Sage Journals: Discover world-class research

Abstract

Patients fulfilling criteria for euthanasia can choose to donate their organs after circulatory death [donors after euthanasia (DCD V)]. This study assesses the outcome of islet cell isolation from DCD V pancreases. A procedure for DCD V procurement provided 13 pancreases preserved in Institut Georges Lopez-1 preservation solution and following acirculatory warm ischemia time under 10 minutes. Islet cell isolation outcomes are compared with those from reference donors after brain death (DBD, n = 234) and a cohort of donors after controlled circulatory death (DCD III, n = 29) procured under the same conditions. Islet cell isolation from DCD V organs resulted in better in vitro outcome than for selected DCD III or reference DBD organs. A 50% higher average beta cell number before and after culture and a higher average beta cell purity (35% vs 24% and 25%) was observed, which led to more frequent selection for our clinical protocol (77% of isolates vs 50%). The functional capacity of a DCD V islet cell preparation was illustrated by its in vivo effect following intraportal transplantation in a type 1 diabetes patient: injection of 2 million beta cells/kg body weight (1,900 IEQ/kg body weight) at 39% insulin purity resulted in an implant with functional beta cell mass that represented 30% of that in non-diabetic controls. In conclusion, this study describes procurement and preservation conditions for donor organs after euthanasia, which allow preparation of cultured islet cells, that more frequently meet criteria for clinical use than those from DBD or DCD III organs.

Keywords

islet transplantation organ donation after euthanasia organ donation after circulatory death organ donation after medical assistance in dying

Introduction

Intraportal (IP) islet cell transplantation allows restoration of a functional beta cell mass (FBM) in type 1 diabetes patients^1–3. With metabolic outcome of clinical implants dependent on a minimal number of implanted beta cells, availability of good quality donor organs is considered a main limitation for islet cell transplantation programs⁴. In a number of countries, including Belgium, Luxemburg, The Netherlands, and some regions in Canada, a legal and ethical framework allows organ and tissue donation after euthanasia^5,6, thus providing the possibility to increase the organ donor pool⁷. These donors are rarely hospitalized or admitted to the intensive care unit and have not underwent an acute neurological insult with its associated cytokine storm, nor episodes of cardiac dysfunction and decreased organ perfusion^8–11. Despite these benefits, organ procurement can only be performed after cessation of circulation, leading to exposure to an initial episode of warm ischemia time (WIT), increasing the risk for organ dysfunction. They are therefore classified as a subtype of donors after circulatory death using a modified Maastricht classification [donors after euthanasia (DCD V)]¹². Our university hospital is strongly involved in end-of-life care with referrals specific for these procedures. This allowed us to evaluate in vitro and in vivo outcome of islet cell isolations from these donors while selecting conditions that have been shown beneficial for DCD organ procurement and preservation, such as the use of Institut Georges Lopez-1 (IGL-1) preservation solution and limiting acirculatory warm ischemia to 10 min¹³.

Materials and Methods

Euthanasia and Organ Donation

As in some other regions worldwide, a legal and ethical framework exists in Belgium allowing organ and tissue donation after euthanasia. The Belgian law stipulates a number of criteria that should be met before euthanasia can be performed [Wet van 28 mei 2002 Betreffende de Euthanasie (Law concerning Eutanasia); Belgisch Staatsblad/Moniteur Belge, June 22, 2002, https://justice.belgium.be]. This is the case for patients that are terminally ill but also for patients that suffer from untreatable neuropsychiatric disorders or dementia. Patients should voluntarily, well-considered and repeatedly express their request for euthanasia without external pressure. After approval of this request, patients may express the will to donate organs. Decisions and procedures regarding euthanasia and organ donation are completely separated, avoiding social or psychological pressure on the patient or physician¹⁴ but also respecting the patients right to self-determination. Organ allocation is done by Eurotransplant (Leiden, The Netherlands) as an independent allocation organism.

Organ Procurement

Donation after euthanasia involves a particular procedure of donation after circulatory arrest and is classified as DCD V in the modified Maastricht classification¹⁵. End-of-life therapy can be performed at the hospitalization unit followed by rapid transportation of the donor to the operating theater during the 5-min no-touch period. Heparin (300 U/kg body weight) is administered immediately before administration of end-of-life medication, typically consisting of a sedation with 15 mg midazolam, followed by 3 g thiopental. In our center, as an alternative and after extensive discussion with the patient, sedation can be performed in the presence of the relatives with subsequent transportation to the operating theater where end-of-life therapy is administered. Also followed by a 5-min no-touch period after cessation of circulation before declaration of death by three independent physicians, this approach avoids rushing to the operating theater in the presence of mourning relatives and is preferred by more than 90% of our patients. A rapid sterno-laparotomy is then performed followed by cold flush, topical cooling, and procurement of the pancreas. Because of its beneficial effect on DCD pancreas preservation for islet cell isolation¹³, IGL-1 is used as cold preservation solution in our DCD V procurement. Total WIT is defined as time between end-of-life therapy and abdominal aorta cannulation initiating cold preservation. It consists of an agonal phase (time till circulatory arrest) and a subsequent acirculatory phase¹⁶. All procedures involved in organ donation after euthanasia were evaluated and approved by the ethical committee of our institution (CME 2013/V7).

