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Keywords:

  • Contrast;
  • Doppler;
  • Feline;
  • Pancreatitis;
  • Ultrasonography

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. References

Background: The diagnosis of feline pancreatic disease is difficult, because clinical abnormalities and routine noninvasive diagnostic tests are unreliable.

Objective: The purpose of this study was to investigate by Doppler ultrasonography if vascularity and blood volume differs in the otherwise ultrasonographically normal and diseased feline pancreas.

Animals: Thirty-six client owned cats.

Methods: The pancreas was examined with B-mode and contrast-enhanced color and power Doppler ultrasonography. Doppler images were analyzed with a computer program: parameter fractional area represents a vascularity index and color-weighted fractional area assesses blood volume.

Results: Based on the B-mode findings, the pancreas was considered normal in 11 clinically healthy cats and diseased in 25 cats of which 4 were clinically healthy and 21 had clinical signs consistent with pancreatic disease. Histologic or cytologic samples were taken in all diseased pancreata. Fifteen samples were of diagnostic quality: purulent or mixed cellular inflammation (8), nodular hyperplasia (4), and neoplasia (3) were identified. Vascularity and blood volume for all Doppler methods was significantly higher in cats with pancreatic disease. Significantly higher Doppler values were detected with power Doppler than with color Doppler, and with postcontrast color and power Doppler than with precontrast Doppler technologies.

Conclusion: Contrast-enhanced Doppler ultrasonography appears feasible in the feline pancreas. Significant differences were found between normal cats and those with evidence of pancreatic pathology. Further studies are needed to evaluate its use for the differentiation of pancreatic disorders and in cats suspected to have pancreatic disease but without B-mode ultrasonographic changes of the pancreas.

Feline pancreatitis, once believed to be rare, is being recognized with increased frequency. The antemortem diagnosis of feline pancreatic diseases is difficult. It requires a combination of clinical signs, physical examination findings, high serum feline pancreatic lipase immunoreactivity concentrations (fPLI), and changes on abdominal ultrasonography consistent with pancreatic disease.1 Key clinical signs commonly seen in humans and dogs with pancreatitis, such as vomiting and abdominal pain, are reported infrequently in cats, and the most commonly reported signs are anorexia and lethargy.2–5 Furthermore, serum amylase and lipase activities, useful for the diagnosis of pancreatitis in humans and dogs, have been shown to lack sensitivity and specificity for pancreatitis in the cat.1,6 In particular, the diagnosis is problematic in cats with less severe pancreatitis, because of the waxing and waning nature of disease, the lack of consistent ultrasonographic changes, and decreased sensitivity of fPLI as a consequence of less enzyme release.7,8 Various diagnostic imaging methods have been investigated for use in the antemortem diagnosis of feline pancreatic disease. B-mode ultrasonography used alone is not very sensitive in detecting abnormalities of the pancreas, and a variety of changes have been reported in cats with pancreatitis, including a normal appearance of the pancreas.3,4,9 The reported sensitivity of abdominal ultrasonography ranges from 20 to 67%.3,4,7 Common ultrasonographic findings in feline pancreatic disease include diffuse hypoechogenicity, hyperechogenicity or heterogeneity, enlargement, irregular borders, nodules, masses, pseudocysts, or abscesses. Concomitant findings may be surrounding hyperechoic mesentery, focal abdominal effusion, lymph node enlargement, corrugation of the duodenum, and signs of extrahepatic biliary obstruction.10 Nodules of up to 1 cm with or without enlargement of the pancreas may indicate nodular hyperplasia and masses >2 cm are suggestive for malignant neoplasia.9 However, a mass-like appearance also may be found with pancreatitis, and vice versa, malignant lesions may appear diffuse. In conclusion, a definitive diagnosis based on sonographic characteristics is rarely possible because sonographic characteristics overlap for different pancreatic diseases. Histopathology usually is required to establish a definite diagnosis in most cases.9

