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Jan 2019
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Imaging the Vasculature of Immunodeficient Mice Using Positron Emission Tomography/Computed Tomography (PET/CT) and 18F-fluorodeoxyglucose Labeled Human Erythrocytes
利用正电子发射断层扫描/计算机断层扫描(PET、CT)和18F-氟脱氧葡萄糖标记人类红细胞 进行免疫缺陷小鼠脉管系统成像   

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Abstract

Nuclear blood pool imaging using radiolabeled red blood cells has been used in the clinical setting for the evaluation of a number of medical conditions including gastrointestinal hemorrhage, impaired cardiac contractility, and altered cerebrovascular blood flow. Nuclear blood pool imaging is typically performed using Technetium-99m-labeled (99mTc) human erythrocytes (i.e., the “tagged RBC” scan) and gamma camera-based planar scintigraphic imaging. When compared to typical clinical planar scintigraphy and single-photon emission computed tomographic (SPECT) imaging platforms, positron emission tomography (PET) provides superior image quality and sensitivity. A number of PET-based radionuclide agents have been proposed for blood pool imaging, but none have yet to be used widely in the clinical setting. In this protocol, we described a simple and fast procedure for imaging the vasculature of immunodeficient mice through a combination of a small animal positron emission tomography/computed tomography (PET/CT) scanner and human erythrocytes labeled with the PET tracer 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG). This technique is expected to have significant advantages over traditional 99mTc -labeled erythrocyte scintigraphic nuclear imaging for these reasons.

Keywords: Blood pool imaging (血池显像 ), Tagged RBC scan (标志式红血球扫描), 18F-FDG (18F-氟脱氧葡萄糖), Human erythrocytes (人红细胞), PET/CT (PET/CT), Immunodeficient mice (免疫缺陷小鼠 )

Background

Gamma camera-based imaging of radiolabeled erythrocyte is often used for visualizing the blood pool in nuclear medicine. For example, 99mTc-labeled human erythrocytes and 99mTc-labeled human serum albumin blood pool imaging with planar scintigraphy and single photon emission computed tomography (SPECT) have been used clinically to evaluate cardiac function (Thrall et al., 1978; Hacker et al., 2006; Mohseni et al., 2015), gastrointestinal bleeding (Sadri et al., 2015), vascular diseases (Liu et al., 2017), and orbital cavernous hemangioma (OCH) (Dong et al., 2017). As PET scanners have superior nuclear tracer sensitivity and image resolution compared to clinical gamma-camera scintigraphy and SPECT scanners (Rahmim and Zaidi, 2008), development of a robust PET-based blood pool imaging method is of significant clinical interest. Various PET tracers have been proposed as blood pool imaging agents, but suffer from certain limitations. For example, 15-oxygen-labeled (15O) H215O, 13-nitrogen-labeled (13N) 13NH3, 11-carbon-labeled (11C) 11C-Acetate and 82-rubidium chloride (82Rb) have been used to investigate myocardial blood flow, but are not widely used clinically, as the short radioactive half-life of 15O (122 seconds), 13N (9.97 minutes), 82Rb (76 seconds), and 11C (= 20.334 minutes) require the presence of a cyclotron in close proximity to the PET scanner or to an 82Rb generator. (Herrero et al., 2006; Nakazato et al., 2013; Lee et al., 2017). As the positron emitter 18-fluorine (18F) has a tracer half-life (108 minutes) well-suited for clinical imaging, 18F-based tracers are considered attractive for PET imaging, especially given the wide spread prevalence of FDG-specific PET imaging for oncology patients, as well as other 18F-based agents, such as 18F-fluorothymidine (18F-FLT) used for visualizing tumor proliferation (Cysouw et al., 2017).

In this protocol, we describe the method for labeling human erythrocytes with FDG and washing the FDG-labeled erythrocytes. We also describe the injection of labeled erythrocytes into NSGTM immunodeficient mice and subsequent imaging of the mouse vasculature using a small animal microPET/CT platform. Given that the erythrocyte labeling and washing procedure is straightforward, and that the FDG leakage rate from labeled erythrocytes is relatively slow (~10% in 46 min), we believe this method allows for robust PET imaging of the mouse vasculature. We believe that this imaging technique would be of interest to investigators seeking to visualize the vasculature of immunodeficient mice for other applications. In addition, we speculate that this technique would offer significant advantages over 99mTc-based nuclear blood pool imaging for the evaluation of occult gastrointestinal bleeding in patients, and may be useful for evaluating other clinical pathologies, including those involving the cerebral and myocardial vasculature.

Materials and Reagents

  1. 1.8 ml cryovial tube (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 343958)
  2. 3.5 ml cryovial tube (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 343958)
  3. 15 ml conical tube (Corning, catalog number: 430790)
  4. 400-600 µl Lithium Heparin tube (BD. Microtainer® catalog number: 365985)
  5. 1 ml sterile syringe (NIPRO Medical Corporation, catalog number: JD+01D2238)
  6. 10 cc BD Luer-LokTM disposable syringe (Thermo Fisher Scientific, catalog number: BD 309604)
  7. 15 ml FalconTM conical centrifuge tube (Thermo Fisher Scientific, catalog number: 14-959-53A)
  8. 10 ml and 25 ml FisherbrandTM Sterile Polystyrene Disposable Serological Pipets (Thermo Fisher Scientific, catalog numbers: 13-676-10F and 13-676-10M)
  9. 22 gauge needle (BDTM Needle 1, BD, catalog number: 305155)
  10. 26 gauge x ¾” mouse and rat tail vein Monoject IV catheter (Patterson Veterinary, catalog number: 07-836-8403)
  11. ParafilmTM M PM996 all-purpose laboratory film
  12. 200 µl and 1,000 µl TipOne sterile filter pipet tips (USA Scientific, catalog numbers: 1120-8810 and 1122-1830)
  13. 0.2 µm Nalgene® bottle top sterile filter unit (Sigma-Aldrich, catalog number: Z370576-12EA)
  14. 4-8 week old male splenectomized NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSGTM) immunodeficient mice (The Jackson Laboratory, stock number: 005557)
  15. 10 ml of human whole blood in standard ACD anticoagulant solution shipped next day (Zen-Bio, Inc, SER-WB10ML-SDS)
  16. 1 ml of 2-deoxy-2-(18F)fluoro-D-glucose (18F-FDG) with specific activity calibrated at a minimum of 20 milliCuries/ml at the time of cell labeling (United States Pharmacopeia (USP) grade, Cardinal Health nuclear pharmacy)
  17. 0.9% Sodium Chloride Injection USP, 100 ml Fill in 150 ml PAB® (B. Braun, NDC number: 00264-1800-32)
  18. 250 ml bottle of IsoThesia (Isoflurane) solution (Henry Schein Animal Health, catalog number: 029405)
  19. Sterile de-ionized water
  20. NaCl (Sigma-Aldrich, catalog number: S7653-1KG)
  21. KCl (Sigma-Aldrich, catalog number: P9333-1KG)
  22. K2EDTA dihydrate (Sigma-Aldrich, catalog number: 03660-1KG)
  23. Heparin sodium salt (Sigma-Aldrich, catalog number: 375095-100KU)
  24. Phosphate Buffered Saline (PBS) solution, pH 7.4 (Thermo Fisher Scientific, catalog number: 10010023)
  25. Filter sterilized 100 ml 5x EDTA solution (see Recipes)
  26. 100ml 1x EDTA solution (see Recipes)
  27. 1 Unit/ml of heparin-PBS solution (see Recipes)

