Proteomics and blood transfusion

Transfusion medicine is a clinical discipline characterized by one of the most advanced quality management systems, which is structured so as to assure the production of blood components and raw materials, for biopharmaceutical fractionation, that are safe, efficient and effective. During the production, pathogen inactivation and storage processes there is the risk of changes in the integrity of blood components, especially at the protein level. These changes could be the cause of some of the negative effects of transfusion therapy. It is therefore a major challenge to identify significant alterations of these products, and, in this context, proteomics can play a potentially relevant role in transfusion medicine to assess the protein composition of blood-derived therapeutics, particularly for identifying modified proteins. Proteomics can also provide a more detailed understanding of the proteins found in plasma derivatives, with particular regard to peptide and protein changes related to the various procedures used for protein purification and pathogen inactivation during the modern plasma industrial factionation. The latter could cause protein modifications and/or degradation and neoantigen production, with the potential to induce adverse effects in recipients. At present, blood component quality control is mainly focused on standardized quantitative assessment, providing relatively limited information about products. Proteomics allows a comprehensive study of protein modifications, qualitative and quantitative analysis, and high-throughput protein identification. Moreover, being the only tool to evaluate structural changes in proteins (or their degradation products) after their manipulation, and having the capacity to identify many new proteins, proteomics seems to be the most promising tool for global quality assessment and possible improvement of the production process of blood components and plasma derivatives. It can also provide comprehensive information about possible contaminants and neoantigens that may influence the immunogenic capacity of blood-derived therapeutics.     

Storage of red blood cell concentrates: Clinical impact

The storage of red blood cells for transfusion purposes induces modifications of biochemical and biological properties. Moreover, these modifications are modulated by the donors’ characteristics and the cell processing. These ex vivo alterations were suspected to decrease the transfusion efficiency and even to induce adverse events. This short article will review the red blood cells storage lesions and the clinical data related to them. In particular, the questions regarding the donors and recipients sex will be discussed.     

Proteomic Feature Maps: A new visualization approach in proteomics analysis

The different steps of a proteomics analysis workflow generate a plethora of features for each extracted proteomic object (a protein spot in 2D gel electrophoresis (2-DE), or a peptide peak in liquid chromatography–mass spectrometry (LC–MS) analysis). Yet, the joint visualization of multiple object features on 2D gel-like maps is rather limited in currently available proteomics software packages. We introduce a new, simple, and intuitive visualization method that utilizes spheres to represent proteomic objects on proteomic feature maps, and exploits the spheres size and color to provide simultaneous visualization of user-selected feature pairs. Our contribution, a unified and flexible visualization mechanism that can be easily applied at any stage of a 2-DE or a LC–MS based differential proteomics study, is demonstrated and discussed using five representative scenarios. The joint visualization of proteomic object features and their spatial distribution is a powerful tool for inspecting and comparing the proteomics analysis results, attracting the users attention to useful information, such as differential expression trends and patterns, and even assisting in the evaluation and refinement of a proteomics experiment.     

Application of two-dimensional electrophoresis in the research of retinal proteins of diabetic rat

Diabetes mellitus （DM） is a chronic disease which is associated with numerous serious health complications such as diabetic retinopathy, and is the leading cause of new cases of blindness in adults at the age of 20-74 years old. The aim of the study was to establish and optimize a two-dimensional polyacrylamide gel electrophoresis （2-DE） technique for retina proteomics to improve the resolution and reproducibility, and to observe the proteomic changes of retinal tissues in diabetic and normal rats. Proteins were extracted from retinal tissues of normal and 8 weeks diabetic SD rats and used in two-dimensional electrophoresis. Various conditions of retina proteomic 2-DE were adjusted, optimized and protein spots of differential expression were obtained through analysis of 2-DE images with PDQuest software. By choosing appropriate sample amount, using pre-cast IPG dry strips （pH 5-8） and casting 12% equal gel, satisfactory 2-DE images of retina were obtained and a steady 2-DE technique was established. In this way, we found 36 spots in 2-DE gel of diabetic retinas that exhibited statistically significant variations, including up-regulation of 5 proteins in diabetic rat retinas, down-regulation of 23, and disappearance of 8, in comparison with normal tissues. The differences of protein expression were observed in retinas between diabetic and normal rats. Our established 2-DE technique of retina proteins could be effectively applied in proteomics of retina diseases. Cellular ＆ Molecular Immunology. 2007;4（1）：65-70.     

