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.     

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.     

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.     

Evaluation of antisera for bloodstain grouping. I. ABH, MN, and Rh

Sixty-eight different commercially available blood grouping antisera and lectins with ABH, MN, and Rh D, C, E, c, and e specificities were serologically evaluated for their applicability to bloodstain antigen determination. The characteristics of the antisera were determined with red cells, with fresh bloodstains, and with series of aging bloodstains. The Rh antisera were tested under a variety of serological conditions and with bloodstains on various substrata. Additionally, studies on optimization of absorption-elution procedure variables were carried out, and some data on the storage characteristics of red cells and blood grouping antisera were gathered.     

Studies on ABH antigen grouping of ammoniacal extracts of bloodstains by absorption-elution

The effects of absorption time, titer of antiserum or lectin in the absorption stage, and elution temperature on relative antibody or lectin yield in elutes in the absorption-elution procedure were studied in bloodstains and ammoniacal extracts of bloodstains using conventional grouping reagents. In addition, monoclonal anti-A and anti-B and affinity-purified Ulex europaeus agglutinin I (UEA I) reagents were employed for comparison in these processes. Ammoniacal extracts of bloodstains and dried bloodstains on cotton substrata behaved comparably with respect to the parameters studied. The monoclonal anti-A and anti-B and UEA I reagents studied yielded satisfactory results, comparable in some cases to conventional reagents, with respect to the parameters studied.     

The detection of A and B antigens on human hair by the absorption-elution technique using LISS and papain-treated test cells

The absorption-elution technique with low ionic strength solution (LISS) and papain-treated test cells previously used for bloodstains was employed for the detection of ABO antigens on human hair. Antigen identification was always possible, with good intensity of agglutination, even in those cases where classic techniques had given false-negative results. It was possible to obtain positive results with fragments of human hair as small as 0.2 cm.     

Orosomucoid (ORM) typing by isoelectric focusing: description of two new alleles in a German population and thermostability in bloodstains

The genetic polymorphism of serum orosomucoid (ORM) was studied in 168 unrelated German individuals using isoelectric focusing followed by immunoprinting. Two new alleles, tentatively designated ORM1*14 and ORM2*13, were identified. The method was successfully applied to demonstrate ORM1 types in dried bloodstains. Each type of ORM1 was also correctly determined in bloodstains heated at 130 degrees C for 30 min. The results indicated that ORM1 is a new powerful genetic marker system for the grouping of bloodstains.     

The ABH reactions of seminal stains

The ABO grouping results from approx. 1000 seminal stains have been collected and analysed. Most of the stains came from rape cases where the ABO and secretor status of both complainant and suspect were known. The results of the survey provided information concerning the usefulness of elution and inhibition as methods of body fluid grouping, the relative strengths of reaction of the A, B and H antigens in body fluids and the interpretation of the ABH reactions of body fluid stains.     

单克隆抗体斑点ELISA快速检测血痕ABO血型

本文利用酶标记单克隆抗A、抗B抗体进行直接斑点ELISA检测血痕ABO血型。此法可检测出含量相当于0.005μl的全血,整个实验过程仅需1.5小时。对48份血痕标本均能正确鉴定。与常规方法相比,具有快速、简便、灵敏等特点,有一定的实用价值。     

The collaborative study on typing group-specific component in casework bloodstains

Casework bloodstains were typed for group-specific component (GC) at eight forensic laboratories. Approximately 600 bloodstains were examined of which a mean of 62.7% gave results. This is comparable to other blood grouping systems in current use. Stains that were over three-months old were successfully typed in six of the laboratories. A wide variety of substrates was examined; these included many items of clothing as well as metal blades, concrete, paint, cement, glass and grass. Of substrates that were examined several times, none consistently gave problems with GC typing. The GC system has been shown, therefore, to be an effective test in operational forensic science.     

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.     

C6 phenotyping in sera and bloodstains by isoelectric focusing followed by electroimmunoblotting

The genetic polymorphism of C6 was investigated in 329 unrelated Japanese individuals using isoelectric focusing in polyacrylamide gels followed by an electroimmunoblotting technique. Besides six common phenotypes C6 A, AB, B, AB2, BB2 and B2, six rare variants were observed. The allele frequencies were: C6*A = 0.4422, C6*B = 0.4757, C6*B2 = 0.0714, C6*A3 = 0.0015, C6*M1 = 0.0046 and C6*B3 = 0.0046. The population data confirmed that the C6*B2 allele is the third common allele characterizing Japanese. The present electroimmunoblotting technique was applied to demonstrate C6 types in dried bloodstains. The C6 types were determined from bloodstains stored at 4 degrees C for up to 10 weeks, at room temperature for up to 2 weeks and at 37 degrees C for up to 4 days. The results show that this component system offers a new powerful means for the medico-legal grouping of bloodstains.     

Identification of fetal bloodstains by enzyme-linked immunosorbent assay for human alpha-fetoprotein

A rapid and highly sensitive enzyme-linked immunosorbent assay (ELISA) for determination of human alpha-fetoprotein (AFP) using commercially available reagents was devised and applied to identification of fetal bloodstains. When experimentally prepared bloodstains, 1 by 2 mm in area, were submitted to analysis, only fetal bloodstains showed positive reactions in the present ELISA. The reactions did not change significantly when these bloodstains were stored at room temperature for one week. The present ELISA seems to be suitable for forensic science practice.     

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。     

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.     

ABO基因型检验及其法医学应用研究

目的采用ＰＣＲ技术对ＡＢＯ血型系统进行基因型检验。方法选择最佳扩增条件进行四引物复合扩增,用限制性内切酶ＫｐｎＩ和ＡｌｕＩ分别酶解扩增产物,电泳分离、银染显色法检验ＡＢＯ基因型。结果对２７０例血斑、２０例混合斑、２０根毛发（有毛囊）、１２份唾液斑等不同的生物检材进行了分型,与血清学方法检验结果相符。结论该方法能够应用于法医学的检验     

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。     

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.