About the HIV virus
About HIV/AIDS:
HIV (human immunodeficiency virus) is a virus spread through certain body fluids that attacks the body’s immune system, specifically the CD4 cells, often called T cells. Over time, HIV can destroy so many of these cells that the body can’t fight off infections and disease. These special cells help the immune system fight off infections. Untreated, HIV reduces the number of CD4 cells (T cells) in the body. This damage to the immune system makes it harder and harder for the body to fight off infections and some other diseases. Opportunistic infections or cancers take advantage of a very weak immune system and signal that the person has AIDS (acquired immune deficiency syndrome). Learn more about the stages of HIV and how to know whether you have HIV.
What is HIV?
} HIV is a virus that gradually attacks the immune system, which is our body’s natural defense against illness.
} There are many different strains of HIV – someone who is infected may carry various different strains in their body. These are classified into types, with lots of groups and subtypes. The two main types are:
HIV-1: the most common type found worldwide
HIV-2: this is found mainly in Western Africa, with some cases in India and Europe.
Both types of HIV weaken the immune system, but HIV-2 tends develop more slowly and is less easy for people to transmit than HIV-1
HIV Virus - Learn about transmission risks and disease symptoms
Human Cell - HIV Virus Structure Labeled
SPECIMEN COLLECTION
Blood needed for an HIV test can be collected either by venepuncture.
Acceptable Sample Tube:
Minimum volume required : 3 ml
Sample Type: Serum
Stability: 4 weeks at 2-8 °C,
7 days at 25 °C,
3 months at -20 °C.
Stability: 4 weeks at 2-8 °C,
7 days at 25 °C,
3 months at -20 °C.
Central Sample Receiving Area:
Ø CSR department will verify the Name, MRN on the tube and same will be match with request form
Ø Test will be requested into the LIS (Laboratory Information system).
Ø Sample will be sent to Virology section for Testing.
Laboratory
Microbiology
Section Virology
HIV Diagnostic Testing
1. HIV Combi PT (Screening) on Cobas.... .machine from Roche Diagnostics for quantities testing less than 1.0 reading is negative and more than 1.0 reading meaning is screening test positive after that test is refer to confirmatory testing for Western Blot/ELISA Technique
2. INNO-LIA HIV I/II Score is a line immunoassay (LIA), to confirm the presence of antibodies against the human immunodeficiency virus type 1 (HIV-1), including group O, and type 2 (HIV-2) in human serum or plasma. The INNO- LIA HIV I/II Score also differentiates between HIV-1 and HIV-2 infections.
Initial and Supplemental HIV Tests
Initial Test
- Enzyme Immunoassay (EIA)
- Chemiluminescent Immunoassay (CIA)
Supplemental Tests
- Western blot
- Indirect Immunofluorescence Assay (IFA)
- Qualitative HIV-1 RNA
- Qualitative HIV-1 RNA
HIV Screening Test
HIV Combi PT Intended use
· Immunoassay for the in vitro qualitative determination of HIV-1 p24 antigen and antibodies to HIV-1, including group O, and HIV-2 in human serum.
· This assay is indicated as an aid in the diagnosis of infection with HIV-1 and/or HIV-2.
· This assay is also indicated in testing serum specimens to screen individual organ donors when specimens are obtained while the donor's heart is still beating (Cadaveric).
· The assay does not discriminate among HIV-1 antibody, HIV-2 antibody, or HIV p24 antigen reactivity.
Confirmatory Testing
Western Blot/ELISA Technique
ELISA (enzyme-linked immunosorbent assay)
An enzyme-linked immunosorbent assay, also called ELISA or EIA, is a test that detects and measures antibodies in your blood. This test can be used to determine if you have antibodies related to certain infectious conditions. Antibodies are proteins that your body produces in response to harmful substances called antigens. An ELISA test may be used to diagnose: HIV, which causes AIDS
Test principle: Recombinant proteins and synthetic peptides from HIV-1 and HIV-2, and a synthetic peptide from HIV-1 group O are coated as discrete lines on a nylon strip with plastic backing. Five HIV-1 antigens are applied: sgp120 and gp41, which detect specific antibodies to HIV-1, and p31, p24, and p17, which may also cross-react with antibodies to HIV-2. HIV-1 group O peptides are present in the HIV-1 sgp120 band. The antigens gp36 and sgp105 are applied to detect antibodies to HIV-2. In addition to these HIV antigens, four control lines are coated on each strip: background control line, ± cut-off line (human IgG), 1+ positive control line (human IgG) and one strong 3+ positive control line which is also the sample addition control line (anti-human Ig). The INNO-LIA HIV I/II Score is based on the enzyme immunoassay principle (EIA). The test sample is incubated in a test trough together with the multiple antigen-coated test strip. HIV antibodies, if present in the sample, will bind to the individual HIV antigen lines on the strip. Afterwards, a goat anti-human IgG labelled with alkaline phosphatase is added and will bind to any HIV antigen/antibody complex previously formed. Incubation with enzyme substrate (BCIP/NBT) produces a dark brown color in proportion to the amount of HIV antibody present in the sample. Color development is stopped with sulfuric acid. If the sample contains no HIV-specific antibodies, the labelled anti human antibody will not be bound to antigen/antibody complex so that only a low standard background color develops.
