Subcellular fractionation of stored red blood cells reveals a compartment-based protein carbonylation evolution.

During blood banking, erythrocytes undergo storage lesions, altering or degrading their metabolism, rheological properties, and protein content. Carbonylation is a hallmark of protein oxidative lesions, thus of red blood cell oxidative stress. In order to improve global erythrocyte protein carbonylation assessment, subcellular fractionation has been established, allowing us to work on four different protein populations, namely soluble hemoglobin, hemoglobin-depleted soluble fraction, integral membrane and cytoskeleton membrane protein fractions. Carbonylation in erythrocyte-derived microparticles has also been investigated. Carbonylated proteins were derivatized with 2,4-dinitrophenylhydrazine (2,4-DNPH) and quantified by western blot analyses. In particular, carbonylation in the cytoskeletal membrane fraction increased remarkably between day 29 and day 43 (P<0.01). Moreover, protein carbonylation within microparticles released during storage showed a two-fold increase along the storage period (P<0.01). As a result, carbonylation of cytoplasmic and membrane protein fractions differs along storage, and the present study allows explaining two distinct steps in global erythrocyte protein carbonylation evolution during blood banking. This article is part of a Special Issue entitled: Integrated omics.

Analysis and clinical relevance of microparticles from red blood cells.

PURPOSE OF REVIEW: The mechanisms involved in the formation of red blood cell (RBC) microparticles in vivo as well as during erythrocyte storage are reviewed, and the potential role of microparticles in transfusion medicine is described. RECENT FINDINGS: Microparticles release is an integral part of the erythrocyte ageing process, preventing early removal of RBCs. Proteomics analyses have outlined the key role of band 3-ankyrin anchoring complex and the occurrence of selective RBC membrane remodelling mechanisms in microparticles formation. The presence of several RBC antigens, expressed on microparticles, has been demonstrated. The potential deleterious effects of RBC microparticles in transfused recipients, including hypercoagulability, microcirculation impairment and immunosuppression, are discussed. SUMMARY: Formation and role of RBC microparticles are far from being completely understood. Combining various approaches to elucidate these mechanisms could improve blood product quality and transfusion safety. Implementation of RBC microparticles as biomarkers in the laboratory routine needs to overcome technical barriers involved in their analysis.

Spatially-resolved eigenmode decomposition of red blood cells membrane fluctuations questions the role of ATP in flickering.

Red blood cells (RBCs) present unique reversible shape deformability, essential for both function and survival, resulting notably in cell membrane fluctuations (CMF). These CMF have been subject of many studies in order to obtain a better understanding of these remarkable biomechanical membrane properties altered in some pathological states including blood diseases. In particular the discussion over the thermal or metabolic origin of the CMF has led in the past to a large number of investigations and modeling. However, the origin of the CMF is still debated. In this article, we present an analysis of the CMF of RBCs by combining digital holographic microscopy (DHM) with an orthogonal subspace decomposition of the imaging data. These subspace components can be reliably identified and quantified as the eigenmode basis of CMF that minimizes the deformation energy of the RBC structure. By fitting the observed fluctuation modes with a theoretical dynamic model, we find that the CMF are mainly governed by the bending elasticity of the membrane and that shear and tension elasticities have only a marginal influence on the membrane fluctations of the discocyte RBC. Further, our experiments show that the role of ATP as a driving force of CMF is questionable. ATP, however, seems to be required to maintain the unique biomechanical properties of the RBC membrane that lead to thermally excited CMF.

Microparticles in stored red blood cells: an approach using flow cytometry and proteomic tools.

BACKGROUND AND OBJECTIVES: Microparticles (MPs) are small phospholipid vesicles of less than 1 microm, shed in blood flow by various cell types. These MPs are involved in several biological processes and diseases. MPs have also been detected in blood products; however, their role in transfused patients is unknown. The purpose of this study was to characterize those MPs in blood bank conditions. MATERIALS AND METHODS: Qualitative and quantitative experiments using flow cytometry or proteomic techniques were performed on MPs derived from erythrocytes concentrates. In order to count MPs, they were either isolated by various centrifugation procedures or counted directly in erythrocyte concentrates. RESULTS: A 20-fold increase after 50 days of storage at 4 degrees C was observed (from 3370 +/- 1180 MPs/microl at day 5 to 64 850 +/- 37 800 MPs/microl at day 50). Proteomic analysis revealed changes of protein expression comparing MPs to erythrocyte membranes. Finally, the expression of Rh blood group antigens was shown on MPs generated during erythrocyte storage. CONCLUSIONS: Our work provides evidence that storage of red blood cell is associated with the generation of MPs characterized by particular proteomic profiles. These results contribute to fundamental knowledge of transfused blood products.

Vacuum assisted venous drainage does not increase trauma to blood cells.

