-
1.
Safety of Intraoperative Cell Salvage in Cancer Surgery: An Updated Meta-Analysis of the Current Literature
Frietsch T, Steinbicker AU, Horn A, Metz M, Dietrich G, Weigand MA, Waters JH, Fischer D
Transfusion medicine and hemotherapy : offizielles Organ der Deutschen Gesellschaft fur Transfusionsmedizin und Immunhamatologie. 2022;49(3):143-157
Abstract
BACKGROUND Allogeneic blood transfusions in oncologic surgery are associated with increased recurrence and mortality. Adverse effects on outcome could be reduced or avoided by using intraoperative autologous blood cell salvage (IOCS). However, there are concerns regarding the safety of the autologous IOCS blood. Previous meta-analyses from 2012 and 2020 did not identify increased risk of cancer recurrence after using autologous IOCS blood. The objective of this review was to reassess a greater number of IOCS-treated patients to present an updated and more robust analysis of the current literature. METHODS This systematic review includes full-text articles listed in PubMed, Cochrane, Cochrane Reviews, and Web of Science. We analyzed publications that discussed cell salvage or autotransfusion combined with the following outcomes: cancer recurrence, mortality, survival, allogeneic transfusion rate and requirements, length of hospital stay (LOS). To rate the strength of evidence, a Grading of Recommendations Assessment, Development and Evaluation (GRADE) of the underlying evidence was applied. RESULTS In the updated meta-analysis, 7 further observational studies were added to the original 27 observational studies included in the former 2020 analysis. Studies compared either unfiltered (n = 2,311) or filtered (n = 850) IOCS (total n = 3,161) versus non-IOCS use (n = 5,342). Control patients were either treated with autologous predonated blood (n = 484), with allogeneic transfusion (n = 4,113), or did not receive a blood transfusion (n = 745). However, the current literature still contains only observational studies on these topics, and the strength of evidence remains low. The risk of cancer recurrence was reduced in recipients of autologous salvaged blood with or without LDF (odds ratio [OR] 0.76, 95% confidence interval [CI]: 0.64-0.90) compared to nontransfused patients or patients with allogeneic transfusion. There was no difference in mortality (OR 0.95, 95% CI: 0.71-1.27) and LOS (mean difference -0.07 days, 95% CI: -0.63 to 0.48) between patients treated with IOCS blood or those in whom IOCS was not used. Due to high heterogeneity, transfusion rates or volumes could not be analyzed. CONCLUSION Randomized controlled trials comparing mortality and cancer recurrence rate of IOCS with or without LDF filtration versus allogeneic blood transfusion were not found. Outcome was similar or better in patients receiving IOCS during cancer surgery compared to patients with allogeneic blood transfusion or nontransfused patients.
-
2.
Should Cell Salvage be Used in Liver Resection and Transplantation? A Systematic Review and Meta-analysis
Rajendran L, Lenet T, Shorr R, Abou Khalil J, Bertens KA, Balaa FK, Martel G
Annals of surgery. 2022
-
-
-
Free full text
-
-
Editor's Choice
Abstract
OBJECTIVE To evaluate the effect of intraoperative blood salvage and autotransfusion (IBSA) use on red blood cell (RBC) transfusion and postoperative outcomes in liver surgery. BACKGROUND Intraoperative RBC transfusions are common in liver surgery and associated with increased morbidity. IBSA can be utilized to minimize allogeneic transfusion. A theoretical risk of cancer dissemination has limited IBSA adoption in oncologic surgery. METHODS Electronic databases were searched from inception until May 2021. All studies comparing IBSA use to control in liver surgery were included. Screening, data extraction, and risk of bias assessment were conducted independently, in duplicate. The primary outcome was intraoperative allogeneic RBC transfusion (proportion of patients and volume of blood transfused). Core secondary outcomes included: overall survival (OS) and disease-free survival (DFS), transfusion-related complications, length of hospital stay, and hospitalization costs. Data from transplant and resection studies were analyzed separately. Random effects models were used for meta-analysis. RESULTS Twenty-one observational studies were included (16 transplant, 5 resection, n=3,433 patients). Seventeen studies incorporated oncologic indications. In transplant, IBSA was associated with decreased allogeneic RBC transfusion (MD -1.81, 95% CI[-3.22, -0.40], P=0.01, I2=86%, very-low certainty). Few resection studies reported on transfusion for meta-analysis. No significant difference existed in OS or DFS in liver transplant (HR=1.12[0.75, 1.68], P=0.59, I2=0%; HR=0.93[0.57, 1.48], P=0.75, I2=0%) and liver resection (HR=0.69[0.45, 1.05], P=0.08, I2=0%; HR=0.93[0.59, 1.45], P=0.74, I2=0%). CONCLUSION IBSA may reduce intraoperative allogeneic RBC transfusion without compromising oncologic outcomes. The current evidence base is limited in size and quality, and high-quality randomized controlled trials are needed.
