RESUMO
Regulatory T cells (Tregs) are a unique subset of lymphocytes that play a vital role in regulating the immune system by suppressing unwanted immune responses and thus preventing autoimmune diseases and inappropriate inflammatory reactions. In preclinical and clinical trials, these cells have demonstrated the ability to prevent and treat graft vs. host disease, alleviate autoimmune symptoms, and promote transplant tolerance. In this review, we provide a background on Treg cells with a focus on important Treg cell markers and Treg subsets, and outline the methodology currently used for manufacturing adoptive regulatory T cell therapies (TRACT). Finally, we discuss the approaches and outcomes of several clinical trials in which Tregs have been adoptively transferred to patients.
Assuntos
Doenças Autoimunes , Doença Enxerto-Hospedeiro , Humanos , Linfócitos T Reguladores , Imunoterapia Adotiva/métodos , Doenças Autoimunes/terapiaRESUMO
BACKGROUND: Red cell (RBC) transfusions are beneficial for patients with sickle cell disease (SCD), but ex vivo studies suggest that inflamed plasma from patients with SCD during crises may damage these RBCs, diminishing their potential efficacy. The hypoxic storage of RBCs may improve transfusion efficacy by minimizing the storage lesion. We tested the hypotheses that (1) The donor RBCs exposed to the plasma of patients in crisis would have lower deformability and higher hemolysis than those exposed to non-crisis plasma, and (2) hypoxic storage, compared to standard storage, of donor RBCs could preserve deformability and reduce hemolysis. STUDY DESIGN AND METHODS: 18 SCD plasma samples from patients who had severe acute-phase symptoms (A-plasma; n = 9) or were at a steady-state (S = plasma; n = 9) were incubated with 16 RBC samples from eight units that were stored either under conventional(CRBC) or hypoxic(HRBC) conditions. Hemolysis and microcapillary deformability assays of these RBCs were analyzed using linear mixed-effect models after each sample was incubated in patient plasma overnight at 37°C RESULTS: Relative deformability was 0.036 higher (p < 0.0001) in HRBC pairs compared to CRBC pairs regardless of plasma type. Mean donor RBC hemolysis was 0.33% higher after incubation with A-plasma compared to S-plasma either with HRBC or CRBC (p = 0.04). HRBCs incubated with steady-state patient plasma demonstrated the highest deformability and lowest hemolysis. CONCLUSION: Hypoxic storage significantly influenced RBC deformability. Patient condition significantly influenced post-incubation hemolysis. Together, HRBCs in steady-state plasma maximized donor red cell ex vivo function and survival.
Assuntos
Anemia Falciforme , Hemólise , Humanos , Adulto , Preservação de Sangue , Eritrócitos/metabolismo , Doadores de Sangue , Deformação EritrocíticaRESUMO
BACKGROUND: Hypothermic storage transforms red blood cells (RBC) from smooth biconcave discocytes into increasingly spherical spiculated echinocytes and, ultimately, fragile spherocytes (S). Individual cells undergo this transformation at different rates, producing a heterogeneous mixture of RBCs at all stages of echinocytosis in each unit of stored blood. Here we investigated how washing (known to positively affect RBC properties) changes morphology of individual RBCs at the single-cell level. STUDY DESIGN AND METHODS: We tracked the change in shape of individual RBCs (n = 2870; drawn from six 4- to 6-week-old RBC units) that were confined in an array of microfluidic wells during washing in saline (n = 1095), 1% human serum albumin (1% HSA) solution (n = 999), and the autologous storage supernatant (control, n = 776). RESULTS: Shape recovery proceeded through the disappearance of spicules followed by the progressive smoothening of the RBC contour, with the majority of changes occurring within the initial 10 minutes of being exposed to the washing solution. Approximately 57% of all echinocytes recovered by at least one morphologic class when washed in 1% HSA (36% for normal saline), with 3% of cells in late-stage echinocytosis restoring their discoid shape completely. Approximately one-third of all spherocytic cells were lysed in either washing solution. Cells washed in their autologous storage supernatant continued to deteriorate during washing. CONCLUSION: Our findings suggest that the replacement of storage supernatant with a washing solution during washing induces actual shape recovery for RBCs in all stages of echinocytosis, except for S that undergo lysis instead.
