NOACs) are superior to warfarin among Asians with nonvalvular atrial fibrillation remains unclear.Whether non–vitamin K antagonist oral anticoagulants (IS/SE), 0.55 (0.43–0.69), 0.82 (0.68–0.98), and 0.81 (0.67–0.97); major bleeding, 0.41 (0.31–0.53), 0.65 (0.53–0.80), and 0.58 (0.46–0.72); and all‐cause mortality, 0.58 (0.51–0.66), 0.61 (0.54–0.68), and 0.57 (0.51–0.65). A total of 3623 (62%), 17 760 (88%), and 26 000 (94%) patients were taking low‐dose apixaban (2.5 mg twice daily), dabigatran (110 mg twice daily), and rivaroxaban (10–15 mg once daily), respectively. Similar to all‐dose NOACs, all low‐dose NOACs had lower risk of IS/SE, major bleeding, and mortality when compared with warfarin. In contrast to other standard‐dose NOACs, apixaban was associated with lower risks of IS/SE (0.45 [0.31–0.65]), major bleeding (0.29 [0.18–0.46]), and mortality (0.23 [0.17–0.31]) than warfarin.In this nationwide retrospective cohort study collected from Taiwan National Health Insurance Research Database, there were 5843, 20 079, 27 777, and 19 375 nonvalvular atrial fibrillation patients taking apixaban, dabigatran, rivaroxaban and warfarin, respectively, from June 1, 2012 to December 31, 2016. Propensity‐score weighting was used to balance covariates across study groups. Patients were followed until the first occurrence of any efficacy or safety outcome or the end date of study. Hazard ratios  comparing apixaban, dabigatran, and rivaroxaban with warfarin were: ischemic stroke/systemic embolism (NOACs were associated with lower risk of IS/SE, major bleeding, and mortality compared with warfarin in the largest real‐world practice among Asians with nonvalvular atrial fibrillation. All low‐dose NOACs had lower risk of IS/SE, major bleeding, and mortality when compared with warfarin. Standard‐dose apixaban caused a lower risk of IS/SE, major bleeding, and mortality compared with warfarin.All  This is the largest population‐based study of Asians with nonvalvular atrial fibrillation to investigate the effectiveness and safety of the non–vitamin K antagonist oral anticoagulants apixaban, dabigatran, and rivaroxaban versus warfarin during an extended follow‐up period.Our results showed that all three non–vitamin K antagonist oral anticoagulants exhibited lower risks of thromboembolic events, all major bleeding, and all‐cause mortality compared with warfarin.There is a high prevalence of low‐dose non–vitamin K antagonist oral anticoagulant use among this large Asian cohort, and this subgroup also had lower risks of thromboembolic events, major bleeding, and mortality compared to warfarin.Apixaban, rivaroxaban, and dabigatran should be considered as first line therapy for Asian patients with nonvalvular atrial fibrillation.Atrial fibrillation (AF) is the most common cardiac arrhythmia with a global prevalence of 2% to 3%. AF significantly increases the risk of thromboembolic events and death.The data, analytical methods, and study materials will not be made available to other researchers for purposes of reproducing the results or replicating the procedure.This study was approved by the Institutional Review Board of Chang Gung Memorial Hospital. Informed consent was waived by the Institutional Review Board of Chang Gung Memorial Hospital because the original identification number of each patient in the National Health Insurance Research Database (NHIRD) was encrypted and deidentified to protect patients' privacy by using a consistent encrypting procedure. The National Health Insurance system is a mandatory universal health insurance program in Taiwan that provides comprehensive medical care coverage to all Taiwanese. As of 2014, there were >23 million enrollees and a >99% coverage rate of the entire population.International Classification of Diseases, Ninth Revision, Clinical Modification [ICD‐9‐CM] codes [427.31] from January 1, 2010, to December 31, 2015, or ICD‐10‐CM codes [I48] from January 1, 2016, to December 31, 2016) were identified. Patients were included who had their first prescription of an NOAC including dabigatran , rivaroxaban , or apixaban , as well as patients who started warfarin treatment from June 1, 2012, to December 31, 2016. The index date was defined as the first date of prescription for any NOAC or warfarin after June 1, 2012, for each group. The follow‐up period was defined from the index date until the first occurrence of any study outcome or the end date of study period , whichever came first.A dynamic cohort with 4 study groups  was used in the study. A flowchart of the study enrollment is shown in Figure ICD‐9‐CM or ICD‐10‐CM codes and enrollment in the Registry of Catastrophic Illness Patient Database, a subpart of the National Health Insurance database. Only patients with end‐stage renal disease on dialysis can be registered in the Registry of Catastrophic Illness Patient Database according to the rules of the National Health Insurance system.Patients were excluded who took more than 1 kind of NOAC during their entire treatment course. To establish a cohort of NVAF patients who took oral anticoagulants for the primary purpose of stroke prevention, those patients were excluded with diagnoses indicating valvular AF , venous thromboembolism (pulmonary embolism or deep vein thrombosis) or joint replacement therapy within 6 months before the index date. Patients were also excluded who had end‐stage renal disease requiring renal replacement therapy because NOACs are contraindicated in such patients in Taiwan. The diagnosis of end‐stage renal disease requiring dialysis in our present study was confirmed by both specific ICD‐9‐CM codes for identifying IS by analyzing the medical records of 1002 consequent AF patients in the inpatient claims database of Chang Gung Memorial Hospital, Linkou, which is the largest medical center in Taiwan, between January 2010 and December 2015. Clinical diagnosis of IS was determined according to the compatible brain imaging. In total, there were 104 confirmed cases of IS at discharge, and 96 can be identified with an ICD‐9‐CM code indicating IS  in the discharge claims. There were only 10 cases with ICD‐9‐CM indicating IS without compatible brain imaging available. The sensitivity, specificity, positive predictive value, and negative predictive value were 0.92, 0.98, 0.91 and 0.99, respectively. ICH was defined with the use of codes for atraumatic hemorrhage. Major GIB was defined as a hospitalized primary code indicating bleeding in the gastrointestinal tract. All major bleeding events were defined as the total hospitalized events of ICH, major GIB, and other critical site bleedings. The diagnosis codes of NHIRD were shifted from ICD‐9‐CM to ICD‐10‐CM after January 1, 2016. The ICD‐9‐CM and ICD‐10‐CM codes used to identify the study outcomes and the baseline covariates are summarized in Table Six study outcomes were defined to determine the efficacy and safety profiles for NOACs and warfarin: ischemic stroke/systemic embolism (IS/SE), intracranial hemorrhage (ICH), major gastrointestinal bleeding (GIB), acute myocardial infarction, all major bleeding events, and all‐cause mortality. All study outcomes were required to be a discharge diagnosis to avoid misclassification. The accuracy of diagnosis indicating IS in the NHIRD was validated previously.2DS2‐VASc score  was adopted to predict the risk of ischemic stroke/thromboembolic events in AF patients, and the HAS‐BLED score  was adopted to predict the risk of bleeding in patients with AF treated with oral anticoagulants.Baseline covariates were referred to any claim record with the above diagnoses or medication codes before the index date. A bleeding history was confined to events within 6 months preceding the index date. A history of prescription for medicine was confined to at least once within 3 months preceding the index date.2DS2‐VASc and HAS‐BLED scores, because CHA2DS2‐VASc and HAS‐BLED scores were a combination of other covariates. Incidence rates were estimated using the total number of study outcomes during the follow‐up period divided by person‐years at risk. The risk of study outcomes for 3 NOACs versus warfarin (reference) was obtained using survival analysis . The balance of potential confounders at baseline (index date) between each study group was assessed using the absolute standardized mean difference rather than statistical testing, because balance is a property of the sample and not of an underlying population. The value of absolute standardized mean difference ≤0.1 indicated an insignificant difference in potential confounders between the 2 study groups.P value <0.05. All statistical analyses were performed using computer software .The propensity score method, which simulates the effect of a randomized clinical trial for observational cohort data,2DS2‐VASc and HAS‐BLED scores, and had a higher proportion of comorbidities than the warfarin group before propensity score weighting , 17 760 (88%), and 26 000 94%) patients were prescribed low‐dose apixaban (2.5 mg twice daily), dabigatran (110 mg twice daily), and rivaroxaban (15 or 10 mg once daily), respectively. Patients who took 3 low‐dose NOACs were older, had higher CHA% patientThis is the largest population‐based study to investigate the efficacy and safety of apixaban, dabigatran, and rivaroxaban versus warfarin with a specific focus on Asians with NVAF during an extended follow‐up period. To date, no previous study has directly compared the effects of these 3 NOACs in Asian patients. Our results showed that all 3 NOACs exhibited lower risks of IS/SE, ICH, all major bleeding, and all‐cause mortality compared with warfarin in a large Asian cohort with AF. We also observed a high prevalence of low‐dose NOACs prescription among the large Asian cohort, with an ≈62%, 88%, and 94% of patients taking low‐dose apixaban, rivaroxaban, and dabigatran, respectively. In contrast to the other 2 standard‐dose NOACs, standard‐dose apixaban (5.0 mg twice daily) demonstrated a lower risk of IS/SE, all major bleeding, and ICH compared with warfarin. All 3 standard‐dose NOACs, apixaban (5.0 mg twice daily), dabigatran (150 mg twice daily), and rivaroxaban (20 mg daily) showed lower mortality than the warfarin group. Three low‐dose NOACs showed similar performance as without subgrouping; that is, the risk of IS/SE, ICH, major bleeding, and mortality were all lower than warfarin.Few studies have directly compared the efficacy and safety of all 3 NOACs versus warfarin in non‐Asian patients. Larsen et alOf