On the detection of minor Rhesus antigens in blood stains

A new method for the detection of minor C, Cw, c, E, e-antigens of the Rhesus system in blood stains has been developed based on the absorption-elution technique with the use of anti-C, anti-Cw, anti-c, anti-E, and anti-e sera and standard erythrocyte preparations preliminarily treated with highly active proteases (protease C or papain). This method makes it possible to determine complete Rhesus phenotype in blood stains and substantially extend the possibilities of their differentiation on material objects (evidence) for the purpose of forensic-biological examination.     

Micromethod for the detection of erythrocyte antigens in blood stains

A micromethod was developed to allow the analysis of blood stains of minor size by the absorption elution technique. The individual absorption, washing, and elution steps were carried out in Beckman tubes containing 5 microliter antiserum. The final agglutination reaction was read through the inverted microscope in microtest plates regularly used for HLA typing. For this final reaction, 2-4 microliter eluate was incubated with 2,000 red blood cells suspended in 1 microliter saline and supplement. For the purpose of standardization, the intensity of agglutination in the microtest plate had to be defined. In comparison to the standard method (tube test and centrifugation), the proposed method proved to be slightly more sensitive with regard to the Rhesus and slightly less sensitive with regard to the AB0 system. With the proposed method very small traces could be successfully analyzed. Thus, two cotton threads 1 mm in length were sufficient for testing antigens A and B, and two cotton threads 2.5 mm in length were enough to detect an Rh antigen.     

恒河猴ABO血型的研究

本文应用A型、B型、AB型血清，及抗－A与抗－B单克隆抗体对113只猕猴的ABO血型进行了测试。结果表明：在恒河猴（Macacamulatta）红细胞表面及血清中均未证明有类人凝集原和凝集素存在。但可根据其唾液中类人ABH型物质存在的情况判定该动物的类人血型。本实验检出的血型表型频率分别为：A型17．70％；B型52.21％；AB型20.36％；O型9.73％。该结果与国外报道的血型分布频率有差异。     

Sensitive Method for the Confident Identification of Genetically Variant Peptides in Human Hair Keratin

Recent reports have demonstrated that genetically variant peptides derived from human hair shaft proteins can be used to differentiate individuals of different biogeographic origins. We report a method involving direct extraction of hair shaft proteins more sensitive than previously published methods regarding GVP detection. It involves one step for protein extraction and was found to provide reproducible results. A detailed proteomic analysis of this data is presented that led to the following four results: (i) A peptide spectral library was created and made available for download. It contains all identified peptides from this work, including GVPs that, when appropriately expanded with diverse hair-derived peptides, can provide a routine, reliable, and sensitive means of analyzing hair digests; (ii) an analysis of artifact peptides arising from side reactions is also made using a new method for finding unexpected modifications; (iii) detailed analysis of the gel-based method employed clearly shows the high degree of cross-linking or protein association involved in hair digestion, with major GVPs eluting over a wide range of high molecular weights while others apparently arise from distinct non-cross-linked proteins; and (v) finally, we show that some of the specific GVP identifications depend on the sample preparation method.     

Comparison of fingernail ridge patterns of monozygotic twins

The ridge patterns on the fingernails of corresponding fingers of a pair of twins were compared microscopically and found to be readily distinguishable from one another. Based on blood grouping in six blood group systems (ABO, Rhesus, Ss, Duffy, Kidd, and Kell), the probability that the twins were monozygotic was calculated to be 89.1%.     

抗人同种异型G1m(3)单克隆抗体研制及特异性鉴定

用快速液相色谱仪从G1m（3）阳性人血浆中纯化IgG1蛋白，用其免疫BALB／C／小鼠．建立了一株分泌抗人G1m（3）单克隆抗体的细胞株（D7E8）。经抑制ELISA，直接ELISA及斑点ELISA分析，证明D7E8抗体具有G1m（3）单一特异性。其培养上清液效价为512倍，并初步应用于法医办案。     

Semi quantitative detection of signature peptides in body fluids by liquid chromatography tandem mass spectrometry (LC–MS/MS)

