Platelet-free plasma does not yet exist
Abstract
Background
Human blood plasma contains clinically relevant information that can be used to develop disease biomarkers. Despite intensive research, >99.9% of initially promising biomarkers never reach the clinic. A possible explanation is the inconsistent composition of plasma samples stored in biobanks. Although believed to be “cell-free”, human plasma still contains 1E5-1E8 platelets and erythrocyte ghosts (ery-ghosts) per mL, even after two centrifugation steps. Moreover, the concentration of remaining cells varies, and depends on both the operator and centrifugation protocol (>200 existing protocols).
Although for some analytes the presence of remaining cells will not matter, for others, such as extracellular vesicle (EV) concentration and EV-microRNA (EV-miRNA) analyses, remaining cells disturb measurements and interpretation of results. Since the concentration of remaining cells is too low for visual confirmation and because downstream effects on other analytes (DNA, proteins) are unknown, this is an unrecognized and possibly underestimated problem.
Main body
We developed a procedure to specifically and simultaneously sort and concentrate platelets and ery-ghosts from double centrifuged human plasma. Cell concentrations pre- and post-sorting were measured by flow cytometry. The presence of platelets and ery-ghosts was confirmed by super-resolution microscopy, using specific capture- and fluorescent detection antibodies.
Using a novel sorting procedure, the concentration of platelets and ery-ghosts increased 64-fold and 40-fold post-sorting, respectively. We confirmed that the sorted particles were platelets and ery-ghosts using direct stochastic optical reconstruction microscopy (dSTORM) imaging. Ery-ghosts were both membrane dye and CD235a positive, whereas platelets showed characteristic pseudopods and were positively labeled with a membrane dye and CD63.
Short conclusion
In summary, we demonstrate that residual platelets and ery-ghosts are still present in so-called "platelet-free" plasma. These findings highlight an overlooked issue, emphasizing the importance of sample quality reporting and the need for novel methods to obtain truly cell-free plasma. Enhancing standardization in plasma sample quality and composition will create a more consistent foundation for reliable biomarker development.