note, these real‐world and pivotal studies all focused primarily on non‐Asian patient groups. Previous studies indicated that Asians with AF are more sensitive to warfarin and have an unacceptably higher risk of ICH than non‐Asians even when international normalized ratios are all ideally maintained between 2 and 3.A subgroup analysis of pivotal trials in Asians showed that NOACs displayed better efficacy and safety in Asians than in non‐Asians.Recent real‐world practice showed a worldwide trend toward the use of low‐dose NOACs.ICD‐9M: 433, 434, or 436; or ICD‐10: I63, I64) as well as previous studies comparing the efficacy of NOACs and warfarin in a real‐world practice. Because the NHIRD does not have brain imaging data, we cannot clarify the issue of whether the reduction of ischemic stroke is contributed from embolic stroke or not. The 3 NOACs prescribed had varying rates of renal excretion and, thus, decisions regarding the use of a specific NOAC or its dosage may have been guided by the renal function and body weight of each patient. Because the NHIRD does not contain creatinine clearance levels, and coding that indicates impaired renal function was dependent on physician's choice, our results may have been biased by the chronic kidney disease population. In addition, we observed a high prevalence of low‐dose NOAC prescriptions in our Asian cohort. The lack of both renal function data and body weights makes it difficult to determine if those patients given low‐dose NOACs were correctly prescribed an adjusted dose or were “off‐label” underdosed. However, the lack of such data is a common limitation of most health insurance databases around the world. Miscoding and misclassification of the underlying comorbidities and outcomes registered by each physician's choice of treatment constitutes an additional limitation of the present study. However, only primary discharge diagnoses were adopted in the present study to improve the outcome accuracy. The 3 NOAC groups also had significantly more comorbidities than the warfarin group. Although inverse propensity score weighting with several variables allowed the matching of comorbidities among the 4 groups, residual confounding by unmeasured variables and selective prescribing behavior could not be excluded in the present study. The Taiwan NHIRD does not have laboratory results, so we had no basis upon which to assess the quality of warfarin anticoagulation among patients. It is possible that the favorable efficacy of NOACs on IS/SE and mortality in our present study was at least partly due to low time in therapeutic range with warfarin. However, this would not explain the reduced risk of IS/SE, ICH, major GIB, and all major bleeding with NOACs at the same time. More importantly, whether quality of warfarin anticoagulation in our study was or was not adequate, it reflects the real‐world practice of anticoagulation quality among Asians taking warfarin. Also, the crude annual risks of IS and major bleeding in the warfarin group in the present study were 2.79% and 3.00%/year, respectively, which were comparable with the Asian post‐hoc analysis of the reported risks with 1.90 and 3.84%/year in the ARISTOLE trial (mean time in therapeutic range of 60.0%), 2.24% and 5.14%/year in the ROCKET‐AF trial (mean time in therapeutic range of 47.1%), and 2.02% and 3.82%/year in the RE‐LY trial (mean time in therapeutic range of 56.5%).The present study had several limitations. We have excluded patients with diagnoses indicating valvular AF, venous thromboembolism, or joint replacement therapy within 6 months before the index date in order to establish a cohort of NVAF taking oral anticoagulants. However, it is not clear that this is sufficiently exclusive. In the present study, we adopted the same definition codes indicating ischemic stroke (Apixaban, rivaroxaban, and dabigatran were associated with reduced risks of IS/SE, ICH, all major bleeding, and all‐cause mortality compared with warfarin in a large Asian cohort with NVAF. There was a high prevalence of low‐dose NOAC prescription among the Asian cohort. In contrast to the other 2 standard‐dose NOACs, standard‐dose apixaban demonstrated a lower risk of IS/SE, all major bleeding, and ICH compared with warfarin. All 3 standard‐dose NOACs showed lower mortality than the warfarin group. Three low‐dose NOACs showed similar performance as without subgrouping.This study was supported by grants 102‐2628‐B‐182‐011‐MY3, 102‐2314‐B‐182A‐053‐MY3, and 105‐2628‐B‐182A‐003‐MY3 from the Ministry of Science and Technology and CMRPG3B0991‐3, CMRPG3E1681, CMRPG3F0041, CMRPG3F0853, CMRPG3D1631, CMRPD1F0252, CMRPG3F0041 and CMRPG3E0291 from the Chang Gung Memorial Hospital, Linkou, Taiwan.None.


To study the outcome and experience of using metallic stents in treating patients with malignant ureteral obstruction (MUO).Seventy-six patients with MUO were assigned to the metallic stent group (MSG) or the ordinary polymer stent group (OPSG) according to the different materials. The success rate of the operation, duration of operation, patency rate serum creatinine values ,postoperative complications and QOL scores were compared between the two groups.P < 0.01), respectively. There was no significant difference between the groups in serum creatinine values at 3 days after the operation (P > 0.05); however, the creatinine values at 3 days after the operation decreased significantly compared with those before the operation (P < 0.01). In the OPSG, there was no significant difference in creatinine values between 3 days and 6 months after operation, while the creatinine values 1 year after operation were increased significantly compared to those at 3 days after the operation (P < 0.05). In the MSG, there was no significant difference among creatinine values at different intervals (P > 0.05). The total rate of post-procedural complication was lower in the MSG than that in the OPSG(P < 0.05). There was no significant difference in the QOL score between the two groups before the operation (P > 0.05); however, the QOL scores at 6 months and 1 year after the operation were higher in the MSG than that in the OPSG(P < 0.05). In the MSG, there was no significant difference in the QOL score between preoperation and 6 months after surgery. Similarly, there was also no difference in the QOL score between 6 months after surgery and 1 year after surgery(P > 0.05). On the contrary, the differences of QOL score in the OPSG group were much significant between disparate time intervals (P < 0.05).In the OPSG and MSG, the success rates of the operation were 95.5% and 96.9%, respectively, and the durations of the operation were 20.6 ± 2.2 min and 50.9 ± 10.3 min (For patients with MUO who require long-term retention of the stent, metallic stents with longer indwelling time are superior to ordinary polymeric stents. Ureteral obstructions secondary to malignant tumour increase each year, mainly due to the following factors. First, primary tumours infiltrate the ureteral wall; second, tumours or metastatic lesions oppress the ureter; third, the swollen lymphonodus wraps the ureter ; fourth,Data were retrospectively obtained from the medical records of patients with MUO who were treated during the period of June 2014 to August 2018 at the Second Hospital of Tianjin Medical University, Lu'an Affiliated Hospital of Anhui Medical University, and Zhejiang Cancer Hospital. This retrospective study was approved by the institutional review board in our hospital. Patients were informed of the risks and benefits of participation in the study; each provided written, informed consent before enrolment. Based on the different ureteral stent materials, 76 patients were divided into the metallic stent group (MSG) and the ordinary polymer stent group (OPSG), as shown in Table In the MSG, which comprised 32 patients, 3 different surgical methods were adopted. One case involved catheterization failure, defined as follows: First, all patients included initially accepted retrograde ureteroscope or cystoscope catheterization. Those who could not receive catheterization accepted the percutaneous nephroscope anterograde catheterization; if that failed, the case was recorded as failed catheterization, and nephrostomy was conducted. Second, retrograde ureteroscope or cystoscope catheterization was successful, but the stent position was not good after re-examination; moreover, renal functional improvement was not ideal. In the OPSG, which comprised 44 patients, there were 2 cases of failed catheterization, followed by nephrostomy. The evaluation criteria for inadequate stent drainage after surgery were as follows: with the exclusion of hypertensive nephropathy, diabetic nephropathy, and other kidney damage factors, the patient’s serum creatinine values increased and hydronephrosis became aggravated, as shown by the urinary colour ultrasound or other imaging examinations. For such patients, the stent should be changed before the expected time; if this fails, a nephrostomy was performed.Ureteroscopy catheterization: In the lithotomy position, the guide wire was passed through the ureter lumen into the kidney pelvis with a ureteroscope, followed by a coaxial introduction of the Resonance® sheath and catheter. The guidewire and catheter were then exchanged for the closed-ended metallic stent through the sheath, with the aid of the catheter as a pusher. The sheath was then finally removed to leave the stent in position.Percutaneous nephroscope anterograde catheterization: In the prone position, guided by ultrasound, the target calyx was punctured. After establishing percutaneous nephroscope channels, the guide wire was anterogradely placed into the bladder with a ureteroscope. Subsequently, the outer sheath along the guide wire was placed into the bladder, and the metallic stent was then placed along the sheath into the bladder and formed a circle in the kidney pelvis.Cystoscope ureteral catheterization: In the lithotomy position, local anaesthesia was conducted. The method of catheterization was similar to that of the ureteroscope approach.For OPSG, ureteroscope and cystoscope catheterization were performed in a manner similar to that of traditional surgical methods. For MSG, the following methods were used:P < 0.05 was considered statistically significant.SPSS 16.0 software  was used to analyse the data. The data were expressed as P < 0.01), according to statistical analysis.The success rate and duration of operation for each group are shown in