Equipment

  1. Inveon PET/CT (Siemens Medical Inc., INVEN, catalog number: 138757)
  2. Centrifuge 5810 R (Eppendorf, catalog number: 108308)
  3. Centrifuge 5810 R (Eppendorf, catalog number: 119548)
  4. 200 µl single channel manual pipetter
  5. 1,000 µl single channel manual pipetter
  6. AtomLabTM 500 dose calibrator (Biodex Medical Systems, Inc)
  7. Tissue culture incubator (Sanyo Scientific, catalog number: 133060)
  8. Biological safety cabinet (The Baker company, SterilGARD, catalog number: 101951)
  9. Rotator platform (Labnet International, Inc GYROMINI)
  10. Blood Glucose Test Strips (TrueTrack from @Nitro Diagnostics TM (SN 7688648), Code 4698, Lot RS4698)
  11. BioVet® (m2m Imaging) physiological monitoring and heating system (m2m imaging Corp. USA. http://www.m2mimaging.com/)

Software

  1. Microsoft Excel program (Microsoft Excel 2010)
  2. Inveon PET/CT small animal imaging platform (Siemens Medical Inc., Knoxville, Tennessee) 
  3. Inveon Workstation Software (Siemens Medical Inc., Knoxville, Tennessee)
  4. BioVet® (m2m Imaging) physiological monitoring and heating system (m2m imaging Corp. USA. www.m2mimaging.com)

Procedure

  1. Human red blood cell (RBC) preparation
    1. Gently pipet 10 cc’s of whole human blood in standard anticoagulant citrate dextrose (ACD) solution (overnight shipping from Zen-Bio, Inc) into a sterile 15 ml conical centrifuge tube with a 10 ml sterile disposable pipet. The original blood sample should have a good quality shown in Figure 1. Blood should be slowly pipetted to minimize mechanical trauma/shear injury to erythrocytes.


      Figure 1. Original anti-coagulated blood. The original anti-coagulated blood sample should have no clot and have good fluidity.

    2. Centrifuge blood at 1,000 x g (RCF) for 10 min in a desktop centrifuge (no brake) at room temperature.
    3. Slowly remove nearly all of the plasma from the red blood cell (RBC) fraction using a 10 ml sterile pipet. The plasma should be close to straw colored or have a slightly reddish tinge as Figure 2. Plasma that has a frankly turbid or significant reddish coloration implies either improper centrifugation or significant pre-existing hemolysis of the original blood sample that may render the sample unsuitable for radiotracer labeling. Slowly transfer 2 ml of pelleted RBCs into a 15 ml conical centrifuge tube using 10 ml sterile pipet. Residual plasma and buffy coat should be avoided during erythrocyte aspiration. Add 8 ml of 1x EDTA solution to RBCs. Close cap on tube and gently invert tube several times to wash cells.


      Figure 2. The original blood after first centrifuge step. Ideally, the plasma should be close to straw colored or have a slightly reddish tinge.

    4. Centrifuge at 1,000 x g for 10 min at room temperature, then slowly aspirate the supernatant which shown in Figure 3 with a 10 ml sterile pipet. Rapid supernatant aspiration will disturb the supernatant/RBC interface and result in aspiration of RBCs.


      Figure 3. The RBCs after washing with 4x volume of 1x EDTA solution

    5. Transfer 250 µl of RBCs with 1,000 µl pipet tip into 1.8 ml cryovials (Labeled “1” and “2”). Care should be taken to avoid pipetting any residual supernatant.
    6. To each RBC cryovial, add 100 µl 5x EDTA solution and 50 µl sterile de-ionized water (total volume = 400 µl) via manual pipette.

  2. 18F-FDG labeling of human erythrocytes
    1. To maximize the radiolabeling efficiency of human RBCs for in vivo PET imaging of the vasculature of NSGTM immunodeficient mice, the specific activity of USP grade 18F-FDG should be calibrated to at least 20 milliCurie (mCi) per ml at the start of cell labeling.
      Note: (The Jackson Laboratory, stock number: 005557) has been used, due to its severe immunodeficiency. Other immunodeficient mouse strains have not been tested, but may be feasible as well.
    2. Add 100 µl 18F-FDG solution to each RBC cryovial (F.V. = 500 µl) behind appropriate radioactivity shielding. Methods for handling radioactive materials for this protocol should follow those established by the radiation safety officer at your institution. Directly count radioactivity in each cryovial using the AtomLabTM dose calibrator (mCi’s) and record the time. The amount of activity should be at least 1 mCi of FDG in each vial. Ensure the cap is tightly screwed on the cryovial. Care should be taken when choosing an appropriate vial for FDG RBC labeling, as some cryovials are composed of certain plastics that do not maintain a good seal about the cap when exposed to higher temperatures and can result in local radioactivity leakage/contamination. A thin strip of ParafilmTM can be wrapped around the margins of the cap to ensure no radioactivity leakage from the tube and reduce water loss from the cryovial lumen.
    3. Gently finger resuspend RBCs. Place vials in an upright position in a tube rack tied/taped to a rotator platform as Figure 4. Incubate rotating samples in an upright position at 37 °C for 30 min at ≤ 60 RPM. Avoid inverting tube during rotation, as this likely increases the amount of dried cells adherent to the tube wall.


