bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of Prothrombinase Induced Clotting Time (Pefakit ®, PiCT) Assay and its Validation in the Monitoring of Anti-Xa and Anti-FIIa Drugs.
Authors: Hoppensteadt Debra A. , Cunanan Josephine , Walenga Jeanine M. , Fareed Jawed
Published: October 2019

Unfractionated heparin (UFH), low molecular weight heparin (LMWH), fondaparinux, danaparoid, direct and indirect (via a cofactor) acting anti-FIIa and anti-Xa anticoagulant drugs are being used in patients for the prophylaxis and treatment of thrombosis. The aPTT does not reliably measure the effects of these drugs. Therefore, there is no simple/reliable method for monitoring patients administered these drugs, particularly those with renal insufficiency and obesity. The newly developed Prothrombinase Induced Clotting Time (PiCT) assay (Pefakit®; Pentapharm, Ltd.; Basel, Switzerland) was developed for monitoring heparins. The PiCT is based on the activation of plasma coagulation by a defined amount of activated FXa, phospholipid and Russell’s viper venom (RVV). The prothrombinase complex formed following recalcification is independent of endogenous thrombin mediated FV activation because the RVV directly activates FV. In this study, the PiCT was used to determine the relative anticoagulant actions of UFH, LMWH, anti-FIIa and anti-FXa drugs. Drugs were supplemented to normal human pooled plasma in a concentration range of 10- 0.08 μg/ml. Plasma samples were analyzed using the PiCT, aPTT, Heptest, thrombin time (TT), ecarin clotting time (ECT), chromogenic anti-FIIa, chromogenic anti-FXa and fibrinopeptide A (FPA) generation assays. In addition, plasma samples from patients treated with these drugs were also tested. The UFH, LMWH, anti-FIIa drugs and the indirect acting anti-FXa drugs produced a concentration-dependent prolongation of the PiCT. Different responses were obtained for each drug. The anti-FIIa drugs gave the strongest dose-response response followed by UFH, LMWH and fondaparinux, in that order. The direct acting anti-FXa agents showed a weak response in the PiCT. Compared to the other assays, the PiCT response showed better sensitivity, reproducibility and linearity over a broader dose range than the aPTT, Heptest, TT, ECT, anti-FIIa and anti-FXa assays. Samples from patients treated with enoxaparin (0.5 mg/kg IV) gave peak concentrations of 0.92± 0.11 U/ml by the PiCT, compared to 0.95± 0.24 U/ml by the anti-FXa assay. Samples from patients treated with bivalirudin, lepirudin or argatroban gave comparable values by the PiCT, aPTT and ECT. The relative prolongation of the PiCT was proportional to the inhibition of thrombin generation as measured by FPA. The PiCT is as simple to perform as the aPTT and can be adapted to any instrument capable of detecting a fibrin clot. Unlike other clot-based tests, the PiCT is sensitive to most old and new anticoagulant drugs and the results are linear through both the therapeutic and interventional dosing ranges. The PiCT may offer the first available single assay that can be universally used for monitoring most of the new anticoagulant drugs regardless of their mechanism of action.
Incidence and Disease Pattern of Venous Thrombembolism after Radical Prostatectomy.
Authors: Beyer Jan , Wessela Sven , Hakenberg Oliver , Wirth Manfred , Schellong Sebastian
Published: October 2019

Background: Little is known about the time of development and distribution pattern of venous thrombembolism (VTE) after radical prostatectomy (RPE) for malignancy.

Patients and Methods: 415 consecutive patients undergoing standard RPE were prospectively assessed regarding the incidence and distribution pattern of VTE, the hemodynamic relevance of pelvic lymphoceles (LC) and other possible risk factors for VTE. All patients underwent RPE and received heparin prophylaxis until discharge, followed by acetylsalicylic acid 100 mg daily. On day -1, 8 and 21 complete compression ultrasound, measurement of pelvic vein flow and pelvic ultrasound were performed.

Results: VTE was found in 17,3% of patients. Most thrombi were limited to calf muscle veins (55,5%). Isolated calf deep vein thrombosis (DVT) was found in 22,2% and proximal DVT in 12,5%. On day 8 and 21 the following incidences of VTE were found: calf muscle vein thrombosis 4.8 and 4.8%; isolated calf vein thrombosis 1.7% and 2.2%; proximal DVT 0.5% and 1.7%; pulmonary embolism 0.5% and 0.5%, respectively. Multivariate analysis revealed surgery time (OR 1.26), number of blood tranfusion (OR 1.73) and venous flow reduction (OR 2.83) as risk factors for the development of VTE. 33.3% of the patients developed a LC postoperatively; of these 5.3% showed a reduction of pelvic vein flow. A venous flow reduction was only seen with LC.

Conclusion: The incidence and distribution pattern of VTE after RPE is comparable to major abdominal surgery for malignancy. The development of LC is a significant risk factor for VTE, when pelvic vein flow is impaired. Since the majority of proximal VTE developed between day 8 and day 21 when the patient is already discharged, the concept of a prolonged heparin prophylaxis after RPE should be evaluated prospectively.
Azacitidine (Vidaza®) Treatment Response Assessed Using Three Alternative Dosing Schedules in Patients with Myelodysplastic Syndromes (MDS).
Authors: Lyons Roger , Cosgriff Thomas , Modi Sanjiv , Lintz Linda , Beach C.L. , Backstrom Jay T.
Published: October 2019

