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This study assessed the active role of the portal vein endothelium in the functional loss of pancreatic islet (PI) grafts, considered in the context of PI transplantation for treatment of type I diabetes mellitus. We hypothesized that PI engraftment may be jeopardized by portal vein endothelial cell-induced activation of host T cells. We designed an in vitro system using portal vein endothelial cells (PVEC) from Lewis (Lew) rats and PI from Brown-Norway (BN) and Lew rats. PI were co-cultured with Lew PVEC for three days. The PI were removed and trypsinized PVEC were divided into three groups: (A) PVEC not exposed to PI; (B) PVEC exposed to syngeneic PI; and (C) PVEC exposed to allogeneic PI. The groups were analyzed by flow cytometry for ICAM-1 and MHC Class I and Class II molecules. Functional assays of lymphocytes (ie, lymphocyte proliferation assay, 51Cr release assay, and cytokine release assay) were also performed. We observed MHC Class I and ICAM-1 upregulation on PVEC after PI contact. MHC Class II molecule was not upregulated on PVEC after PI exposure. IL-6 production was increased non-specifically following PI coculture with PVEC. TNF-a was increased only after allogeneic PI-PVEC coculture. PVEC exposed to either allogeneic or syngeneic PI could not stimulate naïve splenocyte proliferation or cytotoxicity. We conclude that PVEC allo-PI interaction results in increased ICAM-1 and MHC Class I expression on the PVEC. Neither lymphocyte proliferation nor cytotoxicity could be enhanced in response to enhanced MHC Class I and ICAM-1 expression, which was associated instead with non-specific inflammatory cytokine release.

The risk of hemorrhage in infants with severe coagulopathies unresponsive to fresh frozen plasma (FFP) infusions may preclude therapeutic invasive interventional procedures. We describe the successful use of recombinant factor VIIa (rFVIIa) in two such infants, the first with cirrhosis requiring paracentesis and the second with necrotizing enterocolitis requiring laparotomy. This report reviews the current concepts on the mechanism of action of the drug rFVIIa and considers its expanded use in infants unresponsive to FFP replacement.

Human hepatocyte viability and function in vivo in an athymic rat was assessed after transplantation on prevascularized polymer constructs with hepatotrophic stimulation. Sixteen liver biopsy specimens, weighing 5 to 12 g, were obtained from the New England Organ Bank and from the operating room after liver resection. In the laboratory they were catheterized and perfused to obtain liver cell suspensions. From eight of the 16 cell suspensions, only in vitro studies were performed. They showed 40% cell attachment 24 hours after initial cell plating. For patients aged 2, 35, and 60 years, they showed a 20% increase, a 1% decrease, and a 57% decrease (respectively) in cell number from day 2 to day 4, after cell plating. Eight cell suspensions were transplanted into athymic rats. On sections examined histologically, implanted hepatocytes were seen within the fibroblast ingrowth, in the space of the polymer device, until day 21 after cell injection. On day 9 after hepatocyte injection, reorganized hepatic parenchyma was seen on the tissue section. Implanted hepatocyte areas, quantitated through morphometric analysis on days 0, 3, and 7, showed a 36% increase in engraftment 3 days after injection, and a 42% decrease 7 days after injection. At the same time-points, immunoperoxidase staining visualized intracellular albumin, which was specific for the implanted hepatocytes. In conclusion, the authors demonstrated the feasibility of their technique (prevascularized polymer device with hepatotrophic stimulation), using human hepatocytes. Further studies are underway, before implementation of human clinical trials.

