Monitoring of Argatroban in Critically Ill Patients: A Prospective Study Comparing Activated Partial Thromboplastin Time, Point-of-Care Viscoelastic Testing with Ecarin Clotting Time and Diluted Thrombin Time to Mass Spectrometry.

BACKGROUND: The direct thrombin inhibitor argatroban is indicated for the treatment of heparin-induced thrombocytopenia II, but it is also used off-label to treat critically ill patients presenting with heparin resistance, severe antithrombin deficiency, or hypercoagulability. Direct drug monitoring is not routinely available, and argatroban dosing is mainly based on global coagulation assays such as activated partial thromboplastin time (PTT) or diluted thrombin time (TT), both of which have limitations in patients with hypercoagulability. METHODS: Blood samples were obtained from critically ill patients treated with argatroban. Activated PTT and diluted TT were measured with a STA R Max3 analyzer (STAGO Deutschland GmbH, Germany) using an argatroban-calibrated kit. Ecarin clotting time was measured using a point-of-care viscoelastic test device. Liquid chromatography with tandem mass spectrometry was performed using a reversed-phase column, a solvent gradient, and an API4000 mass spectrometer with electrospray. Correlation was described using Pearson correlation coefficient r and Bayesian multilevel regression to estimate relationships between outcomes and covariates. RESULTS: From June 2021 to March 2022, 205 blood samples from 22 patients were analyzed, allowing for 195 activated PTT-liquid chromatography with tandem mass spectrometry comparisons, 153 ecarin clotting time-liquid chromatography with tandem mass spectrometry comparison, and 105 diluted TT-liquid chromatography with tandem mass spectrometry comparisons. Compared to liquid chromatography with tandem mass spectrometry, performance of argatroban quantification was best for diluted TT (r = 0.91), followed by ecarin clotting time (r = 0.58) and activated PTT (r = 0.48). Regression analysis revealed that patients with sepsis were more prone to argatroban overdosing (coefficient, 4.194; 95% credible interval, 2.220 to 6.792). CONCLUSIONS: Although activated PTT monitoring of argatroban is the most commonly used test, in critically ill patients, diluted TT provides more precise measurements. Alternately, point-of-care viscoelastic ecarin clotting time also provides guidance for argatroban dosing to identify overdosing if available. The data also suggested that patients with sepsis are at greater risk for argatroban overdosing.

Peripartum Haemorrhage: Haemostatic Aspects of the Updated Peripartum Haemorrhage Guideline of the German-Speaking Countries.

Background: Peripartum haemorrhage (PPH) is a potentially life-threatening complication. Although still rare, the incidence of peripartal haemorrhage is rising in industrialised countries and refractory bleeding remains among the leading causes of death in the peripartal period. Summary: The interdisciplinary German, Austrian, and Swiss guideline on "Peripartum Haemorrhage: Diagnostics and Therapies" has reviewed the evidence for the diagnostics and medical, angiographic, haemostatic, and surgical treatment and published an update in September 2022 . This article reviews the updated recommendations regarding the early diagnosis and haemostatic treatment of PPH. Keystones of the guideline recommendations are the early diagnosis of the bleeding by measuring blood loss using calibrated collector bags, the development of a multidisciplinary treatment algorithm adapted to the severity of bleeding, and the given infrastructural conditions of each obstetric unit, the early and escalating use of uterotonics, the therapeutic, instead of preventative, use of tranexamic acid, the early diagnostics of progressive deficiencies of coagulation factors or platelets to facilitate a tailored and guided haemostatic treatment with coagulation factors, platelets as well as packed red blood cells and fresh frozen plasma when a massive transfusion is required. Key Messages: Essential for the effective and safe treatment of PPH is the timely diagnosis. The diagnosis of PPH requires the measurement rather than estimation of blood loss. Successful treatment of PPH consists of a multidisciplinary approach involving surgical and haemostatic treatments to stop the bleeding. Haemostatic treatment of PPH starts early after diagnosis and combines tranexamic acid, an initially ratio-driven transfusion with RBC:plasma:PC = 4:4:1 (when using pooled or apheresis PC) and finally a goal-directed substitution with coagulation factor concentrates for proven deficiencies. Early monitoring of coagulation either by standard parameters or viscoelastic methods facilitates goal-directed haemostatic treatment.

