The absorption-elution technique with the use of highly active proteases: a novel technology for the detection of Rhesus D antigen in bloodstains

The proposed method for the specific detection of Rhesus D antigen in 6 month old bloodstains is based on the use of modified immunoglobulins (anti-rhesus D peptides) in the absorption-elution reaction (AER). These peptides were prepared by an original method using polyclonal anti-rhesus D antibodies and standard erythrocytes preliminarily treated with a highly active protease. Specific identification of D antigen of the Rhesus system obviates the problem of poor AER specificity and thereby significantly improves the quality of forensic biological expertise.     

新鲜血痕样本直接扩增试验条件比较

目的探讨新鲜血痕在不同直接扩增试剂盒的试验条件。方法存放2d内的新鲜血痕FTA卡540份和存放1～3m的陈旧性血痕FTA卡270份,各分别随机均分为3组,每份血痕打3片1．0mm纸片,分别用ONATyper^TM15Plus试剂盒、Goldeye^TM20A试剂盒和华夏。”试剂盒在标准条件（说明书条件）、优化条件1（标准条件＋1μLDMSO）和优化条件2（标准条件＋室温浸泡1h）扩增检验,比较在3种条件下各组STR检验成功率。结果陈旧血痕样本用3种直扩试剂盒,在3种条件下,检验成功率均〉97．00％,且均高于新鲜血痕。新鲜血痕在标准条件和优化条件1下,成功率为27．22％～31．67％,在优化条件2下,检验成功率相似（〉97．00％）,与标准条件和优化条件1比较,有统计学差异（P〈0．01）。结论新鲜血痕在加入直接扩增试剂后于室温浸泡1h,可有效提高STR检验成功率。     

An improved method for subtyping Pi in old bloodstains.

Immunofixation procedures were used for detecting alpha-1 antitrypsin protease inhibitor (Pi) phenotypes in bloodstains. Neuraminidase elution of bloodstains, together with isoelectric focusing, immunofixation, and silver staining techniques, makes possible Pi subtyping in old bloodstains. No extra bands appear when the storage time is no longer than three months.     

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。     

Validation studies of an immunochromatographic 1-step test for the forensic identification of human blood.

An immunochromatographic 1-step test for the detection of fecal occult blood was evaluated for applicability for the forensic identification of human blood in stained material. The following experiments were conducted: 1) determination of the sensitivity and specificity of the assay; 2) evaluation of different extraction media for bloodstains (sterile water, Tris buffer pH 7.5 provided in the test kit, 5% ammonia); 3) analysis of biological samples subjected to a variety of environmental insults; and 4) evaluation of casework samples. This immunochromatographic 1-step occult blood test is specific for human (primate) hemoglobin and is at least an order of magnitude more sensitive than previous methods for detecting human hemoglobin in bloodstains. The antigen is insensitive to a variety of environmental insults, except for exposure to certain detergents and household bleaches and prolonged exposure to certain preparations of luminol. The entire assay can be conducted in field testing conditions within minutes. When in the laboratory the supernatant from a DNA extraction is used for the assay, there is essentially no consumption of DNA for determining the presence of human hemoglobin in a forensic sample. The data demonstrate that this test is robust and suitable for forensic analyses.     

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.     

Lewis typing of human bloodstains by enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b)

The Lewis blood grouping of human dried bloodstains could be determined by an enzyme-linked immunosorbent assay (ELISA) using monoclonal anti-Le(a) and anti-Le(b) antibodies with an avidin-biotin complex (ABC). The bloodstains aged 1 year were used as samples, and approximately 1 mg of the stains was enough to type each Lewis antigen reliably by this method. The Lewis substances of 106 individual stains were correctly typed regardless of their ABO blood group system.     

ELISA检测人IgG确定种属的改良方法——酶联免疫在物证检验中的应用研究之二

本实验采用含1/10,000戊二醛的0.05M,pH9.6碳酸盐缓冲液稀释抗体包被反应板,并使酶标记抗体浓度由1/100提高到1/50,使实验时间由常规方法的20多小时缩短到6小时。1∶320,000稀释的标定的人新鲜血痕浸液仍能得出阳性结果。本实验除用聚苯乙烯反应板外,还使用了聚氯乙烯反应板。两种反应板的实验条件及结果相同。聚氯乙烯反应板可以根据实验所需反应孔的多少进行剪裁,从而使实验材料得到节约。这两种反应板都可以预先用抗体包被后,置-20℃冰箱保存备用,从而简化了操作。本方法快速、灵敏、简单、重复性好,便于推广。     

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.     

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。     

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.     

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.     

