Drawing Closer: Alzheimer’s Blood Test for Primary Care

In today’s JAMA Neurology, researchers led by Oskar Hansson, Lund University, Sweden, report how a fully automated immunoassay for plasma Aβ performed when they put it through its paces. Roche Diagnostic’s Elecsys system predicted Aβ-positive individuals with about 80 percent accuracy. That number improved by 5 percent when the researchers took ApoE genotype into consideration. Alzforum first reported on this data when Hansson presented it at AAIC in 2018 (Aug 2018 conference news). Since then, first author Sebastian Palmqvist and colleagues have tested samples in a validation cohort in Germany. Here the test was more accurate, at 86 percent. “I think we are not that far away from a blood-based biomarker that can be implemented in primary care to improve diagnostics of AD,” Hansson told Alzforum.

  • Roche Diagnostic’s Elecsys tested in Swedish and German cohorts.
  • Fully automated plasma test predicts AD with 85 percent accuracy.
  • The immunoassay could offer a screen for people at risk.

The Elecsys system is one of a handful of plasma immunoassays that are vying with mass spectrometry-based methods for use as a blood test in the clinic. Each has its pros and cons. The immunoassays are easier to implement, though not quite as accurate as the mass-spec approaches. Being fully automated, the Elecsys system comes with the added advantage of cutting down on variability that can be introduced by manual steps that are part of mass spec, ELISA, and Simoa immunoassay approaches. It can be scaled up easily for use in primary-care settings, which makes it attractive to Hansson. “A lot of patients with Alzheimer’s disease are not diagnosed, or they get an incorrect diagnosis,” he noted. “A plasma assay could improve this situation and boost the number of people who get proper symptomatic treatments,” he said.

Palmqvist and colleagues first tested the assay using samples from 446 women and 396 men in the Swedish BioFINDER cohort. These volunteers ranged in age from 59 to 88, with an average age of 72. Sixty-four had Alzheimer’s disease, 265 mild cognitive impairment, 513 were healthy controls. All agreed to lumbar taps for cerebrospinal fluid collection, and the researchers used their CSF Aβ42/Aβ40 ratio to determine their brain-amyloid status. All 64 AD patients tested positive, as did 157 of those with MCI and 147 of the controls. Blood samples were collected at the same time.

The plasma Aβ42/Aβ40 ratio tracked lower in amyloid-positive people, in keeping with the premise that the stickier Aβ42 gets trapped in the brain and no longer clears into the blood. The lower plasma ratios predicted amyloid status with an AUC of 0.8. This is not good enough for a stand-alone diagnostic test, the authors note.

To see if they could improve accuracy, they combined this analysis with plasma tests for tau and neurofilament light, and they also took ApoE status into consideration. Individually, the two plasma markers improved the AUC slightly. ApoE brought it to 0.85.

However, while these additional parameters increased the sensitivity of detection, they decreased the specificity. This is not surprising. Tau and NfL levels increase in other neurodegenerative diseases, and ApoE4 is a risk variant for other forms of neurodegeneration as well. Still, the researchers got the best predictive value by adding all three markers to the Aβ ratio, improving accuracy to 0.87. The Aβ assay worked as well in cognitively impaired as in healthy people, but plasma tau and NfL added no value in the latter. Importantly, however, the assay worked as well in younger people as in older. “The data suggest the assay performs equally well in preclinical as in prodromal AD,” said Hansson.

For validation, the researchers turned to a prospective study running in the German cities of Ulm and Hanover. Among 94 volunteers with AD, 109 with MCI, and 34 healthy controls, the Elecsys immunoassay performed slightly better than in the Swedish cohort, predicting amyloid positivity with an AUC of 0.86. Adding plasma tau brought that down to 0.84. Plasma NfL and ApoE genotype were unavailable for this cohort. “Overall, the data show good reproducibility,” said Hansson.

The authors hope better plasma-tau assays will make the test more accurate. Hansson told Alzforum that Roche already has upped the sensitivity of the Aβ assay beyond that reported in this paper; he hopes the improved assay will be finalized within the year.

Sid O’Bryant, University of North Texas Health Science Center, Fort Worth, thinks the work represents a significant advance. “Overall, the findings […] are promising and demonstrate that the field is rapidly moving from ‘if’ blood biomarkers can be used in AD to ‘how’ they can be used,” he wrote in a JAMA Neurology editorial.

These assays could facilitate both primary-care diagnosis and recruitment into clinical trials. In a cost analysis, Palmqvist and colleagues estimated that a plasma test could take $3.2 million off the $9.2 million price tag for screening 1,000 trial volunteers by amyloid PET, assuming they could reduce the number of scans to 200.

O’Bryant believes that there is room for improvement. If these tests are to be used in the clinic, scalability will be a huge concern, he wrote. “The blood collection and processing procedures are not applicable to standard clinical lab practice and will cause substantial barriers to clinical application.”

