Multiorgan Donors Research Articles

Osteopontin Is Elevated in AAA and May Drive Disease Progression

Previous in vitro and animal studies have suggested the importance of osteopontin (OPN), an inflammatory extracellular matrix (ECM) protein, in the formation and growth of abdominal aortic aneurysms (AAAs). The relationship between OPN and the inflammatory cell phenotypes present in the human AAA condition was investigated. Serum OPN concentrations were measured in risk-factor matched non-AAA and AAA patients by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry was used to determine the source of OPN secretion using tissue collected from multiorgan donors and AAA patients undergoing open repair. Vascular smooth muscle cells (VSMCs) were exposed to inflammatory mediators, and the OPN response was evaluated by quantitative RT-PCR and ELISA. The inflammatory nature of OPN and the aortic wall was then determined using a T regulatory cells type 1 (TR1) suppressor cell induction assay. CD45RO– cells were induced to the TR1 suppressor cell phenotype through the action of high-molecular-weight hyaluronan. Inhibition of induction by various compounds was used as a surrogate for inflammatory activity and confirmed by interleukin (IL)-10 ELISA. OPN was elevated in both the plasma (2.08 vs 1.19 ng/mL; P < .05) and the aortic wall homogenate (154.0 vs 38.3 ng/mL; P < .01) of AAAs compared to controls. On immunohistochemistry, OPN localized to the tunica media and VSMCs of the diseased aorta but was not visible in healthy aorta from multiorgan donors. Cigarette smoke extract was the most potent stimulator of OPN secretion in VSMCs and increased supernatant concentration by 2.5-fold and messenger RNA concentration by fourfold over control (P < .05). OPN additionally demonstrated an ability to inhibit the induction of IL-10-secreting TR1 lymphocytes, a depleted population in the AAA patient, from naïve precursors by half (P < .01; Fig). OPN is elevated in the plasma and aortic wall of patients with AAA and is secreted by the VSMCs of the tunica media. It is responsible for inhibition of TR1 cells, decreased IL-10 expression, and an overall proinflammatory environment leading to vessel breakdown and diameter growth. Therefore, inhibition of OPN activity could represent a potential first-in-man pharmaceutical treatment for AAA.

A retrospective database analysis to evaluate the potential of exvivo lung perfusion to recruit declined lung donors.

Ex vivo lung perfusion (EVLP) is currently used for both standard and extended-criteria donor (ECD) lungs. To enlarge the donor pool, we might have to extend the threshold for ECD donation. The purpose of this study was to estimate how many additional ECD lungs could be recruited by EVLP. We reviewed all multi-organ donors (MODs) from our collaborative donor hospitals (January 2010-June 2015). All unused lung donors were categorized using registered donor data and evaluated by two independent investigators to identify which lungs could be transplanted after EVLP. 584 MODs were registered at our transplant center. 268 (45.9%) were declined as lung donor at the moment of registration, and 316 (54.1%) were considered as a donor for lung transplantation. In the latter, lungs from 220 (37.7%) donors were transplanted and 96 donors (16.4%) were not. We identified 78 of 364 declined donors (21.4%) whose lungs could potentially become transplantable after EVLP. With this retrospective database analysis of unused lung donors, we identified a large potential for EVLP to further increase the donor pool in transplant centers where the majority of donor lungs are already extended.

Co-localization of acinar markers and insulin in pancreatic cells of subjects with type 2 diabetes.

To search for clues suggesting that beta cells may generate by transdifferentiation in humans, we assessed the presence of cells double positive for exocrine (amylase, carboxypeptidase A) and endocrine (insulin) markers in the pancreas of non-diabetic individuals (ND) and patients with type 2 diabetes (T2D). Samples from twelve ND and twelve matched T2D multiorgan donors were studied by electron microscopy, including amylase and insulin immunogold labeling; carboxypeptidase A immunofluorescence light microscopy assessment was also performed. In the pancreas from four T2D donors, cells containing both zymogen-like and insulin-like granules were observed, scattered in the exocrine compartment. Nature of granules was confirmed by immunogold labeling for amylase and insulin. Double positive cells ranged from 0.82 to 1.74 per mm2, corresponding to 0.26±0.045% of the counted exocrine cells. Intriguingly, cells of the innate immune systems (mast cells and/or macrophages) were adjacent to 33.3±13.6% of these hybrid cells. No cells showing co-localization of amylase and insulin were found in ND samples by electron microscopy. Similarly, cells containing both carboxypeptidase A and insulin were more frequently observed in the diabetic pancreata. These results demonstrate more abundant presence of cells containing both acinar markers and insulin in the pancreas of T2D subjects, which suggests possible conversion from one cellular type to the other and specific association with the diseased condition.

