bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased Oxygen Supply in Metabolic Syndrome Patients Treated with Gingko Biloba
Authors: Siegel Guenter , Mockenhaupt Felicitas , Ermilov Eugeny
Published: October 2019

Background: In Swiss mountaineers, it was observed that they did not suffer from O2 deficiency even in high altitude with diminished O2 partial pressures, if they had taken gingko. In a pilot study with 11 metabolic syndrome patients, who received as therapy 2 × 120 mg/d Gingko biloba special extract EGb 761 (Rökan novoÒ: Spitzner Arzneimittel, Ettlingen; TeboninÒ: Schwabe Pharmaceutical, Karlsruhe, Germany) over 2 months, it should be proven whether a change of the blood parameters MCH, MCHC, MCV and Hct could eventually supply a mechanistic explanation.

Methods: Photometric methods, commercial ELISAs and EIAs, and clinical routine chemistry were applied.

Results: MCH was increased from 27.7 ± 0.9 to 28.6 ± 1.0 pg (p < 0.0003), a change of +3.2% (p < 0.0044), MCHC from 317.7 ± 2.5 to 333.0 ± 3.1 g/L (p < 0.0), a change of +4.8% (p < 0.001), while Hct was decreased from 0.463 ± 0.011 to 0.407 ± 0.009 vol/vol (p < 0.0), a change of -12.0% (p < 0.001). Additionally, alterations in several biomarkers were measured: SOD +17.7% (p < 0.009), oxLDL/LDL -21.0% (p < 0.002), MPO -29.6% (p < 0.013), hs-CRP -39.3% (p < 0.004), HOMA-IR -14.0% (p < 0.024), CREA -11.3% (p < 0.001), ALT -16.0% (p < 0.0208). The table demonstrates the results of a linear correlation analysis between these biomarker changes and the changes in Hct and MCHC showing calculated Pearson’s correlation coefficients r and error probabilities p.

Discussion: Even though the additional load of the single erythrocyte with an increased hemoglobin concentration seems insignificant, it is, however, decisive for the O2-saturation of the blood. Decrease of the hematocrit from 46.3% to 40.7% signalizes a normalization of the initially elevated value in these metabolic syndrome patients. Possibly, this lowering by 12.0% would not have been so distinct in a normal person. Reduction of oxidative stress, inflammatory parameters as well as decrease in insulin resistance offer an explanation for the increase in MCH and MCHC.

Conclusion: With the intake of ginkgo, a better O2-supply for organs and tissues is guaranteed not only through an increased blood perfusion but also through an improved load of the single erythrocyte with hemoglobin (MCHC) by 4.8% as well as through reduction of the hematocrit (Hct) by 12.0%.

Figure 1 Figure 1.

Disclosures

Siegel: Schwabe Pharmaceutics, Karlsruhe, Germany: Research Funding.
Autoimmune Hemolytic Anemia Induced By Kangaroo Essence Pills (Report of Two Cases)
Authors: Meng Yuesheng , An Zengmei
Published: October 2019

The autoimmune hemolytic anemia (AIHA) is characterized by the autoimmune destruction and shortened life span of red blood cells. The kangaroo essence pills (KEP)are marketed worldwide as an aphrodisiac agent. They may contain muscles and genital tissues of Australian kangaroo as the key active components. We report here two cases of autoimmune hemolytic anemia induced by the KEP. Patient One: A 68 years old female presented with malaise, dizziness, jaundice and deep-colored urine for three weeks. Before this, she had taken three brand names of KEP for several weeks. An anemia was diagnosed in a local hospital but the underlying cause was obscure. Treatment with iron, folic acid and mecobalamin was ineffective. Laboratory analysis revealed anemia with a hemoglobin level of 34.4g/L, reticulocyte count of 3.0%, elevated white blood cell count of 22.7×109/L with neutrophiles of 81.4%, and normal platelet count (98.4×109/L). The urine was positive for occult blood, proteinuria (+++), red blood cells (11-15/HP) and bilirubin (++). Total serum bilirubin was 93.3 umol/L and direct bilirubin 69.7umol/L. Both of direct and indirect antiglobulin tests (Coombs' tests) were positive. Cold agglutinins of IgM were negative. Ham’s test was negative. Activity of Glucose-6-phosphate dehydrogenase was normal. Bone marrow morphology showed erythroid hypercellularity, with a predominance of polychromatic normoblasts and orthochromatic normoblasts and abnormality of double-phases. A diagnosis of warm antibody autoimmune hemolytic anemia was made. Intravenous methylprednisolone with a daily dose of 80 mg was administered immediately in addition to transfusion of saline-washed red blood cells. Due to the severity of anemia, three regimens of daily plasmapheresis were given. Clinical conditions were improved rapidly. Two weeks later, the hemoglobin level was returned to normal range and she was discharged. Follow-up for one year proved no evidence of relapse. The patient’s history had no record of allergy and other health problems. The KEP was the only possible cause to induce hemolysis. Besides kangaroo tissues, some KEP may have other addictives such as ginseng, Chinese wolfberry, deers’ sinew, snowdeer’s penis, yak penis, wild donkey penis, cordyceps sinensis, and so on. It is yet known which ingredient, or even some impurities, were the prime culprit in this case. Patient Two: A 25 year old young woman presented with pale and dizziness for a week. CBC test showed decreased hemoglobin level 76.5g/L, reticulocyte count of 4.5%, increased direct bilirubin 48.2 umol/L, and a positive Coombs' test. Two weeks ago she had tried a brand name of KEP as analeptic agent. She was diagnosed with autoimmune hemolytic anemia and immediately cured with oral methylprednisolone. To our knowledge, the AIHA induced directly by “kangaroo essence pills” has yet been reported. It is suggested that caution should be made to take aphrodisiacs of unknown ingredients.

