1.Analysis of abnormal ALT in blood donors in five Zang autonomous prefectures of Qinghai Province, China: characteristics and screening strategies
Yingnan DANG ; ; Rong TANG ; Liqin HUANG ; Hailin WU ; Tingting CHEN ; Shengju LI ; Yanli SUN ; Xin ZHENG ; Yanxia LI ; Xianlin YE ; Jinfeng ZENG
Chinese Journal of Blood Transfusion 2025;38(4):502-507
[Objective] To investigate the factors associated with alanine aminotransferase (ALT) abnormalities in multi-ethnic blood donors across five Zang autonomous prefectures in the plateau regions of Qinghai Province, and to provide evidence for ensuring blood safety and formulating screening strategies. [Methods] A retrospective analysis was performed on the ALT abnormal test results of blood donors in the Zang autonomous prefectures of Qinghai from 2022 to 2024. The correlations between ALT levels and factors including gender, age, altitude, and infectious markers were investigated. [Results] The overall ALT unqualified rate among blood donors in this region was 9.01%. Significant differences in ALT levels were observed across genders and age groups (P<0.05). Variations in ALT abnormality rates were also noted among different plateau regions (P<0.05). Overall, ALT values exhibited an increasing trend with rising altitude. The average ALT unqualified rates were 11.19% in Zang donors, 7.96% in Han donors, and 4.79% in donors from other ethnic groups (P<0.05). No statistically significant association was observed between ALT abnormality and the presence of HBV/HCV infectious markers (P>0.05). [Conclusion] In the plateau areas of Qinghai, multi-ethnic blood donors have a relatively high ALT levels and ALT unqualified rates, showing distinct regional characteristics. ALT elevation in voluntary blood donors is related to non-pathological factors such as gender, age, and dietary habits, but not to infectious indicators.
2.Serological and molecular biological characteristics of hepatitis B vaccinated donors with asymptomatic chronical infection in Shenzhen
Xianlin YE ; Tong LI ; Ruonan WANG ; Ran LI ; Heng LIU ; Jinfeng ZENG
Chinese Journal of Blood Transfusion 2024;37(1):26-31
【Objective】 To investigate asymptomatic infection of hepatitis B virus(HBV) among hepatitis B vaccinated donors in Shenzhen, and analyze its serological and molecular characteristics. 【Methods】 The HBsAg ELISA positive blood samples of blood donors born after 1992 were collected. HBsAg, anti-HBs, HBeAg, anti-HBe and anti-HBc were further detected by Roche electrochemiluminescence immunoassay (ECL). BCP/PC and S regions were amplified by Nested-PCRs, HBV DNA quantification were adopted by qPCR simultaneously, and the sequences were also analyzed. 【Results】 A total of 46 632 blood samples of donors(31 612 males and 15 020 females) from December 2020 to January 2022 collected, and 99 samples with HBsAg ELISA positive were screened out. After tested by ECL, Nested-PCRs, and real-time fluorescence PCR, 61 were confirmed HBsAg positive, with the positive rate at 0.13% (61/46 632), including 49 males (0.16%, 49/31 612) and 12 females (0.08%, 12/15 020). The HBsAg positive rate of males was higher than that of females (P<0.05). 50 out of 61 sequences for S region were obtained. By phylogenetic analysis, there were 46 cases of type B (92%, 46/50, 38 males and 8 females), 4 cases of type C (8%, 4/50, 3 males and 1 female). The high frequency mutations observed in S region were N40S (8/46,17.39%), G44E (7/46,15.22%), Q129H/R(6/46,13.04%), Y161F/S(7/46, 15.22%), V179A(4/46,8.70%), S53L(2/4,50%), C69T(2/4,50%) and I126S/T(2/4,50%), including the immune escape mutations Q129R and T/I126A/N/S/T. 【Conclusion】 Hepatitis B vaccination can significantly reduce the positive rate of HBsAg and increase the safety of blood transfusion. The high frequency immune escape mutations have become a potential risk of blood safety, and need to be further explored.
