Embee Diagnostics - Featured Section on CRP

C-reactive protein (CRP) has been a measure of acute phase reactions to inflammation for the last 15 years. Recently improved high sensitive and standardized quantitative assays in serum and cerebrospinal fluid (CSF) have allowed a re-evaluation of its potential as a diagnostic laboratory test. CRP is an abnormal serum glycoprotein produced by the liver during acute inflammation. Because it disappears rapidly when inflammation subsides, its detection signifies the presence of a current inflammatory process. Further, by serial measurements important information can be obtained on the resolution or continuation of the inflammatory process.

C-reactive protein was first described by Tillet and Francis in 1930. They concluded that sera of patients suffering from acute infection precipitated with a non-proteic pneumococcus extracts called C polysaccharide in the presence of calcium ions. The protein that caused this reaction was therefore called C-reactive protein (CRP). All acute inflammatory processes (infectious and non-infectious), and certain malignant conditions, result in rise in serum CRP as a non-specific phenomenon. CRP production is a non-specific response to disease and it can never, on its own, be used as a diagnostic test. However if the CRP result is interpreted in the light of full clinical information on the patient, then it can provide exceptionally useful information.

As CRP was the first recognized acute phase reactant, it can bind to a number of molecules, including phosphate esters, lipids, polyanions (DNA polylysin), polycations (histones, protamine) and a variety of polysaccharids. CRP is synthesized by the liver under regulatory control of cytokines. Synthesis of CRP and other acute phase proteins by hepatocytes is modulated by cytokines. Interleukins 1b and 6 and tumour necrosis factor are the most important regulators of CRP synthesis. The intact CRP molecule is a pentameric protein with identical subunits arranged in a doughnut-shaped polymer.

The function of CRP is felt to be related to its role in the innate immune system (Du Clos, Terry V, 2000). Similar to immunoglobulin IgG, it activates complement, binds to Fc receptors and acts as an opsonin for various pathogens. Interaction of CRP with Fc receptors leads to the generation of proinflammatory cytokines that enhance inflammatory response. Unlike IgG, which specifically recognizes distinct antogenic epitopes, CRP recognizes altered self and foreign molecules based on pattern recognition. Thus CRP is thought to act as a surveillance molecule for altered self and certain pathogens. This recognition provides an early defence and leads to a proinflammatory signal and activation of the humoral, adaptive immune system.

Fig.1 CRP binds to molecular groups found on a wide variety of bacteria and act as an opsonin.

Thus a number of functions have been ascribed to CRP, including initiation of opsonization and phagocytosis and activation of complement (Fig.1), neutrophils, and monocyte-macrophage. Collectively these properties imply an important role for CRP in the recognition of microbial organisms and as an immunomodulator in the host defence. CRP may also be important in the recognition of necrotic tissues.

CRP binds to apoptotic cells, protects the cells from assembly of the terminal complement components, and sustains an anti-inflammatory innate immune response (Gershov D et al 2000).

Levels of CRP increase very rapidly in response to trauma, inflammation and infection and decrease rapidly with the resolution of the condition (Fig.2). Since an elevated CRP level is always associated with pathological changes, determination of CRP is of great value in diagnosis, treatment and monitoring of inflammatory conditions. CRP is a more sensitive and reliable indicator of inflammatory processes than the ESR and the leucocyte count. The serum CRP concentrations increase faster than that of the ESR and when the condition subsides, CRP falls very quickly, reaching normal levels several days before the ESR normalises (Fig.2). Measurement of serum has thus emerged as a useful tool to help to answer the following questions: Is the patient getting better? Is he or she getting worse? Are there any complications? Or is there anything wrong with the person? Rises in CRP are only one part of a number of intricate changes in serum proteins and enzymes but it happens to be one that is earliest to measure because it increases so dramatically.

Fig. 2 CRP begins to rise in bacterial infections within 4-6 hours, peaks at 36-50 hours, closely parallels acute response with 4-7 hour half-life, allowing to normal 3-7 days after the stimulus is withdrawn. The ESR shows a slower rise and return to normal than C-reactive protein (CRP).

