Clinicopathological characteristics, outcome and survival were reviewed retrospectively. Results: Donor and recipient characteristics were similar. Rates of post-operative complications and graft dysfunction were significantly higher in the PF group compared with the KF group (episodes of acute rejection within the first year after SPKT: 11 (18%) versus 2 (4.8%); graft pancreatitis: 18 (18%) versus 2 (4.8%), p = 0.04; vascular thrombosis of the pancreas: 9 (14.8%) versus 1 (2.4%), p = 0.03; and delayed graft function of the kidney: 12 (19.6%) versus 2 (4.8%), p = 0.019). The three-month pancreas graft survival was significantly higher in the KF group . No significant difference was observed in pancreas graft survival five years after transplantation . Kidney graft survival was similar between the two groups. Multivariate analysis revealed order of graft implantation as an independent prognostic factor for graft survival three months after SPKT  and five years . Conclusion: Our data indicates that implantation of the pancreas prior to the kidney during SPKT has an influence especially on the early-post-operative outcome and survival rate of pancreas grafts.Background/Objectives: The sequence of graft implantation in simultaneous pancreas-kidney transplantation (SPKT) warrants additional study and more targeted focus, since little is known about the short- and long-term effects on the outcome and graft survival after transplantation. Material and methods: 103 patients receiving SPKT in our department between 1999 and 2015 were included in the study. Patients were divided according to the sequence of graft implantation into pancreas-first (PF,  Simultaneous pancreas-kidney transplantation (SPKT) is an established therapy for patients with insulin-dependent diabetes mellitus, complicated by end-stage renal disease. Successful SPKT leads to euglycemia, which could slow the progression of diabetic microvascular and macrovascular complications and it improves survival rates and recipients’ quality of life compared with patients on dialysis or patients after kidney transplantation alone ,6,7,8,9.However, its success depends on several factors such as the profiles of donors and recipients, methods of implantation techniques, and graft harvesting such as effects of ischemia-reperfusion injuries (IRI) on graft damages ,9,10,11.In this context, pancreas transplantation is associated with a high incidence of post-operative complications and up to 15% graft losses within the first year after SPKT ,9,10,11.Implantation of the pancreas before the kidney seems reasonable to avoid prolonged cold ischemic time and subsequent ischemic reperfusion injury of the pancreas graft, especially since kidney grafts can tolerate cold ischemia better than pancreas grafts . HoweverTherefore, the aim of this study was to determine the impact of graft implantation order on the outcome and survival after SPKT. We analyzed post-transplant outcome characteristics, survival rates, and risk factors for graft failures in SPKT depending on graft implantation order.After approval by the local ethics committee [AZ-Nr: 111-16-14032016] medical data from all patients undergoing SPKT at the University Hospital of Leipzig between 1999 and 2015 were retrospectively analyzed from a prospectively collected electronic data base.Patients were divided into two groups according to the order of graft implantation: (1) pancreas first (PF) and (2) kidney first (KF). The transplantation order was determined based on the ischemia times and implantation time points from the transplantation protocols.Special emphasis was placed on patient and graft characteristics, postoperative complications, metabolic outcomes, renal function, and causes of graft failure depending on graft implantation order.2), cytomegalovirus (CMV)-status, donor cause of death, duration of insulin dependent diabetes mellitus, duration of dialysis, and time on the waiting list. Peri- and post-transplant data included information on cold ischemia time (CIT) and warm ischemia time (WIT) of the grafts, immunosuppressive therapy as well as organ graft function: Duration of operation, rates of re-operation, infectious complications, number of rejection episodes, and delayed graft function (DGF). CIT is defined as time the organ spent in cold preservation solution after removal from the donor. WIT is the time from cross-clamping until cold perfusion, plus the time of implantation (organ out of ice until reperfusion). Surgical complications were defined as the need for relaparotomy within the first three months after transplantation.Characteristics included donor and recipient age, gender, and body mass index  or administration of 8 mg per kg bodyweight anti-thymocyte globulin (ATG) in parallel with increased baseline immunosuppression.DGF of the kidney was defined as the requirement of dialysis in the first week following transplantation .Pancreas graft failure was defined as resumed insulin therapy, removed pancreas, re-transplantation, or patient death.Kidney graft failure was defined as the need for dialysis, removed kidney, re-transplantation, or patient death.Postoperative mortality was considered as in-hospital mortality in all cases.Laboratory parameters of ischemia-reperfusion-injury: Peak of C-reactive protein  and serum lipase (mmol/L) within the first three days; endocrine function: low-density lipoprotein (LDL)-cholesterol/high density lipoprotein (HDL)-cholesterol ratio, HbA1C (%), C-peptide (ng/mL), and renal function: Creatinine (mmol/L) and urea (mmol/L) were analyzed up to five years after transplantation.The procurement and transplantation of pancreas and kidney allografts were performed according to international standards and guidelines as described previously ,16,17,18In short, the pancreas was transplanted into the right iliac fossa using a standard technique with an intraperitoneal location in the right iliac fossa. The Y-graft was anastomosed to the recipient’s common iliac artery, the portal vein was connected to the inferior vena cava of the recipient. Exocrine drainage was carried out with a hand-sutured side-to-side duodenojejunostomy 40 cm beyond the flexure of Treitz ,18. The Immunosuppressive therapy comprised an induction therapy with the interleukin-2 receptor antagonist basiliximab or antithymocyte globulin, followed by a triple maintenance immunosuppression consisting of calcineurin inhibitors (tacrolimus or cyclosporine), and/or antimetabolites (mycofenolate mofetil or sirolimus) and tapered steroids (prednisolone).t-test, χ2, analysis of variance (ANOVA), Kruskal–Wallis, and Wilcoxon–Mann–Whitney test was used. Survival rates were calculated using the Kaplan–Meier analysis and the log-rank test was applied to test statistical significance. Graft survival was calculated as the time from initial transplant to graft failure, censoring for death with a functioning graft, and grafts still functioning at time of analysis. Patient survival is defined as time from transplant to patient death, censoring for patients still alive at time of analysis. If a recipient was alive or lost to follow-up at time of last contact, then survival time was censored at time of last contact. Multivariate analysis was performed with logistic regression analysis. Variables to be entered into the multiple logistic regression analysis were chosen on the basis of the results of univariate analysis. p values < 0.05 were regarded as significant. All statistical analyses were performed by using IBM SPSS Statistics 24.0 .Baseline data are presented as mean values with the standard deviation (SD) such as the proportion percentage (%). For comparison between the two groups, the appropriate statistical significance test including the Student’s p = 0.949). Donor, recipient, and graft characteristics according to the different implantation order are summarized in The overall study population included 103 patients receiving SPKT in our department between 1999 and 2015. In 61 patients (59.2%), the pancreas was implanted before the kidney (PF), and in 42 patients (40.8%) the kidney was implanted first (KF). The mean follow-up period was 9.1 ± 1.2 years , episodes of acute rejection (p = 0.034), rates of graft pancreatitis (p = 0.04), and total rate of vascular thrombosis of the pancreas (p = 0.03) were significantly higher in the PF group.The analysis of post-operative outcome parameters is shown in n = 2) of the patients developed arterial thrombosis and 7.8% (n = 8) of the patients developed venous thrombosis. The majority of thrombosis (n = 8) occurred within four weeks after SPKT. All arterial thrombosis occurred in the PF group (n = 2). Thereby, in one patient the graft could be preserved with re-laparotomy and thrombectomy.In total, 1.9% (p = 0.04). In total, acute rejection occurred in 26 patients (25%) during the complete follow-up period . In 13 patients (50%) acute rejection could be confirmed histologically with renal biopsies. In one case (3.8%) the diagnosis was confirmed with endoscopic biopsies of the duodenal segment of the graft.During the first year after SPKT acute rejection occurred in eleven patients (10.7%) in the PF group and in two patients (1.9%) in the KF group .Within the first three days after transplantation, CRP peak—as an indicator of ischemia-reperfusion injury, such as peak of lipase—was also significantly higher in the PF group in comparison with the KF group  compared with the KF group  (p = 0.496). The causes of death included multiple organ failure (n = 2), septic shock (n = 2) and fatal heart attack (n = 1) in the PF group and septic shock (n = 1), and heart failure (n = 1) in the KF group, respectively.Overall, in-hospital mortality was higher in the PF group   .p = 0.034). The one-, three-, and five-year pancreas graft survival rates in patients after SPKT were 75.3%, 71.6%, and 71.6% in the PF group, respectively, and 90.4%, 87.7%, and 84.8% in the KF groups, respectively (p = 0.104) (p = 0.499) (p = 0.419).Pancreas graft survival was significantly higher when the kidney was implanted first. During the first three months after SPKT, the percentage of pancreas graft loss was 23% in the PF group and 7.9% in the KF group (= 0.104) A. The on= 0.499) B. OveralMultivariate Cox regression analysis of the total study population revealed that donor cause of death, donor recipient age and recipient BMI, duration of pancreas cold ischemia time and order of graft implantation are independent predictors of pancreas graft loss within three months and five years after SPKT. Era of transplantation and recipient gender showed a significant impact on pancreas graft survival at three months only, while they had no significant effect on 5-year graft survival .The current study showed that the implantation of the kidney graft before the pancreas graft during SPKT is associated with reduced rates of post-operative complications and significantly better pancreas graft survival in the early post-operative course.The most frequent postoperative complication after SPKT and subsequent pancreas graft loss remain pancreas graft thrombosis. In our study, the average pancreas graft thrombosis rate was 9.7% , which