Multi-Tracer Studies of Brain Oxygen and Glucose Metabolism Using a Time-of-Flight Positron Emission Tomography – Computed Tomography Scanner

NOTE: The Institutional Review Board and Radioactive Drug Research Committee of Washington University School of Medicine approved all studies based on the protocol described below. All human participants provided informed written consent prior to participating in research studies based on the protocol below. See the Table of Materials for details related to all equipment, materials, and reagents used in this protocol.

1. Participant Selection

1. Inclusion criteria
   1. Include only adult participants. Ensure participants can follow study procedures, including instructions for the inhalation of radiopharmaceutical gases, and are able to provide informed consent or assent with appropriate surrogate consent. Ensure participants can undergo imaging in the supine position for up to 2-3 h with breaks, and continuously for 60 min without breaks.
2. Exclusion criteria
   1. Exclude participants who have contraindications to radioactive drug research, such as pregnancy at the time of PET. Exclude participants with contraindications to MRI, since MRI is required for anatomic image registration, spatial normalization, and partial volume correction.
   2. Exclude participants with any contraindication to radial artery cannulation when radial artery cannulation is to be performed. Exclude participants with significant anemia or who have recently donated blood products, since invasive arterial sampling results in a total blood volume loss that can exceed 100 mL.
   3. Exclude participants with significant pulmonary diseases as their capacity to successfully inhale and exchange labeled [¹⁵O]O₂ and [¹⁵O]CO in the lungs may be impaired. Exclude individuals with ongoing medical illnesses beyond those of interest, which are expected to significantly alter cerebral metabolism and blood flow.
      NOTE: This is dependent on the specific aims and targets of a particular project, for example, if the project is focused on healthy aging, large strokes or sickle cell anemia may be among the exclusion criteria.
   4. Exclude participants who cannot achieve a blood glucose level below 165 mg/dL prior to the administration of [¹⁸F]FDG.

2. MRI for neuroanatomy

1. Schedule MRI in advance of PET scanning. Ensure an MRI protocol for a 3T scanner that includes a magnetization prepared rapid gradient echo (MPRAGE) pulse sequence with isotropic 0.8-1.0 mm resolution and, ideally, prospective methods for motion correction²¹.
   NOTE: Prospective motion correction provides additional optimization of T1w anatomy for use in fusion with PET imaging, but the omission of prospective motion correction is unlikely to be highly consequential for current PET imaging in participants who are adherent to avoiding head movement. High-quality T1w anatomy is significant for partial volume correction of PET^22,23.
2. Acquire neuroanatomical MRI for regional parcellations and segmentations, atlas registrations, and partial volume corrections. Verify successful neuroanatomical MRI before scheduling and subjecting a participant to invasive arterial lines and radiopharmaceuticals.

3. Laboratory orders

1. Order arterial blood gases, hemoglobin, hematocrit, and plasma glucose from clinically certified laboratories. Order quantitative oxyhemoglobin, carboxyhemoglobin, and methemoglobin for smokers and those with medical conditions associated with altered hemoglobins.
2. Measure pulse oximetry using validated devices, such as those designed for critical medical care.

4. Delivery of radiopharmaceuticals

NOTE: Measuring brain oxygen metabolism and AG with PET requires a cyclotron facility capable of producing and delivering ¹⁵O radiopharmaceuticals, which have 122 s half-lives. Transport of radiopharmaceuticals between the cyclotron facility and PET scanner must be sufficiently and reliably rapid to provide adequate dosing at the time of radiotracer administration.

1. Ensure that all radioactivity dose calibrators have power and allow time for sufficient instrument warm-up (typically, at least 60 min).
2. For gaseous radiopharmaceuticals, position a shielded gas canister and its incorporated pico-ammeter dose calibrator near the PET gantry of the PET scanner. Ensure secure attachments of sterilized expandable bellows, particulate filter, large-diameter plastic polymer tubing, clamp, and disposable mouthpiece. See Figure 1.
3. Complete quality assurance procedures for nylon gas lines, 1/8 inch inner diameter, under exhaust, from the cyclotron for delivery of [¹⁵O]CO and [¹⁵O]O₂. Complete quality assurance of purified hydrogen sources and processing units for generating radiopharmaceutical [¹⁵O]H₂O as a derivative of [¹⁵O]O₂, for subsequent injection. See Figure 1.

