Nonuniform Distribution of Occult Blood in Feces RICHARD E. ROSENFIELD, M.D., SHAUL KOCHWA, PH.D.,
KACZERA,
Rosenfield, Richard E., Kochwa, Shaul, Kaczera, Zygmunt, and Maimon, Jonathan: Nonuniform distribution of occult blood in feces. Am J Clin Pathol 71: 204-209, Inhibition of anti-Rh29 by erythrocytic stroma in feces was devised as a specific test for fecal occult blood. The sensitivity of this test was equivalent to that of a standard Hemoccult® test, namely, 108 erythrocytes/g feces. Comparison of results of this test with results of Hemoccult tests of random stool specimens and of stools following ingestion of autologous blood revealed nonuniform distribution of occult blood in feces. The extent of nonuniformity was determined by testing samples of stool specimens following ingestion of 51Cr-labeled autologous blood. This allowed comparison of Hemoccult, inhibition of anti-Rh, and radioactivity, and showed that the three labels could separate in the feces and that some single small samples of feces could be relatively free of blood while blood was readily demonstrable in other portions. The variability of a standard Hemoccult test was somewhat reduced by dispersing the feces in distilled water before performing the test. (Key words: Fecal occult blood.)
Department of Pathology, Mount Sinai School of Medicine, City University of New York, New York
Received October 20, 1977; received revised manuscript and accepted for publication December 27, 1977. Supported by Contract NO1-CB-43870 from National Cancer Institute, NIH, Bethesda, Maryland. Address reprint requests to Dr. Rosenfield: Director, Department of Blood Bank and Clinical Microscopy, The Mount Sinai Hospital, Professor of Pathology, Mount Sinai School of Medicine, City University of New York, 1176 Fifth Avenue, New York, New York 10029. * Smith Kline Diagnostics, Division of Smith Kline Corporation, Philadelphia, Pennsylvania.
AND JONATHAN MAIMON, M.S.
new interest in tests for fecal occult blood. 15,17 To overcome problems posed by intermittent gastrointestinal blood loss and nonhomogeneity of shed blood in feces, three daily tests for blood in stool specimens have been recommended. 315 Since a positive screening test for fecal occult blood leads to costly colonic and radiologic evaluations, a more specific test for occult human blood in human feces was sought. Meat-free diets to reduce the false-positive results of chemical tests and the intravenous administration of 51Cr-labeled autologous blood generally were considered to be unacceptable for mass screening programs. Blood provides a wide variety of protein and cellular antigens that carry identifiable antigenic products. Feces, however, consists of particulate matter, cells, bacteria, and viruses, and contains numerous enzymes such as proteases, lipases, and neuraminidases of both endogenous and exogenous origin. These should, and do, have deleterious effects on human blood elements that carry recognizable antigens. Despite these problems, a serologic test for human blood in human feces was constructed. It consisted of attempting to neutralize Rh antibody with hypotonically treated stool samples after differential centrifugation, first to eliminate large particles and then to recover small ones such as bacteria and erythrocytic ghosts. The Rh specificity used was Rh29, an antigen possessed almost universally, being absent only in the extremely rare Rhnun blood type. 4 Rh29, like other Rh antigens, is expressed only on erythrocytic membranes and not on other cells. 12 Except for a very few nonhuman primates, Rh does not occur in other species. 12 Results of this test were compared with results of both standard Hemoccult tests and Hemoccult tests performed on stool samples dispersed in distilled water.
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Materials and Methods (I) Bovine Serum Albumin (BSA). Powdered bovine serum albumin! was dissolved in 2% concentration in t Armour Pharmaceutical Co., Chicago, Illinois, n Society of Clinical Pathologists
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FECAL OCCULT BLOOD has long been detected by chemical tests of stool for peroxidase activity. These tests, however, are subject to false-positive results from ingested animal and plant peroxidases or peroxidaselike substances." This has led to use of meat-free diets to reduce the incidence of false-positive results. 3 The problems inherent with these tests have been reviewed, 15,18 and many causes of both false-positive14 and false-negative 813 results have been described. It is known from 51 Cr-labeled autologous blood studies that 2 - 4 ml of blood may be lost in the feces daily. 1,2 Thus, bleeding in excess of this amount should represent pathologic fecal occult blood. In the last decade, a commercial product (Hemoccult®*) consisting of stable , dried, guaiac-impregnated filter paper has provided this degree of sensitivity, and has been associated with a relatively low incidence of false-positive reactions. 7,11,13,17 Consideration of population screening tests for the early detection of colonic malignancy has generated
PH.D.,
NONUNIFORM DISTRIBUTION OF OCCULT BLOOD IN FECES
Vol. 71 . No. 2
0.9% NaCl. This solution was stored at 4 C for as long as a month before use. (2) Anti-Rh29. The serum of Mrs. J.R.B. was used. 4 (3) Anti-Rhl. The serum of Mrs. F. was used. 10 (4) Radioactivity Measurements. Measurements were made with a Wallac 500$ gamma counter equipped with a 3-inch beryllium-coated crystal. m
I-Labeling
of
Erythrocytes
il
Cr-Labeled
Erythrocytes for
Ingestion
Twenty-five milliliters of sterile A . C D . blood were labeled with 60 pCi 51Cr as sodium chromate. 9,1 ' Ten milliliters of packed erythrocytes from this labeled blood were washed six times, mixed with 100 ml of grape juice, and swallowed. Stool
Specimens
Feces, usually the total for one day, were collected without preservative and processed on the same day. An occasional specimen was stored at 4 C overnight before processing. Fractionation of Stool
Specimens
Specimens (0.5 or 1.0 g) of feces were dispersed with wooden applicator sticks in ten volumes of distilled water. Large heavy particles were sedimented either by gravity (for 15 or 30 min) or, preferentially, by centrifugation at ~ 50 x g for 5 min (500 rpm in a Sorval GLC-1 centrifuge).** This material is referred to as "50 x g sediment." The supernatant fluid from this separation was then sedimented at 27,000 x g( 15,000 rpm in a Sorval RC-5 refrigerated centrifuge) for 30 min at 4 C, a procedure known to sediment erythrocytic stroma. 10 This sediment is referred to as "final pellet," and the supernatant fluid as "final supernatant." An alternative, simpler, and faster preparation of "final pellet" was deli LKB, Stockholm, Sweden. § Amersham-Searle, Des Plaines, Illinois. ' Mallinckrodt, St. Louis, Missouri. ** Ivan Sorval, Norwalk, Connecticut.
