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.     

A sandwich enzyme-linked immunosorbent assay for ABO blood typing of semen by using anti-p 84 monoclonal antibody as a marker of blood group substance in semen

A blood group substance (BGS), a protein with ABH antigenic activity, was isolated from human seminal plasma and designated as p 84 (Sato, 1995). We have developed a method for determining the ABO blood type of semen by performing a sandwich enzyme-linked immunosorbent assay (ELISA) in which p 84 is captured with an anti-p 84 monoclonal antibody, and evaluated the specificity and sensitivity of this method. Although BGS activity was detected in semen sensitively by this method, it was not detected in saliva, urine, breast milk, blood or vaginal secretions. Since the concentration of p 84 in semen was independent of the secretion status, the status can be determined as non-secretor when p 84 but not BGS activity was detected. To determine the stability of BGS activity on p 84, dried stains of semen on filter paper were kept at 4, 26, and 37 degrees C for 8 months, 2 years and 1 month, respectively, and their BGS activities were examined. After 8 months at 4 degrees C, over 60% of the original BGS activity was recovered from the stain. The activity could be detected even from a square as small as 0.25 by 0.25 cm. After 1 month at 37 degrees C and 2 years at 26 degrees C, 31 and 20% of the BGS activity, respectively, still remained. It could be detected from the pieces of 1.0 by 1.0 cm and 0.5 by 0.5 cm squares, kept for 1 month at 37 degrees C and 2 years at 26 degrees C, respectively. Finally, semen was mixed with saliva or blood at varying volumetric ratios and used for the sources of dried stains. The BGS activity of p 84 could be detected in the stains until the ratio between semen and saliva or blood reached 1:4. We conclude that this sandwich ELISA offers a more sensitive and specific method for determining the ABO blood type of semen samples obtained from sexual assault victims than existing methods, such as the conventional absorption-elution and classical hemagglutination-inhibition tests.     

Determination of MN blood group from blood stains by electrophoresis and immunoblotting

The determination of blood groups from blood stains is extremely important in medicolegal practice, but there is the possibility of an error in the determination of MN phenotypes by the absorption-elution test. We investigated a new method applying electrophoresis and immunoblotting. As a consequence of various experiments, the most appropriate pretreatment of blood stains was as follows. Blood stains were immersed in physiological saline for 0.5 to 1 h and centrifuged. The supernatant was discarded. The sediment was dissolved in sample buffer (TRIS-buffered physiological saline containing 2% sodium dodecyl sulfate) and followed by thermodegradation. It was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to a nitrocellulose membrane by Western blotting, MN phenotypes could be determined accurately from blood stains by an enzyme immunoassay (EIA) using commercially available polyclonal anti-M and anti-N sera. For blood stains more than 1 month old it was not easy to determine the MN phenotypes.     

Comparative studies between counterimmunoelectrophoresis and microprecipitation method of identification of human minute bloodstains

A minute bloodstain on a thread 2 mm in length was tested to identify human origin by counterimmunoelectrophoresis (CIE), modified CIE, and microprecipitation method (MPM), using anti-human HbA serum. The detection limits expressed as the highest dilution of human blood on the thread in positive reaction were 1:160, 1:320, and 1:320 in CIE, modified CIE, and MPM, respectively. About 1 h was required to obtain the results. In CIE and modified CIE, the detection limits diminished according to the reduction of the samples from one half to one-eighth of the thread, but not in MPM.     

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.     

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.     

The investigation of blood and secretion stains by the absorption-elution reaction using anti-H monoclonal antibodies

Possible use of monoclonal antibodies anti-H in absorption-elution reaction was studied. Blood and secretion stains on gauze were analysed. Practical usefulness of monoclonal antibodies anti-H for investigation of human blood and secretions was stated. Differences in interaction of monoclonal antibodies with traces of different origin were found.     

Determination of ABO blood grouping from human oral squamous epithelium by the highly sensitive immunohistochemical staining method EnVision+

Using the highly sensitive immunohistochemical staining method EnVision+, which employs a dextran polymer reagent for the secondary antibody, the detection of the ABH antigens was attempted in the oral squamous epithelium. This new technique uses monoclonal antibody as a primary antibody and it takes about three hours for staining. The time is much shorter than conventional absorption-elution testing or absorption-inhibition testing for the determination of ABO blood grouping. Secretor saliva samples were stained at strong intensity by the antibody, which corresponded to its blood group and anti-H. On the one hand, nonsecretor saliva samples were stained at strong intensity only by the antibody that corresponded to its blood group, and at weak intensity only by anti-H. Since human oral squamous epithelium antigens were stained specifically by this method, we can examine the ABO blood group of saliva samples and perform cytodiagnosis at the same time. Our research suggested that the EnVision+ Method is a useful technique for ABO blood grouping of saliva in forensic cases.     

