*4.1.5. Back-table separation of the liver-pancreas block and further back-table preparation of the pancreas*

The combined liver-pancreas block is taken to the back-table for separation. The aortic segment is divided such that the proximal portion of the SMA remains with the pancreas, whilst the celiac axis remains in continuity with the liver. Superior to the pancreatic head, the portal vein is divided approximately 1 cm from the pancreas, whilst the splenic artery is divided closer to its emergence from the celiac axis [45, 46]. The GDA is ligated and divided prior to entering the pancreas; a longer length remains with the liver. The splenic artery and portal vein associated with the pancreas should be marked with a prolene suture to facilitate identification at the transplant center. The spleen is also routinely removed at the donor hospital, in addition to skeletonization of the pancreas prior to transportation. The Harmonic Scalpel is once again a useful tool that facilitates all pancreas-related back-table work if the graft is to be used for whole pancreas transplantation [41]. Limited back-table perfusion of the pancreas with UW solution is employed to ensure no blood is left within the organ or its vessels, whilst minimizing the risk of graft pancreatitis or edema.

In pancreas retrievals for islet cell isolation, the author's use a similar en bloc technique, with careful mobilization of the pancreas prior to aortic cannulation as per **Figure 3**. However, there is no need for meticulous hemostasis post perfusion and it is not necessary to remove the bulk of the tissues as this can be performed at the islet isolation facility. At the isolation center, the duodenum, spleen, and connective, extracapsular and vascular tissues are removed from the pancreas prior to it being cannulated to allow infusion of the digestive collagenase enzyme for

**Figure 3.** Procurement of the DBD pancreas. (A) Skeletonization of the pancreas, using the spleen as a handle, (B) stapled division of the superior mesenteric pedicle, (C) liver and pancreas ready for en bloc removal, (D) division of the aorta, (E) pancreas appearance after back-table preparation (n.b. Portal vein and superior mesenteric artery), and (F) back-table

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packing of iliac conduits in preservation solution.

Pancreas-alone donors are uncommon in this day and age due to developments in procurement and preservation techniques. Some authors raised concerns that combined liver-pancreas retrieval, in contrast to pancreas retrieval alone, resulted in significant "flush" injury to the pancreas owing to a higher volume of perfusion solution and the utilization of dual aortoportal cannulation in the combined donors [52]. However, other studies clearly demonstrated that multi-organ retrieval, including combined liver-pancreas retrieval, was not detrimental to pancreas transplantation outcomes [53–58]. Another factor that previously precluded combined liver-pancreas procurement was aberrant hepatic arterial anatomy, in particular the presence of an aberrant or accessory right hepatic artery originating from the superior mesenteric artery [58]. Abandoning retrieval of the pancreas due to this situation is now rare, as a preserved length of the right hepatic artery originating from the SMA stump can effectively be anastomosed to the GDA as part of a back-table reconstructive procedure [45, 46]. It is only when the right hepatic artery is within the substance of the pancreas that whole pancreas retrieval should be precluded in favor of the liver [59] but the pancreas can still be retrieved for islet cell isolation as the pancreas can still be readily perfused, and on the back table the vessels readily separated, including if necessary taking them from the body of the pancreas [9]. However, if this is undertaken then care should be taken to not damage the parenchyma of the pancreas as this makes the distension of the pancreas with collagenase for digestion more difficult [9]. Over the last 25 years and more than 1000 retrievals the authors have never found any anatomical vascular anomaly to prevent an *en bloc* liver-pancreas retrieval, although this

162 Organ Donation and Transplantation - Current Status and Future Challenges

is cited as a common reason to decline pancreas retrieval worldwide.

minimizing the risk of graft pancreatitis or edema.

