The Impact of Hemolysis on Cell-Free microRNA Biomarkers.
Author(s): Kirschner MB, Edelman JJ, Kao SC, Vallely MP, van Zandwijk N, Reid G
Publication: Front Genet, 2013, Vol. 4, Page 94
PubMed ID: 23745127 PubMed Review Paper? No
Purpose of Paper
The purpose of this paper was to determine the effect of hemolysis on the levels of microRNA (miRNA, miR) in plasma from healthy patients and those with malignant pleural mesothelioma (MPM) or coronary artery disease (CAD).
Conclusion of Paper
When miRNA levels in hemolyzed and non-hemolyzed plasma specimens from a single blood draw were compared, the majority of miRNAs that were detectable by TaqMan array microfluidic cards displayed higher levels in hemolyzed plasma specimens. Only 25.2%-28.9% of miRNAs were found at comparable levels in case-matched hemolyzed and non-hemolyzed specimens (<1.5 fold change). Although only 11% of detectable miRNAs were identified as differentially expressed (>2-fold change) due to hemolysis in all three diagnostic groups, the authors report that the lack of overlap was largely due to values failing to meet the 2-fold threshold rather than true differences among the groups. Importantly, only 11 of the 136 miRNAs detected were unaffected by hemolysis in any of the 6 specimen pairs. miRNAs affected by hemolysis included 5 species (miR-106a, miR-17, miR-92a, miR-21 and miR-210) that have been previously proposed as biomarkers of disease.
The purpose of this study was to determine the effect of hemolysis on the levels of miRNA in plasma from healthy patients and those with MPM or CAD. Multiple tubes of blood were collected from each of 1 patient with MPM, 3 with CAD and 2 healthy controls. Hemolysis was assessed by spectrophotometry, and one tube was classified as non-hemolyzed (absorbance at 414 (A414)=0.127-0.174) and another, without further manipulation, was classified as hemolyzed (A414=0.25-0.574). To investigate higher levels of hemolysis, RBCs were lysed by freeze-thawing and added in a dilution series to plasma from a healthy individual. Plasma was stored at -80°C until RNA extraction by mirVAna and quantification by TaqMan arrays.
Summary of Findings:
Compared to matched non-hemolyzed specimens, hemolyzed plasma had higher levels (>2-fold) of 40.2% (78 of 194), 47.7% (71 of 149) and 53.9% (49 of 91) of detectable miRNAs in healthy patients, patients with MPM, and patients with CAD, respectively. In addition, matched hemolyzed specimens had 1.5-2.0 fold higher levels of 24.2% (47 of 194), 13.4% (20 of 149), and 17.6% (16 of 91) of detectable miRNAs in healthy patients, patients with MPM, and patients with CAD, respectively. Importantly, only 28.9%, 29.5% and 25.2% of miRNAs were unaffected (<1.5 fold change) by hemolysis in healthy patients, patients with MPM, and patients with CAD, respectively; and, only 6.7% (13 of 194), 9.4% (14 of 149) and 3.3% (3 of 91) of miRNAs were found at lower levels in the hemolyzed specimen in healthy patients, patients with MPM and patients with CAD, respectively. Of the 136 miRNAs identified in at least 4 of the 6 specimens, only 11% (15 of the 136) had higher levels (>2-fold) in hemolyzed specimens in each of the three diagnostic groups, but the authors state the lack of overlap was largely due to values failing to meet the 2-fold threshold in one diagnostic group. Importantly, only 11 of the 136 miRNA were unaffected by hemolysis in any of the 6 specimen pairs. As expected, specimens spiked with increasing concentrations of RBC lysate had higher levels of hemolysis-affected miRNAs (miR-16, miR-15b, miR-451, miR-486-3p, and miR-532-3p) and RBC-specific miRNAs (miR-1255B,miR-636,miR-886-5p, and RNU48), but comparable levels of liver-specific (miR-122) and non hemolysis-dependent miRNAs (miR-1274B,miR-142-3p, miR-146a) when compared to plasma that did not contain RBC lysate. miRNAs affected by even modest hemolysis included the proposed disease biomarkers, miR-106a, miR-17, miR-92a, and miR-210, while miR-21 was among those affected by a high degree of hemolysis.
- Coronary Artery Disease
- Neoplastic - Carcinoma
Analyte Technology Platform RNA Real-time qRT-PCR Cell count/volume Spectrophotometry RNA Low density array
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Hemolysis Absent
Biospecimen Aliquots and Components Biospecimen components 0% RBCs added
0.008% RBCs added
0.016% RBCs added
0.031% RBCs added
0.0625% RBCs added
0.125% RBCs added
Real-time qRT-PCR Specific Targeted nucleic acid miR-16