Hypoxia and hypocapnia storage of γ-irradiated red cell concentrates.

BACKGROUND: γ-irradiation is used to treat red blood cell (RBC) concentrates (RCCs) transfused to immunosuppressed patients. This treatment damages RBCs and increases storage lesions. Several studies have shown the beneficial effect of reducing O2 content during RBC storage. The present research work investigated the effect of γ-irradiation on RCCs stored under normal and hypoxia/hypocapnia conditions. MATERIALS AND METHODS: O2 concentration (measured as oxyhaemoglobin fraction, sO2) and ABO-matched RCCs from whole blood donations, leukoreduced and prepared in phosphate, adenine, glucose, guanosine, saline and mannitol (PAGGSM) were pooled and split in two identical RCCs within 24 h post donation. One bag (Hx) was submitted to O2 and CO2 adsorption for 3 h on an orbital shaker at 22±2 °C and then transferred to a storage bag impermeable to gas. The other bag (Ctrl) was left as it was. The two bags were then stored at 4 °C. γ-irradiation (25 Gy) was applied at day 2 or 14, and the RCCs were stored until day 43. Different parameters (metabolites, haemolysis, morphology) were measured. RESULTS: Starting sO2 values were 63.7±18.4% (n=12) in Ctrl and 20.8±9.8% (n=12) in Hx bags, and reached 90.8±9.1% and 6.6±5.9% at day 43, respectively. As expected, an increase in glycolysis rate was observed after deoxygenation. Extracellular potassium concentrations were identical and reached around 70 mM at expiry with an irradiation-dependent kinetic release. No difference in haemolysis was observed after irradiation on day 2 in either group (<0.40%, p>0.9999). When irradiated at day 14, haemolysis was lower (p=0.033) in RCCs under hypoxia at the end of storage (day 28, 0.67±0.16%) compared to control (1.06±0.33%). Percentages of spherocytes were lower under hypoxia. DISCUSSION: The storage under hypoxia provided equivalent storage when RCCs were irradiated at day 2 and was advantageous when irradiated at day 14. In summary, O2-depletion of RCCs enable a better storage of RBCs, particularly when late irradiation is applied.

Antioxidant power measurement in platelet concentrates treated by two pathogen inactivation systems in different blood centres.

BACKGROUND AND OBJECTIVES: The antioxidant power measurement can be useful to validate the execution of the pathogen inactivation treatment of platelet concentrates. The aim of this study is to evaluate the technology on different blood preparations including INTERCEPT and Mirasol treatments that are in routine use in Belgium and Luxemburg. MATERIALS AND METHODS: The antioxidant power measurement was tested on 78 apheresis platelet concentrates and 54 pools of buffy-coats-derived platelet concentrates before and after INTERCEPT treatment. In addition, 100 Reveos platelet pools were tested before and after Mirasol treatment. The antioxidant power was quantified electrochemically using disposable devices and was expressed as equivalent ascorbic acid concentration. RESULTS: Mean results for apheresis platelet concentrates were of 90 ± 14 and 35 ± 10 µmol/l eq. ascorbic acid before and after INTERCEPT treatment, respectively. The mean results for pools of buffy-coats-derived platelet concentrates were of 81 ± 10 and 29 ± 4 eq. µmol/l ascorbic acid before and after INTERCEPT treatment, respectively. For buffy-coats-derived platelet concentrates treated by Mirasol technology, the mean results were of 98 ± 11 and 32 ± 10 µmol/l eq. ascorbic acid before and after illumination, respectively. CONCLUSION: The antioxidant power significantly decreases with pathogen inactivation treatments for platelet concentrates treated by INTERCEPT or Mirasol technologies.

Sulfenylome analysis of pathogen-inactivated platelets reveals the presence of cysteine oxidation in integrin signaling pathway and cytoskeleton regulation.

