analytical aspects of molecular alzheimer’s disease biomarkers

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ISSN 1752-036310.2217/BMM.12.44 © 2012 Future Medicine Ltd Biomarkers Med. (2012) 6(4), 1–13

Analytical aspects of molecular Alzheimer’s disease biomarkers

A biomarker can broadly be defined as “a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmaco-logic responses to a therapeutic intervention” [1]. There is an increasing demand for biomarkers to be of use as a primary measure for the effective-ness of a drug in a clinical trial. No potential biomarker candidate discovered in the field of neurologic or psychiatric disorders has yet been validated as a predictor of the effect of the treat-ment on the clinical outcome [2]. Biomarkers are considered as continuous (using numerical val-ues) or categorical (with discrete or descriptive values). Ideally, the biomarker has a direct link with the pathophysiology of the disease. In clini-cal trials, biomarkers are used as a diagnostic end point (approximating a characteristic of the con-dition of a patient, reflecting how a patient feels, functions or survives) or as a surrogate end point marker. Surrogate end points are based on sci-entific observations that are expected to predict a clinical benefit, or lack thereof, in a drug trial. In addition, if a novel biomarker is found during data-driven research (e.g., proteomic studies), new possible intervention targets and insight into the disease mechanism may be obtained.

At present, all commercially available assays for quantification of proteins in the cerebro-spinal fluid (CSF) need to be considered as rela-tive quantitative assays (different from definitive quantitative assays), since the calibration is per-formed with a reference standard that is not well characterized, not available in a purified form

or not fully representative of the endogenous biomarker [3]. The qualitative outcome of these tests (diagnosis) is based on absolute concentra-tions of the individual biomarker. The accuracy for these analytes is difficult to investigate since no reference material (e.g., gold standard) is cur-rently available. No standard has been deposited with any standardization organization, hamper-ing the comparison between commercially avail-able assays. However, efforts to generate refer-ence materials and/or methods have recently been initiated; the topic is also covered in this issue of Biomarkers of Medicine (see [4]). In this present review, the focus is on analytical issues connected to the use of the established molecular Alzheimer’s disease (AD) biomarkers in CSF.

Markers reflecting AD pathologyIn 1998, a consensus report was published that listed the properties of an ideal biomarker for AD [5]. Criteria were given for defining, devel-oping and assigning biomarkers in addition to recommendations on how to establish them. For AD, there are presently three CSF biomarkers, b-amyloid (Ab), total tau (T-tau), and phosphor-ylated tau (P-tau), which together with potential blood markers and brain imaging were included in the Alzheimer’s Disease Neuroimaging Initiative (ADNI) with the goal of finding the most useful combination of biomarkers for diag-nosis and monitoring treatment effects [6–8]. The CSF markers are firmly established to the point that the newly revised research criteria for diagnosing AD now partly rely on them [9].

In general, a biomarker has multiple uses such as a diagnostic tool and a method to monitor therapy. The quality of a biomarker depends on how big the difference is between, for example, patients and healthy controls, but also on the capacity of the method used to measure it (the uncertainty in the method should be much less than the difference between the groups). A good biomarker should also be specific towards a disease, allowing for differentiation between clinically related syndromes. In addition, it is of importance that the stability of the methods used is high enough to establish cut-off levels both in individual laboratories and on a global scale. In the field of Alzheimer’s disease, there are currently three cerebrospinal fluid markers that have been verified in multiple studies and the analytical aspects of measuring them will be discussed.

KEYWORDS: accuracy n Alzheimer’s disease n biomarkers n cerebrospinal fluid n pathophysiology n quality control

Ulf Andreasson*1, Eugeen Vanmechelen2, Leslie M Shaw3, Henrik Zetterberg1,4 & Hugo Vanderstichele2

1Institute of Neuroscience & Physiology, Department of Psychiatry & Neurochemistry, The Sahlgrenska Academy, University of Gothenburg, Mölndal, Sweden 2ADx NeuroSciences, Technologiepark 4, 9052 Gent, Belgium 3Department of Pathology & Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA 4UCL Institute of Neurology, Queen Square, London WC1N 3BG, UK *Author for correspondence: Tel.: +46 31 3432410 Fax: +46 31 3432426 [email protected]

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Review Andreasson, Vanmechelen, Shaw, Zetterberg & Vanderstichele Analytical aspects of molecular Alzheimer’s disease biomarkers Review

Biomarkers can be separated into different classes depending on their nature, but only the molecular CSF markers, Ab, T-tau and P-tau, will be covered here. Recent reviews on non-molecular markers such as different types of imaging can be found elsewhere [10,11]. A qualification opinion for using CSF tau and Ab1–42 in clinical trials of predementia AD was recently published by the European Medicines Agency [12].

