Validation of the hemoglobin (Hb) hypsochromic shift assay for determination of the time since deposition (TSD) of dried bloodstains

Abstract

Determining the time since deposition (TSD) of a bloodstain found at a crime scene could prove invaluable to law enforcement investigations, identifying the time frame in which the individual depositing the evidence was present. Although various TSD methods have been proposed, none have gained widespread use due to poor time resolution and weak age correlation. Previously, we examined the UV–visible absorption spectral profile of hemoglobin (Hb) in dried bloodstains of different ages and identified a hypsochromic shift (blue shift, shift to shorter wavelength) of the HbSoret band that demonstrated a high correlation with TSD. While this method demonstrated promise for use with forensic samples, additional validation work was needed. We have further evaluated the effects of temperature and underlying substrate on the ability to obtain reliable TSD measurements using the Hb hypsochromic shift assay. We evaluated bloodstains deposited onto cotton, denim, polyester, and paper incubated at various temperatures for up to 3 months. The effects of temperature were consistent with previous studies and no significant effects from underlying substrate were observed. We further evaluated the use of a portable spectrophotometer which could be used “on-site” at crime scenes. This would allow not only an estimation of the age of the stain but the rapid positive identification of the presence of blood due to the unique visible spectrum of Hb. Good quality DNA profiles can be obtained from the original TSD aqueous extracts.