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Factors affecting human islet of Langerhans isolation yields
Factors Affecting Human Islet of Langerhans Isolation Yields C. Toso, J. Oberholzer, F. Ris, F. Triponez, P. Bucher, A. Demirag, E. Andereggen, L. Buehler, N. Cretin, B. Fournier, P. Majno, Y. Hong, J. Lou, and P. Morel
T
HE CLINICAL results of human islet transplantation are improving.1,2 However, most recipients still need islet preparations isolated from more than one donor to provide a sufficient islet mass to obtain insulin independence. The number of islets in a human pancreas varies between 300,000 to 1.5 million. With present techniques, only 30% to 50% of islets within a pancreas are isolated, a discrepancy that emphasizes the need for improved yields of isolated human islets. The present study investigates the donor characteristics and the technical factors that may affect isolation yields, with special attention to donor selection and timing of procurement. MATERIALS AND METHODS From November 1991 to October 2000, 234 human islet isolations have been performed at our laboratory. The first 50 procedures used to standardize the technique (from November 1991 to October 1993) and 15 islet isolations performed for autologous transplantation (isolation without purification) have been excluded from the present study. Pancreata were retrieved from multiorgan donors harvested in Switzerland and in French centers participating in the GRAGIL cooperative project.3 After aortic cross-clamping, University of Wisconsin (UW) solution was infused and pancreata were harvested as quickly as possible. The organs were brought to the isolation laboratory in UW solution at 4°C in a temperaturecontrolled box (HUG-Box, Safetherm, Geneva, Switzerland). Isolations were performed according to a modification of Ricordi’s semiautomated technique.2,4 Briefly, the pancreas was separated from the duodenum, from adipose tissue, and from the spleen when harvested with the pancreas. Wirsung’s duct was cannulated at the level of the ampulla vater. Collagenase P (2 mg/mL in 250 mL Hank’s solution) at 4°C (for 127 isolations, until 1997) or Liberase HI at 30°C (for 107 isolations, after 1997) was injected manually. The distended pancreas was placed in a digestion chamber (capacity 500 mL) that had been prewarmed to 29°C. A double stainless-steel mesh (with windows ranging from 500 to 1000 m in size) was placed between the lower and the upper portion of the chamber to retain undigested tissue. The chamber was then connected to the tubing of a circulating system activated by a peristaltic pump and warmed to 37°C, using an electronically controlled heating system. The chamber, containing seven glass marbles, was shaken continuously during the digestion. Samples of the preparation were taken regularly from a tap adapted to the chamber itself.5 The macroscopic appearance of the digest was 0041-1345/02/$–see front matter PII S0041-1345(01)02925-6 826
monitored through a window adapter on the chamber. Dithizonestained samples were used to evaluate the microscopic appearance. When the pancreas was soft and the free islets had been released, digestion was stopped by dilution with 6 to 8 L of cold Hank’s solution (4°C), supplemented with 5% newborn calf serum (Seromed, Berlin, Germany) in the first 2 L. The tissue digests were collected, pooled in University of Wisconsin solution (DuPont Pharma, Bad Humburg, Germany), and kept on ice for 30 minutes. The digested tissue was suspended at the lowest level of a discontinuous Euro-Ficoll gradient (Sigma, St Louis, Mo) on a cell separator (Model 2991, Cobe, Lakewood, Col.) using densities of 1.108 at the bottom and 1.096, 1.037, and 1.006 at the top for purification. After purification, samples were taken for counting. Only intact, dithizone-stained islets were counted; the equivalent islet number (EIN) was calculated by normalizing the islets to a standard diameter of 150 m as described previously.6 Because 82 partial pancreata were harvested at the beginning of our experience, we expressed isolation yields as EIN per gram of pancreas wet weight before enzyme injection. The following donor factors were evaluated retrospectively with respect to isolation yields (EIN/g): age; gender; body mass index (weight [kg]/body surface area [m2]); cause of death; length of ICU stay; history of hypotension (mean arterial blood pressure ⬍60 mm Hg); blood glucose value; liver function tests; and serum amylase and lipase. The duration of secondary warm ischemia during the procurement was defined as the time between aortic cross-clamping and the end of pancreatectomy. The duration of cold ischemia was the time between pancreatectomy and collagenase injection. Data are presented as mean values ⫾ standard deviations (SD) unless specified otherwise. Statistical analysis was performed with Student’s t test. P ⬍ .05 was considered statistically significant.
