Human Bloodstains Research Articles

Development of a Radioimmunoassay Technique for the Detection of Human Hemoglobin in Dried Bloodstains

A sensitive radioimmunoassay for the detection of human hemoglobin in dried bloodstains for the purpose of forensic science species identification has been developed. Bloodstains from 13 animal species were tested and found to be negative for human blood. A minimum volume of 0.8 microL of fresh blood is required to produce sufficient stain for successful testing. Bloodstains prepared from newborn and sickle-cell bloods were determined to be human. Bloodstains ranging in age from 1 month to 6 years which had been maintained desiccated at 20 to 25 degrees C were also successfully tested. Positive results were obtained on human bloodstains stored at 24 degrees C with relative humidity ranging from 0 to 98% for a period of 3 weeks. Absolute counts per minute (CPM) decreased with increased humidity. Human bloodstains exposed to bacterial contamination (gram positive or negative species) under humid conditions for 2 weeks also tested positive. Bacterial contamination caused a decrease in CPM, but insufficient to result in an erroneous conclusion as to species of origin. Positive results were also obtained on human bloodstains stored for 6 weeks at various temperatures ranging from -16 to 37 degrees C. No significant decreases in CPM were noted for any of the temperature conditions described.

Glucose phosphate isomerase variants in human bloodstains and seminal stains

Glucose phosphate isomerase (GPI) variants occurring in human red cells were also demonstrated in human semen. Phenotyping was possible from bloodstains of 6 weeks storage and seminal stains of 12 weeks storage. The GPI system may be a supplemental tool for medicolegal individualization of seminal stains.

Human blood typing: A forensic science approach. Part II: Experiments

In this article, the authors describe several experiments that illustrate techniques available to the forensic serologist for typing a human blood stain in the ABH grouping system.

Antigenic properties of human and animal bloodstains studied by enzyme-linked immunosorbent assay (ELISA) using various antisera against specific plasma proteins

Antigenic properties of bloodstains of human and non-human primates as well as other animal bloodstains were investigated by the inhibition ELISA using commercially available anti-human albumin (Alb), alpha 2-macroglobulin (alpha 2-M), fibrinogen, transferrin, and immunoglobulin G. In general, chimpanzee bloodstains showed strong cross-reactions with these antisera, and the extent of the cross-reactions of other animal bloodstains decreased largely with the phylogenic order, i.e., agile gibbon (ape), Old World monkeys (Japanese monkey and hamadryas baboon), New World monkeys (night monkey and tufted capuchin monkey), prosimians (grand galago and ring-tailed lemur) and other animals (rat, cattle, swine, goat, dog, cat, and chicken). Among these antisera, anti-human alpha 2-M showed the weakest cross-reaction with chimpanzee bloodstains, and anti-human Alb showed next.

A method for subtyping group-specific component in bloodstains

A method is described for subtyping group-specific component (Gc) derived from human bloodstains. Bloodstained cuttings were extracted in 6 M urea. The extracts were subjected to ultrathin-layer polyacrylamide gel isoelectric focusing in the pH 4.5–5.4 range. After isoelectric focusing, Gc was detected by immunofixation in cellulose acetate membranes. This method permitted the successful typing of Gc in at least four-month-old bloodstains maintained at room temperature. Bloodstains from 266 liquid blood samples of known origin were subjected to both this method and immunofixation conventional agarose gel electrophoresis with no phenotypic discrepancies observed. The Gc population data for Whites from Baltimore, Maryland, were homogeneous with white sample populations from other geographical locations within the U.S.A.; whlie Gc data from northern U.S.A. black sample populations appeared to be heterogeneous compared with a southern United States black sample population.

A sequence of tests of minute human blood stains for human origin identification and ABO blood grouping

A series of examinations is presented for human origin identification and ABO blood grouping of doubtful minute human blood stains. A blood-stained thread (0.5 cm in length) was first tested to identify human origin by microprecipitation method and then the ABO blood type was determined by both a modified absorption-elution test and a modified mixed agglutination. In the continuous tests, the maximum limits of positive reactions of the microprecipitation method, the modified absorption-elution test, and the modified mixed agglutination were 1:640, 1:160, and 1:2,560 diluted blood, respectively. A and B agglutinogens were more sensitively determined than H agglutinogen. Hemagglutinogens of blood stains on cotton threads were more easily detected than those of polyester ones.

Human blood stain identification and sex determination in dried blood stains using recominant 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.

