Influence of the luminol chemiluminescence reaction on the confirmatory tests for the detection and characterization of bloodstains in forensic analysis

Preliminary tests for the detection of stains at crime scenes aim to focus the police work making them more efficient in the combat of criminality. The application of the luminol chemiluminescence reaction (3-aminoftalhidrazida) in presumptive tests for the detection of bloodstains is known for more than 40 years in forensic science. This reaction is based on the emission of light through the chemical reaction of luminol mixed with hydrogen peroxide and a hydroxide in the presence of a catalytic molecule (iron from the hemoglobin) (Laux [1]).This work evaluates the luminol interference and its effect on subsequent serological and DNA testing. Samples prepared with blood and different concentrations of luminol solution containing luminol, peroxide of hydrogen and sodium carbonate, were analyzed. Additionally, samples of serial dilutions of standard DNA mixed with luminol solution were also analyzed. Although presumptive tests with luminol do not establish the characterization and identification of stains at crime scenes, preliminary results indicated that it is suitable for the detection of invisible bloodstains for forensic analysis, with few detrimental effects on the serological tests and subsequent DNA recovery and typing.     

A novel approach to obtaining reliable PCR results from luminol treated bloodstains

In recent years the forensic scientist has been afforded great advances in technology both in the detection of latent bloodstains and in acquiring reliable DNA typing results from very small pieces of physical evidence. Scientists are now able to detect minute quantities of latent bloodstains by utilizing the luminol reagent, oftentimes indicating that an attempt has been made to conceal any evidence of bloodshed. With the introduction of PCR based technology to the forensic arena, scientists are now routinely able to obtain DNA typing results from previously insufficient amounts of biological material, items as small as a single hair, saliva on a cigarette butt, or a bloodstain the size of a pin head. We present here a merging of these two advances coupled with a new collection medium for post luminol treated latent bloodstains. The forensic scientist is now able to routinely isolate and recover an adequate amount of DNA suitable for PCR typing at all of the Promega GenePrint PowerPlex 1.1 loci. In this study, several dilutions of latent bloodstains were prepared in an effort to simulate transferred bloodstains that are routinely encountered in a crime scene setting. The latent bloodstains were treated with luminol and subsequently collected using conventional cotton tipped swabs as well as a Puritan sponge tipped swab. PCR typing at the Promega GenePrint PowerPlex 1.1 loci was then attempted upon all dilutions of the latent bloodstains for both collection mediums. The results clearly indicate that it is now routinely possible to recover adequate amounts of DNA suitable for PCR typing upon post luminol treated bloodstains.     

ABO基因分型与多重STR联合检测在法医学中的应用

目的建立ABO基因型和Goldeneye16A试剂盒联合检测的方法,并评价其在法医学实践中的应用价值。方法将6种ABO基因型(A/A,A/O,B/B,B/O,A/B,O/O)的序列特异性引物(PCR-SSP)检测方法与Goldeneye16A试剂盒相整合进行同步分型。通过对460份男性个体血痕样本、9947A DNA及90份案件样本进行检测,考察方法的一致性、灵敏度及对法庭科学检材的适用性。结果应用本文方法可同时检出6种ABO基因型和15个常染色体STR基因座及性别决定基因座,检测灵敏度为125pg,其中ABO基因检测灵敏度达63pg。460份男性血痕和90份案件检材证实该联合分型方法用于各类检材结果准确、稳定。结论本文ABO基因分型与多重STR联合检测方法,适用于各类含有核细胞的生物检材,在法庭科学DNA鉴定中有较好的应用前景。     

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.     

