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Emergency Radiology |
1 From the Departments of Radiology (N.F., C.B.S., M.A.B., G.C.) and Surgery (D.F., D.B.H.) and General Clinical Research Center (G.P.S.), University of California at San Diego, 200 W Arbor Dr, San Diego, CA 92103-8756. From the 2001 RSNA Annual Meeting. Received March 31, 2004; revision requested June 8; revision received June 18; accepted July 27. Supported in part by a 2003 scholarship from the American Roentgen Ray Society; National Institutes of Health (NIH) grant 1K08 CA 102158; and NIH grant MO1-RR00827 from the National Center for Research Resources of the NIH for the University of California at San Diego, General Clinical Research Center. Address correspondence to C.B.S. (e-mail: csirlin@ucsd.edu).
| ABSTRACT |
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90 mm Hg) after blunt abdominal trauma.
MATERIALS AND METHODS: The investigational review board approved the study and waived informed consent. The study group consisted of 128 hypotensive patients with blunt abdominal trauma who underwent screening US over a 9-year period. Abdomens were scanned for free fluid and for parenchymal heterogeneity in visceral organs; scans that depicted these were considered positive. Prospective reports were used to calculate diagnostic performance. Patients were retrospectively given a fluid score according to the number of fluid pockets visualized (0, 1, or
2) (consensus by three readers) and were assigned to a low- or high-risk group according to the presence of hematuria and/or axial fracture on radiographs. Screening US results were compared with findings with the best available reference standard (computed tomography [CT]), repeat US, other diagnostic test, laparotomy, autopsy, clinical course). Data were compared by using
2 or Fisher exact test, depending on expected frequencies, with Bonferroni correction for multiple comparisons. Continuous variables were compared by using unpaired Student t test or Mann-Whitney U test, depending on data distribution.
RESULTS: The study included 77 male and 51 female patients (mean age, 42 years). Sensitivity was 85% (44 of 52) for detection of any injuries, 97% (30 of 31) for surgical injuries (ie, injuries requiring surgery), and 100% (10 of 10) for fatal injuries. Specificity was 96% (73 of 76), 82% (80 of 97), and 69% (81 of 118), and accuracy was 91% (117 of 128), 86% (110 of 128), and 71% (91 of 128), for respective injury categories. One nonfatal surgical injury was missed in a high-risk patient. For each injury category, frequency of injury in patients with a fluid score of 2 or more was nine times that in patients with a score of 0 (P < .001 for all comparisons). Frequency of false-negative US findings in high-risk patients was eight times that in low-risk patients (P < .01).
CONCLUSION: In patients who are hypotensive after blunt abdominal trauma and not hemodynamically stable enough to undergo diagnostic CT, negative US findings virtually exclude surgical injury, while positive US findings indicate surgical injury in 64% of cases.
© RSNA, 2005
| INTRODUCTION |
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Ultrasonography (US), although not as accurate overall, has several advantages and continues to be the primary screening modality for blunt abdominal trauma at select centers in the United States, Europe, and Asia (121). Among its advantages are speed and mobility, absence of ionizing radiation, and invulnerability to breathing artifact in dyspneic or minimally cooperative patients. US can be performed at the bedside and directly integrated with patient resuscitation. We believe that these advantages make US particularly suitable for screening hemodynamically unstable patients with blunt abdominal trauma, in whom CT may be problematic or contraindicated. Thus, the purpose of our study was to determine retrospectively the accuracy of screening US in patients with blunt abdominal trauma and hypotension (systolic blood pressure
90 mm Hg).
| MATERIALS AND METHODS |
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Patient Data
Patients had their blood pressure measured and recorded by trained trauma nurses at arrival. Screening US was performed during the secondary trauma survey, usually within 10 minutes after patient arrival. The precise interval from arrival to screening US was not recorded. Patients admitted directly to the operating room (number not recorded) were scanned there rather than in the resuscitation suite.
After screening US, patients were admitted to the trauma service for stabilization, observation, and/or management of injuries. They were followed up by the trauma surgical staff during their hospital admission and at follow-up visits in the clinic. CT and other diagnostic tests were performed at the discretion of the trauma service. In general, patients with positive US findings who could not be stabilized with fluid resuscitation underwent emergent laparotomy, while those who could be stabilized underwent CT for more definitive evaluation. Patients with negative US findings underwent repeat US and/or follow-up CT or were followed up clinically. Autopsies were performed in all patients who died.
