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A Quantitative Immunochemical Fecal Occult Blood Test for Colorectal Neoplasia

Zohar Levi, MD; Paul Rozen, MBBS; Rachel Hazazi, BSc; Alex Vilkin, MD; Amal Waked, BSc; Eran Maoz, MD; Shlomo Birkenfeld, MD; Moshe Leshno, MD, PhD; and Yaron Niv, MD
[+] Article and Author Information

From Rabin Medical Center, Beilinson Hospital, Petach Tikva, Israel; Clalit Health Services, Tel Aviv and Bat-Yam, Israel; and Tel Aviv University Medical School, Tel Aviv, Israel.


Note: These results were presented, in part, at Digestive Diseases Week, Los Angeles, California, 20–25 May 2006.

Acknowledgments: The authors thank the medical and secretarial staff of the endoscopy units and the patients for their cooperation. They also thank Dr. Ester Shabtai and Doron Comaneshter for statistical analyses, Ms. Ziona Samuel for helping with patient enrollment, and Ms. Sally Zimmerman for secretarial assistance.

Grant Support: The Eiken Chemical Company, Alfa Wasserman, and Pharmatrade provided instruments and reagents. Research grants from the Eiken Chemical Company and the Katzman Family Foundation supported other costs.

Potential Financial Conflicts of Interest: Grants received: P. Rozen (Eiken Chemical Co.).

Requests for Single Reprints: Paul Rozen, MBBS, Department of Gastroenterology, Rabin Medical Center, Beilinson Hospital, 39 Jabotinsky Street, Petach Tikvah 49100, Israel; e-mail, paulro@clalit.org.il.

Current Author Addresses: Drs. Levi, Vilkin, and Niv; Ms. Hazazi; and Ms. Waked: Rabin Medical Center, Beilinson Campus, PO Box 85, Petach Tikva 49100, Israel.

Dr. Rozen: Department of Gastroenterology, Rabin Medical Center, Beilinson Hospital, 39 Jabotinsky Street, Petach Tikvah 49100, Israel.

Dr. Maoz: Clalit Health Services, Zamenhof Medical Center, 34 Zamenhof Street, Tel Aviv, Israel.

Dr. Birkenfeld: Clalit Health Services, Bat Yamon Medical Center, 33 Nusinson Street, Bat-Yam, Israel.

Dr. Leshno: Recanti Building, Tel Aviv University, Ramat Aviv, Israel.

Author Contributions: Conception and design: P. Rozen, Y. Niv.

Analysis and interpretation of the data: Z. Levi, P. Rozen, A. Waked, R. Hazazi, M. Leshno, Y. Niv.

Drafting of the article: P. Rozen, R. Hazazi, M. Leshno.

Critical revision of the article for important intellectual content: P. Rozen, M. Leshno, Y. Niv.

Final approval of the article: P. Rozen, R. Hazazi, A. Vilkin, S. Birkenfeld, Y. Niv.

Provision of study materials or patients: A. Vilkin, Z. Levi, E. Maoz, S. Birkenfeld, Y. Niv.

Statistical expertise: M. Leshno.

Obtaining of funding: P. Rozen, Y. Niv.

Administrative, technical, or logistic support: R. Hazazi, A. Vilkin, Z. Levi, A. Waked, Y. Niv.

Collection and assembly of data: Z. Levi, R. Hazazi, A. Vilkin, A. Waked.


Ann Intern Med. 2007;146(4):244-255. doi:10.7326/0003-4819-146-4-200702200-00003
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We performed a detailed evaluation of an immunochemical method to measure the amount of occult blood in fecal samples. When using the hemoglobin threshold of 75 ng/mL or greater to define an abnormal result, the test's sensitivity was 94.1% for detecting colorectal cancer and 67.0% for detecting all clinically significant neoplasia with corresponding specificities of 87.5% and 91.4%, respectively. The amount of fecal hemoglobin in most nonadvanced adenomas was less than 75 ng/mL. This is an advantage, because colonoscopy screening has been criticized for identifying many persons with adenoma who are then entered into a labor-intensive and expensive adenoma follow-up protocol (27). In our heterogeneous study sample, the positive predictive value of 43.9% meant that almost every second colonoscopy performed for a positive immunochemical FOBT result diagnosed a clinically significant neoplasm.

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Figures

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Figure 1.
Study flow diagram.

I-FOBT = immunochemical fecal occult blood test; IBD = inflammatory bowel disease.