PICO Summary
Population
Patients undergoing oncologic and non-oncologic liver surgery (either resection or transplantation), (21 studies, n= 3,433).
Intervention
Any intraoperative blood salvage and autotransfusion (IBSA) device.
Comparison
No IBSA use.
Outcome
Data from transplant and resection studies were analyzed separately. Despite significant heterogeneity, most studies reported lower rates and volumes of intraoperative allogeneic red blood cell transfusion in patients undergoing IBSA. In transplant, IBSA was associated with decreased allogeneic red blood cell transfusion (mean difference: -1.81, very-low certainty). Few resection studies reported on transfusion for meta-analysis. There was no significant difference in overall survival or disease-free survival in liver transplant and liver resection.
-
3.
Clinical prognosis of intraoperative blood salvage autotransfusion in liver transplantation for hepatocellular carcinoma: A systematic review and meta-analysis
Wang Z, Li S, Jia Y, Liu M, Yang K, Sui M, Liu D, Liang K
Frontiers in oncology. 2022;12:985281
Abstract
BACKGROUND Intraoperative blood salvage autotransfusion(IBSA) has been widely used in a variety of surgeries, but the use of IBSA in hepatocellular carcinoma (HCC) patients undergoing liver transplantation (LT) is controversial. Numerous studies have reported that IBSA used during LT for HCC is not associated with adverse oncologic outcomes. This systematic review and meta-analysis aims to estimate the clinical prognosis of IBSA for patients with H+CC undergoing LT. METHODS MEDLINE, Embase, Web of Science, and Cochrane Library were searched for articles describing IBSA in HCC patients undergoing LT from the date of inception until May 1, 2022, and a meta-analysis was performed. Study heterogeneity was assessed by I(2) test. Publication bias was evaluated by funnel plots, Egger's and Begg's test. RESULTS 12 studies enrolling a total of 2253 cases (1374 IBSA and 879 non-IBSA cases) are included in this meta-analysis. The recurrence rate(RR) at 5-year(OR=0.75; 95%CI, 0.59-0.95; P=0.02) and 7-year(OR=0.65; 95%CI, 0.55-0.97; P=0.03) in the IBSA group is slightly lower than non-IBSA group. There are no significant differences in the 1-year RR(OR=0.77; 95% CI, 0.56-1.06; P=0.10), 3-years RR (OR=0.79; 95% CI, 0.62-1.01; P=0.06),1-year overall survival outcome(OS) (OR=0.90; 95% CI, 0.63-1.28; P=0.57), 3-year OS(OR=1.16; 95% CI, 0.83-1.62; P=0.38), 5-year OS(OR=1.04; 95% CI, 0.76-1.40; P=0.82),1-year disease-free survival rate(DFS) (OR=0.80; 95%CI, 0.49-1.30; P=0.36), 3-year DFS(OR=0.99; 95%CI, 0.64-1.55; P=0.98), and 5-year DFS(OR=0.88; 95%CI, 0.60-1.28; P=0.50). Subgroup analysis shows a difference in the use of leukocyte depletion filters group of 5-year RR(OR=0.73; 95%CI, 0.55-0.96; P=0.03). No significant differences are found in other subgroups. CONCLUSIONS IBSA provides comparable survival outcomes relative to allogeneic blood transfusion and does not increase the tumor recurrence for HCC patients after LT. SYSTEMATIC REVIEW REGISTRATION https://www.crd.york.ac.uk/prospero/, identifier CRD42022295479.
-
4.
