Jean-Daniel Tissot

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

Background and Objectivesu2002 Microparticles (MPs) are small phospholipid vesicles of less than 1 µm, 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.

Proteome changes in platelets after pathogen inactivation--an interlaboratory consensus.

Pathogen inactivation (PI) of platelet concentrates (PCs) reduces the proliferation/replication of a large range of bacteria, viruses, and parasites as well as residual leucocytes. Pathogen-inactivated PCs were evaluated in various clinical trials showing their efficacy and safety. Today, there is some debate over the hemostatic activity of treated PCs as the overall survival of PI platelets seems to be somewhat reduced, and in vitro measurements have identified some alterations in platelet function. Although the specific lesions resulting from PI of PCs are still not fully understood, proteomic studies have revealed potential damages at the protein level. This review merges the key findings of the proteomic analyses of PCs treated by the Mirasol Pathogen Reduction Technology, the Intercept Blood System, and the Theraflex UV-C system, respectively, and discusses the potential impact on the biological functions of platelets. The complementarities of the applied proteomic approaches allow the coverage of a wide range of proteins and provide a comprehensive overview of PI-mediated protein damage. It emerges that there is a relatively weak impact of PI on the overall proteome of platelets. However, some data show that the different PI treatments lead to an acceleration of platelet storage lesions, which is in agreement with the current model of platelet storage lesion in pathogen-inactivated PCs. Overall, the impact of the PI treatment on the proteome appears to be different among the PI systems. Mirasol impacts adhesion and platelet shape change, whereas Intercept seems to impact proteins of intracellular platelet activation pathways. Theraflex influences platelet shape change and aggregation, but the data reported to date are limited. This information provides the basis to understand the impact of different PI on the molecular mechanisms of platelet function. Moreover, these data may serve as basis for future developments of PI technologies for PCs. Further studies should address the impact of both the PI and the storage duration on platelets in PCs because PI may enable the extension of the shelf life of PCs by reducing the bacterial contamination risk.

Oxidation of proteins: Basic principles and perspectives for blood proteomics

Protein oxidation mechanisms result in a wide array of modifications, from backbone cleavage or protein crosslinking to more subtle modifications such as side chain oxidations. Protein oxidation occurs as part of normal regulatory processes, as a defence mechanism against oxidative stress, or as a deleterious processes when antioxidant defences are overcome. Because blood is continually exposed to reactive oxygen and nitrogen species, blood proteomics should inherently adopt redox proteomic strategies. In this review, we recall the biochemical basis of protein oxidation, review the proteomic methodologies applied to analyse redox modifications, and highlight some physiological and in vitro responses to oxidative stress of various blood components.

Haemovigilance in a general university hospital: need for a more comprehensive classification and a codification of transfusion‐related events

Background and Objectivesu2002 The purpose of this study was to analyse the transfusion‐related events recorded in a general university hospital.

Improving platelet transfusion safety: biomedical and technical considerations.

Platelet concentrates account for near 10% of all labile blood components but are responsible for more than 25% of the reported adverse events. Besides factors related to patients themselves, who may be particularly at risk of side effects because of their underlying illness, there are aspects of platelet collection and storage that predispose to adverse events. Platelets for transfusion are strongly activated by collection through disposal equipment, which can stress the cells, and by preservation at 22 °C with rotation or rocking, which likewise leads to platelet activation, perhaps more so than storage at 4 °C. Lastly, platelets constitutively possess a very large number of bioactive components that may elicit pro-inflammatory reactions when infused into a patient. This review aims to describe approaches that may be crucial to minimising side effects while optimising safety and quality. We suggest that platelet transfusion is complex, in part because of the complexity of the material itself: platelets are highly versatile cells and the transfusion process adds a myriad of variables that present many challenges for preserving basal platelet function and preventing dysfunctional activation of the platelets. The review also presents information showing--after years of exhaustive haemovigilance--that whole blood buffy coat pooled platelet components are extremely safe compared to the gold standard (i.e. apheresis platelet components), both in terms of acquired infections and of immunological/inflammatory hazards.