This study covers a modified semi-quantitative approach to detecting signature peptides for body fluid identification. A liquid chromatography tandem mass spectrometer normally used for toxicology was adapted to detect target ion transitions for five semen or saliva specific peptides. Peptide concentrations were measured based on a mixture of synthetic peptide standards. Samples were processed using a three-hour trypsin digestion and Microcon membrane filtration. This method generates PCR compatible DNA and peptide fractions that can be typed without any further treatment. Preliminary validation tests covered stains on different substrates, semen/saliva mixtures, limit of detection, and repeatability. All signature peptides were present at different concentrations, varied amongst donors, and were tissue specific. Saliva peptides were detected at lower concentrations and had a higher limit of detection (LOD). Semen peptides had higher concentrations and were detected even as a minor component in a mixture. All semen peptides and all, but one, saliva peptides were detected on the various substrates. Semen peptide concentrations had relative standard deviations (RSDs) lower than 20%, indicating high repeatability, different from saliva where higher RSDs were observed. DNA fractions did not show signs of degradation or PCR inhibition.     

Serological detection of Rhesus antigen D in blood traces from Rh-negative (cde) people

A D-like structure was serologically detected on the internal side of Rh-negative (cde) erythrocyte membrane. This structure is absolutely inaccessible for anti-D antibodies in morphologically intact liquid blood erythrocytes. In hemolyzed erythrocytes of blood stains this antigenic structure, serologically identified as Rh antigen D, is accessible for binding with anti-D antibodies and for detection by the absorption-elution test, particularly so after blood stain treatment with proteases.     

Use of hand-held laser scanning in the assessment of craniometry

The intra- and inter-examiner reliability was evaluated for hand-held 3D laser scanning technology when it was combined with localization of landmarks for craniometry. The data from the laser surface scanning were compared with those of conventional direct measuring. Using thirty unidentified skulls requested for individual identification, measurements were taken of the line distances from lambda to 26 landmarks, and also for seven breadth parameters. For the laser surface scanning, two examiners performed replicate measurements with an interval of 1 week. In the conventional direct measuring, the first examiner took replicate measurements with a 1-week interval. To assess intra- and inter-examiner reliabilities, the intraclass correlation coefficient was used. Analysis of variance with repeated measures for each parameter was performed to compare the conventional method with the 3D scanning method. Both the 3D scanning and conventional methods showed excellent intra-examiner reliabilities, and the 3D laser scanning method also showed excellent inter-examiner reliability. A statistical difference between the two examiners was found only in nasal breadth in the 3D laser scanning method. There was no significant difference between the two measuring methods, though the 3D laser scanning method tended to give a slightly lower reading. Collectively, the 3D laser scanning method with point localization is a useful method with excellent reliability, and it can replace the conventional direct measuring method in craniometry.     

Use of hand-held laser scanning in the assessment of craniometry

The intra- and inter-examiner reliability was evaluated for hand-held 3D laser scanning technology when it was combined with localization of landmarks for craniometry. The data from the laser surface scanning were compared with those of conventional direct measuring. Using thirty unidentified skulls requested for individual identification, measurements were taken of the line distances from lambda to 26 landmarks, and also for seven breadth parameters. For the laser surface scanning, two examiners performed replicate measurements with an interval of 1 week. In the conventional direct measuring, the first examiner took replicate measurements with a 1-week interval. To assess intra- and inter-examiner reliabilities, the intraclass correlation coefficient was used. Analysis of variance with repeated measures for each parameter was performed to compare the conventional method with the 3D scanning method. Both the 3D scanning and conventional methods showed excellent intra-examiner reliabilities, and the 3D laser scanning method also showed excellent inter-examiner reliability. A statistical difference between the two examiners was found only in nasal breadth in the 3D laser scanning method. There was no significant difference between the two measuring methods, though the 3D laser scanning method tended to give a slightly lower reading. Collectively, the 3D laser scanning method with point localization is a useful method with excellent reliability, and it can replace the conventional direct measuring method in craniometry.     

Diagnosis of stress preceding the fatal outcome

Postmortem examinations of cytomedines of internal organs in 181 men showed a possibility to use their (cytomedines') quantitative and qualitative characteristics as diagnostic criteria of stress influencing the human organism shortly before the death onset. Bioregulatory peptides were obtained by using Morozov's and Khavinson's method of acetous extraction. The composition of cytomedines was determined by spectrophotometer, while their biological activity (BA) was studied through affecting the phagocytic activity of blood neutrophils. It was concluded that, under stress, there are a significant inhibition of BA and a reduced count of cytomedines in the internal organs. The found data can be used to detect the stress preceding death.     

