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Nanofiltration to remove microparticles and decrease the thrombogenicity of plasma: in vitro feasibility assessment

Ming-Li Chou, Liang-Tzung Lin, David Devos, and Thierry Burnouf

Transfusion

Background: 

As plasma contains procoagulant microparticles (MPs), removing MPs by 75-nm nanofiltration may decrease plasma in vitro thrombogenicity while maintaining the hemostatic activity from coagulation factors.

STUDY DESIGN AND METHODS:

We defined conditions to nanofilter leukoreduced plasma on a 75-nm hollow-fiber membrane filter. Plasma quality was assessed by coagulation, immunochemical, and electrophoretic assays. MP removal was evaluated by biophysical (flow cytometry, dynamic light scattering, nanoparticle tracking analysis, and tunable resistive pulse sensing) and functional (thrombin generation assay [TGA; Technothrombin], prothrombinase [Zymuphen MP-activity], tissue factor [Zymuphen MP-TF], and procoagulant phospholipid-dependent clotting time [STA-Procoag-PPL] assays) methods. Spiking experiments using platelet MPs were performed to determine extent of removal by nanofiltration.

RESULTS:

Freshly collected leukoreduced, but not previously frozen, plasma could be readily nanofiltered on a 0.01-m2 75-nm nanofilter under conditions preserving protein and lipoprotein profile, coagulation factor content, and global coagulation activity (prothrombin time, activated partial thromboplastin time). Biophysical methods confirmed an extensive removal of MPs during nanofiltration. All functional assays indicated a marked reduction of plasma in vitro thrombogenicity. There was no thrombin generation in nanofiltered plasma tested by TGA assay with "RC-low phospholipid concentration" reagent, while it was similar to that of starting and leukoreduced plasma samples when using "RC-high phospholipid concentration" reagent. More than 9 log of MPs were removed by nanofiltration.

CONCLUSION:

Nanofiltration of 75 nm efficiently removes MPs and decreases in vitro thrombogenicity of plasma without affecting the protein content or the hemostatic activity of coagulation factors. Studies are needed to evaluate the impact of MP removal on in vivo thrombogenic risks and hemostatic efficacy.

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