Keywords
direct oral anticoagulants - dabigatran - rivaroxaban - apixaban - serum - methods
Serum samples are taken from all patients in acute and nonacute clinical situations. On adequate handling, clinical chemical analyses are performed on automated clinical chemistry analyzers. Blood coagulation parameters need to be collected into plastic or siliconized glass tubes containing an anticoagulant such as sodium citrate to inhibit blood coagulation in vitro. Limitations of blood drawing for coagulation parameters include incomplete filling of the tube resulting in an incorrect ratio of anticoagulant to blood leading to incorrect coagulation results and activation of blood clotting during and after blood sampling due to incorrect handling. Correct handling and centrifugation at given temperatures, analysis within a given time frame, and specific caution for freezing and thawing of samples are also required.[1]
Serum samples of patients are often used to determine the concentration of drugs during therapy.[2] Drug levels or other clinical chemical parameters may be requested by clinicians hours or days after original blood collection as an add on test. Therefore, serum samples are frequently stored routinely for several days. This becomes important for patients with drug overdose or intoxication or for forensic purposes.[3]
[4] Heparin, low-molecular-weight heparin, fondaparinux, and danaparoid need antithrombin or heparin-cofactor II for accelerating the inhibition of specific coagulation enzymes.[5] A specific determination of hirudins requires activation through meizothrombin.[6] Antithrombin and meizothrombin are consumed during blood clotting as would take place during preparation of serum. Therefore, antithrombin and meizothrombin-dependent anticoagulant drugs may encounter limitations when analyzed from serum samples.
DOACs bind specifically either to thrombin or to factor Xa. These coagulation proteases are also consumed during blood clotting that occurs during preparation of serum samples. DOAC concentrations are determined by liquid chromatography techniques after extensive purification from plasma proteins.[7]
[8]
[9] These methods require specialized techniques and expert operators, which are rarely available in routine laboratories. UV (ultraviolet)-detection photometry analysis provides an easy, less time consuming, and sensitive analysis for pharmaceuticals. Here, we describe a simple and rapid photometric method for the determination of DOAC in serum. We have compared data obtained by spiking DOAC to human serum and plasma samples as well as from patients on treatment.
Methods
Patients
These studies were approved by the local ethics committee and participants gave written informed consent before blood sampling. Serum and plasma samples were taken from patients under treatment with rivaroxaban 10-mg once daily before and between days 4 to 6 following primary elective total knee and hip replacement surgery 12 hours after intake of medicine, from patients with atrial fibrillation on treatment with dabigatran 110- or 150-mg twice daily (2 or 12 hours after intake of medicine) or treated with apixaban 5-mg twice daily 12 hours after intake of the drug and for control studies from patients not taking any anticoagulant.
Preparation of Samples
Serum Samples
Blood was collected from control persons without intake of an anticoagulant and from patients on treatment with one of the DOACs into kaolin containing plastic tubes to generate and obtain serum. Blood was allowed to clot at room temperature for 30 minutes. Samples were centrifuged at 1,800 g and room temperature for 10 minutes and the supernatant was shock frozen in aliquots and stored at –72°C until analyzed.
Plasma Samples
Blood for platelet poor plasma (PPP) samples were taken during the same venipuncture as for those to obtain serum, collected into plastic tubes containing 3.8% sodium citrate (1/9, v/v, citrate/blood), centrifuged within 30 minutes at 1,800 g and room temperature for 10 minutes and several aliquots of the supernatant were shock frozen and kept at −85°C until analyzed.
Origin and Quality of DOACs
Dabigatran and rivaroxaban were purified from commercially available Pradaxa (Boehringer, Ingelheim, Germany) and Xarelto (Bayer HealthCare, Wuppertal, Germany; optical density at 405 nm) and their purity was characterized by analytical methods as described.[10]
[11] Apixaban was provided by BMS (Plainsboro, NJ).