Immunohistochemical determination of maternal and child blood groups (ABO) in mature placental tissue

Using the indirect immunoperoxidase technique (PAP method), ABO characteristics of mother and child were correctly identified in tissue specimens from 10 mature human placentas. In one case, a weak infantile A reaction was overlooked in the agglutination test but correctly identified by immunohistochemistry. In accordance with the weak expression of ABO characteristics in cord blood, immunohistochemical labeling of infantile erythrocytes with monoclonal and human antibodies, as well as Ulex europeaeus agglutinin I (UEA I), was less pronounced than that of mature erythrocytes. Labeling of the chorionic vessel endothelium, in contrast to that of adult endothelial tissue, was negative with anti-A or anti-B but, regardless of the infantile blood group, pronounced with UEA I. Regular identification of the blood groups was possible in decomposed placental tissue stored at room temperature for 1 week, but not in tissue stored for 2 or more weeks.     

ABO tissue antigens of Egyptian mummies

ABO groups were investigated on skin (and muscle), bone and hair specimens from 14 Egyptian mummies dating from the Roman period. Samples were tested by the AE (absorption-elution), MA (mixed agglutination) and HIF (histo-immunofluorescence) methods, in order to evaluate the reliability of each method. For half of the mummies (7) the results were concordant on all samples (3-9 samples for each mummy) with all employed methods, suggesting an unequivocal blood group conclusion. For the other seven mummies there were discordant results with the different methods and interpretation of the results was thus inconclusive. HIF seems to be the most reliable method as specific blood group substances are identified on specific histologic structures. Failure to detect tissular ABO antigens was mainly due to excessive resin impregnation.     

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。     

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。     

Specific research of antigens of the ABO system in samples of decayed blood

Liquid blood and blood stains were examined after storage under different conditions and temperature regimes ranging from 18 to 26 degrees C. Blood stains were washed by distilled water or heated to 120 degrees C for as long as 4 hours. Then, blood groups were determined by absorption-elution.     

Detection of P1 antigen in blood stains by absorption elution test with protease C

Some lots of goat anti-P1 sera intended for analysis of liquid blood can detect the antigen in blood stains by the absorption-elution test.     

Immunocytochemical determination of ABH and MN antigens on dried blood traces in the nanoliter range.

The absorption-elution test and the mixed cell agglutination reaction are both ultimately based on the ability of indicator cells to agglutinate. This agglutination reaction requires a relatively large amount of antigenic epitopes, and, in addition, a relatively high volume of blood traces. The immunocytochemical demonstration of epitopes requires a lower volume, which, however must be fixed for investigation, thus possibly causing damage to the epitopes and thereby preventing detection. An immunocytochemical method is presented which permits ABH and MN antigen determination on dried blood traces of nanoliter quantities without special fixation. This method is based on immunocytochemical demonstration of antigens directly on the cell membrane in combination with the use of a coated glass slide that ensures maximum economy of epitopes.     

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.     

Sex determination of blood stains with a recombinant DNA probe: comparison with radioactive and non-radioactive labeling methods

A recombinant DNA probe hybridizing specifically to human repeat DNA sequence (pHY10) of which about 3000 copies are present on the Y chromosome was used for sex determination of degraded DNA samples of blood stains. Human blood stains of male and female origin were readily differentiated with the pHY10 DNA probe. This radioactive technique enabled reliable and sensitive sex determination from blood or dried blood stains greater than 20 years old. Less than 1 microliter of blood or 1 piece of 0.5 cm length thread of blood stain from cotton fabric was sufficient for the test using dot blot hybridization. Compared with the radioactive labeling method, the photobiotin labeling method showed one thirtieth to one fiftieth lower sensitivity and presented some problems which are expected to be resolvable.     

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。     

One method of collecting fallen off epithelial cell

In this paper, the comparative analysis of ABO genotyping and serological typing was conducted in 360 unrelated blood samples from northern Chinese Han population using genotyping method and serological typing method, respectively. The results of ABO genotyping were obtained by Goldeneye 16BT STR plus ABO kit. The ABO serological types were determined by the antigen–antibody agglutination test. The ABO types were confirmed by the two methods and no contradiction types were found; two more types were obtained using the ABO genotyping method and the discrimination power was further improved; the information of ABO genotyping and 15 STRs could be obtained at the same time using the Goldeneye 16BT STR plus ABO kit.     

人体组织ABO血型检测方法的研究

探讨人体组织ABO血型检测方法。对已知ABO血型尸体的不同组织,用红细胞粘连试验、吸收—抑制试验和吸收－解离试验进行ABO血型测定。12例尸体的16种组织中均检出与尸体血痕相同的ABH物质。对不同温度保存的组织块进行ABH物质检测的结果显示,4℃保存的组织ABH物质的检出时间长于室温,空腔脏器的检出时间短于实质脏器。三种方法中,红细胞粘连试验简单易行,适用于基层单位,吸收－抑制试验用于组织块的测定时优于吸收－解高试验。