Essentials Cysteine oxidation to sulfenic acid plays a key role in redox regulation and signal transduction. Platelet sulfenylome was studied by quantitative proteomics in pathogen inactivated platelets. One hundred and seventy-four sulfenylated proteins were identified in resting platelets. Pathogen inactivation oxidized integrin ßIII, which could activate the mitogen-activated protein kinases pathway. ABSTRACT: Background Cysteine-containing protein modifications are involved in numerous biological processes such redox regulation or signal transduction. During the preparation and storage of platelet concentrates, cell functions and protein regulations are impacted. In spite of several proteomic investigations, the platelet sulfenylome, ie, the proteins containing cysteine residues (R-SH) oxidized to sulfenic acid (R-SOH), has not been characterized. Methods A dimedone-based sulfenic acid tagging and enrichment coupled to a mass spectrometry identification workflow was developed to identify and quantify the sulfenic acid-containing proteins in platelet concentrates treated or not with an amotosalen/ultraviolet A (UVA) pathogen inactivation technique. Results One hundred and seventy-four sulfenylated proteins were identified belonging mainly to the integrin signal pathway and cytoskeletal regulation by Rho GTPase. The impact on pathogen inactivated platelet concentrates was weak compared to untreated ones where three sulfenylated proteins (myosin heavy chain 9, integrin ßIII, and transgelin 2) were significantly affected by amotosalen/UVA treatment. Of particular interest, the reported oxidation of cysteine residues in integrin ßIII is known to activate the receptor αIIbßIII. Following the pathogen inactivation, it might trigger the phosphorylation of p38MAPK and explain the lesions reported in the literature. Moreover, procaspase activating compound-1 (PAC-1) binding assays on platelet activation showed an increased response to adenosine diphosphate exacerbated by the tagging of proteins with dimedone. This result corroborates the hypothesis of an oxidation-triggered activation of αIIbßIII by the pathogen inactivation treatment. Conclusions The present work completes missing information on the platelet proteome and provides new insights on the effect of pathogen inactivation linked to integrin signaling and cytoskeleton regulation.

Transfusion-transmitted cytomegalovirus: behaviour of cell-free virus during blood component processing. A study on the safety of labile blood components in Switzerland.

BACKGROUND: Nowadays, most blood products are leukocyte-reduced. After this procedure, the residual risk for transfusion transmitted cytomegalovirus (TT-CMV) is mostly attributed to cell-free viruses in the plasma of blood donors following primary infection or viral reactivation. Here, objectives are: 1) to study the behaviour of cell-free CMV through the blood component processing; 2) to determine the anti-CMV seroprevalence, the level of viremia, the window-period in blood donor population; and 3) to identify cases of TT-CMV in bone marrow transplant (BMT) recipients. MATERIALS AND METHODS: Cell-free CMV was injected into blood bags originating from regular donors. Blood components were processed according to either the CompoSelect® or the CompoFlow® (Fresenius Kabi AG) techniques. Samples were analysed at each step for presence of virus DNA using quantitative polymerase chain reaction (PCR). The anti-CMV seroprevalence in our donor population was taken from our donor data system. The viremia was assessed in pooled plasmas samples from routine donations by quantitative PCR. Medical charts of 165 BMT anti-CMV seronegative recipients/anti-CMV seronegative donors who received CMV-unscreened blood products were reviewed. RESULTS: Cell-free CMV passes without any decrease in viral load through all stages of blood processing. The anti-CMV seroprevalence was 46.13%. Four DNA positive samples out of 42,240 individual blood donations were identified (0.009%); all had low levels of viremia (range 11-255 IU/mL). No window-period donation was identified. No TT-CMV was found. DISCUSSION: Cell-free CMV remains a concern with current blood component processing as it passes through all the processes. However, since low levels of CMV DNA were identified in the donations tested, and no BMT recipients had TT-CMV, the residual threat of TT-CMV after leukocyte reduction appears to be very low.

Oxygen in Red Blood Cell Concentrates: Influence of Donors' Characteristics and Blood Processing.

