THE BENZIDINE TEST FOR OCCULT BLOOD IN FAECES

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616.34-008.3-074

THE BENZIDINE TEST FOR OCCULT BLOOD IN FAECES1

B Y C. D. NEEDHAM AND R. G. SIMPSON

(From the Royal Infirmary and Woodend Hospital, Aberdeen)

VARIOUS types of ulcerative lesions of the digestive tract are encountered in clinical practice, but they often elude diagnosis until the disease has progressed to a late stage. In some cases clinical evidence may not appear to provide sufficient grounds to warrant radiological examination, and in others the suspicion of organic disease is aroused, but radiological studies give negative or inconclusive results. I n spite of help which may be obtained from further special investigations, particularly from endoscopic methods, there remain many cases in which some additional diagnostic aid is desirable. The possibility tha t actively progressive lesions of the alimentary tract may betray their presence by causing small amounts of blood to appear in the stool has been considered for many years. Leech (1907) stated tha t Korczynski and Jaworski (1886) suggested the prussian blue reaction as a chemical test for detecting very small amounts of blood in faeces, though it appears that Boas (1901) was the first to use the expression 'occult bleeding', stressing the value of its detection in the diagnosis of carcinoma of the alimentary tract . Since then various chemical tests have been suggested and used in clinical practice, the benzidine and guaiac tests being the most popular in this country.

A practical form of the now commonly used guaiac test had been described by Weber (1893), but Adler and Adler (1904) were among the first to employ the benzidine reaction in testing for occult blood in faeces. This type of benzidine test, which requires a concentrated or saturated solution of benzidine in glacial acetic acid and hydrogen peroxide, has with minor modifications been widely used since that time, and is still the one most frequently advocated (Bell, 1923; Burger, 1934; Marshall, 1938; Murphy, 1939; Johnson and Oliver, 1941; French and Douthwaite, 1945; Harrison, 1947; Hawk, Oser, and Sum-merson, 1947; Gradwohl, 1948; Lambie and Armytage, 1948; Kolmer, 1949; Stewart and Dunlop, 1949). Gregersen (1919) realized tha t the usual form of the test was too sensitive for clinical use, because the faeces from healthy people frequently gave a positive result. He clearly demonstrated tha t the sensitivity of the test depended on the concentration of benzidine in the test solution, an 8 per cent, solution detecting 1 : 20,000 and a 0-5 per cent, solution 1 : 500 blood in faeces. He concluded that a powder containing one part of benzidine to four parts of barium peroxide, made up in 50 per cent, acetic acid to yield a 0-5 per cent, solution of benzidine, and applied direct to faeces on a glass slide, provided the most suitable test, because it would detect

1 Received July 4, 1951.

Quarterly Journal of Medicine, New Series XXI, No. 82, April 1952. K

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124 C. D. NEEDHAM AND R. G. SIMPSON

clinically significant amounts of blood but would not react to the minimal amounts of blood present in some normal faeces. This modification of the benzidine test is described as an additional method by Harrison (1947) and by Kolmer (1949), and is preferred by some other authors (Aaron, 1924; Ogilvie, 1927; Alvarez and Wight, 1929; Stitt, Clough, and Clough, 1939; Kirschen, Sorter, and Necheles, 1942; Bockus, 1944; Todd, Sanford, and Stilwell, 1948; Hutchison and Hunter, 1949).

So many modifications of the benzidine test have been described tha t there is obviously a wide divergence of opinion, not only as to the best method of carrying out the test, but as to its clinical application and interpretation. I t appears that insufficient attention has been directed to the desiderata of a test the function of which is to detect occult blood in faeces. We suggest tha t the essential requirements of such a test are as follows.

1. I ts sensitivity must be standardized, and must be sufficient to detect clinically significant .amounts of blood, but not so great as to react to the minute traces of blood that may be present in normal stools.

