There is a lack of reliable biomarkers for disorders of the central nervous system (CNS), and diagnostics still heavily rely on symptoms that are both subjective and difficult to quantify. The cerebrospinal fluid (CSF) is a promising source of biomarkers due to its close connection to the CNS. Extracellular vesicles are actively secreted by cells, and proteomic analysis of CSF extracellular vesicles (EVs) and their molecular composition likely reflects changes in the CNS to a higher extent compared with total CSF, especially in the case of neuroinflammation, which could increase blood–brain barrier permeability and cause an influx of plasma proteins into the CSF. We used proximity extension assay for proteomic analysis due to its high sensitivity. We believe that this methodology could be useful for de novo biomarker discovery for several CNS diseases. We compared four commercially available kits for EV isolation: MagCapture and ExoIntact (based on magnetic beads), EVSecond L70 (size-exclusion chromatography), and exoEasy (membrane affinity). The isolated EVs were characterized by nanoparticle tracking analysis, ELISA , and proximity extension assay (PEA) using two different panels, each consisting of 92 markers. The exoEasy samples did not pass the built-in quality controls and were excluded from downstream analysis. The number of detectable proteins in the ExoIntact samples was considerably higher (~150% for the cardiovascular III panel and ~320% for the cell regulation panel) compared with other groups. ExoIntact also showed the highest intersample correlation with an average Pearson’s correlation coefficient of 0.991 compared with 0.985 and 0.927 for MagCapture and EVSecond, respectively. The median coefficient of variation was 5%, 8%, and 22% for ExoIntact, MagCapture, and EVSecond, respectively. Comparing total CSF and ExoIntact samples revealed 70 differentially expressed proteins in the cardiovascular III panel and 17 in the cell regulation panel. To our knowledge, this is the first time that CSF EVs were analyzed by PEA. In conclusion, analysis of CSF EVs by PEA is feasible, and different isolation kits give distinct results, with ExoIntact showing the highest number of identified proteins with the lowest variability. There is a great need for CNS biomarkers for diagnostic, monitoring, and patient stratification applications. Current clinical practice largely relies on assessment of patient-reported symptoms, which is highly subjective and difficult to quantify. Novel biomarkers that could identify CNS disorders before symptom onset have the potential to greatly improve treatment of many diseases .Cerebrospinal fluid (CSF) holds great promise as a source for CNS biomarkers, and its analysis is already used widely in clinical decision-making . For exaOne challenge when searching for biomarkers in CSF (as with most body fluids) is that highly abundant proteins such as ALBUMIN can hamper the detection and quantification of less abundant markers . AnotherExtracellular vesicles (EVs) are small, membrane-enclosed particles that likely are released by all cells ,10. EVs PEA is not a comprehensive proteomic analysis but relies on matched antibodies labelled with partially complementary DNA oligonucleotides. Once two matching oligonucleotides are in proximity, they hybridize and are extended by DNA polymerase. These assays are performed on the microfluidic nanoliter-sized chambers. This methodology enables detection of 92 markers from one microliter of plasma. PEA has been applied to EVs derived from various cell cultures, breast milk, and seminal plasma . FurtherIn this study, we included two PEA panels: the “cardiovascular III” panel, which contained several markers that potentially are important for amyotrophic lateral sclerosis (ALS), for example, YKL40/CHI3L1 , MCP-1 , and CHIIt has been demonstrated that different isolation methods yield considerably different EV isolates in terms of total protein, purity, and results in downstream applications . The choThe study overview is depicted in 10 vs. 3–8 × 108)  c. The EV8 × 108) d. Albumi8 × 108) e. The exPEA, like other assays based on sandwich immunoassays, can be subjected to a so-called “high-dose hook effect”  if the c2 values) and the number of identified proteins in each bin; both panels indicate similar results—more identified proteins and a higher signal were obtained in the ExoIntact group compared with the other EV groups . PCA could clearly separate the different groups, indicating differences in the EV isolation methods  in order to identify any high-dose hook effects. The neat exoEasy samples did not meet the quality control standard; however, diluted samples did, which likely is due to the hampering effects of the proprietary elution buffer. A buffer exchange step might be necessary to analyze exoEasy samples by PEA.Although we could not find any previous reports regarding the analysis of CSF-derived EVs by PEA, plasma-derived “neuronal exosomes” were recently analyzed using neurology and neuro-exploratory panels. The authors could distinguish patients with HIV-associated neurocognitive disorder from HIV patients without cognitive impairment .In this study, we could detect a rather large variation in the number of detected proteins between different kits. Unsupervised hierarchical clustering is a common method to reveal subpopulation structures in a dataset and has gained increasing attention for analysis of comprehensive biological datasets in the last two decades . The metThe highest number of detected proteins was in the ExoIntact group. In addition, the signal of detected proteins varied depending on the isolation kit, as depicted in For most proteins, the signal was stronger in total CSF compared with EV samples. However, several proteins were upregulated in EVs. During the sample preparation, the EVs were concentrated 100 times using 100 kDa spin filters. If the proteins were large enough (over 100 kDa), they could also be enriched by the concentration step. However, several of the enriched proteins were smaller than 100 kDa; for example, MEPE (~60 kDa), Azu1 (~16–27 kDa), and ITGb2 (~85 kDa). This strengthens the hypothesis that the proteins were concentrated as EV cargo rather than as free, soluble proteins. Several detected proteins in the CSF EVs were “CNS-enriched”, for example PODXL2, OMG, and SLITRK2, which supports the idea that these EVs reflect CNS.One of the most important questions is whether analyzing CSF EVs instead of total CSF is of value. In this study, we can confidently say that there are differences between the CSF EVs and total CSF, but if this adds diagnostic value or not is still unknown and will be addressed in an upcoming study.In conclusion, we have here for the first time analyzed CSF-derived EVs using PEA. We clearly demonstrated how different isolation kits affect results, both in terms of the number of identified proteins and signal strengths. In this setting, isolation using ExoIntact resulted in the highest number of identified proteins in both the cardiovascular III and cell regulation panels.g, 4 °C for 5 min in the first step, after which the supernatants were then transferred to new tubes and subjected to a second centrifugation step of 10,000× g, 4 °C for 20 min. The supernatant was collected as cleared CSF, aliquoted (per kit: 2 × 1100 µL and 1 × 1210 µL), and stored at −80 °C. EVs were isolated using exoEasy , EVSecond L70 , ExoIntact , and MagCapture  according to the manufacturers’ instructions. Prior to isolation with MagCapture, the cleared CSF was concentrated using ultrafiltration spin filters  to reach a volume of less than 1000 µL. After isolation, the EV suspensions from each kit were concentrated using Vivacon 500 spin filters to volumes less than 11 µL, and PBS was subsequently added to reach a final volume of 1/100 of the starting cleared CSF volume (per kit: 2 × 11 µL and 1 × 12.1 µL). One microliter from each sample was saved, and 1.1 µL of the 12.1 µL samples was used to produce 1:10 and 1:100 diluted samples (10 µL each). In total, 26 samples were analyzed using PEA : 3 × nondiluted samples from each kit (representing three independent isolations), 1 × 1:10 and 1 × 1:100 diluted samples from each kit, 3× total cleared CSF, and 1 × 1:10 and 1:100 diluted samples from total cleared CSF. The 10 µL samples were mixed with 13 µL M-PER  with a 1:100 protease inhibitor cocktail . A lysis buffer with protease inhibitor mixed with PBS at the same final concentration as the samples was included as a negative control. The samples were sequentially added in a random order to a 96-well plate , sealed with a temperature-resistant plate-sealer, and stored at −80 °C. The samples were shipped on dry ice and analyzed at the Olink Proteomics Inc Boston site, MA, USA.Pooled, commercially available CSF from healthy donors was acquired from PrecisionMed Inc.  and Lee BioSolutions Inc. . The samples were used after approval from the institutional review board of the Shonan Research Center at the Takeda Pharmaceutical Company (CS-00001128 (approved 10 February 2016) and CS-00200049 (approved 11 July 2019)). The CSF was cleared by centrifugation: 2000× EVs were analyzed using nanoparticle tracking analysis . For each sample, five videos were recorded and analyzed using Nanoparticle Tracking Analysis software version 2.3 build 0033 .CD63 and CD81 were measured using PS Capture  according to the manufacturer’s instructions. The CD63 antibody was provided with the kit and used at 1:100 dilution. The CD81 antibody  was used at 1:200 dilution. The exoEasy samples were diluted 1:5, while the other EV samples were diluted 1:2.5.Albumin concentration was measured using an ELISA kit  according to the manufacturer’s instructions. The exoEasy samples were diluted 1:200, the other EV samples at 1:50, and total CSF at 1:2000.Initial data clean-up was performed in Microsoft Excel 360. Samples and values that did not meet Olink’s quality control criteria were excluded. We next evaluated high-dose hook effect artifacts and removed markers that had an 80% or higher signal in 1:10 or 1:100 dilutions compared with nondiluted samples.The freely available software platform Perseus v1.6.7.0  was usedVenn diagrams e,f were t-test was used and statistical significance indicated as: * p ≤ 0.05, ** p ≤ 0.01, and *** p ≤ 0.001.Bar charts and statistical analyses were made using Graph Pad Prism version 8 . Error bars indicate standard deviations. Student’s