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Appendix Figure 1.
Folded paper “float” opened (left) and placed in toilet bowl (right).

After defecation and fecal sampling, the participant flushes the float into the toilet.

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Appendix Figure 2.
Stool probe and fecal sample storage tube.

The patient removes the fecal probe that has a serrated tip that accumulates the fecal sample. The probe is then reinserted deeper into the tube past a scraper and through a membrane that removes excess feces. The bottom compartment of the tube contains a 2-mL buffer solution for stabilizing the fecal specimen in the tip serrations.

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Figure 2.
Histograms of fecal hemoglobin density.

Using a log2 scale, the analysis demonstrates statistically significantly different values for advanced polyps, cancer, or clinically significant neoplasia compared with normal and hyperplastic polyps or nonadvanced polyps, with little overlap of the latter 2 conditions. The mean fecal hemoglobin value (SD) and 95% CI are indicated for each category. The 75-ng/mL hemoglobin threshold for calling a test result positive or negative is indicated in each category (dashed lines). The vertical bars represent the proportion that has the specified immunochemical fecal hemoglobin level.

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Figure 3.
Scatterplot illustrating the joint distribution of the first 2 immunochemical fecal occult blood test (I-FOBT) measurements on a log2-transformation scale.

The internal lines are at a 75-ng/mL hemoglobin threshold for calling a test result positive or negative, and numbers in each section show classifications of patients based on this cutoff. Each symbol represents a pair of first and second fecal hemoglobin measurements for one patient. A total of 847 patients without colorectal cancer (CRC) or advanced adenomatous polyps (APs) (open circles) were classified below the 75-ng/mL value as having negative test results for both measurements, while 37 patients with CRC or AP (open triangles) also had negative test results. Twenty-one patients without CRC or AP were classified above the threshold as having positive test results both times, as were 32 patients with CRC or AP. Seventeen and 20 patients without CRC or AP were below the cutoff value for 1 measurement but were above the cutoff value for the other measurement. This also occurred for 11 patients with CRC or AP. The correlation coefficient for the first and second I-FOBT (I-FOBT 1 and I-FOBT 2, respectively) samples was 0.597.

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Figure 4.
Receiver-operating characteristic curves for sensitivity and specificity for colorectal cancer (top) and clinically significant neoplasia (cancer and advanced polyps) (bottom).

Each line represents the highest fecal immunochemical hemoglobin measurement from the specified sequence of fecal samples collected and their added value to provide maximum sensitivity and specificity. I-FOBT 1 = first immunochemical fecal occult blood test collected; I-FOBT 2 = first 2 I-FOBTs collected; I-FOBT 3 = all 3 I-FOBTs collected. Overall, there was no difference between collecting 2 or 3 I-FOBTs. However, the highest sensitivity was obtained when the fecal hemoglobin measurements of all 3 I-FOBTs were used at a 75-ng/mL threshold for calling a test result positive or negative (Appendix Table 2). AUC = area under the curve.

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Figure 5.
The posttest probability after a positive test result (top) and negative test result (bottom) for clinically significant neoplasia (colorectal cancer or advanced adenomatous polyps).

The values are presented as a function of the immunochemical fecal occult blood test (I-FOBT) threshold for calling a test result positive or negative, with alternate pretest probabilities of colorectal cancer or advanced adenomatous polyps of 0.05, 0.10, 0.25, 0.50, and 0.75. For every I-FOBT threshold value, the sensitivity and specificity (also presented on the receiver-operating characteristic curve) were calculated, as well as positive and negative likelihood ratios. Posttest probabilities for a positive I-FOBT result (top) and for a negative I-FOBT result (bottom) were calculated.

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Figure 6.
Fagan nomogram.

The nomogram provides a graphical tool for estimating how much the result of a test changes the probability that a patient has the disease. To use the nomogram, one should first estimate the probability of the disease before testing, which is often the disease prevalence, although it may be increased or decreased on the basis of clinical findings in the patient population or in a particular patient. The next step is to determine the likelihood ratio corresponding to the diagnostic test result. A line is drawn connecting the pretest probability and the point on the middle vertical line corresponding to the likelihood ratio for the test result (represented by a range of test results [boxes]). This line is extended to intersect with the right-hand vertical line, which gives the posttest probability. This point is the new estimate of probability that the patient has the disease. For example, in this heterogeneous-risk, symptomatic study sample, clinically significant neoplasia was found in 91 of 1000 patients (prior probability, 0.091). For a patient with an immunochemical fecal occult blood test (I-FOBT) result greater than 150 ng/mL, the sensitivity and specificity of the test were 54% and 95%, respectively, providing a likelihood ratio of 10.881 (0.54/[1 − 0.95]), and posttest probability was 52% (dashed line). However, a patient with a pretest probability of 0.002, which is similar to that in a low-risk screening population, and an I-FOBT result between 51 and 75 ng/mL, for which the likelihood ratio is 1.92, would have a posttest probability of advanced neoplasia of 0.38% (dotted line). These demonstrate and emphasize how the level of I-FOBT influences the posttest probability of clinically significant neoplasia. The clinical observations behind the likelihood ratios in the nomogram are available in Appendix Table 3.