Study Design and Cohort Selection

The database of our Beta Cell Bank was used to conduct a retrospective analysis of prospectively collected donor and procurement characteristics and associated quality control data of islet cell isolates. Between January 2015 and December 2020, 13 DCD V organs that matched our set criteria were processed and included for analysis. They were compared with our reference cohort of 234 donors after brain death (DBD) from the same time period and a cohort of 29 DCD III (donors after controlled circulatory death) organs selected for acirculatory WIT <10 min and IGL-1 preservation solution, conditions that have been shown to favor DCD islet cell isolation¹³. Reasons for euthanasia request are shown in Table 1. The study was reviewed and approved by the ethical committee of our institution (B.U.N. 143202042685 and CME 2005/136).

Table 1.

Reason for Request for Euthanasia in 13 DCD V Used for Pancreatic Islet Cell Isolation.

n (%)	13
Degenerative neurological disorder	1 (8)
Psychiatric disorder	6 (46)
Pain syndrome	5 (38)
Unbearable suffering, not otherwise specified	1 (8)

DCD V: donors after euthanasia.

Islet Cell Isolation, Purification, Quality Control, and Culture

Islet cells were isolated using a modification of the automated Ricordi method¹⁷ and purified by continuous gradient with Biocoll (Biochrom, Berlin, Germany) and a cooled COBE 2991 cell processor (Terumo BCT, Lakewood, CO, USA). After isolation and purification, the cell preparations were cultured in cell culture flasks T175 (Sarstedt, Nümbrecht, Germany) at 37°C in a humidified incubator (5% CO₂) in a Ham’s F10 based medium (Lonza, Bazel, Switzerland).

Preparations were characterized immediately after purification and after a 1- to 5-day culture period by their beta cell number, insulin content, and insulin purity, as described previously¹⁷. Beta cell number was calculated from the total nuclear count (NucleoCounter YC-100; ChemoMetec, Allerod, Denmark) and the percentage of insulin positive cells [immunocytochemistry with guinea pig anti-insulin (1/2,000, in-house produced) on 1.5 µm araldite sections; >2 × 10³ cells counted; pictures were captured using Nikon Eclipse Ti microscope and analyzed with NIS-Elements AR v5.21 software (Nikon Europe, Amsterdam, The Netherlands; Fig. 1)]. Yield immediately post purification was also expressed as islet equivalent (IEQ), calculated using a volume based method after dithizone staining¹⁹.

Figure 1.

Composition of islet cell preparation. Beta cell number is calculated from the total nuclear count and the percentage of insulin positive cells on immunocytochemistry. This is illustrated by a donor after euthanasia preparation stained for insulin in green (bar indicates 100 µm). Insulin-negative cells include glucagon- or somatostatin-positive cells and non-endocrine cells that consist predominantly of pancreatic duct cells¹⁸.

Clinical Transplantation and Assessment

Preparations are cultured and consecutively combined into a graft of one or multiple donor preparations. Islet cell grafts were defined by beta cell number, insulin content, insulin purity, and insulin biosynthesis capacity¹⁸.

In our protocol, non-uremic type 1 diabetes mellitus patients typically receive two IP²⁰ islet cell transplantations unless they achieve insulin independence or a serum human C-peptide level above 1.0 ng/ml (measured at blood glucose levels below 220 mg/dl) and a glycemia coefficient of variation <25% after a single islet cell infusion¹.

Immunosuppression consists of an induction with anti-thymocyte globulin (bolus of 9 mg/kg followed by 3 mg/kg during 6 days, unless T-lymphocyte count <50/mm³) for a first transplantation or basiliximab (20 mg on day 0 and day 4) for a second transplantation. Patients receive a single injection of 500 mg methylprednisolone immediately before transplantation and anti-tumor necrosis factor (TNF) therapy during a 10-day period after transplantation (etanercept 50 mg on day 1 and 25 mg on days 3, 7, and 10). Maintenance therapy consists of mycophenolate mofetil and tacrolimus aiming at through levels between 8 and 10 ng/ml in the first year following transplantation, and 6 and 8 ng/ml thereafter.

Metabolic function was determined by glycosylated hemoglobin (HbA1c) concentrations, serum C-peptide levels, fasting blood glucose levels, and its coefficient of variation²¹. Daily insulin dose was adjusted to keep blood glucose levels between 70 and 180 mg/dl. Hyperglycemic clamp tests were performed to measure the achieved FBM expressed as percentage of non-diabetic controls^1,22; BETA-2 scores were calculated as described²³. All islet cell recipients gave written consent for use of their data in our clinical studies.

Statistical Analysis

Individual donor and procurement characteristics reported in the Eurotransplant donor and procurement file as well as on pancreas processing, isolation outcome, and culture characteristics were stored in our Filemaker database (Filemaker, Inc., Santa Clara, CA, USA). Results are presented as median [interquartile range (IQR)] if not otherwise specified. Statistical analysis was performed using SPSS (IBM, Armonk, NY, USA). Nonparametric tests (two-tailed Mann-Whitney U test for continuous variables, chi-square test for categorical variables) were used for analysis, unless otherwise specified. Statistical significance was assumed at P < 0.05.