2. The test should not be affected by taking an ordinary diet or commonly used medicines. There persists a widespread belief tha t foodstuffs containing chlorophyll (Bell, 1923; Aaron, 1924; Rosenthal, 1940; French and Douthwaite, 1945; Gradwohl, 1948; Hutchison and Hunter, 1949; Savill, 1950) and medicines containing iron (Bell, 1923; Burger, 1934; Lambie and Armytage, 1948; Hutchison and Hunter, 1949; Stewart and Dunlop, 1949; Price, 1950) will give rise to false positive reactions with the stool benzidine test, although this has been denied by other authors (Adler, 1921; Abrahams, 1923; Ogilvie, 1927; Schwartz and Vil, 1947; Todd, Sanford, and Stilwell, 1948). I t is also usually advised tha t meat should be excluded from the diet for three or four days before collecting the faeces for the benzidine test. Although some authors (Ogilvie, 1927; Alvarez and Wight, 1929; Johnson and Oliver, 1941; Schwartz and Vil, 1947; and Todd, Sanford, and Stilwell, 1948) have stated tha t previous dietary restrictions are not necessary if the Gregersen type of test is used, we have been unable to find any adequate body of evidence to establish this claim. I t appears to be true that meat in the diet may often cause a positive result when tests with a concentrated solution of benzidine are used (White, 1909; Hektoen, Fantus, and Portis, 1919; Bell, 1923; Bramkamp, 1929; Kiefer, 1934; Hoerr, Bliss, and Kauffman, 1949), and this is not very surprising, since Abrahams (1923) and Bell (1923) both showed tha t as little as 1 ml. of blood taken by mouth could cause a positive stool reaction. The consequent dietary restriction which must be imposed renders the test of very limited value, since it cannot be used in the out-patient department, and even in the wards it is scarcely practicable as a daily procedure for diagnostic purposes.

3. A minimum of apparatus should be required, and it should be possible to carry out the test quickly and easily in the test-room of the ward or out-patient department, using faeces either from a specimen-container or from a rectal glove-finger smeared on white absorbent paper. Any test which is complicated or time-consuming will not be done frequently, and so will not be used as a


THE BENZIDINE TEST FOR OCCULT BLOOD IN FAECES 125

routine either in the out-patient department or on serial specimens from selected patients in the ward.

4. The test should be reasonably inoffensive and hygienic; a procedure which involves the transference of a piece of faeces to a test-tube, the addition of water, and boiling, followed by the pouring of some of the faecal suspension into another test-tube and its subsequent disposal, is necessarily objectionable.

For several years there has been in general use in the wards of the Aberdeen Royal Infirmary the modification of the Gregersen (1919) test which is described in the next section of this paper. This test is a clean and simple procedure requiring a minimum of equipment, and the present investigation is designed to decide whether its sensitivity is such that it will detect clinically significant amounts of blood in faeces, but will avoid giving false positive reactions when patients are taking ordinary diets or iron-containing medicines.

The Present Investigation The modified Gregersen benzidine test used throughout the present investiga

tion has been carried out as follows. Individual powders wrapped in wax paper, each consisting of a finely divided mixture of benzidine hydrochloride 25 mg. and barium peroxide 200 mg., are dispensed or bought ready prepared.2 A powder prepared in this way will retain its potency for more than a year, and all that is necessary to make up the test solution is to shake up one of the powders in 5 ml. of glacial acetic acid in a clean test-tube, thus obtaining a 0-5 per cent, solution of benzidine hydrochloride. Then either the examining finger of a rectal glove or a clean orange-stick dipped in a faecal specimen is smeared on white filter-paper, and a little of the benzidine solution is poured over the smear. A blue colour appears if the faeces contain blood. The blue colour may not be apparent in a dark brown or black faecal smear, but then the white paper serves as a very efficient background as the blue or blue-green colour spreads out from the smear into the surrounding white paper. The resulting colour reactions have been graded and interpreted in the following manner:

Positive ( + ): a deep blue colour, appearing within 15 seconds. Weakly positive ( ± ) : a greenish-blue colour, appearing within 30 seconds. Negative (N): no coloration appearing within 30 seconds.

The investigations undertaken in the present study may be grouped under three main headings: they concern the sensitivity of the test, its selective properties, and its routine use in the out-patient department.

