Figure 1.
Hemoglobin UV-VIS spectral shift parameters.
UV-VIS spectral profiles from bloodstains stored at room temperature for 15 minutes (solid line) and 1 year (dashed line) were compared in order to identify potential differences between “fresh” and “old” stains. The putative time-dependent parameters (1–5) involving changes in the Soret band and α and β peaks are indicated (1–5).
Figure 2.
Bloodstain age (TSD) correlates with a blue shift of the Hb Soret band.
Spectral profiles (350–600 nm) were obtained from samples stored at 22°C (blue diamonds) and 37°C (red squares) for 15 minutes to 1 year. The λmaxSoret is plotted as a function of stain age: A) “full range”, 15 minutes –1 year (in days); B) “hours”, 15 minutes –48 hours (in hours); C) “days”, 15 minutes –7 days (in days); D). “weeks”, 15 minutes –1 month (in days) All data points are an average of bloodstains from two biological replicates (two individual donors). The value used for each of the two biological samples was obtained from the average of three technical replicates for each sample. Using a logarithmic regression function (22°C – blue; 37°C - red), a strong degree of correlation was observed (R2>0.95 for most data sets) allowing for differentiation of deposited hours, days, weeks and months prior to collection and analysis. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article).
Figure 3.
Effects of temperature and humidity on λmaxSoret measurements.
To assess the effects of humidity, bloodstains were stored at 22°C (A) at various relative humidity (50% - blue diamonds, 75%-red squares, 80%-yellow asterisks, 85%-purple circles, 90%-green diamonds). For bloodstains stored at constant temperature the Soret band blue shift decreases as the humidity level increases. To assess the effects of temperature, bloodstains were stored at a constant relative humidity of 50% (C) at different temperatures (−20°C-blue diamonds, 4°C–red squares, 22°C–yellow asterisks, 30°C-purple circles, 37°C-green triangles). At constant humidity the Soret band blue shift increases with increasing temperature. For each condition, the λmaxSoret is plotted as a function of stain age (15 minutes –1 week, in hours). All data points are an average of bloodstains from two individual donors (average of triplicate measurements) and the standard error for each measurement is displayed. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article).
Table 1.
Effects of Temperature and Humidity on the Soret Band Blue Shift.
Figure 4.
TSD sample extracts can be used to confirm the presence of human blood and obtain the DNA profile of the bloodstain donor.
The use of a portable spectrophotometer (NanoPhotometer™) permits the rapid identification of the presence of blood via its characteristic Hb absorption spectrum (A, left panel). Further confirmation of the presence of blood and its human origin was provided by a positive result with an immunochromatographic test (ABAcard® HemaTrace®) (A, right panel) using an aliquot of the same TSD extract. DNA was isolated from the remaining extract and a full autosomal DNA profile (PowerPlex® 16 HS, Promega) was obtained using standard amplification conditions (B).