�n b-amyloidAb was identified as the major component in senile plaques [13,14], one of the patho-physiological hallmarks of AD, and it was found that the peptide is derived from a transmem-brane precursor protein [15]. It was shown to be present in CSF [16], but no difference in the con-centration was observed between AD and con-trols when using western blot [17] or ELISA [18,19]. However, several different Ab species that vary in length, most frequently being truncated at the C-terminus, are present in CSF [20], and the 42-amino acid long variant Ab1–42 was shown to be present in lower concentrations in AD [21]. The reduction of Ab1–42 is usually attributed to the notion that it gets stuck in plaques instead of reaching the CSF. Assays measuring Ab species with different N-terminal-specific antibodies, such as 3D6, and Ab antibodies mapping to an internal epitope, 4G8, perform equally as well as Ab1–42 in discriminating AD from controls [22], which is in line with the observation that it is only Ab species starting at position one in the Ab sequence that are relatively abundant in the CSF [20], although truncation at the amino terminus of Ab is one of the early signs of pathol-ogy in the brains of affected subjects [23], and thus more work, preferably using N-truncated specific antibodies, will be necessary.

�n TauTau was first shown to be a factor that is essential for microtubule assembly [24] and later identified in a hyperphosphorylated form in extracellular tangles [25], the second pathophysiological hall-mark of AD. Tau has several potential phospho-rylation sites and is present in CSF both with and without phosphorylation; both forms were found to be elevated in AD [26,27]. The increase in CSF concentrations of T-tau and P-tau are usually attributed to neuronal cell death and tangle pathology, respectively.

Owing to a broad overlap in concentrations for each individual analyte between selected study populations, a multianalyte approach with

clearly defined algorithms was established in order to obtain high clinical accuracy [28–30]. A major obstacle for the diagnostic field is the lack of uniform values when studies that utilized the AD markers are compared, as can be seen for a number of studies in which concentrations were ranked in ascending order for each diagnostic group (Figure 1A). However, if the ranking order is made identical in both diagnostic groups, there is not only a confirmation of the clinical value in each individual study, but also evidence that the differences in mean (or median) values for Ab1–42 or T-tau between healthy controls and AD are very similar (Figure 1B). It may be that the lack of uniformity regarding an optimal cut-off can partly be attributed to factors not related to the assays, such as differences in the cohorts used in the studies, and to the possibility that AD has a multifactorial pathogenesis [31]. However, the range in variability in optimal cut-off limits, as judged from Figure 1B, closely resembles the range when identical samples are run in different laboratories using the same assay [32], suggest-ing that interlaboratory variability is the major contributor.

When used in combination, the three biomar-kers have high (~90%) diagnostic sensitivity and specificity for AD compared with controls [33]. In addition, the markers can aid in differ-entiating AD from other neurological diseases such as Parkinson’s disease and frontotemporal dementia [30,34,35]. However, there is still room for improvement, and the search for other CSF markers is ongoing [36,37]. The more easily acces-sible blood is an attractive body fluid in which to look for new biomarkers, but so far has yet emerged no candidate that has stood the test of time, even though a large number of analytes have been investigated [38–41].

Biomarker quantificationPrecise and accurate quantification of changes in concentrations of tau and Ab in CSF is impor-tant to better understand the pathology of AD with respect to the disease onset, prognosis and progression rate. A variety of assays on different technology platforms have been commercial-ized during the last decade. In general, standard operating procedures are needed to ensure that the statistical power gained by large numbers of samples is not compromised by preanalytical fac-tors; investigators can confirm results from pilot studies; prognostic value of candidate biomarkers can be more easily assessed; and discovery-based biomarker research is better designed from the onset. A data-driven approach is needed to better

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www.futuremedicine.com 3future science group

Analytical aspects of molecular Alzheimer’s disease biomarkers Review

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understand which component can affect the clin-ical value or qualitative statement made from the individual biomarker concentrations. The impact of several parameters on the functional perform-ance of the assay needs to be documented in the development phase of the assay.

