Performance evaluation of a canine C-reactive protein flow-immunosensor measurement system for clinical application

Abstract

We investigated the analytical performance and robustness of a flow-type immunosensor (FIS)-based canine C-reactive protein (CRP) measurement system against analytical interferents. To improve the clinical applicability of the canine FIS CRP measurement system, we reduced the measurement time to 9 min. Statistical analyses, including scatter plots, Bland–Altman plots, and Passing–Bablok regression analyses, were performed to evaluate agreement with a comparative method (particle-enhanced turbidimetric immunoassay). Within-run imprecision (10 replicates) was 8.2% and 6.4% at ~39 and 115 mg/L; between-run imprecision (20 measurement days) was 9.9% and 6.5% at ~39 and 115 mg/L, respectively. The lower limit of quantification in the FIS measurement system was 4.0 mg/L, and quantification linearity was confirmed from 4.0 to 300 mg/L. CRP content was measured in canine heparinized plasma samples (n = 43) using both the comparative method (x) and the FIS (y). The regression equation for our new method (y) as a function of the reference method (x) was y = 0.979x + 0.592 (R² = 0.974). The system was robust against typical interfering components, including hemoglobin, lipids, and bilirubin. The FIS method was not suitable for K₂EDTA plasma. Based on between-run imprecision (20 measurement days) and concentration-specific targeted bias derived from Passing–Bablok regression, the observed total error was 20.3% and 14.6% at ~39 and 115 mg/L, respectively, meeting the American Society for Veterinary Clinical Pathology desirable and minimum performance goals, as well as optimal performance at the higher concentration level.

Keywords

acute-phase proteins C-reactive protein clinical test flow immunosensor

C-reactive protein (CRP) is an acute-phase protein that is frequently used as an inflammatory marker in humans and dogs.^2,4,6,15,19 CRP is a serum protein, with a molecular weight of ~115 kDa in dogs, produced in the liver in response to inflammatory cytokines.²¹ CRP concentrations in healthy dogs are generally low. Using an assay (Point strip canine CRP assay; Ushio Europe) specifically developed for canine CRP measurement, the CRP concentrations in most healthy dogs were <5 mg/L.¹⁴ The RI for canine CRP measured using a particle-enhanced turbidimetric immunoassay (PETIA) reagent (Canine CRP reagent kit; Gentian Diagnostics), which we adopted as the comparative method in our study, has been reported as ≤6.8 mg/L in most healthy dogs.¹¹ In dogs with inflammatory conditions, CRP concentrations measured using the Gentian immunoassay were 907 ± 859 nmol/L.⁹ Based on the molecular weight of canine CRP of ~115 kDa, this corresponds to 104 ± 99 mg/L, reflecting substantial increases during acute inflammation. When inflammation subsides, the blood CRP content decreases rapidly.

The precise half-life of CRP in dogs has not been well established. However, in serial monitoring of dogs with gastrointestinal infections undergoing hospitalization and treatment, CRP concentrations declined to near baseline within a few days as successful treatment progressed.²² Similarly, in dogs with the systemic inflammatory response syndrome, CRP concentrations decreased over the course of hospitalization in patients who responded favorably to treatment.²³ Therefore, a canine plasma CRP measurement system is considered to require a lower limit of quantification (LLOQ) of ~5 mg/L and an upper LOQ of ~300 mg/L. Nonetheless, CRP measurement methods in veterinary medicine are not standardized, and various CRP measurement devices and reagents are used.^12,13,20,24 In many CRP measurement methods, CRP is detected using an antigen–antibody reaction. In large-scale laboratory facilities, measurements are carried out using immunoturbidimetric reagents on automated biochemical analyzers. This method has a high degree of precision and is one of the most reliable routine measurement methods.¹² Other small devices using immunonephelometric and immunoturbidimetric methods are in operation in small- and medium-sized facilities; however, their measurement precision is inferior to that of automated biochemical analyzers.¹⁷

