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Keywords:

  • horse;
  • colic;
  • large colon volvulus;
  • ultrasound;
  • multiple organ dysfunction syndrome;
  • oedema

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Manufacturer's address
  9. References

Reasons for performing study: The post operative response of the large colon wall after a surgically corrected large colon volvulus (LCV) has not been investigated.

Objectives: To use transabdominal ultrasound to monitor the post operative change in large colon wall thickness following surgical correction of LCV.

Hypothesis: A prolonged period to colon wall involution is correlated with an increased rate of post operative morbidity and mortality.

Methods: A prospective clinical study including horses that presented to the North Carolina State University Veterinary Teaching Hospital for colic between September 2006 and March, 2008, had surgically diagnosed and corrected LCV (at least 360°) without resection and recovered from anaesthesia. Ultrasound of the ventral large colon was performed at the time of anaesthetic recovery and every 6–8 h until the colon wall returned to normal thickness (≤5 mm). Outcome was evaluated using a one-way ANOVA to compare average time to colon wall involution between: 1) survivors and nonsurvivors; and 2) horses that developed multiple organ dysfunction syndrome (MODS) during the post operative period and those that recovered without evidence of MODS.

Results: Sixteen horses that recovered without evidence of MODS had a significantly shorter period to colon wall involution (≤5 mm) compared to those diagnosed with MODS (mean ± s.e. 19.6 h ± 2.5 and 39.7 h ± 6.7 respectively, P = 0.006). There was no significant difference in mean period to colon wall involution between survivors and nonsurvivors (26.2 ± 4.9 and 33.2 ± 7.8 h, respectively).

Conclusions: A shorter time to colon wall involution was associated with decreased post operative morbidity in horses presented for surgical correction of large colon volvulus without resection.

Potential relevance: Ultrasonographic monitoring of colon wall involution after surgical correction of LCV may aid in identifying those cases at risk of MODS. Further investigation of colon wall involution time using a larger number of horses is warranted.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Manufacturer's address
  9. References

Large colon volvulus (LCV) is one of the most fatal forms of colic in the horse with published survival rates ranging from 35–65% (Barclay et al. 1980; Harrison 1988; Snyder et al. 1989) and equally high rates of post operative morbidity (Hughes and Slone 1998; Proudman et al. 2005). Depending on the duration and severity of intestinal strangulation, the large colon experiences varying degrees of primary ischaemic and secondary reperfusion injury. Although large colon resection or euthanasia is clearly indicated in cases of obvious ischaemic necrosis, most horses with large colon volvulus have no clear line of demarcation between viable and nonviable intestine, making the site or even need for large colon resection unclear (Southwood 2004).

The gold standard for intraoperative assessment of colon viability and short-term survival remains histological evaluation of pelvic flexure biopsy (van Hoogmoed et al. 2000), but few surgeons find this diagnostic test clinically applicable. As a result, numerous other pre- and intraoperative criteria, including duration of colic, heart rate, packed cell volume (PCV), total protein (TP), plasma lactate, colonic luminal pressure, surface oxygen tension and visual assessment of intestinal colour, oedema, motility and blood flow, have been used to predict colonic viability and patient survival, with varying degrees of accuracy (Hughes and Slone 1998; van Hoogmoed and Snyder 1998; Cook 2001; Southwood et al. 2002; Johnston et al. 2007). Very little work has been done to investigate correlations between post operative parameters and outcome.

Previous work by Pease et al. (2004) demonstrated that a diagnosis of LCV can be made preoperatively using ultrasound to identify oedema in the wall of the ventral colon; however, the post operative response of the large colon wall after surgically corrected LCV has not been investigated. Increased large colon wall thickness, whether the result of oedema and/or inflammation, represents altered gastrointestinal barrier function and is the probable cause of endotoxin absorption in these patients, both preand post operatively. Ultrasound offers a direct, noninvasive and readily available method for monitoring colon wall thickness.

The purpose of this study was to determine which measured variables, if any, were correlated with post operative morbidity and mortality in horses following correction of large colon volvulus≥360°. Parameters evaluated included ventral colon wall thickness, time to colon wall involution (colon wall thickness ≤5 mm), blood lactate (BL), PCV, TP and post operative diagnosis of multiple organ dysfunction syndrome (MODS).