Assuntos
Preservação de Sangue , Forma Celular , Eritrócitos , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas , Análise de Célula Única , Eritrócitos/citologia , Eritrócitos/metabolismo , Humanos , Fatores de TempoRESUMO
BACKGROUND: Great deformability allows red blood cells (RBCs) to flow through narrow capillaries in tissues. A number of microfluidic devices with capillary-like microchannels have been developed to monitor storage-related impairment of RBC deformability during blood banking operations. This proof-of-concept study describes a new method to standardize and improve reproducibility of the RBC deformability measurements using one of these devices. STUDY DESIGN AND METHODS: The rate of RBC flow through the microfluidic capillary network of the microvascular analyzer (MVA) device made of polydimethylsiloxane was measured to assess RBC deformability. A suspension of microbeads in a solution of glycerol in phosphate-buffered saline was developed to be used as an internal flow rate reference alongside RBC samples in the same device. RBC deformability and other in vitro quality markers were assessed weekly in six leukoreduced RBC concentrates (RCCs) dispersed in saline-adenine-glucose-mannitol additive solution and stored over 42 days at 4°C. RESULTS: The use of flow reference reduced device-to-device measurement variability from 10% to 2%. Repeated-measure analysis using the generalized estimating equation (GEE) method showed a significant monotonic decrease in relative RBC flow rate with storage from Week 0. By the end of storage, relative RBC flow rate decreased by 22 ± 6% on average. CONCLUSIONS: The suspension of microbeads was successfully used as a flow reference to increase reproducibility of RBC deformability measurements using the MVA. Deformability results suggest an early and late aging phase for stored RCCs, with significant decreases between successive weeks suggesting a highly sensitive measurement method.
Assuntos
Deformação Eritrocítica/fisiologia , Eritrócitos/citologia , Eritrócitos/fisiologia , Dispositivos Lab-On-A-Chip/normas , Técnicas Analíticas Microfluídicas , Bancos de Sangue/normas , Velocidade do Fluxo Sanguíneo/fisiologia , Preservação de Sangue/efeitos adversos , Preservação de Sangue/métodos , Preservação de Sangue/normas , Criopreservação , Contagem de Eritrócitos/instrumentação , Contagem de Eritrócitos/métodos , Contagem de Eritrócitos/normas , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Citometria de Fluxo/normas , Hemólise , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/normas , Estudo de Prova de Conceito , Reprodutibilidade dos Testes , Fatores de Tempo , Armazenamento de Sangue/métodosRESUMO
BACKGROUND: The isolation of lymphocytes - and removal of platelets (PLTs) and red blood cells (RBCs) - from an initial blood sample prior to culture is a key enabling step for effective manufacture of cellular therapies. Unfortunately, currently available methods suffer from various drawbacks, including low cell recovery, need for complex equipment, potential loss of sterility and/or high materials/labor cost. METHODS: A newly developed system for selectively concentrating leukocytes within precisely designed, but readily fabricated, microchannels was compared with conventional density gradient centrifugation with respect to: (i) ability to recover lymphocytes while removing PLTs/RBCs and (ii) growth rate and overall cell yield once expanded in culture. RESULTS: In the optimal embodiment of the new microfluidic approach, recoveries of CD3+, CD19+ and CD56+ cells (85%, 89% and 97%, respectively) were significantly higher than for paired samples processed via gradient-based separation (51%, 53% and 40%). Although the removal of residual PLTs and RBCs was lower using the new approach, its enriched T-cell fraction nevertheless grew at a significantly higher rate than the gradient-isolated cells, with approximately twice the cumulative cell yield observed after 7 days of culture. DISCUSSION: The standardization of each step of cellular therapy manufacturing would enable an accelerated translation of research breakthroughs into widely available clinical treatments. The high-throughput approach described in this study - requiring no ancillary pumping mechanism nor expensive disposables to operate - may be a viable candidate to standardize and streamline the initial isolation of lymphocytes for culture while also potentially shortening the time required for their expansion into a therapeutic dose.
