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Introduction Extracorporeal photopheresis (ECP) serves as a second line treatment for patients with acute or chronic graft versus host disease (GVHD) and demonstrates efficacy in ameliorating GVHD in this setting. The mechanism by which ECP acts against GVHD has not been fully elucidated yet. Preliminary data point to an association between GVHD and increased neutrophil extracellular trap (NET) formation (NETosis) although much is unknown regarding the pathologic implication of this association. In this study we performed a preliminary assessment of the influence of ECP on NETosis among patients with GVHD. Methods Six patients after allogeneic transplantation treated with ECP for severe GVHD post allogeneic transplant at the Rabin Medical Center in Israel were enrolled to the study. Blood samples were obtained at 3 different time points: before an ECP cycle, immediately after the end of the ECP and 24h after the initiation of the ECP cycle. Neutrophils were obtained from whole blood samples using a percoll gradient. NETosis was assessed by measurement of neutrophil elastase activity using a commercial NETosis assay kit by Cayman Chemical and by immunofluorescence staining for DNA (DAPI (4',6-diamidino-2-phenylindole)) and for citrullinated H3 (H3Cit), a marker of NET formation. All assessments were executed in unstimulated neutrophils and in neutrophils that were stimulated with phorbol myristate acetate (PMA)(100nmol) for 4 hours. Results Six patients (4 males) with chronic GVHD were included in the study. The underlying hematologic disease was acute myeloid leukemia (AML) in four patients, B-cell acute lymphocytic leukemia (B-ALL) and myelodysplastic syndrome (MDS) each in one patient. ECP was executed for moderate to severe GVHD. We observed a sharp increase in the formation of NETs following treatment with ECP among all study participants. The level of neutrophil elastase activity was significantly elevated from a mean value of 2.21mU/mL (±0.6mU/mL) at baseline to a mean value of 13.82mU/mL (±5.54mU/mL) immediately after the treatment (p-value=0.0022). The mean level of neutrophil elastase activity was significantly elevated to a mean peak value of 17.35mU/mL (±10.74mU/mL) 24h following the initiation of the ECP cycle (p-value 0.0063 for the difference between the first and the last time points). Pre-incubation of the neutrophils with PMA yielded similar results, as the mean neutrophil elastase activity was 6.05mU/mL (±3.63mU/mL), 20.16mU/mL (±6.72mU/mL), and 24.57mU/mL (±13.59mU/mL) before the ECP cycle initiation, immediately after treatment, and 24h following ECP onset, respectively (Figure 1). In agreement, the expression of H3cit was also increased in neutrophils derived from patients with GVHD after ECP treatment (Figure 2). Conclusion Our preliminary data indicate that ECP induces NET formation among patients with GVHD. NETosis might play a role in the therapeutic effects of ECP in this setting. These initial results might set the stage for future studies in this field. Figure 1 Figure 1. Disclosures Goldberg: MSD Israel: Consultancy. Yeshurun: Astellas: Consultancy; Janssen: Consultancy. Wolach: Janssen: Consultancy; Abbvie: Consultancy, Honoraria, Research Funding; Astellas: Consultancy; Amgen: Research Funding; Novartis: Consultancy; Neopharm: Consultancy.

