Cerebrospinal fluid (CSF) leakage is a rare condition. Prompt diagnosis and early treatment of CSF leakage minimizes the risk of severe complications such as bacterial meningitis. Different diagnostic modalities are used to detect the site of CSF leakage but often with unreliable results. The literature offers limited evidence-based guidance on the diagnostic approach for rhinorrhea. Correct localization of the defect is the mainstay for successful surgical treatment. Herein, we describe a case of recurrent meningitis due to cranio-nasal fistula and rhinorrhea successfully localized with radioisotope cisternography (RIC). We provide a detailed and practical overview of the RIC procedure and compare different imaging modalities used to detect the site of CSF leakage. CSF leaA 23-year-old man with a history of cranio-cerebral trauma was admitted to the Department of Infectious Diseases and Travel Medicine for torpid headache, nausea, vomiting, and altered mental status. His symptoms started one day before. He had a history of a previous episode of purulent meningitis (7 years earlier).During the initial evaluation, his vital signs were as follows: blood pressure 150/90 mm Hg, pulse was 80 and regular, oxygen saturation 97% on room air, temperature was 38 °C, and respiratory rate 30. Heart, abdomen, and legs were normal. No skin petechiae were observed.On neurologic examination, he was somnolent, disorientated, had nuchal rigidity, and positive Lasegue and Kernig sign. No cranial nerve abnormalities were present. Lumbar puncture (LP) was performed during the first hour of admission. The macroscopic appearance of the CSF was purulent. White blood cell count: >5000 cells/µL (>99% polymorphonuclears), glucose level: 1.75 mmol/L, chloride level: 120 mmol/L, protein level: 3.86 g/L, lactate level: 10.1 mmol/L . He was administered dexamethasone, mannitol (osmotic diuretic), intravenous fluids, and vitamin C as well. Chest X-ray was negative. Computed tomography (CT) of the brain and paranasal sinuses showed small irregularity in the cribriform plate, over the frontal and ethmoidal sinuses, with a small pathological collection inside right frontal, ethmoidal, and maxillary sinus. No brain edema, hydrocephalus, or abscess formation was observed. Despite being on sedative medication, the patient was restless and agitated, and therefore some CT images were blurry and could not be evaluated accurately. Blood and urine cultures were negative. Nasal and throat swabs were positive for Staphylococcus epidermidis. We also conducted a lumbar puncture to gain CSF for microbiological examination. There was a positivity of latex agglutination for Streptococcus pneumoniae in CSF and also positive CSF culture for the same pathogen with good sensitivity to all tested antibiotics. All relevant laboratory results were measured in whole blood/plasma and are summarized in One day after the initiation of antibiotic treatment, the neurological status improved, and the patient was responsive and able to communicate. There was defervescence of fever. Subsequently, the patient started to complain of rhinorrhea. He told the treating physician that he had had intermittent rhinorrhea for years. Neurosurgical consultation suggested RIC as a method for visualization of assumed cranio-nasal communication.111In-DTPA with 42 MBq (2 mL) was introduced into the spinal subarachnoid space via the LP. For the radioisotope to be fully introduced and dispersed into the subarachnoid space, without leaving any residues inside the LP needle and to counteract the backwash through the LP needle, we had to institute additional 3 mL of normo-saline. The whole procedure had to be performed with the help of additional tubing, otherwise the radioisotope would leak out after we disconnected the syringe from the LP needle (At the end of the antibiotic treatment (14th day), in cooperation with the Institute of Nuclear and Molecular Medicine, we decided to perform RIC. The radioisotope P needle . AfterwaEarly scanning (5–6 h) showed symmetrical activity in the ventricular system. There was no activity in the liver, which confirmed correct application of the radioisotope. Sagittal images showed slight atypical activity caudally of the cribriform plate. Except physiological distribution, delayed scanning (>24 h) revealed the focus of an increased activity over the cribriform plate and extremely intense activity extracranially inside right paranasal cavity. Planar images of the abdominal area showed activity in the spinal canal and swallowed activity in the stomach and intestines, which also confirmed liquorrhea. Single photon emission computed tomography (SPECT) scanning clearly confirmed activity over the internal lamina of the frontal bone, as well as over the anterior part of cribriform plate (two defects). There was a posttraumatic discontinuity of the skull base  and a second defect in the anterior part of the cribriform plate. The first signs of the isotope leakage were evident at 6 h post-application. After 24 h, the activity was evident in communicating fistula and inside the right maxillary sinus . FinallyThe patient was then transferred to the Department of Neurosurgery. Following a successful localization of the fistula, the surgeon performed a reconstruction of the anterior cranial fossa floor through right frontal craniotomy. The patient was discharged 12 days after the surgery and has had no recurrence of rhinorrhea since.S. pneumoniae and H. influenzae, and the outcome of these episodes is generally favorable [Bacterial meningitis in patients with CSF leakage can be classified as community acquired, in the case of anatomic defects or contiguous spread of infection, or nosocomial meningitis after surgery for trauma. In patients with rhinorrhea, the diagnosis should first be confirmed by measurement of beta-2-transferrin levels. This protein is specific for CSF, and a small amount of rhinal discharge 0.4 mL) is adequate for the evaluation. During the evaluation of our patient, this laboratory measurement was not conducted because of the clinical picture with suggestive history of recurrent meningitis and actual large volume rhinorrhea. The most common causative pathogens are  mL is adavorable . The parMany imaging methods, including cranial CT, MRI, MR cisternography (MRC), contrast-enhanced MRI (CE-MRI), or RIC, are used to detect the location of CSF leakage, all with their advantages and limitations. HRCT can indicate the leak localization by detecting the bone defect. However, CSF leakage may not be present at the congenital or traumatic defect localization . In our 131I, diethylenetriamine pentaacetic acid (DTPA), 111In-DTPA, 99Tc human serum albumin, and 99Tc pertechnetate can be used. 99Tc-DTPA has been suggested as a better brain scanning agent than 99Tc pertechnetate because its renal clearance from the blood stream is more rapid, presumably resulting in better lesion-to-background ratio at earlier times after injection, and there is no interference from choroid plexus and salivary gland concentration. Clinically, marked reduction in the time delay between injection and imaging to 1 h or less has been advocated because such early studies with DTPA were felt to be at least as reliable as pertechnetate images 2 or more hours after injection [111In over 99Tc because of longer half-life (67 h vs. 6 h), which is advantageous in visualization of intermittent leaks [111In has been rare [111In RIC is 5 mSv at most, while radiation from contrast enhanced CT cisternography may expose the patient to up to eight times more [99Tc-DTPA has been suggested as a better brain scanning agent than 99Tc pertechnetate because its renal clearance from the blood stream is more rapid, presumably resulting in better lesion-to-background ratio at earlier times after injection, and there is no interference from choroid plexus and salivary gland concentration. Clinically, marked reduction in the time delay between injection and imaging to 1 h or less has been advocated because such early studies with DTPA were felt to be at least as reliable as pertechnetate images 2 or more hours after injection [RIC is an invasive study, but LP as a method for instituting the radioactive tracer is a simple and safe procedure. This method is mainly used in cases where there is still diagnostic uncertainty. The cephalad ascent of the radioisotope is not affected by activity or by posture  after the injection; therefore, the patient has no limitation in his activities . Different leaks . Moreovemes more . 99Tc-DTnjection .Localization of the leak to the right or left nasal cavity may be sometimes difficult because of the tendency of the fluid to cross sides and flow from both nostrils. In a study of four patients who underwent RIC, as well as MRI and/or CT for suspected CSF leaks, Thomas et al. found that RIC accurately detected and localized the leaks in all patients . The traAs with all imaging methods, there are some issues with RIC that need to be considered. After an LP, commonly a small amount of CSF may leak from the puncture site. This is called a backwash phenomenon. If the amount of the backwash were large, stronger lumbar epidural radioactivity might be seen in the cisternographic images and could lead to early appearance of radioactivity in the urinary bladder . MultiplIn the spectrum of neurodiagnostic tests in evaluation of the patients with spontaneous CSF leaks, RIC has its own particular indication and is not necessarily a substitute or competitor to other imaging techniques. 111In-DTPA is the preferred radioisotope of choice for its longer half-life and the ability to detect intermittent leaks. Timely detection of anatomical defect thus allows for targeted surgical treatment.For an infectious disease specialist or neurologist, recurrent meningitis should be a sign of possible CSF leakage and should lead to prompt imaging to determine the site of skull-based CSF leak. Although vaccination prevents meningitis, some cases may be caused by streptococcal serotypes not included in the vaccine, as documented in our case. There are many different imaging modalities, each with its advantages and caveats. This case report demonstrates the significance and the practical approach to RIC with SPECT in cases of rhinorrhea, being a method for definitive visualization of cranio-nasal communication.