Objective: Unexpectedly wide distribution (<10 to >90%) of hemoglobin oxygen saturation (sO2) within red cell concentrates (RCCs) has recently been observed. Causes of such variability are not yet completely explained whereas the roles of oxygen and oxidative lesions during the storage of RCCs are known. The objectives of the present study are to characterize sO2 distribution in RCCs produced in a Swiss blood center and to investigate the influence of processing and donors' characteristics. Methods: The level of sO2 was measured in 1701 leukocyte-depleted RCCs derived from whole blood donations in both top-bottom (TB; component filtered, SAGM) and top-top (TT; whole blood filtration, PAGGSM) RCCs. The sO2 value was measured non-invasively through the PVC bag prior to storage by resonance Raman spectroscopy. Gender, age, blood type, hemoglobin level, and living altitude of donors, as well as process method and time-to-process were recorded. Results: Overall, the sO2 exhibited a wide non-Gaussian distribution with a mean of 51.2 ± 18.5%. Use of top-top kits resulted in a 16% higher sO2 (P < 0.0001) than with top-bottom ones. Waiting time before processing only had a modest impact, but the blood processing itself reduced the sO2 by almost 12% (P < 0.0001). sO2 was also significantly affected by some donors' characteristics. RCCs from men exhibited 25% higher sO2 (P < 0.0001) than those donated by women. Multivariate analysis revealed that the apparent correlation observed with hemoglobin level and age was actually due to multicollinearity with the sex variable. Finally, we noticed no significant differences across blood type but found that altitude of residence was associated with the sO2 (i.e., higher in higher living place). Conclusion: These data confirm wide sO2 distribution in RCCs reported recently. The sO2 was impacted by the processing and also by donors' characteristics such as the gender and the living altitude, but not by the hemoglobin level, blood group and donor age. This study provides new hints on the factors influencing red blood cells storage lesions, since they are known to be related to O2 content within the bags, giving clues to better process and to better store RCCs and therefore potentially improve the efficacy of transfusion.

General overview of blood products in vitro quality: Processing and storage lesions.

Blood products are issued from blood collection. Collected blood is immediately mixed with anticoagulant solutions that immediately induce chemical and/or biochemical modifications. Collected blood is then transformed into different blood products according to various steps of fabrication. All these steps induce either reversible or irreversible "preparation-related" lesions that combine with "storage-related" lesions. This short paper aims to provide an overview of the alterations that are induced by the "non-physiological" processes used to prepare blood products that are used in clinical practice.

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.

Chronic miR-29 antagonism promotes favorable plaque remodeling in atherosclerotic mice.

Abnormal remodeling of atherosclerotic plaques can lead to rupture, acute myocardial infarction, and death. Enhancement of plaque extracellular matrix (ECM) may improve plaque morphology and stabilize lesions. Here, we demonstrate that chronic administration of LNA-miR-29 into an atherosclerotic mouse model improves indices of plaque morphology. This occurs due to upregulation of miR-29 target genes of the ECM (col1A and col3A) resulting in reduced lesion size, enhanced fibrous cap thickness, and reduced necrotic zones. Sustained LNA-miR-29 treatment did not affect circulating lipids, blood chemistry, or ECM of solid organs including liver, lung, kidney, spleen, or heart. Collectively, these data support the idea that antagonizing miR-29 may promote beneficial plaque remodeling as an independent approach to stabilize vulnerable atherosclerotic lesions.

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.

Small-Scale Perfusion Bioreactor of Red Blood Cells for Dynamic Studies of Cellular Pathways: Proof-of-Concept.

To date, the development of bioreactors for the study of red blood cells (RBCs, daily transfused in the case of disease or hemorrhage) has focused on hematopoietic stem cells. Despite the fact that mature RBCs are enucleated and do not expand, they possess complex cellular and metabolic pathways, as well as post-translation modification signaling and gas-exchange regulation. In order to dynamically study the behavior of RBCs and their signaling pathways under various conditions, a small-scale perfusion bioreactor has been developed. The most advanced design developed here consists of a fluidized bed of 7.6 mL containing 3·10(9) cells and perfused at 8.5 µL/min. Mimicking RBC storage conditions in transfusion medicine, as a proof-of-concept, we investigated the ex vivo aging of RBCs under both aerobic and anaerobic conditions. Hence, RBCs stored in saline-adenine-glucose-mannitol (SAGM) were injected in parallel into two bioreactors and perfused with a modified SAGM solution over 14 days at room temperature under air or argon. The formation of a fluidized bed enabled easy sampling of the extracellular medium over the storage period used for the quantitation of glucose consumption and lactate production. Hemolysis and microvesiculation increased during aging and were reduced under anaerobic (argon) conditions, which is consistent with previously reported findings. Glucose and lactate levels showed expected trends, i.e., decreased and increased during the 2-week period, respectively; whereas extracellular glucose consumption was higher under aerobic conditions. Metabolomics showed depletion of glycolsis and pentose phosphate pathway metabolites, and an accumulation of purine metabolite end-products. This novel approach, which takes advantage of a fluidized bed of cells in comparison to traditional closed bags or tubes, does not require agitation and limit shear stress, and constantly segragates extracellular medium from RBCs. It thus gives access to several difficult-to-obtain on- and off-line parameters in the extracellular medium. This dynamic bioreactor system does not only allow us to probe the behavior of RBCs under different storage conditions, but it also could be a powerful tool to study physiological or pathological RBCs exposed to various conditions and stimuli.