Although gravity drainage has been the standard technique for cardiopulmonary bypass (CPB), the development of min imally invasive techniques for cardiac surgery has renewed interest in using vacuum assisted venous drainage (VAVD) Dideco (Mirandola, Italy) has modified the D903 Avant oxygenator to apply a vacuum to its venous reservoir. The impact of VAVD on blood damage with this device is analyzed. Six calves (mean body weight, 71.3 +/- 4.1 kg) were con nected to CPB by jugular venous and carotid arterial cannu lation, with a flow rate of 4-4.51 L/min for 6 h. They were assigned to gravity drainage (standard D903 Avant oxygen ator, n = 3) or VAVD (modified D903 Avant oxygenator, n = 3). The animals were allowed to survive for 7 days. A standard battery of blood samples was taken before bypass, throughout bypass, and 24 h, 48 h, and 7 days after bypass. Analysis of variance was used for repeated measurements. Thrombocyte and white blood cell counts, corrected by hematocrit and normalized by prebypass values, were not significantly different between groups throughout all study periods. The same holds true for hemolytic parameters (lactate dehydrogenase [LDH] and plasma hemoglobin). Both peaked at 24 hr in the standard and VAVD groups: LDH, 2,845 +/- 974 IU/L vs. 2,537 +/- 476 IU/L (p = 0.65), respectively; and plasma hemoglobin, 115 +/- 31 mg/L vs. 89 +/- 455 mg/L (p = 0.45), respectively. In this experimental setup with prolonged perfusion time, VAVD does not increase trauma to blood cells in comparison with standard gravity drainage.

Stored red blood cells: a changing universe waiting for its map(s).

The availability of stored red blood cells (RBCs) for transfusion remains an important aspect of the treatment of polytrauma, acute anemia or major bleedings. RBCs are prepared by blood banks from whole blood donations and stored in the cold in additive solutions for typically six weeks. These far from physiological storage conditions result in the so-called red cell storage lesion that is of importance both to blood bankers and to clinical practitioners. Here we review the current state of knowledge about the red cell storage lesion from a proteomic perspective. In particular, we describe the current models accounting for RBC aging and response to lethal stresses, review the published proteomic studies carried out to uncover the molecular basis of the RBC storage lesion, and conclude by suggesting a few possible proteomic studies that would provide further knowledge of the molecular alterations carried by RBCs stored in the cold for six weeks.

Automated segmentation of multiple red blood cells with digital holographic microscopy.

We present a method to automatically segment red blood cells (RBCs) visualized by digital holographic microscopy (DHM), which is based on the marker-controlled watershed algorithm. Quantitative phase images of RBCs can be obtained by using off-axis DHM along to provide some important information about each RBC, including size, shape, volume, hemoglobin content, etc. The most important process of segmentation based on marker-controlled watershed is to perform an accurate localization of internal and external markers. Here, we first obtain the binary image via Otsu algorithm. Then, we apply morphological operations to the binary image to get the internal markers. We then apply the distance transform algorithm combined with the watershed algorithm to generate external markers based on internal markers. Finally, combining the internal and external markers, we modify the original gradient image and apply the watershed algorithm. By appropriately identifying the internal and external markers, the problems of oversegmentation and undersegmentation are avoided. Furthermore, the internal and external parts of the RBCs phase image can also be segmented by using the marker-controlled watershed combined with our method, which can identify the internal and external markers appropriately. Our experimental results show that the proposed method achieves good performance in terms of segmenting RBCs and could thus be helpful when combined with an automated classification of RBCs.

Microparticles from stored red blood cells induce thrombin generation

Background: Microparticles are small phospholipid vesicles of <1 lm shed in blood flow by various cell types including red blood cells. Erythrocyte-derived microparticles (EMPs) accumulate in erythrocyte concentrates (ECs) during their storage time. EMPs are considered as part of storage lesion and as their exact role is not elucidated, they could be involved in these clinical outcomes. Aims: The aim of this study is to evaluate the impact and implication of EMPs isolate from ECs on coagulation. Methods: EMPs were first isolated from erythrocyte concentrates by centrifugation and counted by flow cytometry. Using a calibrated automated thrombogram, EMPs were then added to different type of plasmas in order to evaluate the potential of thrombin generation. Results: We demonstrate that EMPs isolated from ECs are capable to accelerate and amplify thrombin generation in presence of a low exogenous tissue factor concentration, thanks to their negatively charged membrane necessary for the assembly of coagulation complexes. Interestingly, in the absence of exogenous tissue factor, EMPs are also able to trigger thrombin generation. In addition, thrombin generation induced by EMPs is not affected by the presence of anti-TF antibodies. Finally, thrombin generation induced by EMPs is not affected by using plasma samples deficient in factor VII, XI or XII. However, thrombin generation is reduced in plasma deficient in factor VIII or IX and is completely abolished in plasma deficient in factor X, V or II. No thrombin generation was observed in plasma samples without EMPs. Summary/conclusion: Several studies have shown a link between storage time of blood products and post transfusion complications. We provide evidence that EMPs accumulated during storage of erythrocyte concentrates were not only able to accelerate and support thrombin generation in plasma in presence of a low exogenous tissue-factor concentration, but also to trigger thrombin generation in absence of exogenous TF. The impact of those transfused EMs is unknown on recipients, nevertheless it could be hypothesized that under certain circumstances, transfused EMPs could be involved in thrombin generation and could be linked to adverse clinical outcome. Further work is needed to determine whether procoagulant EMPs transfused with erythrocyte concentrate may account for some of the complications occurring after red blood cell transfusion, and more particularly after transfusion of ''older''stored blood, rich in EMPs.