We aimed to develop a compact CRP measurement system with the same analytical performance as an automated biochemical analyzer and an immunoturbidimetric reagent system, which would be sufficiently compact to be installed in small- and medium-sized hospitals. We reported previously¹⁶ the construction of a new system for canine CRP measurement using a flow-type immunosensor (FIS). Although this system performed as well as an automated biochemical analyzer and immunoturbidimetric reagents, it required >1 h for measurement, making it less practical for routine testing. In clinical settings, rapid reporting of CRP test results is desired. According to the manufacturers’ information, the Dri-Chem canine CRP method (Dri-Chem Slide vc-CRP-P; Fujifilm) has a measurement time of ~5 min per sample,⁷ and a PETIA reagent (the Gentian canine CRP immunoassay), run as an open-channel assay on routine clinical chemistry analyzers, can be performed in ~10 min using major clinical chemistry analyzers.⁸

We used a canine CRP measurement system with a FIS developed in a previous study¹⁷ to evaluate the impact on the precision of the analysis of reducing the measurement time to <10 min. We also evaluated the robustness of the system against anticoagulants and interfering substances, such as hemoglobin (Hb), lipids, and bilirubin, which are often problematic in clinical testing.

Materials and methods

Structure and principles of the FIS system

The FIS (DXS-610; Seeds Tec) that we used was originally developed for quantitative measurement of trace amounts of polychlorinated biphenyls in insulating oil. We have adapted this platform for clinical application to quantify canine CRP in plasma samples.¹⁶ The system consists of 3 main components: 1) a sample introduction module, 2) a column cartridge containing CRP antibody-immobilized beads, and 3) an optical detection module that measures fluorescence intensity generated by the binding of CRP in the sample to the fluorescently labeled antibody. In our previous study,¹⁶ 2 μL of plasma was incubated with 2,000 μL of fluorescent antibody solution at room temperature for 60 min. The mixture was then passed through the CRP antibody-immobilized column, where the CRP-antibody complex was captured. Unbound fluorescent antibody was washed off, and the bound fluorescence was detected, which is proportional to the CRP concentration. Each column cartridge is designed for 10 measurements and can be replaced by trained personnel in a clinical setting. Calibration curves were generated using standard solutions before sample measurement to convert fluorescence signals to CRP concentrations.

Analytical conditions for the improved FIS system

FIS analysis was performed under conditions modified from our previous system.¹⁶ The reaction time and temperature were changed from 60 min at room temperature (20°C) to 5 min at 25°C. Preliminary experiments were conducted to optimize these conditions: a 300 mg/L in-house CRP standard solution was reacted for 60 min at 4, 25, and 37°C, and 25°C was selected as a practical operating temperature. For reaction time optimization, 2 standard concentrations (100 and 300 mg/L) were measured in 5-min intervals for up to 60 min; the proportional relationship between concentrations was maintained even at 5 min. Based on these studies, subsequent measurements were performed at 25°C for 5 min. The complete assay can be finished within 10 min, including the reaction between the fluorescent antibody and the plasma sample.

Calibration curves

Calibration curves were prepared using a canine CRP standard solution (Gentian).¹⁶ Each 2 μL of calibrator solution (0, 10, 30, 78, 155, and 310 mg/L) was mixed with 2,000 μL of fluorescein-labeled anti-canine CRP antibody solution in a microtube and incubated for 5 min at 25°C. Subsequently, 200 μL of this sample was introduced into the FIS (DXS-610), and the fluorescence intensity was measured. For calibration, each standard concentration was measured 3 times, and the mean value was used to construct the calibration curve, with the canine CRP concentration on the x-axis and the fluorescence intensity on the y-axis.

