Bloodstain age estimation through infrared spectroscopy and Chemometric models

The chemical profiling of bloodstains is essential to link the suspect with the crime. The current study proposed a proof-of-concept methodology for the investigation of bloodstains by utilizing advanced ATR-FTIR spectroscopy coupled with new generation chemometric methods. Current study provides encouraging data to allow discrimination between human and animal blood though with small sample size. In this study, different models for the age estimation of human bloodstains are developed from the trained data sets of 1–175 days old bloodstains. The models such as curve estimation (CE), multiple linear regression (MLR), and partial least squares regressions (PLSR) are developed to determine the best prediction model for aged human bloodstains. The obtained results on the dating of bloodstains are very encouraging and also tested for unknown samples. The maximum dating errors are observed in the curve estimation models whereas, the other models MLR, PLSR show excellent age estimation of unknown bloodstains. These models represent an error of ~3 ± 1 days and ~4 ± 1 days in actual and estimated date, respectively, which is lowest ever reported so far. The present methodology is expected to provide a valuable insight into forensic society and hence, to the law enforcement community. The present methodology can further be explored for an ideal model by including all other external variables/factors and for more longer aging time.     

A sandwich enzyme immunoassay for human muscle-specific beta-enolase and its application for the determination of skeletal muscle injury

A sensitive sandwich enzyme immunoassay for human beta-enolase was developed and used to examine beta-enolase in blood or bloodstains as a marker for the determination of skeletal muscle injury. Human beta-enolase was purified from human skeletal muscle, and then an antibody against it was prepared. Polystyrene balls coated with rabbit anti-human beta-enolase IgG were incubated with human beta-enolase and then with anti-human beta-enolase Fab'-peroxidase conjugate. Peroxidase activity bound to the polystyrene balls was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as a hydrogen donor. The detection limit for human beta-enolase was 2.6 pg (30 amol) per assay. The degree of cross-reaction of the sandwich enzyme immunoassay for other organs except for heart (1/10) was about 1/150 or less. Moreover, the localization of beta-enolase in various human tissues was examined by Northern blot analysis, and this confirmed that beta-enolase was expressed only in skeletal and cardiac muscle. Antigenic activity in bloodstains containing beta-enolase was recovered well after storage for 60 days at room temperature. The ratio of beta-enolase to total protein in bloodstains made from non-traumatic blood, nasal hemorrhage and menstrual blood, was within the normal range. In contrast, the ratio of beta-enolase in bloodstains from traumatic blood was obviously elevated (10-30 fold) in comparison with non-traumatic blood. Furthermore, the ratio of beta-enolase was proved to be higher in stains adhering to weapons that had passed through skeletal muscle, indicating that detection of beta-enolase in bloodstains could be used to distinguish crime weapons. These results suggest that beta-enolase is a useful marker for identification of skeletal muscle injury as well as for detecting the origin of bleeding.     

Validation studies of rapid stain identification-blood (RSID-blood) kit in forensic caseworks

When bloodstains are detected at crime scene using presumptive tests (e.g. luminol, phenolphthalein, leuchomalachite green), it is important to establish the real human nature of each stain. This is possible using confirmatory tests. One of these is rapid stain identification-blood (RISD-blood) a lateral flow immuno-chromatographic strip test format which allows the identification of human blood by detection of glycophorin A, a red blood cell membrane antigen, using two anti-human glycophorin A (GPA) monoclonal antibodies.The aim of this study is to assess the sensitivity of RSID-blood test in old, degraded bloodstains and in some bloodstains previously treated with BlueStar Forensic, a presumptive test which is often used in crime scene investigations to detect latent bloodstains. The genetic analysis of all bloodstains of confirmed human nature was subsequently performed using the AmpF1STR Identifiler PCR Amplification Kit (Applied Biosystems), to validate the possibility of obtain a consistent and reliable DNA typing results.     

现场血迹推断出血点位置的研究进展

血迹是命案现场最常见的痕迹之一,对血迹形态的分析是一种重建犯罪现场的有效方法,而根据现场血迹推断出血点位置则是血迹形态分析的主要任务之一。随着血迹研究的不断发展,出现了许多不同的方法（如传统拉线法、三角函数法、绘图法、计算机程序法、公式计算法、回归方程法等）用于推断出血点位置。通过结合国内外相关文献,对不同的方法进行了综述,以期为相关研究提供参考。     

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.     

人血痕ORM、Pi、AHSG和GC同步等电聚焦分型

作者在国内外首次建立了血清类粘蛋白、α1－抗胰蛋白酶、α2－HS－糖蛋白和型特异性成份等四种遗传标记同步等电聚焦免疫酶放大分型方法。累计个人识别机率为0．9878，非父排除率为0．6648，是国内外已报道同步电泳分型方法中鉴别能力最高的。本法可对稀释度为1／100的血清进行分型。对于室温保存四周的血痕盯全部正确分型。除Pi外，ORM、AHSG和GC三种遗传标记至少在24周之内皆可正确分型。     

Identification of male bloodstains by dot hybridization of human Y chromosome-specific deoxyribonucleic acid (DNA) probe

The sex determination of bloodstains was performed using a human Y chromosome-specific (DNA) fragment of 1.9-kb length as a hybridization probe. The DNA samples were taken from 1- and 4-week-old bloodstains of males and females, respectively. Strong signals with male DNA were observed by Y-probe, while faint signals with female DNA were detected. In addition, clear signals were observed in the extract samples from male bloodstains (16-week-old) on paper. Dot hybridization of the Y-probe would be widely applicable to studies on sex determination of medicolegal materials such as blood, bloodstains, teeth, and cadaverous parts.     

