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Laboratory diagnosis of HIV infection

You are here : Home/ Blood Bank Zone/ Trasfusion Transmitted Diseases/ 7. Laboratory diagnosis of HIV infection

7. Laboratory diagnosis of HIV infection

The development of specific tests for HIV has made the diagnosis of HI’) and AIDS very simple. The finding of HIV-antibody in patients with previously unexplained immunodeficiency is diagnostic of AIDS. Presence of HIV-antibody in an asymptomatic individual shows the exposure of the person to HI’) and indicates potential infectivity.

The laboratory diagnosis of HiV is based on

1. Detection of specific antibody to HIV
2. Detection of viral antigen.
3. Viral cultures
4. Detection of genetic material by polymerase chain reaction.

The mainstay of diagnosis of HIV and AIDS is the detection of specific antibody to HIV. The tests used are

1. Screening tests
2. Supplemental/confirmatory tests

There are three main kinds of primary screening assay available.

1. Enzyme linked Immunosorbant assay (ELISA)
2. Particle agglutination assays
3. Simple rapid assays

Each laboratory must consider the following features of the test before selecting the most suitable assay system for their laboratory.

1. Sensitivity and specificity of test
2. Scientific principle and complexity
3. Reaction time
4. Availability and cost

Principle of ELISA system

The HIV-antigen preparation is fixed on a solid phase usually wells of a polystyrene microtitre plate or plastic beads.

In indirect (sandwich) ELISA system, the serum to be tested in incubated with the antigen on the solid phase and after appropriate washings, the presence of specific antigen-antibody complex can be demonstrated by an enzyme-labelled antiglobulin. The reaction are revealed by the enzyme reacting with a chromogenic substance.

In competitive immunoassay, an enzyme labelled anti-HIV antibody is added on the serum and antigen coated solid phase. if anti-HIV antibodies are present in the sample, due to completion less of enzyme-labelled anti-HIV antibody is fixed to the immobilized antigen and absence of coloured reaction indicates the presence of anti-HIV in the final stage.

Both the test assay are widely used. However, available competitive ELISA system only detect HIV-1 antibodies whereas indirect ELISA systems can be used for detection of antibody to HIV-1 and HIV-2 both.

Depending upon the source of viral antigen the type of assay system can be graded as :

1st generation assay       Viral lysate
2nd generation assay       Recombinant antigen
3rd generation assay       Synthetic viral oligopeptides

Now a days, test assay systems are available for detection of anti-H1V-1 and anti-HIV-2 in a combined test.

In India, it is mandatory to screen all the blood donation units for antibodies to HIV-1 and I-IIV-2, however the choice of test in any centre depends on :
* technical experience
* infrastructure of the laboratory
* number of tests to be performed
* cost and availability of test system

Indirect ELISA for anti-H1V-1 and anti-H1V-2

Principle
Synthetic peptides representing immunodomination epitopes of HP/-i and HIV-2 are coated onto wells of a microplate. Diluted serum, or plasma samples are added to these wells. If antibodies specific for HIV-1 and/or I-IIV-2 are present in the sample, they will form stable complexes with the HIV peptide antigen on the well. A goat anti-human IgG labelled with horseradish peroxidase is added. If the antigen/antibody complex is present, the peroxidase conjugate will bind to the complex and remain in the well.

Enzyme substrate is then added. During incubation, a blue colour will develop in proportion to the amount of anti-HP) antibody bound to the well. Wells containing samples negative for anti-HP) antibody remain colourless.

An acid stop solution is added to each well and the yellow colour is read on microplate reader at 450 nm.

