Background 

Thrombus imaging of platelets and fibrin has a rich history dating back to the mid 1970s. Regardless of the imaging modality, these probes have only been effective for acute thrombi (<48 hours) [1]. Moreover, prior or concurrent use of anti-coagulation typically negates the efficacy of probes, even to acute thrombus [1]. LVAD thrombi are typically acellular fibrin accretions [2] deposited despite concurrent anticoagulation with warfarin and aspirin [3, 4]. Direct diagnosis and characterization of LVAD thrombus with noninvasive imaging is complicated by a titanium housing that precludes the use of MRI, CT, or ultrasound and favors a nuclear medicine approach. Further complicating the task, the high-shear and high blood flow conditions created by LVADs, such as the HeartMate II (HMII, Abbott-Thoratec spinning at 8,800-10,000 RPM) necessitate a probe with high molecular specificity and extreme binding avidity. Capella Imaging is developing a novel, fibrin-specific 99mTc small tetrameric molecule (99mTc-F4A) able to bind to native aged thrombus under LVAD operational conditions and in the presence of anticoagulation. Preliminary data showed that 99mTc-F4A clears rapidly into urine with minimal serum interference or uptake in off target tissues. 

​

LVAD placement and imaging 

Thoratec, now Abbott, shared an extracorporeal procedure developed by Dr. Hill, a computer LVAD controller, and provided custom inlet/outlet cannulas for the HeartMate II (HMII) LVAD. De-identified LVADs with unknown medical history from patients undergoing heart transplant or pump exchange were rewired for reuse. LVADs were placed externally in ~70 kg bovine calves, as shown in Figure 1. The inlet cannula entered the LV through left atrial appendage was placed just below the mitral valve. This cannula attached to the LVAD inlet through a new sintered stainless steel elbow. A second cannula attached to a new stainless elbow on LVAD outlet and was sutured into the distal thoracic aorta. Animals with LVADs operated at ~8900 RPM were heparinized prior to treatment and throughout the study.  

 

St. Louis Children’s Hospital Department of Radiology shared a portable Digirad ERGO planar gamma camera and an experienced nuclear medicine technologist under a strict protocol for camera use and return to the Hospital. In contradistinction with SPECT cameras, planar panel cameras cannot distinguish the source of gamma emissions.

​

 

​

​

​

​

 

​

​

 

Fig 1 shows the LVAD placed above and slightly offset from the chest incision. Lead shielding was placed between the LVAD and body to isolate the detector from spurious emissions arising from blood pooled around the heart and from flowing through the great vessels along the spine. Lead shielding was placed over the lower abdomen to protect staff from radioactivity excreted into the bladder. Calves did not urinate during these studies. The bulls were not amenable to urinary catheter placement and percutaneous bladder drains were considered but avoided to avoid radioactive blood or urine leaks around the drains.

​

Pharmacokinetics

Five animals received 99mTc-F4A IV doses at 2.5, 5.0 or 10 mCi IV. Timed venous blood samples were drawn and gamma images were serially acquired every 1 min near the time of injection then roughly every 5 to 10 min thereafter. Pharmacokinetic and imaging data were analyzed with GraphPad and MatLab, respectively. LVADs were recovered, allowed to decay, and returned to Thoratec for evaluation (pending).

 

 

 

 

 

 

 

 

 

 

 

 

Fig 2A presents the blood pharmacokinetic (PK) profile of the first 2 animals imaged after receiving 10mCi or 5mCi of 99mTc-F4A, respectively. Compared to preliminary mouse studies, the circulatory half-lives of the probe were markedly decreased. Unexpectedly the PK (%ID/g) curves of the first 2 animals did not align. Compartmental analysis suggested a prolonged third half-life, possibly from unbound 99mTc-tricarbonyl (~3%) interacting with plasma protein. To test and negate this, excess cysteine was added to 99mTc-F4A doses, and the PK studies of the next three calves given 2.5, 5.0, and 10.0 µCi closely aligned (Fig 2B).  99mTc-F4A had 23.2 min β-elimination half-life with a 25.8 L volume of distribution, 0.03 min-1 elimination rate (Ke) and 0.76 L/min clearance rate.