RESULTS
In the present study, 169 isolations were performed over a 7-year period. A mean of 24 (14 to 52) isolations/year were performed, with increasing numbers each year. The mean yield during the entire period was 1689.2 ⫾ 1560 EIN/g. From the Division of Surgical Research, Department of Surgery, University Hospital, University of Geneva, Switzerland. Supported by Grant 3200-061873.00 from the Swiss National Science Foundation and by the Foundation Carlos et Elsie de Reuter. Address reprint requests to Dr Christian Toso, Division of Surgical Research, Department of Surgery, University Hospital, 24 rue Micheli-du-Crest, 1211 Geneva 14, Switzerland. © 2002 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 34, 826 – 827 (2002)
ISLET ISOLATION YIELDS
827
Table 1. Effect of Donor Characteristics and Pancreas Procurement Parameters on Human Isolation Yield EIN/g
Age (years) ⬎20 (n ⫽ 155) ⬍20 (n ⫽ 13) BMI (kg/m2) ⬎25 (n ⫽ 52) ⬍25 (n ⫽ 117) Hypotension No (n ⫽ 99) Yes (n ⫽ 65) Secondary warm ischemia (min) ⬍30 (n ⫽ 13) ⬎30 (n ⫽ 93) Cold ischemia (h) ⬍8 (n ⫽ 135) ⬎8 (n ⫽ 25)
P
1763 ⫾ 1581 1011 ⫾ 981
⬍.01
2135 ⫾ 2142 1508 ⫾ 1147
⬍.01
1873 ⫾ 1751 1512 ⫾ 1217
.06
2434 ⫾ 1871 1625 ⫾ 1996
.05
1906 ⫾ 1037 1037 ⫾ 645
⬍.005
Data expressed as mean ⫾ SD.
During the last 2 years, only whole organs were processed yielding 211,171 ⫾ 145,576 EIN per pancreas. Age had a significant effect on isolation yield; better results were obtained with pancreata from donors ⬎20 years (Table 1). Isolation yields from donors ⬎20 years varied little, even when age exceeded 50 years. The BMI was positively correlated with isolation yield: pancreata from donors whose BMI was ⬎25 kg/m2 had higher yields. Although not statistically significant, occurrence of a hypotensive episode was associated with lower isolation yields. The length of both the secondary warm ischemia and the cold ischemia times had relevant effects on yield. Gender, cause of death, length of ICU stay, and dose of vasopressors (noradrenaline, adrenaline, or dopamine) ongoing at the time of procurement had no detectable effect on the yield. Blood chemistry tests (blood glucose, liver, and pancreas tests) also did not correlate with the yield. DISCUSSION
The increasing interest and demand for islet transplantation challenges the current capacity of islet isolation facilities. The scarcity of donors and the costs of islet isolations require reliable criteria to avoid the exclusion of eventually successful donors and to reduce the number of procedures with insufficient islet yields. The present study investigated donor and procurement parameters that affect islet isolation yields. Donor age had an impact on islet yields, as described previously by other groups.7–9 Lower isolation results were achieved with pancreata from donors of ⬍20 years, possibly due to the presence of a softer parenchyma with less connective tissue surrounding the islets. In the present study, all isolations from donors ⬎20 years provided similar numbers of islets, even when age was ⬎50 years. Other series have observed lower isolation yields and decreased
glucose sensitivity of islets from donors ⬎50 years of age.7,8,10 However, the data presented by the Edmonton group, similar to our experience, revealed no deleterious effect of older donor age on islet graft outcome.1 A high BMI was positively correlated with isolation yields, confirming previous data.7,9 The total islet mass may be increased in overweight patients, due to a higher metabolic demand. In addition, the higher islet yield in obese donors may be explained by a higher content of fat cells in the pancreas parenchyma, a factor that has been demonstrated to increase yield.11 The occurrence of a hypotensive episode was associated with a trend toward poorer isolation results, whereas no correlation was found between the administration of vasoactive drugs and islet yields. This