Erythrocyte sedimentation rate (ESR) is more commonly used as a non-specific marker of disease activity. However, as more is learned about CRP, measuring this parameter could be a better test than the ESR. The ESR, which is an indirect parameter of acute phase protein changes, can be influenced by concentrations of fibrinogen, monoclonal proteins and red cell morphology, whereas CRP has no cross-interfaces. CRP is useful for its negative predictive value as a negative CRP rules out the possibility of an inflammatory or necrotic course. A positive reaction is certainly an indication of a problem, but it is not specific for any single disease.

ESR has several disadvantages that prevent it from being an ideal laboratory test to monitor acute inflammation or tissue injury. However, the ESR remains useful for the detection of paraproteinaemia, which do not necessarily provoke an acute phase response. SLE and progressive systemic sclerosis, even when active, usually cause only a trivial increase in CRP (in the range 1-6 mg%), although the ESR may be very high .The reason for the discrepancy between ESR and CRP is unknown, but indicates the two tests are complementary. A comparison of ESR with CRP is shown in Table1.

CONDITION OR VARIABLE	CRP	ESR
Specimen requirements	Serum or Plasma Stable in stored specimens	Fresh specimen of whole blood Cannot be performed on stored specimen
Method of measurement	Direct quantitation of acute phase response	Indirect measurement of fibrinogen elevation
Magnitude and rate of rise	Elevation begins within 4 to 6 hrs, closely parallels acute response with 4 to 7 hrs. half life, allowing return to normal in 3 to 7 days after stimulus is withdrawn. Peak levels 100-1000% above base line.	Rises more slowly, may not return to normal for weeks, despite clinical improvement. Fibrinogen increases up to 400% above base line.
Effects of anaemia, polycythemia, interaction of proteins and red blood cells, size, shape of red blood cells	Unaffected	False negative or false positive reactions, depending on abnormality
Age and gender	Minimal change from neonate to elderly	Rises with age, higher values in women

Traditional methods for measuring CRP include precipitation and agglutination assays. The latex agglutination assay is a qualitative test with a detection limit of approximately 10 mg/litre, the upper limit of normal. Because CRP levels can increase so rapidly and dramatically, the latex agglutination assay is subject to false-negative reactions due to a prozone-type phenomenon in which all of the antibody combining sites on the latex particles are bound to an excess of CRP so no cross-linking (agglutination) can occur. Consequently the qualitative tests should be performed on several dilutions of serum to avoid negative reactions. If several dilutions are formed, the latex agglutination method can easily be converted to a semi-quantitative assay so distinctions can be made between levels of positivity (e.g. less than 50 mg/litre and more than 150 mg/litre). Such semi-quantitative distinctions would be very useful to the clinician trying to distinguish between bacterial (high CRP levels) and viral infections (normal to slightly elevated CRP).

Highly specific antibodies to CRP permit the development of rapid, specific, and very sensitive assays for this protein. These newer immunoassays include laser nephelometry (the most popular method), RIA, and enzyme immunoassays and have created a renewed interest in CRP testing in a variety of clinical settings. Measurement of CRP may be superior to the erythrocyte sedimentation rate (ESR) and may someday replace it. Recently, instrument manufacturers have developed assay systems that allow random access assays for CRP to be performed virtually on demand with 10 to 20 minutes turn-around-time (TAT).

An ultra-sensitive immunoturbidimetric assay has been developed for CRP. The new assay measures the increased turbidity resulting from antibody-antigen complexes formed when sample and antibody reagent is mixed. The assay has sensitivity of 0.1 mg/litre. The ready-to-use liquid reagents can be placed directly on a chemistry analyser and will yield precise results in minutes (Cortlandt Manor, NY, USA).

As in all serological tests, haemolytic, lipemic or turbid sera may cause incorrect results and should not be used. Drugs that may cause false-positive results include oral contraceptives. Drugs that may cause false-negative results due to suppression of inflammation include NSAIDS, steroids and salicylates. The presence of intrauterine device may cause inflammation, which produces a positive test. Overnight refrigeration of the sample may produce a false-positive result. There is no need to refrigerate samples if the assay is to be performed on the same day. Demographic factors including age, sex and race should be used to adjust the upper reference limit for CRP. Clinicians should be aware of these factors before using CRP to assess inflammatory disease.

