An Assessment Rubric for a Resident Training Program in Surgery: A Single-Institution Experience.

Using a Collaborative Action Research model, our research team established a one-month clinical resident training program for first- and second-year clinical residents. We created and implemented an assessment rubric to assess the residents' progress toward independent practice in surgery, and thereby, to evaluate the program itself. The program included training in three areas: basic techniques and procedures in the operating room, surgical ward management, and academic activities. The rubric measured the residents' performance according to three achievement levels: Level 1 (demonstration), Level 2 (active help) and Level 3 (passive help). The program and rubric implementation began in June 2019 and continued until March 2020, when the program outcomes and shortcomings were analyzed. Among nineteen clinical residents, a total of nine clinical residents participated in the study. Most participants reached achievement Level 3 for their performance of basic techniques in the operating room. Finally, we discussed ideas for improvement and drafted plans for an improved rubric to complete the action research cycle. Our research team found the rubric to be a useful tool in evaluating the status of the new clinical resident training program.

Totally laparoscopic colectomy with intracorporeal anastomosis for colonic liposarcoma: A case report.

Liposarcoma is a type of soft tissue sarcoma. Primary colonic liposarcomas are extremely rare. An 86-year-old man with diarrhea and anorexia visited our outpatient clinic at Okayama City Hospital. Colonoscopy and computed tomography imaging revealed a large submucosal tumor in the descending colon with a maximum diameter of approximately 10 cm. He underwent totally laparoscopic left hemicolectomy with intracorporeal anastomosis. The histopathological diagnosis was a well-differentiated liposarcoma without lymph node metastases. The patient's postoperative course was uneventful. We herein report a rare case of totally laparoscopic colectomy with intracorporeal anastomosis in an elderly patient with colonic liposarcoma.

An experience of total laparoscopic partial colectomy with intracorporeal triangulating anastomosis in an obese patient with descending colon cancer.

A 68-year-old woman was transferred to the emergency room of Okayama City Hospital because of worsening epigastric pain. After the examination, she was diagnosed with descending colon cancer, and laparoscopic colectomy was planned. However, exteriorization of the bowels to produce anastomosis was difficult because the rich adipose tissue of the mesocolon hardly stretched, and the abdominal wall was thick as the patient was obese. Therefore, an intracorporeal triangulating end-to-end anastomosis was performed. The colon was divided at 10 cm either side from the tumor using an endoscopic linear stapler. After the resection of the stumps with staples, the posterior walls were tied with stay sutures and then stapled with an endoscopic linear stapler. The anterior wall was stapled twice in the same manner, and the intracorporeal anastomosis was completed. The patient's postoperative course was uneventful. This technique provided an alternative intracorporeal anastomotic technique as troubleshooting in laparoscopic colectomy.

Prolonged warm ischemia exacerbated acute rejection after lung transplantation from donation after cardiac death in a mouse.

OBJECTIVE: In lung transplantation (LTx) from donation after cardiac death (DCD), the donor lungs are inevitably exposed to warm ischemic time (WIT) between the cardiac arrest and the initiation of cold preservation. We conducted this study to examine the effect of prolonged WIT on lung allograft rejection in a murine model of LTx from DCD. METHODS: Allogeneic BALB/c → B6 LTx from DCD was performed with a WIT of 15 min (WIT15 group, n = 5) or 60 min (WIT60 group, n = 5). Recipients were immunosuppressed by perioperative costimulatory blockade. The lung allografts were analyzed by histology and flow cytometry on day 7 after the LTx. RESULTS: Histologically, the rejection grade in the WIT60 group was significantly higher than that in the WIT15 group (3.4 ± 0.4 vs. 2.2 ± 0.2, P = 0.0278). Moreover, the intragraft CD8+ to CD4+ T cell ratio in the WIT60 group was significantly higher than that in the WIT15 group (2.3 ± 0.12 vs. 1.2 ± 0.11, P < 0.0001). CONCLUSIONS: Prolonged WIT could exacerbate the severity of lung allograft rejection after LTx from DCD. Minimization of the WIT could improve the outcomes after LTx from DCD.

The efficacy of a trauma call system: challenges in managing severe trauma at a rural emergency center without full-time emergency physicians.

