You are here : Home/ Blood Bank Zone/ Trasfusion Transmitted Diseases/ 3. Laboratory diagnosis of HBV infection
Laboratory diagnosis of HBV infection
3. Laboratory diagnosis of HBV infectionAs large amounts of HBsAg is shed into plasma by HBV, it is relatively easy to develop immunossay suitable for mass screening. A number of immunoassays have been developed with a variable degree of sensitivity and specificity.
It is essential to use highly sensitive and specific test assay system to avoid any risk of transmission of HBV
Reverse passive haemagglutinatlon (RPHA)
Although preferrably 3rd generation test assay system should be used for screening purposes, in many transfusion microbiology laboratories RPHA is still being used as a screening procedure.
RBCs of human/animal origia are coated with anti-HBs and on reaction with serum containing HBsAg, the red cells agglutinate in a characteristic scattered cell pattern. Sensitivity of RPHA for detection of HBsAg is of the order of 10-100 ng.ml of serum. Human, Turkey and sheep red cells can be used to prepare RPHA reagents while anti-HBs is usually produced in chimpanzees, guinea pigs, rabbits or sheep.
The test can be performed in U or V welled microtitre plates. A number of commercial kits are available and manufacturer’s instruction should be followed.
Clotted blood or serum samples in leakproof containers.
Microtitre plates having U - shaped wells, (polystyrene)
Automatic pipette 25 ul (O.025m1)
Pipette dropper (disposable) 25 ul (O.025m1)
Reagents (as given in the kit)
* Human red blood cells coated with anti-HBs
* Cell diluent for preparing a suspension
* Specimen diluent
* HBsAg positive control serum (Human)
* HBsAg negative control serum (Human)
Arrange samples according to the laboratory worksheet.
1. Include a negative and a positive control on each test-plate used.
2. Label the microplate to enable identification of test specimens, wipe the surface of the test plate with a damp cloth to remove static electricity.
3. Using a clean 25 ul pipette dropper, deliver 25 ul of specimen diluent to each of required number of test wells. Tap the plate to assure reagents are at bottom of the well.
4. Using a 7 ul pipette, deliver 7 ul of particle free serum into the appropriate well. The tip of the pipette should be immersed in the diluent and mix.
5. Deliver 25 ul of evently suspended reconstituted cells into each well with serum.
6. Place a cover on the test plate & mix throughly.
7. Incubate at RT (22°C) for 2 hours.
8. Take readings
Diffuse cell pattern is indicative of agglutination and a positive result. Compact cell button is indicative of a negative result.
The reaction may be better observed with a concave mirror viewer.
1. False-positive - rheumatoid factor
use of plasma instead of serum
2. Heat inactivated serum may also produce a false-positive pattern.
Enzyme Linked Immunosorbant Assay (ELISA) This test system is very sensitive and specific, and detects level of HBsAg as low as O.5ng/ml comparable to the level of detection by radioimmunoassay (RIA). As RIA is associated with hazards due to the use of radiosotopic reagents, more and more laboratories have now shifted from RIA to ELISA as a very sensitive screening test for HBsAg.
Anti-HBs is bound to the solid phase i.e., the polystyrene microplate well. The test sample is incubated in the antibody coated well. Through washing with buffer removes unreacted substances and leaves antigen if present attached to the surface of the well. A second antibody labelled with enzyme is then used to react with the happed antigen. Again a washing step removes unreacted enzyme-labelled antibody that is not bound to the antigen. The amount of enzyme left in the well is therefore proportional to the amount of antigen in the test specimen. The final step is testing for enzyme activity using the enzyme substrate.
Depending on the enzyme and enzyme substrate used, the assay may be read visually or quantitated by some optical system e.g. spectrophotometer or ELISA reader.
Arrange samples according to laboratory worksheet and follow all instructions according to the manufacturer.
1. Dispense lOOul of negative control, low positive control (undiluted/diluted) and samples into their respective wells.
2. Apply a cover sealer in order to prevent evoporation.
3. Incubate according to the procedure selected:
A 1 hour 37°C
B 18 to 22 hours (overnight) RT.