Component analysis of the presently observed total variability is more complex than initially thought. Currently, it is well accepted that accu-rate biomarker quantification in CSF will only be obtained after extensive standardization at the level of the assay, the sample and the laboratory. The performance of an assay is determined by its robustness in function of sample handling and boundary conditions, the availability of run-validation samples, its lot consistency and long-term stability and the selection, characterization, and stability of the most critical raw materials. The components can have an effect in the pre-analytical, analytical or postanalytical (clinical) phase (Figure 2).

Technology platformsTests for molecular markers can be divided into immuno- and nonimmuno-dependent assays. Most of the currently available assay formats are immunodependent and so make use of anti-bodies. Immunoassays have one fundamental drawback in that they measure the analyte in an indirect manner. This aspect makes them more vulnerable towards confounding factors (e.g., cross-reactivity and matrix interferences)

and, as such, are not optimal as reference meth-ods for the evaluation of reference materials. Both reference methods and reference materials are reviewed in another article of this issue of Biomarkers in Medicine (see [4]).

The current paper provides an overview for the currently commercialized CSF biomarker assays of parameters that are considered to be critical for generating reliable results (see also Figure 3). The development of potentially effec-tive disease-modifying drugs makes the need for early and accurate differential diagnosis of dementia more urgent. It is therefore essential that the biomarker-dependent diagnostic proce-dures, including sample processing and testing, are standardized so that diagnostic conclusions can be drawn, wherever the result is coming from. At present, even when using the same assay, considerable variability in absolute con-centrations of AD biomarkers has been found between different centers, leading to discrep-ant use of cut-off values [42,43]. This variability in results may be due to differences in subject selection (patients and control subjects), as well as other analytical or clinical factors (e.g., diag-nostic procedures). The use of different cut-off values between different centers is hampering comparability in multicenter studies. The vari-ability in established cut-off values may also be due to differences in clinical procedures, such as patient selection, CSF collection, pre-analytical (laboratory) procedures, analytical

AssayNeedRegulatory guidances

NeedReference methodsReference materials

NeedLaboratory SOPs

External QC programsProficiency panelsOperator training

Storage and stabilityQuality control (including batch)Production and purificationSelection and characterization

Run validationLot consistency

Stability

Boundary conditionsSample handling

Robustness Raw materials

Product concept

Sample Laboratory

Accuracy

Figure 2. Accuracy of biomarker quantification. QC: Quality control; SOPs: Standard operating procedures.

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procedures (including assay batches used) and interpretation of results among centers.

�n ImmunoassaysELISAs were developed in two laboratories, inde-pendently from each other, in the beginning of the 1970s [44,45]. The use of two antibodies directed against a different epitope of the analyte contrib-utes to specificity of the assay. One antibody is attached to a solid phase (e.g., microtiter plate, beads and membranes), while the other is cou-pled (directly or indirectly) to a reporter system (e.g., enzyme and fluorophore). The commercially available ELISA-type assays on the market for the AD biomarkers are listed in TABle 1. All of these assays can be run in a 96-well format, but there is a demand for special equipment for some assays, beyond a plate reader for reading absorbance, for data acquisition. The assays differ with respect to the format (single analyte or multiplex assay format), how development is carried out (label type, coupled to either the detector antibody or to [strept]avidin), the sample incubation (simul-taneous or sequential with detector antibody, boundary conditions and volume), the calibrators (numbers, ready-to-use vs stock solution and the matrix) and the curve fit algorithm. In addition, only limited information is available with respect to the stability of the individual components and/or total assay. Only a few vendors have docu-mented the analytical and/or clinical qualification of the assay, such as Innotest® P-tau (Thr181) [30].