Abstract 3429 Introduction: Mechanical hemolysis is a non-immune mediated destruction of red blood cells that can be associated with blood transfusion. Various etiologies include the use of blood warmers, small bore needles or high infusion rates. We report the investigation of hemoglobinuria cases observed post-packed red blood cell (PRBC) transfusion with subsequent changes in management. Case report: Over a time period of 2 months, 6 events of hemoglobinuria and hemolysis were reported in 5 patients in the pediatric hematology/oncology treatment center. Each child presented with abnormally tea-colored urine following PRBC transfusion. Transfusion reaction workup included a direct antiglobulin test (DAT), urinalysis (UA), serum haptoglobin (H), lactate dehydrogenase (LDH), and total bilirubin (T-B). All patients had a negative or unchanged DAT, negative RBC antibody screen, decreased H, increased T-B, and increased LDH (4 out of 6), suggestive of intravascular hemolysis (Table). In spite of hemolysis, hemoglobin (Hgb)/hematocrit(Hct) increased normally. Multiple factors were investigated to determine a cause: collection and processing of blood units; handling of blood at the transfusion service; and blood administration by nursing staff. The only significant change identified was the implementation of a new infusion pump (pump A) replacing a model that was phased out. A hospital-wide retrospective review of urinalysis (UA) was performed over a one month time period, along with prospective UA surveillance in the outpatient unit. Case Date Unit S Age (yr) Diagnosis UA Dipstick Blood/RBC LDH (U/L) H (mg/dL) T-B (mg/dL) DAT 1 6/7/12 CPDA1 F 4 RMS 3+/0-3 637 13 1.4 Neg 2 6/12/12 CPDA1 × 3 M 10 ALL 2+/0-2 331 9 0.5 Neg 3 6/29/12 CPDA1 M 4 DBA 3+/0-3 364 1.6 Pos * 4 7/9/12 AS-5 F 7 ALL Neg/0-3 – 3.3 Neg 5 7/14/12 CPDA1 × 2 F 11 PTLD 2+/0-3 338 2.3 Neg 6 7/18/12 AS-5 × 2 M 10 ALL Trace/0-3 400 2.8 Neg Patient 2 and 6 are identical. Corrective actions: 7/13- Pressure sensors changed from 12 to 6psi; 7/16- CPDA1 units replaced by AS-5 in children over 9 months; 7/23- Pump A replaced by pump B in out-patient unit. Abbreviations: S-Sex; RMS:Rhabdomyosarcoma; ALL-Acute Lymphoblastic leukemia; PTLD-Post Transplant Lymphoproliferative disorder; DBA Diamond Blackfan Anemia; DAT-Direct antiglobulin test; LDH- Lactate Dehydrogenase; H- Haptoglobin; T-B-Total Bilirubin. Normal values: LDH * DAT was unchanged from pre-transfusion. Results: In the 6 cases, a total of 10 irradiated PRBC units (7 with citrate phosphate dextrose buffer (CPDA1, HCT 65–80%) and 3 with additive solution (AS-5, HCT 55–65%)) were transfused. Although Pump A had been validated by the manufacturer with non-irradiated additive solution units, an in vitro study with irradiated CPDA1 RBC units was performed to compare pump A with an alternative pump B. Samples were taken after infusion using the pumps only (no needle or catheter) and dripped directly into the test tube for measurement of free hemoglobin (FHb). Irradiated CPDA1 units infused at a low rate (50ml/hr) showed an increase in FHb level at 1998mg/dL with pump A versus 496 mg/dL with pump B. Non-irradiated AS-5 units tested as controls resulted in a FHb level at 246mg/dL with pump A versus 117mg/dL with pump B. CPDA1 units were subsequently replaced with AS-5 units for transfusion. Finally, pump A was replaced by pump B in the outpatient hematology oncology unit. In vitro studies are still ongoing to determine if irradiation of the PRBC may also play a role in the mechanical hemolysis observed with pump A. One month retrospective UA review found no association between patients with hemoglobinuria and RBC transfusion. The UA prospective surveillance performed in the outpatient unit revealed that 7 of 26 patients had trace hemoglobinuria. Each one had a confounding factor (chronic hemolysis) for hemoglobinuria. Conclusion: Preliminary investigation determined that the infusion of highly concentrated irradiated RBC (CDPA1) using a specific commercial pump was associated with mechanical hemolysis. The change to less concentrated RBC units through an alternative pump has been to date, an effective corrective action. Disclosures: No relevant conflicts of interest to declare.

3142 Poster Board III-79 Background Evolving concerns about storage lesions for red blood cells (RBC) and their effect on patient outcomes have led to ongoing trials evaluating the benefits of transfusing fresher blood (less than 14 days) to acutely ill patients. If improved outcomes with fresh blood are clearly demonstrated, current inventory management strategies may have to be revisited. Methods We evaluated several RBC maximum shelf lives (MSLs) and their impact on RBC availability. First, we determined the average age of the RBC units in our inventory based on the ABO/Rh blood type by analyzing the data set of 18,987 RBCs transfused from April 08, 2008 to April 09, 2009. Secondly, we determined the feasibility of issuing RBC of designated age to patients using the same data set. Results We found that 80% of the RBC inventory was delivered before 14 days of age (DoA), and that 42% versus 65% was issued before 14 DoA versus 21 DoA, respectively. RBC units spent on average 8.6 days on the shelf with a mean age at delivery of 10.2 days and a mean age at issue for transfusion of 18.8 days which varied depending on the ABO blood type as described in [Table 1][1]. | Blood type | A + | A – | B + | B – | AB + | AB – | O + | O – | Mean | Median | |:-------------------:| ---- | ---- | ---- | --- | ---- | ---- | ---- | ---- | ---- | ------ | | AVG age at delivery | 10.5 | 12.1 | 14.6 | 13 | 20.2 | 19 | 8.1 | 10.3 | 10.2 | 8 | | AVG age at issue | 20.4 | 27.7 | 21 | 25 | 29.3 | 32.7 | 14.9 | 21.2 | 18.8 | 17 | | AVG days on shelf | 9.8 | 15.6 | 6.3 | 12 | 9 | 13.8 | 6.7 | 10.9 | 8.6 | 6 | Table 1: Average age at delivery and issue, and average days on shelf by blood type We then defined four scenarios where RBCs have different MSL: Scenario #1, #2, and #3 used a MSL of 7, 14, and 21 days, respectively. Scenario #4 used a combination of different MSLs depending on the category of patients, as follows: 7 days for 12% of patients (i.e., neonates), 14 days for 15% of patients (i.e., adult cardiovascular surgical patients), and 42 days for the 73% remaining patients receiving RBCs for transfusion. The simulations were performed using the original 18,987 data sets and results are summarized in [Table 2][2] showing a significant decrease in the availability of RBC units by 51%, 20%, 10% and 0% for Scenario #1, #2, #3, and #4, respectively. Furthermore, the outdate rate would increase to 3.2%, 2.2%,1.0%, and 4.4% for the respective scenario. | Scenarios | MSL | Available to be issued | Decrease in availability | Outdate rate | |:---------:| --------- | ---------------------- | ------------------------ | ------------ | | # 1 | < 7 days | 9,248 | 9,739 (51%) | 300 (3.2%) | | # 2 | < 14 days | 15,153 | 3,834 (20%) | 334 (2.2%) | | # 3 | < 21 days | 17,192 | 1,795 (10%) | 169 (1.0%) | | # 4 | Combo | 18,987 | 0 (0%) | 893 (4.4%) | Table 2: Decrease in availability with respect to different scenarios Conclusion Changing the maximum shelf life for red blood cell units will require novel approaches to RBC inventory management in order to meet hospital demands without risking a major shortage and putting patients at risk. Disclosures No relevant conflicts of interest to declare. [1]: #T1 [2]: #T2

