*Corresponding author: E-mail: firstname.lastname@example.org;
Phenolphthalein Method as a Simple and Low Cost
Test for Detection of Fecal Occult Blood
Seyedeh Maryam Sharafi1
, Azar Balouti2
, Mahboobeh Hadipour2
, Morteza Yousefi3
and Hossein Yousofi Darni4*
Infectious Diseases and Tropical Medicine Research Center, Isfahan University of Medical Sciences, Isfahan, Iran. 2
Department of Medical Parasitology and Mycology, Isfahan University of Medical Sciences, Isfahan, Iran. 3
Department of Surgery, Kurdistan University of Medical Sciences, Sanandaj, Iran. 4
Cancer Prevention Research Centre, Isfahan University of Medical Sciences, Isfahan, Iran.
This work was carried out in collaboration between all authors. Author SMS performed some lab work and also helped for preparation of the first draft of the paper. Authors AB, MH and HY collected the samples and helped with the performance of lab works. Author MY contributed to the designing of research work and also preparation of the paper. Author HYD designed the research and supervised the lab works and also finalized preparation of the paper. All authors read and approved the final manuscript.
DOI: 10.9734/BJMMR/2017/34677 Editor(s): (1) Mohammed Rachidi, Molecular Genetics of Human Diseases, French Polynesia, University Paris 7 Denis Diderot, Paris, France. (2)Gentian Vyshka, University of Medicine in Tirana, Albania. Reviewers: (1) Cletus Anes Ukwubile, Federal Polytechnic, Nigeria. (2)Somchai Amornyotin, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand. Complete Peer review History:http://www.sciencedomain.org/review-history/20209
Received 5th June 2017 Accepted 19th July 2017 Published 26th July 2017
Background: Colorectal cancer is one of the main causes of cancer related death worldwide.
Different investigation showed that screening with fecal occult blood is able to reduce incidence of colorectal cancer in a given area. To provide a simple and cost effective test for screening of colorectal cancer in this work sensitivity of phenolphthalein method in detection of occult blood has been investigated.
Methods: In this descriptive study alcohol pyramidon and phenolphthalein methods were used to
estimate the minimum amount of human occult blood which can be detected in distilled water and
fecal samples. Then 100 fecal samples were examined by the two tests.
Results: Alcohol pyramidon method detected minimum amount of 0.125 µL blood in 1 ml distilled
water while phenolphthalein methods detected minimum amount of 0.0312 µL blood in 1 mL distilled water. In another experiment Alcohol pyramidon method detected minimum amount of 1 µL blood in 1 mL fecal sample while phenolphthalein methods detected minimum amount of 0.25 µL blood in about 1 mL fecal sample. Phenolphthalein method provided more positive results when 100 fecal samples were tested.
Conclusion: phenolphthalein method is a sensitive, simple and economy test for colorectal cancer
screening which can be used both in labs and at home.
Keywords: Colorectal cancer; occult blood; screening; phenolphthalein method.
Colorectal cancer is one of the leading causes of cancer-related deaths in the United States. Screening by means of fecal occult-blood testing leads to reduction of mortality and incidence of colorectal-cancer . Fecal occult blood and its constituent, fecal occult hemoglobin, is the most abundant component associated with colorectal lesions, so positive fecal occult blood tests may be a good marker for discovery of early colorectal cancers [2-6]. About four decades ago fecal occult blood test was introduced to screen for colorectal cancer [7,8]. After that in several investigations it t has been shown that screening for fecal occult blood significantly reduce the rate of death from colorectal cancer [9-13]. Also screening with fecal occult-blood test, significantly reduces the incidence of colorectal cancer . Fecal occult blood test is able to detect large polyps which are more likely to bleed [15-17] and also to become cancerous .
Chemical fecal occult bold tests are not specific for human blood . In condition of dietary constituent with sufficient peroxidase activity they may give false positive results . On the other hand various chemicals may interfere with the test and lead to providing false negative results [20,21]. However, they are is still the most implemented test for colorectal cancer screening .
Chemical tests such as guaiac , benzidine , Tetra-p-bhenylenediamine & hydrochloride  were used for detection of fecal occult blood. These tests are not specific for human blood . To overcome the problem of non-specificity immunochemical fecal occult blood tests were introduced. These tests seem to have higher specificity compared with chemical tests . Although chemical tests for detection of occult blood are not specific for human blood, they are still the most implemented test for colorectal cancer screening . In order to present a
sensitive, simple and economy chemical test for detection of fecal occult blood, in this work sensitivity of phenolphthalein method has been investigated.
