Identifying people on the verge of dementia before they actually succumb has become somewhat of a Holy Grail for Alzheimer disease research—finding cheap and reliable methods for this even more so. Toward that end, a prospective study involving thousands of French elderly sustains hope that the bewildering hunt for plasma AD biomarkers may not be for naught. Led by Jean-Charles Lambert of the Institute Pasteur in Lille, France, scientists report that seniors diagnosed with dementia at two years of follow-up had low baseline plasma Aβ1-42/Aβ1-40 ratios. The team also measured truncated forms of plasma Aβ, and found that low plasma Aβ(n-42)/Aβ(n-40) ratios were associated with increased risk of mixed/vascular dementia but not AD. Lambert presented these data in July at the International Conference on Alzheimer’s Disease (ICAD) in Vienna (see ARF conference story). The full report appears in the September 15 issue of Neurology.

This study “contributes to the growing evidence that we may be able to find a plasma biomarker, which would be, from a population perspective, much more usable than cerebrospinal fluid (CSF) or brain imaging measures,” Nicole Schupf of Columbia University, New York, told ARF. Though the latter measures, at this point, offer more predictive and diagnostic value, spinal taps and brain scans are expensive and invasive, fueling interest in AD biomarkers that could be evaluated from a simple blood draw.

Thus far, the search for blood-based biomarkers can be characterized by sprinkles of promise awash with confusion. Plasma Aβ concentrations rise with age and are elevated in people with pathogenic mutations in presenilin or amyloid-β precursor protein (APP) (Ringman et al., 2008). However, data from studies of sporadic AD have been all over the map, with various groups reporting that disease risk associates with high plasma Aβ42 levels (e.g., Schupf et al., 2008 and ARF related news story) or with low Aβ1-42/Aβ1-40 ratios (e.g., van Oijen et al., 2006), or that correlations with plasma Aβ were too weak to be useful (e.g., Lopez et al., 2008; Hansson et al., 2008 and ARF related news story).

The current paper was an epidemiological tour de force involving 8,414 non-institutionalized elderly (ages 65 and up) in Bordeaux, Montpellier, and Dijon. Funded primarily by the French government as well as several non-government organizations and pharmaceutical companies, the Three-City (3C) Study was designed to determine the influence of vascular factors in dementia, Lambert said. Scientists collected baseline blood samples from participants randomly chosen from electoral rolls of each city between 1999 and 2001, and did follow-up assessments, including neuropsychological testing, two and four years later.

From the initial cohort, 257 developed incident dementia during the four-year follow-up period, and 1,185 were selected randomly as non-demented controls. Lambert and colleagues used a new xMAP-based assay developed by Innogenetics, Ghent, Belgium, to measure full-length and truncated Aβ peptides in the blood samples. Controlling for various factors including age, gender, education, diabetes, cholesterol, and APOE genotype, they found that people in the upper Aβ1-42/Aβ1-40 tertile had half the risk of developing AD and a 2.9-fold lower risk of mixed/vascular dementia, compared to those in the lowest tertile. However, the upper tertile for truncated Aβ, i.e., n-42/n-40, was only associated with reduced (3.7-fold) risk of mixed/vascular dementia but not AD.

“Maybe these truncated species are more involved in vascular amyloidosis than they are in forming plaques,” Schupf said. In a recent study led by Richard Mayeux at Columbia University, New York, Schupf and colleagues analyzed more than 1,100 seniors in northern Manhattan and found that those with high baseline levels of plasma Aβ42 were three times as likely to develop AD over four to six years of follow-up (Schupf et al., 2008).

At a glance, these findings seem to clash with the 3C Study, which found that high Aβ1-42/Aβ1-40 ratios correlated with lower AD risk. However, the New York study found that conversion to AD coincided with a drop in plasma Aβ42, which Schupf sees as “highly consistent” with the association Lambert and colleagues report at two years of follow-up. “You don't really know where those guys were five years ago. Two years is quite a short period of time. They may have been en route [to dementia/AD],” she told ARF. “It would be of interest to know how many of those who converted to AD had mild cognitive impairment (MCI) at their initial examination.”