Previous CALGB trials published by Silverman et al (JCO2002;20:2429) have shown azacitidine to be efficacious and well tolerated in the treatment of MDS at a dosing schedule of 75 mg/m2/day x 7 days every 28 days. The objective of this phase II, multicenter, randomized, open-label trial was to study treatment response in patients with MDS with 3 alternative, subcutaneous azacitidine dosing regimens, each eliminating the need for weekend injections. The 3 alternative dosing schedules, repeated in 28-day cycles, were AZA 5-2-2 (75 mg/m2/day x 5 days, followed by 2 days of no treatment, followed by 75 mg/m2/day x 2 days), AZA 5-2-5 (50 mg/m2/day x 5 days, followed by 2 days of no treatment, followed by 50 mg/m2/day x 5 days), and AZA 5 (75 mg/m2/day x 5 days). Eligible MDS patients must have had a life expectancy of ≥ 7 months and an ECOG performance status grade of 0–3, with a FAB classification of RA, RARS, RAEB, RAEB-T, or CMML. RA and RARS patients must also have had either hemoglobin < 110 g/L with transfusion requirements, a platelet count < 100 x 109/L, or an ANC < 1.5 x 109/L. Patients experiencing International Working Group response criteria for MDS (Blood2000;96:3671) defined as complete remission, partial remission, stable disease, or hematologic improvement (major or minor) after 6 cycles of treatment were eligible to receive an additional 12 cycles. A total of 44 patients have been randomized to date with 22, 14, and 8 receiving either AZA 5-2-2, AZA 5-2-5, or AZA 5, respectively. As the AZA 5 day dosing schedule was added later to the protocol by amendment, no patients in this arm are currently evaluable for response. Most patients are male (64%) and elderly with a median age of 74 years. Most patients have RA, using either FAB (45%) or WHO (36%) definitions; another 27% and 16% have RAEB or RAEB-1, respectively. As azacitidine is incorporated into DNA/RNA through successive cell cycles, patients often require a minimum of 4 to 6 cycles of azacitidine before responses are observed. Early results show that the efficacy and tolerability observed with the alternative dosing schedules are consistent with the 75 mg/m2/day x 7 days dosing results from previous studies.
Proteasome Inhibitor Bortezomib Can Inhibit Bone Marrow Fibrosis Development in a Murine Model of Myelofibrosis.
Authors: Wagner-Ballon Orianne , Gastinne Thomas , Tulliez Micheline , Lacout Catherine , Pisani Didier , Chagraoui Hedia , Villeval Jean-Luc , Vainchenker William , Giraudier Stephane
Published: October 2019

Several lines of evidence obtained from idiopathic myelofibrosis (IM) studies are in favour of a crucial role of the NF-κB pathway activation in myelofibrosis induction. It has been demonstrated that megakaryocytes, monocytes but also CD34+ cells from IM patients present a spontaneous NF-κB pathway activation associated with transforming growth factor-β1 (TGF-β1) secretion. This growth factor has been previously shown as the main fibrogenic cytokine involved in the myelofibrosis development. Mice exposed to high systemic levels of thrombopoietin (TPO) mediated by a retroviral vector (TPOhigh mice) develop a myeloproliferative disorder featuring numerous aspects of the human disease including bone marrow fibrosis, extramedullary hematopoiesis and dysmegakaryopoiesis. Moreover, TPOhigh mice display high plasma levels of IL-1α suggesting that the NF-κB pathway may play a role in this model of fibrosis. We then conducted a study to investigate whether NF-κB inhibition in this mice model could have an impact on myelofibrosis development using the proteasome inhibitor bortezomib. One month after engraftment with TPO-overexpressing hematopoietic cells, 2 groups of 20 immunocompetent C57BL/6J mice displaying similar myeloproliferation induced by TPO overexpression were constituted. Mice engrafted were treated twice a week, with either bortezomib (1 mg/kg) or a placebo for 4 weeks. At the end of this protocol, mice from both groups were examined for histological and haematological analysis including TGF-β1 and IL-1α levels determination. Here, we demonstrate that: i) the NF-κB pathway is activated in TPOhigh spleen cells and bortezomib treatment is able to inhibit this activation; ii) bortezomib treatment is able to decrease plasma concentration of TGF-β1 and IL-1α in mice as well as TGF-β1 content in extracellular fluids of marrow and spleen; iii) myelofibrosis development is inhibited after bortezomib treatment. These results emphasize the interest of developing bortezomib treatment in IM patients.
Evidence That Fibrinogen Inhibits Leukocyte Adhesion to Fibrin Clot and Immobilized Fibrinogen by Binding to the Substrate but Not to Integrins.
Authors: Lishko Valeryi K. , Ugarova Tatiana P.
Published: October 2019

