McPherson & Pincus: Henry's Clinical Diagnosis and Management by Laboratory Methods, 21st ed.

Copyright 2006 W. B. Saunders Company CHAPTER 22 Laboratory Diagnosis of Gastrointestinal and Pancreatic Disorders Martin H. Bluth, MD PhD Rosemarie E. Hardin, MD Scott Tenner, MD Michael E. Zenilman, MD Gregory A. Threatte, MD KEY POINTS Helicobacter pylori is recognized as the principal cause of gastrointestinal ulcers and gastritis. Serology tests are used to screen for H. pylori and noninvasive breath tests or endoscopic biopsy are used to confirm eradication after treatment. Serum amylase is the analyte of choice to diagnose acute pancreatitis. Other markers, including lipase, trypsinogen (and metabolites), AST and ALT, can aid in determining etiology and/or diagnosis. Routine laboratory testing is of little value in identifying patients with chronic pancreatitis. Diagnosis relies on imaging techniques (ERCP). Laboratory screening for cystic fibrosis should be limited to sweat chloride testing. Genetic analysis should be used as a confirmatory test in specific cases. Chronic diarrhea can often be classified as inflammatory, osmotic or secretory in origin or the result of altered bowel motility. Initial stool tests for blood, microbes, fat and leukocytes help determine subsequent testing algorithms. IgA deficiency should be considered in patients suspected of having celiac sprue and incorporated in the interpretation of anti-gliadin, anti-endomysium, anti-reticulin, or anti-transglutaminase antibody results. Lactose intolerance increases with age. Screening tests for true disaccharidase deficiencies include oral challenge of suspected disaccharides to reproduce the abdominal symptomatology followed by stool analysis. Definitive diagnosis of disaccharidase deficiencies depends on the demonstration of low specific enzyme activity in the mucosa of small intestinal biopsy material. p-ANCA and ASCA can be used to distinguish abdominal pain seen in irritable bowel syndrome from inflammatory bowel disease. Carcinoid tumor is the most common type of neuroendocrine tumor of the gastrointestinal tract. Intraoperative gastrin measurements are useful in identifying whether the abnormal tissue is completely removed in patients undergoing surgery for gastrinomas. Screening with fecal occult blood testing can decrease mortality from colon cancer by 15-35%. A variety of factors can create false-positive or false-negative results.

Over the past two decades the practice of gastroenterology has evolved tremendously. Increased expertise with endoscopic techniques has allowed direct visualization and biopsy of pathological lesions, facilitating confirmation of suspected clinical diagnoses such as gastric

and duodenal ulcer disease. Although these endoscopic evaluations are invaluable diagnostic modalities, they are expensive, invasive and require the specialized skill of a gastroenterologist. In recent years, clinical practices have continued to evolve with the advent of new laboratory evaluations for noninvasive diagnosis of gastrointestinal and pancreatic disorders. Examples include fecal immunochemical and DNA testing for detection of colon cancer and various serum antibody testing to aid diagnosis of inflammatory bowel disease. The latter gives one the ability to differentiate Crohn's disease from ulcerative colitis, which was previously impossible using the available routine laboratory testing. Furthermore, new laboratory tests with enhanced sensitivity and specificity are replacing older, previously established tests for diagnosis of diseases. One such example is the replacement of fecal chymotrypsin with fecal elastase levels to detect pancreatic insufficiency. Knowledge of these tests, their sensitivity and specificity profiles as well as awareness of false positives and negatives of these tests is crucial to the interpretation of the results, especially to the clinician responsible for appropriate disease management. New noninvasive serum tests are also being explored for clinical utility as screening methods, such as pepsinogen tests for detection of atrophic gastritis and early gastric carcinoma. In this chapter we will present clinically relevant disorders and discuss the utility of laboratory methods in confirming disease, guiding therapeutic interventions, predicting prognosis and monitoring therapy. In this edition, we have chosen to adapt laboratory evaluations to those that most closely reflect common clinical practice in the era of endoscopy and with the advent of the newer diagnostic tests. Email to Colleague Print Version Copyright 2007 Elsevier Inc. All rights reserved. - www.mdconsult.com

McPherson & Pincus: Henry's Clinical Diagnosis and Management by Laboratory Methods, 21st ed.
Copyright 2006 W. B. Saunders Company Common Gastroenterological Disorders
Gastric Disorders Peptic Acid Disease

The practical approach to peptic acid disease requires the integration of data supplied by the clinician, endoscopist, radiologist, clinical pathologist, and surgical pathologist. Helicobacter pylori has been recognized as the principal cause of duodenitis and duodenal ulcers, as well as being strongly associated with type B chronic antral gastritis, gastric ulcers, nonulcer dyspepsia, gastric carcinoma, and MALTomas ( Veldhuyzen van Zanten, 1994 ; Wotherspoon, 1998 ; Peterson, 1991 ; Thiede, 1997 ). The use of nonsteroidal antiinflammatory drugs (NSAIDs) causes or aggravates peptic and gastric inflammation and ulceration. Hypersecretory states are a much rarer cause of acid peptic disease. Data gathered by history and physical examination may initially suggest peptic acid disease. Radiologic and/or endoscopic techniques are employed to confirm the diagnoses. Testing for H. pylori and hypersecretory states involves laboratory analysis. (For more information see Ch. 56 .) Since H. pylori has been shown to be the most important causative agent for peptic ulcer disease, and is significantly associated with multiple other types of upper gastrointestinal (GI) pathology, there has been tremendous research involving its detection and treatment, and the confirmation of pathogen eradication. Within the last decade there have been numerous Food and Drug Administration (FDA)-approved and commercially available products for the detection of this bacterium. A cogent argument has been made that all patients found to harbor this organism should be treated ( Graham, 1997 ). Although the numbers and types of tests will likely continue to grow, tissue sampling, breath tests, and serology are currently the mainstay in the diagnostic armamentarium. Testing for H. pylori often utilizes the organism's ability to produce urease. Radioactive and nonradioactive breath hydrogen tests are examples of noninvasive means for detecting active H. pylori infection. Each is sensitive and specific prior to therapy. The incidental use of proton pump inhibitors, antibiotics, or bismuth-containing antacids may lead to false-negative tests. Treatment of H. pylori may not lead to complete eradication of the organism. Hydrogen breath tests may be falsely negative if they are performed too soon after treatment, before the bacterial load is great enough to be detected ( Atherton, 1994 ). Serum antibodies directed against H. pylori can be used to detect exposure to H. pylori. Enzyme-linked immunosorbent assay (ELISA) tests are available and reliable (Feldman, 1995a, b [19] [20]; van de Wouw, 1996 ). Although quantitative levels of these antibodies are not currently routinely utilized in the clinical setting to determine whether there is current or past infection, they have been reported to be highly accurate ( Lerang, 1998 ). At present, serology is generally utilized to screen for H. pylori and breath tests are used to confirm

eradication after treatment unless endoscopy allows collection of tissue for rapid urease testing or histologic review ( Megraud, 1997 ). Urease-based chemical tests are routinely used to detect H. pylori in biopsy specimens obtained via endoscopy. Fresh biopsy specimens obtained via endoscopy are placed into fluids or gels containing urea. The bacterial urease splits the urea, producing ammonia. The change in pH affects a color indicator, thus providing the basis for the detection. Bacterial load will determine the amount of urease present and can affect the rapidity of response. If the load is too low, the test can be falsely negative ( Xia, 1994 ). The test is inexpensive and easy to perform, but it requires endoscopy with its expense and potential risks. Office-based serologic quick-test kits are available. The accuracy of these kits has been shown to be dependent on the antibody preparations used. Immunoglobulin G (IgG) preparations perform most consistently. Other test qualities such as reproducibility, cost, and ease of utilization are factors to be considered when reviewing each of the many available brands marketed today ( Laheij, 1998 ). Histologic review of biopsy specimens stained with WarthinStarry or Giemsa's stain remains one of the most frequently employed techniques to determine active infection. Culture of the organism may be inconsistent and is usually not done in routine clinical settings. Stool studies employing antigen enzyme assays and polymerase chain reaction (PCR) methodologies are also commercially available, but efficacy remains controversial ( Makristathis, 1998 ). Hypersecretory states are suggested by extensive peptic acid disease, especially in the absence of H. pylori, and the use of NSAIDs. Failure to respond to the usual doses of histamine-2 (H2)-receptor blocking agents and proton pump inhibitors also suggests oversecretion of hydrochloric acid. Although gastric analysis remains the gold standard with regard to the amount of acid secreted, it is invasive and used much less frequently. Care must be taken to avoid the use of antisecretory medications for the appropriate time intervals before such testing. H2-receptor blockers should be held for 48 hours and proton pump inhibitors should be avoided for 7 days. H2-receptor blockers are available without prescriptions, so patient education is important and clinicians must remember to review all of the medications their patients utilize. Gastrin levels, with and without secretin stimulation, can be used to diagnose Zollinger Ellison syndrome, in many cases sparing the patient gastric analysis. Serum gastrin levels greater than 150 ng/L (normal < 100 ng/L), especially with simultaneous gastric pH values of < 3, are highly suggestive of a gastrinoma. For equivocal results, secretin can be given (2 U/kg) intravenously and serial gastrin levels can be drawn at 2, 5, 10, 15, and 20 minutes. An increase in gastrin of more than 100 ng/L (normal increase < 50 ng/L or 50%) is considered a positive test. Octreotide, a synthetic form of somatostatin, has been used for localization of the tumor(s). Radioactive-labeled octreotide binds to somatostatin receptors and can be subsequently localized by scintigraphy. If such tumors are surgically removed, gastrin levels can be used to assess potential success or future recurrence.
Pancreatic disorders Macroamylasemia

Macroamylasemia is the term used to describe a condition of persistently elevated serum amylase activity with no apparent clinical symptoms of a pancreatic disorder. It is attributed to the presence of an amylasemacromolecule complex whose larger size precludes its

excretion into urine, prolonging its half-life. Macroamylase is a circulating complex of normal amylase linked to an immunoglobulin in most cases and to a polysaccharide in others. The immunoglobulins involved are IgA and IgG. The composition of macroamylases is heterogeneous. Analysis of the complex after acid dissociation revealed that P-type and Stype isoamylases were present in variable proportions. The molecular weight has been estimated at 150 000 to more than 1 million. Macroamylasemia may also occur in hyperamylasemic patients with undiminished urine amylase and in patients with normal serum and urine amylase activity. Serum lipase may also form a complex with circulating immunoglobulins, resulting in macrolipasemia ( Zaman, 1994 ). Table 22-1 shows the distinguishing features of different types of hyperamylasemia.

Table 22-1 -- Differential Diagnosis of Hyperamylasemia and Macroamylasemia Condition Serum Serum Urinary Cam: Ccr Serum amylase lipase amylase macroamylase Pancreatic hyperamylasemia Salivary hyperamylasemia Macroamylasemia type 1 Macroamylasemia type 2 Macroamylasemia type 3 High High High High Normal High Normal Normal Normal Normal High Low or normal Low Low or normal Normal High Low or normal Very low Low Low or normal Absent Absent High Moderate Trace

Cam : Ccr = amylase clearance : creatinine clearance ratio = (urinary amylase/serum amylase) (serum creatinine/urinary creatinine). After Kleinman DS, O'Brien JF: Macroamylase. Mayo Clin Proc 1986, 61:69, with permission.

Macroamylasemia can occur with a frequency of 1.05% in randomly selected patients, 2.56% among persons with hyperamylasemia, and 0.98% in persons with normal serum amylase ( Klonoff, 1980 ). Macroamylasemia per se is not a disease entity because no clinical symptoms consistently accompany it. It is an acquired and benign condition that may occur in apparently healthy individuals and is found more frequently in men than in women. The age at the time of discovery in most patients is in the fifth through seventh decades. The occurrence of macroamylasemia may be an early sign of disease, either as a marker or as a nonspecific disease-induced dysproteinemia with amylase-binding capability, and it may be regarded as one of the immunoglobulin-complexed enzyme disorders. Clinically, it is important to differentiate macroamylasemia from other conditions associated with hyperamylasemia. A patient with hyperamylasemia, a very low (< 1%) amylase/creatinine clearance ratio, and normal renal function should be considered for the possibility of having macroamylasemia. Definitive identification of macroamylasemia,

however, requires direct demonstration of the existence of macroamylase molecules by ultracentrifugation, chromatography, or other physical techniques. A detection method using chromatography has been in use for many years and a rapid and simple assay based on selective precipitation of macroamylase in a polyethylene glycol solution has also been reported ( Levitt, 1982 ).
Acute Pancreatitis

Since the first description in 1929, serum amylase has remained the universal laboratory diagnostic test in the determination of acute pancreatitis ( Elman, 1929 ). Derived from pancreatic acinar cells, the serum amylase level rises over the first 2-12 hours after the onset of acute pancreatitis, peaks at 48 hours and returns to normal within 3-5 days ( Zieve, 1964 ). In the appropriate clinical setting marked by new-onset sharp, boring epigastric pain radiating to back or flanks associated with nausea and vomiting, the serum amylase helps to confirm the suspected diagnosis of acute pancreatitis with a positive predictive value approaching 100%. Despite high positive and negative predictive values, there are certain clinical situations where the clinician must entertain a degree of skepticism and be aware of the assay's limitations. The sensitivity is limited in patients with hypertriglyceridemia and alcoholism. The specificity is limited by elevations in amylase from inflammatory intra-abdominal processes, parotid and submandibular salivary gland inflammation. Also, decreased clearance can lead to falsely elevated levels in patients with renal insufficiency and normal persons who harbor proteins or polypeptides that are not associated with disease ( Smotkin, 2002 ). Regardless, the serum amylase is accurate in the appropriate clinical setting. Using a cutoff of greater than three times the upper limit of normal will lead to an increased specificity ( Steinberg, 1985 ). Although serum lipase is derived from pancreatic acinar cells, it rises slightly earlier than amylase, 4-8 hours after the onset of acute pancreatitis, and peaks earlier, at 24 hours ( Steinberg, 1985 ). The serum lipase also lasts longer in the serum, 8-14 days. For these reasons, serum lipase is more sensitive and specific than the serum amylase. However, the utility of serum lipase in acute pancreatitis has been shown to vary due to discrepancies in measurement method, patient selection, and cutoff point ( Tietz, 1993 ). There is no additional clinical benefit in the determination of serum lipase in a patient with the clinical symptoms of acute pancreatitis and a serum amylase greater than three times the upper limit of normal. The use of a serum lipase in the diagnosis of acute pancreatitis should be reserved to patients with clinical symptoms consistent with the disease and an amylase that is suspected to be falsely low, such as in alcoholics, patients with hypertriglyceridemia, or presenting late with the disease. Due to the additional cost and lack of benefit in the majority of patients, utilizing serum lipase in conjunction with serum amylase as a routine process in the laboratory evaluation of suspected acute pancreatitis should be considered inappropriate. It is recognized that amylase and lipase may both arise from sources other than the pancreas ( Frank, 1999 ). Thus, utilizing both assays may optimize accuracy ( Corsetti, 1993 ). Others feel that pancreatic isoamylase determination is the most cost-effective method ( Sternby, 1996 ). Due to a lack of a readily available gold standard measurement for the diagnosis of acute pancreatitis and variability of chemical methods, it is difficult to calculate sensitivity and specificity for these enzymes precisely. Recently, urinary dipstick testing for