2. MATERIALS AND METHODS
In this study, 30 spare blood and 60 fecal samples were collected with patients’ informed consent from Alzahra hospital diagnostic lab in Isfahan, Iran (This work was approved by Research Ethic committee of Isfahan University of medical sciences). In order to test the sensitivity of the alcohol pyramidon or phenolphthalein methods, in 5 transparent glass test tubes 1mL distilled water was added. Then 2, 1, 0.5, 0.25 and 0.125 µl blood was added into tubes 1-5. In the next step both alcohol pyramidon or phenolphthalein methods were performed for the above five tubes and the results were recorded.
In the next experiment again to five test tubes, 3 mL distilled water and about 1 ml stool sample was added to each tube. Then 2, 1, 0.5, 0.25 and 0.125 µL blood was added to tubes 1-5 respectively and both alcohol pyramidon or phenolphthalein methods were performed for the above five tubes and the results were recorded.
2.1 Alcohol Pyramidon Method
Alcohol byramidon reagent was prepared by adding 5 gram powder of this alcohol to 100 ml distilled water and mixed well. Other reagents were 10% acetic acid and 5% H2O2. To perform
the test, to test tubes containing diluted fecal samples in distilled water equal volume of Alcohol pyramidon reagent was added and mixed well. After that 5 drops of 10% acetic acid was added and mixed again. Finally, 5 drops of 5% H2O2 was added and appearance of the
2.2 Phenolphthalein Method
To make phenolphthalein reagent, 1 gram phenolphthalein powder and 25 g KOH were dissolved in 100 ml distilled water. The resulting red solution was boiled and 10 g zinc powder was added to get a colorless solution. The reagent was then filtered and equal volume of 96% alcohol was added. In order to perform the test, 5 drops of phenolphthalein reagent was added to tubes containing stool sample in distilled water and mixed. In the last stage 5drops of H2O2 was added and appearance of
red color was monitored. According to the intensity of the red color positive results recorded as 1+ to 4+.
Experiment 1: To 7 transparent glass test tubes
1ml distilled water was poured and 2, 1, 0.5,
0.25, 0.125, 0.0625 and 0.0312 µL blood sample was added to tubes 1-7 respectively. In the next step alcohol pyramidonand
phenolphthaleinmethods were performed on all above tubes. Results of this experiment has been summarized in Table 1.
Experiment 2: In 5 test tubes about 1ml stool
sample was mixed with 3mls distilled water and then 2, 1, 0.5, 0.25 and 0.125 µL blood samples was added to tubes 1-5 respectively. In the next step phenolphthalein and alchoholpyramidon methods were performed on all above tubes. Results of this experiment has been summarized in Table 2.
Experiment 3: one hundred spare stool samples
with order for occult blood test were examined both by alcohol pyramidon and phenolphthalein methods. The results have been summarized in Table 3.
Results of this work showed that phenolphthalein method detected as less as about 0.03 µl blood in 1 ml distilled water. Also this test detected 0.24 µl blood in mixture of 3 ml distilled water and 1 ml feces. This indicates that the test detected about 0.06 µl blood in 1 ml of stool. So it seems that phenolphthalein is a very sensitive test for detection of occult blood in stool samples. Beside sensitivity, other advantage of this test is its simplicity. To perform this test what we need is a 5 ml glass tube, phenolphthalein reagent and H2O2. So it is possible to present this test in a
simple kit which can be used in medical diagnostic labs and also at home by people older than 50 who want to screen themselves for colorectal cancers. Several factors such as the
diet of the patient may influence the result of the occult blood test and lead to providing
false negative results [20,21]. On the other hand these factors may also provide false
positive results and this is a common problem with using all chemical occult blood tests. Because phenolphthalein occults blood test is very simple and easy to do and also
cost effective, with the repetition of the test it is possible to overcome false positive or false
Table 1. Results of alcohol pyramidin or phenolphthalein for test tubes containing 2, 1, 0.5, 0.25, 0.125, 0.0625 and 0.0312 µL blood sample in 1 mL distilled water
Amount of blood 2 µL 1 µL 0.5 µL 0.25 µL 0.125 µL 0.0625 µL 0.0312 µL
Alcohol pyramidon 4+ 4+ 4+ 4+ 2+ - -
Phenolphthalein 4+ 4+ 4+ 4+ 3+ 2+ 1+
Table 2. Results of alcohol pyramidon or phenolphthalein for 5 test tubes containing 2, 1, 0.5, 0.25 and 0.125 µl blood samples in test tubes containing about 1 ml stool sample mixed with
3 mls distilled water
Amount of blood 2 µL 1 µL 0.5 µL 0.25 µL 0.125 µL
Alcohol pyramidon 2+ 1+ - - -
Phenolphthalein 4+ 4+ 2+ 1+ -
Table 3. Results of detection of fecal occult blood using alcohol pyramidon and phenolphthalein methods in 100 stool samples with order for occult blood test
Results of the tests Negative 4+positive 3+positive 2+positive 1+positive
Alcohol pyramidon 87 6 1 2 4
In this research the sensitivity of phenolphthalein method was determined by estimation of minimum amount of blood which can be detected in 1 mL fecal samples. Considering this fact that the consistency of fecal samples is not constant in all patients, so estimation of occult blood in 1 mL feces is not a standard method and there may be some variation among different patients. This was a one of the leading limitation of this research. However, in practice there is no alternative method to overcome this limitation.
