Human salivary Raman fingerprint as biomarker for the diagnosis of Amyotrophic Lateral Sclerosis

C Carlomagno, P I Banfi, A Gualerzi, S Picciolini, E Volpato, M Meloni, A Lax, E Colombo, N Ticozzi, F Verde, V Silani, M Bedoni, C Carlomagno, P I Banfi, A Gualerzi, S Picciolini, E Volpato, M Meloni, A Lax, E Colombo, N Ticozzi, F Verde, V Silani, M Bedoni

Abstract

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease leading to progressive and irreversible muscle atrophy. The diagnosis of ALS is time-consuming and complex, with the clinical and neurophysiological evaluation accompanied by monitoring of progression and a long procedure for the discrimination of similar neurodegenerative diseases. The delayed diagnosis strongly slows the potential development of adequate therapies and the time frame for a prompt intervention. The discovery of new biomarkers could improve the disease diagnosis, as well as the therapeutic and rehabilitative effectiveness and monitoring of the pathological progression. In this work saliva collected from 19 patients with ALS, 10 affected by Parkinson's disease, 10 affected by Alzheimer's disease and 10 healthy subjects, was analysed using Raman spectroscopy, optimizing the parameters for detailed and reproducible spectra. The statistical multivariate analysis of the data revealed a significant difference between the groups, allowing the discrimination of the disease onset. Correlation of Raman data revealed a direct relationship with paraclinical scores, identifying multifactorial biochemical modifications related to the pathology. The proposed approach showed a promising accuracy in ALS onset discrimination, using a fast and sensitive procedure that can make more efficient the diagnostic procedure and the monitoring of therapeutic and rehabilitative processes in ALS.

Conflict of interest statement

The authors declare that they have no competing interests. Dr. Silani is in the Editorial Board of Amyotrophic Lateral Sclerosis, European Neurology, American Journal of Neurodegenerative Diseases, Frontiers in Neurology; received compensation for consulting services and/or speaking activities from AveXis, Cytokinetics, Italfarmaco, and Zambon.

Figures

Figure 1
Figure 1
Average Raman spectra (black line) and standard deviation (grey band) of saliva obtained using laser power of 128, 256 and 512 mW (left column) and acquisition time of 10, 20 and 30 seconds (right column).
Figure 2
Figure 2
Average Raman spectra of saliva analyzed on aluminium foil, glass and calcium fluoride.
Figure 3
Figure 3
Average Raman spectra of saliva mixed with silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs) with ratios NPs:saliva of 5:5 and 9:1 compared with the one acquired on aluminium foil.
Figure 4
Figure 4
Average Raman spectra of saliva without and after filtering with 3, 10, 30 kDa cut-offs.
Figure 5
Figure 5
Average Raman spectra with SD of (A) ALS, (B) PD, (C) AD and (D) CTRL groups.
Figure 6
Figure 6
Subtraction spectra of the average ALS signal versus the (A) PD average signal, (B) AD average signal and (C) CTRL average signal. (D) Overlapped average spectra of the experimental groups.
Figure 7
Figure 7
(A) Principal Component Analysis (PCA) 3 axis distribution (X = PC1; Z = PC2; Y = PC3). (B) Linear discriminant Analysis (LDA) showing the distribution of canonical variable values for the ALS (n = 19), PD (n = 10), AD (n = 10) and CTRL (n = 10). ***p < 0.001, One-Way ANOVA test.

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