Redox Proteomics and Platelet Activation: Understanding the Redox Proteome to Improve Platelet Quality for Transfusion.

Blood banks use pathogen inactivation (PI) technologies to increase the safety of platelet concentrates (PCs). The characteristics of PI-treated PCs slightly differ from those of untreated PCs, but the underlying reasons are not well understood. One possible cause is the generation of oxidative stress during the PI process. This is of great interest since reactive oxygen species (ROS) act as second messengers in platelet functions. Furthermore, there are links between protein oxidation and phosphorylation, another mechanism that is critical for cell regulation. Current research efforts focus on understanding the underlying mechanisms and identifying new target proteins. Proteomics technologies represent powerful tools for investigating signaling pathways involving ROS and post-translational modifications such as phosphorylation, while quantitative techniques enable the comparison of the platelet resting state versus the stimulated state. In particular, redox cysteine is a key player in platelet activation upon stimulation by different agonists. This review highlights the experiments that have provided insights into the roles of ROS in platelet function and the implications for platelet transfusion, and potentially in diseases such as inflammation and platelet hyperactivity. The review also describes the implication of redox mechanism in platelet storage considerations.

Antioxidant power as a quality control marker for completeness of amotosalen and ultraviolet A photochemical treatments in platelet concentrates and plasma units.

BACKGROUND: Pathogen inactivation treatments such as INTERCEPT aim to make sure blood and blood-derived products are free of pathogens before using them for transfusion purposes. At present, there is no established quality control assay that assesses the completeness of the treatment. As INTERCEPT is a photochemical treatment known to generate reactive oxygen species we sought to use the antioxidant power (AOP) of the blood product as a marker of treatment execution. In this perspective, we evaluated an electrochemically based miniaturized system, the EDEL technology, for measuring the AOP in both platelet concentrates (PCs) and plasma. STUDY DESIGN AND METHODS: Aliquots were withdrawn from PCs or plasma units before and after INTERCEPT treatment and a few microliters were directly deposited into the EDEL sensor for the AOP measurement. The result is expressed in EDEL, an arbitrary unit (micromolar equivalent of ascorbic acid). RESULTS: The INTERCEPT treatment resulted in a significant decrease of the AOP. An AOP threshold of 66.5, 89.0, 59.8, and 131.5 EDEL was determined for apheresis PCs collected from female and male donors, buffy coat PCs, and plasma units, respectively. Below the threshold value, INTERCEPT treatment is considered to be executed. Additionally, we showed that the presence of the photosensitizer in combination with the ultraviolet A illumination is required to observe the AOP decrease. CONCLUSION: The measurement of the AOP of PCs and plasma units can be used to document the completeness of the INTERCEPT treatment.

Urinary di-(2-ethylhexyl) phthalate metabolites for detecting transfusion of autologous blood stored in plasticizer-free bags.

BACKGROUND: Autologous blood transfusion (ABT) efficiently increases sport performance and is the most challenging doping method to detect. Current methods for detecting this practice center on the plasticizer di(2-ethlyhexyl) phthalate (DEHP), which enters the stored blood from blood bags. Quantification of this plasticizer and its metabolites in urine can detect the transfusion of autologous blood stored in these bags. However, DEHP-free blood bags are available on the market, including n-butyryl-tri-(n-hexyl)-citrate (BTHC) blood bags. Athletes may shift to using such bags to avoid the detection of urinary DEHP metabolites. STUDY DESIGN AND METHODS: A clinical randomized double-blinded two-phase study was conducted of healthy male volunteers who underwent ABT using DEHP-containing or BTHC blood bags. All subjects received a saline injection for the control phase and a blood donation followed by ABT 36 days later. Kinetic excretion of five urinary DEHP metabolites was quantified with liquid chromatography coupled with tandem mass spectrometry. RESULTS: Surprisingly, considerable levels of urinary DEHP metabolites were observed up to 1 day after blood transfusion with BTHC blood bags. The long-term metabolites mono-(2-ethyl-5-carboxypentyl) phthalate and mono-(2-carboxymethylhexyl) phthalate were the most sensitive biomarkers to detect ABT with BTHC blood bags. Levels of DEHP were high in BTHC bags (6.6%), the tubing in the transfusion kit (25.2%), and the white blood cell filter (22.3%). CONCLUSIONS: The BTHC bag contained DEHP, despite being labeled DEHP-free. Urinary DEHP metabolite measurement is a cost-effective way to detect ABT in the antidoping field even when BTHC bags are used for blood storage.