Reagents & material
I. Microplate (12 x 8 wells)
2. Negative control
3. Positive control
4. Sample diluent
5. Wash solution concentrate
6. Peroxidase conjugate concentrate
7. Conjugate diluent
8. Treatmenthylbenzidine (TMB) reagent
9. Peroxide reagent
10. Stop solution (IN H2S04)

Test tubes and racks
Multipipettors: 100 ul and tips or automated dispenser
Disposable glass pipettes : 1 ml , 5m1, 10 ml

Timer
Disposable gloves
Deionized or distilled water
Microplate washer/dispenser
Sodium hypochiorite
Multichannel micropipette for delivering 50 ul, 75ul, 100 ul, with disposable tips.
Micropipette with disposable tips capable of delivering 5 ul, lOOul
Incubater 37°C
Sodium hypochlorite
Incubation box with wad of wet filter paper
Microwell plate washing system
ELISA Plate Reader (reads at 450mn /492 nm as specified in the kit, as this changes according to the èolour reaction of the conjugate)
Specimen colleciton
Clotted sample/serum sample
Use of heat inactivated serum or plasma is not recommended as this may lead to false-positive results

Procedure

Use the laboratory worksheet for arranging the samples.
1. Remove the microplate and reagents from the refrigerator and allow to come to room temperature (approximately 30 minutes). Remove the plate from its pouch just before use and label.
2. Into clean test tubes, uncoated microplates or other comparable containers, dilute 5 ul of each sample to be tested with 250 ul. Sample diluent and mix well. This method may be performed with an automatic diluting device.
3. Dispense lOOul of the sample diluent into A 1 for use as a substrate blank.
4. Dispense 100 ul of the negative control sample into each of 3 wells and 100 ul of the positive control samples into each of 2 wells. Similarly dispense 100 ul of diluted samples into wells using a separate pipette for each sample.
5. Incubate at room temperature (15° to 30CC) for 30 minutes.
6. Aspirate and wash the plate 5 times with 350 ul per well of wash solution. Automated washer should be adjusted to fill each well completely without overfilling. After the final wash, be sure all of the solutipn is removed from each well. Sharply tap the plate upside down on absorbent paper to remove the last remaining liquid, taking care not to dislodge strips from holder.
7. Dispense 100 ul of diluted peroxidase ocnjugate into each well of the microplate, including Al.
8. Incubate the plate for 30 minutes at room temperature.
9. Aspirate and wash the plate 5 times with 350 uI/well of wash solution. After the final wash, be sure all solution is removed from each well.
10. Add 100 ul of freshly prepared substrate solution into each well.
11. Incubate the substrate-filled plates for 30 minutes at room temperature (15° to 30°C). Start the timing with 3 minutes after the addition of the reagent to the first well.
12. Stop the reaction by additing 100 ul of stop solution into each well in the same order used in the addition of the substrate reagent.
13. After adding the stop solution, the colour developed may be read on a plate reader at 4SOnm. The reader should be blanked at 450 nm against the substrate blank Test must be read within 30 minutes.

Results

Test validity

Three (3) negative and two (2) positive controls must be inlcuded on each run. The control results must be examined before the sample results can be interpreted.
Calculation of Negative control Mean (NCx)
Absorbance of individual negative control values must be less than or equal to 0.150.
Calculation of Positive control mean (PCx)
The positive control mean must be equal to or greater than 0.80. If the mean value is less than 0.80, the run should be repeated.
Cutoff determination

Cutoff = NCx +0.150

Interpretation of sample results

1. If the initial result absorbance value is less than the calculated cutoff value then the sample is considered non-reactive.
2. If the screening value of a sample is equal to or greater than the cutoff retest in duplicate using a fresh dilution of the original sample.
3. If both retest values are less than the cutoff, the interpretation.of the test is non-reactive for HIV 1+2 antibodies.
Reporting of results

Although the terms ‘positive’ and ‘negative’ reaction are frequently used, it is preferrable to report the results of the screening test as reactive/nan-reactive for positive and negative results respectively. Positive and negative test should only be used to discuss the status of the donated blood unit whereas donor status is assessed only after the initial result has been confirmed by further assays. It is important to remember that a number of conditions can give false negative and positive results.

