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DOI: 10.1148/radiol.2292020825
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(Radiology 2003;229:415-420.)
© RSNA, 2003


Gastrointestinal Imaging

CT Predictors of Failed Laparoscopic Appendectomy1

Bettina Siewert, MD, Vassilios Raptopoulos, MD, Shiu-Inn Liu, MD, Richard A. Hodin, MD, Roger B. Davis, ScD and Max P. Rosen, MD, MPH

1 From the Departments of Radiology (B.S., V.R., M.P.R.) and Surgery (S.I.L., R.A.H.), and Division of General Medicine (R.B.D.), Beth Israel Deaconess Medical Center and Harvard Medical School, 330 Brookline Ave, Boston, MA 02215. From the 2001 RSNA scientific assembly. Received July 9, 2002; revision requested August 23; final revision received March 5, 2003; accepted April 7. Address correspondence to B.S. (e-mail: bsiewert@caregroup.harvard.edu).


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: To identify computed tomographic (CT) signs that may help predict possible failure of laparoscopic appendectomy and subsequent conversion to open appendectomy.

MATERIALS AND METHODS: Of 234 consecutive patients who underwent preoperative CT and in whom laparoscopic appendectomy was attempted, 26 required conversion to open appendectomy. Conversion was correlated with the following CT findings: appendix location, appendicolith, cecal wall thickening involving the base of the appendix, lymphadenopathy, and appendiceal diameter. The extent of inflammation was graded by using a six-point scale: 0 meant normal appendix; 1, possibly abnormal appendix (6-mm diameter without other abnormality); 2, abnormal appendix (diameter >= 6 mm with wall enhancement) without adjacent fat stranding; 3, abnormal appendix surrounded by fat stranding; 4, abnormal appendix surrounded by fat stranding and fluid; and 5, inflammatory mass or abscess. Student t and {chi}2 tests were used for statistical analysis of interval and nominal values, respectively.

RESULTS: Although there was a significant difference in appendiceal diameter between the patients in whom laparoscopic appendectomy was successfully completed (11.3 mm ± 3.5 [SD]) and those who required conversion (12.9 mm ± 3.9), no distinct cutoff point was identified. Of the five CT findings evaluated, none was a significant predictor of conversion to open appendectomy. Eleven (7%) of 164 patients with a CT inflammation grade of 0–3 required conversion, whereas 15 (21%) of 70 patients with a grade of 4 or 5 required conversion (P < .04).

CONCLUSION: The majority of patients with appendicitis can be treated with laparoscopic appendectomy. Nevertheless, patients who require conversion to open appendectomy tend to have high CT inflammation grades of 4 or 5, which indicate the presence of periappendiceal fluid or an inflammatory mass or abscess.

© RSNA, 2003

Index terms: Appendicitis, 751.291 • Appendix, CT, 751.12111, 751.12112, 751.12115 • Laparoscopic surgery


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acute appendicitis has generally been treated with open appendectomy. With the advent of laparoscopy, laparoscopic appendectomy has become an increasingly popular surgical alternative (1). Although laparoscopic appendectomy in its current stage of development is not universally regarded as the surgical procedure of choice, reports in the literature demonstrate several advantages over open appendectomy, including lower cost and wound infection rate, shorter hospital stay, less postoperative pain, and faster recovery (2). However, during the laparoscopic procedure, complications may arise or the extent of disease may make safe resection impossible, and these conditions result in the conversion to open appendectomy. Changing to open appendectomy leads to increases in surgery time and higher hospital costs compared with the surgery time and costs associated with initially performing open appendectomy alone. High-risk conditions that may lead to conversion to an open procedure include patient age older than 65 years, diffuse peritonitis at physical examination, and the surgery being performed by surgeons with less experience in laparoscopic procedures (3).

