Christen Corby, Caitlyn Hauke, Brian Gestring, and Lawrence Quarino 1

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Volume 4, Number 1, August 2012 Preliminary Study Analyzing the Halo Effect: Factors Involved in Sequencing the Deposition of Overlapping Bloodstains Caused by Wipe Patterns and Blood Droplets

1 Volume 4, Number 1, August 2012 Preliminary Study Analyzing the Halo Effect: Factors Involved in Sequencing the Deposition of Overlapping Bloodstains Caused by Wipe Patterns and Blood Droplets Christen Corby, Caitlyn Hauke, Brian Gestring, and Lawrence Quarino 1 Abstract Sequencing the order of overlapping bloodstains is an area of crime scene reconstruction that is often overlooked in current evaluation methods. This study examined the patterns observed when blood wipes and droplet bloodstains overlap and how variables such as blood volume and substrate affect such patterns. This research revealed that a red halo of blood often formed around droplets when wipe patterns were produced over blood droplets. This feature was consistently found when droplets were made on several nonabsorbent, smooth substrates with volumes of blood ranging from 5-100µL. Additionally, a substrate-colored halo was often produced around the droplets when droplets were placed on top of blood wipes on several smooth, nonabsorbent substrates. This feature was often found when the volume of the blood swipe was larger (50 or 100µL) and the droplet volume ranged from µL. The presence of halos around blood droplets in overlapping bloodstains may therefore be helpful in determining the order of bloodstain deposition. Keywords: Overlapping bloodstains; Bloodstain sequencing; Bloodstain pattern analysis; Wipe patterns; Blood droplets 1 Forensic Science Program, Cedar Crest College, 100 College Drive, Allentown, PA 18104

2 Introduction Bloodstain patterns are often byproducts of violent crimes and can reveal a wealth of information regarding the events that took place at a crime scene. By analyzing photographs, sketches, physical evidence, and the crime scene itself, some understanding of the events that transpired during the crime may be determined. Crime scene information such as the locations and types of bloodstain patterns along with victim ante-mortem or post-mortem information and DNA testing of bloodstains is necessary for proper interpretation (James, Kish, Sutton, 2005). Bloodstain patterns can tell an investigator where a victim was located in relation to a suspect during an attack, what type of weapon was used, how the weapon was used, what movements took place during the course of the incident by those involved, whether the death was a homicide, and how many people were involved. These aspects are often ascertained by the shape, volume, size, degree of wetness, and position of individual bloodstains (Raymond, 1997). One of the major objectives in studying bloodstains on evidence and at crime scenes is to determine the sequence of events that created those stains (James, Kish, Sutton, 2005). During a violent criminal event, multiple types of bloodstains can be produced through distinctly different mechanisms, allowing analysts to reconstruct the formation of these stains. Often, multiple stains will occur at the same time and in the same location, which can lend to the difficulty of determining which event took place first. The sequencing of overlapping bloodstain patterns has been a neglected area of research thus far in forensic investigative pursuits, and its value is severely underestimated. Properly analyzing the sequence of overlapping bloodstains as a part of crime scene reconstruction could help to order events that occurred during a crime and provide vital information in an investigation. Overlapping stains can result when bloodstain generating events happen sequentially, when a victim or objects at the scene are moved after the incident, or when the victim is taken to another location (Raymond, 1997). Raymond stated that the chronology of bloodstains could be interpreted by looking at features such as disruptions in the continuity of bloodstains, dislodging of dried stains, and changes in direction of blood on a vertical surface (1997). It is important to distinguish the sequence of overlapping bloodstains, as this information can allow the investigator to create a timeline of events that took place during a crime. Anderson also articulated the importance of determining the first stain in a sequence, as it is the bloodstain closest in time and distance to the first criminal event (1993). Hurley and Pex focused on sequencing bloodstains that had been stepped on as well as examining shoeprints with blood spatter on top of them in order to determine if a suspect had been at a crime scene during or after an event (1990). They noted that it was difficult to sequence stains when blood droplets had completely dried prior to being stepped on. In addition, the researchers stated that one should take caution when evaluating overlapping bloodstains via photographs, as it is an unpracticed and difficult skill. This research examined the deposition of overlapping blood wipes and droplet bloodstains, which can occur as a result of crime scene traffic, including movement of the victim and perpetrator, as well as the result of multiple events at a scene. Blood droplets result from blood traveling through the air, while wipe patterns are formed by transferring blood from a moving surface onto another surface (SWGSTAIN, 2009). This study tried to identify any 19

Blood droplet stains of known volume were simply pipetted onto substrates and allowed to dry.