Influence of specific thoracic donor therapy on kidney donation and long-term kidney graft survival.

To analyze the impact of a specific thoracic donor-treatment protocol (including restrictive fluid balance) on kidney donation and on kidney graft survival. A cohort study. Lung Donors and kidney recipients from 2003 to 2008 were the pre-protocol cohort, and those from 2009 to 2013 were the protocol cohort. The main outcome variables were graft survival and rate of kidney donation. Kidney donation rates were similar in both periods (86.2 vs. 86.2 %; p > 0.05). Both donors and kidney recipients were older and with more comorbidities in the protocol group and this is the reason there were more cases of delayed graft function (differences not statistically significant) and with higher sequential creatinine levels of kidney recipients during the protocol period. However, graft survival was similar in both groups. The probability of graft survival 5 years after transplantation was 0.75 (95 % confidence interval 0.65-0.85) in the pre-protocol cohort and 0.81 (0.70-0.92) in the protocol cohort. Specific treatment for multi-organ donors including restrictive fluid balance does not affect kidney donation or kidney graft loss, and has no impact on long-term viability. Hemodynamics must be closely monitored by medical personnel with specific training.

Emotional management and quality of life in mother living versus multi-organ donor renal transplant recipients

The aim of this study was to evaluate psychological differences and quality of life between kidney recipients from living (mother) and multi-organ donor. Overall, 40 patients who had undergone both living (mother) and multi-organ kidney transplantation 3-6 months before were asked to complete four self-report instruments: Toronto Alexithymia Scale, Short Form Health Survey, Regulatory Emotional Self-efficacy, and Attachment Style Questionnaire. A greater difficulty in emotional, social, and mental health functioning was found in recipients receiving kidney from mother living donor. Moreover, in these patients, higher levels of avoidant attachment dimensions were associated with a worse quality of life.

36th International Symposium on Intensive Care and Emergency Medicine : Brussels, Belgium. 15-18 March 2016.

36th International Symposium on Intensive Care and Emergency Medicine : Brussels, Belgium. 15-18 March 2016.

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

Tissue Characterization after a New Disaggregation Method for Skin Micro-Grafts Generation.

Several new methods have been developed in the field of biotechnology to obtain autologous cellular suspensions during surgery, in order to provide one step treatments for acute and chronic skin lesions. Moreover, the management of chronic but also acute wounds resulting from trauma, diabetes, infections and other causes, remains challenging. In this study we describe a new method to create autologous micro-grafts from cutaneous tissue of a single patient and their clinical application. Moreover, in vitro biological characterization of cutaneous tissue derived from skin, de-epidermized dermis (Ded) and dermis of multi-organ and/or multi-tissue donors was also performed. All tissues were disaggregated by this new protocol, allowing us to obtain viable micro-grafts. In particular, we reported that this innovative protocol is able to create bio-complexes composed by autologous micro-grafts and collagen sponges ready to be applied on skin lesions. The clinical application of autologous bio-complexes on a leg lesion was also reported, showing an improvement of both re-epitalization process and softness of the lesion. Additionally, our in vitro model showed that cell viability after mechanical disaggregation with this system is maintained over time for up to seven (7) days of culture. We also observed, by flow cytometry analysis, that the pool of cells obtained from disaggregation is composed of several cell types, including mesenchymal stem cells, that exert a key role in the processes of tissue regeneration and repair, for their high regenerative potential. Finally, we demonstrated in vitro that this procedure maintains the sterility of micro-grafts when cultured in Agar dishes. In summary, we conclude that this new regenerative approach can be a promising tool for clinicians to obtain in one step viable, sterile and ready to use micro-grafts that can be applied alone or in combination with most common biological scaffolds.

Assessment of cryopreserved donor skin viability: the experience of the regional tissue bank of Siena.