Disclosures

No relevant conflicts of interest to declare.
Hepcidin Levels in Patients with Lung Cancer
Authors: Mandadi Mounika , Rios Jorge Arturo , Schade Andrew , Kloecker Goetz H
Published: October 2019

Background:

Hepcidin is a 25 amino acid peptide produced by the liver and plays a key role in iron homeostasis. It regulates the serum iron concentration by binding and inhibiting ferroportin, a transmembrane protein causing iron efflux. Anemia is a common complication in patients with cancer and is caused by the interaction of the tumor cells and the immune system resulting in the expression of various inflammatory cytokines which cause changes in iron metabolism resulting in anemia. Elevated levels of hepcidin have been noted in patients with various cancers. A better understanding of anemia in cancer patients may lead to a more effective and safe treatment of cancer induced anemia, possibly by targeting hepcidin.

Methods:

Blood samples of patients with lung cancer before treatment, with any histology or stage were obtained from the Brown Cancer Center’s Clinical Data and Specimen Repository. Ferritin and hemoglobin level were performed on each sample at the University of Louisville laboratory. The deidentified samples of plasma were then tested by a chemiluminescence kit for determination of the hepcidin level.

Results:

Plasma of 30 patients with lung cancer was available for testing.

There was no correlation seen between the hepcidin level and age, race, sex, lung cancer histology and stage. Hepcidin has a Pearson correlation of 0.66 with ferritin levels. The range of ferritin was 8.7 to 765 ng/ml and hepcidin 349 to 411321 pg/ml.

Conclusions:

Hepcidin correlates with ferritin in lung cancer patients. Targeting hepcidin in lung cancer patients may be a treatment option for cancer induced anemia.

Disclosures

No relevant conflicts of interest to declare.
A Case of Kaposi’s Sarcoma in a HIV Negative Patient with CLL Treated with Rituximab
Authors: Belur Anuradha Avinash , Arumugam Raajasekar Arun Kumar , Nannapaneni Srikant , Chelliah Thandavababu
Published: October 2019

Case Description: - A 76 year old lady was diagnosed with Chronic Lymphocytic Leukemia (CLL) with 11 q deletion after she presented with generalized lymphadenopathy and anemia. She was treated with rituximab 375mg/m2 day1 and bendamustine 60mg/m2 on day 1 and day 2 and completed six cycles of treatment. After the sixth cycle she developed multiple itchy, papular lesions with bleeding on both lower extremities. She was evaluated multiple times by vascular surgery and dermatology without a definitive diagnosis. She underwent a biopsy with staining for HHV-8, CD31 and CD34 which was positive confirming the diagnosis of Kaposi sarcoma. ELISA test for HIV was negative. She was started on treatment with Doxorubicin 20 mg/m2every 3 weeks and with 3 cycles there was significant regression of the lesions.

Discussion-: We describe a case of CLL which was initially started on treatment with rituximab and bendamustine. She tolerated the treatment well, but a few months later presented with skin lesions which on biopsy was diagnosed as Kaposi sarcoma. It is very uncommon for Kaposi sarcoma to develop in a HIV negative patient. This patient was immunocompromised with recent chemotherapy. Rituximab specifically depletes B cells and leads to impaired T cell mediated immunity. This case illustrates the importance of a high index of suspicion in patients treated with rituximab as it is used for a number of hematologic malignancies like leukemia, lymphoma as well as non-malignant conditions like autoimmune disorders. While infusion reactions and reactivation of hepatitis B are side effects physicians are aware of and cautious of while using rituximab, Kaposi’s Sarcoma remains a less known side effect. Awareness of this possibility is important in physicians prescribing rituximab.

Footnotes

* Asterisk with author names denotes non-ASH members.

Disclosures

No relevant conflicts of interest to declare.
Inhibition of Protein Kinase a Induces Platelet Apoptosis
Authors: Zhao Lili , Wang Zhicheng , Zhang Weilin , Chen Mengxing , Dai Kesheng
Published: October 2019

Background:

Platelet apoptosis elucidated by either physical or chemical compound or platelet storage occurs wildly, which might play important roles in controlling the numbers and functions of circulated platelet, or in the development of some platelet-related diseases. However, up to now, little is known about the regulatory mechanisms of platelet apoptosis. Protein kinase A (PKA) is highly expressed and plays central roles in signal transduction in human platelets. The certain level of PKA activity is indispensability for maintaining circulating platelets in resting state. In nucleated cells, PKA stimulated proliferation or promoted cell death via apoptosis in different cell types. However, it is still unclear whether PKA plays a role in platelets apoptosis. The aim of the current study is to investigate the role of PKA in platelets apoptosis.

Methods and Results:

Apoptotic events were assessed in platelets by flow cytometry, western blotting. PKA inhibitor H89 dose-dependently elucidated depolarization of mitochondrial inner membrane potential (ΔΨm), activation of caspase-3, -9, cleavage of gelsolin, phosphatidylserine (PS) exposure and membrane shrinkage in platelets. Incubation of platelets with PKA catalytic subunit-α (PKACα)-siRNA specific reduced the production PKACα and obviously elucidated platelet apoptosis. Platelet aggregation induced by ADP, botrocetin, or α-thrombin was significantly reduced in platelets treated with H89. However, H89 does not incur P-selectin surface expression and PAC-1 binding in platelets. After intraperitoneal injection of PKA inhibitor (Rp-cAMPS), the circulating platelets were reduced at 8 hour time point and recovered within 24 hours. H89 down-regulated the expression of Bad in platelets, and phosphorylation of Bad at Ser 112 was reduced in platelets. Cyclosporin A and ROS inhibitor (NAC) significantly reduced H89-induced platelet ΔΨm depolarization. However, z-DEVE-fmk, GM6001, MDL28170, forskolin, SB203580, BAPTA had no obvious effect on depolarization of ΔΨm.

