Affinophoresis of red blood cells: surface antigen-specific acceleration of electrophoresis

A method employing the technique of affinophoresis to increase the electrophoretic mobility of specific cells according to their surface antigens was developed. Red blood cells were treated consecutively with the maximum subagglutinating dose of an anti-red blood cell serum, a biotinylated second antibody, avidin and finally with a negatively charged biotin-affinophore which was prepared by coupling biotin to polylysine (average degree of polymerization, 270 or 1150), followed by complete succinylation. The electrophoretic mobility of cells was analyzed with an automatic cell electrophoresis analyzer. The use of a homologous anti-serum increased the electrophoretic mobility of rabbit, human and rat blood cells by 2.9, 1.7 and 1.6 times, respectively. A larger affinophore containing fewer biotin moieties was more effective. In the case of a mixture of red blood cells from two species, cells from only one species could be accelerated by using homologous antiserum, e.g., affinophoresis of a mixture of human and rat red blood cells by using either homologous antiserum gave two separate peaks on the histogram, whereas a single peak would be obtained in usual electrophoresis because there is little difference in the original migration velocities of the two cell types.     

Glutathione reductase-catalyzed cascade of redox reactions to bioactivate potent antimalarial 1,4-naphthoquinones--a new strategy to combat malarial parasites

Our work on targeting redox equilibria of malarial parasites propagating in red blood cells has led to the selection of six 1,4-naphthoquinones, which are active at nanomolar concentrations against the human pathogen Plasmodium falciparum in culture and against Plasmodium berghei in infected mice. With respect to safety, the compounds do not trigger hemolysis or other signs of toxicity in mice. Concerning the antimalarial mode of action, we propose that the lead benzyl naphthoquinones are initially oxidized at the benzylic chain to benzoyl naphthoquinones in a heme-catalyzed reaction within the digestive acidic vesicles of the parasite. The major putative benzoyl metabolites were then found to function as redox cyclers: (i) in their oxidized form, the benzoyl metabolites are reduced by NADPH in glutathione reductase-catalyzed reactions within the cytosols of infected red blood cells; (ii) in their reduced forms, these benzoyl metabolites can convert methemoglobin, the major nutrient of the parasite, to indigestible hemoglobin. Studies on a fluorinated suicide-substrate indicate as well that the glutathione reductase-catalyzed bioactivation of naphthoquinones is essential for the observed antimalarial activity. In conclusion, the antimalarial naphthoquinones are suggested to perturb the major redox equilibria of the targeted infected red blood cells, which might be removed by macrophages. This results in development arrest and death of the malaria parasite at the trophozoite stage.     

Lateral-driven continuous dielectrophoretic microseparators for blood cells suspended in a highly conductive medium

This paper presents lateral-driven continuous dielectrophoretic (DEP) microseparators for separating red and white blood cells suspended in highly conductive dilute whole blood. The continuous microseparators enable the separation of blood cells based on the lateral DEP force generated by a planar interdigitated electrode array placed at an angle to the direction of flow. The simplified line charge model that we developed for the theoretical analysis was verified by comparing it with simulated and measured results. Experimental results showed that the divergent type of microseparator can continuously separate out 87.0% of the red blood cells (RBCs) and 92.1% of the white blood cells (WBCs) from dilute whole blood within 5 min simply by using a 2 MHz, 3 Vp-p AC voltage to create a gradient electric field in a medium that conducts at 17 mS cm(-1). Under the same conditions, the convergent type of microseparator could separate out 93.6% of the RBCs and 76.9% of the WBCs. We have shown that our lateral-driven continuous DEP microseparator design is practical for the continuous separation of blood cells without the need to control the conductivity of the suspension medium, overcoming critical drawbacks of DEP microseparators.     

