Proteomics has been largely used in the field of biomarker discovery [30,31] as well as in transfusion medicine [32,33] since proteins are the effective form of genes, and are thus more able to inform about the physiological state. Indeed, contrary to the genome, the proteome composition fluctuates during time, changes among cell population and is physiological state-dependent.

Clinical proteomics is a major asset for diagnosis, prognosis or evolutionary biomarkers. Varieties of improved proteomic technologies allow separation, identification and quantification of proteins, here potential biomarkers. Gel-based technologies (two-dimensional gel electrophoresis, 2DE, and two-dimensional differential gel electrophoresis, 2D-DIGE), as well as chromatographic techniques (e.g., reverse phase liquid chromatography, RPLC, and strong cation exchange liquid chromatography, SCX-LC) have been developed in order to fractionate proteins or peptides from complex samples. Then, proteins can be analyzed by mass spectrometry (MS) consisting in the ionization of proteins or peptides in order to identify them according to their mass-to-charge ratio (m/z). The two commonly used sources of ionization are matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). MALDI source is preferentially combined with time-of-flight (TOF) analyzer, whereas the ESI source is coupled with quadrupole (Q), ion trap (IT), fourier transform ion cyclotron resonance (FTICR), Orbitrap or combinations of these analyzers. Peptide mass fingerprinting (PMF) or de novo sequencing by tandem mass spectrometry (MS/MS or MS²), combined with powerful bioinformatic tools allow protein identification by database interrogation. Protein quantitation [34,35] can be achieved thanks to diverse treatments before or after the sampling step [36] (metabolic stable isotope labeling of amino acids in cell culture, SILAC [37], and chemical addition of isotope-coded affinity tags, ICAT [38], or isobaric tags for relative and absolute quantitation, iTRAQ [39]), by spiking samples before MS analysis with known amount of an isotopically labeled analyte (absolute quantitation of proteins, AQUA [40]) and even by recently developed label-free methods, based on MS or chromatography data-processing (spectral counting [41] or total ion current, TIC, integration [42]).

Surface enhanced lazer desorption/ionization time-of-flight (SELDI-TOF), also known as ProteinChip Array technology, is widely used in biomarker discovery [43,44]. A solid-phase chromatographic surface allows sample decomplexification according to chemical or biochemical properties. Users can design their own chromatographic surface to target specific protein population. Bound proteins are then ionized and analyzed as in classical MALDI-TOF MS analysis. However, SELDI-TOF MS analysis does not lead to protein identification, but only gives a mass list of proteins showing relative abundance differences between samples. Once targeted, these proteins have to be further purified then identified by standard mass spectrometry. Caputo et al. have developed such kind of methods for protein identification on ProteinChip surfaces, i.e., on-chip digestion and MS/MS sequencing by hybrid quadrupole-TOF [45].

Recent advances in all these proteomic techniques have improved reproducibility, but the problem of inter-laboratory reproducibility still subsists. To ensure this reproducibility, a control of the initial state of samples, with standardization of sample collection, storage and preparation conditions, is required. According to Lehmann et al. [46], the following pre-analytical steps have to be controlled: the sampling procedure and the type of container, the temperature and the time before transport (if transport needed), the time between sample collection and sample processing, the process itself (centrifugation, aliquoting, type of secondary container), the storage (temperature and duration) and the pre-treatment just before proteomic analysis.

The general complexity of a proteome, and particularly the huge dynamic range of protein abundance composing the blood proteome, makes necessary the fractionation of blood samples before proteomic analysis, by depletion or fractionation processes. Then, other sample preparation can be required depending on the type of analysis to be performed, and cannot be standardized to all biomarker discovery studies.

Blood donation conditions such as the donor position, the time of tourniquet application, and the needle bore size are important. In upright position, blood macromolecular component rates can be increased, due to a shift in body water from intravascular to interstitial compartment (macromolecules cannot pass through blood vessels and are thus concentrated) [70]. The same effect can be noticed if the tourniquet application exceeds one minute. Indeed, proteins and macromolecules can thus accumulate upstream the tourniquet and be detected at a higher rate than normal by this effect of concentration. Moreover, Cengiz et al. have shown in a recent study that erythrocyte deformability is still altered up to 180 seconds after tourniquet removal, and erythrocyte aggregation is increased up to 30 seconds after tourniquet removal [71]. They also confirmed that tourniquet application induces leukocytes activation.

Concerning the needle bore size, too thin needle can induce hemolysis, thus free hemoglobin and other protein concentrations increase in serum/plasma. It is not problematic when working with blood products obtained from whole blood donation because in case of large blood drawn volume, largest needles are used (16 to 19-gauge needles). In routine venipuncture, smaller blood volumes are drawn and patient comfort is of high importance, thus thinner needles are chosen (21 to 23-gauge needles). However, too thin needles (25-gauge needles) should be avoided since it can induce pre-analytical variability (hemolysis), leading to false interpretation of test results [72,73].