Treatment of Acquired von Willebrand Disease due to Extracorporeal Membrane Oxygenation in a Pediatric COVID-19 Patient with Vonicog Alfa: A Case Report and Literature Review.

Acquired von Willebrand disease (aVWD) is frequently observed in patients with the need for extracorporeal membrane oxygenation (ECMO). aVWD can be treated by plasma-derived concentrates containing factor VIII (FVIII) and/or von Willebrand factor (VWF) and recombinant VWF concentrate as well as adjuvant therapies such as tranexamic acid and desmopressin. However, all of these therapeutic options possibly cause thromboembolism. Therefore, the optimal treatment remains uncertain. This report presents a case of a 16-year-old patient suffering from severe acute respiratory distress syndrome due to coronavirus disease 2019 with the need of ECMO support. Our patient developed aVWD under ECMO therapy characterized by loss of high-molecular-weight multimers (HMWM) and severe bleeding symptoms following endoscopic papillotomy due to sclerosing cholangitis. At the same time standard laboratory parameters showed hypercoagulability with increased fibrinogen level and platelet count. The patient was successfully treated with recombinant VWF concentrate (rVWF; vonicog alfa; Veyvondi) combined with topic tranexamic acid application and cortisone therapy. rVWF concentrate vonicog alfa is characterized by ultra-large multimers and absence of FVIII. Patient could be successfully weaned from ECMO support after 72 days. Multimer analysis 1 week after ECMO decannulation showed an adequate reappearance of HMWM.

Acquired Factor XIII Deficiency in Patients with Multiple Trauma.

INTRODUCTION: Fibrin stabilizing factor (FXIII) plays a crucial role in blood clotting, tissue repair, and immune defense. FXIII deficiency after trauma can lead to prolonged wound healing due to persistent infections or coagulation disorders. The aim of this study was to describe the prevalence of acquired FXIII deficiency after trauma and to provide a description of the time-course changes of important coagulation parameters in relation to FXIII activity. In this context, patient characteristics, laboratory data, and treatment modalities were examined with respect to their influence on FXIII activity. Furthermore, the effects of in vitro administration of FXIII on clot firmness and outcomes in patients with severe traumatic brain injury were investigated. PATIENTS AND METHODS: Two trauma cohorts (A and B) were examined prospectively in a two-center study, and another (cohort C) was examined retrospectively. In cohort A (trauma patients, n=880) routine laboratory tests were conducted, and FXIII activity was measured. In cohort B (polytrauma patients, n=26), additional clinical parameters were collected, and in-vitro FXIII administration and rotational thromboelastometry (ROTEM) analyses were performed. In cohort C (polytrauma patients with severe traumatic brain injury [sTBI], n=84), the impact of initially measured FXIII activity on clinical outcomes after sTBI was investigated using the modified Rankin Scale (mRS) at least 6 months after trauma. RESULTS: The prevalence of FXIII activity <70% in cohort A was 12.4%, with significant differences in age, Hb, fibrinogen, and Hct levels, platelet count, aPTT, and INR (vs. prevalence of FXIII activity >70%). Cohort B showed a decrease in FXIII activity from 85% to 58% after 7 days. FXIII deficiency correlated with time after trauma, aPTT, and fibrinogen level, lactate, and Hb levels. In-vitro administration of FXIII showed a positive influence on clot firmness due to improved maximum clot firmness (MCF in FIBTEM) and reduced maximum lysis (ML in EXTEM). Finally, a significant difference in FXIII activity between patients after sTBI with good and poor clinical outcomes was observed 6 months after trauma. CONCLUSION: We demonstrated that trauma-associated FXIII deficiency is a common coagulation disorder, with FXIII deficiency increasing further in the first 7 days after trauma, the period of early surgical care. In vitro administration of FXIII was able to demonstrate significant clot stabilizing effects. For trauma patients with sTBI, FXIII activity could serve as a prognostic parameter, as it differed significantly between patients with good and poor clinical outcomes.

