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.     

New method of blood typing using analytical magnetapheresis

We report a new method of blood typing based on the agglutination of red blood cell (RBC) with serum-treated magnetic particles in analytical magnetapheresis. Blood typing of ABO was demonstrated. The agglutination patterns of RBCs are different for different blood types and can be used to determine the ABO blood typing in analytical magnetapheresis. Six samples can be tested in each run. The running time was less than 10 min. Magnetic particles were prepared in the laboratory. The amount of RBCs needed for the agglutination test was about 1.0 microl of adult blood. The blood typing of ABO was used to illustrate the capable applications of analytical magnetapheresis to nonmagnetic samples like cells without magnetic labels. Analytical magnetapheresis has a great potential for cell related analysis.     

A novel method for blood‐typing using nitrocellulose

Blood wicking in its steady‐state form, i.e. the uniform distribution of blood cells in plasma, is completely different from that in its coagulated form on a porous surface like paper. The hydrophilic property of the cellulose leads to a significant wicking of the blood cells on paper fibers after rinsing with isotonic solution. The difference in the wicking length of the blood cells in steady state and that in the coagulated form could be considered as a criterion to recognize the blood type in a paper‐based kit. However, owing to the molecular structure of the nitrocellulose, a better process occurs while separating the coagulated blood from the steady‐state form of cells. Therefore, it is possible to use the nitrocellulose for the blood‐typing kit which leads to a simpler way to diagnose a blood type. Two series of experiments were performed on nitrocellulose membrane. First, antibody solutions and blood samples were sequentially absorbed on nitrocellulose strips, allowed to interact, rinsed with an isotonic solution and distilled water, and image processing performed on a digital picture of the remaining blood cells. The efficiency of the agglutinated blood cell fixation was quantified by red color intensity. Then, it was demonstrated that there is no considerable difference in fixation of agglutinated blood cells with rinsing using isotonic and nonisotonic solutions. This fact can be a considerable advantage over paper since it can eliminate the probable mistake from using unisotonic solution for rinsing. Second, owing to the nonwicking property of the blood cells on the hydrophobic nitrocellulose fibers, we employed another diagnostic criterion and investigated nitrocellulose blood‐typing prototypes. The nitrocellulose blood‐typing kit provides more simple, sensitive and trustworthy assay for rapid blood typing in situations with no access to laboratory facilities.     

Phototoxicity of non-steroidal anti-inflammatory drugs: in vitro testing of the photoproducts of Butibufen and Flurbiprofen.

In this work, the phototoxicity of two non-steroidal anti-inflammatory drugs, Butibufen and Flurbiprofen, was examined. Both were unstable to light, to give several photoproducts which were isolated and identified. The different photoproducts were formed by a primary photochemical mechanism which involves an initial cleavage of the C-C bond alpha to the carbonyl group, followed by several secondary processes. The cytotoxic effects of the xenobiotics were evaluated using two well-established biological in vitro tests: (a) enzyme leakage lactate dehydrogenase and glutamate-oxaloacetate transaminase from cultured fibroblasts and (b) lysis of red blood cells. The benzylic alcohols caused extensive leakage from cultured fibroblasts at the different concentrations assayed. The alcohol obtained from Butibufen was a potent lytic agent for human red blood cells. The other photoproducts, Butibufen and Flurbiprofen did not produce observable toxic effects on cells.     