Results

Donor and Procurement Characteristics of DCD V

Baseline characteristics of 13 DCD V are compared with a reference cohort of 234 DBD and to 29 DCD III selected for preservation solution and acirculatory WIT (Table 2). As most DCD V procedures are planned, time between admission to the hospital and organ recovery as well as time at the intensive care unit was nearly absent. There was no need for vasoactive medication, documented cardiac arrest, or episodes of hypotension in DCD V donors. This translated biochemically in normal serum sodium and creatinine levels. Blood glucose levels were normal in all DCD V. Serum lipase levels were significantly higher in DCD V when compared with DBD with levels above 60 U/l (reference value < 60 U/l) in more than 50% of cases (P = 0.014). Most DCD V procedures were performed at our campus and planned during office hours resulting in a median cold preservation time of 2 h, which is significantly shorter than the median preservation time in DBD or DCD III (P < 0.001), that are imported from other procurement hospitals in more than 50%. Pancreas extraction time was also significantly shorter in DCD V. Total WIT in DCD V ranged between 10 and 22 min, consisting of an agonal WIT between 1 and 14 min and acirculatory WIT below 10 min. While average body mass index (BMI) of DCD V tended to be slightly higher, age and North American Islet Donor Score (NAIDS) were similar between donor types.

Table 2.

Donor and Organ Procurement Characteristics for Pancreases Procured From DCD V Compared With DBD and DCD III.

n	DCD V	DBD	P	DCD III	P
n	13	234	P	29	P
Donor characteristics
Age (years)	52 (37–62)	55 (46–63)	0.318	55 (44–61)	0.727
Body mass index (kg/m²)	27 (24–32)	25(23–28)	0.052	25 (23–27)	0.049
Gender (% M/F)	8/92	48/52	0.004	83/17	<0.001
Time in hospital (days)	0 (0–0)	2 (1–4)	<0.001	5 (4–8)	<0.001
Time in intensive care (days)	0 (0–0)	2 (1–4)	<0.001	5 (3–7)	<0.001
Sodium (mmol/l)	140 (139–141)	148 (143–153)	<0.001	144 (142–147)	<0.001
Glucose (mg/dl)	87 (79–100)	179 (148–229)	<0.001	165 (129–197)	<0.001
Lipase (U/l)	90 (43–114)	31 (20–63)	0.001	56 (19–186)	0.461
Alanine-aminotransferase (U/l)	23 (18–41)	35 (20–79)	0.159	65 (37–154)	<0.001
Lactate dehydrogenase (U/l)	427 (378–493)	375 (258–561)	0.435	520 (364–810)	0.118
Creatinine (µmol/l)	68 (53–74)	83 (66–107)	0.004	86 (65–113)	0.012
Procurement characteristics
Agonal WIT (min)	6 (5–8)	NA		7 (2–13)	0.793
Acirculatory WIT (min)	8 (7–9)	NA		8 (7–9)	0.484
Total WIT (min)	13 (12–16)	NA		15 (10–22)	0.515
Pancreatectomy time (min)	24 (23–30)	39 (30–50)	<0.001	34 (25–45)	0.013
Cold ischemia time (h)	2 (2–3)	9 (6–11)	<0.001	6 (4–13)	<0.001
NAID score	72 (61–85)	65 (59–74)	0.113	77 (67–87)	0.667

Data are presented as median (interquartile range); statistical significance (Mann-Whitney U) was assumed at P < 0.05.

DCD V: donors after euthanasia; DBD: donors after brain death; DCD III: donors after controlled circulatory death; WIT: warm ischemia time; NA: not available; NAID: North American Islet Donor Score.

In Vitro Outcome of Islet Cell Isolation From DCD V

Isolation outcome parameters of DCD V are compared with DBD and matched DCD III, and are summarized in Table 3. Pancreas weight, percentage of undigested tissue, and post-digestion cell pellet volume were similar between all groups, while digestion time tended to be shorter for DBD and DCD III. Compared with DBD, DCD V pancreases yielded 50% more beta cells after purification and after 1 to 5 days of culture. When expressed as IEQ, difference in isolation yield after purification was 20%. Subgroup analysis of DBD organs that were preserved using IGL-1 showed higher beta cell yield than organs preserved using histidine-tryptophane-ketoglutarate or University of Wisconsin preservation solution [117 (72–217) vs 96 (44–138) × 10⁶ beta cells after purification and 71 (43–127) vs 55 (30–81) × 10⁶ beta cells after culture, data not shown], but this was still on average 25% lower than what was obtained in DCD V. When compared with DCD III, matched for IGL-1-preservation and acirculatory WIT <10 min, DCD V organs yielded on average two times more beta cells after purification. Beta cell recovery after culture was similar between donor types [DCD V, 61% (52–70); DCD III, 57% (45–73); and DBD, 64% (53–76); P = 0.674 and P = 0.512 vs DCD III and DBD, respectively]. DCD V preparations also contained a significantly higher percentage of insulin positive cells than DBD or DCD III preparations (35% vs 24% and 25%, respectively). Of the 13 donors after euthanasia, 10 islet cell preparations (77%) were used for clinical transplantation, while this was the case for 116 out of 234 DBD preparations (50%, P = 0.055) and 14 out of 29 DCD III preparations (48%, P = 0.083).