Chemotherapy-induced febrile neutropenia (FN) is a life-threatening and chemotherapy dose-limiting adverse event. FN can be prevented with granulocyte-colony stimulating factors (G-CSFs). Guidelines recommend primary G-CSF use for patients receiving either high (> 20%) FN risk (HR) chemotherapy, or intermediate (10–20%) FN risk (IR) chemotherapy if the overall risk with additional patient-related risk factors exceeds 20%. In this study, we applied an EHR text-mining tool for real-world G-CSF treatment evaluation in breast cancer patients.Breast cancer patients receiving IR or HR chemotherapy treatments between January 2015 and February 2021 at LUMC, the Netherlands, were included. We retrospectively collected data from EHR with a text-mining tool and assessed G-CSF use, risk factors, and the FN and neutropenia (grades 3–4) and incidence.A total of 190 female patients were included, who received 77 HR and 113 IR treatments. In 88.3% of the HR regimens, G-CSF was administered; 7.3% of these patients developed FN vs. 33.3% without G-CSF. Although most IR regimen patients had ≥ 2 risk factors, only 4% received G-CSF, of which none developed neutropenia. However, without G-CSF, 11.9% developed FN and 31.2% severe neutropenia.Our text-mining study shows high G-CSF use among HR regimen patients, and low use among IR regimen patients, although most had ≥ 2 risk factors. Therefore, current practice is not completely in accordance with the guidelines. This shows the need for increased awareness and clarity regarding risk factors. Also, text-mining can effectively be implemented for the evaluation of patient care.The online version contains supplementary material available at 10.1007/s00520-022-07343-5. Quantifying healthcare outcomes in clinical practice is a crucial step towards the improvement of cancer patients’ care . The eleFor years, granulocyte colony-stimulating factors (G-CSF) are used to prevent chemotherapy-induced neutropenia in breast cancer patients . NeutropProphylactic use of granulocyte colony-stimulating factors (G-CSF), e.g., filgrastim, has shown to reduce the severity and duration of neutropenia and the incidence of FN by 50–90% , 14, 15.FN incidence is primarily related to the type and intensity of the chemotherapy regimen. Therefore, European Organisation for Research and Treatment of Cancer (EORTC) and National Comprehensive Cancer Network (NCCN) guidelines indicate that primary prevention of FN with G-CSF (PP G-CSF) should be applied if patients receive treatment with a high-risk (HR) > 20%) for FN. For patients receiving a chemotherapy regimen with an intermediate FN risk (IR) of 10–20%, other patient-related risk factors should be considered to define if the overall FN risk exceeds 20%, and thus, PP G-CSF is indicated , 20. Alt% for FN.Even though several guidelines on G-CSF use for clinical practice are present, it is not clear to what extend these are followed. Therefore, the aim of this study is to retrospectively review guideline adherence for HR and IR chemotherapy regimens in a breast cancer population by collecting data from the EHR with text mining.All patients aged 18 years and older with breast cancer were included if they started a HR or IR chemotherapy regimen between January 2015 and February 2021 at Leiden University Medical Center (LUMC), Leiden, the Netherlands. Patients participating in the DIRECT study were excluded, since these patients by study design were not allowed to receive PP G-CSF treatment and therefore might bias results . The stuWe performed the data collection from the EHR with rule-based text-mining software . This tool enables extraction of structured  and unstructured (free-text notes) data and the immediate conversion of results into a dataset. For unstructured notes, it enables to search for (combinations of) keywords and shows all notes matching with these results. We validated the software previously for data extraction to evaluate metastatic renal cell carcinoma treatments, which showed high accuracy (> 90%) for data collection from structured data . All use2), hemoglobin (RF: hemoglobin < 12 g/dl), ALT , AST , eGFR , absolute neutrophil count : age (RF: age > 65 years), sex , length and weight (RF: body surface area < 2.0 m9 cells/L), serum We did not include the following risk factors: prior episodes of FN, since we estimated the risk prior to cycle one; antibiotic prophylaxis, since patients in The Netherlands do not receive antibiotic prophylaxis; and cardiovascular disease, one or more comorbidities, and prior infections, since the high variability in free-text terminology combined with the uncertainty whether these risk factors are noted structurally in the EHR would result in an incomplete, and potentially incorrect risk estimation.9 cells/L, or ANC < 1.0 × 109 cells/L, predicted to fall below 0.5 × 109 cells/L within 48 h, with fever or clinical signs of sepsis. Fever is defined as rise in axillary temperature > 38.5 °C for 1 h [9 cells/L (Common Terminology Criteria for Adverse Events v.5.0). We included cases of FN and neutropenia if they met the definition based on structured data, or when noted in unstructured text by a treating physician.The primary outcome was the incidence of FN from chemotherapy initiation until 21 days after last treatment cycle. FN is defined as the ANC < 0.5 × 10t-test, the number of risk factors between the subgroups was compared. Also, chi-square tests or Fisher’s exact test if expected frequency was lower than five, was performed to compare risk factors between subgroups.Data management and analysis was performed using R 4.1.0 . Descriptive statistics were used to describe patient, treatment, and disease characteristics. The number of patients on HR or IR chemotherapy regimen receiving PP G-CSF that developed neutropenia or FN was summarized in percentages and visualized in a Sankey plot. With the Student’s In total, 190 breast cancer patients, which received an IR or HR regimen between January 2015 and February 2021, were included. All patients were female and had a median age of 52.6 years (± 11.4 years). Baseline patient, tumor, and treatment characteristics are shown in Table Table Table n = 4) had a mean of 5 risk factors, and without PP G-CSF (n = 109) 3.3 (p = 0.13). No significant difference was found between the distribution of individual risk factors between both groups . Furthermore, the risk factor BSA below 2 m2 was more prevalent in the group who did not develop FN  developed FN Table . Figure In this study, we investigated whether EHR text mining could be applied to evaluate G-CSF use among breast cancer patients in clinical practice. The high recall rate both and the results of the within-program manual validation indicate that adherence to guidelines can be performed using EHR text mining. We found that, in general, G-CSFs were not as often administered as primary prophylaxis as indicated in the EORTC guideline. Not all patients (8.8%) who received HR regimens started with G-CSF prophylaxis. Also, even though almost all patients with IR regimens had two or more of the investigated risk factors, only 4% received PP G-CSF. This resulted in an overall neutropenia incidence of 32%, including 11% FN. Our results indicate that prophylactic treatment with G-CSF should be optimized to further prevent the occurrence of (febrile) neutropenia.Despite guidelines indicating 100% of the patients on a HR regimen should receive PP G-CSF, we report a PP G-CSF use of 88%. An undertreatment of the HR population was also found by Gawade et al., who reported 76.4% PP G-CSF use . Also, PAlmost none (4%) of the patients treated with an IR chemotherapy received PP G-CSF. As PP G-CSF should only be administered if the cumulative FN risk per individual patient exceeds 20%, the risk estimation of patient receiving an IR treatment should be based on additional risk factors . The lowNone of the investigated risk factors was significantly related to PP G-CSF prescriptions in our patients. It is remarkable that 20 patients (18%) aged > 65 years did not receive PP G-CSF, since this is the most prominent mentioned risk factor in the guidelines . HoweverGawade et al., who compared mainly comorbidities to PP G-CSF initiation by retrospectively reviewing a large medical claims database, also showed that risk factors seemed not to influence PP G-CSF initiation in the IR group, although this was suggested in the NCCN guidelines . FurtherMoreover, G-CSFs were known to be costly and as a consequence, the choice for treatment above > 20% FN risk only was highly related with the cost-effectiveness . However. However, not all potential risk factors could (fully) be assessed retrospectively. Partially, as the EHR is a secondary source and not all patient data are equally well documented in the EHR, e.g., in this study, we could only report the performance status for approximately 60% of the patients. But also, to some extent, because definitions were unclear or too broad, e.g., one or more comorbidities. We still performed manual validation of the critical end-points G-CSF use and FN incidence after text mining, since these were mainly documented in unstructured text. Since only a selection of the data had to be validated, this process was faster compared to complete manual data extraction. Our study shows that a text-mining tool can be an effective method to review adherence to guidelines and that results can be used as a concrete starting point to optimize patient care.In this study we applied EHR text-mining software. This enabled fast, structured, and pseudonymized patient inclusion and data extraction. Using this method, missing data of the included risk factors, in general, was limitedBy application of text mining to the EHR, we were able to review G-CSF use in daily practice in breast cancer patients. PP G-CSF use among HR regimen patients was high, however not maximal, and undertreatment resulted in a higher incidence of FN. Most IR regimen patients had more than two risk factors and were therefore entitled to the use of PP G-CSF. However, few received PP G-CSF which could have prevented the occurrence of FN and neutropenia. Therefore, current practice is not completely in accordance with the guidelines, in particular for patients treated with IR regimens, and may result in unnecessary toxicity for patients. We conclude that awareness of risk factors related with neutropenia should be enlarged and these risk factors could be more clearly defined in the guidelines. Finally, our study shows that text-mining methods can be effectively implemented to review daily practice for the evaluation and improvement of patient care.Supplementary file1 (XLSX 22 KB) Supplementary file 1: Queries for patient selection and data extraction in CTcueBelow is the link to the electronic supplementary material.


