bluuebunny/crossref_metadata_2025 huggingface dataset:
ADAMTS13 Expressed In Platelets Offers Systemic Protection Against Arterial Thrombosis and Therapeutic Benefits In Murine Models Of Thrombotic Thrombocytopenic Purpura
Authors: Pickens Brandy , Mao Yingying , Zheng X. Long
Published: October 2019

ADAMTS13, a plasma metalloprotease, cleaves von Willebrand factor (VWF) and inhibits arterial thrombosis and inflammatory response. Deficiency of plasma ADAMTS13, due hereditary mutations in ADAMTS13 gene or autoantibodies against ADAMTS13 protease, results in a potentially fatal syndrome, thrombotic thrombocytopenic purpura (TTP). Plasma infusion or exchange is the only effective therapy available to date. To develop novel therapeutics against TTP, we tested a hypothesis that recombinant ADAMTS13, expressed specifically in platelets, may offer systemic protection against arterial thrombosis and therefore provide therapeutic benefit for TTP. To test this hypothesis, we first generated transgenic mice (JAX B6SJL) carrying a human full-length ADAMTS13 gene under a murine platelet glycoprotein 1b (CD41) promoter. The transgenic mice were then bred with Adamts13-/- (CAST/Ei) mice for >4 generations. By Western blotting and activity assay, we show that a full-length human ADAMTS13 protein (∼195 kDa) and its proteolytic activity toward a FRETS-hVWF73 peptide are detected in platelet lysate obtained from transgenic (rA13-PltTG) mice but not in Adamts13-/- mice or wild-type mice. Little to no ADAMTS13 activity was detected in plasma in transgenic mice, suggesting the expressed human ADAMTS13 is stored inside platelets. ADAMTS13 was releasable upon stimulation with thrombin (1 U/ml), collagen (10 µg/ml), and AYPGKF (0.5 mM). More significantly, rA13-PltTG mice had higher baseline platelet count than Adamts13-/- mice, but exhibited a dramatically reduced rate of thrombus formation in mesenteric arterioles after oxidative injury with 10% ferric chloride as compared with Adamts13-/-mice and wild-type mice. Finally, rA13-PltTG mice were protected from Shigatoxin-2 (Stx-2) or murine recombinant VWF (mVWF)-induced “TTP-like” syndrome, as demonstrated by fewer rA13-PltTG mice having thrombocytopenia (defined by a 40% drop in platelet count from the baseline after challenge with Stx-2 or mVWF), faster and more complete recovery of thrombocytopenia, and significantly higher survival rate than Adamts13-/- mice. In summary, we have generated transgenic mice expressing human ADAMTS13 in platelets. Platelet ADAMTS13 is releasable upon stimulation by agonists and biologically functional in proteolytic cleavage of VWF in vitro. The platelet-derived ADAMTS13 offers systemic protection against arterial thrombosis after oxidative injury and provide a therapeutic benefit to murine models of TTP, resulting from hereditary ADAMTS13 deficiency. We are now in the process testing the efficacy of this strategy as a novel therapeutic for acquired TTP with inhibitors.

Disclosures:

No relevant conflicts of interest to declare.
Increased Functional T Cell Defects in Patients with low CD4 Counts after Allogeneic Hematopoietic Stem Cell Transplantation
Authors: Holtick Udo , Frenzel Lukas P. , Alexander Shimabukuro-Vornhagen , Theurich Sebastian , Claasen Julia , Scheid Christof , von-Bergwelt-Baildon Michael , Hallek Michael , Wendtner Clemens , Chemnitz Jens M.
Published: October 2019

Background

The recovery of the host immune system after allogeneic hematopoietic stem cell transplantation is pivotal to prevent infections, relapse and secondary malignancies. In particular, numerical CD4 T-cell reconstitution is delayed and CD4-helper cell function considered impaired as consequence of the transplant procedure and concommitant immunosuppressive medication. From HIV/AIDS patients it is known that numerical and functional CD4 defects increase the risk of opportunistic infections. Therefore, even in the absence of immunosuppressants and graft-vs-host disease, anti-infective prophylaxis is usually given for at least six months. We hypothesized that the numerical CD4 defect in patients may be reflected by immunosuppressive RNA fingerprints previously established for certain immuno-inhibitory molecules and tested whether the functional CD4 capacity was different according to the CD4 cell number.

Methods

RNA was separated from CD4 T-cells of 10 patients with CD4 counts >500/µl, 10 patients with CD4 counts <200/µl and four healthy controls. All patients had to be off immunosuppression and without any clinical signs of graft-vs-host disease. Transcriptional activity was assessed with regard to previously defined fingerprints motives for CTLA-4, IL-10, PD-1, TGF-β and PGE-2. CD4 T-cells from all groups were further tested for their proliferative capacity and cytokine production.

Results

Hierarchical clustering segregated the three groups. Applying the immunosuppressive fingerprints, patients with CD4 T-cells >500/µl were demonstrated to be under the influence of PGE2, whereas patients with CD4 T-cells <200/µl were demonstrated to be under the influence of PGE2 and CTLA-4. In normal controls, no association was found. The proliferative capacity of patient CD4 T-cells upon CD3-CD28-bead stimulation was not significantly different from healthy controls. The production of IL-2 by stimulated CD4 T-cells was significantly downregulated in patients with CD4 T-cells <200/µl, while there was no difference in IFN-ƴ and TNF-α secretion.

Conclusion

The severity of the CD4 numerical defect reflects the state of immunosuppression as demonstrated by RNA immuno-inhibitory fingerprint motives. This partially translates into functional differences as measured by decreased IL-2 secretion. In addition to time after transplant, CD4 T-cell numbers should be considered for the decision to stop or maintain anti-microbial prophylaxis in patients after allogeneic stem cell transplantation.