Effect of Autotransfusion in HCC Surgery on Survival and Recurrence: A Systematic Review and Meta-Analysis
Murtha-Lemekhova A, Fuchs J, Ritscher E, Hoffmann K
Cancers. 2022;14(19)
-
-
-
Free full text
-
Editor's Choice
Abstract
BACKGROUND The chronic blood shortage has forced clinicians to seek alternatives to allogeneic blood transfusions during surgery. Due to anatomic uniqueness resulting in a vast vasculature, liver surgery can lead to significant blood loss, and an estimated 30% of patients require blood transfusions in major hepatectomy. Allogeneic transfusion harbors the risk of an immunologic reaction. However, the hesitation to reinfuse a patient's own blood during cancer surgery is reinforced by the potentiality of reintroducing and disseminating tumor cells into an individual undergoing curative treatment. Two methods of autotransfusions are common: autotransfusion after preoperative blood donation and intraoperative blood salvage (IBS). We aim to investigate the effect of autotransfusion on recurrence and survival rates of patients undergoing surgery for HCC. METHODS The protocol for this meta-analysis was registered at PROSPERO prior to data extraction. MEDLINE, Web of Science and Cochrane Library were searched for publications on liver surgery and blood salvage (autologous transfusion or intraoperative blood salvage). Comparative studies were included. Outcomes focused on long-term oncologic status and mortality. Hazard ratios (HR) estimated outcomes with a fixed-effects model. Risk of bias was assessed using ROBINS-I, and certainty of evidence was evaluated with GRADE. Separate analyses were performed for liver transplantation and hepatectomies. RESULTS Fifteen studies were included in the analysis (nine on transplantation and six on hepatectomies), and they comprised 2052 patients. Overall survival was comparable between patients who received intraoperative blood salvage (IBS) or not for liver transplantation (HR 1.13, 95% CI [0.89, 1.42] p = 0.31). Disease-free survival also was comparable (HR 0.97, 95% CI [0.76, 1.24], p = 0.83). Autotransfusion after prior donation was predominantly used in hepatectomy. Patients who received autotransfusion had a significantly better overall survival than the control (HR 0.71, 95% CI [0.58, 0.88], p = 0.002). Disease-free survival was also significantly higher in patients with autotransfusion (HR 0.88, 95% CI [0.80, 0.96], p = 0.005). Although overall, the certainty of evidence is low and included studies exhibited methodological heterogeneity, the heterogeneity of outcomes was low to moderate. CONCLUSION Autotransfusion, including intraoperative blood salvage, does not adversely affect the overall or disease-free survival of patients with HCC undergoing resection or transplantation. The results of this meta-analysis justify a randomized-controlled trial regarding the feasibility and potential benefits of autotransfusion in HCC surgery.
PICO Summary
Population
Patients undergoing liver surgery for hepatocellular carcinoma (15 studies, n= 2,052).
Intervention
Autologous transfusion (including intraoperative blood salvage).
Comparison
No autologous transfusion.
Outcome
Overall survival was comparable between patients who received intraoperative blood salvage or not for liver transplantation (Hazard ratio (HR): 1.13, 95% CI: 0.89-1.42). Disease-free survival also was comparable (HR: 0.97, 95% CI: 0.76-1.24). Autotransfusion after prior donation was predominantly used in hepatectomy. Patients who received autotransfusion had a significantly better overall survival than the control (HR, 0.71, 95% CI: 0.58-0.88). Disease-free survival was also significantly higher in patients with autotransfusion (HR, 0.88, 95% CI: 0.80-0.96). Although overall, the certainty of evidence is low and included studies exhibited methodological heterogeneity, the heterogeneity of outcomes was low to moderate.
-
5.