Novel mutation and expanding phenotype in IRF2BP2 deficiency.

OBJECTIVES: Inborn errors of immunity manifest with susceptibility to infection but may also present with immune dysregulation only. According to the European Society for Immunodeficiencies Registry about 50% of inborn errors of immunity are classified as common variable immunodeficiencies (CVID). In only few CVID patients are monogenic causes identified. IFN regulatory factor-2 binding protein 2 (IRF2BP2) is one of 20 known genes associated with CVID phenotypes and has only been reported in two families so far. We report another IRF2BP2-deficient patient with a novel pathogenic variant and phenotype and characterize impaired B cell function and immune dysregulation. METHODS: We performed trio whole-exome sequencing, determined B cell subpopulations and intracellular calcium mobilization upon B cell receptor crosslinking in B cells. T cell subpopulations, T cell proliferation and a type I IFN signature were measured. Colonoscopy and gastroduodenoscopy including histopathology were performed. RESULTS: The 33-year-old male presented with recurrent respiratory infections since childhood, colitis and RA beginning at age 25 years. We identified a novel de novo nonsense IRF2BP2 variant c.1618C>T; p.(Q540*). IgG deficiency was detected as consequence of a severe B cell differentiation defect. This was confirmed by impaired plasmablast formation upon stimulation with CpG. No serum autoantibodies were detected. Intracellular cytokine production in CD4+ T cells and CTLA4 expression on FOXP3+ Tregs were impaired. Type I IFN signature was elevated. CONCLUSION: The identified loss-of-function variant in IRF2BP2 severely impairs B cell development and T cell homeostasis, and may be associated with colitis and RA. Our results provide further evidence for association of IRF2BP2 with CVID and contribute to the understanding of the underlying pathomechanisms.

Extreme obesity is a strong predictor for in-hospital mortality and the prevalence of long-COVID in severe COVID-19 patients with acute respiratory distress syndrome.

Acute Respiratory Distress Syndrome (ARDS) is common in COVID-19 patients and is associated with high mortality. The aim of this observational study was to describe patients' characteristics and outcome, identifying potential risk factors for in-hospital mortality and for developing Long-COVID symptoms. This retrospective study included all patients with COVID-19 associated ARDS (cARDS) in the period from March 2020 to March 2021 who were invasively ventilated at the intensive care unit (ICU) of the University Hospital Dresden, Germany. Between October 2021 and December 2021 patients discharged alive (at minimum 6 months after hospital discharge-midterm survival) were contacted and interviewed about persistent symptoms possibly associated with COVID-19 as well as the quality of their lives using the EQ-5D-5L-questionnaire. Long-COVID was defined as the occurrence of one of the symptoms at least 6 months after discharge. Risk factors for mortality were assessed with Cox regression models and risk factors for developing Long-COVID symptoms by using relative risk (RR) regression. 184 Patients were included in this study (male: n = 134 (73%), median age 67 (range 25-92). All patients were diagnosed with ARDS according to the Berlin Definition. 89% of patients (n = 164) had severe ARDS (Horovitz-index < 100 mmHg). In 27% (n = 49) extracorporeal membrane oxygenation was necessary to maintain gas exchange. The median length of in-hospital stay was 19 days (range 1-60). ICU mortality was 51%, hospital mortality 59%. Midterm survival (median 11 months) was 83% (n = 55) and 78% (n = 43) of these patients presented Long-COVID symptoms with fatigue as the most common symptom (70%). Extreme obesity (BMI > 40 kg/m2) was the strongest predictor for in-hospital mortality (hazard ratio: 3.147, confidence interval 1.000-9.897) and for developing Long-COVID symptoms (RR 1.61, confidence interval 1.26-2.06). In-hospital mortality in severe cARDS patients was high, but > 80% of patients discharged alive survived the midterm observation period. Nonetheless, most patients developed Long-COVID symptoms. Extreme obesity with BMI > 40 kg/m2 was identified as independent risk factor for in-hospital mortality and for developing Long-COVID symptoms.Trial registration DRKS-ID DRKS00027856.

