March 2011 Clinical Laboratory News: Fecal Occult Blood Tests
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March 2011: Volume 37, Number 3

Fecal Occult Blood Tests
Life Savers or Outdated Colorectal Screening Tools?

By Callum G. Fraser, PhD

Recently, interest in colorectal cancer screening has exploded. Worldwide, this cancer is the third most common in men after lung and prostate cancers and ranks second in women after breast cancer. Nearly 60% of cases occur in developed countries: in the U.S., nearly 154,000 cases and 5,000 deaths were reported in 2008 (1). Significant colorectal neoplasia, defined as advanced adenomatous polyps and cancer, occurs mostly in individuals older than age 50, with incidence and mortality substantially higher in men than in women.

But there is good news, too. Increased awareness, lifestyle changes, and better detection methods have decreased incidence in the U.S., likely due to removal of pre-cancerous polyps during colonoscopy. While this procedure is generally considered the “gold standard,” there are a number of options for screening (2), including fecal occult blood testing (FOBT), flexible sigmoidoscopy alone or combined with FOBT, double contrast barium enema, and, more recently, fecal DNA testing and computed tomographic colonography.

Some of these modalities have a more robust evidence base supporting their use in colorectal cancer screening; however, experts agree that the existing data are insufficient to determine a single most efficient and effective screening strategy. Furthermore, current recommendations and guidelines produced by authoritative groups do not agree.

Fecal tests, particularly those that detect occult blood, remain a favored screening modality. In this article, the pros and cons of both traditional and newer FOBT will be discussed from a laboratory professional’s point of view.

Traditional FOBT

Many screening programs still use the traditional, low-sensitivity, guaiac FOBT (gFOBT). Because adenomatous polyps and cancers are thought to bleed intermittently, most gFOBT cards require two samples from three different feces. The individual applies the samples to six windows on the test card that contains filter paper impregnated with guaiac gum (Figure 1).

Figure 1
Example of a Traditional gFOBT Card

Individuals collect samples from three stools and place them directly onto a test card. The test detects heme from bleeding colorectal polyps. Shown is a hema-screen card manufactured by Immunostics, Inc.

Used with permission of the manufacturer.

The chemical reaction that takes place on the paper is based on the pseudoperoxidase activity of hemoglobin’s heme moiety. When peroxidase activity is present, the hydrogen peroxide in the alcohol developer solution releases oxygen, which then reacts with a component of the guaiac gum, producing an unstable blue or blue-green color.

gFOBT has a number of advantages. The test cards are inexpensive and widely available from many different manufacturers and suppliers in a range of different formats. Many cards also have integral positive and negative controls that identify user or test card problems. Another advantage is that heme in feces is relatively stable, at least after application to the card. Although light and very high temperature can affect the test cards and cause false-positive results, the analytical performance characteristics are very well documented.

Most importantly, gFOBT has the best documented evidence as a screening method for colorectal cancer. The authors of a comprehensive Cochrane review found substantial evidence in support of gFOBT, including a 16% reduction in the relative risk of colorectal cancer mortality (3).

However, gFOBT also has significant disadvantages (4). The guaiac reaction is not specific for human hemoglobin. Foodstuffs, particularly non-human blood and certain vegetables containing peroxidase activity, may cause false-positive results. Individuals therefore might be asked to follow dietary restrictions before collecting samples. A recent systematic review concluded, however, that these dietary restrictions do not affect the positivity rate. Consequently, dietary restrictions prior to sampling are not required in order to minimize the number of individuals who might not complete the test (5). Moreover, researchers have shown that the effect of any dietary plant peroxidases can be minimized by waiting at least 48 hours after collection to develop the test.

The effect of dietary iron on gFOBT remains controversial. Some researchers claim iron causes false-positive results, while others say it has no effect. Large amounts of ascorbic acid (vitamin C) in the diet also can potentially cause false-positive results, and individuals should discontinue taking this type of supplement before collecting fecal samples.

Several other controversies exist regarding causes of false-positives. For example, aspirin, non-steroidal anti-inflammatory drugs, and warfarin also may cause false-positives and lower the positive-predictive value of gFOBT. Therefore, some experts suggest that patients discontinue anti-coagulants if clinically feasible when collecting fecal samples.

Practical Matters

To ensure valid test results, screening program participants need to place fecal samples directly onto gFOBT cards. Heme in native feces is unstable and, if samples are collected in traditional laboratory sampling devices and then sent for testing, the delay will cause false-negative results and potentially result in a missed case of significant neoplasia.

Feces should be allowed to dry on the card before development takes place. If the sample is wet, the number of false-positives increases. One randomized, controlled trial used rehydrated fecal samples on gFOBT cards (3), and as expected, more disease was found but so were greater numbers of false-positives.