(UH, LPF and CW, JMC contributed equally to this work.)

Disclosures:

No relevant conflicts of interest to declare.
Monitoring Early CMV-Specific CD8+ T-Cell Responses After Allogeneic Stem Cell Transplantation By Pentamer and Streptamer Staining
Authors: Beloki Lorea , Ciaurriz Miriam , Ramirez Natalia , Zabalza Amaya , Mansilla Cristina , Bandres Eva , Calvillo Mercedes Rodriguez , Olavarría Eduardo
Published: October 2019

We present the results of a pilot study using pentamer (PM) and streptamer (ST) multimer complexes for monitoring CMV-specific CD8+ T-cells (CTLs). We analysed 15 patients that underwent allogeneic Stem Cell Transplantation (HSCT). Patient characteristics are summarized in Table 1. All patients and donors were positive for the HLA-A*02:01 allele. PM and ST were directed against the epitope NLVPMVATV (495-503) of the CMV phosphoprotein 65 (pp65). Samples were obtained at 15-day intervals until day +90 and monthly thereafter.PatientCMV status (D/R)GenderAgeDiagnosisDonorConditioningEngraftment (day)Follow up (months)GVHD (day)1+/+F43NHLSIBRIC2415-2-/+F33MDSURDRIC1912-3+/+M55AMLSIBMAC1714474-/+F41AMLURDMAC2121-5+/+F32AMLSIBMAC2013256+/+F64MDSSIBRIC2714897-/+M58CLLURDRIC2313928+/+M57ALLURDMAC155-9+/+F39AMLURDMAC2012-10-/+M42ALLURDMAC2125-11-/+M44AMLURDMAC12815312+/+F65AMLSIBRIC258-13-/+M27AMLSIBMAC3031814+/+M65AMLSIBRIC1966215+/-F30SAASIBRIC133-

Three patterns were observed. In 3 patients (20%) no CMV-specific-CTLs could be detected despite several CMV reactivations, requiring prolonged cumulative antiviral therapy (median 68 days; range 67-136).

In 7 patients (47%) CMV reactivation occurred at a mean of 41 days (10-94) and triggered a rapid increase of CMV-specific-CTLs with a median of 22.7 x 105/L (range 1.3-279.7). The CMV-PCR became immediately negative and antiviral therapy was stopped promptly after a median of 15.5 days (6-23).

Finally, 5 patients (33%) showed an early immune reconstitution with CMV-specific-CTLs detected with a median of 0.7 x 105/L (range 0.2-2.8) in the absence of CMV-PCR reactivation at a median of 21 days (10-34) post-SCT. No CMV-PCR reactivation was observed in this group with a median follow-up of 12 months (5-14).

Discussion

Monitoring CMV-specific-T-cells might be able to distinguish patients at higher risk of recurrent virus reactivation and in need of prolonged antiviral therapy. Patients with increasing CMV-specific-CTLs detectable at the time of CMV-PCR reactivation may only need a short course of antiviral therapy, while those with early CMV-specific-CTLs may be protected from CMV reactivation.

Conclusion

Using Multimer-based (Pentamer and Streptamer) monitoring of CMV-specific T-cell immune reconstitution after allogeneic HSCT may contribute to the clinical decision regarding when and for how long to commence anti-CMV therapy.

Disclosures:

No relevant conflicts of interest to declare.
C-Reactive Protein As a Predictor Of Bacterial Disease In Children With Sickle Cell Disease and Fever
Authors: Olivares Maria , Richardson Matthew W , Visintainer Paul
Published: October 2019

Introduction

Children with sickle cell disease (SCD) are at increased risk of serious bacterial disease compared to children without SCD. Children with SCD and fever are recommended to undergo prompt evaluation, including a physical exam, complete blood count, blood culture, and sometimes chest x-ray. These children often receive empiric parenteral antibiotics until blood cultures show no bacteremia for forty-eight hours. Studies in this population have identified clinical and laboratory features that are associated with low rates of bacteremia or that predict bacteremia. C-reactive protein is an acute phase reactant that has been studied as a predictor for bacteremia in a variety of clinical situations. Crp, however, has not been evaluated as a predictor of serious bacterial disease in children with SCD.

Method

Retrospective chart review of children age 0 – 18.9 year old admitted to Baystate Children’s Hospital with a diagnosis of SCD and fever who had crp and blood culture drawn between January 1, 2009 and December 31, 2011. SCD included homozygous SS disease, SC disease, or Sβ-thalassemia. Bacterial disease was defined as bacteremia, osteomyelitis, pneumonia/ acute chest syndrome, pyelonephritis. Parental report of fever was accepted. Age, maximum temperature, white blood count (WBC), absolute neutrophil count (ANC), percent of neutrophils (ANC/WBC), and crp were evaluated.

Results

80 cases met inclusion criteria. 18 cases had bacterial disease. Compared to children without bacterial disease, those with bacterial disease were older (mean age 11 vs. 4.8 yr, p < 0.001), had higher mean crp (5.7 vs. 1.6 mg/dL, p <0.001), higher mean WBC (20.9 vs. 15.6 x 103/mm3, p = 0.004), and higher mean ANC (13.5 vs. 8.7 x 103/mm3, p = 0.002). There were no significant differences between maximum temperature and percent neutrophils between the two groups. After regression analysis for all variables, age and crp remained significant between the two groups (p = 0.001 for both). A receiver operator characteristic (ROC) curve using crp and age produced an area under the curve of 0.87.