Assuntos
Separação Celular/métodos , Centrifugação com Gradiente de Concentração/métodos , Filtração/métodos , Microfluídica/instrumentação , Microfluídica/métodos , Linfócitos T/citologia , Transferência Adotiva/métodos , Plaquetas/citologia , Contagem de Células , Sobrevivência Celular , Células Cultivadas , Eritrócitos/citologia , HumanosRESUMO
Washed red blood cells (RBCs) are indicated for immunoglobulin A (IgA) deficient recipients. Centrifugation-based cell processors commonly used by hospital blood banks cannot consistently reduce IgA below the recommended levels, hence double washing is frequently required. Here, we describe a prototype of a simple, portable, disposable system capable of washing stored RBCs without centrifugation, while reducing IgA below 0.05 mg/dL in a single run. Samples from RBC units (n = 8, leukoreduced, 4-6 weeks storage duration) were diluted with normal saline to a hematocrit of 10%, and then washed using either the prototype washing system, or via conventional centrifugation. The efficiency of the two washing methods was quantified and compared by measuring several key in vitro quality metrics. The prototype of the washing system was able to process stored RBCs at a rate of 300 mL/hour, producing a suspension of washed RBCs with 43 ± 3% hematocrit and 86 ± 7% cell recovery. Overall, the two washing methods performed similarly for most measured parameters, lowering the concentration of free hemoglobin by >4-fold and total free protein by >10-fold. Importantly, the new washing system reduced the IgA level to 0.02 ± 0.01 mg/mL, a concentration 5-fold lower than that produced by conventional centrifugation. This proof-of-concept study showed that centrifugation may be unnecessary for washing stored RBCs. A simple, disposable, centrifugation-free washing system could be particularly useful in smaller medical facilities and resource limited settings that may lack access to centrifugation-based cell processors.
Assuntos
Preservação de Sangue/métodos , Centrifugação , Eritrócitos , Imunoglobulina A/sangue , Preservação de Sangue/instrumentação , Deformação Eritrocítica , Hemoglobinas/análise , Humanos , Hidrogéis , Membranas Artificiais , Estudo de Prova de ConceitoRESUMO
OBJECTIVE: RBCs suspended in plasma form multicellular aggregates under low-flow conditions, increasing apparent blood viscosity at low shear rates. It has previously been unclear, however, if RBC aggregation affects microvascular perfusion. Here, we analyzed the impact of RBC aggregation on perfusion and 'capillary' hematocrit in an AMVN at driving pressures ranging from 5 to 60 cm H2 O to determine if aggregation could improve tissue oxygenation. METHODS: RBCs were suspended at 30% hematocrit in either 46.5 g/L dextran 40 (D40, non-aggregating medium) or 35 g/L dextran 70 (D70, aggregating medium) solutions with equal viscosity. RESULTS: Aggregation was readily observed in the AMVN for RBCs suspended in D70 at driving pressures ≤40 cm H2 O. The AMVN perfusion rate was the same for RBCs suspended in aggregating and non-aggregating medium, at both 'venular' and 'capillary' level. Estimated 'capillary' hematocrit was higher for D70 suspensions than for D40 suspensions at intermediate driving pressures (5-40 cm H2 O). CONCLUSIONS: We conclude that although RBC aggregation did not affect the AMVN perfusion rate independently of the driving pressure, a higher hematocrit in the 'capillaries' of the network for D70 suspensions suggested a better oxygen transport capacity in the presence of RBC aggregation.
Assuntos
Agregação Eritrocítica , Microvasos/fisiologia , Modelos Cardiovasculares , Perfusão , Viscosidade Sanguínea , Hematócrito , Humanos , Oxigênio/metabolismoRESUMO
OBJECTIVE: Hct in narrow vessels is reduced due to concentration of fast-flowing RBCs in the center, and of slower flowing plasma along the wall of the vessel, which in combination with plasma skimming at bifurcations leads to the striking heterogeneity of local Hct in branching capillary networks known as the network Fåhraeus effect. We analyzed the influence of feeding Hct and perfusion pressure on the Fåhraeus effect in an AMVN. METHODS: RBC suspensions in plasma with Hcts between 20% and 70% were perfused at pressures of 5-60 cm H2 O through the AMVN. A microscope and high-speed camera were used to measure RBC velocity and Hct in microchannels of height of 5 µm and widths of 5-19 µm. RESULTS: Channel Hcts were reduced compared with Hctfeeding in 5 and 7 µm microchannels, but not in larger microchannels. The magnitude of Hct reduction increased with decreasing Hctfeeding and decreasing ΔP (flow velocity), showing an about sevenfold higher effect for 40% Hctfeeding and low pressure/flow velocity than for 60% Hctfeeding and high pressure/flow velocity. CONCLUSIONS: The magnitude of the network Fåhraeus effect in an AMVN is inversely related to Hctfeeding and ΔP.