Conclusions:

These data indicate that inhibition of PKA results in ROS-mediated platelet apoptosis. The findings suggest a novel mechanism for PKA in regulating platelet numbers and functions, which has important pathophysiological implications for thrombosis and hemostasis.

Disclosures

No relevant conflicts of interest to declare.
Xanthine Oxidase Mediates High Fat Diet-Induced Vascular Permeability via Junction Adhesion Molecule a Tyrosine Phosphorylation and Tight Junction Disruption
Authors: Chattopadhyay Rima , Kundamani-Sridharan Venkatesh , Tinnikov Alexander , Dyukova Elena , Singh Nikhlesh K , Kotla Sivareddy , Rao Gadiparthi N
Published: October 2019

Previously we have shown that 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE), the 15-LO metabolite of arachidonic acid (AA), affects endothelial cell (EC) barrier function. In this study, we have examined the role of xanthine oxidase (XO) in 15(S)-HETE-induced EC barrier dysfunction. 15(S)-HETE induced JamA phosphorylation on tyrosine residues 164, 218 and 280 involving XO-mediated reactive oxygen species (ROS) production and Src and Pyk2 activation, resulting in its dissociation from Occludin and causing tight junction disruption with increased EC barrier permeability and enhanced leukocyte and monocyte transmigration in vitro using EC monolayer and ex vivo using arteries as models. The phosphorylation of JamA on tyrosine residues 164, 218, and 280 appear to be critical for its role in 15(S)-HETE-induced EC barrier dysfunction, as mutation of any one of these amino acid residues prevented its dissociation from Occludin and restored TJ integrity and barrier function. Feeding WT mice but not 12/15-LO-/- mice with HFD for 3 months resulted in enhanced XO expression and its activity in the artery, which was correlated with increased aortic EC TJ disruption and its barrier permeability with enhanced leukocyte adhesion. These observations provide additional support for the role of 12/15-LOX in the dysregulation of vascular permeability in response to HFD.

Disclosures

No relevant conflicts of interest to declare.
Subcutaneous Immunoglobulin (SCIG) in Responders to Intravenous Therapy with Chronic Immune Thrombocytopenia (ITP)
Authors: Iuliano Francesco , Iuliano Eleonora , Luci Maria , Perricelli Alessia , Pomillo Angelo , Abruzzese Elisabetta
Published: October 2019

Introduction. Immune thrombocytopenia (ITP) is characterized by platelet destruction due to the presence of platelet antibodies. This phenomenon is associated with impaired platelet production by bone marrow. Short-course corticosteroids and high-dose intravenous immunoglobulins remain the first-line treatments. Aim of this study was to test if SCIG is equally effective and can be less expensive than hospital-based IVIG in treating chronic-ITP

Methods

Seven-ITP patients aged between 13-52 years were given liquid intravenously immunoglobulin (IVIg) stabilized with proline in a dose of 400 mg/kg per day for 5 consecutive days.The median time since ITP diagnosis was 18 months (range, 16 – 38) Median (range) baseline platelet count 19 (3 – 32) x 109/L.,median of 2 (1 – 3) prior ITP therapies. Furthermore, none of of the subjects was a good candidate for TPO-receptor agonists (romiplostim and eltrombopag) because of age or other comorbidities.6 / 6 had a good response to the first 5 day course of gammaglobulin therapy, (platelet count ≥50 × 10(9) /l and ≥2× baseline). The peak platelet count occurred within 7 days from the beginning of the therapy. Only in one patient the platelet count peak took place after 12 days. During the first year of treatment, only one of seven initial responder patients achieved CR(defined as platelet count ≥ 100 * 10^9/L measured on two occasions > 7 days apart and the absence of bleeding) and was excluded from the study. After a loading dose of 2 g/kg over 2 days of IVIg, home SCIG of 8 gr total weekly dose was started by 87,3% (6/7) to maintain platelet counts in the target range of 50–100x109/L.

Results

5/6 patients reached the target. One patient achieved CR during the maintenance therapy.No untoward reactions necessitating cessation of therapy were encountered during this study. The most common side effect observed was headache.

Conclusion

Application of SCIG was well tolerated, easy to manage, and led to stabilization of the disease course.The therapy facilitates home therapy, as the infusion technique is easy for children, adults and elderly people to learn and there is no need for venous access. SCIG home therapy leads to significantly improved life situations for the patients; the SCIG home therapy regimen in particular reduces the costs of treatment. Overall costs per patient were strongly reduced in SCIG in comparison to IVIg.SCIG may represent an effective new therapeutic option in chronic ITP.

Disclosures

Abruzzese: novartis, bristol myers squibb, ariad, pfizer, takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees.
five Int22h Homologous Copies in Association with Intron22 Inversion Type 3
Authors: Lannoy Nathalie , Ravoet Marie , Grisart Bernard , Fretigny Mathilde , Hermans Cedric R.
Published: October 2019

Introduction

F8 intron22 inversion is the causative gene defect in up to 45% of severe hemophilia A (HA) patients mediated by recombination between three highly homologous copies located in intron 22 (int22h-1) and two other extragenic copies (int22h-2 and int22h-3) positioned more telomerically outside the gene. Intrachromosomal rearrangement between int22h-1 and int22h-3 provide the F8 type 1 inversion while the F8 type 2 inversion could be explained by the participation of the int22-h2 sequence in the homologous recombination. However, a third type, known since 1993*, was explained by the existence of a duplicated copy of int22h-3 or int22h-2 in case of intron22 inversion type 3A or 3B respectively.

