Anti-Thrombin IgA in a Patient with Multiple Myeloma Leading to In Vitro Interference in Multiple Coagulation Tests and Confounding Diagnosis.

BACKGROUND: We report the case of a 59-year-old multiple myeloma patient in whom an anti-human thrombin IgA antibody led to prolonged in vitro coagulation times, suggesting inhibitors to all intrinsic coagulation factors in the absence of spontaneous bleeding. METHODS: Routine and extensive special coagulation tests, in vivo bleeding time, and specific antibody testing were performed. RESULTS: Although the patient did not suffer from spontaneous bleeding and had a normal in vivo bleeding time, the anti-human thrombin IgA autoantibody affected all coagulation assays involving human thrombin in vitro, mimicking inhibitors to intrinsic coagulation factors. As the IgA paraprotein and the IgA antibody virtually disappeared after autologous stem cell transplantation, the coagulation tests also largely normalized. CONCLUSION: Antibodies to human thrombin may interfere with all coagulation assays involving thrombin, imitating a severe coagulopathy. However, in vivo they do not necessarily lead to strongly increased bleeding tendency. Complex and ambiguous coagulation abnormalities should be evaluated and treated in an interdisciplinary setting, including a highly specialized coagulation laboratory, from the beginning.

Evaluation of two fully automated ADAMTS13 activity assays in comparison to manual FRET assay.

INTRODUCTION: The objective of the present study was to evaluate and compare the validity and utility of two fully automated ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) activity assays for clinical diagnostic decision-making and to compare their performance. METHODS: Two automated ADAMTS13 activity assays (Werfen HemosIL® AcuStar ADAMTS13 Activity, Technoclone Technofluor ADAMTS13 Activity) were compared with a manual FRET assay (BioMedica ACTIFLUOR ADAMTS13 Activity). The following samples were used: 13 acute phase TTP (thrombotic thrombocytopenic purpura) samples from 11 different patients, one sample from a patient with congenital ADAMTS13 deficiency, 16 samples from control patients, three follow-up samples from TTP patients in long-term remission and one sample from a patient with stem cell transplantation related thrombotic microangiopathy (TMA). The WHO 1st International Standard for ADAMTS13 and several dilutions of normal plasma with ADAMTS13-depleted normal plasma were also tested. Statistical analysis included descriptive statistics, sensitivity and specificity, Passing & Bablok regression and Bland-Altman plot. RESULTS: The quantitative comparison between the HemosIL® (x) and Technofluor (y) methods showed a strong correlation (Pearson r = 0.98, n = 49). When considering an ADAMTS13 activity of <10% as a hallmark for the diagnosis of TTP, two fully automated assays were both able to identify all TTP- and non-TTP-samples correctly, resulting in sensitivities and specificities of 100%. CONCLUSION: Both fully automated ADAMTS13 activity assays showed a good diagnostic performance and quantitative correlation among themselves, discriminating reliably between TTP- and non-TTP-patients.

Major cardiac surgery with recombinant FIX Fc fusion protein replacement in hemophilia B: a case report.

The introduction of extended factor IX (FIX) products has significantly facilitated the treatment of hemophilia B patients. However, optimal perioperative management remains a topic of hot debate, particularly in surgeries with high bleeding risk. For the first time, we report here a patient with mild hemophilia B and degenerative aneurysms of aortic root and ascending aorta undergoing elective Bentall's operation with full cardiopulmonary bypass, who was successfully managed with eftrenonacog alfa (Alprolix®), a recombinant FIX Fc fusion protein (rFIXFc). rFIXFc could safely be monitored using the Pathromtin SL aPTT-reagent. No significant bleeding was noted intraoperatively despite systemic heparinization as well as postoperatively. Higher doses of rFIXFc were inevitable to reach target FIX levels intraoperatively, whereas in the post-surgery setting stable FIX concentrations were maintained with only few rFIXFc injections facilitating fast wound healing and remobilization of the patient.

Intermittent retinal artery occlusions as the first clinical manifestation of polycythemia vera: a case report.