Table 1. Percentage of Recovery of I-labeled Stroma from Feces—Comparison of Three Methods to Remove Heavy Stool Particles Erythrocytes Added to Feces Method to Remove Heavy Particles
1.03 x 109
1.30 x 108
4.00 x 10'
50 x g 15 minutes settling 30 minutes settling
86 96 90
81 96 85
86 90 98
vised later. This consisted of centrifugation of 0.5-ml volumes of the 50 x g supernatant fluid at —12,000 x g for 15 min in a Beckman-Spinco Microfuge.tt Final pellets thus obtained were adequate for single serologic tests and were fully as reliable as material sedimented at 27,000 x g. Final pellets were resuspended in a volume of 0.9% NaCl that corresponded to the volume (or weight) of the sample of stool from which they had been obtained. Recovery of
l25
I-Labeled
Stroma
The efficiency of recovery of known erythrocytic stroma was determined by preparing artificial mixtures. Three 1-g aliquots of Hemoccult-negative stool were mixed with 107, 108, and 109 erythrocytes. Each aliquot also received 3 x 107 of 125I-labeled erythrocytes. Final pellets were counted for recovery of labeled stroma, and the percentage of recovery was adjusted to total radioactivity precipitated by trichloracetic acid. The results, shown in Table 1, indicate approximately 90% recovery. Hemoccult
tests.
Stool specimens were tested for occult blood with Hemoccult slides by the use of the standard method described by the manufacturer. "Hemoccult-Water"
Test
One gram of feces was dispersed in 10 ml of distilled water and homogenized with an applicator stick. A drop of the suspension was tested on a Hemoccult slide as described by the manufacturer. Serologic Tests for Rh in Stool Pellets Two Rh specificities were studied for the capacity of erythrocytes in feces to inhibit or neutralize Rh antibody. In each case, the anti-Rh was diluted in 2% BSA so that it would modestly agglutinate ficin-treated Rhpositive erythrocytes. 5 For this test equal volumes of diluted anti-Rh and a 2% suspension of erythrocytes in t t Spinco, Palo Alto, California.
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A mixture was prepared with 100 p\ 0.1 N HC1, 200 (JL\ 0.6 M NaCl, 2 pi 0.02 M IC1 solution, and 5 pi 125I (0.5 mCi).§ This solution was stable, but after the addition of 50 juJ 1 M glycine buffer, pH 9.0, it was used immediately (solution A). A 10% suspension (0.4 ml) of washed erythrocytes in 0.9% NaCl was added to solution A and incubated at room temperature for 3 min with gentle swirling. Another freshly prepared solution A was then added, after which the erythrocytes were centrifuged and washed repeatedly with 0.9% NaCl until the washings were free of radioactivity.
205 125
A.J.C.P. • February 1979
ROS ENFIELD ET AL.
206
Table 2, Detection of Blood in Artificial Mixtures of Erythrocytes in Feces Erythrocytes/g Stool1 6
107
108
Test
RBC Used
0
10
Hemoccult-water Rh antibody neutralization Anti-Rhl
Rh:l or Rh: - 1
Negative
Negative
Trace
Moderate
Strong
Rh:l Rh:- 1 Rh:l or Rh: - 1
Negative Negative Negative
Negative Negative Negative
Doubtful Negative Doubtful
Moderate Negative Moderate
Strong Negative Strong
Anti-Rh29
Distribution of Swallowed Blood in Stool To assay the distribution of swallowed blood in feces, feces were collected daily for five days from a volunteer who swallowed 10 ml of autologous, washed, packed erythrocytes labeled with 51Cr9 to carry 3.2 x 106cpm. All stool samples were weighed, and 1-g portions were prepared from each. Twenty such portions were prepared on day 1, and ten each were prepared on days 2 and 3. All were tested as follows: (a) standard Hemoccult; (b) radioactivity and Hemoccult-water test after dispersal of the sample in distilled water; (c) radioactivtt Serofuge, Clay Adams Co., Division of Becton, Dickinson and Co., Parsippany, New Jersey.
ity of 50 g sediment; (d) radioactivity of final pellet. The capability of the final pellet to neutralize anti-Rh29 was assayed in eight of the 20 samples of the first day's specimen and all samples of the second and third' days' specimens. The format of analysis allowed for the detection of three independent markers: peroxidase activity by two differing Hemoccult tests, erythrocytic membrane Rh antigen activity by neutralization of anti-Rh29, and 51 Cr-labeled j3 chains of hemoglobin by radioactivity, the latter adjusted as required by the geometry of counting. In addition, the distributions of radioactivity in the 50 x g sediment, final pellet, and final supernatant were calculated. Results Serologic Testing for Blood in Artificial Mixtures of Erythrocytes in Stool When artificial mixtures of erythrocytes in stool were prepared as described in Materials and Methods, the stool samples dispersed in distilled water were consistently positive by Hemoccult in accordance with the number of erythrocytes added to the 1-g stool sample. Final pellets of these samples, when incubated with anti-Rh, neutralized agglutinating activity in similar accordance with numbers of added erythrocytes. Typical results are shown in Table 2, where either Rh: 1 (Rh0 or D positive) or Rh:-1 erythrocytes added to feces neutralized anti-Rh29, but only Rh: 1 cells neutralized anti-Rhl. Complete neutralization was observed with 109 erythrocytes/g feces, and almost complete neutralization with 10* erythrocytes. Doubtful neutralization was seen with 107 erythrocytes, and no neutralization could be detected with 106 erythrocytes/g feces. Parallel Hemoccult tests of the same well-mixed feces at 1:10 concentration in distilled water were negative with 106 erythrocytes/g feces, trace positive with 107 erythrocytes, moderately positive with 108 erythrocytes, and strongly positive with 109 erythrocytes/g feces. The results portrayed in Table 2 were very reproducible for freshly prepared mixtures of erythrocytes in feces. However, when artificial mixtures of erythro-
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0.9% NaCl were mixed, incubated for 15 min at 37 C, and briefly centrifuged (1,000 x g for 10 sec).tt The extent of agglutination used was + + + (numerous large clumps of cells) on a scale ranging from + (very weak but discernible agglutination under x6 magnification) to + + + + (solid agglutination, all cells in one clump after gentle shaking). One of the antisera used was anti-Rh29; the other was anti-Rhl (Rh0 or D). Stool specimens from normal volunteer subjects, found to be free of blood by repeated standard Hemoccult tests, were used for evaluation. From each specimen, five 1-g aliquots were prepared. One of these was untreated, while the others were mixed with 0.1 ml of a cell suspension in 0.9% NaCl that contained 106, 107, 108, or 109 erythrocytes/ml. The last was essentially 0.1 ml packed cells. After the suspension was mixed with wooden applicator sticks, a 10-ml volume of distilled water was added, and each stool sample was tested by Hemoccult-water test and then processed to prepare final pellets. To 0.1 ml of each final pellet suspension, 0.1 ml diluted anti-Rh was added. Additionally, 0.1 ml anti-Rh was mixed with 0.1 ml packed erythrocytes (109 erythrocytes) and another 0.1 ml antibody was mixed with 0.1 ml 0.9% NaCl. All seven antibody mixtures were incubated at 37 C for an hour and centrifuged at —12,000 x g for 4 min. One drop of each supernatant fluid was then added to one drop of a 2% suspension of ficin-treated erythrocytes to test for residual antibody activity.