Simultaneous forward and reverse ABO blood group typing using a paper-based device and barcode-like interpretation

A new platform of a paper-based analytical device (PAD) for simultaneous forward and reverse ABO blood group typing has been reported. This platform can overcome the discrepancy results as influenced by the individual haematocrit. The test and the control of non-haemagglutination on each channel were performed in parallel. The PAD was fabricated by printing six parallel channels with wax onto Whatman No. 4 filter paper. An LF1 blood separation membrane was used for the separation of plasma from whole blood for reverse grouping. The blood group was identified by haemagglutination of the corresponding antigen–antibody. For forward grouping, Anti-A, -B and –A,B were treated on the test line of PAD, and inactivated Anti-A, -B and –A,B were immobilized on the control line. For reverse grouping, 30% standard A-cells, B- and O- were added to the test channel after plasma separation, and O-cells were used as a control. Then, 0.9% normal saline (NSS) containing 1% Tween-20 was bi-functionally used for dilution of the blood sample and elution of the non-agglutinated RBCs within the channels. The distance of agglutinated RBCs in each test line was compared with the distance of non-agglutinated RBCs in the parallel control line. The forward and reverse patterns of blood groups A, B, AB and O were a barcode-like chart in which the results can be visually analysed. The PAD has excellent reproducibility when 10 replications of the A, B, AB or O blood groups were performed. The results of both forward and reverse grouping were highly correlated with conventional methods compared with the slide method and tube method, respectively (n = 76). Thus, this ABO typing PAD holds great potential for future applications in blood typing point-of-care testing.     

Recent and future trends in blood group typing

Blood group typing is the process of testing red blood cells to determine which antigens are present and which are absent. It is standard practice to test for A, B, and D (Rh) antigens and to perform tests for other antigens in selected cases. ABO blood group typing is confirmed by reverse grouping that detects expected isoagglutinins. Unexpected antibodies can be demonstrated by antibody screening tests. For transfusion, donor units compatible with the patient are selected. Prior to transfusion, a crossmatch is performed as a final check for incompatibility. This article describes the recent and future methods of blood group typing and testing of serological compatibility. In addition, methods for blood bank automation are presented.     

A novel enhancement assay for immunochromatographic test strips using gold nanoparticles

The immunochromatographic assay is a well-known and convenient diagnostic system. In this report, the development of a novel enhancement assay for the test strips is described. Additionally, this highly sensitive immunochromatographic assay was applied to detect human chorionic gonadotropin hormone (HCG) as the model case. The primary antibody-conjugated gold nanoparticles were used as the enhancer of the standard method. The primary antibodies were immobilized within a defined detection zone (test line) on the diagnostic nitrocellulose membrane. The secondary antibodies were conjugated with colloidal gold nanoparticles. In combination with an effective sample pretreatment, the gold-conjugated antibodies and the primary antibodies formed a sandwich complex with the target protein. Within the test line, the sandwich complex was immobilized, and furthermore, concentrated by the enhancer resulting in a localized surface plasmon resonance (LSPR) phenomenon and a distinct red color on the test line. The intensity of color of the red test line (signal intensity), which correlated directly with the concentration of the target protein in the standard or spiked samples, was assessed visually and by computer image analysis using a three-determination analysis. Under optimum conditions, the limit of detection (LOD) for HCG assay was 1 pg/mL. When using human serum, 10 pg/mL of HCG could be detected. We have also spiked total prostate-specific antigen (TPSA) in female serum. The LOD for TPSA was determined as 0.2 ng/mL. With this method, the quantitative determination of the target protein could be completed in less than 15 min. Our novel immunochromatographic strips using the enhancing method based on LSPR of gold nanoparticles are useful as a rapid and simple screening method for the detection of important analytes for medical applications, environmental monitoring, food control, and biosecurity.      

体外细胞模型和高效液相质谱联用分析预测黄芪中的活性成分

利用体外细胞模型模拟体内细胞对中药有效成分的特异性吸收,结合高效液相色谱/质谱分析筛选中药黄芪中的生物活性成分。将中药黄芪提取液与Caco-2细胞及红细胞分别混合培养,破碎与药材结合后的细胞,使之释放出结合的药材中的成分。运用高效液相色谱/飞行时间质谱(HPLC-ESI/TOFMS)分析中药黄芪提取液与活性细胞有结合的成分,并对其进行结构鉴定。结果显示:黄芪中有10个化合物与Caco-2细胞结合,14个化合物与红细胞结合。本方法可用于预测口服药物在体内的吸收以及与特定靶细胞的结合情况,特异性地筛选中药复杂体系中的药效物质基础。     