Conclusions

Children with SCD hospitalized for fever and diagnosed with bacterial illnesses are older and have higher crp compared to those without bacterial illness. The ROC curve based age and crp has characteristics of a good screening test. Because children with SCD and fever will usually receive empiric antibiotics regardless and the repercussions of a missed bacterial illness are great, using crp to determine antibiotic administration is likely limited. Rather, a low crp, especially in a younger child, may be helpful to determine patients who are candidates for early discharge or those who can be managed as an outpatient. Prospective use of crp for fever in this population, including outpatients, is warranted in order to identify the best sensitivity and specificity.

Disclosures:

No relevant conflicts of interest to declare.
Hemoglobinopathies Of Difficult Diagnosis. Case Reports From a Main Referral Pediatric Hospital In Venezuela
Authors: Bello Adriana C.
Published: October 2019

Hemoglobinopathies, sickle cell disease and thalassemias, are the most common genetic disorders worldwide, affecting millions of individuals. Diagnosis can take place very early in life, ideally through newborn screening, sometimes later in life. Disease modifying, acute and long term management strategies are provided for specific genotypes, and can be challenging for the treating hematologists.

It is essential the adequate diagnosis for the optimal management of these patients. Most diagnosis are made through hemoglobin electrophoresis, high performance liquid chromatography (HPLC) in the majority of cases of thalassemias. However, in some cases the correct diagnosis cannot be made by these methods, and more complex studies are needed, at the molecular level to genetically characterize the disorder.

We report a series of 12 patients from the pediatric hematology department from Hospital de Ninhos JM de los Rios, affected by severe congenital hemolytic anemias, who where assessed and managed at this tertiary Referral Center, who did not have definitive diagnoses by conventional methods, in September 2010. They all had presented symptoms from severe anemia since very early age, and where of difficult management through strategies like chronic transfusions, splenectomy or hydroxycarbamide, because lack of definitive diagnosis.

Samples were shipped after appropriate consentment to a highly specialized referral laboratory, (Hemoglobin Diagnostic Reverence Laboratory, Boston University School of Medicine), free from any charge, for its molecular characterization.

We obtained very accurate and complex results, also very interesting cases, like one unstable hemoglobinopathy from Heinz bodies (Hb Volga) and never before described thalassemic mutations, both homozygous and heterozygous. This was assuring, also reflected in a more appropriate and safer management of our patients.

We conclude it is always necessary to establish adequate diagnosis in our patients affected by hemoglobinopathies, for their adequate characterization and management. This will be reflected in a better quality of life.

Disclosures:

No relevant conflicts of interest to declare.
Iron Overload Occurs Very Early In Newly-Diagnosed Patients With Congenital, Transfusion-Dependent Anemias
Authors: Kattamis Antonios , Delaporta Polyxeni , Papassotiriou Ioannis , Kyriakopoulou Dimitra , Tourkantoni Natalia , Kostaridou Stavroula
Published: October 2019

Systematic transfusions are lifesaving for patients with severe congenital anemias, but they eventually lead to iron overload and the indispensable necessity of iron chelation therapy. Current official guidelines for the starting time of chelation therapy derives from data obtained with the use of desferrioxamine, which has been shown to have significant toxicity in very young patients, especially when used in low iron burden. No data exist on the use of the oral iron chelators in this setting. The purpose of this study was to evaluate the changes of iron parameters at the initial period of transfusion therapy in newly diagnosed patients with congenital anemias

Methods

Nine patients participated in this study. One patient was diagnosed with Diamond-Blackfan anemia, one patient with severe alpha-thalassemia, while 7 had beta-thalassemia. Three of the beta-thalassemia started transfusions at 1.5, 2 and 5 years, respectively. All others started transfusions between 2-4 months of age. Iron, transferrin saturation, ferritin levels. serum transferrin receptors (sTfR), were estimated by standard methods, while labile plasma iron (LPI) by the FeROS LPI kit (Aferrix, Ltd, Israel). The main results of the study show that: 1) transferrin saturation increases rapidly with transfusions (mean levels after 4 transfusions 49.2% (range: 23.8-90.5%), mean after 6 transfusions 69.1% (range: 39.5-112%)), though it has significant diversity in between patients, as indicated also by one patient that continued to have transferrin saturation at 65% even after 12 sessions. Transferrin saturation significantly correlates with ferritin levels (r=0.763, p<0.0001), with the number of previous transfusions (r=0.486, p=0.002) and with the levels of sTfR, which is also an index of the degree of erythropoiesis (r=0.550, p<0.001). The increase of ferritin correlates also with the sTfR levels (r=0.697, p<0.0001), while the rate of increasing transferrin saturation per transfusion correlates to sTfR levels (r=0.486, p=0.002). LPI levels appears early in the transfusion history and correlates with transfusion saturation.

Discussion

The results of the study indicate that iron overload starts early in the transfusion history of young patients with transfusion-dependent anemias. These findings dispute current guidelines suggesting starting chelation therapy, when the patients have already received 10 transfusions or when ferritin levels reach more than 1000ug/dl.

