Why C-reactive protein is one of the most requested tests in clinical laboratories?

Introduction

C-reactive protein (CRP) is an acute-phase protein discovered by Tillet and Francis in 1930, which is synthesized by the liver in response to the secretion of several inflammatory cytokines including interleukin 6 (IL-6), IL-1 and tumor necrosis factor (TNF) [1]. CRP, named for its capacity to precipitate the somatic C-polysaccharide of Streptococcus pneumoniae, was the first acute-phase protein to be described and to be adopted in clinical laboratories as a sensitive systemic marker of inflammation and tissue damage [2]. CRP belongs to the pentraxin family of calcium-dependent ligand-binding plasma proteins and is composed of five identical nonglycosylated polypeptide subunits, each containing 206 aminoacid residues. Human CRP binds with highest affinity to phosphocholine residues, but also to a variety of other autologous and extrinsic ligands, and it aggregates or precipitates the cellular, particulate, or molecular structures bearing these ligands. Autologous ligands include native and modified plasma lipoproteins, damaged cell membranes, a number of different phospholipids and related compounds, small nuclear ribonucleoprotein particles, and apoptotic cells. Extrinsic ligands include many glycans, phospholipids, and other constituents of microorganisms, such as components of bacteria, fungi, and parasites [3]. When aggregated or bound to macromolecular ligands, CRP is recognized by the complement component 1q (C1q) and potently activates the classical complement pathway, engaging C3, the main adhesion molecule of the complement system, and the terminal membrane attack complex, C5-C9. The secondary effects of CRP that follow ligand binding resemble some of the key properties of antibodies, thus contributing to host defense against infection and playing a role of proinflammatory mediator.

Circulating CRP concentration

Circulating CRP is produced only by hepatocytes, predominantly under transcriptional control by IL-6, and to slighter extent by IL-1β and TNF-alpha, although other sites of local CRP synthesis and secretion have been suggested. De novo hepatic synthesis starts very rapidly after a single stimulus, serum concentrations rising above 5 mg/L by about 6 h and peaking around 48 h. The plasma half-life of CRP is about 19 h and is constant under all conditions of health and disease: the sole determinant of circulating CRP concentration is, therefore, the synthesis rate that reflects the intensity of the pathological processes stimulating CRP production [4]. If the stimulus for increased production ceases, circulating CRP levels fall rapidly, at almost the plasma CRP clearance. Subjects in the general populations tend to have stable CRP concentrations characteristic for each individual, apart from occasional spikes related to minor or subclinical infections, inflammation, or trauma. CRP values tend to increase with age, reflecting the increasing incidence of subclinical pathologies. The median CRP approximately doubles with age, from approximately 1 mg/L in the youngest decade to approximately 2 mg/L in the oldest, and tends to be higher in females [5]. Negligible diurnal difference and no significant seasonal variation in baseline CRP concentrations have been described [6]. The biological variability of CRP is crucial in both providing a fundamental prerequisite for the correct definition of analytical performance specifications (APS) and for the right clinical interpretation of concentration changes in serial analyses. However, the biological variability of CRP is still a matter of concern. As highlighted by Braga and Panteghini [7] not only the data base compiled by Carmen Ricos et al. [8] suffered of important limitations but a systematic review of the literature performed until 2012 led to the conclusion that there was “a paucity of robust data on biological CRP variability in serum” [7]. More recently, the European Biological Variation Study (EuBIVAS) provided more and valuable information on the biological variation (BV) estimates for many proteins, including CRP [9]. In basal conditions, serum CRP was found to be stable and in extreme low plasma concentrations (<0.5 mg/L) in many individuals, but even small inflammatory episodes did cause a 10- to 20-fold increase in 25% of subjects making difficult, if not impossible, to imagine a real steady state for this protein. Within-subject BV (CV_I) and Between-subject BV (CV_G) were found to be 42.2 and 76.3%, respectively. These BV estimates should be taken into consideration only when CRP is used as a cardiovascular risk indicator and may suggest that the BV model should not be used for to calculate APS, and that this protein should be assigned to the clinical outcome model [10]. In another recent study in athletes, the authors observed that “a high percentage of outliers, from 21-84%, had to be removed to reach homoscedasticity, a requirement for ANOVA analysis”, thus confirming that subclinical inflammatory processes may distort the steady-state situation [11]. Therefore, while an evidence-based reference change value (RCV) cannot be suggested due to the excessively high BV, the dynamics of CRP is increasingly recognized as a clinical information with higher diagnostic ability than a single measurement [12, 13]. CRP is found in two conformations, circulating pentameric CRP (pCRP) and monomeric CRP (mCRP), which present different pro-inflammatory or anti-inflammatory effects. pCRP stimulates the classical complement pathway, provokes phagocytic activities, and encourages the process of apoptosis. In contrast to pCRP, mCRP boosts the chemotaxis, assembles leukocytes from circulation to inflammation areas and thereby interrupts apoptosis [14]. Two different mechanisms have been proposed to explain the nature of mCRP: according to the first, mCRP is mainly expressed locally by cells. The second mechanism considers that mCRP is obtained by local CRP dissociation. Accumulating evidence suggests that mCRP can be separated from pCRP at the inflammatory site, exposing its proinflammatory activities and mCRP appears to be a more specific marker of underlining pathological processes than pCRP because of its tight association with inflammation. Therefore, while pCRP is a basic structural form, mCRP is the active structural form and the transition from pCRP to mCRP is a key regulator of its proinflammatory activity [15].