On this point, Lambert noted that MCI was still a relatively new concept at the time the 3C Study was designed, making systematic diagnoses extremely challenging for large epidemiological studies. “However, we can postulate that some of the individuals who developed dementia at two or four years of follow-up likely presented MCI at inclusion,” he wrote in an email to ARF. In the future, baseline MCI status could likely be addressed in one of the three cities to make the sample size more manageable, he noted.

While large sample size is clearly a strength of the 3C Study, it can also cause problems for interpretation. “Usually more is better, but I wonder if it holds any meaning for individual diagnosis,” Anne Fagan of Washington University, St. Louis, Missouri, said of the new work. “If you study enough people, you'll find an association somewhere.” She suggested that timing may be critical in studies of plasma Aβ and dementia. “I found it curious that the associations were restricted to individuals diagnosed at two years of follow-up and not at four years,” Fagan told ARF. She notes previous work showing that CSF levels of the β-secretase enzyme BACE1 are elevated in MCI, relative to healthy people, but dip during conversion to dementia (see ARF related news story). “This plasma story could be the same thing,” Fagan said. “Maybe you have to catch it at a certain timepoint in the course of disease in order for it to be meaningful diagnostically and predictively.” Schupf agrees, stressing that plasma Aβ studies need to be longitudinal, with repeated peptide measures in elders who start off free of cognitive impairment. “If Aβ was high and then goes down with onset, it’s not going to look different from non-demented in a cross-sectional study,” she said.

Another problem with plasma Aβ studies is that variations in the techniques for measuring plasma Aβ make it hard to compare results from different labs. Such challenges were highlighted in a recent study showing highly variable recovery rates for Aβ40 and Aβ42 in spiked samples sent to various U.S. labs using different protocols to measure Aβ peptide concentrations (Okereke et al., 2009 and ARF related news story). The use of different assays may not necessarily cause problems, though. Lambert noted that the 3C findings essentially reproduced those of a similar, large Dutch study that did not use xMAP-based technology to measure plasma Aβ (van Oijen et al., 2006).

Barring methodological concerns, a fundamental problem with plasma Aβ studies is the uncertainty about what is being measured. “We don't know, in fact, whether the plasma Aβ peptides are coming directly from brain, from vascular cells, or from other organs in the body,” Lambert said. New data by Fagan and colleagues seem to suggest that plasma Aβ measurements may not reflect changes in brain Aβ as closely as CSF measures or positron emission tomography (PET) using the amyloid tracer Pittsburgh compound-B (PIB). Consistent with past work by her group (Fagan et al., 2006) and others, “everybody who was PIB-positive had low CSF Aβ42 levels, but the majority of PIB-negative people had high CSF Aβ42,” Fagan said of her new study, which is currently in press at EMBO Molecular Medicine. However, plasma Aβ concentrations did not seem to correlate with brain amyloid load assessed by PIB-PET, or with CSF Aβ, she said.

These data would seem to strengthen the case for relying on brain scans and CSF samples for AD biomarkers. “But when the day comes where we have treatments, we're going to want to have large-scale screening of predictors that allow us to intervene before people develop symptoms,” Schupf said. “On a population basis, that really is a blood test. I don't think we're there yet. I think this [study] helps to suggest that we might get there.”

Masood Kamali-Moghaddam of Uppsala University, Sweden, expects the new data to intensify efforts “to detect and identify other toxic isomers of Aβ, such as Aβ oligomers in blood.” (See full comment below.) Scientists described some attempts in this direction at the ICAD meeting in July (see ARF related conference story).—Esther Landhuis


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  1. The paper is well written and the study seems to be carried out very well, at least in regard to the data analysis. (Though an experimental scientist that I am, I was a bit surprised to see only half of the name of one of the used kits—the INNO-BIA kit—without further information on the experimental procedures.) In addition, the study is one of the biggest of its kind, and similar to the Rotterdam Study, although with different methodology.