应用DNA工作站进行批量血斑STR分型的研究

目的建立对大批量血斑样品PCR-STR基因分型检测的自动化新方法。方法应用自动化DNA工作站改良优化Chelex-100法和DNAIQ磁珠法的实验条件,建立两种批量血斑的自动化DNA提取方法;筛选确定PCR-STR反应体系的构建和PCR-STR产物测序电泳分析前处理程序。结果1104份血斑样品经Chelex-100法批量提取、Profiler Plus试剂盒扩增均一次检出9个STR基因座,定量PCR测定模板浓度均值为0.43ng/ml,荧光检测信号在200~800RFU之间;对其中114份血斑样品用DNAIQ磁珠法批量提取、同试剂盒扩增均一次检出9个STR基因座,定量PCR测定模板浓度均值为0.7ng/ml,荧光检测信号在1000~2000RFU之间;对其中50份血斑进行自动和手动Chel-ex-100检验法比较,成功率分别为100%和98%,且前者等位基因峰高信号更均衡。结论本文建立的自动化DNA工作站批量检测方法,在成功率、稳定性、均一性等方面具有优势。     

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.     

用双色荧光原位杂交技术鉴定血痕性别

目的 探讨荧光原位杂交技术在血痕性别鉴定中应用及其价值。方法对20例新鲜人血及20例1-2年人血痕的X、Y染色体采用双色荧光探针进行原位杂交分析。结果 人新鲜血,男性X、Y信号检出的完整率为98.8％,其中Y信号的检出率为100％,女性X、X信号检出的完整率为96.5％;1-2年人血痕,男性X、Y信号检出的完整率为88％,其中Y信号的检出率为90％,女性X、X信号检出的完整率为80％;40例血液(痕)性别检测的符合率为100％。结论双色荧光原位杂交技术可以鉴定血痕性别。     

A Comparison of Four Presumptive Tests for the Detection of Blood on Dark Materials

Detection of blood on dark materials is difficult for crime scene examiners so presumptive tests are used to assist. This study compared the ability of luminol, leuko crystal violet, tetramethylbenzidine, and Combur Test®E to detect whole, diluted blood (1:100) and a key‐shaped blood transfer stain (1:10), on dark cotton sheeting, tea towel, socks, synthetic carpet, and car mats. Powdered bleach was used to evaluate specificity of the blood detection tests. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall misclassification rate (OMR) assessed the quality of the blood tests. Luminol was the preferred test for diluted blood having the highest sensitivity (79%–96%), NPV (66%–93%), and the lowest OMR (3%–15%). Luminol was also found to be most efficient with a testing time on 25 items of 2 h 50 min compared with up to 8 h. Overall, luminol was the most effective method, also providing information on bloodstain patterns.     

The use of monoclonal anti-M and anti-N antibodies in forensic medicine

Anti-M and anti-N monoclonal antibodies (MA) may be useful for bloodstain analysis by absorption-elution reaction. In order to detect N antigen in bloodstains aged up to 4 weeks the material tested must be treated by methanol. The material fixation is not recommended for analysis of "aged" bloodstains as well as for M antigen detection. Anti-M MA may be used for analysis of liquid blood using agglutination reaction.     

The application of visible wavelength reflectance hyperspectral imaging for the detection and identification of blood stains

Current methods of detection and identification of blood stains rely largely on visual examination followed by presumptive tests such as Kastle–Meyer, Leuco-malachite green or luminol. Although these tests are useful, they can produce false positives and can also have a negative impact on subsequent DNA tests. A novel application of visible wavelength reflectance hyperspectral imaging has been used for the detection and positive identification of blood stains in a non contact and non destructive manner on a range of coloured substrates. The identification of blood staining was based on the unique visible absorption spectrum of haemoglobin between 400 and 500 nm. Images illustrating successful discrimination of blood stains from nine red substances are included. It has also been possible to distinguish between blood and approximately 40 other reddish stains. The technique was also successfully used to detect latent blood stains deposited on white filter paper at dilutions of up to 1 in 512 folds and on red tissue at dilutions of up to 1 in 32 folds. Finally, in a blind trial, the method successfully detected and identified a total of 9 blood stains on a red T-shirt.     

