Fecal Occult Blood Test Kit (OX-Toluidine Method) - BioReagent, high purity

Cat. No.: D1354183

GRADE & PURITY BioReagent

Overview

  Chemical examination of feces includes tests for pH reaction, occult blood, bile pigments, and fat determination, among which the occult blood test holds the most significant clinical value. When upper gastrointestinal bleeding is less than 5 mL, there is no visible blood in the feces, and red blood cells are destroyed and cannot be detected by microscopy, such bleeding is termed "occult." Tests to detect this blood in feces are called fecal occult blood tests (FOBT). Chemical methods commonly use reagents such as o-tolidine, pyramidon, guaiac, colorless malachite green, and Metol.

  The detection principle of this Fecal Occult Blood Qualitative Detection Kit (o-Tolidine Method) is based on the fact that heme in hemoglobin possesses peroxidase-like activity. It catalyzes H₂O₂ as an electron acceptor to oxidize the chromogenic substrate o-tolidine into o-tolidine blue, producing a color change from green to blue. The intensity of the color is proportional to the hemoglobin concentration. Based on the speed of color development, the hemoglobin (i.e., occult blood) content can be semi-quantified. Therefore, this test can also be referred to as a semi-quantitative fecal occult blood test. It is primarily used for examining human or animal fecal hemoglobin. It should be noted that trace bleeding from the normal human or animal digestive tract, bleeding caused by irritation, or animal feed containing fish meal, iron supplements, etc., may lead to false-positive results. This method is characterized by high sensitivity and a high false-positive rate, capable of detecting Hb at 0.2–1.0 μg/mL. It can be used in conjunction with the pyramidon method, which offers moderate sensitivity and specificity.

This product is intended for research use only and is not suitable for clinical diagnosis or other purposes.

D1354183	Component	100 T	300 T	Storage
D1354183A	O-Tolidine Solution	10 ml	30 ml	2-8℃. Store in the dark.
D1354183B	Oxidizing Agent	10 ml	30 ml	2-8℃

Materials to Be Prepared by User

• Fecal specimens
• Wooden sticks or toothpicks
• Cotton swabs or white porcelain plates
• Droppers or pipettes

Protocol (For Reference Only)

1. Fecal specimens should be collected and tested promptly to avoid decreased sensitivity due to prolonged storage. Avoid contact with water.

2. Use a wooden stick or toothpick to collect a small amount of feces (10–50 mg, approximately the size of a mung bean). Smear it onto a cotton swab or white porcelain plate.

3. Add 2 drops (approximately 0.1 mL) of O-tolidine Solution onto different areas of the fecal sample.

4. Add 2 drops (approximately 0.1 mL) of Oxidizing Agent. Immediately start timing and observe the color change.

5. Complete the interpretation within 2 minutes. If a blue color appears within 2 minutes, it indicates the presence of hemoglobin (Hb) in the feces, i.e., a positive fecal occult blood test.

6. Please use the reagent promptly after opening to prevent affecting subsequent experimental results.

Result Interpretation

Color Change	Interpretation
Dark blue-black appears immediately after adding reagents.	(4+)
Blue-brown appears immediately after adding reagents, gradually turning dark brown-black.	(3+)
Initially light green after adding reagents, gradually turning distinctly blue-brown.	(2+)
Changes from light green to green after 10 seconds.	(+)
No color develops within 2 minutes.	Negative

Precautions

1. During specimen collection, transportation, and kit usage, personnel should take appropriate protective measures to avoid human contact and prevent potential contamination and self-infection.

2. Trace bleeding from the normal human or animal digestive tract or bleeding caused by irritation may sometimes lead to false-positive results with this method.

3. For asymptomatic individuals where occult bleeding is suspected, it is recommended to collect samples for at least 3 consecutive days and perform two tests on samples taken from different parts of the specimen.

4. Consumption of animal blood, meat, liver, chlorophyll-rich vegetables, iron supplements, traditional Chinese medicines, or other specific drugs should be stopped for at least 3 days before testing to avoid false-positive reactions.

5. For experimental animals in colitis models, the feed should not contain fish meal, iron supplements, or other specific drugs to avoid false-positive results.

6. The O-tolidine Solution contains acetic acid and is highly irritating. Operations should be performed in a fume hood.

7. The Oxidizing Agent should be stored sealed at 4°C. Its efficiency may decrease after 6 months. Commercially available hydrogen peroxide (30%) or urea peroxide can be used to prepare a 1–3% solution as an alternative.

8. If necessary, gradient control experiments can be performed using blood or hemoglobin solution.

9. The kit should not be stored together with the Oxidizing Agent. Avoid direct sunlight, high temperatures, and freezing.

Appendix Tables

Table 1: Interference Factors and Evaluation for Chemical FOBT Methods

Interference Factors	False Negative	False Positive
Specimen	Decreased sensitivity due to aged stool; prolonged intestinal transit degrading Hb/heme.	Occult blood from non-gastrointestinal sources (e.g., gingival bleeding, epistaxis, menstrual blood).
Food and Drugs	High doses of Vitamin C or other reducing substances.	Hemoglobin-containing animal blood (fish, meat, liver); Chlorophyll-rich fresh vegetables (peroxidase); Iron/Bismuth supplements; Drugs causing GI bleeding (aspirin, corticosteroids, NSAIDs, etc.).
Materials/Reagents	Low concentration or inactive H₂O₂; Improper reagent storage (freezing, light, heat) causing degradation.	Contamination of equipment with copper ions, iron ions, disinfectants, bromine, iron, boric acid, peroxidase, etc.
Operation	Insufficient reaction time; Inaccurate color interpretation.	Adding water to the specimen before testing (actually increases false positives).

Table 2: Methodological Evaluation of Chemical FOBT Methods

Method	Characteristics	Evaluation
o-Tolidine Method	High Sensitivity High False Positive Rate	Detects Hb at 0.2–1.0 mg/L; can detect bleeding of 1–5 mL in the digestive tract. Due to its high sensitivity,微量 bleeding can yield positive results. A negative result with this highly sensitive test reliably rules out occult blood.
Pyramidon Method	Moderate Sensitivity Moderate Specificity	Detects Hb at 6–10 mg/L; detects bleeding of ~20 mL in the digestive tract. Less affected by food and drugs, lower false positive rate. A positive result with this less sensitive test reliably indicates occult blood.

Color Development Comparison with Different Hemoglobin Concentrations

Comparison of color development with different hemoglobin concentrations (from left to right: 10 μg/mL, 50 μg/mL, 200 μg/mL)

Storage and Shipping

Storage

Store at 2-8°C,Protected from light

Shipped In

Wet ice

Stability And Storage

D1354183A ortho toluidine solution: Long term storage (12 months) at 2-8 ℃. Store in the dark. D1354183B oxidizer: Long term storage at 2-8 ℃ (12 months). Store in the dark.

Contents & Storage

D1354183	Component	100 T	300 T	Storage
D1354183A	O-Tolidine Solution	10 ml	30 ml	2-8℃. Store in the dark.
D1354183B	Oxidizing Agent	10 ml	30 ml	2-8℃