Anticoagulant effects of different calcium supplementation pathways in calcium-free RCA-CVVH: a study protocol for a prospective randomized cross-over trial

Background When regional citrate anticoagulation used in continuous renal replacement therapy, one of the key aspects to achieve safe and effective extracorporeal circulation is the management of calcium ions. For calcium-free RCA-CVVH, the anticoagulant effects of different calcium supplementation pathways have not yet been explored. In this trial, we would test our hypothesis that compared with the SCV, when calcium was infused through the VL-FV, the arterial iCa2+ was lower. Methods This is a prospective randomized cross-over trial involving 24 patients undergoing RCA-CVVH. The patients were randomly divided into two groups: VL-FV—SCV group and SCV—VL-FV group. The difference of iCa2+ between arterial iCa2+ and post-ltration iCa2+ was compared. Secondary indicators included the incidence rates of catheter dysfunction and hypocalcemia. Discussion This is the rst trial on the anticoagulant effects of calcium-free RCA-CVVH through different calcium supplement routes. We will conrm that the arterial iCa2 + level is slightly lower when calcium is infused in the VL-FV than in the SCV, and the incidence rates of catheter dysfunction and hypocalcemia will help us to determine which site is safer.


Background
Anticoagulation in vitro is an important part of continuous renal replacement therapy (CRRT). The commonly anticoagulants in clinic are heparin, low molecular weight heparin and citrate, etc. In recent years, a good deal of studies has con rmed that citrate has the advantages of good tolerance, low risk of bleeding and good biocompatibility, as well as no effect on hemo ltration. In 2012, KDIGO recommended citrate as the rst choice of anticoagulant agent in renal replacement therapy for acute kidney injury (AKI) [1] , and since then, regional citrate anticoagulation (RCA) has been widely used in clinic.
When RCA used in CRRT (Referred to as RCA-CRRT), one of the key aspects to achieve safe and effective extracorporeal circulation is the management of calcium ions. The combination of citrate and ionized calcium inhibits the coagulation process, which can achieve regional anticoagulation during the extracorporeal circulation of RCA. When the blood returns to the body, part of the citrate is removed by the lter in the form of calcium citrate chelate, and the remaining part of the systemic circulation is metabolized by the tricarboxylic acid cycle, at the same time , the calcium ions is released [2] . In a sense, if we want to ensure normal coagulation function in the body, you must supplement some of the calcium removed by the lter.
In the process of CRRT, as long as enough calcium is added before blood transfused back into the body, the coagulation function of patients will not be affected. Meanwhile, the aim of local anticoagulation in vitro can also be achieved.
Due to the different types of dialysate and replacement uid, the pathway of calcium supplementation is also different in clinic. A number of related foreign studies [3][4] have shown that the appropriate ratio of calcium-containing dialysate and/or replacement uid can be su cient to achieve effective and safe anticoagulation and calcium supplementation, and to meet the requirements of calcium balance without additional calcium supplementation. For calcium-free RCA, additional calcium supplements are required.
There are generally two ways to do this: the deep vein and the venous line [5][6] . A common clinical problem is how to choose between the two ways when calcium supplementation is given. According to the process of blood circulation, extracorporeal anticoagulation mechanism of RCA and the characteristics of blood lter catheters, etc., the two pathways are reasonable and feasible in clinic.
However, whether it has effects on pre-ltration calcium ion concentration (iCa 2+ ), post-ltration iCa 2+ or the arterial iCa 2 + , whether it has effects on the rate of calcium supplementation and the regulation of citrate, which is still blind spots without support for relevant data in actual clinical work.
The purpose of this study is: (1) To explore the difference in anticoagulant effects of two different calcium supplementation pathways in calcium-free RCA-CRRT; (2)To explore the safety of the two pathways to increase the evidence for individualized calcium supplementation, so that clinical staff can exibly adjust the calcium supplementation rate in actual work according to different calcium supplementation pathways.

Study design
This is a prospective, single-center, randomized cross-over trial. This study was approved by Medical Ethics Committee of the First Hospital of Lanzhou University (LDYYLL2018-165) and registered at chictr.org.cn (ChiCTR1800020046). The trial is still being conducted at the First Hospital of Lanzhou University. The study protocol is reported according to the Standard Protocol Items: Recommendations for Interventional Trials guidelines ( Figure 1). In addition, the nal report will adhere to the Consolidated Standards of Reported Trials (CONSORT) statement.

Study population
Adults patients (age≥18 years), who are received RCA-CVVH (Continuous venous-venous hemo ltration) with hemo ltration catheter punctured in femoral vein (FV). The exclusion criteria are patient refusal; patients with intravenous calcium or oral calcium before enrollment; patients with hyperkalemia (iCa 2+ >1.35mmol/L) before enrollment.

Ethics, consent, and permission
This study follows the tenets of the Declaration of Helsinki, which was approved by Medical Ethics Committee of the First Hospital of Lanzhou University, and written informed consent will be obtained from all participants.

Randomization
The trial follows the CONSORT 2010 ow chart form randomization (Fig.2). A randomization code was generated with a computerized system by the nurse who is not involved with this trial. When eligible patients enter this trial, the nurse will notify the group allocation to investigators. Based on the different calcium supplementation pathways, patients will be randomly assigned to two groups (Group A: VL-FV-SCV (Subclavian vein) group; Group B: SCV-VL-FV group) with 1:1 ratio. . The initial calcium infusion rate is set following the doctors' advice and adjusted to achieve the arterial iCa 2+ at 1.0mmol/L 1.2mmol/L after each iCa 2+ measurement by 1-2ml/h (in accordance with clinical experience and without a pre-speci ed scheme). Relevant parameters (including blood ow, transmembrane pressure (TMP), ultra ltration rate (UFR), replacement uid rate, dosage of citrate, etc.) and initial arterial iCa 2+ will be recorded at the beginning of CRRT.