Methods

Three unrelated HA patients with intron22 type 3A/3B were identified by southern blotting. To appreciate the length of the int22h extragenic duplicated, genomic hybridisation was performed using Affimetrix CytoScan High-Density array.

Results

Breakpoint analyses by CGH-array in all patients show same duplication of approximately 180kb delimited between intron 1 of CLIC2 gene and distal repeat int22h-3reflecting the presence of five genomic int22 copies. A fourth non-hemophiliac case was added since analysis by CGH technique identified duplication delineated by same boundaries.

Conclusion

This study suggests the existence of genotypes harboring five int22h copies mediated by 180 kb duplication at Xq28 locus probably not associated with HA. We propose that this duplication has happened by tandem inversed duplication. Such genotype is to be considered as polymorphism and could be associated with the two kinds of F8 intron22 inversion type 3 when intrachromosomal recombination has occurred between homologous copies.

*Antonarakis SE and the international consortium study. Factor VIII gene inversions in severe hemophilia A: results of an international consortium study. Blood 1995; 86: 2206-2212

Disclosures

No relevant conflicts of interest to declare.
Influence of Factor V Deficiency on ROTEM® Clotting Time
Authors: Haas Thorsten , Cushing Melissa M. , Asmis Lars M
Published: October 2019

Introduction

Acquired factor V (FV) deficiency is repeatedly mentioned to be a major driver of trauma induced coagulopathy. Viscoelastic point-of-care tests such as ROTEM® seem to be advantageous in detection and guidance of trauma related bleeding management. Physiologic effect of FV can be characterized by an increase in thrombin building, which can be detected by ROTEM® clotting time (CT). However, the impact of varying levels of factor V activity in otherwise normal plasma on ROTEM® CT is not yet published. The aim of this in vitro investigation is to assess if mild to moderate factor V deficiency can be detected by ROTEM® CT.

Methods

Factor V deficient plasma (Code 0020011500; Instrumentation Laboratories, Bedford, MA, USA) and normal human plasma (pooled, FV content 96%) was mixed to generate plasma with 0, 10, 16, 25, 50 and 100% FV content. The FV deficient plasma is rendered FV deficient by immunologic means and has FV activity of ≤1%.

ROTEM® (TEM International GmbH, Munich, Germany) measurements were performed in each plasma dilution to assess impact of different levels of FV on thrombin generation. Measurements were repeated in triplicate. Clotting time (CT) was plotted against FV activity. Data is depicted as mean +/- SD.

Results

ROTEM® measurements showed a prolongation of clotting time with decreasing FV activity. A decrease of factor V activity from 100% to approximately 25% was accompanied by a slight change in CT from 50 sec to approximately 65 sec. If factor V activity drops significantly to less than 10%, a much more pronounced prolongation in CT of more 140 sec was observed.

Discussion

Most publications dealing with acquired factor V deficiency following trauma related bleeding reference a prospective study demonstrating that factor V becomes critically low in about 20% of all cases. Those levels of below 30% factor activity were linked to an increase in TEG® r time, which is comparable to the ROTEM® CT, similar to the results in our study.

Notably, a case control study demonstrated that factor V remained fairly stable (77% ± 29%) in trauma patients with no decrease in thrombin generation. Likewise, another case control study in trauma patients demonstrated that EXTEM CT remained within reference range (median 64 sec (IQR 55,75 sec)).

Conclusion

Moderate to severe factor V deficiency can be detected by prolongation of ROTEM® CT in platelet poor plasma (PPP). Further studies will help to determine if platelets added to PPP may further decrease ROTEM® CT, as approximately 20% of the entire FV pool is stored in platelet α-granules in a partially activated form. In addition, the occurrence and the potential clinical impact of factor V deficiency in trauma related bleeding management needs to be further investigated.

Disclosures

Haas: CSL Behring: Honoraria; Octapharma: Honoraria; TEM International: Honoraria.
Delivering a Hemophilic Newborn with No Intracranial Hemorrhage: Is Caesarian Section the Answer?
Authors: Nazir Hanan , Allawati Tuqa , Elbeshlawi Ismail , Al-Khabori Murtadha K. , Alkindi Sara , Alriyami Wafa , Wali Prof. Yasser
Published: October 2019

Birth is not only considered the first hemostatic challenge for a child with hemophilia A, but also it can result in one of the most devastating and often preventable type of bleeding in hemophilic newborns. Three to four percent of infants with hemophilia experience a head bleed at delivery. The optimal mode of delivery of the hemophilic carrier expecting an affected child is still a matter of uncertainty and debate.

The aim of this study was to evaluate the frequency of major bleeding in the neonatal period and to examine the association between mode of delivery and neonatal intracranial hemorrhage (ICH) in an Arab cohort of infants with hemophilia A.

Subjects and methods:

A retrospective multi-center study of children with severe hemophilia A (age 0-18 years) was conducted. Five hemophilia treatment centers distributed across four Arabian Gulf countries participated in the study. Two centers from Oman and one center of each of KSA, UAE and Kuwait were included.