3.Perioperative outcomes of single-lumen versus double-lumen endotracheal tubes in totally thoracoscopic cardiac surgery: A retrospective cohort study
Zhenzhong WANG ; Shuo XIAO ; Dou FANG ; Yuxin LI ; Jinfeng WEI ; Yingxian YE ; Jianjun ZHANG ; Xiaogang GUO ; Qingshi ZENG ; Huanlei HUANG
Chinese Journal of Clinical Thoracic and Cardiovascular Surgery 2024;31(11):1606-1612
Objective To investigate the relationship between two-lung ventilation (TLV) with single-lumen endotracheal tube (SLT), one-lung ventilation (OLV) with double-lumen endotracheal tube (DLT) and postoperative pulmonary complications (PPCs) after total thoracoscopic cardiac surgery. Methods The clinical data of patients who underwent totally thoracoscopic cardiac surgeries in the Guangdong Provincial People’s Hospital from October 2019 to October 2021 were retrospectively analyzed. The patients were divided into 2 group according to the type of endotracheal tube, including a SLT group and a DLT group. Baseline data, surgical variables and PPCs were compared. The influencing factors of PPCs in the two groups were analyzed by binary logistic regression analysis. Results Finally 349 patients were enrolled, including 180 males and 169 females with an average age of (50.0±14.8) years. There were 219 patients in the SLT group and 130 patients in the DLT group. There was no statistical difference in baseline data, surgical variables or PPCs between the two groups (P>0.05). Binary logistic regression analysis showed that PPCs were related to body mass index in the SLT group (OR=0.778, 95%CI 0.637 to 0.951, P=0.014) and preoperative smoking history in the DLT group (OR=0.058, 95%CI 0.004 to 0.903, P=0.042). Conclusion For the patients who undergo totally thoracoscopic cardiac surgery, TLV with SLT and OLV with DLT show no significant association with PPCs. At the same time, PPCs are associated with body mass index in the SLT group, while associated with preoperative smoking history in the DLT group.
4.Chinese expert consensus on blood support mode and blood transfusion strategies for emergency treatment of severe trauma patients (version 2024)
Yao LU ; Yang LI ; Leiying ZHANG ; Hao TANG ; Huidan JING ; Yaoli WANG ; Xiangzhi JIA ; Li BA ; Maohong BIAN ; Dan CAI ; Hui CAI ; Xiaohong CAI ; Zhanshan ZHA ; Bingyu CHEN ; Daqing CHEN ; Feng CHEN ; Guoan CHEN ; Haiming CHEN ; Jing CHEN ; Min CHEN ; Qing CHEN ; Shu CHEN ; Xi CHEN ; Jinfeng CHENG ; Xiaoling CHU ; Hongwang CUI ; Xin CUI ; Zhen DA ; Ying DAI ; Surong DENG ; Weiqun DONG ; Weimin FAN ; Ke FENG ; Danhui FU ; Yongshui FU ; Qi FU ; Xuemei FU ; Jia GAN ; Xinyu GAN ; Wei GAO ; Huaizheng GONG ; Rong GUI ; Geng GUO ; Ning HAN ; Yiwen HAO ; Wubing HE ; Qiang HONG ; Ruiqin HOU ; Wei HOU ; Jie HU ; Peiyang HU ; Xi HU ; Xiaoyu HU ; Guangbin HUANG ; Jie HUANG ; Xiangyan HUANG ; Yuanshuai HUANG ; Shouyong HUN ; Xuebing JIANG ; Ping JIN ; Dong LAI ; Aiping LE ; Hongmei LI ; Bijuan LI ; Cuiying LI ; Daihong LI ; Haihong LI ; He LI ; Hui LI ; Jianping LI ; Ning LI ; Xiying LI ; Xiangmin LI ; Xiaofei LI ; Xiaojuan LI ; Zhiqiang LI ; Zhongjun LI ; Zunyan LI ; Huaqin LIANG ; Xiaohua LIANG ; Dongfa LIAO ; Qun LIAO ; Yan LIAO ; Jiajin LIN ; Chunxia LIU ; Fenghua LIU ; Peixian LIU ; Tiemei LIU ; Xiaoxin LIU ; Zhiwei LIU ; Zhongdi LIU ; Hua LU ; Jianfeng LUAN ; Jianjun LUO ; Qun LUO ; Dingfeng LYU ; Qi LYU ; Xianping