is comparable to published data ,20. Therp = 0.022) [To our knowledge, only two other studies have previously examined the effect of graft implantation order on short- and long-term outcomes in SPKT. In a retrospective single-center analysis of 151 patients after SPKT, Salzedas–Netto et al. showed a significantly higher three-month pancreas graft survival when the kidney was implanted first . Additionally, the frequency of technical graft failures was significantly lower in this group  [In contrast, in a 2016 published register (Scientific Registry of Transplant Recipients) data analysis of 12,700 patients by Niclauss et al., the rate of pancreas graft loss within three months after SPKT was significantly lower in the PF group  . Beyond The reasons for an increased rate of complications and early graft loss in the PF group of our study remains speculative. One reason could be the mechanical stress after graft positioning and surgical retractor adjustment for the consecutive kidney transplant procedure. Salzedas–Netto et al. assumed that implantation of the kidney before the pancreas graft could reduce the risk of intra-operative damage from retractors to the pancreas and pancreatic edema. Unfortunately, no details on intraoperative findings and rector problems are available to us, that could support this thesis. Furthermore, different surgical access routes and procedures were used in the studies. In the study by Salzedas–Netto et al., organs were implanted intra-peritoneally (pancreas), as well as extra-peritoneally (kidney) by two surgical teams. In our analysis and the study by Salzedas–Netto et al., all patients underwent systemic drainage with side-to-side enteric anastomoses . In contp = 0.08). Moreover, the time difference between the implantation of both organs was lower in the KF group  is the phenomenon whereby brief episodes of ischemia and reperfusion applied in distant tissues like the lower extremity render organs subject to transplantation, more resistant to ischemia. The underlying mechanism of RIP are not fully understood, however, experimental studies suggest that a combination of circulating mediators and neuronal signaling might be responsible for conditioning of the targeted organ ,32. FurtIn the event of simultaneous pancreas-kidney transplantation the successive implantation of both organs requires a sequential (two-step) temporary clamping of external iliac vessels and by nature, imitate the procedure of RIP for the second organ to be transplanted. The assumption that pancreatic allografts may benefit from RIP if transplanted second to a kidney is highly speculative but spurs further research.There are some limiting factors of this study. First, the low number of patients in each group and the retrospective non-randomized design should be mentioned. Second, the long investigation period and different surgical teams restricted data evaluation, thus making further controlled and prospective studies necessary.In conclusion, our results suggest that the sequence of graft implantation during SPKT influences the early post-operative course. In our analysis, KF patients seem to have a slight advantage compared with patients receiving PF during SPKT. We would recommend a kidney first transplant if two parallel working teams are available, one doing the back-table preparation, one doing the transplant procedure. If one team is responsible for both back-table and the transplant procedure we would recommend a pancreas transplant first, since the pancreas is more sensitive to ischemia and one or two hours might matter. However, we also recommend a careful surgical retractor adjustment not to compromise the pancreas allograft. Further investigation is required in larger series to determine how the order of graft implantation correlates with post-transplant graft function. Our two main floated hypotheses, that (1) mechanical stress may harm the first and (2) remote ischemic preconditioning may have beneficial effects on the second organ subject to transplantation. Both assumptions may stimulate further investigation.


The aim of the present systematic review was to investigate the effect of residual bone height (RBH) and vertical bone gain on new bone formation (NBF) and graft shrinkage after lateral sinus lifts using different biomaterials. Methods: An electronic search was conducted on three databases to identify randomized controlled trials (RCTs) published until January 2021 with at least one follow-up at 6 months and at least five patients treated, comparing biomaterials used for maxillary sinus augmentation with a lateral approach. Graft volumetric changes, RBH, vertical bone gain, implant failure, and post-operative complications were evaluated. The risk of bias was assessed using the Cochrane tool. Results: We used 4010 identified studies, of which 21 were RCTs. Overall, 412 patients and 533 sinuses were evaluated. Only three publications had an overall low risk of bias. After 6 months, xenograft (XG) showed the least volume reduction (7.30 ± 15.49%), while autogenous graft (AU) was the most reabsorbed (41.71 ± 12.63%). NBF appeared to not be directly correlated with RBH; on the contrary, the overall linear regression analysis showed that NBF significantly decreased by 1.6% for each mm of postoperative vertical graft gain. This finding suggests that the greater the augmentation, the lower the NBF. A similar tendency, with a regression coefficient even higher than the overall one, was also observed with alloplast (AP) and XG. Conclusions: The present results suggested that NBF was essentially independent of preoperative bone height. On the contrary, the smaller the volume was of the graft placed, the higher the amount of new bone formed, and the smaller the graft shrinkage was. Minimizing the augmentation volume might be beneficial to graft healing and stability especially when using AP and XG. In the posterior maxilla, where the possible lack and/or low quality of bone associated with the pneumatisation of the sinus represent a risk for implant osseointegration ,2,3, theIn 1978, Tatum was the first to practise bone grafting under the sinus membrane [Even if biological complications occur with this technique ,11, stanAs an alternative to autogenous bone graft (AB), several biomaterials such as allograft (AG), xenograft (XG), and alloplastic material (AP), either alone or in combination, have been proposed as grafting materials for sinus lifts . While oXG has demonstrated its effectiveness in maxillary sinus floor augmentation, both alone and combined with AB ,26,27. XAlloplastic materials such as polymers, calcium sulphates, hydroxyapatite (HA), calcium phosphates, and bioactive glass are inorganic osteoconductive bone substitutes ,24. An hDemineralized, freeze-dried bone allograft (DFDBA) and FDBA are space-occupying osteoconductive biomaterials that have only slight osteoinductive properties ,32. In aSeveral recent reviews based on histomorphometric data comparing different graft materials used in SL agree on the supremacy of AB to induce new bone formation ,35,36, aThe aim of the present review was therefore to analyse the effects of residual bone height (RBH) and vertical bone gain on the new bone formation (NBF) and the graft shrinkage after lateral sinus lifts using different biomaterials. A second aim was to test if the dimensional baseline variables such as the residual bone height and the initial size of the graft may play a role on the shrinkage and the new bone formation.The present systematic review followed the guidelines of transparent reporting of systematic reviews and meta-analyses (PRISMA), and the protocol was registered on Prospero with the code CRD42020199261.The following focused questions were analysed in accordance with the PICO strategy:1. What was the effect of residual bone height and post-operative vertical change on new bone formation in sinus augmentation procedures 6 months after loading? Population: Healthy patients with an atrophic posterior maxilla requiring lateral sinus augmentation;Intervention: Sinus augmentation with any biomaterial;Comparison: Any comparison between materials included in the intervention or with augmentation without adding biomaterials;Outcomes: Review of new bone formation, implant survival, and complications.2. What was the shrinkage of different bone substitutes when used in sinus augmentation procedures 6 months after surgery?Population: Healthy patients with an atrophic posterior maxilla requiring lateral sinus augmentation;Intervention: Sinus augmentation with any biomaterial;Comparison: Any comparison between materials included in the intervention or with augmentation without adding biomaterials;Outcomes: Graft volumetric changes measured with CBCT, implant survival, and complications.British Dental Journal, the British Journal of Oral and Maxillofacial Surgery, Clinical Implant Dentistry and Related Research, Clinical Oral Implants Research, Clinical Oral Investigations, the European Journal of Oral Sciences, Implant Dentistry, the International Journal of Oral and Maxillofacial Implants, the International Journal of Oral and Maxillofacial Surgery, the International Journal of Oral Implantology , the International Journal of Periodontics and Restorative Dentistry, the Journal of Clinical Periodontology, the Journal of Dental Research, the Journal of Dentistry, the Journal of Maxillofacial and Oral Surgery, the Journal of Oral and Maxillofacial Surgery, the Journal of Periodontal Research, and the Journal of Periodontology.The research of the articles was performed online using three main databases: PubMed/MEDLINE, Scopus, and the Cochrane Central Register of Controlled Clinical Trials (CENTRAL). The investigation began in April 2020 and ended in January 2021. The survey did not exclude any article by year of publication or language and was performed using the following search strategy: “(maxillary sinus lift OR maxillary sinus augmentation OR maxillary sinus elevation OR maxillary sinus grafting OR maxillary sinus surgery OR sinus graft) AND dental implants” that was modified for each database. A by-hand search was also performed in the following journals: the http://clinicaltrials.gov/; http://www.centerwatch.com/clinicaltrials/; http://www.clinicalconnection.com/; www.greylit.org; www.opengrey.eu; accessed on 1 October 2021).The reference lists of all identified RCTs and relevant systematic reviews were scanned for any additional studies. Online registries providing information about in-progress clinical trials and the grey literature were checked , both split-mouth and parallel, that included a test and a control group or two or more comparisons were included. Only studies in English with at least one post-surgery follow-up at 6 months and at least 5 patients treated were included. Non-randomized clinical studies, case series, pilot studies, retrospective studies, cohort studies, reviews, animal or in vitro studies, and any publications using duplicate data were excluded. Studies using trans-sinus implants and comparing the use of short implants (not associated with the sinus lift technique) with long implants  were not considered.Selection