Latent evidence detection using a combination of near infrared and high dynamic range photography: an example using bloodstains

In this paper, we use bloodstains to illustrate an approach for identifying latent evidence on dark cloth using near infrared (NIR) photography combined with high dynamic range (HDR) photography techniques. NIR photography alone has been used to capture latent evidence that cannot be seen in normal ambient light. HDR techniques combine multiple bracketed photographs of the same image to increase the dynamic range of the photograph which can provide greater contrast. Using NIR photography alone, we were able to detect a bloodstain up to a 1/16 dilution, an improvement over previous studies. Combining NIR photography with the HDR process resulted in a noticeable increase in visibility up to 1/16 dilution when compared to NIR photographs alone. At 1/32 dilution, we were able to detect bloodstains that were not visible using NIR alone. NIR is a useful tool for imaging latent evidence, and combining NIR with HDR consistently provides better results over NIR alone.     

Statistical Evaluation of Alternative Light Sources for Bloodstain Photography

Bloodstain photography is important in forensic applications, especially for bloodstain pattern analysis. This study compares the enhancement effect of bloodstain photography using three different types of light source: fluorescent white light, near‐ultraviolet (UV) light‐emitting diode (LED) light, and 410 nm LED light. Randomized complete block designs were implemented to identify the lighting that would statistically produce the best enhancement results for bloodstains on different types of surfaces. Bloodstain samples were prepared on white cotton, brown carpet, tar road, and wood. These samples were photographed in darkroom conditions using a Canon EOS 50D digital SLR camera, with Canon EFS 60 mm f/2.8 Macro USM lens. Two‐way analysis of variance and Fisher's least significant difference test were used to analyze the contrast of the images. The statistical analysis showed that 410 nm light is the best among the tested lights for enhancing bloodstains on the tested surfaces, where the contrast of bloodstain to background was the highest.     

Alteration of expirated bloodstain patterns by Calliphora vicina and Lucilia sericata (Diptera: Calliphoridae) through ingestion and deposition of artifacts

Abstract: Bloodstain pattern analysis can provide insight into a sequence of events associated with a violent crime. However, bloodstain pattern analysis can be confounded by the feeding activity of blow flies. We conducted two laboratory experiments to investigate the relationships between Lucilia sericata (green bottle fly) and Calliphora vicina (blue bottle fly), expirated bloodstains, and pooled bloodstains on a range of surfaces (linoleum, wallpaper, textured paint). C. vicina and L. sericata changed bloodstain pattern morphology through feeding and defecation. They also deposited artifacts in rooms where blood was not present originally. Chemical presumptive tests (Hemastix^®, phenolphthalein, leucocrystal violet, fluorescein) were not able to differentiate between insect artifacts and bloodstains. Thus, C. vicina and L. sericata can confound bloodstain pattern analysis, crime scene investigation, and reconstruction. Crime scene investigators should be aware of these fundamental behaviors, and the effects that blow flies can have on expirated and pooled bloodstain patterns.     

The Morphology of Fecal and Regurgitation Artifacts Deposited by the Blow Fly Lucilia cuprina Fed a Diet of Human Blood

Fly feces and regurgitation deposits may be mistaken for bloodstain patterns at a crime scene, potentially compromising event reconstruction and/or misdirecting police resources. In some instances, these artifacts contain sufficient human biological material to generate a full DNA profile, sometimes 2 years after deposition. Clearly, it is important that investigators can make the distinction between artifacts and bloodstains. This study examined 6645 artifacts deposited on a smooth, nonporous surface after Lucilia cuprina were fed human blood. Artifacts were also compared with bloodstains on a variety of other surfaces. Both similarities and differences were found between artifacts and bloodstains, highlighting the need for an identification system to assist personnel with little training in bloodstain pattern analysis. The morphology of the artifacts has been described so that these deposits may be more clearly distinguished from bloodstains, targeted by crime scene personnel as potential sources of human DNA, and/or identified as potential evidence contaminants. Flowcharts have been devised to facilitate the analysis.     

