2. In the semiquantitative test the reactions obtained

are roughly equivalent to those which would occur

in a tube test.

3. Agglutinins are found in high proportion of normal

individuals and titers less than 1:80 are of doubtful

significance. A rising titer is more significant than a

single high titer.

4. False positive reactions may occur in sera of patients

infected with Pasteurella tularensis or vaccinated

with Vibrio cholerae.

5. False positive results are likely if the test is read more

than 1 minute after mixing on slide test.

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

correlated with the clinical findings to arrive at the

final diagnosis.

7. Prozoning may sometimes be encountered in serum

containing very high titers on slide test.

8. Since techniques and standardization vary from

laboratory to laboratory one tube difference in titers

can be expected.

SLIDE SCREENING TEST FOR BRUCELLA

ANTIBODIES (BRUCEL-RB)®

(Courtesy: Tulip Group of Companies)

Reagent

The BRUCEL-RB reagent contains smooth, killed buffered

suspensions of Brucella abortus strain 99, colored with

rose bengal, standardized against the 2nd International

preparation, having specific reactivity towards antibodies

to Brucella.

Reagent Storage and Stability

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

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

mentioned on the reagent vial labels.

Each batch of reagents undergoes rigorous quality

control at various stages of manufacture for its specificity,

sensitivity, and performance.

Serology/Immunology 637

Possible causes Solutions

1. Past history of immunization, inapparent infection or prior disease False positive reactions may occur in sera of patients infected with

Pasteurella tularensis of vaccinated with Vibrio cholerae

Also check the patient’s history

2. Prolonged rocking of the slide causes drying of the test material Agglutination should be observed within 1 minute

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

temperature at 2–8°C

Turbid and contaminated serum should not be used for testing

5. Error in interpreting results. Any debris or dirt in the slide/test tube

could be mistaken for agglutination

Clean and dry glassware should be used for testing

6. Single high titer is interpreted as positive Agglutinins are found in high proportion of normal individuals and

titers less than 1:80 are of doubtful significance. A rising titer is more

significant than a single titer

Troubleshooting

Problem: False positive results

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 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. Prozoning effect In serum with very high titers, prozoning may be observed

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

5. Rotation of the slide too fast may break up agglutinating clumps,

which lead to false negative in borderline cases

Rock the slide gently back and forth and observe for agglutination

macroscopically within 1 minute

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

give false negative results

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

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

Problem: False negative results

Principle

The smooth, colored, killed BRUCEL-RB antigen suspension

is mixed with the patient serum. Specific antibodies to

Brucella antigens if present in titres > 120, in the patient

serum will react with the antigen suspension to produce

an agglutination reaction. No agglutination indicates the

absence of detectable levels of specific antibodies to Brucella.

Note

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

2. The reagent contains 0.01% thimerosal as preservative.

Avoid contact with skin and mucosa. On disposal,

flush with large quantities of water.

Sample Collection and Storage

1. No special preparation of patient is required prior to

sample collection by approved techniques. Do not use

hemolyzed serum samples.

2. Clean and dry glassware free from detergents must be

used for sample collection.

3. Do not heat/inactivate the serum.

638 Concise Book of Medical Laboratory Technology: Methods and Interpretations 4. Though freshly collected serum is preferred, samples

can be stored at 2 to 8°C, for 24 hours, or frozen for 8

days should a delay in testing occur.

Material Provided with the Kit

BRUCEL-RB Brucella rose bengal colored antigens.

Additional Material Required

Stop watch, positive control, isotonic saline, glass slide

with clear/white background, appropriate pipettes/

micropipettes, mixing sticks and a high intensity direct

light source.

Procedure

Bring all reagents to room temperature.

Shake and mix the BRUCEL-RB antigen suspension

well before dispensing.

Slide Test Method

Qualitative Method

1. Place one drop of positive control onto the reaction

circle of glass slide.

2. Place 80 µL of saline onto the next reaction circle of

the glass slide.

3. Place 80 µL of patient serum to be tested onto the next

reaction circle.

4. Add one drop of well mixed BRUCEL-RB antigen

suspension in each of the above circles containing

positive control, isotonic saline and the patient serum

to be tested.

5. Mix contents of each circle uniformly over the entire

circle with separate mixing sticks.

6. Gently rock the slide back and forth, observe for

agglutination macroscopically, at one minute against

a white background.

Semiquantitative Method

1. Using a pipette, place 80 µL, 40 µL, 20 µL, 10 µL, and

5 µL of patient serum to be tested on 5 different circles

on the glass slide. The corresponding titers obtained

will be 1:20, 1:40, 1:80, 1:160, and 1:320 respectively.

2. Place one drop of BRUCEL-RB antigen suspension to

each circle.

3. Mix contents of each circle uniformly over the entire

circle with separate mixing sticks.

4. Gently rock the slide back and forth, observe for

agglutination macroscopically at 1 minute against a

white background.

Interpretation of Results

Qualitative Method

Agglutination is a positive test result and indicates the

presence of antibodies to Brucella in titers > 1:20 in the

patient serum.

No agglutination is a negative test result and indicates

absence of antibodies to Brucella in titers 1:20 in the

patient serum.

Semiqualitative Method

Agglutination is a positive test result. The titer of patient

serum corresponds to the visible agglutination in the test

circle with the minimum amount of serum sample.

Remarks

1. Turbid and contaminated serum should not be used

for testing.

2. Agglutinins are found in high proportion of normal

individuals and titers less than 1:80 are of doubtful

significance. A rising titer is more significant than a

single high titer.

3. False positive reactions may occur in sera of patients

infected with Pasteurella tularensis or vaccinated with

Vibrio cholerae.

4. False positive results are likely if the test is read more

than 1 minute after mixing on the slide.

5. It is recommended that results of the test should be

correlated with the clinical findings to arrive at the

final diagnosis.