Proteomics and Transfusion Medicine: the bet is open

In the last few years, the clinical and academic settings have continuously been dialoguing about the need of improved and reliable strategies to assess the quality of the production and handling processes of blood components and plasma derivatives, with the shared goal to guarantee their safety and effectiveness for the whole health care system¹. Transfusionists, through recent retrospective and highly-debated studies, are growingly asking for improved tools to assess the quality of transfusion-relevant products. This is relevant in the light of the need of State or government agencies to assess the quality of all the procedures performed at blood banks, from collection, processing, testing, production processes. In this respect, although the statistical appropriateness and validity of recent retrospective studies² has been highly condemned³ and prospective double-blind trials are currently in fieri^4,5, new strategies have to be developed in order to complement clinical observations. At its dawn, proteomics emerged as a potential candidate for in-depth investigations of blood components and plasma derivatives. Proteomic technologies have the potential to serve as a preclinical tool for assessing new preparation methods for blood products or newly designed blood derived therapeutics prior to their use in clinical trials. As its maturity is now at hand, the proteomics expertise seems to be ready to be massively transferred to the clinical setting, where it could be potentially used as a valid tool to test, from bench to bedside, the quality of collected blood components prior to or during storage. A creative alliance of researchers specialized either in proteomics or transfusion medicine has the potential for major improvements in the treatment of patients with blood derived therapeutics.

This special issue is the second attempt, besides the Blood Proteomics issue appeared on Journal of Proteomics, edited by Elsevier, to bridge the gap between independent laboratories and regroup recent findings in the promising field of blood related proteomics, with the declared intent to regroup laboratories which have lately branched out from the same research root. To this regard, the Tuscia University of Viterbo and the Italian National Blood Centre (Centro Nazionale Sangue [CNS]) made an agreement to study the validity of proteomics approaches in investigating red cell protein lesions, by monitoring their oxidative decoration and damage.

The main goal of this collaboration is to address red blood cell storage lesions, through the quality assessment of current standards for storage of erythrocyte concentrates and the individuation of alternative strategies, with the final aim to know and then to prevent these lesions before they had occurred.

Together they organized the first meeting in Viterbo (Italy), on the 12^th–14^th October 2009, with the hope to organize a second one in 2010 in some other location. The XXXIX SIMTI Congress is a good opportunity to spread out the second special issue on this field published by Blood Transfusion journal, which collect about twenty reviews and articles written by specialists from all over the word. In this special issue, Hess asserted that if we better understood the changes that occur with the storage of red cells, platelets and plasma, we could both design better storage systems and regulate storage more effectively. If we knew more about the viruses and bacteria and the cytokines and blood breakdown products that threaten blood safety we could institute controls to further improve blood safety. Thus, better understanding of the basic biology and better tests on which to base better standards are needed. His review explores some specific examples.

Thiele et al. in his review reported how proteomic applications are valuable tools for the study of blood derivatives and how they have improved our understanding of the impact of production, processing and storage of blood products. The Authors summarize major advancements in proteomic investigations on blood products and discuss the potential role of proteomics as an innovative tool to address some of the current “hot topics” in transfusion medicine. Both tools, namely high-tech proteomic analyses as well as clinical studies, should be exploited in order to pursue the main goal of clinical transfusion medicine, as to deliver to patients safer and more effective blood products. In line with this statement, the article by Sparrow provides a perspective of the potential relationship between the RBC storage lesions and the occurrence of transfusion-related immunomodulation (TRIM). This concept has recently emerged as a potentially explanation to numerous clinical observations that suggest that RBC transfusion is associated with increased pro-inflammatory or immunosuppressive effects that may increase morbidity in at least some groups of patients. Platelets and vascular endothelial cells also potentially contribute to the “response” by the recipient, as both cell types are highly responsive to inflammatory signals, and when activated release significant quantities of potent bioactive mediators. Thus, in situations of heightened inflammation or breach of vascular integrity, the immune and thrombotic systems are likely to be intricately linked in a complex network of signalling and response. The role of proteomics in advancing our understanding of the RBC storage lesions as well as in providing insights into the biological mechanisms of TRIM are also discussed. On the other hand, Tissot’s group reviewed the potential pro-coagulant effect of erythrocyte microparticles (MPs) and their potential adverse effect after blood transfusion. The Authors assess that there is an urgent need of large clinical studies with the aim to determine precise effects (if any) of MPs in blood recipients. It is also stressed the importance of determining how MPs are involved in the coagulation cascade, by either passively acting through the provision of additional negatively charged membrane surface, or rather by allowing the expression of TF or other proteins implicated in the coagulation process. Furthermore, studies of MPs from platelets, endothelial cells or monocytes and comparison with erythrocyte-derived MPs are needed. Proteomic analysis, as well as quantitative proteomic evaluation of MP protein content will open new avenues in haematology, because it will help to decipher the complexity of the hypercoagulable states and the effect of erythrocyte concentrate transfusion in routine clinical practice. Combining various approaches that are haemostatic investigations, MPs characterisation either by flow cytometry or by proteomics, clinical studies will certainly help to secure transfusion medicine.

