dispensed Excess reagent dispensed gives false positive results at borderline concentrations. Ensure that exactly one drop of reagent is dispensed onto the slide

 

654 Concise Book of Medical Laboratory Technology: Methods and Interpretations Accessories

Glass slide with six reaction circles, sample dispensing

pipettes, mixing sticks, rubber teat.

Additional Material Required

Stopwatch, high intensity direct light source, isotonic

saline, pipettetes, test tubes.

Test Procedure

Bring reagent and samples to room temperature before

testing.

Qualitative Method

1. Pipettete one drop of test sample onto the glass slide

using a disposable pipette provided with the kit.

2. Add one drop of RHELAX ASO latex reagent to the drop

of test sample on the slide.

3. Using a mixing stick, mix the serum and the RHELAX

ASO latex reagent uniformly over the entire circle. Do

not let the dropper tip touch the liquid on the slide.

4. Immediately start a stopwatch. Rock the slide

gently, back and forth, observing for agglutination

macroscopically at 2 minutes.

Semiquantitative Method

1. Using isotonic saline, prepare serial dilutions of the

serum sample positive in the qualitative method 1:2,

1:4, 1:8, 1:16 and so on.

2. Pipette the diluted specimens onto the slide. Start with

the 1:2 diluted test specimen.

3. Add a drop of RHELAX ASO reagent to it and mix

well. Spread the mixture uniformly over the entire

circle.

4. Immediately start a stopwatch. Rock the slide gently, back

and forth, observing for agglutination macroscopically

at 2 minutes. Proceed similarly with each dilution as test

specimen.

Interpretation of Test Results

Qualitative Method

Agglutination is a positive test result and indicates the

presence of detectable levels of antistreptolysin O in the

test specimen.

No agglutination is a negative test result and indicates

the absence of detectable levels of antistreptolysin O in the

test specimen.

Semiquantitative Method

Agglutination in the highest serum dilution corresponds to

the amount of ASO in IU/mL present in the test specimen.

The concentration of ASO can be calculated as follows:

ASO (IU/mL) = S × D

where S = Sensitivity of the reagent, i.e. 200 IU/ ml.

D = Highest dilution of serum showing agglutination.

Remarks

1. Markedly lipemic, hemolyzed and contaminated

serum samples could produce non-specific results.

2. Serum samples having markedly higher protein

content may produce nonspecific reagent aggregation.

3. Use of plasma rather than serum can lead to false

positive results.

4. Do not read results beyond 2 minutes.

5. It is recommended that all positive test results should

be further tested with methods enabling quantitation

of ASO titers.

6. It is recommended that results of the tests should be

correlated with clinical findings to arrive at the final

diagnosis.

Possible causes Solutions

1. Plasma is used as a test specimen Only serum must be used for testing

2. Samples are stored for a long period Should a delay in testing occur, store samples at 2–8°C. Samples can

be stored for upto a week at 2–8°C

3. Cross contamination due to the usage of the same mixing stick Separate mixing stick should be used for mixing the controls and the

sample

4. Markedly lipemic, hemolyzed and contaminated serum samples

could produce non-specific results

Avoid using lipemic, hemolyzed and contaminated serum samples for

testing

Troubleshooting

Problem: False positive results

Contd...

Serology/Immunology 655

Possible causes Solutions

5. Drying of the reagent on the slide Do not read results beyond 2 minutes. The test should not be carried

out directly under the fan

6. Presence of dust or debris on the glass slide used Dust or debris could be misinterpreted as agglutination therefore only

clean and dry glass slides must be used for testing

7. Latex particles contaminated with positive control/positive

sample

Care must be taken to see that the latex reagent dropper tip does not

touch the sample or control taken on the slide during dispensing of

the reagent

8. Wrong dropper used for dispensing the sample Accessories provided with the kit only must be used for optimum

results

9. Increase in drop size, thereby leading to excess reagent

dispensed

Excess reagent dispensed gives false positive results at borderline

concentrations. Ensure that exactly one drop of reagent is dispensed

onto the slide

10.Reagent dropper not held vertically while dispensing To ensure accurate dispensing of the reagent, hold the reagent

dropper vertically while dispensing the reagent

11.Cross contamination due to the usage of the same mixing stick Separate mixing stick should be used for mixing the controls and the

sample.

12.Dried latex particles observed in the latex reagent

• During slide test with negative control

•  In the dropper of the vial (due to freezing of the latex reagent

during storage)