Effect of emicizumab on activated clotting time performed on i‐ STAT Alinity analyzer

Emicizumab is a monoclonal bispecific antibody mimicking the activated form of coagulation Factor VIII (FVIII), approved in prophylaxis in severe and moderate hemophilia A (HA). It is administered subcutaneously every 7 or 14 days and allows a constant level of coagulation equivalent to that obtained with 10–20 UI/dL FVIII.1 Emicizumab has previously been shown to interfere with numerous coagulation assays based on the activation of the intrinsic pathway (i.e., activated partial thromboplastin time [aPTT], FVIII coagulometric assay). Despite normal coagulation level being not fully restored, emicizumab strongly shortens aPTT.2 Activated clotting time (ACT) is a whole blood coagulation assay measured on a point of care analyzer in the operating room to monitor unfractionated heparin (UFH) activity in several settings (e.g., cardiopulmonary bypass [CPB] and cardiac catheterization). Like aPTT, ACT is triggered after activation of the contact pathway but unlike aPTT, ACT is not performed in citrated plasma but in native whole blood. Given the similarities between both assays, ACT might be subject to a shortening, potentially leading to an underestimation of heparin-induced anticoagulation. Currently, there are three main manufacturers of ACT analyzers (Werfen with the Hemochron analyzers series, Medtronic with the ACT+/ACTII, and Abbott with the i-STAT analyzers). Unlike the Hemochron and the ACT+/ACTII analyzers where clotting is mechanically detected, the i-STAT Alinity (Abbott) analyzer uses an electrochemical detection of blood coagulation using a specific thrombin substrate with an amide bond whose cleavage generates an electroactive compound detected by amperometry. Interestingly, some authors investigated the impact of emicizumab on ACT. Capdevilla et al.3 previously reported no impact of emicizumab on UFH-induced ACT prolongation using the Hemochron Signature Elite analyzer (Werfen). Conversely, Tanaka et al.4 reported a decrease in ACT with emicizumab using the ACTII device (Medtronic). With a similar protocol to that developed by Capdevilla et al., we investigated if emicizumab interferes with the ACT using the i-STAT Alinity analyzer. For that purpose, we performed ACT on whole blood samples from adult male healthy donors (HDs) spiked with UFH and/or emicizumab and from adult male patients with severe HA without inhibitor under emicizumab after in vitro spiking with UFH ± FVIII. Additionally, blood samples from 3 HDs were spiked with a FVIII inhibitor without and with emicizumab in the presence or not of UFH to also assess the impact of decrease of FVIII. Our study (Emistat) was registered on clinicaltrial.org on April, 2023 (NCT 05856903) and approved by an ethics committee (Commission de protection des personnes Ouest III, record number 23.01540.000187). The patients gave oral consent after being informed. Blood was collected at the same time as routine tests performed for their medical follow-up. In accordance with national legislation and institutional requirements, written consent was not required for this study. Healthy volunteers gave written consent for their blood being used for scientific research purposes, when donating blood to the blood bank of the French Blood Establishment (EFS). Patient and healthy donor characteristics are depicted in Table 1. Blood samples were collected in vacutainers tubes containing 0.109 M trisodium citrate. To simulate standard conditions for ACT that are normally performed immediately after drawing blood without sodium citrate, citrated whole blood spiked with UFH and/or emicizumab and/or FVIII was recalcified (final calcium chloride concentration at 12.5 mM) immediately before performing the assay. This concentration was chosen based on bibliographic data5, 6 and on in vitro assessment of the optimal concentration (see Supplementary Figure 1). ACTs were measured with ACT kaolin cartridges on two i-Stat Alinity devices in prewarm mode according to the manufacturer's instructions. Statistical analyses were performed with Graphpad Prism 5.0 software (GraphPad Software Inc). Paired Wilcoxon rank tests were used to compare ACT of HDs samples spiked with emicizumab to the baseline (without emicizumab) and to compare ACT of HA patients spiked with FVIII to the baseline (without FVIII). Mann Whitney tests were used to compare ACT of HA patients to HDs. Two-sided significance tests were used throughout. The first part of the study consisted in spiking citrated whole blood samples from 8 HDs with a vehicle (buffer) or emicizumab (10, 25, and 50 μg/mL) and with a vehicle (saline) or UFH (0.5, 1, 2.5, and 5 IU/mL). Emicizumab concentrations in whole blood were chosen based on the reference range of steady trough levels established in plasma at 25–80 μg/mL.2 The concentration of emicizumab working solution dedicated to spiking was checked after spiking a commercial plasma from a severe HA using a coagulometric assay performed on a STAR-Max analyzer (Stago) with specific calibrators (R2-diagnostic). We found that emicizumab induced a dose dependent shortening of ACT whatever the UFH level with an impact not clinically relevant at 1 IU/mL and below, but relevant at 2.5 IU/mL and above (Figure 1, Supplementary Table 1). The second part of the study consisted in spiking citrated whole blood samples from 8 severe HA patients under emicizumab with UFH (0, 0.5, 1, 2.5, and 5 IU/mL) ± plasma derived FVIII (pdFVIII, Factane) at 