Protein profile of purified plasma- and aqueous humor-derived extracellular vesicles from patients with ocular toxoplasmosis

ABSTRACT Purpose: To characterize the extracellular vesicle protein cargo in the aqueous humor and plasma of patients with ocular toxoplasmosis. Methods: Aqueous humor and plasma were collected from six patients with active ocular toxoplasmosis and six patients with cataract. Extracellular vesicles were isolated, and western blotting and mass spectrometry were performed for protein analysis. Results: All plasma samples from patients with ocular toxoplasmosis and cataract were positive for the tetraspanins CD63 and TSG101. However, the aqueous humor from patients with ocular toxoplasmosis was positive only for CD63. Sixty-seven new unreported proteins were identified in the aqueous humor and plasma of patients with the ocular toxoplasmosis and cataract. Of the 67 proteins, 10 and 7 were found only in the cataract and ocular toxoplasmosis groups, respectively. In general, these proteins were involved in immune system activation and retina homeostasis and were related to infections and retina-associated diseases. Conclusion: The distinct protein signatures between ocular toxoplasmosis and cataract may be helpful in the differential diagnosis of ocular toxoplasmosis. However, more studies are needed to better understand the role of these proteins in the pathogenesis of ocular toxoplasmosis.

Plasmonic Nanogap-Enhanced Tunable Three-Dimensional Nanoframes in Application to Clinical Diagnosis of Alzheimer's Disease.

Advancements in nanotechnology led to significant improvements in synthesizing plasmon-enhanced nanoarchitectures for biosensor applications, and high-yield productivity at low cost is vital to step further into medical commerce. Metal nanoframes via wet chemistry are gaining attention for their homogeneous structure and outstanding catalytic and optical properties. However, nanoframe morphology should be considered delicately when brought to biosensors to utilize its superior characteristics thoroughly, and the need to prove its clinical applicability still remains. Herein, we controlled the frameworks of double-walled nanoframes (DWFs) precisely via wet chemistry to construct a homogeneous plasmon-enhanced nanotransducer for localized surface plasmon resonance biosensors. By tuning the physical properties considering the finite-difference time-domain simulation results, biomolecular interactions were feasible in the electromagnetic field-enhanced nanospace. As a result, DWF10 exhibited a 10-fold lower detection limit of 2.21 fM compared to DWF14 for tau detection. Further application into blood-based clinical and Alzheimer's disease (AD) diagnostics, notable improvement in classifying mild cognitive impairment patients against healthy controls and AD patients, was demonstrated along with impressive AUC values. Thus, in response to diverse detection methods, optimizing nanoframe dimensions such as nanogap and frame thickness to maximize sensor performance is critical to realize future POCT diagnosis.

Phosphoproteomic subtyping of gastric cancer reveals dynamic transformation with chemotherapy and guides targeted cancer treatment.

There are only a few effective molecular targeted agents for advanced unresectable or recurrent advanced gastric cancer (AGC), which has a poor prognosis with a median survival time of less than 14 months. Focusing on phosphorylation signaling in cancer cells, we have been developing deep phosphoproteome analysis from minute endoscopic biopsy specimens frozen within 20 s of collection. Phosphoproteomic analysis of 127 fresh-frozen endoscopic biopsy samples from untreated patients with AGC revealed three subtypes reflecting different cellular signaling statuses. Subsequent serial biopsy analysis has revealed the dynamic mesenchymal transitions within cancer cells, along with the concomitant rewiring of the kinome network, ultimately resulting in the conversion to the epithelial-mesenchymal transition (EMT) subtype throughout treatment. We present our investigation of intracellular signaling related to the EMT in gastric cancer and propose therapeutic approaches targeting AXL. This study also provides a wealth of resources for the future development of treatments and biomarkers for AGC.

Standard of care versus standard of care plus Ericksonian hypnosis for percutaneous liver biopsy: Results of a randomized control trial.