Trained trauma research assistants prospectively entered patients records into a computerized trauma registry, including arrival blood pressure and results of all imaging and other diagnostic examinations and procedures. Database information was updated regularly by the trauma service as new tests were performed.
To monitor potential postdischarge complications, the trauma service participated in a county-level auditing program. The county coroner and trauma surgeons from each level I center in the county met monthly. Diagnostic errors and delays were discussed, tabulated, and entered into each institutions trauma registry. To our knowledge, this is the only systematic county-level trauma-auditing program in the United States.
US Examinations
Each screening US examination was performed during resuscitation by one of 15 certified sonographers (225 years experience) with one of two US scanners (HDI 3000, Advanced Technologies Laboratories, Bothell, Wash; 128-XP, Acuson, Mountain View, Calif), as described elsewhere (20). Seven regions were examined for fluid, including the right and left upper quadrants, epigastrium, pelvis, right and left paracolic gutters, and retroperitoneum. Visceral organs were evaluated for parenchymal abnormalities. Cross-sectional and longitudinal scanning was performed through the liver, spleen, and each kidney. At least two representative images of each organ were captured. Although scanning times were not routinely recorded, they ranged from 3 to 5 minutes.
Initially, US was available 24 hours a day. Later, because of a shortage in sonographers, US availability was curtailed to 17 hours (on weekdays) or 9 hours (on weekends) a day. Patients who presented at times when US was not available underwent CT or other tests and were not included. Thus, our study population was not a consecutive sample.
US Interpretation
Until April 2001, US studies were filmed (Image Link; Eastman Kodak, Rochester, NY) and reviewed on an alternator. After April 2001, US studies were archived digitally on a picture archiving and communication system (IMPAX; Agfa, Ridgefield Park, NJ) and reviewed on a 19-inch color monitor with 1024 x 1024-pixel resolution (MWD 421; Barco Display Systems, Kortrijk, Belgium).
US studies were interpreted prospectively and in consensus by a resident (minimum experience, 6 months) and staff radiologist on the US service at the time of the scan. Scans obtained after hours were interpreted preliminarily by the resident on call; the final interpretation was then rendered by the staff radiologist the following morning. US findings were classified as positive if free fluid was visible or if a parenchymal abnormality that could be secondary to trauma was present.
Given the findings of previous studies, any quantity of fluid was considered positive (23), except for anechoic fluid measuring less than 3 cm in maximum anteroposterior dimension and isolated to the pelvic recesses in reproductive-age women, which was considered physiologic in the absence of other suspicious findings (17). In general, parenchymal abnormalities consisted of any solid organ lesion that was not clearly a simple cyst or a calcification and included intraparenchymal masses, hematomas, lacerations, and/or geographic zones of echotextural heterogeneity. In the presence of medical ascites (eg, cirrhosis or other cause of nontraumatic intraperitoneal fluid), free fluid was considered positive because hemoperitoneum could not be excluded. Indeterminate parenchymal abnormalities were also considered positive even in the absence of free fluid. Well-visualized nontraumatic lesions such as simple cysts were considered negative findings.
Data Collection and Reference Standard
The trauma registry, medical records, autopsy reports, and imaging reports were read retrospectively by a research fellow (N.F., 1 year of experience) and an attending specialist in body imaging (C.B.S., 9 years of experience) in consensus and were entered into a computerized database. As explained elsewhere, patients were retrospectively assigned to a high-risk or a low-risk group (20). High-risk patients were those with hematuria or at least one fracture or dislocation of the sixth through 12th ribs, lumbar spine, or pelvic bones evident on radiographs. Low-risk patients had none of these predictors.
In injured patients, the specific organ(s) injured were recorded, along with the number, general location (intra-, extra-, or combined intra- and extraperitoneal), and severity of injuries (nonsurgical, surgical, or fatal). Nonsurgical injuries were defined as those that required no intervention or that were considered minor at autopsy. Surgical injuries were defined as those in which laparotomy was performed. The classification of injuries as surgical versus nonsurgical was based on the method of injury management. Our trauma surgeons were consistently conservative in their management of minor splenic and hepatic lacerations over the 9-year period, and management algorithms did not change; therefore, we made no attempt to reclassify injuries retrospectively. Fatal injuries were defined as those in which abdominal injury contributed to death. The classification of injuries as nonsurgical, surgical, or fatal was based on the review of all clinical records but was made without knowledge of US results. Because patients did not always undergo the same follow-up procedure(s), the best available data were used as the reference standard for each patient.
