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.     

4种溶液对陈旧斑迹胶体金试剂条ABO检测结果比较

目的比较4种不同溶液用于陈旧斑迹ABO胶体金试剂条分型检验的结果。方法采集已知血型的静脉血127份,唾液73份制备斑迹,放置1~2年;分别用去离子水、PBS、PBS(含2%吐温20)、PBS(含2%吐温20、1%吐温80)4种溶液浸泡,再用ABO胶体金试剂条检测其血型,观察结果的清晰度及准确度。结果用不同溶液浸泡后进行ABO分型检测结果中,去离子水和PBS溶液分型检测线不清晰,难以判型;含吐温的PBS溶液分型检测线比较清晰,所测样本结果均准确,其中同时含吐温20和吐温80的溶液结果更佳。结论根据本文结果,在陈旧斑迹的ABO血型检验中可选择使用含有吐温的PBS溶液作为斑迹浸泡溶液。     

Blood grouping in mixed blood stains and excretions

Blood grouping in mixed blood stains and excretions can often be a problem. It is related with the fact that, when blood and excretions of persons having different groups get mixed, the determinants of blood antigens of one person interact with the active centers of excretions' antibodies of another one and vice versa. The authors suggest a method to do away with the phenomenon.     

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.     

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.     

Analysis of DNA in minute volumes of blood from stains and crusts

As the first step, the locus D1S80 was amplified by the polymerase chain reaction technique from genomic DNA extracted from artificial bloodstains and crusts with different amount of blood (32 microl, 16 microl, 8 microl, 4 microl, 2 microl, and 1 microl). In all samples of bloodstains and crusts, identification by DNA analysis was possible. As the second step, the locus HLA-DQA1 was amplified from genomic DNA extracted from diluted blood samples (640, 320, 160, 80, 40, 20, 10, and 5 leukocytes). DNA amplification was possible in diluted blood samples with at least 10 leukocytes. Considering the conditions in which the present study was carried out, it was possible to conclude that 1 microl of bloodstains or crusts was enough for identification. It was also concluded that five leukocytes are not enough material to render consistent DNA identification.     

Dual examinations for identification of urine as being of human origin and for DNA-typing from small stains of human urine

Concurrent methods for identification of urine as being of human origin, and for DNA-typing from small stains of human urine were examined. A urine stain was extracted with phosphate-buffered saline (PBS), and the extract was filtered using a Centricon-100 device. The filtrate was subjected to electrospray ionization liquid chromatography-mass spectrometry (ESI-LC-MS) for identification of human urine and a DNA-typing sample was obtained by dialfiltration of the residue using a DNA purification kit. After the purified residue was treated with an AmpflSTR Profiler PCR amplification kit, the DNA-types were analyzed by capillary electrophoresis using a Genetic Analyzer. It was possible to identify a urine stain as being of human origin, and complete DNA profiles could be successfully obtained from a urine stain which had been created by 50 microL of female urine. Serial analyses of urine stains found at a crime scene provide effective information for forensic investigation. This method is recommended for stain identification and for DNA-typing from a urine stain.     

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.     

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.     

Zinc test as a new tool for identification of human seminal stains

An extremely simple qualitative method for identification of seminal stains based on high levels of zinc in human semen is described. It uses reaction of 1-(2-pyridylazo)-2-naphthol with zinc to develop a deep red color. The data are presented on the sensitivity, stability and specificity of the present method. We can recommend it for identification of human semen especially in old or denatured samples.     

Studies and observations on Lewis grouping of body fluids and stains

Leb positive individuals may phenotypically express both Lea and Leb in their secreted body fluids. Therefore, the interpretation of a Le(a + ,b-), non-secretor result is dependent on the absence of Leb. This study emphasises the importance of accurate procedure and biased selection of antisera such that Leb is preferentially detected in comparison with Lea. The relationship of the ABO group to the expression of Le is discussed in conjunction with the selection of samples for testing antisera and inclusion as control standards.     

