The CARBOGEN detects antilipoidal antibodies in

serum or plasma. As against the conventional VDRL

reagents, test samples do not require heat inactivation.

Each batch of reagent undergoes rigorous quality

control at various stages of manufacture for its specificity,

sensitivity and performance.

Reagent Storage and Stability

Store the reagent at 2 to 8°C. Do not freeze. The shelf-life

of the reagent is as per the expiry date mentioned on the

reagent vial label. Avoid exposure to elevated temperatures

and air as the reagent is highly sensitive to denaturation

and drying.

Principle

During the test procedure, the specimen, serum or plasma

is mixed with CARBOGEN reagent and allowed to react

for 8 minutes. If antilipoidal antibodies are present in the

specimen,theywillreactwithCARBOGENreagentforming

visible black floccules. If antilipoidal antibodies are not

present in the specimen, there will be no flocculation.

Note

1. In vitrodiagnostic reagent for laboratory or professional

use only. Not for medicinal use.

2. The reagent contains sodium azide 0.1% as preservative.

Avoid contact with skin and mucosa. On disposal,

flush with large quantities of water.

3. CARBOGENRPR/Carbonantigenshouldbe gentlybut

thoroughly mixed before testing to disperse the carbon

particles uniformly and improve test readability.

4. Performance of the reagent must be verified with

positive and negative controls and it is recommended

that controls be run with each test series.

5. Accessories provided with the kit only must be used

for optimum results.

Sample Collection and Storage

1. Nospecial preparation of the patient is required prior to

sample collection by approved techniques. Hemolyzed

or lipemic samples are not suitable for testing.

2. Fresh serum or plasma should be used for testing.

3. Samples not tested immediately may be stored at 2 to

8°C for up to 48 hours.

4. Hazy samples should be centrifuged. Use the clear

supernatant for testing.

Possible causes Solutions

7. Serum or plasma stored for a long period of time is

used for testing

Fresh serum or plasma should be used for testing. Samples not tested immediately

may be stored at 2–8°C for upto 48 hours. Hazy samples should be centrifuged.

Use clear supernatant for testing

8. Antigenic suspension has settled down in the vial Shake the reagent vial well before use to disperse latex particles uniformly and

improve test readability

9. Cold reagents are used for testing Bring all the reagents and samples to room temperature before commencing the

testing procedure

10. Expired reagents are used for testing Check the expiry date of the reagents before use

11. Error in interpreting the test results Agglutination results should be interpreted carefully after comparing with positive

and negative controls

Contd...

Serology/Immunology 619

Material Provided with the RPR Kit

1. Carbon antigen

2. Positive control, reactive with the reagent

3. Negative control, nonreactive with the reagent

4. Disposable slides with eight reaction circles

5. Disposable sample/control dispensing pipettes

6. Mixing sticks

7. Rubber teats

8. Reagent dropper for dispensing the carbon antigen.

Additional Material Required

Stop watch, high intensity light source, isotonic saline,

pipettes, test tubes, mechanical rotor at 180 rpm

circumscribing a circle 2 cm in diameter on a horizontal

plane.

Note: For CARBOGEN Carbon Antigen: Item Nos. 2 to 7

listed above under RPR kit, would be required additionally.

Test Procedure

Bring reagent and samples to room temperature before

testing.

Thoroughly mix the CARBOGEN reagent suspension by

gentle agitation before testing.

Qualitative Method

1. Place one drop of the test specimen, positive and

negative controls onto separate reaction circles of the

disposable slide using a sample dispensing pipette.

2. Addonedropofwell-mixedCARBOGENreagentnextto

the test specimen, positive control and negative control

by using the reagent dropper provided with the kit. Do

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

3. Using a mixing stick, mix the test specimen and the

CARBOGEN reagent thoroughly spreading uniformly

over the entire reaction circle.

4. Immediately start a stopwatch. Rotate the slide gently

and continuously either manually or on a mechanical

rotor at 180 rpm.

5. Observe for flocculation macroscopically at 8 minutes.

Quantitative Method

1. Using isotonic saline, prepare serial dilutions of the

test specimen positive in the qualitative method 1:2,

1:4, 1:8, 1:16, 1:32, 1:64, 1:128 and so on.

2. Perform the qualitative test procedure using each

dilution as a test specimen.

3. The titer is reported as the reciprocal of the highest

dilution which shows a positive test result.

Interpretation of Test Results

Qualitative Method

1. Large and medium black floccules against white

background : Reactive

2. Small black floccules against white background:

Weakly reactive

3. No floccules, even grey background: Nonreactive.

Flocculation is a positive test result and indicates

presence of antilipoidal antibodies in the test specimen.

No flocculation is a negative test result and indicates

absence of antilipoidal antibodies in the test specimen.

Quantitative Method

The titre of antilipoidal antibodies is the highest dilution of

the test specimen giving a positive test result.

Remarks

1. Quantitative procedure must be performed to

determine response to treatment and detect reinfection.

2. False-positive reactions occur not infrequently and

have been attributed to a variety of acute and chronic

conditions.

3. In absence of supporting clinical, historical or

epidemiological evidence, reactive result must be

confirmed with more specific Treponema tests.

4. It is strongly recommended that results of the test

should be correlated with clinical findings to arrive at

the final diagnosis.

5. Dispose all used and contaminated material as per

Standard Biohazard Safety Guidelines.

6. The reagent dropper provided for dispensing the

carbon antigen should be thoroughly cleaned with

distilled water and air dried after use, to ensure that it

does not contaminate the reagent during subsequent

use.

7. Very slight roughness should be interpreted as a

negative test result.

620 Concise Book of Medical Laboratory Technology: Methods and Interpretations Possible causes Solutions

1. Carbogen antigen contaminated with positive control/positive test

sample

Care must be taken to ensure that the dropper tip does not touch the

liquid on the slide while dispensing

2. Drying of the reagent on the slide. Do not interpret test results beyond 8 minutes. Testing should not be

carried out under the fan or under conditions where drying is enhanced

3. False positives may occur due to overspill from one back circle to

another while rotating

Care must be taken to see that there is no overspill during rotation

of the card

4. False positive reactions can also be attributed to a variety of acute

and infectious mononucleosis, hepatitis, systemic lupus erythematosus and rheumatoid arthritis

Check the history of the patient. The test results must be correlated

with clinical findings and all positive results must be further confirmed

by using Treponemal tests

Possible causes Solutions

1. Negative control contaminated with positive control/

positive sample

Validate the carbon antigen by using known negative (saline) and positive control. If proper results are obtained, with known saline and positive controls then

the negative control is contaminated and should not be used

Problem: Negative control giving false positive reaction

Problem: False negative results

Possible causes Solutions

1. Excess serum dispensed (prozoning) Pipette exactly 50 µL of serum or positive or negative control to black

circle of the RPR card

2. Excess antigen dispensed (postzoning) Dispense exactly one drop of Carbogen reagent using the reagent

dropper provided with the kit

3. Reagent is contaminated because of unclean dropper and hence

not suitable for subsequent use

The reagent dropper provided for dispensing the Carbon antigen

should be thoroughly cleaned with distilled water and air dried after

use, to ensure that it does not contaminate the reagent during subsequent use

4. The reagent may be exposed to elevated temperatures, air and direct sunlight,