Autologous Immunomagnetic Selected cd133+ Cells in the Treatment of No-option Critical Limb Ischemia: Clinical and Ceus Assessed Results.

Type of the study. Prospective single centre not randomized. Aim of the study. To assess the safety, feasibility and efficacy of local intramuscular administration of autologous selected CD 133+ cells in patients suffering from CLI.

All the patients enrolled were suffering from CLI according to the TASC 2 definitions and had no revascularization option, on the basis of contrast CT or angiography imaging. A detailed informed consent, approved by our EC, had been required. An age lower than 18 years and upper than 70 years because of a poor marrow responsiveness to drug simulation in elderly people Clinical unsteadiness of the CLI, such as gangrene requiring major amputation and a poor life expectancy were introduced as exclusion criteria for supposed latency of the EPCs action. Severe systemic illness was judged to increase the risk of the marrow stimulation. Patients with allergic diathesis, child-bearing age, previous EPCs muscular implant, previous medical experimental protocol and any conflict of interest with the study were excluded.

Patients: we enroll patients with a history of Rutherford stage 4 (rest pain) or 5 (small ischemic lesions) PAD. All the patients have previous (contrast CT, MR or angiography) vascular imaging excluding revascularization options, both endovascular and open and encountered the enrolment and exclusion criteria. Every patient undergo routine physical and instrumental examination including electrocardiogram, chest X-ray and blood sample analysis. Younger patients met the Buerger disease criteria, whereas others had pure atherosclerotic lesions.

CEUS Imaging Protocol. Two operators (F.C. and A.G., with 20 and 3 years experience, respectively) who are blinded to treatment perform contrast-enhanced US for all patients. Two US scanner (Philips iU22 (Bothell, WA, USA) with linear array L9-3 transducer and GE Logiq e9 (GE Healthcare, Milwaukee, WI, USA) with linear array L6-9 transducer are used.

The imaging parameters are: reduced transmit power (mechanical index <0.06), at approximately 7-10 frames per second and one focus well below the level of the target to ensure a more uniform pressure field. Dual-mode presentation of a grayscale image side-by-side with the contrast image facilitated real-time identification of anatomic structures and ROI selection. Image loops of approximately 60 seconds were acquired. Effort was made to have a uniform gain across the image and to avoid gain saturation. The TGC (time gain compensation) was set such that before contrast arrival a uniform black image was shown. A vial of contrast agent (SonoVue BR1; Bracco, Milan, Italy) is prepared at a concentration of about 2x108 sulfur hexafluoride-filled microbubbles per milliliter, according to the manufacturer's recommendations. Prior to each injection, microbubbles are resuspended by shaking the vial. The position of the probe is recorded for each patient in order to maintain the same position during follow up. The contrast injection consists of an intravenous bolus of 4.8 ml of contrast agent injected in antecubital vein in 2 seconds followed by a saline flush of 5 ml. The injection is made with the patient supine and after 10 minute of rest to avoid exercise related micro-vascular dilatation. The radiologist maintains a constant image plane with the aid of the tissue (fundamental image) of the "Contrast Side/Side" imaging mode.