PURPOSE: The purpose of this study was to compare levels of pain and anxiety during percutaneous ultrasound-guided liver biopsy between patients receiving standard of care and those receiving standard of care plus the support of Ericksonian hypnosis. MATERIALS AND METHODS: This prospective, single-center, single-blind, randomized controlled superiority trial included 70 participants. Participants were randomly assigned to either the standard of care group and received oral anxiolytic medications with reassuring conversational support, or to the experimental group, and received Ericksonian hypnosis (i.e., conversational hypnosis) in addition to standard of care. The primary outcome was the level of pain experienced during the biopsy, measured on a 10-point visual analog scale (0 indicating no pain to 10 indicating excruciating pain). Secondary outcomes included anxiety level during the biopsy, pain level within one hour of the biopsy measured using the same 10-point visual analog scale, amount of analgesic medication taken in the 24 h following the biopsy, and patient willingness to undergo another ultrasound-guided percutaneous liver biopsy in the future. RESULTS: Thirty-six participants were included in the standard of care group, and 34 were included in the experimental group. The mean score of pain experienced during the biopsy was lower in the experimental group (2.4 ± 1.9 [standard deviation (SD)]) compared to the standard of care group (4.4 ± 2.6 [SD]) (P = 0.001). The level of anxiety experienced during the biopsy was lower in the hypnosis group (2.1 ± 1.8 [SD]) compared to the standard of care group (4.8 ± 2.4 [SD]) (P < 0.001). No significant differences in other secondary outcomes were observed between the two groups. CONCLUSION: The addition of Ericksonian hypnosis to standard of care reduces the pain experienced by patients during percutaneous ultrasound-guided percutaneous liver biopsy by comparison with standard of care alone.

Defining the Etiology of Renal Allograft Dysfunction Using Banff 2019 Classification: Correlation with Post-Transplant Duration and Creatinine Levels-A Comprehensive Analysis of 200 Renal Biopsies at a Tertiary Care Medical Center Hospital.

OBJECTIVE: Chronic kidney disease is a growing global health issue, contributing significantly to morbidity and mortality. The incidence of end-stage renal disease (ESRD) is approximately 100 per million population. Renal transplantation remains the cornerstone treatment for ESRD, with a projected 20-year survival rate of 60%. We aim to define the etiology of renal allograft dysfunction using the Banff 2019 classification by analyzing 200 renal allograft biopsies in correlation with creatinine levels across post-transplant time frames. METHODOLOGY: 200 renal allograft biopsies are analyzed using the recent Banff 2019 classification with creatinine levels and post-transplant duration correlation. RESULTS: The study included 150 (75%) male patients and 50 (25%) female patients, with the majority 78 (39%) representing the age group of 16-30 years. 36 (18%) biopsies were within 3-month post-transplant, while 92 (46%) were 2-year post-transplant. According to the Banff 2019 classification, 92 (46.0%) transplant rejection biopsies were identified, with most 54 (27%) exhibiting antibody-mediated rejection (Category 2), including 40 (20%) active acute antibody-mediated rejection (ABMR) and 14 (7.0%) chronic active ABMR. T-cell-mediated rejection (TCMR; Category 4) represented 12 (6%) biopsies, including 10 (5%) acute TCMR and 2 (1%) chronic active TCMR. Category 5, the miscellaneous group, represented 100 (50%) biopsies, out of which 32 (16%) exhibited calcineurin inhibitor (CNI) toxicity, 38 (19%) acute tubular necrosis, and 8 (4%) thrombotic microangiopathy. A notable variation in the dysfunction distribution across different post-transplant time frames indicated a temporal evolution in the underlying causes of allograft dysfunction. Specific Banff categories showed a robust association with renal dysfunction, potentially contributing to the elevation of creatinine levels and renal function deterioration. CONCLUSION: Our study highlights the intricate pathophysiology of renal allograft dysfunction. Most biopsies were attributed to ABMR whereas one-third of biopsies exhibited mixed lesions (ABMR and TCMR or ABMR and calcineurin inhibitor toxicity (CNIT)). Additionally, this study suggests that renal allograft rejection remains a significant contributor to graft dysfunction. A complex interplay between histological findings, Banff classification, and renal function is noted. A significant difference in the distribution of dysfunction across post-transplant time frames is noted suggesting a temporal evolution in the etiology of allograft dysfunction. Certain Banff categories demonstrate a stronger association with renal dysfunction that may influence creatinine level increase and renal function deterioration. In correspondence to the recent Banff 2019 guidelines for diagnosing ABMR, we emphasize the role of C4d staining on immunofluorescence or immunohistochemistry in allograft biopsies as imperative for timely diagnosis and immunosuppressant therapy adjustment, ultimately enhancing graft survival. Further research is needed to elucidate the underlying mechanisms driving renal dysfunction in different Banff categories, ultimately informing personalized management strategies for patients with renal allograft dysfunction. In line with the Banff 2019 guidelines for diagnosing ABMR, this study highlights the critical role of C4d staining through immunofluorescence or immunohistochemistry in allograft biopsies for early diagnosis and timely adjustment of immunosuppressive therapy, ultimately improving graft survival.

