Name: magnetic levitation coupled with portable imaging and analysis for disease diagnostics
Uploaded: 2017-02-19T15:00:00
Description: Watch this Scientific Journal Video about Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics at JoVE.com

The authors would like to acknowledge Dr. Matthew Heeney of Boston Children's Hospital/Dana-Farber Cancer Institute and Dr. Farzana Pashankar of Yale-New Haven Hospital for providing sickle cell patient samples. The authors would like to thank Chu H. Yu and Ashwini Joshi for their assistance in testing these samples and compiling the data.
S.T. acknowledges the American Heart Association Scientist Development Grant (15SDG25080056) and the University of Connecticut Research Excellence Program award for financial support of this research. S.K. acknowledges that this material is based upon work supported by the National Science Foundation Graduate Research Fellowship (DGE-1247393).

Ethical Statement: All procedures involving human blood samples were carried out according to the institutional regulations. All protocols were reviewed and approved by the Institutional Review Board. An informed consent was given by all participants.
1. Sample Preparation for Sickle Cell Disease Diagnosis5,8
<ol>
	<li>Prepare a 50 mM solution of gadobutrol in Hank&#39;s Balanced Salt Solution (HBSS).</li>
	<li>Dissolve 10 mM sodium ...

<ol>
	<li>Tasoglu, S., Khoory, J., Tekin, H., Thomas, C., Karnoub, A., Ghiran, I., Demirci, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Levitational+Image+Cytometry+with+Temporal+Resolution.">Levitational Image Cytometry with Temporal Resolution.</a> Advanced Materials. 27 (26), 3901-3908 (2015).</li><li>Tasoglu, S., Yu, C. H., Liadudanskaya, V., Guven, S., Migliaresi, C., Demirci, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+Levitational+Assembly+for+Living+Material+Fabrication.">Magnetic Levitational Assembly for Living Material Fabrication.</a> Advanced Healthcare Materials. 4 (10), 1469-1476 (2015).</li><li>Tasoglu, S., Yu, C. H., Gungordu, H. I., Guven, S., Vural, T., Demirci, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+and+magnetic+self-assembly+of+magnetoceptive+gels.">Guided and magnetic self-assembly of magnetoceptive gels.</a> Nature Communications. 5, 4702 (2014).</li><li>Amin, R., Knowlton, S., Yenilmez, B., Hart, A., Joshi, A., Tasoglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smart-phone+Attachable,+Flow-Assisted+Magnetic+Focusing+Device.">Smart-phone Attachable, Flow-Assisted Magnetic Focusing Device.</a> RSC Advances. 6, 93922-93931 (2016).</li><li>Knowlton, S. M., Sencan, I., Aytar, Y., Khoory, J., Heeney, M. M., Ghiran, I. C., Tasoglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sickle+Cell+Detection+Using+a+Smartphone.">Sickle Cell Detection Using a Smartphone.</a> Sci Rep. 5, 15022 (2015).</li><li>Knowlton, S., Yu, C. H., Jain, N., Ghiran, I. C., Tasoglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Smart-Phone+Based+Magnetic+Levitation+for+Measuring+Densities.">Smart-Phone Based Magnetic Levitation for Measuring Densities.</a> PLoS One. 10 (8), e0134400 (2015).</li><li>Yenilmez, B., Knowlton, S., Tasoglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-Contained+Handheld+Magnetic+Platform+for+Point+of+Care+Cytometry+in+Biological+Samples+.">Self-Contained Handheld Magnetic Platform for Point of Care Cytometry in Biological Samples .</a> Advanced Materials Technologies. 1, 1600144 (2016).</li><li>Yenilmez, B., Knowlton, S., Yu, C. H., Heeney, M., Tasoglu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Label-Free+Sickle+Cell+Disease+Diagnosis+Using+a+Low-Cost,+Handheld+Platform.">Label-Free Sickle Cell Disease Diagnosis Using a Low-Cost, Handheld Platform.</a> Adv Mat Tech. 1 (5), 1600100 (2016).</li><li>Bender, M. A., Douthitt Seibel, G., Pagon, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> GeneReviews. , (1993).</li><li>Kaul, D. K., Fabry, M. E., Windisch, P., Baez, S., Nagel, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erythrocytes+in+sickle+cell+anemia+are+heterogeneous+in+their+rheological+and+hemodynamic+characteristics.">Erythrocytes in sickle cell anemia are heterogeneous in their rheological and hemodynamic characteristics.</a> J Clin Invest. 72 (1), 22-31 (1983).</li><li>Joiner, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gardos+pathway+to+sickle+cell+therapies?.">Gardos pathway to sickle cell therapies?.</a> Blood. 111 (8), 3918-3919 (2008).</li><li>Finch, J. T., Perutz, M. F., Bertles, J. F., D&#246;bler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Sickled+Erythrocytes+and+of+Sickle-Cell+Hemoglobin+Fibers.">Structure of Sickled Erythrocytes and of Sickle-Cell Hemoglobin Fibers.</a> Proc Natl Acad Sci. 70 (3), 718-722 (1973).</li><li>Lew, V. L., Etzion, Z., Bookchin, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dehydration+response+of+sickle+cells+to+sickling-induced+Ca(++)+permeabilization.">Dehydration response of sickle cells to sickling-induced Ca(++) permeabilization.</a> Blood. 99 (7), 2578-2585 (2002).</li><li>Ernst, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> NCCLS Procedures for the Collection of Diagnostic Blood Specimens by Venipuncture: Approved Standard-Sixth Addition. 27 (26), (2007).</li></ol>

