Predicting Amputation using Local Circulating Mononuclear Progenitor Cells in Angioplasty-treated Patients with Critical Limb Ischemia

Although angioplasty improves blood flow in critical limb ischemia, some patients will still progress to limb amputation. Compared to prognosis based on traditional risk factors and intrinsic vascular features, the number of local MPCs can be more predictive of the clinical outcome, regarding endothelial dysfunction and limb amputation. The number of local MPCs represents a promising indicator of the prognosis of vascular events, such as ischemia heart disease, and lower limb ischemia, with potential application in risk stratification and prevention enforcement.

Demonstrating the procedures will be Eduardo Vera-Gomez, Alejandro Hernandez-Patricio, Carolina Aranda-Rodriguez, Juan Ariel Gutierrez-Buendia, Atzin Sua Ruiz-Hernandez, Mario Antonio Tellez-Gonzalez, and Gabrielle Alexandra Dominguez-Perez. PhD's from the laboratories of experimental metabolism and tissue regeneration, as well, will be Gabrielle Hernandez-De Rubin and Oscar Antonio Loman-Zuniga. And these from the vascular surgery department.

To determine the pre and post angioplasty FMD, use a vascular linear transducer to measure the diameter of the brachial artery of the patient. Next place, the cuff of a sphygmomanometer above the measurement site in the forearm, and insufflate the cuff 50 millimeters of mercury above the systolic blood pressure for five minutes before deflating. Within 60 seconds of deflating, measure the brachial artery diameter again, and use the equation to estimate the FMD.

To evaluate the clinical severity of limb ischemia, according to the Rutherford classification, place an 18 gauge needle into the blood vessel at the selected groin site, and place an introducer over the needle. Advance a flexible guidewire into the introducer and replace the guide with a six French introducer. Using fluoroscopic guidance, inject contrast medium to identify the artery trajectory and blocked vascular sites.

Introduce two, 0.014 French navigation guidewires and two, 0.014 French support guidewires into the vessel, and advance the guide wires to the blocked site. Next, introduce one five French catheter, and then one three French catheter, and collect 10 milliliters of blood from the site closest to the vascular obstruction. Place the sample on ice and advance a guidewire again.

When the guidewire is in place, introduce an angioplasty balloon catheter, containing an inflatable balloon located at the end of the catheter, and advance the angioplasty balloon catheter to the site of the lesion. Inflate the balloon against the blocking plaque, located at the vascular wall, and verify restoration of the blood flow. 30 minutes after performing the angioplasty, advance a catheter to the site closest to the vascular block and collect 10 milliliters of blood.

Then, remove all of the guidewires and provide the appropriate post-operative care. Two weeks after the angioplasty, evaluate the clinical severity of the limb ischemia, the resolving rest pain, lower ischemia, and functional foot preservation, according to the Rutherford classification, and identify those cases requiring a major amputation due to an unfavorable outcome. Within one hour of collection, transfer six milliliters of each collected blood sample into new 15 milliliter tubes, and dilute the samples at a one-to-one ratio in PBS.

Add two milliliters of density gradient medium to each of three test tubes, and add three equal volume aliquots of the diluted blood to each tube to no more than three quarters full. Separate the blood cells by density gradient centrifugation, and use a pipette to collect the cells of the interface of the resulting layers. Pull the cells from each sample into a single 15 milliliter tube, and wash each sample six times, with two milliliters of PBS per wash.

After the last wash, re-suspend the MPC containing pellets in one milliliter of PBS per sample for counting, and add one times 10 to the six cells per tube to the appropriate number of five milliliter flow cytometry tubes per sample. Pellet the cells by centrifugation, and re-suspend each cell sample in 100 microliters of the antibody cocktail of interest for 10 seconds, before incubating the cells for 20 minutes at four degrees Celsius, protected from light. At the end of the incubation, use isotype-matched control antibodies to set up the appropriate forward and side scatter gates for analysis, and use a tube containing a high number of CD45 and CD34 positive cells to gate the NPCs.

To select for double positive immuno phenotypes, create new plots and gate-identify the KDR, CD133 and CD184 positive, CD34 positive, CD45 positive cells, then gate to identify the main MPC subpopulations. The number of MPCs can then be correlated with the baseline FMD value, and post angioplasty delta of FMD, and the proportion of patients requiring major amputation of the lower limb. In this representative analysis, 30 days after lower limb angioplasty, the baseline number of CD45 positive, CD34 positive, KDR positive MPCs, negatively correlated with the FMD.

Whereas the change in CD45 positive, CD34 positive, CD133 positive, CD184 positive MPCs, after angioplasty, significantly correlated with FMD improvement. An increased baseline number of MPCs CD45 positive, CD34 positive, KDR positive subpopulations, as well as post angioplasty reduction in the MPC CD45 positive, CD34 positive, CD133 positive, CD184 positive cells, were also observed in patients, who evolved to limb amputation, During the vascular approach, be sure to collect the blood from the site closest to the vascular obstruction.