The effect of different concentrations of heparin sealing liquid on thrombosis after peripherally inserted Central Catheter catheterization in tumor patients (HEALTH): protocol for a prospective, single-blind, randomized, controlled trial

Background: Malignant tumor patients with peripherally inserted central catheter (PICC) are at high risk of developing deep venous thrombosis. Different concentrations of sealing liquid result in different outcomes. However, there is a lack of evidence of large randomized controlled trials to show the different incidences of deep vein thrombosis using different concentrations of sealing liquid. The aim of this study is to compare the difference in the incidence of deep vein thrombosis of the upper extremity in cancer patients with PICC using physiological saline, 10 U/ml heparin, and 50 U/ml heparin, separately. Methods: A three-arm, single-center, single-blind, randomized controlled trial will be performed. We plan to recruit a total of 639 cancer patients within 12 months with a follow-up period of six months. Patients will be randomized at 1:1:1 ratio by centralized service allocation concealment. Sealing liquid with different concentrations, i.e. physiological saline, 10 U/ml heparin, and 50 U/ml heparin will be administered. Primary outcome is the incidence of upper extremity venous thrombosis. Secondary outcomes include the time of thrombus and the severity of thrombosis with three grades. All randomized patients will be analyzed by intention-to-treat. Chi-square test will be used to compare the incidence of upper extremity venous thrombosis. Kaplan-Meier survival curve and log-rank test will be presented to compare thrombus in relation to time. A Cox regression will test the effect of group on thrombus, with hazard ratio adjusted for signicant clinical variables. Discussion: The trial will help to resolve the uncertainty of the clinical practice of PICC catheterization liquid concentration, and to provide the basis for the implementing the clinical intervention and effectively reducing PICC-related venous thrombosis. The present study is ongoing, and data will be published in peer-reviewed journals after its conclusion.