Definitions
US findings were classified as true-positive, false-positive, true-negative, or false-negative, as previously described (2,24). Briefly, a true-positive finding was defined as a positive US finding for which follow-up tests confirmed an injury. A false-positive finding was a positive US finding for which subsequent studies showed no evidence of injury. Medical ascites was considered a false-positive finding because additional studies were required to exclude injury definitively.
A true-negative finding was a negative US finding for which all subsequent imaging and other diagnostic studies were negative and/or the patients clinical course was benign after hemodynamic stabilization and clinical observation, without evidence of missed injury during admission, at follow-up clinic appointments, or during monthly audits of county trauma centers. A false-negative finding was a negative US finding for which any subsequent study showed evidence of abdominal injury (direct depiction of visceral injury, hemoperitoneum, free air, or unexplained simple free fluid). Positive US findings corresponding to CT abnormalities that were not definitely traumatic were considered false-positive for the purposes of this study. This was a conservative decision to minimize classification bias in favor of US.
Image Review
True-positive, false-positive, and false-negative sonograms were retrospectively reviewed by three body imaging faculty in consensus (G.C., C.B.S., and M.A.B., with 22, 9, and 8 years of experience, respectively), without knowledge of the reference standard assessment. The number of fluid pockets visualized retrospectively was recorded. A fluid pocket was defined as a region of fluid in a particular quadrant, gutter, or abdominopelvic recess (eg, right upper quadrant, left upper quadrant, right renal fossa, left renal fossa, right paracolic gutter, left paracolic gutter, pelvis).
Patients were then assigned a fluid score based on the number of visualized fluid pockets (0, 1, or
2; groups were combined because of low numbers), as described elsewhere (24). In one patient, US was terminated to expedite laparotomy after visualization of a single pocket of fluid; this patient was excluded from the fluid score analysis but included in the diagnostic performance analysis. Another patient, in whom US findings had been prospectively interpreted as negative, had an obvious pocket of free fluid at retrospective image review. This case was counted as false-negative in the assessment of diagnostic performance (error of observation), but the patient was assigned a fluid score of 1 for the fluid score analysis.
Statistical Analysis
The sensitivity, specificity, accuracy, positive predictive value (PPV), negative predictive value (NPV), and likelihood ratios of positive and negative findings were calculated (along with 95% confidence intervals) for all abdominal injuries, surgical injuries, and fatal injuries. The PPVs for these categories of injury were recalculated after patients were stratified into low- and high-risk groups. Sensitivities and specificities were recalculated after the exclusion of patients with parenchymal abnormalities but without fluid. The frequency of injury (any, surgical, and fatal) was also calculated for each fluid score. Finally, sensitivity and specificity were recalculated after patients were reclassified into three 3-year cohorts according to admission date (April 1994 through December 1996, January 1997 through December 1999, January 2000 through July 2003) to assess for temporal trends in diagnostic performance.
Proportions were compared by means of the
2 or Fisher exact test, depending on expected frequencies. Bonferroni correction was performed for multiple comparisons. Continuous variables were compared by means of the unpaired Student t test if normally distributed and by means of the Mann-Whitney U test if not. For comparisons that did not achieve statistical significance, the power to detect 20% differences in injury frequency was retrospectively calculated with actual sample sizes. Statistical analyses were performed by a trained statistician (G.P.S.) from the clinical research center at our institution by using software (SPSS; SPSS, Chicago, Ill).
| RESULTS |
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There were 77 male and 51 female patients. The mean patient age was 42 years, and mean ages in male and female patients were not significantly different (P > .10, unpaired Student t test). The systolic blood pressures at arrival ranged from 0 to 90 mm Hg for both male and female patients, with a median of 80 mm Hg. The median systolic blood pressures for male and female patients were not significantly different (P > .09, Mann-Whitney U test). Patient demographics, mechanism of injury, and distribution of arrival systolic blood pressure are summarized in Table 1, Table 2, and Table 3, respectively. Of the 128 patients, 59% (76 of 128) had no injuries, and 41% (52 of 128) had injuries. Injuries in 24% (31 of 128) required surgery, and those in 8% (10 of 128) were fatal.
