How I use… C-reactive protein in daily practice

Introduction

C-reactive protein (CRP) is an acute phase protein (APP), a term that describes proteins whose concentration changes in response to inflammation or immune system stimulation, regardless of the cause. CRP is generated quickly, with significant increases observed just a few hours after the inflammatory stimulus (e.g., 4 hours after a surgical intervention), and peak concentrations are reached at approximately 24 hours. This reaction is part of the innate immune response, which means that it is rapid and non-specific, and it can be triggered by any process that damages the animal [1]. After more than 20 years of using and providing external services for CRP measurement at our laboratory, we have found that practitioners who regularly use CRP consider it to be one of the most important biomarkers of inflammation. Included as a part of their routine analytical profiles, CRP assay is employed for periodic check-ups, diagnostics, treatment monitoring, and predicting the outcome of inflammatory disease. This review will provide updated information and practical examples on how CRP can be employed in clinical practice, and will follow the initial consensus based on a seven-point plan established some years ago about the use of CRP in companion animals [1].

Always use a validated assay 

Any method used to assess CRP in practice must be validated to ensure the results can be trusted. Various manufacturers currently offer certified in-house (“benchtop”) canine-specific tests, and the larger clinical pathology laboratories also provide assays. Although certain human assays may not be suitable for dogs, others can be effective and provide a cost-efficient means of measuring CRP [2]. However, it is always to be recommended that standardization of the assay (using purified protein or pools of acute phase serum) and control samples should be of canine origin. 

Serum, EDTA or heparinized plasma can be employed for CRP measurements, and because the protein is fairly stable, samples can be refrigerated for several days or frozen for long-term preservation. It is important to know the effect of hemolysis, lipemia and bilirubinemia on the CRP values obtained, as the effects can vary depending on the method used. In our laboratory, the reference range for CRP in healthy dogs is below 12 mg/L, and whilst this value can vary between laboratories, it is usually not higher than 20 mg/L. There are no evident changes in reference ranges due to age or gender, although in pregnant bitches CRP increases at 21 days after fertilization, coinciding with embryonic implantation.

Other APPs can be used alongside CRP 

The possibility of using CRP along with other APPs will be considered later, but the different APPs will be described here. APPs that increase concentration after an inflammatory stimulus are known as POSITIVE APPs, while others that decrease after such a stimulus are called NEGATIVE APPs (Figure 1).

Positive APPs are further classified into two groups, namely major and moderate: Major APPs in dogs are CRP and serum amyloid A (SAA). While these proteins are present in low level in healthy animals, their concentration can increase by 10-100 times when stimulated. Moderate APPs are haptoglobin (Hp), ferritin and fibrinogen. Their concentrations increase 2-10-fold following stimulation. Major APPs exhibit a rapid increase in concentration followed by a steep decline, usually within hours, while moderate APPs take longer to both increase from and return to normal levels.

Albumin and paraoxonase-1 (PON-1) are examples of negative APPs. During inflammation, the albumin serum concentration decreases. This may be because albumin is the most abundant protein in serum, and its decrease can promote the synthesis of other proteins related to inflammation. PON-1 has an antioxidant function, and its decrease in inflammatory processes is possibly due to the associated oxidative stress that consumes this protein.

CRP for infection and inflammation 

The main reason to use a CRP assay is to confirm or rule out an inflammatory or infectious process. Whilst the most widely used tool for detecting inflammation is the evaluation of white blood cells (WBCs), ideally CRP should be measured and interpreted at the same time. Moreover, CRP has several advantages over WBCs in detecting and evaluating the severity of inflammation, especially as it has better sensitivity. For example, higher sensitivity of CRP has been reported in cases of babesiosis (Figure 2) [3] or various surgical procedures [4], and this is particularly important where there is decreased bone marrow activity. In addition, CRP is highly stable, allowing for long-term storage of samples, which is not possible with WBCs.

Whilst elevated CRP levels are very useful in detecting inflammation in the dog, it is important to note that a CRP within the reference range is also a significant clinical finding, as it suggests the absence of an acute inflammatory or infectious process. In addition to detect or rule out inflammation, CRP can also provide additional information for the clinician, as shown in Table 1.

Table 1. What information can CRP provide beyond detecting inflammation?

CRP may aid identification of a possible etiology 

Although CRP cannot identify the cause of inflammation, due to its non-specific nature, its magnitude of increase can help to narrow down the list of possible causes and guide the diagnostic process (Table 2; Figure 3). This can lead to various practical applications, as outlined in Table 3. In addition, it is important to note that there are situations where CRP may significantly aid in differentiating between the causes of a problem, such as:

• Nasal disease: slight increases in CRP values have been observed in cases of aspergillosis, non-specific rhinitis, or neoplasia [17]. However, if there is a marked elevation in CRP levels in a dog with signs of nasal disease (Figure 4), it may indicate an underlying inflammatory problem that is of clinical significance.
• Gastrointestinal (GI) disease: some conditions that cause chronic GI signs in dogs, such as inflammatory bowel disease (IBD), parasitic infections, dietary and antibiotic responsive diarrhea, GI neoplasia or presumed motility disorders may not show marked increases in CRP [9]. Also, intestinal malabsorption caused by exocrine pancreatic insufficiency, wheat-sensitive enteropathy or anaerobic bacterial overgrowth has been reported not to produce increases in CRP in dogs [18]. Therefore, if there is a noticeable increase in CRP associated with any of these conditions, it can indicate the presence of a complicating systemic inflammatory condition.