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.

In vitro evaluation of pathogen-inactivated buffy coat-derived platelet concentrates during storage: psoralen-based photochemical treatment step-by-step.

BACKGROUND: The Intercept Blood SystemTM (Cerus) is used to inactivate pathogens in platelet concentrates (PC). The aim of this study was to elucidate the extent to which the Intercept treatment modifies the functional properties of platelets. MATERIAL AND METHODS: A two-arm study was conducted initially to compare buffy coat-derived pathogen-inactivated PC to untreated PC (n=5) throughout storage. A four-arm study was then designed to evaluate the contribution of the compound adsorbing device (CAD) and ultraviolet (UV) illumination to the changes observed upon Intercept treatment. Intercept-treated PC, CAD-incubated PC, and UV-illuminated PC were compared to untreated PC (n=5). Functional characteristics were assessed using flow cytometry, hypotonic shock response (HSR), aggregation, adhesion assays and flow cytometry for the detection of CD62P, CD42b, GPIIb-IIIa, phosphatidylserine exposure and JC-1 aggregates. RESULTS: Compared to fresh platelets, end-of-storage platelets exhibited greater passive activation, disruption of the mitochondrial transmembrane potential (Δψm), and phosphatidylserine exposure accompanied by a decreased capacity to respond to agonist-induced aggregation, lower HSR, and CD42b expression. The Intercept treatment resulted in significantly lower HSR and CD42b expression compared to controls on day 7, with no significant changes in CD62P, Δψm, or phosphatidylserine exposure. GPIIbIIIa expression was significantly increased in Intercept-treated platelets throughout the storage period. The agonist-induced aggregation response was highly dependent on the type and concentration of agonist used, indicating a minor effect of the Intercept treatment. The CAD and UV steps alone had a negligible effect on platelet aggregation. DISCUSSION: The Intercept treatment moderately affects platelet function in vitro. CAD and UV illumination alone make negligible contributions to the changes in aggregation observed in Intercept-treated PC.

The clinical and biological impact of new pathogen inactivation technologies on platelet concentrates.

Since 1990, several techniques have been developed to photochemically inactivate pathogens in platelet concentrates, potentially leading to safer transfusion therapy. The three most common methods are amotosalen/UVA (INTERCEPT Blood System), riboflavin/UVA-UVB (MIRASOL PRT), and UVC (Theraflex-UV). We review the biology of pathogen inactivation methods, present their efficacy in reducing pathogens, discuss their impact on the functional aspects of treated platelets, and review clinical studies showing the clinical efficiency of the pathogen inactivation methods and their possible toxicity.

LC-MS/MS analysis and comparison of oxidative damages on peptides induced by pathogen reduction technologies for platelets.