Skin allografts from cadaver donors are an important resource for treating extensive burns, slow-healing wounds and chronic ulcers. A high level of cell viability of cryopreserved allografts is often required, especially in burn surgery, in Italy. Thus, we aimed to determine which conditions enable procurement of highly viable skin in our Regional Skin Bank of Siena. For this purpose, we assessed cell viability of cryopreserved skin allografts procured between 2011 and 2013 from 127 consecutive skin donors, before and after freezing (at day 15, 180, and 365). For each skin donor, we collected data concerning clinical history (age, sex, smoking, phototype, dyslipidemia, diabetes, cause of death), donation process (multi-tissue or multi-organ) and timing of skin procurement (assessment of intervals such as death-harvesting, harvesting-banking, death-banking). All these variables were analysed in the whole case study (127 donors) and in different groups (e.g. multi-organ donors, non refrigerated multi-tissue donors, refrigerated multi-tissue donors) for correlations with cell viability. Our results indicated that cryopreserved skin allografts with higher cell viability were obtained from female, non smoker, heartbeating donors died of cerebral haemorrhage, and were harvested within 2h of aortic clamping and banked within 12 h of harvesting (13-14h from clamping). Age, cause of death and dyslipidaemia or diabetes did not appear to influence cell viability. To maintain acceptable cell viability, our skin bank needs to reduce the time interval between harvesting and banking, especially for refrigerated donors.

The incidence and morphology of Monckeberg's medial calcification in banked vascular segments from a monocentric donor population.

Little is known about the real incidence and the clinical relevance of the enigmatic Monckeberg's medial calcification in the patency of the femoral artery allograft. Here we present a retrospective study on 143 multiorgan donors (mean age 38years, range 14-59years), to describe the incidence and the morphological features of vascular calcifications in banked femoral arteries suitable for clinical use. In the present series, focal vascular calcifications were present in 36 (25%) cases, 23 cases localized in the intima, 7 in the media, and 6 were mixed. No correlation was found between the incidence of calcifications and the classical cardiovascular clinical risk factors (n=9); only hypertension correlated with the medial localization, but not with the incidence, of the calcification (P=0.017). While the macroscopic exclusion criteria of vascular grafts include atheromatous and not-atheromatous lesions, we ignore the actual impact of Monckeberg's medial calcification on vessel transplantation and allograft life. In our opinion this is a very important topic, since when the histological criteria for Monckeberg's calcification diagnosis are used, 25% of our young donors population was affected. Whether Monckeberg's medial calcification is a stable arterial condition, apparently underestimated in the general population, or a dynamic process evolving with age and atherosclerosis, or a banking-related vascular alteration, still remain an open issue deserving further studies with subjects of different ages.

Characterization of Islets from Chronic Calcific Pancreatitis Patients of Tropical Region with Distinct Phenotype

Background and Objective: Islet autotransplantation is performed to preserve endocrine function in patients undergoing pancreatic resections for painful chronic pancreatitis. We characterized islets isolated from chronic pancreatitis patients (CP) of tropical region. Patients and Methods: Pancreatic tissues were obtained from CP patients with and without diabetes undergoing pancreatic resections (n = 35) and brain-dead multi organ donors (n = 6; considered as controls). Islets isolated were assessed for yield, purity, viability and in vitro islet function (Glucose stimulated insulin release, GSIR) as per standard protocols. Results: Islets from CP patients without diabetes were similar to controls in yield (control 4120 - 6100 IE/g, CP 3550 - 5660 IE/g), purity (control 78% ± 12%, CP 70% ± 8.2%) and viability (control 85% ± 8%, CP 81% ± 10%) and islets from CP patients with diabetes showed decreases in yield (3002 - 2300 IE/g), purity (61% ± 16%) and viability (62% ± 21%). Islets measuring 50 - 200 μ were similar in abundance in controls (94.74% ± 3.2%) and CP patients with and without diabetes, 86.31% ± 4.9%, 91.03% ± 3.8%. GSIR of islets from CP patients and controls were similar at 5.5 mM glucose (2.8 - 3.1 μU/ml). However, GSIR at 16.5 mM glucose was decreased in CP patients (control 18.5 ± 0.6, CP without diabetes 11.8 ± 0.3, CP with diabetes 4.3 ± 0.3 μU/ml). Conclusion: Our results demonstrate suitability of islets isolated from CP patients of tropical region for autotransplantation.