Influencing Factors and Differences in Born Aggregometry in Specialized Hemostaseological Centers: Results of a Multicenter Laboratory Comparison.

Introduction Light transmission aggregometry (LTA) is regarded as the gold standard in platelet function diagnostics. However, there is a relevant degree of interlaboratory variability in practical applications. Objective The aim of the present study was to develop a practicable laboratory comparison on LTA and to analyze differences and influencing factors in regard to standardization in five specialized hemostaseological centers. Methods The study was performed on 30 patients in total. Each center performed LTA on blood samples from six healthy volunteers (three men and three women) using the inductors collagen (Col), adenosine diphosphate (ADP), arachidonic acid (ARA), and ristocetin. The LTA was performed three times using different methods as follows: (1) International Society on Thrombosis and Haemostasis recommendations with identical reagents, (2) in-house protocols and the identical reagents; and (3) in-house protocols and in-house reagents. Results A total of 396 measurements of 30 probands were performed. Even after standardization of the protocol and using identical reagents, there were significant differences between the centers regarding the final and maximum aggregation ( p = 0.002 and <0.001) and further significant differences in the maximum and final aggregation according to the wavelength of the device used to measure the LTA (PAP-8: 430 nm, APACT 4004: 740 nm [ p < 0.001 each]). Using identical reagents but individual inductor concentrations and laboratory protocols also resulted in different maximum and final aggregation. The largest differences were seen with Col and ristocetin; there were significant influences from the reagents' manufacturers in the results of aggregometry for the inductor Col ( p < 0.01) but not for ADP, ARA, and ristocetin. Conclusion In this study, we proved that there are significant influences from the used aggregometers, inductors concentrations, and manufacturers. These results illustrate the challenges and importance of standardization of LTA.

Prospective validation of a biomarker-driven response prediction model to romiplostim in lower-risk myelodysplastic neoplasms - results of the EUROPE trial by EMSCO.

The EUROPE phase 2 trial investigated the predictive value of biomarkers on the clinical efficacy of single agent romiplostim (ROM) treatment in patients with lower-risk myelodysplastic neoplasms (LR-MDS) and thrombocytopenia within the 'European Myelodysplastic Neoplasms Cooperative Group' (EMSCO) network. A total of 77 patients with LR-MDS and a median platelet count of 25/nl were included, all patients received ROM at a starting dose of 750 µg by SC injection weekly. Thirty-two patients (42%) achieved a hematologic improvement of platelets (HI-P) with a median duration of 340 days. Neutrophil (HI-N) and erythroid (HI-E) responses were observed in three (4%) and seven (9%) patients, respectively. We could not confirm previous reports that HI-P correlated with baseline endogenous thrombopoietin levels and platelet transfusion history, but SRSF2 mutation status and hemoglobin levels at baseline were significantly linked to HI-P. Sequential analysis of variant allelic frequency of mutations like SRSF2 did not reveal an impact of ROM on clonal evolution in both responders and non-responders. In summary, our study confirms the safety and efficacy of ROM in LR-MDS patients and may allow to better define subgroups of patients with a high likelihood of response.

Predictive ability of viscoelastic testing using ClotPro® for short-term outcome in patients with severe Covid-19 ARDS with or without ECMO therapy: a retrospective study.

