**4. Conclusion**

the high variability of the results of biomarkers measured by different commercial kits can be explained, by the use of different antibodies, the nature of the calibrator, the calibration method and many others factors as for example the nature of standard. Increasing data during years and by incorporation of new centers (since this first report concerning 40 laboratories, in summer of 2012, 64 laboratories were participating at this program) will permit to better identify the major sources of variability in analytical steps. Thus, we can just list the different

The pipetting mode (inverse pipetting...) is not specified by the manufacturer. Using a single tip can influence the standard curve accuracy. However, the magnitude of this effect, if any,

For lyophilised standard, accurate solubilization and accurate pipetting is critical. Moreover, since for INNOTEST Ab42, the first point of the curve calibration must be adjusted depending of the set value, accurate pipeting is absolutely needed. The type of curve fitting used and the

**c.** Reagent handling and adhesion of biologists to the manufacturer standard operating

The adhesion of routine laboratories to the manufacturer SOP is absolutely needed to reduce the part of the variability found in CSF biomarkers analysis. For that, a great effort must be done by the different manufacturers to limit individual interpretation of the technical instruc‐ tions. The best example consists in the definition of the «room temperature» which can mainly vary from the north to the south of Europe. The maintenance of laboratory equipment is a crucial point to ensure the accuracy of pipeting volumes, the accuracy of temperatures, the

Implementing these techniques in the laboratory needs a training program ensured by the manufacturer. Moreover, habilitation and qualification of the laboratory staff must be done.

Different means are used to ensure validation of results. The definition of the criteria of acceptance of results must be strict. They include the calibration curve parameters, the CV of the duplicate samples and the use of an internal quality control program. For the CV criteria acceptance, in our experience, it seems that they are to be adequately defined since, the recommendation of CV < 20% done in the INNOTEST documentation, is not acceptable all along the dynamical range of the assay, in particular when the concentration level is near the clinical cut-off. Moreover, in the absence of QC samples in the kit, the biologist needs to implement its own QC program with different crucial points to resolve: the nature of the

software for data calculation were shown as possible factors of variability [43].

accuracy of detection signals and the quality and reproducibility of washing steps.

**d.** Familiarization with the method and Competency Train

**e.** Validation criteria of runs for rejecting data

points to be further investigated.

186 Understanding Alzheimer's Disease

**a.** Pipetting

**b.** Calibration

procedure (SOP)

**In the laboratory, the biologist will take care for:**

should be tested, to provide a better basis for recommendation [43].

The present chapter highlights two main issues responsible for the lack of harmonization of CSF AD biomarkers cut-offs values: the lack of standardization of the pre-analytical steps and the high variability of results linked to the analytical step. This latter issue can be explained by the absence of transferability of results between the different platforms but also by the high inter laboratory dispersion within the same assays. Previous consensus guidelines for preanalytical factor standardization gave the way to resolve this issue, evidencing the need to standardize sampling and storage tubes, the type of the needle for the CSF puncture and the long term storage. Establishing SOPs for sample processing would allow to compare diagnostic conclusions between different laboratories. The implementation of those SOPs in the clinical community may reduce part of the variability found in the analysis of AD CSF biomarkers. Antibody purification, coating surfaces, preparation of standards, manufacturers instructions are also sources of variation, which need to be decreased and requires increased efforts by kit manufacturers. The optimal approach is a collaborative effort between commercial kit and instrument platform manufacturers, laboratories concerned by those methods, and reference standardization programs.
