The supernatant was collected and passed through a Protein G Sepharose 4 Fast Flow?(Cytiva Life Sciences) affinity chromatography column for IgG isolation. to plasma of VKA-treated controls. This effect was most evident on point-of-care test INR measurements, while the recombinant Quick reagent exhibited a lesser degree of interference. In contrast, tissue-derived thromboplastin reagents (Owren STA-Hepato Prest and Quick STA-NeoPTimal) remained largely unaffected by these antibodies, both in pooled normal plasma and VKA anticoagulated control plasma. Among these reagents, the Owren INR Cobimetinib hemifumarate reagent exhibited the lowest sensitivity to the influence of LA antibodies. This observed difference in sensitivity is independent of the plasma dilution factor or the presence of factor V or fibrinogen in Owren reagent. Conclusion INR reagents that utilize recombinant human thromboplastin are more sensitive to the presence of monoclonal and patient-derived antibodies with LA activity. Consequently, APS patients positive for LA should be monitored using tissue-derived thromboplastin reagents, given its reduced susceptibility to interference by LA-causing antibodies. Keywords: anticoagulants, antiphospholipid syndrome, international normalized ratio, lupus coagulation inhibitor, warfarin Essentials ? Lupus anticoagulant (LA) may interfere with international normalized ratio tests. ? The interference of LA-causing antibodies on international normalized ratio measurements was tested. ? Recombinant human Cobimetinib hemifumarate thromboplastin reagents are more sensitive to LA. ? Anticoagulation in LA patients can be monitored safely using tissue-derived thromboplastins. 1.?Introduction Antiphospholipid syndrome (APS) is an acquired autoimmune disorder Rabbit polyclonal to PIWIL2 defined by thrombosis and adverse pregnancy outcome in the presence of persistent antiphospholipid antibodies (aPL) [1]. The current laboratory criteria for the classification of APS include 3 aPL types: anticardiolipin (aCL) autoantibodies, antiCbeta-2-glycoprotein I (a2GPI) autoantibodies, and lupus anticoagulants (LAs). LAs refer to a heterogeneous group of autoantibodies that interfere with phospholipid-dependent coagulation tests for 10 minutes, and the plasma was stored at??80 C until further use. Pooled normal plasma (PNP) was prepared from healthy volunteers, as previously described [30]. In short, blood was collected from Cobimetinib hemifumarate healthy controls by venipuncture (3.2% sodium citrate; Beckton Dickinson) after obtaining informed consent. Platelet-poor plasma was obtained from the supernatant fraction after double centrifugation at 2840? for 10 minutes at room temperature. Afterward, the plasma was pooled, and aliquots were stored at??80 C until further use. 2.3. Purification of antigen-specific patient IgG antibodies The study protocol was approved as nonmedical Research Involving Human Subjects Act research by the medical ethics committee Cobimetinib hemifumarate of the Erasmus MC, Rotterdam, the Netherlands (MEC 2021-0131). Fresh apheresis plasma was obtained from APS patients undergoing regular therapeutic plasma exchange for anticoagulant-refractory APS. IgG antibodies were precipitated from the apheresis plasma by gradually adding solid ammonium sulfate to a final concentration of 50%, followed by incubation for at least 1 hour at room temperature and centrifugation at 4200? at 4 C for 20 minutes. The pellet obtained after the centrifugation was resuspended in 0.05 M sodium phosphate buffer (pH 7.0), and the Cobimetinib hemifumarate conductivity was adjusted to 28.5 mS/cm using Milli-Q water. IgM antibodies were precipitated by the addition of polyethylene glycol 6000 to a final concentration of 8%, followed by a centrifugation step at 4000? at room temperature for 30 minutes. The supernatant was collected and passed through a Protein G Sepharose 4 Fast Flow?(Cytiva Life Sciences) affinity chromatography column for IgG isolation. The Protein G column was washed using 0.01 M sodium phosphate buffer (pH 7.0) containing 0.15 M NaCl. Bound IgG antibodies were eluted with 0.1 M glycine (pH 3.0), and the pH of the protein-containing fractions was neutralized immediately using 1.0 M Tris (pH 8.5). The purity.