Published: February 2014
Acute pancreatitis (AP) is an inflammatory condition of the pancreas that can extend to extrapancreatic tissues. AP is broadly classified as mild or severe. Mild AP is often referred to as interstitial pancreatitis, based on its radiographic appearance. The pancreatic blood supply is preserved in interstitial pancreatitis. Severe AP implies persistent failure of one or more end organs.
Different types of local complications can develop in the setting of AP. These include acute peripancreatic fluid collection, acute pseudocyst, pancreatic necrosis and walled off pancreatic necrosis (WOPN). Pancreatic necrosis forms when there is a disruption of the pancreatic blood supply. Walled off pancreatic necrosis refers to a collection containing pancreatic and peripancreatic necrotic debri and liquefied dead tissues encased by a fibrous tissue wall. WOPNs usually develop over 4 weeks in necrotizing pancreatitis.
Acute fluid collections can form in the peripancreatic areas and are not encapsulated by a fibrous wall. Acute pseudocysts are well-developed peripancreatic fluid collections of pancreatic juice encapsulated by a nonepithelialized wall of granulation tissue (Figure 1). Pseudocysts typically form 4 weeks after an episode of AP.
Terms such as pancreatic phlegmon, hemorrhagic pancreatitis and pancreatic abscess are no longer used in the revised Atlanta classification.1
When acute pancreatitis occurs on two or more occasions, it is classified as acute recurrent pancreatitis. In some cases, acute recurrent pancreatitis progresses to chronic pancreatitis, characterized by parenchymal fibrosis and loss of exocrine function.
The yearly incidence of AP in the United States is approximately 32-44 new cases per 100,000 popuation and has increased over the last decade. This increase in incidence has been observed worldwide. Acute pancreatitis is the most common cause of GI related hospitalizations, with more than 274,000 hospitalizations in 2012. Eighty percent of cases of AP are mild; the remaining 20% are severe. The overall mortality rate for AP is around 5%.
Gallstones and alcohol are the two most common causes of AP in Western countries, accounting for 80% of cases. Gallstone (biliary) pancreatitis results from transient obstruction of the ampulla of Vater by small stones or crystals (microlithiasis). Clinical features suggesting biliary pancreatitis include preceding biliary colic, the presence of cholelithiasis or biliary dilation on gallbladder ultrasound, and liver function test abnormalities. Biliary pancreatitis typically does not recur after cholecystectomy or endoscopic therapy (biliary sphincterotomy and stone extraction). Alcohol is the second leading cause of AP. Acute recurrent pancreatitis from alcohol often leads to chronic pancreatitis.
Numerous less-common causes have been described (Table 1). Hypertriglyceridemia produces acute pancreatitis if triglyceride levels are above 1000 mg/dL. Therefore, triglyceride level should be routinely checked in patients presenting with AP within 24 hours of presentation. Markedly elevated triglyceride levels may be encountered in the setting of diabetes, alcoholism, and inherited disorders of lipoprotein metabolism (Fredrickson types I, II, and V). Hypercalcemia produces AP through calcium-mediated activation of trypsinogen . Hypercalcemia-associated AP can occur in the setting of primary and secondary hyperparathyroidism, malignancy, and metabolic bone disease. Certain medications (e.g., sulfa drugs, 6-mercaptopurine, didanosine, furosemide, and valproate) have also been implicated as causes of acute pancreatitis.
|Causes of Acute Pancreatitis|
|After endoscopic retrograde cholangiopancreatography|
Obstruction of the pancreatic duct can produce acute or chronic pancreatitis. Causes of obstructive AP include ductal adenocarcinoma, ampullary tumors and polyps, neuroendocrine and cystic pancreatic tumors, and intraductal papillary mucinous tumors. Congenital abnormalities such as pancreas divisum and annular pancreas can also result in obstructive AP.
The pathogenesis of AP has been studied extensively using animal models. Although different etiologies produce distinct inciting events, the final common pathway is premature activation of enzymes within the acinar cell. Ordinarily, pancreatic proenzymes become activated on release within the duodenum. Pancreatitis results when activation of pancreatic enzymes occurs early within the acinar cells, producing autodigestion of the pancreas and surrounding tissues. Exposure of trypsinogen to lysosomal enzymes such as cathepsin B has been shown as a mechanism for early trypsin activation.