​

Planar Gamma Imaging

Planar imaging of 99mTc-F4A in large animals (70 kg) demonstrated that the probe targeted aged fibrin in LVADs spinning at ~ 8900 RPM (~5L/min). Acute fibrin formed in the new inlet elbows along the outer curvature despite anticoagulation with heparin. A similar but lower degree of clotting was noted in outlet steel elbows. In 4 of 5 animals, 99mTc-F4A bound to chronic thrombus on the inlet and sometimes outlet bearing. In one animal, the LVAD was thrombus free. Imaging was performed for up to 4 hours in some animals, but analysis showed that adequate fibrin contrast was typically appreciated in less than 1-hour post injection. At 65 minutes, shown in Fig 3, the free probe in blood pool had undergone 3 half-lives or 88% clearance.

 

 

 

 

 

 

 

 

 

 

​

At variable times beyond 90 minutes, the bound probe signal diminished suggesting in vivo probe metabolism. This late loss of signal will permit serial imaging studies over days without dose-to-dose interference. At this time, the preferred dose is 10 mCi with cysteine/animal.

​

Significance

These results provide proof of concept that 99mTc-F4A in a large animal (calf, 70kg) model implanted with an excised HMII LVAD operating at 8900 RPM can target aged fibrin in the presence of anticoagulation. A marked reduction in circulating half-life was appreciated in the large animals versus mice. The addition of cysteine to the probe neutralized a small residual of unchelated 99mTc.  While planar imaging was these extracorporeal LVAD studies, clinical trials will require high-resolution, high-sensitivity SPECT imaging, perhaps using a dedicated portable nanoSPECT-like pinhole camera placed immediately over the skin above the underlying LVAD.  Although beyond the current scope, local experienced investigators have a conceptual design plan.

 

 

1. Lanza G, Cui G, Schmieder A, Zhang H, Allen J, Scott M, et al. An unmet clinical need: The history of thrombus imaging. J Nucl Cardiol 2017; DOI 10.1007/s12350-017-0942-8.

2. Prasad S, Robertson J, Itoh A, Joseph S, Silvestry S. Histologic analysis of clots in explanted axial continuous-flow left ventricular assist devices. J Heart Lung Transplant. 2015; 34: 616-8.

3. Meyer AL, Malehsa D, Budde U, Bara C, Haverich A, Strueber M. Acquired von Willebrand syndrome in patients with a centrifugal or axial continuous flow left ventricular assist device. JACC Heart failure. 2014; 2: 141-5.

4. Adatya S, Bennett MK. Anticoagulation management in mechanical circulatory support. Journal of thoracic disease. 2015; 7: 2129-38.

 

Publications and Presentations

1) Cui G, Akers WJ, Scott MJ, Nassif M, Allen JS, Schmieder AH, Paranandi KS, Itoh A, Beyder DD, Achilefu S, Ewald GA, Lanza GM. Diagnosis of LVAD thrombus using a high-avidity fibrin-specific 99mTc probe. Theranostics 2017: (In minor Revision)

2) Hartupee JC, Cui G, Scott MJ, Itoh A, Achilefu S, Lanza GM, Ewald G. Detection of Left Ventricular Assist Device thrombosis using a fibrin specific probe. AHA National Meeting. 11/12/2017. Anaheim, S3218 (abstract)

3) Invited Presentation: Lanza GM, Cui G, Akers W, Scott MJ, Allen JS, Itoh A, Nassif M, Hartupee J, Beyder D, Achilefu S, Ewald GA. A Look to the Future:  Imaging thrombus using nuclear imaging and nanotechnology. Baylor University Medical Center, State of The Art Imaging in MCS, 9/15/2017