LYU ; Aijun MA ; Liqiang MA ; Shuxuan MA ; Xainjun MA ; Xiaogang MA ; Xiaoli MA ; Guoqing MAO ; Shijie MU ; Shaolin NIE ; Shujuan OUYANG ; Xilin OUYANG ; Chunqiu PAN ; Jian PAN ; Xiaohua PAN ; Lei PENG ; Tao PENG ; Baohua QIAN ; Shu QIAO ; Li QIN ; Ying REN ; Zhaoqi REN ; Ruiming RONG ; Changshan SU ; Mingwei SUN ; Wenwu SUN ; Zhenwei SUN ; Haiping TANG ; Xiaofeng TANG ; Changjiu TANG ; Cuihua TAO ; Zhibin TIAN ; Juan WANG ; Baoyan WANG ; Chunyan WANG ; Gefei WANG ; Haiyan WANG ; Hongjie WANG ; Peng WANG ; Pengli WANG ; Qiushi WANG ; Xiaoning WANG ; Xinhua WANG ; Xuefeng WANG ; Yong WANG ; Yongjun WANG ; Yuanjie WANG ; Zhihua WANG ; Shaojun WEI ; Yaming WEI ; Jianbo WEN ; Jun WEN ; Jiang WU ; Jufeng WU ; Aijun XIA ; Fei XIA ; Rong XIA ; Jue XIE ; Yanchao XING ; Yan XIONG ; Feng XU ; Yongzhu XU ; Yongan XU ; Yonghe YAN ; Beizhan YAN ; Jiang YANG ; Jiangcun YANG ; Jun YANG ; Xinwen YANG ; Yongyi YANG ; Chunyan YAO ; Mingliang YE ; Changlin YIN ; Ming YIN ; Wen YIN ; Lianling YU ; Shuhong YU ; Zebo YU ; Yigang YU ; Anyong YU ; Hong YUAN ; Yi YUAN ; Chan ZHANG ; Jinjun ZHANG ; Jun ZHANG ; Kai ZHANG ; Leibing ZHANG ; Quan ZHANG ; Rongjiang ZHANG ; Sanming ZHANG ; Shengji ZHANG ; Shuo ZHANG ; Wei ZHANG ; Weidong ZHANG ; Xi ZHANG ; Xingwen ZHANG ; Guixi ZHANG ; Xiaojun ZHANG ; Guoqing ZHAO ; Jianpeng ZHAO ; Shuming ZHAO ; Beibei ZHENG ; Shangen ZHENG ; Huayou ZHOU ; Jicheng ZHOU ; Lihong ZHOU ; Mou ZHOU ; Xiaoyu ZHOU ; Xuelian ZHOU ; Yuan ZHOU ; Zheng ZHOU ; Zuhuang ZHOU ; Haiyan ZHU ; Peiyuan ZHU ; Changju ZHU ; Lili ZHU ; Zhengguo WANG ; Jianxin JIANG ; Deqing WANG ; Jiongcai LAN ; Quanli WANG ; Yang YU ; Lianyang ZHANG ; Aiqing WEN
Chinese Journal of Trauma 2024;40(10):865-881
Patients with severe trauma require an extremely timely treatment and transfusion plays an irreplaceable role in the emergency treatment of such patients. An increasing number of evidence-based medicinal evidences and clinical practices suggest that patients with severe traumatic bleeding benefit from early transfusion of low-titer group O whole blood or hemostatic resuscitation with red blood cells, plasma and platelet of a balanced ratio. However, the current domestic mode of blood supply cannot fully meet the requirements of timely and effective blood transfusion for emergency treatment of patients with severe trauma in clinical practice. In order to solve the key problems in blood supply and blood transfusion strategies for emergency treatment of severe trauma, Branch of Clinical Transfusion Medicine of Chinese Medical Association, Group for Trauma Emergency Care and Multiple Injuries of Trauma Branch of Chinese Medical Association, Young Scholar Group of Disaster Medicine Branch of Chinese Medical Association organized domestic experts of blood transfusion medicine and trauma treatment to jointly formulate Chinese expert consensus on blood support mode and blood transfusion strategies for emergency treatment of severe trauma patients ( version 2024). Based on the evidence-based medical evidence and Delphi method of expert consultation and voting, 10 recommendations were put forward from two aspects of blood support mode and transfusion strategies, aiming to provide a reference for transfusion resuscitation in the emergency treatment of severe trauma and further improve the success rate of treatment of patients with severe trauma.