Table 3.

Isolation Outcome for Pancreases Procured From DCD V Compared With DBD and DCD III.

n	DCD V	DBD	P	DCD III	P
n	13	234	P	29	P
Isolation characteristics
Pancreas weight (g)	110 (98–127)	100 (83–113)	0.096	113 (96–120)	0.914
Digestion time (min, >35°C)	16 (16–19)	14 (11–17)	0.046	14 (11–19)	0.276
Undigested pancreas (%)	15 (12–26)	19 (13–26)	0.348	17 (14–22)	0.515
Digest pellet volume (ml)	25 (20–35)	25 (20–30)	0.294	25 (20–33)	0.936
Isolation outcome
Beta cell
Purity (% insulin)	35 (26–40)	24 (18–33)	0.015	25 (17–31)	0.040
Number after purification (×10⁶)	164 (88–194)	109 (63–175)	0.142	86 (50–151)	0.058
Number after culture (×10⁶)	90 (56–129)	62 (36–107)	0.208	60 (31–93)	0.081
IEQ
Purity (% DTZ)	66 (57–75)	60 (50–70)	0.096	52 (46–67)	0.007
Number after purification (×10³)	161 (111–211)	130 (79–199)	0.401	130 (62–173)	0.159
Insulin content (µg/10⁶ beta cells)
After purification	22 (17–26)	18 (13–26)	0.212	20 (14–27)	0.494
After culture	17 (15–30)	14 (11–20)	0.059	15 (10–23)	0.204

Data are presented as median (interquartile range); statistical significance (Mann-Whitney U) was assumed at P < 0.05.

DCD V: donors after euthanasia; DBD: donors after brain death; DCD III: donors after controlled circulatory death; IEQ: islet equivalent; DTZ, dithizone.

Functional Capacity of DCD V Islet Cell Preparation

The functional capacity of a DCD V islet cell preparation following transplantation was demonstrated by one DCD V islet cell preparation that fulfilled our quantitative release criteria for clinical transplantation of 2.0 × 10⁶ beta cells/kg body weight (1,900 IEQ/kg body weight) after overnight culture. It contained 39% insulin positive cells with an insulin biosynthesis capacity of 35 pmol/10⁶ beta cells/2 h (Table 4). It was transplanted in a patient who received an IP transplant 10 years earlier (IP 1 and 2) but with progressive decline in FBM to 6% of non-diabetic controls since 5 years (Table 1). While still on maintenance immune suppressive therapy, she underwent two new IP grafts of which the second IP (IP 4) transplant contained the single donor DCD V islet cell preparation (Table 1). Its in vivo outcome was demonstrated 3 months post-transplant by a hyperglycemic clamp test: this second implant increased FBM by 30% leading to a total FBM of 55% of non-diabetic controls. The obtained FBM was associated with an improved metabolic control as illustrated by HbA1c levels of 5.3%, a decrease in daily insulin requirements to 0.08 U/kg, and a drop in glycemia coefficient of variability from 19.5% to 11.5%. Implant function was also demonstrated in summary tracings of continuous glucose monitoring, with a mean glycemia of 106 ± 29 mg/dl and a time in range (percentage of time between 70 and 180 mg/dl) of 94%. Serum C-peptide levels were maintained above 3 ng/ml (Fig. 2B) during 3-year follow-up.

Table 4.

Recipient, Donor, and Graft Characteristics of a Patient That Received an IP Islet Cell Graft Prepared From a Single DCD V 9 Months After an IP Implant From a DBD.

Recipient
Age (years)	48
Gender	Female
Weight (kg)	62
Donor	IP3		IP4
Donor type	DBD		DCD V
Age (years)	63		27
BMI (kg/m²)	26		39
Cold ischemia time (h)	6		1
Culture time (days)	6		1
Graft	IP3		IP4
Beta cell mass (10⁶ beta cells/kg)	1.8		2.0
Beta cell purity (% insulin)	37		39
Alpha cell purity (% glucagon)	10		8
Islet mass (IEQ/kg body weight)	NA		1,900
Insulin content (µg/10⁶ beta cells)	22		20
Insulin biosynthesis (pmol/10⁶ beta cells)	17		35
Outcome	Pretransplant	PT month 2	Pretransplant	PT month 3
Hyperglycemic clamp (% NDC)	6	23	26	55
C-peptide (ng/ml)	0.45	1.42	1.94	3.27
Daily insulin requirement (U/kg)	0.41	0.22	0.25	0.08
HbA1c (%)	6.3	5.3	5.4	5.3
BETA-2 score	4.2	13.2	12.5	21.8
Mean glycemia (mg/dl)	153 ± 65	120 ± 39	120 ± 37	106 ± 29
Glycemic coefficient of variation (%)	27	15	20	11.5
Time in range (%)	69	88	89	94

IP: intraportal; DCD V: donors after euthanasia; DBD: donors after brain death; BMI: body mass index; IEQ: islet equivalent; NA: not available; PT: post-transplantation; NDC: non-diabetic control.

Figure 2.