AIMS: There have been some reports about the efficacy of trauma team activation. In November 2015, we implemented a trauma call system, wherein a general surgeon, neurosurgeon, and orthopedic surgeon are called to the emergency department when severe trauma patients are transferred to our emergency department. In this study, we evaluated the efficacy of this trauma call system. METHODS: The purpose of the present study was to evaluate the efficacy of a trauma call system for trauma cases with an Injury Severity Score ≥16. We compared the mortality of trauma cases and the time from arrival to the start of the examination and intervention before and after implementing this trauma call system. RESULTS: There was no significant difference in the mortality rates before and after the implementation of the trauma call system. The median time from arrival to the start of contrast-enhanced computed tomography or transcatheter arterial embolization improved from 54 to 19 min (P = 0.015) and 171 to 84 min (P = 0.030), respectively, after the implementation of the trauma call system. CONCLUSION: Our trauma call system did not significantly improve the mortality of trauma patients with an Injury Severity Score ≥16. However, it was effective for reducing the time from the arrival to the start of contrast-enhanced computed tomography or transcatheter arterial embolization.

SPRED2 deficiency may lead to lung ischemia-reperfusion injury via ERK1/2 signaling pathway activation.

PURPOSE: Inflammatory changes during lung ischemia-reperfusion injury (IRI) are related to the activation of the extracellular signal-regulated kinase (ERK)1/2 signaling pathway. Sprouty-related EVH1 (enabled/vasodilator-stimulated phosphoprotein homology 1)-domain-containing proteins (SPREDs) are known inhibitors of ERK1/2 signaling. The role of SPRED2 in lung IRI was examined in a left hilar clamp mouse model. METHODS: C57BL/6 wild-type (WT) and Spred2-/- mice were used in the left hilar clamp model. Experimental groups underwent 30 min of left hilar clamping followed by 1 h of reperfusion. U0126, an ERK1/2 inhibitor, was administered to Spred2-/- mice with reperfused lungs. RESULTS: The partial pressures of oxygen of the Spred2-/- mice after reperfusion were significantly worse than those of WT mice (p < 0.01). Spred2-/- mice displayed more severe injuries than WT mice with increased neutrophil infiltration observed by a histological evaluation and flow cytometry (p < 0.001). This severe inflammation was inhibited by U0126. In addition, the rate of ERK1 activation was significantly higher in the lungs of Spred2-/- mice after reperfusion than in WT mice according to a Western blot analysis (p < 0.05). CONCLUSION: The activation of the ERK1/2 signaling pathway influences the severity of lung IRI, causing inflammation with neutrophil infiltration. SPRED2 may be a promising target for the suppression of lung IRI.

[Clinicopathological Study of Small Intestinal Carcinoma].

We analyzed the clinicopathological characteristics, preoperative diagnosis, surgical operations, chemotherapy regimens, and prognoses of 6 patients with primary small intestinal carcinomas that were resected at our hospital between January 2004 and December 2014. The patients(3 men and 3 women)were 65 to 77 years old(mean: 70 years old). We were able to diagnose 3 patients pathologically before surgery via double balloon endoscopy and endoscopy of the large intestine. We performed partial resection of the jejunum in 3 patients, partial resection of the ileum in 1 patient, laparoscopic ileocecal resection in 1 patient, and right hemicolectomy in 1 patient. The histological type was well-differentiated adenocarcinoma in 2 patients, moderately differentiated adenocarcinoma in 2 patients, papillary adenocarcinoma in 1 patient, and poorly differentiated adenocarcinoma in 1 patient. The tumor depth was T2 in 1 patient, T3 in 2 patients, and T4 in 3 patients. The pathological stage was I in 1 patient, II A in 1 patients, II B in 2 patient, III A in 1 patient, and III B in 1 patient. The postoperative median duration of follow-up was 44 months(range: 10-127). Regarding prognosis, 5 patients are alive without recurrence, and 1 patient died of peritoneal dissemination. The overall 5-year survival rate was 75%. We suggest that it is very important to perform radical resection with lymph node dissection for patients without distant metastases.

Perioperative supplementation with bifidobacteria improves postoperative nutritional recovery, inflammatory response, and fecal microbiota in patients undergoing colorectal surgery: a prospective, randomized clinical trial.

