bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reticulocyte Folate Concentration: A Tool To Monitor Immediate Folate Availability
Authors: Miller Peter J. , Agnew Elizabeth B. , Knovich Mary Ann , Farland Andrew M. , Waldron Johanna , Owen John 
Published: October 2019

The utilization of folic acid during erythropoiesis is widely known and accepted in medical literature and deficiency is known to cause a characteristic megaloblastic anemia resulting from the inhibition or ineffective synthesis of DNA. Although the resultant megaloblastic anemia may take considerable time before evidence or symptoms present, there are more acute changes visualized on peripheral smear that are representative of a functional folate deficiency. In addition to erythrocyte macrocytosis, hypersegmentation of neutrophils can also be seen. Often times, despite these visualized changes measurement of serum folate and/or total red cell folate yields a result within the accepted “normal” range of the assay. It has been demonstrated that these visualized characteristics represent a functional folate deficiency and can be overcome with folic acid supplementation regardless of the measured folic acid levels indicating a yet to be understood mechanism of folate utilization. Here, we sought to measure the folic acid levels in the earliest erythrocyte progenitors in peripheral circulation, the reticulocytes.

Methods
Reticulocytes were isolated using anti-CD71 (the transferrin receptor) coated magnetic beads. After separation, a sample slide was made utilizing methylene blue for visual confirmation of reticulocyte isolation. The reticulocytes were then lysed with citric acid and a Nanodrop-1000 spectrophotometer was used to determine absorbance at 413 nanometers. This absorbance was used to determine the sample hemoglobin concentration from a simple calibration curve. The sample folic acid level was then determined using the lysing method utilizing mouse monoclonal anti-folate binding protein, paramagnetic particles coated in anti-mouse IgG, human serum albumin and milk folate binding protein. Results were calculated as nanogram of folate per gram of hemoglobin.

Results
Twenty-five samples from normal individuals, not taking folate supplements, were analyzed. The range of results was 2.51 to 17.38 with a mean level of 9.61.

Conclusion
This protocol effectively and efficiently allows for isolation of reticulocytes in numbers high enough for measurement of folate in nanogram per gram of hemoglobin. By this method we show the normal reticulocyte folate level to be approximately 3-15 ng/g of Hgb. This figure is consistent with the normal red cell folate concentration. Further analysis is planned for comparing these results in patients with a suspected functional folate deficiency but “normal” red cell folate levels. Efficient isolation of reticulocytes and measurement of folate levels will allow us to probe the underlying cause of acute folate deficiency seen in very sick patients.

Disclosures:
No relevant conflicts of interest to declare.
Recovery From Hemoglobin Of 2g/Dl Without Prbc Transfusion: A Lesson Learned From a Jehovah’s Witness With Cold Agglutinin Disease
Authors: Gohari Pouyan , Chandra Abhinav B. , Slatkin Richard , Lipshitz Jay S. 
Published: October 2019

Supportive management of critically ill patients with anemia often involves packed red blood cell transfusion. Jehovah’s Witness’ refusal of blood products because of their religious beliefs can render these situations challenging. Successful adaptation of medical management in Jehovah’s Witness’ with severe anemia provides physicians with a distinct opportunity to question common transfusion practices and learn about anemia. We present a case of a 43 year-old Jehovahs Witness who presented with autoimmune hemolytic anemia – cold agglutinin disease. His hemoglobin nadired at 2g/dL. The patient was successfully managed to recovery with corticosteroids, rituximab, folic acid and an erythropoeitin stimulating agent. To our knowledge this is the lowest Hgb that a patient with life threatening cold agglutinin disease survived without the use of any blood product transfusion. Such cases afford a deeper appreciation of the body's ability to adapt to and recover from anemia without the use of exogenous blood.

Disclosures:
No relevant conflicts of interest to declare.
Systematic Evaluation Of Chronic Organ Damage In Adult Sickle Cell Patients. A Seven-Year Follow-Up Study
Authors: van Tuijn Charlotte F.J. , Schimmel Marein , van Beers Eduard J. , Biemond Bart J. 
Published: October 2019

Introduction
The occurrence of organ damage in sickle cell disease (SCD) is a crucial determinant for both medical treatment and prognosis. In a previous study, we systematically analyzed the prevalence of SCD related organ damage and complications in adult sickle cell patients in a tertiary teaching hospital in the Netherlands. We now describe a seven-year follow-up of this patient cohort, to provide an insight into the course of the various forms of organ damage and SCD related complications.