Calcium supplementation procedure
When CVVH is started, arterial iCa 2+ and post-ltration iCa 2+ will be measured every 1-2h. The calcium infusion rate will be adjusted to achieve the desired iCa 2+ target range. Ca 2+ is monitored until its level became stable and then researchers continuously check the arterial iCa 2+ three times under the rate remaining unchanged. At that moment, researchers will record the rate of calcium infusion and the speci c time. After that, the calcium supplementation pathway should be changed from the design (Group A is to the SCV; Group B is to the venous line) holding the previous rate. After 1 hour, arterial iCa 2+ and post-ltration iCa 2+ are measured again. If phosphate and carbonate drugs are required in intravenous infusion during RCA-CVVH, they are stipulated to be transfused through peripheral veins.

Withdrawal, dropout, and discontinuation
If participants voluntarily withdraw their informed consent at any time during the study, they will be withdrawn from the trial and exclude from data analysis. The trial will be discontinued if the following events occur during the procedure: the patient dies; the patient gets better and CRRT will be stopped; the patient gives up the treatment; the patient goes out for examination; the treatment is be compelled to be quitted due to the equipment performance.

Data collection
Demographic data will be collected before CVVH. The data collected includes age, sex, diagnosis, indication for RRT, APACHE II score, mechanical ventilation. CVVH relevant parameters will also be collected, which contains blood ow, TMP, PA, PV, UFR, dosage of citrate. As the main measurement index, arterial iCa 2+ and post-ltration iCa 2+ will be measured by blood gas analyzer, of which the blood sample size is 1mL. The PH value, K + , tHbc and iCa 2+ are also recorded at the same time.

Data management
The numbered data are double-input by EpiData3.1 software and managed by Excel software. During the trial, the personal information and related information of all the subjects are kept secret, which are only used for the statistical analysis of this study.

Statistical analysis
Analyses will be performed with SPSS 22.0. Calcium concentration, the main evaluating indicator, will be expressed as mean ± standard deviation or median (interquartile range) and be compared to the twostage cross-over ANOVA or Wilcoxon signed-rank test based on results of the Shapiro-Wilk test. All categorical variables will be expressed as numbers or percent and be analyzed with the chi-square test.
Multivariate linear regression will be used to analyze the in uence factors of iCa 2+ , in which backward stepwise regression is used to screen variables, =0.05 =0.10. All statistical tests are conducted by bilateral test, and P≤0.05 is considered signi cant.

Discussion
This is the rst prospective trial on the anticoagulant effects of different calcium supplementation pathways in calcium-free RCA-CVVH. All patients will use double-lumen hemo ltration catheters.
In the process of CRRT, the combination of ionized calcium and citrate, infused into arterial line, inhibits the coagulation process through reducing the local free calcium, which can achieve regional anticoagulation during the extracorporeal circulation. When the blood returns to the body, part of the citrate is removed by the lter membrane with the action of convection and diffusion in the form of calcium citrate chelate, and the remaining entering the systemic circulation is metabolized into carbon dioxide and water by the tricarboxylic acid cycle in mitochondria of the liver, muscle tissue and kidney, at the same time, the calcium ions is released [7] . In a sense, if we want to ensure normal coagulation function in the body, you must supplement some of the calcium removed by the lter. In the process of CRRT, as long as enough calcium is added before blood transfused back into the body, the coagulation function of patients will not be affected. Meanwhile, the aim of local anticoagulation in vitro can also be achieved.
For patients receiving RCA-CVVH with double-lumen hemo ltration catheters, additional calcium supplements are required to ensure the safe and effective extracorporeal circulation. There are generally two ways in clinic: the deep vein and the venous line. According to the process of blood circulation, extracorporeal anticoagulation mechanism of RCA and the characteristics of blood lter catheters, etc., the two pathways are reasonable and feasible in clinic. There is a hole on each line (arterial line and venous line) of the catheter, which are located in the FV together, which could bring on a phenomenon-"recycle" [7] . If the calcium is supplied in the rst way mentioned above, the adjustment of citrate may be affected by the re-entry of iCa 2+ from the venous line into the arterial line to participate in the extracorporeal cyclic process. On the other hand, if the second scheme is implemented, the recycle of iCa 2+ can be reduced appropriately for which uses the other deep veins instead of the venous lines of dialysis catheters. We will hypothesized that compared with the deep vein, when calcium was infused through the venous line, the arterial iCa 2+ was lower. We will test our hypothesis by comparing the before and after effects of calcium supplementation in two different locations (the SCV and the FV-VL).
During RCA-CVVH, poor management of calcium concentration, for instance, insu cient supplementation and citrate accumulation, may result in hypocalcemia during or after dialysis, and serious complications, such as hypocalcemia tetany, hypotension, arrhythmia, and even sudden cardiac arrest and death. On another face, excessive supplementation would lead to hyperkalemia or tissue calcium deposition [8] , the increase of risk of dysfunction of catheter and coagulation of the catheter in vitro. At present, in academic circle, it is generally believed that the condition is feasible and safe when post-ltration iCa 2+ is at 0.25mmol/L 0.45mmol/L and arterial iCa 2+ is at 1.0mmol/L 1.2mmol/L [9][10][11] . We will compare the incidence of catheter dysfunction and hypocalcemia between the two locations in order to explore their clinical signi cance for anticoagulant safety.
In summary, in this randomized cross-over trial, we will hypothesized that compared with the SCV, when calcium was infused through the FV-VL, the arterial iCa 2+ was lower. In addition, comparisons of the incidence of catheter dysfunction and hypocalcemia will help clarify which position is safer and more effective for calcium supplementation of calcium-free RCA-CVVH.