Investigating Novel Methods for Estimating Time Since Deposition (TSD) of Bloodstains in Forensic Samples

Date of Submission


Document Type


Degree Name

Master of Science in Forensic Science


Forensic Science


Claire L. Glynn

Committee Member

David San Pietro

Committee Member

Christina Zito




Spectrophotometry, Alkaline Phosphatase


Bloodstains--Analysis, Criminal investigation--Methodology

Call No. at the Univ. of New Haven Library

AS36.N29 For. Sci. 2018 no.7


Blood is the most commonly encountered biological fluid found at violent crimes and can provide probative evidence in terms of DNA profiles and pattern analysis. However, these stains can also reveal previously untapped potential in determining the time of deposition of a bloodstain resulting from a trauma or event. The ability to estimate Time Since Deposition (TSD) of bloodstains has been researched in the past utilizing many different methods, yet, results varied with no complete agreement on one method for implementation into real casework. The aim of this study was to investigate a variety of methods which exhibit potential for TSD estimation. These include investigating over time; enzyme activity, the quantification and spectrophotometric observation of total protein, and the degradation of two RNA species.

Following Institutional Review Board (IRB) approval, venous blood was collected from volunteers with informed consent into sterile EDTA vacutainer tubes. 100 uL of blood was deposited on white cotton cloth, in triplicate, and allowed to age in a cool, dark environment for 24 hours, 48 hours, 1 week, 2 weeks, 1 month, 3 months, 6 months, 9 months, and 1 year. Enzyme activity of Alkaline Phosphatase (ALP) was determined using a colorimetric reaction which was measured using a Nanodrop® OneC UV-Vis spectrophotometer. Total protein was extracted, quantified, and viewed spectrophotometrically also using the UV-Vis spectrophotometer. Total RNA was extracted using the RNeasy mini kit, quantified, and expression analysis was performed using RT-PCR targeting beta-actin and 18 S RNA.

After spectrophotometrically observing the enzymatic activity of ALP, the concentration was determined using a standard curve. It was found that from fresh blood to 1-year blood the concentration dropped from 178.9 U to 0 U. When examining the amount of quantified total protein, it was found to have not decreased as drastically, ranging from 5.912 mg/mL to 2.783 mg/mL in the same 1-year period. When each sample was spectrophotometrically observed, three specific peaks were seen at 412 nm designated lambda, 541 nm designated beta, and 576 nm designated alpha. However, two additional peaks at 225 nm and 295 nm were observed. Quantifiable amounts of total RNA were extracted from all samples ranging from 22.159 ng/uL to 7.000 ng/uL, with no real trend observed with an increase or decrease over time. Both Beta-actin and 18S RNA were detected in all samples and have shown a general trend of consistency in expression for up to three months, however, the samples showed inconsistency after six months.

This study has shown, using several different methods, the great potential present in the ability to estimate the TSD of bloodstains. It is the authors belief that not one single method will provide the answer, more so the utilization of multiple methods in concert with each other will ultimately provide the investigator with greater accuracy.