Pre-Analytical Modification of Serum miRNAs: Diagnostic Reliability of Serum miRNAs in Hemolytic Diseases.
Author(s): Takada Y, Shibuta T, Hatano M, Sato K, Koga M, Ishibashi A, Harada T, Hisatomi T, Shimura H, Fukushima N, Leecharoenkiat K, Chamnanchanunt S, Svasti S, Fucharoen S, Umemura T
Publication: J Clin Med, 2021, Vol. 10, Page
PubMed ID: 34768564 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to investigate potential effects of hemolysis introduced during blood draw or delayed centrifugation, or due to hemolytic disease on levels of miRNA in serum. The authors also compared different markers of hemolysis and analyzed hemolysis in leftover serum specimens.
Conclusion of Paper
Spectrophotometric quantification of oxyhemoglobin was more sensitive marker of hemolysis than free hemoglobin. Hemolysis quantified by two different methods, spectrophotometric quantification of oxyhemoglobin and miR-451a real-time amplification, were strongly correlated. When blood was transferred to an EDTA tube, significantly higher levels of the red blood cell miRNAs (miR-451a, miR-486-5p and miR-16) were observed, but levels of the granulocyte (miR-223) and the platelet-related miRNA (miR-126) only increased non-significantly. Levels of miR-451a, miR-486, miR-126, miR-16 and miR-223 in serum increased when blood was stored at room temperature for up to x h. All of the 116 previously collected and frozen serum specimens examined were hemolyzed based upon spectrophotometric quantification of oxyhemoglobin and elevated levels of miR-451a. Retrospective serum specimens also had abnormally high levels of lactate dehydrogenase, AST and potassium. Importantly, levels of miR-451a were still significantly higher in patients with hemolytic disease than in leftover serum specimens or control specimens.
Studies
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Study Purpose
The purpose of this study was to investigate potential effects of hemolysis introduced during blood draw, delayed centrifugation, or due to hemolytic disease on levels of miRNA in serum. The authors also compared different markers of hemolysis and analyzed hemolysis in leftover clinical serum specimens. Blood was collected from healthy patients (n=8), and patients with autoimmune hemolytic anemia (n=2), beta thalassemia (n=13), alpha-thalassemia (n=1), and malaria (n=3) into vacuum tubes containing coagulation stimulators. After 30 min at room temperature for clot formation, serum was separated by centrifugation at 3500 rpm for 10 min and residual debris were removed with a 0.45 µm filter. Serum was stored at -80°C for up to 7 days. miRNA were extracted using the Nucleospin miRNA Plasma Extraction Kit and levels were quantified by real-time PCR using TaqMan miRNA Assay Kits. To identify markers of hemolysis, packed red blood cells from EDTA blood were hemolyzed by freezing at -80°C and serially diluted. Hemolysis was then quantified by the spectrophotometric detection of free hemoglobin at 540 nm and oxyhemoglobin at 414 nm, and by real-time PCR amplification of miR-451a. To detect artifacts of hemolysis during blood draw case-matched EDTA plasma and serum specimens were isolated from blood collected from 8 individuals; by centrifugation at 3500 rpm for 10 min. To investigate the effects of delayed processing, blood was stored for 1, 6 and 24 h at room temperature, before isolation of serum. Levels of hemolysis, LD, AST, potassium and miR-451a were investigated in 116 previously obtained serum specimens (details not provided).
Summary of Findings:
Spectrophotometric quantification of oxyhemoglobin was more sensitive marker of hemolysis than free hemoglobin. Hemolysis quantified by two different methods, spectrophotometric quantification of oxyhemoglobin and miR-451a real-time amplification, were strongly correlated(r=0.885). When blood was transferred to a second EDTA vacuum tube, significantly higher levels of red blood cell miRNAs including miR-451a (1.86-fold, P < 0.02), miR-486-5p (1.85-fold, P < 0.02), and miR-16 (1.97-fold, P < 0.02) were observed compared to the remaining blood in the original tube, but levels of the granulocyte miRNA, miR-223, and the platelet -related miRNA, miR-126, increased non-significantly (1.39 and 1.26 -fold, respectively). Storage of blood at room temperature, resulted in increased levels of miR-451a and miR-486 beginning after 1h (1.62-fold and 1.65-fold, respectively), increased miR-126 after 6 h (1.91-fold) and increased miR-16 and miR-223 after 24 h (1.54 and 1.81-fold, respectively). All 116 previously frozen serum specimens were hemolyzed based upon spectrophotometric quantification of oxyhemoglobin and elevated levels of miR-451a (2.6-fold, P<0.05), as well as abnormally high levels of lactate dehydrogenase (average 162.8 U/L), AST (average 22.9 U/L) and potassium (average 4.2 U/L). Importantly, levels of miR-451a were significantly higher in patients with hemolytic disease than in previously frozen or healthy control specimens (P<0.05, both).
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Other diagnoses
- Normal
Platform:
Analyte Technology Platform RNA Real-time qRT-PCR Protein Clinical chemistry/auto analyzer Protein Spectrophotometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Preaquisition Diagnosis/ patient condition Healthy
Autoimmune hemolytic anemia
Beta thalassemia
Alpha-thalassemia
Malaria
Storage Storage duration 0 h
6 h
24 h
Biospecimen Aliquots and Components Hemolysis Hemolysate added
Spectrophotometry Specific Technology platform Real-time PCR amplification of miR-451a