ABO and Lewis typing of secretion stains on nitrocellulose membranes using a new dot-blot-ELISA technique

A new method for ABO and Lewis typing of body fluids is described. It combines the advantages of a good antigen binding to nitrocellulose membranes, the need of only very small amounts of stain material and the high sensitivity of an enzyme-linked immunosorbent assay for antigen detection. This is of special interest because conventional ABO and Lewis typing of secretion stains need relatively large stain dimensions. The method is very easy to handle, does not need any expensive equipment and gives a permanent record. Furthermore the high sensitivity offers the possibility of analyzing even sweat and urine stains without the need of concentrating these extracts.     

Identification in blood stains through DNA typing with C4 and HLA-DR probes

A restriction fragment length polymorphism analysis using double digestion of DNA preparations with XbaI and BglII restriction enzymes and hybridization with C4 and HLA-DR probes is described. The typing conditions selected reveal extensive individual variation in both C4 and DR gene regions. In our panel of 46 unrelated individuals, 37 different phenotypic patterns were recognized when both probes were used, and preliminary discriminative power values of 0.865 and 0.914 were calculated for C4 and DR beta, respectively. The probability of a chance match using both systems is probably about 1.5.10(-2). The potential of this method for individual identification of blood stains was demonstrated on DNA prepared from 6-month-old dried blood stains from seven panel individuals. The seven individuals were all identified when comparing stain DNA patterns with panel control patterns. No RFLP pattern changes were observed following storage of blood stains. Based on these experiments with C4 and DR beta DNA typing under laboratory conditions, it is concluded that DNA typing with such probes may become a powerful tool in future stain identification analyses.     

Rapid and sensitive typing of forensic stains by PCR amplification of polymorphic simple repeat sequences in case work.

After failure of conventional typing and multilocus DNA fingerprinting methods to compare a minute vaginal swab stain with blood from a murder victim and a suspect, we used enzymatic DNA amplification (polymerase chain reaction, PCR) to discriminate the DNAs by typing of simple sequence lengths polymorphisms. A mixed dinucleotide locus in the HLA-DRB gene region and three novel tetranucleotide polymorphisms located autosomally as well as on the human Y chromosome were used to exclude a jailed subject from a case of murder.     

Differex~（TM）与Chelex-100法在混合斑检验中的应用比较

目的探讨DifferexTM系统法在法医学性侵犯案件混合斑检验中的应用价值,并与传统Chelex-100法进行比较。方法对日常性侵犯案件中的生物检材分别通过DifferexTM系统法与Chelex-100法检验来比较两种方法的优劣。结果采用Chelex-100法提取时,在所检验样本中仅有床单全部获得单一分型,而采用DifferexTM系统法则只有一条内裤的分型为混合型,其它均为单一分型。应用DifferexTM系统法还可以同时提取女性上皮细胞DNA,成功率为100%。采用Chelex-100法时出现双尖峰的频率也明显超过DifferexTM系统法。结论 DifferexTM系统法在法医学性侵犯案件混合斑检验中比Chelex-100法具有更明显的优势,是一种非常有应用前景的方法。     

Applicability of two commercially available kits for forensic identification of saliva stains

The RSID-saliva test and the SALIgAE-saliva test are two recently developed forensic saliva detection kits. In this study, we compared the sensitivity and the specificity of the two test kits with the Phadebas amylase test by analyzing amylases from various sources including human, animals, plants, and micro-organism. The data demonstrate that the RSID-saliva test and the SALIgAE-saliva test offer higher sensitivity and specificity for the detection of saliva than the Phadebas amylase test. The detection limits of the RSID-saliva test, the SALIgAE-saliva test, and the Phadebas amylase test equate to 10, 4, and 1000 nL, respectively for human saliva. The RSID-saliva test and the SALIgAE-saliva test were further evaluated by analyzing semen, vaginal secretion, breast milk, blood, urine, sweat, and feces. The results of the two tests are in good agreement. The two tests reacted with urine, breast milk, and feces, but not with semen, vaginal secretion, blood, and sweat.     