The sensitivity of the test. 1. Serial dilutions of normal blood in water and in faeces were tested, and the results are shown in Table I. The aqueous solutions were poured on to white filter-paper and the test solution added. The procedure for the faecal dilutions requires further explanation. Three separate specimens of benzidine-negative stools of medium brown colour and of average consistency were each weighed out in six exact 0-5 gm. amounts on watch-glasses. In to

2 These powders may be bought ready prepared from Messrs. Allen and Hanbury.



these portions there was intimately mixed 0-5 ml. of oxalated normal blood in serial aqueous dilutions, so that for each stool a series of faecal emulsions was obtained containing 2 per cent. (1: 50), 1 per cent. (1 : 100), 0-5 per cent. (1:200), 0-2 per cent. (1:500), 0-1 per cent. (1 : 1,000), and 0-05 per cent. (1 : 2,000) respectively of whole blood. The test was then carried out in the usual manner

TABLE I

The Modified Gregersen Test: Ranges of Sensitivity to Blood in Graded Dilutions in Water and in Faeces

Serial dilutions of blood Nature of , ~ * ^

diluent 1 : 50 1 : 100 1 : 200 1 : 500 1 : 1,000 1 : 2,000 1 : 10,000 1 : 50,000 1 : 100,000 1 : 500,000 Tap water + + .. . . + + to ± ± N N Faeces:

Sample I + ± N Sample II + + + .. ± N Sample III + + + ± N N

on each of the faecal emulsions. The test was found to be sensitive up to approximately 0-2 per cent. (1 : 500) of blood in faeces.

2. After the establishment of the in vitro sensitivity of the test, it was necessary to determine the smallest quantity of blood which must usually be introduced into the stomach in order to produce a positively-reacting stool. Seventeen ward patients were studied in this way, only those subjects being chosen in whom potential sources of spontaneous bleeding into the ahmentary tract could be reasonably excluded. I t was found particularly important to inquire and examine for recent epistaxis, bleeding gums, haemorrhoids, and anal fissures. In women the dates of menstruation had to be taken into account. From each of the patients selected for the study a representative sample of every stool that was passed was saved in a waxed carton, the date and time being noted on each. All subjects continued their accustomed—usually full— hospital diets, but were not allowed to have liver or black puddings.3 Brushing of the teeth was forbidden, because many hospital patients have easily-bleeding gums. As soon as consistently negative stools had been obtained for four consecutive days, a measured quantity of blood, freshly obtained from the patient's arm and diluted with 5 to 10 ml. of normal saline, was injected into the stomach through a Ryle's tube which had been swallowed immediately beforehand. Care was taken to apply only the gentlest aspiration in making certain that the tube was in the stomach. The blood was followed by a further 10 ml. of normal saline in order to ensure complete emptying of the tube, which was then withdrawn. No further blood was given until the patient 's stool benzidine reaction had reverted to negative, and had remained so for at least four days after the previous feeding of blood. The results of this part of the investigation are shown in Table I I . I t was unfortunately not possible to do a full series of tests on each patient, but the results seem to show that 3 to 5 ml. of blood is necessary to cause a positive (-f) reaction.

3 Blood constitutes one-third to one-quarter by weight of a black pudding, each pudding weighing approximately five ounces.


T H E BENZIDINE TEST FOR OCCULT BLOOD IN FAECES 127

The selective properties of the test. The next group of experiments was designed to show whether there are substances other than blood which, when taken by mouth, can cause a positive benzidine reaction in the stool. The types of substances investigated were (1) certain medicinal preparations of iron salts, and (2) a wide variety of common foodstuffs.

TABLE I I

Results of Stool Benzidine Tests in 17 Patients after Intragastric Administration of Measured Quantities of Blood*

Quantities of blood given by Ryle's tube

Case , number 1 ml. 2 ml.

1 2 3 4 5 (i 7 8 9

10 11 12 13 14 15 16 17

N" N N N N ± i ± N N

N N

NT N

N

3 ml.

N N N

±

N

N

+

±

5 ml. N

N

+ ± ± ± -f-+ + ± + + +

10 ml.

+ ± -j-

15 ml

+

* Each symbol in the Table signifies the maximum strength of the stool benzidine reaction during a period of four days after each separate administration of blood.