Advances in proteomic techniques for biomarker discovery in COPD

Chronic obstructive pulmonary disease (COPD) is a disorder characterized by chronic inflammation of the lung with airflow obstruction and progressive deterioration of pulmonary function. The need to discover and validate biomarkers as prognostic tools of development and progression of the disease has received further support with the advent of proteomic techniques. Liquid chromatography-mass spectrometry (LC/MS) and gel electrophoresis-mass spectrometry (2-DE/MS) have been applied to investigate the proteome of a number of lung-origin samples, including sputum, bronchoalveolar lavage fluid, exhaled-breath condensate, cells and biopsies from COPD patients. In particular, 2-DE and MS are the main proteomic approaches with 2-DE presenting the major approach for quantitative proteomics. The molecules identified as potential biomarkers of COPD may represent a preliminary step for better comprehension of the mechanisms involved in the onset/progression of the disease.     

Proteomic strategies and challenges in tumor metastasis research

The rapidly evolving field of proteomics offers new approaches to understanding the pathogenesis of cancer and metastatic disease. Although numerous tumor markers have been identified with different genomic methods in the past, most are either not specific or sensitive enough to be used in routine clinical setting. The rationale for proteomic profiling is based on the fact that proteins represent the dynamic state of the cells, reflecting pathophysiological changes in the disease more accurately than genomic and epigenetic alterations. Emerging proteomic techniques allow simultaneous assessment of a large number of proteins at one time. The study of protein profiles in complex systems, such as plasma, serum or tissues of cancer patients is likely to become valuable for monitoring the response of patients during treatment or for detecting recurrence of the disease.     

Concise review: trends in stem cell proteomics

Gene expression analyses of stem cells (SCs) will help to uncover or further define signaling pathways and molecular mechanisms involved in the maintenance of self-renewal, pluripotency, and/or multipotency. In recent years, proteomic approaches have produced a wealth of data identifying proteins and mechanisms involved in SC proliferation and differentiation. Although many proteomics techniques have been developed and improved in peptide and protein separation, as well as mass spectrometry, several important issues, including sample heterogeneity, post-translational modifications, protein-protein interaction, and high-throughput quantification of hydrophobic and low-abundance proteins, still remain to be addressed and require further technical optimization. This review summarizes the methodologies used and the information gathered with proteome analyses of SCs, and it discusses biological and technical challenges for proteomic study of SCs. Disclosure of potential conflicts of interest is found at the end of this article.     

Proteomic analyses of murine macrophages treated with Bacillus anthracis lethal toxin

Bacillus anthracis is the etiological agent of anthrax and the bacterium produces a tripartite anthrax toxin composed of protective antigen (PA), lethal factor (LF) and edema factor (EF). PA represents the binding domain of the toxin and acts in concert with either LF, a metalloprotease, or EF, an adenylate cyclase, to form lethal toxin (LeTx) or edema toxin (EdTx), respectively. We analyzed the proteomics response of two murine macrophage cell lines (J774.1A and RAW264.7) following B. anthracis LeTx treatment to detect unique host proteins involved in anthrax infection using difference in-gel electrophoresis (DIGE) followed by nanoLC-MS for identification of the proteins. The comparative proteomics approach identified a set of proteins in each cell line that was consistently upregulated when the two macrophage cell lines were treated with LeTx. The upregulated proteins include those involved in energy metabolism, cytoskeleton structure and stress response. A subset of five proteins (ATP synthase beta subunit, beta-actin, Hsp70, vimentin, and Hsp60 homolog) was identified that were commonly upregulated in both cell lines. The proteomic data suggest the involvement of reactive oxygen species (ROS) in cell lysis as seen by the upregulation of proteins that lead to the production of ROS in both the cell lines used in our study. However, proteins that afford protection against ROS may play an important role in the survival of the macrophage to LeTx infection as shown by the differences in proteomic responses of the two cell lines to the action of LeTx. These identified proteins may have the potential to be used as biomarkers for diagnostics and therapeutics.     