The Use of Near‐Infrared Photography to Image Fired Bullets and Cartridge Cases

An imaging technique that is capable of reducing glare, reflection, and shadows can greatly assist the process of toolmarks comparison. In this work, a camera with near‐infrared (near‐IR) photographic capabilities was fitted with an IR filter, mounted to a stereomicroscope, and used to capture images of toolmarks on fired bullets and cartridge cases. Fluorescent, white light‐emitting diode (LED), and halogen light sources were compared for use with the camera. Test‐fired bullets and cartridge cases from different makes and models of firearms were photographed under either near‐IR or visible light. With visual comparisons, near‐IR images and visible light images were comparable. The use of near‐IR photography did not reveal more details and could not effectively eliminate reflections and glare associated with visible light photography. Near‐IR photography showed little advantages in manual examination of fired evidence when it was compared with visible light (regular) photography.     

The use of Polilight in the detection of seminal fluid, saliva, and bloodstains and comparison with conventional chemical-based screening tests

Biological stains can be difficult to detect at crime scenes or on items recovered from crime scenes. The use of a versatile light source may assist in their detection. The ability of Polilight to locate potential semen, saliva, and blood stains on a range of substrates and at different dilutions was tested. We also tested the use of Polilight in comparison with conventional chemical-based presumptive screening tests such as acid phosphatase (AP), Phadebas, and luminol, often used in casework for detecting potential semen, saliva, and blood stains, respectively. The Polilight was able to locate stains that were not apparent to the naked eye. The color of the material on which a stain is deposited can have an effect on the detectibility of the stain. The Polilight was found to be comparable with the AP and Phadebas tests in terms of its sensitivity. In a comparative study between the AP test and Polilight on 40 casework exhibits, one false-negative result was observed when using the Polilight. On a series of mock casework exhibits it was determined that the Polilight can be used successfully to locate saliva stains for DNA analysis. The sensitivity of luminol for detecting potential bloodstains was greater than that of Polilight; however the Polilight has particular application in instances where a bloodstain may have been concealed with paint. Overall, the Polilight is a relatively safe, simple, noninvasive, and nondestructive technique suitable for use in forensic casework.     

Preliminary Observations on the Ability of Hyperspectral Imaging to Provide Detection and Visualization of Bloodstain Patterns on Black Fabrics

Abstract: The analysis of bloodstain patterns can assist investigators in understanding the circumstances surrounding a violent crime. Bloodstains are routinely subjected to pattern analysis, which is inherently dependent upon the ability of the examiner to locate and visualize bloodstain patterns on items of evidence. Often, the ability to properly visualize bloodstain patterns is challenging, especially when the stain patterns occur on dark and/or patterned substrates. In this study, preliminary research was performed to better understand how near‐infrared reflectance hyperspectral imaging (HSI) could be used to observe bloodstain patterns on commonly encountered black fabrics. The ability of HSI to visualize latent bloodstains on several commonly encountered substrates is demonstrated. The images acquired through HSI are of sufficient quality to allow for differentiation between stains produced from an impact mechanism or a transfer mechanism. This study also serves as a proof of concept in the differentiation of multiple staining materials. Because of its ability to generate spectral data, the data provide a preliminary separation of stains where more than one type of stain existed.     

The detection of Y chromosomes in bloodstains--a reevaluation

A method has been described for detecting Y chromosomes in the leukocytes of human bloodstains prepared on a variety of substrates. The factors that influence the proportion of chromosomes exhibiting a Y spot (the Y cell index) in a bloodstain are considered, including the subjective nature of assessment of the Y chromosome fluorescence, the substrate, and the age of bloodstain. In contrast to previous workers no decay in Y cell index with the age of the stain was observed. The results of a blind trial involving stains derived from case work, where from other evidence there was no doubt as to the sex of the donor, are presented. Sixty-five percent of the male bloodstains were correctly identified and no females were wrongly reported as male.     

Multivariate analysis for estimating the age of a bloodstain

Our objective is to provide crime laboratories with a technique for estimating the age of a bloodstain. Toward that goal, we have used multiplexed, real-time RT-PCR (or qPCR) to determine the relative stability of different-sized segments of the same RNA species as well as differences in stability between two different RNAs' change over time in bloodstains. Our results indicate that a multivariate analysis of the changing ratio of the different RNA segments can be used to differentiate between samples of different ages in the defined population. Bloodstains from 29 of 30 donors could be partitioned into different ages using this technique. Although further improvements will be required before this approach can be implemented in crime laboratories, the multivariate analysis holds promise of providing a reliable approach for temporally linking a bloodstain to the commission of a crime or excluding a bloodstain as being irrelevant to the case in question.     

