Development of Quantitatively Determining the Microparticles from Different Sources in Human Blood Plasma by Nine-color Flow Cytometry_West China Medical Journal

Authors：

 LiYang ^1,2 , HuangQiaorong ¹ , LiXue ¹ , ZengTingting ³ , ZhuHuanling ² ,  MengWentong ¹

1. Laboratory of Stem Cell Biology, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;
2. Department of Hematology, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;
3. Department of Laboratory Medicine, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, P. R. China;

Corresponding author：

MengWentong, Email: mwentong@sina.com

Keywords：

Nine-color flow cytometry; Microparticle; Platelet poor plasma

DOI：

10.7507/1002-0179.201600546

Video：

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Abstract Full text Figures/Tables Video References Cited by

Objective To develop a method to quantitatively determine the microparticles (MP) from different sources in plasma by nine-color flow cytometry. Methods Annexin-V and 8 antibodies including CD235a, CD41a, CD45, CD34, CD66b, CD20, CD3 and CD14 were used to establish nine-color flow cytometric panel.Platelet poor plasma samples were single-stained and stained with 1 of 8 antibodies lacking respectively, and then we determined the detector voltages and compensations.From December 2014 to January 2015, we detected and analyzed 10 plasma samples from normal adults, and repeatability test and dilution tests were done. Results In staining lacking 1 of 8 antibodies, the percentage of positive MP populations change was all less than 15% based on the population number in single-stained experiment.In dilution tests, there were good linear correlations between MPs from platelets and erythrocytes.In repeatability test, the coefficient of variation of MP from erythrocytes, platelets and granulocytes was all less than 10%.In the platelet poor plasma samples from normal adults, MP from platelets, erythrocytes, endotheliocytes, monocytes, granulocytes, B and T lymphocytes could be detected, and the average concentration of them were respectively 132.6/μL[(60.6-288.9)/μL], 35.4/μL[(22.0-99.7)/μL], 21.6/μL[(3.3-45.5)/μL], 13.9/μL[(7.3-35.1)/μL], 60.0/μL[(22.5-101.2)/μL], 21.9/μL[(6.0-33.4)/μL]and 1.2/μL[(0.7-2.8)/μL]. Conclusions Quantitatively determining MP from different sources in plasma by nine-color flow cytometry has been successfully developed.This method is simple and fast, and can be applied in clinical detection.

Citation： LiYang, HuangQiaorong, LiXue, ZengTingting, ZhuHuanling, MengWentong. Development of Quantitatively Determining the Microparticles from Different Sources in Human Blood Plasma by Nine-color Flow Cytometry. West China Medical Journal, 2016, 31(12): 1989-1994. doi: 10.7507/1002-0179.201600546 Copy

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10.	黄巧容, 余建华, 蒋能刚, 等.急性白血病患者八色流式免疫表型分析起始管方案的建立[J].华西医学, 2012, 27(4):555-559.
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13.	Lacroix R, Robert S, Poncelet PA. Overcoming limitations of microparticle measurement by flow cytometry[J]. Semin Thromb Hemost, 2010, 36(8):807-818.
14.	Bertolini F, Shaked Y, Mancuso P, et al. The multifaceted circulating endothelial cell in cancer:towards marker and target identification[J]. Nat Rev Cancer, 2006, 6(11):833-845.
15.	Wood B. 9-color and 10-color flow cytornetry in the clinical laboratory[J]. Arch Pathol Lab Med, 2006, 130(5):680-690.
16.	Mahnke YD, Roederer M. Optimizing a multicolor immunophenotyping assay[J]. Clin Lab Med, 2007, 27(3):469-485.
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1. Wu ZH, Ji CL, Li H, et al. Membrane microparticles and diseases[J]. Eur Rev Med Pharmacol Sci, 2013, 17(18):2420-2427.
2. Kriebardis A, Antonelou M, Stamoulis K, et al. Cell-derived microparticles in stored blood products:innocent-bystanders or effective mediators of post-transfusion reactions[J]. Blood Transfus, 2012, 10(Suppl 2):s25-s38.
3. Marsh A, Schiffelers R, Kuypers F, et al. Microparticles as biomarkers of osteonecrosis of the hip in sickle cell disease[J]. Br J Haematol, 2015, 168(1):135-138.
4. Mooberry MJ, Key NS. Microparticle analysis in disorders of hemostasis and thrombosis[J]. Cytometry A, 2016, 89(2):111-122.
5. Baron M, Boulanger CM, Staels B, et al. Cell-derived microparticles in atherosclerosis:biomarkers and targets for pharmacological modulation[J]. J Cell Mol Med, 2012, 16(7):1365-1376.
6. Barteneva NS, Fasler-Kan E, Bernimoulin M, et al. Circulating microparticles:square the circle[J]. BMC Cell Biol, 2013, 14:23.
7. Gelderman MP, Simak J. Flow cytometric analysis of cell membrane microparticles[J]. Methods Mol Biol, 2008, 484:79-93.
8. van der Heyde HC, Gramaglia I, Combes V, et al. Flow cytometric analysis of microparticles[J]. Methods Mol Biol, 2011, 699:337-354.
9. Preffer F, Dombkowski D. Advances in complex multiparameter flow cytometry technology:applications in stem cell research[J]. Cytometry B Clin Cytom, 2009, 76(5):295-314.
10. 黄巧容, 余建华, 蒋能刚, 等.急性白血病患者八色流式免疫表型分析起始管方案的建立[J].华西医学, 2012, 27(4):555-559.
11. Yuana Y, Bertina RM, Osanto S. Pre-analytical and analytical issues in the analysis of blood microparticles[J]. Thromb Haemost, 2011, 105(3):396-408.
12. Diamant M, Tushuizen ME, Sturk A, et al. Cellular microparticles:new players in the field of vascular disease[J]. Eur J Clin Invest, 2004, 34(6):392-401.
13. Lacroix R, Robert S, Poncelet PA. Overcoming limitations of microparticle measurement by flow cytometry[J]. Semin Thromb Hemost, 2010, 36(8):807-818.
14. Bertolini F, Shaked Y, Mancuso P, et al. The multifaceted circulating endothelial cell in cancer:towards marker and target identification[J]. Nat Rev Cancer, 2006, 6(11):833-845.
15. Wood B. 9-color and 10-color flow cytornetry in the clinical laboratory[J]. Arch Pathol Lab Med, 2006, 130(5):680-690.
16. Mahnke YD, Roederer M. Optimizing a multicolor immunophenotyping assay[J]. Clin Lab Med, 2007, 27(3):469-485.
17. Lockett SJ. Speeding up image cytometry[J]. Cytometry A, 2015, 87A (2):99-100.

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West China Medical Journal

Development of Quantitatively Determining the Microparticles from Different Sources in Human Blood Plasma by Nine-color Flow Cytometry

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