WIDAL TEST
A 20year old male patient complaining of continuous fever for the past 10 days got
admitted in the medical ward. The serological test done is given, interpret the
results
Aim
To perform serological diagnosis of enteric fever
Principle – Tube agglutination test
During enteric fever, antibodies are formed against O and H antigens of
Salmonella species at the end of first week. When patient’s serum containing
antibodies reacts with O and H antigens, agglutination will occur that is positive
Requirements
1. Patient’s serum
2. WIDAL antigens – TO, TH, AH and BH
3. Positive control serum
4. Physiological saline
5. Test tubes
Test Procedure
Quantitative tube test procedure
Master Dilution preparation:
2.3ml Normal saline + 0.2ml or 200µl Patient serum
2.3ml + 0.2 ml = 2.5 ml
( 1: 12.5 dilution)
• 4 rows of tubes are arranged and each row contains eight tubes (1-8)
• To add 200µl of Normal saline all tubes (1-8)(4 rows)
• To add 200µl of Master Dilution- First tube of all 4 rows
• Then mix and take 200µl from first tube to second tube,mix second tube and
transfer 200µl to third tube and continue the same till seventh tube
• Discard 200µl from seventh tube
• 8th tube in all rows acts as a saline control (or) negative control
• Repeat the same dilution step in all rows
• The dilution would be obtained from 1st to 7th tube = 1:50, 1:100, 1:200,
1:400, 1:800, 1:1600, 1:3200
• It should be done in all 4 rows
• Then 200µl of O Ag for first rows for all 8 tubes
• 200µl of H Ag for second rows for all 8 tubes
• 200µl of AH Ag for third rows for all 8 tubes
• 200µl of BH Ag for fourth rows for all 8 tubes mix well
• Incubate all tubes at 370C overnight
Interpretation
The highest dilution showing agglutination is titre
Significant O titre = 1: 100 or more
Significant H titre = 1: 200 or more
• O agglutination – granular deposits ( in tube method-Felix tube)
• H agglutination – cottony woolly clumps (in tube method-Dreyers tube)
• Saline control – compact button (in tube method)
Demonstration of four fold titre increase at an interval of 7-10 days between paired
sera is more meaningful
ASO TEST
A 10-year-old girl suffering from migratory polyarthritis with repeated history of
sore throat. The serological test done is given, interpret the results.
AIM
To perform for the serological diagnosis of Streptococcus pyogenes infection and
their relative effects such as rheumatic fever and acute glomerulonephritis
Principle – Passive Latex agglutination test
During Streptococcus pyogenes infection, antibodies (ASO) are formed against
streptolysin O of the causative organism. When patient’s serum containing
antibodies reacts with latex particles coated with streptolysin-O, agglutination
occurs that is positive.
An elevated ASO titre of more than 200 IU/ml may indicate an acute streptococcal
infection
Requirements
ASO Latex reagaent
ASO Positive control
ASO Negative control
Test procedure
Qualitative method
1. Pipette one drop of test sample onto the glass slide
2. Add one drop of ASO latex reagent to the drop of test sample on the slide
3. Mix it with mixing stick
4. Rock the slide gently and observe for agglutination macroscopically in 2
minutes
Semiquantitative method
1. Prepare serial dilutions of the positive serum sample using saline to get the
following dilutions –
1:2, 1:4, 1:8, 1:16 and so on
2. Pipette the diluted specimens onto the slide
3. Add a drop of ASO reagent to it
4. Mix well and rock the slide
5. Observe for agglutination macroscopically in 2 minutes
Interpretation of results
Qualitative method
Agglutination – presence of clumps is Positive indicating the presence of detetable
levels of Anti-streptolysin O in sample
No agglutination - absence of clumps is Negative indicating the absence of
detectable levels of Anti-streptolysin O in sample
Semi quantitative method
Highest serum dilution showing agglutination corresponds to the amount of ASO
in IU/ml present in sample
The concentration of ASO can be calculated as follows:
ASO (IU/ml) = S x D
Where S = Sensitivity of the reagent i.e., 200 IU/ml
D= Highest dilution of serum showing agglutination
CRP TEST
A baby delivered 12 hours ago by forceps after prolonged labor presents with weak
cry, tachypnoea, refusal of feeds and lung crepitations. The serological test done is
given, interpret the results.
Aim
To perform for the serological diagnosis of inflammation, infection and tissue
destruction
Principle - Reverse Passive (Latex) agglutination test
C-reactive protein (CRP) is a normal alpha globulin, which increases in
inflammatory processes and infection, hence its increasing levels indicates a
variety of infections and inflammations.
The patient’s serum containing detectable levels of CRP reacts with latex particles
coated with anti-CRP, agglutination occurs that is positive reaction.
