In this video, a protocol for continuous blood sampling is provided. Blood radioactivity will be measured in parallel to a PET/CT using an extracorporeal arteriovenous shunt. By doing this, the arterial input function for subsequent quantification of PET/CT data is generated and allows later on a precise biokinetic modeling.
The main advantages of this protocol are that it's reproducible, generates minimal blood loss, and facilitates a high accuracy in temporal resolution of the arterial input function. It is essential to cross-calibrate all of the measurement devices to be used for the determination of the activity concentration of the blood. Demonstrating the procedure will be Anne Moller, a technician from our lab.
After confirming a lack of response to toe pinch in a 12-hour fasted rat, shave the leg and groin on the surgical side of the animal and cleanse the exposed skin with additional disinfectant. Apply ointment to the animal's eye, and place a rectal probe for continuous monitoring and maintenance of body temperature. Tape the limbs of the rat to the work surface, and disinfect the operating site with a mucosal disinfectant.
Make an approximately 20-millimeter incision at the groin, and dissect the fine skin layers to expose the femoral vein, artery, and nerve. Place two fine filaments under the femoral vein and artery, and ligate the vessels with the distal filaments. Holding the vessels under tension with a bulldog clamp, use the proximal suture filaments to tense the vessel with the bulldog clamps without a knot.
Block the vein with a proximal aneurysm clamp two to three millimeters distal from the suture with the bulldog clamp, and use corneal scissors to make an incision that is 1/3 of the vessel diameter. The vessel incision is the most critical step in our protocol. Please ensure to use microsurgical instruments, like corneal scissors, and ensure to cut the vessel carefully in a 45-degree angle.
Use a sterile cotton swab to remove the leaking blood, and dilate the vein with a dull forceps. Holding the vessel open, insert the sharpened catheter into the vein in the proximal direction, up to the aneurysm clip. Open the aneurysm clip, and push the catheter another two to three centimeters.
If the catheter has been placed correctly, blood will flow into the catheter. Secure the catheter with the two proximal knots. Use an insulin syringe to flush and aspirate 100 microliters of heparinized saline through the catheter to check the functionality of the catheter.
Then place a catheter into the artery, as just demonstrated for the vein, and close the leg with sutures. Demonstrating the procedure will be Joanna Forster, a technician from our laboratory. For image acquisition, place the animal in a head-prone position on the shuttle bed pallet and move the shuttle bed to the extended bed position for injection.
Connect the catheters to the shunt system, and start the peristaltic pump with a flow rate of 1.52 milliliters per minute to fill the shunt system with the blood of the animal. Move the shuttle bed to the center of the field of view of the positron emission tomography, or PET, detection ring, and start the online blood sampling system. Then start the PET/computed tomography, or CT, workflow.
After 60 seconds, inject 0.5 to one milliliter of an approximately 22-megabecquerel dose of 18F-fluorodeoxyglucose intravenously via the T-piece. Flush the T-piece with about 150 microliters of heparinized saline solution. For the PET emission acquisition, set the acquire by time option to 3, 600 seconds and select F-18 as the study isotope.
Use 350 to 650 kiloelectron volts as the energy level and 3, 438 nanoseconds as the timing window. For the CT acquisition, select attenuation scan in the acquisition option, and in the projection settings field select 180 projection for a half total rotation. For the field of view and resolution settings, select low as the magnification and four by four for the binding with a 275-millimeter axial scanning length and 3, 328 pixels as the transaxial charge coupled device size.
In the exposure settings field, set 500 microamps for the current, 80 kilovolts for the voltage, and 180 milliseconds for the exposure time. Then acquire a dynamic PET over the next 60 minutes, followed by a CT scan at the end of the PET imaging. For a PET emission histogram, set a series of 20 frames as the dynamic framing and select subtract for the delays.
In the advanced settings field, select 128 as the sinogram width, three as the span, and 79 as ring difference and dead time correction. For PET reconstruction, use a two-dimensional ordered subset expectation maximization and apply and save scatter sonogram with four iterations and Fourier for rebinning as the reconstruction algorithm. Then select 128 by 128 as the matrix size, use one as the image zoom, and select all for the frames and all for the segments.
For manual blood sampling 30, 60, 90, 600, and 1, 800 seconds after starting the imaging acquisition, open the first three-way valve. Collect 100 microliters of arterial blood into an EDTA capillary blood collection tube 30 seconds after the tracer injection. To measure the activity of the whole blood, load the sample into a well counter to allow calculation of the activity of the whole blood for each time point of the manual blood sampling in kilobecquerel per milliliter.
For online blood sampling, use the tube guide to place the tube into the detector, and start the blood sampler software. Open the acquisition interface, and confirm that the computer of the online blood sampling setup and of the PET/CT are time synchronized. Press the Start button exactly 60 seconds before the tracer is injected to acquire enough data for the background correction.
Save the raw data via the Save button in the PMOD database after the measurement. For correction and calibration of the online blood data, switch to the correction interface and enable the decay correction. Select 18 F, and define the start time of image acquisition, enabling the Average button to perform background correction.
Activate the calibration, and enter the calibration factor. Save the corrected and calibrated blood data using the Save TAC button, and select the blood. crv file.
At the beginning of the continuous blood sampling, an initial peak in the radioactivity concentration can be observed starting at about five seconds after tracer injection in the online analysis. After this peak, the activity in the blood declines rapidly, reaching a plateau at about 15 minutes. In the manual blood sampling data, the detected peak is smaller and the plateau is not easily defined.
In the image-derived data, the peak and the starting point of the plateau are clearly visible. Nevertheless, the maximum of the peak is smaller compared to the continuous blood sampling data. In this sub-optimal continuous blood sampling, no data acquisition was possible during the first 3 1/2 minutes of the sampling due to blood clotting.
After clearing the clot, the flow in the tube system was restarted and the measurement was continued, revealing a peak at about four minutes that did not record the maximum radioactivity in the blood. Manual blood sampling and image-derived analyses were still possible in this sub-optimal sampling, however, and demonstrated outcomes comparable to those observed for correct continuous blood sampling outcomes. Keep the welfare of the animal in mind during the whole procedure, prepare the shunt system exactly as recommended, and try to reduce the length of the tubing.
The protocol can be used for the variation of new diagnostic PET traces, the characterization of new disease models, and the assessment of therapy response of new therapeutics in the preclinical setting.