Appropriate blood component usage

Blood transfusion (which includes FFP, platelets, cryoprecipitate and any other blood-derived product) remains an important modality of treatment across all clinical disciplines. A blood transfusion is deemed appropriate when used in an evidence-based fashion and where the effects of the transfusion are felt to outweigh any potential risks and complications that may arise from the transfusion. In certain cases, it may be the best treatment option available, for example plasma exchange in thrombotic thrombocytopenic purpura. However, blood transfusion can result in acute or delayed complications, as well as the risk of transmission of infectious agents. The inappropriate use of blood and blood products increases the risk of transfusion-related complications and adverse events to recipients. It also contributes to shortages of blood products and the possibility of it not being available when required for other patients in an appropriate setting. It is therefore necessary to reduce the unnecessary transfusions through the appropriate clinical use of blood, avoiding unnecessary transfusions, and use of alternatives to transfusion.     

The storage lesions: From past to future

Red blood cell (RBC) concentrates are stored in additive solutions at 4 ^oC for up to 42 days, whereas platelets concentrates (PCs) are stored at 22 ^oC with continuous agitation for up to 5 to 7 days, according national regulations, and the use or not of pathogen inactivation procedures. Storage induces cellular lesion and alters either RBC or platelet metabolism, and is associated with protein alterations. Some age-related alterations prove reversible, while other changes are irreversible, notably following protein oxidation. It is likely that any irreversible damage affects the blood component quality and thus the transfusion efficiency. Nevertheless, there still exists a debate surrounding the impact of storage lesions, for both RBCs and PCs. Uncertainty is not completely resolved. Several studies show a tendency for poorer outcomes to occur in patients receiving older blood products; however, no clear significant association has yet been demonstrated. The present short review aims to promote a better understanding of the occurrence of storage lesions, with particular emphasis on biochemical modifications opening discussions of the future advancement of blood transfusion processes. The paper is also an advocacy for the implementation of an independent international organization in charge of planning and controlling clinical studies in transfusion medicine, in order to base transfusion medicine practices both on security principles, but also on clinical evidences.     

Zastosowanie cytometrii przepływowej oraz analizy proteomicznej w ocenie erytrocytarnych białek błonowych podczas przechowywania koncentratów krwinek czerwonych

For over 100 years the erythrocyte cell membrane attracted interest of transfusiologists mainly due to the antigens localized on their surface and associated risks of patient alloimmunisation and therefore the need of serological selection of donor's and recipient's blood. Presently it is known that RBC antigens and other membrane proteins play important transport and protective functions, and are involved in adhesion, maintenance of cell shape and in the process of aging and phagocytosis. Since the available results of retrospective clinical observations suggest an adverse effect of transfusion on selected groups of patients, it is important to undertake studies on the changes taking place within the cell membrane of erythrocytes stored in blood banks. Flow cytometric analysis of stored leucodepleted or non-leucodepleted erythrocyte concentrates, revealed significant changes in the level of expression of many RBC surface molecules: CD44, CD47, CD55, CD58, CD59, CD235a (GPA). In parallel, a significant development of proteomic analysis of stored RBCs is observed. Stored RBCs offer less variability of biological material, caused by drugs, illnesses, etc. when compared with clinical proteomics studies; however, the complexity of the methodology and the lack of uniform and comparable procedures may cause misinterpretation and even create artifacts. Still, modern research methods offer hope for the elaboration of aging biomarkers of stored RBCs as well as for raising the level of transfusion medicine quality.     

