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.     

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.     

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.     

An inexpensive thread-based system for simple and rapid blood grouping

This study investigates the use of thread as a flexible and low-cost substrate for the rapid grouping of blood. The use of a capillary substrate such as thread for blood grouping utilises the sensitivity of the flow resistance of large particles in narrow capillary channels to separate agglutinated red blood cells (RBCs) from plasma. Large and discrete particles formed in a continuous liquid phase do not provide capillary wicking driving force and fall behind the capillary wicking front, leading to their separation from the wicking liquid. The capillary substrate therefore provides a very promising but different mechanism for the separation of the agglutinated RBCs and the blood serum phase compared to most existing blood grouping methods. The principle of chromatographic separation is also exploited in this study via the use of suitable dyes to enhance the visual detection of the agglutinated RBCs and the serum phase; surprising and encouraging outcomes are obtained. Using a thread-based device, the ABO and Rh groups can be successfully determined with only 2 μL of whole blood from a pricked finger tip within 1 min and without pre-treatment of the blood sample. It is hoped that a new, inexpensive, rapid and simple method may provide an easy-to-use blood grouping platform well suited to those in developing or remote regions of the world.     

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.     

Integrated separation of blood plasma from whole blood for microfluidic paper-based analytical devices

Many diagnostic tests in a conventional clinical laboratory are performed on blood plasma because changes in its composition often reflect the current status of pathological processes throughout the body. Recently, a significant research effort has been invested into the development of microfluidic paper-based analytical devices (μPADs) implementing these conventional laboratory tests for point-of-care diagnostics in resource-limited settings. This paper describes the use of red blood cell (RBC) agglutination for separating plasma from finger-prick volumes of whole blood directly in paper, and demonstrates the utility of this approach by integrating plasma separation and a colorimetric assay in a single μPAD. The μPAD was fabricated by printing its pattern onto chromatography paper with a solid ink (wax) printer and melting the ink to create hydrophobic barriers spanning through the entire thickness of the paper substrate. The μPAD was functionalized by spotting agglutinating antibodies onto the plasma separation zone in the center and the reagents of the colorimetric assay onto the test readout zones on the periphery of the device. To operate the μPAD, a drop of whole blood was placed directly onto the plasma separation zone of the device. RBCs in the whole blood sample agglutinated and remained in the central zone, while separated plasma wicked through the paper substrate into the test readout zones where analyte in plasma reacted with the reagents of the colorimetric assay to produce a visible color change. The color change was digitized with a portable scanner and converted to concentration values using a calibration curve. The purity and yield of separated plasma was sufficient for successful operation of the μPAD. This approach to plasma separation based on RBC agglutination will be particularly useful for designing fully integrated μPADs operating directly on small samples of whole blood.     

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.     

Preparation of latex reagents combined with IgM and its F(ab')2 fragment from commercial ABO blood grouping reagent

To achieve a rapid assay for ABO blood grouping using a latex reagent, two latex reagents were produced, one of which combined with mouse monoclonal immunoglobulin M (IgM) isolated from commercial ABO blood grouping reagent, and the other of which combined with its F(ab')2 fragment prepared by cold pepsin digestion. The latex reagent adsorbing the F(ab')2 fragment was able to detect the 1000-fold diluted saliva and provided much better sensitivity than that of IgM. This suggests that the difference in sensitivity between the two latex reagents is responsible for adsorption orientation of the antigen site on the latex particles. The new assay successfully completed the ABO blood grouping of cigarette ends within 30 min.     

A glass microfluidic chip for continuous blood cell sorting by a magnetic gradient without labeling

This paper presents a microfluidic chip for highly efficient separation of red blood cells (RBCs) from whole blood on the basis of their native magnetic properties. The glass chip was fabricated by photolithography and thermal bonding. It consisted of one inlet and three outlets, and a nickel wire of 69-microm diameter was positioned in the center of a separation channel with 149-microm top width and 73-microm depth by two parallel ridges (about 10 microm high). The two ridges were formed simultaneously during the wet etching of the channels. The nickel wire for generating the magnetic gradient inside the separation channel was introduced from the side of the chip through a guide channel. The external magnetic field was applied by a permanent magnet of 0.3 T placed by the side of the chip and parallel to the main separation channel. The RBCs were separated continuously from the 1:40 (v/v) diluted blood sample at a flow rate in the range 0.12-0.92 microL/min (9-74 mm/min) with the chip, and up to 93.7% of the RBCs were collected in the middle outlet under a flow rate of 0.23 microL/min. The cell sedimentation was alleviated by adjusting the specific density of the supporting media with bovine serum albumin. Quantum dot labeling was introduced for visual fluorescence tracking of the separation process. The uneven distribution phenomenon of the blood cells around the nickel wire was reported and discussed.     