Changes in the morphology and presumptive chemistry of impact and pooled bloodstain patterns by Lucilia sericata (Meigen) (Diptera: Calliphoridae)

Bloodstain pattern analysis can be critical to accurate crime scene reconstruction. However, bloodstain patterns can be altered in the presence of insects and can confound crime scene reconstruction. To address this problem, we conducted a series of controlled laboratory experiments to investigate the effect of Lucilia sericata (Meigen) on impact bloodstains and pooled bloodstains in association with three combinations of common surfaces (linoleum/painted drywall, wood floor/wallpaper, and carpet/wood paneling). L. sericata fed from the pooled bloodstains and added insect stains through regurgitation and defecation of consumed blood. L. sericata formed defecatory trails of insect stains that indicated directionality. Defecatory stains fluoresced when viewed at 465 nm with an orange filter. These observations differed from Calliphora vicina insect stains because feeding on blood spatter was not observed and trails of insect stains were formed by L. sericata. The fluorescence of defecatory stains can be used as a method to detect insect stains and discriminate them from real bloodstains.     

Exsanguinated blood volume estimation using fractal analysis of digital images

The estimation of bloodstain volume using fractal analysis of digital images of passive blood stains is presented. Binary digital photos of bloodstains of known volumes (ranging from 1 to 7 mL), dispersed in a defined area, were subjected to image analysis using FracLac V. 2.0 for ImageJ. The box-counting method was used to generate a fractal dimension for each trial. A positive correlation between the generated fractal number and the volume of blood was found (R(2) = 0.99). Regression equations were produced to estimate the volume of blood in blind trials. An error rate ranging from 78% for 1 mL to 7% for 6 mL demonstrated that as the volume increases so does the accuracy of the volume estimation. This method used in the preliminary study proved that bloodstain patterns may be deconstructed into mathematical parameters, thus removing the subjective element inherent in other methods of volume estimation.     

二次溅落状血迹形态的模拟实验观察

目的观察二次溅落状血迹的形态特征,探讨其形成机理及与行为方式的关系。方法建立室内现场环境,用10mL血液,以单滴连续滴落或一次性倾倒方式,从10～160 cm高度以10 cm为间隔,制作二次溅落状血迹模型;用棍棒打击及撞击方式制作低位打击及头部撞击溅落血迹模型。观察各种血迹分布,并分析比较其形态差异。结果二次溅落状血迹和头部撞击溅落状血迹在垂直面上均形成倒三角形空白区域的分布形态,但前者分布面积较后者小;低位打击溅落在垂直面上的血迹无此特征,且打击飞溅血滴的飞行角度大于二次溅落血迹。结论利用二次溅落状血迹的形态特征,对于鉴别低位溅血、分析行为方式、证实相关证词具有重要价值。     

5种免提取试剂盒检验滤纸血痕结果的比较

目的探讨5种免提取试剂盒对滤纸血痕样本检验的效果。方法陈旧滤纸血痕(存放时间12～14个月)及新鲜滤纸血痕(存放时间小于1个月)各920份,分别随机分为5组。应用AGCU 17+1、Goldeneye 20A、Powerplex16HS、Identifiler Plus、Identifiler Direct 5种免提取试剂盒进行检验,对比各组检验结果。结果陈旧滤纸血痕5种试剂盒的检验成功率为98.91%～100%,各组间无差异(P>0.05);新鲜滤纸血痕,Identifiler Plus和Identifiler Direct试剂盒检验成功率高于AGCU17+1、Goldeneye 20A及Powerplex 16HS试剂盒(P<0.01);将样本做陈旧化处理后再用成功率较低的3种试剂盒进行检验,成功率分别升至100%、99.46%、99.46%;Identifiler Plus试剂盒扩增循环27次效果优于28次。结论本文5种试剂盒均可用于滤纸血痕的直接扩增检验,但使用AGCU17+1、Goldeneye 20A及Powerplex 16HS试剂盒需将新鲜血痕做陈旧化处理;Identifiler Plus试剂盒需将循环次数降为27次。     

Gm(11) grouping of dried bloodstains

An absorption inhibition method for the detection of gamma marker Gm(11) in dried bloodstains is described. Particular reference is made to the association of Gm(11) with Gm(-1, -2). When a dried bloodstain fails to inhibit anti-Gm(1) and anti-Gm(2), this may represent a true Gm(-1, -2) result or there may be insufficient material to inhibit either antibody. The detection of Gm(11) in a bloodstain extract provides an objective means of confirming the apparent absence of Gm(1) and Gm(2) as representing a true Gm(-1, -2) result. This antigen compares very well with other blood group systems with regard to the amount of bloodstain required for analysis and its stability. No evidence is available for preferential loss of Gm(1) and Gm(2) relative to Gm(11) in dried bloodstains.     