Methods
At baseline in 2006, 110 adult patients visiting the outpatient clinic of our hospital were enrolled. All enrolled patients from the primary analysis were included for follow-up. Patients were screened for sickle cell related manifestations during visits to the outpatient clinic biannually. Various forms of sickle cell related organ damage and complications (presence of microalbuminuria, renal failure, pulmonary hypertension retinopathy, iron overload, cholelithiasis, avascular osteonecrosis, leg ulcers, acute chest syndrome, stroke, priapism and admissions for painful crises) were routinely screened according to international guidelines. Patients with genotype HbSS/HbSβ0 and HbSC/HbSβ+were grouped for further analysis.

Results
Of all originally included patients (N=110), nine were lost to follow-up (N=9). The mean age of the current study cohort is 37 years (IQR 27-46). Overall, 59 patients (58%) developed a new form of organ damage or new complication since baseline analysis, including eight patients who deceased (7 due to a sickle cell disease related death). In the HbSS/HbSβ0 genotype group (N=60) we found an increase in the percentage of patients who have had an Acute Chest Syndrome (29% to 47%) or have been diagnosed with avascular osteonecrosis (15% to 24%), retinopathy (23% to 34%) or pulmonary hypertension (31% to 48%). In the HbSC/HbSβ+ (N=35) group we found an increase in the occurrence of avascular osteonecrosis (9% to 14%), retinopathy (59 % to 70%) and pulmonary hypertension (9% to 19%). Furthermore, the use of hydroxycarbamide increased in both genotype groups and the frequency of admissions for painful crises remained stable for both genotype groups.

Conclusion
In the past period of seven years 58% of the patients in a previously well descript cohort of adult SCD patients developed a new sickle cell related complication. For some forms of organ damage or complications a substantial increase occurred dependent of a patient’s genotype.

Disclosures:
No relevant conflicts of interest to declare.
Thrombosis Risk Factors Assessment In Children Treated For Acute Lymphoblastic Leukemia
Authors: Smalisz-Skrzypczyk Katarzyna , Klukowska Anna , Pawelec Katarzyna , Matysiak Michal 
Published: October 2019

The aim of the study was to evaluate factors predisposing to thrombosis in children treated for acute lymphoblastic leukemia according to ALL IC BFM 2002.
An analysis of 30 cases of thrombosis in 210 children (14%) treated due to ALL in the Department in years 2007-2011 was carried out. Age at onset, location, cause and treatment methods were taken into consideration. All analyzed patients underwent screening for congenital thrombophilia.
The age of patients ranged from 1.5 to 16 years with the median of 7.5 years. Arterial thrombosis occurred in 1 person (3%), venous thrombosis in 29 (97%) patients. Thromboembolism was usually related to the central venous catheter (n = 27, 90%). Massive thrombosis occurred in 6 patients (20%). Relapses occurred in 2 children.
The most common causes of thrombosis were: the presence of the central catheter (n = 27, 90%) and L-asparaginase treatment (n = 16, 53%). The factor V Leiden mutation was diagnosed in 1 patient, protein C deficiency in 1 patient as well, and elevated levels of factor VIII were detected in 3 cases.
Five children (16%) underwent systemic thrombolysis with recombinant tissue plasminogen activator and 17 patients (52%) had local thrombolysis. Anticoagulation with warfarin following low-molecular-weight heparin discontinuation was used in 6 patients (20%). The complete resolution of the thrombus was observed in the whole group of patients.

Conclusions
1. A risk factor for thrombosis in children with ALL is the treatment of L-asparaginase and the presence of central venous catheter.
2. Screening for congenital thrombophilia in children treated for ALL should not be done routinely since the coexistence of this two conditions is rare.
Thrombosis in children with ALL responds well to treatment.

Disclosures:
Klukowska: Octapharma AG: Investigator Other.
Convergent Squint- a Symptom Of Thrombosis
Authors: Laguna Pawel , Szymanska Krystyna , Milewska Kamila , Sawionek Iwona , Pawelec Katarzyna , Matysiak Michal 
Published: October 2019

