Sensitivity & Specificity

Two important parameters in serological tests :Sensitivity: The ability of the test to detect even very minute quantities of antigen or antibody.

When the test is highly sensitive, false negative results may be absent or minimal.

Specificity: The ability of the test to detect reactions between homologous Ags & Abs only, and with no other.

In highly specific test, false positive reactions are absent or minimal.

Sensitivity of various immunoassays

VDRL Test

The Venereal Disease Research Laboratory (VDRL) tests are slide tests for syphilis that use an antigen containing cardiolipin, lecithin, and cholesterol.

The antigen, suspended in a buffered saline solution, forms flocculates when combined with lipoidal antibodies in serum or cerebrospinal fluid from syphilis patients.

VDRL TestThe VDRL tests are fast, easy to

perform, and excellent for screening of samples.

The VDRL tests measure IgM and IgG antibodies to lipoidal material released from damaged host cells as well as to lipoprotein-like material, and possibly cardiolipin released from the treponemes.

VDRL TestThe antilipoidal antibodies are

antibodies that are not only produced as a consequence of syphilis and other treponemal diseases, but also may be produced in response to nontreponemal diseases of an acute and chronic nature in which tissue damage occurs.

Without some other evidence for the diagnosis of syphilis, a reactive nontreponemal test does not confirm T. pallidum infection.

VDRL TestSpecimen:Only serum and cerebrospinal fluid (CSF) are

appropriate specimens for the VDRL tests. a) Serum specimens that are excessively

hemolyzed, grossly contaminated with bacteria or otherwise extremely turbid are unsatisfactory for testing. A specimen is too hemolyzed for testing when printed matter cannot be read through it.

b) Spinal fluid-An acceptable CSF specimen should not contain particulate matter that would interfere with reading test results. CSF specimens with even traces of blood are unsuitable for testing.

VDRL TESTSpecimen Preparation : Serum Heat the serum in a 56oC water bath for 30

minutes. Remove the serum from the water bath and

examine for debris. Recentrifuge all serum specimens containing particulate debris.

Reheat serum at 56oC for 10 minutes if testing is delayed more than 4 hours.

8. Specimens must be at room temperature, 23o -29oC when tested.

If testing is to be delayed more than 4 hours, stopper the specimen tube and store serum at refrigerator temperature (2� -8�C).

VDRL CSF:Centrifuge at 1000-1200 x g for 10 + 5

minutes and transfer to a clean labeled Tube.

Do not heat spinal fluid before testing. If testing is to be delayed more than 4

hours, refrigerate the CSF specimen (2o 8oC). If testing is to be delayed more than 5 days, freeze the specimen at temperatures at or below -20oC. Avoid repeated freezing -thawing of specimens.

Specimens must be at room temperature, 23o -29oC when tested.

VDRLReagents :VDRL antigen: A colorless alcohol

solution containing 0.03% cardiolipin, 0.9% cholesterol, and sufficient purified lecithin to produce standard reactivity.

VDRL-Buffered Saline pH 6.0 ±0.1 (1.0% NaCl).

Control serum samples. Reactive (R), weakly reactive (W) and nonreactive (N) sera .

Test Specimen(s).

VDRLBefore testing all Equipments should be

calibrated and tested for accuracy.Pipettes, Volume should be checked by

using pure dionized water and high sensitive balace, where a known volume of water will be placed on the balance and there should not be a significant difference.

Centrifuges, Speed should be checked and calibrated if needed by Biomedical Engs.

Rotators, Time and Speed should be checked and calibrated if needed by Biomedical Engs.

VDRLQUALITY CONTROL:Positive and negative

controls should be run along with test specimens

1. Strong positive control.2. Weak positive control3. Negative control.

VDRL Procedure: Patients’ serum is inactivated by

heating at 56 oC for 30 minutes in a water bath to remove non-specific inhibitors (such as complement).

The test can be performed both qualitatively and quantitatively. Those tests that are reactive by qualitative test are subjected to quantitative test to determine the antibody titres.