This point was echoed by Hugo Vanderstichele, ADx Neurosciences, Zwijnaarde, Belgium. He also thinks it is important to understand how assays relate to biology. “Now we need to develop Precision Qualified Assays,” he said. These marry assay analytical performance to biology that’s relevant for specific contexts of use. They demand better standardization than currently available, he said. He also thinks Aβ plasma assays could be improved by taking isoforms besides Aβ40 and Aβ42 into account, by incorporating other markers, and by better understanding biological factors that might shift plasma Aβ levels, such as exercise, the body-mass index, and medications (see comment below).

Could other assay modalities do better than Elecsys? Perhaps. The system falls short of the accuracy of recently described mass-spec assays, which claim AUCs of 0.84 to 0.97 depending on assay and cohort (Feb 2018 news; Jul 2017 conference news). “We really need head-to-head comparisons running the different assays on the same set of samples,” said Hansson.

Such studies are underway. Co-author Kaj Blennow, University of Gothenburg, Sweden, has completed a round-robin study. It collected one set of samples and shared them among labs for analysis on different assay platforms. Part of the difficulty with such studies is collecting enough material to share, but Blennow told Alzforum they have 80 individual samples, all treated exactly the same way and divided into identical aliquots. They have been tested in 10 different laboratories using 11 different plasma assays, including three SIMOA immunoassays, two ELISAs, four immunoprecipitation/mass spec methods, one immunomagnetic reduction method, and the fully automated Elecsys immunoassay.

All have measured plasma Aβ42, Aβ40, and the ratio. Blennow and colleagues are currently analyzing the data and will present them in July at AAIC.—Tom Fagan

Comments

  1. Drawing closer: Alzheimer’s blood test for primary care

    This study of Palmqvist et al. showed with fully automated immunoassays that the plasma Aβ1-42/Aβ1-40 ratio can predict amyloid plaque load (amyloidopathy) in the brain (BioFinder study). In addition, at the upcoming AAIC meeting, Inge Verberk from Amsterdam University Medical Center will present a follow-up study of a previously published Simoa immunoassay approach (Verberk et al., 2018) using newly developed AMYBLOOD Simoa assays. She will report comparable clinical performance using a different patient cohort and technology. The Simoa assays to be presented by Charlotte Teunissen's group at Amsterdam UMC are developed in close collaboration with ADx NeuroSciences.

    The potential value of the plasma Aβ42/Aβ40 ratio has been increasingly recognized over the last several years by:

    • Performing studies focused on a specific context of use (~Aβ-PET imaging);
    • The availability of a reference method (FDA-approved Aβ-PET imaging with visual interpretation);
    • Better selection and characterization of subjects for inclusion in the study (CSF biomarker profile, Aβ-PET imaging);
    • Use of larger sample sizes and integration of qualification, followed by validation cohorts;
    • Use of technology platforms with better precision (automation, random access) and lower analytical sensitivity.

    Before a plasma Aβ immunoassay can be used to rule out the need for a costly Aβ-PET scan, the test should achieve a high sensitivity (>85 percent) and a high negative predictive value for a specific clinical context, for instance for pharma trials.

    Suggestions for the use of the plasma Aβ42/Aβ40 ratio were already published more than a decade ago based on classical ELISAs and supported recently by the more labor-intensive mass-spectrometry technology (Nakamura et al., 2018; Ovod et al., 2017). However, with their current design, it seems immunoassays cannot reach the same diagnostic accuracy for Aβ1-42/Aβ1-40 as mass spectrometry, pointing to the need to (i) extend the algorithm for blood testing by integration of other proteins or protein isoforms, (ii) have a better understanding of the characteristics of monoclonal antibodies that are used in the assay design, (iii) improve analytical performance of immunoassays, and (iv) generate standard operating procedures for collection and storage of blood samples.

    Several obvious plasma biomarkers (e.g. Neurofilament Light, tau, BACE1 protein, YKL-40) (Vergallo et al., 2019) are not able to fill that clinical accuracy gap between mass spectrometry-based studies and immunoassay data (Feb 2019 news). However, ongoing longitudinal studies in (pre)clinical study cohorts, such as from the SCIENCe project, the pre-insight AD cohort (Verberk et al., 2018; Vergallo et al., 2019), or the AIBL study cohort, can potentially confirm and validate that a simple plasma test might help to identify a stage of AD before MCI and thus might aid in the setup of new clinical and prevention trials.

    It is important that biology and assay performance are more closely linked to each other. Precision Qualified Assays (PQAs) will provide a solution in the future by combining clearly defined analytical performance requirements of an assay with the observed effects in patients (see also Biomarker Qualification: Evidentiary Framework). This requires a high(er) level of standardization of biomarker assays than done now. Not only are extensive standardizations needed at the level of the lab, the sample, and the assay, but also the biological variation or biological differences emerging for a specific context of use need to be taken into account.

