bluuebunny/crossref_metadata_2025 huggingface dataset:
The Effects Of Oral Iron Treatment On Reactive Oxygen Radicals, Protective Enzymes and Lipid Peroxidation In Women With Iron Deficiency Anemia
Authors: Kadikoylu Gurhan , Ozkan Arzu , Yenisey Cigdem , Yavasoglu Irfan , Bolaman Zahit
Published: October 2019

Iron deficiency anemia (IDA) is the commonest of deficiency disease in the world. In IDA, hemoglobin synthesis is impaired, nonetheless the production of iron (Fe2) containing proteins such as cytochrom, myoglobin, catalase (CAT), and peroxidase is affected. While oxidative stress can play an important role on the development of IDA, lipid peroxidation may be induced. The aim of the study is to investigate the effects of oral iron treatment on reactive oxygen radicals (ROR), protective enzymes from ROR, and lipid peroxidation in women with IDA. This prospective Phase-IV study was approved by ethical committees of university and the Ministry of Health. Sixty-seven women (mean age 34±11 years) with IDA were enrolled to the study. Inclusion criteria were hemoglobin level < 11 g/dl, serum ferritin level < 16 ng/ml, and iron saturation <15%. Twenty-seven helathy women (mean age 27±7 years) were accepted as control group. Exclusion criteria of control group were anemia, acute and chronic infection, acute and chronic bleeding, pregnancy, malignancy, chronic inflammatory, and renal diseases. In both group, whole blood counts, hydrogen peroxide (H202) as ROR, glutathione (GSH), superoxide dismutase, glutathione peroxidase, glutathione reductase, CAT for protective enzymes from ROR, and malondialdehyde (MDA) for lipid peroxidation were evaluated. Ferrous fumarate 200 mg/day were given to the patients for 4 weeks. At the end of 4 weeks, all parameters were repeated. All results were compared with SPSS 15.0 using independent and paired t-tests. P values <0.05 were accepted as significant. In women with IDA, the levels of GSH (p<0.001), H202 (p<0.001), and MDA (p<0.05) were significantly higher than controls, but other parameters were not different in both groups (p>0.05). At the end of treatment, GSH and H202 levels significantly decreased (p<0.001 for both), but other parameters did not changed (p>0.05). In women with IDA, while H202 increased, lipid peroxidation was induced. Compensation of oxidative stress were ensured by GSH. Iron treatment reduced oxidative stress, but it did not affect lipid peroxidation and protective enzymes from ROR.

Disclosures:

No relevant conflicts of interest to declare.
Risk Factors For Cerebral Vasculopathy In a Sickle Cell Disease Newborn Cohort
Authors: Sommet Julie , Alberti Corinne , Holvoet Laurent , Haouari Zinedine , Missud Florence , Ithier Ghislaine , Verlhac Suzanne , Elmaleh Monique , Baruchel Andre , Benkerrou Malika
Published: October 2019

Children with sickle cell disease (SCD) have a significant vascular morbidity responsible of pain and stroke. They are at high risk of cerebral vasculopathy (CV) that can be detected in these children by transcranial Doppler. The aim of this study was to identify risk factors for CV using longitudinal data in the 375 newborn cohort of Robert Debre Pediatric Sickle Cell Referral Center. Among the 59 children presented CV, seven had a stroke. There was a total of 2677 patient-years of follow up. Overall, the incidence rate of CV was 2.20/100 patient-years (95% CI 1.64-2.76) and the incidence rate of stroke was 0.26/100 patient-years (95% CI 0.07-0.46). The cumulative risk of CV by age 14 years was 26.0% (95% CI 20.0%-33.3%).To take into account longitudinal data, risk factors of CV were assessed by a survival Cox model built in two steps. The final Cox multivariable analyze for CV retained five variables. Upper airways obstruction (HR 1.41 95%CI 1.00-1.98), bronchial obstruction (HR 2.13 95%CI 1.77-2.57), acute anemic event (HR 1.15 95%CI 1.01-1.32) and reticulocyte count (HR 1.41 per 20000/mm3 increase 95%CI 1.16-1.71) were independent risk factors whereas hydroxycarbamide therapy (HR 0.03 95%CI 0.01-0.35) was protective. Evaluation and specific treatment for symptoms of asthma or sleep apnea or nocturnal respiratory abnormalities could potentially reduce the risk of CV and stroke after treatment. Our findings suggest that hydroxycarbamide could protect from CV.

Disclosures:

No relevant conflicts of interest to declare.
Exchange Transfusion In Adult Patients With Sickle Cell Disease With Refractory Vaso-Occlusive Crises
Authors: Moffett Padmini , Moffett Bryan K
Published: October 2019