      Figure 4. RBCs labeled with 100 µl 18F-FDG solution on rotator platform and placed in an upright position in the 37 °C incubator

    4. Centrifuge RBC samples at 1,000 x g for 10 min (no brake) at 4 °C. The present sample is shown in Figure 5.


      Figure 5. The free 18F-FDG fraction (upper clear half of solution) and the precipitated RBCs fraction (red bottom half of solution)

    5. Place samples in a ventilated laboratory hood behind appropriate radioactivity shielding and gently transfer the supernatant (about 250 µl) to a 3.5 ml cryovial labeled“SUPE 1 and 2”.
    6. Gently resuspend each 250 µl RBCs cell pellet in 1 ml 1x EDTA solution via manual pipette. Care should be taken to resuspend cells slowly to minimize pipet-induced mechanical damage/shear injury to cells. 
    7. Repeat centrifugation of RBC samples at 1,000 x g for 10 min (no brake) at 4 °C. The centrifuged sample is shown in Figure 6.


      Figure 6. Centrifuged 18F-FDG-labeled RBC sample after washing with 1 ml 1x EDTA solution

    8. Transfer 2nd supernatant to an appropriate “SUPE” tube.
    9. Gently resuspend 250 µl RBCs cell pellets again in 1 ml 1x EDTA solution, as described above.
    10. Repeat centrifugation of RBC samples at 1,000 x g 10 min (no brake) at 4 °C. 
    11. Transfer supernatant to an appropriate “SUPE” tube. Carefully pipet residual supernatant from the interface with the cell pellet, leaving only a tiny meniscus overlying the pellet. 
    12. Directly count SUPE and RBC pellet cryovials using AtomLab detector via dose calibrator (µCurie) and record time. Typical amounts of intracellular radiotracer activity are limited by the total allowable volume of cell solution (250 µl pelleted RBCs) that can be injected into a mouse per day as per our institutional IACUC guidelines, but usually range from ~150 to 300 µCuries, and can be higher depending upon the chosen 18F-FDG specific activity at the start of cell labeling. Gently resuspend the pellet in 250 µl of 1x EDTA solution. The labeling of two RBC samples allows for titration of a larger volume of labeled RBCs, as needed.
    13. Transfer the RBC suspension to a 1 ml syringe using a 22 gauge needle. Any residual clumps of cells should not be aspirated, as these likely represent damaged/coagulated cells. Cells should be slowly aspirated into the syringe lumen to minimize mechanical damage/shear injury to cells. The radioactivity in the syringe should be measured before and after animal injection to determine net activity injected in vivo, as there will be residual activity left in the syringe after injection.

  3. Small animal PET/CT imaging
    1. Prepare 4-6 week-old male splenectomized NODSCIDgamma (NSGTM) immunodeficient mice for experiment.
    2. The mouse can be fasted the night before PET/CT imaging to encourage shifting of mouse metabolic activity from glucose to fatty acids, thus minimizing myocardial uptake of any free FDG in the labeled cell preparation. The mouse can also be placed on a very low carbohydrate diet for this purpose as well, as needed. Draw 500 µl of mouse blood through retro-orbital venous plexus puncture, or other approved venous blood draw at your institution, as seen in the example shown in Figure 7. Blood should be collected into heparinized blood collection tube. If the blood is drawn too slowly, there is a risk of significant coagulation occurring, rendering the blood sample unsuitable for FDG labeling. Measure blood glucose level with a blood glucose monitor for PET calibration.


      Figure 7. Example of drawing blood through rat leg vein

    3. Anesthetize the mouse via a nosecone manifold under 2-4% inhalational isoflurane. The lowest level of inhalational isoflurane possible is recommended, as isoflurane may induce vasodilation of the mouse vasculature. Record the level of inhalational isoflurane used per mouse.
    4. Warm the mouse tail with either a warm soaked towel to stimulate dilation of the mouse tail veins. Insert a tail vein micro-catheter into one of the dilated mouse tail veins. Flush tail vein catheter with 1 U/ml heparin-PBS solution. See example shown in Video 1.

      Video 1. Standard microcatheter insertion into mouse/rat tail (This video was made at University of south Florida, according to guidelines from the University of South Florida on Animal Care and approved by the Animal Research Ethics Board of University of South Florida under protocol IS00004376.)

    5. Secure the mouse onto the micro-PET/CT scanner bed under 2-4% inhalational isoflurane anesthesia via nose-cone manifold as shown in Figure 8. The animal can be secured to the micro-PET/CT scanner bed by gently wrapping the animal in bubble packaging to minimize animal movement and preserve body warmth. Care must be taken when wrapping the animal, as tight wrapping may impede animal respiratory motion. 


      Figure 8. Placement of animal in microPET/CT scanner bed for 18F-FDG RBC imaging of the whole body vasculature

    6. Slowly inject 500 µl of FDG-labeled human erythrocyte suspension into the mouse through the tail vein microcatheter over a course of 1 min to minimize shear injury to cells from passage through the microcatheter lumen. See the example shown in Figure 9.


      Figure 9. Example of injection of FDG-labeled RBCs through tail vein microcatheter

    7. Acquire ECG-gated whole-body images of the mouse, followed by CT calibration images. PET/CT image acquisition parameters will depend upon the particular experimental indications and unique imaging platform setup at a given institution; as such, the following is a description of the protocol used at our institution. 
      1. Place electrocardiogram (ECG) leads on two front limbs and one hind limb of the mouse (ground lead on a rear leg) for ECG gated PET imaging. The signals detected by these electrodes are recorded during the 10 minute time period by BioVet® (m2m Imaging) physiological monitoring and heating system. 
      2. Set the threshold for TTL cardiac gating signals in a rising mode of R-wave peak.
      3. Reconstruct the PET list-mode data using 3D-OSEM iterative algorithm with four iterations and eight subsets, with a final image volume of 256 x 256 x 256 voxels. Set effective voxel effective dimensions at 1.4 x 1.4 x 1.4 mm. 
      4. For each animal there are three data sets: standard 3-dimensional (3D) PET reconstruction, resulting in a motion–time average 3D PET image; dynamic 3D PET reconstruction with 30 frames; and the phase-based 4-dimensional (regular 3-dimensional plus time, 4D) PET cardiac reconstruction, with four cardiac gate binning. Please see Figures 10 and 11. In all cases, CT attenuation correction is applied to the PET images (Choi et al., 2019).


