Ultrasound and biochemical indicators will be used as outcome measures. The primary outcome consists of the total PV and the grey-scale median (GSM), and intima-media thickness (IMT), lipid levels, apolipoprotein A-IV level, platelet count (PLT), fibrinogen (FIB) and platelet aggregation rate (PAR) are the secondary outcomes. Biological specimens will be stored at -80 degrees Celsius after collection. All the biological specimens will be analyzed in the current trial and for future use in ancillary studies
Primary outcome
Vessel plaque quantification (VPQ) is preferred for observing plaque morphological characteristics as a non-invasive assessment method [21]. The vessel plaque quantification technique has a substantial advantage over traditional two-dimensional ultrasound, which has been used for vascular event risk prediction. There are two aspects to be measured by the same sonologist: the total PV of the carotid artery on each side and the grey-scale median (GSM), which can indicate the average hardness of plaques proportionally [22, 23]. Both the PV and GSM will be assessed on weeks 0, 12, and 24 on each side.
Secondary outcomes
Regular carotid ultrasound
For diagnosing atherosclerosis, every participant will be required to undergo regular carotid ultrasound for screening before entry. Intima-media thickness (IMT) will be measured at study entry, exit and follow-up. A prior study has documented that people with higher IMT are more likely to suffer from cardiovascular disease (CVD), especially stroke [24]. Aside from IMT, the maximum thickness of a single plaque, which is calculated by Crouse integration, will be measured throughout the study at each assessment.
Lipid level testing
As a previous study described, dyslipidaemia, such as high TGs and high total cholesterol (TC), usually occurs along with atherosclerotic plaques. Therefore, the modification of blood lipids, including TGs, TC, HDL, and LDL, seems to play a vital role in controlling the progression of atherosclerosis. Patients will undergo three blood tests to estimate their lipid level before and after the treatment, as well as after the follow-up period. The blood sample collection will be performed with the participants in a fasting state before 10 am.
Apolipoprotein A-Ⅳ testing
Apolipoprotein A-Ⅳ is a type of HDL that can activate LCAT and participate in the antidromic transport of TGs. Prior to the work of Ruby Xu et al. [25], apolipoprotein A-Ⅳ was considered a new-found ligand of αIIbβ3 integrin on platelets. In other words, apolipoprotein A-Ⅳ can antagonize the combination between platelets and fibrinogen. Consequently, the study will use apolipoprotein A-Ⅳ as one of the secondary outcomes to determine whether acupuncture could inhibit platelet aggregation by improving apolipoprotein A-Ⅳ levels in the blood.
Coagulation testing
Several coagulation-related measures are also involved in this protocol; these measures consist of platelet count (PLT), fibrinogen (FIB) and PAR. Thus far, platelet aggregation and hypercoagulability are regarded as the initial factors of atherothrombosis that can ultimately cause myocardial infarction or cerebrovascular accidents.
Sample size
This is a three-arm study. In our search for previous studies of the effects of acupuncture on carotid atherosclerotic plaque, it was found that most of the studies were two-arm studies of acupuncture versus drugs. There is a lack of high-quality clinical research. In addition, the effect of acupuncture exhibits a significant correlation between acupoints. Therefore, it is not appropriate to use the previous acupuncture clinical research literature data to estimate the sample size. Furthermore, this is a pilot study to guide future randomized controlled trials and to test the feasibility of the protocol. Considering that there are many clinical blood tests in the study, we will choose the clinical minimum sample size of 30 individuals for each group to reduce the burden of detection of the subjects. The maximum dropout rate within the intervention is expected to be approximately 15%. The total number of patients needed to be randomized is therefore 105 (35 for each group).
Safety assessment
Acupuncture may cause some adverse reactions, such as bleeding, redness, fainting, and pain. All adverse events need to be recorded in common reporting format (CRF) tables. Adverse event records should include time, symptoms, extent, and their relationship with acupuncture. The investigator should handle and record adverse events. Serious adverse events should be reported to the Guangdong Provincial Hospital Ethics Committee. Participants who are unable to continue participating in the study due to adverse events will be excluded.
