Detection of Hepatitis C Virus (HCV) by
Rapid Method
Aim: To detect HCV by Rapid Method
Introduction
Hepatitis C Virus was identified in 1989 as the main aetiological agent of non-A, non-B
hepatitis (NANBH) accounting for greater than 90% of post-transfusion hepatitis cases.
HCV is a spherical virus of about 30-60 nm in diameter with single positive stranded RNA
and is related to the family flaviviridae. It is considered to be the major cause of acute
chronic hepatitis, liver cirrhosis and hepatocellular carcinoma throughout the world. It is
therefore necessary to correctly diagnose Hepatitis C infection.
The test for antibodies to HCV was proved to be highly valuable in the diagnosis and study
of the infection, especially in the early diagnosis of HCV after transfusion. The diagnosis of
hepatitis C can be easily made by finding elevated serum ALT levels and presence of
anti-HCV in serum/plasma (Fig.1).
Recently recombinant DNA techniques have been used to encode the genome of HCV.
The genome encodes for three structural proteins (capsid protein, envelope glycoproteins
E1 & E2) and several non-structural proteins (NS2, NS3, NS4 & NS5)
Recently the 4th generation assay for testing of anti-HCV has been established. The 4th
Generation HCV TRI-DOT utilizes a unique combination of modified HCV antigens from the
putative core, NS3, NS4 & NS5 regions of the virus to selectively identify all subtypes of
Hepatitis C Virus in human serum/plasma with a high degree of sensitivity and specificity.
The antigens used are chemically treated and unfolded in a special way to make them
more reactive & specific to different epitopes of core & NS3 region thereby minimizing the
chances of crossreactivity & enhancing the specificity. Also,the superior sensitivity of the
test allows for the significantly earlier detection of antibodies during sero-conversion
following HCV infection, thereby reducing the incidence of post transfusion hepatitis and
providing a safer blood supply
Principle of the Assay
1. HCV antigens are immobilized on a porous immunofiltration membrane.
Sample and the reagents pass through the membrane and are absorbed
into the underlying absorbent pad (Fig. 4).
2. As the patient's sample passes through the membrane, HCV antibodies if present
in serum/plasma, bind to the immobilized antigens. In the subsequent washing step,
unbound serum/plasma proteins are removed (Fig. 4).
3. In the next step, the protein-A conjugate is added which binds to the Fc portion of
the HCV antibodies to give distinct pinkish purple dot against a white background at
the test region (“T1”&/ or “T2”). At the control region (“C”) a “Built-in Quality Control
Dot” has been devised to confirm the proper functioning of the device, reagent and
correct procedural application.
KIT COMPONENTS
The kit contains sufficient reagent and devices for the number of tests as mentioned on the
pack. All kit components should be stored at 2-8oC. DO NOT FREEZE KIT
COMPONENTS.
HCV TRI-DOT Device : Individually quality checked, packed & sealed device. It is marked
with "C" for Control Dot and “T1” & “T2” for Test Dots.
Buffer Solution : Buffer containing BSA and Sodium Azide. Ready to use.
Protein- A Conjugate: Protein- A Conjugate in liquid form containing Sodium Azide. Ready
to use.
Sample Dropper : Long disposable plastic dropper provided for adding the sample.
SAMPLE / SPECIMEN COLLECTION & STORAGE
Collect blood in a clean dry sterilized vial and allow it to clot. Separate the serum by
centrifugation at room temperature.
It is recommended that FRESH samples should be used. If serum is not to be assayed
immediately it should be stored at 2-8oC or frozen at -20oC. Serum may be stored at
2-8oC for upto 3 days and stored frozen at -20oC for 3 months. Only serum or plasma
should be used for the test.
Haemolysed specimen or specimen with microbial contamination should be discarded and
fresh aliquot should be collected.
SAMPLE / SPECIMEN PROCESSING
Though HCV TRI-DOT works best when used with fresh samples, however the frozen or
viscous samples can also perform well if the following instructions are strictly adhered to :
A. Frozen samples
i)Allow the sample to thaw in a vertical position in the rack. Mix the sample thoroughly. If
particles are seen, allow them to settle at the bottom or if a centrifuge is available, the
sample can be centrifuged at 10,000 r.p.m. for 15 minutes.
(ii) Insert the dropper just below the top surface of the sample and withdraw one drop of the
sample.
B. Thick or viscous samples Whenever possible, clear specimen should be used. However,
viscous, thick or turbid samples which may sometimes take more than 40-60 seconds to
flow through the membrane should be centrifuged at 10,000 r.p.m. for 15 minutes and
retested on a fresh device to avoid inconsistent results.
C. Transportation
(i) The WHO guidelines for the safe transport of specimen (WHO/ EMC/97.3) should be
read carefully by the laboratory staff as these guidelines hold equally good for Hepatitis
samples.
(ii) If the specimen is to be transported, it should be packed in compliance with the current
Government regulations on transport of aetiologic agents.
Observation
The appearance of only one dot at control region was observed.
Result and Conclusion
The given patient sample is HCV negative
