scholarly journals In vitro migratory capacity of CD34+ cells is related to hematopoietic recovery after autologous stem cell transplantation

Blood ◽  
2001 ◽  
Vol 97 (3) ◽  
pp. 799-804 ◽  
Author(s):  
Carlijn Voermans ◽  
Marisha L. K. Kooi ◽  
Sjoerd Rodenhuis ◽  
Hans van der Lelie ◽  
C. Ellen van der Schoot ◽  
...  
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Migratory Capacity ◽  
Hematopoietic Recovery ◽  
Cd34 Cells ◽  
Spontaneous Migration

Abstract To investigate whether the migratory ability of peripheral blood-derived CD34+ cells of patients undergoing autologous peripheral blood stem cell transplantation is related to the homing efficiency of these cells, the migration in vitro of these cells was determined and correlated with in vivo hematopoietic recovery. Large inter-individual differences of the in vitro migratory ability of the CD34+ cells were observed, ranging from 1.1% to 16.4% for spontaneous migration and 6.2% to 40.8% for SDF-1–induced (100 ng/mL) migration. Significantly faster hematologic recovery was observed in those patients who received transplanted CD34+cells that showed high spontaneous and SDF-1–induced migration in vitro (P < .05). Moreover, CD34+ cells from healthy G-CSF–mobilized donors exhibited significantly higher spontaneous and SDF-1–induced (P < .01) migration than CD34+ cells from patients mobilized with chemotherapy and G-CSF. The lower migratory capacity in vitro of patient-derived CD34+ cells was not due to lower expression of CXCR-4 but probably reflected decreased motogenic behavior of the cells. These results indicate that the migratory capacity of the cells is important for hematopoietic recovery. The data suggest that the engraftment potential of autologous stem cells is more or less impaired by treatment before or during the mobilization procedure and might possibly be restored by in vitro manipulation of the cells. In addition, an exponential relation between CXCR-4 expression and number of CD34+ cells that mobilized to the peripheral blood was found (P < .001), suggesting that CXCR-4 expression plays a role in the mobilization of CD34+ cells.

Abstract 32: Intravenous Mobilized Autologous Peripheral Blood CD34+ Stem Cell Transplantation for Angina

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Hadyanto Lim ◽  
Lindarto Dharma ◽  
Zein Umar ◽  
Hariaji Ilham
Keyword(s):  
Stem Cell ◽  
Angina Pectoris ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Exercise Tolerance ◽  
Exercise Stress ◽  
Peripheral Blood Cd34 ◽  
Cd34 Cells ◽  
Angina Frequency

Background: Intramyocardial CD34+ stem cell therapy for patients with refractory angina shows that this is safe and feasible. We aimed to determine whether intravenous transplantation of mobilized autologous peripheral blood CD34+ stem cells provides beneficial effects for patients with angina pectoris. Methods: We administered granulocyte colony stimulating factor (G-CSF, 5.0 μg/kg/day) subcutaneously once a day for 4 days to 15 patients (4 women and 11 men aged 50-78 years) with intractable angina pectoris (Canadian Cardiovascular Society functional class III-IV) for mobilization of CD34+ cells into the peripheral blood. Ischemia was assessed by exercise stress testing. Leukapheresis procedure was started on the day 4 of G-CSF using the Spectra Optia cell separator. Circulating and intravenous transplantation of autologous CD34+ cells after leukapheresis were measured by flow cytometry. The effects of G-CSF on blood were measured by hematology analyzer and semi-auto chemistry analyzer. Results: Intravenous peripheral blood CD34+ cells increased after leukapheresis (from 1.12±0.48 cells/μL to 107.42±23.83 cells/μL, p<0.001) and total white blood cells count (from 7.82 ± 2.63x10 3 /μl to 37.47±15.07 x10 3 /μl, p<0.001). Indices of hsCRP, platelets, hemoglobin, alanine aminotransferase, lactic dehydrogenase, and uric acid were not changed by treatment. At week 4, angina frequency was significantly lower after intravenous CD34+ cells (from 15.07±4.03 to 3.27±1.49, p<0.001). Similarly, improvement in exercise tolerance was significantly higher by stem cell transplantation (from 5.90±2.53 minutes to 8.41±2.49 minutes, p<0.001). Most patients reported mild myalgia which were easily managed with acetaminophen. Conclusions: Intravenous autologous CD34+ stem cell transplantation improved angina frequency and exercise tolerance. The cell mobilization and leukapheresis procedures were found safe and tolerable in patients with angina pectoris.