3 patterns which may give rise when wipe patterns are deposited over blood droplets and when blood droplets are deposited over wipe patterns. In the case of this research, wipe patterns were simulated by placing a known volume of blood on a substrate and smearing the blood with a gloved finger. Blood droplet stains of known volume were simply pipetted onto substrates and allowed to dry. Results will show that red halos of blood typically form around blood droplets when wipe patterns were created over blood droplets on smooth, non-absorbent substrates (Figure 1A). Conversely, a substrate-colored halo typically formed around the droplets when droplets were deposited over wipe patterns (Figure 1B). Figure 1A: Red halo on treated wood (left) Figure 1B: Substrate-colored halo on treated wood (right) Materials and Methods Defibrinated sheep blood (Hemostat Laboratories, Dixon CA) was used for all experiments in this study. Before use, the blood was placed into a beaker which was placed in a hot water bath and heated to 37 C to simulate body temperature. Room temperature and relative humidity were monitored with a thermometer equipped with a humidity meter during this research. Substrates used in the study included floor tile; glass; metal; painted dry wall; and treated (stained) wood. The substrates were cleaned with a 5% bleach solution prior to use. Bleach residue was then removed by rinsing the substrate with deionized water. Test samples prepared on each substrate included a base-stain (either a droplet or a wipe stain) and a corresponding top-stain (the converse of the base stain). All test samples were prepared horizontally except for those on painted drywall which were prepared vertically. The resulting pattern was then observed to determine if the predicted halo formed. Known volumes of blood droplets were deposited on the substrate using a P20 or P200 PIPETMAN Classic TM pipette (Gilson, Inc, Middleton WI) which dispensed blood droplets very close to the surface. Blood droplets used as the base-stain were simply placed on the surface of the substrate. Wipe patterns were produced by placing a known volume of blood surface on the substrate and then smearing across the surface with a latex-gloved finger. Although the act of smearing the 20

4 bloodstain likely led to a small change in the volume of the wipe due to the transfer of blood onto the latex glove, it was not deemed significant. Prior to application of the top-stain, the base-stain was allowed to dry completely. Resulting patterns were photographed using a speckfinder visual imaging system (Dazor Manufacturing Corporation, St. Louis MO). When the droplet stain was the base-stain on a substrate, an equal volume of a wipe pattern was placed on top of it for all substrates tested. Volumes tested included 1ul, 5ul, 10ul, 20ul, 50ul, and 100ul. However, varying volumes of the droplet stain were tested when placed over a wipe pattern although the volume of the droplet stain never exceeded that of the wipe pattern. Base-stain wipe patterns containing 1ul of blood were tested with 1ul blood droplets; 5ul wipe patterns were tested with 1ul and 5ul blood droplets; 10ul wipe patterns were tested with 1ul, 5ul, and 10ul blood droplets; 20ul wipe patterns were tested with 1ul, 5ul, 10ul, and 20ul blood droplets; 50ul wipe patterns were tested with 1ul, 5ul, 10ul, 20ul, and 50ul blood droplets; and 100ul wipe patterns were tested with 1ul, 5ul, 10ul, 20ul, 50ul, and 100ul blood droplets. Five replicates for each sample set were normally tested although in some test samples on the floor tile, ten replicates were examined. Results Substrate-colored halo formation with droplet over wipe patterns An examination of the data in Table 1 clearly shows that substrate-colored halo formation is more likely to occur at higher base-stain (wipe patterns) volumes for each substrate. Samples with base-stain volumes of 1ul did not show any halo formation for any substrates tested, and only painted drywall and glass showed an appreciable number of halos formed with base-stain volumes of 5ul and 10ul. In addition, the halos were typically more pronounced at higher basestain volumes (Figure 2). A trend toward a greater percentage of halo formation was also observed with increasing volume of top-stain (droplets). A graphical presentation of the relationship between top-stain (droplet) and base-stain (wipe) volume for all substrates combined for substrate-colored halo formation can be observed in Figure 3. Photographs of substratecolored halos on painted drywall, metal, glass, and treated wood can be found in Figure 4. 21