Anticoagulative treatment of children has always presented problems due to a lack of generally approved clinical standards. We present the case of a 5 -year-old boy with bilateral convergent strabismus. It was preceded by otitis, which was treated with antibiotics for a few days before admission to hospital. In the last two days before admission his condition worsened, he complained of a headache and started vomiting. In neurological examination on the second day of his stay, the boy presented with decreased spontaneous activity and hypersomnia. This muscle tone, reflexes were decreased and he had, poor motor coordination. There were no abnormal reflexes and cerebellar testing showed no aberrations. There was 1 cm nuchal rigidity. Examination of cranial nerves showed sixth nerve palsy on both sides. Ophtalmological examination revealed bilateral papilledema. Neuroimaging showed thrombosis of the transverse and sigmoid sinus. A lumbar puncture was not performed because anticoagulative treatment, had been started. The final diagnosis was: bilateral sixth nerve palsy due to sinovenosous thrombosis as a complication of otitis in a patient with a genetic disorder The patient received anticoagulative, antiinflammatory, antibiotic and anti - edemal treatment. He responded to therapy, showing clinical improvement followed by normalisation in neuroimaging. On examination after 1.5 months his general condition was good. Motor coordination was poor and muscle tone was lowered as before. His comprehension was good. Speech was limited to the first syllables of words, as before. He had convergent strabismus in his left eye, without sixth nerve palsy symptoms. Movements of the right eye were normal. In thrombophilic investigation the presence of factor V Leiden mutation was found. The boy is now in outpatient care. He is undergoing vision therapy and speech therapy. Convergent squint can be a symptom of thrombosis in children. Neuroimaging is essential in diagnosis.

Disclosures:
No relevant conflicts of interest to declare.
Rapid and Effective Isolation Of (T) Cell Populations By a New Method Avoiding Magnetic Beads
Authors: Stadler Herbert , Przibilla Sabine , Dreher Stefan , Stemberger Christian , Germeroth Lothar , Busch Dirk 
Published: October 2019

In the last two decades, conventional isolation of therapeutic cells has been carried out using high affinity antibodies and magnetic bead technology. Although good purity and reasonable yields can be obtained in many cases, major disadvantages remain comprising biological interference of non-reversible selection reagents (e.g. stimulation, receptor blockade etc.), the difficulty to purify complex multi-parametric cell populations by positive selection, time consuming protocols as well as limitations in high-throughput processing. We report here on the development of a new non-magnetic and fast cell selection technology applying immune affinity chromatography. Therefore, a matrix consisting of beads coated with Streptactin and low-affinity recombinant Fab-fragments (Fab-Streptamers) directed against defined T cell surface antigens has been generated. Stable and specific target cell retention is achieved by passing whole blood or PBMCs over the affinity matrix. After binding and washing, target cells can be gently retrieved by D-biotin administration. Eluted cells are then passed over a second matrix removing D-biotin and free Fab-Streptamers, subsequently yielding in a label-free authentic cell population for further use. Most importantly, total cell processing times can be kept extremely short; depending on the sample size even down to several minutes. Sequential isolation steps are possible and allow positive selection of complex cell populations like regulatory T cells or central memory T cells defined by several markers in high yields and purities. We are currently integrating this approach into a fully closed separation device for clinical cell purification. In addition, high throughput cell selection for diagnostic or basic research applications can be achieved by embedding the matrix into pipette tips and the use of suitable pipetting robots.

Disclosures:
No relevant conflicts of interest to declare.
Non-Irradiation Approach To Study Hematopoiesis and Leukemogenesis In a De Novo Murine Model Of Hematopoietic/Leukemia Stem Cell Transplant
Authors: Liu Xiaofei , Yang Dan , Fang Shumin , Geng Yang , Zhao Qianhao , Yang Pengjie , Peng Siying , Shen Yuelei , Pan Guangjin , Wang Jinyong 
Published: October 2019

Abstract
Traditional mouse models for hematopoietic stem cell transplant (HSCT) require whole body irradiation to ablate the hematopoietic cells in recipients, with the defect of disturbing non-hematopoietic cells and introducing potential tumorgenesis in a nonautonomous manner. Here, we use a novel approach to produce mice that whole hematopoietic-specific ablation can be conditionally achieved without global body irradiation. Briefly, a Diphtheria toxin receptor (DTR)-GFP reporter element was targeted into the ROSA26 locus to produce DTR-GFP reporter mice, with a loxp-stop-loxp cassette. Then the DTR-GFP reporter mice were crossed to Vav-Cre mice to produce double transgenic mice (DTR-GFP mice). We injected DT to ablate the bone marrow cells from DTR-GFP mice, and transplant WT bone marrow cells into them. Our data showed that all hematopoietic cells including hematopoietic stem cells, Myeloid, lymphoid lineages are GFP positive in DTR-GFP mice. A single dose of DT can kill all the hematopoietic cells from DTR-GFP mice. One month later, WT bone marrow cells were successfully engrafted into the DTR-GFP recipients without irradiation. We are using this model to re-evaluate some leukemia models that irradiated bone marrow niches might be involved in the tumorigenesis.Thus, we establish a de novel HSCT approach without irradiated myeloablation, which will benefit studies of hematopoiesis, leukemogenesis, hematopoietic stem cell niche, as well as other types of tissue transplants that need ablation of recipient hematopoietic system or immune system.