The Importance of the Number of Transplanted Cells with Dipeptidyl Peptidase-4 Expression on the Hematopoietic Recovery and Lymphocyte Reconstitution in Patients with Multiple Myeloma after Autologous Hematopoietic Stem-Cell Transplantation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5415-5415
Author(s):  
Anna Kopinska ◽  
Malgorzata Krawczyk-Kulis ◽  
Joanna Dziaczkowska-Suszek ◽  
Katarzyna Bieszczad ◽  
Krystyna Jagoda ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Hematopoietic Stem ◽  
Dipeptidyl Peptidase 4 ◽  
Hematopoietic Recovery ◽  
Cd34 Cells

Abstract Introduction Autologous hematopoietic stem cell transplantation (AHSCT) remains the treatment of choice in multiple myeloma (MM) patients (pts). In earlier research it has been suggested that the expression of dipeptidyl peptidase-4 (DPP4, CD26) influences both the homing and lymphocyte reconstitution after AHSCT in pts with lymphoproliferative neoplasms. The aim of the study is to investigate the effect of transplanted CD26 positive cells of the hematopoietic recovery and lymphocyte reconstitution in MM pts after AHSCT. Patients and methods Forty eight pts with MM with median age 56 (range 21-76) were undergoing AHSCT in our center in years 2011-2013. Conditioning regimen was Melphalan 200. Number of all CD26+ cells, CD26+ lymphocytes, CD26+ monocytes and CD26+ and CD34+ cells were measured in harvested material. Number of lymphocyte's subpopulations (all lymphocytes CD3+, helpers CD3+CD4+, suppressors CD3+CD8+, natural killer (NK) CD3-CD16+CD56+, cytotoxic NK CD3+CD16+CD56+, lymphocytes B CD3-CD19+) were measured in peripheral blood during regeneration period after AHSCT. In both flow cytometry was used. The hematopoietic regeneration was measured as following: the day of white blood cells' regeneration when WBC count reached >1,0x109/L, the day of granulocytes' regeneration when ANC >0,5x109/L and the day of platelets' regeneration when PLT >20x109/L. Results All pts successfully engrafted. The results of AHSCT are shown in table nr 1. Table 1. The number of transplanted cells and regeneration during the procedure AHSCT in pts with MM. Parameter Median Range Mean Standard deviation Number of transplanted WBCx108/kg b.w. 4,26 0,73-18,8 5,43 4,36 Number of transplanted CD34+cells x106/kg b.w. 3,36 2,2-8,2 3,52 1,28 Number of transplanted CD26+ lymphocytes [109/L] 46,5 9-148 53,6 30,8 Number of transplanted CD26+ monocytes [109/L] 3,65 0-82 8,03 13,05 Number of all transplanted CD26+ cells [109/L] 50,42 9,6-213 62,5 23,2 Regeneration WBC >1x109/L (day) 13 10-20 13 2,64 ANC >0.5x109/L (day) 13 9-20 13,3 2,16 PLT >20x109/L (day) 14 11-20 14,1 2,18 As regards regeneration of hematopoietic cells after AHSCT it was shown that a higher number of transplanted CD26+ monocytes improves the reconstitution of suppressor (p=0,019) and NK lymphocytes (p=0,0237). A higher number of all transplanted CD26+ lymphocytes has a positive impact of the reconstitution of suppressor lymphocytes (p=0,0054), whereas a higher number of all transplanted the CD26+ cells improves the regeneration of cytotoxic NK (p=0,0126) and helper lymphocytes (p=0,0261). There were no confirmed adverse effects of the number of CD26+ cells on the hematopoietic regeneration0 and lymphocytes B reconstitution after AHSCT. Discussion Our research shows that the number of transplanted CD26-positive cells may improve immune reconstitution after AHSCT in patients with multiple myeloma, which was not clearly demonstrated before. As is well known faster lymphocyte reconstitution after AHSCT is associated with improved patient survival. Therefore, the greater the number of transplanted autologous CD26-positive cells may be associated with improved survival, which, however, needs further investigation. Disclosures No relevant conflicts of interest to declare.

