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The Principles and Techniques of Biopsy
PATHOLOGY
to be, but the higher the chance of morbidity. Conversely, the less tissue taken, the lower the morbidity and mortality, but the diagnostic difficulty will be greater for the reporting pathologist, and the chance of an inconclusive report will be increased. It is clear that there is a fine balance to be achieved when taking a biopsy to maximize the diagnostic yield and keep the risk to the patient as low as possible. Endoscopic biopsy is a routine procedure in everyday clinical practice, generating an enormous amount of work for clinicians as well as pathologists (Figures 1 and 2). It is therefore essential for both the endoscopist and pathologist to have a thorough understanding of the various requirements and difficulties involved in all of the aspects of the procedure. The opportunity to take biopsies is one of the main distinguishing features between endoscopic and radiological examinations in the gut. Occasionally, an endoscopy is done solely to obtain diagnostic tissue: for example, a duodenal biopsy for the diagnosis of coeliac disease.
The Principles and Techniques of Biopsy With Special Reference to Endoscopic Biopsy Linmarie Ludeman Roland M Valori Neil A Shepherd
The word biopsy derives from Greek and may be loosely translated as a view of the living (bios = life, opsis = sight). The tissue for examination may be obtained from virtually any part of the human body, using a wide variety of techniques. The tissue samples are then examined either histologically, cultured for microbiology or analysed biochemically, depending on the clinical indications. Samples may also be used for cytogenetic analysis or for molecular studies, although these procedures are, at present, uncommon in routine practice. This contribution will concentrate specifically on endoscopic biopsy, discussing the indications, techniques and pathological considerations relevant to clinical practice, as it is one of the most common special investigations in clinical practice today. An overview of various aspects of biopsy may be found in Surgery 1999; 17(10): iii–vii. The amount of tissue required for diagnosis varies greatly, depending on the type of tissue, the biopsy technique and the indication for the biopsy. The more tissue available for diagnosis, the more accurate the eventual diagnosis is likely
1 Forceps biopsy of normal colonic mucosa (note normal vascular pattern above) viewed via the colonoscope.
Linmarie Ludeman is a Specialist Registrar in Histopathology at Gloucestershire Royal Hospital, Gloucester, UK. She qualified from the University of the Orange Free State, Bloemfontein, South Africa. She started her pathology training in Sheffield, UK and is currently part of the Bristol rotation in the UK. Roland M Valori is Consultant Gastroenterologist at Gloucestershire Royal Hospital, Gloucester, UK. He was formerly a Senior Lecturer at University College and Middlesex Hospitals, London, UK. He trained in Gastroenterology at the Royal London Hospital, London, UK, and St Mark’s Hospital, Birmingham, UK. His research interest is in the application of evidence-based medicine to gastroenterology. Neil A Shepherd is Consultant Histopathologist at Gloucestershire Royal Hospital, Gloucester, UK, and Cheltenham General Hospital, Cheltenham, UK, and Visiting Professor at the University of Cranfield, UK. His research interests are largely in the pathology and molecular biology of gastro-intestinal neoplasia.
SURGERY
2 Endoscopic view of numerous gastric polyps, one being biopsied. Histologically, these were fundic gland cyst polyps, common benign lesions of the stomach.
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Coeliac disease In the past it has been necessary to use a Crosby capsule to obtain sufficient tissue to enable a diagnosis of coeliac disease. It is now accepted that three to four endoscopic biopsies taken from the second part of the duodenum or beyond is sufficient. The pathologist should be warned if the endoscopist is unsure about the limit of the examination. This is because proximal duodenal biopsies may be difficult to interpret, leading to a false-positive diagnosis.
Endoscopic biopsy Any suspicious or unusual lesion identified at endoscopy should be biopsied. This is particularly the case for possible malignant lesions (ulcers and polypoid lesions, Figure 2). The exception to this rule is when the lesion appears vascular, in which case endoscopic biopsy may be dangerous. To maximize the benefit from endoscopic biopsy it is helpful to have a protocol for biopsy in specific situations.
