Detection of Antiphospholipid Antibodies by Flow Cytometry: Rapid Detection of Antibody Isotype and Phospholipid Specificity

Stewart, Michael W.; Etches, Wai S.; Russell, A. S.; Percy, J. S.; Johnston, Carol A.; Chew, Christina; Gordon, Philip A.

doi:10.1055/s-0038-1649636

Cited by 33 publications

(23 citation statements)

References 18 publications

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“…Binding of C5b-9 to phospholipid-coated beads was detected as follows. Polystyrene beads were coated with phospholipid (CL or PS) as originally described (Stewart et al, 1993) and blocked with either 10% (v/v) fetal bovine serum (Gibco, Burlington, Ontario) or 0·3% gelatin (Sigma, St Louis, Mo.). Test sera were incubated with the phospholipid-coated beads in the presence of 1 mM CaCl 2 for 30 min at RT.…”

Section: Measurement Of Platelet Activation In Response To Apa Seramentioning

confidence: 99%

Antiphospholipid antibody‐dependent C5b‐9 formation

1997

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The relationship between the presence of antiphospholipid antibodies (APA) and the production of the terminal membrane attack complex (MAC) of complement (C5b‐9) was studied. Serum samples from known high positive APA patients induced platelet activation and destruction which was inhibited by heat‐inactivation of the sera. The response was restored if the heat‐inactivated APA‐positive sera were supplemented with normal sera. Adsorption of the APA‐positive sera with phospholipid (PL)‐coated polystyrene beads inhibited platelet destruction. Addition of monoclonal antibody (mAb) to C5b‐9 (aE11) also inhibited platelet destruction, suggesting that the APA‐dependent platelet destruction might be complement‐mediated. Purified APA, in the presence of normal serum, induced C5b‐9 formation and binding to PL‐coated beads in a dose‐dependent manner as detected by flow cytometry. Prospective analysis of 200 serum samples for C5b‐9 production showed that all sera testing negative for the presence of APA also tested negative for C5b‐9 production. All sera with high levels of IgG binding to PL (GPL) showed evidence of C5b‐9 production. Sera with low or moderate GPL values showed varying levels of C5b‐9 production. These data suggest that complement may play a key role in APA‐dependent platelet activation, in vivo.

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Section: Measurement Of Platelet Activation In Response To Apa Seramentioning

confidence: 99%

Antiphospholipid antibody‐dependent C5b‐9 formation

1997

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“…The ability of the flow cytometer to discriminate between individual microspheres on the basis of size, fluorescent intensity, and/or fluorescent wavelength makes possible multianalytical assays. The use of microspheres of different sizes for multiplex assays has been described for different analytes in numerous publications (1,15,16,18,23,24). However, discrimination of microspheres by fluorescence has been documented only recently (8,14).…”

mentioning

confidence: 99%

Flow Cytometric Microsphere-Based Immunoassay: Analysis of Secreted Cytokines in Whole-Blood Samples from Asthmatics

Camilla

Mély

Magnan

et al. 2001

Clin Diagn Lab Immunol

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The ability of flow cytometry to resolve multiple parameters was used in a microsphere-based flow cytometric assay for the simultaneous determination of several cytokines in a sample. The flow cytometer microspherebased assay (FMBA) for cytokines consists of reagents and dedicated software, specifically designed for the quantitative determination of cytokines. We have made several improvements in the multiplex assay: (i) dedicated software specific for the quantitative multiplex assay that processes data automatically, (ii) a stored master calibration curve with a two-point recalibration to adjust the stored curve periodically, and (iii) an internal standard to normalize the detection step in each sample. Overall analytical performance, including sensitivity, reproducibility, and dynamic range, was investigated for interleukin-4 (IL-4), IL-6, IL-10, IL-12, gamma interferon (IFN-␥), and tumor necrosis factor alpha. These assays were found to be reproducible and accurate, with a sensitivity in the picograms-per-milliliter range. Results obtained with FMBA correlate well with commercial enzyme-linked immunosorbent assay data (r > 0.98) for all cytokines assayed. This multiplex assay was applied to the determination of cytokine profiles in whole blood from atopic and nonatopic patients. Our results show that atopic subjects' blood produces more IL-4 (P ‫؍‬ 0.003) and less IFN-␥ (P ‫؍‬ 0.04) than the blood of nonatopic subjects. However, atopic asthmatic subjects' blood produces significantly more IFN-␥ than that of atopic nonasthmatic subjects (P ‫؍‬ 0.03). The results obtained indicate that the FMBA technology constitutes a powerful system for the quantitative, simultaneous determination of secreted cytokines in immune diseases.It has been known for years that fluorescent flow cytometric detection combined with the use of sized latex microspheres allows one to perform specific and quantitative immunoassays of soluble analytes (9). The ability of the flow cytometer to discriminate between individual microspheres on the basis of size, fluorescent intensity, and/or fluorescent wavelength makes possible multianalytical assays. The use of microspheres of different sizes for multiplex assays has been described for different analytes in numerous publications (1,15,16,18,23,24). However, discrimination of microspheres by fluorescence has been documented only recently (8,14).The routine use of this attractive technology faces three distinct hurdles. First, the software commercialized with cytometers is complex and more appropriate for the qualitative cellular analysis of individual samples than for the batch mode of sampling required for the quantitative assay of several analytes. Second, reagent development faces unique analytical difficulties, such as the calibration of each individual assay in a multiplex assay and the quality of complex reagents with multiple components. Third, the concept of multiplex quantitative assays, albeit very attractive in principle, has yet to demonstrate its usefulness compared with well-acce...

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“…For the sake of completeness, we also mention the use of FACS analysis for the determination of aCL [31].…”

Section: Immunological Testsmentioning

confidence: 99%

Antiphospholipid Syndrome

Schössler

2000

Int Arch Allergy Immunol

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Antiphospholipid syndrome (APS) is a disease characterized by venous and arterial thromboses or spontaneous abortions and the repeated detection of antiphospholipid antibodies (aPL). APS may be associated with another autoimmune disease (secondary APS), particularly systemic lupus erythematosus (SLE), or unrelated to an underlying disease (primary APS). APS affects almost all organs. In addition to the clinical criteria, lupus anticoagulant testing and immunological aPL determinations are required to establish the diagnosis of APS.

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Detection of Antiphospholipid Antibodies by Flow Cytometry: Rapid Detection of Antibody Isotype and Phospholipid Specificity

Cited by 33 publications

References 18 publications

Antiphospholipid antibody‐dependent C5b‐9 formation

Antiphospholipid antibody‐dependent C5b‐9 formation

Flow Cytometric Microsphere-Based Immunoassay: Analysis of Secreted Cytokines in Whole-Blood Samples from Asthmatics

Antiphospholipid Syndrome

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