Identification of human skin from a tissue fragment by detection of squamous cell carcinoma-related antigen using an enzyme immunoassay

A new method of identifying human skin from a tissue fragment by detection of squamous cell carcinoma-related (SCC) antigen, using an enzyme immunoassay, was developed. When an extract was prepared from 0.1 g human skin homogenized with 1 ml of phosphate buffered saline, this method was able to detect SCC antigen in extracts diluted 10²-fold. There was no difference in the detection limit between individuals. Species specificity was good, and there was no cross reaction observed with skins from animals. Our method could also discriminate between skin and other organs or tissues, except for esophagus and lung. A practical case to which this method was applied is presented.     

SMCY性别特异融合抗原的克隆表达及其抗体制备

目的利用蛋白检测的快速性优势,研究不同性别在SMCY抗原氨基酸序列的差异,筛选出特异性氨基酸序列,并克隆表达性别特异融合抗原,制备相应抗体,建立一种快速鉴别法医物证性别的方法。方法通过对人SMCY和SMCX进行序列分析,发现了三段差异片段,采用搭桥PCR方法获得差异片段全长基因,连接入p ET-28a载体进行原核表达,用Ni柱纯化后的性别特异融合抗原免疫制备多克隆抗体,用ELISA法和western blot检测SMCY多抗与抗原的反应特异性,制作胶体金试纸条检测样本。结果筛选出具有性别特异性的氨基酸序列,获得SMCY性别特异融合抗原,成功制备出多克隆抗体及胶体金试纸条。结论获得SMCY性别特异融合抗原具有很好的抗原活性,制备的多克隆抗体可以与抗原特异性地结合,用于性别鉴定。     

The rapid determination of the ABO group from body fluids (or stains) by dot enzyme-linked immunosorbent assay (dot-ELISA) using enzyme-labeled monoclonal antibodies

Using ABH enzyme-labeled monoclonal antibodies, the authors could rapidly detect the ABO group from body fluids and body fluid stains by the dot enzyme-linked immunosorbent assay (dot-ELISA). In this test, the antigen was immobilized on nitrocellulose paper; the entire piece of paper was coated with an appropriate dilution of enzyme-labeled McAb directly against the antigen of interest; and, finally, 3,3'-diaminobenzidine (DAB) substrate solution was added. The site of a positive reaction is clearly visible as a brown spot. We analyzed 521 samples and got satisfactory results. We also analyzed 99 practical case samples by this method and achieved the same results as those obtained by other researchers using other methods. This method is accurate, simple, direct, rapid, and sensitive; it also produces easily observed results, requires no equipment, and can be completed in 30 min. The test proved to be clearly more sensitive for the detection of the ABO blood group in secretor saliva than the conventional hemagglutination inhibition test. Also saliva diluted 10(-4) to 10(-5) and the ABO group of nonsecretor saliva and urine could be easily detected by this method.     

FAM羧基荧光素修饰引物检测D1S80基因座遗传多态性

目的建立用FAM羧基荧光素修饰引物检测DIS80基因座的方法。方法采用5-FAM修饰引物,通过PCR扩增,DNA全自动遗传分析仪进行片段长度分析,检测129名黑龙江汉族无关个体DIS80基因座遗传多态性。结果在所调查的129名黑龙江汉族无关个体样本中,DIS80基因座有21个等位基因,56个基因型,基因频率在0.0039-0.1667之间。杂合度(H)为0.9097,个人识别机率(PD)为0.9691,非父排除概率(PE)为0.8113,多态性信息总量(PIC)为0.8988。群体内基因型频率分布符合Hardy-Weinberg平衡。结论FAM羧基荧光素修饰引物检测DIS80基因座的方法,简便、灵敏、稳定性好,DIS80基因座用于个体识别及亲权鉴定具有很高的实用价值。     