The recruitment of phagocytic leukocytes to sites of injured vessel wall plays an important role in thrombus remodeling during normal vascular repair and in the pathophysiology of thrombosis. Fibrin and fibrinogen, present in the thrombus, are potent adhesive substrates for neutrophils and monocytes. They support cellular attachment by binding cell surface receptors that belong to the β2 subfamily of integrins. Adhesive interactions of neutrophils and monocytes with polymerized fibrin and insoluble fibrinogen matrix in vivo occur in the presence of high concentrations of circulating plasma fibrinogen (~2–4 mg/ml). One important property of fibrinogen that would have a major bearing on leukocyte adhesion is its capacity to form complexes with fibrin. Therefore, by virtue of its binding to the fibrin clot and/or immobilized fibrinogen, soluble plasma fibrinogen can influence leukocyte adhesion to these substrates. In this study, the possibility that soluble fibrinogen could protect fibrin from adhesion of leukocytes was examined. Fibrinogen was an efficient inhibitor of adhesion of U937 monocytoid cells and neutrophils to fibrin gel and immobilized fibrin(ogen). An investigation of the mechanism by which fibrinogen exerts its influence on leukocyte adhesion indicated that it did not block integrins but rather associated non-covalently and weakly with fibrin(ogen) substrates. Consequently, leukocyte integrins that engage fibrinogen molecules loosely bound to the fibrin(ogen) matrix are not able to consolidate their grip on the substrate; subsequently, cells detach. This conclusion is based on the evidence obtained in adhesion studies using various β2-integrin bearing cells and performed under static and flow conditions. Furthermore, surface plasmon resonance studies, undertaken to determine the Kd of fibrinogen-fibrin interactions under flow conditions, indicated that fibrinogen formed complexes with fibrin(ogen) with micromolar affinities. Thus, these findings reveal a new role of fibrinogen in integrin-mediated leukocyte adhesion. They also imply that the anti-adhesive effect of fibrinogen may protect the thrombus from an excessive leukocyte accumulation and premature dissolution at the early stages of wound healing when hemostatic plug integrity is critical for preventing blood loss.
Survival of Limited Stage Peripheral T-Cell Lymphoma Is Similar To Diffuse Large B-Cell Lymphoma.
Authors: Savage Kerry J. , Chhanabhai Mukesh , Voss Nicholas , Tamara Shenkier , Gascoyne Randy D. , Connors Joseph M.
Published: October 2019

Background: Peripheral T-cell lymphomas (PTCL) represent a heterogeneous group of diseases with an overall poor prognosis. Little information is available regarding the outcome of PTCL patients who present with limited stage disease. We sought to determine the outcome of PTCL patients presenting with limited disease in comparison with a cohort of patients with limited stage diffuse large B-cell lymphoma (DLBCL).

Methods: In a retrospective analysis we identified all patients with limited stage (non-bulky (<10cm) stage I/II disease no symptoms) PTCL diagnosed at the British Columbia Cancer Agency (BCCA) between 1983 and 2004. Patients were excluded if they had cutaneous anaplastic large cell lymphoma (CutALCL) (n=13), NK/T-cell lymphoma nasal type (n=9) or primary CNS/ocular involvement (n=6).

Results: Thirty-seven patients with PTCL were identified according to the World Health Organization Classification: ALK-neg ALCL 8 (22%); PTCL-unspecified (PTCLUS) 28 (78%); enteropathy associated TCL (EATL) 1 (3 %). The majority received CHOP-type chemotherapy (n=31, 86%), most with brief chemotherapy followed by involved-field radiation (n=19, 61%). The 5 y OS and PFS was similar between PTCLUS and ALK-neg ALCL. There was no difference in survival between extranodal and nodal cases. The outcome of PTCL patients (including ALK-neg ALCL and PTCLUS) was compared to a cohort of limited stage DLBCL patients (excluding CNS/ocular lymphoma) (n=305) diagnosed over the same time period and treated similarly. There was no difference in 5 y OS or PFS (Figure 1,2). Interestingly, there were no late relapses observed in PTCLUS, in marked contrast to DLBCL.

Conclusions: Limited stage PTCL is rare, however outcomes appear to be comparable to early stage DLBCL, supporting that they should be treated in a similar manner. Unlike limited stage DLBCL where late relapses occur, a plateau in the progression-free survival curve is observed, highlighting a distinct natural history for limited stage PTCL.

Overall Survival Limited Stage PTCL vs DLBCL p=.18 Overall Survival Limited Stage PTCL vs DLBCL p=.18 Progression-Free Survival Limited Stage PTCL vs DLBCL p=.07 Progression-Free Survival Limited Stage PTCL vs DLBCL p=.07
Extended Thromboprophylaxis with Fondaparinux after Major Orthopedic Surgery of the Lower Limbs: The EXPERT Study.
Authors: Singelyn Francois
Published: October 2019

Background: The benefit-to-risk ratio of extended fondaparinux therapy was demonstrated in patients undergoing hip fracture surgery, but not in patients undergoing major joint arthroplasty. In addition, no data on the use of fondaparinux in patients in whom a continuous neuraxial/deep peripheral nerve blockade is performed are available.

Objective: The EXPERT trial was an open-label, international study in patients undergoing major orthopedic surgery designed to evaluate the overall efficacy and safety of 2.5 mg fondaparinux when (I) given daily for 4±1 weeks after surgery, and (II) a “therapeutic window” is applied due to concomitant use of a neuraxial/deep peripheral indwelling catheter.

Patients/Methods: Patients undergoing major orthopedic surgery received once-daily subcutaneous 2.5 mg fondaparinux starting post-surgery for 4±1 weeks. In patients with a neuraxial/deep peripheral indwelling catheter, one of the planned doses of fondaparinux was omitted, the catheter was removed 36 hours after the previous fondaparinux dose, and the next fondaparinux dose administered 12 hours after catheter removal. The primary endpoints were symptomatic venous thromboembolism (VTE) and major bleeding 5±1 weeks after surgery.

Results: 5704 patients (mean age ± SD: 66.1 ± 11.9 years) were recruited into the intention-to-treat (ITT) population between July 2003 and October 2004. A neuraxial/deep peripheral indwelling catheter was placed in 1630 (28.6%) of them. They underwent surgery for total hip replacement (51.6%, n=2941), total knee replacement (39.7%, n=2263), hip fracture (6.2%, n=353), or other indications (2.6%, n=148). Fondaparinux was given for a median of 35 (range: 1–105) days. In the ITT population, the rate of adjudicated confirmed symptomatic VTE was 1.0% (54/5387). It was 0.8% (13/1535) in patients with catheter and 1.1% (41/3852) in patients without catheter, with an odds ratio of 0.79 (90% CI: 0.47 to 1.34; p=0.47) in favor of patients with a catheter. The upper limit of the 90% CI was below the predetermined criterion of 1.75 for the non-inferiority of patients with a catheter versus patients without a catheter. Comparable results were observed in the per-protocol population. The overall rate of major bleeding was 0.8% (42/5382). It was 0.9% (35/3850), and 0.5% (7/1532) in patients with and without catheter, respectively. No spinal or perineural hematomas occurred. At 5±1 weeks, 23 (0.40%) patients had died.