        Figure 10. Example for Micro PET acquisition set-up


        Figure 11. Example for Micro CT acquisition set-up using Siemens Inveon platform

Data analysis

  1. Micro-PET/CT analysis
    PET/CT image processing/analysis parameters will depend upon the particular experimental indications and unique imaging platform setup at a given institution; as such, the following is a description of the protocol used at our institution.
    1. Analyze the whole-body PET images of the mouse using Inveon Workstation Software (Siemens Medical Inc., Knoxville, Tennessee). 
    2. Select the vendor software-supplied Patlak compartment plot option for kinetic modeling. 
    3. For 3D PET and 4D PET data sets, manually select multiple volumes of interest (VOI) based on corresponding CT images for the following organs (as needed): heart, leg muscle, liver, kidney and brain. Voxel activities are represented in standardized uptake values (SUV). Plot dynamic activity curves for VOIs using dynamic 3D PET data set for each animal. The 4D PET data are used for defining cardiac function. First, segment the heart on CT images based on anatomical features, then transfer the segmented volume (cardiac PET VOI) for image co-registration, As seen with the example shown in Figure 12. Images are represented as maximum intensity projection (MIP) reconstructions of the source data. ECG guided binning of PET MIP images can be performed to obtain pseudo-dynamic images of mouse cardiac contractility, as shown in Video 2.


      Figure 12. Example of basic parameter setup for microPET/CT reconstruction in Siemens Inveon workstation

      Video 2. Rebinned cardiac cine imaging of ECG-gated whole body microPET images of NSG mouse injected with 2.2 MBq (250 μl) of FDG-labeled human RBCs (This video was made at University of south Florida, according to guidelines from the University of South Florida on Animal Care and approved by the Animal Research Ethics Board of University of South Florida under protocol IS00004376.)

  2. Labeling efficiency calculation
    Labeling efficiency = (radioactivity dose of RBC pellet)/(radioactivity dose of SUPE + radioactivity dose of RBC pellet).

Recipes

  1. Filter sterilized 100 ml 5x EDTA solution
    700 mM NaCl
    20 mM KCl
    12.5 mM K2EDTA dihydrate
    Add sterile de-ionized water to final volume of 100 ml
    Pass final solution through 0.2 micrometer (µm) Nalgene bottle top sterile filter unit 
  2. 100 ml 1x EDTA solution
    20 ml of filter-sterilized 5x EDTA solution
    80 ml of sterile di-ionized water
    Pass final solution through 0.2 micrometer (µm) Nalgene bottle top sterile filter unit 
  3. 1 Unit/ml of heparin-PBS solution
    Heparin sodium salt dissolved in sterile Phosphate Buffered Saline (PBS) solution, pH 7.4

Acknowledgments

We kindly appreciate the suggestions and comments to this work by Dr. Mikalai Budzevich at Moffitt Cancer Center. This protocol was adapted for Choi et al. (2019).

Competing interests

A provisional international patent application was submitted by the H. Lee Moffitt Cancer Center and Research Institute describing the imaging technique outlined in the manuscript (WO Application number WO2017123666A2; PCT/US2017/013063 filed on 2017-01-11). No additional declarations or potential competing interests are made with regards to the patent application by the authors of the manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Ethics

All experiments procedures were approved by University of South Florida (USF) Institutional Animal Care and Use Committee (IACUC). All experiments were performed in accordance to federal regulations and USF IACUC principles (protocol IS00004376) and procedures.

References

  1. Choi, J. W., Budzevich, M., Wang, S., Gage, K., Estrella, V. and Gillies, R. J. (2019). In vivo positron emission tomographic blood pool imaging in an immunodeficient mouse model using 18F-fluorodeoxyglucose labeled human erythrocytes. PLoS One 14(1): e0211012.
  2. Cysouw, M. C. F., Kramer, G. M., Frings, V., De Langen, A. J., Wondergem, M. J., Kenny, L. M., Aboagye, E. O., Kobe, C., Wolf, J., Hoekstra, O. S. and Boellaard, R. (2017). Baseline and longitudinal variability of normal tissue uptake values of [18F]-fluorothymidine-PET images. Nucl Med Biol 51: 18-24.
  3. Dong, H., Zhang, Z., Guo, Y., Zhang, H. and Xu, W. (2017). The application of technetium-99m-red blood cell scintigraphy in the diagnosis of orbital cavernous hemangioma. Nucl Med Commun 38(9): 744-747.
  4. Hacker, M., Hoyer, X., Kupzyk, S., La Fougere, C., Kois, J., Stempfle, H. U., Tiling, R., Hahn, K. and Stork, S. (2006). Clinical validation of the gated blood pool SPECT QBS processing software in congestive heart failure patients: correlation with MUGA, first-pass RNV and 2D-echocardiography. Int J Cardiovasc Imaging 22(3-4): 407-416.
  5. Herrero, P., Kim, J., Sharp, T. L., Engelbach, J. A., Lewis, J. S., Gropler, R. J. and Welch, M. J. (2006). Assessment of myocardial blood flow using 15O-water and 1-11C-acetate in rats with small-animal PET. J Nucl Med 47(3): 477-485. 
  6. Lee, B. C., Moody, J. B., Weinberg, R. L., Corbett, J. R., Ficaro, E. P. and Murthy, V. L. (2017). Optimization of temporal sampling for 82rubidium PET myocardial blood flow quantification. J Nucl Cardiol 24(5): 1517-1529. 
  7. Liu, M., Zhao, Z. Q., Fang, W. and Liu, S. (2017). Novel approach for 99mTc-labeling of red blood cells: Evaluation of 99mTc-4SAboroxime as a blood pool imaging agent. Bioconjug Chem 28(12): 2998-3006.
  8. Mohseni, S., Kamali-Asl, A., Bitarafan-Rajabi, A., Entezarmahdi, S. M., Shahpouri, Z. and Yaghoobi, N. (2015). Effects of filtration on right ventricular function by the gated blood pool SPECT. Ann Nucl Med 29(4): 384-390.
  9. Nakazato, R., Berman, D. S., Alexanderson, E. and Slomka, P. (2013). Myocardial perfusion imaging with PET. Imaging Med 5(1): 35-46.
  10. Rahmim, A. and Zaidi, H. (2008). PET versus SPECT: strengths, limitations and challenges. Nucl Med Commun 29(3): 193-207.
  11. Sadri, K., Momenypoor, S., Dabbagh Kakhki, V. R., Sadeghi, R., Aryana, K., Johari Daha, F., Zakavi, S. R. and Jaafari, M. R. (2015). Nano liposomes labeled with 99mTc-HMPAO, a novel agent for blood pool imaging. Iran J Pharm Res 14(4): 981-988.
  12. Thrall, J. H., Freitas, J. E., Swanson, D., Rogers, W. L., Clare, J. M., Brown, M. L. and Pitt, B. (1978). Clinical comparison of cardiac blood pool visualization with technetium-99m red blood cells labeled in vivo and with technetium-99m human serum albumin. J Nucl Med 19(7): 796-803.

