Serology/Immunology 625

Each batch of reagents undergoes rigorous quality

control at various stages of manufacture for its specificity

and performance.

Reagent Storage and Stability

1. Store the reagents at 2 to 8°C. Do not freeze.

2. The shelf-life of reagents is as per the expiry date

mentioned on the reagent bottle labels.

Principle

When the colored, smooth suspension of attenuated

Typhochek antigen suspensions are incubated with

patient serum, anti-Salmonella antibodies present in the

patient’s serum react with the antigen suspensions to

produce an agglutination. Agglutination is a positive test

result, indicating presence of Salmonella antibodies in

the patient’s serum. No agglutination is a negative test

result indicating absence of Salmonella antibodies in the

patient’s serum.

Note

1. In vitro diagnostic reagent for laboratory and professional use only. Not for medicinal use.

2. The S. typhi ‘O’ reagent contains phenol 0.5%, S. typhi

‘H’, S. paratyphi ‘AH’, S. paratyphi ‘BH’ reagents contain

formaldehyde 0.3% and S. paratyphi ‘AO’, S. paratyphi

‘BO’ reagents contain ethanol 0.7% as preservatives.

Avoid contact with skin and mucosa. On disposal, flush

with large quantities of water.

Sample Collection and Storage

1. No special preparation of the patient is required prior

to sample collection by approved techniques. Do not

use hemolyzed samples.

2. Clean and dry glassware free from detergents must be

used for sample collection.

3. Do not heat/inactivate the serum.

4. Though freshly collected serum is preferable, store

samples at 2 to 8°C in case of delay in testing, for up

to 72 hours.

Material Provided with the Kit

Reagent Pack

Typhochek 4 × 50 mL set contains item Nos. 1, 2, 5 and 6

mentioned below.

1. Typhochek S. typhi ‘O’ Antigen suspension

2. Typhochek S. typhi ‘H’ Antigen suspension

3. Typhochek S. paratyphi ‘AO’ Antigen suspension

4. Typhochek S. paratyphi ‘BO’ Antigen suspension

5. Typhochek S. paratyphi ‘AH’ Antigen suspension

6. Typhochek S. paratyphi ‘BH’ Antigen suspension.

Note: Item Nos. 1 to 6 each is available as individual

reagent packs.

Additional Material Required

Timer, Kahn tubes/test tubes, pipettes (0.1 mL, 1 mL),

isotonic saline, incubator (37°C), test tube rack.

Procedure

a. Bring reagents to room temperature before testing.

b. Shake and mix antigens well before dispensing.

c. Carefully label test tubes for sample and reagent

identity when more than one antigens is used during

test procedure.

a. Standard Tube Test Method

1. Take appropriate number of sets (as required; one

set for each antigen suspension) of 8 Kahn tubes/test

tubes and label them 1 to 8.

2. Pipette into tube No. 1 of all sets 0.9 mL of isotonic

saline.

3. To each of the remaining tubes (2 to 8 of each set),

add 0.5 mL of isotonic saline.

4. To tube No. 1 of all sets, add 0.1 mL of serum sample

to be tested and mix well.

5. Transfer 0.5 mL of the diluted serum sample from

tube No. 1 to tube No. 2 and mix well.

6. Transfer 0.5 mL of the diluted serum sample from

tube No. 2 to tube No. 3 and mix well. Continue this

serial dilution till tube No. 7 in each set.

7. Discard 0.5 ml of the diluted serum from tube No. 7

of each set.

8. Tube No. 8 in all the sets serves as a saline control.

9. To all the tubes of the respective sets, add 0.5 mL of

the respective Typhochek antigen suspensions and

mix well.

10. This will give final dilutions in tube 1 to 7 as 1:20,

1:40, 1:80, 1:160, 1:320, 1:640, 1:1280.

11. Cover and incubate at 37°C overnight (approximately

18 hours).

12. Dislodge the sedimented button gently and observe

for agglutination macroscopically.

Interpretation of Results

The titer of the patient serum using Typhochek antigen

suspensions is the highest dilution of the serum sample

that gives a visible agglutination.

626 Concise Book of Medical Laboratory Technology: Methods and Interpretations Remarks

1. TAB vaccinated patients may show a high titer of

antibodies to each of the antigens.

2. ‘O’ being a somatic antigen brings about a coarse,

compact, granular agglutination, whereas ‘H’ being a

flagellar antigen brings about larger, loose, flocculant

agglutination.

3. Apart from the pattern of sedimented antigens, in the

tube test method a decrease in opacity as compared

to the saline control must also be considered while

judging the degree of agglutination.

4. While the ‘O’ antigen is species specific, the ‘H’ antigen

is specific to the serotype.

Possible causes Solutions

1. Past history of immunization, inapparent infection or prior disease Positive reaction will be seen in all three ‘H’ antigens in such cases

whereas in case of infection, antibodies will be seen only against the

infecting species. Also the patient’s history

2. Anamnestic response to other vaccines and unrelated fevers in the

case of persons who have had a prior infection or immunization

The anamnestic fever can be differentiated from enteric fever by repetition of the test after a week. The anamnestic response shows only

a transient rise, while in enteric fever the rise is sustained

3. If reagents have been exposed to excessively high temperatures,

precipitates could be formed

Ensure that the reagents are not exposed to high temperatures and

are stored properly at 2–8°C

4. Contamination of serum could lead to false positives Ensure that clean and dry glassware free from detergents are used for

sample collection. If samples are not tested immediately, store them

at 2–8°C. Turbid serum should not be used for testing

Possible causes Solutions

1. Infection is in very early stages, when antibody titer is very low The agglutinin titer depends on the stage of the disease. Agglutinins

usually appear by the end of the 1st week, so that the blood taken earlier

may give a negative test result. The testing should be repeated after a

week in such cases. Demonstration of a rise in titer of antibodies by testing two or more serum samples is more meaningful than a single test

2. Patients on antibiotic therapy during the testing phase Check the history of the patient for administration of the antibiotics.

3. Insufficient quantity of serum is used for testing hence leading to

postzone effect

Pipette one drop of patient serum on the four reaction circles, in case

of slide test. In case of tube test, carry out the dilutions carefully and

correctly as per the instructions given in the pack insert

4. Hemolyzed samples may be have been used Avoid using hemolyzed samples for testing

5. Reagents not brought to room temperature. Cold reagents could

give false negative results

All reagents must be brought to room temperature prior to commencing the testing procedure

6. The tube is shaken very vigorously while observing for agglutination Shake the tube gently after incubation and observe for agglutination.

‘O’ antigen will show coarse, compact, granular agglutination whereas ‘H’ antigen will show large, loose, flocculant agglutination

7. Insufficient reagent present in the vial Ensure sufficient reagent is present in the vial before retrieving

Troubleshooting

Problem: False positive results

Problem: False negative results

5. Turbid and contaminated sera should not be used for

testing.

6. Generally, antibody titers of 180 or more are considered

clinically and diagnostically significant. However,

the significant titer may vary from population to

population and needs to be established for each area.

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

correlated with clinical findings to arrive at the final

diagnosis.