Quantitative Biomarker Tests in Multiple Sclerosis Research

The Quanterix Simoa HD-1 Analyzer uses digital ELISA to reach unparalleled sensitivity when calculating the blood biomarkers and cerebrospinal fluid (CSF) connected to neurodegenerative diseases such as Multiple Sclerosis (MS), an autoimmune disease where demyelination of the central nervous system is a typical feature, eventually causing irreversible disability¹.

There are four types of MS: progressive-relapsing MS (PRMS), primary-progressive MS (PPMS), secondary-progressive MS (SPMS), and relapsing-remitting MS (RRMS). The expanded disability status scale (EDSS) evaluates impairment in eight categories (including motor function, vision, and cerebellar) to assess the severity of MS².

With a score of zero meaning there is no impairment, and scores above five showing moderate to severe impairment, EDSS scores vary from zero to ten in increments of 0.5. A diagnosis given by a physician along with the EDSS score are the present method for evaluating the gravity of the disease in patients.

This study investigated the correlation of disease severity in patients with RRMS, accounting for more than 85% of the categories of MS, with ultrasensitive measurements of human Tau protein, ubiquitin carboxyl terminal hydrolase L1 (UCH-L1), neurofilament light protein (NF-Light), and glial fibrillary acidic protein (GFAP).

As MS demyelination comes with axonal damage, releasing Tau and NF-Light into extracellular fluid and thereby raising CSF levels³, the following Simoa Assays were selected. Studies have demonstrated that Tau and NF-Light levels can be associated with the rate of brain atrophy⁴, MS onset and progression⁵, and MS severity⁶.

UCH-L1 is the most plentiful protein in the brain and sustains neural ubiquitin levels⁷. A decrease in UCH-L1 function has been related to neurodegenerative disease, and the ensuing ubiquitinated protein aggregates are typical of diseases such as Alzheimer’s and Parkinsons⁷.

Finally, an increase in GFAP levels are an indication of chronic lesion formation and astrogliosis, both signs of MS⁸. Similar to NF-Light and Tau, the latest studies have demonstrated that baseline levels of GFAP are correlated with MS severity and can be employed to foresee the rate of future neurological decline⁸.

Experimental Design

Human samples (plasma EDTA) were supplied by Bioreclamation from 12 evidently healthy donors and 16 with RRMS. RRMS patients were classed as mild, moderate, or severe according to a physician’s diagnosis with EDSS scores generally less than three for mild, more than five for severe, and three to five for moderate RRMS.

Figure 1: Concentration in healthy and RRMS donors for a) GFAP and b) NF-Light. Error bars represent the mean and SEM.

Figure 2: Concentration in healthy versus MS stratified by severity according to EDSS score for a) GFAP and b) NF-Light. Error bars represent the mean and SEM.

All samples were tested on Human Neurology 4-Plex “A” (UCH-L1, NF-Light, Tau, and GFAP), item 102153, and were prepared in line with the package insert for automated analysis by the  HD-1 Analyzer. Concentration results were generated for all four biomarkers after around 2.5 hours.

Results

Mean concentrations for healthy and RRMS donors for Tau (2.02±0.277 vs. 1.31±0.288) and UCH-L1 (8.96±1.93 vs. 20.3±8.57) did not represent a notable increase in RRMS when compared with healthy donors (p > 0.05). UCHL1 in a single donor exhibited a significant increase in concentration (147 pg/mL) when compared with other healthy and RRMS donors.

Mean concentrations for GFAP and NF-Light demonstrated a large increase in RRMS when compared with healthy donors, with p <0.005 for GFAP (62.2±5.82 vs. 117±14.4, Figure 1A) and p <0.05 for NF-Light (5.14±0.623 vs. 12.6±2.89, Figure 1B). The stratification of RRMS severity displayed further significance for NF-Light and GFAP (Table 1 and Figure 2), and to a lesser extent UCHL1 (Table 1).

A large increase above the healthy control was noticed in both severe RRMS (18.2±4.87 above healthy, p < 0.005) and moderate RRMS (5.31±1.47 above healthy, p < 0.005) for NF-Light. NF-Light additionally displayed a small increase in mild RRMS (2.43±1.33 above healthy). However this was not statistically significant (p = 0.09).

Moreover, NF-Light demonstrated an increase in mean concentration for moderate versus mild RRMS (2.88±2.22) and severe versus moderate RRMS (12.9±8.87). Similarly, GFAP also displayed a large increase in measured concentration in moderate RRMS (72.2±14.0 above healthy, p < 0.001) and severe RRMS (113±15.0 above healthy, p < 0.001).

Dissimilar to NF-Light, no difference was noted between healthy control and mild RRMS, but an increase in severe versus moderate RRMS was observed (40.8±26.1). However this was not significant.

Conclusions

In this small population of RRMS samples categorized by the severity of the disease, two of the four biomarkers, GFAP and NF-Light, in the Neurology 4-Plex A Simoa kit demonstrated a substantial increase in measured analyte as the severity of the disease progressed.

While UCH-L1 showed promise in additional quantifying measurable differences in disease severity, it would necessitate a larger population of samples to support whether this noted increase for a single patient is relevant to the disease progression, or is simply correlated with a flare-up.

A quantitative biomarker test is beneficial to gain a more precise initial diagnosis along with monitoring the treatment for specific patients with MS because of the inherently subjective nature of the EDSS score. The 4-plex provides more information than the individual assays alone can give to determine this information better.

TABLE:1

Table 1. Mean concentrations (pg/mL) measured in healthy donors and patients with MS, stratified by severity relating to EDSS score.

*Individual results for severe RRMS in UCH-L1: 7.95, 13.4, 15.5 and 147 pg/mL.

It is important to note that samples from patients diagnosed with RRMS in this study were active recipients of medical treatment which may affect the results.

References and Further Reading

  1. Bartosik-Psujek H and Stelmasiak Z. The CSF levels of total-tau and phosphotau in patients with   relapsing-remitting multiple sclerosis. J Neural Transm. 2006 113: 339–345
  2. Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an expanded disability status   scale (EDSS).Neurology. 1983 33(11):1444-52.
  3. Fialova L, Bartos A, Svarcova J, Malbohan I. Increased intrathecal high-avidity anti-tau   antibodies in patients with multiple sclerosis. PLoS One. 2011 6(11): e27476.
  4. Mellergard J, Tisell A, Blystad I, et al. Cerebrospinal fluid levels of neurofilament and tau   correlate with brain atrophy in natalizumab-treated multiple sclerosis. European Journal of   Neurology. 2016 24(1): 112-21.
  5. Martinez AM, Olsson B, Bau L, et al. Glial and neuronal marker sin cerebrospinal fluid predict   progression in multiple sclerosis. Multiple Sclerosis Journal. 2015 21(5): 550-61.
  6. Salzer J, Svenningsson A, and Sundstrom P. Neurofilament light as a prognostic marker in   multiple sclerosis. Mult Scler. 2010 16(3): 287-92
  7. Jara JH, Frank DD, and Ozdinler HP. Could dysregulation of UPS be a common underlying   mechanism for cancer and neurodegeneration? Lessons from UCHL1. Cell Biochem Biophys.   2013 67: 45-53.
  8. Axelsson M, Malmestro C, Nilsson S. Glial fibrillary acidic protein: a potential biomarker for   progression in multiple sclerosis. J Neurol 2011 258: 882–88.

Last updated: Aug 16, 2019 at 4:47 AM

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