Fluorescent particles in the antibody solution result in false TF- and CD14-positive microparticles in flow cytometric analysis

Abstract

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 TFprotein 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 21 ) 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 21 (American Diagnostica); $15,000 events mL 21 (BD)]. These TF-positive events did not exert any TF-activity, and centrifugation (17,000g, 30 min, 48C) 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. ' 2011 International Society for Advancement of Cytometry

Measurement of microvesicle levels in human blood using flow cytometry

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“…This includes sheath solutions, all buffers used in sample preparation, antibodies, and other probes, etc. Antibodies aggregates can also be removed by ultracentrifugation (34). As a quality control, background counts should be measured in sheath solutions, buffers, and all other reagents after filtration to demonstrate low counts are maintained.…”

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