Abstract
Citrate is the preferred anticoagulant for blood collection, as EDTA damages platelets and heparin modifies their function (1). Citrate allows the rapid generation of platelet-rich plasma (PRP), with a high yield of platelets; however, this method has certain disadvantages. In particular, the PRP preparation has a limited stability (no longer than 2 h) and contains plasma proteins, including enzymes. In addition, human platelet-rich plasma (PRP) prepared from blood collected into trisodium citrate (3.8% w/v) has a depressed ionic calcium concentration, which can cause platelet aggregation and release of substances during centrifugation (2). To overcome these different problems, a centrifugation technique has been developed for the isolation and washing of platelets from human or rodent blood anticoagulated with acid-citrate-dextrose (ACD). The cells are resuspended in a physiological buffer under well-defined conditions, notably the presence of plasmatic ionic calcium concentrations (2 mM) and the absence of coagulation factors or other plasma components.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Ludlam, C. (1981) Assessment of platelet function. In Thrombosis and Haemostasis (Bloom, A. L., and Thomas, D. P., eds.), Churchill Livingstone, New York, pp. 775–795.
Kinlough-Rathbone, R. L., Packham, M. A., and Mustard, J. F. (1983) Platelet aggregation. In Methods in Hematology (Harker, L. A. and T.S, Z., eds.), Vol. 8, Churchill Livingstone, New, York, pp. 64–91.
Cazenave, J. P., Hemmendinger, S., Beretz, A., Sutter-Bay, A., and Launay, J. (1983) Platelet aggregation: a tool for clinical investigation and pharmacological study. Methodology. Ann. Biol. Clin. (Paris) 41, 167–179.
Mustard, J. F., Perry, D. W., Ardlie, N. G., and Packham, M. A. (1972) Preparation of suspensions of washed platelets from humans. Br. J. Haematol. 22, 193–204.
Ardlie, N. G., Perry, D. W., Packham, M. A., and Mustard, J. F. (1971) Influence of apyrase on stability of suspensions of washed rabbit platelets. Proc. Soc. Exp. Biol. Med. 136, 1021–1023.
Han, P. and Ardlie, N. G. (1974) The influence of pH, temperature, and calcium on platelet aggregation: maintenance of environmental pH and platelet function for in vitro studies in plasma stored at 37 degrees C. Br. J. Haematol. 26, 373–389.
Kinlough-Rathbone, R. L., Mustard, J. F., Packham, M. A., Perry, D. W., Reimers, H. J., and Cazenave, J. P. (1977) Properties of washed human platelets. Thromb. Haemost. 37, 291–308.
Molnar, J. and Lorand, L. (1961) Studies on apyrases. Arch. Biochem. Biophys. 93, 353.
Ardlie, N. G., Glew, G., and Schwartz, C. J. (1966) Influence of catecholamines on nucleotide-induced platelet aggregation. Nature 212, 415–417.
Gachet, C., Stierle, A., Ohlmann, P., Lanza, F., Hanau, D., and Cazenave, J. P. (1991) Normal ADP-induced aggregation and absence of dissociation of the membrane GPIIb-IIIa complex of intact rat platelets pretreated with EDTA. Thromb. Haemost. 66, 246–253.
Lanza, F., Beretz, A., Stierle, A., Hanau, D., Kubina, M., and Cazenave, J. P. (1988) Epinephrine potentiates human platelet activation but is not an aggregating agent. Am. J. Physiol. 255, H1276–H1288.
Baurand, A., Eckly, A., Bari, N., Leon, C., Hechler, B., Cazenave, J. P., et al. (2000) Desensitization of the platelet aggregation response to ADP: differential down-regulation of the P2Y1 and P2cyc receptors. Thromb. Haemost. 84, 484–491.
Rolf, M. G., Brearley, C. A., and Mahaut-Smith, M. P. (2001) Platelet shape change evoked by selective activation of P2X1 purinoceptors with α,β-methylene ATP. Thromb. Haemost. 85, 303–308.
Sinakos, Z. and Caen, J. P. (1967) Platelet aggregation in mammalians (human, rat, rabbit, guinea-pig, horse, dog). A comparative study. Thromb. Diath. Haemorrh. 17, 99–111.
Walsh, P. N., Mills, D. C., and White, J. G. (1977) Metabolism and function of human platelets washed by albumin density gradient separation. Br. J. Haematol. 36, 287–296.
Corash, L., Tan, H., and Gralnick, H. R. (1977) Heterogeneity of human whole blood platelet subpopulations. I. Relationship between buoyant density, cell volume, and ultrastructure. Blood 49, 71–87.
Tangen, O., McKinnon, E. L., and Berman, H. J. (1973) On the fine structure and aggregation requirements of gel filtered platelets (GFP). Scand. J. Haematol. 10, 96–105.
Lages, B., Scrutton, M. C., and Holmsen, H. (1975) Studies on gel-filtered human platelets: isolation and characterization in a medium containing no added Ca2+, Mg2+, or K+. J. Lab. Clin. Med. 85, 811–825.
Aster, R. H., and Jandl, J. H. (1964) Platelet sequestration in man. I. Methods. J. Clin. Invest. 43, 843.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc.
About this protocol
Cite this protocol
Cazenave, JP., Ohlmann, P., Cassel, D., Eckly, A., Hechler, B., Gachet, C. (2004). Preparation of Washed Platelet Suspensions From Human and Rodent Blood. In: Gibbins, J.M., Mahaut-Smith, M.P. (eds) Platelets and Megakaryocytes. Methods In Molecular Biology™, vol 272. Humana Press. https://doi.org/10.1385/1-59259-782-3:013
Download citation
DOI: https://doi.org/10.1385/1-59259-782-3:013
Publisher Name: Humana Press
Print ISBN: 978-1-58829-101-1
Online ISBN: 978-1-59259-782-6
eBook Packages: Springer Protocols