The computed tomographic (CT) findings of acute appendicitis have been well established, and CT has an important role in the diagnostic process (411). The purpose of this study was to identify CT signs that may help predict possible failure of laparoscopic appendectomy and subsequent conversion to open appendectomy.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Between July 1996 and June 2000, 595 consecutive patients underwent laparoscopic appendectomy at our institution; 58 of these patients required conversion to open appendectomy. Two hundred thirty-four of these 595 patients underwent preoperative CT and were included in this study. During this period, we noticed a substantial increase in the use of CT to examine patients who had undergone appendectomy: from 33% of patients in 1997 to 59% in 2000 (12). There were 99 male and 135 female patients, and their mean age was 38 years ± 15 (SD) (age range, 17–81 years). Institutional review board approval for this study was obtained. Informed patient consent was not required. All imaging examinations were performed for accepted clinical indications and are considered to be acceptable imaging procedures for patient care.

CT Imaging
CT imaging was performed by using a HiSpeed Advantage unit (GE Medical Systems, Milwaukee, Wis). As part of our routine scanning protocol, a nonfocused study was performed and transverse images were obtained. To ensure optimal bowel opacification, 600–900 mL of a 2.1% barium sulfate suspension (Readi-Cat 2; E-Z-Em, Westbury, NY) was administered orally at least 45 minutes prior to scanning, according to patient tolerance. No contrast material was administered rectally. Patients also received 150 mL of 43% iodinated contrast material (various agents) intravenously at a rate of 2 mL/sec. Helical scanning of the abdomen and pelvis was performed during two sequential breath holds by using 7-mm collimation and a pitch of 1.5 following a 50-second delay after the beginning of the intravenous contrast material injection (11).

Image Review and Comparison of Imaging and Surgical Findings
The CT images were reviewed on film hard copy (n = 76) or with a picture archiving and communication system, or PACS (n = 158). Measurements were made with manual calipers on the hard-copy images and with electronic calipers on the PACS images. CT findings were assessed by consensus between two radiologists (B.S., V.R.), who were blinded as to whether laparoscopic appendectomy was successful or nonsuccessful, in which case conversion to open appendectomy was necessary.

The following CT findings were evaluated retrospectively: appendiceal diameter and wall thickness measurements, appendix location (ie, right lower quadrant, retrocecal, or pelvic), cecal wall thickening involving the base of the appendix, periappendiceal or ileocecal lymphadenopathy larger than 5 mm in diameter on the short axis (13), appendicolith, and appendiceal wall enhancement. The latter was used for CT grading of inflammation (13). An appendiceal diameter (DApp) of 6 mm or greater was considered abnormal. Appendiceal wall thickness was calculated as follows: (DApp - LApp)/2, where LApp is the thickness of the appendiceal lumen (in millimeters).

The extent of inflammation was graded by using imaging features seen anywhere along the course of the appendix and in the periappendiceal region. A six-point scale was used: A grade of 0 indicated a normal appendix; grade 1, a possibly abnormal appendix—that is, one at least 6 mm in diameter without intraluminal fluid or wall enhancement or one containing an appendicolith; grade 2, an abnormal appendix—that is, one 6 mm or larger in diameter with wall enhancement—without adjacent fat stranding; grade 3, an abnormal appendix with surrounding fat stranding; grade 4, an abnormal appendix with surrounding fat stranding and fluid; and grade 5, an inflammatory mass or abscess. We previously found this CT grading system to correlate well with the severity of appendiceal inflammation at surgery (12). Thus, the accuracy of grading was not tested in this study.

The surgery notes on the patients who required conversion to open appendectomy were reviewed by a single surgeon (S.I.L.), and the reasons for conversion were compared with the CT findings in consensus by this surgeon and a radiologist (B.S.). The surgeons’ experience was not evaluated in this study. Laparoscopic appendectomy is a popular procedure at our institution, and by the time CT had gained acceptance, all surgeons were well trained in the technique.

Data Evaluation
The 234 patients were separated into two groups: those in whom appendectomy was performed completely laparoscopically (n = 208) and those in whom laparoscopic excision had to be converted to open appendectomy (n = 26). The CT findings in each group were compared to assess their significance as a sole predictor of conversion from laparoscopic to open appendix resection. Statistical analysis of the interval values, including appendiceal wall thickness and diameter, was performed by using the Student t test. The {chi}2 test was used for statistical analysis of the nominal data, including appendix location and the presence of an appendicolith, a periappendiceal lymphadenopathy, or cecal wall thickening involving the base of the appendix.