Clinical applications

In 1983, the seminal paper by Pepys and Baltz represented a milestone in elucidating not only the structure, synthesis, biological properties and function of CRP as the main “acute phase protein”, but also the value of its measurement in clinical practice [16]. As shown in Table 1, CRP was found to be associated with several conditions, including infections, malignancies, ischemic necrosis and trauma.

The authors, in addition, recognized the evidence that CRP levels rises modestly despite active tissue-damaging inflammatory processes in some disorders, including systemic lupus erythematosus, scleroderma, dermatomyositis, Sjogren’s syndrome, ulcerative colitis, graft-versus-host disease and leukemia. On the basis of these findings and the development of more sensitive, accurate and automated methods, the measurement of CRP has been increasingly adopted in clinical practice, and the list of clinical applications is continuously updated, including for example the diagnosis and monitoring of COVID-19, as shown in Table 2.

As a matter of fact, CRP is a preferred serologic marker of acute inflammatory conditions because its faster kinetics and shorter half-life that result in a quick fall once inflammation resolves, thus being helpful not only for diagnosis but also for evaluating the response to treatment. A meta-analysis on 19 diagnostic accuracy studies, in fact, reported a combined diagnostic sensitivity of 0.79 (CI 0.69–0.87) and 0.70 specificity (CI 0.69–0.79) in acute inflammatory conditions [18]. Another recently published meta-analysis indicated that CRP has a relatively greater diagnostic accuracy for inflammation and infection diseases, particularly for post-operative infectious complications even if other conditions that cause tissue damage are associated with the rise of this protein [19]. CRP should be defined an “old wine in new bottles” as recently published studies have provided new insights on its clinical value. A Cochrane systematic review demonstrated that the use of point-of-care testing (POCT) of CRP reduced the antibiotic prescription from 516 to 397 in 1,000 participants of both intervention and control groups, while it did not increase the number of recovered participants nor the total mortality within 28 days follow-up [17]. The rise of CRP and its kinetics after 48 h from the baseline value was found to be a simple and accessible strategy for predicting respiratory deterioration and intubation in COVID-19 patients [12]. A machine learning approach to patients with suspected myocarditis, using CRP measurement was found to provide valuable information, even if it showed negligible importance in predicting patients’ outcomes [20]. Another paper emphasized the clinical value of CRP measurement and its association with tumor location in patients with colorectal cancer [21], thus highlighting the need for further research on a better utilization of CRP measurement in clinical practice.

High sensitivity CRP assays and cardiovascular risk

The story of CRP methods is a paradigmatic example of the impressive technological development experienced in laboratory medicine in the last decades. The measurement of CRP in serum/plasma was initially performed with methods that provided only a qualitative results (positive/negative), moving to semiquantitative assays, and finally methods which assure quantitative results. Initially, clinical laboratories adopted radialimmunodiffusion (RID) techniques, electrophoresis in agarose gel, latex-agglutination (LA), and later on electroimmunoassay (EIA), immunoturbidimetric (IT), laser nephelometry (LN) and immunofluorimetric (IF) methods. Analytical advances allowed to achieve both an increased analytical sensitivity and a significant reduction of the analytical turnaround time (TAT) [22], [23], [24]. More recently, point-of-care (POCT) tests have been developed and introduced in laboratory medicine to guide antibiotic prescribing for lower respiratory tract infections [25].

Since the early 1990s, a growing body of evidence has supported the idea that cardiovascular diseases (CVD), including coronary heart disease, ischemic stroke, and acute myocardial infarction, as well as peripheral vascular diseases (PVD), are related to inflammation [26]. The concept of the involvement of inflammation in atherosclerosis has spurred the discovery and adoption of inflammatory biomarkers for cardiovascular risk prediction. CRP has proved to be the most robust biomarkers as it is “an excellent analyte with negligible diurnal variation, not depend on food intake, has a long half-life and a remarkable dynamic range” [27]. According to a seminal paper by Peter Libby “CRP integrates inflammatory signals that arise from a variety of sources and predicts a variety of clinical outcomes” [28]. However, the prospect of using CRP as a predictor of future vascular risks faced initially a big obstacle because existing assay methods, such as latex agglutination and capillary immunoprecipitation with 3–8 mg/L of a limit of detection (LOD), were not sensitive enough to detect very low-levels of CRP in serum. Therefore, assays with higher analytical sensitivity (hs-CRP) have been developed and adopted in clinical laboratories. These assays assure an analytical detection limit of about 0.00016 mg/L with a analytical coefficient of variation (CV) <15% at 0.2 mg/L, thus allowing an accurate measurement of the protein in serum/plasma samples even at very low levels [29] to eventually provide evidence of the association of serum CRP with the incidence of major coronary heart disease (CHD) events [30]. More recently, high-sensitivity POCT assays have been developed to measure CRP in whole blood samples, thus further reducing the TAT and making possible its measurement in decentralized settings [31]. CRP, therefore is the paradigm of a measurand which may provide different clinical values moving from qualitative to quantitative and ultimately high-sensitive methods, as shown in Figure 1. Other measurands which provide different clinical information are, for example, cardiac troponin, which with the first-generation methods was detectable only in patients with acute myocardial infarction while with high-sensitivity assays are now used for risk stratification in general population, and urinary albumin which is no more used to detect only “gross proteinuria”.