Magic-TT (Magnetism-induced cell target transplantation) Enhanced the CD45+ Cells Target Migration, in Situ Proliferation and Promotion of Hematopoietic Recovery after Transplantation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5404-5404
Author(s):  
Qianli Jiang ◽  
Hao Huang ◽  
Yongjun Zhou ◽  
Qiuxia Zhang ◽  
Sun Xiaowei ◽  
...  
Keyword(s):  
Magnetic Field ◽  
Bone Marrow ◽  
Stem Cell ◽  
Cell Transplantation ◽  
In Vitro Study ◽  
Confocal Microscope ◽  
Hematopoietic Recovery ◽  
Donor Cells

Abstract Background: In our previous work (56th ASH poster, No.2416), we developed a novel cell transplantation system named MagIC-TT. The purpose of this study is to explore whether the MagIC-TT can promote hematopoietic recovery in the mice experiment and illustrate it¡¯s mechanism both in vivo and in vitro. Methods: 1) In vivo study: With regard to auto-transplantation, the C57BL/6 CD45-GFP cells were sorted and magnetized from the bone marrow of C57BL/6-Tg(CAG-EGFP) mice. Forty C57BL/6 female mice (2 groups, twenty mice each group) were transplanted into the femur cavity with or without magnetic field (M or W group), after 7.5Gy irradiation. Following transplantation, the survival of mice, hematopoiesis as well as GFP+ cells in different tissues, such as peripheral blood, bone marrow, liver, spleen, thymus and lung etc. were observed. Femurs of recipients were decalcified with our own derived semi-solid decalcification (SSD) technique to illustrate the distribution, proliferation of donor cells and the relationship between recipients and donor cells. Allo-transplantation: The C57BL/6 CD45-GFP cells were injected into the femur cavity of FVB mRFP transgenic mice (sponsored by Prof. XH Wu, Fudan University, Shanghai, China) after 7.5Gy irradiation. GVHD was observed in addition to what was done in auto-transplantation. 2) In vitro study: Magnetized CD45-GFP cells and non-magnetized BMSC-RFPs were cultured respectively or co-cultured with or without magnetic field (M or W group). The magnetic field was added to the top or the bottom of cell culture dish. Cell morphology, cell proliferation, cell viability, as well as cell migration, transwell migration and matrigel migration assays induced by magnetism were studied. The interaction of CD45-GFP cells and BMSC-RFPs was observed by confocal microscope, electronic microscope, immunohistochemical staining, western blot, real-time PCR and deep sequencing. Results: 1) In vivo study: During the first few hours after transplantation, lots of magnetized CD45-GFP cells resided within the femur and knee joints in M group while few in W group. Many GFP cells migrated into the lung soon after transplantation in the W group (P =0.046), followed by other organs such as kidney and skin (Fig.1). FACS showed that more GFP+ cells resided within the target femurs than the controls (Table.1). With SSD, frozen sections, confocal microscope and Lightsheet Z.1 Microimage (Carl Zeiss); transplanted GFP+ cells and their micro-environment were all well demonstrated (Fig.1). On removal of magnetic field, CD45-GFP cells were observed to migrate into the spleen, kidney, gut and other organs, showing the slow release of target transplanted cells from femur. GVHD on skin and lung etc. were observed in C57BL/6 to FVB allogenic transplanted mice (Fig. 1). The hematopoietic recovery in M group occurs much earlier than the controls, especially for the platelets, 10.67d ¡À 1.53d vs 14.75d ¡À 2.06d (M vs W group, P =0.035). 2) In vitro study: With the help of MagIC-TT, CD45-GFP cells can migrate through the matrigel and transwell membranes much more efficiently. The magnetized CD45-GFP cells advance toward the inner roof of petri dish in the culture medium, and attach to BMSC-RFP growing on the inner roof of dish and proliferate in the niche composed by BMSC-RFP under the effect of magnetic field (Fig.2). Conclusion: MagIC-TT could enhance CD45+ cells target migration, improve stem cell homing and proliferation efficiency, as well as promotion hematopoietic recovery in vivo. This study would shed light on current Hematological Stem Cell Transplantation (HSCT) and other cell therapies. Table 1. The FACS results of femurs of CD45-GFP cells injected into C57 mice, at 0.5h, 24h and 72h respectively. group 0.5h£¨%£© p 24h£¨%£© p 72h£¨%£© p *LC **RT *LC **RT *LC **RT BMM 0.017¡À0.006 0.497¡À0.151 0.040 0.080¡À0.026 1.573¡À0.508 0.030 0.190¡À0.139 1.960¡À0.809 0.049 BMW 0.017¡À0.012 0.050¡À0.017 0.184 0.013¡À0.006 0.027¡À0.015 0.184 0.023¡À0.015 0.320¡À0.434 0.368 P 1.000 0.007 0.013 0.006 0.108 0.036 *LC: Control femur without magnetic field (W group); **RT: Treated femur with magnetic field (M group). Disclosures No relevant conflicts of interest to declare.