Conclusions: 2.5 mg fondaparinux given daily for 4±1 weeks after major orthopedic surgery was both effective and safe in routine practice. This high benefit-to-risk ratio was maintained in patients in whom a 48-hour “therapeutic window” was applied due to placement of a neuraxial/deep peripheral indwelling catheter.
Successful Pregnancy Outcome in 5 Patients with Thrombotic Thrombocytopenic Purpura.
Authors: Scully Marie , Starke Richard , Lee Richard , Mackie Ian , Machin Samuel J. , Cohen Hannah
Published: October 2019

Pregnancy is an initiating event for acute thrombotic thrombocytopenic purpura (TTP). Patients with a history of TTP have a high risk of relapse during pregnancy with associated maternal and fetal morbidity/mortality. We report 5 TTP patients who underwent serial clinical and laboratory monitoring (haemoglobin, platelets, blood film, reticulocytes, lactate dehydrogenase (LDH), ADAMTS-13 activity and IgG antibodies) and treatment within a specialist obstetric/haematology clinic, resulting in successful pregnancy outcomes. Patients: Cases 1 and 2 presented with TTP in their first pregnancy and had second trimester fetal losses. Case 3 had four TTP relapses and soon after achievement of clinical remission became pregnant. Case 4 had a single acute episode of TTP 6 years prior to pregnancy and normal ADAMTS 13 activity at the onset of pregnancy. Case 5 presented with acute TTP and a left sided stroke at 16 weeks gestation. ADAMTS-13 activity was <5% at onset of pregnancy in cases 1–3 and at presentation of TTP during pregnancy in case 5. Only case 3 had IgG antibodies to ADAMTS13. Cases 1–3 received regular plasma exchange (PEX) every 2 weeks initially, starting during the first trimester, with ADAMTS 13 activity levels maintained >15% during pregnancy (range 15–78%, normal range 66–126%) by collagen binding assay. Case 4 had normal laboratory parameters, including serial ADAMTS 13 activity levels, throughout pregnancy, requiring no specific therapy for TTP. Case 5 received intensive PEX (84 procedures) and pulsed methylprednisolone during pregnancy. Rituximab (375mg/m2, weekly for 4 weeks) was given at 28 weeks gestation with no observed toxicity except asymptomatic transient neonatal neutropenia. All 5 cases continued low dose aspirin (75 mg daily) throughout pregnancy. Case 1 and 2 received prophylactic low molecular weight heparin (LMWH) and cases 3 and 5 received treatment doses of LMWH for pulmunory emboli and TTP associated stroke at presentation. Live healthy infants were delivered in all 5 cases in the third trimester (31–41 weeks). These data suggest successful pregnancy outcome is achievable in patients with a history of TTP/acute TTP with therapy based on close clinical and laboratory monitoring. Regular PEX in patients with a history of TTP and severely reduced ADAMTS 13 activity/IgG antibodies to ADAMTS 13 at the onset of pregnancy was associated with successful pregnancy outcome. Serial ADAMTS 13 activity and antibody levels during pregnancy and post-partum provided a useful adjunct to decision making on intensity of treatment.
Stepwise Development of Committed Progenitors in the Bone Marrow That Generate Functional T Cells in the Absence of the Thymus.
Authors: Dejbakhsh-Jones Sussan , Garcia-Ojeda Marcos E. , Chatterjea Devavanii , Mukhopadhyay Aditi , Weissman Irving L. , Strober Samuel
Published: October 2019

We identified committed T cell progenitors (CTPs) in the mouse bone marrow that have not rearranged the TCR β gene, express a variety of genes associated with commitment to the T cell lineage including GATA-3 and TCF-1, Cβand Id2, and show a surface marker pattern (CD44+CD25-CD24+CD5-) that is similar to the earliest T cell progenitors in the thymus. More mature committed intermediate progenitors (CIPs) in the marrow have rearranged the TCR gene loci, express Vαand Vβgenes as well as CD3ε, but do not express surface TCR or CD3 receptors. CTPs, but not progenitors from the thymus, reconstituted the αβ T cells in the lymphoid tissues of athymic nu/nu mice. These reconstituted T cells vigorously secreted IFN- γ after stimulation in vitro, and protected the mice against lethal infection with murine cytomegalovirus (MCMV). Also CTPs from the parent strain can reconstitute an F1 MHC haplotype mismatched host. In conclusion, CTPs in wild-type bone marrow can generate functional T cells via an extrathymic pathway.
High Plasma Cell Labeling Index (PCLI) Is an Adverse Prognostic Factor in Bortezomib-Treated Patients with Relapsed Myeloma.
Authors: Gidron Adi , Tariman Joseph , Shabbir Munira , Mehta Jayesh , Singhal Seema
Published: October 2019

Del(13) is a powerful adverse prognostic factor in patients with myeloma. A small subset analysis from a randomized multi-center study comparing dexamethasone to bortezomib for relapsed myeloma suggested that the outcome of bortezomib-treated patients was not affected by del(13) (Jagannath et al, ASCO 2005). We studied patients with relapsed myeloma treated with bortezomib on whom complete karyotype data were available to determine the effect of del(13) on response rates and survival. To ensure consistency, this analysis was confined to 47 patients (44–85 y; median 62) who received all their therapy at Northwestern and in whom all cytogenetic analyses were performed in a single laboratory. 40 had failed prior thalidomide and 32 had failed prior high-dose therapy. All patients were treated with bortezomib as a single agent in the standard fashion initially. Dexamethasone ± thalidomide were added if there was no response. Progression after initial response or lack of response to bortezomib ± thalidomide ± dexamethasone was classified as treatment failure. Overall response rate (≥50% decline in paraprotien) was 72%. Complete clearance of paraprotein was seen in 32%. The median event-free (EFS) and overall (OS) survival durations from the start of therapy were 227 and 777 days. OS was higher in responders (777 vs 373 days; P=0.015) as was EFS (259 vs 120 days; P=0.0001). As the table shows, del(13) did not affect response rates.

