Kent R. Olson MD Clinical

• Professor, Departments of Medicine and Pharmacy, University of California, San Francisco
• Medical Director, San Francisco Division, California Poison Control System

https://publichealth.berkeley.edu/people/kent-olson/

The visible fluorescent light is collected by the objective in the normal way prehypertension occurs when quizlet purchase cheap adalat line, passes to the eyepiece and any excitation rays bouncing back from the slide and coverglass are reflected back along their original path to the source and prevented from reaching the observer heart attack usher mp3 order adalat cheap online. The objective in this system also acts as a condenser arteria peronea buy cheap adalat 30 mg on-line, so the illumination and objective numerical apertures are the same pulse pressure 64 cheap adalat 30 mg, optically correct blood pressure chart age wise buy adalat line, and in their most efficient condition blood pressure medication with little side effects adalat 20mg. Light of all wavelengths pass from the source through a heat-absorbing filter, into a second filter which removes red light, and then through a wavelength selection filter which allows only the desired excitation wavelength(s) to pass. On passing through the specimen, the objective collects both exciting and fluorescent wavelengths. This is useful when normal stains cannot be used for fear of masking any fluorescent reactions. Brighter images are seen if dichroic mirrors are used as up to 90% of the excited energy can reach the preparation and 90% of the resultant visible light can be presented to the eye. Oil and water immersion objectives in low and high powers have been developed, they have higher numerical apertures and can gather more light, avoiding much of the lost stray light reflected from coverslips. The filters and light sources used in fluorescence microscopy in modern systems rely on digital image capture, and these images are monochromatic, i. The highly colored fluorescence images which appear in publications are the result of pseudo coloring composite images. The confocal microscope Using conventional epifluorescence microscopes in fluorescence microscopy, the fluorochrome present in the field of view will be excited whether it is in, or out of focus. The out of focus fluorescence will reduce the contrast and resolution of the image. The confocal system uses a pinhole stop to observe the specimen, excluding the out of focus portion of the image. With modern computer technology and software, a series of optical sections can be recombined to create a 3D image of a cell or structure even when using multiple labeling techniques. It also provides some of the formulas for these fixatives currently used in pathology, histology and anatomy. It is fair to say that the appropriate fixation of tissues for histological examination is central to all histology tests, as without this process all tissues would degrade and analysis would be useless. The last century has seen the development of a range of fixatives, with few recent modifications. The mechanisms and principles by which specific fixatives act fall into several broad groups. These include the covalent addition of reactive groups and cross-links, dehydration, the effects of acids, salt formation, and heat. When choosing a fixative there is a balance between the advantages and disadvantages which each fixative possesses. The major objective of fixation in pathology is to maintain clear and consistent morphological features (Eltoum et al. The development of specific fixatives has usually been empirical, although much of the understanding of the mechanisms of fixation has been based upon information obtained from leather tanning and vaccine production. Kim Suvarna microanatomy of stained tissue sections, the original microscopic relationships between cells, cellular components. Many tissue components are soluble in aqueous acid or other liquid environments and to reliably view the microanatomy and microenvironment of these tissues the soluble components must not be lost during fixation and tissue processing. Each fixative, combined with the tissue processing protocol, maintains some molecular and macromolecular aspects of the tissue better than other fixative/processing combinations. If soluble components are lost from the cytoplasm of cells, the color of the cytoplasm on hematoxylin and eosin (H&E) staining will be reduced or modified and aspects of the appearance of the microanatomy of the tissue. Similarly, immunohistochemical evaluations of structure and function may be reduced or lost. Almost any method of fixation induces shrinkage or swelling, hardening of tissues and color variations in various histochemical stains (Sheehan & Hrapchak, 1980; Horobin, 1982; Fox et al. Various methods of fixation always produce some artifacts in the appearance of tissue on staining. However, for 40 Types of fixation 41 diagnostic pathology it is important that such artifacts are consistent, predictable and understood. The chosen fixative acts by minimizing the loss or enzymatic destruction of cellular and extracellular molecules, maintaining macromolecular structures and protecting tissues from destruction by microorganisms. The fixative should also prevent the subsequent breakdown of the tissue or molecular features by enzymatic activity and/or microorganisms during long term storage. These tissues removed from patients are an important resource which may at a later stage be subjected to further specialized tests. A fixative not only interacts initially with the tissue in its aqueous environment but it also has ongoing reactivity with any unreacted fixative and the chemically altered tissues. Fixation interacts with all phases of processing and staining from dehydration to staining of tissue sections using histochemical, enzymatic or immunohistochemical stains (Eltoum et al. It follows that any stained tissue section, produced after specific fixation combined with tissue processing, is a compromise of fixed tissue changes formed from the natural living tissue. Fixatives are therefore selected based on their ability to produce a final product needed to demonstrate a specific feature of a specific tissue (Grizzle et al. In diagnostic pathology, the fixative of choice for most pathologists has been 10% neutral buffered formalin (Grizzle et al. An important constraint in using formaldehyde has been the loss of antigen immunorecognition due to this type of fixation combined with processing the tissue to paraffin wax (Eltoum et al. However, from a clinical perspective the advent of heat-induced epitope retrieval methods, instigated in the early 1990s, have overcome many of these limitations (Shi et al. All widely used fixatives are therefore selected by compromise, with their positive aspects balancing against their less desirable features. The most important characteristic of a fixative is to support high quality and consistent staining with H&E, both initially and after storage of the paraffin blocks for at least a decade, although new guidelines within the United Kingdom recommend that paraffin processed blocks are now kept for 30 years. The fixative must have the ability to prevent short and long term destruction of the micro-architecture of the tissue by stopping the activity of catabolic enzymes and hence autolysis, minimizing the diffusion of soluble molecules from their original locations. Another important characteristic of a good fixative, which helps maintain tissue and cellular integrity, is the fixation and inactivation of infectious agents. It is also important to have good toxicological and flammability profiles which permit the safe use of the fixative (Grizzle & Fredenburgh, 2005). Other important characteristics of an ideal fixative include being useful for a wide variety of tissue types, including fat, lymphoid and neural tissues. It should preserve small and large specimens and support histochemical, immunohistochemical, in situ hybridization and other specialized procedures. The fixative should penetrate and fix tissues rapidly, have a shelf life of at least one year and be compatible with modern automated tissue processors. It should be readily disposable or recyclable, support long term tissue storage to give excellent microtomy of paraffin blocks and should be cost effective (Dapson, 1993). Types of fixation Fixation of tissues can be accomplished by physical and/or chemical methods. Each component is less soluble in water after heat fixation than the same component of a fresh