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Manufacturer's address
  9. References

Database

This prospective study was performed at the North Carolina State University Veterinary Teaching Hospital between September 2006 and March 2008. The study population consisted of mature horses (age >2 years) that presented for colic, had a surgical diagnosis and correction of LCV (>360°) without resection and recovered from anaesthesia. This project was approved by the North Carolina State University Animal Care and Use Committee and client permission was obtained for patient participation in the study. Participation in the study had no impact on client financial responsibility nor on post operative management, which remained at the discretion of the attending clinician and owner.

Abdominal ultrasonography

Following anaesthetic recovery, colon wall measurements were taken immediately post operatively and then every 6–8 h until the colon wall thickness returned to normal (≤5 mm). Colon wall thickness was measured via ultrasonographic evaluation of the ventral abdomen as described by Pease et al. 2004. Images were obtained using a Megas ES1 ultrasound machine and an 8–5 MHz curvilinear probe. Prior to evaluation, the ventral abdomen was prepared just caudal to the xiphoid using isopropyl alcohol and acoustic coupling gel. Because the site had been clipped routinely for surgical preparation, additional clipping was not required. If a protective belly bandage was in place, it was removed to obtain the ultrasound images and then reapplied immediately; however, the ventral large colon was often successfully imaged without removal of the abdominal bandage. At least 3 longitudinal images of the ventral colon wall were obtained approximately 2 cm caudal to the xiphoid (with the depth set at 10–12 cm), either by a veterinarian or trained technician, and were stored digitally. These images and measurements were evaluated and confirmed by a board certified radiologist (A.P.P.). Measurements were obtained using electronic callipers that were part of the built in ultrasound software and the thickest value measured was used for statistical evaluation.

Additional parameters

Blood was collected via jugular vein catheter every 6–8 h for measurement of PCV, TP and BL. Additionally, each case had a complete examination (heart rate, temperature, mucous membrane colour, capillary refill time, peripheral pulse pressure and visual pain score) recorded every 6–8 h throughout hospitalisation.

Outcome evaluation

The medical record was examined by one of the authors (M.K.S.) for diagnosis of MODS. MODS was identified in any horse having 2 or more of the following during the post operative period: neurological dysfunction detected as stupor and incoordination, haemodynamic dysfunction detected as congested mucus membranes with increased capillary refill time, haemostatic dysfunction detected as jugular vein thrombosis and/or platelet count <100 × 10 9/l, renal dysfunction as indicated by creatinine >20 mg/l, intestinal dysfunction identified by nasogastric reflux >3 l and decreased motility on abdominal ultrasound, hepatic dysfunction indicated by bilirubin >60 mg/l, clinical signs of laminitis and respiratory distress (persistent tachypnoea and nostril flare) (Mackay 2008). M.K.S. was also present for daily post operative monitoring and treatment of horses included in the study; therefore relevant physical examination findings were recorded in the medical record and haematological and biochemical data were evaluated at least every 72 h. General abdominal ultrasound was performed at the time of ventral colon wall measurement and evidence of ileus was noted in the medical record. Additional ultrasound was performed following colon wall involution only if clinically indicated (i.e. inappetance, colic, fever).

Statistical analysis

Outcome was evaluated using a one-way ANOVA with a post hoc pairwise multiple comparison procedure (Holm-Sidak method) to compare average time to colon wall involution between: 1) horses discharged from the hospital (survivors) and horses that died or were subjected to euthanasia following recovery from anaesthesia (nonsurvivors); and 2) horsesethat developed MODS during the post operative period (complicated) and those that recovered without evidence of MODS (uncomplicated).

