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Impact of Anticoagulation and Sample Processing on the Quantification of Human Blood-Derived microRNA Signatures.

Author(s): Mussbacher M, Krammer TL, Heber S, Schrottmaier WC, Zeibig S, Holthoff HP, Pereyra D, Starlinger P, Hackl M, Assinger A

Publication: Cells, 2020, Vol. 9, Page

PubMed ID: 32824700 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare miRNA levels in EDTA, citrate, and CTAD plasma subjected to room temperature or refrigerated centrifugation delays. Effects of hemolysis on miRNA levels and correlation of miRNA levels with markers of platelet activation were also investigated. Venous blood from six non-fasting healthy donors was collected using a 24-gauge needle into pre-chilled (4°C) and room temperature CTAD, 3.8% sodium citrate, and K₂EDTA tubes. Blood was stored at room temperature or 4°C for <30 min, 2 h, 6 h, or 24 h before processing. Plasma was obtained by centrifugation at 1,000 x g for 10 min followed by 10,000 x g for 10 min. Plasma was aliquoted and stored at -80°C until RNA extraction and real-time RT-PCR using the thrombomiR microRNA Analysis Kit with the addition of glycogen in the RNA precipitation step. To control for each step in the extraction and amplification, UniSp4 was added prior to extraction, cel-miR-39 prior to reverse transcription, and UniSp3 before PCR amplification. Hemolysis was evaluated using the ratio of miRNA-23a-3p to miRNA-451a. Levels of sCD62P, TSP-1, and PF4 in the plasma were measured using ELISA kits.

   Summary of Findings:

   Although there were no differences in levels of the 12 miRNAs evaluated between CTAD and citrate plasma when plasma was processed within 30 min, EDTA plasma had higher levels of miR-191-5p, miR-21-5p, miR-451a, and miR-320a than CTAD plasma (P<0.05, P<0.05, P<0.001, and P<0.001; respectively) and higher levels of miR-451a and miR-320a than citrate plasma (P<0.01 and P<0.05, respectively). CTAD plasma had lower inter-individual variability. Plasma concentration of PF4 was weakly correlated with miR-320a and miR-451a (R² approximately 0.15), and modestly correlated when used in combination with TSP-1 and sCD62P (R² approximately 30%), indicating that the elevation of these miRNAs in EDTA plasma may reflect platelet activation. Collection tube temperature only significantly affected miR-451a (higher at room temperature) and miR-320a (lower in CTAD plasma at room temperature). Levels of miRNAs tended to increase during refrigerated storage, with ANOVA finding significant differences in miR-223-3p (P=0.0006), miR-150-5p (P<0.0001), miR-320a (P=0.0002), miR-21-5p (P< 0.0001), miR-24-3p (P=0.0034), and miR-451a (P=0.003). Significant increases were observed at 6 h for miR-191-5p and miR-451a and at 24 h for miR-223-3p, miR-150-5p, miR-320a, and miR-24-3p (P<0.05, all). An association between storage time and anticoagulant was only found for miR-21-5p (P=0.0235). Refrigerated storage of blood resulted in larger differences between EDTA and CTAD plasma in miR-191-5p, miR-320a, miR-126-3p, miR-21-5p, and miR-451a (P<0.0001, all) and miR-24-3p levels (P<0.05); larger differences between EDTA and citrate plasma in miR-191-5p, miR-320a, miR-21-5p and miR-451a levels (P<0.001 - P<0.0001, all); and significant differences between citrate and CTAD plasma in miR-191-5p and miR-126-3p levels (P<0.05 and P<0.01, respectively). Storage of blood at room temperature resulted in significant increases in miR-223-3p (P<0.0001), miR-197-3p (P=0.0001), miR-150-5p (P=0.0063), miR23a-3p (P=0.0003), miR-191-5p (P<0.0001), miR-320a (P=0.0002), mi-R24-3p (P=0.0002), miR-21-5p (P=0.031), miR27b-3p (P=0.0058), and miR-451a (P=0.0008). While differences in miR-191-5p, miR-320a, miR-21-5p, and miR-451a were similar to those observed at 4°C, miR-126 3p was less affected by room temperature storage and miR-223-3p, miR-197-3p, and miR-27-3p were more affected, particularly in EDTA plasma. The differences between anticoagulant types with room temperature storage were the same as with refrigerated storage. While none of the miRNAs were affected by addition of 0.016% lysed red blood cells to CTAD plasma, significant increases in the levels of miR-150-5p (P<0.05), miR-191- 5p (P<0.01), miR-320a (P<0.01), miR-21-5p (P<0.05), miR-27-3p (P<0.05), and miR-451a (P<0.05) were observed with addition of 0.125% lysed red blood cells to CTAD plasma and all of the investigated miRNAs increased with the addition of 2% lysed RBCs.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	ELISA
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Hemolysis	Hemolysate added No hemolysate added
   Biospecimen Acquisition	Anticoagulant	Citrate-theophylline-adenosine-dipyridamole Potassium EDTA Sodium citrate
   Storage	Storage duration	<30 min 2 h 6 h 24 h
   Storage	Storage temperature	4°C Room temperature
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-223-3p miR-197-3p miR-150-5p miR-191-5p miR-23a-3p miR-320a miR-24-3p miR-21-5p miR-126-3p miR-27-3p miR-28-3p miR-451a
   ELISA Specific	Targeted peptide/protein	PF4 TSP-1 sCD62P

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