Haemolysis Detection in MicroRNA-Seq from Clinical Plasma Samples

Abstract

The abundance of cell-free microRNA (miRNA) has been measured in blood plasma and proposed as a source of novel, minimally invasive biomarkers for several diseases. Despite improvements in quantification methods, there is no consensus regarding how haemolysis affects plasma miRNA content. We propose a method for haemolysis detection in miRNA high-throughput sequencing (HTS) data from libraries prepared using human plasma. To establish a miRNA haemolysis signature we tested differential miRNA abundance between plasma samples with known haemolysis status. Using these miRNAs with statistically significant higher abundance in our haemolysed group, we further refined the set to reveal high-confidence haemolysis association. Given our specific context, i.e., women of reproductive age, we also tested for significant differences between pregnant and non-pregnant groups. We report a novel 20-miRNA signature used to identify the presence of haemolysis in silico in HTS miRNA-sequencing data. Further, we validated the signature set using firstly an all-male cohort (prostate cancer) and secondly a mixed male and female cohort (radiographic knee osteoarthritis). Conclusion: Given the potential for haemolysis contamination, we recommend that assays for haemolysis detection become standard pre-analytical practice and provide here a simple method for haemolysis detection.

Specific plasma microRNA profiles could be potential non-invasive biomarkers for biochemical pregnancy loss following embryo transfer

“…MiR-363 is reported to regulate angiogenesis during pregnancy, and its expression is associated with preeclampsia [ 43 ] and RIF [ 44 ]. While miR-363-3p is reported to be a hemolysis-susceptible miRNA [ 45 ], we suggest caution in the interpretation of miR-363-3p as a reproductive biomarker. MiR-98 is involved in rat embryo implantation during the peri-implantation period [ 46 ]; miR-98 in bovine intrauterine extracellular vesicle (EV) regulates endometrial immune responses for implanting conceptuses [ 47 ].…”

OncoUroMiR: Circulating miRNAs for Detection and Discrimination of the Main Urological Cancers Using a ddPCR-Based Approach

“…The assessment of miRNAs levels in plasma samples is challenging, with several conditions having the possibility of introducing bias. For instance, hemolysis alters plasma miRNA content and may confound biomarker discovery and assessment [ 50 , 51 , 52 ]. Thus, we established a verification step for hemolysis and excluded all cases in which it was apparent, since miRNA traces expressed in red blood cells are released by hemolysis.…”

Integration of circulating microRNAs and transcriptome signatures identifies early‐pregnancy biomarkers of preeclampsia

“…We set a threshold cycle value ( C t ) of < 30 as the cut‐off to define an abundance of miRNA; all miRNAs with an expression level ≥30 were excluded. To monitor potential haemolysis in the samples, we first ruled out any difference in expression abundance of hsa‐miR‐23a and hsa‐miR‐451 between participants who developed PE and those with a pregnancy with no adverse outcomes 33 . Then, haemolysis was examined using ΔCq, calculated as the mean Cq of hsa‐miR‐23a minus the mean Cq of hsa‐miR‐451a.…”

“…To monitor potential haemolysis in the samples, we first ruled out any difference in expression abundance of hsa‐miR‐23a and hsa‐miR‐451 between participants who developed PE and those with a pregnancy with no adverse outcomes. 33 Then, haemolysis was examined using ΔCq, calculated as the mean Cq of hsa‐miR‐23a minus the mean Cq of hsa‐miR‐451a. A ΔCq value greater than 5 was considered indicative of possible haemolysis, while a ΔCq value exceeding 7 conferred a high risk of haemolysis affecting the obtained data.…”