1. Using both the modified Gregersen test and the more commonly used type of benzidine test, we examined the following substances:

A. Iron and ammonium citrate: (i) a standard ward mixture containing 30 grains to the fluid ounce, both with and without the addition of 50 per cent. v/v of N/10 HC1; (ii) a thick emulsion of blood-free faeces in the standard ward mixture. No positive result was obtained.

B. Pil. ferrous sulphate, 3 grains: (i) the whole pill crushed; (ii) the green coating crushed; (hi) the inner portion crushed. No positive result was obtained.

C. Stools containing iron. We examined a series of stool specimens from each of five subjects, of whom three had been taking iron and ammonium citrate, 60 grains daily, and the other two pil. ferrous sulphate, 9 to 18 grains daily, for at least 10 days before the experiment began. Although in every instance the stools were dark-grey or black, a positive benzidine reaction was never obtained. The muddy discoloration imparted to the benzidine solution in the usual form of the test should not be mistaken for a true positive colour reaction.

2. Using the modified Gregersen test, we examined the possible effect of dietary constituents on the stool benzidine reaction.

A. Random samples of various items of hospital food were crushed and



rubbed on white filter-papers and tested in the usual manner. The results obtained were as follows.

(i) Positive ( + ): black pudding, raw or fried; liver, raw or fried; roast mut-

TABLE I I I

Results of Study of 187 Consecutive Out-Patients who had Eaten Meat during Preceding Three Days

Numbers of patients f IV ,

Stool Potential No apparent Not followed benzidine *'source of cause for to final

test bleeding bleeding diagnosis Totals + 49 7f 0 56 ± 20 6 0 26 N 18 67 20 105

Totals 87 80 20 187 * Including conditions listed under Group A and Group B, Table IV. t This number includes four who had eaten black pudding and one other who had eaten

approximately 4 ounces of fried liver.

Negative (N)'105

Total number of cases '187

No apparent cause For bleeding

apparent cause tor bleeding, but positive test explained on dietary grounds

/ J Potential source of bleeding present

Not Followed to final diaqnosis

FIG. 1. Analysis of results of the benzidine test in 187 patients who had eaten meat. The positive results explained on dietary grounds occurred in the faeces of four patients who had eaten black pudding and of another who

had eaten approximately four ounces of fried liver.

ton ; meat stew; rabbit, stewed; chicken, boiled; fish, fried or boiled; tinned luncheon meat ; turnip, raw; potato, raw.

(ii) Weakly positive ( ± ) : green peas, boiled; lettuce, raw; tripe, jellied. (hi) Negative (N): turnip, boiled and mashed; potato, boiled or roasted;

carrot, raw or boiled; tomato, raw; cabbage, raw or boiled; spinach, t inned; kale, raw or boiled; onion, raw; meat and vegetable broth; various brands of meat extract, including oxo and bovril; lemon juice; orange juice; apples, raw or stewed; prunes, stewed; egg custard; curds and whey; egg white; egg yolk; milk; cheese; chutney; red-currant jelly; infusion of tea ; coffee, essence, powder, or grounds; cocoa powder.


THE BENZIDINE TEST FOR OCCULT BLOOD I N FAECES 129

B. The above results, together with the fact tha t the patients in the blood-

feeding experiment were taking normal diet throughout prolonged periods of

daily observation, suggested that foodstuffs other than liver and black pudding,

TABLE IV

Results of Stool Benzidine Test performed on 603 Out-patients

Number of cases

Group A: 1. Chronic duodenal ulcer . . . . . 2. Chronic gastric ulcer . . . . . . 3. Clinical duodenal ulcer (barium meal X-ra}' negative) 4. Gastric cancer . . . . . . . 5. Oesophageal (peptic) ulcer or hiatal hernia 6. Oesophageal cancer . . . . . . 7. Pancreatic cancer . . . . . . 8. Non-specific colitis . . . . . . 9. Diverticulitis . . . . . . .

10. Colonic cancer . . . . . . . 11. Other organic conditions (including bleeding gums,

haemorrhoids, and anal fissure) . . . .

Total, Group A .

Group B : 1. Hepatic cirrhosis . . . . . . 2. Congestive heart failure . . . . . 3. Pernicious anaemia . . . . . .

