Objective: To present the first Egyptian clinical practice guideline for kidney transplantation (KT).Methods: A panel of multidisciplinary subspecialties related to KT prepared this document. The sources of information included updates of six international guidelines, and review of several relevant international and Egyptian publications. All statements were graded according to the strength of clinical practice recommendation and the level of evidence. All recommendations were discussed by the panel members who represented most of the licensed Egyptian centres practicing KT.Results: Recommendations were given on preparation, surgical techniques and surgical complications of both donors and recipients. A special emphasis was made on the recipient’s journey with immunosuppression. It starts with setting the scene by covering the donor and recipient evaluations, medicolegal requirements, recipient’s protective vaccines, and risk assessment. It spans desensitisation and induction strategies to surgical approach and potential complications, options of maintenance immunosuppression, updated treatment of acute rejection and chemoprophylactic protocols. It ends with monitoring for potential complications of the recipient’s suppressed immunity and the short- and long-term complications of immunosuppressive drugs. It highlights the importance of individualisation of immunosuppression strategies consistent with pre-KT risk assessment. It emphasises the all-important role of anti-human leucocyte antigen antibodies, particularly the donor-specific antibodies (DSAs), in acute and chronic rejection, and eventual graft and patient survival. It addresses the place of DSAs across the recipient’s journey with his/her gift of life.Conclusion: This guideline introduces the first proposed standard of good clinical practice in the field of KT in Egypt.Abbreviations: Ab: antibody; ABMR: Ab-mediated rejection; ABO: ABO blood groups; BKV: BK polyomavirus; BMI: body mass index; BTS: British Transplantation Society; CAN: chronic allograft nephropathy; CDC: complement-dependent cytotoxicity; CKD: chronic kidney disease; CMV: cytomegalovirus; CNI: calcineurin inhibitor; CPRA: Calculated Panel Reactive Antibodies; (dn)DSA: (de novo) donor-specific antibodies; ECG: electrocardiogram; ESWL: extracorporeal shockwave lithotripsy; FCM: flow cytometry; GBM: glomerular basement membrane; GN: glomerulonephritis; HIV: human immunodeficiency virus; HLA: human leucocyte antigen; HPV: human papilloma virus; IL2-RA: interleukin-2 receptor antagonist; IVIg: intravenous immunoglobulin; KT(C)(R): kidney transplantation/transplant (candidate) (recipient); (L)(O)LDN: (laparoscopic) (open) live-donor nephrectomy; MBD: metabolic bone disease; MCS: Mean channel shift (in FCM-XM); MFI: mean fluorescence intensity; MMF: mycophenolate mofetil; mTOR(i): mammalian target of rapamycin (inhibitor); NG: ‘not graded’; PAP: Papanicolaou smear; PCN: percutaneous nephrostomy; PCNL: percutaneous nephrolithotomy; PKTU: post-KT urolithiasis; PLEX: plasma exchange; PRA: panel reactive antibodies; PSI: proliferation signal inhibitor; PTA: percutaneous transluminal angioplasty; RAS: renal artery stenosis; RAT: renal artery thrombosis;:rATG: rabbit anti-thymocyte globulin; RCT: randomised controlled trial; RIS: Relative MFI Score; RVT: renal vein thrombosis; TB: tuberculosis; TCMR: T-cell-mediated rejection; URS: ureterorenoscopy; (CD)US: (colour Doppler) ultrasonography; VCUG: voiding cystourethrogram; XM: cross match; ZN: Ziehl–Neelsen stain This document integrates recent international guidelines with Egyptian experience and environmental and socioeconomic circumstances. The latter are dominated by a rich bioecological environment, specific demographics and social constraints, limited financial resources and exclusively live-donor transplantation due to inability to perform deceased donor kidney transplantation (KT) in Egypt at the current time. It is focussed on the adult recipient. While we touch on general issues related to children, pregnant women and elderly patients, we have kept this information brief, and cited selected comprehensive position statements that provide more details on these special populations.Six guidelines (including their latest updates), namely Kidney Disease Improving Global Outcomes (KDIGO), European Association of Urology (EAU), European Renal Best Practice (ERBP), British Transplantation Society (BTS), National Institute of Health Research (NIHR), and Australian Transplantation guidelines .Review of several guides, position statements, meta-analyses and leading institutional protocols .Relevant Egyptian publications.@ software) to which the current and past leading nephrologists of Egyptian Ministry of Health-licensed transplant centres were recruited. A total of 40 nephrologists were invited, of whom 28 responded.Results of a specifically designed 17-question online survey , marital status, and consanguinity with the recipient (1B).We strongly recommend social and psychological assessment, and illicit drug testing. (1 C).We strongly recommend routine urine analysis and culture (1 C).We strongly recommend 24-h urine collection for protein and creatinine (1 C).We recommend Ziehl–Neelsen stain (ZN) and PCR for tuberculosis (TB) in urine for suspected cases (2 C).We strongly recommend routine blood tests including full and differential blood counts, renal function tests, liver function tests, blood electrolytes, lipid profile, viral profile testing for hepatitis B, hepatitis C, cytomegalovirus (CMV), and human immunodeficiency virus (HIV) (1 C).We strongly recommend radiological investigations in terms of as grey-scale ultrasonography (US), plain urinary tract X-ray, chest X-ray, CT angiography, CT urography, and radioisotope renography (1 C).We strongly recommend an electrocardiogram (ECG) (1 C).We strongly recommend taking a detailed history including age, gender, BMI, marital status, duration of dialysis, original kidney disease and amount of urine output per day, history of previous surgery, and history of medications (1B).We strongly recommend routine urine analysis and culture (1B).We suggest ZN and PCR for TB in urine for suspected cases (3 C).We strongly recommend routine blood tests including full and differential blood counts, renal function tests, liver function tests, blood electrolytes, lipid profile, viral profile testing for hepatitis B, hepatitis C, CMV and HIV (1B).We suggest examination of sputum for TB by ZN and PCR for suspected cases (3 C).We recommend radiological profile including abdominal US, abdominopelvic CT, chest X-ray and micturition cystourethrogram (in cases with suspected voiding dysfunction or VUR) (2B).We recommend ECG, echocardiography and vascular assessment including vessels of the neck, pelvis and lower limbs as appropriate (2B).We suggest full urodynamic studies and urethrocystoscopy as appropriate (3 C).We suggest upper and/or lower gastrointestinal tract endoscopy as appropriate (3 C).We suggest biopsy from kidneys, liver, rectum as appropriate (3 C).We recommend exclusion of malignant diseases by thyroid evaluation, mammography in females, Papanicolaou smear (PAP) smear in females, and DRE and PSA in males (2B).It is compulsory and mandated by the Egyptian Code for Organ and Tissue Transplantation that hospitals seeking to perform organ transplantation to be licensed by the Egyptian Ministry of Health.It is compulsory to obtain a fully informed consent from the donors and recipients.It is compulsory to obtain formal approval from the Egyptian Supreme Committee for Organ Transplantation for every case of KT.H. Influenzae, seasonal influenza, meningococcus and pneumococcus (1 C).We strongly recommend all kidney transplant candidates to receive the inactive immunisation vaccines and boosters prior to transplantation for hepatitis B, We suggest assessing immunogenicity by measuring available serum antibody (Ab) titres rather than previous history of infection or vaccination (3 C).We suggest giving live vaccines  if scheduled or National Health alerts and recommendations are issued, and to be at least 6 weeks prior to KT (2 C).We strongly recommend quadrivalent meningococcal conjugate vaccine for renal transplant candidates with previous splenectomy or planned to receive complement inhibitors (1 C).We suggest human papilloma virus (HPV) vaccine to be given to both sexes within the age range of 9 to 26 years (3 C).We recommend KT recipients (KTRs) to start or resume immunisation vaccines and boosters 6 months after KT except for seasonal flu (2 C).We strongly recommend seasonal flu vaccine for KTRs to be not earlier than 1 month after KT and before the onset of influenza season then annually (1 C).We strongly recommend avoiding all live vaccines for KTRs (1D).We suggest considering family and caregivers immunisation for enhancing KTRs protection (3 C).The risk of community-acquired infection is significantly increased in KTRs, in parallel with the extent of immunosuppression. The incidence of pneumonia and influenza is multiplied many fold in KTRs compared to that in the general population. In the typical subtropical environment, including Egypt, KTRs are exposed to many other infections such as hepatitis, TB, typhoid, and others.H. Influenzae, HBV, S. Typhi, etc., the level of protection can be measured by specific Ab levels, which determines the need for booster doses. Pre-transplant vaccination with live attenuated vaccines should be completed 4 weeks before KT to avoid activation upon immunosuppression, or 2 weeks with inactivated vaccines to provide enough time for acquiring adequate immunity. Vaccination is of confirmed value in reducing KTRs’ susceptibility to pneumococcal pneumonia, H. influenzae, seasonal influenza, hepatitis B new viral infection and meningococcal meningitis. The benefit of other vaccinations  typing of KTCs and donors, using molecular methods preferably Ab titration methods to remove inhibition (1B). Typing should include at all loci (1D) including DQ (1D) and DP in sensitised recipients (2D).We strongly recommend testing for anti-HLA Abs in all KTRs by solid phase assay (1B).We strongly recommend a pre-transplant assessment of the recipient’s past and present immunological risk factors, including DSAs (1D).In the re-transplant population, we recommend a higher risk score for historic DSAs to repeat mismatch especially at class II compared to other recognised sources of sensitisation (1 C).We recommend using the recipient’s immunological risk for individualisation of immunosuppressive therapy and post-KT monitoring (2D).We suggest not routinely testing KTCs for non-HLA Abs (2 C).There is evidence that the incidence of acute rejection and the eventual long-term graft outcomes are related to pre-transplant recipient’s immunological risk factors. Most of the relevant recent literature, as well as 89.3% of our survey respondents, recommend pre-transplant risk assessment including historic data, and detection of circulating anti-HLA Abs by complement-dependent cytotoxicity (CDC) and solid-phase techniques (Luminex®). Unfortunately, a quantitative tool for measuring the collective recipient’s immunological risk is not yet available. Pending the development of such a validated tool, the main players may be categorised into five classes .Table 3.We recommend offering desensitisation to KTRs with ‘high’ to ‘very high’ immunological risk .We strongly recommend offering desensitisation for KTRs with immunological barrier to their potential donors due to both/either ABOi (1B) or HLAi (1 C).We strongly recommend