Development of a time-resolved immunofluorometric assay for quantifying platelet-derived microparticles in human plasma
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Published on: January 31, 2018
Annika E Michelsen1, Ragnhild Wergeland, Oddvar Stokke
1Research Institute for Internal Medicine, University of Oslo, Rikshospitalet, N-0027 Oslo, Norway. a.e.michelsen@biokjemi.uio.no
Abstract:
Platelet-derived microparticles (PMPs) are considered a marker of platelet activation. They vary considerably in size, and flow cytometry, the predominant method used to assay PMPs, is only detecting larger PMPs (>0.1 microm). We describe here a method that quantifies the amount of PMP-located GPIIb antigen in detergent-treated platelet-free plasma (PPP) by means of a one-step time-resolved immunofluorometric assay (TR-IFMA). This assay uses a streptavidin-coated microwell plate and two different monoclonal antibodies to GPIIb (CD41), one conjugated to biotin and the other labeled with europium ion. A wide linear range standard curve with low background and a high sensitivity was obtained. Pre-assay ultracentrifugation or filtration of PPP extensively reduced the fluorometric signal, indicating that the GPIIb antigen is mainly particle-located. A strong correlation between the amount of GPIIb and PMP as detected by flow cytometry was found. Consequently, the assay can be used to study PMP-related phenomena and, in contrast to flow cytometry, can be used on frozen samples and is independent of PMP size.
Insights
This study introduces a new assay to quantify platelet-derived microparticles (PMPs) by measuring GPIIb antigen. This method offers a sensitive and size-independent alternative to flow cytometry for PMP analysis.
Area of Science:
- Hematology
- Biochemistry
- Analytical Chemistry
Background:
- Platelet-derived microparticles (PMPs) are key indicators of platelet activation.
- Current flow cytometry methods for PMP detection are limited, primarily identifying larger microparticles (>0.1 microm).
Purpose of the Study:
- To develop and validate a novel assay for quantifying PMPs.
- To overcome the size limitations of existing PMP detection methods.
Main Methods:
- A one-step time-resolved immunofluorometric assay (TR-IFMA) was developed to quantify GPIIb antigen in platelet-free plasma (PPP).
- The assay utilizes a streptavidin-coated microwell plate with biotin and europium-labeled anti-GPIIb (CD41) monoclonal antibodies.
- Detergent treatment of PPP was employed to access particle-located antigens.
Main Results:
- The TR-IFMA demonstrated a wide linear range, high sensitivity, and low background.
- Ultracentrifugation or filtration significantly reduced the signal, confirming the particle-bound nature of the GPIIb antigen.
- A strong correlation was observed between GPIIb antigen levels and PMPs detected by flow cytometry.
Conclusions:
- The developed TR-IFMA is a sensitive and reliable method for quantifying PMPs.
- This assay is independent of PMP size and can analyze frozen samples, offering advantages over flow cytometry.
- The assay facilitates the study of PMP-related phenomena.