Background
Peripherally inserted Central Catheter (PICC) is a method of catheterization of the peripheral vein (the main vein, the median vein of the elbow, the cephalic vein, etc.), so that the tip of the catheter is located in the superior vena cava [1].PICC was introduced to China in the late 1990s and is now widely used in cancer intravenous chemotherapy, long-term infusion, total parenteral nutrition (TPN), and the construction of nutritional pathways in preterm infants [2][3][4][5].Although the placement of the PICC can produce fewer complications than other central venous catheters, as a foreign body in the venous lumen, PICC also could result in complications, such as phlebitis, infection, catheter blockage and thrombosis.
Among them, catheter-related venous thrombosis is a serious complication [7].PICC-related venous thrombosis refers to the process of forming a blood clot in the inner wall of the vessel and the wall of the catheter where the PICC is located due to multiple factors such as puncture or direct injury of the intima of the vessel and the patient's own state after PICC catheterization [8].The incidence of PICC-related deep venous thrombosis varies greatly depending on the population, screening methods, and catheter type.
The incidence of pulmonary embolism caused by upper extremity venous thrombosis is 35% [10], and the incidence of tumor patients is more than twice that of the general population, reaching 51.4% [11,21,23].
The main risk factors included individual patient factors, catheter placement, and catheter-related factors [11,21].Therefore, how to effectively prevent PICC thrombosis is the focus of clinical nursing work.It is also an urgent problem to be solved.
Sealing is one of the basic conditions to ensure the smoothness of the PICC catheter.When the infusion is completed or is during the intermittent period, the tube should be sealed once a week to ensure smooth drainage.According to the "Intravenous Therapy Technical Practices" implemented in China since May 1, 2014, the maintenance of intravenous catheters requires sealing with the use of a saline or heparin saline, the volume of which should be twice the total volume of a catheter plus an extension tube, and with positive pressure.The concentration of heparin saline can be 100 U/ml in the infusion port, or from 0 to 10 U/ml in PICC and CVC.Since January 2014, we have used 0.9% saline as a sealing liquid for patients with cancer in our center with indwelling three-way membrane PICC.10U/ml heparin saline was used as a sealing liquid for patients with terminal open PICC.The results showed that the incidence of catheterrelated venous thrombosis was higher, at 29.17% [24], which increased the number of hospital stays and treatment costs.Studies of the meta-analysis of the effectiveness and safety of heparin saline and saline have shown that heparin saline was more effective than saline in reducing the rate of venous catheter occlusion and in preventing the formation of venous thrombosis [25], but the concentration of heparin solution was not studied in the research.The study from Yang and colleagues [26] reported that the use of high-concentration heparin saline (heparin 50mg plus saline 4ml) sealed tube can effectively prevent deep vein thrombosis caused by femoral vein catheterization, with no signi cant increase in the bleeding rate.Patients with malignant tumors are hypercoagulable and are at high risk of venous thrombosis.However, there is insu cient evidence on this population.Therefore, it is necessary to evaluate the difference of thrombus formation in PICC tumor patients with different concentrations of heparin sealing liquid, to provide a scienti c basis for the prevention of thrombosis by PICC sealing with the use of sealing liquid in different concentrations.
The current standard for the use of normal saline and heparin concentrations is based on the 2011 edition of the American Infusion Association (INS) guidelines and the Code of Practice for Intravenous Therapy Care Technology, which was implemented on May 1, 2014 in China.Maintenance of the catheter requires the use of normal saline the volume of which is twice the total volume of a catheter plus an extension tube or a 10 U/ML heparin saline positive pressure seal.However, there is no uniform standard in practical applications.At present, the most commonly used methods for sealing liquid concentration in China and other countries have their own advantages and disadvantages.These include (1) saline sealing tube: when sealing the tube solution, it is not restricted by disease type, especially for patients with hemorrhagic tendency, coagulation mechanism disorder and liver and kidney dysfunction.Normal saline, which is now widely used in clinical practice, is a 0.9% sodium chloride solution the osmotic pressure of which is equal to that of the human plasma.However, Jonker et al [27] found that saline as a sealing liquid also has its shortcomings, because physiological saline has no anticoagulant effect, although it can play a role in maintaining catheter patency, it also increases the probability of thrombosis.
(2) Heparin brine seal: Heparin has strong physicochemical properties with strong negative charge, and can effectively resist blood coagulation in vivo and in vitro, mainly through the interaction with antithrombin III (AT-III) in plasma.AT-III is an inhibitor of activated coagulation factors XIIa, XIa, Xa, IXa and thrombin (factor IIa).Heparin can catalyze the interaction of AT-III with many coagulation factors, prevent the aggregation and destruction of the platelet, and impede the formation of Thrombin; prevent prothrombin from becoming thrombin; inhibit thrombin, and prevent brinogen from becoming brin, thereby exerting anticoagulant effects.Because of its strong anti-coagulation effect in vivo and in vitro, and low toxicity, a small amount of heparin solution has no effect on the human body, so it is used to prevent the serious complications of post-tube thrombosis [28].The main adverse reaction of heparin is that excessive use of the drug can cause spontaneous bleeding.The long-term use of heparin by patients with Hepatic insu ciency can cause thrombocytosis tendency due to depletion of antithrombin-III.And the use of heparin is prohibited in Heparin-induced thrombocytopenia (HIT).
Studies in China have shown that different types and concentrations of sealing liquids have different effects in tube formation, catheter patency and thrombosis.A controlled trial conducted by Zhang et al [17] showed that PICC-negative tumor patients were sealed with three concentrations (50, 125, 250 U/ml) of heparin dilution, and the incidence of blockage was 70%, 40%, and 15%, respectively.Two different concentrations and doses of heparin sealing liquid were compared by Yu and colleagues [19].It was found that inpatients who received intravenous indwelling needles were best treated with 50 U/mL heparin solution to maintain venous catheter patency and reduce extubation rate.Xiao et al [20] did a 1:1 parallel randomized trial among patients with PICC-negative nasopharyngeal carcinoma in T3-T4 stage.One group was sealed with 20 ml of normal saline pulse positive pressure, and the other group was sealed with 20 ml of 20 U/mL heparin dilution.There was a signi cant difference in thrombus formation rates between the two groups, with the rate of the heparin group lower than that of the saline group (11.4% vs 48.6%).Zeng et al. conducted a systematic review of the effects of two different sealing liquids in prevention of PICC thrombosis and catheter occlusion in China.The results of ve randomized controlled trials showed that the concentration range was 10 U/ml-125 U/ml for the heparin group.The effect of preventing PICC catheter thrombosis and tube occlusion is better in the group sealing with heparin than the group with sterile saline [22].
However, the literature review from the Cochrane System Review database [14] showed that there was no signi cant statistical difference in the incidence of infection and mortality between adult patients undergoing CVC (central venous catheters) catheterization sealed using 0.9% saline and 0.9% heparin solution.There was consistency in patency rate between Leukemia patients undergoing PICC catheterization in a home environment using a 100 U/ml catheter and a 10 U/ml heparin solution in sealing tube [15].Another RCT study showed that there was also no statistically signi cant difference in the patency rate between hemodialysis patients using 0.9% saline and 1000 IU heparin [16].
In summary, there is a difference among the studies' results from the population and treatment therapies.In China, there is also a lack evidence of large randomized controlled trial to show the difference.This project studies the question existing in the above research in China and abroad.In this study, PIMC patients with malignant tumors would be sealed with different concentrations of sealing liquid in three different groups in a 1:1:1 randomized controlled trial to compare the difference in the incidence of deep vein thrombosis of the upper extremity in tumors patients with PICC using physiological saline, 10 U/ml heparin solution, and 50 U/ml heparin solution.Thrombosis would be screened at different catheter indwelling time, with follow-up observation until extubation, to investigate the exact occurrence and differences of PICC-related upper extremity venous thrombosis in different groups of sealing liquids, and to provide guidance for clinical intervention and effective reduction of thrombosis rate.

