SOP-CM-005 — Stool Rapid Immunochromatographic Tests (Helicobacter pylori Stool Antigen and Qualitative Fecal Occult Blood Test / FOBT) SOP

1.0 Purpose

This SOP sets out the procedure for rapid qualitative lateral-flow immunochromatographic assays on human fecal specimens at Tarangnan Community Hospital. It covers two gastrointestinal screening examinations: Part A, the detection of active Helicobacter pylori infection using the RightSign HPG-C61 stool antigen cassette; and Part B, the detection of lower gastrointestinal bleeding using the qualitative immunochemical fecal occult blood test (iFOBT / FIT) cassette.

2.0 Scope

This procedure applies to all fecal rapid examinations performed in the Clinical Microscopy Section of Tarangnan Community Hospital. It covers patient preparation, medication withholding rules, specimen collection, buffer extraction, lateral-flow inoculation, procedural timing, visual line interpretation, correlation with serology, and clinical reporting.

3.0 Definitions

• Helicobacter pylori Stool Antigen (HpSA): A direct non-invasive marker detecting structural antigens of viable H. pylori* organisms shed in feces, reflecting current active gastric colonization.
• Immunochemical Fecal Occult Blood Test (iFOBT / FIT): A rapid lateral-flow immunoassay using monoclonal antibodies specific to intact human hemoglobin (globin moiety), eliminating dietary restrictions associated with legacy guaiac tests.
• Proton Pump Inhibitor (PPI) Clearance Window: A mandatory two-week medication withholding period before stool antigen collection required to prevent false-negative results caused by pharmacological suppression of bacterial density.
• Antibiotic Clearance Window: A mandatory four-week medication withholding period following systemic antibiotic or bismuth therapy required before assessing H. pylori eradication.
• Spiral Extraction Applicator: A grooved plastic wand integrated into the collection buffer tube cap designed to sample a standardized quantity of feces from multiple sites without scooping.
• Globin Degradation: The proteolytic breakdown of human hemoglobin by digestive proteases in the upper gastrointestinal tract, rendering iFOBT predominantly sensitive to lower gastrointestinal bleeding sources.

4.0 Responsibility

4.1 Hospital Director

• Grants final approval for this analytical SOP.
• Ensures the hospital procures validated H. pylori stool antigen and iFOBT rapid test kits.

4.2 Pathologist

• Provides clinical consultation on discordant, equivocal, or conflicting gastrointestinal laboratory findings.
• Correlates H. pylori stool antigen findings with serological antibody titers, endoscopic biopsies, or referral investigations.

4.3 Chief Medical Technologist / Laboratory Quality Manager

• Maintains and reviews this SOP, ensuring Medical Technologists complete documented training and annual competency assessments.
• Verifies kit storage conditions (2 to 30 °C), monitors procedural control performance, and reviews lot-to-lot verification logs.
• Investigates test invalidation rates and clinician feedback per LAB-QM-010.

4.4 Medical Technologists

• Verify patient compliance with pre-analytical medication withholding instructions (PPIs, bismuth, antibiotics) and collection criteria upon specimen receipt.
• Execute standardized multi-site stool extraction using the spiral applicator rod.
• Dispense exact extracted buffer volumes, time chromatographic migration, and read test bands strictly within manufacturer-specified windows.
• Record internal procedural control band validity before authorizing results in the LIS.
• Correlate stool antigen findings with serology records when clinicians request it.

4.5 Laboratory Aides

• Receive sealed stool specimens and transport them to the Clinical Microscopy biohazard processing station.
• Clean and sanitize workstation surfaces, tube racks, and vortex mixers with 0.5% sodium hypochlorite solution daily.
• Dispose of used extraction buffer tubes, cassettes, and biohazard packaging into labeled yellow infectious waste containers.
• Do not perform extractions, inoculate test cassettes, interpret result lines, or release reports.

5.0 Policy / Procedure

5.1 General Analytical Framework and Workstation Biosafety

` Stool Rapid Immunoassay Workflow | +---> Check Patient Requisition & Clinical Medication History: | H. pylori Antigen: Off PPIs/Bismuth >= 2 weeks; Off Antibiotics >= 4 weeks | iFOBT / FIT: No active menses/hemorrhoids; Off heavy NSAIDs/alcohol >= 48 h | +---> Part A: H. pylori Stool Antigen (RightSign HPG-C61) | 3-6 random stabs into stool (~50 mg feces in spiral rod) | Shake vigorously in extraction buffer; stand for 2 minutes | Dispense 2 DROPS (~80 µL) into Well (S) | Read at 10 MINUTES (Strict limit: Do NOT read after 20 minutes) | +---> Part B: Qualitative Immunochemical FOBT (iFOBT / FIT) 3-6 random stabs into stool across specimen Shake vigorously in extraction buffer; break dropper tip Dispense 2-3 DROPS (~80-100 µL) into Well (S) Read at 5-10 MINUTES (Strict limit: Do NOT read after 10 minutes) `