It is important to note that a positive gFOBT does not necessarily indicate colon cancer. Because heme is relatively stable, a positive gFOBT also might be due to bleeding from the upper gastrointestinal tract, not just the colon or rectum. Interestingly, a recent systematic review concluded that the current body of evidence is insufficient to recommend for or against routine esophagogastroduodenoscopy for gFOBT-positive, but colonoscopy-negative individuals. Clinical judgment in these cases is recommended (6).

Analytical Issues

Unfortunately, there’s no way to automate analysis of gFOBT cards. In addition, interpreting the often subtle color changes on cards with positive samples is difficult, especially for the inexperienced. Often, and wrongly, nurses, physicians, and others develop the cards. Indeed, one study raised concerns that clinical errors are being made solely because of problems in interpretation (7).

Manufacturers set the analytical sensitivity or detection limit of their gFOBT cards and therefore the positivity rate. Consequently, manufacturers also set the demand for colonoscopy, which is a vital consideration in many countries.

More importantly, however, gFOBT has less than ideal clinical specificity and sensitivity for significant neoplasia. False-positive (normal colonoscopy) and false-negative (interval cancer) results are common. A systematic review of the literature on repeated annual or biennial gFOBT revealed that the reported sensitivity for colorectal cancer varied from 51–100%, and specificity varied from 90–97%. Furthermore, the positive-predictive value—the percentage of positive tests that are true positive results—ranged from 2.4–17.0% (8).

Use of gFOBT in Practice

Unfortunately, gFOBT is oftentimes performed incorrectly in healthcare settings. A recent survey showed that family physicians in the U.S. do not routinely adhere to published guidelines on the use of these tests (9). Many still collect a single sample during a digital rectal examination (DRE), although this practice misses many cases of colorectal neoplasia. Moreover, they also reported using traditional gFOBT with all its disadvantages rather than newer tests with greater analytical sensitivity. In addition, some physicians repeated the test rather than referring individuals with positive results for colonoscopy.

An Australian study performed in a hospital reported similar misuse of the test (10). The authors found that the test was applied in clinically inappropriate settings without consideration of the confounding issues, often leading to inappropriate clinical decisions and considerable cost to the hospital and patient. They concluded that gFOBT had no utility in an acute hospital setting.

Evidence-based clinical guidelines in the U.K. unanimously recommend against using gFOBT to investigate symptomatic patients and instead advise clinicians to refer these patients directly for bowel visualization (4). Consequently, many hospital laboratories no longer perform gFOBT and actively discourage its use in wards, clinics, and by general practitioners.

Obsolete for Screening?

Today, many laboratorians and other health care workers have concluded that the disadvantages of traditional gFOBT outweigh the advantages. Furthermore, the value of the results are questionable considering how many clinicians misuse the test. Therefore, it is logical to ask whether traditional gFOBT is an outdated test?

Currently, although guidelines differ, one preferred option for colorectal cancer screening in the U.S. is high-sensitivity FOBT, either guaiac-based or immunochemical. Screening is conducted quite differently in Europe (11). Depending on local labor costs and the mechanism of kit distribution and collection, the authors of a recent article comparing U.S. and European strategies suggest that gFOBT might prove more practical and affordable in Europe than the recommended fecal immunochemical test (FIT).

Despite its shortcomings, gFOBT will likely be used for the foreseeable future. Therefore, it is important that all users be knowledgeable of the well-documented problems relating to diet, sample collection, handling and storage of test cards, and analytical procedures. In a recent article, I described why I believe that gFOBT should be treated exactly like other point-of-care laboratory tests (4).

Newer Fecal Occult Blood Tests

High-sensitivity gFOBT has been available for a number of years, and some guidelines advocate their use (12). These tests are not commonly used worldwide, however, and less is known about their analytical and clinical performance characteristics than traditional gFOBT (13). Although the evidence is convincing that the high-sensitivity tests detect more significant colorectal neoplasia, they also produce more false-positive test results and probably suffer from some of the disadvantages of traditional gFOBT.

Much more attention has been paid recently to FIT, sometimes termed the immunochemical fecal occult blood test (iFOBT). The former name is preferred since FIT and gFOBT have many differences and using dissimilar names emphasizes this fact.

Fecal Immunochemical Tests

A variety of guidelines in the U.S., Europe, and elsewhere now advocate using FIT for colorectal cancer screening (11–13). The test is based on monoclonal or polyclonal antibodies raised against the globin moiety of human hemoglobin that detect intact human hemoglobin or its very early degradation products. Manufacturers offer FIT in both qualitative (positive/negative) and quantitative (measurement of fecal hemoglobin content) formats. The analytical sensitivity of qualitative FIT is about 10 times lower than gFOBT. Furthermore, the test cards are more expensive, although reimbursement in the U.S. is greater.