In vivo functional beta cell mass. Functional beta cell mass in intraportal implants as determined by hyperglycemic clamp area under curve (AUC) and expressed as percentage of non-diabetic controls. Comparison of in vivo effect of cultured islet cells prepared from DBD organs (full circles) or from a DCD V organ (open circles). (A) Following transplantation in five different patients. (B) Following consecutive transplantations in the same patient: IP3—single donor DBD, IP4—single donor DCD V: arrows indicate time of injection. DBD: donors after brain death; DCD V: donors after euthanasia; PT: post-transplantation.

We also compared the obtained FBM with the four patients receiving a 16- to 40-h cultured single DBD islet cell graft that contained on average 4.2 × 10⁶ beta cells/kg recipient body weight (range = 2.2–5.1 × 10⁶) at 28% insulin positivity (range = 25%–51%) and with an insulin biosynthesis capacity of 22 pmol/10⁶ beta cells/2 h (range = 15–25 pmol/10⁶ beta cells/2 h). They showed an increase in FBM that ranged between 6% and 22% (median 12%) (Fig. 2A) 2 months post-transplantation.

Discussion

Availability of donor pancreases remains a major limitation for clinical islet cell transplantation⁴. Some centers have therefore expanded their donor pool with DCD III^24–28, but this is at the cost of lower isolation yields, which can at least partially be attributed to a period of warm ischemia before cold perfusion^13,24,29. In Belgium, an ethical and legal framework is established for organ donation after euthanasia (DCD V), including in case of unbearable suffering due to neuropsychiatric disorders, unbearable pain, or dementia³⁰. Like DCD III, this involves organ procurement after circulatory arrest which is associated with initial agonal and acirculatory WIT¹². While in DCD III the agonal phase is a passive process of therapy withdrawal, in DCD V this is intentionally accelerated by the use of end-of-life medication. Consequently, agonal WIT and acirculatory WIT are shorter in DCD V^8,13. Reports from DCD V liver transplantation show that despite shorter WIT, these organs are, as DCD III organs, at increased risk for non-anastomotic biliary strictures^31,32.

Organ donation after euthanasia allows optimal conditions during daytime with a fully staffed procurement and islet cell isolation team. These donors have not underwent brain death nor associated cytokine storm⁹, which have a negative impact on isolation yield and in vivo function¹⁰. DCD V lung recipients showed good early graft function³³, even when the procedure is initiated outside the hospital³⁴. A recent study reported equal short- and long-term outcome as for DCD III and DBD³⁵.

DCD V procedures constitute a minority (<10%) of all DCD procedures, but it has been suggested that DCD V organ donation has the potential to double the total number of donor organs available for transplantation⁷. This led us to compare DCD V islet isolation with the golden standard of DBD using in vitro and in vivo outcome parameters. At variance with reports of DCD III^13,28, DCD V isolation yield was on average 50% higher than from our DBD cohort when expressed as beta cell number and 20% when expressed as IEQ. DCD V islet cell preparations also contained a significantly higher percentage of insulin positive cells and were more frequently used for clinical transplantation.

The semi-planned nature of euthanasia procedures allowed us to procure DCD V pancreases under conditions (acirculatory WIT ≤ 10 min, IGL-1 cold preservation solution) that have been shown to maintain islet cell isolation yield in DCD III at levels achieved for DBD¹³, further supporting the value of these conditions. When we compared isolation yield in DCD V with a cohort of DCD III from the same time period and matched for these beneficial conditions, beta cell yield after euthanasia was still 50% higher.

Our DCD V also exhibited donor profiles, including for BMI, time in hospital, and sodium and blood glucose levels that favor islet isolation yield^36–38. This was not reflected in higher global NAID scores due to negative scoring of a short cold preservation time, which was below 3 h in more than 75% of cases in our DCD V cohort: While previous data emphasize on limiting cold ischemia time of pancreases procured for islet isolation to 8 to 12 h^39,40, negative influences of shorter cold ischemia time have been reported³⁷.

We did notice higher lipase values in DCD V, which has been reported to be associated with pancreas damage leading to a lower isolation yield^36,37. However, in our DCD V cohort, lipase elevation may be related to the use of psychopharmaceutic drugs or opioid analgesics and therefore not influencing islet cell yield. Euthanasia requests and procedures for patients suffering from neuropsychiatric disorders have a predominance of female patients⁴¹. This was also the case in our DCD V cohort and might positively influence isolation yield¹³.

In our clinical program, islet cell isolates from multiple donors are cultured and combined to meet our quantitative release criteria for clinical transplantation¹. Multiple injections of multi-donor islet cell isolates are usually needed to achieve metabolic goals, which complicates studying individual donor variables by in vivo outcome in patients^1,42,43. Out of 10 DCD V preparations used for clinical transplantation, we identified one that was used as a single donor graft. Its in vivo function was demonstrated by a significant increase in FBM assessed by hyperglycemic clamp before and 3 months after islet cell infusion in this patient that received a previous IP DBD implant. This outcome parameter correlates well with decreasing glycemic variability and insulin needs²¹. Recently, and like others, we aim to continue exogenous insulin at a low dose to support the beta cell implant⁴⁴. While exogenous insulin was not completely withdrawn, FBM and metabolic effect during 3-year follow-up was preserved at a level that has been reported to support a state of insulin independence²¹.