A deep learning approach to case prioritisation of colorectal biopsies.

AIMS: To create and validate a weakly supervised artificial intelligence (AI) model for detection of abnormal colorectal histology, including dysplasia and cancer, and prioritise biopsies according to clinical significance (severity of diagnosis). MATERIALS AND METHODS: Triagnexia Colorectal, a weakly supervised deep learning model, was developed for the classification of colorectal samples from haematoxylin and eosin (H&E)-stained whole slide images. The model was trained on 24 983 digitised images and assessed by multiple pathologists in a simulated digital pathology environment. The AI application was implemented as part of a point and click graphical user interface to streamline decision-making. Pathologists assessed the accuracy of the AI tool, its value, ease of use and integration into the digital pathology workflow. RESULTS: Validation of the model was conducted on two cohorts: the first, on 100 single-slide cases, achieved micro-average model specificity of 0.984, micro-average model sensitivity of 0.949 and micro-average model F1 score of 0.949 across all classes. A secondary multi-institutional validation cohort, of 101 single-slide cases, achieved micro-average model specificity of 0.978, micro-average model sensitivity of 0.931 and micro-average model F1 score of 0.931 across all classes. Pathologists reflected their positive impressions on the overall accuracy of the AI in detecting colorectal pathology abnormalities. CONCLUSIONS: We have developed a high-performing colorectal biopsy AI triage model that can be integrated into a routine digital pathology workflow to assist pathologists in prioritising cases and identifying cases with dysplasia/cancer versus non-neoplastic biopsies.

Does the standard proteinuria cut-off for renal biopsy in lupus nephritis as per the current guidelines hold good for Asian population? A single-centre study from South India.