BACKGROUND: SARS-CoV-2 infections are suspected to trigger the coagulation system through various pathways leading to a high incidence of thromboembolic complications, hypercoagulation and impaired fibrinolytic capacity were previously identified as potentially mechanisms. A reliable diagnostic tool for detecting both is still under discussion. This retrospective study is aimed to examine the prognostic relevance of early viscoelastic testing compared to conventional laboratory tests in COVID-19 patients with acute respiratory distress syndrome (ARDS). METHODS: All mechanically ventilated patients with COVID-19 related ARDS treated in our intensive care unit (ICU) between January and March 2021 were included in this study. Viscoelastic testing (VET) was performed using the ClotPro® system after admission to our ICU. Prevalence of thromboembolic events was observed by standardized screening for venous and pulmonary thromboembolism using complete compression ultrasound and thoracic computed tomography pulmonary angiography at ICU admission, respectively. We examined associations between the severity of ARDS at admission to our ICU, in-hospital mortality and the incidence of thromboembolic events comparing conventional laboratory analysis and VET. ECMO related coagulopathy was investigated in a subgroup analysis. The data were analyzed using the Mann-Whitney U test. RESULTS: Of 55 patients enrolled in this study, 22 patients required treatment with ECMO. Thromboembolic complications occurred in 51% of all patients. Overall hospital mortality was 55%. In patients with thromboembolic complications, signs of reduced fibrinolytic capacity could be detected in the TPA assay with prolonged lysis time, median 460 s (IQR 350-560) vs 359 s (IQR 287-521, p = 0.073). Patients with moderate to severe ARDS at admission to our ICU showed increased maximum clot firmness as a sign of hypercoagulation in the EX-test (70 vs 67 mm, p < 0.05), FIB-test (35 vs 24 mm, p < 0.05) and TPA-test (52 vs 36 mm, p < 0.05) as well as higher values of inflammatory markers (CRP, PCT and IL6). ECMO patients suffered more frequently from bleeding complications (32% vs 15%). CONCLUSION: Although, the predictive value for thromboembolic complications or mortality seems limited, point-of-care viscoelastic coagulation testing might be useful in detecting hypercoagulable states and impaired fibrinolysis in critically ill COVID-19 ARDS patients and could be helpful in identifying patients with a potentially very severe course of the disease.

Enhancing Anticoagulation Monitoring and Therapy in Patients Undergoing Microvascular Reconstruction in Maxillofacial Surgery: A Prospective Observational Trial.

BACKGROUND: In reconstructive surgery, loss of a microvascular free flap due to perfusion disorders, especially thrombosis, is a serious complication. In recent years, viscoelastic testing (VET) has become increasingly important in point-of-care (POC) anticoagulation monitoring. This paper describes a protocol for enhanced anticoagulation monitoring during maxillofacial flap surgery. OBJECTIVE: The aim of the study will be to evaluate, in a controlled setting, the predictive value of POC devices for the type of flap perfusion disorders due to thrombosis or bleeding. VET, Platelet monitoring (PM) and standard laboratory tests (SLT) are comparatively examined. METHODS/DESIGN: This study is an investigator-initiated prospective trial in 100 patients undergoing maxillofacial surgery. Patients who undergo reconstructive surgery using microvascular-free flaps will be consecutively enrolled in the study. All patients provide blood samples for VET, PM and SLT at defined time points. The primary outcome is defined as free flap loss during the hospital stay. Statistical analyses will be performed using t-tests, including the Bonferroni adjustment for multiple comparisons. DISCUSSION: This study will help clarify whether VET can improve individualized patient care in reconstruction surgery. A better understanding of coagulation in relation to flap perfusion disorders may allow real-time adaption of antithrombotic strategies and potentially prevent flap complications.

