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Impact of collection, isolation and storage methodology of circulating microvesicles on flow cytometric analysis

  • Authors:
    • Fancong Kong
    • Liming Zhang
    • Hongxiang Wang
    • Guolin Yuan
    • Anyuan Guo
    • Qiubai Li
    • Zhichao Chen
  • View Affiliations / Copyright

    Affiliations: Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, P.R. China, Department of Hematology, Jingzhou Central Hospital, Jingzhou, Hubei 434020, P.R. China, Department of Hematology, Wuhan Central Hospital, Wuhan, Hubei 430012, P.R. China, Department of Hematology, Xiangyang Central Hospital, The Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, Hubei 441021, P.R. China, Department of Biomedical Engineering, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, Hubei 430074, P.R. China
    Copyright: © Kong et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Pages: 2093-2101
    |
    Published online on: September 25, 2015
       https://doi.org/10.3892/etm.2015.2780
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Abstract

Microvesicles (MVs) in body fluids participate in a variety of physical and pathological processes, and are regarded as potential biomarkers for numerous diseases. Flow cytometry (FCM) is among the most frequently used techniques for MV detection. However, different handling methods unavoidably cause pre‑analytical variations in the counts and sizes of MVs determined by FCM. The aim of the present study was to investigate the effect of centrifugation, storage conditions and anticoagulant on MV measurements. Blood samples were obtained from 13 healthy donors, including 4 women and 9 men. Calcein‑AM staining was used to label MVs and assess the impact of pre‑analytical preparation, including centrifugation, and storage conditions on MV measurements obtained using FCM. The range of factors investigated for comparison included: Platelet‑free plasma (PFP) stored at ‑80˚C for 1 or 4 weeks; MVs stored at 4˚C for 3‑4 days or 1 week; MVs frozen at ‑80˚C for 1 or 4 weeks; and anticoagulants, either heparin or ethylenediaminetetraacetic acid (EDTA). No statistically significant differences in MV counts were detected between the two centrifugation speeds (16,000 and 20,500 x g) or among the three centrifugation times (15, 30 and 60 min) investigated. Similarly, no significant differences were noted in MV counts between the two anticoagulants tested (heparin and EDTA). However, the storage of PFP or MVs in heparin‑anticoagulated plasma for different periods markedly affected the detected MV counts and size distribution. The counts and sizes of MVs from EDTA‑anticoagulated plasma were only affected when the MVs were frozen at ‑80˚C for 4 weeks. In conclusion, calcein‑AM is able to efficiently identify MVs from plasma and may be an alternative to Annexin V for MV staining. EDTA preserves the MV counts and size more accurately compared with heparin under calcein‑AM staining. PFP centrifuged at 16,000 x g for 15 min is sufficient to isolate MVs, which enables the batch processing of samples. PFP, rather than MVs alone, appears to be the preferable mode of sample storage, as MVs stored in PFP were less affected by storage temperature and duration. The present study provides a methodology for MV collection, storage and isolation, to facilitate further investigation of MVs as biomarkers in disease.
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Copy and paste a formatted citation
Spandidos Publications style
Kong F, Zhang L, Wang H, Yuan G, Guo A, Li Q and Chen Z: Impact of collection, isolation and storage methodology of circulating microvesicles on flow cytometric analysis. Exp Ther Med 10: 2093-2101, 2015.
APA
Kong, F., Zhang, L., Wang, H., Yuan, G., Guo, A., Li, Q., & Chen, Z. (2015). Impact of collection, isolation and storage methodology of circulating microvesicles on flow cytometric analysis. Experimental and Therapeutic Medicine, 10, 2093-2101. https://doi.org/10.3892/etm.2015.2780
MLA
Kong, F., Zhang, L., Wang, H., Yuan, G., Guo, A., Li, Q., Chen, Z."Impact of collection, isolation and storage methodology of circulating microvesicles on flow cytometric analysis". Experimental and Therapeutic Medicine 10.6 (2015): 2093-2101.
Chicago
Kong, F., Zhang, L., Wang, H., Yuan, G., Guo, A., Li, Q., Chen, Z."Impact of collection, isolation and storage methodology of circulating microvesicles on flow cytometric analysis". Experimental and Therapeutic Medicine 10, no. 6 (2015): 2093-2101. https://doi.org/10.3892/etm.2015.2780
Copy and paste a formatted citation
x
Spandidos Publications style
Kong F, Zhang L, Wang H, Yuan G, Guo A, Li Q and Chen Z: Impact of collection, isolation and storage methodology of circulating microvesicles on flow cytometric analysis. Exp Ther Med 10: 2093-2101, 2015.
APA
Kong, F., Zhang, L., Wang, H., Yuan, G., Guo, A., Li, Q., & Chen, Z. (2015). Impact of collection, isolation and storage methodology of circulating microvesicles on flow cytometric analysis. Experimental and Therapeutic Medicine, 10, 2093-2101. https://doi.org/10.3892/etm.2015.2780
MLA
Kong, F., Zhang, L., Wang, H., Yuan, G., Guo, A., Li, Q., Chen, Z."Impact of collection, isolation and storage methodology of circulating microvesicles on flow cytometric analysis". Experimental and Therapeutic Medicine 10.6 (2015): 2093-2101.
Chicago
Kong, F., Zhang, L., Wang, H., Yuan, G., Guo, A., Li, Q., Chen, Z."Impact of collection, isolation and storage methodology of circulating microvesicles on flow cytometric analysis". Experimental and Therapeutic Medicine 10, no. 6 (2015): 2093-2101. https://doi.org/10.3892/etm.2015.2780
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