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.     

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.     

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.     

A sequence of tests of minute human blood stains for human origin identification and ABO blood grouping

A series of examinations is presented for human origin identification and ABO blood grouping of doubtful minute human blood stains. A blood-stained thread (0.5 cm in length) was first tested to identify human origin by microprecipitation method and then the ABO blood type was determined by both a modified absorption-elution test and a modified mixed agglutination. In the continuous tests, the maximum limits of positive reactions of the microprecipitation method, the modified absorption-elution test, and the modified mixed agglutination were 1:640, 1:160, and 1:2,560 diluted blood, respectively. A and B agglutinogens were more sensitively determined than H agglutinogen. Hemagglutinogens of blood stains on cotton threads were more easily detected than those of polyester ones.     

Examination of the correlation of groupings in blood and semen

The grouping of blood/saliva samples from a male so as to predict his semen groups is only justified if there is a strict correlation between the groupings in these body fluids. This correlation has been examined in the ABO, phosphoglucomutase (PGM1) and glyoxalase I (GLO) grouping systems in blood and semen samples collected from more than 250 individuals. Though no results proved inconsistent with this correlation, a number of semen gave inconclusive grouping results. Reasons for this are discussed as well as the relevance of the results to semen stain analysis. Semen amylase activities are also reported.     

Rapid direct PCR for ABO blood typing

Many different molecular typing methods have been reported to complement routine serological ABO blood typing in forensics. However, these ABO genotyping methods are often time-consuming and call for an initial DNA isolation step that requires the use of expensive kits or reagents. We report here a rapid direct ABO genotyping method that eliminates the need for DNA extraction from fresh blood, hair, and body fluid stains before PCR. Using a fast PCR instrument and an optimized polymerase, the genotyping method-which employs a multiplex allele-specific primer set for the simultaneous detection of three single-nucleotide polymorphism (SNP) sites (nucleotides 261, 526, and 803)-identifies A, B, O01/O02, O03, and cis-AB01 alleles in around 70 min from sample collection to electropherogram. Not only will this ABO genotyping method be efficiently used in forensic practice for rapid screening of samples before full-blown multilocus short tandem repeat profiling, but it will also demonstrate an example of rapid direct genotyping of SNPs that offers the advantages of time- and cost-efficiency, convenience, and reduced contamination during DNA analysis.     

ABO基因分型及其在法医学中的应用

为建立一种ABO血型系统基因分型方法，采用PCR-RFLP技术，成功地将ABO系统区分为AA，AO，AB，OO，BB，BO六种基因型。对240名中国汉族无关个体血样的ABO（基因型频率调查结果表明，6种基因型的频率分布为0．0125～0．3834，符合Hardy－Weinbeng遗传平衡法则（P＞0．1），其DP值为0．8161。家系分析表明，亲代a、b、o基因传递遵守孟德尔遗传规律。对法医学中常见的血痕、混合斑、骨组织及毛发根部等生物样品进行检测，均能准确判定ABO基因型，并可在实际案件鉴定中应用。     

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.     

A modification of the microplate method for reverse ABO typing of bloodstains and additional validation studies

The results of additional validation studies of a sensitive microplate hemagglutination assay for ABO reverse grouping of bloodstains are presented. The results of the validation study demonstrate the reliability of the microplate assay for use in routine serological casework. Based on these studies, the microplate assay has now replaced the Lattes crust test for ABO reverse grouping of bloodstains in the FBI Laboratory.     

Evaluation of an enzyme linked immunosorbent assay (ELISA) method for ABO and Lewis typing of body fluids in forensic samples.

An ELISA for the detection of the ABO group and secretor status of body fluids and stains other than blood is described, together with the validation procedures employed before its introduction into forensic casework. Criteria for the interpretation of results have been formulated for the method in use in this laboratory. The method was found to be reliable and to have a higher success rate than the haemagglutination techniques previously employed.     

