Safranin O without fast green is the best staining method for testing the degradation of macromolecules in a cartilage extracellular matrix for the determination of the postmortem interval.

Methods for the determination of the postmortem interval (PMI) include methods that monitor the postmortem changes of cells and molecules in different tissues. The rate of pathological degradation of macromolecules in the extracellular matrix (ECM) of hyaline cartilage could be verified by assessing the intensity of collagen and proteoglycan (PG) staining. In the presented in vitro pilot study, this methodology was used for the first time to determine PMI. The osteochondral samples of three donors were stored at 11 °C and 35 °C and analyzed on day 1, day 12, and day 36 postmortem. The intensity of staining using Masson's trichrome and Sirius red for collagen, and Alcian blue and Safranin O dyes for PG was estimated ten times according to the modified Bern grading scale. Statistical analysis showed that the Safranin O without Fast green method is the most appropriate (raters agreement 0.5541) for up to 36 days postmortem, and that the influence of time is more important (p = 0.023) than the influence of temperature (p = 0.061) on the degradation of the ECM macromolecules. The described method, which is simple and can be performed in any histological laboratory, should be verified in corpore conditions, on a large number of donors, and using an objective method for assessing the intensity of cartilage macromolecule staining for PMI determination.