desensitisation in the context of direct kidney donation (Ab reduction) or Kidney Paired Donation program (Ab avoidance) (1 C).We suggest escalating individual desensitisation protocols  + plasma exchange (PLEX) + rituximab ± others) to achieve pre-defined acceptable CDC, flow cytometric and solid-phase parameters, taking individual centre experience and total cost into consideration (NG).We suggest for individual transplant centres to set their own acceptable immunological targets to be achieved by desensitisation before proceeding to KT (NG).We suggest to follow-up DSAs after KT in desensitised KTRs (NG).We suggest avoiding desensitisation and searching for a different donor in recipients who remain in the ‘highest risk’ category  (NG).Desensitisation is the logical clinician’s response to the increasing disclosure of preformed anti-HLA Abs in KTRs . It has There are many protocols for desensitisation, which should be tailored to the magnitude of DSAs in terms of their number and MFIs. Most popular desensitisation protocols use either high-dose IVIg (2 g/kg), or PLEX (or immunoadsorption) + low dose IVIg (100 mg/kg after each session). Other protocols add, as yet off-label, rituximab, bortezomib, obinutuzumab, imlifidase, eculizumab, tocilizumab, or IgG endopeptidase, etc. DSAs must be measured again after desensitisation to ensure achievement of pre-set targets. Owing to the considerable inter-laboratory variability of current MFI measurements, each centre is required to set its own targets.We strongly recommend transplanting the kidney with the lesser function and/or minor anomaly (1 C).We recommend considering a right donor nephrectomy in female donors of the child-bearing age to avoid potential right hydronephrosis during pregnancy (2D).We strongly recommend that open live-donor nephrectomy (OLDN) be performed by experienced transplant urologists in centres where laparoscopic equipment and experts are not available (1A).We strongly recommend performing laparoscopic LDN (LLDN) in well-equipped highly specialized centres with trained staff only (1A).We suggest offering long-term annual monitoring of all living kidney donors by physical examination and measuring urinary protein excretion, serum creatinine and estimated GFR (eGFR) (3 C).OLDN is still a valid approach in KT; however, the open approach is associated with an increased risk of wound complications, as well as poor cosmetic outcome. Although the warm ischaemia time is longer in LLDN when compared to OLDN, there is no difference in final graft function ,21.LLDN is as safe as OLDN regarding donor and graft survival, rejection, and urological complications in addition to the superior results in terms of shorter hospital stay and time to return to work, and less intraoperative blood loss . The risWe strongly recommend that the KT surgery be performed by a well-trained team lead by a highly experienced transplant urologist (1A).We strongly recommend open KT as the standard surgical approach (1A).We suggest perivascular lymphatics to be carefully ligated (NG).We suggest meticulous mobilisation of appropriate segments of both the iliac artery and vein (NG).We suggest cooling the surface of the kidney with ice bags/slush during implantation (NG).We strongly recommend that the renal vein  is anastomosed to the external iliac vein end-to-side (1B).In case of a short renal vein , we recommend tension-free anastomosis by ligating and dividing the recipient’s internal iliac vein (2B).We strongly recommend repairing any intimal rupture or flap in either donor or recipient arteries prior to anastomosis to avoid iliac artery dissection (1A).We recommend a single renal graft artery to be anastomosed to internal iliac artery (end-to-end) or to the external or common iliac artery (end-to-side) (1B).We suggest that two, equal size, graft arteries can be anastomosed separately or anastomosed together to create one ostium (3 C).We suggest that two, unequal size, graft arteries can be anastomosed separately (3 C).We suggest that anastomosis is achieved using 5/0 or 6/0 non-absorbable monofilament polypropylene sutures (3 C).We recommend that time of vascular anastomosis and hence graft re-perfusion time should be kept to minimum for better graft function (2B).We strongly recommend that native ureter should be kept to an appropriate length to avoid kinking or redundancy in addition to preserving its periureteric fat to preserve its blood supply (1B).We strongly recommend that ureter is anastomosed to the bladder by an extravesical non-refluxing (Lich–Gregoir) uretero-neocystostomy (1A).We recommend stenting the uretero-neo-cystostomy (2A).We recommend removing the stent within 30 days (2 C).The kidney (whether right or left) can be placed in either the right or left iliac fossa. An extraperitoneal iliac fossa ‘hockey-stick’ Gibson incision is the standard approach . The fatTension-free anastomosis of the donor renal vein is mandatory. Transposition of the recipient iliac artery and vein can help to achieve that. The renal vein can also be lengthened using either donor gonadal vein or recipient saphenous vein .The internal iliac artery is more frequently affected by atherosclerosis than the external or common iliac arteries and so less preferred for end-to-end anastomosis with the donor artery. Multiple renal graft arteries should be handled carefully to ensure proper graft reperfusion. In case of two renal graft arteries and one is small it can be either sacrificed (if possible), anastomosed to the main renal artery (end-to-side), or anastomosed to the inferior epigastric artery (end-to-end). For multiple arteries, reject the donor or combine more than one of the previously mentioned techniques. Finishing the vascular anastomosis, unclamp the renal vein first then the artery .In a normal bladder with no underlying abnormality, the well-vascularised graft ureter can be anastomosed to the bladder by either an extravesical (Lich–Gregoir) or intravesical (Politano–Leadbetter) uretero-neo-cystostomy using monofilament absorbable sutures with the former more preferred for its reduced overall complications and less UTIs. When the graft ureter is short, ischaemic, or denuded, the surgeon should use the native ureter for ureteroureterostomy or pyeloureterostomy if they are completely in a healthy condition. Although ureteric stenting is preferable as it may help to prevent major urological complications, it may be omitted to avoid another endoscopic procedure to be removed . Duplex We suggest image-guided drainage or exploration for large post-KT haematomas causing graft pressure or haemodynamic instability (3 C).We recommend performing colour Doppler US (CDUS) if RVT is suspected (2B).We recommend surgical exploration if graft shows impaired perfusion on CDUS (2B).We recommend performing thrombectomy if RVT is confirmed with a viable graft. Similarly, we suggest performing a graft nephrectomy if RVT is confirmed with an unviable graft (2B).We strongly recommend not using routine prophylactic anticoagulation to prevent RVT in low-risk patients (1B).Renal artery thrombosis (RAT)We recommend performing CDUS if RAT is suspected (2B).We recommend urgent surgical exploration if the graft shows impaired perfusion on CDUS (2B).We recommend performing thrombectomy if RAT is confirmed with a viable graft (2B).We recommend performing graft nephrectomy if RAT is confirmed with an unviable graft (2B).We strongly recommend performing CDUS if RAS is suspected with MR or CT angiography performed for equivocal cases (1A).We suggest performing percutaneous transluminal angioplasty (PTA)/stenting as first-line for RAS (3 C).We suggest performing surgery for failed PTA, or long, multiple, or narrow RAS (3 C).Haematomas occur frequently after KT (0.2–25%) being mostly minor and asymptomatic and requiring no intervention. Larger haematomas may be associated with graft dysfunction due pressure, or haemodynamic instability requiring image-guided drainage or exploration ,30.prophylaxis ) is the outcome of high-quality evidence and extensive clinical implementation worldwide (1A). It is the preferred protocol by 88.9% of our survey’s respondents. Long-acting tacrolimus and mycophenolic acid are as safe and effective as short-acting tacrolimus and mycophenolate mofetil respectively, with certain pharmacokinetic or pharmacodynamic advantages.de novo CNI-free protocols. There are concerns about the early use of proliferation signal inhibitors (PSIs) regarding the increased incidence of biopsy-confirmed acute rejection when used without CNIs, delayed wound healing, proteinuria, anaemia, pneumonia, etc.There are acknowledged alternatives to standard Tac/MMF/P protocol, which are not inferior regarding graft survival at least at 1 year . HoweverCNIs have a narrow therapeutic window, hence the need for frequent blood level monitoring. Achievement of the target blood levels  is of fuMycophenolate, on the other hand, has a wider therapeutic window, has fewer drug–drug interactions, and its blood level is less volatile. Therefore, the need for monitoring is less pressing than with CNIs (2D).The only exception to the 3-month immunosuppression stability rule is the steroid elimination protocol. The latter requires complete steroid withdrawal by 1 week post-KT in order to avoid subsequent acute rejection. If prednisolone is used beyond the first week, we suggest continuation for at least 1 year (2 C). Although these recommendations are adopted by most centres, there are other less popular protocols as shown in In stable grafts, we strongly recommend that CNIs be continued, rather than withdrawn, unless clearly indicated (1B).We strongly recommend female KTRs who wish to become pregnant to be switched from mycophenolate to azathioprine at least 6 weeks before attempting pregnancy (1A).Female KTRs must switch mycophenolate to azathioprine once she discovers a confirmed pregnancy (NG).We suggest reducing CNI blood level targets in KTRs aged >65 years with stable grafts, taking the patient’s immunological risk grade into consideration (NG).If a steroid elimination protocol is contemplated, we recommend limiting its use to low-risk KTRs, using biological induction, and complete steroid withdrawal by 1 week post-KT (2 C) and to be considered for paediatric recipients.We recommend intensifying maintenance immunosuppression following recovery from an acute rejection, either by increasing the target drug blood levels, or by switching patients on cyclosporine to tacrolimus and/or azathioprine to MMF (2 C).In patients with rising serum creatinine attributed to the use of CNIs, we suggest switching to a PSI-based protocol .We recommend that male KTRs be advised that m-TOR inhibitors reduce the male sperm count and are counselled accordingly (1 C).There is evidence that long-term standard triple immunosuppression is effective for an indefinite duration. Even though CNIs may induce interstitial fibrosis, this rarely progresses to graft failure despite a slowly rising serum creatinine. So, stable grafts may remain on this protocol indefinitely, which is the preferred policy in 67.9% of our survey respondents. As few as 10.7% and 7.1% preferred CNI withdrawal at 3 and 12 months respectively, while 14.3%, 7.1% and 14.3%% preferred steroid withdrawal at 3, 4, and 12 months post-KT, respectively.Nevertheless, there are a few exceptions where minimisation or elimination of one or more of the triple immunosuppression components is required for a variety of reasons. The latter include age limitations, pregnancy, drug side-effects or idiosyncrasy, significant