Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

The goal of the following experiment is to detect the presence of cancer before formation of secondary tumors. This is achieved by first obtaining and separating blood samples from stage four melanoma patients. As a second step, the blood is introduced into a photoacoustic flow chamber, which allows detection and isolation of melanoma cells.

Next, the captured cells are imaged to confirm the presence of detected melanoma. The results show the system's ability to accurately and rapidly detect and isolate melanoma cells based on photoacoustic wave forms and cell imaging techniques. The main advantage of our technique over existing methods that detect circulating melanoma cells, such as R-T-P-C-R, biomes Magnetic Cell Sorting and other methods, is that our method is label free, and it allows for capture of circulating melanoma cells without altering their surface or securing them to a substrate.

To prepare a sample for metastatic cancer detection begin by drawing approximately 10 milliliters of whole blood from a stage four cancer patient In two five milliliter vacutainer centrifuge, the tubes for 10 minutes. At 3000 RPM, the blood sample will separate into three different layers. The bottom layer comprises the erythrocytes.

The middle layer, also called the buffy coat, contains leukocytes and melanoma cells, and the top layer contains plasma and platelets. Remove as much plasma as possible without disturbing the Buffy coat. Add 250 microliters of his opaque 10 77 to two milliliter troop tubes.

Using a nine inch pastel pipette extract the remaining plasma, the entire Buffy coat, and the top layer of erythrocytes. Making sure to collect less than 500 microliters. Transfer the solution into the previously prepared wind Trobe tubes.

The blood will again separate, but this time the erythrocytes will be separated by the hispa, allowing for easy extraction of the Buffy coat. Use a nine inch Pesa pipette to isolate the entire Buffy coat and place it in a two milliliter eend orph tube. Then suspend it in 1.5 milliliter of PBS with 2%volume per volume between 20.

To construct a flow chamber, start with an eight millimeter long acrylic cylinder with a 20 millimeter outer diameter and a 17 millimeter in a diameter. Next drill, three five millimeter diameter holes through the side of the cylinder at 90 degree angles from each other. Then cut three pieces of polymer tubing and place them into the drilled holes.

Stretch para film across the bottom of the acrylic ring to make a watertight seal. The para film will form a shallow bowl with the acrylic ring and will hold the acrylamide solution that will form a flow chamber. Suspend a 1.6 millimeter diameter wire through the pieces of tubing directly across from one another, making sure that the wire alone is visible through the middle of the ring into the third piece of tubing.

Insert a 1.6 millimeter diameter wire and position the tube one millimeter away from the suspended wire. These wires prevent acrylamide from entering the tubing. Prepare five milliliters of acrylamide.

Then quickly pour the mixture into the acrylic ring, filling it to the top. After a few seconds, the solution will gel. Making a flow chamber mold carefully pull the wires out while making sure not to disturb the acrylamide gel, leaving a cylindrical flow path and a place to position an optical fiber focused onto the flow path.

The flow chamber is now ready for use using ultrasound gel acoustically. Couple a polyvinyl ledine fluoride piso electric acoustic transducer to a flow chamber directly above the flow path. Using a bayonet neo cons or BNC cable, connect the transducer to the input of a broadband receiver.

Connect the output of the filter to the oscilloscope to trigger the oscilloscope. Set up a photo diode near the transducer and use a B NNC cable to plug it into the oscilloscope. Extract the small tubing of the flow chamber until a small pocket of air forms and couple it with the transducer.

Then insert the optical fiber and fill the pocket with water to reduce interface signals and increase the amount of laser light applied to the flow path. Due to the numerical aperture of the optical fiber and the short distance to the flow path. The laser will only irradiate one slug of sample at any given time, ensuring that the sample directly in front of the laser is the same sample creating photo acoustic signals.

Next, attach a T connector to one arm of the flow chamber and two syringe pumps to the other branches of the connector. One syringe contains air while the other will contain the previously prepared cell suspension. Then start both syringe pumps.

When the two admissible fluids reach the T junction, they'll be partitioned into homogeneous slugs and flow past the transducer. Irradiate the flowing sample with a nanosecond pulse laser to generate photoacoustic waves in the melanoma cells. As melanin is a broadband optical absorber, any visible laser wavelength can be used.

A signal appearing on the oscilloscope when a slug is atop the transducer indicates that the slug contains melanoma. Track it through the system before dropping it into a collection vial. To prepare samples for immuno cyto chemistry, place glass slides into cyto funnels and load them into the cyto spin for centrifuge to help the captured cells stick to the glass slides.

Put 100 microliters of 1%BSA in PBS into each funnel. Then centrifuge at 600 RPM for three minutes. After centrifugation, add the cell samples for the CyTOF funnels, then centrifuge at 600 RPM for three minutes.

Spray the slides with an ethanol based fixative. Then wash the slides in PBS for 10 minutes twice to block non-specific binding. Incubate each slide with a mixture of 10 microliters of goat serum and 90 microliters of PBS for one hour at room temperature.

After the incubation, decant the slides and add primary antibody solution. Each slide should contain 1%rabbit mark, one 1%mouse CD 45 and 10%goat serum in PBS incubate for one hour in a humidified chamber at room temperature. Next, wash the slides in PBS for 10 minutes three times.

Then incubate the slides in the appropriate fluorescent secondary antibodies in the dark for one hour at room temperature, decant the slides. Then wash them in PBS for five minutes, three times to stain the nuclei, incubate the cells with 0.1 micrograms per milliliter of DPI for one minute. Then decant the slides and wash in PBS.

Finally, using a resin based mounting medium mount a cover slip over the cell sample seal the cover slip with clear nail polish and image with a fluorescent microscope. The photoacoustic wave forms from white blood cells and melanoma cells are shown here on the left. The white blood cells having no inherent pigmentation produce no photoacoustic waves and manifest as a flat line of electronic noise on the right.

However, the pigmented melanoma cells produce a robust photoacoustic wave. After immuno cyto, chemical staining, cultured melanoma cells show dappy signals in blue, one marked one is highlighted in green overlaying images for DPI and mart one with CD 45 as an indicator of leukocytes. This figure shows melanoma cells among several white blood cells After its development.

Our technique will pave the way for researchers in the field of cancer detection to explore the phenomenon of metastasis to both improve cancer detection and ultimately cure its fatal spread.