简介

使用放射性标记的红细胞的核血池成像已被用于临床评估许多医疗条件,包括胃肠道出血、心肌收缩力受损和脑血管血流改变。核血池成像通常使用锝99m标记(99mtc)的人红细胞(即>,标记红细胞扫描)和基于伽玛照相机的平面闪烁成像。与典型的临床平面闪烁显像和单光子发射计算机断层显像(spect)成像平台相比,正电子发射断层显像(pet)具有更高的成像质量和灵敏度。许多基于pet的放射性核素药物已被提出用于血池显像,但还没有一种药物在临床上得到广泛应用。在这个方案中,我们描述了一个简单而快速的程序,通过小动物正电子发射断层扫描/计算机断层扫描(pet/ct)和用pet示踪剂2-脱氧-2-(18f)氟-d-葡萄糖标记的人红细胞联合成像免疫缺陷小鼠的血管。(18F-FDG)。由于这些原因,这项技术有望比传统的99mTc标记的红细胞闪烁核显像具有显著的优势。【背景】基于γ照相机的放射性标记的红细胞成像经常被用于可视化核医学中的血池。例如,SUP> 99mTC标记的人红细胞和“SUP> 99m TC标记的人血清白蛋白血池平扫显像和单光子发射计算机断层显像(SPECT)已用于临床评价心功能(Surryet al/e>),1978;Hux等<>,2006;MoHSENI 等>2015)消化道出血(Sadri等2015),血管疾病(刘AL2017),眼眶海绵状血管瘤(OCH)(董等< ,2017)。由于PET扫描仪与临床伽玛相机闪烁成像和SPECT扫描仪(Ramim和Zadii,2008)相比具有优越的核示踪剂敏感性和图像分辨率,因此开发基于PET的健壮的血池成像方法具有重要的临床意义。各种PET示踪剂已被提出作为血池成像剂,但遭受一定的局限性。例如,15的氧标记(15 O)H<2 15 O,13个氮标记( 13 < /SUP> N) 13 NH>子> 3 ,11个碳标记( 11 C) 11 C-乙酸乙酯和82氯化铷( ROOS RB)已被用于研究心肌血流量,但在临床上未被广泛应用,随着SUP> 15 O(122秒)的短放射性半衰期,13 N(9.97分钟), 82 RB(76秒), 11 C(=20.334分钟)要求在PET扫描仪附近存在回旋加速器,或到 82 RB发生器。(Helro>2006);Nakazato 等人>2013;李等人>,2017)。正电子发射体18氟(18 F)具有良好的示踪半衰期(108分钟),适合于临床影像学, 18 F-示踪剂被认为是PET成像的诱因,特别是考虑到FDG特异性PET显像在肿瘤患者中的广泛流行,以及其他 18 F基药物,如~(18)氟荧光素(18 F- FLT)用于肿瘤增殖的可视化(CySouw等2017)。



在该协议中,我们描述了用FDG标记人红细胞和洗涤FDG标记红细胞的方法。我们还描述了用小动物MPET/CT平台将标记红细胞注入NSG TM免疫缺陷小鼠和随后对小鼠脉管系统的成像。鉴于红细胞标记和洗涤过程是简单的,并且来自标记红细胞的FDG泄漏率相对较慢(在46分钟内为10%),我们相信这种方法允许对小鼠脉管系统进行健壮的PET成像。我们相信,这种成像技术将感兴趣的研究人员试图可视化免疫缺陷小鼠的血管的其他应用。此外,我们推测,该技术将比 99m 基于TC的核血池显像对评估隐匿性消化道出血的患者具有显著优势,并可用于评估其他临床病理学,包括涉及脑和心肌血管的病变。

关键字:血池显像 , 标志式红血球扫描, 18F-氟脱氧葡萄糖, 人红细胞, PET/CT, 免疫缺陷小鼠

材料和试剂

  1. 1.8毫升冷冻瓶管(赛默飞世尔科学公司,赛默科学公司TM,目录号:343958)
  2. 3.5毫升冷冻瓶管(赛默飞世尔科学公司,赛默科学公司TM,目录号:343958)
  3. 15ml锥形管(康宁,目录号:430790)
  4. 400-600微升锂肝素管(bd.microtainer?产品目录号:365985)
  5. 1毫升无菌注射器(NIPro医疗公司,产品目录号:JD+01D2238)
  6. 10 cc bd luer loktm一次性注射器(Thermo Fisher Scientific,目录号:bd 309604)
  7. 15毫升FalconTM锥形离心管(赛默飞世尔科学公司,目录号:14-959-53A)
  8. 10毫升和25毫升FisherbrandTM无菌聚苯乙烯一次性血清学吸管(Thermo Fisher Scientific,目录号:13-676-10F和13-676-10M)
  9. 22号针(bdtm针1,bd,目录号:305155)
  10. 26号x 3/4“小鼠和大鼠尾静脉单项目IV导管(帕特森兽医,目录号:07-836-8403)
  11. 副膜tmm pm996通用实验室膜
  12. 200微升和1000微升Tipone无菌过滤吸管头(美国科学,目录号:1120-8810和1122-1830)
  13. 0.2μm Nalgene?瓶顶无菌过滤装置(Sigma-Aldrich,目录号:Z370576-12EA)
  14. 4-8周龄雄性脾切除术nod.cg-prkdcscidil2rgtm1wjl>/szj(nsgtm)免疫缺陷小鼠(杰克逊实验室,库存编号:005557)
  15. 第二天装运的标准ACD抗凝剂溶液中的10毫升人类全血(Zen Bio,Inc,SER-WB10毫升-SDS)
  16. 1毫升2-脱氧-2-(18f)氟-d-葡萄糖(18f-fdg),细胞标记时比活性至少校准为20毫居里/毫升(美国药典(USP)级,基本健康核药房)
  17. 0.9%氯化钠注射液USP,100毫升,加入150毫升PAB?(B.Braun,NDC编号:00264-1800-32)
  18. 250毫升一瓶等速(异氟醚)溶液(Henry Schein动物健康,目录号:029405)
  19. 无菌去离子水
  20. 氯化钠(Sigma-Aldrich,目录号:S7653-1kg)
  21. KCL(Sigma-Aldrich,产品编号:P9333-1kg)
  22. K2EDTA二水合物(Sigma-Aldrich,目录号:03660-1kg)
  23. 肝素钠(Sigma-Aldrich,目录号:375095-100KU)
  24. 磷酸盐缓冲盐水(PBS)溶液,pH值7.4(Thermo Fisher Scientific,目录号:10010023)
  25. 过滤灭菌100毫升5x EDTA溶液(见配方)
  26. 100ml 1x EDTA溶液(见配方)
  27. 1单位/ml肝素PBS溶液(见配方)

