A new method for anti‐negative interference of calcium dobesilate in serum creatinine enzymatic analysis

Abstract Background Serum creatinine is a widely used biomarker for evaluating renal function. Sarcosine oxidase enzymatic (SOE) analysis is currently the most widely used method for the detection of creatinine. This method was negatively interfered with by calcium dobesilate, causing pseudo‐reduced results. The aim of this study was to explore a new method to alleviate the negative interference of this drug on creatinine detection. Method We formulated eight drug concentrations and 12 creatinine concentrations from serum. The SOE method, the new method, and the Jaffe method were used for detection in five systems. Creatinine biases were analyzed under the conditions with or without the interference of calcium dobesilate, at consistent or inconsistent creatinine concentrations. Creatinine concentrations were also analyzed at three medical decision levels (MDLs). Results Calcium dobesilate had negative interference in creatinine SOE analysis. With the increase in calcium dobesilate concentrations, the negative bias increases. The new BG method showed an anti‐negative interference effect. In the Roche system, the BG method reduced the negative bias from −71.11% to −16.7%. In the Abbott system, bias was reduced from −45.15% to −2.74%. In the Beckman system, the bias was reduced from −65.36% to −7.58%. In the Siemens system, the bias was reduced from −58.62% to −7.58%. In the Mindray system, the bias was reduced from −36.29% to −6.84%. Conclusion The new method alleviated the negative interference of calcium dobesilate in creatinine SOE detection. The negative bias could be reduced from −60% or −70% to less than −20%.


| Instruments and reagents
Three different reagents were used to analyze creatinine concentration in five automatic biochemical analyzers in local laboratories: Tables S1-S5. The new BG reagent and APA reagent were supplied by the company of Zhongshan BGH Biochem. Co., Ltd, with complete technical support. The new BG reagent was subjected to stability analysis before the experiment. Calcium dobesilate powder was obtained from Meryer Chemical Technology Co., Ltd. (Lot NO: 76827018). For instrument parameter settings, see Tables S1-S5.  11 In order to ensure the reliability of the test data, the five tests were all carried out under the condition of internal quality control. All the prepared specimens were divided into five aliquots and frozen at −80°C before the test. All tests were repeated twice to get the average.

| Preparation of calcium dobesilate in eight concentrations
Eight concentrations of calcium dobesilate were determined according to the pharmacokinetics: 0, 2, 4, 8, 16, 24, 32, 48, and 64 μg/ml. 12 Concentration preparation method was as follows: The serum in each concentration of creatinine was first prepared to contain calcium dobesilate 65 μg/ml, and then, the concentration of 65 μg/ml was diluted with serum without calcium dobesilate to obtain other concentrations. In people, after a single oral 500 mg calcium dobesilate administration, after 3-6 h, blood drug concentration can reach the peak concentration of about 12.83-23.15 μg/ml and was maintained for 10 h. 13 In human body, the trough drug concentration is 2.66-8.33 μg/ml, after which it slowly decreases and is undetectable after 24-h administration. It was mainly excreted in urine and feces in the form of prototype, F I G U R E 1 New BG reagent showed an anti-negative interference effect on creatinine analysis in the Roche system. Three different reagents (system-matched SOE reagent, new enzymatic BG reagent, and Alkaline picric acid [APA] reagent) were employed to detect creatinine concentration with the interference of calcium dobesilate at various concentrations ranging from 2 to 64 μg/ml.

| Statistical analysis
All statistical analyses were performed using GraphPad Prism 8.0 software. It has been reported that calcium dobesilate does not interfere with the results of creatinine detection by analysis of alkaline picrate method, and the APA method can be used as an internal control for anti-interference effect. The percentage deviations (y-axis) were calculated based on the concentration of the drug-free specimen without calcium dobesilate interference and were plotted vs.
the calcium dobesilate concentrations (x-axis) or vs. the creatinine concentrations (x-axis).