Quantitative IgD measurement for discrimination of human bloodstains

The IgD concentrations of eluates of artificial bloodstains and of the corresponding sera from 40 subjects of different ages were measured by single radial immunodiffusion. IgD was found in bloodstains stored up to 53 days, i.e. IgD storage stability is sufficient for forensic purposes. Since serum IgD concentrations of individuals, in particular of adults, are almost invariable and serum levels of different individuals can vary by more than a 1000-fold, the discrimination of bloodstains on the basis of IgD is generally possible. Thus IgD constitutes a further marker in antibody profiling of bloodstains.

Transferrin subtyping of human bloodstains

A method was described for subtyping transferrin derived from human bloodstains. Bloodstain cuttings were extracted in 0.5% ferrous ammonium sulfate. The extracts were subjected to ultrathin-layer polyacrylamide gel isoelectric focusing. After isoelectric focusing, transferrin was detected by silver staining. This method permitted the successful typing of Tf in 6-month-old blood stains maintained at −20°C and room temperature and 3-month-old bloodstains maintained at 37°C.

Isotachophoretic Analysis of Bloodstains: Differentiation of Human, Menstrual, Bovine, and Ovine Bloods

Isotachophoresis, a technique to separate components by constant current electrophoresis, was used to differentiate between bloodstains of male, female, menstrual, bovine, and ovine bloods on cotton cloth and filter paper. Bloodstain analysis by isotachophoresis of stains from male and female subjects showed identical cationic patterns, but gave different profiles in the anionic system. Plasma had one extra peak in the anionic system when compared to the profile of serum. This extra peak is due to the presence of fibrinogen in plasma. Some hemoglobin peaks overlapped with serum protein peaks, but these could be identified by comparisons at lower concentrations. Menstrual blood had a much different pattern than normal human blood as was expected since many more compounds are found in menstrual blood than in normally circulating blood. Human, bovine, and ovine bloodstains showed different profiles both in the cationic and anionic systems. These results indicate that isotachophoresis can be used for the rapid and simple analysis of bloodstains to differentiate reliably human male, female, and menstrual blood and also to distinguish human bloodstains from those of cattle or sheep.

A method for dissociating group‐specific component from protein complexes in human bloodstains and its significance in forensic biology

AbstractIn some bloodstains group‐specific component (Gc) forms complexes with blood platelet membrane proteins such as actin which, during isoelectric focusing (IEF), migrate with a lower isoelectric point (pI) than free Gc. When this association occurs it is difficult and sometimes impossible to identify the phenotypes of the Gc system. The problem has been overcome by extracting the bloodstain with 6 M urea prior to IEF. It is then possible to detect and to type Gc in bloodstains that would otherwise not be amenable to Gc typing.

Typing of esterase D by isoelectric focusing

AbstractA method is described for obtaining reproducible resolution of Esterase D (EsD) phenotypes from human bloodstains by ultrathin‐layer polyacrylamide gel isoelectric focusing. By employing a pH 4.5–5.5 gradient and an electrode wick distance of 9.5 cm, all the common EsD variants were separated in 85 min. Electrofocusing of EsD took 53 and 60 min on the Cold Focus Apparatus and Ultrophor, respectively, when the distance separating the electrodes was 8.0 cm. Bloodstains up to 4 weeks old could be readily typed for EsD using this method.

Phosphoglucomutase‐1 subtyping of human bloodstains on ultrathin‐layer polyacrylamide gels

AbstractA method is described for obtaining nondistorted, reproducible phosphoglucomutase‐1 subtyping patterns from fresh human bloodstains by ultrathin‐layer polyacrylamide gel isoelectric focusing. Electrofocusing of phosphoglucomutase on 0.2 mm thick gels took 52 and 80 min on the Cold Focus Apparatus and Ultrophor, respectively, when the distance separating the electrodes was 8.0 cm, whereas a 9.5 cm distance between electrode wicks required 95 min on the Multiphor to complete isoelectric focusing. The gels with 8.0 cm between electrode wicks provided sharper band patterns compared with the gels with 9.5 cm between electrode wicks.

Species Identification of Blood and Saliva Stains by Enzyme-Linked Immunoassay (ELISA) Using Monoclonal Antibody

An indirect enzyme-linked immunoassay (ELISA) method for the identification of human blood and saliva stains is reported. The method uses a monoclonal antibody which reacts with human immunoglobulin G (IgG) in extracts of blood and saliva stains up to 16 months old. Semen stain extracts gave weak or negative results. For routine screening purposes dilutions of 1:1000 for bloodstain extracts and 1:100 for saliva stain extracts would be suitable. Of 32 other animal species tested, only chimpanzee, mouse, rat, and eel cross-reacted significantly, and the presence of the last three was clearly indicated by appropriate controls. The monoclonal antibody gave poor results in the crossover and gel diffusion techniques.