Quality control

For QC of the FIS analysis, we used 3 samples: the 10 mg/L Gentian canine CRP standard solution used for calibration curve preparation, and 2 in-house precision evaluation samples designated as level 1 (~40 mg/L) and level 2 (~115 mg/L). The in-house precision evaluation samples were prepared from pooled canine heparinized plasma. The nominal CRP concentrations for levels 1 and 2 were assigned based on a single measurement using the comparative method, consisting of an automated clinical chemistry analyzer (Model 3100; Hitachi High-Tech) and a PETIA reagent (canine CRP reagent kit; Gentian). The in-house precision evaluation samples were aliquoted into screwcap microtubes and stored at −30°C. For performance monitoring during the study, acceptance limits were defined as the $\bar{x}$ ± 2 SD based on 10 replicate FIS measurements after the first calibration. Before measurement, each aliquot was thawed and used, and no aliquot was used across multiple days or refrozen. QC measurements were performed at the start of each measurement day. The procedure for preparing the analytical samples was the same as that used for the calibration.

After replacing the CRP antibody-immobilized column cartridge, only the 10 mg/L Gentian calibrator was measured. If any measurement fell outside the predefined acceptable range, the column was replaced, and recalibration was conducted if necessary. During our 1-mo study period, no recalibration was required.

Canine plasma samples

Canine plasma was collected from residual samples at the Veterinary Clinical Laboratory (Dobutsu Kensa, Kanagawa, Japan) and Biomedical Science Examination and Research Center (Okayama University of Science, Ehime, Japan). Individual identification was not available because we did not access patient information. Plasma from blood samples containing lithium heparin or sodium citrate as anticoagulants was separated at the local clinic through centrifugation at 1,700 × g for 10 min and 2,000 × g for 10 min, respectively. Blood samples containing K₂EDTA were transported to the laboratory as whole blood. All samples were maintained at 4°C during transport. The requested clinical tests were conducted immediately. After the tests, the K₂EDTA-blood was centrifuged (1,700 × g, 10 min) to obtain plasma. Residual plasma was frozen at −30°C on the same day. For FIS analysis, the samples were thawed immediately before measurement. We used plasma stored frozen for <6 mo. According to previous studies,¹¹ up to 4 freeze-thaws do not affect canine CRP levels.

Patient health information was not accessible; however, all samples were submitted by primary or secondary care veterinary hospitals and therefore originated from dogs that may have had clinical conditions. We collected no additional blood from animals specifically for research purposes. Therefore, our study was exempt from the ethical considerations of the Ethics Committee on Clinical Research of the Okayama University of Science (2020-0007).

Precision of the FIS system

We evaluated the analytical precision of the FIS system using 2 in-house precision evaluation samples, levels 1 and 2. The procedure for preparing the analytical samples was the same as that used for the calibrators. We performed 10 consecutive measurements on the same day under identical conditions to evaluate the within-run imprecision (repeatability) of the FIS system. According to American Society for Veterinary Clinical Pathology (ASVCP) guidelines, 20 consecutive measurements are generally recommended to assess within-run imprecision. However, we replaced the CRP antibody-immobilized bead cartridge every 10 measurements, limiting the evaluation of repeatability to 10 consecutive runs under the same conditions. To evaluate the between-run imprecision, measurements were performed for 20 d. According to the ASVCP total tolerable error guidelines, 10 traditional quality specifications based on biological variation for canine CRP include an optimal analytical CV (CVopt) of 6.08% and a desirable analytical CV (CVdes) of 12.2%. We assessed precision based on these criteria.

Quantifiable range

The LLOQ was determined based on the CV% of repeated low-concentration CRP standard solution measurements. A precision profile diagram was constructed to evaluate the reproducibility of measurements. The ASVCP guidelines recommend replicate measurements at each concentration; we performed 3 replicates.³ The LLOQ was defined as the lowest concentration at which the CV% of repeated measurements was ≤10%.