Expert opinion on biologic stains. Determination of status, future trends

In this paper an attempt is made to critically review the literature, with special emphasis on bloodstain analysis. One essential aim is the integration of this field into casework. Three basic components in skillful assessment of stains are described: (1) analysis of stain morphology, (2) discriminating and attributing analyses, (3) individualization. Regarding the first, the analysis of stain morphology is based upon the extensive experimental literature published since 1895--mainly in continental Europe. Since 1971 there have also been publications in the American literature. The large family of stain forms and their dependency on multiple variables are described, especially regarding the modes of formation, the energy of impact, and the physical properties of the substrate. The essential elements for reconstruction of the crime are described. The areas of application are arranged in case groups. Since in case work the stain pattern is complicated by many artifacts and overlaps, forensic pathologists are considered the ideal experts for the analysis of bloodstain patterns, as they have a profound knowledge of the type and sequence of injuries. If this is not the case, the forensic pathologist should at least be integrated into the investigating team. In practical application, the stain form is not always adequately analysed. The education and training of pathologists should be improved to achieve this standard. Analysis of the stain morphology and a subsequent selection of stains are also essential prerequisites for meaningful further investigations. By the use of discriminating and attributing analyses, one can as a rule arrive at a definite answer by using only one test. This is true for basic questions such as the identification of blood type, as well as proof of exclusion. One can distinguish between traditional methods, the new field of immunochemistry and rarely used methods. Immunochemistry has permitted success in recent years in determination of the blood group from hair. It is recommended that reference laboratories be established for training in these rare methods. Individualization analyses are subdivided into two large fields: non-DNA individualization and DNA individualization. It is postulated that in the future stain laboratory both areas will coexist. In non-DNA individualization, essential progress has been made. The detection of protein polymorphisms by blotting and subsequent visualization by antibody-linked enzyme/substrate reactions has led to a considerable increase in sensitivity and specificity.(ABSTRACT TRUNCATED AT 400 WORDS)     

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     

汗潜指印的STR分型检测

目的探索汗潜指印的荧光STR复合扩增检测的方法。方法采用Chelex-100和Microco-100浓缩柱,提取汗潜指印中DNA,STR复合扩增荧光电泳检测。结果 105例汗潜指纹STR分型可明确判读5个以上基因座的占30.3％,个体之间的差异、捺印指印时用力大小以及指印遗留在客体上时间的长短均影响检测成功率。结论该汗潜指印的DNA提取方法步骤简单,方法较为稳定,使单枚汗潜指印可望获得DNA分型。     

HLA 及其在法医学上的应用

HLA 是迄今人类最复杂的遗传多态性系统,其在法医学上的应用,包括亲子鉴定和个人识别的重要性日益被认识。作者等对血痕、血清和唾液的 HLA 测定的研究结果证明:(1)血痕中存在较强的抗补体作用。应用淋巴细胞毒抑制试验测定血痕的 HLA 时,增加补体用量可使测定的正确率达100%,经90天的血痕仍能完全正确检出 HLA 型。(2)血清的 HLA 测定是可行的,这对远道送检血清及尸体血清测定 HLA 有一定价值。(3)唾液中有可溶性 HLA 抗原存在。     

Specimen preparation for ABO determination of various kinds of blood stains

Trying to optimize the preparation of blood stains, we found methanol fixation not to produce very good results for the determination of ABO blood group antigens. It is advantageous to transfer blood stains before testing to cotton cloth. This transfer is also of practical use if blood stains are to be saved on a smooth surface for lateral determination. We testet on 35 different carrier materials, on which blood stains in casework often were found, whether blood grouping gave better results on either the original material or after transfer. Results are shown on a table. The test revealed, that solubility of the stain in aqua dest is a good sign for a successful transfer. Blood stains on pine-wood soil, soil and loam were not suited for ABO grouping.     

Predicting the position of the source of blood stains for angled impacts

Droplets of pig's blood were dropped onto paper at different angles to the horizontal to produce blood stains. Impact velocities varied from 1.82 to 5.76 m/sec, drop size from 3.7 to 5.0 mm in diameter, and the surface sloped at angles between 22.7 degrees and 90 degrees to the horizontal. From the data a single equation relating stain size to drop size and velocity for all impact angles was produced; ab = 111.74 (Re(1/2)We(1/4))(0.75)D(o)D(o) + 0.00084 with R(2) = 0.88, where a is the stain width, b the stain length, Re the Reynolds number, and We the Weber number. A second equation related the number of spines, N, to drop size, velocity, and surface slope for all impact angles as N = 0.76 We(0.5) sin(3)theta with R(2) = 0.9, where theta is the impact angle. Combining these equations the impact velocity can be determined and hence the position of the stain's source.     

Identification and age estimation of blood stains on colored backgrounds by near infrared spectroscopy

Non-destructive identification and subsequent age estimation of blood stains are significant steps in forensic casework. The latter can provide important information on the temporal aspects of a crime. As previously shown, visible spectroscopy of blood stains on white backgrounds can successfully be used for their identification and age estimation. The use of this technique however, is hampered by dark backgrounds. In the present study the feasibility to use near infrared (NIR) spectroscopy was evaluated for blood stain identification and age estimation on dark backgrounds. Using NIR reflectance spectroscopy, blood stains were distinguished from other substances with 100% sensitivity and 100% specificity. In addition, Partial Least Squares Regression analysis was applied to estimate the age of blood stains on colored backgrounds. The age of blood stains up to 1 month old was estimated successfully with a root mean squared error of prediction of 8.9%. These findings are an important step toward the practical implementation of blood stain identification and age estimation in forensic casework, where a large variety of backgrounds can be encountered.     