Disclosures:

No relevant conflicts of interest to declare.
Low Serum Vitamin B12 With Supplement In Pernicious Anemia
Authors: Jajeh Ahmad
Published: October 2019

Pernicious anemia is commonly associated with autoimmune disorders. This abstract will describe three cases with low serum Vitamin B12 even with 2 weeks Vitamin B12 inrtamuscular injection of 1000 micrograms. First patient is thirty nine 39 years old white male with known history of pernicious anemia and Alopecia Totalis with prior history of Methotrexate and cyclosporin treatment. The patient had low serum iron and low iron saturation at initial presentation. He was given IV Iron supplement to saturate iron stores with good result. However, his serum vitamin B12 remain border line low normal below 300 pg/ml ( patient’s level is 210, normal range 180-941 pg/ml), even with every two weeks IM injection for more than six months. Patient was also given oral supplement with no increase in the serum vitamin B12 level. The second patient is sixty eight 68 years old white female with history of colon cancer that underwent resection and history of Hashimoto’s Thyroiditis who was on Thyroid hormonal supplement. Her serum Vitamin B12 level remain borderline low below 300. The third patient is forty 40 years old white female with history of Hypothyroidism and breast cancer that had received adjuvant chemotherapy post Lumpectoy and radiation therapy. This patient was treated with IV intravenous iron supplement for iron deficency with good result. All three patients had blocking antibodies to intrinsic factor and presented with various degree of iron deficency and treated with oral as well as IV iron supplement. Peripheral blood smear were reviewed on all the three patients. Hypersegmentation of neutrophils was seen with normocytic normochromic red cell morphology. One patient had low plateletes count. All patients had white blood cell counts below ten thousands. Two patients showed excess of apoptotic neutrophils. None of the patients had enlarged spleen or adenopathy or evidence of bone marrow hypofunction. All of the above patients improved clinically interm of sense of well being and increased level of energy with vitamin B12 supplement. None of the above patients had abnormal liver or kidney function. All patients had low serum vitamin B12 with one monthly dose IM injection plus oral supplement. All patients had normal LDH level and normal Folic acid level.

In conclusion

The low serum vitamin B12 levels with adequate supplement that should saturate the liver reserve open an interesting question about the possiblility of Transcobalamine receptor abnormality interm of abnormal function or deficency. Antibodies blocking the receptors or rapid neutrophils turn over is another possibility. Further study needed to solve this delema.

Disclosures:

No relevant conflicts of interest to declare.
Absolute Monocyte Count Trichotomizes Chronic Lymphocytic Leukemia Into High Risk Patients With Immune Dysregulation, Disease Progression and Poor Survival
Authors: Herishanu Yair , Kay Sigi , Deutsch Varda , Ben-Ezra Jonathan , Naparstek Elizabeth , Perry Chava , Katz Ben-Zion
Published: October 2019

Peripheral blood absolute monocyte count (AMC) has been reported to correlate with clinical outcome in different types of cancers. This association may relate to alteration in circulating monocytic subpopulations and tumor infiltrating macrophages. In this study we evaluated the clinical significance of peripheral blood AMC in 80 treatment naive patients with CLL. The measurement of AMC was based on morphological enumeration using an automated microscope (CellaVision DM1200), due to our findings that automated complete blood count data may yield incorrect monocytes enumeration values in CLL. The median AMC in patients with CLL was not different from normal controls, however the AMC range exceeded the values of healthy individuals (median of 458/µL with a of range 0-4868/µL in CLL vs. median of 310/µL with a range of 105-1154 in healthy controls). The AMC divided the patients into 3 distinct sub-groups with different characteristics and outcomes. Patients with high AMC (>750/µL) were younger and also had higher absolute lymphocytes counts, while patients with low AMC (<250/µL) had prominent immune dysregulation (lower serum IgA levels, susceptibility to infections and a tendency for positive direct anti-globulin test). The low and high AMC patients had a shorter time to treatment compared to the intermediates AMC subgroup (250-750/µL), whereas low AMC was associated with increased mortality caused by infectious complications. In conclusion, AMC quantification during the disease course classifies CLL patients into subgroups with unique clinical features and outcomes.

Disclosures:

No relevant conflicts of interest to declare.
A Pilot Study To Examine The Effect Of Extracorporeal Membrane Oxygenation (ECMO) On Plasma Factor XIII Levels
Authors: Sanchez Dimarys , Stine Kimo , Crary Shelley E. , Fiser Richard , Schmitz Michael , Prodham Parthak , Nick Todd , Tang Xinyu
Published: October 2019

Background

Children with acute, severe cardiopulmonary compromise may require support with ECMO. Adequate anticoagulation without inducing life-threatening hemorrhage plays a critical role in the management of these patients. Recent data from adult studies suggest that patients undergoing cardiac bypass for coronary artery bypass grafting (CABG) often have reduced Factor XIII levels (FXIII). This relative FXIII deficiency may be predictive of hemorrhage or severe blood loss. Such studies have never been conducted in the pediatric population. Our primary objective will be to determine if a relative deficiency in FXIII occurs in children undergoing ECMO and to determine any time dependency of these changes. Our secondary objective will be to determine if blood loss and/or transfusion requirements during ECMO correlates with FXIII levels.

Methods

Single center ongoing study utilizing a prospective design involving pediatric patients (ages 0-18) undergoing ECMO. Samples for FXIII, D-Dimers, Fibrinogen and Thrombin Time are being drawn at designated time points as follows: prior to or during ECMO cannulation, 2 hours, 24 hours, 3-5 days and 14-21 days after initiation of ECMO and 1-3 days after discontinuation of ECMO. Mixed effects analysis of covariance (ANCOVA) will be used for data analysis.

Results

To date, fifteen patients have been enrolled and analyses have been conducted for n=7 patients with a total of 36 time points of data. Figure 1a and 1b show the mean and standard deviation (SD) of FXIII and Fibrinogen, respectively. Figure 1c shows the mean ratio of FXIII to Fibrinogen. Error bars represent 1 SD around the mean (central point).