Five subjects (1.5%) presented CSF biomarkers that were compatible with AD: one was diagnosed with AD, whereas the others showed HIV encephalitis, multiple sclerosis, cryptococcal meningitis, and neurotoxoplasmosis. Regardless of confounders, 79.6% of study participants presented normal CSF AD biomarkers. Isolated abnormalities in CSF beta amyloid 1–42 (7.9%) and tau (10.9%) were associated with age, biomarkers of intrathecal injury, and inflammation, although no HIV-specific feature was associated with abnormal CSF patterns. CSF levels of AD biomarkers very poorly overlapped between HIV-positive clinical categories and AD controls. Despite the correlations with neurocognitive performance, the inter-relationship between amyloid and tau proteins in PLWH seem to differ from that observed in AD subjects; the main driver of the isolated increase in tau seems represented by non-specific CNS inflammation, whereas the mechanisms underlying isolated amyloid consumption remain unclear.People living with HIV (PLWH) age with an excess burden of comorbidities that may increase the incidence of age-related complications. There is controversy surrounding the hypothesis that HIV can accelerate neurodegeneration and Alzheimer’s dementia (AD). We performed a retrospective study to analyze the distribution of cerebrospinal fluid (CSF) AD biomarkers  in adult PLWH (on cART with undetectable viremia,  With the increasing life expectancy of people living with HIV (PLWH), the diagnosis and management of co-morbidities is crucial in HIV care; indeed, health-adjusted life expectancy is shorter in females and males living with HIV ,2. In coDespite the fact that, just few decades ago, HIV-associated dementia was a common, severe diagnosis in AIDS patients, nowadays, age-related neurodegenerative disorders in ageing PLWH may be as common as in the general population or, as some evidence suggests, they may be even more prevalent ,6,7. ForIn line with the recent hypothesis of an infectious trigger in AD pathogenesis , in vitrHerpesviridae , and the antimicrobial role of amyloid beta against viruses, bacteria, and fungi invading the central nervous system (CNS) has been recently described ) and ptau (81.5 pg/mL [67.5–103.4]) were extremely higher compared to the three HIV-positive groups (p < 0.001 for both), whereas BA42 was significantly lower  were male and the median age was higher than the HIV-positive counterpart (70 years (60–76), p < 0.0005), and not with BA42 , nor age . CSF ptau and BA42 showed a very mild correlation with each other  and none with age . Interestingly, age did not correlate with any of the AD biomarker CSF levels in the HIV-negative control group, and only CSF tau levels mildly correlated with age in the whole HIV+ study population , two cases had no neurological/neurocognitive issue , and the last had asymptomatic CSF escape.Half of the four cases with CSF pattern C were related to CSF escape (one symptomatic and one asymptomatic), whereas the other half was detected in neurocognitive impaired subjects (one symptomatic and one asymptomatic).p = 0.048), and no other demographic, clinical, or viro-immunological parameter was significantly associated with CSF pattern B in this clinical group  and CSF cell count  were the only factors associated with increased odds of detecting a CSF pattern C among undetectable subjects during univariate analysis, but no factor was independently associated with isolated increased tau during multivariate analysis in this clinical group , BA42 , CSAR , age , and proteins , CSAR , age . BA42 correlated with CSAR  and CSF proteins only , Frontal assessment battery , Digit symbol , and Grooved Pegboard for dominant  and non-dominant hand , as shown in p = 0.022), and Mini-Addenbrooke’s Cognitive Examination test . Similarly, CSF ptau correlated with semantic verbal fluency . BA42 weakly correlated with the adjusted score of Trail Making test part A only  patients underwent neurocognitive assessment. CSF tau correlated with age-, sex- and education-adjusted scores during the Free and Cued selective reminding test delayed part .Of the 14 subjects with isolated increased tau, one had primary HIV infection, four had neurological complaints in the presence of a high CSF HIV-RNA (>100.000 cp/mL) but no CSF escape, six had no neurocognitive/neurological issue (three were in the presence of a high CSF HIV-RNA), and three had neurocognitive impairment (one asymptomatic).p = 0.001), CSF neopterin , as Tourtelotte index increased , and as CSF glucose decreased . With the multivariate model, which also included CSF proteins , increased age  and CSF glucose  were independently associated with increased and decreased odds of detecting CSF pattern B, respectively , Tourtelotte index , CSF cells  and proteins  were all associated with increased odds of detecting an isolated high tau; at multivariate, only CSF neopterin was independently associated with the odds of finding CSF pattern C , ptau , neopterin , cells  and proteins  and Tourtelotte index  and IHDS  and the immediate copy of the Rey–Osterrieth complex figure , phonemic verbal fluency , and Grooved Pegboard for non-dominant hand  underwent neurocognitive assessment. CSF tau correlated with the adjusted score of the Free and Cued Selective reminding test delayed part  and males . Using multivariate analysis, including the HIV acquisition route  and CSAR , no parameter independently predicted differential odds of detecting CSF pattern B in this clinical group (p = 0.007) and IgG intrathecal synthesis , but no variable confirmed an independent association with the odds of an isolated increased tau in patients with CNS confounding , but not with BA42, and with current CD4+ T-cells count , CSF HIV-RNA , neopterin , length of HIV infection , and length of viral suppression . Instead, CSF ptau did correlate with BA42 , as well as with the length of viral suppression . BA42 did not correlate with any assessed variable, but for ptau. None of the three CSF AD biomarkers correlated with age.CSF tau correlated with ptau  underwent neurocognitive evaluation. None of the three CSF biomarkers correlated with any score of the 16 tests or with the two screenings administered  presented a CSF biomarker profile that was compatible with, and typical of AD; out of those five patients, 1 patient had undetectable viremia, another had detectable viremia, and 3 patients had CNS confounding. Only the first was diagnosed with AD in 2018 and in depth previously described [The 41 year-old man with detectable viremia suffered from HIV encephalitis with acute clinical onset in the advanced stages of HIV infection . The brain MRI showed diffuse and symmetrical T2-weighted hyperintensities within the white matter of centrum semiovale, corona radiata, internal capsula and the brain stem. After the introduction of cART, he ameliorated, showing normalization of imaging, symptoms, CSF BA42, and reduction of tau: 14 months after the acute episode, CSF BA42 moved from 365.0 to 1029.0, tau from 811.0 to 515.0 pg/mL, and CSF HIV-RNA from 375,412 to 227 cp/mL.The last three subjects suffered from the first flare of new-onset multiple sclerosis , cryptococcal meningitis  and neurotoxoplasmosis , respectively. All of them observed a normalization of CSF biomarkers after the introduction of the respective specific treatments at a mean follow-up of 3 months.Therefore, the overall prevalence of actual AD among our cohort was 0.3%.Regardless of neurocognitive impairment, antiretrovirals, viral replication, immunological status, or CNS confounding, most of the study participants presented normal CSF AD biomarkers . No single HIV-related variable was associated with abnormal CSF biomarker patterns. More importantly, CSF median levels of AD biomarkers very poorly overlapped between HIV-positive clinical categories and HIV-negative AD controls, suggesting a reliable power of discrimination between HIV-related neurological processes and AD.Despite several hypothetical parallelism between AD pathogenesis and HIV-related CNS phenomena, such as the role of chronic inflammation within CNS which was observed in both disorders ,34,35, oIsolated low BA42, which was uncommonly found in the overall population 7.9%), as in each clinical category, did not differ according to CSF viral replication, cART administration nor the presence of other neurological infections or disorders. After excluding neurological confounding, we observed an association between isolated low BA42 and increasing age in both detectable and undetectable patients. This may primarily reflect physiological changes in BA metabolism with ageing, but also the fact that age can behave as a proxy of the length of continuous exposure of the brain to HIV proteins. Molecular mechanisms triggering amyloid deposition and their associated toxicity are not yet completely clarified, but over the last decades, emerging evidence highlighted that AB plays a role as innate immune protein that protects against fungal, bacterial, and viral infections %, as in . As for The main limitation in assessing CSF BA42 is represented by the cross-sectional design. Longitudinal studies could be more accurate in deciphering at which point the subject is at with regard to the hypothetical evolution of BA42 levels in response to HIV infection, as it is likely that, after an infectious trigger, CSF concentrations of BA42 change over time, increasing and decreasing according to the type, amount, and duration of the trigger itself. In potential agreement with this, the association between normal/higher CSF glucose levels and lower odds of presenting isolated low BA42 in subjects with the replicating virus may identify in CSF glucose a more sensitive and long-lasting representative marker of intrathecal chronic and subtle inflammatory processes affecting BA metabolism . On theA mild contribution in increased total tau and ptau was given by CNS inflammatory infectious or non-infectious processes (10.9%). Indeed, we observed an increasing prevalence of isolated high CSF tau in the three clinical groups, with the highest being in those presenting CNS confounding conditions. Both in detectable and undetectable patients, an isolated increase in CSF tau (and ptau) was associated with common and aspecific markers of intrathecal inflammation: CSF cells and proteins, neopterin, and Tourtelotte index. Once again, the observed infrequent alterations in markers of tauopathies highlight aspecific mechanisms underlying the increase in tau and ptau, rather than HIV-related processes. In line with this finding, being markers of axonal injury, tau and ptau have been found to be elevated in several CNS infections during the acute flare ,45,46. EStrengths of this study are represented by the largest sample size and the most extensive assessment  compared with previous studies on CSF AD biomarkers in PLWH, by the novelty of grouping patients according to CSF patterns rather than analyzing single biomarkers, by the presence of a negative control group for AD, as well as by the length of HIV infection in the cohort (median 8 years), that enhanced the likelihood to detect chronic and subacute processes, if any, despite the cross-sectional design. Lastly, compared to previous smaller studies on AD biomarkers in PLWH, our cohort enrolled subjects that better represent the current prototypes of patients in care.As with other limitations, we performed a retrospective snapshot without the capacity to describe or predict the eventual trajectory of the CSF AD biomarker levels of PLWH at the age of HIV-negative AD controls, which was indeed compatible with AD’s epidemiology, and therefore, about 20 years older than in our HIV-positive participants; this remains one of the main issues for directly comparing and translating AD biomarker levels and cut-off in PLWH. Furthermore, to date, findings describing CSF biomarkers of AD in PLWH should be considered of limited potential for a direct translation into routine clinical practice as the immunoassays adopted for measuring BA and tau proteins have never undergone a laboratory validation in the HIV-positive population.In conclusion, clustering PLWH according to canonical patterns of CSF AD biomarkers did not detect any HIV-specific association; the inter-relationship between amyloid and tau proteins seem to differ from that observed in AD subjects and although the main driver of alterations in CSF tau proteins seem represented by non-specific inflammation within the CNS, the mechanisms underlying the rare isolated BA42 consumption remain unclear. Longitudinal studies are warranted to better depict long-term effects of HIV infection as well as of antiretroviral therapy on CNS amyloid and tau metabolism.