Inflammatory bowel disease Conventional mucosal biopsies are sufficient for the pathologist to diagnose inflammatory bowel disease. The site of the biopsies needs to be clearly identified because the distribution of inflammation is an important determinant of the final diagnosis. To facilitate this the biopsies can be mounted on a cellulose acetate strip. Each biopsy is mounted in order, with the biopsies abutting each other in a straight line, so that all the biopsies can be mounted on one slide. It is important for at least one of these biopsies to be taken from the rectum. It can be helpful to obtain biopsies from the same level in the colon if patchy inflammation is identified because varying degrees of chronic inflammation at the same level of the colon are highly suggestive of Crohn’s disease. Patients who have had ulcerative colitis affecting the entire colon for more than 10 years are at increased risk of colorectal cancer. These patients are often offered surveillance colonoscopy with multiple biopsies. There are no agreed guidelines on the frequency of examination or the number and sites of biopsies. A two-year interval is the most popular and the more biopsies taken the greater the likelihood of identifying dysplasia or malignancy. It is possible to obtain two biopsies with each passage of a conventional biopsy forceps while some biopsy forceps are able to take multiple biopsies. It is recommended that a minimum of 24 biopsies be taken from 12 paired sites at 5–10 cm intervals from the caecum to the rectum. It is important to take multiple biopsies from any suspicious lesions. Inflammatory polyps are common in chronic inflammatory bowel disease, particularly if there has been severe inflammation previously. These polyps often have a filiform shape with bifid forms. They are usually a deeper red than adenomatous polyps. It is not necessary to remove these polyps unless they look atypical.
Barrett’s oesophagus Barrett’s oesophagus is a true metaplasia in which the normal squamous mucosa of the oesophagus is replaced by columnarlined epithelium as a result of gastric (and probably duodenal) content reflux. Barrett’s oesophagus is associated with an increased risk of oesophageal adenocarcinoma, particularly if there is associated dysplasia. Dysplasia can be detected only microscopically. Therefore, when Barrett’s oesophagus is identified, biopsies should be taken at quadrantic and segmental (every 2 cm) intervals above the level of the lower oesophageal sphincter. Sometimes it can be difficult to identify this level in the presence of a hiatus hernia. The upper limit of vertical folds in the cardia of the stomach identifies the anatomical junction between the oesophagus and stomach. In view of the increased risk of oesophageal carcinoma, surveillance endoscopy with multiple biopsies has been recommended. The benefit of surveillance depends on the annual incidence of malignancy in Barrett’s oesophagus. An incidence of more than 1% makes surveillance worthwhile, whereas an incidence below 0.5% would not. The overall incidence of malignant transformation in Barrett’s oesophagus probably lies somewhere between these two limits. Currently, there is no reliable way of identifying high-risk groups, apart from those known to have dysplasia, those with peptic stricturing and ulceration and those with long segment disease. Ulceration in the upper gut Ulceration of the first part of the duodenum is invariably benign and associated with Helicobacter infection or the use of aspirin or other non-steroidal anti-inflammatory drugs (NSAIDs). Therefore, it is not usually necessary to biopsy duodenal ulcers directly. However, it may be important to take antral biopsies for histology and urease testing when an ulcer is identified in order to identify Helicobacter infection. Distal duodenal ulcers should be biopsied because they are more likely to be malignant. All gastric ulcers should be biopsied and brushings must be taken for cytology. Biopsy of the ulcer base is unhelpful because this will show only granulation tissue or exudate. Ideally, gastric ulcers should be biopsied at the ulcer rim with a minimum of four biopsies, one from each quadrant. Brushings taken for cytology should also be taken from the ulcer edge. Patients with gastric ulcers should have repeat endoscopy after an interval (3–6 months) to ensure healing. Unhealed ulcers should be followed up indefinitely until healed because of the risk of undetected cancer and the high risk of malignant transformation. Antral biopsies for identification of Helicobacter may also be taken, although it has been argued that there are more economic means of diagnosing Helicobacter infection than histological assessment.
SURGERY
Colonic polyps Colonic polyps are usually removed with diathermy techniques (Figure 3). The purpose of diathermy is to ablate any underlying adenomatous tissue that has not been removed and to cauterize blood vessels to minimize bleeding. Small polyps (less than 5 mm in diameter) can be removed with ‘hot’ biopsy (see page vii) diathermy forceps. The polyp is grasped by the biopsy forceps, using a conventional biopsy technique. The mucosa is lifted and tented and a small current applied until the mucosa adjacent to the biopsy forceps blanches. Care must be taken not to apply too much current because the very small contact area leads to intense current density and heating which increase the risk of perforation, particularly in the right colon. Snare polypectomy is used for larger polyps. A snare is
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a
a A multilobulated tubulo-villous adenoma viewed via the colonoscope. It was snared and removed. b The site of the polyp showing the diathermy burn.
b
3
looped over the polyp and tightened around the stalk or base. Current is then applied as the snare is gently closed. High diathermy settings may be required for broad-based and thickstalked polyps. However, as the snare burns and the electrical contact area reduces, it may be necessary to turn the diathermy setting down to reduce the risk of perforation. Once the polyp has been removed it is important to retrieve it for histological examination. This can be done by sucking it through the biopsy channel of the instrument into a mucous trap or into a piece of gauze introduced in the suction circuit outside the colonoscope. Otherwise, the polyp needs to be removed with the diathermy snare or a polyp basket. Removing the polyp in this way requires withdrawal of the instrument and re-insertion if further polyps need to be removed or further biopsies taken. Large, broad-based polyps can be removed more easily and safely by lifting them off the submucosa with saline injections around and within the polyp. If the polyp does not lift off the underlying tissue easily there is an increased likelihood of associated malignancy. In this situation it may be better to biopsy the polyp and consider surgical resection.