Assuntos
Pressão Sanguínea , Capilares/metabolismo , Eritrócitos/metabolismo , Modelos Cardiovasculares , Capilares/citologia , Eritrócitos/citologia , Hematócrito , HumanosRESUMO
BACKGROUND: Higher hematocrit increases the oxygen-carrying capacity of blood but also increases blood viscosity, thus decreasing blood flow through the microvasculature and reducing the oxygen delivery to tissues. Therefore, an optimal value of hematocrit that maximizes tissue oxygenation must exist. STUDY DESIGN AND METHODS: We used viscometry and an artificial microvascular network device to determine the optimal hematocrit in vitro. Suspensions of fresh red blood cells (RBCs) in plasma, normal saline, or a protein-containing buffer and suspensions of stored red blood cells (at Week 6 of standard hypothermic storage) in plasma with hematocrits ranging from 10 to 80% were evaluated. RESULTS: For viscometry, optimal hematocrits were 10, 25.2, 31.9, 37.1, and 37.5% for fresh RBCs in plasma at shear rates of 3.2 or less, 11.0, 27.7, 69.5, and 128.5 inverse seconds. For the artificial microvascular network, optimal hematocrits were 51.1, 55.6, 59.2, 60.9, 62.3, and 64.6% for fresh RBCs in plasma and 46.4, 48.1, 54.8, 61.4, 65.7, and 66.5% for stored RBCs in plasma at pressures of 2.5, 5, 10, 20, 40, and 60 cm H2 O. CONCLUSION: Although exact optimal hematocrit values may depend on specific microvascular architecture, our results suggest that the optimal hematocrit for oxygen delivery in the microvasculature depends on perfusion pressure. Therefore, anemia in chronic disorders may represent a beneficial physiological response to reduced perfusion pressure resulting from decreased heart function and/or vascular stenosis. Our results may help explain why a therapeutically increasing hematocrit in such conditions with RBC transfusion frequently leads to worse clinical outcomes.
Assuntos
Hematócrito , Microvasos/fisiologia , Modelos Cardiovasculares , Oxigênio/metabolismo , Anemia/sangue , Anemia/fisiopatologia , Pressão Sanguínea , Viscosidade Sanguínea , Humanos , Consumo de OxigênioRESUMO
Although effective low-cost interventions exist, child mortality attributable to sickle cell disease (SCD) remains high in low-resource areas due, in large part, to the lack of accessible diagnostic methods. The presence of dense (ρ > 1.120 g/cm(3)) cells is characteristic of SCD. The fluid, self-assembling step-gradients in density created by aqueous multiphase systems (AMPSs) identifies SCD by detecting dense cells. AMPSs separate different forms of red blood cells by density in a microhematocrit centrifuge and provide a visual means to distinguish individuals with SCD from those with normal hemoglobin or with nondisease, sickle-cell trait in under 12 min. Visual evaluation of a simple two-phase system identified the two main subclasses of SCD [homozygous (Hb SS) and heterozygous (Hb SC)] with a sensitivity of 90% (73-98%) and a specificity of 97% (86-100%). A three-phase system identified these two types of SCD with a sensitivity of 91% (78-98%) and a specificity of 88% (74-98%). This system could also distinguish between Hb SS and Hb SC. To the authors' knowledge, this test demonstrates the first separation of cells by density with AMPSs, and the usefulness of AMPSs in point-of-care diagnostic hematology.
Assuntos
Anemia Falciforme/diagnóstico , Separação Celular/métodos , Anemia Falciforme/genética , Contagem de Células , Separação Celular/economia , Centrifugação , Variação Genética , Humanos , Sistemas Automatizados de Assistência Junto ao Leito/economia , Curva ROC , Fatores de TempoRESUMO
BACKGROUND: The shape of human red blood cells (RBCs) deteriorates progressively throughout hypothermic storage, with echinocytosis being the most prevalent pathway of this morphologic lesion. As a result, each unit of stored blood contains a heterogeneous mixture of cells in various stages of echinocytosis and normal discocytes. Here we studied how the change in shape of RBCs following along the path of the echinocytic transformation affects perfusion of an artificial microvascular network (AMVN). STUDY DESIGN AND METHODS: Blood samples were obtained from healthy consenting volunteers. RBCs were leukoreduced, resuspended in saline, and treated with various concentrations of sodium salicylate to induce shape changes approximating the stages of echinocytosis experienced by RBCs during hypothermic storage (e.g., discocyte, echinocyte I, echinocyte II, echinocyte III, spheroechinocyte, and spherocyte). The AMVN perfusion rate was measured for 40% hematocrit suspensions of RBCs with different shapes. RESULTS: The AMVN perfusion rates for RBCs with discocyte and echinocyte I shapes were similar, but there was a significant decline in the AMVN perfusion rate between RBCs with shapes approximating each subsequent stage of echinocytosis. The difference in AMVN perfusion between discocytes and spherocytes (the last stage of the echinocytic transformation) was 34%. CONCLUSION: The change in shape of RBCs from normal discocytes progressively through various stages of echinocytosis to spherocytes produced a substantial decline in the ability of these cells to perfuse an AMVN. Echinocytosis induced by hypothermic storage could therefore be responsible for a similarly substantial impairment of deformability previously observed for stored RBCs.
