Ignite Creation Date:
2024-05-05 @ 12:07 PM
Last Modification Date:
2024-10-26 @ 9:20 AM
Study NCT ID:
NCT00245830
Status:
COMPLETED
Last Update Posted:
2015-01-19
First Post:
2005-10-26
Brief Title:
Ischemic Preconditioning of Liver in Cadaver Donors
Sponsor:
University of Medicine and Dentistry of New Jersey
Organization:
Rutgers The State University of New Jersey
Study Overview
Official Title:
Ischemic Preconditioning of Liver in Cadaver Donors
Status:
COMPLETED
Status Verified Date:
2015-01
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
The long-term goals of this proposal are to develop clinical protocols of donor preconditioning to improve liver graft function and ameliorate complications of poor graft function after liver transplantation Achievement of these objectives would improve liver recipient outcomes increase utilization of livers and alleviate the current critical shortage of livers for transplantation More stringent liver donor selection intended to decrease the complications of poor graft function conflicts directly with efforts to maximize the use of donor livers Ischemic preconditioning IPC of liver attenuates hepatic ischemia reperfusion injury IRI in animals Preliminary data show hepatic IPC effectively decreases IRI following hepatic resection in humans
The specific aims of this project are AIM 1 To test the hypothesis that 10 minutes of hepatic ischemic preconditioning in deceased donors would improve liver graft function and decrease injury in the early post transplant period AIM 2 To test the hypothesis that ischemic preconditioning of deceased donor livers would decrease systemic inflammatory response in liver recipients in the early post transplant period AIM 3 To examine whether ischemic preconditioning of deceased donor livers decreases early post transplant pulmonary edema and acute rejection and shortens hospital stay
The specific aims of this project are AIM 1 To test the hypothesis that 10 minutes of hepatic ischemic preconditioning in deceased donors would improve liver graft function and decrease injury in the early post transplant period AIM 2 To test the hypothesis that ischemic preconditioning of deceased donor livers would decrease systemic inflammatory response in liver recipients in the early post transplant period AIM 3 To examine whether ischemic preconditioning of deceased donor livers decreases early post transplant pulmonary edema and acute rejection and shortens hospital stay
Detailed Description:
Specific Aims
1 To test the hypothesis that 10 minutes of hepatic ischemic preconditioning in deceased donors would improve liver graft function and decrease injury in the early post transplant period
To accomplish this aim we will compare International Normalized Ratios of prothrombin time INRPT and serum aspartate AST and alanine aminotransferase ALT and total bilirubin TB levels immediately post transplant and on day 1 3 7 14 and 30 and injury score of reperfusion liver biopsies in recipients of livers from IPC and No IPC donors
2 To test the hypothesis that ischemic preconditioning of deceased donor livers would decrease the systemic inflammatory response in liver recipients in the early post transplant period
To accomplish this aim we will compare plasma levels of cytokines tumor necrosis factor-alpha TNF-alpha interleukins-6 8 and 10 IL-6 8 and 10 and soluble L-selectin expression levels of adhesion molecules CD11betaCD18 and L-selectin and oxidative burst of neutrophils platelet P-selectin and platelet-neutrophil complexes PNC in the peripheral blood 3 12 24 and 48 hours following reperfusion in recipients of livers from IPC and No IPC donors
3 To examine whether ischemic preconditioning of deceased donor livers decreases early post transplant pulmonary edema and acute rejection and shortens hospital stay
To accomplish this aim we will compare interstitial and alveolar edema in chest radiographs done after transplant and on post operative days 1 2 and 3 biopsy confirmed acute rejection within 30 days post transplant and number of days to discharge after transplantation in recipients of livers from IPC and No IPC donors
