A test for occult blood is one of the most informative methods for diagnosing diseases of the gastrointestinal tract. This study allows you to detect microscopic bleeding that is not visible to the naked eye, which is critical for the early diagnosis of ulcers, polyps and oncological processes. However, the reliability of the result directly depends not only on the quality of the reagents in the laboratory, but also on how correctly the patient collected and preserved the biomaterial until delivery.
One of the most common questions that arises from patients is shelf life the collected material. Many people mistakenly believe that feces, like many foods, can remain unchanged in the cold for a long time. In fact, biological samples are subject to active processes of fermentation and decay, which can distort the chemical picture. The optimal delivery time is considered to be 2 to 4 hours after defecation, but when using special preservatives, this period can be increased to 12-24 hours.
In this article We will analyze in detail how the temperature regime affects the safety of red blood cells in the sample, why the material should not be frozen, and what errors most often lead to false positive or false negative results. Understanding these nuances will help you avoid repeated testing and get an accurate diagnosis the first time.
Why storage time is critical for analysis
Feces are a complex mixture of undigested food debris, bacteria, epithelial cells and waste products of microorganisms. Immediately after leaving the body, active biochemical processes continue in the sample. The enzymes contained in the digestive juice begin to break down proteins, and bacteria begin to actively multiply. If the right conditions are not provided, erythrocytes (red blood cells), which are what laboratory technicians are looking for, begin to deteriorate.
The destruction of blood cells occurs under the influence of the aggressive environment of the intestines and bacteria. The longer the sample is at room temperature, the fewer intact (whole) red blood cells remain in it. When conducting a chemical test (for example, the Gregersen method), this can lead to a false negative result: there is blood in the body, but in the laboratory it is no longer “seen”, since hemoglobin has changed its structure.
In addition, long-term storage contributes to a change in the acidity (pH) of the environment. This is especially important for immunochemical methods, which are more sensitive to environmental conditions than classical reactions with benzidine. A change in pH can activate or, conversely, suppress the reaction of reagents with hemoglobin, which will make the result uninformative.
Temperature: can it be frozen material
The question of what temperature to store a container at often causes controversy. The standard recommendation for most biological fluids is cooling, but with feces for occult blood the situation has its own characteristics. The basic rule is: freezing is strictly prohibited. Ice crystals formed during freezing destroy the cell walls of red blood cells. After thawing, the sample will contain a mess of cellular debris, and it will be impossible or extremely difficult to determine the presence of blood.
The optimal storage mode is the temperature of the refrigerator, but not the freezer. The temperature range should be from +2 to +8 degrees Celus. Under such conditions, the processes of bacterial reproduction and fermentation slow down significantly, which allows the structure of hemoglobin to be preserved for a longer period. However, even in the refrigerator, the sample cannot be kept forever.
⚠️ Attention: Do not place the container with biomaterial on the shelf of the refrigerator door. When the door is opened, the temperature there fluctuates sharply, which can lead to partial thawing of the condensate or, conversely, local freezing near the wall. Use the middle shelves of the main chamber.
If you are using special transport containers with refrigerants (often provided by paid laboratories), follow the instructions on the package. Some modern systems allow sample storage at room temperature thanks to stabilizing buffers, but this is the exception and not the rule. In a standard plastic container from a pharmacy, cold is necessary.
Dependence of the period on the type of reagent
The period during which the analysis remains reliable, directly depends on the research method used by the laboratory. There are two main approaches: chemical (Gregersen reaction) and immunochemical (iFOBT). They have different requirements for the stability of hemoglobin.
The chemical method is based on the ability of hemoglobin, having peroxidase activity, to oxidize reagents. This method is less specific, but more durable in terms of timing if the sample is refrigerated. However, it reacts to both human and animal hemoglobin and therefore requires a strict diet. The immunochemical method looks for specific antigens of human hemoglobin. It is more accurate, but the antigen protein itself undergoes denaturation faster if stored incorrectly.
The table below shows the dependence of the permissible storage time on the conditions and type of analysis:
| Storage conditions | Chemical method (Gregersen) | Immunochemical method (iFOBT) |
|---|---|---|
| Room temperature (+20...+24°C) | No more than 3 hours | No more than 2 hours |
| Refrigerator (+2...+8°C) | Up to 6-8 hours | Up to 12 hours (maximum 24) |
| With preservative (special container) | Up to 3-5 days | Up to 7-14 days |
| Frozen (-18°C and below) | Unacceptable | Unacceptable |
The table shows that the presence of a preservative radically changes the situation. If you are tested as part of screening programs (for example, mailed kits), you will most likely be given a tube with a stabilizer liquid. In this case, the storage rules may be more lenient, but still require clarification in the instructions for a specific kit.
