CHAPTER
22 LABORATORY DIAGNOSIS
OF GASTROINTESTINAL AND
PANCREATIC DISORDERS
Haseeb A. Siddiqi,* Martin J. Salwen,* Mohammad F. Shaikh, Wilbur B. Bowne
PANCREATIC DISORDERS, 306
Pancreas in Systemic Disease, 306
Inflammatory Diseases of the
Pancreas, 307
GASTROENTEROLOGIC
DISORDERS, 310
Peptic Ulceration, 310
Zollinger-Ellison Syndrome, 311
KEY POINTS
• Almost all patients with duodenal ulcers and most with
chronic gastritis have demonstrable Helicobacter pylori
infection. H. pylori stool antigen assays and urea breath tests
are useful in diagnosis and in monitoring for eradication after
treatment.
• Acute pancreatitis presents with abdominal pain and elevated levels
of serum amylase or lipase. Reversible causes must be excluded in
patients with recurrent episodes of acute pancreatitis. Routine
laboratory testing is of limited value in diagnosing chronic
pancreatitis.
• Sweat chloride determination is the necessary initial test in the
workup for cystic fibrosis. Genetic testing can be used to identify the
mutations associated with this disease.
• Patients with chronic diarrhea should be evaluated for fecal blood,
fat, leukocytes, and stool pathogens (bacterial culture on routine
media, ova, and parasite examination).
• Clostridium difficile should be considered a cause of diarrhea in
patients on antibiotic therapy or hospitalized for more than
3 days.
• Diagnostic evaluation of a patient suspected of celiac disease should
be initiated with anti–tissue transglutaminase immunoglobulin A and
total serum immunoglobulin A before placing the patient on a
gluten-free diet.
• Primary lactose intolerance is common in adults, and secondary
lactose intolerance may occur in infection and in inflammatory bowel
disease.
• Positivity of perinuclear antineutrophil cytoplasmic antibody is most
often associated with ulcerative colitis and that of anti–Saccharomyces
cerevisiae antibody with Crohn’s disease.
• Endoscopy has replaced gastric acid aspiration for diagnosis. Gastric
acid output testing is useful when acid levels are very high or very
low.
• Gastrin, the most powerful gastric acid stimulator, varies inversely
with gastric acid secretion. Serum gastrin levels are elevated in gastric
atrophy, and gastric acid levels are reduced.
• Secretin stimulates gastrin production in patients with
a gastrinoma but not in patients with other causes of
hypergastrinemia.
• Intraoperative gastrin measurements are useful in identifying whether
the abnormal tissue is completely removed in patients undergoing
surgery for gastrinomas.
• A fecal occult blood test is used to screen for colon cancer.
306
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Diarrhea and Malabsorption, 312 Macroscopic Examination, 322
Gastrointestinal Tumors, 318 Microscopic Examination, 322
Gastrointestinal Bleeding, 320 SELECTED REFERENCES, 322
Markers for Gastrointestinal
and Pancreatic Tumors, 321
STOOL COLLECTION AND
EXAMINATION, 321
Collection, 321
Diagnosis of pancreatic and gastrointestinal disease is guided by the
patient’s history and the significant signs and symptoms. Findings with
strong negative predictive values exclude some possible causes and focus
the differential diagnosis. Initially, noninvasive procedures are preferen-
tially performed. Patient preparation is as important as correct selection
of the diagnostic tests or procedures indicated. Endoscopy, when war-
ranted, can provide direct visualization of the entire gastrointestinal lumen
and permits biopsy. Imaging-assisted invasive techniques may be required
in the critically ill with gastrointestinal bleeding or obstruction. To ensure
interpretable endoscopic results and to avoid false-positive and false-
negative results, stringent patient preparation is required. Similarly, testing
requires appropriately collected specimens. Emphasis is given in this
chapter to frequently used diagnostic tests.
PANCREATIC DISORDERS
PANCREAS IN SYSTEMIC DISEASE
Cystic Fibrosis
Cystic fibrosis (CF) is characterized by abnormally viscous mucous secre-
tions from the various exocrine glands of the body, including the pancreas,
salivary glands, and peritracheal, peribronchial, and sweat glands. Involve-
ment of the intestinal glands may result in the presence of meconium ileus
at birth. Two thirds of cases are diagnosed before 1 year of age. Chronic
lung disease and malabsorption resulting from pancreatic insufficiency are
the major clinical problems of those who survive beyond infancy, but intel-
ligence and cognitive functions are unaffected (Cheng et al, 1990).
CF is the most common genetic disorder in Caucasian North Ameri-
cans. It is an autosomal recessive disease of ion transport affecting the CF
transmembrane conductance regulator (CFTR) gene on chromosome 7
that encodes an epithelial chloride channel protein. Heterozygotes have
no recognizable clinical symptoms. Homozygotes fully express the syn-
drome of recurrent pulmonary infection, pancreatic insufficiency, steator-
rhea, and malnutrition. CF is due to defective epithelial chloride transport
across membranes, which causes abnormally dehydrated tenacious secre-
tions of all exocrine glands. The viscid inspissated mucous plug ducts cause
chronic inflammation with atrophy of acini, fibrosis, dilation, and cystic
duct changes.
CF is often fatal in childhood, occurring in 1 in 3500 newborns.
Advances in treatment have increased the life span of CF patients; if
current mortality rates continue, the average female patient will be
expected to live until 37 years of age, and the average male patient will live
until age 40 (MacKenzie et al, 2014). Ninety percent die from pulmonary
complications.
*Corresponding authors. Each author contributed equally to this chapter.
 Overall, the prevalence in the United States is 1 in 3900, with the
highest ethnic subgroup being Caucasians (1 in 2500), Ashkenazi Jews (1
in 2300) and Native American tribes (1 in 10,900) (Palomaki et al, 2004).
The Pueblo tribe in particular has been described as having a similar
incidence as Caucasians. It is also frequent in Hispanics but is uncommon
in Asians and blacks. More than 25,000 Americans have CF, and almost
1000 new cases are diagnosed each year. The incidence is 1 in 1600 Cau-
casian births and 1 in 17,000 African American births in the United States.
Approximately 1 in every 20 Caucasians is a carrier of one of the alleles.
Nearly 2000 mutations of the CFTR gene have been identified. Available
probes can be used to test for 70 mutations that account for over 90% of
cases of CF. Genetic testing can identify the mutations associated with CF
(Weiss et al, 2005).
The degree of the defect depends on the nature of the mutation.
Several characterized mutations lead to a milder form of the disease. The
classic δ F508 mutation leads to CF when two copies of the gene are
inherited.
Because of multiple alleles at the cystic fibrosis gene, the demonstration
of increased chloride in the sweat is a necessary initial test in the diagnosis
of CF. Until 1996, sweat chloride testing was employed most often in
response to clinical developments or family history.
More recently, screens for CF have been developed that involve first
an immunoassay for immunoreactive trypsinogen (IRT). Trypsinogen is
the inactive, proenzyme form of trypsin secreted by pancreatic acinar cells;
the active enzyme form is produced by proteolytic cleavage by proteases,
including activated trypsin itself, of an N-terminal peptide segment result-
ing in the active form of this enzyme. There are several forms of trypsino-
gen, one of which is IRT; there are two forms of proenzyme: a cationic
form (IRT1) and an anionic form (IRT2). Both forms have been found to
become elevated in a number of different gastrointestinal diseases such as
CF, meconium ileus, and diseases of the pancreas, including pancreatic
steatorrhea and pancreatic carcinoma. ELISA tests have been developed
for each form (Lindau-Sheppard & Pass, 2010), although most antibodies
used for the assay recognize both forms.
With the widespread adoption of newborn screening for CF (e.g., IRT,
with or without DNA analysis for known CFTR mutations), sweat chlo-
ride testing is now typically performed only when an abnormal result for
these screens is found (Farrell et al, 2008).
Using the long-established Gibson-Cooke method, 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 and is weighed, diluted with water, and analyzed for sodium and
chloride concentrations. Total body sweating in patients with cystic fibrosis
is hazardous, and a number of deaths from the procedure have been
reported.
When performed properly in duplicate, the sweat test has a sensitivity
of 90% to 99%. High rates of incorrect results have been attributed to
problems associated with sweat specimen sample collection and test analy-
sis (National Committee for Clinical Laboratory Standards [NCCLS],
2000; LeGrys et al, 2007; Farrell et al, 2008).
More than 99% of children with CF have concentrations of sweat
chloride greater than 60 mmol/L. The sweat chloride may not be as dra-
matically increased in adolescent or adult patients. The test needs to be
performed with care (LeGrys et al, 2007).
In children, chloride concentrations greater than 60 mmol/L in sweat
on at least two occasions are diagnostic. Levels of between 50 and
60 mmol/L are suggestive in the absence of adrenal insufficiency. Patients
in whom CF is suspected on the basis of indeterminate sweat electrolyte
results may undergo confirmatory testing following administration of a
mineralocorticoid such as fludrocortisone. In those patients with CF, elec-
trolyte 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 CF, but
the reverse is true in normal subjects. Sweat chloride concentrations
greater 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, muco-
polysaccharidosis, and fucosidosis. These disorders usually can be easily
differentiated from CF by their clinical symptoms. False-negative sweat
test results have been seen in patients with CF in the presence of hypo-
proteinemic 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 to 10 days before the onset of menses.
Peak values were slightly less than 65 mmol/L. Men showed random
fluctuations up to 70 mEq/L. For this reason, interpretation of sweat
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electrolyte values in adults must be approached with caution (Rosenstein
& Cutting, 1998; NCCLS, 2000).
Pancreatic abnormalities occur in more than 80% of cases. The clinical
manifestations are varied. Islets of Langerhans are usually spared. No cure
is available. Persons heterozygous for the R117H mutation may develop
pancreatic insufficiency as the result of plugging of ducts, causing idio-
PART 2
pathic chronic pancreatitis (Durie, 2000).
Hemochromatosis
Excessive body iron accumulation from any source is directly toxic to cells
and causes fibrosis. Symptoms include the triad of bronze coloration of
the skin, cirrhosis, and diabetes. Humans have no major iron excretory
pathway. In this disease, the pancreas is slightly enlarged and deep brown
as the result of accumulated hemosiderin, the iron-containing pigment.
When untreated, progressive fibrosis of the pancreas with atrophy occurs.
Iron is deposited in the acinar and duct cells and in the β cells of the islets.
Other cells of the islets appear spared. Similar pigments are noted in the
skin. β-cell loss results in bronze diabetes. Hypogonadism with pituitary
dysfunction is present in half of cases, and cardiomegaly and osteoarthritis
are present in most cases. Cirrhosis is seen in 70% of cases. Hepatocellular
carcinoma (HCC) occurs in 30% of cases
Secondary hemochromatosis is typically seen in anemia caused by mul-
tiple blood transfusions, hemolytic anemias, or increased oral iron intake,
which result in excess iron storage.
Hereditary hemochromatosis (HH) is a human leukocyte antigen
(HLA)–linked autosomal recessive defect in duodenal iron absorption
regulation. As discussed in Chapters 21 and 23, the HFE gene is on the
short arm of chromosome 6. In this common genetic disease, the homo-
zygosity frequency is 1 in 220. When HH is diagnosed, other family
members should be screened; one-quarter of siblings will test positive
(Powell et al, 1996; Bulaj et al, 2000; Beutler et al, 2002). HCC has
become a chief cause of death in HH (Barton et al, 1998).
A number of diagnostic strategies exist (Jensen, 2004). Because the liver
is the major site of the body’s iron storage, liver biopsy remains the gold
standard of diagnosis. Given the possibility of complications associated
with this invasive procedure, and the potential patient coagulopathy, other
screening methods are first performed, with biopsy only employed as a
confirmatory measure (Powell, 2002; Jensen, 2004). The screening test
consists of transferrin saturation (TS) = serum iron ÷ total iron binding
capacity × 100. Results are interpreted as abnormal if greater than 60% in
women and greater than 50% in men (Powell, 2002). Other tests, such as
serum ferritin, serum ferritin iron, hepcidin, and imaging with MRI are
also employed (Jensen, 2004; Konz et al, 2014). In HH, genetic testing for
mutations in HFE, transferrin receptors, ferroporitin, and hepcidin can
help with diagnosis.
INFLAMMATORY DISEASES OF THE PANCREAS
Pancreatitis is an inflammation of the pancreas caused by injury to acinar
cells due to activation of digestive enzymes within the pancreatic paren-
chyma; it is characterized by significant morbidity and mortality. Clinical
manifestations of pancreatitis are highly variable.
Acute Pancreatitis
Acute reversible inflammation is due to enzymatic necrosis. Acute pancre-
atitis occurs at any age—usually 30 to 70 years—but is rare in children. Its
diagnosis is based on the presence of two out of the following three condi-
tions: abdominal pain consistent with acute pancreatitis (typically acute
onset, epigastric, with other causes ruled out); serum amylase and/or lipase
activity greater than triple the upper limit of the normal reference range;
and imaging evidence of acute pancreatitis (Morinville et al, 2012). Clini-
cal suspicion is supported by findings of elevated serum amylase and/or
lipase (Table 22-1).
In 30% of patients, the diagnosis of acute pancreatitis was not suspected
and was made only at autopsy (Wilson et al, 1985). Many causes have been
identified. Gallstones continue to be the leading cause (30% to 60%), and
alcohol is the second most common cause (responsible for 15% to 30% of
cases). Other causes include duct obstruction due to tumors or parasites,
duct anomalies such as pancreas divisum, infections (mumps, coxsackievi-
rus A), blunt trauma or post endoscopic retrograde cholangiopancreatog-
raphy (ERCP), many drugs (diuretics, sulfonamides), organophosphates,
methyl alcohol, nitrosamines, hypertriglyceridemia, and hypercalcemia.
Amylase
Amylase in serum and urine is stable for 1 week at ambient temperature
and for at least 6 months under refrigeration in well-sealed containers.
307
 Plasma specimens that have been anticoagulated with citrate or oxalate be 62% and 93%, respectively (Hofmeyr et al, 2014). Serum amylase levels
22 LABORATORY DIAGNOSIS OF GASTROINTESTINAL AND PANCREATIC DISORDERS