Assuntos
Eritrócitos/citologia , Microvasos/fisiologia , Perfusão , Animais , Viscosidade Sanguínea/fisiologia , Forma Celular , Deformação Eritrocítica/fisiologia , Eritrócitos/fisiologia , Humanos , Perfusão/instrumentação , Perfusão/métodos , RatosRESUMO
Plasma sodium concentration is normally held within a narrow range. It may however vary greatly under pathophysiological conditions. Changes in osmolality lead to either swelling or shrinkage of red blood cells (RBCs). Here we investigated the influence of suspension osmolality on biophysical properties of RBCs and their ability to perfuse an artificial microvascular network (AMVN). Blood was drawn from healthy volunteers. RBC deformability was measured by osmotic gradient ektacytometry over a continuous range of osmolalities. Packed RBCs were suspended in NaCl solutions (0.45, 0.6, 0.9, 1.2, and 1.5 g/dL), resulting in supernatant osmolalities of 179 ± 4, 213 ± 1, 283 ± 2, 354 ± 3, and 423 ± 5 mOsm/kg H2O. Mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC) were determined using centrifuged microhematocrit. RBC suspensions at constant cell numbers were used to measure viscosity at shear rates ranging from 0.11 to 69.5s(-1) and the perfusion rate of the AMVN. MCV was inversely and MCHC directly proportional to osmolality. RBC deformability was maximized at isosmotic conditions (290 mOsm/kg H2O) and markedly decreased by either hypo- or hyperosmolality. The optimum osmolality for RBC suspension viscosity was shifted toward hyperosmolality, while lower osmolalities increased suspension viscosity exponentially. However, the AMVN perfusion rate was maximized at 290 mOsm/kg H2O and changed by less than 10% over a wide range of osmolalities. These findings contribute to the basic understanding of blood flow in health and disease and may have significant implications for the management of osmotic homeostasis in clinical practice.
Assuntos
Eritrócitos/citologia , Hemoglobinas/química , Microvasos , Reologia , Adulto , Idoso , Viscosidade Sanguínea , Contagem de Eritrócitos , Deformação Eritrocítica/fisiologia , Índices de Eritrócitos , Voluntários Saudáveis , Homeostase , Humanos , Microcirculação , Microfluídica , Pessoa de Meia-Idade , Concentração Osmolar , Perfusão , Viscosidade , Adulto JovemRESUMO
BACKGROUND: Prolonged storage of red blood cells (RBCs) leads to storage lesions, which may impair clinical outcomes after transfusion. A hallmark of storage lesions is progressive echinocytic shape transformation, which can be partially reversed by washing in albumin solutions. Here we have investigated the impact of this shape recovery on biorheologic variables. STUDY DESIGN AND METHODS: RBCs stored hypothermically for 6 to 7 weeks were washed in a 1% human serum albumin (HSA) solution. RBC deformability was measured with osmotic gradient ektacytometry. The viscosity of RBC suspensions was measured with a Couette-type viscometer. The flow behavior of RBCs suspended at 40% hematocrit was tested with an artificial microvascular network (AMVN). RESULTS: Washing in 1% albumin reduced higher degrees of echinocytes and increased the frequency of discocytes, thereby shifting the morphologic index toward discocytosis. Washing also reduced RBC swelling. This shape recovery was not seen after washing in saline, buffer, or plasma. RBC shape normalization did not improve cell deformability measured by ektacytometry, but it tended to decrease suspension viscosities at low shear rates and improved the perfusion of an AMVN. CONCLUSIONS: Washing of stored RBCs in a 1% HSA solution specifically reduces echinocytosis, and this shape recovery has a beneficial effect on microvascular perfusion in vitro. Washing in 1% albumin may represent a new approach to improving the quality of stored RBCs and thus potentially reducing the likelihood of adverse clinical outcomes associated with transfusion of blood stored for longer periods of time.
