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.     

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.     

Development of a radioimmunoassay technique for the detection of human hemoglobin in dried bloodstains

A sensitive radioimmunoassay for the detection of human hemoglobin in dried bloodstains for the purpose of forensic science species identification has been developed. Bloodstains from 13 animal species were tested and found to be negative for human blood. A minimum volume of 0.8 microL of fresh blood is required to produce sufficient stain for successful testing. Bloodstains prepared from newborn and sickle-cell bloods were determined to be human. Bloodstains ranging in age from 1 month to 6 years which had been maintained desiccated at 20 to 25 degrees C were also successfully tested. Positive results were obtained on human bloodstains stored at 24 degrees C with relative humidity ranging from 0 to 98% for a period of 3 weeks. Absolute counts per minute (CPM) decreased with increased humidity. Human bloodstains exposed to bacterial contamination (gram positive or negative species) under humid conditions for 2 weeks also tested positive. Bacterial contamination caused a decrease in CPM, but insufficient to result in an erroneous conclusion as to species of origin. Positive results were also obtained on human bloodstains stored for 6 weeks at various temperatures ranging from -16 to 37 degrees C. No significant decreases in CPM were noted for any of the temperature conditions described.     

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.     

Evaluation of four deoxyribonucleic acid (DNA) extraction protocols for DNA yield and variation in restriction fragment length polymorphism (RFLP) sizes under varying gel conditions.

This study originated from discussions and recommendations of the Technical Working Group on DNA Analysis Methods (TWGDAM). Four bloodstain deoxyribonucleic acid (DNA) extraction protocols and five semen stain DNA extraction protocols were evaluated. Nine laboratories participated in the extraction of DNA from 20 bloodstains and 20 semen stains using each protocol. All blood and semen stains originated from a single donor and were prepared under uniform conditions to permit the direct comparison of DNA yields and restriction fragment lengths. The extracted DNA from approximately 600 bloodstains and 700 semen stains was quantified by yield gel analysis and a slot blot hybridization technique. The extracted DNA was digested and restriction fragment length polymorphism (RFLP) patterns were generated using three single-locus probes. The RFLP sizing data produced from the blood and semen stains were evaluated with respect to (1) DNA extraction method, (2) gel length, (3) agarose type, (4) presence or absence of ethidium bromide in the gel, and (5) fragment sizes obtained from DNA isolated directly from the donor's liquid blood. This study demonstrates conclusively that high-molecular-weight DNA can be isolated using either organic or nonorganic DNA extraction protocols and that the resulting RFLP sizes are highly reproducible regardless of gel length, agarose type, or presence/absence of ethidium bromide.     

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.     

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.     

应用单细胞凝胶电泳测定人血痕淋巴细胞降解的实验研究

目的探讨血痕形成时间与其DNA降解的淋巴细胞出现率的关系。方法应用单细胞凝胶电泳(SCGE)技术结合荧光显微镜和专业的计算机图像分析技术,测定离体72h内不同时间段的血痕中彗星样淋巴细胞出现率。结果获得人离体血液经不同放置时间间隔后,经LUC IA图像分析系统采集得到荧光图像,并获得体现DNA降解趋势的二项式回归方程y=-0.0178X2+2.4623X+8.1098,r2=0.9522,具有高度的统计学意义。结论72h内的人血DNA降解的淋巴细胞出现率与血痕形成时间之间具有高度的线性相关。     

Bloodstain pattern analysis--casework experience

The morphology of bloodstain distribution patterns at the crime scene carries vital information for a reconstruction of the events. Contrary to experimental work, case reports where the reconstruction has been verified have rarely been published. This is the reason why a series of four illustrative cases is presented where bloodstain pattern analysis at the crime scene made a reconstruction of the events possible and where this reconstruction was later verified by a confession of the offender. The cases include various types of bloodstains such as contact and smear stains, drop stains, arterial blood spatter and splash stains from both impact and cast-off pattern. Problems frequently encountered in practical casework are addressed, such as unfavourable environmental conditions or combinations of different bloodstain patterns. It is also demonstrated that the analysis of bloodstain morphology can support individualisation of stains by directing the selection of a limited number of stains from a complex pattern for DNA analysis. The complexity of real situations suggests a step-by-step approach starting with a comprehensive view of the overall picture. This is followed by a differentiation and analysis of single bloodstain patterns and a search for informative details. It is ideal when the expert inspecting the crime scene has also performed the autopsy, but he definitely must have detailed knowledge of the injuries of the deceased/injured and of the possible mechanisms of production.     

D20S161和D8S384两个基因座在法医学中的应用

评估D2 0S16 1和D8S384两个基因座在法医学中的应用价值。用自制的D2 0S16 1和D8S384两个DNA分型试剂盒 ,对人血、人精液、人唾液、动物血、人血与动物血的混合检材和人血痕、人精液斑、人唾液斑、动物血痕、人血与动物血的混合斑痕检材 ,以及陈旧血痕检材进行检测分型 ,并用这两个基因座PCR引物序列与DNA数据库进行联网对比分析。自制的D2 0S16 1和D8S384两个DNA分型试剂盒能对人血、人精液、人唾液、人血与动物血的混合检材分型 ,而动物血没有PCR产物 ;自制的D2 0S16 1和D8S384两个DNA分型试剂盒能对人血痕、人精斑、人唾液斑和人血与动物血的混合斑痕检材正确分型 ,而动物血痕没有PCR产物 ;斑痕检材分型结果与对应体液检材分型结果无差异 ;5 0份陈旧血痕检材全部获得阳性分型结果。DNA数据库联网比较提示 ,D2 0S16 1和D8S384基因座引物除了能与各自的模板序列发生特异性扩增外 ,理论上不能与DNA数据库中 6 0 6 36 4种已知序列产生PCR产物。D2 0S16 1和D8S384两个基因座具有高度的种属特异性 ,抗污染能力强 ,不易受降解的影响 ,是解决法医现场生物检材个人识别和亲子鉴定的理想手段     

用Y—染色体特异DNA探针鉴识微量干血痕性别的研究

法医鉴识干血痕性别,通常是用盐酸阿地平染色观察 Y—小体的方法。重组 DNA 技术的发展与应用,为法医物证检验开辟了新的领域。本文用 Y—染色体特异 DNA 探针鉴识干血痕性别的成功,为法医的血痕性别签定,提供了一种新的检验方法。     

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.     