Research Design A prospective randomized and single masked liver recipients study will be conducted in one liver transplant center over a period of two years Deceased liver donors will be randomized in equal proportions to one of two organ recovery procedures 1 IPC or 2 No IPC Allocation will be stratified by age 50 or age 50 to facilitate examination of the effects of age on IPC As the trial will enroll donors over a two-year period treatment assignments will be balanced over blocks of length 12 16 and 20 where the length will be selected at random to control for time varying factors Recovered livers are transplanted into recipients 18 years of age All other aspects of donor management organ recovery and preservation recipient selection graft implantation and post transplant management including immunosuppression will be according to standard practice
II Research Methods
1 Liver Recovery and IPC Donors are positioned supine mechanically ventilated and administered 100 oxygen and a muscle relaxant The abdomen and chest are opened in the midline Briefly round and falciform ligaments are divided and the gallbladder is incised and irrigated The gastroduodenal artery is ligated and junction of splenic and superior mesenteric veins or when pancreas is procured the inferior mesenteric vein is prepared for canulation The supraceliac aorta is exposed just below the diaphragm The infrarenal abdominal aorta is isolated for canulation No mobilization of the kidneys is performed before organ perfusion Thoracic organs are mobilized either simultaneously or before mobilization of abdominal organs Five hundred unitskg of heparin is given intravenously and ascending thoracic and infrarenal abdominal aorta are canulated Another canula is inserted either into splenic vein or inferior mesenteric vein The supraceliac abdominal aorta is clamped and the inferior vena cava is vented in the pericardium UW solution 40C is infused into abdominal aorta and portal vein 2 liters each The abdominal cavity is packed with ice slush Following completion of organ perfusion heart lungs liver and pancreas and kidneys are removed in sequence Livers are placed in plastic bags containing a liter of UW solution 40C and packed in ice until the time of transplantation
Ischemic preconditioning is performed soon after opening the abdomen by clamping the hepatic hilum with a vascular clamp for five minutes which is repeated again after five minutes of reperfusion During IPC the peritoneal cavity is inspected for bleeding congestion and edema of the pancreas and small intestine
2 Graft Implantation Recipient hepatectomy is done in a standard manner with caval preservation and without utilization of venovenous bypass The liver graft is implanted in a piggyback manner88 The allograft is reperfused after completion of the suprahepatic caval and portal vein anastomoses The graft is flushed with approximately 300 ml of recipient blood which is vented via the donors infrahepatic vena cava after which this structure is ligated Arterial and bile duct anastomoses are completed after graft reperfusion
3 Liver Biopsy and Reperfusion Injury A wedge and a needle biopsy of the right lobe is performed immediately upon opening the donors abdomen and before IPC Another wedge and needle biopsy is done 90 min after recipient reperfusion Liver biopsy after transplantation is performed only when clinically indicated Formalin fixed specimens are embedded in paraffin sectioned five microns thin and stained with hematoxylin and eosin The amount of fat in donor biopsies is graded semi-quantitatively as follows89 No fat grade 0 1-15 grade I 16-30 grade II 31-45 grade III 45 grade IV Reperfusion injury is graded 0-10 in increasing severity by examining 30 high power fields 600x of each post perfusion biopsy Apoptotic cells are enumerated and hepatocyte swelling zone 3 hemorrhage and necrosis are assigned a semi-quantitative score90 The reperfusion scores in the proposed study are expected to range 05 to 5 based on our previous work36 The pathologist is masked regarding the group assignment of all biopsies
4 Immunosuppression and Acute Rejection All patients receive tacrolimus and steroids for immunosuppression In patients with hepatorenal syndrome tacrolimus use is delayed for several days and may be used at a much lower dose than the standard use Pharmacological immunosuppression will be quantified by determining the mean dose of steroid per day the mean of the weekly median tacrolimus level for the first five weeks and the proportion of recipients in each group that might receive other agents such as mycophenolate and IL-2 receptor antibody Immunosuppresion caused by recipients liver failure will be assessed by their model for end stage liver disease MELD score at the time of transplantation Following transplantation liver biopsies are performed only when clinically indicated Acute cellular rejection is diagnosed when at least two of the three following criteria are present portal mononulclear infiltration bile duct inflammationdamage and subendothelial inflammation of the portal venules andor terminal hepatic venules Severity of the rejection is graded based on the rejection activity index as follows 0-2 no rejection 3 borderline 4-5 mild 6-7 moderate and 8-9 severe91 Mild rejection is treated initially with escalation of prograf dose alone or in combination with steroids Steroid resistant rejection upon confirmation with a biopsy is treated with muromonab OKT3