Critical Steps within the Protocol 
Critical factors in this process include the proper alignment of the magnets. If the magnets become dislodged or separated more than normal within the device, this can affect the results. To control for this fault or others in the process, a density-controlled particle, such as polystyrene microspheres, can be used periodically to control for changes over time. Further, levitation time is important to allow the cells to reach equilibrium. For red blood cells, 10 min is sufficient ...

Here, we present a technology platform and a technique which uses magnetic levitation coupled with automated imaging and analysis to analyze the density distribution of a patient&#39;s cells as an indicator for disease. This versatile approach for density-based cytometric analysis can ultimately be applied to a range of disease diagnostics. However, in order to be compatible with point-of-care testing and use in developing countries, the technique must satisfy requirements for low cost, portability, and usability. The device and consumables must be easily obtained at a low cost. The sample preparation must be simple, analysis should be automated with minimal requirements for user input or interpretation, and results should be returned quickly. Further, the device must be compact and portable to be useful in clinical settings as well as developing countries. Thus, we have developed a device and method to use magnetic levitation in point-of-care-compatible technology by coupling automated imaging and image analysis to return results regarding the density distribution of a population of a patient&#39;s cells.
Point-of-care technologies offer a notable advantage over current clinical laboratory testing procedures. The technology currently available is too expensive to be owned by a clinician or too complex to be carried out by medical staff. Many of these procedures require labor-intensive protocols which must be carried out by a trained technician. For these reasons, patient samples such as blood or urine are generally collected in the physician&#39;s office then transferred to a remote, centralized testing laboratory for clinical testing, which may take several days for the physician to receive the results of the test. This can cause delays or complications in the course of treatment in some cases, makes this testing very costly and inefficient (causing a financial burden on insurance payers), and further makes many diagnostics inaccessible in low-resource settings and developing countries.
Here, we present a magnetic levitation technique coupled with automated imaging and analysis in both a device with embedded imaging and processing (Figure 1) and a smartphone-compatible device (Figure 2). These magnetic levitation-based devices represent a broadly applicable platform technology which has the potential to be applied to a range of different medical diagnostic applications. The magnetic levitation approach functions based on an equilibrium between two forces: a magnetic force and a buoyancy force1,2,3. When a particle is suspended in a paramagnetic medium and inserted into a magnetic field generated by two magnets with like poles facing each other, a magnetic force acts on the particle in the direction toward the centerline between the two magnets. The buoyancy force is caused by the relative density of the particle compared to the suspending medium and is upward in the case of particles less dense than the medium and downward in the case of particles denser than the surrounding medium. Based on these two forces, particles will reach an equilibrium levitation position in the field which balances these two forces; this position is directly related to the density of the particle, with denser particles levitating lower in the field than less dense particles. An imaging module, either a built-in smartphone camera4,5,6 or independent optical components equipped with a magnifying lens7,8, are used to visualize the positions of the particles. Image processing, either through a smartphone application4,5,6 or an embedded processing unit7,8, then processes the captured images to quantify the spatial distribution and, therefore, the density distribution of the population. In order to analyze larger samples (such as those with only a few particles of interest per milliliter, flow can be integrated directly into the device such the particles are levitated and analyzed as they pass through the imaging region (Figure 2).
<img alt="Figure 1" src="/files/ftp_upload/55012/55012fig1.jpg" /> 
Figure 1: Self-contained Magnetic Levitation Platform. (a) Compact magnetic levitation device including a magnetic focusing module, imaging components (a light-emitting diode (LED), an optical lens, and a camera detector), and a processing unit with a display screen. (b) Magnetic field strength in the cross-section of the area between the magnets where the sample is inserted. The field strength is greatest at the surface of the magnets and approaches zero at the centerline between them. (c) Particles, such as cells, within the magnetic field experience several forces: a magnetic force (Fm) toward the centerline between the magnetics, with magnitude varying based on the position of the particle; a gravitational force (Fg&#39;) which depends on the particle density relative to that of the suspending medium, and a drag force (Fd) resisting the particle motion. Reproduced, with permission, from Yenilmez, et al.8 <a href="http://cloudflare.jove.com/files/ftp_upload/55012/55012fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55012/55012fig2.jpg" /> 