En bloc pediatric kidney (EBPK) allografts are a potential solution to expand the organ donor pool; however, EBPK transplantation has been traditionally considered suboptimal due to concerns of perioperative vascular and urologic complications. Accidental organ or vasculature injury during harvest is not uncommon; however, this does not necessarily mean that the organ should be discarded. Careful vascular reconstruction can be performed using donor vascular grafts, salvaging the organ without stenosis or thrombosis of the vessels. We report an extensive vascular reconstruction of the right renal artery, aorta, and inferior vena cava of a damaged EBPK allograft using a donor pediatric aorta vascular patch with the goal of avoiding postoperative vascular complications. Renal transplantation remains the best treatment option for patients with end-stage renal disease (ESRD). However, few ESRD patients on the waiting list receive a kidney transplant due to the wide gap between potential candidates and organ donors . En blocTransplantation of small pediatric kidney allografts  has been implemented more frequently over the years, but the increased risk of postoperative vascular and urologic complications, delayed graft function (DGF), or even primary graft nonfunction has made their utilization challenging in both pediatric and adult recipients –4. AdditWe report the transplantation of an inadvertently injured EBPK in an adult recipient after extensive vascular reconstruction in the back-table operation. There was a damaged donor right renal artery reconstructed using a donor aortic vascular patch from the same pediatric donor.The donor was a 13-month-old male deceased infant who died of anoxia secondary to unwitnessed cardiac arrest. His weight was 12.7 kg, with a terminal creatinine (Cr) level of 0.24 mg/dL and KDPI of 60%. The right and left kidneys both measured 5 cm long and 4 cm wide. During the kidney retrieval, the aorta and inferior vena cava (IVC) were divided cranially at the level of the takeoffs of both renal arteries and veins with damage to the takeoff of the right renal artery at its ostium . Since aThe recipient was a 28-year-old male who weighed 74 kg. He was on hemodialysis for 32 months. The recipient's external iliac vessels were dissected free, and the EBPK allograft was removed from the pulsatile perfusion machine and kept on ice.The superior cap used for the aorta was fashioned to cover the right renal artery injury at the takeoff, preventing stenosis of the ostium. No increased velocities were identified on postoperative Doppler ultrasound. The vascular reconstruction was performed using the donor's own vessel grafts procured during multiorgan procurement that were not used for the primary transplants of other abdominal organs as the liver. These vessels are preserved in conservation solutions, flushed with heparin, and kept preserved at a temperature of 4°C.The aorta and IVC were anastomosed end-to-side to the recipient's right external iliac vessels in a tension-free manner using running 6-0 Prolene® sutures. Finally, ureteroplasty was accomplished by side-to-side anastomosis of both distal ureters with 6-0 PDS interrupted suture and the Miami Transplant Institute extravesical ureteroneocystostomy with ureteral stent-free technique was performed onto the recipient bladder dome using 6-0 PDS interrupted sutures .Z-velocity) in both the external iliac and graft arteries were also recorded. The patient had an uneventful recovery showing a Cr level of 1.20 mg/dL at a 3-month follow-up.The warm ischemia time was 19 minutes, and the cold ischemia time was 1420 minutes. Surgical drainage or ureteral stent were not used. Postoperative Doppler ultrasound showed no collections in the perinephric space or signs of obstructive uropathy. Laminar blood flow and normal parameters (i.e., resistive index, peak systolic velocity and ratio, and Since the first pediatric donor kidney transplantation in 1964, several advancements in surgical techniques, immunosuppression, and organ preservation technology have permitted the use of pediatric kidneys . Recent There are several concerns in using EBPK, one of them is the increased risk of primary graft nonfunction, which could be secondary to a hyperfiltration injury. According to this theory described by Brenner , an adulAnother significant concern is related to vascular complications, Fananapazir et al.  found a In order to reduce the risk of these vascular complications, we describe the use of cap-shaped patches from the same donor as a conduit in both proximal ends of the allograft aorta and IVC and at the distal allograft vascular end, providing increased length to reduce excessive traction and subsequent thrombosis and/or stenosis , 17. TheIn regard to urologic complications, a retrospective study by Fananapazir et al.  demonstrThe continuous shortage of organ offers and the increase in the kidney transplant waiting list demand for the more aggressive use of extended criteria donors including EBPK , 19. Gro


Studies have suggested that a more liberal criterion of donor–recipient weight ratio (DRWR) is associated with superior waitlist survival without compromising posttransplant outcomes in selected critically ill patients. Successful transplantation of an extremely oversized donor heart into a small recipient is herein described. A 2-year-old girl accepted a size-mismatched adult donor heart offer (DRWR of 4.4) due to frequent complications with a left ventricular assist device. During the immediate postoperative period, spatial constraints within the thoracic cavity compromised graft function. Computed tomography revealed severe compression of the left bronchus due to the oversized allograft with lobar collapse of the left lung. With temporary extracorporeal membrane oxygenation support, graft function improved within 1 month after transplantation. Subsequent adaptive size remodeling of the transplanted heart with concomitant left bronchus re-expansion was observed within 6 months after transplantation. Despite a complicated posttransplant recovery, the patient was discharged home with minimal respiratory sequelae. Our report describes an alternative strategy for managing early morbidities related to an oversized graft and supports extending the criteria of size matching in pediatric heart transplantations. Case report; Heart transplant; Pediatric; Size mismatch. Accepti210.13039/100000738VA ECMO) support for suspected primary graft dysfunction (PGD) and left bronchus compression.A 2-year-old girl with dilated cardiomyopathy received an urgent paracorporeal continuous-flow LVAD implantation in INTERMACS  profile 1. Organ recovery following VAD implantation was encouraging, but her ambulatory rehabilitation was complicated by repeated episodes of embolic stroke due to pump thrombosis, which necessitated frequent pump exchange. Accordingly, we accepted an offer from a size-mismatched donor. The donor was a 36-year-old female victim of a traffic accident. The DRWR was 4.4 (53 kg–12 kg) and the donor-recipient height ratio was 2.0 (166 cm–82 cm). The maximal transverse diameter of the donor heart was 11.2 cm, and the maximal transverse diameter of the left hemithorax in the recipient was 9.4 cm, as measured using computed tomography (CT). Before implantation, the bilateral pleura were opened widely to accommodate the oversized allograft within the chest. The biatrial anastomosis technique was used. The donor's aorta was anastomosed to the lesser curvature of the aortic arch of the recipient. After operation, the recipient was placed on central venoarterial extracorporeal membrane oxygenation (2 retention was frequently noted while weaning from VA ECMO. CT revealed the heart deviated the tracheobronchial tree to the right, compressed left bronchus, and occupied almost the whole left hemithorax (In the first week after transplantation, bradycardia following COmithorax . Sternal310.13039/100004811CT had clearly demonstrated the process of adaptive size remodeling of an extremely oversized donor heart, with the associated resolution of airway compression within 6 months after transplantation, which was rarely published in the field of pediatric heart transplant (Although evidence suggests that oversized cardiac allografts undergo size adaptation to accommodate the reduced requirements in small recipients within a few months after transplantation, a disproportionately large graft might lead to “big heart” hyperperfusion syndrome and compression of intrathoracic organs . In our ansplant . Posttra4Compared to the well-recognized risks of PGD from undersized donor hearts, the association between PGD and oversized donor hearts remains elusive , 7. In t5In pediatric heart transplantation, the so-called “big heart” or hyperperfusion syndrome caused by a disproportionately large donor heart is a well-recognized short-term complication of oversized grafts . A vigor6The spatial constraints in small children could increase the risk of compression of other intrathoracic organs by oversized donor hearts. Recipients of hearts from donors of significantly larger size experienced more transient pulmonary atelectasis than recipients of hearts from more closely size-matched donors. However, studies have reported that clinical impairment of lung function is less likely to occur as a result of an oversized donor heart . Electiv7We presented a successful transplant of an extremely oversized donor heart into a small child following initial struggles with PGD and left bronchus compression. Remodeling of the oversized donor heart was clearly demonstrated by serial CT. Our report described an alternative strategy for managing early morbidities from oversized grafts and supports extending the criteria of size matching in pediatric heart transplants.All authors listed have significantly contributed to the investigation, development and writing of this article.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.Data will be made available on request.The authors declare no conflict of interest.No additional information is available for this paper.