INTRODUCTION: Current rheumatology and nephrology society guidelines in lupus nephritis do not recommend renal biopsy for proteinuria of less than 500 mg/24 h. This might lead to a significant delay in the early diagnosis of lupus nephritis. AIM: The main aim of this study is to determine the nature of renal lesions in patients with low-grade proteinuria and to analyze the predictors for clinically significant lupus nephritis. METHODS: This was a single-center, retrospective study. All consecutive patients of lupus nephritis, with low-grade proteinuria (200 mg to 500 mg/24 h) undergoing renal biopsy were enrolled in this study. The renal biopsies were classified into significant lesions (Class III/IV/V) and non-significant lesions (Class I and II). Treatment naïve groups and treatment-modified groups were analyzed separately. Predictive factors for significant renal lesions were determined by univariate and multivariate analysis. RESULTS: We identified 183 patients of lupus with proteinuria between 200 and 500 mg / 24 h. Mean (SD) age was 30.2 (11.39) years with 167 (91.2%) of them being females. The mean (SD) baseline proteinuria was 351.03 (98.1) mg/24 h 85 patients (46.5%) had proliferative lupus nephritis where whereas 17 patients (9.3%) had membranous nephropathy. Crescents and fibrinoid necrosis were seen in 10 (5.46%) and 24 (13.11 %) patients respectively. Isolated proteinuria without any other sediments was seen in 95 patients (51.9%) of which 29 patients had proliferative lupus nephritis. Elevated Anti-double stranded DNA (anti-dsDNA), low C3, low C4 and the presence of urinary sediments were significantly associated with significant renal lesions in biopsy. CONCLUSION: Significant renal lesions were seen in around half of the patients with low-grade proteinuria underscoring the importance of performing a renal biopsy in this set of patients. Low C3 and C4, urinary sediments, and elevated anti-dsDNA were predictors for significant renal lesions.

Etiological Spectrum of Lymphadenopathy Among Children on Lymph Node Biopsy.

Background Pediatric peripheral lymphadenopathy is commonly a benign condition and most cases resolve spontaneously; however, it can be a manifestation of a serious underlying disease. This study aimed to determine the etiological spectrum of persistent pediatric lymphadenopathy on excisional biopsy in a tertiary care children's hospital in a low-middle-income country and to make recommendations regarding evaluation, diagnostic testing, and surgical interventions best suited to the population. Methodology A prospective cross-sectional study was conducted on 243 pediatric patients between the ages of one to 12 years undergoing excisional biopsy for persistent lymphadenopathy (more than four weeks duration) from April 1, 2021, to March 31, 2024. Patient demographic data along with signs, symptoms, and results of investigations including histopathological diagnosis were documented on a structured proforma. Results Patients' age range was two to 12 years (mean = 7.29 ± 2.30 years). The male-to-female ratio was 1:53. The Mean duration of lymphadenopathy was 35.89 ± 6.95 days (range = 25 to 57 days). The average size of lymph nodes ranged from 1 cm to a complex nodal mass of 7 cm. Histopathology showed reactive hyperplasia (40.32%, n = 98), tuberculosis (TB) (33.7%, n = 82), lymphoma (10.3%, n = 25), atypical mycobacterial adenitis (6.99%, n = 17), chronic granulomatous inflammation-histiocytosis (6.2%, n = 15), and Langerhans cell histiocytosis (2.5%, n = 6). The most common site was cervical. Sputum GeneXpert for TB had a true-positive rate of 78.84% while PPD was positive in only 13 TB patients. Atypical mycobacterial adenitis was successfully treated with excision and antibiotics. Supraclavicular nodes were strongly associated with lymphoma (p = 0.008). Conclusions Persistent pediatric lymphadenopathy is most commonly caused by TB followed by lymphoma. Positive sputum GeneXpert for TB with a suggestive clinical picture in endemic regions may be sufficient to start empiric therapy without the need for excisional biopsy in mycobacterial TB adenitis with negative PPD results and normal chest X-ray. In all other cases, excisional biopsy remains the gold standard for diagnosis. However, further studies should be conducted to formulate less invasive management algorithms.

Clinical Oversight and Delayed Diagnosis of a Pathological Compression Fracture Causing Paraplegia.

While osteoporosis is the primary cause of vertebral compression fractures (VCFs), it's crucial to promptly recognize pathological fractures through comprehensive diagnostic tests, including vertebral biopsies, to determine the exact etiology. For instance, a 66-year-old male with osteoporosis experienced worsening lower limb weakness and back pain after an initial vertebroplasty for a T12 compression fracture. Subsequent MRI revealed severe circumferential extradural compression at T12, leading to further surgeries that eventually uncovered metastatic adenocarcinoma from a pancreatic tumor. This case highlights the importance of precise diagnosis through vertebral biopsy and the necessity of sufficient ventral decompression or corpectomy, coupled with extensive laminectomy, to address severe neurological impairments like paraplegia. Prompt and accurate interventions can significantly improve patient outcomes and quality of life.