trypsinogen-2 was shown to have a sensitivity of 94% and a specificity of 95% as compared to serum amylase with a sensitivity of 85% and a specificity of 91% with 300 U/L as the upper limit ( Kemppainen, 1997 ). This may provide a rapid screening test under the correct clinical circumstance. A sensitive assay that detects plasma calcitonin precursors is another method that is currently being investigated for the determination of severity of an acute episode of pancreatitis. Abnormal levels can be detected upon admission, usually within hours after the onset of abdominal pain ( Ammori, 2003 ). Plasma calcitonin precursors have been demonstrated to rise significantly with the onset of severe infection and systemic inflammation, as occurs with acute pancreatitis. Furthermore, this rise occurs in a predictable stepwise fashion allowing this serum assay to potentially serve as a marker for disease severity. Although a considerable number of other enzymes have been examined for their potential clinical role in the diagnosis and prognosis of acute pancreatitis, none has gained widespread clinical use. Additionally, urinary amylase offers no advantage over serum testing and urinary clearance of amylase is not specific ( Lankisch, 1977 ). Laboratory testing can help distinguish the etiology in patients with acute pancreatitis. Management decisions to prevent a recurrence of disease depend on the ability to determine the etiology. A meta-analysis showed that an alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) of more than 150 IU/dL (a threefold elevation) had a positive predictive value of 95% in predicting gallstones as the underlying cause. Despite the high specificity, only half of the patients with gallstone pancreatitis demonstrated a significantly elevated AST/ALT ( Tenner, 1994 ). Due to a combination of low sensitivity and low specificity, it appears that the bilirubin and alkaline phosphatase have a limited role in the diagnosis of gallstone acute pancreatitis. However, in a patient with gallstone pancreatitis, persistent elevations of the serum bilirubin may signal the presence of a persistent common bile duct stone warranting endoscopic retrograde cholangiopancreatography (ERCP) and stone extraction. Because of decreased clearance, the alkaline phosphatase remains elevated for weeks beyond an acute event involving the biliary tree. Unlike the normal pancreas that becomes inflamed in patients with gallstone pancreatitis, the pancreas in a patient with alcohol-induced acute pancreatitis has been damaged over years of alcohol consumption. The ducts have been altered by the deposition of proteinaceous plugs. The gland itself typically has altered architecture. For this reason, the disease is different. One in four patients with alcohol-induced acute pancreatitis present with a normal amylase ( Spechler, 1983 ). The gland becomes burned out. Although the amylase is affected, the lipase is not as affected. There appears to be four to five times more lipase in the pancreas than amylase ( Tietz, 1993 ). Thus, the lipase/amylase ratio appears to predict alcoholinduced pancreatitis ( Tenner, 1992 ). Using multiples of the upper limit of normal, a ratio of greater than 3 is predictive, while greater than 5 is diagnostic for acute alcohol-induced acute pancreatitis. In addition to the lipase/amylase ratio, carbohydrate deficient transferrin (CDT) appears useful in the determination of alcoholism. A person who consumes large amounts of alcohol will have a CDT elevation regardless of whether they have been consuming alcohol during the past several days. It is an ideal marker in a patient suspected of being an alcoholic, who denies alcohol use when the alcohol level is normal ( Le Moine, 1994 ).

In the management of acute pancreatitis, difficulty in the early determination of severity complicates the management of a significant proportion of patients. Multiple laboratory tests have been studied in an attempt to define severity early in the course of the disease. Despite intense study, only two tests, trypsinogen activation peptide (TAP) and hematocrit appear to be useful early in the course of the disease. Inappropriate early activation of trypsin in the acini of the pancreas leads to the release of trypsinogen activation peptide. This protein product is typically not seen at significant levels in the blood or urine. In patients with acute pancreatitis, TAP levels rise. A TAP greater than 30 mmol/L has been shown to be associated with severe disease, with a negative predictive value of 100% ( Tenner, 1997 ). A new ELISA is available from Biotrin (Dublin, Ireland) that can assist in the determination of severity through the use of urine samples. A hematocrit above 44 or rising over the first 24 hours has been shown to be associated with pancreatic necrosis ( Baillargeon, 1998 ). This is likely related to hemoconcentration from a combination of severe third space losses, fluid sequestration, and poor intravenous hydration. A serum C-reactive protein is useful later (after 36-48 hours after the onset of symptoms) in determining the presence of pancreatic necrosis ( Buchler, 1986 ). Refer to Table 22-2 for a summary of laboratory tests used in the evaluation of acute pancreatitis.

Table 22-2 -- Laboratory Tests in Acute Pancreatitis Laboratory test Purpose Usage and limitations Amylase Diagnosis Accurate over three times the upper limit of normal, decreased specificity in renal failure, normally elevated in macroamylasemia, test interference in hypertriglyceridemia, elevated from other sources such as salivary gland and/or intra-abdominal inflammation (not above 3), can be normal in alcohol-induced acute pancreatitis Diagnosis Decreased specificity in renal failure, immune complex creates false positives, elevated from salivary gland and intraabdominal inflammation Diagnosis Limited use, unclear if superior to amylase/lipase Etiology Etiology Etiology If greater than three times upper limit of normal, gallstones present as etiology in 95% of cases. Low sensitivity Greater than 5 is diagnostic for alcohol acute pancreatitis. Low sensitivity Useful in patients who deny alcohol, remains elevated for weeks after binge drinking Greater than 30 mmol/L in 6- to 12-hour urine, 100% negative predictive value Greater than 44 on admission, or rising over initial 24 hours associated with pancreatic necrosis

Lipase

Trypsinogen 2 AST/ALT Lipase/amylase ratio Carbohydrate deficient transferrin (CDT)

Trypsinogen Severity activation peptide (TAP) Hematocrit Severity

Laboratory test

Purpose

Usage and limitations Values over 200 IU/L associated with pancreatic necrosis. Useful after first 3648 hours

C-reactive protein Severity

Chronic Pancreatitis

Chronic pancreatitis is marked by progressive destruction of islet cells and acinar tissue, the latter responsible for the maldigestion associated with this disease, due to loss of enzyme secretion responsible for digestion of foodstuffs within the small intestine. Routine laboratory testing is of little value in patients suspected as having chronic pancreatitis. Although the amylase and lipase may be elevated in acute exacerbations, the absence of these enzyme elevations in the serum does not rule out an attack of pain from chronic pancreatitis. Chronic pancreatitis is suspected in the correct clinical setting and presents as mild glucose intolerance to frank diabetes mellitus, chronic abdominal pain, and/or maldigestion/malabsorption. Hyperglycemia may result from disease progression with subsequent pancreatic endocrine dysfunction. As malnutrition progresses, the serum albumin may fall. A serum beta-carotene may be found to be low as malabsorption for lipids develops. The clinical diagnosis of chronic pancreatitis depends on the finding of structural abnormalities in ductal anatomy found on imaging, typically ERCP. The simplest method of functional testing of the pancreas is assessing the presence of fat in the stool. Unfortunately, maldigestion of fat occurs after 90% of pancreatic lipase secretory capacity is lost. Serum trypsinogen assays are available and may have a diagnostic utility when values are below 20 ng/mL but the levels are only found to be low in patients with advanced disease (typically when steatorrhea is already present) ( Jacobsen, 1984 ). Because of inadequate delivery of fecal elastase to the duodenum, a low level of pancreatic elastase in the stool can be used in the diagnosis of chronic pancreatitis. Although initial studies suggested that the test could not detect chronic pancreatitis in the absence of steatorrhea, more novel tests have shown that the test is accurate in the evaluation of less advanced disease. A novel ELISA for fecal elastase, a pancreas-specific enzyme that is not degraded during intestinal transport and reaches concentration in fecal matter five to six times that found in duodenal juice, has been developed and marketed and appears to be very sensitive for chronic pancreatitis ( Loser, 1996 ). One clinical study found sensitivities of 63%, 100% and 100% for patients with mild, moderate and severe pancreatic insufficiency respectively. If symptoms suggest this disorder, the anatomy of the gland is reviewed radiographically and insulin and exocrine pancreatic enzymes are replaced as necessary. There is little clinical need to estimate the percentage of exocrine or endocrine function. When pancreatic maldigestion is suspected as the cause for diarrhea, many clinicians will attempt an empiric course of exogenous pancreatic enzymes. If this works, the diagnosis is likely in the appropriate clinical setting.
Cystic Fibrosis

Cystic fibrosis (mucoviscidosis) of the pancreas is an autosomal recessive disease with an incidence of 1 in 1600 Caucasian births and 1 in 17 000 African American births in the United States. Approximately 1 in every 20 Caucasians is a carrier. Cystic fibrosis is characterized by abnormal secretion from the various exocrine glands of the body, including the pancreas, salivary glands, peritracheal, peribronchial, and peribronchiolar glands, lacrimal

glands, sweat glands, mucosal glands of the small bowel, and bile ducts. Involvement of the intestinal glands may result in the presence of meconium ileus at birth. Chronic lung disease and malabsorption resulting from pancreatic involvement are the major clinical problems of those who survive beyond infancy. Because of the multiple alleles at the cystic fibrosis gene (see Ch. 69 ), laboratory diagnosis still depends largely on the demonstration of increased sodium and chloride in the sweat. Unfortunately, unless the sweat test is correctly performed, it probably is the least reliable test and has a high proportion of false-positive and false-negative results. In children, chloride concentrations of over 60 mmol/L of sweat on at least two occasions are diagnostic. Levels of between 50-60 mmol/L are suggestive in the absence of adrenal insufficiency. Patients in whom cystic fibrosis is suspected on the basis of indeterminate sweat electrolyte results may undergo confirmatory testing by having the sweat electrolytes test repeated following administration of a mineralocorticoid such as fludrocortisone. In these patients, the electrolyte values would remain unchanged, whereas normal controls would show a decrease in sweat electrolytes. Sodium concentrations in sweat tend to be slightly lower than those of chloride in patients with cystic fibrosis, but the reverse is true in normal subjects. Sweat chloride concentrations of more than 60 mmol/L may be found in some patients with malnutrition, hyperhidrotic ectodermal dysplasia, nephrogenic diabetes insipidus, renal insufficiency, glucose-6-phosphatase deficiency, hypothyroidism, mucopolysaccharidosis, and fucosidosis. These disorders are usually easily differentiated from cystic fibrosis by their clinical symptoms. False-negative sweat test results have been seen in patients with cystic fibrosis in the presence of hypoproteinemic edema. Sweat electrolytes in about half of a group of premenopausal adult women were shown to undergo cyclic fluctuation, reaching a peak chloride concentration most commonly 5-10 days prior to the onset of menses. Peak values were slightly under 65 mmol/L. Men showed random fluctuations up to 70 mEq/L. For this reason, interpretation of sweat electrolyte values in adults must be approached with caution.
Intestinal Disorders Chronic Diarrhea

Acute diarrhea is self-limited, typically viral, and resolves quickly. Chronic diarrhea is a common complaint of patients presenting to physicians. The differential diagnosis is complex and a variety of laboratory tests can be found to be useful. The definition of chronic diarrhea is greater than three loose stools per day for more than 4 weeks' duration and/or daily stool weight greater than 200 g/day ( Thomas, 2003 ). Using a definition of chronic diarrhea as excessive stool frequency without associated abdominal pain, the prevalence of this disorder in western population is estimated to be approximately 4-5% ( Thomas, 2003 ). Laboratory testing begins with randomly collected stool specimens submitted for blood, fat and microbes (ova and parasites). In addition, detecting the presence of leukocytes in the stool is of importance in determining whether the diarrhea is inflammatory in nature (ulcerative colitis, Crohn's disease, ischemic colitis, invasive microbes). Detection of fecal leukocytes with Wright's stain on microscopy is also of importance. However, a newer method utilizing lactoferrin may be more accurate in the identification of leukocytes and appears to be more sensitive ( Guerrant, 1992 ). The principal function of the colon is to absorb water from the fecal stream. Approximately 90% of the water that enters the colon is removed during transit. The rectosigmoid colon also

stores stool until it is possible to defecate in a socially acceptable fashion. Diarrhea occurs when the amount of water in the colonic lumen (which is the sum of the water reaching it from the small bowel and the water secreted by the colonic mucosa) exceeds the amount of water capable of being absorbed by the colonic mucosa. It can also result from irritation or inflammation of the colon, which interferes with the colon's ability to store feces. An absent or significantly abbreviated colon ensures large volume and loose stools. The causes of diarrhea are often divided into four major pathogenic groups. These major groups are the inflammatory diarrheas, the osmotic diarrheas, the secretory diarrheas, and the diarrheas that result from altered bowel motility. Specific causes of diarrhea may do so by more than one pathogenic means, and more than one diarrheal etiology can be present in a single patient simultaneously. Although many clinicians use the category factitious as a fifth pathogenic classification for those who self-induce diarrhea, the method or methods employed by the patient involve one of the four aforementioned types. A brief mention of factitious diarrhea is warranted because it is not uncommon. Analysis of laxatives should be done early in the evaluation. Stool water should be analyzed for osmolality and electrolytes. The osmotic gap is calculated from electrolyte concentrations in stool water by the following formula: 290 - [2 (Sodium + Potassium)]. The sum of the sodium and potassium concentrations is multiplied by a factor of two to account for associated anions. The osmolality of the stool within the distal intestine is estimated to be 290 mOsm/kg (equilibrates with plasma osmolality). If findings suggest secretory diarrhea, osmotic gap less than 50, the patient may have ingested a laxative causing a secretory diarrhea, such as sodium phosphate (Fleet phosphosoda). If stool electrolyte analysis suggests osmotic diarrhea, osmotic gap > 125 mOsm/kg, magnesium laxatives (Maalox) should be suspected. Similarly, iatrogenic diarrhea is not a separate pathogenic category. Although multiple drugs and other therapies induce diarrhea as an unwanted side effect, the mechanism by which they do so involves inflammation, osmotic load, secretion, altered motility, or some combination thereof. Rectal incontinence is often incorrectly reported as diarrhea. Diarrhea may precipitate incontinence in someone who can control defecation with formed stool. The management of incontinence may be quite different from the management of diarrhea, so it is important to distinguish between the two. Inflammatory or exudative diarrhea is often bloody, but it does not have to be. The presence of fecal leukocytes on microscopic evaluation may be the only clue to inflammation. In some cases of inflammatory diarrhea, the mucosa of the bowel appears grossly normal, while histologic review of biopsy specimens demonstrates inflammation. A frankly exudative process classic for inflammatory diarrhea is not present in these cases. The overlap of the pathogenic types of diarrhea is thus demonstrated. Semantic arguments aside, it is most reasonable to determine whether blood or fecal leukocytes are present in the stool of a patient with diarrhea. Their presence suggests that inflammation is playing a role in the patient's diarrhea. Crohn's disease, ulcerative colitis, ischemic colitis, invasive infectious organisms, and radiation-induced colitis are common causes of inflammatory diarrhea. The atypical inflammatory bowel diseases such as microscopic colitis or collagenous colitis do not produce exudative diarrhea. Hypersecretion or lessened absorption of water may be the means by which these entities produce diarrhea. Classification via pathogenesis remains controversial.