should be avoided for amylase determination because amylase is a calcium- do not correlate with cause or severity of pancreatitis. The pancreas con-
containing enzyme. Heparinized plasma specimens do not interfere with tributes 40% of the total serum amylase; the rest comes mostly from the
the amylase assay. salivary glands (Halangk & Lerch, 2005).
Diagnosis is confirmed by detection of elevated serum amylase three- Although a variety of reliable amylase methods are available, care is
fold above normal. It peaks in 20 to 30 hours, often at 10 to 20 times the required in specimen handling. Caution must be exercised to avoid con-
upper reference limit (Papachristou & Whitcomb, 2005). Amylase returns tamination of specimens with saliva, because its amylase content is approxi-
to normal in 48 to 72 hours. Elevated values persisting longer than this mately 700 times that of serum. Red cells contain no amylase, so hemolysis
suggest continuing necrosis or possible pseudocyst formation. Amylase does not affect most methods, except those coupled-enzyme methods in
continues to be a first-line test for acute pancreatitis in current clinical which the released peroxide is determined by a coupled-peroxidase
practice despite certain problematic issues. Serum amylase has poor sen- reaction.
sitivity for pancreatitis; it is not increased in about 20% of patients with The urine amylase activity rises promptly, often within several hours
pancreatitis. Serum amylase increases nonspecifically in many acute of the rise in serum activity, and may remain elevated after the serum level
abdominal conditions. In hyperlipidemic patients with pancreatitis, normal has returned to the normal range. Values greater than 1000 Somogyi units/
serum and urine amylase levels are frequently encountered. The spuriously hour are seen almost exclusively in patients with acute pancreatitis. In a
normal levels are believed to be the result of suppression of amylase activity majority of patients with acute pancreatitis, serum amylase activity is ele-
by triglyceride or by a circulating inhibitor in serum. A recent series found vated, and a concomitant increase in urine amylase activity occurs.
the sensitivity and specificity for serum amylase for acute pancreatitis to Increased renal clearance of amylase can be used in the diagnosis of acute
and relapsing pancreatitis, but the ratio of amylase clearance to creatinine
clearance expressed as a percentage adds little to the diagnosis, because
elevated ratios may be found in unrelated conditions.
Lower than normal serum amylase activity may be found in patients
TABLE 22-1 with chronic pancreatitis and has been seen in such diverse conditions as
Laboratory Tests in Acute Pancreatitis congestive heart failure, pregnancy (during the second and third trimes-
ters), gastrointestinal (GI) cancer, bone fracture, and pleurisy.
Laboratory Test Purpose Usage and Limitations Serum amylase may be elevated in patients with pancreatic carcinoma
but often too late to be diagnostically useful. Serum amylase activity may
Amylase Diagnosis Accurate over 3× the upper normal
also be elevated in patients with cholecystitis, peptic ulcer, renal transplant,
limit; decreased specificity in renal
viral hepatitis, or ruptured ectopic pregnancy, or after a gastrectomy.
failure; elevated in
macroamylasemia;
Increased ascites fluid amylase levels have been seen in patients with
hypertriglyceridemia interferes; pancreatitis, a leaking pancreatic pseudocyst, pancreatic duct rupture, pan-
elevated from other sources such creatic cancer, abdominal tumors that secrete amylase, and perforation of
as salivary glands and/or a hollow viscus. Fractionation of amylase in serum, urine, and other body
intraabdominal inflammation (not fluids can be done by physical means, such as electrophoresis, chromatog-
above 3×); can be normal in raphy, or isoelectric focusing; each isoenzyme is then quantitated by direct
alcohol-induced pancreatitis densitometry.
Lipase Diagnosis Decreased specificity in renal failure;
Macroamylasemia
immune complexes create false
positives; elevated from salivary Macroamylasemia is not a disease, but an acquired benign condition that
glands and intraabdominal is more frequent in men and is usually discovered incidentally in the fifth
inflammation through seventh decades (Remaley & Wilding, 1989). A persistent increase
Trypsinogen 2 Diagnosis Limited use; unclear if superior to in serum amylase is seen without clinical symptoms. Urine amylase is
amylase/lipase normal or low. Macroamylases are heterogeneous complexes of normal
AST/ALT Etiology If greater than 3× upper normal amylase (usually salivary isoenzyme) with immunoglobulin (Ig)G, IgA, or
limit, gallstones present as polysaccharide (Van Deun et al, 1989). Because of their large size, mac-
etiology in 95% of cases. Low roamylases cannot be filtered through the glomerulus and are retained in
sensitivity the plasma; they are not present in urine. Plasma amylase activity is often
Lipase/amylase Etiology >5 is diagnostic for alcohol-induced increased two- to eightfold. Serum lipase is normal. Macroamylasemia is
ratio acute pancreatitis. Low sensitivity found in about 1% of randomly selected patients. Renal function is normal,
and the amylase/creatinine clearance ratio is low (Table 22-2).
CDT Etiology Useful in patients who deny alcohol;
remains elevated for weeks after Lipase
binge drinking; not widely
available The pancreas is the major and primary source of serum lipase. Human
pancreatic lipase is a glycoprotein with a molecular weight of 45 kDa.
Hematocrit Severity >44% on admission, or rising over
Lipase is not present in the salivary glands. Lipases are defined as enzymes
initial 24 hrs; associated with
pancreatic necrosis
that hydrolyze preferentially glycerol esters of long-chain fatty acids at the
carbon 1 and 3 ester bonds, producing 2 moles of fatty acid and 1 mole of
C-reactive protein Severity >150 mg/L associated with
β-monoglyceride per mole of triglyceride. After isomerization, the third
pancreatic inflammation. Useful
fatty acid can be split off at a slower rate. Lipolysis increases in proportion
after first 36-48 hrs
to the surface area of the lipid droplets, and the absence of bile salts in
ALT, Alanine aminotransferase; AST, aspartate aminotransferase; CDT, carbohydrate- duodenal fluid with resultant lack of emulsification renders lipase
deficient transferrin. ineffective.

TABLE 22-2
Differential Diagnosis of Hyperamylasemia and Macroamylasemia
Condition Serum Amylase Serum Lipase Urinary Amylase Cam :Ccr Serum Macroamylase

Pancreatic hyperamylasemia High High High High Absent

Salivary hyperamylasemia High Normal Low or normal Low or normal Absent
Macroamylasemia High Normal Low Low High

Adapted from Kleinman DS & O’Brien JF: Macroamylase, Mayo Clin Proc 61:669–670, 1986.
Cam:Ccr = amylase clearance:creatinine clearance ratio = (urinary amylase/serum amylase) × (serum creatinine/urinary creatinine).