*pancreas*

*4.1.5. Back-table separation of the liver-pancreas block and further back-table preparation of the* 

The combined liver-pancreas block is taken to the back-table for separation. The aortic segment is divided such that the proximal portion of the SMA remains with the pancreas, whilst the celiac axis remains in continuity with the liver. Superior to the pancreatic head, the portal vein is divided approximately 1 cm from the pancreas, whilst the splenic artery is divided closer to its emergence from the celiac axis [45, 46]. The GDA is ligated and divided prior to entering the pancreas; a longer length remains with the liver. The splenic artery and portal vein associated with the pancreas should be marked with a prolene suture to facilitate identification at the transplant center. The spleen is also routinely removed at the donor hospital, in addition to skeletonization of the pancreas prior to transportation. The Harmonic Scalpel is once again a useful tool that facilitates all pancreas-related back-table work if the graft is to be used for whole pancreas transplantation [41]. Limited back-table perfusion of the pancreas with UW solution is employed to ensure no blood is left within the organ or its vessels, whilst

In pancreas retrievals for islet cell isolation, the author's use a similar en bloc technique, with careful mobilization of the pancreas prior to aortic cannulation as per **Figure 3**. However, there is no need for meticulous hemostasis post perfusion and it is not necessary to remove the bulk of the tissues as this can be performed at the islet isolation facility. At the isolation center, the duodenum, spleen, and connective, extracapsular and vascular tissues are removed from the pancreas prior to it being cannulated to allow infusion of the digestive collagenase enzyme for

**Figure 3.** Procurement of the DBD pancreas. (A) Skeletonization of the pancreas, using the spleen as a handle, (B) stapled division of the superior mesenteric pedicle, (C) liver and pancreas ready for en bloc removal, (D) division of the aorta, (E) pancreas appearance after back-table preparation (n.b. Portal vein and superior mesenteric artery), and (F) back-table packing of iliac conduits in preservation solution.

islet cell isolation [9]. As such there is also no need for preservation of pancreas vasculature, which can be given wholly to the liver when separated on the back-table.

*4.2.1. Perfusion route*

process [66].

*4.2.2. Perfusion fluid types*

pancreas during CS:

stream effects;

• Cellular edema;

• Acidosis;

nate accumulation;

• Altered redox potentials (RP);

• Accumulation of reactive oxygen species (ROS);

• Adenosine triphosphate (ATP) depletion; and,

• Disruption in glycolytic pathways [67–69].

In order to achieve adequate *in situ* pancreatic perfusion during abdominal organ perfusion as a whole, the aorta must be securely cannulated and flushed using pressure such that perfusion media can flow into the pancreas via the superior mesenteric, gastroduodenal, and splenic arteries. Once perfusion fluid has traversed the pancreas, it must be allowed to exit the donor's vasculature via the systemic and/or portal routes to prevent graft edema. Aortic-only perfusion is routinely performed by our center, and subsequent venous venting is usually undertaken via the IVC in the thorax. In the event that dual aorto-portal perfusion is employed for combined liver-pancreas retrievals, portal venous access via an inferior mesenteric cannula can impede pancreatic outflow, and reduce the physiologic arterial-portal pressure difference that is required for pancreatic perfusion/flow [42, 60]. As such, in these cases, the portal vein may instead be accessed after dividing it superior to the pancreas, thereby also allowing unobstructed pancreatic venous drainage via the proximal aspect of the transected portal vein [60, 61]. A further back-table flush of the pancreas at the donor center is sometimes conducted via the splenic artery and SMA, although this step may be omitted [45, 62–65]. Evidence for or against either approach is currently lacking in both the case of whole pancreas and cellular transplantation. But preference in the cellular setting appears to favor not having any over-perfusion or edema as this can impede and dilute the infusion of the collagenase used for digestion of the pancreas in the isolation

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In general, the same final fluid employed for the final *in situ* flush of the pancreas is then utilized for preservation of the organ in a bag of cold preservation fluid (CS). The preservation fluid utilized must maintain the organ at a hypothermic temperature (0–4°C), whilst simultaneously ameliorating the consequences of cold ischemia and prolonged organ immersion in fluid. As such, cold organ preservation fluids should ideally have the following properties that aim to minimize and/or reverse cellular and subcellular processes occurring within the