drug–drug interactions, infections or other post-KT complications.The CARMEN study showed the benefit and safety of early steroid withdrawal by the first week post-KT. This concurs with an Egyptian study where steroids were administered for only 3 days .Some centres prefer to switch even with stable grafts to a CNI-free protocol for the sake of better graft function . EveroliCNI replacement with Belatacept has also been shown to improve graft function and histology and prolong patient survival . HoweverWe strongly recommend intravenous ‘pulse’ (‘bolus’) corticosteroids for the initial treatment of acute T-cell-mediated rejection (TCMR) followed by gradual tapering of oral prednisolone. (1D)We recommend adding lymphocyte-depleting Abs for acute TCMR that do not respond to corticosteroids, those above Banff Grade I, and for recurrent TCMRs (2 C).We recommend treating acute ABMR with IVIg and plasma exchange with or without anti-CD20, corticosteroids, or lymphocyte-depleting Abs (2 C) guided by biopsy findings and clinical response (NG).We strongly recommend checking for compliance to immunosuppression treatment in patients with acute rejection (1A) particularly in late rejection (NG).We recommend intensifying maintenance immunosuppression following an acute rejection (see Box 13).We recommend adding or restoring maintenance prednisone in patients not on steroids who have a rejection episode (2D).We recommend adding or restoring maintenance tacrolimus in patients not on CNIs who have a rejection episode (2D).Initial treatment of acute rejection with pulse steroids remains a ‘gold standard’. While response is awaited, the result of graft biopsy should be available, thereby confirming the diagnosis, categorising the rejection as being cellular, Ab-mediated or mixed, Banff scoring, and excluding other causes of acute graft dysfunction. In the absence of adequate response to steroids in cell-mediated rejection, or if the Banff score is above Grade A, a T-cell depleting Ab should be used. If the biopsy shows an ABMR, PLEX or immunoadsorption should be used, to be followed by low-dose IVIg (100 mg/kg) or high-dose IVIg (2 g/kg) with or without steroids have become the standard of care. Anti-CD20 (rituximab) is a second-line despite mixed evidence of its added benefit.Since acute rejection constitutes an additional immunological risk, it is imperative to review the patient’s compliance to immunosuppressive drugs, restore steroids and CNIs if withdrawn or minimised, and increase the target blood levels of CNIs. (See Box 12)We strongly recommend antimicrobial prophylaxis in all recipients , to be individually tailored according to the recipient’s age, history of previous infection(s), comorbid conditions, status of specific immunity, and strength of immunosuppression (NG).Chemoprophylaxis is a routine recommendation in most KT guidelines and experiences worldwide. It targets organisms that are often quiescent due to natural or vaccine-boosted immunity and may recrudesce with immunosuppression. The most commonly used agents are shown in We suggest monitoring graft function by measuring the urine volume, urinary protein excretion, serum creatinine (and eGFR), and performing US examination at a decremented frequency with progression of post-KT duration .We recommend female KTRs attempting pregnancy to be at least 1 year post-KT (2 C) AND fulfilling the following:Stable kidney function with serum creatinine not exceeding 1.5 mg/dL without rejection episodes in the past year (2 C).Proteinuria not exceeding 1 g/day (2 C).Well controlled blood pressure.No active CMV infection or at least 6 months following last active CMV infection.We strongly recommend modifying the immunosuppression protocol in women in the setting of attempted or realised pregnancy as mentioned in Box 13We strongly recommend a graft biopsy in the setting of persisting delayed graft function (1 C).We strongly recommend a graft biopsy following treatment of acute rejection episodes if serum creatinine does not return to baseline (1 C).We suggest that all very-high- and high-risk KTRs undergo a scheduled graft biopsy (with staining for C4d) at 3–6 months post-KT and 1–3 times thereafter for the first 2 years (NG).We suggest at least one graft biopsy (with staining for C4d) in intermediate-risk KTRs during the first year  (NG).de novo DSA, or with one biopsy confirmed ABMR episode or evidence of non-adherence to undergo at least one protocol biopsy (NG).We suggest that any KTR with detected We suggest that a protocol biopsy be obtained prior to a shift in the maintenance immunosuppression protocol and at 3 months thereafter (NG).We suggest that a protocol biopsy to be obtained in KTRs with a high risk of recurrence of their original disease (NG).de novo glomerulonephritis (GN). The potential benefit of early treatment is opposed by potential risk of injury to a healthy graft. For this reason, individual centres diverge in including surveillance biopsy in their routine protocols. In our survey, 48% of responders favour protocol biopsies. Evidence continues to show a negative impact on graft survival in patients with subclinical TCMR and ABMR, thereby tipping the balance in favour of routine protocol biopsy at least until non-invasive rejection biomarkers have been developed, as predicted by 16% of our survey respondents.The objective of a surveillance biopsy is to detect subclinical rejection, certain viral infections, drug toxicity or recurrence/We recommend that a serum sample should be sent at the time of renal biopsy (for acute or chronic graft dysfunction) to look for HLA-specific antibodies (2 C).We suggest that monitoring of post-KT DSA be performed in very high- and high-risk patients during the first 2 years post-KT  (NG).We suggest screening for anti-HLA Abs in intermediate- and low-risk KTRs at 3 months post-KT, then once annually for the first 2 years post-KT with a concurrent biopsy if positive  (NG).We suggest screening for anti-HLA Abs in recipients under minimisation protocols every 3 months for 1 year, and once annually for the first 2 years post-KT (NG).de novo DSA (dnDSA). Recent literature reports ~20% incidence of dnDSA in immunological low- or moderate-risk patients. This occurs within the first year after KT with a range of 3–24 months. The incidence increases with non-adherence to immunosuppressive medication. dnDSA could also be associated with both clinical and subclinical TCMR.Acute ABMR can occur at any time after KT due to the development of It is recommended to stain all biopsies for C4d and to send a serum sample for detection of HLA-specific Abs, in order to facilitate the diagnosis of ABMR in the setting of C4d negativity. This is consistent with joint British Society for Histocompatibility and Immunogenetics (BSHI)/BTS guidelines and those of the Transplantation Society. It is also in agreement with the local survey with a 56% positive response for DSA screening.We recommend considering the diagnosis of acute rejection, as well as other causes of decline of graft function, in KTRs who meet the criteria of acute kidney injury (NG).We strongly recommend graft biopsy to confirm the clinical diagnosis of acute rejection before initiating its treatment, unless the biopsy is contraindicated or will substantially delay treatment (1 C).We recommend that two cores of renal tissue should be obtained at KT biopsy to increase the sensitivity of the investigation (2 C).We recommend early testing for serum anti-HLA Abs in patients with acute rejection (2B).We suggest testing for non-HLA Abs in patients with biopsy evidence of microvascular inflammation without C4d deposits and negative serum DSAs (NG).Emphasis is made in most recent guidelines to confirm the diagnosis of acute rejection by biopsy (85.7% of survey respondents concur) and to simultaneously test for serum anti-HLA Abs (see guideline 9.9). Of the survey respondents, 21.4% required two or more of the following criteria to diagnose acute rejection: Clinical symptoms and signs (46.4%), rise of serum creatinine (75%), imaging (25%), and biomarkers (7.1%).We suggest standard clinical, laboratory and imaging evaluation to categorise the cause of chronic graft dysfunction function (NG).We suggest graft biopsy in KTRs with non-obstructive progressively declining graft function (NG).We recommend testing for anti-HLA Abs in KTRs with histological evidence of chronic active rejection (2 C).We suggest chronic kidney disease (CKD) staging in patients with chronic graft dysfunction and implementing standard management protocols accordingly (NG).de novo GN, infection, graft arterial stenosis or other kidney disease. Graft biopsy is mandatory to establish the diagnosis. Traditionally, little can be done to help those with chronic rejection. When chronic ABMR is recognised, histologically and/or serologically, as a common cause of chronic rejection, more targeted treatment protocols, including steroids anti-CD20 or even anti-CD19 and other biological agents have been applied with variable responses, albeit being so far generally unfavourable.Progressive non-obstructive decline of graft function may be attributed to chronic rejection, immunosuppressive drug toxicity, recurrent/de novo GN in KTRs who develop proteinuria or other urinary abnormalities and/or progressively deteriorating graft function. (NG)We suggest considering recurrence of the original disease or de novo GN (NG).We suggest performing non-invasive laboratory tests  for diagnosis and follow-up of recurrent disease or de novo GN is suspected. (NG) OR if sustained new onset proteinuria develops  (2 C).We suggest obtaining a graft biopsy when recurrence or de novo GN.Many kidney diseases may recur in the graft, particularly those of immunological nature. The chances and average timing of recurrence of different diseases are fairly well known, hence the feasibility of early detection even by routine follow-up and surveillance biopsy. The same applies to We suggest routine checking for the potential adverse reactions of the immunosuppressive drugs (NG).We suggest, whenever applicable, to measure the blood level of drugs known to interact with CNIs for initial dose adjustment and with changes in CNI doses or blood levels (NG).It is imperative to monitor KTRs for clinical side-effects, blood counts, liver and kidney functions for adverse drug reactions. Blood levels of CNIs must be monitored as explained in Box 12. The mTORi blood levels are also mandatory, and MMF levels preferable. Therapeutic blood level monitoring extends beyond the immunosuppressive drugs to many others that utilise the cytochrome P450/P-glycoprotein system for their absorption and metabolism. If measurement of blood levels is not feasible, careful clinical observation for adverse reactions is mandatory.Known previous chronic infectionWe suggest assessment of KTRs for re-activation of pre-existing chronic infection at 3 months post-KT and as frequently as necessary thereafter (NG).Cytomegalovirus (CMV)In those who had received induction therapy, we suggest screening all KTRs (except if recipient negative/donor negative [R–/D–]) for active CMV infection every 3 months (1A–C) by detecting serum CMV-specific IgM, four-fold rise in IgG, pp65 rapid antigen (NG).For 1 year if R+/D+For 2 years if R–/D+We suggest repeat screening for CMV infection 3 months after stopping universal prophylaxis  (NG).BK Polyomavirus (BKV)We recommend screening all KTRs for BKV viral load or by performing urine microscopy for decoy cells or by PCR on urine or serum (2 C) monthly for the first 9 