There is a lot of talk about cerebrospinal fluid biomarkers for Alzheimer’s disease, but no robust test has arrived at local doctors’ offices yet. Before such tests can move to medical care clinics around the world from the research centers in which they are currently being used, they have to perform reliably across different settings. This sounds like a formality, but achieving it is a complex process that engages multiple international groups. To get there, one early step is developing accurate ways to determine the absolute amount of Aβ and other biomarkers in a given sample of cerebrospinal fluid (CSF). In the May 19 issue of Clinical Chemistry, researchers led by Kaj Blennow at the University of Gothenburg, Mölndal, Sweden, describe a candidate protocol for quantifying CSF Aβ42 using mass spectrometry. The method is under review by the regulatory bodies that have taken on CSF tests for Alzheimer’s. If it passes muster, it will become the first approved reference measurement procedure for CSF Aβ42. Other, similar methods are expected to follow suit. With these procedures in hand, researchers will be able to produce a definitive reference CSF material with a known quantity of Aβ42 that any manufacturer can use to calibrate commercial measurement kits. At that point, CSF Aβ42 will finally be ready for prime time.
The paper represents part of an international initiative to bring AD fluid biomarkers up to clinical standards. In 2011, Maria Carrillo at the Alzheimer’s Association convened the Global Biomarkers Standardization Consortium (GBSC) to spearhead this effort (see Aug 2012 news story). The group includes leading scientists and groups in the field, such as Blennow and his colleague Henrik Zetterberg; Leslie Shaw at the University of Pennsylvania, Philadelphia; and Piotr Lewczuk at the University Hospital Erlangen, Germany. In addition to academic researchers, the GBSC includes scientists from companies that develop fluid assays, ranging from those that currently make assays, such as Innogenetics and Meso Scale, to firms that are developing new, automated assays, such as Roche. Pharmaceutical companies that evaluate and use those assays, like Bristol Myers Squibb, are members as well.
The group’s leaders have persuaded international certification and reference bodies to join the cause (see Aug 2012 news story). The International Federation of Clinical Chemistry and Laboratory Medicine appointed a working group to tackle the problem of AD fluid biomarker reproducibility. The membership of this working group largely overlaps with that of the GBSC. Meanwhile, the Institute for Reference Materials and Measurements (IRMM) of the Joint Research Centre of the European Commission in Geel, Belgium, a measurement agency similar to the U.S. National Institute of Standards and Technology (NIST), agreed to help the GBSC produce certified reference materials for Aβ42, tau, and phospho-tau. This is the process by which fluid-based tests such as those for insulin are developed. The institute’s Ingrid Zegers manages the project and also sits on the IFCC working group.
Blennow and colleagues focused on developing a mass spectrometry method for measuring Aβ42. Mass spectrometry is more accurate than antibody-based methods, as the latter may be confounded by cross-reactivity, Blennow told Alzforum. The researchers first add a known quantity of synthetic, labeled Aβ42 to the CSF sample. This serves as an internal calibrator, and because the labeled Aβ weighs more than endogenous Aβ, mass spectrometry can differentiate the two. Next, the CSF sample is treated with guanidine hydrochloride, a denaturing agent that liberates Aβ from other proteins and breaks up aggregates. This allows mass spectrometry to measure total Aβ, not just free peptide, Blennow said. Finally, researchers run the sample through high-performance liquid chromatography tandem mass spectrometers, which determine the absolute amount of Aβ42. The method is accurate at concentrations from 150 to 4,000 pg/ml, and the measurement varies by only about 5 percent from one assay to the next, even when performed at different labs on different machines, the paper reports.
Blennow has submitted his data for approval to the Joint Committee for Traceability in Laboratory Medicine (JCTLM), the regulatory body that evaluates reference methods. This committee represents a collaboration between the IFCC, the International Committee for Weights and Measures, and the International Laboratory Accreditation Cooperation.
Simultaneously, other members of the IFCC working group are developing similar mass spectrometry methods. Blennow’s group participated with three other labs in a round-robin study to compare these protocols. The other participants included Shaw and colleagues at UPenn; the research organization Pharmaceutical Product Development (PPD); and mass spectrometer manufacturer Waters Corp., in collaboration with Pfizer. The four groups received a common set of 12 CSF samples. All used the same method of preparing the sample, but other aspects of the protocol differed, Shaw told Alzforum—the groups used different mass spectrometry systems, handled the calibrator differently, and in some cases measured multiple Aβ peptide products, such as Aβ40 and Aβ38.
All four groups obtained internally reproducible results that varied less than 5 percent between assays, Shaw said. The variability between groups was about 12 percent—a good result considering the differences in protocols. “We were gratified to see that we had a reasonable degree of agreement across the four centers. We’ve shown that we have the basis for an accurate and precision-based methodology,” Shaw said.
Blennow expects that the JCTLM will approve multiple mass spectrometry methods as reference measurement procedures. The IRMM can then use these methods to determine the quantity of Aβ42 in a pooled, 5-liter CSF sample that will serve as a reference material. IRMM will test the reference sample periodically to make sure the Aβ measurement remains stable over time. Once this reference material has been validated, IRMM will provide aliquots to companies that manufacture kits for testing CSF Aβ, for use in calibration. This may still be two or three years away, Blennow said. Tau measurements lag behind Aβ, with mass spectrometry methods for measuring this protein still in development.
The need for such calibrators remains great. Blennow also oversees the Alzheimer's Association’s CSF Quality Control Program, in which numerous labs around the world measure the same CSF samples using currently available research-grade commercial kits (see Nov 2009 news story). Despite efforts to standardize protocols in the five years since this program began, results still vary by 15 to 20 percent between centers, Blennow said. These numbers have improved only slightly since 2009, and are not tight enough to support multicenter clinical trials, diagnostic purposes, or to track how a person’s disease responds to a medication. In other fields, biomarker measurement variation dropped sharply once kits with calibrators become available, Blennow noted. The AD field hopes to follow that example.—Madolyn Bowman Rogers
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- Worldwide Quality Control of CSF Biomarkers—How Does it Work?
- Paris: Standardization a Hurdle for Spinal Fluid, Imaging Markers
- Little by Little—Standardizing, Validating Those Biomarkers
- Optimizing CSF Aβ42 ELISA for Trials and Regulatory Approval
- Leinenbach A, Pannee J, Dülffer T, Huber A, Bittner T, Andreasson U, Gobom J, Zetterberg H, Kobold U, Portelius E, Blennow K, IFCC Scientific Division Working Group on CSF proteins. Mass spectrometry-based candidate reference measurement procedure for quantification of amyloid-β in cerebrospinal fluid. Clin Chem. 2014 Jul;60(7):987-94. Epub 2014 May 19 PubMed.