VDRLQualitative test: 0.05 ml of inactivated serum is

taken into one well. 1/60th ml (or 1 drop from 18 gauge needle) of the cardiolipin antigen is then added with the help of a syringe to the well and rotated at 180 rpm for 4 minutes.

Every test must be accompanied with known reactive and non-reactive controls. The slide is then viewed

Under low power objective of a microscope for flocculation. The reactive and non-reactive controls are looked first to verify the quality of the antigen.

Depending on the size the results are graded as weakly reactive (W) or reactive (R).

Reactive samples are then subjected to quantitative test.

VDRLQuantitative test: This is performed to determine the antibody

titers.The serum is doubly diluted in saline from 1in

2 to 1:256 or more. 0.05 ml of each dilution is taken in the well and 1/60 ml of antigen is added to each dilution and rotated in a rotator. Rotate the slide for 8 minutes at 180 ±2 rpm.

The results are then checked under the microscope. The highest dilution showing flocculation is

considered as reactive titer. the qualitative test may be non-reactive. If the

clinical findings are strongly suggestive of syphilis, a quantitative test may be directly performed on the serum specimen.

VDRLCSF -VDRL: VDRL test may also be performed on CSF

samples in the diagnosis of neurosyphilis.Quantitative VDRL is the test of choice on

CSF specimens. However, there are some variations in this test.

The VDRL antigen is diluted in equal volumes with 10% saline, CSF must not be heated (or inactivated), the volume of antigen solution taken is 0.01 ml (or 1 drop from 21 gauge needle) and rotation time is 8 minutes.

Rest of the procedure remains same.

VDRL

VDRLSOURCES OF ERROR:Reactivity is decreased if the temperature of the

testing area, specimens, or reagents is less than 23°C ; test reactivity is increased if the temperature is greater than 29°C.

If hemolyzed, contaminated, or extremely turbid serum specimens are tested, the reliability of test results will be questionable.

If outdated or inadequately tested antigen is used, test results may be erroneous.

If the specimens and antigen are not rotated for the correct speed and length of time, test results may be incorrect.

If the antigen suspension is frequently forced through the syringe and needle, the suspension may lose reactivity.

VDRLTEST LIMITATIONS :Plasma cannot be used for the VDRL test. False-positive reactions can occur with

cardiolipin antigens, mainly in specimens from persons who abuse drugs; who have diseases such as lupus erythematosus mononucleosis, malaria, leprosy or viral pneumonia; or who have recently been immunized.

It is therefore recommended that any reactive samples are retested using a specific treponemal test such as TPHA or FTA-Abs.

VDRLTEST LIMITATIONS :The VDRL may be reactive in persons from

areas where yaws is endemic. As a rule, residual titers from these infections will be <1:8.

Non-treponemal test titers of persons treated in latent or late stages of syphilis or who have become reinfected do not decrease as rapidly as do those from persons

in the early stages of their first infection. In fact, these persons may remain “serofast,” retaining a low-level reactive titer for life.

VDRLPerformance Characteristics:

RPRThe rapid plasma reagin (RPR) 18-mm

circle card test is a macroscopic, nontreponemal flocculation card test used to screen for syphilis.

The antigen is prepared from a modified Venereal Disease Research Laboratory (VDRL) antigen suspension containing choline chloride to eliminate the need to heat inactivate serum, ethylenediaminetetraacetic acid (EDTA) to enhance the stability of the suspension, and finely divided charcoal particles as a visualizing agent

RPRThe RPR test measures IgM and IgG

antibodies to lipoidal material released from damaged host cells as well as to lipoprotein-like material, and possibly cardiolipin released from the treponemes.

The antilipoidal antibodies are antibodies that are produced not only as a consequence of syphilis and other treponemal diseases, but also in response to nontreponemal diseases of an acute and chronic nature in which tissue damage occurs.

Without some other evidence for the diagnosis of syphilis, a reactive nontreponemal test does not confirm T. pallidum infection.