Sickle cell disease occurs in 1/500 African-American births. Pain is one of the most common complications of sickle cell disease and is associated with depression, anxiety, decreased quality of life and poor sleep patterns. Vaso-occlusive crises resulting in three or more hospitalizations per year occur in 48% of patients with sickle cell disease (Annals of Emergency Medicine - May 2009 (Vol. 53, Issue 5, Pages 587-593). Hydroxyurea has been shown to ameliorate the frequency of painful vaso-occlusive crises in sickle cell anemia (Charache et al, Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia. Investigators of the Multicenter Study of Hydroxyurea in Sickle Cell Anemia NEJM. 1995), unfortunately many eligible patients are not treated due to psychosocial reasons and fear of teratogenicity or malignancy or have painful crisis refractory to hydroxyurea. A panel of experts has suggested that RBC exchange transfusion in these patients may decrease ED visits and subsequent hospitalizations based on anecdotal evidence [Best practices for transfusion for pateitns with sickle cell disease. T Wun, K Hagel. Hematology Review 2009, July 1; 1(22); e22]. We present the case of a 31 year old African-American male with Hgb S/beta thalassemia + with complications of sickle cell disease including Parvovirus B19 induced aplastic anemia, iron overload secondary to multiple simple transfusions, multiple vaso-occlusive crises as well as a left lower extremity ulcer precipitated by treatment with hydroxyurea. He was treated with folic acid as well as deferasirox for iron chelation. He began PRBC exchange transfusions every 6 weeks in March of 2011. In the one year prior to exchange transfusion initiation he had 13 ED visits and 4 hospitalizations for vaso-occlusive crises. After initiation of exchange transfusions he had 11 ED visits and 2 hospitalizations for vaso-occlusive crises the following year. An elevated WBC is associated with poor outcomes in sickle cell disease (Miller et al Predictors of adverse outcomes in children with sickle cell disease. N Engl J Med 2000). His average WBC level was 13 x 109/L, which decreased to 11 x 109/L after initiation of exchange transfusion. The patient underwent extended typing for E,C, and Kell RBC antigens to minimize development of antibodies. Though monthly exchange transfusions are costly and carry the attendant risk of antibody formation, this may be offset by reducing the financial and psychological burden of frequent hospitalizations for management of refractory vaso-occlusive crises. Exchange transfusion may also ameliorate the cost associated with long-term iron chelation. Further investigation is warranted to determine whether a program of monthly exchange transfusions can curtail ED visits and hospitalizations in adult patients with sickle cell disease and multiple, refractory vaso-occlusive crises.

Disclosures:

No relevant conflicts of interest to declare.
The Function Of Transcription Elongation In Hematopoietic Stem Cell Development
Authors: Zhou Ting , Cook Jennifer , Liu Xiuli , Bai Xiaoying
Published: October 2019

The specification of hematopoietic stem cells (HSCs) in vertebrate embryos is tightly regulated by RNA polymerase II (Pol II)-mediated transcription, which proceeds through multiple steps including initiation/activation, elongation and termination. Increasing evidences in recent years have demonstrated that transcriptionally engaged Pol II often pauses shortly after initiation by pausing factors DSIF and NELF, and its release requires positive elongation factors such as p-TEFb. Little is known about how pausing-to-elongation switch contribute to cell fate determination. In a zebrafish spt5 mutant, we detected a dramatic loss of runx1+ HSCs in the AGM region in 36-hour embryos. Spt5 is an essential subunit of DSIF, which pauses Pol II in early elongation stage but stimulates Pol II elongation upon phosphorylated by p-TEFb. The zebrafish mutant carries a single amino acid change in the Spt5 protein that disrupts the pausing function but leaves the stimulatory function intact, suggesting a requirement of Pol II pausing in HSC formation. Similarly, knocking down the other pausing factor NELF by morpholinos also greatly reduced the number of runx1+ HSCs in zebrafish embryos. This loss of HSCs is not caused by general developmental defects, as markers for vessels and aorta endothelium remain normal in mutants. We further demonstrated that reducing Pol II elongation by either p-TEFb inhibitors or morpholino knockdown of CDK9, the essential subunit of p-TEFb, could rescue the HSC defect in spt5 mutants. The rescue by cdk9 morpholino is dose-dependent. Low-dose morpholino that presumably caused partial knockdown of CDK9 was able to rescue spt5 mutants, whereas high-dose morpholino completely wiped out HSCs in both wild type and mutant embryos. In conclusion, our data suggested that HSC formation during embryonic development requires a balanced transcription that is regulated by both negative and positive elongation factors. Such mechanisms may also be employed in other tissues to regulate cell fate determination.

Disclosures:

No relevant conflicts of interest to declare.
Myocardial Iron overload In Thalassemia Major. how Early To Check?
Authors: Borgna-Pignatti Caterina , Meloni Antonella , Guerrini Giulia , Filosa Aldo , Ruffo Giovan Battista , Casini Tommaso , Romano Nicola , Maggio Aurelio , Chiodi Elisabetta , Positano Vincenzo , Lombardi Massimo , Pepe Alessia
Published: October 2019

Introduction

It is still controversy in thalassemia major (TM) if Cardiovascular Magnetic Resonance (CMR) T2* screening should be initiated before the 10 years. To answer this question, we studied retrospectively the prevalence of cardiac iron and function and myocardial fibrosis by CMR in a consistent cohort of TM patients younger than 10 years.

Methods

From the 2171 patients enrolled in the MIOT (Myocardial Iron Overload in Thalassemia) network, we retrospectively selected the 35 TM patients aged less than 10 years who had undergone at least one MRI scan. Myocardial iron overload (MIO) was measured by T2* multislice multiecho technique. Biventricular function parameters were quantitatively evaluated in a standard way by cine images. To detect myocardial fibrosis, late gadolinium enhancement images were acquired.

Results

Patients’ age ranged from 4.2 to 9.7 years. All MRI scans were performed without sedation. Nine patients (25.7%) showed no myocardial iron overload (MIO),22 patients (62.9%) showed an heterogeneous MIO with a T2* global value ≥ 20 ms; 2 patients (5.7%) showed an heterogeneous MIO and a T2* global value < 20 ms and 2 patients (5.7%) had a homogeneous MIO. No patients showed myocardial fibrosis.

Table 1 reports the data of the 4 patients (3 males and 1 female) with significant myocardial iron overload (global heart T2* < 20 ms). The youngest was 6 years old, all showed no heart dysfunction and in all the iron transfused was less than 35 g.

Conclusion

The first cardiac T2* assessment should be performed as early as possible without sedation and it is mandatory whenever poor compliance is suspected or if chelation has been started late. The use of the contrast medium to detect myocardial fibrosis can be postponed until after 10 years of age. A more sensitive segmental approach to detected heart iron should be considered. Therapy can be modified on the basis of the MRI results and serious organ damage can be prevented.

