Investigation of the usefulness of a bile duct biopsy and bile cytology using a hyperspectral camera and machine learning.

To improve the efficiency of pathological diagnoses, the development of automatic pathological diagnostic systems using artificial intelligence (AI) is progressing; however, problems include the low interpretability of AI technology and the need for large amounts of data. We herein report the usefulness of a general-purpose method that combines a hyperspectral camera with machine learning. As a result of analyzing bile duct biopsy and bile cytology specimens, which are especially difficult to determine as benign or malignant, using multiple machine learning models, both were able to identify benign or malignant cells with an accuracy rate of more than 80% (93.3% for bile duct biopsy specimens and 83.2% for bile cytology specimens). This method has the potential to contribute to the diagnosis and treatment of bile duct cancer and is expected to be widely applied and utilized in general pathological diagnoses.

Verifying a novel bile cytology scoring system.

BACKGROUND: The scoring system for bile cytology (SSBC) aims to improve bile cytology diagnostic accuracy. Here, the practicality of SSBC was verified by multiple cytotechnologists. METHODS: Bile cytological specimens were evaluated by 24 cytotechnologists using SSBC. The samples were assessed before using the SSBC (first-time assessment) according to three categories: benign, indeterminate, and malignant. A first scoring evaluation (FSE) was then performed using SSBC; each item in the scoring system was classified as present or absent. After distributing an instruction sheet with diagnostic criteria, a second scoring evaluation (SSE) was performed using SSBC. Each method was evaluated using diagnostic accuracy and interobserver and intraobserver agreement. RESULTS: Several samples were assessed as indeterminate in the first-time assessment. Although the specificity of the SSE improved, the sensitivity and accuracy decreased compared with those of the FSE. The overall interobserver agreement was fair for all parameters, including abnormal chromatin, irregular internuclear distances, irregularly overlapped nuclei, irregular cluster margins, and final evaluation in the FSE and SSE. The final evaluation by histological type exhibited slight agreement for well-differentiated tubular adenocarcinoma and almost perfect agreement for poorly differentiated tubular adenocarcinoma in the FSE and SSE. For moderately differentiated tubular adenocarcinoma, agreement was moderate in the FSE and fair in the SSE. For cholangitis, a slight agreement was observed in the FSE, which improved to fair in the SSE. CONCLUSIONS: Although the SSBC is expected to improve specificity, there exists ambiguity regarding SSBC criteria and interindividual assessment differences. Therefore, the objective assessment method should be revised.

Application of enhancer of zeste homolog 2 immunocytochemistry to bile cytology.

BACKGROUND: Bile cytology has low diagnostic sensitivity and requires ancillary techniques. This study assessed the utility of enhancer of zeste homolog 2 (EZH2) immunocytochemistry (ICC) in bile cytology. METHODS: A total of 141 bile cytology specimens from 141 patients were evaluated retrospectively. Papanicolaou-stained slides were immunostained with an antibody to EZH2. After calculation of the EZH2 labeling index (LI), the cutoff value was determined via receiver operating characteristic curve analysis. Cytological performance with and without EZH2 ICC was evaluated with reference to the final diagnosis. RESULTS: The area under the curve for the EZH2 LI was 0.955, and the cutoff value for identifying benign bile samples versus malignant ones was 24.0%. The sensitivity and specificity values for malignancy were 53.4% and 100% for routine cytology only, 89.0% and 95.7% for EZH2 ICC only, and 89.8% and 95.7% for a combination of routine cytology and EZH2 ICC. The sensitivities of EZH2 ICC only and a combination of routine cytology and EZH2 ICC were significantly improved in comparison with routine cytology only (P < .001). EZH2 ICC alone had a sensitivity of 68.0% and a specificity of 85.7% in bile samples with atypical cytology, a sensitivity of 87.0% in samples that were suspicious for malignancy, and a sensitivity of 85.7% and a specificity of 100% in samples that were negative for malignancy. CONCLUSIONS: EZH2 ICC improved the diagnostic sensitivity for pancreatobiliary adenocarcinoma in bile cytology. This method is particularly meaningful in samples of indeterminate cytology and may be useful as an initial assessment to ensure that no cancer cells are missed.

Roles of Bile-Derived Exosomes in Hepatobiliary Disease.