months (2D) then every 3 months until 2 years post-KT (2B), whenever there is an unexplained rise in serum creatinine and after treatment for acute rejection (2D).We recommend that renal biopsies with SV40 staining should be done in patients with suspected polyomavirus nephropathy or in chronically deteriorating graft function (2 C).We recommend reduction of immunosuppressive medications in patients with BKV viral load of >1000 copies/mL sustained for 3 weeks or >10 000 copies/mL (2 C).Epstein–Barr virus (EBV)We recommend monitoring high-risk KTRs (R–/D+) for EBV by nucleic acid test (2 C) once in the first week, then monthly for the first 3–6 months and every 3 months until the end of the first post-KT year (2D).We recommend monitoring high-risk KTRs for EBV following treatment of acute rejection (2D).Hepatitis B virus (HBV)We suggest measuring the serum level of HB Ab titre in HB-negative or -positive KTRs and maintaining a level >10 IU/mL by vaccination, or administration of booster doses .The list of bacterial, viral, fungal and parasitic  diseasesWe strongly recommend screening all non-diabetic KTRs with fasting plasma glucose, oral glucose tolerance testing, and/or HbA1c (1 C) at least:Weekly for 4 weeks (2D);Every 3 months for 1 year (2D); andAnnually, thereafter (2D).We recommend screening for new-onset diabetes after KT with fasting glucose, oral glucose tolerance testing, and/or HbA1c after starting, or substantially increasing the dose, of CNIs, mTORi, or corticosteroids (2D).Pre-transplant diabetes is often ameliorated in CKD due to impaired clearance of insulin and many glucose lowering agents, despite the peripheral tissue insulin resistance. This perturbation is corrected as kidney function is restored by a successful KT. Unfortunately, corticosteroids, CNIs (particularly tacrolimus), and mTORis are diabetogenic, thereby leaching the benefit of restored kidney function, and prompting the development of new onset diabetes in non-diabetic KTRs.We strongly recommend measuring blood pressure at each clinic visit (1 C).We suggest assessment for cardiovascular disease in all KTRs before, 3 months after KT and then annually (NG).We suggest assessment for obesity in all KTRs before, 3 months after, and 6-monthly thereafter including (NG):Measure height and weight and calculating BMI at each visit, at each visit.Measurement of a complete lipid profile in all adult and adolescent KTRs.Measurement of a complete lipid profile 2–3 months after a change in treatment or other conditions known to cause dyslipidaemiasCardiovascular events occur in 3.5–5% of KTRs every year, reflecting a significantly high risk compared to the general population. This is attributed to a large number of ‘traditional’ and ‘non-traditional’ risk factors that tend to persist after KT. The latter adds a further drug-related risk, attributed to corticosteroids and CNIs. Therefore, it is mandatory to include event and risk factor monitoring of KTRs.In patients in the immediate post-KT period, we strongly recommend measuring serum calcium and phosphate at least weekly, until stable (1B) then every 3–6 months for as long as necessary depending on the stage of post-KT CKD stage, patient’s age, and the extent of metabolic bone disease (MBD).We suggest measuring serum vitamin D (2 C) and parathormone levels (NG), every 3–6 months for as long as necessary depending on the stage of post-KT CKD stage, patient’s age, and the extent of MBD.We suggest measuring bone mineral density by dual-energy X-ray absorptiometry (DXA) or CT scanning at 3 months post-KT to estimate fracture risk (NG).We suggest post-KT measurement of bone mineral density every 6 months for high-fracture risk KTRs and annually for low-fracture risk patients (NG).We recommend avoiding, or using the lowest therapeutic doses, of immunosuppressive drugs that have a negative impact on bone health (NG).CKD-associated bone disease continues after KT and often worsens despite normal graft function, owing to the use of corticosteroids. CNIs may increase serum parathormone levels. Sirolimus is incriminated in deterioration of post-KT osteodystrophy. On the other hand, combination of everolimus and MMF in a steroid-free, CNI-free protocol seems to be bone-protective ,6566].de novo, which is mainly attributed to the use of corticosteroids. The role of other immunosuppressive drugs in this complication is uncertain.Post-KT osteodystrophy is often associated with mineral abnormalities, hypovitaminosis D, and persistent hyperparathyroidism, hence the mentioned monitoring recommendations. In addition, osteonecrosis may develop We recommend pre-KT and at least one annual post-KT tumour screening by clinical examination , fecal blood and tumour markers as per local guidelines.We recommend chest imaging prior to KT in all KTRs (2 C).We recommend chest CT for current or former tobacco users with a > 30 pack-year history, as per local guidelines, and chest radiograph for other KTRs (2 C).We strongly recommend screening for RCC with US for KTRs at increased risk, such as a long time on dialysis, family history of renal cancer, acquired cystic disease, and analgesic nephropathy (1D).We strongly recommend screening for bladder carcinoma using urine cytology or cystoscopy for KTRs at increased risk, such as schistosomiasis, previous cyclophosphamide use or history of heavy smoking (>30 pack-year) (1D).We strongly recommend screening for hepatocellular carcinoma in KTRs with cirrhosis prior to KT using techniques  (1 C).We strongly recommend screening for bowel cancer in KTRs with inflammatory bowel disease as per local guidelines (1 C).We recommend screening for cervical cancer (PAP smear), for female recipients annually after KT (1 C).We recommend breast cancer screening in all female recipients aged 50–69 years every 1–2 years (1 C).The incidence of post-KT myeloproliferative disease is multiplied many folds post-KT. This is attributed to immunosuppression , the use of T-cell depleting agents and to certain viral infections .The occurrence of other cancers is also significantly increased in KTRs, with increasing incidence with advancing age >65 years. The most notable are non-melanoma skin cancers and bronchial, urinary, hepatic and bowel malignancy.Obviously, early detection of these cancers is mandatory for adequate specific treatment and modification of immunosuppressive therapy. The latter often includes switching to mTORis [66]. Unfortunately, only 20% of our survey’s respondent included routine monitoring for tumour markers.We suggest that every transplant centre establishes a multispecialty annual review clinic for the appraisal of its KTRs at their KT anniversariesThe high success rate of KT has alleviated a lot of concerns in children and the elderly. Many female KTRs can complete successful pregnancies and deliver normal babies while on immunosuppressive therapy. However, there are issues that must be taken into consideration regarding vaccination, risk assessment, induction, choice of immunosuppressive drugs and their doses, and subsequent monitoring.We have alluded to some recommendations concerning these populations in the various sections of this Guideline. However, it is strongly recommended to collaborate with respective specialists and refer to specific guidelines for the adequate management of individual cases.


Wx) gene, which also affects the diversity of amylose content. The present study assessed the grain quality profiles, starch fine structure, and crystallinity characteristics of the near-isogenic lines Q11(lvWx), NIL(aWx), and NIL(bWx) in the indica rice Q11 background containing different Wx alleles. Q11(lvWx) rice contained a relatively higher amylose level but very soft gel consistency and low starch viscosity, compared with rice lines carrying aWx and bWx. In addition, starch fine structure analysis revealed a remarkable decrease in the relative area ratio of the short amylopectin fraction but an increased amylose fraction in Q11(lvWx) rice. Chain length distribution analysis showed that Q11(lvWx) rice contained less amylopectin short chains but more intermediate chains, which decreased the crystallinity and lamellar peak intensity, compared with those of NIL(aWx) and NIL(bWx) rice. Additionally, the starches in developing grains showed different accumulation profiles among the three rice lines. Moreover, significant differences in starch gelatinization and retrogradation characteristics were observed between near-isogenic lines, which were caused by variation in starch fine structure. These findings revealed the effects of lvWx on rice grain quality and the fine structure of starch in indica rice.Amylose largely determines rice grain quality profiles. The process of rice amylose biosynthesis is mainly driven by the waxy ( The main edible part of rice is the endosperm, which is mainly composed of starch, accounting for more than 80% of milled rice. Starch in the rice endosperm is generally divided into two types, amylose and amylopectin. Rice quality performance, such as eating and cooking quality, and nutrition quality, is mainly determined by the rice grain composition and the fine structure of starch ,2. GenerWx gene encodes granule-binding starch synthase  in Wx are associated with variations in the amylose content (AC) in rice cultivars. To date, a series of Wx alleles have been identified, including lvWx, aWx, inWx, bWx, mw/laWx, mpWx, mqWx, op/hpWx, and Wx [aWx and lvWx alleles; however, the lvWx allele was identified as the ancestral type of the Wx gene, contributing to a higher AC than that of the aWx allele. Moreover, this allele also contributes to a very low starch viscosity and a soft gel consistency because of ungelatinized starch granules [Wx alleles, inWx results in an intermediate AC (about 20%); bWx contributes to a low AC (about 15%); mw/laWx also contributes to a low AC (about 14%); mpWx, mqWx, and op/hpWx contribute to a very low AC (8–12%) [waxy rice, generally, the 23-bp deletion in the Wx locus  is responsible for glutinous rice [lvWx is distributed mainly in wild rice and aus subspecies, whereas aWx is distributed mainly in indica rice subspecies, and bWx is distributed mainly in japonica rice subspecies [The synthesis of rice starch involves a series of enzymes that are involved directly in starch synthesis and transcription factors that regulate the process indirectly. Among them, the , and Wx ,10,11,12granules . As rega (8–12%) ,11,12,13AC about %; Wxmp, ous rice . Among tbspecies . Wx gene and studying their expression profiles (enzyme activities) and relationships with AC using natural rice varieties, near-isogenic lines (NILs), and chromosome segment substitution lines [Wx allelic variation contributes to AC variation, but also causes changes in the amylopectin fine structure [Wx alleles  on the fine structure and physicochemical properties of rice starch. There has been great progress made in identifying natural allelic variations in the on lines ,15,16,17tructure ,19. HoweWx locus in both japonica and indica rice backgrounds, and we investigated the effects of lvWx on rice grain cooking and eating quality profiles in the japonica rice background; however, limited information is available in the indica rice background, because we only constructed one NIL(aWx) in the background of indica rice Q11(lvWx) [Wx allelic variation on rice grain quality profiles, we further constructed a NIL of the bWx allele, NIL(bWx), in the same indica background. Thus, this study aimed to compare the allelic variation of these Wx alleles, especially lvWx, taking into account