Methods/design
Trial design This study is prospective, single-center, single-blind, randomized, 1:1:1 parallel controlled trial, aiming at comparing the difference in the incidence of upper extremity venous thrombosis in tumor patients with PICC using physiological saline, 10 U/ml heparin solution, and 50 U/ml heparin solution.The study will respect the Consolidated Standards of Reporting Trials (CONSORT) as show in the Figure 1

Hypothesis
This study has three research hypotheses: First, there is a statistical signi cance in comparing the difference of the incidence of thrombus between the saline group and the 10 U/ml heparin solution group ( rst main hypothesis).
Second, there is a statistical signi cance in comparing the difference of the incidence of thrombus between the saline group and the 50 U/ml heparin solution group (second main hypothesis).
Third, there is a statistical signi cance in comparing the difference of the incidence of thrombus between the 10 U/ml heparin solution group and the 50 U/ml heparin solution group (third main hypothesis).

Setting and sample (population)
Selection of research subjects About 639 patients with PICC will receive one of the three different concentrations of the sealing liquid in the Department of Oncology, Guangzhou First People's Hospital.The subjects will be randomly assigned in a ratio of 1:1:1 to receive one of the three concentrations of the sealing liquid.The enrollment period was from July 2017 to June 2018, and patients with malignant tumors who were placed in PICC at the First People's Hospital of Guangzhou will be screened.Patients who meet the following inclusion criteria and no exclusion criteria listed in Table 1 will be eligible.Patients who have serious adverse events or unplanned extubation will be exit.All subjects who have signed an informed consent form and screened for entry into the trial have the right to withdraw from the trial at any time.Those who do not complete the observation cycle speci ed by the protocol will be reported.

Informed Consent:
Informed consent forms will be signed by tumor patients who are placed in PICC in the First People's Hospital of Guangzhou and who meet the criteria listed in Table1.

Outcome measures and de nitions Primary outcome
The primary outcome is incidence of upper extremity venous thrombosis (%).The term is de ned as the rate of patients with upper extremity venous thrombosis who were placed in a PICC.The Denominator of the rate is number of cases that have been placed in a PICC per unit time.The Numerator is the number of cases of thrombosis happened in lateral upper limb per unit time.