A microfluidic device integrated with multichamber polymerase chain reaction and multichannel separation for genetic analysis

This work describes a microfluidic device integrated with multichamber polymerase chain reaction (PCR) and multichannel separation for parallel genetic analysis. The microdevice consists of three functional units: temperature control, multiple PCR (four chambers PCR), and multiple channel separation (four separation channels, each channel connected to a PCR chamber). Platinum (Pt)/titanium (Ti) microheater was used to ensure homogeneous temperature field, and Pt-chip sensor was used for temperature monitoring. The interface between chip-PCR and chip separation was simplified by connecting the PCR chamber with separation channel directly. After chip-PCR, PCR products were introduced into parallel separation channels for subsequent separation/detection by applying an electric field automatically. This microdevice was successfully applied for detection of pathogens including hepatitis B virus (HBV) and Mycobacterium tuberculosis (MTB), and genotyping of human leucocyte antigen (HLA)-B27 as well, demonstrating the feasibility of the integrated microdevice for parallel genetic analysis.     

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.     

Explorations of ABO-Rh antigen expressions on erythrocyte dielectrophoresis: changes in cross-over frequency

A quadrupole dielectrophoretic microdevice was utilized to examine the ABO-Rh dependencies on erythrocyte polarizations. This important step toward medical microdevice technology would transform key clinical blood tests from the laboratory into the field. Previous work in dielectrophoretic microdevices demonstrated that the large number of ABO antigens on erythrocyte membranes impacts their dielectrophoretic signature at 1 MHz. This work explores the dielectrophoretic behavior of native human erythrocytes categorized by their ABO-Rh blood types and directly compares these responses to the same erythrocyte sample modified to remove the A and B antigens. A β(1-3)-galactosidase enzyme was utilized to cleave the ABO polysaccharide backbone at the galactosidase bonds. The enzymatic reaction was optimized by comparing agglutination of the native and modified blood cells in addition to UV-Vis and HPLC analysis of the reaction effluent for saccharide residues. Next, the dielectrophoretic behaviors of the native and modified erythrocytes were visually verified in a quadrupole electrode microdevice over a frequency range from 100?kHz to 80?MHz. The lower cross-over frequency (COF), which transitions from negative to positive dielectrophoresis, for ABO blood types tested (A+, A-, B+, B-, AB+, O+ and O-) differed over the range from 17 to 47?MHz. The COFs of the corresponding enzyme-modified erythrocytes were also determined and the range narrowed to 29-41 MHz. A second COF in the 70-80?MHz range was observed and was reduced in the presence of the transmembrane Rhesus factor. These results suggest that antigen expression on erythrocyte membrane surfaces influence cell polarizations in nonuniform AC fields.     

β-环糊精衍生物毛细管气相色谱固定相的研究

将全甲基-β-环糊精、全戊基-β-环糊精及二者的混合液分别静态涂渍在氯化钠粗糙化的弹性石英毛细管内壁.为提高固定相的色谱性能,将环糊精衍生物与OV-1701按1∶4的质量比相混合,制备手性柱.对毛细管的特性、固定相的极性进行了测试,并对手性化合物(±)茚满醇、DL-丙氨酸、DL-缬氨酸、(±)乙酸二氢香芹酮等难分离物质对进行了拆分.结果表明,三种手性固定相均属于中等极性,具有较好的分离性能和成膜能力.分离效果表明,混合固定相对手性化合物及难分离物质对的分离存在协同效应.     