成都地区汉族群体C_3表型频率调查与血痕中C_3表型的检测

用醋酸纤维素薄膜电泳免疫固定法,调查了成都地区汉族群体 C_3表型频率的分布。在400例无关健康献血员中发现三种 C_3表型,SS 型397例,FS 型2例,SS_(var)型1例,其基因频率是 C_3~S=0.9963,C_3~F=0.0025,C_3~(Svar)=0.0013。表现型观察值与期望值吻合(p>0.95)。37℃和室温中放置2天的血痕,4℃中放置23天的血纱布和放置35天的玻璃上血痕,-20℃中放置至少87天血液的 C_3表型,可全部检出。人血清在室温中放3天,4℃中放13天,-20℃中至少放106天,可以全部检出 C_3表型。5例亲子鉴定案中检查了 C_3系统。     

Haptoglobin typing of human bloodstains using a specific DNA probe

The stability of DNA in human bloodstains and various post mortem tissues has been investigated. High molecular weight (HMW) DNA was usually recovered from dried bloodstains, even those up to a few years old, but very rapid degradation was found to occur post mortem in the liver, pancreas, spleen and kidney. Other tissues such as the heart, thyroid and skeletal muscle were found to give a reasonable yield of HMW DNA during the first few days after death. The feasibility of using DNA extracted from forensic bloodstain specimens for the detection of DNA polymorphisms was explored using a human haptoglobin (Hp) alpha chain specific probe. Using HindIII and XbaI digests the Hp genotypes Hp2, Hp1F and Hp1S were distinguished by Southern blot analysis in DNA prepared from 1 cm2 bloodstains up to 15-18 months old.     

广州人群红细胞GLOI表型的分布调查及血痕GLOI的检测

本文报道了广州地区人群的GLOI表型分布,基因频率为:GLOI~1=0.1716,GLOI~2=0.8284。室温下(25-30℃)保存的血痕20天内可全部正确分型;日晒8小时,露天过夜,4℃保存105天的血痕均可正确分型;流水冲洗2小时的血痕不能分型;室内腐败血标本9天内检测结果可靠。在实际应用时应考虑这些因素。     

Differentiation between human and chimpanzee in bloodstains by enzyme-linked immunosorbent assay (ELISA) using antihuman serum

An enzyme-linked immunosorbent assay (ELISA) for species identification of human bloodstains using two commercially available antisera against human serum is described. Human bloodstains were to be distinguished from those of chimpanzees and other animals using raw antisera, and the differentiation between human and chimpanzee became more evident when those antisera were absorbed with a small amount of chimpanzee plasma. Human bloodstains could clearly be identified by the present method even after 4 weeks of aging at room temperature.     

Antigenic properties of human and animal bloodstains studied by enzyme-linked immunosorbent assay (ELISA) using various antisera against specific plasma proteins

Antigenic properties of bloodstains of human and non-human primates as well as other animal bloodstains were investigated by the inhibition ELISA using commercially available anti-human albumin (Alb), alpha 2-macroglobulin (alpha 2-M), fibrinogen, transferrin, and immunoglobulin G. In general, chimpanzee bloodstains showed strong cross-reactions with these antisera, and the extent of the cross-reactions of other animal bloodstains decreased largely with the phylogenic order, i.e., agile gibbon (ape), Old World monkeys (Japanese monkey and hamadryas baboon), New World monkeys (night monkey and tufted capuchin monkey), prosimians (grand galago and ring-tailed lemur) and other animals (rat, cattle, swine, goat, dog, cat, and chicken). Among these antisera, anti-human alpha 2-M showed the weakest cross-reaction with chimpanzee bloodstains, and anti-human Alb showed next.     

酶标2G8单克隆抗体斑点ELISA快速检验人血痕G2m(23)因子

利用自制的抗人 G2m(23)酶标2G8酶标单克隆抗体,首次应用直接斑点 ELISA 方法检测血痕中 G2m(23)因子。该法最小检出量为0.000048μl 血清;整个试验可在15分钟内完成。对常见的13种动物血和8种鱼血无交叉反应;盲测450例干血痕结果全部正确。该方法具有快速、准确、灵敏、简便等特点,有较大的实用价值。     

抗人Tf及抗人Hb试剂盒的比较

目的研究比较抗人Tf(转铁蛋白)和抗人Hb(血红蛋白)试剂盒的实验方法。方法采用胶体金标记单克隆抗体结合免疫层析技术,对不同稀释度的人血、动物血进行检测,并对保存时间、溶解度等影响因素进行研究。结果抗人Tf和抗人Hb试剂盒同样具有简单、快速、灵敏、稳定、特异性好的优点,但抗人Tf试剂盒能检测出陈旧及难以溶解血痕。结论抗人Tf试剂盒适用于法医物证的血痕种属检验。     