Assuntos
Preservação de Sangue/métodos , Transfusão de Eritrócitos , Eritrócitos/efeitos dos fármacos , Albumina Sérica/farmacologia , Adenina/farmacologia , Trifosfato de Adenosina/sangue , Soluções Tampão , Forma Celular , Índices de Eritrócitos , Eritrócitos/citologia , Eritrócitos Anormais/ultraestrutura , Glucose/farmacologia , Glutationa/sangue , Guanosina/farmacologia , Hemorreologia , Humanos , Técnicas In Vitro , Manitol/farmacologia , Modelos Anatômicos , Oxirredução , Perfusão , Plasma , Refrigeração , Cloreto de Sódio/farmacologia , SoluçõesRESUMO
A critical accomplishment in the rapidly developing field of regenerative medicine will be the ability to foster repair of neurons severed by injury, disease, or microsurgery. In C. elegans, individual visualized axons can be laser-cut in vivo and neuronal responses to damage can be monitored to decipher genetic requirements for regeneration. With an initial interest in how local environments manage cellular debris, we performed femtosecond laser axotomies in genetic backgrounds lacking cell death gene activities. Unexpectedly, we found that the CED-3 caspase, well known as the core apoptotic cell death executioner, acts in early responses to neuronal injury to promote rapid regeneration of dissociated axons. In ced-3 mutants, initial regenerative outgrowth dynamics are impaired and axon repair through reconnection of the two dissociated ends is delayed. The CED-3 activator, CED-4/Apaf-1, similarly promotes regeneration, but the upstream regulators of apoptosis CED-9/Bcl2 and BH3-domain proteins EGL-1 and CED-13 are not essential. Thus, a novel regulatory mechanism must be utilized to activate core apoptotic proteins for neuronal repair. Since calcium plays a conserved modulatory role in regeneration, we hypothesized calcium might play a critical regulatory role in the CED-3/CED-4 repair pathway. We used the calcium reporter cameleon to track in vivo calcium fluxes in the axotomized neuron. We show that when the endoplasmic reticulum calcium-storing chaperone calreticulin, CRT-1, is deleted, both calcium dynamics and initial regenerative outgrowth are impaired. Genetic data suggest that CED-3, CED-4, and CRT-1 act in the same pathway to promote early events in regeneration and that CED-3 might act downstream of CRT-1, but upstream of the conserved DLK-1 kinase implicated in regeneration across species. This study documents reconstructive roles for proteins known to orchestrate apoptotic death and links previously unconnected observations in the vertebrate literature to suggest a similar pathway may be conserved in higher organisms.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiologia , Proteínas de Ligação ao Cálcio/metabolismo , Caspases/metabolismo , Regeneração Nervosa , Neurônios/fisiologia , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Animais Geneticamente Modificados/fisiologia , Apoptose , Axônios/metabolismo , Axônios/patologia , Axônios/fisiologia , Axotomia , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Cálcio/metabolismo , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/genética , Calreticulina/metabolismo , Caspases/genética , Ativação Enzimática , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Plasmídeos/genética , Plasmídeos/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Imagem com Lapso de TempoRESUMO
Quantification of sickle hemoglobin (HbS) in patients with sickle cell disease (SCD) undergoing hydroxyurea or chronic transfusion therapy is essential to monitoring the effectiveness of these therapies. The clinical monitoring of %HbS using conventional laboratory methods is limited by high per-test costs and long turnaround times usually associated with these methods. Here we demonstrate a simple, rapid, inexpensive paper-based assay capable of quantifying %HbS in blood samples from patients with SCD. A 20 µL droplet of whole blood and hemoglobin solubility buffer was deposited on chromatography paper. The relative color intensities of regions of the resulting blood stain, determined by automated image analysis, are used to estimate %HbS. We compared the paper-based assay with hemoglobin electrophoresis (comparison method) using blood samples from 88 subjects. The test shows high correlation (R(2) = 0.86) and strong agreement (standard deviation of difference = 7%HbS) with conventional Hb electrophoresis measurement of %HbS, and closely approximates clinically predicted change in %HbS with transfusion therapy (mean difference 2.6%HbS, n = 5). The paper-based assay can be completed in less than 35 min and has a per-test cost less than $0.25. The assay is accurate across a wide range of HbS levels (10-97%) and hemoglobin concentrations (5.6-12.9 g/dL) and is unaffected by high levels of HbF (up to 80.6%). This study demonstrates the feasibility of the paper-based %HbS assay. The paper-based test could improve clinical care for SCD, particularly in resource-limited settings, by enabling more rapid and less expensive %HbS monitoring.
