Collection & Handling

We have just reviewed the complex journey of digestion and elimination that culminates in the formation of feces. Now, we must confront a topic that is often uncomfortable but is absolutely paramount to the accuracy of any stool test: the pre-analytical phase of collection and handling

This phase is uniquely challenging for several reasons. The collection is performed by the patient, often at home, which introduces a huge potential for error and non-compliance. The sample itself is aesthetically unpleasant, which can lead to improper or incomplete collection. Furthermore, feces is a biologically complex and “live” sample, containing a massive microbiome and active enzymes, making it highly susceptible to degradation

A single error in this phase can render the most sophisticated laboratory test invalid. A stool sample collected from the toilet bowl water will yield a false-negative result for parasites. A delay in transport can cause a false-positive result for occult blood. As laboratory professionals, our primary role in this phase is to provide crystal-clear, unambiguous patient instructions and to enforce strict specimen acceptance criteria upon receipt. There is no such thing as a “minor” error in stool handling

Patient Instructions: The Foundation of a Quality Sample

Success or failure is determined before the sample ever reaches the lab. The patient education we provide is critical

1. Collection Container

• Requirement: The patient must be given a clean, dry, wide-mouthed container with a tight-fitting, leak-proof lid.
• Rationale
  • Clean & Dry: To prevent contamination with cleaning agents, deodorizers, or water, all of which can interfere with tests or kill pathogens
  • Wide-Mouthed: For ease of collection
  • Tight-fitting Lid: To prevent leakage during transport and contain odor. Many labs use containers that have an integrated collection spoon built into the lid, which is ideal
• Patient Education: The patient must be instructed never to use containers from home like old pill bottles or food jars, which may contain interfering residues

2. Collection Procedure: Avoiding Contamination

This is the most common point of failure

• The Golden Rule: The stool specimen MUST NOT be contaminated with urine or toilet water.
• Why is this CRITICAL?
  • Urine: Urine is acidic and has a high salt concentration, which can destroy trophozoites (the motile, feeding stage of protozoan parasites), making them undetectable
  • Toilet Water: Contains a host of problems
    • Free-living Protozoa & Nematodes: The water may contain non-pathogenic organisms that look very similar to human parasites, leading to a disastrous false-positive identification
    • Disinfectants/Deodorizers: Chemicals in the bowl (e.g., from “blue water” cleaners) can interfere with chemical tests (like occult blood) and kill pathogens
    • Dilution: It dilutes the sample, potentially lowering the concentration of a target analyte (like an antigen or toxin) below the limit of detection
• Acceptable Collection Methods (Patient Instructions)
  1. “Hat” Collector: Provide the patient with a clean plastic collection device that fits inside the toilet bowl under the seat. This is the preferred method
  2. Plastic Wrap: Instruct the patient to stretch a clean piece of plastic wrap loosely across the rim of the toilet bowl
  3. Clean, Dry Pan: For infants in diapers, a clean, disposable diaper can be placed inside-out (plastic side in) to prevent the stool from being absorbed into the diaper material
• Lab Action: Upon receipt, if there is any sign of water contamination (e.g., the sample is very liquidy at the bottom), the specimen should be rejected

3. Sample Selection: Collecting the “Right” Part

• Instruction: Using the collection spoon or tongue depressors, the patient should collect a representative sample, especially any areas that look abnormal
• Specifics for Different Tests
  • For Ova & Parasite (O&P) Exam: The patient should be instructed to specifically sample any bloody, slimy, or watery areas of the stool, as this is where parasites and white blood cells are most likely to be found
  • For Occult Blood: The patient should sample from the center of the formed stool to avoid contamination from external hemorrhoids

4. Quantity of Sample

• General Guideline: A “walnut-sized” portion for a formed stool, or about 5-10 mL for a liquid stool, is usually sufficient
• Overfilling: Patients often provide far too much sample. This is not only a biohazard and odor issue for the lab but can also interfere with the proper mixing of the sample with preservative in O&P vials

Handling, Transport, & Preservation: The Race Against Degradation

Once collected, the clock starts ticking

Time & Temperature

• The Rule of Thumb: Many analytes degrade quickly at room temperature. The specimen should be delivered to the lab as soon as possible.
• Specific Timeframes for Unpreserved Stool
  • Examination for Trophozoites (e.g., Giardia, E. histolytica): This is a STAT situation. A liquid or soft stool must be examined within 30 minutes of passage. Trophozoites are extremely fragile and will disintegrate rapidly
  • General O&P Exam: A formed stool (which would only contain the more robust cysts) should be examined within 2-3 hours
  • Bacterial Culture: Should be processed within 2 hours. Overgrowth of normal flora can mask the true pathogen
  • Viral Testing: Can tolerate slightly longer delays
• Refrigeration: If a delay is unavoidable, the specimen should be refrigerated at 4°C. This slows down bacterial overgrowth and can preserve parasite morphology for a short time. NEVER FREEZE a stool sample for O&P or culture, as this will destroy the organisms

Preservatives: The Gold Standard for O&P

Because of the extreme lability of trophozoites, for any routine O&P exam, the specimen must be placed into a preservative immediately after collection. The lab provides a two-vial kit

• Vial 1: 10% Formalin
  • Purpose: An excellent fixative that preserves the morphology of parasite cysts, larvae, and eggs for direct wet mount examination and concentration procedures. It also works well for immunoassay techniques
  • Limitation: It is not a good preservative for trophozoite morphology on a permanent stained smear
• Vial 2: Polyvinyl Alcohol (PVA) or an Equivalent
  • Purpose: This is the critical component for preserving the fragile trophozoite stage. PVA is an adhesive that helps the specimen stick to the slide and contains a fixative (like mercuric chloride in the classic formula, or safer, non-mercury alternatives now) that preserves the delicate nuclear and cytoplasmic details needed for identification on a permanent trichrome stain
  • Newer Alternatives: Many labs now use single-vial systems with proprietary, non-mercury fixatives (e.g., TOTAL-FIX, Ecofix) that can be used for all O&P procedures, including immunoassays
• Patient Education: The patient must be carefully instructed on how to add the stool to the vials (up to the “fill line”) and to mix thoroughly until the stool is completely suspended in the preservative

Dietary & Medication Restrictions

For certain tests, the patient’s diet or medications can cause significant interference. This is most critical for the Guaiac-based Fecal Occult Blood Test (gFOBT).

• Restrictions for gFOBT (72 hours prior to collection)
  • AVOID: Red meat (contains animal hemoglobin), certain raw fruits and vegetables (e.g., broccoli, turnips, horseradish, which contain plant peroxidases), and Vitamin C supplements (>250 mg/day, which is a reducing agent and can cause a false negative). Aspirin and other NSAIDs should also be avoided as they can cause gastric irritation and true bleeding
• Lab Responsibility: These restrictions must be clearly communicated to the patient. A failure to follow them is a major cause of false-positive or false-negative results. Note These restrictions do not apply to the more modern immunochemical-based occult blood tests (FIT), which are specific for human globin

Conclusion

The pre-analytical phase for fecal testing is a partnership between a well-informed patient and a vigilant laboratory. It is our duty to provide instructions that are so clear and simple that error is minimized. Upon receipt, it is our duty to act as the final quality control checkpoint, scrutinizing the specimen for signs of contamination, improper transport, or incorrect preservation. A specimen that is compromised must be rejected. By upholding these rigorous standards, we ensure that the results we generate are a true and accurate reflection of the patient’s gastrointestinal health