of studies:Two reviewers (G.G. and L.M.) independently read the titles, the abstracts, and the full texts of the selected articles to ensure they met the inclusion criteria. In cases of disagreement, a third co-author (P.P.) was consulted.After the selection of the articles to be included in the review, the data were collected by two authors (L.M. and G.G.) using a Microsoft Excel spreadsheet . The following information was extracted: year and journal of publication, authors, title, type of study , country in which the study was performed, presence of sponsors, number of operators, presence or absence of smoking patients, total number of patients and maxillary sinuses treated and evaluated, age and sex of patients, total number of implants inserted and type , follow-up period in months, outcomes, grafting materials used, use or not of a covering membrane, number of implants inserted in each group, number of failed implants with relative timing, any post-operative complications found, residual vertical bone dimension prior to surgery, volumetric bone gain, and finally, histomorphometric data on the amount of new bone obtained. The biomaterials used in the various studies were reported based on the category they belong to, i.e., autologous graft (AU), xenograft (XG), allograft (AG), alloplastic material (AP), autologous tissue graft (ATG), biological material (BIO), or for spontaneous healing (only membrane elevation) without the use of any material (SH). If the article contained the total linear measurements before and after surgery, the actual bone gain was calculated by subtracting the preoperatively measured value from the post-operative one; this measure was reported in mm ± SD. 3 ± SD along with the percentage of contraction with respect to immediate post-surgery graft volume. For the volumetric measurement, the volume found at least 6 months after surgery was reported in mmIn cases of missing or unclear data, the authors of the various articles were contacted by e-mail to obtain further details.Cochrane Handbook for Systematic Reviews of Interventions [The risk of bias (RoB) assessment was conducted according to the Cochrane criteria, ventions . All theIf a potential bias could cause a serious weakness of confidence in the results, the associated risk was defined as high. Otherwise, the risk of bias was classified as “low” if it did not alter the results, or as “unclear” if it raised some doubts about the results of the study.It was planned to undertake a meta-analysis of the graft shrinkage for each comparison between materials if at least three studies with the same comparison were found. However, no meta-analysis could be performed as there were no more than two studies for each type of comparison. Therefore, no meta-analysis was performed. Linear regression analysis was undertaken between outcome variables  and baseline data . GraphPad Prism 5.1  was used as the statistical software. To obtain some missing information, the authors of 10 articles were contacted ,57,58,59The 21 articles included in the review presented a comparison among at least two different biomaterials: 8 of them were parallel studies ,54,55,57Most of the publications were not sponsored, and only six articles were financed ,49,50,54In total, 412 patients and 533 sinuses were analysed with 319 for the first PICO question and 301 for the second one. In 12 studies, smokers were excluded from the patient’s selection ,55,57,58Four studies presented a population with a mean age below 50 years ,49,50, 1In only two studies, the implant insertion was performed simultaneously with the lateral sinus lift procedure ,48; in 1While in most RCTs, the surgical procedure involved the use of a resorbable membrane covering the lateral window, three studies did not consider their use ,56,59, aHistomorphometrical and vertical height data were reported by 13 studies, including 5 studies that compared different types of heterologous grafts ,50,52,53Volumetric changes were considered in 11 articles. Three studies compared the use of alloplastic material with autologous bone and/or their combination ,54 whileNine studies did not specify the number of implants placed after the lateral sinus lift ,53,54,56The main characteristics of the included studies are summarized in The evaluation of the risk of bias (RoB) of the included studies  showed tThirteen studies presenting histomorphometric analyses, that mean residual (pre-operative) bone height (RBH), and the postoperative bone height (PBH) for both test and control groups were included. In 10 studies, the biopsies were taken 6 months after sinus lift ,53,56,592 = 0.04 and a slope of 2.11 (95% CI = −2.19 to 6.43), meaning that NBF increases by 2.1% per each mm increase in residual bone height; therefore, the NBF was essentially independent of preoperative bone height. In other words, the observed increase in new bone formation with the increasing RBH was not significant (p = 0.32). In the first analysis, all the histomorphometric data of new bone formation percentage (NBF%) values  were correlated with the respective mean RBH. The hypothesis was that the NBF% would increase when the RBH increased. The overall linear regression analysis showed a slight agreement with the hypothesis  with a r2 = 0.33 and a slope of −1.56 (95% CI: −2.50 to −0.61), meaning that NBF significantly decreased when postoperative vertical graft dimension increased. Per each mm of vertical height, NBF changed by about 1.6%. The trend was highly significant (p = 0.0023). In the second analysis, the NBF% was correlated with the postoperative height  with the hypothesis that the higher the graft, the lower the new bone formation inside the graft. The overall linear regression analysis showed a tendency in agreement with the hypothesis  with a r2 = 0.37 for alloplasts and R2 = 0.48 for xenografts).It seemed interesting to investigate if the correlation was the same or different among the different materials. Since the number of samples per material was limited, a comparison could only be attempted between alloplasts (n = 6) and xenografts (n = 11) . The twoThe postoperative bone height (PBH), which was correlated to new bone formation, was obtained by adding the RBH and the graft vertical dimension itself. Given that the graft dimension was dependent on the amount of graft inserted by the operator at the surgical time, it was a variable that could be controlled to increase the expected new bone formation, as opposed to the residual bone height.3 were normalized to the percentage of the reduction and dividing by the baseline values. Other grafts composed by combined materials  were not considered for comparison because each of them was investigated by a single study.Regarding graft volume changes, eleven RCTs were considered with a total of 302 sinuses. Graft volume was estimated using CBCT, and volume change was calculated by subtracting the value at the 6-month follow-up from the baseline value. Data were grouped by grafting material. Five groups were considered including autogenous graft , xenograft , alloplast , allograft , and autogenous + alloplast . The data of volume reduction in mmp = 0.009) and between AG and XG (p = 0.048)  while the one that resorbed most was AU (41.71 ± 12.63%). For the other biomaterials, a volumetric contraction of 27.82 ± 15.58% was evaluated for the AP, 30.23 ± 1.61 for the AG, and finally, 26.68 ± 11.03% for AU + AP . Signifi= 0.048) . An unpaFrom the analysis of the data extrapolated from the articles, only 10 implants, among 620 inserted, failed: three of those were placed in sinuses grafted with AG ,57, threPost-operative complications were not reported in five articles ,46,51,56Da Silva et al.  found foIn the split-mouth study by Chackartchi et al. , a patieThe aim of the present systematic review based on RCTs had been to investigate if there was an effect of residual bone height (RBH) and vertical bone gain on new bone formation (NBF) as well as the shrinkage of the graft when using different biomaterials. Graft volumetric shrinkage occurring after SLs using different biomaterials ,54,60,61The resorption rate of AP, AG, and AU + AP indicated that greater graft stability may be achieved using bone substitutes or a composite graft, which then added AP to the patient’s bone, compared to using AU alone. Similar conclusions were drawn by Shanbhag et al. in their review, which further stipulated that neither two-stage implant placements nor survival rates of simultaneously placed implants were affected by graft volume reductions . Some authors suggested that the amount of new bone formation (%) was a reliable parameter of bone graft performance since the greater that the quality of the newly formed bone was, the greater the BIC (bone-implant contact) will be, and, eventually, the implant survival . SeveralThe NBF, as well as the material used in the sinus lift, may also be affected by sinus morphology and dimension; reduced osteogenic potential occurs in larger sinus cavities where more time is required to obtain adequate bone formation because of the greater distance between the sinus walls and the augmented region ,70. In nOur results, revealing that residual bone height was not a remarkable factor affecting graft maturation, are in accordance with those reported by previous histomorphometric studies analysing biopsies retrieved 6 months after lateral sinus lift had been performed with XG  and AP . PignatoKühl et al. considered overaugmentation preferential in two-stage sinus lifts to compensate for expected graft volumetric reduction at the time of implant placement . On the Regarding the correlation between PBH and NBF, further studies are needed to investigate these hypotheses further and to understand the clinical significance of reducing the graft size in order to increase the NBF.It has long been shown that NBF occurs in the maxillary sinus via sinus mucosa lifting without using any graft type ,77,78, sOne of the limits of the present review is the great variability of sinus morphology as sinus dimensions can influence the percentage of new bone formation, as has been shown by Stacchi et al. .In addition, these conclusions could not be confirmed or denied as the current literature lacks RCTs, reviews, and meta-analyses assessing the correlation between vertical bone gain and histomorphometric outcomes. Within the limits of the present systematic review, the following conclusions could be drawn:Six months after lateral sinus lift, the material showing the lower volume reduction was the XG while the one that resorbed most was the AU, according to the current literature. Since the analysis of the correlation between NBF and RBH had revealed a non-significant increase in new bone formation when residual bone height increased, NBF is essentially independent of preoperative bone height.NBF significantly decreased when postoperative vertical graft dimension increased, supporting the hypothesis that the higher the graft height, the lower the new bone formation within it. Therefore, at surgical time, the operators should carefully control the amount of grafted biomaterial applied to optimize the expected NBF.


