Preparation of Patient’s Serum (Inactivation)

Heat clear serum sample in a water bath at 56°C for 30

minutes. Examine all serum samples after removing from

the water bath and those found to contain particulate

debris should be recentrifuged. Serum samples to be

tested more than 4 hours after inactivation should be

reheated at 56°C and allowed to cool to room temperature.

Reagents Usually Supplied

¾ Reagent 1: VDRL antigen

¾ Reagent 2: Buffered saline diluent

¾ Reagent 3: Positive control serum

¾ Reagent 4: Negative control serum.

Preparation of Working Solutions

Working Antigen Suspension

a. Pipette 0.4 mL of Buffered Saline Diluent into a glass

stoppered bottle (with concave or flat inner bottom)

and make the reagent completely cover the inner

bottom as a thin layer.

b. Add 0.5 mL of VDRL Antigen from the lower half of a

1.0 mL pipette, which is graduated to the tip, directly

Serology/Immunology 609

to the Buffered Saline Diluent while continuously but

gently rotating the bottle. Add the antigen drop by drop

but rapidly, allowing 6 seconds for completing the

addition of 0.5 mL of the antigen. The pipette tip should

remain in the upper third of the bottle and rotation

should not be vigorous enough to splash Buffered

Saline Diluent on to the pipette. The proper speed

of rotation is obtained when the center of the bottle

circumscribes a 5 cm diameter circle approximately

three times a second.

c. The last drop of the antigen should be blown from the

pipette tip so that the pipette tip does not touch the

surface of the contents of the bottle.

d. Continue rotation of the bottle for 10 more seconds.

e. Now add 4.1 mL of Buffered Saline Diluent to the

bottle. Close the bottle with the stopper and shake it

for approximately 10 seconds. The working antigen

suspension is now ready for preliminary testing.

Note

There is some indication that maturation of the antigen

increases the sensitiveness and this is almost complete

within 15 to 30 minutes after preparing Working Antigen

suspension. This may then be used within 24 hours.

Under condition of high temperature and low humidity,

the Working Antigen should be stored in a refrigerator,

but should be brought to room temperature before use.

Mix the working antigen suspension gently each time it is

used. Do not mix it by forcing it back and forth through

the syringe and needle as this may lead to breakdown of

antigen particles which results in loss of activity.

Preliminary Testing of the Working

Antigen Suspension

1. Each time the Working Antigen Suspension is

prepared, it has to be tested with negative and positive

control sera by means of slide qualitative test method

described under Procedure, using the controls in place

of test sera.

2. The Working Antigen Suspension should give expected

typically reactive and nonreactive results respectively.

Also, the size and number of antigen particles per

microscopic field in the nonreactive serum should

be optimum. If the antigen particles in nonreactive

serum appear too large, the fault will usually be in

the preparation of the Working Antigen Suspension

although other factors are sometimes responsible.

3. The antigen control (suspension in saline) should

be smooth in appearance with antigen particles well

dispersed.

4. Do not use Working Antigen Suspension if it does not

give satisfactory performance in the preliminary testing.

Storage and Stability

The VDRL Antigen and Buffered Saline Diluent are to

be stored exclusively in a cool and dark place at room

temperature (preferably at 23–29°C); and at these conditions,

the reagents are stable till the expiry date mentioned.

The control sera are stable at 2 to 8°C till the expiry date

mentioned.

Working Antigen Suspension prepared for any day

must NOT under any circumstances be kept and used for

the subsequent days.

Procedure

Qualitative Test

1. Pipette 0.05 mL of patient’s inactivated serum into the

concavity of the VDRL slide.

2. Pipette 0.05 mL each of positive and negative control

sera into other two concavities of the VDRL slide.

3. Add one drop (1/75 mL) of the Working Antigen

Suspension to each of the above concavities, with a

calibrated 23-gauge needle without bevel.

4. Rotate the slide for 4 minutes with hand on a flat

surface (this movement should circumscribe roughly

about 5 cm diameter circle 120 times per minute) or

on a VDRL rotator.

5. Read the tests immediately under a low power

objective of a microscope.

Quantitative Test

For quantitative test with sera reacting strongly in qualitative

test, the following procedure should be followed:

1. Prepare different dilutions of test serum, in test tubes,

in the range of 1:2, 1:4, 1:8, 1:16, 1:32 or more with

normal saline.

2. Transfer 0.05 mL of each of the above diluted sera into

separate concavities of the VDRL slide.

3. With the help of a 23-gauge needle and syringe, add

one drop (1/75 mL) of Working Antigen Suspension

to each of the above concavities.

4. Rotate the slide for 4 minutes with hand on a flat

surface (this movement should circumscribe roughly

about 5 cm diameter circle 120 times per minute) or

on a VDRL rotator.

5. Read the test immediately under a low power objective

of a microscope.

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

The results of controls should be satisfactory for validating

the results of tests.

Interpretation of Test Results

The antigen particles are seen as small fusiform needles which

remain more or less evenly dispersed in case of a nonreactive

serum and aggregate into clumps with reactive serum. The

conclusions can be drawn from the observations as follows:

 Observation Conclusion

i. No clumps or very Nonreactive

 slight roughness

ii. Small clumps Weakly reactive

iii. Medium/large Reactive

 clumps

Notes