C- Reactive Protein Latex Agglutination Test: Principle, Procedure and Result

Principle:

Latex particles coated with antibody to CRP are reacted with patient serum. In this case, the CRP is acting as the antigen. If CRP is present above normal threshold levels, the antigen–antibody combination will result in a visible agglutination reaction. An elevated CRP level is a sensitive, although nonspecific, indicator of inflammation.

Reagents:

(Kit from Wampole, Remel, or other manufacturers)

• CRP latex reagent, which contains a 1 percent suspension of polystyrene latex particles coated with anti–human CRP produced in goats or rabbits
• Positive human serum control with a concentration of approximately 20 mg/dL of CRP
• Negative human serum control
• Disposable sampling pipettes
• Disposable test slides
• Not in kit but needed:
• Timer; disposable stirrers; serological pipettes; test tubes, 12 x75 mm

CAUTION:The human serum used in the preparation of controls is tested by an FDA-approved method for the presence of antibodies to HIV as well as for hepatitis B surface antigen and HCV and found to be negative. However, because no test method can offer complete assurance that HIV, hepatitis B, hepatitis C, or other infectious agents are absent, the reagent should be handled with the same care as a clinical specimen. Reagents in the kit contain sodium azide as a preservative. Sodium azide may form lead or copper azide in laboratory plumbing. An explosion may occur upon percussion. Flush drains thoroughly with water after disposing of fluids containing sodium azide.

Specimen Collection:

Collect blood aseptically by venipuncture into a clean, dry, sterile tube and allow it to clot. Separate the serum without transferring any cellular elements. Do not use grossly hemolyzed, excessively lipemic, or bacterially contaminated specimens. Fresh nonheat inactivated serum is recommended for the test. However, if the test cannot be performed immediately, serum may be stored between 2°C and 8°C for up to 2 days. If there is any additional delay, freeze the serum at –20°C or below

Procedure:

Qualitative Slide Test

1. Be sure reagents and specimens are at room temperature.
2. Using one of the pipettes provided, fill it about twothirds full with undiluted serum. While holding the pipette perpendicular to the slide, deliver one free-falling drop to the center of one oval on the slide. If a calibrated pipetter is used instead of the pipettes provided, adjust the pipetter to deliver 0.05 mL (50 mL).
3. Using the squeeze-dropper vials provided, add one drop of positive control and one drop of negative control to separate ovals on the slide. Note: A positive and a negative control should be run with each test.
4. Resuspend the latex reagent by gently mixing the vial until the suspension is homogeneous. Place one drop of CRP latex reagent next to each serum specimen and to each control.
5. Using separate stirrers, mix each specimen and control until the entire area of each oval is filled.
6. Tilt the slide back and forth, slowly and evenly, for 2 minutes. Place the slide on a flat surface and observe for agglutination using a direct light source.
7. The CRP positive control serum must show distinct agglutination, and the negative control must be nonreactive. If the reagent fails to agglutinate with the positive control, or does agglutinate with the negative control, it should be discarded.

Semiquantitative Slide Test

1. If a positive reaction is obtained, the specimen may be serially diluted with a glycine-saline buffer in order to obtain a semiquantitative estimate of the CRP level.
2. Begin with a 1:2 dilution of patient serum obtained by mixing equal parts specimen and glycine-saline buffer. Blend the tube contents thoroughly.
3. Add 0.1 mL of buffer to the desired number of test tubes. Add 0.1 mL of 1:2 dilution to the first tube; mix and transfer 0.1 mL to the next additional tube. Continue until all tubes are diluted.
4. Perform a slide agglutination test on each dilution by repeating the procedure (steps 3 through 7) as above, and look for agglutination.

Results:

• A positive reaction is reported when the specimen shows agglutination, indicating the presence of CRP in the serum at a level equal to or greater than 0.6 mg/dL.
• The titer is represented by the last dilution that shows a positive reaction.
• A negative reaction is characterized by a lack of visible agglutination in the undiluted specimen.

Comments:

The latex agglutination test for CRP is a screening test for elevated levels of CRP in serum. A level of 0.6 mg/dL or higher gives a positive result with the undiluted specimen. Normal levels range from 0.1 mg/dL in newborns to 0.5 mg/dL in adults. Usually, with the onset of a substantial inflammatory stimulus, such as infection, myocardial infarction, or surgical procedures, the CRP level increases very significantly (>tenfold) above the value reported for healthy individuals. Following surgery, CRP levels rise sharply and usually peak between 48 and 72 hours. Levels decrease after the third postoperative day and should return to near normal between the fifth and seventh postoperative day. Thus, CRP levels can be used to monitor the outcome of surgery. CRP testing can also be used to monitor graft rejection, drug therapy with anti-inflammatory agents, and recurrence of malignancies. For patients with rheumatoid arthritis, elevated CRP can be used as an indicator of the active stage of the disease. In most situations, however, it is desirable to have more than one determination so that base levels can be established.

Limitations of Procedures:

1. Reagent, controls, and test specimens should be brought to room temperature and gently mixed before using. 
2. Reagent and control should not be used after the expiration date indicated on the outside kit label.
3. Do not use the CRP reagent if there is evidence of freezing.
4. False-negative reactions may be due to high levels of CRP in undiluted specimens. A 1:5 dilution should always be run for this reason. False-positive reactions may occur with a reaction time longer than 2 minutes or with specimens that are lipemic, hemolyzed, or contaminated with bacteria. Therefore, any visibly contaminated, lipemic, or hemolyzed specimen should not be used.
5. Discard buffer if contaminated (evidence of cloudinessor particulate material in solution)