Introduction: Patients treated with direct Xa inhibitors may require an urgent surgery. A reversal agent for Xa inhibitors was recently approved for major bleeding but not prior to urgent surgery. Administration of prothrombin complex concentrate (PCC) in this setting is a common practice; however, it is based on limited experience in healthy volunteers. Objective: To characterize the population receiving PCC for apixaban/rivaroxaban reversal prior to an urgent surgery/procecdure and evaluate its efficacy and safety. Methods: A retrospective study in two tertiary hospitals. Bleeding was evaluated by surgical reports, hemoglobin drop and use of packed red blood cells or additional PCC during 48h. Safety measures were thrombotic complications and 30-day mortality. Results: Sixty-two patients, aged 80.7±9, received PCC prior to urgent invasive procedures ;39 (63%) received apixaban and 23( 37%) rivaroxaban. Ninety percent of them received anticoagulation due to atrial fibrillation. Most urgent procedures were abdominal surgery (61%), orthopedic surgery (13%) or transhepatic cholecystostomy insertion (10%). Mean dose of PCC was 26.6±8 U/kg. Fourteen patients (23%) received 1 gram of tranexamic acid. Bleeding was reported by surgeons in 3(5%) patients and no patient required additional PCC. Sixteen (26%) patients received packed cells (median 1 unit, range 1-5). Thirty day mortality and thrombosis were 13(21%) and 2(3%) respectively. Cause of death was related to the primary disease or sepsis. No patient died due to bleeding/thrombosis. Conclusions: PCC is safe and effective for the reversal of Xa inhibitors prior to an urgent surgery/procedure. Disclosures No relevant conflicts of interest to declare.

Several human histo-blood groups are glycosphingolipids, including P/P1/P(k). Glycosphingolipids are implicated in HIV-host-cell-fusion and some bind to HIV-gp120 in vitro. Based on our previous studies on Fabry disease, where P(k) accumulates and reduces infection, and a soluble P(k) analog that inhibits infection, we investigated cell surface-expressed P(k) in HIV infection. HIV-1 infection of peripheral blood-derived mononuclear cells (PBMCs) from otherwise healthy persons, with blood group P(1)(k), where P(k) is overexpressed, or blood group p, that completely lacks P(k), were compared with draw date-matched controls. Fluorescence-activated cell sorter analysis and/or thin layer chromatography were used to verify P(k) levels. P(1)(k) PBMCs were highly resistant to R5 and X4 HIV-1 infection. In contrast, p PBMCs showed 10- to 1000-fold increased susceptibility to HIV-1 infection. Surface and total cell expression of P(k), but not CD4 or chemokine coreceptor expression, correlated with infection. P(k) liposome-fused cells and CD4(+) HeLa cells manipulated to express high or low P(k) levels confirmed a protective effect of P(k). We conclude that P(k) expression strongly influences susceptibility to HIV-1 infection, which implicates P(k) as a new endogenous cell-surface factor that may provide protection against HIV-1 infection.

During the last decade the frequency of therapy-related acute leukemia (t-leuk) and myelodysplastic syndrome (t-MDS) has been increasingly observed. Over the past 15 years, we treated 56 patients with t-leuk who had received prior chemotherapy (39%), radiotherapy (11%), or both (45%). The drugs received included alkylating agents and topoisomerase II inhibitors. The primary tumors included hematological malignancies (49%) and solid tumors such as breast or ovarian cancer. The median age at diagnosis of the primary tumor was relatively young (43 years +/- 18). Twelve patients had more than one primary tumor and 31 patients had a family history of malignancy. Karyotypic abnormalities were found in 91% of the patients. Prognosis was uniformly poor, with an overall median survival of 10 months. Twelve of the 18 patients examined (67%) had a multidrug resistance phenotype. P53 genes of the leukemic cells, as well as the original tumors, were analyzed in 21 patients using polymerase chain reaction (PCR) with single-stranded conformation polymorphism analysis followed by sequencing. P53 mutations were identified in 38% of these patients, a relatively high prevalence compared with other forms of MDS or de novo acute myeloid leukemia. Mutations were nongermline and restricted to the leukemic cells. We identified different p53 mutations in the various primary tumors of individual patients. The presence of a mutator phenotype was assessed by PCR analysis of microsatellites in eight loci (one trinucleotide repeat sequence, four dinucleotide, and three mononuclear repeat sequences). Microsatellite instability in two to seven loci were found in 15 of 16 (94%) of the patients. This instability is compatible with a mutator phenotype, which predisposes the patients to the development of malignancies including t-leuk.