Prophylactic infusion of cytomegalovirus-specific cytotoxic T lymphocytes stimulated with Ad5f35pp65 gene-modified dendritic cells after allogeneic hemopoietic stem cell transplantation

Blood ◽  
2008 ◽  
Vol 112 (10) ◽  
pp. 3974-3981 ◽  
Author(s):  
Kenneth P. Micklethwaite ◽  
Leighton Clancy ◽  
Upinder Sandher ◽  
Anna M. Hansen ◽  
Emily Blyth ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hemopoietic Stem Cell ◽  
Safety And Efficacy ◽  
Hemopoietic Stem Cell Transplantation

Abstract Cytomegalovirus (CMV) and its therapy continue to contribute to morbidity and mortality in hemopoietic stem cell transplantation (HSCT). Many studies have demonstrated the feasibility of in vitro generation of CMV-specific T cells for adoptive immunotherapy of CMV. Few clinical trials have been performed showing the safety and efficacy of this approach in vivo. In this study, donor-derived, CMV-specific T cells were generated for 12 adult HSCT patients by stimulation with dendritic cells transduced with an adenoviral vector encoding the CMV-pp65 protein. Patients received a prophylactic infusion of T cells after day 28 after HSCT. There were no infusion related adverse events. CMV DNAemia was detected in 4 patients after infusion but was of low level. No patient required CMV-specific pharmacotherapy. Immune reconstitution to CMV was demonstrated by enzyme linked immunospot assay in all recipients with rapid increases in predominantly CMV-pp65 directed immunity in 5. Rates of graft-versus-host disease, infection, and death were not increased compared with expected. These results add to the growing evidence of the safety and efficacy of immunotherapy of CMV in HSCT, supporting its more widespread use. This study was registered at www.anzctr.org.au as #ACTRN12605000213640.

scholarly journals Ex vivo expansion and maturation of peripheral blood CD34+ cells into the myeloid lineage

Blood ◽  
1992 ◽  
Vol 80 (6) ◽  
pp. 1405-1412 ◽  
Author(s):  
DN Haylock ◽  
LB To ◽  
TL Dowse ◽  
CA Juttner ◽  
PJ Simmons
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Ex Vivo ◽  
Fold Increase ◽  
Hematopoietic Stem ◽  
Cell Production ◽  
Peripheral Blood Cd34 ◽  
Cd34 Cells