308
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 Serum lipase has been described as a better first-line test for diagnosis
of acute pancreatitis than serum amylase. It has a sensitivity and specificity
of 92% and 91%, respectively (Hofmeyr et al, 2014). Serum lipase increases
in 4 to 8 hours and remains elevated for 8 to 14 days. Increased lipase
activity rarely lasts longer than 14 days; prolonged increases suggest a poor
prognosis or the presence of a pancreatic cyst. Hyperglycemia and elevated
bilirubin concentrations may be present, and leukocytosis is frequently
reported.
Pancreatic lipase must be differentiated from lipoprotein lipase, alies-
terase, and arylester hydrolase, which are related but different enzymes.
The activities of these enzymes may be included in the measurement of
lipase activity unless 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 higher concen-
trations 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 water/substrate
interface. Similar to 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 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 40° C. The optimal pH is 8.8, but other values
ranging from 7.0 to 9.0 have been reported. This difference probably is
due to the effects of differences 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.
Both serum lipase and amylase are useful in ruling out acute pancre-
atitis. Although determination of serum lipase has diagnostic advantages
over serum amylase for acute pancreatitis, this value is not specific for acute
pancreatitis. Serum lipase may also be elevated in patients with chronic
pancreatitis, obstruction of the pancreatic duct, and nonpancreatic condi-
tions, including renal disease, acute cholecystitis, intestinal obstruction or
infarction, duodenal ulcer, and liver disease, as well as alcoholism and
diabetic ketoacidosis, and in patients who have undergone ERCP. Patients
with trauma to the abdomen uniformly have increases in both serum
amylase and lipase. Elevation of serum lipase activity in patients with
mumps strongly suggests significant pancreatic involvement by the disease.
Trypsinogen
Trypsin is produced in the exocrine pancreas as two proenzymes, known
as trypsinogen 1 and trypsinogen 2. These proenzymes are activated in the
duodenum by an enterokinase that yields trypsin 1 and trypsin 2, respec-
tively. Trypsin present within the peripheral circulation is inactivated by
complexing with α-2-macroglobulin or α-1-antitrypsin (AAT). Trypsin,
unlike amylase, is produced solely by the pancreatic acinar cells and there-
fore is 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 et al, 2001). Currently, levels of all forms of trypsin are deter-
mined by specific immunoassays.
Trypsin assays are currently used to differentiate the cause of an acute
episode of pancreatitis. One study demonstrated that trypsinogen 2 and
trypsin-2-AAT are increased in all forms of acute pancreatitis but are more
elevated in alcohol-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-
2-AAT to trypsinogen 1 was determined to be the best discriminator
between biliary and alcoholic pancreatitis (Andersen et al, 2001). Another
study supported the use of trypsin assays for the diagnosis of acute pan-
creatitis, because the determined time course profile of trypsinogen 2 and
trypsin-2-AAT is appropriate for diagnostic purposes. These enzymes are
elevated within hours of onset of the acute episode and therefore are
already elevated upon admission; this is 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, which is
extremely useful for diagnosing acute pancreatitis upon admission, for
predicting severity of illness, and for monitoring disease progression
(Kemppainen et al, 2000). Elevated trypsin-1-ATT has also been demon-
strated in patients with biliary tract cancer (Andersen et al, 2001).
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Serum trypsinogen 2 levels rise rapidly, showing a tenfold to twentyfold
increase. Urinary concentrations are even more steeply elevated. A recent
meta-analysis of 18 trials investigating the diagnostic utility of urinary
trypsinogen-2 in acute pancreatitis demonstrated a pooled sensitivity and
specificity of 80% and 92%, respectively. The pooled sensitivity and speci-
ficity for post-ERCP pancreatitis in this same analysis were 86% and 94%,
PART 2
respectively (Jin et al, 2013). Irrespective of the cause, all origins allow
activation of the inactive proenzyme trypsinogen to trypsin, which then
activates most of the other digestive enzymes and produces tissue damage
and necrosis of the pancreas, surrounding fat, and adjacent structures.
Other enzymes that have been proposed as diagnostic tools include
pancreatic isoamylase, phospholipase A, elastase 1, and trypsinogen 2
(Forsmark & Baillie, 2007). Other tests (aspartate aminotransferase,
alanine aminotransferase, C-reactive protein [CRP], hematocrit,
carbohydrate-deficient transferrin [CDT], and trypsinogen activation
peptide [TAP]) have shown low sensitivity for diagnosing acute pancreati-
tis. CDT is a marker for chronic alcoholism. Urinary TAP is a valuable
marker for severity of pancreatitis. Markers of inflammatory response (e.g.,
CRP) peak, following interleukin (IL)-1 and IL-6 increases, on day 3 after
onset of abdominal pain; this is useful in predicting the severity of pancre-
atitis (Smotkin & Tenner, 2002).
A computed tomography (CT) scan is the most useful test to establish
the diagnosis, with characteristic radiologic findings of enlarged edematous
and inflamed pancreas with or without surrounding fluid collection, with
or without necrosis. An ultrasonogram may be useful in showing a diffusely
enlarged, hypoechoic pancreas, and may show the presence of gallstones
in the gallbladder, indicating a possible cause. A CT severity score (the
Balthazar score) is based on the degree of necrosis, inflammation, and fluid
collection. A 23% mortality rate is associated with any degree of pancreatic
necrosis, and a strong association has been noted between necrosis and
morbidity and mortality. After initial assessment, a CT scan need not be
repeated unless one suspects development of a complication such as pan-
creatic necrosis. Magnetic resonance imaging (MRI) is being used increas-
ingly to detect pancreatitis and to characterize the pancreatic necrosis seen
on CT into peripancreatic necrotic fluid collection, necrotic pancreatic
parenchyma, and hemorrhagic foci. MRI can also detect pancreatic duct
disruption, seen early in the course of acute pancreatitis.
Serum and urine amylase elevations occur in many conditions other
than pancreatitis, such as renal failure, parotitis, and diabetic ketoacidosis.
Patients with acidemia may have spurious elevations of serum amylase.
This explains why patients with diabetic ketoacidosis may have marked
elevations of serum amylase without evidence of acute pancreatitis. No
data indicate that measuring both amylase and lipase adds significant diag-
nostic accuracy. Once the diagnosis is established, daily measurement of
amylase or lipase provides little value in gauging the clinical course or the
prognosis.
Predictors of severe acute pancreatitis include hematocrit greater than
44% with failure to decrease at 24 hours (this is indicative of pancreatic
necrosis and is predictive of organ failure) and C-reactive protein greater
than 150 mg/L. Serum creatinine greater than 2.0 mg/dL or marked
hyperglycemia (>150 mg/dL) is predictive of mortality (Lankisch et al,
2001). A strong association has been found between the extent of blood
urea nitrogen (BUN) increase and mortality at 24 hours. Each increase in
BUN of 5 mg/dL was associated with a corresponding increase in mortal-
ity. A reduction in blood urea was associated with significantly improved
survival (Wu et al, 2009) (Table 22-3). One serum marker of interest is
cytokeratin 8, a cytoskeletal protein and marker of apoptosis. Higher
cytokeratin 8 levels have been shown to be associated with a milder clinical
course of acute pancreatitis (Koruk et al, 2012).
Hemorrhagic pancreatitis, a severe form of acute pancreatitis, results
from necrosis within and around the pancreas with hemorrhage that may
cause shock and death. Initially, necrosis is coagulative, but necrotic cells
rapidly undergo liquefaction. Biliary tract disease with gallstones or
inflammation of the gallbladder or bile ducts, or alcoholism, is present in
about 80% of patients. The male/female ratio is 1 : 3 in acute pancreatitis
associated with biliary tract disease and 6 : 1 in alcoholism. Pancreatic
microlithiasis may be responsible for many cases.
The sequence of changes following release of activated intrapancreatic
enzymes in acute pancreatitis consists of microvascular leakage causing
edema, necrosis of fats, and acute inflammatory reaction. Proteolytic
destruction of pancreatic tissue and blood vessels causes edema and focal
dilation of acini with variable amounts of hemorrhage. In fat necrosis,
neutral fats are broken down, glycerol is reabsorbed, and fatty acids
combine with calcium salts to form soaps (saponification) with a zone of
acute inflammation around the foci of necrosis. After a few days, secondary
infection with suppuration and abscesses may occur.
309
 TABLE 22-3
22 LABORATORY DIAGNOSIS OF GASTROINTESTINAL AND PANCREATIC DISORDERS
Laboratory Findings in Acute Pancreatitis
At Presentation At 48 Hours
Age >55 Hematocrit Fall by >10%
Leukocyte >16,000/mm3 Urea nitrogen Increase by >5 mg/
count dL despite fluids
Blood glucose >200 mg/dL Serum calcium <8 mg/dL
LD 350 U/L pO2 <60 mmHg
AST >250 U/L Base deficit >4 mEq/L
Fluid sequestration >6000 mL
ALT, Alanine aminotransferase; AST, aspartate aminotransferase; LD, lactate dehy-
drogenase; pO2, partial pressure of oxygen.
In 15% to 30% of those with pancreatic necrosis, poorly defined areas
of acute fluid collection occur, along with fibrosis. The liquefied areas are
walled off, and pseudocysts form. Pseudocysts contain pancreatic fluid
enclosed in fibrous tissue with no epithelial lining; they often communicate
with a pancreatic duct and continue to increase in mass.
Complications of Acute Pancreatitis
Hypocalcemia and mild jaundice may appear after 24 hours as the result
of biliary obstruction. A sepsis-like syndrome due to digestive enzymes in
the systemic circulation may cause the release of inflammatory cytokines,
a systemic immune response syndrome with severe systemic complications.
About 75% of patients with acute pancreatitis have a benign course and
recover rapidly. No treatment has proven to interrupt the inflammatory
process effectively.
Idiopathic acute pancreatitis occurs in about 10% to 20% of patients
with pancreatitis. It is believed that many cases are germline mutations of
cationic trypsinogen (PRSS1) (see earlier) or serine protease inhibitor,
kazal type 1 (SPINK1). There is high risk for development of endocrine
or exocrine insufficiency and pancreatic adenocarcinoma. These mutations
can cause an autosomal recessive hereditary acute or chronic pancreatitis
with onset in childhood or early adulthood. PRSS1 abrogates the inactiva-
tion of trypsinogen for cleavage of trypsin. SPINK1 mutation inactivates
pancreatic secretory trypsin inhibitor (Howes et al, 2005; Schneider,
2005).
Patients with these disorders typically have recurrent acute pancreatitis
sometime between infancy and the fourth decade. Chronic pancreatitis and
pancreatic cancer develop at a relatively young age. No specific treatment
is known for the prevention or treatment of hereditary pancreatitis. In
concert with standard pancreatitis laboratory testing and imaging, genetic
testing for the aforementioned mutations can lead to this diagnosis. Ancil-
lary diagnostic modalities include ERCP with secretin stimulation or
sphincter of Oddi manometry. These tests can help identify sphincter of
Oddi dysfunction that could contribute to recurrent acute pancreatitis
(Testoni, 2014).
Chronic Pancreatitis
It is the irreversible damage and often-progressive inflammation with
irregular fibrosis, duct dilation, and loss of pancreatic parenchyma that
characterize chronic pancreatitis. This occurs after repeated bouts of acute
pancreatitis, obstruction of pancreatic duct by mechanical blockage or
congenital defect or by neoplasm, gallstone duct obstruction, or alcohol-
ism. Early in the course, the pancreas becomes enlarged. Some cases
develop pseudotumor mass lesions. Subsequently, as the result of scarring,
the gland usually shrinks with loss of acini and still later loss of ductules.
Preserved or even increased islets are seen in the fibrous scar. Patients seek
medical attention for abdominal pain or maldigestion.
Maldigestion/malabsorption and steatorrhea are due to pancreatic
insufficiency with loss of enzymes, glucose intolerance or diabetes, and
islet damage. A low fecal elastase 1 (i.e., concentration of <15 μg/g) has
been associated with pancreatic insufficiency with a sensitivity and specific-
ity of 93.3% and 81.5%, respectively, in nonoperated patients (Benini et al,
2013). Clinically, recurrent or chronic pain is reported at a lower incidence
than in acute pancreatitis, but this is now increasing in frequency. The
incidence is greater in males than in females, and the average age of onset
is 40 years. It is more prevalent in tropical countries, and the main form
is chronic calcifying pancreatitis with duct calcifications. In temperate
areas, chronic alcoholism is reported in more than half of cases. No caus-
ative factor is apparent in 40% of cases.
310
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The central enzyme involved in activation of all digestive proenzymes
is trypsin, which is synthesized and maintained as inactive trypsinogen in
secretory granules in the pancreatic acinar cell. After release into the
pancreatic duct, trypsinogen is cleaved by enterokinase on the brush
border of the duodenum to active trypsin. Trypsin is stabilized in the
pancreatic acini by a serine protease inhibitor, SPINK1. Mutations in
SPINK1 increase the risk of chronic pancreatitis almost twelvefold by
impairing the ability of acinar cells to counteract and inhibit the damaging
effects of intracellular trypsin (Schneider et al, 2004; DiMagno &
DiMagno, 2005). PRSS1 mutations involving codons 29 and 122 cause
autosomal dominant forms of hereditary pancreatitis (Whitcomb, 2000;
Cohn et al, 2005).
GASTROENTEROLOGIC DISORDERS
PEPTIC ULCERATION
Helicobacter pylori has been recognized as the principal cause of duodenitis
and duodenal ulcers, and it has been strongly associated with chronic antral
gastritis, gastric ulcer, nonulcer dyspepsia, gastric carcinoma, and mucosa-
associated lymphatic tissue lymphoma (Peterson, 1991; Veldhuyzen van
Zanten & Sherman, 1994; Thiede et al, 1997; Wotherspoon, 1998). The
use of nonsteroidal anti-inflammatory drugs (NSAIDs) causes or aggra-
vates peptic and gastric inflammation and ulceration. Hypersecretory
states are a much rarer cause of peptic ulcer disease. Data gathered by
history and physical examination may initially suggest peptic ulcer disease.
Radiologic and/or endoscopic techniques are employed to confirm the
diagnoses. Testing for H. pylori and hypersecretory states involves labora-
tory analysis.
Because H. pylori has been shown to be the most important cause of
peptic ulcer disease and is significantly associated with multiple other types
of upper GI pathology, a great deal of research has focused on its detection
and treatment and on confirmation of pathogen eradication. Within the
last decade, numerous products used for the detection of this bacterium
have become commercially available. The American College of Gastroen-
terology recommends H. pylori testing in individuals with peptic ulceration
but leaves the clinician considerable discretion in choosing the diagnostic
modality, because no one test is recognized as the gold standard (Chey
et al., 2007). Although the numbers and types of tests will likely continue
to grow, tissue sampling, breath tests, and fecal antigen detection are cur-
rently the mainstay in the diagnostic armamentarium. The incidental use
of proton pump inhibitors (PPIs), antibiotics, or bismuth-containing ant-
acids may lead to false-negative tests.
Some diagnostic testing relies on the fact that the organism has urease
activity, which metabolizes urea to bicarbonate and ammonia. The tests
that employ this key characteristic are rapid urease testing and urea breath
testing.
Rapid urease testing involves taking a specimen from an antral biopsy
and placing it on an agar gel or reaction strip with urea and a pH-sensitive
color-changing indicator. A change in color would indicate a change in
pH, which in turn represents urease activity and thus suggests presence of
H. pylori.
The urea breath test also exploits the presence of urease in the H. pylori
organism. It has the advantage of not requiring an endoscopically obtained-
tissue sample. Labeled urea is ingested and labeled CO2 is detected and
quantified in expired breath. The label can be either nonradioactive 13C
or radioactive 14C. Another nonendoscopic urease test works by detecting
labeled bicarbonate in a blood sample (Ahmed et al, 2005).
Although not included in the latest guidelines on H. pylori diagnosis,
we discuss hydrogen breath tests here for historical interest. Radioactive
and nonradioactive hydrogen breath tests are examples of noninvasive
means for detecting active H. pylori infection. Each is sensitive and specific
before therapy. 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 & Spiller, 1994).
Serum antibodies directed against H. pylori can be used to detect expo-
sure to H. pylori. Enzyme immunoassay (EIA) tests are available and reli-
able (Feldman et al, 1995; Feldman & Evans, 1995; van de Wouw et al,
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 et al, 1998). At present, serology is generally used to screen for
H. pylori, and breath tests are used to confirm eradication after treatment.
Alternatively, endoscopy allows collection of tissue for rapid urease testing
or histologic examination (Megraud, 1997).
 Office-based serologic quick-test kits are available. The accuracy of
these kits has been shown to be dependent on the antibody preparations
used. 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
et al, 1998). Histologic review of biopsy specimens stained with Warthin-
Starry or Giemsa stain remains one of the most frequently employed
techniques to detect active infection. Culture of the organism may be
inconsistent and usually is not done in routine clinical settings. If endos-
copy must be performed for other reasons, a rapid urease test is the least
expensive means of documenting the presence of H. pylori.
Hypersecretory states are suggested by extensive peptic ulcer disease,
especially in the absence of H. pylori, and by the use of NSAIDs. Failure
to respond to the usual doses of histamine-2 (H2)-receptor blocking agents
and PPIs also suggests oversecretion of hydrochloric acid. 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 PPIs should be avoided for 7 days. H2-receptor blockers are
available without a prescription, so patient education is important, and
clinicians must remember to review all of the medications utilized by their
patients.
ZOLLINGER-ELLISON SYNDROME
This syndrome is defined by the triad of peptic ulceration, hyperchlorhy-
dria, and non–β islet cell tumors (gastrinomas). Duodenal ulcers do not
occur in achlorhydric individuals but are present in those with extreme
hyperchlorhydria. Gastrinomas may occur in the body or tail of the pan-
creas or in the upper duodenum; they may be multiple and malignant.
About 25% of Zollinger-Ellison (ZE) patients have multiple endocrine
neoplasia (MEN) 1 with hyperparathyroidism (Hung et al, 2003).
Gastrin levels, with and without secretin stimulation, can be used to
diagnose ZE syndrome. Serum gastrin levels greater than 150 ng/L (refer-
ence, <100 ng/L), especially with simultaneous gastric pH values of less
than 3, are highly suggestive of a gastrinoma. For equivocal results, secre-
tin (2 U/kg in 10 mL 0.9% sodium chloride) can be given intravenously
in 30 seconds, and serial gastrin levels can be drawn at 0, 2, 5, 10, 15, 20,
and 30 minutes. An increase in gastrin of 200 ng/L or greater within 15
minutes of injection is considered a positive test result. Although these
criteria are typically applied, it is important for the clinician to bear in
mind that a nonelevated result does not preclude presence of a gastrinoma.
Indeed, there have been reports of gastrinomas with normal serum gastric
levels. These cases have been attributed to the fact that some tumors
secrete bioactive gastrin precursors, some of which go undetected by avail-
able testing modalities (Rehfeld et al., 2012). Octreotide, a synthetic form
of somatostatin, has been used for localization of tumors. Radiolabeled
octreotide binds to somatostatin receptors and can be subsequently local-
ized by scintigraphy (Zimmer et al, 1995). Somatostatin (SST) receptor
scintigraphy (SRS) is now the imaging modality of choice for these lesions,
with a reported mean sensitivity and specificity of 72% and 86%, respec-
tively (Gibril & Jensent, 2005). The underlying mechanism for the effec-
tiveness of SRS is that neuroendocrine tumors, such as gastrinomas, have
increased density of SST receptors (SSTRs), specifically subtypes SSTR2
and SSTR5 (Warner & O’dorisio, 2002). If such tumors are surgically
removed, gastrin levels can be used to assess potential success or future
recurrence.
Gastrin is a primary GI hormone that is produced mainly by the antral
G cells; it regulates gastric acid secretion and stimulates growth of the
gastric mucosa, among other functions. To a lesser extent, gastrin is pro-
duced by G cells of the proximal small intestine and δ 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 motil-
ity. Other functions include stimulation of gastric pepsinogens and intrin-
sic factor secretion, release of secretin from the small intestine, and
secretion of pancreatic enzymes as well as bicarbonate (Hill, 2006). This
hormone is secreted from antral distention, mainly after the detection of
digested protein products. Maximal stimulation of gastrin secretion occurs
within a pH range of 5 to 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 (Hill, 2006). This serves to protect the stomach
from overacidification caused by 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 are found in human blood and tissues:
G34, G17, and G14, known as big gastrin, little gastrin, and mini gastrin,
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respectively. Each form has a different assay sensitivity. All gastrins origi-
nate from a single precursor, preprogastrin, which is cleaved by the action
of trypsin. It is interesting to note that 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
PART 2
such cases, only little gastrin would be detectable in serum (Goetze &
Rehfeld, 2003).
Laboratory determination of gastrin levels with radioimmunoassay
(RIA) or EIA is indicated for the confirmation of suspected gastrin-
secreting tumors—namely, gastrinomas or ZE 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 cho-
lecystokinin (CCK) peptides. Before 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, alter-
ing the results of the assay. Specimens must be frozen immediately because
gastrin is unstable in serum. Because of the action of proteolytic enzymes,
50% of the specimen’s immunoreactivity may be lost within 48 hours at a
temperature of 4° C. It is recommended that specimens should be kept in
a freezer at a temperature of –70° C without a self-defrosting cycle if long-
term storage is required. Specimens must be analyzed immediately after
thawing, while avoiding refreezing and thawing.
Fasting serum gastrin levels are increased with increasing age, espe-
cially in patients older than 60 years, in part because of gastric mucosal
atrophy. Approximately 15% of individuals older than 60 years may have
gastrin levels between 100 and 800 ng/L (Hill, 2006). Reference intervals
for infants and children differ from those for adults; interpretation should
use age-specific reference ranges. Gastrin concentration greater than
1000 ng/L with gastric acid hypersecretion (basal acid secretion >15 mmol/
hour) is diagnostic of gastrinoma. The secretin stimulation test is a pro-
vocative biochemical test that can help confirm the diagnosis of ZE
in questionable cases. Infused secretin should cause a drop in gastrin
levels in normal individuals. However, in patients with ZE, a dramatic
increase in gastrin level is seen after secretin infusion. 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 blood flow to the tumor (Ashley et al, 1999). Limitations
include altered results from conditions that may lead to elevated gastrin
levels, such as gastric ulcer disease, chronic renal failure, hyperparathy-
roidism, pyloric obstruction, vagotomy, retained gastric antrum, short
bowel syndrome, and pernicious anemia. Certain medications, such as
antacids, H2-blocking agents, and proton pump inhibitors, can also increase
gastrin measurements; all of these agents are commonly used in the treat-
ment of patients with peptic ulcer disease. However, the elevations are
moderate and certainly are not as high as in a patient with a gastrin-
secreting tumor.
Intraoperative testing for gastrin is of potential use because gastrinomas
can be multiple and are often difficult to locate; they can be distributed
widely in the stomach, pancreas, and duodenum or periaortic lymph nodes.
Intraoperative gastrin measurement is of potential use because gastrin has
a short half-life of approximately 10 minutes. The catabolic breakdown of
most peptide hormones follows first-order exponential decay. Therefore,
if the entire hormone-secreting tissue is surgically resected, only approxi-
mately 12.5% of the baseline concentration would be present in serum
after three half-lives. When patients with ZE or gastrinoma were evaluated
with intraoperative gastrin assays, a drop in gastrin levels to within refer-
ence values within 20 minutes of resection was indicative of cure (Sokoll
et al, 2004). Gastrin and secretin levels remain the most sensitive methods
to detect recurrence (Norton & Jensen, 2007).
Pepsin and Pepsinogen
Pepsinogens are the biologically inactive proenzymes of pepsins that are
produced by 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 inhib-
ited by gastric inhibitory peptide (GIP), anticholinergics, histamine H2-
receptor antagonists, and vagotomy (Hill, 2006). 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 in the duodenum. The ratio of
concentration of PGI to PGII in the 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 auto-
catalytically. Both groups of pepsinogens are activated at an acid pH below
5 and are destroyed by alkaline pH. Both types can be detected in blood.
311
 Only type I pepsinogens are present in the urine. Pepsins are responsible
22 LABORATORY DIAGNOSIS OF GASTROINTESTINAL AND PANCREATIC DISORDERS
for the hydrolysis of proteins to polypeptides. The pepsinogen released
from the gastric mucosa 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 (Hill, 2006). Immunoassay is the method
used to detect serum pepsinogen. However, the PGI isoform is commonly
analyzed in the clinical laboratory because it is the isoform commonly
associated with disease.
Serum levels of pepsinogen I provide an accurate estimate of parietal
cell mass and correlate with the acid-secretory capacity of the stomach.
Increased pepsinogen levels and associated activity are observed in patients
with disease states that lead to increased gastric output or with increased
parietal cell mass—namely, gastrinoma, ZE syndrome, duodenal ulcer
disease, and acute and chronic gastritis. Decreased levels of pepsinogen are
associated with decreased parietal cell mass, atrophic gastritis, and gastric
carcinoma, as well as with myxedema, Addison’s disease, and hypopituita-
rism (Hill, 2006). The PGI/PGII ratio decreases linearly with worsening
atrophic gastritis. Absence of pepsinogen is noted in patients with achlor-
hydria. PGI levels measured by immunoassay usually range from 20 to
107 μg/L, and PGII levels usually range from 3 to 19 μg/L.
Pepsinogen assays are being explored for their utility in the noninvasive
identification of patients with chronic atrophic gastritis and to obtain 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 with healthy individuals (Miki
et al, 2003). It is hoped that this finding will help to identify a subgroup
of individuals with chronic atrophic gastritis who would benefit from
endoscopic evaluation for detection of early-stage gastric tumor. 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 et al, 2003). These authors recommended that cri-
teria for diagnosing chronic atrophic gastritis should include persons with
PGI less than 70 μg/L and a PGI/PGII ratio less than 3.0. In Japan, the
pepsinogen serum screening test has been demonstrated to detect a higher
percentage of early cancers compared with conventional methods, and a
considerable number of patients have subsequently been candidates for
treatments with endoscopic surgery (Miki et al, 2003). The most sensitive
test for fundic atrophic gastritis is considered to be the PGI/II serum ratio,
with 99% sensitivity and 94% specificity (Hill, 2006), and 64% sensitivity
and 61% specificity for antral and pangastric atrophic gastritis (Lee et al,
2014). Furthermore, PGII 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 et al, 2000). Pepsinogen assays there-
fore may prove useful as a serum screening method for detection of gastric
carcinoma among high-risk individuals.
DIARRHEA AND MALABSORPTION
Diarrhea
About 8 to 10 L of fluid enters the duodenum every 24 hours. Much of
this fluid is absorbed in the small intestine, and about 1.5 L enters the
large intestine, but only 100 to 150 mL is voided in stools. Increased fluid
secretion or decreased fluid absorption in the small or large intestine may
result in diarrhea. One of the most important parameters defining diarrhea
in an individual patient is a change in the usual bowel habit to more fre-
quent, looser stools. Diarrhea is the passage of three or more loose or
liquid stools per day, or more frequently than is normal for the individual
(World Health Organization [WHO], 2013). A decrease in fecal consis-
tency (i.e., increased fluidity) is more important than the number of stools.
However, this property is difficult to measure, so increased stool weight,
frequency, and duration are used in defining diarrhea.
The diagnosis of diarrhea starts with a thorough history to character-
ize the condition. The WHO guidelines categorize diarrhea into four
clinical umbrellas: acute watery, acute bloody, persistent, and diarrhea
with malnutrition. Additional clinical questions to further characterize
the condition include: Is the diarrhea bland or bloody (dysentery)? Are
there constitutional symptoms? What is the duration of the illness? Self-
limited, acute diarrhea (<2 weeks in duration) without bleeding or con-
stitutional symptoms rarely requires diagnostic testing. Chronic diarrhea,
the passage of blood, and constitutional symptoms all suggest the need
for a specific diagnosis. History is the key to developing the differential
312
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diagnosis and guiding the laboratory evaluation (Table 22-4). The physi-
cal examination, although usually less helpful than the history, must be
comprehensive.
Acute diarrheas of less than 4 weeks generally have an infectious origin.
Those with a course of longer than 4 weeks are considered chronic diar-
rheas (Fine & Schiller, 1999) and are categorized as osmotic, secretory, or
inflammatory. Hypermotility or shortened gut may reduce the transit time
and absorptive surface, resulting in diarrhea and/or some degree of
malabsorption.
Osmotic diarrhea results from unabsorbable or poorly absorbed solutes.
These include polyethylene glycol in colon-cleansing solutions, magne-
sium salts (magnesium citrate in cathartics, magnesium hydroxide in some
antacids), sorbitol in chewing gum, lactulose in the treatment of hepatic
encephalopathy, and lactose in lactase-deficient individuals. These osmoti-
cally active substances in the lumen alter the osmotic gradient that nor-
mally favors Na+ absorption, drawing fluid into the lumen.
Fasting or cessation of consumption of the suspected solute stops
osmotic diarrhea. Stool pH values of less than 5.6 are consistent with
carbohydrate malabsorption (Fine & Schiller, 1999). Sodium and potas-
sium concentrations in stool water are measured to calculate the fecal
osmotic gap, which estimates the contributions of electrolytes and non-
electrolytes to water retention in the gut lumen. Osmotic gap is useful in
differentiating osmotic from secretory diarrhea; it is best calculated as 290
– 2 × ([Na+] + [K+]), where 290 represents the stool osmolality that approxi-
mates plasma osmolality, and a factor of 2 is used to account for associated
anions. Osmotic gaps of greater than 125 mOsm/kg characterize osmotic
diarrhea, and those of less than 50 mOsm/kg are seen in secretory diarrhea
(Fine, 1999).
Stool osmolality must be measured on a freshly collected specimen.
Because of continued degradation of stool carbohydrates, osmolality of the
specimen increases over hours. Continued diarrhea during a 48-hour fast
is suggestive of a secretory process, although fecal weight may decrease
because of increasing dehydration (Fordtran, 1967; Fine & Schiller, 1999).
Disaccharidase Deficiency
Because the small intestinal mucosa is impermeable only to disaccharides,
special enzymes are required for the breakdown of these molecules. Defi-
ciency leads to malabsorption and thus dietary intolerance manifested in
diarrhea. The important enzymes are lactase-phlorizin hydrolase, sucrase-
isomaltase, and maltase-glucoamylase (Clinton et al, 2012). Disaccharide
absorption may also be diminished from primary alactasia or primary
trehalase deficiency, or from secondary disaccharidase deficiencies due to
celiac disease, tropical sprue, acute viral gastroenteritis, or drugs such as
orally administered neomycin, kanamycin, and methotrexate. These sec-
ondary 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% to 80% of African
Americans, and an even greater percentage of Asians 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 thus 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 symptoms, 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 and 7 days of age
commonly have high stool pH. Stools can be analyzed for sugars by chro-
matography 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 to 0.5 g/dL is regarded as
suspicious; and more than 0.5 g/dL is considered abnormal. In patients
with intolerance to sugar, the amount of total reducing substance 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%
to 30% false-positive results were noted following administration of
TABLE 22-4
Laboratory Tests in the Differential Diagnosis of Diarrhea
Test Method Use

