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Bile duct cytology
Example alt text
Adenocarcinoma of bile duct
Definition
Study of bile duct cells for diagnosing diseases
Specialty
  • Cytopathology
  • Gastroenterology
Imaging techniques
  • Endoscopic retrograde cholangiopancreatography (ERCP)
  • Endoscopic ultrasound (EUS)
  • Magnetic resonance cholangiopancreatography (MRCP)
Sample collection methods
  • Bile aspiration
  • Bile duct lavage
  • Brush biopsy
Cytogenic examination
  • Fluorescence in situ hybridisation (FISH)
  • Next-generation sequencing (NGS)
  • microRNA
Classification of Results
  • Benign
  • Malignancy
  • Dysplasia

Bile duct cytology is a pathological diagnostic procedure used to examine cell samples obtained from the bile duct to identify structural and genetic abnormalities[1][2]. They provide insights into diagnoses of hepatobiliary diseases, for example, bile duct obstruction, cholangitis or cholangiocarcinoma[3][4].

To obtain cells from the bile duct epithelium, one must visualise the area using endoscopic retrograde cholangiopancreatography (ERCP), endoscopic ultrasound (EUS) and magnetic resonance cholangiopancreatography (MRCP)[5][6][7]. Biliary cells are obtained by numerous methods, for example, bile duct aspiration, bile duct biopsy and obtaining bile duct lavage[8][9][10]. Complementary cytogenetic techniques such as fluorescence in situ hybridisation (FISH), next generation sequencing (NGS), and examining microDNA are employed to support primary findings[5][11].

Following a thorough examination of the specimen by pathologists, in conjunction with cytology reports detailing specific biomarkers, doctors can confirm whether the tissue sample is benign or malignant.

However, there results could be misidentified or misclassified, giving rise to wrong results and false diagnoses[12][13].

The emergence of bile duct cytology and visualisation techniques can be traced back to the 1950s, with the introduction of Intravenous cholangiography, percutaneous transhepatic cholangiography (PTC) and secretin test[14][15][16]. In 1975, the first ever documented attempt of ERCP and brush cytology was made[17].

Modern Endoscopic Evaluation of the Bile Duct

[edit]

Visualising the bile duct is essential for collecting cells from the bile duct for examination. Endoscopic retrograde cholangiopancreatography (ERCP), endoscopic ultrasound (EUS) and magnetic resonance cholangiopancreatography (MRCP) are widely adopted tools in examining biliary structures. Though they serve the same purpose, different biliary diseases and individuals call for different imaging techniques, often case-sensitive[18]

Diagram showing how the endoscope is inserted into the bile duct during ERCP.

Endoscopic Retrograde Cholangiopancreatography (ERCP)

[edit]

ERCP is an established practice for bile duct endoscopy[5]. It combines endoscopy and fluoroscopy to observe and treat problems within the liver, pancreas and gallbladder. The individual is first put into deep sedation with general anesthesia such as propofol[19][20]. Then, a contrast agent is injected into the individual, and radiographic guidance reveals any abnormal strictures in the biliary tree. A duodenoscope further aids the discovery of minute anomalies within the duct.

Image of a radial echoendoscope.


Endoscopic Ultrasound (EUS)

[edit]

EUS comprises endoscopic visualisation and ultrasound imaging, in observing the bile duct and the surrounding vascular network and lymphatics[6]. Evaluation and curing of pancreaticobiliary disease often necessitate EUS. Commonly used echoendoscopes are radial EUS and linear EUS. Similar to computed tomography (CT), radial EUS provides a 360-degree view in the plane perpendicular to the direction the scope[6]. Alternatively, linear EUS displays an oblique image running parallel to the scope. If the stenosis within the bile duct does not permit passage of standard EUS scopes, mini-probes are used. EUS is considered a salvage therapy when ERCP has failed or when the bile duct had been surgically altered or completely blocked[21].

Image showing MRCP of common bile duct (CBD) stones,

Magnetic Resonance Cholangiopancreatography (MRCP)

[edit]

Contrary to the invasive nature of ERCP and EUS, MRCP does not require injecting contrast agents or using ionising radiation. Overall, it is accurate in evaluating the shapes and sizes of bile duct stones (round or prismatic) and aberrant tissue obstruction within the bile duct[7][22]. Recent advancement in MRCP is tridimensional imaging, which enhances image resolution and allows for a three-dimensional, spatial depiction of the relevant bile duct structures[7].


Cytological Techniques in Bile Duct Analysis

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Sample Collection

[edit]

Sample collection is an essential step for examining cells in the bile duct. The most commonly employed methods currently include bile aspiration, bile duct lavage, and brush biopsy[5][8][9].