Pathological considerations in endoscopic biopsy The pathological request form Despite recent advances in technology and new inventions within the medical world, one of the most important parts of any procedure requiring histological evaluation remains the request form and the information contained on it. Sadly, this is an area much neglected by clinicians who may not appreciate the importance of adequate and relevant information as a part of the histological assessment of a biopsy. The word ‘Biopsy’ scribbled somewhere on an otherwise empty form is not sufficient information in any instance. Although the pathologist may be tempted to issue a report saying: ‘Yes, it is’, this is of no benefit to the patient. It has to be remembered that the pathology request form is a specialist referral from one consultant to another, and it is often essential in the making of a diagnosis. For instance, the diagnosis of microscopic colitis requires normal endoscopic appearances with a history of chronic diarrhoea, and cannot be made if the endoscopic findings are not known. Here, it might be useful to include a copy of the endoscopic report with the histology request form. In the same way, the level of biopsies taken from the oesophagus is essential in the diagnosis of Barrett’s oesophagus and often the diagnosis cannot be made if the level is not known.
Safety considerations in endoscopic biopsy Any instrument passed through the biopsy channel of an endoscope must be done under direct vision. If a clear endoscopic view cannot be obtained, great care must be exercised when advancing the biopsy forceps. This is because it is possible to perforate the gut, particularly if the wall is very thin and fragile, as it is in the oesophagus and right colon. More advanced biopsy techniques, such as polypectomy, should be performed only by fully trained personnel or trainees under direct supervision. Modern disinfection techniques are capable of sterilizing re-usable equipment. However, the risk of variant CJD (vCJD) infection from endoscopic biopsies is considered to be higher than from other types of biopsies because of the high concentration of lymphoid tissue in the gut. Therefore, it is likely that all endoscopy units will be required to use disposable biopsy equipment in the near future.
SURGERY
Biopsy orientation Orientation is of utmost importance in assessing some features, such as villous architecture in coeliac disease and completeness of excision for dysplastic lesions. Endoscopic biopsies are often very difficult to orientate and attempts to do so may cause damage to the biopsy, due to trauma through handling or air-drying. In cases where multiple colonic biopsies are taken from the same patient, the biopsies may be placed in order on a piece of cellulose acetate with the mucosal surface uppermost. Some laboratories place the biopsies directly in cassettes with six chambers, and some routinely cut sections at multiple levels through the tissue block to aim for at least some degree of orientation.
v
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PATHOLOGY
Special techniques and staining All biopsy sections are routinely stained with haematoxylin and eosin. Oesophageal, gastric and duodenal biopsy sections are stained with AB/PAS (alcian blue/periodic acid-Schiff) to enhance the demonstration of acid and neutral mucins (Figure 4) in the diagnosis of intestinal metaplasia and mucin-secreting carcinomas, as well as fungi and Whipple’s disease in the duodenum. All gastric biopsy sections should be routinely stained to detect Helicobacter pylori by using modified Giemsa, Warthin-Starry, Gimenez, Genta or half Gram stains. Special stains for other organisms can be used, depending on the clinical indication. Immunohistochemistry is often used to differentiate different kinds of tumours. Tissue samples can also be cultured to reveal the presence of micro-organisms, analysed biochemically and used for cytogenetic analysis or for molecular studies depending on the clinical suspicion, though these last two procedures are at present uncommon in routine practice.
propria. The lamina propria may show a vacuolated appearance if the peroxide with which the instruments were cleaned comes into contact with the mucosa. Inflation: it is not uncommon to find gas in the lamina propria, and even in the submucosa (termed pseudolipomatosis coli) as a result of inflation of the colon with air. It is a common finding of no significance. Pseudolipomatosis may superficially resemble adipose tissue within the lamina propria but this does not usually create diagnostic difficulty. Conversely, inadequate bowel inflation may lead to a macroscopic appearance of general erythema, resulting in a biopsy that will range from normal to slightly congested. Diathermy artefact: this is the most common artefact, and readily recognizable microscopically (Figure 5). The diathermy line is useful to confirm the complete excision of an adenomatous polyp, and the presence of lesional tissue at the diathermy line is a clear indication of incomplete excision (Figure 5). The artefactual distortion of the biopsy may vary from a small amount of nuclear condensation, to a frazzled appearance of the entire biopsy, rendering it all but impossible for diagnosis.
