Guidance for Industry and FDA Staff - Review Criteria for Assessment of Qualitative Fecal Occult Blood In Vitro Diagnostic Devices
Document issued on: August 8, 2007
This document supersedes “Review Criteria for Assessment of Fecal Occult Blood In Vitro Diagnostic Devices” issued on July 29, 1992
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U.S. Department of Health and Human Services
Contains Nonbinding Recommendations
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Table of Contents
- Risks to Health
- Types of FOB Tests
- Analytical Performance Characteristics
- Method Comparison
- Clinical Studies
Guidance for Industry and FDA Staff
Review Criteria for Assessment of Qualitative Fecal Occult Blood In Vitro Diagnostic Devices
|This guidance represents the Food and Drug Administration's (FDA's) current thinking on this topic. It does not create or confer any rights for or on any person and does not operate to bind FDA or the public. You can use an alternative approach if the approach satisfies the requirements of the applicable statutes and regulations. If you want to discuss an alternative approach, contact the FDA staff responsible for implementing this guidance. If you cannot identify the appropriate FDA staff, call the appropriate number listed on the title page of this guidance.|
This guidance is intended to provide device manufacturers and FDA staff with recommendations for 510(k) submissions for various types of fecal occult blood (FOB) tests. This document is a revision of the guidance, “Review Criteria for the Qualitative Assessment of Fecal Occult Blood In Vitro Diagnostic Devices,” issued on July 29, 1992. It is updated to address issues associated with new developments for FOB tests, including automated FOB analyzers.
FDA's guidance documents, including this guidance, do not establish legally enforceable responsibilities. Instead, guidances describe the Agency's current thinking on a topic and should be viewed only as recommendations, unless specific regulatory or statutory requirements are cited. The use of the word should in Agency guidances means that something is suggested or recommended, but not required.
The Least Burdensome Approach
The issues identified in this guidance document represent those that we believe should be addressed before your device can be marketed. In developing the guidance, we carefully considered the relevant statutory criteria for Agency decision-making. We also considered the burden that may be incurred in your attempt to follow the guidance and address the issues we have identified. We believe that we have considered the least burdensome approach to resolving the issues presented in the guidance document. If, however, you believe that there is a less burdensome way to address the issues, you should follow the procedures outlined in the document “A Suggested Approach to Resolving Least Burdensome Issues”.
A manufacturer who intends to market an FOB test should conform to the general controls of the Federal Food, Drug and Cosmetic Act (the Act), including the premarket notification requirements described in 21 CFR 807 Subpart E, and obtain a substantial equivalence determination from FDA prior to marketing the device. This guidance document identifies the classification regulation and product code for FOB tests and describes measures that, when followed by manufacturers, will generally lead to a timely 510(k) review and clearance. This document supplements other FDA documents regarding the specific content requirements of a 510(k) submission. You should also refer to 21 CFR 807.87 and other FDA resources on this topic, such as “Premarket Notification: 510(k)”.
As explained in “The New 510(k) Paradigm - Alternate Approaches to Demonstrating Substantial Equivalence in Premarket Notifications”, a manufacturer may submit either a Traditional 510(k) or an Abbreviated 510(k). An Abbreviated 510(k) provides a means to streamline the review of data in a 510(k) through a reliance on FDA-recognized consensus standards, special controls, or FDA guidance documents. Guidance on the content and format for abbreviated and traditional 510(k)s is available at "Format for Traditional and Abbreviated 510(k)s". Information regarding the use of standards can be found in Section 514(c)(1)(B) of the Act and the FDA guidance, “Use of Standards in Substantial Equivalence Determinations”. The Special 510(k) is available for manufacturers considering modifications to their own cleared devices. Information on how to prepare a Special 510(k) is available at "How To Prepare A Special 510(k)".
The scope of this document is limited to tests used to detect fecal occult blood. These tests are classified and identified under 21 CFR 864.6550, Occult blood test.
(a) Identification. An occult blood test is a device used to detect occult blood in urine or feces. (Occult blood is blood present in such small quantities that it can only be detected by chemical tests or by microscopic or spectroscopic examination.)
(b) Classification. Class II (performance standards).