A preliminary study on the stabilization of blood typing antibodies sorbed into paper

This study investigated the stability of the primary blood typing antibodies (Anti-A, Anti-B and Anti-D IgM) on paper. This knowledge is critical to manufacture a new type of paper-based blood typing device where blood group antibodies must be kept active on paper for extended periods. Two strategies were explored. The first involved mixing additives such as polyvinylpyrrolidone (PVP), dextran and glycerol, with antibodies before sorption onto paper. While all the additives tested improved the antibody stability on paper, their protection for storage at room temperature was limited; dextran provided the longest protection, followed by PVP and then glycerol. The second strategy relied on freeze-drying to stabilize the antibodies in paper. Freeze dried antibodies sorbed into paper could be stored for long periods at ambient conditions without significantly loss of their activity. The thermal stability of antibodies in paper was also improved by freeze-drying. Our work shows that the use of additives and freeze-drying are effective approaches to retain the activities of IgM blood group antibodies on paper. These approaches will be further explored for the large scale development of a new generation of clinical and home-care blood testing devices.     

Triethylamine-Mediated Transformation of Phosphonates into Phosphonamidates

Organophosphorus compounds such as phosphonamidates are gaining attention across different fields of chemistry, with interesting applications as pharmaceuticals, or pesticides. However, practical application of phosphonamidates is complicated by their difficult syntheses which often involve expensive or unstraightforward reagents and harsh conditions. To remedy these issues, we present a flexible, room temperature synthesis for novel P-alkylphosphonamidates without the need for intermediary purification. Commonly available phosphonates are first chlorinated by use of oxalyl chloride and phosphonylaminium salts are used to mediate the harsh reactivity of phosphonochloridates, giving rise to the desired products. We demonstrate the compatibility of our protocol with primary and secondary amines, as well as with different phosphonate esters. The proposed pathway also enables the synthesis of primary phosphonamidates using ammonium acetate as a cheap and safe alternative for ammonia. In future research, this protocol will also enable the synthesis of bioactive targets that are incompatible with current protocols.     

Selenoxide Elimination Triggers Enamine Hydrolysis to Primary and Secondary Amines: A Combined Experimental and Theoretical Investigation

We discuss a novel selenium-based reaction mechanism consisting in a selenoxide elimination-triggered enamine hydrolysis. This one-pot model reaction was studied for a set of substrates. Under oxidative conditions, we observed and characterized the formation of primary and secondary amines as elimination products of such compounds, paving the way for a novel strategy to selectively release bioactive molecules. The underlying mechanism was investigated using NMR, mass spectrometry and density functional theory (DFT).     

Antifungal activity from 14-helical beta-peptides

We have discovered that short beta-peptides (9 or 10 residues) designed to adopt globally amphiphilic helical conformations display significant antifungal activity. The most promising beta-peptides cause little lysis of human red blood cells at concentrations that kill Candida albicans, a common human fungal pathogen. Since fungi are eukaryotes, discrimination between fungal and human cells is a significant finding. Our beta-peptides are active under assay conditions that mimic physiological ionic strength; in contrast, alpha-helix-forming host-defense alpha-peptides are inactive against C. albicans under these conditions.     

Classifying Human Haptoglobin Phenotypes on Native‐PAGE Using a Modified Coomassie Brilliant Blue R 250 Staining

There are three types of human Haptoglobin, Hp 1‐1, Hp 2‐1, and Hp 2‐2, each characterized by a distinct combination of the two α chains of the holoprotein. A modified Coomassie Brilliant Blue R 250 (mCBB‐R250) staining method for detecting the phenotypes of human blood haptoglobin molecules is presented. Addition of excess hemoglobin to the sample allowed specific formation of different haptoglobin‐hemoglobin complexes, which, in turn can be separated into distinctive migration pattern on native‐PAGE and stained by CBB‐R250. The typing results are consistent with that using Western blotting. In comparison to the existing methods for haptoglobin typing, our method is comparable in accuracy, and is easier to carry out. The results on typing of 29 plasma samples from Taipei Blood Center were also presented.     