设备

  1. Inveon PET/CT(西门子医疗公司,Inven,目录号:138757)
  2. 离心机5810R(Eppendorf,产品目录号:108308)
  3. 离心机5810 R(Eppendorf,目录号:119548)
  4. 200微升单通道手动移液器
  5. 1000微升单通道手动移液器
  6. AtomLabTM500剂量校准器(Biodex医疗系统公司)
  7. 组织培养箱(三洋科技,目录号:133060)
  8. 生物安全柜(Baker公司,消毒池,目录号:101951)
  9. 旋转平台(Labnet International,Inc Gyromini)
  10. 血糖测试条(TrueTrack来自@Nitro Diagnostics TM(SN 7688648),代码4698,批号RS4698)
  11. Biovet?(M2M成像)生理监测和加热系统(M2M成像公司,美国)http://www.m2m imaging.com/“target=”blank“>http://www.m2mimaging.com/)

软件

  1. Microsoft Excel程序(Microsoft Excel 2010)
  2. Inveon PET/CT小动物成像平台(西门子医疗公司,田纳西州诺克斯维尔)
  3. Inveon工作站软件(西门子医疗公司,田纳西州诺克斯维尔)
  4. Biovet?(M2M Imaging)生理监测和加热系统(M2M Imaging Corp.USA.www.m2mimaging.com)