Hepcidin as a new biomarker for detecting autologous blood transfusion.

Autologous blood transfusion (ABT) is an efficient way to increase sport performance. It is also the most challenging doping method to detect. At present, individual follow-up of haematological variables via the athlete biological passport (ABP) is used to detect it. Quantification of a novel hepatic peptide called hepcidin may be a new alternative to detect ABT. In this prospective clinical trial, healthy subjects received a saline injection for the control phase, after which they donated blood that was stored and then transfused 36 days later. The impact of ABT on hepcidin as well as haematological parameters, iron metabolism, and inflammation markers was investigated. Blood transfusion had a particularly marked effect on hepcidin concentrations compared to the other biomarkers, which included haematological variables. Hepcidin concentrations increased significantly: 12 hr and 1 day after blood reinfusion, these concentrations rose by seven- and fourfold, respectively. No significant change was observed in the control phase. Hepcidin quantification is a cost-effective strategy that could be used in an "ironomics" strategy to improve the detection of ABT.

In vitro study of platelet function confirms the contribution of the ultraviolet B (UVB) radiation in the lesions observed in riboflavin/UVB-treated platelet concentrates.

BACKGROUND: Platelet inactivation technologies (PITs) have been shown to increase platelet storage lesions (PSLs). This study investigates amotosalen/ultraviolet (UV)A- and riboflavin/UVB-induced platelet (PLT) lesions in vitro. Particular attention is given to the effect of UVB alone on PLTs. STUDY DESIGN AND METHODS: Buffy coat-derived PLT concentrates (PCs) were treated with amotosalen/UVA, riboflavin/UVB, or UVB alone and compared to untreated PCs throughout storage. In vitro PLT function was assessed by blood gas and metabolite analyses, flow cytometry-based assays (CD62P, JC-1, annexin V, PAC-1), hypotonic shock response, and static adhesion to fibrinogen-coated wells. RESULTS: In our experimental conditions, riboflavin/UVB-treated PCs showed the most pronounced differences compared to untreated and amotosalen/UVA-treated PCs. The riboflavin/UVB treatment led to a significant increase of anaerobic glycolysis rate despite functional mitochondria, a significant increase of CD62P on Day 2, and a decrease of JC-1 aggregates and increase of annexin V on Day 7. The expression of active GPIIbIIIa (PAC-1) and the adhesion to fibrinogen was significantly increased from Day 2 of storage in riboflavin/UVB-treated PCs. Importantly, we showed that these lesions were caused by the UVB radiation alone, independently of the presence of riboflavin. CONCLUSION: The amotosalen/UVA-treated PCs confirmed previously published results with a slight increase of PSLs compared to untreated PCs. Riboflavin/UVB-treated PCs present significant in vitro PSLs compared to untreated PCs. These lesions are caused by the UVB radiation alone and probably involve the generation of reactive oxygen species. The impact of these observations on clinical use must be investigated.

Molecular screening of cancer-derived exosomes by surface plasmon resonance spectroscopy.

We report on a generic method to detect and identify the molecular profile of exosomes either derived from cultured cell lines or isolated from biofluids. Exosomes are nanovesicles shed by cells into their microenvironment and carry the molecular identity of their mother cells. These vesicles are actively involved in intercellular communication under physiological conditions and ultimately in the spread of various diseases such as cancer. As they are accessible in most biofluids (e.g., blood, urine, or saliva), these biological entities are promising tools for cancer diagnostics, offering a non-invasive and remote access to the molecular state of the disease. The composition of exosomes derived from cancer cells depends on the sort and state of the tumor, requiring a screening of multiple antigens to fully characterize the disease. Here, we exploited the capacity of surface plasmon resonance biosensing to detect simultaneously multiple exosomal and cancer biomarkers on exosomes derived from breast cancer cells. We developed an immunosensor surface which provides efficient and specific capture of exosomes, together with their identification through their distinct molecular profiles. The successful analysis of blood samples demonstrated the suitability of our bioanalytical procedure for clinical use.

In vitro assays and clinical trials in red blood cell aging: Lost in translation.