Test combinations such as CRP, IL-6 and procalcitonin have been found useful in the diagnosis of pneumonia in children. Using the combination of IL-8 and/or CRP to restrict antibiotic therapy in truly infected infants reduces unnecessary antibiotic therapy and is cost effective. Measurement of CRP levels and white blood count has an additional diagnostic value in the diagnosis of acute appendicitis.

Well-known laboratory parameters indicating infections include white blood count, immature-to-total neutrophil ratio, CRP, ESR and procalcitonin. The immature-to-total neutrophil (IT) ratio is calculated as the sum of immature granulocytes divided by the sum of all neutrophil granulocytes. The IT ratio is considered to be elevated if it is more than 0.20 (Russel GAB et al 1992).

In recent years, several new markers of infection have been investigated, such as tumour necrosis factor-alpha, soluble tumour necrosis factor receptor, interleukin (IL-6), IL-1b, IL-8, IL-1 receptor antagonist, soluble intracellular adhesion molecule, granulocyte colony-stimulating factor, soluble IL-2 receptor and neopetrin, markers of complement-activation, leucocyte-a1-proteinase inhibitor, and most recently CD116 as a cell surface marker (Weirch E et al 1998). None of these markers has yet made the progress from the laboratory to clinical application.

Numerous reviews on CRP have been published prior to 1997 (Deodhar S, 1989), yet some conclusions require modification in view of the many recent investigations that used more advanced quantitative methods to study new and more carefully defined clinical conditions in larger patient populations. In addition several studies utilised serial CRP determinations rather than a single value at time of initial assessment (Jaye DL, Waits KB 1997).

In newborns and premature babies, the symptoms of bacterial infections are often highly uncharacteristic in the first few days of life. Infectious diseases such as bacterial meningitis, sepsis and pneumonia can frequently be severe in newborn babies. Neonates, especially born preterm, often fail to induce elevations in temperature and white cell counts that are hallmarks of infection in older children. Determination of serum CRP can be used to help to confirm or rule out bacterial infections in the neonatal period, for even premature babies have the capacity to synthesise CRP in the liver if they contract an infection.

Da Silva, et al 1995 reviewing the use of CRP as a tool for diagnostic neonatal sepsis, concluded that CRP is probably the best available diagnostic test. Further, Yentis SM, Soni N, Sheldon JC, 1995 found daily measurements of CRP to correlate with resolution of sepsis, specifically, “A decrease in CRP by 25% or more from previous day’s level was a good indicator of resolution of sepsis, with a sensitivity of 97%, specificity of 95% and predictive value of 97%”.

CRP elevation in neonates has been documented in non-infectious conditions including meconium aspiration, respiratory strea syndrome, foetal hypoxia and intraventricular haemorrhage. These disorders may mimic bacterial infections clinically.

Thus CRP is not useful alone in the primary diagnosis of neonatal sepsis, but may be helpful as a part of screening panel. Further helpful for monitoring response to therapy.

Meningitis is of particular interest in view of its potential severity and the importance of rapid diagnosis and appropriate treatment.

Some studies using serum CRP have described almost perfect discrimination between bacterial versus viral meningitis in children. Bacterial meningitis is associated with much higher serum CRP levels at presentation than cases of aseptic or proven viral meningitis. The latter frequently have CRP concentrations within the normal range or which are only very slightly raised, unless they develop secondary bacterial infective complications. Patients with meningitis in whom CRP values are determined at least 12 hours after the onset of fever are less than 2 mg/dL are far less likely to have bacterial meningitis. False-negative cases among CRP test results were found to be examined too early in bacterial meningitis (Tatara R, Imen H, 2000). Patients with tuberculosis meningitis seem to fall in between. Appropriate therapy for either bacterial or tuberculous meningitis causes the CRP level to fall, and especially in infants and children, this simple CRP test can be used to monitor objectively the response to treatment with many advantages over repeated lumbar puncture.

The serum CRP levels monitoring in children with bacterial meningitis represents useful and objective information about the clinical evaluation. The procedure is inexpensive and suitable for use in endemic areas lacking sophisticated laboratory facilities (Dias LR, Alves Ribeiro M, Farhat CK, 1999).

Newer standardised quantitative assessments of CRP can be very useful in distinguishing between bacterial and other forms of meningeal irritation during the first few days of hospitalisation. Many studies have indicated that by serial measurements important information on the resolution or continuation of inflammatory processes can be obtained.