rice grain physicochemical properties in the indica rice background. In addition, we investigated the starch fine structure profiles during the development of the endosperm. The results of the present study will deepen our knowledge concerning the influence of Wx allelic variation on the physicochemical properties and starch structure of indica rice grains.In our previous work, we constructed several NILs for the 11(Wxlv) . To obtaaWx) on a background of indica cultivar Q11  was generated by introgressing the aWx allele from indica cultivar Guichao 2 after six backcrosses [Wxb allele, harbored by indica cultivar 9311, was introgressed after six backcrosses using marker-assisted selection to generate NIL(Wxb). The three rice lines Q11(lvWx), NIL(aWx), and NIL(Wxb) were cultivated at an experimental farm of the Yangzhou University . The three lines were each grown randomly in triplicate, during summer, and under the same management and climatic conditions.The NIL was determined using a halogen moisture analyzer . Briefly, 1 g of sample was weighed in the weighing pan, incubated at 120 °C for 10 min, and then the water loss rate was calculated via auto calculation by the instrument. A Kjeltec 2300 nitrogen determination instrument (Foss Tecator) was used to determine the rice flour crude protein content (PC) from different NILs. The apparent amylose content (AAC) was measured colorimetrically as described by Zhang et al. .A Rapid Visco Analyzer (RVA)  was used to assess the pasting properties of rice starch and flour, as described previously . The RVAH), conclusion temperature (Tc), peak temperature (Tp), and onset temperature (To). Triplicate measurements were performed for all analyses. Differential scanning calorimetry  was used to determine the thermal properties as described previously . Briefly3, and heated in a metal bath at 85 °C for 12 h. Thereafter, the sample was left to cool to room temperature, centrifuged at 4000× g for 10 min, and then 1.5 mL of the supernatant was transferred to a sample vial. For each sample, 100 μL of the solution was injected into the GPC system by the autosampler. The mobile phase used for the GPC system was also DMSO, and the flow rate of the measurement was 0.8 mL/min. The GPC data were subjected to detection, recognition, and identification (DRI) analyses, and the molecular size distributions were reported as the dextran-equivalent molecular weight (Mw). The molecular weight distribution curves were analyzed, and the following GPC parameters were extracted: amylopectin (AP), amylopectin short chains (AP1), amylopectin long chains (AP2), and amylose chains (AM). Each of the fractions was calculated using the total integrated area of each GPC peak. Besides, quantitative analysis of debranched starch was also carried out following the method of Zhu et al. [Isoamylase  was used to debranch rice starch, as described previously . Gel peru et al.  in a higqo) was used to calculate the SAXS parameter, Bragg spacing (D), which represents the lamellar distance, using D = 2π/qo. X-ray diffraction (XRD)  was used to analyze the starch supra-molecular structure according to a previously described method . From theviously . The posp < 0.05.Three technical replicates were performed for all measurements in this study, and all data are shown as means ± the standard deviation (SD). SPSS 16.0 statistical software  was used to carry out a one-way analysis of variance (ANOVA) on the data. Statistical significance was accepted at Wx alleles on rice grain phenotype, we assessed the appearance of brown and milled grains from Q11(lvWx), NIL(aWx), and NIL(bWx). All three rice lines showed a very similar grain size and appearance (lv)Wx showed the highest AAC among the three samples, which was consistent with our previous results that rice carrying lvWx tends to have a higher AAC than those carrying other Wx alleles [lvWx) had the highest AAC level and exhibited a very soft GC, compared with the other lines. In fact, previous studies proposed that rice varieties with this characteristic of high AAC but soft GC were good germplasm resources for rice grain quality improvement [lvWx cannot be gelatinized completely during heating, and thus the gelatinized paste contains many partially swollen granules, resulting in high flowability (higher GC) [To investigate the effect of pearance . The phypearance . However alleles . It is g alleles ,23. Howerovement ,25. Howegher GC) . Moreovegher GC) .aWx) and NIL(bWx) rice, Q11(lvWx) rice starch showed a significantly lower AP1 peak, whereas the AM peak was the highest among the three samples. We further compared the ratio of each fraction, in which starch from Q11(lvWx) rice exhibited the lowest AP1 fraction among the three samples (lvWx) starch was higher than the AP2 fraction of NIL(aWx) starch. In terms of the branching degree, Q11(lvWx) rice starch showed the lowest degree of branching, which was consistent with our previous results that rice with higher AC tended to have a low branching degree [lvWx) rice showed a relatively higher AM fraction  comprised significantly lower levels of A chains (DP 6–12) but higher levels of B chains (DP ≥ 12), compared with those in NIL(aWx) (lvWx) starch exhibited lower numbers of A chains, and higher numbers of B chains, to that of NIL(bWx) rice; however, the degree of change was relatively high. Similarly, previous studies in maize and rice found that the Wx gene led to changes in both amylose and amylopectin fine structures [Wx functions to determine the amylopectin molecular structure, at least in part.The chain length distribution (CLD) of the branched starch from the three rice lines was further analyzed using HPAEC. Generally, amylopectin chains can be classified as A chains (DP 6–12), B1 chains (DP 13–24), B2 chains (DP 25–36), and B3+ chains (DP ≥ 37) according to the classical amylopectin cluster model . The ricNIL(Wxa) B. Similaructures ,19,27. TbWx) rice had a significantly higher AP1 fraction throughout endosperm development. In addition, Q11(lvWx) and NIL(aWx) starch showed similar AP1 levels at the early stage of endosperm development (5 and 10 DAF); however, from 15 DAF, Q11(lvWx) starch exhibited the lowest AP1 fraction, compared with the other samples. In terms of the AP2 fraction, in the three rice lines, little change was observed during endosperm development (bWx) rice also showed a relatively higher AP2 fraction throughout endosperm development, whereas there were no significant changes in the AP2 fraction between Q11(lvWx) and NIL(aWx) before 15 DAF. From 20 DAF, the AP2 fraction in Q11(lvWx) starch increased to a greater extent than that of NIL(aWx) starch. Comparison of the AM fractions showed that Q11(lvWx) starch contained relatively high amounts of AM throughout the whole filling stage  starch was the highest among the three rice lines during the whole filling stage, which could be attributed to its higher GBSSI enzyme activity, as reported previously [Wx gene during the filling stage [Starch accumulation is crucial for the development of the rice endosperm. It is well known that the accumulation of starch increases rapidly with the grain filling process, and this process is sensitive to the environmental temperature ,29. Howeeviously . Moreoveng stage . lvWx) rice had the lowest RC, while NIL(bWx) starch showed the highest RC among the three samples (lvWx) had a higher amylose content and a lower number of longer chains, resulting in lower relative crystallinity.Starch’s semi-crystalline layer comprises crystalline and amorphous regions arranged as lamellae, with a repeating distance of 90–100 Å. Moreover, amylopectin branched chains form double helices, thus determining the crystalline region, in which the amylopectin branched chains are arranged horizontally into a lattice . It is b samples . It is blvWx) starches had significantly lower Imax values, while the D value was higher in Q11(lvWx) among the three samples. It is believed that short chains of amylopectin form ordered semi-crystalline structures, the number of which determines the Imax value [lvWx) starch, because the lamellar distance has been proven to correlate positively with the amylose content.SAXS was used to assess the starches’ supramolecular structure, with the aim of determining whether starch structural changes affect the ordered structure of starch. Similar single peaks were observed in all three samples ; howeverax value . Our preax value . Thus, vlvWx was cloned initially based on the very low starch viscosity curve measured by RVA in the japonica rice background [indica rice background, rice flour and starch from the three lines were used for RVA analysis. As shown in lvWx) exhibited a markedly lower RVA curve than those of NIL(aWx) and NIL(bWx), which was consistent with the results in the japonica background [lvWx) rice flour were significantly lower than those of the NIL(aWx)  and NIL(bWx)  flours. Moreover, the Q11(lvWx) rice flour showed lower SBV (−3.50) and PT (6.33) values than the NIL(bWx) flour  but higher values than NIL(aWx) flour . To determine if the RVA curve was affected by the starch structure, the RVAs of starch samples were determined. Similarly, lower RVA curves and parameters including PKV, HPV, BDV, and CPV were observed for Q11(lvWx) starch, compared with those of the other samples (aWx) and NIL(bWx) were different between rice flour and starch, which indicated that factors within rice flour affect the RVA parameters [lvWx) rice showed a normal high AC level. It has been reported that a higher PKV can result from a lower AC [lvWx) rice is the main reason for the low PKV.The ckground . To furtckground . Moreove samples . In addirameters . Previourameters . Howeverlower AC ,37. GeneWx allelic variation. Q11(lvWx) and NIL(aWx) starches showed a marked shift in the endothermic peak (lvWx) and NIL(aWx) starch showed similar DSC curves, the DSC parameters differed between these two samples. Q11(lvWx) starch exhibited relatively higher To, Tp, and Tc but lower ΔH values, compared with those from NIL(aWx) and NIL(bWx). Studies have found that the gelatinization of starch is an endothermic process, which starts from the glass transition stage in the amorphous region, leaches from the amylose molecules, and then proceeds via dissociation of the amylopectin crystallites [To, Tp, and Tc) correlated positively with AC, whereas ΔH correlated negatively with increasing AC [lvWx) rice, led to higher To, Tp, and Tc values, but lower ΔH values among the three samples.DSC was employed to determine the NIL-derived starch gelatinization characteristics associated with mic peak A, which tallites . In additallites ,39. Moreasing AC ,36,40. TlvWx) rice exhibited significantly higher gelatinization properties, including ΔH, Tc, Tp, and To, than those from NIL(aWx) and NIL(bWx) (aWx) starch also showed relatively higher ΔH, Tc, Tp, and To values than those of NIL(bWx) starch. It is believed that amylose usually forms a double helix during the retrogradation process, and the outermost short branches combine to enable amylopectin crystallization [lvWx) and NIL(aWx) starches have the lowest amounts of short-branch amylopectin, they showed a relative higher ΔH, implying that during starch retrogradation, amylose makes a higher contribution to the formation of double-helical structures. Similar results have been found in other rice cultivars having the same genetic background [The retrogradation profiles of the gelatinized starch samples were subsequently measured using DSC. Compared with that of natural starch, all three starch samples displayed a lower retrogradation peak B. HoweveNIL(Wxb) . Moreovelization . Althougckground .Wx allelic variation on rice grain physicochemical properties, starch structural variations, and their relationships with the physicochemical properties. Rice carrying the lvWx allele had a higher AC but a softer GC than those carrying the aWx and bWx alleles. Moreover, starch fine structure analysis, determined using GPC, showed that the starch from Q11(lvWx) rice contained significantly lower AP1 and AP2 fractions but a higher AM fraction among the three rice lines. Additionally, evidence from developing seeds suggested that the accumulation of both the AP1 and AM fractions is different among the three rice lines. Moreover, HPAEC analysis showed that Q11(lvWx) rice contains fewer short-chain amylopectins and more intermediate chain amylopectins, resulting in decreased crystallinity and lamellar peak intensity than those of NIL(aWx) and NIL(bWx) rice. An assessment of the rice starch retrogradation properties, gelatinization properties, and pasting properties also revealed that the starch fine structure, as well as the starch’s ordered structure, affect these physicochemical properties. Overall, this work increased our understanding of the effects of variations of the Wx allele, especially lvWx, on rice grain quality profiles between rice NILs with the same genetic background. In addition, the present study provides a resource for breeding high-amylose but low-starch-viscosity rice.The present study clarified the role of


