Most people will remember the Ice Bucket Challenge better than its purpose, to fund a cure for amylotrophic lateral sclerosis (ALS). ALS is a neurodegenerative disease, also known as motor neuron disease. Brain diseases can produce confusingly similar symptoms in the early stages therefore a non-invasive, discriminating test would be hugely beneficial to treatment and research. Scientists in Milan recently reported a convenient new way to potentially detect and monitor the progression of the ALS based on the Raman spectroscopy of saliva rather than an invasive biopsy.
ALS has a range of initial symptoms ranging from difficulty swallowing to muscle weakness in the arms and legs and muscle twitching. These are caused by increasing failure of voluntary muscle control. In the final stages of the disease patients may require mechanical ventilation to breath and may only be able to communicate by eye movements.
Eventually symptomatic tests diagnose ALS and differentiate the condition from other neurodegenerative diseases such as Parkinson’s Disease (PD) or Alzheimer’s Disease (AD). Unfortunately such tests make early diagnosis and potential treatment impossible. Spinal fluid analysis is used to identify ALS but this is invasive and carries some risks to the patient.
In a recent publication in Nature Scientific Reports a team from the University of Milan, Italy reported the development of a saliva test for ALS. A saliva test for any disease is immediately attractive due to its non-invasive nature and potential for simple repeat testing. The report describes method development and application to three patient groups and a control group. The four groups comprised ALS (19 individuals), PD (10 individuals), AD (10 individuals) plus 10 control individuals with none of these diseases. Patients in the study groups were initially diagnosed using established benchmark tests. The goal of the Milan team was to establish reliable methods and protocols to obtain characteristic molecular fingerprints of components in the saliva and examine these fingerprints to look for subtle but significant differences between the four patient groups.
The Milan team found that saliva samples dried onto aluminium substrates produced the best results but that the saliva samples themselves had to be stripped of high molecular weight components that inhibited the detection of the molecules of most interest. Raman spectroscopy gives a fingerprint of the vibrations of molecules, which means spectra typically contain a lot of information. Unfortunately it usually has low sensitivity unless an enhancing agent such as silver or gold nanoparticles are used to increase the size of the Raman signal many thousands of times. The Milan groups discovered that the nanoparticles they used to enhance the Raman signal were inhibited by the non-distinct high weight components. Following a key innovative step, when these were filtered out, the Raman spectra of the purified saliva samples yielded much more information.
With informative datasets, the Milan spectroscopists set about analysing the results. A statistical method known as Principal Component Analysis (PCA) was used to pick out similarities and differences in the saliva sample spectra from the 49 study participants. The mathematical method takes no account of the spectroscopy, the molecules in the samples or the patients in the study. PCA is essentially blind to the expertise of the scientists and the identity of the patients but finds a number of spectra-like components that when added together in simple linear combinations can represent the spectra of each and any Raman spectrum of saliva from the 49 study participants.
What they found was that the relative proportions of three Principal Components PC1, PC2 and PC3 clustered more or less into four groups. These four groups were the saliva samples from ALS, PD, AD and control participants.
The Milan team have developed a useful Raman assay protocol that can distinguish saliva samples from ALS patients from other patients with neurodegenerative diseases and healthy individuals. The method needs to be validated in larger studies and applied to longitudinal projects but the University of Milan have innovated a promising tool for disease research and treatment evaluation.
The full Nature Scientific Report can be found here.
Time magazine has a reminder of the Ice Bucket Challenge phenomenon.