Pathological artefacts of biopsy When considering the nature of the method of obtaining the biopsies, it is clear that the fragments of tissue are subject to a varying degree of trauma, from the initial preparation of the area of biopsy, such as bowel preparation, to the endoscopic procedure, the actual taking of the biopsy and its retrieval. The tissue sample is subsequently fixed in formalin and embedded in paraffin wax. Sections are then cut and stained before the slides are ready for microscopic examination. All of these processes can lead to artefactual distortion of the sample, and the pathologist needs to be aware of this possibility when interpreting the findings. Crush artefact: in cases where the tissue is grasped with forceps, there may be traumatic distortion of the tissues. This is particularly common and well described in duodenal biopsies, when the Brunner’s gland secretions may be ‘squirted’ into the lamina propria and onto the luminal surface, thereby strongly resembling either Whipple’s disease or even a tumour (Figure 4a). These secretions are PAS positive (Figure 4b) as are the inclusions found in Whipple’s disease, thereby luring the unwary pathologist into making an incorrect diagnosis of Whipple’s disease. In the same manner, mucous cells that have become detached from the basement membrane may resemble the cells of a signet ring carcinoma. These cases can be problematic and distinction from true carcinoma may be difficult. The use of immunohistochemical markers will be of no value, as both epithelial and tumour cells show cytokeratin positivity.
aa
Bowel preparation: for all colonoscopic procedures, the colon is prepared before the passing of the endoscope, and various methods are currently used. All preparation methods will influence the histological appearance of the biopsy, to varying degrees. If irritant preparations are used to prepare the bowel there may be enhanced apoptosis and a mild inflammatory infiltrate within the superficial epithelial cells. In this case, these appearances should not be interpreted as an inflammatory condition unless there is more definite evidence such as crypt damage. More vigorous preparation techniques may lead to mucin depletion, oedema and focal congestion of the lamina
bb Duodenal biopsies showing traumatic distortion of the tissues. a Brunner’s gland secretions are ‘squirted’ into the lamina propria and onto the luminal surface, thereby resembling Whipple’s disease. b The secretions are PAS positive.
4 SURGERY
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PATHOLOGY
more friable, with poor intercellular adhesion. This so-called ‘carry over’ should be distinguished from true neoplasm, and this should always be excluded should a single group of tumour cells be found that are not attached to underlying tissues. Cutting multiple levels through the block may be of value in such circumstances. u
The latter is particularly a problem where small polyps are ‘hot’ biopsied (see page iv).
FURTHER READING Cotton P B, Williams C B. Practical Gastrointestinal Endoscopy. 4th edition. Oxford: Blackwell Science, 1998. Haboubi N Y, Geboes K, Shepherd N A, Talbot I C. Gastrointestinal Polyps. London: Greenwich Medical Media, 2002. Schiller K F, Cockel R, Hunt R H, Warren B F. Atlas of Gastrointestinal Endoscopy and Related Pathology. 2nd edition. Oxford: Blackwell Science, 2002.
Contamination: during the process of fixation, cellular debris from other samples may contaminate the biopsy sample. Blocks from tumours are often the culprits because the tumour cells are
CROSS REFERENCE Shepherd N A, Birch P A, Earnshaw J J. The principles and techniques of biopsy. Surgery 1999; 17(10): iii–vii.
5 Diathermy artefact is seen as a haematoxyphilic blush (centrally). There is an adenomatous focus (appearing dark blue to the left of the midline) and this reaches the diathermy mark. Excision of the small adenoma is incomplete.
Forthcoming Topics in Surgery 20:9 September 2002 • Anatomy of the kidney and ureter • Disorders of coagulation and haemostasis • Blood transfusion in surgery • The spleen and indications of splenectomy • Surgery in the immunocompromised patient (excluding AIDS) • Tourniquets: uses and precautions • Perioperative aneamia and its management • Applied physiology of haemopoiesis 20:10 October 2002 • Anatomy of the prostate and male urethra • Acute pancreatitis • Chronic pancreatitis • Gastric cancer • Investigation of abdominal masses • What is new in colorectal cancer? • Staging of gastrointestinal cancer
Urological diagnosis: history, investigations Haematuria Urinary incontinence in adults Pathology of tumours of the male genital tract
• • • • • • • •
20:12 December 2002 Bladder cancer Management of urinary calculi Pain and swelling of scrotum, epididymitis and torsion Management of testicular tumours Prostate cancer Renal tumours Pathology of tumours of the urinary tract
21:1 Janurary • Cancer of the oesophagus • Investigation of dysphagia • Reflux oesophagitis: pathophysiology and management • Postoperative complications: prevention, recognition and management • Interpretation of preoperative cardiorespiratory investigation • Postoperative analgesia • The pathology of upper gastrointestinal tumours
20:11 November 2002 • Benign prostatic hyperplasia • Urinary retention • Urinary tract infection • Urinary tract trauma
SURGERY
• • • •
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to be, but the higher the chance of morbidity. Conversely, the less tissue taken, the lower the morbidity and mortality, but the diagnostic difficulty will be greater for the reporting pathologist, and the chance of an inconclusive report will be increased. It is clear that there is a fine balance to be achieved when taking a biopsy to maximize the diagnostic yield and keep the risk to the patient as low as possible. Endoscopic biopsy is a routine procedure in everyday clinical practice, generating an enormous amount of work for clinicians as well as pathologists (Figures 1 and 2). It is therefore essential for both the endoscopist and pathologist to have a thorough understanding of the various requirements and difficulties involved in all of the aspects of the procedure. The opportunity to take biopsies is one of the main distinguishing features between endoscopic and radiological examinations in the gut. Occasionally, an endoscopy is done solely to obtain diagnostic tissue: for example, a duodenal biopsy for the diagnosis of coeliac disease.