    There is a need for: 

    • More detailed reporting of study results, including characteristics (e.g. selectivity, specificity) of antibodies used in each assay (which protein isoform is detected?);
    • Documented performance characteristics of assays and validation approaches (see also Points to Consider Document: Scientific and Regulatory Considerations for the Analytical Validation of Assays Used in the Qualification of Biomarkers in Biological Matrices); 
    • A better understanding of confounding factors that have an impact on Aβ concentrations in samples (e.g. exercise, BMI, medication) or a better understanding of the mechanism of clearance of Aβ in peripheral fluids;
    • Comparison of clinical performance when using other Aβ isoforms, including but not limited to Aβ34, Aβ-3, … or using other proteins (e.g. total tau, NfL);
    • Open sharing of results obtained in the larger patient cohorts with different assay formats;
    • Standard operating procedures for sample collection and storage. Since Aβ1-42 in plasma is not as stable as in the CSF, more critical review of the sample collection and storage protocol is required. A specific sample type (e.g. EDTA, plasma, serum) can give you an identical or a different outcome;
    • Harmonization and generation of certified reference materials. The field will gain by initiation of harmonization of plasma measurements by production of certified reference materials using plasma samples. The round-robin study on 70 EDTA-plasma samples, organized by Professor Kaj Blennow at the University of Gothenburg, Sweden, and including different technology platforms, will improve our current understanding of how changes in assay design might affect the result. The latter study is more designed to verify correlations in concentration between analytes and is less focused on clinical application of the assays. It is a very good first step in bringing the results of the plasma studies to another level. Such insights are essential and will further streamline efforts to use the different biomarkers in different stages of evaluation of prevention and treatment options. The field will progress faster if the learnings from past experiences in CSF will be integrated in the workflow. All stakeholders will have to work more closely together in a shorter period of time to make the assays available for implementation in a routine clinical environment;
    • Integration of technology platforms with better multiplexing possibilities, low sensitivity, and more easily upscaled for possible worldwide use.

    References:

    Nakamura A, Kaneko N, Villemagne VL, Kato T, Doecke J, Doré V, Fowler C, Li QX, Martins R, Rowe C, Tomita T, Matsuzaki K, Ishii K, Ishii K, Arahata Y, Iwamoto S, Ito K, Tanaka K, Masters CL, Yanagisawa K. High performance plasma amyloid-β biomarkers for Alzheimer's disease. Nature. 2018 Feb 8;554(7691):249-254. Epub 2018 Jan 31 PubMed.

    Ovod V, Ramsey KN, Mawuenyega KG, Bollinger JG, Hicks T, Schneider T, Sullivan M, Paumier K, Holtzman DM, Morris JC, Benzinger T, Fagan AM, Patterson BW, Bateman RJ. Amyloid β concentrations and stable isotope labeling kinetics of human plasma specific to central nervous system amyloidosis. Alzheimers Dement. 2017 Aug;13(8):841-849. Epub 2017 Jul 19 PubMed.

    Vergallo A, Mégret L, Lista S, Cavedo E, Zetterberg H, Blennow K, Vanmechelen E, De Vos A, Habert MO, Potier MC, Dubois B, Neri C, Hampel H, INSIGHT-preAD study group, Alzheimer Precision Medicine Initiative (APMI). Plasma amyloid β 40/42 ratio predicts cerebral amyloidosis in cognitively normal individuals at risk for Alzheimer's disease. Alzheimers Dement. 2019 Jun;15(6):764-775. Epub 2019 May 18 PubMed.

    Verberk IM, Slot RE, Verfaillie SC, Heijst H, Prins ND, van Berckel BN, Scheltens P, Teunissen CE, van der Flier WM. Plasma Amyloid as Prescreener for the Earliest Alzheimer Pathological Changes. Ann Neurol. 2018 Nov;84(5):648-658. Epub 2018 Oct 4 PubMed.

    View all comments by Hugo Vanderstichele View all comments by Eugeen Vanmechelen

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References

News Citations

  1. With Sudden Progress, Blood Aβ Rivals PET at Detecting Amyloid
  2. Closing in on a Blood Test for Alzheimer’s?
  3. Finally, a Blood Test for Alzheimer’s?

Further Reading

No Available Further Reading

Primary Papers

  1. Palmqvist S, Janelidze S, Stomrud E, Zetterberg H, Karl J, Zink K, Bittner T, Mattsson N, Eichenlaub U, Blennow K, Hansson O. Performance of Fully Automated Plasma Assays as Screening Tests for Alzheimer Disease-Related β-Amyloid Status. JAMA Neurol. 2019 Jun 24; PubMed.

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