The patients in each group were assigned discriminating values of appendiceal diameter—specifically, 9, 10, 11, 12, or 13 mm—and CT grades—specifically, grade 3 or higher or grade 4 or higher—and the distribution of patients with appendiceal diameters and CT grades higher or lower than these values in each group was tested by using contingency tables at {chi}2 analysis. A P value of .05 or less was considered to indicate statistical significance.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Of the 234 patients in whom laparoscopic appendectomy was started, 208 (89%) underwent the procedure to completion, whereas 26 (11%) required conversion to open appendectomy. When we compared the size of the appendix between the groups, there was a significant difference in the diameters of the organs, with a mean diameter of 11.3 mm ± 3.5 in the patients who underwent successful laparoscopic appendectomy versus 12.9 mm ± 3.9 in those who required conversion (P < .03). However, no discriminating size could be identified. An appendix with a diameter of 13 mm or greater was present in 45 (22%) of 208 patients in whom laparoscopic appendectomy was completed, as compared with 10 (38%) of 26 patients who required conversion to an open procedure (P = .096). The difference in predicting conversion was not statistically significant when appendiceal diameters of 9, 10, 11, and 12 mm were tested.

At surgery, only one appendix, measuring 21 mm in diameter, could not be excised laparoscopically because of its size (Fig 1). There was no significant difference in appendiceal wall thickness between the two groups: 2.6 mm ± 1.4 in the patients in whom complete laparoscopic appendectomy was performed compared with 3.2 mm ± 1.5 in those who required conversion to open appendectomy.



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Figure 1. Transverse CT image shows 21-mm-diameter appendix (solid arrow) in 39-year-old man with acute appendicitis. There is periappendiceal stranding and an enlarged regional lymph node (dotted arrow). Initial attempt to perform laparoscopic appendectomy resulted in conversion to open procedure because the large appendix was filled with pus.

 
Of the five CT signs evaluated, none was statistically significant in predicting conversion to open appendectomy in the 208 patients in whom laparoscopic appendectomy was completed compared with the 26 patients who required conversion to open appendectomy (Table 1). With regard to the location of the appendix, it was noted to be retrocecal in 16 (8%) of the patients who underwent completely laparoscopic appendectomy (group 1) compared with five (19%) of the patients in whom the procedure was changed to open appendectomy (group 2) (Fig 2). The appendix was located in the pelvis in seven (3%) patients in group 1 compared with one (4%) patient in group 2, and in the right lower quadrant in 185 (89%) patients in group 1 compared with 20 (77%) patients in group 2.


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TABLE 1. Correlations between CT Signs and Successfully Completed Laparoscopic Appendectomy

 


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Figure 2. Transverse CT image shows retrocecal appendix (arrow) in 34-year-old woman with acute appendicitis. Periappendiceal stranding is present (grade 3). Excision of the retrocecal appendix in this patient required conversion from laparoscopic to open appendectomy owing to inability to visualize the appendix laparoscopically. However, neither grade 3 nor retrocecal appendix was associated with higher incidence of conversion to open procedure in this series.

 
An appendicolith was seen in 45 (22%) patients in group 1 compared with eight (31%) patients in group 2 (Fig 3). Cecal wall thickening involving the base of the appendix was seen in 40 (19%) patients in group 1 compared with five (19%) patients in group 2 (Fig 4). Lymphadenopathy was seen in 12 (6%) patients in group 1 compared with four (15%) patients in group 2 (Fig 1).



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Figure 3. Transverse CT image in 46-year-old woman with acute appendicitis shows an appendicolith (arrow) at the base of the appendix. Successful laparoscopic appendectomy was performed.

 


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Figure 4. Transverse CT image in 35-year-old man with acute appendicitis shows cecal wall thickening (arrow) involving the base of the appendix. This region was found to be necrotic at laparoscopy, and conversion to open appendectomy was necessary.

 
CT grading of disease severity was helpful in predicting high risk of conversion to open appendectomy. There was a higher incidence of conversion from laparoscopic to open appendectomy in the patients with higher CT grades (Table 2). Only 11 (7%) of the 164 patients with a CT grade of 0–3 required conversion, whereas 15 (21%) of the 70 patients with a CT grade of 4 or 5 required conversion (P < .04) (Figs 2, 5).