The use of probiotics has been widely documented to benefit human health, but their clinical value in surgical patients remains unclear. The present study investigated the effect of perioperative oral administration of probiotic bifidobacteria to patients undergoing colorectal surgery. Sixty patients undergoing colorectal resection were randomized to two groups prior to resection. One group (n=31) received a probiotic supplement, Bifidobacterium longum BB536, preoperatively for 7-14 days and postoperatively for 14 days, while the other group (n=29) received no intervention as a control. The occurrences of postoperative infectious complications were recorded. Blood and fecal samples were collected before and after surgery. No significant difference was found in the incidence of postoperative infectious complications and duration of hospital stay between the two groups. In comparison to the control group, the probiotic group tended to have higher postoperative levels of erythrocytes, hemoglobin, lymphocytes, total protein, and albumin and lower levels of high sensitive C-reactive proteins. Postoperatively, the proportions of fecal bacteria changed significantly; Actinobacteria increased in the probiotic group, Bacteroidetes and Proteobacteria increased in the control group, and Firmicutes decreased in both groups. Significant correlations were found between the proportions of fecal bacteria and blood parameters; Actinobacteria correlated negatively with blood inflammatory parameters, while Bacteroidetes and Proteobacteria correlated positively with blood inflammatory parameters. In the subgroup of patients who received preoperative chemoradiotherapy treatment, the duration of hospital stay was significantly shortened upon probiotic intervention. These results suggest that perioperative oral administration of bifidobacteria may contribute to a balanced intestinal microbiota and attenuated postoperative inflammatory responses, which may subsequently promote a healthy recovery after colorectal resection.

[Clinicopathological Outcome of Perforated Colorectal Cancer].

We investigated the clinicopathological findings of 90 patients with colorectal perforation who underwent emergency surgery between January 2008 and July 2015.T he patients were divided into 2 groups, namely those with perforation due to colorectal cancer(colorectal cancer group, n=20)and those with perforation due to benign colorectal disease(non-colorectal cancer group, n=70).We investigated the clinicopathological findings of the 2 groups.The SOFA score was significantly lower in the colorectal cancer group than in the non-colorectal cancer group.Of the 20 cases of primary cancer, 11 were located in the sigmoid colon; 5, in the rectum; 2, in the transverse colon; 1, in the ascending colon; and 1, in the cecum.The perforation occurred at the tumor site in 8 patients and at the oral site of cancer in 12.Eleven patients had stage II cancer, 1 had stage IIIa, and 8 had stage IV.Ten patients underwent curability A resection; 1, curability B resection; and 8, curability C resection.Recurrence was observed in 6 of the 10 patients who had undergone curability A resection and in 1 patient who had undergone curability B resection.The initial recurrence site was the liver in 3 cases, the peritoneum in 2 cases, and a local site in 2 cases.Even if the patents underwent curative operation, the recurrence rate was high.Therefore, we conclude that adjuvant chemotherapy is required along with careful follow-up.

Early Growth Response-1 Plays an Important Role in Ischemia-Reperfusion Injury in Lung Transplants by Regulating Polymorphonuclear Neutrophil Infiltration.

BACKGROUND: Early growth response-1 (Egr-1) has been shown to be a trigger-switch transcription factor that is involved in lung ischemia-reperfusion injury (IRI). METHODS: Mouse lung transplants were performed in wild-type (WT) C57BL/6 and Egr1-knockout (KO) mice in the following donor → recipient combinations: WT → WT, KO → WT, WT → KO, and KO → KO to determine whether the presence of Egr-1 in the donor or recipient is the most critical factor for IRI. Pulmonary grafts were retrieved after 18 hours of ischemia after 4 hours of reperfusion. We analyzed graft function by analyzing arterial blood gas and histology in each combination and assessed the effects of Egr1 depletion on inflammatory cytokines that are regulated by Egr-1 as well on polymorphonuclear neutrophil (PMN) infiltration. RESULTS: Deletion of Egr1 improved pulmonary graft function in the following order of donor → recipient combinations: WT → WT < WT → KO < KO → WT < KO → KO. Polymerase chain reaction assays for Il1B, Il6, Mcp1, Mip2, Icam1, and Cox2 showed significantly lower expression levels in the KO → KO group than in the other groups. Immunohistochemistry demonstrated clear Egr-1 expression in the nuclei of pulmonary artery endothelial cells and PMN cytoplasm in the WT grafts. Flow cytometry analysis showed that Egr1 deletion reduced PMN infiltration and that the extent of reduction correlated with graft function. CONCLUSIONS: Both graft and recipient Egr-1 played a role in lung IRI, but the graft side contributed more to this phenomenon through regulation of PMN infiltration. Donor Egr-1 expression in pulmonary artery endothelial cells may play an important role in PMN infiltration, which results in IRI after lung transplantation.