Secondary outcomes 1. The time of thrombus
The time of thrombus is de ned as the time after replacement the time of diagnosis of thrombosis by color Doppler ultrasound.
2. The severity of thrombosis, including grade I, grade II, and grade III.It is de ned as: 1) Grade I: After the catheter is placed in the venous lumen, there is a small hill-shaped mass echo or a small agglomeration echo outside the catheter, mainly in isolated type.It is examined by color Doppler ow imaging, and the venous blood ow is smoothly presented.There is a 1-30% narrow surface.Under direct vision, it can be found that there are lower echoes in the blood vessels and the wall, and the blood ow is small, and the color blood ow cannot reach full lling.
2) Grade II: Through examination, it is found that there is a thrombus formation around the catheter or in the venous lumen, and there are many places.It is found by CDFI that there is relatively unobstructed venous blood ow, and there is a narrow surface of 31-50% in the cross section of the blood vessel.It can be seen that the amount of echo in the blood vessel and the lower wall is moderate, and the color vessel lling state has a relatively serious defect.
3) Grade III: The thrombus is completely blocked, and the thrombus around the catheter and the venous cavity is more common, mainly of the fusion type.The venous thrombosis is mainly composed of fusion, and is presented in a large piece.The thrombus is basically lled with a large area tube.The cavity, through CDFI examination, found that there is no blood ow signal or a small amount of blood ow signal in the narrow channel, and the cross section of the blood vessel has more than 50% of the narrow surface.Under direct vision, there are more mid-echo and low echo in the blood vessel.The blood cannot present a colored blood ow in the blood vessels and cannot collapse the blood vessel wall.
Adverse Events: The incidence of adverse reactions will be assessed among three groups.Adverse reactions include bleeding and coagulopathy.Adverse reactions were based on activated partial thromboplatin time (aPTT), prothrombin time (PT), brinogen (Fbg), and plasma thrombin time (TT) to assess whether the patient's coagulation function is normal, with or without bleeding tendency.Serious adverse reactions will be monitored and reported to the Ethics Committee in a timely manner.

Recruitment, randomization, allocation concealment and blinding
Since the catheterization was mainly performed during working hours, this study used a full-time research nurse to screen patients.For each patient who receives informed consent, the research nurse will train them and their families to fully understand the research protocol.After the patient has obtained informed consent, the research nurse will contact the randomizer of the trial to obtain a random number to which study group for the patient.The randomization scheme is generated by the SAS statistical analysis software random grouping module, and the random number is stored in an envelope and managed by the research randomizer.Patients will be randomized to a 1:1:1 ratio, 1) saline group, 2) 10 U/ml heparin solution group, 3) 50 U/ml heparin solution group.This study was set up as single blind, i.e. the patient did not know the concentration of the sealing liquid used.Because in the research nurses need to know the concentration of the sealing liquid used during the operation, it is di cult that the nurses are blinded.
But statistician will be blinded.

Procedure
The study includes four stages.They are 1) Pre-screening stage: to make PICC catheterization for tumor patients; 2) Screening period: to con rm whether the subject meets the study quali cation.This stage begins after the signing of the informed consent form (ICF) and continues until the random assignment (up to 2 weeks); 3) Intervention period: from the time of randomization to the occurrence of upper extremity venous thrombosis.Participant will be given different concentrations of sealing liquid to seal the tube; 4) Follow-up period: Start from the rst visit (de ned as the rst day when the subject receives the dispensed sealant); perform a vascular ultrasound every week for the rst month, and thereafter every month A vascular ultrasound is performed until a thrombus occurs or the study is over.In addition, the last vascular ultrasound was performed on the day before extubation of the subject to assess whether the subject developed a thrombus.
Interventions Sealing: In addition to the three concentrations of the three sealing liquids, all the other operations follow the same standardized operating procedures.

