Importance of the Pre-analytical Procedure and Standardisation of Methods
The most important issues are the adoption of standardized protocols during sample collection and other pre-analytical processes, method validation including establishing prospective quality control protocols. Up to now, the determination for reliable biomarkers for AD in peripheral blood is very challenging due to difficulties of the standardization of the methods of analysis. To validate plasma or serum biomarkers, assays should be run using validated and standardized protocols because the determination of all oxidative stress biomarkers requires a rigorous pre-analytical procedure in order to avoid artefacts. The blood sample must be drawn very carefully in order to avoid haemolysis which can interfere in some assay (e.g. determination of oxidized LDL). Once drawn, the blood has to be immediately centrifuged at 3000 rpm during 15 min if possible at 4 °C. Indeed, the concentration of some antioxidants such as vitamin C, p-carotene and reduced glutathione may rapidly decrease within 30 min if the whole blood is kept at room temperature before centrifugation. For both vitamin C, isoprostanes and oxidized glutathione, it is also necessary to mix the plasma respectively with metaphosphoric acid, butylhydroxy- toluene (BHT) and N-ethylmaleimide in order to prevent spontaneous oxidation of the markers in the tube. At least, plasma or serum must be immediately frozen on dry ice and then kept at -80 °C until analysis which must be performed as soon as possible. Lack of standardization of procedures for blood collection, processing, and storage (time in storage, number of subsequent freeze-thaw cycles) leads to preanalytical variability (incubation times before separating plasma (or serum) from cells, type of blood collection tubes, temperature for collection and storage) causing inconsistencies in downstream analysis and results [140, 141].
In 2014, the international working group of the Standards for Alzheimer’s Research in Blood biomarkers (STAR-B) and Blood-Based Biomarker Interest Group (BBBIG) have published the first set of guidelines in order to standardize preanalytical variables for blood-based biomarker studies [142, 143]. The principle of the guidelines for preanalytical methods for blood-based AD biomarker by BBBIG/STAR-B working group follows the regulatory good laboratory practice as defined by Clinical Laboratory Improvement Amendment in United States or international standards of Clinical Laboratory Standards Initiative. For the progress of blood-based biomarker studies in AD, the adoption of guidelines to standardize preanalytical methods across cohorts and laboratories is required. Therefore, the BBBIG/STAR-B guidelines are a good starting point toward standardized methods that will be essential to move putative blood-based biomarkers forward in future studies.
The downstream analytical methods should be validated on the same matrix (e.g., serum, plasma, urine...). Analytical validation should include calibration curves, intra- and interprecision and accuracy. Serum and plasma contain high amounts of albumin and immunoglobulins, successful protein biomarker discovery requires enrichment techniques for low abundance proteins and sensitive technological platforms to facilitate detection and quantification of protein biomarkers. Sample size for controls and AD groups should be large enough. The identification of new biomarkers should be validated, replicated and compared with existing gold standards and validated in larger studies and validated in different groups. Demographic information (age, gender, education.), APOE status should be included. Further complications in the interpretation of plasma profiles then arise from the use of different types of medication for patients with AD. For instance, a study with limited number of patients (10 controls and 21 AD) showed that the medication including Rivastigmine and Donepezil-Memantin could modify the pattern of oxidative markers . Patients with AD have impaired systemic availability of several nutrients which could impact on peripheral antioxidant levels. Finally, the potential issue is the variability in results depending on the statistical or computational methods used to identify these biomarkers.