程序

  1. 人红细胞制剂
    1. 用10毫升无菌一次性移液管将10毫升全血用标准抗凝血枸橼酸葡萄糖(ACD)溶液(从Zen Bio,Inc.公司运输过夜)轻轻移液至15毫升无菌锥形离心管中。原始血样应具有图1所示的良好质量。血液应缓慢地输送,以减少对红细胞的机械损伤/剪切损伤。
      < BR>
      图1。原抗凝血样。原抗凝血样应无凝块,流动性好。
      < BR>
    2. 在室温下,在台式离心机(无制动器)中以1000x g(rcf)离心血液10分钟。
    3. 用10毫升无菌移液管缓慢移走红细胞(rbc)部分几乎所有的血浆。等离子体应该接近稻草色或有一点微红的色调,如图2所示。有明显浑浊或明显红色的血浆意味着离心不当或原始血液样本存在明显溶血,可能使样本不适合放射性示踪剂标记。用10毫升无菌移液管将2毫升颗粒红细胞缓慢移入15毫升锥形离心管。红细胞抽吸时应避免血浆残留和皮肤发黄。向红细胞中加入8毫升1X EDTA溶液。关闭试管盖,轻轻翻转试管几次,清洗试管。
      < BR>
      图2。离心第一步后的原始血液。理想情况下,血浆应接近稻草色或略带红色。
      < BR>
    4. 在室温下以1000x g离心10分钟,然后用10毫升无菌移液管缓慢吸出图3所示的上清液。快速吸取上清液会干扰上清液/红细胞界面,导致红细胞吸取。
      < BR>
      图3。用4x体积的1X EDTA溶液清洗后的红细胞 < BR>
    5. 用1000微升移液管将250微升红细胞移入1.8毫升冷冻瓶中(标记为“1”和“2”)。应注意避免用移液管移取任何残留上清液。
    6. 在每个红细胞冷冻瓶中,通过手动移液管加入100微升5x EDTA溶液和50微升无菌去离子水(总体积=400微升)。
      < BR>
  2. 人红细胞f-fdg标记
    1. 为了最大限度地提高人红细胞的放射性标记效率,在NSG TM免疫缺陷小鼠的血管中,用E/Em> PET显像,在细胞标记开始时,USP级 18 F-FDG的特异性活性应被校准到至少每毫升20毫居里(MCI)。< BR> 注:(杰克逊实验室,库存编号:005557)已被使用,因为其严重的免疫缺陷。其他免疫缺陷小鼠菌株尚未进行测试,但也可能是可行的。>
    2. 在适当的放射性屏蔽后,将100μl18f-fdg溶液添加到每个红细胞冷冻瓶(f.v.=500μl)中。本议定书中放射性物质的处理方法应遵循贵机构辐射安全官员制定的方法。用AtomLab试剂盒直接测定放射性活度,记录时间。每个小瓶中的活性量应至少为1 mci的fdg。确保盖子拧紧在冷冻瓶上。当选择适当的小瓶用于FDG RBC标记时,应小心,因为一些低温小孔由某些塑料组成,当暴露在较高温度下时,它们不能保持帽的良好密封,并且可能导致局部放射性泄漏/污染。一个薄条的PARFLAM TM可以被包裹在盖子的边缘,以确保从管中没有放射性泄漏,并减少冷冻管腔的水损失。
    3. 轻轻地用手指恢复红细胞。如图4所示,将小瓶垂直放置在与旋转器平台捆绑/捆绑的管架上。在37°C的垂直位置孵育旋转样品,在小于60转/分的转速下孵育30分钟。避免在旋转过程中倒置管子,因为这可能会增加粘附在管壁上的干细胞的数量。 < BR>
      图4。RBC用100 L L 18F-FDG溶液在旋转平台上标记,在37°C培养箱中放置直立位置<强> >强> <强> BR/> < BR>
    4. 在4℃下离心分离RBC样品1000℃>EX×G>10分钟(无制动)。本样品如图5所示。 < BR>
      图5。自由< > > 18个>上半部分(上半透明溶液)和沉淀红细胞组分(溶液的红下半部)>强>强> >强> > > > > < BR>
    5. 将样品放置在通风的实验室罩中,在适当的辐射屏蔽之后,将上清液(约250升)轻轻转移到标示“Supe 1和2”的3.5毫升冰冻瓶中。
    6. 在1毫升1X EDTA溶液中,用手动移液器轻轻地将每250 L的红细胞颗粒重新沉淀。应注意缓慢地重新培养细胞,以尽量减少移液管对细胞造成的机械损伤/剪切损伤。
    7. 在4℃下重复离心1000次<×g > 10分钟(无制动)。离心样品如图6所示。
      < BR>
      图6。用1 mL 1X EDTA溶液洗涤18F FDG标记的红细胞样品<<强> <强> <强> Br/> < BR>
    8. 将2nd上清液转移到适当的“supe”管中。
    9. 如上所述,在1 mL 1X EDTA溶液中,再次将250μL的红细胞细胞小球重新悬浮。
    10. 在4°C下重复离心RBC样品1000X G>10分钟(无制动)。
    11. 将上清液转移到适当的“SUPE”管中。小心地从与细胞颗粒的界面上吸管残余的上清液,只留下一个微小的弯月面覆盖在球团上。
    12. 使用原子探测器通过剂量校准器(居里)直接计数SUPE和RBC球团冷冻池,并记录时间。细胞内放射性示踪物的典型量受细胞溶液(250μL颗粒化红细胞)的总允许体积的限制,可以按照我们的机构IACUC指南每天注射到小鼠体内,但通常范围从150到300个居里,在细胞标记开始时,根据所选择的 18 F-FDG特异性的活性可以更高。在1X EDTA溶液中250μL温和地重新沉淀球团。两个红细胞样品的标记允许根据需要滴定更大体积的标记的红细胞。
    13. 用22号针头将红细胞悬液转移到1毫升注射器中。不应吸入任何残留的细胞团,因为这些细胞团可能代表受损/凝固的细胞。细胞应缓慢吸入注射器腔,以尽量减少对细胞的机械损伤/剪切损伤。注射前和注射后应测量注射器内的放射性,以确定体内注射的净放射性,因为注射后注射器内会残留放射性。
      < BR>
  3. 小动物pet/ct成像
    1. 制备4~6周龄雄性脾切除术后nodscidgamma(nsgtm)免疫缺陷小鼠进行实验。
    2. 在pet/ct显像前一天晚上,可以禁食小鼠,以促进小鼠代谢活动从葡萄糖转移到脂肪酸,从而使标记细胞制剂中任何游离fdg的心肌摄取量降至最低。根据需要,也可以将小鼠置于非常低碳水化合物的饮食中。如图7所示,通过眶后静脉丛穿刺抽取500微升小鼠血,或在您的机构抽取其他经批准的静脉血。血液应收集到肝素化的血液收集管中。如果采血太慢,有发生明显凝血的风险,使血液样本不适合作fdg标记。使用血糖监测仪测量血糖水平,以便进行PET校准。
      < BR>
      图7。大鼠腿部静脉采血例 < BR>
    3. 在吸入2-4%异氟醚的情况下,通过鼻锥歧管麻醉小鼠。建议尽可能低水平吸入异氟醚,因为异氟醚可能导致小鼠血管舒张。记录每只小鼠吸入异氟醚的水平。
    4. 用一条浸过水的毛巾加热鼠尾,刺激鼠尾静脉扩张。将尾静脉微导管插入扩张的小鼠尾静脉中。用1u/ml肝素pbs溶液冲洗尾静脉导管。参见视频1中的示例。
    5. 在2-4%异氟醚吸入麻醉下,通过鼻锥管将小鼠固定在微型PET/CT扫描床上,如图8所示。通过用气泡包装轻轻地将动物包裹在微型PET/CT扫描仪床上,可以将动物固定在床上,从而最大限度地减少动物的活动,保持身体的温暖。包装动物时必须小心,因为紧密的包装可能会妨碍动物的呼吸运动。
      < BR> 视频1。标准微导管插入小鼠/鼠尾(此视频由南佛罗里达大学制作,根据南佛罗里达大学动物护理指南和南佛罗里达大学动物研究伦理委员会根据协议IS00004376批准。)


      图8。动物在micropet/ct扫描床上进行全身血管f-fdg-rbc显像 < BR>
    6. 通过尾静脉微导管将500微升FDG标记的人红细胞悬液缓慢注入小鼠体内,持续1分钟,以减少通过微导管腔对细胞的剪切损伤。参见图9所示的示例。

      图9。经尾静脉微导管注射fdg标记红细胞的例子 < BR>
    7. 采集小鼠心电门控全身图像,然后采集ct定标图像。PET/CT图像采集参数将取决于特定机构的特定实验适应症和独特的成像平台设置;因此,以下是我们机构使用的协议说明。
      1. 将心电图(ECG)导联放置在小鼠的两个前肢和一个后肢(后肢接地导联)上,用于ECG门控PET成像。这些电极检测到的信号在10分钟的时间内由biovet?(m2m成像)生理监测和加热系统记录。
      2. 在R波峰值上升模式下设置TTL心脏门控信号的阈值。
      3. 用3D-OSEM迭代算法重建PET列表模式数据,四次迭代,八个子集,最终图像体积为256 x 256 x 256体素。将有效体素有效尺寸设置为1.4 x 1.4 x 1.4 mm。
      4. 每只动物都有三个数据集:标准三维(3D)PET重建,生成运动时间平均的3D PET图像;30帧动态3D PET重建;基于相位的四维(常规三维加时间,4D)PET心脏重建,有四个心脏门。请参见图10和图11。在所有情况下,CT衰减校正应用于PET图像(Choi等,2019年)。
        图10。微型PET采集设置示例 < BR>
        图11。使用西门子Inveon平台设置微型CT采集的示例
    < BR>