Leptomeningeal metastasis (LM) refers to the dissemination of malignant cells in the subarachnoid space, pia, and arachnoid mater and is a severe condition associated with metastatic solid tumors. The most common solid tumor that develops into LM is lung cancer and the incidence increased in patients with advanced non‐small‐cell lung cancer (NSCLC) with targetable mutations. However, tissue biopsy of LM is inaccessible, leading to the paucity of genomic profiles of LM to guide targeted treatments and explore biological mechanisms. In recent years, liquid biopsy is considered a minimally invasive and dynamic method to trace the genomic alterations of cancer cells and some studies started to perform sequencing of cerebrospinal fluid (CSF) in patients with LM to reveal the targeted mutations and genomic profiles. In this review, we focused on studies performed sequencing of CSF in NSCLC patients with LM and summarized the sequencing results and their commonality. As the only way to reveal the genomic landscapes of LM, our review provided evidence that sequencing of CSF is a promising management method in LM patients to dynamically guide target therapy and monitor intracranial tumor response. Furthermore, it reveals a unique genomic profile of LM including driver genes, drug‐resistant mutations, and a number of copy number variations. Sequencing of CSF in LM patients seems to provide more comprehensive genomic information than we expected and the biological significance behind the genomic alternations needs further study. The frequency of CNVs and alternations of the reported genes. Information regarding the following characteristics was extracted: author, year, institution, topic, number of patients, treatment history of the tyrosine kinase inhibitors (TKI), sequencing platform and method, sequencing sample, paired plasma, and paired tissue. Alternated genes were analyzed per study and on a per patient basis. Alternations, including single nucleotide variations (SNVs), insertion–deletion mutations, frameshift mutations, and copy number variations (CNVs) of the reported genes, were obtained. When the mutations were not included in the above types, the data were excluded. The results of multiple sampling at the same time and different sequencing samples from one patient were considered as a whole, which were analyzed together to represent the genomic alterations of the patient. The detection rates and the concordance rates were recorded if they were reported in the original studies. If the rates were not reported explicitly, they were presented in the form of a ratio through manual extraction. We compared the alternated genes with the list of Cancer Gene Census (CGC) and pan‐lung cancer data in The Cancer Genome Atlas (TCGA) database. CGC is an ongoing effort to catalog alternated genes that have been causally implicated in cancer and explain how the dysfunction of these genes drives cancer. The data can be obtained from the websites directly. Representative cases of CSF sequencing in guiding treatment were also summarized.2.3Only descriptive statistics were used. The frequency was defined as the number of patients with detected mutations divided by the total number of patients.33.1EGFR) mutations and the otherEGFR subtypes, so the information of the two studies was merged and duplicated patients were removed. Patients with other types of primary tumors were also reported in three studiesEGFR mutations in the primary tumor and one study included patients with ALK receptor tyrosine kinase (ALK) fusions. The history of TKI treatments was illustrated in 238 patients and 192 of them were TKI exposed. Three hundred and twenty‐two patients had sequencing results from paired plasma, and 212 patients had sequencing results from paired biopsy tissues. Eight studies performed sequencing through different cancer‐related gene panels, and nine studies sequenced only predesignated genes including EGFR, KRAS proto‐oncogene (KRAS), B‐Raf proto‐oncogene (BRAF), erb‐b2 receptor tyrosine kinase 2 (ERBB2), and MET proto‐oncogene (MET). Cell‐free DNA (cfDNA) in CSF was sequenced in 14 studies, cells in CSF were sequenced in four studies, and circulating tumor cells (CTC) in CSF were sequenced in three studies. The next‐generation sequencing (NGS) and the droplet digital PCR (ddPCR) were the most commonly used methods and were used in 10 and five studies, respectively. Most studies used various Illumina platforms for NGS and BioRad QX20TM system for ddPCR  Table . Four st3.33.3.1EGFR mutations. Fourteen studies reported subtypes of EGFR mutations. The most frequent subtypes were classical sensitive exon21 L858R and exon19 del mutations, which occurred in 158/377 and 137/351 patients, and accounted for 90% (288/320) of all the activating EGFR mutations, which was similar to the prevalence in NSCLC.A total of 525 patients in 15 studies performed sequencing of CSF and 377 of them had detected The most common secondary mutation T790M was detected in 42/377 patients. Of the 42 patients, the treatment history of 34 patients were known, and 67% (23/34) patients had received TKI treatment. C797S mutations were detected in seven patients and all of them were exposed to the third generation of TKI, indicating the secondary drug‐resistant mutations in CSF were also relevant to TKI treatment Figure .3.3.2EGFR mutations, other driver genes and potential drug‐resistant mutations were also revealed in the genomic profile, including MET, ALK, ERBB2, KRAS, ROS proto‐oncogene 1 (ROS1), BRAF, ret proto‐oncogene (RET), and neurotrophic receptor tyrosine kinase 1 (NTRK1). Overall, 155 mutations of at least one of the above genes were detected in 201 patients from 11 studies (Table MET amplification (37/155). ALK was the following mutated gene and was found in 33 patients, but this result needs to be interpreted cautiously because all the enrolled patients in the study of Zheng et al were ALK‐mutated.NTRK1 (23/155), ERBB2 (18/155), and BRAF (14/155). ERBB2 amplification was found in six patients. NTRK fusions and BRAF V600E are the rare driver mutations of NSCLC, while in the sequencing results of CSF, nearly all types of NTRK1 and BRAF mutations were copy number amplifications .In addition to es Table . The mosTP53) frequently occurred in CSF, with a detection rate of around 50%.TP53 loss of heterozygosity (LOH) in CSF than in plasma .TP53 LOH or mutations, higher frequencies of genomic alternations in CNVs, EGFR, MET, and RB transcriptional corepressor 1 (RB1) were discovered. This profile was unique in CSF and might be related to resistance and the emergence of LM.Tumor protein p53 , MET, RB1, cyclin‐dependent kinase inhibitor 2A (CDKN2A), NTRK1, NK2 homeobox 1 (NKX2‐1), cyclin‐dependent kinase 4 (CDK4), fibroblast growth factor receptor 1 (FGFR1), RPTOR independent companion of MTOR complex 2 (RICTOR), BRAF, SMAD family member 4 (SMAD4), cyclin D1 (CCND1), fibroblast growth factor 3 (FGF3), cyclin‐dependent kinase 6 (CDK6), and POM121 transmembrane nucleoporin like 12 (POM121L12) , and fibroblast growth factor 19 (FGF19), and the cyclin family related genes such as CCND1, CDK4, and CDK6 showed only copy number amplifications. Since somatic CNVs affect a larger fraction of the cancer genome than any other type of alterations does, the profile of CNVs in CSF of LM patients warrants further study.Even though detecting CNVs is more challenging than SNVs due to technology issues,) Figure . Most of3.3.4EGFR, the most frequently mutated gene was TP53. Ninety‐four genes mutated in two or more patients and 25 genes mutated in more than 10 patients , olfactory receptor family 4 subfamily A member 15 (OR4A15), casein kinase 2 alpha 1 (CSNK2A1), olfactory receptor family six subfamily F member 1 (OR6F1), BMP/retinoic acid inducible neural specific 3 (BRINP3), and thrombospondin type 1 domain containing 7A (THSD7A). These genes may promote oncogenic transformation and indicate a role in LM but need extensive available evidence.In order to dig more information on genomic alternations in LM patients, we summarized the sequencing results from the studies which performed sequencing in gene panels instead of sequencing in several predefined genes. In summary, seven studies reported 120 genes with variations. Except for s Figure . Compare3.4Liquid biopsy is considered a dynamic method to trace genomic alternations and guide targeted treatment. Here, we summarized 14 representative cases Table  and mostEGFR mutations in CSF dropped, while after that, the subsequent change was the increase of T790M level in plasma, which occurred earlier than that in CSF.In the studies, a higher detection rate of T790M was found in extracranial lesions and plasma than in CSF.4In this review, we summarize the existing literature on the topic of sequencing of CSF in NSCLC patients with LM. We found sequencing of CSF performed well and revealed a unique genomic profile. We also summarized the cases that used CSF sequencing to guide treatments and suggested that it was better to combine the sequencing of plasma together.Plasma and CSF have been widely compared as samples for liquid biopsy in LM patients. Here, we found CSF performed better than plasma. The phenomenon attributes to some possible reasons. First, more interference of background gene fragments from normal blood cells existed in plasma than in CSF. Second, LM tumor cells are bathed by CSF, so the DNA fragments are released into CSF directly. However, the cells and cfDNA in CSF need to be absorbed through the arachnoid villi into the superior sagittal sinus every 4 h and then get into the systemic circulation. Third, the interference from extracranial lesions could be prevented by the blood–brain barrier and blood–CSF barrier.OR4A15 and OR6F1 belong to the olfactory receptor family, which can pair with the molecular secreted from neighboring neurons and is also highly expressed in different cancer tissues.NAV3 and BRINP3 are predominantly expressed in the nervous system and involved in neuron proliferation and regeneration. It is important to explore whether these mutations are interference of background genes in CSF or an implication of connections between LM tumor cells and neurons.Tumor metastasis is an evolutionary process, and the acquired genomic alterations of metastatic carcinoma during disease evolution induce treatment resistance and accelerate dissemination,TP53 LOH uniquely occurred in CSF rather than plasma, accompanied by more genomic mutations and CNVs. Aneuploidy, another trigger of genomic instability, is also widely detected in patients in CSF from LM patients.MET amplification, which was found associated with treatment resistance and brain metastasis in NSCLC. Most genes with CNVs also mutated, emphasizing the importance of genomic alterations in these genes and their relevant pathways. While the genes belonging to the fibroblast growth factor family and cyclin family showed a specific amplification, which indicated an enhanced function in the cell cycle and survival activities in LM.A common characteristic across studies is the number of CNVs detected in CSF, although the large fragment alternations were difficult to recognize during sequencing. We speculated that indicated the potential existence of genomic instability and might be the underlying cause of the difference in LM. EGFR changes in CSF along with treatments. Besides, mutations in CSF might provide clues to different treatment responses, such as T790M in TKI‐naive patients and bypass activations including MET amplification and ERBB2 amplification.EGFR subtypes in CSF and plasma were not simultaneous and T790M occurred earlier in plasma than in CSF after first‐ or second‐generation TKI therapy. The phenomenon may attribute to the followings: (1) the intracranial lesions were not exposed to the concentration of first‐ or second‐generation TKI that was enough to kill the cancer cells due to the obstruction of the blood–CSF barrier and (2) the blood–brain barrier and blood–CSF barrier also obstruct T790M mutations into CSF from systemic circulation. Furthermore, the difference in genomic alternations between CSF and plasma can be affected by clinical features, radiotherapy history, or TKI treatment of patients,In the representative cases, most patients improved in both brain imaging and CSF cytology after receiving TKI. Dynamic monitoring revealed mutation subtypes and AFs of TP53 LOH, a number of CNVs, and more mutated genes than plasma, indicating the potential existence of genomic instability in LM tumors. The sequencing of CSF in LM patients seems to provide more comprehensive information than we expected and the biological significance behind the genomic alternations needs further study.In conclusion, like tissue biopsy, sequencing of CSF in LM patients can guide targeted treatments and reveal resistance mutations and also provides a dynamic method to monitor intracranial tumor response. Furthermore, it reveals a unique genomic profile including mutations in driver genes and drug‐resistant genes, Tianqi Gao: Have made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; been involved in drafting the manuscript or revising it critically for important intellectual content; agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Fengxi Chen: have made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; been involved in drafting the manuscript or revising it critically for important intellectual content. Man Li: have made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; Given final approval of the version to be published. Each author should have participated sufficiently in the work to take public responsibility for appropriate portions of the content; Agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.No potential conflict of interest relevant to this article was reported.Our study is secondary research, and all data presented were from published studies. So, there is no need for ethical approval.Appendix S1Click here for additional data file.Table S1Click here for additional data file.


