A sensitive enzyme-linked immunosorbent assay used to differentiate type AB specimen from a mixture of type A and type B specimens

A sensitive enzyme-linked immunosorbent assay (ELISA) for detection of ABO blood substances in human body fluids was devised. The ELISA plates coated with purified human anti-A or anti-B serum effectively captured the blood substances, and these were then analysed by the combination of rabbit anti-A and goat anti-B. This capture ELISA could differentiate the type AB specimen from a mixture of the type A and the type B specimens, and the method was applied to rape cases to make the ABO typing of the criminal.     

Distribution of A and B antigens in organs of blood group AB individuals: observations disclosed by a double immunoenzymatic labeling method

The localization of A and B antigens in the organs of blood group AB individuals has been studied using a double immunoenzymatic labeling method. Both A and B antigens were found in the various epithelial cells of these organs, but the epithelial cells could be classified into the following four types depending on the reaction pattern with anti-A and anti-B sera: type 1: cells that stained positive with both anti-A and anti-B sera; type 2: cells that stained positive with anti-A serum only; type 3: cells that stained positive with anti-B serum only; type 4: cells that were negative with both sera. The distribution of each of these epithelial cell types varied considerably, even in the same tissue and individual. Our results seem to suggest that a dissociation in the conversion to the A and B antigens occurs in the tissue of individuals belonging to blood group AB and that the degree of this dissociation varies from tissue to tissue and from cell to cell.     

Congenital tetragametic blood chimerism explains a case of questionable paternity

Human chimerism is the presence of ≥ 2 cell populations in one person that contain genetic material from more than one zygote. Chimerism may be either acquired by transfusion or transplantation of donor cells, or congenital arising from embryo fusion or dizygotic twin-twin transfusion. We encountered a 4-year-old boy with developmental hip dysplasia whose preoperative (serologic) blood group was AB, but whose red cell agglutination was atypical ("mixed field") and caused us to study the patient's parents' ABO blood groups. Parental blood groups (AB and O) suggested possible nonparentage. An alternative explanation of the findings was that the child was chimeric or mosaic. Molecular cloning and genotyping of his ABO locus in leukocytes revealed two heterozygous genotypes: A102/O01 and B101/O01. Other loci, each of which possessed three distinct alleles, unambiguously showed transmission of two alleles from either the child's mother (e.g., HLA-A) or two alleles from the child's father (e.g., D8S1179). Findings indicate that the child is a tetragametic chimera.     

Immunoenzyme AB0 blood group determination using a single hair. I

The immunoenzyme technique was used to determine the ABO blood group of strands of human scalp hair. The hair was obtained from 168 individuals of known blood groups (A1: n = 58; A2: n = 11; B: n = 28; O: n = 46; A1B: n = 16; A2B: n = 9). Immunostaining was carried out by using monoclonal anti-A, anti-B and anti-H as primary antibodies. Group-specific staining was clearly observed within the medulla of the hair. The ABO blood group of all hair samples was determined correctly by the Sternberger (PAP) or APAAP (immunoalkaline phosphatase) technique. The present study indicates that immunoenzyme techniques can be regarded as practical methods for determining ABO blood group of hair.     

ABO genotyping by mutagenically separated polymerase chain reaction

ABO blood groups were determined by the mutagenically separated polymerase chain reaction (MS-PCR). The products from two sets of PCR reactions using the same program for the nucleotides at positions 261 and 703 from cDNA at the ABO locus were used to distinguish A, B and O alleles. Two forward mutagenic allele-specific primers of different lengths for the ABO polymorphic site were paired with the same reverse primer in each PCR reaction. The 216 bp fragment of the PCR products for the 261th nucleotide was A or B allele-specific and the 195 bp fragment was O allele-specific. The 126 bp fragment of the PCR products for the 703th nucleotide was B allele-specific and the 106 bp fragment was A or O allele-specific. The ABO genotypes were determined by the intersection of the predicted alleles from these two PCR reactions. The PCR products were obtained using 10 ng of DNA in 50 μL of PCR reaction mixture, and electrophoresed in 4% agarose gel. In this study, 265 ABO-phenotype known samples (A: 31, B: 48, AB: 6 and O: 180) in Chinese were used. The results of ABO genotypes were AA: 1, AO: 30, BB: 2, BO: 46, AB: 6 and OO: 180. These results were confirmed by the PCR-RFLP ABO genotyping method. This technique is a simple, rapid, and reliable method for ABO genotyping.     

An unusual variant of blood group A

A blood specimen from a forensic science case appeared to violate Landsteiner's Rule. The red cells failed to react with anti-A, anti-B, or O serum while reacting strongly with Ulex europaeus lectin but not other anti-A lectins. The saliva from the person involved was found to contain both A and H blood group substances in a ratio of 4:1. The blood group was determined to be type Am.     