egg. Picking up a frozen section on a warm microscope slide, both attaches the section to the slide and partially fixes it by heat and dehydration. Even though adequate morphology could be obtained by boiling tissue in normal saline, heat is primarily used to accelerate other forms of fixation as well as the other steps of tissue processing. The exception is the use of dry heat fixation of microorganisms prior to Gram staining. Most methods of fixation used in the processing of tissue for histopathological diagnoses rely on chemical fixation carried out by liquid fixatives. Reproducibility of the microscopic appearances of tissues after H&E staining is the prime requirement of the fixatives used for diagnostic pathology. Several chemicals, or their combinations, can act as good fixatives and accomplish many of the stated goals of fixation. Some fixatives add covalent reactive groups which may induce cross-links between proteins, individual protein moieties within nucleic acids and between nucleic acids and proteins (Horobin, 1982; Eltoum et al. Another approach to fixation is to use agents which remove free water from tissues and precipitate and coagulate the proteins. These agents denature proteins by breaking the hydrophobic bonds responsible for maintaining the tertiary structure of proteins. Some fixatives are mixtures of reagents and are referred to as compound fixatives. Microwave fixation Microwave heating can reduce times for fixation of some gross specimens and histological sections from more than 12 hours to less than 20 minutes (Kok & Boon, 2003; Leong, 2005). Microwaving tissue in formalin results in the production of large amounts of dangerous, potentially explosive vapors. In the absence of a hood for extraction or a microwave processing system designed to handle these vapors, this may cause safety problems. Freeze-drying and freeze substitution Freeze-drying is a useful technique for studying soluble materials and small molecules. These methods of fixation are used primarily in the research environment and are rarely used in the clinical laboratory setting. Boiling or poaching an egg precipitates the proteins and, on cutting, Chemical fixation this utilizes organic or non-organic solutions to maintain adequate morphological preservation. Chemical fixatives can be considered as members of three major categories: coagulant, cross-linking, and compound (Baker, 1958). Physical methods of fixation 43 Coagulant fixatives Both organic and non-organic solutions may coagulate proteins making them insoluble. Cellular architecture in vivo is maintained primarily by lipoproteins and fibrous proteins such as collagen. Coagulating these proteins maintains tissue histomorphology at the light microscope level. Unfortunately, because coagulant fixatives result in cytoplasmic flocculation and poor preservation of mitochondria and secretory granules, these fixatives are not useful in ultrastructural analysis. Alcohol denatures protein differently depending on the choice and concentration of alcohol, the presence of organic and non-organic substances and the pH and temperature of fixation. The protein denaturing effect of ethanol is > phenols > water and polyhydric alcohols > monocarboxylic acids > dicarboxylic acids (Bhakuni, 1998). Dehydrant coagulant fixatives the most commonly used in this group are alcohols. The removal and replacement of free water from tissue by any of these agents has several potential effects on proteins within the tissue. Water molecules surround hydrophobic areas of proteins and, by repulsion, force hydrophobic chemical groups into closer contact with each other stabilizing hydrophobic bonding. Similarly, molecules of water participate in hydrogen bonding in hydrophilic areas of proteins, and therefore removal of water destabilizes this hydrogen bonding. Additionally, with the water removed the structure of the protein may become partially reversed, with hydrophobic groups moving to the outside surface of the protein. Once the tertiary structure of a soluble protein has been modified, the rate of reversal to a more ordered soluble state is slow and most proteins after coagulation remain insoluble even if returned to an aqueous environment. Even though most proteins become less soluble in organic environments, up to 13% of protein may be lost. These acids may also insert a lipophilic anion into a hydrophilic region and disrupt the tertiary structures of proteins (Horobin, 1982). Picric acid or trinitrophenol dissolves slightly in water to form an acid solution (pH 2. In reactions it forms salts with basic groups of proteins causing the proteins to coagulate. Picric acid fixation produces brighter staining, but the low pH solution may cause hydrolysis and the loss of nucleic acids. Non-coagulant cross-linking fixatives Several chemicals were selected as fixatives secondary to their potential actions of forming cross-links both within and between proteins and nucleic acids. The side chains of peptides or proteins which are most reactive with methylene hydrate have the highest affinity for formaldehyde; these include lysine, cysteine, histidine, arginine, tyrosine and the reactive hydroxyl groups of serine and threonine (Table 4. Aldehyde groups are chemically and biologically reactive and are responsible for many histochemical reactions. French and Edsall (1945) and Fraenkel-Conrat and his colleagues (1948a, 1948b, 1949) meticulously identified most of the reactions of formaldehyde with amino acids and proteins using simple chemistry. In an aqueous solution, formaldehyde forms methylene hydrate, a methylene glycol as the first step in fixation (Singer, 1962). If the formalin is washed away, reactive groups may rapidly return to their original states, but any bridging which has already occurred may remain. Washing for 24 hours removes approximately half of the reactive groups and after 4 weeks up to 90% are removed (Helander, 1994). During long term storage in formalin, the reactive groups may be oxidized to the more stable groups. Returning the specimen to water or alcohol following fixation therefore reduces the further fixation of the specimen because the reactive groups produced by the initial reaction with formalin may reverse and be removed. When the current relatively short fixation times are used with 10% neutral buffered formalin (hours to days), the formation of hydroxymethyl side chains is the primary and characteristic reaction and the formation of actual cross-links may be rare.

It is reasonable not to offer any further treatment following successful endoscopic decompression in the elderly as there is a high death rate (~80% at two years) from causes other than recurrent volvulus pulse pressure variation formula purchase adalat on line. In elderly patients with co-morbidities and recurrent episodes of volvulus arteria coronaria sinistra adalat 30mg mastercard, the options are resection or two-point fixation with combined endoscopic/percutaneous tube insertion (gastrostomy tubes are frequently used for this purpose) pulse pressure measurement purchase adalat 20 mg without a prescription. Sometimes hypertension 24 purchase 20mg adalat mastercard, this can only be achieved after decompression of the caecum using a needle prehypertension causes and treatment adalat 30mg free shipping. Investigation Plain abdominal radiography confirms the presence of large bowel distension blood pressure percentile buy cheap adalat. Surgical management after resuscitation depends on the underlying cause and the relevant chapters in this book should be consulted. Functional disease requires colonoscopic decompression in the first instance and conservative management. In the presence of large bowel disease, the point of greatest distension is in the caecum, and this is heralded by the onset of pain. The use of an enhanced recovery programme with early introduction of fluids and solids is, however, becoming increasingly popular. The resultant stasis leads to accumulation of fluid and gas within the bowel, with associated distension, vomiting, absence of bowel sounds and absolute constipation. Varieties the following varieties are recognised: If a primary cause is identified this must be treated. There is no convincing evidence for the use of prokinetic drugs to treat postoperative adynamic ileus. Otherwise the decision to take a patient back to theatre in these circumstances is always difficult. The need for a laparotomy becomes increasingly likely the longer the bowel inactivity persists, particularly if it lasts for