Age estimation of bloodstains: a preliminary report based on aspartic acid racemization rate

This study describes an innovative application of a well-established method of age determination. The conventional method of aspartic acid racemization (AAR) is based on estimation of the d-l-aspartic acid ratio in slow turnover tissues, such as tooth tissue, to reflect the age of an individual. This method has been recently applied to age estimation in forensic investigations, and is also widely used for archeological dating of fossils. We suggest that the aspartic acid racemization method could be applied to a significant, although unresolved, forensic issue: that of bloodstain dating. Standard kinetic experiments were used to describe the characteristics of the racemization reaction in bloodstains, which were then employed to estimate the age of various samples. The soluble protein fraction of a bloodstain produced a stronger correlation between elapsed time and d-aspartic acid content than total amino acid fractions. According to our preliminary results, the time lapse after the creation of a bloodstain can be determined ex vivo by measuring the extent of aspartic acid racemization. Our analysis highlights the need for further study into the preservation and composition of bloodstains to assist in further development of this pioneering application.     

Chemical Enhancement Techniques of Bloodstain Patterns and DNA Recovery After Fire Exposure*

Abstract: It is common in forensic casework to encounter situations where the suspect has set a fire to cover up or destroy possible evidence. While bloodstain pattern interpretation, chemical enhancement of blood, and recovery of deoxyribonucleic acid (DNA) from bloodstains is well documented in the literature, very little information is known about the effects of heat or fire on these types of examinations. In this study, a variety of known types of bloodstain patterns were created in a four‐room structure containing typical household objects and furnishings. The structure was allowed to burn to flashover and then it was extinguished by firefighters using water. Once the structure cooled over night, the interior was examined using a bright light. The bloodstains were evaluated to see if the heat or fire had caused any changes to the patterns that would inhibit interpretation. Bloodstain patterns remained visible and intact inside the structure and on furnishings unless the surface that held the blood was totally burned away. Additionally, a variety of chemical techniques were utilized to better visualize the patterns and determine the possible presence of blood after the fire. The soot from the fire formed a physical barrier that initially interfered with chemical enhancement of blood. However, when the soot was removed using water or alcohol, the chemicals used, fluorescein, luminol, Bluestar^®, and Hemastix^®, performed adequately in most of the tests. Prior to DNA testing, the combined phenolphthalein/tetramethyl benzidine presumptive test for the presence of blood was conducted in the laboratory on samples recovered from the structure in an effort to assess the effectiveness of using this type of testing as a screening tool. Test results demonstrated that reliance on obtaining a positive presumptive result for blood before proceeding with DNA testing could result in the failure to obtain useful typing results. Finally, two DNA recovery methods (swabbing the stain plus cutting or scraping the stain) were attempted to evaluate their performance in recovering samples in an arson investigation. Recovery of DNA was more successful in some instances with the swabbing method, and in other instances with the cutting/scraping method. Therefore, it is recommended that both methods be used. For the most part, the recovered DNA seemed to be unaffected by the heat, until the temperature was 800°C or greater. At this temperature, no DNA profiles were obtained.     

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、96 h(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、96 h后,微量生物物证提取套装...     

Comparative studies between counterimmunoelectrophoresis and microprecipitation method of identification of human minute bloodstains

A minute bloodstain on a thread 2 mm in length was tested to identify human origin by counterimmunoelectrophoresis (CIE), modified CIE, and microprecipitation method (MPM), using anti-human HbA serum. The detection limits expressed as the highest dilution of human blood on the thread in positive reaction were 1:160, 1:320, and 1:320 in CIE, modified CIE, and MPM, respectively. About 1 h was required to obtain the results. In CIE and modified CIE, the detection limits diminished according to the reduction of the samples from one half to one-eighth of the thread, but not in MPM.     

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.     