10"
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207
NONUNIFORM DISTRIBUTION OF OCCULT BLOOD IN FECES
cytes in feces were incubated at 37 C, serologic ability to neutralize anti-Rh was present at 24 hours, but absent after 48 hours unless 0.1% sodium azide was included as a preservative. With added sodium azide, Rh antigenic activity was preserved at 37 C for as long as five days. False-positive and False-negative Fecal Occult Blood
Results of Tests for
Unreliability of Tests for Occult Blood in Stool Specimens Stool specimens obtained from more than a hundred unselected patients were tested for blood both by the standard Hemoccult method and by determination of the capacity of the final pellet material to inhibit antiRh29. Whenever these tests were performed, artificial mixtures of blood in Hemoccult-negative feces from the volunteer were prepared and tested as standards known to contain 0, 107, 108, 109 erythrocytes/g feces. Although these standards always provided parallel and concordant results between Hemoccult-water tests and Rh serology tests, very few patient samples were in agreement for both Rh serology and the standard Hemoccult test. The results are summarized in Table 3, where, of 18 stool samples found positive by either test, only three were equally positive by both. Indeed, four samples were strongly positive by Rh serology and negative by standard Hemoccult test, while six others were strongly positive by the standard Hemoccult test but negative by Rh serology. A volunteer swallowed autologous blood and collected stool specimens for four subsequent days to provide feces that might contain blood and permit a more valid comparison of the two test procedures. Two experiments were performed. In one, the volunteer swal-
Number of Patients
Hemoccult
Serology
3 1 4 4 6 >100
+ + ± +
+ ± + + -
+ = strongly positive. ± - weakly positive. - - negative.
lowed 10 ml of blood, and concordant positive results were obtained on the second and third days after blood ingestion. In the other experiment, a 20-ml volume of blood was swallowed, and concordant results were observed on the second postingestion day. In both experiments, stools on subsequent days were negative by both tests, and in both experiments stools on day one were discordantly positive. After ingestion of 10 ml of blood, the first day's stool was standard Hemoccultpositive but negative by Rh serology; after 20 ml the reverse was observed: the standard Hemoccult test was negative, but the Rh serologic test was positive. Autologous Ingested Blood in Stool: Distribution of Three Independent Markers To determine the uniformity of the distribution of ingested blood in feces, a volunteer swallowed 10 ml of packed autologous erythrocytes previously labeled with 51Cr. The details of the experiment are given in Materials and Methods. Table 4 summarizes the average percentage of distribution of 51Cr in 50 x g sediments, final pellets, and final supernatants for all three days of stool collection, during which time frame approximately 90% of total radioactivity was recovered in the stools. As expected, relatively little radioactivity was recovered in final pellets where erythrocytic membranes were collected; erythrocytic membranes accept rather little chromation, more than 90% binding to the /3 chains of hemoglobin. 6 An increase from 8.9% on day 1 to 18.9% on day 3 was observed, and might have indicated increasing adsorbance of hemoglobin or hemoglobin fragments by bacteria. Such adsorbance had obviously occurred on larger fibers and cellular particles because the 50 x g sediments carried about 40% of total radioactivity on all three days. As shown in Table 1, stroma labeled with 125I were not sequestered to such an extent of the 50 x g sediment but were found in 90% concentration in the final pellet. Very few final supernatants contained more than 50% of total radioactivity. Table 4 also summarizes the distributions of radioactivity in the various aliquots assayed on each day.
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Neither meat nor animal blood inhibited anti-Rh, but both caused positive Hemoccult tests. However, when a normal volunteer deliberately ate as much as 1 kg of very rare beef, standard Hemoccult tests were negative. Hemoccult tests of feces in distilled water were only faintly positive after ingestion of 1 kg of beef and were negative after ingestion of only 500 g. As reported, 14 scraped tomato skin, but not tomato pulp, was found Hemoccult-positive. Also as reported, 8 ascorbic acid in stool was found to abolish a known positive Hemoccult test, but did so only at a level of at least 5 mg/g feces and only when the pH of the stool was less than 7.0. Indeed, when the pH was less than 5.0, even the inhibition of anti-Rh was reduced because, as reported, 10 the Rh antigens of erythrocytic stroma are totally destroyed at low pH. Such low pH values, however, seem rather unlikely to occur in feces.
Table 3. Results of Tests for Occult Blood in Unselected Patient Stool Samples
R0SENFIELD£7"/1L.
208
Table 4. Distribution of 51Cr within Stool Specimens
Total, expressed as 1010 erythrocytes Mean Standard deviation Coefficient of variation Percentage within portion of specimen 50 x g sediment Mean Standard deviation Final pellet Mean Standard deviation Final supernatant Mean Standard deviation
Day 1 (n = 20)
Day 2 (n = 10)
Day 3 (n = 10)
5.2411 1.3194 25.2
3.1973 0.7791 24.4
0.9000 1.1054 122.8
39.1 1.94
41.1 2.55
42.4 2.35
8.9 1.32
15.2 1.38
18.9 4.1
52.0 1.97
43.7 1.80
38.7 3.06
Discussion Specimens of stool vary in consistency and quantity. They contain undigested matter in the form of fibers and clumps of cellular particles. They also contain nu-
merous bacteria, viruses, and a broad variety of enzymes. A single stool sample can be of any size, but is often about 250 g of very heterogeneous material. If this were to contain 5 ml of fully dispersed whole blood, there would be only about 108 erythrocytes/g stool, a very small fraction of the total sample. Furthermore, depending at least in part on the site of blood loss, the erythrocytes could be present intact, as stroma, or as fragments. Therefore, the feces had to be fractionated in a fashion designed to recover stroma and stromal fragments. A serologic test for human occult blood in human feces was developed to avoid alleged pitfalls of paperimpregnated guaiac tests. This serologic test consisted of testing the capacity of a stool fraction to neutralize appropriately diluted anti-Rh29, the immune response of a person with Rhnull phenotype. To perform this test, feces were dispersed in ten volumes of distilled water, a step that hemolyzed erythrocytes but allowed for the sedimentation of unwanted large heavy fibers and particles. Stroma, bacteria, and other small particles were then pelleted by high-speed centrifugation, resuspended, and mixed with the anti-Rh in the presence of bovine serum albumin. After an hour at 37 C, pellet solids were again sedimented at high speed, and supernatant fluid was tested for its residual anti-Rh activity. This test consistently identified 108 erythrocytes added artificially to one gram of Hemoccult-negative Table 5. Distribution of Positive Tests* for Blood in Stool Specimens Day 1 (8 samples) All four tests H + HW + C H + HW + Rh HW + C + Rh H + C + Rh H + HW H + C H + Rh HW + C HW + Rh C + Rh H HW C Rh None Total positive tests by each method
f1 HHW I Rh
Ic
Day 2 (10 samples)
Day 3 (10 samples)
4 3 0 0 0 0 0 0 1 0 0 0 0 0 0 0
6 2 0 1 0 0 0 0 1 0 0 0 0 0 0 0
0 1 0 1 1 1 0 0 0 0 0 0 0 0 3 3
7 8 4 8
8 10 5 10
3 3 5 3
* Tests: Standard Hemoccult (H), Hemoccult test of feces in water (HW), inhibition of anti-Rh29 indicative of &]0W erythrocytes/g feces (Rh), radioactivity indicative of
>3 x 107 erythrocytes/g feces (C).
