An efficient method for isolation of representative and contamination-free population of blood platelets for proteomic studies

Abstract

To date, there has been no ideal method for blood platelet isolation which allows one to obtain a preparation devoid of contaminations, reflecting the activation status and morphological features of circulating platelets. To address these requirements, we have developed a method which combines the continuous density gradient centrifugation with washing from PGI₂-supplemented platelet-rich plasma (PRP). We have assessed the degree of erythrocyte and leukocyte contamination, recovery of platelets, morphological features, activation status, and reactivity of isolated platelets. Using our protocol, we were able to get a preparation free from contaminations, representing well the platelet population prior to the isolation in terms of size and activity. Besides this, we have obtained approximately 2 times more platelets from the same volume of blood compared to the most widely used method. From 10 ml of whole citrated blood we were able to get on average 2.7 mg of platelet-derived protein. The method of platelet isolation presented in this paper can be successfully applied to tests requiring very pure platelets, reflecting the circulating platelet state, from a small volume of blood.

Keywords:

• Density gradient centrifugation
• platelet isolation
• platelet proteomics
• platelets

Declaration of interest

The authors report no conflict of interest

Acknowledgment

We would like to acknowledge the assistance of Ewa Sitkiewicz from Mass Spectrometry Laboratory in Institute of Biochemistry and Biophysics of Polish Academy of Sciences, Warsaw who carried out the proteomic analysis of our platelet preparations. We are also grateful to Beata Celuch from the Department of Haematology, Blood Neoplasms and Bone Marrow Transplantation, Wroclaw Medical University, Ewa Korecka with the team from Central Analytical Laboratory, Independent Public Clinical Hospital, Wroclaw, for the excellent technical help.