• All fecal specimens shall be handled as potentially infectious biohazards capable of transmitting enteric pathogens (Salmonella, Shigella, Campylobacter, norovirus, hepatitis A, and intestinal parasites).
• Personnel shall wear disposable nitrile gloves, fluid-resistant laboratory coats, and eye protection throughout specimen extraction and handling.
• Extractions shall be performed on clean, absorbent bench paper that is replaced immediately if soiled and discarded at the end of each shift.
• Separate and dedicated test cassettes shall be used for H. pylori antigen and FOBT testing. The extraction buffer from an H. pylori collection tube shall never be applied to an FOBT cassette, and FOBT buffer shall never be applied to an H. pylori cassette.

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5.2 Part A: Helicobacter pylori Stool Antigen Rapid Immunoassay (RightSign HPG-C61)

5.2.1 Clinical and Analytical Principle

• Helicobacter pylori is a microaerophilic, spiral, Gram-negative bacterium colonizing the gastric mucosa, strongly etiologic in chronic gastritis, peptic ulcer disease, gastric mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric adenocarcinoma.
• The RightSign H. pylori Antigen Rapid Test Cassette (REF HPG-C61) is a qualitative, lateral-flow immunochromatographic assay detecting H. pylori antigens in human feces.
• Monoclonal anti-H. pylori antibodies are immobilized on the test line (T) zone. Extracted stool antigen reacts with gold-conjugated anti-H. pylori antibodies, migrates across the nitrocellulose membrane, and binds to the test line, producing a visible pink/red band.
• Excess conjugate continues migrating to the control line (C) zone, where it binds to goat anti-mouse antibodies, generating a mandatory procedural control band.
• Unlike serological antibody testing (which detects past exposure and remains positive for months to years post-cure), stool antigen testing identifies active infection and is an approved test-of-cure following eradication therapy.

5.2.2 Critical Pre-Examination Medication Hold Rules

Attending physicians and nursing staff shall verify that patients observe the following pharmacological withholding intervals before stool collection:

• Proton Pump Inhibitors (PPIs) and Bismuth Compounds: Must be discontinued for at least two (2) weeks before stool collection. Agents such as omeprazole, pantoprazole, lansoprazole, esomeprazole, and bismuth subsalicylate suppress H. pylori bacterial load and urease activity, shifting the organism into a coccoid non-shedding state that causes false-negative antigen results.
• Systemic Antimicrobial Therapy: Must be discontinued for at least four (4) weeks before stool collection. Antibiotics (clarithromycin, amoxicillin, metronidazole, tetracycline, levofloxacin) drastically reduce bacterial density below analytical detection limits.
• Post-Treatment Eradication Assessment: When used as a test of cure, stool antigen testing shall be scheduled no earlier than four (4) weeks following the completion of eradication therapy.

5.2.3 Reagents and Kit Contents (RightSign HPG-C61)

• Individually pouched test cassettes containing desiccant.
• Specimen collection tubes containing 2 mL of extraction buffer with integrated spiral applicator wand.
• Package insert.
• Storage: Store kits at 2 to 30 °C in sealed pouches until the printed expiration date. Do not freeze.

5.2.4 Specimen Collection, Extraction, and Testing Sequence

• Collect fresh feces in a clean, dry, leak-proof container without preservatives or transport media. Refrigerate at 2 to 8 °C if testing is not performed immediately; test within 72 hours.
• Bring the test cassette, extraction tube, and stool specimen to room temperature (15 to 30 °C) before testing.
• For solid stool extraction, unscrew the cap of the specimen collection tube containing extraction buffer. Randomly insert the spiral applicator rod into the fecal specimen at at least three (3) to six (6) different sites. Ensure feces fills the spiral grooves of the wand, equivalent to approximately 50 mg of stool (roughly one-fourth the size of a green pea). Do not scoop, scrape, or overload the wand, as excess stool causes high viscosity and blocks capillary migration.
• For liquid or diarrheal stool extraction, hold a disposable transfer pipette vertically and aspirate approximately 80 µL (two drops) of liquid stool, dispensing directly into the extraction buffer tube.
• Reinsert the applicator wand into the tube and tighten the screw cap securely. Shake the collection tube vigorously by hand for 20 seconds to disperse the stool specimen into the extraction buffer. Allow the tube to stand upright for two (2) minutes to permit large particulate debris to settle to the bottom.
• Remove the RightSign HPG-C61 cassette from its sealed pouch, place it on a clean level bench surface, and label with two patient identifiers. Snap off or twist open the dispensing tip of the extraction tube. Invert the collection tube vertically over the circular sample well (S) and dispense exactly two (2) full drops (approximately 80 µL) of extracted supernatant into the sample well (S), avoiding air bubbles.
• Start the digital timer immediately upon dispensing. Read the result at ten (10) minutes. Do not read or interpret the test after twenty (20) minutes, as drying artifacts may yield non-specific colored lines.
• If the extracted liquid fails to migrate across the membrane window within two minutes due to particulate obstruction, centrifuge the extraction tube at 1000 × g for 3 minutes, aspirate the clear supernatant, and re-test with a fresh cassette.

