Comparison of assays measuring extracellular vesicle-tissue factor in plasma samples: Communication from the ISTH SSC Subcommittee on Vascular Biology.

BACKGROUND: Scientific and clinical interest in extracellular vesicles (EVs) is growing. EVs that expose tissue factor (TF) bind factor VII/VIIa and can trigger coagulation. Highly procoagulant TF-exposing EVs are detectable in the circulation in various diseases, such as sepsis, COVID-19 or cancer. Many in-house and commercially available assays have been developed to measure EV-TF activity and antigen but only a few studies have compared some of these assays. The ISTH SSC Subcommittee on Vascular Biology initiated a multicenter study to compare the sensitivity, specificity and reproducibility of these assays. MATERIALS AND METHODS: Platelet-depleted plasma samples were prepared from blood of healthy donors. The plasma samples were spiked either with EVs from human milk, or EVs from TF-positive and TF-negative cell lines. Plasma was also prepared from whole human blood with or without LPS stimulation. Twenty-one laboratories measured EV-TF activity and antigen in the prepared samples using their own assays representing 18 functional and 9 antigenic assays. RESULTS: There was a large variability in the absolute values for the different EV-TF activity and antigen assays. Activity assays had higher specificity and sensitivity compared to antigen assays. In addition, there was a large intra-assay and inter-assay variability. Functional assays that used a blocking anti-TF antibody or immunocapture were the most specific and sensitive. Activity assays that used immunocapture had a lower coefficient of variation compared to assays that isolated EVs by high-speed centrifugation. CONCLUSION: Based on this multicenter study, we recommend measuring EV-TF using a functional assay in the presence of an anti-TF antibody.

A sensitive tissue factor activity assay determined by an optimized thrombin generation method.

BACKGROUND: Tissue factor (TF) is the principal activator of the coagulation system, but an increased concentration in the blood in cancer and inflammatory diseases has been suggested to play a role increasing the risk of venous thromboembolism. However, measurement of the TF concentration is difficult, and quantitation of activity is the most valid estimation. The objective of this study was to establish a sensitive method to measure TF activity based on thrombin generation. METHODS: The assay is based on thrombin generation (TG) measured on the Calibrated Automated Thrombogram (CAT). Various low concentrations of TF were prepared from reagents containing 1 pM TF and 4 µM phospholipid (PPL), and no TF and 4 µM PPL, and a calibration curve was produced from Lagtime vs TF concentration. TF in blood samples was measured after isolation and resuspension of extracellular vesicles (EVs) in a standard plasma from which EVs had been removed. The same standard plasma was used for the calibrators. RESULTS: Contact activation of the coagulation system was avoided using CTI plasma samples in Monovette tubes. EVs contain procoagulant phospholipids but addition of PPL only reduced lagtime slightly at very low concentrations of TF resulting in overestimation to a lesser extent at 10 fM but no interference at 30 fM or higher. Addition of EVs to the TG analysis induced a small unspecific TF-independent activity (i.e., an activity not inhibited by antibodies against TF) which also may result in a smaller error in estimation of TF activity at very low levels but the effect was negligible at higher concentrations. It was possible to measure TF activity in healthy controls which was found to be 1-6 fM (EVs were concentrated, i.e. solubilized in a lower volume than the original volume plasma). Coefficient of variation (CV) was below 20% at the low level, and below 10% at a level around 100 fM TF. However, the step with isolation of EVs have a higher inherent CV. CONCLUSION: A sensitive and rather precise one-stage TG-based method to measure TF activity has been established.

Sulfate and acid-base balance.

It has been acknowledged for years that compounds containing sulfur (S) are an important source of endogenous acid production. In the metabolism, S is oxidized to sulfate, and therefore the mEq sulfate excreted in the urine is counted as acid retained in the body. In this study we show that pH in fluids with constant [Na] and [HEPES] declines as sulfate ions are added, and we show that titratable acidity increases exactly with the equivalents of sulfate. Therefore, sulfate excretion in urine is also acid excretion per se. This is in accordance with the down-regulation of proximal sulfate reabsorption under acidosis and the observation that children with distal renal tubular acidosis may be sulfate depleted. These results are well explained using charge-balance modeling, which is based only on the three fundamental principles of electroneutrality, conservation of mass, and rules of dissociation as devised from physical chemistry. In contrast, the findings are in contrast to expectations from conventional narratives. These are unable to understand the decreasing pH as sulfate is added since no conventional acid is present. The results may undermine the traditional notion of endogenous acid production since in the case of sulfur balance, S oxidation and its excretion as sulfate exactly balance each other. Possible clinical correlates with these findings are discussed.

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.

The effect of pH on thrombin generation-An unrecognized potential source of variation.

BACKGROUND: When CO2 escapes from plasma, the pH of the plasma increases. In samples left open or kept in long-term storage, the pH may increase considerably. Assays in which the ratio of plasma sample relative to the total volume including reagents is high may be sensitive to the pH of the plasma sample. OBJECTIVE: The aim was to investigate the effect of the pH of plasma samples used in the calibrated automated thrombin generation (CAT) assay in which the ratio (plasma sample) / (total volume) is high. METHODS: Plasma pH was increased by allowing CO2 to escape in open beakers before the CAT analysis. The effect of pH was also investigated by mixing plasma with buffers with different pH levels. RESULTS: At a pH close to 8.0, endogenous thrombin potential (ETP) and peak decreased considerably, whereas lagtime and time-to-peak were modestly increased. Mixtures of plasma and buffer with pH levels between 7 and 8 showed that ETP and peak decreased at alkaline pH; lagtime and time-to-peak were higher at acidic pH levels but were shortened, partly in contrast to first results, at alkaline pH levels. The addition of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer to plasma with a high pH attenuated the effects; however, the effect was most significant if added before the CO2 escaped. CONCLUSION: Modifications of plasma pH can significantly alter thrombin generation. In alkaline samples, for example, after lengthy storage in a freezer where pH can increase considerably, thrombin generation is lowered. To minimize this effect, plasma should be stored in tubes filled to the maximum volume.