We assessed the upper limit of the reportable range by evaluating linearity using serial dilutions of high-concentration heparin plasma samples (300 mg/L as determined by the Gentian canine CRP reagent). Each dilution was measured in triplicate, and the mean concentration was used for analysis. Linearity was evaluated using Passing–Bablok regression analysis, with theoretical concentrations on the x-axis and measured concentrations on the y-axis. The slope, intercept, and coefficient of determination (R²) were used to assess agreement with the expected linear relationship (slope = 1, intercept = 0). Residuals were examined to ensure that no systematic deviations were apparent. All analyses were performed using validation support software (JSCC; Japan Society for Clinical Chemistry).

Comparison of methods between the FIS system and the reference method

The relationship between the FIS measurements and the comparative method was assessed with canine heparin plasma samples (n = 43), using Passing–Bablok regression analysis and Bland–Altman difference plot analysis. Frozen plasma samples were thawed and immediately used for FIS and comparative analysis on the same day. The R² of the regression was calculated to quantify the degree of fit between measured and predicted values. Residuals from the regression were examined to detect any systematic or proportional bias.

Bland–Altman plots were constructed (Excel for Mac v.16.65; Microsoft), with the x-axis as the mean of FIS and comparative method measurements and the y-axis as the percentage differences between the 2 methods, calculated as: ([FIS – comparative]/mean of FIS and comparative) × 100%. These plots were used to evaluate the agreement between the 2 methods. To verify the validity of the 95% limits of agreement (LOAs), the differences between the FIS and comparative method measurements were tested for normality using the Shapiro–Wilk test. Because the differences were not normally distributed (p ≤ 0.05), the 95% LOAs were calculated non-parametrically using the 2.5th and 97.5th percentiles of the differences. The slope and intercept of the regression, along with their 95% CIs calculated using the bootstrap method, were reported to quantify the precision and reliability of the regression estimates. Regression analyses were performed using Validation Support/Excel v.64 (Special Committee on Quality Control, JSCC).

Total observed error

Analytical performance of the FIS system was evaluated using observed total error (TEobs), which was calculated as: TEobs (%) = |targeted bias (%)| + 2 × CV (%). For TEobs calculation, between-run imprecision (20 measurement days) of the 2 in-house precision evaluation samples (levels 1 and 2) was used as the CV. Concentration-specific targeted bias at each concentration level (levels 1 and 2) was estimated from the Passing–Bablok regression equation (x, comparative method; y, FIS) by back-calculating the predicted comparative value x_pred = (y – intercept)/slope and computing targeted bias (%) = (y – x_pred)/x_pred × 100%. TEobs values were compared with ASVCP performance goals (optimal, desirable, and minimum total errors) for canine CRP (TEopt = 14.8%, TEdes = 29.6%, and TEmin = 44.4%, respectively) to assess whether analytical performance was acceptable.¹⁰

Interference of blood components

The potential interference of Hb, unconjugated bilirubin (Bil-F), conjugated bilirubin (Bil-C), and lipids on CRP measurement using the FIS was evaluated. Interference Check A Plus (Sysmex) was used, and test solutions were prepared in accordance with the manufacturer’s instructions. We used pooled heparinized plasma from multiple dogs, with a CRP concentration of 33 mg/L. The concentrations of each interfering substance were set according to the kit manual in 5 steps as follows: Hb, 980, 1,960, 2,940, 3,920, and 4,900 mg/L; Bil-F, 42, 84, 125, 167, and 209 mg/L; Bil-C, 40, 80, 121, 161, and 201 mg/L; and lipids, 302, 604, 906, 1,208, and 1,510 formazin turbidity units (FTUs). Test solutions were prepared by adding the stock solution of the interfering substance to the pooled plasma, followed by stepwise dilution using the same pooled plasma. Untreated plasma served as the baseline sample. Although the ASVCP guidelines recommend using multiple individual samples (e.g., n = 5) to enhance the generalizability of interference testing,³ our use of pooled plasma provided reasonable estimation of the interference effects. Each concentration of the added interferent was measured once, and interference was calculated as the percentage bias between the test and baseline samples:

\begin{matrix} i n t e r f e r e n c e e f f e c t (%) = [\frac{(t e s t v a l u e - b a s e l i n e v a l u e)}{b a s e l i n e v a l u e}] \\ \times 100 % \end{matrix}

