15 November 2012. Even as Alzheimer’s disease biomarkers are wending their way through a lengthy standardization and qualification process, individual labs are experimenting with deploying them toward the next challenge, that is, to improve the differential diagnoses of related dementias. In the November Archives of Neurology, scientists led by Oskar Hansson, Skåne University Hospital, Malmo, Sweden, suggest that adding measures of cerebrospinal fluid (CSF) α-synuclein and neurofilament light chain to established markers of AD can help clinicians distinguish among disorders on the dementia-parkinsonism spectrum. In the October 17 Journal of Alzheimer’s Disease, members of that same team, this time led by Kaj Blennow of the University of Gothenburg in Molndal, Sweden, propose a technique that uses mass spectrometry to skirt the matrix effect that confounds current methods of estimating Aβ. Both papers highlight the rising importance of fluid biomarkers, which researchers hope will offer a means of diagnosing and tracking neurodegenerative disease progression.
A Multiple Biomarker Assay
While CSF Aβ42, phosphorylated tau, and total tau are different between people with Alzheimer’s disease (AD) and controls, those markers alone do not distinguish AD from other disorders with similar pathology. For instance, dementia with Lewy bodies (DLB) and Parkinson’s disease with dementia (PDD) tend to come with amyloid plaques, though the distribution, time course, and overall pathology differ from AD. To test if additional markers could sharpen differential diagnoses, Hansson and colleagues examined CSF from 453 people with various neurodegenerative disorders. They found that taking into account the concentrations of α-synuclein and neurofilament light chain—a component of the neuronal cytoskeleton—might make for a better diagnostic. Researchers find α-synuclein pathology in about half of AD patients, where it appears as a component of amyloid plaques. Previous research also reported more soluble α-synuclein in AD brain tissue (see ARF related news story). In addition, neurofilament light chain has been shown to be a marker of neuronal injury (see ARF related news story).
The researchers used a novel bead-based immunoassay to test four biomarkers simultaneously—Aβ42, total tau, phosphorylated tau, and α-synuclein. They measured neurofilament light chain separately by ELISA. Not only were CSF tau levels higher and Aβ42 levels lower in AD patients relative to those who had PDD or DLB, but AD patients had higher CSF α-synuclein levels as well, possibly because it leaked from degenerating neurons, the authors wrote. These combined biomarkers separated disease categories with 90 percent sensitivity and 81 percent specificity.
Because clinical manifestations and pathologies overlap among these dementias, it is hard to tell them apart based solely on clinical tests or limited biomarker panels. Correct diagnosis is important now, both to give a prognosis and to avoid medication errors. For instance, people with DLB are prone to hallucinations and should avoid certain antipsychotics prescribed for AD. Differential diagnosis will be critical in the future. “When we have disease-modifying treatments that target individual pathophysiologies, it will be important to correctly diagnose patients early on,” said Annika Öhrfelt, University of Gothenburg, co-first author with Sara Hall, Lund University, Sweden.
The research team further found that higher levels of CSF neurofilament light chain distinguished people with atypical parkinsonian disorders such as multiple system atrophy (MSA), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD) from those who had idiopathic Parkinson’s disease (PD). Neurofilament light chain could reflect more severe neurodegeneration inflicted by these atypical disorders, suggested the authors. The protein may eventually make a good biomarker to exclude PD, something that the field “urgently” seeks, said Brit Mollenhauer, Paracelsus-Elena-Klinik, Kassel, Germany. “Even movement disorder experts incorrectly diagnose 20 percent of patients at the earliest stages of Parkinson’s,” she told Alzforum. A solid biomarker would help diminish that margin of error, Mollenhauer said, though she agreed that neurofilament light chain itself needs more study and validation.
The particular four-biomarker test used in this study is no longer available from Innogenetics in Gent, Belgium, which developed the assay and was recently sold to the Japanese diagnostics company Fujirebio. Even so, this study provides proof of concept that may lead to the development of other assays that look at these biomarkers simultaneously, said Öhrfelt.
Antibody-Free Aβ Detection
The second paper by Blennow and colleagues proposes a way to measure CSF Aβ that avoids matrix effects. This is where concentration-dependent binding of the peptide to different proteins in the CSF compromises analysis, as sequential dilution does not proportionally free up Aβ (see ARF related news story). The new method, involving mass spectrometry, does not depend on antibody binding. It measures Aβ directly, even in the presence of other proteins, and can distinguish among Aβ38, Aβ40, and Aβ42.
First author Josef Pannee, University of Gothenburg, and colleagues froze CSF samples from two separate sets of 15 AD patients and 15 healthy controls. Just before analysis, the team thawed the specimens and denatured the proteins with guanidine hydrochloride. The researchers then added known quantities of “heavy” standards for Aβ38, Aβ40, and Aβ42, all of which incorporated a heavier isotope of nitrogen, as internal controls before mass spectrometry analysis.
The method detected about twice the Aβ42 as an ELISA done in parallel, and it more accurately identified AD patients and controls based on the Aβ42/Aβ40 ratio. However, the method is laborious and time-consuming, meaning high-throughput use on a par with ELISA is not yet feasible, Pannee told Alzforum. “The next generation of mass spectrometers coming out now are much faster and have much better sensitivity,” said Pannee. He plans to use this method to detect other Aβ peptides, and expand its use to other analytes. However, this method will not distinguish different oligomeric forms of Aβ, which are widely believed to be the most toxic species. Organizations, including the Institute for Reference Materials and Measurements and the International Federation of Clinical Chemistry, are taking steps to standardize CSF assays of AD biomarkers (see ARF related news story).
“We have a golden opportunity to once and for all measure CSF Aβ directly, free from matrix effects and with adequate sensitivity,” wrote Les Shaw, University of Pennsylvania Medical Center, Philadelphia, to Alzforum in an e-mail. Shaw agreed that these methods are not yet ready to be widely used in the clinic, but suggested that if companies decide to invest in this technology, more efficient mass spectrometry methods could be on the horizon (see full comment below).—Gwyneth Dickey Zakaib.
Hall S, Ohrfelt A, Constantinescu R, Andreasson U, Surova Y, Bostrom F, Nilsson C, Widner H, Decraemer H, Nägga K, Minthon L, Londos E, Vanmechelen E, Holmberg B, Zetterberg H, Blennow K, Hansson O. Accuracy of a Panel of 5 Cerebrospinal Fluid Biomarkers in the Differential Diagnosis of Patients With Dementia and/or Parkinsonian Disorders. Arch Neurol. 2012 Aug 27:1-8. Abstract
Pannee J, Portelius E, Oppermann M, Atkins A, Hornshaw M, Zegers I, Höjrup P, Minthon L, Hansson O, Zetterberg H, Blennow K, Gobom J. A Selected Reaction Monitoring (SRM)-Based Method for Absolute Quantification of Aβ38, Aβ40, and Aβ42 in Cerebrospinal Fluid of Alzheimer's Disease Patients and Healthy Controls. J Alzheimers Dis. 2012 Oct 17. Abstract