The xMAP® technology has evolved from flow cytometry and utilizes internally dyed beads as

the solid phase. Multipexing is obtained by using beads with a different ratio of two dyes. The reporter system is a fluorophore (phycoerythrin). Two lasers in the Luminex® instrument provide information on the identity of the bead (linked to the analyte of interest) and the intensity of the phycoerythrin signal. As the xMAP is an open platform, different vendors (e.g., Innogenetics, Invitrogen and Millipore) have developed AD biomarker assays.

Meso Scale Discovery has developed a plat-form that utilizes electrochemiluminescence [46] for detection that requires one of their instru-ments for reading the plate. Even though the platform is capable of multiplexing, no such assays exist at present for all the AD markers.

The newest member to the family of immunoassay platforms for the AD markers is from Perkin Elmer, who has assays for Ab and T-tau. Their assays are built on the AlphaScreen® technology, which utilizes beads both for cap-turing and detection. Upon illumination, one of the bead types releases highly reactive but short-lived singlet oxygen. If during its lifetime the singlet oxygen reaches the other bead, there is a formation of light, which is then the signal used for quantitation [47]. AlphaScreen assays are homogeneous, that is, they do not include washing steps in the test procedure.

Selected reaction monitoringSeveral efforts have been made to validate the quantitative changes of Ab1–42 in CSF using methods other than ELISA [48–51]. All of these

Review parameters

Supplier

Clinical value TechnologySample

Instrumentation Antibodies

TypeEpitope TypeMatrix Supply

CalibratorProviderFlexibility

Raw materials(critical)

Components(buffers)Analytical

Run acceptance

Proficiency panel

Run validationcontrols

Algorithm Calibrator

Concentrationrange

Matrix (buffer, CSF)

Calibration

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IP

Performance Assay

Figure 3. Summary of information that should be publicly available for commercialized assays for cerebrospinal fluid biomarker analysis in Alzheimer’s disease. CSF: Cerebrospinal fluid; IP: Intellectual property.

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Table 1 Commercial immunoassays for Alzheimer’s disease biomarkers.

Company Analyte Catalog number

Antibodies† Solid phase Detection principleCoating Capturing

Type Clone Epitope Type Clone Epitope

Invitrogen Ab1–42T-tauP-tau (Thr181)P-tau (Ser199)P-tau (Thr231)P-tau (Ser396)Ab1–42P-tau (Thr181)T-tau