The age of erythrocyte concentrates (EC) in transfusion medicine and the adverse outcomes when transfusing long-term-stored EC are highly controversial issues. Whereas the definition of a short-term-stored EC or a long-term-stored EC is unclear in clinical trials, data based on in vitro storage assays can help defining a limit in addition of the expiration date. The present review merges together these data in order to highlight an EC age cut-off and points out potential misleading consideration. The analysis of in vitro data highlights the presence of reversible and irreversible storage lesions and demonstrates that red blood cells (RBC) exhibit two limits during storage: one around 2 weeks and another one around 4 weeks of storage. Of particular importance, the first lesions to appear, i.e. the reversible ones, are per se reversible once transfused, whereas the irreversible lesions are not. In clinical trials, the EC age cut-off for short-term storage is in general fewer than 14 days (11 ± 4 days) and more disperse for long-term-stored EC (17 ± 13 days), regardless the clinical outcomes. Taking together, EC age cut-off in clinical trials does not totally fall into line of in vitro aging data, whereas it is the key criteria in clinical studies. Long-term-stored EC considered in clinical trials are not probably old enough to answer the question: "Does transfusion of long-term-stored EC (older than 4 weeks) result in worse clinical outcomes?" Depending on ethical concerns and clinical practices, older EC than currently assayed in clinical trials should have to be considered. These two worlds trying to understand the aging of erythrocytes and the impact on patients do not seem to speak the same language.

Differences between calcium-stimulated and storage-induced erythrocyte-derived microvesicles.

Microvesicles (MVs), or microparticles, are a complex, dynamic and functional part of cells. Red blood cell (RBC)-derived MVs are naturally produced in vivo (during normal aging processes or in several diseases) as well as ex vivo during cold storage of RBCs, or in vitro by ATP depletion or treatment with Ca(2+) and calcium ionophore. All these MVs are equivalently classified according to their size and/or surface markers. Nevertheless, their content in proteins can differ and a few differences in terms of lipid raft proteins, notably stomatin and flotillin-2, have been reported. Based on two-dimensional gel electrophoreses, the present study highlights the differences between MVs induced during storage of RBCs (storage-MVs) and MVs stimulated by Ca(2+) entry (Ca-MVs). Upon treatment, Ca-MVs are formed following a clear recruitment of Ca(2+)-binding proteins (sorcin, grancalcin, PDCD6) and particularly annexins (4 and 5). Therefore, it emerges that different molecular pathways are available to produce similar MVs by disturbing the membrane/cytoskeleton interactions. Interestingly, these differences provide non-negligible pieces of information on the parent cells, and the mechanisms and modes of actions involved in the formation of MVs. In addition to biophysical characterization, protein analysis is important to classify these cellular corpuscles and evaluate their potential impacts in diseases or transfusion medicine.

α-Synuclein in central nervous system and from erythrocytes, mammalian cells, and Escherichia coli exists predominantly as disordered monomer.

Since the discovery and isolation of α-synuclein (α-syn) from human brains, it has been widely accepted that it exists as an intrinsically disordered monomeric protein. Two recent studies suggested that α-syn produced in Escherichia coli or isolated from mammalian cells and red blood cells exists predominantly as a tetramer that is rich in α-helical structure (Bartels, T., Choi, J. G., and Selkoe, D. J. (2011) Nature 477, 107-110; Wang, W., Perovic, I., Chittuluru, J., Kaganovich, A., Nguyen, L. T. T., Liao, J., Auclair, J. R., Johnson, D., Landeru, A., Simorellis, A. K., Ju, S., Cookson, M. R., Asturias, F. J., Agar, J. N., Webb, B. N., Kang, C., Ringe, D., Petsko, G. A., Pochapsky, T. C., and Hoang, Q. Q. (2011) Proc. Natl. Acad. Sci. 108, 17797-17802). However, it remains unknown whether or not this putative tetramer is the main physiological form of α-syn in the brain. In this study, we investigated the oligomeric state of α-syn in mouse, rat, and human brains. To assess the conformational and oligomeric state of native α-syn in complex mixtures, we generated α-syn standards of known quaternary structure and conformational properties and compared the behavior of endogenously expressed α-syn to these standards using native and denaturing gel electrophoresis techniques, size-exclusion chromatography, and an oligomer-specific ELISA. Our findings demonstrate that both human and rodent α-syn expressed in the central nervous system exist predominantly as an unfolded monomer. Similar results were observed when human α-syn was expressed in mouse and rat brains as well as mammalian cell lines (HEK293, HeLa, and SH-SY5Y). Furthermore, we show that α-syn expressed in E. coli and purified under denaturing or nondenaturing conditions, whether as a free protein or as a fusion construct with GST, is monomeric and adopts a disordered conformation after GST removal. These results do not rule out the possibility that α-syn becomes structured upon interaction with other proteins and/or biological membranes.

