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Plasma (Original Mix) ~UPD~

A single point mutation of the factor V (FV) gene, leading to the substitution Arg506Gln in the FV molecule (FV-Leiden) and hence resistance to its breakdown by activated protein C (APC), is the most prevalent risk factor for venous thrombosis in the Caucasians. A ratio determined by activated partial thromboplastin time (APTT) of test plasma in the presence or absence of exogenous APC (the APC ratio), is the method widely used to screen individuals with this risk factor for thrombosis. Because of functional defects of vitamin K-dependent clotting factors in patients on oral anticoagulant therapy, this method cannot be applied to those patients without modification. One modification is to mix test plasma (1:5 or 1:10) with FV-deficient plasma so that 80-90% of functioning vitamin K-dependent factors are supplied by the FV-deficient plasma. Even with 10-20% of FV in the mixture, APC-resistance still can be demonstrated. In this report, we present our results of the modified APC-sensitivity assay using FV-deficient plasma from different commercial sources. APC ratios determined by the original method in which test plasma is not mixed with FV-deficient plasma can be significantly different from those determined by the modified method in which test plasma is diluted 1:5 with FV-deficient plasma. This difference between methods was observed not only in normal individuals, but also in FV-Leiden positive individuals, and in patients on warfarin therapy. Further, APC ratios varied significantly depending on the commercial source of the FV-deficient plasma. The modified method is apparently suitable to identify APC-resistance in patients on warfarin therapy, as well as in individuals not receiving anticoagulant treatment. However, one must be aware that APC-resistance ratios obtained with the modified method are likely to be different from those established with the original method, and the source of FV-deficient plasma can be a factor influencing the ratios in the former cases.

Plasma (Original Mix)

Original CellBrite Cytoplasmic Membrane Stains are lipophilic carbocyanine dyes. These dyes undergo an increase in fluorescence when they insert into lipid bilayers. Lipophilic carbocyanine dyes stably label the plasma membrane and other intracellular membranes of cells. They also can be used to stain fixed cells or artificial lipid bilayers. CellBrite cytoplasmic membrane stains are lipophilic carbocyanine dyes. These dyes undergo an increase in fluorescence when they insert into lipid bilayers. Lipophilic carbocyanine dyes stably label the plasma membrane and other intracellular membranes of cells. They also can be used to stain artificial lipid bilayers. Immediately after staining cultured cells, the dyes primarily localize to the plasma membrane. If cells are cultured over time after staining, the labeled membranes are internalized and staining gradually becomes mostly intracellular.

Trans-acting programmed death-ligand 1 (PD-L1) derives from malignant cells in three known forms. High levels of secreted splice variant PD-L1 (sPD-L1), ADAM10/ADAM17-shed sPD-L1, and PD-L1-positive extracellular vesicles (evPD-L1) each predict poor prognosis and limited response to PD-(L)1 checkpoint inhibitors in cancer. To our knowledge, no clinical intervention has reduced any of these circulating forms of extracellular PD-L1. Here, we explore therapeutic plasma exchange (TPE) as a treatment to reduce circulating extracellular PD-L1.

Here, we report the first known clinical intervention to remove either sPD-L1 or evPD-L1 from plasma in vivo. TPE reduces plasma sPD-L1 and evPD-L1 in vivo and may have a role in treatment with immunotherapy. TPE may also prove useful in patients with other extracellular vesicle-related conditions.

Patients undergoing therapeutic plasma exchange (TPE) are compared by starting soluble programmed death-ligand 1 (sPD-L1) level above or below survival cut-off established in patients with melanoma (0.277 ng/mL). For categorical variables, n (%) is given. For continuous variables, mean (quartiles) is given.