Response to bortezomib by del(13) status (P=0.69) Paraprotein response Del(13) (n=13) No del(13) (n=34) Any response vs no response; P=0.31 No response 5 (38%) 8 (24%) 50–94% decline 3 (23%) 10 (29%) 95–99% decline 2 (15%) 4 (12%) 100% decline 3 (23%) 12 (35%)

No variable influenced response significantly in Cox analysis. However, there was a trend (P=0.07) towards lower response rates in patients relapsing after prior transplant. When PCLI (available in 34 patients) was included in the model, there was a trend (P=0.08) towards lower likelihood of 100% responses with del(13). Response to bortezomib and age ≤ 60 were associated with significantly better EFS with/without PCLI in the model. PCLI ≥1% was the only factor associated with poorer OS when included in the model. When PCLI was excluded, response to bortezomib was associated with significantly superior OS.

Figure Figure

While OS and EFS did not seem to be influenced by del(13), the curves shown below suggest that differences may become apparent with longer follow-up in a larger number of patients.

Figure Figure

Thus, small patient numbers in this study and that of Jagannath et al, and a trend towards a lower likelihood of complete paraprotein clearance with del(13) in our experience suggests that more data are needed before any definitive conclusions can be drawn on the impact of del(13) on bortezomib therapy.
Generation of a Novel Mouse Model of Cooley’s Anemia.
Authors: Huo Yongliang , McConnell Sean , Sun Chiao-Wang , Wu Li-Chen , Ryan Thomas M.
Published: October 2019

A novel mouse model of Cooley’s Anemia (CA) has been generated by targeted gene replacement of the adult murine α-globin genes with human α-globin and the adult mouse β-globin genes with a human γ- to β-globin gene switching cassette containing a β0 thalassemic allele. A positive-negative gene replacement construct was designed to simultaneously delete both of the adult mouse α-globin genes by inserting a 3.8kb human α1-globin gene and a hygromycin marker gene flanked by loxP sites in murine embryonic stem (ES) cells. Both adult murine β-globin genes were deleted by insertion of an Hprt marker gene that was later replaced by a 5.6kb human Aγ-globin gene, 4.1kb human β0-globin gene, and a loxP flanked hygromycin marker gene by a “tag and exchange” strategy. The human β0-globin knock-in allele contains a single G to A nucleotide mutation in the first base of intervening sequence 1 [β0-IVS1(GtoA)-globin]. This single base change destroys the splice donor site of IVS-1 resulting in the recruitment of several cryptic splice sites. Use of these cryptic splice sites produces a frameshift in the mRNA that results in no functional β-globin polypeptide synthesis from this allele. This β0-IVS1(GtoA)-globin gene mutation is a naturally occurring β0 thalassemia allele found in Mediterranean populations. Chimeric mice were generated from both the α- and β-globin targeted cells lines. After germline transmission the α- and β-globin targeted mice were bred to cre recombinase transgenic mice to remove the marker genes. Heterozygous CA mice exhibit β thalassemia intermedia. The α- and β-globin targeted mice were interbred to produce animals homozygous for the human α1- and γβ0-globin knock-in alleles. Instead of dying early in fetal life as all current homozygous β0 thalassemia mouse models, these novel homozygous CA mice survive solely on high levels of human fetal hemoglobin (α2γ2) throughout fetal development. Newborn homozygous CA mice are blood transfusion dependent similar to β thalassemia major infants. This novel model of CA has multiple improvements over existing models of β thalassemia. Namely, CA mice express 100% human hemoglobin in their RBCs, mimic the human γ- to β-globin gene switch, synthesize no functional β-globin chains after birth, have a single mutant human β0-globin knock-in allele at each β-globin locus, and are blood transfusion dependent for life after birth.
16189 Mitocondrial Variant and Iron Overload.
Authors: Salvador Maria , Gonzalez Fernando A. , Ropero Paloma , Olga Briceño , Eduardo Anguita , Ana Villegas
Published: October 2019

Hereditary haemochromatosis(HH) is one of the most common autosomal recessive diseases in Europe, this is characterized by iron overload. There are two predominant mutations associated with the disease in the HFE gene: C282Y and H63D. The majority of haemochromatosis patients are homozygous for C282Y mutation and a small proportion are compound heterozygous C282Y/H63D. Not all C282Y homozygotes and compound heterozygous will show signs of iron loading. Several studies have shown the incomplete penetrance of haemochromatosis and suggest that iron overload is influenced by both enviromental and genetic factors wich may modify the phenotipic expression of the disease.

Iron loading is associated with impaired mitochondrial function. It has been reported that the T16189C variant of mitochondrial DNA may act as a modifier increasing iron loading in HH. 16189 variant lies in the first hypervariable region (HV1), close to the sequences controlling replication and transcription.