Amyloid plaques contain many proteins in addition to beta amyloid (Aβ). Previous studies examining plaque-associated proteins have shown these additional proteins are important; they provide insight into the factors that drive amyloid plaque development and are potential biomarkers or therapeutic targets for Alzheimer’s disease (AD). The aim of this study was to comprehensively identify proteins that are enriched in amyloid plaques using unbiased proteomics in two subtypes of early onset AD: sporadic early onset AD (EOAD) and Down Syndrome (DS) with AD. We focused our study on early onset AD as the drivers of the more aggressive pathology development in these cases is unknown and it is unclear whether amyloid-plaque enriched proteins differ between subtypes of early onset AD. Amyloid plaques and neighbouring non-plaque tissue were microdissected from human brain sections using laser capture microdissection and label-free LC–MS was used to quantify the proteins present. 48 proteins were consistently enriched in amyloid plaques in EOAD and DS. Many of these proteins were more significantly enriched in amyloid plaques than Aβ. The most enriched proteins in amyloid plaques in both EOAD and DS were: COL25A1, SMOC1, MDK, NTN1, OLFML3 and HTRA1. Endosomal/lysosomal proteins were particularly highly enriched in amyloid plaques. Fluorescent immunohistochemistry was used to validate the enrichment of four proteins in amyloid plaques  and to compare the amount of total Aβ, Aβ40, Aβ42, phosphorylated Aβ, pyroglutamate Aβ species and oligomeric species in EOAD and DS. These studies showed that phosphorylated Aβ, pyroglutamate Aβ species and SMOC1 were significantly higher in DS plaques, while oligomers were significantly higher in EOAD. Overall, we observed that amyloid plaques in EOAD and DS largely contained the same proteins, however the amount of enrichment of some proteins was different in EOAD and DS. Our study highlights the significant enrichment of many proteins in amyloid plaques, many of which may be potential therapeutic targets and/or biomarkers for AD.The online version contains supplementary material available at 10.1186/s40478-022-01356-1. Amyloid plaques are a neuropathological hallmark of Alzheimer’s disease and primarily consist of the protein beta amyloid (Aβ). However, it is often overlooked that amyloid plaques also contain hundreds of proteins in addition to Aβ. These include proteins that directly interact with Aβ (e.g. apolipoprotein E ), proteiPrevious studies have typically used immunohistochemistry to identify amyloid plaque proteins. Mass spectrometry-based proteomics is an alternative approach that allows efficient quantification of thousands of amyloid plaque proteins simultaneously. Proteomics also offers additional advantages of allowing discovery of novel plaque proteins due to its unbiased nature and bypassing complications due to antibody sensitivity and specificity issues. Given these significant advantages, we recently developed a localized proteomics approach to analyze the proteome of neuropathological lesions in AD such as plaques and neurofibrillary tangles –13.The significant heterogeneity in the clinical and neuropathological phenotype of AD suggests that multiple subtypes of AD exist. Previous studies have used various approaches to define AD subtypes –17. SomeAPP, PSEN1 and PSEN2 mutations. The cause of the remaining ~ 90% of EOAD cases is unknown and these cases are therefore characterized as sporadic EOAD. It is currently unclear if the same molecular mechanisms drive sporadic EOAD cases and late-onset AD [The aim of this study was to compare the amyloid plaque proteome in two subtypes of early onset AD: sporadic early onset AD (EOAD) and Down Syndrome (DS) with AD. Between 5 and 10% of AD cases are considered early onset . Of thesonset AD . DS withonset AD . People onset AD . Intraneonset AD , which ionset AD , 23. Matonset AD . The proonset AD , albeit onset AD  and a hionset AD . Multiplonset AD –31, howeHere, we show that amyloid plaques in DS and EOAD are enriched in many proteins besides Aβ including a common core group of 48 proteins that are enriched in plaques in both AD subtypes. While similar proteins were enriched in both DS and EOAD, some proteins were enriched to a greater extent in plaques in a particular subtype of AD, providing new evidence that some distinctions in plaque protein composition are present.All procedures were performed under protocols approved by the Institutional Review Board at New York University Alzheimer Disease Center, NY, USA. In all cases, written informed consent for research was obtained from the patient or legal guardian, and the material used had appropriate ethical approval for use in this project. All patients’ data and samples were coded and handled according to NIH guidelines to protect patients’ identities.APP, PSEN1 or PSEN2, tissue formalin fixation time < 6 months. Inclusion criteria for DS cases was ABC neuropathological score of A3, B3, C3, formalin fixation time < 6 months. Formalin fixed paraffin embedded tissue blocks containing the hippocampus and surrounding entorhinal/temporal cortex that were collected and processed as part of routine autopsy procedures were used in this study. This region was selected because it contains a high amount of amyloid pathology in EOAD and in DS with AD. N = 3 cases of EOAD, DS, late onset sporadic AD (LOAD) and cognitively normal, age matched controls were included in immunohistochemistry validation studies. Case specific information for the human tissue samples used in this study is included in Table N = 5 cases of early onset sporadic Alzheimer’s disease (EOAD) and n = 5 cases of DS with Alzheimer’s disease were included for proteomic experiments. Inclusion criteria for EOAD included age < 65 years, ABC neuropathological score of A3, B3, C3 , no muta2 solution (Promega) and 4.2 µl Betaine (Sigma). Cycling conditions were at 98 °C for 4 min and 40 cycles at 98 °C/10 s, 63 °C/1 min and 72 °C/1 min 10 s, followed by 72 °C 10 min. All the amplified fragments were resolved on 2% agarose gels, stained with GelRed 10,000X (Biotium) and visualized under UV exposure. DNA was purified from the agarose gel using the Illustra™ GFX™ PCR DNA and Gel Band Purification Kit (Cytiva) as indicated by the manufacturer, and DNA concentration was quantified using nanodrop One (Thermo Scientific). The second endpoint PCR was performed using the purified DNA with the conditions described previously, except for the concentration of the primers, which was reduced to 0.15 µM. Unpurified PCR products were submitted to Genewiz for Sanger sequencing, and the sequences were analyzed using SnapGene 5.3.1 software  or frozen tissue (FT) for the cases where it was available , Aβ40 , Aβ42 . The peptides were gradient eluted directly into an Orbitrap Fusion Lumos mass spectrometer using a 145-min gradient. The Orbitrap Fusion Lumos mass spectrometer acquired high resolution full MS spectra with a resolution of 240,000 (at m/z 200), AGC target of 1e6, with a maximum ion time of 50 ms, and scan range of 400–1500 m/z. Following each full MS data-dependent low resolution HCD MS/MS spectra were acquired. All MS/MS spectra were collected using the following instrument parameters: rapid ion trap scan rate, ACG target of 2e4, maximum ion time of 150 ms, one microscan, 0.7 m/z isolation window, fixed first mass of 150 m/z and NCE of 32.H. Sapiens proteome database  using the Andromeda search engine [Protein quantitation was performed using MaxQuant software suite v. 1.6.3.4 . Raw dath engine . A list h engine . Missingh engine  using deh engine . To do tp value of p < 0.05 (paired t-test). Fold change difference was selected as the primary determinant of enrichment/depletion in plaques as this correlated best with immunohistochemistry studies, which is the gold standard approach for identifying plaque enriched proteins. Uncorrected p-values were included to provide an indication of variance within a group, however plaque-enriched proteins identified by p-values alone did not correlate as well with prior gold-standard immunohistochemistry studies.Plaque enriched/depleted proteins were determined as those with a fold change difference between plaques and non-plaques > 1.5 fold and an uncorrected t-test and proteins were deemed significantly different based on a combination of p < 0.05 and fold change difference > 1.5.Direct comparison of plaque protein levels in DS and EOAD was performed using plaque protein levels that were normalized to the neighboring non-plaque tissue for each individual case. For this, normalized plaque protein levels were calculated as the ratio of protein intensity in plaques:non-plaques for each case. Differences in normalized plaque protein levels between DS and EOAD were identified using an unpaired General data manipulations and grouping were performed in R v4.0.2  using th. [. was filtered to identify plaque-enriched proteins that were identified by at last 2 peptides, had a fold-change difference of > 1.5 fold between plaques and non-plaques for AD versus controls or preclinical AD versus controls and did not include the word “keratin” or “immunoglobulin” in the protein name to make their data comparable with ours. Proteins with an abundance in the bottom 10% in sAD plaques or preclinical plaques were excluded. Uniprot ID was used to match proteins between studies.Systematic literature searches were used to identify plaque enriched proteins that have been validated in previous studies. A protein was designated a “known plaque protein” if there was published evidence of enrichment in amyloid plaques in human tissue using immunohistochemistry or mass spectrometry. Additional literature searches were used to determine if a protein was functionally associated with either Aβ or APP in instances where there was no immunohistochemistry evidence of presence in plaques. Key words used in these pubmed searches were: “Alzheimer’s and gene ID” or “Alzheimer’s and protein name”. Plaque enriched proteins identified by mass spectrometry were determined by comparison with Xiong et al. , which iChange in brain protein expression in AD versus controls was determined using our in-house developed database—NeuroPro—which combines results from 33 previous studies that used proteomics to identify consistent protein differences between AD and control human brain tissue , 44–73.Proteins were selected for validation studies based on the following criteria: enrichment in both EOAD and DS plaques, protein abundance in the top 50% in amyloid plaques, high fold change enrichment in plaques, appropriate commercial antibody available and limited/no previous evidence of presence in plaques by immunohistochemistry. Based on these criteria the following proteins were selected for immunohistochemistry validation studies: MSN, EZR, SMOC1 and ARL8B. 8 µm formalin-fixed paraffin embedded tissue sections containing the hippocampus and surrounding cortex were used for immunohistochemistry validation studies using the fluorescent immunohistochemistry method described above. Primary antibodies used for these validation studies included: MSN , EZR , SMOC1 , ARL8B , Aβ . The combined formic acid and citrate buffer antigen retrieval method (described above) was used for all validation immunohistochemistry studies. 