KHB3544KHB0041KHO0631

KHB7041

KHB7051

KHB7031

LHB3441LHB7051

LHB0041

M‡

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡

1‡

‡

‡

‡

‡

‡

181

‡

MPP

P

‡

P

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡

42‡

181

199

‡

396

‡

‡

‡

Plastic surface

Bead-based

Colorimetric

Fluorometric

Innogenetics Ab1–42P-tau (Thr181)T-tau

Ab1–42P-tau (Thr181)T-tau

8032480317

80323

80584§

MM

M

MM

M

21F12HT7

AT120

4D7A3AT270

AT120

42 (43)159–163

218–224

42181

‡

MM

M

MM

M

3D6AT270

BT2 and HT73D6HT7

HT7

1181

193–198 and 159–1631159–163

159–163

Plastic surface

Bead-based

Colorimetric

Fluorometric

Millipore Ab1–42Ab1–42P-tau (Thr231)T-tau

EZHS42HNDG4-36KHND1MAG-39K§

MMP

M

‡

‡

‡

‡

42‡

‡

‡

MMM

M

‡

‡

‡

‡

4–1017–24231

‡

Plastic surfaceBead-basedBead-based

ColorimetricFluorometricFluorometric

Meso Scale Discovery

Abx-42P-tau (Thr231)T-tau

K150FUEK151DRD

K151DSD

‡

‡

‡

‡

‡

‡

‡

‡

‡

M‡

‡

4G8‡

‡

17–24‡

‡

Carbon surface Electro-chemiluminescence

Covance Ab1–42Ab1–42

SIG-38953SIG-38942

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡

Plastic surfacePlastic surface

ChemiluminescenceColorimetric

Cusabio Ab1–42T-tau

CSB-E10684hCSB-E12011h

M

M

‡

‡

‡

‡

M

P

‡

‡

‡

‡

Plastic surface

Colorimetric

EIAab Ab1–42T-tau

E0946hE1983h

PM

‡

E0838142Mid-tau

PP

‡

‡

1‡

Plastic surface Colorimetric

Life Sciences Advanced Technologies

T-tau¶

Abx-42¶

E01T0024E01A0050

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡


MyBioSource Ab1–42T-tau

MBS703888MBS700382

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡

‡


Perkin Elmer Ab1–42T-tau

AL203AL271

MM

12F4BT2

42199–202

MM

82E1tau 12

1–59–18

Bead-based AlphaScreen® technology

†Questions about the antibody identities were sent to all listed companies. ‡No answer was recieved or the company was not willing to share the information.§Multiplex assay.¶Competitive assay.Ab: b-amyloid; M: Monoclonal antibody; P-tau: Phosphorylated-tau; P: Polyclonal antibody; T-tau: Total tau.

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methods rely on immunoaffinity and thus may have a bias towards specific forms of Ab depend-ing on the characteristics of the anti-Ab antibody used. On the other hand, selected reaction moni-toring (SRM), also called multiple reaction moni-toring, is a mass spectrometric method where the specificity of the assay is produced by different mass filters in sequence instead of using antibod-ies. Quantification is made possible by the use of a stable isotopically labeled internal standard that is added to the sample prior to any treatments that need to be performed before injection into the mass spectrometer. The SRM method is exe-cuted using a triple quadropole mass spectrometer, where two of the quadropoles act as mass filters and one is used for fragmentation. When the sam-ple is injected as a spray into the spectrometer, the liquid is evaporated and the ions enter the first quadropole where only ions with the correct mass (e.g., of the analyte of interest) pass through. Since there might be several substances with the same mass that pass the first quadropole, the ions are fragmented in the second one and another selection is made on the resulting fragments in the third quadropole, after which a detector is located. To reduce the complexity and to increase the specificity, the sample is usually run through a high-performing liquid chromatography column coupled to the inlet of the mass spectrometer. To further minimize the complexity and to concen-trate the sample prior to injection into the col-umn, it is possible to use solid-phase extraction. As long as the internal standard is added at the very beginning of the protocol, possible losses should be equal for the endogenous and exogenous pep-tides, which will allow for the signals in the mass spectrometer for these species to be compared and, therefore, the substance in the sample quan-tified. The described method was used in a recent publication of a SRM method for Ab1–42 where

the sensitivity approached that of an ELISA [52]. Mass spectrometry is less efficient on proteins that usually need to be enzymatically processed prior to analysis. For the different forms of tau, with molecular weights in the range of 37–46 kDa [53], this may pose a problem since information on the six different splicing variants might be lost and the signal is spread out over many peptides that are recognized as only one in immunoassays. In addition, the lower molar concentration of tau compared with Ab42 might be too challenging for the sensitivity of the present state-of-the-art instruments. This is reflected in the fact that it took 3 ml of CSF to be able to identify some of the tau isoforms using immunoprecipitation-coupled mass spectrometry [54].

Assay comparisonAlthough different commercial assays for the AD markers often claim that their assay meas-ures the same protein or peptide, there is still a substantial difference in the concentration range when assays are compared. In particular, the assay range covered by classical ELISA technol-ogy encompasses only 2 log units (see Figure 4). Newer bead-based or carbon-surface combined with fluorescence, electrochemiluminescence or AlphaScreen technology assays have a much broader concentration working range (~4 log units). However, the reported values come from the manufacturers and do not by necessity reflect upper and lower limits of detection. In addition, the lack of traceable certified reference materials makes the absolute calibration of the standard in the kits difficult. The absence of an equivalent concentration for each analyte when tested on a different technology platform is not new in the field. This aspect of the assays has also been documented for other analytes [55]. Diagnostic companies generally commercialize

Table 1. Commercial immunoassays for Alzheimer’s disease biomarkers (cont.).

