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HCV Test 

Hepatitis C virus (HCV ) rapid test positive as shown above image. The 4th Generation HCV  test i.e. HCV TRI-DOT is a rapid, visual, sensitive, and qualitative in vitro diagnostic test for the detection of antibodies to Hepatitis C Virus in human serum or plasma. The 4th Generation HCV TRI-DOT has been developed and designed with increased sensitivity for core and NS3 antibodies using a unique combination of modified HCV antigens. They are for the putative core (structural), protease/helicase NS3 (non-structural), NS4 (non-structural), and replicate NS5 (non-structural) regions of the virus in the form of two test dots “T1” and “T2” to provide a highly sensitive and specific diagnostic test

Principle of HCV Test

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.  As the patient’s sample passes through the membrane, HCV antibodies if present in the blood, bind to the immobilized antigens. In the subsequent washing step, unbound serum/plasma proteins are removed.  In the next step, the protein-A conjugate is added which binds to the Fc portion of the HCV antibodies to give a distinct pinkish-purple dot against a white background at the test region (“T1” and/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.

Test Requirements

• Test Kit contains

1. HCV TRI-DOT test device
2. Buffer Solution
3. Protein-A conjugate
4. Sample Dropper

• Extra we need

1. Infectious Wastebin
2. Gloves
3. Timer

• Specimen: serum/plasma

Test Procedure of HCV Test

1. Add 3 drops of Buffer Solution to the center of the device.
2. Hold the dropper vertically downwards and add 1 drop of the patient’s sample (50 µl serum or plasma) using the sample dropper provided. (use a separate sample dropper for each specimen to be tested).
3. Add 5 drops of Buffer Solution.
4. Add 2 drops of Protein- A Conjugate.
5. Add 5 drops of Buffer Solution.
6. Read result immediately and discard the device considering it to be potentially infectious

Result Interpretation of HCV test

• Non-Reactive: Only on dot at the control region
• Reactive test result:  Appearance of two dots, one at the control region
  “C” & others at the test region “T1” indicates that the sample is REACTIVE for antibodies to HCV.  The appearance of two dots, one at the control region
  “C” & others at the test region “T2” indicates that the sample is REACTIVE for antibodies to HCV.  The appearance of all the three dots, one each at “C”
  “T1” and “T2” region indicates that the specimen is REACTIVE for antibodies to HCV.
• Invalid test result:  If no dot appears after the completion of the test, either with clear background or with complete pinkish/purplish background the test indicates ERROR. This may indicate a procedural error or deterioration of specimen/reagents or particulate matter in the specimen. The specimen should be retested on a fresh device.

Keynotes on HCV Test

1. Performance characteristics of HCV tri-dot test kits of  J. Mitra & Co. Pvt. Ltd has a sensitivity of 100% and a specificity of 98.9%.
2. It is important to allow each solution to soak in the test device before adding the next solution as in the video.
3. This 4th generation HCV TRI-DOT has been developed and designed using modified HCV antigens representing the immunodominant regions of HCV antigen.

Limitations of HCV test

Limitations of the HCV-Tri-dot test are as follows-

1.  The 4th Generation HCV TRI-DOT detects anti-HCV in human serum or plasma and is only a screening test. All reactive samples should be confirmed by supplemental assays like RIBA. Therefore for a definitive diagnosis, the patient’s clinical history, symptomatology as well as serological data, should be considered. The results should be reported only after complying with the above procedure.
2.  The test is only validated for serum and plasma from individual bleeds and not for pools of serum or plasma or other body fluids.
3.  A non-reactive result does not exclude the possibility of exposure to or infection with HCV.
4. It should be noted that repeated false reactive results may occur due to the non-specific binding of the sample to the membrane.
5. The presence of anti-HCV does not imply a Hepatitis C infection but may be indicative of recent and/or past infection by HCV.
6.  Patients with auto-immune liver diseases, Renal disorders, and Antenatal samples may show false reactive results.
7.  The kit works best when used with fresh samples and when all the kit components are at room temperature (20-25°C). Samples that have been frozen and thawed several times contain particulates that can block the membrane, hence resulting in an improper flow of reagents and high background color which may make the interpretation of results difficult.
8.  Optimum test performance depends on strict adherence to the test procedure as described in this manual. Any deviation from the test procedure may lead to erratic results.

References

1. http://jmitra.co.in/download/Procedure/Manual-HCV-Tri-Dot.pdf
2.  Caypers, H.T.M. Wiakel, I.N. Vander Poel, C.L. etal (1971)
   J. of Hepatology, 13, 5.1.
3. Halfon, P. et al (1997) J. Medical Virology. 52:391-395.
4. Sarin, S.K. & Hess. G. (1998). Transfusion-associated Hepatitis.
5.  Sayers, M.H. & Gretch D.R. (1993). J. Transfusion 30,809-13.