Three sweet potato varieties with purple-, yellow-, and white-fleshed root tubers were planted in four growing locations. Starches were isolated from their root tubers, their physicochemical properties  were determined to investigate the effects of variety and growing location on starch properties in sweet potato. The results showed that granule size  ranged from 12.1 to 18.2 μm, the iodine absorption parameters varied from 0.260 to 0.361 for OD620, from 0.243 to 0.326 for OD680 and from 1.128 to 1.252 for OD620/550, and amylose content varied from 16.4% to 21.2% among starches from three varieties and four growing locations. Starches exhibited C-type X-ray diffraction patterns, and had ordered degrees from 0.634 to 0.726 and lamellar thicknesses from 9.72 to 10.21 nm. Starches had significantly different swelling powers, water solubilities, pasting viscosities, and thermal properties. Native starches had rapidly digestible starch (RDS) from 2.2% to 10.9% and resistant starch (RS) from 58.2% to 89.1%, and gelatinized starches had RDS from 70.5% to 81.4% and RS from 10.8% to 23.3%. Two-way ANOVA analysis showed that starch physicochemical properties were affected significantly by variety, growing location, and their interaction in sweet potato. Ipomoea batatas), an essential root tuber starchy crop, is widely cultivated in over 100 countries. There are over 90 million tonnes in annual production , Rugao (RG) , Yangzhou (YZ) , and Zunyi (ZY) . All varieties in every growing location were planted repeatedly in three experimental plots. The soil in each growing location was given the same nitrogen treatment of 15 kg/ha applied in the ridge before planting. The fertilizers included urea, superphosphate, and potassium sulfate. Temperature variation of growing location during sweet potato growth stage is presented in Starch was isolated from fresh root tuber of sweet potato using water homogenization, sieving, and treatment of 0.2% NaOH and anhydrous ethanol following the processes of Guo et al. [Starch and water slurry was analyzed with a laser particle size analyzer  following the processes of Guo et al. [Starch was dissolved in dimethyl sulfoxide containing 10% 6.0 M urea (UDMSO) and stained with iodine solution following the processes of Man et al.  exactly.The amylose content (AC) was measured using concanavalin A (Con A) precipitation method with an amylose/amylopectin assay kit  following its assay procedures. For measuring principle, starch is first dispersed completely into amylose and amylopectin, and separated into two aliquots. The Con A specifically precipitates the amylopectin in an aliquot, and the remaining amylose in the supernatant is quantified. The dispersed amylose and amylopectin in another aliquot are also quantified. The AC is the ratio of amylose to both amylose and amylopectin.Starch was analyzed using an XRD spectrometer  following the method of Guo et al.  exactly.−1 resolution from 4000 to 800 cm−1. The spectrum was deconvoluted using Lorentzian line shape with 19 cm–1 half-width and 1.9 resolution enhancement factor after baseline correction from 1200 to 900 cm−1.Starch was analyzed using a FTIR spectrometer  following the method of Guo et al.  exactly.Starch was analyzed using a SAXS system  following the method of Cai et al.  except tStarch was analyzed using a RVA  following the method of Guo et al.  exactly.2SO4 method were used to evaluate the water solubility , and the swelling power was the ratio of precipitated starch to total starch with subtraction of soluble carbohydrates.The swelling power and water solubility of starch at 95 °C were measured following the method of Guo et al.  exactly.Starch was analyzed using a DSC  following the method of Guo et al. [Aspergillus niger amyloglucosidase  following the method of Guo et al. [The digestibilities of native and gelatinized starches were evaluated using both porcine pancreatic α-amylase  and o et al.  exactly.The data difference among different starches with Tukey’s test and the effects of variety, growing location, and their interaction on starch properties with two-way ANOVA test were analyzed with SPSS 19.0.Three sweet potato varieties grown in four locations were used to investigate the effects of variety and growing location on starch physicochemical properties in sweet potato. The granule size, iodine absorption parameters and AC of starch were influenced significantly by variety, growing location, and their interaction. C-type starches in root tubers had different proportions of A- and B-type crystallinities in different varieties and growing locations. The ordered degree and lamellar thickness of starch were affected by variety and growing location of sweet potato. The pasting properties, swelling power, water solubility, thermal properties, and digestibility exhibited significant differences among different varieties and growing location. Therefore, in consideration of the applications of starch, it is very important to choose an appropriate variety for planting sweet potato in a specific growing location.


A new etherified starch, δ-hydroxybutyl starch (δ-HBS), was prepared by utilising 4-chlorobutan-1-ol as the etherifying reagent. The method of Zeisel gas chromatography for the determination of the molar substitution was described. This technique offers a simple and rapid method for quantitative analysis with reproducible results. Meanwhile, the mechanism of the Zeisel reaction was also investigated. The increasing industrial importance of hydroxyalkyl starch has created interest in methods for its analysis. Depending on the reaction conditions, the complex substitution pattern differs with respect to DS and MS, and regioselectivity. Among them, the molar degree of substitution (MS) values, i.e., the average number of hydroxyalkyl groups substituted per anhydroglucose unit, directly influence the physicochemical properties ,2,3,4,5,Several analytical methods have been developed over the years for the determination of the molar substitution ratio of hydroxyalkyl starch. Zeisel gas chromatography has been employed as an analytical technique for the determination of the hydroxyethyl group in hydroxyethyl starch with the quantitative conversion of the substituted alkoxyl unit to the corresponding iodide by reaction with hydriodic acid ,7,8,9, tIn recent years, synthesis and characterisation of 2-hydroxybutyl starch have been studied, but there is no similar report on δ-hydroxybutyl starch (δ-HBS), i.e., the hydroxyl group in the terminal of butyl . MeanwhiCorn starch, δ-hydroxybutyl starch (made in a lab by ourselves), 1-iodobutane, 2-iodobutane, toluene, o-xylene, hydriodic acid (55%), tetrahydrofuran, 1, 4-butanediol  were obtained from Aladdin Industrial Corporation, Shanghai, China.The gas chromatography  was equipped with an FID detector, and the capillary chromatographic column was SPB-5 (30 m × 0.32 mm × 0.25 μm). The GC operating condition was as follows: The injector was held at 200 °C, and the initial oven temperature of 60 °C was held for 4 min, which was then increased to 230 °C at 30 °C/min. This temperature was also held for 4 min. The injection volume was 2 µL. The detector temperature was 250 °C.Iodobutane, toluene, o-xylene were used for GC blank analysis, respectively. Toluene was used as the internal standard. Toluene of 1.0 mL was dissolved in o-xylene and made into a 200 mL solution in a volumetric flask. A set of standards was prepared by injecting 1, 2, 3, 6, 10, 18, 28, and 40 μL of iodobutane into test tubes stoppered with Suba Seals containing 2.0 mL of internal standard solution. The tubes were mixed and shaken vigorously and then allowed to stand for 10 min. The exact weight of iodobutane was recorded. Each solution was used for GC analysis.Approximately 50 mg of δ-hydroxybutyl starch or unmodified starch, accurately weighed, was transferred to a 25 mL reactive vial containing 200–250 mg of adipic acid. Two millilitres of internal standard solution and 5 mL of hydriodic acid were added to the vial, which was immediately tightly capped with a Mininert valve and weighed. Vials were placed in a preheated block at 140 °C for 10 h. After being heated, the vials were removed from the block, shaken, cooled to room temperature, and reweighed to determine if any loss due to leakage had occurred. Then, 0.1 mL of the upper organic layer was transferred to a sampler vial containing 1.0 mL of o-xylene. The sampler vial was capped tightly and mixed for 5 s. The obtained sample would be used for GC analysis.Calculation of the MS of hydroxybutyl starch is historically based on the butyl oxide unit, C4H8O. The MS ratio, which is defined as the mole fraction of epoxybutane per anhydroglucose unit, is calculated by the following equations:P in formula 1 equals the weight of iodobutane and m equals the weight of sample; the values 184.02 and 72.11 in equations equal the molecular weight of iodobutane and C4H8O, and 162.14 equals the molecular weight of anhydroglucose.The Based on the percent alkoxyl substitution anticipated in the prepared samples, the quantity of respective 2-iodobutane needed for calibration was determined according to the graph in n = 6), which indicates that the method is sufficiently precise. The repeatability, linear range, and detection limits were studied for the GC method. The RSD for six replicate runs was 1.11% -O) was shifted and split, resulting in a multiple-peak separation between 5.1 and 5.5 ppm. The clear broadening of anomeric proton’s peak for δ-HBS is due to the substitution at the O-2 position [2-CH2-C group in -OCH2CH2CH2CH2OH. The spectrograms indicated successful etherification.A typical  4.1 ppm A correspposition ,26. The A new class of hydroxybutyl starch was synthesised by utilising 4-chlorobutan-1-ol as the etherifying reagent. Zeisel gas chromatography was adopted for the analysis of the MS of δ-HBS. For consideration of the potentials of a relatively new, cheap, underutilised but abundant starch resource, further investigations are currently underway in our laboratory.