Impact of prolonged cold preservation on the graft function and gene expression levels in an experimental lung transplantation model.

PURPOSE: Ischemia reperfusion injury (IRI) remains a significant cause of morbidity and mortality after lung transplantation. Early growth response-1 (EGR1) drives the expression of inflammatory mediators and has an important role in IRI. We hypothesized that the severe IRI caused by a long preservation induces a specific expression pattern of EGR1 and its target genes which would correlate with the lung graft function. METHODS: SD rat lungs were preserved at 4 °C for 3 or 18 h, then transplanted and reperfused. Pulmonary grafts were evaluated for the blood gas oxygenation and pathological findings. The intra-graft mRNA levels of EGR1 and its downstream target genes were measured by real-time PCR. A Western blotting analysis of the EGR1 expression was used to validate the changes in the protein level. RESULTS: There was upregulation of EGR1, MIP-2 and PAI-1 when there was prolonged hypothermic preservation. The expression levels of MIP-2 and PAI-1 were observed to increase for up to 4 h in the 18 h preserved lungs. There were no differences in the expression levels of IL-1ß and ICAM-1 between the lungs subjected to short and long periods of ischemia. CONCLUSIONS: Our data showed that prolonged hypothermic graft preservation deteriorates the pulmonary graft function, which was associated with the induction of EGR1 and its downstream target genes, which may aggravate IRI following lung transplantation.

Cutting edge: Pseudomonas aeruginosa abolishes established lung transplant tolerance by stimulating B7 expression on neutrophils.

The mechanisms that link bacterial infection to solid organ rejection remain unclear. In this study, we show that following the establishment of lung allograft acceptance in mice, Pseudomonas aeruginosa airway infection induces a G-CSF-dependent neutrophilia that stimulates acute rejection. Graft-infiltrating neutrophils sharply upregulate the B7 molecules CD80 and CD86, but they do not express CD40 or MHC class II in response to P. aeruginosa infection. Neutrophil B7 promotes naive CD4(+) T cell activation and intragraft IL-2(+), IFN-γ(+), and IL-17(+) T lymphocyte accumulation. Intravital two-photon microscopy reveals direct interactions between neutrophils and CD4(+) T cells within pulmonary allografts. Importantly, lung rejection in P. aeruginosa-infected recipients is triggered by CD80/86 on neutrophils and can be prevented by B7 blockade without affecting clearance of this pathogen. These data show that neutrophils enhance T cell activation through B7 trans-costimulation and suggest that inhibiting neutrophil-mediated alloimmunity can be accomplished without compromising bacterial immune surveillance.

Five-year update on the mouse model of orthotopic lung transplantation: Scientific uses, tricks of the trade, and tips for success.

It has been 5 years since our team reported the first successful model of orthotopic single lung transplantation in the mouse. There has been great demand for this technique due to the obvious experimental advantages the mouse offers over other large and small animal models of lung transplantation. These include the availability of mouse-specific reagents as well as knockout and transgenic technology. Our laboratory has utilized this mouse model to study both immunological and non-immunological mechanisms of lung transplant physiology while others have focused on models of chronic rejection. It is surprising that despite our initial publication in 2007 only few other laboratories have published data using this model. This is likely due to the technical complexity of the surgical technique and perioperative complications, which can limit recipient survival. As two of the authors (XL and WL) have a combined experience of over 2500 left and right single lung transplants, this review will summarize their experience and delineate tips and tricks necessary for successful transplantation. We will also describe technical advances made since the original description of the model.

Egr1: a novel target for ameliorating acute allograft rejection in an experimental lung transplant model.