Aim: to determine the frequency of the 16189 variant in normal population and in a group of HH pacients with mutations in HFE gene or iron loading due to Mayor or Intermediate b-Thalassemia.

Materials and methods: We stablished the frequence of 16189 variant in normal population by studying it in a group of 102 blood donors which did not have any mutation in HFE gene. We determined the variant in 27 C282Y homozygous, 32 compound heterozygous C282Y/H63D and 31 H63D homozygous and 16 β-thalassemics aswell.

DNA was extracted from peripheral blood, HV1 region was amplified by PCR using primers:

F(15800–15819)CAAGTAGCATCCGT ACTATA; R(16344–16325)GTAATGTGCTATGTACGGTA The product of amlification was digested with MnlI acording to manufacturer’s specifications. In wild type there is a restriction site at position 16189 which is absent in mutant allele. All samples lacking MnlI site were directly sequenced in a ABI PRISM 310 Genetic Analyzer.

Statistical analysis was performed using the SPSS package.

Results: shown in table 1

Conclusions: Our results do not show significant differences between the groups analyzed, but no significant increased frequency of the variant in the group of compound heterozygous. With this study we cannot conclude that mitochondrial function influences iron overload. This is could be dued to the short number of samples analyzed. In β-Thalassemia iron loading is modifed by an increased absorption(TM y TI) and transfusions(TM). At the present we are studying other factors that could play an important role in iron homeostasis: Transferrin receptor, Hepcidine and Hemojuveline.

Table 1. Results Controls C282Y/C282Y C282Y/H63D H63D/H63D β-Thalassemia 16189C 12% 11.1% 18.7% 6.4% 12.5%
Ca 15-3 a Marker for the Megaloblastic Anaemia?.
Authors: Mettivier Vincenzo , Pezzullo Luca , Finizio Olimpia , Rocco Stefano , Bene Lucia , De Rosa Carlo
Published: October 2019

Ca 15-3 is a glycoprotein present in the cells of the mammary carcinoma and in some epithelial cells. Is a marker used for the monitoring of the breast and gastrointestinal carcinoma. The megaloblastic anaemia is an anaemia characterized by deficit of absorption of Vitamin B12 and is associated with gastritis atrophic and the target cell is the parietal gastric cell.

In our institution, from June 2003 to December 2004, the level of Ca15-3 and of others tumour markers (CEA; Ca125; Ca19.9; alfa-FETO) they have been tested in the serum of 16 patients (9 male and 7 female with median age of 64,5 and range of 37–80 years) with de novo megaloblastic anaemia, 2 patients were gastrectomized. In all patients has been effected: esophagogastroduodenoscopy and control anti-parietal gastric cells antibody (APCA).

Increase level in the serum of CA 15-3 with normal level of other tumour markers have been found in 14/16 patient with median value of 61 U/ml (range 35–100 U/ml) in two patients (gastrectomized) the value of CA 15-3 was normal. Besides in 12/14 patients have shown positivity for the APCA, only in two patients has been diagnosed a gastritis atrophic, in the other patients has been observed a normal gastric mucous.

After a median observation of 24 months any patient has developed a mammary or gastro-intestinal carcinoma

These results indicate what the increased level of CA 15-3 antigen in patient with megaloblastic anaemia is positively correlated with APCA and with the presence of a normal gastric mucous. These clinical conditions make to suppose the destruction from the APCA of the parietal gastric cells with the liberation of this glycoprotein and this is shown in two patients gastrectomized with presence of APCA and not increased level of the CA 15-3.

In conclusion the CA15-3 antigen is probably an specific marker for the diagnosis of megaloblastic anaemia and is probably associated with the destruction of parietal gastric cells.
MP4, a Human Hemoglobin Modified with Maleimide-Peg, Reduces Sickling of Red Blood Cells in an In Vitro Model.
Authors: Winslow Robert , Oliva Mariana , Intaglietta Marcos , Vandegriff Kim , Baker Dale , Malavalli Ashok
Published: October 2019

Human hemoglobin surface-modified with maleimide polyethylene glycol (MP4) has been shown to be effective in transporting O2 and maintaining tissue perfusion in several animal models. MP4 appears to be free of vasoconstriction, and clinical trials have shown MP4 to be free of the side-effects commonly associated with hemoglobin-based products. Since MP4 is effective in delivering O2 to capillaries in the hamster microcirculation, our hypothesis is that it would be of therapeutic benefit in Sickle Cell Disease. In order to test this hypothesis, we measured pressure that develops as dilute sickle cells pass through a filter with and without MP4. Cells from patients with sickling disorders were washed with phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) and suspended at 1% hematocrit in the presence and absence of MP4 (2 g/dL). The suspension was then pumped through a hollow fiber oxygenator (Living Systems Instrumentation, Model Number OXR) using an array of 162 polypropylene fibers, 300 mm OD, 240 mm ID, surface area 0.0115 m2. After emerging from the oxygenator the suspension passed through a filter (Millipore Isopore, pore size 5.0 mm, 13 mm diameter) at a pump rate of 1 mL/min. All experiments were performed at 21oC. After achieving a steady flow rate, the oxygenator gas was abruptly changed to N2, and the PO2 and pressure of the prefilter suspension was continuously monitored. As PO2 fell, pressure began to rise in all experiments, but in the presence of MP4, the pressure rise was offset, being lower by about 10 mm Hg, indicating a favorable effect on sickling at constant PO2. If MP4 was saturated with CO, sickling was inhibited, even in the absence of O2. These results indicate that MP4 has a significant effect on sickling, apart from any effect it might have in promoting capillary perfusion. Furthermore, the results confirm that despite its low P50 (~5 mm Hg), MP4 does not “steal” O2 from sickle cells. Supported by grants HL64395 and HL076163 from the NHLBI
Inter-Site Validation of a Single Breath Hold T2* MR Technique for Heart and Liver Iron Measurement.
Authors: Kirk Paul , Anderson Lisa J. , Tanner Mark A. , Galanello Renzo , Matta Gildo , Tan Ru S. , Ng Ivg , Lai Angie , Chen Kevin , Firmin David N. , Pennell Dudley J.
Published: October 2019