5 Plasma TNF-alpha IL-6 and 8 and soluble L-selectin Plasma is separated from systemic venous blood taken 3 12 24 48 hours after reperfusion from recipients of livers with and without IPC and samples are stored at -700C TNF-alpha IL-6 and IL-8 and soluble L-selectin levels are quantitated with commercially available enzyme-linked immunosorbent assay kits R D Systems Minneapolis MN in duplicates The assays are performed according to manufacturers instructions
6 Neutrophil CD11betaCD18 and L-selectin Platelet P-selectin and Platelet-Neutrophil Complexes and Oxidative burst of Neutrophils in peripheral blood Systemic venous blood is collected into heparinized 10 Uml syringes 3 12 24 and 48 hours after reperfusion from recipients of livers with and without IPC Fifty microL of blood are added to saturating concentrations of fluorescein isothiocyante FITC or phycoerythrin PE conjugated monoclonal antibodies Dako Corp Carpenteria CA to CD 11betaCD18 CD 62 L L-selectin CD 42 platelet von Willebrand factor receptor and CD 62P P-selectin Following 10 min of incubation at room temperature red cells are lysed by the addition of 200 microL of FACSlyse Becton Dickinson Franklin Lakes NJ After 10 min of incubation 250 microL of 02 formaldehyde in PBS is added Samples are analyzed in a FasCalibur flow cytometry Becton Dickinson Fair Lakes NJ Fluorescence of FITC is measured at 515 nm and PE at 580 nm Neutrophils are distinguished by their readily identifiable characteristics on forward and side-scatter plot A minimum of 5000 neutrophil events are counted Events staining positive for both CD 11beta and CD 42 are considered to be PNC as described by Peters et al72 To minimize the risk of identifying individual platelets and neutrophils as PNC flow rate is adjusted to result in 4500 eventssec Results are considered positive if the fluorescence intensity exceeds 98 of isotype matched control antibodies Results are expressed as median fluorescence intensity MFI Oxidative burst is measured by utilization of oxidation of dihydrorhodamine to florescent rhodamine by neutrophil generated reactive oxygen species as described by Vowells et al92 Briefly 300 microL of heparinized whole blood is added to 4 ml of red cell lysis buffer 370C After five minutes of incubation the pellet is resuspended in HBSS containing 5 fetal calf serum 18 microL of DHR in DMSO is added and incubated at room temperature for 10 min followed by flow cytometric analysis Stimulation with PMA 1microgml is used as a positive control
7 Pulmonary Edema Chest radiographs are performed in an antero-posterior projection with the patient in semi-erect position upon completion of the transplant and on postoperative days 1 2 and 3 Pulmonary edema is assessed by the presence or absence of interstitial edema and alveolar edema as per accepted criteria93 The radiologist is masked regarding group assignment of subjects
1 To test the hypothesis that 10 minutes of hepatic ischemic preconditioning in deceased donors would improve liver graft function and decrease injury in the early post transplant period
To accomplish this aim we will compare International Normalized Ratios of prothrombin time INRPT and serum aspartate AST and alanine aminotransferase ALT and total bilirubin TB levels immediately post transplant and on day 1 3 7 14 and 30 and injury score of reperfusion liver biopsies in recipients of livers from IPC and No IPC donors
2 To test the hypothesis that ischemic preconditioning of deceased donor livers would decrease the systemic inflammatory response in liver recipients in the early post transplant period
To accomplish this aim we will compare plasma levels of cytokines tumor necrosis factor-alpha TNF-alpha interleukins-6 8 and 10 IL-6 8 and 10 and soluble L-selectin expression levels of adhesion molecules CD11betaCD18 and L-selectin and oxidative burst of neutrophils platelet P-selectin and platelet-neutrophil complexes PNC in the peripheral blood 3 12 24 and 48 hours following reperfusion in recipients of livers from IPC and No IPC donors
