestinal strangulation obstruction in ponies. Am J Vet Res 1983; 44 (11): 21152122.
  • 19
    Kawcak CE, Baxter GM, Getzy DM, et al. Abnormalities in oxygenation, coagulation, and fibrinolysis in colonic blood of horses with experimentally induced strangulation obstruction. Am J Vet Res 1995; 56 (12): 16421650.
  • 20
    Moore JN. Endotoxemia: part II, biological reactions to endotoxin. Compend Contin Educ Pract Vet 1981; 10: S392S399.
  • 21
    Paloma MJ, Paramo JA, Rocha E. Endotoxin-induced intravascular coagulation in rabbits: effect of tissue plasminogen activator vs urokinase on PAI generation, fibrin deposits and mortality. Thromb Haemost 1995; 74 (6): 15781582.
  • 22
    Warr TA, Rao LVM, Rapaport SI. Disseminated intravascular coagulation in rabbits induced by administration of endotoxin or tissue factor: effect of anti-tissue factor antibodies and measurement of plasma extrinsic pathway inhibitor activity. Blood 1990; 75: 14811489.
  • 23
    Marshall JC. Inflammation, coagulopathy, and the pathogenesis of multiple organ dysfunction syndrome. Crit Care Med 2001; 29 (7): S99S10.
  • 24
    Bull BS, Bull MH. Hypothesis: disseminated intravascular inflammation as the inflammatory counterpart to disseminated intravascular coagulation. Proc Natl Acad Sci 1994; 94: 81908194.
  • 25
    Wada H, Minamikawa K, Wakita Y, et al. Hemostatic study before onset of disseminated intravascular coagulation. Am J Hematol 1993; 43: 190194.
  • 26
    Dolente BA, Wilkins PA, Boston RC. Clinicopathologic evidence of disseminated intravascular coagulation in horses with acute colitis. J Am Vet Med Assoc 2002; 220 (7): 10341038.
  • 27
    Kojima M, Shimamura K, Mori K, et al. A histological study of microthrombi in autopsy cases of DIC. Bibl Haematol 1983; 49: 95106.
  • 28
    Morris DD. Recognition and management of disseminated intravascular coagulation in horses. Vet Clin North Am: Eq Pract 1988; 4 (1): 115143.
  • 29
    Nijsten MW, Ten Duis HJ, Zijlstra JG, et al. Blunted rise in platelet count in critically ill patients is associated with worse outcome. Crit Care Med 2000; 28 (12): 38433846.
  • 30
    Akca S, Haji-Michael P, De Mendonca A, et al. Time course of platelet counts in critically ill patients. Crit Care Med 2002; 30 (4): 753756.
  • 31
    Hinchcliff KW, Kociba GJ, Mitten LA. Diagnosis of EDTA-dependent pseudothrombocytopenia in a horse. J Am Vet Med Assoc 1993; 203 (12): 17151716.
  • 32
    Vincent JL, Yagulshi A, Pradier O. Platelet function in sepsis. Crit Care Med 2002; 30 (5): S313S317.
  • 33
    Pizzo SV. Serpin receptor1: a hepatic receptor that mediates the clearance of antithrombin III–proteinase complexes. Am J Med 1989; 87 (3): B10SB14S.
  • 34
    De Papp E, Drobatz KJ, Hughes D. Plasma lactate concentration as a predictor of gastric necrosis and survival among dogs with gastric dilatation-volvulus: 102 cases (1995–1998). J Am Vet Med Assoc 1999; 215 (1): 4952.