This technique allows separation of various cell types and cells undergoing various processes based solely on density with little to no manipulation in a short period of time. The main advantages of this method are that it as minimally invasive, requires small volumes, provides fast results, does not depend on classic readouts, and specialized personnel is not required. This technique can be applied for detecting multiple diseases, for example anemia and sepsis.
To begin use the one-click lancing device to prick the finger of the rhesus factor positive blood donor, and collect 10 microliters of blood in one milliliter of DPBS. Add one microliter of fluorescent dye in one milliliter suspension of the rhesus factor positive cells to fluorescently stained the plasma membrane and incubate at 37 degrees Celsius for 15 minutes. Pellet the cells by spinning at 5, 600 times G for 15 seconds and wash the pellet three times using one milliliter of DPBS.
Resuspend the pellet in one milliliter of HBSS with calcium and magnesium. Use the one-click lancing device to prick the finger of the rhesus factor negative blood donor and collect two microliters of blood. To prepare the experimental tube containing only beads, add 174 microliters of HBSS with calcium and magnesium, one microliter of IgG control beads, one microliter of heavy beads with a 1.2 gram per milliliter density, and 24 microliters of 500 millimolar gadolinium.
To prepare the experimental control tube containing IgG, add 172 microliters of HBSS with calcium and magnesium, one microliter of IgG control beads, one microliter of heavy beads, one microliter of rhesus factor negative blood, one microliter of stained rhesus factor positive blood suspension, and 24 microliters of 500 millimolar gadolinium. To prepare the sample experimental tube, add 172 microliters of HBSS with calcium and magnesium, one microliter of anti-RhD coated beads, one microliter of heavy beads, one microliter of rhesus factor negative blood, one microliter of stained rhesus factor positive blood suspension, and 24 microliters of 500 millimolar gadolinium. Set up the magnetic levitation device.
And load 50 microliters of the sample into a capillary tube until the tube is filled. Seal the ends of the capillary tube with a capillary sealant ensuring that there are no bubbles. Place the capillary tube in the capillary holder between the top and bottom magnets, adjust the stage and focus for optimal viewing.
Wait approximately five to 20 minutes for the cells and beads to settle at their magnetic equilibrium position. Using this protocol, the blood cells were separated depending upon the levitation height using the magnetic levitation device. Low and high density beads were used to provide density levitation heights and size references.
The polymorphonuclear cells were levitating above the red blood cells at 21 millimolar gadolinium ion concentration. Old blood cells were levitating at 60 millimolar gadolinium ion concentration. The red blood cells, polymorphonuclear cells, and lymphocytes were separated at 40 millimolar gadolinium ion concentration based on their intrinsic densities.
The rhesus factor antigen was detected on the red blood cells using IgG and anti-RhD antibody coated beads. The bead red cell complexes were not generated in the IgG control sample, but the fluorescently stained red blood cells captured by the rhesus factor positive beads were detected. The binding of the anti-RhB to the rhesus factor positive cells created a bead cell complex in the center at intermittent density of the unbound beads and the negative red blood cells.
Further, the complex formation was confirmed by observing the emitted fluorescence. The B bead complexes formed between the high and low density beads coated with the antibodies for CR1 and CD47 indicate the presence of red blood cell derived extracellular vesicles. Capping the capillary without introducing bubbles is the most challenging step and requires practice.
This method could provide insight for hematology, especially focused on red blood cell diseases.