We deemed the interference to be acceptable if it was below the recommended optimal total error of canine CRP, which is 10%.¹⁰

Interference by blood anticoagulants

The FIS system is primarily designed for use with heparinized plasma, the standard sample type for canine biochemical testing in Japan. However, evaluation of EDTA and citrate plasma was performed to assess the feasibility of using these sample types when heparin plasma is unavailable because of limited blood volume. For the lithium heparin addition experiment, pooled heparinized plasma was used; for the K₂EDTA addition experiment, pooled EDTA plasma was used; and for the sodium citrate addition experiment, pooled sodium citrate plasma was used. The CRP concentration in each pooled plasma was ~30 mg/L. The concentrations of each anticoagulant were set based on standard clinical usage, with 3 stepwise levels corresponding to 2×, 4×, and 8× the baseline plasma concentration. The baseline concentrations in the respective pooled plasmas were 5 U/mL for heparin, 1.5 mg/mL for EDTA, and 0.32% for sodium citrate. Test solutions were prepared by spiking the respective stock solutions into the pooled plasma, followed by stepwise dilution using the same pooled plasma. Pooled plasma without additional anticoagulant served as the control. Each concentration of the added anticoagulant was measured once; the values of the original pooled plasma were used as a reference, and deviations from the measured values of <10% were assessed as having no effect. In companion animal veterinary practice in Japan, plasma is generally preferred over serum for routine biochemical testing. Therefore, our evaluations were based on plasma samples.

Results

Calibration curves

A calibration curve was generated from the fluorescence intensities of standard solutions (0, 10, 30, 78, 155, and 310 mg/L; Fig. 1 ). Calibration was performed at the beginning of the study, and the 10 mg/L standard was measured after each column replacement to verify the stability of the system. All measurements consistently fell within the predefined acceptance range, and no recalibration was required during our 1-mo study.

Figure 1.

Calibration curve of canine C-reactive protein (CRP) using a flow-type immuno sensor.

Quality control

During our study, we monitored the performance of the FIS system according to the protocol described above using 3 QC samples. These included the Gentian 10 mg/L calibrator and 2 in-house precision evaluation samples, level 1 (~40 mg/L) and level 2 (~115 mg/L). QC measurements were performed at the start of each day or following column replacement, as appropriate. The within-run measurements (10 replicates) obtained during initial calibration yielded $\bar{x}$ CRP concentrations (±SD) of 10.7 ± 1.2 mg/L for the 10 mg/L calibrator, 39.2 ± 3.2 mg/L for level 1 sample, and 115 ± 7 mg/L for level 2 sample. Over the 1-mo course of our study, the between-run imprecision, expressed as CV%, was 12.4% for the 10 mg/L calibrator, 9.9% for level 1 sample, and 6.5% for the level 2 sample. All QC measurements remained within the predefined acceptance limits ( $\bar{x}$ ± 2 SD) throughout our study period, and no recalibration was required.

Precision of the FIS system

Analytical precision was evaluated using the same in-house precision evaluation samples as for QC (levels 1 and 2), allowing a consistent assessment across within-run and between-run measurements. Within-run imprecision, assessed over 10 replicates, yielded CVs of 8.2% for level 1 and 6.4% for level 2 ( $\bar{x}$ ± SD; 39.2 ± 3.2 mg/L and 115 ± 7 mg/L, respectively). Between-run imprecision over 20 measurement days was 9.9% for level 1 and 6.5% for level 2 ( $\bar{x}$ ± SD; 39.4 ± 3.9 mg/L and 118 ± 8 mg/L, respectively).