Table 2. Clinical interpretation of the magnitude of serum CRP increase in dogs (from [11]).

Table 3. Potential usefulness of CRP in narrowing the list of causes and guiding diagnosis.

This point only refers to CRP measurement as a stand-alone test, but additional examples of how CRP can be used in combination with other APPs to identify the cause of certain conditions are discussed below.

CRP helps monitor response to treatment 

CRP values returning to normal levels in an infectious-inflammatory disease indicates that a dog is responding to treatment and implies a good prognosis. This has been demonstrated in numerous inflammatory diseases, such as acute pancreatitis, where CRP can be a useful tool for monitoring clinical progression and response to treatment [19]. CRP has been shown to better reflect the evolution of inflammation after treatment compared to WBCs in conditions such as immune-mediated polyarthritis, indicating periods of relapse and remission of the disease [20]. Examples of interpretation are:

• Positive response → decrease in CRP: If the dog’s initial CRP levels are high, a return to the reference range along with clinical improvement indicates that treatment can be stopped. In this way, antibiotic treatments can be greatly optimized. For instance, in a study involving dogs with bacterial pneumonia, the duration of antibiotic administration was significantly reduced when normalization of serum CRP was used to guide the treatment [21]. In this study, antibiotics were discontinued 5-7 days after serum CRP returned to normal, as opposed to the conventional approach of treating for 1-2 weeks beyond the resolution of alveolar density on thoracic radiographs. This reduction in treatment duration did not increase the number of relapses.
• Negative response → little or no decrease in CRP: The absence of a decrease in CRP after treatment would indicate that either the diagnosis or the treatment was inadequate, and should necessitate a thorough evaluation of the patient. This could be considered a “red-light” alert. For example, CRP levels after surgery are typically low by the time sutures are removed [4]. Therefore, an increase in CRP during the postoperative period may indicate the presence of complications, such as incision site infection that can appear after surgical treatment for pyometra.

CRP can predict the presence of an inflammatory-infectious disease

CRP’s high sensitivity and fast stimulation of response allow detection of subclinical inflammation, with changes in APPs occurring before clinical signs develop. Therefore, this protein is highly suitable for routine health checks, where an increase in APPs in an apparently healthy animal can indicate the presence of a subclinical disease or predict the development of active disease in the near future. For example, in a study on dogs infected with Babesia gibsoni, high concentrations of CRP were documented despite the absence of obvious clinical signs or parasitemia [22]. Similarly, high CRP values are reported in asymptomatic dogs infected with Leishmania infantum – and it can be assumed that these animals may develop clinical signs at a later stage [23].

The future: APPs profiles

Divergences between major and moderate APPs can provide useful clinical information, so if possible, it is recommended to use a laboratory profile that includes at least one major and one moderate APP. The term “divergence” describes a situation where APPs of the two groups do not change as expected in the presence of an inflammatory stimulus. Some relevant examples are:

• ↑ Hp & ↔ CRP (increased Hp concentration with CRP values remaining in the reference range). Given that CRP usually shows a greater increase than Hp in an inflammatory situation, detecting this profile in a dog with no history of glucocorticoid treatment could indicate increased production of endogenous steroids. Glucocorticoids stimulate increases in haptoglobin and decreases in CRP [24], making this profile a potential indicator of canine hyperadrenocorticism. If a dog diagnosed with hyperadrenocorticism shows an increase in CRP, it indicates the presence of a severe inflammatory stimulus that can overcome the inhibitory effect of the endogenous corticosteroids. This may suggest the need to investigate potential inflammatory complications such as severe sepsis, urinary infection, deep pyoderma, severe mastitis or immune-mediated hemolytic anemia [24].
• ↔ Hp & ↑ CRP (steady or decreased levels of Hp accompanied by an increase in CRP levels). This profile may indicate hemolysis or internal hemorrhage leading to hemolysis. This is because Hp binds to hemoglobin released from damaged erythrocytes to facilitate its degradation and prevent the oxidative stress caused by hemoglobin; therefore, Hp can be consumed after hemolysis. As an example, in dogs naturally infected with B. canis, the concentration of CRP increased (mean value of 170 mg/L), while the concentration of Hp remained within the reference range (mean value of 2.7 g/L). The haptoglobin values could reflect the mixed effects of inflammation, which is associated with moderate increases, and hemolysis, which reduces the haptoglobin concentration [3].
• ↑ CRP & ↔ Ferritin (increased CRP and normal levels of ferritin). In canine pyometra there is a significant increase in serum CRP, indicating a severe inflammatory condition. However, serum ferritin, despite being a moderate positive APP, does not show a significant increase. This divergence in the dynamics of these APPs could be a useful tool for suspecting cases of this disease [25].

Conclusion

CRP is a valuable diagnostic tool in routine canine practice and is an excellent assay to incorporate when testing both in disease situations and in routine health screens. It is expected that the information present in this paper will assist practitioners already using CRP to maximize its benefits, and encourage those unfamiliar with CRP to consider its applications [1].