Several factors could have added to this metabolic outcome. The preparation contained a high percentage of insulin and glucagon positive cells, which has been shown in preclinical models to positively correlate with implant function^45,46. It is conceivable that a prior period of 10-year immune suppression facilitated survival of the implant. It is likely that the survival of the previous islet cell implant contributed to the metabolic effect achieved by the DCD V implant. These variables also operate in transplants of DBD and DCD III organs, which further underlines the need for multifactorial analysis of well-defined study groups. The present study demonstrates that DCD V organs deserve their place in such analysis. It reports procurement and preservation conditions that more frequently resulted in cultured islet cell preparations meeting the criteria for use in our clinical transplant protocol.

Footnotes

Acknowledgements

The authors thank all procurement teams affiliated with the Eurotransplant network and all collaborators at the Diabetes Research Center of Vrije Universiteit Brussel (VUB) and Universitair Ziekenhuis Brussel (UZ Brussel).

Ethical Approval

Ethical committee approval was obtained from the Comity for Medical Ethics Universitair Ziekenhuis Brussel (UZ Brussel) and Vrije Universiteit Brussel (VUB), and is acknowledged within the text of the submitted manuscript. All procedures involved in organ donation after euthanasia were evaluated and approved (CME 2013/V7). Clinical islet transplantation was evaluated and approved (CME 2005/136). Retrospective study of prospectively collected data on donor, procurement, and isolation characteristics was evaluated and approved (B.U.N. 143202042685).

Statement of Human and Animal Rights

All procedures in this study were conducted in accordance with the Comity for Medical Ethics Universitair Ziekenhuis Brussel (UZ Brussel) and Vrije Universiteit Brussel (VUB) approved protocols.

Statement of Informed Consent

Written informed consent was obtained from the patients for their anonymized information to be published in this article. Written informed consent could not be obtained from deceased organ donors, but their use was according to Eurotransplant Guidelines and approved by the Comity for Medical Ethics Universitair Ziekenhuis Brussel (UZ Brussel) and Vrije Universiteit Brussel (VUB).

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request. The data are not publicly available due to privacy or ethical restrictions.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by grants from the European Commission (FP7 241883, H2020 681070), the Flemish Government (IWT 130138), the Juvenile Diabetes Research Foundation (17-2013-296, 2-SRA-2019-708-S-B), and the “Wetenschappelijk Fonds Willy Gepts” from Universitair Ziekenhuis Brussel (UZ Brussels).

ORCID iD

Daniel Jacobs-Tulleneers-Thevissen

References

Keymeulen

Gillard

Mathieu

Movahedi

Maleux

Delvaux

Ysebaert

Roep

Vandemeulebroucke

Marichal

’t

Veld

, et al. Correlation between beta cell mass and glycemic control in type 1 diabetic recipients of islet cell graft. Proc Natl Acad Sci U S A. 2006;103(46):17444–49.

Lablanche

Vantyghem

Kessler

Wojtusciszyn

Borot

Thivolet

Girerd

Bosco

Bosson

Colin

Tetaz

, et al. Islet transplantation versus insulin therapy in patients with type 1 diabetes with severe hypoglycaemia or poorly controlled glycaemia after kidney transplantation (TRIMECO): a multicentre, randomised controlled trial. Lancet Diabetes Endocrinol. 2018;6(7):527–37.

Pipeleers

Chintinne

Denys

Martens

Keymeulen

Gorus

. Restoring a functional beta-cell mass in diabetes. Diabetes Obes Metab. 2008;10(Suppl 4):54–62.

Nano

Kerr-Conte

Scholz

Engelse

Karlsson

Saudek

Bosco

Antonioli

Bertuzzi

Johnson

PRV

Ludwing

, et al. Heterogeneity of human pancreatic islet isolation around Europe: results of a survey study. Transplantation. 2020;104(1):190–96.

McCormack

Fléchais

. The role of psychiatrists and mental disorder in assisted dying practices around the world: a review of the legislation and official reports. Psychosomatics. 2012;53(4):319–26.

Mulder

Sonneveld

Healey

Van Raemdonck

. The first international roundtable on “organ donation after circulatory death by medical assistance in dying” demonstrates increasing incidence of successful patient-driven procedure. Am J Transplant. 2022;22(3):999–1000.

Bollen

van Smaalen

ten Hoopen

van Heurn

Ysebaert

van Mook

. Potential number of organ donors after euthanasia in Belgium. JAMA. 2017;317(14):1476–77.

van Reeven

Gilbo

Monbaliu

van Leeuwen

Porte

Ysebaert

van Hoek

Alwayn

IPJ

Meurisse

Detry

Coubeau

, et al. Evaluation of liver graft donation after euthanasia. JAMA Surg. 2020;155:917–24.

Schwarz

Custódio

Rheinheimer

Crispim

Leitão

Rech

. Brain death-induced inflammatory activity is similar to sepsis-induced cytokine release. Cell Transplant. 2018;27(10):1417–24.

10.

Contreras

Eckstein

Smyth

Sellers

Vilatoba

Bilbao

Rahemtulla

Young

Thompson

Chaudry

Eckhoff

. Brain death significantly reduces isolated pancreatic islet yields and functionality in vitro and in vivo after transplantation in rats. Diabetes. 2003;52(12):2935–42.

11.

