Platelet-derived microparticles (PMP) are nano-sized fragments (100-1000 nm) released from platelets under various physiological and pathological conditions (Nieuwland and Sturk 2002).
Many diseases have now been found to be associated with high platelet-derived microparticles (PMP) count in blood, like myocardial infarction, acute coronary syndrome, stroke, venous thrombo-embolism, thrombocytopenic purpura, preeclampsia, fungal (candida albicans) sepsis, hereditary thrombophilia, thalassemia, antiphospholipid antibody syndrome, SLE, rheumatoid arthritis, psoriasis etc (Nieuwland and Sturk 2002, Italiano, Mairuhu et al. 2010, Bucciarelli, Martinelli et al. 2012, Marques, Campos et al. 2012, Woth, Tokes-Fuzesi et al. 2012, Tantawy, Adly et al. 2013, Breen, Sanchez et al. 2015, Campello, Spiezia et al. 2015, Kailashiya, Singh et al. 2015, Campello, Radu et al. 2016, Fortin, Cloutier et al. 2016, Papadavid, Diamanti et al. 2016, Sun, Zhao et al. 2016).
PMP estimation and characterization may be used as a potential biomarker and risk assessment tool in these conditions (Kafian, Mobarrez et al. 2015, Kailashiya, Singh et al. 2015). Thus sensitive and accurate estimation of PMP becomes crucial. Owing to their small size, diverse structure, functions, and polydispersed nature, special techniques and skills need to be applied for their accurate estimation and characterization.
Blood/samples should be collected in atraumatic fashion in citrate-theophylline-adenosine-dipyridamole (CTAD), sodium citrate, or acid-citrate-dextrose anticoagulants, instead of heparin and EDTA (Kim, Song et al. 2002, Lacroix, Robert et al. 2010, Yuana, Bertina et al. 2011, Coumans, Brisson et al. 2017). Hemolyzed or clotted blood should be discarded, and the sample should be processed preferably within 1 hour (Kim, Song et al. 2002, Coumans, Brisson et al. 2017). From anticoagulated whole blood, platelet poor plasma (PPP) containing microparticles (MP) can be prepared by differential centrifugation.
To further differentiate PMP from other MP, specific surface protein labeling like fluorescent tagged CD41, CD31 antibodies, and immuno-gold labeling can be used, especially for flow cytometry and electron microscopy based estimation (Ayers, Kohler et al. 2011). Purified PMP can be generated from washed platelets (WP) also by stimulation with platelet agonists like thrombin (Ayers, Kohler et al. 2011, Aatonen, Ohman et al. 2014, Kailashiya, Singh et al. 2015). Ultracentrifugation, ultrafiltration with size exclusion chromatography and affinity chromatography can be used for further separation (Nordin, Lee et al. 2015, Pocsfalvi, Stanly et al. 2016, Coumans, Brisson et al. 2017).
Flow cytometry (FCM) is the most widely used technique for estimation and characterization of PMP. Absolute count, surface and content characterization, procoagulant activity etc can be analyzed by FCM (Mobarrez, Antovic et al. 2010, Macey, Enniks et al. 2011, Helmond, Catalfamo et al. 2013). Size distribution analysis is not usually recommended by FCM as it lacks accuracy (Lacroix, Robert et al. 2010). Fluorescent-labeled monoclonal antibodies against CD41, CD62P (P-selectin), CD61, PAC1 antibody etc can be used for specific detection of PMP, distinguishing them from other MP (Nieuwland and Sturk 2002, Mobarrez, Antovic et al. 2010, Kailashiya, Singh et al. 2015). Most advantageous facts about FCM based assay are need of very small sample volume and multiple parameter (including phenotyping) analysis of PMP.