Signs and Symptoms
Acute pancreatitis manifests with the sudden onset of epigastric pain radiating to the back. The pain may be severe and worsens after meals. Abdominal pain can last for days and is associated with anorexia, nausea, and vomiting. In alcohol-related AP, pain occurs from hours to days after binge drinking. Most patients present to the emergency department. However, occasional patients manage their symptoms at home by minimizing oral intake for a few days.
Physical examination often reveals systemic signs such as fever, tachycardia, and hypotension. Abdominal examination reveals epigastric tenderness, with localized guarding and rebound. Sluggish or absent bowel sounds indicate coexisting ileus. Less-frequent findings signal complications, including Grey Turner’s (flank ecchymosis) or Cullen’s (umbilical ecchymosis) signs suggesting retroperitoneal hemorrhage, a palpable mass suggesting a pseudocyst, panniculitis suggesting subcutaneous fat necrosis, and dullness to percussion of lung fields suggesting pleural effusion. The differential diagnosis of upper gastrointestinal bleeding in acute pancreatitis includes erosion of a pseudocyst into the splenic artery (hemosuccus pancreaticus) or bleeding from gastric varices that arise secondary to splenic vein thrombosis.
The diagnosis of AP is made when any two of the following three criteria are met. 1) Classic abdominal pain 2) Radiographic evidence of acute pancreatitis 3) Elevation of amylase and/or lipase three times upper limit of normal. Pancreatic enzyme levels are elevated because of leakage from pancreatic acinar cells into the interstitial space and subsequent absorption into the circulation. The amylase level becomes elevated within hours of the development of pain and can remain elevated for 3 to 5 days. The differential diagnosis for hyperamylasemia includes intestinal obstruction, visceral perforation, tubo-ovarian abscess, renal failure, and salivary gland disease. Macroamylasemia is a condition in which amylase is chronically elevated because of its binding to an abnormal serum protein, leading to delayed clearance. Serum lipase has higher specificity for pancreatic disease, but its level may be elevated in other conditions as well. The severity of pancreatitis does not correlate well with the magnitude of elevation of the serum amylase and lipase levels. There is no value in following daily trends of serum amylase and lipase levels because they do not correlate with recovery or prognosis.
Laboratory abnormalities encountered in AP include hyperglycemia, hypocalcemia, leukocytosis, and mild elevations of liver function test results. Elevation of the serum alanine aminotransferase level to greater than 80 U/mL is highly specific and poorly sensitive for biliary pancreatitis. Laboratory markers that reflect intravascular volume depletion – such as elevated BUN >20mg/dL and hemoconcentration have been recognized as important markers of severity in AP.2-4 Following these markers may also gauge the adequacy of fluid resuscitation.
Plain films of the chest and abdomen are appropriate for the initial radiographic assessment of AP. An abdominal radiograph is helpful for excluding other causes of acute abdominal pain, such as obstruction and perforation. In AP, the abdominal radiograph is typically normal or may demonstrate a localized upper quadrant ileus. A chest radiograph can detect pulmonary complications of AP such as atelectasis, pleural effusions (most commonly left-sided), or infiltrates suggesting acute respiratory distress syndrome.
Although transabdominal ultrasound is poorly reliable for imaging the pancreas itself, it is the best initial radiographic test for the evaluation of mild AP. Transabdominal US detects gallstones, sludge, and biliary dilation indicating a biliary source, and rules out acute cholecystitis.
Contrast-enhanced computed tomography (CT) of the abdomen is the preferred test for detecting complications. CT features in interstitial pancreatitis include homogenous contrast enhancement; diffuse or segmental pancreatic enlargement; irregularity, heterogeneity, and lobularity of the pancreas; and obliteration of the peripancreatic fat planes. Contrast enhanced CT detects areas of pancreatic necrosis (Figure 2), which carries significant morbidity and increased mortality if infected or associated with organ failure. A CT should not be routinely ordered for all patients with AP; however, the American College of Gastroenterology (ACG) practice guidelines state that “a dynamic contrast-enhanced CT is recommended at some point beyond the first 3 days in clinically severe acute pancreatitis (on the basis of a high APACHE score or persistent organ failure) to distinguish interstitial from necrotizing pancreatitis.” It is unlikely that intravenous contrast worsens or precipitates pancreatic necrosis, and abdominal CT should generally not be withheld on this basis. A CT may also be considered for those in whom a localized pancreatic complication is suspected (e.g., pseudocyst, splenic vein thrombosis, splenic artery aneurysm). A CT is also appropriate 4 to 6 weeks after resolution of AP to exclude a tumor if the cause of the attack is unclear.