5. Arterial lines

1. Placement and management
   1. Cannulate the radial artery by consultation with a physician with specialty training for routine placement of arterial lines, for example, interventional radiology, anesthesiology, or critical care medicine.
      1. Make available an ultrasound device with a probe appropriate for imaging the radial artery.
      2. Supply the interventional physician with their preferred consumables for arterial line placement, most commonly, a kit with a guidewire that implements the Seldinger technique.
      3. Make every effort to insert the angiocatheter in the participant's non-dominant hand.
   2. Evaluate and manage the participant throughout radial artery cannulation using the services of an experienced nurse or equivalent healthcare professional.
      1. Ensure collateral arterial circulation of the hand by ultrasound.
      2. Prepare and manage the sterile field during arterial cannulation.
      3. Assist the interventional physician with sterile cannulation procedures.
      4. Prime rigid pressure lines and supply normal saline pressurized to 300 mm Hg.
   3. Perform continuous arterial line management during the 2-3 h protocol, including routine monitoring of arterial pressure waveforms and manipulation of arterial line circuits. For continuous arterial sampling, switch in-line stopcocks from supplying back pressure for the radial artery to extraction of arterial blood by peristaltic pumping to flushing and maintaining radial artery access. When priming line segments that never flow into the radial artery, flush with heparinized solutions (up to 10 U/mL) to help maintain patency.
      NOTE: Minimize using heparin when possible for maintaining the radial artery catheter given the uncertain evidence of its efficacy and to avoid the risk of provoking heparin-induced thrombocytopenia.
   4. Use a moldable, yet rigid, arm board to help secure the radial artery catheter and lines. Ensure that repeated manipulations of stopcocks do not disturb the radial artery catheter.
   5. Confirm normal arterial pressure waveforms prior to each radiotracer administration to ensure patency of the radial artery catheter.
2. Gamma detectors for arterial samples
   NOTE: Use a gamma detection probe device and peristaltic pump for arterial input function measurement. Although hand-drawn samples are feasible for [¹⁸F]FDG and other longer half-life radiotracers, employing a sequence of four rapid ¹⁵O scans is greatly facilitated by automated sampling using a peristaltic pump and a gamma detector sufficiently compact to place alongside the participant's hand.
   1. Position arterial-line assemblies as close as feasible to the radial artery catheter to make measurements with minimal dispersion and delay at the gamma detection probe. Ensure that the path from the cannulation site through line assemblies to the gamma detection probe is consistent with calibration data available for the line assembly.
   2. Supply continuous power through an uninterruptible power supply to ensure the stability of photomultipliers for coincidence gamma detectors. For coincidence detection, use 100 ns timing windows and ensure that the nominal detection sensitivity is at least 2.4 cps kBq^-1 mL^-1 with <1% linearity through 10 kcps.
      NOTE: Liquid-filled waveguides for coincidence detection are fragile, requiring care when handling and transporting from storage areas to the PET suite.