Packed cell volume, TP and BL were compared between the designated groups of horses at 0, 6, 12 and 18 h post operatively using a one-way ANOVA with a post hoc multiple comparison procedure (Fisher LSD method). Correlation between colon wall thickness and PCV, TP and BL was assessed by determining Pearson's correlation coefficient. For each statistical test, a power analysis was performed. For all tests, a value of P<0.05 was considered statistically significant.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Manufacturer's address
  9. References

Clinical findings

A total of 16 horses (11 mares, 3 geldings, 2 stallions) fit the inclusion criteria during this time period. Breeds included one Arabian, 2 Warmbloods, 2 Friesians, one Walking Horse and 10 Quarter Horses. Sixty-eight percent of horses (11/16) survived to discharge and 56% (9/16) recovered without complication. Of the 7 horses diagnosed with MODS during the post operative period, 2 survived to discharge; all horses that recovered without complication survived to discharge. Post operative complications experienced in this sample population were similar to those in previous reports (van Hoogmoed et al. 2000; Johnston et al. 2007) and commonly included small intestinal ileus, laminitis, jugular vein thrombosis, thrombocytopenia and respiratory distress. Less commonly reported complications identified on post mortem examination included 2 cases of focal large colon necrosis secondary to venous thrombosis and 2 cases of fungal pneumonia with concurrent evidence of pulmonary thromboembolism. Table 1 provides a summary of the diagnosed surgical lesion, clinical outcome and large colon viability. Large colon viability was based on survival or histopathological assessment at necropsy by board certified pathologists. Tissues were considered nonviable if loss of glandular epithelium and disruption of tissue architecture was extensive (van Hoogmoed et al. 2000).

Table 1. Summary of surgical diagnosis and clinical outcome of 16 horses undergoing surgical correction for large colon volvulus. A complicated outcome was defined as a horse that developed multiple organ dysfunction syndrome (MODS) during the post operative period, whereas an uncomplicated recovery as a horse that recovered without evidence of MODS. Nonsurvivors were horses that died or were subjected to euthanasia following recovery from general anaesthesia. Survivors were horses that survived to hospital discharge. Colon viability was based on survival or histopathology at the time of necropsy
Degree volvulusNo. of horsesOutcomeViable colon
UncomplicatedComplicatedSurvivorNonsurvivor
  • *

    One nonsurvivor unavailable for post mortem examination.

360972819
540101010
720624334*

Ultrasound

All horses had increased colon wall thickness (>5.0 mm) at the time of anaesthetic recovery. Mean ± s.e. time to colon wall involution in horses that recovered without evidence of MODS was significantly shorter (19.6 ± 2.5 h; Fig 1) than in horses diagnosed with MODS (39.7 ± 6.9 h; Fig 2: P = 0.006). There was no significant difference in time to colon wall involution between survivors and nonsurvivors, (26.2 ± 4.9 h and 33.2 ± 7.8 h, respectively) (Fig 3). However, the power of the performed test was 0.048, which is below the desired power of 0.8. Colon wall thickness was not significantly different between groups at any time point.

image

Figure 1. Percutaneous abdominal ultrasound images obtained just caudal to the xiphoid. These images demonstrate the rapid decrease in colon wall oedema seen in a horse that experienced no post operative complications. a) Colon wall thickness was 20.3 mm immediately post operatively (t=0 h); b) colon wall thickness was 2.8 mm 18 h after recovery from anaesthesia (t=18 h). Serosal and mucosal borders are depicted by white and black arrows, respectively.

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image

Figure 2. Percutaneous abdominal ultrasound was used to monitor changes in colon wall thickness post operatively. These images demonstrate the persistent colon wall oedema seen in a horse that experienced multiple organ dysfunction postoperatively. a) Colon wall thickness was 9.9 mm immediately postoperatively (t=0 h); b) the colon wall remained thickened at 12.3 mm 24 h after recovery from anaesthesia (t=24 h). Serosal and mucosal borders are depicted by white and black arrows, respectively.

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image

Figure 3. Time (mean±s.e.) to colon wall involution (5 mm thickness) in horses recovering from surgical correction of large colon volvulus. Horses that recovered without evidence of MODS had a significantly shorter time to colon wall involution (19.6±2.5 h) compared to horses diagnosed with MODS post operatively (39.7±6.9 h) (*P=0.006). There was no significant difference in mean time to colon wall involution between survivors and nonsurvivors, (26.2±4.9 and 33.2±7.8, h, respectively).