Abstract 2221 Neonatal alloimmune thrombocytopenia (NAIT) is a life-threatening bleeding disorder in the fetus/ neonate caused by maternal alloantibodies directed against fetal platelet antigens inherited from the father. Of the 21 allonatigens described up to date http://www.ebi.ac.uk/ipd/hpa/table2.html, the human platelet antigen (HPA) −1 defined as a leucine /proline polymorphism at residue 33 in b3 integrin is the immune-dominant antigen leading to severe NAIT. Immunization to HPA-1a occurs only in 10% of HPA-1bb pregnant women, 90% of them carrying histocompatibility leukocyte antigen (HLA) DRB3*0101. The aims of the present study were: 1. to prospectively study whether in addition to HLA DR B3*0101 there other potential HLA DR antigens involved in the immune process. 2. To analyse whether the response to IVIgG treatment correlates with maternal HLA DR genotype 3. to determine whether, anti HLA antibodies in addition to HPA1a antibodies, resulted in a more severe thrombocytopenia. We enrolled 21 pregnant women who had previously given birth to newborns with NAIT due to HPA1 incompatibility and had antibodies directed to their spouses HPA1 phenotype (leucine or proline polymorphism ) at the Sheba Medical Center from May 2001 to January 2011. The control group consisted of 21 women who delivered at term newborns with thrombocytopenia and a diagnosis of NAIT was ruled out. Twelve women of 21 (57%) in the study group carried both HLADRB3*0101 and DRB4*0101phenotype. But in none of the women in the control group such combination was detected. Interestingly is the observation that of 20 women who gave birth once more following IVIgG treatment, four did not respond to IVIgG and 3 (75%) of them harbor both DRB3*0101 and DRB4*0101. When lymphocytotoxic cross match analysis was performed in eighteen couples of the study group seven (39%) of them had positive lymphocytotoxic cross match. However the presence of both HLA and HPA1 antibodies against their partner phenotype did not affect platelets' counts in the newborn. Taken together we postulate that the presence of both HLADRB3*0101 and HLADRB4*0101 in the mother increases the risk of NAIT development when HPA1 incompatibility between the couples is found and can serve as a diagnostic tool. The presence of HLA antibodies against the spouse did not alter response to IgG treatment. Disclosures: No relevant conflicts of interest to declare.

Positive direct antiglobulin tests (DAT) may be non-specific or associated with auto-antibodies and malignancies, including multiple myeloma and lymphoproliferative disorders. Screening blood donations for DAT is not routinely performed. Furthermore, it is unknown whether a positive DAT in healthy donors is associated with an increased risk of malignancy. All blood donations at MDA National Blood Services in Israel undergo Autocontrol (AC) testing using automated blood grouping equipment (Autogrouper© 1986–2000, and Olympus PK7200© from 2000-present). AC-positive donations are tested for DAT using tubes and/or Diamed gel cards with anti-IgG, anti-C3 and anti-IgG/C3. Donors with positive DAT tests were identified from 1999–2003 and matched in a 1:4 ratio to DAT-negative donors for sex, year of donation and age (by 5 year categories). Cancer incidence was ascertained through July 30, 2006 via the Israel Cancer Registry. We compared the incidence of all cancers, hematopoietic, and solid tumors among DAT+ and DAT– donors. We also compared cancer rates in DAT+ donors with the expected rates in the general Israeli population, controlling for age, sex and ethnicity. Results: The final study population included 586 DAT+ and 2344 DAT– donors. In both groups 62.6% were males, and the mean age of the DAT+ donors was 34.5 (range: 17–67) compared to 32.0 (11–74) years in DAT– donors. Malignancies occurred in 17 (2.9%) of the DAT+ and 27 (1.2%) of the DAT– donors during the follow-up period; of these, three donors in the DAT+ group were diagnosed with hematopoietic cancer within 12 months of their donation (Hodgkin lymphoma within 2 months, non-Hodgkin lymphoma within 4 months and multiple myeloma within 8 months). Even after excluding these early cases and with a mean follow up of 66 months (range: 14–91 months), the relative risk (RR) of developing cancer was 2.14 (95% confidence interval (CI): 1.13–4.10) comparing donors with positive DAT to those with negative DAT, while the RR for hematopoietic cancer was 8.3 (95% CI: 1.5–43.2). Comparing DAT+ blood donors with the general population, the standardized incidence ratios (observed/expected cases) were elevated at 2.11 (95% CI: 1.15–3.54, p=0.016) for all malignancies and 8.03 (95% CI: 2.2–20.6, p=0.003) for hematologic malignancies, while they were not increased in the DAT negative group. Among DAT+ donors, the most frequent malignancies were lymphoma, multiple myeloma, thyroid, prostate and gastrointestinal tumors (2 cases in each category). Conclusion: There is evidence of a significantly increased risk of cancer, especially hematologic malignancies, among blood donors with a positive DAT, within a short follow-up period. A positive direct antiglobulin test thus appears to be a risk marker for malignancy. Further studies are necessary to confirm these findings, assess their significance, and determine whether the DAT would be a useful aid to early detection of cancer in healthy populations.