Disclosures:

No relevant conflicts of interest to declare.
Haemophagocytic Lymphohistiocytosis (HLH) Associated With a Heterozygous PRF1 Mutation Not Previously Described As Pathogenic
Authors: Arbelaez Alejandro , Catley Laurence
Published: October 2019

Case Presentation

A 16 year old male presented with three weeks of fatigue, nausea, vomiting, anorexia, arthralgias, and pancytopaenia. He was febrile, with mouth ulcers, hepato splenomegaly, and palpable non tender supraclavicular and axillary lymphadenopathy. Neurological examination was normal. Remarkable abnormal tests: Hb 77 g/L, platelets 29 x109/L, WCC 0.7 x109/L (neutrophils 0.33), normal PT and APTT but hypofibrinogenaemia 0.8 g/L. Ferritin was elevated 56 061 ug/L (20– 150). Liver function tests showed a mixed pattern with elevated LDH (2137 U/L), mild hypertriglyceridaemia (4.6 mmol/L) and low haptoglobin (< 0.06 g/L), EBV IgM was equivocal with reactive IgG. Bone marrow showed evidence of haemophagocytosis (Fig 1). All HLH-2004 criteria were fulfilled except elevated sCD25 (unavailable). NK cell chromium release assay revealed severely suppressed NK cell function. In addition, perforin gene (PRF1) sequencing for mutation screening showed a missense mutation Ala437Val (1310 C>T) PRF1 heterozygous, a previously unclassified variant. There was no evidence of an underlying cause of HLH. Our patient received induction therapy with etoposide 150 mg/m2 and dexamethasone 10 mg/m2 with dosing and frequency as suggested by the HLH-94 protocol. Pancytopaenia recurred and an increase in ferritin concentration was interpreted as relapse of the condition. At that point, cyclosporine was added on in conjunction with maintenance etoposide and dexamethasone obtaining partial remission. He underwent allogeneic SCT successfully in another centre.Figure 1Bone marrow aspirate of this patient showing evidence of haemophagocytosis (H & E). A and B show large macrophages undergoing phagocytosis of different cellular elements of the bone marrow.Figure 1. Bone marrow aspirate of this patient showing evidence of haemophagocytosis (H & E). A and B show large macrophages undergoing phagocytosis of different cellular elements of the bone marrow.

Discussion

Several genes have been implicated in the genesis of primary HLH; all of them have in common impaired cytotoxic function by NK and T cells. It has been reported that mutations of the PRF1 gene comprise approximately 20 to 30% of the cases of primary HLH. This variant has been reported as a polymorphism and was not conclusive for the diagnosis of perforin deficiency as the cause of HLH in this case. In addition, the mutation was heterozygous. Although HLH is usually a recessive disorder, there are reports of potential disease associations in the heterozygous state. There is significant overlap between primary and secondary HLH and the role of some heterozygous mutations remain to be investigated.

Disclosures:

No relevant conflicts of interest to declare.
Proteasome Inhibitor Based Protocol For Antibody Mediated Rejection In Kidney Transplantation
Authors: Sanchorawala Vaishali , Quillen Karen , Gautam Amitabh , Francis Jean M
Published: October 2019

Background

Rapid and complete elimination of donor-specific anti-human leukocyte antigen antibodies (DSA) during antibody-mediated rejection (AMR) of renal transplantation is rarely achieved with traditional antihumoral therapies. Current antihumoral therapies do not target the mature antibody producing plasma cells. Bortezomib is a first in class proteosome inhibitor for the treatment of plasma cell derived tumors like multiple myeloma. Proteasome inhibitor-based therapy has been shown to effectively treat refractory AMR. Here, we report on our experience with plasma cell targeted therapy (bortezomib) as an antirejection strategy for refractory early and delayed AMR post renal transplantation.

Methods

Adult kidney transplant recipients with AMR, diagnosed by Banff criteria, received a bortezomib-based regimen after failing at least 2 traditional treatments for AMR. Bortezomib therapy was administered (1.3 mg/m2 IV D1,4,8,11) with plasmapheresis performed immediately before each dose of Bortezomib and a single dose of rituximab (375 mg/m2) given with the first bortezomib dose. Methylprednisolone was administered along with Bortezomib at 100 mg IV on D1,4 and 50 mg IV on D8,11. All patients received valganciclovir prophylaxis for prevention of herpes zoster and cytomegalovirus reactivation. Monitoring included serial donor-specific antihuman leukocyte antigen antibody (DSA) levels, serum creatinine levels, urine protein excretion and repeated allograft biopsies.

Results

Ten episodes of AMR in eight renal transplant recipients were treated with bortezomib, rituximab, methylprednisone and plasmapheresis.

Post treatment reduction in immunodominant DSA was observed and this data will be available at the meeting. None of the patients experienced peripheral neuropathy from bortezomib and only one patient had GI toxicity with nausea and diarrhea, which was transient. None of the patients had reactivation of herpes zoster infection.

In conclusion, bortezomib, rituximab, methylprednisone and plasma exchange therapy provided reversal of AMR rejection post renal transplantation with improvement in renal function, proteinuria and histology, at a median follow-up of 15 months.

Disclosures:

No relevant conflicts of interest to declare.
Increased Levels Of Tissue Factor, Adhesion Molecules, Nitric Oxide and Adiponectin In End Stage Renal Disease. A Complex Interplay
Authors: Fareed Jawed , Kaneva Kristiyana , Hoppensteadt Debra , Bansal Vinod
Published: October 2019

Introduction

End stage renal disease (ESRD) represents the fifth (final) stage of chronic kidney disease characterized by an established kidney failure (GFR <15 mL/min/1.73 m2). To further understand the pathophysiology of ESRD, this study was designed to measure the circulating levels of tissue factor (TF), adhesion molecules, such as p-selectin (P-Sel), soluble ICAM (s-ICAM), nitric oxide and adiponectin (AD).