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One of three false-positive findings was based on questionable splenic parenchymal heterogeneity not corroborated with follow-up imaging studies. Another was a case of cirrhosis and medical ascites prospectively interpreted as a nontraumatic abnormality; this finding is classified as false-positive because follow-up CT was required to exclude hemoperitoneum definitively. In the third false-positive finding, CT confirmed the presence of free fluid in the splenorenal fossa but also showed periportal and pericholecystic fluid; these findings were considered nontraumatic for the purposes of this study, as they may have been due to third-spacing of fluid related to vigorous fluid resuscitation rather than injury. Classifying this finding as false-positive rather than true-positive was a conservative analytic decision.
Overall, 30 of 47 patients (64%) with positive US findings underwent CT (23 patients, 49%), repeat US (two patients, 4%), or diagnostic peritoneal lavage (five patients, 11%) rather than exploratory laparotomy as the next diagnostic test, suggesting that their condition stabilized enough after arrival to enable additional testing despite the positive US findings. Another three patients (6%) died before further testing could be performed. Their injuries were confirmed at autopsy. The remaining 14 patients (30%) with positive US findings underwent therapeutic laparotomy without confirmatory tests.
Statistical Results
Table 6 summarizes the performance of screening US according to the type of injury sustained. The sensitivity of US was 85% (44 of 52 patients) for detection of injuries overall, 97% (30 of 31) for that of surgical injuries, and 100% (10 of 10) for that of fatal injuries. The corresponding specificities were 96% (73 of 76 patients), 82% (80 of 97), and 69% (81 of 118), and the corresponding accuracies were 91% (117 of 128), 86% (110 of 128), and 71% (91 of 128).
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The evaluation of organ parenchyma increased the sensitivity of US for detection of overall injury from 83% (43 of 52 patients) to 85% (44 of 52) and decreased the specificity from 97% (73 of 75) to 96% (73 of 76). It did not change the sensitivity for surgical or fatal injuries. The sensitivity (P = .66) and specificity (P = .31) were statistically similar for all 3-year cohorts; the sensitivity and specificity were 81% (13 of 16 patients) and 100% (24 of 24), respectively, for 19941996; 78% (seven of nine) and 96% (27 of 28) for 19971999; and 89% (24 of 27) and 91% (21 of 23) for 20002003.
The PPVs of screening US for all injuries, surgical injuries, and fatal injuries were then stratified by patient risk group (Table 7). In low-risk patients with positive US findings, the risk was 83% (15 of 18 patients) for overall injury, 61% (11 of 18) for surgical injury, and 11% (two of 18) for fatal injury. By comparison, the high-risk patients had higher frequencies for each injury category: 100% (29 of 29), 66% (19 of 29), and 28% (eight of 29), respectively. These differences did not reach statistical significance, however (P > .050 for all). Retrospectively, the power to detect a 20% difference in injury frequency with actual sample sizes ranged from 5% to 53%.
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2) (Table 8). The frequency of injury in patients with no fluid (including one with true-positive findings and one with false-positive findings due to parenchymal abnormality) was 10% for all injuries (eight of 82 patients), 1% for surgical injuries (one of 82), and 0% for fatal injuries (none of 82). Patients with two or more fluid pockets had significantly higher injury frequencies than those with no fluid for each injury category: 97% (32 of 33 patients), 79% (26 of 33), and 24% (eight of 33), respectively (P < .001 for all). Patients with a single fluid pocket also had higher injury frequencies: 92% (11 of 12 patients) (P < .001), 25% (three of 12) (P < .01), and 17% (two of 12) (P < .02), respectively.
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| DISCUSSION |
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The ability rapidly to exclude injury requiring surgery or other intervention in hypotensive patients with blunt abdominal trauma is of fundamental clinical importance. If such injury is excluded, the surgeon is alerted that the patients hypotension is due to extraabdominal causes. The surgeons attention then is correctly redirected to other anatomic regions, expediting appropriate management. Our findings suggest that US can play an important role in this clinical decision-making process. In our experience, abdominal injury requiring intervention is highly unlikely in hypotensive patients with negative US findings. The only surgical injury missed in the 9-year study period was a nonfatal mesenteric injury detected 37 minutes after US. No fatal injuries or nonsurgical injuries requiring transarterial embolization were missed.