Identification of specific proteins in different lymphocyte populations by proteomic tools

The solubilized proteins of purified CD19+ (B), CD8+ (T) as well as CD4+ (T) lymphocytes were separated by high resolution two‐dimensional polyacrylamide gel electrophoresis, and the gels were analyzed using Melanie 3.0. Nine gels were studied, three for each lymphocyte population. After image analysis, 1411 ± 73 spots (mean + SD) were detected. The protein pattern of B lymphocytes segregated from the one of T lymphocytes by ascendant heuristic clustering analysis. In addition, computer analysis separated CD8+ from CD4+ lymphocytes. When a search was performed in order to detect subsets of specific spots (presence vs. absence), a group of three spots, detected in the area of the protein maps corresponding to isoelectric point (pI) of 5.2 to 5.4 and molecular weight (Mr) of 50 to 51 kDa, were found in both CD8+ and CD4+ cells, but not in CD19+ cells. Mass spectrometry analysis revealed that these spots were associated with several proteins such as vimentin, tubulin, desmin and cytokeratin. Two spots, located in the area of the gel corresponding to pI of about 5.0 and a Mr of 30 kDa, appeared as CD8+ cell associated. Mass spectrometry analysis showed that the two spots were related to the same non‐identified protein. Moreover internal peptides sequences matched with two human expressed sequence tags: gi|9759776, gi|12798420. No spots were found as only B cell associated.

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. Am. J. Hematol. 91:467–472, 2016.

MONO‐OLIGOCLONAL PRODUCTION OF IMMUNOGLOBULINS IN A CHILD WITH THE WISKOTT‐ALDRICH SYNDROME

We describe a patient with WAS, who developed severe haemorrhagic eczema and thrombocytopenia at the age of 6 months. He also suffered from multiple episodes of otitis and brochitis. At the age of 4.5 years, a splenectomy was performed and two enlarged hilar lymph nodes composed of monoclonal λ-positive B lymphocytes were found

Red blood cells ageing markers: a multi-parametric analysis

BACKGROUNDnRed 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.nnnMATERIALS AND METHODSnFive 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.nnnRESULTSnThe 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.nnnDISCUSSIONnVarious 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.

Autologous Blood Transfusion in Sports: Emerging Biomarkers

Despite being prohibited by the World Anti-Doping Agency, blood doping through erythropoietin injection or blood transfusion is frequently used by athletes to increase oxygen delivery to muscles and enhance performance. In contrast with allogeneic blood transfusion and erythropoietic stimulants, there is presently no direct method of detection for autologous blood transfusion (ABT) doping. Blood reinfusion is currently monitored with individual follow-up of hematological variables via the athlete biological passport, which requires further improvement. Microdosage is undetectable, and suspicious profiles in athletes are often attributed to exposure to altitude, heat stress, or illness. Additional indirect biomarkers may increase the sensitivity and specificity of the longitudinal approach. The emergence of -omics strategies provides new opportunities to discover biomarkers for the indirect detection of ABT. With the development of direct quantitative methods, transcriptomics based on microRNA or messenger RNA expression is a promising approach. Because blood donation and blood reinfusion alter iron metabolism, quantification of proteins involved in metal metabolism, such as hepcidin, may be applied in an ironomics strategy to improve the detection of ABT. As red blood cell (RBC) storage triggers changes in membrane proteins, proteomic methods have the potential to identify the presence of stored RBCs in blood. Alternatively, urine matrix can be used for the quantification of the plasticizer di(2-ethyhexyl)phthalate and its metabolites that originate from blood storage bags, suggesting recent blood transfusion, and have an important degree of sensitivity and specificity. This review proposes that various indirect biomarkers should be applied in combination with mathematical approaches for longitudinal monitoring aimed at improving ABT detection.