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Standard deviations of these samples, expressed as coefficients of variation, were about 25% on days 1 and 2, and 123% on day 3. Thus, radioactivity of labeled swallowed blood was not distributed uniformly in the feces. Table 5, summarizes the comparisons of all positive tests for blood in feces. Since all stool samples contained some 51Cr, adjusted radioactivity was scored as positive only when it indicated the presence of S3 x 107 erythrocytes/g feces. Here, too, marked variations were observed, especially on day 3. But even on days 1 and 2 little more than half the samples were positive by all four tests. On days 1 and 2, radioactivity and the Hemoccult test of stool in distilled water were the most efficient tests, and inhibition of anti-Rh29 appeared to be only 50% as efficient. On day 3, however, Rh serology was the best test, providing 50% detection, while the other three tests were less efficient. The semiquantitative interpretations of both Hemoccult results and the inhibition observed in inhibition tests of anti-Rh29 were ranked and, with the ranks of total radioactivity, Spearman's rank correlation coefficients (rs) were employed to find significant correlations between any two tests. This statistical analysis is not shown in the tables. In six comparisons of paired strengths of reactions on three days of stool collection, no significant correlation was found on days two and three (P > 0.05). On day 1 the standard Hemoccult test versus the Hemoccult-water test and the standard Hemoccult test versus radioactivity correlated, P = 0.03 and 0.005, respectively.
A.J.C.P. • February 1979
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NONUNIFORM DISTRIBUTION OF OCCULT BLOOD IN FECES
stool, and false-positive results were not encountered. Theoretically, however, false-negative results could occur because both proteases and lipases may have deleterious effects on erythrocytic membranes, particularly with prolonged exposure. In addition,pH values of less than 5.0 can destroy the Rh antigens of stroma. 10 Similarly, a false-positive result could occur if the final pellet were to have, for example, a profound enzymatic effect on the anti-Rh, despite the protection of 2% BSA. Neither false-positive nor false-negative results were observed, but the test was evaluated with only several hundred stool samples.
of stool were tested with either two or more standard Hemoccult tests on different parts of the specimen or a Hemoccult-water test of a larger portion. On days 1 and 2 (Table 5), the Hemoccult-water test was positive for all samples. These were not positive by standard Hemoccult test. Unfortunately, the Hemoccult-water test is not as useful for mass screening. In addition, the intermittent nature of gastrointestinal bleeding would still necessitate testing on several days. Acknowledgment: Dr. Sidney Winawer, Director, Diagnostic Gastrointestinal Unit, Sloan-Kettering Memorial Hospital and Associate Professor of Medicine, Cornell University Medical College, New York, advised the authors in the preparation of this report.
References 1. Cameron AD: Gastro-intestinal blood loss measured by radioactive chromium. Gut 1:177-182, 1960 2. Ebaugh FG Jr, Clemens T Jr, Rodnan G, et al: Quantitative measurement of gastrointestinal blood loss. I. The use of radioactive Cr51 in patients with gastrointestinal hemorrhage. Am J Med 25:169-181, 1958 3. Gregor DM: Occult blood testing for detection of asymptomatic colon cancer. Cancer 28:131-134, 1971 4. Haber GV, Bastani A, Arpin PD, et al: RhnuU and pregnancy complicated by maternal anti-"total Rh." I. Anti-Rh29 (Rh). Transfusion 7:389, 1967 5. Haber GV, Rosenfield RE: Ficin treated red cells for hemagglutination studies, Festskrift. Edited by Anderson PH, Copenhagen, Munksgaard, 1957, pp 45-50 6. Heisterkamp D, Ebaugh FG Jr: Site of attachment of the chromate ion to the haemoglobin molecule. Nature 193:12531255, 1962 7. Humphery TJ, Goulston K: Chemical testing of occult blood in faeces: "haematest", "occultest", and guaiac testing correlated with 51 chromium estimation of faecal blood loss. M e d J A u s t 1:1291-1293, 1969 8. Jaffe RM, Kasten B, Young DS, et al: False-negative stool occult blood tests caused by ingestion of ascorbic acid (vitamin C). Ann Intern Med 83:824-825, 1975 9. Kaplan E, Hsu KS: Determination of erythrocyte survival in newborn infants by means of Cr^-labelled erythrocytes. Pediatrics 27:354-361, 1961 10. Kochwa S, Rosenfield RE: Immunochemical studies of the Rh system. I. Isolation and characterization of antibodies. J Immuno 92:682-692, 1964 11. Ostrow JD, Mulvaney CA, Hansell JR, et al: Sensitivity and reproducibility of chemical tests for fecal occult blood with an emphasis on false-positive reactions. Digestive Diseases 18:930-940, 1973 12. Rosenfield RE, Allen FH Jr, Rubinstein P: Genetic model for the Rh blood-group system. Proc Natl Acad Sci USA 70: 1303-1307, 1973 13. Stroehlein JR, Fairbanks VF, McGill DB, et al: Hemoccult detection of fecal occult blood quantitated by radioassay. Am J Dig Dis 21:841-844, 1976 14. Wiener SL, Wiener J: Red fruits causing false positive occult blood tests in stool. N Engl J Med 408, 1975 15. Winawer SJ, Sherlock P, Schottenfeld D, et al: Screening for colon cancer. Gastroenterology 70:783-789, 1976 16. Winawer SJ: Fecal occult blood testing. Am J Dig Dis 21:885888, 1976 17. Winawer SJ, Leidner SD, Miller DG, et al: Results of a screening program for the detection of early colon cancer and polyps using fecal occult blood testing. Gastroenterology 72:(Abstr) A-127/1150, 1977
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The Rh serologic test described is not an office procedure. It requires about four hours to compete and an experienced immunohematology technician to interpret the result. Therefore, the test is not practical unless warranted by very special considerations. Although anti-Rh29 was used in these experiments, the more commonly available anti-Rh 17 (Hr 0 , made by Rh: 1,-17 [-D-] persons) would be almost equally serviceable. The original reason to construct this serologic test, namely, false-positive paper-impregnated guaiac tests for fecal occult blood, seems to have rather minimal importance. The published frequency of false-positive results is
KACZERA,
Rosenfield, Richard E., Kochwa, Shaul, Kaczera, Zygmunt, and Maimon, Jonathan: Nonuniform distribution of occult blood in feces. Am J Clin Pathol 71: 204-209, Inhibition of anti-Rh29 by erythrocytic stroma in feces was devised as a specific test for fecal occult blood. The sensitivity of this test was equivalent to that of a standard Hemoccult® test, namely, 108 erythrocytes/g feces. Comparison of results of this test with results of Hemoccult tests of random stool specimens and of stools following ingestion of autologous blood revealed nonuniform distribution of occult blood in feces. The extent of nonuniformity was determined by testing samples of stool specimens following ingestion of 51Cr-labeled autologous blood. This allowed comparison of Hemoccult, inhibition of anti-Rh, and radioactivity, and showed that the three labels could separate in the feces and that some single small samples of feces could be relatively free of blood while blood was readily demonstrable in other portions. The variability of a standard Hemoccult test was somewhat reduced by dispersing the feces in distilled water before performing the test. (Key words: Fecal occult blood.)