Figure 2: Smartphone-compatible Flow-assisted Magnetic Levitation Platform. (a-c) Front (a), side (b), and back (c) views of magnetic levitation device (d) The components of the device include: 1) Magnetic levitation module, including permanent magnets, a magnifying lens, and an LED and light diffuser, 2) smartphone case, 3) electronics, including a microcontroller, pump driver, and Bluetooth receiver, 4) micro-pump holder, 5) adjustable orifice, 6) waste tube holder, 7) battery holder, 8) sample holder, 9) dual-purpose stand and cover. (e) Flow schematic, showing pumping of the sample through the magnetic field. (f) Cross-section of the magnetic levitation module, showing how particles of different densities will align as they are pumped through the field; less dense particles, such as Particle 1, will equilibrate at a higher levitation height than denser particles, such as Particle 2. Reproduced, with permission, from Amin, et al.1 <a href="http://cloudflare.jove.com/files/ftp_upload/55012/55012fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The minimum requirements for use of any sample for density distribution analysis in this system include the ability to obtain a suspension of cells or particles greater than approximately 5 &#181;m and less than approximately 250 &#181;m in size (for imaging and image processing) and its compatibility with mixing in a solution of a paramagnetic solution such as the gadobutrol used here. For disease diagnostics, compatible applications include those in which (i) cells of interest inherently have an altered density when they carry a disease compared to healthy controls, (ii) a density change can be induced in the cell by addition of a reagent or some alternative treatment for a short incubation time, or (iii) different cell types are being identified in a single sample and inherently (or via some treatment) have unique characteristic densities.
Sickle cell disease is a genetic disorder causing a mutated form of hemoglobin, HbS, to be produced in a person&#39;s red blood cells (RBCs), which can result in intermittent vaso-occlusive events and chronic hemolytic anemia9. It is diagnosed using either hemoglobin isoelectric focusing, high-performance liquid chromatography (HPLC) fractionation, or hemoglobin electrophoresis which are highly accurate but must be performed in a clinical testing laboratory because they are incompatible with point-of-care settings. Solubility and paper-based tests for sickle cell disease have been proposed, but generally require subjective user interpretation and confirmatory testing. Here, we use a density-based approach to identify sickle RBCs, which attain a higher density than RBCs from people without sickle cell disease. The mechanism involves polymerization of the mutated form of hemoglobin, HbS, which causes RBC dehydration in sickle cell disease RBCs under deoxygenated conditions10,11,12,13.
This density-based approach can also be applied to separate cells of different types on the basis of density: white blood cells (WBCs) and RBCs7. WBCs are generally responsible for fighting infections in the body. WBC cytometry can be used to quantify the number of these cells in the blood and serves as a useful diagnostic tool. WBC counts higher than normal (generally considered greater than 11,000 cells per &#181;L) may indicate infection, immune system disorders, or leukemia. WBC counts below the normal range (around 3,500 cells per &#181;L) may be caused by autoimmune disorders or conditions which damage bone marrow. Unlike alternative technologies, the process presented here does not rely on lysis of the RBCs or stains in order to identify WBCs. This cell-based test takes advantage of the unique inherent densities of the two cell types to perform separation, as the WBC population density has been reported to be lower than that of the RBC population as calculated previously using density gradient centrifugation1,5,8.
Compared to alterative testing at remote locations, this test is rapid, with simple sample preparation (Figure 3), separation of cells in the device within 10 - 15 min, and automated imaging and analysis which requires less than 1 min. In this way, the device can return results quickly to better inform medical decisions, allow treatment to be administered immediately to alleviate physical and psychological pain, and reduce the risk of complications associated with a delay in medical care. This technique can be performed on-site either in clinical settings due to simple sample preparation and automated imaging and analysis which returns a result with minimal user input or interpretation. Because of the use of a simple approach using permanent magnets for sample analysis and the use of either a smartphone or simple electrical components for imaging and image processing, the device as well as the per-test costs are minimal compared to some sophisticated testing procedures.

