HCV by PCR Neelam Gajjar 7/26/2009

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

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

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

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.

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

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

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

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