Endoscopic ultrasound (EUS) and magnetic resonance cholangiopancreatography (MRCP) are emerging as potentially valuable tests in the evaluation of AP. Both are helpful in detecting stones in the common bile duct and in directly assessing the pancreatic parenchyma. Magnetic resonance imaging is similar or superior to contrast CT in its ability to stage AP and detect necrosis and complications, and it does not require intravenous contrast.
Assessment of Severity
An important initial step in management is the assessment of severity by clinical and radiographic criteria. Severe AP implies persistent organ failure with/without infected pancreatic necrosis and carries a mortality rate exceeding 30%. Although in the original Atlanta classification definition of severe AP included those with acute pseudocyst and pancreatic necrosis, it is now recognized that sterile pancreatic necrosis or pseudocyst without organ failure, has a mortality rate comparable to interstitial AP. Therefore, patients with sterile local complications (i.e. pseudocyst, necrosis or acute fluid collection), are categorized “moderately” severe due to the high morbidity but low mortality rate. Early recognition of severe pancreatitis improves outcomes by prompting aggressive fluid resuscitation and transfer of the patient to an intensive care unit.
Several clinical and radiographic severity scores have been proposed. The Ranson score was developed for alcoholic AP and comprises five clinical criteria measured at admission and six clinical criteria measured at 48 hours (see Table 2). The criteria measured at admission reflect the local inflammatory effects of pancreatic enzymes; those measured at 48 hours represent the later systemic effects. Three or more Ranson criteria predict a severe course and increased mortality. The APACHE II score (acute physiology, age, chronic health evaluation: http://www.mdcalc.com/apache-ii-score-for-icu-mortality/) is generated from multiple physiologic and disease parameters. The calculation of APACHE scores helps predict severity from the day of admission and may be recalculated on a daily basis. More recently, Bedside Index of Severity in Acute Pancreatitis (BISAP) was developed as a simpler and equally accurate prediction tool (see Table 3).5 This severity index comprises of five clinical variables that are routinely available on hospital admission and has been prospectively validated. A score of 3 or more was associated with significantly increased mortality with equal discrimination as APACHE.
Table 2. Ranson Criteria for Severity of Acute Pancreatitis
|Age >55 yr|
|LDH >350 IU/L|
|AST >250 IU/L|
|Glucose >200 mg/dL|
|At 48 Hours|
|Hematocrit decrease >10%|
|BUN increase >5 mg/dL|
|Calcium <8 mg/dL|
|Pao2 <60 mm Hg|
|Base deficit >4 mg/dL|
|Fluid sequestration >6 L|
AST, aspartate aminotransferase; BUN, blood urea nitrogen; LDH, lactate dehydrogenase; Pao2, partial pressure of arterial carbon dioxide; WBC, white blood cell.
Table 3. BISAP score
|Clinical variables – 1 point allocated per variable|
|BUN >25mg/dL (1)|
|Impaired mental status (1)|
2 or more of following:
|Age >60 (1)|
|Pleural effusion (1)|
|Possible scores and corresponding mortality (%)|
|0 : 0.2|
|1 : 0.6|
|2 : 2|
|3 : 5-8|
|4 : 13-19|
|5 : 22-27|
In addition to formal scoring systems, patients should be followed closely for other markers of increased severity, including signs of hemodynamic instability or organ failure. Respiratory failure can occur through the development of large pleural effusions or acute respiratory distress syndrome. Rarely, hemorrhage into retroperitoneal tissues causes further hemodynamic compromise. Extra vigilance for these complications will result in more timely and aggressive management and improve patient outcomes.
The primary goals of therapy in AP are meticulous supportive care and prevention of pancreatic necrosis, infection, and organ failure. The primary treatment is pancreatic rest and analgesia. Patients should be given nothing by mouth, and intravenous fluids should be given with careful attention to volume status. The ACG guidelines state, “all patients should receive close supportive care including pain control, fluid resuscitation, and nutritional support.” A therapeutic algorithm closely based on the ACG guidelines is shown in (Figure 3).