Point-of-Care Assessment of Direct Oral Anticoagulation in Acute Ischemic Stroke: Protocol for a Prospective Observational Diagnostic Accuracy Study.

BACKGROUND: Treatment of ischemic stroke with recombinant tissue plasminogen activator for intravenous thrombolysis (IVT) must be delivered within a narrow time window after symptom onset. This effective hyperacute treatment can be administered after ruling out active anticoagulation with direct oral anticoagulants (DOACs). Whenever this is impractical, e.g., due to aphasia, plasmatic DOAC levels are measured with a consequent delay in the IVT decision-making process ranging from 30 to 60 minutes of time. This study will test the hypothesis that hyperacute point-of-care assessment of clotting time in the patient's whole blood has sufficient diagnostic accuracy to determine immediately whether stroke patients are pretreated with DOAC. METHODS AND DESIGN: This will be a prospective single-center diagnostic accuracy study in 1,850 consecutive acute ischemic stroke patients at a tertiary stroke center in Saxony, Germany. Presence of active anticoagulation with DOAC will be determined by point-of-care quantification of clotting time via whole blood viscoelastic testing (ClotPro) using Russell venom viper and ecarin assay compared with high-performance liquid chromatography-tandem mass spectrometry as the reference standard. DISCUSSION: Viscoelastic point-of-care assessment of clotting time in whole blood might improve swift delivery of time-sensitive hyperacute treatment with IVT in stroke patients.

Point of care coagulation management in anesthesiology and critical care.

Point of care (POC) devices are increasingly used in the ICU and in anesthesia. Besides POC-devices for blood gas analysis, several devices are available for coagulation measurements. Although basic principles for thromboelastographic measurements are not novel, some promising developments were made during the last decade improving both user-friendliness and measurement reliability. For instance, POC measurements of activated clotting time (ACT) for heparin monitoring is still regarded as standard-of-care in cardiac interventions and surgery. In the field of anesthesia and intensive care medicine, POC-devices for thromboelastographic and platelet aggregation measurements are widely used. Their impact in case of bleeding and patient blood management for cardiothoracic and trauma surgery is well known. Moreover, there are promising concepts for anticoagulation monitoring including new oral anticoagulant drugs. Coagulation POC-devices may also identify patients at specific risk for thromboembolic events quickly. On the other hand, benefits of POC-devices need to be balanced against limitations, which include technical restrictions and operator related errors, mainly affecting reproducibility and interpretation of results. Therefore, it is recommendable to consider results of POC-coagulation testing in comparison to standard laboratory tests (SLT). Nevertheless, in urgent or emergency situations POC results enable fast decision making to optimize patient care.

Development and validation of an analytical method for the determination of direct oral anticoagulants (DOAC) and the direct thrombin-inhibitor argatroban by HPLC-MS/MS.

Since direct oral anticoagulants (DOAC) are administered frequently to an elderly, co-morbid population, medical emergencies including trauma, acute bleeding or organ failure are not uncommon. In these situations, the type, dosage or the time of last intake of anticoagulants is often unknown and single substance analysis by functional tests is only possible if the substance contained in the sample is known. A reliable and validated toxicology screen of DOAC and argatroban would be helpful inform not only attending physicians in the emergency department but also law enforcement and courts of justice. After precipitation with acetone, HPLC separation was achieved on a Phenomenex Luna Pentafluorophenyl Colum using acetonitrile-water (90:10, v/v) as mobile phase system. Detection was performed using a 3200 Q Trap mass spectrometer (AB Sciex). For analysis MRM Scans (MS/MS) with positive ionization were chosen. The method was validated for blank serum as the matrix of choice. Limits of detection are between 0.5 and 1.0 ng/mL, limits of quantification are between 1.9 and 3.6 ng/mL and recoveries are above 60%. The applicability of the method was demonstrated by the determination of DOAC in body fluids from forensic cases and in therapeutic drug monitoring. The rapid simultaneous detection and quantification of apixaban, argatroban, dabigatran etexilate, dabigatran, edoxaban and rivaroxaban in body fluids by HPLC-MS/MS closes an important gap in emergency toxicology.