Qualitative FIT has many important benefits (4). It detects more significant colorectal neoplasia than gFOBT, and it is more user-friendly. In general, individuals only need to sample one or two stools, thereby increasing test compliance. The test is available with card or tube-collection devices, with the latter format being more user-friendly and potentially enhancing compliance. The analytical specificity of the test means that dietary interferences are not a problem. FIT is also more specific for lower gastrointestinal bleeding since blood from the upper gastrointestinal tract is digested before feces are formed. Furthermore, laboratory analysis is simple and reliable using the widely familiar immunochemical test cassettes that typically have a positive control embedded.

Although qualitative FIT has many advantages compared to gFOBT, it also has some disadvantages. Analysis takes somewhat longer than gFOBT and cannot be automated. As with gFOBT, fecal samples must be placed directly onto or into the collection devices since fecal hemoglobin is very unstable. Furthermore, the stability of hemoglobin in tube collection devices is generally poor, so samples must be analyzed within a few days of collection.

Perhaps the biggest drawback of qualitative FIT is that the positivity rates are significantly greater than traditional gFOBT. Therefore, using qualitative FIT as a first-line screening test would result in large numbers of referrals to colonoscopy and would create untenable demands on some healthcare systems, especially in countries with limited resources.

Many studies have been published on FIT (13). A recent important study in The Netherlands directly compared automated FIT with gFOBT (14). This randomized, controlled trial included 20,623 participants from which a single sample was collected for FIT. The researchers found the positivity rates to be significantly higher for FIT (5.5%) compared to the gFOBT (2.4%), while the number of participants requiring colonoscopy to find one cancer was similar for both tests. However, and most importantly, as shown by many others (13), FIT detected significantly more adenomas and cancers.

Practical Matters and Automated Analysis

Before adopting any qualitative FIT, laboratorians need to be aware that commercially available tests differ widely. German researchers found that qualitative FIT from different manufacturers gave very different positivity rates, likely reflecting their different analytical sensitivity (15). The positivity varied from 6.4–46.8%, and overall agreement between the different tests was only poor to moderate. In addition to the variation in positivity rates, the tests also had different clinical performance characteristics.

Some of the disadvantages of qualitative FIT for large-scale screening programs can be alleviated using analytical systems. There are a number of these available (Figure 2) and they have been used for many years in Japan and other East Asian countries and, more recently, in Australia, Italy, and elsewhere. Many other countries have or are undertaking pilot studies that revolve around this screening strategy, often with the aim of changing from gFOBT to FIT.

Figure 2
Example of an Automated Quantitative FIT System

FIT analytical systems such as this one, the OC Sensor Diana (Eiken Chemical Company), facilitate high throughput of samples for colorectal cancer screening.

Used with permission of the manufacturer.

FIT analytical systems facilitate high throughput of samples, with lower imprecision, and eliminate analyst bias. They have tube-based collection devices that individuals find more acceptable (13). However, hemoglobin may not be very stable in the buffer, which may cause some cases of significant disease to be missed (16). Another major advantage is that screening program organizers can set the hemoglobin cut-off concentration to meet local requirements.

As expected, higher cut-off concentrations increase clinical specificity but decrease sensitivity. A number of excellent studies have shown this relationship between cut-off concentration and positivity rate and clinical performance (13). The number of samples that a patient must collect for analysis is also an important consideration. Not only does this factor into the number of samples the laboratory must process, but it also impacts the tradeoff between specificity and sensitivity. However, in a multicenter study of 20,596 average-risk subjects that investigated the effect of using different cut-off concentrations, as well as the effect of analyzing one versus two samples, researchers found that no strategy was clearly superior (17). More recent evidence also indicates that low hemoglobin cut-off concentrations detect advanced adenomatous polyps very well (13).

Here to Stay?

Today, various colorectal cancer guidelines recommend traditional gFOBT for asymptomatic populations screening programs. Evidence shows that their use does reduce mortality from the disease; however, evidence also has accumulated regarding significant problems collecting and analyzing fecal samples.

Despite the negative aspects of gFOBT, its use as a colorectal screening test will most likely continue for some time. Increasingly, however, FIT is becoming a widely favored option for replacing gFOBT. As stated in a recent editorial, the evidence is “in” regarding the benefits and potential of FIT (18). In particular, automated qualitative FIT is likely to become the favored approach worldwide, at least for large scale screening programs, because of its significant advantages (19). In particular, this test format detects adenoma, as well as colorectal cancer, from a single sample of feces with a high degree of sensitivity, and programs can set the cut-off hemoglobin concentration to give the most appropriate positivity rate, and thereby the desired clinical characteristics.


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Callum G. Fraser, PhD, is a senior advisor to the Scottish Bowel Screening Programme, honorary professor at the University of Dundee, and honorary consultant clinical biochemist to NHS Tayside, Scotland.


Disclosure: The author discloses that he has received honoria/expenses from Immunostics, Inc., Ocean, N. J. and Alpha Labs Ltd, Eastleigh, Hants, U.K.

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