1) Time of sealing
After each infusion, the tube is rst ushed and then sealed; in the intermittent period, the tube is ushed and sealed once a week.(1) ush tube with pulse: use 0.9% saline 10-20ml and push-stop-push bolus method, so that the physiological saline could form a vortex in the catheter, and the residual drug in the catheter could be rinsed clean; (2) seal tube with positive pressure: ush tube with 0.9% saline or heparin saline 5ml with positive pressure, when the syringe remains 0.5-1ml sealing liquids, push while pushing the needle until it is completely withdrawn PICC puncture placement and maintenance methods: 1) Each signed an informed consent form before the PICC was placed.
2) Measure the base arm circumference at 10 cm above the elbow joint of the upper limb where the tube was placed, and use the color Doppler ultrasonography to examine the condition of the blood vessel to be punctured, including the diameter, elasticity, valve, blood ow velocity, blood vessel curvature, and vessel wall thickness of the blood vessel.
3) Traditional blind puncture method: the patient takes a supine position, and the upper limb and the trunk are stretched by 90°.Measure the total length from the puncture point to the right sterno-lock joint and then down to the third intercostal space, which is the catheter placement length; disinfect the upper arm skin to establish a maximized sterile area; The assistant assists in preparing tools to be used in the placement of the tube and placing them on the sterile table, pre-ushing the catheter, and preparing tools for the puncturing.The venipuncture was performed at an angle of 15°-30°.After returning blood, the blood vessel was inserted into the puncture sheath in parallel with the blood vessel and the needle core was withdrawn; The catheter with a predetermined length was inserted along the puncture sheath, and the guide wire was removed; the chest piece was taken to determine the position of the catheter.
4) B-ultrasound guided by Seldinger technology: Place a sterile towel to create a maximized sterile area.Apply the aseptic mixture to the B-ultrasound probe with the help of the assistant, and put on the sterile probe cover.Perform the cross-sectional scan under B-ultrasound, and nd the thickest vein in the lower third of the venous side.Note that the vein should be distinguished from the artery, that the vein is easily crushed.Move the probe, place the vein of the cross section in the center of the horizontal scale, select different guide needles according to the depth, put the needle into the guide needle, and repeat the previous positioning.Hold the probe with your left hand, insert the needle with your right hand, and look at the B-ultrasound interface.When you see the needle tip enters the blood vessel, you will see blood ow from the top of the puncture needle.Remove the B-ultrasound, place the guide wire along the puncture needle, and remove the puncture needle when it is placed approximately 20-30 cm.At the puncture point, perform local anesthesia by injecting lidocaine, use the knife to expand the skin.The cutting depth of the cutting knife was determined according to the fatness of the patient.Insert the guiding sheath along the guide wire, then withdraw the guide wire and introduce the sheath core.Insert the PICC into the vein along the guiding sheath to the same length of the preset tube, then remove the guide wire; determine the position of the catheter using the chest piece.
5) Immediately after catheterization, check whether the blood vessels placed in the PICC catheter have thrombosis by performing color Doppler ultrasound, and record relevant information.
6) PICC maintenance: including replacement of the applicator and heparin cap.10ml Pulse tube with positive pressure sealing tube by using a 0.9% injection of physiological saline, 1-2 times a week.
Selection of catheters and Seldinger puncture bags: Catheters: 4Fr single-chamber three-way valve PICC and the open-end high-pressure single-chamber PICC from Bard.Seldinger puncture bags: the MST puncture kit from Bard.

Research equipment:
Ultrasound used in Seldinger technology: U.S. Sonor's L25 two-dimensional real-time portable bedside Doppler color ultrasound diagnostic instrument and Site-Rite*5B ultrasound guidance system produced by Bard.The 5-10MHz broadband probe was used.
Con rmed thrombosis B-ultrasound: IE33 color ultrasonic diagnostic instrument produced by Philips, USA.The probe is 10L.

Data collection and storage
previous report [11] and the preliminary study in our center [24], we assumed that the incidence of upper extremity venous thrombosis was 30-40% in the saline group, 25% in the 10 U/ml heparin solution group, and 15% in the 50 U/ml heparin solution group.To test the rst main hypothesis of the difference of the incidence of thrombus between the saline group and the 10 U/ml heparin solution group, we estimated that we would need a total of 639 participants with 213 at each group to achieve 80% power at a twosided 1.7% (5%/3, Bonferroni method) signi cance level, with an enrolment period of one year and a follow up period of a minimum of half a year, and taking into account the 5% dropout rate.Under these sample size, we have as least 99.99% and 72% power to test the other two hypotheses (second main hypothesis, third main hypothesis, respectively).We did not plan to do an interim analysis.

Study population
All randomised patients will be analysed by intention-to-treat (ITT).Assessment for baseline characteristics will be analysed using full analysis set.E cacy assessment will be analysed by full analysis set and per protocol set.Safety analysis will use safety set including patients receiving PICC in the study.