Quantifiable range

The CV% of the measured value was within 10% for CRP solutions >4.0 mg/L ( Fig. 2 ). Therefore, the LLOQ of this measurement system was 4.0 mg/L.

Figure 2.

Precision profile diagram of canine C-reactive protein (CRP) quantification values in the low concentration range.

Canine CRP samples at 4.0, 8.0, 10, 30, 80, 150, and 300 mg/L were measured in triplicate, and the mean values were plotted against theoretical concentrations ( Fig. 3 ). Passing–Bablok regression analysis yielded a slope of 0.986, an intercept of 0.816, and R² of 1.00, indicating excellent linearity across the measured range. Residuals were randomly distributed, with no apparent systematic or proportional deviations. Our results confirm that the FIS-based canine CRP measurement system offers accurate and linear quantification over the range of 4.0–300 mg/L.

Figure 3.

Linearity of quantitative canine C-reactive protein in the flow-type immunosensor.

Comparison of methods between the FIS system and the reference method

Scatter plots of CRP concentrations in canine heparin plasma (n = 43) measured by the comparative (x-axis) and FIS (y-axis) methods were in strong agreement ( Fig. 4A ). Passing-Bablok regression analysis yielded the equation: y = 0.979x + 0.592 (R² = 0.974), with 95% CI (0.918–1.01) for the slope and (–0.853 to 2.51) for the intercept, which included the ideal values of 1 for the slope and 0 for the intercept, indicating minimal proportional and systematic bias.

Figure 4.

Relationship between quantitative canine C-reactive protein (CRP) values in the comparative method and flow-type immunosensor (FIS). A. Scatterplots comparing assay system (x) and FIS (y) measurements using autoanalyzer and immunoturbidimetric reagents (n = 43). The regression equation of the relationship between the comparative method (x) and the FIS method (y) measurements was y = 0.979x + 0.592 (R² = 0.974). B. Bland–Altman plot of canine CRP measurements by FIS versus the comparative method. The y-axis is the percentage difference between the 2 methods, calculated as: ([FIS − comparative]/mean of FIS and comparative) × 100, and the x-axis is the mean of the 2 measurements. The central line indicates the mean difference (bias), and the dashed lines are the 95% limits of agreement (–22.2 to 17.8%).

In the Bland–Altman plots, the percentage difference between the 2 methods ([FIS − comparative]/mean × 100%) tended to increase slightly at higher mean CRP concentrations ( Fig. 4B ). Differences between methods were assessed for normality using the Shapiro–Wilk test (p ≤ 0.05), confirming a non-normal distribution. Accordingly, the 95% LOAs were calculated using a non-parametric approach, yielding a mean difference (bias) of −2.52 mg/L (95% LOAs: −26.8 to 24.2 mg/L). When expressed as a percentage of the mean concentration, the bias was −2.17% (95% LOAs: −22.2 to 17.8%).

Regression analysis of the Bland–Altman residuals was performed to assess fixed (systematic) and proportional bias. The intercept was 1.54% (p = 0.58), and the slope was −0.046 (p = 0.07), indicating no statistically significant fixed or proportional bias.

Total observed error

TEobs (%) was calculated as: |targeted bias (%)| + 2 × CV (%). Between-run imprecision (20 measurement days) of the in-house precision evaluation samples (levels 1 and 2) was used as the CV. Concentration-specific targeted bias was estimated from the Passing-Bablok regression equation (x, comparative method; y, FIS). Targeted bias was −0.5% at 39.2 mg/L (level 1) and −1.6% at 115 mg/L (level 2), yielding TEobs values of 20.3% (0.5 + 2 × 9.9) and 14.6% (1.6 + 2 × 6.5), respectively. According to ASVCP performance goals for canine CRP (TEopt = 14.8%, TEdes = 29.6%, TEmin = 44.4%), both TEobs values met TEdes and TEmin; the higher-level TEobs also met TEopt. Relative to the ASVCP goals, TEobs corresponded to 137%, 69%, and 46% at ~39 mg/L, and 99%, 49%, and 33% at ~115 mg/L (TEopt, TEdes, and TEmin, respectively).