Exosomes are vesicles with a diameter of 30-150 nm produced by living cells and secreted into the extracellular matrix. Exosomes mediate cellular communication by carrying active molecules, such as nucleic acids, proteins, and liposomes. Although exosomes are found in various body fluids, little is known about bile-derived exosomes. This review is the first to summarize the methods of bile storage and isolation of biliary exosomes, highlighting the roles of bile-derived exosomes, especially exosomal noncoding RNAs, in physiological and disease states and discussing their potential clinical applications.

Bile cytology: A new scoring system for improving diagnostic accuracy.

BACKGROUND: Benign and malignant cells need to be distinguished in any cytological examination of bile. Here, we report an original scoring system to improve the diagnostic accuracy of bile cytology. METHODS: The study used 158 bile aspiration samples obtained for cytological examination. Fourteen cytological findings were used to differentiate benign and malignant samples. Statistical significance tests and multivariate analysis were used to determine and quantify significant findings and develop a scoring system. RESULTS: Four cytological findings were significant in discriminating between benign and malignant cells: abnormal chromatin, irregularly arranged nuclei, irregularly overlapped nuclei, and irregular cluster margins. Our newly developed scoring system based on these four cytological findings yielded excellent results with a sensitivity of 87%, specificity of 98%, and an odds ratio of 329. CONCLUSIONS: The use of our new scoring system is expected to contribute to the diagnostic accuracy of cytological evaluations of bile samples.

Clues to predict incidental gallbladder cancer.

BACKGROUND: Consequences of incidental gallbladder cancer (iGBC) following cholecystectomy may include repeat operation (depending on T stage) and worse survival (if bile spillage occurred), both avoidable if iGBC were suspected preoperatively. METHODS: A retrospective single-institution review was done. Ultrasound images for cases and controls were blindly reviewed by a radiologist. Chi-square and Student's t tests, as well as logistic regression and Kaplan-Meier analyses were used. A P ≤ 0.01 was considered significant. RESULTS: Among 5796 cholecystectomies performed 2000-2013, 26 (0.45%) were iGBC cases. These patients were older (75.61 versus 52.27 years), had more laparoscopic-to-open conversions (23.1% versus 3.9%), underwent more imaging tests, had larger common bile duct diameter (7.13 versus 5.04 mm) and higher alkaline phosphatase. Ultrasound imaging showed that gallbladder wall thickening (GBWT) without pericholecystic fluid (PCCF), but not focal-versus-diffuse GBWT, was associated significantly with iGBC (73.9% versus 47.4%). On multivariable logistic regression analysis, GBWT without PCCF, and age were the strongest predictors of iGBC. The consequences iGBC depended significantly on intraoperative bile spillage, with nearly all such patients developing carcinomatosis and significantly worse survival. CONCLUSIONS: Besides age, GBWT, dilated common bile duct, and elevated alkaline phosphatase, number of preoperative imaging modalities and the presence of GBWT without PCCF are useful predictors of iGBC. Bile spillage causes poor survival in patients with iGBC.

The usefulness of adding p53 immunocytochemistry to bile drainage cytology for the diagnosis of malignant biliary strictures.

BACKGROUND: Obstructive jaundice is frequently caused by bile duct strictures. Determination of malignant strictures is crucial for the initiation of appropriate treatment. Cytologic examination of bile drainage fluid is an easy and reproducible method of detecting malignant cells. This method, however, frequently yields indeterminate results, such as atypia or suspicious of malignancy, due to difficulties in differentiating malignancy from benign atypia. Immunocytochemical assessment of p53 expression by cells in bile drainage fluid may enhance the ability to detect malignancy. METHODS: A total of 139 samples of bile drainage fluid were obtained from 80 patients. Following cytologic examination, the samples were incubated with antibody to p53. The performance of cytology with and without p53 immunocytochemistry was evaluated, with reference to surgical or clinical findings of benign and malignant biliary strictures. RESULTS: Bile drainage cytology alone had a sensitivity of 31.6% and a specificity of 98.4% in the identification of malignant strictures, whereas the combination of p53 immunocytochemistry and bile drainage cytology had a sensitivity of 80.3% and a specificity of 92.1%. P53 immunocytochemistry alone had a sensitivity of 64.5% and a specificity of 92.7% for the identification of malignant strictures in bile drainage samples with atypical cytology, and a sensitivity of 85.0% and a specificity of 100.0% in samples with suspicious of malignancy. CONCLUSION: The addition of p53 immunocytochemistry to bile drainage cytology can be useful in identifying malignant strictures in samples showing indeterminate results on bile drainage cytology. Diagn. Cytopathol. 2017;45:592-597. © 2017 Wiley Periodicals, Inc.