荧光标记复合扩增检测4个STR基因座多态性

目的建立荧光标记复合扩增D1S2142,D13S1492,D14S306,D15S659基因座检测分型方法,并对成都汉族群体4个基因座的遗传多态性进行调查。方法用6-FAM标记D1S2142和D15S659引物,HEX、TMR分别标记D14S306和D13S1492引物,PCR复合扩增,310基因分析仪电泳自动收集电泳结果数据,GeneScan Analysis Software3.7NT软件计算扩增产物片段相对大小,Genotyper(3.7NT软件进行样本基因型分型,建立了荧光标记复合扩增检测4个STR基因座基因型的方法,对145名成都汉族无关个体样本进行分型。结果荧光标记复合扩增D1S2142,D13S1492,D14S306,D15S659基因座,每个STR基因座都获得了清晰的基因型分型结果。145份样本,4个STR基因座分别检出10,14,7,12个等位基因和22,54,21,39种基因型,其基因型分布均符合Hardy-W e inberg平衡。4个基因座在成都汉族群体的杂合度分别依次为0.7793,0.8345,0.7793和0.8345;多态信息含量分别依次为:0.7656,0.8730,0.7470和0.8312。累计非父排除率为0.9783,累计个人识别机率为0.9999 917。结论荧光标记复合扩增D1S2142,D13S1492,D14S306,D15S659基因座,可实现对每个基因座准确分型;成都汉族群体该4个基因座的遗传学数据,可为群体遗传学和法医学研究与应用提供基础资料。     

Bite mark documentation and analysis: the forensic 3D/CAD supported photogrammetry approach

Bite mark identification is based on the individuality of a dentition, which is used to match a bite mark to a suspected perpetrator. This matching is based on a tooth-by-tooth and arch-to-arch comparison utilising parameters of size, shape and alignment. The most common method used to analyse bite mark are carried out in 2D space. That means that the 3D information is preserved only two dimensionally with distortions. This paper presents a new 3D documentation, analysis and visualisation approach based on forensic 3D/CAD supported photogrammetry (FPHG) and the use of a 3D surface scanner. Our photogrammetric approach and the used visualisation method is, to the best to our knowledge, the first 3D approach for bite mark analysis in an actual case. The documentation has no distortion artifacts as can be found with standard photography. All the data are documented with a metric 3D measurement, orientation and subsequent analysis in 3D space. Beside the metrical analysis between bite mark and cast, it is possible using our method to utilise the topographical 3D feature of each individual tooth. This means that the 3D features of the biting surfaces and edges of each teeth are respected which is--as shown in our case--very important especially in the front teeth which have the first contact to the skin. Based upon the 3D detailed representation of the cast with the 3D topographic characteristics of the teeth, the interaction with the 3D documented skin can be visualised and analysed on the computer screen.     

A solid-phase immunoenzyme method for the quantitative determination of ephedrine

Competitive ELISA method for ephedrine assay in biological fluids is developed. Anti-ephedrine antibodies obtained by rabbit immunization with conjugated antigen ephedrine-protein and ephedrine complex with horseradish peroxidase were used as reagents. ELISA in combination with chromatographic methods may be used in medicolegal and narcological practice.     

Detection of Lea substance in saliva stains by enzyme-linked immunosorbent assay (ELISA) using anti-gum arabic serum

It is known that rabbit anti-gum arabic (GA) serum has cross-reactivity with Lea antigen, and that, by using this cross-reactive anti-Lea antibody, the presence of Lea antigen in red blood cells and saliva can be demonstrated with accuracy. We have devised a rapid and highly sensitive method for detecting Lea substance in human saliva by the enzyme-linked immunosorbent assay (ELISA) method using an anti-Lea antibody isolated from anti-GA serum by affinity chromatography on Synsorb Lea. The ELISA plate, coated with the specific anti-Lea antibody, adsorbed the Lea substance in saliva which was subsequently identified by adding enzyme labeled anti-Lea IgG in that order. The method could detect the Lea substance in Le(a+) saliva stains as small as 0.1 by 0.1 cm in size that had been stored at room temperature for three weeks and in Le(a+) saliva stains 0.7 by 0.7 cm in size that had been stored for ten years. This method seems to be useful for quantitative analyses of the Lea substance in various body fluids.