<table border="0" cellpadding="0" cellspacing="0" width="938">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Gadavist (Bayer)</td>
			<td style="width:257px;">Jefferson Medical and Imaging</td>
			<td style="width:119px;">2068062</td>
			<td style="width:241px;">Gadavist contains 1M gadobutrol, a chelate of gadolinium. We purchased 2 mL vials with 15/ca.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Square glass microcapillary tubes</td>
			<td style="width:257px;">Vitrocom</td>
			<td style="width:119px;">8270</td>
			<td>50 mm length is sufficient</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:321px;">Sodium metabisulfite</td>
			<td style="width:257px;">Sigma-Aldrich</td>
			<td style="width:119px;">S9000</td>
			<td>Chemical formula: Na2S2O5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Leica Microsystems Critoseal tube sealant</td>
			<td style="width:257px;">Fisher Scientific</td>
			<td style="width:119px;">02-676-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Hank&#39;s Balanced Salt Solution</td>
			<td style="width:257px;">Sigma-Aldrich</td>
			<td style="width:119px;">H9269&#160;SIGMA</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Trypsin-EDTA</td>
			<td style="width:257px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4049</td>
			<td>Or other reagent as recommended for the cell type used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">MICROLET 2 Adjustable Lancing Device</td>
			<td style="width:257px;">Walgreens</td>
			<td style="width:119px;">246567</td>
			<td>Any lancing device is acceptable when used according to biosafety protocols</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Microlet Lancets</td>
			<td style="width:257px;">Walgreens</td>
			<td style="width:119px;">667474</td>
			<td>Must be dispoable and not reused</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Hausser Bright-Line Phase Hemacytometer</td>
			<td style="width:257px;">Fisher Scientific</td>
			<td style="width:119px;">02-671-6</td>
			<td>Or any preferred method for cell counting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">ACK Lysing Buffer</td>
			<td style="width:257px;">ThermoFisher</td>
			<td style="width:119px;">A1049201&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

magnetic levitation coupled with portable imaging and analysis for disease diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

For cell density distribution analysis, which is the technique used for sickle cell disease diagnosis, the aim is to identify the width of the distribution of the cell population. Blood cells from patients without sickle cell disease will be confined within a predictable width. Cells from patients with sickle cell disease will be distributed throughout a wider region, with a downward skew in the cell distribution (see Figure 4.) For any particular application, a threshold...

Watch this Scientific Journal Video about Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics at JoVE.com

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for ...

