C-Reactive Protein (CRP) : Principle, Procedure & Test results
C - reactive protein test commonly abbreviated crp test is a qualitative detection and semi-quantitative estimation of C - reactive protein in human serum samples.
C - reactive protein is an acute phase protein of human serum which is synthesized by liver (hepatocytes). It is normally present only in trace amounts in serum. The level of CRP can rapidly increase by as much as l000-fold in response to inflammation or infection.
The level of CRP increases significantly after most forms of bacterial and viral infections, tissue injuries, inflammation and malignant neoplasms. Hence the clinical measurement of CRP in serum, is a valuable screening test for organic disease and a sensitive index of disease activity in inflammatory, ischemic and infective conditions.
Principle of C - reactive protein test
The principle of C-Reactive-Protein test is based on the immunological reaction between human C - reactive protein found in patient’s serum or control serum and the corresponding antihuman C - reactive protein antibodies bound to latex particles. So if the serum mixed with latex reagent contains CRP, there will be a distinctly visible agglutination of the latex particles in the test cell of the slide.
CRP Test Sensitivity: The minimum detection limit is 6 mg /liter of serum.
The appropriate specimen for this test is serum not plasma
- Collect 2 mL of venous blood in a plain red topped vacutainer tube.
- Allow the tube to stand for 30 minutes and separate the serum by centrifugation at 2500–3000 rpm for 5–10
- Test tubes
- Test slides
- Disposable stirrers
- CRP Latex reagent : A suspension of uniform polystyrene particles coated with monospecific antihuman CRP (goat) in buffer
- CRP Negative control
- CRP Positive control
Required Equipment for Rheumatoid arthritis test
- Rotary shaker
- Test tubes
- Serological pipettes
- Isotonic saline (0.9%)
Procedure for Rheumatoid arthritis test
CRP Qualitative Test
- Bring the CRP latex reagent, specimen and the controls to room temperature before the test.
- Shake the latex suspension well before use and thoroughly clean and dry the test slide before use.
- Pipette 40 μL of test samples (Serum) in the test slide fields.
- Add one drop of RF Latex Reagent to each field.
- Stir to spread reaction mixture over the entire area of the field using an applicator stick.
- Tilt the slide back and forth for 2 minutes in a rotary shaker so that the mixture rotates slowly.
- Observe for agglutination after 2 minutes under bright artificial light.
Results and Interpretation
A negative reaction is indicated by no agglutination
A positive reaction is indicated by any observable
Agglutination in the reaction mixture.
CRP semi-quantitative test
- Prepare serial dilutions of the positive sample using diluted normal saline, i.e., 1:2, 1:4, 1:8 and 1:16.
- Add 50 μL of each dilution of the test sample to individual wells and spread the diluted test serum over the entire area of the test circle using separate applicator sticks.
- Gently mix CRP latex suspension for uniform resuspension of the latex particles and add one drop (50 μL) to test field.
- Mix well with separate applicator sticks used in the previous step and spread mixture over the entire area of the particular cell and then tilt the slide back and forth for 2 minutes, so that the mixture rotates slowly inside the cells or gently rock the slide in a rotary shaker for two minutes and read immediately under direct bright light.
Rheumatoid arthritis test Quality control
Include positive and negative controls with each test batch. Acceptable performance is indicated when a uniform milky suspension with no agglutination is observed with the CRP negative control and agglutination in the form of large aggregates is observed with the CRP positive control
Interpretation of results
A positive reaction is indicated by any observable agglutination in the reaction mixture. Record the last dilution showing a positive reaction. Most normal healthy humans have CRP concentrations of less than 6 mg/liter. In patients with high serum concentrations continued monitoring of the CRP levels can give a good indication of patient response to therapy during inflammatory disorders.
- Always ensure the reagents and specimens are at room temperature before use and store the kits at 2–8°C after use.
- Do not use lysed or contaminated serum for the test.
- Do not use severely lipemic serum as it interferes with the state of latex particles.
- Process the specimen on the same day within 2 hours of collection.
- If analysis is not done on the same day of collection, separate the serum and store it at 2–8 °C for up to 3 days.
Possible sources of variability
- CRP latex reagent should be shaken well before use as otherwise erroneous results will be obtained.
- Ensure the CRP latex reagent is uniform without visible clumping. When stored refrigerated, a slight sedimentation may occur and should be considered normal.
- Do not use the latex reagent or controls if they become contaminated.
- Ensure that the sample is free of fibrin/particles by centrifuging.
- Reaction time is critical. If reaction time exceeds 3 minutes, drying of the reaction mixture may cause false positive results.
- Freezing the CRP latex reagent will result in spontaneous agglutination.
- Intensity of agglutination is not necessarily indicative of relative CRP concentration; therefore, screening reactions should not be graded.
- A false negative can also be due to prozone phenomena (antigen excess). Therefore, it may be necessary to check even negative sera by retesting at a 1:10 dilution with glycine buffer
Sankara Nethralaya’s, CRP Test, Manual of Medical Laboratory Techniques page 346