Initial Screening Tests

PART 2
Fecal leukocytes Wright’s stain or methylene blue Identify inflammatory diarrhea
Fecal occult blood test Immunochemical Detect blood
Fecal osmotic gap 290 − 2 × (fecal Na+ + K+) Distinguish secretory vs. osmotic diarrhea
Stool alkalinization Color change after adding NaOH to stool/urine Phenolphthalein laxative ingestion
Infectious Causes
Stool bacterial culture Routine culture and sensitivity Identify Shigella, Salmonella
Stool special culture Specialized culture and serotyping Identify E. coli 0157:H7, Yersinia, Campylobacter
Stool C. difficile toxin assay EIA for toxins A and B Pseudomembranous colitis
HIV serology EIA, Western blot HIV enteritis
Stool rotavirus screen EIA for antigen Rotavirus enteritis
Stool ova and parasites Concentration and stains Enteric parasitic infection
Stool mycobacteria Acid-fast stain, culture and sensitivity, PCR Mycobacterium, antibiotic selection
Stool E. histolytica Ag EIA for antigen Entameoba histolytica
Stool Giardia Ag EIA for antigen Giardia lamblia
Stool Cryptosporidium Ag EIA for antigen Cryptosporidium parvum
Endocrine Causes
Urine 5-HIAA or blood serotonin HPLC Carcinoid syndrome
Serum VIP RIA VIPoma
Serum TSH, free T4 Immunoassay Hyperthyroidism
Serum gastrin RIA Zollinger-Ellison syndrome
Serum calcitonin RIA Hypocalcemia-related diarrhea
Serum somatostatin RIA Somatostatinoma
Malabsorption
Lactose tolerance test See text Lactase deficiency
Stool reducing sugars Clinitest tablets Carbohydrate intolerance
Sweat chloride See text Cystic fibrosis
D-Xylose absorption test See text Evaluate pancreatic and jejunal function
Fecal fat stain Fat stain Lipid malabsorption
Serum carotene Spectrophotometry Lipid malabsorption
14
C-glyceryl trioleate test malabsorption See text Lipid malabsorption
Serum IgA Nephelometry Rule out IgA deficiency
Antitissue transglutaminase antibody EIA Celiac disease
Hydrogen breath test Electrochemical hydrogen Carbohydrate malabsorption
monitorinomatography g
Bacterial colony count Small bowel aspirate and quantitative culture Bacterial overgrowth
Other
Serum ionized calcium Ion-specific electrode Hypocalcemia-related diarrhea
Serum protein and albumin Nephelometry, photometry IBD, protein-losing enteropathy
Stool alpha-1-antitrypsin Nephelometry Protein-losing enteropathy
Quantitative immunoglobulins Nephelometry Agammaglobulinemia
7α-Hydroxy-4-cholestin-3-one HPLC Bile salt malabsorption
Fecal elastase or pancreolauryl test EIA Pancreatic insufficiency
Intestinal biopsy Endoscopic or open biopsy Whipple’s disease, MAI, abetalipoproteinemia, neoplasia,
lymphoma, amyloidosis, eosinophilic gastroenteritis,
agammaglobulinemia, intestinal lymphangiectasia, Crohn’s
disease, tuberculosis, graft-versus-host disease, Giardia, other
parasitic infections, collagenous colitis, microscopic colitis
Extraintestinal causes See text Hyperthyroidism, diabetes, hypoparathyroidism, adrenal
cortical insufficiency, hormone-secreting tumors

5-HIAA, 5-Hydroxyindoleacetic acid; Ab, antibody; Ag, amtigen; EIA, enzyme immunoassay; HIV, human immunodeficiency virus; HPLC, high-performance liquid chroma-
tography; IBD, inflammatory bowel disease; MAI, Mycobacterium avium-intracellulare; PCR, polymerase chain reaction; RIA, radioimmunoassay; TSH, thyroid-stimulating
hormone; VIP, vasoactive intestinal peptide.

lactose—that is, a flat tolerance curve and less than 20 mg/dL (1.1 mmol/L)
increase in blood sugar (Krasilnikoff et al, 1975). Delayed gastric emptying
appears to be the cause of the false-positive result because duodenal instil-
lation of lactose eliminates the flat tolerance curve.
With our increased understanding of the genetic basis of certain inher-
ited disaccharidase deficiencies, the role of genetic testing has been inves-
tigated. When testing a saliva or serum sample could avoid sedation and
instrumentation of an infant (for tissue diagnosis), it should be considered.
A recent genetic analysis of infants with congenital sucrose-isomaltase (SI)
deficiency suggests that genetic testing of saliva or serum for four specific
mutations in the SI gene could lead to a valid diagnosis in 83% of these
infant patients of European extraction (Uhrich et al, 2012).
Definitive diagnosis of disaccharidase deficiency depends on the dem-
onstration of low specific enzyme activity in the mucosa of small intestinal
biopsy material. An assay for disaccharidase has been published (Dahlqvist,
1968).