Mechanisms of red blood cells agglutination in antibody-treated paper

Recent reports on using bio-active paper and bio-active thread to determine human blood type have shown a tremendous potential of using these low-cost materials to build bio-sensors for blood diagnosis. In this work we focus on understanding the mechanisms of red blood cell agglutination in the antibody-loaded paper. We semi-quantitatively evaluate the percentage of antibody molecules that are adsorbed on cellulose fibres and can potentially immobilize red blood cells on the fibre surface, and the percentage of the molecules that can desorb from the cellulose fibre surface into the blood sample and cause haemagglutination reaction in the bulk of a blood sample. Our results show that 34 to 42% of antibody molecules in the papers treated with commercial blood grouping antibodies can desorb from the fibre surface. When specific antibody molecules are released into the blood sample via desorption, haemagglutination reaction occurs in the blood sample. The reaction bridges the red cells in the blood sample bulk to the layer of red cells immobilized on the fibre surface by the adsorbed antibody molecules. The desorbed antibody also causes agglutinated lumps of red blood cells to form. These lumps cannot pass through the pores of the filter paper. The immobilization and filtration of agglutinated red cells give reproducible identification of positive haemagglutination reaction. Results from this study provide information for designing new bio-active paper-based devices for human blood typing with improved sensitivity and specificity.     

A Safe and Efficient Strategy for the Rapid Elimination of Blood Lead In Vivo Based on a Capture–Fix–Separate Mechanism

Lead poisoning is an important problem because of its serious effects on human health. Yet a solution is not available due to an incomplete understanding of the state of lead ions in blood. Since most blood lead binds to hemoglobin (Hb) in red blood cells, identifying and capturing lead‐contaminated Hb in RBCs is important. Herein, a magnetic blood lead remover with hyperbranched poly(amidoamine)s (HPAM) as template/co‐adsorbent and core–shell mesoporous structure was synthesized. Lead‐containing Hb was selectively captured and then fixed by mesoporous channels. The magnetic separation technology was used to separate the magnetic remover from blood. A related blood lead clean‐up apparatus was used to remove lead from the blood of a pig in vivo. Results of physical/chemical characterizations, biocompatibility experiments, animal tests, and theoretical simulation verify the safety and efficiency of this removal strategy and the high efficiency of the blood lead clean‐up apparatus.     

UVA‐Induced DNA Damage and Apoptosis in Red Blood Cells of the African Catfish Clarias gariepinus

Ultraviolet‐A light (UVA)‐induced DNA damage and repair in red blood cells to investigate the sensitivity of African catfish to UVA exposure is reported. Fishes were irradiated with various doses of UVA light (15, 30, and 60 min day⁻¹ for 3 days). Morphological and nuclear abnormalities in red blood cells were observed in the fish exposed to UVA compared with controls. Morphological alterations such as acanthocytes, crenated cells, swollen cells, teardrop‐like cells, hemolyzed cells, and sickle cells were observed. Those alterations were increased after 24 h exposure to UVA light and decreased at 14 days after exposure. The percentage of apoptosis was higher in red blood cells exposed to higher doses of UVA light. No micronuclei were detected, but small nuclear abnormalities such as deformed and eccentric nuclei were observed in some groups. We concluded that exposure to UVA light induced DNA damage, apoptosis, and morphological alterations in red blood cells in catfish; however, catfish were found to be less sensitive to UVA light than wild‐type medaka.     

Study on the discrete dielectrophoresis for particle–cell separation

This paper presents the application of the discrete dielectrophoretic force to separate polystyrene particles from red blood cells. The separation process employs a simple microfluidic device that is composed of interdigitated electrodes and a microchannel. The discrete dielectrophoretic force is generated by adjusting the duty cycle of the applied voltage. The electrodes make a tilt angle with the microchannel to change the moving direction of the red blood cells. By adjusting the voltage magnitude and duty cycle, we investigate the deflection of red blood cells and the variation of cell velocity along electrode edge under positive dielectrophoresis. The experiments with polystyrene particles show that the enrichment of the particles is greater than 150 times. The maximum separation efficiency is 97% for particle-to-cell number ratio equal to 1:2000 in the sample having high cell concentration. Using the appropriate applied voltage magnitude and duty cycle, the discrete dielectrophoretic force can prevent the clogging of microchannel while successfully separating the particles from the cells with high enrichment and efficiency. The proposed principle can be readily applied to dielectrophoresis-based devices for biomedical sample preparation or diagnosis such as the separation of rare or infected cells from a blood sample.     