Identification of Human Blood Stains by Radioimmunoassay

A radioimmunoassay (RIA) for the platelet-specific protein, β-thromboglobulin (BTG) was assessed for use as a sensitive marker of human blood. Samples of blood (0·1 ml) from human and animal sources were spotted on a towel, allowed to dry, then eluted with distilled water. Only the eluates of the human blood stains reacted in the assay system. The sensitivity of the assay system was assessed by rinsing a 0·lml dried blood stain successively with 1·0ml of distilled water. The first three eluates all reacted positively in the standard assay system while further dilutions required the more sensitive assay. The stability of the human BTG antigen was assessed under dried stain conditions and it was found to remain immunoreactive for up to four months. The radioimmunoassay for β-thromboglobulin represents a unique approach to the identification of small samples of blood of an unknown origin. Confirmation that it is blood and is of human origin can be made in as little as three hours in a simple one-step procedure that is at least an order of magnitude more sensitive than conventional tests currently being employed.

The typing of group-specific component (Gc protein) in human blood stains

It is known that the typing of group-specific component (Gc protein) in human blood stains is difficult since Gc protein of the extracts of blood stains migrates more anodally to the α 1-globulin region in agar-gel immunoelectrophoresis, while Gc protein in liquid blood normally migrates to the α 2-globulin region. We have reported that the Gc protein found in the α 1-region is the result of binding of actin to Gc protein (Shinomiya, K., Kimura, H., Yoshida, K., and Shinomiya, T., J. Biochem., 92 (1982) 1163–1171, which renders it difficult to determine the Gc-phenotypes in the blood stains. On the basis of the above findings, we developed the method of phenotyping the Gc protein of human blood stains by agar-gel immunoelectrophoresis. Since the binding activity of actin to Gc protein is lost after treatment with a high concentration of guanidine HCl, the extracts of blood stains were treated with 4 M guanidine HCl to dissociate Gc protein and actin and then dialyzed to remove guanidine HCl. By this method we are able to determine the phenotypes of Gc protein in blood stains. The method we have developed is a useful tool in the forensic laboratory.

The typing of α1-antitrypsin in human bloodstains by isoelectric focusing

A polyacrylamide gel isoelectric focusing (PAGIF) technique is described for the determination of α 1-antitrypsin (Pi) phenotypes in bloodstains. The time limits for Pi type determination of bloodstains kept under different storage conditions are given. The resolution of PAGIF in the typing of Pi phenotypes in human bloodstains in investigated.

Application of enzyme-linked immunosorbent assay (ELISA) to identification of Anopheles mosquito bloodmeals

The microplate method of the ELISA described by Voller et al. (1974) was manipulated for identification of Anopheles bloodmeals. Blood samples collected from man and from laboratory animals were put in small amounts (1 to 2 μl) on filter paper. Also bloodmeals from A. stephensi fed on human volunteers and guinea-pig were smeared on filter paper one, six 12, 24 and 48 hours after ingestion. The samples were tested by a modified microplate ELISA, using alkaline phosphatase anti-human IgG conjugate. Results showed that ELISA is a quite specific and sensitive technique for the detection of a small amount of human blood, even 24 hours after ingestion by anopheline mosquitoes. The ELISA seems to be a more practical and reproducible method than the precipitin test for identification of arthropod blood-meals, and could also be used for identification of blood spots in forensic laboratories. The anthropophilic index of anopheline mosquitoes is an important factor in the role of different Anopheles mosquitoes in malaria transmission. The technique which has been most commonly used for determination of the source of Anopheles bloodmeals is the precipitin test which was first employed by Uhlenhuth et al. (1908) as a medicolegal procedure for the identification of human blood stains. Later it was used and modified for determining the sources of bloodmeals in mosquitoes and other insects ( King & Bull, 1923 ; Rice & Barber, 1935 ). Although the precipitin test is quite a specific and reliable test, the microplate indirect method of enzyme-linked immunosorbent assay (ELISA) developed by Voller et al. (1974) may be more practical for the identification of human blood in engorged anopheline mosquitoes. The ELISA test has, therefore, been modified for mosquito bloodmeal identification in this study.

The Identification of Human Bloodstains by Means of Thin-Layer Immunoassay: A Preliminary Report

The application of thin-layer immunoassay to the problem of human bloodstain identification is described. The differentiation of human, dog and rabbit bloodstains has been achieved by this method with the use of anti-human haemoglobin serum.

The Identification of Human Bloodstains by Means of a Micro-Thin-Layer Immunoassay Procedure

A method for the identification of human bloodstains using a micro-thin-layer immunoassay (TIA) procedure is presented. The equivalent of approximately 0.01 micro /leter of human blood may be detected using this method with anti-human hemoglobin. Blind studies and stability studies indicate that the method is sufficiently specific and sensitive to be of forensic science value.