OBJECTIVES: Acute allograft rejection is one of the significant complications occurring in lung transplant recipients. Early growth response-1 (Egr-1), zinc-finger-type transcription factor, is known as a master switch regulator of diverse chemical mediators. We used an orthotopic mouse model of left lung transplant to elucidate the function of Egr-1 in acute pulmonary rejection. METHODS: Left lung grafts retrieved from C57BL/6 wild mice or C57BL/6 Egr-1-null mice were orthotopically transplanted into BALB/c mice; the lungs were harvested at day 1, 3, 5 or 7 after lung transplantation. The grade of acute rejection was histopathologically evaluated. The intragraft gene expression levels of Egr-1 and downstream target mediators were quantitatively measured by real-time polymerase chain reaction. Immunohistochemical analysis was used to determine the location and distribution of the Egr-1 protein in the pulmonary graft. RESULTS: Severe acute rejection was observed in allografts from wild-type mice at 5 days after transplantation. Only minimal rejection was seen in the lung graft from Egr-1-null donor mice at 5 days after transplantation. Strong upregulation of Egr-1 mRNA transcripts was observed at day 1, which then decreased during the next 5 days. The mRNA of Egr-1 target mediators [interleukin-1-beta (IL-1ß), monocyte chemotactic protein-1 (MCP-1) and plasminogen activator inhibitor-1] reached maximal levels at day 5. Egr-1-null allografts exhibited significantly lower expressions of IL-1ß and MCP-1 mRNA (P < 0.05). CONCLUSIONS: Our study showed that deletion of Egr-1 in lung allografts ameliorates severe acute rejection with the reduction of expression levels of chemical mediators, implying a new possible strategy for treating acute pulmonary allograft rejection.

Peculiar mechanisms of graft recovery through anti-inflammatory responses after rat lung transplantation from donation after cardiac death.

BACKGROUND: Although lung transplantation from donation after cardiac death (DCD), especially uncontrolled DCD, is limited by warm ischemic periods, the molecular mechanism of warm ischemia-reperfusion-injury (IRI) has not been well elucidated. The purpose of this study was to clarify the particular longitudinal mechanisms of molecular factors involved in warm IRI. METHODS: Cold ischemic-time (CIT)-group lungs were retrieved and subjected to 3-h of cold preservation, whereas warm ischemic-time (WIT)-group lungs were retrieved after 3-h of warm ischemia. Orthotopic rat lung transplantation was performed and the grafts were reperfused for 1 or 4-h. The graft functions, gene expression, and activation of inflammatory molecules in the grafts were analyzed. Exhaled-carbon-monoxide-concentration (ExCO-C) was measured during reperfusion. RESULTS: Only the WIT-group showed obvious primary graft dysfunction at 1-h reperfusion, but the graft function was recovered during 4-h reperfusion. Most of pro-inflammatory cytokines and stress-induced molecules showed different expression and activation patterns between CIT and WIT groups. In the WIT-group, the expressions of anti-inflammatory molecules, IL-10 and HO-1, were significantly increased at 1-h reperfusion compared to the CIT-group, and these high levels were maintained through 4-h reperfusion. Furthermore, ExCO-C levels in the WIT-group increased immediately after reperfusion compared to the CIT-group. CONCLUSIONS: This study indicates that warm IRI may involve a different mechanism than cold IRI and anti-inflammatory pathways may play important roles in the graft recovery after lung transplantation from uncontrolled DCD.

Emergency granulopoiesis promotes neutrophil-dendritic cell encounters that prevent mouse lung allograft acceptance.

The mechanisms by which innate immune signals regulate alloimmune responses remain poorly understood. In the present study, we show by intravital 2-photon microscopy direct interactions between graft-infiltrating neutrophils and donor CD11c(+) dendritic cells (DCs) within orthotopic lung allografts immediately after reperfusion. Neutrophils isolated from the airways of lung transplantation recipients stimulate donor DCs in a contact-dependent fashion to augment their production of IL-12 and expand alloantigen-specific IFN-γ(+) T cells. DC IL-12 expression is largely regulated by degranulation and induced by TNF-α associated with the neutrophil plasma membrane. Extended cold ischemic graft storage enhances G-CSF-mediated granulopoiesis and neutrophil graft infiltration, resulting in exacerbation of ischemia-reperfusion injury after lung transplantation. Ischemia reperfusion injury prevents immunosuppression-mediated acceptance of mouse lung allografts unless G-CSF-mediated granulopoiesis is inhibited. Our findings identify granulopoiesis-mediated augmentation of alloimmunity as a novel link between innate and adaptive immune responses after organ transplantation.