Spiking of Serum and Plasma Samples with DOAC
Serial dilutions of dabigatran, rivaroxaban, and apixaban were added to 6 (rivaroxaban and apixaban) or 4 (dabigatran) individual serum and plasma samples from persons not taking an anticoagulant. Samples were analyzed within 2 hours for the content of the DOAC by the methods described later. The mean of the six determinations was calculated for serum and plasma samples of each DOAC. The concentration of each sample was calculated from the optical density at 405 nm (OD) concentration curve for serum and plasma samples of each DOAC using SAS release 9.3 program (SAS Institute Inc., Cary, NC).
Analysis of Dabigatran
Serum Samples
Dabigatran was determined from serum samples by a colorimetric method[12] (international patent application No PCT/EP2012/002540). The assays were run on a microtiter plate reader with duplicate samples (Multiskan FC, Thermo Fisher Scientific, Langenselbold, Germany) connected to the software program SkanIt 3.1 (Thermo Fisher Scientific, Germany). Initially, the assay was developed by using different amounts of the reagents and incubation periods (data not shown). The final assay procedure was as initially described also for plasma samples.[13] Ten µL serum and 100 µL tris buffer (0.05 mol Tris, 0.075 mol EDTA, 0.175 mol NaCl, 37 KIU aprotinin/mL, pH 8.4) were incubated for 5 minutes followed by addition of 100 µL human thrombin (Sigma Aldrich, Deisenhofen, Germany, 0.74 NIH units/mL tris buffer), for 60 seconds at 37°C. Fifty µL chromogenic substrate S2238 (1.59 mmol/L in distilled water, Chromogenix, Essen, Germany) were added and incubated for 5 minutes. The reaction was stopped by adding 25 µL 20% acetic acid. For all samples, a background OD at 405 nm was used by pipetting the reagents from backward, that is, first acetic acid, to ensure a similar final matrix but with no activity. Absorption of samples was read at 405 nm and the concentration of dabigatran of the samples was calculated from a concentration/optical density curve using the software program and prepared from pooled human serum samples (obtained from 20 volunteers) spiked with dabigatran ranging from 25 to 500 ng/mL.
Plasma Samples
The assay was performed with the same solution and incubation steps as for serum samples using the software program SkanIt 3.1 for Multiskan FC.
Analysis of Rivaroxaban and Apixaban
Serum Samples
Rivaroxaban and apixaban were determined by a colorimetric method[14] in the presence of human factor Xa and a substrate (patent No GB1019674.9). The samples and reagents were pipetted into microtiter plates and after a special time period the results of the reaction were measured in a special reader (Multiskan FC, Thermo Fisher Scientific, Langenselbold, Germany) connected to the software program SkanIt 3.1 (Thermo Fisher Scientific, Germany).
The optimal assay conditions were developed using serial dilutions of the reagents and incubation times (data not shown). The final assay was conducted as follows: 25 µL serum (diluted 1:15 with tris-buffer) and 25 µL factor Xa (7.1 nkat/mL) were incubated for 2 minutes followed by addition of 50 µL chromogenic substrate S2222 (3.37 mmol/L distilled water, Chromogenix, Essen, Germany). After 20 minutes of incubation, the reaction was stopped by adding 25 µL of 20% acetic acid. For all samples, a background OD at 405 nm was used by pipetting the reagents from backward, that is, first acetic acid. Absorption of samples was read at 405 nm and the concentration of rivaroxaban/apixaban of the samples was calculated from a concentration/optical density curve prepared from pooled human serum samples (obtained from 20 volunteers) spiked with rivaroxaban/apixaban using the software program.
Plasma Samples
The antifactor Xa activity was performed with the same assay conditions as described for serum samples to determine rivaroxaban/apixaban and using the software program SkanIt 3.1 for Multiskan FC.
Statistical Analysis
Data are given as mean and standard deviation (SD). Box plots were used for the figures of concentration of DOAC in serum and plasma samples. Differences between groups were calculated using the SAS release 9.3 software program. SAS mixed procedure was used to analyze differences between control and patients groups. The level of significance was determined by the npar one-way procedure. The level of significance was set at p < 0.01.
