Study population
This multicenter, prospective, observational study in NVAF patients treated with rivaroxaban or apixaban was carried out in the National Heart Center Singapore and Khoo Teck Puat Hospital, and approved by the Domain Specific Review Board (Study reference number: 2017/00815). Patients were recruited between 5 October 2017 and 6 February 2020. After providing their signed informed consent, NVAF patients who were at least 21 years of age and on rivaroxaban or apixaban for at least 3 continuous days were included in the study. Pregnant or breastfeeding women or women who had given birth in the past 90 days and patients enrolled in another drug or device study were excluded. A total of 106 rivaroxaban and 40 apixaban patients were recruited. The choice and dose of DOAC prescription was based on their physician’s discretion.
Baseline characteristics including gender, ethnicity, age, height, weight, smoking history, alcohol consumption, serum creatinine, co-morbidities and concomitant medications were recorded. Body mass index (BMI) and CHA2DS2-VASc scores were computed based on the collected records. Creatinine clearance (CrCl) was estimated via the Cockcroft-Gault equation [19]. Patients were followed up by phone at week 2, months 1, 3, 6 and 12. At each check-point, patients were interviewed on compliance and clotting or bleeding events.
Determination of drug plasma concentrations
Trough plasma samples were obtained at 24 hours or 12 hours after last administered dose of rivaroxaban or apixaban respectively. Peak plasma samples were obtained on the same day of trough plasma samples, at 3 hours after intake of medication with food.
Plasma concentrations for rivaroxaban and apixaban were measured using high performance liquid chromatography-mass spectrometry (HPLC-MS). The lower limits of quantification (LLOQ) and upper limits of quantification (ULOQ) were 5 ng/mL and 1000 ng/mL for rivaroxaban, and 1 ng/mL and 500 ng/mL for apixaban.
Measured plasma concentrations below the LLOQs of the analytical methods were set to half of the LLOQ (LLOQ/2) of the respective analytical methods
Determination of clinical endpoints of bleeding and SSE
Bleeding events were categorized into major and minor bleeding, by the International Society on Thrombosis and Haemostasis (ISTH) criteria [20]. Major bleeding was defined as (1) fatal bleeding, or (2) bleeding in a critical area or organ, for example intracranial, intraspinal, intraocular, retroperitoneal, intra-articular, pericardial, or intramuscular with compartment syndrome, or (3) bleeding causing a fall in hemoglobin level of ≥ 20 g L− 1 (1.24 mmol L− 1) or leading to transfusion of ≥ 2 units of whole blood or red cells. Minor bleeding was defined as any overt bleeding that did not fall under the ISTH major bleeding criteria.
Major bleeding and SSE events were adjudicated by the study team comprising a cardiologist and a cardiology specialist pharmacist based on doctors’ diagnoses obtained from electronic medical records (EMRs). Minor bleeding events were likewise obtained from EMRs or based on clinical signs and symptoms self-reported by patients during the follow-up phone interviews.
Statistical analysis
For the descriptive analysis, Shapiro-Wilk test was used to determine normality of the variables for n < 50, while Kolmogorov-Smirnov test was used to determine normality of the variables for n ≥ 50. Normally distributed continuous variables were reported as mean and standard deviation (SD). Ordinal variables and non-normally distributed continuous variables were reported as median and inter-quartile range (IQR) values. Categorical variables were reported as frequencies and percentages.
For the analysis for plasma concentrations and bleeding events, patients were categorized into four classes (Class I, II, III and IV) based on equal quartiles of the populations’ range of plasma concentrations adapted from Testa et al [12, 13], with Class I corresponding to the group of patients whom plasma concentrations fall within the lowest quartile and Class IV corresponding to the group of patients whom plasma concentrations fall within the highest quartile.
Statistical tests were performed using the Independent-sample’s t-test for normally distributed continuous variables, Mann-Whitney U test for ordinal variables and non-normally distributed continuous variables, and Chi-square test or Fisher’s exact test for categorical variables, to identify predictors for bleeding and SSE events.
Univariable logistic regression was performed to estimate relative risk for bleeding and SSE. Results were reported as odds ratio (OR) and 95% confidence interval (CI). Multivariable logistic regression was performed for covariates with a p-value < 0.1 from the univariable analysis to adjust for confounders.
Statistical analyses were performed with the IBM SPSS Statistics 27 for Windows. A p-value of < 0.05 was considered to be statistically significant.