Phyllostachys heterocycla) shoots/culms/branches are connected together into a close inter-connecting system in which nutrients are transported and shared among each organ. However, the starch storage and utilization mechanisms during bamboo shoot growth remain unclear. This study aimed to reveal in which organs starch was stored, how carbohydrates were transformed among each organ, and how the expression of key genes was regulated during bamboo shoot growth and developmental stages which should lay a foundation for developing new theoretical techniques for bamboo cultivation.Both underground rhizomes/buds and above-ground Moso bamboo  and starch hydrolase (α-amylase and β-amylase) activities exhibited the same dynamic change patterns as those of the starch content. From S0 to S3, the activity of starch synthesis-related enzyme and starch amylase in bamboo rhizome was significantly higher than that in bamboo shoot, while the NSC content in rhizomes was obviously lower than that in bamboo shoots. It was revealed by the comparative transcriptome analysis that the expression of starch synthesis-related enzyme-encoding genes were increased at S0, but reduced thereafter, with almost the same dynamic change tendency as the starch content and metabolism-related enzymes, especially during S0 and S1. It was revealed by the gene interaction analysis that AGPase and SBE were core genes for the starch and sucrose metabolism pathway.Bamboo shoots were the main organ in which starch was stored, while bamboo rhizome should be mainly functioned as a carbohydrate transportation channel and the second carbohydrate sink. Starch metabolism-related genes were expressed at the transcriptional level during underground growth, but at the post-transcriptional level during above-ground growth. It may be possible to enhance edible bamboo shoot quality for an alternative starch source through genetic engineering.The online version contains supplementary material available at 10.1186/s12870-021-03257-2. AGPS1 , AGPL2 (Adenosine diphosphate glucose pyrophosphorylase lager subunit 2), SSII (Soluble starch synthase II), SSIII (Soluble starch synthase III), and ISA (Isoamylase) genes participate in starch metabolism in rice, wheat and potato  to 1 g frozen sample, which was then homogenized with a pestle and mortar on ice. The homogenate was centrifuged at 10,000×g for 10 min at 4 °C. Next, the supernatant was kept at 2 °C, and then used as the crude enzyme solution (AGPase and SBE) to be measured. The above enzyme extract was added to the precipitate and used as the GBSS crude enzyme solution to be kept at 2 °C and measured. The activities of AGPase and GBSS of crude enzyme solution were measured according to the methods of Nakamura et al. [The activity of starch synthase was determined by spectrophotometry. Crude extracts of starch synthase were prepared according to the methods of Nakamura et al. . Adding a et al.  and Dai a et al. . Activita et al. .0.1 g sample was homogenized with 1 mL distilled water. The homogenate was extracted for 15 min at room temperature. The homogenate was shaken once every 5 min to make it fully extracted. Centrifuge for 10 min at 25 °C for 3000 g. adding distilled water to the supernatant, then it is crude amylase extract solution. α-amylase and β-amylase reagent kits  were used to determine the activities of amylase. The specific operation about α-amylase determined is as follows: 75 μL crude extract was taken, bathed in water at 70 °C for 15 min, then 75 μL sodium citrate solution (including soluble starch) was added after cooling. Then 150 μL 3, 5-dinitrosalicylic acid, NaOH, potassium sodium tartrate solution was added in water bath at 95 °C for 5 min. After cooling, the absorbance was measured at 540 nm. Distilled water was used as the control. β-amylase determined as follow: Amylase diluent was obtained by 1 mL of the crude amylase solution, adding 4 mL of distilled water and shaking it well for the determination of the total activity of (α + β) amylase. Seventy-five microliter amylase diluent, add 75 μL sodium citrate solution (including soluble starch), keep the temperature at 40 °C for 5 min, add 150 μL 3, 5-dinitrosalicylic acid, NaOH, potassium sodium tartrate solution, water bath at 95 °C for 5 min, cool down and measure the absorbance at 540 nm. The soluble protein determination method complied with the Bradford Coomassie brilliant blue method .www.majorbio.com).Two gram samples of the top and basal tissues of bamboo shoots were taken, and total RNA was extracted by the TRIzol  method. The concentration and purity of the RNA were then detected by a Nanodrop 2000 instrument, the integrity of the RNA was detected via AGE (agarose gel electrophoresis), and the RIN value was determined by an Agilent 2100 instrument . An RNA library was constructed by a TruSeq™ RNA Sample Preparation Kit . The library was based on the NovaSeq 6000 sequencing platform  for high-throughput sequencing. Sequencing was conducted by Shanghai Majorbio Bio-pharm Technology Co., Ltd., and the sequencing data were analysed on the free online platform of the Majorbio Cloud Platform  . The specific primer pairs used for qRT-PCR were designed by Shanghai Majorbio Bio-pharm Technology Co., Ltd.  to compared the NSC content and starch metabolism-related enzyme activities different growth and developmental stages of Moso bamboo shoot at top, middle, bottom and rhizomes . The results were expressed as the means ± standard deviations. Figures were constructed with Origin 2018 software, which ordinate is the mean of the three biological measurements, and the error bar is the standard deviation. Pearson’s correlation analysis (p < 0.05) was conducted among starch metabolic enzyme activity, amylose and amylopectin content of Moso bamboo shoot.One-way analysis of variance (ANOVA)  or Kruskal-Wallis test  was used by SPSS 21.0 software and Duncan post hoc test  and base (right) of Moso bamboo shoots.


The Donor Rejected Organ Pre-transplantation (DROP) Score was proposed: − 2.68 +  + (0.03*age) + (0.04*weight)-(0.03*height) + (0.29 if diabetes) + (1.65 if anti-HCV-positive) + (0.27 if HBV core) − (0.69 if hypotension) + (0.09*creatinine) + (0.38*log10AST) + (0.34*log10ALT) + . At validation, the DROP Score showed the best AUCs for the prediction of liver-related graft discard  and macrovesicular steatosis ≥ 30% . Patients exceeding the DROP 90th centile had the worse post-LT results .The DROP score represents a valuable tool to predict the risk of liver function-related graft discard, steatosis, and early post-LT graft survival rates. Studies focused on the validation of this score in other geographical settings are required.Several studies have explored the risk of graft dysfunction after liver transplantation (LT) in recent years. Conversely, risk factors for graft discard before or at procurement have poorly been investigated. The study aimed at identifying a score to predict the risk of liver-related graft discard before transplantation. Secondary aims were to test the score for prediction of biopsy-related negative features and post-LT early graft loss. A total of 4207 donors evaluated during the period January 2004–Decemeber 2018 were retrospectively analyzed. The group was split into a training set (The online version contains supplementary material available at 10.1007/s13304-022-01262-0. Liver transplantation (LT) is the best therapeutic strategy for managing more than 50 pathologies causing end-stage liver disease . One of Therefore, the current focus is on identifying predictive criteria to guide the safe use of liver grafts , since iIn recent years, many studies have focused on the risk of early graft dysfunction after transplantation –9, whileThis study aimed at identifying and validating a score to predict the risk of liver-related graft discard from donors after brain death (DBD). The secondary aim was to test the score for prediction of biopsy-related features and graft loss at 3 months after transplantation.n = 2,694), and the three University Centers of Rome . Only DBDs offered for a primary transplant were included. DBD with missing clinical information (n = 17) and livers used for secondary (n = 118) or ABO-incompatible transplants (n = 30) were excluded from analysis so that the final sample numbered 4,207 cases.We performed a retrospective analysis of 4,372 DBDs evaluated for liver graft donation from January 1st 2004 to December. Four Italian centers joined the project: University of Pisa, Italy (This group was split into a Training Set of 3156 candidates (75.0%) and a Validation Set of 1051 candidates (25.0%) using a causal number generator randomization. A flowchart reporting the selection process is reported in Supplementary Fig. 1.In Italy, liver donors are allocated on a regional basis except for urgencies , pediatric recipients, and patients with a model for end-stage liver disease score ≥ 29. If a local center declines a graft before or during procurement surgery, it is offered at the national level via the Italian National Center for Transplantation Office. Decline criteria are varied across centers, and liver graft biopsy is left at the discretion of the surgical procurement team. With the intent to avoid center-related biases, only donors that were declined both locally and nationally were considered in the present study.We categorized the causes of graft discarding in two groups, namely liver-related versus liver-unrelated. Liver-related reasons for discard included any of the following: pre-procurement liver blood tests and/or imaging; gross anatomy; procurement histology, and poor perfusion. Liver-unrelated reasons for graft discard were donor tumors, donor infections, and pre-procurement donor cardiac arrest.A liver graft biopsy was performed on demand, depending on surgical evaluation at procurement. The time of biopsy was before organ procurement. Biopsies review was not centralized, but performed by the different Pathology services on a rota basis.Donor hypotension was defined as any episode of mean arterial pressure < 60 mm Hg for more than 1 h during the intensive care unit (ICU) stay.The vasoactive-inotropic score was calculated according to the formula:dopamine (mcg/Kg/min) + dobutamine (mcg/Kg/min) + vasopressin  + noradrenaline (mcg/Kg/min × 100) + adrenaline (mcg/Kg/min × 100).U test and Fisher's exact test were used to compare continuous and categorical variables, respectively.Continuous variables were reported as medians and inter-quartile ranges (IQR). Dummy variables were reported as numbers and percentages. We used the maximum likelihood estimation method for managing missing data . For modp value < 0.20 were used for the multivariable model. Odds ratios (OR) and 95% confidence intervals (95%CI) were reported.A competing-risk analysis using a cause-specific logistic regression model was constructed to identify the risk factors for liver-related graft discard. The competing event  was censored in the model. The analysis was performed on the Training Set data. Thirty-one variables were initially tested in a univariable model. All the covariates with a The model's accuracy was assessed through c-statistic analysis, with the intent to evaluate its ability to predict a liver-related discarded graft. In the Training Set, validation was eventually performed using a bootstrap approach based on 100 generated samples deriving from the original set.Areas under the curve (AUCs) and 95%CIs were reported. The model's accuracy was compared in both sets with previous scores, namely the Discard Risk Index (DSRI) , the donp < 0.05 were considered statistically significant. We used the SPSS statistical package version 24.0 .Survival probabilities were estimated using the Kaplan–Meier method. Survival rates comparisons were estimated using the log-rank method. Variables with a n = 660; 15.7%); pre-procurement liver function tests and/or imaging ; poor