The product code for fecal occult blood tests is: KHE – Reagent, Occult Blood
The recommendations in this guidance are intended for tests that detect analytes that indicate the presence of occult blood in feces. It is not intended for devices that test fecal material for the presence of cancer-specific analytes (e.g., tumor antigens, epigenetic biomarkers, or genotyping).
Failure of an FOB test to perform as indicated, or error in interpretation of results, may lead to improper patient management, including misdiagnosis and inappropriate treatment. For example, false negative results from an FOB test in patients who have asymptomatic cancer, adenoma, or other conditions could potentially give false reassurance and cause a delay in reporting symptoms and thus delay initiating treatment. False positive results could lead to performing unnecessary procedures (e.g., colonoscopy). The measures recommended to mitigate these risks are provided in this guidance document.
When designing, testing, or preparing labeling for the device, manufacturers should consider conditions that are known to lead to false test results:
- Intermittent bleeding and irregular distribution of blood in the feces may contribute to false negative results.
- False positive guaiac test results may occur from a diet of red or rare meats due to the pseudoperoxidase activity of myoglobin or blood present in the meat. Therefore, the American Cancer Society recommends avoiding red meats for three days before testing. The dietary restrictions on intake of raw fruits and vegetables containing peroxidases (e.g., cantaloupe, horseradish, radish, turnip, and broccoli) are recommended before guaiac testing, however, the value of such restrictions has not been established (Reference 1).
- Certain drugs such as aspirin and non-steroidal anti-inflammatory drugs may increase gastrointestinal bleeding and cause positive results. Iron compounds have been reported to cause false positive results. Ascorbic acid (vitamin C) taken at levels greater than 250 mg/day can cause false negative guaiac test results. Vitamin C in excess of 250 mg from either supplements or citrus fruits and juices should be avoided for three days before testing (Reference 1).
- The American Cancer Society cautions that a single FOB test done during a digital rectal exam in the doctor’s office is not sufficient for screening. The risk of a false negative result is mitigated by performing three separate tests on three consecutive bowel movements.
We recommend that you conduct a risk analysis prior to submitting your premarket notification to identify any other risks specific to your device. If you identify additional risks, or choose to use an alternative approach to address a particular risk identified in this document, you should provide sufficient information to support the approach you have used to address the risk(s).
There are two types of FOB tests that have been cleared by FDA:
The devices may be for prescription (clinical laboratories and physician offices) and/or over-the-counter use. The features of these FOB test types are described below.
The guaiac FOB slide test is a qualitative test. Guaiac is a phenolic compound derived from the wood resin of Guaiacum trees. The test principle is based on the oxidation of colorless phenolic compounds present in guaiac, to quinones, resulting in the production of a blue color. If blood is present in the stool sample, the hematin portion of the hemoglobin molecule functions as a pseudoenzyme, catalyzing the release of oxygen from hydrogen peroxide which in turn causes the oxidation of Guaiac and subsequent blue color.
Stool samples collected by the patient are smeared onto filter paper contained in the slides. The filter paper is allowed to dry. Once all the samples are collected, the slides are either returned to the physician’s office for development, or for over-the-counter tests, developed at home. Rehydration of stored guaiac slides does increase test sensitivity, however, rehydration also increases false positive results. Therefore, rehydration is not recommended (Reference 2).
“In-the-bowl” FOB tests work using the same principle as guaiac slide tests. The test consists of a tissue paper film coated with a chromogenic dye and peroxide. When the paper is dropped into the toilet bowl after a bowel movement, a color reaction detects the presence of blood from the stool.
False positive guaiac results may occur from a diet of red or rare meats and/or dietary peroxidases. Vitamin C (ascorbic acid) may cause false negative results.
Immunochemical tests for FOB utilize monoclonal and/or polyclonal antibodies to specifically detect the presence of human hemoglobin in feces. These tests may be automated or performed manually. Collected feces are scraped with a sampling probe and placed into a sample bottle containing extraction buffer. The sample bottle containing the resulting fecal extract is then tested.
