SOP-S-002 — Hepatitis B Surface Antigen (HBsAg) Rapid Qualitative Immunoassay and Confirmatory Referral SOP

1.0 Purpose

This procedure covers the qualitative detection of Hepatitis B surface antigen (HBsAg) in human serum, plasma, or whole blood using authorized rapid immunoassay kits at Tarangnan Community Hospital, including method-specific steps, internal control checks, and confirmatory referral of reactive specimens.

2.0 Scope

This procedure applies to all qualitative HBsAg screening by Medical Technologists in the Clinical Laboratory using Method A (AllTest/Trimera REF IHBSG-402) or Method B (SD BIOSENSOR STANDARD Q REF QHBS01G). It covers specimen processing, strict method separation, result interpretation, and confirmatory referral to Samar Provincial Hospital. General serological operations are governed by Serology Section Workflow Quality Control and Result Reporting Sop.

3.0 Definitions

• Hepatitis B Surface Antigen (HBsAg): The major outer envelope glycoprotein of the Hepatitis B virus, serving as the primary serological marker for acute or chronic active infection.
• Lateral-Flow Immunoassay: Rapid diagnostic membrane strip using immobilized antibodies and colloidal gold conjugates to detect specific antigens by capillary flow.
• Procedural Control Line (C Line): Built-in quality indicator band that must develop color to demonstrate adequate liquid migration and reagent viability.
• Confirmatory Referral: Direct transmission of reactive screening specimens to an accredited tertiary reference facility for definitive confirmation by ELISA or neutralization assay.

4.0 Responsibility

4.1 Hospital Director

• Approves this procedure and maintains agreements with referral centers.

4.2 Pathologist

• Oversees serology assays and reviews reactive and referred findings.

4.3 Chief Medical Technologist / Laboratory Quality Manager

• Maintains method-specific instructions, verifies reagent lot performance, and oversees proficiency testing.
• Ensures all testing technologists are competency-assessed on both authorized HBsAg methods.

4.4 Medical Technologists

• Verify test kit identity and use only the procedure matching the kit in hand.
• Follow the method-specific specimen volumes, buffer requirements, and timing windows strictly.
• Validate the procedural C line on every cassette and initiate confirmatory referral for reactive specimens.

4.5 Laboratory Aides

• Clean analytical surfaces, log daily refrigerator temperatures, and assist with referral package transport.
• Shall not perform testing, dispense specimens, interpret test lines, or authorize patient results.

5.0 Policy / Procedure

5.1 Analytical Principle and Method Separation

The analytical screening, procedural validation, duplicate retesting, and confirmatory referral algorithm follows the established protocol (see Figure 1).

Figure 1 — HBsAg rapid screening, duplicate retest, and confirmatory referral algorithm

• Both authorized methods are qualitative lateral-flow chromatographic immunoassays detecting HBsAg. As specimen flows through the membrane, anti-HBsAg antibody-gold conjugates bind to HBsAg, forming colored complexes captured at the test line (T).
• The two methods use distinct sample volumes, buffer requirements, and timing windows. Technologists shall never mix components or substitute procedures between the two products.

5.2 Specimen Requirements and Preparation

• Acceptable specimen types include serum, plasma (heparin, EDTA, or citrate), or fresh whole blood.
• For serum collection, draw blood into plain red-top or gold-top SST tubes. Allow complete clotting for 30–60 minutes, then centrifuge at 1,000–1,200 × g for 10 minutes.
• Inspect specimen condition: use clear, non-hemolyzed serum. Discard grossly hemolyzed or contaminated specimens per Phlebotomy Workflow Unacceptable Specimens and Adverse Reactions Sop.
• Equilibrate refrigerated serum and test pouch to room temperature (15–30 °C) before running tests.

5.3 Method A: AllTest / Trimera HBsAg Rapid Cassette (REF IHBSG-402)

• Check the foil pouch to verify that the expiration date is valid and the seal is intact. Open the pouch and place the cassette horizontally on a clean, dry bench.
• Dispense serum into specimen well S and start digital timer: - For serum or plasma: hold the provided disposable dropper vertically and dispense exactly 3 drops (approximately 75 µL) of serum or plasma into the specimen well (S). Do not add buffer. - For venipuncture whole blood: dispense exactly 3 drops (approximately 75 µL) of whole blood into the specimen well (S), then immediately add 1 drop (approximately 40 µL) of assay buffer. - For fingerstick capillary blood: transfer 75 µL of capillary blood into the specimen well (S), then immediately add 1 drop (approximately 40 µL) of assay buffer.
• Start a digital timer immediately upon specimen loading. Read and interpret results between 15 and 30 minutes. Do not interpret results after 30 minutes, as background drying may cause false lines.
• Interpret visual findings: - Control line C present and visible: the control line (C) must appear for any test to be considered valid. - Test invalid; repeat immediately with a fresh cassette: if the control line (C) fails to appear, the test is invalid and null regardless of the test region; repeat immediately with a fresh cassette. - Test line T visible (reactive or faint): two distinct colored lines appear: one in the control region (C) and one in the test region (T). Any visible intensity of the test line (T), no matter how faint, indicates a reactive result. - Non-Reactive (Negative): only one colored line appears in the control region (C). No visible line appears in the test region (T).