• Disrupted ionic pumps and ion accumulation and/or depletion, with subsequent down-

• Mitochondrial dysfunction, including reversal of the electron transport chain, and succi-

#### *4.1.6. Pancreatic inspection and graft assessment*

The pancreas must be closely inspected during the retrieval process, and any concerns regarding organ quality and/or integrity should be fully conveyed to the implanting surgeon. Graft assessment should include qualities inherent to the donor pancreas, in addition to any retrieval-related damage, and should be made both *in situ* and on the back-table as per **Figure 3**. The pancreas should be assessed for parenchymal damage, capsular breach, and/or hematoma(s). Furthermore, other important factors that may preclude further transplantation individually or in combination should be identified, including fibrosis, mass(es), high intra-parenchymal fat content, calcification, edema, and/or significantly diseased arteries [40, 46, 59]. It is important to note that much of this assessment is highly subjective, and an "acceptable" pancreatic appearance and/or texture will vary from center-to-center. Obviously some of the co-factors such as high intra-parenchymal fat content, calcification, edema, and/ or significantly diseased arteries do not preclude the pancreas from being used for cellular transplantation. As an example, high intra-parenchymal fat content has been shown to be an advantage when performing islet isolation. Additionally, calcification, edema, and/or significantly diseased arteries do not affect the pancreas when used for islet cell isolation as all blood vessels and extraneous tissues are stripped from the pancreas prior to its use. The pancreas should not be discarded without direct consultation with the recipient team and exploration of its use for cellular transplantation if precluded from whole organ transplantation [9].

#### *4.1.7. Packaging the organ for transport*

Following perfusion, back-table dissection, and final inspection, the pancreas can then be packed into a suitable transport container along with perfusate solution to ensure ongoing exposure to cold preservation solution. Our unit uses the sterile triple plastic bag technique whereby the organ and a suitable volume of organ perfusion fluid is instilled into the first sterile plastic bag, without dilution from iced slush. All air is removed from the bag, prior to sealing it with a zip-tie or heavy tie. This bag is then placed inside a second sterile plastic bag filled with iced slush, ensuring close and adequate cooling of the perfusate-filled inner bag. These two bags are then placed inside a third sterile plastic bag that is securely sealed, double tied, and appropriately labeled to identify the organ and contents of the bags. Additional vessels retrieved for back-table reconstruction of the whole pancreas may also be packed into the triple sterile plastic bag set with the pancreas, or alternatively are placed inside a sterile vessel jar filled with preservation solution as per **Figure 3**, which is then double-bagged in sterile plastic bags, the first of which contains iced slush. The sealed pancreas and vessels are thence transported in a suitable, insulated iced shipping container. The container is labeled with its contents along with the contact details for both the donor and recipient coordinators.

#### **4.2. In situ perfusion and cold static preservation**

The function of *in situ* perfusion of the pancreas, as with other organs, is to achieve rapid removal of residual blood, whilst simultaneously cooling the organ and exposing it to preservation fluid media for subsequent cold static storage (CS).

#### *4.2.1. Perfusion route*

islet cell isolation [9]. As such there is also no need for preservation of pancreas vasculature,