Immunochemical tests for FOB are more specific and are not subject to interference from dietary peroxidases, animal blood and Vitamin C (ascorbic acid) as occurs with guaiac tests (Reference 3). However, hemoglobin protein tends to be altered chemically as it passes through the gastrointestinal tract, changing its antigenicity. The result is that a test may detect occult blood when it is the result of lower gastrointestinal tract bleeding, but may fail to detect upper gastrointestinal tract bleeding, producing a false negative result. In addition, monoclonal antibodies recognize single antigenic determinants and may not react positively with genetic variants of human hemoglobin, such as occur in sickle cell disease.
Sensitivity of Immunological Tests to Hemoglobin Variants
For immunological monoclonal antibody tests, you should demonstrate that the monoclonal antibody specific for hemoglobin equivalently recognizes variants of hemoglobin (e.g., hemoglobin SS and hemoglobin CC) in whole blood samples from persons with hemoglobinopathies that are prevalent in the United States (e.g., sickle cell disease). Otherwise, you should indicate in the Limitations section of the package insert that the test has not been validated for testing of patients with hemoglobinopathies.
Monoclonal and polyclonal antibodies that recognize human proteins may cross-react with epitopes on related molecules from other species. For monoclonal and polyclonal antibody-based immunological tests, you should demonstrate that the following immunologically-related substances do not interfere with the test:
- hemoglobin from beef, chicken, fish, horse, pig, rabbit, goat, and sheep
- myoglobin or ground up meat extracts prepared from beef, chicken, fish, horse, pork or ham, rabbit, goat, and sheep
If a specific type of meat extract yields a positive result with your immunological test, you should include cautions in the package insert against ingesting that type of meat before or during the testing period. Alternatively, you may choose to determine if cooked meat produces the same results. If the test yields negative results in this case, you should include clear instructions in the package insert that only meat that is well-cooked may be eaten. If cooked meat produces a false positive result, you may want to test people who have eaten meat to determine if digested meat still causes a false positive result after passing through the intestinal tract.
You should evaluate all “in-the-bowl” tests, as appropriate, for potential interference from peroxidase activity in foods that contain peroxidase, e.g., horseradish, red radish, raw turnip, cauliflower, broccoli, parsnip, and cantaloupe. Typically, these foods are acknowledged as contraindicated for guaiac tests and include a statement in the Limitation section of the package inserts with regard to this knowledge. Therefore, interference testing for peroxidase activity is not needed for guaiac tests.
You should evaluate all “in-the-bowl” tests and any FOB test where the sample is taken from toilet water, or potentially compromised by contact with toilet water, for potential interference from the following reagents commonly found in toilet water: toilet bowl refreshers, bluing agents, cleaners, deodorizers, fluoride, chloride, iron and other metals. Alternatively, if your FOB test is not an “in-the-bowl” test, you should demonstrate that your device includes step-by-step collection procedures that prevent contact with toilet water.
You should evaluate all FOB tests for potential interference from drugs that might be commonly used by patients tested for fecal occult blood, e.g., Vitamin C (ascorbic acid), therapeutic iron preparations.
You should include a cautionary statement in the Limitations section of the package insert specifying the types of potential interferents for which your test has not been evaluated.
For all FOB tests except “in-the-bowl” tests, you should demonstrate that the positive threshold, or cut-off, of your FOB test is the same as, or comparable to the cut-off of the predicate FOB test for which clinical data is available. If, for example, your test had a significantly lower cut-off than the predicate, samples that were considered negative for the predicate could be considered positive for the new test, and the clinical sensitivity and specificity of the predicate are not applicable to the new device. Re-evaluating the discrepant results using a third method is considered inappropriate (refer to “Guidance for Industry and FDA Staff - Statistical Guidance on Reporting Results from Studies Evaluating Diagnostic Tests”).
Comparison of the cut-off for the new device with the predicate cut-off may be accomplished by testing increasing dilutions of normal human hemoglobin in water or stool in side-by-side tests with the predicate test and comparing the results. The number of dilutions should be adequate to determine that the cut-off between the new test and the predicate test are comparable and reproducible. For this purpose, we recommend that you combine the reproducibility studies (described below) and the cut-off comparison study.