Department of Pathology, Mount Sinai School of Medicine, City University of New York, New York
Received October 20, 1977; received revised manuscript and accepted for publication December 27, 1977. Supported by Contract NO1-CB-43870 from National Cancer Institute, NIH, Bethesda, Maryland. Address reprint requests to Dr. Rosenfield: Director, Department of Blood Bank and Clinical Microscopy, The Mount Sinai Hospital, Professor of Pathology, Mount Sinai School of Medicine, City University of New York, 1176 Fifth Avenue, New York, New York 10029. * Smith Kline Diagnostics, Division of Smith Kline Corporation, Philadelphia, Pennsylvania.
AND JONATHAN MAIMON, M.S.
new interest in tests for fecal occult blood. 15,17 To overcome problems posed by intermittent gastrointestinal blood loss and nonhomogeneity of shed blood in feces, three daily tests for blood in stool specimens have been recommended. 315 Since a positive screening test for fecal occult blood leads to costly colonic and radiologic evaluations, a more specific test for occult human blood in human feces was sought. Meat-free diets to reduce the false-positive results of chemical tests and the intravenous administration of 51Cr-labeled autologous blood generally were considered to be unacceptable for mass screening programs. Blood provides a wide variety of protein and cellular antigens that carry identifiable antigenic products. Feces, however, consists of particulate matter, cells, bacteria, and viruses, and contains numerous enzymes such as proteases, lipases, and neuraminidases of both endogenous and exogenous origin. These should, and do, have deleterious effects on human blood elements that carry recognizable antigens. Despite these problems, a serologic test for human blood in human feces was constructed. It consisted of attempting to neutralize Rh antibody with hypotonically treated stool samples after differential centrifugation, first to eliminate large particles and then to recover small ones such as bacteria and erythrocytic ghosts. The Rh specificity used was Rh29, an antigen possessed almost universally, being absent only in the extremely rare Rhnun blood type. 4 Rh29, like other Rh antigens, is expressed only on erythrocytic membranes and not on other cells. 12 Except for a very few nonhuman primates, Rh does not occur in other species. 12 Results of this test were compared with results of both standard Hemoccult tests and Hemoccult tests performed on stool samples dispersed in distilled water.
0002-9173/79/0200/0204 $00.80 © Ai
204
Materials and Methods (I) Bovine Serum Albumin (BSA). Powdered bovine serum albumin! was dissolved in 2% concentration in t Armour Pharmaceutical Co., Chicago, Illinois, n Society of Clinical Pathologists
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FECAL OCCULT BLOOD has long been detected by chemical tests of stool for peroxidase activity. These tests, however, are subject to false-positive results from ingested animal and plant peroxidases or peroxidaselike substances." This has led to use of meat-free diets to reduce the incidence of false-positive results. 3 The problems inherent with these tests have been reviewed, 15,18 and many causes of both false-positive14 and false-negative 813 results have been described. It is known from 51 Cr-labeled autologous blood studies that 2 - 4 ml of blood may be lost in the feces daily. 1,2 Thus, bleeding in excess of this amount should represent pathologic fecal occult blood. In the last decade, a commercial product (Hemoccult®*) consisting of stable , dried, guaiac-impregnated filter paper has provided this degree of sensitivity, and has been associated with a relatively low incidence of false-positive reactions. 7,11,13,17 Consideration of population screening tests for the early detection of colonic malignancy has generated
PH.D.,
NONUNIFORM DISTRIBUTION OF OCCULT BLOOD IN FECES
Vol. 71 . No. 2
0.9% NaCl. This solution was stored at 4 C for as long as a month before use. (2) Anti-Rh29. The serum of Mrs. J.R.B. was used. 4 (3) Anti-Rhl. The serum of Mrs. F. was used. 10 (4) Radioactivity Measurements. Measurements were made with a Wallac 500$ gamma counter equipped with a 3-inch beryllium-coated crystal. m
I-Labeling
of
Erythrocytes
il
Cr-Labeled
Erythrocytes for
Ingestion
Twenty-five milliliters of sterile A . C D . blood were labeled with 60 pCi 51Cr as sodium chromate. 9,1 ' Ten milliliters of packed erythrocytes from this labeled blood were washed six times, mixed with 100 ml of grape juice, and swallowed. Stool
Specimens
Feces, usually the total for one day, were collected without preservative and processed on the same day. An occasional specimen was stored at 4 C overnight before processing. Fractionation of Stool
Specimens
Specimens (0.5 or 1.0 g) of feces were dispersed with wooden applicator sticks in ten volumes of distilled water. Large heavy particles were sedimented either by gravity (for 15 or 30 min) or, preferentially, by centrifugation at ~ 50 x g for 5 min (500 rpm in a Sorval GLC-1 centrifuge).** This material is referred to as "50 x g sediment." The supernatant fluid from this separation was then sedimented at 27,000 x g( 15,000 rpm in a Sorval RC-5 refrigerated centrifuge) for 30 min at 4 C, a procedure known to sediment erythrocytic stroma. 10 This sediment is referred to as "final pellet," and the supernatant fluid as "final supernatant." An alternative, simpler, and faster preparation of "final pellet" was deli LKB, Stockholm, Sweden. § Amersham-Searle, Des Plaines, Illinois. ' Mallinckrodt, St. Louis, Missouri. ** Ivan Sorval, Norwalk, Connecticut.