The pancreas must be closely inspected during the retrieval process, and any concerns regarding organ quality and/or integrity should be fully conveyed to the implanting surgeon. Graft assessment should include qualities inherent to the donor pancreas, in addition to any retrieval-related damage, and should be made both *in situ* and on the back-table as per **Figure 3**. The pancreas should be assessed for parenchymal damage, capsular breach, and/or hematoma(s). Furthermore, other important factors that may preclude further transplantation individually or in combination should be identified, including fibrosis, mass(es), high intra-parenchymal fat content, calcification, edema, and/or significantly diseased arteries [40, 46, 59]. It is important to note that much of this assessment is highly subjective, and an "acceptable" pancreatic appearance and/or texture will vary from center-to-center. Obviously some of the co-factors such as high intra-parenchymal fat content, calcification, edema, and/ or significantly diseased arteries do not preclude the pancreas from being used for cellular transplantation. As an example, high intra-parenchymal fat content has been shown to be an advantage when performing islet isolation. Additionally, calcification, edema, and/or significantly diseased arteries do not affect the pancreas when used for islet cell isolation as all blood vessels and extraneous tissues are stripped from the pancreas prior to its use. The pancreas should not be discarded without direct consultation with the recipient team and exploration of its use for cellular transplantation if precluded from whole organ transplantation [9].

Following perfusion, back-table dissection, and final inspection, the pancreas can then be packed into a suitable transport container along with perfusate solution to ensure ongoing exposure to cold preservation solution. Our unit uses the sterile triple plastic bag technique whereby the organ and a suitable volume of organ perfusion fluid is instilled into the first sterile plastic bag, without dilution from iced slush. All air is removed from the bag, prior to sealing it with a zip-tie or heavy tie. This bag is then placed inside a second sterile plastic bag filled with iced slush, ensuring close and adequate cooling of the perfusate-filled inner bag. These two bags are then placed inside a third sterile plastic bag that is securely sealed, double tied, and appropriately labeled to identify the organ and contents of the bags. Additional vessels retrieved for back-table reconstruction of the whole pancreas may also be packed into the triple sterile plastic bag set with the pancreas, or alternatively are placed inside a sterile vessel jar filled with preservation solution as per **Figure 3**, which is then double-bagged in sterile plastic bags, the first of which contains iced slush. The sealed pancreas and vessels are thence transported in a suitable, insulated iced shipping container. The container is labeled with its

contents along with the contact details for both the donor and recipient coordinators.

The function of *in situ* perfusion of the pancreas, as with other organs, is to achieve rapid removal of residual blood, whilst simultaneously cooling the organ and exposing it to preser-

which can be given wholly to the liver when separated on the back-table.

*4.1.6. Pancreatic inspection and graft assessment*

164 Organ Donation and Transplantation - Current Status and Future Challenges

*4.1.7. Packaging the organ for transport*

**4.2. In situ perfusion and cold static preservation**

vation fluid media for subsequent cold static storage (CS).

In order to achieve adequate *in situ* pancreatic perfusion during abdominal organ perfusion as a whole, the aorta must be securely cannulated and flushed using pressure such that perfusion media can flow into the pancreas via the superior mesenteric, gastroduodenal, and splenic arteries. Once perfusion fluid has traversed the pancreas, it must be allowed to exit the donor's vasculature via the systemic and/or portal routes to prevent graft edema. Aortic-only perfusion is routinely performed by our center, and subsequent venous venting is usually undertaken via the IVC in the thorax. In the event that dual aorto-portal perfusion is employed for combined liver-pancreas retrievals, portal venous access via an inferior mesenteric cannula can impede pancreatic outflow, and reduce the physiologic arterial-portal pressure difference that is required for pancreatic perfusion/flow [42, 60]. As such, in these cases, the portal vein may instead be accessed after dividing it superior to the pancreas, thereby also allowing unobstructed pancreatic venous drainage via the proximal aspect of the transected portal vein [60, 61]. A further back-table flush of the pancreas at the donor center is sometimes conducted via the splenic artery and SMA, although this step may be omitted [45, 62–65]. Evidence for or against either approach is currently lacking in both the case of whole pancreas and cellular transplantation. But preference in the cellular setting appears to favor not having any over-perfusion or edema as this can impede and dilute the infusion of the collagenase used for digestion of the pancreas in the isolation process [66].

