**Pre-Analytical and Analytical Critical Factors Influencing the High Variability of the Concentrations Levels of Alzheimer Disease Biomarkers in Cerebral Spinal Fluid**

Armand Perret-Liaudet, Aline Dorey, Yannick Tholance, Benoit Dumont and Isabelle Quadrio

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55512

**1. Introduction**

Alzheimer's disease (AD) is a fatal neurodegenerative disorder characterized by a progressive neuronal death and loss of cognitive functions. AD is the most common type of dementia and its incidence rise to 10% in people aged over 90 [1]. Due to Increased longevity, it has been estimated that the number of people suffering from this neurodegenerative disorder will rise from 26.6 million cases in 2006 to 106,8 million worldwide in 2050 [2].

Although clinical intervention to halt the disease is inefficient, the clinical and psychological cares are likely known to significantly improve the quality of life of the patient but also those of the family. At the prodromal stage of the disease (Mild cognitive Impairment linked to AD), there are no sufficient evidences that treating the patient improves the patient outcome. This lack of evidence poses in some cases an ethical problem that is to announce the diagnosis of AD at an autonomous patient who will shift irreversibly in the coming years to the dementia stages. However, as reported in new criteria established by the National Institute on Aging (NIA) and the Alzheimer's Association, core clinical criteria could be used by healthcare providers without access to advanced imaging techniques or cerebrospinal fluid analysis. Criteria including these last advanced tools still remain in the research field [3]. On the contrary, the diagnosis is highly aimed to be accurate at least at the clinical stage of mild dementia, to detect the AD pathology. Core clinical criteria seems to be enough to ensure the AD diagnosis and the use of biomarkers (imaging or CSF biomarkers) can only increase the certainty that the basis of the clinical dementia syndrome is the AD pathophysiological process

© 2013 Perret-Liaudet et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

in a patient presenting the core clinical diagnosis [4]. The CSF biomarker panel of AD is a picture of the neurodegeneration, the neuronal loss, the tangle formation and Aβ-amyloid<sup>42</sup> (Aβ42) peptide accumulation in the brain. Indeed, the core CSF biomarkers for AD diagnosis are a decrease of Aβ42 levels and more recently a decrease of the ratio of Aβ-amyloid42 / Aβamyloid40 (Aβ42/Aβ40) which reflect senile plaques pathology as well as an increase of total tau (T-tau) and phosphorylated tau (P-tau) which reflect axonal degeneration [5,6]. The use of AD biomarker tests for routine diagnostic purposes at the present time, is only proposed as optional for use in patients with dementia when deemed appropriate by the clinician. From the several reasons for this limitation, the workgroup with the task of revising the 1984 criteria for Alzheimer's disease (AD) dementia, highlighted the limited standardization of biomarkers from one locale to another [4]. Despite a decrease in the number of side effects associated with the puncture, lumbar puncture remains an invasive procedure that is clearly the main factor preventing the wide dissemination of these biomarkers in the routine. However, we cannot ignore that the significant variability in measured biomarkers levels found in various studies, resulting in a high variability of both the diagnostic accuracy [7] and of the clinical cut-off for the diagnostic of AD [8], is a hindrance to the spread of these markers and their integration in the diagnostic criteria [3]. The cut-offs obtained in Europe for CSF total tau and beta-amyloid measured by the ELISA assays from the same manufacturer, were reported highly diverse, with two to three fold differences between the highest and lowest reported values [8]. Three major explanations are proposed in this report: first, the inter-laboratory comparisons are very difficult, as some laboratories have adopted the cut-off values from the research literature whereas others have established their own controls, these last controls being likely different in neuropsychology evaluation, neuroimaging and the follow-up. Secondly, the lack of standardized material between the different assays but also the lack of standardized protocols, seem to be a major source of this variation. Finally, pre-analytical factors are those factors that contribute to the variation of the laboratory results before the analysis of the sample. One consensus report has already established the main pre-analytical factors that should be standardized for CSF AD biomarkers analysis [9]. However, the importance of some preanalytical confounding factors highlighted in this report remained to be elucidated. The aim of this report is to discuss and focus on main critical points in the different preanalytical steps likely to be responsible of data variability. For analytical steps, the introduction since 2009 of an external quality control at a large scale gave an overview of the «desaster», in the same line that prior results. We will discuss rapidly the prior results reported in 2011 and we will underline the urgent need for standardization.

vivo» factors are those biological factors that are linked directly to the patient, the «in vitro»

Pre-Analytical and Analytical Critical Factors Influencing the High Variability of the Concentrations Levels of...

http://dx.doi.org/10.5772/55512

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Answering this issue needs to know if a nycthemeral cycle exists that could modify the concentrations levels of AD CSF biomarkers during the day. Although a lack of standardiza‐ tion in the diagnostic strategy of the patient still exists, in most cases, after a first examination including a clinical and a neuropsychological evaluation, if needed, the lumbar puncture is generally scheduled in a second visit with morphological brain imaging in the same time, with the aim to minimize the duration of the hospitalization. As the time of the lumbar puncture is highly dependent of the coordinated organization of the clinical memory centre, of the biological laboratory and of the imaging department associated with it (waiting homeostasis

Previous results have suggested the existence of a large diurnal variability in Aβ levels during a time period of 36 hours, but without significant differences between the hours all along the day period [13]. Following these amazing and unexplained data, recent studies were unable to demonstrate the existence of a temporal fluctuation in CSF biomarker levels, not only for Aβ, but also for T-tau and P-tau [10, 14, 15]. Therefore, there is no need to standardize a specific time interval during the day for CSF collection dedicated to the AD biomarkers assays.

At our knowledge, there are no study that has analyzed the influence of fasting on AD CSF biomarkers. The comparison of patients with and without fasting would give a set of indirect and biased data without clear conclusion. Moreover, for ethical reasons, it seems to be impossible to start a research study focused on this topic, as this study would imply a protocol with the realization of successive lumbar punctures in a short delay. Therefore, it is not possible to answer scientifically this issue. Nevertheless, it has been shown that, independently of the patient food intake, Aβ levels in plasma are very stable [10]. As there is a lack of data concerning this topic, as those kind of data could probably never be obtained, and taking account of the large diversity in the locale organization, it is not logical to recommend fasting for the analysis

Due to the possible decreased rostro-caudal concentration gradient, the site of CSF withdrawal must be also standardized. At our knowledge, there is no study reporting any difference between AD biomarkers concentrations obtained by a ventricular puncture and those obtained by lumbar puncture. Therefore, it is not recommended to analyse these markers in the

factors are linked to the procedure of sample handling and processing.

*2.1.1. Is there a specific time of day needed to collect the CSF?*

results, scheduling imaging...), this question is highly relevant.

*2.1.2. Is fasting able to modify the concentrations levels of AD biomarkers ?*

**2.1.** *In vivo* **factors**

of AD biomarkers in CSF.

*2.2.1. Localization of the puncture*

**2.2.** *In vitro* **factors**