脑脊液蛋白质组学高丰度蛋白去除方法的建立与评价

目的建立和评价脑脊液蛋白质组学中高丰度蛋白的去除方法。方法使用Oasis HLB（hydrophilic—lipophilic balance）过滤柱。运用反相固相法，根据蛋白质不同的疏水性分馏脑脊液，去除高丰度蛋白和盐。将含有低丰度蛋白的过滤液冻干，利用双向凝胶电泳（2-DE）分析评价脑脊液蛋白层析去除高丰度蛋白的效果。结果通过Oasis HLB柱处理可将大部分高丰度蛋白去除。2-DE图谱中低丰度蛋白质分辨率明显增加，点数增多59．86％。结论本研究建立的脑脊液高丰度蛋白去除方法可有效地应用于疾病的脑脊液蛋白质组学研究。     

Inactivation des pathogènes des produits sanguins labiles : entre enjeux financiers et conséquences possibles à long terme

Pathogen inactivation of blood products represents a global and major paradigm shift in transfusion medicine. In the next near future, it is likely that most blood products will be inactivated by various physicochemical approaches. The concept of blood safety will be challenged as well as transfusion medicine practice, notably for donor selection or biological qualification. In this context, it seems mandatory to develop analytical economic approaches by assessing costs-benefits ratio of blood transfusion as well as to set up cohorts of patients based on hemovigilance networks allowing rigorous scientific analysis of the benefits and the risks of blood transfusion at short- and long-term.     

Détection du génome viral du parvovirus B19 dans les pools de plasmas servant à la préparation du plasma frais congelé traité pour viro-atténuation par solvant–détergent : expérience de l’Afssaps et de l’EFS Aquitaine-Limousin

Since 1998, the Aquitaine-Limousin branch of the French Blood Institute has set up a parvovirus B19 (PV B19) systematic screening on each unit of plasma to be treated by solvent–detergent procedure for virus inactivation. Parvovirus B19 nucleic acid systematic testing in plasma pools became mandatory since 2005 (European monograph “Human plasma” – pooled and treated for virus inactivation). The French competent state authority (AFSSAPS) has decided to introduce this test as a part of the external quality control of labile blood products. This process is related to the harmonization of quality control practice realised on blood products in Europe even if the human plasma pooled and treated for virus inactivation by solvent–detergent is considered in France as a blood labile component. Implementation of this test required a validation step and a close cooperation between AFSSAPS and Aquitaine-Limousin blood transfusion centre. Validation consisted in perfecting a semi-quantitative, real-time nucleic acid testing method with automated extraction. This collaborative study leads us to control 1642 plasma pools. All the results were under the threshold of 10,0 IU/μL. AFSSAPS's results were in agreement with those of Aquitaine-Limousin's blood transfusion center who carry out the parvovirus B19 screening both on fresh frozen plasma units composing the pool and on plasma pools.     

Plasmodium falciparum infection-induced changes in erythrocyte membrane proteins

Over the past decade, advances in proteomic and mass spectrometry techniques and the sequencing of the Plasmodium falciparum genome have led to an increasing number of studies regarding the parasite proteome. However, these studies have focused principally on parasite protein expression, neglecting parasite-induced variations in the host proteome. Here, we investigated P. falciparum-induced modifications of the infected red blood cell (iRBC) membrane proteome, taking into account both host and parasite proteome alterations. Furthermore, we also determined if some protein changes were associated with genotypically distinct P. falciparum strains. Comparison of host membrane proteomes between iRBCs and uninfected red blood cells using fluorescence-based proteomic approaches, such as 2D difference gel electrophoresis revealed that more than 100 protein spots were highly up-represented (fold change increase greater than five) following P. falciparum infection for both strains (i.e. RP8 and Institut Pasteur Pregnancy Associated Malaria). The majority of spots identified by mass spectrometry corresponded to Homo sapiens proteins. However, infection-induced changes in host proteins did not appear to affect molecules located at the outer surface of the plasma membrane. The under-representation of parasite proteins could not be attributed to deficient parasite protein expression. Thus, this study describes for the first time that considerable host protein modifications were detected following P. falciparum infection at the erythrocyte membrane level. Further analysis of infection-induced host protein modifications will improve our knowledge of malaria pathogenesis.     

