Systems and methods for producing a calibration standard for an optical analysis system (e.g., a diagnostic reader) from a live test sample are disclosed. The calibration standard may include an image reproduced on a substrate. The reproduced image may be a replication of a digital image of a live test sample captured using the optical analysis system and then digitally processed to be reproduced on the substrate. The image reproduced on the substrate may include at least one optical feature digitally added to the replication of the digital image of the live test sample. The added optical features may be used to allow for more robust calibration using the calibration standard.