C-REACTIVE PROTEIN(CRP)

MOHAMMAD ZEESHANRESIDENT MICROBIOLOGY

HISTORY• First Acute Phase Reactant (APR)

• 1930 Tillet and Francis -- Substance in the serum of patient with acute inflammation that react with the C-polysaccharides of the cell wall of Pneumococcus and form precipitate.

• 1941 found to be protein (c-reactive protein).

• Earliest assay for CRP – Qualitative,Semiquatitative precipitin tests.

ARTICLE INTERACTIONS OF C-REACTIVE PROTEIN WITH THE COMPLEMENT SYSTEM : I. PROTAMINE-INDUCED CONSUMPTION OF COMPLEMENT IN ACUTE PHASE SERA

Protamine sulfate was found to consume large amounts of C selectively during preincubation with sera of individuals in the "acute

phase". Marked depletion of C1, C4, and C2 with minimal, if any, depletion of C3-9, was observed. The consumption was time and

temperature dependent, occurring most rapidly and extensively at 37°C, 0.10 M relative salt concentration and pH 7.5–8.0; it required calcium ions. It was mediated by a heat-stable nondialyzable factor

which separated with C-reactive protein (CRP) during fractionation and purification, correlated with serum CRP levels, and, like other known reactivities of CRP, was inhibited by phosphoryl choline. Preparations of CRP purified either from serum or ascites resulted in consumption of large amounts of C1, C4, and C2 when preincubated with normal serum and protamine. -------------------------

The Journal of Experimental Medicine 1974

• Various analytical methods are available for CRP determination:

• ELISA.• IMMUNOTURBIDOMETRY.• RADIAL IMMUNODIFFUSION.

• BETTER SENSITIVITY AND SPECIFIITY

• QUANITATIVE

• EARLY RESULTS

GENETIC & BIOCHEMISTRY• The CRP geneThe CRP gene first

chromosome (1q21-q23)

• Globulin ,nonspecific

• Composed of 5 identical 23-kDa polypeptide subunits arranged in a cyclic pentameter shape.

• Each of these subunits contains one binding site for a phosphocholine molecule & 2 binding sites for Ca+2

SITES ENABLE CRP TO RECOGNIZE AND BIND TO A

VARIETY OF MICROORGANISMS,

CELLULAR DEBRIS, AND NUCLEAR MATERIAL FROM

DAMAGED CELLS

• C-Reactive protein binds not only with the polysacchrides present in many bacteria, fungi and protozoal parasites but also with:

In the presence of Ca+2 ions: Phosphorylcholine, Phosphatidylicholine Polyanion (Nucleic Acid)

In the absence of Ca+2 ions: Polycation (Histones)

FORMATION OF CRP-LIGAND COMPLEXES

FUNCTION OF CRP CRP-ligand complexes activate the complement

system, thereby facilitating phagocytosis and the removal of materials released from damaged cells as well as potentially toxic materials from invading microorganisms

CRP-ligand complexes bind directly to neutrophils, macrophages, and other phagocytic cells, stimulating an inflammatory response and the release of cytokines

• The fetus is able to produce CRP and other acute-phase reactant proteins as early as 4 to 5 weeks of gestation.

• Paired mother and infant sampling shows that CRP does not cross the placenta, and although maternal risk factors can exert an effect on the fetus, there is no correlation between maternal and infant CRP levels at birth.

CRP as an Acute-Phase Protein:CRP as an Acute-Phase Protein:

• CRP is the most sensitive of the APR, with levels rising as much as 1000-fold during the acute inflammatory processes.

• Levels begin to rise within 4 to 6 hours of the onset of signs of infection or tissue injury and peak 24 to 48 hours later and also rapidly disappear with the resolution of event.

• Rise in serum may be dependent on the amount of tissue damage present.

• Useful serum marker to assess and monitor the presence, severity, and course of the inflammatory response in infectious and noninfectious disorders, (MI, angina, malignancies, RA, IBD, burns, and trauma, and post surgery).

Laboratory Methods to Measure CRP

Comparison of CRP testing methodsTest Type What Is

Measured How the Test Is

Performed How the Results Are

Reported

Qualitative The presence or absence of CRP-antibody complexes within a given amount of serum sample

A measured amount of serum + latex reagent = signs of agglutination ??

Positive: Presence of any agglutination (concentration of CRP >6 mg/L)

latex reagent polystyrene or plastic beads coated with anti-human CRP antibodies.

Negative: Absence of agglutination (concentration of CRP <6 mg/L)

Semiquantitative

Anapproximate amount of CRP-antibody complexes within a given amount of serum sample.