50 IU/dL (final concentration) in whole blood to get a concentration approximatively at 100 IU/dL in plasma. Patients were on prophylaxis with emicizumab for more than 4 weeks and with no FVIII infusion ≥5 days. Emicizumab plasma level was determined as previously described (Table 1). We observed that emicizumab slowed down the ACT prolongation in the presence of supra-therapeutic levels of UFH (Figure 2, Supplementary Table 2): at very high levels of UFH (5 IU/mL), ACT from HA patients under emicizumab were far shorter than those from HDs without emicizumab (median: 509 [465–601, IQR] vs. 901 s [775–1000, IQR], p < 0.001). Conversely, we observed a slightly longer ACT at ≤1 IU/mL UFH in HA patients compared to HDs. Spiking of blood from HA patients under emicizumab with FVIII shortened ACT when UFH is ≤2.5 IU/mL (Figure 2, Supplementary Table 2). The third part of the study consisted in spiking citrated whole blood samples from 3 HDs with FVIII inhibitor at 70 μg/mL (monoclonal antibody, clone ESH-4 [Cryopep], preincubated 2 h at 37°C to decrease FVIII level. These samples were then spiked with emicizumab [50 μg/mL] and UFH, 1 and 2.5 IU/mL). FVIII level before and after spiking with FVIII-inhibitor and before spiking with emicizumab was determined with a coagulometric assay using STA-CK Prest reagent on a STAR-Max analyzer (Stago) (Supplementary Figure 2). Unexpectedly, it resulted only in a moderate decrease in mean FVIII level from 114 (78–128, range) to 29 (18–36) IU/dL. It induced a prolongation in mean ACT from 153 (149–155, range) to 210 (205–215, range) s at baseline, the former being shortened at 136 (129–144) s when adding emicizumab (50 μg/mL) (Supplementary Figure 2). In the presence of UFH, FVIII inhibitor increased the mean ACT from HDs from 421 (409–429) to 879 (750–1000) s at 2.5 IU/mL, the former being strongly shortened to 385 (378–388) s when adding emicizumab (50 μg/mL). During CPB, ACT above 400–450 s are usually recommended7 that can be obtained with high doses of UFH. Empirically, the target anti-Xa level would be at least 2.5 IU/mL in whole blood corresponding to 3.3 IU/mL in plasma with a 25% hematocrit.8 For catheter or surgical ablation of atrial fibrillation, the recommended ACT target is at least 300 s.9 CPB in HA patients are usually performed after FVIII replacement.10 This supports our testing on blood from HDs and after in vitro FVIII level correction in HA patients. However, the recently reported case of a CPB performed without FVIII replacement in a patient with severe HA on prophylaxis with emicizumab and presenting with major risk factors of thrombosis11 encouraged us to also test the impact of UFH on ACT in samples from patients treated with emicizumab without correcting FVIII level. Here, we report for the first time that emicizumab interferes with UFH-induced ACT increase when using ACT kaolin cartridges on i-STAT Alinity analyzers. No interference had been observed by Capdevilla et al.3 with the Hemochron signature elite analyzer contrary to Tanaka et al.4 with the ACTII device (Medtronic). Isaacs et al.11 reported ACT in the expected values when performing CPB in a patient with the Hepcon system (Medtronic). Discordant results between the different ACT analyzers might be explained by the different composition of the reagents: only kaolin as activator for ACTII (Medtronic) and ACT-kaolin (i-STAT Alinity), a mix of kaolin and silica for ACT-HR (Hemochron) and celite for LR-ACT (Hemochron). Our study had some limitations. First, the assays were not performed on native whole blood but on citrated whole blood after recalcification. Second, the ACT were performed on spiked blood samples. Third, the assays were all performed on samples with normal hematocrit while lower hematocrit may have affect the results. Despite no clear recommendation exists about the upper limit of the ACT target range, some authors indicate a target range of 400–600 s for CPB.12 Physicians must be aware of the potent underestimation of ACT in the presence of emicizumab, when using the i-STAT Alinity analyzer. In this context, underestimation of ACT may lead of heparin overdosing during CPB. All ACT reagents and analyzers have not the same sensitivity to emicizumab. R. Marlu designed the study. L. Seyve and J. B. Prigent performed the experiments. R. Marlu and L. Seyve analyzed the data. R. Marlu performed the statistical analysis and wrote the initial draft of the manuscript. R. Marlu and C. Barro recruited the patients. C. Lo Presti and G. Szymanski provided technical support. All authors critically reviewed the manuscript and approved the final version. We thank Z. Baidi and the DRCI (Direction de la Recherche Clinique, CHU Grenoble Alpes) for their assistance in regulatory and administrative aspects. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The data that supports the findings of this study are available in the supplementary material of this article. Supplementary Figure 1 Activated clotting time of citrated normal whole blood samples recalcified at different levels of calcium chloride. Supplementary Figure 2 Impact of emicizumab spiking on samples from healthy donors pre-incubated with a FVIII inhibitor. Supplementary Table 1 Impact of emicizumab and unfractionated heparin on activated clotting time in whole blood from healthy donors. Supplementary Table 2 Comparison of activated clotting times from hemophiliac patients under emicizumab to healthy volunteers. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.