Data collection and management
This study will collect population data from eligible participants before the first treatment; these data include name, age, duration, occupation, blood pressure, sex, and body mass index (BMI). Drug and acupuncture adverse reactions will be recorded during the treatment and follow-up periods based on the reaction of the participants. At weeks 0, 12, and 24, data on carotid ultrasound, blood lipids, platelet aggregation, fibrinogen, platelet count, and apolipoprotein A-IV will be collected. The data collection will be performed by three staff members who will not take part in acupuncture treatment, index evaluation, statistical analysis, or grouping. The data collection and entry will be performed independently by two of the staff members and finalized by a third staff member. The main investigators, acupuncturist, sonographers, and laboratory staff will not be involved in data collection.
All data must be saved in paper with identification codes in seal, while electronic data is saved on the ResMan Research Manager of the Clinical Trial Management Public Platform, and photo formats are also named with identification codes to ensure authenticity and integrity. All data must be saved in paper, electronic and photo formats to ensure authenticity and integrity. The data should be kept for at least 5 years after the article is published. A data monitoring committee was also set up in this study, composed of experts with good clinical research experience in the Department of Acupuncture of Guangdong Provincial Hospital of Traditional Chinese Medicine. Experts will regularly monitor research progress, data, participant management, distribution, and more. The committee is independent from the fund sponsor and has no conflicts of interest.
Quality control
Before the study, all researchers will be required to accept uniform training. Researchers should be fully aware of the purpose and composition of this study. All acupuncturists will be required to have obtained a licence and have worked for more than 3 years. Before the study, acupuncturists will be trained to clarify the acupuncture standard in this study. During the study, the main investigator will check the CRF every week. The grouping, data collation, and data analysis will be carried out by specific staff members, and there will be no work intersect between these members. The adverse events that might occur during the study will be recorded in detail. The participants’ personal data will be kept by the researcher, and no one other than the researcher is allowed to access the data.
Statistical analysis
This study will use per-protocol (PP) and intention-to-treat (ITT) analysis principles. ITT will include all subjects who participated in at least 1 treatment, including participants who dropped out. Missing data for participants who dropped out will be supplemented with the most recent data. The PP principle will analyse all participants’ data that conform to the study protocol, and those of individuals who did not conform to the protocol will not be included.
The data will be analysed by professional statisticians with SPSS 20.0 software (IBM SPSS Statistics, IBM Crop, Somers, New York, USA). First, we will describe the baseline data, such as age, blood pressure, course of disease, BMI, and sex, and compare the baseline data between the three groups. The continuous variables will be presented as the mean ± standard deviation (SD), and the categorical variables will be presented as the composition ratio and rate.
Continuous variables that conform to a normal distribution will be analysed by ANOVA, and the non-conforming data will be analysed using the rank sum test. Categorical variables will be analysed using the chi-square (χ2) test or Fisher’s exact test. The confidence interval (CI) will be estimated at 95%, and the significance level will be set at 0.05. A P value༜0.05 will be considered statistically significant. The adverse data will be recorded and analysed for the relationship with acupuncture. We will calculate the drop-out rate and analyse the reasons for dropping out.
Ethics and dissemination
The study was planned in accordance with the Declaration of Helsinki. This study was approved by the Institutional Ethics Committee of Guangdong Provincial Hospital of Traditional Chinese Medicine (approval NO. YF2018-107-01). The trial has been registered at the Chinese Clinical Trial Registry (ChiCTR1800019259). If there is any change to the clinical research protocol and informed consent, the researchers will be asked to report the modifications to both ethics committee and registry centre timely.
Before the participants are enrolled in the study, the researchers will fully inform the patient of the treatment and tests that need to be completed and fully inform the participant regarding the risks that may exist in the study and their power to withdraw from the study. Patients are also informed enough to understand the possible adverse reactions of collecting biological specimens. All treatments and tests in this study will be provided for free. Throughout the research process, we will provide free medical advice and guidance to all participants. After all follow-ups, we will provide 3 months of free acupuncture treatment for participants in the SA group. The participants will also be paid for part of their travel expenses to and from the hospital, all of which will be offered to enhance participant compliance.
Patients and public involvement
An acupuncture patient originally proposed acupuncture treatment for carotid atherosclerotic plaque. None of the other participants in the study participated in the design or evaluation. The burden of the intervention will be assessed by the patients themselves. The results will be disseminated to study participants via email or phone messages.
Trial status
The trial is currently in the recruitment phase. The protocol is registered on November1st, 2018. The protocol number is ChiCTR1800019259. The recruitment started on July1st, 2018 and as of October 2019, a total of 42 people had participated in the study. The expected date of trial completion is 30 June 2020.