Interference of blood components

The interference of adding Bil-F, Bil-C, lipids, and Hb on CRP measurements in dogs was <10%, up to the maximum concentration of each interfering component; therefore, the effects of all tested interferents at all tested concentrations were acceptable ( Fig. 5 ).

Figure 5.

Effects of interfering measurement components in blood measurements: A. unconjugated bilirubin; B. conjugated bilirubin; C. lipids (formazin turbidity units, FTU); D. hemoglobin.

Interference by blood anticoagulants

Heparin and sodium citrate did not significantly affect the CRP measurements in dogs, with effects <10% even when the maximum concentration was added; K₂EDTA caused differences ≤30% ( Fig. 6 ).

Figure 6.

Effects of interfering blood anticoagulants: A. lithium heparin; B. sodium citrate; C. K₂EDTA.

Discussion

The within-run imprecision (CV%) was 8.2% for the level 1 in-house precision evaluation sample (~40 mg/L) and 6.4% for the level 2 sample (~115 mg/L). For between-run imprecision over 20 d, the CV% values were 9.9% for level 1 and 6.5% for level 2 in-house precision evaluation samples. In comparison, a study using a 60-min reaction time reported within-run CVs of 1.8% and 2.4% and between-run imprecision of 3.6% and 4.4% for the corresponding concentrations,¹⁶ indicating that shortening the reaction time to 5 min resulted in higher variability, particularly at lower concentrations.

According to the ASVCP total allowable error guidelines, the recommended CVopt is 6.08%, and the recommended CVdes is 12.2% for traditional quality specifications based on biological variation in canine CRP content.¹⁰ The within-run and between-run imprecisions of our in-house precision evaluation samples exceeded the CVopt (6.08%) for both level 1 and level 2 samples; however, they remained within the CVdes range (12.2%).

The within-run precision of the FIS system for level 1 samples (~40 mg/L) was 8.2%, which is higher than that for level 2 samples (~115 mg/L; CV = 6.4%). The increase in CV% at lower CRP concentrations is a known phenomenon, as reported in studies of various canine CRP measurement systems.^1,5,13 For example, low- and middle-concentration CV% values for several point-of-care testing devices and autoanalyzer systems have been reported as follows: 5.4–21.8% for the Point strip canine CRP assay (5–50 mg/L),¹⁴ 5.7 and 6.4% for the Idexx Catalyst CRP (37.1 and 49.1 mg/L, respectively),⁵ 4.6% for the Fuji DriChem CRP (52.7 mg/L),¹ and 7.1% for the Randox canine CRP (7.7 mg/L).¹ Compared with these reported data, the FIS system had comparable precision in the low- and middle-concentration range. Furthermore, the analytical performance in the low concentration range is supported by the LLOQ of 4.0 mg/L determined from the CV of repeated measurements. Thus, the observed increase in CV% for level 1 samples remains within a range considered acceptable for clinical interpretation. In addition, the TEobs for levels 1 and 2 samples fell within ASVCP performance goals (TEopt = 14.8%, TEdes = 29.6%, TEmin = 44.4%),¹⁰ supporting the conclusion that the FIS system has analytically acceptable performance versus the comparative method across the clinically relevant concentration range. Therefore, although the CV% is higher at lower CRP concentrations, this does not pose a significant problem for clinical use.