The Principles and Techniques of Biopsy With Special Reference to Endoscopic Biopsy Linmarie Ludeman Roland M Valori Neil A Shepherd
The word biopsy derives from Greek and may be loosely translated as a view of the living (bios = life, opsis = sight). The tissue for examination may be obtained from virtually any part of the human body, using a wide variety of techniques. The tissue samples are then examined either histologically, cultured for microbiology or analysed biochemically, depending on the clinical indications. Samples may also be used for cytogenetic analysis or for molecular studies, although these procedures are, at present, uncommon in routine practice. This contribution will concentrate specifically on endoscopic biopsy, discussing the indications, techniques and pathological considerations relevant to clinical practice, as it is one of the most common special investigations in clinical practice today. An overview of various aspects of biopsy may be found in Surgery 1999; 17(10): iii–vii. The amount of tissue required for diagnosis varies greatly, depending on the type of tissue, the biopsy technique and the indication for the biopsy. The more tissue available for diagnosis, the more accurate the eventual diagnosis is likely
1 Forceps biopsy of normal colonic mucosa (note normal vascular pattern above) viewed via the colonoscope.
Linmarie Ludeman is a Specialist Registrar in Histopathology at Gloucestershire Royal Hospital, Gloucester, UK. She qualified from the University of the Orange Free State, Bloemfontein, South Africa. She started her pathology training in Sheffield, UK and is currently part of the Bristol rotation in the UK. Roland M Valori is Consultant Gastroenterologist at Gloucestershire Royal Hospital, Gloucester, UK. He was formerly a Senior Lecturer at University College and Middlesex Hospitals, London, UK. He trained in Gastroenterology at the Royal London Hospital, London, UK, and St Mark’s Hospital, Birmingham, UK. His research interest is in the application of evidence-based medicine to gastroenterology. Neil A Shepherd is Consultant Histopathologist at Gloucestershire Royal Hospital, Gloucester, UK, and Cheltenham General Hospital, Cheltenham, UK, and Visiting Professor at the University of Cranfield, UK. His research interests are largely in the pathology and molecular biology of gastro-intestinal neoplasia.
SURGERY
2 Endoscopic view of numerous gastric polyps, one being biopsied. Histologically, these were fundic gland cyst polyps, common benign lesions of the stomach.
iii
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PATHOLOGY
Coeliac disease In the past it has been necessary to use a Crosby capsule to obtain sufficient tissue to enable a diagnosis of coeliac disease. It is now accepted that three to four endoscopic biopsies taken from the second part of the duodenum or beyond is sufficient. The pathologist should be warned if the endoscopist is unsure about the limit of the examination. This is because proximal duodenal biopsies may be difficult to interpret, leading to a false-positive diagnosis.
Endoscopic biopsy Any suspicious or unusual lesion identified at endoscopy should be biopsied. This is particularly the case for possible malignant lesions (ulcers and polypoid lesions, Figure 2). The exception to this rule is when the lesion appears vascular, in which case endoscopic biopsy may be dangerous. To maximize the benefit from endoscopic biopsy it is helpful to have a protocol for biopsy in specific situations.
