Background: Emicizumab is administered at a fixed dose for prophlaxis of hemorrhage in hemophilia A, monitoring drug levels is not indicated. Measurement of drug levels may be useful in patients experiencing breakthrough bleeding episodes for assessment of compliance or development of an anti-drug antibody. On therapy trough levels have been reported to range from 35 to 55 micrograms/mL (Pipe et al, Lancet Haematol 2019;6: e295–305).

Aims: Compare analytical performance characteristics of one stage (OSA) and chromogenic (CSA) Emicizumab assays.

Methods: A modified FVIII:C OSA using HemosIL^® SynthASil Reagent (Instrumentation Laboratory [IL], Bedford, MA) and CRYOcheck™ Factor VIII Deficient Plasma (F8DP) (Precision BioLogic, Dartmouth,NS, Canada) and human reagent based FVIII:C CSA assay (Hyphen Biomed, Neuville-sur-Oise, France) and a drug specific calibrator (r² Diagnostics, South Bend, IN, USA) were developed on the ACL TOP 700 (IL).

Results: Precision of OSA and CSA was <10%CV for two levels of control [repeatability (n=9) and within laboratory assessments (n=20)]. Accuracy compared to known (emicizumab spiked) targets (n=9): OSA yielded: m = 1.05, b = -0.021, R² = 0.99. CSA yielded: m = 0.81, b = -2.78, R² = 0.97. Overall deviation from assigned value: OSA = 2% and CSA = -31%. Assays performed on patient samples demonstrated a similar negative bias. LoB, mean of F8DP (n=5, target <1.0 mcg/mL) for OSA was 0.18 mcg/mL, CSA was 1.16 mcg/mL. LoQ mean/ %CV of dilute control (n=5, target 4.88 mcg/mL) for OSA was 4.86 mcg/mL/ 1.1% and CSA 2.62 mcg/mL/ 3.2%. Residual FVIII (>10%) artifactually raises emicizumab level in CSA more than the OSA. Preanalytic heat inactivation was examined; emicizumab recovery was not consistently maintained.

Table 1: Precision

Figure 1: Accuracy

Conclusions: Although both assays have acceptable precision, the OSA has superior performance characteristics. Heat inactivation of sample is not reliable solution for accurate measurement of emicizuamb in the presence of FVIII for either method.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/emicizumab-assays-one-stage-vs-chromogenic-stage-assay/