MicroRNA 320a and Membrane Antigens as Tools to Evaluate the Pathophysiology of Platelets Stored in Blood Banks

Abstract

Our research group, through the analysis of miRNomes in platelet concentrates (PCs) stored in blood banks, identified and validated the miR-127 and miR-320a miRNAs as biomarkers of platelet storage lesions (PSLs) in PCs. In order to validate the miRNAs 127 and 320a methodologically, as PSL biomarkers in a large number of PC bags, we also evaluated important immunological markers involved in the platelet activation/aggregation process-the CD62P receptor (P-selectin), the surface glycoproteins (GP) IIb/IIIa, and the purinergic P2Y12 receptor-via flow cytometry. The miRNAs miR-127 and miR-320a were quantified by real-time quantitative PCR (RT-qPCR). To carry out this study, 500 collection tubes were used at the upper edge of the PC bags containing platelets. Each tube was divided into seven equal parts (totaling 3500 samples) for platelet analysis from 7 different storage days, where the 1st day represents the high-quality control, and the 7th day corresponds to the low-quality control of the platelets. After analyzing all parameters during storage days, it was concluded that the relative quantification of miR-320a below 0.50 and the CD62P receptor below 27.92% are reliable indicators of the absence of storage lesions in blood banks. We believe that the values found in the expression of the CD62P receptor legitimize the use of the miR-320a and miR-127 miRNAs to build a kit capable of accurately measuring whether the stored platelets are suitable for transfusion.

Keywords: biomarkers; membrane antigens; miR-127; miR-320a; platelet concentrate; storage lesion.

Figure 1

Expression (%) of the CD62P receptor in platelet concentrates during the 7 days of storage. The error bars represent the variability of the data that were calculated from the standard deviation; * p < 0.05.

Figure 2

Expression (%) of glycoproteins IIb (CD41) and IIIa (CD61) in platelet concentrates during the 7 days of storage. The error bars represent the variability of the data that were calculated from the standard deviation; * p < 0.05.

Figure 3

(a) Correlogram showing the miRNAs and membrane receptors that most correlated during the 7 days of PC storage. The scales with a blue gradient are positively correlated, while those with a red gradient are negatively correlated; the white gradient represents insignificant correlation (p > 0.01). (b) Heatmap showing the patterns of expression of miRNAs and receptors during the 7 days of storage of PCs. Z-score was the metric applied to infer the best clustering patterns. Gradients with a tendency towards a red color indicate a lower Z-score, and gradients with a tendency towards a blue color indicate a greater Z-score. (c) Variation in the levels of expression of miR-127 and miR-320a during the 7 days of storage.