Hepatitis C - RNA Viral Load

This page provides viral load testing information for Hepatitis C virus at Public Health Ontario (PHO).


  • Testing indications have been updated.

Testing Indications

HCV RNA testing is intended to diagnose HCV infection in individuals with evidence of HCV antibody and/or evidence of liver diseases. Detection of HCV RNA indicates an active HCV replication and is evidence of an active infection.

HCV RNA testing is also intended for the following:

  • As an aid in the management of HCV-infected patients undergoing anti-viral therapy
  • To determine spontaneous HCV clearance
  • Transplant recipient work-up and post-transplant monitoring
  • Rule out acute HCV infection in patients with negative or inconclusive HCV antibody
  • Immunocompromised patients
  • Patients with high-risk activities (e.g. Injection drug users, HIV-positive patients engaging in MSM activities)  and risk exposures
  • Children between the ages of 6 weeks and 18 months of age who were born to HCV-positive mothers

HCV RNA testing in Dried Blood Spots (DBS) is intended for:

  • Remote/isolated areas where access to laboratory facilities may not be available or, if available, may have limited capacity for processing
  • Inability to provide venous-collected blood specimens

Acceptance/Rejection Criteria

  1. DBS specimens are accepted for testing provided the following conditions are met:
    • Positive laboratory HCV antibody test in Ontario
    • Positive HCV antibody test by Point of Care (POC) test
    This information must be indicated on the requisition under “other relevant and clinical information” field.
  2. Blood specimens received that are not collected or processed using the specifications on this test information sheet will be rejected.
  3. Requests received other than outlined in the testing indication will be rejected, unless approved by microbiologists.
  4. Requests with incomplete information on the requisition will be rejected.

Specimen Collection and Handling

Specimen Requirements

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

HCV RNA Viral Load


2.5 mL

Serum separator tube (SST); vacutainer tubes without additives

HCV RNA Viral Load


4 DBS per patient sample to allow for additional testing if required

DBS placed in an individual resealable bag with a desiccant sachet in each bag

Submission and Collection Notes


Freshly drawn whole blood specimens collected in an SST or other vacutainer tubes without additives may be stored and/or transported at 2°C to 25°C for up to 24 hours before centrifugation. Following centrifugation, remove serum from cells immediately. Serum specimens may be stored and/or transported at 2°C to 8°C for up to 6 days or at ≤-18°C for up to 12 weeks. If HCV genotyping is requested, serum specimens may be stored and/or transported at 2°C to 8°C for up to 72 hours or at ≤-18°C for up to 6 weeks. If more extended storage specimens is required, it must be frozen at ≤-60°C.


Submit a DBS card containing at least four (4) circles fully delineated with the patient’s blood. Allow the spots to dry properly before submitting the card to PHO. DBS may be stored and/or transported at 2°C to 25°C for up to 30 days.


Complete all fields on the requisition. Indicate patient history and all other relevant clinical information and select whether the request is for diagnostic, pre-treatment, on-treatment, or post-treatment monitoring purposes. The date and time of collection must be recorded on the test requisition.


Label the specimen container with the patient’s full name, date of collection and one other unique identifier such as the patient’s date of birth or Health Card Number. Failure to provide this information may result in rejection or testing delay.


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

HCV RNA viral load testing is not intended for donor screening.

DBS samples are not a replacement for venous-collected samples as the sensitivity for detecting HCV RNA from DBS is lower than serum-collected specimens.

Storage and Transport

Place the specimen in a biohazard bag and seal. To avoid delays in testing, it is recommended to ship specimens for testing to PHO’s laboratory immediately after collection or processing. Whole blood that has not been centrifuged must be received at PHO’s laboratory within 24 hours of collection, before 2:00 p.m. Monday - Friday. Serum stored at 2°C to 8°C must be shipped with ice packs within 3 days of separation. Frozen serum specimen must be shipped on dry ice.

Filter cards containing DBS specimens should be shipped to the PHO’s laboratory in individual re-sealable bags containing a desiccant sachet. When the sample is appropriately prepared, it will be stable at room temperature for up to 30 days. Do not refrigerate samples. Do not ship on weekends.

Shipping of specimens shall be done by Transportation of Dangerous Goods (TDG) certified individuals in accordance with TDG regulations.

Special Instructions

Please note that PHO does not provide supplies or Kits for DBS.

Requisitions and Kit Ordering

Test Frequency and Turnaround Time (TAT)

HCV RNA viral load testing for serum and DBS is performed daily Monday to Friday. Turnaround time is up to 6 workdays from receipt by PHO’s laboratory.

Test Methods

Specimens for HCV RNA viral load are tested using Roche cobas® HCV Assay on the cobas® 6800/8800 system. The cobas® HCV assay is an in vitro nucleic acid amplification test for the quantitation of HCV in serum of HCV-infected individuals. The assay detects HCV genotypes 1-6.


First-time HCV antibody reactive patients will be automatically tested for HCV RNA viral load. All specimens must meet PHO’s acceptance criteria for HCV RNA testing.

HCV Genotyping will be tested on the first pre-treatment (i.e. baseline) specimen submitted for HCV RNA viral load testing if the HCV viral load is ≥125 IU/mL. No additional specimen is usually required if sufficient specimen volume is received.


Results should be interpreted in the context of the patient’s clinical presentation, risk factors, and exposure history. The table below provides a guide for interpreting HCV RNA viral load results.

Serum Results

DBS Results1


Not Detected

Not Detected

HCV RNA not detected. 2

<1.50E+01 IU/mL

<1.00E+03 IU/mL

HCV RNA detected below the lower limit of quantitation. Unable to quantify.
(An exact value cannot be calculated as it is below the lower limit of quantitation of 15 IU/mL (serum) or 1000 IU/mL (DBS).)

1.50 E+01 to 1.00 E+08 IU/mL

1.00 E+03 to 1.00 E+08 IU/mL

Viral load will be reported in IU/mL.

>1.00E+08 IU/mL

>1.00E+08 IU/mL

HCV RNA detected above the upper limit of quantitation. Unable to quantify.
(An exact value cannot be calculated as it is above the upper limit of quantitation of 1.0E+08 IU/mL.)

1Based on internal validation studies performed at PHO’s laboratory, HCV RNA testing conducted on DBS is less sensitive than venous-collected samples. The lower limit of detection (LLOD) of HCV RNA using one DBS per test (1000 IU/mL) ; thus, DBS samples should NOT be used to rule out active HCV infection or to determine whether a patient on treatment has achieved an undetectable HCV RNA level.

2Repeat HCV viral load testing is recommended within 6 months for patients with recent exposure or ongoing risk for HCV infection.

No further testing for HCV is recommended for individuals with two inconclusive HCV antibody and not detected HCV RNA results at least 6-8 weeks apart unless clinically warranted.


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

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Updated 30 March 2023