Abstract
Radiofrequency ablation is a safe and effective treatment for primary and secondary liver malignancies and has a low complication rate; however, there are various radiofrequency ablation-related complications which can occur from the thorax to the pelvis. Although most of these complications are usually minor and self-limited, they may become fatal if diagnosis and treatment are delayed. It is important for radiologists performing radiofrequency ablation to have a perspective regarding the possible radiofrequency ablation-related complications and their risk factors as well as the radiologic findings for their timely detection and increase of the treatment efficacy, and thereby encouraging the use of the radiofrequency ablation technique. This article illustrates the various imaging features of common and rare radiofrequency ablation-related complications as well as offers technical tips in order to avoid these complications.
Introduction
Radiofrequency (RF) ablation is a treatment technique designed to cause tumor destruction by the localized application of thermal energy. RF ablation is well-known as a safe, well-tolerated, and effective treatment for primary and secondary liver malignancies. It also has important advantages, including its minimal invasiveness, easy repeatability in the event of recurrences, and wider indication for those patients who are not surgical candidates (1–5). Although RF ablation has evolved as a promising modality for tumor ablation, very diverse RF ablation-related complications can occur anywhere from the thorax to the pelvis. Although most complications are usually self-limited with conservative therapy, they may become fatal if the diagnosis and treatment are delayed. The complication rate reported in published literature reports is low with the mortality rate varying from 0 to 1.6% and the complication rate between 0 and 17% (6–9). Several complications, including bleeding, infection, and injury to blood vessels, bile ducts, the diaphragm, or abdominal organs, are prone to occur in patients with risk factors (6). Knowledge regarding these risk factors for various RF ablation-related complications and their radiologic findings may allow physicians to detect complications in a timely manner and to, therefore, increase the treatment efficacy, thus promoting the use of the RF ablation technique. The purpose of this article is to illustrate the various imaging features of common and rare RF ablation-related complications as well as to offer technical tips for avoiding these complications. Complications are classified as being early (≤2 weeks) or delayed (>2 weeks).
Early complications (≤2 weeks)
Thoracic complications
Right pleural effusion commonly occurs following RF ablation. In a previous report, the incidence of symptomatic pleural effusion after RF ablation was reported as 1.8%, but all of these patients improved with thoracentesis and/or diuresis within 3 weeks (10). The reflexion of the diaphragm attaches at the midaxillary line of the 12th rib, inferiorly. Therefore, thoracic complications, including hemothorax or pneumothorax, can occur by traversing of the diaphragm and the pleura with an RF electrode, especially when managing a mass located in the hepatic dome when using a percutaneous intercostal approach (Fig. 1) (1,11). Careful selection of a safe window and needle pathway with real-time monitoring is important in order to avoid diaphragmatic and pleural injury. Although rare, pericardial injury may also occur. Chest radiography or a computed tomography (CT) scan is recommended if dyspnea or chest pain occurs following RF ablation. Most thoracic complications are self-limited when using conservative therapy, however, interventional treatment should be considered on a case-by-case basis.
Hemothorax in a 62-year-old man who underwent RF ablation for hepatocellular carcinoma. (a) Immediate post-ablation CT scan shows right hemothorax; (b) angiogram shows contrast leakage (arrow) from the right, ninth intercostal artery, which was embolized with gelfoam and microcoils.
Vascular complications
Portal vein or hepatic vein thrombosis usually manifests soon after RF ablation (12). In one published study, portal vein thrombosis occurred at a rate of 1.7% (13). In cases of portal vein thrombosis, small-caliber (<4 mm) vessels are prone to thrombosis because the heat-sink effect depends on the size of the vessel and the amount of blood flow through it (12,14,15). However, if the blood flow is decreased, thermal damage can cause thrombosis even in relatively large diameter vessels. A centrally located tumor, compression of the vein, and mechanical damage of the vessel by an electrode can also be considered as risk factors. CT may depict the thrombus as a filling defect in the portal vein, commonly with segmental arterial hyperperfusion of the hepatic parenchyma peripheral to the affected portal vein. The mechanism involved in hepatic vein thrombosis is thought to be similar to that in portal vein thrombosis. The incidence of hepatic vein thrombosis has been reported as 1.4% (13). CT scans obtained during the portal venous phase may reveal a lack of the opacification of the hepatic vein. Portal vein branches usually lie in the boundary of a wedge-shaped area of decreased enhancement (16). This feature may be useful for differentiating hepatic venous congestion from portal ischemia. Vascular complications are conservatively managed in most patients, sometimes with the prophylactic use of antibiotics.