3 To examine whether ischemic preconditioning of deceased donor livers decreases early post transplant pulmonary edema and acute rejection and shortens hospital stay
To accomplish this aim we will compare interstitial and alveolar edema in chest radiographs done after transplant and on post operative days 1 2 and 3 biopsy confirmed acute rejection within 30 days post transplant and number of days to discharge after transplantation in recipients of livers from IPC and No IPC donors
Research Design A prospective randomized and single masked liver recipients study will be conducted in one liver transplant center over a period of two years Deceased liver donors will be randomized in equal proportions to one of two organ recovery procedures 1 IPC or 2 No IPC Allocation will be stratified by age 50 or age 50 to facilitate examination of the effects of age on IPC As the trial will enroll donors over a two-year period treatment assignments will be balanced over blocks of length 12 16 and 20 where the length will be selected at random to control for time varying factors Recovered livers are transplanted into recipients 18 years of age All other aspects of donor management organ recovery and preservation recipient selection graft implantation and post transplant management including immunosuppression will be according to standard practice
II Research Methods
1 Liver Recovery and IPC Donors are positioned supine mechanically ventilated and administered 100 oxygen and a muscle relaxant The abdomen and chest are opened in the midline Briefly round and falciform ligaments are divided and the gallbladder is incised and irrigated The gastroduodenal artery is ligated and junction of splenic and superior mesenteric veins or when pancreas is procured the inferior mesenteric vein is prepared for canulation The supraceliac aorta is exposed just below the diaphragm The infrarenal abdominal aorta is isolated for canulation No mobilization of the kidneys is performed before organ perfusion Thoracic organs are mobilized either simultaneously or before mobilization of abdominal organs Five hundred unitskg of heparin is given intravenously and ascending thoracic and infrarenal abdominal aorta are canulated Another canula is inserted either into splenic vein or inferior mesenteric vein The supraceliac abdominal aorta is clamped and the inferior vena cava is vented in the pericardium UW solution 40C is infused into abdominal aorta and portal vein 2 liters each The abdominal cavity is packed with ice slush Following completion of organ perfusion heart lungs liver and pancreas and kidneys are removed in sequence Livers are placed in plastic bags containing a liter of UW solution 40C and packed in ice until the time of transplantation
Ischemic preconditioning is performed soon after opening the abdomen by clamping the hepatic hilum with a vascular clamp for five minutes which is repeated again after five minutes of reperfusion During IPC the peritoneal cavity is inspected for bleeding congestion and edema of the pancreas and small intestine
2 Graft Implantation Recipient hepatectomy is done in a standard manner with caval preservation and without utilization of venovenous bypass The liver graft is implanted in a piggyback manner88 The allograft is reperfused after completion of the suprahepatic caval and portal vein anastomoses The graft is flushed with approximately 300 ml of recipient blood which is vented via the donors infrahepatic vena cava after which this structure is ligated Arterial and bile duct anastomoses are completed after graft reperfusion
3 Liver Biopsy and Reperfusion Injury A wedge and a needle biopsy of the right lobe is performed immediately upon opening the donors abdomen and before IPC Another wedge and needle biopsy is done 90 min after recipient reperfusion Liver biopsy after transplantation is performed only when clinically indicated Formalin fixed specimens are embedded in paraffin sectioned five microns thin and stained with hematoxylin and eosin The amount of fat in donor biopsies is graded semi-quantitatively as follows89 No fat grade 0 1-15 grade I 16-30 grade II 31-45 grade III 45 grade IV Reperfusion injury is graded 0-10 in increasing severity by examining 30 high power fields 600x of each post perfusion biopsy Apoptotic cells are enumerated and hepatocyte swelling zone 3 hemorrhage and necrosis are assigned a semi-quantitative score90 The reperfusion scores in the proposed study are expected to range 05 to 5 based on our previous work36 The pathologist is masked regarding the group assignment of all biopsies