CSF CRP concentrations are seven fold lower than those of serum. This difference is explained by direct hepatic release of CRP into plasma, which then undergoes ultra filtration to form CSF. Meningeal irritation stimulates CRP production. Once CRP enters the CSF it binds to damaged tissue. Minimal CSF inflammation may be apparent in patients undergoing lumbar puncture very early in the course of the disease, especially in neonates with rapidly developing meningitis in whom bacterial multiplication can outpace the ability of liver to mount a CRP response (Benjamin DR et al, 1984). These aspects of CRP metabolism in the central nervous system, poorly defined normal ranges, the lack of evidence of de novo synthesis in CSF and the impracticality of testing multiple samples of CSF as often as serum for monitoring response to treatment favour the use of serum CRP analysis instead of CSF. Still some authors recommend CSF CRP as an important tool in differential diagnosis of meningitis (Abrahamson JS, et al 1985). Yet others gave opposite opinions perhaps because of their different test design (Donald PR, et al 1985).

Measurements of the CRP levels with WBC have an additional diagnostic value in the diagnosis of acute appendicitis (Erkasap S, et al 2000).

Serum CRP cannot differentiate bacterial and viral aetiology of community acquired pneumonia in children in primary health settings (Heishanen-Kosma T, Korppi M, 2000). However, (Toikka P et al, 2000) have found that in some patients with very high serum CRP, Interlukin-6 and procalcitonin values, bacterial pneumonia is probable.

It is often difficult to diagnose abdominal infections in pregnant women. The ESR is regularly above normal during pregnancy, and therefore of limited diagnostic value in these situations. Since CRP is usually at a normal level in pregnant women, increased CRP concentrations strongly indicate infectious complications.

Acute systemic Gram-positive and Gram-negative bacterial infections are among the most potent stimuli for CRP production. In chronic bacterial infections such as tuberculosis and leprosy they are usually lower, though still markedly raised. Malaria, especially with P. falciparum is associated with high CRP values.

The combination of IL-8 and/or CRP is a reliable and early test for the diagnosis of nosocomial bacterial infection in newborn infants. Using the combination of IL-8 and/or CRP to restrict antibiotic therapy to truly infected infants reduces unnecessary antibiotic therapy and is cost-effective (Franz AR et al 1999).

Localization of infection within the urinary tract influences decisions regarding the choice and route of antimicrobial therapy, and what follow-up is needed. Clinical assessment alone cannot reliably distinguish cystitis from pyelonephritis in every case, particularly in very young children.

Urinary tract infection triggers the mucosal cytokine response; hence ESR and CRP might be valuable in identifying serious infections (Benson M et al. 1994). Some investigators, using cut-off values of 25 to 50 mg/liter have suggested that raised CRP can be of value in predicting whether a child has cystitis or pyelonephritis.

In general, clinical symptoms of urinary tract infection associated with a high CRP level (usually above 50 mg/liter) indicates pyelonephritis, while a normal to slightly elevated CRP level indicates an uncomplicated lower urinary tract infection.

Recently the use of CRP and fever for distinguishing children with septic arthritis and for determining whether arthrocentesis was required has been advocated. CRP rises rapidly and decreases to normal ranges within 1 week of treatment in most cases of haematogenous osteomyelitis without septic arthritis (Roine I et al 1995).

Serial measurements of CRP are a logical option for routine use in conjunction with other clinical and laboratory data in evaluating children with acute haematogenous osteomyelitis. A secondary rise may be an important warning sign of recurrence of both septic bone and joint infections.

CRP values correlate better than ESR with the severity of clinical disease activity as well as radiological findings in rheumatoid arthritis and are ideal for following active inflammation in this condition as well as its resolution and responses to anti-inflammatory treatment. In contrast, CRP may be only moderately increased or even absent in seronegative arthropathies, scleroderma and dermatomyositis, making it less useful for monitoring these conditions.

Correlation of CRP concentrations with disease activity in SLE has been less promising than for rheumatoid arthritis. Some persons with severe active disease have little or no elevation. In SLE, measurement of CRP may be most useful for diagnosis of infectious complications and monitoring response to antimicrobial therapy rather than assessing disease activity (Barland P and Lipstein E, 1996).