5.Cost-benefit analysis of hepatitis B virus serological and nucleic acid testing in blood donors
Xianlin YE ; Wen XIONG ; Tong LI ; Ran LI ; Liqing HUANG ; Jinfeng ZEN
Chinese Journal of Blood Transfusion 2023;36(1):56-59
【Objective】 In an effort to prevent transfusion-transmitted hepatitis B infection, universal HBsAg screening, HBsAg+ MP nucleic acid test(NAT) for HBV and HBsAg + individual(ID) NAT were analyzed for cost-effectiveness. 【Methods】 On the basis of screening data and the documented parameter, the number of window period infections, chronic infections and occult infections was constructed, and cost-benefit analysis was conducted. 【Results】 Of 132 208 donations, the yield rate of ID NAT for HBsAg-/DNA+ (0.11%) was significantly higher than HBsAg+ MP NAT(0.058%). Furthermore, the predicted preventing transfusion transmitted HBV cases by ID NAT is 1.25 times as that by MP-6 NAT, so did the benefits. The cost-benefit of the three screening models were 1∶63.6、1∶28.6 and 1∶53.4. 【Conclusion】 Universal HBsAg in combination with ID HBV NAT screening was the most effective among all screening strategy. It is necessary to applied HBsAg and ID HBV NAT screening for the safety of blood transfusion.
6.Eeffect of ultra-low dose CT scanning on the diagnosis of ankle fracture and the quality of a three-dimensional printing model
Meng ZHANG ; Ming LEI ; Fenghuan LIN ; Jingzhi YE ; Yanxia CHEN ; Jun CHEN ; Jinfeng LIU ; Mengqiang XIAO
Chinese Journal of Radiological Medicine and Protection 2022;42(6):475-480
Objective:To investigate the effect of ultra-low dose (ULD) computed tomography (CT) scanning on the diagnosis of ankle fractures and the quality of a three-dimensional printing (3DP) model.Methods:This study was a prospective study. A total of 61 patients with clinical ankle fractures treated conservatively in Zhuhai Hospital of Guangdong Hospital of Traditional Chinese Medicine from November 2019 to January 2022 were included in this study. Patients underwent standard dose (SD) CT scan and ultra-low dose (ULD) CT scan, respectively. The tube voltage/tube current of SD and ULD were 120 kV/100 mAs and 80 kV/10 mAs, respectively. Two senior radiologists evaluated the presence of ankle fractures. The effective radiation dose ( E), noise, signal-to-noise ratio (SNR), contrast signal-to-noise ratio (CNR), and CT value of bone cortex minus CT value of peripheral fat (CTc) were compared. The radiologists also evaluated the discoverability, diagnosability, and overall image quality of the fracture line according to Likert′s 5-point scoring method. Two senior orthopedists subjectively evaluated the quality of each 3DP model (model clarity and operation guidance). A score ≥ 3 indicated that the quality of the CT diagnostic image and 3DP model were acceptable. Results:The interval between the two CT scans was (9.23 ± 1.92) d. A total of 94 fracture sites were found. There were no missed diagnosis or misdiagnosis based on the SD and ULD scans.Noise, SNR, and CNR were better on the SD CT scanning ( F=5.92, 9.70, 8.32, P=0.00), however, CTc was higher on the ULD scans ( F=27.55, P<0.01). The image scores of the SD and ULD scans were (4.97 ± 0.18) and (4.21 ± 0.71), and the quality scores of the 3DP model (4.99 ± 0.01) and (4.87 ± 0.34), respectively. The SD scans were better than the ULD scans with respect to CT image quality and 3DP model quality ( Z=-6.88, -2.91, P<0.01), but both were considered suitable to meet clinical needs (all ≥ 3 points). The E associated with SD and ULD scannings were (34.68 ± 4.96) μSV and (1.04 ± 0.10) μSV, respectively. The latter was thus significantly better than the former ( F=38.77, P =0.00). Conclusions:The E value of ULD scanning is about 3.00% of SD scanning E, which can meet the needs of clinical diagnosis of ankle fracture and 3DP model printing diagnosis.