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TABLE 2. CT Grading in Patients Who Underwent Laparoscopic and Open Appendectomies

 


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Figure 5. Transverse CT image in 61-year-old man with acute appendicitis shows inflammatory mass (arrow) with early abscess formation (grade 5). Laparoscopic appendectomy was not possible, and conversion to an open procedure was necessary.

 
At comparison of the CT and surgical findings, we noted that 15 of 20 patients in whom laparoscopic appendectomy had to be converted to an open procedure because of severe inflammatory changes had a CT score of 4 or 5 and that all 20 had a score of 3 or higher. However, a CT grade of 3 did not contribute to the identification of patients who may have been at higher risk of requiring conversion to open appendectomy. Of 66 patients with a CT grade of 0–2, two (3%) required conversion to open appendectomy; these results are not significantly different from the 14% (24 of 168) of patients with a CT grade of 3–5 who required conversion. We analyzed several combinations of findings, including CT grade and appendix location, none of which helped to further narrow down the group of patients who might have required conversion to open appendectomy.

Review of the surgery notes revealed that of the 26 patients who required conversion from laparoscopic to open appendectomy, 20 had severe inflammatory changes, whereas technical factors were responsible for the conversion in the remaining six patients. The inflammatory changes included extensive inflammation in the right lower quadrant or pelvis in 16 patients and inflammation at the base of the appendix in three. In one patient, the inflamed appendix was large (21 mm in diameter) and pus filled, and these combined conditions made laparoscopic excision impossible. The CT grade in these 20 patients was 3 or higher: grade 3 in five, grade 4 in nine, and grade 5 in six patients. Many patients with a CT grade of 3 or higher underwent successful laparoscopic appendectomy, however (Table 2).

The diameters of the appendix in the patients who required conversion ranged from 8 to 21 mm (mean diameter, 14 mm ± 4), with the 21-mm appendix being the one that was impossible to resect laparoscopically. The technical difficulties at appendectomy in six patients were problems visualizing the entire appendix with laparoscopy in three patients, uncontrollable hemorrhage in two patients, and inability to maintain pneumoperitoneum in one patient. Of these six patients, two had a CT grade of 2 and four a CT grade of 3. These patients had a mean appendiceal diameter of 11 mm ± 2 (range, 10–16 mm), which is not significantly different from the mean appendiceal diameter (13 mm ± 4; range, 7–21 mm) in the 20 patients in whom inflammatory changes were the cause of the failed laparoscopic procedure (P = .92).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The rate of conversion from laparoscopic to open appendectomy varies, with most investigators reporting a conversion rate of around 10% (14,15), which is similar to the 11% conversion rate in our series. Although conversion rates as low as 3.3% have been reported, this value may represent a significant bias related to the selection of patients who undergo an initial attempt at laparoscopic appendectomy (16,17). Conversion to open appendectomy is influenced by several factors related to differences in patient population, surgical technique, and surgeon skill. Surgeons who perform fewer laparoscopic procedures have been found to have higher conversion rates (3).

The reasons for conversion that are related to the individual patient can be divided into those involving the focal abnormality and those involving technical factors (1,2,1417). Technical factors include the following: (a) the inability to identify the appendix laparoscopically due to an unusual position of the appendix—for example, in the case of a retrocecal appendix—or to malrotation of the small bowel; (b) the inability to laparoscopically remove the appendix in its entirety; (c) uncontrollable hemorrhage or injury to the small bowel; (d) the inability to maintain adequate pneumoperitoneum; and (e) hypotension due to the Trendelenburg position.

In our series, technical difficulties necessitated conversion in six (23%) of the 26 patients who required conversion to open appendectomy. These patients had low CT grades (2 or 3) and relatively small appendiceal diameters. To this patient group we assigned two patients who had unpredictable and uncontrollable hemorrhage and one patient in whom pneumoperitoneum could not be maintained. In three patients, the technical difficulties were related to the inability to laparoscopically visualize a retrocecal appendix. We identified a retrocecal appendix at CT in 16 (8%) of 208 patients who underwent successful laparoscopic appendectomy and in five (19%) of 26 patients who required conversion (P = .115), and this finding alone did not preclude successful laparoscopic resection. However, it should be emphasized that it is important to report a retrocecal location of an appendix to the surgeon, because this may explain atypical symptoms or represent a challenge for laparoscopic resection.

