C-reactive protein (CRP) is a major acute phase protein and is synthesized by the liver in response to factors related to inflammation. The concentration of serum CRP is elevated up to several hundred folds within 24 ~ 48 hours during various inflammatory conditions like infection, tissue damage, myocardial infarction, pulmonary disorders, panniculitis, acute pancreatitis, polyarthritis, hemangiosarcoma and metabolic syndrome. Measurement of CRP levels is mainly used as a marker of inflammation diagnosis in human medicine^[1-4]. In veterinary medicine, canine CRP is also known to rapidly increase in acute phase response similar to human beings. It has been reported that the level of canine CRP is increased in diverse diseases such as babesiosis, parvovirus infection, tumors, pyometra, and arthritis. So canine CRP test can be used for diagnosis, monitoring, and screening of canine patients, and is especially useful for prognosis of inflammation even when there is no alteration of white blood cell or band neutrophil count^[5]. The detection of canine CRP has been developed various methods as enzyme-linked immunosorbent-assay (ELISA), electro immuno- assay, time-resolved immunofluorometric assay (TR-IFMA) and turbidimetric immunoassay (TIA)^[6,7]. However, these methods require large amounts of blood and time for the assay and may have different results depending on the user’s proficiency. Point-of-care testing (POCT) is a diagnostic test that complements the disadvantages of these conventional methods and it enables real-time measurements at near the site of patients^[8]. Many POCT technologies have been developed for decades and high-quality POCT devices with improved sensitivity and specificity are constantly being released in the market.

  SelexOn™ (Infopia, Anyang, Korea) is POCT analyzer utilizing chromatographic immunoassay to quantitatively or qualitatively detect the concentration of more than 30 cardiac, cancer, thyroid and infection markers in human blood such as cardiac troponin I (cTnI), alpha-fetoprotein (AFP), C-reactive protein (CRP), thyroid-stimulating hormone (TSH)^[9] and has been working authentically for a long time in the field. Recently SelexOn™ canine CRP is developed and it is a quantitative device capable of measuring CRP in dogs. We evaluated the clinical utility of SelexOn™ canine CRP in dogs and compared with Life Assays canine CRP test (Life Assays, Lund, Sweden).

Materials and Methods

Animals and sample collection
  The dogs (n = 200) which were brought to Seoul local veterinary hospital by the client for different diseases or for the health assessments were used in this study. Primarily blood samples were collected in test tube containing EDTA for diagnosis or assessments purpose. Secondarily, the remaining blood was used for this study and the clients were informed before about that. SelexOn™ canine CRP device (Infopia, Korea) was used to evaluate CRP by using blood. Then the remaining blood was centrifuged (3000 RPM for 5 minutes). Then prepared serum was used for CRP analysis by Life Assays canine CRP® test (Life Assays, Sweden). Measurement of CRP concentration was performed within 0 - 3 hours after sampling. The samples were stored either at 4°C or room temperature for the short-term study.

Test devices and protocols
  The following two POCT analyzers were selected for this study: Life Assays Canine CRP® test kit (IDEON Science Park, Lund, Sweden) and SelexOn™ Canine CRP (Infopia, Anyang, Korea). Life Assays Canine CRP® test kit is a latex agglutination test for determination of canine CRP and is measured quantitatively through Life Assays® Vet Reader instrument. According to the Life Assays® manual, the measuring range of canine CRP is 10 – 210 mg/L and the reference range is reported as less than 20 mg/L. SelexOn™ Canine CRP is a sandwich assay using two monoclonal antibodies specific to canine CRP and is quantified by the thickness of the test line depending on the color of gold nanoparticle. EDTA-treated venous whole blood diluted with buffer is injected into the blood inlet area of test strip and then the erythrocyte is separated and only plasma is transferred. Target analyte in plasma binds to gold-nanoparticle labeled antibody and then when gold-nanoparticle labelled antibody-target analyte complexes pass through the test line that is immobilized antibody specific to analyte, these produce visible red lines on nitrocellulose membrane. The optical system of the SelexOn™ meter measures the intensity of red lines and the integrated software converts the signal intensity to a quantitative result and shows it on the display. According to the SelexOn™ manual, the measuring range of canine CRP is 10 – 200 mg/L and the reference range is reported as less than 20 mg/L.