#### *4.2.2. Perfusion fluid types*

In general, the same final fluid employed for the final *in situ* flush of the pancreas is then utilized for preservation of the organ in a bag of cold preservation fluid (CS). The preservation fluid utilized must maintain the organ at a hypothermic temperature (0–4°C), whilst simultaneously ameliorating the consequences of cold ischemia and prolonged organ immersion in fluid. As such, cold organ preservation fluids should ideally have the following properties that aim to minimize and/or reverse cellular and subcellular processes occurring within the pancreas during CS:


There are multiple preservation fluids currently in existence. These can be broadly classified as those that are intracellular and extracellular/intermediate in nature, based largely upon the solution's potassium content, or low viscosity compared to high viscosity solutions [70]. Common components include colloid and/or impermeants to counteract cellular edema, antioxidants for protection against ROS generation, ATP precursors to allow replenishment upon reperfusion, and buffers to retard the acidosis attendant with organ ischemia [70].

A "pre-flush" is defined as a crystalloid fluid, such as Hartmann's solution, that is perfused *in situ* prior to the final flush/preservation fluid, such as UW. The pre-flush can be employed safely in pancreatic procurement, although it is not commonly utilized. The function of this pre-flush in the context of pancreas procurement is to potentially (1) reduce the amount of UW required, thereby reducing retrieval costs, and (2) to clear all blood from the vasculature such that any residual blood does not aggregate with the hydroxyethyl starch in UW [77, 78]. UW is traditionally perfused in much lower volumes in comparison to HTK, and this is also reflected in the various pancreas retrieval guidelines in existence. This is largely related to the higher viscosity of UW, in addition to the larger volume and time for HTK perfusion to achieve equilibration of electrolyte content with the extracellular milieu [79, 80]. Australian guidelines recommend a 2–4 L crystalloid/low viscosity solution *in situ* pre-flush, followed by a UW flush of at least 1–2 L; a volume range for HTK is not specified [45]. In contrast, UK guidelines state a UW flush of 50–70 ml/kg should be employed via the aorta, whilst Eurotransplant allows for 50–100 ml/kg UW or 150–300 ml/kg HTK [74, 75]. Published reports may deviate from this; perfusion volumes utilized in aortic-only perfusion range from 0.8–

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5.6 L, 4.9–9.7 L, and 0.8–7.9 L for UW, HTK, and Celsior respectively [81].

creas retrieval, although there is certainly enthusiasm for this approach [83, 85].

preservation of the pancreas preceding islet isolation [85].

Heparin is a standard additive to the *in situ* perfusion fluid during DCD organ retrievals, including for the pancreas. Additionally, thrombolytics such as streptokinase or tissue plasminogen activator (tPA) can be added to the *in situ* perfusion fluid, or alternatively our approach is to directly inject tPA into the aorta before commencement of the cold *in situ* flush; the aim of this is to achieve a higher quality vascular flush through the clearance of microthrombi [82–84]. However no comparative evidence exists for or against the use of thrombolytics in DCD pan-

Great focus has remained on improving the quality of pancreas transport to the islet transplant centers, including novel ways to provide oxygen rich media to the graft whilst in cold storage during shipping. In late 1988 Kuroda et al. was the first to report the use of the Two-Layer Method (TLM) for shipping of the pancreas prior to islet cell isolation [86]. The TLM uses a perfluorochemical (PFC) and the organ perfusion fluid; initially Euro-Collins' solution was used but was replaced by UW solution. The benefits of the use of the PFC are due to it being a biologically inert liquid that acts as an oxygen-supplying media. A pancreas preserved using the TLM is theoretically oxygenated through the PFC and substrates are supplied by the UW solution. This allows the pancreas preserved using the TLM to generate adenosine triphosphate during storage, prolonging the preservation time [87]. Strong debate still remains over its benefits, if any, when compared to the use of UW solution during CS [88, 89] and a recent publication of guidelines recommended against the use of the TLM for

