Blood Cell Research Group, Section for Research, Department of Medical Biochemistry, Oslo University Hospital, Ullevål, Oslo, Norway.
Cytometry A. 2011 Dec;79(12):990-9. doi: 10.1002/cyto.a.21147. Epub 2011 Oct 11.
Tissue factor (TF)-positive microparticles (MPs) are highly procoagulant, and linked to thrombosis in sepsis and cancer. MP-associated TF may be assayed by immunological or functional methods. Several reports have demonstrated discrepancies between TF-protein and TF-activity, which have been explained by antibody binding to "encrypted" or degraded forms of inactive TF-protein. Our goal was to evaluate the possible interference of fluorescent antibody aggregates in solutions containing antibodies against TF and CD14 in flow cytometric analysis. Using monocyte-derived microparticles (MPs) released from human monocytes, incubated with or without lipopolysaccharides (LPS) in vitro, we measured MP-associated TF-protein (flow cytometry) and TF-activity (clot formation assay). MPs released from monocytes exposed to LPS (1 ng mL(-1) ) had ∼14 times higher TF-activity than MPs originated from monocytes exposed to only culture medium. However, using untreated anti-TF antibodies (American Diagnostica and BD) in the flow cytometric analysis, MPs released from unstimulated monocytes had a similar number of TF-positive events as MPs secernated from LPS-stimulated monocytes [∼45,000 events mL(-1) (American Diagnostica); ∼15,000 events mL(-1) (BD)]. These TF-positive events did not exert any TF-activity, and centrifugation (17,000g, 30 min, 4°C) of the antibody solutions prior to use effectively removed the interfering fluorescent events. Removal of fluorescent interference, probably in the form of fluorescent antibody aggregates, from the antibody solutions by centrifugation is essential to prevent the occurrence of false positive flow cytometric events. The events can be mistaken as MP-associated TF-protein, and interpreted as a discrepancy between TF-protein and TF-activity.
组织因子（TF）阳性微粒（MPs）具有高度促凝性，与败血症和癌症中的血栓形成有关。MP 相关 TF 可通过免疫学或功能方法进行检测。几项报告表明 TF 蛋白和 TF 活性之间存在差异，这可以通过抗体与无活性 TF 蛋白的“加密”或降解形式结合来解释。我们的目标是评估在流式细胞分析中含有针对 TF 和 CD14 的抗体的溶液中荧光抗体聚集体可能产生的干扰。使用单核细胞来源的 MPs（从人类单核细胞释放，体外与或不与脂多糖（LPS）孵育），我们测量了 MP 相关的 TF 蛋白（流式细胞术）和 TF 活性（凝块形成测定）。与仅暴露于培养基的单核细胞释放的 MPs 相比，暴露于 LPS（1ng mL(-1)）的单核细胞释放的 MPs 的 TF 活性高约 14 倍。然而，在流式细胞分析中使用未经处理的抗 TF 抗体（美国诊断学公司和 BD）时，未受刺激的单核细胞释放的 MPs 具有与 LPS 刺激的单核细胞分泌的 MPs 相似数量的 TF 阳性事件[约 45,000 个事件 mL(-1)（美国诊断学公司）；约 15,000 个事件 mL(-1)（BD）]。这些 TF 阳性事件没有发挥任何 TF 活性，并且在使用之前将抗体溶液离心（17,000g，30min，4°C）可以有效去除干扰的荧光事件。通过离心从抗体溶液中去除荧光干扰（可能以荧光抗体聚集体的形式）对于防止假阳性流式细胞术事件的发生至关重要。这些事件可能被误认为是 MP 相关的 TF 蛋白，并被解释为 TF 蛋白和 TF 活性之间的差异。