Recently, a [18F]fluoride labeled PSMA-inhibitor, [18F]PSMA-1007, has been introduced, and it has ensured large-scale productions, overcoming this limitation of [68Ga]PSMAs. In this study, PSMA-1007 has been labeled with low (A), medium (B) and high (C) starting activities of [18F]fluoride, in order to verify if radiochemical yield, radiochemical purity and stability of [18F]PSMA-1007 were affected. These parameters have been measured in sixty-five consecutive batches. In addition, the estimation of [18F]PSMA-1007 production costs is provided.Prostate-specific membrane antigen is overexpressed in prostate cancer and it is considered a good target for positron emission tomography/computed tomography imaging of primary cancer and recurrent/metastatic disease, as well as for radioligand therapy. Different PSMA-analogues labeled with [18F]fluoride was 52%, while for the high one it decreased to 40%. The radiochemical purity was 99% for all three activities. [18F]PSMA-1007 did not show radiolysis up to 8 h after the end of synthesis, confirming that the radiopharmaceutical is stable and suitable to perform diagnostic studies in humans for a long period of time after the end of radiolabeling. Furthermore, radiochemical stability was demonstrated in fetal bovine serum at 4 °C and 37 °C for 120′.The radiochemical yield for low and medium activities of [18F]fluoride of 90 GBq (B) seems to be the best option enabling a final amount of about of 50 GBq of [18F]PSMA-1007, which is promising as it allows to: (a) perform a large number of scans, and/or (b) supply the radiopharmaceutical to any peripheral diagnostic centers in need.A starting activity of [ Prostate cancer (PC) is the second most common diagnosed cancer in men and the major cause of mortality and morbidity worldwide  is a transmembrane protein and it is considered a valuable marker for PC. PSMA is upregulated in poorly differentiated, metastatic, and hormone-refractory PCs, being not or poorly expressed in inflammatory and/or benign prostatic tissues, as well as other normal organs . Recently, 18F-labeled PSMA targeted radiotracers have been introduced and they are increasingly being used in clinical practice since then  and larger productions by cyclotron might also allow providing the distribution to several PET centers that are not more than 4 h away from the producing facility, equipped with cyclotron and radiopharmacy  at end of synthesis (EOS), stability of the radiolabeled PSMA over a 8-h period of time and the analysis of the production costs for a nuclear medicine center with its own cyclotron/radiopharmacy facility.The purpose of this research is to find the best parameters to achieve the maximal amount of fluoride was produced with a 18/9 MeV cyclotron  by irradiating a standard volume of 2.6 mL of [18O]H2O . The parameters of the beam, such as time and target current, were adjusted according to the desired final activity.[18F]PSMA-1007 were performed using a fully automated radiosynthesizer  as was previously described , dimethyl sulfoxide (DMSO) for precursor, ethanol, Phosphate Buffered Saline (PBS), Tetrabutylammonium Carbonate (TBA-HCO3) water/ethanol solution 0.075 M. These reagents were purchased as a single kit .The reagents used for each synthesis of PSMA-1007 was also tested for bacterial endotoxin through kinetic chromogenic Limulus Amebocyte Lysate (LAL) method .[18F]PSMA-1007 was tested using different starting amounts of radioactivity, to verify possible effects, if any, on the final product. Samples from product vial (at room temperature) were analyzed by radio-HPLC, to detect the presence of degradation impurities or free [18F]fluoride, at different times  after EOS.The radiochemical stability of [18F]PSMA-1007 were performed in Fetal Bovine Serum  at 4 °C and 37 °C up to 2 h, to simulate blood stream conditions.In vitro stability studies of [18F]PSMA-1007 were added to FBS to reach a final volume of 1 mL, keeping a constant activity of 37.0 ± 3.7 MBq, and the radio-HPLC was performed at different times after incubation  for both temperatures.Different volumes of [18F]PSMA-1007 and relative quality controls we have evaluated the use of three different starting activities of [18F]fluoride, identified as: low , medium  and high activity . These activities of [18F]fluoride were obtained by monitoring both essential parameters of the cyclotron: time/length of the beam  and constant target current of 60 μA.In a series of sixty-five consecutive syntheses of [18F]PSMA-1007 at the EOS was of 19 mL, with a mean activity of 28.71 ± 0.56 GBq for A, 46.84 ± 2.54 GBq for B and 65.70 ± 4.23 GBq for C; it appeared clear, colorless, particle-free, sterile with a pH ranging between 5 and 7.The final volume of [18F]PSMA-1007, that is fully automated, has been shown to be very reliable. In fact, the measured RCY at EOS, not corrected for decay and related to a synthesis time of about 40′, was similar for A (52.09 ± 7.10%) and B (52.66 ± 3.40%), while for C it decreased to 40.26 ± 1.76% , performed by radio-HPLC after each production, was ≥ 99% Table ; the ret18F]PSMA-1007 was analyzed up to 8 h after EOS, and the radiochemical purity was > 99% with no evidence, on the chromatograms, of degradation impurities and detectable amounts of free [18F]fluoride , the radiopharmaceutical was tested in FBS up to 120′, at 4 °C and 37 °C, showing absence of measurable radiolysis in both conditions, as reported by radio-HPLC analysis in Fig. 18F]PSMA-1007 biodistribution, by PET/CT, were found in patients who received the radioligand at different times from EOS  occurred only in two cases and it was more likely related to mechanical errors within the module rather than to chemical failure during radiosynthesis. The flow of [18F]fluoride activity was monitored through the geigers of the synthesizer during the entire radiolabeling process.Thus, the analysis to define and to optimize all possible variables in the process of fluoride did not show significant differences. In details, RCY was 52% for batches labeled with activities A and B, resulting slightly higher than the average of [18F]PSMA-1007 yield, declared by module’s manufacturer: ≥ 40 ± 10% (not corrected for decay); whilst a RCY was reduced to 40% for batches labeled with activities C.The trend of RCY and RCP, using three different activities of [18F]fluoride from the activity due to [18F]PSMA-1007). At the end, the final product activity was quantified only in the dose dispenser.For each batch production reported, we evaluated the trend of critical steps of synthesis process that can be monitored by sensors: QMA cartridge and reactor  were measured after 8 h from EOS. The activity detected on each of these components was respectively 0.37%, 1.48%, 16.7% and 12.3% of the [18F]fluoride starting activity. This justify only a part of the radioactivity loss.There are no significant differences of the trends on activity sensors for the three activities A, B and C. To quantify the loss of the residual radioactivity in the synthesis process, the critical components of the synthetic device . Moreover, stability studies were serially performed on [18F]PSMA-1007 in the period included from 2 to 8 h after EOS, showing no degradation of the radiolabeled PSMA.The RCP values were substantially similar for all the three different starting activities of [18F]fluoride (B) coupled two advantages: a relatively short time of beam/radiolabeling (about 60′) and a better synthesis performance in terms of RCY (52.66 ± 3.4%)  along with an increase of the time of beam was observed (55′), without a significant gain in the amount of final available activity of [18F]PSMA-1007.According to these results, the medium starting activity of PSMA-1007, disposable/reagents, personnel, technologies and maintenance, radioactive waste dismission and general costs. On these bases, a reasonable price lower than 30 € for 37 MBq should be justified.In our site, the costs for the production of batches of 46.84 GBq of [18F]PSMA-1007. In fact, they showed, in addition to [18F]fluoride, other radioactive contaminants with longer physical half-lives, including: [58Co]cobalt, [57Co]cobalt, [51Cr]chromium, [54Mn]manganese, etc. Thus, according to the current laws in European countries (Euratom BSS Council Directive 2013/59/Euratom), the removal and the wasting of these disposables follows very restricted rules. In fact, they cannot be eliminated very quickly, but they can be released after γ-ray spectrometry analysis and a prolonged stockage or released to authorized and specialized contractors.In the analysis of the costs, we accounted also those related to the wasting of the disposables used for the synthesis of [18F]PSMA-1007 have been evaluated in order to define the best operating procedures. Suggested starting activity of [18F]fluoride should range from to 55 to 90 GBq, showing the following advantages:Short time of beam (30′).Highest radiochemical yield (> 50%) and purity (> 99%) at EOS.18F]PSMA-1007.Relatively low costs of production with many available doses of [Methodological aspects for an optimal production of [18F]PSMA-1007 produced following this approach, may satisfy the growing requests of PSMA PET/CT in clinical practice, guaranteeing high quality radiopharmaceutical.All these aspects strongly indicate that [


Trypanosoma cruzi enable the identification of thousands of antigens recognized by serum samples from patients with Chagas disease. Because most of these antigens are hypothetical proteins, gPhage provides evidence of their expression during infection. We built and validated a comprehensive map of Chagas disease antibody response to show how linear and putative conformation epitopes, many rich in repetitive elements, allow the parasite to evade a buildup of neutralizing antibodies directed against protein domains that mediate infection pathogenesis. Thus, the gPhage platform is a reproducible and effective tool for rapid simultaneous identification of epitopes and antigens, not only in Chagas disease but perhaps also in globally emerging/reemerging acute pathogens.Large-scale mapping of antigens and epitopes is pivotal for developing immunotherapies but challenging, especially for eukaryotic pathogens, owing to their large genomes. Here, we developed an integrated platform for genome phage display (gPhage) to show that unbiased libraries of the eukaryotic parasite   •Genomic shotgun phage display (gPhage) of eukaryotes is feasible and promising.•gPhage allows rapid antigen ID and epitope mapping, including 3D structures.•Conformation epitopes can be identified and validated by using the gPhage platform.