Table 1. Percentage of Recovery of I-labeled Stroma from Feces—Comparison of Three Methods to Remove Heavy Stool Particles Erythrocytes Added to Feces Method to Remove Heavy Particles
1.03 x 109
1.30 x 108
4.00 x 10'
50 x g 15 minutes settling 30 minutes settling
86 96 90
81 96 85
86 90 98
vised later. This consisted of centrifugation of 0.5-ml volumes of the 50 x g supernatant fluid at —12,000 x g for 15 min in a Beckman-Spinco Microfuge.tt Final pellets thus obtained were adequate for single serologic tests and were fully as reliable as material sedimented at 27,000 x g. Final pellets were resuspended in a volume of 0.9% NaCl that corresponded to the volume (or weight) of the sample of stool from which they had been obtained. Recovery of
l25
I-Labeled
Stroma
The efficiency of recovery of known erythrocytic stroma was determined by preparing artificial mixtures. Three 1-g aliquots of Hemoccult-negative stool were mixed with 107, 108, and 109 erythrocytes. Each aliquot also received 3 x 107 of 125I-labeled erythrocytes. Final pellets were counted for recovery of labeled stroma, and the percentage of recovery was adjusted to total radioactivity precipitated by trichloracetic acid. The results, shown in Table 1, indicate approximately 90% recovery. Hemoccult
tests.
Stool specimens were tested for occult blood with Hemoccult slides by the use of the standard method described by the manufacturer. "Hemoccult-Water"
Test
One gram of feces was dispersed in 10 ml of distilled water and homogenized with an applicator stick. A drop of the suspension was tested on a Hemoccult slide as described by the manufacturer. Serologic Tests for Rh in Stool Pellets Two Rh specificities were studied for the capacity of erythrocytes in feces to inhibit or neutralize Rh antibody. In each case, the anti-Rh was diluted in 2% BSA so that it would modestly agglutinate ficin-treated Rhpositive erythrocytes. 5 For this test equal volumes of diluted anti-Rh and a 2% suspension of erythrocytes in t t Spinco, Palo Alto, California.
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A mixture was prepared with 100 p\ 0.1 N HC1, 200 (JL\ 0.6 M NaCl, 2 pi 0.02 M IC1 solution, and 5 pi 125I (0.5 mCi).§ This solution was stable, but after the addition of 50 juJ 1 M glycine buffer, pH 9.0, it was used immediately (solution A). A 10% suspension (0.4 ml) of washed erythrocytes in 0.9% NaCl was added to solution A and incubated at room temperature for 3 min with gentle swirling. Another freshly prepared solution A was then added, after which the erythrocytes were centrifuged and washed repeatedly with 0.9% NaCl until the washings were free of radioactivity.
205 125
A.J.C.P. • February 1979
ROS ENFIELD ET AL.
206
Table 2, Detection of Blood in Artificial Mixtures of Erythrocytes in Feces Erythrocytes/g Stool1 6
107
108
Test
RBC Used
0
10
Hemoccult-water Rh antibody neutralization Anti-Rhl
Rh:l or Rh: - 1
Negative
Negative
Trace
Moderate
Strong
Rh:l Rh:- 1 Rh:l or Rh: - 1
Negative Negative Negative
Negative Negative Negative
Doubtful Negative Doubtful
Moderate Negative Moderate
Strong Negative Strong
Anti-Rh29
Distribution of Swallowed Blood in Stool To assay the distribution of swallowed blood in feces, feces were collected daily for five days from a volunteer who swallowed 10 ml of autologous, washed, packed erythrocytes labeled with 51Cr9 to carry 3.2 x 106cpm. All stool samples were weighed, and 1-g portions were prepared from each. Twenty such portions were prepared on day 1, and ten each were prepared on days 2 and 3. All were tested as follows: (a) standard Hemoccult; (b) radioactivity and Hemoccult-water test after dispersal of the sample in distilled water; (c) radioactivtt Serofuge, Clay Adams Co., Division of Becton, Dickinson and Co., Parsippany, New Jersey.
ity of 50 g sediment; (d) radioactivity of final pellet. The capability of the final pellet to neutralize anti-Rh29 was assayed in eight of the 20 samples of the first day's specimen and all samples of the second and third' days' specimens. The format of analysis allowed for the detection of three independent markers: peroxidase activity by two differing Hemoccult tests, erythrocytic membrane Rh antigen activity by neutralization of anti-Rh29, and 51 Cr-labeled j3 chains of hemoglobin by radioactivity, the latter adjusted as required by the geometry of counting. In addition, the distributions of radioactivity in the 50 x g sediment, final pellet, and final supernatant were calculated. Results Serologic Testing for Blood in Artificial Mixtures of Erythrocytes in Stool When artificial mixtures of erythrocytes in stool were prepared as described in Materials and Methods, the stool samples dispersed in distilled water were consistently positive by Hemoccult in accordance with the number of erythrocytes added to the 1-g stool sample. Final pellets of these samples, when incubated with anti-Rh, neutralized agglutinating activity in similar accordance with numbers of added erythrocytes. Typical results are shown in Table 2, where either Rh: 1 (Rh0 or D positive) or Rh:-1 erythrocytes added to feces neutralized anti-Rh29, but only Rh: 1 cells neutralized anti-Rhl. Complete neutralization was observed with 109 erythrocytes/g feces, and almost complete neutralization with 10* erythrocytes. Doubtful neutralization was seen with 107 erythrocytes, and no neutralization could be detected with 106 erythrocytes/g feces. Parallel Hemoccult tests of the same well-mixed feces at 1:10 concentration in distilled water were negative with 106 erythrocytes/g feces, trace positive with 107 erythrocytes, moderately positive with 108 erythrocytes, and strongly positive with 109 erythrocytes/g feces. The results portrayed in Table 2 were very reproducible for freshly prepared mixtures of erythrocytes in feces. However, when artificial mixtures of erythro-
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0.9% NaCl were mixed, incubated for 15 min at 37 C, and briefly centrifuged (1,000 x g for 10 sec).tt The extent of agglutination used was + + + (numerous large clumps of cells) on a scale ranging from + (very weak but discernible agglutination under x6 magnification) to + + + + (solid agglutination, all cells in one clump after gentle shaking). One of the antisera used was anti-Rh29; the other was anti-Rhl (Rh0 or D). Stool specimens from normal volunteer subjects, found to be free of blood by repeated standard Hemoccult tests, were used for evaluation. From each specimen, five 1-g aliquots were prepared. One of these was untreated, while the others were mixed with 0.1 ml of a cell suspension in 0.9% NaCl that contained 106, 107, 108, or 109 erythrocytes/ml. The last was essentially 0.1 ml packed cells. After the suspension was mixed with wooden applicator sticks, a 10-ml volume of distilled water was added, and each stool sample was tested by Hemoccult-water test and then processed to prepare final pellets. To 0.1 ml of each final pellet suspension, 0.1 ml diluted anti-Rh was added. Additionally, 0.1 ml anti-Rh was mixed with 0.1 ml packed erythrocytes (109 erythrocytes) and another 0.1 ml antibody was mixed with 0.1 ml 0.9% NaCl. All seven antibody mixtures were incubated at 37 C for an hour and centrifuged at —12,000 x g for 4 min. One drop of each supernatant fluid was then added to one drop of a 2% suspension of ficin-treated erythrocytes to test for residual antibody activity.