Conclusion and Significance

This is the first pediatric study evaluating FXIII activity in children undergoing ECMO. Firm conclusions cannot be drawn with the interim analyses conducted to date. If variations in FXIII are found, our results will provide important information towards determining if children undergoing ECMO could potentially benefit from FXIII replacement, in turn minimizing bleeding complications and/or transfusion requirements often encountered with this treatment modality.

Disclosures:

No relevant conflicts of interest to declare.
Plasma FXIII LEVEL Variations During Menstrual CYCLE
Authors: Halimeh Susan , Talat Sharief Lava A. , Lawrie Andrew S. , Kappert Guenther , Mackie Ian J. , Peyvandi Flora , Kadir Rezan A.
Published: October 2019

Introduction

Factor XIII (FXIII) has an important role in the control of bleeding through fibrin cross-linking. While many physiological factors affect plasma FXIII level, the effect of the menstrual cycle is not fully understood.Aim: The study aimed to assess possible changes in plasma FXIII activity during the normal menstrual cycle and to assess any correlation between FXIII activity during the menstrual phase and the menstrual blood loss.

Methods

In this longitudinal study, a total of 32 women of reproductive age were recruited. Menstrual blood loss was measured using the pictorial blood-assessment chart (PBAC). A bleeding questionnaire and score were also completed. Blood samples were taken during menstrual phase (day 1-5), proliferative phase (day 6-11), periovulatory phase (day 12-17), secretory phase (days 18-23), and premenstrual phase (day 24-29) for assessment of FXIII level using quantitative ammonia release assay.

Results

Mean FXIII level was significantly lowest during menstrual and periovulatory phases of the cycle (114 IU/dL) compared to secretory (121 IU/dL) and premenstrual (122 IU/dL) phases (p=0.036). No significant correlation between FXIII activity during menstrual phase and women age (p= 0.53) or PBAC score (p=0.53). Among 14 women with PBAC score ≥ 100, the median FXIII activity during the menstrual phase of the cycle was 116 IU/dL, this was not statistically different from group with PBAC score <100 (113 IU/dL) (p = 0.72).

Conclusion

FXIII activity was lowest during the menstrual and periovulatory phases of the cycle. Understanding these changes in FXIII during menstrual cycle will help clinicians to perform blood tests during appropriate phase of the menstrual cycle and to capture the baseline level.

Disclosures:

Halimeh: Octapharma AG: Investigator Other, Research Funding.
Molecular Diagnosis Of One Chinese Patient With Hemophilia B
Authors: Zhou Rong-Fu , Gao Bo , Ou-yang Jian
Published: October 2019

FTo investigate the molecular mutation leading to Hemophilia B in one patient.

Methods

FOne-stage method was used to detect APTT, PT, TT, Fibrinogen and FVIII:C, FIX:C. The genomic DNA was extracted from the peripheral blood of proband. All exons and their flanks of Factor IX gene were amplified by polymerase chain reaction (PCR). The PCR products were sequenced directly.

Results

FThe proband was a 20-month boy presenting with scalp hematoma after trauma. Regular coagulation tests showed that his APTT was 135.1s, PT 11.9s, TT 15.6s and Fibrinogen 2g/l. Normal mixed plasma could correct the prolonged APTT to 35s. His FIX:C was 6.4% and FVIII:C was normal. Direct sequencing of PCR products suggested that there was a 5085T>C mutation (NG_007994.1) in Exon1, and a 36060G>C mutation in Exon8 of F9 gene. The former mutation caused the substitution of Leu19 by Pro, which lies in -28th in signal peptide. The later mutation lead to the substituion of Gln370 by His.

Conclusion

FMutations of 5085T>C in Exon1 and 36060G>C in Exon8 might be the causes of coagulation factor IX defiency for the patient. These mutations are de novo ones according to the database presenting in http://www.factorix.org/.

Disclosures:

No relevant conflicts of interest to declare.
Evaluation Of Factor V Leiden, Prothrombin Mutations and The Homozygous mutation 677T In The MTHFR Gene In Patients With Venous Thrombosis
Authors: Pizzuti Michele , Santagostino Alberto , Rizzo Maria Antonietta , Dragonetti Daniela , Pietrafesa Maria Grazia , Attolico Immacolata , Amendola Angela , Filardi Nunzio , Cimminiello Michele , Matturro Angela , Vertone Domenico , Nuccorini Roberta , Pascale sara Pasquina , Coluzzi Sabrina
Published: October 2019

Over the last few years there has been increased interest in laboratory tests for thrombophilia. Testing is predominantly performed to identify asymptomatic individuals at risk for a first thrombosis or patients (pts) at risk of relapse. The results are not always concordant, and the impact on clinical practice is not yet defined, especially for heterozygous forms.

We evaluated heterozygous and homozygous Factor V Leiden (FVL) and prothrombin (FII) mutations and the homozygous C677T mutation in the methylenetetrahydrofolate reductase (MTHFR) gene in 98 pts with venous thrombosis distinguishing idiopathic forms from forms that are secondary to malignancy, surgery, forced immobilization or hormone treatments.

FII mutation was present in 17% of idiopathic and 5.8% of secondary forms. FVL mutation was present in respectively 17% and 15.8% and MTHFR homozygous state in 31.9% and 21.5%. At least one of FII or FVL mutations was present respectively in 31.9% and 19%, at least one of the three mutations examined was present in 57.4% and 36.2%.