Figure 1:

Clinical value of CRP measurement according to different analytical sensitivities.

Why CRP is one of the most requested tests in clinical laboratories?

Figure 2 shows why CRP is one of the most requested tests in laboratory medicine. There are two main reasons for explaining the “popularity” of this test. First, the wide range of clinical conditions in which CRP provides useful information including inflammatory diseases, sepsis, malignancies, as well as its role in evaluating the risk of cardiovascular events. Second, the analytical advantages of this “robust biomarker” that provides similar results in fresh, stored or frozen samples, does not depend on food intake, presents negligible diurnal and seasonal variation, and a definite half-life. In addition, CRP concentrations are measured in biological samples with automated, and not expensive methods easily available to potentially all clinical laboratories in high, medium and low-income countries. The standardization of its measurement is still in progress and other articles in this issue of the Journal should provide more information on this issue.

Figure 2:

Main reasons of the high demand for CRP measurement in clinical practice.

Minimum retesting interval and CRP velocity

Many data are available to confirm that CRP is still one of the most requested laboratory tests in many countries, as demonstrated by recently published papers in Denmark, United Kingdom, The Netherlands, Croatia and Italy, with data obtained both in hospital and primary care settings [32], [33], [34], [35], [36]. The volume of tests ordered and the variability of the demand, however, represent a matter of concern and efforts to improve appropriateness, avoiding unnecessary and repeated requests have been carried out. The minimum retesting interval (MRI) is a popular demand management solution for the identification and reduction of over-utilized tests [37]. Guidelines recommend CRP should not be repeated within a 24 h period with the eception of requests in neonates and therefore the introduction of a rule avoiding the repetition of CRP before 48 h, particularly when introduced using automated IT-based systems was found to significantly reduce unnecessary repeat CRP test, resulting in cost saving, more efficient use of laboratory resources without compromising the quality of care and patient safety [32], [33], [34], [35], [36]. The concept of CRP dynamics, based on serial measurements of the protein, has been found to improve its clinical value in several conditions and has been termed “CRP velocity” (CRPv). Estimated CRP velocity is defined as “the level of the first CRP measurement divided by the time from the patient’s first reported symptom to the CRP being measured and expressed as the velocity of CRP measurement in mg/L/h” [13]. From a practical viewpoint, it is obtained by evaluating the dynamics of the first two CRP measurements from admission divided by the time (in hours) between the two serial tests. The idea behind this concept is that particularly in patients with low CRP level at admission a single measurement can mislead clinicians leading to a wrong rule-out of a bacterial infection. The dynamic features of CRP and the advantage of measuring its rate of rise in order to improve its diagnostic ability has been recently reported in several papers and different clinical conditions, including cardiovascular diseases [38], [39], [40]. The knowledge of the kinetics and dynamics of the protein may therefore improve the appropriateness of test request in the pre-analytical phase, and its diagnostic value using these metrics in the post-analytical phase, thus highlighting two faces of the same coin.

Conclusions

CRP was the first acute-phase protein to be described and adopted in clinical laboratories as an exquisitely sensitive systemic marker of inflammation and tissue damage. Over the time, the impressive advances in analytical methods (from qualitative to high-sensitivity assays), automation and availability of results in a short time, not only translated in increasing demand for the right management of systemic inflammatory diseases but also in subclinical inflammatory processes underlying atherothrombotic events. The emphasis in cardiovascular medicine on “high-sensitive CRP” led to a false impression that is somehow a different analyte from “conventional” CRP. The CRP analyte measured with “high-sensitivity” methods, in fact, is the same protein regardless of the assay range. It has been long postulated that CRP may have significant proinflammatory effects and, in particular, the rat model myocardial infarction provided direct evidence of its role in exacerbating tissue damage [41]. However, this is not a matter of discussion in the context of this paper. In conclusion, CRP, an old but valuable inflammatory biomarker that is still receiving interest and new applications as demonstrated in the recent CIVID-19 pandemic, is and will be one of the most requested laboratory tests.