C-REACTI VE PROTEI N

(CRP)
MOHAMMAD ZEESHAN
RESIDENT 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
37C, 0.10 M relative salt concentration and pH 7.58.0; 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 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 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 methods


Test 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)



Semiquanti
tative
An
approximate
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 healthy
individuals based on high-sensitivity CRP and LDL-cholesterol levels
High-sensitivity CRP compared with all levels of Framingham
Risk 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.