The early and accurate in vivo diagnosis of sporadic Creutzfeldt–Jakob disease (sCJD) is essential in order to differentiate CJD from treatable rapidly progressive dementias. Diagnostic investigations supportive of clinical CJD diagnosis include magnetic resonance imaging (MRI), electroencephalogram (EEG), 14-3-3 protein detection, and/or real-time quaking-induced conversion (RT-QuIC) assay positivity in the cerebrospinal fluid (CSF) or in other tissues. The total CSF tau protein concentration has also been used in a clinical setting for improving the CJD diagnostic sensitivity and specificity. We analyzed 182 CSF samples and 42 olfactory mucosa (OM) brushings from patients suspected of having sCJD with rapidly progressive dementia (RPD), in order to determine the diagnostic accuracy of 14-3-3, the total tau protein, and the RT-QuIC assay. A probable and definite sCJD diagnosis was assessed in 102 patients. The RT-QuIC assay on the CSF samples showed a 100% specificity and a 96% sensitivity, significantly higher compared with 14-3-3 (84% sensitivity and 46% specificity) and tau (85% sensitivity and 70% specificity); however, the combination of RT-QuIC testing of the CSF and OM samples resulted in 100% sensitivity and specificity, proving a significantly higher accuracy of RT-QuIC compared with the surrogate biomarkers in the diagnostic setting of patients with RPD. Moreover, we showed that CSF blood contamination or high protein levels might interfere with RT-QuIC seeding. In conclusion, we provided further evidence that the inclusion of an RT-QuIC assay of the CSF and OM in the diagnostic criteria for sCJD has radically changed the clinical approach towards the diagnosis. Prion disorders are a group of rare, rapidly progressive, and fatal neurodegenerative diseases, which occur in sporadic, genetic, or acquired forms. They are caused by a conformational conversion of the normal cellular prion protein (PrP) into an abnormal misfolded pathological form, termed disease-associated PrP.Disease-associated PrP can acquire different conformations, which, in combination with the status of methionine (M)/valine (V) polymorphism at codon 129 of the gene coding PrP, define the different molecular subtypes of sporadic Creutzfeldt–Jakob disease (sCJD), with distinctive clinical and neuropathological features.sCJD commonly presents as rapidly progressive dementia (RPD), mimicking a number of potentially reversible forms, acronymized under the term VITAMINS , includiConversely, although not included in the diagnostic criteria for sCJD, the determination of the CSF levels of the microtubule-associated protein tau is helpful in diagnostic practice  and, in Recently, in vitro prion amplification techniques, including the protein misfolding cyclic assay (PMCA) and real-time quaking-induced conversion (RT-QuIC) assays, have been set up for CJD diagnosis . IntriguThe aim of the present study was to compare the diagnostic value of the old CSF biomarkers, 14-3-3 and tau, and the RT-QuIC amplification assay in sCJD. We evaluated prospectively the CSF 14-3-3 positivity, tau levels, and CSF RT-QuIC in 182 patients with possible sCJD, in addition to OM testing in 42 patients. We assessed the utility and the diagnostic value of each single marker in sCJD diagnosis.A total of 182 CSF samples and 42 OM samples were analyzed from patients with RPD and suspected sCJD.Among the CSF samples, 130 resulted 14-3-3 positive, 111 had tau levels >1300 pg/mL, and 98 were RT-QuIC positive, while among the 42 OM samples, 32 were RT-QuIC positive; the surrogate biomarkers and RT-QuIC testing in the CSF and OM of each sample are reported in At follow-up, 102 patients were diagnosed with sCJD. In detail, 61 patients were diagnosed with definite sCJD (sample code #1 to #61) and 41 patients received a diagnosis of probable sCJD . The other 69 patients in the non-CJD group comprised cases with an alternative clinical diagnosis  or showed a clinical evolution incompatible with a prion disease .Eighty patients  and 91%  in OM. The specificity was 100% , indicating the high reliability of the assay. Conversely, the 14-3-3 protein showed a sensitivity of 85%  and a specificity of 46% , and the tau sensitivity was 85%  and the specificity was 70% .Nineteen cases with a probable (twelve) and definite (seven) sCJD diagnosis, with a CSF positive to RT-QuIC testing showed biomarker levels not orienting to sCJD clinical diagnosis . In partWhile in some CSF samples one or two biomarkers could be considered altered if not completely positive, three patients  had negative 14-3-3 and normal tau levels (<350 pg/mL). However, in all of these three samples, the RT-QuIC was positive both in the CSF and OM. Patient #100 was 14-3-3 negative and showed borderline normal tau level (373 pg/mL); this patient was found to be RT-QuIC positive in the CSF.One CSF sample (#83) was 14-3-3 positive, but with low levels of tau.Intriguingly, in the surrogate markers misleading group, we found the only three sCJD subjects with negative OM RT-QuIC ; they were 14-3-3 negative in two cases and positive in one case, while they all had tau levels under the diagnostic cut-off of 1300 pg/mL.Four patients displayed a typical clinical presentation and evolution with a disease duration of about two months, clinically classified as classical sCJD. The supporting investigations showed an EEG with periodic sharp wave complexes, and a hyperintense signal in the basal ganglia and cortical ribbon in the diffusion weighted imaging (DWI) was observed in the MRI. CSF was 14-3-3 positive and had tau levels higher than 1300 pg/mL. Unexpectedly, RT-QuIC was negative in CSF and, as a diagnostic confirmation, the patients who underwent nasal brushing and RT-QuIC in the OM sample had positive results. Three cases were autopsy confirmed .These CSF samples showed increased levels of total protein (>45 mg/dL) and albumin (>35 mg/dL); sample #33 was also blood contaminated. To determine if any interference for RT-QuIC by the CSF matrix effect or by pathological PrP in itself, in a dose dependent way, was present in the CSF samples, a RT-QuIC analysis was repeated on neat, 1:10-, and 1:20-fold diluted CSF in PBS. Unfortunately, only CSF samples #33 and #42 were available after the first CSF biomarker screening. As reported in To prove if in the sCJD RT-QuIC negative OM samples the same interferences effects as for CSF could be avoided by sample dilution, we tested these samples at a 1:5, 1:10, 1:20, and 1:50 dilution. However, no seeding reaction was observed after the dilution of the OM samples.The RT-QuIC assay has already been shown to have a high diagnostic value, which is independent of the disease evolution . In thisIn particular, the prion strain type of disease-associated PrP did not influence the RT-QuIC seeding reactions or the diagnostic accuracy. In particular, we did not observe differences of RT-QuIC sensitivity among CJD subtypes, as a variability in the RT-QuIC reactions was observed in the samples belonging to the same CJD molecular subtype. In addition, the use of a specific PrP substrate to improve the RT-QuIC sensitivity among sporadic CJD subtypes was not demonstrated, but we showed that we reached 100% sensitivity when CSF is coupled with OM brushing.Conversely, the surrogate biomarkers have a potential prognostic value related to their levels, as they are found to be normal in sCJD with a long disease duration , and theIn this prospective study, we found a high overall diagnostic accuracy for CSF RT-QuIC, in line with previous studies, ,9,11,12,We previously showed that 14-3-3 was positive in the CSF of neuroleptic-treated patients , as wellAlthough prion diseases are characterized by important neuronal damage, we found several CSF samples with negative 14-3-3 and with normal tau levels, resulting in a final sensitivity of 84% and 85% for the 14-3-3 and tau assays, respectively. These results are far from what we reported in our previous study on 127 definite sCJD cases, where we showed a diagnostic sensitivity of 96% for CSF 14-3-3 detection and 92% tau determination . We coulIn three patients, the CSF samples were negative for both 14-3-3 and tau and were completely non-diagnostic. However, an MRI scan showed a hyperintense signal in the cortical ribbon at DWI, which progressed to diffused brain involvement in the subsequent MRI scans. The CSF was RT-QuIC positive in all of these subjects.These findings indicate that in presence of normal biomarkers, such as a negative 14-3-3 and normal tau, sCJD diagnosis might not be excluded. In these cases, the early diagnosis of a prion disorder is important for clinicians in order to avoid additional and unnecessary diagnostic investigations, a relatively common condition before the introduction of RT-QuIC. Besides a reduction in the cost of the diagnosis, a precise diagnosis has relevant ethical implications for both the patient and the family. Paradoxically, four sCJD cases with a typical disease phenotype were RT-QuIC negative in the CSF, but this test was not relevant for the diagnosis.In the four cases with negative RT-QuIC in the CSF, 14-3-3 was positive and tau was >1300 pg/mL. Among them, the three definite sCJD cases showed onset and clinical signs, MRI, and EEG, suggesting typical sCJD, and had similar short duration. However, the negative RT-QuIC result in the CSF was likely related to blood contamination or a high protein concentration, which might interfere with the prion seeding reactions. In fact, when these samples were diluted, a positive response to the RT-QuIC assay was obtained. Inhibition in ThT fluorescence by blood has been recently confirmed by Foutz et al., showing a direct interference by increasing the blood concentration in th RT-QuIC reaction ,22; in pNevertheless, these sCJD cases with an apparent negative CSF RT-QuIC received a diagnosis of CJD, because the OM samples of the same patients have been tested positive. These data confirm that OM RT-QuIC may, however, widely fill the gap in the case of negative CSF RT-QuIC, corroborating what we previously reported ,9. DespiIn conclusion, we showed that, by exploiting its extremely high sensitivity and specificity, CSF RT-QuIC, alone or in combination with OM RT-QuIC, can give an early and accurate in vivo diagnosis of sCJD, avoiding dangerous false negative results by surrogate biomarkers.Moreover, considering that, in less than one week, RT-QuIC gives such sensitive and specific results, and in most cases at the early stages of the disease, it needs to be underlined how a first screening by RT-QuIC can help in a reduction of the cost of diagnosis of a prion disorder, avoiding long and expensive, less accurate instrumental and molecular investigations.Finally, given the absolute specificity of RT-QuIC, a positive result may be considered as a definite diagnosis, and this implication may lead to considering and evaluating if autopsy is really necessary. In negative RT-QuIC cases, however, very few cases of sporadic or variant CJD  cannot bThis study included 182 patients presenting with RPD, referred to the Neuropathology Laboratory in Azienda Ospedaliera Universitaria Integrata of Verona, Verona, Italy, because of the clinical suspicion of CJD, between January 2015 and December 2017. The final diagnosis of these patients was established at death or when a definite alternative diagnosis was available. The diagnosis of human prion disease was made according to internationally established criteria .The ethical committees of the Azienda Ospedaliera Universitaria Integrata of Verona, Italy, approved the study (Prot. n. 28917 15 June 2012). Written informed consent for participation in the research was obtained in accordance with the Declaration of Helsinki (1964–2008) , and theOne hundred and eighty-two CSF samples were collected by lumbar puncture, were aliquoted as soon as collected, and were stored at −80 °C.Forty-two OM specimens were obtained by nasal brushing and processed as previously described . Out of Written informed consent from each patient or their representatives was obtained before each CSF and OM sampling.PRNP coding for PrP and to determine M/V polymorphism at codon 129 for molecular classification.After informed consent, genetic analysis was carried out on the DNA extracted from the blood specimens to exclude mutations of the gene CSF 14-3-3 was detected by immunoblot. For each sample, the equivalent of 25 μL of CSF was loaded onto a 13% polyacrylamide gel and transferred to polyvinylidene fluoride membranes, as previously described . Thee me® hTAU Ag ELISA kit , according to the manufacturer’s instructions. The absorbance values were obtained with a microplate reader and the tau concentrations were estimated from standard curves made for each assay.The CSF tau protein concentrations were measured in duplicate by sandwich ELISA, using the INNOTESTThe recombinant PrP substrates were prepared, as previously described ,9. The RSc was detected by a 3F4 anti-human PrP monoclonal antibody. The blots were developed with an enhanced chemiluminescence system  and PrP visualized on autoradiographic films .To determine the definite sCJD diagnosis, samples from the frontal, occipital cortex, and cerebellum were collected from autopsied brains, to be tested for the presence of prion proteinase K-resistant fragment, as previously described . Brain tStatistical comparisons of mean relative demographical data of different patient groups were performed by the t-test. The sensitivity, specificity, and relative 95% confidence intervals (CI) of the RT-QuIC, 14-3-3, and tau protein were calculated.