Results
Spiking of DOAC to Serum and Plasma Samples
Spiking of serial dilutions of the DOAC showed no differences for serum and plasma concentrations for dabigatran (r = 0.9997), rivaroxaban (r = 0.9991), and apixaban (r = 0.9998), respectively ([Fig. 1A–C]).
Fig. 1 Comparative concentrations of direct oral anticoagulants (DOACs) for serum and plasma samples of healthy volunteers (n = 6, mean, SD) spiked with different concentrations of dabigatran (A), rivaroxaban (B), and apixaban (C), as measured by test procedure described in text and OD at 405 nm.
Patients on Treatment with Dabigatran
Patients not on treatment with an anticoagulant displayed serum and plasma concentrations of 16.6 ± 18.7 ng/mL and 8.4 ± 7.7 ng/mL, respectively (p = 0.0001) ([Fig. 2A]). In patients on therapy, the concentrations of dabigatran in serum (n = 219) and plasma (n = 363) were 75.9 ± 70.2 ng/mL and 112.9 ± 85.8 ng/mL, respectively (p = 0.0223) ([Fig. 2B]). The p values for differences of serum samples of control versus patients was 0.0085 and for plasma samples less than 0.0001. The correlation of serum and plasma values was r = 0.165.
Fig. 2 Box plots of concentrations of dabigatran in serum and plasma samples of control persons not treated with an anticoagulant (A), and on treatment with dabigatran (B).
Patients on Treatment with Rivaroxaban
Control patients without intake of an anticoagulant had serum (n = 143) and plasma (n = 144) concentrations for rivaroxaban of 18.0 ± 17.6 ng/mL and 14.5 ± 12.4 ng/mL respectively (p = 0.36) ([Fig. 3A]). In patients on therapy, the concentrations of rivaroxaban in serum (n = 132) and plasma (n = 135) were 81.7 ± 40.3 ng/mL and 66.5 ± 40.7 respectively (p = 0.0003) ([Fig. 3B]). The p values for differences of serum and plasma samples of controls versus patients were both less than 0.0001. Values of serum and plasma samples displayed a correlation of 0.7251.
Fig. 3 Box plots of concentrations of rivaroxaban in serum and plasma samples of control persons not treated with an anticoagulant (A) and of patients on therapy with rivaroxaban (B).
Patients on Treatment with Apixaban
Control patients without intake of an anticoagulant had serum (n = 48) and plasma (n = 48) concentrations for apixaban of 24.4 ± 18.1 ng/mL and 16.8 ± 23.2 ng/mL, respectively (p = 0.0031) ([Fig. 4A]). In patients on therapy, the concentrations of apixaban in serum (n = 58) and plasma (n = 69) were 263.6 ± 141.7 ng/mL and 190.7 ± 82.4 respectively (p = 0.0003) ([Fig. 4B]). The p values for differences of serum and plasma samples of controls versus patients were both less than 0.0001. The correlation of serum and plasma values in patients was r = 0.709.
Fig. 4 Box plots of concentrations of apixaban in serum and plasma samples of control persons not treated with an anticoagulant (A) and of patients treated with apixaban (B).
Discussion
The results of the present investigations demonstrate that dabigatran, rivaroxaban, and apixaban can be determined from serum samples after spiking samples with the DAOC. The correlations of serum and plasma samples were very high and in the same range as reported for other drugs.[15] The correlation of values using serum and plasma samples of patients on treatment was lower but also in the range reported for other drugs.[16] Dabigatran showed lower relation between serum and plasma levels using samples of patients on treatment compared with rivaroxaban or apixaban. Instability of drugs in serum has been reported for other compounds.[17] The reason of a higher variability of serum compared with plasma concentrations of patients on treatment with the thrombin inhibitor dabigatran may be manifold and remain to be investigated.