RPR Specimen: Serum and plasma are both suitable specimens for the

qualitative test; however serum is the preferred sample for the quantitative test. Test plasma samples within 48 hrs after collection.

An acceptable specimen should not contain particulate matter that would interfere with reading test results. Specimens that are excessively hemolyzed, grossly contaminated with bacteria, chylous or otherwise extremely turbid are unsatisfactory. A specimen is too hemolyzed for testing when printed matter cannot be read through it.

Not all unsuitable specimens should be discarded or not analyzed. When an unsatisfactory sample is received in the laboratory, notify the requesting physician and discuss whether that specimen should be tested. If the ordering physician still desires a test result, then the condition of the sample must be stated on the report, and a notation made of any limitation on interpretation of the test result.

RPR Handling 1. Serum : Allow sufficient time (approximately 20 minutes) at

room temperature for the specimen to clot. Centrifuge the specimen at room temperature at 1000

to 1200 x g for at least 5 minutes to sediment cellular elements.

Keep serum specimens in the original collection tube if testing will be performed within a few hours. Remove serum from clot and store at refrigerator temperature (2° -8°C) if testing is to be delayed. If a delay of more than 5 days is anticipated before testing, freeze the specimen at -20°C or lower. Avoid repeated freeze-thawing of specimens. Although unheated serum specimens may be used, serum may be heated at 56°C for 30 minutes without affecting test outcome. Specimens must be at room temperature (23° -29°C) at the time of testing.

RPRHandling 2. Plasma:Centrifuge the specimen at room

temperature at 1000 to 1200 x g for at least 5 minutes to sediment cellular elements. Plasma may be retained in the original collection tube if the test is to be performed immediately. If not, plasma should be removed from cellular elements.

Store plasma specimens at refrigerator temperature (2° -8°C) and test within 48 hours. Plasma samples must be at 23° -29°C at the time of testing. Do not heat plasma.

RPRMATERIALS:

RPR antigen suspension Control serum samples. 0.9% Saline. Mechanical rotator Disposable latex gloves

RPRCalibration:Before testing all Equipments should be

calibrated and tested for accuracy.Pipettes, Volume should be checked by

using pure dionized water and high sensitive balace, where a known volume of water will be placed on the balance and there should not be a significant difference.

Centrifuges, Speed should be checked and calibrated if needed by Biomedical Engs.

Rotators, Time and Speed should be checked and calibrated if needed by Biomedical Engs.

RPRQuality ControlIt is the responsibility of the laboratorian to

ensure that reagents are of good quality and standard reactivity. Chemicals and distilled water should be of high quality, and solutions should be prepared according to the directions specified for each technique.

Test each new lot of RPR antigen for the RPR 18-mm circle card test in parallel with a reference reagent to verify that the two antigens (Positive and negative) are comparable before placing the new antigen into routine use.

Positive and negative controls should be runs along with test specimens

RPRProcedure: Qualitative Test Place 50 μl of serum or plasma onto a 18-mm

circle of the RPR test card, using a disposable Dispenstir or a safety pipetting device.

Using the inverted Dispenstir (closed end) or flat toothpicks, spread the serum or plasma to fill the entire circle. Do not spread the specimen beyond the confines of the circle.

Gently shake the RPR antigen dispensing bottle to resuspend the particles.

Holding the dispensing bottle and needle in a vertical position, dispense several drops to clear the needle of air. Then add exactly 1 free-falling drop (17 μl) of antigen suspension to each circle containing serum or plasma. Do not mix.

RPRProcedure: contPlace the card on the mechanical rotator

under a humidifying cover. Rotate the card for 8 minutes at 100 ∀ 2 rpm.

Immediately remove the card from the rotator; briefly rotate and tilt the card by hand (three or four to-and-fro motions) to aid in differentiating nonreactive from minimally reactive results.

Perform the quantitative test on serum specimens showing any degree of reactivity (clumping) or Aroughness

RPR Quantitative test: Dilute to an endpoint titer all serum specimens with rough

nonreactive results in the qualitative test. Test each specimen undiluted (1:1), and in 1:2, 1:4, 1:8, and 1:16 dilutions.

