Effect of wavelength and filter set choices on fluorogenic thrombin generation assay: Considerations for interlaboratory differences

Introduction Insufficient agreement between results of various in-house and commercial thrombin generation (TG) assays complicates TG adoption and use. This can be caused by different assay variables, for example, volumes and concentrations of reagents. Although most modern assays employ the same fluorogenic thrombin substrate Z-Gly-Gly-Arg 7-amino-4-methylcoumarin (ZGGR-AMC), the excitation (Ex) and emission (Em) wavelengths used for fluorescence signal measurements can be different. Objectives We sought to investigate the impact of wavelengths on the TG curves in commercial normal, hemophilia A, and antithrombin-deficient plasma samples. Methods Fluorescence of ZGGR-AMC and AMC and TG curves were measured using different Em/Ex pairs to mimic settings used by common in-house and commercial TG methodologies. Results The peak fluorescence of free AMC was measured at an Ex/Em of 360 nm/440 nm. The uncleaved substrate ZGGR-AMC produced a low fluorescence signal, which yielded a maximum value at an Ex/Em of 300 nm/390 nm. Interference of ZGGR-AMC fluorescence with that of AMC is abrogated at Em above 430 nm. Calibrated thrombin peak heights were higher at Ex/Em of 340 nm/440 nm and 360 nm/460 nm, but the overall difference in peaks between either wavelength pairs was less than 20%. Further, we found that the inner filter effect depended on the choice of Ex/Em wavelength pairs, demonstrating variable distortion in the shape of the TG curves, which was consistent with reduced linearity of calibrator curves. Conclusion These data demonstrate that the choice and quality of fluorescence filters may have limited but measurable effects on the outcomes of TG analysis.