In human medicine, computed tomography (CT) and ultrasonography are the most commonly utilized imaging modalities for evaluation of pancreatic pathologies.11,12 CT is considered the gold standard for staging acute severe pancreatitis in humans and allows detection of pancreatic necrosis and fluid accumulation.13 However, for the diagnosis of feline pancreatitis, a recent study detected that using abdominal ultrasonography and fPLI in combination were far superior to CT.7 Contrast-enhanced ultrasonography using carbon dioxide microbubbles is a sensitive technique that can be used to examine vascularization of the pancreas in humans. It has been used to differentiate benign from malignant or focal inflammatory processes.14–16 Malignant pancreatic lesions may be further differentiated with contrast-enhanced ultrasonography in human patients. Adenocarcinomas often were hypovascularized whereas neuroendocrine tumors were hypervascularized lesions. Masses associated with pancreatitis showed a different vascularization pattern depending on the degree of inflammation and necrosis.14–19

The purpose of the present study was 1st to assess the feasibility of contrast-enhanced Doppler ultrasonography in the feline pancreas. Second, vascularity and blood volume of the ultrasonographically normal pancreas in clinically healthy cats was determined with contrast-enhanced Doppler ultrasonography. Values were compared with Doppler measurements in cats with histologically or cytologically confirmed B-mode ultrasonography evidence of pancreatic disease.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. References

Patient Selection

Animals were divided into 2 groups. Group A included clinically healthy cats in which the pancreas appeared normal on B-mode ultrasonography. The normal feline pancreas was defined as hypo-to iso-echoic in comparison with the liver, with a uniform architecture, regular contours, and normal dimensions.20 Criteria for inclusion of cats in the normal group were lack of a history of vomiting, diarrhea, anorexia, lethargy, or weight loss for the past 3 months. CBC, serum biochemistry, and urinalysis had to be within normal limits. Group B included cats in which pancreatic disease was diagnosed with B-mode ultrasonography and ultrasound-guided aspirates or histopathologic samples taken via tru-cut biopsy or at necropsy were available. Cats were either presented for signs related or unrelated to pancreatic disease. All cats underwent a physical examination, abdominal radiography, abdominal routine, and contrast ultrasonography, as well as urine, hematologic, and serum biochemical analyses. Because blood viscosity increases with increasing hematocrit and blood flow velocity is inversely proportional to the viscosity of blood,21,22 hematocrit, hemoglobin, and red blood cell count were recorded to assess their influence on the Doppler parameters. Owner consent was obtained for the study, which was approved by the Animal Ethics Council of the Canton of Zürich, Switzerland.

For each ultrasonographic procedure, hair over the abdomen was clipped, and acoustic gel applied to the skin. If necessary, cats were sedated or anesthetized with various protocols. The entire abdomen was scanned by B-mode ultrasonography 1st using a 5–8 MHz curved array transducer.a The pancreas also was scanned with a 5–12 MHz linear transducer.a Echogenicity, texture, and contour of the pancreas were evaluated. The thicknesses of the right and left pancreatic lobes, duodenal wall, and major duodenal papilla, as well as the width of the right and left pancreatic ducts and the common bile duct were measured. The presence of free abdominal fluid, hyperechoic mesentery, lymphadenomegaly, or other abdominal diseases also was recorded. For the ultrasonographic contrast procedure, the pancreas was examined with color Doppler and power Doppler ultrasonography before and after IV injection of a 1st generation microbubble contrast agent at a dosage of 80 mg/kgb using a 5–12 MHz transducer.a For color and power Doppler ultrasonography, settings were kept constant for all examinations (79% color gain, medium wall filter, and 700 Hz pulse repetition frequency for color Doppler and 81% color gain, medium wall filter, and 500 Hz pulse repetition frequency for power Doppler ultrasonography). Five color and 5 power Doppler images each were captured pre and postcontrast for computerized image analysisc similar to a method described previously.23,24 A region of interest (ROI) was drawn around the pancreatic lobe without including adjacent tissue. Two measurements were computed for each ROI: fractional area (FA) and color-weighted fractional area (CWFA). FA represents a vascularity index and indicates the percentage area of the pancreas occupied by blood vessels. CWFA determines the mean blood flow through the ROI in color Doppler or blood volume within the tissue in power Doppler ultrasonography. Computerized parameters were determined by calculating the median of 5 images for pre and postcontrast color and power Doppler ultrasonography.