Postablation syndrome
Postablation syndrome is defined as a combination of flulike symptoms, including low-grade fever, malaise, chill, pain at the treatment site, nausea, and vomiting. The reported prevalence of these symptoms after RF ablation for hepatic tumors is approximately 36–95% (17,18). The symptoms usually present within 1–3 days of the procedure (17,18). Large tumor volume, ablated tissue volume, the difference between the two, multiple ablations in a single session, and post-ablation increases in aspartate aminotransferase concentration has been associated with the occurrence of post-ablation syndrome (17,18). These symptoms are usually mild and self-limiting, tending to resolve within 2 weeks with or without supportive treatment (19). Patients should be informed that these symptoms may occur, that these complications are usually not serious, but rather a self-limiting side-effect of the procedure. However, it is important to distinguish post-ablation syndrome from other complications, such as concurrent infection, liver abscess formation, and intestinal perforation. If fever is unresolved or shows delayed onset, patients should be carefully evaluated for the occurrence of other complications (13,20).
Abscess
Bacterial contamination of an ablated hepatic tumor or the parenchyma may lead to abscess formation. The incidence of liver abscess after RF ablation is between 0.3–2.4% (20–22). Important risk factors for abscess include colonization of the biliary tract caused by bilioenteric anastomosis, external biliary drainage, a functionally incompetent sphincter of Oddi, an immunocompromising condition such as diabetes, tumor with retention of iodized oil from previous transcatheter arterial chemoembolization, and treatment with an internally cooled electrode system (21,22). Abscesses often result in a prolonged hospital stay and may even be fatal (21,22). Therefore, an operator should keep in mind the risk factors for abscess formation as well as the importance of aseptic techniques during the entire procedure. The prophylactic use of antibiotics to prevent an abscess remains controversial (21,23). Because fever frequently occurs after RF ablation in the absence of infection, the clinical diagnosis of post-RF ablation abscess is sometimes delayed.
The imaging finding of abscess that develops after RF ablation does not differ from that of the usual type of hepatic abscess. CT shows a hypoattenuating lesion which contains gas, and a double-target sign may be observed on contrast-enhanced CT scanning. Minimal air bubbles within the ablation zone are seen in 63% of CT scans performed immediately after RF ablation and they usually resolve within 1 month (24). However, new intralesional gas bubbles and lesion enlargement on follow-up imaging may indicate infection (22). Most post-RF ablation abscesses can be successfully managed by simple aspiration and treatment with suitable antibiotics, but some may require percutaneous catheter drainage. Pseudoaneurysm formation, a rare complication of hepatic abscess, may complicate the clinical course of these patients (25).
Bleeding
Bleeding is a common complication which can occur both during and immediately after RF ablation, especially in cirrhotic patients with coagulopathy. The bleeding usually occurs from direct mechanical injury to the blood vessels by the RF electrode rather than resulting from RF thermal injury. Bleeding may also originate from an incompletely coagulated subcapsular tumor, a tear of the liver parenchyma, a hepatic rupture due to an intrahepatic hematoma, or delayed rupture of a hepatic artery pseudoaneurysm (26). Because needle electrodes of a large diameter (17–14 gauge) are used, bleeding after RF ablation may result in massive blood loss if diagnosis and treatment are delayed.
Coagulopathy is the most important risk factor related to bleeding after RF ablation (20). Therefore, all patients should undergo screening for coagulopathy and, if present, it should be corrected before the RF ablation procedure. Other risk factors include the use of multiple punctures or multiple electrodes and the location of the targeted lesion behind a major blood vessel. Safe placement of the RF electrode with real-time monitoring during the entire procedure and avoiding needle repositioning can reduce the chance of bleeding. In addition, traversing sufficient normal liver parenchyma to the lesion as well as cauterization of the electrode tract is strongly recommended (2,27).