Inflammatory bowel disease Conventional mucosal biopsies are sufficient for the pathologist to diagnose inflammatory bowel disease. The site of the biopsies needs to be clearly identified because the distribution of inflammation is an important determinant of the final diagnosis. To facilitate this the biopsies can be mounted on a cellulose acetate strip. Each biopsy is mounted in order, with the biopsies abutting each other in a straight line, so that all the biopsies can be mounted on one slide. It is important for at least one of these biopsies to be taken from the rectum. It can be helpful to obtain biopsies from the same level in the colon if patchy inflammation is identified because varying degrees of chronic inflammation at the same level of the colon are highly suggestive of Crohn’s disease. Patients who have had ulcerative colitis affecting the entire colon for more than 10 years are at increased risk of colorectal cancer. These patients are often offered surveillance colonoscopy with multiple biopsies. There are no agreed guidelines on the frequency of examination or the number and sites of biopsies. A two-year interval is the most popular and the more biopsies taken the greater the likelihood of identifying dysplasia or malignancy. It is possible to obtain two biopsies with each passage of a conventional biopsy forceps while some biopsy forceps are able to take multiple biopsies. It is recommended that a minimum of 24 biopsies be taken from 12 paired sites at 5–10 cm intervals from the caecum to the rectum. It is important to take multiple biopsies from any suspicious lesions. Inflammatory polyps are common in chronic inflammatory bowel disease, particularly if there has been severe inflammation previously. These polyps often have a filiform shape with bifid forms. They are usually a deeper red than adenomatous polyps. It is not necessary to remove these polyps unless they look atypical.
Barrett’s oesophagus Barrett’s oesophagus is a true metaplasia in which the normal squamous mucosa of the oesophagus is replaced by columnarlined epithelium as a result of gastric (and probably duodenal) content reflux. Barrett’s oesophagus is associated with an increased risk of oesophageal adenocarcinoma, particularly if there is associated dysplasia. Dysplasia can be detected only microscopically. Therefore, when Barrett’s oesophagus is identified, biopsies should be taken at quadrantic and segmental (every 2 cm) intervals above the level of the lower oesophageal sphincter. Sometimes it can be difficult to identify this level in the presence of a hiatus hernia. The upper limit of vertical folds in the cardia of the stomach identifies the anatomical junction between the oesophagus and stomach. In view of the increased risk of oesophageal carcinoma, surveillance endoscopy with multiple biopsies has been recommended. The benefit of surveillance depends on the annual incidence of malignancy in Barrett’s oesophagus. An incidence of more than 1% makes surveillance worthwhile, whereas an incidence below 0.5% would not. The overall incidence of malignant transformation in Barrett’s oesophagus probably lies somewhere between these two limits. Currently, there is no reliable way of identifying high-risk groups, apart from those known to have dysplasia, those with peptic stricturing and ulceration and those with long segment disease. Ulceration in the upper gut Ulceration of the first part of the duodenum is invariably benign and associated with Helicobacter infection or the use of aspirin or other non-steroidal anti-inflammatory drugs (NSAIDs). Therefore, it is not usually necessary to biopsy duodenal ulcers directly. However, it may be important to take antral biopsies for histology and urease testing when an ulcer is identified in order to identify Helicobacter infection. Distal duodenal ulcers should be biopsied because they are more likely to be malignant. All gastric ulcers should be biopsied and brushings must be taken for cytology. Biopsy of the ulcer base is unhelpful because this will show only granulation tissue or exudate. Ideally, gastric ulcers should be biopsied at the ulcer rim with a minimum of four biopsies, one from each quadrant. Brushings taken for cytology should also be taken from the ulcer edge. Patients with gastric ulcers should have repeat endoscopy after an interval (3–6 months) to ensure healing. Unhealed ulcers should be followed up indefinitely until healed because of the risk of undetected cancer and the high risk of malignant transformation. Antral biopsies for identification of Helicobacter may also be taken, although it has been argued that there are more economic means of diagnosing Helicobacter infection than histological assessment.
SURGERY
Colonic polyps Colonic polyps are usually removed with diathermy techniques (Figure 3). The purpose of diathermy is to ablate any underlying adenomatous tissue that has not been removed and to cauterize blood vessels to minimize bleeding. Small polyps (less than 5 mm in diameter) can be removed with ‘hot’ biopsy (see page vii) diathermy forceps. The polyp is grasped by the biopsy forceps, using a conventional biopsy technique. The mucosa is lifted and tented and a small current applied until the mucosa adjacent to the biopsy forceps blanches. Care must be taken not to apply too much current because the very small contact area leads to intense current density and heating which increase the risk of perforation, particularly in the right colon. Snare polypectomy is used for larger polyps. A snare is
iv
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PATHOLOGY
a
a A multilobulated tubulo-villous adenoma viewed via the colonoscope. It was snared and removed. b The site of the polyp showing the diathermy burn.
b
3
looped over the polyp and tightened around the stalk or base. Current is then applied as the snare is gently closed. High diathermy settings may be required for broad-based and thickstalked polyps. However, as the snare burns and the electrical contact area reduces, it may be necessary to turn the diathermy setting down to reduce the risk of perforation. Once the polyp has been removed it is important to retrieve it for histological examination. This can be done by sucking it through the biopsy channel of the instrument into a mucous trap or into a piece of gauze introduced in the suction circuit outside the colonoscope. Otherwise, the polyp needs to be removed with the diathermy snare or a polyp basket. Removing the polyp in this way requires withdrawal of the instrument and re-insertion if further polyps need to be removed or further biopsies taken. Large, broad-based polyps can be removed more easily and safely by lifting them off the submucosa with saline injections around and within the polyp. If the polyp does not lift off the underlying tissue easily there is an increased likelihood of associated malignancy. In this situation it may be better to biopsy the polyp and consider surgical resection.
