1. HCV by PCR
Neelam Gajjar
7/26/2009

2. In this presentation: Objectives Overview Specimen type and handling
Procedure
Instruments Used
Treatment
In this presentation:

3. Overview HCV is a major, growing health problem worldwide
Serological tests are useful for screening, however molecular approaches are the mainstay of HCV tests
Qualitative Molecular Tests:
Confirmation of positive screening tests
Quantitative Molecular Tests:
Prognostic information regarding likelihood of response to therapy
Monitoring of treatment efficacy

4. Specimen Type and Handling
Human Serum or Plasma
Serum – Blood collected in SST tube [non- hemolysed serum only]
Plasma – EDTA as anticogulant [heparin is not acceptable]
Whole blood must be transported at 2-25o C OR frozen at 20-80o C.
Process within 6 hrs of collection
Stored at room temperature for 24 hrs
Refrigerate for up to 5 days

5. Procedure
Starts with Specimen and control preparation, extraction followed by PCR reagent preparation, amplification(real-time PCR), DETECTION 650 uL of vortexed and spun  and unclotted specimens are transferred in to Sample-tube which is loaded in to sample rack onto COBAS Ampliprep instrument  for processing with low and high pos control as well as negative control.
 Prepare K-carrier from your processed specimen
Prepare HCV-ASR  MMX  by adding Maganese AND then add MMX  to processed specimen in to K carrier.
Load the K-carrier in to Taq Man Analyzer
The COBAS Taq Man analyser automatically determines the HCV RNA titer for the sample .The HCV  RNA titer is expresse in IU/mL.

6. Procedural Information
Analytical reportable range for quantitative HCV ASR:
10 – 5,000,000 IU/mL
Material and Process Information
Each CAP TNAI -Kit contains sufficient reagent for 24 specimen runs
Specimen preparation reagents  cassettes are packaged as 48 test runs and HCV ASR MMX COMES IN PACKAGE OF 2 x 24 TEST RUNS
One or Two Day Process

7. COBAS AmpliPrep Features and benefits
72 samples can be loaded at once and processed within 4 hours to meet high-volume testing demands
Continuous sample and reagent loading without operation interruption minimizes downtime and maximizes hands-off time for the operator
Bar-coded sample racks and reagents provide positive sample identification and reagent tracking to reduce hands-on time
Dedicated and disposable sample processing units minimize the potential for cross- contamination
Ready-to-use, compact reagent cassettes are designed to eliminate manual preparation time and prevent manual preparation errors

8. COBAS TaqMan 48 System Features and benefits
Real-time TaqMan PCR methodology provides unmatched accuracy, superior linear range, and the potential to run one test for quantitative and qualitative requirements
Run sizes of 6 to 48 samples provide flexibility and maximum efficiency
The utility channel adds flexibility and cost effectiveness by allowing the laboratory to run IVD and user-defined assays on one fast, easy, and accurate platform
Two independently operating thermal cyclers provide approximate run times of 90 to 120 minutes to increase workflow flexibility and minimize the need for multiple platforms
• AMPLILINK Software is Windows driven for easy navigation, and provides the security of positive sample ID throughout the run, as well as enhanced LIS capabilities

9. Treatment HCV approx 170 million worldwide
HCV RNA testing is used to diagnose acute and chronic Hepatitis C
HCV genotype indicates:
Antiviral therapy with the combination of pegylated interferon alfa and ribavirin
Duration of treatment
Dose of ribavirin
Virological monitoring procedure