Isotype controls in phenotyping and quantification of microparticles: A major source of error and how to evade it

Trummer, Arne; Rop, Christiane De; Tiede, Andreas; Ganser, Arnold; Eisert, R.

doi:10.1016/j.thromres.2008.01.005

Cited by 34 publications

(49 citation statements)

References 20 publications

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“…Typically isotype controls are run to evaluate nonspecific binding of antibodies. Due to variations in the nonspecific binding of isotype controls versus specific antibodies, it has been suggested that antigen negative microvesicles may represent an alternate method for evaluating nonspecific binding (36). Plasma controls should be run to evaluate staining, gating, and final microvesicle counts.…”

Section: Interference and Assay Optimizationmentioning

confidence: 99%

Measurement of microvesicle levels in human blood using flow cytometry

Chandler

2016

Cytometry Part B Clinical

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Microvesicles are fragments of cells released when the cells are activated, injured, or apoptotic. Analysis of microvesicle levels in blood has the potential to shed new light on the pathophysiology of many diseases. Flow cytometry is currently the only method that can simultaneously separate true lipid microvesicles from other microparticles in blood, determine the cell of origin and other microvesicle characteristics, and handle large numbers of clinical samples with a reasonable effort, but expanded use of flow cytometric measurement of microvesicle levels as a clinical and research tool requires improved, standardized assays. The goal of this review is to aid investigators in applying current best practices to microvesicle measurements. First pre-analytical factors are evaluated and data summarized for anticoagulant effects, sample transport and centrifugation. Next flow cytometer optimization is reviewed including interference from background in buffers and reagents, accurate microvesicle counting, swarm interference, and other types of coincidence errors, size calibration, and detection limits using light scattering, impedance and fluorescence. Finally current progress on method standardization is discussed and a summary of current best practices provided. V C 2016 Clinical Cytometry Society

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Section: Interference and Assay Optimizationmentioning

confidence: 99%

Measurement of microvesicle levels in human blood using flow cytometry

Chandler

2016

Cytometry Part B Clinical

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“…When analyzing very small particles, accuracy depends on the proper discrimination of EVs from other non cell derived particles and on thorough removal of background noise. Prior publications have noted difficulties associated with using FCM to analyze EVs, includ ing false positive signals arising from EV mimicking immune complexes (55,56), self aggregation of antibodies due to agita tion (57), and limited applicability of traditionally used FCM controls such as FMO (fluorescence minus one) and/or anti body isotypes (58). EV sample collection and processing is yet another area in which standardization is needed, yet no con sensus exists on an optimal protocol (59 61).…”

Section: Nonementioning

confidence: 99%

“…However, our attempts to replicate the background of an antibody with its isotype proved to be difficult, as it was impossible to know whether the signal was true background or simply an artifact caused by the differences in spectral properties between the two stains. Indeed, a number of publications have noted similar issues associated with using isotype controls for this purpose (58,59). Isotype gates can vary widely depending on a number of factors including: antibody supplier, fluorochrome:protein ratio, antibody concentration, propensity for aggregation, and antibody subclass (7,48,58).…”

Section: Ev Gating Strategiesmentioning

confidence: 99%

“…Indeed, a number of publications have noted similar issues associated with using isotype controls for this purpose (58,59). Isotype gates can vary widely depending on a number of factors including: antibody supplier, fluorochrome:protein ratio, antibody concentration, propensity for aggregation, and antibody subclass (7,48,58). Though these variables can be accounted for/controlled to a certain degree, it is difficult if not impossible to match perfectly the background fluores cence of a fluorochrome conjugated antibody to that of its isotype.…”

Section: Ev Gating Strategiesmentioning

confidence: 99%

“…Of all of the controls we tested (FMO, unstained, isotype, and lysed), lysed samples proved to be the most consistently reli able as an indicator of background fluorescence across all markers when samples were left unwashed after staining. Other researchers have reported the use of antigen negative EVs as negative controls for setting background fluorescence gates (58). However, because background fluorescence can often vary from individual to individual, it may be inappro priate to apply the gates created from a single sample of entirely different origin to all clinical samples in the study.…”

Section: Ev Gating Strategiesmentioning

confidence: 99%

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Does RBC Storage Age Effect Inflammation, Immune Function and Susceptibility to Transfusion Associated Microchimerism in Critically Ill Patients? Adverse Effects of RBC Storage in Critically Ill Patients

Spinella¹,

Norris²

2015

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

et al. 2011

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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

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Isotype controls in phenotyping and quantification of microparticles: A major source of error and how to evade it

Cited by 34 publications

References 20 publications

Measurement of microvesicle levels in human blood using flow cytometry

Measurement of microvesicle levels in human blood using flow cytometry

Does RBC Storage Age Effect Inflammation, Immune Function and Susceptibility to Transfusion Associated Microchimerism in Critically Ill Patients? Adverse Effects of RBC Storage in Critically Ill Patients

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

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