Focal appendiceal abnormalities include (a) extensive inflammatory changes surrounding the appendix, including severe inflammation or necrosis at the appendiceal base, which can make safe application of the laparoscopic stapler impossible; (b) dense adhesions; (c) localized perforation or diffuse peritonitis, which requires an open approach to achieve complete irrigation of the abdominal cavity and thus prevent abscess formation; and (d) appendiceal tumor, which requires an open approach to investigate the abdominal cavity for metastatic disease.

The extent of inflammatory changes, including local perforation, is reflected in the CT grading: Although uncomplicated appendicitis is characterized by a low CT grade (grade 0–3), with the only findings being a thickened appendix 6 mm or larger in diameter and periappendiceal fat stranding (in grade 3), complicated appendicitis is characterized by periappendiceal fluid involvement or an inflammatory right lower quadrant mass.

In our study, the majority of patients (n = 153, 74%) who underwent successful laparoscopic appendectomy had a CT grade of 0–3, whereas 15 (58%) of the 26 patients who required conversion to open appendectomy had a CT grade of 4 or 5. Twenty-four of the 26 patients had a CT grade of 3 or higher. Furthermore, 15 (21%) of the 70 patients with a CT grade of 4 or 5 required conversion, whereas only 11 (7%) of the 164 patients with a grade of 0–3 required conversion. Thus, periappendiceal fluid accumulation, as well as an inflammatory mass or abscess in the right lower quadrant, was more commonly seen in the patients who required conversion. Such information is helpful in choosing the surgical procedure, or it could be used to stratify patients into surgical and medical management groups since some authors report that patients with CT findings of abscess or phlegmon initially can be treated nonsurgically (18).

Periappendiceal and ileocecal lymphadenopathies, defined as lymph nodes larger than 5 mm in diameter on the short axis (14), were seen in four (15%) patients who required conversion versus 12 (6%) who underwent successful laparoscopic surgery. The presence of a lymphadenopathy in a periappendiceal region, where usually no large lymph nodes are identified, is believed to be related to extensive inflammatory changes in the area.

Inflammation at the appendiceal base is specifically listed in the surgical literature as a reason for conversion (3). The closest CT correlate is the arrowhead sign, which was initially reported by Rao et al (19) to be seen in 30% of their study patients. The arrowhead sign consists of a thickened and narrow appendiceal base with contrast agent collection in the upper portion of the cecum following rectal administration. Because our CT protocol for the evaluation of suspected appendicitis involves oral contrast agent administration, we analyzed the presence of cecal wall thickening involving the base of the appendix instead. Cecal wall thickening involving the base of the appendix was present in 40 (19%) of the 208 patients who underwent successful laparoscopy and in five (19%) of the 26 who required conversion (P = .792). The presence of a certain amount of inflammation must be assumed with any appendicitis causing obstruction of the lumen and thus propagating the inflammatory process; however, the inflammation rarely interferes with the laparoscopic resection of the appendix.

Although the detection of an appendicolith at CT is significantly associated with the presence of appendicitis (20), with a positive predictive value of 0.74, the detection of an appendicolith should not be the sole basis of a diagnosis of appendicitis (20). Furthermore, the presence of an appendicolith is an unreliable predictor of laparoscopic appendectomy failure and thus should not influence the choice of surgical approach. In our study, one or more appendicoliths were found in 45 (22%) patients who underwent laparoscopic appendectomy versus eight (31%) who required conversion to open appendectomy (P = .351).

