(Courtesy: Tulip Group of Companies)

Reagent

Syphfinal reagent is a ready-to-use, uniform suspension

of polystyrene latex particles coated with cardiolipin,

suspended in a suitable buffer of proprietary composition.

Though Syphfinal reagent, in performance, corresponds

to the other USRs, it accords better readability to the test

results, thereby giving better confidence in reporting

results. As against the conventional VDRL tests, the

samples do not require heat inactivation.

Each batch of reagent undergoes rigorous quality

control at various stages of its manufacture for its

sensitivity, specificity and performance.

Reagent Storage and Stability

Store the reagent at 2–8°C. Do not freeze. The shelf life of the

reagent is as per the expiry date mentioned on the reagent

vial labels. Avoid exposure to elevated temperatures, air

and direct sunlight as the reagents are highly sensitive to

denaturation, drying and microbial contamination.

Principle

Syphfinal, latex VDRL reagent, is based on the principle

of agglutination. The test specimen, serum or plasma is

mixed with Syphfinal latex VDRL reagent and allowed to

react for 6 minutes. If antilipoidal antibodies are present in

the specimen, they will react with the latex reagent forming

a visible agglutination. If antilipoidal antibodies are not

present in the specimen, then no agglutination is observed.

616 Concise Book of Medical Laboratory Technology: Methods and Interpretations Note

1. In vitro diagnostic reagent for laboratory and 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. All reagents derived from human source are tested

for HBsAg and anti-HIV antibodies and found to

be nonreactive. However, handle the material as if

infectious.

4. Syphfinal latex VDRL reagent should be gently but

thoroughly mixed before testing to disperse the latex

particles uniformly and improve test readability.

5. Performance of the reagents must be verified with

positive and negative controls provided with the kit

and it is recommended that controls be run with each

test series.

Sample Collection and Storage

No special preparation of the patient is required prior to

sample collection by approved techniques. Do not use

hemolyzed samples. Fresh serum or plasma should be

used for testing. Samples not tested immediately may be

stored at 2 to 8°C for up to 48 hours. Hazy samples should

be centrifuged. Use clear supernatant for testing.

Material Provided with the Kit

Latex VDRL reagent, positive control reactive with the reagent,

negative control nonreactive with the reagent, disposable

slides with eight reaction circles, disposable sample/control

dispensing pipettes, mixing sticks, rubber teats.

Additional Material Required

Stopwatch, high intensity direct light source, isotonic

saline, pipettes, test tubes.

Test Procedure

Bring all reagents and samples to room temperature

before testing.

Qualitative Method

1. Pipette one drop of test sample onto one of the

reaction circles of the disposable slide using a sample

dispensing pipette. The disposable slides and the

sample dispensing pipettes are provided with the kit.

2. Repeat the procedure with positive and negative

controls.

3. Add one drop of well-mixed Syphfinal latex reagent to

the test sample, positive control and negative control

respectively. Do not let the dropper tip touch the liquid

on the slide.

4. Using a mixing stick, mix the test sample and Syphfinal

reagent thoroughly spreading uniformly over the

entire reaction circle.

5. Immediately start a stopwatch. Rotate the slide

gently and continuously, observing for agglutination

macroscopically at 6 minutes.

Quantitative Method

1. Using isotonic saline prepare serial dilutions of the test

sample, e.g. 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, etc.

2. Perform qualitative test procedure using each dilution

as test specimen.

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

dilution which shows a positive test result.

Interpretation of Test Result

Qualitative Method

Agglutination is a positive test result and indicates the

presence of antilipoidal antibodies in the test sample.

No agglutination is a negative test result indicating the

absence of detectable levels of antilipoidal antibodies in

the test specimen.

Quantitative Method

The titre of antilipoidal antibodies is the highest dilution of

the test sample giving a positive test result (i.e. agglutination).

Remarks

1. Quantitative procedure must be performed to

determine the 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 the absence of supporting clinical, historical or

epidemiological evidence, reactive results must be

confirmed with more specific Treponema tests.

4. It is recommended that the results of the test should

be correlated with the clinical findings to arrive at the

final diagnosis.

5. Dispose all used and contaminated materials as per

Standard Biohazard Safety Guidelines.

Serology/Immunology 617

Troubleshooting

Problem: False positive results

Possible causes Solutions

1. Reagent is contaminated because of unclean dropper and hence

not suitable for subsequent use

The reagent dropper provided for dispensing the reagent should be

thoroughly cleaned with distilled water and air-dried after use, to ensure that it does not contaminate the reagent during subsequent use.

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

touch the liquid on the slide while dispensing

2. Drying of the reagent on slide Do not interpret test results beyond 6 minutes. Testing should not

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

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

and chronic conditions like leprosy, malaria, infectious mononucleosis, hepatitis, systemic lupus erythematosus and rheumatoid

arthritis

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

with clinical findings

4. False positives may occur due to overspill from one circle to

another while rotating

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

of the card

Possible causes Solutions

1. Negative control contaminated with positive control/positive

sample

Validate the latex reagent by using known negative (saline) and positive controls. If proper results are obtained, with known negative (saline) and positive controls then the negative control is contaminated

and should not be used for further testing

Problem: Negative control giving positive reaction

Problem: False negative results

Possible causes Solutions

1. The reagent may be damaged by exposure to elevated

temperatures, air and direct sunlight, as it is highly

sensitive to denaturation, drying and microbial contamination

Check the performance of the reagent with positive and negative controls provided

with the kit. Avoid exposure of the reagent to light. The vial must be closed properly after use

2. The reagent is in a frozen condition The reagent should not be frozen. It should be stored at 2–8°C

3. Weak agglutination may be interpreted as negative The latex reagent should be gently but thoroughly mixed before testing to disperse

the latex particles uniformly and improve test readability. The serum and the reagent should be mixed thoroughly

4. Excess sample dispensed/reagent dispensed leading

to prozoning/postzoning.

Pipette exactly one drop of test sample using the sample-dispensing pipette. Similarly, dispense one drop of well-mixed latex reagent using the reagent dropper

provided with the kit

5. Hemolyzed or lipemic samples may have been used

for testing

Avoid using hemolyzed or lipemic samples for testing

6. Reagent is contaminated because of unclean dropper

and hence not suitable for subsequent use

The reagent dropper provided for dispensing the reagent should be thoroughly

cleaned with distilled water and air-dried after use, to ensure that it does not contaminate the reagent during subsequent use

Contd...

618 Concise Book of Medical Laboratory Technology: Methods and Interpretations RAPID PLASMA REAGIN (RPR) CARD TEST/

CARBON ANTIGEN FOR SYPHILIS TESTING

(CARBOGEN)

(Courtesy: Tulip Group of Companies)

Summary

The Rapid Plasma Reagin (RPR)/Carbon Antigen test is

a macroscopic non-Treponema flocculation test for the

detection and quantitation of antilipoidal antibodies.

Non-Treponema tests like CARBOGEN are of great value

when used for screening and follow-up of therapy.

Reagent

1. CARBOGEN reagent:Aparticulate carbon suspension

coated with lipid complexes.

2. Positive control,reactivewiththeCARBOGENreagent.

3. Negative control, nonreactive with the CARBOGEN

reagent.