Electrochemical cytosensors for non-invasive liquid biopsy: Detection procedures and technologies for circulating tumor cells.

Each year, millions of new cancer cases and cancer-related deaths underscore the urgent need for effective, affordable screening methods. Circulating tumor cells (CTCs), which derived from tumors and shedding into bloodstream, are considered promising biomarkers for liquid biopsy due to their unique biological significance and the substantial volume of supporting research. Among many advanced CTCs detection methods, electrochemical sensing is rapidly developing due to their high selectivity, high sensitivity, low cost, and rapid detection capability, well meeting the growing demand for non-invasive liquid biopsy. This review focuses on the entire procedure of detecting CTCs using electrochemical cytosensors, starting from sample preparation, detailing bio-recognition elements for capturing CTCs, highlighting design strategies of cytosensor, and discussing the prospects and challenges of electrochemical cytosensor applications.

Nucleic acid liquid biopsies in cardiovascular disease: Cell-free DNA liquid biopsies in cardiovascular disease.

Cardiovascular disease (CVD) is the leading cause of death worldwide, and despite treatment efforts, cardiovascular function cannot always be restored, and progression of disease be prevented. Critical insights are oftentimes based on tissue samples. Current knowledge of tissue pathology typically relies on invasive biopsies or postmortem samples. Liquid biopsies, which assess circulating mediators to deduce the histology and pathology of distant tissues, have been advancing rapidly in cancer research and offer a promising approach to be translated to the understanding and treatment of CVD. The widely understood elevations in cell-free DNA during acute and chronic cardiovascular conditions, associate with disease, severity, and offer prognostic value. The role of neutrophil extracellular traps (NETs) and circulating nucleases in thrombosis provide a solid rationale for liquid biopsies in CVD. cfDNA originates from various tissue types and cellular sources, including mitochondria and nuclei, and can be used to trace cell and tissue type lineage, as well as to gain insight into the activation status of cells. This article discusses the origin, structure, and potential utility of cfDNA, offering a deeper and less invasive approach for the understanding of the complexities of CVD.

Percutaneous transthoracic needle biopsy of lung lesions is a safe method associated with a very low risk of pleural recurrence.

AIM: Percutaneous transthoracic needle biopsy (PTNB), an alternative to bronchoscopic confirmation of lung lesions, is today being associated with a risk of pneumothorax and hemorrhage. Further, there are no data on the possible risk of malignant disease spreading to the pleura at the site of the PTNB. Previous studies have dealt with this risk in stage I non-small cell lung cancer only. The aim of this study was thus to assess the risk of pleural recurrence for all types of lung lesions. Secondary objectives included assessment of diagnostic yield and safety with respect to the incidence of pneumothorax and hemorrhage. METHODS: Clinical data of all patients from the University Hospital in Pilsen who had undergone PTNB of lung lesions between 1.1.2018 and 31.12.2022 were included in this retrospective study. RESULTS: Following PTNB, ipsilateral pleural effusion occurred in 4.8% of patients without prior pleural infiltration. The effusion was confirmed as malignant in one patient (0.7%). Diagnostic yield of the method was 86.6%. We recorded pneumothorax or hemorrhage in the lung parenchyma or pleural space requiring medical intervention in 3.4% and 1.1% of patients, respectively. CONCLUSION: In our study, percutaneous transthoracic needle biopsy of lung lesions showed high sensitivity and low degree of acute complications requiring an invasive solution. The risk of pleural recurrence after a biopsy was very low. Consequently, we continue to consider this method to be an alternative to bronchoscopy biopsies.

Ultrasound-guided core needle biopsy of deep fascia: A cadaveric study evaluating feasibility, accuracy and reliability.