Red blood cell-derived microparticles isolated from blood units initiate and propagate thrombin generation.

BACKGROUND: Red blood cell-derived microparticles (RMPs) are small phospholipid vesicles shed from RBCs in blood units, where they accumulate during storage. Because microparticles are bioactive, it could be suggested that RMPs are mediators of posttransfusion complications or, on the contrary, constitute a potential hemostatic agent. STUDY DESIGN AND METHODS: This study was performed to establish the impact on coagulation of RMPs isolated from blood units. Using calibrated automated thrombography, we investigated whether RMPs affect thrombin generation (TG) in plasma. RESULTS: We found that RMPs were not only able to increase TG in plasma in the presence of a low exogenous tissue factor (TF) concentration, but also to initiate TG in plasma in absence of exogenous TF. TG induced by RMPs in the absence of exogenous TF was neither affected by the presence of blocking anti-TF nor by the absence of Factor (F)VII. It was significantly reduced in plasma deficient in FVIII or F IX and abolished in FII-, FV-, FX-, or FXI-deficient plasma. TG was also totally abolished when anti-XI 01A6 was added in the sample. Finally, neither Western blotting, flow cytometry, nor immunogold labeling allowed the detection of traces of TF antigen. In addition, RMPs did not comprise polyphosphate, an important modulator of coagulation. CONCLUSIONS: Taken together, our data show that RMPs have FXI-dependent procoagulant properties and are able to initiate and propagate TG. The anionic surface of RMPs might be the site of FXI-mediated TG amplification and intrinsic tenase and prothrombinase complex assembly.

Red blood cell microparticles: clinical relevance.

SUMMARY: Microparticles are small phospholipid vesicles of less than 1 µm released into the blood flow by various types of cells such as endothelial, platelet, white or red blood cells. They are involved in many biological and physiological processes including hemostasis. In addition, an elevated number of microparticles in the blood is observed in various pathological situations. In the context of transfusion, erythrocyte-derived microparticles are found in red blood cell concentrates. Their role is not elucidated, and they are considered as a type of storage lesion. The purpose of this review is to present recent data showing that erythrocyte-derived microparticles most likely play a role in transfusion medicine and could cause transfusion complications.

Proteomics of blood and derived products: what's next?

Proteomics has changed the way proteins are analyzed in living systems. This approach has been applied to blood products and protein profiling has evolved in parallel with the development of techniques. The identification of proteins belonging to red blood cell, platelets or plasma was achieved at the end of the last century. Then, the questions on the applications emerged. Hence, several studies have focused on problems related to blood banking and products, such as the aging of blood products, identification of biomarkers, related diseases and the protein-protein interactions. More recently, a mass spectrometry-based proteomics approach to quality control has been applied in order to offer solutions and improve the quality of blood products. The current challenge we face is developing a closer relationship between transfusion medicine and proteomics. In this article, these issues will be approached by focusing first on the proteome identification of blood products and then on the applications and future developments within the field of proteomics and blood products.

Electrochemical reactions and ionization processes.

Electrochemical or photo-electrochemical reactions in both electrospray ionization and laser desorption ionization are discussed stressing the role of the electrode reaction in influencing the ionization process. In particular, upon application of a high voltage during electrospray ionization, the emitter includes a working electrode, where redox reactions are observed, such as electro-generation of benzoquinone and metal ions. In contrast, the target plate in laser-induced desorption ionization also acts as a photo-electrode, especially when modified with a mesoporous semiconductor. We illustrate here how these electrochemical reactions can be used for tagging purposes, and for oxidative or reductive dissociation reactions.

Biomarker analysis of stored blood products: emphasis on pre-analytical issues.

