In some clinical situations such as sepsis, DIC, and cancer, the plasma itself could contain enzymes that might be able to hydrolyze S-2222. If that happened, background activity could be seen and would therefore lead to an underestimation of the heparin level in the sample. To rule out background activity in the sample, the assay can be run without FXa and instead, and equal volume of buffer is substituted. The absorbance obtained is then subtracted from the absorbance from the normal run.