*4.2.3. Additive(s) to perfusate*

*4.2.4. Two-layer method*

University of Wisconsin (UW) solution remains the most popular pancreatic preservation fluid, and was initially developed specifically for this purpose [71]. It is an intracellular solution with a high potassium content and high viscosity as it contains hydroxyethyl starch, a particularly important component for pancreas preservation [24]. UW contains other components that fulfill many ideal criteria that should be exhibited by preservation fluids, including the addition of impermeants such as raffinose, the ATP precursor adenosine, and anti-oxidants such as allopurinol. [68] Histidine-tryptophan-ketoglutarate (HTK) is another commonly utilized preservation fluid for the pancreas. In contrast to UW, HTK it is an "intermediate" solution with a significantly lower potassium and sodium concentration, thereby in effect preventing ongoing organ metabolism. HTK also has low viscosity, theoretically allowing higher flow rates, and the histidine component of HTK provides it with significant buffering capacity [68, 70]. The next most commonly studied and clinically utilized pancreas perfusion and preservation fluid is Celsior, which has similar potassium content to HTK in addition to containing histidine as a buffer. It differs from HTK in that it has much higher sodium content; furthermore, it incorporates some of the advantageous constituents of UW, including similar impermeants and one shared anti-oxidant [68, 70]. Most recently, the use of Institut Georges Lopez (IGL-1) solution has been reported in pancreatic transplantation [72]. This solution has similar constituents to UW, except the sodium and potassium concentrations are reversed such that it more closely resembles the extra-cellular environment [68]. A number of other more recently developed perfusion fluids have shown good effect in the preservation of pancreata for islet cell transplantation in particular the ET-Kyoto perfusion fluid. This fluid has a high sodium:low potassium ratio, and contains trehalose to protect the cell membrane against hypothermia and the nitric oxide donor nitroglycerin that facilitates vasodilatation [73].

National guidelines and/or protocols differ with respect to recommended perfusion and preservation fluids for the pancreas [45, 60, 74, 75]. UW and HTK solutions are the two most frequently recommended solutions for pancreas retrieval by such guidelines, although their utilization and volumes vary significantly. UK guidelines stipulate that *in situ* UW perfusion must be undertaken for pancreas retrieval, whilst Eurotransplant, German, and Australia/ New Zealand guidelines allow for either UW or HTK. Furthermore, none of these guidelines preclude dual perfusion when the pancreas is being retrieved, although German standards stipulate portal venous perfusion via a catheter inserted directly into the portal vein above the pancreas/duodenum [45, 60, 74, 75]. The use of Celsior or IGL-1 solution has not yet been incorporated into major National or Regional guidelines, although both have been employed in the clinical context [64, 65, 72, 76].

A "pre-flush" is defined as a crystalloid fluid, such as Hartmann's solution, that is perfused *in situ* prior to the final flush/preservation fluid, such as UW. The pre-flush can be employed safely in pancreatic procurement, although it is not commonly utilized. The function of this pre-flush in the context of pancreas procurement is to potentially (1) reduce the amount of UW required, thereby reducing retrieval costs, and (2) to clear all blood from the vasculature such that any residual blood does not aggregate with the hydroxyethyl starch in UW [77, 78].

UW is traditionally perfused in much lower volumes in comparison to HTK, and this is also reflected in the various pancreas retrieval guidelines in existence. This is largely related to the higher viscosity of UW, in addition to the larger volume and time for HTK perfusion to achieve equilibration of electrolyte content with the extracellular milieu [79, 80]. Australian guidelines recommend a 2–4 L crystalloid/low viscosity solution *in situ* pre-flush, followed by a UW flush of at least 1–2 L; a volume range for HTK is not specified [45]. In contrast, UK guidelines state a UW flush of 50–70 ml/kg should be employed via the aorta, whilst Eurotransplant allows for 50–100 ml/kg UW or 150–300 ml/kg HTK [74, 75]. Published reports may deviate from this; perfusion volumes utilized in aortic-only perfusion range from 0.8– 5.6 L, 4.9–9.7 L, and 0.8–7.9 L for UW, HTK, and Celsior respectively [81].