The overall goal of this process is to perform density-based cellular analysis for various medical diagnostic applications by measuring the altered distribution of a cell population or by separating cell types based on their characteristic densities. This method can help solve a key challenge in biomedical engineering to perform important cell ways medical diagnostics in clinical settings or developing countries. The main advantage of this technique is that it does not require a lot of equipment or preparation and it can be done by almost anyone.The implications of this technique extend toward sickle cell diagnosis because this device can quantify the increased density and decreased levitation rate caused by low oxygen conditions in sickle cells. Though this method is useful for medical diagnostic tests as demonstrated here, it can also be applied to other density based cytometric tests by altering the sample and modifying the software. First, prepare 10 milliliters of a 50 millimolar solution of gadobutrol and Hanks balanced salt solution.Then add sodium metabisulfite to achieve a 10 millimolar concentration in the gadolinium solution. Place 100 microliters of the gadolinium sodium metabisulfite stock solution in a microcentrifuge tube. Obtain a blood sample by standard venipuncture techniques or by finger stick.To collect the fingerstick sample, apply pressure near the pierced site without squeezing the tissue. Wipe the pierced site at least three times, and then collect the blood sample with a micropipettor. Mix up to one microliter of the blood sample with the gadolinium sodium metabisulfite solution in the tube to obtain the sample for sickle cell disease diagnosis.First, prepare both 25 and 250 millimolar solutions of gadobutrol and Hanks balanced salt solution. Then, acquire a blood sample using the previously described fingerstick or venipuncture techniques. Dilute about one microliter of the blood sample by a factor of 1000 with a 25 millimolar gadobutrol solution to obtain the sample for white blood cell cytometry.To prepare an additional sample for determining the levitation range of white blood cells alone add 5 microliters of the blood sample to 500 microliters of red blood cell lysis buffer. Incubate the mixture at room temperature for three to five minutes. Then, prepare a solution of one part 250 millimolar gadolinium solution and nine parts lysate to obtain the lysate sample solution with a gadolinium concentration of 25 millimolar.To begin sample analysis, turn on the device and place it on a level surface. Draw the sample into a square glass capillary tube. Once the tube is half full, seal the end with capillary tube sealant.Insert the capillary into the magnetic levitation module of the device, so that only one centimeter of the tube remains visible. Allow the sample to sit undisturbed in the device for 10 minutes to equilibrate. Then, check that the cells of interest are visible on the screen.Press the single measurement button to save the image. Record the analysis results automatically displayed on screen. If the device indicates that too many or too few cells are onscreen, move the capillary slightly in or out of the device and equilibrate the sample for another 10 minutes.After saving the first image, move the capillary in or out by about 0.5 centimeters. Allow the sample to equilibrate, and then acquire another image for analysis. Acquire data from at least five images from different locations on the sample to avoid errors.Save the images to the device or to a USB drive. Once finished, remove the sample from the device and dispose of it appropriately. First, fit a smartphone into the magnetic levitation device.Ensure that the smartphone camera is in line with the magnetic levitation module. Secure the smartphone upright on a stand on a level surface. Insert the capillary into the levitation module and attach the micropump tubing to each end.Place the sample vial in the sample holder. Then, launch the smartphone app. Turn on the micropump and set the parameters for flow rate and LED brightness on the first screen.Once the sample is flowing smoothly, swipe left to enter image capture mode. Acquire at least five different videos. Then, in the analysis section of the app, select flow assisted analysis and open each file to be analyzed.When subjected to magnetic levitation, blood cells from patients without sickle cell disease are confined to a predictable width. Cells from patients with sickle cell diseases are distributed over a wider region that trends downwards. This is reflected in the analysis of the confinement widths.The healthy control cells appear in a narrow band, whereas the sickle cells appear in a broader band with far more variation in overall width. Individual cell types with discrete density ranges can also be differentiated by this method with the best separation achieved at low gadolinium concentrations. Here, the levitation height of white blood cells was determined from a sample of which red blood cells had been lysed before analysis.This known levitation height was used for white blood cell counting. To calculate the number of white blood cells per microliter of a sample diluted by one to 1000, the number of cells observed in the image is multiplied by 2000. Once mastered, this technique can be done in only 10 minutes if it's performed properly.Following this procedure, other researchers can use this approach to develop medical diagnostic tests for a range of medical conditions.

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