Study design and animals
The study was developed using guidelines for evaluation of clinical chemistry methods [13] and National Committee for Clinical Laboratory Standards (NCCLS) Approved guideline for method comparison and bias estimation [14]. The American Society of Veterinary Clinical Pathology (ASVCP) guidelines: allowable total error guidelines for biochemistry were utilized for bias estimation [15]. Sample collection and animal use was approved by the institutional research ethics review committee of College of veterinary medicine and agriculture (Reference no VM/ERC/09/01/12/2020).
Study animals and area
The animal populations used for conducting the study were male cattle and goats aged greater than one year visiting veterinary clinics from Arba Minch, a city located 500 kilometers south of Addis Ababa. The animals were selected using a simple random sampling method.
Sample size determination
The sample size was determined based on the concepts and practices in the evaluation of clinical chemistry methods [14] and National Committee for Clinical Laboratory Standards (NCCLS) Approved guideline for method comparison and bias estimation using patient samples [15]. It could be evaluated using 40 to 100 samples analyzed using both methods under investigation (two field methods), or using one tested method and a reference method, or using both instruments on the same day over a length of 8 to 20 days (preferably within 4 hours). Accordingly the current study utilized 120 serum samples, 60 from each animal species.
Blood Collection and Processing
5ml blood sample was aseptically collected from jugular vein of cattle and goats by sterile needle of 20 gauge needle using vacationer tubes. After clotting, the serum was separated by low-speed centrifugation at 2500 revolution per minute for 10 minutes. The serum samples that don't have any obvious abnormalities (clots, hemolysis) were transferred into sterile cryogenic vials that bear the species name and number.
Laboratory analysis
Instrumental setup
Spectroscopic analysis was conducted using the instrument EMP-168 semi-automated chemistry analyzer (EMP-168 Chengdu Empsun Medical Technology Co., Ltd., China). The instrument was calibrated using calibrator and quality control samples for normal (N) and pathological (P) were run for validation before running samples for tests. While for refractometry, Portable refractometer with triple scale that give specific gravity, total serum protein and refractive index. The refractometer had a scale interval of 0.2 g/dl and a measuring range of 0 to 12 g/dl. Prior to measuring serum protein, distilled water was used to verify the refractometer's calibration. The interval between the serum protein determinations with the refractometer and the biuret method for each sample was less than two hours and each sample analyzed twice with each method. All measurements were taken at room temperature.
Biuret method
This method is based on colorimetric principle, in which the copper ions from the biuret reagent react with the amide groups from the proteins at strong alkaline pH, creating a violet color. The total protein concentration in the sample was calculated using formula: optical density of sample divided by optical density of standard and the result was multiplied by standard concentration. The standard concentration used was 6 g/dl [16, 17].
Refractometery
This method is based on measurement of refractive index produced by a serum sample due to the combined concentration of all its solute. A drop (10 µl) of serum is used for this measurement, and the angle corresponds to the border line between the dark and the light area, which is measured by the image detector. The measured angle is converted to total serum protein concentration in grams per deciliter (g/dl) [8].
Ethical considerations
The College of Veterinary Medicine and Agriculture at Addis Ababa University's institutional animal research ethical review committee gave its approval for sample collection and animal use (Certificate reference number VM/ERC/09/01/12/2020).
Data management and analysis
The data generated from laboratory investigation was recorded in a Microsoft Excel spreadsheet and analyzed using SPSS V. 20 software. Serum protein concentration was expressed as mean ± standard deviation (g/dl). The statistical differences between both methods were analyzed using a paired t-test and the correlation was determined by Pearson’s correlation test. A P < 0.05 was considered significant. The normality of the data was tested with the Shapiro–Wilk normality test. For both species, scatterplots and Bland-Altman plots were generated. A t-test for paired samples was run to evaluate the significance of differences between total protein values determined by the biuret method and the refractometer for both cattle and goats.