•Most Chagas disease antigens are hypothetical proteins rich in repetitive elements. Parasitology; Sequence analysis; Systems biology Candida auris)  , are exaLarge-scale mapping of antibody repertoires—especially against pathogens with large genomes—can be particularly challenging. Such endeavors have long relied on a wide range of techniques, from assays such as enzyme-linked immunosorbent assay (ELISA), to cDNA libraries and microarray platforms . All thePhage display has initially been developed and used to map antibody epitopes , 2019, iTrypanosoma cruzi and afflicts millions of patients worldwide , many of which are still used today for diagnostic purposes . Phage dT. cruzi induces a strong antibody and T cell response that partially controls parasitism, subsequently leading to a low-grade, chronic infection. Over the course of several decades, up to one-third of infected individuals eventually develop cardiomyopathy , with patients dying of heart failure and/or cardiac arrhythmia; ∼60% remain asymptomatic and ∼10% develop digestive motility disorders , which encode the periplasmic export leader peptide and the reading frame for gene VIII, respectively F. Sequen protein G, we stip value > 0.05)  from patients at different stages of the disease . For each stage of the disease, IgG from individual patients was combined into pools to produce two biological replicas  A. Asympt > 0.05) B. IgG fr > 0.05) A (Faulin > 0.05) . After 3 > 0.05) C, and al > 0.05) . Sequenc > 0.05) , a requiT. cruzi-derived peptides, upon selection, there was considerable enrichment of particles displaying a peptide (except for control cohort B)   did not display any peptides (out-of-frame inserts) and only 8.9% displayed ohort B) D and 2E.e genome F. Converctively) F. Asymptctively) A, as expT. cruzi-derived antigens . However, most of them represent fragments from the same protein with minor variations in length or in amino acid sequence, presumably owing to the high number of multigene families and repetitive elements (cruzi protein (antigen)  .p value < 2.2 × 10−16): the larger clusters were also the most abundant, strongly suggesting that both values could be used as proxies for immunogenicity (Epitopes were subsequently quantified in two ways: (1) the number of fragments or distinct peptides in each cluster and (2) their overall abundance  . We obsegenicity D.www.iedb.org) for known Chagas-disease-associated antigens. All Chagas disease antigens and epitopes were downloaded from the IEDB database and compared with each individual peptide antigen identified in our study by using a pairwise sequence alignment algorithm. We observed that 469 (21%) of the epitopes were identical to epitopes in our working data set, including B13, gp85/trans-sialidases, mucin, and SAPA, among others  were not associated with any epitopes in the IEDB. Almost three-quarters of all epitopes identified (74.5%) with gPhage belonged to hypothetical proteins , including epitopes belonging to hypothetical proteins  protein . Epitope regions G. We use regions H. In som regions . Only twT. cruzi antibody response of infected patients is heterogeneous and directed toward a multitude of antigens  were recognized by IgG antibodies in ELISA: 9 were recognized by all patient groups, in addition to the immunodominant epitope B13 , which reacted strongly with the IgG pools A. Four pantigens D. CollecT. cruzi antigens (B13 and cluster #0) , were recognized specifically by IgG from the two pools of patients with severe cardiomyopathy; however, the immobilized plastic-bound synthetic peptide was not recognized in ELISA C. This pster #0) G.We tested the binding of PPHTRRVTVRCGPPSCADE phage to IgG from each individual patient in the pools (cohort A and B) and found that it bound to IgG from a single patient (#8), which had been inadvertently added to both pools of IgG from patients with severe CCC H. Again,T. cruzi genome contains a high number of repetitive elements and multigene families, which seem to play a role in infection  , we used the numbers of unique sequences in each cluster as proxies for antigenicity. We reasoned that the different sequences within the individual clusters likely represent variants of the same epitope shared by different member of each multigene family. In total, 60, 6, and 17 clusters of epitopes could be unequivocally mapped to members of the gp85/trans-sialidase, mucin, and MASP families, respectively A and 5B. number) , data suT. cruzi-neutralizing monoclonal antibody (H1A10) that recognizes a subset of the gp85/trans-sialidase family  that have helped to close this gap and to produce better annotated genomes , the number of unique peptides that compose the epitope cluster, and their frequency in each cohort of patients used for the biopanning . For insWe have also included the quantification of all epitopes using two criteria: their overall abundance in the biopanning and the number of unique peptides that comprise the antigenic cluster, which showed good linear correlation. It is important to note, however, that quantification of individual antigens/epitopes based on their abundance might be distorted by the immune response of individual patients when IgG from several patients are combined during the selection process, as it was carried out here (cohorts of 10 patients). For instance, IgG from patient #8 (present in both cohorts of severe CCC) capture in large numbers of phage displaying peptides belonging the same epitope: peptides belonging to cluster #7 (consensus sequence PPHTRRVTVRCGPPSCADERA) correspond to 40% and 81% of all recovered phage in both cohorts A and B of severe CCC, respectively. It means that epitopes recognized by IgG from the other 9 patients were squeezed into the remaining 60% and 19% of the recovered phage for each cohort A and B, respectively. We did not observe other clusters with such high abundance in the remaining cohorts (asymptomatic and mild CCC), so this problem should be limited to patients with severe CCC. To avoid this bias in future studies, we recommend that gPhage selection should be performed using IgG of individual patients and not pool of patients.−5%), which is not far from the lowest-abundance clusters (∼10−6%), indicating that other low-frequency epitopes might also be true antigens. Again, whether all these low-abundance epitopes are genuine antigens or not, they have all been include in the final data set along with their abundance in each cohort. For instance, there are 209 epitopes in our list which are recognized only by the control IgG, all low-abundance epitopes (10−6 to 10−5%) represented by a single peptide (only 4 of them are clusters with 2–4 peptides). They may represent cross-reactive T. cruzi epitopes (or mimotopes) recognized by natural antibodies present in the general population. We opted to do this rather than set up specific cutoff values, such as abundance or number of peptides that compose the epitope cluster. The reason for that is because at least some of the low-abundance epitopes correspond to previously validated epitopes such the MASP immunodominant C-terminal epitope   and may be a better indication of a true epitope compared with the remaining 2,476 single-peptide epitopes. Certainly, because phage selection using patients’ IgG cohort A was less stringent than with cohort B, a significant proportion of the former epitopes are represented by low-abundance single peptides. But, it is also difficult to assess whether the selection with cohort B was actually too stringent to the point of missing epitopes, enriching in immunodominant epitopes. Here, we validated at least one epitope   or antigeptides) .T. cruzi ribosome illustrates how the combination of antigen identification and epitope mapping can provide insight into protein domains and/or their 3-dimensional structure.As a proof-of-principle, we performed two large-scale epitope mapping studies with this data set to illustrate how the gPhage platform can be used to build a comprehensive atlas of the antibody response of a large cohort of patients and, critically, pinpoint many of the epitopes from these antigens to specific protein regions/domains. The strong antibody response of patients with Chagas disease toward the carboxy-terminal domain of members of gp85/trans-sialidase multigene family seems to protect the parasite by preventing the production of high-titer neutralizing antibodies against the other two functional domains , which have been implicated in cell infection , which map to cluster #3782, have been implicated in cardiomyopathy , attesting to the validity of gPhage as a new platform for antigen/epitope discovery.Our gPhage data may also shed light on a gap in knowledge regarding the antibody response during the course of Chagas disease. Anti-solution . Treatmereatment . Antibodefficacy B, none pLeishmania sp and others belonging to the family Trypanosomatidae. It could also be applied to pathogenic fungi with relatively simple genomes containing very few introns, such as those of Candida sp (Candida (C. auris), gPhage could be an effective tool for studying the antibody response against this organism. The Plasmodium falciparum genome is similar in size to that of T. cruzi and only about half of the genes in P. falciparum contain introns, which are usually smaller than exons and less numerous than more complex life forms  in a few cases. Moreover, in most cases, these same epitopes may also share high similarity to antigens belonging to other T. cruzi strains with more abundantly annotated genomes . Indeed, in this report, each individual epitope was compared by pairwise alignment to curated member of these families to study the immune response against members of the multigene families. Finally, discrepancies in antigen assignment may conceivably reflect genomic differences among the various strains of T. cruzi.As in any novel enabling platform methodology and initial proof-of-concept, full validation of the putative epitopes and full antigens serially selected and isolated from our patient discovery cohort should ideally be experimentally confirmed in an independent patient validation cohort. Within this framework, the tentative antigens presented  should bT. cruzi strains  and comprehensive genome databases online and improvements in genomic annotation software should help mitigate these practical issues.Therefore, we would recommend that for follow-up studies, each individual epitope should be carefully curated with an appropriate software algorithm (and ultimately investigator supervision and discretion) for proper candidate antigen assignment. Of course, the actual clinical strains of parasites that infected the human patients used in this study are bound to be genetically different from the experimental standard strain used to build the gPhage library and those available in databases required for antigen assignment. Thus, our list of tentative epitopes and candidate antigens may reflect such biological diversity; in other words, a collection of epitope/antigen pairs that might share high similarity with the original immunogenic parasite protein in first place. Future studies with gPhage libraries build with clinical Given that this initial methodology and proof-of-concept report has also become a potential resource for other investigators, we have reasoned that we should include all candidate epitopes . Ongoing and future studies will certainly be required to assess whether these low-abundance single-peptide epitopes represent genuine antigens or not. Thus, we have opted instead of use an arbitrary experimental cutoff  to allow independent researchers to study the full data set by using their own criteria. On this rationale, we have tabulated the data set into an easily online-searchable spreadsheet file with all available data generated in our study .Finally, we have identified at least one putative conformational epitope, which could not be validated by ELISA by using a synthetic peptide but which adopts a similar conformation in solution or in the context of the phage capsid protein. Thus, it is possible, perhaps even likely, that other selected epitopes from our list , which wgiordano@iq.usp.br).Further information and requests for resources and reagents should be directed to R.J.G  were classified as having either mild cardiomyopathy, when LVEF was higher than 40% (LVEF> 40%), or severe cardiomyopathy, when LVEF was lower or equal 40% (LVEF ≤ 40%). Patients with no EKG alteration were considered asymptomatic. Serum samples were pooled into sets of 10 patients  and the immunoglobulins purified by affinity chromatography by using sepharose-G protein resin, following a