6. Scanning

1. Minimize head movements
   1. Prior to scanning, educate the participant on the importance of avoiding head movements during scanning.
2. Study participant positioning
   1. Ensure that PET-CT hardware and software configurations permit scanning with head-first or feet-first positioning of the study participant. Whenever possible, prefer feet-first positioning for PET, such that the participant's extremities and torso lie beyond the PET gantry, the participant's head remains centered within the PET gantry, and the CT gantry remains unoccupied (Figure 1).
   2. Guide and position the participant to lie on the lowered gantry table in accordance with all pre-existing protocols for human PET-CT scanning, such as ensuring recent urinary voiding, wearing appropriate garments, and removing eyeglasses, jewelry, or hair ornaments incompatible with the scanner.
   3. Ensure the integrity of all arterial and venous lines as the gantry table moves into the scanner bore. Ensure continuous connection of arterial lines with pressurized saline sources. Ensure disconnection of peristaltic pumps, gamma detection probes, and any other devices that cannot pass through the scanner bore and reconnection subsequently.
   4. Ensure the comfort of the participant, especially adequate cushions for positioning of head, neck, spine, hips, and leg flexion. Ensure comfortable head positioning upon freely positioning foam headrests, and then secure the participant's forehead to the gantry table with elastic, self-adhesive, self-removable wrapping as a reminder to the participant to avoid head motion. Ensure sufficient blankets for thermal comfort. Confirm the participant's understanding of the necessity of avoiding head movement throughout CT and PET scanning.
      NOTE: The headrests and elastic wrapping substantially limit head motion even in cognitively impaired participants.
   5. Ensure that the gantry table passes through the scanner bore until the participant's extremities and torso lie beyond the CT gantry and the participant's head is centered in the CT gantry. Align the participant's canthomeatal line with vertical laser markers upon entry into the CT gantry, then angle the chin slightly inferiorly. Perform CT as described below. Confirm on CT that this positioning captures the entire cerebrum and cerebellum within the PET field-of-view.
   6. Ensure that the gantry table continues through the scanner bore until the participant's head is centered in the PET gantry. Ensure adequate access to arterial and venous lines as well as adequate wrist extension to accommodate the radial artery cannulation. Ensure that ceiling exhaust vents can effectively remove inadvertently exhaled radiotracer gases near the edge of the PET gantry. Perform CT and PET as described below in steps 6.3-6.5.
   7. Routinely monitor the participant during PET, assessing the participant's comfort with minimal verbal communication during scanning.
      NOTE: Feet-first positioning facilitates the administration of radiotracer gases with improved communications and improved manipulations of inhalation tubing. However, for most gantry tables, feet-first positioning may be incompatible with using thermoplastic head-retention masks. Feet-first positioning may also be incompatible with devices that shield the head from emissions originating from the body inferior to the head. Such shields can otherwise reduce randoms originating from the body.
3. CT for attenuation correction
   1. Obtain a low radiation-dose head CT appropriate for attenuation correction. If the participant requires a break during scanning and leaves the scanner, repeat the CT scan prior to resuming the PET session to allow for console-based reconstruction of subsequent PET imaging. Useful CT parameters include tube current 75 mA, rotation time 0.5 s, spiral pitch 1.5, and tube voltage 120 kVp, with vendor-recommended convolution kernels and reconstruction to 512 x 512 x 88 matrix, 0.98 x 0.98 x 3.00 mm³ resolution.
4. Administration of gaseous radiopharmaceuticals
   1. Instruct participants on the procedure of inhalation prior to the first gas administration, with emphasis on inhaling and exhaling through the tube and not breathing through their nose.
   2. Prepare the shielded gas-storage container equipped with a pico-ammeter dose calibrator. Within the gas container, connect expandable bellows to the rigid narrow-diameter nylon line from the cyclotron as well as to a semi-rigid large-diameter plastic polymer tube that is kept clamped except during administration. Place a virus-capturing particulate filter in line with the large-diameter tube according to infection control requirements formulated for the Covid-19 pandemic. Attach a disposable plastic mouthpiece to the end of the tubing.
   3. Request that the cyclotron facility deliver gaseous radiopharmaceuticals to the gas container.
   4. Prepare to administer all gaseous radiopharmaceuticals as boluses. The procedure for inhalation is identical in all cases.
      1. Monitor the activity of each gas delivered to the expandable bellows to peak activity, and then wait for the activity to fall below the maximum dose allowed by the Radioactive Drug Research Committee (55 mCi). Monitor activities using pico-ammeter dose calibrators. Initiate scanner acquisition of emissions.
      2. Commence scanning immediately prior to the start of inhalation to ensure the acquisition of the start of the time activity curve. Obtain 6-7 min of emissions with 3 s frames x 23, 5 s frames x 6, 10 s frames x 20, and then 30 s frames for the remainder of the scan.
      3. Instruct the participant to exhale fully and lower the participant's mask.
      4. Place the mouthpiece in the participant's mouth. Ask the participant to form a tight seal around the mouthpiece with their lips and then inhale as much as possible. Ask the participant to hold their breath for a few seconds to facilitate their lungs absorbing the gaseous radiopharmaceutical.
      5. Ask the participant to exhale again through the tube, blowing residual gaseous radiopharmaceutical back into the bellows. Re-clamp the tube and retrieve it from the participant.
      6. Monitor the gas activity in the bellows from the start of inhalation to the end of exhalation. Calculate the total administered dose by the difference in activities. Aim for the total administered dose to exceed 20 mCi.
   5. Administer one dose of [¹⁵O]CO. Monitor arterial measurements for nominal time activity curves. If arterial measurements are technically insufficient, administer a replacement dose of [¹⁵O]CO. Allow intravascular [¹⁵O]CO to attain steady state, requiring approximately 1-2 min¹⁷.
   6. Administer two sequential doses of [¹⁵O]O₂. Monitor arterial measurements for nominal time activity curves.
      NOTE: This procedure is robust even with mildly cognitively impaired participants. However, participants incapable of performing this procedure, due to severe cognitive impairment or facial weakness, are excluded to minimize the risk of inadequate radiotracer administration and leakage of gas into the ambient air during exhalation. For continuous administration and use of masks for scavenging gaseous radiotracers, consult alternative methods reported and referenced by Iguchi et al.²⁴ Repetition of dosing [¹⁵O]O₂ in duplicate is recommended because of the lower signal-to-noise ratios (unfavorable noise equivalent counts) typically obtained with [¹⁵O]O₂.
5. Administration of injected radiopharmaceuticals
   1. Request that the cyclotron facility deliver intravenous radiopharmaceuticals to the scanner bay.
   2. Prepare to administer all intravenous radiopharmaceuticals as boluses. The procedure for intravenous injection is similar for all cases.
      1. Monitor the dose in a well-counter dosimeter until it falls below the maximum allowable (25 mCi for [¹⁵O]H₂O and 6 mCi for [¹⁸F]FDG).
      2. Commence scanning immediately prior to intravenous injection to ensure the acquisition of the start of the time activity curve. For [¹⁵O]H₂O, obtain 6-7 min of emissions with 3 s frames x 23, 5 s frames x 6, 10 s frames x 20, and then 30 s frames for the remainder of the scan. For [¹⁸F]FDG, obtain 60 min of emissions with 3 s frames x 23, 5 s frames x 24, 20 s frames x 9, 60 s frames x 13, 300 s frames x 7, and a 351 s frame x 1.
      3. Promptly inject the intravenous dose. Measure the residual radioactivity in the syringe to calculate the administered dose by differences. Ensure that the total administered dose is not below the minimum dose allowed (15 mCi for [¹⁵O]H₂O and 4 mCi for [¹⁸F]FDG).
      4. Monitor arterial measurements for nominal time activity curves. For [¹⁵O]H₂O, if arterial measurements are technically insufficient, administer a replacement dose.
         NOTE: Administration of [¹⁵O]H₂O is in some respects simpler than the administration of gases as the former can be injected intravenously. However, due to the short half-life of [¹⁵O]H₂O, this still requires careful orchestration, depending on the PET scanner location and where [¹⁵O]H₂O is produced and obtained.