Donor-derived malignancies after solid organ transplantation have an estimated incidence of 0.02–0.2%, and donor-derived leukemia is particularly uncommon –3. We prThe patient was a 65-year-old man whose past medical history included nonalcoholic steatohepatitis with cirrhosis, multifocal hepatocellular carcinoma treated twice with radiofrequency ablation and transarterial chemoembolization, severe coronary artery disease with multiple drug-eluting stents complicated by recurrent restenosis, hypertension, type II diabetes mellitus, and obstructive sleep apnea. Three and a half years prior to the current presentation, he underwent liver transplantation with primary non-function of the allograft, requiring a second liver transplant days later. The hospitalization was complicated by shock and repeated ischemic acute kidney injury, resulting in dialysis-dependent end-stage renal disease.9/L, normal hemoglobin, platelet count 27 × 109/L, and INR 1.7. With immediate allograft function, the patient’s creatinine reached a nadir of 1.1 mg/dL. Immunosuppressive agents included basiliximab, tacrolimus, mycophenolate mofetil, and prednisone.Three years after liver transplantation, the patient received a kidney transplant from a 54-year-old deceased male donor who had died from a cerebrovascular accident. The donor’s initial labs included WBC 11.5 × 10Five months after kidney transplantation, the patient experienced intermittent gross hematuria and gradual rise in creatinine to 2.3 mg/dL. Urinalysis showed >50 WBCs/hpf and >20 RBCs/hpf. Biopsy of the allograft kidney initially suggested mild acute cellular rejection based on light microscopy; however, immunofluorescence and electron microscopy showed a monomorphic infiltrate. A myeloid neoplastic process was subsequently favored based on electron micrographs containing cells with an immature myeloid appearance and rare Auer rods Fig. . ImmunohPeripheral blood flow cytometry detected a suspicious blast population (0.04% of leukocytes) immunophenotypically consistent with the abnormal myeloid blast population in the kidney. Marrow aspirate flow cytometry identified a similar abnormal myeloid blast population (0.02% of leukocytes). Bone marrow aspirate and biopsy morphology was normocellular with trilineage hematopoiesis. Cytogenetics showed normal male karyotype. We sought additional leukemia cells for molecular and chimerism studies, but further testing of kidney tissue was limited by the sample type, quantity obtained, and prior processing. Given the patient’s pyuria and myeloid sarcoma in the kidney allograft, we hypothesized there could be leukemia cells in his urine. Although not routinely performed, flow cytometry of cells derived from the urine indeed demonstrated the same abnormal myeloid blast population (17% of leukocytes). Chimerism studies on CD117-positive flow-sorted cells from urine resulted as 71% kidney donor and 29% host, consistent with donor-derived origin.Hours after chimerism results were obtained, the patient developed severe dyspnea in the outpatient setting which progressed to cardiac arrest and pulseless electrical activity. Resuscitation efforts by paramedics were unsuccessful. On postmortem examination, immediate cause of death was attributed to cardiac arrest due to coronary artery disease, with up to 95% stenosis in the left anterior descending coronary artery. The kidney allograft showed multifocal green-colored masses throughout Fig. ; histoloA few days later, results of fluorescent in situ hybridization (FISH) testing from the second kidney biopsy returned positive for PML-RARα t gene rearrangements consistent with APL, as well as gains of 11q and 21q.We were concerned about possible transmission to other patients who received transplanted organs from the same deceased donor and reported our case to the United Network for Organ Sharing, discovering that there was a second kidney recipient and a heart recipient from the same donor being followed by other institutions. The second kidney recipient experienced worsening renal function starting 5 months post-transplant; the heart recipient was asymptomatic with no cardiac abnormalities on imaging. We advised that peripheral blood of these patients be sent for RT-PCR testing for PML-RARα transcripts, which returned positive in both patients. The second kidney recipient was initially treated with venetoclax and azacitidine; we advised switching to all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), and the patient was subsequently treated with ATRA alone due to contraindications to ATO. The heart recipient was treated with ATRA and ATO. Both patients are currently in remission with no PML-RARα transcripts detectable by RT-PCR.In general, reports of donor-derived leukemia after solid organ transplantation typically describe lack of evidence of leukemia in the donor, 2 or more years between transplantation and development of leukemia in the organ recipient, and lack of leukemic disease in other recipients with organs transplanted from the same donor. Thus, it has been hypothesized that nonmalignant hematopoietic stem cells may be carried over with the solid organ, subsequently undergoing malignant transformation in the recipient –7. In coOne other case of donor-derived leukemia presenting in multiple organ recipients from the same donor has been reported . There i


The aim of this systematic review was to evaluate existing literature on inflammatory markers in CSF from patients with hydrocephalus and identify potential markers capable of promoting hydrocephalus development and progression. rd 2020 were identified from PubMed, Embase, and reference lists. Studies were screened for eligibility using the predefined inclusion and exclusion criteria. Data from eligible studies were extracted, and sources of bias were evaluated. We included articles written in English investigating inflammatory markers in CSF from patients with hydrocephalus and control subjects. The review was conducted according to the PRISMA guidelines by three independent reviewers.  Relevant studies published before December 3β, and LRG compared with control subjects, whereas the CSF of PHH patients had increased levels of IL-6, IL-18, and VEGF. CSF from patients with “other hydrocephalus diagnoses” had elevated IFN-γ compared to control subjects, and VEGF was increased in congenital hydrocephalus, spina bifida, and hydrocephalus associated with tuberculous meningitis compared with controls.  Twenty-two studies analyzed CSF from 311 patients with idiopathic normal pressure hydrocephalus (iNPH), 178 with posthemorrhagic hydrocephalus (PHH), 151 with other hydrocephalus diagnoses, and 394 control subjects. Fifty-eight inflammatory markers were investigated. The CSF of iNPH patients had increased CSF levels of IL-6, IL-1β, LRG, IL-18, VEGF, and IFN-γ are elevated in CSF from patients with hydrocephalus and may be involved in promotion of hydrocephalus development and progression. They may serve as novel disease biomarkers, and their signaling pathways may represent targets for pharmacological management of hydrocephalus. IL-6, IL-1 Hydrocephalus is characterized by excessive amounts of cerebrospinal fluid (CSF) accumulating within the brain ventricles resulting in an increased intracranial pressure . The CSFThe systematic literature review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement , but is Studies were included in the review if they were (1) original research, (2) written in English, and (3) available in full text. Studies were excluded from the review if they (1) were reviews, meta-analyses, conference abstracts, comments, notes (i.e. gray literature), case reports, duplicates, or not published in English; (2) were not available in full text; (3) were animal or postmortem studies; (4) did not focus on patients with hydrocephalus; (5) did not analyze CSF for presence of inflammatory markers; (6) did not include a control group; (7) did not separate patients with hydrocephalus of different etiologies into separate groups; or (8) did not make a statistical comparison between patients with hydrocephalus and control subjects .rd 2020. The following search terms were applied in all four searches: “hydrocephalus” AND “cerebrospinal fluid” OR “CSF” OR “cerebrospinal fluid” AND “inflammation” OR “inflammatory” OR “inflammatory marker” OR “biomarker” OR “marker” OR “cytokine.” A unique search term was, in addition, applied to each of the four searches.PubMed and Embase were searched for relevant studies published before December 3Search 1 “normal pressure hydrocephalus” OR “NPH” OR “idiopathic normal pressure hydrocephalus” OR “iNPH.” The search was limited to studies published after January 1st 2005 where the international iNPH guidelines were first presented and later published [Search 2 “hemorrhage” OR “bleeding” OR “posthemorrhagic” OR “post-hemorrhagic” OR “post hemorrhagic.”Search 3 “trauma” OR “traumatic” OR “post-traumatic” OR “posttraumatic” OR “post traumatic” OR “post head injury.”Search 4 “meningitis” OR “infection” OR “infectious” OR “bacterial” OR “viral” OR “encephalitis.” Additional relevant studies were identified and manually curated from reference lists.The following data were extracted from each study: first author's name, publication year, study design, patient groups, number of patients in each group, diagnostic criteria including inclusion and exclusion criteria, age, sex, type of medication given, type of surgery performed, CSF source, preanalytical CSF handling, analytical method, CSF marker(s) analyzed, and result of CSF analysis.Authors SDL, NR, MJ, AS, and NM designed the protocol and search strategy.Initially, authors SDL and NR independently screened the studies by title and abstract. Studies that clearly violated the eligibility criteria were excluded. Afterwards, SDL and NR independently assessed the studies for eligibility by full-text review. After the independent assessment, SDL and NR collectively selected the studies for the final analysis. Authors SDL and EKO extracted data from relevant studies independently. After independent data collection, SDL and EKO collectively screened the extracted data, discussed any disagreements, and corrected any inconsistencies.most evidence when two or more studies reported consistent results, and a maximum of one study reported a contradictory result. Inflammatory markers were categorized as having limited evidence when results were reported from a single study.Inflammatory markers were separated into two categories based on the degree of consistency in results among studies. Inflammatory markers were categorized as having The potential risk of bias was assessed by the evaluation of the following data from each study: study design, sample size, age and sex, diagnostic criteria, inclusion and exclusion criteria, medication, circumstances of CSF sampling, pre-analytical CSF handling, selection of inflammatory markers for CSF analysis, and analytical methods.The PubMed and Embase search identified 1198 potential studies for the review, and 3 additional studies were identified from relevant reference lists. The initial search thus revealed 1201 potential studies for the review. Screening by title and abstract resulted in an exclusion of 1002 studies. Full-text review for eligibility of the remaining 199 studies led to the exclusion of 177 further studies. Hence, 22 studies were included in the final analysis .N = 311), PHH patients (N = 178), and patients with other hydrocephalus diagnoses (N = 151). The latter patient group included hydrocephalus associated with tuberculous meningitis (N = 44), spina bifida (N = 28), aqueduct stenosis (N = 25), acute hydrocephalus of unknown origin (N = 12), congenital hydrocephalus (N = 9), hydrocephalus following embolization of unruptured intracranial aneurysms (N = 8), fetal onset hydrocephalus (N = 7), late onset hydrocephalus (N = 7), arrested hydrocephalus (N = 6), and hydrocephalus with brain atrophy (N = 5). The CSF from patients with hydrocephalus was compared to CSF from control subjects (N = 394). The 22 studies investigated the presence of 58 inflammatory markers in total.The 22 studies included in the review were cross-sectional, case-control studies. Five studies were prospective –25, whilThe 22 studies applied different analytical methods to detect presence of inflammatory markers in the CSF samples. The analytical methods included enzyme-linked immunosorbent assays (ELISAs) , 23–35, CSF from iNPH patients was screened for presence of 36 inflammatory markers spread across 11 studies , 42. Detβ , and LRG showed most evidence of increased levels in CSF from iNPH patients compared to control subjects  iNPH Patients. In 2005, English international guidelines for the clinical diagnosis of iNPH were published [ublished , 39. Twoublished , 33 refeublished , 46. Fivublished –38, 42. ublished , 34.