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

Summary

Lower limb amputation may occur even after angioplasty of obstructed vessels in Critical Limb Ischemia (CLI). Mononuclear Progenitor Cells (MPCs) reflect vascular repair. The present protocol describes the quantification of MPCs from circulation close to angioplasty, and its relationship with endothelial dysfunction and prediction of lower limb amputation.

Abstract

Critical limb ischemia (CLI) represents an advanced stage of the peripheral arterial disease. Angioplasty improves the blood flow to the lower limb; however, some patients irreversibly progress to limb amputation. The extent of vascular damage and the mechanisms of vascular repair are factors affecting post-angioplasty outcome. Mononuclear Progenitor Cells (MPCs) are reactive to vascular damage and repair, with the ability to reflect vascular diseases. The present protocol describes quantification of MPCs obtained from blood circulation from vessel close to the angioplasty site, as well as its relationship with endothelial dysfunction and its predictive ability for limb amputation in the next 30 days after angioplasty in patients with CLI.

Introduction

Peripheral Arterial Disease (PAD) is characterized by a chronic and progressive vascular obstruction with limitation of blood supply¹. At a global scale, PAD of the lower limbs affects around 10% of the elderly population, while up to 7% of such cases are submitted to limb amputation^2,3.

Critical Limb Ischemia (CLI) represents the most serious presentation of PAD¹. Patients usually experience pain at rest, ulcers, or gangrene attributable to occluded arteries; while clinical prognosis is unfavorable and marked by a 30% risk of limb amp....

Protocol

The institutional research ethics committee from Centro Médico Nacional “20 de Noviembre” ISSSTE approved this prospective protocol, all enrolled patients provided written informed consent.

1. Evaluation of vascular block of lower limb, blood sampling and balloon angioplasty

NOTE: The study sample used for this experiment comprised of 20 diabetic patients, aged 68 years old and 10 out of 20 were males. Half of the sample were smokers and most prevalent co-morbidities were type 2 diabetes mellitus, systemic arterial hypertension and/or dyslipidemia. The sample was intended to be standardized for age....

Results

Blood samples from blocked arteries, at the site addressed for angioplasty, were collected from 20 diabetic patients, aged 68 years old and 10 out of 20 were males. Half of sample population were smokers. Vascular lesions were mainly scored as Rutherford class VI; whereas patients showed a higher prevalence of type 2 Diabetes Mellitus (100%), hypertension (70%) and dyslipidemia (55%).

A 30 days clinical follow up after lower limb angioplasty was carried out. The percentage of MPC subpopulation.......

Discussion

Blood collection at the precise site of the vascular block may show technical difficulties; therefore, we performed blood collection in the proximity to vascular block. Likewise, the amount of MPCs close to the vascular plaque seems to be highly dynamic and may originate variations before and after angioplasty. According to our observations, it is recommended to evaluate baseline- and 30min-post-angioplasty changes in the number of MPCs, since they may reflect several pathophysiological processes occurring within vascula.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
BSA	Roche	10735086001	Bovine Serum Albumin (BSA) as a buffering agent, stabilizer, standard and for blending.
Calibration Beads	Miltenyi Biotec / MACS	#130-093-607	MACQuant calibration beads are supplied in aqueous solution containing 0.05% sodium azide. 3.5 ml for up to 100 tests
CD133/1 (AC133)-PE	Milteny Biotec / MACS	#130-080-801	Antibody conjugated to R-Phycoerythrin in PBS/EDTA buffer
CD184 (CXCR4)-PE-VIO770	Miltenyi Biotec / MACS	#130-103-798	Monoclonal, Isotype recombinant human IgG1, conjugated
CD309 (VEGFR-2/KDR)-APC	Miltenyi Biotec / MACS	#130-093-601	Antibody conjugated to R-Phycoerythrin in PBS/EDTA buffer
CD34-FITC	Miltenyi Biotec / MACS	#130-081-001	The monoclonal antibody clone AC136 detecs a class III epitope of the CD34
CD45- VioBlue	Miltenyi Biotec / MACS	#130-092-880	Monoclonal CD45 Antibody, human conjugated
Conical Tubes	Thermo SCIENTIFIC	#339651	15ml conical centrifuge tubes
Cytometry Tubes	FALCON Corning Brand	#352052	5 mL Polystyrene Round-Bottom Tube. 12x75 style. Sterile.
EDTA	BIO-RAD	#161-0729	Heavy metals, (as Pb) <10ppm, Fe<0.01%, As<1ppm, Insolubles<0.005%
Improved Neubauer	Without brand	Without catalog number	Hemocytometer for cell counting. (range 0.1000mm, 0.0025mm2)
K2 EDTA Blood Collection Tubes	BD Vacutainer	#367863	Lilac plastic vacutainer tube (K2E) 10.8mg, 6 mL.
Lymphoprep	Stemcell Technologies	01-63-12-002-A	Sterile and checked on the presence of endotoxins. Density: 1.077±0.001g/mL
Paraformaldehyde	SIGMA-ALDRICH	#SZBF0920V	Fixation of biological samples, (powder, 95%)
Pipette Transfer 1,3mL	CRM Globe	PF1016, PF1015	The transfer pipette is a tool that facilitates liquid transfer with greater accuracy.
Test Tubes	KIMBLE CHASE	45060 13100	Heat-resistant test tubes. SIZE/CAP 13 x 100 mm