Efficacy of intraoperative cell salvage in spine surgery: a meta-analysis
Cheriyan J, Cheriyan T, Dua A, Goldstein JA, Errico TJ, Kumar V
J Neurosurg Spine. 2020;:1-9
-
-
-
Full text
-
Editor's Choice
Abstract
OBJECTIVE Intraoperative cell salvage systems, or cell savers, are widely used for the management of blood loss in patients undergoing spine surgery. However, recent studies report conflicting evidence of their efficacy. The purpose of the meta-analysis was to investigate the efficacy of cell savers in reducing blood transfusion requirements in patients undergoing spine surgery. METHODS Both retrospective and prospective studies that investigated the efficacy of cell savers in reducing transfusion requirements in spine surgery patients when compared with control patients were identified from MEDLINE, Embase, Cochrane Collaboration Library, Google Scholar, and Scopus databases. Outcome data extracted included number of patients receiving allogenic transfusions (transfusion rate); units of allogenic transfusions; postoperative hemoglobin; costs; operative time; and complications. RevMan 5 software was used to perform statistical analyses. A random-effects model was used to calculate pooled odds ratios (with 95% CIs) and weighted mean differences (WMDs [95% CI]) for dichotomous and continuous variables, respectively. RESULTS Eighteen studies with 2815 patients in total were included in the meta-analysis. During spine surgery, the use of intraoperative cell salvage did not reduce the intraoperative (OR 0.66 [95% CI 0.30, 1.41]), postoperative (OR -0.57 [95% CI 0.20, 1.59]), or total transfusion (OR 0.92 [95% CI 0.43, 1.98]) rate. There was a reduction in the number of allogenic units transfused intraoperatively by a mean of 0.81 (95% CI -1.15, -0.48). However, there were no differences in the number of units transfused postoperatively (WMD -0.02 [95% CI -0.41, 0.38]) or the total units transfused (WMD 0.08 [95% CI -1.06, 1.22]). There were also no differences in operative time (WMD 19.36 [95% CI -2.43, 42.15]) or complications reported (OR 0.79 [95% CI 0.46, 1.37]) between groups. A difference in postoperative hemoglobin (WMD 0.54 [95% CI 0.11, 0.98]) between both groups was observed. CONCLUSIONS Cell saver is efficacious at reducing intraoperative allogenic units transfused. There is no difference in transfusion rates, postoperative units transfused, and the total number of units transfused. Further cost analysis studies are necessary to evaluate the cost-effectiveness of this method of blood conservation. CLASSIFICATION OF EVIDENCE Type of question: therapeutic; study design: meta-analysis; strength of recommendation: low.
PICO Summary
Population
Patients undergoing spine surgery (18 studies, n=2815).
Intervention
Use of intraoperative cell salvage.
Comparison
Control.
Outcome
The use of intraoperative cell salvage did not reduce the intraoperative, or total transfusion rate. There was a reduction in the number of allogenic units transfused intraoperatively by a mean of 0.81. However, there were no differences in the number of units transfused postoperatively or the total units transfused. There were also no differences in operative time or complications reported between groups. A difference in postoperative hemoglobin between both groups was observed.
-
6.
[Safety of cell salvage in tumor surgery : Systematic review with meta-analysis]
Frietsch T, Steinbicker AU, Hackbusch M, Nguyen XD, Dietrich G
Anaesthesist. 2020
Abstract
BACKGROUND Allogeneic blood transfusion is avoidable in many oncological interventions by the use of cell salvage or mechanical autotransfusion (MAT). As irradiation is elaborate and expensive, the safety of leucocyte depletion filters (LDF) for autologous blood from the surgical field might be a more acceptable alternative for the prevention of cancer recurrences. A previous meta-analysis could not identify an increased risk of cancer recurrence. The aim of this review article is to provide an update of a previous meta-analysis from 2012 as well as a safety analysis of cell salvage with LDF due to the improved data situation. MATERIAL AND METHODS This systematic review included all studies in PubMed, Cochrane, Cochrane Reviews and Web of Science on cell salvage or autotransfusion combined with outcomes, e.g. cancer recurrence, mortality, survival, blood transfusion, length of hospital stay (LOS) after the use of MAT without irradiation and with or without LDF. The grades of recommendations (GRADE) assessment of underlying evidence was applied. RESULTS A total of seven new observational studies and seven meta-analyses were found that compared unfiltered or filtered cell salvage with autologous predeposition, allogeneic transfusion or without any transfusion. No randomized controlled trials have been completed. A total of 27 observational and cohort studies were included in a meta-analysis. The evidence level was low. The risk of cancer recurrence in recipients of autologous salvaged blood with or without LDF was reduced (odds ratio, OR 0.71, 95% confidence interval, CI 0.58-0.86) as compared to non-transfused subjects, allogeneic or predeposited autologous transfusion. The transfusion rate could not be assessed due to the substantial selection bias and large heterogeneity. Cell salvage does not change mortality and LOS. Leucocyte depletion studies reported a removal rate of cancer cells in the range of 99.6-99.9%. CONCLUSION Randomized controlled trials on a comparison of MAT and allogeneic blood transfusion as well as LDF and irradiation would be desirable but are not available. From observational trials and more than 6300 subjects and various tumors, cell salvage in cancer surgery with or without LDF appears to be sufficiently safe. The efficacy of leucocyte depletion of autologous salvaged blood is equivalent to irradiation. Unavailability of radiation is not a contraindication for cell salvage use in cancer surgery. By usage of leucocyte depleted salvaged autologous blood, the risks of allogeneic transfusion can be avoided.