Maintenance of IKKß activity is necessary to protect lung grafts from acute injury.

BACKGROUND: Signaling pathways that target I-κB kinase ß (IKKß) activation stimulate the expression of nuclear factor (NF)-κB-dependent genes and are thus believed to primarily promote inflammation and injury in solid organ grafts. METHODS: We examined the role of IKKß in a mouse model of lung transplantation-mediated ischemia-reperfusion injury using NF-κB essential modulator (NEMO)-binding domain (NBD) peptide to pharmacologically inhibit IKK activation. As myeloid cells are primarily responsible for the production of acute inflammatory mediators after lung transplantation, we also investigated the effects of myeloid cell-specific IKKß gene deletion on acute lung graft injury by transplanting mutant mice. RESULTS: When NBD was administered at a dose that partially inhibits IKKß activation, we observed attenuated lung graft injury and blunted expression of intragraft proinflammatory mediators. Surprisingly, when the dose of NBD was increased to a level that ablates intragraft IKKß activation, graft inflammation, and injury were significantly worse compared with recipients treated with control peptide. Similar to lung recipients with pharmacologically ablated IKKß activity, donor-recipient transplant combinations with a myeloid cell-specific IKKß gene deletion had marked intragraft inflammation and poor lung function. CONCLUSIONS: Our data show maintenance of IKKß activity is critical for promoting graft homeostasis with important implications for targeting NF-κB-dependent signaling pathways for treating acute lung injury.

Bcl3 prevents acute inflammatory lung injury in mice by restraining emergency granulopoiesis.

Granulocytes are pivotal regulators of tissue injury. However, the transcriptional mechanisms that regulate granulopoiesis under inflammatory conditions are poorly understood. Here we show that the transcriptional coregulator B cell leukemia/lymphoma 3 (Bcl3) limits granulopoiesis under emergency (i.e., inflammatory) conditions, but not homeostatic conditions. Treatment of mouse myeloid progenitors with G-CSF--serum concentrations of which rise under inflammatory conditions--rapidly increased Bcl3 transcript accumulation in a STAT3-dependent manner. Bcl3-deficient myeloid progenitors demonstrated an enhanced capacity to proliferate and differentiate into granulocytes following G-CSF stimulation, whereas the accumulation of Bcl3 protein attenuated granulopoiesis in an NF-κB p50-dependent manner. In a clinically relevant model of transplant-mediated lung ischemia reperfusion injury, expression of Bcl3 in recipients inhibited emergency granulopoiesis and limited acute graft damage. These data demonstrate a critical role for Bcl3 in regulating emergency granulopoiesis and suggest that targeting the differentiation of myeloid progenitors may be a therapeutic strategy for preventing inflammatory lung injury.

Suppression of inflammatory cytokines during ex vivo lung perfusion with an adsorbent membrane.

BACKGROUND: Lung grafts can be perfused ex vivo for 2 hours without edema formation; however, prolonged ex vivo lung perfusion (EVLP) eventually induces lung injury. This study evaluated the change in proinflammatory cytokines of the perfusate during EVLP and investigated the effect of cytokine removal using an adsorbent membrane. METHODS: Porcine heart-lung blocks were harvested after electrically induced cardiac arrest and underwent 12-hour EVLP with an adsorbent membrane (membrane group: n = 5) and without an adsorbent membrane (control group: n = 6). RESULTS: In the control group, both tumor necrosis factor-alpha and interleukin 8 levels were elevated in the perfusate 2 hours after perfusion. Although tumor necrosis factor-alpha and interleukin 8 levels were significantly lower in the membrane group than in the control group during the EVLP period, there was no significant difference in oxygenation, pulmonary vascular resistance, edema formation, or myeloperoxidase activity between the two groups. CONCLUSIONS: Tumor necrosis factor-alpha and interleukin 8 levels of the perfusate were elevated during EVLP. Although adverse effects of these inflammatory cytokines were anticipated, removal of inflammatory cytokines by the adsorbent membrane did not improve lung function during prolonged EVLP. Factors other than the cytokines may play a major role in causing lung injury during EVLP. Further research is needed to investigate the real mechanism of lung graft injury during prolonged EVLP and to establish longer EVLP duration for graft treatment. This strategy could contribute to the salvage of potentially damaged lungs, especially from cardiac death donors, and to expansion of the donor pool.