Reproducibility (Qualitative Tests)
You should evaluate the reproducibility of your test results in the setting for which your device is intended (i.e., in laboratories, in physician’s offices, or by lay users) based on the claims for your device.
You should have the intended users test replicates of serial dilutions of known concentrations of normal human hemoglobin in water or stool. The dilutions should be spaced sufficiently close around the cut-off to adequately determine the reproducibility of the results.
The following is an example of a protocol that may be performed to evaluate reproducibility. As mentioned above, the protocol used to evaluate reproducibility may be designed to simultaneously evaluate the cut-off. Dilute hemoglobin in distilled water to the following concentrations: 1 mg/mL, 2 mg/mL, 4 mg/mL, and 6 mg/mL (equivalent to 0.1, 0.2, 0.4, and 0.6 grams of hemoglobin per 100 grams of occult blood negative stool, respectively, and approximately 1, 2, 4, and 6 mL of blood per 100 grams of negative stool). Use these dilutions to test the sensitivity of the test and compare the reactions of this test to those of at least one other legally marketed test. We recommend you follow “User Protocol for Evaluation of Qualitative Test Performance; Approved Guideline,” CLSI/NCCLS document EP12-A.
Prozone (Hook) Effect (Immunological Tests)
In tests designed to measure the level of analyte using antibodies, the possibility for prozone effect exists. This is a phenomenon that occurs when the concentration of analyte is in such excess of the substrate that the formation of immune complexes is prevented. This results in the test incorrectly reporting a low level of analyte for the patient specimen.
You should determine the susceptibility of your test to prozone effects by testing samples with very high concentrations of occult hemoglobin. (It is not necessary to test a visibly bloody specimen.) You should state in the package insert the highest concentration of hemoglobin tested that yields a positive result with your device, i.e., the level below which no interference from prozone effect was observed.
You should describe the protocols for validating the expiration dates of your device. You should conduct real-time stability testing of your FOB test under conditions the test will be stored prior to use. You should perform studies to evaluate performance of the FOB test following exposure to various conditions such as elevated temperatures and multiple freeze/thaw cycles since these types of fluctuations can occur during shipping. You should perform these evaluations with multiple lots. You should provide the acceptance criteria for each study.
Specimen collection and handling
You should substantiate the recommendations in your labeling regarding specimen collection, storage, and transport by demonstrating that the device produces the same results at the extremes of the storage times and temperatures recommended to users. For example, an appropriate study may compare the results obtained from matched specimens evaluated at various time intervals, exposed to different temperatures, and that have undergone a number of freeze/thaw cycles consistent with what is specified in the package insert. You should evaluate these conditions using samples that are close to the cut-off. You can use spiked samples for these analyses. You should state the acceptance criteria for your results as well as the temperature ranges (and other environmental conditions) you recommend in the package insert.
Over the Counter Tests
For over the counter tests, we recommend that you conduct studies to demonstrate that at least 50 lay persons over the age of 50 with various educational backgrounds can understand the package insert and interpret the test results as accurately as laboratory professionals. Package inserts for over-the-counter tests should be written for a 7th grade reading level. We recommend that you have each person collect their own specimen and use the test following only the package insert directions, i.e., without additional explanation from the manufacturer. We recommend that you have each individual provide the results of his or her testing to the manufacturer and then have a health care professional familiar with the test procedure evaluate the specimen to determine whether the specimen was adequately collected.
We also recommend that you have each lay user respond to questions that evaluate how well he/she understood the information provided in the package insert. We suggest that you contact FDA prior to beginning your study to review your package insert and questions in order to obtain feedback on whether they appropriately assess the user’s ability to understand the test.
Lay users should also be asked to interpret the results of a variety of test specimens, including borderline positive samples. Duplicate specimens should be interpreted by personnel experienced in evaluating the type of device being tested and the results compared to those of the lay reader.
It may be helpful to refer to “Guidance on Medical Device Patient Labeling; Final Guidance for Industry and FDA Reviewers”, and the guidance document “Human Factors Principles for Medical Device Labeling” for additional information related to this subject.
For software controlled devices, including automated analyzers, we recommend that you provide validation and verification testing, as well as a hazard analysis as described in FDA’s “Guidance for the Content of Premarket Submissions for Software Contained in Medical Devices”. You may also refer to “Medical Device Use-Safety: Incorporating Human Factors Engineering into Risk Management”, or “General Principles of Software Validation”.