-
7.
Meta-analysis comparing autologous blood-derived products (including platelet-rich plasma) injection versus placebo in patients with achilles tendinopathy
Lin M T, Chiang C F, Wu C H, Hsu H H, Tu Y K
Arthroscopy : the Journal of Arthroscopic & Related Surgery : Official Publication of the Arthroscopy Association of North America and the International Arthroscopy Association. 2018;34((6):):1966-1975 e5
Abstract
PURPOSE To compare the effectiveness of autologous blood-derived products (ABP) injection with that of placebo (sham injection, no injection, or physiotherapy alone) in patients with Achilles tendinopathy. METHODS Electronic databases, including PubMed, Scopus, EMBASE, and Cochrane Library were searched up to June 2017. All published or unpublished randomized controlled trials (RCTs) were included. Two independent raters assessed the risk of bias of RCTs with the Cochrane Risk of Bias Tool. The primary outcome was Victorian Institute of Sports Assessment-Achilles (VISA-A) score. Weighted mean differences (WMDs) were used for random effect meta-analysis. RESULTS Seven RCTs were enrolled in meta-analysis. The ABP injection and placebo revealed equal effectiveness in VISA-A score improvement at 4 to 6 weeks (short term, WMD 2.29, 95% confidence interval [CI]: -1.69, 6.27), 12 weeks (medium term, WMD 2.63, 95% CI: -1.72, 6.98), 24 weeks (long term, WMD 4.61, 95% CI: -1.25, 10.47), and 48 weeks (very long term, WMD 4.16, 95% CI: -6.82, 15.14). In meta-regression, there was no association between change in VISA-A score and duration of symptoms at 4 to 6 weeks (short term), 12 weeks (medium term), and 24 weeks (long term). CONCLUSIONS This meta-analysis revealed that ABP injection was not more effective than placebo (sham injection, no injection, or physiotherapy alone) in Achilles tendinopathy and that no association was found between therapeutic effects and duration of symptoms. LEVEL OF EVIDENCE Level I, meta-analysis of Level I studies.
-
8.
Evaluation of Autologous Blood in Pterygium Surgery With Conjunctival Autograft
Zeng W, Dai H, Luo H
Cornea. 2018;38((2):):210-216.
Abstract
PURPOSE Autologous blood has been used exploratively with conjunctival autograft in pterygium surgery. However, it is controversial whether autologous blood performed better than other fixation methods, including fibrin glue and sutures. This meta-analysis was conducted to evaluate the effectiveness of using autologous blood in pterygium surgery with conjunctival autograft. METHODS The study was conducted according to the PRISMA guidelines. The MEDLINE, Cochrane library, and Embase databases were systematically searched from their establishment until April 1, 2018. Randomized controlled trials comparing autologous blood with fibrin glue/suture in pterygium surgery with conjunctival autograft were included. The methodological quality of the included studies was assessed using the Cochrane risk of bias tool. Outcome measurements were recurrence, graft displacement, graft retraction, and surgical duration. Review Manager 5.3 (Cochrane Community, Cochrane Collaboration, London, UK) was used to perform the statistical analysis. When I < 50%, statistical heterogeneity was considered acceptable, and a fixed-effects model was adopted; alternatively, the random-effects model was used. RESULTS Seven randomized controlled trials including 516 patients were finally included in the meta-analysis. Four studies with 379 patients compared autologous blood and fibrin glue. Autologous blood was inferior to fibrin glue with respect to surgical duration, graft retraction, and graft displacement. However, there was no statistical difference between the 2 groups in terms of the recurrence rate. Four studies with 152 patients compared autologous blood and traditional suturing. Autologous blood was superior to sutures in terms of surgical duration and inferior to sutures in terms of graft retraction. No difference was detected in terms of graft displacement and recurrence rate. CONCLUSIONS In conclusion, autologous blood is an appropriate method for graft fixation in pterygium surgery. Current research suggests that autologous blood derivatives may be a promising approach after pterygium excision. However, this requires further confirmation.