(2) PHH Patients. Patients diagnosed with PHH are heterogeneous in relation to age of onset, underlying pathological event, and clinical presentation. The internationally accepted diagnostic criteria for PHH are yet to be established [ablished . Howeverablished , 40, 41.ablished . Of the ablished , 29, 31,ablished , 28.(3) Patients with Other Hydrocephalus Diagnoses. Seven studies investigated patients with other hydrocephalus diagnoses [iagnoses , 40, 42.(1) All Patients with Hydrocephalus. The majority of the studies enrolled patients with hydrocephalus according to a certain diagnostic criteria. Only two studies specified the inclusion criteria that extended beyond the diagnostic criteria [criteria , 42. Onecriteria . Four stcriteria , 27, 28.criteria , 28, precriteria , 24, devcriteria , 42.(2) Control Subjects. The ideal control group should be the representative of the population from which patients arise [ts arise . Of the ts arise , 40–42. ts arise , 27, 38.ts arise , 33, 37.ts arise , 36. Onets arise . Of the ts arise , 38–42. ts arise , 42. Addts arise , 28, 39.Ideally, patients should not receive medication that is expected to influence the levels of the investigated inflammatory markers . However(1) CSF Sampling. It remains controversial whether a protein concentration gradient exists from the ventricular to the lumbar space [ar space –53. To rar space . Ten stuar space –39, 42, ar space , 40, 41.ar space , whereasar space . A thirdar space . Two stuar space , 34. Twoar space , 36.(2) Preanalytical CSF Handling. Preanalytical factors such as centrifugation, time from sampling to storage, storage material, and storage temperature are known to influence CSF analyses [g for 10 min at 4°C or room temperature prior to storage at -80°C [g/2500-3000 rpm for 6-10 min) [analyses –58. CSF at -80°C . Fourteeat -80°C , 33, 39 at -80°C , 37, 38.-10 min) , 37, 38.-10 min) . Eight s-10 min) –30, 32, -10 min) . The rem-10 min) –58. Howe-10 min) , 37–39. -10 min) .(3) Selection of Inflammatory Markers for CSF Analysis. The majority of the studies provided information in the introduction of their publication explaining why the inflammatory markers, or at least why some of them, were selected for analysis. Eight studies stated that some of the selected inflammatory markers had been detected previously in CSF from patients with hydrocephalus [cephalus –38, 41. cephalus . The stucephalus . Biased cephalus .β, and LRG were associated with the most evidence of significantly increased CSF levels. For PHH patients, IL-6, IL-18, and VEGF were associated with the most evidence of significantly increased CSF levels. For patients with other hydrocephalus diagnoses, IFN-γ and VEGF were significantly increased in several types of hydrocephalus compared to controls.In the current review, we systematically evaluated the existing literature on inflammatory markers in CSF from patients with hydrocephalus. We sought to identify promising inflammatory markers for future experimental research that aims to elucidate whether inflammatory markers can alter CSF dynamics and contribute to hydrocephalus development and progression. If neuroinflammation is involved in the pathogenesis of hydrocephalus, inflammatory markers may serve as novel disease biomarkers. Modulation of inflammatory pathways affects a range of systemic illnesses , 60 and κB pathway was recently demonstrated to cause CSF hypersecretion and subsequent hydrocephalus in rats [κB pathway [κB pathway. IL-1β is secreted primarily by immune cells in situations of infection, cell injury, and inflammation [κB pathway can be initiated by IL-1β [β also appears to have the prerequisites to promote choroidal CSF hypersecretion and cause hydrocephalus development.Activation of the NF- in rats . IL-6 dr in rats , 62, whe in rats . IL-6 ha pathway . IL-6 maammation . Similarby IL-1β . Hence, α2-glycoprotein) is produced by various cell types including hepatocytes, astrocytes, neutrophils, and epithelial cells in the inflamed tissue. LRG levels rise in inflammatory conditions such as meningitis, tuberculosis, and rheumatoid arthritis and are therefore suggested to be a marker of inflammation similar to acute-phase proteins [β [β levels. LRG signals by binding to endoglin (ENG), a component of the receptor complex for TGF-β, and LRG is therefore proposed to modulate TGF-β signaling [β signaling pathway has been reported to crosstalk with the NF-κB pathway [β, would initiate such crosstalk and hence potentially promote choroidal CSF hypersecretion and hydrocephalus development. IL-18 is secreted by immune cells such as macrophages and nonimmune cells such as epithelial cells, keratinocytes, and osteoblasts [β and therefore shares properties related to the receptor structure and signaling pathways, including activation of the NF-κB pathway [β, VEGF is capable of activating the NF-κB pathway [γ is secreted primarily by immune cells [γ intensifies the inflammatory response. The elevated IFN-γ levels in patients with other hydrocephalus diagnoses suggest presence of an ongoing inflammatory condition. Although IFN-γ primarily activates the JAK/STAT pathway [γ is also capable of activating the NF-κB pathway [γ has the prerequisites to promote choroidal CSF hypersecretion similarly to IL-6, IL-1β, IL-18, and VEGF.LRG . However, as some studies failed to report an age distribution or compared CSF from a mix of children and adults, results separated according to age would likewise have been associated with certain limitations.Another limitation of this review is the heterogeneity in sampling and handling of CSF across the included studies. Several studies directly compare lumbar CSF with ventricular CSF which may have influenced the results due to differences in the protein content from the ventricles to the lumbar space. The influence of sample time on CSF protein concentration remains debatable . HoweverThe review included inflammatory markers both directly and indirectly linked to inflammation. The definition of an indirect inflammatory marker falls within a grey area. Hence, some of the indirect inflammatory markers might be more relevant than others. The current review included indirect inflammatory markers that appeared relevant based on the identified studies in the original search. Regarding medication, it is surprising that only one study comment on the use of medication in relation to the investigated outcome . It can β, LRG, IL-18, VEGF, and IFN- γ, which were associated with the most evidence of increased levels in CSF from patients with hydrocephalus compared to control subjects. Future research should investigate whether these markers directly contribute to hydrocephalus development and progression. Such research will elucidate whether inflammatory markers may serve as novel disease biomarkers and whether their signaling pathways represent potential targets for pharmacological management of hydrocephalus. Future studies investigating the presence of inflammatory markers in CSF from patients with hydrocephalus should strive to eliminate the potential sources of bias presented in the current review. Efforts should be made to minimize patient heterogeneity. Patient diagnosis should occur in accordance with published international guidelines, and the inclusion and exclusion criteria should be clearly stated. Control subjects should, if possible, resemble healthy individuals. Studies should avoid small sample sizes, and patients should be matched on age and sex. CSF samples should preferably originate from the same space, be handled in the same manner, and if possible, be analyzed with the same analytical method. Furthermore, inflammatory markers should preferably be selected for analysis in an unbiased manner. The efforts done to eliminate the sources of bias may result in the identification of additional inflammatory markers, which are potentially involved in hydrocephalus development and progression.Despite the limitations, this systematic review presents a currently needed overview of the relevant literature and enables identification of promising inflammatory markers for future experimental research. In the current systematic review, we evaluated the existing literature on inflammatory markers in CSF from patients with hydrocephalus. We identified six inflammatory markers, IL-6, IL-1


BCR-ABL1 kinase domain (KD) mutation testing have a well consolidated role in the routine management of chronic myeloid leukemia (CML) patients, as they provide precious information for therapeutic decision-making. Molecular response levels are used to define whether a patient has an “optimal”, “warning”, or “failure” response to tyrosine kinase inhibitor (TKI) therapy. Mutation status may be useful to decide whether TKI therapy should be changed and which alternative TKI (or TKIs) are most likely to be effective. Real-time quantitative polymerase chain reaction (RQ-qPCR) and Sanger sequencing are currently the gold standard for molecular response monitoring and mutation testing, respectively. However, in recent years, novel technologies such as digital PCR (dPCR) and next-generation sequencing (NGS) have been evaluated. Here, we critically describe the main features of these old and novel technologies, provide an overview of the recently published studies assessing the potential clinical value of dPCR and NGS, and discuss how the state of the art might evolve in the next years.Molecular monitoring of minimal residual disease (MRD) and  BCR-ABL1 fusion gene, resulting from the t chromosomal translocation and encoding a deregulated tyrosine kinase responsible for leukemic transformation [Chronic myeloid leukemia (CML) is regarded as a paradigm of precision medicine. The hallmark of the disease, the m the t9;2 chromosBCR-ABL1 transcript levels (molecular response (MR)). It was soon realized that MR measurement by RT-qPCR could best stratify patients in terms of “depth” of response as compared to the classical hematologic and cytogenetic  translocation was detectable) assessments. Over the years, time-dependent MR milestones have been defined that harbor prognostic significance, draw the ideal trajectory toward optimal response, and lay the foundations for TFR [3; defined as a 3 log reduction of BCR-ABL1 transcript levels from the standardized baseline defined by the International Scale (IS), that is, BCR-ABL1 < 0.1%) and deep molecular response (DMR), further stratified into MR4 (4 log-reduction or BCR-ABL1 < 0.01%), MR4.5 (4.5 log-reduction or BCR-ABL1 < 0.032%), MR5 (5 log-reduction or BCR-ABL1 < 0.001%). Since the early 2000s, intensive international cooperative efforts have been devoted to the technical optimization of the assays and to the standardization of MR definitions and acceptability criteria, so as to maximize precision, accuracy, and interlaboratory reproducibility of results.Optimization of CML treatment throughout the years has been made possible by the forward-looking implementation and standardization of molecular monitoring strategies. Since the International Randomized study of Interferon-alpha plus cytarabine  versus STI571 (imatinib mesylate) (IRIS) study, which led to the approval of the first TKI, imatinib , real-tih the t9;2 translo for TFR . They inBCR-ABL1 that impair TKI binding. Detailed lists of mutations conferring resistance to each TKI are currently available [BCR-ABL1 KD mutations are, at present, the only “actionable” one, since the detection of a