数据分析

  1. 微型pet/ct分析
    pet/ct图像处理/分析参数将取决于特定机构的特定实验适应症和独特的成像平台设置;因此,以下是对我们机构使用的协议的描述。
    1. 使用Inveon工作站软件(西门子医疗公司,田纳西州诺克斯维尔)分析鼠标的全身PET图像。
    2. 选择供应商软件提供的用于动力学建模的Patlak隔间图选项。
    3. 对于3d pet和4d pet数据集,根据以下器官(如需要)的相应ct图像,手动选择多个感兴趣的体积(voi):心脏、腿部肌肉、肝脏、肾脏和大脑。体素活动以标准化摄取值(suv)表示。使用每只动物的动态3d pet数据集绘制vois的动态活动曲线。4d pet数据用于定义心功能。首先,根据解剖特征在CT图像上分割心脏,然后转移分割的体积(心脏PET VOI)进行图像配准,如图12所示。图像表示为源数据的最大强度投影(mip)重建。心电引导下pet-mip图像的二值化可以获得小鼠心脏收缩力的伪动态图像,如视频2所示。
      < BR>
      图12。西门子Vieon工作站微PET/CT重建基本参数设置实例<强> >强> /强> BR/> < BR> < BR> 视频2。用2.2μq(250μl)FDG标记的人红细胞对NSG小鼠ECG门控全身微PET图像进行心脏电影成像(此视频是在南佛罗里达州大学制作的)根据南佛罗里达州大学动物护理学的指导方针,由南佛罗里达州大学动物研究伦理委员会通过ISO 044366认证。
    < BR>
  2. 标记效率计算< BR> 标记效率=(RBC颗粒的放射性剂量)/(红细胞颗粒的SuPE+放射性剂量的放射性剂量)。

食谱

  1. 过滤灭菌100毫升5X EDTA溶液< BR> 700毫米氯化钠
    20毫米kcl
    12.5毫米k2edta二水合物
    加入无菌去离子水至最终体积100毫升
    通过0.2微米(nm)Nalgin瓶顶无菌过滤装置进行最终的解决方案;
  2. 100毫升1x EDTA溶液
    20毫升过滤消毒的5X EDTA溶液< BR> 80毫升无菌去离子水
    通过0.2微米(nm)Nalgin瓶顶无菌过滤装置进行最终的解决方案;
  3. 1单位/ml肝素PBS溶液
    肝素钠溶于无菌磷酸盐缓冲液(PBS)中,pH值7.4

致谢

我们非常感谢Moffitt癌症中心的Mikalai Budzevich博士对这项工作的建议和评论。该协议适用于Choi 等>。(2019)。

竞争利益

H.Lee Moffitt癌症中心和研究所提交了一份临时国际专利申请,描述了手稿中概述的成像技术(WO申请号:WO2017123666A2;PCT/US2017/013063,申请日期:2017-01-11)。没有额外的声明或潜在的竞争利益,关于专利申请的作者的手稿。这并没有改变我们对plos-one共享数据和材料政策的坚持。

伦理学

所有实验程序均经南佛罗里达大学(USF)机构动物护理和使用委员会(IACUC)批准。所有实验均按照联邦法规和USF IACUC原则(协议IS00004376)和程序进行。

工具书类

  1. Choi,J.W.,Budzevich,M.,Wang,S.,Gage,K.,Estrella,V.和Gillies,R.J.(2019年)。使用18F-氟脱氧葡萄糖标记的人红细胞在免疫缺陷小鼠模型中进行体内正电子发射断层血池成像。plos-one>14(1):e021012。
  2. Cysouw,M.C.F.,Kramer,G.M.,Frings,V.,De Langen,A.J.,Wondergem,M.J.,Kenny,L.M.,Aboagye,E.O.,Kobe,C.,Wolf,J.,Hoekstra,O.S.和Boellaard,R.(2017年)。nucl-med-biol>51:18-24。
  3. Dong,H.,Zhang,Z.,Guo,Y.,Zhang,H.和Xu,W.(2017)。锝-99m-红细胞闪烁显像在眼眶海绵状血管瘤诊断中的应用。nucl-med common>38(9):744-747。
  4. Hacker,M.,Hoyer,X.,Kupzyk,S.,La Fougere,C.,Kois,J.,Stempfle,H.U.,Tiling,R.,Hahn,K.和Stork,S.(2006年)。充血性心力衰竭患者门控血池spect-qbs处理软件的临床验证:与muga的相关性,首次通过RNV和二维超声心动图。Int J心血管成像>22(3-4):407-416。< BR>
  5. Herrero,P.,Kim,J.,Sharp,T.L.,Engelbach,J.A.,Lewis,J.S.,Gropler,R.J.和Welch,M.J.(2006年)。使用15o-水和1-11c-醋酸盐评估小动物宠物大鼠的心肌血流量。j nucl-med>47(3):477-485;
  6. Lee,B.C.,Moody,J.B.,Weinberg,R.L.,Corbett,J.R.,Ficaro,E.P.和Murthy,V.L.(2017年)。优化82铷pet心肌血流定量的时间采样。j nucl cardiol>24(5):1517-1529。
  7. Liu,M.,Zhao,Z.Q.,Fang,W.和Liu,S.(2017)。红细胞标记的新方法:99mtc-4saboroxime作为血池显像剂的评价。bioconjug chem>28(12):2998-3006。
  8. Mohseni,S.,Kamali Asl,A.,Bitarafan Rajabi,A.,Entezarmahdi,S.M.,Shahpouri,Z.和Yaghoobi,N.(2015年)。门控血池spect对右心室功能的滤过作用。ann-nucl-med>29(4):384-390。
  9. Nakazato,R.,Berman,D.S.,Alexanderson,E.and Slomka,p.2.(2013.report).myocardial perfusion imaging with pet.imaging med5(=1:35-46). Rahmim,A.and Zaidi,H.(,2008).pet versus spect:strengths,limitations and challenges.anucle med commun29(>3:193-207). Sadri,K.,Momenypoor,S.,Dabbagh Kakhki,V.R.,Sadeghi,R.,Aryana,K.,Johari Daha,F.,Zakavi,S.R.and Jaafari,M.R.(*)pubmed//>26664365“target=bulank>nano liposomes labeled with99mThrall,J.H.,Freitas,J.E.,Swanson,D.,Rogers,W.L.,Clare,J.M.,Brown,M.L.and Pitt,B.(1978).target=Blank>clinical comparison of cadiacc blood pool visualization with technetium-999m red blood cells labeled in vivoand with technetium-999m human serum albumin<><>a/>
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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Wang, S. and Choi, J. W. (2019). Imaging the Vasculature of Immunodeficient Mice Using Positron Emission Tomography/Computed Tomography (PET/CT) and 18F-fluorodeoxyglucose Labeled Human Erythrocytes. Bio-protocol 9(19): e3391. DOI: 10.21769/BioProtoc.3391.
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