Osmotic diarrhea occurs when the osmotic load of the fecal stream favors excess water loss. In other words, the osmotic gradient drives water into the colonic lumen creating looser, more voluminous stools. One can calculate a stool osmotic gap by first measuring stool osmolality, sodium, and potassium. A value of greater than 100 mmol/L suggests the presence of a large number of unmeasured osmotic particles causing fluid to be drawn into the colonic lumen. The stool specimen from which these measurements are derived must be very fresh. Fecal bacteria continue to produce osmotic particles as a result of digestion while the specimen awaits processing. These bacterial breakdown products can falsely elevate the fecal osmotic load. The most practical way of determining that the diarrhea is osmotic in the cooperative patient is to fast the patient. A strict fast causes osmotic diarrhea to subside. Uncooperative patients or patients with factitious diarrhea who continue to ingest osmotically active substances will continue to have diarrhea. Such patients may have to be observed during the fast. Secretory diarrheas result from the active secretion of water into the fecal stream that overwhelms the absorptive process. Multiple toxins, hormones, and medications can cause an active secretion of water and electrolytes in the colonic lumen. Such diarrheas can cause dehydration, electrolyte depletion, and even death. The classic secretory diarrhea is cholera. In developed countries, medications are probably the most common cause of secretory diarrhea. A variety of hormonal causes such as gastrinoma (ZollingerEllison syndrome), carcinoid syndrome, medullary thyroid carcinoma, mastocytosis, vasoactive intestinal polypeptide (VIP)-producing tumors (e.g., VIPoma syndrome), and villous adenoma of the rectosigmoid colon have been identified. Analysis of the urine for 5-hydroxyindoleacetic acid for carcinoid syndrome, vanillylmandelic acid for pheochromocytoma and histamine for mastocytosis is rarely helpful. However, if the clinical suspicion exists, the tests should be submitted for analysis. Unlike osmotic diarrheas, the conditions continue to generate diarrhea even when the patient invokes a strict fast. In fact, these patients can dehydrate quickly without continued fluid intake, and such fasting should be under observation when secretory diarrhea is suspected. Motility disorders can hurry the fecal stream and thwart complete water absorption. Irritable bowel syndrome may involve excessive neural stimulation with resultant decreased stool transit times. Short gut syndromes (e.g., postsurgical) reduce the amount of absorptive colon and can result in diarrhea. Motility disorders are, by far, the most difficult to characterize and quantify. Most current diagnostic methodologies alter the colonic milieu and, presumably, alter motility. Barium studies or the ingestion of radiopaque markers may assist in estimating colonic transit time, but consensus normal values are lacking. Unless there are obvious structural defects, or the diagnosis of irritable syndrome is clear, motility disorders are often suspected by exclusion. The diagnosis of diarrhea starts with a very thorough history. Whether the diarrhea is bland or bloody, the presence or absence of constitutional symptoms and the duration of the illness are major points in determining the subsequent evaluation. Self-limited, acute diarrhea (less than 2 weeks in duration) without bleeding or constitutional symptoms rarely requires diagnostic testing. Chronic diarrhea, the passage of blood, and constitutional symptoms all suggest the need for making a specific diagnosis. The history is the key to narrowing down the potential diagnosis. The physical examination, although usually less helpful than the history, must still be very comprehensive.

A critical aspect in the evaluation of diarrhea revolves around immunocompetence. In patients with the acquired immunodeficiency syndrome (AIDS), or who have been significantly immunosuppressed, the diagnostic evaluation must consider unusual infections. In any patient with chronic diarrhea it is wise to consider establishing human immunodeficiency virus (HIV) status. Patients with diarrhea must be queried about their medications, diet, and water supply. Duration of symptoms, stool frequency, urgency, incontinence, daily stool patterns, stool consistency, and stool volume should be estimated. Travel histories, sexual practices, and family histories may be useful. Constitutional symptoms such as fever, weight loss, arthralgias/arthritis, rashes, and the like, may give strong clues as to the diarrheal etiology. If the patient is among others who develop diarrhea simultaneously, a common infectious source should be considered (see Ch. 56 ). Antibiotic usage, recent surgery, radiation, or chemotherapy, and any change in a patient's usual regimen may shed light on the situation. The clinician should always inquire about potential similar episodes in the past and determine if the diarrhea is recurrent. It helps to know if there are outbreaks of diarrheal illnesses in the community. In patients with similar diarrheal illnesses occurring over the same time period, an infectious cause or common toxin can be suspected. Infectious agents may be sought. Stool culture, enzymes for rotavirus and giardiasis, and ova and parasite examinations should be done in the appropriate clinical setting (see Ch. 61 ). Table 22-3 shows recommended tests that can be used in the evaluation strategy.

Table 22-3 -- Laboratory Tests in the Differential Diagnosis of Diarrhea Test Method Use Initial screening tests Fecal leukocytes Hemoccult test Fecal osmotic gap Wright's stain or methylene blue Peroxidase reaction for hemoglobin FOG = fecal osmolality 2 (fecal Na + K) Color change after adding NaOH to stool Routine culture and sensitivity Identify inflammatory diarrhea Identify hemorrhagic diarrhea Distinguish secretory vs. osmotic diarrhea

Stool alkalinization

Phenolphthalein laxative ingestion

Infectious causes Stool bacterial culture Identify Shigella, Salmonella, Campylobacter Identify E. coli 0157:H7, Yersinia, Vibrio Pseudomembranous colitis HIV enteritis Rotavirus enteritis

Stool special culture Specialized culture and serotyping Stool C. difficile toxin assay HIV serology Stool rotavirus Tissue culture cytotoxicity ELISA Antigen enzyme

Test screen Stool ova and parasites Stool mycobacteria Stool protozoans E. histolytica Ab titers Stool Giardia antigen Endocrine causes Urine 5-HIAA Blood serotonin Serum VIP Serum TSH, free T4 Serum gastrin Serum calcitonin Serum somatostatin Maldigestion Lactose tolerance test Sweat chloride Stool reducing sugars Malabsorption

Method immunoassay Wet mount Acid-fast stain and culture, PCR Iodine or modified acid-fast stain Serology Enzyme immunoassay HPLC HPLC RIA Immunoassay RIA RIA RIA See text See text Clinitest tablets

Use Enteric parasitic infection M. tuberculosis, MAI Cryptosporidium, Isospora belli Entameoba histolytica Giardia lamblia

Carcinoid syndrome Carcinoid syndrome VIPoma Hyperthyroidism ZollingerEllison syndrome Hypocalcemia-related diarrhea Somatostatinoma Lactase deficiency Pancreatic insufficiency Cystic fibrosis Carbohydrate intolerance

D-Xylose absorption See text test 72-Hour fecal fat content Fecal fat stain Serum carotene
14

Evaluate surface area of intestinal mucosa Lipid malabsorption Lipid malabsorption Lipid malabsorption

Saponification and titration Sudan stain Spectrophotometry

CO2 breath test as a test for lipid (fat) malabsorption See text Endomysial Serology

Lipid malabsorption Celiac disease

Test antibody Gliadin antibody H2 breath test Bacterial colony count

Method Serology Expired H2 by gas chromatography

Use Celiac disease Carbohydrate malabsorption

Small bowel aspirate Bacterial overgrowth and quantitative culture Ion-specific electrode Hypocalcemia-related diarrhea Biuret reaction, anionic dyes See text Nephelometry Endoscopic biopsy Endoscopic or open biopsy IBD, protein-losing enteropathy Protein-losing enteropathy Agammaglobulinemia Neoplasia, lymphocytic colitis, collagenous colitis Whipple's disease, MAI, abetalipoproteinemia, lymphoma, amyloidosis, eosinophilic gastroenteritis, agammaglobulinemia, intestinal lymphangiectasia, Crohn's disease, tuberculosis, graft-versus-host disease, Giardia, other parasitic infections, collagenous colitis, microscopic colitis

Other and miscellaneous Serum ionized calcium Serum protein and albumin Stool alpha-1antitrypsin Quantitative immunoglobulins Colon biopsy Intestinal biopsy

PCR = polymerase chain reaction; HPLC = high-performance liquid chromatography; RIA = radioimmunoassay; IBD = inflammatory bowel disease; MAI = Mycobacterium aviumintracellulare; HIV = human immunodeficiency virus; ELISA = enzyme-linked immunoabsorbent assay; 5-HIAA = 5-hydroxyindoleacetic acid; Ab= antibody.

Any diarrheagenic medication that can be stopped should be, especially if it was started or the dosage was increased around the time that the diarrhea began. It should be remembered that the active drug may not be responsible for the diarrhea, but that the carrier substance (e.g., sorbitol) may be. Stools may be alkalinized to test for phenolphthalein if surreptitious laxative use is suspected. It must be remembered that many readily available substances can cause diarrhea, and stool alkalinization, although widely written about, is often of little practical value. It is simple and inexpensive, however, and should be considered under the right clinical conditions. Stool can be tested for blood, electrolytes, leukocytes, and osmolality. Infectious agents may be sought via enzymatic testing, culture, or direct microscopic evaluation. Fecal fat testing is

also relatively simple. The standard method for detecting fat in the stool is using Sudan stain. Sensitivity varies based on the level of observer skill and experience. An alternative method of assessing fecal fat is semiquantitative, the steatocrit. This method correlates well with quantitative fat output as measured using the van de Kamer method ( Sugal, 1994 ). Although 48- to 72-hour quantitative testing of stool for fecal fat remains the gold standard, for practical considerations, this test has been largely abandoned. A fast may be very helpful. Once these data have been collected it is usually possible to classify the diarrhea and begin to find the specific diagnosis, if it has not become evident already. Complicated and expensive diagnostic evaluations for secretory diarrhea should generally not be undertaken unless other more likely causes have been ruled out or unless signs and symptoms are suggestive. Breath testing is becoming increasingly utilized for the evaluation of chronic diarrhea, abdominal bloating and pain. The most common tests use a probe for carbon-14 or a nonradioactive fermentable sugar. Breath testing assists in the evaluation of a person's difficulty in metabolizing lactose, sucrose and glucose (secondary to bacterial overgrowth). The exact methodology depends on the sugar studied and the sensitivity and specificity desired. The most frequent tests for lactase insufficiency rely on the ingestion of 25 g of lactose.
HIV-Related Diarrhea

The actual causes of diarrhea in the patient with HIV are related to the aforementioned pathophysiologic mechanisms. However, the specific etiologic agents (especially the infectious ones) often differ greatly from those in the immunocompetent patient. Thus, in all patients with chronic diarrhea, it is prudent to consider the possibility of AIDS.
Nosocomial Diarrhea

Clostridium difficile, a Gram-positive, spore-forming anaerobic bacillus, is the most important cause of nosocomial diarrhea in adults with greater than 300 000 cases per year in the US ( Malnick, 2000 ). It is thought to be associated with approximately 25% of all antibiotic-associated cases of diarrhea and 50-75% of cases involving antibiotic-associated colitis ( Malnick, 2000 ). It may present clinically, from a mild watery diarrhea to lifethreatening pseudomembranous colitis and toxic megacolon. This can lead to colonic perforation and peritonitis, with a mortality rate as high as 38% ( Poutanen, 2004 ). Patients can present with watery diarrhea, lower abdominal pain/cramping, systemic symptoms such as fever and malaise, or can have occult GI bleeding. The pathogenesis of this disease entity usually involves disruption of the normal colonic flora, typically following a course of antibiotic therapy in hospitalized patients, followed by exposure to a toxigenic strain of C. difficile. Broad-spectrum antibiotics such as penicillin, clindamycin and cephalosporins have been particularly implicated; however, any antibiotic can lead to development of C. difficile colitis ( Malnick, 2000 ). Clinical suspicion of the disease is confirmed with detection of C. difficile toxin A or B virulence factors in stool samples. Both toxin A and B lead to increased vascular permeability and have potential to cause hemorrhage. They induce the production of tumor necrosis factor alpha and inflammatory interleukins that are responsible for the inflammatory response and pseudomembrane formation ( Poutanen, 2004 ). Direct visualization of the colonic mucosa with the aid of endoscopy is required for diagnosis of pseudomembranous colitis associated with C. difficile. However, endoscopy should be avoided in cases of suspected fulminant colitis because of the risk of perforation. Laboratory

methods are available for confirmation of C. difficile infection. Tissue culture cytotoxicity assays, which take at least 48 hours to complete, for detecting C. difficile cytotoxin B in stool specimens are considered the gold standard, with a sensitivity ranging between 94-100% and specificity of approximately 99% ( Malnick, 2000 ). This tissue culture assay can detect as little as 10 pg of toxin in stool specimens ( Malnick, 2000 ). Rapid enzyme immunoassays, which can be completed within several hours, have been developed for the detection of toxin A or B from stool specimens. However, the sensitivity and specificity of these immunoassays are decreased, 65-85% and 95-100% respectively, compared with cytotoxic assays, The ELISA can detect 100-1000 pg of toxin in stool specimens ( Malnick, 2000 ). In hospitalized patients with greater than six stools per day, ELISA is the optimal diagnostic test ( Malnick, 2000 ). Stool cultures can also be performed but require up to 96 hours for completion. Latex agglutination tests that detect glutamate dehydrogenase, a common clostridial protein, can also be performed but has much lower sensitivity and specificity compared to other available tests, limiting its clinical utility. PCR methods for detection of C. difficile toxin A or B are currently being developed with similar sensitivity and specificity profiles compared to cytotoxic assays ( Poutanen, 2004 ). However, PCR is unable to distinguish between asymptomatic carriage and symptomatic infection. It is currently recommended that these tests be performed on diarrheal stool specimens; in most cases one stool sample is sufficient for diagnosis of C. difficile infection ( Poutanen, 2004 ). However, multiple samples may be required for confirmation, and empiric treatment with oral antibiotics may be indicated in patients with clinical evidence of C. difficile infection. Refer to Table 22-4 for laboratory tests available for the diagnosis of C. difficile-associated diarrhea.

Table 22-4 -- Laboratory Tests Available for the Diagnosis of Clostridium DifficileAssociated Diarrhea Test Advantages Disadvantages C. difficile cytotoxin assay Excellent specificity (99 100%) Decreased diagnostic sensitivity (8090%) Test results not available until after 48 h Requires tissue culture facility Detects only toxin B Immunoassay for detection of toxin A or toxins A and B Good specificity (95100%) Reduced sensitivity (65 85%) as compared with Test results available cytotoxin assay within 4 h Technically simple Stool culture to isolate C. difficile with subsequent cytotoxin assay of isolate Excellent sensitivity (> 90%) Results not available for at and specificity (> 98%) least 7296 h Enables typing of strain Labor-intensive for outbreak Requires tissue culture investigation facility

Reprinted from CMAJ 06-Jul-04; 171(1), Pages 5158 by permission of the publisher. 2004 CMA Media Inc. Clostridium difficile-Associated diarrhea in adults

Malabsorption Syndromes

Malabsorption results from either inadequate mucosal absorption of carbohydrates, proteins, fats vitamins or minerals or from the presence of substances in the bowel that cannot be absorbed, for example nonabsorbable sugars such as lactulose and sorbitol. Maldigestion results from an intraluminal defect that leads to the incomplete breakdown of nutrients into their absorbable substrates. This can occur with pancreatic insufficiency and loss of exocrine function. Normal absorption, therefore, cannot occur. These conditions may result in increased osmotic load of the colon, resulting in diarrhea. In addition, patients can have selective malabsorption/maldigestion of specific nutrients resulting in associated clinical sequelae. Hepatic maldigestion results from interference or obstruction of bile flow. Loss of bile salts interferes with fat emulsification, diminishing the surface area available for lipolytic action. In addition, bile salt activation of lipase activity is lost. Patients are usually jaundiced, pass dark urine, and have other signs of liver disease. Hepatic steatorrhea may coexist with pancreatic steatorrhea, as in patients with a neoplasm obstructing the ampulla of Vater. Malassimilation or the inability to assimilate fats and proteins due to maldigestion also occurs in patients with vasculitis, diabetes mellitus, carcinoid syndrome, hypogammaglobulinemia, and relative vitamin B6 or B12 deficiency. Enteric malabsorption comprises a variety of conditions that have in common normal digestion but inadequate net assimilation of foodstuffs. This may result from competition by bacteria or altered bacterial flora, as in the blind loop syndrome or diverticulosis of the small bowel, and from obstruction to the flow of lymph, as in lymphoma. It may also result from diseases affecting the small bowel mucosa, such as amyloidosis, inflammation following irradiation (radiation enteritis), diminished mucosal surface area as in gastroileostomy (gastric bypass), or small bowel resection. Depending on the location within the intestinal tract of such pathology, preferential loss of specific substrates may occur. One of the most common clinical scenarios encountered is regional enteritis localized to the distal ileum, the site of vitamin B12 and bile acid absorption, which will result in vitamin B12 deficiency as well as a decreased pool of circulating bile acids for metabolism. The classic malabsorption syndromes, celiac disease and Whipple's disease, are described below. Patients with malabsorption syndromes may remain symptomatic. If symptoms arise, the clinical presentation may be specific to the malabsorbed substrate such as with lactase deficiency causing lactose intolerance, or may be a general consequence of the increased osmotic load to the colon. For example, steatorrhea is a hallmark finding in patients with malabsorption, resulting in fluid, semifluid, or soft and pasty, pale, bulky and foul smelling stools. These stools may be foamy due to the high fat content and may tend to float on water. However, the latter may occur with stools from healthy individuals and is therefore, a nonspecific sign of malabsorption. Normal individuals with a normal fat intake excrete up to 5 g of lipid daily. Steatorrhea may be defined as the presence of more than 5 g of lipid