Nanoparticle Tracking Analysis (NTA) should be preferred for absolute count and size distribution analysis of PMP in pure samples (Aatonen, Ohman et al. 2014). Very small sample volume (400-500 uL), properly diluted, is required for NTA based analysis. NTA equipped with fluorescence module can be used to analyze phenotypic characteristics of selectively fluorescent labeled PMP in mixed MP samples (Dragovic, Gardiner et al. 2011).
Electron Microscopy (EM) gives an advantage of detecting even smallest size particles and is considered the gold standard for imaging MP. Both Transmission Electron Microscopy (TEM) and Scanning Electron Microscopy (SEM) can be utilized to characterize MP for size distribution and structure analysis (van der Pol, Hoekstra et al. 2010). Absolute count is not preferably done with EM. Cryo-EM is used to avoid structure distortion during sample preparation, thus facilitating morphological analysis and spatial visualization by 3D imaging of MP (Yuana, Koning et al. 2013). Atomic force microscopy (AFM) is suitable for size distribution analysis of polydispersed samples and 3D image generation as the structure of MP is preserved due to lack of drying/dehydrating steps in this technique.
Resistive pulse sensing (RPS) is used for counting and sizing of PMP. This technique is quick and convenient but maintenance of instrument apertures is strenuous. Dynamic Light Scattering (DLS) is also used for size estimation of particles, but it cannot resolve mixtures of microvesicles and exosomes (Dragovic, Gardiner et al. 2011, Yuana, Bertina et al. 2011). Enzyme-linked immune sorbent assay (ELISA) has also been reported for a quantitative assay which facilitates simple spectrophotometric or fluorimetric detection of PMP (Osumi, Ozeki et al. 2001). The advantage of this technique is that multiple samples of small volume can be analyzed by ELISA at the same time making it suitable for clinical applications.
Biosensors are newer tools for convenient assay. Glassy carbon disc electrodes with graphene oxide and PAC1 antibody, nanosilica-PAC1, P-selectin antibody and conjugated HRP on ITO (Indium Tin Oxide) electrode and an ‘on-chip’ NanoBioAnalytical platform based on combination of Surface Plasmon Resonance (SPR) and AFM based biosensors have been reported for estimation of PMP levels (Kailashiya, Singh et al. 2015, Obeid, Ceroi et al. 2017, Singh, Srivastava et al. 2017).
Functional and content assay of PMP can also be performed to explore their roles in physiological and disease conditions. PMP proteins analysis can be performed using by proteomics approaches like total protein quantity, SDS-PAGE, 2D gel electrophoresis, Western blotting and Mass Spectrometry (Choi, Kim et al. 2013, Aatonen, Ohman et al. 2014, Kreimer, Belov et al. 2015, Pienimaeki-Roemer, Kuhlmann et al. 2015). Small amounts of mRNAs present in PMP can be assayed by RT-PCR, microarray and gene sequencing techniques (Choi, Kim et al. 2013, Aatonen, Ohman et al. 2014).
Raman spectroscopic techniques (Laser-Tweezers Raman Spectroscopy, Surface-Enhanced Raman Scattering, etc) have also been reported for analysis of biochemical composition (Cholesterol, lipids, surface proteins, carotenoids, nucleic acids etc) of microvesicles (Tatischeff, Larquet et al. 2012, Buzas, Gardiner et al. 2017). Procoagulant activity, Thrombin generation, Tissue factor activity assay, fibrinolysis etc can be assayed by ELISA based methods and kits (Hemker, Giesen et al. 2002, Hellum, Ovstebo et al. 2012, Vila-Liante, Sanchez-Lopez et al. 2016, Coumans, Brisson et al. 2017). Many pre-analytical, analytical, and post-analytical factors affect PMP analysis, so it is necessary to implement all possible precautions and follow reported recommendations to minimize errors while working with PMP.
These findings are described in the article entitled Platelet-derived microparticles analysis: Techniques, challenges and recommendations, recently published in the journal Analytical Biochemistry. This work was conducted by Jyotsna Kailashiya from Banaras Hindu University, Varanasi.
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