Over the past few decades, researchers and clinicians have dedicated significant attention to fascial tissues. Current interest focuses on their anatomical and pathophysiological features. Breakthroughs in ultrasound (US) and magnetic resonance imaging (MRI) have enhanced our ability to study the dynamics and alterations of the tissue structures. However, a microscopic perspective is also essential for a comprehensive understanding of some pathologies of the fasciae. The aim of this study was to investigate, using a cadaveric study: (1) the ease of visualization of the landmarks used for the US-guided fascial core needle biopsy (CNB); (2) the consistency and accuracy of needle placement inside fascial layers using US guidance and confirmed by histological examination; (3) inter-rater reliability. We assessed the feasibility of US-guided CNB in different topographical regions of human cadavers: the thoracolumbar fascia (TLF), fascia lata (FL), and crural fascia (CF). The results, confirmed by histological examination, revealed no significant difference in needle placements between the in-plane approaches in the long and short axes for all locations and fasciae studied (long axis: 91.88%; short axis: 96.22%); p > 0.05. US-guided core needle biopsy with the in-plane approach is feasible, consistent and reliable. It could provide most or all of high-quality fascial tissue samples required for pathological examination. It could also reveal changes in fascial pathologies, capturing the exact site of pathology thanks to US guidance, in particular in patchy diseases such as eosinophilic fasciitis.

How i do it: Robot-assisted transcerebellar stereotactic approach for brainstem lesion.

BACKGROUND: Stereotactic approaches to diffuse intrinsic pontine gliomas (DIPGs) remain essential due to advances in molecular biology and management, necessitating tissue sampling. Here we present an effective technique with a biopsy by robot-assisted transcerebellar approach. METHOD: Our procedure was performed using the ROSA robotic system and the OARM CT scan, which provided stereotactic conditions for this transcerebellar approach to brainstem lesions. CONCLUSION: The robot-assisted transcerebellar stereotactic approach remains essential to provide complications for biopsy of brainstem lesions.

Unveiling Cas12j Trans-Cleavage Activity for CRISPR Diagnostics: Application to miRNA Detection in Lung Cancer Diagnosis.

Cas12j, a hypercompact and efficient Cas protein, has potential for use in CRISPR diagnostics, but has not yet been used because the trans-cleavage activity of Cas12j is veiled. Here, the trans-cleavage behavior of Cas12j1, 2, and 3 variants and evaluate their suitability for nucleic acid detection is unveiled. The target preferences and mismatch specificities of the Cas12j variants are precisely investigated and the optimal Cas12j reaction conditions are determined. As a result, the EXP-J assay for miRNA detection by harnessing the robust trans-cleavage activity of Cas12j on short ssDNA is developed. The EXP-J method demonstrates exceptional detection capabilities for miRNAs, proving that Cas12j can be a pivotal component in molecular diagnostics. Furthermore, the translational potential of the EXP-J assay is validated by detecting oncogenic miRNAs in plasma samples from lung cancer patients. This investigation not only elucidates the trans-cleavage characteristics of Cas12j variants, but also advances the Cas12j-based diagnostic toolkit.

The utility of disc space and vertebral body specimens cell count differential for the diagnosis of native vertebral osteomyelitis: a prospective cohort study.