Requirements:
1. Distilled Water
2. Disposable gloves
3. Precision pipettes with disposable tips capable of delivering 10µL, 20 - 200µL, and 200 - 1000µL, respectively.
4. Orbital mixer or rocker (shaker machine)
5. Graduated cylinders: 10, 25, 50, and 100mL
6. Tweezers for strip handling.
7. Timer
8. Gum
9. Dry incubator at 37°C
10. Vacuum aspirator which contains 5% sodium hypochlorite solution in a waste bottle.
11. Sample Diluent
12. Specimen - serum
13. Controls (Positive, Negative)
14. Test Strips
15. Conjugate solution Ready-to-use
16. Substrate solution Ready-to-use
17. Stop Solution Ready-to-use
18. Washing - (Tris buffer containing sodium chloride, detergent and 0.02% bromo-nitro-dioxane as preservative, to be diluted 5x in distilled water. Diluted wash solution is stable for 2 weeks if kept at 2 - 8°C.)
19. Incubation tray With 11 troughs each.
20. Adhesive sealers
21. Data reporting sheet
22. Reading card - identification of reactive antigen lines
23. Storage of developed strips
24. Not Book for reporting
Note: All reagents and the strip tube should be returned to the refrigerator (2 - 8°C) immediately after use
Safety and environment:
Please refer to the Safety Data Sheet (SDS) and product labeling for information on potentially hazardous components
Specimen (collection and handling):
a. The INNO-LIA HIV I/II Score may be performed on human serum or plasma collected in tubes containing citrate, heparin or EDTA as anticoagulants.
b. Before storage, serum or plasma should be separated from blood clot or blood cells by centrifugation.
c. Store the specimens at 2 - 8°C. For storage longer than one week, freeze at -20°C or lower.
Procedure: (3 hours sample incubation)
S.No. | Reagent/sample | Volume | Times | Minutes |
1 | Sample Diluent | 1 ml | ||
2 | Specimen | 20µL | ||
3 | Controls | 20µL | ||
4 | Test strips | 3 hours | ||
5 | Washing Solution | 1 ml | 3 | 6 minutes |
6 | Conjugate | 1 ml | 30 minutes | |
7 | Washing Solution | 1 ml | 3 | 3 minutes |
8 | Substrate | 1 ml | 30 minutes | |
9 | Stop solution | 1 ml | 10 to 30 minutes |
Aspirate the stop solution.
Remove the strips from the test troughs and place them, membrane side upwards, on absorbent paper using tweezers. As soon as the strips have dried completely, results can be interpreted. To accelerate the drying process, place strips in a dry incubator at 37°C for 30 minutes or use a hair dryer for 1 minute. Developed strips will retain their color if stored in the dark. After that paste the stripes on the data reporting sheet by gum on the proper way
Note:
• The strip must be completely dried to avoid any misinterpretation due to faintly visible bands appearing after addition of stop solution.
• Do not place paper on top of the strips as long as they are wet.
• Weak control bands can be observed for samples containing high IgG levels (above the normal IgG range).
HIV-1 Western Blot Antigens
Strip Reading:
The identity and location of the antigens and controls coated on the strip are
as follows:
The intensity of the reaction on the control lines on each strip is used to assign the reactivity ratings for each antigen on that strip:
Intensity of antigen line reaction (R) Rating
Lower than ± R <± -
Equal to ± R = ± ±
Higher than ±, but lower or equal to 1+ ± < R ≤ 1+ +
Higher than 1+ but lower than 3+ 1+ < R < 3+ 2+
Equal to 3+ R = 3+ 3+
Higher than 3+ R > 3+ 4+
A reactivity rating must be made separately for each strip. To facilitate the interpretation, a reading card is enclosed. The alignment of the developed strip with the 3+ control line on the reading card makes the identification of the lines easier.
Interpretation of the results
Confirmation
Extensive evaluations have shown that results may be interpreted as follows:
A sample is NEGATIVE for HIV antibodies:
- If all lines are negative (< ±)
- If one line has a rating of ±, the other lines are negative.
A sample is INDETERMINATE for HIV antibodies:
- If two or more lines have a ± rating
- If one line has a positive rating (≥ 1+), the other lines are negative or ±
- If two or more lines have a positive rating (≥ 1+), but the conditions for HIV positivity, as described below are not fulfilled.
A sample is POSITIVE for HIV antibodies:
- If two lines have a rating ≥ 1+:
• Two envelope lines of the same HIV type (sgp120 + gp41 or sgp105 + gp36).
• One envelope antigen (sgp120, gp41, sgp105 or gp36) and the p24 antigen line.
- If at least three lines have a rating of ≥ 1+:
• One of them must be an envelope antigen (sgp120, gp41, sgp105 or gp36).
Reporting of HIV result
· Negative sample result verified negative in the LIS
· HIV Final by western Blot for confirmation - Positive sample result will be send to our consultant pathologist.
· Confidential result form will be filled for Positive specimen and same to be forwarded to department pathologist.
· Department pathologist will inform the concern physician.
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