Phototriggered Secretion of Membrane Compartmentalized Bioactive Agents

A strategy for the light‐activated release of bioactive compounds (BODIPY, colchicine, paclitaxel, and methotrexate) from membrane‐enclosed depots is described. We have found that membrane‐permeable bioagents can be rendered membrane impermeable by covalent attachment to cobalamin (Cbl) through a photocleavable linker. These Cbl‐bioagent conjugates are imprisoned within lipid‐enclosed compartments in the dark, as exemplified by their retention in the interior of erythrocytes. Subsequent illumination drives the secretion of the bioactive species from red blood cells. Photorelease is triggered by wavelengths in the red, far‐red, and near‐IR regions, which can be pre‐assigned by affixing a fluorophore with the desired excitation wavelength to the Cbl‐bioagent conjugate. Pre‐assigned wavelengths allow different biologically active compounds to be specifically and unambiguously photoreleased from common carriers.     

Separation of parasites from human blood using deterministic lateral displacement

We present the use of a simple microfluidic technique to separate living parasites from human blood. Parasitic trypanosomatids cause a range of human and animal diseases. African trypanosomes, responsible for human African trypanosomiasis (sleeping sickness), live free in the blood and other tissue fluids. Diagnosis relies on detection and due to their often low numbers against an overwhelming background of predominantly red blood cells it is crucial to separate the parasites from the blood. By modifying the method of deterministic lateral displacement, confining parasites and red blood cells in channels of optimized depth which accentuates morphological differences, we were able to achieve separation thus offering a potential route to diagnostics.     

Engineering paper as a substrate for blood typing bio-diagnostics

The effect of paper structure on blood typing visualization was quantified and analyzed to engineer low-cost diagnostics. Commercial and experimental papers varying in fibre composition, basis weight, density and porosity were investigated for their ability to separate agglutinated (blood interacted with specific antibodies) from non-agglutinated (blood interacted with non-specific antibodies) red blood cells (RBCs). Antibodies solutions and blood samples were sequentially absorbed on paper, allowed to interact, eluded with a saline solution, and the intensity of the remaining blood spot was quantified by image analysis. The efficiency and clarity of RBC separation was quantified with the relative intensity (R.I.) index defined as the intensity ratio of the non-specific test over the specific system; the lower the R.I., the better is the separation between a positive from a negative test. Thick and dense papers are improper for blood typing as they retain indiscriminately both agglutinated and non-agglutinated RBCs. Thin and porous papers provided the best performance. The R.I. index (the lower the better) increased fairly linearly with paper density and thickness but inversely proportionally with paper pore size. The type of fibres played a minor role. The paper structure is critical in the design of blood typing assay. However, it is only one element of the diagnostic system to engineer with the interactions RBC-antibody-paper.     

Disposable integrated microfluidic biochip for blood typing by plastic microinjection moulding

