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.