Disclosures:
Peng: Biocytogen: Employment, Membership on an entity’s Board of Directors or advisory committees. Shen:Biocytogen: Employment, Membership on an entity’s Board of Directors or advisory committees.
GPI-80 Defines Self-Renewal Ability In Hematopoietic Stem Cells During Human Development
Authors: Prashad Sacha L. , Calvanese Vincenzo , Yao Catherine , Kaiser Joshua , Sasidharan Rajkumar , Magnusson Mattias , Mikkola Hanna K.A 
Published: October 2019

Abstract
Advances in pluripotent stem cell and reprogramming technologies have provided hope of generating transplantable hematopoietic stem cells (HSC) in culture. However, better understanding of the identity and regulatory mechanisms that define the self-renewing HSC during human development is required. We discovered that the glycophosphatidylinositol-anchored surface protein GPI-80 (Vanin-2), previously implicated in neutrophil diapedesis, distinguishes a functionally distinct subpopulation of human fetal hematopoietic stem and progenitor cells (HSPC) that possess self-renewal ability. CD34+CD90+CD38-GPI80+ HSPCs were the only population that could maintain proliferative potential and undifferentiated state in co-culture on supportive stroma, and displayed engraftment potential in sublethally irradiated NSG mice. GPI-80 expression also enabled tracking of human HSC during development as they migrate across fetal hematopoietic niches, including early fetal liver and bone marrow. Microarray analysis comparing CD34+CD90+CD38-GPI80+ HSPC to their immediate progeny (CD34+CD90+CD38-GPI80-) identified novel candidate self-renewal regulators. Knockdown of GPI80, or the top enriched transcripts encoding surface proteins (ITGAM) or transcription factors (HIF3a) documented the necessity of all three molecules in sustaining human fetal HSC self-renewal. These findings provide new insights to the poorly understood regulation of human HSC development and suggest that human fetal HSCs utilize common mechanisms with leukocytes to enable cell-cell interactions critical for HSC self-renewal.

Disclosures:
No relevant conflicts of interest to declare.
Characteristics Of Paroxysmal Nocturnal Hemoglobinuria Clones In Colombian Patients
Authors: Londoño Mónica , Arenas-Mantilla Mario , Henao-Uribe Alicia Maria , Novoa Ricardo , Naranjo Andrea , Bedón Fernanda 
Published: October 2019

Abstract

Introduction
Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder, characterized by the clonal expansion of hematopoietic stem cells lacking glycosylphosphatidylinositol-anchored proteins. PNH clones are frequently found in patients with aplastic anemia (AA), myelodysplastic syndrome (MDS), unusual site thrombosis and non-inmume hemolysis. In Colombia, the DECF laboratory analyses blood samples from all over the country, summited by the APEC (association of patients with complement diseases) for PNH screening.

Methods
We reviewed the results of flow cytometry (FC) analyses from 1448 patients screened for PNH in DECF laboratory, between 2010 and 2013, and evaluated the association between clinical characteristics and distribution of PNH clone sizes. Clinical characteristics were considered as referred by treating physicians in the FC analysis request forms. All patients included gave written informed consent.

Results
Mean age of the study population was 44.6±18.5 years and 60.5% were female patients. The most frequent indications for screening were thrombosis (23.5%) and unexplained cytopenias (21.8%). Only 14% of the samples were PNH positive. Table 1 shows the results of FC analysis according to the indications for screening. Median clone sizes were 0.1% (interquartile range (IQR): 0-5.8%) in erythrocytes, 4.2% (IQR: 1.1-53.9%) in granulocytes and 3.8% (IQR: 0.7-64.1%) in monocytes. PNH clone size in granulocytes showed strong correlation with clone size in monocytes (r=0.86, p<0.001) and moderate correlation with clone size in erythrocytes (r=0.65, p=0.001). Figure 1 depicts the distribution of PNH granulocyte clone sizes among indications for screening. In the group of patients with PNH granulocyte clone sizes >5%, those with AA had smaller clone sizes than the rest (Figure 2), while patients with hemolysis showed larger clone sizes than the rest (Figure 3). No significant differences in clone size distribution were found within patients with PNH granulocyte clone sizes ≤5%.