Methods

This study included 119 ESRD patients undergoing maintenance hemodialysis in conjunction with an ongoing IRB approved protocol on the profiling of inflammatory markers in this syndrome. Citrated blood plasma samples were collected from these patients prior to the routine dialysis session. Nitric oxide levels (NO) were measured using a commercial kit from R&D systems (Minneapolis, Minnesota) and ELISA based methods for TF, P-Sel, s-ICAM and adiponectin were also purchased from R&D systems. Chromogenic and thrombin substrate method were used to measure the anti-Xa activity thrombin generation.

Results

Tissue factor levels were found to be increased in the ESRD group (20.4±6.1pg/ml) vs the control (11.9±2.8pg/ml). The nitric oxide level was markedly higher in the ESRD group (32±17uM) vs the controls (7±3uM). The p-selectin levels were also elevated in the ESRD group (46±20ng/ml) vs the control (31±3ng/ml). The soluble ICAM levels were higher in the ESRD group (250±112ng/ml) vs the control (180±19ng/ml). Interestingly, the adiponectin levels were also increased in the ESRD group (19.2±9.3ug/ml) vs the control (11.2±4.1ug/ml). The pre-dialysis samples of the ESRD patients exhibited detectable levels of heparin.

Summary/Conclusions

These studies suggest that TF, NO, p-selectin and s-ICAM levels are increased in the ESRD patient. It is of interest to note that despite that a significant number of ESRD patients were diabetic; the AD levels were increased in this group. These results suggest that while ESRD represents a pro-inflammatory/hypercoagulable state, the repeated administration of heparin and other drugs may contribute to the regulation of the hemostatic process and inflammatory balance.

Disclosures:

No relevant conflicts of interest to declare.
Serotonin and Its Metabolism Enhances Megakaryopoiesis and Proplatelet Formation
Authors: Ye Jie yu , Meng Fan yi , Jiang Qianli , Li Su yi , Liang En yu , Chong Beng , Yang Mo
Published: October 2019

Serotonin (5-HT) has been recently identified as a novel growth factor. We previously demonstrated that 5-HT enhances murine megakaryopoiesis via 5-HT2 receptors and has promotor effect on hematopoiesis(Yang M et al, Stem Cells, 2007). However, the molecular mechanism remains under explored. In the terminal stage of mammalian megakaryocyte development, platelets are released from proplatelet protruding from megakaryocytes via cytoskeleton reorganization. 5-HT is shown to modulate cell migration and remolding by activating cytoskeleton reorganization, but the effects of 5-HT on proplatelet formation have not been investigated. Our results showed that 5-HT significantly promoted human CFU-MK formation and reduced apoptosis on human megakaryocytes through phosphorylation of Akt. These effects were attenuated by addition of ketanserin, a 5HT2 receptor inhibitor. 5-HT also stimulated proplatelet formation through activating the p-Erk1/2 expression and F-actin reorganization. Melatonin, the metabolism of 5-HT, promoted the recovery of platelets and the formation of bone marrow colony forming units in irradiated mice. Our findings suggested that 5-HT and melatonin plays an important role in human megakaryopoiesis. Interaction of 5-HT and 5-HTR2B induced downstream activation of PI3-k/Akt signal pathway leading to human MK cell proliferation. In addition, activation of 5-HTR2B also induced Erk1/2 phosphorylation, which then promoted cytoskeleton reorganization and subsequent proplatelet formation. We also proved that melatonin exerts a protective effect on MK and platelets in the irradiation mice model.

Disclosures:

No relevant conflicts of interest to declare.
The Role Of Mycophenolate Mofetil In Patients With Refractory ITP
Authors: Atmakusuma Tubagus Djumhana , Hardi Fransiska
Published: October 2019

Background

Referring to the ASH 2011 Guideline for ITP, several immune suppression agents are considered off-label use due to insufficient data for any specific agent to be recommended. In Indonesia mycophenolate mofetil (MMF) has been used as an alternative in ITP patients who failed to any recommended treatment in the ASH 2011 Guideline.

Methods

A retrospective study has been conducted to review data of ITP patients in three hospitals in Jakarta Indonesia. All ITP patients have been excluded from any potential causes of thrombocytopenia, including anamnesis, physical examination and laboratory tests for SLE, hepatitis C, hepatitis B and HIV.

Results

52 ITP patients have been assessed which comprised 38 female (73,1 %) and 14 male (26.9%) with mean age 36.31 years old (ranged between 18 to 67 years old).13 of 52 patients have received MMF. 8 of 13 patients received MMF after failure to prednisone, 3 of 13 patients received MMF after failure to prednisone and azathioprine, 1 of 13 patients received MMF after failure to prednisone, then received thrombopoietin receptor agonist. Mean platelet count at diagnosed was 28.211/µL (ranged between 1,000 to 86,000/µL).

6 of 13 patients achieved good response (increased platelet count > 100,000/µ) and 3 of 13 patients achieved intermediate response (increased platelet count > 30,000 to < 100,000/µL). 4 other patients had no response to MMF (platelet count remained < 30,000/µL) in whom 1 patient has previously received prednisone, followed by MMF, 1 patient has previously received prednisone, then azathioprine, followed by MMF, 1 patient has previously received prednisone, then azathioprine , subsequently MMF followed by vincristine, and 1 patient has previously received prednisone, then azathiprone, subsequently MMF followed by eltrombopage.

Conclusion

9 of 13 patients (almost 70 %) have responded to MMF. The remaining 30 % had no response to MMF.