Bile Aspiration

[edit]

Bile aspiration is the most established and convenient method of obtaining cells from the biliary tree for cytodiagnosis. With the aid of a guidewire, the catheter scrapes tissue across the stricture to collect samples. A central suction line is connected to the catheter to aspirate and retrieve samples[8]. Fine needle aspiration is a newer method with improved accuracy. The steel needle has a diameter of 0.8mm and can be advanced up to 10 cm beyond its sheath[23]. By moving back and forth, it collects tissue from the lesion. Afterwards, the needle is withdrawn and proceeds with subsequent staining.

Bile Duct Lavage

[edit]

Bile duct lavage is a standard procedure following brush cytology. A catheter injects sterile solution into the bile duct to wash out any fluid and debris[9]. Saline is administered at regular intervals, with 10-15 ml of normal saline injected into the gallbladder. This is followed by a series of saline flushes in 2-3 ml aliquots to irrigate the gallbladder and biliary ducts[24]. It is beneficial as it provides more cells for effectively diagnosing the cytology of indeterminate biliary strictures[25].

Brush Biopsy

[edit]
Image showing retrieval of cells after brush cytology.

Brush biopsy is a common sampling technique in cytological examinations[10]. It uses a specially designed brush that obtains cells from all epithelial layers[26]. The method involves opening the brush to expose bristles and carefully swiping it along the tissue layer. The brush with desired cells is cut from the catheter and placed in fixative, allowing it to agitate to dislodge tissues[26].

Slide Preparation

[edit]

Immediately after sample extraction, the brush, containing the samples, is smeared directly onto a glass slide. It is followed by wet fixation using absolute alcohol[27], which stabilises the cells, prevents loss of cell contents and aids permeation of stains[28]. Staining creates drastic contrasts in cellular images, allowing pathologists to examine delicate structures closely. Both hematoxylin and eosin stain and Papanicolaou stain are well-adopted staining methods in cytological examination[27][29].

Cytogenic Examination

[edit]

Bile duct cytogenetic examination analyses genetic material from bile duct cells, aiding diagnosis of biliary disorders and malignancies. Common approaches include fluorescence in situ hybridisation (FISH), next-generation sequencing (NGS), and investigation of microRNA.

Image showing fluorescent DNA in FISH.

Fluorescence in situ hybridisation (FISH)

[edit]

FISH is a molecular technique used to detect specific genetic abnormalities at a chromosomal level. It uses fluorescently labeled probes designed to bind to particular DNA sequences within the chromosomes of cells. When these probes attach to their target regions, they emit fluorescence, detected and visualised under a fluorescence microscope. FISH is particularly valuable for identifying aneuploidies, which are deviations from the normal number of chromosomes (46), as well as large inversions, deletions or duplications of chromosomal segments[1]. It commonly observes two types of chromosomal abnormalities, polysomy or monosomy[2].


Next-generation sequencing (NGS)

[edit]

NGS is the process of using a DNA sequencer to read nucleotide base sequences. The latest models of DNA sequencers have high throughput, allowing the screening of thousands of genes simultaneously with great precision. A geneticist then interprets the sequences obtained to identify any pathogenic gene variants that predispose the subject to cancer. Common target genes of NGS in oncopathological studies are KRAS and TP53, which are tumour-suppressing genes. Often, a combination of mutations is found in malignant tumours[5][30].

MicroRNA

[edit]

MicroRNAs (miR) are small, non-coding molecules that regulate mRNA expression, abundantly found in extracellular vesicles[11]. To obtain miRs, mRNA is first isolated from brush cytology samples and transcribed into miR using reverse transcription kits. Next, to reveal the amount of miR present in the sample, quantitative miR expression analysis is performed[31]. Certain miRs are specific to certain malignant, making them a distinctive biomarker in bile duct cancer with high diagnostic potential.

Other Methods

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Immunocolorimetric ELISA

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ELISA is a biochemistry assay that detects and quantifies the amount of target protein by making use of specific identification structures called antigens. Colour change occurs once antigens are detected in the reaction mixture. The colour intensity of the solution is measured using a colorimeter and can thus be used to infer the concentration of antigens[32]. Minichromosome maintenance proteins (MCM) are notable targets in cancer research. Given their involvement in DNA synthesis initiation, any dysregulation is highly indicative of premalignant or malignant tumours[5].

Result analysis in Bile Duct Cytology

[edit]

Cellular Characteristics

[edit]
Benign bile ducts with considerable inflammatory cell infiltration.

Benign cells

[edit]

Benign cells are non-cancerous, slow-growing, and non-invasive[33]. They typically exhibit a picket-fence arrangement, with basally located nuclei and columnar, non-mucinous cytoplasm[34]. Such conditions are often linked to infiltrating inflammation, bile duct stones, and necrosis[3]. Looking at benign tissues as a whole, they tend to have smooth borders with tapered margins[7].



Microscopic image of bile duct dysplasia.

Dysplasia

[edit]

Dysplasia conditions precede the formation of cancer, which is often referred to as premalignant lesions[34]. Common features include nuclear pseudostratification, distorted cell polarity, cribriforming, and abnormally large nuclei[35]. It is recommended to conduct bile duct brushing of the remaining bile duct to assess for any sub-clinical carcinomatous changes[36].