CRP concentrations are significantly higher in persons with inflammatory bowel disease than in unaffected controls. The CRP may have some role in the differential diagnosis of ulcerative colitis, where values tend to be higher than in Crohn’s disease (Thompson D et al 1992).

CRP has been found useful in the diagnosis, the presence and the extent of tissue necrosis.

Myocardial infarction is invariably associated with a major CRP response. The peak value of CRP occurs about 50 hours after the onset of pain in myocardial infarction and correlates closely in magnitude, though clearly not in timing, with the peak serum level of cardiac isoenzymes such as creatinine kinase MB. In patients who recover uneventfully the CRP falls rapidly towards normal in the usual exponential fashion (Danesh J et al 1998).

However, complications such as persistent cardiac dysfunction, further infarction, aneurysm formation, intercurrent infection, thromboembolism are associated with either persistently raised CRP levels or secondary increase after the initial decrease.

Angina without infarction and invasive investigation, such as coronary arteriography do not stimulate CRP production, whereas some other causes of chest pain such as pulmonary embolism, pleurisy or pericarditis are usually associated with raised CRP levels. Routine assays of CRP after infarction or in patients with chest pain may thus assist in diagnosis and the recognition and management of complications.

Over the last three years, CRP assays have been tested in a series of large-scale prospective clinical studies which demonstrated the value of this marker in predicting risk of future heart attack, stroke, and peripheral vascular disease in otherwise healthy men and women. In 1997, it was already reported that levels of CRP were elevated at baseline among apparently healthy individuals who subsequently developed first-ever heart attacks compared to those who did not. The men in the highest CRP quartile had three times the risk of myocardial infarction, two times the risk of ischemic stroke and four times the risk of developing severe peripheral artery disease compared to men in the lowest quartile. Similar data were reported in 1998 concerning healthy middle-aged women. Moreover, in both these studies, the clinical use of CRP significantly added to the predictive value of total and HDL cholesterol.

It is interesting to note that most of the beneficial effect of aspirin could be observed in subjects with the higher CRP levels. This raises the likelihood that this is due to its general anti-inflammatory effect rather than as an inhibitor of platelet activation.

Recent studies suggested that elevated concentration of CRP and cardiac Troponin I in patients with an acute coronary syndrome are associated with a high risk of cardiac events. It was demonstrated that in the patient group with unstable angina or NQMI (non-Q wave myocardial infarction) abnormal CRP concentration on admission and elevated concentration of Troponin I are important for predicting the incidence of major cardiac complications within six months.

Recent reports indicate that inflammation may be associated with atherosclerosis, and low levels of CRP may already be present as an indication of atherosclerosis. Myocardial infarction is frequently at the end of a long process of inflammation-mediated atherosclerosis. Thus the inflammation is believed to have a role in pathogenesis of cardiovascular events, measurement of markers of inflammation has been proposed as a method to improve the prediction of these events.

CRP may be used as a marker of subclinical atherosclerosis and cardiovascular risk. Specifically CRP has been positively linked to future cardiovascular events in healthy women, healthy men and elderly patients (Gracia-Moll X et al 2000).

CRP and cardiovascular disease is linked by complement: CRP induces adhesion molecule expression in human endothelial cells in the presence of serum. These findings support the hypothesis that CRP may play a direct role in promoting the inflammatory component of atherosclerosis and present a potential target for the treatment of atherosclerosis (Pasceri V et al 2000).

Half of all myocardial infarctions occur in persons in whom plasma lipids are normal. The study by Ridker PM et al 2000 showed that the addition of measurement of C-reactive protein (CRP) to the screening based on lipid levels may provide an improved method of identifying women at risk of cardiovascular events. Cardiovascular events were defined as death from coronary heart disease, non-fatal myocardial infarction or stroke or the need for coronary revascularisation procedures.

Other clinical studies validate the use of CRP assays in the prediction of future cardiovascular disease. The study by (Kitpatric ES et al 2000) suggests that some of the risk factors associated with coronary heart disease in Type 1 diabetes patients are also independently predictive of high CRP concentrations.