The collected data were date of birth, maternal parity, mode of delivery, duration of labor, admission to neonatal intensive care unit, symptomatic ICH, and other bleeding episodes. Review of electronic patient record along with telephone interviews when needed were the tools used for data collection.

Results:

A total of 153 patients were found. Most of them were born through spontaneous vaginal delivery (SVD) (125 patients; 82 %). Vacuum extraction was adopted in six patients (4%), while twenty two patients were offered caesarian section (CS) (14%).

ICH was reported in a total of four patients (2.6%). All of them were full term babies with variable time interval between the onset of the bleed and birth. One patient presented at birth, another presented at the age of 4 weeks while the remaining two had the bleed at the age of 2 weeks.

Vacuum extraction was associated with the highest risk of ICH (two patients; 33 %).The other two patients were born by SVD (1.6 %). CS was not associated with ICH.

Conclusions:

Normal vaginal delivery is still considered a safe journey through the birth canal for hemophilic newborns. Larger prospective studies are needed to define an evidence-based optimal mode of delivery for the hemophilia carrier expecting an affected child.

Disclosures

No relevant conflicts of interest to declare.
Evaluation of Thrombin Generation Assay in Patients with Hemophilia
Authors: Karimi Mehran , Haghpanah Sezaneh , Bazrafshan Asghar , Eatemadfar Peyman , Dehghani Javad , Afrasiabi Abdolreza
Published: October 2019

Background: Diagnosis of hemophilia is based on detection of factor level and coagulation screening test. These screening tests can not reflect global hemostatic balance. Thrombin generation (TG), as a focal point and key process, is an important step in hemostasis and thrombosis. Recent evidence has indicated that TG assay is a better predictor of the coagulation capacity and subsequently overall assessment of hemostasis compared with traditional coagulation tests. The parameters of the thrombogram are helpful in the evaluation of bleeding or thrombotic risk and also in the management of these situations.

Objective: The aim of this study is to evaluate the correlation between TG parameters with bleeding symptoms and disease severity in patients with hemophilia.

Methods: In this cross-sectional study, 59 male patients with hemophilia were randomly selected from those already registered at Shiraz Hemophilia Center of southern Iran, from February to December 2012. Also, 38 healthy age-matched men were considered as a control group from those referred for check up. Informed written consent was obtained from all participants. The proposal was approved by the Medical Ethics Committee of Shiraz University of Medical Sciences. Disease severity was defined based on the activity level of deficient factor (severe <1%, moderate from 1 to 5%, and mild >5% IU/dL). Bleeding score (BS) was calculated by performing a clinical evaluation using a modified questionnaire based on Tosetto et al- questionnaire. Correlation of TG parameters with disease severity and BS were determined.

Results: From 59 patients with hemophilia (52 hemophilia A and 7 hemophilia B), 40 patients (68%) had severe disease, 14 (24%) moderate and 5 (8%) mild disease. All TG parameters showed statistically significant differences between patients and controls (P<0.001). Only Lag time showed a statistically significant correlation with BS (rs= 0.316, P =0.016). All TG parameters except peak showed association with disease severity (P<0.05). In addition, endogenous thrombin potential showed a significant correlation with factor activity level (rs= 0.459, P <0.001). Both Lag time and start tail showed significant negative correlations with factor activity level (rs= -0.488, P <0.001 and rs=- 0.289, P <0.026 respectively).

Conclusion: Based on our results, although most of the TG parameters evaluated were not significantly correlated with bleeding score of hemophilia patients, the majority of TG parameters were significantly associated with factor activity level and disease severity. In patients with hemophilia, plasma factor activity level has a poor predictive value in the evaluation of efficacy of treatment; but it seems that TG assay is an appropriate tool for assessment of global hemostasis and better reflection of clotting function in the management of patients with hemophilia.

Disclosures

No relevant conflicts of interest to declare.
Severe Spontaneous Intracranial Bleeding in 11 Months Old Boy with Congenital Afibrinogenemia with Novel Mutation - Deletion aα 6477A in Fibrinogen Gene
Authors: Zapletal Ondrej , Blatny Jan , Kotlin Roman , Dyr Jan E
Published: October 2019

Congenital afibrinogenemia is very rare inherited bleeding disorder, caused by absence of fibrinogen in plasma. Serious spontaneous bleeding including intracranial haemorrhage may occur at any age.

We present a case report of a boy with afibrinogenemia caused by novel mutation – homozygous deletion Aα 6477A. Diagnosis was set after bleeding post cleft lip and palate plastic surgery. Spontaneous intracranial haematoma in occipital region occurred when the boy was 11 month old. Replacement therapy with plasma derived fibrinogen concentrate successfully covered neurosurgical evacuation of hematoma. The patient was then commenced on regular prophylaxis with fibrinogen concentrate two times a week. No further bleeding episodes occurred until present time.

Spontaneous intracranial bleeding in patients with inherited bleeding disorders is often life threatening. Immediate replacement therapy is crucial, together with diagnostics and surgery, when necessary. Prophylactic substitution therapy after a severe bleeding episode is recommended.

Disclosures

No relevant conflicts of interest to declare.
Sub-Clinical Activation of Thrombosis May Predict Survival in Cancer Patients
Authors: Batra Anupam , Mehdi Syed A. , Brodzik Frank , Pasquale Donald
Published: October 2019

Background: Approximately 10% of newly diagnosed cancer patients will develop thromboembolic disease (TED) in the first year following diagnosis. We hypothesize that a subgroup of cancer patients at diagnosis will have subclinical changes in their coagulation system that will indicate increased risk for subsequent TED and/or predict increased adverse cancer outcome.