Table P > 0.05); however, the creatinine values at 3 days after the operation decreased significantly compared with those before the operation (P < 0.01). In the OPSG, there was no significant difference in creatinine values between 3 days and 6 months after surgery, while creatinine values at 1 year after surgery were increased compared with those at 3 days after surgery (P < 0.05). In the MSG, there were no significant differences among creatinine values at 3 days, 6 months, and 1 year after the operation (P > 0.05). The average duration of follow-up was 12 months, during which ordinary polymer stents were generally replaced within 1–3 months and metallic stents were replaced within approximately 12 months; the longest retention time was approximately 2 years. In the MSG, outpatient re-examinations showed no fracture, translocation, or dysfunction in the metallic stents. Among all patients who were followed-up, six experienced intermittent haematuria, four experienced repeated urinary tract infections after surgery, and one exhibited stones attached to metallic stents.Serum creatinine values before the operation and 3 days, 6 months, and 1 year after the operation are shown in Table In the OPSG, the patency rates at 3 days, 6 months, and 1 year after the operation were 100% (42/42), 83.8% (31/37), and 40.0% (12/30), respectively . There was no significant difference in QOL score between the two groups before the operation (P > 0.05); however, the QOL scores at 6 months and 1 year after the operation were higher in the MSG than that in the OPSG (P < 0.05). In the MSG, there was no significant difference in the QOL score between preoperation and 6 months after surgery. Similarly, there was also no difference in the QOL score between 6 months after surgery and 1 year after surgery (P > 0.05). On the contrary, the differences of QOL score in the OPSG group were much significant between disparate time intervals (P < 0.05).The post-procedural complication and QOL score of different stages for each group are shown in Tables The Cox multivariate analysis of potential survival risk did not yield any endpoints of statistical significance including gender, age at diagnosis, tumour location, site of ureteral obstruction, preoperative creatinine, stent type, and anaesthesia in spite of reduced survival rates with prolonged follow-up Table .Table 8CMalignant tumours causing ureteral obstruction are mainly from the abdomen, pelvis, and retroperitoneum; these include rectal cancer, colon cancer, gastric cancer, cervical cancer, ovarian cancer, bladder cancer, and prostate cancer. Methods for removing malignant external compressive ureteral obstructions include ureteral stenting and nephrostomy. Since nephrostomy causes inconvenience and trauma, ureteral stenting is often used. Three technical parameters are critical for ideal ureteral stent performance: design, materials, and surface coating , and thuIn this study, we used the Resonance® metallic ureteral stent, which is a hollow, spiral-shaped, full-length metal stent with the same shape as the double J catheter. It has the following advantages: first, it is made of nickel-cobalt-chromium-molybdenum alloy, which has high biocompatibility and less capsid, such that it is not prejudiced against MRI examination; second, its dense, hollow, spiral structure can inhibit endogenous tissue growth and can provide double drainage from the spiral outer surface and the lumen; third, its radial compressive resistance is > 68 kg, which is much higher than that of traditional stents (4~11 kg) and other metal stents ; fourth,P > 0.05), while creatinine values at 3 days after the operation in each group were significantly lower than those before the operation (P < 0.01). This finding indicated that catheterization of either metallic stents or ordinary polymeric stents could enable effective hydronephrosis drainage and improve short-term renal function. As the statistical analysis shows, there was no significant difference in creatinine values among 3 days, 6 months, and 1 year after operation in the MSG (P > 0.05). Furthermore, in the OPSG, there was no significant difference in creatinine values between 3 days and 6 months after operation (P > 0.05); however, the creatinine values at 1 year after operation were significantly higher than those at 3 days after operation (P < 0.05). The patency rate decreased to 40.0%, which was in striking contrast to the 91.7% observed in the MSG; this finding indicated that over 6 months, both the metallic stent and ordinary polymeric stent maintain a high drainage patency rate. However, 1 year later, even ordinary polymeric stents with regular replacement could not assure a higher patency rate, and the replacement success rate was gradually decreased. The advantages of metallic stents are notable. Although the duration of the operation of metallic stent catheterization was longer than that of the ordinary polymeric stent (P < 0.01), this difference may be related to the surgeon’s proficiency. Overall, therefore, metallic stents are preferred for patients with malignant external compressing ureteral obstructions.The patency rate of metallic stents for the treatment of MUO lies between 37% and 100% –21, and Thus far, reports of surgical experience sharing regarding metallic stent catheterization are not too much. To our knowledge, the success rate of catheterization has no relation to the type of stent but is related to the patient’s objective condition. The main reasons for failed catheterization are as follows: first, bladder shape is altered and the ureteral orifice moves with pelvic tumour compression, or after uterine and rectal tumour surgery, thereby causing an undiscoverable ureteral orifice Figs.  and 6; sP < 0.05). The outpatient re-examinations showed no fracture, translocation, or dysfunction in the metallic stents. There was no significant difference in the QOL score between the two groups before the operation(P > 0.05). However, the QOL scores at 6 months and 1 year after the operation were higher in the MSG than that in the OPSG (P < 0.05). In the MSG, there was no significant difference in the QOL score between preoperation and 6 months after surgery. Similarly, there was also no difference in the QOL score between 6 months after surgery and 1 year after surgery(P > 0.05). On the contrary, the differences of QOL score in the OPSG group were much significant between disparate time intervals (P < 0.05). We think there were several reasons for the lower QOL score of most patients with ordinary polymer stents. Firstly, because the QOL score was greatly influenced by the progress of primary tumour and individual psychological factors, frequent replacement of the stent might result in worry and fear. Secondly, nephrostomy leads to a low quality of life for those patients with failed catheterization due to inconvenience and trauma. Thirdly, family conflicts may occur due to repeated hospitalizations. Lastly, postoperative complications were the important factors. The Cox multivariate analysis of potential survival risk did not yield any endpoints of statistical significance including gender, age at diagnosis, tumour location, site of ureteral obstruction, preoperative creatinine, stent type, and anaesthesia in spite of reduced survival rates with prolonged follow-up (Fig. Those who have internal metallic stents need regular follow-up and should replace the stents within 1 year to avoid stone formation. Some patients who have rapid tumour progression or infiltrated ureteral lumen, or who are susceptible to stones should replace the stents within 6 months . During -up Fig. . AdditioWe conclude that the Resonance® metallic stent is safe and effective for treating MUO. For patients who require long-term stent retention, metallic ureteral stents with good drainage effect, longer indwelling time, and lower cost are superior to ordinary polymeric stents.


GEJ‐NENs) are rare and heterogeneous tumors. We aim to analyze the clinicopathlogical features and prognostic factors of GEJ‐NENs and to compare the outcome of GEJ‐NENs with other gastric NENs.Gastroesophageal Junction neuroendocrine neoplasms  database.A total of 297 GEJ‐NENs among different races were different. All Chinese patients had GEJ‐NENs of grade 3, with 67.7% of poorly differentiated NEC and 32.3% of MANEC. In SEER database, 70.8% of white, 62.5% of black, and 87.5% of AP patients had poorly differentiated/undifferentiated tumors. In Cox multivariate analysis, NEC/MANEC , lymph node metastasis , and distant metastases  are independent predictors of overall survival. Surgical resection showed a median OS improvement of 13.1‐73.3 months . Adjuvant therapy did not improve survival for postoperative GEJ‐NEN patients (P = 0.141). GEJ‐NENs were larger, higher grade, more distant metastasis, and worse prognosis than other gastric NENs.The clinical characteristics of GEJ‐NENs were mostly poorly differentiated carcinomas, and all of Chinese patients were NEC/MANEC. The outcome of MANEC was preferable to NECs. Both lymph nodes metastasis and distant disease were independent predictors of prognosis. Surgical resection can improve survival, but postoperative adjuvant therapy had no additional benefit. GEJ‐NENs have worse survival than other gastric NENs. The primary aim of this study was to investigate the clinical and pathological features of Chinese patients with GEJ‐NEN by comparing with those from Surveillance, Epidemiology, and End Results (SEER) Cancer Registry, and to study the prognostic predictors for GEJ‐NENs using a multicenter cohort from China. A second aim was to characterize the GEJ‐NENs compared with other gastric NENs using a population‐based registry.2Clinical data of patients with pathology confirmed GEJ‐NENs from 2000 to 2017 were retrieved from 10 hospitals in China. All of these hospitals were representative centers which located in different parts of China. This study was approved by the hospital institutional review board.The study cohort included all patients registered in the SEER database from 2000 to 2013. Individual cases were retrieved with the SEER*Stat software . Because of the SEER database's inclusion of unidentifiable patient information, this study was exempted for approval by the Office of Human Subjects Research of the National Institutes of Health. We identified patients with NETs using the following ICD‐O‐3 codes: 8240‐8249. 