98
`
`Definition
`
`Tests for fecal occult blood detect blood in the stool that is
`not visible on gross inspection, usually less than 50 mg of
`hemoglobin per gram of stool . Normal adults usually show
`less than 2 to 3 mg/gm . Increased amounts are associated
`with a variety of benign and malignant gastrointestinal dis-
`eases, especially colonic neoplasms, and the tests are most
`often used in screening patients for such lesions .
`
`Technique
`
`Benzidine-based tests (e .g ., Hematest) have been virtually
`eliminated from use because of their excessive sensitivity,
`which results in a high frequency of false-positive reactions,
`as well as the carcinogenicity of the reagent .
`The current worldwide standard is the Hemoccult slide
`test (Smith-Kline Diagnostics), a guaiac-based test, in which
`one side of a guaiac-impregnated paper is smeared with
`stool and tested by addition of a few drops of developer
`solution (stabilized peroxide reagent) to the opposite side
`of the paper . In screening programs, the patient recovers
`stool from the toilet bowl using a wooden applicator, smears
`a small portion onto two windows of the card, and closes
`the cover. This is done on three successive days, and the
`cards are mailed for testing, as there is little degradation of
`reactive hemes in the dry, smeared specimens over a period
`of I week (fewer than 15% of samples) .
`Appearance of unequivocal blue color, of any intensity,
`within 30 seconds, is considered a positive test ; greenish
`colors, caused by oxidation of fecal bilirubin to biliverdin,
`should not be read as positive . Positive and negative control
`spots are included in the card to be sure that the reagents
`are reactive with substrate and not reactive in the absence
`of substrate . It has been found that diffusion of the hematin
`from the smeared stool into the paper is important, so that
`the reaction can be enhanced by wetting the smeared side
`of the paper with a few drops of water several minutes
`before adding the developer . However, such rehydration
`of specimens also increases the rate of false-positive reac-
`tions from plant peroxidases and meat hemes in the diet,
`and is not recommended unless there is rigid adherence to
`a restricted diet, outlined in Table 98 .1 .
`A modification of the Hemoccult test has been marketed
`(ColoScreen, Helena Laboratories) in which a selective in-
`hibitor of nonheme vegetable peroxidases has been added
`in hopes of diminishing the false-positive reactions from
`this source . Another group of tests is based on the immu-
`nologic detection of human hemoglobin or globin in the
`stool, but the tests developed thus far have been unreliable
`because of over- or undersensitivity and poor reproduci-
`bility . Both approaches might improve specificity, but nei-
`ther solves the problem of major and variable degradation
`
`LABORATORY
`
`Tests for Fecal Occult Blood
`
`J . DONALD OSTROW
`
`of hematin or globin in the intestinal lumen, a problem
`shared by Hemoccult .
`A new, promising, and almost quantitative approach
`(HemoQuant, Bioscience Labs) is to measure fecal hemes
`by the specific fluorescence of their porphyrin derivatives,
`after extraction of the porphyrins from the specimen to
`remove interfering substances. A variable (1 to 98%) pro-
`portion of unabsorbed hemoglobin/hematin is converted to
`porphyrins by intestinal bacteria (so-called intestinally con-
`verted fraction, or ICF) ; this fraction can be extracted into
`an organic solvent in the presence of citric acid . The re-
`maining intact hemes can be chemically converted to por-
`phyrins by heating in a test tube in the presence of oxalic
`acid and ferrous sulfate, and then extracted, along with the
`ICF, to yield total converted porphyrins as a measure of
`total unabsorbed hemes .
`HemoQuant is little affected by the variable degradation
`of hemes in the intestinal lumen or by the degree of hy-
`dration of the stool ; is insensitive to nonheme peroxidases,
`oral iron, and cimetidine ; and is almost quantitative . It is
`therefore highly likely to become the future test of choice,
`if it can be simplified and its cost reduced . The early hope,
`that the ICF would help to distinguish upper from lower
`gastrointestinal blood loss, has not been realized .
`Neither HemoQuant, any of the variants of the other
`new tests, nor the numerous commercial imitations of Hem-
`occult, have undergone the extensive evaluation in large
`screening programs that has documented the efficacy and
`identified the limitations in interpretation of the Hemoccult
`test . Moreover, the appropriate upper normal limits for
`fecal hemes have not been established for these new tests .
`For the present, therefore, Hemoccult is the only validated
`test appropriate for use in detection of fecal occult blood .
`
`Table 98.1
`Strict Low-peroxidase Diet for Occult Blood Screening
`
`Carrot
`Cauliflower
`Cucumber
`Grapefruit
`Green beans
`
`Mushrooms
`Parsnip
`Pumpkin
`Turnips
`Zucchini
`
`Do not ingest
`Rare red meat
`Bloodwurst
`Horseradish
`Broccoli
`Cabbage
`Cantaloupe
`Nonsteroidal anti-inflammatory agents
`Ascorbic acid
`Aspirin
`O .K . to ingest
`Well-cooked fish and chicken
`Cooked fruits and vegetables
`Canned tuna fish
`Strawberries
`
`Bran cereals
`Peanuts
`Popcorn
`Oranges (1 a day)
`
`°Less than 50 g of the foods listed have peroxidase activity equivalent to
`1 .0 ml blood (1 mg hemoglobin per gram of stool) .