Statistical Analyses

Distribution of data (continuous variables) within groups A and B was analyzed with box plots, scattergrams, and histograms and tested for normality with the 1-sample Kolmogorov-Smirnov test. Although data were normally distributed and the 1-sample Kolmogorov-Smirnov test was nonsignificant for all continuous variables (P= .07–.99), nonparametric statistical analyses were performed because groups A and B were rather small. To assess the association of pancreatic vascularity (FA) and blood volume (CWFA) of pre and postcontrast color and power Doppler with the continuous variables (weight, age, hematocrit, hemoglobin concentration, RBC count, thicknesses of the right and left pancreatic lobe, right and left pancreatic duct, the common bile duct, and duodenal wall), Spearman's rank correlations were calculated. The Mann-Whitney U-test was used for the categorical variables sex and the 2 groups of cats (group A and B) with respect to the continuous variables. To compare findings of post and precontrast Doppler ultrasonography, the differences between post and precontrast FA and CWFA were calculated within each Doppler method (color and power Doppler). To compare power and color Doppler vascularity (FA), the difference between them also was calculated. The means of the differences were computed together with the 95% confidence intervals (CI) and compared by a paired t-test. Results are given as odds ratios with 95% CI. Commercially available software was used.d The level of significance was set at P <.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. References

Thirty-six cats were included in the study: group A comprised 11 animals and group B 25 cats. In group B, 21 cats had clinical signs suspicious for pancreatic disease and 4 cats were presented with signs unrelated to pancreatic disease. Median age of group A cats was 6 years (range 2–15 years) and differed significantly from the median age of 14 years in group B (range 9–21 years) (P= .0009). Median hematocrit, hemoglobin, and RBC were within normal reference ranges for all animals and did not significantly differ between groups A and B. Abdominal radiographs in group A were unremarkable. In group B, abdominal effusion was evident in 1 cat. Mild to moderate hepatomegaly was present in 5 cats. B-mode findings of the pancreas in group B are summarized in Tables 1 and 2. Ultrasound-guided fine needle aspirates were taken in 19 cats from group B. Ten samples were nondiagnostic. In 6 cats, either purulent or mixed cellular inflammation with the presence of neutrophils, histiocytes, and lymphocytes was seen. Malignant lymphoma was diagnosed in 1 cat and neuroendocrine carcinoma was suspected in another animal. Nodular pancreatic hyperplasia was diagnosed in 1 case based on the presence of large numbers of epithelial cells often with weakly granulated but hyperbasophilic cytoplasm and mild variation of cell and nuclear size. In 1 cat, an ultrasound-guided tru-cut biopsy sample was taken and adenocarcinoma of the exocrine pancreas was diagnosed. Histopathologic samples were taken at necropsy in 5 cats. Nodular hyperplasia was diagnosed in 3 cases and occurred concomitantly with mild lymphocytic interstitial pancreatitis in 1 cat, with severe diffuse amyloidosis of the islet cells in 1 animal and with malignant lymphoma in 1 case. In 2 cats, severe purulent or mixed cellular inflammation with the presence of neutrophils, histiocytes, and lymphocytes was seen (Fig 1).

Table 1.   B-mode findings in group B (25 diseased cats).
 Number
  • a

    A nodule was defined as a focal solid lesion up to 1 cm whereas a mass represented a lesion >2 cm.10

Abnormal hypoechogenicity of pancreas to liver25
Irregular contour of the pancreas19
Presence of masses or nodules within the pancreasa10
Presence of masses or nodulesa and cysts within the pancreas7
Hyperechoic mesentery18
Inhomogeneous pancreas17
Free fluid6
Table 2.   B-mode measurements (in mm) in group B (25 diseased cats).
 MedianMinimumMaximum
Size of right pancreatic lobe8.65.015.8
Size of left pancreatic lobe8.65.014.0
Right pancreatic duct1.30.75.4
Left pancreatic duct1.20.43.3
Common bile duct2.51.110.0
Major duodenal papilla2.71.56.0
Duodenal wall thickness2.61.93.5
image

Figure 1.  (A): Longitudinal B-mode image of a normal left pancreatic limb. To the left, the stomach is identified with a star. (B): Longitudinal B-mode image of the left limb of the pancreas with cytologically confirmed mixed cellular inflammation. The pancreas is hypoechoic and enlarged with irregular contours, the surrounding mesentery is hyperechoic suggesting focal peritonitis. (C): Longitudinal power Doppler image of the left limb of the pancreas with histologically confirmed neoplasia (adenocarcinoma of the exocrine pancreas). Marked enlargement of the pancreatic limb with undulating surface and surrounding hyperechoic mesentery can be seen.