more than seven days or if bowel activity recommences following surgery and then stops again. Postoperative ileus may be prolonged in the presence of hypoproteinaemia or metabolic abnormality (see below). Infection: intra-abdominal sepsis may give rise to localised or generalised ileus. Reflex ileus: this may occur following fractures of the spine or ribs, retroperitoneal haemorrhage or even the application of a plaster jacket. Pseudo-obstruction this condition describes an obstruction, usually of the colon, that occurs in the absence of a mechanical cause or acute intra-abdominal disease. It is associated with a variety of syndromes in which there is an underlying neuropathy and/or myopathy and a range of other factors. Clinical features Paralytic ileus takes on a clinical significance if, 72 hours after laparotomy: Colonic pseudo-obstruction this may occur in an acute or a chronic form. Abdominal radiographs show evidence of colonic obstruction, with marked caecal distension being a common feature. If this is ineffective, intravenous neostigmine should be given (1 mg intravenously), with a further 1 mg given intravenously within a few minutes if the first dose is ineffective. During this procedure, it is best to sit the patient on a there has been no return of bowel sounds on auscultation; there has been no passage of flatus. Radiologically, the abdomen shows gas-filled loops of intestine with multiple fluid levels (if an erect film is felt necessary). Management Nasogastric tubes are not required routinely after elective intra-abdominal surgery. Rarely, an endoscopically placed tube colostomy is used as a vent for patients with a chronic unremitting condition. Systematic review and meta-analyis of the diagnostic and therapeutic role of water-soluble contrast agent in adhesive small bowel obstruction. Benefits and harms of adhesion barriers for abdominal surgery: a systematic review and meta-analysis. Ceresoli M, Coccolini F, Catena F, Montori G, Di Saverio S, Sartelli M, Ansaloni L. Water-soluble contrast agent in adhesive small bowel obstruction: a systematic review and meta-analysis of diagnostic and therapeutic value. Reduction in adhesive smallbowel obstruction by Seprafilm stop adhesion barrier after intestinal resection. Adhesive small bowel obstruction after laparoscopic and open colorectal surgery: a systematic review and meta-analysis. Closure of mesenteric defects in laparoscopic gastric bypass: a multicentre, randomised, parallel, openlabel trial. Abdominal examination should pay attention to tenderness and peritonism over the caecum and as with mechanical obstruction, caecal perforation is more likely if the caecal diameter is 14 cm or greater. Appendicitis is sufficiently common that appendicectomy (termed appendectomy in North America) is the most frequently performed urgent abdominal operation and is often the first major procedure performed by a surgeon in training. Advances in modern radiographic imaging have improved diagnostic accuracy; however, the diagnosis of appendicitis remains essentially clinical, requiring a mixture of observation, clinical acumen and surgical science and as such it remains an enigmatic challenge and a reminder of the art of surgical diagnosis. Although much more uncommon, the appendix also has a propensity to the formation of tumours which, despite humble and innocuous beginnings, may disseminate widely with dramatic clinical consequences. Aside from its tendancy to cause surgical pathology the appendix, long thought to be a vistigial organ, may also have important roles in both immune function and maintaining the gut microbiota. The putative role of the appendix in the pathogenesis of ulcerative colitis (appendicectomy seems to be protective) for example, may be explained by its interaction with the intestinal flora and gut immune function. The vermiform appendix is present only in humans, certain anthropoid apes and the wombat. It is a blind muscular tube with mucosal, submucosal, muscular and serosal layers. Morphologically, it is the undeveloped distal end of the large caecum found in many lower animals. At birth, the appendix is short and broad at its junction with the caecum, but differential growth of the caecum produces the typical tubular structure by about the age of 2 years (Condon). In approximately one-quarter of cases, rotation of the appendix does not occur, resulting in a pelvic, subcaecal or paracaecal position. Occasionally, the tip of the appendix becomes extraperitoneal, lying behind the caecum or ascending colon. Rarely, the caecum does not migrate A wombat is a nocturnal, burrowing Australian marsupial. Sometimes, as much as the distal one-third of the appendix is bereft of mesoappendix. The appendicular artery, a branch of the lower division of the ileocolic artery, passes behind the terminal ileum to enter the mesoappendix a short distance from the base of the appendix. Four, six or more lymphatic channels traverse the mesoappendix to empty into the ileocaecal lymph nodes. Microscopic anatomy during development to its normal position in the right lower quadrant of the abdomen. The position of the base of the appendix is constant, being found at the confluence of the three taeniae coli of the caecum, which fuse to form the outer longitudinal muscle coat of the appendix. At operation, use can be made of this to find an elusive appendix, as gentle traction on the taeniae coli, particularly the anterior taenia, will lead the operator to the base of the appendix. The mesentery of the appendix or mesoappendix arises from the lower surface of the mesentery or the terminal ileum the appendix varies considerably in length and circumference. In the base of the crypts lie argentaffin cells (Kulchitsky cells), which may give rise to carcinoid tumours (see below). The appendix is the most frequent site for carcinoid tumours, which may present with appendicitis due to occlusion of the appendiceal lumen. While no discernible change in immune function results from appendicectomy, the prominence of lymphatic tissue in the appendix of young adults seems to be important in the aetiology of appendicitis (see below). There is usually abundant lymphoid tissue in the mucosa, especially in younger individuals. Soon afterwards, Charles McBurney described the clinical manifestations of acute appendicitis including the point of maximum tenderness in the right iliac fossa that now bears his name. The incidence of appendicitis seems to have risen greatly in the first half of this century, particularly in Europe, America and Australasia, with up to 16% of the population undergoing appendicectomy. In the past 30 years, the incidence has fallen dramatically in these countries, such that the individual lifetime risk of appendicectomy is 8. Acute appendicitis is relatively rare in infants and becomes increasingly common in childhood and early adult life, reaching a peak incidence in the teens and early 20s. Aetiology There is no unifying hypothesis regarding the aetiology of acute appendicitis. Decreased dietary fibre and increased consumption of refined carbohydrates may be important. As with colonic diverticulitis, the incidence of appendicitis is lowest in societies with a high dietary fibre intake. In resource-poor countries that are adopting a more refined western-type diet, the incidence continues to rise. This is in contrast to the dramatic decrease in the incidence of appendicitis in western countries observed in the past 30 years. No reason has been established for these paradoxical changes; however, improved hygiene and a change in the pattern of childhood gastrointestinal infection related to the increased use of antibiotics may be responsible. While appendicitis is clearly associated with bacterial proliferation within the appendix, no single organism is responsible. A fibrotic stricture of the appendix usually indicates previous appendicitis that resolved without surgical intervention. Obstruction of the appendiceal orifice by tumour, particularly carcinoma of the caecum, is an occasional cause of acute appendicitis in middle-aged and elderly patients. Intestinal parasites, particularly Oxyuris vermicularis (pinworm), can proliferate in the appendix and occlude the lumen. Pathology Obstruction of the appendiceal lumen seems to be essential for the development of appendiceal gangrene and perforation. However, in many cases of early appendicitis, the appendix lumen is patent despite the presence of mucosal inflammation and lymphoid hyperplasia. Occasional clustering of cases among