7.Analysis of NAT non-reactive results implicated in HBsAg ELISA reactive blood donors by multi-assays
Xiaoxuan XU ; Xianlin YE ; Xin ZHENG ; Tong LI ; Binghuan YU ; Yu ZHAO ; Ran LI ; Jinfeng ZENG
Chinese Journal of Blood Transfusion 2021;34(12):1310-1313
【Objective】 To investigate NAT non-reactive results implicated in HBsAg ELISA reactive voluntary blood donors in Shenzhen. 【Methods】 HBsAg ELISA+ but NAT-blood samples were collected, and HBsAg was further retested by TRFIA, Roche ECLIA and neutralization test. HBV DNA of individual donation was detected by commercial Roche MPX and Uultrio Elite, and virus nucleic acid was extracted via 2.5 mL. Molecular characterizations of HBsAg+ /NAT-samples were determined by quantitative polymerase chain reaction(qPCR) and nested PCR amplifification of the precore and core promoter regions and HBsAg(S) region. HBV serological markers were detected, and the samples with suspicious results were followed up and detected by multi-assay. 【Results】 Among 67 602 samples, 73(0.11%) HBsAg ELISA+ and NAT-blood samples were enrolled in the study. 15(20.5%, 15/73) were confirmed HBsAg+ by TRFIA, ECLI and five alternative DNA assays, and the other 2(2.7%, 2/73) were further identified as HBsAg+ by follow-up study. In 17 confirmed HBsAg+ samples, the viral loads ranged undetectable to 378 IU/mL, with the median of 10.1 IU/mL. Weak correlation was found between HBsAg and HBV DNA load(R2=0.394 4). 【Conclusion】 Some Hepatitis B virus infected blood samples may miss even with different HBsAg assays. Multi-assays with high sensitivity should be combined for blood screening to ensure blood safety..The inconsistent results should be followed up and further tested for hepatitis B serological markers to assist the confirmation.
8.Molecular characteristics of hepatitis B infection with low level HBsAg and nucleic acid testing non-reactive results in blood donors
Xiaoxuan XU ; Xianlin YE ; Xia WANG ; Tong LI ; Yu ZHAO ; Ran LI ; Heng LIU ; Jinfeng ZENG
Chinese Journal of Blood Transfusion 2021;34(8):827-831
【Objective】 To investigate HBV infection with low level of HBsAg and nucleic acid testing(NAT) non-reactive results in blood donors, and analyze molecular characteristics. 【Methods】 Low level HBsAg but NAT-nonreactive samples were collected and tested for HBsAg by Abbott chemiluminescent microparticle immunoassay (CMIA)., HBsAg, anti-HBs, HBeAg, anti-HBe and anti-HBc were further detected by Roche electrochemiluminescence immunoassay(ECLI). BCP/PC and S regions were also amplified by Nested-PCRs and qPCR for HBV DNA quantity were adopted simultaneously. 【Results】 Of 100 363 donations, 60(0.054%) low level HBsAg and NAT-nonreactive blood samples were enrolled the study. In which, 54/60(90%) and 57/60(95%) were WanTai HBsAg ELISA and DiaSorin HBsAg ELISA reactive respectively. Of 33 cases genotyped, genotype B were 87.9%( 29/33), including adw2 96.6%(28/29) and adw1 3.4%(1/29), C was observed in 4(12.1%) with sero-type adrq+. Mutations in S gene of genotype B such as Q101R, Q129H, T131I, M133L/T, F134L, G145R, V168A, L175S and V177A were observed as notable mutations, which can affect HBsAg diagnosis. A high frequency mutation C1799G(87.5%, 21/24)were detected in BCP/PC and would reduce the replication of virus. The median viral load measured by qPCR was 49.6(0~628)IU/mL. 【Conclusion】 A small part of donations with low-level HBsAg and NAT-nonreactive can not be deferred by one isolated ELISA screening assay. It is necessary to apply more sensitive and specific HBsAg assays and NAT in blood screening, and improve the ability to detected mutants.