Lakey

JRT

Warnock

Rajotte

Suarez-Almazor

Shapiro

Kneteman

. Variables in organ donors that affect the recovery of human islets of Langerhans. Transplantation. 1996;61(7):1047–53.

12.

Detry

Le Dinh

Noterdaeme

De Roover

Honoré

Squifflet

Meurisse

. Categories of donation after cardiocirculatory death. Transplant Proc. 2012;44(5):1189–95.

13.

De Paep

Van Hulle

Ling

Vanhoeij

Pirenne

Keymeulen

Pipeleers

Jacobs-Tulleneers-Thevissen

. Lower beta cell yield from donor pancreases after controlled circulatory death prevented by shortening acirculatory warm ischemia time and by using IGL-1 cold preservation solution. PLoS ONE. 2021;16(5):e0251055.

14.

Bollen

Ten Hoopen

Ysebaert

van Mook

van Heurn

. Legal and ethical aspects of organ donation after euthanasia in Belgium and the Netherlands. J Med Ethics. 2016;42(8):486–89.

15.

Evrard

. Belgian modified classification of Maastricht for donors after circulatory death. Transplant Proc. 2014;46(9): 3138–42.

16.

Thuong

Ruiz

Evrard

Kuiper

Boffa

Akhtar

Neuberger

Ploeg

. New classification of donation after circulatory death donors definitions and terminology. Transpl Int. 2016;29(7):749–59.

17.

Ling

Pipeleers

. Prolonged exposure of human beta cells to elevated glucose levels results in sustained cellular activation leading to a loss of glucose regulation. J Clin Investig. 1996;98(12):2805–12.

18.

Brandhorst

Johnson

PRV

Korsgren

Brandhorst

. Quantifying the effects of different neutral proteases on human islet integrity. Cell Transplant. 2017;26(11):1733–41.

19.

Keymeulen

Ling

Gorus

Delvaux

Bouwens

Grupping

Hendrieckx

Pipeleers-Marichal

Van Schravendijk

Salmela

Pipeleers

. Implantation of standardized beta-cell grafts in a liver segment of IDDM patients: graft and recipient characteristics in two cases of insulin-independence under maintenance immunosuppression for prior kidney graft. Diabetologia. 1998;41(4):452–59.

20.

Maleux

Gillard

Keymeulen

Pipeleers

Ling

Heye

Thijs

Mathieu

Marchal

. Feasibility, safety, and efficacy of percutaneous transhepatic injection of beta-cell grafts. J Vasc Interv Radiol. 2005;16(12):1693–97.

21.

Gillard

Hilbrands

Van de Velde

Ling

Lee

Weets

Gorus

De Block

Kaufman

Mathieu

Pipeleers

, et al. Minimal functional beta-cell mass in intraportal implants that reduces glycemic variability in type 1 diabetic recipients. Diabetes Care. 2013;36(11):3483–88.

22.

DeFronzo

Tobin

Andres

. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Am J Physiol. 1979;237(3):E214–23.

23.

Forbes

Oram

Smith

Lam

Olateju

Imes

Malcolm

Shapiro

Senior

. Validation of the BETA-2 score: an improved tool to estimate beta cell function after clinical islet transplantation using a single fasting blood sample. Am J Transplant. 2016;16(9):2704–13.

24.

Markmann

Deng

Desai

Huang

Velidedeoglu

Frank

Liu

Brayman

Lian

Wolf

Bell

, et al. The use of non-heart-beating donors for isolated pancreatic islet transplantation. Transplantation. 2003;75(9):1423–29.

25.

Andres

Kin

O’Gorman

Livingstone

Bigam

Kneteman

Senior

Shapiro

. Clinical islet isolation and transplantation outcomes with deceased cardiac death donors are similar to neurological determination of death donors. Transpl Int. 2016;29(1):34–40.

26.

Clayton

Swift

Turner

James

RFL

Bell

PRF

. Non-heart-beating organ donors: a potential source of islets for transplantation? Transplantation. 2000;69(10):2094–98.

27.

Zhao

Muiesan

Amiel

Srinivasan

Asare-Anane

Fairbanks

Persaud

Jones

Ashraf

Littlejohn

, et al. Human islets derived from donors after cardiac death are fully biofunctional. Am J Transplant. 2007;7(10):2318–25.

28.

Doppenberg

Nijhoff

Engelse

de Koning

EJP

. Clinical use of donation after circulatory death pancreas for islet transplantation. Am J Transplant. 2021;21:3077–87.

29.

De Paep

Jacobs-Tulleneers-Thevissen

. Warm ischemia time influences human islet cell isolation yield when assessed as beta cell number but not as islet equivalent number. Am J Transplant. 2021;21:3814–15.

30.

Ysebaert

Van Beeumen

De Greef

Squifflet

Detry

De Roover

Delbouille

Van Donink

Roeyen

Chapelle

Bosmans

, et al. Organ procurement after euthanasia: Belgian experience. Transplant Proc. 2009;41(2): 585–86.

31.

Gilbo

Jochmans

Sainz

Pirenne

. Reducing non-anastomotic biliary strictures in donation after circulatory death liver transplantation: cold ischemia time matters! Ann Surg. 2017;266(6):e118–19.

32.