Company Analyte Catalog number

Antibodies† Solid phase

Detection principleCoating Capturing

Type Clone Epitope Type Clone Epitope

DRG Ab1–42 COL3995 M ‡ 1 P ‡ 42 Plastic surface

Colorimetric

IBL Ab1–42 27711 M 44A3 38–42 M 82E1 1–5 Plastic surface

Colorimetric

WAKO Ab1–42 296-64401 M BAN50 1 M BC05 42 Plastic surface

Colorimetric

†Questions about the antibody identities were sent to all listed companies. ‡No answer was recieved or the company was not willing to share the information.§Multiplex assay.¶Competitive assay.Ab: b-amyloid; M: Monoclonal antibody; P-tau: Phosphorylated-tau; P: Polyclonal antibody; T-tau: Total tau.

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immunoassays on technology platforms that are suitable for regulatory approval and integration into centralized laboratory testing. The overall performance of an assay, which is linked to the intended use of the product, is highly depend-ent on the quality of the individually selected materials, the possibility to establish in-process quality control procedures, the shelf life and its long-term availability. It has been shown to be very difficult to design nonfunctional characteri-zation methods to qualify raw materials so that the outcome of the nonfunctional test can be related directly to the analytical and/or clinical performance of the product.

There are several possible contributors to the deviating results. The assay design for measure-ment of the same analytes on different technol-ogy platforms is not always fully comparable, even when tests are provided by a vendor on more than one technology platform. The vendor does not always provide a protocol to generate

identical output values, as such limiting its worldwide integration [56]. It remains difficult to compare results due the lack of an accepted worldwide reference method or reference materi-als. Differences include the selection and com-bination of the antibodies, their use for coating or detection and binding conditions (time, tem-perature and shaking). In addition, the antibod-ies can be a source of unspecific effects [57], such as there may be differences in cross-reactivity between different pairs of antibodies used.

Monoclonal antibodies have been preferred in the past as they bind to defined epitopes of the molecule, provide more specific and accurate tests, can be produced in unlimited quantities, allow for good manufacturing practice produc-tion and have the intrinsic property of providing better lot-to-lot consistency. With respect to the antibodies, several process parameters can affect the lot consistency, such as the integration in the production process of a new batch of the mono-clonal antibodies, derived through a modified purification process or from another hybridoma cell line (e.g., subclone and serum-free adapta-tion). Batch control for the secondary conjugate is considered of less importance.

The calibrators used in the kits are most likely from different sources and, even if the stated amount is the same, their true concentration, as well as the amount of aggregates present, might differ owing to nonharmonized methods of syn-thesis and concentration determination. This problem can be dealt with if the kit producers have access to a certified reference material that can be used for assigning values to their cali-brator. Highly purified, well-characterized cali-brators are essential to achieving reproducible and robust assay standardization. Calibrations of the assays are achieved using synthetic pep-tides or recombinant proteins. In addition, it is widely recognized that working with Ab1–42

is

especially challenging as it is prone to aggrega-tion. Owing to ethical, practical and technical reasons, calibrators are not prepared in human CSF. Instead, calibrators are typically prepared in a buffer matrix that is different from the CSF test sample, but no genuinely acceptable artificial CSF preparation is currently available. Immunodepleted CSF is impractical on a com-mercial scale because of its lack of availability in sufficient quantities or its inconsistency with respect to the production process.

A difference in composition of the sam-ple diluent and the fluid under investigation, often called the matrix effect, can be a major source of the variation. For example, dilution

Figure 4. Reported assay range for a selected set of commercial assays. Graphical presentation of assay range for (A) Ab1–42 and (B) T-tau making a distinction between ELISA-based assays and other technologies. Commercial assays such as Covance, Life Sciences Advanced technologies, MyBioSource, Cusabio and DRG provided minimal details on their assays. Phosphorylated-tau assays were not included because different calibrators were used and the identity of this calibrator was often not known or described. Ab: b-amyloid; T-tau: Total tau.