Pathological considerations in endoscopic biopsy The pathological request form Despite recent advances in technology and new inventions within the medical world, one of the most important parts of any procedure requiring histological evaluation remains the request form and the information contained on it. Sadly, this is an area much neglected by clinicians who may not appreciate the importance of adequate and relevant information as a part of the histological assessment of a biopsy. The word ‘Biopsy’ scribbled somewhere on an otherwise empty form is not sufficient information in any instance. Although the pathologist may be tempted to issue a report saying: ‘Yes, it is’, this is of no benefit to the patient. It has to be remembered that the pathology request form is a specialist referral from one consultant to another, and it is often essential in the making of a diagnosis. For instance, the diagnosis of microscopic colitis requires normal endoscopic appearances with a history of chronic diarrhoea, and cannot be made if the endoscopic findings are not known. Here, it might be useful to include a copy of the endoscopic report with the histology request form. In the same way, the level of biopsies taken from the oesophagus is essential in the diagnosis of Barrett’s oesophagus and often the diagnosis cannot be made if the level is not known.
Safety considerations in endoscopic biopsy Any instrument passed through the biopsy channel of an endoscope must be done under direct vision. If a clear endoscopic view cannot be obtained, great care must be exercised when advancing the biopsy forceps. This is because it is possible to perforate the gut, particularly if the wall is very thin and fragile, as it is in the oesophagus and right colon. More advanced biopsy techniques, such as polypectomy, should be performed only by fully trained personnel or trainees under direct supervision. Modern disinfection techniques are capable of sterilizing re-usable equipment. However, the risk of variant CJD (vCJD) infection from endoscopic biopsies is considered to be higher than from other types of biopsies because of the high concentration of lymphoid tissue in the gut. Therefore, it is likely that all endoscopy units will be required to use disposable biopsy equipment in the near future.
SURGERY
Biopsy orientation Orientation is of utmost importance in assessing some features, such as villous architecture in coeliac disease and completeness of excision for dysplastic lesions. Endoscopic biopsies are often very difficult to orientate and attempts to do so may cause damage to the biopsy, due to trauma through handling or air-drying. In cases where multiple colonic biopsies are taken from the same patient, the biopsies may be placed in order on a piece of cellulose acetate with the mucosal surface uppermost. Some laboratories place the biopsies directly in cassettes with six chambers, and some routinely cut sections at multiple levels through the tissue block to aim for at least some degree of orientation.
v
© 2002 The Medicine Publishing Company Ltd
PATHOLOGY
Special techniques and staining All biopsy sections are routinely stained with haematoxylin and eosin. Oesophageal, gastric and duodenal biopsy sections are stained with AB/PAS (alcian blue/periodic acid-Schiff) to enhance the demonstration of acid and neutral mucins (Figure 4) in the diagnosis of intestinal metaplasia and mucin-secreting carcinomas, as well as fungi and Whipple’s disease in the duodenum. All gastric biopsy sections should be routinely stained to detect Helicobacter pylori by using modified Giemsa, Warthin-Starry, Gimenez, Genta or half Gram stains. Special stains for other organisms can be used, depending on the clinical indication. Immunohistochemistry is often used to differentiate different kinds of tumours. Tissue samples can also be cultured to reveal the presence of micro-organisms, analysed biochemically and used for cytogenetic analysis or for molecular studies depending on the clinical suspicion, though these last two procedures are at present uncommon in routine practice.
propria. The lamina propria may show a vacuolated appearance if the peroxide with which the instruments were cleaned comes into contact with the mucosa. Inflation: it is not uncommon to find gas in the lamina propria, and even in the submucosa (termed pseudolipomatosis coli) as a result of inflation of the colon with air. It is a common finding of no significance. Pseudolipomatosis may superficially resemble adipose tissue within the lamina propria but this does not usually create diagnostic difficulty. Conversely, inadequate bowel inflation may lead to a macroscopic appearance of general erythema, resulting in a biopsy that will range from normal to slightly congested. Diathermy artefact: this is the most common artefact, and readily recognizable microscopically (Figure 5). The diathermy line is useful to confirm the complete excision of an adenomatous polyp, and the presence of lesional tissue at the diathermy line is a clear indication of incomplete excision (Figure 5). The artefactual distortion of the biopsy may vary from a small amount of nuclear condensation, to a frazzled appearance of the entire biopsy, rendering it all but impossible for diagnosis.