Detection of thyroglobulin in blood stains as an aid in the diagnosis of mechanical asphyxia

Mechanical forces applied to the neck region are known to release certain amounts of thyroglobulin into circulation. In this experiment, an attempt was made to detect thyroglobulin in bloodstains as an aid in the diagnosis of mechanical asphyxia. Experimental bloodstains containing thyroglobulin at concentrations of 1, 2, 5 and 10 mu g/mL were prepared on a sheet of filter paper. Small pieces of bloodstains, measuring approximately 2.4 cm2 in area, were extracted with 0.1 mL of distilled water and the extracts were tested against an antihuman thyroglobulin serum by precipitation-electrophoresis. Bloodstains containing more than 1 mu g/mL of thyroglobulin formed distinct precipitin lines for up to one month of storage, while bloodstains containing more than 5 mu g/mL of thyroglobulin formed distinct precipitin lines for up to three months of storage. The present results suggest that the bloodstains can be utilized in the diagnosis of mechanical asphyxia.     

A new marker for estimation of bloodstain age by high performance liquid chromatography.

The applicability of a new marker for estimation of bloodstain age by reverse-phase high performance liquid chromatography (HPLC) is described. Using a microBondasphere C18 column with a two step linear gradient of 10.5-46.25% acetonitrile in 0.1% trifluoroacetic acid, an intriguing peak (unidentified) at a retention time of about 5 min was observed on chromatograms from human adult bloodstains and designated as 'X'. The area of this peak, which could be detected in extracts of bloodstains, but not in their fresh whole blood, increased with time. The ratios of the X area to heme area in bloodstains stored at room temperature and 4 degrees C for up to 52 weeks old linearly correlated with stain age by plotting on a double logarithmic scale. In bloodstains exposed to fluorescent light at room temperature, the regression equation calculated from the ratios (Rx) and the ages of stains in weeks (W) is ln(1000.Rx) = 1.1084 + 0.3937.ln(7.W), and the coefficient of correlation (r) is 0.9776 (n = 144, P < 0.001). When stains were stored at 37 degrees C, the ratio transformed into logarithms correlated linearly with stain age. The regression equation describing the relationship in bloodstains exposed to fluorescent light at 37 degrees C is ln(1000.Rx) = 2.4477 + 0.0866.W (r = 0.9826, n = 144, P < 0.001). The results of the present study suggest that the HPLC method may be applicable to the estimation of bloodstain age.     

A highly specific and sensitive sandwich enzyme immunoassay for human hemoglobin A

A highly specific and sensitive sandwich enzyme immunoassay for human hemoglobin A (Hb A) is described. A rabbit anti-human Hb A IgG-coated polystyrene ball was incubated with human Hb A and subsequently with affinity-purified anti-human Hb A Fab'-horseradish peroxidase conjugates, which had been prepared before and after absorption with Japanese monkey Hb-Sepharose 4B and dog Hb-Sepharose 4B. Bound peroxidase activity was measured by fluorimetry using 3-(4-hydroxyphenyl)propionic acid as a substrate. The detection limits of human Hb A using the conjugate before and after the absorption were 0.65 pg/tube (3 X 10(10)-fold dilution of whole blood) and 2.0 pg/tube (1 X 10(10)-fold dilution of whole blood), respectively. Human Hb A could be discriminated from Hb of animals such as Japanese monkey, dog, cat, pig, horse, sheep, chicken and cow by measuring bound peroxidase activity in the presence and absence of the conjugates prepared before and after the absorption. Human Hb A in bloodstains on cotton gauze could be discriminated from Hb of animals described above even after seven washings. Human Hb A in 220,000-fold diluted bloodstains on cotton gauze could also be discriminated from Hb of animals described above.     

Isotachophoretic analysis of bloodstains: differentiation of human, menstrual, bovine, and ovine bloods

Isotachophoresis, a technique to separate components by constant current electrophoresis, was used to differentiate between bloodstains of male, female, menstrual, bovine, and ovine bloods on cotton cloth and filter paper. Bloodstain analysis by isotachophoresis of stains from male and female subjects showed identical cationic patterns, but gave different profiles in the anionic system. Plasma had one extra peak in the anionic system when compared to the profile of serum. This extra peak is due to the presence of fibrinogen in plasma. Some hemoglobin peaks overlapped with serum protein peaks, but these could be identified by comparisons at lower concentrations. Menstrual blood had a much different pattern than normal human blood as was expected since many more compounds are found in menstrual blood than in normally circulating blood. Human, bovine, and ovine bloodstains showed different profiles both in the cationic and anionic systems. These results indicate that isotachophoresis can be used for the rapid and simple analysis of bloodstains to differentiate reliably human male, female, and menstrual blood and also to distinguish human bloodstains from those of cattle or sheep.     

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.