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Hematocrit, hemoglobin concentration, RBC, and weight did not significantly correlate with vascularity (FA) or blood volume (CWFA) of pre and postcontrast power or color Doppler ultrasonography (r=−0.28–0.13, P= .16–.75). For all animals, age correlated moderately with CWFA and FA of postcontrast color and power Doppler ultrasonography (r= 0.42–0.5, P= .001–.01). However, age did not correlate with the Doppler parameters within groups A or B (r=−0.35–0.44, P= .3–.79). With increasing thickness of the left or right pancreatic lobe, FA and CWFA significantly increased for all of the Doppler methods (r= 0.3–0.6, P≤ .0001–.04). With increasing width of the diameter of the common bile duct, pre and postcontrast color and power Doppler CWFA significantly increased (r= 0.39–0.44, P= < .02–.04). Left pancreatic duct size correlated significantly with postcontrast power Doppler FA and CWFA (r= 0.46–0.5, P= .004–.01). However, within groups A or B, thickness of the left or right pancreatic lobe, diameter of the common bile duct, and diameter of the left pancreatic duct did not significantly correlate with any of the Doppler parameters (r=−0.33–0.53, P= .06–.6). All Doppler parameters (FA and CWFA of pre and postcontrast color and power Doppler) were significantly greater in diseased cats compared with normal cats (P= .001–.007) (Fig 2). If only cats with cytologically or histologically confirmed pancreatic disease were included in the analyses, FA and CWFA of pre and postcontrast color Doppler and postcontrast power Doppler were significantly different from values measured in normal cats (P= .007–.02). Although precontrast power Doppler FA and CWFA were also higher in diseased cats, the difference was not significant (P= .07 and .09) (Table 3).

image

Figure 2.  Longitudinal postcontrast power Doppler ultrasonographic images (A): left limb of the pancreas with cytologically confirmed purulent and histiocytic pancreatitis with hemorrhage. (B): left pancreatic limb with cytologically suspected neuroendocrine carcinoma. Note the increased vascularity and blood volume in comparison with the right pancreatic limb in a clinically healthy cat (C).

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Table 3.   Doppler values for the pancreas of normal cats and diseased cats with cytologic or histologic diagnosis.
 Group A (11 Healthy Cats)Fifteen Diseased Cats with Cytologic or Histologic DiagnosisP-Value
MedianMinimum–MaximumMedianMinimum–Maximum
  • a

    Fractional area (FA), vascularity [%]; Color-weighted fractional area (CWFA), blood volume.

Precontrast color Doppler FAa0.30.0–9.55.40.0–31.6.02
Precontrast color Doppler CWFAa0.50.0–12.98.30.0–55.2.02
Postcontrast color Doppler FAa2.70.0–12.010.00.1–38.5.009
Postcontrast color Doppler CWFAa4.20.0–14.115.00.1–71.6.007
Precontrast power Doppler FAa0.60.0–15.05.00.0–45.0.07
Precontrast power Doppler CWFAa0.20.0–7.02.40.0–32.1.09
Postcontrast power Doppler FAa4.20.1–13.016.00.0–62.5.01
Postcontrast power Doppler CWFAa1.00.0–7.87.50.0–45.4.02

Findings of postcontrast Doppler were compared with those of precontrast Doppler ultrasonography (Fig 3). Compared with values obtained before administration of contrast medium, a significant increase of FA and CWFA of power Doppler ultrasonography was detected after contrast administration (Table 3). The mean difference for FA and CWFA was 9.1% (95% CI 4.1–14.1%, P= .0008) and 6.5 (95% CI 3.0–10.1, P= .0008). The same analysis was performed for color Doppler ultrasonography. Again, FA and CWFA significantly increased after administration of contrast medium (mean difference for FA = 5.4, 95% CI 2.3–8.6%, P= .001; mean difference for CWFA = 9.1, 95% CI 3.4–14.8, P= .003). The increase in FA was not as great as that identified for power Doppler ultrasonography because of the single injection of contrast medium. Power Doppler images were always taken immediately after the injection of the contrast medium, and color Doppler images were recorded subsequently.