(measured as fatty acids) in feces per 24 hours. Although the source of fecal lipid is largely dietary, gastrointestinal excretions, cellular desquamation, and bacterial metabolism also contribute. Lipids are normally present as soaps and triglycerides. In addition, lipoids are present, including higher alcohols, paraffins, and vegetable carotenoids. Although diet has some effect on it, the pattern of lipids excreted may be very different from the lipids ingested in the diet, and the quantity of fat ingested by a normal individual has a relatively small effect on the total output of fat. According to one study, fecal lipid is equal to a constant (2.93 g) plus 2.1% of the dietary fat intake. On a fat-free diet, the output of fat normally varies from 14 g/day. Another clinical presentation of malabsorption is the development of fat-soluble vitamin (A, D, E, and K) deficiencies. Primary and secondary alterations of the bowel mucosa may also result in deficiencies of water-soluble vitamins. Other evidence of nutritional deficiencies, such as hypoprothrombinemia, glossitis, anemia, edema, ascites, and osteomalacia may be evident in these individuals. These patients are also liable to experience significant weight loss due to diarrhea-induced, large caloric losses leading to cachexia in severe cases. Classic fat malabsorption is diagnosed by revealing excessive fecal fat. Spot stool specimens can be stained with Sudan stain for detection of fecal fat. The low sensitivity of this test limits its clinical application. However, if positive, it may prompt further evaluation with a 72-hour stool collection ( Romano, 1989 ). A false-negative rate as high as 25% may result with use of the qualitative Sudan III fat staining if steatorrhea is less than 10 g per 24 hours ( Romano, 1989 ). The gold standard however, remains quantifying the amount of fecal fat per 24 hours in a 72-hour stool collection after consumption of a high-fat diet. Normal fat absorption requires normal mucosa, pancreatic enzymes and bile acids. Intraluminal defects as occur with maldigestion or mucosal abnormalities will result in abnormal fat excretion, detectable in the stool specimen. Therefore, the 72-hour fecal stool fat measurement is an accurate diagnostic test for identification of maldigestion/malabsorption with high sensitivity and specificity. The evaluation of stool for fecal fat content remains the definitive test for steatorrhea, an indicator of malabsorption. However, other diagnostic tests include determination of levels of carotenoid, the main precursor of vitamin A in humans, which requires the normal absorption of dietary fat for proper absorption; as well as the breath test and titrimetric methods for detection of malabsorption; and the d-xylose test for differentiation of pancreatic malabsorption from enteric malabsorption. These tests are described in full detail in the latter part of this chapter.
Celiac Sprue

Celiac sprue is a disorder characterized by intestinal malabsorption of nutrients due to sensitivity to the alcohol-soluble portion of gluten known as gliadin. Wheat, rye, barley and, to a lesser extent, oats contain this protein substance and can induce mucosal damage in the gut causing nonspecific villous atrophy of the small intestine mucosa. The prevalence is not clear but estimated to be between 1:300 and 1:1000 (Catassi, 1994; Not, 1998 ). Most patients with celiac sprue are asymptomatic. The diagnosis is often made by an astute clinician that notes a patient with thin stature, iron deficiency anemia, weight loss, chronic bloating and/or diarrhea. This disease has variable clinical manifestations and can lead to severe symptoms such as profound malabsorption, steatorrhea, and wasting. There are associations between celiac sprue and type 1 diabetes mellitus, Down syndrome, dermatitis herpetiformis, IgA deficiency, autoimmune thyroid disease, and others ( Barr, 1998 ). Uncontrolled celiac sprue

appears to predispose patients to gut carcinomas and lymphomas ( Nehra, 1998 ). There is a genetic predisposition and it is most common in Caucasians of Northern European descent. Due to the enteropathy associated with the disorder, multiple hematologic and biochemical abnormalities may be found in persons with untreated celiac sprue, including iron deficiency, folate deficiency, and vitamin D deficiency. The peripheral blood film may reveal nonspecific target cells, siderocytes, crenated red cells, HowellJolly bodies, and Heinz bodies. Similarly, small bowel absorptive testing will be abnormal, including oral d-xylose testing and fecal fat evaluation. The gold standard for diagnosis remains biopsy of the small bowel mucosa and identification of classic histologic changes ( Trier, 1998 ). This is done via endoscopy. The lesions may be patchy, and sampling errors can occur. Biopsy is reserved for patients in whom the diagnosis is suspected based on signs or symptoms of the disease, especially in higher-risk populations. Owing to the fact that these patients must utilize a gluten-free diet for the rest of their lives in order to control symptoms and mitigate cancer risk, histologic diagnosis is very important. In current clinical practice, there are four serologic studies used to assist in the diagnosis of celiac sprue ( Table 22-5 ). These include testing for antibodies to gliadin (AGA-IgA and AGA-IgG), endomysium (EMA-IgA), reticulin (ARA-IgA), and transglutaminase (tTG-IgA), all of which are commercially available. Results of serological testing for celiac disease must be analyzed with caution because this disease is associated with selective IgA deficiency that will give rise to false-negative serum IgA antibody tests ( Thomas, 2003 ). Therefore, IgG serology or total IgA levels should be checked if there is a high clinical suspicion of celiac disease. The sensitivity and specificity of these tests are extremely high when compared to a gold standard of flattened small bowel villi responding to dietary changes ( Farrell, 2001 ). Endomysial antibodies have the best sensitivity and specificity, but they are currently detected via immunofluorescence of sections of monkey esophagus or human umbilical cord and are costly, cumbersome, and subject to interobserver interpretive variability.

Table 22-5 -- Ranges of Sensitivities and Specificities for Commercially Available Serologic Tests for Celiac Sprue Adults Children Sensitivity (%) Specificity (%) Sensitivity (%) Specificity (%) AGA-IgA 31100 AGA-IgG 4695 EMA-IgA 89100 85100 8798 95100 90100 91100 100100 86100 67100 100100

ARA-IgA 4192 95100 29100 98100 From Murray JA: Serodiagnosis of celiac disease. Clin Lab Med 1997; 17:452, with permission.

Wheat storage protein, gliadin, is available to be used as an antigen in an ELISA. Although serum IgA and IgG AGA levels are frequently elevated in untreated celiac sprue, these tests are of only moderate sensitivity and specificity. The IgG AGA testing is particularly useful in

the 2% of patients with celiac disease who appear to be IgA deficient. However, these tests have largely been replaced by EMA. EMA binds to connective tissue surrounding smooth muscle cells. Most laboratories use sections of human umbilical cord. Serum IgA EMA binds to the endomysium to produce a characteristic staining pattern seen on indirect immunofluorescence. The antibody is very sensitive and specific. However, after treatment the titers fall quickly to undetectable levels ( Volta, 1995 ). The epitope against which EMA is directed has been shown to be tissue transglutaminase. Use of IgA anti-tTG assays has been shown to be highly sensitive and specific for the diagnosis of celiac sprue ( Dieterich, 1998 ). An ELISA for IgA anti-tTG is widely available, less costly and easier to perform than the older immunofluorescence assay for IgA EMA. Although IgG endomysium and IgG tTG antibodies may be suitable for serological diagnosis of celiac disease, they cannot be used to monitor the response to dietary modification. Endomysium IgA antibodies disappear following treatment of celiac sprue with a gluten-free diet.
Whipple's Disease

Whipple's disease is a very rare multisystem disease that often presents with arthralgias, diarrhea, and weight loss. It is caused by the Gram-positive rod Tropheryma whippelii, an organism yet to be cultured. As this disease can be treated and is no longer uniformly fatal, it is important to make the diagnosis. PCR testing of infected tissue or cerebrospinal fluid (CSF) is the optimal way to confirm the diagnosis and monitor treatment ( von Herbay, 1997 ). Biopsy of the duodenum with periodic acidSchiff (PAS) staining had been considered pathognomonic for Whipple's disease. It is now recognized that PAS-positive macrophages may be seen in AIDS patients with Mycobacterium avium complex. Thus, PCR has gained even more importance in the management of this entity. Long-term antibiotic therapy with central nervous system (CNS) penetration is used to treat patients with Whipple's disease ( Singer, 1998 ; Ramzan, 1997 ).
Disaccharidase Deficiency

Many of the previously listed conditions causing malabsorption may also be associated with intolerance to disaccharides. Disaccharide absorption is diminished either from primary disaccharidase deficiencies such as sucraseisomaltase deficiency, lactase deficiency, primary alactasia, primary trehalase deficiency, or secondary disaccharidase deficiencies due to celiac disease, tropical sprue, acute viral gastroenteritis, or drugs such as orally administered neomycin, kanamycin, and methotrexate. These secondary disaccharidase deficiencies are usually transient and involve more than one enzyme. Although the incidence of lactose intolerance due to congenital lactase deficiency is low, the prevalence of lactose intolerance in adults is quite high. About 10% of Caucasians, 70-80% of African Americans, and an even greater percentage of Asian people manifest some degree of lactose intolerance even though they were able to digest lactose well as infants. In these disorders, intestinal bacteria ferment unhydrolyzed and unabsorbed carbohydrates, producing gas, lactic acid or other organic acids. Normally, absorption of digested carbohydrates is rapid and fairly complete in the proximal small intestine. Unhydrolyzed disaccharides or monosaccharides unabsorbed because of deficiencies in transport are osmotically active and hence cause secretion of water and electrolytes into the small and large intestines. This can result in protracted diarrhea as well as complaints of bloating and flatulence. Screening tests for disaccharidase deficiencies include oral challenge of suspected disaccharides to reproduce the abdominal symptomatology, followed by stool analysis. The

stools are usually watery, acidic, explosive, and fermentative. Stool pH of less than 5.5 is suggestive, but the measurement of pH is not valid if the patient is taking oral antibiotics. High pH does not exclude the diagnosis. Normal infants between 3-7 days of age commonly have high stool pH. Stools can be analyzed for sugars by chromatography or by one of the semiquantitative nonspecific tests for urinary sugar adapted for stool analysis. The Clinitest tablet (Bayer Diagnostics, Australia) is suitable for this purpose. The presence of 0.25 g/dL reducing substances is considered normal; from 0.25-0.5 g/dL is regarded as suspicious; more than 0.5 g/dL is considered abnormal. In patients with intolerance to sugar, the amount of total reducing substances in the stool usually exceeds 0.25 g/dL feces. An oral tolerance test using a specific sugar such as lactose or sucrose can be used to establish a specific carbohydrate intolerance. Although the oral tolerance test is fairly specific and sensitive, in some instances, 23-30% false-positive results were noted following administration of lactose that is, a flat tolerance curve and less than a 20 mg/dL (1.1 mmol/L) increase in blood sugar ( Krasilnikoff, 1975 ). Delayed gastric emptying appears to be the cause of the false-positive result, because duodenal instillation of lactose eliminates the flat tolerance curve. Definitive diagnosis of disaccharidase deficiencies depends on the demonstration of low specific enzyme activity in the mucosa of small intestinal biopsy material. An assay for disaccharidase has been published ( Dahlqvist, 1968 ).
Inflammatory Bowel Disease

Immunologic mechanisms within the colon are involved in the pathogenesis of inflammatory bowel disease. The underlying antigenic challenge to the immunologic response is not clearly understood. Over the last decade, two antibody tests have become available that assist in the laboratory evaluation of patients with inflammatory bowel disease. Perinuclear-antineutrophil cytoplasmic antibody (p-ANCA) and anti-Saccharomyces cerevisiae antibody (ASCA) can be used to distinguish abdominal pain seen in irritable bowel syndrome from inflammatory bowel disease; and subtype inflammatory bowel disease as either ulcerative colitis or Crohn's disease ( Sendid, 1998 ; Shanahan, 1994 ) (see Table 22-6 ). These tests have limitations, and interpretation requires careful understanding of the tests. Whereas few normal persons with irritable bowel syndrome will have ANCA, 70% of persons with ulcerative colitis and 20% of persons with Crohn's disease will have significant titers. In patients with inflammatory bowel disease, 65% of patients with Crohn's disease will have ASCA, whereas only 20% of patients with ulcerative colitis will have significant titers. Given the low sensitivity and specificity, the use of these tests should be dependent upon the clinical circumstance. For example, a person with diarrhea and equivocal biopsy findings found to have a positive ANCA is more likely to have inflammatory bowel disease than irritable bowel syndrome. Likewise, if a person with what appears to be ulcerative colitis is found to have a positive ASCA, Crohn's colitis may be present.

Table 22-6 -- Markers for Inflammatory Bowel Disease Percent frequency p-ANCA ASCA Irritable bowel syndrome (normal patients) < 5 <5

Percent frequency p-ANCA ASCA Ulcerative colitis Crohn's disease 70 20 15 65

p-ANCA = perinuclear antineutrophil cytoplasmic antibody; ASCA: anti-Saccharomyces cerevisiae antibody.

Neuroendocrine Tumors

Neuroendocrine tumors of the gastrointestinal tract are relatively rare neoplastic lesions with protean clinical manifestations ( Table 22-7 ). Abdominal pain and diarrhea are two of the commoner clinical symptoms. Due to the fact that they exhibit amine precursor uptake and decarboxylation (APUD) they are also known as APUDomas. The most common of these tumors are the carcinoid tumors, constituting 50% of the total. Gastrinomas comprise 25% of the APUDomas, while 15% are insulinomas and 6% are VIPomas. Glucagonomas are quite rare and make up 2-5% of series. Other even rarer types, such as somatostatinomas, have been identified ( Perry, 1996 ). The chemical measurement of the secretagogues of these tumors generally suggests the diagnosis in the right clinical setting. Localizing the tumors can be quite challenging. Ultrasonography, CT scanning, magnetic resonance imaging (MRI) operative exploration, endoscopy, angiography, and octreotide scanning all have significant technical limitations. With the exception of ultrasonography and MRI, there can be potential morbidity from the testing as well. Although it was initially hoped that octreotide scanning would replace other modes of localization, recent data suggest that it is useful in determining the extent of carcinoids and gastrinomas, while of little use in finding insulinomas or nonfunctional tumors ( Kisker, 1997 ). These tumors are often small, frequently multifocal, and can be located in a variety of organs. They can be malignant or benign. They are similar on histologic examination, so histologic staining is employed to distinguish the types ( Perry, 1996 ). As in all neoplastic lesions, biopsy is essential to confirm the diagnosis. Table 22-7 shows the distinguishing features of the various APUDomas.

