Hepatitis C – Diagnostic Serology

This page provides serology testing information for Hepatitis C Virus at Public Health Ontario (PHO).

Testing Indications

Testing for HCV antibodies is indicated for the work-up of patients with suspected chronic or acute viral hepatitis or those with unexplained elevated liver function tests (e.g. ALT, AST), for screening those at high risk of infection (e.g. intravenous drug users, individuals in correctional facilities, etc.), for determining past exposure, needle-stick or occupational exposure, and as part of an epidemiologic investigation (e.g. infection control breach or outbreak situation).

Acceptance/Rejection Criteria

  • Donor testing is not available through PHO’s laboratory. Specimens from patients being screened as potential donors (e.g. organ, tissue, cells, fertility, etc.) should be referred to a laboratory that performs donor screening assays. Specimens received for donor screening at PHO’s laboratory will be rejected.
  • Specimens received more than 7 days post collection will not be tested.

Specimen Collection and Handling

Specimen Requirements

Test Requested Required Requisition(s) Specimen Type Minimum Volume Collection Kit

Hepatitis C


1.5 mL

Serum Separator Tube (SST)

Submission and Collection Notes


Complete all fields of the requisition form, including:

  • Test(s) requests and reason for testing
  • Patient setting, specimen type and site
  • Relevant clinical information

Label the specimen container(s) with the patient’s first and last name, date of collection, and one other unique identifier such as the patient’s date of birth or Health Card Number. For additional information see: Criteria for Acceptance of Patient Specimens. Failure to provide this information may result in rejection or testing delay.


Refer to instructions for ordering Hepatitis C testing for how to complete the “Hepatitis Serology” section of the general test requisition.


Submit centrifuged serum separator tubes (SST) for serological assays. Centrifugation within 24 hours of collection is optimal for HCV RNA reflex testing.


One FULL 5 ml SST is required for testing a combination of hepatitis virus, HTLV, syphilis and rubella. If a full tube cannot be drawn, submit two tubes.


Do not submit glass tubes.


Haemolysed, icteric, lipemic or microbially contaminated serum is not recommended for testing.

Storage and Transport

Place specimen tube in biohazard bag and seal. Place completed General Test Requisition in the pouch at the front of the biohazard bag.

Specimen tubes should be stored at 2-8°C following collection and centrifugation, and should be shipped to PHO’s laboratory on ice packs within 3 days of collection. If delayed shipping is anticipated, remove serum from clot and store frozen at -20°C or colder, and ship on dry ice.

All clinical specimens must be shipped in accordance to the Transportation of Dangerous Good Act.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

HCV serology testing is performed daily Monday to Friday.

Turnaround time is up to 3 business days from receipt of the specimen at PHO’s laboratory for non-reactive specimens and up to 6 business days for reactive and indeterminate specimens.

Test Methods

HCV serology testing is performed using two different tests:

  1. HCV antibody screen: chemiluminescent microparticle immunoassay (CMIA) for the qualitative detection of HCV antibody in human serum. This test does not distinguish between IgG and IgM.
  2. HCV antibody supplemental test: chemiluminescent immunoassay (CLIA) for qualitative detection of HCV antibody in human serum. This test only detects IgG.


All serum specimens submitted for HCV antibody testing are screened by CMIA. Non-reactive CMIA specimens are reported as non-reactive and no supplemental testing is performed.

Reactive CMIA specimens:

  • with a signal-to-cutoff S/CO value below PHO’s internally established threshold are tested using the HCV antibody supplemental test.
  • with a S/CO above PHO’s internally established threshold are considered reactive and no supplemental antibody testing is done. 

All first-time HCV antibody reactive specimens are automatically tested for HEPC RNA Viral Load.


Results should be interpreted in the context of the clinical history, signs and symptoms of the patient. The table below provides a general overview of possible interpretations. A PHO Microbiologist is available for questions about HCV serology results and interpretations.


HCV Antibody Screening Result

HCV Antibody Supplemental Result

Possible Interpretations/Comments




Not tested

  • No evidence of HCV antibody.
  • No prior exposure to HCV.
  • Acute HCV infection window period. HCV RNA testing is recommended for patients who have had recent exposure or are suspected to be in the window period.1






Not tested



  • Inconclusive result for HCV antibody.
  • HCV RNA testing is recommended.1
  • For patients with repeatedly inconclusive HCV antibody results, no further testing is recommended if two inconclusive HCV antibody and not detected HCV RNA viral load results are obtained 6-8 weeks apart.
  • For infants, the HCV IgG antibody detected may represent transfer of maternal antibodies which may persist for up to 18 months.






Not tested2

  • Evidence of HCV antibody.
  • The presence of HCV antibody does not indicate immunity but suggests prior exposure to HCV.
  • For infants, the HCV IgG antibody detected may represent transfer of maternal antibodies which may persist for up to 18 months.
  • Patients that are first-time HCV antibody reactive are automatically tested for HCV RNA viral load.

1Testing for HCV RNA earlier than 6 weeks of age is not recommended as assay sensitivity is limited before that age.
2HCV antibody supplemental testing will not be performed if the S/CO value is above PHO’s internally established threshold or if the patient has a history of a reactive HCV antibody result.


Results are reported to the physician, authorized health care provider (General O. Reg 45/22, s.18) or submitter as indicated on the requisition.

Specimens that are reactive for HCV antibody are reported to the Medical Officer of Health as per the Ontario Health Protection and Promotion Act.

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Updated 9 May 2023