standard manufacturer protocol (Thermo Fischer). All information regarding patients used in this study, including age and gender, can be found in The Institutional Review Board at the University of São Paulo Medical School approved the use of serum samples used in this study . All patients with Chagas disease (N = 59) had at least two positive results for the presence of anti-T. cruzi was grown in Liver Infusion Broth medium at 28°C  (E. coli strain TG1 (Lucigen) and DH10B (Invitrogen) were used for phage display and cloning experiments, respectively.The epimastigote form of ingales) . E. coliAF130864.1) was provided by Dr. Lars Frykberg   .T. cruzi was grown in Liver Infusion Broth medium at 28°C (strain Sylvio X10) (The epimastigote form of vio X10) . The celThe purified genomic DNA was subjected to fragmentation in the COVARIS S2 equipment (Covaris Inc.). Aliquots of 30 μg in 130 μl of TE were fragmented at the following conditions: duty cycle, 10%; intensity, 4; cycles/burst, 200; time, 80 seconds. The fragments were subjected to electrophoresis in low-melting-point agarose gel and the region between 100 and 500 bp was selected and purified by extractions with phenol and chloroform. For DNA end repair, we treated the fragments with T4 DNA polymerase (Thermo Fisher Scientific) following a standard manufacturer protocol.E. coli strain DH10B (Invitrogen) and cultured in LB medium for 16 h. Plasmid DNA was isolated by using QIAGEN Plasmid Maxi Kit, following a standard manufacturer protocol. The material was subjected to two consecutive cesium chloride (CsCl)/ethidium bromide gradient purification, followed by extractions with n-butanol to remove ethidium bromide and dialysis against TE buffer for salt removal.Plasmid pG8SAET was propagated into T. cruzi DNA fragments were ligated to the vector by using T4 DNA ligase (Thermo Scientific) following a standard manufacturer protocol of the enzyme. Several vector:insert molar ratios were tested to determine the best condition. In the final ligation reaction, we used 100 μg of the digested vector and a vector:insert ratio of 1:30. The DNA was transformed into electrocompetent E. coli bacteria (strain DH10B). The transformed bacteria were recovered and propagated overnight (ON) in LB medium containing carbenicillin (50 ug/mL). Serial dilutions were plated on LB agar medium to estimate the number of transformants in the library. In the next day, plasmid DNA from the library was purified, resuspended in TE and kept at -20°C until use. The plasmid library was transformed into E. coli (strain TG1 – Lucigen) and infected with the helper phage M13KO7 for phage production. All phage display selections were then performed using the same pool of gPhage library kept at -20°C  to minimize unwanted bias owing to phage amplification.The pure pG8SAET vector was digested with the enzyme Eco105I (Thermo Fisher Scientific) and dephosphorylated with FastAP (Thermo Fisher Scientific) following a standard manufacturer protocol. The vector was subsequently purified by phenol:chloroform extraction and ethanol precipitation to remove enzymes and other contaminants. The 8 unique inserts with average size of 143 bp. Sequencing data showed that ∼75% of the clones (∼3x108) contained a T. cruzi insert with average size of 143 bp, which corresponds to a total of ∼4.3x1010 bp of the T. cruzi haploid genome (3x107 bp) or ∼1,400 times. However, not all inserts result in peptide display and not all displayed peptides correspond to T. cruzi proteins . After dilution in LB media, a small aliquot was separated for quantification of phage recovery by colony count, and the remaining bacteria was then infected with helper phage at a multiplicity of infection of 20 and culture in LB media containing carbenicillin (100 μg/ml) . On the following day, cells were centrifuged 10,000 g for 10 min, and phage particles were recovered from the supernatant by the NaCl/PEG method (Purified IgG (1 μg in 100 μl PB) was immobilized on microtiter plates (96 microwells) ON at 4°C, washed three times with PBS, blocked with PBS containing 1% bovine serum albumin (PBS/BSA) for 1 h at room temperature (RT), and incubated with phage library  by using BlastN  and the first peptide (seed) removed from the list and compared with all remaining peptides (termed Levenshtein distance) by using the FuzzyWuzzy package (http://hmmer.org/). Hydrophilicity analysis was performed by using available criteria  were identified by using XSTREAM    were dowmuscle/) , and seqmuscle/)  and mucimuscle/)  were remmuscle/) .T. cruzi proteins (Genbank). Antigens that match T. cruzi ribosomal proteins were selected and aligned to proteins (blue) from the human ribosome (PDB #4UG0 and 4V6X—for the P2 protein). To locate their position in the structure, epitopes were visualized using Chimera (v1.11)  .Synthetic peptides (Chinese Peptide Company) were immobilized on 96-well microtiter plate (50 μg/mL) in 50 mM carbonate buffer, pH 9.0 (OV at 4°C). Wells were washed with PBS supplemented with Tween-20 (0.05%) (PBST), blocked for 2 h at RT with PBS/BSA and incubated with patient sera in PBS . After 2 h at RT, wells were washed with PBST, incubated with goat anti-human IgG conjugated to horseradish peroxidase (Sigma Aldrich), washed with PBST and developed with OPD substrate (Sigma).9 transducing units of phage PPHTRRVTVRCGPPSCADE or negative control insertless phage (pG8SAET) in 100 μl PBS 1% BSA for 2 h at RT. For the competition assays, phage was incubated in the presence of synthetic peptide (10 μg/ml). Wells were washed and incubated with rabbit antibacteriophage IgG . Washed and incubated with goat anti-rabbit IgG conjugated to IRDye 680LT (LiCOR Biosciences) . Wells were washed and phage bound to the wells quantified using the Odyssey system (LiCOR).IgG from individual patients  were immobilized on 96-well microtiter plates . The wells were washed with PBS, blocked with PBS/BSA (1 h at RT) and incubated with 10p-values are reported along with each result as appropriate.All statistical inference tests were performed by using GraphPad Prism 6. Tests used and corresponding


A high model of end-stage liver disease (MELD) score (>30) adversely affects outcomes even if patients receive prompt liver transplantation (LT). Therefore, balanced allocation of donor grafts is indispensable to avoid random combinations of donor and recipient risk factors, which often lead to graft or recipient loss. Predictive models aimed at avoiding donor risk factors in high-MELD score recipients are urgently required to obtain satisfactory outcomes.Data of patients with MELD score >30 who underwent LT at three transplantation institutes between 2015 and 2018 were retrospectively reviewed. Early allograft dysfunction (EAD), length of intensive care unit (ICU) stay, and graft loss were recorded. Corresponding independent risk factors were analyzed using stepwise multivariable regression analysis. A prediction model of graft loss was developed, and discrimination and calibration were measured.After applying the exclusion criteria, 778 patients were enrolled. The incidence of EAD was 34.8% (271/778). Donor graft macrovesicular steatosis, graft-to-recipient weight ratio (GRWR), warm ischemia time (WIT), cold ischemia time (CIT), and ABO blood incompatibility, together with donor serum albumins, were independent predictors of EAD. The incidence of ICU stay over 10 days was 64.7% (503/778). Donor age, recipient's MELD score, Child score, and CIT were independent predictors of ICU stay. The 3-year graft survival rates (GSRs) in the training and validation cohorts were 64.2 and 59.3%, respectively. The independent predictors of graft loss were recipient's Child score, ABO blood type incompatibility, donor serum total bilirubin over 17.1 μmol/L, and cold CIT. A nomogram based on these variables was internally and externally validated and showed good performance . For a recipient with a high MELD score, the avoidance of ABO blood type incompatibility and CIT ≥6 h would achieve a 3-year GSR of up to 78.4%, whereas the presence of the aforementioned risk factors would decrease the GSR to 35.4%.The long-term prognosis of recipients with MELD scores >30 could be greatly improved by avoiding ABO blood type incompatibility and CIT ≥6 h. Liver transplantation (LT) is an effective and curative treatment for patients with end-stage liver disease (ESLD). However, the pre-transplant state of these patients and the shortage of donor livers largely limit the development of LT. Many patients die due to long waiting lists or the unavailability of a suitable standard criteria donor (SCD) graft. Therefore, the allocation of organ resources has recently gained much attention. The model for end-stage liver disease (MELD) score of ESLD severity was adopted as a criterion for organ allocation , 2. As aThe concept of extended criteria donor (ECD) has long been introduced to bridge the gap between the demand and supply of liver donor grafts. ECD is defined as donor age >60 years, hepatitis C virus (HCV)-positive donor, liver with cold ischemia time (CIT) >12 h, donor after cardiac death (DCD), or donor liver with macrovesicular steatosis (MVS) >30%. While liver grafts with MVS, DCD, or long CIT are being used for low-acuity recipients , 8 whethThe aim of this multicenter study was to evaluate donor risk factors for early allograft dysfunction (EAD), length of ICU stay, and graft loss in patients with high MELD scores. In addition, we aimed to identify the risk factors for complicated outcomes and higher costs. To strengthen the results of the analysis, a nomogram was established to predict graft loss. These findings may be used in the selection of liver graft donors and recipients and to predict graft survival (GS) in the high acuity cohort, thus improving post-transplantation prognosis.We reviewed medical records of patients who received deceased donor liver transplant (DDLT) at three transplantation centers in China, namely the First Affiliated Hospital of Zhejiang University School of Medicine, the Qingdao University Affiliated Hospital, and the Shulan (Hangzhou) Hospital, from January 2016 to July 2019. These records were collected and maintained by the China Liver Transplant Registry database.Recipients with a MELD score >30 before LT were enrolled in this study. Cases of pediatric LT, split/reduced-size LT, re-transplantation, mortality during LT, abdominal multi-visceral transplantation, and combined liver and kidney transplantation were excluded.To determine predictors of early allograft function, length of ICU stay, and GS, the following donor variables were analyzed: donor age, sex, body mass index (BMI), ABO blood type, CIT, warm ischemia time (WIT), and results of pre-procurement serologic tests , albumin [ALB), and total bilirubin [TB) levels).LT was approved by the Liver Transplantation Committee of each institution and was performed after informed consent was obtained from all patients. This study was approved by the ethics committee and was performed according to the ethical principles of the Declaration of Helsinki. the requirement for informed consent related to the study was waived due to its retrospective nature. No organs from prisoners were used in the study.EAD was defined by one or more of the following criteria: (I) TB ≥10 mg/dL on postoperative day (POD) 7; (II) international normalized ratio (INR) ≥1.6 on POD 7; or (III) ALT or AST levels >2,000 U/L within the first 7 PODs. Each case was classified as “EAD” or “non-EAD” according to the criteria above. Fatty infiltration of the liver was differentiated into macrovesicular and microvesicular steatosis. Macro-vesicular