Table 22-7 -- Neuroendocrine Tumors APUDoma Hormones Clinical sequelae Carcinoid Gastrinoma Insulinoma VIPomas Glucagonoma Serotonin (many others possible) Gastrin Insulin Vasoactive intestinal polypeptide Glucagon Abdominal pain, flushing, diarrhea, asthma, edema Ulcers, diarrhea Hypoglycemia Diarrhea, hypokalemia, acidosis, hypochlorhydria Necrolytic migratory erythema, weight loss, diabetes, depression, deep venous thromboses Gallstones, diabetes. diarrhea, hypochlorhydria

Somatostatinoma Somatostatin

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McPherson & Pincus: Henry's Clinical Diagnosis and Management by Laboratory Methods, 21st ed.
Copyright 2006 W. B. Saunders Company Common Tests, Methods and Clinical Applications
Amylase (Total Serum and Urine)

Amylase is a stable enzyme. In serum and urine it is stable for 1 week at room temperature and for at least 6 months under refrigeration in well-sealed containers. It may be kept in the frozen state much longer without appreciable loss of activity. Plasma specimens that have been anticoagulated with citrate or oxalate should be avoided for amylase determination because amylase is a calcium-containing enzyme, and falsely low enzyme activities are obtained from such specimens. Heparinized plasma specimens do not interfere with the amylase assay. While there are a variety of amylase methods available, regardless of the method chosen, caution must be exercised to avoid contamination of specimens with saliva, because its amylase content is approximately 700 times that of serum. Red cells contain no amylase, so hemolysis generally presents no problem with most of the methods except those coupledenzyme methods in which the released peroxide is determined by a coupled-peroxidase reaction.
Interpretation

Elevations of serum and urine amylase are observed in a wide variety of disorders. Most of the elevations of serum amylase are due to increased rates of amylase entry into the bloodstream, decreased rates of clearance, or both. Serum amylase activity rises within 6-48 hours of the onset of acute pancreatitis in about 80% of patients, but is not proportional to the severity of the disease. Values of over 600 Somogyi units/dL, or over four times the upper limit of normal, are highly suggestive of the diagnosis. Activity usually returns to normal in 3-5 days in patients with the milder edematous forms of the disease. Elevated values persisting longer than this suggest continuing necrosis or possible pseudocyst formation. The urine amylase activity rises promptly, often within several hours of the rise in serum activity, and may remain elevated after the serum activity has returned to the normal range. Values of over 1000 Somogyi units/h are seen almost exclusively in patients with acute pancreatitis. False-negative results are often seen when the urine specimen is taken too soon or too late or in patients with fulminating necrosis in which the production of amylase is decreased or has ceased. In a majority of patients with acute pancreatitis, serum amylase activity is elevated and there is a concomitant increase in urine amylase activity. There may be instances, however, in which the elevated urine amylase is not accompanied by a concomitant increase in serum amylase.

Increased renal clearance of amylase can be used in the diagnosis of acute and relapsing pancreatitis, and the ratio of amylase clearance to creatinine clearance expressed as a percentage has been used diagnostically. This ratio (Cam/Ccr) can be calculated by the following formula:

The normal ratio averages 1-4%, whereas that for patients with pancreatitis usually exceeds 4% and is often in the range of 7-15%. Unfortunately, about one-third of patients with pancreatitis have normal ratios, and elevated ratios may be found in patients with burns, ketoacidosis, renal insufficiency, heart disease, and duodenal perforation, as well as following thoracic surgery. Thus, the ratio adds little to the diagnostic armamentarium. Approximately 20% of patients with pancreatitis have normal or near-normal amylase activity. In hyperlipemic patients with pancreatitis, normal serum and urine amylase levels are frequently encountered. The spuriously normal levels are believed to be the result of suppression of amylase activity by triglyceride or by a circulating inhibitor in serum. Lower than normal serum amylase activity may be found in patients with chronic pancreatitis and has also been seen in such diverse and unexpected conditions as congestive heart failure, pregnancy (during the second and third trimesters), gastrointestinal cancer, bone fractures, and pleurisy. Serum amylase may be elevated in patients with pancreatic carcinoma, but too late to be diagnostically useful. It is also elevated frequently (in over 60% of cases) in patients with diabetic ketoacidosis. Polyacrylamide gel electrophoresis has demonstrated that in this condition, it is usually salivary rather than pancreatic amylase that is elevated. Serum amylase activity may also be elevated in patients with cholecystitis or peptic ulcer, or following gastric resection, renal transplant, viral hepatitis, or ruptured ectopic pregnancy. Very high activity has been reported in patients with carcinoma of the lung. Fewer hyperamylasemic patients may be found to have intestinal obstruction, mesenteric thrombosis, and peritonitis. In some of these patients, pancreatic secretions find their way into the peritoneal cavity and are absorbed into the bloodstream. In others, there may be inflammation involving the pancreas. Increased ascites fluid amylase levels have been seen in patients with pancreatitis, a leaking pancreatic pseudocyst, pancreatic duct rupture, pancreatic cancer, abdominal tumors that secrete amylase, and perforation of a hollow viscus.
Lipase

The pancreas is the major and primary source of serum lipase. Human pancreatic lipase is a glycoprotein with a molecular weight of 45 000 Da. In contrast to amylase, which is present in both the pancreas and the salivary glands, lipase is not present in the salivary glands. Lipases are defined as enzymes that hydrolyze preferentially glycerol esters of long chain fatty acids at the carbon 1 and 3 ester bonds, producing 2 mol of fatty acid and 1 mol of monoglyceride per mole of triglyceride. After isomerization, the third fatty acid can be split off at a slower rate. Lipolysis increases in proportion to the surface area of the lipid droplets,

and the absence of bile salts in duodenal fluid with a resultant lack of emulsification renders lipase ineffective. The presence of colipase and bile salt is required for full catalytic activity and the greatest specificity of the pancreatic lipase. Proteins, bile acids, and phospholipids inhibit serum lipase; colipase reverses this inhibition. Both lipase and colipase are secreted by the pancreas and are, therefore, present in the serum. Colipase is present in the blood of patients with pancreatitis but in variable concentrations and usually below normal and below the amount needed to activate pancreatic lipase fully. To determine accurately and fully the pancreatic lipase activity in patients with pancreatitis it is essential to add colipase to the reagent pack. Pancreatic lipase must be differentiated from lipoprotein lipase, aliesterase, and arylester hydrolase, which are related but different enzymes. These enzymes' activities may be included in the measurement of lipase activity unless the suitable assay conditions for pancreatic lipase are adapted. Lipase is also present in liver, stomach, intestine, white blood cells, fat cells, and milk. Calcium is necessary for maximal lipase activity, but at a concentration higher than 510-3M, it has an inhibitory effect. It is speculated that the inhibitory effect is due to its interference with the action of bile salts at the watersubstrate interface. Like serum albumin, bile salts prevent the denaturation of lipase at the interface. Heavy metals and quinine inhibit lipase activity. Lipase is filtered by the glomeruli owing to its low molecular weight; it is normally completely reabsorbed by the proximal tubules and is absent from normal urine. In patients with failure of renal tubular reabsorption caused by renal disorders, lipase is found in the urine. Urine lipase activity in the absence of pancreatic disease is inversely related to the creatinine clearance. Serum lipase is stable up to 1 week at room temperature and may be kept stable longer if it is refrigerated or frozen. The optimal reaction temperature is about 40C. The optimal pH is 8.8, but other values ranging from 7.0-9.0 have been reported. This difference probably is due to the effect of the difference in types of substrate, buffer, incubation temperature, and concentrations of reagents used. Serum is the specimen of choice for blood lipase assays. Icterus, lipemia, and hemolysis do not interfere with turbidimetric lipase assays. In acute pancreatitis serum lipase activity tends to become elevated at about the same time as, if not earlier than, the elevation of serum amylase, and it remains elevated for about 7-10 days. Increased lipase activity rarely lasts longer than 14 days, and prolonged increases suggest a poor prognosis or the presence of a cyst. The combined use of serum lipase and serum amylase is effective in ruling out acute pancreatitis. Although determination of serum lipase has diagnostic advantages over serum amylase for acute pancreatitis, it is not specific for acute pancreatitis. Serum lipase may also be elevated in patients with chronic pancreatitis, obstruction of the pancreatic duct, and nonpancreatic conditions including renal diseases, various abdominal diseases such as acute cholecystitis, intestinal obstruction or infarction, duodenal ulcer, and liver disease, as well as alcoholism, and diabetic ketoacidosis, and in patients who have undergone endoscopic retrograde cholangiopancreatography. Patients with trauma to the abdomen uniformly have increases in both serum amylase and lipase, whereas those with primarily head injury or

manipulation of the parotid gland during surgery have a significant increase in serum amylase only. Elevation of serum lipase activity in patients with mumps strongly suggests significant pancreatic as well as salivary gland involvement by the disease. Serum lipase and amylase tests have been and continue to be widely used in the diagnosis of acute pancreatitis. Both serum amylase and serum lipase are elevated in many patients who have inflammatory or other disorders of organs in the abdominal cavity but no evidence of pancreatitis. It is concluded that the pancreas is exquisitely sensitive to inflammatory or metabolic disturbances in the peritoneum and nearby organs.
Isoamylases

The amylase present in blood and urine of normal individuals is predominantly of pancreatic and salivary origin. Amylase of pancreatic and salivary origin is abbreviated to P-type and Stype amylase (isoenzyme, isoamylase), respectively, whenever such a distinction is needed. These two types of amylase are closely related enzymes, but have organ-specific variations. They have the same amino acid composition and yield similar but not identical peptide maps. Each appears to consist of a single polypeptide chain without subunits. Pancreatic amylase has a molecular weight of 54 000. Higher molecular weights have been reported for salivary amylase. Both amylases contain sulfhydryl groups. Amylases are metalloenzymes containing at least one atom of calcium per molecule; they require this metal for their catalytic activities. The pH for optimal activity ranges from 6.9-7.0. The pH optimum for salivary amylase varies with the anion used as activator, of which chloride is the most important. Optimal chloride concentration is 10 mmol/L, and the activation is allosteric. Bromide and iodide ions also activate amylase. The isoelectric points (pI) have been reported to be 7.6 and 7.2 for P-type, and 6.4 and 5.8 for S-type isoamylases. The fractionation of amylase in serum, urine, or other body fluids may be achieved by physical means, such as electrophoresis, chromatography, and isoelectric focusing, and each isoenzyme is then quantitated either by direct densitometry or by amyloclastic or saccharogenic techniques. A simplified, readily adaptable, chromatographic method has been described by Fridhandler (1980) . A chemical inhibition assay employing a salivary amylasespecific protein inhibitor is also being used for isoenzyme determinations and is commercially available. The chemical inhibition of isoamylase determination is simple, fast, and suitable for emergency situations. An immunoinhibition method using a monoclonal antibody to inhibit the salivary amylase and subsequently quantitate the remaining pancreatic amylase has been reported ( Mifflin, 1985 ). Because of its simplicity, acceptable analytical precision, and good correlation with the electrophoretic isoamylase method, this method should be further investigated for clinical application. A great number of reports have supported the finding that in acute pancreatitis, P-type amylase is invariably elevated in both serum and urine. The S-type isoenzyme, however, is decreased to 0-15% of the total activity of serum hyperamylasemia in patients with acute pancreatitis, to 12-25% in those with chronic relapsing pancreatitis, and to near zero in those with carcinoma of the head of the pancreas. The P-type isoenzyme is also elevated in chronic relapsing pancreatitis, hypoparathyroidism, and glomerulonephritis. S-type amylase is increased in the serum of patients with chronic pancreatitis, mumps, pancreatic insufficiency, Sjgren's syndrome, cholelithiasis, common duct narrowing, alcohol ingestion, acute gastroenteritis, acute respiratory insufficiency, chronic renal failure, lung cancer, and with other cancer-associated hyperamylasemias. Isoenzyme studies on serum, urine, and duodenal

fluid from patients with cystic fibrosis revealed that two-thirds of the patients had no or little pancreatic amylase. The relative activity of P-type isoamylase has been reported to be highly useful as a diagnostic index of pancreatic pseudocysts ( Warshaw, 1980 ). The P1 isoamylase (the slowest migrating or least anodic) normally accounts for 80-90% of total amylase activity: P2 and P3 account for 0-4% in both serum and pancreatic juice. The mean ratios of P2/P1 and P3/P1 in fresh pancreatic juice, normal serum, acute pancreatitis serum, chronic pancreatitis serum, and pancreatic cancer serum were always less than 0.25 and less than 0.04, respectively. This ratio was elevated in about 90% of sera from patients with proven pseudocysts but not from others. In several cases, this isoamylase analysis ruled out a pseudocyst correctly, whereas ultrasound or CT scan erroneously indicated the presence of a pseudocyst.
Gastrin

Gastrin is a primary gastrointestinal hormone, produced mainly by the antral G cells, that regulates gastric acid secretion and stimulates growth of the gastric mucosa among other functions. To a lesser extent, gastrin is produced by the G cells of the proximal small intestine and delta cells of the pancreas. Gastrin acts on the parietal cells located in the fundus of the stomach, stimulating the secretion of gastric acid. Gastrin also increases blood flow to the stomach and is responsible for increased gastric and intestinal motility. Other functions include stimulation of gastric pepsinogens and intrinsic factor secretion, release of secretin from the small intestine and secretion of pancreatic enzymes as well as bicarbonate ( Henderson, 1994 ). This hormone is secreted mainly after the detection of digested protein products as well as from antral distention. Maximal stimulation of gastrin secretion occurs within a pH range of 5-7. An acid environment serves as a negative-feedback mechanism for the release of gastrin with 80% reduction in secretion at a pH of 2.5 ( Henderson, 1994 ). This serves to protect the stomach from overacidification from excess stimulation of gastrin. For this reason, individuals on acid suppression therapy for peptic ulcer disease may have elevated gastrin levels. Three main forms of gastrin exist in human blood and tissues: G34, G17, and G14, known as big gastrin, little gastrin and mini gastrin respectively. All gastrins originate from a single precursor, preprogastrin that is cleaved by the action of trypsin. Preprogastrin yields progastrin, which is subsequently processed to yield glycine-extended gastrin (G34 Gly and G17 Gly) before conversion to the amidated forms G34 and G17 ( Wang, 1999 ). Interestingly, in pathologic cases of increased gastrin production, as with achlorhydric gastritis or gastrinomas, larger molecular forms of gastrin and incompletely processed precursors are present and are beyond the scope of detection by conventional assays. In such cases only little gastrin would be detectable in serum ( Goetze, 2003 ). Laboratory determination of gastrin levels with available RIA or ELISA assays is indicated for the confirmation of suspected diagnosis of gastrin-secreting tumors, namely, gastrinomas or ZollingerEllison syndrome. The antibodies present in these assays are specific for the biologically active C-terminal of the gastrin molecule and they have minimal cross-reactivity with CCK peptides. Prior to determination of gastrin levels, a patient must be fasting for 12 hours because the concentration of G34 doubles and the concentration of G17 quadruples following a meal, altering the results of the assay. Specimens must be frozen immediately because gastrin is unstable in serum. Due to the action of proteolytic enzymes, 50% of the