尿液及尿斑DNA分型的研究

目的研究尿液及尿斑的DNA提取及其检验。方法用Chelex100法及QIAampMiniKit提取尿液及尿斑样本中的DNA,进行PCR扩增及STR检验。结果新鲜的及存放时间在12h以内的尿液样本能得到较好的分型结果;存放2d左右的尿液样本有50%能检出基因型;存放7d及更长时间的尿液样本全部不能检出基因型;尿斑样本的分型成功率很低。结论较新鲜的尿液样本均能进行DNA分型,在法医检案中具有应用价值。     

Quantitative determinations of Hp, Pi, Tf and GC in stored blood stains

Quantitative analysis of serum proteins in blood stains by rocket immunoelectrophoresis showed the following results: Long elution times in processing blood stains do not produce a significantly higher yield of serum proteins in stains, and they increase the risk of structural alteration of protein molecules. The amount of proteins detected decreases with increasing age of blood stains. Some proteins are already altered in stains after a short storage time and are no longer useful for phenotyping. Our results confirm that blood stain material should be processed as soon as possible.     

Determination of the Lewis blood group substances in stains of forensically relevant body fluids

Forensic investigations often demand a clear definition of secretor status. Lewis-typing of secretion stains may help to verify non-secretor results and to identify mixtures of secretions from Le (a-b-) persons and secretors (or non-secretors). Furthermore it gives an additional check on secretor status, determined by ABO-grouping. Few problems may arise, when testing prepared saliva or semen stains. Therefore our interest was focussed on the possibility of Lewis-typing in stains appearing in forensic case work such as cigarette tips, stamps and envelope flaps, semen stains and vaginal swabs, nasal secretion, sweat and urine stains. All stains with the exception of sweat and urine were successfully Lewis-typed. In saliva stains Lewis substances could be determined even after 5 years and in semen stains for at least up to 40 days.     

Evaluation of Commercial Kits for Dual Extraction of DNA and RNA from Human Body Fluids, ,

STR typing of DNA evidence can identify the donor with a high power of discrimination but cannot identify the tissue origin of a body‐fluid stain. Using RNA to attribute a crime scene stain to a particular tissue may aid in reconstruction efforts. With blood from 10 donors, four DNA and RNA coextraction kits were evaluated by measuring yields and STR and mRNA profiles. T tests indicated some significant differences in kit performance. The Zymo Research ZR‐Duet^? kit performed best based on average DNA (41.4 ng) and mRNA (4.07 ng) yields and was the only kit to provide complete DNA/RNA profiles for all samples. The consistency of this kit was challenged by data from additional blood and saliva donors. Further testing is advised before a superior kit is unequivocally chosen. Stand‐alone DNA or RNA purification generally offers higher yield, but coextraction may still allow successful STR profiling and tissue source identification.     

人体组织黄递酶Ⅲ分型

应用超薄层PAGIEF对人体组织作DIA3分型，结果显示睾丸、卵巢、子宫肌和牙髓组织均可检出DIA3多型带。脑、肝、肾、肾上腺、脾、肠、胰腺、心肌、肺、皮肤和骨骼肌以及血液、阴道液和毛根均未检测出DIA3。应用紫外分光光度法测定人体不同组织，血液和阴道液中DIA相对活性，结果各组织DIA活性相对值与PAGIEF中DIA显色谱带密度基本一致。作者认为，用PAGIEF对生殖器官和牙髓进行DIA3分型在法医的个人识别中有一定意义，在混合斑鉴定中可以不考虑阴道液对精液DIA3分型的干扰。     

短串联重复分型异常

短串联重复序列(STR)已被广泛应用于法医学的个体识别和亲子鉴定中。大多数情况下,STR的分型均得到了准确、可靠的结果,但是在应用中也发现了一些异常的分型现象。引起这些异常现象原因很多,有DNA的遗传性变异、DNA模板质量不好、分型试剂或方法的不同、PCR反应中非特异性反应以及异常的电泳行为等。这些异常的结果会干扰STR分型结果的解析。     

Gm/Km typing of bloodstains in U-bottom microtiter plates

A technique was developed for Gm/Km typing of bloodstains and sera in U-bottom microtiter plates. Gm/Km typing of sweat and urine samples was also attempted with limited success.     

Sensitivity of various study technics in blood stains

Quadratic pieces of fleece measuring 16 mm2 were soaked with 10 different blood-samples in the dilution steps of 1:1, 1:10, 1:100, 1:1000, respectively, and were tested in blood group typing and identification tests of forensic serology. The above spezified dilutions correspond with 5 microliters, 0.5 microliter, 0.05 microliter and 0.005 microliter of blood, respectively. The detection limit of the microspectrometric test for blood was the dilution 1:10, of the porphyrine test a dilution above 1:100, whereas the preliminary test for blood (peroxidase) succeeded always up to a dilution of 1:1000 and the species determination by the radial immunodiffusion test in agar gels succeeded in most cases op to a dilution of 1:1000. The detection limit of the anti-human globulin inhibition test was between the dilution steps 1:10 and 1:100 when non-titrated and undiluted anti-human globulin serum was used. Gc- and ABO-grouping were possible up to a dilution of 1:100 and were thus the most sensitive grouping systems. Phenotyping of the enzyme-systems and the Gm/Km-system usually required stains with considerably higher blood concentrations i.e. stains of undiluted blood.