| Calcium dobesilate has obvious negative interference in creatinine enzymatic analysis in five auto-biochemical analysis systems
In every concentration, creatinine was negatively affected by calcium dobesilate in all five systems. In the Roche system, with the increase in concentrations of calcium dobesilate, the negative interference became more and more obvious ( Figure 1). Interestingly, in a chart, with increased creatinine concentration, the interference effect decreased in the SOE method; that is, when creatinine concentration goes up, the negative interference by calcium dobesilate goes down. In the Roche system, negative interference bias ranged from −1.85% to −71.11%. The same situation was also observed in the Abbott system (Figure 2), where bias ranged from −0.41% to −45.15%; in the Beckman system ( Figure S1), bias ranged from −1.54% to −64.78%; in the Siemens system ( Figure S2), bias ranged from −0.67% to −58.62%; and in the Mindray system ( Figure S3), bias ranged from −0.20% to −39%, revealing relatively lower negative interference compared with other systems.

| New BG enzymatic method showed a superior advantage in anti-negative interference of calcium dobesilate in five systems
In five systems, we analyzed creatinine concentrations by using the BG method to compare with the original SOE method (matching

| DISCUSS ION
A number of previous studies have reported on calcium dobesilate's interference with creatinine detection based on the Trinder reaction. 13,[15][16][17] Because this reaction interferes with creatinine quantification, this is very detrimental to the evaluation of renal function, and the detection result is lower than the actual result due to negative interference. 15 This can be interpreted as the patient's kidney function is improving or that the medication is responding, thus leading to a clinical misjudgment. Therefore, it is necessary to develop new reagents that can resist the negative interference of calcium dobesilate.
When creatinine concentration was detected by the SOE method, hydrogen peroxide (H 2 O 2 ) promoted 4-amino-aminopyrine (4-AAP) reaction to produce quinoneimine chromogenic agent. The color intensity of the resulting quinoneimine chromogen is proportional to the creatinine concentration. However, as H 2 O 2 is a strong oxidant in this process, it can be easily interfered by strong reducing substances. Calcium dobesilate is a potent reducing drug, which reduces H 2 O 2 and causes a reduction in color-developing substances, which, in turn, leads to the pseudo results and the formation of negative interference. This is an essential factor in the results of all Trinder's chemical reaction patterns. In collaboration with F I G U R E 2 New BG reagent showed an anti-negative interference effect on creatinine analysis in the Abbott system. Three different reagents (system-matched SOE reagent, new enzymatic BG reagent, and alkaline picric acid [APA] reagent) were employed to detect creatinine concentration with the interference of calcium dobesilate at various concentrations ranging from 2 to 64 μg/ml.  Consequently, this new method was not suitable for Roche cobas c701 when calcium dobesilate was above 32 μg/ml. In response to this phenomenon, we consulted some application engineers who suggested that this may be due to parameter settings, when performing BG new reagent testing on Roche Cobas 701, the first absorbance reading dot maybe should not be set to the 19th point; the Roche kit is the first calculated absorbance value read at the 22nd point to eliminate some background interference (Table S1). As can be seen from Figure 3, the anti-negative interference effect was effective when the calcium dobesilate was in low concentration. We wondered whether there were not enough anti-interference substances. Accordingly, we further explored the concentration of laccase to test whether increasing the amount of it would work in the item, but cannot cover all kinds of drugs. When judging test results, clinicians need to analyze the patient's condition from many aspects, and it is necessary to integrate multiple items of the same clinical significance for comparison so as to reach a more scientific conclusion. At the same time, due to the differences between the detection systems, it is recommended not to frequently change the detection methods and detection systems when judging the disease or efficacy.

ACK N OWLED G EM ENTS
We wish to thank all the patients for collecting their serum for medical serum tests. We also wish to thank five medical units for their help in this experiment and Zhongshan BGH Biochem. Co., Ltd for supplying calcium dobesilate used to prepare the new antiinterference reagent for this experiment and for providing us with steady technology support in the experiment.

CO N FLI C T O F I NTE R E S T
All authors declare that they have no potential conflicts of interest.
There is no commercial conflict of interest in this article. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

AUTH O R CO NTR I B UTI O N S
Hailan Shen contributed to this research design, data acquisition and interpretation, graph and this article. Kena Chen contributed to this research design, specimen collection, specimen testing, and data analysis. Dr Ju Cao contributed to the conception and design of the research and manuscript review.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data are included in this article.