In pts with limb venous thrombosis (n-64), the percentages for each mutation are respectively: FII-23.5% and 6.6%, FVL-20.5% and 6.6%, MTHFR 35.2% and 16.6% .At least one mutation between FII and FVL is present respectively in 42.1% and 13.3% (P = 0,028) while at least one of the three mutations is present in 61.7% and 30.5% (P = 0,022)

In our pts with pulmonary thromboembolism (n-36) FII mutation was absent while FVL mutation was present in 7.6% of idiopathic and 21.7% of the secondary thrombosis, MTHFR in 30.7% and in 30.4%. At least one of the three examined mutations was present in 38.4% and 47.8%.

The mutations we have studied are generally more frequent in idiopathic forms. The difference reaches statistical significance in limb venous thrombosis where they seem to have a significant relevance, although in a heterozygous state, and to promote thrombosis even in absence of other causes.

In pulmonary thromboembolism (PTE) pts FII mutation was absent, FVL mutation is less frequent in pts with idiopathic thrombosis and MTHFR homozygous state is equivalent between the two subgroups. Thus, they seem to not influence PTE pathogenesis that would be likely sought in different causes.

Disclosures:

No relevant conflicts of interest to declare.
Thrombin infusion in endotoxin-treated rabbits reduces the plasma levels of plasminogen activator inhibitor: evidence for a protein-C- mediated mechanism [see comments]
Authors: Colucci M , Triggiani R , Cavallo LG , Semeraro N
Published: October 2019

Plasminogen activator inhibitors (PAIs) play a pivotal role in the control of fibrinolysis. The mechanisms regulating the plasma levels of PAI(s) are still unknown. We report here that the infusion of bovine thrombin (1 U/kg/min, over 60 minutes) in rabbits treated with 0.5 microgram/kg endotoxin (to induce an increase in circulating fast- acting PAI) causes a marked reduction of PAI (50% of preinfusion value), as indicated by functional assay and reverse fibrin autography. Moreover, blood clots prepared from samples obtained after thrombin infusion lysed faster than preinfusion clots when exposed, in vitro, to tissue plasminogen activator. Donor-receiver transfusion experiments showed that the half-life of circulating PAI activity was shorter in thrombin-infused rabbits than in controls (4.1 minutes versus 7.4 minutes), suggesting an accelerated clearance. As expected, thrombin infusion resulted also in activation of protein C (PC). The following observations suggest a close relationship between PC activation and PAI reduction. (1) Infusion of thrombin in rabbits made deficient in vitamin K-dependent plasma proteins by warfarin treatment did not result in modification of PAI activity. (2) Treatment of the latter animals with a barium citrate eluate (PE) of rabbit plasma restored both the anticoagulant and profibrinolytic response to thrombin. (3) Short infusion of thrombin-activated PE (containing activated PC, PCa), but not of unactivated PE, induced both anticoagulation and reduction of PAI activity. In vitro, incubation of PAI-rich rabbit serum with thrombin-activated PE and phospholipids resulted in a progressive disappearance of PAI activity with a t1/2 of 30 minutes. However, this slow inactivation rate does not fully explain the results obtained in vivo. Our data suggest that thrombin infusion in rabbits causes a reduction of circulating PAI activity and that activation of PC is the intermediary mechanism involved in this phenomenon.
Gene transfer into normal human hematopoietic cells using in vitro and in vivo assays
Authors: Dick JE , Kamel-Reid S , Murdoch B , Doedens M
Published: October 2019

The ability to transfer new genetic material into human hematopoietic cells provides the foundation for characterizing the organization and developmental program of human hematopoietic stem cells. It also provides a valuable model in which to test gene transfer and long-term expression in human hematopoietic cells as a prelude to human gene therapy. At the present time such studies are limited by the absence of in vivo assays for human stem cells, although recent descriptions of the engraftment of human hematopoietic cells in immune-deficient mice may provide the basis for such an assay. This study focuses on the establishment of conditions required for high efficiency retrovirus- mediated gene transfer into human hematopoietic progenitors that can be assayed in vitro in short-term colony assays and in vivo in immune- deficient mice. Here we report that a 24-hour preincubation of human bone marrow in 5637-conditioned medium, before infection, increases gene transfer efficiency into in vitro colony-forming cells by sixfold; interleukin-6 (IL-6) and leukemia inhibitory factor (LIF) provide the same magnitude increase as 5637-conditioned medium. In contrast, incubation in recombinant growth factors IL-1, IL-3, and granulocyte- macrophage colony-stimulating factor increases gene transfer efficiency by 1.5- to 3-fold. Furthermore, preselection in high concentrations of G418 results in a population of cells significantly enriched for G418- resistant progenitors (up to 100%). These results, obtained using detailed survival curves based on colony formation in G418, have been substantiated by directly detecting the neo gene in individual colonies using the polymerase chain reaction. Using these optimized protocols, human bone marrow cells were genetically manipulated with a neo retrovirus vector and transplanted into immune-deficient bg/nu/xid mice. At 1 month and 4 months after the transplant, the hematopoietic tissues of these animals remained engrafted with genetically manipulated human cells. More importantly, G418-resistant progenitors that contained the neo gene were recovered from the bone marrow and spleen of engrafted animals after 4 months. These experiments establish the feasibility of characterizing human stem cells using the unique retrovirus integration site as a clonal marker, similar to techniques developed to elucidate the murine stem cell hierarchy.
Effect of single amino acid substitutions on the formation of the PlA and Bak alloantigenic epitopes
Authors: Goldberger A , Kolodziej M , Poncz M , Bennett JS , Newman PJ
Published: October 2019

