Thrombin generation measured on ST Genesia, a new platform in the coagulation routine lab: Assessment of analytical and between-subject variation

Background: The thrombin generation (TG) assay, which measures global coagulation, has mainly been used as a research tool to investigate thrombotic and bleeding disorders. Recently, Diagnostica Stago launched the ST Genesia, a fully automated system to perform “routine version” of this assay. The objectives of this study were to evaluate the imprecision compared with the previous method, Thrombinoscope CAT, and to establish reference intervals. Methods: Thrombin generation was measured in platelet-poor citrated plasma from 20 normal controls (fresh and after freezing at −80°C up to 12–13 weeks) on CAT and ST Genesia in duplicate to estimate the total variation, and within and between variations. The reference intervals were estimated nonparametrically in 30 men, 30 women taking combined oral contraceptives (COCs), and 30 women not taking COCs. These were sampled in both Vacutainer and Monovette tubes (i.e., tubes with a high and minimal contact activation, respectively). Results: Freezing had minimal effects. Imprecision was comparable between the ST Genesia and CAT, with a strong correlation between the two methods. TG was higher when sampled in Vacutainer than in Monovette. We observed a distinct difference between women taking and not taking COCs, whereas men and women not taking COC were quite similar. Conclusions: Thrombin generation on ST Genesia showed an analytical variation similar to that of CAT. The results depended on the type of sample tubes; thus, reference intervals must be established for the collection tubes used in each laboratory. Furthermore, a considerable difference was observed between women using and not using COCs

Extracellular vesicle-associated procoagulant phospholipid and tissue factor activity in multiple myeloma

<div><p>Multiple myeloma (MM) patients have increased risk of developing venous thromboembolism, but the underlying mechanisms and the effect on the coagulation system of the disease and the current cancer therapies are not known. It is possible that cancer-associated extracellular vesicles (EV), carrying tissue factor (TF) and procoagulant phospholipids (PPL) may play a role in thrombogenesis. The aim of this study was to perform an in-depth analysis of procoagulant activity of small and large EVs isolated from 20 MM patients at diagnosis and after receiving first-line treatment compared with 20 healthy control subjects. Differential ultracentrifugation at 20,000 × <i>g</i> and 100,000 × <i>g</i> were used to isolate EVs for quantitative and phenotypical analysis through nanoparticle tracking analysis, Western blotting and transmission electron microscopy. The isolated EVs were analyzed for procoagulant activity using the calibrated automated thrombogram technique, a factor Xa-based activity assay, and the STA Procoag-PPL assay. In general, MM patients contained more EVs, and immunoelectron microscopy confirmed the presence of CD9- and CD38-positive EVs. EVs in the 20,000 × <i>g</i> pellets from MM patients exerted procoagulant activity visualized by increased thrombin generation and both TF and PPL activity. This effect diminished during treatment, with the most prominent effect observed in the high-dose chemotherapy eligible patients after induction therapy with bortezomib, cyclophosphamide, and dexamethasone. In conclusion, the EVs in patients with MM carrying TF and PPL are thus capable of exerting procoagulant activity.</p></div

Serum NMR-Based Metabolomics Profiling Identifies Lipoprotein Subfraction Variables and Amino Acid Reshuffling in Myeloma Development and Progression

Multiple myeloma (MM) is an incurable hematological cancer. It is preceded by monoclonal gammopathy of uncertain significance (MGUS)—an asymptomatic phase. It has been demonstrated that early detection increases the 5-year survival rate. However, blood-based biomarkers that enable early disease detection are lacking. Metabolomic and lipoprotein subfraction variable profiling is gaining traction to expand our understanding of disease states and, more specifically, for identifying diagnostic markers in patients with hematological cancers. This study aims to enhance our understanding of multiple myeloma (MM) and identify candidate metabolites, allowing for a more effective preventative treatment. Serum was collected from 25 healthy controls, 20 patients with MGUS, and 30 patients with MM. 1H-NMR (Nuclear Magnetic Resonance) spectroscopy was utilized to evaluate serum samples. The metabolite concentrations were examined using multivariate, univariate, and pathway analysis. Metabolic profiles of the MGUS patients revealed lower levels of alanine, lysine, leucine but higher levels of formic acid when compared to controls. However, metabolic profiling of MM patients, compared to controls, exhibited decreased levels of total Apolipoprotein-A1, HDL-4 Apolipoprotein-A1, HDL-4 Apolipoprotein-A2, HDL Free Cholesterol, HDL-3 Cholesterol and HDL-4 Cholesterol. Lastly, metabolic comparison between MGUS to MM patients primarily indicated alterations in lipoproteins levels: Total Cholesterol, HDL Cholesterol, HDL Free Cholesterol, Total Apolipoprotein-A1, HDL Apolipoprotein-A1, HDL-4 Apolipoprotein-A1 and HDL-4 Phospholipids. This study provides novel insights into the serum metabolic and lipoprotein subfraction changes in patients as they progress from a healthy state to MGUS to MM, which may allow for earlier clinical detection and treatment.This work was funded by grants from the Danish Research Council for Independent Research (4183-00268) and the Obel Family Foundation (26145).Scopu