O’Neill

Roebuck

Khoo

Wigmore

Harrison

. A meta-analysis and meta-regression of outcomes including biliary complications in donation after cardiac death liver transplantation. Transpl Int. 2014;27(11):1159–74.

33.

Van Raemdonck

Neyrinck

Dupont

Coosemans

Decaluwé

De Leyn

Nafteux

Verleden

. Transplantation of lungs recovered from donors after euthanasia. J Heart Lung Transplant. 2011;30(4):S16.

34.

Healey

Cypel

Pyle

Mills

Heffren

Katz

Smith

Teranishi

Lavery

Beitel

MacLean

, et al. Lung donation after medical assistance in dying at home. Am J Transplant. 2021;21(1):415–18.

35.

Ceulemans

Vanluyten

Monbaliu

Schotsmans

Fieuws

Vandervelde

De Leyn

Decaluwé

Van Veer

Depypere

Van Slambrouck

, et al. Lung transplant outcome following donation after euthanasia. J Heart Lung Transplant. 2022;S1053-2498(22):01393–6.

36.

O’Gorman

Kin

Murdoch

Richer

McGhee-Wilson

Ryan

Shapiro

JAM

Lakey

JRT

. The standardization of pancreatic donors for islet isolations. Transplantation. 2005;80(6):801–806.

37.

Wang

Kin

O’Gorman

Shapiro

AMJ

Naziruddin

Takita

Levy

Posselt

Szot

Savari

Barbaro

, et al. A multicenter study: North American Islet Donor Score in donor pancreas selection for human islet isolation for transplantation. Cell Transplant. 2016;25(8):1515–23.

38.

Luis

Bilbao

Omori

Rawson

McFadden

Juan

Nair

Mullen

El-Shahawy

Dafoe

, et al. Sodium levels of human pancreatic donors are a critical factor for determination of islet efficacy and survival. Am J Physiol Endocrinol Metab. 2015;308(5):E362–69.

39.

Nano

Clissi

Melzi

Calori

Maffi

Antonioli

Marzorati

Aldrighetti

Freschi

Grochowiecki

Socci

, et al. Islet isolation for allotransplantation: variables associated with successful islet yield and graft function. Diabetologia. 2005;48(5):906–12.

40.

Goto

Eich

Felldin

Foss

Källen

Salmela

Tibell

Tufveson

Fujimori

Engkvist

Korsgren

. Refinement of the automated method for human islet isolation and presentation of a closed system for in vitro islet culture. Transplantation. 2004;78(9):1367–75.

41.

Thienpont

Verhofstadt

Van Loon

Distelmans

Audenaert

De Deyn

. Euthanasia requests, procedures and outcomes for 100 Belgian patients suffering from psychiatric disorders: a retrospective, descriptive study. BMJ Open. 2015;5(7):e007454.

42.

Balamurugan

Naziruddin

Lockridge

Tiwari

Loganathan

Takita

Matsumoto

Papas

Trieger

Rainis

Kin

, et al. Islet product characteristics and factors related to successful human islet transplantation from the collaborative islet transplant registry (CITR) 1999-2010. Am J Transplant. 2014;14(11):2595–606.

43.

Hering

Clarke

Bridges

Eggerman

Alejandro

Bellin

Chaloner

Czarniecki

Goldstein

Hunsicker

Kaufman

, et al. Phase 3 trial of transplantation of human islets in type 1 diabetes complicated by severe hypoglycemia. Diabetes Care. 2016;39(7):1230–40.

44.

Wassmer

Perrier

Combescure

Pernin

Parnaud

Cottet-Dumoulin

Brioudes

Bellofatto

Lebreton

Berishvili

Lablanche

, et al. Impact of ischemia time on islet isolation success and posttransplantation outcomes: a retrospective study of 452 pancreas isolations. Am J Transplant. 2021;21(4):1493–502.

45.

Jacobs-Tulleneers-Thevissen

Bartholomeus

Suenens

Vermeulen

Ling

Hellemans

In’t Veld

Pipeleers-Marichal

Pipeleers

. Human islet cell implants in a nude rat model of diabetes survive better in omentum than in liver with a positive influence of beta cell number and purity. Diabetologia. 2010;53(8):1690–99.

46.

Jacobs-Tulleneers-Thevissen

Chintinne

Ling

Gillard

Schoonjans

Delvaux

Strand

Gorus

Keymeulen

Pipeleers

. Sustained function of alginate-encapsulated human islet cell implants in the peritoneal cavity of mice leading to a pilot study in a type 1 diabetic patient. Diabetologia. 2013;56(7):1605–14.

Utility of Islet Cell Preparations From Donor Pancreases After Euthanasia

Abstract

Keywords

Introduction

Materials and Methods

Euthanasia and Organ Donation

Organ Procurement

Study Design and Cohort Selection

Islet Cell Isolation, Purification, Quality Control, and Culture

Clinical Transplantation and Assessment

Statistical Analysis

Results

Donor and Procurement Characteristics of DCD V

In Vitro Outcome of Islet Cell Isolation From DCD V

Functional Capacity of DCD V Islet Cell Preparation

Discussion

Footnotes

Acknowledgements

Ethical Approval

Statement of Human and Animal Rights

Statement of Informed Consent

Data Availability Statement

Declaration of Conflicting Interests

Funding

ORCID iD

References