You should provide a description of the procedural or built-in controls for your FOB test. Over-the-counter FOB tests must incorporate positive and negative controls. You should identify the components of the internal control and describe the function of the control in detail.
Examples of descriptions include:
- The control determines if chemicals are working properly.
- The control determines if an adequate amount of sample was added.
- The control determines if the proper procedure was followed.
You should validate that the controls perform as expected and correctly monitor the appropriate variables as claimed in the labeling.
For guaiac tests and immunological tests, you may compare your device to a predicate device with a similar test methodology by performing the reproducibility and cut-off studies described above. However, for immunological tests, you should also demonstrate that the results obtained with your device are substantially equivalent to those obtained with a predicate device that has known clinical outcome data. Results are typically presented as positive, negative, and overall percent agreement between the two methods.
For some FOB tests, test results may need to be compared directly to clinical outcome data to establish substantial equivalence (see Section 8, Clinical Studies below).
In some cases, method comparison to a predicate is not sufficient. For “in-the-bowl” tests, devices with new technologies, or tests claiming to have greater analytical sensitivity than existing tests, we recommend that you provide clinical data, i.e., testing of actual clinical specimens from patients with a confirmed diagnosis. We suggest you contact the FDA (the contact information of an appropriate staff member is provided at the beginning of this document) for more information on whether clinical data is required for your test.
When performing clinical studies, you should compare results obtained with the new device using patient samples to the results obtained with a reference clinical method such as colonoscopy, sigmoidoscopy, and/or biopsy, as appropriate. You should include specimens from patients with a variety of gastrointestinal disorders, (e.g., cancer, polyps, adenomas, anemia, diverticulitis,) and summarize the results in terms of clinical sensitivity and specificity.
You should describe the following in the 510(k):
- All clinical protocols and statistical analyses.
- A clear description of the samples and patient inclusion and exclusion criteria, including whether samples are chosen from patients with specific clinical outcomes or risk profiles pertinent to the indications for use. The test population should include subjects for whom the test is intended, e.g., men and woman age 50 and over, and individuals with risk factors.
- Statistical support for the sample size for each sub-group (clinical or demographic) you include in your study. You should test a minimum of 120 confirmed normal specimens. We recommend you follow “How to Define and Determine Reference Intervals in the Clinical Laboratory; Approved Guideline,” CLSI/NCCLS document C28-A2.
FDA offers an opportunity to obtain input about the studies you are planning to conduct to support your premarket submission. This process is referred to as a pre-IDE review. You may contact the Division of Immunology and Hematology to inquire whether it may be beneficial for you to gain feedback on your study plan prior to initiating clinical studies.
The premarket notification should include labeling in sufficient detail to satisfy the requirements of 21 CFR 807.87(e). The following suggestions are aimed at assisting you in preparing labeling that satisfies the requirements of 21 CFR 807.87(e)1.
The intended use statement should be compatible with the performance characteristics of the assay. It should include the type of FOB test, state that the test is qualitative, indicate where the test may be performed (laboratory, physician office, or over-the counter) and specify the patient population for which the device is indicated.
Directions for use
You should include clear and concise instructions that describe the technological features of the device and how the device is to be used by patients.
Specimen Collection and Patient Preparation
The following list describes the types of information you should provide in the labeling:
- Instructions for collecting the sample, e.g., a stool sample may be collected in a clean cup, on collection paper, on toilet paper, from the toilet bowl.
- Instructions as to how many times the specimen is to be sampled, e.g., “Randomly insert the Applicator Stick into the fecal sample two times.” Also provide instructions as to how much sample is necessary, for example, “A thin smear should be applied to each of two guaiac windows.”
- Information as to how many times the test should be performed. The American Cancer Society recommends home collection of two samples from three consecutive specimens for a total of 6 samples. (Reference 1).