image

Figure 3.  Longitudinal power Doppler ultrasonographic images of a histologically confirmed moderate to severe multifocal, chronic-active pancreatitis with fatty tissue necrosis in a cat: (A): Image obtained before contrast medium application. (B): Postcontrast image: note the increase in vascularity and blood volume in comparison with the precontrast image.

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To compare power and color Doppler ultrasonography, the difference between precontrast power Doppler FA and precontrast color Doppler FA was found to be highly significant (P<.0001). The difference between postcontrast power Doppler FA and postcontrast color Doppler FA also was highly significant (P= .0003). However, this calculation is again biased because of the single injection of contrast agent.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. References

In the present study, color and power Doppler ultrasonography with and without administration of contrast medium was shown to be feasible for the assessment of vascularity and blood volume of the normal and diseased feline pancreas. Doppler values were determined for a group of normal cats and vascularity and blood volume was found to be low. Cats with cytologically or histologically confirmed sonographic evidence of pancreatic disease showed significantly greater vascularity and blood volume values. Increased Doppler values therefore may represent an additional ultrasonographic feature of feline pancreatic disease. Several studies in humans have demonstrated the usefulness of contrast-enhanced Doppler ultrasonography in the differentiation of pancreatic lesions.13,19,25,26 A recent study showed that contrast-enhanced ultrasonography is comparable to contrast-enhanced CT, the 1st choice method in humans for the assessment of patients with acute pancreatitis.25 Contrast-enhanced ultrasonography was able to accurately determine the severity of acute pancreatitis and predict clinical outcome.25 Another study comparing conventional ultrasonography, nonenhanced and contrast-enhanced power Doppler ultrasonography showed that pancreatitis-associated tumors were correctly diagnosed in 85% by contrast-enhanced power Doppler ultrasonography.19 Good results also were found for contrast-enhanced power Doppler ultrasonography in the differentiation of neuroendocrine tumors.26 Whether vascularity and blood volume differs between neoplastic and inflammatory diseases of the feline pancreas could not yet be investigated in the present study due to the small number of cases. Another limitation of the present study was that vascularity and blood volume were only investigated in cats with pre-existing B-mode ultrasonographic changes of the pancreas. Cats with suspected pancreatic disorders but with normal ultrasonographic appearance of the pancreas have been described in the literature and were not included. One may assume that increased vascularity and blood volume may represent an early sign of inflammation or neoplasia and therefore, contrast-enhanced Doppler ultrasonography could be of diagnostic value in those patients with an otherwise ultrasonographically normal pancreas. However, this question was not assessed in the present study and should be a goal of future studies.

There are certain limitations with fine needle aspirates of the pancreas. In the present study, the majority of diagnoses were made using ultrasound-guided fine needle aspirations and 10 of 19 samples were nondiagnostic. This is a lower success rate than reported in the literature. In 1 study, ultrasound-guided fine needle aspiration achieved a diagnosis in 8 out of 10 samples. However, only exocrine pancreatic tumors and no other diseases were included.27 Also, a negative fine needle aspiration result does not rule out underlying malignancy.28 Additionally, necrosis and fibrosis are major problems for the differential diagnosis of ductal carcinomas and pancreatitis-associated masses. Because these tissues are not vascularized, necrotic pancreatitis may be interpreted falsely as ductal carcinoma.19 Therefore, histopathology is considered the method of choice to diagnose pancreatic disease in humans and animals. However, it is invasive, more expensive, and potentially dangerous in hemodynamically unstable cats.1 Furthermore, in a recent study of dogs with experimentally induced pancreatitis, pancreata were removed and sectioned every 2 cm to determine if pancreatitis was a focal or regional disease.29 In half of the dogs with acute pancreatitis and two thirds of the dogs with chronic pancreatitis, evidence of pancreatic inflammation was found in <25% of the sections.29 These results illustrate the potentially focal nature of the disease and emphasize the fact that although histologic evidence of pancreatitis proves pancreatitis, the lack of it does not rule out pancreatitis or a malignant process.29