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Serum biochemical parameters

Mean ± s.e. TP concentration was significantly (P<0.02) lower at 12 and 18 h post operatively in horses with a complicated recovery (43.9 ± 1.9 g/l and 45.3 ± 2.2 g/l, respectively) compared to horses with an uncomplicated recovery (53.6 ± 3.7 g/l and 57.0 ± 4.0 g/l, respectively). TP was also significantly (P<0.05) lower at 0, 12 and 18 h post operatively in nonsurvivors (38.7 ± 3.3 g/l, 42.8 ± 1.5 g/l and 43.0 ± 2.1 g/l, respectively) compared to horses that survived to hospital discharge (50.2 ± 2.2 g/l, 50.4 ± 2.6 g/l and 54.0 ± 2.7 g/l, respectively: Fig 4). Preoperatively, mean BL concentration was significantly (P < 0.02) higher in nonsurviving horses (10.14 ± 2.3 mmol/l) compared to survivors (6.68 ± 1.36 mmol/l). Preoperative BL concentration was also significantly (P<0.001) higher in horses diagnosed with MODS post operatively (10.87 ± 1.72 mmol/l) compared those that recovered without complications (5.34 ± 1.21 mmol/l: Fig 5). PCV was significantly (P = 0.002) higher preoperatively in nonsurvivors compared to survivors, with a mean of 0.54 l/l compared to 0.43 l/l respectively. TP and colon wall thickness were negatively correlated (r =−0.342) in horses diagnosed with MODS post operatively (P = 0.0246: Fig 6). There was no correlation between colon wall thickness and TP in horses that did not develop MODS post operatively (r = 0.124). No other correlation was found between colon wall thickness and the additional measured variables.

image

Figure 4. Total protein concentration in horses diagnosed with large colon volvulus which was corrected surgically. Total protein concentration was significantly (*P < 0.02) lower at 12 and 18 h post operatively in horses diagnosed with MODS postoperatively (complicated) compared to horses that recovered without evidence of MODS (uncomplicated). Total protein concentration was significantly (*P < 0.05) lower at 0, 12 and 18 h post operatively in horses that died or were subjected to euthanasia after recovery from general anaesthesia (died) compared to horses that survived to hospital discharged (survived).

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image

Figure 5. Blood lactate concentration in horses diagnosed with large colon volvulus, which was surgically corrected. Blood lactate concentration was significantly (*P < 0.001) increased preoperatively in horses diagnosed with MODS post operatively (complicated) compared to horses that recovered without evidence of MODS post operatively (uncomplicated) and horses that died or were subjected to euthanasia during the post operative period (died) compared to horses that survived to hospital discharge (survived) (*P<0.02).

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image

Figure 6. Correlation between colon wall thickness and TP concentration in horses following surgical correction of large colon volvulus. Total protein and colon wall thickness were negatively correlated (r=−0.342) in horses diagnosed with MODS post operatively (P=0.0246).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Manufacturer's address
  9. References

Previous reports have described transabdominal ultrasound as a useful imaging modality for identifying changes in colon wall thickness in the horse due to right dorsal colitis, intestinal lymphoma and large colon volvulus (Scharner et al. 2002; Jones et al. 2003; Pease et al. 2004; Sheats et al. 2008). To gain a better understanding of changes in the intestinal wall of the large colon following naturally occurring strangulation, ultrasound was used to monitor colon wall thickness from the ventral acoustic window, as described by Pease et al. (2004). It was hypothesised that the oedema/inflammation, represented by increased colon wall thickness, in the intestinal wall of a viable colon would resolve more rapidly than in a severely compromised or nonviable colon, and that a shorter period to colon wall involution would correlate with a higher rate of survival.

The study population demonstrated similar survival and complication rates to previously published reports, with 68% surviving to discharge and 56% recovering without complication (Barclay et al. 1980; Harrison 1988; Snyder et al. 1989; Hughes and Slone 1998). Although specific treatment decisions were at the discretion of the attending clinician, routine post operative management of horses diagnosed with LCV included 5–7 days of i.v. antibiotic drugs, anti-endotoxic and anti-inflammatory therapies and hetastarch. Horses' feet were routinely iced in an effort to prevent laminitis.

