Ultraviolet 365 as an Alternative Light Source for Detection of Blood Serum

Blood, Plasma, and Serum

Human blood was obtained from healthy volunteers by the finger-stick method using a Health Lancing device (CVS pharmacy®, USA) fitted with a microlancet (CVS pharmacy®, USA). Blood was dropped onto Parafilm® M Laboratory Film (Bemis Company, Inc., Oshkosh, WI) and transferred to a 1.5 mL Eppendorf® microcentrifuge tube (Eppendorf, Hamburg, Germany) using a 10–100 µL Eppendorf® micropipettor (Hamburg, Germany), with the volume set at 100 µL; blood was then added to material as needed, using the same micropipettor with the volume set at 20 µL. For clot transfer experiments, blood was transferred from the Eppendorf tube to a fresh piece of Parafilm® M Laboratory Film, or to skin, using an Eppendorf® micro pipettor. After a certain amount of time, filter paper or cloth was placed directly onto the blood pool with very slight pressure. Twenty microliter of blood was typically used for each group. For purification of blood plasma and cellular fractions, 400 µL of whole blood was placed in a 1.5 mL Eppendorf tube and spun in a microcentrifuge (Qualitron Inc., Gyeonggi-Do, South Korea) for 1 min immediately after collection. The supernatant was removed and transferred to a new tube; this cycle was repeated 2× until no red material was visible. Purification of blood serum was performed in an identical manner, except that blood was first allowed to clot for at least 2 h prior to centrifugation.

Ultraviolet Light Sources

The UV light systems used in these studies were as follows: a LED UV flashlight, 395 nm, 00F-51UV-001A (Esco-Lite, Shenzhen, China), referred to as Esco-Lite 395; a handheld UV lamp, 365 nm, UVP UVL-4FUV Lamp (Analytik Jena, Upland, CA), referred to as Analytik Jena 365; a LED UV flashlight, 365 nm, LED-UV301-356 nm (Shenzhen Lightfe Light Limited, Shenzhen, China), referred to as Shenzhen Lightfe 365; and a LED portable 3 watt UV light bulb, 365 nm, UV-3W-365UV-E27-AC (Golden Gadgets, South El Monte, CA), referred to as Golden Gadgets 365. Photographs were taken using a Sony RX100 digital camera.

Fabrics

Specific fabrics that were used in this study include a black leather wallet (make/company unknown); a Nike® (Beaverton, Oregon) white leather tennis shoe; a black nylon backpack, the Wanderer style by Altura Photo (Florida, USA); linen that was handwoven from natural, unprocessed flax (Vavstuga, Shelburne Falls, MA) in a 3:1 herringbone pattern by professional weaver Tess Farley (Columbia, SC); Whatman® 1 and 3 mm filter paper (Whatman, Maidstone, Kent, UK); and a blue/green/black patterned 100% silk tie treated to be stain resistant from the manufacturer (Croft & Barrow®: Kohl’s, Menomonee Falls, WI). All fabrics were obtained from the author’s personal collection.

UV Detection of Blood Components

Initially, the detection of blood plasma and serum purified by microcentrifugation was examined using a handheld UV fluorescent lamp (Analytik Jena 365). As shown in Fig. 1, whole blood and the cellular fraction appeared relatively dark under UV, whereas detection of serum and plasma was enhanced (Fig. 1). By visual inspection, fluorescence of blood plasma and serum appeared to be of relatively similar intensities (Fig. 1). Next, the fluorescence of serum applied to various articles was evaluated using LED flashlights at two different wavelengths, UV365 and UV395. As demonstrated, serum was essentially invisible under normal light or UV395 on certain objects (interior wall, rock), but was revealed using UV365 (Fig. 2). Unlike whole blood, serum was not effectively detected using BLUESTAR®FORENSIC (Fig. 3). Taken together, these results demonstrate that UV365 is useful for the detection of blood serum on a variety of substances.

Development and Detection of Serum Halos/Edges

Next, the development and detection of serum “halos” or edges, which may form at the periphery of the bloodstain during the clotting process was evaluated. Of particular interest was to determine whether serum edges were specific for clotting blood that was transferred, or whether these could also be observed when blood was added directly to material. The appearance of serum edges in blood imprints was time-dependent (Fig. 4); in this system using relatively small volumes of blood (~25 µL), it was noted that after 50–60 min, only minimal transfer of bloodstains was observed (data not shown). Serum edges were also detected when blood was placed onto skin, and clotting allowed to proceed prior to transfer (Fig. 5). Unlike what was observed with imprints of clotting blood, when blood was added directly to material, no serum edges were apparent using either filter paper or linen (Figs 6 and 7). These results were confirmed using a variety of implements to apply blood, as shown in Fig. 8. Blood that had clotted for several hours was vigorously stirred and added to filter paper, with no serum edge being visible (Fig. 9); when dried blood that was several days old was ground in a mortar and pestle, rehydrated, and added to material, a similar result was obtained (Fig. 9). Collectively, these results demonstrate that blood added directly to filter paper or linen did not result in the presence of a serum edge, detectable by UV light. Serum edges were only observed when blood was imprinted during the clotting process.

Linen is described as the world’s strongest natural fiber and, like filter paper, is quite absorbent, with linen able to retain approximately 20% of its own weight (10). To evaluate properties of serum edges on less absorbent material, a similar set of experiments was performed using a silk tie, which had been treated during manufacture to become stain resistant, and a leather wallet. Samples were evaluated under a stereomicroscope using both normal (white) light and UV365. As shown, a serum halo was detectable on blood that had been directly added to leather, with the bloodstain maintaining a round shape (Fig. 10). A serum edge was also present in clot transfer samples on leather, although more spread out, which followed the irregular shape of the stain (Fig. 10). As with leather, a serum edge was observed in both direct addition and clot transfer samples using treated silk (Fig. 10). Thus, it is possible to observe a serum halo/edge when blood is directly added, depending upon the surface of the material.

Finally, experiments were done to determine, if under any circumstances, a fluorescent halo/edge might be present in blood samples added directly to paper. As shown in Fig. 11, like serum, juice from multiple fruits fluoresced under UV365, (Fig. 11), as did (undiluted) honey, which was particularly bright (Fig. 11). To determine whether bloodstains containing serum halos/edges could be mimicked by the addition of such substances, blood mixtures were created and added directly to filter paper. As demonstrated in Fig. 12, serum halos/edges were formed that were similar in appearance to those observed in clotting imprints (Fig. 12). Taken together, these results illustrate that juices from certain common fruits exhibit fluorescence, which could result in an inaccurate determination of serum detection with UV365. In addition, these data show that serum halos/edges can be mimicked by direct addition of blood to filter paper, with the inclusion of certain additives.