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。     

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。     

Alleles responsible for ABO phenotype-genotype discrepancy and alleles in individuals with a weak expression of A or B antigens

ABO types obtained from evidentiary samples have been used effectively to obtain the initial information leading to the apprehension of culprits in Japanese criminal investigations. A simple ABO genotyping method using multiplex sequence-specific PCR and capillary electrophoresis was developed as a supplement to serological ABO typing. Limitations in predicting a phenotype based on genotype were evaluated using 1134 randomly selected Japanese peripheral blood samples. A concordance rate of 99.82% (1132/1134 samples) was found between genotypes and phenotypes defined as Groups A, B, AB, and O. Sequencing analysis revealed that one discrepant sample contained an O allele having a previously unreported point mutation at the primer binding site in exon 6, and another discrepant sample contained an O allele lacking the guanine deletion at nt 261 (the O301 allele). Therefore, the existence of such alleles must be given some consideration when predicting phenotype based on genotype.     

人类精子ABO血型抗原的间接免疫荧光研究

应用间接免疫荧光技术,对40份精子标本进行 ABO 血型抗原检测。A 型人精子上存在 A 抗原;B 型人精子上存在 B 抗原;AB 型人精液中,一部分精子带有 A 抗原,另一部分精子带 B 抗原。各血型人的精子均有 H 抗原。精子血型抗原为本身所固有,并非来源于精浆。不同人的精子血型抗原含量各不相等,与其供体是否为分泌状态或强弱无直接关系。精子 ABO 血型抗原主要存在于精子的颈部和顶体等区域。     

ABO blood grouping of hairs using an avidin-biotin-peroxidase complex technique

Fifty-nine hair specimens obtained from human autopsies and volunteers were used for the determination of ABO blood group substances using the ABC (Avidin-Biotin Complex) technique. Positive staining for A, B and H blood group substances was detected only in the medulla of the hairs. Blood group antigens could not be detected in seven hair specimens because they possessed no medulla. Forty-seven specimens obtained from fresh cadavers and volunteers gave the correct results corresponding to the blood group of the donor, but some specimens from individuals of blood group A2, Le(a + b-) showed weak reaction with anti-A and strong reaction with anti-H. The staining intensity with anti-B and -H in some individuals of blood group AB was stronger than with anti-A serum. Five hair specimens obtained from decomposed bodies were also examined. The blood group antigens could be specifically detected in hairs obtained from two exhumed and one putrid body, but no positive reactions were obtained from two cases of drowning where the bodies had been in the sea for about 6 months. In a blind trial, hair specimens from 28 individuals were also examined. Twenty-two specimens which possessed a medulla gave the correct result. Six specimens gave no result because they possessed no medulla.     

用ELISA法进行AB型体液（斑）与A型、B型混和体液（斑）的血型测定和鉴别

在法医物证检案工作中,经常会遇到ABO型血型的判定,一般通过吸收试验、解离试验、以及混和凝集试验就能够进行分型.但在实际工作中,遇到的检材多数并非单一的体液,而是两种或者两种以上体液的混和斑.特别是强奸案的鉴定,阴道内提取的检材常常是由男性精液和女性阴道分泌物的混合物,要从混和斑中判断各自的血型,目前仍较困难.国内外许多学者都进行了有关的实验,试图     

Examinations of ABO bloodgroupings of human hair

In order to bring into play the function of human hair ABO bloodgrouping in the field of medicolegal expertise on material evidence and raise its accuracy, the author, through a test on human hair of 500, makes some emphatic researches on the disposal of hairs, on the application of anti-A and anti-B serum, on the selection of red blood cell indicator and on the elution temperature as well. Five hundred samples of human hair have been examined upon the basis of the improved operation method and through the application of anti-A and anti-B serum, the titer of which being 1:128, and therein, fine results have been obtained.     

Light-microscopic examination of ABH and Lewis antigens in human tracheal and epiglottic glands using the avidin-biotin-peroxidase complex technique

The localization of ABH and Lewis antigens was examined in formalin-fixed, paraffin-embedded human tracheal and epiglottic glands using monoclonal anti A, B, H, Lea and Leb antibodies. The mucous cells of the glands showed reactivity with antibodies corresponding to the respective ABO blood groups of the tissue donors. The mucous cells from one blood group A, Le(a-b-) individual showed no reactivity with any antibodies and those from another blood group A, Le(a-b-) individual showed reactivity only with anti A antibody. In individuals from blood group Le(a + b-) of all ABO groups, the mucous cells reacted exclusively with anti Lea. In blood group O, Le(a-b+) individuals, the mucous cells showed intense reaction with anti H and Leb antibodies and weak to moderate reactivity with anti Lea. In Le(a-b+) individuals of A1, B and A1B blood groups, the mucous cells showed strong reactivity with anti A and/or B antibodies, moderate with anti Leb, weak or no activity with anti Lea and absent with anti H. In blood group A2 Le(a-b+) individuals, the mucous cells stained with anti A were weakly stained or completely unstained with anti H antibody, but cells negative with anti A gave strong positive reactions with anti H antibody.     