Abstract Hematopoietic reconstitution (HR) after peripheral blood stem cell transplantation is characterized by a delay of 8 and 12 days for recovery to safe levels of neutrophils and platelets even in patients with the most rapid engraftment. We postulate that a further enhancement in the rate of HR may be achieved by transplanting with an expanded postprogenitor cell population that can provide mature functional cells within days of infusion. In this study we investigated the ability of combinations of hematopoietic growth factors (HGF) to generate nascent granulocyte-macrophage colony-forming units (CFU-GM) in a 7-day suspension culture of peripheral blood CD34+ cells. A combination of 6 HGF, ie, interleukin-1 beta (IL-1), IL-3, IL-6, granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage- CSF (GM-CSF), and stem cell factor (SCF), was identified as the most potent combination of those tested. Subsequently, large volume suspension cultures of CD34+ cells from the same patients using the same 6-factor combination were established and monitored for 21 days. An exponential rate of nucleated cell production (mean 1,324-fold increase) occurred during culture. CFU-GM production paralleled nucleated cell production until day 10, peaked at day 14 (mean 66-fold increase), and was then maintained until day 21. Cells produced in culture were predominantly neutrophil precursors and developed normally as assessed by morphology, immunophenotype, and superoxide generation. This stroma-free, cytokine-driven culture system can achieve a degree of amplification, which suggests the feasibility of ex vivo culture of hematopoietic progenitor cells as an adjunct to hematopoietic stem cell transplantation.

Impact of CD34+ Cell Dose in Peripheral Blood Stem Cell Transplantation from Unrelated Donors for Acute Leukemia Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2065-2065
Author(s):  
R. Marks ◽  
A. Spyridonidis ◽  
G. Ihorst ◽  
H. Bertz ◽  
J. Finke
Keyword(s):  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Peripheral Blood Stem Cell ◽  
Hazard Ratio ◽  
Blood Stem Cell ◽  
Cd34 Cells ◽  
Unrelated Donors ◽  
Blood Stem Cell Transplantation

Abstract In a retrospective study we analysed the impact of the graft composition and several clinical criteria on the outcome of adult AML (n=104) and ALL (n=29) patients undergoing first allogeneic peripheral blood stem cell transplantation from unrelated donors at the University Hospital in Freiburg. The median age of the patients was 52 years (range 18–74) and of the donors 35 years (range 20–58). All patients above 55 years of age received a preparative regimen consisting of fludarabine, melphalan and carmustin (FBM) whereas patients <55y received standard busulphan and cyclophosphamide. Graft-versus-host disease (GvHD) prophylaxis consisted of rabbit ATG (40–60 mg/kg; Fresenius) and cyclosporine combined with MTX in BuCy treated patients or mycophenolate mofetil in patients treated with FBM. There was a trend in the younger donors to mobilize more CD34+ cells than older ones (r=−0.147, p=0.11). A median of 6.1x106 CD34+ cells/kg (range 1.5–17.0) and 3.1 x108CD3+ cells/kg (range 1.0–20.0) were infused. There was no association between the numbers of CD34+ and CD3+ cells in the transplanted peripheral blood stem cell graft (r=0.097, p=0.29). The dose of CD3+ cells infused correlated with the occurence of acute GvHD (aGvHD) II–IV (p=0.02) but not with the development of chronic GvHD (cGvHD). The risk of chronic or acute GvHD was not different between patients receiving more than the 75. percentile (p75) of CD34+ cells (>8x106cells/kg) compared to those receiving < p75 CD34+ cells. In multivariate analysis of all leukemia patients, acute GvHD, chronic GvHD and age were independent prognostic factors in OS. But in older patients (>50y), higher (>p75) CD34+ cell dose in the PB graft was associated with a trend to better OS at one year both in univariate (hazard ratio 0.508, 95% CI, 0.231 to 1.114, p=0.0908) and multivariate analysis (hazard ratio 0.521, 95% CI, 0.235 to 1.155, p=0.1084), while CD34+ cell dose did not have any impact in the OS of younger (<50y) patients. Univariate analysis of 104 patients with myeloid diseases revealed that patients sex, disease status at HCT, HLA match, or the preparative regimen (BuCy versus FBM) did not have any recognizable effect on OS, resulting in a 1-year survival of 65–70% in all analysed subgroups. Multivariate analysis showed that occurence of aGvHD was associated with significantly increased mortality in younger patients (BuCy treated, hazard ratio 3.287, 95% CI 1.126 to 9.596, p=0.03) as compared to FBM patients (hazard ratio 1.52, 95% CI 0.552 to 4.188, p=0.41). Relapse rates were lower in patients who experienced cGvHD as compared to those without cGvHD resulting in a significantly better OS (hazard ratio 0.276, 95%CI, 0.102 to 0.750, p=0.011). In conclusion the data show that in leukemia patients treated with peripheral blood stem cell transplantation from unrelated donors, older patients receiving grafts with higher CD34+ cell counts show a trend to better survival, while in the same group FBM conditioning does not increase the treatment related mortality.