An immediate color Doppler study performed following removal of the RF electrode may be helpful for the early detection of arterial bleeding along the needle tract (2). Furthermore, careful monitoring of patients’ vital signs and laboratory indicators, including the serum hemoglobin level, is essential for the postprocedural care of patients (1,3–5). If bleeding is suspected, CT is the modality of choice for revealing the bleeding into the peritoneal cavity. An acute post-RF ablation hematoma is usually seen as a crescentic or biconvex lesion along the hepatic surface near the entry site of the RF electrode and with higher attenuation than that of water. Most venous bleeding is successfully managed by conservative treatment or transfusion, although more intensive therapy, such as transcatheter arterial embolization, may be necessary for arterial bleeding which could be life-threatening (1,3–5).
In addition to bleeding, an arteriovenous fistula or intrahepatic arterial pseudoaneurysm can also develop from direct traumatic injury to the vessels caused by an RF electrode (Fig. 2). Because patients with intrahepatic pseudoaneurysm run the risk of lethal complications such as rupture of the aneurysm and bleeding into the bile duct or peritoneal cavity, clinicians keep in mind the possibility of such complications. Using a cone-shaped electrode may be helpful in order to decrease the chance of arterial injury and to avoid traumatic pseudoaneurysms.
Pseudoaneurysm along the needle tract in a 74-year-old man with rectosigmoid colon cancer after RF ablation for hepatic metastasis. Immediate post-ablation CT scan shows opacification of the irregular intrahepatic pseudoaneurysm (short arrows) apart from the ablation site (long arrow), but also along the course of the needle tract. This was presumed to have occurred due to direct injury to the vessel.
Adjacent organ injury
Injury of adjacent organs, including the gallbladder, gastrointestinal tract, and the diaphragm, is primarily caused by thermal damage resulting from heating, and can occur especially when the edge of a thermal lesion is <1 cm from the surface of the liver (11). Adhesion due to prior abdominal surgery, intra-abdominal inflammation, or percutaneous therapy may also be potential risk factors.
Gallbladder injury
Cholecystitis can develop after RF ablation of a mass adjacent to the gallbladder. While minimal wall thickening of the gallbladder on immediate post-RF ablation CT is a common finding in gallbladder injury, symptomatic cholecystitis or gallbladder perforation is rare. It has been proposed that the fluid content within the gallbladder lumen may have a role in heat dissipation (28). In some instances, gallbladder perforation may be associated with other complications such as colonic injury. In this type of combined injury, gas in the biliary system may be an indicator. Percutaneous or endoscopic drainage is helpful in the management of gallbladder perforation.
Hemobilia and blood in the gall bladder can occur as the complication of RF ablation. The creation of fistulae between vascular structures and the intrahepatic bile ducts or thermal necrosis of vessels and bile ducts have been proposed as possible etiologies (29). The incidence of the hemobilia following RF ablation has been reported to be between 0 and 0.3% (10,30–33). The sonographic appearance of a hemocholecyst is clumps of echogenic material within the gallbladder. A CT scan can show a high-attenuation, mass-like lesion in a distended gallbladder. However, in only 30% of patients, endoscopic retrograde cholangiopancreatography can identify bleeding evidence at the duodenal ampulla. Hemobilia can be successfully managed by percutaneous transhepatic biliary drainage or percutaneous cholecystostomy; however, in some patients open or laparoscopic cholecystectomy is required (Fig. 3).
Hemocholecyst and hemobilia in a 61-year-old man who underwent RF ablation for hepatocellular carcinoma. He had uncorrected coagulapathy before the RF ablation. Unenhanced, immediate post-ablation CT scan shows acute hemocholecyst and hemobilia seen as layered high attenuation in the gallbladder (asterisk) and the common bile duct (short arrow). The ablated lesion (long arrow) adjacent to the gallbladder is also noted.
Bowel injury
Bowel perforation has been identified as one of the most serious complications associated with RF ablation. The incidence of bowel perforation after RF ablation has been reported to be between 0.1–0.3% (13,20,30). The colon seems to be at risk for perforation because of its relatively thin wall and less mobility rather than other gastrointestinal tract (Fig. 4). Conversely, the thick gastric wall and small bowel peristalsis are judged to be protective (20). Minimal injury produces thickening of the bowel wall, fat stranding, and ascites around the injured gastrointestinal tract site (24,34). Free air in the abdominal cavity may be an indicator of bowel perforation. Bowel injury occasionally causes a fistula between the RF ablation zone and bowel, and thus resulting in contamination and abscess. However, an abscess at the periphery of the liver can cross the hepatic capsule to the adjacent bowel wall as it becomes larger.