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Appendix Figure 3.
The desktop OC-MICRO instrument.

The instrument is 32 cm wide, 53 cm deep, and 42 cm high. The top left bottle contains diluting solution, the center bottle contains cleaning fluid, and the right bottle contains distilled water. Two trays are loaded into the instrument. One tray holds 10 patient-prepared immunochemical fecal occult blood test tubes, and the other tray is for tubes where the immunochemical fecal occult blood test–antibody reaction occurs. The hemoglobin value is automatically calculated from the resulting turbidity. (The OC-MICRO is manufactured by Eiken Chemical Co., Tokyo, Japan.)

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Comments

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Immunochemical Fecal Occult Blood Test
Posted on March 1, 2007
Tetsuji Fujita
Jikei University School of Medicine
Conflict of Interest: None Declared

TO THE EDITOR: In 1000 consecutive ambulatory patients at moderate to high risk for colorectal disease, Dr. Levi and colleagues showed that the quantitative immunochemical fecal occult blood test (FOBT) had good sensitivity and specificity to detect clinically significant neoplasia (1). The authors are to be congratulated on making the nomogram, by which we could estimate the posttest probability of clinically significant neoplasia not only in symptomatic patients but also in the general population.

A colonoscopy was completed in 93 % of their patients, but the rate of complication related to colonoscopy is not described in the article. Although colorectal cancer incidence rates increase with age and the diagnostic yield of colonoscopy is higher in the elderly group (2), the lifetime risk of colorectal cancer death actually decreases in persons with advancing age and increasing comorbidity (3). The risk of colonoscopy complications appears to increase with patient age and cormorbidity (4). Therefore, in elderly patients with poor health, the risks of invasive colorectal cancer screening may outweigh the benefits. I think the nomogram should not be used to determine whether to offer colonoscopy to these patients whose life expectancy is relatively limited.

Tetsuji Fujita, MD Jikei University School of Medicine Tokyo, Japan 105-8461

References

1. Levi Z, Rosen P, Hazazi R, Vilkin A, Waked A, Maoz E, et al. A quantitative immunochemical fecal occult blood test for colorectal neoplasia. Ann Intern Med. 2007; 146: 244-55.

2. Karajeh MA, Sanders DS, Hurlstone DP. Colonoscopy in elderly people is a safe procedure with a high diagnostic yield: a prospective comparative study of 2000 patients. Endoscopy. 2006; 38: 226-30.

3. Ko CW, Sonnenberg A. Comparing risks and benefits of colorectal cancer screening in elderly patients. Gastroenterology. 2005; 129: 1163-70.

4. Mandelblatt JS, Schechter CB, Yabroff KR, Lawrence W, Dignam J, Extermann M, et al. Toward optimal screening strategies for older women. J Gen Intern Med. 2005; 20: 487-96.

Conflict of Interest:

None declared

Quantitative Immunochemical Fecal occult blood test for diagnosing Colorectal neoplasia
Posted on March 27, 2007
Yogesh M Shastri
Medizinische Klinik I-ZAFES, J. W. Goethe-University Hospital,
Conflict of Interest: None Declared

Dear Editor

We read with great interest the article by Levi et al(1) and must congratulate them for a nice piece of work. They have introduced really a unique concept of offering the possibility of having an option of different positivity thresholds for performing quantitative immunochemical fecal occult blood test (IFOBT). We have certain queries and comments to them. In the introduction section of their article they have mentioned that none of the English language publication study has systematically compared colonoscopy with the fecal immunochemical method for diagnosing CRN (colorectal neoplasia).We would like to bring to their attention that our group has published a study on the similar theme in 2006(2).

