A Novel Point-of-Care Whole Blood Coagulation Assay to Monitor Emicizumab Therapy in Patients with Hemophilia

Introduction: Emicizumab is a humanized, bispecific antibody against activated Factor IX (FIX/IXa) and FX/Xa that mimics the cofactor function of activated FVIII (FVIIIa) by spatially relocating FIXa and FX to the appropriate position in the tenase complex. Though the bleeding rate in patients on emicizumab is remarkably reduced, hemostatic monitoring is important in the event of breakthrough bleeding, development of anti-drug antibodies, and for surgery. An APTT is markedly shortened in the presence of small amounts of emicizumab, and, hence not useful. The thrombin generation assay (TGA), thromboelastometry, and clot waveform analysis measure the hemostatic effect of emicizumab, both in vivo and ex vivo. However, these assays are time-consuming, need expert interpretation, and not widely available. We have developed a novel, point-of-care, whole blood assay based on a dielectric microsensor (ClotChip) that can obtain global blood coagulation assessment in a miniaturized, portable measurement platform. The aim of this study was to assess the sensitivity of ClotChip to detect the addition of variable concentrations of emicizumab ex vivo into hemophilic whole blood and the feasibility of monitoring emicizumab therapy in vivo using ClotChip .

Methods: After IRB approval, patients accrued for the study were adults with severe hemophilia A with inhibitors starting emicizumab therapy (n=2), and a child with severe hemophilia A without inhibitors (n=1). Blood samples were obtained by venipuncture into collection tubes containing 3.2% sodium citrate anticoagulant. Samples were collected both prior to (n=2), and at time points of 30 min (n=1), 1 hour (n=1), 1 week 1 (n=1), and 8 weeks (n=1) after initiation of emicizumab therapy. Whole blood samples from hemophilia patients not on emicizumab were spiked with different concentrations of emicizumab, with and without the addition of replacement therapy. Whole blood samples were then tested with the ClotChip. Coagulation was induced with CaCl₂ addition. ClotChip is based on the electrical technique of dielectric spectroscopy (DS) integrated into a low-cost (material cost < $1), small- sized (26mm × 9mm × 3mm), and disposable microfluidic biochip with miniscule sample volume (< 10 µL). ClotChip curves were calculated as blood permittivity at 1MHz, and the time to reach a peak in permittivity (T_peakFig 1A) was taken as an indication of coagulation time. TGA using low tissue factor concentration was also performed on blood samples according to the manufacturer's direction.

Results: We observed a decrease in the ClotChip T_peak parameter for post-therapy samples (30 min and 1 hr.) compared to baseline (pre-therapy) for hemophilia patients with inhibitors on emicizumab therapy (Fig 1B). A time-dependent decrease was observed in ClotChipT_peak after emicizumab administration with week 1 and 8 samples showing normal values. Ex vivo spiking with emicizumab in blood from patients with hemophilia with and without inhibitors showed a concentration dependent decrease in ClotChip T_peak parameter (Fig 1C). Addition of rFVIII or rFIX in emicizumab-spiked blood from the patient without inhibitors further decreased ClotChipT_peak in a concentration dependent manner (Fig 1D). Similarly, addition of rFVIII to the emicizumab spiked blood from the inhibitor patient further decreased ClotChip Tpeak compared to emicizumab alone (Fig 1E). ClotChip T_peak exhibited strong negative correlation with ETP (r_s = 0.81, Fig 1F) and peak thrombin (r_s = -0.82) from TGA assays.

Conclusions: Our studies demonstrate the feasibility of monitoring emicizumab therapy in patients with hemophilia with and without inhibitors, using a novel, microfluidic, dielectric sensor (ClotChip), allowing whole blood assessment of hemostasis in a single disposable sensor. ClotChip has potential to fulfill an unmet clinical need to assess global coagulation potential in hemophilia patients on emicizumab therapy, especially when additional hemostatic therapy is needed for treatment of breakthrough bleeds. Additional spiking studies to assess the addition of bypassing agents (rFVII or FEIBA) to blood from emicizumab-treated patients are underway.

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