Assays for the Measurement of Dabigatran
Assays for the Measurement of Dabigatran
Thirty-five pairs (trough and peak levels) of samples were drawn for each enrolled participant. The laboratory testing matrix included three ECAs on two different analyzers, one dTT assay, one ECT, and one LC/MS-MS method Table 1.
For the chromogenic ecarin assays, Pearson correlation was 0.948, 0.984, and 0.979 for laboratories A, B, and C, respectively Figure 1A, but all sites varied significantly (P < .001) from the predicate BI-MS method. Bland-Altman bias plots demonstrate that most of these result differences are directional, indicating that overall, the ECA method underestimates drug level compared with the BI-MS method Figure 1B. There was no statistical improvement when comparing the range of expected trough dabigatran levels Table 2. When comparing the change in drug concentration between trough and peak drug concentration, only laboratory B demonstrated no significant differences (P = .10) (when compared with changes between drug concentrations as measured by BI-MS). When comparing among laboratories that report the ECA, there were no significant differences between values reported between laboratories A and B (P = .10).
(Enlarge Image)
Figure 1.
A, Regression analysis between laboratories A, B, and C performing the ecarin chromogenic assay (ECA); laboratory D performing dilute thrombin times (dTT); and laboratory E performing liquid chromatography/tandem mass spectrometry (LC-MS/MS) for determining dabigatran levels and predicate measurements using Boehringer-Ingelheim (Ingelheim, Germany) mass spectrometry (BI-MS). Laboratory A = 0.85x + 11.8; R = 0.948. Laboratory B = 0.92x – 2.58; R = 0.984. Laboratory C = 0.78x – 4.16; R = 0.979. Laboratory D = 0.81x – 5.56; R = 0.937. Laboratory E = 1.06x + 8.28; R = 0.977. B-D, An overlay of Bland-Altman–style plots demonstrating the bias between (B) each respective laboratory site's ECA-determined dabigatran concentration and predicate mass spectrometry quantitation of dabigatran concentration, (C) dTT-determined dabigatran concentration and predicate mass spectrometry quantitation of dabigatran concentration, and (D) LC/MS-MS–determined dabigatran concentration and predicate mass spectrometry quantitation of dabigatran concentration.
There was borderline correlation (R = 0.937) and statistically significant differences (P < .001) between the commercial dTT kit (laboratory D) and BI-MS–measured dabigatran levels (Figure 1A). The bias plots indicate that values determined using the dTT method are primarily lower than BI-MS measurements, with increasing bias as drug concentration increases Figure 1C. There was no observed improvement in correlation or drug concentration differences between this dTT and predicate BI-MS when evaluating those samples in the expected trough range of results (~40–240 ng/mL) or the observed changes between trough and peak drug levels (Table 2). Results obtained with the dTT method demonstrated no significant differences (P = .54) between this method and the chromogenic ecarin method for only one of the three laboratories that performed the chromogenic method.
There was acceptable correlation (R = 0.977) for laboratory E using LC/MS-MS compared with the predicate BI-MS values (Figure 1A). Bias plots indicate that the LC/MS-MS method of laboratory E overestimates drug concentration (P < .001) compared with BI-MS Figure 1D. There was no statistical improvement between methods when evaluating drug levels from 40 to 240 ng/mL, but no statistically significant difference between methods (P = .57) was observed when assessing the change between trough and peak drug concentrations (Table 2).
There was acceptable correlation (R = 0.970) between BI-MS and the ECT, but a relatively flat slope Figure 2 was evident. As expected, there were significant differences between BI-MS, which measures drug concentration, and ECT, which merely reports time in seconds. These differences did not improve when evaluating expected trough levels or differences between trough and peak samples. There was good correlation (R = 0.962) but significant differences (P < .001) between dabigatran levels obtained from calibrated ECT and BI-MS measurements. The ECT-dabigatran results correlated with other laboratory methods, with R values ranging from 0.874 (laboratory A) to 0.934 (laboratory E). There were no significant differences between ECT-dabigatran levels and those levels reported by laboratory C using ECA (P = .43) and laboratory D using LC-MS/MS (P = .23).
(Enlarge Image)
Figure 2.
Regression analysis of a single site performing both classic ecarin clotting time (ECT); y = 0.311x + 28.0; R = 0.970) and dabigatran levels obtained from calibrated ECT; y = 0.920x – 25.48; R = 0.962) compared with dabigatran levels measured by Boehringer-Ingelheim (Ingelheim, Germany) mass spectrometry (BI-MS).