1 10 100 1000 10,000 100,000

ELISA-based assaysOther technologies

Invitrogen

Aβ1–42 (pg/ml)

Invitrogen

Innogenetics

Innogenetics

Millipore

Millipore

MesoScale Perkin Elmer

EIAab

IBL

WAKO

1 10 100 1000 10,000 100,000

Invitrogen

T-tau (pg/ml)

Co

mp

any

Co

mp

any

Invitrogen

Innogenetics

Innogenetics

Millipore

Perkin Elmer

EIAab

Discovery

MesoScale Discovery

ELISA-based assaysOther technologies

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of CSF samples with detergent-containing buffer markedly increased the measured con-centration of Ab42 in all assays tested [22]. In addition, the low recovery (<60%) for Ab42 in CSF is a strong indicator of interference [58], which needs to be solved using alternative test procedures. However, better recovery figures were later obtained for all three AD markers [59]. There is also a difference in test instruc-tions (time and temperature), as well as in the amount of CSF or its dilution factor added in each well. The CSF dilution can affect con-centrations in CSF owing to the fact that the analyte might be released from matrix proteins, resulting in higher values.

There may be platform-dependent differences in measured levels of Ab42, T-tau and P-tau in CSF as judged by the fact that factors were needed to harmonize the results from ELISA and xMAP, even though both assays were from the same manufacturer and therefore most likely employed the same sources for the calibrators [60]. These limitations have added to the com-plexity of comparisons between laboratories, research groups and publications, and even within a single research group over a multiyear time period. Statistical data are available to show that it is very difficult to obtain a constant con-version factor, covering the whole assay range for each analyte. Although strictly for research purposes, interconversion of these data might be of interest, but it is not a recommended prac-tice to report, as a final result, concentration data produced by one format after conversion using a factor that relates the two different methodologies.

As with other immunoassay methods, optimal assay performance depends on the level of opera-tor training and experience with the technology. A recent report of an international workshop observed striking differences between perform-ance of operators, even when all operators per-formed the assays in the same laboratory and with the same kits and/or samples [61]. As with any analytical technique, careful attention to the details of these multistep procedures is essential.

The active participation in proficiency test-ing is an essential requirement for assessment of performance over time, and for the AD CSF biomarkers, this can be achieved by participa-tion in the Alzheimer’s Association-sponsored CSF international quality control (QC) pro-gram [32,101]. One could also introduce the use of the ‘method performance chart’ approach for operator qualification. QC samples are essential to evaluate stability over time of assay

calibration. There are currently no widely avail-able CSF QC materials to validate a given run. No run-validation samples are included in most of the assays and, if available, the performance of these samples in the kit is not always identical to the performance using CSF samples. A better approach is the use of a proficiency panel gen-erating on-site information on selectivity, spe-cificity, precision and laboratory differences in the use of recipients. At present, each laboratory must prepare their own suitable QC samples to validate each plate of data. This can be achieved by pooling large volumes of CSF (typically 50–100 ml at a time) and aliquoting for this purpose. Ideally, CSF pools should be prepared to span the dynamic range of the assay(s). Target ranges should be established during the analyti-cal validation process prior to routine analysis of test samples.

There is a need for established guidelines that define different aspects of CSF collection and/or storage when used for AD diagnosis. However, bringing together all these recom-mendations in a single specific guidance backed up by scientific evidence is essential for its use in the field of AD biomarker detection and to ensure future collaboration between different research centers [62].

Despite differences in absolute concentrations in analytes, the relative difference between ana-lyte concentrations in AD CSF between different clinical contrast groups is remarkably consist-ent (Figure 1). Thus, future collaborative efforts comparing the different assays and technologies with respect to analytical aspects will largely benefit the diagnostic performance studies of these biomarkers. Furthermore, this comparison should not be restricted to analytical aspects of the assay, but should also take into consideration preanalytical aspects in the different laboratories using the assay and assay handling [63], so that clinical performance in several studies can be compared over time.

Introduction of a new product on the marketIt is obvious from this overview that the cur-rently available commercial assays for biomar-ker quantification in CSF can be considered as ‘precision-based’ assays, made as fit for pur-pose, missing at least one of the key elements of the performance requirements in order to obtain regulatory approval. One of the limiting factors for pharmaceutical integration is the absence of dilutional linearity for (incurred) samples. This problem could be overcome in

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the future (in part or totally) by the inclusion in the assays of a calibrator series prepared in CSF, the identification and integration of an artificial CSF matrix that is fully compatible with the immunoassay formats and that can be used to dilute the samples or by using a totally new approach (assay format or technology), resulting in an investment cost for its integra-tion in the market. The new format will most probably result in changed concentrations of some of the analytes, which cannot be solved at present by the use of an international refer-ence standard. A new analytical qualification, as well as clinical validation of the new version of the product, will be required in order to generate not only a worldwide acceptance of the product, but also a higher probability of obtaining regulatory approval. There is a need to find a balance between excellent analyti-cal performance and validated clinical utility (Figure 5).