Background

Approximately 60,000 people are born with thalassaemia major every year. The average life expectancy of thalassaemia major patients is 35 years due to iron overload Cardiomyopathy. The cardiomyopathy is reversible when treated early, but once heart failure is established it is often rapidly progressive, and unresponsive to treatment. The single breath hold (SBH) T2* technique has been validated as the most robust and reproducible non-invasive measurement of myocardial and iron load. Our aim in this study was to validate the transferability and reproducibility of this technique in different scanners worldwide.

Methods

We aim to compare the reproducibility in six different sites worldwide as part of an NIH funded grant (R01-DK66084-01). So far, two of these sites have been validated: Singapore (Siemens Sonata, 1.5T scanner) and Cagliari, Italy (GE Signa, 1.5 T scanner). At both validation sites, 10 patients were scanned for heart and liver T2*, and scans were repeated for interstudy reproducibility. All patients then flew to London to be rescanned on our reference Siemens Sonata scanner.

Results

Of the 20 patients scanned, 70% had myocardial iron loading (T2* <20ms) and in 10% the myocardial iron loading was severe.

Liver iron loading was present in 65% of patients and in 30% this was severe.

The coefficient of variation (COV) for the heart T2* measurements between the local sites and London was 5.9% and 4.9% yielding an average coefficient of variation across both sites of 5.4% (figure 1).

The coefficient of variation (COV) for the liver T2* measurements between the local sites and London was 11.3% and 3.9% yielding an average coefficient of variation across both sites of 7.6% (figure 2).

There was no significant correlation between liver and myocardial loading.

Conclusion

These are the first data demonstrating the transferability of the SBH T2* technique and the clinical validation from the 2 collaborating centers were excellent for both heart and liver measurements. Further MR sites confirmed for validation include Children’s Hospital of Philadelphia (USA); Ramathibodi Hospital, Bangkok (Thailand); and Chinese University Hong Kong.

Figure 1 Figure 1. Figure 2 Figure 2.
Modulation of Platelet Adhesion by Antithrombin.
Authors: Loncar Robert , Stoldt Volker , Thomas Volker , Zotz Reiner B. , Scharf Rudiger E.
Published: October 2019

In this study, we evaluated the influence of antithrombin on platelet adhesion onto immobilized fibrinogen using an in vitro system simulating venous and arterial flow. Platelets in anticoagulated whole blood (PPACK, 40μM) were labeled with mepacrine (10μM). Adhesion of platelets onto fibrinogen-coated glass cover slips was assessed in a rectangular flow chamber (shear rates of 13 s−1 to 1500 s−1). Platelets were visualized at 15 sec, 1 and 5 min following perfusion using a fluorescence laser-scan microscope. In parallel, the effects of supraphysiological supplementation of blood with antithrombin (2.8 IU/ml of blood) on platelet adhesion rates was evaluated. During perfusion, platelet adhesion onto fibrinogen linearly increased with exposure time and shear rates. Within the first min of perfusion, an inverse correlation between platelet adhesion and plasma antithrombin activity was observed at shear rates of 13 s−1 and 50 s−1 (r=−0.48 and r=−0.7, p each <0.05). Significant differences in platelet adhesion (1786±516 U vs. 823±331 U, p<0.05) related to low (92±3.3%) and high (117±4.1%) antithrombin activity was observed at a flow rate of 13s−1 within first minute. Further supplementation of anticoagulated whole blood with antithrombin (activity up to 280 %) decreased the rate of platelets adhesion (ratio of adhesion at 1 and 5 min) about 35% when compared to nonsupplemented blood (1.25 ± 0.17 vs. 1.95 ± 0.4, p=0.008). Application of heparine as anticoagulant did not enhance the antiadhesion properties of antithrombin. Our findings are in accordance with the “low shear phenomenon” of arterial thrombus progression, i.e. thrombus enlargement at distal areas with reduced flow or even stasis. Moreover, the observation that antithrombin significantly suppressed platelet adhesion onto immobilized fibrinogen under low flow conditions is of therapeutic interest and needs further evaluation.
New Coagulation Assays Reference Ranges for Healthy Adults Using the Modern Sysmex CA-1500 Coagulometer.
Authors: Wakeman Lisa , Munro Roger , Dorward Nick , Benton Ann , Gibb Andy , Al-Ismail Saad
Published: October 2019

Reference ranges (RRs) in coagulation are applicable only to specific analyser and reagent combinations and frequently need to be re-established if any of these are changed. In no other sphere of clinical laboratory practice are RRs more affected by such a wide range of multiple demographic and pre-analytical variables. For most routine clinical laboratories therefore, the collection of multiple, separate RRs is not feasible so a representative group of healthy adults such as laboratory staff frequently constitute the reference population from which these limits are calculated. Early morning venous samples were collected into glass B-D Vacutainers (Ref: 367691) from 221 healthy laboratory personnel (F= 159; M = 62) aged 20–63 yrs for both gender. Age groups were equally represented. Samples were processed on a Sysmex CA-1500 analyser within 1 hour of collection. Appropriate NCCLS guidelines were followed throughout. Reagents employed were - Actin FSL (APTT); Innovin (PT); Dade-Behring reference, calibration and deficient plasmas (factor assays); Dade-Behring kit ref: OWWR15 (ATIII); Chromogenix kit ref: 82209863 (Protein C). Outliers were excluded, data examined for normal distribution from histograms and significance levels calculated from the Anderson - Darling test of normality. RRs for normally distributed parameters were calculated using means ± 2SDs. RRs for non-normally distributed parameters were calculated using the log natural transformation and the antilog of 2.5- and 97.5- percentiles. Italicised parameters shown below are non-normally distributed.