Methods: We performed a prospective, non-interventional study of recently diagnosed cancer patients who were scheduled to start chemotherapy. We obtained a single plasma sample at the time of diagnosis and prior to therapy, and measured prothrombin 1.2 fragment (F1.2), a measure of thrombin activation, and plasma microparticle levels (MP), a measure of platelet, endothelial, and other cell activation. We reviewed subsequent clinical outcomes including survival and incidence of venous thrombosis. We accrued 22 patients with mean age 63 years (range 39 to 89; 20 males, 2 females), 12 with lung, 6 with colorectal, and 4 with head and neck cancer. Mean follow-up was 24 months (range 3 to 57). Plasma F1.2 and MP were assayed using commercially available ELISA kits.

Results:

T1.2 concentrations were significantly higher in the deceased subgroup (table 1; p<0.002), and in patients with venous thrombosis (table 2; p<0.05). MPs were not informative. Sensitivity and specificity for prediction of survival and venous thrombosis (table 3) were determined by comparing patients in highest quartile of levels with lower quartiles combined and by using threshold value of mean +2.5 SD’s determined by measuring levels in 8 healthy controls.

Table 1. Survival: F1.2 and MP (Mann-Whitney) Deceased Patients (N=7) Alive Patients (N=15) Mean +/- SD Median Mean +/- SD Median p-value F1.2 (pM) 436+/-214 355 121+/-117 105 <0.002 MP (nM) 10+/-10 3 8.8+/-2.8 2.8 Not Significant

Table 2. Venous Thrombosis: F1.2 and MP (Mann-Whitney) Thrombosis (N=4) No Thrombosis (N=18) Mean +/- SD Median Mean +/- SD Median p-value F1.2 (pM) 368+/-267 313 189+/-191 163 <0.05 MP (nM) 5.9+/-5.3 3.9 9.8+/-15.6 2.7 Not Significant

Table 3. Sensitivity/Specificity Analysis >mean + 2.5 SD’s Highest Quartile MP T1.2 MP T1.2 Death SENSITIVITY 43% 43% 43% 71% Death SPECIFICITY 87% 100% 87% 93% Thrombosis SENSITIVITY 25% 25% 25% 50% Thrombosis SPECIFICITY 78% 89% 78% 78%

Conclusion: T1.2 levels measured at time of diagnosis of cancer correlated with increased mortality and development of venous thrombosis. MP levels were not informative, possibly due to the limited size of the study population. Additional studies are needed to elucidate whether these biomarkers may be useful in identifying a higher risk population for death and/or venous thrombosis, and to help guide enhanced therapy for such higher risk patients.

Disclosures

No relevant conflicts of interest to declare.
Flow Cytometry Screening for Paroxysmal Nocturnal Hemoglobinuria Abnormal Clones in Saudi Arabia
Authors: AlGhasham Nahlah , Abulkhair Yasmeen , Khalil Salem
Published: October 2019

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare disease with insidious process, chronic course and life-threatening condition. PNH is clinically defined by the deficiency of the endogenous glycosyl phosphatidylinositol (GPI)-anchored complement inhibitory protein. It has always aroused interest in the medical profession rendering screening and proper diagnosis by flow cytometry (FCM) technology a priority

We reviewed all samples submitted for PNH/ FCM screening for the past 2 years (2012-2013) at hematology section, Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center (General Organization). We collected the positive cases and reviewed them for age, gender, indication for screening, sample type, size of the PNH clone and cell type affected. Immunophenotypic analysis was performed using gating antibodies CD45, CD15, CD33, CD235a GPI-linked antibodies, CD59, CD14, and CD24 as well as fluorescent Aerolysin (FLAER).

In a total of 366 peripheral blood samples submitted for PNH/ FCM screening fifteen samples (4%) were positive for PNH clones but only 12 patients were available for analysis. The median age for our patients was 34 years with approximately equal male to female distribution.

12 cases showed type II and III clones within the RBCs with clone size ranging between 0.04% and 56%. Analysis of granulocytes and monocytes revealed type III clone in 8 cases, type II and III clone in 3 cases and non in one case. The percentage of the clone varies between the granulocytes and monocytes and ranges from 1% up to 100%.

Of 12 positive PNH cases, 8 (66.7%) patients were diagnosed as having aplastic anemia (AA), 1 (8.3%) patient with Budd-Chiari syndrome, 1 (8.3%) patient has chronic immune thrombocytopenia (ITP), and 2 (16.7%) patients presented with pancytopenia.

This study confirms the rarity of the disease since only 4% of the submitted samples for analysis turned to be positive for PNH. The detection limit for a PNH clone by FCM in the RBC or WBC is 0.01%. Identification of small PNH clone is greater FCM sensitivity relative to old test used for the same purpose (Ham test). The use of the FLAER allowed us to detect granulocytic PNH clone, however, granulocytes PNH clone detection alone without RBCs clone detection is not recommended. This review confirms the previous percentage of positive cases (5.9%) reported from this center on a smaller number of cases during the past few years.

Disclosures

No relevant conflicts of interest to declare.
Metastatic Breast Cancer in a Patient with Fanconi Anemia
Authors: Graham Jeffrey , Grenier Debjani , Ponnampalam Arjuna
Published: October 2019

Fanconi anemia (FA) is a rare inherited disorder characterized by progressive bone marrow failure, congenital malformations and a propensity for developing malignancies at an early age. The underlying genetic defect in FA creates a state of cellular hypersensitivity to many traditional chemotherapy agents, making the treatment of malignancies in this population particularly challenging.