For a new assay concept, it is an advantage if the assay is ‘easy to use’, has enough flex-ibility for integration of novel biomarkers in the near future, generates high-quality output values in a short period of time and can be sup-plied at a relatively low cost for an implementa-tion in the laboratory. The community needs to be supplied with all details related to ‘assay

qualification/validation’, including full details on protocols and/or results. The supplier of the assay must have the willingness to modify the product concept by inclusion of the ‘voice of the customer’. Clinical accuracy of the assay could be qualified, followed by validation, using CSF samples collected under standardized conditions in different consortia.

Performance evaluation must include detailed results on selectivity, specificity, precision (e.g., intrarun, inter-run, intercenter, test–retest and lot-to-lot variability), accuracy (e.g., linearity, parallelism, proportional linearity and recovery) and test robustness, as well as stability data on total kit or kit components. Together with a description of all critical components required to perform the test, it will allow each labora-tory to generate standard operating procedures. The kits should be provided to the customer with well-described kit inserts, containing all the relevant information to perform the test according to specifications defined during the development phase of the product. Analytical performance issues related to the use of the kits are linked to lot variability, matrix inter-ference resulting in increased output signals upon dilution, inaccurate robustness of the test performance(s) and the measurement bias with nonequivalent effects on aqueous-based calibrators and CSF matrix when dilutions are performed.

In addition to the ongoing community and manufacturer work, there are several scientific community-based opportunities to address gaps that are not currently being investigated, such as the urgent need for reference materials and methods, assay acceptance criteria and availabil-ity of artificial CSF for calibrators to establish a nonbiased standard curve.

Future perspectiveAdvancing age is the greatest known risk factor for AD, and since life expectancy is predicted to increase, so will the socioeconomic costs associated with the disease. However, a lot of effort has been put into the task of develop-ing disease-modifying drugs, and when these become available, biomarkers will have an important role to play in the making of early and correct diagnoses in addition to monitor-ing treatment. It will be advantageous if uni-versal cut-off levels for the CSF biomarkers are in place prior to the advent of efficient thera-pies. This task has potential to be solved with the aid of emerging reference methods and materials in combination with QC programs.

Per

form

ance

Excellent

Regulatoryacceptance

GoodRedesign andredevelopment

Analyticalqualification

Clinicalvalidation

Marginal

None Qualified Validated

Clinical utility

Figure 5. The road to regulatory approval of cerebrospinal fluid biomarker assays.

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Financial & competing interests disclosureE Vanmechelen and H Vanderstichele are cofounders of the biotechnology company ADx NeuroSciences. This publication was funded by the Swedish Research Council, and is a part of the BIOMARKAPD project in the EU Joint Neurodegenerative Disease Research programme. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

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Executive summary

Markers reflecting Alzheimer’s pathology

� There are presently three cerebrospinal fluid biomarkers for Alzheimer’s disease – b-amyloid 1–42, total-tau and phosphorylated-tau –which are firmly established to the point that they recently have been included in the research criteria for the disease.

Biomarker quantification

� Several companies provide commercial ELISA-type assays for Alzheimer’s disease biomarkers.

� The lack of gold standards hampers the harmonization of assays from different companies.

Technology platforms

� There are assays on several different technical platforms for the Alzheimer’s disease biomarkers.

Selected reaction monitoring

� Selected reaction monitoring is a strong candidate as a reference method for b-amyloid 1–42.

� Bridging with immunoassays is required.

Assay comparison

� The measured concentration of biomarkers using different assays varies significantly.

Introduction of a new product on the market

� New assays should be ‘easy to use’, flexible, fast, generate high-quality output and be relatively inexpensive.

� Kit manufacturers should aim at making as much information public as possible for the end user to make an informed decision on which assay to choose.

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analytical aspects of molecular alzheimer’s disease biomarkers

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