All containers have to be tested for a possible interference with analyses to be performed. Indeed, containers can salt out plastic components, which can be detected in analysis by mass spectrometry. The presence of polymers in an MS analysis induces effect of signal suppression: the high amount of ionized polymer saturates the detector, and the other sample minority components ions cannot be detected anymore. In 2004, Drake et al. have demonstrated the presence of polymers on mass spectra from extracts of saline solution incubated in different blood sample containers, from two different manufacturers [74].

Investigating serum/plasma proteomes, Hsieh et al. have shown that there was no significant difference between samples collected in glass red-top tubes and samples collected in serum separator tiger-top tubes [75]. However, comparing the nature of the anticoagulant used, they have found high significant differences in plasma protein profiles. Most common anticoagulants are EDTA, heparin and sodium citrate. Their different action ways lead to these differences in proteome composition. Moreover, they are also known to interfere with subsequent analysis. EDTA is known to interfere in enzymatic activity assays, whereas heparin is not advised for some ELISA-based assay kit.

First of all, the time between blood donation and preparation of the labile products is of high importance. Banks et al. have shown in 2005 that this duration induces differences in SELDI-TOF profiles of plasma collected with different anticoagulants and serum samples [76].

A study of West-Nielson et al., based on principal component analysis (PCA) of MALDI-TOF spectra, reveals that there was no significant difference between spectra of serum from blood left to clot up to 24 h at 4 °C and from blood left to clot up to 4 h at 24 °C [77]. However, when blood is left to clot for longer times at 24 °C (8 h to 24 h), clearly changes appear in mass spectra, which was confirmed later [75]. In 2007, Timms et al. have shown that both transport time and temperature play a role on SELDI-TOF spectra profiles of sera [78].

Studying RBC microparticles, Rubin et al. have encountered some pre-analytical caveats. Indeed, our group has shown that sample processing temperature and vortexing duration affect microparticles counting [79]. Dealing with centrifugation force (1500 or 3000 × g) and duration (15 vs. 30 minutes), serum proteome profiles do not appear to be affected [75].

Protein degradations, such as fragmentation by proteolysis, and protein modification, such as oxidation, have been described as storage lesions (see part 2). However, these alterations can be induced during sample manipulation, and cannot be differentiated from storage lesions. The addition of protease inhibitors to prevent handling-related proteolysis is thus of high importance. If protease inhibitors are omitted, it has been shown that low molecular weight compounds appear and accumulate during sample preparation [80]. It is necessary to prevent proteolytic cleavages because generated peptides could thus be mistaken for potential biomarker. Indeed, analyzing peptidome variations of pathological sera, Davis et al. have shown that modifications in low molecular weight compounds reveal disease-induced hemostatic dysregulation rather than direct protease activity of a given pathology [81]. Indirectly pathologically-induced peptidome variations are not specific enough to be considered as good biomarkers and have thus to be avoided by use of protease inhibitors, even if it is not known whether all protease activities are repressed.

RBCs possess a set of enzymes responsible for antioxidant defense (catalase, superoxide dismutase, peroxiredoxins and glutathione peroxidase). However, these enzymes can be inactivated by excess of reactive oxygen species (ROS), and additional protein oxidation can thus occur during sample processing. In order to prevent this phenomenon, particularly prior to cystein oxidation assessment in stored RBC redox proteomic studies, cell-permeable cystein-specific reagents, such as iodoacetamide (IAA) or N-ethylmaleimide (NEM) alkylating agents, are commonly used [82,83].

Storage conditions are important to preserve quality of labile blood products and to ensure the efficiency of transfusion therapy. Thus, it is known that erythrocyte concentrates are preferentially stored at 4 °C whereas platelet concentrates are stored at 22 °C to prevent cold activation of platelets. Finally, fresh frozen plasma is stored at −25 °C at least.

Thus, whole blood storage cannot ensure optimal preservation of each blood components. In labile blood products aging field, samples are ECs, PCs or FFPs, and are stored according to blood banking legislation. In pathological-case biomarker research, samples are either serum or plasma, and are not subject to any storage legislation. Several studies aimed at investigating the reproducibility of analysis after storage at −20/−30 °C or −80 °C, and after freeze-thaw cycles [75,84,85]. It appears that storage at −20 °C instead of the recommended −80 °C storage does not induce loss of information. Samples stored at −20/−30 °C can thus be used for biomarker discovery [86].

Some proteins have been shown to be highly temperature-sensitive: cryoproteins. Described for the first time in the 1930’s by Wintrobe [87] as myeloma’s patient sera precipitating at cold temperature, cryoproteins are serum or plasma proteins having the property to precipitate below the physiological temperature. Most of the time composed of immunoglobulins (mainly IgM) these precipitates are called cryoglobulins (CG), and are classified under three categories according to the universally adopted classification of Brouet [88]. Other proteins have this particularity to precipitate at low temperature, as cryofibrinogen (CF) (for instance, see the study on clinical cases of patients with either CF alone or combined CF plus CG [89]) and cold agglutinins. Proteomic techniques are well adapted to determine the protein composition of cryoprecipitates [90]. Cryoproteins are potential biomarkers, and pre-analytical precautions have to be taken for their investigation. Indeed, blood must be sampled in anticoagulant-free, 37 °C pre-heated container, handled and centrifuged at 37 °C, in order to prevent cryoprotein elimination by precipitation before analysis.