Severe Bleeding Diathesis in Siblings with Platelet Dysfunction due to a Novel Nonsense RASGRP2 Mutation.

Next-generation sequencing is increasingly applied during the diagnostic work-up of patients with bleeding diathesis and has facilitated the diagnosis of rare bleeding disorders such as inherited platelet function disorders. Mutations in RAS guanyl releasing protein 2 (RasGRP2), also known as calcium- and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), underlie a recently described platelet signal transduction abnormality. Here we present the case of a consanguineous family originating from Afghanistan with two siblings affected by recurrent severe mucocutaneous bleedings. Platelet function testing demonstrated a marked reduction of aggregation induced by collagen and adenosine diphosphate. Whole exome sequencing revealed a novel homozygous nonsense RASGRP2 mutation segregating with the bleeding disorder in the family.

Bleeding signs due to acquired von Willebrand syndrome at diagnosis of chronic myeloid leukaemia in children.

A considerable proportion of patients with chronic myeloid leukaemia (CML) may present at diagnosis with high platelet counts. This may result in thrombosis or bleeding complications due to binding of von Willebrand factor (VWF) multimers to platelets. Paediatric CML is very rare and no systematic investigation on clinical complications of elevated platelets has been reported. Data on platelet count and associated haemostaseological complications were retrospectively analysed in a cohort of 156 children with CML. Fifty-one percent (81/156) patients presented with thrombocytosis (platelet count> 500 × 109 /l), and were extreme (>1 000 × 109 /l) in 23/156 (16%). There were no cases of thrombosis but mild bleeding signs were present in 12% (n = 9) children with thrombocytosis. Bleeding occurred without correlation to elevated platelet counts and was associated with reduced large VWF multimers, indicating a diagnosis of acquired von Willebrand syndrome (AVWS), which resolved after initiation of CML treatment. Patients with paediatric CML frequently exhibit high platelet counts not resulting in thrombosis. In patients with thrombocytosis mild bleeding signs due to a low percentage of large VWF multimers can be demonstrated. AVWS may be underdiagnosed in paediatric CML (Clinical-Trials.gov NCT00445822, 9 March 2007).

Expression of Glypican 3 is an Independent Prognostic Biomarker in Primary Gastro-Esophageal Adenocarcinoma and Corresponding Serum Exosomes.

Exosomes are nano-sized membranous vesicles of endosomal origin that carry nucleic acids, lipids and proteins. The cargo of exosomes is cell origin specific and the release of these exosomes and uptake by an acceptor cell is seen as a vital element of cell-cell communication. Here, we sought to investigate the diagnostic and prognostic value of the expression of glypican 3 (GPC3) on primary gastro-esophageal adenocarcinoma (GEA) tissue (tGPC3) and corresponding serum exosomes (eGPC3). Circulating exosomes were extracted from serum samples of 49 patients with GEA and 56 controls. Extracted exosomes were subjected to flow cytometry for the expression of eGPC3 and GPC3 expression on primary GEA tissue samples was determined by immunohistochemistry and correlated to clinicopathological parameters. We found decreased eGPC3 levels in GEA patients compared to healthy controls (p < 0.0001) and high tGPC3 expression. This was significantly associated with poor overall survival (high vs. low eGPC3: 87.40 vs. 60.93 months, p = 0.041, high vs. low tGPC3: 58.03 vs. 84.70 months, p = 0.044). Cox regressional analysis confirmed tGPC3 as an independent prognostic biomarker for GEA (p = 0.02) and tGPC3 expression was validated in two independent cohorts. Our findings demonstrate that eGPC3 and tGPC3 can be used as potential diagnostic and prognostic biomarkers for GEA.