Disclosures:

Off Label Use: Mycophenolate mofetil.
Long-Term Outcomes Of Splenectomy-Sparing Therapies For Immune Thrombocytopenia
Authors: Varma Mala , Grossbard Michael L.
Published: October 2019

Background

Anti-Rh(D) (Cooper Blood 2002), rituximab (Godeau Blood 2008), and dapsone (Sharma ASH 2006) have been shown to be splenectomy-sparing agents for the treatment of immune thrombocytopenia (ITP). In these studies, patients treated with anti-Rh(D) and rituximab were followed for 2 years; patients were treated with dapsone for 6 to 48 months.

Specific Aims

To determine long-term outcomes in unsplenectomized patients with ITP treated with anti-Rh(D), rituximab, or dapsone.

Methods

Institutional Review Board approval was obtained. The charts of 52 consecutive patients with ITP followed in a hematology practice between 07/01/03 and 06/30/13 were reviewed. Response was defined as a platelet count (PLT) > 100,000/µL. Relapse was defined as PLT < 30,000/µL.

Results

Twenty-one patients have been treated with anti-Rh(D), rituximab, or dapsone. Some patients treated with anti-Rh(D) or rituximab received brief concurrent steroids. For the entire cohort the mean duration of ITP was 3.50 years (y), range 0.008 to 19 y and the mean duration of follow-up was 3.84 y, range 0.10 to 8.81 y. Among 16 patients who received anti-Rh(D), 85% responded to the initial infusion. Among the 10 responders who have had follow-up exceeding 1 year, 4 have had a sustained response following the first infusion (mean follow-up duration 3.35 y, range 2.66 y to 5.98 y; six have relapsed, all have responded to retreatment. Among 7 patients who received rituximab, 71% responded to the initial treatment. All 5 responders have had follow-up exceeding 1 year, among whom 2 have had a sustained response following the first infusion (mean follow-up duration 2.24 y, range 2.19 y to 6.67 y); three have relapsed, all have responded to retreatment. Among 3 patients who received dapsone, 66.7% responded; the response is partial in 1 patient with PLT 50,000/µL (mean follow-up duration 3.94 y, range 0.11 y to 8 y). Toxicities have included: with anti-Rh(D) 1 case each of grade II uterine pain, grade II chills, and grade II non-cardiac chest pain; with rituximab 1 case of grade II back pain. Therapeutic switches have included 2 cases of anti-Rh(D) to rituximab and 1 case each of anti-Rh(D) to rituximab to dapsone, anti-Rh(D) to rituximab to dexamethasone, anti-Rh(D) to romiplostim, and dapsone to rituximab. No patients have undergone splenectomy.

Conclusion

Anti-Rh(D), rituximab, and dapsone are splenectomy-sparing therapies for ITP over an extended period. For patients treated with anti-Rh(D) or rituximab who relapse, retreatment is effective.

Disclosures:

Off Label Use: Rituximab and dapsone therapies of ITP.
Effect Of Purified Poloxamer 188 and Various Dextrans On Erythrocyte Sedimentation Rate In Healthy Subjects and Patients With Sickle Cell Disease
Authors: Hoppensteadt Debra , Emanuele Martin , Molnar Joann , Iqbal Omer , Fareed Jawed
Published: October 2019

Introduction

Purified poloxamer188 (P188) (Mast Therapeutics) is a non-ionic, linear block copolymer composed of a central chain of hydrophobic polyoxypropylene and two flanking chains of hydrophylic polyoxyethylene (MW 8.5 kDa). This agent has hemorheologic properties which result in improved microvascular blood flow. P188 has been investigated in a number of indications and is currently under study in an international phase 3 clinical trial in sickle cell patients with vaso-occlusive crisis. Dextrans represent branched polysaccharides of 10-70 kDa that have been used as antithrombotic agents and plasma expanders. Sickle cell disease (SCD) represents a complex hemorheologic condition due to RBC aggregation and cell-fibrin/fibrinogen interactions. The erythrocyte sedimentation rate (ESR) is reflective of RBC and plasma interactions. This study was designed to compare the effect of P188 and dextrans on ESR’s in blood obtained from healthy subjects and patients with sickle cell disease who were seen at Loyola University Medical Center clinics.

Material and Methods

Whole EDTA blood collected from normal individuals (n=8) and sickle cell patients confirmed by electrophoresis (n=11) were supplemented with P188 or dextran 10K, 18K , 40K and 70K at various concentrations (or saline control). ESR was measured using standard laboratory technique.

Results

The ESR’s for sickle cell patients (26.4 ± 7.1 mm/hr) were significantly higher in comparison to the ESR’s for healthy subjects (14.6 ± 2.1 mm/hr). Supplementation of P188 decreased ESR’s in both populations. Normal blood ESR’s decreased to 9.1 ± 1.3 mm/hr (38%), whereas the sickle cell patient values decreased to 14.1 ± 4.6 mm/hr (47%).  At comparable concentrations, none of the dextrans changed ESR’s in healthy subjects or patients with sickle cell disease.

Discussion

These results demonstrate that ESR in SCD patients are elevated compared to healthy subjects. P188 supplementation decreased (up to 50%) ESR’s in both the healthy subjects and sickle cell patients. This may be due to the inhibition of rouleaux formation resulting from P188 effects on RBC membranes or cell-protein interactions. None of the dextrans produced a similar decrease, suggesting that the observed lowering of ESR by P188 is unlikely to be due to a non-specific effect related to polymer molecular weight.

Clinical Implications

P188 is a potential therapeutic agent which may facilitate blood flow and reduce cell-fibrin/fibrinogen interactions in a variety of hemorrheologic disorders. The observed decrease in ESR both in normal and sickle cell blood samples by P188 may primarily be due to increased membrane hydration, fibrinogen dispersion and anti-adhesive effects of this agent.

