Standard Operating Procedure for Widal Test

STANDARD OPERATING PROCEDURE (SOP): WIDAL AGGLUTINATION TEST

Document ID: [________________]
Effective Date: [________________]
Revision Number: [________________]
Department: Laboratory Services / Clinical Pathology

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1. PURPOSE

The purpose of this Standard Operating Procedure (SOP) is to provide a standardized, accurate, and reproducible methodology for performing the Widal Agglutination Test to aid in the laboratory diagnosis of Typhoid and Paratyphoid fevers.

2. SCOPE

This procedure applies to all qualified laboratory personnel performing serological testing on patient serum samples at [_________________ Laboratory Name].

3. RESPONSIBILITY

The Laboratory Manager is responsible for ensuring compliance with this SOP. Laboratory Technicians are responsible for the precise execution of the test, documentation of results, and reporting to the requesting physician.

4. MATERIALS AND REAGENTS

• Reagents: Widal Antigens (Salmonella typhi 'O', 'H'; S. paratyphi 'AH', 'BH') obtained from [_________________ Manufacturer].
• Equipment: Centrifuge, Serological Pipettes, Test Tubes/Slide, Timer, Incubator (if required).
• Controls: Known Positive and Negative control sera.

5. PROCEDURE

5.1 Specimen Collection

• Patient Name: [_________________]
• Patient ID/MRN: [_________________]
• Collection Date/Time: [_________________]
• The specimen must be serum collected in a [_________________ tube type] and stored at [_________________ temperature] until testing.

5.2 Test Methodology (Slide/Tube)

1. Preparation: Bring all reagents to room temperature ([____]°C).
2. Slide Method (Screening):
   • Place [____] µL of patient serum onto the designated reaction circles.
   • Add one drop of each Widal antigen reagent to the corresponding circles.
   • Rotate the slide for [____] minutes.
   • Observe for agglutination under a high-intensity light source.
3. Tube Method (Confirmatory):
   • Prepare serial dilutions of patient serum in saline (e.g., 1:20, 1:40, 1:80, 1:160).
   • Add equal volumes of the respective antigens.
   • Incubate at [____]°C for [____] hours.
   • Read for the presence of a granular or flocculent deposit.

6. QUALITY CONTROL

• Positive Control Result: [_________________]
• Negative Control Result: [_________________]
• Lot Number of Reagents: [_________________]
• Expiry Date of Reagents: [_________________]

7. RECORDING AND REPORTING

Results shall be recorded as the highest dilution showing agglutination. Significant titers defined by the clinical director are:

• Typhi O: [_________________]
• Typhi H: [_________________]
• Paratyphi A/B: [_________________]

Observations/Discrepancies: [____________________________________________________________________]

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8. AUTHORIZATION AND SIGNATURES

Prepared By:
Name: ___________________________
Title: ___________________________
Signature: ___________________________
Date: ___________________________

Reviewed and Approved By (Laboratory Director):
Name: ___________________________
Title: ___________________________
Signature: ___________________________
Date: ___________________________

Quality Assurance/Compliance Officer:
Name: ___________________________
Signature: ___________________________
Date: ___________________________

Template Document: Standard Operating Procedure for Widal Test

• Document owner: [Role or team]
• Effective date: [Date]
• Version: [Version number]
• Applies to: [Team, department or process]

3. RESPONSIBILITY

ratory Manager is responsible for overall oversight. Medical Laboratory Technologists are responsible for sample collection, preparation, running the assay, and recording results. Quality Control Officers are responsible for validating reagent performance and reviewing documentation.

4. MATERIALS AND REAGENTS

• Widal antigen suspensions (Salmonella typhi O, Salmonella typhi H, Salmonella paratyphi AH, and Salmonella paratyphi BH)
• Patient serum sample
• Positive and negative control sera
• Glass slide or Kahn tubes for test tube method
• Micropipettes and tips
• Saline solution
• Agglutination viewing tile and timer

5. PROCEDURE

1. Collect venous blood from the patient into a plain collection tube and separate the serum via centrifugation. (Responsible: [Role name])
2. Bring all Widal antigen suspensions, controls, and test sera to room temperature before testing. (Responsible: [Role name])
3. Label the reaction circles on the glass slide or tubes clearly with the patient ID and specific antigen types. (Responsible: [Role name])
4. Pipette measured volumes of patient serum, such as [Volume amount], onto the designated test circles or tubes. (Responsible: [Role name])
5. Add one drop of Salmonella typhi O antigen suspension to the first test circle or tube. (Responsible: [Role name])
6. Add one drop of Salmonella typhi H antigen suspension to the second test circle or tube. (Responsible: [Role name])
7. Add one drop of Salmonella paratyphi AH and BH antigen suspensions to their respective test positions. (Responsible: [Role name])
8. Run positive and negative control tests alongside the patient sample using the provided control sera. (Responsible: [Role name])
9. Mix each mixture thoroughly using a clean wooden applicator stick or by gently agitating the slide for [Number] seconds. (Responsible: [Role name])
10. Place the slide on a mechanical rotator or let it stand undisturbed for exactly [Number] minutes. (Responsible: [Role name])
11. Observe the reaction under a good light source for visible agglutination and record the titer where clumping occurs. (Responsible: [Role name])

6. QUALITY CONTROL

Verify that positive controls show distinct agglutination and negative controls show no visible clumping before reporting any patient results. Document all lot numbers and expiration dates of the reagents.

7. RECORDING AND REPORTING

Record all observed agglutination titers in the laboratory information system and sign off on the report sheet.

8. AUTHORIZATION AND SIGNATURES

Records and revision

• Prepared by: [Full name]
• Reviewed by: [Full name]
• Date completed: [Date]
• Signature: ______________________
• Date signed: [Date]