Millions of blood products are transfused every year; many lives are thus directly concerned by transfusion. The three main labile blood products used in transfusion are erythrocyte concentrates, platelet concentrates and fresh frozen plasma. Each of these products has to be stored according to its particular components. However, during storage, modifications or degradation of those components may occur, and are known as storage lesions. Thus, biomarker discovery of in vivo blood aging as well as in vitro labile blood products storage lesions is of high interest for the transfusion medicine community. Pre-analytical issues are of major importance in analyzing the various blood products during storage conditions as well as according to various protocols that are currently used in blood banks for their preparations. This paper will review key elements that have to be taken into account in the context of proteomic-based biomarker discovery applied to blood banking.

Iontophoretic fraction collection for coupling capillary zone electrophoresis with matrix-assisted laser desorption/ionization mass spectrometry.

An automated fraction collection interface has been developed for coupling capillary electrophoresis (CE) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). This fraction collection approach is based on electromigration and diffusion and does not rely on the presence of a liquid junction, sheath-liquid, electro-osmotic flow, or a superimposed hydrodynamic flow. Neutrally coated capillary with negligible electro-osmosis can thus be used to provide high-efficiency separations of biological compounds. The in-capillary separation resolution is totally independent from the spotting process. CE-separated species can be collected either in a multiwell plate or directly on a MALDI target. In the present work, an eight-protein mixture, submitted to trypsin proteolysis, has been used as a sample test and separations have been conducted in 50 microm i.d. neutrally coated capillaries. As compared to direct MALDI MS analysis, the integration of CE improved the number of detected peptides from 36 to 87 and the average sequence coverage from 24% to 38%. Internal calibration was used, and an average mass accuracy of 16.1 ppm is reported. Finally, diffusion-migration numerical simulations of the iontophoretic fraction collection process have been carried out.

Electrospray micromixer chip for on-line derivatization and kinetic studies.

An electrospray microchip for mass spectrometry comprising an integrated passive mixer to carry out on-chip chemical derivatizations is described. The microchip fabricated using UV-photoablation is composed of two microchannels linked together by a liquid junction. Downstream of this liquid junction, a mixing unit made of parallel oblique grooves is integrated to the microchannel in order to create flow perturbations. Several mixer designs are evaluated. The mixer efficiency is investigated both by fluorescence study and mass spectrometric monitoring of the tagging reaction of cysteinyl peptides with 1,4-benzoquinone. The comparisons with a microchip without a mixing unit and a kinetic model are used to assess the efficiency of the mixer showing tagging kinetics close to that of bulk reactions in an ideally mixed reactor. As an ultimate application, the electrospray micromixer is implemented in a LC-MS workflow. On-line derivatization of albumin tryptic peptides after a reversed-phase separation and counting of their cysteines drastically enhance the protein identification.

Proteomics of Stored Red Blood Cell Membrane and Storage-Induced Microvesicles Reveals the Association of Flotillin-2 With Band 3 Complexes.

The storage of erythrocyte concentrates (ECs) induces lesions that notably affect metabolism, protein activity, deformability of red blood cells (RBCs), as well as the release of oxygen. Band 3 is one of the proteins affected during the ex vivo aging of RBCs. This membrane protein is an anion transporter, an anchor site for the cytoskeleton and other membrane proteins as well as a binding site for glycolytic enzymes and bears blood group antigens. In the present study, band 3 complexes were isolated from RBCs stored for 7 and 42 days in average (n = 3), as well as from microvesicles (n = 3). After extraction of membrane proteins with a deoxycholate containing buffer, band 3 complexes were co-immunoprecipitated on magnetic beads coated with two anti-band 3 antibodies. Both total membrane protein extracts and eluates (containing band 3 complexes) were separated on SDS-PAGE and analyzed by bottom-up proteomics. It revealed that three proteins were present or absent in band 3 complexes stemming from long-stored or short-stored ECs, respectively, whereas the membrane protein contents remained equivalent. These potential markers for storage-induced RBC aging are adenylosuccinate lyase (ADSL), α-adducin and flotillin-2, and were further analyzed using western blots. ADSL abundance tended to increase during storage in both total membrane protein and band 3 complexes, whereas α-adducin mainly tended to stay onto the membrane extract. Interestingly, flotillin-2 was equivalently present in total membrane proteins whereas it clearly co-immunoprecipitated with band 3 complexes during storage (1.6-fold-change, p = 0.0024). Moreover, flotillin-2 was enriched (almost threefold) in RBCs compared to microvesicles (MVs) (p < 0.001) and the amount found in MVs was associated to band 3 complexes. Different types of band 3 complexes are known to exist in RBCs and further studies will be required to better understand involvement of this protein in microvesiculation during the storage of RBCs.