Blood typing is the most important test for both transfusion recipients and blood donors. In this paper, a low cost disposable blood typing integrated microfluidic biochip has been designed, fabricated and characterized. In the biochip, flow splitting microchannels, chaotic micromixers, reaction microchambers and detection microfilters are fully integrated. The loaded sample blood can be divided by 2 or 4 equal volumes through the flow splitting microchannel so that one can perform 2 or 4 blood agglutination tests in parallel. For the purpose of obtaining efficient reaction of agglutinogens on red blood cells (RBCs) and agglutinins in serum, we incorporated a serpentine laminating micromixer into the biochip, which combines two chaotic mixing mechanisms of splitting/recombination and chaotic advection. Relatively large area reaction microchambers were also introduced for the sake of keeping the mixture of the sample blood and serum during the reaction time before filtering. The gradually decreasing multi-step detection microfilters were designed in order to effectively filter the reacted agglutinated RBCs, which show the corresponding blood group. To achieve the cost-effectiveness of the microfluidic biochip for disposability, the biochip was realized by the microinjection moulding of COC (cyclic olefin copolymer) and thermal bonding of two injection moulded COC substrates in mass production with a total fabrication time of less than 20 min. Mould inserts of the biochip for the microinjection moulding were fabricated by SU-8 photolithography and the subsequent nickel electroplating process. Human blood groups of A, B and AB have been successfully determined with the naked eye, with 3 microl of the whole sample bloods, by means of the fabricated biochip within 3 min.     

Coupling chemiluminescence with capillary electrophoresis to analyze single human red blood cells

In this paper, we describe a new method for determination of hemoglobin of single red blood cells by coupling chemiluminescence with capillary electrophoresis (CL-CE). The chemiluminescent detection is based on the catalytic effects of hemoglobin on the luminol-hydrogen peroxide reaction. The conditions of chemiluminescent reaction and capillary electrophoresis were investigated. Hemoglobin in human blood samples was detected with the present method, the linear range from 1.7 μg mL⁻¹ to 6.8 μg mL⁻¹ was tested, and the correlation coefficient of 0.997 and low detection limit of 0.17 μg mL⁻¹ (approximately 2.2 pg, S/N = 3) were obtained. Cell injection procedure was improved, and the method was successfully used to determine hemoglobin of single red blood cells and the statistical result of the average content of hemoglobin in 26 human red blood cells was 23.6 pg. Compared to other current methods, CE with CL system is simple, sensitive and will become an attractive alternative method for single cell analysis.     

Examination of the in vitro degradation of [14C] pentaerythritol tetranitrate in rat and human blood with an improved thin-layer radiochromatographic procedure

Improvements were made on a reported thin-layer radiochromatographic assay for the determination of [14C]pentaerythritol tetranitrate (PETN) and its metabolites in whole blood, using methanol instead of dioxane as the extracting solvent. Recovery of total radioactivity for the entire work-up procedure was greater than 90%, and the distribution of PETN and its metabolites in degraded blood samples was found to be reproducible. This modified method appeared simpler and yielded better recovery of radioactivity than the literature method. In vitro metabolism of [14C]PETN in rat and human blood was examined by incubation of the drug with fresh blood at 37 degrees C for 60 min. In rat blood, the half-life of PETN degradation was about 15 min producing the trinitrate, dinitrate and mononitrate metabolites. Human blood was also capable of degrading PETN in vitro, but at a lower rate than rat blood, yielding only the trinitrate metabolite in quantifiable amounts during the incubation period. Equilibrium of PETN between plasma and red blood cells was observed within 1 min after PETN addition to both rat and human blood. The apparent plasma/red blood cells partition ratios of PETN were 1.1 and 1.7 for rat and human blood, respectively. PETN degradation was approximately ten times slower in rat plasma than in rat blood, suggesting that enzymes in erythrocytes are important for PETN metabolism in rat whole blood.     

Electrochemical Red Blood Cell Counting: One at a Time

We demonstrate that the concentration of a red blood cell solution under physiological conditions can be determined by electrochemical voltammetry. The magnitude of the oxygen reduction currents produced at an edge‐plane pyrolytic graphite electrode was diagnosed analytically at concentrations suitable for a point‐of‐care test device. The currents could be further enhanced when the solution of red blood cells was exposed to hydrogen peroxide. We show that the enhanced signal can be used to detect red blood cells at a single entity level. The method presented relies on the catalytic activity of red blood cells towards hydrogen peroxide and on surface‐induced haemolysis. Each single cell activity is expressed as current spikes decaying within a few seconds back to the background current. The frequency of such current spikes is proportional to the concentration of cells in solution.