Identification of rare levels of methylated tumor DNA fragments using an optimized bias based pre-amplification-digital droplet PCR (OBBPA-ddPCR).

BACKGROUND: The analysis of aberrant DNA methylations is used for the diagnosis of cancer as significant changes in the gene methylation pattern are often detected during early carcinogenesis. In this study, we evaluated the performance of a two-step method that combines pre-amplification with ddPCR technique. RESULTS: By using ddPCR, the dependence of amplification efficiency for methylated and unmethylated DNA fragments on the relevant MgCl2 concentration and the annealing temperature was established in addition to the primer design. We found that the efficiency can be adjusted toward methylated sequences by using primers covering one to four CpG sites under appropriately selected MgCl2 concentration and annealing temperature. Applying a PCR bias between 85% and 95%, five copies of methylated tumor DNA fragments were detected against a background of 700,000 copies of unmethylated DNA fragments with a high signal-to-noise ratio. The analysis of serum samples from patients with prostate cancer showed a significantly improved performance of the new method in comparison with the MS-HRM technique, ddPCR alone, or ddPCR in combination with an unbiased pre-amplification using methylation-independent primers. CONCLUSIONS: We define this method as an optimized bias-based pre-amplification-digital droplet PCR (OBBPA-ddPCR) technique. This novel method is recommended for the early detection of cancer-specific DNA methylation biomarkers in the form of a liquid biopsy.

Novel parameters of extended complete blood cell count under fluorescence flow cytometry in patients with sepsis.

BACKGROUND: We first describe a patient who developed urosepsis from an ordinary urinary tract infection. In this case, the new hematological parameters of immature leukocytes, that is, the high-fluorescence lymphocyte cell (HFLC) and immature granulocyte (IG) counts peaked early, whereas the established infection parameters, that is, C-reactive protein (CRP) and total white blood cell count showed less dynamic regarding infection and therapy. METHODS: To investigate this phenomenon in greater detail, the novel parameters HFLC and IG counts are investigated retrospectively in a cohort of 38 patients who were admitted to the anesthesia intensive care unit. Three groups of patients have been analyzed and compared: patients without signs of infection, patients with limited infections, and patients with sepsis. Data were collected with a Sysmex XE-5000 hematological analyzer. RESULTS: In patients (n = 22) without any signs of infection, both values are very low. In patients with limited local infections (n = 10), moderate elevations of the IG and HFLC counts are seen. In patients with sepsis (n = 6), the IG and HFLC counts are significantly higher. CONCLUSION: The total IG count seems to be useful for distinguishing a septic patient from a nonseptic (P < 0.004). Hematological parameters have the advantage of being measured easily during routine blood cell analysis.

Regulation of thrombomodulin expression in prostate cancer cells.

In carcinomas the expression of thrombomodulin (TM) is inversely correlated with tumour progression and metastasis. In the present study a decreased TM expression in human prostate cancer cell lines, LNCaP, DU-145, and PC-3, in relation to normal prostate epithelial cells (PrEC) is shown. Sequencing and methylation-specific high resolution melting (MS-HRM) analyses of bisulphite-modified genomic DNA indicates a high degree of methylation in DU-145 cells and lesser degrees in PC-3 and LNCaP cells, whereas in PrEC the TM promoter is unmethylated. The expression of TM is negatively regulated by NF-κB- and GSK3-ß-dependent signalling pathways and positively regulated by retinoic acid and transcription factor Sp1 in PrEC, LNCaP and PC-3 cells, but not in DU-145 cells. However, exposure of DU-145 cells to the demethylating agent, 5-aza-2'deoxycytidine, restores the TM expression and its control by retinoic acid, NF-κB- and GSK3-ß-dependent signalling. In conclusion, the study establishes that in prostate cancer cell lines relative to PrEC the TM is down-regulated and that the TM promoter is hypermethylated, which seems to be responsible for the down-regulation and failed regulation of TM expression in DU-145 cells.