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Multiplex Method for Measuring Biomarkers of Alzheimer Disease in Cerebrospinal Fluid

Posted on: Saturday, 12 February 2005, 03:00 CST

Alzheimer disease (AD), the most common form of dementia, is a neurodegenerative disorder characterized by neuronal and synaptic loss. Despite its high prevalence and great cost to society, there is no definitive premortem test for AD (1, 2). The diagnosis of probable or possible AD is based on a history of insidious onset and progressive impairment of memory as well as loss of other cognitive functions (3). The most important clinical task is the exclusion of reversible causes of dementia, most commonly vascular dementia. It is of little comfort to treating physicians and family members that postmortem examination of the brain by the pathologist is the only reliable diagnostic test for AD. Typically, microscopic examination of stained sections of brain taken from cortical and limbic brain regions reveals the hallmark changes of AD, namely, accumulation of intracellular neurofibrillary tangles and extracellular amyloid plaques. Neurofibrillary tangles are composed mainly of abnormally hyperphosphorylated tau protein (P-TAU). The extracellular amyloid plaques are composed of the 40-and 42-amino acid-long β- amyloid peptides [Aβ(1-40) and Aβ(1-42)]. Several neuropathologic subtypes of AD have been described, depending principally on the cortical regions most affected by neuronal loss, and the rates of disease progression may vary among them (4). It is becoming increasingly clear that the clinical and classical neuropathologic diagnosis of AD is actually a lumping of several etiologically distinct diseases with similar end-stage phenotypes (5).

The following questions arise. Of those elements evaluated in the postmortem examination, are there some that can be measured during life that have at least a moderate correlation with postmortem measurements? If so, can these tests be adapted for general use? Currently, clinicians must make a clinical diagnosis of AD (cAD) and treat presumptively (6). Although expert clinicians are quite skilled at making the diagnosis in moderately to severely affected individuals [there is very good correlation between cAD and neuropathologic (pAD) diagnoses late in the disease process], this is much less the case early in the disease process. Because the pharmacologic interventions currently available are aimed at slowing or compensating for neuronal loss and its effects, aids to making a more accurate cAD diagnosis earlier in the disease process are highly sought. This need is likely to be even more keenly felt as newer therapies become available.

What tests are available to the clinician for making a diagnosis of cAD? Aside from imaging studies, analysis of cerebrospinal fluid (CSF) for biomarkers of AD is the current state of the art (7). The assays are currently performed in a plate-based ELISA format with antibodies directed against Aβ(1-42), total tau protein (T- TAU), and P-TAU. In AD groups (compared with control groups), Aβ(1-42) concentrations in the CSF are markedly decreased (possibly because it has become entrapped within the cortex as part of the amyloid plaques), whereas both T-TAU and P-TAU are increased. These tests have empirical virtue in that they measure the same elements in the CSF that the neuropathologist analyzes in tissue.

In this issue of Clinical Chemistry, Olsson et al. (8) report on their adaptation of a flow cytometry platform to improve the sensitivity and throughput of the analyses of these CSF biomarkers. The microsphere-based Luminex xMAP(TM) technology involves the covalent coupling of a capture antibody to spectrally specific microspheres. Each microsphere is labeled with a precise concentration ratio of red- and orange-emitting fluorochromes, giving it a unique spectral signature. Classification of each bead is made with excitation at 635 nm and measurement of the emission wavelengths, together with the intensities, of each of the dyes. Quantification can be achieved by addition of a second biotinylated antigen-directed antibody and a third fluorochrome [phycoerythrin (PE)] coupled to streptavidin. Simultaneous measurement of the PE signal, using a second laser exciting at 532 nm, quantifies the amount of antigen bound at the microsphere surface.

Although the fundamental mechanism of biomarker detection is the same (antigen-antibody affinity), the flow cytometry platform offers several advances compared with the ELISA platform. These include simultaneous biomarker analyses, leading to smaller sample size requirements, and reduced assay time, leading to a potential decrease in labor-associated costs. The authors compared the performance of the xMAP technology with currently available ELISA tests and found that the two were at least equivalent, with some increases in detection limits seen with the xMAP tests.