BACKGROUND: Polycythemia vera (PV) is a myeloproliferative neoplasm with increased hemoglobin, hematocrit, platelet count and leukocytosis, resulting in increased blood viscosity. PV which is initially presenting with ocular symptoms is rare, but irreversible retinal vessel occlusions leading to the diagnosis of PV have been described in literature. CASE PRESENTATION: We describe a patient with PV, initially presenting with attacks of monocular temporary loss of vision due to intermittent retinal artery occlusions of different retinal arteries. The patient was immediately treated with phlebotomy and the impaired arterial retinal perfusion could be restored without permanent retinal ischemia. We were able to document these transient arterial occlusions with fundus photography as well as fluorescein angiography. To the best of our knowledge, a case like this has never been documented before. CONCLUSION: This report is pertinent, in order to raise awareness among clinicians for polycythemia vera, as it can in fact be used as a differential diagnosis for patients with retinal artery occlusion. We would like to stress that early therapy might reverse the vessel complications.

Increased levels of NETosis in myeloproliferative neoplasms are not linked to thrombotic events.

Morbidity and mortality of Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs) are mainly determined by thromboembolic complications. Thrombus formation is facilitated by a neutrophil-specific form of cell death linked to neutrophil extracellular trap (NET) formation (NETosis). Preclinical and clinical data suggested a potential link between NETosis and thrombosis in MPNs. In this study, we aimed to define the impact of NETosis on clinical end points in a large MPN cohort. NETosis was induced in vitro by ionomycin and quantified by enzyme-linked immunosorbent assay-based nucleosome release assays as well as fluorescent staining of free DNA in samples from 103 MPN patients and 28 healthy donors. NETosis rate was correlated with a broad set of clinical data, such as MPN subtype, mutational status, laboratory variables, history of thrombotic events, and treatment types. Triggered NETosis levels were clearly higher in MPN patients than in healthy donors. Positivity for JAK2 V617F or exon 12 as well as CALR mutations correlate with increased NET formation. However, neither JAK2 allelic burden nor history of thromboembolic complication nor the presence of other risk factors for thrombosis (eg, leukocytosis) were associated with the rate of NETosis. In addition, none of the analyzed laboratory parameters nor the type of treatment significantly impacted the rate of NETosis formation. The biology of MPNs has an impact on NET formation because genetic driver mutations favor induction of NETosis, but this does not seems to translate into important clinical end points such as thromboembolic complications. Therefore, NETosis may play a role in facilitating thrombosis, but it is not a sole causative determinant in MPN-associated thrombophilia.

Differential procoagulatory response of microvascular, arterial and venous endothelial cells upon inflammation in vitro.

INTRODUCTION: Inflammation induces a procoagulant phenotype of endothelial cells (EC) with the exposure of tissue factor (TF), a potent initiator of the extrinsic coagulation cascade. Although systemic inflammation affects the whole vascular system, thrombotic lesions occur particularly in microcirculation. This raises the question of whether TF-procoagulant activity (TF-PCA) differs between EC from arterial, venous, and microvascular beds. MATERIALS AND METHODS: Functional coagulation tests, including TF-PCA, and inflammatory responses were investigated on arterial, venous and microvascular endothelial cells. Interleukin-6 (IL-6) and TF-levels were determined in cohort of 59 septic patients. RESULTS: We found that tumor necrosis factor alpha (TNFα), lipopolysaccharide, and interleukin-1ß induce a solid, dose-dependent increase in TF-PCA, which is highest in microvascular EC. A positive correlation of interleukin-6 (IL-6) with TF levels was observed in a cohort of 59 septic patients. In contrast, TF-PCA was independent of IL-6 concentrations in vitro. Re-analysis of publicly available gene expression data revealed that among the top 50 genes annotated to coagulation, TF is one of three regulated genes common to the three investigated EC subtypes. The response to inflammatory stimuli in terms of exposure of leukocyte-endothelial- and platelet-endothelial adhesion molecules (E-selectin and PECAM-1), remodeling of adherens junctions, co-exposure of negatively charged surfaces nor breakdown of the glycocalyx was comparable between the EC subtypes and did not explain the higher TF-PCA on microvascular cells. We found that the ratio of TF and TFPI exposure on the endothelial membrane significantly differs between the EC subtypes. CONCLUSIONS: These findings indicate that the ratio of TF to its inhibitor TFPI is a determinant of endothelial TF-PCA, which is most pronounced on microvascular endothelial cells and might explain why the microvascular system is particularly susceptible to inflammation-induced thrombosis.

