Depletion series of footwear impressions in blood were deposited on black cotton fabric after which they were lifted using alginate and subsequently enhanced using protein stains amido black (AB), Crowle’s stain (CS), coomassie blue (CB), and Hungarian red (HR). Other factors that were considered during this study were the age of the impression and the temperature of the environment. A novel score system for the enhancement of footwear impressions was introduced, which used the product of scores for size and detail of the impression.

The study showed that temperatures between 8 °C and 37 °C did not impact chemical enhancement, whereas the age of the impression did. An impression aged for 7 days yielded higher enhancement scores than impressions aged for 1 or 28 days, especially for AB. The results of depletions 1 to 5 were similar to the results of only depletion 5. However, at depletion 5, AB was the best-performing protein stain.

CB and AB yielded the highest level of enhancement of the impressions, whereas CS and HR resulted in poorer quality enhancements. AB was the preferred protein stain of use because AB was the most sensitive protein stain used in this study and there were fewer health risks involved in using water-based AB than in using methanol-based CB.