Requirements
CRP latex reagent
Patient’s serum
Positive control serum
Negative control serum
Glass Slide
Plastic droppers
Mixing sticks
Test procedure
Qualitative method
1. Add one drop of patient’s serum on the glass slide
2. Add one drop of CRP latex reagent to the drop of serum
3. Mix it with mixing stick
4. Rock the slide gently and observe for agglutination macroscopically in 2
minutes
Semiquantitative method
1. Prepare serial dilutions of the positive serum sample using saline to get the
following dilutions
1:2, 1:4, 1:8, 1:16 and so on
2. Pipette each dilution of the test specimen onto separate reaction circles
3. Add one drop of CRP latex reagent to the drop of test specimen
4. Mix it with separate sticks
5. Rock the slide gently and observe for agglutination macroscopically in 2
minutes
Interpretation of results
Qualitative method
Agglutination – presence of clumps is Positive indicating the presence of
detectable levels of CRP in the test sample
No agglutination –absence of clumps is Negative indicating the absence of
detectable levels of CRP in the test sample
Semiquantitative method
The highest serum dilution showing agglutination corresponds to the amount of
CRP in mg/dl present in the specimen
Calculations
Concentration of CRP can be calculated as follows:
CRP (mg/dl) = S x D
Where, S = Sensitivity of the reagent i.e. 0.6 mg/dl (or) 6mg/L
D = Highest dilution of serum showing agglutination
RA TEST/ RF TEST
A 45-year-old lady suffering from pain and swelling of small joints of feet and
hand with morning stiffness for the past 6 months. The serological test done is
given, interpret the results.
Aim
To perform for the serological diagnosis of Rheumatoid arthiritis (autoimmune
disease) by detecting rheumatoid factor in patient’s serum
Principle - Reverse Passive (Latex) agglutination test
In rheumatoid arthirits, rheumatoid factor (autoantibodies) are developed in almost
80% of the cases. When patient’s serum containing rheumatoid factor reacts with
latex particles coated with denatured human IgG, agglutination occurs that is
positive
Requirements
Normal saline
Patient’s serum
Commercially available RA Kit with positive and negative control serum
Test slide
Mixing sticks
Test procedure
Qualitative slide test
Place one drop of test specimen on the slide
Add one drop of latex gammaglobulin reagent
Mix it well and rock the slide gently
Look for agglutination in 2 minutes
Results and Interpretation
Coarse clumps seen - positive
No clumps - Negative
Semiquantitative test
Prepare serial dilutions of positive sample using normal saline to get the following
dilutions -
1:2, 1:4, 1:8, 1:16, 1:32, 1:64
Place one drop of diluted sample in each circle of the glass slide
Add one drop of latex reagent in each of these circle
Mix well with applicator stick
Rock the slide gently and examine for agglutination in 2 minutes
Calculation
Concentration of Rheumatoid factor (RF) can be determined as follows:
RF IU/ml = Sensitivity IU/ml x Titre
Where: Sensitivity = 8 IU/ml
Titre = Highest dilution showing clear cut agglutination
RPR TEST
A 26-year-old female attending antenatal clinic with bad obstetric history having
one abortion and one still birth. The serological test done is given, interpret the
results.
Aim
To perform for the serological diagnosis of syphilis (STD)
Principle: Slide flocculation test
During syphilis infection, antibodies (reagins) are formed against
Treponema pallidum.
When patient’s serum containing antibodies reacts with RPR antigen (cardiolipin
coated carbon particles), black clumps or floccules will appear macroscopically
and it is called reactive (positive)
Requirements
Patient’s serum
RPR antigen
Positive control serum
Negative control serum
Physiological saline
Applicator sticks
Plastic slides
Dropper with needle
Test procedure
Qualitative slide test
1. Add 50µl of serum sample, 50 µl of positive control serum and 50 µl of negative
control serum on separate circles of the slide
2. Add each one drop of RPR antigen suspension
3. Mix well with separate sticks
4. Rock the slide gently for 8 minutes on a mechanical rotator
Result and Interpretation
Medium and large aggregates against white background – reactive
Small agglutinates around periphery – weak reactive
Button formation at the centre of circle – Non-reactive
Semiquantitative test
Positive samples are subjected to the semi-quantitative test
Place 50 µl diluted saline buffer onto each of five circles of the slide
Add 50 µl of serum sample to the drop of saline buffer in 1st circle and mix well
Aspirate 50µl from 1st circle and transfer to 2nd circle
Repeat the same upto 5th circle
Aspirate and discard 50µl from 5th circle
Dilutions obtained would be as 1:2, 1:4, 1:8, 1:16, 1:32
Then add 1 drop of RPR antigen suspension to the circles
Mix and rock the slide gently for 8 minutes on a mechanical rotator
Interpretation
The titre of reagin antibodies is the highest dilution of the test sample which is
reactive