    Previously, different studies have reported increased Aβ1-40 and decreased Aβ1-42, and also decreased Aβ1-42/Aβ1-40 ratios in CSF, but there are conflicting data on the level of these Aβ species and the ratio of these species in blood.

    Another striking thing is the decreased level of Aβ1-42 with progression of dementia (that has not been commented on in the paper), which is usually speculated to be due to absorption to the senile plaques in the brain. But some recent studies have demonstrated that this might depend on the methodology used. In an ELISA-based assay, the Aβ1-42 level is decreased, while in a denaturing Western blot experiment, the level of Aβ1-42 is increased in the same model material.

    The main part of the results in this paper conforms with the data from the Rotterdam Study, with some differences in the details, for instance, finding that the Aβ1-42/Aβ1-40 ratio in this study "may be associated with the risk of dementia only in individuals diagnosed at two years of follow-up," while in the case of the Rotterdam Study this time period is eight years. The current study has also included the measurement of other Aβ pieces (Aβn-40 and Aβn-42), which might shed light on some details.

    Overall, my belief is that this paper, by confirming other well-done studies such as the Rotterdam Study, takes us one step closer to be convinced that blood Aβ might reflect brain and CSF Aβ. This, in turn, might take us to another level to try harder to detect and identify other toxic isomers of Aβ, such as Aβ oligomers in blood, which is a more non-invasive approach compared to the CSF. Of course, this might require that more powerful tools be developed and applied in the field. Having said that, I do not believe that the outcome of this paper will change the current situation and won't clarify much whether blood Aβ is a promising AD biomarker.

  2. First, we would like to thank Dr Kamali-Moghaddam for his comments. Regarding the methodology, we apologize about the name of the kit used which is not fully written (INNO-BIA plasma Aβ forms). The strength of such a study is the use of a commercially available kit. Thus, experimental procedures are the manufacturer’s instructions. We did not think that it was necessary to add them in the materials and methods. However, for Alzforum readers, here is the experimental procedure. All plasma samples were diluted 1/3 with a buffer containing detergent before analysis.

    Quantification of Aβ isoforms in plasma was performed using INNO-BIA plasma Aβ forms assays (Innogenetics, Ghent, Belgium), a multiplex microsphere-based Luminex xMAP technique that allows simultaneous analysis of Aβ1-40 and Aβ1-42 (module A) an Aβn-40 and Aβn-42 (module B).

    The monoclonal antibodies (MAbs) 21F12 and 2G3, which specifically bind Aβ peptides ending at 42 and 40, respectively, were used as capture antibodies. The capture MAbs were covalently coupled to carboxylated beads of different regions (region 104 for Aβ42;, region 105 for Aβ40;, and region 102 for a non-Aβ binding MAb). MAb 3D6, which specifically binds Aβ peptides starting at Asp1, and MAb 4G8 which react with all N-terminally truncated Aβ peptides up to Val18, were used as detector antibodies.

    In short, Aβ isoforms ending either at Aβ40 or Aβ42 were selectively captured by beads that have been coated with either MAb 21F12 for Aβ42 or MAb 2G3 for Aβ40. A third class of beads was coated with MAb AT120, used to measure matrix effects due to heterophilic antibodies in the plasma sample. In module A, biotinylated MAb 3D6, which selectively binds Aβ peptides starting at Aβ1, is used as detector antibody, providing specific quantification of Aβ1-42 and Aβ1-40 isoforms. In module B, biotinylated MAb 4G8, which binds all N-terminally truncated Aβ peptides up to those starting at Aβ18, is used as detector antibody, providing specific quantification of Aβn-42 and Aβn-40 isoforms.