Disclosures:

Emanuele: Mast Therapeutics: Employment. Fareed:Mast Therapeutics: Research Funding.
Decreased Levels Of Procoagulant Phospholipids In Bleeding Patients With Overcoagulation By Vitamin K Antagonists
Authors: Van Dreden Patrick , François Dominique , Mathieu Emmanuel , Grusse Matthieu , Vasse Marc
Published: October 2019

Background

The major adverse effect of vitamin K antagonists (VKA) is the increased risk for bleeding complications. In addition to well-identified risk factors such as age, prior gastrointestinal tract bleeding, or hypertension, the intensity of anticoagulation evaluated by the International Normalized Ratio (INR) measurement is a major determinant of VKA-induced bleeding. However, for the same degree of VKA overcoagulation and comorbidities, some patient will bleed, whereas others remain asymptomatic. Therefore, identifying specific biological markers that identify patients at high risk of bleeding would have great clinical impact. Microparticles, derived from different cellular origins (endothelium, red blood cells, leukocytes, platelets or apoptotic tissues), can be detected in plasma and express procoagulant phospholipids (PPL). The presence of PPL has been associated with various diseases complicated by an hypercoagulable state. Therefore, we hypothesize that the procoagulant activity of PPL could protect against haemorrhage in patients with VKA overcoagulation.

Patients and methods

53 consecutive patients who were referred to the emergency department of our institution and with an INR > 5 were enrolled in the study: 22 (10 females; 12 males, median age 82 years) were symptomatic (20 cases of minor bleeding, 2 cases of non-fatal major bleeding), whereas 31 (18 females, 13 males, median age 78 years) were asymptomatic. Median INR was 7.36 (range: 5 – 22.6) and 6.3 (range: 5 – 10.7) in symptomatic and asymptomatic patients, respectively (p = 0.17, not significant). PPL were evaluated using a factor Xa-based coagulation assay (STA-Procoag-PPL, Diagnostica Stago) in which shortened clotting times are associated with increased levels of PPL. We also quantified thrombomodulin (TM) by an ELISA assay (Asserachrom Thrombomodulin, Diagnostica Stago) and by a functional assay based on the ability of TM to activate Protein C in the presence of thrombin, since high plasma levels of TM were previously identified as a predictor of bleeding complications.

Results

Clotting times were significantly lower in asymptomatic patients than in bleeding patients [respective median values 36.5 seconds (range: 27.1 – 72.2) and 47.2 seconds (range : 30.5 – 72.8); p = 0.03. In contrast, there were no significant differences for TM levels, whatever the assay used (functional or immunological).

Conclusion

Increased PPL could contribute to decrease the haemorrhagic risk of patients treated by VKA. It is not clear if the decrease of PPL is directly responsible of the hemorrhagic syndrom, or if PPL are decreased because of a consumption during the hemorrhagic episode. In order to answer this question, it could be of interest to analyse if a prospective follow-up of this parameter could help to identify patients with an increased hemorrhagic risk when treated by VKA.

Disclosures:

No relevant conflicts of interest to declare.
Effect Of Recombinant Human Soluble Thrombomodulin On Clinical Outcomes Of patients With Coagulopathy After Hematopoietic Stem Cell Transplantation
Authors: Chi SungGi , Ikezoe Takayuki , Takeuchi Asako , Anabuki Kazuki , Kim Tsukie , Sakai Mizu , Taniguchi Ayuko , Togitani Kazuto , Yokoyama Akihito
Published: October 2019

Recombinant human soluble thrombomodulin (rTM) comprises the active, extracellular domain of thrombomodulin (TM), and inactivates coagulation by binding to thrombin. In addition, the thrombin-rTM complex activates protein C to produce activated protein C (APC), which inactivates factors VIIIa and Va in the presence of protein S, further inhibiting thrombin formation. The use of rTM for the treatment of DIC was approved in Japan in in 2008, and since then it has proved effective in individuals with DIC complicated by a variety of underling diseases including sepsis and acute leukemia. Of note, rTM possesses anti-inflammatory and cytoprotective effects and the use of rTM successfully rescued individuals with sinusoidal obstruction syndrome, engraftment syndrome and transplantation-associated microangiopathy complicated by hematopoietic stem cell transplantation (HSCT). The present study retrospectively investigated whether the use of rTM improved the clinical outcome of individuals who received HSCT in our institution.

From 2001 to 2012, 71 individuals with hematological diseases received HSCT in our institution. Of these, 41 developed coagulopathy in association with various underlying conditions. The patients who developed coagulopathy after 2008 (n=23) were treated by rTM, and the others (n=11) were treated by either heparin and/or antithrombin III concentrate. Seven patients did not receive any anticoagulant therapy. Of note, treatment of coagulopathy by rTM significantly improved clinical outcomes of patients at day 100 and dramatically prolonged their overall survival (p=0.044). Taken together, rTM is useful to improve clinical outcomes of transplant recipients with coagulopathy.

Disclosures:

No relevant conflicts of interest to declare.
Platelet RNA-Seq Identifies Genes Expressed Differentially After Clopidogrel Treatment
Authors: Prakasa Kalpana Rao , Becker Lewis , Faraday Nauder , Becker Diane , Qayyum Rehan
Published: October 2019

Background

Clopidogrel is an antiplatelet agent that acts through direct inhibition of ADP-dependent platelet activation. Several studies have also found an inhibitory effect of clopidogrel on inflammation. Although platelets are known to participate in inflammatory processes, the mechanism(s) through which clopidogrel might modify platelet-mediated inflammation is unknown. We examined the platelet transcriptome to gain insight into the mechanism(s) through which clopidogrel modulates inflammation.