Rules for collecting biomaterial for maximum accuracy
Even if you perfectly observe the temperature regime, violation of the collection technique can nullify all efforts. The container must be sterile and dry. The use of baby food jars or matchboxes is unacceptable, since residual detergents or chemicals may react with reagents.
The defecation process should take place in a clean container (a vessel, a potty or cling film stretched over the toilet). Contact with water from a toilet containing bleach or disinfectants means is strictly prohibited. Chlorine can give a false positive reaction or destroy red blood cells. Also, urine must not be allowed to enter, as it may contain enzymes that distort the result.
To collect the material, use a special spoon built into the lid of the container, or a wooden spatula. It is enough to take the material in 3-4 places of one portion of feces, especially if suspicious inclusions (mucus, streaks) are noticeable. The volume of the sample should be approximately the size of a walnut or 1/3 of the container.
☑️ Checklist for preparing for collection
Diet and medications: preparation affects storage
Although the topic of the article concerns storage, it is impossible not to mention that the composition of feces changes even before the moment of collection. If you plan to store the sample for even a minimal time, it is important that the source material is “clean” from external factors. Some foods and medications can simulate the presence of blood or, conversely, hide it.
When using chemical methods (Gregersen), a strict diet is required 3 days before the test. It is necessary to exclude meat (beef, pork, lamb), fish, liver, as their hemoglobin will react. Also excluded are foods containing peroxidase (apples, cucumbers, cauliflower, horseradish) and iron supplements. With the immunochemical method, a diet is usually not required, since the reagents are specific only to human hemoglobin.
⚠️ Attention: Taking aspirin, nonsteroidal anti-inflammatory drugs (NSAIDs) and anticoagulants can cause microbleeding in the gastrointestinal tract, which will lead to a positive result. Discuss with your doctor the need to discontinue these medications 5-7 days before the test.
If you are taking bismuth medications (for example, to treat gastritis), the stool turns black. This is not blood, but may make the technician's visual assessment difficult. In such cases, storing the sample in a transparent container may cause the patient to panic if they see a black color, although this is just a consequence of taking medication.
What to do if you accidentally ate a steak before the test?
If you used an immunochemical method, most likely the result will be accurate. If it is chemical, there is a high risk of a false positive response. It is better to move the date of the test 3 days ahead by following a diet than to get an incorrect diagnosis and undergo an unnecessary colonoscopy.
Common mistakes when transporting to the laboratory
The last stage is delivery. This is where the temperature chain is most often disrupted. In the summer, when it’s hot, even 20 minutes on the road by car or on public transport can warm the container to room temperature or higher. In winter, on the contrary, the sample can become hypothermic if carried in your hands without a thermal bag.
A common mistake is waiting in line at the registration desk. When you hand the container over to the nurse, you do not know what conditions it will be in before the laboratory begins work. If the office is hot, the destruction of red blood cells will accelerate. Therefore, the “bring it, hand it over” rule works best. Try to arrive when the laboratory opens or hand over the material through special reception windows where there are refrigerators.
Labelling is also considered an error. The container must be signed immediately after collection. If you forget to do this and mix up samples (for example, taking tests for the whole family), the consequences can be fatal to your health. The label should include your full name, date and time of collection.
Frequently asked questions (FAQ)
Is it possible to test stool for occult blood if it has been left in the refrigerator all night?
Taking such a test is risky. Although cold slows down the destruction of red blood cells, within 8-10 hours (nighttime sleep time) some of the cells may be destroyed, which will lead to a false negative result. If there is no other choice, pass, but be prepared to retake. For overnight storage, it is necessary to use a preservative.
Does menstruation affect the test result in women?
Yes, it does have a critical effect. Even a minimal amount of menstrual blood entering the sample will give a sharply positive result, which will not be associated with gastrointestinal diseases. Women are advised to take the test no earlier than 3 days after the end of menstruation. It is also not recommended to take the test immediately after a colonoscopy or X-ray with barium.
What to do if it is not possible to get to the laboratory right away?
If you have collected the material, but cannot get there within 3-4 hours, place the container in the refrigerator (on the middle shelf, not in the freezer!). This will buy you time. However, try not to exceed the 6-8 hour limit for a regular container. It is better to call the laboratory and find out if they accept material collected the night before (some centers accept until 20:00).
Is it possible to use stool collected through an enema?
No, you cannot. An enema washes out the contents of the rectum, dilutes the sample with water and can injure the mucous membrane, which will lead to the appearance of artificial blood. The analysis will be uninformative. Defecation should be natural.