We describe a 42-year-old female who presented with a solitary mass in her left breast. Core biopsy revealed an invasive ductal carcinoma that did not express estrogen (ER) or progesterone receptors (PR), but did express human epidermal growth factor receptor 2 (HER2). Staging work-up revealed diffuse skeletal metastatic disease. At her initial consultation with medical oncology, she was discovered to be pancytopenic. Further history revealed a sibling with aplastic anemia and that she had undergone chromosomal breakage testing for FA in the past, which was subsequently confirmed to be positive. She underwent a bone marrow aspirate and biopsy that showed metastatic marrow infiltration by non-hematopoietic cells. In addition there was morphological evidence of dyserythropoiesis and cytogenetic abnormalities on karyotyping, features suggestive of FA. She was initially started on trastuzumab monotherapy. Low dose radiation therapy was added due to local tumor progression. Combined HER2 directed therapy was to be implemented, but was held due to a functional decline in the patient. To date, she has not received definitive genetic testing to determine which FA subgroup she belongs to.

This case highlights two important aspects of FA. The first is the inherent increase in susceptibility to neoplasms in this group, including solid tumors such as breast cancer. The genes associated with FA are involved in deoxyribonucleic acid (DNA) repair pathways, including mutations in the breast cancer susceptibility gene, BRCA2. The second is the heightened sensitivity to the toxic effects of many standard chemotherapy and radiation treatments. This creates unique challenges in the treatment of malignancies in this population and stresses the importance of targeted therapies.

Disclosures

No relevant conflicts of interest to declare.
Calr Gene Mutation in Patients with JAK2-Negative Myeloproliferative Neoplasms
Authors: Jingyan Xu , Zhou Rong-Fu , Chen Bing , Ouyang Jian
Published: October 2019

Methods Genomic DNA from bone marrow and peripheral blood cells were extracted from 40 patients with MPD(male 26,female 14),Aged 13 to 76 years old, JAK2V617F mutation and CALR genetic mutations was identified by PCR- direct sequencing.

Results The number of MPD(PV15,ET25) 40 cases in patients with JAK2V617F mutation rate was 60%,among them the polycythemia vera ( PV) positive rate was 66. 7% ( 10 /15) ,and essential thrombocythemia ( ET ) positive rate was 56% ( 14 /25) . The number of JAK2V617F -negative MPD 16 cases in patients with CALR mutation rate was 12. 5% ( 2 /16),Two of the 11 patients with ET were CALR mutation positive(18.2%). Two novel mutation in CALR exon 9,c.1099_1150del ( p. chr19F12915572-12915623del) was detected in patient Tao c.1099-1151delinsT( p.chr19:12915572_12915624delinsT) was detected in patient Xu. These mutation was absent in the controls,Two novel mutation in CALR have not been reported so far.

Conclusion CALR gene mutations testing helps to JAK2V617F negative MPD diagnosis, makes the MPD early detection and treatment.

Disclosures

No relevant conflicts of interest to declare.
Inhibition of Siah2 Ubiquitin Ligase By Vitamin K3 Attenuates Hypoxia and Blocks K562-R Cells Resistance
Authors: Xu Na , Xiao Yajuan , Zhou Xuan , Li Lin , Huang Fen , Xu Bing , Xiaoli Liu
Published: October 2019

Background and objective: Hypoxia has been shown to favor the self-renewal of murine and human hematopoietic stem cells. Hypoxia as a key feature of the “stem cell niches” in vivo, and studies found that hypoxia modified proliferation and differentiation of chronic myeloid leukemia (CML) stem cells.The E3 ubiquitin ligase Siah2 is an important regulator of the hypoxic response,which has been implicated in the regulation of the hypoxia response, as well as in the control of Ras, JNK/p38/NF-κB, MAPK signaling pathways. In the present study, we identified that SIAH2 induced k562 cells resistance to imatinib by hypoxia-inducible factor (HIF)-1a activated vascular epithelial growth factor (VEGF) pathway in hypoxia micro environment. In this study we show that SIAH2/Hif-1α induced K562 cell remain in G0 stage and resistance to imatinib, and we verified that vitamin K3 (SIAH2 inhibitor) reversed K562-R drug resistance in hypoxia microenvironment.

Methods: We detected Siah2 expression levels in K562-wild type (K562-W) and K562-imatinib-resistance type (K562-R) cell lines by western blot analysis. Those two cell lines were further cultured for 24 h and 48 h under the condition of normal and hypoxia concentration of oxygen (1%, 5%), and treated K562-R with 0A5A15A30mM vitamin K3 for 72 hours in hypoxia concentration, explored cell cycle and apoptosis by flow cytometry (FCM) dyed by Annexin-V; analyzed the expression levels of Siah2, HIF-1α respectively by real-time PCR and western blot.

Results: The protein of Siah2 and HIF-1α was significantly higher in K562-R compared with K562-w cells (P<0.01). Cell cycle analysis showed a 3% increase in K562-W G0/G1 cells in 1% O2 compared with normal O2, and 7% in K562-R. Under hypoxia condition, the cellular apoptotic ratio of K562-R was 5.46%, much less than 11.08% in K562-W cells. G0 cell proportion increased significantly with the long time Hypoxia (P<0.01); After being cultured in 1% oxygen concentration for 24 hours, we confirmed Siah2,HIF-1α were all up regulated in both cell lines, moreover, it was more obvious in K562-R cells. Siah2 protein expression increased along treated with vitamin K3 concentration (0, 5, 15, 30 mM) (P<0.05), on the contrary ,HIF-1α protein expression decreased with vitamin K3 concentration (P<0.05). the proportion of G0 cell was decreased in K562-R cells treated with Vitamin K3 (15mM)for 48h Under the condition of 1% oxygen concentration compared with control group (P=0.02).