63× images of fluorescent immunohistochemistry were collected using a confocal microscope Zeiss 700 with the ZEN Black 2.3 SP1 acquisition software. ARL8B immunoreactivity in neurons, microglia and astrocytes was tested using the same method as above with the following primary antibodies: GFAP , IBA1  and MAP2 . A negative control was included in all immunohistochemistry experiments, which consisted of a section of AD hippocampal tissue that underwent the same method with the primary antibody omitted.The percentage of amyloid plaques co-localized with ARL8B or SMOC1 was quantified using whole slide fluorescent scans that were collected using the Aperio VERSA digital slide scanner (Leica) with the 10× objective. Images were visualized and analyzed using the software Aperio ImageScope ver. 12.4.3 (Leica). Plaques co-stained with SMOC1 or ARL8B and Aβ or plaques stained only with Aβ in the hippocampal region were manually counted and then the ratio of co-stained plaques versus total plaques was calculated . The proportion of the plaques was obtained by plotting total number of plaques compared to the plaques co-stained by SMOC1 or ARL8B and Aβ, using the “grouped” layout of GraphPad Prism 8. Significant differences between groups were identified using one-way ANOVA followed by Tukey multiple comparison’s analysis, using GraphPad Prism 8 software.Amyloid plaques in DS and EOAD had similar amounts of total Aβ, Aβ40 and Aβ42  or extracellular proteins (enrichment FDR: 3.34 × 10−8). The enrichment of vesicle proteins was predominantly driven by endosome or lysosome proteins .Examination of the proteins that had the highest enrichment in plaques in both DS and EOAD included many proteins less studied in the AD field Table A, B. TheOur results suggest that that major plaque enriched proteins in EOAD and DS were largely the same. The consistency of protein enrichment in plaques was even noted at an individual case level Fig. C, D. HowWe also directly compared plaque protein levels in DS and EOAD. For this analysis, plaque protein levels that were normalized against background protein levels for each individual case were used. 38 proteins were significantly different between DS and EOAD plaques after correction for background protein differences. 25 proteins were significantly higher and 13 proteins were significantly lower in DS plaques in comparison to EOAD plaques  was selected as it was the most abundant novel plaque protein identified in our study. ARL8B was selected as a representative plaque-enriched lysosomal protein that had no prior immunohistochemistry evidence of presence in amyloid plaques. Moesin (MSN) and SMOC1 were selected as both have only one prior publication confirming their presence in plaques using immunohistochemistry, but no immunohistochemistry evidence of enrichment in plaques in EOAD or DS. Fluorescent immunohistochemistry confirmed that ezrin, moesin, ARL8B and SMOC1 were enriched in amyloid plaques in comparison to surrounding non-plaque tissue in DS, EOAD and late-onset sporadic AD. Moesin Fig. , Ezrin , and SMOSMOC1 strongly co-localized with amyloid fibrils only in a subset of amyloid plaques Fig. . The proARL8B was also abundant in amyloid plaques in all subgroups Fig. . In contWe also validated the presence or absence of these four plaque proteins in vascular amyloid pathology. MSN, EZR and SMOC1 immunoreactivity occasionally co-localized with CAA or in plaques which were in direct contact with blood vessels. However, ARL8B immunoreactivity was absent in vascular amyloid pathology, which is consistent with its weak direct colocalization with Aβ in amyloid plaques Fig. .Fig. 10COur results show that amyloid plaques in DS and EOAD are highly enriched in many proteins in addition to Aβ. Here, we have identified a core group of 48 proteins that are consistently enriched in plaques in comparison to neighboring non-plaque tissue in DS and EOAD. Many of these enriched plaque proteins have been validated in previous studies to colocalize with plaques or correlate with Aβ pathology in typical late onset AD, suggesting that this core group of enriched plaque proteins is consistent in both early and late onset AD subtypes. We also identified 15 novel proteins that were consistently enriched in plaques in both DS and EOAD. Our immunohistochemistry studies showed that while similar proteins are present in plaques in DS and EOAD the relative abundance of some of these proteins  is distinct in plaques in DS and EOAD.Our unbiased proteomics approach highlighted the striking enrichment of many proteins in amyloid plaques that have not been extensively studied in the context of AD such as COL25A1, SMOC1, NTN1, MDK, OLFML3 and HTRA1. The small number of previous studies examining the role of these proteins in AD suggest that these proteins likely have an important role in AD pathology. All of these highly enriched plaque proteins were also enriched in amyloid plaques in typical late onset AD  and 5/6 One of these highly enriched plaque proteins—SMOC1—was the focus of our immunohistochemistry validation studies. The role of SMOC1 in AD and its function in the brain is currently unknown. Single cell RNAseq studies suggest that SMOC1 is enriched in oligodendrocyte precursor cells in the brain  and prevWe hypothesize that the proteins that are highly enriched in amyloid plaques have an important mechanistic role in AD pathology. A common criticism regarding the pathological importance of proteins that accumulate in plaques is that they may not have a mechanistic role in driving pathology and are simply present in plaques by chance. However, a comprehensive review of the literature does not support this criticism. 60% of the 48 proteins commonly enriched in plaques in EOAD and DS have already been confirmed to have a mechanistic role in driving AD pathology in transgenic mouse models or in vitro Table . PreviouThe core group of 48 proteins enriched in plaques in both DS and EOAD were highly enriched in extracellular proteins and endosomal-lysosomal system proteins. The enrichment of extracellular proteins is expected given the extracellular location of amyloid plaques. However, the significant enrichment of endosomal-lysosomal system proteins in plaques was intriguing. A growing body of evidence convincingly shows that Aβ accumulates intraneuronally within endolysosomal vesicles at early stages of AD , 136. EnArl8b (encoded by the gene ARL8B) is an example novel lysosomal protein that we identified as enriched in amyloid plaques in both DS and EOAD. Arl8 is a small GTPase located on lysosomes that facilitates lysosomal trafficking along axons by acting as the linking molecule between lysosomes and kinesin-1 , 143. DiOur immunohistochemistry and literature validation studies showed that amyloid plaque enriched proteins had different colocalization patterns in amyloid plaques. For example, endolysosomal proteins typically have punctate/granular localization in plaques. This staining pattern was observed for ARL8B in our study, which was identical to the staining pattern seen for other lysosomal proteins in past studies such as cathepsin D , cathepsDirect comparison of the amyloid plaque proteome in EOAD and DS showed that amyloid plaques in the two subtypes of younger onset AD had a very similar plaque protein composition. This shows that despite the different disease initiating factors, the resulting amyloid plaques still largely contain the same proteins. While some proteins were enriched to a much greater extent in amyloid plaques in either DS or EOAD (e.g. SMOC1), the trend for enrichment in both subtypes was highly similar. It is still unclear how these relative plaque protein differences influence AD pathogenesis. Future mechanistic studies examining how each of these proteins influence Aβ aggregation or clearance are needed. Future proteomic studies examining whether these major plaque enriched proteins are also enriched in other subtypes of AD  would also potentially provide insight into differences into the rate, topography or type of plaque pathology between these subtypes.In conclusion, we provide a new resource for the AD field that comprehensively characterizes proteins that are enriched in amyloid plaques in multiple subtypes of AD. We propose that these consistently enriched amyloid plaque proteins provide insight into the mechanisms driving amyloid plaque development in AD and are potentially novel drug targets and/or biomarkers for AD.Additional file 1: Table S1. Total imputed dataset. Table S2. Unimputed data. Table S3. Proteins enriched in plaques in both EOAD and DS  Schematic diagram of the APOE genotyping methodology. Six scrolls of 8 µm were sectioned from FFPE blocks and DNA was isolated with the automated QIAsymphony SP. An endpoint PCR was performed, samples were resolved in a 2% agarose gel and amplified DNA was purified from the gel. A second PCR with the purified DNA was performed and un-purified PCR products were sequenced to determine the APOE genotype. (B) Representative gel of EOAD and DS samples used for sequencing. APOE band is located at 348 bp. (C) Sanger sequencing chromatogram showing the nucleotides located in the single-nucleotide polymorphisms (SNPs) rs429358 and rs7412, which determine the APOE variant ε3.