The canine CRP LLOQ in the measurement system constructed in our study was 4.0 mg/L, comparable to the performance of a 60-min reaction FIS system (3.9 mg/L).¹⁶ We confirmed linearity across the range of 4.0–300 mg/L, allowing reliable CRP quantification within the clinically relevant concentrations for dogs as reported in prior studies.^9,13

Interference by blood components, with the addition of up to 20.9 mg/dL Bil-F, 20.1 mg/dL Bil-C, 1,510 FTU lipids, and 490 mg/dL Hb, did not significantly affect the CRP measurements (<10%). Similarly, interference from anticoagulants was not significant up to concentrations of 40 U/mL lithium heparin and 2.56% sodium citrate. However, K₂EDTA at low concentrations affected the measurement and is therefore unsuitable for use with FIS. Hb, bilirubin, and lipids have been reported to cause errors in some solid-phase sandwich immunoassay CRP measurement methods.¹⁸ Conversely, studies have shown that when using an analytical system that combines a biochemical autoanalyzer and the Gentian canine CRP PETIA reagent, the presence of color or turbidity caused by these substances at high concentrations does not affect the measured values.^12,13 Only a limited number of studies have evaluated the effects of interfering substances in blood samples on canine CRP measurement systems, making it difficult to compare the performance of our FIS method with that of other methods. Nonetheless, the performance of the FIS system may be similar to that of the automated analyzer equipped with the Gentian canine CRP PETIA reagent used as the comparative method in our study.

The FIS system and the conventional comparative method were in good overall agreement across the measured CRP range in canine heparin plasma. Analysis using Passing–Bablok regression indicated minimal proportional and systematic bias, supporting the reliability of the FIS measurements. Bland–Altman analysis confirmed that the differences between the 2 methods were largely random. The mean difference (bias) was −2.52 mg/L (95% LOAs: −26.8 to 24.2 mg/L), corresponding to a −2.17% bias (95% LOAs: −22.2 to 17.8%) relative to the mean CRP concentration. Regression analysis of the Bland–Altman residuals found no statistically significant fixed (intercept = 1.54%; p = 0.58) or proportional (slope = –0.046; p = 0.07) bias. These findings suggest that the FIS system may offer a practical approach for CRP measurement in routine clinical settings, with minor deviations unlikely to influence clinical interpretation in a meaningful way.

Using between-run imprecision and concentration-specific targeted bias derived from Passing–Bablok regression, TEobs was 20.3% at 39.2 mg/L and 14.6% at 115 mg/L, meeting the ASVCP performance goals for TEdes and TEmin and, at the higher level, TEopt. Given these results, the FIS system had analytically acceptable agreement with the comparative method across the clinically relevant concentration range.

Our study has some limitations. First, although we included a comparison with a conventional immunoturbidimetric method, recovery experiments to evaluate potential systematic errors caused by matrix components (spike-and-recovery tests) were not performed. The good agreement observed between the FIS and comparative methods across the clinically relevant concentration range suggests that such experiments were not essential for the purpose of our study. Second, RIs for canine CRP were not established because patient health information was not accessible for the anonymized residual plasma samples used in our study. These limitations should be addressed in future studies to further validate the method.

Regarding clinical application, the FIS system is compact and capable of providing results in <10 min. However, its proper use requires personnel with adequate knowledge of QC and clinical laboratory procedures. Sample preparation involves manual handling, requiring an accurate pipetting technique, and users should understand concepts such as device calibration and QC measurements. These requirements, however, are standard competencies expected of veterinary clinical laboratory staff and are not specific to the FIS system. Therefore, when operated by trained personnel, the FIS system can offer rapid and reliable CRP measurements in canine clinical settings, including small- and medium-sized veterinary hospitals. If the FIS system becomes commercially available, having dedicated calibrators together with standardized verification materials would facilitate routine performance monitoring and harmonized implementation across sites.

Footnotes

Declaration of conflicting interests

Noboru Fujitani received research funding from Seeds Tec; Hideki Toita runs Seeds Tec; Norio Tateishi is an employee of Seeds Tec; and Nobutoshi Kanaki runs Doubutsu Kensa.

Funding

This study was funded by Seeds Tec.

ORCID iD

Akihisa Hata

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