Parameter Reference Range Anderson Darling P-Value P-value for normal distribution Mann Whitney U-test (M versus F) *=significant difference PT sec 10.0 – 11.8 <0.005 0.003* APTT sec 24.7 – 31.7 0.006 0.232 TCT sec 13.8 – 17.4 0.035 0.198 Fib g/L Clauss 1.6 – 4.2 0.190 t-test not significant Fib g/L Derived 2.1 – 4.9 0.200 t-test not significant II % 82 – 133 <0.005 0.019* V% 70 – 150 0.021 0.303 VII % 60 – 164 0.008 0.037* X% 75 – 147 0.539 t-test not significant VIII % 48 – 204 <0.005 0.520 IX % 65 – 142 <0.005 0.275 XI % 61 – 142 <0.005 0.394 XII % 59 – 133 0.088 t-test not significant Protein C % 75 – 160 0.036 0.024* ATIII % 86 – 128 0.329 t-test not significant

Kruskal Wallis tests on our data indicate that all coagulation factors are positively associated with age except factors IX and XII. Significant differences (p=0.014) in factor VIIIc was found between those of blood group O and non group O. Significant correlation was found between declining APTTs and associated increasing factor VIIIc when measured in individual volunteers.
Cooperative Action of 1alpha,25-Dihydroxyvitamin D3 and Retinoic Acid in NB4 Acute Promyelocytic Leukemia Cell Differentiation Is Transcriptionally Controlled.
Authors: Bastie Jean-Noel , Balitrand Nicole , Guillemot Isabelle , Chomienne Christine , Delva Laurent
Published: October 2019

All-trans-retinoic acid (RA) and 1alpha,25-dihydroxyvitamin D3 (1,25D3) are involved in the control of hematopoiesis and have been suggested to play a role in cellular differentiation and are as such potent inducers of differentiation of myeloid leukemia cells. In this study, we have shown that in promyelocytic NB4 cells, addition of 1,25D3 enhances terminal granulocytic RA-dependent differentiation concomitant with the enhanced activation of the RA-transcriptional activity through an RARbeta promoter. By EMSA and ChIP assays, we further demonstrate that while both VDR and RAR are bound to the RARbeta promoter in NB4 cells, addition of 1,25D3 increases VDR binding to this promoter while that of RA induces the release of VDR and increases the binding of RAR. Thus, contrary to normal myeloid cells, 1,25D3 does not act as a transrepressor of RA-transcriptional activity in leukemic cells suggesting that transcriptional regulation of RA-target genes may be modified in malignant cells. In promyelocytic leukemic cells the combination of 1,25D3 and RA results in both enhanced transactivation and differentiation.
Simultaneous Administration of AMN107 and Imatinib in the Treatment of Imatinib-Sensitive and Imatinib-Resistant Chronic Myeloid Leukemia.
Authors: Griffin James D. , Weisberg Ellen L.
Published: October 2019

Chronic myelogenous leukemia (CML) and Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) are caused by the Bcr-Abl tyrosine kinase oncogene. The Abl inhibitor imatinib is an effective, frontline therapy for early, chronic phase CML. However, accelerated or blast crisis phase CML and Ph+ ALL patients often relapse because of drug resistance that results from the emergence of imatinib-resistant point mutations within the Bcr-Abl kinase domain. The aminopyrimidine ATP-competitive inhibitor, AMN107, was designed to fit into the ATP-binding site of the Bcr-Abl protein in such a way as to exhibit higher efficacy against imatinib-resistant Bcr-Abl point mutants. AMN107 is active against many imatinib-resistant Bcr-Abl mutants in vitro and in vivo, and is significantly more potent than imatinib against wild-type Bcr-Abl. AMN107 is currently showing promise in phase I/II clinical trials involving CML patients who are unresponsive to imatinib, and thus could potentially be used as a single agent in selected patients resistant or intolerant to imatinib. Alternatively, the use of more than one inhibitor of Abl should effectively lower the number of residual Bcr-Abl-expressing cells having the potential to undergo mutation, and therefore could potentially suppress the emergence of drug-resistant Bcr-Abl mutations. Thus, AMN107 and imatinib could be administered together to achieve higher responsiveness in CML patients. In the current study, we investigated the combination of imatinib and AMN107 in a panel of wild-type and imatinib-resistant Bcr-Abl-expressing cell lines, including 32D.p210, K562, F486S-Ba/F3, F317L-Ba/F3, M351T-Ba/F3, and T315I-Ba/F3. We found evidence of additive to synergistic effects in several of the cell lines examined. In addition, the combination of AMN107 and imatinib was studied in vivo using a bioluminescent Bcr-Abl model of CML. Mice harboring murine 32D.p210 cells engineered to stably express firefly luciferase were treated with vehicle, AMN107 alone (15mg/kg), imatinib alone (75mg/kg), or both AMN107 and imatinib at their respective doses. Mice treated with both agents were observed to carry an overall lower tumor burden (as measured by levels of total body bioluminescence and percent spleen weights) than vehicle-treated mice and mice treated with each agent alone. These results suggest that the combination of imatinib and AMN107 may be a more effective treatment for CML than either agent alone.