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22 LABORATORY DIAGNOSIS OF GASTROINTESTINAL AND PANCREATIC DISORDERS
Lactose Tolerance Test
A lactose tolerance test provides a presumptive diagnosis of lactase defi-
ciency. In patients with alactasia or hypolactasia, whether primary or sec-
ondary to mucosal disease, oral lactose results in an insignificant rise in
blood glucose. Following an overnight fast, a blood sample is drawn, and
50 g of lactose dissolved in 400 mL of water is orally administered. A 100-g
lactose dose has been reported to yield more definitive results. Blood
samples are collected at 30, 60, and 120 minutes after lactose ingestion.
An optional 5-hour stool specimen can be collected, and its appearance,
consistency, and pH noted.
Patients with lactase deficiency exhibit a peak rise of less than 20 mg/
dL of reducing substances, expressed as glucose. In individuals with flat
tolerance curves, the test should be repeated in 2 days and the less abnor-
mal of the two curves used for interpretation. In patients with lactase
deficiency, the nonabsorbed lactose, upon reaching the colon, is degraded
to gas and lactic acid, which inhibits salt and water absorption, resulting
in abdominal discomfort and diarrhea. In children, the oral dose of lactose
or other sugars is 2 g/kg body weight. Lactase can also be detected in a
mucosal biopsy specimen.
Secretory diarrhea is evoked by a variety of exogenous and endogenous
secretagogues. The hypersecretion of isotonic fluid exceeds the absorptive
capacity of the colon. Cholera toxin activates mucosal cyclic adenosine
monophosphate (cAMP) that results in outpouring of vast quantities of salt
and water in jejunum with normal structure. In Verner-Morrison syn-
drome (pancreatic cholera), vasoactive intestinal peptide also activates
cAMP. Thus secretory diarrhea remains unrelieved despite fasting.
Diarrhea also occurs in hypergastrinemic states (e.g., ZE syndrome).
Sustained hypersecretion of acid lowers intestinal pH, denatures pancreatic
enzymes (causing steatorrhea), and precipitates bile salts (causing bile salt
malabsorption); the latter induces water secretion in the colon. In patients
with mastocytosis, excessive histamine release stimulates acid hypersecre-
tion, but plasma gastrin concentrations remain low or normal. Gastroin-
testinal dysfunction caused by release of mast cell mediators can be
observed in both cutaneous and systemic mastocytosis (Liu et al., 2010).
Signs and symptoms include abdominal pain, diarrhea, nausea, vomiting,
peptic ulcer disease, and GI bleeding. Diarrhea can result from increased
motility induced by prostaglandin D2 secretion or from decreased rectal
compliance and overactive rectal contractility (Jensen, 2000).
Drug-Induced Diarrhea
Prokinetic agents (metoclopramide, domperidone, cisapride), proton
pump inhibitors, and antibiotics (erythromycin and other macrolides)
produce loose stools or diarrhea. Erythromycin binds motilin receptors on
the GI smooth muscle membranes (motilin agonist). When used as a
prokinetic agent, intravenous administration is more effective than oral
dosage, and long-term use downregulates motilin receptors.
Other Causes of Diarrhea
Diarrhea may occur in patients with hypothyroidism and hyperthyroidism,
diabetes and adrenal insufficiency, tumors (villous adenoma, small bowel
carcinoid), and infiltrative disorders (scleroderma, reactive amyloidosis,
gut lymphoma). Ethanol abuse, ischemic bowel disease, and radiation
enteritis also produce diarrhea. Heat-labile enterotoxin of Escherichia coli
and toxins of Staphylococcus aureus and Clostridium perfringens activate bio-
chemical events that induce a secretory response.
Gut Peptides in Diarrhea
In acute infectious diarrheas, the plasma concentrations of glucagon, PYY,
and motilin may be increased, contributing most likely to altered gut
motility and promoting mucosal repair. Patients with Crohn’s disease have
an elevated pancreatic polypeptide, GIP, motilin, and glucagon, and in
ulcerative colitis, a modest elevation is observed in pancreatic polypeptide,
GIP, motilin, and gastrin, the last in response to the associated hypochlor-
hydria. No demonstrable abnormalities in gut peptides account for disor-
dered motility in the irritable bowel syndrome.
Vasoactive Intestinal Peptide
VIPomas are rare tumors that secrete vasoactive intestinal polypeptide
(VIP). VIP suppresses gastric acid secretion, resulting in secretory diarrhea
with stool volume exceeding 700 mL/day in all patients (even during
fasting) and 3 L/day in approximately 70%. The stools are tea-colored and
odorless, with features of a secretory diarrhea such as persistence with
fasting, high sodium concentration, and a low stool osmotic gap. Most
patients with VIPoma have the watery diarrhea–hypokalemia–hypochlor-
hydria (WDHA) syndrome. The diagnosis of VIPoma is established by the
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presence of an otherwise unexplained high-volume secretory diarrhea and
a serum VIP concentration in excess of 75 pg/mL. It is important to
replace fluid losses in these patients, and octreotide may be used to control
diarrhea.
Patients usually present between 30 and 50 years of age; 90% of cases
are primary pancreatic tumors, most commonly arising in the body or tail.
Metastatic spread has already occurred in 60% to 70% at presentation.
Long-acting somatostatin analogs control symptoms in greater than 90%
of cases (Kapoor et al, 2009).
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 stimu-
lator of water and electrolyte secretion, acting through stimulation of
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.
Laboratory evaluations for determination of VIP levels are clinically
relevant for diagnosis of VIPomas. These VIP-secreting tumors, most
commonly of pancreatic origin, account for 10% of neuroendocrine
tumors of the gastrointestinal tract. Approximately 60% of VIPomas are
malignant, and 6% are associated with MEN type 1. This syndrome was
first described in 1958 by Verner and Morrison, and is characterized by
WDHA.
VIPomas produce a severe, secretory diarrhea that has been termed
pancreatic cholera. Diarrhea may be present for several years before the
diagnosis of VIPoma. Patients typically produce more than 3 L of watery
stools per day, although volume can be as high as 30 L/day. Diarrhea is
not controlled with fasting, and an average secretion of 300 mmol of potas-
sium 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, 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 electro-
lyte disturbances with potassium depletion and acidosis. The typical meta-
bolic profile seen in these patients is hypokalemia with hyperchloremic
metabolic acidosis. The diagnosis of VIPoma 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 and associated
with VIP production.
The biochemical assay of VIP is sensitive and specific. The normal
value for circulating VIP levels is less than 170 pg/mL. For patients with
functional VIP-secreting tumors, values range from 675 to 965 pg/mL
(Thomas et al, 2003). Appropriate handling of the serum sample is critical
for the accuracy of the VIP assay because VIP has a half-life of 1 minute.
The serum must be added immediately to aprotinin, a protease inhibitor
that prevents breakdown of VIP. The sample must be separated within 10
minutes and frozen to –20° C. Some of the non-VIP products of the
precursor molecule are secreted at higher levels than VIP; however, com-
mercially 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. In
addition, the serum pancreatic polypeptide level should be determined at
the time of the VIP assay; this value will be elevated if the VIPoma is
located within the pancreas.
CT scan, MRI, and abdominal ultrasound are useful imaging modali-
ties 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.
Antibiotic-Associated Diarrhea
This term is typically reserved for Clostridium difficile, a gram-positive,
spore-forming anaerobic bacillus that is the most important cause of
nosocomial diarrhea in adults; more than 300,000 cases are reported per
year in the United States. C. difficile is thought to be associated with
approximately 25% of all antibiotic-associated cases of diarrhea and 50%
to 75% of cases involving antibiotic-associated colitis (Malnick & Zimhony,
2000). It represents a significant clinical burden in the United States, with
recent inquiries of national databases revealing an increase in incidence
over the last decade (Halabi et al, 2013; Pant et al, 2015). Halabi and col-
leagues queried the Nationwide Inpatient Sample and found over 2.5
million discharges of C. difficile colitis over the period from 2001 to 2010.
Pant and colleagues queried the Nationwide Emergency Room Sample
and found over half a million ER visits over half that time period, 2006 to
2010. They also found a 47% and 24% increase in the number of inpatients
and emergency room visits, respectively, for this diagnosis over the study TABLE 22-5
periods. Clearly, this is an important problem requiring clinical attention. Laboratory Tests for the Diagnosis of Clostridium difficile–
It may manifest clinically, from a mild, watery diarrhea to life-threatening Associated Diarrhea
pseudomembranous colitis and toxic megacolon. This can lead to colonic
perforation and peritonitis, with a mortality rate as high as 38% (Poutanen Test Advantages Disadvantages
& Simor, 2004). Patients can present with watery diarrhea, lower abdomi-

PART 2
nal pain/cramping, or systemic symptoms such as fever and malaise, or can Clostridium difficile Excellent specificity Decreased diagnostic
have occult gastrointestinal bleeding. The pathogenesis of this disease cytotoxin assay (99% to 100%) sensitivity (80% to
entity usually involves disruption of the normal colonic flora, typically 90%); test results not
available until after
following a course of antibiotic therapy in hospitalized patients, followed
48 hours; requires
by exposure to a toxigenic strain of C. difficile. Broad-spectrum antibiotics
tissue culture facility;
such as penicillin, clindamycin, and cephalosporins have been particularly detects toxin B only
implicated; however, any antibiotic can lead to development of C. difficile
Immunoassay for High negative Toxin assay required
colitis (Malnick & Zimhony, 2000). Clinical suspicion of the disease is
glutamate predictive value because GDA does
confirmed with detection of C. difficile toxin A or B virulence factors in
dehydrogenase without additional not distinguish
stool samples. Toxins A and B lead to increased vascular permeability and antigen (GDA) of C. tests toxigenic and
have the potential to cause hemorrhage. They induce the production of difficile nontoxigenic strains
tumor necrosis factor-α and inflammatory interleukins that are responsible
Immunoassay for Good specificity (95% Reduced sensitivity
for the inflammatory response and pseudomembrane formation (Poutanen
detection of toxin A to 100%); test (65% to 85%) as
& Simor, 2004). or toxins A and B results available compared with
Endoscopic visualization of the colonic mucosa is required for diagno- within 4 hours; cytotoxin assay
sis of pseudomembranous colitis associated with C. difficile. However, technically simple
endoscopy should be avoided in cases of suspected fulminant colitis because
Stool culture to isolate Excellent sensitivity Results not available for
of the risk of perforation. Laboratory methods are available for confirma- C. difficile with (>90%) and at least 72-96 hours;
tion of C. difficile infection. Tissue culture cytotoxicity assays, which take subsequent cytotoxin specificity (>98%); labor-intensive;
at least 48 hours to complete, are considered the gold standard for the assay of isolate enables typing of requires tissue culture
detection of C. difficile cytotoxin B in stool specimens, with a sensitivity strain for outbreak facility
ranging between 94% and 100% and a specificity of approximately 99%. investigation
This tissue culture assay can detect as little as 10 pg of toxin in stool Nucleic acid Excellent sensitivity Useful only in acute
specimens. Rapid EIAs, which can be completed within several hours, have amplification assay (93% to 100%); disease; false
been developed for the detection of toxin A or B from stool specimens. for toxigenic C. useful in positives of concern
However, the sensitivity and specificity of these immunoassays are 65% to difficile confirmation of
85% and 95% to 100%, respectively, compared with cytotoxic assays. The results of GDA or
EIA can detect 100 to 1000 pg of toxin in stool specimens. In hospitalized toxin immunoassays
patients with more than six stools per day, EIA is the optimal diagnostic
test (Malnick & Zimhony, 2000). Stool cultures can also be performed but Adapted from Poutanen SM, Simor AE: Clostridium difficile–associated diarrhea in
adults, Can Med Assoc J 171:51–58, 2004; Fenner L, et al: Rapid and reliable
require up to 96 hours for completion. Polymerase chain reaction (PCR)
diagnostic algorithm for detection of Clostridium difficile, J Clin Microbiol 46:328–
methods for detection of C. difficile toxin A or B are currently being devel- 330, 2008; Surawicz CM, et al: Guidelines for diagnosis, treatment, and preven-
oped with similar sensitivity and specificity profiles compared with cyto- tion of Clostridium difficile infections, Am J Gastroenterol 108:478–498, 2013.
toxic assays (Poutanen & Simor, 2004). Traditional PCR techniques can
still take 3 to 4 hours but have high sensitivities and specificities (83% to
95% and 97% to 99%, respectively) (Putsathit et al, 2015). Even more
recently, automated PCR assays utilizing the Taq Man hybridization probe
for the tcdB gene for Toxin B have cut down processing time to 10 minutes less than 100 cells per microliter are at risk for opportunistic infections
per 10 samples and maintain high sensitivities and specificities (94% to that are typically chronic, such as C. parvum, MAC, cytomegalovirus,
97% and 97% to 99%, respectively) (Putsathit et al, 2015). Isospora belli, or microsporidia.
PCR is unable to distinguish between asymptomatic carriage and symp- An epidemiologic history with a focus on travel history (Entamoeba
tomatic infection. It is currently recommended that these tests be per- histolytica, Giardia lamblia), sexual exposure (history of unprotected anal
formed on diarrheal stools; in most cases, one stool sample is sufficient for intercourse suggesting transmission of herpes simplex virus, Neisseria gon-
the diagnosis of C. difficile infection (Poutanen & Simor, 2004). However, orrhoeae, Chlamydia trachomatis, or, occasionally, E. histolytica), and food
multiple samples may be required for confirmation, and empirical treat- associations (lactose intolerance) should be sought. In patients who are
ment with oral antibiotics may be indicated in patients with clinical evi- taking highly active antiretroviral therapy, medication-induced diarrhea
dence of C. difficile infection. Diarrheic stools can also be screened by an (nelfinavir, ritonavir) should be considered, particularly when diarrhea is
immunoassay for glutamate dehydrogenase antigen, a C. difficile–specific the sole presenting symptom. Clostridium difficile should be considered
antigen, and those positive should be tested for toxins A and B (Fenner because most patients with HIV are given antibiotics for the treatment of
et al, 2008). Refer to Table 22-5 for laboratory tests available for the various infections (Sanchez et al, 2005).
diagnosis of C. difficile–associated diarrhea.
Malabsorption Syndromes
HIV-Related Diarrhea Malabsorption is the pathologic state of impaired nutrient absorption in
Diarrheal disease in human immunodeficiency virus (HIV)–infected indi- the gastrointestinal tract. Normal nutrient absorption occurs in three
viduals is frequently caused by infectious agents but may also be due to steps: luminal and brush border processing, absorption into the intestinal
infiltrative diseases, such as lymphoma or Kaposi’s sarcoma. Common mucosa, and transport into the circulation. Disruption in any one or a
enteric pathogens with sufficient virulence that cause disease in healthy combination of these steps can result in inadequate mucosal absorption of
hosts can also cause diarrheal disease in HIV-infected individuals with carbohydrates, proteins, fats, vitamins, or minerals. Malabsorption can also
intact or compromised immunologic function. Less virulent pathogens result from the presence of substances in the bowel that cannot be absorbed
such as Cryptosporidium parvum, which appear to require some immune (e.g., lactulose, sorbitol). Maldigestion results from an intraluminal defect
compromise to establish disease, are more common in patients with more that leads to the incomplete breakdown of nutrients into their absorbable
advanced HIV infection or acquired immunodeficiency syndrome (AIDS). substrates. This can occur with pancreatic insufficiency and loss of exocrine
Mycobacterium avium-intracellulare complex (MAC) is predominantly function, resulting in increased osmotic load of the colon and diarrhea. In
associated with lung infection in immunocompetent patients. MAC may addition, patients can have selective malabsorption/maldigestion of spe-
also produce disseminated disease with bowel infiltration and malabsorp- cific nutrients, resulting in associated clinical sequelae. Irrespective of the
tion in patients with severe immune compromise and should be considered cause, diarrhea, especially steatorrhea, is the most common feature of
in the differential diagnosis. In these cases, blood should also be obtained malabsorption.
in fungal isolator tubes for culture. Hepatic maldigestion results from interference or obstruction of bile
In patients with HIV, it is important to ascertain the nature and chro- flow. Loss of bile salts interferes with fat emulsification, diminishing the
nicity of symptoms and the CD4 cell count. Patients with CD4 cell counts surface area available for lipolytic action. In addition, bile salt activation