children and young adults suggests an infective agent, possibly viral, which initiates an inflammatory response. Seasonal variation in the incidence is also observed, with more cases occurring between May and August in northern Europe than at other times of the year. Lymphoid hyperplasia narrows the lumen of the appendix, leading to luminal obstruction. Once obstruction occurs, continued mucus secretion and inflammatory exudation increase intraluminal pressure, obstructing lymphatic drainage. Oedema and mucosal ulceration develop with bacterial translocation to the submucosa. Resolution may occur at this point either spontaneously or in response to antibiotic therapy. If the condition progresses, further distension of the appendix may cause venous obstruction and ischaemia of the appendix wall. Finally, ischaemic necrosis of the appendix wall produces gangrenous appendicitis, with free bacterial contamination of the peritoneal cavity. Alternatively, the greater omentum and loops of small bowel become adherent to the inflamed appendix, walling off the spread of peritoneal contamination and resulting in a phlegmonous mass or paracaecal abscess. Rarely, appendiceal inflammation resolves, leaving a distended mucus-filled organ termed a mucocele of the appendix. It is the potential for diffuse peritonitis that is the great threat of acute appendicitis. Peritonitis occurs as a result of free migration of bacteria through an ischaemic appendicular wall, frank perforation of a gangrenous appendix or delayed perforation of an appendix abscess. Factors that promote this process include extremes of age, immunosuppression, diabetes mellitus and faecolith obstruction of the appendix lumen, a free-lying pelvic appendix and previous abdominal surgery that limits the ability of the greater omentum to wall off the spread of peritoneal contamination. In these situations, a rapidly deteriorating clinical course is accompanied by signs of diffuse peritonitis and systemic sepsis syndrome. This is due to mid-gut visceral discomfort in response to appendiceal inflammation and obstruction. Central abdominal pain is associated with anorexia, nausea and usually one or two episodes of vomiting that follow the onset of pain (Murphy). The patient often gives a history of similar discomfort that settled spontaneously. A family history is also useful as up to one-third of children with appendicitis have a first-degree relative with a similar history. The cardinal features are those of an unwell patient with low-grade pyrexia, localised abdominal tenderness, muscle guarding and rebound tenderness. Inspection of the abdomen may show limitation of respiratory movement in the lower abdomen. The patient is then asked to point to where the pain began and where it moved (the pointing sign). Asking the patient to cough or gentle percussion over the site of maximum tenderness will elicit rebound tenderness. Periumbilical colic Pain shifting to the right iliac fossa Anorexia Nausea With progressive inflammation of the appendix, the parietal peritoneum in the right iliac fossa becomes irritated, producing more intense, constant and localised somatic pain that begins to predominate.

The ulcers typically also have a yellow necrotic floor blood pressure 90 over 60 buy adalat without prescription, from which blood and pus exude blood pressure medication sweating order genuine adalat online. In the majority of cases they are confined to the distal sigmoid colon and the rectum heart attack song cheapest adalat. A pericolitis is not uncommon and results in adhesions and may cause intestinal obstruction heart attack cpr cheap generic adalat uk. It is important to emphasise blood pressure keeps dropping buy adalat 30mg with visa, however blood pressure chart what is too low purchase adalat 20 mg online, that the presence of the parasite does not indicate that it is pathogenic. Endoscopic dilatation may be performed in expert hands as an alternative to surgical resection. There will be areas of normal colon or rectum in between areas of inflamed mucosa that are irregular and ulcerated, with a mucopurulent exudate. The earliest appearances are aphthous ulcers surrounded by a rim of erythematous mucosa. There may be stricturing, and it is important to exclude malignancy in these sites. Rectal agents can be particularly effective if the disease activity is localised to the rectum. Diloxanide furoate is effective against chronic infections associated with the passage of cysts in stools. When severe, antibiotics and indeed hospitalisation and intravenous fluids may be needed. Colonic diverticula Diverticula (hollow out-pouchings) are a common structural abnormality. Diverticula are found in the left colon in around 75% of over 70 year olds in the Western world. The condition is overwhelmingly found in the sigmoid but can affect the whole colon. Interestingly, in South-East Asia right-sided diverticular disease is more common. Diverticula are most often asymptomatic (diverticulosis) and found incidentally, but they can present clinically with sepsis or haemorrhage. In addition, invasion of the systemic circulation, which is a characteristic feature of salmonellosis, may cause severe gram-negative sepsis and septic shock may develop. Some patients may develop metastatic sepsis, including septic arthritis and osteomyelitis, meningitis, encephalitis and pancreatitis. Treatment is directed towards the responsible organism and surgery should be avoided. Aetiology Epidemiological studies indicate that diverticular disease is a consequence of a refined Western diet, deficient in dietary fibre. The combination of altered collagen structure with ageing, disordered motility and increased intraluminal pressure, most notably in the narrow sigmoid colon, results in herniation of mucosa through the circular muscle at the points where blood vessels penetrate the bowel wall. The rectum has a complete muscular coat and a wider lumen and is thus very rarely affected. Diverticular disease is rare in Africa and Asia where the diet is high in natural fibre. Clostridium difficile Clostridium difficile is a toxin-producing gram-positive bacillus that is an increasing concern in many hospitals. Although normally present in around 2% of the population, it proliferates after antibiotic treatment (especially cephalosporins). Infection may progress to pseudomembranous colitis, so called because on visualisation of the bowel, plaques of inflammatory exudate between oedematous mucosa are seen. Diagnosis is usually made by detection of the toxin in stool samples, rather than by culture. Complications of diverticular disease the majority of patients with diverticula are asymptomatic but historical studies suggest that somewhere between 10 and 30% will have symptomatic complications (Summary box 70. Rarely, diverticular disease may perforate into the retroperitoneum, leading to a psoas abscess, and even groin fistulation. Classification of contamination the degree of infection has a major impact on outcome in acute diverticulitis. Patients with inflammatory masses have a lower mortality than those with perforation (3% versus. Classification systems have been developed for acute diverticulitis to try and rationalise the literature, the most commonly used being the Hinchey classification (Table 70. On identification of abscesses in stable patients, drainage may be carried out percutaneously, avoiding the need for laparotomy/laparoscopy. Contrast studies and endoscopy are usually avoided for 6 weeks after an acute attack for fear of causing perforation. They are used subsequently, however, to exclude a coexisting carcinoma and assess the extent of diverticular disease. Diverticulitis Abscess Peritonitis Intestinal obstruction Haemorrhage Fistula formation Clinical features In mild cases, symptoms such as distension, flatulence and a sensation of heaviness in the lower abdomen may be indistinguishable from those of irritable bowel syndrome. These symptoms are thought to result from a combination of increased luminal pressure affecting wall tension and increased visceral hypersensitivity. Surgical treatment is rarely, if ever appropriate for diverticular disease in the absence of complications. Diverticulitis typically presents as persistent lower abdominal pain, usually in the left iliac fossa. The lower abdomen is tender, especially on the left, but occasionally also in the right iliac fossa if the sigmoid loop lies across the midline. The sigmoid colon may be tender and thickened on palpation and rectal examination