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NONUNIFORM DISTRIBUTION OF OCCULT BLOOD IN FECES
cytes in feces were incubated at 37 C, serologic ability to neutralize anti-Rh was present at 24 hours, but absent after 48 hours unless 0.1% sodium azide was included as a preservative. With added sodium azide, Rh antigenic activity was preserved at 37 C for as long as five days. False-positive and False-negative Fecal Occult Blood
Results of Tests for
Unreliability of Tests for Occult Blood in Stool Specimens Stool specimens obtained from more than a hundred unselected patients were tested for blood both by the standard Hemoccult method and by determination of the capacity of the final pellet material to inhibit antiRh29. Whenever these tests were performed, artificial mixtures of blood in Hemoccult-negative feces from the volunteer were prepared and tested as standards known to contain 0, 107, 108, 109 erythrocytes/g feces. Although these standards always provided parallel and concordant results between Hemoccult-water tests and Rh serology tests, very few patient samples were in agreement for both Rh serology and the standard Hemoccult test. The results are summarized in Table 3, where, of 18 stool samples found positive by either test, only three were equally positive by both. Indeed, four samples were strongly positive by Rh serology and negative by standard Hemoccult test, while six others were strongly positive by the standard Hemoccult test but negative by Rh serology. A volunteer swallowed autologous blood and collected stool specimens for four subsequent days to provide feces that might contain blood and permit a more valid comparison of the two test procedures. Two experiments were performed. In one, the volunteer swal-
Number of Patients
Hemoccult
Serology
3 1 4 4 6 >100
+ + ± +
+ ± + + -
+ = strongly positive. ± - weakly positive. - - negative.
lowed 10 ml of blood, and concordant positive results were obtained on the second and third days after blood ingestion. In the other experiment, a 20-ml volume of blood was swallowed, and concordant results were observed on the second postingestion day. In both experiments, stools on subsequent days were negative by both tests, and in both experiments stools on day one were discordantly positive. After ingestion of 10 ml of blood, the first day's stool was standard Hemoccultpositive but negative by Rh serology; after 20 ml the reverse was observed: the standard Hemoccult test was negative, but the Rh serologic test was positive. Autologous Ingested Blood in Stool: Distribution of Three Independent Markers To determine the uniformity of the distribution of ingested blood in feces, a volunteer swallowed 10 ml of packed autologous erythrocytes previously labeled with 51Cr. The details of the experiment are given in Materials and Methods. Table 4 summarizes the average percentage of distribution of 51Cr in 50 x g sediments, final pellets, and final supernatants for all three days of stool collection, during which time frame approximately 90% of total radioactivity was recovered in the stools. As expected, relatively little radioactivity was recovered in final pellets where erythrocytic membranes were collected; erythrocytic membranes accept rather little chromation, more than 90% binding to the /3 chains of hemoglobin. 6 An increase from 8.9% on day 1 to 18.9% on day 3 was observed, and might have indicated increasing adsorbance of hemoglobin or hemoglobin fragments by bacteria. Such adsorbance had obviously occurred on larger fibers and cellular particles because the 50 x g sediments carried about 40% of total radioactivity on all three days. As shown in Table 1, stroma labeled with 125I were not sequestered to such an extent of the 50 x g sediment but were found in 90% concentration in the final pellet. Very few final supernatants contained more than 50% of total radioactivity. Table 4 also summarizes the distributions of radioactivity in the various aliquots assayed on each day.
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Neither meat nor animal blood inhibited anti-Rh, but both caused positive Hemoccult tests. However, when a normal volunteer deliberately ate as much as 1 kg of very rare beef, standard Hemoccult tests were negative. Hemoccult tests of feces in distilled water were only faintly positive after ingestion of 1 kg of beef and were negative after ingestion of only 500 g. As reported, 14 scraped tomato skin, but not tomato pulp, was found Hemoccult-positive. Also as reported, 8 ascorbic acid in stool was found to abolish a known positive Hemoccult test, but did so only at a level of at least 5 mg/g feces and only when the pH of the stool was less than 7.0. Indeed, when the pH was less than 5.0, even the inhibition of anti-Rh was reduced because, as reported, 10 the Rh antigens of erythrocytic stroma are totally destroyed at low pH. Such low pH values, however, seem rather unlikely to occur in feces.
Table 3. Results of Tests for Occult Blood in Unselected Patient Stool Samples
R0SENFIELD£7"/1L.
208
Table 4. Distribution of 51Cr within Stool Specimens
Total, expressed as 1010 erythrocytes Mean Standard deviation Coefficient of variation Percentage within portion of specimen 50 x g sediment Mean Standard deviation Final pellet Mean Standard deviation Final supernatant Mean Standard deviation
Day 1 (n = 20)
Day 2 (n = 10)
Day 3 (n = 10)
5.2411 1.3194 25.2
3.1973 0.7791 24.4
0.9000 1.1054 122.8
39.1 1.94
41.1 2.55
42.4 2.35
8.9 1.32
15.2 1.38
18.9 4.1
52.0 1.97
43.7 1.80
38.7 3.06
Discussion Specimens of stool vary in consistency and quantity. They contain undigested matter in the form of fibers and clumps of cellular particles. They also contain nu-
merous bacteria, viruses, and a broad variety of enzymes. A single stool sample can be of any size, but is often about 250 g of very heterogeneous material. If this were to contain 5 ml of fully dispersed whole blood, there would be only about 108 erythrocytes/g stool, a very small fraction of the total sample. Furthermore, depending at least in part on the site of blood loss, the erythrocytes could be present intact, as stroma, or as fragments. Therefore, the feces had to be fractionated in a fashion designed to recover stroma and stromal fragments. A serologic test for human occult blood in human feces was developed to avoid alleged pitfalls of paperimpregnated guaiac tests. This serologic test consisted of testing the capacity of a stool fraction to neutralize appropriately diluted anti-Rh29, the immune response of a person with Rhnull phenotype. To perform this test, feces were dispersed in ten volumes of distilled water, a step that hemolyzed erythrocytes but allowed for the sedimentation of unwanted large heavy fibers and particles. Stroma, bacteria, and other small particles were then pelleted by high-speed centrifugation, resuspended, and mixed with the anti-Rh in the presence of bovine serum albumin. After an hour at 37 C, pellet solids were again sedimented at high speed, and supernatant fluid was tested for its residual anti-Rh activity. This test consistently identified 108 erythrocytes added artificially to one gram of Hemoccult-negative Table 5. Distribution of Positive Tests* for Blood in Stool Specimens Day 1 (8 samples) All four tests H + HW + C H + HW + Rh HW + C + Rh H + C + Rh H + HW H + C H + Rh HW + C HW + Rh C + Rh H HW C Rh None Total positive tests by each method
f1 HHW I Rh
Ic
Day 2 (10 samples)
Day 3 (10 samples)
4 3 0 0 0 0 0 0 1 0 0 0 0 0 0 0
6 2 0 1 0 0 0 0 1 0 0 0 0 0 0 0
0 1 0 1 1 1 0 0 0 0 0 0 0 0 3 3
7 8 4 8
8 10 5 10
3 3 5 3
* Tests: Standard Hemoccult (H), Hemoccult test of feces in water (HW), inhibition of anti-Rh29 indicative of &]0W erythrocytes/g feces (Rh), radioactivity indicative of
>3 x 107 erythrocytes/g feces (C).