8240, carcinoid tumor; 8241, enterochromaffin; 8242, enterochromaffin‐like; 8243, goblet; 8244, mixed adenoneuroendocrine carcinoma; 8245, adenocarcinoid; 8246, neuroendocrine carcinoma; and 8249, atypical carcinoid. We selected NENs with primary of stomach (site code: C16.0‐16.9). Exclusion criteria included age less than 18 years, NEN as the second primary malignancy, NENs diagnosed at autopsy or death, and diagnoses without microscopic confirmation.The following variables were included in the analysis: age at diagnosis, race, sex, year of diagnosis, primary tumor location, tumor grade and differentiation, AJCC staging, nodal status, distant metastasis, type of surgery performed, and OS. Tumor grade according WHO 2010 classification based on Ki‐67 index and mitotic count was analyzed from Chinese cohort.2.1t test, χ2 test (or Fisher exact test) and Mann‐Whitney method were used. Overall survival (OS) time was measured from the date of initial diagnosis until the date of death or last follow‐up. Survival analysis was performed with OS as the primary outcome measure. Survival was evaluated using Kaplan‐Meier estimates and Cox proportional hazard regression. Statistical tests used two‐tailed P values, and P < 0.05 was considered statistically significant. All statistical analyses were performed in SPSS .To investigate the clinicopathological characteristics of the study patients, Student's 33.1We retrospectively analyzed clinical and pathologic features of 297 patients with histological confirmed GEJ‐NENs from 10 hospitals. The entire group had a median age of 63 (35‐85), and 87.2% were male (n = 259). Based on the WHO‐2010 grading classification, all the patients with GEJ‐NENs were grade 3, and the proportion of poorly‐differentiated NEC and MANEC was 67.7% and 32.3%. Regional lymph node metastasis was found in 155 (52.2%) patients and 78 (26.2%) had distant metastasis at diagnosis. According to the AJCC/UICC staging system, 2 (0.7%) patients were classified as stage I, 46 (15.5%) as stage II, 171 (57.6%) as stage III and the other 78 (26.2%) as stage IV, respectively.P = 0.002), curative operations , and more lymphatic metastasis , but were less associated with distant metastasis . The MANEC and NEC groups were statistically similar in other clinicopathological characteristics, including gender, age, Ki67 index, and tumor size. The comparison of clinicopathological characteristics between NEC and MANEC of the gastroesophageal junction is shown in table Compared with their NEC counterparts, the MANECs were more highly associated with early stage (3.2P < 0.000). Surgical of primary tumor was performed in most of the patients in different race groups. Table In total, 297 and 274 patients with GEJ‐NENs were included, respectively, from the Chinese cohort and SEER database. The clinicopathological characteristics of GEJ‐NENs among different races were distinct. The mean ages were 62.7, 64.5, 58.5, and 61.5, respectively, in Chinese, white, black patients, and Asian/Pacific Islander (AP) patients. Except for black patients, male patients were more frequent. In Chinese patients, tumor size was larger than that in other groups. Chinese patients were poorly differentiated NEC and MANEC. In SEER database, 70.8% of white, 62.5% of black and 87.5% of AP patients had poorly differentiated/undifferentiated tumors. Distant metastasis at the time of presentation was more frequent in white and black patients than Chinese patients    underwent surgical resection, of which 89.6% (n = 206) were curative and 10.4% (n = 24) were palliative. Surgical resection offered a survival advantage with HR 0.21 (95%CI 0.14‐0.33) and the median OS improved from 13.1 to 73.3 months (P = 0.002). On multivariate analysis, NEC/MANEC     , and the subgroup of MANEC had longer survival than NEC  and in white patients . Patients with GEJ‐NEN were more frequently male than female . Tumors of GEJ‐NENs were larger  and predominantly poorly differentiated and undifferentiated tumors . GEJ‐NENs were highly invasive with more distant metastases , whereas non‐GEJ NENs had more localized lesions . Patients with GEJ‐NENs were less likely to be treated with resection , but more likely to receive radiotherapy . Table A total of 3152 patients with gastric NENs were identified in the SEER database from 2000 to 2013, including 274 GEJ‐NEN patients and 2878 non‐GEJ NEN patients. GEJ‐NENs were more commonly diagnosed at an older age    Figure B; in reg) Figure C&D.4Gastroesophageal NENs account for a relatively small and heterogeneous population with an aggressive course; however, limited information is available regarding characteristics because of its rarity.The current study indicated that all the Chinese patients with GEJ‐NENs were poorly differentiated carcinomas, and 96 cases (32.3%) were MANEC with both adenocarcinoma and neuroendocrine differentiation. According to gastric NENs clinical classification,Because inadequate understanding of GEJ‐NENs, it is controversial of the prognostic value of their histologic classification. Compared with the adenocarcinoma, NEC was more aggressive with poorly differentiated morphology.The importance of surgical resection of gastric NENs on survival has been reported in the previous literature.Although classified as similar entities in classification schemes, tumors of gastroesophageal have distinct characteristics compared with those located elsewhere in the stomach, which has been observed in gastric adenocarcinoma.It has been reported that the biological behavior and clinical outcome of patients with gastric carcinoma varies among different human races.It is acknowledged that this study has limitations. The primary limitation is that the SEER database does not include information of Ki‐67 index and data on recurrence or disease‐free survival. The chemotherapy and specific treatment regimens were not included. The second limitation is the retrospective nature of our study. The pathological data from achieves were according to the 2010 WHO classification, although a new term of mixed neuroendocrine‐non‐neuroendocrine neoplasm(MiNEN) was proposed, considering the morphological and biological heterogeneity of gastrointestinal mixed tumors.5GEJ‐NENs were highly invasive with frequent distant metastases, with predominantly poorly differentiated and undifferentiated tumors, thus showed worse survival than other gastric NENs. The clinicopathological characteristics of GEJ‐NENs among different races were distinct. Chinese patients with GEJ‐NENs are all NEC or MANEC, and the latter show distinct metastatic patterns and better survival. Surgical resection improved the survival, and there was no additional survival benefit to adjuvant chemotherapy, and prospective studies using defined diagnostic criteria are necessary to determine optimal management.None declared.


Subclinical mastitis (SCM) and intramammary infection (IMI) increase the sodium (Na) concentration and electrical conductivity (EC) and decrease the potassium (K) and calcium (Ca) concentrations in glandular secretions of lactating dairy cattle.Low‐cost portable Na, K, Ca, and EC meters are clinically useful cow‐side tests for diagnosing SCM and IMI.One hundred fifteen dairy cows at dry off and 92 cows within 4‐7 days postcalving.Quarter foremilk samples were obtained and the somatic cell count (SCC) was measured using a DeLaval cell counter with SCM defined as SCC ≥ 200 000 cells/mL. Microbiological culture of foremilk samples was used to diagnose IMI. Cisternal milk Na, K, and Ca concentrations and EC were measured using portable ion‐selective meters. Logistic regression was used to determine the area under the receiver operating characteristic curve (AUC) and the optimal cut point was determined using Youden's index. Diagnostic test performance was evaluated by comparing the AUC and calculating the sensitivity, specificity, and positive likelihood ratio (+LR) at the optimal cut point for SCM and IMI.Diagnostic test performance was much better when the meters were used to diagnose SCM as compared to IMI. Cisternal milk Na concentration provided the most accurate method for identifying quarters with SCM or IMI. However, AUC was <0.90 and +LR was <10 for all diagnostic test evaluations.Cisternal milk Na, K, and Ca concentrations and EC were not sufficiently predictive of SCM or IMI to be recommended as clinically useful diagnostic tests. Consequently, the net effect of increased milk Na and Cl concentrations, even in the presence of decreased milk K concentration, is an increase in milk EC.The LAQUAtwin meters  are hand‐held direct  ion‐selective electrode meters that recently have become available for measuring Na, K, and Ca concentrations and EC in biological fluids. The low‐cost (approximately $250 US) and portability of the meters, as well as their ability to measure cation concentrations or EC in uncentrifuged milk samples, make them very attractive for on‐farm or cow‐side use. Studies conducted by our laboratory over the past 5 years have validated the accuracy of Na and K LAQUAtwin meters in measuring unbound Na and K concentrations in bovine cisternal milk.22.1A prospective observational study was conducted at the University of Illinois Dairy Research Farm using a convenience sample of 115 cows sampled during the last week of lactation and 92 cows sampled 4 to 7 days postpartum over a period of 13 months between July 1, 2015 and July 31, 2016. A total of 130 animals calved during the course of the study; samples at freshening were not obtained from 38 of these animals because of workload constraints or the availability of analytical equipment that met calibration requirements. All methods were evaluated and approved by the University of Illinois Institutional Animal Care and Use Committee. The study reported here was part of a series of studies investigating the diagnosis of SCM and IMI in lactating dairy cattle.A farm visit was performed at least once a week to collect foremilk  samples from each quarter separately in the same week of drying off between 12:00 and 16:00 and at 4 to 7 days postpartum  between 12:00 and 14:00. Samples therefore were collected 8 to 12 hours after the previous milking at dry off and 7 to 9 hours after the previous milking at freshening. Somatic cell counts at these time intervals after the previous milking are similar to those obtained immediately before milking, which is the recommended time for accurate interpretation of somatic cell count (SCC) values.2.2The reference method for determining SCC in our study was the DCC , which is a