9.Effect of rhubarb-free anthraquinone in the treatment of acute pancreatitis with adipose-derived stem cell transplantation in rats
Junjie ZHENG ; Weijun TIAN ; Jinfeng WANG ; Kui YE
Chinese Journal of Tissue Engineering Research 2019;23(12):1390-1396
BACKGROUND: Studies have shown that rhubarb-free anthraquinone can protect the stability of the intestinal mucosa cell structure and function. However, the combination of rhubarb-free anthraquinone with adipose-derived stem cells to treat acute pancreatitis in rats has not yet been reported. OBJECTIVE: To investigate the effects of rhubarb-free anthraquinone on the treatment of acute pancreatitis by fat stem cell transplantation in rats. METHODS: Cryopreserved mouse adipose-derived stem cells (provided by the Chinese Academy of Medical Sciences) were rapidly recovered and cultured in vitro. The survival cells were labeled using PKH-26, and then the cell survival rate of adipose-derived stem cells was determined by MTT colorimetry. Sprague-Dawley rats provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. were equally randomized into four groups. Animal models of acute pancreatitis were made in all rats through intestinal wall puncture for retrograde administration of 5% sodium taurocholate to the pancreaticobiliary tube, and then the model rats were given tail vein injection of L-DMEM complete medium (0.5 mL) in the model group, intragastric administration of 200 mg/kg rhubarb-free anthraquinone in the rhubarb-free anthraquinone group, tail vein injection of adipose-derived stem cells (1×107 cells/L, 0.5 mL) in the cell transplantation group, and intragastric administration of 200 mg/kg rhubarb-free anthraquinone plus tail vein injection of 1×107 adipose-derived stem cells (0.5 mL) in the combined treatment group. All interventions in each group were performed once a day, for 3 continuous days. RESULTS AND CONCLUSION: (1) The levels of serum amylase and interleukin-6 were significantly reduced in the rhubarb-free anthraquinone group and cell transplantation group compared with the model group (P < 0.05) , while the level of trypsinogen activation peptide significantly increased (P < 0.05). In the combined treatment group, the levels of three indicators mentioned above were significantly decreased compared with the rhubarb-free anthraquinone group and cell transplantation group (P < 0.05). (2) The results of hematoxylin-eosin staining showed that the pathological changes in the pancreatic tissue of rats were significantly reduced in the rhubarb-free anthraquinone group and cell transplantation group, such as fatty degeneration, hemorrhage, cell necrosis and inflammatory cell infiltration. The remission of pathological changes was more obvious in the combined treatment group. (3) The number of positive cells labeled by PKH-26 was the highest in the combined treatment group, followed by the cell transplantation group, while there were no PKH-26-positive cells in the rhubarb-free anthraquinone group and model group (P < 0.05). (4) Compared with the model group, the number of apoptotic cells in the pancreatic tissue was significantly reduced in the rhubarb-free anthraquinone group and cell transplantation group, and lowest in the combined treatment group (P < 0.01). (5) The expression of transforming growth factor-β1 and Smad2/3 at gene and protein levels was highest in the model group, followed by the rhubarb-free anthraquinone group and cell transplantation group (P < 0.05) , and lowest in the combined treatment group (P < 0.01). To conclude, the combined intervention of rhubarb-free anthraquinone and adipose-derived stem cell transplantation could effectively improve the blood biochemical index level in the rats with acute pancreatitis, significantly relieve the degree of inflammatory response, pancreatic tissue morphology, and apoptosis of pancreatic cells, which may be related to the reduction of the transforming growth factor-β/Smad signal pathway.
10. Current status and influencing factors of job burnout in rural preschool teachers in Sichuan Province
Jianguo CHEN ; Yunli YE ; Hui LI ; Rong ZHANG ; Yuqi CHEN ; Jinfeng LAI ; Ya LIU
China Occupational Medicine 2019;46(06):723-731
OBJECTIVE: To understand the current status and influencing factors of job burnout in rural preschool teachers. METHODS: A stratified cluster random sampling method was used to investigate the job burnout and social support of 756 rural preschool teachers in Sichuan Province using the Maslach Burnout Inventory, Educators Survey and Social Support Revalued Scale. RESULTS: The positive rate of job burnout was 22.1%(167/756), and the positive rates of emotional exhaustion, reduced personal achievement, and depersonalization were 13.1%, 10.7% and 1.2%, respectively. The results of unconditional logistic regression analysis showed that post status, daily working hours, subjective support and utilization of support were all related to job burnout(P<0.05). Among them, in-post and daily working hours >10 hours were risk factors of job burnout, high subjective support and utilization of support were the protective factors of job burnout. CONCLUSION: The situation of job burnout in rural preschool teachers in Sichuan Province is not serious, post status, daily work hours and social support can affect their job burnout.

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