Combining US results with risk group assessment further improves the ability to exclude surgical injury: Only two of 59 low-risk patients (3%) with a fluid score of 0 sustained injury, and the injury was not surgical or fatal in either patient. Thus, even in profoundly hypotensive patients, a fluid score of 0 and the absence of injury predictors effectively exclude surgical injury. Knowledge of this helps not only to expedite appropriate management of nonabdominal injuries but also to reduce the rate of unnecessary laparotomy. In our series, no hypotensive patient with suspected blunt abdominal trauma underwent unnecessary exploratory laparotomy.
Although screening US may fail to depict nonsurgical injuries, this failure is not clinically important in the emergent resuscitation setting, since acute management is unaltered. However, the trauma surgeon and the radiologist need to be aware of this possibility and obtain ancillary tests if indicated after the cause of the hypotension is identified and appropriately treated.
Although nationally there has been a shift toward more conservative treatment of hepatic and splenic injuries, we made no attempt to reclassify injuries retrospectively according to current algorithms. We believe this decision was justified, as our trauma surgeons had a consistently conservative approach throughout the 9-year study period.
Recently, objective predictors (hematuria, axial fracture) of false-negative examination results and missed injuries have been reported (20). Follow-up CT or other testing was recommended routinely in all patients with one or more injury predictors, even if results of screening US were negative. With these new guidelines, six of seven patients with nonsurgical injuries and one patient with a surgical injury missed at screening US in this study would have been correctly triaged for additional evaluation. The only injury that would have been missed with use of this algorithm was a minor nonsurgical splenic injury (American Association for the Surgery of Trauma grade 1), in a patient without hemoperitoneum, that was detected with CT 2
hours after initial US.
As discussed elsewhere (20), high-risk patients had false-negative US findings eight times more frequently than low-risk patients. This emphasizes that US is a screening test for blunt abdominal trauma, not a definitive test. In the presence of objective predictors, hypotensive patients with trauma and negative findings at US should undergo confirmatory CT to exclude missed injury if they can be stabilized. If stabilization is not possible, US should be repeated because it may take time for hemoperitoneum to accumulate. Diagnostic peritoneal lavage or exploratory laparotomy may be necessary.
In contrast, the large majority (94%) of hypotensive patients with blunt abdominal trauma and positive US findings had injuries, but only 64% had surgical injuries. There are likely two reasons for the relatively low PPV for surgical injury. First, to maximize the sensitivity of screening US, radiologists at our institution have been trained to use a low threshold for calling into question any potential abnormality. Consequently, scans that demonstrated questionable, probably nontraumatic, or artifactual findings were prospectively read as positive. Second, and more important, not all hypotensive patients with actual abdominal injuries require surgical intervention. This emphasizes that hypotensive patients with positive US findings should undergo confirmatory tests, if possible, prior to laparotomy.
Assessment of fluid score can further help to delineate patient risk and guide management in patients with blunt abdominal trauma and positive findings at screening US. In our series, a fluid score of 1 was associated with a 25% risk of surgical injury, and that risk increased to 79% with a fluid score of 2 or higher. Thus, although hypotensive patients with a fluid score of 2 are at high risk of having surgical injury, 21% were successfully managed without surgery. This result suggests that even these patients should undergo confirmatory CT prior to laparotomy if they can be stabilized. A fluid score of 1 was not as useful in predicting surgical injury; although a large majority (92%) of these patients had injuries, only 25% required surgery.
Given our findings, we now propose a modified triage algorithm that incorporates patient stability, fluid score, and risk group assessment (Figure). Hemodynamically unstable patients are initially screened with US. If the US findings are negative, other sources of hypotension should be considered and treated. Although the risk of surgical injury in this group is extremely low, CT can be performed after successful stabilization, especially if risk predictors for injury are known or suspected. If the US findings are positive and the patients condition can be stabilized, CT is performed to guide management and identify patients who require surgery or other intervention.
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One limitation of our study was that only the arrival blood pressure was documented in the trauma registry; follow-up blood pressure values were not available. Thus, there was no prospective record of how many patients with positive US findings could or could not be stabilized during resuscitation. Moreover, we could not identify patients who became hypotensive after arrival in the resuscitation suite. Hence, there were likely additional unstable patients with blunt abdominal trauma who were not included in our study population.