In the particular case of RBC, Papassideri and coworkers focused on the current knowledge on aging and death signaling-pathways operating both in vivo systems and stored red blood cells and suggested future directions in the preservation science, helpful for addressing what seems to be the current critical question in transfusion medicine. The future research efforts in red blood cells preservation science should be enriched with emerging principles, techniques and knowledge regarding the red blood cell interactome, the formation of lipid rafts and other functionally important multi-protein complexes, the repair/destroy mechanisms, the response to oxidative stress, the post-translational processing, the protein sorting into the vesicles and the membrane as a place of execution of the senescence-related apoptosis-like events. This advanced, sophisticated approach would contribute to the understanding of the mechanisms that define life and death of RBCs in vivo, in the plastic bags and in the circulation after transfusion. It also represents a safer way to the successful optimization of red blood cell preservation protocols for transfusion purposes.

Bosman’s review describes the main molecular events occurring during storage in blood bank conditions, when erythrocytes undergo various morphological, biochemical and functional changes: the so-called storage lesions. The development of these storage lesions is associated with changes in band 3, the major protein of the erythrocyte membrane, as indicated mostly by immunoblot data showing band 3 aggregation and degradation. Band 3 is the central protein in a large, multifunctional membrane complex, and plays a pivotal role in erythrocyte homeostasis. He reviewed the data supporting the theory that changes in band 3 structure play a pivotal role in the storage lesions in the blood bank, including vesicle formation. Bosman et al. also reported relations between changes in band 3 structure and the fate of erythrocytes after transfusion, as band 3-related changes might affect red cell survival and thus transfusion outcome either immediately by the binding of physiological auto-antibodies to senescent cell-specific antigens, or by increasing the susceptibility to biochemical and mechanical stress that the erythrocyte experiences during its passage through the body. Especially the latter may contribute to the removal of a considerable fraction of the erythrocytes within the first 24 hours after transfusion, and thereby to the development of untoward transfusion side effects.

Contiguously Castagnola et al. assert that during the last two decades many experimental data concerning the specific interaction of haemoglobin and various enzymes of the glycolytic pathway with the cytoplasmatic domain of band 3 protein, brought Giardina’s group to formulate the hypothesis of a modulation of the erythrocyte metabolism driven by the free energy connected to the R to T haemoglobin transition. The phosphorylation of the specific residues of the cytoplasmatic domain of band 3 protein may have a role in such erythrocyte modulation.

As alternative or complementary approach, Mozzarelli et al. reported an interesting summary about recent trends in haemoglobin-based oxygen carriers as blood substitutes. Indeed, although blood transfusion is the elective therapy in the treatment of many hypo-oxygenation pathologies and is generally considered safe as long as defined guidelines are followed, increasing evidence indicates that the aging of blood⁶, well before the conventional limits for blood storage, can undermine transfusion efficacy and may cause adverse effects. Moreover, the increasing shortage of healthy donors in Western countries and the constant lack of blood in Third World and East European countries call for the development of alternatives to donated blood. Among these alternatives, genetically or chemically modified haemoglobins (haemoglobin-based oxygen carriers) have been deeply investigated in the last fifty years as alternative therapy.

Regarding platelets, Schubert and Devine reported that platelet transfusions are life-saving medical procedures for patients undergoing major surgery or suffering from diseases such as cancer or thrombocytopenia. Research efforts in transfusion science are applied to the banking of platelets in areas ranging from donor genotyping to the improvement of the blood component processing procedure and determination of product quality. After production, platelet concentrates are stored at room temperature (20–24°C) with agitation for only 4–7 days. This shelf-life restriction is imposed by health regulatory authorities in order to ensure safety and quality of the blood product owing to the risk of bacterial growth and the loss of platelet viability during storage commonly referred to as the “platelet storage lesion” (PSL). In line, Egidi et al. focused on platelet storage temperatures and the main issues which affect both current (22°C) and alternative (4°C) storage protocols for platelet concentrates. In particular, the authors herein discussed the initial events which trigger morphological and physiological changes upon cold activation, partially distinguishable from physiological activation. A glance is also given at recent proteomic approaches, which promise to improve current knowledge on blood components of transfusion interest, mainly addressing current technical hurdles (such as the analysis of membrane proteins and their reciprocal interaction).

An original and interesting review has been provided by Alves et al., who reported their experience at the Brazilian Blood Transfusion Service of Rio de Janeiro. Due to the increasing number of cases of cancer in Brazil, the growing of Blood Transfusion Service of National Cancer Institute was absolutely necessary, absorbing all the new technology related to blood banks. It was the first Service to perform aphaeresis using a continuous flow centrifuge device in Brazil in 1970. Still today, the number of voluntary non-remunerated healthy blood donors is insufficient, which results in the need to optimize the use of donated blood and to find strategies to recruit an increased number of donors. The Authors also described the profile of the donors and the outcome of the tests performed to detect blood transmissible diseases in 2009. The implications of the (late) coming Nucleic Acid Test versus serology tests have been discussed, along with the desire to apply proteomics technology to monitor platelets degradation.

In this Special Issue, the interested reader will also find reviews dealing with proteomics application to proteomics application to quality control processes in plasma-derivative production and biomarker discovery.