According to recent estimates, about 8.2 million deaths and 14.1 million new cancer cases occurred in 2012 worldwide and over the years, the burden of cancer has shifted to developing countries . Colorectal cancer with over 1.2 million new cancer cases and 608,700 deaths in 2008 is the third most commonly diagnosed cancer in males and the second in females . In the time period 1998–2002 the rate of colorectal cancer per 100000 populations were 4.1 in India to 59.1 in Czech among men and 3.6 in India to 39.9 in New Zealand among females .
Screening measures lowers colorectal cancer mortality by detecting tumors at earlier stages [29-31]. In the United States the increased use of screening measures has been considered as one of the most important factors responsible for the recent decline in colorectal cancer rates [32,33]. Considering importance of screening methods for prevention from colorectal cancer, it seems that phenolphthalein method described in this study is an appropriate method for screening colorectal cancer especially in developing countries.
Phenolphthalein method is a sensitive, simple and low price test for screening of colorectal cancer especially in low income countries which can be performed easily not only in diagnostic labs but also at home by patients.
As per international standard or university standard, patient’s written consent has been collected and preserved by the authors.
As per international standard or university standard, written approval of Ethics committee
has been collected and preserved by the authors.
This work was financially supported by grant from Isfahan university of medical sciences.
Authors have declared that no competing interests exist.
1. Schoen RE, Pinsky PF, Weissfeld JL,
Yokochi LA, Church T, Laiyemo AO, et al. Colorectal-cancer incidence and mortality with screening flexible sigmoidoscopy. New England Journal of Medicine. 2012; 366(25):2345-57.
2. Greegor DH. Diagnosis of large-bowel
cancer in the asymptomatic patient. Jama. 1967;201(12):943-5.
3. Greegor DH. Detection of silent colon
cancer in routine examination. CA: A Cancer Journal for Clinicians. 1969;19(6): 330-7.
4. Greegor DH. Occult blood testing for
detection of asymptomatic colon cancer. Cancer. 1971;28(1):131-4.
5. Greegor DH. Detection of colorectal
cancer using guaiac slides. CA: A Cancer Journal for Clinicians. 1972;22(6):360-3.
6. Hastings JB. Mass screening for colorectal
cancer. The American Journal of Surgery. 1974;127(2):228-33.
7. Stroehlein JR, Fairbanks VF, McGill DB,
Go VL. Hemoccult detection of fecal occult blood quantitated by radioassay. Digestive Diseases and Sciences. 1976;21(10):841-4.
8. Winawer SJ. Fecal occult blood testing.
Digestive Diseases and Sciences. The American Journal of Digestive Diseases. 1976;21(10):885-8.
9. Bertario L, Russo A, Crosignani P, Sala P,
Spinelli P, Pizzetti P, et al. Reducing colorectal cancer mortality by repeated faecal occult blood test: A nested case– control study. European Journal of Cancer. 1999;35(6):973-7.
colorectal cancer mortality: A case-control study. British Journal of Cancer. 1999; 79(3-4):680.
11. Kronborg O, Fenger C, Olsen J, Jørgensen
OD, Søndergaard O. Randomised study of screening for colorectal cancer with faecal-occult-blood test. The Lancet. 1996; 348(9040):1467-71.