5.2.5 Interpretation of H. pylori Stool Antigen Results

• POSITIVE: Two distinct colored bands appear, with one line in the Control (C) zone and one line in the Test (T) zone. Any visible pink or red line in the test zone, regardless of intensity, indicates a positive result (active H. pylori infection).
• NEGATIVE: Only one colored band appears in the Control (C) zone. No band is visible in the Test (T) zone. This indicates the absence of detectable H. pylori antigen above the cutoff.
• INVALID: The Control (C) band fails to appear. The test is invalid regardless of test line presence. Review collection instructions, ensure proper drop volume, and repeat using a new cassette from an unopened pouch.

5.2.6 Correlation with H. pylori Serum Antibody Testing

• TCH performs both H. pylori stool antigen testing (Clinical Microscopy) and H. pylori serum antibody testing (Serology per SOP-S-001).
• Stool antigen detects active, viable bacterial colonization in the stomach. Serum antibody testing detects circulating IgG antibodies reflecting immunological response to past or present infection.
• In patients who have successfully completed antibiotic therapy, serum IgG antibodies may remain elevated for months or years, whereas stool antigen becomes negative within 1 to 2 weeks post-eradication.
• When clinical discordance occurs between serology and stool antigen, the Medical Technologist shall document the analytical targets and refer findings to the Pathologist for clinical synthesis.

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5.3 Part B: Qualitative Immunochemical Fecal Occult Blood Test (iFOBT / FIT)

5.3.1 Clinical and Analytical Principle

• Occult gastrointestinal bleeding is a critical clinical finding in colorectal cancer, adenomatous polyps, bleeding peptic ulcers, diverticular disease, ulcerative colitis, and Crohn's disease.
• The qualitative immunochemical fecal occult blood test (iFOBT / FIT) uses a rapid lateral-flow chromatographic strip incorporating monoclonal anti-human hemoglobin antibodies.
• Analytical Sensitivity: Detects as low as 50 ng/mL human hemoglobin (equivalent to approximately 6 µg hemoglobin per gram of feces).
• Superiority Over Legacy Guaiac Methods: Guaiac-based tests rely on the pseudoperoxidase activity of heme, causing extensive cross-reactivity with dietary animal blood (beef, pork), dietary peroxidase enzymes (broccoli, turnips, horseradish, cauliflower), and chemical interference from therapeutic ascorbic acid (vitamin C false-negatives).
• In contrast, the modern immunochemical test is highly specific for intact human globin. No dietary restrictions (red meat, vegetables, or vitamin C) are required.
• Lower vs. Upper GI Bleeding: Globin is rapidly degraded by gastric and duodenal digestive proteases (pepsin, trypsin, chymotrypsin). Consequently, intact globin reaches the stool primarily from bleeding sites in the lower gastrointestinal tract (colon, rectum), making iFOBT an optimal screening tool for colorectal lesions. Extensive upper GI bleeding may yield positive iFOBT results only when blood transit is rapid.

5.3.2 Pre-Examination Interferences and Patient Guidance

Although dietary restrictions are eliminated, patients shall observe the following clinical collection precautions:

• Menstrual Bleeding: Do not collect stool specimens during active menstruation or within three (3) days following the cessation of menstrual flow.
• Active Anorectal Bleeding: Defer collection if the patient has actively bleeding hemorrhoids, acute anal fissures, or visible blood in the urine (hematuria).
• Medication Interference: Heavy consumption of non-steroidal anti-inflammatory drugs (NSAIDs, e.g., aspirin, ibuprofen, naproxen), corticosteroids, or anticoagulants can induce gastrointestinal mucosal microbleeding; where clinically permissible, attending physicians should withhold these medications for 48 hours before specimen collection.
• Alcohol: Heavy alcohol intake causing transient gastric irritation should be discontinued for 48 hours before testing.