Dot—ELISA在用酶标单克隆抗体检验体液斑和血痕ABH血型物质中的应用研究

本文应用Dot-ELISA法用酶标单克隆抗体,直接检验出稀释5万倍的精斑、10万倍的唾液斑及阴道分泌物等体液斑和血痕中ABH物质,还可检验出1～17年的体液斑中ABH物质。全部操作在室温中进行,以肉眼观察颜色变化判断结果,试验周期30～60分钟,准确率100％,实验结果可长期保存。     

Evaluation of ABO and Lewis genotypes using primer extension preamplification

There are some difficulties with blood typing from ABO variant bloodstains and Lewis negative samples using serologic methods. In these samples, DNA analysis should be employed simultaneously to avoid errors in typing. Primer extension preamplification (PEP) produces copies of template DNA. The minimum quantity to examine nucleotide substitutions of ABO and Lewis genotypes by PCR ranged from 1 to 3 ng DNA. The PCR products with or without PEP treatment showed identical ABO and Lewis genotyping results. Performing both serologic and PCR testing served to crosscheck the ABO and Lewis grouping of such specimens. Errors in ABO and Lewis typing can be avoided as discrepancies are investigated further. The application of the PEP method to limited amounts of DNA samples for ABO and Lewis blood groupings is useful.     

混合斑中精斑ABO血型的检验

<正> 1988年,壹岐裕志等报告了用吸附抗α_2-SGP 血清的硝化纤维素膜(NCF)检验混合斑中的精斑 ABO 血型,但耗时。本文作者通过对此方法的改进,采用常彩琴等研制的抗人精特异蛋白血清(anti-human seminalpeculiar protein,ASPP),采用蛋清粘片热解离法检验混合斑中精斑 ABO 血型,耗时短,效果好。现介绍如下。     

应用斑点ELISA快速进行体液(斑)的血型测定

<正> 在法医学上体液(斑)的血型测定一般常规应用中和试验、吸收试验、解离试验及混合凝集反应。这些试验既耗时,又需较高的技术,且要新鲜标准红细胞指示结果。本实验系应用我们自己提纯标酶的抗-A,-B与抗-H单克隆抗体A,利用斑点ELISA来进行体液(斑)的血型测定。通过颜色变化直接判断血型。     

ABO genotyping by polymerase chain reaction.

ABO blood group system's genotyping by polymerase chain reaction in genomic DNA level is developed. The positions of nucleotide 258 and 700 of cDNA from A transferase were used to distinguish A, B, and O alleles by restriction enzyme digestion. To identify the 258th nucleotide, a 199- or 200-bp DNA fragment was amplified by PCR and digested with Kpn I. For the 700th nucleotide, a 128-bp PCR amplified fragment was designed and digested with Alu I. By examining the DNA fragment digested patterns, ABO genotypes were easily determined. Results obtained using this method on 20 ABO-known peripheral blood samples showed that this new technique could provide accurate ABO genotype. Biologic forensic samples, such as, blood stains, saliva stains, semen stains, hair, bone tissue, and semen contaminated with vaginal secretion were also successfully typed. This rapid, sensitive and reliable method should be applicable not only in forensic identification but also in medical examination.     

型特异性沉淀素血清检验人唾液斑精斑ABO血型的研究与应用

本文介绍了利用型特异性沉淀素血清环状沉淀法检验人唾液斑、精液斑ABO血型的方法与实验结果,并与中和试验及解离试验进行了比较。实验结果表明,本法操作简便,对多种干扰条件下的唾液斑、精斑均具有高度的型特异性,并能从分泌液与血液的混合斑中准确地鉴别出分泌液的血型。本法仅需0.4cm的分泌斑纱线即可进行血型鉴定,其灵敏度高于中和试验而略低于热解离试验,并能有效地检出陈旧分泌液斑中的型物质,因此适于在实际检案中应用。     

Detection of group-specific antigens of the ABO system in fish blood

The subject of the case study was blood of 8 fish species. It was examined within the ABO system. 68 tests of stains of fish blood mixed with human blood were made. It was shown as possible to differentiate between human blood and fish blood in mixed stains.     

一种改良酸性磷酸酶试验鉴定精斑的方法

本文报道了一种改良的酸性磷酸酶试验方法。用该法鉴定精斑，可克服非特异性反应，排除其它体液和植物汁液的影响。     

A microplate method for reverse ABO typing of bloodstains

A sensitive and reliable hemagglutination assay, using V-bottom microplates, is described for the detection of the ABO blood group alloantibodies in bloodstained material. When used in conjunction with an absorption-elution procedure, the microplate assay resulted in a 300% increase in the number of conclusive grouping results when compared to the Lattes crust test. The use of the microplate reverse grouping assay permits 24 specimens to be assayed conveniently on a single plate and eliminates the tedious and time-consuming microscopic examination required for the Lattes crust test.