C-reactive protein

Introduction

C-reactive protein (CRP) is a sensitive biomarker of inflammation, synthesized by the liver in response to cytokines, especially interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-1 (IL-1). CRP plays a role in the immune response by binding to phosphocholine on damaged or dead cells and some microorganisms, promoting their elimination through phagocytosis and complement activation.

• Acute-Phase Response: CRP is part of the acute-phase response, rising quickly (6–8 hours post-stimulus) and peaking at 48 hours. Its levels decrease rapidly once inflammation subsides.
• Clinical Utility: CRP measurement helps identify acute infections, chronic inflammatory conditions, and tissue damage.

 

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Principle of the Test

The CRP test relies on antigen-antibody interaction, where CRP in the patient’s serum reacts with antibodies specific to CRP, leading to agglutination. The intensity of agglutination reflects the CRP level.

Key Assay Techniques:

1. Slide Agglutination (Qualitative or Semi-Quantitative):
   • Detects the presence or rough estimation of CRP levels.
2. Turbidimetric/Immunoturbidimetric:
   • Measures the turbidity caused by agglutination, proportional to CRP concentration.
3. Nephelometric:
   • Measures light scattering due to immune complexes, providing precise CRP levels.
4. Enzyme-Linked Immunosorbent Assay (ELISA):
   • A highly sensitive method used for specific and quantitative CRP detection.
5. High-Sensitivity CRP (hs-CRP):
   • Specialized assay for detecting low levels of CRP to assess cardiovascular risk.

 

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Requirements

Sample Collection

• Type: Serum (preferred) or plasma (EDTA/heparinized).
• Volume: 2–3 mL of venous blood.
• Handling:
  • Separate serum promptly by centrifugation to avoid hemolysis.
  • If testing is delayed, store serum at 2–8°C (up to 72 hours). For longer storage, freeze at −20°C.
  • Avoid multiple freeze-thaw cycles, as this can degrade CRP.

 

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Reagents and Materials

1. CRP Latex Reagent:
   • Polystyrene latex particles coated with anti-CRP monoclonal or polyclonal antibodies.
2. Diluent:
   • Normal saline or phosphate-buffered saline (PBS).
3. Positive Control:
   • Serum with a known CRP concentration (used to validate the test).
4. Negative Control:
   • CRP-free serum or saline (ensures test specificity).
5. RBC Suspensions (for older hemagglutination-based methods):
   • Used in outdated methods but less common in modern assays.

 

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Equipment

• Glass Slides (for slide tests).
• Rotator (for mixing during slide agglutination).
• Cuvettes (for turbidimetric or nephelometric assays).
• Spectrophotometer/Nephelometer (for quantitative analysis).
• Pipettes and calibrated tips.

 

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Procedure

Qualitative Test (Slide Agglutination)

1. Place a drop (50 µL) of undiluted serum on a clean glass slide.
2. Add an equal drop (50 µL) of CRP latex reagent.
3. Mix gently using a disposable stick or slide rotator.
4. Observe for visible agglutination within 2–5 minutes.

Interpretation:

• Positive: Visible clumping (CRP present above the detectable limit, often >6 mg/L).
• Negative: No visible clumping (CRP absent or below detectable limit).

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Semi-Quantitative Test

1. Prepare serial dilutions of the serum (e.g., 1:2, 1:4, 1:8, etc.) in saline or PBS.
2. Add 50 µL of each dilution to separate wells or slide areas.
3. Add 50 µL of CRP latex reagent to each dilution.
4. Mix and observe for agglutination.
5. The highest dilution showing agglutination indicates the approximate CRP titer.

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Quantitative Test (Immunoturbidimetric or Nephelometric)

1. Prepare a calibration curve using CRP standards of known concentrations.
2. Mix serum with anti-CRP antibody reagent in a cuvette.
3. Incubate the mixture at 37°C for the specified time.
4. Measure turbidity (optical density) or light scatter using a spectrophotometer or nephelometer.
5. Calculate CRP concentration from the calibration curve.

 

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Results and Interpretation

Normal CRP Levels

• Standard CRP Test: < 10 mg/L indicates no significant inflammation.
• hs-CRP (for Cardiovascular Risk):
  • < 1 mg/L: Low risk.
  • 1–3 mg/L: Moderate risk.
  • > 3 mg/L: High risk.

Elevated CRP Levels

1. Mild Elevation (10–40 mg/L):
   • Mild infections (e.g., viral infections).
   • Pregnancy or obesity-related inflammation.
2. Moderate Elevation (40–200 mg/L):
   • Active bacterial infections and autoimmune diseases (e.g., rheumatoid arthritis).
3. High Elevation (>200 mg/L):
   • Severe bacterial infections (sepsis, pneumonia) and significant tissue damage (trauma, burns).

 

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Clinical Significance

1. Infections

• • Elevated CRP levels help distinguish bacterial infections (high CRP) from viral infections (lower CRP).

2. Autoimmune Diseases

• • CRP is elevated in diseases like rheumatoid arthritis, lupus, and vasculitis, serving as a marker of disease activity and treatment response.

3. Cardiovascular Diseases

• • hs-CRP predicts cardiovascular risk by detecting low-level chronic inflammation involved in atherosclerosis.

4. Monitoring

• • CRP levels guide treatment decisions for infections, autoimmune diseases, and post-surgical recovery.

 

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Limitations

1. Non-Specific:
   • Elevated CRP does not pinpoint the source of inflammation. Clinical correlation is essential.
2. False Positives:
   • Elevated CRP in pregnancy, obesity, smoking, or hormone replacement therapy.
3. Transient Rise:
   • CRP levels peak rapidly and normalize quickly; the timing of sample collection is critical.
4. Low Sensitivity for Chronic Conditions:
   • Conditions with low-grade inflammation (e.g., diabetes, metabolic syndrome) require hs-CRP for detection.

 

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Precautions

1. Sample Quality: Avoid hemolyzed or lipemic samples, which may interfere with results.
2. Reagent Handling: Store reagents as specified to maintain stability and accuracy.
3. Interpretation: Always correlate results with clinical history, physical examination, and other laboratory findings.