Colonic perforation in a 46-year-old man who underwent RF ablation for recurrent hepatocellular carcinoma after left lateral segmentectomy. (a) Portal-phase CT scan obtained 4 days after RF ablation shows a large number of air bubbles in the RF ablation zone (long arrow), a large abscess (asterisk) in the subhepatic space, and discontinuation of the colon wall (short arrows). Colonic perforation resulting in a perihepatic and hepatic abscess is highly suspected. (b) Photograph of the gross specimen, obtained from segmentectomy of the colon, shows focal perforation in the hepatic flexure of the colon.
To minimize bowel injury, the pathway of an RF electrode must be completely traced and the distance between the electrode and the bowel wall should be monitored. A straight electrode is superior to the expandable type for this purpose (1). When there is an insufficient distance between the RF ablation zone and the adjacent bowel, the use of an open or laparoscopic approach, instillation of 5% D/W into peritoneal cavity to create artificial ascites, placement of a balloon between the tumor and the gastrointestinal tract or aspiration of bowel gas and fluid have recently been applied in order to decrease the chance of gastrointestinal tract injury (35–37). The ingestion of cold water can also be attempted as it is practical, safe, and effective for minimizing heat injury to the stomach. Management of gastrointestinal tract injury includes fasting, antibiotic therapy, abscess drainage, and surgical closure of the fistula.
Diaphragm injury
Thermal injury of the diaphragm is rare, but it can occur after RF ablation of a mass in the hepatic dome close to the diaphragm by a percutaneous intercostal approach due to poor visibility of the tumor and collateral thermal injury to the diaphragm (38). Although diaphragmatic thermal injury is usually self-limiting, it may result in focal defect of the diaphragm, thereby causing a hernia of structures in the abdominal cavity after hepatic shrinkage (Fig. 5) (39). Hernia can make the size of the perforation larger which would then require surgical repair. Diaphragmatic paresis has also been reported (20,31). Creation of artificial ascites for the ablation of tumors located just beneath the diaphragm has been reported as a useful method for avoiding the diaphragm injury (40,41). Laparoscopically separating the diaphragm under laparoscopic ultrasound guidance can also be helpful for avoiding diaphragm injury.
Hernia from a diaphragm injury in a 60-year-old man after RF ablation for subcapsular hepatocellular carcinoma. Portal-phase CT scan obtained 1 month after RF ablation shows omental herniation (arrow) through the focal diaphragmatic defect near the subcapsular ablation zone.
Hepatic infarction
Hepatic infarction is very uncommon because the liver receives its dual blood supply from the hepatic artery and the portal vein. Although the mechanism is not well understood, extensive hepatic infarction may be attributed to overestimation of the heat-sink effect of major hepatic vessels, and thus leading to extensive portal vein thrombosis. The incidence of liver infarction is in the range of 0.07–2.3% (42). Aging and large tumor size are risk factors. A well-defined, wedge-shaped area of low attenuation that extends to the liver surface, as seen on contrast-enhanced CT images, is considered to indicate hepatic infarction (Fig. 6) (43). Pneumatosis is frequently observed in the peripheral portal vein branches of the infarcted area. The management is conservative and may include the prophylactic use of antibiotics. Nevertheless, instances of fatal liver failure have not infrequently been reported (11,13,26).
Extensive hepatic infarction in a 49-year-old man after RF ablation for poorly differentiated adenocarcinoma of the liver. Portal-phase CT scan obtained 1 month after RF ablation shows a round, ablated tumor (asterisk) in segment VIII of the liver and a well-defined, wedge-shaped area of low attenuation extending to the liver surface (arrows) and suggesting extensive hepatic infarction.
Skin burn
The incidence of skin burns after RF ablation is in the range of 0.1–3.2% for severe skin burn (second- to third-degree) and 5–33% for mild skin burn (11,44,45). The use of high currents for a prolonged period of time during the RF ablation procedure and patients with decreased peripheral circulation due to peripheral vascular disease increase the risk of ground pad skin burn. This complication can be avoided by using sufficiently large grounding pads, placing the pads horizontally to allow a longer leaking edge, and maintaining equidistance from the electrode to pad in order to prevent excessive heating of the nearest pad (3). Above all, paying careful attention to a patient's complaint is most important.