Methods

Three individuals with subclinical atherosclerosis and family history of early onset coronary artery disease were enrolled. Blood was collected for RNA-seq at baseline and one-week after clopidogrel 75mg daily. RNA was extracted from leukocyte-depleted platelet-rich plasma and approximately 70 million 100bp paired-end reads per sample were obtained using the Illumina HiSeq 2500. RNA-seq analysis was performed using Bowtie/TopHat/Cufflinks software pipeline. Genes with expression ≥ 0.3 fragments per kilobase of transcript per million reads (FPKM) were considered to be expressed. We set the threshold for significant differential expression as ≥ 2-fold change in gene expression after clopidogrel therapy with a false discovery rate of 0.05.

Results

The study sample consisted of 2 women and 1 man. Among the 12,297 expressed genes, the most highly expressed nonubiquitous genes included PPBP, PF4, and TUBB1, confirming platelets as the source of the RNA. Comparing samples before and after clopidogrel, treatment was associated with up-regulation of 733 genes and down-regulation of 387 genes. Pathway analyses found that genes related to platelet activation, degranulation, and aggregation were up-regulated, while genes related to immune system processes, such as B- and T-cell activation and innate immune response, were down-regulated. Examples of down-regulated genes include the toll-like receptor family genes, TLR2, TLR4, TLR7, and TLR8, the chemokine receptor genes CCR2, CCR5,CCR6, and CCR7, and the interleukin receptor genes, IL4R, IL7R, and IL13RA.

Conclusion

One week of clopidogrel therapy up-regulates transcription of pro-thrombotic platelet genes and down regulates transcription of inflammation-related platelet genes. These data suggest that clopidogrel may exert a direct anti-inflammatory action by inhibiting the ability of platelets to participate in inflammatory processes. The relationship of variability in clopidogrel-induced modulation of these pathways to clinical outcomes is unknown.

Disclosures:

No relevant conflicts of interest to declare.
UV-C Treatment Of Intermediates Of Biologicals To Inactivate Viruses Without Denaturing Desired Protein
Authors: Groener Albrecht , Dreier Christine
Published: October 2019

Dedicated virus reduction steps implemented in the manufacturing process of biologicals, either isolated from human plasma or produced as recombinant proteins, are essential safety measures to assure that a potential virus contamination of the source material will not be transmitted to patients requiring these therapeutic proteins.

Currently applied virus reduction steps as solvent/detergent treatment and virus filtration are very effective virus reduction methods with inherent method-dependent gaps regarding the reduction capacity for a very wide range of viruses of diverse physico-chemical characteristics: solvent/detergent treatment does not inactivate non-enveloped viruses and, depending on the pore size of the virus filter, small viruses are not removed when the desired protein is large and has to pass the filter. Therefore, another virus inactivation method was studied which is considered especially effective for small viruses: UV-C treatment using the UVivatec system provided by Sartorius Stedim Biotech GmbH, Göttingen, Germany.

Experiments were performed to study the impact of UV-C treatment on the integrity of proteins employing fibrinogen as an example for a large protein and on the inactivation capacity for poliovirus (a small non-enveloped virus). The integrity of fibrinogen was assessed by comparing the untreated fibrinogen with the UV-C treated fibrinogen using HPLC, Clauss assay and thromboelastometry. Virus inactivation was studied in a bioassay using a sensitive cell culture infectivity assay employing a cynomolgus cell line.

The results show that UV-C treatment inactivates viruses and modifies fibrinogen in a dose dependent manner; the monomer, dimer and polymer peak in the fibrinogen preparation studied changed from approx. 75% to 60%, 17% to 25% and 8% to 13%, respectively, at a UV-C intensity of 400 J/m² demonstrated by HPLC measurement. In order to protect fibrinogen from modifications, the antioxidant glutathione was added to the fibrinogen preparation. At an UV-C intensity of approx. 300 J/m², sufficient to effectively inactivate viruses studied, a modification of fibrinogen was not any longer detectable.

Disclosures:

Groener: CSL Behring: Employment.
Are We Appropriately Using Intravenous Immunoglobulin In The Saskatoon Health Region?
Authors: Manna Mita , Karim Safiya , Brose Kelsey , Dallas Karen L
Published: October 2019

Intravenous Immunoglobulin (IVIG) is a plasma protein used in the treatment of various diseases. The cost of IVIG is approximately $50-$80 per gram. In 2005, Saskatchewan's usage of IVIG was above the national average, at 100g per 1000 persons compared to 95g per 1000 persons. IVIG is currently licensed by Health Canada for use in six diseases; however, IVIG is often used for off-label indications. There are several published guidelines that outline recommended uses of IVIG. These are largely based on case studies and expert opinion. Despite these guidelines, IVIG is often used in multiple settings where there is no supporting evidence.

We aimed to evaluate adherence to published guidelines by quantifying IVIG usage in the Saskatoon Health Region (SHR). We conducted a retrospective chart review of patients who received IVIG in 2011 and 2012, classifying the usage based on diagnosis and prescribing specialty. Our primary endpoint was the percentage of appropriate use of IVIG in comparison to published guidelines. Our secondary endpoint was adverse reactions seen in patients receiving IVIG.

In 2011, 227 patients received IVIG totalling 50,528g and 2.78 million dollars. In 2012, 216 patients received IVIG totalling 63,155g and 3.49 million dollars. According to published guidelines, 36% of the total usage in 2011 and 21% of the total usage in 2012 was not indicated (Figure 1). IVIG was prescribed the most by neurologists, hematologists, and rheumatologists (Figure 2). Despite the recent guidelines, the results show that there was no significant improvement in usage of IVIG between 2011 and 2012 (p-value 0.079, odds ratio 1.46). No adverse reactions were documented.