Errors in the determination of the point of origin of bloodstains

Bloodstain pattern analysts are sometimes called upon to determine the point of origin of a pattern of bloodstains. A derivation of expressions for the uncertainties deltaX and deltaY in the coordinates of the point of origin P(X, Y) of two bloodstains on a surface has recently been published. These uncertainties were expressed in terms of the uncertainties in the measured distance between the bloodstains and the uncertainties in the angles of impact of the bloodstains. This paper extends the derivations in the previous work by expressing the uncertainties in the coordinates of the point of origin of two bloodstains in terms of the uncertainties in the length and width measurements from which the angles of impact of the bloodstains are calculated.     

Lewis blood group determination in bloodstains by planimetric measurement of eluted monoclonal antibodies

Planimetric measurements were employed for reading the results of an elution test to determine Lewis blood groups in dry human bloodstains. In the absorption-elution test, two varieties of indicators were used to detect eluted Lewis antibodies. First, 64 blood-stains aged between 2 to 8 months were tested with glutaraldehyde (GLA)-treated erythrocytes (planimetric hemagglutination assay, PMHA). This method demonstrated that dry stains weighing approximately 0.4 mg (equivalent to 3 microliters of whole blood) were sufficient for detection of Lea or Leb antigen. Results were obtained within 1 h. Then, 37 of these stains were tested with Lewis substance-coated latex particles (planimetric latex agglutination assay, PMLA). The presence of Lea and Leb antigen were detected from dry stains weighing 0.1 mg (equivalent to 1 microL of whole blood) within 3 h. Both these assays are faster and simpler with accuracy than the enzyme-linked immunosorbent assay (ELISA). Latex particles coated with Lewis substance are, in particular, strongly agglutinated and show agglutination patterns more clearly than erythrocytes. The blind tests using these two methods properly classified 7 Le(a + b-) and 23 Le(a-b + ) bloodstains; whereas, 5 Le(a-b-) stains were undetermined by the criteria for these tests. These results indicate the usefulness of the PMHA and PMLA for typing Lewis blood groups from small 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.     

放射免疫分析法测定性激素判断血痕的性别

本文对测定血痕中睾酮量(T)和全血蛋白量(P),及T/P的比值来判断血痕性别的可能性进行了探讨.通过测定102名健康成年人血痕(男性57名,女性45名),得出本法对男性血痕的肯定率为80.7%,对女性血痕的肯定率为88.9%.实验中还观察到时间因素及某些环境因素(霉变)能使两性血痕T/P值降低.     

Esterase D typing of bloodstains by non-equilibrium focusing

Non-equilibrium focusing in a pH 4-6 gradient in ultra-thin polyacrylamide gels has been shown to be a reliable and reproducible method for detecting the six common esterase D phenotypes (EsD 1,2-1,2,5-1,5-2 and 5) in dried bloodstains. Successful typing is dependent on both the age and phenotype of the stain in question. The effects of age on the isozyme pattern of each phenotype are described and illustrated. In a comparative trial using 100 simulated and 300 authentic casework bloodstains, non-equilibrium focusing was shown to be more efficient than thin-layer starch gel electrophoresis for the typing of esterase D.     

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.     

被洗织物上潜血痕迹发现方法的比较

目的探索被洗涤后衣物上潜血痕迹的发现方法。方法用联苯胺法、鲁米诺法和新型仪器TL-445激光生物检材发现仪寻找不同织物上不同洗涤强度的潜在血迹。结果联苯胺仅能发现部分纯棉织物上潜血,鲁米诺能显示全部纯棉织物上血迹,TL-445激光生物检材发现仪能显示所有纯棉、纯化纤织物上血迹。结论用TL-445激光生物检材发现仪检测相比联苯胺法和鲁米诺法更适合洗涤后织物上潜血痕迹的发现。     

A sensitive sandwich enzyme immunoassay for gamma-seminoprotein and its application to sex discrimination of blood and bloodstains.

A sensitive sandwich enzyme immunoassay for gamma-seminoprotein (p30, prostate-specific antigen) is described for sex discrimination of blood and bloodstains. A polystyrene ball coated with rabbit anti-gamma-seminoprotein IgG was incubated with gamma-seminoprotein and, after washing, with affinity-purified rabbit anti-gamma-seminoprotein Fab'-horseradish peroxidase conjugate. Peroxidase activity bound to the polystyrene ball was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as hydrogen donor. The detection limit of gamma-seminoprotein was 0.15 pg per assay. Blood levels of gamma-seminoprotein, measured using 1-10 microliters of blood, were at least 3.3-fold higher in male adults than in female adults. The ratio of gamma-seminoprotein in terms of pg to hemoglobin in terms of mg was significantly higher in male adults than in female adults. Thus, the measurement of gamma-seminoprotein or both gamma-seminoprotein and hemoglobin was useful for the discrimination of blood and bloodstains of male and female adults, although with some limitations.