Dynamics of hemoglobin in human erythrocytes and in solution: influence of viscosity studied by ultrafast vibrational echo experiments

Ultrafast spectrally resolved stimulated vibrational echo experiments are used to measure the vibrational dephasing of the CO stretching mode of hemoglobin-CO (HbCO) inside living human erythrocytes (red blood cells), in liquid solutions, and in a glassy matrix. A method is presented to overcome the adverse impact on the vibrational echo signal from the strong light scattering caused by the cells. The results from the cytoplasmic HbCO are compared to experiments on aqueous HbCO samples prepared in different buffers, solutions containing low and high concentrations of glycerol, and in a solid trehalose matrix. Measurements are also presented that provide an accurate determination of the viscosity at the very high Hb concentration that is found inside the cells. It is demonstrated that the dynamics of the protein, as sensed by the CO ligand, are the same inside the erythrocytes and in aqueous solution and are independent of the viscosity. In solutions that are predominantly glycerol, the dynamics are modified somewhat but are still independent of viscosity. The experiments in trehalose give the dynamics at infinite viscosity and are used to separate the viscosity-dependent dynamics from the viscosity-independent dynamics. Although the HbCO dynamics are the same in the red blood cell and in the equivalent aqueous solutions, differences in the absorption spectra show that the distribution of a protein's equilibrium substates is sensitive to small pH differences.     

Rare‐Cell Enrichment by a Rapid,Label‐Free,Ultrasonic Isopycnic Technique for Medical Diagnostics

One significant challenge in medical diagnostics lies in the development of label‐free methods to separate different cells within complex biological samples. Here we demonstrate a generic, low‐power ultrasonic separation technique, able to enrich different cell types based upon their physical properties. For malaria, we differentiate between infected and non‐infected red blood cells in a fingerprick‐sized drop of blood. We are able to achieve an enrichment of circulating cells infected by the ring stage of the parasite over nonparasitized red blood cells by between two and three orders of magnitude in less than 3 seconds (enabling detection at parasitemia levels as low as 0.0005 %). In a second example, we also show that our methods can be used to enrich different cell types, concentrating Trypanosoma in blood at very low levels of infection, on disposable, low‐cost chips.     

Rare‐Cell Enrichment by a Rapid,Label‐Free,Ultrasonic Isopycnic Technique for Medical Diagnostics

One significant challenge in medical diagnostics lies in the development of label‐free methods to separate different cells within complex biological samples. Here we demonstrate a generic, low‐power ultrasonic separation technique, able to enrich different cell types based upon their physical properties. For malaria, we differentiate between infected and non‐infected red blood cells in a fingerprick‐sized drop of blood. We are able to achieve an enrichment of circulating cells infected by the ring stage of the parasite over nonparasitized red blood cells by between two and three orders of magnitude in less than 3 seconds (enabling detection at parasitemia levels as low as 0.0005 %). In a second example, we also show that our methods can be used to enrich different cell types, concentrating Trypanosoma in blood at very low levels of infection, on disposable, low‐cost chips.     

High-performance liquid chromatographic determination of cyanide in human red blood cells by pre-column fluorescence derivatization.

A method for the determination of cyanide in human red cells has been developed. Cyanide was extracted from red cells by adding water and methanol, and then derivatized with 2,3-naphthalene-dialdehyde and taurine to give a fluorescent product, which was determined by reversed-phase high-performance liquid chromatography with fluorescence detection. The recovery of cyanide from red cells was ca. 83%, and the limit of detection was 100 pmol/ml. The mean concentrations of red cell cyanide from ten smokers and from ten non-smokers were 705 and 466 pmol/ml, respectively. The method was also applicable to whole blood.     