In conclusion, our data analysis shows that IVIG usage in the SHR does not adhere well to guidelines. Pre-printed order sets outlining appropriate indications for IVIG are currently being introduced in the SHR to help improve adherence to guidelines. After the order sets have been implemented for one year, we will re-evaluate the usage of IVIG in the SHR to see if improvements have been made. Our goal is to ensure there is appropriate resource allocation of IVIG and to reduce risks to the patient and financial costs to the health care system.

Disclosures:

No relevant conflicts of interest to declare.
A New Method For Electronic Crossmatch: ABO /Rh Blood Group Confirmation and Antibody Screening Concomitantly With Serologic Crossmatch
Authors: Demirkan Fatih , Gunal Veli , Dereli Yasar
Published: October 2019

Introduction

Altough electronic crossmatch (E-XM) is cost effective and reduce laboratory workload, practical difficulties of determining 2 consecutive ABO/Rh in patients, requirement for the verification of the donor ABO group, concerns for errors in patient identification/specimen labeling and lack of reimbursement for performing ABO confirmation testing on a second separately collected specimen could avoid its widespread implementation in some countries.

Method

Using gel santrifuge method, ABO/Rh grouping and immuno-hematologic tests are done by a fully automatic system and recorded in blood bank information system. For patients and donors who do not have a previous blood group and antibody screen in the system, study steps are: 1. ABO/Rh group is performed with the 1st specimen and recorded 2. Donor blood group and antibody screen are done in the same gel card. 3. A second specimen is required at the time of transfusion request for regular antiglobulin crossmatch and antibody screen. ABO confirmation of patient and donor is done concomitantly in the same card with XM. 4. When 2 or more units are requested, first unit is selected by serologic XM, subsequent units are selected by E-XM from the available donor pool. Donor pool for electronic selection is fed by serologically compatible but undelivered units. Computer system is capable of questioning availability for E-XM eventhough serologic XM is not performed and could show the missing parameters needed for E-XM.

Results

After 6 months of validation process, between 1.01.2012- 31.07.2013, 36.7% of erythrocytes were reserved by E-XM using this method (table 1). Evaluation of the efficacy of the XM process revealed that 37.5% of the serologically confirmed units were undelivered and those supported the pool for E-XM (table 2).

Conclusions

1. This method implements a double specimen system for ABO/Rh verification to ensure the transfusion security and guarantees the selection of at least 1 unit serologically. 2. Reduces work load and cost effective even in centers who are currently using serologic XM.

Disclosures:

No relevant conflicts of interest to declare.
Differentiation in B-precursor acute lymphoblastic leukemia cell populations with CD34-positive subpopulations
Authors: Re GG , Estrov Z , Antoun GR , Felix EA , Pinkel DP , Zipf TF
Published: October 2019

B-precursor acute lymphoblastic leukemia bone marrow specimens that contained subpopulations of cells with immunophenotypes corresponding to early (CD34) and late (CD20) and (CD22) stages of normal B-cell differentiation were studied. Subpopulations of cells were isolated according to immunophenotype and then analyzed by both a clonogenic assay and molecular genetic methods. Clonal equivalence of the early and late immunophenotypic subpopulations was confirmed for each case by the demonstration of identical lg gene rearrangements. The in vitro colony-forming assay consistently showed a growth advantage for the CD34+ subpopulations over the CD34- subpopulations. CD34 mRNA was detected readily in these isolated precursor cells. When two specimens in which virtually all of the leukemia cells were CD34+ and CD34+CD20+ and CD34+CD22+ subpopulations were also present the CD34 mRNA was limited to the cells without the late-stage differentiation antigens on their surface. Furthermore, the c-myb mRNA was found only in the subpopulations that also contained CD34 mRNA. Our results show that a limited program of differentiation reminiscent of normal B-cell development may be present in this leukemia.
Differential regulation of primitive human hematopoietic cells in long- term cultures maintained on genetically engineered murine stromal cells
Authors: Sutherland HJ , Eaves CJ , Lansdorp PM , Thacker JD , Hogge DE
Published: October 2019

Various growth factors are known to stimulate both early and late stages of human hematopoietic cell development in semisolid assay systems, but their role as microenvironmental regulators is poorly understood. To address this problem, we developed a novel coculture system in which highly purified primitive human hematopoietic cells were seeded onto an irradiated feeder layer of cells from a murine marrow-derived stromal cell line (M2–10B4) previously engineered by retroviral-mediated gene transfer to produce specific human factors. Effects on cells at very early, intermediate, and late stages of hematopoiesis were then evaluated by assessing the number of clonogenic cell precursors (long-term culture initiating cells [LTC-IC]), clonogenic cells, and mature granulocyte and macrophage progeny present in the cultures after 5 weeks. In the absence of any feeders, cells at all stages of hematopoiesis decreased to very low levels. In contrast, maintenance of LTC-IC was found to be supported by control murine stromal cells as effectively as by standard human marrow adherent layers. The presence of granulocyte colony-stimulating factor (G-CSF) and interleukin-3-producing M2–10B4 cells in combination was able to further enhance the maintenance and early differentiation of these cells without a decline in their proliferative potential as measured by the clonogenic output per LTC-IC. However, this effect was lost if granulocyte-macrophage CSF (GM-CSF)-producing feeders were also present. On the other hand, in the presence of GM-CSF-producing feeders, the output of mature granulocytes and macrophages increased 20- fold. These findings show that it is possible to selectively improve the maintenance of very primitive human hematopoietic cells in vitro or their output of mature progeny by appropriate manipulation of the long- term marrow culture system. Further exploitation of this approach should facilitate investigation of the mechanisms operative within the human marrow microenvironment in vivo and the design of protocols for in vitro manipulation of human marrow for future therapeutic applications.