Direct current insulator-based dielectrophoretic characterization of erythrocytes: ABO-Rh human blood typing

A microfluidic platform developed for quantifying the dependence of erythrocyte (red blood cell, RBC) responses by ABO-Rh blood type via direct current insulator dielectrophoresis (DC-iDEP) is presented. The PDMS DC-iDEP device utilized a 400 x 170?μm2 rectangular insulating obstacle embedded in a 1.46-cm long, 200-μm wide inlet channel to create spatial non-uniformities in direct current (DC) electric field density realized by separation into four outlet channels. The DC-iDEP flow behaviors were investigated for all eight blood types (A+, A-, B+, B-, AB+, AB-, O+, O-) in the human ABO-Rh blood typing system. Three independent donors of each blood type, same donor reproducibility, different conductivity buffers (0.52-9.1?mS/cm), and DC electric fields (17.1-68.5?V/cm) were tested to investigate separation dependencies. The data analysis was conducted from image intensity profiles across inlet and outlet channels in the device. Individual channel fractions suggest that the dielectrophoretic force experienced by the cells is dependent on erythrocyte antigen expression. Two different statistical analysis methods were conducted to determine how distinguishable a single blood type was from the others. Results indicate that channel fraction distributions differ by ABO-Rh blood types suggesting that antigens present on the erythrocyte membrane polarize differently in DC-iDEP fields. Under optimized conductivity and field conditions, certain blind blood samples could be sorted with low misclassification rates.     

Silica microbeads capture fetal nucleated red blood cells for noninvasive prenatal testing of fetal ABO genotype

ABO hemolytic disease of the newborn (ABO-HDN), which may cause neonatal jaundice and polycythemia, or even stillbirth or neonatal death, is widespread in China. Prenatal testing for the fetal ABO blood group can reduce unnecessary concerns or ensure prompt treatment. Herein, we presented a method to employ high-density silica microbeads (SiO₂ MBs) for capturing fetal nucleated red blood cells (fnRBCs) in maternal peripheral blood, and we detected the ABO genotype of the fetus using these captured cells. We evaluated 52 patients using the SiO₂ MBs. Among 26 pregnant women with type O blood, 8 (30.8%) of the fetuses had type A blood, 5 (19.2%) had type B blood, and 13 (50%) had type O blood. SRY genes were detected in all 27 male fetuses. This study represents a simple and effective method for noninvasive prenatal detection of the fetal ABO genotype. We believe that this method has great potential for noninvasive prenatal testing of the fetal Rh blood group and other fetal diseases as well.     

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.     

Separation mixed semen of two individuals using magnetic beads coupled ABH blood group antibody

In sexual assault cases, one of the most common samples collected is a mixed semen stain, which is often found on the vagina, female underwear, or bed sheets. However, it is usually difficult to identify the perpetrator based on this sample alone. One technique that has been developed to address this issue is magnetic bead-based separation. This method involves using modified magnetic microspheres to capture and enrich specific target cells, in this case, sperm cells. In this study, we utilized magnetic beads coupled with ABH blood group antibody to isolate sperm cells from an individual of a single ABO blood type. Subsequently, polymerase chain reaction amplification and capillary electrophoresis were employed to perform the genotyping the short tandem repeat (STR) loci. This approach allows for the identification of different individuals in a mixed seminal stain sample from two individuals, by first separating sperm cells based on ABH antigen differences and subsequently utilizing autosomal STR typing on the enriched single blood group cells.     

The pressure induced B1-B2 phase transition of alkaline halides and alkaline earth chalcogenides. A first principles investigation

In this work, we considered the pressure induced B1-B2 phase transition of AB compounds. The DFT calculations were carried out for 11 alkaline halides, 11 alkaline earth chalcogenides and the lanthanide pnictide CeP. For both the B1 and the B2 structures of each compound, the energy was calculated as a function of the cell volume. The transition pressure, the bulk moduli and their pressure derivatives were obtained from the corresponding equations of state. The transition path of the Buerger mechanism was described using roots of the transition matrix. We correlated the computed enthalpies of activation to some structure defining properties of the compounds. A fair correlation to Pearsons hardness of the ions was observed.     

Electrochemical typing of blood using affinity membranes

Human blood group substance of the ABO system was immobilized in a mebrane matrix. The membrane-bound blood group substance retained its binding ability against the corresponding antibody (agglutinin) in serum. The transmembrane potential changed drastically with the agglutination of the membrane-bound blood group substance. Electrochemical typing of blood was performed with sera, using a pair of membranes with immobilized A- and B-type blood group substances. The blood type was determined by measuring the transmembrane potential across these membranes before and after the agglutination reaction. A possible re-use of the membrane-bound blood group substance by treatment with galactose is discussed.     

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.