portable automatic cell counter with a reported measurement range of 10 000 to 4 000 000 cells/mL and repeatability (coefficient of variation [CV]) of 12% at 100 000 cells/mL and 8% at 400 000 cells/mL. The DCC method has been validated for bovine milk at 4 and 37°C against the Fossomatic and direct microscopic methodsSamples were measured using the DCC at the University of Illinois Dairy Research Farm as recommended by the manufacturer. Approximately 1 μL of foremilk was drawn into the single‐use cassette and inserted into the DCC analyzer. The result was shown on the display after 45 seconds and indicated the number of cells/μL of milk; this number was multiplied by 1000 to provide cells/mL of milk.Milk EC and electrolyte concentrations were measured on quarter milk samples within 4 hours of collection; the delay was related to workload constraints during the course of the study. Milk samples were transported back to the laboratory and placed in water bath at 37°C for at least 30 minutes to approximate milk temperature when used as a cow‐side or on‐farm test. Portable meters were used for measuring milk EC , and Na , K , and Ca  concentrations in quarter foremilk samples according to the manufacturer's recommendations. These hand‐held meters were suitable for cow‐side or on‐farm use  as described elsewhere.2.3Milk culturing was performed based on National Mastitis Council recommendations2.4P < .05 was considered significant. A minimum sample size of 300 quarters at dry off and freshening was identified as a goal based on what was logistically possible because of time and resource constraints balanced against the need for an adequate number of quarters to satisfactorily evaluate the diagnostic test performance of a new test for SCM or IMI. The measured ionized Ca concentration (cCa2+m) was corrected for milk pH using the slope value ([ΔcCa2+] /ΔpH = 2.714) from an equation developed for cow's milk at 37°C relating cCa2+ to measured pH (pHm)pH = 6.60) therefore was: cCa2+pH = 6.60 = cCa2+m + 2.71 × (pHm − 6.60).Statistical software programs  were used for all analyses and cCa2+pH = 6.60 measured by Horiba meters on quarter milk samples.The least squares mean cisternal milk EC, ionized Na, K, and Ca concentrations, and the 95% confidence interval (CI) for the same 4 variables in healthy quarters (SCC < 100 000 cells/mL)Corynebacterium spp. infection). Intramammary infection was defined on a quarter basis as the presence of MG and SCC ≥ 100 000 cells/mL; our definition for IMI therefore reflects the isolation of a pathogen from glandular secretion with evidence of inflammation. The SCC cut point for our IMI definition was selected because quarter SCC < 100 000 cells/mL reflects an internationally accepted definition of udder quarter health.Subclinical mastitis was defined on a quarter basis as SCC ≥ 200 000 cell/mL because it is the most frequently used method and cutoff for diagnosing SCM, providing maximum sensitivity (Se) and Sp and minimal diagnostic error.cCa2+pH = 6.60 measured by the Horiba meters at dry off and freshening. Logistic regression was conducted on a quarter basis without consideration of cow effect reflecting interdependence of quarters. This analytical approach was selected because the practical implementation of selective dry cow antimicrobial administration should be based on the results of diagnostic testing of quarter samples without adjustment for cow factors.Binary logistic regression (PROC LOGISTIC) was used to characterize the relationship between SCM  or IMI , and milk EC, as well as Na and K concentrations and P‐value of <.05 indicates that the test result is significantly better than a chance result. The AUC values for different predictors were compared using a nonparametric approach.Receiver operating characteristic (ROC) curves were constructed for each logistic regression model and the area under ROC curve (AUC) and 95% Wald confidence limits for the AUC were calculated. The AUC provides a global index of diagnostic test performance and a Wald 33.1Quarter milk samples were obtained at dry off from 115 cattle, comprising 102 Holstein Friesian, 9 Jersey, 2 Ayrshire, 1 Brown Swiss, and 1 Milking Shorthorn. The median SCC at dry off  was 356 000 cells/mL. Quarter milk samples were obtained at freshening from 92 cattle, comprising 81 Holstein Friesian, 8 Jersey, 1 Ayrshire, 1 Brown Swiss, and 1 Milking Shorthorn. The median SCC at freshening  was 113 000 cells/mL.Of the 459 quarters sampled at dry off, 14% (n = 63) were considered healthy (SCC < 100 000 cells/mL), 68% (n = 312) had SCM, and 25% (n = 113) had IMI. The percentages summed to >100% because quarters could have both SCM and IMI. Of the 364 quarters sampled at freshening, 46% (n = 166) were considered healthy, 33% (n = 120) had SCM, and 17% (n = 61) had IMI. The percentages summed to <100% because some quarters had a SCC ≥ 100 000 cells/mL but <200 000 cells/mL with no MG.cCa2+pH = 6.60 and SCC are provided at dry off  mS/cm, 28  mmol/L, 36  mmol/L, and 3.0  mmol/L, respectively  mS/cm, 17  mmol/L, 44  mmol/L, and 2.9  mmol/L, respectively .The median and 2.5%‐97.5% CI for milk EC, Na and K concentrations, and  4.5 CI, .5‐5.6 mS3.2P = .06) from that for EC at dry off but was greater (P = .01) than that for EC at freshening. The AUC for milk K concentration was lower (P = .02) than that for EC at freshening. The AUC for milk Na concentration for diagnosing SCM was greater than that for milk K concentration at dry off (P = .004) and freshening (P < .001). The AUC for cCa2+pH = 6.60 at dry off and freshening were lower than that of EC, Na concentration, and K concentration (P < .001). Furthermore, the Wald P values for AUC for cCa2+pH = 6.60 at dry off (P = .21) and freshening (P = .66) indicated that ionized Ca concentration was not predictive of SCM. The κ values indicated moderate agreement among cisternal milk EC > 5.2 mS/cm, Na concentration > 39 mmol/L, and K concentration <29 mmol/L as diagnostic tests for SCM at dry off, and moderate agreement between milk EC > 4.9 mS/cm and Na concentration >24 mmol/L as diagnostic tests for SCM at freshening.Values for the optimal cut point, AUC, Se, Sp, +LR, and κ for diagnosing SCM based on the results of logistic regression analysis are presented in Table 3.3P = .02). The κ values indicated poor agreement for cisternal milk EC >5.0 mS/cm, Na concentration >33 mmol/L, and K concentration <23 mmol/L as diagnostic tests for IMI at dry off, fair agreement for cisternal Na concentration >24 mmol/L as a diagnostic test for IMI at freshening, and poor agreement between cisternal milk EC >4.7 mS/cm and K concentration <39 mmol/L as diagnostic tests for IMI at freshening.Values for the optimal cut point, AUC, Se, Sp, +LR, and κ for diagnosing IMI based on the results of logistic regression analysis are presented in Table 3.4Klebsiella pneumoniae and Serratia marcescens) and Gram‐positive cocci . Ten quarters had Prototheca spp. isolated, 8 quarters had coagulase‐negative staphylococci isolated, 4 quarters had yeast, 3 quarters had a Gram‐positive rod isolated , and 2 were considered contaminated. The median milk EC, Na and K concentrations, and cCa2+pH = 6.60 in quarters with clinical mastitis were 5.8 mS/cm, 52 mmol/L, 33 mmol/L, and 2.0 mmol/L, respectively . The primary isolates in the 74 quarters were Gram‐negative rods . This association does not necessarily mean EC is a useful diagnostic test, because such tests should have sufficiently high Se and Sp and therefore +LR >10 to provide clinically useful predictions of the presence of SCM. Our results indicated that the median EC for milk from healthy quarters at 37°C was 4.5 mS/cm, and that EC provided a moderately accurate method for diagnosing SCM at an optimal cut point of >5.2 mS/cm at dry off (moderate agreement based on κ = 0.47) and >4.9 mS/cm at freshening (moderate agreement based on κ = 0.40). Our findings agreed with another report that the mean milk EC of healthy cows was 4.9 mS/cm at temperatures approximating rectal and milk temperature, whereas mean milk EC increased to 5.4 and 6.4 mS/cm in cows with SCM or clinical mastitis, respectively.A positive relationship between milk EC and SCC has been demonstrated in many studies,Sodium is a plasma electrolyte that passes into milk at low levels of cellular injury,Staphylococcus aureus‐infected quarters with SCC of 250 000‐500 000 and >750 000 cells/mL milk, respectively.Potassium is the predominant mineral in milk from healthy quarters on a mmol/L basis.Mammary secretory cells have pumps in their basolateral membranes that pump Na into the extracellular fluid and K into the cells, whereas Na and K are passively transported across the apical membrane of secretory cells into milk.Our results indicated that the cisternal milk ionized Ca concentration corrected to a standardized pH of 6.60 was not predictive of SCM. Milk pH increases with increased milk SCC,We elected not to report predictive values of a positive test (PV+) or a negative test (PV−) in our study. These 2 indices are influenced by the prevalence of disease in the sample population and consequently PV+ and PV− can only accurately summarize test performance when the study population is representative of the total population. In contrast, estimates for Se, Sp, and +LR are characteristics of the test itself and are not influenced by disease prevalence.The major limitation of our study was that it was based on only 1 herd with the most common isolates being environmental mastitis pathogens. We sampled foremilk after removal of approximately 10 mL of milk but before alveolar milk could mix with cisternal milk.The proportion of cattle of various dairy breeds in our study approximates that of the US dairy industry, and consequently our results should be generalizable to dairy cattle in the United States. However, a potential limitation of our study was that we ignored the effect of sample interdependence as a result of quarters being clustered within cows. As such, the reported CI for Se and Sp in our study underestimated the true CI obtained using random effects logistic regression analysis.It is of interest to compare the results of the study reported here for diagnosing quarters with SCM to our findings in this study populationAuthors declare no conflict of interest.Authors declare no off‐label use of antimicrobials.The study had University of Illinois at Urbana IACUC approval number 15080.Authors declare human ethics approval was not needed for this study.