5 Table 1: Frequency of substrate-colored halos for each substrate at each base-stain (wipe) volume and top-stain (droplet) volume Volume of base-stain (wipe) Volume of top-stain (droplet) Tile 1 Metal 2 Painted Drywall 3 Treated Wood 4 Glass 5 1ul 1ul 0/5 6 0/5 0/5 0/5 0/5 5ul 1ul 0/5 0/5 2/5 0/5 1/5 5ul 1/5 0/5 5/5 0/5 2/5 10ul 1ul 0/5 0/5 2/5 0/5 0/5 5ul 1/5 0/5 2/5 0/5 2/5 10ul 0/5 2/5 3/5 2/5 2/5 20ul 1ul 0/5 0/5 0/5 0/5 0/5 5ul 3/5 2/5 0/5 0/5 3/5 10ul 3/5 2/5 0/5 0/5 4/5 20ul 0/5 1/5 0/5 0/5 4/5 50ul 1ul 0/10 0/5 1/5 0/5 0/5 5ul 4/10 1/5 0/5 2/5 3/5 10ul 4/10 2/5 1/5 3/5 3/5 20ul 6/10 2/5 3/5 5/5 4/5 50ul 2/10 3/5 1/5 5/5 5/5 100ul 1ul 2/10 4/5 2/5 2/5 0/5 5ul 5/10 4/5 2/5 3/5 1/5 10ul 5/10 4/5 1/5 1/5 4/5 20ul 1/10 5/5 1/5 3/5 4/5 50ul 1/10 5/5 1/5 4/5 5/5 100ul 4/10 5/5 3/5 3/5 4/5 Total (42/160).44 (46/105).29 (30/105).31 (33/105).49 (51/105) 1 Room Temperature: 20 C; Relative Humidity: 50% 2 Room Temperature: 24 C; Relative Humidity: 70% 3 Room Temperature: 23 C; Relative Humidity: 43% 4 Room Temperature: 23 C; Relative Humidity: 43% 5 Room Temperature: 24 C; Relative Humidity: 50% 6 represents the total number of samples with halos divided by the total number of samples 7 represents the frequency of substrate-colored halo formation 8 One-Way Anova testing at the 95% confidence interval for the combined data from all substrates, No significant difference between substrates, F(2, 100) = 1.99, p=.10 22

6 Figure 2: Substrate-colored halos on tile (top 20ul droplet placed on 20ul wipe; middle 20ul droplet on 50ul wipe; bottom 20ul droplet on 100ul wipe). The halo is pronounced proportionally with increased volume of blood smear 23

7 Volume of Base-Stain (wipe) Figure 3: Percentage of substrate-colored halo formation as a function of base-stain (wipe) and top-stain (droplet) volume 24

8 Figure 4: Examples of substrate-colored halos (top left painted drywall, 5ul droplet on 5ul wipe placed vertically; top right glass, 20ul droplet on 20ul wipe; lower left metal, 50ul droplet on 50ul wipe; lower right treated wood, 100ul droplet on 100ul wipe) 25

