Haemolysis during sample preparation alters microRNA content of plasma

Our plain-English summary of Haemolysis during sample preparation alters microRNA content of plasma, by M.B. Kirschner, S.C. Kao, J.J. Edelman, et al (PloS One, 2011).

The study

• Authors: M.B. Kirschner, S.C. Kao, J.J. Edelman, N.J. Armstrong, M.P. Vallely, N. van Zandwijk, G. Reid
• Journal: PloS One
• Published: 2011;6(9):e24145
• Cited by: 478 articles, as counted by NIH iCite
• Read it: PubMed · Publisher · Free full text

Abstract

The presence of cell-free microRNAs (miRNAs) has been detected in a range of body fluids. The miRNA content of plasma/serum in particular has been proposed as a potential source of novel biomarkers for a number of diseases. Nevertheless, the quantification of miRNAs from plasma or serum is made difficult due to inefficient isolation and lack of consensus regarding the optimal reference miRNA. The effect of haemolysis on the quantification and normalisation of miRNAs in plasma has not been investigated in great detail. We found that levels of miR-16, a commonly used reference gene, showed little variation when measured in plasma samples from healthy volunteers or patients with malignant mesothelioma or coronary artery disease. Including samples with evidence of haemolysis led to variation in miR-16 levels and consequently decreased its ability to serve as a reference. The levels of miR-16 and miR-451, both present in significant levels in red blood cells, were proportional to the degree of haemolysis. Measurements of the level of these miRNAs in whole blood, plasma, red blood cells and peripheral blood mononuclear cells revealed that the miRNA content of red blood cells represents the major source of variation in miR-16 and miR-451 levels measured in plasma. Adding lysed red blood cells to non-haemolysed plasma allowed a cut-off level of free haemoglobin to be determined, below which miR-16 and miR-451 levels displayed little variation between individuals. In conclusion, increases in plasma miR-16 and miR-451 are caused by haemolysis. In the absence of haemolysis the levels of both miR-16 and miR-451 are sufficiently constant to serve as normalisers.

In plain English

This Sydney laboratory study looked at how haemolysis affects measurements of microRNAs in blood plasma. Haemolysis is the bursting of red blood cells in a blood sample. MicroRNAs are small RNA molecules proposed as possible blood markers of disease. The study did not examine asbestos exposure. Plasma came from healthy volunteers, patients with malignant mesothelioma and patients with coronary artery disease. In plasma without obvious haemolysis from 37 people, levels of miR-16, a common reference standard, varied by only 2 PCR cycles. Nearly all miR-16 and miR-451 in whole blood came from red blood cells. In haemolysed samples their copy numbers were up to 8-fold higher. When burst red blood cells were added to plasma, levels rose before any colour change was visible. Excluding samples with absorbance above 0.2 at 414 nm significantly reduced variation in miR-451 and miR-16. The authors concluded that even minimal haemolysis makes these molecules unsuitable as reference standards. They suggested that measuring free haemoglobin might be a simple test of whether a sample suits microRNA analysis that uses one of the body's own microRNAs as the reference.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory methods study of human blood plasma samples, including an experiment in which burst red blood cells were added to plasma
• Who was studied: Plasma from 9 healthy volunteers, 18 patients with malignant mesothelioma and 10 patients with coronary artery disease (37 people) for the first comparison. A later series came from 9 healthy people, 20 mesothelioma patients and 16 coronary artery disease patients. Samples were collected under ethics approval from Sydney hospitals. Collection dates were not given.
• Exposure: No asbestos exposure was studied. The factor studied was haemolysis (bursting of red blood cells) in plasma samples, measured as free haemoglobin.
• Main results:
  • In plasma without obvious haemolysis from the healthy, mesothelioma and coronary artery disease groups, miR-16 levels varied by only 2 PCR cycles.
  • miR-16 measured in plasma taken on three separate occasions from the same individual varied by less than 1 cycle.
  • In visibly haemolysed (pink) samples, miR-451 and miR-16 were significantly higher than in non-haemolysed samples from the same blood collection, with up to 8-fold more copies per ml of plasma.
  • Most miR-16 and miR-451 in whole blood came from red blood cells. White blood cells (PBMCs) and plasma together contributed less than 1% of copies.
  • When burst red blood cells were added to plasma, colour change was visible only above 0.125% (v/v). At 0.031% (v/v), where haemolysis was not visible, miR-16 copies were already above the mean level seen in 25 non-haemolysed samples.
  • Using an absorbance at 414 nm above 0.2 as a cut-off to remove haemolysed samples significantly decreased the variability of miR-451 (p = 0.008) and miR-16 (p = 0.026). It did not significantly reduce the variability of miR-15b (p = 0.893) or miR-24 (p = 0.086).
  • When miR-16 was used as the reference standard, the results falsely suggested that miR-92a and miR-155 were lower in haemolysed than in non-haemolysed plasma from the same patient.
• The authors' conclusion: The authors concluded that changes in miR-16 and miR-451 caused by even minimal haemolysis make these molecules inappropriate as reference standards between samples. They suggested that measuring free haemoglobin might be a simple test of whether a sample is suitable for microRNA analysis that uses the body's own microRNAs as the reference.

Topics

Medical terms in this study, explained in our Lexicon: Biomarker, Malignant Mesothelioma.

Related studies

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• Restoring expression of miR-16: a novel approach to therapy for malignant pleural mesothelioma — G. Reid, M.E. Pel, et al. (2013)
• Tumor Suppressor microRNAs Contribute to the Regulation of PD-L1 Expression in Malignant Pleural Mesothelioma — S.C. Kao, Y.Y. Cheng, et al. (2017)
• Increased circulating miR-625-3p: a potential biomarker for patients with malignant pleural mesothelioma — M.B. Kirschner, Y.Y. Cheng, et al. (2012)
• Mechanism of haemolysis by chrysotile fibres — M.C. Jaurand, L. Magne, et al. (1983)
• Safety and activity of microRNA-loaded minicells in patients with recurrent malignant pleural mesothelioma: a first-in-man, phase 1, open-label, dose-escalation study — N. van Zandwijk, N. Pavlakis, et al. (2017)

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