-
9.
Cell salvage used in scoliosis surgery: is it really effective?
Liu JM, Fu BQ, Chen WZ, Chen JW, Huang SH, Liu ZL
World Neurosurgery. 2017;101:568-576
Abstract
BACKGROUND Scoliosis surgery is usually associated with large volume of intraoperative blood loss and cell salvage is commonly used to filter and retranfusion autologous blood to patients. However, the efficacy of using cell salvage in scoliosis surgery is still controversial. OBJECTIVE The purpose of this study is to make clear that intraoperative use of cell salvage is effective to decrease the volume of perioperative allogenic blood transfusion in scoliosis surgery. METHOD A meta-analysis was conducted to identify the relevant studies from PubMed, EMbase, Medline, Cochrane library and Google scholar till July 2016. All randomized trials and controlled clinical studies comparing the clinical outcomes of using cell salvage versus noncell salvage in scoliosis surgery were retrieved for the meta-analysis. The data were analyzed by RevMan 5.3. RESULTS A total of 7 studies with 562 patients were included in this meta-analysis. Based on the analysis, the volumes of perioperative and postoperative allogenic RBCs transfusion in cell salvage group were significantly less than those in control group (P = 0.04 and P = 0.01). However, no significant difference was detected in the amount of intraoperative allogenic RBCs transfusion and the risk of patients needing allogenic blood transfusion between the two groups (P = 0.14 and P = 0.61). Both the hemoglobin and hematocrit levels on the first day after surgery were significantly higher in cell salvage group than those in control group (P = 0.002 and P < 0.001). But no significant differences were noted in neither hemoglobin nor hematocirt level at the time of discharge between the two groups (P = 0.76 and P = 0.32). One of the included study reported the number of patients with complications related to transfusion in the two groups, which was not significant different (P = 0.507). CONCLUSIONS Cell salvage significantly reduced the volumes of perioperative and postoperative allogenic RBCs transfusion in scoliosis surgery and increased the hemoglobin and hematocrit levels on the first day postoperatively. In addition, it seemed not increase the rate of transfusion complications during the surgery.
-
10.
Review: Autologous blood transfusion drainage compared with no drainage in total knee arthroplasty: a meta-analysis and systematic review
Hong KH, Pan JK, Xie H, Guo D, Yang WY, Su HT, Xu SC, Liu J
Pakistan Journal of Pharmaceutical Sciences.. 2017;30((6)):2321-2327.
Abstract
This is the first meta-analysis to assess the clinical efficiency, safety and potential advantages regarding the use of ABT drains compared with no drainage which is controversial in total knee arthroplasty (TKA).A comprehensive literature search was carried out in March 2015 using the PubMed, Embase, and Cochrane Library databases. A metaanalysis was carried out on two retrospective comparative studies (RCSs) and five randomized controlled trials (RCTs). The number of patients receiving homologous blood transfusion was the primary outcome of the meta-analysis; the secondary outcome measure was the mean drop in Hb level in comparison to the mean pre-operative HB level, the range of flexion of the knee joint, and infections of the wound after surgery. A total of 868 patients, who were included in two retrospective studies and five RCTs, were distributed into subgroups for the meta-analysis. This pooled data showed no benefit of ABT drainage compared no drainage in the homologous blood transfusion rate (13.05% and 16.91%, OR:0.73[0.47,1.13], Z=1.41, P=0.016; and 3.49% and 6.54%, OR: 0.50[0.12,2.01], Z=0.98,P=0.033,respectively in subgroups), Hb drop (Weight mean differences (WMD): 0.20[-0.28,0.68], Z=0.82, P=0.41; WMD:0.16[-0.41,0.55], Z=0.93, P=0.35, respectively), range of flexion of the knee joint (WMD:-0.82 [-3.35,1.70], Z =0.64,P=0.52)and wound infection (OR:0.25[0.61,10.20]; Z =1.28, p=0.2) after TKA surgery. Our findings do not recommend the routine use of postoperative ABT drainage in total knee arthroplasty. Well-designed RCTs with large sample sizes, longer term measures and extensive follow-up period should be performed in the future to update the findings of this study.