TKI-resistant mutation mandates a change of therapy and the type of mutation may guide the selection of the second- or subsequent-line TKI [BCR-ABL1 KD mutation screening. However, this sequencing technique has a relatively poor sensitivity. Moreover, in cases who have failed multiple lines of therapy and may harbor two or more mutations, Sanger sequencing must often be preceded by a cumbersome process of cloning in order to assess whether mutations are on the same (“compound”) or on different  BCR-ABL1 molecules. Assessment of clonal configuration is important, because compound mutations have been shown to be highly resistant to first-, second-, and in some cases even third-generation TKIs [Molecular testing is important also in the setting of patients who do not achieve an optimal response to TKI therapy. Such patients may have acquired point mutations in the kinase domain (KD) of vailable . Althougline TKI . Sanger ion TKIs .The flow chart of routine assessments in CML is reported in Technologies for MR monitoring and mutation testing have, in the meantime, evolved. Here, we describe some of the novel technologies that are going to impact most the way we measure response to therapy and identify TKI-resistant mutations, and we critically review the results of the studies that have already been performed to evaluate their application in CML.BCR-ABL1 transcript, the need for a standard curve, or the sensitivity to inhibitors), both improving its performance [6, with a low thermal mass and the possibility to perform a fully integrated process within a single instrument that combines consumable sample loading, thermal cycling image acquisition, and analysis software. This innovation creates a streamlined workflow that reduces contaminations and human handling errors and has a unique capability to decrease the time to result [In the last few years, many studies have been conducted in order to overcome the intrinsic limitations of “classical” quantitative PCR (RT-qPCR) ,9  study, which enrolled subjects on imatinib therapy for more than 2 years and with undetectable BCR-ABL1 by RT-qPCR for at least 18 months, Mori et al. demonstrated that a more successful selection of candidates for TKI discontinuation (TFR) was offered by the application of the Fluidigm dPCR platform. This platform is a first-generation dPCR relying on a relatively small number of multiple parallel micro-reactions. Overall, the large majority of cases which remained in stable undetectable DMR [In recent years, different groups approached dPCR for quantification of the able DMR  were dPCable DMR . Moreoveable DMR .BCR-ABL1 transcript measurement. In the ENEST next trial, which evaluated the rate of DMR in patients treated with nilotinib as first-line therapy, dPCR detected residual BCR-ABL1 transcripts in 39.4% of samples that were estimated to be in confirmed MR4.5 [BCR-ABL1 transcript levels using dPCR [The application of the Fluidigm platform was also experienced by other groups, confirming the good accuracy and sensitivity of dPCR in the setting of ed MR4.5  by RT-qPing dPCR .BCR-ABL1 transcripts by dPCR [4, showing the increase in BCR-ABL1 transcripts 3 months earlier than RT-qPCR in 4/10 cases [Later, Goh and colleagues confirmed the capability of nanofluidic dPCR to monitor the continuous decline in MRD levels even after they became undetectable by conventional RT-qPCR. Indeed, when 62 samples were screened by RT-qPCR and dPCR, the latter, preceded by a pre-amplification step, showed a 2–3 log improvement in sensitivity, with 75% of samples defined as negative by RT-qPCR that showed detectable  by dPCR . In anot10 cases .BCR-ABL1 not on complementary DNA (cDNA) but on genomic DNA (gDNA), which might be relevant if we consider that CML leukemic stem cells hidden in the hypoxic bone marrow niche are unable to synthesize BCR-ABL1 messenger RNA (mRNA) and the corresponding fusion protein [ABL1 and BCR breakpoint cluster regions are both positioned in genomic regions rich in Alu sequences, which might impair the perfect match of primers and the accurate quantitation of BCR-ABL1 transcript [The application of dPCR also improved MRD monitoring in pediatric CML cases. An interesting approach demonstrated the feasibility of using dPCR for the amplification of relatively long amplicons, thus allowing to monitor  protein . Differeanscript . Of 687 anscript .BCR-ABL1 on gDNA rather than on cDNA is also very interesting in adults, because it was previously reported that patients in long-term TFR who do not show detectable BCR-ABL1 transcripts on cDNA can be positive on gDNA, especially if the latter is extracted from the lymphoid compartment where B cells are more often BCR-ABL1-positive than T cells [BCR-ABL1 fusion gene [The possibility of detecting  T cells . A gDNA-ion gene ,31.BCR-ABL1 transcripts was recently set up by our group. Bernardi and colleagues underlined the capability of dPCR to offer a precise, sensitive, and accurate quantification of BCR-ABL1 transcripts in both peripheral blood cells and circulating exosomes [The application of a chip-based dPCR platform for the detection of exosomes ,33  identified by the CD26 surface marker. The authors observed that both dPCR and LSC quantitation by flow cytometry were more sensitive than RT-qPCR in identifying patients with residual disease, scoring as positive a series of samples where the BCR-ABL1 transcript was undetectable by RT-qPCR [dPCR also proved its value when patients in DMR were analyzed in parallel using RT-qPCR and dPCR for the monitoring of  RT-qPCR .BCR-ABL1 transcripts down to a 0.01% abundance, with high reproducibility across multiple replicates [Very recently, a third-generation dPCR chip-based platform was also tested for MRD monitoring in CML. Indeed, the novel microfluidic array partitioning device was capable of precisely quantifying plicates .BCR-ABL1 transcripts, another pitfall of RT-qPCR seems to lie in the different efficiency of amplification of the e14a2 transcript variant as compared to the shorter e13a2—a problem that might negatively impact the clinical management of CML patients [In addition to the technical limitation in the detection of very low levels of patients . This hypatients . These rpatients .BCR-ABL1 transcript detection by dPCR. The QXDx BCR-ABL %IS by Bio-Rad is the first dPCR-based in vitro diagnostics product receiving the United States (US) Food and Drug Administration (FDA) clearance and the European Conformity (CE-IVD) mark. It is suitable for droplet-based platforms and its performance was very recently independently evaluated and reported [r = 0.996) when quantifying BCR-ABL1 transcript levels ranging from 20% to 0.002%. These results further support the introduction of dPCR as the standard of care for CML patient monitoring. In addition, some panels of experts recently underlined that coordinated efforts should be made for optimization and standardization of CML MRD monitoring by dPCR [Considering these encouraging results, it is not surprising that new commercial assays have become available for reported . The QXD by dPCR ,45.p < 0.0001). Indeed, 14.3% of samples already defined to be in MR3.0 by RT-qPCR appeared to have higher levels of BCR-ABL1 transcripts by dPCR. On the other hand, 20.9% of cases in MR4.0 by RT-qPCR were not found to be in DMR by dPCR. This has a relevant clinical impact, considering that only patients who are in MR4 or better can be candidates for TFR [At the last American Society of Hematology (ASH) congress held in 2019, a discrepancy in the “molecular class” positioning when dPCR was used in comparison to RT-qPCR was reported  [5.0, compared to 90.9% at MR4.5, 90.9% at MR4.7, and 72.7% at MR5.0 of RT-qPCR (p < 0.05). Interestingly, the interlaboratory coefficient of variability of dPCR was lower than that measured for RT-qPCR (23.6% vs. 44.4% down to MR4.0) (p < 0.05) [At the 2020 meeting of the European Society of Hematology (EHA), a multicenter international study confirmed that dPCR is a valid alternative to RT-qPCR, demonstrating a low interlaboratory variation and a high assay linearity. Indeed, dPCR showed a detection rate of 90.9% at MR1 level) , and 81% < 0.05) .BCR-ABL1 transcript and potentially eligible for stopping TKI therapy. In fact, the biased diagnostic performance of the BCR-ABL1 molecular detection and quantification may impact the inclusion of CML patients in therapy-stopping trials [These data are constantly confirmed by new evidence and altogether stress the utility to move to dPCR for the monitoring of MRD in CML patients, particularly in subjects presenting a low level of g trials ,50, whicg trials ,51. A coBCR-ABL1 KD mutation testing provides important information whenever therapeutic reassessment has to be considered. In this context, both next-generation sequencing (NGS) and dPCR have been explored as alternatives to Sanger sequencing.As mentioned above, BCR-ABL1 transcript with a sensitivity that can theoretically achieve 1%, to follow the kinetics of mutant transcripts over time and in relation to therapy, and to discriminate between compound and polyclonal mutations in cases of multiple substitution falling on the same sequence reads.When benchtop next-generation sequencers for diagnostic use first landed on the market, BCR-ABL1 KD mutation screening was one of the first applications to be explored in hematology given that mutation status was already recognized at those times as an essential piece of information in the clinical decision algorithms for TKI-resistant patients . The powBCR-ABL1 KD mutation screening within a lab network and to compare Sanger sequencing and NGS performance in a consecutive series of 236 CML patients eligible for mutation screening because of failure or warning response according to the 2013 ELN recommendations [BCR-ABL1 transcripts).Between 2013 and 2016, a series of retrospective studies were published that aimed to investigate the use of NGS in patients who had developed resistance to TKI therapy. In particular, these studies engaged in the reanalysis by NGS of serial samples from patients who had acquired TKI-resistant mutations and had already been studied by Sanger sequencing. They demonstrated that NGS holds the potential to identify emerging mutations up to several months earlier than Sanger sequencing ,53. NGS ndations . This stBCR-ABL1 mutation testing [Taken together, all the data summarized above have already resulted in NGS being included in position papers on  testing ,60, as w testing .Nevertheless, efforts aimed at enhancing our mutation-testing capabilities continue. On one hand, they have been and are being directed toward more sensitive and accurate NGS-based screening. Strategies of error-corrected sequencing have been described . MoreoveJAK2 [IDH1/IDH2 [MYD88 [B-RAF [c-KIT [On the other hand, dPCR has been considered as a complementary or even alternative method. In the hematological scenario, dPCR has already been employed for detection of hotspot mutations in many genes, such as JAK2 , IDH1/IDDH1/IDH2 ,64, MYD82 [MYD88 , B-RAF [8 [B-RAF ,66, and F [c-KIT . Each dPF [c-KIT . All patF [c-KIT  , dasatinib-resistant, nilotinib-resistant, dasatinib- and bosutinib-resistant, nilotinib- and bosutinib-resistant), in order to provide an indication as to which TKI (or TKIs) should be avoided, if any. The study confirmed the advantage of NGS over Sanger sequencing; however, it also highlighted that, in the setting of failures, low-level mutations detectable by NGS could have impacted the choice of the subsequent TKI in 9% of the patients. In samples positive for second-generation TKI-resistant mutations, a very good concordance was observed between dPCR and NGS, irrespective of type of mutation and variant allele frequency, as well as when reactions started from very low cDNA amounts (as little as 30 ng). In patients with a warning response, the aim of mutation testing is to identify those who need a change in therapy rather than a “watch and wait” approach, since detection of a TKI-resistant mutation is an indication for therapeutic switch. Bearing in mind that more than 50 different mutations may confer resistance to