Effectiveness and Safety of Romiplostim Among Patients with Newly Diagnosed, Persistent and Chronic Immune Thrombocytopenia in European Clinical Practice.

INTRODUCTION: Romiplostim has been approved in Europe since 2009 to treat patients with chronic primary immune thrombocytopenia (ITP). Using real-world data from seven European countries, we measured the effectiveness and safety outcomes within 24 weeks following romiplostim initiation by duration of ITP: less than 3 months ("newly diagnosed"), 3-12 months ("persistent"), and more than 12 months ("chronic"). METHODS: Adults with ITP and ≥ 1 romiplostim administration between 2009 and 2012 were included. Endpoints included durable platelet response, median platelet count, rescue therapy, bleeding and adverse events. We used inverse probability of censoring weighted estimators to estimate cumulative risk of each outcome. There were 64 newly diagnosed, 50 persistent, and 226 chronic ITP patients at romiplostim initiation. RESULTS: Durable platelet response at 24 weeks ranged from 32% [confidence interval (CI): 18-46%] in newly diagnosed patients to 53% (CI 37-68%) in persistent patients. Median platelet count during follow-up ranged from 88 (CI 80-96) × 109/L in chronic patients to 131 (CI 102-160) × 109/L in newly diagnosed patients. CONCLUSION: Regardless of ITP duration, over half of patients discontinued concomitant ITP medications. Few adverse events were observed. Although only approved for chronic patients, estimates of the romiplostim treatment effect were similar across patients being managed in European clinical practice, regardless of ITP duration at romiplostim initiation.

Case report: successful perioperative management of patients with haemophilia A using an extended half-life factor VIII (Efmoroctocog alfa) during neurosurgical procedures.

Patients with haemophilia A (HA) undergoing neurosurgical procedures have a high risk of haemorrhage with potential fatal outcome. Here, we present a successful perioperative haemostatic concept applying an extended half-life factor VIII (EHL FVIII), Efmoroctocog alfa, in two patients with HA undergoing neurosurgery for paramedian right-sided disc herniation (case 1) and astrocytoma (case 2). After adequate EHL FVIII treatment the surgical procedures were performed without any bleeding complications despite the high-risk interventions. Laboratory measurements confirmed stable FVIII levels throughout the hospital stay. We suggest close interdisciplinary collaboration between involved clinicians as mandatory prerequisite for an optimized perioperative management in patients with HA. The presented cases indicate, that the increased stability, safety and fewer injections provide a rationale to use EHL FVIII products in HA patients undergoing surgical interventions with a very high bleeding risk.

Pain management in hemophilia: expert recommendations.

INTRODUCTION: As a typical consequence of bleeding into muscles and joints, patients with severe hemophilia suffer from acute and chronic pain. In spite of its high prevalence, pain in this patient group is not always sufficiently considered or treated in an effective manner. AIM: The recommendations presented in this paper address possible improvements in pain management in hemophilia patients and particularities that have to be taken into account in this patient group. METHOD: The manifold aspects of pain management in hemophilia patients were discussed within the framework of an expert meeting. Based on the available literature and the experts' clinical experience, the participants developed a set of recommendations presented in this paper. RESULTS: Pain management in patients with hemophilia is often insufficient, a fact that not only influences the patients' quality of life but also implies the risk of difficult to manage chronic pain. Both the prevalent polypharmacy (due to comorbidities) as well as the underlying disease itself present special challenges to pain therapy in this patient group. The present review and recommendations are intended to support medical professionals in recognising the risks of pain chronicity, applying basic principles of multimodal pain therapy, including the options of psychological intervention and modalities of physical medicine in therapy concepts, and reaching a comprehensive understanding of the range of analgesic options available.

Molecular characteristics of BRCA1/2 and PALB2 mutations in pancreatic ductal adenocarcinoma.

