Biochemistry Archives • Page 3 of 3 • Microbe Online

C-reactive protein (CRP) is one of several ‘acute-phase’ proteins formed by the liver and released into the blood circulation within a few hours after injury or response to inflammation. CRP is not an antibody (which are γ-globulins) but rather a β-globulin. C-reactive protein activates the complement pathway, modulates the activity of phagocytic cells, and helps in the opsonization of infectious agents and dead or dying cells. C-reactive protein was named for its ability to bind with a C-polysaccharide in the cell wall of Streptococcus pneumoniae. The normal CRP level of human body is less than 10mg/L. CRP is an "acute-phase" protein that is elevated as much as 1000-fold in acute inflammation. CRP concentration peaks within 48 hours of the inflammatory stimulus and when the stimulus for production stops, CRP decreases quickly. CRP is a nonspecific indicator of inflammation. The increased high level of CRP in blood gives a sign that there can be inflammatory processes occurring in the body. CRP test helps in the detection, diagnosis, and prognosis involving tissue damage and inflammation. Methods of CRP determination include capillary precipitation, Ouchterlony immunodiffusion, radial immunodiffusion, particle immunoassay, and particle agglutination of the latex particles. Principle of CRP Test Laboratories use various CRP test to measure CRP levels in the blood. C-reactive protein (CRP) has been most widely measured using ELISA, immunoturbidimetry, or antibody-based nephelometric assays. These tests are typically sensitive to concentrations of 5-20 mg/L. High-sensitivity CRP (hs-CRP) assays can detect lower levels of CRP (0.5-10 mg/L). Traditionally C-reactive protein test is based on the principle of latex agglutination. The reaction occurs between the human C-reactive protein and the corresponding anti-human CRP antibodies. In the case of a positive reaction, within 2 minutes, visible agglutination of latex particles can be observed. Materials required for CRP test Materials required to perform CRP assay depend on the type of method used. These laboratory equipment and supplies are necessary to detect CRP levels using the agglutination test method. Serum sample Positive and negative control Glass slide with six reaction circle Mixing sticks Test tube Saline A uniform suspension of polystyrene latex particles coated with anti-CRP antibodies. Procedure In this post, we have outlined the procedure of latex agglutination test both for qualitative assay as well as a semi-quantitative assay. Qualitative Method Take all reagents and serum samples to room temperature before the test starts and mix latex reagent gently before use. Do not dilute the controls and serum. Place one drop of serum, positive control, and negative control on a separate glass slide using a (disposable pipette) dropper. Add one drop of CRP latex reagent to the drop of test specimen (serum) on each slide. Using the mixing stick mix the serum and CRP latex reagent uniformly over the entire circle. Immediately start a stopwatch, and rock the slide gently back and forth for 2 minutes. Observe the clump (agglutination) macroscopically. Semi-quantitative Method Prepare the serial dilution of samples 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and so on by using normal saline. Pipette each dilution of the serum sample and control (positive control and negative control) on to separate reaction circle. Add one drop of CRP latex reagent to each drop of diluted serum (sample) and controls. Using the mixing stick mix the sample and latex reagent uniformly over the entire circle. Immediately start a stopwatch, and rock the slide gently back and forth (120 rpm/minute) for 2 minutes. Observe the clump (agglutination) macroscopically. Results and interpretation of CRP test A positive reaction is indicated by agglutination. False-negative results may occur when there is an excess antigen. In such conditions, the test should be repeated. When such a prozone effect is suspected, a diluted serum sample should be used. The presence of agglutination indicates a CRP concentration equal to or greater than the reagent sensitivity (mg/L CRP). The titer, in the semi-quantitative method, is defined as the highest dilution showing a positive result. Calculation: The approximate CRP concentration in the patient sample is calculated as follows: Sensitivity (Indicated on the label of the latex vial) x CRP Titer = mg/L For example, sensitivity (indicated on the label of the latex reagent vial) is 6 mg/L the semi-quantitative method, multiplication of dilution factor with 6 mg/L will yield the approximate level of CRP in the serum sample. Dilution(mg/L) 1:1 61:2 121:4 241:8 481:16 961:32 192 C-reactive protein (CRP) level in blood C-reactive protein is synthesized in the liver and a trace level of CRP had been reported in the sera of apparently healthy adults and normal children. The normal adult level of CRP is reported to be less than 10 mg/L. However, CRP levels less than 10 mg/L do not rule out an inflammatory or infectious process. During infections or inflammatory disease states, CRP levels may rise rapidly within the first 6 to 8 hours and peak at levels of up to 350-400 mg/L after 48 hours. CRP levels are unaffected by anemia, protein levels, red blood cell shape, or patient age or sex. However, in women, CRP concentrations tend to be higher late in pregnancy. Patients with high CRP levels are at increased risk for coronary artery disease. Elevated CRP levels have been associated with: acute bacterial infections tumors myocardial infarction post-operative necrosis acute rheumatic fever rheumatoid arthritis Uses CRP is a useful marker for monitoring disease activity. Quantification of CRP is done for the early detection of bacterial infections in a wide variety of clinical settings. CRP testing is useful in the differential diagnosis of pyelonephritis versus cystitis, bacterial versus viral infections, necrotizing pancreatitis versus edematous interstitial pancreatitis CRP test is used for following disease prognosis and effectiveness of therapy in a number of chronic diseases such as rheumatoid arthritis and inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease). CRP may be used to monitor the activity of infections that require long-term antibiotics and which are difficult to monitor clinically (e.g., osteomyelitis or prosthetic joint infections). Limitation Limitation of CRP testing by agglutination method; A reaction time longer than two minutes may lead to false-positive results due to the drying effect. Only fresh serum specimens should be used, plasma must not be used since fibrinogen may cause non-specific agglutination of the latex particles. False-negative results can occur due to the prozone phenomenon. So, all negative sera need to be checked by retesting at a 1:10 dilution. References and further readings Vanderschueren, S., Deeren, D., Knockaert, D. C., Bobbaers, H., Bossuyt, X., & Peetermans, W. (2006). Extremely elevated C-reactive protein. European Journal of Internal Medicine, 17(6), 430–433. https://doi.org/10.1016/j.ejim.2006.02.025 C-Reactive Protein Test: Purpose, Procedure, and Results. (2022). Retrieved 3 May 2022, from https://www.healthline.com/health/c-reactive-protein