imatinib, NGS remains the best approach to highlight emerging mutations that are going to lead to treatment failure in patients with a warning response to imatinib. In contrast, in patients with a warning response to second-generation TKIs, dPCR might represent an attractive alternative. However, patients who would benefit most from dPCR are those who fail TKI therapy. Here, mutation testing aims to a timely and rational TKI switch  and only a limited spectrum of mutations impacts the selection of the second- or subsequent-line TKI .At the same meeting, Vannuffel et al.  presenteAt the 2020 EHA meeting, we further confirmed the value of dPCR as a fast and effective technique for detecting the T315I mutation in another series of 44 CML samples. The sensitivity of dPCR was 0.02%, and overall dPCR allowed us to identify the T315I mutation in 16% of samples that were defined as wildtype by other methods. Indeed, when dPCR and Sanger sequencing results were compared, 25 samples showed concordant results, five were mutated by dPCR but not by Sanger, and no samples were wildtype by dPCR but mutated by Sanger. When dPCR was compared to NGS, 19 samples were concordant, two cases were mutated by NGS but wildtype by dPCR, and two additional cases were wildtype by NGS but mutated by dPCR. The variant allele frequencies (VAFs) in these two cases were 0.43% and 0.39%—values well below the detection limit of NGS (3%). Interestingly, out of the two cases where dPCR failed to detect the T315I on cDNA, one was found positive when gDNA was analyzed. Indeed, when applying dPCR to gDNA instead of cDNA, in 2/16 samples, the T315I mutation was detected only on gDNA . Thus, our experience demonstrated the value of dPCR in detecting the T315I mutation, with a sensitivity and accuracy not inferior to and sometimes better than those offered by NGS .In summary, while the lower limit of detection of Sanger sequencing is routinely 20%, that of NGS is between 1% and 3%, and that of dPCR may be as low as 0.01%. The turnaround time also favors dPCR over both sequencing technologies: 2 working days vs. 6 and 11 working days for Sanger sequencing and NGS, respectively. Cost per sample is around 200 EUR for Sanger sequencing, 90–100 EUR for NGS (when pooling eight samples on an Illumina MiSeq or Ion Torrent PGM), and from 3.5 to 11 EUR for dPCR.The main advantages and disadvantages of Sanger sequencing, NGS, and dPCR for BCR-ABL1 KD mutation testing are shown in In CML, we currently stand at a turning point between old and novel methods for molecular testing. Indeed, in the hematology/oncology field, CML has been one of the first settings where novel technologies like dPCR and NGS have been explored for routine diagnostic use. This is mainly due to the fact that, over the past 20 years, sensitive molecular monitoring and mutation testing have become indispensable for optimal patient management. The development of different technologies in CML management and the number of PubMed citations reflecting their usage over the years are represented in BCR-ABL1 transcript levels, especially when DMR (a sine qua non condition to identify patients who are candidates for TFR) has to be assessed. As reported above [1.0 to MR4.0 of dPCR results as compared to RT-qPCR results. Similar observations were previously reported by an Italian collaborative group [dPCR has proven to be a robust, sensitive, and accurate alternative to RT-qPCR in the monitoring of ed above , the prove group ,72. ThesAs far as BCR-ABL1 KD mutation testing is concerned, over the past decade, NGS has proven to hold several advantages over Sanger sequencing in retrospective and prospective studies, such that the ELN 2020 recommendations have already incorporated the use of NGS for the assessment of patients not responding adequately to TKIs [The power of NGS might, in the future, be exploited well beyond the identification of TKI-resistant mutations in BCR-ABL1. Very recently, whole=exome sequencing and RNA-sequencing identified novel gene mutations and gene rearrangements or fusion transcripts in newly diagnosed CP CML patients, particularly those who ultimately experienced disease progression and poor outcome . Indeed,As technology evolves at a rapid pace, there are also several innovative approaches on the horizon. Some of them might be implemented in the diagnostic panorama of CML in the near future, which will be characterized by precise and personalized medicine . Single-


The striatum, the main input structure of the basal ganglia, is critical for action selection and adaptive motor control. To understand the neuronal mechanisms underlying these functions, an analysis of microcircuits that compose the striatum is necessary. Among these, cholinergic interneurons (ChIs) provide intrinsic striatal innervation whose dysfunction is implicated in neuropsychiatric diseases, such as Parkinson’s disease and Tourette syndrome. The ability to experimentally manipulate the activity of ChIs is critical to gain insights into their contribution to the normal function of the striatum and the emergence of behavioral abnormalities in pathological states. In this study, we generated and tested CAV-pChAT-GFP, a replication-defective canine adenovirus type 2 (CAV-2) vector carrying the green fluorescent protein (GFP) sequence under the control of the human choline acetyltransferase (ChAT) promoter. We first tested the potential specificity of CAV-pChAT-GFP to label striatal ChIs in a rat before performing experiments on two macaque monkeys. In the vector-injected rat and monkey striatum, we found that GFP expression preferentially colocalized with ChAT-immunoreactivity throughout the striatum, including those from local circuit interneurons. CAV-2 vectors containing transgene driven by the ChAT promoter provide a powerful tool for investigating ChI contributions to circuit function and behavior in nonhuman primates. Numerous studies have suggested that the striatum, the main recipient of afferents to the basal ganglia, has a critical function in motor control and motivation. This structure mostly consists of GABAergic spiny projection neurons (SPNs) that target the output nuclei of the basal ganglia. The activity of SPNs is dependent upon excitatory inputs from the cortex and thalamus, under the control of dopaminergic and cholinergic modulation and different types of GABAergic interneurons  and canine adenovirus type 2 (CAV-2) vectors are widely used in rodents and primates to express transgenes in neurons into various brain regions. Each platform has advantages and disadvantages. Due to the CAV-2 vector’s capacity to harbor expression cassettes of 7–30 kb  vector carrying the green fluorescent protein (GFP) gene under the control of the human choline acetyltransferase promoter (pChAT), we report its ability to restrict GFP expression to striatal ChIs.Macaca fascicularis, (monkey P), 10.7 kg and one rhesus, Macaca mulatta, (monkey C), 11 kg] were used in this study. Monkeys C was chronically implanted with a head-fixation device and a recording chamber placed above the striatum. In this animal, injection coordinates were chosen based on electrophysiological mapping of the striatum we performed over several weeks before injections. Typically, the dorsal border of the striatum was identified by an increase of background noise and irregular spike activity after passing the cortex and underlying white matter. Striatal neurons (mostly output neurons or SPNs) were identified by their characteristic low-frequency discharge. These are different from neighboring structures, such as globus pallidus and adjacent fiber bundles .All experimental procedures were approved by the Institutional Animal Care and Use Committee of INT (Permission Number: 14675-2018041009396760) and complied with the rules of the European Community Council Directive (2010/63/EU) for the care and use of laboratory animals. One adult male Long Evans rat (300 g) and two adult male macaques [one cynomolgus, CAV-pChAT-GFP and CAV-mCherry are E1/E3-deleted replication-defective vectors. The constructs used in this study are schematically shown in 9 pp/μl) and CAV-pChAT-GFP (109 pp/μl) were pressure-injected into the dorsal striatum of respectively the left and right hemispheres. Two injections (0.5 μl each for CAV-mCherry and 1 μl each for CAV-pChAT-GFP) were performed for each vector. After surgery, the skin was sutured, prophylactic antibiotics were injected , and the animal was replaced in its home cage for a survival period of 7 days.The rat was anesthetized by an intraperitoneal injection of ketamine (100 mg/kg)/xylazine (10 mg/kg) solution and prepared for stereotaxic injections of the viral vectors. CAV-mCherry  delivered in one hemisphere and 20 μl of CAV-mCherry (2 × 109 pp/μl) delivered into 4 sites  in the other hemisphere with tracks into the putamen at two rostrocaudal levels and two different depths per track. The distances between the injection sites were 7 mm along the rostrocaudal direction and 6 mm in depth. After each injection, the needle was left in place an additional 5 min to minimize reflux along the injection tract. When the injections were completed, the holes were filled with Spongel and the skin was sutured.Both monkeys received injections of CAV-2 vectors into the putamen (same constructs as those used in the rat). Monkey P had injections in both sides of the brain and monkey C on one side . Under general anesthesia (sevoflurane 2%) and aseptic conditions, the head of monkey P was fixed in a stereotaxic frame and small holes were drilled into the skull at chosen coordinates above the striatum. After puncturing the dura, a 30-gauge needle connected to a 50 μl Hamilton syringe was slowly lowered through drilled holes to the target depth. We injected a single volume of 50 μl of CAV-pChAT-GFP vector (2 × 109 pp/μl) was injected into the putamen at two rostrocaudally different levels: one single 50 μl injection (5 μl/min) and 15 μl divided in three 5 μl boluses (2 μl/min) along the same track. Also, 15 μl of CAV-mCherry (2 × 109 pp/μl) divided into three 5 μl injections (2 μl/min) separated by 2 mm along the same track were delivered at a more anterior level. After injections, the chamber was sealed with a cap. The two monkeys were monitored during recovery from anesthesia and then returned to their home cages for a survival period of 10 days.Monkey C was injected unilaterally into the putamen using a microdrive fixed on the chamber and we replaced the recording electrode with a 30-gauge needle connected to a 50 μl Hamilton syringe. The needle was positioned inside a metal guide tube designed to penetrate the dura. A total of 65 μl of the CAV-pChAT-GFP vector  prepared in 0.12 M sodium phosphate buffer, pH 7.4 (PB). After perfusion, blocs of the brain containing the striatum were post-fixed and processed for sectioning . One out of every ten sections was stained with cresyl violet to localize the injection sites within the striatum and to determine the general histological characteristics of the tissue throughout the rostrocaudal extent of the striatum.Selected sections at the level of the striatum were processed for simultaneous immunohistofluorescence detection of GFP, mCherry, and ChAT according to a previously described protocol  surrounding the injection sites previously localized on cresyl violet staining adjacent sections. For each section, an image of the entire striatal region was obtained from a single confocal slice with 20× objective and sequential acquisition of the different wavelength channels . The analysis was then performed with Neurolucida software  as followed: for each confocal image, all GFP-labeled neurons were identified on the green channels and examined for co-localization with ChAT in the blue channels and/or mCherry in the red one. Then, the relative percentages of double-labeled neurons for GFP and ChAT, and mCherry and ChAT, were determined. A total of 76 GFP-labeled and 20 mCherry-labeled neurons were counted in the rat. In monkey P, 86 GFP-labeled and 30 mCherry-labeled neurons were counted.Coronal sections of the rat brain injected with CAV-pChAT-GFP and CAV-mCherry into the