INTRODUCTION: Poly-(ADP)-ribose polymerase (PARP) inhibitors are successfully used for treatment of BRCA-mutated (mut) breast cancers and are under extensive evaluation for BRCA- and PALB2-mutated pancreatic ductal adenocarcinoma (PDAC). However, the optimal treatment regimen for BRCA/PALB2-mutated PDCA has yet to be established. Moreover, limited data are available on the association of BRCA/PALB2 gene alterations with other comutations and immunological biomarkers. MATERIAL AND METHODS: Tumour samples of 2818 patients with PDAC were analysed for BRCA1/2 PALB2 mutations and other genes by next-generation sequencing (NGS) (MiSeq on 47 genes, NextSeq on 592 genes). TMB was calculated based on somatic non-synonymous missense mutations. MSI-H/dMMR was evaluated by NGS, and PD-L1 expression was determined using immunohistochemistry. RESULTS: In 4.2% (n=124) of all PDAC samples BRCA mutations have been detected. BRCA2 mutations were more commonly observed than BRCA1 mutations (3.1%(n=89) vs 1.1% [n=35], p<0.0001). BRCA2 mutation was associated with an older age (64 vs 61 years for wild-type (wt), p=0.002) and PALB2 mutation was observed more frequently in female than in male patients. BRCA and PALB2 mutations were associated with MSI-H/dMMR compared with wt (BRCA: 4.8% vs 1.2%, p=0.002; PALB2: 6.7% vs 1.3 %, p=0.18), PD-L1 expression of >1.0% (BRCA: 21.8% vs wt 11.2%, p<0.001, PALB2: 0.0% vs 12.4 %, p=0.38) and high TMB (BRCA: mean 8.7 vs 6.5 mut/MB, p<0.001; PALB2: 10.6 mut/Mb vs 6.6 mut/Mb, p=0.0007). Also, PD-L1 expression and TMB differed between BRCA and PALB2 mutation and wt samples in MSS tumours (p<0.05). BRCA-mutated and PALB2-mutated PDACs were characterised by a different mutational profile than was observed in wt tumours. CONCLUSIONS: BRCA and PALB2 mutations were found in a significant subgroup of PDACs. These mutations were associated with a distinct molecular profile potentially predictive for response to immune-checkpoint inhibitor therapy. Therefore, these data provide a rationale to evaluate PARP inhibitors in combination with immune-checkpoint inhibitors in patients with BRCA/PALB2-mutated PDAC.

Dietary lipids fuel GPX4-restricted enteritis resembling Crohn's disease.

The increased incidence of inflammatory bowel disease (IBD) has become a global phenomenon that could be related to adoption of a Western life-style. Westernization of dietary habits is partly characterized by enrichment with the ω-6 polyunsaturated fatty acid (PUFA) arachidonic acid (AA), which entails risk for developing IBD. Glutathione peroxidase 4 (GPX4) protects against lipid peroxidation (LPO) and cell death termed ferroptosis. We report that small intestinal epithelial cells (IECs) in Crohn's disease (CD) exhibit impaired GPX4 activity and signs of LPO. PUFAs and specifically AA trigger a cytokine response of IECs which is restricted by GPX4. While GPX4 does not control AA metabolism, cytokine production is governed by similar mechanisms as ferroptosis. A PUFA-enriched Western diet triggers focal granuloma-like neutrophilic enteritis in mice that lack one allele of Gpx4 in IECs. Our study identifies dietary PUFAs as a trigger of GPX4-restricted mucosal inflammation phenocopying aspects of human CD.

Lessons learned from immunoadsorption for hyperviscosity in IgM multiple myeloma-A case report.

We report the case of a 63-year-old Caucasian woman with multiple relapsed IgM multiple myeloma (MM) and elevated free kappa light chains (fκLC). Due to hyperviscosity syndrome with visual impairment, regular plasma exchanges were performed. As part of her 11th line of therapy, an experimental protocol consisting of pembrolizumab, pomalidomide, and dexamethasone was initiated. To reduce fκLC and immunoglobulin (Ig) M, we performed immunoadsorption (IA) using columns containing recombinant single domain camelid antibody fragments as ligands. We measured pembrolizumab (humanized IgG4 kappa anti-PD1 antibody) levels before and after each IA session and found a 98.1% reduction from baseline with five sessions of IA. Comparable elimination kinetics were observed for serum IgG, whereas fκLC and IgM were eliminated to a substantially lesser extent. These findings highlight that in hyperviscosity syndrome due to IgM MM, broad spectrum IA columns might be only moderately effective compared to total plasma exchange or double filtration plasmapheresis. Monoclonal antibodies are efficiently reduced by extracorporeal therapies and re-dosing is necessary to provide sufficient efficacy. In the case of serious adverse events such as immune-related adverse events, IA might be used to eliminate the monoclonal antibody. Measuring IgG levels might be a reasonable strategy for monitoring drug levels of monoclonal antibodies during IA.