Cryptorchidism is a common disorder in boys that has been widely studied both experimentally and clinically. The role of the gubernaculum, a mesenchymal tissue extending from the fetal testis and epididymis to the developing scrotum, is still unclear. Even the name is debated: ‘gubernaculum epididymis’ or ‘gubernaculum testis’. This review does not aim to provide a global overview of competing theories on testicular descent, but focuses on the role of the gubernaculum in epididymo-testicular descent. We identified four major pitfalls of gubernaculum research: the role of the gubernaculum, of insulin-like peptide 3, anti-Müllerian hormone, and androgens. The major critical issues were that the gubernaculum plays a guiding role for the epididymis, descending prior to the testis and expanding the inguinal canal; insulin-like peptide 3 is not as important for the process of descent in humans as the rate of insulin-like peptide 3 mutations is low; anti-Müllerian hormone plays no significant role in epididymo-testicular descent; androgens and gonadotropins play a crucial role in epididymo-testicular descent. The role of the epididymis in the complex process of gubernaculum, epididymis, and testis migration is underestimated and should be included in future research. Cryptorchidism, one of the most common disorders in boys, has been widely studied both experimentally and clinically. Many review articles have been published, but the main conclusions remain debatable. In trying to understand testicular descent, one of the most controversial issues is the gubernaculum. Though it may seem that this topic is all sorted out, upon a closer look, even the name ‘gubernaculum testis’ or ‘gubernaculum epididymis’ needs to be decided . Of courThe gubernaculum, the mesenchymal tissue extending from the fetal epididymis and testis to the developing scrotum, was originally named by John Hunter . Later, processus vaginalis and in rodents it remains open with a fat pad on the epididymis, which helps prevent inguinal hernias [Gubernaculum research is mostly conducted in rodents. The rodent gubernaculum differs from human gubernaculum in terms of structure, migration time, and last step of descent. A major difference is that the rodent gubernaculum is fibrous with a circumferential cremaster muscle, but the human gubernaculum is jelly mesenchymatous . In addi hernias ,8.In this review, we discuss the four major pitfalls met earlier and still potentially misleading the research on this common congenital disorder in boys:The role of the gubernaculum;The link between hypogonadism and epididymo-testicular descent;The role of insulin-like peptide 3 (Insl3) in epididymo-testicular descent;The role of anti-Müllerian hormone (AMH) in epididymo-testicular descent.Literature to review was found in the PubMed database excluding case reports, editorials, and opinions.The human gubernaculum consists of a fibrous structure rich in collagen and elastic fibers without contractile potential and extracellular matrix . It undeIn 1978, Bergh et al. reported that testis descent can occur with a severed proximal gubernaculum, whereas testicular descent is prevented when the distal gubernaculum is cut. In addition, descent of the epididymis can occur in the absence of a testis . These fprocessus vaginalis and more proximal location of the testis [Pax2/8) and fibroblast growth factor [processus vaginalis’. Furthermore, no extension of the gubernaculum into the bottom of the scrotum exists [Going back to the findings reported by Bergh et al. and taking a closer look at the epididymis reveals interesting discoveries. In 2012, Rachmani et al. showed that the degree of nonfusion between the testis and epididymis is associated with a higher position of the testis. Total nonfusion reliably interferes with descent . In 2014e testis . In genee testis ,19. An ih factor ,1. In 19m exists ,22. In 2m exists . This suWnt4, Insl3, Ar, Lgr8, Hoxa10, Amh, Dmrt1, and Fst. In addition to spermatogenic failure and gubernacular defects, E2F1-null cryptorchid mice have a significant decrease in epididymal size compared to wild-type individuals [Genetic studies on the etiology of cryptorchidism also support the importance of the epididymis for testicular descent. Recent research by Jorgez et al. showed that E2F transcription factor-1 (E2F1) regulates genes required for testicular descent, such as ividuals .Both gubernaculum and cremaster muscle are a derivative of mesenchyme and have a distinct myosinic pattern . The epiThe next topic of debate is the role of hypogonadism and androgens in epididymo-testicular descent and whether it is staged. Clarnette et al. stated that, ‘in conditions of androgen insensitivity, the testis is located in the inguinal region, indicating that the first phase of descent is normal, but the inguinoscrotal descent has failed to occur’ . AntiandWT1) may be associated with testicular maldescent and genital abnormalities consistent with defects in the androgen pathway of testis descent [WT1 have shown that androgens are important for both intra-abdominal and inguinoscrotal descent, but multiple other factors may modulate this process [androgen receptor and WT1 expression has been found in prostate cancer cell lines, together with WT1 repression of the androgen receptor promoter [Mutations in Wilms’ tumor gene  for cryptorchidism in humans? When trying to replicate the motivating results, many studies were looking for INSL3 gene mutations in former cryptorchid boys and men; surprisingly, it appeared at extremely low rates. The cumulative incidence of INSL3 gene mutations in eight studies with a total of 859 patients was 2.3% . SimilarAMH is another factor commonly discussed in the process of testicular descent. It is produced by Sertoli cells and causes the regression of Müllerian ducts . ClarnetMost experimental evidence supports the leading role of gonadotropins and androgens in epididymo-testicular descent. However, the role of the epididymis is underestimated. Future research should not omit the role of the epididymis in the complex process of gubernaculum, epididymis, and testis migration.


The HSA-mediated retention time of each PSMA ligand generated by this novel ‘albumin-mediated size exclusion chromatography’ (AMSEC) was converted to a ligand-specific apparent molecular weight (MWapp), and a normalization accounting for unspecific interactions between individual radioligands and the SEC column matrix was applied. The resulting normalized MWapp,norm. could serve to estimate the blood clearance of renally excreted radioligands by means of their influence on the highly size-selective process of glomerular filtration (GF). Based on the correlation between MW and the glomerular sieving coefficients (GSCs) of a set of plasma proteins, GSCcalc values were calculated to assess the relative differences in the expected GF/blood clearance kinetics in vivo and to select lead candidates among the evaluated radioligands. Significant differences in the MWapp,norm. and GSCcalc values, even for stereoisomers, were found, indicating that AMSEC might be a valuable and high-resolution tool for the preclinical selection of therapeutic lead compounds for clinical translation.A meticulously adjusted pharmacokinetic profile and especially fine-tuned blood clearance kinetics are key characteristics of therapeutic radiopharmaceuticals. We, therefore, aimed to develop a method that allowed the estimation of blood clearance kinetics in vitro. For this purpose,  Quality control via radio-TLC was performed using 1.0 m NH4OAc/DMF  on TLC silica gel 60 F254 plates  or 0.1 m sodium citrate buffer on iTLC-SG chromatography paper . Radioligands were used for experiments if the radio chemical purity (RCP) was > 95%.Radiolabeling with no-carrier-added [rocedure  with mol177Lu-labeled species of PSMA ligands. For ease of readability, the lutetium complex is omitted in the designation of all radioligands in running text, figures, and tables .All data presented in this work were obtained with the All size exclusion chromatographic (SEC) experiments were carried out using the above-mentioned HPLC system equipped with a Superdex 75 Increase 10/300 GL gel filtration size exclusion column . Eluents at room temperature, a constant flow rate of 0.8 mL/min, and an acquisition time of 35 min were generally applied. The column was calibrated as recommended by the manufacturer using a commercially available set of proteins . Retention times of calibration proteins and calculated calibration parameters are given in the All retention times of radio signals were corrected for the offset between the UV-vis detector and radio-detector (Δt = 0.084 min). Throughout the development of the AMSEC method, three gel filtration columns of the same type were used for experiments (most data were acquired using column 1). As calculations were based on absolute empirical retention times that inherently differed slightly from column to column, all retention times determined on column 2 and column 3 were mathematically converted to an equivalent retention time on column 1 using the columns’ calibration parameters. A detailed description of these calculations is provided in the All numerical retention times explicitly given in running text or tables and plotted in diagrams are already corrected for the offset between UV-vis and radio-detector and, if initially determined on columns 2 or 3, converted to the equivalent retention time on column 1  as a mobile phase. Upon injection of approximately 1.0 MBq (5–10 µL) of a radioligand, the elution profile was monitored via radioactivity detection, and the retention times of observed radiopeaks  were determined via semi-automated peak integration. By means of calibration, retention times were translated into a ligand-specific raw apparent molecular weight  as a parameter to assess the extent of HSA binding  in PBS (pH 7.4) at physiological concentration  corresponded to the maximal MWapp,raw, theoretically observable in AMSEC runs in the case of continuous binding of a radioligand to HSA . The retention time of acetone  served as a reference for a ligand with no interaction with HSA in AMSEC runs  and an actual physical molecular weight below the column fractionation range .For reference purposes, conventional SEC runs of HSA  and acetone  with PBS (pH 7.4) as a mobile phase were executed. The retention time of HSA  of a radioligand executed in analogy to the AMSEC run but using PBS (pH 7.4) without HSA as a mobile phase. The determined retention time tR,blank, together with the retention time tR,AMSEC from the AMSEC run, tR,HSA, and tR,acetone determined in R,norm. for every radioligand  and corresponding glomerular sieving coefficients (GSCs) of 12 human plasma proteins  was exe−5 .The values of bottom asymptote A1 and top asymptote A2 were set to 4.2∙10n et al. ) and 1  radioligands for RLT with improved therapeutic efficacy.In conclusion, AMSEC might offer the unique possibility to gain insight into human blood clearance kinetics, a fundamental aspect in the pharmacokinetics of radioligands already in vitro at the preclinical stage. If clinical data confirm this novel preclinical approach, AMSEC could furthermore contribute to continually ameliorated animal protection, as preclinical in vivo experiments  could be reduced to pre-selected compounds that exhibit favorable MW