specimen's immunoreactivity may be lost within 48 hours at a temperature of 4C. It is recommended that specimens should be kept in a freezer at a temperature of -70C without a self-defrosting cycle, if long-term storage is required. Specimens must be analyzed immediately after thawing, avoiding refreezing and thawing. Normal reference gastrin levels for fasting individual range up to 100 ng/L. However, it should be noted that fasting serum gastrin levels are increased with increasing age, especially in patients older than 60 years, in part due to unrecognized gastric mucosal atrophy. Approximately 15% of individuals older than 60 years may have gastrin levels between 100800 ng/L ( Henderson, 1994 ). It should also be noted that reference intervals for infants and children differ from those for adults, so values should be compared to an age-specific reference range for accuracy. Gastrin concentration greater than 1000 ng/L with gastric acid hypersecretion (basal acid secretion > 15 mmol/h) is diagnostic of gastrinomas. In patients with ZollingerEllison syndrome (ZE) there is usually a marked elevation, up to 2000 times the normal gastrin level. The secretin stimulation test is a provocative biochemical test that can help confirm the diagnosis of ZE in questionable cases. Infused secretin should cause a drop in gastrin levels, as occurs in normal patients. However, in patients with ZE, there is a dramatic increase in gastrin level, confirming the diagnosis. The mechanism by which secretin stimulates an increase in gastrin levels in these patients is poorly understood; however, it is thought to be due to a direct local effect on the blood flow to the tumor ( Ashley, 1999 ). Limitations include altered results from conditions that may lead to elevated gastrin levels such as gastric ulcer disease, chronic renal failure, hyperparathyroidism, pyloric obstruction, vagotomy, retained gastric antrum, short bowel syndrome, and pernicious anemia. Certain medication can also increase gastrin measurements such as antacids, H2blocking agents and proton pump inhibitors, all commonly used in the treatment of patients with peptic ulcer disease. However, these elevations are moderate and certainly not as high as in a patient with a gastrin-secreting tumor. Assays have traditionally been used to detect elevated gastrin levels associated with diseases such as ZE or gastrinoma. However, the clearly established benefit of rapid intraoperative PTH assays (see Ch. 15 ) has prompted similar interest in gastrin as an intraoperative guide for therapeutic management, either to confirm adequate removal of gastrin-secreting tissue or to prompt further dissection and possibly more elaborate procedures. Intraoperative testing is of potential use because gastrinomas can be multiple and are often difficult to locate because they can be distributed widely, among for example, stomach, pancreas, and duodenum or periaortic lymph nodes. Gastrin is an appropriate hormone for rapid intraoperative testing because it has a short analyte half-life, approximately 10 minutes, so that changes can be detected shortly after resection of hypersecreting tissue, rapid analysis that can be completed within the time frame of the operative procedure, and a positive clinical utility ( Sokoll, 2004 ). The catabolic breakdown of most peptide hormones follows first-order exponential decay. Therefore, if the entire hormone-secreting tissue is surgically resected, only approximately 12.5% of the baseline concentration would be present in serum after three half-lives ( Sokoll, 2004 ). In one clinical study, patients with ZE or gastrinomas were evaluated with intraoperative gastrin assays, and a drop of gastrin levels to within reference values within 20 minutes of resection was indicative of cure. The sensitivity for intraoperative gastrin assays has been estimated to be 88%. This test may help to identify patients who may benefit from more extensive dissections or operative procedures such as duodenopancreatectomy, a procedure not advocated on all patients because of associated morbidity and mortality ( Sokoll, 2004 ). However, gastrin assays intraoperatively may help to identify patients who

would benefit from this surgical intervention, a remarkable advancement in the management of patients with gastrinomas.
Pepsin and Pepsinogen

Pepsinogens are the biologically inactive proenzymes of pepsins that are produced by the chief cells and other cells in the gastric mucosa and are found in two distinct types, pepsinogen I (PGI), also known as pepsinogen A, and pepsinogen II (PGII), also known as pepsinogen C. Pepsinogen secretion is stimulated by the vagus nerve, gastrin, secretin and CCK, and is inhibited by gastric inhibitory peptide (GIP), anticholinergics, histamine H2receptor antagonists and vagotomy ( Henderson, 1994 ). PGI is produced in the chief cells and mucous cells of oxyntic glands; PGII is produced in mucous cells in oxyntic and pyloric regions and the duodenum. The ratio of concentration of pepsinogen I to pepsinogen II in serum or plasma of healthy individuals is approximately 4:1 ( Samloff, 1982 ). Pepsinogen is converted to the active form, pepsin, by gastric acid that can activate additional pepsinogen autocatalytically. Both groups of pepsinogen are activated at an acid pH below 5 and destroyed by alkaline pH. Both types can be detected in blood. Only type I pepsinogens are present in the urine, and type II is present in semen ( Henderson, 1994 ). Pepsins are responsible for the hydrolysis of proteins to polypeptides. The pepsinogen released from the gastric mucosa is predominantly secreted and constitutes a major component of gastric fluid. Only approximately 1% gets into the peripheral blood. Active pepsin is rapidly inactivated in the bloodstream, whereas pepsinogen is stable in the blood. Pepsinogen is then filtered by the kidneys and is excreted in the urine, where the slightly acidic pH converts the pepsinogen, now called uropepsinogen to uropepsin ( Henderson, 1994 ). Immunoassay is the method used to detect serum pepsinogen. However, the PGI isoform is commonly analyzed in the clinical laboratory since it is the isoform commonly associated with disease. Serum levels of pepsinogen I are an accurate estimate of parietal cell mass and correlate with acid-secretory capacity of the stomach. Increased pepsinogen levels and associated activity is observed in patients with disease states that lead to increased gastric output or with increased parietal cell mass, namely, gastrinomas, ZollingerEllison syndrome, duodenal ulcer disease and acute and chronic gastritis ( Henderson, 1994 ). Decreased levels of pepsinogen are associated with disease states marked by decreased parietal cell mass, namely atrophic gastritis and gastric carcinoma as well as in patients with myxedema, Addison's disease and hypopituitarism ( Henderson, 1994 ). The PGI/PGII ratio decreases linearly with worsening atrophic gastritis. Absence of pepsinogen is noted in patients with achlorhydria. This must be kept in mind when analyzing serum pepsinogen levels, possibly limiting clinical utility of this serum assay. Pepsinogen I levels measured by immunoassay usually range from 20-107 and pepsinogen II levels usually range from 3-19 g/L. Pepsinogen assays are being explored for their utility in the noninvasive identification of patients with chronic atrophic gastritis as well as to provide an estimate of the extent of atrophic gastritis, a known precursor of gastric carcinoma. Severe atrophic body gastritis causes a four- to fivefold increase in the risk of gastric carcinoma compared to healthy individuals ( Sanduleanu, 2003 ). This will hopefully identify a sub group of individuals with chronic atrophic gastritis that would benefit from endoscopic evaluations for detection of early-stage gastric tumors. These assays are currently utilized in Japan, an area marked by high prevalence of gastric cancer, as a potential method for widespread screening of high-risk individuals ( Miki, 2003 ). Miki recommended that criteria for diagnosing chronic atrophic gastritis be persons with PGI < 70 g/liter and PGI/PGII ratio < 3.0. All patients fitting these

criteria should then be referred to a gastroenterologist for further endoscopic evaluations. In Japan, the pepsinogen serum screening test has been demonstrated to detect a higher percentage of early cancers compared to conventional methods, and a considerable number of patients have subsequently been candidates for treatments with endoscopic surgery ( Mikki, 2003 ). The most sensitive test for fundic atrophic gastritis is considered to be the PGI/II serum ratio, with 99% sensitivity and 94% specificity ( Henderson, 1994 ). Furthermore, pepsinogen II levels may be a useful marker of prognosis, serving as an independent predictor of tumor biology and survival in patients with gastric carcinoma. The absence of PGII production has been associated with aggressive tumor behavior and shorter overall survival in gastric cancer patients ( Fernandez, 2000 ). Pepsinogen assays may therefore prove to be a useful serum screening method for detection of gastric carcinoma among highrisk individuals.
Trypsinogen

Trypsin is produced in the exocrine pancreas as two proenzymes, known as trypsinogen 1 and trypsinogen 2. The proenzymes are activated in the duodenum by an enterokinase that yields trypsin 1 and trypsin 2 respectively. Trypsin present within the peripheral circulation is inactivated by complexing with either alpha-2-macroglobulin or alpha-1-antitrypsin (AAT). Trypsin, unlike amylase, is solely produced by the pancreatic acinar cells and is therefore a specific indicator of pancreatic damage. Premature activation of the proenzyme to active trypsin within the pancreatic parenchyma is thought to be a key mechanism in the development of acute pancreatitis ( Andersen, 2001 ). Currently, levels of all forms of trypsin are determined by specific immunoassays. Trypsin assays have found clinical utility in the diagnosis of acute pancreatitis. Pancreatitis, discussed in detail in the previous section, is frequently the result of either excessive alcohol consumption or a sequela of gallstone disease. The differentiation between these two etiologies is aided by careful history and imaging studies, in particular ultrasonography. However, in approximately 5-10% of acute pancreatitis cases, a specific etiology is never determined. This is important, because although the initial management and resuscitation of a patient with pancreatitis follows the same management logarithm for both etiologies, patients with biliary pancreatitis, resulting from gallstones, may benefit from surgical intervention. Elective cholecystectomy should be performed in these patients after the pancreatitis resolves, to avoid a recurrent episode of a potentially fatal disease. Therefore, biochemical markers to differentiate the etiologies would be clinically invaluable. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), amylase, and lipase levels are useful in diagnosing acute pancreatitis, and are sometimes suggestive of one etiology versus the other. However, trypsin assays are currently being utilized for the purpose of differentiating the cause of an acute episode of pancreatitis. One study demonstrated that trypsinogen 2 and trypsin-2AAT are increased in all forms of acute pancreatitis but were more elevated in alcoholic-associated pancreatitis than in biliary pancreatitis. Trypsinogen 1, amylase and lipase were found to be more elevated in patients with biliary pancreatitis. Furthermore, the ratio of serum trypsin-2AAT to trypsinogen 1 was determined to be the best discriminator between biliary and alcoholic pancreatitis ( Andersen, 2001 ). Interestingly, another study supported the use of trypsin assays for diagnosis of acute pancreatitis because the determined time course profile of trypsinogen 2 and trypsin-2AAT is appropriate for diagnostic purposes. These enzymes are elevated within hours of the onset of the acute episode, and therefore already elevated upon admission, followed by a rapid rise. Both enzyme levels remain elevated longer than amylase and the magnitude of elevation

corresponds to the severity of pancreatic inflammation, extremely useful for diagnosing acute pancreatitis upon admission, predicting severity of illness and for monitoring disease progression ( Kemppainen, 2000 ). Elevated trypsin-1ATT has also been demonstrated in patients with biliary tract cancer ( Andersen, 2001 ).
Vasoactive Intestinal Peptide (VIP)

Vasoactive intestinal peptide (VIP), a neuropeptide, has a molecular weight of 3381, consists of 28 amino acids and is a member of the secretinglucagon family ( Radebold, 2003 ). It is initially synthesized as a 170-amino-acid precursor pre-pro-VIP, which post-translationally, is modified to yield the 28-amino-acid VIP ( Vinik, 2004 ). Vasoactive intestinal peptide, released in response to gut distention, is a potent vasodilator and is responsible for the relaxation of vascular and nonvascular smooth muscle of the intestinal tract. It is also a potent stimulator of water and electrolyte secretion, acting through stimulation of cyclic AMP (cAMP). Similar to glucagons, VIP stimulates breakdown of glycogen and lipid stores and inhibits histamine-stimulated acid secretion in the stomach, resulting in hypochlorhydria or achlorhydria. Diagnostic laboratory evaluations for determination of VIP levels are clinically relevant for diagnosis of VIPomas, VIP-secreting tumors, most commonly of pancreatic origin, accounting for 10% of neuroendocrine tumors of the gastrointestinal tract. Most of these tumors are sporadic and may also occur in the adrenal glands, mediastinum, retroperitoneum, lung and jejunum. The incidence of the tumors is estimated to be between 0.05-0.2/million per year. Approximately 60% of VIPomas are malignant and 6% are associated with multiple endocrine neoplasia (MEN) type I. This syndrome was first described in 1958 by Verner and Morrison, characterized by watery diarrhea, hypokalemia and achlorhydria (WDHA), and was later discovered to be caused by tumors secreting large amounts of vasoactive intestinal peptide hormone ( Radebold, 2003 ). In this section, we will focus on the GI manifestations of VIPomas. VIPomas produce a severe, secretory diarrhea that has been termed pancreatic cholera. Diarrhea may be present for several years prior to the diagnosis of VIPoma. Patients typically produce more than 3 L of watery stools per day though volume can be as high as 30 L/day. Diarrhea does not improve with fasting and an average secretion of 300 mmol of potassium per 24 hours occurs ( Vinik, 2004 ). Stool volume of less than 700 mL/day excludes the diagnosis of VIPoma. If the diagnosis is missed or delayed, as is often the case, the chronic diarrhea results in severe fluid and electrolyte imbalances that produce a myriad of clinical symptoms, the most severe of which is sudden death from cardiac arrhythmias resulting from electrolyte disturbances, namely potassium depletion and acidosis. The metabolic profile present in these patients is typically hypokalemic, hyperchloremic metabolic acidosis. The diagnosis of VIPomas is made with confirmation of raised fasting VIP levels in association with secretory diarrhea and the presence of a lesion, most commonly located in the pancreas associated with VIP production. If patients have mild or intermittent symptoms, common in the early stage of tumor growth, random VIP levels may be normal, delaying the diagnosis. Under these circumstances, VIP levels should be obtained during a bout of diarrhea when active secretion of VIP can be detected. Confirming the biochemical diagnosis of VIPoma involves a highly sensitive and specific radioimmunoassay to detect raised VIP levels. The normal value for circulating VIP levels is less than 170 pg/mL. Patients with functional VIP-secreting tumors have ranges from 675-

965 pg/mL ( Thomas, 2003 ). It is important to note that appropriate handling of the serum sample is critical to the accuracy of the VIP radioimmunoassay because VIP has a half-life of 1 minute. The serum must be immediately added to aprotinin, a protease inhibitor that prevents breakdown of VIP. In addition, the sample must be separated within 10 minutes and frozen to -20C. Some of the non-VIP products of the precursor molecule are secreted at higher levels than VIP; however, commercially available assays are not available and clinical utility has not been established. False-positive elevations of VIP can be observed in patients with small bowel ischemia or severe low-flow states resulting from diarrhea and subsequent dehydration not associated with VIP-producing lesions ( Vinik, 2004 ). In addition, serum pancreatic polypeptide level should be determined at the time of the VIP RIA; this value will be elevated if the VIPoma is located within the pancreas. CT scan, MRI and abdominal ultrasound are useful imaging modalities for VIPoma tumor localization. Angiography can be used to localize smaller tumors. Various nuclear scans have been utilized for localization of VIPomas, including 123I VIP receptor scintigraphy, which is currently under investigation.
Sweat Chloride

Pilocarpine is introduced into the skin by iontophoresis to stimulate locally increased sweat gland secretion. The resulting sweat is absorbed by filter paper or gauze, weighed, diluted with water, and analyzed for sodium and chloride concentrations. The method is painless and reliable if performed properly. Using the Wescor Macroduct Sweat collection system (Wescor Inc., Logan, UT), volumes of sweat up to 100 L can be obtained and the chloride can be measured directly using 20 L samples with a chloride analyzer such as the Corning 925 (Chiron Diagnostics, Ltd., East Walpole, MA). This reduction of steps in the procedure leads to greater reproducibility. Total body sweating in patients with cystic fibrosis is hazardous, and a number of deaths from the procedure have been recorded. When performed properly in duplicate, the sweat test has a sensitivity of 90-99%. Unacceptably high rates of incorrect results have been attributed to problems associated with sweat specimen sample collection and test analysis. Methodologic unreliability, technical errors, inadequate and inappropriate collection of sweat, inexperience of laboratory workers, lack of appropriate quality controls, and misinterpretation of test results were found to be sources of errors ( LeGrys, 1994 ). To provide the best possible quality of sweat testing, laboratories are referred to the document Sweat Testing: Sample Collection and Qualitative Analysis: Proposed Guidelines (document C34-P, 1993), developed by the National Committee for Clinical Laboratory Standards (NCCLS) (Villanova, PA) to improve the performance of the sweat test for the diagnosis of cystic fibrosis ( LeGrys, 1994 ). This NCCLS document includes a discussion of sweat stimulation, qualitative measurements of sweat chloride and sodium, and quality control issues. The CAP and the Cystic Fibrosis Foundation have jointly developed the External Proficiency Testing Survey for Sweat Test Analysis (Set SW) for laboratories to further improve the quality of the sweat test. The Cystic Fibrosis Foundation recommends that laboratories that perform few sweat tests per year should refer patients to a cystic fibrosis center ( LeGrys, 1994 ).
Apt Test for Neonatal Bleeding

When blood is found in the gastrointestinal tract or stool of neonates, a determination of whether the blood is swallowed blood of maternal origin or secondary to disease in the

newborn must be made ( Guritzky, 1996 ). The Apt test is a qualitative test that is used to make this determination in grossly bloody stools or hematemesis from a newborn. Nursing infants may ingest maternal blood, as well, if there is nipple cracking or bleeding. The sample is first mixed with water and centrifuged. The supernatant, which should be pink, is then mixed with 1% sodium hydroxide in a ratio of 5:1. If the blood is of maternal origin, the mixture turns yellow-brown after several minutes. Fetal blood remains pink. This test, based on the fact that HbF is more resistant to alkali denaturation than adult hemoglobin, has a relatively low sensitivity and the result must be interpreted with caution ( McRury, 1994 ).
Tests for Steatorrhea
Screening Tests

Screening tests for detection of steatorrhea include microscopic examination of feces for fat globules and determination of serum carotenoid. Carotenoids are a group of compounds that are the major precursors of vitamin A in humans. Absorption of carotenoid in the intestines depends on the presence of dietary fat and its normal absorption. Because carotenoids are not stored in the body to any appreciable degree, lack of carotenoids in the diet or disturbances in absorption of lipids from the intestine can result in decreasing levels of serum carotenoid. This is a simple and useful screening test for steatorrhea. In addition to steatorrhea and poor dietary intake, liver disease and high fever may also cause a low level of serum carotenoid. Elevated serum carotenoid levels are seen in patients with hypothyroidism, diabetes, hyperlipidemia, and excessive intake of carotene.
Breath Test.