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 of lipase activity is lost. Patients are usually jaundiced, pass dark urine, and
22 LABORATORY DIAGNOSIS OF GASTROINTESTINAL AND PANCREATIC DISORDERS
have other signs of liver disease. Hepatic steatorrhea may coexist with
pancreatic steatorrhea, as in patients with a neoplasm obstructing the
ampulla of Vater. The inability to assimilate fats and proteins due to mal-
digestion 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 nutrients.
This may result from competition by bacteria or altered bacterial flora, as
in the blind loop syndrome or diverticulosis of the small bowel, or from
obstruction to the flow of lymph. It may also result from diseases affecting
the small bowel mucosa, such as amyloidosis, inflammation following irra-
diation (radiation enteritis), diminished mucosal surface area as in gastro-
ileostomy (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 salt absorption, which will result in vitamin B12
deficiency, as well as a decreased pool of circulating bile salts for
metabolism.
Steatorrhea is a hallmark finding in patients with malabsorption, result-
ing in fluid, semifluid, or soft and pasty, pale, bulky, and foul-smelling
stools. These stools may be foamy because of the high fat content and may
float on water. However, the latter may occur with stools from healthy
individuals and therefore is a nonspecific sign of malabsorption.
In patients with steatorrhea, unabsorbed fecal dietary fat is passed in
stools above and beyond the normal 1% to 9%, along with as much as
40% of ingested fat. The quantity of fecal fat depends on the dietary fat
intake. Thus dietary fat intake must be known for proper interpretation of
fecal fat, which is expressed as the percentage of dietary fat, allowing
assessment of variation in an individual patient. Normally, greater than
93% of dietary fat is absorbed, but diarrhea of any cause may lead to a
slight increase in fecal fat content.
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 hypo-
prothrombinemia, glossitis, anemia, edema, ascites, and osteomalacia, may
be evident in these individuals. These patients may experience significant
weight loss due to diarrhea, leading to cachexia in severe cases.
Quantitative fecal fat measurement has many limitations and should be
abandoned (Holmes & Hill, 1988; Hill, 2001). Sample collection is known
to be incomplete (Ditchburn et al, 1971; West et al, 1981). Also, there is
poor precision in the analytic performance, making interpretation uncer-
tain (Duncan & Hill, 1998). Newer tests provide improved sensitivity and
specificity for the diagnosis of malabsorption (Hill, 2001): 14C-glycerol
trioleate breath test (Turner et al, 1987) and mixed-chain triglyceride
breath test are widely available (Vantrappen et al, 1989; Amarri et al,
1997). However, these tests have limited reliability in diabetes, obesity,
hyperthyroidism and hypothyroidism, and chronic respiratory insuffi-
ciency and should not be performed in pregnancy.
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 pancreatic insufficiency or other causes
results in decreased absorption of triglycerides. This in turn results in a
decrease in expired carbon dioxide (CO2) derived through the metabolism
of triglyceride fatty acids.
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 scintil-
lation counter, and the results are reported as a percentage of the dose of
14
CO2 excreted per hour.
To distinguish pancreatic insufficiency from other causes of steator-
rhea, 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 14CO2 is measured as previously described. The second
stage of the test is performed 5 to 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.
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Agammaglobulinemia
X-linked agammaglobulinemia is a primary humoral immunodeficiency
characterized by recurrent bacterial infection of the respiratory tract and
increased susceptibility to enteroviral infection. Absence of humoral
immunity makes the patient susceptible to bacterial gastroenteritis.
Abetalipoproteinemia
Abetalipoproteinemia is a rare autosomal recessive disorder that is charac-
terized by defective assembly and secretion of apolipoprotein B (apoB) and
apoB-containing lipoproteins, resulting from mutations in the gene encod-
ing the microsomal triglyceride transfer protein; the serum β lipoprotein
is absent. Abetalipoproteinemia causes defective absorption of lipids.
Patients may have neurologic manifestations, acanthocytes, fat malabsorp-
tion, steatorrhea, and associated fat-soluble vitamin deficiencies (Gregg
et al., 1994).
Tests for Steatorrhea
Screening tests for detection of steatorrhea include microscopic examina-
tion 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 carotenoids in the intestines depends
on the presence of dietary fat. Because carotenoids are not stored in the
body to any appreciable degree, lack of carotenoids in the diet or distur-
bances 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 cause a low level of serum carotenoid. Elevated serum
carotenoid levels are seen in patients with hypothyroidism, diabetes,
hyperlipidemia, and excessive intake of carotene.
Tests for Malabsorption
When a diagnosis of malabsorption is being entertained, it is important to
distinguish pancreatic maldigestion from enteric malabsorption. In chil-
dren, the main cause of pancreatic malabsorption is CF, 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
d-xylose absorption test.
The cellobiose-mannitol sugar permeability test and the lactulose-
mannitol test have been used in the diagnosis of celiac disease. Modern
evaluation of this disorder has been described earlier. 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 the Schilling test for vitamin B12 absorption, which tends
to be abnormal in patients with enteric steatorrhea in whom the abnormal-
ity is not correctable with intrinsic factor. Endoscopy, radiologic studies,
and biopsy have replaced these methods in many cases.
Fecal Elastase. Elastase-1 is a proteolytic enzyme produced by the
pancreas. Pancreatic elastase survives intestinal transit intact and is five- to
sixfold concentrated in the feces (Lankisch, 2004). Reduced pancreatic
elastase-1 in feces indicates pancreatic insufficiency in infants older than
2 weeks of age with CF and in older children with the disorder (Phillips
et al, 1999; Cade et al, 2000; Leus et al, 2000).
This EIA is unaffected by pancreatic enzyme replacement therapy.
Although sensitive for detection of severe pancreatic insufficiency, it lacks
sensitivity for detection of milder forms. Fecal elastase is better than fecal
chymotrypsin, para-aminobenzoic acid, bentiromide, and pancreolauryl
tests (Lankisch, 2004). Single analysis of a 100-mg stool sample is adequate
for determination of fecal elastase levels. If borderline values are detected,
a repeat sample may be useful. This test should be performed only on
formed stool. With a cutoff of 200 μg/g stool, the positive predictive value
of fecal elastase determination is estimated to be approximately 50% (Lüth
et al, 2001).
Xylose Absorption Test. The d-xylose absorption test is a valu-
able test for the differential diagnosis of malabsorption. In this proce-
dure, a 25-g dose of pentose sugar in water is administered orally, and
the amount excreted in urine over a 5-hour period is determined. If the
amount excreted is less than 3 g, the diagnosis is most likely enteroge-
nous malabsorption, because pancreatic enzymes are not required for
absorption of d-xylose. d-Xylose is passively absorbed in the small intes-
tine 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 renal excretion. Therefore, in patients with renal disease,
xylose should be quantified in blood 2 hours after its oral administration
because urine values are difficult to interpret in the absence of reference TABLE 22-6
values. Biomarkers in the Diagnosis of Celiac Disease and Monitoring
Isotopic techniques and the starch tolerance test have been used as Compliance to Gluten-Free Diet
alternatives to the d-xylose test. Quantitative specific fecal trypsin and
chymotrypsin assays may be helpful, as may the Schilling test, which is Biomarker Method Comments
used to assess the function of the terminal ileum. An oral dose of radioac-

PART 2
tive vitamin B12 is followed by an intramuscular large dose of nonradioac- Antireticulin IFA (rat kidney) Lack optimal sensitivity
tive vitamin B12, and radioactivity in urine is measured. Urinary radioactivity antibodies—IgG/IgA and specificity for
reflects the absorbed amount of vitamin B12. A repeat test to diagnose routine diagnostic use
pernicious anemia or gastric pathology in a patient with steatorrhea Total IgA Quantitative Useful in ruling out IgA
involves coadministration of intrinsic factor and vitamin B12. An abnormal nephelometry deficiency; specific IgG
result indicates ileal disease. antibodies need to be
tested in IgA-deficient
Laxative Abuse individuals
Surreptitious laxative abuse is a frequently overlooked cause of chronic Antigliadin antibodies— Quantitative EIA Low sensitivity and
diarrhea and is the final diagnosis for chronic diarrhea in 15% to 26% of IgG/IgA specificity; useful in
patients at referral centers (Bytzer et al, 1989; Duncan et al, 1992). In monitoring dietary
compliance
Munchausen’s syndrome by proxy, adults administered laxatives surrepti-
tiously to young children (Duncan, 2000). The main prerequisite for Antideaminated gliadin Quantitative EIA Inferior performance
making the diagnosis of surreptitious laxative abuse is clinical suspicion. antibodies—IgG/IgA relative to other
Analysis of urine and fecal samples taken during diarrhea is necessary. diagnostic assays
Phenolphthalein is less frequently found since over-the-counter sales were Antiendomysial IFA (rhesus monkey High sensitivity and
banned. Senna, aloin, and cascara are colonic stimulants that are abused antibodies—IgG/IgA esophagus; specificity in CD;
and can be detected by thin-layer chromatography. human umbilical observer bias limits
cord) usefulness
Celiac Disease Antitissue glutaminase— Quantitative EIA Assays using purified
Celiac disease (gluten-sensitive enteropathy) is a disorder precipitated, in IgG/IgA human or recombinant
genetically predisposed individuals, by the ingestion of gluten, the major human tTG are more
storage protein of wheat and similar grains, characterized by intestinal sensitive than those
using guinea pig tTG;
malabsorption of nutrients due to sensitivity to the alcohol-soluble portion
useful in both diagnosis
of gluten known as gliadin. Wheat, rye, and barley contain this protein
and monitoring dietary
and can induce mucosal damage in the gut, causing nonspecific villous
compliance
atrophy of the small intestinal mucosa. Celiac disease does not develop
HLA-DQ2/HLA-DQ8 PCR-based assays High negative predictive
unless a person has alleles that encode for HLA-DQ2 or HLA-DQ8
value; not affected by
proteins, products of two of the human leukocyte antigen genes. This
dietary gluten; found
genetic predisposition is most common in Caucasians of Northern Euro-
in ≈30% of general
pean descent. The prevalence is not clear but is estimated to be as high as population
1% in some countries, and the condition is being increasingly recognized
(Green & Cellier, 2007; Sabatino & Corazza, 2009).
Some patients remain asymptomatic, but an astute clinician may
suspect this disorder when patients present with thin stature, iron defi-
ciency anemia, weight loss, chronic bloating, and/or diarrhea. In severe are currently detected via immunofluorescence of sections of monkey
cases, one may see malabsorption, steatorrhea, and wasting. Associations esophagus or human umbilical cord and are costly, cumbersome, and
have been noted between celiac disease and type 1 diabetes mellitus, Down subject to interobserver interpretive variability.
syndrome, dermatitis herpetiformis, IgA deficiency, autoimmune thyroid Wheat storage protein, gliadin, is available to be used as an antigen in
disease, and other disorders (Barr & Grehan, 1998). Because of enteropa- an EIA. Although serum IgA and IgG AGA levels are frequently elevated
thy associated with the disorder, multiple hematologic and biochemical in untreated celiac disease, these tests are of only moderate sensitivity and
abnormalities may be found in persons with untreated celiac disease, specificity. IgG AGA testing is particularly useful in the 2% of patients
including deficiencies of iron, folate, or vitamin D. The peripheral blood with celiac disease who appear to be IgA deficient. However, these tests
film may reveal nonspecific target cells, siderocytes, crenated red cells, have largely been replaced by EMA. EMA binds to connective tissue sur-
Howell-Jolly bodies, and Heinz bodies. Similarly, small bowel absorptive rounding smooth muscle cells. Most laboratories use sections of human
testing will be abnormal, including oral d-xylose testing and fecal fat umbilical cord. Serum IgA EMA binds to the endomysium to produce a
evaluation. characteristic staining pattern seen on indirect immunofluorescence. The
The gold standard for diagnosis remains histologic examination of antibody is highly sensitive and specific. However, after treatment, the
multiple biopsies of the affected small bowel mucosa for the identification titers fall quickly to undetectable levels (Volta et al., 1995). The epitope
of villous atrophy and crypt hyperplasia. The lesions may be patchy, and against which EMA is directed has been shown to be tissue transglutamin-
sampling errors can occur (Green & Cellier, 2007; Ensari, 2010). Biopsy ase. Use of IgA anti-tTG assays has been shown to be highly sensitive and
is reserved for patients in whom the diagnosis is suspected on the basis of specific for the diagnosis of celiac disease (Dieterich et al., 1998). An EIA
signs or symptoms of the disease, especially in higher-risk populations with for IgA anti-tTG is widely available, less costly, and easier to perform than
supporting serologic findings. These patients must be maintained on a the older immunofluorescence assays for IgA EMA.
gluten-free diet for the rest of their lives to control symptoms and mitigate Antigliadin antibody serology is best avoided in the diagnosis of celiac
cancer risk (Table 22-6). disease because of frequent false positives. A second generation of antiglia-
In current clinical practice, four serologic studies are used to assist in din antibody test based on the potentiation of toxic gliadin peptides by
the diagnosis of celiac disease. These include testing for antibodies to tTG enzymatic activity is used to monitor dietary compliance. These IgA
gliadin (AGA-IgA and AGA-IgG), endomysium (EMA-IgA), reticulin and IgG deamidated gliadin peptide (DGP) assays appear similar to tTG
(ARA-IgA), and transglutaminase (tTG-IgA), all of which are commer- IgA or IgG in diagnostic accuracy, leading to the belief that strongly posi-
cially available. Results of serologic testing for celiac disease must be tive tTG IgA in conjunction with positive DGP serology may be used as
analyzed with caution because this disease is associated with selective IgA confirmation of celiac disease without the need for biopsy histology.
deficiency that will give rise to false-negative serum IgA antibody tests Although IgG endomysium and IgG tTG antibodies may be suitable
(Thomas et al., 2003). Transient IgA deficiency may be seen in patients on for serologic diagnosis of celiac disease, they cannot be used to monitor
phenytoin, penicillamine, or sulfasalazine. Therefore, total IgA levels the response to dietary modification. Endomysium IgA antibodies disap-
should be checked or specific IgG serology performed if there is a high pear following treatment of celiac sprue with a gluten-free diet.
clinical suspicion of celiac disease. The sensitivity and specificity of these The HLA-DQ2 allele is identified in 90% to 95% of patients with
tests are extremely high when compared with a gold standard of flattened celiac disease and HLA-DQ8 in most of the remaining patients. These
small bowel villi responding to dietary changes (Farrell & Kelly, 2001). alleles occur in 30% to 40% of the general population, and the absence of
Endomysial antibodies have the best sensitivity and specificity, but they these alleles is important for its high negative predictive value. Thus the