    The procedure could be described as follows: after sonication and vortexing, a mixture (100 μL/well) of the beads (either Module A or B) were added to 96 well filter plates (Millipore Corporation, Bedford, Massachusetts). After draining the wells using a vacuum manifold (Millipore Corporation, Bedford, Massachusetts), standards, blanks, or diluted (1:3) plasma samples were added (75 μL/well) in duplicate together with the biotinylated detector MAb (25 μL/well) (MAb 3D6 for Module A and MAb 2G3 for Module B) and incubated overnight at 2-8ºC in the dark (plates covered with aluminum foil) on a plate shaker (600 rpm). After washing, phycoerythrine-labeled streptavidine was added (100 μL/well) and incubated for one hour on a plate shaker. After a second wash step, 100 μL of phosphate-buffered saline was added. The assays were analyzed on a Luminex 200 IS instrument (Luminex, Austin, Texas). For each set of microspheres, 100 beads were analyzed, and the median fluorescence intensity (MFI) was used for quantification.

    A ready-to-use calibrator series is included in duplicate in each assay run and, using the calibration curve constructed with the median fluorescence values for each of the standards, concentrations were determined by sigmoidal curve fitting. Moreover, in each series two run-validation control samples are also included.

    I hope that this is helpful.


News Citations

  1. Vienna: New Genes, Anyone? ICAD Saves Best for Last
  2. Hot Plasma—Blood Aβ a Risk Marker for AD?
  3. Plasma Aβ Testing Beset by Questions of Assays, Biology, Timing
  4. BACE1 Gets Attention as Potential Alzheimer’s Biomarker
  5. Studies Reveal New Hope, Old Problems With AD Biomarkers
  6. Vienna: New Tack to See Amyloid Oligomers in Body Fluids

Paper Citations

  1. . Biochemical markers in persons with preclinical familial Alzheimer disease. Neurology. 2008 Jul 8;71(2):85-92. PubMed.
  2. . Peripheral Abeta subspecies as risk biomarkers of Alzheimer's disease. Proc Natl Acad Sci U S A. 2008 Sep 16;105(37):14052-7. PubMed.
  3. . Plasma Abeta(1-40) and Abeta(1-42) and the risk of dementia: a prospective case-cohort study. Lancet Neurol. 2006 Aug;5(8):655-60. PubMed.
  4. . Plasma amyloid levels and the risk of AD in normal subjects in the Cardiovascular Health Study. Neurology. 2008 May 6;70(19):1664-71. PubMed.
  5. . Evaluation of plasma Abeta(40) and Abeta(42) as predictors of conversion to Alzheimer's disease in patients with mild cognitive impairment. Neurobiol Aging. 2010 Mar;31(3):357-67. Epub 2008 May 19 PubMed.
  6. . Performance characteristics of plasma amyloid-beta 40 and 42 assays. J Alzheimers Dis. 2009;16(2):277-85. PubMed.
  7. . Inverse relation between in vivo amyloid imaging load and cerebrospinal fluid Abeta42 in humans. Ann Neurol. 2006 Mar;59(3):512-9. PubMed.

Further Reading


  1. . Biochemical markers in persons with preclinical familial Alzheimer disease. Neurology. 2008 Jul 8;71(2):85-92. PubMed.
  2. . Peripheral Abeta subspecies as risk biomarkers of Alzheimer's disease. Proc Natl Acad Sci U S A. 2008 Sep 16;105(37):14052-7. PubMed.
  3. . Performance characteristics of plasma amyloid-beta 40 and 42 assays. J Alzheimers Dis. 2009;16(2):277-85. PubMed.
  4. . Evaluation of plasma Abeta(40) and Abeta(42) as predictors of conversion to Alzheimer's disease in patients with mild cognitive impairment. Neurobiol Aging. 2010 Mar;31(3):357-67. Epub 2008 May 19 PubMed.
  5. . Biomarkers of Alzheimer's disease. Neurobiol Dis. 2009 Aug;35(2):128-40. PubMed.

Primary Papers

  1. . Association of plasma amyloid beta with risk of dementia: the prospective Three-City Study. Neurology. 2009 Sep 15;73(11):847-53. PubMed.