Skin burn may also occur at a contact surface with forceps holding an electrode with a peel-off coating at the holding site or with a peel-off coating needle shaft (Fig. 7) (46).
Burn caused by an uncoated shaft of an RF electrode in a 54-year-old man after RF ablation for hepatic metastasis from pancreatic cancer. (a) Photograph of the electrode insertion site shows skin burn seen as black color with peripheral reddish swelling, and occurring immediately after the RF ablation. (b) Photograph of the RF electrode, retrospectively checked, reveals peel-off of the needle shaft coating approximately 1 cm in length and just before the handle (arrow).
Completeness of the needle shaft coating must be checked before the RF ablation and listening to a patient’s complaints during the procedure is very important.
Rupture of exophytic tumor
Rupture of exophytic tumor during RF ablation can occur with a huge explosive sound (Fig. 8) (47). The exact mechanism of this exceptional condition is not fully understood. During monitoring of RF ablation using a single, internally cooled electrode, ultrasound usually shows that high echogenic focus first occurs at the tip of the electrode and then the second echogenic focus occurs at the shaft 3.0–3.5 cm from the tip. The area between these two points becomes the ablation zone. If the second point is located at the capsule, capsular rupture can occur, especially in a capsulated tumor. The high power of the RF generator can also cause the sudden appearance of a huge amount of vapor, and thus leading to a phreatic explosion. Therefore, gradual increase of the power of the RF generator and with careful placement of the electrode is recommended.
Tumor rupture during RF ablation in a 42-year-old man with hepatic metastasis from renal cell carcinoma. During RF ablation, a “pop” sound was heard. Portal phase, immediate post-ablation CT scan shows incomplete ablation of the exophytic tumor, tumor rupture (arrow), and hemoperitoneum.
Delayed complications (>2 weeks)
Bile duct injury
Heat produced by RF energy can damage the bile duct which is vulnerable to heat damage due to its slower bile flow resulting in less of a cooling effect than that of blood flow (Fig. 9). The incidence rate of bile duct injury occurring after RF ablation is 1–12% (6,20,23,48). RF ablation for lesions near the hepatic hilum is challenging because complete ablation of the tumor and protection of the major bile ducts and portal venous branches are frequently incompatible goals. Although major bile ducts near the hepatic hilum are considered to be protected from thermal damage by the heat-sink effect of the portal vein, the risk of injury to the major bile ducts may increase as the operator attempts to overcome the heat-sink effect (2,43). Therefore, the risk of thermal damage to the bile duct should be considered in terms of the potential risk versus benefit. RF ablation therapy combined with transarterial chemoembolization or percutaneous ethanol injection can be considered as an alternative treatment for patients with a hilar mass adjacent to major hepatic ducts.
Bile duct injury in a 65-year-old man who underwent RF ablation for clear cell carcinoma of the liver. (a) Portal-phase CT scan obtained 1 year after RF ablation shows bile duct dilatation (short arrows) around the ablation zone (long arrow), and suspected to be bile duct injury caused by the RF ablation. (b) MR cholangiogram obtained 1 year after RF ablation shows dilatation and separation of the right anterior and posterior bile duct due to bile duct stricture near the ablation zone (arrow).
The clinical manifestations vary according to the onset and the location of the injured bile duct. Acute bile duct necrosis may result in intrahepatic or extrahepatic biloma, sometimes with secondary infection. Upstream bile duct dilatation develops as a result of bile duct stenosis developing after RF ablation. While stenosis of the peripheral bile duct tends to be subclinical, stenosis of the central duct may cause severe jaundice requiring percutaneous transhepatic biliary drainage (23).