317
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 presence of HLA-DQ2 and HLA-DQ8 is important for determining
22 LABORATORY DIAGNOSIS OF GASTROINTESTINAL AND PANCREATIC DISORDERS
which family members should be screened with serologic testing (Kauki-
nen et al, 2002). Uncontrolled celiac disease appears to predispose patients
to intestinal carcinomas and lymphomas (Nehra, 1998).
Whipple’s Disease
Whipple’s disease is a rare multisystem disease that often presents with
arthralgias, diarrhea, malabsorption, and weight loss. It is predominantly
found in males, and about 15% of patients do not present with classical
signs and symptoms of this disease (Fenollar et al, 2007). It is caused by
Tropheryma whipplei, a bacillus that does not stain well with Gram stain,
although it is classified with gram-positive bacteria based on 16S rRNA
sequencing (Marth & Raoult, 2003). This disorder can affect the central
nervous system and cause endocarditis. Demonstration of periodic acid–
Schiff (PAS)–positive material in the lamina propria and villous atrophy of
the small intestine are diagnostic. A prothrombin time should be checked
before biopsy because of the frequent occurrence of vitamin K
malabsorption.
T. whipplei has been cultured from the stools of a patient with Whipple’s
disease, using a specific axenic medium and specific techniques (Raoult
et al., 2006). PCR testing of infected tissue or cerebrospinal fluid has been
used to confirm the diagnosis and monitor treatment (von Herbay et al,
1997). Biopsy of the duodenum with 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-intracellulare complex. Thus PCR has gained even more importance
in the management of this entity. Long-term antibiotic therapy with
central nervous system penetration is used to treat patients with Whipple’s
disease (Ramzan et al, 1997; Singer, 1998).
Inflammatory Bowel Disease
Immunologic mechanisms within the colon are involved in the pathogen-
esis of inflammatory bowel disease. The underlying antigenic challenge to
the immunologic response is not clearly understood. Over the past decade,
two antibody tests have become available that assist in the laboratory evalu-
ation of patients with inflammatory bowel disease (Rutgeerts & Vermeire,
2000). The combination of clinical findings, endoscopy, radiologic imaging,
and blood work may help differentiate the subtypes of inflammatory bowel
disease. Perinuclear-antineutrophil cytoplasmic antibody (p-ANCA) and
anti–Saccharomyces cerevisiae antibody (ASCA) can be used to help distin-
guish abdominal pain seen in irritable bowel syndrome from inflammatory
bowel disease and can help distinguish ulcerative colitis from Crohn’s
disease (Sendid et al., 1998; Shanahan, 1994) (Table 22-7). These tests
have limitations, and interpretation requires careful understanding of the
tests. Although 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. Among patients with
inflammatory bowel disease, 65% of those with Crohn’s disease will have
ASCA, whereas only 20% of patients with ulcerative colitis will have sig-
nificant titers. Given their low sensitivity and specificity, use of these tests
should be dependent on 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.
GASTROINTESTINAL TUMORS
Pancreatic Adenocarcinoma
Ductal adenocarcinomas of the exocrine pancreas are malignant epithelial
tumors composed of mucin-producing glandular structures. They consti-
TABLE 22-7
Markers for Inflammatory Bowel Disease
FREQUENCY (%)
p-ANCA ASCA
Irritable bowel syndrome (normal patients) <5 <5
Ulcerative colitis 70 15
Crohn’s disease 20 65
ASCA, Anti–Saccharomyces cerevisiae antibody; p-ANCA, perinuclear antineutrophil
cytoplasmic antibody.
318
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tute 85% of all pancreatic tumors and are the fourth most frequent cause
of death from cancer. They originate in small ducts and progress from
noninvasive pancreatic intraepithelial lesions to invasive cancers.
Precursor lesions for pancreatic adenocarcinoma include intraepithelial
pancreatic mucinous neoplasms (IPMNs) and pancreatic intraepithelial
neoplasia (PanIN) (Hruban et al, 2005; Distler et al, 2014). PanIN is the
most common of these precursor lesions. First described in 1998, PanIN
are a series of histological descriptions of lesions initially found in close
proximity to invasive adenocarcinoma (Brat et al, 1998). This series traces
the progression and development of exocrine pancreatic ductal adenocar-
cinoma from normal pancreatic ductal epithelium through PanIN grades
1-A, 1-B, 2, 3, and then to infiltrating pancreatic ductal adenocarcinoma
(Recavarren et al, 2011). Histologically, this is manifested by changes from
low-columnar or cuboidal epithelium with normal nuclei to papillary and
micropapillary architecture with nuclear irregularities, abnormal mitoses,
and luminal necrosis. Ki-67 staining also increases with PanIN grade
(Klein et al, 2002).
Little change in their incidence or survival rate has been seen in the
past 20 years; 5-year survival is less than 5%. A slight male predominance
has been noted, especially in younger age groups. This is a disease mostly
of late life; 80% of patients are 60 to 80 years of age, although cases in the
third decade are not uncommon. This cancer is more common in African
Americans and Native Americans. A two- to threefold greater risk has been
reported among cigarette smokers. Patients with chronic pancreatitis and
diabetes mellitus are at increased risk. High intake of meat and fat appears
to be a risk factor, as well as intake of nitrosamines and polycyclic hydro-
carbons. The neoplastic mechanism remains obscure.
Clinical presentation depends largely on the location of the tumor:
60% arise in the head, often with painless jaundice; 10% in the body and
10% in the tail are often silent until quite large or widely disseminated;
20% are diffuse at presentation. Familial clusters have been reported, but
no distinct genetic abnormality has been described.
The ampullary region is invaded by carcinomas of the head of the
pancreas, causing bile duct obstruction. Acute painless dilation of the
gallbladder (Courvoisier’s sign) and jaundice occur as the result of common
bile duct obstruction by tumor in most cases. Carcinomas of the body and
tail do not impinge on the biliary tract. Migratory thrombophlebitis
(Trousseau’s sign) develops in 10% of patients with pancreatic cancer,
especially those with tumors of the body and tail, probably because of
platelet-aggregating factors and procoagulants from the tumor. Anorexia,
conspicuous weight loss, and gnawing epigastric pain that radiates to the
back are frequent clinical features.
Early diagnosis of pancreatic carcinoma is unusual. These tumors are
typically silent until they impinge on a vital structure or on the posterior
wall of the abdomen and then cause severe pain. Pain is usually the first
symptom. Less than 20% of the tumors are resectable at the time of
presentation.
No morphologic difference is known between carcinomas of the head
of the pancreas and those of the body and tail. These tumors are usually
moderately or poorly differentiated mucin-producing adenocarcinomas.
They have abortive tubular structures and dense stromal fibrosis and
aggressive infiltrative growth, often with perineural invasion. Tumor cells
are cuboidal or columnar and are usually poorly differentiated. The adja-
cent pancreatic parenchyma often demonstrates foci of ductal dysplasia and
intraductal tumor growth.
Point mutations in KRAS and in p16 are found in more than 90% of
cases, and mutations in p53 cause inactivation in 50% to 70% of cases.
Both carcinoembryonic antigen (CEA) and CA 19-9 (tumor marker for
pancreatic cancer) are elevated in some pancreatic adenocarcinomas.
According to the most recent guidelines from the American Society of
Clinical Oncology, clinicians are advised to use these tumor markers very
cautiously (Locker et al, 2006). CA 19-9 is not recommended as a marker
for screening for pancreatic cancer, nor for determination of operability.
Routine use of CA 19-9 is not recommended for monitoring treatment
response. However, it can be measured at the initiation of treatment and
then every 1 to 3 months thereafter. An elevation should prompt investiga-
tion for advanced metastatic disease. However, their increases can be used
to monitor for recurrence (Table 22-8).
Neuroendocrine Tumors
Gastroenteropathic neuroendocrine tumors (GEP-NET) are relatively
rare neoplasms with varied clinical manifestations. Abdominal pain and
diarrhea are two of the more common clinical symptoms. Carcinoid tumor
was the term chosen, more than 100 years ago, to distinguish these tumors
from carcinomas of the GI tract. Because these tumors are rare, they were
lumped together. Recently, with greater knowledge of their development
TABLE 22-8
Pancreatic Cancer–Associated Genetic Syndromes
Genetic Syndrome Gene(s)
Hereditary breast and ovarian cancer BRCA2
syndrome BRCA1
Peutz-Jeghers syndrome STK11/LKB1
Familial adenomatous polyposis APC
Familial atypical multiple melanoma CDKN2A
Hereditary nonpolyposis colorectal cancer Mismatch repair genes
syndrome
Hereditary pancreatitis PRSS1
SPINK1
Familial pancreatic cancer Unknown
From Shi C, Hruban RH, Klein AP: Familial pancreatic cancer, Arch Pathol Lab Med 133:365–374, 2009.
and biological behavior, the World Health Organization classification of
GEP-NET was published in 2000. Specific markers of normal and neo-
plastic neuroendocrine cells are the hormones that occur in the GEP
system. The organ in which a certain hormone-producing tumor develops
appears to make a difference (e.g., the differing malignant potential of
duodenal compared with pancreatic gastrinoma) (Klöppel et al., 2004).
These tumors exhibit amine precursor uptake and decarboxylation (APUD)
and are therefore known as APUDomas. Cells of the GEP-NET belong
to the system of disseminated neuroendocrine cells called APUD cells.
These cells are scattered throughout the mucosa of the GI tract and the
islets of the pancreas. Insulinomas and gastrinomas are the most common
pancreatic endocrine tumors. Glucagonomas are rare and make up 2% to
5% of series. Other, even rarer types, such as somatostatinomas and
VIPomas, have been identified (Perr & Vinik, 1996). Chemical measure-
ment of the secretagogues of these tumors generally suggests the diagnosis
in the right clinical setting.
Localizing these tumors can be challenging. Ultrasonography, CT
scanning, MRI, endoscopy, angiography, and octreotide scanning all have
significant technical limitations. With the exception of ultrasonography
and MRI, potential morbidity can result from the testing as well. Soma-
tostatin receptor scintigraphy (SRS) is widely used in the evaluation of
GEP-NETs (Warner & O’dorisio, 2002) and is a useful tool in the arma-
mentarium of the diagnostician. The underlying basis of this imaging
modality is that NETs have a greater density of SSTRs on the cell surface.
As such, this diagnostic modality is value-added in utility because it can
identify those patients who would be susceptible to therapy with SST
analogs. The SST analogs used in SRS typically have high affinity for
receptor subtypes SSTR2 and SSTR5, low affinity for SSTR3, and no
affinity for SSTR1 and SSTR4 (Sclafani et al, 2011). These tumors are
often small, frequently are 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 used to distinguish the types (Perry
& Vinik, 1996). A recent retrospective analysis comparing SRS positivity
with IHC analysis of SSTR2 and SSTR5 expression discovered a high
concordance rate (70%) but a nonnegligible (50%) number of SRS-
negative tumors that stained positively for SSTR2. This subpopulation of
patients is important because they may be susceptible to radiopharamaceu-
ticals (Sclafani et al, 2011). These findings highlight the fact that, as with
all neoplastic lesions, biopsy is essential to confirm the diagnosis.
GEP-NETs make up about 10% of pancreatic neoplasms and are clas-
sified as functioning or nonfunctioning tumors. These tumors are single
or multiple, and benign or malignant. They are often solitary, circum-
scribed lesions, amenable to surgical resection. Immunohistochemistry is
needed for demonstration of endocrine activity. Hormone assays are useful
for the diagnosis of pancreatic endocrine tumors (Modlin & Tang, 1997;
Eriksson et al, 2000; Barakat et al, 2004).
Cytologic criteria are unreliable in diagnosis. The diagnosis of malig-
nancy requires demonstration of metastases and vascular or tissue invasion.
These tumors are often slow growing. Functioning tumors cause recogniz-
able clinical syndromes due to excess peptide hormone secretion. Non-
functioning tumors have a worse prognosis and typically present with
metastases or local symptoms.
GI endocrine tumors may be part of the syndrome of MEN 1 with
parathyroid disease and pituitary and pancreatic tumors. Islet cell tumors
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Pancreatic Cancer Risk Increase Histopathologic Features
3.5- to 10-fold Ductal adenocarcinoma
2-fold
PART 2
132-fold Intraductal papillary mucinous neoplasm
Up to 4-fold Ductal adenocarcinoma, intraductal papillary
neoplasm, pancreatoblastoma
13- to 22-fold Ductal adenocarcinoma
Increased Medullary carcinoma
53-fold Ductal adenocarcinoma
9- to 32-fold
may secrete various normal hormones that are not ordinarily produced in
the pancreas, including adrenocorticotropic hormone (ACTH), parathy-
roid hormone, calcitonin, and vasopressin.
Insulinoma
Insulinomas are derived from β cells and produce insulin that induces
clinically significant hypoglycemia. Factitious disease due to exogenous
insulin is part of the differential diagnosis. Insulinomas are usually found
in the pancreas and are typically small and solitary and often contain
amyloid; 10% are malignant. Diagnosis is made by demonstrating elevated
plasma insulin associated with hypoglycemia and measurement of
C-peptide. C-peptide is equimolar to insulin and is needed to exclude
increases in insulin from exogenous sources.
Gastrinoma
Gastrinomas are gastrin-secreting non–β cell pancreatic tumors that cause
a syndrome of intractable peptic ulcer disease and gastric acid hypersecre-
tion. Gastrin binds cholecystokinin-B receptors on parietal cells of the
stomach to stimulate acid secretion and also has a trophic effect on gastric
mucosal parietal and enterochromaffin-like cells, causing mucosal hyper-
trophy (McIntyre et al, 2002). The mean age at onset is 50 years, with
male preponderance. At the time of diagnosis, 70% to 90% of the tumors
are malignant with metastases or local invasion. Up to 60% of gastrinomas
are associated with MEN 1, and about one-half of MEN 1 patients develop
gastrinomas. Most gastrinomas arise in the gastrinoma triangle: duode-
num, pancreatic head, and hepatoduodenal ligament.
Gastrin is not usually detected in the adult pancreas but is present in
the fetal pancreas. Gastrinomas induce peptic ulcer disease refractory to
treatment, esophagitis, and diarrhea. High levels of plasma gastrin are
noted with high basal gastric acid secretion. Increased gastrin levels
are also caused by gastric atrophy and by acid antisecretory drugs such as
H2 blockers. Some gastrinoma patients are hypercalcemic because of
PTH-related protein production or because patients with MEN 1 may
develop hyperparathyroidism. About 50% of patients with gastrinomas
have metastases, mainly to the liver and nodes. The 5-year survival is
20% with liver metastases, and without metastases, 80% will survive for
5 years.
Gastrinomas can cause high-volume diarrhea through an increased rate
of gastric acid secretion, resulting in a volume that cannot be fully reab-
sorbed. The acid exceeds the neutralizing capacity of the pancreatic bicar-
bonate and inactivates the pancreatic digestive enzymes; this interferes
with the emulsification of fat by bile salts, resulting in steatorrhea. A secre-
tory component to the diarrhea has also been observed. Diagnosis is
established by measuring serum gastrin levels; a level greater than 1000
pg/mL is virtually diagnostic. The secretin stimulation test can differenti-
ate patients with gastrinomas from those with the many other causes of
hypergastrinemia; this test should be performed in every patient suspected
to have the ZE syndrome who has a nondiagnostic fasting serum gastrin
concentration (Berna et al, 2006).
Glucagonoma
This is a rare tumor, mostly of pancreatic origin, that secretes glucagon (α
cells), which induces glycogenolysis and gluconeogenesis in the liver and
raises blood glucose levels. It causes a syndrome of mild diabetes,
319
 necrotizing migratory erythematous rash, anemia, and venous thrombosis,
22 LABORATORY DIAGNOSIS OF GASTROINTESTINAL AND PANCREATIC DISORDERS
and it is associated with severe infection. Onset occurs at 40 to 70 years
of age, with slight female predominance (Bloom & Polak, 1987). These
are large, locally invasive tumors that histologically resemble trabecular
and solid patterns of insulinomas; two thirds are malignant. Plasma gluca-
gon levels are elevated up to 30 times above normal.
Somatostatinoma
These usually solitary tumors are derived from δ cells that produce soma-
tostatin, which inhibits pituitary release of growth hormone and secretion
by α, β, and δ cells. This peptide also regulates glucose homeostasis and
causes a syndrome of mild diabetes, gallstones, steatorrhea, and hypochlor-
hydria, due to the inhibitory effect of somatostatin on the other islet and
gastrointestinal neuroendocrine cells (Krejs et al., 1979). Somatostatin
measurement in a fasting serum specimen may aid diagnosis; patients with
somatostatinomas may have 1000-fold elevated concentrations. These
tumors may also secrete calcitonin or ACTH, and most are malignant with
metastases at diagnosis.
VIPoma
These tumors are presented in the previous section on diarrhea and
malabsorption.
Carcinoid Syndrome
Pancreatic carcinoids are rare malignant neoplasms resembling intestinal
carcinoids. They synthesize serotonin and motilin. When confined to the
pancreas, these tumors may induce an atypical carcinoid syndrome that
consists of facial flush, hypotension, periorbital edema, and lacrimation.
Cases with liver metastases cause a classical carcinoid syndrome. Diagnosis
is aided by demonstration of increased 5-hydroxyindoleacetic acid
(5-HIAA) levels in 24-hour urine specimens.
Amyloidosis
GI amyloidosis is mainly of the reactive type (secondary or amyloid-
associated [AA] amyloidosis) and results from mucosal or neuromuscular
infiltration. It may result in GI bleeding from vascular friability. The
patient may also have diarrhea from a combination of altered intestinal
motility, bacterial overgrowth, and protein-losing enteropathy. Liver
involvement can be seen in primary (amyloid light chain) or AA amyloi-
dosis. Common symptoms include involuntary weight loss, fatigue, and
abdominal pain. Common physical findings consist of hepatomegaly,
ascites, purpura, and splenomegaly. The most common laboratory abnor-
mality is an elevated alkaline phosphatase. The diagnosis of multiple
myeloma should be confirmed by tissue biopsy of duodenal or colorectal
mucosa. Although GI complications can result in significant morbidity,
they usually are not the cause of death, which is most often due to renal
failure, restrictive cardiomyopathy, or ischemic heart disease (Ebert &
Nagar, 2008).
GASTROINTESTINAL BLEEDING
Throughout most of the GI tract, the lumen of the gut is separated from
the capillary blood supply by only a single layer of epithelial cells. Thus
even minor injury to the epithelial lining may result in GI bleeding. Occult
GI bleeding is bleeding unknown to the patient. Iron deficiency anemia
should be considered to be the result of GI bleeding until excluded
(Rockey, 2010).
Hemoglobin and hematocrit are usually low, and their levels may have
some relation to blood loss. A low mean cell volume (MCV) may indicate
that the patient is iron deficient and that chronic blood loss has occurred.
An elevated MCV may indicate a folate or vitamin B12 deficiency with
macrocytosis and raises the possibility of ethanol abuse, chronic liver
disease, gastric cancer in association with pernicious anemia, or regional
enteritis involving the terminal ileum.
If a clotting defect exists, prompt correction of the deficiency is crucial.
Extensive transfusion dilutes platelets and clotting factors, particularly
factors V and VII. Also, a high proportion of patients who bleed while
taking therapeutic anticoagulants do so from a clinically significant lesion.
It is important to evaluate these patients for a GI pathologic condition and
to correct their clotting status. Leukocytosis may accompany acute GI
bleeding, but it is usually not in excess of 15,000 white blood cells/mm3.
Leukocytosis should not be attributed to acute blood loss without comple-
tion of a search for sources of infection.
Elevated BUN in a patient whose BUN was recently normal, or whose
serum creatinine concentration is normal, suggests an upper GI bleed. The
BUN rise may be due to the hypovolemia of acute blood loss, but digestion
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of blood proteins in the small intestine and absorption of nitrogenous
products may be the cause.
Fecal Occult Blood Testing
The fecal occult blood test (FOBT) depends on the peroxidase activity of
hemoglobin. Reagents used include guaiac (Hemoccult), benzidine (mar-
keting restricted by federal regulation because of its carcinogenicity),
orthotoluidine (Hematest), and ortho-dianisidine. The reagents differ in
sensitivity, although all are associated with frequent false-positive and
false-negative results, mostly due to the specimen tested. Specimens col-
lected by digital rectal examination have a high frequency of false positives
caused by injury to the rectal mucosa.
The normal person loses 0.5 to 1.5 mL of blood into the GI tract daily
in association with the normal shedding of epithelial cells. Commercial
tests are designed to turn positive with blood loss greater than 5 to 10 mL
per day. This corresponds to 5 to 10 mg of hemoglobin/gram of feces,
assuming a blood hemoglobin of 15 g/dL and an average daily 150-g stool.
In addition to sample handling and testing, diet is very important in
compromising these tests. The high rate of false positives is due to the lack
of specificity of the reagents and the presence of peroxidase activity in
other fecal constituents, as well as the sensitivity of the different chromo-
gens to peroxidase activity. The myoglobin and hemoglobin of ingested
meat and fish have peroxidase activity that may falsely indicate the presence
of occult blood. Bacteria in the bowel, as well as ingested vegetables and
fruits, such as horseradish, turnips, bananas, black grapes, pears, and
plums, have peroxidase and can falsely elevate fecal peroxidase activity.
False-negative tests occur in the presence of large amounts of vitamin C
and other antioxidants.
Patient preparation and specimen collection are important in ensuring
valid test results. For 72 hours prior, patients should be on a diet free of
exogenous peroxide activity (meat, fish, turnips, horseradish), GI irritants
(aspirin, NSAIDs), and vitamin C. Variations in testing technique produce
errors due to stool consistency, the application of large aliquots of stool,
and, particularly, inaccurate timing of the reaction.
Immunochemical tests for hemoglobin are more sensitive and specific
and yield fewer false negatives. They are not affected by diet, animal
hemoglobin, or human myoglobin. Dietary peroxidases do not affect
immunochemical test results. They are useful only for screening blood of
the lower intestinal tract and are insensitive to upper GI bleeding because
globin is destroyed in the small intestine.
Colon cancer is a leading cause of cancer-related deaths in the United
States, 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 FOBT to detect these cancers at an earlier stage, potentially resulting
in decreased mortality. Because of the generally favorable clinical biology
of these tumors when detected earlier (with an 80% to 90% survival rate
with local confined disease) (Helm et al, 2003) and the relatively inexpen-
sive, noninvasive nature of FOBT, this may serve as a useful screening
technique. Several professional organizations recommend annual or bien-
nial FOBT, but universally accepted screening protocols have not been
established. The American Cancer Society guidelines for colorectal cancer
screening, the most widely utilized, recommend annual FOBT and flexible
sigmoidoscopy every 3 to 5 years, beginning at age 50, in asymptomatic,
average-risk individuals (Marshall, 1996). Limitations of this testing
include the high numbers of false-positive and false-negative results. The
sensitivity of FOBT has been estimated at between 30% and 50%. The
true sensitivity of FOBT is difficult to determine because individuals who
test negative do not undergo further colonoscopic evaluation to determine
whether the FOBT is a true negative. Only approximately 5% to 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.
Occult blood can be detected by chemical (guaiac), hemoporphyrin, or
immunologic methods. Occult blood may arise anywhere along the intes-
tinal 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 fis-
sures, inflammatory bowel disease, peptic ulcer disease, or angiodysplasias
of the colon. Laboratory diagnosis of the presence of fecal occult blood
generally involves a guaiac-smear test (Hemoccult, Seracult, Coloscreen),
the most commonly used method at present. Guaiac is a naturally occur-
ring phenolic compound that is oxidized to quinone by hydrogen peroxi-
dase, resulting in a detectable color change. These tests detect the
pseudoperoxidase activity of heme as intact hemoglobin or as free heme
(Allison et al, 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 more
than 5 to 10 mL/day of blood in the stool results in a positive guaiac smear.
Stool specimens should be received from three consecutive stools. Two
slides should be prepared for each stool sample. Slides should not be
rehydrated and should be developed within 7 days of collection. To ensure
quality testing, some medical institutions now require that stool samples
be sent to the laboratory for guaiac testing, rather than having residents
or nurses perform tests on the ward. In addition, the test must be per-
formed under appropriate conditions that serve to limit its sensitivity.
Factors that can cause inaccuracies in the results include the presence of
bleeding gums, for example, or ingestion of large amounts of red meat
before testing. In addition, patients may be using certain medications that
will influence the results of FOBT. For example, drugs that can cause GI
irritation and subsequent bleeding, such as anticoagulants, aspirin,
NSAIDs, or colchicines, may lead to false-positive results. Other drugs
that have been implicated include reserpine and oxidizing drugs such as
iodine. Ingestion of large amounts of vitamin C can cause false-negative
results. Patients should avoid such medications or food products before
FOBT. A positive FOBT prompts further evaluation for a GI lesion.
Additional evaluations usually include 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 neo-
plasia. If a patient is presenting with signs and symptoms suggestive of
colon cancer, further evaluation is warranted, even in the presence of nega-
tive FOBT.
Fecal DNA testing is a promising technique for colorectal cancer
screening. This test involves the collection of a single stool specimen,
which is then screened for DNA markers. PCR is used to amplify the fecal
DNA. These tests are useful because DNA is shed continuously and
remains stable in stool, and minute amounts are detectable. Clinical studies
have shown that fecal DNA testing has high specificity and sensitivity for
cancer detection, considerably more than chemical guaiac testing. Several
markers have been investigated; however, further evaluation and improved
specificity and sensitivity are needed for their widespread use (Itzkowitz
et al, 2008; Lieberman, 2009; Mandel, 2008).
Blood in Newborn Feces (Apt Test for
Swallowed Blood)
When blood is found in the GI tract or stool of neonates, usually on the
second or third day of life, a determination must be made of whether it is
swallowed blood of maternal origin or is secondary to disease in the
newborn (Guritzky & Rudnitsky, 1996). The Apt test is a qualitative test
that is used to make this determination in grossly bloody stools or vomitus
(hematemesis) from a newborn. Infants may ingest maternal blood during
birth or from a fissured or bleeding nipple. The sample first is mixed with
water and then is centrifuged. The supernatant, which should be pink, is
then mixed with 1% sodium hydroxide in a 5 : 1 ratio. If the blood is of
maternal origin, the mixture turns yellow-brown after several minutes.
Fetal blood remains pink because fetal hemoglobin is more resistant to
alkali denaturation than adult hemoglobin. The test has relatively low
sensitivity, and the result must be interpreted with caution (McRury &
Barry, 1994).
MARKERS FOR GASTROINTESTINAL AND
PANCREATIC TUMORS
Tumor markers are substances produced by tumor cells or other body cells
in response to neoplastic or certain nonneoplastic conditions; they are
found in tissues or body fluids. Different tumor markers are found in dif-
ferent types of cancers, and the same tumor marker may be found in more
than one type of cancer. Tumor markers are not expressed in all people
with cancer, and some are expressed in patients with benign conditions.
Identification or measurement of tumor markers is used in the detection,
diagnosis, or management of some types of cancer. Although an abnormal
tumor marker level may suggest cancer or limited efficacy of therapy, this
alone is not enough to establish diagnosis and is usually combined with
other tests, such as biopsy (Bigbee & Herberman, 2003; Chan et al, 2006;
Locker et al, 2006).
Enzymes
Enzymes were one of the first groups of tumor markers identified, and
their elevated levels were linked with cancer. Most changes in enzyme
levels are not specific or sensitive enough to be used to identify the cancer
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type or the specific organ involved. Lactate dehydrogenase, a glycolytic
pathway enzyme, is released as a result of cell damage, and its elevation in
malignancy is nonspecific (Schwartz, 1982). Neuron-specific enolase
(NSE) is found in neural tissue and in cells of the diffuse neuroendocrine
system. NSE is found in association with tumors of neuroendocrine origin,
and its serum levels are of prognostic significance (Zeltzer et al, 1986).
PART 2
Urokinase-plasminogen activator, a serine protease system, is a prognostic
marker in colorectal cancer (Duffy et al, 1999). Tumor-associated trypsin
inhibitor (TATI) is identical to pancreatic secretory trypsin inhibitor, also
known as the kazal inhibitor. It is strongly expressed in pancreatic cells and
in low concentrations in normal GI and urogenital tissues. TATI is a useful
marker for GI, urologic, and pancreatic cancers (Stenman, 2002).
Hormones
Hormones are normally produced in endocrine organs but may also be
produced in nonendocrine tissue. They are assayed by EIA using mono-
clonal antibodies or by RIA. Multiple endocrine neoplasia (MEN 2A and
MEN 2B) syndromes synthesize several polypeptide hormones such as
ACTH, calcitonin, gastrin, glucagon, and insulin. Vasoactive intestinal
peptide is produced by some endocrine tumors (Perry, 1996).
ACTH may be the result of pituitary or ectopic production. Elevated
ACTH levels have been reported from pancreatic, gastric, and colonic
carcinomas. Patients with serotonin-producing carcinoid tumors often
have marked tenfold increases in urinary 5-HIAA excretion. Ingestion of
serotonin-rich foods, such as bananas, chocolate, plums, or walnuts, or
medications containing guaifenesin may produce false-positive elevations
(Kema et al, 1992).
Other Protein Markers
Oncofetal antigens are proteins produced in fetal life that decrease to low
levels or disappear after birth but reappear in cancer patients. The discov-
eries of α-fetoprotein (AFP) and carcinoembryonic antigen (CEA) in the
1960s had a profound effect on the use of tumor markers. AFP is a marker
for hepatocellular and germ cell (nonseminomatous) carcinomas. CEA, a
glycoprotein, is a marker for colorectal, GI, lung, and breast carcinomas.
CEA testing may be useful as an adjunct to clinical staging (Sikorska et al,
1992). Amplification of Her2/neu, a tyrosine kinase receptor of the epi-
dermal growth factor family, is seen in GI, breast, and ovarian tumors.
bcl-2, a protein known to inhibit apoptosis, appears early in colorectal
cancer. C-peptide is a marker for insulin production.
Blood Group Antigens
Some of the blood group carbohydrates are tumor markers with elevated
levels in specific types of cancer. CA 19-9, a sialylated Lewis antigen, is
elevated in patients with colorectal and pancreatic carcinomas (Lamerz,
1992). Its levels are elevated in benign conditions such as Mirizzi’s syn-
drome (Robertson & Davidson, 2007). It is absent in individuals with
pancreatic tumors who are Lewis negative. Another related Lewis antigen,
C50, is used as a pancreatic tumor marker but is considered less sensitive
than CA 19-9. CA 242 and CA 72-4 are markers for GI and pancreatic
tumors, but CA 72-4 is considered of greater prognostic value in pancreatic
cancer (Louhimo et al, 2004).
Genetic Markers
Two classes of genes are implicated in cancer: cell activator genes and cell
suppressor genes. Most oncogenes code for proteins that activate cell
proliferation. Mutated K-ras is present in 95% of pancreatic cancers, in
40% of colon cancers, and in lower percentages of other tumors (Chan et
al, 2006). Mutations of ras oncogenes have been detected in the stools of
9 of 15 patients with curable colorectal tumors (Sidransky et al, 1992).
Any genetic testing requires that the physician clearly understand how
to interpret the results and how the results may affect patient management;
patients and their family members must be counseled appropriately (Grody
et al, 2001; LeGrys et al, 2007).
Tumor Suppressor Genes
Oncogenicity derives from loss of the gene, rather than its activation, as
with the p53 gene. Three-fourths of colon carcinomas show deletion in
one p53 allele and a point mutation in the other allele.
STOOL COLLECTION AND EXAMINATION
COLLECTION
Uninstructed patients sometimes exhibit considerable ingenuity in collect-
ing stool specimens, but a few simple instructions are likely to produce
321
 more satisfactory specimens. A scoured, well-rinsed bedpan is a convenient MICROSCOPIC EXAMINATION
22 LABORATORY DIAGNOSIS OF GASTROINTESTINAL AND PANCREATIC DISORDERS