Assuntos
Anemia Falciforme/sangue , Testes Hematológicos/instrumentação , Testes Hematológicos/métodos , Hemoglobina Falciforme/metabolismo , Processamento de Imagem Assistida por Computador , Papel , Feminino , Humanos , Masculino , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: Complement system is activated in patients with trauma. Although complement activation is presumed to contribute to organ damage and constitutional symptoms, little is known about the involved mechanisms. Because complement components may deposit on RBCs, we asked whether complement deposits on the surface of RBC in trauma and whether such deposition alters RBC function. DESIGN: A prospective experimental study. SETTING: Research laboratory. SUBJECTS: Blood samples collected from 42 trauma patients and 21 healthy donors. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: RBC and sera were collected from trauma patients and control donors. RBCs from trauma patients (n = 40) were found to display significantly higher amounts of C4d on their surface by flow cytometry compared with RBCs from control (n = 17) (p < 0.01). Increased amounts of iC3b were found in trauma sera (n = 27) (vs 12 controls, p < 0.01) by enzyme-linked immunosorbent assay. Incubation of RBC from universal donors (type O, Rh negative) with trauma sera (n = 10) promoted C4d deposition on their surface (vs six controls, p< 0.05). Complement-decorated RBC (n = 6) displayed limited their deformability (vs six controls, p < 0.05) in two-dimensional microchannel arrays. Incubation of RBC with trauma sera (n = 10) promoted the phosphorylation of band 3, a cytoskeletal protein important for the function of the RBC membrane (vs eight controls, p < 0.05), and also accelerated calcium influx (n = 9) and enhanced nitric oxide production (n = 12) (vs four and eight controls respectively, p < 0.05) in flow cytometry. CONCLUSIONS: Our study found the presence of extensive complement activation in trauma patients and presents new evidence in support of the hypothesis that complement activation products deposit on the surface of RBC. Such deposition could limit RBC deformability and promote the production of nitric oxide. Our findings suggest that RBC in trauma patients malfunctions, which may explain organ damage and constitutional symptoms that is not accounted for otherwise by previously known pathophysiologic mechanisms.
Assuntos
Cálcio/sangue , Ativação do Complemento/fisiologia , Eritrócitos/metabolismo , Óxido Nítrico/sangue , Fragmentos de Peptídeos/sangue , Ferimentos e Lesões/sangue , Adulto , Idoso , Estudos de Casos e Controles , Complemento C3b/análise , Complemento C4b , Ensaio de Imunoadsorção Enzimática , Feminino , Citometria de Fluxo , Humanos , Escala de Gravidade do Ferimento , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Ferimentos e Lesões/complicaçõesRESUMO
The significant biological and functional differences between small and large platelets suggested by recent studies could have profound implications for transfusion medicine. However, investigating the relationship between platelet size and function is challenging because separating platelets by size without affecting their properties is difficult. A standard approach is centrifugation, but it inevitably leads to premature activation and aggregation of separated platelets. This paper describes the development and validation of a microfluidic device based on controlled incremental filtration (CIF) for separating platelets by size without the cell damage and usability limitations associated with centrifugation. Platelet samples derived from whole blood were used to evaluate the dependence of the CIF device separation performance on design parameters and flow rate, and to compare the properties of PLT fractions generated by the CIF device with those produced using a centrifugation protocol in a split-sample study. This was accomplished by quantifying the platelet size distribution, mean platelet volume (MPV), platelet-large cell ratio (P-LCR) and platelet activation before and after processing for all input and output samples. The 'large platelet' fractions produced by the CIF device and the centrifugation protocol were essentially equivalent (no significant difference in MPV and P-LCR). Platelets in the 'small platelet' fraction produced by the CIF device were significantly smaller than those produced by centrifugation (lower MPV and P-LCR). This was because the CIF 'small platelet' fraction was contaminated by much fewer large platelets (â¼2-times lower recovery of >12 fL platelets) and retained the smallest platelets that were discarded by the centrifugation protocol. There was no significant difference in platelet activation between the two methods. However, centrifugation required a substantial amount of additional anticoagulant to prevent platelet aggregation during pelleting. Unlike centrifugation, the CIF device offered continuous, flow-through, single-step processing that did not cause platelet aggregation. Such a capability has the potential to accelerate the basic studies of the relationship between platelet size and function, and ultimately improve transfusion practice, particularly in the pediatric setting, where the need for low-volume, high-quality platelet transfusions is most urgent.
