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Urinalysis & Body Fluids

Chemical Testing

The physical examination of feces provides us with powerful clues about overt pathology - we can see the blood from a lower GI bleed or the pale color of a biliary obstruction. However, many of the most significant gastrointestinal diseases leave a more subtle, invisible trail. This is where chemical testing becomes indispensable

Fecal chemical analysis allows us to detect occult (hidden) pathology. We are searching for microscopic amounts of blood, quantifying the failure to absorb nutrients like fat, and measuring the biochemical byproducts of maldigestion. These tests are the cornerstone of colorectal cancer screening and the definitive workup for malabsorption syndromes. Success in this area, however, is critically dependent on a deep understanding of the test principles and a fanatical adherence to pre-analytical patient preparation

Fecal Occult Blood Testing (FOBT): The Search for Hidden Bleeding

This is the most frequently performed chemical test on feces. Its primary purpose is for the screening of colorectal cancer, which often bleeds intermittently and in small amounts long before it causes any symptoms

There are two fundamentally different types of FOBT, and you must master the distinction

Type A: Guaiac-based FOBT (gFOBT)

This is the older, traditional method

  • Principle: Pseudoperoxidase Activity
    • The test is not specific for blood.: It is based on the fact that the heme component of hemoglobin acts as a catalyst (a pseudoperoxidase)
    • The Reaction
      1. A stool sample is smeared onto paper impregnated with guaiac, a phenolic compound from a tree resin
      2. A developer solution containing hydrogen peroxide (H₂O₂) : is added
      3. If heme is present, it will catalyze the reaction between the H₂O₂ and the guaiac, oxidizing the guaiac
      4. Oxidized guaiac turns blue.
    • Summary: Heme + H₂O₂ + Guaiac (colorless) → Oxidized Guaiac (blue)
  • CRITICAL Section: Interferences & Dietary Restrictions
    • Because the test relies on a non-specific chemical reaction, it is plagued by interferences. Strict patient preparation is required for 3 days prior to and during the collection period.
    • Causes of FALSE POSITIVES
      • Dietary Heme: Ingestion of red meat (beef, lamb), which contains hemoglobin and myoglobin
      • Dietary Peroxidases: Certain raw vegetables and fruits have their own peroxidase enzymes that can catalyze the reaction (e.g., broccoli, cauliflower, radishes, turnips, melons)
      • Drugs: Aspirin and other NSAIDs can irritate the gastric lining, causing minor bleeding that is not related to cancer
    • Causes of FALSE NEGATIVES
      • Vitamin C (Ascorbic Acid): This is a powerful reducing agent. It chemically reverses the oxidation of the guaiac, preventing the blue color from forming even if blood is present. Ingestion of >250 mg/day of Vitamin C can invalidate the test

Type B: Immunochemical-based FOBT (iFOBT or FIT - Fecal Immunochemical Test)

This is the modern, superior method that has largely replaced the gFOBT

  • Principle: Specific Antibody Reaction
    • This is an immunoassay, not a chemical reaction.
    • The test uses highly specific polyclonal or monoclonal antibodies that are targeted against the globin: portion of human hemoglobin.
  • CRITICAL Section: The Advantages of FIT/iFOBT
    • 1. High Specificity: Because the antibodies only recognize human globin, the test is not affected by:
      • Dietary heme from red meat
      • Plant peroxidases
      • Therefore, NO DIETARY RESTRICTIONS ARE REQUIRED.: This dramatically improves patient compliance and accuracy
    • 2. No Vitamin C Interference: The test is an antibody-antigen reaction, not an oxidation-reduction reaction, so Vitamin C has no effect
    • 3. Specificity for Lower GI Bleeding: The globin protein is digested by proteases as it passes through the upper GI tract. Therefore, the FIT is most sensitive for detecting bleeding from the colon and rectum - the primary sites of colorectal cancer. It is less likely to pick up clinically insignificant bleeding from the stomach
  • Methodology: Often involves a probe collection device that the patient uses to sample the stool and place into a buffer tube. The test is then run on an automated immunoassay platform or a qualitative cassette device in the lab

Tests for Malabsorption (Steatorrhea)

Steatorrhea is the presence of excess fat in the feces, a hallmark of malabsorption. Chemical tests allow us to screen for and definitively quantify this condition