The subunits that comprise the platelet-specific integrin alpha IIb beta 3 are polymorphic in nature, with several allelic forms present in the human gene pool. Minor changes in the secondary and tertiary structures of platelet membrane glycoproteins (GP) IIb and IIIa encoded by these alleles can result in an alloimmune reaction after transfusion or during pregnancy. To better understand the molecular structure of the PlA alloantigen system, located on GPIIIa, and the Bak alloantigen on GPIIb, we used a heterologous mammalian expression system to express these integrin subunits in their known polymorphic forms. An expression vector containing the PlA1 form of a GPIIIa cDNA, which encodes a leucine at amino acid 33 (Leu33), was modified to express the PlA2- associated form encoding a proline at amino acid 33 (Pro33). Similarly, a Baka GPIIb cDNA expressing an isoleucine at amino acid 843 (IIe843) was modified to express the Bakb form containing a serine at the same position (Ser843). Transfection of these vectors into COS cells resulted in the synthesis of GPIIb and GPIIIa molecules that were identical in size to those present in platelet lysates. Immunoprecipitation of the GPIIIa-transfected COS lysates with PlA)- specific alloantisera indicated that the Leu33 form was recognized only by anti-PIA1 sera while the Pro33 form was bound only by anti-PlA2 sera, showing that single amino acid polymorphisms are necessary and sufficient to direct the formation of the PlA1 and PlA2 alloepitopes. Similar experiments with Bak allele-specific expression vectors indicated that while the amino acid polymorphism (IIe843 in equilibrium Ser843) was necessary, posttranslational processing of pro-IIb was required for efficient exposure of both the Baka and Bakb alloepitopes.
Treatment for acute myelocytic leukemia with allogeneic bone marrow transplantation following preparation with BuCy2
Authors: Copelan EA , Biggs JC , Thompson JM , Crilley P , Szer J , Klein JP , Kapoor N , Avalos BR , Cunningham I , Atkinson K
Published: October 2019

One hundred twenty-seven patients with acute myelocytic leukemia (AML) were given busulfan 4 mg/kg on each of 4 days and cyclophosphamide 60 mg/kg on each of 2 days (BuCy2) followed by allogeneic bone marrow transplantation from an HLA-identical or one antigen disparate sibling. For 71 patients in first complete remission, 23 in second complete remission or initial relapse, and 33 patients with primary refractory disease, second or subsequent relapse, or a preceding hematologic disorder, the 3-year leukemia-free survival (LFS) is 63.1%, 32.6%, and 24.2% respectively. The actuarial probability of relapse for each group is 14.1%, 40.6%, and 61.0%. In multivariate analyses, relapse and decreased LFS were associated with advanced disease phase and with M4/M5 French-American-British classification. The LFS of first remission patients was adversely associated with a short time interval from diagnosis to transplantation. This study indicates that BuCy2 is an attractive preparative regimen for marrow transplantation in patients with AML and that prognostic factors for relapse and LFS are similar those described for regimens containing total body irradiation.
Cytomegalovirus infection causes delayed platelet recovery after bone marrow transplantation
Authors: Verdonck LF , de Gast GC , van Heugten HG , Nieuwenhuis HK , Dekker AW
Published: October 2019

The pathogenic effect of cytomegalovirus (CMV) infection on the hematopoietic recovery after bone marrow transplantation (BMT) was retrospectively studied in 87 recipients of (nonpurged) autologous BMT and in 56 recipients of allogeneic BMT from HLA-identical siblings. Indications for autologous BMT were lymphomas or acute leukemias and for allogeneic BMT various malignancies or aplastic anemia. Patients were divided for the study in two groups, CMV-positive and CMV-negative on the basis of the CMV status pretransplant, and CMV-negative patients were kept CMV-negative by the local transfusion policy. In allogeneic BMT recipients, platelet recovery was significantly slower in CMV- positive patients than in CMV-negative patients (platelets greater than 50,000 cells/microL after 41 days v 27 days, P = .007). This difference held true when patients with acute graft-versus-host disease above grade I were excluded (platelets greater than 50,000 cells/microL after 42 days v 24 days, P = .01). In autologous BMT, the negative effect on platelet recovery was present in patients with lymphomas, but absent in patients with acute leukemias. Patients with acute leukemias had a very delayed recovery of platelets and granulocytes after autologous BMT, irrespective of the CMV status, probably due to the original stem cell disorder. Platelet recovery was significantly slower in CMV-positive autologous BMT recipients with lymphomas than in those not infected (platelets greater than 50,000 cells/microL after 36 days v 24 days, P = .0002). The presence of CMV infection had no effect on the recovery of granulocytes in autologous or allogeneic BMT. These data show that CMV infection causes delayed platelet recovery after BMT; however, in autologous BMT, the underlying disease (ie, acute leukemia) is more determinant for hematopoiesis after BMT.
Recurrent deletion in the human antithrombin III gene
Authors: Grundy CB , Thomas F , Millar DS , Krawczak M , Melissari E , Lindo V , Moffat E , Kakkar VV , Cooper DN
Published: October 2019

Eight unrelated patients with recurrent thromboembolism, a family history of thrombosis, and plasma antithrombin III (ATIII) activity/antigen levels consistent with a diagnosis of heterozygous type I ATIII deficiency were studied by polymerase chain reaction/direct sequencing of ATIII gene exon-coding regions. Frameshift mutations of one base and two bases, respectively, were found to have occurred in two unrelated patients at the same GAG codon (Glu 245) within exon 4 of the ATIII gene. A literature search showed six further hitherto unrecognized deletion “hotspots” in four other human genes. These deletion-prone sites exhibited sufficient sequence homology with each other to derive a consensus sequence (T G A/G A/G G A/C), suggesting that deletion in human genes may not only be non- random but also sequence-directed.
Immunolocalization of the Bcl-2 protein within hematopoietic neoplasms
Authors: Zutter M , Hockenbery D , Silverman GA , Korsmeyer SJ
Published: October 2019