striatum of the right and left hemispheres, respectively , were prThe right striatum injected with CAV-pChAT-GFP displayed many GFP-labeled neurons. These neurons were distributed at a distance of up to 600 μm surrounding the injection site. Qualitatively, it appeared that transduced cells were more rarely observed near the injection site  arrows. In the left striatum, many mCherry-labeled cells were observed close to the injection site and within 600 μm . mCherryWe then injected CAV-pChAT-GFP and CAV-mCherry into the striatum of two macaques. In monkey C, the two viral vectors were injected in the same hemisphere. In this monkey, only the injection sites at the posterior levels  were properly located in the striatum . Sectionn = 5 sections).To address this possibility, monkey P received injections of CAV-pChAT-GFP and CAV-mCherry in the right and left hemispheres, respectively. As in the rat striatum, the right striatum contained neurons expressing GFP with a large cell body and several dendrites with multipolar or bipolar orientation . These mIn the present experiment, we developed a cell-type-specific gene expression approach using a CAV-2 vector carrying a ChAT promoter to investigate the role of ChIs in nonhuman primates. The results of this pilot study indicate that CAV-2 vectors containing a human ChAT promoter can be a valuable tool to preferentially target transgene expression to the ChI population of the striatum in monkeys. This represents a promising outcome, particularly for primate models where genetic resources are underdeveloped.Previous studies have shown that CAV-2 is an effective gene transfer tool in both rodents and primates .ME, PA, and EK conceived and designed experiments. ML and EK produced the virus vectors. A-CM, HE, JS, AG, ME, and PA performed experiments. ME and A-CM analyzed data and made figures. PA, ME, and EK wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


With the k-ε renormalization group turbulence model, the drag reduction mechanism of three- dimensional spherical crown microstructure of different protruding heights distributing on the groove surface was studied in this paper. These spherical crown microstructures were divided into two categories according to the positive and negative of protruding height. The positive spherical crown micro-structures can destroy a large number of vortexes on the groove surface, which increases relative friction between water flow and the groove surface. With decreasing the vertical height of the spherical crown microstructure, the number of rupture vortexes gradually decreases. Due to the still water area causes by the blocking effect of the spherical crown microstructure, it was found that the shear stress on the groove surface can be reduced, which can form the entire drag reduction state. In another case, the spherical crown microstructures protrude in the negative direction, vortexes can be generated inside the spherical crown, it was found that these vortexes can effectively reduce the resistance in terms of pressure and friction. In a small volume, it was shown that the surface drag reduction rate of spherical crown microstructures protrudes in negative directions can be the same as high as 24.8%. Nowadays microfluidics has been used in a wide range of areas, such as health detection , chemicaIt is often found that using microstructure can provide better performance improvement . Past reIn recent years, micro-riblets on the skin of Shark were found to be effective in the reduction of flowing resistance. It was observed that these riblets on the surface of objects can hinder the horizontal translation of vortexes through the surface, and reduced vortex jets and outer turbulence ,10 in thAfter decades of continuous exploration, the surface drag reduction technology using bionic surface microstructure has been applied in many fields. Choi et al.  studied Due to their simple lateral interface structure, some typical two-dimensional groove surfaces, such as V-shaped grooves, fan-shaped grooves, and blade-shaped grooves are selected to thoroughly study surface microstructure models of their grooves in the flowing direction . HoweverIn this paper, a new model combining the traditional V-shaped groove surface with a uniformly distributed spherical crown structure was proposed, the turbulent drag reduction mechanism of these microstructures was analyzed in the following paper.It is generally believed that the valley bottom of the groove can effectively limit the turbulence of the fluid flows and reduce the energy exchange between the fluid and the wall, the V-shaped groove can achieve a good drag reduction effect. However, due to the vortices that formed from the fluid flows through the tip of the V-shaped groove, the drag reduction effect of the entire structure will weaken. In this work, the RNG k-ε model was used to accurately simulate the influence of the vortices at the bottom of the groove.The RNG k-ε model has a more accurate description of the dissipation rate of turbulent kinetic energyThe RNG k-ε model considers the influence of eddy currents on turbulence and improves the accuracy of vortices flowThe RNG k-ε model provides an analytical formula for the turbulent Prandtl number, which can more accurately simulate the problem of turbulent boundary layer flow.The RNG k-ε is a model derived from the instantaneous Naiver-stokes equation with the method of the re-normalization group . Compareρ is the fluid density, p is hydrostatic pressure, iu and ju are velocity component of a fluid element, ijτ is sheer stress, if is unit mass force, ix and jx are components of displacement, t is the time.The N-S equation is expressed as :(1)∂∂t is [k is turbulent kinetic energy, tμ is molecular viscosity coefficient, kP is turbulence generation term.In the RNG tion) is :(2)∂∂t(ρk-ε model-specific tuning parameters:The turbulent energy dissipation rate equation (ε equation) is :(3)∂∂t and (3):  = 0.012 .In this paper, a special groove microstructure was selected for investigating the surface drag reduction mechanism, which is a number of spherical crowns set on the surface of the V-shaped groove. This special groove microstructure with the crown was improved from a typical V-shaped groove similar to the triangular groove structure of shark skin size.The dimensions of the V-shaped groove used in this paper were measured by other researchers . Based oThe flow region illustration in modeling about the special groove microstructure with spherical crowns shows in The flow region with two different surface shows in (1) The upper wall and the lower wall of this flow region are set as smooth surface, because these two surfaces simulate the groove surface which needs to be smooth.(2) Two surfaces in the Z-direction are set as the inlet surface and the outlet surface. The boundary condition at the inlet surface is set as a velocity condition, and the outlet surface is set as free flow.(3) The last two surfaces which perpendicular to the flow direction are set as the symmetrical boundary.This model simulates a small region in the entire flow field, using a symmetrical boundary can ensure that the simulated region reflects the characteristics of the entire flow field.In order to evaluate the effect of V-shaped groove surface with a spherical crown on the drag reduction mechanism, different flowing velocity is selected to calculate and analyze with a finite element method, while the height of spherical crowns on V-shaped groove surface would be changed. So, the turbulent kinetic energy and surface shear stress on this grooved surface can be obtained and explored in detail .In this paper, it is focused on discriminate the influence of the spherical crown groove structure with different heights on the overall drag reduction rate of the surface. The turbulent kinetic energy and the surface shear stress on the V-shaped groove surface with a spherical crown of different height can be achieved, the drag reduction mechanism of positive spherical crowns and negative spherical crowns will be respectively discussed, and some difference about the drag reduction mechanism of two models can be defined.The drag reduction rate of the traditional V-shaped groove model from the shark skin surface is generally about 6.5%, which is undesirability. Based on this, the two-dimensional V-shaped groove structure is partially modified in this model, which distributes positive spherical crowns of different heights on the surface of the V-shaped groove. For the spherical crown structure of the V-shaped groove surface, when the protruding direction of the spherical crown is positive, the curve of the drag reduction rate with the flow speed is shown in The drag reduction potential of the groove is determined by the effective penetration height of the crowns on the surface . Figure In addition, due to the blocking effect of the spherical crown on the water flow, there is a still water area within a certain range behind the crown, which shows in In The influences of different height of positive spherical crown on V-shaped groove surface have some difference for the flow velocity and the drag reduction rate, To compare with the positive spherical crowns’ microstructure, the drag reduction effect of the V-shaped groove structure with the concavity microstructure named negative spherical crown is explored. When the protruding spherical crown on the surface of the V-shaped groove becomes concavity as the negative spherical crown, the drag reduction effect is completely different from the positive spherical crowns’ microstructure at the same flowing speed. The curve of the drag reduction rate with the flow velocity shows in In order to investigate the drag reduction effect of the negative spherical crown on the groove surface, an observation line which the position as same as In the field of hydrokinetics, it is well known that the wall surface resistance is mainly composed of two aspects of friction of vortexes with the surface and the pressure resistance of the water with the surface. Because of the existence of riblets, vortexes locate above the groove, interacting only with the peak of the grooved surface, and lateral flow velocity pulsation in the groove is much smaller than the lateral flow of riblets’ top . In thisIt is generally believed that the increase in wetting area in the groove surface structure increases the pressure resistance , but thiThe negative spherical crown microstructure of the V-shaped groove structure has a very clear effect of drag reduction, and In this paper, the flow characteristics of water in different V-shaped groove surface structures with the spherical crown microstructure was investigated. The effect of different spherical crown structures on drag reduction rates was analyzed by comparing the surface Reynolds shear stress, fluid velocity, near-wall turbulent kinetic energy and the distribution of intensity of vortexes. it leads to the following conclusions:(1). Both the positive and negative protrusions of the spherical crown structure contribute similarly to reducing surface resistance, but there are also some differences. Two kinds of protrusions have a higher drag reduction effect for the flowing liquid with a low speed, and the drag reduction rate of two microstructures decreases with the increasing speed of flow water.(2). The drag reduction effect of the negative spherical crown is generally better than the positive spherical crown microstructure.(3). The microstructure of the spherical crown is protruding positive, the surface drag reduction rate of the spherical crown which the crown height +4μm is the lowest. As the height of the spherical crown decreases, the drag reduction rate of the groove surface also increases.(4). The structure of the spherical crown is protruding negative, the surface drag reduction rate of the spherical crown which the crown height −4μm is the highest. As the height of the spherical crown increases, the drag reduction rate of the groove surface also increases. The negative spherical crown structure has good surface drag reduction characteristics, and the highest drag reduction rate can reach 24.8%.


