Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

The overall goal of this procedure is to simulate near infrared fluorescence imaging applications in breast conserving surgery using tissue like breast phantoms. This is accomplished by first creating tumor like fluorescent inclusions of different size and shape. The second step is to integrate the fluorescent inclusions in breast shaped phantoms with optical characteristics similar to human breast tissue.

Next, the near infrared fluorescence imaging system is applied to guide the surgeon intraoperatively in localizing and removing the fluorescent tumor like inclusion. The final step is to scan the surgical cavity margin for the presence of residual disease following removal of the fluorescent tumor like inclusion. If applicable, the surgeon can then excise remaining tumor tissue guided by the near infrared fluorescence imaging system.

The main advantage of this technique over existing methods like bed CT or MRI, is that near infrared fluorescence. Optical imaging provides high resolution images in real time and can easily be applied in an intraoperative setting. Moreover, this technique is inexpensive and makes use of non-ionizing radiation.

Demonstrating the procedure will be graduate student from our research group. To begin, collect solid items of the desired shape and size that can serve as models for tumor simulating inclusions such as beads or marbles. Thoroughly clean the tumor models and remove any irregularities.

Place each model in a separate thin walled square plastic box with a smooth surface. If necessary, fixate the model to the bottom of the box to keep it in position. Then pour the required amount of silicone component A into a mixing bowl and add silicone component B in a 10 to one ratio by weight.

Mix both components thoroughly. Gently pour the silicone mixture in the plastic box to prevent trapping air bubbles. Let the silicone mixture solidify for at least six hours before cutting the mold and removing the tumor model.

Optionally, the silicone mold can be cut in a zigzag pattern to allow it to fit back together cleanly. To create fluorescent inclusions, add two grams of aros to 50 milliliters of tris buffered saline, or TBS, which has been prepared with sodium azide as described in the text protocol. Heat the aros slurry using a microwave until the boiling point is reached.

Stir thoroughly until the aros is completely dissolved. Add 1.1 grams of hemoglobin and five milliliters of intralipid, 20%to 50 milliliters of TBS, and add this to the aros mixture under constant stirring to resemble the optical characteristics of the surrounding breast. Phantom tissue add 20.0 milligrams of the fluorescent dye incy and green to 83.8 milliliters of deionized water.

Pipette five milliliters from this solution and add it to the aros mixture. To obtain a final concentration of 14 micromolar. Gently fill the silicone molds with the H aros mixture using a syringe.

Let the fluorescent inclusion solidify at room temperature for approximately one hour. Protect the inclusions from light by covering the entire mold with aluminum foil. After solidification, gently open the mold and press out the inclusion.

Protect the Aros inclusions from light and dehydration by wrapping them in aluminum foil and store them in a humidified storage container at four degrees Celsius. Obtain a cup shaped mold to create breast phantoms of the desired size and volume to create a breast phantom. With the volume of 500 milliliters, add 50 grams of gelatin, 250 bloom to 500 milliliters of TBS.

Heat the gelatin slurry to 50 degrees Celsius under constant stirring. Once the gelatin is completely dissolved, let the gelatin mixture gradually cool down and maintain it at a constant temperature of 35 degrees Celsius. Using a hot water bath under constant stirring, add 5.5 grams of bovine hemoglobin to simulate the absorption of photons, and 25 milliliters of intralipid.

20%to simulate the scattering of photons in the tissue. Pre chill the cup shaped mold at four degrees Celsius for at least one hour. Once cool.

Pour the gelatin mixture in the mold to a level that corresponds to the predefined depth of the aros tumor. Simulating inclusion. Let the gelatin mixture solidify at four degrees Celsius for 30 minutes to one hour.

After solidification position a tumor simulating fluorescent aros inclusion on the surface of the phantom and temporarily fixate the inclusion with a small needle. Pour the remainder of the warm gelatin mixture in the remaining mold volume, allowing for adherence of both layers without creating refraction artifacts. Mark the location of the fluorescent tumor simulating inclusions on the mold before letting the phantom solidify overnight at four degrees Celsius.

Once solidified, remove the needles used for temporary fixation of the inclusions and gently remove the breast phantom from its mold. Protect the breast phantom from light and dehydration by wrapping it in aluminum foil and storage it in a humidified storage container at four degrees Celsius. A near infrared fluorescent camera system for intraoperative application is required for simulating targeted fluorescence imaging in breast cancer surgery.

See the text protocol for setup of the fluorescent camera system. Take the tissue simulating breast phantom from its container and place it on a flat, non fluorescent surface. Next position the fluorescent imaging device above the breast phantom, leaving a sufficient working distance for excision of the tumor.

Simulating inclusions, localize the tumor simulating fluorescence inclusion using palpation and or fluorescence imaging of the phantom breast in case no fluorescent signal can be detected. The inclusion is either positioned too deep in the phantom for detection or the image acquisition time should be increased once the inclusion is localized in size, the phantom breast and remove the tumor. Simulating inclusion under real-time fluorescence guidance using conventional surgical equipment directly after removal of the tumor simulating inclusion image, the surgical cavity for any remaining fluorescent activity indicating inadequate excision in case of any remaining fluorescent activity.

Excise the inclusion remnant under direct fluorescence guidance until no fluorescent signal is left image. The excised phantom fragments to simulate for fluorescent guided macroscopic margin status. Assessment representative results of fluorescence guided intraoperative tumor localization are shown here.

Fluorescent tumor like inclusions were detected up to a depth of approximately two centimeters. Deeper seeded inclusions were detected after incision of overlaying phantom tissue. The surgeon was continually guided by the fluorescent signal originating from the fluorescent tumor like inclusion.

During breast conserving surgery on the phantoms after removal of the tumor like inclusion, the surgeon evaluated the extent of surgery by scanning the surgical cavity. Any remaining fluorescent signal indicated incomplete tumor excision and the tumor remnant was removed during the same surgical procedure. Following excision of the tumor like inclusion, the excised lump was imaged to evaluate gross surgical margin status.

Fluorescence imaging might aid evaluation of microscopic margin status by a pathologist through pinpointing areas that are suspect for close or positive margins. The implications of this technique extend toward better outcomes for breast conserving surgery by conjugating near infrared fluorescence, optical imaging agents to targeted Myers such as monoclonal antibodies. Targeted delivery of fluorescent agents can be obtained for intraoperative imaging applications.