C 30 minutes, 37°C
Before ending of incubation
4. Prepare washing solution by dissolving 20 ml wash buffer in 500 ml distilled water sufficient for one plate. Storage life of washing solution is 1 week when stored at 2°C to 8°C.
5. Prepare diluted Enzyme conjugate by dissolving 0.2 ml (200 ul) in 10 ml Enzyme conjugate diluent.
6. At the end of the incubation, remove and discard the cover sealer. Aspirate the liquid and rinse each well five times with 0.3 ml wash buffer. Avoid overflow from the reactrion wells. The rinsing buffer shoud be aspirated throughly.
When either an automatic or semiautomatic washing device is used, each dispensing step should be followed by an aspirating step after 30 seconds to allow complete filling up of the wells. After rinsing, turn the strips mouth down on to blotting paper and gently tap to remove any excess buffer. Leave the strips overtuned on blotting paper until they are all drained.
7. Dispense 0.lml (lOOul) of enzyme conjugate, diluted 1:50 into all wells. Repeat steps 2 and 3.
8. Prepare chromogen / substrate just before the end of second incubation by adding 0.2 ml chromogen in 10 ml diluent. Use within 1 hour.
9. Repeat step 6.
10. Dispense 0.1 ml (lOOul) chromogen/substrate into all wells.
11. Incubate for 30 minutes at room temperature, protecting from intense light.
12. Dispense 0.2m1 (200ul) blocking reagent into all wells in the same order as for chromogen/ substrate, maintaining the same time interval.
13. Read the colour produced by the samples visually or with an instrument at 450 nm.
Interpretation of results by visual reading
The procedure should be considered valid If:
* the colour produced by the diluted low positive control is clearly different from that of negative control, which must remain nearly colourless.
* the colour produced by the positive control is clealry more intense than that of diluted low positive control.
The samples producing a colour more intense than or equal to that of low positive control should be considered reactive for HBsAg.
The samples producing a less intense colour than that of diluted low positive control should be considered negative for HBsAg.
Calculation of results from readings by ELISA reader
Blank the instrument with air for 450 mn filter.
Record the absorbance at 450 nm for each specimen and calculate the mean absorbance value for controls.
The presence or absence of HBsAg is determined by relating the absorbance value of unknown samples to that of the cut-off value. The unknown samples whose absorbance is greater than or equal to the cut-off value should be considered reactive for HBsAg. The unknown samples whose absorbance Is less than the cut-off value should be considered nonreactive. Ensure absolute cleanliness of the well bottom in order to avoid abnormal absorbance values.
Calculation for determining the cut-off vlaue
The cut-off value is obtained by adding the mean absorbance of the negative control values (NC) to the mean absorbance of the low positive control values (LPC) multiplied by 0.2.
Cut-off value = NC+(LPC x 0.2)
The calculation of cut-off value may differ depending upon the kit. Follow the manufacturer’s instructions.
The mean absorbance for low positive control (LPC) should be at least 3 times that for the negative control (NC), when blanking the spectrophotometer with a well containing 100 ul chromogen/substrate and 200 ul blocking reagent. If not, this may not be valid and the test should be repeated.
Strict adherence to the instructions is necessary to obtain reliable results
Non-repeated positive results can occur due to technical errors such as
* Interchange of vital caps
* Use of the same tip for withdrawing different reagents or dispensing different samples.
* Leave the vials open for too long
* Exposure of reagents to intense heat, light or storing sources of bacterial contamination.
* Inadequate rising of wells
* Contamination of well rims by conjugate or samples
* Use of reagents from different master lots
All tests which are positive by ELISA once are repeated in duplicate and if possible a confirmatory neutralization test may be done. The blood unit found ELISA reactive is segregated from the blood storage and appropriately discarded.
Blood bank zone Next ArticlesYou are here : Home/ Blood Bank Zone/ Trasfusion Transmitted Diseases/ 3. Laboratory diagnosis of HBV infection
© 2007 bloodindex