微量生物物证提取套装在现场微量血痕DNA检验中的应用

目的探讨微量生物物证提取套装应用于提取现场微量血痕DNA的检验效果。方法将静脉血制成地面血痕。分别应用微量生物物证提取套装法和普通法提取血痕。分别于恒温摇床放置2、24、48、72、96h(每组50份)进行血痕DNA检验,对比各组检验结果。结果恒温摇床上分别放置24、48、72、96h后,微量生物物证提取套装法的DNA检出率(分别为100.00%、100.00%、100.00%、96.00%)均高于普通法(分别为62.00%、26.00%、10.00%、0),差异均具有统计学意义(P<0.05)。恒温摇床上放置2h,两种方法的DNA检出率差异无统计学意义(P>0.05)。结论微量生物物证提取套装可有效提升放置时间较久的现场微量血痕的检出率和检出时限。     

应用ELISA检测人IgG进行血痕种属鉴别的研究——酶联免疫在物证检验中的应用研究之一

本文应用 ELISA-双抗体夹心法,通过检出血中的人 IgG 鉴定人血痕。双抗体夹心法是常用来检测抗原的一种方法,但在法医学上用于测定血痕种属尚少报道。我们建立的这种方法,新鲜人血痕的阳性结果可测到64万倍。保存三年的陈旧血痕仍可测出。马、牛、羊、狗、猪、鸡、鸭、鸽、兔、驴、骡和鹌鹑均为阴性。由于本法灵敏度高、特异性好、试剂易得,勿须贵重仪器,在物证检验中便于推广。     

Improving the point of origin determination in bloodstain pattern analysis

In bloodstain pattern analysis, it is important to know the point of origin (PO) of an impact pattern. This point can be estimated by means of the stringing method, the tangent method, or by commercially available computer programs. In this study, the accuracy of two computer programs was investigated under different conditions. Impact patterns were created by means of a modified mouse trap, and subsequently the PO was calculated. By examining the characteristics of single bloodstains, the influence on the deviation could be determined. To improve the estimation of the PO, it is important to select bloodstains that lie close to the presumable location of the blood source, that are large (width >1.5 mm) and that show an elliptical form. If possible, bloodstains from different walls should be taken into account. Our recommendations may improve the PO determination of impact patterns.     

Determination of the age of bloodstains using immunoelectrophoresis.

The technique of immunoelectrophoresis was used to determine the age of bloodstains. The immunoelectrophoretic patterns (IEP) of bloodstains ranging from 15 days to one year old were obtained by the use of high titer anti-whole human serum. The IEPs revealed gradual disappearance of beta-globulins and gamma-globulin with increase in the age of bloodstains. A comparative study of the IEP of normal human serum with those of the experimental bloodstains showed the absence of some of the corresponding proteins. The absence of a particular serum protein in the IEP of a given bloodstain will indicate the age of that bloodstain.     

Improved Area of Origin Estimation for Bloodstain Pattern Analysis Using 3D Scanning

An important component of crime scene reconstruction is bloodstain pattern analysis (BPA). Where BPA concerns impact patterns, estimating the area of origin is critical information for scene reconstruction. Traditionally, this is achieved by measuring individual bloodstains and performing trigonometric calculations; however, 3D scanning has been proposed as a viable alternative for overcoming logistical and practical concerns with the manual method. Therefore, this project aimed to establish whether the FARO Focus 3D scanner and FARO Zone 3D software can improve the accuracy of area of origin estimates relative to the manual method. We created a series of eight bloodstain impact patterns and performed paired analysis using the two methods to estimate areas of origin for each pattern. Our data suggested that FARO-derived estimates were generally more accurate than using the manual method. FARO-estimated heights of origin areas were generally closer to the true distance. Both methods underestimated the distance from the wall for most patterns originating 150mm or greater from the wall, but overestimated distances for patterns originating closer to the wall. The degree to which distances were underestimated increased significantly the further the blood source was from the wall and was greater for FARO-derived estimates. The results of this research contribute to the validation of these instruments for operational implementation for BPA and should be considered alongside the practical benefits of 3D scanning relative to manual methods. Further, 3D scanning can provide reliable BPA reconstruction documentation for technical review and court presentation.