Post-transplant liver fibrosis (PTLF) is a common and severe complication in liver recipients. In this study, we assessed the impact of donor liver genetics on the development of PTLF. A total of 232 patients undergoing liver transplantation were included. Twenty-two single nucleotide polymorphisms (SNPs) associated with liver fibrosis were analyzed. Univariate analysis revealed seven donor SNPs to be associated with PTLF. In a multivariate analysis, independent risk factors of PTLF were genetic variation of donor GRP78 rs430397 , GSTP1 rs1695 , miRNA-196a rs12304647 , and TNF-α rs1800630 ; blood tacrolimus levels at maintenance > 7 ng/ml ; and post-transplant diabetes mellitus . A predictive model that included donor SNPs showed better prognostic ability for PTLF than a model with only clinical parameters . Given that donor gene SNPs are associated with an increased risk of PTLF, this model integrated with donor gene polymorphisms may help clinicians predict PTLF. Liver fibrosis (LF) can result from hepatic virus B (HBV) infection, excess alcohol-based liver disease, non-alcoholic fatty liver disease, autoimmune liver diseases, and hereditary diseases . LF can Liver transplantation (LT) is a therapy for end-stage liver disease. However, HBV recurrence, diabetes, immunosuppressors, and alcohol intake after LT will cause LF again. Post-transplant liver fibrosis (PTLF) can also lead to new liver cirrhosis, which causes further life-threatening complications such as carcinoma or liver failure. Liver grafts also have poor survival due to the progression of liver fibrosis to graft cirrhosis . ClinicaDonor graft genetics play a vital role in LF and can reduce the survival of the graft as well as patient prognosis. Recent studies have shown that single nucleotide polymorphisms (SNPs) involved in liver fibrosis or cirrhosis is associated with the development of LF and may also increase PTLF occurrence. Those studies have identified a number of novel LF-susceptibility genes, including some inflammatory factors. Cytokines play an important role in mediating several immune responses, which are associated with an increased risk of LF. IL-1β is a proinflammatory cytokine and interleukin 1β (IL-1β) rs1143627 has functional importance in IL-1β biological activity . Tumor nOther research studies have also suggested that polymorphisms are responsible for LF. For example, the Cytochrome P4502E1 (CYP2E1) gene plays a key role in dimethylformamide metabolizing pathways. In CYP2E1 rs2031920, the minor T allele has a higher transcriptional and enzyme activity. Thus, this polymorphism may be a risk factor for LF . The vitThe GRP78 pathway is one of the most important responders to disease-associated stress. The glucose-regulated protein 78 (GRP78) rs430397 mutations lead to a variety of reactive oxygen species and cause LF , 18. TheDNA repair mechanisms also play a role in LF development. Genetic variants of the human oxoguanine glycosylase 1 (hOGG1) rs1052133—important enzymes in the BER pathway—reduces DNA repair activity and increases the susceptibility to LF . The X-rLi et al. verified that the programmed cell death-1 (PD1) rs10204525 polymorphisms were associated with altered circulating TNF-α and interferon (IFN) -γ levels in the liver, and caused LF in the Han Chinese . ESR1 rsHowever, the impact of liver allograft genetic polymorphisms on graft fibrosis has not been fully investigated. The aim of this study was to investigate whether the 23 donor SNPs associated with liver fibrosis affects PTLF development.The incidence rate of PTLF was 18.1% (42/232). Clinical characteristic comparisons between the LF and the non-LF groups are shown in The distribution of donor SNPs associated with PTLF is shown in The donor liver graft with GRP78 rs430397, TNF-β rs909253, hOGG1 rs1052133, miRNA-196a rs12304647, and TNF-α rs1800630 polymorphism showed a remarkably higher risk of PTLF. LT patients with donor liver graft GSTP1 rs1695 and HLA-DQ rs2856718 polymorphisms had a reduced risk of PTLF .Clinical values and donor SNPs that showed statistical significance in univariate analysis  were anaThe predictive capacity of the risk factors on PTLF was further assessed by logistic regression analysis. Different predictive models were established by different inclusion criteria. Model 1 only included clinical values while Model 2 contained donor genetic mutations as risk factors. AUROC was used to evaluate the model’s predictive power on PTLF .Model 2 demonstrated better predictive power than Model 1 by using AUROC  containing Japanese samples showed that rs2856718 within the HLA-DQ locus was related to hepatitis B persistence . The C gGSTP1, a member of the GST superfamily, impacts hepatic conditions in patients with cryptogenic cirrhosis because of its expression in the biliary epithelium. Rs1695 has been found in GSTP1 genes, some of which increased the occurrence of liver cirrhosis . This stIn addition, there was crosstalk between the transplanted liver and recipient. PTDM—especially type 2 diabetes mellitus (T2DM)—has been demonstrated to increase LF occurrence . T2DM paThe clinical status, such as blood tacrolimus concentration and PTDM, can injure the liver and lead to LF. We found that genetic mutations of the donor liver rs430397, rs1695, rs12304647, and rs1800630 could also result in LF occurrence. Several studies have shown that gene polymorphisms are involved in increasing the rate of LF occurrence in humans , 32, 33.One limitation of the study is that it was based on a relatively small number of Han Chinese samples, and the majority of individuals had HBV-related liver disease. This research should be validated in a large-sample size study with multiple etiologies of liver diseases, such as hepatitis C virus, NAFLD, and alcohol. Another limitation of this analysis was the diagnosis of LF using liver biopsy, which is the standard method for assessing fibrosis, but is not favored by recipients due its invasive nature. Nonetheless, we are optimistic that new onsets of early warning predictors of fibrosis can be found in the future using non-invasive techniques, allowing precise assessment of fibrosis in individuals after LT.We demonstrated that donor rs430397, rs1695, rs12304647, and rs1800630 polymorphisms are associated with an increase of PTLF and can serve as potential clinical biomarkers for the prediction of LF. For patients with donor livers, those genetic variants, blood tacrolimus concentrations at maintenance, and PTDM may also contribute to PTLF. Furthermore, the integrated model with donor gene polymorphisms can be a potential tool for predicting PTLF.We prospectively enrolled 328 adult patients who underwent LT due to HBV-related diseases. The donor deaths occurred in our center between October 2015 and October 2017. Liver recipients who passed away or had graft failure within two years after LT were excluded. Those with biliary complication-induced fibrosis at the two year check point were also excluded. This resulted in 232 patients that were included in our study and their characteristics are shown in This study was approved by the Ethics Committee of The First Affiliated Hospital of Zhejiang University on the basis of the Regulations on Human Organ Transplant and national legal requirements. This study complies with the guidelines of China’s Ethical Committee and the Helsinki Declaration. All donors and recipients gave informed consent before transplantation. No donor organs were obtained from executed prisoners.The end point of this study was taken two years after LT and assessed graft fibrosis by using the fibrosis-4 index (FIB-4) as well as the aspartate aminotransferase -to-platelet ratio index (APRI) score , 40.Data were collected and analyzed for patient age, gender, body mass index (BMI), blood type, ischemia time, recipient primary disease (HCC or not), comorbidities, immunosuppressive protocol, post-transplant complications, and mortality.9/L] × (ALT [U/L])1/2) [9/L] × 100 [The criterion for PTLF is both an APRI score >1.50 and FIB-4 score >3.25. The FIB-4 values were defined as the formula age (years) × AST [U/l]/(platelets [10/L])1/2) . The APRL] × 100 .Genomic DNA was extracted from freshly frozen donor liver tissues and SNPs were detected using Applied Biosystems SnaPShot technology as mentioned previously . Twenty-https://www.snpstats.net/snpstats/start.htm?q=snpstats/start.htm). A P value < 0.05 was considered statistically significant. The predictive model instruction and risk score calculation are described in our previous research [Quantitative variables are presented as the mean ± SD or median  and were compared using the Student’s t-test or Mann-Whitney test. Categorical variables were presented as values (percentages) and compared using the Chi-square test. Cumulative survival comparison was performed using the Kaplan-Meier method. Risk factors were identified using logistic regression analysis. Variables selected for univariate analysis were entered into a step-by-step multivariate regression model. A P value of < 0.05 was regarded as statistically significant. Data analysis was performed using SPSS version 23.0  and SNP States (research . The areSupplementary Table 1