Biology of Cholangiocytes: From Bench to Bedside.

Cholangiocytes, the lining epithelial cells in bile ducts, are an important subset of liver cells. They are activated by endogenous and exogenous stimuli and are involved in the modification of bile volume and composition. They are also involved in damaging and repairing the liver. Cholangiocytes have many functions including bile production. They are also involved in transport processes that regulate the volume and composition of bile. Cholangiocytes undergo proliferation and cell death under a variety of conditions. Cholangiocytes have functional and morphological heterogenecity. The immunobiology of cholangiocytes is important, particularly for understanding biliary disease. Secretion of different proinflammatory mediators, cytokines, and chemokines suggests the major role that cholangiocytes play in inflammatory reactions. Furthermore, paracrine secretion of growth factors and peptides mediates extensive cross-talk with other liver cells, including hepatocytes, stellate cells, stem cells, subepithelial myofibroblasts, endothelial cells, and inflammatory cells. Cholangiopathy refers to a category of chronic liver diseases whose primary disease target is the cholangiocyte. Cholangiopathy usually results in end-stage liver disease requiring liver transplant. We summarize the biology of cholangiocytes and redefine the concept of cholangiopathy. We also discuss the recent progress that has been made in understanding the pathogenesis of cholangiopathy and how such progress has influenced therapy.

Cytological Findings of 140 Bile Samples from Dogs and Cats and Associated Clinical Pathological Data.

BACKGROUND: Cholecystocentesis can be part of the diagnostic workup of hepatobiliary disease in small animals, but literature on cytological evaluation of bile is scant. OBJECTIVES: To determine the diagnostic utility of cytological assessment of bile aspirates. ANIMALS: Fifty-six and 78 client-owned dogs and cats, respectively, with bile collected by cholecystocentesis and submitted to our diagnostic laboratory between 1999 and 2014. METHODS: Retrospective study describing cytological findings of bile, concurrent bacterial culture results, hematological and serum biochemical data, gallbladder biopsy results, as well as final diagnosis and complications after cholecystocentesis. RESULTS: Infectious agents were found in 30% of canine and 22% of feline bile aspirates, and inflammation in 5% and 19% respectively. Presence of microorganisms was more often detected on cytological examination (24%) than by culture (21%). The most common bacterial isolates were Escherichia coli and Enterococcus spp., isolated from 14.8% and 6.7% of cultured samples respectively. Only increased canine pancreatic lipase immunoreactivity concentration (cPLI) was significantly associated with the presence of microorganisms, inflammatory cells, or both in bile. Clinically relevant complications of cholecystocentesis occurred in 2 dogs. The majority of the animals undergoing cholecystocentesis suffered from hepatic, pancreatic, gastrointestinal disease, or a combination thereof. CONCLUSIONS AND CLINICAL IMPORTANCE: Cytological examination of bile is inexpensive and straightforward, and yields diagnostically relevant information that precedes and complements bacterial culture.

Clonorchis sinensis ova in bile juice cytology from a patient with severe hyperbilirubinemia and portal vein thrombosis.

Infection with the trematode Clonorchis sinensis is the most common human fluke infection in East Asian populations. Although this infection is associated with obstructive jaundice or choledocholithiasis, portal vein thrombosis has not been reported. Here, we report the first case of a 60-year-old man who had both C. sinensis infection and portal vein thrombosis with severe hyperbilirubinemia (75.4 mg/dl). He initially presented with abdominal pain and jaundice. Computed tomography revealed gallstones, common bile duct calculus, and thrombus in the left main branch of the portal vein. A nasobiliary tube was inserted under endoscopic retrograde cholangiography. Cytology of the bile juice revealed many C. sinensis eggs. The abdominal pain and jaundice improved following choledocholithotomy and combination treatment with a chemotherapeutic agent and anti-coagulant. This case suggests that inflammation around the portal vein as a result of C. sinensis infection has the potential to evoke portal vein thrombosis. Such cases should be treated with both a chemotherapeutic agent and anti-coagulant therapy. In conclusion, the possibility of infection with C. sinensis should be considered in patients presenting with hyperbilirubinemia and portal vein thrombosis, particularly in East Asian populations.