Nodular hyperplasia is a common incidental finding in old dogs and cats. It involves the exocrine tissue only and occurs in many foci. Histologically, hyperplastic nodules can be difficult to differentiate from adenomas. The hyperplastic nodules are not encapsulated and do not compress the surrounding parenchyma.30 Acinar cell metaplasia is common in chronic pancreatitis in humans. The changes in the acinar cells as well as the ductular epithelial changes are considered preneoplastic lesions, and a gradual progression from mild to severe metaplasia and concurrent neoplasia has been well documented.

Power Doppler ultrasonography was found to be superior to color Doppler ultrasonography. This finding is in accordance with previous publications in dogs and humans demonstrating that power Doppler was more sensitive for the detection of small vessels.23,31,32 It can be explained by the lack of angle dependence and the way random noise is depicted. Power Doppler ultrasonographic images are derived from the energy of the reflected signals, and random noise has low energy; this feature permits higher gain settings and increased sensitivity for flow detection.33 The sensitivity of Doppler ultrasonography can be increased by echo-enhancers, such as Levovist, a 1st generation galactose-based contrast medium. It consists of stabilized air microbubbles, which basically act as scattering reflectors. With high intensity of the ultrasound beam (eg, as with Doppler ultrasonography), the shell of the microbubbles is disrupted and a transient, strong, nonlinear echo is emitted.14,25 In the present study, contrast-enhanced Doppler ultrasonography measured greater values of vascularity and blood volume and was found to be superior to precontrast Doppler ultrasonography. However, ultrasonography contrast agents are rather expensive and may not be affordable in every patient. Also, not every ultrasound machine is equipped with power Doppler. Because values of all Doppler technologies were significantly higher in the investigated diseased cats, nonenhanced color or power Doppler ultrasonography may provide satisfactory results for the investigation of the feline pancreas.

In the present study, diseased cats were significantly older than normal cats. This finding may be explained by the higher probability of pancreatic disorders in older cats.34 A recent study investigating prevalence and histopathology of pancreatitis in cats identified a significant correlation between age and chronic pancreatitis.35 The significant positive correlation of age with vascularity and blood volume for all animals could not be confirmed within group A or B. Thus, values scattered widely. Therefore, age dependency is thought to be a result of the significantly different ages of the 2 groups. Ideally, the study should have been performed in age-matched groups. Similarly, we hypothesize that the significant positive association of thickness of the left or right pancreatic lobe, diameter of the common bile duct, and diameter of the left pancreatic duct with some of the Doppler parameters for all animals but not within groups also is only a consequence of the difference between groups.

An antemortem diagnosis of pancreatitis in cats is difficult. For this reason, radioimmunoassays to determine feline trypsin-like immunoreactivity (fTLI) and fPLI have been developed. However, results for fTLI were disappointing, showing a sensitivity of only 33% and a specificity of <80%.4,36,37 Only 1 study evaluating fPLI to diagnose pancreatitis in cats is available, and the results are very promising.7 Therefore, measurement of fPLI in combination with abdominal ultrasonography seems the most reliable approach to the diagnosis of feline pancreatitis.7 Ideally, measurement of fPLI to support the diagnosis of pancreatitis in the cats of this study should have been included. Unfortunately, this assay was not performed.

In conclusion, contrast-enhanced Doppler ultrasonography appears to be a feasible technology for the evaluation of the feline pancreas. Results indicate a significant difference in vascularity and blood volume in cats with or without evidence of pancreatic pathology. Further studies are needed to evaluate its use for the differentiation of various pancreatic disorders and in cats suspected of having pancreatic disease but without B-mode changes of the pancreas.

Footnotes

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. References

aATL 5000, Philips AG, Zurich, Switzerland

bLevovist (400 mg/mL), Schering AG, Baar, Switzerland

cQwin, Leica Microsystems AG, Glattbrugg, Switzerland

dStatView 5.0.1, SAS Institute Inc, Cary, NC and SPSS 10.0, SPSS Schweiz AG, Zurich, Switzerland

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Footnotes
  7. References