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Standard deviations of these samples, expressed as coefficients of variation, were about 25% on days 1 and 2, and 123% on day 3. Thus, radioactivity of labeled swallowed blood was not distributed uniformly in the feces. Table 5, summarizes the comparisons of all positive tests for blood in feces. Since all stool samples contained some 51Cr, adjusted radioactivity was scored as positive only when it indicated the presence of S3 x 107 erythrocytes/g feces. Here, too, marked variations were observed, especially on day 3. But even on days 1 and 2 little more than half the samples were positive by all four tests. On days 1 and 2, radioactivity and the Hemoccult test of stool in distilled water were the most efficient tests, and inhibition of anti-Rh29 appeared to be only 50% as efficient. On day 3, however, Rh serology was the best test, providing 50% detection, while the other three tests were less efficient. The semiquantitative interpretations of both Hemoccult results and the inhibition observed in inhibition tests of anti-Rh29 were ranked and, with the ranks of total radioactivity, Spearman's rank correlation coefficients (rs) were employed to find significant correlations between any two tests. This statistical analysis is not shown in the tables. In six comparisons of paired strengths of reactions on three days of stool collection, no significant correlation was found on days two and three (P > 0.05). On day 1 the standard Hemoccult test versus the Hemoccult-water test and the standard Hemoccult test versus radioactivity correlated, P = 0.03 and 0.005, respectively.
A.J.C.P. • February 1979
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NONUNIFORM DISTRIBUTION OF OCCULT BLOOD IN FECES
stool, and false-positive results were not encountered. Theoretically, however, false-negative results could occur because both proteases and lipases may have deleterious effects on erythrocytic membranes, particularly with prolonged exposure. In addition,pH values of less than 5.0 can destroy the Rh antigens of stroma. 10 Similarly, a false-positive result could occur if the final pellet were to have, for example, a profound enzymatic effect on the anti-Rh, despite the protection of 2% BSA. Neither false-positive nor false-negative results were observed, but the test was evaluated with only several hundred stool samples.
of stool were tested with either two or more standard Hemoccult tests on different parts of the specimen or a Hemoccult-water test of a larger portion. On days 1 and 2 (Table 5), the Hemoccult-water test was positive for all samples. These were not positive by standard Hemoccult test. Unfortunately, the Hemoccult-water test is not as useful for mass screening. In addition, the intermittent nature of gastrointestinal bleeding would still necessitate testing on several days. Acknowledgment: Dr. Sidney Winawer, Director, Diagnostic Gastrointestinal Unit, Sloan-Kettering Memorial Hospital and Associate Professor of Medicine, Cornell University Medical College, New York, advised the authors in the preparation of this report.
References 1. Cameron AD: Gastro-intestinal blood loss measured by radioactive chromium. Gut 1:177-182, 1960 2. Ebaugh FG Jr, Clemens T Jr, Rodnan G, et al: Quantitative measurement of gastrointestinal blood loss. I. The use of radioactive Cr51 in patients with gastrointestinal hemorrhage. Am J Med 25:169-181, 1958 3. Gregor DM: Occult blood testing for detection of asymptomatic colon cancer. Cancer 28:131-134, 1971 4. Haber GV, Bastani A, Arpin PD, et al: RhnuU and pregnancy complicated by maternal anti-"total Rh." I. Anti-Rh29 (Rh). Transfusion 7:389, 1967 5. Haber GV, Rosenfield RE: Ficin treated red cells for hemagglutination studies, Festskrift. Edited by Anderson PH, Copenhagen, Munksgaard, 1957, pp 45-50 6. Heisterkamp D, Ebaugh FG Jr: Site of attachment of the chromate ion to the haemoglobin molecule. Nature 193:12531255, 1962 7. Humphery TJ, Goulston K: Chemical testing of occult blood in faeces: "haematest", "occultest", and guaiac testing correlated with 51 chromium estimation of faecal blood loss. M e d J A u s t 1:1291-1293, 1969 8. Jaffe RM, Kasten B, Young DS, et al: False-negative stool occult blood tests caused by ingestion of ascorbic acid (vitamin C). Ann Intern Med 83:824-825, 1975 9. Kaplan E, Hsu KS: Determination of erythrocyte survival in newborn infants by means of Cr^-labelled erythrocytes. Pediatrics 27:354-361, 1961 10. Kochwa S, Rosenfield RE: Immunochemical studies of the Rh system. I. Isolation and characterization of antibodies. J Immuno 92:682-692, 1964 11. Ostrow JD, Mulvaney CA, Hansell JR, et al: Sensitivity and reproducibility of chemical tests for fecal occult blood with an emphasis on false-positive reactions. Digestive Diseases 18:930-940, 1973 12. Rosenfield RE, Allen FH Jr, Rubinstein P: Genetic model for the Rh blood-group system. Proc Natl Acad Sci USA 70: 1303-1307, 1973 13. Stroehlein JR, Fairbanks VF, McGill DB, et al: Hemoccult detection of fecal occult blood quantitated by radioassay. Am J Dig Dis 21:841-844, 1976 14. Wiener SL, Wiener J: Red fruits causing false positive occult blood tests in stool. N Engl J Med 408, 1975 15. Winawer SJ, Sherlock P, Schottenfeld D, et al: Screening for colon cancer. Gastroenterology 70:783-789, 1976 16. Winawer SJ: Fecal occult blood testing. Am J Dig Dis 21:885888, 1976 17. Winawer SJ, Leidner SD, Miller DG, et al: Results of a screening program for the detection of early colon cancer and polyps using fecal occult blood testing. Gastroenterology 72:(Abstr) A-127/1150, 1977
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The Rh serologic test described is not an office procedure. It requires about four hours to compete and an experienced immunohematology technician to interpret the result. Therefore, the test is not practical unless warranted by very special considerations. Although anti-Rh29 was used in these experiments, the more commonly available anti-Rh 17 (Hr 0 , made by Rh: 1,-17 [-D-] persons) would be almost equally serviceable. The original reason to construct this serologic test, namely, false-positive paper-impregnated guaiac tests for fecal occult blood, seems to have rather minimal importance. The published frequency of false-positive results is