Gaso-Sokac and Josic showed how proteomics technology is a very useful tool for validation of existing processes for production of therapeutic proteins from human plasma, and for the development and fine-tuning of new production processes. Furthermore, this technology can be used for characterization and quality control of final products, and for detection and minimizing of batch-to-batch variations.

Magni et al. reviewed various functionalized magnetic beads used for peptide and protein capture present in different biological fluids for the sake of biomarker discovery. Functionalized magnetic beads are widely applied as an off-line pre-fractionation step in the field of mass spectrometry-based clinical proteomics. The general aim in these studies is to detect differences in protein/peptide profiles for the purpose of discovering biomarkers. The review will go through the technical aspects concerning both the purification step and MALDI-TOF mass spectrometry analysis. Furthermore, examples of clinical applications of this approach using serum, plasma, urine and other body fluids are provided.

While this Special Issue holds a considerable body of reviews dealing with how proteomic applications have improved our understanding of processing and storage of blood, original investigations are included as well which further stress an increasingly likely translational role for proteomics in transfusion-related issues and as a preclinical tool for the assessment of new preparation methods for blood and plasma-derivative products.

Marrocco et al. by a pilot proteomic study confirmed proteomics as a strategy for plasma marker detection in patients suffering from HBV-associated liver cirrhosis. Their findings confirmed the potential utility of gelsolin either as a prognostic marker or a replacement therapeutic agent to alleviate liver injury. Thus, developing a early diagnosis plasma biomarkers can substitute invasive liver biopsy, presently the best means of diagnosing cirrhosis.

Pavone et al. reported an investigation on protein carbonylation, which is an irreversible and not reparable reaction which is caused by the introduction into proteins of carbonyl derivatives such as ketones and aldehydes, generated from direct oxidation processes or from secondary protein reaction with reactive carbonyl compounds. By redox proteomics the Authors revealed significantly increased levels of reactive carbonyl compounds in blood from uremic patients, particularly those undergoing chronic haemodialysis. Thus, the present study suggests that biomaterials used for fabrication of haemodialysis membrane may affect the carbonyl balance in chronic uremic patients.

Cortelazzo et al. studied changes in human plasma proteome from coagulopathies, commonly encountered in victims of viper envenomation which were treated with an administration of immunoglobulin. Since Echis carinatus Venom (EV) toxins mainly acts both directly and indirectly on fibrinogen, Authors planned to establish a suitable analysis of its beta (FIBB) e gamma (FIBG) chains. The study helped to understand the mechanism of envenomation and to find alternative treatments other than the common treatment with the administration of IgG.

Pasini et al. performed a proteomic investigation of Macaques red blood cells. Notably enough, while this species is the closest evolutionary relative of humans and is routinely used in basic and applied biomedical research, the macaque red blood cell proteome has been so far under-investigated. Their genetic, physiological, immunological and metabolic similarity to humans makes macaques invaluable models of human disease. The comparison between Macaques and human red blood cells shed light on several open issues in red blood cell biology and provided a departure point for more comprehensive understanding of red blood cell function. These similarities also mean that macaques are often the only experimental models available for evaluating increasingly specific drugs in development, and as a proof-of-concept bridge can help reduce the numbers of compounds that fail in clinical pharmaceutical research.

Finamore et al. performed a proteomic analysis of platelets from blood of healthy donors (n=6) collected by venipuncture in Vacutainer. Comparative analysis for differential enrichment of Gene Ontologies has been also performed to evaluate the differential functional representation of the molecular repertoire investigated with these two orthogonal approaches. The overall molecular functional classification revealed differences between the two proteomic approaches. In particular, they found significant differences in cytoskeletal proteins (19.65% 2-DE versus 45.60 Shotgun) and receptors (0,92% 2-DE versus 6.90% Shotgun). Results indicated that molecular profiling performed with two orthogonal proteomics analytic methodologies, enhanced the possibility to obtain deeper information on the biology and patho-physiology of the systems under investigation. This becomes particularly relevant with platelets, in light of their multiple physiological functions and their involvement in a variety of human diseases.

In agreement, Timperio et al. analyzed two commercial products of plasma-derived factor VIII (FVIII) with the final aim to collect information regarding the presence of contaminants. The investigation showed the presence of von Willebrand Factor, fibrinogen in excess, and other substances that could be considered as contaminants or impurities.

In conclusion, this Special Issue is meant to represent none but just another brick in the wall of proteomics application to transfusion medicine, a further attempt to keep laboratories working on this hot topic worldwide connected, while temptatively attracting a clinical audience from the other side of the bridge. While the road ahead is still long, progresses in this co-operation are yet encouraging, as proteomics strategies have already been demonstrated to be valuable tools for improving our understanding of the impact of processing and storage of blood products, as well as suited to production of blood components on the protein fractions, or to discover peculiar biomarkers readily adoptable for targeted evaluation of blood-component integrity or functionality. Although the technical background is in continuous and rapid expansion, proteomics spread in routine clinical practice has been hitherto hampered by high costs for dedicated facilities and specialized personnel. Nonetheless, the bet is still open.