Another reason for conversion to open appendectomy is the unexpected finding of an appendiceal tumor during laparoscopic appendectomy. Specific CT findings of appendiceal tumors that present as acute appendicitis have been reported to occur in 87% of patients and include a focal soft-tissue mass and cystic dilatation of the appendix, each in 43% of cases (21). Thirteen percent of patients with appendicitis who have diffuse wall thickening may have a tumor that is indistinguishable from acute appendicitis. Patients with appendiceal tumors have a greater mean appendiceal wall thickness (1.1 cm; range, 0.4–2.0 cm) compared with the patients with acute appendicitis in our study (0.3 cm ± 0.2; range, 0.1–0.7 cm). Likewise, the mean appendiceal diameter in patients with appendiceal tumor is larger (2.9 cm; range, 1.1–6.0 cm) than that in the patients with acute appendicitis in our study (1.1 cm ± 0.4; range, 0.4–2.1 cm).

Our study data show that the mean diameter of the appendix in the patients who required conversion from laparoscopic to open appendectomy was significantly larger (12.9 mm ± 3.9) than that in the patients who underwent laparoscopic appendectomy (11.3 mm ± 3.5, P < .03), but no discriminating diameter could be identified. A trend was noted among the 55 patients with an appendiceal diameter of 13 mm or greater: 10 (18%) of these patients versus 16 (9%) of 179 patients with an appendiceal diameter of less than 13 mm required conversion to open appendectomy (P = .096). According to the data reported by Pickhardt et al (21), these 55 patients would automatically include the patient population with appendiceal tumors. The importance of the appendiceal diameter was confirmed by Horrow et al (22), who reported larger appendiceal diameter to be correlated with perforation at surgery.

One limitation of our study was verification bias (23,24). By choosing only those patients in whom laparoscopic appendectomy was attempted, we introduced verification bias. Such selection may be related to the known high accuracy of CT in the diagnosis of appendicitis. We do not know how many patients in our study underwent open appendectomy partially or solely on the basis of the CT findings. This may be the reason that some of the features that previously have been shown to be important in the decision to change to an open procedure were not statistically significant predictors of conversion in our preselected patient population. These features include retrocecal appendix, appendicolith, cecal wall thickening, and lymphadenopathy. A CT grade of 4 or higher was significant, but it is a recently described finding, and thus, in contrast to the other CT findings, it was not considered when choosing the surgical approach.

Another limitation of our study was that the experience of the surgeons, which has been shown to be related to successful laparoscopic appendectomy (3), was not evaluated. We judged our surgeons to be experienced in laparoscopic appendectomy on the basis of our institutional practices and surgical trends.

Negative appendectomies for right lower quadrant pain (ie, those that reveal a normal appendix at pathologic examination) are still quite common in cases in which preoperative imaging is not performed (5). Preoperative CT enables one to establish the diagnosis; it also depicts unusual anatomy (eg, retrocecal or pelvic appendix and malrotation of small bowel) and the extent of inflammatory changes. These factors might influence patient treatment in terms of which surgical approach is chosen. Our study data show that in patients with appendicitis, preoperative CT not only can be used for diagnosis, but it also may alert the surgeon to possible difficulties and help guide surgical planning.

In conclusion, no isolated CT finding, such as retrocecal appendix, appendicolith, cecal wall thickening involving the base of the appendix, regional lymphadenopathy, or appendiceal diameter of 13 mm or greater can be used to predict failure of laparoscopic appendectomy. Patients who have CT findings of appendicitis with periappendiceal fluid and/or an inflammatory mass or abscess are more likely to require conversion from laparoscopic to open appendectomy than are those with only appendiceal enlargement or periappendiceal fat stranding.


    FOOTNOTES
 
Author contributions: Guarantors of integrity of entire study, V.R., B.S.; study concepts, V.R., B.S.; study design, V.R., M.P.R., B.S.; literature research, V.R., B.S.; clinical studies, S.I.L., R.A.H.; data acquisition, V.R., B.S., S.I.L., R.A.H.; data analysis/interpretation, B.S., V.R., M.P.R., R.B.D.; statistical analysis, B.S., V.R., R.B.D.; manuscript preparation, B.S., V.R., M.P.R.; manuscript definition of intellectual content, V.R., B.S.; manuscript editing, B.S., V.R., M.P.R.; manuscript revision/review, B.S., V.R., S.I.L., R.A.H., M.P.R.; manuscript final version approval, B.S., V.R., M.P.R.


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 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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