finding suggests that successful stabilization of vital signs is the most important goal, regardless of the dose of amines, and that a more liberal use of vasoactive drugs in modern donor management does not affect yield. Secondary warm ischemia and cold ischemia times provided relevant parameters for islet isolation7–9,12; every effort should be made to shorten both times. The pancreas must be harvested quickly, before the kidneys and, if possible, before the liver. The retrieval must take care to preserve the capsule intact.9 Finally, procuring hospitals should be within a short distance from the islet isolation facility to allow initiation of digestion within 8 hours of aortic clamping. A better selection of donors and improved procurement procedures should give better isolation yields and decrease the variability between isolations, which in turn may facilitate new developments in the isolation process. Recent clinical successes1 are a stimulus to give pancreas procurement the same attention as with other solid organs to increase the chances of satisfactory islet isolation. REFERENCES 1. Shapiro AM, Lakey JR, Ryan EA, et al: N Engl J Med 343:230, 2000 2. Oberholzer J, Triponez F, Mage R, et al: Transplantation 69:1115, 2000 3. Benhamou PY, Oberholzer J, Toso C, et al: Diabetologia 44:859, 2001 4. Ricordi C, Lacy PE, Finke EH, et al: Diabetes 37:413, 1988 5. Buhler L, Deng S, Mage R, et al: Transplant Proc 26:628, 1994 6. Ricordi C, Gray DW, Hering BJ, et al: Acta Diabetol Lat 27:185, 1990 7. Lakey JR, Warnock GL, Rajotte RV, et al: Transplantation 61:1047, 1996 8. Zeng Y, Torre MA, Karrison T, et al: Transplantation 57:954, 1994 9. Brandhorst D, Hering BJ, Brandhorst H, et al: Transplant Proc 26:592, 1994 10. Benhamou PY, Watt PC, Mullen Y, et al: Transplantation 57:1804, 1994 11. Mahler R, Franke FE, Hering BJ, et al: J Mol Med 77:87, 1999 12. Ketchum RJ, Nicolae M, Jahr H, et al: Transplant Proc 26:596, 1994
T
HE CLINICAL results of human islet transplantation are improving.1,2 However, most recipients still need islet preparations isolated from more than one donor to provide a sufficient islet mass to obtain insulin independence. The number of islets in a human pancreas varies between 300,000 to 1.5 million. With present techniques, only 30% to 50% of islets within a pancreas are isolated, a discrepancy that emphasizes the need for improved yields of isolated human islets. The present study investigates the donor characteristics and the technical factors that may affect isolation yields, with special attention to donor selection and timing of procurement. MATERIALS AND METHODS From November 1991 to October 2000, 234 human islet isolations have been performed at our laboratory. The first 50 procedures used to standardize the technique (from November 1991 to October 1993) and 15 islet isolations performed for autologous transplantation (isolation without purification) have been excluded from the present study. Pancreata were retrieved from multiorgan donors harvested in Switzerland and in French centers participating in the GRAGIL cooperative project.3 After aortic cross-clamping, University of Wisconsin (UW) solution was infused and pancreata were harvested as quickly as possible. The organs were brought to the isolation laboratory in UW solution at 4°C in a temperaturecontrolled box (HUG-Box, Safetherm, Geneva, Switzerland). Isolations were performed according to a modification of Ricordi’s semiautomated technique.2,4 Briefly, the pancreas was separated from the duodenum, from adipose tissue, and from the spleen when harvested with the pancreas. Wirsung’s duct was cannulated at the level of the ampulla vater. Collagenase P (2 mg/mL in 250 mL Hank’s solution) at 4°C (for 127 isolations, until 1997) or Liberase HI at 30°C (for 107 isolations, after 1997) was injected manually. The distended pancreas was placed in a digestion chamber (capacity 500 mL) that had been prewarmed to 29°C. A double stainless-steel mesh (with windows ranging from 500 to 1000 m in size) was placed between the lower and the upper portion of the chamber to retain undigested tissue. The chamber was then connected to the tubing of a circulating system activated by a peristaltic pump and warmed to 37°C, using an electronically controlled heating system. The chamber, containing seven glass marbles, was shaken continuously during the digestion. Samples of the preparation were taken regularly from a tap adapted to the chamber itself.5 The macroscopic appearance of the digest was 0041-1345/02/$–see front matter PII S0041-1345(01)02925-6 826
monitored through a window adapter on the chamber. Dithizonestained samples were used to evaluate the microscopic appearance. When the pancreas was soft and the free islets had been released, digestion was stopped by dilution with 6 to 8 L of cold Hank’s solution (4°C), supplemented with 5% newborn calf serum (Seromed, Berlin, Germany) in the first 2 L. The tissue digests were collected, pooled in University of Wisconsin solution (DuPont Pharma, Bad Humburg, Germany), and kept on ice for 30 minutes. The digested tissue was suspended at the lowest level of a discontinuous Euro-Ficoll gradient (Sigma, St Louis, Mo) on a cell separator (Model 2991, Cobe, Lakewood, Col.) using densities of 1.108 at the bottom and 1.096, 1.037, and 1.006 at the top for purification. After purification, samples were taken for counting. Only intact, dithizone-stained islets were counted; the equivalent islet number (EIN) was calculated by normalizing the islets to a standard diameter of 150 m as described previously.6 Because 82 partial pancreata were harvested at the beginning of our experience, we expressed isolation yields as EIN per gram of pancreas wet weight before enzyme injection. The following donor factors were evaluated retrospectively with respect to isolation yields (EIN/g): age; gender; body mass index (weight [kg]/body surface area [m2]); cause of death; length of ICU stay; history of hypotension (mean arterial blood pressure ⬍60 mm Hg); blood glucose value; liver function tests; and serum amylase and lipase. The duration of secondary warm ischemia during the procurement was defined as the time between aortic cross-clamping and the end of pancreatectomy. The duration of cold ischemia was the time between pancreatectomy and collagenase injection. Data are presented as mean values ⫾ standard deviations (SD) unless specified otherwise. Statistical analysis was performed with Student’s t test. P ⬍ .05 was considered statistically significant.
RESULTS
In the present study, 169 isolations were performed over a 7-year period. A mean of 24 (14 to 52) isolations/year were performed, with increasing numbers each year. The mean yield during the entire period was 1689.2 ⫾ 1560 EIN/g. From the Division of Surgical Research, Department of Surgery, University Hospital, University of Geneva, Switzerland. Supported by Grant 3200-061873.00 from the Swiss National Science Foundation and by the Foundation Carlos et Elsie de Reuter. Address reprint requests to Dr Christian Toso, Division of Surgical Research, Department of Surgery, University Hospital, 24 rue Micheli-du-Crest, 1211 Geneva 14, Switzerland. © 2002 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 34, 826 – 827 (2002)
ISLET ISOLATION YIELDS
827
Table 1. Effect of Donor Characteristics and Pancreas Procurement Parameters on Human Isolation Yield EIN/g
Age (years) ⬎20 (n ⫽ 155) ⬍20 (n ⫽ 13) BMI (kg/m2) ⬎25 (n ⫽ 52) ⬍25 (n ⫽ 117) Hypotension No (n ⫽ 99) Yes (n ⫽ 65) Secondary warm ischemia (min) ⬍30 (n ⫽ 13) ⬎30 (n ⫽ 93) Cold ischemia (h) ⬍8 (n ⫽ 135) ⬎8 (n ⫽ 25)
P
1763 ⫾ 1581 1011 ⫾ 981
⬍.01
2135 ⫾ 2142 1508 ⫾ 1147
⬍.01
1873 ⫾ 1751 1512 ⫾ 1217
.06
2434 ⫾ 1871 1625 ⫾ 1996
.05
1906 ⫾ 1037 1037 ⫾ 645
⬍.005
Data expressed as mean ⫾ SD.