7. Arterial measurements

1. After positioning the study participant in the scanner, prepare all devices needed for arterial measurements of radiopharmaceutical activities.
   1. Ensure the integrity of connections between the radial artery angiocathether, rigid pressure lines, stopcocks, pressurized bag for supply of normal saline, extension catheter sets for gamma detectors, and extension catheter sets for peristaltic pumps. Ensure correct priming so that no air bubbles can be introduced into the radial artery and that no air bubbles will interfere with peristaltic pumping for extracting arterial blood.
      1. For the gamma detector, use microbore catheter extension sets with no more than 48 cm length and no more than 0.6 mL priming volume.
      2. For the peristaltic pump, set the maximum allowable occlusion pressure limit.
      3. If the peristaltic pump is an infusion pump operating with reversed flow directions, select the minimum flow rate to keep vessel open (KVO).
2. Immediately prior to administering radiopharmaceuticals, close the stopcock to the pressure bag and pressure monitor, and operate the pump at 300 mL/h. Confirm the extraction of blood from the radial artery and its passage through extension catheter sets for gamma detectors and extension catheter sets for the pump. Confirm continuous measurements of radiopharmaceutical activity in the gamma detectors throughout the bolus passage of radiopharmaceuticals through the arterial circulation²⁵.
3. Verify simultaneous acquisition of time activity curves from the PET scanner.
4. Continue pumping at 300 mL/h and verify measurements of radiopharmaceutical activity in the gamma detectors for at least 300 s after inhalation of [¹⁵O]CO, 120 s after inhalation of [¹⁵O]O₂, 120 s after injection of [¹⁵O]H₂O, and 300 s after injection of [¹⁸F]FDG.
   1. For [¹⁸F]FDG, continue pumping blood at reduced pump rates no lower than 20 mL/h until the end of PET scanning.
5. After each arterial measurement, reconfigure the arterial lines to flush the radial artery catheter; then, flush the line circuit that supplies the gamma detectors and pump. For the isolated circuit through the pump, set pump rates at 300 mL/h. Optionally, use a second bag of heparinized saline under gravity to provide additional flows for clearing blood out of the pump.
   NOTE: Given the prolonged imaging protocol and repeated sampling of the arterial blood, the risk of line occlusion can be high, requiring ongoing vigilance by the investigator team and nurse. Use strict sterility measures and maintain a closed circuit that is configured only by stopcocks.

8. Venous sampling

1. In advance of scanning, establish two sites for intravenous access, contralateral to radial artery cannulation for radiopharmaceutical injection, and ipsilateral to radial artery cannulation for venous sampling. Prefer antecubital access.
2. In advance of scanning, prepare syringes and syringe caps; indelibly mark syringes and caps, cap all syringes, and weigh capped syringes with an analytical balance to 0.0001 g precision. Record times of venous sampling at the start and completion of drawing at least 2 mL of blood. At room temperature (20-25 °C), centrifuge 1 mL of blood at an RCF of 3,300 × g for at least 60 s to extract plasma. Count activities in whole blood and plasma using a well counter calibrated for 68 Ge.
   NOTE: Venous sampling is most appropriate for measurements of [¹⁸F]FDG 30-60 min after injection. Venous samples obtained during ¹⁵O scanning are technically very challenging and difficult to control and they do not correlate well with arterial samples.