Modulator binding protein antagonizes activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport of red blood cell membranes.

Red blood cells contain a protein that activates membrane-bound (Ca2+ + Mg2+)-ATPase and Ca2+ transport. The red blood cell activator protein is similar to a modulator protein that stimulates cyclic AMP phosphodiesterase. Wang and Desai [Journal of Biological Chemistry 252:4175--4184, 1977] described a modulator-binding protein that antagonizes the activation of cyclic AMP phosphodiesterase by modulator protein. In the present work, modulator-binding protein was shown to antagonize the activation of (Ca2+ + Mg2+)-ATPase and Ca2+ transport by red blood cell activator protein. The results further demonstrate the similarity between the activator protein from human red blood cells and the modulator protein from bovine brain.     

Analysis of the phospholipid composition of plasmodium knowlesi and rhesus erythrocyte membranes

Methods for the separation and analysis of the phospholipid classes have been studied. The lipid extracts of normal and Plasmodium knowlesi- infected rhesus erythrocytes and of the parasite itself have been examined for phospholipid composition on an animal-to-animal basis. Several differences were apparent between the phospholipids of parasites and infected host cells. Phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine represented larger percentages in the parasite than in the host; the average phosphatidylinositol content was 1.8 % in infected host cells and 4.3 % in parasites. Sphingomyelin and phosphatidylserine were also strikingly different in the two membranes; in the parasite they averaged less than 20 % and 33 % respectively of their level in the infected red blood cell.     

Secondary metabolites, cytotoxic response by neutral red retention and protective effect against H2O2 induced cytotoxicity of Sedum caespitosum

The EtOAc, n-BuOH and H20 subextracts of the crude MeOH extract of the aerial parts of Sedum caespitosum (cav.) Dc. were screened for cytotoxicity using the neutral red assay in Chinese hamster ovary cells as well as their protective effect against H2O2 induced cytotoxicity in human red blood cells. While the extracts did not show cytotoxicity, they displayed a protective effect compared to a blank and ascorbic acid. Gallic acid (1), kaempferol 3-O-alpha-rhamnopyranoside (2), quercetin 3-O-beta-glucopyranoside (3), quercetin 3-O-alpha-rhamnopyranoside (4) and myricetin 3-O-alpha-rhamnopyranoside (5) were isolated from the EtOAc extract and identified by 1D- and 2D-NMR. The protective effects of the isolated compounds against H2O2 induced cytotoxicity in human red blood cells were evaluated and 5 was the most active.     

基于错流过滤原理的微流控细胞分离芯片的研制

首次提出并制备了一种错流过滤式细胞分离微流控芯片.     

Determination of nucleotides, nucleosides and nucleobases in cells of different complexity by reversed-phase and ion-pair high-performance liquid chromatography

Procedures are presented for the analysis of profiles of purine and pyridine compounds in human and rabbit red blood cells by reversed-phase high-performance liquid chromatography and in Ehrlich ascites tumour cells of mouse by ion-pair high-performance liquid chromatography. These compounds are present in rabbit erythrocytes in higher concentrations than in human blood cells, and in rabbit reticulocytes the concentration of purine compounds is still higher. During glucose-free incubation, human red cells accumulate adenosine and adenine in the presence of coformycin owing to the inhibition of adenosine and AMP deamination. Ehrlich ascites tumour cells lose major portions of purine mono-, di- and triphosphates between the seventh and eleventh day after inoculation into mouse peritoneal cavities.     

