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Updated: May 28, 2026
IP-FCM: Immunoprecipitation Detected by Flow Cytometry
Published on: December 2, 2010
Fluorescent particles in the antibody solution result in false TF- and CD14-positive microparticles in flow
Hans Christian D Aass1, Reidun Øvstebø, Anne-Marie S Trøseid
1Blood Cell Research Group, Section for Research, Department of Medical Biochemistry, Oslo University Hospital, Ullevål, Oslo, Norway. H.C.Aass@medisin.uio.no
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 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.
Insights
Fluorescent antibody aggregates can cause false positives in flow cytometry assays for tissue factor (TF)-positive microparticles (MPs). Centrifugation removes these aggregates, ensuring accurate measurement of TF-protein and TF-activity in thrombosis research.
Area of Science:
- Hematology
- Immunology
- Biotechnology
Background:
- Tissue factor (TF)-positive microparticles (MPs) are crucial in thrombosis, particularly in sepsis and cancer.
- Assaying MP-associated TF can involve immunological or functional methods, but discrepancies between TF-protein and TF-activity have been reported.
- These discrepancies may arise from antibody interactions with inactive TF forms or interfering substances.
Purpose of the Study:
- To investigate the interference of fluorescent antibody aggregates in flow cytometric analysis of TF-positive MPs.
- To evaluate the impact of such interference on the accurate quantification of MP-associated TF-protein and TF-activity.
Main Methods:
- Human monocytes were stimulated with lipopolysaccharides (LPS) to induce TF-positive MPs.
- MP-associated TF-protein was measured using flow cytometry, and TF-activity was assessed via a clot formation assay.
- Antibody solutions were analyzed before and after centrifugation to identify and remove interfering fluorescent events.
Main Results:
- LPS-stimulated MPs exhibited significantly higher TF-activity (approximately 14-fold) compared to unstimulated MPs.
- Flow cytometry using untreated anti-TF antibodies showed similar numbers of TF-positive events for both stimulated and unstimulated MPs, indicating false positives.
- Centrifugation of antibody solutions effectively removed these interfering fluorescent events, which were likely antibody aggregates.
Conclusions:
- Fluorescent antibody aggregates can lead to false-positive results in flow cytometric detection of MP-associated TF-protein.
- Pre-centrifugation of antibody solutions is essential to eliminate interference and ensure accurate assessment of TF-protein and TF-activity.
- Accurate measurement is critical for understanding the role of TF-positive MPs in thrombotic conditions.
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