Immuno-monitoring of CD8+ T cells in whole blood versus PBMC samples.

The study of natural T cell responses against pathogens or tumors, as well as the assessment of new immunotherapy strategies aimed at boosting these responses, requires increasingly precise ex vivo analysis of blood samples. For practical reasons, studies are often performed using purified PBMC samples, usually cryopreserved. Here, we report on FACS analyses of peripheral blood T cells, performed by direct antibody staining of non-purified total blood. For comparison, fresh PBMC, purified by Ficoll, were analysed. Our results show that the latter method can induce a bias in subpopulation distribution, in particular of CD8+ T cells, and sometimes lead to inaccurate measurement of antigen specific CD8+ T cell responses. Direct analysis of total blood can be applied to longitudinal immuno-monitoring of T cell-based therapy. While the need to purify and cryopreserve PBMC for subsequent studies is obvious, the use of whole blood has the advantage of providing unbiased results and only small amounts of blood are used.

Red blood cell microparticles: clinical relevance.

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.

Red blood cell microparticles and blood group antigens: an analysis by flow cytometry.

BACKGROUND: The storage of blood induces the formation of erythrocytes-derived microparticles. Their pathogenic role in blood transfusion is not known so far, especially the risk to trigger alloantibody production in the recipient. This work aims to study the expression of clinically significant blood group antigens on the surface of red blood cells microparticles. MATERIAL AND METHODS: Red blood cells contained in erythrocyte concentrates were stained with specific antibodies directed against blood group antigens and routinely used in immunohematology practice. After inducing erythrocytes vesiculation with calcium ionophore, the presence of blood group antigens was analysed by flow cytometry. RESULTS: The expression of several blood group antigens from the RH, KEL, JK, FY, MNS, LE and LU systems was detected on erythrocyte microparticles. The presence of M (MNS1), N (MNS2) and s (MNS4) antigens could not be demonstrated by flow cytometry, despite that glycophorin A and B were identified on microparticles using anti-CD235a and anti-MNS3. DISCUSSION: We conclude that blood group antigens are localized on erythrocytes-derived microparticles and probably keep their immunogenicity because of their capacity to bind specific antibody. Selective segregation process during vesiculation or their ability to elicit an immune response in vivo has to be tested by further studies.

Increased numbers of circulating polyfunctional Th17 memory cells in patients with seronegative spondylarthritides

OBJECTIVE: A distinct subset of proinflammatory CD4+ T cells that produce interleukin-17 was recently identified. These cells are implicated in different autoimmune disease models, such as experimental autoimmune encephalomyelitis and collagen-induced arthritis, but their involvement in human autoimmune disease has not yet been clearly established. The purpose of this study was to assess the frequency and functional properties of Th17 cells in healthy donors and in patients with different autoimmune diseases. METHODS: Peripheral blood was obtained from 10 psoriatic arthritis (PsA), 10 ankylosing spondylitis (AS), 10 rheumatoid arthritis (RA), and 5 vitiligo patients, as well as from 25 healthy donors. Synovial tissue samples from a separate group of patients were also evaluated (obtained as paraffin-embedded sections). Peripheral blood cells were analyzed by multiparameter flow cytometry and immunohistochemistry. Cytokine production was examined by enzyme-linked immunosorbent assay and intracellular cytokine staining using specific monoclonal antibodies. Synovial tissue was examined for infiltrating T cells by immunohistochemical analysis. RESULTS: We found increased numbers of circulating Th17 cells in the peripheral blood of patients with seronegative spondylarthritides (PsA and AS), but not in patients with RA or vitiligo. In addition, Th17 cells from the spondylarthritis patients showed advanced differentiation and were polyfunctional in terms of T cell receptor-driven cytokine production. CONCLUSION: These observations suggest a role of Th17 cells in the pathogenesis of certain human autoimmune disorders, in particular the seronegative spondylarthritides.