9 Red-colored halo formation with wipe patterns over droplets Although 1ul volumes of wipe patterns and blood droplets did not produce red-colored halos on any substrate tested, the percentage of red-halos formed was very high for all other volumes tested (Table 3). For all substrates combined, wipe and droplet volumes of 5ul yielded red-halos in 64% of test samples, wipe and droplet volumes of 10ul yielded red-halos in 84% of test samples, wipe and droplet volumes of 20ul yielded red-halos in 96% of test samples, and wipe and droplet volumes of 50ul and 100ul both yielded red-halos in 88% of test samples. At each volume beginning with 5ul, the percentage of red-halos formed far exceeded the percentage of substrate-colored halos produced with similar volumes of base-stain. Figure 5 provides photographs of red-halos formed on each tested substrate. Table 2: Frequency of red-halos that were formed on all substrates Volume of basestain (droplet) and top-stain (wipe) Tile 1 Metal 2 Painted Drywall 3 1µL 0 (0/5) 0 (0/5) 0 (0/5) 0 (0/5) 0 (0/5) 5µL.80 6 (4/5) 7.80 (4/5) 1.0 (5/5) 0 (0/5).60 (3/5) 10µL 1.0 (5/5).80 (4/5).60 (3/5).80 (4/5) 1.0 (5/5) 20µL 1.0 (5/5).80 (4/5) 1.0 (5/5) 1.0 (5/5) 1.0 (5/5) 50µL 1.0 (5/5).40 (2/5) 1.0 (5/5) 1.0 (5/5) 1.0 (5/5) 100µL 1.0 (5/5).40 (2/5) 1.0 (5/5) 1.0 (5/5) 1.0 (5/5) Treated Wood 4 Glass 5 Total 8.80 (24/30).53 (16/30).77 (23/30).63 (19/30).77 (23/30) 1 Room Temperature: 20 C; Relative Humidity: 50% 2 Room Temperature: 24 C; Relative Humidity: 70% 3 Room Temperature: 23 C; Relative Humidity: 43% 4 Room Temperature: 23 C; Relative Humidity: 43% 5 Room Temperature: 24 C; Relative Humidity: 50% 6 represents the frequency of red-colored halo formation 7 represents the total number of samples with halos divided by the total number of samples 8 One-Way Anova testing at the 95% confidence interval for the combined data from all substrates, No significant difference between substrates, F(4, 25) = 0.45, p=.77 26

Figure 4: Examples of red-colored halos (upper left tile, 5ul wipe on 5ul

middle left metal, 20ul wipe on 20ul droplet; middle right glass, 50ul wipe on

Effect of substrate on halo formation Using the data in Tables 1 and 2, one-way

determine if the effect of substrate on the frequency of both substrate-colored

The variance between each substrate (the combined data for each substrate

10 Figure 4: Examples of red-colored halos (upper left tile, 5ul wipe on 5ul droplet; upper right drywall, 10ul wipe on 10ul droplet placed vertically; middle left metal, 20ul wipe on 20ul droplet; middle right glass, 50ul wipe on 50ul droplet; bottom center treated wood, 100ul wipe on 100ul droplet). Effect of substrate on halo formation Using the data in Tables 1 and 2, one-way Anova (analysis of variance) at the 95% confidence interval was used to determine if the effect of substrate on the frequency of both substrate-colored and red-colored halos formation was statistically significant. The variance between each substrate (the combined data for each substrate regardless of base-stain and topstain volume) was divided by the variance within the data for each substrate. For both substrate- 27