Statistical analysis
  Statistical analysis was done by using the Prism 5.03 software (GraphPad Software Inc., San Diego, CA, USA) for the of the data. Results are expressed as mean ± standard deviation of the mean (SD). The paired Student’s t-test was used and correlated with the Spearman’s rank correlation coefficient. The level of significance was set at p < 0.05.

Results and Discussion

  A total of 200 dogs were included in this study after informing the owners, of these 137 dogs was purebred, including 29 different breeds, and 63 were mixed-breed. 91 of the dogs were male (37 castrated and 54 intact) and 109 of the dogs were female (38 spayed and 71 intact). 183 dogs had been diagnosed with various diseases while 17 of the dogs were healthy. All types of dogs were used for sample collection such as different breeds, sex, disease, or healthy were tested and the results were within expected levels because of the assessed CRP levels in 0 - 220 mg/L^[7].

  All samples were analyzed by SelexOn™ Canine CRP (blood) and Life Assays Canine CRP® (serum) device. The lowest CRP concentration measured by the Life Assays Canine CRP® was 10 mg/L whereas at the same sample it was 11.21 mg/L. The height CRP concentration measured by the Life Assays Canine CRP® was 86 mg/L whereas at the same sample it was 91.12 mg/L. The averages were 21.55 ± 32.53 (SD) mg/L and 22. 46 ± 40.84 (SD) mg/L, respectively (Figure 1). Thus, it is clear that all the data either lower, upper or average values of CRP between these two groups were very close in these two devices.

Figure 1: The average CRP level of SelexOn™ canine CRP instruments compared to the Life Assays (n = 200). The averages were 21.55 ± 32.53 mg/L and 22.46 ± 40.84 mg/L, respectively. The data were expressed by mean ± SD (standard deviation). There is no statistical significant difference between two groups.

  The correlation coefficient (Spearman’s) between the SelexOn™ Canine CRP (blood) and Life Assays Canine CRP® was 0.9380 for the 200 samples that were examined. The calculated association was significant (p < 0.0001) (Figure 2) indicating its accuracy and reliability. In general, the SelexOn™ Canine CRP (blood) assay tended to yield higher CRP concentrations than Life Assays Canine CRP. It’s might be due to sample type, as CRP was measured directly from blood SelexOn™ Canine CRP (blood) assay.

  In addition, CRP was measured directly from blood by SelexOn™ Canine CRP, thus it can save extra 5 minutes which is needed for serum sample preparation for Life Assays Canine CRP. So, using SelexOn™ Canine CRP is convenient, easy and faster than Life Assays Canine CRP. Canine CRP measurement is performing to screen diseases and to monitor the efficacy of anti-inflammatory and anti-infectious drugs and to detect the subclinical immune diseases like the human^[7]. Elevation of CRP level is a specific indication of inflammatory processes than traditional inflammatory markers rather than diagnosis a specific disease. So, a single evaluation of CRP level did not correlate with clinical outcomes and prognosis, for this repeating CRP monitoring is required^[10-12]. In this regards by SelexOn™ Canine CRP is really convenient, friendly and save time as canine CRP can be determined directly from blood not from serum within short time. So, SelexOn™ Canine CRP could help to diagnose or to monitor inflammatory activity during and after treatment by measuring Canine CRP values easily.

Figure 2: Spearman’s rank correlation between SelexOn™ canine CRP instruments compared to the Life Assays of CRP in dogs (n = 200).

  Therefore, SelexOn™ canine CRP device is suitable to use for clinical test and alternatives to traditional laboratory devices for the virtue of its accuracy, time savings, and easiness.

References