macroscopic aspect of the organ at surgery ; and poor perfusion during procurement .The characteristics of the entire population, Training and Validation Sets are reported in Table n = 111; 2.6%); bacterial infections ; pre-procurement cardiac arrest .Among the liver-unrelated discard causes the most frequent were: tumors ; anti-HCV-positive donor ; weight , and age . Anti-HBV core positive donor  and DM2  were also risk factors. On the opposite, previous hypotension episodes  and donor height  showed a protective effect for the risk of liver-related graft discard.Table p = 0.055), AST , total bilirubin , and ALT  were slightly below the level of statistical significance.Peak values of serum creatinine  Score = − 2.68 +  + (0.03*age years) + (0.04*weight kg)—(0.03*height cm) + (0.29 if DM2) + (1.65 if anti-HCV positive) + (0.27 if anti-HBV core positive)—(0.69 if hypotension episode) + (0.09*serum creatinine peak) + (0.38*log10 AST peak) + (0.34*log10 ALT peak) + .p < 0.001) concerning the other tested scores in both validation processes. For instance, DSRI AUC was 0.66–0.68, while donor BMI and donor age AUCs were 0.62 and 0.59–0.61, respectively  showed the best DOR 33.48) with a sensitivity of 26.4 and a specificity of 97.1  > 30% and any rate of fibrosis and necrosis. In both the sets, DROP AUC and AIC performed better than the tested scores Table . Supplemn = 2,642) were stratified in four sub-classes according to the following thresholds of DROP: <50th centile (<− 0.79); 50th–75th centile (− 0.79 to 0.66); 75th–90th centile (0.66–1.32); and, >90th centile (>1.32). At survival analyses, patients >the 90th centile of DROP showed 3-month worse survival versus the <50th centile   and based entirely on pre-transplant variables [A score focusing on the risk of liver graft discarding—namely, the discard risk index (DSRI)—has recently been generated from a large donor population  and validated on a Training Set of 1,051 donors, DROP was entirely based on variables available at the time of donor reporting and included not only donors discarded after intra-operative evaluation but also those declined before procurement as per their clinical chart data. Since graft discard criteria may greatly vary across transplant centers due to local experience and waitlist dynamics, the current study included only liver grafts that were declined at a national level. However, this might not have entirely offset the bias of initial graft discard on the eventual decline by other centers, as highlighted in the international literature [We developed the DROP score able to solve the above limitations. Created on an extensive, interregional experience  or ≥ 30% (true positive). Also the AIC was the best one among the different tested scores. AIC estimates the relative amount of information lost by a given model: the less information a model loses, the higher the quality. In other terms, the smallest the AIC value, the smallest the loss of information, the better the quality of the model. It is fascinating to note that a mathematical score that can be obtained with data available at the time of donor reporting can predict the risk of MaS with similar diagnostic performances of gross evaluation of expert procurement surgeons or radiological examinations performable only during procurement , 30.The practical use of this score should present several beneficial effects, mainly in the setting of a more appropriate donor–recipient matching. In Italy, in fact, the transplant centers are not strictly bound to a MELD-based allocation system, presenting a percentage of cases in which a proper allocation of marginal offers to fitter recipients is done and vice versa . The effThe further amelioration of the donor–recipient match vesiculated by the early identification during the donation process of grafts with a relevant risk of discard should better consent to allocate them only to specific sub-groups of recipients presenting a beneficial effect in receiving even more marginal grafts .The study presents some limitations. First, it is based on retrospective and multicenter data. Nevertheless, these biases are shared by all the studies focusing on this topic , 12, 13.Third, the liver biopsies performed before organ procurement were not evaluated by the same pathologists, and an interrater variability assessment for macrovesicular steatosis should be considered. Unfortunately, the possibility to perform a centralized revision of the biopsies was impossible due to the retrospective nature of the study.Lastly, we are not able to assert if the two Italian regions considered in the present study had a different rate of graft discard respect to the national mean value. This datum should impact mainly on the role of the variable “regional sharing” as a risk factor for liver-related graft discard. Further studies involving more centers are required for better detailing this aspect.In conclusion, the DROP score might be a useful tool to predict the risk of liver-related graft discard. The score is also able to predict several histological variables like steatosis, fibrosis, and necrosis. More studies aimed at investigating this score in other geographical settings are required.Supplementary file1 (DOC 29 KB)Supplementary file2 (TIF 329 KB)Supplementary file3 (TIF 317 KB)Supplementary file4 (TIF 423 KB)Supplementary file5 (TIF 7323 KB)Below is the link to the electronic supplementary material.


P < 0.001). Moreover, our team also studied the association between ABCC3 expression and clinical features of glioma patients, finding that differential expression of ABCC3 was remarkably related to age, 1p19q codeletion, PRS type, chemo status, grade, IDH mutation state, and histology. Overall, our findings suggested ABCC3 might be a novel prognosis marker in glioma.Glioma is the most common malignant primary brain tumor with an inferior survival period and unsatisfactory prognoses. Identification of novel biomarkers is important for the improvements of clinical outcomes of glioma patients. In recent years, more and more biomarkers were identified in many types of tumors. However, the sensitive markers for diagnoses and prognoses of patients with glioma remained unknown. In the present research, our team intended to explore the expression and clinical significance of ABCC3 in glioma patients. Sequential data filtration  was completed, which gave rise to the determination of the relationship between glioma and the ABCC3 gene. Clinical assays on the foundation of CGGA and TCGA datasets unveiled that ABCC3 expression was distinctly upregulated in glioma and predicted a shorter overall survival. In the multivariable Cox analysis, our team discovered that the expression of ABCC3 was an independent prognosis marker for both 5-year OS (HR = 1.118, 95% CI: 1.052–1.188;  Glioma is the most commonly seen malignancy primary cerebroma and the most fatal type of cerebroma in adults . Of the The progress in biological information and high-flux sequencing has realized the determination of various cancer markers which might assist the prognostic accurateness of GBM, which might give rise to more valid interventions in this regard , 8. Tan Herein, we searched CGGA datasets and identified many survival-related genes based on several conditions. Finally, we identified 132 genes which may be the most important survival-related genes in glioma. Among those genes, our attention focused on ABCC3. Recently, some studies have discovered the dysregulation of ABCC3 in many cancers, including glioma –18. HoweWe collected 1018 glioma sufferers for the following investigation. Clinic feature data and transcriptomic sequencing results of CGGA microarray and RNA-sequencing cohorts were acquired from the CGGA dataset . Our teaP < 0.001 being significant on statistics.Survival and survminer packages were used in R program , and K–MThe information of differentially expressed survival-associated genes between tumor and matched normal tissues was from TCGA and GTEx databases. GEPIA was applied to analyze the expressions of the survival-associated genes in GBM .P result was <0.05.To determine the impact of ABCC3 expression on prognoses, our team has to evaluate if the ABCC3 expression was related to the rest of the factors clinically, such as sex, age, IDH1 variant phase, and cancer WHO gradation. For that reason, univariable and multivariable Cox proportion assays were finished to identify the independent prognosis effects of ABCC3 with the forward stepwise procedure. The ABCC3 expression and clinical factors were considered to be independence factors when the modified P < 0.05.The data of gene expressions acquired from ROC curve filtration and the relevant clinic data were studied via R program and subjected to filtration via Genetic expression and relevant clinic data acquired from ROC curve filtration were studied in R to abstract the clinic data related to the ABCC3 gene. The association between the expression of ABCC3 and a variety of clinic features was identified via beeswarm.P < 0.05 was considered statistically significant.All the statistical analyses were performed using R version 3.4.2 software. A two-tailed P < 0.05 being the threshold. As shown in Figures P < 0.001), HIST1H2BK8 (P < 0.001), ANXA1 (P < 0.001), AK2 (P < 0.001), ABCC3 (P < 0.001), and ABRACL (P < 0.001).We performed the K–M, univariable Cox method, and multivariable Cox analyses to screen the survival-related genes. Then, AUC >0.7 was taken as the liminal value for ROC curve analyses , PRS types, histological status, gradation, ages, and chemotherapy were factors related to higher risks and IDH variant and 1p19q codeletion were related to lower risks (P < 0.001) was related to OS in an independent way, which unveiled that ABCC3 could serve as an independence marker for the clinical outcome of this disease. Moreover, PRS types, gradation, ages, chemotherapy, IDH variant, and 1p19q codeletion might be independent prognosis factors as well (To investigate the prognostic value of the expression of ABCC3 in glioma patients, our team performed univariable Cox analyses and observed that ABCC3 (HR = 1.369; 95% CI = 1.306–1.435; er risks . Multiva as well . ROC cur as well . Finally as well , 1p19q c as well , PRS typ as well , chemo s as well , grade ( as well , IDH mut as well , and his as well . Our finAmongst inhomogeneous primary cancers of the CNS, gliomas are the most common type, with GBM featured by the most unsatisfactory prognoses , 23. In In this study, we analyzed CGGA datasets and identified 132 possible survival-related genes with a high score of ROC. Among the 132 genes, we showed the top 6 genes, including MED8, ABCC3, ABRACL, AK2, ANXA1, and HIST1H2BK. However, only ABCC3, HIST1H2BK, AK2, and ANXA1 exhibited a high level in GBM. Previously, several studies have reported the expressing pattern and function of HIST1H2BK and ANXA1 in glioma , 32. ForPreviously, some studies have reported the effects of ABCC3 in many types of tumors. For instance, ABCC3 was found to be involved in the regulation of the sensitivity of doxorubicin in triple-negative breast cancer . ABCC3 wABCC3 is upregulated in patients with glioma. Its abnormal expression can be utilized as an independent diagnostic and prognostic biomarker for this tumor. Nevertheless, its mechanisms and other effects remain unknown. Moreover, this study is limited by its small sample. Hence, further studies are needed.


In the article titled “Artificial Liver Support System Improves Short-Term Outcomes of Patients with HBV-Associated Acute-on-Chronic Liver Failure: A Propensity Score Analysis” , some inThe authors would like to thank Jian-Rong Huang, Shao-Jie Xin, Zhong-Ping Duan, Yu-Ming Wang, Qing Xie, Zhi-Liang Gao, Jian-He Gan, Tao Han, Shu-Mei Lin, Chen Pan, Li Zhuang, and Yong-Ping Chen for patient data collection. This work was supported by the Science & Technology Key Program of Zhejiang China (#2017C03051).