Methods for determination of drugs in serum samples are required if plasma samples are not available (3). Serum samples are preferred for determination of drugs because of the absence of many proteins that relates mainly to coagulation proteins. Purification of drugs from serum samples is therefore easier to perform.[18] DOACs can also be the determined from serum samples using sophisticated liquid chromatography techniques (7, 8, 9). With these analyses, we demonstrated that DOACs can be determined also by adding an excess of thrombin or factor Xa followed by addition of an enzyme-specific substrate that releases quantitatively a chromophor from the substrate negatively related to the presence of the DOAC (12, 14). These assays can be performed in laboratories equipped with coagulation platforms to determine anticoagulants.
Clinical trials with DOACs were performed without determination of the anticoagulant effect of dabigatran,[19] rivaroxaban,[20] or apixaban.[21] Pharmacokinetic analyses were computed using a population-based model for dabigatran[22]
[23] and rivaroxaban.[24]
[25]
[26] Markers for activation of blood coagulation were reported from a clinical study using apixaban.[27] Few reports were published for small patient groups treated with dabigatran[28]
[29]
[30] or rivaroxaban.[29]
[ 31]
[ 32]
[ 33] The reported values for subjects not on DOACs are in the same range as reported in our patients using plasma and serum samples. For apixaban, similar ranges of steady state plasma concentrations were reported for volunteers as in our patients.[34]
There were some differences between the results of the three DOACs when determined in serum and in plasma samples from patients on treatment. For dabigatran, control values were higher in serum than in plasma samples. In patients, however, dabigatran concentrations were lower in serum than in plasma samples. However, differences were significant between persons not on dabigatran and patients on treatment in both cases. Therefore, patients on treatment can be separated from control values using serum samples as well as plasma samples. One reason for the lower values in serum samples of patients on treatment may that dabigatran is consumed during coagulation of blood after withdrawal to obtain serum. This may explain in part also the low correlation of serum and plasma values in patients on therapy with dabigatran. The mechanism of fibrin formation in serum samples in the presence of dabigatran remains to be investigated.
Values for controls without treatment of rivaroxaban were not different but somewhat higher for apixaban in serum compared with plasma samples. In patients on treatment, serum values were higher for rivaroxaban and apixaban when determined from serum samples compared with plasma samples. The mechanisms of the interaction of these inhibitors on fibrin formation and the difference to dabigatran remain to be determined. Importantly, serum samples are significantly higher in patients on therapy compared with controls and serum and plasma concentrations show a good correlation.
The amount of anticoagulant present in patient may be necessary to be determined in deterioration of renal function, in the elderly or oldest population, for surgery or in acute clinical situations, during recurrent events or during bleeding complications, or to check compliance.[35]
[36] The concentration of DOACs can be accurately determined by high-pressure liquid chromatography methods. These techniques are, however, not suitable for clinical routine use. Thrombin- and factor Xa-dependent coagulation or chromogenic substrate assays are developed to determine the concentration or activity of dabigatran, rivaroxaban, and apixaban.
The limitations of this investigation are that the determination DOAC in serum samples remains to be validated in clinical studies. However, a validation of the plasma concentrations of DOACs also has to be undertaken in relation to the incidences of thrombotic or bleeding events. The time of blood sampling after the intake of the last dose of the anticoagulant plays an important role and remains to be standardized in general. The strength of the investigations is that the feasibility of using serum samples for the determination of DOACs was shown using a simple and reliable method that can be applied to a coagulation platform connected with a photometer.
In conclusion, the determination of dabigatran, rivaroxaban, and apixaban from serum samples of patients may offer an additional tool in specific clinical situations such as lack of withdrawal of plasma samples or facilitated freezing compared with plasma. The suitability of the determination of DOACs from serum samples by other anti-factor Xa chromogenic substrate assays remains to be proven. If this results in positive findings, adaption of the assays to specific coagulation platforms may reduce the variability of the results.[37]