Conclusions
Our findings are comparable to those reported in other countries. PNH granulocyte clone sizes had stronger correlation with PNH monocyte clones than with PNH erythrocyte clones. About one third of the patients with AA and one fourth of those with hemolysis were PNH positive. Over 50% of patients with AA showed PNH clones sizes <5%. Patients with hemolysis have larger clones than the rest. Further studies are needed to establish the association between PNH clone sizes and clinical outcomes of related disorders.

Disclosures:
No relevant conflicts of interest to declare.
Defects In DNA Double-Strand Break Repair and Increased Alternative Non-Homologous End Joining Pathways In Multiple Myeloma: Implications For Genome Stability
Authors: Herrero Ana B , San Miguel Jesus F , Gutierrez Norma 
Published: October 2019

Abstract
Multiple myeloma (MM) is a hematological malignancy characterized by frequent chromosome abnormalities. However, the molecular basis for this genome instability remains unknown. Since DNA rearrangements can be generated thought an improper repair of double strand breaks (DSBs), we investigated the functionality of DSB repair in MM cells. We found that four out of seven MM cell lines analyzed exhibited clear defects in the repair of ionizing radiation (IR)-induced DSBs, revealed by a slow kinetics of g-H2AX disappearance, a prolonged G2/M DNA damage checkpoint activation and an increased sensitivity to IR. An analysis of the proteins that participate in DSB repair revealed no lowered amounts of proteins of the classical NHEJ pathway. However, increased levels of proteins involved in homologous recombination (HR), and in an alternative NHEJ subpathway (Alt-NHEJ) were found in all MM cell lines compared to controls. Interestingly, the Alt-NHEJ protein DNA ligase III was also overexpressed in three out of five samples of plasmatic cells isolated from patients with MM. In vivo assays using a digested plasmid as a substrate revealed slight or non-significant defects in extrachomosomal NHEJ in MM repair-deficient cells, however increased HR was detected by a high percentage of cells with endogenous and IR-induced Rad51 foci. Importantly, the Alt-NHEJ pathway, known to play a role in the generation of deletions and translocations leading to cancer progression, was also found upregulated in MM cells, as revealed by larger deletions and higher sequence microhomology at repair junctions compared to control cells. Taken together, our results uncover aberrant DSB repair in MM. Consequences for genome instability, progression and treatment of the disease will be discussed.

Disclosures:
No relevant conflicts of interest to declare.
NPM-RAR Binding To Tradd Selectively Blocks TNF-Induced Apoptosis While Allowing TNF Activation Of JNK
Authors: Redner Robert L. , Chattopadhyay Anuja , Hood Brian L. , Conrads Thomas P. 
Published: October 2019

Abstract
The t(5;17)(q35;q21) variant of acute promyelocytic leukemia fuses nucleophosmin (NPM) to the retinoic acid receptor alpha (RARA). Expression of the NPM-RAR fusion protein leads to a leukemic phenotype similar to that caused by the t(15;17)(q22;q21) PML-RAR fusion. Using a proteomic approach to identify proteins that bind NPM-RAR, we previously demonstrated that TRADD, amongst other proteins, is a distinct binding partner for NPM-RAR. TRADD serves as an adapter protein that mediates TNF-receptor-mediated activation of the caspase, NFkB, and JNK pathways. We reported that NPM-RAR inhibits TNF-activation of caspase 8 and caspase 3, with subsequent impairment of TNF-induced apoptosis. Extending these findings, we assayed activation of JNK kinase in NPM-RAR-expressing subclones of HELA and U937 cells. We find that TNF activated JNK kinase in NPM-RAR HELA and U937 cells to a similar extent as nontransfected parental controls. This indicates that NPM-RAR binding to TRADD selectively inhibits TNF’s ability to activate caspases, without impairing its ability to activate JNK. In this regard, NPM-RAR effects on TRADD is similar to the EBV oncogenic viral protein LMP1, which binds to the death domain of TRADD, to selectively inhibit caspase activity, while still allowing TNF-mediated activation of survival pathways. Our studies identify a novel mechanism through which NPM-RAR may impact leukemogenesis.

Disclosures:
No relevant conflicts of interest to declare.