The test is based on the measurement of 14CO2 in expired air following the ingestion of various 14C-labeled triglycerides (triolein, tripalmitin, and trioctanoin). Steatorrhea from either pancreatic insufficiency or other causes results in a decreased absorption of triglycerides by the digestive system. This in turn results in a decrease in expired CO2 derived from metabolism of triglyceride fatty acids.
Procedure.

After an overnight fast the patient consumes 14C-labeled triglyceride. Periodically, breath CO2 is collected in a trapping solution containing an indicator that changes color when a predetermined amount of CO2 is in solution. The radioactivity of the 14CO2 is then measured in a liquid scintillation counter, and the results are reported as a percentage of the dose of 14 CO2 excreted per hour.
Comment.

To distinguish pancreatic insufficiency from other causes of steatorrhea some investigators have developed a two-stage breath test ( Goff, 1982 ). In the first stage of the test the patient consumes a 14C-labeled triglyceride, and the 14CO2 is measured as previously described. The second stage of the test is performed 5-7 days later and is the same as the first stage except that the patient is given an oral dose of pancreatic enzymes along with the dose of 14C-labeled triglyceride. In patients with steatorrhea due to pancreatic insufficiency the amount of 14CO2 expired should increase relative to the amount of 14CO2 expired in the first stage of the test. Patients with steatorrhea from other causes should show no significant change in the amount of 14CO2 expired following the oral administration of pancreatic enzymes.

Definitive Test for Steatorrhea

The definitive test for steatorrhea is the fecal fat determination. The amount of fat in feces may be determined and expressed as a percentage by weight of wet stool, a percentage by weight of dry stool, a percentage of ingested fat retained (absorbed), or a chemically determined amount of fat per 24-hour stool collection. Because of wide variations in water content of stool, wet weight concentration is the least informative. Dry weight concentration is only slightly less variable because of the effect of diet on bulk. Total output of fat per 24 hours, based on chemical analysis of at least a 3-day stool collection, is the most reliable measurement. For this purpose, the patient is placed on a standard diet containing 100 g fat per day. In infants and children, for whom the standard 100-g diet cannot be used, percent coefficient of fat retention is a more useful expression. This is the difference between fecal fat and ingested fat expressed as a percentage of the ingested fat:

The coefficient of fat retention in normal children and adults is 95% or higher, although in premature infants it may be much lower than this. A low value otherwise is indicative of steatorrhea. The normal fat content of feces consists primarily of fatty acids, fatty acid salts (soaps), and neutral fats, with higher alcohols, paraffins, sterols, and vegetable carotenoids present in significantly smaller amounts. Fractionation of total lipids into free fatty acids and neutral fats was thought formerly to aid in assessment of the exocrine functions of the pancreas. However, owing to the presence of bacterial lipase and the spontaneous hydrolysis of neutral fats, fractionation of total lipids provides no additional information about the cause of steatorrhea. Several laboratory procedures are available for the evaluation of fat malabsorption. Titrimetric methods quantitate various chemical forms of fatty acids, whereas gravimetric and microscopic procedures evaluate total fecal fat. The titrimetric method has been the most widely used procedure for the quantitation of fecal fats. Breath tests represent a more recent approach to the diagnosis of fat malabsorption. In these tests the specific radioactivity of 14 CO2 is measured after the ingestion of a test meal containing carbon-14 (14C)-labeled triglycerides.
Titrimetric Method.

The titrimetric method serves as the laboratory procedure for the definitive diagnosis of steatorrhea. In this method fats and fatty acids are converted to soap (saponified) by boiling feces with alcoholic potassium hydroxide, yielding a solution that contains soaps derived from neutral fats and fatty acids and soaps originally present in the stool. After cooling, excess hydrochloric acid is added to convert soaps to fatty acids. These are extracted with petroleum ether. An aliquot is evaporated, taken up in neutral alcohol, and titrated with sodium hydroxide. Fats are calculated as fatty acids. In some cases of malabsorption, the coefficient of fat retention can be improved by substituting medium-chain fatty acids for long-chain fatty acids in the diet. The titrimetric method does not quantitatively recover medium-chain fatty acids. An improved recovery of medium-chain fatty acids from feces can be obtained by slightly modifying the titrimetric

procedure. The amount of water used during saponification can be reduced and the excess alcohol distilled prior to extraction, resulting in complete recovery of medium-chain and long-chain fatty acids.
Tests for Malabsorption

When a diagnosis of malabsorption is being entertained, it becomes important to distinguish pancreatic maldigestion from enteric malabsorption. In children, the main cause of pancreatic malabsorption is cystic fibrosis, and the sweat chloride determination should be used when clinical evidence warrants it. Screening tests based on absent stool trypsin have also been used. One of the most valuable differential diagnostic tests, especially in adults, is the dxylose absorption test. However, poor kidney function may also result in low excretion, making the test difficult to interpret in patients with renal disease. If the test is performed in these circumstances, blood values should also be assayed. High blood values coupled with low urine values are expected in renal disease. Because reference values in this situation are not available, the test is better avoided in patients with renal disease. The cellobiosemannitol sugar permeability test and lactulosemannitol test have been used in the diagnosis of celiac disease. The modern evaluation of this disorder has been described above. Isotopic techniques and the starch tolerance test have been used as alternatives to the d-xylose test. Quantitative specific fecal trypsin and chymotrypsin assays may be helpful, as may be the Schilling test for vitamin B12 absorption, which tends to be abnormal in patients with enteric steatorrhea and in which the abnormality is not correctable with intrinsic factor. Endoscopy, radiologic studies, and biopsy have replaced these methods in many cases.
D-Xylose

Test

The d-xylose absorption test is a valuable test for the differential diagnosis of malabsorption. In this procedure a 25-g dose of pentose sugar in water is administered orally, and the amount excreted over a 5-hour period in the urine is determined. If the amount excreted is less than 3 g, the diagnosis is mostly likely enterogenous malabsorption because pancreatic enzymes are not required for absorption of d-xylose. d-Xylose is passively absorbed in the small intestine and is not metabolized by the liver, although a portion of an orally or intravenously administered dose is destroyed. The accuracy of the method depends not only on the rate of absorption of d-xylose but also on the rate of excretion by the kidneys. It is therefore advisable in patients with renal disease to collect a blood sample for xylose quantitation 2 hours after the administration of xylose.
Testing and Examination of Feces
Collection

Uninstructed patients sometimes exhibit considerable ingenuity in collecting stool specimens, but a few simple instructions are likely to produce more satisfactory specimens. A scoured, well-rinsed bedpan is a convenient collection container. If the patient does not own one, a carefully cleaned, rinsed, and boiled glass jar of suitable size is a satisfactory alternative. Patients should be warned against passing urine at the same time into the bedpan or container because, among other things, urine has a harmful effect on protozoa. Tongue depressors or pieces of cardboard are reasonably convenient instruments for transferring the stool from bedpan to transport vessel, for which plastic, cardboard, and glass containers are available. We prefer two 2-oz ointment jars with screw caps for small stool samples because they are odor-free, leak proof, and easy to transport. Patients should be instructed not to contaminate

the outside of the container and not to overfill the container. Gas, which frequently accumulates, should be released gradually by carefully loosening of the cap. Failure to observe this simple precaution, especially in the case of an overfilled container, can result in an explosive release of contents. Fecal matter left on the physician's gloved finger at the time of a rectal examination may be transferred to a piece of filter paper for inspection and testing for occult blood. Because of wide variation in bowel habits, intestinal transit time, and bulk of stool, special consideration must be given to methods of timed stool collection. For collection of timed urine specimens, the urinary bladder can be emptied before and at the end of the collection period. The gastrointestinal tract, however, cannot be emptied completely at will. Therefore, the amount of stool collected in a 24-hour period usually correlates very poorly with the amount of food ingested during a similar period of time. For determining the 24-hour fecal excretion of any substance, stool should be collected over a period of at least 3 days, and calculations should be based on the entire specimen divided by the number of days of collection. The accuracy of this method can be enhanced somewhat by having the patient ingest carmine dye (0.3 g) at the beginning and charcoal (1 g) at the end of a collecting period, and collecting the stools from the beginning of the appearance of the dye to the beginning of the appearance of the charcoal. However, Salmonella cubana outbreaks in Massachusetts and California were traced to carmine dye. Another method of signaling the collection period involves the use of inert, nonabsorbable stool markers. These are taken in divided uniform doses for several days prior to the beginning of the collection, continuing through the collection period. The concentration of the material found in the stool specimen is then used to determine the quantity of stool containing 1 day's ingestion of the material as an indication of the 24-hour output. For this purpose, chromium sesquioxide (Cr2O3) has been used, and its concentration in the feces is determined chemically. The substitution of radioactive chromium or zirconium isotope has made it possible to determine concentration by measuring the radioactivity of the stool, but these methods, as currently used, are too time-consuming for routine determinations. Fecal specimen sample collection at home is by no means simple and easy unless the patient has been instructed properly. Hoffman (1973) has described a collection method that has the advantages of ease of transportation and storage, absence of requirements for special equipment, and acceptability to patients and laboratory staff. A pediatric method described by Jelliffe (1973) includes the use of a thick-walled glass tube, which is lubricated by dipping into water and then inserted into the young child's rectum. In about two-thirds of cases, a core of feces can be obtained, which can be poked out with an applicator stick into the container.
Macroscopic Examination of Feces

Inspection of the feces is important because it may lead to a diagnosis of parasitic infestation, obstructive jaundice, diarrhea, malabsorption, rectosigmoidal obstruction, dysentery, ulcerative colitis, or gastrointestinal tract bleeding. The quantity, form, consistency, and color of the stool should be noted. Normally, 100-200 g of stool is passed per day. When diarrhea is present the stool is watery. Passage of large amounts of mushy, foul-smelling, gray stool that floats on the water is characteristic of steatorrhea. Constipation may be associated with passage of small, firm, spherical masses of

stool (scybala). Constipation most often results from the irritable colon syndrome in patients with anxiety or from overuse of laxatives. In such patients, repeated tests for occult blood are called for to detect more serious organic problems such as carcinoma, which may also afflict those patients. A narrow, ribbon-like stool suggests the possibility of spastic bowel or rectal narrowing or stricture. Clay color suggests diminution or absence of bile or the presence of barium sulfate. Blood, especially blood originating from the lower gut, may cause the stool to be red; beets in the diet may mimic this. Bleeding from the upper gastrointestinal tract is more likely to cause the stool to be black and have a tarry consistency. Bismuth, iron, and charcoal may also cause a black color. Stool that is allowed to stand in the air for a time may darken on the surface. Green stools may result from ingestion of spinach and other green vegetables or calomel, or it may result from the presence of biliverdin, seen in patients taking antibiotics orally. It is not unusual to see seeds and vegetable skins. Parasites are considered in Chapter 61 .
Mucus.

The presence of recognizable mucus in a stool specimen is abnormal and should be reported. Translucent gelatinous mucus clinging to the surface of the formed stool suggests spastic constipation or mucous colitis. It is seen in stools of emotionally disturbed patients and may result from excessive straining. Bloody mucus clinging to the fecal mass suggests neoplasm or inflammatory processes of the rectal canal. Mucus associated with pus and blood is found in stools of patients with ulcerative colitis, bacillary dysentery, ulcerating diverticulitis, and intestinal tuberculosis. Patients with villous adenoma of the colon may pass copious quantities of mucus, amounting to 34 L in 24 hours. They frequently develop severe dehydration and electrolyte disturbances, especially hypokalemia.
Pus.

Patients with chronic ulcerative colitis and chronic bacillary dysentery frequently pass large quantities of pus with the stool, the recognition of which requires microscopic examination. This also occurs in patients with localized abscesses or fistulas communicating with the sigmoid colon, rectum, or anus. Large amounts of pus seldom accompany the stools of patients with amebic colitis and its presence is evidence against this diagnosis. No inflammatory exudate is seen in the watery stools of patients with viral gastroenteritis.
Microscopic Examination of Feces Fat.

The crudest technique is microscopic examination using Sudan III, Sudan IV, or Oil Red O stain. The procedure has been widely employed for screening because of its simplicity. In our experience, results have correlated well with quantitative measurements when aliquots of the same homogenized stool have been analyzed. For this purpose, a small aliquot of stool suspension is placed on a slide and mixed with two drops of 95% ethanol; then two drops of saturated ethanolic solution of Sudan III are added, with further mixing. A coverslip is then applied. Under these conditions fatty acids are present as lightly stained flakes or as needlelike crystals that do not stain and therefore may be missed. Soaps also do not stain but appear as well-defined amorphous flakes or as rounded masses or coarse crystals. Neutral fats, however, appear as large orange or red droplets. When 60 or more stained droplets of neutral fats per high-power field (hpf) are seen, one may be reasonably certain that the patient has steatorrhea. Caution is advisable in interpretation, however, because mineral oil or castor oil

may mimic neutral fat. The procedure is then repeated, adding several drops of 36% (v/v) acetic acid to the stool mixture and warming the slide several times over a flame until slight boiling occurs. This converts neutral fats and soaps to fatty acids and melts the fatty acids, causing them to form droplets that stain strongly with Sudan III. The slide is then examined while warm. After this procedure, the presence of up to 100 stained droplets per high-power field is considered normal. Patients with steatorrhea of pancreatic origin are likely to have greater increases in fatty acids and soaps. Some have advocated using Oil Red O because it permits substitution of isopropanol for ethanol.
Meat Fiber.