Pathological artefacts of biopsy When considering the nature of the method of obtaining the biopsies, it is clear that the fragments of tissue are subject to a varying degree of trauma, from the initial preparation of the area of biopsy, such as bowel preparation, to the endoscopic procedure, the actual taking of the biopsy and its retrieval. The tissue sample is subsequently fixed in formalin and embedded in paraffin wax. Sections are then cut and stained before the slides are ready for microscopic examination. All of these processes can lead to artefactual distortion of the sample, and the pathologist needs to be aware of this possibility when interpreting the findings. Crush artefact: in cases where the tissue is grasped with forceps, there may be traumatic distortion of the tissues. This is particularly common and well described in duodenal biopsies, when the Brunner’s gland secretions may be ‘squirted’ into the lamina propria and onto the luminal surface, thereby strongly resembling either Whipple’s disease or even a tumour (Figure 4a). These secretions are PAS positive (Figure 4b) as are the inclusions found in Whipple’s disease, thereby luring the unwary pathologist into making an incorrect diagnosis of Whipple’s disease. In the same manner, mucous cells that have become detached from the basement membrane may resemble the cells of a signet ring carcinoma. These cases can be problematic and distinction from true carcinoma may be difficult. The use of immunohistochemical markers will be of no value, as both epithelial and tumour cells show cytokeratin positivity.
aa
Bowel preparation: for all colonoscopic procedures, the colon is prepared before the passing of the endoscope, and various methods are currently used. All preparation methods will influence the histological appearance of the biopsy, to varying degrees. If irritant preparations are used to prepare the bowel there may be enhanced apoptosis and a mild inflammatory infiltrate within the superficial epithelial cells. In this case, these appearances should not be interpreted as an inflammatory condition unless there is more definite evidence such as crypt damage. More vigorous preparation techniques may lead to mucin depletion, oedema and focal congestion of the lamina
bb Duodenal biopsies showing traumatic distortion of the tissues. a Brunner’s gland secretions are ‘squirted’ into the lamina propria and onto the luminal surface, thereby resembling Whipple’s disease. b The secretions are PAS positive.
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more friable, with poor intercellular adhesion. This so-called ‘carry over’ should be distinguished from true neoplasm, and this should always be excluded should a single group of tumour cells be found that are not attached to underlying tissues. Cutting multiple levels through the block may be of value in such circumstances. u
The latter is particularly a problem where small polyps are ‘hot’ biopsied (see page iv).
FURTHER READING Cotton P B, Williams C B. Practical Gastrointestinal Endoscopy. 4th edition. Oxford: Blackwell Science, 1998. Haboubi N Y, Geboes K, Shepherd N A, Talbot I C. Gastrointestinal Polyps. London: Greenwich Medical Media, 2002. Schiller K F, Cockel R, Hunt R H, Warren B F. Atlas of Gastrointestinal Endoscopy and Related Pathology. 2nd edition. Oxford: Blackwell Science, 2002.
Contamination: during the process of fixation, cellular debris from other samples may contaminate the biopsy sample. Blocks from tumours are often the culprits because the tumour cells are
CROSS REFERENCE Shepherd N A, Birch P A, Earnshaw J J. The principles and techniques of biopsy. Surgery 1999; 17(10): iii–vii.
5 Diathermy artefact is seen as a haematoxyphilic blush (centrally). There is an adenomatous focus (appearing dark blue to the left of the midline) and this reaches the diathermy mark. Excision of the small adenoma is incomplete.
Forthcoming Topics in Surgery 20:9 September 2002 • Anatomy of the kidney and ureter • Disorders of coagulation and haemostasis • Blood transfusion in surgery • The spleen and indications of splenectomy • Surgery in the immunocompromised patient (excluding AIDS) • Tourniquets: uses and precautions • Perioperative aneamia and its management • Applied physiology of haemopoiesis 20:10 October 2002 • Anatomy of the prostate and male urethra • Acute pancreatitis • Chronic pancreatitis • Gastric cancer • Investigation of abdominal masses • What is new in colorectal cancer? • Staging of gastrointestinal cancer
Urological diagnosis: history, investigations Haematuria Urinary incontinence in adults Pathology of tumours of the male genital tract
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20:12 December 2002 Bladder cancer Management of urinary calculi Pain and swelling of scrotum, epididymitis and torsion Management of testicular tumours Prostate cancer Renal tumours Pathology of tumours of the urinary tract
21:1 Janurary • Cancer of the oesophagus • Investigation of dysphagia • Reflux oesophagitis: pathophysiology and management • Postoperative complications: prevention, recognition and management • Interpretation of preoperative cardiorespiratory investigation • Postoperative analgesia • The pathology of upper gastrointestinal tumours
20:11 November 2002 • Benign prostatic hyperplasia • Urinary retention • Urinary tract infection • Urinary tract trauma
SURGERY
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