Total, Group B

Group C: 1. Vague dyspepsia (results of investigations negative) 2. Diseases of liver and bile-passages (excluding hepatic

cirrhosis) . . . . . . . Genito-urinary disorders . . . . . Respiratory tract disorders . . . . . Cardiovascular disorders (excluding congestive heart

failure) . . . . . . . . Neurological d i s o r d e r s . . . . . . Locomotor disorders . . . . . . Anxiety neuroses and psychoneuroses .

Totals

151 24 62 20

6 1 1 8 7 1

27

308

4 19 5

Stool ben

+

64 10 16 10

4 1

5 1 1

11

123

3 3 2

zidine

±

22 2 5 5 2

1 2 2

6

47

4

result!

N

65 12 41

5

1 4

10

138

1 12

3

28

44

16

43

11 13 34

11 20 46 21 67

267

1

3 1

12

*18

1

1

1

7

10

10 12 33

11 19 43 20 48

239

9. Unclassified sundry conditions

Total, Group C .

Grand Total 603 149 61 393 * This figure includes six patients who had eaten black pudding, and one other who had

eaten approximately four ounces of fried liver.

when eaten in customary quantities, were unlikely to exert a significant effect

on the stool benzidine reaction. We therefore fed five ward patients with

measured quantities of liver and of black pudding, applying the same rigid

precautions as in the blood-feeding experiments. A positive ( + ) reaction was

obtained from the stools of all five patients after they had eaten two black

puddings, one black pudding, eight ounces, five ounces, and three ounces of

fried liver respectively.


130 C. D. NEEDHAM AND R, G. SIMPSON

C. A random series of patients attending the medical out-patient department were asked whether they had eaten any meat in the previous three days. The stool benzidine results of the 187 who had eaten meat were recorded, and all except 20 of the patients were followed to final diagnosis. The results are summarized in Table I I I and in Fig. 1. In only 13 of the 187 patients was a positive reaction of any degree obtained in the absence of a proved source of

Negative (i\l)^3,31

Total number of

cases - 603

Positive M = 149

No apparent cause . for bleeding

3 Wo apparent cause For \ bleeding, but positive test explained on dietary grounds

U ~7~\ Potential source of I / I bleeding present

FIG. 2. Analysis of results of the benzidine test in 603 patients. The positive results explained on dietary grounds occurred in the faeces of six patients who had eaten black pudding and of another who had eaten approximately four

ounces of fried liver.

bleeding. Of these 13 subjects, five out of the seven who gave a positive (-}-) reaction had eaten black pudding or liver the previous day.

The routine use of the test in the out-patient department. In order to evaluate the usefulness and limitations of the modified Gregersen test for occult blood in clinical practice, we have analysed the medical records of 603 out-patients whose stools we have tested during the past two years. In every instance the faecal smear was made direct from the gloved finger used in the rectal examination, and was tested immediately by the method we have described. The stool benzidine results together with the final diagnoses are set out in Table IV and Fig. 2. For the sake both of clarity and of economy of space we have classified diagnoses under system groups. The results show tha t a high proportion of positive (-|-) results was explained by the presence of alimentary tract lesions. Of the remaining 26 positive ( + ) results seven could be explained on dietary grounds, and eight others were found in the stools of patients suffering from the conditions hsted in Group B (Table IV) which appear to be liable to cause alimentary bleeding. Thus of 603 stool examinations 149 gave positive ( + ) results, and of these only 11 (one in every 13 or 14) appeared to have no clinical significance.



Discussion The modified Gregersen test used in this investigation is a simple procedure,