The detection of Rh antigens (D,C,c,E,e) on bloodstains by a micro-elution technique using low ionic strength solution (LISS) and papain-treated red cells

Ninety experimental bloodstains, were examined, with the intention of detecting the principal Rh antigens, by using a micro-elution method improved by the use of low ionic strength solution (LISS) and papain-treated red cells. This method makes it possible to employ most commercially produced sera in routine forensic haematology laboratory work.The antigens could regularly be detected in stains of the following ages: D, C and c in stains of at least 6 months, E in stains of at least 4 months, and e in stains of at least 2 months.     

Fundamental studies of bloodstain formation and characteristics

A detailed understanding of blood droplet impact dynamics and stain formation is an essential prerequisite to the interpretation of both individual bloodstains and spatter patterns. The current literature on theoretical models for the spreading and splashing of liquid drops on surfaces relevant to the forensic context of bloodstain formation has been reviewed. These models have been evaluated for a paper substrate using experimental data obtained as function of droplet size, impact velocity and angle. It is shown that for perpendicular impact there are fairly simple mathematical models for the spreading diameter and the number of scallops or spines formed around the stain though these have quite limited ranges of validity in their basic form. In particular, predictions for the diameter are best for small droplets impacting at high velocity and the number of spines saturates for higher impact velocities. In the case of spreading, a modification to the energy conservation model is found to provide excellent agreement with experimental stain diameters across a wide range of impact velocities. For non-perpendicular impact, the width of stains is found to depend principally on the normal component of impact velocity and may be predicted by an appropriate modification to the expression for the perpendicular case. Limitations in the calculation of impact angle from the stain aspect ratio are identified and a theoretical basis for the prediction of spines around an elliptical stain is proposed. Some key issues for future research are identified which include a systematic, quantitative study of the effect of surface properties on bloodstain formation.     

Quantitative Differentiation of Bloodstain Patterns Resulting from Gunshot and Blunt Force Impacts

Bloodstain pattern analysis (BPA) provides significant evidentiary value in crime scene interpretation and reconstruction. In this work, we develop a quantitative methodology using digital image analysis techniques to differentiate impact bloodstain patterns. The bloodstain patterns were digitally imaged and analyzed using image analysis algorithms. Our analysis of 72 unique bloodstain patterns, comprising more than 490,000 individual droplet stains, indicates that the mean drop size in a gunshot spatter pattern is at most 30% smaller than the mean drop stain size in blunt instrument patterns. In contrast, we demonstrate that the spatial distribution of the droplet stains—their density as a function of position in the pattern—significantly differs between gunshot and blunt instrument patterns, with densities as much as 400% larger for gunshot impacts. Thus, quantitative metrics involving the spatial distribution of droplet stains within a bloodstain pattern can be useful for objective differentiation between blunt instrument and gunshot bloodstain patterns.     

Determination of C3 phenotype in dried bloodstains using isoelectric focusing

Fresh whole blood and bloodstains were analyzed by isoelectric focusing (IEF) to determine the C3 phenotype of the blood donor. Three common phenotypes exist as a result of two autosomal alleles. The three phenotypes can be identified in fresh serum or in serum samples which had been stored at -20 degrees C for more than a year. Bloodstains maintained in a desiccator at 25 or at 37 degrees C retained the native form of C3 which could be detected for at least two weeks. Beyond two weeks of storage, stains became difficult to phenotype due to decreased banding intensity. Bloodstains aged longer than one month could not be phenotyped. C3 could not be detected in human semen by the serological methods employed.     

Transferrin subtyping of human bloodstains

A method was described for subtyping transferrin derived from human bloodstains. Bloodstain cuttings were extracted in 0.5% ferrous ammonium sulfate. The extracts were subjected to ultrathin-layer polyacrylamide gel isoelectric focusing. After isoelectric focusing, transferrin was detected by silver staining. This method permitted the successful typing of Tf in 6-month-old blood stains maintained at -20 degrees C and room temperature and 3-month-old bloodstains maintained at 37 degrees C.     

Factor B subtyping in sera and bloodstains by isoelectric focusing and immunoblotting

The polymorphism of BF was investigated in 765 unrelated Japanese individuals by isoelectric focusing and immunoblotting. Besides five common subtypes three rare variants were observed. The allele frequencies were: BF*S = 0.8078, BF*FA = 0.1797, BF*FB = 0.0105, BF*Var. = 0.0020. The above method was successfully applied to subtyping BF in stored bloodstains. The determination limits were: at 4 degrees C 8 weeks, at room temperature 2 weeks and at 37 degrees C only 2 days after storage. The BF subtyping is of practical use in medicolegal individualization of unknown bloodstains.     

C7 typing of blood stains using isoelectric focusing and immunoblotting

By means of isoelectric focusing and immunoblotting C7 types were clearly demonstrated from bloodstains which had been stored at 37 degrees C for up to three weeks, at room temperature for up to six weeks and at 4 degrees C for over ten weeks. The C7 typing is practically useful in medicolegal individualization of unknown bloodstains.