Conclusions: Hypoxia up-regulated of Siah2 and Hif-1α expression in K562-R, promoted cell apoptosis and arrested cells in G0 stage, and reduced cell sensitivity to Imatinib. Vitamin K3 could inhibit Siah2 and lowered Hif-1α in K562-R. These findings reveal an effective treatment for the identification of Siah2 inhibitors and would reverse TKI resistance for CML patients.

Disclosures

No relevant conflicts of interest to declare.
Favorable Clinical Outcome and Unique Characteristics in Association with Twist1 overexpression in De Novo Acute Myeloid Leukemia
Authors: Huang Cih-En , Chen Chih-Cheng , Gau Jyh-Pyng , You Jie-Yu , Chen Yi-Yang , Chou Hui-Ju , Lung Jrhau , Yang Muh-Hwa
Published: October 2019

Epithelial-mesenchymal transition (EMT) is a critical process for inducing stem-like properties of epithelial cancer cells. However, the role of EMT inducers in hematologic malignancies is unknown. Twist1, an EMT inducer necessary for cell migration, has recently been found to have transcriptionally regulatory activity on the expression of Bmi1, and these two are capable of promoting tumorigenesis in a synergized manner. Knowing that Bmi1 expression is essential for maintenance of leukemic stem cells, we speculate that Twist1 might govern the pathogenesis of acute myeloid leukemia (AML) development as well.

We used AML cell lines KG1a and THP1 cells, which had low and high expression levels of Twist1, respectively to demonstrate that upregulated Twist1 increased Bmi1 expression in AML. Bmi1 overexpression endued leukemic cells a higher proliferative potential and increased resistance to apoptosis. In primary AML samples, there was strong positive correlation between the expression levels of Twist1 and Bmi1. AML Patients whose leukemic blasts harbored overexpressed Twist1 had a more aggressive clinical phenotype, but they were more likely to have a better clinical outcome after standard therapy. In vitro studies confirmed that Twist1-overexpressing leukemic cells were more susceptible to cytarabine, but not daunorubicin, cytotoxicity.

Our findings suggest that, in a subset of AML patients, Twist1 plays a prominent role in the pathogenesis of the disease that leads to unique clinical phenotypes. The result indicates that Twist1 could represent a powerful biomarker that serves as a prognostic as well as therapeutic guide for patients with AML.

Disclosures

No relevant conflicts of interest to declare.
Retrospective Analysis of 59 Cases FLT3-ITD Mutation Positive Acute Myeloid Leukemia
Authors: Sun Jie , Hu Juan , Ying Shuangwei , Cai Zhen , He Jingsong , Zheng Weiyan , Luo Yi , Wei Guoqing , Zhang Jie , Ye Xiujin , Xie Wanzhuo , Huang He
Published: October 2019

Objective: Between October 2010 to May 2014, 59 patients who diagnosed as FLT3-ITD positive aAML were analyzed. All the patients were signed informed consents to agree to provide their clinical information to this study. Informed consents were approved by Ethics Committee. Peripheral white cell count at diagnose; complete remission (CR) rate; the percentage of progressing to refractory leukemia and the response to allogenic stem cell transplantation (allo-SCT) were analyzed. Better therapeutic strategy was explored.

Methods: Diagnosis was made according to the French-American-British classification system or World Health Organization diagnostic classification criteria. FLT3-ITD mutation was detected by polymerase chain reaction (PCR) and gel electrophoresis. Refractory leukemia is defined as fail to achieve CR after two courses of conventional chemotherapy, or with a short (<6–12 months) CR1 duration, or patients who have relapsed at least twice.

Results: 59 patients were diagnosed as Flt3-ITD mutation positive aAML in our centre during the last 44months, including 30 males and 29 females, with an average age at 42.2y. Flt-ITD mutation can be detected in 1/59 of M0 (1.7%); 6/59 of M1 (10.1%); 20/59 of M2 (33.8%); 9/59 of M3 (15.2%); 5/59 of M4 (8.5%); 14/59 of M5 (23.7) and 1/59 of M6 (1.7%). 23.7% (14/59) patients had high white blood cell counts (＞100*10E9/L) at diagnose. After induction chemotherapy, 50% (27/54) cases achieved CR. 53.4% (23/43, 16 cases were lost follow-up) patients met the criteria of refractory leukemia. 12 patients received allo-SCT, including 8 refractory leukemia cases. The average time from diagnose to transplantation is 6.5 months (3 to 12 months). 1 patient relapsed at 3 months after allo-SCT who was diagnosed refractory leukemia before allo-SCT, all other 11 cases remained CR with the longest follow-up time at 26 months (3 to 26 months). For the left 15 refractory leukemia cases who did not received allo-SCT, the 1-year OS rate for the 12 cases is 50% (6/12), but the 1-year EFS rate is 0 (0/12) (3 cases were lost follow-up).

Conclusion: FLT3-ITD mutation was reported to be detected in around 30% of aAML. Flt3-ITD mutation is an independent high-risk factor for aAML. Based on retrospective data from 59 cases in our centre, FLT3-ITD mutation positive aAML patients can be characterized as high white blood cell count at diagnose, lower CR rate and higher refractory leukemia percent. allo-SCT can achieve persistent remission in part of patients, even in refractory leukemia patients. Our study also implied that the CR1 is the ideal stage for FLT3-ITD mutation positive aAML patient to receive allo-SCT.

Disclosures

No relevant conflicts of interest to declare.