Classical and intermediate monocytes scavenge non-transferrin-bound iron and damaged erythrocytes.

Myelomonocytic cells are critically involved in iron turnover as aged RBC recyclers. Human monocytes are divided in 3 subpopulations of classical, intermediate, and nonclassical cells, differing in inflammatory and migratory phenotype. Their functions in iron homeostasis are, however, unclear. Here, we asked whether the functional diversity of monocyte subsets translates into differences in handling physiological and pathological iron species. By microarray data analysis and flow cytometry we identified a set of iron-related genes and proteins upregulated in classical and, in part, intermediate monocytes. These included the iron exporter ferroportin (FPN1), ferritin, transferrin receptor, putative transporters of non-transferrin-bound iron (NTBI), and receptors for damaged erythrocytes. Consequently, classical monocytes displayed superior scavenging capabilities of potentially toxic NTBI, which were augmented by blocking iron export via hepcidin. The same subset and, to a lesser extent, the intermediate population, efficiently cleared damaged erythrocytes in vitro and mediated erythrophagocytosis in vivo in healthy volunteers and patients having received blood transfusions. To summarize, our data underline the physiologically important function of the classical and intermediate subset in clearing NTBI and damaged RBCs. As such, these cells may play a nonnegligible role in iron homeostasis and limit iron toxicity in iron overload conditions.

ASH Meeting 2016: developments in hemostaseology.

During the annual meeting of the American Society of Hematology (ASH) in San Diego/California, novel developments in the field of hemostaseology were presented. Alternative treatment strategies besides factor replacement were discussed for patients with hemophilia. One of the highlights of the meeting in this year's plenary session was the presentation of successful adeno-associated virus mediated gene transfer in patients with hemophilia B leading to sustained elevation of factor IX:C (FIX:c). Other alternative treatment approaches in patients with hemophilia A may include bispecific antibodies mimicking factor VIIIa (FVIIIa) activity or disrupting anticoagulant proteins. Focusing on anticoagulation, data on the use of direct oral anticoagulants (DOACs) in cancer patients with atrial fibrillation as well as treatment of superficial vein thrombosis with rivaroxaban were presented. In this short review, we try to highlight the most important presentations during the ASH meeting 2016.

Observational retrospective study of vascular modulator changes during treatment in essential thrombocythemia.

Essential thrombocythemia (ET) patients are at risk of developing thrombotic events. Qualitative platelet (PLT) abnormalities and activation of endothelial cells (ECs) and PLTs are thought to be involved. Microparticles (MPs) can originate from PLTs (PMPs), ECs (EMPs), or red cells (RMPs). Previous studies have indicated that MPs contribute to ET pathophysiology. Endothelial modulators (eg, nitric oxide [NO], adrenomedullin [ADM], and endothelin-1 [ET-1]) are also involved in the pathophysiology of this condition. We hypothesized that treatments for reducing PLT count might also indirectly affect MP generation and endothelial activity by altering endothelial modulator production. The rationale of this study was that hydroxyurea (HU), a cytostatic drug largely used in ET, induces the production of a potent vasoactive agent NO in ECs. An observational retrospective study was designed to investigate the relationship between MPs, NO, ADM, and ET-1 in ET patients on treatment with HU, anagrelide (ANA), aspirin (ASA), and a group of patients before treatment. A total of 63 patients with ET diagnosis: 18 on HU + ASA, 15 on ANA + ASA, 19 on ASA only, and 11 untreated patients, and 18 healthy controls were included in this study. Blood samples were analyzed for MP (absolute total values) and functional markers (percentage values) by flow cytometry. PLT-derived MPs were studied using CD61, CD62P, CD36, and CD63, whereas endothelial-derived MPs were studied using CD105, CD62E, and CD144. Endothelial modulator markers (NO, ADM, and ET-1) were measured by ELISA. Total MP count was higher in the group treated with ANA + ASA (P < 0.01). MP markers modified in ET patients returned to levels of healthy controls following treatment, in particular, in patients on ANA treatment. NO and ADM values were higher in the HU group (P < 0.001). HU and ANA treatment also affected MP production in a cell origin-specific manner. HU and ANA, although acting via different pathways, have similar final effects. For instance, HU causes vasodilatation by increasing NO and ADM levels, whereas ANA impairs vasoconstriction by reducing ET-1. In conclusion, therapy with HU cytostatic drugs and ANA can reduce PLT count in ET, and also affect endothelial modulatory agents, with HU sustaining vasodilation and prothrombotic MP concentration, whereas ANA decreases vasoconstriction.

