All procedures performed in this study involving animals were in accordance with the ethical standards of the Animal Ethics Committee of Faculty of Veterinary Medicine, University of Zagreb, approval no: 640–01/20–02/09; 251–61-01/139–20-27.
Animals
The dogs enrolled in this study were patients admitted during the morning (9–12 a.m.), on the day the first measurement was performed, at the University Veterinary Hospital at Faculty of Veterinary Medicine in Zagreb. All dogs were chosen randomly and the owners gave a written consent for participation in the study. The dogs were admitted due to various reasons that were not known to the examiners at the time of sampling. History data, physical examination findings, complete blood count and biochemical analysis were noted for all enrolled animals to determine their health status. The analysis of samples was performed in July, September and October 2021.
Sampling
Blood from each dog was collected on the same day in span of 3 hour, after admission, from the cephalic vein into 3 biochemistry 4 mL tubes with clot activator (LT BURNIK d.o.o., Vodice, Slovenia). The samples were left at room temperature until a clot formed after which the serum was separated by centrifugation for 10 minutes at 3500 x g. Immediately after centrifugation the serum was separated into 5 aliquots (1 ml) in micro test tubes. Aliquot’s analysis to determine c-CP concentration was done at T0 (4 hours after serum centrifugation), at T1 (after 8 weeks of storage) and at T2 (after 16 weeks of storage). Serum samples that were analyzed at T1 and T2 were frozen at − 80 °C (±2 °C) upon centrifugation. Prior to being analyzed samples at T1 and T2 were thawed at room temperature (21 °C). All samples were subjected to homogenization by a vortex.
Analysis
Serum concentrations of calprotectin were determined with sandwich enzyme-linked immunosorbent assay (ELISA) for in vitro quantitative measurement of canine calprotectin concentration in serum, plasma and other biological fluids – Canine Calprotectin ELISA Kit CALPRO, RK00520 (AbClonal technology, Woburn, USA). The ELISA assay was done manually according to recommended procedure defined by the manufacturer. The negative and positive controls were included in this ELISA kit. All measurements were done on the same lot of reagents.
All sample analysis for determination of stability were done in duplicates. For determination of within-run precision, the two patients’ serum samples were analyzed in 20 replicates. Imprecision was assessed by analyzing 20 replicates on one plate on two levels of calprotectin: one sample (anticipated low level of calprotectin) from a dog without GI symptoms and one sample (anticipated high level of calprotectin) from a dog with gastric dilation/volvulus in which high concentration of calprotectin was anticipated. The two dogs were chosen amongst 22 animals. Coefficient of variation (CV) was calculated as follow:
$$\begin{array}{c}\text{CV}\;\left(\%\right)=\left(\text{*SD}/\text{mean}\right)\;\text{x}\;100\\\ast SD\;-\;standard\;deviation\end{array}$$
(1)
Manufacturer has declared CV < 10% for the within-run Precision (estimated on 20 replicates at low, middle and high level of calprotectin, on one plate). In this study, it has been used 10% as a performance criterion for imprecision.
The stability was expressed as a percentage difference (PD%) between the baseline results (initial measurement) and results measured at T1 and T2 and was calculated by equation (Eq. 2), for each time point:
$$\textrm{PD}\%=\left(\textrm{T}0\hbox{-} \textrm{TX}\right)/\textrm{T}0\ \textrm{x}\ 100$$
(2)
Deviation from the baseline value was expressed as absolute (μg/L) and relative (%) bias.
Total error (TE) was used to define performance criteria for stability. Calculation of TE was done by Eq. 3:
$$\textrm{TE}=\%\,\textrm{Bias}+\left(1.96\ \textrm{x}\,\%\,\textrm{CV}\right)$$
(3)
The manufacturer did not declare any data about calprotectin stability. Declared accuracy (bias) for serum sample was 9% (manufacturer data).
Statistics
Shapiro-Wilk test was used to test normality of distribution on all data obtained by measuring c-CP with ELISA. All data were distributed normally, and the results were presented as range and median with interquartile range (IQR).
Statistical and exploratory data analyses were performed using R software v.4.0.4 R Core Team (2019). R: A language and environment for statistical computing (R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org/), TIBCO Statistica® 13.3.0 (TIBCO Software Inc., Palo Alto, USA; 2017), Microsoft Excel (Microsoft Corporation, Washington, USA, 2019) and MedCalc® Statistical Software version 20.026 (MedCalc Software Ltd., Ostend, Belgium; https://www.medcalc.org; 2022).