In our study 389 patients from 4 different centers in Germany, who were referred for colonoscopy, underwent 3 stool tests; a Guaiac based FOBT, a quantitative ELISA based IFOBT and a bedside qualitative Immunochemical strip test. The performance characteristics of the quantitative IFOBT in both these studies(1, 2) were quite similar, a sensitivity of 67% vs. 64% and specificity of 91% vs. 96% for diagnosing significant CRN in Levi's and our study respectively.

However there were some differences in both these studies (1, 2). Although in the quantitative ELISA based IFOBT in our study we did not evaluate the different range of cut offs threshold as evaluated by Levi. The fixed cut off threshold for positivity of ELISA in our study was 10 ng Hemoglobin/ml as against a range of cut-off 50 ng/ml to 150 ng/ml as that of in Levi's study. While the qualitative strip test evaluated by us was a manual bedside test(2, 3) with a fixed threshold of detection of up to 10ng/ml of Hemoglobin in stool.

Another apprehension about their study method(1) is the need for 3 stool sample collection. It's an accepted fact that increased number of stool sample will reduce the compliance of the screening as its cumbersome and may shy away many of the screenees. Also, it might pose the issue of acceptability for the patients and also might pose difficulties logistically at field level when this test is applied to the masses during CRC screening for average risk population.

As the cost of the test for performing CRC screening is also a very important factor. Some more elaboration on the costs incurred in performing this automated quantitative IFOBT would be appreciated. Does the $20 cost mentioned in the paper include the net charges to the patient? Does it also consider the other expenditure like the recurring cost of reagents, administrative costs and one time cost of the machine (analyzer) etc?

Sincerely - Yogesh Shastri, MD, DNB

- Jurgen Stein, MD, PhD

References: 1. Levi Z, Rozen P, Hazazi R, et al. A quantitative immunochemical fecal occult blood test for colorectal neoplasia. Ann Intern Med. 2007;146(4):244-55. 2. Hoepffner N, Shastri YM, Hanisch E, et al. Comparative evaluation of a new bedside faecal occult blood test in a prospective multicentre study. Aliment.Pharmacol.Ther. 2006;23(1):145-154. 3. Trojan J, Povse N, Schroder O, Stein J. A new immunological test strip device for the rapid, qualitative detection of faecal occult blood. Z.Gastroenterol. 2002;40(11):921-924.

Conflict of Interest:

None declared

IN RESPONSE:
Posted on April 18, 2007
Paul Rozen
Rabin Medical Center
Conflict of Interest: None Declared

April 17, 2007

IN RESPONSE: We thank Drs. Shastri and Stein for their comments and their very relevant questions which provide us with an opportunity to address several important clinical issues. Firstly, our comment on "systematic evaluation" refers to the automated developed and quantitative immunochemical FOBT (I-FOBT) (1). As they point out, they, we and others have evaluated the office-developed I-FOBT immunochemical test (2,3). However, this test uses a fixed threshold, set by the manufacturer, and the hand-development is not conducive for large scale population screening. Evaluating the I-FOBT as a laboratory test, with a quantified result, gives the treating physician the ability to use his clinical judgement as he would for any other laboratory test. Secondly, the number of requested stool samples definitely influences compliance. In our study population, who are used to collecting three guaiac FOBTs, this was not an issue. However, we are now completing a study on an average-risk population and this will allow us to evaluate compliance in that segment of the population who, to date, have not participated in FOBT screening. Thirdly, the maximum cost of FOBT screening is set by our Ministry of Health as this is a test available for the total population through their own HMO. The $20 cost is the total cost per patient that was estimated by the local agent for this I-FOBT. This will need to be budgeted by the HMO which is required to provide, at no cost, an annual FOBT to their average- risk members. References:

1. Levi Z, Rozen P, Hazazi R, Vilkin A, Waked A, Maoz E, Birkenfeld S, Leshno M, Niv Y. A quantitative immunochemical fecal occult blood test for colorectal neoplasia. Ann Intern Med. 2007;146:244-55. 2. Hoepffner N, Shastri YM, Hanisch E, Rosch W, Mossner J, Caspary WF, Stein J. Comparative evaluation of a new bedside faecal occult blood test in a prospective multicentre study. Aliment Pharmacol Ther 2006;23:145- 154. 3. Rozen P, Knaani J, Samuel Z., Comparative screening with a sensitive guaiac and specific immunochemical occult blood test within an endoscopy study. Cancer 2000;89:46-52. Paul Rozen, MB.BS Zohar Levi, MD Yaron Niv, MD, FACG, AGAF

Conflict of Interest:

None declared

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