`
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`490
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`VI . THE GASTROINTESTINAL SYSTEM
`
`quinone . False-positive reactions are given by other mate-
`rials that can catalyze the peroxidase reaction (Table 98 .1),
`including dietary peroxidases in vegetable and fruits, hemo-
`globin and myoglobin in red meats (especially if rare or
`raw) ; oral iron, once thought to be a problem, has been
`shown clearly not to affect the Hemoccult test . Cimetidine
`also gives false-positive reactions, but only in aspirates of
`gastric and duodenal juice, because most of this drug is
`absorbed before reaching the colon . False-negative tests oc-
`cur if high concentrations of reducing agents are present
`in the diet, especially ascorbic acid in citrus fruits and juices
`in doses of 750 mg or more daily . Drugs such as aspirin
`and nonsteroidal anti-inflammatory agents, which often
`cause erosions and bleeding of otherwise normal gastric
`mucosa, also lead to positive tests that may falsely suggest
`the presence of an intrinsic lesion . It is therefore important
`that intake of these interfering substances be eliminated for
`3 days before and 3 days during collection of the stools to
`be tested .
`
`Clinical Significance
`
`Colonic neoplasms and other gastrointestinal lesions bleed
`intermittently and in variable amounts from day to day .
`Therefore, if any of the six windows (2 per stool x 3 stools)
`yields a blue reaction, the patient should undergo more
`intensive diagnostic study to determine the source of blood
`loss . In Western nations, Hemoccult screening of truly
`asymptomatic populations aged 45 years or more has yielded
`3 to 5% positive tests on an unrestricted diet and less than
`2% positive tests on the restricted diet (Table 98 .1) . On the
`latter diet, 10 to 15% of those with positive tests will be
`found to have carcinoma and 20 to 30% to have benign
`neoplasms (e .g ., polyps), usually in the colon . Another 30
`to 60% of positive subjects will reveal other gastrointestinal
`lesions that might be responsible for occult bleeding (e .g .,
`hemorrhoids, diverticulosis, inflammatory bowel disease,
`peptic ulcer, gastritis, esophagitis, and esophageal varices) .
`Less than 15%, and usually less than 10%, of positive sub-
`jects will reveal no lesions, and most of these probably have
`false-positive tests due to vegetable peroxidases from the
`diet .
`Sixty-five to 80% of colorectal carcinomas and 20 to 40%
`of benign colonic adenomas are detected by the three-stool
`Hemoccult test . Thus, any one positive stool is an indication
`for a full work-up of the entire colon, including either air-
`contrast barium enema plus flexible (60 cm) sigmoidoscopy,
`or full colonoscopy . Flexible sigmoidoscopy or rigid proc-
`toscopy alone are satisfactory as complementary tests with
`Hemoccult in screening programs but are inadequate for
`
`Figure 98 .1
`Degradation of hemoglobin in the gastrointestinal tract and meth-
`ods to detect hemoglobin and its metabolites in feces .
`
`Basic Science
`
`As shown in Figure 98 .1, erythrocytes entering the gas-
`trointestinal lumen are usually lysed to free hemoglobin,
`which is converted to free hematin (Fes+ state) plus globin .
`Up to 15% of the hematin is absorbed and converted to
`bilirubin by microsomal heme oxygenase in the enterocytes .
`The remaining 85% may be further degraded, principally
`by colonic bacteria, to form porphyrins . The globin is di-
`gested by pancreatic and intestinal mucosal proteases to
`yield peptides and amino acids .
`Many tests have been designed to detect hemoglobin or
`its intestinal metabolites in the stool (Figure 98 .1) . Thus,
`blood released into the gastrointestinal tract can be detected
`as hemoglobin ( 51 Cr-labeled red cells, immunoassay), he-
`matin (peroxidase activity), porphyrins (fluorescence), or
`globin (immunoassay) . Most such tests have inherent un-
`reliability, however, because the proportion of hemoglobin
`degraded varies greatly among individuals . The only truly
`quantitative tests are : (1) radioassay of 51 Cr in the stool after
`intravenous administration of autologous red cells labeled
`with 51Cr-chromate on the R-chain of hemoglobin (the chro-
`mate is neither degraded nor absorbed in the intestine) ;
`and (2) measurement of total fecal porphyrins after chem-
`ical conversion of all fecal hemoglobin to porphyrins
`(HemoQuant test) . Unfortunately, these two tests are com-
`plex, cumbersome, time-consuming, and expensive . Con-
`sequently, the most widely used, established methods rely
`on the detection of undegraded hematin and hemoglobin
`by virtue of their peroxidase-like activity.