Place 50 μl of 0.9% saline in circles numbered 2 through 5. Do not spread the saline.

Using a safety pipette device, place 50 μl of serum in circle 1 and 50 μl of serum in circle 2.

Mix the saline and the serum in circle 2 by drawing the mixture up and down in a safety pipette eight times. Avoid forming bubbles.

Transfer 50 μl from circle 2 (1:2) to circle 3, and mix. Transfer 50 μl from circle 3 (1:4) to circle 4, and mix. Transfer 50 μl from circle 4 (1:8) to circle 5 (1:16), mix,

and then discard the last 50 μl.

RPR Using the broad end of a clean Dispenstir, spread the

serum dilution to fill the entire surface of circle 5, the highest dilution (1:16). Using the same Dispenstir, repeat for circle 4(1:8), 3(1:4), 2(1:2), and 1).

Gently shake the dispensing bottle to resuspend the antigen particles.

Holding the RPR antigen dispensing bottle in a vertical position, dispense 1 or 2 drops to clear the needle of air. Then add exactly 1 free-falling drop (17 μl) of antigen suspension in each circle. DO NOT MIX.

Place the card on the rotator under the humidifying cover and rotate the card for 8 minutes at 100 ∀ 2 rpm.

Immediately remove the card from the rotator; briefly rotate and tilt the card by hand (three or four to-and-fro motions) to aid in differentiating nonreactive from minimally reactive results.

If the highest dilution tested (1:16) is reactive, continue your serial dilution till 1:512

RPRReading of Results:Read the test reaction under a

high-intensity incandescent lamp for qualitative and Qualitative tests.

Report the results in terms reactive or non reactive for qualitative test.

Report the results in terms of the highest dilution that has given a reactive result, including a minimally reactive result.

RPRPerformance Characteristics:

RPRSOURCES OF ERROR: If the temperatures of the sera, reagents, or

testing area are less than 23°C, test reactivity decreases; if temperatures are greater than 29°C test reactivity increases.

If the speed of the mechanical rotator is too fast or too slow, improper antigen-antibody interaction will cause unpredictable test results.

If the time of rotation is too long test reactivity may be increased, or if too short test reactivity may be decreased.

If the card is excessively rotated and tilted (to-and-fro motions) by hand after removal from the rotator, a false-reactive result may occur.

RPRSOURCES OF ERROR:If lighting produces a glare on the card, the

reactions may be obscured. If the RPR antigen is outdated or not adequately

tested for standard reactivity, the results may be inaccurate.

If the serum is unevenly spread in the circle, the antigen and antibody may not mix properly.

If hemolyzed, contaminated, or improperly collected serum or plasma specimens are tested, the reaction may be masked.

If the moistened humidifying cover is not used to cover tests as they are being rotated, proper humidity will not be maintained, and test components may dry on card giving rise to false reactive results.

RPRTEST LIMITATIONS:The RPR card test cannot be used to test

spinal fluids. A prozone reaction may be encountered

occasionally. In a prozone reaction, complete or partial inhibition of reactivity occurs with undiluted serum (maximum reactivity is obtained only with diluted serum).

The RPR card test may be reactive in persons from areas where yaws, pinta or nonvenereal syphilis is endemic. Generally, residual titers from these infections will be <1:8.

RPRBiological false-positive (BFP) reactions

occur occasionally with cardiolipin antigens, mainly in specimens from persons who abuse drugs; who have diseases such as lupus erythematosus, mononucleosis, malaria, leprosy, or viral pneumonia; or who have recently been vaccinated.

Nontreponemal test titers of persons who have been treated in latent or late stages of syphilis or who have become reinfected do not decrease as rapidly as do those of the persons in the early stages of their first infection. In fact, these persons may remain Aserofast,@ retaining a low-level reactive titer for life.

Next Lecture: Precipitation Light-scattering

techniques