Brightfield and fluorescence in-channel staining of thin blood smears generated in a pumpless microfluidic (original) (raw)
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Gascoyne 2004 Microfluidic approaches to malaria detection
Microfluidic systems are under development to address a variety of medical problems. Key advantages of micrototal analysis systems based on microfluidic technology are the promise of small size and the integration of sample handling and measurement functions within a single, automated device having low mass-production costs. Here, we review the spectrum of methods currently used to detect malaria, consider their advantages and disadvantages, and discuss their adaptability towards integration into small, automated micro total analysis systems. Molecular amplification methods emerge as leading candidates for chip-based systems because they offer extremely high sensitivity, the ability to recognize malaria species and strain, and they will be adaptable to the detection of new genotypic signatures that will emerge from current genomic-based research of the disease. Current approaches to the development of chip-based molecular amplification are considered with special emphasis on flow-through PCR, and we present for the first time the method of malaria specimen preparation by dielectrophoretic field-flow-fractionation. Although many challenges must be addressed to realize a micrototal analysis system for malaria diagnosis, it is concluded that the potential benefits of the approach are well worth pursuing.
12th International Conference on Miniaturized Systems for Chemistry and Life Sciences, MicroTAS 2008, 2008
A novel, point-of-care immunoassay format is demonstrated for malarial diagnosis. The assay system consists of a disposable card with integrated dry reagent storage and an external reader that conducts fluidic actuation and assay quantification. Reagents flow through a porous membrane encased in the card's polymeric laminates to conduct a colorimetric sandwich assay on the membrane's surface. The system produces quantitative results with detection limits on the order of ELISA in under 9 minutes.
A microfluidics-based instrument for cytomechanical studies of blood
1st Annual International IEEE-EMBS Special Topic Conference on Microtechnologies in Medicine and Biology. Proceedings (Cat. No.00EX451), 2000
A complete instrument for the measurement of erythrocyte (red blood cell) flow in microchannels is presented. The instrument measures circa 1500 cells on a cell-by-cell basis in an array of microchannels. Microchannel dimensions and operating pressures are physiologically-analogous. Resulting hi-variate data, obtained by real-time image processing, describes cell flow with a unique volume-velocity space representation which provides detailed insights into both cell flow resistance (independent of cell volume) and the presence of pathological sub-populations. We commence by discussing the instrument's design, with particular attention to the microflnidics. We proceed to report analyses o f the instrument's reproducibility, the characterisation of normal blood, and our ability to detect artificial sub-populations of cells with rigid membranes produced by chemically modifying a fraction o f the analyte. Having validated the performance of the instrument, we then present a clinical result from a transfused thalassaemic displaying highly irregular microrheological metria.
Development of a film-based immunochromatographic microfluidic device for malaria diagnosis
Biomedical Microdevices, 2019
In this study, a novel film-based immunochromatographic microfluidic device (IMD) has been developed for malaria diagnosis. A microfluidic channel was patterned on a polyethylene terephthalate (PET) double-sided adhesive film using a plotting cutter and was assembled with a polycarbonate (PC) film. The PC film used for the probe immobilization layer was activated using oxygen plasma treatment to modify the film surface with avidin-biotin linker to immobilize a capture antibody. A fluorescent labeled Pan type mAb conjugate was prepared for signal indicator after undergoing a sandwich enzyme-linked immunosorbent assay (ELISA). Target antigens include Plasmodium falciparum (P. falciparum) lactate dehydrogenase (LDH) and Plasmodium vivax (P. vivax) LDH which were injected into the sample inlet. Target antigens combined with the conjugate and then flowed to the detection chamber where two test dots and a control dot (Ctrl) exist. In the presence of P. falciparum LDH, three detection dots including test dot 1 (T1), test dot 2 (T2) and Ctrl revealed fluorescence signals where P. falciparum mAb, Pan type pLDH mAb and goat anti-mouse IgG were immobilized, respectively. When P. vivax LDH was present, T2 and Ctrl dots showed fluorescence signals while no signal was detected with the negative control. P. falciparum LDH and P. vivax LDH were successfully detected on the IMD with a detection limit of 50 ng/mL and 100 ng/mL, respectively. The IMD provides a point-of-care diagnosis platform which is able to analyze pathogenic bacteria and viruses that can be applied in the field of clinical diagnosis and food safety testing.
