Effects of speed and time of centrifugation and time of freezing on the amount of produced microparticles from concentrates platelet

authors:

avatar tahereh manoochehrabadi , avatar Zohreh Sharifi ORCID , * , avatar fatemeh yari , avatar elham rezvani broujeni , avatar hamideh mirshafiei , avatar mahin nikougoftar zarif , avatar ghasem hasan nejad

Koomesh: Vol.17, issue 3; 686-691
published online: September 28, 2016
article type: Research Article
received: May 07, 2015
accepted: October 06, 2015

how to cite: manoochehrabadi T, Sharifi Z, yari F, rezvani broujeni E, mirshafiei H, et al. Effects of speed and time of centrifugation and time of freezing on the amount of produced microparticles from concentrates platelet. koomesh. 2016;17(3):e151217. 

Abstract

Introduction: The main method of separating platelet microparticles (PMP) is based on the centrifugation speed and time. Due to the high cost of determining the number of PMP via micro-particles (micro bead) and also the necessity of using an expensive device such as a flow cytometer, it seems that Bradford method would be rather an inexpensive, fast and efficient way to determine the concentration of PMP.  Therefore, in this study the effect of different factors, such as speed and time of centrifugation and time of freezing on the concentration of PMP in the platelet concentrates bags was studied. Materials and Methods: We studied two different speeds of centrifugation for separating PRP. In the first protocol for preparation of PRP, the platelet bags were centrifuged at 1500g for 15min and in the second protocol they were centrifuged at 5000g for the same duration.  To evaluate the effect of time, microparticles were separated in 16000g for 20 and 2 min. To determine the concentration of PMP, Bradford method was used. To evaluation the effect of freezing, the PRP was prepared at 300g for 20 min, and then it was freezed in -80˚c for five days. Flow cytometery analysis was performed for microparticles identification. Results: PMP concentrates with the 1500g centrifugation speed showed higher concentration (P

References

  • 1.

    Piccin A, Murphy WG, Smith OP. Circulating microparticles: pathophysiology and clinical implications. Blood Rev 2007; 21: 157-71.

  • 2.

    Burnier L, Fontana P, Kwak BR, Angelillo-Scherrer A. Cell-derived microparticles in haemostasis and vascular medicine. Thromb Haemost 2009; 101: 439-51.

  • 3.

    Kowalska MA, Ratajczak J, Hoxie J, Brass LF, Gewirtz A, Poncz M, Ratajczak MZ. Megakaryocyte precursors, megakaryocytes and platelets express the HIV coreceptor CXCR4 on their surface: determination of response to stromalderived factor1 by megakaryocytes and platelets. Br J Haematol 1999; 104: 220-9.

  • 4.

    Trummer A, De Rop C, Tiede A, Ganser A, Eisert R. Isotype controls in phenotyping and quantification of microparticles: A major source of error and how to evade it. Thromb Res 2008; 122: 691-700.

  • 5.

    Yin W, Ghebrehiwet B, Peerschke EI. Expression of complement components and inhibitors on platelet microparticles. Platelets 2008; 19: 225-233.

  • 6.

    Janowska-Wieczorek A, Majka M, Kijowski J, Baj-Krzyworzeka M, Reca R, Turner AR, et al. Platelet-derived microparticles bind to hematopoietic stem/progenitor cells and enhance their engraftment. Blood 2001; 98: 3143-3149.

  • 7.

    Esmaili MA, Yari F, Sharifi Z, Nikougoftar M, Fadaei R. Effects of platelet microparticles on the activation of B cells. Modares J Med Sci Pathobiol 2013; 15: 1-10. (Persian).

  • 8.

    Mobarrez F, Antovic J, Egberg N, Hansson M, Jrneskog G, Hultenby K, Walln H. A multicolor flow cytometric assay for measurement of platelet-derived microparticles. Thromb Res 2010; 125: e110-e116.

  • 9.

    Baj-Krzyworzeka M, Baran J, Siedlar M, Szatanek R. Application of Flow Cytometry in the Studies of Microparticles: INTECH Open Access Publisher; 2012.

  • 10.

    Nielsen MH, Beck-Nielsen H, Andersen MN, Handberg A. A flow cytometric method for characterization of circulating cell-derived microparticles in plasma. J Extracell Vesicles 2014; 3.

  • 11.

    Roback JD. Technical Manual: AMER ASSN OF BLOOD BANKS; 2014.

  • 12.

    Robert S, Poncelet P, Lacroix R, Arnaud L, Giraudo L, Hauchard A, Sampol J, Dignat-George F. Standardization of plateletderived microparticle counting using calibrated beads and a Cytomics FC500 routine flow cytometer: a first step towards multicenter studies? J Thromb Haemost 2009; 7: 190-197.

  • 13.

    Baj-Krzyworzeka M, Baran J, Weglarczyk K, Szatanek R, Szaflarska A, Siedlar M, Zembala M. Tumour-derived microvesicles (TMV) mimic the effect of tumour cells on monocyte subpopulations. Anticancer Res 2010; 30: 3515-3519.

  • 14.

    Chandler WL. Microparticle counts in platelet-rich and platelet-free plasma, effect of centrifugation and sample-processing protocols. Blood Coagul Fibrinolysis 2013; 24: 125-132.

  • 15.

    Shah MD, Bergeron AL, Dong J-F, Lpez JA. Flow cytometric measurement of microparticles: pitfalls and protocol modifications. Platelets 2008; 19: 365-372.