Overweight and obese patients may be maintaining a state of low-grade systemic inflammation, increasing their risk for cardiovascular disease. The clue is their consistently above-normal blood concentrations of CRP, a sensitive marker of systemic inflammation (Visser M et al 1999).

The prevalence of elevated CRP levels increased with increasing BMI in both men and women. Obese men were 2 times more likely and obese women 6 time more likely to have elevated CRP levels than their counterparts of normal weight. These findings remained clinically significant after adjusting for age, race, waist-to-hip ratio, inflammatory disease and other factors known to influence CRP concentrations (e.g. smoking status).

Serum CRP levels closely reflect the severity and progress of acute pancreatitis providing a better guide to intra-abdominal events than other markers such as leucocyte counts, ESR and temperature. A CRP concentration greater than 100 mg/litre at the end of the first week of illness is associated with a more prolonged subsequent course and a higher risk of the development of a pancreatic collection. Serial CRP measurements therefore can serve as a useful guide to the need of appropriate imaging techniques and finally to confirm resolution before discharge from hospital.

The CRP concentration always rises after significant trauma, surgery or burns, peaking after 2 days and then falling towards normal with recovery and healing. Infections or other tissue-damaging complications alter this “normal” pattern of CRP response and the failure of CRP to continue falling or the appearance of a second peak may precede clinical evidence of intercurrent infection by 1-2 days.

CRP usually increases more than 100 mg/litre by 48 to 72 hours. In the absence of complications values decline thereafter and reach normal concentrations 3 to 7 days later. CRP elevation persists for a much longer time after surgery when the postoperative course is complicated by infection or other processes involving tissue necrosis, suggesting its value for monitoring outcomes (Deodhar S, 1989).

CRP increases significantly in patients with extensive burns versus those with minor burns. A second later peak of CRP develops if infection occurs as a later complication of the burn suggesting the value of CRP to monitor the course of healing.

Most malignant tumours, especially when they are extensive and metastatic, induce an acute phase response. This is particularly so with those neoplasms which cause systemic symptoms such as fever and weight loss, for example, Hodgkin’s disease and renal cell carcinoma. However, given the non-specific nature of the acute phase response, a definite role of CRP measurements in the management of cancer patients, other than in cases of intercurrent infection has not yet been established.

Approximately 40% of cancer patients with fever and neutropenia develop culture-proved bacterial infection. Fever, however can also be caused by viral infections or a number of other non-infectious causes. Because of significant morbidity and mortality associated with infections in this patient group, there is aggressive use of antibiotics. Fever may be the only sign of serious infection in these patients in whom there is a minimal inflammatory response to infection. Therefore additional tests are needed. CRP is not affected by chemotherapy or transfusions, factors that may influence the ESR. Pronounced elevations of CRP do not occur in malignancies without other concomitant stimuli for synthesis such as intercurrent infections (Santolaya M E et al 1994). WBC did not always change in parallel with CRP in patients with malignant lymphoma and rheumatoid arthritis in elderly patients.

CRP is an independent survival determinant in advanced non-small-cell lung cancer. Median survival time in patients with normal CRP (0.2 mg/dL) and high positive (73 mg/dL) was 24.9 months and 3.7 months respectively.

CRP is elevated during most rejection episodes in transplant recipients. The CRP is a sensitive indicator of renal but not cardiac allograft rejection. A raised CRP may not differentiate between infection and graft rejection but has been able to differentiate between infection and graft versus host disease. However, contradictory results were reported by some workers, so a firm conclusion is difficult to draw regarding use of CRP in this setting.

Studies by Eisenberg MS et al 2000 suggest that elevated levels of CRP are associated with subsequent graft failure in cardiac transplant recipients.

The body of literature concerning studies of the application of CRP measurements in the paediatric and adult populations continues to grow. Based on current data, serial CRP measurements appear to be most useful for monitoring patient response to therapy after the primary diagnosis of invasive infections or inflammatory diseases, for monitoring patients after major surgical procedures and those with serious burns. Monitoring CRP over time may be used to assess for recurrent disease, a secondary process or ineffective therapy. In addition, CRP appears to be suited to most applications for which the ESR is used but offers many advantages.

Clinical applications of measurement of serum CRP concentration fall into four main categories:

CRP levels can be quantified using the following kits available at Embee Diagnostics :

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