The importance of vigorous hydration to optimize outcomes has been increasingly recognized. The ACG guidelines stress, “Patients with evidence of significant third-space losses require aggressive fluid resuscitation.” Many patients sequester substantial amounts of fluid into the retroperitoneal space, producing very high fluid requirements. Intravascular volume depletion can lead to tachycardia, hypotension, renal failure, elevated BUN, hemoconcentration, and generalized circulatory collapse. More than 6 L of fluid sequestration within the first 48 hours is considered a marker of increased severity, according to the Ranson criteria (Table 2). Patients with evidence of hemoconcentration resulting from intravascular water loss appear to be at increased risk for the development of pancreatic necrosis and organ failure. In addition to maintenance fluid requirements, the amount sequestered should be monitored and replaced with isotonic fluids such as normal saline or lactated ringers. Fluid therapy should be goal directed, with close monitoring of urine output and BUN levels. Studies have shown that in patients with elevated BUN, failure to decrease over 24 hours was associated with significant mortality. Some patients require as much as 250 to 350 mL/hour, particularly in the early phases of AP. Of course, the aggressiveness of fluid replacement must be tempered in the elderly and in the presence of underlying cardiac or renal disease.
Oral intake should be severely limited initially and then carefully advanced as pain subsides and hunger returns. A nasogastric tube is necessary only in the presence of vomiting or ileus. Intravenous narcotics by injection or by patient-controlled analgesia should be used liberally during the attack and tapered as the diet is advanced to enable prompt bowel recovery. Carbohydrates are best for early refeeding because they do not stimulate the pancreas as much as fats and proteins.
The ACG guidelines advise nutritional support if NPO status is maintained for longer than 5 to 7 days. Patients with severe AP should receive early nutritional support because of its inherently high level of stress and hypercatabolism. Nasojejunal feeding past the ligament of Treitz does not stimulate the pancreas and is preferred over parenteral feeding in AP. The clinical benefits of enteral over parenteral feeding has been shown in multiple studies. A recent Cochrane meta-analysis found a significantly reduced rate of multi-organ failure, systemic infection, need for operative intervention and mortality.6 Physiologic basis for such benefits include improved humoral and cellular immunity, decreased systemic inflammatory response, and decreased bacterial translocation for enteral feeding compared with parenteral nutrition. The presence of a severe intestinal ileus or delay in tube placement can limit the use of enteral feeding. Many patients with AP develop gastric and colonic ileus but maintain adequate small bowel motility for enteral feeding.
Prevention of Infection
Superinfection of pancreatic necrosis dramatically increases the mortality rate of AP . The ACG guidelines recommend that “in patients with necrotizing pancreatitis associated with organ failure, it is reasonable to initiate treatment with antibiotics with good spectrum of activity against aerobic and anaerobic bacteria.” However, few randomized trials have shown a benefit for ‘prophylactic’ broad-spectrum antibiotics in necrotizing AP. Potential drawbacks of prophylactic antibiotics include the development of resistant organisms and fungal infections. If antibiotics are used, antibiotics with good pancreatic tissue penetration, such as imipenem (500 mg IV every 8 hours), cefuroxime (1.5 g IV every 8 hours), or ciprofloxacin (400 mg IV every 12 hours) are favored in this setting.
Endoscopic Retrograde Cholangiopancreatography (ERCP)
Although the ACG guidelines state that “patients with severe pancreatitis caused by gallstones should undergo urgent ERCP,” there has been much recent debate over the benefit of early endoscopic removal of common bile duct stones in suspected gallstone pancreatitis. This is because most stones pass spontaneously and ERCP can further exacerbate acute pancreatitis. There is strong evidence to suggest a benefit for ERCP with papillotomy and stone extraction in the setting of stone impaction and cholangitis.7 However, randomized trials of early ERCP in the management of all patients with suspected gallstone pancreatitis have shown conflicting results. Guidelines from the British Society of Gastroenterology advise that “severe gallstone pancreatitis in the presence of increasingly deranged liver function tests and signs of cholangitis (fever, rigors, and positive blood cultures) require an immediate and therapeutic ERCP.” If there is only moderate suspicion of retained stones, EUS and MRCP are less risky alternatives to ERCP, with excellent sensitivity for the detection of common bile-duct stones.
