Abstract
The transition from relapsing–remitting multiple sclerosis (RRMS) to secondary progressive MS (SPMS) represents a huge clinical challenge. We previously demonstrated that serum metabolomics could distinguish RRMS from SPMS with high diagnostic accuracy. As differing sample-handling protocols can affect the blood metabolite profile, it is vital to understand which factors may influence the accuracy of this metabolomics-based test in a clinical setting. Herein, we aim to further validate the high accuracy of this metabolomics test and to determine if this is maintained in a ‘real-life’ clinical environment. Blood from 31 RRMS and 28 SPMS patients was subjected to different sample-handling protocols representing variations encountered in clinics. The effect of freeze–thaw cycles (0 or 1) and time to erythrocyte removal (30, 120, or 240 min) on the accuracy of the test was investigated. For test development, samples from the optimised protocol (30 min standing time, 0 freeze–thaw) were used, resulting in high diagnostic accuracy (mean ± SD, 91.0 ± 3.0%). This test remained able to discriminate RRMS and SPMS samples that had experienced additional freeze–thaw, and increased standing times of 120 and 240 min with accuracies ranging from 85.5 to 88.0%, because the top discriminatory metabolite biomarkers from the optimised protocol remained discriminatory between RRMS and SPMS despite these sample-handling variations. In conclusion, while strict sample-handling is essential for the development of metabolomics-based blood tests, the results confirmed that the RRMS vs. SPMS test is resistant to sample-handling variations and can distinguish these two MS stages in the clinics.
Highlights
The transition from relapsing–remitting multiple sclerosis (RRMS) to secondary progressive MS (SPMS) represents a huge clinical challenge
While metabolic perturbations introduced by freeze–thaw and delayed centrifugation have been previously described in healthy individuals[20,21,23,24,26,28], there is a paucity of studies addressing how these factors may affect the accuracy of a diagnostic metabolomics test in a clinical environment
To ensure that our RRMS vs. SPMS diagnostic test is applicable in a clinical setting, it is vital that the high diagnostic accuracy is maintained even in instances when the sample-handling protocol may vary
Summary
The transition from relapsing–remitting multiple sclerosis (RRMS) to secondary progressive MS (SPMS) represents a huge clinical challenge. In CSF, a panel of 250 proteins has been identified which is able to distinguish between RRMS and progressive (SPMS and PPMS combined) MS with an accuracy of 89.4% in a validation cohort[19] While such a test could be useful, monitoring of the transition from RRMS to SPMS in a clinical setting may be challenging due to the high cost of measuring large panels of proteins coupled with the invasive nature of CSF sampling. To ensure that our RRMS vs SPMS diagnostic test is applicable in a clinical setting, it is vital that the high diagnostic accuracy is maintained even in instances when the sample-handling protocol may vary In this cross-sectional study, we further validate our metabolomics diagnostic test on an independent, prospective and well-characterised set of RRMS and SPMS samples (using the sample-handling protocol currently well-accepted in metabolomics) and investigate the impact of two of the most common sources of variation in sample-handling identified in our clinic: (1) freeze–thaw, and (2) delayed centrifugation
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