may reveal a tender mass if an abscess has formed. Distinguishing between diverticulitis and abscess formation is difficult on clinical grounds alone and radiological imaging is essential. Generalised peritonitis as a result of free perforation presents in the typical manner with systemic upset and generalised tenderness and guarding. Bleeding from the sigmoid will be bright red with clots, whereas right-sided bleeding will be darker. Torrential bleeding is fortunately rare and, in fact, more commonly due to angiodysplasia, but diverticular bleeding may persist or recur requiring transfusion and resection. The presentation of a fistula resulting from diverticular disease depends on the site. The most common colovesical fistula results in recurrent urinary tract infections and pneumaturia (flatus in the urine) or even faeces in the urine. Excluding a carcinoma may not always be possible and may represent an indication for resection. Primary anastomosis should be used selectively but is appealing in a young fit patient without gross contamination or overwhelming sepsis. There is good evidence that simple defunctioning with a proximal stoma is associated with higher mortality than a resection. There may be a role for emergency laparoscopy in diverticular disease with washout if there is no faecal contamination. Diverticular fistulae can only be cured by resecting the affected bowel, although a defunctioning stoma can ameliorate symptoms. In colovesical fistula the sigmoid can often be pinched off the bladder and the sigmoid resected. If an anastomosis is performed, it is wise to place an omental pedicle between the bowel and bladder to prevent recurrent Management Patients are frequently recommended to take a high-fibre diet and bulk-forming laxatives, although the evidence for their effectiveness in diverticulosis or after an attack of diverticulitis is limited. Acute diverticulitis is treated by intravenous antibiotics (to cover gram-negative bacilli and anaerobes) alongside appropriate resuscitation and analgesia. A diameter of 5 cm is frequently regarded as the cut off between an abscess likely to settle with antibiotics and one likely to require intervention. Laparotomy for diverticular disease in the acute setting has considerable risk with mortality in most series of 15% and, in the case of faecal peritonitis, mortality approaches 50%. Alongside operative technique, resuscitation, anaesthesia and postoperative management should be optimised. These procedures can be technically challenging and ureteric stents are commonly required to reduce the risk of ureteric injury. Partial cystectomy may be required and assistance from a urological surgeon is often very helpful Haemorrhage from diverticular disease should be distinguished from angiodysplasia. It usually responds to conservative management and only occasionally requires resection. If the source cannot be located, then subtotal colectomy and ileostomy may be the safest option. Indications for surgery in an elective setting, in the absence of complications of the disease, are controversial. There are undoubtedly a small number of patients with recurrent attacks who should be offered an elective sigmoid colectomy (with anastomosis). This could be performed laparoscopically in experienced hands with a likely swifter recovery as well as improved cosmesis. Cohort studies suggest that in patients under 50 years old admitted with diverticulitis, 25% will have a further episode. This may be used as an argument for offering elective resection but equally suggests that 75% will not get another severe attack. Many surgeons would discuss the pros and cons of elective surgery after two emergency admissions, although general health must be carefully considered. There has been an increasing tendency, in recent years, to treat even patients with recurrent attacks of diverticulitis conservatively in the absence of complications. The lesions are only a few millimetres in size and appear as reddish, raised areas at endoscopy. If this fails, a technetium-99m (99mTc)-labelled red cell scan may confirm and localise the source of haemorrhage. Colonoscopy may allow cauterisation to be carried out and an argon laser can be helpful. If it is still not clear exactly which segment of the colon is involved, then a subtotal colectomy may be necessary. Ischaemic colitis Ischaemia of the colon typically results from thrombosis or embolism. Sudden embolic events present with severe pain out of proportion to the degree of peritonism, bloody diarrhoea, haemodynamic instability and shock. Clinical features In the majority of cases, the symptoms are subtle and patients can present with anaemia. Edward Heyde, American internist, published his findings on the association between aortic valve stenosis and angiodysplasia in a letter to the New England Journal of Medicine in 1958. Thrombotic occlusion usually occurs in the context of global atherosclerosis and the presentation tends to be less dramatic with abdominal pain and rectal bleeding. The left colon and, in particular, the splenic flexure are usually the worst affected. In some cases, ulceration at the splenic flexure associated with ischaemic colitis may heal with stricturing and present with subsequent large bowel obstruction. The point at which the colon is brought to the surface must be carefully selected to allow a colostomy bag to be applied without impinging on the anterior-superior iliac spine. Loop colostomy A transverse loop colostomy has in the past been used to defunction an anastomosis after an anterior resection. It is now less commonly employed, as it is difficult to manage and potentially disrupts the marginal arterial supply to the anastomosis. A loop left iliac fossa colostomy is still sometimes used to prevent faecal peritonitis developing following traumatic injury to the rectum, to facilitate the operative treatment of a high anal fistula, for incontinence and to defunction an obstructing low rectal cancer prior to long course chemoradiotherapy. A temporary loop colostomy is made by bringing a mobilised loop of colon to the surface, where it is held in place by a plastic bridge passed through a mesenteric window. When firm adhesion of the colostomy to the abdominal wall has taken place, the bridge can be removed. Following healing of the distal lesion for which the temporary stoma was constructed, the colostomy can be closed. It is usual to perform a contrast examination (proctogram) to check that there is no distal obstruction or continuing problem at the site of previous surgery. Colostomy closure is most easily and safely accomplished if the stoma is mature, typically after the colostomy has been established for at least 2 months. Closure is usually possible with a circumstomal incision, which avoids a full laparotomy, but it is important for patient and surgeon to consider the risks of closure carefully as it does involve a bowel anastomosis. In most hospitals, a stoma care service is available to offer advice to patients, to acquaint them with the latest appliances and to provide the appropriate psychological and practical help. Altering medication or addition of laxatives can be helpful for drug-related constipation and correction of underlying illness is clearly ideal where possible. Benzodiazepines Carbamazepine Chlorpromazine Cholestyramine Iron Opiates, particularly codeine and morphine Tricyclic antidepressants Statins Complications of stomas Stoma complications are underestimated and common (Summary box 70. Sometimes, this can be achieved with an incision immediately around the stoma but on occasion reopening the abdomen and freeing up the stoma may be necessary. Simple suture is associated with an almost 100% risk of recurrence and transfer to the opposite side of the abdomen, or insertion of a piece of prosthetic material within the abdominal wall around the stoma may be necessary. There is some evidence that stoma trephine reinforcement with mesh at the time of initial stoma formation may reduce the incidence of parastomal herniation, which may be as high as 50% over the long term. Some will have obstructed defaecation (a syndrome of impaired rectal emptying associated with pelvic floor dysfunction). Others will have slow colonic transit, a disorder usually seen in women, which may have been present since childhood or may suddenly follow abdominal or pelvic surgery. Defaecating proctography may demonstrate impaired pelvic floor relaxation, rectal intussusception and/or rectocoele if they are causing obstructed defaecation.