Successful immune tolerance induction using turoctocog alfa in an adult haemophilia A patient.

We present here the first known case of successful immune tolerance induction (ITI) using recombinant factor VIII (rFVIII), turoctocog alfa, in a patient with severe haemophilia A. The 38-year-old patient with a long-standing inhibitor required urgent surgery for severe arthropathy. rFVIII was administered throughout the surgical period. Surgery was considered successful, but on day 7 after surgery, an increased level of FVIII inhibitors were detected. ITI was attempted immediately thereafter according to the Bonn protocol. Inhibitors were no longer detected on day 17; 13 months later, successful ITI was achieved. This case suggests that a long-time interval between inhibitor appearance and the start of ITI therapy may not necessarily indicate poor prognosis.

Characterization of Microvesicles in Septic Shock Using High-Sensitivity Flow Cytometry.

PURPOSE: Endothelial pathology is considered to play a key role in septic shock. Since endothelial-derived microvesicles (MV) are elevated in various diseases associated with endothelial pathology, they are considered surrogate markers of the endothelial state. By analyzing the signature of circulating MV with high-sensitivity flow cytometry (hsFC), we wanted to test the hypothesis whether endothelial-derived MV are increased in septic shock. METHODS: MV in blood from healthy volunteers and patients with septic shock treated in a medical intensive care unit were quantified by hsFC, which has an improved detection limit of approximately 0.3 µm. RESULTS: Patients with septic shock (n = 30) showed 3-fold higher levels of CD31+/CD41- MV (58.5 (26.4-101.2) [median (25th-75th percentile)] vs. 19.5 (12.8-25.4) MV/µL; P <0.001) compared with healthy volunteers (n = 18). Absolute counts of CD144+, CD62E+, and CD106+ MV, specific for endothelial-derived MV, were low in all groups. The number of CD31+/CD41- MV correlated significantly with leukocyte count (rs = 0.64; P <0.001). Platelet-derived CD41+ MV were significantly elevated in the group dying within 48 h after inclusion (639.1 (321.3-969.7) vs. 221.5 (119.5-456.9) MV/µL; P = 0.037). Patients dying within 48 h had also significantly higher levels of CD31+/CD41-/AnnexinV- MV (51.9 (24.9-259.8) vs. 18.9 (9.7-31) MV/µL; P = 0.028). CONCLUSIONS: Despite an improved detection limit for MV by using hsFC, counts of endothelial-specific MV are unexpectedly low in patients with septic shock. Increased amounts of CD41+ and CD31+/CD41-/AnnexinV- MV indicate release by activated platelets and possibly leukocytes correlating with unfavorable outcome.

Akacid medical formulation induces apoptosis in myeloid and lymphatic leukemic cell lines in vitro and in vivo.

Akacid medical formulation (AMF) is an oligoguanidine that exerts biocidal activity against airborne and surface microorganisms including bacteria, viruses, fungi, and molds, while showing relatively low toxicity to humans. We have previously shown that AMF exerts antiproliferative effects on a variety of solid tumor cell lines. In this study we raised the question whether AMF could also substantially inhibit cell growth or induce apoptosis in cell lines derived from hematologic malignancies such as leukemia or lymphoma. We found that AMF has antiproliferative effects on various hematologic cell lines derived from human leukemia and lymphoma. Additionally, we show that AMF induces apoptosis in leukemia cell lines not only via the extrinsic and intrinsic pathway, but also in a caspase-independent manner. This effect was found also in G0-arrested cells. Finally, in our animal experiments utilizing male nu/nu Balb/c mice we found a significant growth retardation, which was immunohistochemically associated with a significantly lower number of KI67-positive cells and caspase-3 induction in AMF-treated mice.