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.     

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.     

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.     

PCR－RFLP技术鉴定ABO基因型的法医学应用研究

目的建立PCR－RFLP、非变性PAG胶垂直电泳和银染技术进行ABO基因分型的方法体系,并对200名广东汉族人群ABO基因型频率进行了调查。方法用Chelex－100和酚、氯仿抽提法处理样本,PCR扩增后用非变性聚丙烯酰胺凝胶垂直电泳和银染技术检测分型。结果ABO位点特异性扩增片段长度为175bp～210bp,6种基因型频率分布为0.0250～0.4300,杂合度H值为0.5162,个体识别力DP值为0.7111。结论该方法可成功运用于血液、血痕、精斑、毛发、骨组织和混合斑等检材的个体识别及亲权鉴定的检验。     

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。     

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.     

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.     

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.     

Information from penile swabs in sexual assault cases

A survey has been made to assess the evidential value of tests carried out on 660 casework penile swabs. Most were from suspects in sexual assaults and were examined to see if the donor had had recent anal, oral or vaginal intercourse. The swabs were tested for one or more of the following: blood, faeces, saliva, vaginal secretions, semen. Blood was seldom found, it was usually weak and insufficient for grouping. Faeces were only identified on a pair of swabs from a dead homosexual showing that proof of buggery by this means is rare. Amylase, suggestive of saliva and oral intercourse, was occasionally detected. Glycogen-rich epithelial cells were sometimes present indicating vaginal intercourse. Semen was frequently found but its presence may not result from a recent sexual act. An ABO group different from the donor was obtained from a fifth of the swabs typed. Grouping in other blood group systems was rarely attempted or successful. Penile swabs provided a means of detecting a victim's ABO blood group on a suspect when it would not have been possible to demonstrate the suspect's group on samples from the victim. They also had value in assaults involving more than one offender. The main limitation of penile swabs was the paucity of material on them and the sampling site affected the interpretation of the results.     

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.     

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.     

Cytochemical detection of ABH antigens in human body fluids

The use of the peroxidase-anti-peroxidase (PAP) technique has been described previously for the detection of cellular antigens and in particular ABO antigens from tissue samples (Pedal and Hülle 1984; Pedal and Baedeker 1985; Pedal et al. 1985). In this survey, the PAP method has been employed to study the detection of ABO antigens in cells from body fluids of particular interest to forensic science, namely buccal cells and vaginal cells. Also tested, but in a limited number, were mixtures of body fluids and semen samples. No false reactions were obtained from buccal cells, all samples corresponding to the ABO blood type of the donor. Preliminary results from vaginal cells, vaginal/buccal cell mixtures, and semen were encouraging but must be treated with caution due to the limited number tested. Vaginal smears contaminated with semen showed varying degrees of nonspecificity.     

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.     

Aberrent group B reactions detected in mixtures of semen and vaginal secretions possibly due to acquired B

Routine ABO grouping tests performed on vaginal swabs from a group O female showed the presence of blood group B activity as well as the expected A and H activity after intercourse with her group A partner. The source of the B activity was readily detectable using the Laboratory's routine elution test, but it was not detectable when a monoclonal anti-B was used and detection by the inhibition technique was possible only with a selected antiserum. A possible explanation of the reactions observed is that the acquired B is capable of reacting with only a portion of the antibodies present in normal polyclonal anti-B sera.     

Effect of blood group active micro-organisms on the ABO grouping of human whole saliva

Three saliva samples with false positive ABO grouping results were assayed for blood group active organisms, using a variety of selective media to isolate representative strains from the salivary microflora. Eight out of 40, 8 out of 40 and 4 out of 30 strains from the three samples, respectively, showed blood group activity, which correlated well with the false positive specificities of the saliva samples. In all cases the false reaction only lasted a few days. Investigation of one of these samples before and after the appearance of the false positive activity yielded only one out of 40 blood group active organisms, using the same methods. Similar investigation of two "normal" saliva samples found none out of 40 and one out of 40 blood group active organisms, respectively. It is concluded that occasional false positive ABO grouping reactions of saliva samples are probably caused by the presence of unusually high numbers of blood group active micro-organisms, due to disturbances in the ecological balance of the salivary microflora.     

Practical GC/MS analysis of oxidation dye components in hair fiber as a forensic investigative procedure

The purpose of this study was to improve the reliability of discrimination (or identification) of dyed hair by analyzing chemical substances present in the hair, as an addition to the conventional macroscopical and microscopical examinations and ABO blood group examination. Oxidation hair-dye components such as p-phenylenediamine (PPDA), toluylene-2,5-diamine (T-2,5-DA), o-aminophenol (OAP), m-aminophenol (MAP), p-aminophenol (PAP) and p-amino-o-cresol (PAOC) were selected as the object of study. After alkaline-digestion, hair samples were adjusted to a pH of 12.6 to 12.8, and applied onto an Extrelut column. After 15 min, the components were simultaneously extracted and derivatized with n-hexane including 1% heptafluoro-n-butyryl (HFB) chloride. Their HFB derivatives within a condensed sample were diluted in ethyl acetate, and analyzed by gas chromatography-mass spectrometry (GC-MS) with full mass scanning or selected ion monitoring. For estimating their levels, 2,4,6-trimethylaniline was used as the internal standard. Standard curves obtained by preparing a 5 cm segment of control hair spiked with authentic substances were linear, ranging from 0.1 to 4.0 micrograms for PPDA and T-2,5-DA, and from 0.01 to 0.4 microgram for OAP, MAP, PAP and PAOC. The coefficient of variation of inter-day precisions (each n = 4) was below 16% for PPDA, below 20% for OAP and PAP and below 24% for T-2,5-DA, MAP and PAOC. These components were detectable even at 6 ng for PPDA, T-2,5-DA, MAP and PAP, 8 ng for OAP, and 4 ng for PAOC. Recovery percents using this procedure ranged from 54 to 86%. By using actual dyed hair samples this method was shown to provide high sensitivity for accurate detection.     

An improved means of enzyme typing of hair roots using isoelectric focusing.

An improved method of grouping hair, based on the alleles of PGM observed by isoelectric focusing, has been described. The increased discriminating power of this system (0.77) compared to that obtained by the starch gel technique (0.55) provides a new and more sensitive means of typing hair.     

Blood group frequencies of the population of Trinidad and Tobago, West Indies.

The results of grouping tests performed on blood samples collected over a five-year period at the Trinidad and Tobago Forensic Science Centre are presented. The samples were tested using the ABO, PGM, EAP and GLO blood group systems. Phenotypic frequencies and allele frequencies for each system were calculated for the two major ethnic groups of the population, the African and East Indian. Matching probabilities, which can be used in the interpretation of physical evidence in forensic cases, were also calculated.     

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.     

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。