Statistical analysis
All data analyses will be conducted using SAS 9.4 statistical software.Missing data will not be imputed.
An as-treated analysis will assess the effect of protocol violations.Baseline descriptive characteristics will be presented using means and SD or medians and interquartile range (IQR) for continuous variables and counts/percentages for categorical variables.ANOVA or Kruskal-Wallis test will used to compare continuous variables of baseline characteristics, radiological features and experimental data and multiple comparisons between two groups will be performed using t test or Wilcoxon test.Ordinal categorical data will be compared using Kruskal-Wallis test among three groups and Wilcoxon test between two groups.
Non-ordinal categorical variables will be compared using Chi-square test or Fisher exact test.Primary analysis will be the comparison of incidence of upper extremity venous thrombosis using Chi-square test.Kaplan-Meier survival curves will be presented to compare group failure in relation to dwell time, and the log-rank test will be used to compare the survival curves between study groups.A Cox regression will test the effect of group on failure, with hazard ratios calculated and adjustment for signi cant patient, device and clinical variables.No subgroup analysis and interim analysis will be performed in the data analysis.Due to the short period of trial, we did not set data monitoring committee during the trial procedure.P values <0.05 will be statistically signi cant.The study statistician will be blinded.This RCT study evaluates the difference in thrombus formation between PICC patients with cancer using different concentrations of heparin sealing liquids, which will help to resolve the uncertainty of the clinical practice of PICC catheterization liquid concentration, and to provide the basis for the implementing the clinical intervention and effectively reducing PICC-related venous thrombosis.

Discussion
The study has some limitations.According to the hypothesis that there is a statistically signi cant difference in the incidence of thrombosis between the 50 U/ml heparin solution group and the 10 U/ml heparin solution group, this study shows that only 72% of the tests could detect the difference.In this study, the sample size is large when the rst and second main research hypotheses are met.However, to make the hypothesis achieve more than 80% of the third main hypothesis, a larger sample is needed, and it is hoped that more samples can be collected for veri cation.In addition, the study was set to be singleblind because the nurses were unable to do blindness due to actual operational needs.However, in order to avoid bias, we conducted uniform training for the daily maintenance of the catheter, including the operation procedures such as ushing, sealing, changing dressings and infusion joints.
with the content for the schedule of enrolment, interventions and assessments.The study will also respect the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) as shown in Additional le 1, respectively.

2 ) 2 4)
Sealing methods A. saline sealing: SAS (S, saline; A, administration; S, saline) B. 10 U/ml heparin saline sealing: SASH (S physiological saline; A administration; S physiological saline; H heparin saline) C. 50 U/ml heparin saline: SASH (S saline; A administration; S saline; H heparin saline) 3) Sealing liquid volume Sealing liquid volume = (catheter volume + additional device volume) X Sealing methods: pulsed ush the tube and then seal it with positive pressure After the training, research nurses must pass two tests to do the work.According to the CDC Guidelines for Prevention of Intravascular Catheter-Related Bloodstream Infections, the relevant procedures and guidelines for PICC care have been developed and improved.Finally, this is a single-center study and hopes to be multicentered in the future.Declarations Trial status Patient recruitment is currently underway according to the protocol version 1.0.The study started at 4th Jan 2018 and is expected to end at 1st July 2018.List of abbreviations PICC: peripherally inserted central catheter; TPN: total parenteral nutrition; RCT: randomized controlled trial; INS: American Infusion Association; AT-III: antithrombin III; HIT: Heparin-induced thrombocytopenia; CVC: central venous catheters; CONSORT: Consolidated Standards of Reporting Trials; SPIRIT: Standard Protocol Items: Recommendations for Interventional Trials; ICF: informed consent form; SAS: saline, administration, saline; SASH: physiological saline, administration, physiological saline, heparin saline; WBC: white blood cell; RBC: red blood cell, HGB: hemoglobin; PLT: platelet count; PT: Prothrombin time; aPTT: activated partial thromboplastin time; FDP: brin(-ogen) degradation product; Fbg: brinogen; ECOG: Eastern Cooperative Oncology Group; ITT: intention-to-treat; IQR: interquartile range; TT: plasma thrombin time.
heparin sealing liquids may have different effects on thrombosis in PICC patients with cancer.However, at present, in China it is still controversial about what kind of concentration sealing liquid should be used for PICC catheterized patients.
PICC catheterization is widely used in clinical practice.Catheter-related venous thrombosis is a serious complication.Effective prevention of PICC thrombosis is the focus of clinical nursing work.Sealing is one of the basic conditions to ensure the smoothness of PICC catheters.It has been reported that different concentrations of