Assuntos
Plaquetas , Agregação Plaquetária , Humanos , Criança , Centrifugação , Filtração , Dispositivos Lab-On-A-Chip , Separação Celular/métodosRESUMO
The majority of adoptive cellular therapies are produced from peripheral mononuclear cells obtained via leukapheresis and further enriched for the cells of interest (e.g., T cells). Here, we present a first-of-its-kind closed system, which effectively removes ~85% of monocytes and ~88% of platelets, while recovering ~88% of concentrated T cells in a separate output stream, as the leukapheresis sample flows through a microfluidic device at 5 mL/min. The system is driven by a common peristaltic pump, enabled by a novel pressure wave dampener, and operates in a closed bag-to-bag configuration, without requiring any specialized, dedicated equipment. When compared to standard density gradient centrifugation on paired samples, the new system demonstrated a 1.5-fold increase in T cell recovery and a 2-fold reduction in inter-sample variability for this separation outcome. The T cell-to-monocyte ratio of the leukapheresis sample was increased to 20:1, whereas with density gradient processing it decreased to 2:1. As a result of superior purity and/or gentler processing, T cells enriched by the system showed a 2.7-times higher fold expansion during subsequent culture, and an overall 3.5-times higher cumulative yield. This centrifugation-free and label-free closed system for enriching lymphocytes could significantly simplify and standardize the manufacturing of life-saving cellular therapies.
RESUMO
Microfluidic lab-on-a-chip technologies enable the analysis and manipulation of small fluid volumes and particles at small scales and the control of fluid flow and transport processes at the microscale, leading to the development of new methods to address a broad range of scientific and medical challenges. Microfluidic and lab-on-a-chip technologies have made a noteworthy impact in basic, preclinical, and clinical research, especially in hematology and vascular biology due to the inherent ability of microfluidics to mimic physiologic flow conditions in blood vessels and capillaries. With the potential to significantly impact translational research and clinical diagnostics, technical issues and incentive mismatches have stymied microfluidics from fulfilling this promise. We describe how accessibility, usability, and manufacturability of microfluidic technologies should be improved and how a shift in mindset and incentives within the field is also needed to address these issues. In this report, we discuss the state of the microfluidic field regarding current limitations and propose future directions and new approaches for the field to advance microfluidic technologies closer to translation and clinical use. While our report focuses on using blood as the prototypical biofluid sample, the proposed ideas and research directions can be extrapolated to other areas of hematology, oncology, biology, and medicine.
Assuntos
Técnicas Analíticas Microfluídicas , Microfluídica , Microfluídica/métodos , Técnicas Analíticas Microfluídicas/métodos , Dispositivos Lab-On-A-Chip , Pesquisa Translacional BiomédicaRESUMO
BACKGROUND: The measurement of hemoglobin concentration ([Hb]) is performed routinely as a part of a complete blood cell count to evaluate the oxygen-carrying capacity of blood. Devices currently available to physicians and clinical laboratories for measuring [Hb] are accurate, operate on small samples, and provide results rapidly, but may be prohibitively expensive for resource-limited settings. The unavailability of accurate but inexpensive diagnostic tools often precludes proper diagnosis of anemia in low-income developing countries. Therefore, we developed a simple paper-based assay for measuring [Hb]. METHODS: A 20-µL droplet of a mixture of blood and Drabkin reagent was deposited onto patterned chromatography paper. The resulting blood stain was digitized with a portable scanner and analyzed. The mean color intensity of the blood stain was used to quantify [Hb]. We compared the performance of the paper-based Hb assay with a hematology analyzer (comparison method) using blood samples from 54 subjects. RESULTS: The values of [Hb] measured by the paper-based assay and the comparison method were highly correlated (R(2) = 0.9598); the standard deviation of the difference between the two measurements was 0.62 g/dL. The assay was accurate within 1 g/dL 90.7% of the time, overestimating [Hb] by ≥1 g/dL in 1.9% and underestimating [Hb] by ≥1 g/dL in 7.4% of the subjects. CONCLUSIONS: This study demonstrates the feasibility of the paper-based Hb assay. This simple, low-cost test should be useful for diagnosing anemia in resource-limited settings, particularly in the context of care for malaria, HIV, and sickle cell disease patients in sub-Saharan Africa.