Serial serum/saline dilutions are mixed with a latex reagent and examined for the presence of agglutination. The highest dilution in which agglutination is visualized corresponds to the approximate amount of CRP.

The highest dilution in which agglutination occurs is reported as an approximate titer of CRP in mg/dL or mg/L

(1:6 ratio = 6 to 12 mg/L 1:12 ratio = 12 to 24 mg/L 1:24 ratio = 24 to 48 mg/L 1:48 ratio >48 mg/L)

Test Type What Is Measured

How the Test Is Performed

How the Results Are Reported

Quantitative amount of serum CRP-antibody complexes.

Diluted serum samples + reagent containing monoclonal AB.= CRP-antibody complexes

mg/L or mg/dL

Directly measures CRP-antibody complexes marked with an enzyme or fluorescent tracer.

Enzyme-linked immunoassay (ELISA), Immunofluorescence

Directly measures the amount of agglutination or precipitation caused by the presence of CRP-antibody complexes

Immunoturbidity, nephelometry, or radial immunodiffusion

Test Type What Is Measured How the Test Is Performed How the Results Are Reported

IMPORTANT CLINICAL SCENARIOS AND CRP

NEONATAL SEPSIS.

CARDIOVASCULAR DISEASE.

Neonatal sepsis and CRP• Occurrence – 01to 21 per 1000 live births

• Mortality rate – 30% to 69%

• Diagnosis of Sepsis:– Clinical sign and symptoms.– Septic workup.

• Blood culture ( GOLD STANDARD)• White blood cell count (WBC) with differential • Calculation of the immature to total neutrophil (I:T) ratio and

absolute neutrophil count (ANC) • Platelet count • Erythrocyte sedimentation rate • C-reactive protein (CRP)

Controversies Surrounding the Use of CRP in Infants??• Use of serial CRP levels as an early diagnostic tool for

confirming the presence of sepsis.

• Screening tool to rule out the presence of sepsis.

• Use of CRP to monitor therapy and determine the length of antibiotic treatment.

• Conflicting Cutoff values.

Sensitivity, Specificity, and Clinical Utility of CRP Levels??• Elevated CRP levels combine with other tests support

diagnosis of sepsis while awaiting culture results.

• CRP and IL-8 levels -- if both levels were elevated, a sensitivity of 93% to 100% and specificity of 80% to 83% was achieved.

• A CRP level that returns to the normal range (<10 mg/L) may indicate the adequacy of antibiotic treatment duration, allowing earlier discontinuation of antibiotics.

• 2 CRP levels <10 mg/L have a negative predictive value of 99% in accurately identifying infants not infected or with a resolved infection.

• CRP has a high predictive value when it remains normal 24 to 48 hours after the onset of signs of infection.

• Serial measurements of CRP levels drawn every 24 to 48 hours after the onset of signs of infection have an increased sensitivity between 78.9% and 98%, specificity of 84% to 97%, and a negative predictive value of 99% in detecting sepsis.

Clinical Application of CRP Testing in Infants With Suspected Sepsis:

1. Carefully review the maternal and infant's history and physical and clinical course to assess for confounding factors that could increase CRP levels.

2. Use serial CRP levels in conjunction with other established sepsis workup tests, including WBC count with differential and blood culture.

3. Begin drawing quantitative CRP measurements 24 hours after the onset of signs and symptoms of infection. Repeat the level 24 hours later to capture the maximal rise.

4. Obtain at least 2 normal CRP levels (</=10 mg/L) 24 hours apart to identify infants unlikely to be infected.

5. Consider discontinuing antibiotics at 48 hr if at least 2 CRP levels are normal.

CARDIOVASCULAR DISEASE AND CRP

• Atherosclerosis ---- process of endothelial dysfunction and vascular inflammation.

• Cardiovascular events occur in individuals who have no identifiable traditional risk*

• Need for novel cardiovascular risk factor that help in primary prevention strategies.

• CRP play a role in the pathogenesis and prognosis and risk assessment of cardiovascular disease.

• CDC/AHA recommended the use of hsCRP as a marker for evaluation of cardiovascular disease.

* JAMA 2003

*Circulation. 2003

Predictive value of high-sensitivity

C-reactive protein:

Cardiovascular event-free survival among apparently healthyindividuals based on high-sensitivity CRP and LDL-cholesterol levels

High-sensitivity CRP compared with all levels of FraminghamRisk Score.

CONCLUSION• CRP is an important marker of inflammation.

• Can be used as therapeutic and prognostic marker.

• Solely its significance decrease specially in therapeutic situation. It should be correlates with clinical history, physical examination and other diagnostic parameters.