The Bcl-2 proto-oncogene was discovered at the t(14;18) breakpoint found in most follicular B-cell lymphomas and some diffuse large-cell lymphomas. Bcl-2 is unique among proto-oncogenes, being localized to mitochondria and extending cell survival by blocking programmed cell death. We examined Bcl-2 protein expression in 82 hematologic malignancies and reactive lymphoid processes. All lymphomas with Bcl-2 rearrangement demonstrated high levels of Bcl-2 protein. However, most follicular and diffuse lymphomas without Bcl-2 rearrangement also displayed intense Bcl-2 staining. In these cases, mechanisms other than classic translocation may be deregulation Bcl-2. The pattern of Bcl-2 staining in follicular lymphoma is the inverse of the pattern in reactive hyperplasia, confirming a role for Bcl-2 immunolocalization in routine diagnosis. Small lymphocytic malignancies, including small lymphocytic lymphoma, mantle zone lymphoma, and chronic lymphocytic leukemia, expressed intermediate levels of Bcl-2. Bcl-2 protein varied in plasma cell dyscrasias. Bcl-2 protein levels in T-cell lymphomas reflected their corresponding stage of development. No substantial Bcl- 2 was present in the Reed-Sternberg cells of nodular sclerosing Hodgkin's disease. Chronic myelogenous leukemia was strongly positive for Bcl-2, consistent with the presence of Bcl-2 in normal myeloid progenitors. Immunohistochemistry identified an expanded spectrum of hematopoietic neoplasms in which Bcl-2 may provide a cell survival advantage.
Sex, gestational age, and weight dependancy of adult hemoglobin concentration in normal newborns
Authors: Galacteros F , Guilloud-Bataille M , Feingold J
Published: October 2019

We have measured by cation exchange high pressure liquid chromatography adult hemoglobin (HbA) concentration at birth in 6,123 unselected single newborn individuals. Probably because of the high precision of the analytical method used, we could demonstrate clear a relationship between HbA concentration and, respectively, gestational age and birth weight. We also demonstrated a significant difference in the Hb switching process between male and female newborns. Reference percentile distribution curves are given that could be used to define more precisely those children having slow or fast Hb switching.
Correlation of occult clonogenic leukemia drug sensitivity with relapse after autologous bone marrow transplantation
Authors: Miller CB , Zehnbauer BA , Piantadosi S , Rowley SD , Jones RJ
Published: October 2019

Despite initial complete remission rates exceeding 70%, the majority of patients with acute myeloid leukemia (AML) and adults with acute lymphocytic leukemia (ALL) eventually relapse. Improving the therapeutic results in acute leukemia requires detecting, and understanding the biology of, the minimal residual leukemia remaining after therapy and responsible for relapse. To investigate the biologic relevance of an in vitro assay for clonogenic leukemia (leukemia colony- forming units [CFU-L]) as a measure of minimal residual leukemia, we studied 58 consecutive patients with acute leukemia in complete remission undergoing autologous bone marrow transplantation (BMT) with cyclophosphamide-based therapy. CFU-L were cultured from the pretransplant remission marrows in 45 of 58 patients: 35 of 43 patients with AML and 10 of 15 with ALL. Clonal rearrangements, identical to the patients' overt leukemia when available, were detected in the occult CFU-L from four of the eight patients with ALL in whom adequate DNA for analysis could be obtained from the CFU-L. None of the uncultured pretransplant remission marrows from the 15 ALL patients showed clonal gene rearrangements. We also determined the in vitro sensitivity of the occult CFU-L to 4-hydroperoxycyclophosphamide (4HC), and correlated these results with the outcome of the patients. The sensitivity of the occult CFU-L to 4HC was the only factor that predicted relapse following BMT. The actuarial probability of relapse was 18% in the 23 patients whose CFU-L were sensitive to 4HC compared with 77% in the 22 patients whose CFU-L were resistant (P less than .001). The only factor that influenced the CFU-L sensitivity to 4HC was the type of leukemia. The CFU-L from the AML patients were more sensitive to 4HC than the CFU- L from the ALL patients (P = .001). Occult CFU-L genetically and functionally represent occult leukemia. Therefore, the CFU-L assay should provide a means for studying the biology of minimal residual leukemia and improving the therapeutic results in patients with acute leukemia.
The spectrum of genetic defects in a panel of 40 Dutch families with symptomatic protein C deficiency type I: heterogeneity and founder effects
Authors: Reitsma PH , Poort SR , Allaart CF , Briet E , Bertina RM
Published: October 2019

Heterozygosity for protein C deficiency is associated with thromboembolic episodes, but clinical symptoms are nonrandomly distributed among protein C deficient families. This finding has led to the provisional definition of clinically dominant and clinically recessive protein C deficiency. We report here the molecular basis of hereditary, clinically dominant protein C deficiency in a panel of 40 Dutch probands from apparently independent families. All but one subject was a heterozygote for a point mutation in the protein C gene. These 39 subjects shared 15 mutations, six of which occurred in more than one proband (between two and nine). The diversity in the 15 mutations, together with the observation that the most frequent Dutch mutation was also found in a Swedish family with clinically recessive protein C deficiency, makes it unlikely that the molecular basis of protein C deficiency will be different between the clinically dominant and recessive forms. The recurrence of one of the mutations is most likely due to a founder effect, which suggests that when an additional hereditary factor is involved in the clinical severity of protein C deficiency this factor may remain linked to the protein C gene over many generations.