False negative results are due to - Operations errors
- Low titre antibodies to env coded glycoproteins and gag encoded core proteins
- Complete immunosuppression
- Inability of assay to detect IgM antibody efficiently

False positive results

(Initial Reactive / Not reproducible)

Technical problems

- Improper washing
- Cross contamination of samples
- Edge effect (microtitre plate)
- Incorrect reaction temperature

False positive results are due to :

- Haemolysed or lipaemic sample
- HLA antibodies
- Nonspecific adherence to immunoglobulin
- Repeated freezing & thawing of specimens
- Stickiness of stored sera from malarial regions
- Unexplained factors in parenteral drug abusers
- Persons with alcoholic liver disease
- Cross reactions with other retroviruses

Problems with WV ELISA

Some problems may occur while performing the ELISA technique.
1. Back ground colour at the end of test in all wells

Reasons

* Improper washing of wells.
* Microwells or substrate exposed to strong light

3. No colour in the microwells at the end of test

4. No colour in negative/positive control wells

Reasons

* Failure to add the conjugate or improper identification of reagents
* Failure to add negative/positive controls
* Substrate nor correctly prepared i.e. failure to add the particular reagent or improper identification of ragents.
* Reagents not at room temperature (200C - 24°C) before use (allow it to warm up to room temperature, specially in winter keep the kit and wash buffer in incubator he. at 37°C for 10 minutes before use)
* Improper identification of reagents
* Contamination of wash buffer
* Contamination of distilled water
* Change in the pH of distilled water

5. Unsatisfactory cutoff value i.e too low in direct ELISA or too high in indirect ELISA test. To eliminate this check, test validity as given in kit insert.

Reasons

* Controls may be contaminated
* Mistake in calculation
* Wrong filter used to take the reading
* ELISA reader may be defective

6 Electrical equipment is not working

Reasons

* Check the safety fuse
* Connect the equipment to other electrical point
* Check connections in the plug

Particle agglutination assays

The assay is generally performed in microplates which simply acts as miniaturized test tubes as they are not coated with any antigen. The test principle is similar to haemagglutination based on the fact that sensitized particles made out gelatin or plates will be agglutinated by a specific antibody.

Advantages

1. Does not require use of expensive equipment
2. Simple to perform
3. Visual reading
4. Suitable for smaller number of samples

Only kits available for HIV 1+2 should be used and manufacturer’s instructions should be followed.

Simple rapid tests

Simple tests combine the simplicity of particle agglutination assays together with the technology of EIA. They are relatively easy to perform and are quicker. A number of different formats exist, all using the basic principle in which antigen is immobilized on porous/semiporous membrane or strip and to which the test sample and other reagents supplied along with the kit are added.

Since the test systems available are changing, the choice of test should be based on updated knowledge. The xhosen method should be sensitive enough to detect low-dose antibody carriers and its specificity should be broad to pick up HIV variants also. Although high sensitivity is the most important requirement for a screening test, too many false-positive can lead to loss of blood units from already deficient blood stocks in blood transfusion services.

Immuno dot assay

Principle

Bi-Spot HIV-1 and HIV-2 kit is based on the principle of the solid-phase enzyme immunoassay. The plastic immunocomb card functions as the solid phase, the card possesses 12 projections (teeth) sensitized with synthetic peptide antigens of l-IIV-1 (lower spot), HIV-2 (middle spot) and with antibodies to human immunoglobulin (upper spot), serving as an internal control to verify proper functioning of the kit reagents.

Serum or plasma specimens are deposited into the first row of well of the developing plate. The antigen-antibody reaction takes place with the insertion of the immunocomb card into the wells. Antibodies to HIV-1 and/or HIV-2, if present in the specimens, bind to the antigen spots on the teeth; immunoglobulins present in the specimen also bind to the spot on the upper position of the teeth.

After a wash step, the card is transferred to a compartment containing enzyme-labelled antibodies to human IgG which bind with the antibody complex on the top of the teeth. The card undergoes two successive wash steps and is moved to the chromogeneic substrate compartment for visualization of bound antibodies.

Immunocomb card

Each card possesses 12 projections (teeth), each sensitized with:

1. Synthetic HIV-1 peptides (conserved sequences of gp4l and gp 120) as lower spot.
2. Synthetic HIV-2 peptide (conserved sequence of gp36) as middle spot.
3. Goat anti-human immunoglobulin as an upper spot.

The card may be separated into individual portions, as required in the assay run.

Developing plate

Each developing plate contains 6 reagent solutions (compartment A-F) ready for use in all steps of the test. Each compartment is divided into 12 wells.

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