High-purity isolation platelets by gradient centrifugation plus filtration.

INTRODUCTION
Platelets can be used as a liquid biopsy source to provide rapid, up-to-date, and relevant information on tumor pathology and treatment response. However, there is still a lack of high efficiency methods for platelet isolation with high purity.


METHODS
Three platelet isolation methods were evaluated by platelet recovery and purity. The platelet inhibition cocktail (PIC) was added into peripheral blood, or was not allowed to access the effect of the platelet activation. The CD61, CD45, and CD62P labelled platelets, leukocytes and activated platelets were detected by flow cytometry. Quantitative polymerase chain reaction (qPCR) and next-generation sequencing (NGS) were employed to determine the gene expression levels. A time-dependent experiment combined with qPCR was used to determine the time limit for platelet isolation at room temperature.


RESULTS
Compared to the gradient centrifugation alone, and gradient centrifugation plus filtration and magnetic beads separation, gradient centrifugation plus filtration was the preferred method for more efficient and high-purity platelet isolation, with a recovery rate of 9.1% and a purity of 99.98%. Furthermore, there was no difference in platelet activation level, regardless of whether PIC was used. Moreover, the rate of platelet RNA degradation did not differ when platelets were isolated within 48 h of blood collection.


CONCLUSION
Gradient centrifugation plus filtration at room temperature within 48 h of blood collection, without PIC, is a novel protocol with high recovery and purity rate to isolate platelets.