Quantitation of ribavirin in human plasma and red blood cells using LC–MS/MS

LC–MS/MS has been applied for the rapid determination of the nucleoside analogue ribavirin in human plasma and red blood cells. The incorporation of ribavirin to the erythrocytes has been assayed after in vitro incubation of the cells at different concentrations of the antiviral drug. After protein precipitation, samples were injected into a C₈ column, achieving a complete separation of ribavirin from the endogenous isobaric compound uridine. Calibration ranges varied from 10 to 10 000 ng/mL in plasma and from 0.2 to 200 ng/cell pellet in red blood cells. Precision and accuracy values were always below 10 and 13%, respectively, in all assayed matrices. Ribavirin was demonstrated to remain unchanged after short and long time storage. No matrix effects could be assessed for the analyzed matrices. The developed method has been fully validated. Monitoring of ribavirin concentration in red blood cells in addition to the classic plasma monitoring of the drug could help to explain its efficacy and safety profiles in patients.     

Paper-based device for rapid typing of secondary human blood groups

We report the use of bioactive paper for typing of secondary human blood groups. Our recent work on using bioactive paper for human blood typing has led to the discovery of a new method for identifying haemagglutination of red blood cells. The primary human blood groups, i.e., ABO and RhD groups, have been successfully typed with this method. Clinically, however, many secondary blood groups can also cause fatal blood transfusion accidents, despite the fact that the haemagglutination reactions of secondary blood groups are generally weaker than those of the primary blood groups. We describe the design of a user-friendly sensor for rapid typing of secondary blood groups using bioactive paper. We also present mechanistic insights into interactions between secondary blood group antibodies and red blood cells obtained using confocal microscopy. Haemagglutination patterns under different conditions are revealed for optimization of the assay conditions.     

Blood separation on microfluidic paper-based analytical devices

A microfluidic paper-based analytical device (μPAD) for the separation of blood plasma from whole blood is described. The device can separate plasma from whole blood and quantify plasma proteins in a single step. The μPAD was fabricated using the wax dipping method, and the final device was composed of a blood separation membrane combined with patterned Whatman No.1 paper. Blood separation membranes, LF1, MF1, VF1 and VF2 were tested for blood separation on the μPAD. The LF1 membrane was found to be the most suitable for blood separations when fabricating the μPAD by wax dipping. For blood separation, the blood cells (both red and white) were trapped on blood separation membrane allowing pure plasma to flow to the detection zone by capillary force. The LF1-μPAD was shown to be functional with human whole blood of 24-55% hematocrit without dilution, and effectively separated blood cells from plasma within 2 min when blood volumes of between 15-22 μL were added to the device. Microscopy was used to confirm that the device isolated plasma with high purity with no blood cells or cell hemolysis in the detection zone. The efficiency of blood separation on the μPAD was studied by plasma protein detection using the bromocresol green (BCG) colorimetric assay. The results revealed that protein detection on the μPAD was not significantly different from the conventional method (p > 0.05, pair t-test). The colorimetric measurement reproducibility on the μPAD was 2.62% (n = 10) and 5.84% (n = 30) for within-day and between day precision, respectively. Our proposed blood separation on μPAD has the potential for reducing turnaround time, sample volume, sample preparation and detection processes for clinical diagnosis and point-of care testing.     

Optimization of malaria detection based on third harmonic generation imaging of hemozoin

The pigment hemozoin is a natural by-product of the metabolism of hemoglobin by the parasites which cause malaria. Previously, hemozoin was demonstrated to have a very high nonlinear optical response enabling third harmonic generation (THG) imaging. In this study, we present a complete characterization of the nonlinear THG response of natural hemozoin in malaria-infected red blood cells, as well as in pure isostructural synthesized hematin anhydride, in order to determine optimal imaging parameters for detection. Our study demonstrates the wavelength range for optimal pulsed femtosecond laser excitation of THG from hemozoin crystals. In addition, we show the hemozoin crystal detection as a function of crystal size, incident laser power, and the emission response of the hemozoin crystals to different incident laser polarization states. Our systematic measurements of the nonlinear optical response from hemozoin establish detection limits, which are essential for the optimal design of malaria detection technologies that exploit the THG response of hemozoin.