[Mixed adenoneuroendocrine carcinoma of the gallbladder diagnosed by endoscopic transpapillary catheterization using bile cytology assessed by the cell block method].

A 65-year-old woman was suspected of having advanced gallbladder cancer based on imaging results. This was considered inoperable because it was accompanied by possible liver metastasis. To confirm the diagnosis prior to chemotherapy, endoscopic transpapillary catheterization in the gallbladder (ETCG) was performed. The bile cytology was analyzed by the cell block method, which revealed mixed adenoneuroendocrine carcinoma. Based on the cytological results, extended cholecystectomy and partial hepatectomy were performed for the metastatic lesions. Bile cytology by ETCG with the cell block method was useful for deciding the therapeutic strategy in this patient with metastatic gallbladder cancer.

Brushing cytology of the biliary tract: bile juice from the ERCP sheath tube provides cell-rich smear samples.

OBJECTIVES: To improve the diagnostic accuracy of bile smear cytology, we assessed two cell-yielding procedures. STUDY DESIGN: One hundred and forty-one patients with biliary stricture underwent endoscopic retrograde cholangiopancreatography (ERCP) and conventional brush sampling. The cytologist cut the brush head off the support wire, centrifuged it directly in tissue culture medium for 1 min at 3,000 rpm, centrifuged the medium again and then smeared the cell pellet onto slides. The remaining sheath tube was then cut into 12-cm segments, which were centrifuged in a centrifuge tube for 1 min at 3,000 rpm, collected and submitted for cytospin preparation. RESULTS: The final histopathological diagnoses based on surgery, biopsy or clinical progression were evaluated for sensitivity, specificity and accuracy. Using conventional smears alone, the sensitivity, specificity and accuracy in patients with biliary stricture were 66.1, 80.7 and 68.8%, respectively. For conventional smears, brush washing and sheath tube contents together, the sensitivity improved to 73.9%, specificity to 100% and accuracy to 78.7%. In the patients with bile duct carcinoma, the sensitivity, specificity and accuracy were 87.3, 100 and 90.7%, respectively. CONCLUSION: Superior diagnostic accuracy was achieved when conventional smear procedures were combined with the two new procedures.

Can endoscopic ultrasound-guided fine needle aspiration offer clinical benefit for thick-walled gallbladders?

BACKGROUND: No previous studies have compared cytology obtained under endoscopic transpapillary gallbladder drainage (ETGD) and EUS-guided fine needle aspiration (EUS-FNA) for thick-walled gallbladders. AIM: The present study investigated the diagnostic yield of bile cytology under ETGD and EUS-FNA for gallbladder tumors. METHODS: A total of 69 patients were diagnosed as having gallbladder wall thickening. Among these patients, 28 patients were diagnosed by clinical follow-up, solely by imaging such as computed tomography or by histological examination of surgical specimens. The remaining 41 patients underwent ETGD and/or EUS-FNA. In these 41 patients, the clinical data collected included gender, age, diameter of gallbladder wall, site of gallbladder wall thickening, final diagnosis, adverse events, and diagnostic yield of ETGD and EUS-FNA. RESULTS: Cyto-histological diagnosis with EUS-FNA was higher than that with ETGD, with a sensitivity of 100 versus 71%, specificity of 100 versus 94%, and accuracy of 100 versus 88%, respectively, in the two groups. In addition, the sampling adequacy of EUS-FNA was 100%. Adverse events were seen in five patients in the ETGD group (mild pancreatitis), although no adverse events were seen in the EUS-FNA group (P = 0.08). CONCLUSION: Our results suggest that EUS-FNA can be safely performed for the diagnosis of gallbladder lesions. Further, this procedure may be the diagnostic method of choice over cytology of bile juice obtained via ETGD to obtain histological evidence of gallbladder cancer.