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Cell-derived microparticles : composition and function
Biró, É.
Publication date
2008
Link to publication
Citation for published version (APA):
Biró, É. (2008). Cell-derived microparticles : composition and function.
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S
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Measuring circulating cell-derived
microparticles: A flow cytometric method
of microparticle analysis
Éva Biró, Rienk Nieuwland, Augueste Sturk
J Thromb Haemost 2004; 2: 1843-1844.
CHAPTER 2
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o elucidate the role of microparticles under physiologic and pathophysiologic conditions, our group has performed several studies. Our results and those of others (reviewed in [1,2]) show unequivocally that microparticles play a role in blood coagulation, cell activation, transport of bioactive molecules, and inflammation. However, the exact mechanisms involved await further investigation.
Studies on microparticles can be wrought with pitfalls. Appropriate sampling conditions, processing and storage of samples are essential. Also, care must be taken that control samples are collected and handled in exactly the same way as the studied material. When drawing blood, prolonged use of a tourniquet should be avoided and a needle of 19 G diameter or larger should be used to avoid cell activation or damage. We mostly use citrate (0.32% final concentration) as the anticoagulant, but EDTA or heparin can also be used. The blood should be gently mixed with the anticoagulant, and centrifuged within 15 min at 1550 × g for 20 min at 20°C to pellet the cells. These centrifugation conditions provide an optimal yield of microparticles, while platelet contamination is minimal. The plasma containing the microparticles is carefully aspirated, leaving a layer of about 1 cm undisturbed on top of the cells. The cell-free plasma is aliquotted (250 μL portions), snap-frozen in liquid nitrogen, and stored at −80°C until analysis, when it is thawed on melting ice. Snap-freezing and thawing on melting ice ensure the best possible preservation of microparticle structure and function. When snap-freezing of clinical samples is not possible, control samples should be handled accordingly, and results interpreted with caution.
For measuring cell-derived microparticles ex vivo, our method of choice is flow cytometry. This method enables the analysis of thousands of microparticles in one sample, with the simultaneous determination of multiple markers. Even small subpopulations can be identified and analyzed. Furthermore, all microparticles present are measured, whereas in capture assays only microparticles positive for the surface binding marker are captured and analyzed. Flow cytometry gives fairly quantitative results, although in this respect other methods might be superior. Major disadvantages of flow cytometry are its labor-intensiveness and costs.
For flow cytometric analysis, microparticles in cell-free plasma are pelleted by centrifugation at 18000 × g for 30 min at 20°C. Afterwards, the upper 90% (225 μL) of the plasma is removed and replaced by phosphate-buffered saline containing 0.32% citrate (PBS-citrate). The microparticles are resuspended and pelleted again. Then, the upper 90% of the supernatant is again removed. The microparticles are resuspended and diluted 4-fold with PBS-citrate. Should the remaining plasma (2.5%) in the microparticle suspensions pose a problem, an extra washing step can be introduced, with the inevitable loss of some microparticles. Of the microparticle suspensions, 5 μL aliquots are incubated with monoclonal antibodies (see Table 1) and/or annexin V in a final volume of 50 μL PBS containing 2.5 mmol/L CaCl2 (PBS-Ca), for 15 min at 20°C in the dark. We use PBS
containing 1.4 mmol/L phosphate, to avoid calcium-phosphate precipitates. If
T
MICROPARTICLE ANALYSIS
43
2
2
fluorochrome-conjugated probes are used, 900 μL PBS-Ca is added after incubation, and the sample measured. If unlabeled probes are used, the microparticles are washed once with 200 μL PBS-Ca, then the secondary, fluorochrome-conjugated probe is added. The sample is again incubated for 15 min at 20°C in the dark, then diluted by the addition of 900 μL PBS-Ca, and measured. An unstained sample, a sample labeled with annexin V in the absence of calcium, and appropriate isotype controls must be included in the panel. Isotype controls (clones used by us: IgG1 X40; IgG2a G155-178; IgG2b MCG2b) should have
concentrations and dye/protein ratios similar to that of the specific antibodies and must be from the same species. It is essential to titrate all the probes on appropriate microparticle samples before use, and batch-to-batch variations should also be taken into consideration. For instrument standardization, settings and maintenance, we refer to the appropriate manuals.
Our methods of sample collection, processing, storage, and measurement by flow cytometry provide reproducible results. Other possibilities given by the flow cytometer that have not yet been fully exploited, e.g. the measurement of intravesicular markers and the sorting of microparticles before subsequent analysis by other techniques, will also further our insight into the role and significance of cell-derived microparticles in health and disease.
Table 1. Monoclonal antibodies used to detect microparticles derived from specific cell types. Cellular source of
microparticles
Marker Activation marker Clone
Platelets CD61 (GPIIIa) CD62P (P-selectin) CD63 VI-PL2 Y2/51 CLB-Thromb/6 CLB-gran12 Erythrocytes Glycophorin A clone JC159 T helper cells CD4 CLB-T4/2, 6D10 T cytotoxic cells CD8 SK1 B cells CD20 L27 Granulocytes CD66E CD66B CLB-gran/10, IH4Fc 80H3 Monocytes / macrophages CD14 MφP9 CRIS-6 Endothelial cells CD31 CD144 CD62E (E-selectin) WM-59 BMS158F1 1.2B6 HAE-1f
CHAPTER 2
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References
[1] VanWijk MJ, VanBavel E, Sturk A, Nieuwland R. Microparticles in cardiovascular diseases.
Cardiovasc Res 2003; 59: 277-287.
[2] Freyssinet JM. Cellular microparticles: what are they bad or good for? J Thromb Haemost 2003; 1: 1655-1662.