5.3.3 Specimen Collection, Extraction, and Testing Sequence

• Collect fresh stool in a clean, dry, leak-proof container and submit to the laboratory promptly. The specimen may be refrigerated at 2 to 8 °C for up to 72 hours before extraction.
• Equilibrate the FOBT test cassette, extraction buffer tube, and stool specimen to room temperature (15 to 30 °C). Unscrew the cap of the FOBT extraction buffer tube and insert the spiral applicator wand into the stool specimen at three (3) to six (6) different random locations, coating the spiral grooves with fecal matter.
• Reinsert the applicator wand into the collection tube and tighten the screw cap securely. Shake the tube vigorously by hand for 20 seconds to disperse feces into the buffer, then allow the tube to stand upright for two (2) minutes to permit settling of coarse fecal matter.
• Remove the FOBT cassette from its sealed foil pouch, place it on a clean horizontal surface, and label with two patient identifiers. Break off or unscrew the dropper dispensing tip of the extraction tube.
• Invert the extraction tube vertically above the sample well (S) and dispense two (2) to three (3) full drops (approximately 80 to 100 µL) of extracted buffer into the sample well (S).
• Start the digital timer immediately. Read the result at five (5) to ten (10) minutes.
• Do not read or interpret the FOBT cassette after ten (10) minutes. Prolonged incubation can lead to non-specific background coloration or false-positive ghost lines.

5.3.4 Interpretation of FOBT Results

• POSITIVE (Reactive): Two distinct colored bands appear, with one line in the Control (C) zone and one line in the Test (T) zone. This indicates the presence of human hemoglobin at or above 50 ng/mL. Report as POSITIVE.
• NEGATIVE (Non-Reactive): Only one colored band appears in the Control (C) zone. No visible band appears in the Test (T) zone. This indicates no detectable human occult blood above the cutoff. Report as NEGATIVE.
• INVALID: The Control (C) band fails to appear. The test is invalid regardless of test line presence. Discard cassette and repeat using a newly opened test cassette and freshly extracted specimen.

5.4 Internal Quality Control Governance

• Internal Procedural Control Band (C): A visible colored line in the Control (C) region of every H. pylori and FOBT cassette confirms proper sample volume addition, complete membrane capillary migration, and reagent binding. No patient result shall ever be released from a cassette lacking a valid C band.
• External Quality Control: Positive and negative liquid external controls shall be tested: - Upon receipt of each new shipment or kit lot number. - When storage temperatures exceed the 2 to 30 °C tolerance range. - Whenever cassette results are clinically questionable or unexpected. - Record control results and lot verification details in the electronic QC log under Quality Control Policy (registered under F-QM-002-B-V0).

5.5 Clinical Limitations and Technical Cautions

• Non-Diagnostic of Cancer Alone: A positive FOBT result indicates gastrointestinal bleeding but does not distinguish benign lesions (polyps, hemorrhoids, diverticulosis) from malignant neoplasms (colorectal adenocarcinoma). Further clinical diagnostic workup (colonoscopy, endoscopy) is required.
• Intermittent Bleeding: Not all gastrointestinal polyps or carcinomas bleed continuously. A single negative FOBT result does not completely exclude colorectal disease; serial testing over multiple days increases diagnostic sensitivity.
• Upper GI Bleeding Globin Degradation: Because iFOBT detects intact globin, substantial upper gastrointestinal hemorrhage (bleeding peptic ulcers, esophageal varices) may occasionally test negative if globin is thoroughly digested before excretion.
• Antibiotic-Induced False Negatives in H. pylori: Testing performed within four weeks of antibiotic therapy or two weeks of PPI therapy frequently produces false-negative stool antigen results. Attending physicians shall be advised to observe mandatory clearance intervals.

5.6 Result Reporting Format

Results shall be encoded in the LIS or transcribed onto the official TCH Clinical Microscopy Report:

• H. pylori Stool Antigen: - Helicobacter pylori Stool Antigen Rapid Test: POSITIVE (Reactive) - Helicobacter pylori Stool Antigen Rapid Test: NEGATIVE (Non-Reactive)
• Fecal Occult Blood Test: - Fecal Occult Blood Test (Immunochemical / iFOBT): POSITIVE (Reactive) - Fecal Occult Blood Test (Immunochemical / iFOBT): NEGATIVE (Non-Reactive)
• Record manufacturer, kit lot number, expiration date, and examining Medical Technologist signature.

6.0 Records

7.0 References

• DOH Clinical Laboratory QMS Tool, Section 3 — Service Delivery
• DOH Administrative Order No. 2021-0037, Annex B1 — Technical Standards for Clinical Laboratories
• ISO 15189:2022, Section 7.3 — Examination processes
• RightSign H. pylori Antigen Rapid Test Cassette (Feces) Package Insert
• Qualitative Fecal Occult Blood Rapid Test Cassette (Feces / iFOBT) Package Insert
• CLSI M28-A2, Procedures for the Recovery and Identification of Parasites from the Intestinal Tract

Companion policies:

• Scope of Service and Laboratory User Information Policy — Laboratory test menu and diagnostic service scope
• LAB-QM-PRE-001 — Specimen collection, acceptance criteria, and rejection management
• Laboratory Reporting Policy — Result review, validation, and diagnostic report release