The costs of laparoscopic surgery are blood pressure is lowest in purchase adalat 20 mg with mastercard, however heart attack 5 stents buy 20mg adalat mastercard, generally higher and this may particularly relevant where funds are limited blood pressure medication first line purchase 20mg adalat visa. It is not possible to palpate lesions arteria nutricia effective 20 mg adalat, so if laparoscopic surgery is planned it is useful to tattoo the lesion at prior colonoscopy heart attack 6 hours buy 20mg adalat. Specimen retrieval and bowel anastomosis can then be performed via small incisions blood pressure medication heartburn buy adalat online. Dedicated training in laparoscopic colorectal surgery is important, as there is a relatively long learning curve. Antithrombosis measures should be continued as discussed in the preoperative section and are currently recommended for 28 days postoperatively. Wound infections are relatively common after colonic surgery and may well be more frequent than the 10% usually quoted. The possibility should be borne in mind in any patient not progressing as expected or with unexplained cardiac abnormalities, fever or worsening abdominal pain. In the presence of sepsis or peritonitis, early return to theatre and taking down the leaking anastomosis with the formation of stomas is usually advised. Prolonged nasogastric drainage, intravenous fluid therapy and cautious introduction of oral fluid and diet represented traditional postoperative practice. It is important to appreciate that these programmes require multiple interventions to be instituted and considerable time, effort and education from the surgical, anaesthetic and ward teams. If the lesion is right sided, it is usually possible to perform a right hemicolectomy and anastomosis in the usual manner. If there has been perforation with substantial contamination or if the patient is unstable, it may be advisable to bring out an ileocolostomy rather than forming an anastomosis. This has the advantage of converting an emergency operation with a high chance of a stoma to a situation that can be managed semi-electively by resection and anastomosis. Although early studies cast doubt on the benefits of colorectal stenting, more recently evidence has emerged that stenting leads to a reduction in stoma rates. There is evidence that adjuvant chemotherapy improves outcome after surgery in patients with node-positive disease (Dukes C). Metastatic disease Hepatic metastases can be resected and series have demonstrated 5-year survival of over 30% in resectable disease. Liver surgeons are increasingly aggressive in treatment and the only absolute limitation on what can be resected relates to leaving behind sufficient functioning liver, although this clearly has to be moderated by patient factors. It is important not to biopsy potentially resectable hepatic metastases as this may cause tumour dissemination. The role of chemotherapy and the timing of colonic and hepatic surgery in synchronous metastases is still a matter of debate and such cases should be carefully discussed by a multidisciplinary team. Isolated lung metastases may occasionally be suitable for resection but they are more commonly accompanied by metastases elsewhere. In patients with widespread disease, palliative chemotherapy is offered alongside symptomatic treatment and support by a palliative care team. Colorectal cancer follow-up Since the advent of safe liver resection for metastases the outcome benefit of follow-up has been clearly demonstrated. Follow-up aims to identify synchronous bowel tumours (present in 3%) that were not picked up at the time of original diagnosis due to emergency presentation or incomplete assessment. Similarly, 3% of patients will develop a metachronous (at a different time) colonic cancer and surveillance colonoscopy is designed to diagnose these. The most important determinant of prognosis is tumour stage and, in particular, lymph node status. Patients with disease confined to the bowel wall (Dukes stage A) will usually have cure by surgical resection alone and over 90% will have disease-free survival at 5 years. The disease remains confined to the rectum in 90% of cases but proctitis may extend proximally. Severe and/or extensive colitis may result in anaemia, hypoproteinaemia and electrolyte disturbances. The more extensive the disease the more likely extraintestinal manifestations are to occur. Extensive colitis is also associated with systemic illness, characterised by malaise, loss of appetite, and fever. Patients with severe colitis have a reduction in the number of anaerobic bacteria and in the variability of bacterial strains in the colon, but no causative link with any specific organism has been identified. Pathology In virtually all cases the disease starts in the rectum and extends proximally in continuity. Colonic inflammation is diffuse, confluent and superficial, primarily affecting the mucosa and superficial submucosa. High-grade dysplasia is regarded as an indication for surgery as 40% of colectomy specimens in which highgrade dysplasia was detected will have evidence of a colorectal cancer. In contrast, optimum management of low-grade dysplasia is currently controversial. Ten to twenty per cent of patients with low-grade dysplasia will have a cancer at colectomy. The progression rate of low-grade dysplasia to invasive cancer is unclear and many cancers in patients with low-grade dysplasia probably develop without high-grade dysplasia. Mild disease is characterized by fewer than four stools daily, with or without bleeding. Moderate disease corresponds to more than four stools daily, but with few signs of systemic illness. Inflammatory markers, including erythrocyte sedimentation rate and C-reactive protein, are often raised. Severe disease corresponds to more than six bloody stools a day and evidence of systemic illness, with fever, tachycardia, anaemia and raised inflammatory markers. This is a very significant finding, suggestive of disintegrative colitis, and an indication for emergency surgery if colonic perforation is to be avoided. Extraintestinal manifestations Arthritis occurs in around 15% of patients and is typically a large joint polyarthropathy, affecting knees, ankles, elbows and wrists. Cholangiocarcinoma is an extremely rare association and its frequency is not influenced by colectomy. Acute colitis Approximately 5% of patients present with severe acute (fulminant) colitis. Intensive medical treatment leads to remission in 70% but the remainder require urgent surgery. Toxic dilatation should be suspected in patients who develop severe Symptoms Clinical presentation depends in large part on the extent of disease. Plain abdominal radiographs should be obtained daily in patients with severe colitis, and a progressive increase in colon diameter despite medical therapy is an indication for urgent surgery. Cancer risk in colitis the risk of cancer in ulcerative colitis increases with duration of disease. Patients with pancolitis (defined as the presence of inflammation proximal to the splenic flexure) of more than ten years duration should be entered into screening programmes in order to detect clinically silent dysplasia, which is predictive of increased cancer risk. Where there has been remission and relapse, there may be regenerative mucosal nodules or pseudopolyps. Clostridium difficile colitis may need to be considered in populations at risk of this disease (see below). The monoclonal antibodies infliximab and adalimumab both act against antitumour necrosis factor alpha, which has a central role in inflammatory cascades. A stool chart should be kept and a plain abdominal radiograph is taken daily and inspected for dilatation of the transverse colon. The presence of mucosal islands or intramural gas on plain radiographs, increasing colonic diameter or a sudden increase in pulse and temperature may indicate a colonic perforation. Fluid and electrolyte balance is maintained, anaemia corrected and adequate nutrition is provided, sometimes intravenously in severe cases. The patient is treated with intravenous hydrocortisone four times daily, as well as rectal steroids. Regular and joint review by gastroenterologist and surgeon is essential to identify patients who are failing to make anticipated progress and to ensure that surgery is neither inappropriately delayed nor undertaken. Gastroenterologists will use azathioprine, cyclosporin or infliximab in severe acute attacks to attempt to induce remission. This involves the gastroenterologist, nurses, nutritionist, enterostomal therapists and, occasionally, clinical psychologists and social workers as well as the surgeon. They act as inhibitors of the cyclo-oxygenase enzyme system and are formulated to protect the aspirin-related drug from degradation before reaching the colon. The rectosigmoid stump is left long and can either be brought out as a mucous fistula or closed just beneath the skin. This operation has the advantages that the patient avoids a pelvic operation while unwell, that colonic histology can be assessed and restorative surgery can be contemplated at a