Invasive management of AP is indicated in two clinical settings: infected pancreatic necrosis and gallstone pancreatitis.
Infected Pancreatic Necrosis
Infected Pancreatic Necrosis. Confirmation of infected pancreatic necrosis is important because an intervention to debride necrotic tissue or provide drainage to control sepsis is indicated. Diagnosis of infected pancreatic necrosis may be suspected clinically or radiographically (e.g. gas in pancreatic bed), or can be made through ultrasound- or CT-guided aspiration of areas of pancreatic necrosis. This procedure is safe and reliable, and it has been recommended for patients with CT criteria for pancreatic necrosis and evidence of sepsis or organ failure. Presence of gas within the pancreatic or peripancreatic collection is also considered strongly suggestive of infection.
Management of infected pancreatic necrosis has evolved dramatically over the last few decades from primary open debridement early in the course of disease to a delayed minimally invasive approach, utilizing radiologic, laparoscopic or endoscopic techniques. Studies — including a recent randomized control trial — have shown that delayed, minimally invasive interventions lead to better outcomes and fewer complications.8-10 Postponing debridement allows encapsulation of the area and adequate separation of necrotic and vital areas. Therefore, when possible, necrosectomy should be postponed for 3-4 weeks from symptom onset. All patients with suspected or proven infected necrosis should receive broad-spectrum antibiotics Debridement involves resection of all devitalized pancreatic and surrounding tissue – which can be achieved through open surgery, laparoscopically or endoscopically. Multiple procedures may be required for adequate debridement. However, in some patients with infected necrosis, percutaneous or endoscopic drainage alone may postpone or even spare the need for necrosectomy.
Cholecystectomy is indicated to prevent recurrence of gallstone pancreatitis. In mild disease, an early cholecystectomy performed during the same hospitalization is favored. In severe gallstone pancreatitis, cholecystectomy may be delayed until the patient has clinically improved.
Idiopathic Acute Pancreatitis
Ten percent of pancreatitis cases are idiopathic (IAP). Potential underlying causes include biliary microlithiasis, sphincter of Oddi dysfunction, and undiagnosed genetic defects.
Biliary microlithiasis has been implicated as a common cause of IAP. Recurrent acute episodes can develop in the absence of gallstones on ultrasound, with or without elevated liver enzyme levels. Repeat ultrasound examinations might eventually reveal biliary sludge or small stones. The finding of cholesterol monohydrate or calcium bilirubinate crystals on microscopic bile analysis strongly supports the diagnosis of microlithiasis; however, it is not completely sensitive. Laparoscopic cholecystectomy prevents recurrence in patients with IAP and should be considered in all patients with acute recurrent pancreatitis of unclear cause. Endoscopic sphincterotomy or stone dissolution therapy with ursodiol (8-10 mg/kg/day, in two divided doses) are valid alternatives in patients with high surgical risk. Stone dissolution therapy is effective only for noncalcified, cholesterol monohydrate stones smaller than 1 cm in diameter.
MRCP is a useful first line test for detecting obstructive pathology of the pancreatic duct, such as ductal strictures, mucinous ductal ectasia, common bile duct stones, and pancreas divisum. Endsocopic ultrasound (EUS) is another sensitive structural test for detecting ampullary, biliary, and pancreatic causes of AP. When ductal pathology is found, ERCP may be considered to provide therapy such as stone removal and dilation and stenting of strictures. ERCP with sphincter of Oddi manometry and sphincterotomy may be considered when sphincter of Oddi dysfunction is strongly suspected.
There are no published guidelines for the approach to recurrent IAP. Most clinicians do not favor extensive evaluation for the first episode of IAP, because it does not recur in most patients after the first episode; however, CT after resolution is probably reasonable for excluding pancreatic cancer. It is best to tailor the diagnostic approach individually based on patient characteristics. One suggested approach to the patient with idiopathic recurrent acute pancreatitis is demonstrated in Figure 4.
Mortality rates for hospitalized patients vary from 5% to 10% in most series. It is now recognized that the most important determinants of mortality in acute pancreatitis are persistent organ failure and infected necrosis. In patients with uncomplicated interstitial pancreatitis and sterile necrosis without associated organ failure, mortality is close to zero.11-13 However, mortality rate is higher in patients with infected local complications (e.g. infected necrosis or pseudocyst) and exceeds 30% in patients with both persistent organ failure and infected necrosis.14
- The management of acute pancreatitis includes meticulous supportive care and aggressive but careful volume resuscitation.