12. Mandel JS, Bond JH, Church TR, Snover
DC, Bradley GM, Schuman LM, et al. Reducing mortality from colorectal cancer by screening for fecal occult blood. New England Journal of Medicine. 1993; 328(19):1365-71.
13. Mandel JS, Church TR, Ederer F,
Bond JH. Colorectal cancer mortality: Effectiveness of biennial screening for fecal occult blood. Journal of the National Cancer Institute. 1999;91(5):434-7.
14. Mandel JS, Church TR, Bond JH, Ederer
F, Geisser MS, Mongin SJ, et al. The effect of fecal occult-blood screening on the incidence of colorectal cancer. New England Journal of Medicine. 2000; 343(22):1603-7.
15. Hope R, Chu G, Hope A, Newcombe R,
Gillespie P, Williams S. Comparison of three faecal occult blood tests in the detection of colorectal neoplasia. Gut. 1996;39(5):722-5.
16. John DJBS, Young GP, Alexeyeff
MA, Deacon MC, Cuthbertson AM,
Macrae FA, et al. Evaluation of new occult blood tests for detection of colorectal neoplasia. Gastroenterology. 1993;104(6): 1661-8.
17. Zhou D, Feng F, Zhang Y, Lai Z, Zhang W,
Li L, et al. Comparison of Shams' test for rectal mucus to an immunological test for fecal occult blood in large intestinal carcinoma screening. Analysis of a
check-up of 6480 asymptomatic subjects.
Chinese Medical Journal. 1993;106(10): 739-42.
18. Demers RY, Stawick LE, Demers P.
Relative sensitivity of the fecal occult blood test and flexible sigmoidoscopy in detecting polyps. Preventive medicine. 1985;14(1):55-62.
19. Illingworth D. Influence of diet on occult blood tests. Gut. 1965;6(6):595-8.
20. Barnett RN. The guaiac test: correlation with clinical findings. Gastroenterology. 1952;21(4):540-3.
21. Markman HD. Errors in the guaiac test for occult blood. JAMA. 1967;202(8):846-7.
22. Van Rossum LG, Van Rijn AF, Laheij
RJ, Van Oijen MG, Fockens P,
Van Krieken HH, et al. Random
comparison of guaiac and
immunochemical fecal occult blood tests for colorectal cancer in a screening population. Gastroenterology. 2008;135(1): 82-90.
23. Needham C, Simpson R. The benzidine
test for occult blood in faeces. QJM. 1952;21(2):123-34.
24. Deadman N. Evaluation of chromogens
suitable for occult blood in faeces. Clinica Chimica Acta. 1971;35(2):273-6.
25. Young GP, St John DJB, Winawer SJ, Rozen P. Choice of fecal occult blood tests for colorectal cancer screening:
Recommendations based on performance characteristics in population studies. The American Journal of Gastroenterology. 2002;97(10):2499-507.
26. Torre LA, Bray F, Siegel RL, Ferlay J, Lortet‐Tieulent J, Jemal A. Global cancer statistics. CA: A Cancer Journal for Clinicians. 2015;65(2):87-108.
27. Jemal A, Bray F, Center MM, Ferlay J,
Ward E, Forman D. Global cancer statistics. CA: A cancer Journal for Clinicians. 2011;61(2):69-90.
28. Center MM, Jemal A, Smith RA, Ward E.
Worldwide variations in colorectal cancer. CA: A Cancer Journal for Clinicians. 2009;59(6):366-78.
29. Baxter NN, Goldwasser MA, Paszat LF,
Saskin R, Urbach DR, Rabeneck L. Association of colonoscopy and death from colorectal cancer. Annals of Internal Medicine. 2009;150(1):1-8.
30. Hewitson P, Glasziou P, Watson E,
Towler B, Irwig L. Cochrane systematic review of colorectal cancer screening
using the fecal occult blood test
(hemoccult): An update. The American
Journal of Gastroenterology.
31. Walsh JM, Terdiman JP. Colorectal cancer
screening: scientific review. Jama. 2003; 289(10):1288-96.
32. Espey DK, Wu XC, Swan J, Wiggins C,
33. Levin B, Lieberman DA, McFarland B, Smith RA, Brooks D, Andrews KS, et al. Screening and surveillance for the early
detection of colorectal cancer and
adenomatous polyps, 2008: A joint
guideline from the American Cancer
Society, the US Multi‐Society Task Force on Colorectal Cancer, and the
American College of Radiology. CA: A Cancer Journal for Clinicians. 2008; 58(3):130-60.
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