Biomedical Microdevices, 2011
This paper reports on a microfluidic platform to isolate and study avian red blood cells (RBCs) infected to various degrees by the malaria parasite Plasmodium gallinaceum. The experimental findings point to the feasibility of using the morphological changes on the surface of the malaria infected avian RBC (miaRBCs) as biomarkers for diagnosis. A glass substrate with a controlled surface roughness was used as part of a polydimethylsiloxane (PDMS) microfluidic channels. When wholeblood samples were introduced into the channels, the miaRBCs would be preferentially slowed and eventually become immobilized on the roughened surface. The surface lesions and furrow-like structures on the miaRBC surfaces offered a markedly higher probability to interact with the roughened substrate and allowed the cells to become imobilized on the surface. The captured miaRBCs were from blood samples at various degrees of infection at 3.2%, 3.9%, 9.1%, 13.4%, 20.1%, 28%, and 37%. It was observed that the miaRBCs could be selectively captured under a wall shear rate between 2.1 to 3.2 s −1 , which was directly proportional to the flow rate through the channels. This capture rate could be improved by increasing the channel length and finer flow control. It was also found that a roughened glass substrate with ten-point-height larger than the depth of surface lesions and furrow-like structures of miaRBCs showed a substantial enhancement on the number of immobilized infected RBCs. These findings indicated that surface morphologies, including surface lesions and furrow-like structures, can serve as an alternative biomarker for malaria diagnosis.
Malaria Journal, 2014
Background: Understanding of malaria pathogenesis caused by Plasmodium falciparum has been greatly deepened since the introduction of in vitro culture system, but the lack of a method to enrich ring-stage parasites remains a technical challenge. Here, a novel way to enrich red blood cells containing parasites in the early ring stage is described and demonstrated. Methods: A simple, straight polydimethylsiloxane microchannel connected to two syringe pumps for sample injection and two height reservoirs for sample collection is used to enrich red blood cells containing parasites in the early ring stage (8-10 h p.i.). The separation is based on the non-inertial hydrodynamic lift effect, a repulsive cell-wall interaction that enables continuous and label-free separation with deformability as intrinsic marker. Results: The possibility to enrich red blood cells containing P. falciparum parasites at ring stage with a throughput of~12,000 cells per hour and an average enrichment factor of 4.3 ± 0.5 is demonstrated. Conclusion: The method allows for the enrichment of red blood cells early after the invasion by P. falciparum parasites continuously and without any need to label the cells. The approach promises new possibilities to increase the sensitivity of downstream analyses like genomic-or diagnostic tests. The device can be produced as a cheap, disposable chip with mass production technologies and works without expensive peripheral equipment. This makes the approach interesting for the development of new devices for field use in resource poor settings and environments, e.g. with the aim to increase the sensitivity of microscope malaria diagnosis.
The use of fluorescence enhancement to improve the microscopic diagnosis of falciparum malaria
Malaria Journal, 2007
Background: Giemsa staining of thick blood smears remains the "gold standard" for detecting malaria. However, this method is not very good for diagnosing low-level infections. A method for the simultaneous staining of Plasmodium-parasitized culture and blood smears for both bright field and fluorescence was developed and its ability to improve detection efficiency tested.
Aim: The present study is planned to compare acridine orange (A.O) staining with Giemsa staining by using light microscopy with IF and also with fluorescent microscopy for detection of parasites in peripheral blood of patients suffering from clinically suspected cases of malaria. Place and Duration: In the Pathology department of Allama Iqbal Medical College, Lahore/ Jinnah Hospital Lahore for one-year duration from April 2019 to April 2020. Methods: 200 patients with fever and chills were included. General tests such as Hb, TLC, and platelets were performed by the sysmex K-1000. Blood thin and thick membranes were prepared and stained according to the protocol given, ie, by Giemsa and AO staining, and the slides were examined with various microscopes, ie light microscopy, IFS light microscope and fluorescence microscope. Results: Of the 200 patients, 170 (85%) patients tested positive for parasitemia and 30 (15%) patients were negative for the malaria parasite. fib, TLC and platelet counts were all decreased compared to MP negative cases. Conclusion: IFS microscopy with acridine staining showed early detection of malaria parasites by counting fewer fields compared to light microscopy with Giemsa staining. The time spent detecting parasites was also significantly reduced in the IFS microscope thanks to the use of AO dyes.
Journal of Pharmaceutical Research International, 2021
Objective: The objective of the study is to compare three techniques, routinely used rapid diagnostic tests (lateral flow immune chromatography) versus nucleic acid amplification test (NAT) versus Paper-based microfluidics for DNA diagnostics of Malaria, in terms of their sensitivity and specificity as diagnostic tests in detecting malarial infection among febrile illnesses, suspected of malaria, as well as to compare their cost-effectiveness. Methodology: Three seventy febrile cases suspected of malaria with negative results with RDT will be screened by real-time PCR and DNA microfluidics techniques, sensitivity and specificity of these as screening tests will be compared. The number of extra positive cases detected by NAT gives us the yield. Cost-effectiveness analysis will be done by calculating the incremental cost-effectiveness ratio (ICER) and average cost-effectiveness ratio (ACER) for the tests. Statistical Analysis: Statistical analysis will be done using SPSS version 21. ...