Of the 5 horses that did not survive to discharge, 4 were subjected to euthanasia and one died. Reasons for euthanasia included severe laminitis, disseminated intravascular coagulation, acute respiratory distress syndrome and sepsis unresponsive to therapy; these horses had evidence of multi-organ disease, with no horse subjected to euthanasia due to financial constraints alone. Of the 4 horses available for post mortem examination, 2 had focal ischaemic necrosis due to colonic vein thrombosis and 2 had a viable colon wall based on histopathology; demonstrating that colon viability was not the only variable responsible for poor outcome in this population of horses. This observation is consistent with previous reports in which horses diagnosed with LCV were subjected to euthanasia due to severe post operative complications, despite evidence of colonic viability on either intraoperative pelvic flexure biopsy or post mortem examination (van Hoogmoed et al. 2000; Johnston et al. 2007).

All horses that experienced colon wall involution within 18 h of surgery survived to discharge; however, overall, there was no significant difference in mean time to colon wall involution between survivors and nonsurvivors. This lack of statistical significance is probably due to the limited number of horses that did not survive to discharge (n = 5) and is reflected in the low power of the performed test. Therefore, it is possible that a difference could not be detected. The time to colon wall involution in horses that recovered without complication was found to be significantly shorter than in horses diagnosed with MODS. Based on this result one might hypothesise that time to colon wall involution is determined by the severity of initial injury and that increased colon wall thickness in a minimally compromised colon wall simply represents transmural oedema that resolves rapidly following manual correction. It also follows that minimal mucosal disruption results in a decreased risk of ongoing endotoxemia and secondary complications during recovery. However, one must also consider that the time to colon wall involution is not determined by the severity of the initial insult alone, but also by systemic parameters such as colloid osmotic pressure, vascular leak syndrome and degree of sepsis/ endotoxaemia.

While it is tempting to conclude, based on the results of this study, that delayed colon involution causes sepsis and multiple organ dysfunction due to increased gastrointestinal barrier permeability, it is important to consider the alternative: that sepsis and multiple organ dysfunction cause systemic changes that result in delayed colon healing. In future studies, comparison of pelvic flexure biopsy at the time of surgery, to colon wall involution time, could provide a better understanding of the relationship between initial morphological injury and post operative response of the colon wall.

This study failed to document any correlation between absolute colon wall thickness and outcome and it was commonly observed that colon wall thickness was highly variable among horses. This finding was consistent with the conclusion reported by van Hoogmoed et al. (2000), that oedema scores on intraoperative pelvic flexure biopsies are not useful for predicting survival in horses with naturally occurring large colon volvulus.

Although ultrasound was routinely performed preoperatively in order to diagnose large colon volvulus (Pease et al. 2004), these measurements were not recorded or evaluated based on outcome due to variability in case position during ultrasound (i.e. standing vs. lateral recumbency). Prior to performing this project, 5 horses that had surgically corrected large colon displacement were evaluated post operatively using transabdominal ultrasound. No increased wall thickness was found, confirming that surgical manipulation does not routinely result in detectable colon wall oedema.

Hypoproteinemia is a commonly reported complication in horses following surgical correction of LCV and, if persistent, is a poor prognostic indicator (Hughes and Slone 1998; Southwood et al. 2002). In the current study population, TP was significantly lower post operatively in the nonsurvivor and complicated recovery groups. Delayed colon wall involution was also more common in these 2 groups and may represent either the cause of the hypoproteinaemia (due to severe protein losing enteropathy) or, alternatively, the consequence of low oncotic pressure. One must also consider the potential confounding effect of administration of synthetic colloids (i.e. hetastarch) on serum TP. Because hetastarch expands the intravascular volume by increasing the plasma oncotic pressure, it causes a dose dependent decrease in TP (as well as PCV) ranging from a 20% decrease in healthy horses to 34% in sick horses (Schusser et al. 2007). Perhaps as a result of this confounding factor, little correlation was found between TP and colon wall thickness, except in horses diagnosed with MODS post operatively. In these horses TP and colon wall thickness were negatively correlated, meaning that as TP increased, colon wall thickness decreased. An increase in TP has been documented during resolution of colon wall inflammation in horses with right dorsal ulcerative colitis (Jones et al. 2003). An alternative explanation for the change in TP is that horses diagnosed with MODS were more likely to receive repeated plasma transfusions, causing an artificially elevated serum protein. With so many questions remaining about the role of serum protein and oncotic pressure in formation and/or resolution of post operative oedema in horses diagnosed with LCV, future studies should assess oncotic pressure directly by measuring plasma colloid osmotic pressure in addition to TP.

