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Haemolysis during sample preparation alters microRNA content of plasma.

Author(s): Kirschner MB, Kao SC, Edelman JJ, Armstrong NJ, Vallely MP, van Zandwijk N, Reid G

Publication: PLoS One, 2011, Vol. 6, Page e24145

PubMed ID: 21909417 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine if hemolysis affects the miRNA levels in plasma, to identify a hemolysis threshold of effect, and to determine which blood components express miR-16 and miR-451. Between 1 and 3 tubes of K3EDTA blood were obtained from 9 healthy patients, 16 patients with coronary artery disease and 20 patients with malignant mesothelioma. Plasma and RBCs were stored at -80°C, but PBMC were processed immediately. RBC lysate was produced by freeze-thawing and vortexing the RBC pellet. RNA was isolated using the mirVana PARIS miRNA isolation kit and miRNA was stored at -80°C. Only 4 patients were used for the comparisons of hemolyzed and non-hemolyzed plasma from the same blood draw.

   Summary of Findings:

   High levels of miR-16 were present in plasma, with little variation (2 cycles) among patients despite three different diagnostic categories. Levels of miR-15b and miR-24 were comparably lower than miR-16 and more variable (7 cycles) among patients. miR-451 levels and variability were intermediate to those of miR-16, and those of miR-15b and miR-24. Levels of miR-451 and miR-16 were 2-8 fold higher in hemolyzed plasma compared to non-hemolyzed specimens from the same blood collection, which corresponded to the fold increase in hemoglobin. Similarly, miR-92a (a marker of heart disease) was 2-4 fold higher in hemolyzed specimens than matched non-hemolyzed specimens from the same 4 individuals. However, levels of another heart disease biomarker, miR-155, were only elevated in 2 of the 4 hemolysed specimens, and the increase did not correspond to the fold increase in hemoglobin. Hemolysis was detectable by spectrophotometry with a RBC concentration of as low as 0.016% (v/v), while visible hemolysis was only possible with a RBC concentration of 0.125% or greater. While ALT and AST only increased in specimens with visible hemolysis (0.125%), miR-451, miR-16, and miR-92a were elevated (~2-fold) when RBC concentrations reached 0.031%. Levels of miR-155 and miR-625 were not affected by RBC concentrations of up to 0.125%. When a hemolysis cut-off of <0.2 absorbance units at 414 nm was applied, variability in miR-451 and miR-16 levels decreased significantly (p<0.008 and p<0.026, respectively), but there was no change in variability of miRNAs unaffected by hemolysis (miR-24 and miR-15b). Levels of miR-16 and miR-451 were highest in RBC, as less than 1% of the remaining levels observed in whole blood were attributable to plasma and PBMC.

   Biospecimens

   • Bodily Fluid - Plasma
   • Cell - Red Blood Cells
   • Bodily Fluid - Blood

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Neoplastic - Carcinoma
   • Coronary Artery Disease
   • Normal

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR
   Cell count/volume	Spectrophotometry
   Protein	Spectrophotometry
   Protein	Clinical chemistry/auto analyzer

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Hemolysis	Absent Present Hemolysate added
   Biospecimen Aliquots and Components	Biospecimen components	0% RBCs 0.002% RBCs 0.004% RBCs 0.008% RBCs 0.016% RBCs 0.031% RBCs 0.063% RBCs 0.125% RBCs 0.25% RBCs 0.5% RBCs 1% RBCs 2% RBCs
   Real-time qRT-PCR Specific	Targeted nucleic acid	miR-15b miR-16 miR-24 miR-451 miR-155 miR-92a
   Biospecimen Aliquots and Components	Blood and blood products	Whole blood Plasma Peripheral blood mononuclear cells Red blood cells
   Preaquisition	Diagnosis/ patient condition	Malignant mesothelioma Coronary artery disease Healthy

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