`Peroxidase-based tests (Figure 98 .2) involve the addition
`to a stool sample of a peroxide as substrate and guaiac,
`benzidine, or related compounds as a redox indicator . The
`peroxidase activity of hematin and/or hemoglobin catalyzes
`the oxidation of the colorless indicator compound to a blue
`
`Figure 98 .2
`Principle of peroxidase-based tests .
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`98 . TESTS FOR FECAL OCCULT BLOOD
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`491
`
`further evaluation of a Hemoccult-positive stool because
`over one-third of colonic neoplasms are proximal to the
`rectosigmoid . If only benign lesions or no lesions are dis-
`covered after full examination of the colon, upper gastroin-
`testinal endoscopy is probably indicated, though its cost
`effectiveness has not been validated . If still nothing is found,
`it is probably best to evaluate if the Hemoccult test is a false-
`positive, using 51 Cr-labeled erythrocytes or the HemoQuant
`test to quantify fecal blood loss . If the presence of bleeding
`is confirmed, one may consider performance of a small
`bowel series, mesenteric angiography, and/or abdominal
`scintiscan after intravenous administration of --Tc-labeled
`erythrocytes . Unfortunately, these tests have a low yield in
`the patient with occult blood loss . The fluorescein string
`test for upper gastrointestinal blood loss has a high fre-
`quency of false-positive results and should not be used .
`Most of the colorectal neoplasms detected with Hem-
`occult screening will be either benign adenomas or Duke's
`stage A or B 1 carcinomas, with an 80% or more 5-year
`survival after resection . By contrast, in patients with symp-
`tomatic carcinoma, the proportion of 5-year survivals is less
`than 40% . Because of built-in biases in screening programs,
`however, one may merely be making the diagnosis at an
`earlier stage in the disease, without actually postponing the
`time of demise . Therefore, it is not yet proven that screen-
`ing for colon cancer actually improves survival ; long-term,
`randomized, prospective studies, well under way in New
`York and Minneapolis, should answer this important ques-
`tion .
`
`References
`
`Ahlquist DA, McGill DB, Schwartz S, et al . HemoQuant, a new
`quantitative assay for fecal hemoglobin . Comparison with Hem-
`occult . Ann Intern Med 1984 ;101 :297-302 .
`Ahlquist DA, McGill DB, Schwartz S, et al . Fecal blood levels in
`
`health and disease. A study using HemoQuant . N Engl J Med
`
`1985 ;312 :1422-28 .
`Caligiore P, MacRae FA, St . John DJB, et al . Peroxidase levels in
`food : relevance to colorectal cancer screening . Am J Clin Nutrit
`1982 ;35 :1487-1489 .
`Eddy DM, Nugent FW, Eddy JF, et al . Screening for colorectal
`cancer in a high-risk population . Results of a mathematical model.
`Gastroenterology 1987 ;92 :682-92 .
`Ellefson BS, Larson A, Schwartz S, et al . The HemoQuant test,
`remodelled for the busy clinical laboratory . Clin Chem
`1986 ;32 :1142-43 .
`Gilbertsen VA, McHugh RB, Schuman L, et al . The earlier detec-
`tion of colorectal cancers . A preliminary report of the results
`of the occult blood study . Cancer 1980 ;45 :2899-2901 .
`McDonnell, WM, Ryan JA, Soeger, DM, Elta, GH . Effect of iron
`
`on the guaiac reaction . Gastroenterology 1989 ;96 :74-78 .
`MacRae FA, St . John, DJB . Relationships between patterns of
`bleeding and Hemoccult sensitivity in patients with colorectal
`cancers or adenomas . Gastroenterology 1982 ;82:891-98 .
`MacRae FA, St John, JB, Caligiore P, et al . Optimal dietary con-
`ditions for Hemoccult testing . Gastroenterology 1982 ;82 :899-
`903 .
`Morris DW, Hansell JR., Ostrow JD, et al . Validity of fecal occult
`blood tests in hospitalized patients . Am J Dig Dis 1976 ;21 :845-
`52 .
`Ostrow JD, Mulvaney CA, Hansell JR, et al . Sensitivity and re-
`producibility of chemical tests for fecal occult blood . Am J Dig
`Dis 1973 ;18 :930-40 .
`Peterson WL, Fordtran JS . Editorial . Quantitating the occult . N
`Engl J Med 1985 ;312 :1448-50 .
`Schwartz S, Dahl J, Ellefson, M, et al . The HemoQuant test : a
`specific and quantitative determination of heme (hemoglobin)
`in feces and other materials . Clin Chem 1983 ;29 :2061-67 .
`Simon JB : Occult blood screening for colorectal carcinoma : a crit-
`ical review. Gastroenterology 1985 ;88 :820-37 .
`Winawer SJ, Andrews M, Flehinger B, et al . Progress report on
`controlled trial of fecal occult blood testing for the detection of
`colorectal neoplasia . Cancer 1980 ;45 :2959-64 .
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