Comparative characteristics cultural properties of adherent cells derived from bone marrow of multiorgan donors and tissues donors

Purpose of work - compare cultural properties of adherent cells derived from bone marrow of multiorgan donors and tissues donors, to assess the possibility of using these cells in clinical practice. Materials and methods. Adherent culture cells from cadavers bone marrow were obtained using standart methods of isolation, scaling, and passaging cells. In culture, counted the number of CFU-F, proliferating activity of cell , their complies with the minimal criteria for defining multipotent mesenchymal stromal cells (hMSC) and compatibility with allogeneic tissue grafts. The Results. In cultures of cells derived from multiorgan donors and tissues donors the number of CFU-F was 0,0004% and 0,00024%, while the duration of the phase "rest proliferative" was 3-4 and 10-12 days respectively. All examined cell culture after the second passage have gained a high degree of homogeneity, and the cells were complies the minimum criteria hMSC. Conclusion: bone marrow of multiorgan donors and tissues donors can be used for obtain hMSC, suitable for creating a combined cellular-tissue grafts for clinical use.

Building a Lung Transplant Program

Building a Lung Transplant Program

The clinical use of cryopreserved human skin allografts for transplantation

Abstract Background The biological recovery of human skin allografts is the gold standard for preservation in Skin Banks. However, there is no worldwide consensus about specific allocation criteria for preserved human skin allografts with living cells. A report is presented on the results of 5 years of experience of using human skin allografts in burned patient in the Skin and Tissue Bank at Instituto Nacional de Rehabilitacion . Material and methods The human skin allografts were obtained from multi-organ donors, processed and preserved at −80 °C for 12 months. Allocation criteria were performed according to blood type match, clinical history, and burned body surface. Results Up to now, the Skin and Tissue Bank at Instituto Nacional de Rehabilitacion has processed and recovered 125,000 cm 2 of human skin allografts. It has performed 34 surgical implants on 21 burned patients. The average of burn body surface was 59.2%. More than two-thirds (67.7%) of recipients of skin allografts were matched of the same to type blood of the donor, and 66.6% survived after 126 days hospital stay. Conclusion It is proposed to consider recipient's blood group as allocation criteria to assign tissue; and use human skin allografts on patiens affected with burns over 30% of body surface (according the “rule of the 9”).

Protective ventilation and negative fluid balance strategy in potential multi-organ donor patients

Protective ventilation and negative fluid balance strategy in potential multi-organ donor patients

Expression and Cellular Localization of 15-Hydroxy-Prostaglandin-Dehydrogenase in Abdominal Aortic Aneurysm.

PGE2 has been implicated in abdominal aortic aneurysm (AAA) associated hypervascularization. PGE2-metabolism involves 15-hydroxyprostaglandin-dehydrogenase (15-PGDH) the expression of which in AAA is unknown. The aim of this study was to examine the expression and cell distribution of 15-PGDH in AAA. Here, we show that 15-PGDH mRNA levels were significantly higher in aorta samples from patients undergoing AAA repair than in those from healthy multiorgan donors. Consequently, the ratio of metabolized PGE2 secreted by aortic samples was significantly higher in AAA. AAA production of total PGE2 and PGE2 metabolites correlated positively with PGI2 production, while the percentage of metabolized PGE2 correlated negatively with the total amount of PGE2 and with PGI2. Transcript levels of 15-PGDH were statistically associated with leukocyte markers but did not correlate with microvascular endothelial cell markers. Immunohistochemistry revealed 15-PGDH in the areas of leukocyte infiltration in AAA samples, mainly associated with CD45-positive cells, but not in normal aorta samples. We provide new data concerning 15-PGDH expression in human AAA, showing that 15-PGDH is upregulated in AAA and mainly expressed in infiltrating leukocytes. Our data suggest that microvasculature was not involved in PGE2 catabolism, reinforcing the potential role of microvasculature derived PGE2 in AAA-associated hypervascularization.

El uso clínico de aloinjertos de piel humana criopreservados con fines de trasplante

BackgroundThe biological recovery of human skin allografts is the gold standard for preservation in Skin Banks. However, there is no worldwide consensus about specific allocation criteria for preserved human skin allografts with living cells.A report is presented on the results of 5 years of experience of using human skin allografts in burned patient in the Skin and Tissue Bank at the “Instituto Nacional de Rehabilitacion” Material and methodsThe human skin allografts were obtained from multi-organ donors. processed and preserved at -80°C for 12 months. Allocation criteria were performed according to blood type match, clinical history, and burned body surface. ResultsUp to now, the Skin and Tissue Bank at ‘Instituto Nacional de Rehabilitacion” has processed and recovered 125,000cm2 of human skin allografts. It has performed 34 surgical implants on 21 burned patients. The average of burn body surface was 59.2%. More than two-thirds (67.7%) of recipients of skin allografts were matched of the same to type blood of the donor, and 66.6% survived after 126 days hospital stay. ConclusionIt is proposed to consider recipient's blood group as allocation criteria to assign tissue; and use human skin allografts on patiens affected with burns over 30% of body surface (according the “rule of the 9”).