steatosis (MVS) was categorized qualitatively into four groups according to the pathological examination: no MVS, mild (<30%), moderate (30–60%), or severe steatosis (≥60%). Livers with severe MVS were not used for LT to avoid primary non-function and graft loss.Patients in this cohort underwent orthotopic LT or piggyback LT based on hemodynamic stability, without splenectomy. During the operation, intravenous methylprednisolone and basiliximab were administered as immunosuppressants in all patients. After LT, triple immunosuppression based on a regimen including tacrolimus, mycophenolate mofetil (MMF), and methylprednisolone was administered. All patients infected with hepatitis B virus (HBV) were treated with either entecavir or tenofovir combined with hepatitis B immunoglobulin to maintain a high concentration of HBsAb after the operation.t-tests and chi-square tests. Kaplan-Meier survival analysis was used to evaluate GS rates (GSR), and differences among survival curves were tested by log-rank tests. Two multivariable logistic regression models were applied to identify predictive factors for EAD and length of ICU stay over 10 days, using the predictive factors with P < 0.1 in the univariable analysis. The enrolled patients were divided into training and validation cohorts at a ratio of 1:1. Similarly, a multivariable Cox regression model was used to identify predictive factors for GSR. Based on the multivariable analysis, the nomogram for GS was formulated using the rms package in R. The discrimination of the nomogram was evaluated using the concordance index (C-index). Bootstraps with 1,000 resamples were used to validate the nomogram and construct a calibration curve. The Hosmer-Lemeshow (HL) goodness-of-fit test was used to assess the calibration of the model. The software program SAS (version 9.1) and R software (version 3.6.1) were used for statistical analysis, and statistical significance was set at P < 0.05.The distribution of variables was assessed using the Shapiro-Wilk tests. Parametric continuous variables are presented as mean±standard deviation (SD), non-parametric distribution data as median and interquartile ranges (Q1–Q3) and discrete variables as numbers and percentages. The baseline characteristics were compared using During the study period, 2,210 patients with hepatocellular carcinoma (HCC) and liver cirrhosis at various stages were enrolled in the three institutions. A total of 939 patients had a MELD score >30, and 1,271 patients with MELD scores ≤ 30 were excluded. Furthermore, 46 cases of pediatric LT, 15 of split/reduced-size LT, 46 of re-transplantation, 6 of mortality during LT, 13 of combined liver and kidney transplantation, and 35 missing essential data were also excluded. After applying the inclusion and exclusion criteria, a total of 778 patients with MELD score >30 were collected and analyzed. The baseline characteristics of the patients are presented in P < 0.1, The incidence of EAD was 34.8% (271/778) in the entire cohort. According to the univariable logistic regression analysis, donor graft MVS, graft-to-recipient weight ratio (GRWR), recipient sex, WIT, CIT, serum K > 5.5 mmol/L, ALB <28 g/L, and ABO blood incompatibility were identified as potential risk factors for EAD P < 0.1, . All sigP < 0.1, The incidence of ICU stay over 10 days was 64.7% (503/778) for this high MELD score cohort. Donor age, BMI, recipient age, MELD score, Child score, and CIT were identified as potential risk factors for ICU stay over 10 days according to the univariable logistic regression analysis P < 0.1, . After iP < 0.1, Two hundred and fifty-nine recipients encountered graft loss during follow-up  would achieve a 3-year GSR of up to 78.4 %; however, those given an ABO blood type incompatible liver graft with CIT >6 h (n = 141) would have a lower 3-year GSR of 35.4% (P < 0.01). Donor graft MVS did not affect the GSR, whether in mild or moderate degree, nor did the other donor serum parameters before liver procurement.Subgroup analysis was performed on the basis of the Cox regression analysis. Recipients given an ABO blood type compatible liver graft with CIT <6 h (of 35.4% . CorrespP < 0.01). In addition, the 3-year GSR was 58.7 and 68.4%, respectively, for patients with or without longer length of ICU stay (P < 0.01).The 3-year GSR was 55.1 and 64.6%, respectively, in patients with or without EAD  , or the Since high MELD score and ABO-incompatible (ABO-I) liver grafts are both independent risk factors for graft dysfunction and loss, combining these factors would negatively affect GS. It has also been observed that regardless of the combination, increasing the number of ECD graft factors would worsen the post-LT graft GS. This risk becomes more pronounced in high-risk patients, such as those with a high MELD score.ABO-I LT without preoperative management leads to a cascade of cellular and antibody-mediated reactions that ultimately result in graft rejection. With the advent of B cell desensitization and reduction of preformed anti-donor ABO antibodies, ABO-I LT has become an important therapeutic option to increase the donor pool. ABO-I LT is usually limited to emergent situations when an ABO-compatible LT is unavailable, as it has been associated with an increased risk of graft loss. According to the OPTN/SRTR 2017 Annual Data Report, patients requiring urgent LT with MELD score >35 are already at increased risk of mortality while on the waiting list, and the use of an ABO-I ECD graft for LT compensates for the increased risk of low GS and EAD  can be improved by keeping the CIT below 6 h, as far as possible, and avoiding ABO-I LT. The use of ECD donors with factors such as old age, MVS, and other serological indicators may be allowed under close postoperative monitoring, which greatly improves the utilization and outcomes of ECD and reduces mortality on the waiting list.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.MY, AK, DL, XW, CX, QW, and XX conceived and designed the experiments. MY, AK, CX, RW, BP, and BC collected the data. MY, AK, DL, XW, and ZZ analyzed the data. MY, AK, and DL wrote the paper. JC, SZ, and XX supervised the paper. All authors contributed to the article and approved the submitted version.This research was partially supported by National Natural Science Funds for Distinguished Young Scholar of China (No. 81625003); Key Program, National Natural Science Foundation of China .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Ureteral obstruction of the graft kidney is a common complication of kidney transplantation. However, ureteral obstruction caused by inguinal hernia has rarely been reported. We present a rare case of ureteral obstruction with allograft dysfunction caused by an inguinal hernia.A 76-year-old man, who was a renal transplant recipient, presented with bilateral pitting oedema, reduced urine output, and right inguinal hernia.Abdominal computed tomography revealed severe hydroureteronephrosis of the kidney allograft. A right inguinal hernia with ureteral incarceration was observed.The patient underwent graft percutaneous nephrostomy, followed by antegrade insertion of a double-J tube (DJ). Gradual improvement was observed in his renal function. Right inguinal herniorrhaphy was performed 5 days later.The renal function returned to normal after percutaneous nephrostomy and insertion of the DJ. A right inguinal direct-type hernia with ureter adhesion to the hernial sac was observed during the surgery. The posterior wall defect was repaired by the McVay technique. The DJ was removed after 1 month. The patient's renal function remained stable at 6-month follow-up.The orientation of the graft kidney has a significant influence on the location of the ureter. Upward orientation of the hilum will result in superficial location of the ureter, rendering it close to the hernial sac and susceptible to incarceration. The transplant surgeon should be aware of such a presentation of graft dysfunction with inguinal hernia to prevent a delay in the diagnosis and graft loss. Herein, we present a special case of allograft dysfunction, wherein the transplant ureter inguinal hernia was related to the special position of the graft kidney.Ureteral obstruction is a common complication of renal transplantation. However, ureteral obstruction caused by inguinal hernia is a rare complication, and there are limited reports in the literature. The common causes of inguinal herniation of the transplant ureter are redundancy of transplant ureter2A 76-year-old man visited our clinic with complaints of swelling and indentation upon pressing in both legs for 2 weeks. He had past medical history of one-vessel coronary artery disease, biliary stone had undergone endoscopic retrograde cholangiopancreatography, and end-stage renal disease secondary to analgesic nephropathy. He had undergone living-donor kidney transplantation 3 years ago in mainland China.Physical examination revealed a right-sided inguinal hernia and bilateral lower limbs grade 2/4 pitting oedema. His serum creatinine level increased to 1.2 mg/dL , and bedside renal echography revealed graft kidney hydronephrosis. Abdominal computed tomography was performed, which revealed severe hydroureteronephrosis of the kidney allograft  and vein (5.4 cm), due to which the hilum faced upwards. Therefore, the ureter was located superficially downwards and was prone to inguinal herniation.Possible risk factors include redundant ureter, The majority of the cases in the literatures describe the insertion of a percutaneous nephrostomy tube before surgery to prevent any more graft loss.–4,6–16 Antegrade pyelography can further confirm the presence of hydroureteronephrosis and ureter entrapment in the hernial sac; however, the clinical management does not change.Ultrasound is the first-line diagnostic imaging modality. Ultrasound usually reveals hydronephrosis; however, may not enable visualization of the full length of the ureter. computed tomography can provide information on the level of ureter obstruction and ureter entrapment in the inguinal hernia. Although most of the case reports related to this topic had no DJ insertion prior to surgery, there is no data regarding their operation time and the risk of ureter injury during surgical exploration. We propose that preoperative ureteral stent insertion may be a good option to avoid ureteral injury during herniorrhaphy of the transplant ureter inguinal hernia.In the present case, the DJ was inserted in the antegrade direction preoperatively, which significantly enabled identification of the ureter during surgical exploration of the inguinal canal. In our case, the ureter was located deep in the posterior wall of the inguinal canal and close to the neck of the hernial sac. To our knowledge, this is one of the few studies to have preoperative DJ insertion.4Transplant ureter inguinal hernia is a rare condition that leads to graft hydronephrosis and subsequent acute renal failure. General surgeons and urologists should consider ureteral inguinal hernia in the differential diagnosis of cases with a previous history of renal transplantation. Furthermore, transplant surgeons should understand the importance of orientation of the graft kidney. The orientation not only affects the graft vessel anastomosis but also the susceptibility of the ureter to compression by the hernial sac.The authors thank the patient and his family for permitting use of the medical data and other information that led to successful completion of the present article.Resources: Ping-Chin Lai.Supervision: Wei-Ching Lin, Chao-Hsiang Chang.Writing – original draft: Tian You Chang.