11 colored and red-colored halos, the effect of substrate was not statistically significant. For both data sets, the calculated p-value (0.10 for the substrate-colored halo data; 0.77 for the red-colored halo data) greatly exceeded the threshold 0.05 value showing statistical insignificance (Corel Corporation, ). Effect of temperature and humidity Given the Anova results, it can also be concluded that any effect of humidity and temperature recorded during data collection in this study on both substrate-colored and redcolored halo formation was not statistically significant. It should, however, be noted that only two of five replicates at 50ul and 100ul base-stain and top-stain volumes on metal tested at 70% relative humidity produced red-halos. All other substrates were examined at a relative humidity of 50% or less and all produced red-halos on all five samples at these stain volumes. Perhaps this is just happenstance since the 70% humidity did not affect substrate-colored halo formation on metal given that the frequency of substrate-colored halo formation on metal was the second highest of all substrates tested. Discussion This study showed that given the experimental parameters used in this study, halo formation can occur when a blood droplet overlaps a dried blood wipe and vice versa. Given that blood wipes are a type of contact stain, it is likely that similar results would be observed with contact stains are well. Since it was important to know the effect of blood volume on halo formation, blood wipes appeared to be the logical choice for use in the study. The study was conducted on non-porous substrates. Preliminary work was performed on porous and semi-porous substrates but halo formation did not occur because the base-stain simply absorbed into the substrate. Examination of video using the speckfinder provided some information into the mechanism of halo formation. Substrate-colored halos appear to form as the result of the topstain blood droplet shrinking upon drying and consequently removing blood from the base-stain wipe as it dried. Red-halos appear to form due to the pooling of blood around already dried blood when the top-stain wipe is added. The authors hypothesized this at the start of this part of the study which is why only equal volumes of base-stain and top-stain were used. It was assumed that as long as the base volume was large enough to cause the pooling the red halo would form. Sequencing the deposition of overlapping bloodstains will remain a difficult task for those engaged in crime scene reconstruction. The base-stains employed in this study were dry because preliminary data indicated that wet bloodstains regardless of type simply mixed together. It is likely that in many violent encounters, the time interval between the impact of two overlapping bloodstains will not be of sufficient duration for the base-stain to dry. However, in those situations where it may be applicable, the observation of the halos will provide meaningful information for the reconstruction of a crime. It stands to reason that blood droplets and contact patterns will almost always be found at violent crime scenes. Our preliminary study suggests 28

12 that when a red halo is observed, the droplet was the first stain deposited. Conversely, our preliminary study suggests that when the color of the substrate appears around the droplet, the contact stain was the first deposited. Observation of the halo-effect may also be of value for stain-selection for future DNA testing. For instance, the type of halo present may allow an investigator to better hypothesize which bloodstain is more likely to come from a victim and which bloodstain is more likely to come from a suspect. Limitations of Study and Future Research The study does have limitations which need to be addressed in future study. The first is that the study was performed with defibrinated sheep blood. Although it has been reported that animal blood and human blood are similar enough that animal blood was a suitable alternative for human blood in experimentation (Raymond, Smith, Liesegang, 1996), differences in cell structure and density warrant verifying these results with human blood. Furthermore, the extent to which clotting factors may affect the results in this study is not known since the blood used was defibrinated. A second limitation is that blood droplets were deposited onto substrates very close to the surface which is not likely to occur in actual crime scene casework. How blood droplets traveling different distances or falling from different heights affect halo formation needs to be explored. Thirdly, the lack of significant difference between substrates for both types of halo formation may be the result of a limited number of replicates at each data point. Increasing the number of samples may provide greater specificity into the effect of substrate type as well as provide deeper insight into the role that volume of blood droplets and wipes have on halo formation. With this study, a trend toward halo formation was noted at larger volumes for all substrates. Having a larger pool of samples will allow for statistical testing between different volumes of blood droplets and wipes at varying substrates to see if this trend is significant. Finally, the applicability of this study to actual casework can only be done with future blind studies where participants attempt to determine the sequence of bloodstain deposition based on the presence of either red or substrate-colored halos. 29

13 References Anderson, J. W. (1993). Capillarity distortion analysis. International Association of Bloodstain Pattern Analysts Newsletter, 9(4), Corel Corporation ( ). Quattro Pro (version 9) [Computer software]. Mountain View, CA. Hurley, N. M., & Pex, J. O. (1990). Sequencing of bloody shoe impressions by blood spatter and blood droplet drying times. International Association of Bloodstain Pattern Analysts Newsletter, 3(2), James, S. H., Kish, P. E., & Sutton, T. P. (2005). Principles of bloodstain pattern analysis: theory and practice. Boca Raton, FL: Taylor & Francis Group, CRC Press Inc. Raymond T. (1997). Crime scene reconstruction from bloodstains. Australian Journal of Forensic Science, 29(2), Raymond, M.A., Smith, E.R., & Liesegang, J. (1996). The physical properties of blood- forensic considerations. Science and Justice, 36(3), Scientific Working Group on Bloodstain Pattern Analysis (2009). Scientific Working Group on Bloodstain Pattern Analysis: Recommended terminology. Forensic Science Communications, 11(2), 30

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