The technique for sampling is identical to that used for Sudan preparations for detection of fecal fat. The stool is mixed thoroughly on a slide with a 10% alcohol solution of eosin, allowed to stain for 3 minutes, and then examined for muscle fibers. The entire area under the coverslip is examined, and only rectangular fibers with clearly evident cross-striation are counted. It appears that examination for meat fibers yields results that correlate well with chemical determination of fat excretion.
Leukocytes.

A small fleck of mucus or a drop of liquid stool is placed on a glass microscopic slide with a wooden applicator stick. Two drops of Loffler methylene blue are added and mixed thoroughly and carefully. A coverslip is placed on the mixture, which is allowed to stand for 2-3 minutes for good nuclear staining. Using low-power scanning, rough quantitative counts are made by approximating the average number of leukocytes and erythrocytes. All differential counts should be made under high power, counting 200 cells when possible. Only those cells clearly identified as either mononuclear or polymorphonuclear are included in the differential count. Macrophages and epithelial cells that cannot be clearly identified are ignored. The initial cell counts should be performed at the time of presentation of the specimen.
Fecal Occult Blood Testing

Colon cancer is a leading cause of cancer-related deaths in the US, accounting for approximately 55 000 deaths annually. According to recent cancer statistics, approximately 150 000 new colon cancer cases were diagnosed in 2003. Evidence has demonstrated the clinical usefulness of fecal occult blood testing (FOBT) to detect these cancers at an earlier stage, potentially resulting in decreased mortality. Three randomized, controlled trials have demonstrated that FOBT decreases mortality from colon cancer by 15-35% ( Clinical Guideline, 1997 ). Due to the generally favorable clinical biology of these tumors when detected earlier, with an 80-90% survival rate with local confined disease ( Helm, 2003 ) and the relatively inexpensive, noninvasive nature of FOBT, this may serve as a useful screening technique. Several professional organizations recommend annual of biennial FOBT but universal accepted screening protocols have not been established. The American Cancer Society (ACS) guidelines for colorectal cancer screening, the most widely utilized, recommend annual FOBT and flexible sigmoidoscopy every 3-5 years beginning at age 50 in asymptomatic, average-risk individuals ( Marshall, 1996 ). The College of American Pathologists Laboratory Testing Strategy Task Force also recommends annual stool blood screening as a standard of practice. The limitations of this testing include the high number of false-positive and false-negative results. The sensitivity of FOBT has been estimated between

30-50%. The true sensitivity of FOBT is difficult to determine because individuals who test negative, do not undergo further colonoscopic evaluation to determine if the FOBT is a true negative. Only approximately 5-10% of positive reactions prove to be caused by an occult malignancy ( Simon, 1998 ). However, following a stepwise approach to colon cancer screening should minimize the clinical effects of limited sensitivity and specificity and offer valuable information nonetheless. Occult blood can be detected by chemical (guiac), hemoporphyrin or immunological methods. Occult blood may arise anywhere along the intestinal tract and is often the first warning sign of GI malignancy. Other potential sources of occult blood may arise from bleeding esophageal varices, polyps, esophageal or gastric inflammation, hemorrhoids or fissures, IBD, PUD or angiodysplasias of the colon. Laboratory diagnosis of the presence of fecal occult blood generally involves a guiac-smear test (Hemoccult, Searcult, Coloscreen), the most commonly used method at present. Guiac is a naturally occurring phenolic compound that is oxidized to quinone by hydrogen peroxidase, resulting in a detectable color change. These tests detect the pseudoperoxidase activity of heme, either as intact hemoglobin or as free heme (Allison, 1996). These tests are not specific for human hemoglobin, and hemoglobin from red meat, peroxidase from fruits and vegetables and certain medications can lead to false-positive results. The presence of greater than 20 mL/day of blood in the stool results in a positive guiac smear. Stool specimens should be received from three consecutive stools. Two slides should be prepared for each stool sample. Studies have shown that slides should not be rehydrated and should be developed within 7 days of collection. This is a standard protocol for FOBT, recommended by randomized clinical trials. Interestingly, medical institutions are now requiring that stool samples be sent to the laboratory for guiac testing rather than having residents perform the test on the wards, to ensure compliance with this protocol. Most guiac tests performed by residents are completed after a single digital rectal examination. Rehydration increases test sensitivity for detection of colorectal cancer but decreases specificity, resulting in a 10% or greater increase in false-positive results. This prompts further unnecessary invasive and expensive colonoscopic evaluations, making screening impractical. In addition, the test must be performed under appropriate conditions that serve to limit the sensitivity of this test. Factors that can cause inaccuracies in the results include the presence of bleeding gums for example, or ingestion of large amounts of red meat prior to testing. In addition, patients may be using certain medications that will influence the result of FOBT. For example, drugs that can cause GI irritation and subsequent bleeding such as anticoagulants, aspirin, NSAIDs, colchicines or iron supplements may lead to falsepositive results. Other drugs that have been implicated include reserpine and oxidizing drugs such as iodine. On the contrary, ingestion of large amounts of vitamin C can lead to a falsenegative result. Therefore, patients must be informed of these effects and ideally should avoid such medications or food products prior to FOBT; if not clinically feasible, FOBT should then be interpreted with caution, or avoided altogether as a screening modality in these patients. A positive result on FOBT should be defined as positivity in one or more slide windows, maximizing the sensitivity of the screening procedure. A positive FOBT then prompts further evaluation for suspected colonic neoplasia. A middle-aged patient determined to have a positive FOBT, without slide rehydration, has an estimated 7-14% probability of having an early stage, Dukes A or B colon cancer ( Clinical Guideline, 1997 ). Further evaluations usually include either sigmoidoscopy with barium enema or full colonoscopy, the latter being the preferred modality. A negative FOBT, however, cannot definitely rule out the presence of

colonic neoplasia. If a patient is presenting with signs and symptoms suggestive of colon cancer, further evaluation is warranted, even in the presence of negative FOBT. Fecal immunochemical testing for human hemoglobin, such as HemeSelect or InSure, has been developed to improve the sensitivity and specificity of guiac testing to detect colonic neoplasia. HemeSelect testing is based on an antigenantibody reaction involving fixed chicken red blood cells coated with anti-human-hemoglobin antibody (Allison, 1996). Samples demonstrating agglutination are interpreted as positive for occult human blood. InSure is a test which uses monoclonal mouse antihuman hemoglobin antibody with subsequent colorimetric detection and is sensitive enough to detect 50 gHb/g feces (Quest Diagnostics). These tests do not react with nonhuman hemoglobin or peroxidase, so food restrictions are not necessary. Immunochemical InSure tests target the globin portion of hemoglobin, which does not survive passage through the upper GI tract. Therefore, these tests are more specific for lower gastrointestinal, colonic bleeding (Quest Diagnostics). Fecal DNA testing is a promising technique for detection of occult blood; however, widespread use is not current practice. This test involves collection of a single stool specimen which is then screened for DNA markers originating from cells of cancers present in the gastrointestinal tract that are shed into the stool ( Helm, 2003 ). PCR is used to amplify the fecal DNA to yield an extremely sensitive assay. This is a useful test because neoplastic DNA remains stable in stool, whereas the cells shed from normal epithelium are degraded by enzymes as part of normal cell death ( Helm, 2003 ). APC and p53 are examples of genes serving as DNA markers in stool testing because they control colorectal cell growth and are often affected by colonic neoplasia. Clinical studies have shown that fecal DNA testing has increased specificity, ranging from 93-100% and sensitivity ranging from 71-91% for detection of cancer, considerably more than chemical guiac testing, promising improved laboratory diagnosis of colorectal malignancy. Refer to Figure 22-1 for an algorithm for fecal occult blood testing for early detection of colon cancer.

Figure 22-1 Screening for occult colorectal malignancy with fecal occult blood testing (FOBT).

Fecal Elastase

Elastase 1 is a proteolytic enzyme, produced by the pancreas, with a molecular weight of 28kD, and represents approximately 6% of pancreatic enzyme secretions. It was initially isolated in 1975 and called protease E ( Lankisch, 2004 ). Pancreatic elastase survives intestinal transit intact and is concentrated in the feces, five- to sixfold the concentration present in pancreatic juice ( Lankisch, 2004 ). Fecal elastase 1 determination is a novel fecal enzyme test, used for indirect assessment of pancreatic function to aid diagnosis of exocrine pancreatic insufficiency. This test may replace fecal chymotrypsin levels. Interestingly, elastase 1 is increased in acute and relapsing chronic pancreatitis, more so than serum amylase ( Henderson, 1994 ). In addition, elevation of elastase 1 persists longer than serum amylase levels and, therefore, may be more specific

for detection of pancreatitis and more accurately follow the clinical course of the disease ( Henderson, 1994 ). Continued investigation will likely define appropriate utility for fecal elastase analysis in particular clinical settings; until then, caution is warranted in interpretation of results. This ELISA test uses monoclonal antibodies that react with human pancreatic elastase (PE) ( Lankisch, 2004 ). It is therefore, unaffected by pancreatic enzyme replacement therapy, making it a useful diagnostic test. The test is highly sensitive for detection of severe pancreatic insufficiency; however, it lacks high sensitivity for detection of milder forms, a problem common to indirect tests of pancreatic function. Fecal elastase has diagnostic superiority over fecal chymotrypsin levels as well as older test of exocrine pancreatic function, namely, para-aminobenzoic acid (PABA) bentiromide test and pancreolauryl test ( Lankisch, 2004 ). A single analysis of a 100-mg stool sample is adequate for determination of fecal elastase levels. If borderline values are detected, then a repeat sample may be useful. This test should only be performed on formed stool, important in specimen collection and processing. Based on previous testing, values greater than 200 gPE-1/g are considered normal, values ranging from 100-200 gPE-1/g indicate mild to moderate pancreatic insufficiency and values below 100 indicate severe insufficiency ( Lankisch, 2004 ). Using these cutoff estimations, the fecal elastase enzyme test is estimated to be 57-90% specific. Sensitivity of this test is based on severity of pancreatic insufficiency. This test is estimated to be 100% sensitive for detection of severe disease. However, this decreases to between 33-89% for moderate disease and between 0-65% for mild pancreatic insufficiency as per current preliminary studies. Using a cutoff of 200 g/g stool, the positive predictive value of fecal elastase determination is estimated to be approximately 50% ( Luth, 2001 ). Fecal elastase determination may be more useful than fecal chymotrypsin levels for diagnosis of pancreatic insufficiency. However, it lacks sensitivity for detecting mild to moderate disease and cannot diagnose chronic pancreatitis with certainty. In addition it is unable to differentiate between pancreatic and nonpancreatic steatorrhea, limiting its clinical utility. Refer to Table 22-8 for summary of the clinical application of fecal elastase-1 estimation for diagnosing chronic pancreatitis and exocrine pancreatic insufficiency.

Table 22-8 -- Value of Fecal Elastase-1 Estimation in Diagnosing Chronic Pancreatitis and Exocrine Pancreatic Insufficiency Fecal Morphologic elastase-1 procedures (US, estimation EUS, CT, ERCP) Interpretation of fecal elastase-1 estimation[*] Normal Abnormal Normal Normal Severe exocrine pancreatic insufficiency excluded; mild to moderate impairment possible Exocrine pancreatic insufficiency may or may not be present; test not helpful, especially in differentiating steatorrhea/diarrhea Test confirms chronic pancreatitis but does not indicate whether steatorrhea, and thus pancreatic enzyme substitution-requiring insufficiency, is present

Abnormal

Abnormal

Fecal elastase-1 estimation Normal

Morphologic procedures (US, EUS, CT, ERCP) Abnormal

Interpretation of fecal elastase-1 estimation[*] Two interpretations possible: The patient has chronic pancreatitis on the basis of morphological procedures, but mild to moderate exocrine pancreatic insufficiency was not detected; fecal elastase-1 estimation falsely normal The abnormal morphologic examination is due to scars following acute pancreatitis; fecal elastase1 estimation is correctly normal

CT = computed tomography; EUS = endoscopic ultrasound; ERCP = endoscopic retrograde cholangiopancreatography; US = ultrasound. After Lankisch PG: Now that fecal elastase is available in the United States, should clinicians start using it? Curr Gastroenterol Rep 2004; 6:126131, with permission.
*

Interpretation based on suggestion that diagnosis of chronic pancreatitis should be made on a combination of abnormal results of morphologic examinations and abnormal exocrine pancreatic functions tests.

Lactose Tolerance Test

Following an overnight fast, administer orally 50 g of lactose dissolved in 400 mL of water. Draw fasting blood and blood samples at 30, 60, and 120 minutes after ingestion as for a glucose tolerance test. An optional 5-hour stool specimen can be collected, examining and recording the appearance, consistency, and pH. Patients with lactase deficiency exhibit a peak rise of less than 20 mg/dL in reducing substances expressed as glucose. In all persons with flat tolerance curves, the test should be repeated within 2 days and the less abnormal of the two curves used for interpretation. A control test may be performed, using 25 g glucose and 25 g galactose if the lactose test indicates malabsorption. Some investigators use a 100-g dose, which has been reported by some to yield more definitive results. It may cause symptoms in patients with mild lactase deficiency. In children, the dose of lactose or other sugars is 2 g/kg body weight.
Genetic Markers for Gastrointestinal Disease

The molecular defect underlying cystic fibrosis results in alterations in epithelial cell electrolyte transport. The defect is an autosomal recessive mutation in the CFTR gene located on chromosome 7. The degree of the defect depends on the nature of the mutation. There are several characterized mutations that lead to a milder form of the disease. The classic delta F508 mutation leads to cystic fibrosis when two copies of the gene are inherited. Persons heterozygous for the R117H mutation may develop pancreatic insufficiency. These persons may present as idiopathic chronic pancreatitis ( Durie, 2000 ). The central enzyme involved in activation of all the digestive proenzymes is trypsin. Trypsin is synthesized and maintained as inactive trypsinogen in secretory granules in the pancreatic

acinar cell. Trypsin is stabilized in the pancreatic acini by a serine protease inhibitor, SPINK1. After release into the pancreatic duct, trypsinogen is cleaved by enterokinase on the brush border of the duodenum to active trypsin. Several mutations in trypsin and SPINK1 exist that have recently gained importance in the evaluation of pancreatitis and pancreatic cancer. Cationic trypsinogen (PRSS1) mutations involving codon 29 and 122 cause autosomal dominant forms of hereditary pancreatitis ( Whitcomb, 2000 ). The number of mutations is limited, and the type of mutation appears to alter the protein, leading to decreased ability to prevent inactivation. Thus a series of events leading to active intra-acinar trypsin occurs. Mutations in the SPINK1 molecule lead to a similar problem, as the native form leads to stabilization of trypsin. Mutations have been described (N34S) that decrease the ability to promote trypsin degradation. The presence of increased acinar trypsin leads to activation of proenzymes and thus pancreatitis. Patients with these disorders typically have recurrent acute pancreatitis sometime between infancy and the 4th decade. Chronic pancreatitis and pancreatic cancer develop at a relatively young age. No specific treatment exists for the prevention or treatment of hereditary pancreatitis. Clinical testing is available for the disorders described ( Etemad, 2001 ). There are regulations developed by the Food and Drug Administration for such testing. Ordering genetic testing requires that the physician clearly understand how to interpret the results and anticipate how the results will affect patient management. Although many other markers are currently being evaluated, none has achieved widespread use at this time. A more extensive discussion of tumor markers is found in Chapter 74 . Email to Colleague Print Version Copyright 2007 Elsevier Inc. All rights reserved. - www.mdconsult.com

McPherson & Pincus: Henry's Clinical Diagnosis and Management by Laboratory Methods, 21st ed.
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