Pathogen reduction technologies (PRT) are photochemical processes that use a combination of photosensitizers and UV-light to inactivate pathogens in platelet concentrates (PCs), a blood-derived product used to prevent hemorrhage. However, different studies have questioned the impact of PRT on platelet function and transfusion efficacy, and several proteomic analyses revealed possible oxidative damages to proteins. The present work focused on the oxidative damages produced by the two main PRT on peptides. Model peptides containing residues prone to oxidation (tyrosine, histidine, tryptophane, and cysteine) were irradiated with a combination of amotosalen/UVA (Intercept process) or riboflavin/UVB (Mirasol-like process). Modifications were identified and quantified by liquid chromatography coupled to tandem mass spectrometry. Cysteine-containing peptides formed disulfide bridges (R-SS-R, -2 Da; favored following amotosalen/UVA), sulfenic and sulfonic acids (R-SOH, +16 Da, R-SO3H, +48 Da, favored following riboflavin/UVB) upon treatment and the other amino acids exhibited different oxidations revealed by mass shifts from +4 to +34 Da involving different mechanisms; no photoadducts were detected. These amino acids were not equally affected by the PRT and the combination riboflavin/UVB generated more oxidation than amotosalen/UVA. This work identifies the different types and sites of peptide oxidations under the photochemical treatments and demonstrates that the two PRT may behave differently. The potential impact on proteins and platelet functions may thus be PRT-dependent.

Gene network and proteomic analyses of cardiac responses to pathological and physiological stress.

BACKGROUND: The molecular mechanisms underlying similarities and differences between physiological and pathological left ventricular hypertrophy (LVH) are of intense interest. Most previous work involved targeted analysis of individual signaling pathways or screening of transcriptomic profiles. We developed a network biology approach using genomic and proteomic data to study the molecular patterns that distinguish pathological and physiological LVH. METHODS AND RESULTS: A network-based analysis using graph theory methods was undertaken on 127 genome-wide expression arrays of in vivo murine LVH. This revealed phenotype-specific pathological and physiological gene coexpression networks. Despite >1650 common genes in the 2 networks, network structure is significantly different. This is largely because of rewiring of genes that are differentially coexpressed in the 2 networks; this novel concept of differential wiring was further validated experimentally. Functional analysis of the rewired network revealed several distinct cellular pathways and gene sets. Deeper exploration was undertaken by targeted proteomic analysis of mitochondrial, myofilament, and extracellular subproteomes in pathological LVH. A notable finding was that mRNA-protein correlation was greater at the cellular pathway level than for individual loci. CONCLUSIONS: This first combined gene network and proteomic analysis of LVH reveals novel insights into the integrated pathomechanisms that distinguish pathological versus physiological phenotypes. In particular, we identify differential gene wiring as a major distinguishing feature of these phenotypes. This approach provides a platform for the investigation of potentially novel pathways in LVH and offers a freely accessible protocol (http://sites.google.com/site/cardionetworks) for similar analyses in other cardiovascular diseases.

Extracellular matrix secretion by cardiac fibroblasts: role of microRNA-29b and microRNA-30c.

RATIONALE: MicroRNAs (miRNAs), in particular miR-29b and miR-30c, have been implicated as important regulators of cardiac fibrosis. OBJECTIVE: To perform a proteomics comparison of miRNA effects on extracellular matrix secretion by cardiac fibroblasts. METHODS AND RESULTS: Mouse cardiac fibroblasts were transfected with pre-/anti-miR of miR-29b and miR-30c, and their conditioned medium was analyzed by mass spectrometry. miR-29b targeted a cadre of proteins involved in fibrosis, including multiple collagens, matrix metalloproteinases, and leukemia inhibitory factor, insulin-like growth factor 1, and pentraxin 3, 3 predicted targets of miR-29b. miR-29b also attenuated the cardiac fibroblast response to transforming growth factor-ß. In contrast, miR-30c had little effect on extracellular matrix production but opposite effects regarding leukemia inhibitory factor and insulin-like growth factor 1. Both miRNAs indirectly affected cardiac myocytes. On transfection with pre-miR-29b, the conditioned medium of cardiac fibroblasts lost its ability to support adhesion of rat ventricular myocytes and led to a significant reduction of cardiac myocyte proteins (α-actinin, cardiac myosin-binding protein C, and cardiac troponin I). Similarly, cardiomyocytes derived from mouse embryonic stem cells atrophied under pre-miR-29 conditioned medium, whereas pre-miR-30c conditioned medium had a prohypertrophic effect. Levels of miR-29a, miR-29c, and miR-30c, but not miR-29b, were significantly reduced in a mouse model of pathological but not physiological hypertrophy. Treatment with antagomiRs to miR-29b induced excess fibrosis after aortic constriction without overt deterioration in cardiac function. CONCLUSIONS: Our proteomic analysis revealed novel molecular targets of miRNAs that are linked to a fibrogenic cardiac phenotype. Such comprehensive screening methods are essential to define the concerted actions of miRNAs in cardiovascular disease.