4 Immunosuppression and Acute Rejection All patients receive tacrolimus and steroids for immunosuppression In patients with hepatorenal syndrome tacrolimus use is delayed for several days and may be used at a much lower dose than the standard use Pharmacological immunosuppression will be quantified by determining the mean dose of steroid per day the mean of the weekly median tacrolimus level for the first five weeks and the proportion of recipients in each group that might receive other agents such as mycophenolate and IL-2 receptor antibody Immunosuppresion caused by recipients liver failure will be assessed by their model for end stage liver disease MELD score at the time of transplantation Following transplantation liver biopsies are performed only when clinically indicated Acute cellular rejection is diagnosed when at least two of the three following criteria are present portal mononulclear infiltration bile duct inflammationdamage and subendothelial inflammation of the portal venules andor terminal hepatic venules Severity of the rejection is graded based on the rejection activity index as follows 0-2 no rejection 3 borderline 4-5 mild 6-7 moderate and 8-9 severe91 Mild rejection is treated initially with escalation of prograf dose alone or in combination with steroids Steroid resistant rejection upon confirmation with a biopsy is treated with muromonab OKT3
5 Plasma TNF-alpha IL-6 and 8 and soluble L-selectin Plasma is separated from systemic venous blood taken 3 12 24 48 hours after reperfusion from recipients of livers with and without IPC and samples are stored at -700C TNF-alpha IL-6 and IL-8 and soluble L-selectin levels are quantitated with commercially available enzyme-linked immunosorbent assay kits R D Systems Minneapolis MN in duplicates The assays are performed according to manufacturers instructions
6 Neutrophil CD11betaCD18 and L-selectin Platelet P-selectin and Platelet-Neutrophil Complexes and Oxidative burst of Neutrophils in peripheral blood Systemic venous blood is collected into heparinized 10 Uml syringes 3 12 24 and 48 hours after reperfusion from recipients of livers with and without IPC Fifty microL of blood are added to saturating concentrations of fluorescein isothiocyante FITC or phycoerythrin PE conjugated monoclonal antibodies Dako Corp Carpenteria CA to CD 11betaCD18 CD 62 L L-selectin CD 42 platelet von Willebrand factor receptor and CD 62P P-selectin Following 10 min of incubation at room temperature red cells are lysed by the addition of 200 microL of FACSlyse Becton Dickinson Franklin Lakes NJ After 10 min of incubation 250 microL of 02 formaldehyde in PBS is added Samples are analyzed in a FasCalibur flow cytometry Becton Dickinson Fair Lakes NJ Fluorescence of FITC is measured at 515 nm and PE at 580 nm Neutrophils are distinguished by their readily identifiable characteristics on forward and side-scatter plot A minimum of 5000 neutrophil events are counted Events staining positive for both CD 11beta and CD 42 are considered to be PNC as described by Peters et al72 To minimize the risk of identifying individual platelets and neutrophils as PNC flow rate is adjusted to result in 4500 eventssec Results are considered positive if the fluorescence intensity exceeds 98 of isotype matched control antibodies Results are expressed as median fluorescence intensity MFI Oxidative burst is measured by utilization of oxidation of dihydrorhodamine to florescent rhodamine by neutrophil generated reactive oxygen species as described by Vowells et al92 Briefly 300 microL of heparinized whole blood is added to 4 ml of red cell lysis buffer 370C After five minutes of incubation the pellet is resuspended in HBSS containing 5 fetal calf serum 18 microL of DHR in DMSO is added and incubated at room temperature for 10 min followed by flow cytometric analysis Stimulation with PMA 1microgml is used as a positive control
7 Pulmonary Edema Chest radiographs are performed in an antero-posterior projection with the patient in semi-erect position upon completion of the transplant and on postoperative days 1 2 and 3 Pulmonary edema is assessed by the presence or absence of interstitial edema and alveolar edema as per accepted criteria93 The radiologist is masked regarding group assignment of subjects
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None