later date when the patient is no longer on steroids and has fully recovered. The mesentery is divided close to the bowel and the omentum should be preserved if possible. Dissection of the left colon is continued to divide the sigmoid at a level that will comfortably reach the skin as a mucous fistula. The temptation to close the rectal stump and leave it stapled off in the pelvis should be avoided if at all possible. The diseased rectum may disintegrate, causing a pelvic abscess and severe sepsis, with potentially fatal consequences. An emergency subtotal colectomy can be performed laparoscopically, provided the surgeon and theatre team has adequate experience. Elective surgery the indications for elective surgery include: 1 failure of medical therapy/steroid dependence; 2 growth retardation in the young; 3 extraintestinal disease (polyarthropathy and pyoderma gangrenosum respond to colectomy); 4 malignant change. It is indicated for patients who are not candidates for restorative surgery due to sphincter problems or patient preference. Provided there is no concern regarding rectal cancer, a close rectal dissection may be performed to minimise damage to the pelvic nerves, avoiding erectile and bladder dysfunction. An intersphincteric excision of the anus is undertaken, which results in a smaller perineal wound and fewer healing problems. The position of the ileostomy should be carefully chosen by the patient with the help of a stoma care nurse specialist. It is reserved for patients with adequate anal sphincters and 15 cm (a) (b) Segmental resections are not recommended as even when the right side is not obviously involved there is a high recurrence rate in the remaining colon. A pouch, a completion proctectomy and even an ileorectal anastomosis can be considered at a future date. A substitute rectum is made from joined folds of ileum to form an expanded pouch of small intestine. The pouch is then joined directly to the anus at the level of the dentate line, all other rectal mucosa having been removed. In the earliest operations, the mucosa from the dentate line up to mid-rectum was stripped off the underlying muscle, but it is now known that a long muscle cuff is not needed. Although mucosectomy of the upper anal canal with an anastomosis at the dentate line is claimed to remove all of the at-risk mucosa and any problem of subsequent cancer, it may also increase the risk of incontinence with nocturnal seepage. The alternative is an anastomosis double-stapled to the top of the anal canal, preserving the upper anal mucosa. Continence appears to be better, but there is a theoretical risk of leaving inflamed mucosa behind. The procedure can be carried out in stages and a covering loop ileostomy is virtually always used. Frequency of evacuation is determined by pouch volume, completeness of emptying, reservoir inflammation and intrinsic small bowel motility, but is typically between three and eight evacuations in each 24-hour period. Increased frequency, urgency and faecal incontinence are common (20%, 5% and 5%, respectively), but usually reduce with time. Approximately 50% of patients with ileoanal pouches have a very good quality of life, whereas 35% of patients are less satisfied but choose to retain their pouches. The main reasons for failure are pelvic infection (50%), poor function (30%) and pouchitis or inflammation of the pouch (10%). It is also important for women of reproductive age to be advised that they may suffer from reduced fertility, as well as vaginal dryness, due to denervation of the secretory glands of the vaginal mucosa. Women who have not completed their families may elect for a colectomy with ileostomy and a pouch later. Pouchitis describes an inflammatory condition, which may affect 30% of patients with an ileoanal pouch for colitis. It is characterised not only by the presence of inflammation in the pouch (which is common and frequently asymptomatic) but also by symptoms of pouch dysfunction (increased frequency, tenesmus, bleeding, purulent discharge) and systemic illness (malaise, fever, raised inflammatory markers). The cause of pouchitis is unknown but it appears to relate to inflammatory bowel disease (pouchitis does not usually occur in pouches created for other indications).

The Michel medium reversibly denatures proteins within the tissue and the washing step restores the proteins to their former state arteria iliolumbalis order cheapest adalat and adalat. Remove the tissue from the wash solution and place onto a glass slide to remove excess wash solution blood pressure levels chart buy discount adalat line. This ensures that the biopsy is completely covered and has some protection from dehydration hypertension definition adalat 20mg with visa. Circle the biopsy section on the slide with an isolator hydrophobic marker pen to prevent mixing of adjacent antiserum and label the slide with the antibody specificity heart attack sam tsui chrissy costanza of atc order adalat 30 mg without prescription. The slide should be wiped around the isolator ring if required but care should be taken not to wipe the section off the slide blood pressure medication lotrel order adalat on line amex. Flood the section with working-strength antibody or conjugated antibody and incubate for 30 minutes arrhythmia only at night buy adalat 30 mg otc. The slide should be wiped around the isolator ring if required, 370 19 Immunohistochemical and immunofluorescent techniques no explosion risk, are non-toxic and do not have warm-up or cool-down times. Excitation and emission filters act complementary to each other and have transmission ranges which are appropriate for the fluorochrome being used. Colored glass filters were originally used but these have been superseded by broad or narrow-band interference filters. Unlike glass filters, the interference filters have a high transmission and a near vertical cut-off. The excitation filter allows light corresponding to the excitation wavelength of the fluorochrome to be directed onto the tissue section and all other wavelengths removed. Barrier filters are selected to absorb reflected excitation light and prevent it reaching the eyepieces but allow transmission of the emitted light from the fluorochrome. Most fluorescent microscopes use a system of epiillumination rather than transmitted light dark-ground illumination. The excitation light in epi-illumination systems is directed through the objective lens and onto the section. Fluorescent light passes from the sample through the same objective lens and is viewed through the eyepiece. A dichroic mirror between the objective and eyepiece allows selective reflection of the excitation light onto the section and then selective transmission of the fluorescent light wavelength only. The dichroic mirror only allows excitation light to pass one way and so prevents reflection of this wavelength from the tissue sample back to the eyepiece. The objective lens also acts as a condenser, so the area viewed is equal to the area illuminated and so increasing the objective magnification will give a brighter image and more fluorescence intensity. Epiillumination has several advantages over transmitted light illumination: the light path passes through fewer glass surfaces and so less light is lost, oil immersion of the objective lens is not required, and the use of excitation and barrier filters with a dichroic mirror allows rapid changeover if a two-color fluorescence technique is being used. A drawback of immunofluorescence microscopy is that fluorescence from the chromophore fades with time, especially if it is exposed to excitation light 7. Flood the section with the second stage conjugated antibody and incubate for 30 minutes. Immunofluorescence microscopy the fluorescent microscope should deliver light of a specific wavelength to cause excitation of the fluorochrome and then collect the emitted light for viewing through the eyepiece. This is achieved by applying excitation energy at the maximum absorption wavelength of the fluorochrome so that the maximum amount of light is emitted. The light source and filter arrangement of the microscope are important factors in achieving satisfactory results. The energy output of a fluorochrome is relatively low and a light source capable of delivering sufficient excitation wavelength photons to produce visible fluorescence is essential. Many fluorescent microscopes use mercury vapor or xenon as a light source and these are contained in quartz capsules under pressure. The light output for both of these bulbs diminishes over time due to blackening of the quartz capsule and a change in the spectral emission profile. If a mercury light source is used, it should be regularly checked for uniform fluorescence across the microscope field and the bulb realigned if this is not satisfactory. The slides cannot therefore be used to provide a permanent record of the staining results and so photographic documentation/ digitization of the tissue sections is necessary. Good images can be obtained where the sections show unequivocal staining against a darker background. Anti-fade reagents in the mounting