not requiring special laboratory facilities. The use of white filter-paper for the faecal smear has a double advantage over mixing on a glass slide in that the colour change spreading into the white background is very easily seen and, the paper being expendable, there is no risk of faecal contamination in its disposal. Whether glacial acetic acid or 50 per cent, acetic acid is used probably makes very little difference. I t is not, however, as a convenient second best that we advocate the modified Gregersen test, but as a method superior to the more usual form of the benzidine test by reason of its standardized concentration of benzidine, nicely balanced sensitivity, ready availability, and cleanliness. Much of the confusion still evident in current literature concerning the necessity or otherwise of dietary restriction prior to testing the faeces is due to the still prevalent use of the more usual and over-sensitive type of benzidine test. Even with this method it is difficult to see how a positive faecal reaction could be caused by the ingestion of chlorophyll-containing foods or of medicines containing iron salts, since these substances do not yield a positive result even when subjected to direct testing. We believe that the amount of meat taken in the ordinary diet will seldom cause false positive reactions when the modified Gregersen test is used, because (1) the patients used for studying the effect of feeding varying amounts of blood were given ordinary ward diet throughout the period of the tests, but their faeces gave consistently negative results until blood had been administered; (2) we found it necessary to feed patients with rather large amounts of meat rich in blood (liver and black pudding) before the faeces gave a positive reaction; (3) the evidence from the out-patient series as a whole (Table IV) and in particular from the 187 cases in which the previous diet was reviewed in detail (Table I I I ) suggests that eating meat seldom accounted for a positive reaction. The next question that arises is whether this modified Gregersen test is sufficiently sensitive to detect clinically significant alimentary bleeding. Our findings regarding the in vitro sensitivity of the modified Gregersen test (Table I) are in substantial agreement with those of Gregersen himself (1919), who considered it to be sensitive enough for clinical use. Other observers (Abrahams, 1923; Bell, 1923) have shown that the usual form of the benzidine test becomes positive after feeding 0-5 to 1 ml. of blood, but we found, using the modified Gregersen test, tha t at least 3 ml. of blood was needed to produce a positive reaction (Table II) . In spite of this lesser degree of sensitivity of the modified Gregersen test, the results with outpatients, listed in Table IV, suggest that it is sensitive enough to detect clinically significant bleeding in most instances where it is occurring. Furthermore, experience with routine daily testing in the wards has shown that, when each patient's results are considered as a series, the test is more informative than when used on a single stool specimen, because one is not misled by the occasional positive result in an otherwise negative series, or vice versa. Repeated stool tests—or stool series—are practicable only when a simple form of benzidine test is used.



We do not wish to suggest that this is an infallible screening test for organic disease of the alimentary tract. Even ulcerating lesions may bleed only intermittently, and it is probable that blood arising from a lesion, particularly in the more proximal alimentary tract, may be sufficiently altered before it reaches the stool to render it incapable of giving a positive benzidine reaction. That destruction of blood does occur in its passage through the gastro-intestinal tract is suggested by the work of Andrews and Oliver-Gonzalez (1942) who fed approximately two ounces of calf blood to each of four subjects, and found by a quantitative method that only 30 to 85 per cent, of the blood was passed in the faeces. This fact, coupled with the relative insensitivity of the test for blood diluted in faeces as opposed to water (Table I), probably explains why we had to feed rather large amounts of blood in order to obtain a positive benzidine reaction in the stool (Table II). The single examination possible in the out-patient department inevitably gives some positive results which remain unexplained after fuller investigation (one in every 13 or 14 in our series). This error is largely excluded in in-patient studies by repeated tests, but it appears that the routine use of the test in out-patient practice will, with the minimum of labour and expense, give considerable help in the diagnosis of gastro-intestinal disorders, provided that one takes care to exclude the presence of bleeding from the nose, the gums, haemorrhoids, anal fissure, and menstruation. I t is concluded that the modified Gregersen test can give useful information in out-patient as well as in-patient work if interpreted in conjunction with all the other available evidence.

A cknowledgement

We should like to thank the nursing staff for their willing co-operation in the

in-patient studies.

Summary

1. The literature relating to the development of the benzidine test is briefly reviewed.

2. The desirable properties of a test for small quantities of blood in faeces are considered.

3. Some of the limitations of the benzidine reaction as usually employed are discussed.

4. A modification of the Gregersen type of stool benzidine test is described. 5. In vitro experiments and clinical investigations designed to explore the

suitability of this test for clinical use are described. 6. I t is concluded that this modification of the benzidine test offers significant

advantages over other forms of the test, and will yield useful information when used in either out-patient or in-patient practice.

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THE BENZIDINE TEST FOR OCCULT BLOOD IN FAECES