Total knee arthroplasty is regarded as the most effective treatment for severe knee osteoarthritis. The influential factors of blood loss in total knee arthroplasty remain controversial. The study aims to explore the influential factors of blood loss in total knee arthroplasty comprehensively.Three hundred and four osteoarthritis patients undergoing unilateral primary total knee arthroplasty were enrolled. Demographic characteristics, laboratory results, surgical protocol, and hemostatic and anticoagulation drugs were collected. Estimation of blood loss was calculated using the Gross equation. Multivariable stepwise linear regression analysis was performed to find out the influential factors.P < 0.05). Male appeared to suffer more surgical blood loss than female. Posterior cruciate stabilizing prosthesis might lead to more surgical blood loss than posterior cruciate retaining prosthesis. Tranexamic acid could effectively reduce total blood loss while drainage might increase bleeding. Gender and anticoagulation drugs were correlated with hidden blood loss (both P < 0.05). Low molecular weight heparin resulted in less hidden blood loss than rivaroxaban.Total blood loss reached the biggest volume (1346 ± 671 mL) in the post-operative third day. Hidden blood loss reached 465 ± 358 mL. Gender, tranexamic acid, prosthesis type, and drainage were proven to be positively correlated with the total blood loss (all Posterior cruciate retaining prosthesis and topical use of tranexamic acid were preferred to reduce total blood loss. Drainage was not recommended due to the risk of increasing bleeding. Low molecular weight heparin was recommended to prevent venous thrombosis. Symptomatic knee osteoarthritis (OA) is highly prevalent amongst older people worldwide, and the morbidity even reached 30%, especially in rural regions . Total kIdentifying influential factors of surgical blood loss is an important step toward establishing an effective blood management strategy and further reducing the need for perioperative blood transfusion. However, the existing results of influential factors in OA patients undergoing TKA remain controversial. Cushner and Friedman found that gender of the patients was positively related to blood loss, but excluded the correlation of age, diagnosis, and operative time . AnotherIn the present study, 304 cases undergoing unilateral TKA in our department from the year 2011 to 2016 were enrolled in the study. Excluding from the influence of diagnosis, we only included patients diagnosed as having knee osteoarthritis. Gross equation was adopted to estimate the volume of surgical blood loss. Multivariable stepwise linear regression analysis was performed to explore the influential factors, in which we included as many risk factors as possible. The potential risk factors include gender, BMI, coagulation indexes, electrolyte, surgical protocol, and hemostatic and anticoagulation drugs. The study aims to comprehensively explore the exact factors influencing blood loss in total knee arthroplasty surgery.Patients who were diagnosed with knee OA and underwent unilateral TKA from January 2011 to June 2016 were included in the study. Those with the following conditions were excluded, including rheumatoid arthritis, ankylosing spondylitis, Kashin Beck disease, active infection, tumor, severe cerebrovascular disease, liver disease with severe abnormal liver function, and hematologic diseases like coagulation dysfunction (such as hemophilia) and thrombocytopenia. In addition, patients with long-term use of anticoagulant drugs were also excluded. For hypertensive patients, the preoperative blood pressure was controlled to beneath 140/90 mmHg.2, which was defined as overweight. The different protocol of the patients was described in Table Finally, 304 patients consisting of 61 males and 243 females were included in the study. The average age was 56.8 years, ranging from 42 to 80. The average BMI was 26.46 kg/mSevere pain affecting daily activity, dysfunction of joint motion, and radiography evidence that narrowing joint space with cystic change of subchondral bone, bone sclerosis and formation of osteophyma, were the main indications for OA patients to receive TKA after invalid conservative treatment.Standard surgical methods were adopted for all patients , and all the surgeries were performed in the supine position by the same experienced team. General anesthesia or spinal anesthesia was performed. Tourniquet was commonly used during the operation, and the pressure was set in 60 kPa. Cephalosporins were routinely applied for 24 h after operation to prevent infection. Two kinds of prostheses included posterior cruciate retaining (CR) prosthesis and posterior cruciate stabilizing (PS) prosthesis. The material of plugging the location hole of femur included cement and bone. Autologous re-transfusion drainage regimen was applied in the study, which had beneficial effects of higher post-operative hemoglobin and lower allogenic transfusion rate , 17. ThePhysical exercise and continuous passive motion exercise device (CPM) were applied on post-operative day 1 until discharge. Physical exercises were performed twice daily according to standard guidelines , 19, incAge, sex, height, and weight were recorded. Both preoperative hemoglobin (Hb) and hematocrit (Hct) and post-operative results in the first, third, and fifth days after operation were collected. Preoperative laboratory test results including prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen concentration (Fbgc), albumin (ALB), kalium, natrium, calcium, phosphorus, and magnesium were collected. Prosthesis type, material of plugging the location hole of femur, administration of TXA, HCA, and anticoagulant drug were also recorded.2 × (preoperative Hct – post-operative Hct)/(preoperative Hct + post-operative Hct). In which PBV = k1 × [height (m)]3 + k2 × body weight (kg) + k3. For males, k1 = 0.3669, k2 = 0.0329, and k3 = 0.6041; for females, k1 = 0.3561, k2 = 0.033 08, and k3 = 0.1833. The total blood losses of the first, third, and fifth days after the operation were calculated. In addition, the increased blood losses between the first day and third day after operation were calculated, which could be considered as hidden blood loss after operation.Total blood loss was estimated with Gross equation . The actBoth allogenic transfusion and autologous transfusion were employed during the operation and post-operatively, which was based on the condition of patients. The post-operative draining blood was collected for autologous transfusion that was performed 6 h after operation with an autologous negative pressure drainage device, ConstaVac TM CBC II . The allogenic transfusion triggers were based on the British guideline  and clinTwenty-two kinds of factors were divided into quantitative and quantitative variables. Multivariable stepwise linear regression was used to find out the impact factors of blood loss. In consideration of the peak of post-operative blood loss, the blood loss in the third day after TKA was chosen as the dependent variable. The continuous blood loss between the first day and third day after operation also attracted our attention. That was also set as a dependent variable.Fifteen quantitative variables were included: BMI, PT, APTT, TT, Fbgc, preoperative Hb, preoperative HCT, ALB, kalium, natrium, calcium, phosphorus, magnesium, age, and operation time. Seven qualitative variables were included in the multiple linear regression analysis. For the gender, the male was set as “1” and female was “0.” For the different prosthesis type, “CR” was set as “0” and “PS” was set as “1.” For the utilization of drainage, “without drainage” was set as “0” and “with drainage” was set as “1.” For the material of plugging the location hole of femurs, “nothing” was set as “0,” “cement” was set as “1,” and “bone” was set as “−1.” For the use of tranexamic acid, four subgroups were set. “Not use” was set as “1”, and the rest was a dummy variable. Same method was used for “topical application,” “intravenous application,” and “combined application.” For the use of hemocoagulase agkistrodon, “not use” was set as “0” and “use” was set as “1.” For the application of anticoagulation drugs, “LMWH” was set as “1” and “Rivaroxaban” was set as “0.”The total blood loss in the first, third, and fifth days after TKA and the increased blood loss volume were described in Table More than half of the patients (51.55%) have received transfusion within 3 days after operation, including allogenic transfusion, autologous transfusion, and both. The allogenic transfusion rate reached 35.09%, and the average volume was 0.9 u (0~6 u). 31.68% of the patients received autologous transfusion that reached 169.09 mL in average, of which 49 patients received allogenic transfusion as well. No complications associated with transfusion were found in the study.P < 0.001), without TXA (P < 0.001), drainage (P = 0.019), and prosthetic type (P < 0.001) were positively correlated with total blood loss of TKA, but the rest of the potential risk factors presented a negative result. Male appeared to suffer more surgical blood loss than female. PS prosthesis might lead to more surgical blood loss than CR prosthesis. Tranexamic acid could effectively reduce total blood loss while drainage might increase bleeding. However, different strategies of using TXA showed no effect on blood loss. The regression model was shown in Fig. The result of the multivariable stepwise linear regression analysis showed that gender P < 0.00, withoutP < 0.001) and different anticoagulation drugs (P = 0.009) were correlated with the hidden blood loss. Similarly, more blood loss was found in the male. Patients who received rivaroxaban suffered more post-operative bleeding. The regression model was shown in Fig. Another result showed that gender  than the control group, and the incidence reached 6.3% while it was 3.8% with topical use . In addiThe continuous blood loss after operation, or named hidden blood loss, is also a problem that cannot be neglected. The hidden blood loss reached 465 mL in our study. Some scholars suggested that hidden blood loss might be correlated with hemolysis, blood infiltration into tissue compartments, and the use of anticoagulant drugs , 39. To Different from rivaroxaban that inhibits clotting factor Xa, the major anticoagulant effect of heparin is through its interaction with antithrombin-III (AT-III). This interaction markedly accelerates the ability of AT-III to inactivate the coagulation enzymes thrombin, factor Xa, and factor IXa. Moreover, LMWH, the fragments of standard commercial grade heparin produced by either chemical or enzymatic depolymerization, have reduced ability to catalyze the inactivation of thrombin relative to their ability to inhibit factor Xa because the inactivation of thrombin by heparin is critically dependent on molecular size . ResearcThe total blood loss reached 1346 ± 671 mL in the third day after TKA. The potential influential factors of total blood loss included gender, TXA, prosthesis type, and drainage. Male was considered to suffer more surgical blood loss than female. Perioperative blood management should be enhanced in male patients. Posterior cruciate retaining (CR) prosthesis might be superior in reducing total blood loss of TKA. Drainage was not recommended due to the risk of increasing bleeding. Tranexamic acid was proven to effectively reduce total blood loss, and the topical use was preferred. The hidden blood loss was 465 ± 358 mL, and its impact factors mainly included gender and the different use of anticoagulation. As for the selection to prevent DVT between the low molecular heparin and rivaroxaban, the former was recommended since it might lead to less hidden blood loss.