用酶标抗体免疫组化法测定性交后阴道内容物中精子的ABO血型

应用间接酶标抗体免疫组化法测出了53例新鲜精液、5例陈旧精斑及40例阴道分泌液中的精子与阴道脱落上皮细胞的ABO血型,30例精子与不同血型分泌型阴道分泌液孵育,未发现精子吸附阴道液中血型物质的现象,同时发现人类睾丸曲精细管中部份生精细胞、精子细胞,精子;直细精管部份上皮细胞、精液、精子;睾丸网大部份上皮细胞及副睾管中的精液与精子均含ABH抗原,故认为精子上的ABH抗原主要是精子固有抗原,13例性交后阴道内容物中精子的ABO型测定结果:7例与供者血型吻合,6例不吻合。6例中5例从O型精子中测出了女方分泌型阴道分泌液中的A或AB物质,1例B型精子未测出B及H抗原,文中对这种现象进行了讨论。     

ABO基因分型与多重STR联合检测在法医学中的应用

目的建立ABO基因型和Goldeneye16A试剂盒联合检测的方法,并评价其在法医学实践中的应用价值。方法将6种ABO基因型(A/A,A/O,B/B,B/O,A/B,O/O)的序列特异性引物(PCR-SSP)检测方法与Goldeneye16A试剂盒相整合进行同步分型。通过对460份男性个体血痕样本、9947A DNA及90份案件样本进行检测,考察方法的一致性、灵敏度及对法庭科学检材的适用性。结果应用本文方法可同时检出6种ABO基因型和15个常染色体STR基因座及性别决定基因座,检测灵敏度为125pg,其中ABO基因检测灵敏度达63pg。460份男性血痕和90份案件检材证实该联合分型方法用于各类检材结果准确、稳定。结论本文ABO基因分型与多重STR联合检测方法,适用于各类含有核细胞的生物检材,在法庭科学DNA鉴定中有较好的应用前景。     

Human-type blood group activities on chimpanzee erythrocytes with special reference to M and N

Human-type blood group activities on the red blood cells (RBCs) of three chimpanzees were individually examined with commercial mouse monoclonal antibodies (anti-A, -B, -H, -M, -N, -Lea, and -Leb) as well as lectins (UEA-I and VGA) and conventional polyclonal antisera for the systems ABO, MN, Lewis, Rh-Hr, P, Kell, Kidd, Duffy, and Lutheran. For further analysis of the MN antigens, treatment of the RBCs with sialidase, trypsin, and chymotrypsin were employed. The activities recognized among the three chimpanzees were A, H, M, N, Leb, c, S, k, and Jka. The RBCs of the three individuals possessed the A antigen which showed the same serologic activity as the human A1. Those chimpanzee RBCs showed higher H-activity than the human A1 RBCs. The Lewis b activity was revealed by the absorption-elution method. The RBCs of the three individuals showed a reactivity to the polyclonal anti-M reagents, which was affected by both the sialidase and trypsin treatment. The RBCs of two individuals were agglutinated with the monoclonal anti-N. The receptor was sensitive to sialidase and chymotrypsin. The RBCs of the three individuals, however, did not react with the monoclonal anti-M or with one of the polyclonal anti-N. These results indicate structural differences in the glycophorins and MN antigens between the human and chimpanzee.     

Determination of ABO antigens in fingernails using the APAAP (immunoalkaline phosphatase) technique

Fingernail specimens with adherent nail-bed were taken from autopsy material with blood groups A, AB, B and O. Frozen 4-5-microns sections were submerged and floated carefully during each working step. Portions of fingernails were contaminated with blood and buccal cells, respectively. Furthermore, fingernail fragments of 8 volunteers were embedded in a biocomponent adhesive according to Grieve and Kotowski (Forensic Sci. Soc., 26 29-34) (1986) and cut by the usual microtome technique. APAAP staining is a proper method for demonstrating blood group antigens in fingernails from groove to margin. Frozen sections as well as smallest specimen embedded in a suitable adhesive are applicable for staining procedures. Using freshly prepared artificial stains, blood group constellations of red blood cells and/or buccal cells adherent on the surface of fingernails may be distinguished from the nail matrix.     

The ABO blood grouping of a minute hair sample by the immunohistochemical technique

The unlabeled antibody (PAP) immunoperoxidase technique was applied to the ABO blood grouping of human scalp hairs. Hair samples were subjected to longitudinal- or cross-sectioning, thus obtaining suitable samples for subsequent immunostaining. The immunostaining was carried out using rabbit anti-A and anti-B sera as the primary antibodies. With this technique, the group-specific staining which is revealed as a dark brown precipitate was clearly observed within the medullae of the hair shaft, and depending on the presence or absence of these precipitates, respective blood groups of unknown hair samples were determined. At the hair root, on the other hand, positive stainings were observed not only in medullary cells but also in some cortical cells of the keratogenous zone. From the present study, it can be safely said that this technique is of practical use for the ABO blood grouping from a minute (less than 3 mm) hair sample.     

The use of anti-A and anti-B monoclonal antibodies in forensic medicine

Anti-A and anti-B monoclonal antibodies (MCA) may be used in the analysis of liquid blood, blood and saliva traces in order to detect ABO blood group antigens A and B using common methods of evidence investigation. Use of MCA and isohaemagglutinins anti-A and anti-B in absorbtion-elution reaction during the analysis of minute saliva traces enhances the possibility of establishing the origin of the saliva at the expense of nonsecretor.