Imaging of bile duct malignancy.

Malignant cells

[edit]

Malignant cells are cancerous cells that divide uncontrollably and invade other tissues[4]. They present with features such as enlarged nuclei, anisonucleosis, meaning abnormal size of nucleus, loss of cellular polarity, and crowding or overlapping of cells[34]. Looking at malignant tissues as a whole, they are mostly irregular with shouldered margins[7]. To confirm the extent of malignancy and determine the stage of cancer, immediate imaging studies such as magnetic resonance imaging and endoscopic retrograde cholangiopancreatography should be conducted[37].


Background Assessment

[edit]

Besides carcinoma, bile duct cytology also reveals background complications such as inflammation or necrosis of bile duct epithelial cells[3]. Inflammation of the bile duct is caused by reactive cells, such as cysts and infections[38]. Although the histological findings of bile duct cells are not specific, reactive bile duct cells are observed in sheets that maintain a monolayer appearance, exhibiting slight nuclear overlap, enlarged nuclei, smooth nuclear outlines, finely granular chromatin, and small, visible nucleoli[39].

Bile duct necrosis is a serious complication of localised death of bile duct epithelial cells[40]. Under microscopic view, a necrotic bile duct appears as a dark brown tubular structure with a large dark green cystic mass-like lesion[41]. It causes significant coagulation on the lumen, which exacerbates necrosis of peri-bile duct tissue[41]. The expansive spread of necrotic tissue may cause thrombosis of the portal vein, which runs along the bile duct[41]. Under endoscopic examination, thrombosis can be identified as a filling of the lumen by an unusual mass[42].

Diagnostic Challenges and Improvements

[edit]

Sampling Error

[edit]

Sampling errors remain the major reason for insensitivity or false positive results in brush biopsy[12]. Malignant cells cannot be identified because of technical challenges, including difficult anatomic sites, extensive fibrosis, poor visualisation, and the presence of benign epithelium covering the tumor[13]. A combination of brush cytology and forceps biopsy or endoscopic needle aspiration may be employed to improve diagnostic yield[13][43]. Utilising long cytobrushes to sample larger and longer areas of the biliary tract can increase cellularity to reduce false positives[43]. The cell-collecting technique should be optimised by determining whether to push or pull the brush through the sheath[13].

Misidentification of Malignancy

[edit]

Misidentifying malignancy is a significant diagnostic challenge, particularly with well-differentiated carcinomas such as mucinous and papillary tumors[13]. These tumors can morphologically present as benign, leading to potential false-negative diagnoses[44]. Additionally, smear backgrounds with necrotic debris can obscure viable malignant cells, presenting another diagnostic pitfall[13]. Abundant inflammatory exudate can complicate the differentiation between necrosis related to tumors and inflammatory changes[13]. Cells that appear to be undergoing degenerative changes due to inflammation may also be mistakenly overlooked in malignant conditions[45]. With this ambiguity in mind, cytologists should pay attention to identifying susceptible cytological features to detect malignancy accurately.

History of Bile Duct Visualisation and Cytology

[edit]

Development in visualising the bile duct

[edit]

Intravenous Cholangiography

[edit]

Introduced in 1953, intravenous cholangiography involves injecting hexa-iodo organic compounds into the bloodstream and visualising the biliary structure with laminography[46][14]. However, it is contraindicated in subjects with impaired hepatic functions, as such contrasting agents must undergo hepatic metabolism before being excreted by the body[14].

Percutaneous Transhepatic Cholangiography (PTC)

[edit]

To tackle the above constraint, percutaneous transhepatic cholangiography (PTC) was proposed in 1956 as an alternative to intravenous cholangiography[15]. The subject is first put under anesthesia, and an organic iodine compound is injected into the biliary duct. X-ray visualises the bile duct and the image is examined for any abnormal occlusions[47][48]. This procedure risks puncturing the extrahepatic biliary system and passage of bile into blood, which may result in bile peritonitis and septic shock. One case of death has been recorded post-operation[47].

Development in collecting bile duct samples

[edit]

Secretin Test

[edit]

Secretin test has been utilised to test cholangiopancreatic function since the early 20th century[16]. Secretin is injected intravenously to stimulate pancreatic secretions and diagnose the condition within the biliary duct[49]. Cells are isolated from the secretion for staining, while the biliary pigment concentration is examined. Low pigment concentration indicates potential bile duct occlusion[50]. As newer methodologies emerge, this method is rendered obsolete in modern routines.

Endoscopic Retrograde Cholangiopancreatography with Bile Duct Cytology

[edit]

The diagnostic potential of endoscopic retrograde cholangiopancreatography (ERCP) became apparent in 1975, when Osnes first performed ERCP with brush cytology[17][51]. Cells of the bile duct were collected directly for cytological examinations. The minimally invasive nature gains traction over time in detecting dysplastic cells before evolving into malignant carcinoma[17].

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[edit]
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