Results
Thirty-five pairs (trough and peak levels) of samples were drawn for each enrolled participant. The laboratory testing matrix included three ECAs on two different analyzers, one dTT assay, one ECT, and one LC/MS-MS method Table 1.
For the chromogenic ecarin assays, Pearson correlation was 0.948, 0.984, and 0.979 for laboratories A, B, and C, respectively Figure 1A, but all sites varied significantly (P < .001) from the predicate BI-MS method. Bland-Altman bias plots demonstrate that most of these result differences are directional, indicating that overall, the ECA method underestimates drug level compared with the BI-MS method Figure 1B. There was no statistical improvement when comparing the range of expected trough dabigatran levels Table 2. When comparing the change in drug concentration between trough and peak drug concentration, only laboratory B demonstrated no significant differences (P = .10) (when compared with changes between drug concentrations as measured by BI-MS). When comparing among laboratories that report the ECA, there were no significant differences between values reported between laboratories A and B (P = .10).
(Enlarge Image)
Figure 1.
A, Regression analysis between laboratories A, B, and C performing the ecarin chromogenic assay (ECA); laboratory D performing dilute thrombin times (dTT); and laboratory E performing liquid chromatography/tandem mass spectrometry (LC-MS/MS) for determining dabigatran levels and predicate measurements using Boehringer-Ingelheim (Ingelheim, Germany) mass spectrometry (BI-MS). Laboratory A = 0.85x + 11.8; R = 0.948. Laboratory B = 0.92x – 2.58; R = 0.984. Laboratory C = 0.78x – 4.16; R = 0.979. Laboratory D = 0.81x – 5.56; R = 0.937. Laboratory E = 1.06x + 8.28; R = 0.977. B-D, An overlay of Bland-Altman–style plots demonstrating the bias between (B) each respective laboratory site's ECA-determined dabigatran concentration and predicate mass spectrometry quantitation of dabigatran concentration, (C) dTT-determined dabigatran concentration and predicate mass spectrometry quantitation of dabigatran concentration, and (D) LC/MS-MS–determined dabigatran concentration and predicate mass spectrometry quantitation of dabigatran concentration.
There was borderline correlation (R = 0.937) and statistically significant differences (P < .001) between the commercial dTT kit (laboratory D) and BI-MS–measured dabigatran levels (Figure 1A). The bias plots indicate that values determined using the dTT method are primarily lower than BI-MS measurements, with increasing bias as drug concentration increases Figure 1C. There was no observed improvement in correlation or drug concentration differences between this dTT and predicate BI-MS when evaluating those samples in the expected trough range of results (~40–240 ng/mL) or the observed changes between trough and peak drug levels (Table 2). Results obtained with the dTT method demonstrated no significant differences (P = .54) between this method and the chromogenic ecarin method for only one of the three laboratories that performed the chromogenic method.
There was acceptable correlation (R = 0.977) for laboratory E using LC/MS-MS compared with the predicate BI-MS values (Figure 1A). Bias plots indicate that the LC/MS-MS method of laboratory E overestimates drug concentration (P < .001) compared with BI-MS Figure 1D. There was no statistical improvement between methods when evaluating drug levels from 40 to 240 ng/mL, but no statistically significant difference between methods (P = .57) was observed when assessing the change between trough and peak drug concentrations (Table 2).
There was acceptable correlation (R = 0.970) between BI-MS and the ECT, but a relatively flat slope Figure 2 was evident. As expected, there were significant differences between BI-MS, which measures drug concentration, and ECT, which merely reports time in seconds. These differences did not improve when evaluating expected trough levels or differences between trough and peak samples. There was good correlation (R = 0.962) but significant differences (P < .001) between dabigatran levels obtained from calibrated ECT and BI-MS measurements. The ECT-dabigatran results correlated with other laboratory methods, with R values ranging from 0.874 (laboratory A) to 0.934 (laboratory E). There were no significant differences between ECT-dabigatran levels and those levels reported by laboratory C using ECA (P = .43) and laboratory D using LC-MS/MS (P = .23).
(Enlarge Image)
Figure 2.
Regression analysis of a single site performing both classic ecarin clotting time (ECT); y = 0.311x + 28.0; R = 0.970) and dabigatran levels obtained from calibrated ECT; y = 0.920x – 25.48; R = 0.962) compared with dabigatran levels measured by Boehringer-Ingelheim (Ingelheim, Germany) mass spectrometry (BI-MS).