Many blood group antigens belong to a class of lipid-sugar conjugate molecules known as glycosphingolipids (GSLs), including ABH, Lewis and P blood group systems. GSLs are implicated in HIV-host cell fusion, and several bind to HIV-gp120 in vitro. In addition, inhibiting glycolipid biosynthesis has been shown to prevent HIV membrane fusion. The Pk blood group antigen, also known as globotriaosylceramide (Gb3), has been shown to augment HIV fusion in in vitro models; however its exact role in HIV infection is not known. We have previously developed a soluble analogue of Pk, a highly effective gp120 ligand, which inhibits HIV-1 and HIV-2 membrane fusion. We hypothesize that Pk influences susceptibility to HIV infection, inhibiting at the level of membrane fusion and entry. We have presently investigated the effects of soluble and expressed Pk on HIV-1 infection in vitro. Soluble Pk effects were considered by pre-treating monocyte (M)-tropic HIV-1Ba-L or T-cell (T)-tropic HIV-1IIIB with soluble Pk prior to infection of PHA or PHA/IL2 activated peripheral blood-derived mononuclear cells (PBMCs) or Jurkat T-cells. Productive M-tropic and/or T-tropic HIV-1 infection of PBMCs and Jurkat cells was inhibited by more than 90%. Inhibition of membrane fusion and entry was visualised by transmission electron microscopy. Soluble Pk had minimal effects on cell viability or HIV receptor (CD4) and co-receptor (CCR5 or CXCR4) expression as measured by flow cytometry. Exogenously introduced Pk in Jurkat T-cells was accomplished by liposome fusion, and susceptibility to T-tropic HIV-1IIIB infection was determined. Pk transfer in Jurkat T-cells also reduced productive HIV-1 infection by 50%, without affecting cell viability or HIV receptor and co-receptor expression. Effects of differential Pk expression on HIV susceptibility was investigated using PBMCs with Pk accumulation, from patients with Fabry disease and P1k blood group individuals, or which lack Pk, from p blood group individuals. PBMCs representative of these two groups were infected with M-tropic HIV-1Ba-L or T-tropic HIV-1IIIB. PBMCs with Pk accumulation from patients with Fabry disease were resistant to productive M-tropic, but not T-tropic HIV-1 infection. These PBMCs had significantly decreased expression levels of the M-tropic HIV-1 co-receptor CCR5. Pk accumulation in P1k blood group PBMCs shows resistance to T-tropic and M-tropic HIV-1 infection, unrelated to levels of chemokine receptors. PBMCs lacking Pk from p blood group individuals were hypersensitive to M-tropic HIV-1 infection, while T-tropic HIV-1 infection levels were variable. Interestingly, p blood group PBMCs showed significantly higher levels of the CCR5 co-receptor. Our findings suggest Pk is inhibitory to HIV infection, which may translate into natural resistance in individuals that express high levels of Pk. This may be linked to interactions with CCR5, through receptor trafficking to the cell surface, or associations within lipid rafts. Furthermore, soluble Pk or potential mechanisms to increase cellular Pk expression, provide novel strategies for prevention of HIV infection.