Pegfilgrastrim Compared with Filgrastim after Autologous Hematopoietic Peripheral Blood Stem Cell Transplantation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5287-5287
Author(s):  
Gaetan Vanstraelen ◽  
Pascale Frère ◽  
Marie-Christine Ngirabacu ◽  
Evelyne Willems ◽  
Georges Fillet ◽  
...  
Keyword(s):  
Stem Cell ◽  
Clinical Outcome ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Peripheral Blood Stem Cell ◽  
Blood Stem Cell ◽  
Cell Counts ◽  
Cd34 Cells ◽  
Blood Stem Cell Transplantation

Abstract In order to assess the effect of Pegfilgrastim on the duration of neutropenia and clinical outcome of patients after autologous peripheral blood stem cell transplantation (PBSCT), we compared 20 consecutive patients with lymphoma or multiple myeloma receiving a single 6 mg dose of Pegfilgrastim on day 1 posttransplant to a historical control group of 60 patients receiving daily Filgrastim 5 μg/kg starting on day 1 posttransplant. There were 54 M and 26 F, 30 patients with lymphoma and 50 with myeloma, 26 in CR and 54 not in CR. Mean age was 55±10 yrs and 25 had already received a previous autologous transplant. The two groups were matched for disease and disease status, transplant number, age and sex. Cell dose infused tended to be higher in the Pegfilgrastim group (7.16±3.82 vs 10.03±6.25 x106 CD34+ cells/kg, p=0.0575). There were no differences (p&gt;0.05) in time to 0.5 (8 vs 9 days) or 1 (9 vs 9 days) x109/L neutrophils; to 1 % reticulocytes (13 vs 15 days) or 9 (12 vs 14 days) or 10 (30 vs 25 days) g/dL Hb; to 20 (9 vs 9 days) or 100 (20 vs 31 days) x 109/L platelets. The number of days with fever (2.7±2.3 vs 2.3±2.4 days), incidence of infections (all infections; bacteremia; bacterial, fungal or viral infections; FUO), duration of antibiotic therapy (8.7±5.9 vs 8.4±5.9 days), RBC (1.1±1.6 vs 0.9±1.6) and platelet (1.0±1.7 vs 1.2±1.8) transfusions, and time to hospital discharge (14.5±5.3 vs 15.4±5.8 days) were similar in the Pegfilgrastim compared to the Filgrastim group. However, after initial hematopoietic recovery, several differences between the groups became apparent, with the group always showing higher counts compared to the Filgrastim group (p values &lt;0.05 to &lt;0.001). Neutrophils remained significantly higher in the Pegfilgrastim group between days 14–30, lymphocytes between days 56–90, monocytes between days 21–24, reticulocytes between days 17–42 and platelets between days 35–90, respectively. These differences had no impact on clinical outcome after day 30 due to the low incidence of infectious events after engraftment. We conclude that Pegfilgrastim administrated on day 1 posttransplant facilitates early hematopoietic reconstitution comparable to daily Filgrastim. However, despite a trend towards fewer CD34+ cells transplanted, the Pegfilgrastim group enjoyed higher trilineage cell counts for some time after initial engraftment. This should be further tested in prospective randomized trials.

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