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Preanalytical considerations for clinical assays of circulating human miRNA-451a, miRNA-423-5p and miRNA-199a-3p for diagnostic purposes.

Author(s): Chandel DS, Tom WA, Jiang C, Krzyzanowski G, Fernando N, Olou A, Fernando MR

Publication: PLoS One, 2024, Vol. 19, Page e0303598

PubMed ID: 38768135 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study identified the cellular distribution of miR-451a, miR-423-5p, and miR-199a-3p and compared levels of these miRNAs in PFP obtained from hemolyzed versus unhemolyzed blood, after storage of blood or platelet-rich plasma (PRP) at room temperature or 4°C for up to 7 days; and PFP stored at room temperature for up to 72 h or frozen at -20°C or -80°C for up to 7 days. Blood was collected from healthy volunteers into 10 mL K3EDTA tubes. PRP was separated by centrifugation at 1600 x g for 10 min before separation of PFP by two centrifugations at 1600 x g for 10 min. RNA was extracted using the miRNeasy Serum/ Plasma Kit, quantified using the QubitRNA HS Assay Kit and stored at -80°C.  miR-451a, miR-423-5p, and miR-199a-3p were quantified by ddRT-PCR.  To investigate the cellular distribution of miR-451a, miR-423-5p, and miR-199a-3p, RBCs, WBCs, platelets, PRP, and PFP were obtained (details for RBC, WBC and platelet separation not provided) from the blood of five volunteers. To investigate the effects of hemolysis, aliquots of whole blood from four volunteers were freeze-thaw cycled and miR-451a, miR-423-5p, and miR-199a-3p were compared in matched PFP from non-hemolyzed and hemolyzed specimens. To investigate the stability of miRNAs in blood and PRP, whole blood and PRP from seventeen volunteers (miR-451) or eight volunteers (miRNA-423-5p and miRNA-199a-3p) were stored at room temperature or 4°C for 0, 1, 2, 3, or 7 days before centrifugation to obtain PFP.  To investigate the stability of miRNAs in blood and PFP during short-term storage, whole blood and PFP from six volunteers were stored at room temperature or 4°C for 2, 6, 1, 24, 36, 48, or 72 h before analysis of PFP. To investigate the stability of miRNAs in PFP during short-term storage, PFP from six volunteers was stored at -20°C or at -80°C for 0, 1, 2, 3, or 7 days.

   Summary of Findings:

   As expected, the vast majority of miR-451a was localized to RBCs (99.91%), with a very small percentage of miR-451a found in WBCs (0.055%) and even less localized to platelets (0.012%), PRP (0.007%), and PFP (0.0028%). Similarly, miR-423-5p was mostly found in RBCs (97.96%), with a small percentage of copies found in platelets (1.2%), PRP (0.5%), WBCs (0.34%) and PPP (0.008%). In contrast, miR-199a-3p was mostly localized to platelets (58.3%%), but was also found in RBCs (20%), PRP (12.2%), WBCs (9.4%), and to a lesser extent in PPP (0.06%). Both miR-451a and miR-423-5p were much higher in PFP from hemolyzed (freeze-thaw cycled) than matched unhemolyzed blood (P=0.0001, both), but levels of miR-199a-3p were comparable in PFP from hemolyzed and unhemolyzed blood. Levels of miR-451 in PFP were unaffected by storage of blood for 3 days at either temperature but were significantly higher in PFP isolated after 7 days at room temperature or 4°C than in PFP isolated immediately (P≤0.0001 and P≤0.04, respectively). In contrast, levels of miR-423-5 in PFP increased rapidly when blood was stored at either temperature, with significant increases observed after ≥1 day at room temperature or ≥2 days at 4°C (P≤0.002 and P≤0.0001, respectively). Levels of miR-199a in PFP increased rapidly with room temperature storage of blood and a bit slower with storage at 4°C, with significant increases relative to immediately isolated PFP noted after storage of blood for ≥ 1 day at room temperature (P≤0.02) or ≥3 days at 4°C (P≤0.02). Storage of PRP prior to PFP separation, resulted in significant declines in miR-451a levels in PFP after ≥1 day at either room temperature or 4°C (P≤0.0001 and P≤0.04, respectively) but significant increases in miR-423-5p levels in PFP after ≥2 days at room temperature or ≥7 days at 4°C (P≤0.002, both) and miR-199a after ≥2 days at room temperature or ≥3 days at 4°C (P≤0.008 and P≤0.02, respectively). In a second cohort PFP from blood stored at room temperature rather than processed within 2 h displayed significant increases in miR-423-5 and miR-199a after ≥36 h (P≤0.02, both) and miR-451a after 72 h (P≤0.0015). PFP stored at room temperature rather than frozen within 2 h displayed significant decreases in miR-451a after ≥12 h (P≤0.04) and miR-423-5 after ≥24 h (P≤0.04, both), but no significant effects of storage of PFP for up to 72 h at room temperature were observed on miR-199a levels. All three miRNAs were unaffected by storage of PFP at -80°C for up to 7 days, but miR-451a levels were slightly lower in PFP stored for 2 or 7 (not 3) days at -20°C than in PFP stored for 0 days (unclear if frozen) (P≤0.02).

   Biospecimens

   • Bodily Fluid - Plasma
   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Digital PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated
   Biospecimen Aliquots and Components	Hemolysis	Not induced Freeze/thaw-induced
   Biospecimen Aliquots and Components	Blood and blood products	Red blood cells White blood cells Platelet free plasma Platelet-rich plasma Platelets
   Storage	Storage duration	2 h 6 h 12 h 24 h 36 h 48 h 72 h 0 days 1 day 2 days 3 days 7 days
   Storage	Storage conditions	As whole blood As PRP As PFP
   Storage	Storage temperature	Room temperature 4°C -20°C -80°C

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