- Special dietary precautions, as appropriate. For example, “All raw or red meat, as well as high peroxidase containing fruits and vegetables, can cause false positive results. Therefore, the patient should avoid all raw and rare red meats, horseradish, raw fruits and vegetables like broccoli, cauliflower, red radish, cantaloupe, parsnips and turnips, or other high peroxidase containing vegetables beginning three days prior to testing and continuing throughout the test period. It is recommended that the patient consume a high residue diet (high fiber, high bulk) because it provides roughage to help uncover silent lesions that may bleed intermittently, while also increasing the likelihood of a soft stool for greater ease in obtaining the sample. An acceptable diet may include cooked fruit and vegetables such as spinach and corn as well as lettuce, prunes, grapes, and apples. Cereal and well cooked fish and fowl are also acceptable. These dietary measures will help improve the accuracy of the results obtained.”
- Instructions for avoiding erroneous results due to physical conditions that may interfere with the test. For example, “Specimens should not be collected while the patient has bleeding hemorrhoids or constipation, cuts on hands, or during or immediately after a menstrual period. Hands and test area should be kept clean and free from blood to avoid false positive results.”
- Instructions for patients to avoid interfering substances such as non-steroidal anti-inflammatory drugs, e.g., ibuprofen, naproxen, or aspirin (more than one adult aspirin per day) for seven days prior to testing, and vitamin C in excess of 250 mg from either supplements or citrus fruits and juices, for three days prior to testing (Reference 1).
- Instructions regarding stability of the specimens, including storage, handling or shipping instructions for the protection and maintenance of specimens. For example, “Guaiac slides prepared by the patient should be allowed to dry before development. No more than 4 to 6 days should elapse between preparation and testing. Patients should be instructed to return all slides to the physician or laboratory as soon as possible. The stored pad containing the sample should be protected from excessive heat, sunlight, fluorescent light, volatile chemicals, and humidity.”
You should thoroughly discuss the limitations of the assay. You should list important test limitations and all known contraindications previously noted as well as any interfering conditions mentioned under the Specimen Collection section. You should also discuss any recommended practice or other expert recommendations concerning the limitations of FOB tests for the intended use. Examples of such limitations are listed below:
- Results are not conclusive evidence of the presence or absence of gastrointestinal bleeding or pathology.
- Test results can only be regarded as a preliminary screening or as an aid to diagnosis. It is not intended to replace other diagnostic procedures such as colonoscopy, sigmoidoscopy, or other x-ray studies.
- Because gastrointestinal lesions may bleed intermittently and blood in feces is not distributed uniformly, a negative test result does not assure absence of lesions.
- Any positive result should be followed up with further studies to establish the source of bleeding.
- Guaiac slide tests should not be rehydrated before development because this causes excessive false positive results.
- A protocol for collecting and testing 2 samples from three consecutive stools (total of 6 samples) at home is recommended.
- FOB testing is recommended annually by the American Cancer Society (2001) for average-risk women and men, 50 years of age and older. However, patients with significant risk factors such as family history of colorectal cancer should be screened earlier and more often.
Interpretation of Results
You should explain how to interpret positive and negative results, including their clinical significance. You should provide instructions for reading results, e.g., “Read results within five (5) to ten (10) minutes. Do not read after 10 minutes.”
You should provide directions for the interpretation of the results of controls (performance monitors). Provide a statement that if controls do not perform as expected, assay results are invalid.
1. Smith RA, von Eschenbach AC, Wender R, Levin B, Byers T, Rothberger D, Brooks D, et al. American Cancer Society Guidelines on Screening and Surveillance for the Early Detection of Adenomatous Polyps and Colorectal Cancer. CA-A Cancer J Clin. 2001; 51:44-54.
2. American College of Physicians. Suggested technique for fecal occult blood testing and interpretation in colorectal cancer screening. Ann Intern Med 1997; 126:808-10.
3. Lang CA, Ransohoff DF. Fecal occult blood screening for colorectal cancer. Is mortality reduced by chance selection for screening colonoscopy? JAMA 1994; 271:1011-3.
1Although final labeling is not required for 510(k) clearance, final labeling must comply with the requirements of 21 CFR §801 (or 21 CFR § 809.10) before a medical device is introduced into interstate commerce. Labeling recommendations in this guidance are consistent with the requirements of part 801 and section 809.10.