INFLUENCE OF VARIOUS FACTORS UPON DECISION ON PANCREAS ALLOGRAFT SUITABILITY

AAim. To identify factors influencing upon decision on pancreas allograft suitability for transplant and their clinical significance. Materials and methods. We reviewed 95 multiorgan donors under the age of 45, who were considered as pancreas donor between January 2010 and December 2013. 28 pancreata were procured (Group I), 67 pancreata were refused (Group II). Demographic, clinical and laboratory data, anatomical hepatopancreatoduodenal varieties were taken into account. Results. We found that only three factors have an effect on pancreas allograft refusal probability. According to our data, non-transplantation of the liver from the same donor increases the pancreas graft refusal in 4 times. Elevated donor’s sodium and urea blood levels also increase the probability of donor pancreas denial for transplantation. For example, the probability of pancreas graft refusal from the donor with sodium level 145 mmol/l and urea level 6.0 mol/l is only 32%. As compared to the donor with sodium level 160 mmol/l and urea 12.0 mol/l where probability reaches 85%. Other factors: demographic, laboratory, clinical indicators, gepatopancreatoduodenal blood supply variations were not predictive for the procurement decision. Conclusion. Main predictors of pancreas allograft refusal to be taken into account, appropriate correction of donor metabolic disturbances and sufficient experience of the surgeon performing the procurement can increase the availability of pancreas transplantation.

Effect of Recipient Sensitization (Peak Panel Reactive Antibodies) on 15-Year Survival After Kidney Transplantation

BackgroundDespite dynamic development within the field of transplantology, the immunization of a potential organ recipient remains an important issue for transplant teams. Panel reactive antibodies (PRA) identification in the serum of the recipient remains routine practice in the majority of transplantation centers. The influence of peak PRA levels on graft function is a well known fact. The aim of this study was to determine the effect of peak PRA on long-term survival after renal transplantation. MethodsThe study was conducted on a group of 232 kidney recipients from multiorgan donors, transplanted in 6 transplant centers in Poland from 1995 to 1997. Data analyzed in this study included recipients' age, sex, PRA, HLA, number and time of hemodialyses after the transplantation, cold ischemia time, and etiology of end-stage renal disease. The effect of data examined in this study on mortality was evaluated at set time points at 5, 10, and 15 years after transplantation. The statistical methods included monofactorial and multifactorial Kaplan-Meier survival analysis and Cox proportional hazards model for mortality prediction. A P value of <.05 was considered to be statistically significant. ResultsAmong all of the analyzed factors, only peak PRA concentrations significantly correlated with increased mortality among renal transplant recipients. The results were analyzed in all of the set time points: P = .007 at 5 years, P = .014 at 10 years, and P = .05 at 15 years after transplantation. ConclusionsThe increased level of PRA in kidney recipients is a risk factor increasing mortality after the transplantation.

Influence of the Hemodynamic Status of Multiorgan Donors on Long-Term Kidney Graft Survival—A Multivariable Analysis

BackgroundThe quality of transplanted organ and timing of the initiation of its effective function depends on many factors potentially causing graft dysfunction. The aim of this study was to evaluate the influence of the cardiovascular status of multiorgan donors on the long-term kidney graft survival over a 15-year observation period. MethodsIn 2007, the authors of this study published a multicenter prospective study evaluating the influence of the hemodynamic status of multiorgan donors on the early function of transplanted kidney. The results of that study showed that mean arterial pressure, central venous pressure, and pulmonary capillary wedge pressure values of the donor importantly influence the frequency of delayed graft function after renal transplantation. The present analysis covers the effect of the donor's hemodynamic status parameters on graft function time within the 15-year observation period. Univariate and multivariate analyses using the Cox regression proportional hazard model were performed to evaluate the prognostic parameters for overall survival and renal graft survival time. P < .05 was considered to be significant. ResultsThe univariate analysis showed a significantly shorter time of graft survival in the group of recipients who had a kidney retrieved from donors with lower pulmonary capillary wedge pressure values (P = .038) and lower cardiac index values (P = .039). The same results were obtained for the multifactorial Cox logistic regression analysis. ConclusionsThe filling of the intravascular bed of the donor as determined by pulmonary capillary wedge pressure and maintained donor tissue perfusion as determined by cardiac index, impose important factors influencing long-term kidney graft survival.