Red blood cells ageing markers: a multi-parametric analysis.

BACKGROUND: Red blood cells collected in citrate-phosphate-dextrose can be stored for up to 42 days at 4 °C in saline-adenine-glucose-mannitol additive solution. During this controlled, but nevertheless artificial, ex vivo ageing, red blood cells accumulate lesions that can be reversible or irreversible upon transfusion. The aim of the present study is to follow several parameters reflecting cell metabolism, antioxidant defences, morphology and membrane dynamics during storage. MATERIALS AND METHODS: Five erythrocyte concentrates were followed weekly during 71 days. Extracellular glucose and lactate concentrations, total antioxidant power, as well as reduced and oxidised intracellular glutathione levels were quantified. Microvesiculation, percentage of haemolysis and haematologic parameters were also evaluated. Finally, morphological changes and membrane fluctuations were recorded using label-free digital holographic microscopy. RESULTS: The antioxidant power as well as the intracellular glutathione concentration first increased, reaching maximal values after one and two weeks, respectively. Irreversible morphological lesions appeared during week 5, where discocytes began to transform into transient echinocytes and finally spherocytes. At the same time, the microvesiculation and haemolysis started to rise exponentially. After six weeks (expiration date), intracellular glutathione was reduced by 25%, reflecting increasing oxidative stress. The membrane fluctuations showed decreased amplitudes during shape transition from discocytes to spherocytes. DISCUSSION: Various types of lesions accumulated at different chemical and cellular levels during storage, which could impact their in vivo recovery after transfusion. A marked effect was observed after four weeks of storage, which corroborates recent clinical data. The prolonged follow-up period allowed the capture of deep storage lesions. Interestingly, and as previously described, the severity of the changes differed among donors.

Multitrack electrospray chips.

Multitrack electrospray chips (MTEC) were fabricated by UV-photoablation of polyethylene terephthalate (PET) substrates. They are composed of an array of up to six microchannels that are successively used as electrospray ionization (ESI) emitters for mass spectrometry (MS). There is no requirement for alignment of the different spraying microchannels with the mass spectrometer orifice. The MTEC is thus fixed in front of the mass spectrometer and the successive MS analyses are performed without moving the chip. The sequential electrospraying by successive application of an identical high voltage in each off-axis microchannel was evaluated for the fast screening of peptides and proteins. The counting of cysteines in peptides through chemical modification and the relative quantification of a peptide in two samples are presented herein as two original strategies based on this new analytical tool.

Proteomics of the red blood cell carbonylome during blood banking of erythrocyte concentrates.

PURPOSE: Transfusion of red blood cells (RBCs) is a daily medical procedure. Erythrocyte concentrates (ECs) can be stored up to 56 days at 4 °C in saline additive solution mainly composed of adenine and sugar. Such nonphysiological conditions induce the occurrence of storage lesions, such as alterations of metabolism, protein oxidation, and deterioration of rheological properties. Their accumulation tends to decrease the main EC therapeutic property, that is, the oxygenation capacity. Protein carbonylation is a marker of oxidative stress and aging, and its occurrence during RBC storage was earlier characterized as a time-dependent and cellular compartment dependent modification. EXPERIMENTAL DESIGN: Three ECs from independent donations were followed. The carbolynome was here characterized in soluble and membrane extracts (n-dodecyl ß-D-maltoside-based extraction buffer) of RBCs stored for 6, 27, and 41 days, through biotin hydrazide derivatization, biotin-avidin affinity purification, SDS-PAGE separation, and LC-MS/MS analyses. RESULTS: A total of 142 and 20 proteins were identified as carbonylated in soluble and membrane extracts, respectively. Particularly, a time-dependent evolution of 26.8% of the soluble carbonylome was observed. CONCLUSIONS AND CLINICAL RELEVANCE: Affected cellular mechanisms involve antioxidant defenses, metabolism pathways, and proteasomal degradation. To better store RBCs those functions have to be preserved, which opens new routes of investigation in transfusion medicine.