PurposeThe primary outcome measure of this study was to determine the effect of hamstring graft size on the functional outcome of arthroscopic anterior cruciate ligament reconstruction (ACL-R) and the secondary outcome was to ascertain the effect on revision surgery at the two-year follow-up.MethodsThis is a prospective comparative study of 144 consecutive patients undergoing primary ACL reconstruction using a hamstring autograft. All patients underwent graft harvesting and ACL reconstruction with the standard technique. The graft diameter was recorded intraoperatively using a graft sizer. All patients were followed up with the Knee Injury and Osteoarthritis Outcome Score (KOOS) recorded at preop, six weeks, six months, one year, and two years and whether they underwent revision during this period was documented.ResultsThe mean KOOS for patients with a ≤ 7mm graft diameter was 80.5±13.1, which was significantly lower compared to those with graft > 7 mm of 88.3±8.5, respectively (p<0.001) at the two years follow-up. Patients with graft ≤ 7mm did poorly, especially with mean KOOS subscores of sports and recreation and quality of life (p<0.05). Twenty-three point one percent  of patients with a graft < 7mm underwent revision, whereas only 5.8% and 2.6% of patients underwent revision with a graft diameter of 7.1-8.0 and 8.1-9.0 (p=0.027).ConclusionsThe smaller Hamstring graft diameter leads to poorer functional outcomes of the patient’s ACL reconstruction. Though the number of revisions was high among those with a graft diameter of ≤ 7mm, multicentric studies with many revisions are required to confirm the relation. The incidence of anterior cruciate ligament (ACL) injuries has increased, and its surgical reconstruction has become a routinely practiced procedure worldwide by many orthopedic surgeons over the past two decades -2. The aVarious graft options exist for ACL reconstruction like hamstring, bone-patellar tendon, quadriceps, etc., and multiple factors influence finalizing the decision of the ideal graft choice for the patient. It depends on whether the graft can re-create the anatomical and biomechanical properties of the native ligament, assures safer fixation, faster biological integration, and minimal donor site morbidity. Also, the training and experience of the surgeon harvesting a particular graft are important ,9-10. HaRevision of ACL reconstruction is technically demanding with poor outcomes and its incidence is found to decrease with an increase in the graft size. Graft size is considered one of the major determining factors in predicting the revision rates in ACL-R procedures -19. GrafHence, the primary outcome measure of this study was to determine whether the graft size of the hamstring graft (STG) plays a role in determining the functional outcome of arthroscopic anterior cruciate ligament reconstruction and the secondary outcome measure is to determine whether it increases the need for revision surgery at the two-year follow-up.Among 233 patients, 144 who underwent isolated ACL reconstruction using a hamstring autograft between January 2015 to January 2020 in our hospital were included for evaluation. Patients with a multi-ligament injury, associated meniscal tear requiring intervention, previous trauma to the knee, bilateral ACL tear, revision surgeries, neuromuscular disorder, associated neurovascular injury, and graft amputation while harvesting the graft were excluded. Patients who were not available for follow-up for at least one year were also excluded. All patients included underwent ACL reconstruction using a hamstring graft with either a bio screw/titanium on the tibial side and an Endobutton on the femoral side as a fixation device. An accessory anteromedial portal with a femoral and tibial jig was used for the tunnel. All patients underwent surgery with valid informed consent. Institutional ethical committee approval was taken to conduct the study. Demographic data and clinical findings were documented, and MRI was used to confirm the diagnosis of all patients. Preoperative functional knee scores were documented using the Knee Injury and Osteoarthritis Outcome Score (KOOS) scoring system.Operative techniqueAll grafts were harvested by the senior surgeon or in his presence by other fellowship-trained specialists with more than two years of experience of doing ACL reconstruction using a 2-3 cm vertical anteromedial incision on the proximal tibia, at the level of the insertion of the semitendinosus and gracilis muscles. Following this, the tendon of the STG muscle was dissected individually, stripped against the bone, and the ends were whip-stitched using a non-absorbable 2-0 Ethibond suture; the graft was removed using a closed graft stripper. After clearing muscle, fat, synovium, and whipstitching the other end, the diameters of the STG grafts were measured using the graft sizer available from the ConMed /Stryker  graft sizer, which measured from a range of 3 mm to 12 mm with 0.5 mm increments. The minimum diameter that allowed the smooth passage of the entire graft through the tube completely was taken as the graft diameter. For the sake of consistency, any semitendinosus graft above 20 cm length was tripled as a rule unless a shorter length mandated it to be doubled. Gracilis was doubled in all cases irrespective of the length obtained. For ACL reconstruction, the same technique was used in all cases in which the femoral tunnel was drilled using a femoral jig at the footprint via the accessory anteromedial portal; the tibial tunnel was drilled using an appropriate tibial-zig at the footprint for anatomic single-bundle ACL reconstruction. The graft was fixed on the femur side using an Endobutton and the tibial side using an interference screw (titanium or bioabsorbable).Following the surgery, a standard rehabilitation protocol was used for all patients, and patients were recalled for follow-up at six weeks, six months, one year, and two years. The patient’s knee range of motion, KOOS score, clinical tests, complications, and status of revision surgery were documented at each follow-up visit as part of data collection for the institutional knee registry. For patients who were not able to visit the hospital because of the COVID pandemic, a telephonic consultation was used to collect relevant data.Data analysisAll characteristics were summarized descriptively. For continuous variables, the summary statistics of mean ± standard deviation (SD) were used. For categorical data, the number and percentage were used in the data summaries and diagrammatic presentation. The chi-square (χ2 ) test was used for the association between two categorical variables. The difference in the means of analysis variables between two independent groups was tested by an unpaired t-test. The difference in the means of analysis variables between more than two independent groups was tested by analysis of variance (ANOVA) and the F test of testing equality of variance. If the p-value was < 0.05, the results were considered to be statistically significant. Data were analyzed using SPSS software v.23  and Microsoft Office 2007 .Out of the 144 patients enrolled in the study, 83.4% (120 of 144) of participants were males, and the remainder were females . The mean age of the patient was 30.89 ± 9.3 years (range 16-56). The graft size was divided into four ranges, that is, < 7, 7.1-8.0, 8.1-9, and > 9 mm. The mean age in each graft size range < 7, 7.1 -8, and 8.1-9 mm are 27.6 ± 10.3 years, 30.9 ± 9.9 years, 29.7± 7.8, and 30.5 ± 7.5 years, respectively. There was no statistically significant difference between the demographic variables between groups. The mean graft size in males and females was 8.23 ± 0.56 and 7.88 ± 0.54 (p=0.017), respectively. Most patients of both sexes had a graft between 7.1 and 8 mm. All the patients were followed up at least for one year to be included in the evaluation and the mean overall follow-up was 18.4 ± 4.5 months, and there was no difference in the mean follow-up period amongst patients with different graft diameters (p>0.05).In our study, it was observed that patients with graft size < 7 mm reported an inferior postoperative functional outcome using the KOOS score at a follow-up period of one and two years in comparison to other graft sizes (p= 0.008 and p=0.007). No significant statistical differences were seen in the early postoperative phase in the outcome between the four groups of the graft sizes at the six weeks and six months follow-up. The functional outcome of patients with graft size 7.1-8.0, 8.1-9, and > 9 mm were similar, and there wasn’t much difference in the mean KOOS score, and the mean KOOS score for those > 7 mm was 88.3±8.5 (p<0.001) , respectively, whereas there was no statistically significant difference in the subscale score of pain and symptoms. The patients with graft diameter 7.1-8.0, 8.1-9, and > 9mm did well on all the KOOS subscale scores, and there were no differences between the means of subscale scores Table . In our One of the major drawbacks of a hamstring autograft is the variability in its size and the inability to accurately predict preoperatively. Though some authors have recommended a graft size of at least 7 mm, the ideal size of hamstring graft for ACL reconstruction has not been documented in the literature -24. ACL Magnussen et al., in one of the earlier studies, evaluated the effect of the size of the graft used in the revision as an indicator of graft failure. They concluded that hamstring autografts of size 8 mm in diameter or less are linked with an increase in the rate of revision. A major limitation conceded by them was that they did not consider the patient with graft failure and failed to undergo revision. Moreover, functional outcome was not determined in the study . Park etMariscalco et al., in their retrospective study of 263 patients, considered the effect of graft size on KOOS. They concluded that smaller graft diameters were associated with poorer KOOS at the end of two years of follow-up. They noticed that there was a proportionate increase of 3.3-point in the KOOS pain scale, a 3-point increase in KOOS activity of daily living, and a 5.2-point increase in the KOOS sports function scale for every 1 mm increase in graft size . In our Most of the studies in the literature have considered the number of revision surgeries as the primary outcome measure to evaluate the effect of hamstring diameter among people undergoing ACL surgery. Park et al. observed no revisions in any of the patients with a graft diameter of 8 mm or more, whereas the revision risk was 5.2% among those with a graft diameter of less than 8 mm. Magnussen et al. had 14.3% (17 out of 119 patients) of cases undergoing revisions . Among tThe smaller hamstring graft diameter leads to poorer functional outcomes of patients’ ACL reconstruction, especially in sports and recreation. Though the number of revisions was high among those with a graft diameter of ≤7 mm, multicentric studies with a larger number of revisions are required to confirm the relation.