miRNA extracted from extracellular vesicles is a robust biomarker of amyotrophic lateral sclerosis
Banack SA, Dunlop RA, Stommel EW, Mehta P, Cox PA. miRNA extracted from extracellular vesicles is a robust biomarker of amyotrophic lateral sclerosis. J Neurol Sci. 2022;442. doi:10.1016/j.jns.2022.120396
Promising Biomarker Found for Early-Stage ALS Diagnosis: miRNA Extracted from Extracellular Vesicles
Amyotrophic lateral sclerosis (ALS) affects tens of thousands of people worldwide, but diagnosing the disease accurately can be challenging. However, recent research has shown that miRNA extracted from extracellular vesicles (EVs) can be a reliable biomarker for early-stage ALS diagnosis. EVs are small, non-replicating particles released by cells that contain material from the source cell within their protective lipid bilayer membrane. This makes EVs a promising material for potential biomarkers, and researchers have discovered eight early-stage ALS-diagnostic miRNAs extracted from EVs enriched with L1CAM by immunoaffinity purification.
Internal Quality Control Measures Ensure Reproducibility of miRNA Biomarkers for Standard Clinical Use
However, for biomarkers to be useful in standard clinical use, they must be reproducible over time in different labs and with samples collected using different collection and storage protocols. The researchers have included internal quality control measures, such as melt-curves, no-template controls, and spike-in controls for RNA extraction and cDNA synthesis, to ensure the reproducibility of the miRNA biomarkers.
Utilizing Archived Samples from National ALS Biorepository for Reliable ALS Diagnosis
To obtain blood plasma samples for the study, archived samples from the U.S. National ALS Biorepository were utilized. The Biorepository is part of the U.S. National ALS Registry and is maintained by the Centers for Disease Control and Prevention and the Agency for Toxic Substances and Disease Registry. The blood plasma samples were collected from 50 patients with confirmed or probable ALS at different times and locations throughout the USA by phlebotomists. The recommended protocol included collecting the blood in K2 EDTA tubes and inverting the tubes ten times. The blood specimens were then packed in Styrofoam-insulated shippers with reusable cold-packs to maintain a temperature between 4 and 8 °C for overnight shipping to a central location for processing and storage.
Proper Collection and Storage of Blood Plasma Samples Crucial for ALS Biomarker Reproducibility
In the original study, the samples were processed within 1 hour of collection by being spun-down in a refrigerated centrifuge and then stored and transported at −80 °C. As controls were not available from the Biorepository, 50 samples of blood plasma from individuals without an ALS diagnosis (FDA Approval, #3003372368, Innovative Research Inc.) were used as the control population. The study was reviewed by Advarra IRB Pro00053269, and since de-identified participant data were used, the study was determined not to meet the DHHS definition of human subjects research under 45 CFR 46 and did not require IRB oversight.
Potential ALS Biomarkers Identified, But Further Research Required for Selectivity and Sensitivity Determination
The authors tested the generalizability of the biomarkers by using a cross-section of 50 ALS patients and found that 80% of the original biomarkers were statistically significant. However, larger sample sizes and comparisons with blood samples from ALS-mimic diseases are required to determine the biomarkers‘ selectivity and sensitivity.
Overall, the identification of eight miRNAs as potential biomarkers for ALS diagnosis using L1CAM for immunoaffinity purification and polymer-based precipitation for EV extraction is a significant development in the field. With more research, these biomarkers could become useful in the clinic for early-stage diagnosis and rate of progression prediction. Therefore, it is crucial to collect and store samples properly and follow preanalytic measures to ensure the biomarkers‘ reproducibility and reliability over time.