Human blood stain identification and sex determination in dried blood stains using recombinant DNA techniques

DNA was extracted from human and non-primate dried blood stains. Human male and female specimens were readily distinguished by analysis with a Y-chromosome specific DNA probe. Human and non-primate blood stains were also readily differentiated using a repeat sequence (Alu) DNA probe. The potential power of recombinant DNA analysis in forensic science is discussed.     

The application of visible wavelength reflectance hyperspectral imaging for the detection and identification of blood stains

Current methods of detection and identification of blood stains rely largely on visual examination followed by presumptive tests such as Kastle–Meyer, Leuco-malachite green or luminol. Although these tests are useful, they can produce false positives and can also have a negative impact on subsequent DNA tests. A novel application of visible wavelength reflectance hyperspectral imaging has been used for the detection and positive identification of blood stains in a non contact and non destructive manner on a range of coloured substrates. The identification of blood staining was based on the unique visible absorption spectrum of haemoglobin between 400 and 500 nm. Images illustrating successful discrimination of blood stains from nine red substances are included. It has also been possible to distinguish between blood and approximately 40 other reddish stains. The technique was also successfully used to detect latent blood stains deposited on white filter paper at dilutions of up to 1 in 512 folds and on red tissue at dilutions of up to 1 in 32 folds. Finally, in a blind trial, the method successfully detected and identified a total of 9 blood stains on a red T-shirt.     

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.     

Genetic markers in human bone: II. Studies on ABO (and IGH) grouping.

A combination absorption-elution, two-dimensional absorption-inhibition procedure was used to determine the ABH antigen composition of a series of human bone specimens of known ABO type that had been aged up to nine months under dry and humid conditions at ambient temperature, 37 degrees C, and 56 degrees C; at ambient temperature in dry and wet soil; and buried in soil outdoors. Grouping data for the separate elution and inhibition testing, as well as for the combination procedure, are given. The combination method was found to be a highly reliable procedure for bone tissue ABH typing. Some data on microbial contaminants of human bone specimens aging in soil, and their effects on ABH typing results, are presented. No direct correlation between the properties of microbial contaminants and specific changes in the ABH antigenic composition of aging bone tissue specimens could be ascertained. Data on IGH antigen determination and on the quantitation of immunoglobulin G (IgG) in human bone tissue extracts indicated that immunoglobulin levels were typically too low to expect routinely successful Gm antigen testing results. However, these factors can sometimes be determined in fresh bone tissue extracts, particularly if the extracts are concentrated.     

Detection and use of salivary hemagglutinins for forensic blood grouping

A sensitive method for the detection of anti-A and anti-B hemagglutinins in fresh saliva has been developed. The method utilizes a bromelin treated erythrocyte suspension as indicator cells and includes a simple procedure to concentrate these hemagglutinins. Antiserum directed against immunoglobulin A enhances the hemagglutination assay. We find that these salivary hemagglutinins are present in over 90% of the population and that their titer remains stable over a period of two months. These hemagglutinins can be used to blood type the donor of a saliva sample and can be used in a confirmatory test that complements the commonly used absorption-inhibition test which is used to detect salivary blood group agglutinogens. In preliminary studies we have determined that hemagglutinins can be successfully isolated and analyzed from dried saliva stains.     

间接免疫酶组化法检测人指纹ABO血型

建立一套测定人指纹 ABO血型的方法体系。运用间接免疫酶技术和免疫印迹技术 ,对指纹胶纸提取的指纹、银粉显现提取的指纹、直接转移至硝酸纤维素膜 (NC膜 )上的指纹、银粉显现转移至 NC膜上的指纹、“5 0 2”胶熏显转移至 NC膜上的指纹等进行 ABO血型检测。上述 5种方法采集的 2 12枚指纹 ,绝大部分能正确地检出 ABO血型 ,检出率为 90 %～ 93.8% ;未能检出的样本为非分泌型人指纹。应用间接免疫酶技术及免疫印迹技术测定人指纹的 ABO血型的方法具有特异性强、准确可靠、检测方便等优点 ,在法庭科学中易推广应用 ,并能发挥重要的作用