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 reason-
ably convenient instruments for transferring the stool from bedpan to
transport vessel, for which plastic, cardboard, and glass containers are
available. We prefer to use two 2-ounce ointment jars with screw caps for
small stool samples because they are odor free, leakproof, 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 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
specimen collection at home is by no means simple and easy unless the
patient has been instructed properly.
MACROSCOPIC EXAMINATION
The quantity, form, consistency, and color of the stool should be noted.
Normally, 100 to 200 g of stool is passed per day. Diarrheic 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).
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 GI 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.
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 exces-
sive 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 3 to 4 L in 24 hours. They frequently develop severe dehy-
dration 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 diag-
nosis. No inflammatory exudate is seen in the watery stools of patients
with viral gastroenteritis.
Fat
The simplest technique is microscopic examination using one of the Sudan
stains. This procedure has been widely employed for screening because of
its simplicity. In our experience, results have correlated well with quantita-
tive 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 is 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 applied. Under these conditions, fatty acids are
present as lightly stained flakes or as needle-like 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 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 by 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 has been performed, the presence of up to 100 stained
droplets per high-power field is considered normal. Patients with steator-
rhea of pancreatic origin are likely to have greater increases in fatty acids
and soaps.
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
solution of eosin in 10% ethanol; it is then allowed to stain for 3 minutes
and is examined for muscle fibers. The entire area under the coverslip is
examined, and only rectangular fibers with clearly evident cross-striations
are counted. More than 10 fibers per high-power field suggests maldiges-
tion and/or hypermotility. It appears that examination for meat fibers
correlates well with chemical determination of fat excretion (Moore et al,
1971).
Leukocytes
A small fleck of mucus or a drop of liquid stool is placed on a glass micro-
scopic slide with a wooden applicator stick. Two drops of methylene blue
are added and mixed thoroughly and carefully. A coverslip is placed on the
mixture, which is allowed to stand for 2 to 3 minutes for good nuclear
staining. Using low-power scanning, rough quantitative counts are made
by approximating the average numbers 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.
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