Simultaneous determination of eight catechins and four theaflavins in bottled tea by liquid chromatography-tandem mass spectrometry for forensic analysis

Tea, and particularly bottled tea, is widely consumed worldwide and is often encountered at crime scenes in poisoning cases or used in place of urine in drug abuse monitoring. Tea is a rich source of polyphenols, such as catechins and theaflavins, and these compounds are useful for identification of trace quantities of tea samples. However, information on the contents of catechins and theaflavins in bottled tea is limited. In this study, a method was developed for simultaneous analysis of eight catechins and four theaflavins in tea using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The concentrations of these polyphenols were determined in bottled black, oolong, and green teas after a simple pretreatment process by the standard addition method. The developed LC-MS/MS method was rapid and all tested polyphenol compounds were separated within ~14 min. All tea types contained all the catechins, at varying concentrations, but not all the theaflavins were present in all the tea types. This indicates that the theaflavin composition reflects the degree of the fermentation and could be used for discrimination among different types of tea. All the green tea samples contained all eight catechins; however, the concentrations of these compounds varied among the tea samples. Principal component analysis and hierarchical cluster analysis were useful for discrimination of samples. It has been unclear whether the variations of chemical components are useful for forensic discrimination. Our results demonstrate that, in addition to identification of tea varieties, catechins and theaflavins can be used for the discrimination of bottled tea samples.     

The presumptive reagent fluorescein for detection of dilute bloodstains and subsequent STR typing of recovered DNA

A presumptive reagent for dilute blood detection other than luminol is fluorescein. The sensitivity of fluorescein approaches the sensitivity of detection levels of luminol. The fluorescein detection method offers the advantages of working in a lighted environment, and the reaction persists longer than luminol. A series of diluted bloodstains, ranging from neat to 1:1,000,000, was placed on a variety of substrates. Three sets were made per substrate. One set was exposed to fluorescein, one set was exposed to luminol, and one set served as an uncontaminated control. The fluorescein signal persisted longer than luminol. However, background staining for fluorescein was observed on some substrates within 30 s to 1 min, and no background staining was observed for luminol. Stains on non-absorbent surfaces were detectable at 1:100,000 dilutions, and stains on absorbent surfaces were detectable usually at no more than 1:100. The sensitivity of detection of fluorescein was comparable to that of luminol in this study. In all cases, where sufficient DNA was recovered, typeable results at all 13 core CODIS STR loci were obtained from treated bloodstains and controls. The results from STR typing indicate that there was no evidence of DNA degradation.     

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.     

Detection of latent bloodstains beneath painted surfaces using reflected infrared photography

Bloodstain evidence is a highly valued form of physical evidence commonly found at scenes involving violent crimes. However, painting over bloodstains will often conceal this type of evidence. There is limited research in the scientific literature that describes methods of detecting painted-over bloodstains. This project employed a modified digital single-lens reflex camera to investigate the effectiveness of infrared (IR) photography in detecting latent bloodstain evidence beneath a layer or multiple layers of paint. A qualitative evaluation was completed by comparing images taken of a series of samples using both IR and standard (visible light) photography. Further quantitative image analysis was used to verify the findings. Results from this project indicate that bloodstain evidence can be detected beneath up to six layers of paint using reflected IR; however, the results vary depending on the characteristics of the paint. This technique provides crime scene specialists with a new field method to assist in locating, visualizing, and documenting painted-over bloodstain evidence.     

Improving Luminol Blood Detection in Forensics

The aim of this study was to develop chemical improvements to the original Weber protocol, in order to increase the intensity and time length of light emission and to eliminate false‐positive reactions. The intensity and duration of light were measured on serial blood dilutions using a plate reader chemiluminometer. Blood stains of various concentrations were impregnated in pure cellulose, dried, and luminol solution was added with/without the potential enhancers. An in silico study was also conducted, aiming to demonstrate the enhancing mechanism of hemoglobin denaturation using 8 M urea. The luminol blood detection test revealed important improvements after urea pretreatment or in the presence of monochloro‐triazinyl‐β‐cyclodextrin. This approach also eliminated the false‐positive reaction from sodium hypochlorite. These improvements could provide a higher sensitivity under particular circumstances such as old or washed blood stains, leading to a better localization for further DNA typing and higher quality photographic analysis.