Purified CD34+ cells versus peripheral blood mononuclear cells in the treatment of angiitis-induced no-option critical limb ischaemia: 12-Month results of a prospective randomised single-blinded non-inferiority trial

Abstract

Background: Peripheral blood mononuclear cells (PBMNCs) and purified CD34⁺ cells (PCCs) are increasingly being used at treating no-option critical limb ischaemia (NO-CLI). We aimed to compare the efficacies and uncover the advantages associated with each treatment approach.

Methods: A randomised single-blinded non-inferiority trial (Number: NCT 02089828) was performed. NO-CLI patients were 1:1 randomised to the PBMNCs and PCCs groups, and compared in relation to safety and efficacy outcomes. The primary efficacy outcomes included major amputation and total amputation over 12 months. The major amputation-free survival (MAFS) and total amputation-free survival (TAFS) rates were calculated.

Findings: Fifty patients (25 per group, 47 with thromboangiitis obliterans and 3 with other angiitis) were enrolled, with a median follow-up period of 24.5 months (interquartile range: 17-34 months). One patient in the PCCs group was lost at 2 months and one major amputation occurred in the PBMNCs group at 3 months post-transplantation. The total amputation rates at 6 months post-transplantation were 28.0% in the PCCs group and 16.0% in the PBMNCs group (p = 0.343), and remained unchanged at 12 months. The groups did not differ regarding the MAFS and TAFS (Breslow-Wilcoxon test: p = 0.3014 and p = 0.3414). The PCCs group had a significantly higher probability of rest pain relief than the PBMNCs group (Breslow-Wilcoxon test: p = 0.0454).

Interpretation: PCCs was not inferior to PBMNCs at limb salvage in the treatment of angiitis-induced NO-CLI and appeared to induce earlier ischaemia relief. Each cell type had specific advantages. These outcomes require verification from longer-term trials involving larger numbers of patients. FUND: Training program for outstanding academic leaders of Shanghai health and family planning system (Hundred Talent Program，Grant No. 2018BR40); China National Natural Science Funds (Grant No. 30801122); The excellent core member training programme at Zhongshan Hospital, Fudan University, China (Grant No. 2015ZSYXGG02); and Zhongshan Funds for the Institute of Vascular Surgery, Fudan University, China.

Clinical trial registration: This study is registered with ClinicalTrials.gov (NCT 02089828).

Trial registration: ClinicalTrials.gov NCT02089828.

Keywords: Cell therapy; Critical limb ischaemia; Limb salvage; Peripheral blood mononuclear cells; Purified CD34(+) cells.

Fig. 1

Trial design. PCCs, purified CD34⁺ cells; PBMNCs, peripheral blood mononuclear cells.

Fig. 2

Kaplan-Meier curves showing the probabilities of (A) major amputation-free survival and (B) total amputation-free survival in both groups. The P values were calculated using the Breslow-Wilcoxon test.PCCs, purified CD34⁺ cells, PBMNCs, peripheral blood mononuclear cells.

Fig. 3

Longitudinal pictures of the treated limb of patient 3 in the PCCs group. The patient had gangrene on the second toe and an ulcer on the dorsum of his left foot before cell therapy (A). He underwent a foot debridement during the cell transplantation (B). The wound area reduced quickly in one month (C). The foot healed in the following two months (D, E) and sustained non-ischemic for 3 years (F).

Fig. 4

Longitudinal changes in the (A) Wong-Baker FacesPain Rating Scale(WBFPS) and (B)probability of rest pain relief. (A) The longitudinal changes in the WBFPS in both groups are depicted as linear graphs that show the mean values and thestandard deviation bars. *represents p < 0.05, ** represents p < 0.01 (intra-group comparison with baseline, based on a general linear mixed model).(B) Theprobability of rest pain relief is depicted as Kaplan-Meier curves, and the p value was calculated using the Breslow-Wilcoxon test. PCCs, purified CD34⁺ cells; PBMNCs, peripheral blood mononuclear cells; WBFPS, Wong-Baker Faces Pain Rating Scale.

Fig. 5

Longitudinal changes in blood perfusion restoration and functional improvement. The assessments of blood perfusion restoration included the (A) ankle-brachial index,(B) toe-brachial index, and (C) transcutaneous oxygen pressure, and the functional improvement was assessed using (D) the pain-free walking time. The values are presented in linear graphs that show the means and standard deviations. *represents p < 0.05, **represents p < 0.01 (intra-group comparison with baseline, based on a general linear mixed model),and Φ represents p < 0.05 for the interaction between a time point and the group, based on a general linear mixed model.ABI, ankle-brachial index; TBI, toe-brachial index; TcPO₂, transcutaneous oxygen pressure; PFWT, pain-free walking time,PCCs, purified CD34⁺ cells; PBMNCs, peripheral blood mononuclear cells.

Fig. 6

Quality of life at baseline and at 1-year post-transplantation. The quality of life was assessed using the Short Form-36 scoring system (version 2) in the (A) purified CD34⁺ cellsgroup and (B) peripheral blood mononuclear cellsgroup. *representsp<0.05 (intra-group comparison with baseline, based on the Wilcoxon signed-rank test).PCCs, purified CD34⁺ cells; PBMNCs, peripheral blood mononuclear cells;QoL, quality of life; PF, physical functioning; RP, role-physical; BP, bodily pain; GH, general health; VT, vitality, SF, social functioning, RE, role-emotional, MH, mental health.

Fig. 7

In vivo neovascularization potential of the transplants using a non-obese diabetic/severe combined immunodeficiency mouse limb ischaemia model.The longitudinal changes in blood perfusion restoration in the purified CD34⁺ cells, peripheral blood mononuclear cells, CD34⁻ cells, and the control groupsare presented as (A) laser speckle contrast images and(B) linear graphs of the blood perfusion indexesin which the data are presented as the means and standard deviations. Microvascular density comparisons among groups are presented as (C) immunostaining for mouse-specific CD31 in the ischaemic gastrocnemius muscle on day 8 post-transplantation and (D) bar graphsshowing the mean and standard deviation values. *representsp<0.05 (inter-group comparison with the control group at specific time points, based on Dunnett's t-test). PCCs, purified CD34⁺ cells; PBMNCs, peripheral blood mononuclear cells.