Principle: | Clinical Significance: | Specimen: | Materials: | Reagents: | Standardization: N.A. | Procedure: | Results Derivation: | Expected Result(s) | Quality Control: | Reference:
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The CMS Sure-Vue Rubella test is based on a passive latex agglutination. Latex is sensitized using solubilized rubella virus antigens. This latex reagent when mixed with serum containing rubella antibodies on a card surface will agglutinate forming visible clumps. In the absence of antibody, or if antibody concentration is insufficient to react, the latex will remain smooth and evenly dispersed.
II. Clinical Significance:
Rubella is usually a mild childhood disease, but if infection occurs in the pregnant patient, severe congenital disease may result. At least 95% of vaccinations result in a detectable titer and immunity is probably life-long. Failed immunization is rare.
- 200 µl of serum.
- Specimens may be stored at 2 - 8°C up to 8 days. For long term storage, the specimens should be frozen at -20°C. After thawing frozen specimens, be sure to mix the samples thoroughly by inversion prior to testing.
- Hemolysis could affect the test.
- 25 µl Rainin pipette and tips
- Humidifying rotator
- High intensity incandescent lamp
CMS Sure-Vue Rubella kit (CMS Fisher #23-038004)
- Latex antigen Store at 2 - 8°C.
- Diluent buffer Store at 2 - 8°C.
- High positive control (not used in qualitative assay) Store at 2 - 8°C.
- Low positive control Store at 2 - 8°C.
- Negative control Store at 2 - 8°C.
- Test cards (120 tests) Store at room temperature.
- Plastic stirrers
VI. Standardization: N.A.
- Remove the reagents from the refrigerator 5 minutes prior to use; warm the latex reagent to room temperature by rolling the vial between hands.
- Label a circle on the test card for low positive control, negative control and every sample being tested. (Each card has 15 circles. The cards can be cut if less than 15 tests are to be run. The maximum number of tests the rotator can hold at one time is 15.)
Note: Cards must be flat for proper reactions. If necessary, flatten cards by bowing back in a direction opposite to that of the curl. Care should be taken not to finger mark the test areas, since this may result in an oily deposit and improper test results. Use cards once and discard.
- Dispense 1 drop of the low positive control to the circle labeled for the low positive control.
- Dispense 1 drop of the negative control to the circle labeled for the negative control.
- Using a 25 µl Rainin pipette and the reverse pipetting technique, pipette 25 µl of patient sample to appropriately labeled circles.
- Using a new plastic stirrer end for each circle, spread the serum to fill the entire circle.
- Gently invert the dispensing bottle containing the latex antigen several times to thoroughly mix.
- While holding the bottle in a vertical position, dispense several drops of latex antigen into the bottle cap until a drop of uniform size has been formed. Dispense one free-falling drop of latex antigen onto each circle being used for testing. Recover the predropped latex antigen from the bottle cap.
- Place the card on a rotator and rotate for eight minutes under a moistened humidifying cover. (Recommended rotator speed is 100 rpm, but rotation between 95 and 110 rpm does not significantly affect the results obtained).
- Immediately following rotation, observe the card under a high intensity incandescent lamp. Gently tilt the card back and forth several times to help differentiate weak agglutination from no agglutination.
- The low positive control should show agglutination; the latex will have clumps of particles of varying size with some clearing of the background. The negative control should show no agglutination; the latex will be uniform white and smooth looking in appearance. Record control results on the worklist.
- Patient circles showing any amount of agglutination should be reported as “immune”.
- Patient circles showing no agglutination need to be repeated using a 1:10 dilution of the specimen. (Reduction in the degree of agglutination has been reported with rare high titered specimens). Dilute specimen with the diluent buffer.
Note: When testing specimens at a 1:10 dilution, the low positive control should be used diluted 1:10 by following steps A-C. The negative control need not be diluted for testing. The low positive control shows agglutination different from the uniform appearance of the negative control. If no agglutination occurs, the test should be repeated with the 1:5 dilution of the low positive control prepared as in Step A below. If there is positive reaction, continue testing specimens as the low positive control is formulated to produce agglutination at a titer or 1:10 ± one dilution. If there is no positive reaction the kit should be discarded.
- Prepare a 1:5 dilution of the sample and the low positive control on the qualitative disposable slide by pipetting 100 µl of the dilution (reverse pipetting technique) buffer and 25 µl of the sample (forward rinse pipetting technique) into the square section of the slide and mix several times with the same pipette.
- Place 25 µl of the dilution buffer on the circle beside the square section (reverse pipetting technique).
- Transfer 25 µl of the 1:5 dilution from the square section into the dilution buffer (forward rinse pipetting technique) and mix several times with the same pipette. Discard 25 µl from the circle and repeat the test starting with step 7 above.
- Upon repeat if no agglutination is observed report as “not immune”.
- Upon repeat if agglutination is present report as “immune”.
VIII. Results Derivation:
Refer to section VII.12.&13 Procedure.
IX. Expected Result(s) and /or Critical Values:
It is the opinion of the Immunization Practices Advisory Committee that any detectable antibody is indicative of immunity and protection against subsequent viremic infection.
- Endogenous Control: N.A.
- Exogenous Control:
- A low positive and a negative control are run with every run
- The low positive control should show agglutination; the latex will have clumps of particles of varying size with some clearing of the background.
- The negative control should show no agglutination; the latex will be uniform white and smooth looking in appearance.
- These control trials must generate the expected reaction response. Record results on the Rubella Test log.
- If a control fails to generate the expected reaction response, do not use the kit; complete the following procedure:
- Repeat the Q.C. test.
- Document the failed test result and repeat test in the Rubella Test log, notify the Site Supervisor and the office of the UCL Technical Director immediately.
- Indicate in the designated Rubella Test log who was called along with the date and time of the call.
- When a new kit is opened, record the lot number in the designated laboratory testing log. Check the new kit lot # against the previous kit lot #. Indicate on the log if the lot # is the same as the current lot or if it is a new lot #.
- Refer to “Reagent Overlap, Serology” in the Quality Assurance Manual for the proper overlap protocol.
- Package insert for CMS Sure-Vue Rubella kit, Curtin Matheson Scientific, January 1998.
- Package insert for CMS Sure-Vue Rubella kit, Curtin Matheson Scientific, 1/1/04.
- Package insert for CMS Sure-Vue Rubella kit, Curtin Matheson Scientific, 05/09.
- November 1998 S. Jaeger
- June 2004 S. Raymond/L. McGovern (Revised: language/protocol updated for CMS status)
- January 2012 W. Goedken (Revised: VII.13.Note added., 13.A.; XI.3.)
February 2010 L. Cooksley (Revised: VII.13.pipetting techniques)
March 2011 K. Gourley (Revised: III.3.; VII.1.)
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