This post hoc analysis of an 18‐week randomized trial explored the utility of calculating baseline glycated haemoglobin (HbA1c), postprandial glucose (PPG) increments and nocturnal glucose change in predicting efficacy and safety outcomes in response to bolus insulin intensification in people with type 2 diabetes (T2D). Analyses were conducted on 236 participants with T2D receiving metformin: 116 received fast‐acting insulin aspart (faster aspart) basal–bolus therapy and 120 received basal‐only insulin. Participants were grouped according to baseline HbA1c, PPG increments and nocturnal glucose change variables; analyses were performed on the end‐of‐trial treatment differences between “high” and “low” baseline values. The change from baseline in end‐of‐trial mean HbA1c and mean PPG increments was in favour of faster aspart across all subgroups. Significantly greater treatment differences were observed in participants with high (vs. low) baseline nocturnal glucose change and PPG increments. For baseline HbA1c, significantly greater treatment differences were observed for change in end‐of‐trial PPG increments, but not end‐of‐trial HbA1c. In conclusion, both nocturnal glucose change and PPG increments may be more useful than HbA1c for identifying subgroups of people with T2D who would most benefit from bolus intensification. This post hoc analysis explored the utility of HbA1c, PPG increments and nocturnal glucose change, all at baseline, in predicting efficacy and safety outcomes in participants who intensified basal insulin treatment with mealtime faster aspart.2ClinicalTrials.gov: NCT01850615), an 18‐week, multicentre (51 sites across six countries), open‐label, randomized trial that compared the efficacy and safety of fast‐acting insulin aspart (faster aspart) intensification in a basal–bolus regimen (n = 116) versus continued basal‐only insulin (n = 120), both in combination with metformin.The present study was a post hoc analysis of the onset 3 trial  on clinical outcomes in T2D. Clinical outcomes included in this analysis were change from baseline in HbA1c and mean PPG increments at the visit 18 weeks post‐randomization and the rates of treatment‐emergent severe or blood glucose‐confirmed hypoglycaemia. “Treatment‐emergent” was defined as an event that had an onset up to 1 day after the last day of randomized treatment, but excluded any events that occurred during the run‐in period. Severe or blood glucose‐confirmed hypoglycaemic episodes were defined with a plasma glucose value <3.1 mmol/L (56.0 mg/dL), with or without symptoms consistent with severe hypoglycaemia.To explore the predictive utility of baseline HbA1c, PPG increments and nocturnal glucose change, analyses were performed on the treatment outcome differences  at week 18, comparing “high” and “low” baseline values of each variable. “High” and “low” were defined as being above or below the median value at baseline, respectively. The median values for each variable at baseline were: 61.7 mmol/mol (7.8%) for HbA1c; 2.42 mmol/L (43.6 mg/dL) for PPG increment; and 3.11 mmol/L (56 mg/dL) for nocturnal glucose change. The correlation between these variables at baseline was also investigated.The PPG increments were calculated as the difference between SMBG readings 2 hours post‐meal and pre‐meal, and these were recorded from seven‐point profiles over a 3‐day period, and averaged over time. Nocturnal glucose change was calculated as the difference between SMBG at bedtime and pre‐breakfast the following day. From seven‐point SMBG profiles over a 3‐day period, two values could be calculated with a complete dataset, which were averaged to calculate mean nocturnal glucose change.Definitions of the analysis sets, and details of the statistical models and tests, are provided in 33.1At baseline, there was a positive correlation between PPG increment and nocturnal glucose change (r = 0.66) and between HbA1c and nocturnal glucose change (r = 0.20). There was a small correlation between PPG increment and HbA1c (r = 0.11).3.2P = 0.0006) and PPG increment (P = 0.0099) than in those with “low” values. There was no statistically significant treatment difference in change in HbA1c (P = 0.533) between participants with “high” and “low” baseline HbA1c values , nocturnal glucose change (P < 0.0001) and PPG increment (P = 0.0141) than those with “low” baseline values  and nocturnal glucose change measurement >3.11 mmol/L (56 mg/dL) experienced greater benefit, in terms of reduction in HbA1c and PPG increment at end‐of‐trial, with basal–bolus therapy versus basal‐only therapy compared with those participants with measurements under these cut‐off values. Those with baseline HbA1c >61.7 mmol/mol (7.8%) also experienced greater benefit from basal–bolus therapy, but only in terms of reduction in PPG increment. These findings suggest, in this study population, that baseline values of nocturnal glucose change and PPG increment may be better predictors for response to intensification with bolus insulin than baseline HbA1c.A proof‐of‐concept study, exploring the potential of the nocturnal glucose change measure, termed “BeAM”, as an indicator to intensify treatment targeting postprandial hyperglycaemia in people inadequately controlled on basal insulin, concluded that BeAM values between 2.5 and 3.1 mmol/L (45 and 55 mg/dL) should trigger the consideration of intensification.In addition to monitoring HbA1c, healthcare professionals should consider measuring nocturnal glucose change and/or PPG increment as these factors may provide additional information and, based on the preliminary findings here, appear to be predictive of positive glycaemic response to bolus intensification. Nocturnal glucose change is easier to obtain and calculate in clinical practice than PPG increment. Because of the correlation between PPG increment and nocturnal glucose change demonstrated in the present study, nocturnal glucose change might be a reasonable alternative to measuring PPG increments. This would be less inconvenient to the patient and of great value to physicians. Although nocturnal glucose change is easily calculated, a number of factors could influence the bedtime and pre‐breakfast values ; therefore, physicians would still need to instruct the patient on how best to obtain the values necessary to calculate nocturnal glucose change and maintain consistency in this technique as it is utilized to monitor dose titration.There are some study limitations to consider. The analysis is exploratory in nature and based on one study alone. The findings are based on clinical trial data with faster aspart, a new prandial insulin analogue, on a background of three different basal insulins, and thus results may not be easily generalized. The influence of other factors on the bedtime and pre‐breakfast glucose measurements (listed above), and thus on the nocturnal glucose change value, was not analysed. Nevertheless, the findings reported in the present paper warrant further research into the use of nocturnal glucose as an accessible and clinically useful predictor for insulin intensification. Further studies would benefit from a standardized approach to measuring nocturnal glucose change, preferably using continuous glucose monitoring to capture the full range of glycaemic values while on treatment.In conclusion, in the present trial population, the analysis suggests that PPG increment and nocturnal glucose change measurements may be more useful predictors than HbA1c for identifying people with T2D on basal insulin therapy, with or without oral antidiabetic drugs, who would most benefit from intensification with bolus insulin. Further, nocturnal glucose change may indeed perform just as well as PPG increment, while being easier to measure. The clinical applicability and potential use of nocturnal glucose change as a predictor for insulin intensification in T2D warrants further exploration.A.L.P. has served on advisory boards for Abbott Diabetes Care, Becton Dickinson, Bigfoot, Eli Lilly and Company, Lexicon, Livongo, Mannkind, Medscape, Merck, Novo Nordisk, Omada Health, Sanofi and Zafgen, has served on a speakers' bureau for Novo Nordisk and received research funding from AstraZeneca, Dexcom and Mannkind. M.P. has participated in advisory panels for Novo Nordisk, Boehringer Ingelheim and Eli Lilly. J.E. is an employee of and holds stock in Novo Nordisk. K.S.‐S. is an employee of and holds shares in Novo Nordisk. J.S. is an employee of and holds stock in Novo Nordisk. K.B. has participated in advisory panels for AstraZeneca, Boehringer Ingelheim, Eli Lilly, Merck, Novo Nordisk, Sanofi, Johnson & Johnson, and speakers' bureau for Novo Nordisk and Sanofi.A.L.P. was the principal investigator. A.L.P. and K.B. are the guarantors of this work and, as such, had full access to all data in the study and take responsibility for the integrity of the data and accuracy of the data analysis. J.E. was the responsible statistician. All authors had access to the study data, take responsibility for the accuracy of the analysis, reviewed and contributed to the content of this manuscript, and had authority in the decision to submit this manuscript for publication, in collaboration with Novo Nordisk. All authors approved the manuscript for publication.Figure S1 Onset 3 trial design.Figure S2: Severe or BG‐confirmed hypoglycaemic episodes stratified by baseline parameters.Table S1: Baseline characteristics at randomization of the onset 3 trial population.Appendix S1 Supporting InformationClick here for additional data file.