In agreement with a previous report (Johnston et al. 2007), this study found that BL levels were significantly higher preoperatively in nonsurvivors compared to survivors. While it is certain that ischaemia of the ascending colon contributes to the severely elevated BL concentration documented in these horses, elevated BL probably also reflects the severity of haemodynamic shock and systemic tissue hypoxia experienced secondary to dehydration, endotoxaemia and sepsis (Weil and Afifi 1970). Although Johnston et al. (2007) reported that horses with a nonviable ascending colon had greater increases in BL concentrations than horses with a viable colon, they readily commented that it is impossible to conclude that colonic ischaemia was the cause of hyperlactataemia. In the present study, BL was also significantly higher preoperatively in horses diagnosed with MODS compared to those that recovered without complication. The definition of MODS was based on evidence of physiological derangement in 2 or more organ systems (Mackay 2008). Specific complications seen in the present study population included persistent tachycardia and tachypnoea, altered mucus membrane colour and decreased capillary refill time, ileus with nasogastric reflux, laminitis, jugular and colonic vein thrombosis, diarrhoea and pulmonary thromboembolism. The cause of MODS in these cases is unknown. Certainly the most widely accepted cause is activation of the systemic inflammatory cascade due to endotoxaemia and sepsis, as loss of the mucosal barrier can lead to bacterial translocation and absorption of endotoxin into circulation (Wilson et al. 1994). Alternatively, it may be that horses with the greatest degree of haemodynamic shock at the time of presentation are at greatest risk of developing MODS due to altered organ perfusion and secondary systemic inflammatory changes. Probably it is a combination of both the severity of initial cardiovascular compromise and the degree of endotoxaemia and sepsis experienced.

Based on necropsy findings of nonsurvivors, normal colon wall thickness (≤5 mm) as identified ultrasonographically was associated with normal colon wall thickness and histopathological colon viability at the measured location. However, normal colon wall thickness at one location did not predict overall colon viability, as evidenced by 2 nonsurviving horses that experienced successful colon wall involution at the measured site, but were ultimately diagnosed with colonic vein thrombosis with focal segments of ischaemic necrosis and thickening of the left ventral colon at post mortem examination. Interestingly, in both cases, these regions of increased colon wall thickness were identifiable ante mortem using transabdominal ultrasound. While these cases identify a potential limitation in using ultrasound to assess colon viability at one site post operatively; they also give an example of its potential usefulness in identifying nonviable colon.

Based on the present study results, ultrasonographic monitoring of colon wall involution time in horses recovering from manual correction of LCV may aid in identifying cases at increased risk of MODS. This technique offers clinicians an additional way to monitor these horses and provides an owner with information that may help is assessing the financial implications of intensive post operative care. To determine if survivability can be assessed using transabdominal ultrasound of the colon wall, further investigation into the predictive value of colon wall involution time, among larger numbers of horses, is warranted.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Manufacturer's address
  9. References

The authors would like to thank the United States Equestrian Federation for funding this study.

Manufacturer's address

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Manufacturer's address
  9. References

1 Biosound, Universal Ultrasound, Bedford Hills, New York, USA.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Manufacturer's address
  9. References

Author contributions The initiation, conception and planning for this study were by A.P., V.C. and M.K.S. All authors contributed to the pathology and writing. Execution was by A.P., V.C., M.K.S. and S.J., with statistics by A.P., V.C., M.K.S. and A.B.