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  • Slide 1
  • 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.
  • Slide 2
  • Sensitivity of various immunoassays
  • Slide 3
  • 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.
  • Slide 4
  • VDRL Test The 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.
  • Slide 5
  • VDRL Test The 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.
  • Slide 6
  • VDRL Test Specimen: 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.
  • Slide 7
  • VDRL TEST Specimen Preparation : Serum Heat the serum in a 56 o C 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 56 o C for 10 minutes if testing is delayed more than 4 hours. 8. Specimens must be at room temperature, 23 o - 29 o C 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).
  • Slide 8
  • 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 (2 o 8 o C). If testing is to be delayed more than 5 days, freeze the specimen at temperatures at or below -20 o C. Avoid repeated freezing -thawing of specimens. Specimens must be at room temperature, 23 o - 29 o C when tested.
  • Slide 9
  • VDRL Reagents : 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).
  • Slide 10
  • VDRL 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.
  • Slide 11
  • VDRL QUALITY CONTROL: Positive and negative controls should be run along with test specimens 1. Strong positive control. 2. Weak positive control 3. Negative control.
  • Slide 12
  • VDRL Procedure: Patients serum is inactivated by heating at 56 o C 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.
  • Slide 13
  • VDRL Qualitative 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.
  • Slide 14
  • VDRL Quantitative 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.
  • Slide 15
  • VDRL CSF -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.
  • Slide 16
  • VDRL
  • Slide 17
  • VDRL SOURCES OF ERROR: Reactivity is decreased if the temperature of the testing area, specimens, or reagents is less than 23C ; test reactivity is increased if the temperature is greater than 29C. 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.
  • Slide 18
  • VDRL TEST 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.
  • Slide 19
  • VDRL TEST LIMITATIONS : The VDRL may be reactive in persons from areas where yaws is endemic. As a rule, residual titers from these infections will be

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