Therapeutic plasma exchange (TPE) significantly reduces plasma soluble programmed death-ligand 1 (sPD-L1) levels. (A) A model of the TPE procedure in which patient plasma is separated and replaced to extract non-cellular substances confined to the plasma. (B) A diagram of the present study in which 24 patients undergo plasma exchange. (C) All plasma levels of sPD-L1 immediately prior to (pre) and after (post) TPE using albumin replacement fluid are plotted. TPE significantly reduced sPD-L1 levels in patient plasma by Wilcoxon signed-rank test (p

Most patients undergoing TPE did not have an active cancer diagnosis. Baseline sPD-L1 levels in all patients were compared with matched normal controls and patients with melanoma (online supplementary fig 1), and some patients exhibited sPD-L1 above the clinically significant cut-off level determined in the retrospective melanoma cohort. Patients with high baseline sPD-L1 levels were significantly more anemic than patients with lower baseline sPD-L1 even when controlling for the higher number of female subjects in the high sPD-L1 group (female-only mean Hgb 11.4 vs 14.1, p=0.04; male-only mean Hgb 11.8 vs 14.3, p=0.03). Groups were otherwise similar. TPE significantly reduced plasma sPD-L1 levels in patients receiving albumin-only (ie, no FFP) replacement fluid (figure 2C, p

A representative individual patient treatment course showing sPD-L1 reduction over four successive TPE sessions is also shown (figure 2D). All individual patient TPE courses, including sessions involving donated human blood products (eg, fresh frozen plasma or FFP), are shown in online supplementary fig 2. Pre-TPE and post-TPE sPD-L1 levels for all sessions are also shown (online supplementary fig 3). TPE significantly reduced plasma sPD-L1 even when sessions requiring donated FFP were included (p

Plasma exchange efficiently reduces total, programmed death-ligand 1 (PD-L1)-positive, and ADAM10-positive extracellular vesicle (EV) levels in vivo. Plasma levels of total EVs immediately prior to (pre) and after (post) therapeutic plasma exchange (TPE) are plotted. TPE significantly reduced (A) total (p

Individual patient courses showing total plasma, PD-L1-positive, ADAM10-positive, and CD61-positive EV levels before and after each TPE session are shown in online supplementary fig 5 with exemplary nanoflow plots in online supplementary fig 6. Three successive TPE sessions consistently depleted total, PD-L1-positive, and ADAM10-positive (but not CD61-positive) EVs. These trends were less pronounced when sessions in which patients received donor FFP were included (online supplementary fig 7). In normal control FFP donors, blood type did not correlate with plasma EV concentrations (online supplementary fig 8).

Flow cytometry for EVs was performed as previously published.27 In brief, plasma samples were centrifuged twice at 2000g to deplete platelets. Resultant platelet-free plasma were analyzed using an A60-Micro Plus Nanoscale Flow Cytometer (Apogee FlowSystems) gating for mid-intensity light angle light scatter and markers of interest. Anti-PD-L1 (Genentech: atezolizumab), ADAM10 (R&D Systems: clone 163003), and CD61 (BioLegend: clone VI-PL2) antibodies were conjugated to fluorophores (Life Technologies: Alexa-647, PE phycoerythrin, and Alexa-488) and titrated prior to use. Nanoscale flow cytometer calibration was performed using a standard reference bead mix as previously published. Flow cytometry was performed by team members blinded to the identity of the samples.

This work presents an original way for preparing TiO-based coatings by thermal spraying. As titanium monoxide is oxidized by the mere trace of oxygen contained in hydrogen, it is obvious that plasma spraying of TiO powder does not lead easily to the formation of a titanium monoxide coating. However, thermodynamical calculations show that the conditions necessary for the preparation of TiO can be reached, at the titanium melting temperature (Tm=1933 K), when oxygen reacts with liquid metal in the presence of excess carbon. These results have led to experiments in which TiO-based coatings have been prepared by spraying a stoichiometric mixture of graphite and titanium grains onto cast iron in air. In optimal conditions, a gas-tight hard coating (1000 15 HV3) of a TiO-based phase (composition: TiO0.810.06C0.040.02) has been obtained. 041b061a72


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