Another limitation is that our study population was nonconsecutive. US was not available 24 hours a day throughout the study period; patients who presented when US was unavailable underwent other ancillary tests and were not included. In facilities where 24-hour in-house US is not available, hypotensive patients should undergo CT if possible.
A third limitation is that a surgical reference standard was not available in the majority of our patients. McGahan and Richards (8) argued that the use of clinical improvement as a reference standard is imperfect because minor injuries improve clinically with medical treatment and may be missed. To optimize clinical follow-up, we limited our study to patients who met Major Trauma Outcome Study entry criteria (22). Thus, all surviving patients were admitted to the intensive or intermediate care units, where continuous monitoring is possible, or to the surgical ward for a minimum of 72 hours. Patients who were discharged after screening US without admission did not meet Major Trauma Outcome Study entry criteria and were not included. Unfortunately, these patients are not recorded in the trauma registry, and it is not possible to determine their number retrospectively.
Finally, all county trauma centers were audited monthly to detect potential missed injuries after discharge. Although some discharged patients with missed injuries may have presented to institutions outside the county, this was probably uncommon. More important, our patient population consisted of patients who were hypotensive at arrival in the trauma suite. In these patients, nonsurgical injuries undetected with US during the acute resuscitation period are of little consequence, as long as the trauma surgeon and the radiologist are aware of the potential limitations of US. Although differences in equipment over a 9-year study period may theoretically be a study limitation, sensitivities and specificities were similar among the three 3-year cohorts, which suggests that any equipment-related bias was likely small.
As shown previously, US screening for free fluid is of limited use for detection of injury with no or delayed hemoperitoneum (2,2729). For this reason, our US protocol includes organ parenchymal evaluation, unlike some protocols used by others (511,15,27,28,30). In this study, the benefit of parenchymal evaluation was small; evaluation of organ parenchyma increased overall sensitivity from 83% to 85% but did not change the sensitivity for surgical or fatal injuries. Because the amount of additional time required by experienced sonographers to evaluate solid organs is small but not negligible, this observation brings into question the incremental cost-effectiveness of parenchymal evaluation in hypotensive patients, which needs to be addressed prospectively.
Similarly, US has been unreliable for detection of retroperitoneal injuries (31,32). In our study, 19 injuries in eight patients were missed, of which 12 were extraperitoneal. CT later demonstrated each injury. These retroperitoneal injuries were not life-threatening, however, and it is not clear that earlier detection would have changed management or improved patient outcome.
In conclusion, our findings show that US is a highly sensitive and accurate test for the detection of injuries requiring intervention in the hemodynamically unstable patient with blunt abdominal trauma. Given the potential for rapid deterioration in these patients, CT can be problematic or contraindicated. We report that screening US, sonographic fluid score assessment, and risk group analysis are useful for predicting the risk of surgical injury and therefore can be used in an algorithm to guide clinical decision making and patient care. A completely negative sonogram virtually excludes abdominal injury requiring intervention, allows the trauma surgeon to search for nonabdominal causes of hypotension, and minimizes the risk of nontherapeutic laparotomy. In contrast, a patient with positive US findings and a fluid score of 2 or more is highly likely to require surgery. If the condition of a patient with positive US findings can be stabilized with fluid resuscitation, follow-up CT or other imaging is recommended for more definitive assessment of injuries and to guide appropriate management. When fluid resuscitation cannot be achieved, however, patients should undergo emergent exploratory laparotomy, especially if multiple pockets of fluid are seen.
| FOOTNOTES |
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Authors stated no financial relationship to disclose.
Author contributions: Guarantors of integrity of entire study, C.B.S., N.F.; study concepts, C.B.S., N.F., G.C., M.A.B., D.F., D.B.H.; study design, C.B.S., N.F., G.C., G.P.S.; literature research, C.B.S., N.F., M.A.B.; clinical studies, C.B.S., G.P.S., D.B.H., M.A.B.; data acquisition, C.B.S., N.F., M.A.B., D.F., D.B.H.; data analysis/interpretation, C.B.S., N.F., G.C., G.P.S.; statistical analysis, C.B.S., N.F., G.P.S; manuscript preparation, C.B.S., N.F., G.P.S.; manuscript definition of intellectual content, C.B.S., N.F., G.C., D.F., D.B.H., G.P.S.; manuscript editing, C.B.S., N.F., G.C.; manuscript revision/review and final version approval, all authors
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