5.4 Method B: SD BIOSENSOR STANDARD Q HBsAg Test (REF QHBS01G)

• Open the foil pouch and immediately inspect the silica gel desiccant packet: - Yellow desiccant beads indicate a valid, dry test device. - Green desiccant beads indicate moisture exposure; discard the device immediately and open a fresh pouch.
• Dispense specimen into round sample well (S): - Use either a calibrated micropipette or the provided 100 µL disposable dropper filled exactly to the black indicator line. - Dispense exactly 100 µL of serum, plasma, or whole blood into the round sample well (S). - No chase buffer is used for this method.
• Start a digital timer immediately. Read the result at exactly 20 minutes. Do not interpret results after 30 minutes.
• Interpret test device bands: - Reactive (Positive): both control line (C) and violet-to-red test line (T) are visible. - Non-Reactive (Negative): only the control line (C) appears. - Invalid: the control line (C) fails to appear. Discard and repeat with a new device.

5.5 Method Comparison and Strict Operational Differences

Technologists shall consult this table to verify method parameters before applying specimens:

5.6 Quality Control and Limitations

• Internal control validation: a visible C line is mandatory on every single cassette. Missing C line automatically voids the test.
• External QC testing: run commercial positive and negative controls on every new shipment and every new reagent lot before clinical use.
• Note analytical limitations: - A non-reactive result does not entirely exclude HBV infection if viral load is below the analytical detection limit (analytical sensitivity approximately 0.5–1.0 IU/mL). - Rapid cassettes are screening tools and do not establish quantitative antigen concentration or chronic carrier stage.

5.7 Result Reporting and Confirmatory Referral Protocol

• Report Non-Reactive for Hepatitis B Surface Antigen HBsAg: - Report as: "Non-Reactive for Hepatitis B Surface Antigen (HBsAg)." - HBsAg screening complete upon technical verification and recording in the LIS. - Include standard report disclaimer: "Rapid screening immunoassay. Non-reactive result does not rule out early acute infection below assay detection limits."
• Retest initial reactive in duplicate using fresh cassettes from original tube: - Retest initial reactive in duplicate using fresh cassettes from original tube: any initial reactive or faint test line (T) shall be retested in duplicate using fresh cassettes from the original patient specimen tube. - Either or both duplicate retests reactive: if either or both duplicate retests are reactive, confirm the reactive screening status. Report the preliminary result as "Reactive (Initial Screening Test Only; Specimen Referred for Confirmation)" and immediately log it into the External Referral Logbook. - Both duplicate retests non-reactive (discordant): if both duplicate retests are non-reactive, the result is discordant. Report as: "Discordant / Indeterminate — Initial Reactive, Retest Non-Reactive." Do NOT report as Non-Reactive. Inspect the specimen for hemolysis or lipemia that may have affected the initial test, request a recollected specimen when needed, and refer the serum aliquot to SPH or SACCL for confirmatory testing (ELISA/CLIA) following the dispatch steps below. - Dispatch serum aliquot to SPH or SACCL for confirmatory testing: separate at least 1.0–1.5 mL of clear serum, store at 2–8 °C, package in triple packaging with frozen cool packs per Referral Laboratory Policy, and dispatch serum aliquot to Samar Provincial Hospital (SPH) or the National Reference Laboratory (SACCL) for confirmatory testing. - Notify the attending physician and record notification in the patient record. - Maintain patient confidentiality per Confidentiality Policy and submit mandatory infectious disease surveillance reports under RA 11332.

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
• WHO Guidelines for the Prevention, Care and Treatment of Persons with Chronic Hepatitis B Infection

Companion policies:

• Serology Section Workflow Quality Control and Result Reporting Sop — Serology section general workflow, QC, and result reporting
• Referral Laboratory Policy — Specimen referral, packaging, and transmission procedures
• Laboratory Retention Schedule Policy — Master retention schedule for serological records and referral documents