TheREDGENreagent suspensionshould be gently but

thoroughly mixed before testing to disperse the dye

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 should be used for

optimum results.

Sample Collection and Storage

1. No special preparation of the patient is required

prior to sample collection by approved techniques.

Hemolyzed or lipemic samples are not suitable

for testing. In case of oxalated blood samples, it is

advisable to avoid excess of oxalate as it may interfere

with the test results.

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

supenatant for testing.

Material Provided with the Kit

The TRUST antigen, 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,reagent dropperfor dispensing the REDGEN reagent

suspension.

Additional Material Required

Stopwatch, high intensity light source, isotonic saline,

pipettetes, test tubes, mechanical rotor at 180 rpm circumscribing a circle 2 cm in diameter on a horizontal plane.

Test Procedure

Bring all reagents and samples to room temperature

before testing.

Thoroughly mix the REDGEN reagent suspension by

gentle agitation before testing.

Qualitative Method

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

negative controls onto separate reaction circles of the

disposable slide using a sample dispensing pipette.

2. Add one drop of well mixed REDGEN reagent next

to the test sample or controls by using the reagent

dropper provided with the kit. Do not let the dropper

tip touch the liquid on slide.

3. Using a mixing stick, mix the test sample andREDGEN

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.

614 Concise Book of Medical Laboratory Technology: Methods and Interpretations 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 Redgen 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

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 circle to

another while rotating

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

of the slide

4. 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 and all positive results must be further confirmed by using Treponemal tests

Quantitative Method

1. Using isotonic saline, prepare serial dilutions of the

test sample 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 specimen.

3. The titer is reported as reciprocal of the highest

dilution which shows a positive test result.

Interpretation of Test Results

Qualitative Method

1. Large and medium RED colored floccules against

white background: Reactive

2. Small RED colored floccules against white background:

Weakly reactive

3. No floccules, smooth pink background : Nonreactive.

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.

Quantitative Method

The titer of antilipoidal antibodies is the highest dilution of

test sample giving a positive test result.

Remarks

1. Quantitative procedure must be performed to

deter-mine 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 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

REDGEN 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.

Troubleshooting

Problem: False positive results

Serology/Immunology 615

Problem: False negative results

Possible causes Solutions

1. Negative control contaminated with positive control/positive

sample

Validate the antigen by using known negative (saline) and positive

control. 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

Possible causes Solutions

1. The reagent may be exposed 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

2. Hemolyzed or lipemic samples may have been used for testing Avoid using hemolyzed or lipemic samples for testing

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

and improve test readability

4.  Weak flocculation may be interpreted as negative The antigen should be gently but thoroughly mixed before testing to

disperse the dye particles uniformly and improve test readability. The

serum and reagent should be mixed properly. Small red floccules

against a white background indicate a weakly reactive test result

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

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

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

with positive and negative controls

Note: Very slight roughness should be interpreted as a negative result

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

9. 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: Negative control giving false positive reaction

LATEX SLIDE TEST FOR VDRL SYPHFINAL