Hepatitis C - RNA Viral Load

*Uncontrolled print copy. Valid only on day of print: 08 Aug 2022.

Testing Indications

Hepatitis C virus (HCV) RNA testing should not be used as a diagnostic test for diagnosing hepatitis C infection. It is most commonly used for measuring viremia (i.e., amount of virus in the blood) in patients who are HCV antibody positive, or when assessing patients with inconclusive HCV antibody results. HCV RNA should be ordered at the following times and/or for the following indications:

As a baseline after a positive HCV antibody result in order to determine the infectious status of the patient.
Prior to and during antiviral treatment for hepatitis C.
Post-treatment to determine if the patient has cleared the virus and achieved a sustained virologic response (SVR).
During the assessment and investigation of immuno-compromised HCV antibody negative individuals.
Follow-up of children between the ages of 6 weeks and 18 months of age who were born to HCV positive mothers.

Specimen Collection and Handling

Specimen Requirements

Test Requested	Required Requisition(s)	Specimen Type	Minimum Volume	Collection Kit
HCV RNA Viral Load	Hepatitis PCR Requisition: Hepatitis C RNA and/or Hepatitis B DNA Viral Load	Serum¹	2.5 ml	Blood, clotted - serum separator tube (SST)
HCV RNA Viral Load	Hepatitis PCR Requisition: Hepatitis C RNA and/or Hepatitis B DNA Viral Load	Whole Blood²	5 mL	SST or any tubes without additive²
HCV RNA Viral Load	Hepatitis PCR Requisition: Hepatitis C RNA and/or Hepatitis B DNA Viral Load	Dried Blood Spot (DBS)³	4 DBS per patient sample to allow for additional testing if required³	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 a serum separator tube (SST) 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, and transfer serum into screw-capped cryovial tubes. . 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 of serum specimens is required, it must be frozen at ≤-60°C.

Unspun whole blood must be received at PHO’s Laboratory within 24 hours of collection before 2:00 p.m. Monday – Friday.

Refer to LAB-SD-123 Hepatitis C Virus (RNA) detection in Dried Blood Spots (DBS) for collection criteria and instructions for RNA testing using DBS. DBS samples, when prepared appropriately, will be stable at room temperature for up to 30 days. HCV genotyping will be tested automatically if the HCV Viral load meets testing criteria and has sufficient DBS.

Limitations

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

HCV viral load testing should not be used to screen for HCV in blood or blood products or as a diagnostic test to confirm the presence of HCV infection.

DBS samples are not a replacement for venous-collected samples as the sensitivity for detecting HCV RNA from DBS is lower than serum-collected samples. Use of DBS for HCV RNA testing should only be considered when standard methods are not possible.

Preparation Prior to Transport

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.

Place specimen in a biohazard bag and seal. It is recommended to ship specimens for testing to PHO’s laboratory immediately after collection or processing to avoid delays in testing.

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

Shipping of specimens shall be done by TDG certified individuals in accordance with TDG regulations.

Special Instructions

Please 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

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 days from receipt by PHO’s laboratory.

Reporting

Results are reported to the ordering physician or health care provider as indicated on the requisition.

Test Methods

Specimens for HCV RNA viral load are tested using Roche cobas® HCV Quantitative 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.

Algorithm

For patients with a detectable HCV RNA viral load, genotyping of their HCV may be useful in evaluating the likelihood of response to some currently available anti-viral therapies and for epidemiologic purposes. However, with currently available direct acting anti-HCV therapies, the utility of genotyping has become less important for choosing a treatment regimen or for determining the duration of therapy. Refer to the Hepatitis C – Genotyping/Subtyping Test Information Sheet for additional information.

Interpretation

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 the interpretation of HCV RNA viral load results.

Serum Results	DBS Results¹	Interpretation
Not Detected	Not Detected	HCV RNA not detected
<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 for HCV viral load 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 for HCV viral load 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 Laboratory, HCV RNA testing conducted on DBS is less sensitive than venous-collected samples. The lower limit of detection (LLOD) of HCV RNA using two DBS per test (103 IU/mL) is approximately 1.6 to 2.0 logs higher than a concomitantly tested EDTA plasma or serum sample (LLOD = 15 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.

No additional sample is usually required for HCV genotyping, provided there is sufficient volume. The first pre-treatment (i.e. baseline) sample submitted for HCV RNA viral load testing will be used to automatically perform HCV genotyping if the HCV viral load is ≥125 IU/mL. Refer to the HCV Genotyping Test Information Sheet for more information.