medium reduce the rate of fluorescence fade and so can give shorter exposure time, which is especially useful if multiple exposures of a particular area of tissue are required. The camera system attached to the fluorescent microscope should ideally allow all the available light to enter the camera and not employ beam splitters for simultaneous observation and photography with the subsequent reduction in available light. When using a goat primary antibody an appropriate biotinylated secondary antibody raised against the goat species must be used. Immunohistochemical staining techniques Avidin-biotin techniques In these techniques either peroxidase or alkaline phosphatase may be used as the enzyme label. Labeled streptavidin/streptavidin-biotin complex technique for monoclonal antibodies Method 1. Incubate in optimally prepared labeled streptavidin or streptavidin-biotin complex for 30 minutes. When using a streptavidin-biotin complex the reagents should be mixed 30 minutes before use in order for the complex to form. Modifications required for rabbit primary antibody: in step 5 change to biotinylated swine anti-rabbit secondary. Many of the commercial streptavidinbiotin kits are supplied with a multi-species Novolink polymer detection technique Method 1. The move to automation with continual processing means it is now less appropriate to use batch controls. There is now also a focus towards finding ways of standardizing the controls themselves, and making them more clinically relevant (Torlakovic et al. For those tests where the level of expression is variable, it is important to use controls which contain at least a high and low expressing example of the target protein. Polyclonal antibodies usually contain antibodies specific for several antigenic determinants on the antigen and, as many related molecules have components in common. Monoclonal antibodies potentially eliminate this problem but epitope similarities are seen between some molecules and unwanted cross-reaction can occur. The criteria for specificity have been outlined by Nairn (1976), and problems relating to specificity discussed by Petrusz et al. Incubate in optimally diluted unconjugated rabbit anti-mouse bridge antibody for 30 minutes. As the alkaline phosphatase label is usually intestinal, it is resistant to blocking with levamisole at the concentrations described and hence it is included in the substrate mixture. Data sheets and instructions with the wide range of commercially available substrates/ chromogens are included with the products. Quality control in immunohistochemistry 373 Immunohistochemistry is now also able to provide prognostic or predictive information such as the likely response to specific treatments. Consequently, it is vital that these investigative procedures are properly monitored for both internal and external quality control purposes, the latter if the department is performing these tests for other laboratories. It is important to ensure the correct internal quality control measures are in place. There should be staff experienced in identifying and resolving associated diagnostic procedural problems in order to provide effective and efficient quality control within the laboratory. In addition to technical understanding, the laboratory scientist should also have knowledge of the expected staining patterns for the antibodies in both pathological and non-pathological tissues. Good communication between the laboratory scientist and the pathologist must be maintained, particularly during the introduction and validation of new antibodies and procurement of positive control material. Detailed documentation and an audit trail throughout the process are necessary for potential back-tracking and troubleshooting. Such audit trail details can include antigen retrieval methods, antibody dilution data, control tissue samples, temperatures and incubation times. These automated platforms generally use standardized protocols for antigen retrieval and staining procedures, which makes overall control of the process easier. The generation and storage of automated run logs by these platforms make full reagent traceability possible. The logs can also be interrogated in the event of abnormal staining to identify errors such as missed steps due to low reagent levels. The purpose of fixation is to preserve tissue and prevent further degradation by the action of tissue enzymes or microorganisms. As discussed in Chapter 4, good fixation requires tissue to have adequate time in the fixative to allow the solution to penetrate whilst retaining uniform cellular detail throughout the tissue. However, in the routine laboratory this ideal may be compromised as it is difficult to define a standard tissue size, fixation time and fixative for each specimen type. Tissues need to be adequately, but not over-fixed, so that antigenicity is preserved without excessive alteration of the protein structures of the tissue. Lack of adequate fixation, or delay in fixation, may also be equally detrimental to labile antigens (Donhuijsen et al. It is recommended that paraffin wax with a low temperature melting point be used for this reason. There is no standard paraffin wax processing protocol for the optimal demonstration of all antigens (Williams et al. Other factors including the processor, type and quality of reagents, time in clearing agent and use of vacuum, had been suggested as possible causes of poor processing (Horikawa et al. Microwave processing is now being introduced into some laboratories to reduce the processing time and improve turnaround times for diagnostic specimens; it has been used successfully in conjunction with routine antibody staining. Acceptable staining was achieved when compared to tissues processed in a conventional processor (Emerson et al. As with all processing, if the tissue is not completely fixed then artifacts will be introduced. It is important that any control tissues used in the laboratory are processed using the same protocols used for the patient samples. Recently, alcohol-based fixatives have been considered as an alternative to formalin (van Essen et al. Depending upon the type of fixative used, the protocols may require slight modifications. This results in the targeted epitope being exposed, allowing the antigen binding site to be available to the primary antibody. The revolution of reversing the hydrogen crossbonds formed by formalin was introduced by Shi et al. There are now numerous methods for epitope retrieval including enzyme digestion techniques or, more commonly, heating the slides in a buffered solution. Tissue which is inadequately processed will potentially produce poor quality sections, with poor adhesion to the slides, especially fatty tissue such as breast and skin. Modern tissue processors all have the option to include vacuum and temperature variation at each step, allowing greater optimization of the procedure. Quality control in immunohistochemistry 375 the solution most commonly used in standardized retrieval methods. These methods all successfully demonstrate a much greater range of antigens in tumors, including proliferation markers and oncogene expression. The use of automated immunostainers has brought greater standardization of retrieval methods, as these use standard retrieval solutions with defined reproducible protocols. Non-automated laboratories may have a number of variables which require internal standardization in the antigen retrieval technique, including the choice of heating method. Equipment commonly used to perform epitope retrieval includes the modified pressure cooker, initially reported by Norton et al. Some automated platforms have on-board retrieval where individual slide bays can be heated with the appropriate solution on the slide. Other factors required for successful retrieval include the proper drying and complete removal of water from slides. In addition to avoiding wrinkles or tears in the tissue, these factors will all assist the adhesion of the tissue to the slide. The concentration of enzyme required is dependent on the proteolytic qualities of the product being used. The concentration, pH, and temperature are then usually held constant, while the time of digestion is varied. The time required for optimal digestion will vary, depending on the antigen under investigation, the quality (proteolytic capabilities) of the trypsin and the length of formalin fixation. The time for optimal digestion of antigens which are only present in small amounts. Reagent factors Production of high quality staining is dependent upon the correct storage, handling and application of the reagents used. Once a protocol has been developed, it is important to ensure the reproducibility of the stain. To achieve this, the storage conditions and expiration dates of in-house and commercial reagents must be monitored as the preparation and use of each reagent must be consistent. Details of the storage and preparation of all reagents used in each staining run must be documented as part of the audit trail to allow backtracking and troubleshooting.

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