During the last 2 years, only whole organs were processed yielding 211,171 ⫾ 145,576 EIN per pancreas. Age had a significant effect on isolation yield; better results were obtained with pancreata from donors ⬎20 years (Table 1). Isolation yields from donors ⬎20 years varied little, even when age exceeded 50 years. The BMI was positively correlated with isolation yield: pancreata from donors whose BMI was ⬎25 kg/m2 had higher yields. Although not statistically significant, occurrence of a hypotensive episode was associated with lower isolation yields. The length of both the secondary warm ischemia and the cold ischemia times had relevant effects on yield. Gender, cause of death, length of ICU stay, and dose of vasopressors (noradrenaline, adrenaline, or dopamine) ongoing at the time of procurement had no detectable effect on the yield. Blood chemistry tests (blood glucose, liver, and pancreas tests) also did not correlate with the yield. DISCUSSION
The increasing interest and demand for islet transplantation challenges the current capacity of islet isolation facilities. The scarcity of donors and the costs of islet isolations require reliable criteria to avoid the exclusion of eventually successful donors and to reduce the number of procedures with insufficient islet yields. The present study investigated donor and procurement parameters that affect islet isolation yields. Donor age had an impact on islet yields, as described previously by other groups.7–9 Lower isolation results were achieved with pancreata from donors of ⬍20 years, possibly due to the presence of a softer parenchyma with less connective tissue surrounding the islets. In the present study, all isolations from donors ⬎20 years provided similar numbers of islets, even when age was ⬎50 years. Other series have observed lower isolation yields and decreased
glucose sensitivity of islets from donors ⬎50 years of age.7,8,10 However, the data presented by the Edmonton group, similar to our experience, revealed no deleterious effect of older donor age on islet graft outcome.1 A high BMI was positively correlated with isolation yields, confirming previous data.7,9 The total islet mass may be increased in overweight patients, due to a higher metabolic demand. In addition, the higher islet yield in obese donors may be explained by a higher content of fat cells in the pancreas parenchyma, a factor that has been demonstrated to increase yield.11 The occurrence of a hypotensive episode was associated with a trend toward poorer isolation results, whereas no correlation was found between the administration of vasoactive drugs and islet yields. This finding suggests that successful stabilization of vital signs is the most important goal, regardless of the dose of amines, and that a more liberal use of vasoactive drugs in modern donor management does not affect yield. Secondary warm ischemia and cold ischemia times provided relevant parameters for islet isolation7–9,12; every effort should be made to shorten both times. The pancreas must be harvested quickly, before the kidneys and, if possible, before the liver. The retrieval must take care to preserve the capsule intact.9 Finally, procuring hospitals should be within a short distance from the islet isolation facility to allow initiation of digestion within 8 hours of aortic clamping. A better selection of donors and improved procurement procedures should give better isolation yields and decrease the variability between isolations, which in turn may facilitate new developments in the isolation process. Recent clinical successes1 are a stimulus to give pancreas procurement the same attention as with other solid organs to increase the chances of satisfactory islet isolation. REFERENCES 1. Shapiro AM, Lakey JR, Ryan EA, et al: N Engl J Med 343:230, 2000 2. Oberholzer J, Triponez F, Mage R, et al: Transplantation 69:1115, 2000 3. Benhamou PY, Oberholzer J, Toso C, et al: Diabetologia 44:859, 2001 4. Ricordi C, Lacy PE, Finke EH, et al: Diabetes 37:413, 1988 5. Buhler L, Deng S, Mage R, et al: Transplant Proc 26:628, 1994 6. Ricordi C, Gray DW, Hering BJ, et al: Acta Diabetol Lat 27:185, 1990 7. Lakey JR, Warnock GL, Rajotte RV, et al: Transplantation 61:1047, 1996 8. Zeng Y, Torre MA, Karrison T, et al: Transplantation 57:954, 1994 9. Brandhorst D, Hering BJ, Brandhorst H, et al: Transplant Proc 26:592, 1994 10. Benhamou PY, Watt PC, Mullen Y, et al: Transplantation 57:1804, 1994 11. Mahler R, Franke FE, Hering BJ, et al: J Mol Med 77:87, 1999 12. Ketchum RJ, Nicolae M, Jahr H, et al: Transplant Proc 26:596, 1994