Tumor seeding
Tumor seeding, such as needle tract seeding or peritoneal seeding is a rare but very serious complication which can occur after RF ablation (Fig. 10) (49). The incidence of tract seeding after RF ablation is very low at around 0.2–0.9% (2,20,31,50). Risk factors for tumor seeding include preprocedural biopsy, a perpendicular approach to access a subcapsular tumor, high α-fetoprotein level, poor tumor differentiation, multiple treatment sessions, and the placement of multiple electrodes. In one previous report, risk factors for seeding after RF ablation including subcapsular tumor location, high α-fetoprotein level, and preprocedural biopsy were assessed (50). This article concluded that only preprocedural biopsy was significantly associated with tumor seeding. Tumor seeding may be prevented by minimizing the number of punctures and repositioning the RF electrode and by avoiding a direct approach to an exophytic tumor; rather, normal hepatic parenchyma should be traversed before accessing the tumor (11,51). Similarly, an electrode should not penetrate the hepatic capsule through a tumor when the latter is attached to the posterior hepatic capsule, as tumor seeding along the omentum from the subcapsular tumor can occur due to a perpendicular approach to the tumor. However, the most important technique for preventing tumor seeding is burning of the needle track after finishing nodule ablation. In two previous reports with very high prevalence of tumor seeding (4% and 12.5%), the needle tract was not routinely coagulated (52,53). In general, as the tumor tissue attached to an internally cooled electrode is not killed due to low temperature, this viable tumor tissue may contaminate the needle tract. Therefore, the radiologist performing RF ablation should keep track ablation technique in mind.
Tumor seeding along the needle tract in a 61-year-old woman who underwent RF ablation for hepatocellular carcinoma in segment V of the liver. Arterial-phase CT scan obtained 3 months after RF ablation shows enhancing nodules (arrows) along the needle tract in the muscular layer of the abdomen and attached to the peritoneum.
Fistula
Various RF ablation-related fistulae, including entero-pleural, hepato-pericardial, and various biliary fistulae, can occur (Fig. 11) (54–59). The imaging findings include abscess and air within the biliary tree. These fistulae can be resolved by antibiotics and percutaneous abscess drainage, with or without endoscopic drainage; however, extensive surgery could be required. A safe window of needle positioning by a skilled operator is the only way to avoid these complications. The candidates for RF ablation must be carefully selected, especially when a patient has undergone biliary surgery or has bile duct dilation. It is also necessary for physicians to have better knowledge of the contraindications of RF ablation, particularly for treatment of a large tumor near the main bile ducts.
Biliocutaneous fistula in a 62-year-old man who underwent RF ablation for hepatocellular carcinoma. (a) Portal-phase CT scan obtained 2 months after RF ablation shows an irregular-shaped ablation zone in segment V of the liver and extending to the capsule (arrows). (b) Photograph shows a small hole in the abdominal wall at the RF electrode entrance site. Substantial bile leakage from this hole was noted.
Rapid tumor growth
Anticancer therapy including RF ablation is thought to be one of the causes of rapid tumor growth (Fig. 12). The incidence of rapid tumor growth was reported to be 0.1–2.9% (60,61). The mechanism remains unknown, and is only presumed that RF ablation induces a phenotypic conversion such as sarcomatous change in the remaining viable tumor cells or that insufficient RF ablation can promote rapid growth of the residual tumor through the angiogenesis induced by hypoxia inducible factor-1α/vascular endothelial growth factor A (62,63). Therefore, complete necrosis with a sufficient safety margin is indispensable as part of the treatment of tumors.
Rapid growth of a recurrent tumor in a 50-year-old man who underwent RF ablation for hepatocellular carcinoma. (a) Portal-phase, immediate post-ablation CT scan shows well-ablated zone (asterisk) in segment III of the liver. (b) Portal-phase CT scan obtained 1 month after RF ablation shows a rapidly growing lesion (arrows) in the previous ablation zone.
Conclusion
RF ablation is of widespread use in hepatic tumor treatment due to its effectiveness and safety. Although the complication rate of RF ablation is low, a physician performing it should be aware of the broad spectrum of complications in order to ensure careful patient selection as well as early detection and proper management of the possible complications. Complications after RF ablation are more common in Child-Pugh class C patients than in Child-Pugh class A or B patients. Bleeding, skin burn, and visceral damage occur in subcapsular tumors, even though without significant difference in previous reports (48,64). RF ablation of a central tumor carries a higher risk of biliary tract and central vessel damage (48). Seeding and thermal damage to neighboring organs are found exclusively when using the percutaneous approach. Therefore, so as to minimize the complications of RF ablation, knowledge of these risk factors and prevention methods is required and of great benefit. In addition, because early and accurate diagnosis is necessary for the proper management of complications, radiologists should be familiar with the imaging features of various complications. Proper management of complications is essential for successful treatment using RF ablation.
Footnotes
Funding
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.
