1. Zonalreactions occasionally occurin serological tests.

In such cases, a strongly reactive serum may show a

weak or atypical reaction when undiluted serum is

tested (prozone phenomenon). A completely negative

reaction with very strongly reactive sera is extremely

rare.

2. A reactive or weakly reactive test result indicates the

presence of reagin, which almost invariably is formed

in Treponema infection, but which may be produced

by a variety of other conditions. In actual practice, a

reactive result in the presence of clinical symptoms

is considered as a confirmatory evidence of syphilitic

infection. However, in the absence of clinical findings,

test reactivity can represent any of the following: (a)

Latent syphilis, (b) A biological false-positive reaction,

either temporary or chronic or (c) A technical or clinical

error.

Limitation of the Test

1. Acute or chronic infections such as malaria, leprosy,

infectious mononucleosis and upper respiratory

diseases as well as collagen and immunologic diseases

such as rheumatoid arthritis and lupus erythematosus

can produce false-positive reagin tests.

2. Other less well-known conditions include tissue

regeneration, pregnancy, heroin addiction and the

use of certain drugs for hypertension.

3. Reliable test results require strict attention to details

of technique, including proper identification of

specimens, accurate measurement, temperature

control, correct timing and observation of principles

of quality control.

MODIFIED VDRL REAGENT TREPOLIPIN®

(Courtesy: Tulip Group of Companies)

Reagent

1. TREPOLIPIN reagent A ready to use stabilised

emulsion of cardiolipin, lecithin and cholesterol.

2. Positive control,reactive with TREPOLIPIN reagent.

3. Negativecontrol,nonreactivewithTREPOLIPIN reagent.

The TREPOLIPIN detects antilipoidal antibodies in

serum, plasma and cerebrospinal fluid (CSF). As against the

conventional VDRL reagents, test samples do not require

heat inactivation.

Each batch of reagents undergoes rigorous quality

control at various stages of manufacture for its specificity,

sensitivity and performance.

Reagent Storage and Stability

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

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

mentioned on the reagent vial label. Avoid exposure

to elevated temperature and air as the reagent is highly

sensitive to denaturation and drying.

Principle

When serum, plasma or cerebrospinal fluid (CSF) containing

antilipoidal antibodies is reacted with TREPOLIPIN

reagent, a flocculation reaction is produced.

Flocculation is a positive test result and indicates

presence of antilipoidal antibodies in the sample. No

flocculation is a negative test result and indicates absence

of antilipoidal antibodies in the sample.

Note

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

2. Reagent contains sodium azide 0.1% as preservative.

Avoid contact with skin and mucosa. On disposal,

flush with large quantities of water.

3. The antigen suspension should be gently but thoroughly mixed by swirling before testing to homogenize

the reagent 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.

Serology/Immunology 611

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. Fresh serum, plasma or CSF should be used for testing.

3. Hematogenous CSF should not be used for testing.

For cloudy samples, centrifuge and use the clear

supernatant for testing.

Material Provided with the Kit

1. Stabilized cardiolipin suspension.

2. Reagent dropper assembly for dispensing the antigen

suspension.

3. Positive control, reactive with reagent.

4. Negative control, nonreactive with reagent.

Additional Material Required

Conventional VDRL cavity slide (glass), microscope (with

magnification of 100 x), Pasteur pipettes, mechanical rotor

(180 rpm), isotonic saline.

Note: For TREPOLIPIN 5 × 5 mL kit. Known reactive

and nonreactive samples would be required additionally.

Test Procedure

Bring reagent and samples to room temperature before

testing.

1. Thoroughlymix theTREPOLIPIN reagent suspension

by gentle agitation before testing.

2. With cerebrospinal fluid, the test specimen volume is

0.01 mL.

3. For use with cerebrospinal fluid, each drop of

TREPOLIPIN reagent should be diluted with 0.02 mL

of good isotonic saline before testing.

Qualitative Method

1. Pipette 0.05 mL of serum or plasma to the VDRL slide

cavity.

2. Dispense one drop of TREPOLIPIN reagent to the

surface of the test sample in the same cavity using the

reagent dropper provided.

3. Rotate the slide continuously at 180 rpm for 4 minutes,

observing for flocculation.

4. Read the results macroscopically or microscopically

at 4 minutes.

5. All positive test results may be further tested by the

quantitative test procedure.

Quantitative Method

1. Pipette 0.1 mL of isotonic saline into seven test tubes.

2. Pipette 0.1 mL of the test sample into the first test tube.

3. Transfer 0.1 mL of the diluted test sample from the first

tube to the second tube.

4. Continue the serial dilution of the test sample till dilutions

of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128 are achieved.