Qualitative Screen: Sudan III/IV or Oil Red O Stain

  • Purpose: A rapid microscopic screen to detect the presence of excess fecal fat
  • Methodology (Two-Part Slide)
    1. Neutral Fat Stain: A small amount of stool is emulsified with saline on a slide, stained with Sudan dye, and examined. The dye stains the neutral fats (triglycerides), which appear as large orange-red droplets
    2. Split Fat Stain: Another aliquot of stool is mixed with acetic acid and heated. This splits the fatty acid salts (soaps) to release free fatty acids. The slide is then stained and examined
  • Interpretation
    • Increased Neutral Fats: Suggests a maldigestion problem. The pancreas is not producing enough lipase to break down the triglycerides. This is seen in pancreatic insufficiency (e.g., cystic fibrosis, chronic pancreatitis)
    • Increased Split Fats (Fatty Acids): Suggests a malabsorption problem. The fat was digested correctly, but the small intestine failed to absorb the resulting fatty acids. This is seen in diseases like Celiac disease

Quantitative Gold Standard: 72-Hour Fecal Fat Test

  • Purpose: The definitive, quantitative test to confirm the diagnosis of steatorrhea
  • Methodology: This is one of the most demanding tests for both the patient and the lab
    1. Strict Patient Preparation: The patient must consume a diet containing a constant and known amount of fat (typically 100 grams/day) for 2-3 days before and during the collection period
    2. Collection: The patient must collect every single stool specimen for a continuous 72-hour period into a large, pre-weighed, sealed container
    3. Lab Analysis: The entire, massive collection is weighed, homogenized (an unpleasant task), and then chemically analyzed for total fat content
  • Interpretation
    • Normal: A healthy individual absorbs >95% of dietary fat. The normal excretion is < 7 grams of fat per 24 hours.
    • Steatorrhea: Excretion of > 7 g/day is diagnostic of steatorrhea

Tests for Carbohydrate Malabsorption

These tests are most often used in the workup of infants and children with unexplained osmotic diarrhea

  • Fecal pH
    • Physiology: If carbohydrates (especially lactose) are not absorbed in the small intestine, they travel to the colon, where they are fermented by bacteria. This fermentation process produces large amounts of gas (H₂) and short-chain fatty acids (lactic acid), which lowers the pH of the stool
    • Interpretation: Normal fecal pH is neutral to slightly alkaline (7.0-8.0). A pH < 5.5 is highly suggestive of carbohydrate malabsorption
  • Fecal Reducing Substances (Clinitest)
    • Principle: Uses the Clinitest tablet (copper reduction method) to detect the presence of reducing sugars (glucose, galactose, fructose, lactose) in a liquid stool extract
    • Interpretation: A result of > 0.5 g/dL is considered abnormal and indicates the presence of undigested sugars, confirming carbohydrate malabsorption. This is the classic test for diagnosing inherited disaccharidase deficiencies in infants

Other Key Differentiating Tests

  • Fecal Elastase-1
    • Purpose: A modern, highly specific, non-invasive test for pancreatic exocrine insufficiency.
    • Physiology: Elastase-1 is an enzyme produced exclusively by the pancreas. Crucially, it passes through the GI tract undigested. Its concentration in the stool is therefore a direct measure of the pancreas’s secretory capacity
    • Advantage: It is far easier for the patient and lab than a 72-hour fecal fat test and has replaced it for many indications. A low fecal elastase level is diagnostic for pancreatic insufficiency
  • Stool Osmotic Gap
    • Purpose: A calculation used to differentiate between secretory and osmotic diarrhea
    • Calculation: 290 – [2 x (fecal Na⁺ + fecal K⁺)]
      • (290 is the assumed normal plasma osmolality)
    • Interpretation
      • Low Gap (< 50 mOsm/kg): The fecal electrolytes account for almost all of the osmolality. This means the intestine is actively secreting electrolytes and water. This is the profile of secretory diarrhea.
      • High Gap (> 125 mOsm/kg): There is a large gap, meaning there is some other osmotically active substance in the stool that is not an electrolyte (e.g., lactose, magnesium citrate). This is the profile of osmotic diarrhea.

Conclusion

Fecal chemical testing provides a powerful lens into the hidden functions and dysfunctions of the GI tract. From the life-saving early detection of colorectal cancer with FIT testing to the definitive diagnosis of steatorrhea with a 72-hour fat collection, these assays translate complex physiological processes into actionable clinical data. Success in this field hinges on a deep understanding of the principles behind each test and, above all, a rigorous enforcement of the pre-analytical requirements that ensure the sample’s integrity