BACKGROUND: Diagnostic methods for native vertebral osteomyelitis (NVO) often yield inconclusive results. Image-guided spine biopsies for culture are specific but diagnose NVO in only 50% of cases. Pre-exposure to antimicrobials further reduces diagnostic yield. Our study assesses the value of neutrophil percentage in disc space fluid and vertebral body (DS/VB) samples for diagnosing NVO. METHODS: Adults referred for spine biopsy at Mayo Clinic from August 2022 to September 2023 were consented and enrolled at the time of biopsy. Following routine specimen collection, the biopsy needle was rinsed in saline into an EDTA tube for cell analysis. NVO diagnosis required organism identification in spine tissue or blood and/or positive histopathology, and consistent symptoms and imaging. RESULTS: Sixty-eight patients were prospectively enrolled, comprising 14 with NVO and 54 with alternative diagnoses. The median biopsy sample polymorphonuclear (PMN) percentage for NVO patients was 80.5% (IQR 72.5-85.2), compared to 64.5% (IQR 54.0-69.0) for those without NVO (p < 0.001). Nine (64.3%) NVO patients received antibiotics within 10 days prior to spine biopsy. As a continuous measure, PMN differential showed a moderately strong ability in classifying NVO status with an area under ROC curve of 0.795; an optimal point on the curve of 71.5% corresponded to a sensitivity of 78.6%, specificity of 79.6%, negative predictive value of 93.5% and positive predictive value of 50.0%. CONCLUSION: PMN differential in DS/VB biopsies may serve as an effective diagnostic tool in the evaluation of patients with NVO particularly in ambiguous cases with an initially negative spine biopsy. Future efforts will aim to implement these findings within routine clinical practice.

Galileo-an Artificial Intelligence tool for evaluating pre-implantation kidney biopsies.

BACKGROUND: Pre-transplant procurement biopsy interpretation is challenging, also because of the low number of renal pathology experts. Artificial intelligence (AI) can assist by aiding pathologists with kidney donor biopsy assessment. Herein we present the "Galileo" AI tool, designed specifically to assist the on-call pathologist with interpreting pre-implantation kidney biopsies. METHODS: A multicenter cohort of whole slide images acquired from core-needle and wedge biopsies of the kidney was collected. A deep learning algorithm was trained to detect the main findings evaluated in the pre-implantation setting (normal glomeruli, globally sclerosed glomeruli, ischemic glomeruli, arterioles and arteries). The model obtained on the Aiforia Create platform was validated on an external dataset by three independent pathologists to evaluate the performance of the algorithm. RESULTS: Galileo demonstrated a precision, sensitivity, F1 score and total area error of 81.96%, 94.39%, 87.74%, 2.81% and 74.05%, 71.03%, 72.5%, 2% in the training and validation sets, respectively. Galileo was significantly faster than pathologists, requiring 2 min overall in the validation phase (vs 25, 22 and 31 min by 3 separate human readers, p < 0.001). Galileo-assisted detection of renal structures and quantitative information was directly integrated in the final report. CONCLUSIONS: The Galileo AI-assisted tool shows promise in speeding up pre-implantation kidney biopsy interpretation, as well as in reducing inter-observer variability. This tool may represent a starting point for further improvements based on hard endpoints such as graft survival.

Integrating allele-specific PCR with CRISPR-Cas13a for sensitive KRAS mutation detection in pancreatic cancer.

BACKGROUND: The clustered regulatory interspaced short palindromic repeats (CRISPR)-Cas13a system has strong potential for highly sensitive detection of exogenous sequences. The detection of KRASG12 point mutations with low allele frequencies may prove powerful for the formal diagnosis of pancreatic ductal adenocarcinoma (PDAC). RESULTS: We implemented preamplification of KRAS alleles (wild-type and mutant) to reveal the presence of mutant KRAS with CRISPR-Cas13a. The discrimination of KRASG12D from KRASWT was poor for the generic KRAS preamplification templates and depended on the crRNA design, the secondary structure of the target templates, and the nature of the mismatches between the guide and the templates. To improve the specificity, we used an allele-specific PCR preamplification method called CASPER (Cas13a Allele-Specific PCR Enzyme Recognition). CASPER enabled specific and sensitive detection of KRASG12D with low DNA input. CASPER detected KRAS mutations in DNA extracted from patients' pancreatic ultrasound-guided fine-needle aspiration fluid. CONCLUSION: CASPER is easy to implement and is a versatile and reliable method that is virtually adaptable to any point mutation.