Following liver transplantation, patients require immunosuppressive medications to prevent graft rejection. However, these drugs can have significant side effects including severe anemia. Tacrolimus is now commonly used in pediatrics patients after liver transplantation. Case reports indicate that tacrolimus rarely can cause microangiopathic hemolytic anemia and also hemolytic anemia due to cold reactive IgM antibodies (cold agglutinin disease). We have seen 4 patients with symptomatic cold agglutinins thought to have been triggered by tacrolimus. Four patients, 6 to 26 months post-ABO-compatible liver transplant, presented with severe anemia (hemoglobin < 6g/dL), indirect hyperbilirubinemia, reticulocytosis and low serum haptoglobin (Table 1). Peripheral blood smear exhibited marked autoagglutination but no evidence of microangiopathic changes. The direct antiglobulin test (DAT) initially was strongly positive in all 4 patients (Table 2). After warm saline washes, the DAT remained positive for complements in 3 out of 4 patients. Cold agglutinin titers were low (1:4 – 1:16) but thermal amplitude studies were positive at 37°C in the two tested patients. Viral serologies significant for cold agglutinin syndrome were negative. None of the patients had clinical evidence of post-transplant lymphoproliferative disorder. All 4 patients had brisk hemolysis requiring multiple uncrossmatched packed red blood cells (PRBC) transfusions. Favorable resolution of the hemolytic anemia occurred following steroids, plasmapheresis and withdrawal of tacrolimus. Subsequently, patients were placed on cyclosporin and none exhibited liver rejection or recurrence of their hemolytic anemia. The above observation indicates that acute cold-agglutinin induced hemolysis occurs in patients following liver transplantation and these events appear to have temporally associated with tacrolimus administration. Patient Profiles Patient Age Diagnosis Months after transplant Presenting hemoglobin (g/dL) Reticulocyte (%/Abs k/μL Total/Direct Bilirubin Treatments (in addition to withdrawal of tacrolimus) ND=not done. MP=methylprednisolone 1 8 mos Neonatal iron storage disease 6 2.8 41%/469 4.2/0.6 MP 4mg/kg/d plus 30mg/kg/d x 3 days. Plasmapheresis x 6. 2 13 mos Biliary atresia Idiopathic 6 5.3 8%/250 3.2/0.4 MP 4mg/kg/d. Plasmapheresis x 15. 3 2 yrs 5 mos fulminant hepatic failure 14 4.6 19.5%/237 2.6/0.3 MP 10mg/kg/d. Plasmapheresis x 10. 4 4 yrs Biliary atresia 26 5.3 5%/ND 8.8/0.5 MP 2mg/kg/d→ 4mg/kg/d. Plasmapheresis x 10. Immunohematology Profiles Admission DAT Patient 1 Patient 2 Patient 3 Patient 4 Admission DAT Pre/post saline wash Pre/post saline wash Pre/post saline wash Pre/post saline wash DAT-Direct antiglobulin test, M-microscopically, NT-not tested, +-positive, 0-negative, Polyspecific 3+/0 1+/M+ 4+/4+ 3+/1+ Anti-IgG 2+/0 M+/0 4+/1+ 3+/1+ Anti-C3 2+/0 2+/1+ 4+/4+ 3+/1+ Saline Control 2+/0 1+/0 1+/0 1+/0 Eluate 0 NT Panreactive 1+ Panreactive 1+ Cold agglutinin titer at 4°C in saline NT 4 8 16 Thermal amplitude screen at 30°C NT NT Reactive Reactive Donath-Landsteiner Test NT NT Negative Negative Admission peripheral blood smear 4+ polychromatophilia. 4+ microcytosis 4+ autoagglutination. 3+ polychromatophilia. 3+ spherocytes. 3+ microcytes 3+ autoagglutination. 3+ polychromatophilia. 3+ spherocytes. 2+ macrocytes 4+ autoagglutination. 4+ spherocytes. 2+ polychromatophilia. 2+ macrocytes