CRP test: Introduction, testing procedure, normal range and clinical

CRP Test: Introduction, Testing Procedure, Normal Range and Clinical Significance

Introduction of CRP Test

CRP stands for C-Reactive Protein and it is a carbohydrate reactive protein. CRP test was so named because it was first identified as a substance in the serum of patients with acute inflammation that reacted with the somatic ‘C’ carbohydrate antigen of Pneumococcus. Discovered by Tillett and Francis in 1930. Synthesized by hepatocytes. CRP is an annular pentameric protein found in plasma, whose levels rise in response to inflammation. Molecular Weight 25,106 Dalton. An acute-phase protein of hepatic origin that increases following interleukin-6 secretion by macrophages as well as adipocytes in response to a wide range of acute and chronic inflammatory conditions such as bacterial, viral, or fungal infections; rheumatic and other inflammatory diseases; malignancy; and tissue injury and necrosis. Elevated in 6-10 hours of inflammation and rise up to thousands of times Plasma half-life- 19 hours and fall within few days. CRP binds to the phosphocholine expressed on the surface of dead or dying cells and some bacteria. This activates the complement system via the C1Q complex, promoting phagocytosis by macrophages, which clears necrotic and apoptotic cells and bacteria. It plays a role in innate immunity as an early defense system against infections.

Reference Range

0-10 mg/L

Conditions associated with elevated level of CRP-

Moderate elevation (0-10mg/dL)

Myocardial infarction (MI)
Malignancy
Pancreatitis
Mucosal infections( bronchitis, cystitis)
Connective tissue disease

Marked elevation(>10 mg/dL)

Acute bacterial infections
Major trauma
Systemic vasculitis

The risk of developing cardiovascular disease is quantified as follows:

low: hs- under 1.0 mg/dL

average: between 1.0 and 3.0 mg/dL

high: above 3.0 mg/dL

An especially high CRP reading of greater than 10 mg/L may indicate:

A bone infection, or osteomyelitis
An arthritis flare-up
Inflammatory bowel disease
Tuberculosis
Lupus, another connective tissue disease, or another autoimmune disease
Cancer, especially lymphoma
Pneumonia

Various methods to measure CRP test are as follows-

A. ELISA

B. immunoturbidimetry

C. nephelometry

D. rapid immunodiffusion

E. visual agglutination

A high-sensitivity CRP (hs-CRP) test measures low levels of CRP using laser nephelometry. The test gives results in 25 minutes with a sensitivity down to 0.04 mg/dL.

Latex agglutination method for CRP test

Principle

The method was introduced by Singer, et. al., in 1957. This is a slide agglutination test for the qualitative and semi-quantitative detection of C-Reactive Protein (CRP) in human serum. Latex particles coated with goat IgG anti-human CRP are agglutinated when mixed with samples containing CRP. When latex particles coated with human anti-CRP are mixed with a patient’s serum containing C – reactive proteins, this results in visible agglutination within 2 minutes.

Requirements for CRP Test

The test kit contains-
Negative control
Positive control
Latex particle
Card
Mixing plastic sticks
extra we need –
Test sample-Blood
Centrifuge
Visual aid i.e. light source/table lamp
Micropipette and tips/ droppers

CRP Test Procedure

(Qualitative)

Bring all reagents and serum samples to Room Temperature and mix latex reagent gently prior to use. Do not dilute the controls and serum. Place 50 ul each of serum, positive control, and negative control on separate reaction circles. Then add CRP latex reagent 1 drop to each of the circles. Mix with separate mixing sticks and spread the fluid over the entire area of the cell. Tilt the slide back and forth slowly for 2 minutes observing preferably under artificial light.

Result and Interpretation of CRP Test

Positive: Agglutination of latex particles, indicating the presence of C – reactive protein at a significant and detectable level.
Negative: No Agglutination

Negative Control(NC): Negative

Positive Control(PC): Positive

Test: showing clumping/ agglutination, therefore the test is positive as shown above figure.

Semi-Quantitative Test

Using isotonic saline prepare serial dilutions of the patient’s serum (1/2, 1/4, 1/8, 1/16, 1/32, 1/64, and so on). Transfer 50ul of each serum dilution to a test circle on the slide. Shake the reagent, then add one drop of suspension to the test circle. Mix the drops using a disposable stirrer ensuring coverage of the test circle with the mixture. Place the test slide on the bench and leave for 2 minutes. Gently and evenly, rock and rotate the test slide once and place the test slide on the bench once more. One minute later, read the test slide for the presence of agglutination.

Calculation Titre

CRP mg/dl = 6 (can vary) x D,
where D is the highest dilution of serum showing agglutination and 6 is the sensitivity in mg/L.

Limitations of CRP latex agglutination Test

The strength of the agglutination reaction is not indicative of the CRP concentration. Weak reactions may occur with slightly elevated or markedly elevated concentrations.
Antigen excess may cause false negatives. It is advisable, therefore, to check all negative sera by retesting at a 1:10 dilution.
Reaction times longer than specified may produce apparent false reactions due to a drying effect.
Strongly lipemic or contaminated sera can cause false-positive reactions.
Only serum should be used in this test.
A quantitative titration procedure on positive specimens is required to observe increasing or decreasing levels.
Patients with high titers of rheumatoid factors may give positive results.

universe84a