5. Transfer 0.05 mL each dilution of the test sample from

tubes 1 to 7 to a conventional VDRL slide.

6. Dispense one drop of TREPOLIPIN reagent to each

dilution of the sample on the VDRL slide.

7. Rotate the slide continuously at 180 rpm for four

minutes.

8. Observe for flocculation macroscopically or microscopically at 4 minutes.

Interpretation of Results

Qualitative Method

Flocculation is a positive test result and indicates presence

of antilipoidal antibodies in the test sample.

No flocculation is a negative test result and indicates

absence of antilipoidal antibodies in the test sample. The

strength of flocculation may vary, depending upon the

degree of positivity of the test sample.

Quantitative Method

The antilipoidal antibody titer is the highest dilution of the

test sample giving a positive test result (flocculation).

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 results of the test should be

correlated with clinical findings to arrive at the final

diagnosis.

5. Microscopic evaluation of test results requires welltrained and experienced professional. It is recommended

that a few known negatives should be run with each batch

of the tests so as to familiarize and differentiate effectively

the appearance of nonreactive samples from the reactive

ones.

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

1. Exposure of reagents to high temperature Exposure to high temperatures of the reagents can cause autoflocculation of reagents. Also since the reagent is alcohol based prolonged exposure to high temperature may lead to evaporation. Therefore, avoid exposure of the reagents to

elevated temperatures

2. Cloudy CSF used for testing Hematogenous CSF should not be used for testing. For cloudy samples, centrifuge

at 1000 rpm for 1 minute and use the clear supernatant for testing

3. Serum is allowed to dry prior to addition of antigen To avoid drying of the serum, pipette 50 µL of the serum to the VDRL slide cavity

and immediately add one drop of Trepolipin reagent to the test sample

4. Slide was rotated for too long. Drying has taken place Rotate the slide continuously at 180 rpm for 4 minutes, observing for flocculation.

Do not perform the test directly under a fan

5. False positives may occur due to overspill from one

cavity to another while rotating

Care must be taken to see that there is no overspill of the test mixture during

rotation of the slide

6. 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 results must be correlated with clinical

findings and all positive results must be further confirmed by using Treponemal

tests

Troubleshooting

Problem: False positive results

Possible causes Solutions

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

VDRL slide cavity

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

dropper provided with the kit

3. Serum, plasma or CSF stored for a long period of time is used for

testing

Fresh serum, plasma or CSF should be used for testing

4. The volume of CSF used for testing is incorrect. In CSF testing,

the Trepolipin reagent has not been diluted

Test specimen volume in CSF is 10 mL. Each drop of the Trepolipin

reagent has to be diluted with 20 mL of isotonic saline for CSF testing

5. Hemolyzed samples may have been used for testing Do not use hemolyzed samples

6. Antigenic suspension has settled down in the vial Shake the Trepolipin reagent vial well before use to disperse the

reagent particles uniformly and improve test readability

7. Cold reagents are used for testing Bring all reagents and samples to room temperature before

commencing the testing procedure

8. Expired reagents Check the expiry date of the reagents before use

9. Error in interpreting the test results Flocculation results should be interpreted carefully after comparing

with positive and negative controls. The strength of flocculation may

vary, depending upon the degree of positivity of the test sample

10. False negatives are obtained in the tertiary stage of the disease The test should be confirmed with FTA (fluorescent treponemal

antibody) or TPHA (treponema pallidum hemagglutination) tests

Problem: False negative results

Serology/Immunology 613

TOLUIDINE RED UNHEATED SERUM TEST FOR

RAPID SERODIAGNOSIS OF SYPHILIS REDGEN®

(Courtesy: Tulip Group of Companies)

Reagent

1. REDGENreagent:Aparticulate suspensioncontaining

a red micronised dye coated with lipid complexes.

2. Positive control, reactive with the REDGEN latex

reagent.

3. Negative control, non-reactive with theREDGEN latex

reagent.

REDGEN detects antilipoidal antibodies in serum or

plasma. As against the conventional VDRL reagents, test

samples do not require heat inactivation.

Each batch of reagents undergoes rigorous quality

control at various stages of manufacture for its specificity,

sensitivity and performance.

Reagent Storage and Stability

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

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

mentioned on the reagent vial label.

Avoid exposure to elevated temperature 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 REDGEN reagent and allowed to react for

eight minutes. If antilipoidal antibodies are present in the

specimen, they will react with REDGEN reagent forming

visible red floccules against the white background of the

reaction card. If antilipoidal antibodies are not present in

the specimen, there will be no flocculation, resulting in an

even pink mat on the reaction circle.

Note

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

2. The reagent contains thiomersal 0.1% as preservative.

Avoid contact with skin and mucosa. On disposal,

flush with large quantities of water.

3.