Broad-based proteomic strategies: a practical guide to proteomics and functional screening

Proteomics, the study of the proteome (the collection of all the proteins expressed from the genome in all isoforms, polymorphisms and post-translational modifications), is a rapidly developing field in which there are numerous new and often expensive technologies, making it imperative to use the most appropriate technology for the biological system and hypothesis being addressed. This review provides some guidelines on approaching a broad-based proteomics project, including strategies on refining hypotheses, choosing models and proteomic approaches with an emphasis on aspects of sample complexity (including abundance and protein characteristics), and separation technologies and their respective strengths and weaknesses. Finally, issues related to quantification, mass spectrometry and informatics strategies are discussed. The goal of this review is therefore twofold: the first section provides a brief outline of proteomic technologies, specifically with respect to their applications to broad-based proteomic approaches, and the second part provides more details about the application of these technologies in typical scenarios dealing with physiological and pathological processes. Proteomics at its best is the integration of carefully planned research and complementary techniques with the advantages of powerful discovery technologies that has the potential to make substantial contributions to the understanding of disease and disease processes.     

Where will pathogen inactivation have the greatest impact?

Blood safety has always been a major task in transfusion medicine. A strategy to obtain this aim should include donor education, donor selection, and testing of blood donations. Pathogen inactivation adds another level of safety. In the fractionation industry, pathogen inactivation methods are mandatory. Several countries also use pathogen‐inactivated plasma – from pools or single donors. Concerning the cellular blood components, there is still no method available for red cell concentrates, whereas methods for platelet concentrates are available in some countries and others are in the pipeline for commercialization. The efficiency of the ‘old’ methods to increase blood safety and the costs of the methods seem to be major obstacles for the introduction of the systems. There are also concerns on product quality and loss of volume during the inactivation process. As the importance of pathogen inactivation is largest in countries with blood donors who carry infections it is impossible to protect against, either due to high incidence of the infection or due to shortage of tests, cost will be a major question when pathogen inactivation is considered. Pathogen inactivation of red cell concentrates will also be a necessity. When pathogen inactivation methods are available for all blood components, they will have great impact to protect the patients in countries where a high percentage of the population is infected by agents transmissible through blood transfusion, and in all situations to protect against new pathogens and ‘old’ pathogens that become more virulent. The total risk of contracting infectious diseases through blood transfusion will probably be important when implementation of new methods for pathogen inactivation is considered.     

bFGF诱导的心肌微血管内皮细胞蛋白质组变化

目的:研究碱性成纤维细胞生长因子(bFGF)对培养的心肌微血管内皮细胞(MMEC)蛋白质表达变化的影响。方法:将培养的MMEC随机分为二组(n均=3):(1)bFGF诱导组(bFGF组):向培养瓶内加入终浓度10ng/ml的bFGF,置细胞培养箱内常氧培养24h。(2)对照组:常规37℃培养箱孵育。二组均提取细胞总蛋白质双向凝胶电泳后选取8个差异表达蛋白点进行胶内酶切、肽质量指纹图谱分析。结果:双向凝胶电泳可分离出约500±10个蛋白质。与对照组相比,bFGF组MMEC特异表达8种蛋白,上调1种蛋白表达,下调3种蛋白表达(bFGF组和对照组蛋白点的Volume值相差≥2.0倍)。结论:bFGF能诱导MMEC蛋白质差异表达。