After being capped on the rat sciatic nerve stump in vivo, rodent behaviors and tissue structures are compared via autotomy scoring and histological analyses. The PRGD/PDLLA capped group gains lower autotomy score and improves the recovery, where inflammatory infiltrations and excessive collagen deposition are defeated. Transmission electron microscopy images of the regeneration of myelin sheath in both groups show that abnormal myelination is only present in the uncapped rats. Changes in related genes  are monitored quantitative real‐time polymerase chain reaction (qRT‐PCR) for mechanism investigation. The PRGD/PDLLA capping conduits not only act as physical barriers to inhibit the invasion of inflammatory infiltration in the scar tissue but also provide a suitable microenvironment for promoting nerve repairing and avoiding neuroma formation during nerve recovery.Neuroma formation after amputation as a long‐term deficiency leads to spontaneous neuropathic pain that reduces quality of life of patients. To prevent neuroma formation, capping techniques are implemented as effective treatments. However, an ideal, biocompatible material covering the nerves is an unmet clinical need. In this study, biocompatible characteristics presented by the poly/arginylglycylaspartic acid (RGD peptide) modification of poly{(lactic acid)‐ The terminal nerve stump is shortened to a non‐neuroma site, but a new neuroma is prone to relapse at the new lesion site.Recently, a biocompatible RGD‐functionalized artificial conduit was synthesized for nerve regeneration, in which no neuroma was formed after a 5 week implantation. Additionally, the functionalized RGD peptide showed the ability to attenuate inflammation reaction, which was crucial for painful neuroma development.22.1FigureP < 0.01), especially from the 5th to the 8th week. The average autotomy score in PRGD/PDLLA capped conduit group was 1.0 ± 0.2, while the average score of noncapped group (2.5 ± 0.3) was a nearly two times higher value. The trend of α‐SMA expression was mainly consistent with the autotomy score assessment. As a neuropathic pain related marker, quantitative real‐time polymerase chain reaction (qRT‐PCR) results indicated that its transcriptional level was distinctly lower in PRGD/PDLLA capped group  as compared to noncapped group  at both intervals  was significantly larger than that of PRGD/PDLLA capped group . On the contrary, the number of macrophages in noncapped group was increased from the 2nd to 8th weeks  and was higher than PRGD/PDLLA capped group  was distinctly higher than PRGD/PDLLA capped group , respectively, at both time intervals. The expression of IL‐1β demonstrated a different pattern. A higher level of IL‐1β was detected in PRGD/PDLLA conduit group at the 2nd week and subsequently downregulated . In noncapped group, IL‐1β was upregulated .Immunohistochemistry was employed to determine the degree of inflammation infiltration at the lesion site, in which T cells and macrophages were analyzed Figure2A. The2.3FigureMasson's trichrome staining showed that the nerve fibers in noncapped group were arranged irregularly and haphazardly, appearing with an expansion of collagen deposition (blue) and clotted blood (red) at 2 weeks Figure3A2,A2'Figure2 and 0.91 ± 0.06 mm2) was significantly higher than that of PRGD/PDLLA capped group  in both intervals  was still smaller than that of noncapped group  while it was downregulated and similar values were achieved in both groups at the 8th week.Sirius red staining was utilized to evaluate collagen formation and distribution by polarized light microscopy Figure4A. Res2.4Figure2 were randomly selected for analysis. The ratio of unmyelinated and myelinated fiber, diameter of myelinated fiber, and thickness of myelin sheath were measured from the selected micrographs and statistically analyzed. The diameters of axons in PRGD/PDLLA capped group (3.71 ± 0.36 µm to 4.10 ± 0.14 µm) were longer than that of noncapped group (2.90 ± 0.33 µm to 3.10 ± 0.19 µm) at both intervals  imaging to evaluate effects of PRGD/PDLLA capping conduits on remyelination Figure5A. MicFigureP < 0.01), while the expression of MBP in noncapped group notably decreased at the 8th week . For gene Krox20, expression in capped group was slightly lower than control at the 2nd week , while it became higher than noncapped group  at the 8th week .To investigate whether the expressions of myelin maturation related genes were changed after capping with PRGD/PDLLA conduit, qRT‐PCR analyses were carried out and genes MBP, Krox20, MPZ, and MAG were tested. 3Since the first successful report of peripheral nerve repair in 1836, it has been recognized that not all the nerve injures were amenable to direct end‐to‐end repair as the elasticity was limited.In the present work, a rat nerve terminal resection model was established and autotomy scoring was employed to evaluate the effects of PRGD/PDLLA capping conduits on pain suffering. As a response to denervation in the sciatic nerve, rats lick, scratch, and self‐mutilate their limb, which could be quantified via a modified Wall scale.The analysis of inflammation reactions in the two groups showed that the role of inflammation for peripheral nerve injury was binary that moderate inflammation was essential during the process of regeneration; however, it was also the main source of pain. At the early stage of inflammation, the release of chemokines and increase in the permeability of blood–nerve barrier were accomplished simultaneously.Due to the close connection between neuroma formation and fibrosis, histological samples in the two groups (PRGD/PDLLA capped group and noncapped group) were investigated. Without the protection of PRGD/PDLLA conduit, typical characteristics of neuroma could be demonstrated in the control rats: haphazard patterns of regenerated axons and high degree of collagen deposition, which were the sequel of extraordinary fibrosis.[[qv: 33b]] As another essential event for wound healing, the development of fibrosis was supposed to maintain the origin architecture of tissue and provide mechanism support for axons sprouting.Effects of PRGD/PDLLA conduit on remyelination were also evaluated. Conduits were able to defend the nerve fiber, and the axons were protected by myelin within the complicated cable. Myelin sheath wrapped axons in a spiral fashion and acted as an insulator.4We have evaluated the effects of PRGD/PDLLA capping conduits on inhibiting the formation of painful neuroma by the analysis of behavior, histological structure, and relative gene expression. The results indicated that inflammatory response, autotomy score, and scar deposition were reduced in the conduit group. Moreover, the myelin morphology of nerve stump capped with the PRGD/PDLLA capping conduits was thick and compact in TEM observation and the relative genes of myelin maturity were also upregulated. These factors contribute to the inhibition of painful neuroma. These changes may be due to the incorporation of RGD‐modified capping materials, which provided a favorable microenvironment for the nerve terminal recovery by inhibiting inflammatory amplification. Therefore, PRGD/PDLLA capping conduits could be a promising alternative used in capping nerve ends to inhibit neuroma formation.5Materials: Poly (PDLLA) (Mw: 250 000) was synthesized in house. Gly‐Arg‐Gly‐Asp‐Gly (RGD) and 1, l‐carbonyldiimidazole were purchased from GL Biochem (China). 1‐Ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide, Masson's trichrome, and sirius red were purchased from Sigma‐Aldrich. Other chemicals were analytical grade. 3‐0 and 6‐0 monofilament nylon sutures were purchased from Ethicon, Inc. (Johnson & Johnson). SD rats were obtained from the Center for Disease Control and Prevention of Hubei Province (China). Epon 812 was purchased from Electron Microscopy Sciences . Paraformaldehyde, paraffin, glutaraldehyde solution, osmium tetroxide, lead citrate, and uranyl acetate were purchased from Sinopharm Chemical Reagent Co., Ltd (China).Preparation of PDLLA/PRGD Capping Conduit: The RGD‐modified poly{(lactic acid)‐co‐[(glycolic acid)‐alt‐(L‐lysine)]} (PRGD) was synthesized. First, (3S)‐3‐[4‐(benzyloxycarbonylamino) butyl] morpholine‐2, 5‐dione (BMD) was synthesized by bromoacetyl bromide and Ne‐(benzyloxycarbonyl)‐L‐lysine. Second, poly{(lactic acid)‐co‐[(glycolic acid)‐alt‐(Ne‐benzyloxycarbonyl‐L‐lysine)]} was obtained by copolymerization of D,L‐lactide and BMD. Then, poly{(lactic acid)‐co‐[(glycolic acid)‐alt‐(L‐lysine)]} (PLGL) was synthesized by the catalytic hydrogenation of PLGL. Finally, PLGL was modified with RGD peptide. PRGD (0.05 g) and PDLLA (0.95 g) were dissolved in ethyl acetate (5 wt%). The PRGD/PDLLA membrane was produced by a model mold and then rolled to fabricate PRGD/PDLLA capping conduit with one end closed. The final length of the capping conduit was 10 mm with an inner diameter of 2 mm and a tube wall thickness of 200 µm. The nerve capping conduits were sterilized with ultraviolet light for subsequent experiments and implantations.Rat Model and Surgical Procedure: Experiments undertaken in this study were approved by the Animal Care and Use Committees of Wuhan University of Technology and conformed to NIH guidelines. Adult Sprague‐Dawley rats (180–200 g) were randomly divided into two groups each with 12 rats: Group A: noncapped control group; Group B: PRGD/PDLLA capped group. Each rat was anesthetized with an intraperitoneal injection of 50 mg kg−1 body weight pentobarbital sodium. The right sciatic nerve was exposed and sharply transected into proximal and distal segments at the center of the right thigh. In the control group A, the proximal nerve stump was put aside without any treatment, but the distal stump was removed for 1 cm to avoid spontaneous nerve regeneration. In group B, the proximal stump was sutured with 6–0 monofilament nylon sutures to a depth of 2 mm into the PRGD/PDLLA capping conduit; the distal stump was also removed as in group A. In both groups, the muscle and skin layers were re‐approximated with 3–0 nylon sutures , and then were dehydrated with ethanol, embedded in paraffin, cut into 5 µm thickness, and stained with Masson's trichrome and sirius red for imaging by inverted microscopy . To analyze myelination morphology, the nerve specimens were fixed in glutaraldehyde solution (2.5% v/v). The samples were postfixed with osmium tetroxide (1% w/w) for another 2–3 h, dehydrated by graded ethanol, embedded in Epon 812, and sliced to 60 nm. The sections were stained with lead citrate and uranyl acetate, and observed by a TEM . Photographs of each nerve sample were quantitatively analyzed by ImageJ software .Immunohistochemical Analyses: The nerve paraffin sections of proximal stumps were used to detect T lymphocyte and macrophage via CD3 and CD68 immunohistochemical staining. The nerve paraffin sections were cut into 5 mm thickness, dewaxed in xylol, and dehydrated in ethanol, followed by blocking with goat serum at 24 °C for 30 min. The sections were, respectively, incubated in a solution containing anti‐CD3 primary antibody  and anti‐CD68  at 4 °C overnight, and subsequently incubated with biotinylated secondary antibodies, respectively, for 20 min at 37 °C and visualized by diaminobenzidine. Nuclei were counterstained with hematoxylin, and the nerve sections were imaged under an inverted microscope. The positive number of CD3 and CD68 was analyzed by an image analysis system.Quantitative Real‐Time PCR: At the 2nd and 8th weeks after implantation, proximal nerve segments were collected, and changes in the transcription of genes α‐SMA, Krox20, Mag, Mbp, Mpz, TNF‐α, and IL‐1β were studied via qRT‐PCR.TableCt method.Statistical Analysis: Experimental data were processed using PASW Statistics 18 software . All numerical data were expressed as mean ± standard deviation. T‐test was used to determine the significance of the differences between the groups. P < 0.05 was considered as statistically significant.The authors declare no conflict of interest.