- Assessment of severity is an important initial step in the care of all patients with acute pancreatitis.
- A contrast-enhanced pancreatic CT scan should be considered for patients with severe acute pancreatitis.
- Endoscopic retrograde cholangiopancreatography should be performed in patients with gallstone pancreatitis and signs of ongoing biliary obstruction or cholangitis.
- Nutritional support should be administered to all patients with prolonged NPO status or severe acute pancreatitis.
- Surgical consultation and percutaneous aspiration of pancreatic necrosis should be considered for patients with clinical deterioration or multiorgan system failure.
- Lee SP, Nicholls JF, Park HZ: Biliary sludge as a cause of acute pancreatitis. N Engl J Med 1992;326:589-593.
- American College of Gastroenterology: Practice Guidelines: Acute Pancreatitis, 2006. Available at gi.org/physicians/guidelines/AcutePancreatitis.pdf.
- van Brunschot S, Bakker OJ, Besselink MG, et al. Treatment of necrotizing pancreatitis. Clin Gastroenterol Hepatol. 2012;10(11):1190-1201.
- Yadav D, Whitcomb DC. The role of alcohol and smoking in pancreatitis. Nat Rev Gastroenterol Hepatol. 2010;7(3):131-145.
- Banks PA, Bollen TL, Dervenis C, et al. Classification of acute pancreatitis–2012: Revision of the atlanta classification and definitions by international consensus. Gut. 2013;62(1):102-111.
- Brown A, Orav J, Banks PA. Hemoconcentration is an early marker for organ failure and necrotizing pancreatitis. Pancreas. 2000;20(4):367-372.
- Wu BU, Johannes RS, Sun X, Conwell DL, Banks PA. Early changes in blood urea nitrogen predict mortality in acute pancreatitis. Gastroenterology. 2009;137(1):129-135.
- Wu BU, Bakker OJ, Papachristou GI, et al. Blood urea nitrogen in the early assessment of acute pancreatitis: An international validation study. Arch Intern Med. 2011;171(7):669-676.
- Wu BU, Johannes RS, Sun X, Tabak Y, Conwell DL, Banks PA. The early prediction of mortality in acute pancreatitis: A large population-based study. Gut. 2008;57(12):1698-1703.
- Al-Omran M, Albalawi ZH, Tashkandi MF, Al-Ansary LA. Enteral versus parenteral nutrition for acute pancreatitis. Cochrane Database Syst Rev. 2010;(1):CD002837. doi(1):CD002837.
- Tse F, Yuan Y. Early routine endoscopic retrograde cholangiopancreatography strategy versus early conservative management strategy in acute gallstone pancreatitis. Cochrane Database Syst Rev. 2012;5:CD009779.
- Bakker OJ, van Santvoort HC, van Brunschot S, et al. Endoscopic transgastric vs surgical necrosectomy for infected necrotizing pancreatitis: A randomized trial. JAMA. 2012;307(10):1053-1061.
- van Santvoort HC, Besselink MG, Bakker OJ, et al. A step-up approach or open necrosectomy for necrotizing pancreatitis. N Engl J Med. 2010;362(16):1491-1502.
- van Santvoort HC, Bakker OJ, Bollen TL, et al. A conservative and minimally invasive approach to necrotizing pancreatitis improves outcome. Gastroenterology. 2011;141(4):1254-1263.
- Talukdar R, Clemens M, Vege SS. Moderately severe acute pancreatitis: Prospective validation of this new subgroup of acute pancreatitis. Pancreas. 2012;41(2):306-309.
- Tenner S, Sica G, Hughes M, et al. Relationship of necrosis to organ failure in severe acute pancreatitis. Gastroenterology. 1997;113(3):899-903.
- Singh VK, Bollen TL, Wu BU, et al. An assessment of the severity of interstitial pancreatitis. Clin Gastroenterol Hepatol. 2011;9(12):1098-1103.
- Petrov MS, Shanbhag S, Chakraborty M, Phillips AR, Windsor JA. Organ failure and infection of pancreatic necrosis as determinants of mortality in patients with acute pancreatitis. Gastroenterology. 2010;139(3):813-820.