The real value of the xMAP technology, however, perhaps remains to be achieved. By multiplexing the analysis of three biomarkers, the authors achieved savings in analysis time and sample volume compared with a series of single-biomarker ELISA tests. This begs the question, if three biomarkers are good, would more be better? Previous studies suggest other candidates that could provide additional diagnostic power in separating AD from other causes of dementia: S100β, N-terminally truncated variants of Aβ peptides, α-synuclein, heart fatty acid-binding protein, gelatinase B (MMP-9), and interleukin-6 (9-11). The point is that there are likely numerous CSF proteins whose concentrations might vary in relation to the presence or absence of AD and its subtypes. The only limit on the number of additional biomarkers one might analyze would appear to be the availability of specific antibodies available for these various antigens (two epitopically distinct antibodies are required for each antigen to be quantified by use of PE emission intensities). In a sense, this positions the xMAP technology on the continuum between single-protein measurement (ELISA) and total protein/peptido measurement (mass spectroscopy/ proteomics) tests. As has been seen with ovarian carcinoma serum analysis (12), it may be possible to measure a pattern of altered expression in a limited set of proteins and to match that pattern with one or more neurodegenerative disease states.

Indeed, if ongoing proteomics-based investigations of CSF in neurodegenerative disease identify a diagnostically relevant set of biomarkers and these are easily adapted to the xMAP platform, the best may well be yet to come.

References

1. Dickson DW. Neuropathological diagnosis of Alzheimer's disease: a perspective from longitudinal clinicopathological studies. Neurobiol Aging 1997;18:S21-6.

2. Ritchie K, Lovestone S. The dementias. Lancet 2002;360:1759- 66.

3. Markesbery WR. Neuropathological criteria for the diagnosis of Alzheimer's disease. Neurobiol Aging 1997;18:S13-9.

4. Gallon CJ, Patterson K, Xuereb JH, Hodges JR. Atypical and typical presentations of Alzheimer's disease: a clinical, neuropsychological, neuroimaging and pathological study of 13 cases. Brain 2000;123:484-98.

5. Mattson MP. Pathways towards and away from Alzheimer's disease. Nature 2004;430:631-9.

6. McKhann G, Drachmen D, Folstein M, Katzman R, Price D, Stadlan EM. Clinical diagnosis of Alzheimer's disease: report of the NINCDS- ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology 1984;34:939-44.

7. Blennow K, Hampel H. CSF markers for incipient Alzheimer's disease. Lancet Neurology 2003;2:605-13.

8. Olsson A, Vanderstichele H, Andreasen N, de Meyer G, Wallin A, Holmberg B, et al. Simultaneous measurement of β-amyloid^sub (1- 42)^ total tau, and phosphorylated tau (Thr^sup 181^) in cerebrospinal fluid by the xMAP technology. Clin Chem 2005;51:336- 45.

9. Steinacker P, Mollenhauer B, Bibl M, Cepek L, Esselmann H, Brechlin P, et al. Heart fatty acid binding protein as a potential diagnostic marker for neurodegenerative diseases. Neurosci Lett 2004;370:36-9.

10. Lorenzl S, Albers DS, Relkin N, Ngyuen T, Hilgenberg SL, Chirichigno J, et al. Increased plasma levels of matrix metalloproteinase-9 in patients with Alzheimer's disease. Neurochem Int 2003;43:191-6.

11. Arosio B, Trabattoni D, Galimberti L, Bucciarelli P, Fasano F, Calabresi C, et al. Interleukin-10 and interleukin-6 gene polymorphisms as risk factors for Alzheimer's disease. Neurobiol Aging 2004;25:1009-15.

12. Petricoin EF, Ardekani AM, Hitt BA, Levine PJ, Fusaro VA, Steinberg SM, et al. Use of proteomic patterns in serum to identify ovarian cancer. Lancet 2002;359:572-7.

Bradley B. Miller1,2

James W. Mandell1*

Departments of 1 Pathology (Neuropathology)

and 2 Neurology

University of Virginia Health System

Charlottesville, VA

* Address correspondence to this author at: Department of Pathology (Neuropathology), University of Virginia Health System, Charlottesville, VA, 22908. Fax 434-924-9177; e-mail jwm2m@virginia.edu.

DOI: 10.1373/clinchem.2004.044016

Copyright American Association for Clinical Chemistry Feb 2005

Source: Clinical Chemistry

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