Lysis-on-chip of single target cells following forced interaction with CTLs or NK cells on a dielectrophoresis-based array.

Guiding the interaction of single cells acting as partners in heterotypic interactions (e.g., effectors and targets of immune lysis) and monitoring the outcome of these interactions are regarded as crucial biomedical achievements. In this study, taking advantage of a dielectrophoresis (DEP)-based Laboratory-on-a-chip platform (the DEPArray), we show that it is possible to generate closed DEP cages entrapping CTLs and NK cells as either single cells or clusters; reversibly immobilize a single virus-presenting or tumor cell within the chip at a selected position; move cages and their content to predetermined spatial coordinates by software-guided routing; force a cytotoxic effector to physically interact with a putative target within a secluded area by merging their respective cages; generate cages containing effector and target cells at predetermined E:T ratios; accurately assess cytotoxicity by real-time quantitation of the release kinetics of the fluorescent dye calcein from target cells (>50 lytic events may be tested simultaneously); estimate end points of calcein release within 16 min of initial E:T cell contact; simultaneously deliver Ab-based phenotyping and on-chip lysis assessment; and identify lytic and nonlytic E:T combinations and discriminate nonlytic effector phenotypes from target refractoriness to immune lysis. The proof of principle is provided that DEPArray technology, previously used to levitate and move single cells, can be used to identify highly lytic antiviral CTLs and tumor cells that are particularly refractory to NK cell lysis. These findings are of primary interest in targeted immunotherapy.

Programmable interactions of functionalized single bioparticles in a dielectrophoresis-based microarray chip.

Manipulating single biological objects is a major unmet challenge of biomedicine. Herein, we describe a lab-on-a-chip platform based on dielectrophoresis (DEP). The DEParray is a prototypal version consisting of 320 × 320 arrayed electrodes generating >10,000 spherical DEP cages. It allows the capture and software-guided movement to predetermined spatial coordinates of single biological objects. With the DEParray we demonstrate (a) forced interaction between a single, preselected target cell and a programmable number of either microspheres or natural killer (NK) cells, (b) on-chip immunophenotypic discrimination of individual cells based on differential rosetting with microspheres functionalized with monoclonal antibodies to an inhibitory NK cell ligand (HLA-G), (c) on-chip, real-time (few minutes) assessment of immune lysis by either visual inspection or semiautomated, time-lapse reading of a fluorescent dye released from NK cell-sensitive targets, and (d) manipulation and immunophenotyping with limiting amounts (about 500) cells. To our knowledge, this is the first report describing a DEP-based lab-on-a-chip platform for the quick, arrayed, software-guided binding of individually moved biological objects, the targeting of single cells with microspheres, and the real-time characterization of immunophenotypes. The DEParray candidates as a discovery tool for novel cell:cell interactions with no prior (immuno)phenotypic knowledge.

Highlights from the 2010 BAS/BSCR spring meeting: New Frontiers in Cardiovascular Research.

The British Atherosclerosis Society (BAS)/British Society for Cardiovascular Research (BSCR) spring meeting was held in Manchester, UK, on 7-8 June 2010. Experts in the field of systems biology, proteomics, metabolomics and miRNAs presented how these techniques can be used to discover 'New Frontiers in Cardiovascular Research'. The conference was attended by over 150 participants, mainly from the UK. A total of 2 days of presentations and a poster session with 55 posters provided the possibility to discuss the latest research results and showed the opportunities that new techniques can offer in cardiovascular research.

Electrochemical aspects of electrospray and laser desorption/ionization for mass spectrometry.

Soft-ionization methods, namely electrospray ionization and laser desorption/ionization, are widely used to transfer large molecules as intact gas-phase ions either from a solution or from a solid substrate. During both processes, in-source electrochemical and photoelectrochemical reactions occur. These electrode reactions, which take place at interfaces, play important roles in influencing the ionization products, but they have received little attention. We show that having good control over both types of electrochemical reactions can lead to new analytical applications. Examples include online tagging by grafting of mass tags and in-source photooxidation of peptides.