9. PET image reconstruction and data storage

1. Reconstruct images from the PET-CT, preferring 3D ordinary-Poisson ordered subsets of expectation maximization (OSEM) with 4-8 iterations of five subsets, TOF, delayed randoms, model-based absolute scatter scaling, attenuation correction, all-pass filtering (no filtering), without point-spread function models, decay correction to start of scanning, rescaling to Bq mL^-1, matrix size of 220, zoom 2, and isotropic voxels of 1.65 mm length.
   NOTE: Zoom 2 reduces the transverse field of view of image reconstruction by one-half, thereby ignoring ambient space between the participant's head and the PET detector rings. Stated parameters are starting values for [¹⁵O]O₂ gas, which is highly susceptible to scattering variability, and requires model-based absolute scatter scaling; historically, the choice of models for scattering have strongly impacted the quality of reconstruction of ¹⁵O tracers. Furthermore, interactions of vendor-implemented scattering models with other models such as point-spread function models are currently poorly understood. Consequently, this protocol implements only scattering models without point-spread models.
2. Review reconstructed PET data on the scanner console to ensure adequate radiotracer administration and minimal motion.
   1. Save DICOM files for reconstructed PET, CT, norms data, and listmode data.
      NOTE: Listmode data are essential for optimized reconstructions, which are computationally expensive, and cannot typically be performed at the scanner console. Listmode data are very large (>40 GB per PET session) and adequate storage devices must be reserved.

10. Discharge and participant follow-up

1. Line removal
   1. Remove the radial arterial catheter using sterile technique and manually apply direct radial artery pressure (this should be done by a qualified healthcare professional).
   2. Apply pressure for 15 min using the patent hemostasis technique to minimize the risk of radial artery occlusion.
   3. Inspect the catheter tip for clots or breaks.
   4. After ensuring that adequate hemostasis has been achieved, apply a pressure dressing with sterile gauze and elastic self-adhesive wrapping. Inspect the hand for any changes in color, temperature, sensation, or function.
2. Home-care instructions
   1. Instruct participants to maintain pressure dressings for 2 h following discharge.
      1. Instruct the participants to examine the cannulation site for bleeding or bruising at 2 h, and then place over-the-counter bandaging.
      2. Instruct the participants to avoid bending or wetting the affected wrist/arm for 24 h and avoid strenuous activity involving the wrist/arm for 48 h.
   2. Instruct the participants to inspect their affected wrist/arm for indications of infection, injury, or bleeding during the 48 h after discharge.
   3. Provide multiple methods of contacting the study team as needed.
   4. Provide reassurances for the common complications of mild bruising and transient discomfort.
   5. Contact the study participant 24-48 h after discharge to ensure that no additional complications have arisen.

11. Calibrations

1. Cross calibration
   1. Use rod reference sources of ⁶⁸Ge or ²²Na for absolute calibration of well counters.
   2. Thereafter, cross-calibrate using solutions of 10-37 MBq of [¹⁸F]FDG in 30-670 mL of water with 2% acetonitrile or similar organic solvent. Use rigid media bottles (polyethylene terephthalate or high-density polyethylene) that provide radioactivity containment with negligible gamma attenuation. Use smaller-volume bottles to accommodate less activity for cross-calibrating in the scanner bore. Cross-calibrate all other gamma detection instruments using aliquots drawn from the bottle.
2. Catheter calibration
   1. Perform calibration studies of dispersion and delay arising from the use of catheters and lines between the radial artery and the gamma detection probe.
      1. Use expired blood products from the local blood bank, a controlled temperature bath, and a controlled variation of the hematocrit of blood products.
      2. Assemble compartmental devices to rapidly alternate between unlabeled and [¹⁸F]FDG-labeled blood products, which are delivered to the assembly of catheters, lines, gamma detector probe, and peristaltic pump. Deliver Heaviside "step-function" inputs to the assembly and measure radioactivity over time.
      3. Estimate the convolution kernel for dispersion and delay. Parameterize kernels to vary with hematocrit. Perform all calibrations with the source of blood products at 37 °C. Reuse the kernel for all human studies employing the identical assembly.
         NOTE: These calibrations account only for dispersion and delay through external line assemblies, not internal anatomy.