The collaborative study on typing group-specific component in casework bloodstains

Casework bloodstains were typed for group-specific component (GC) at eight forensic laboratories. Approximately 600 bloodstains were examined of which a mean of 62.7% gave results. This is comparable to other blood grouping systems in current use. Stains that were over three-months old were successfully typed in six of the laboratories. A wide variety of substrates was examined; these included many items of clothing as well as metal blades, concrete, paint, cement, glass and grass. Of substrates that were examined several times, none consistently gave problems with GC typing. The GC system has been shown, therefore, to be an effective test in operational forensic science.     

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

A polyacrylamide gel isoelectric focusing (PAGIF) technique is described for the determination of α₁-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.     

Group specific component subtyping in bloodstains by separator isoelectric focusing in micro-ultrathin polyacrylamide gels followed by immunoblotting

The identification of group specific component (Gc) subtypes derived from blood-stains by separator isoelectric focusing in micro-ultrathin polyacrylamide gels (interelectrode distance: 50 mm) containing 4.5 to 5.4 pharmalytes is described. The separation achieved between Gc 1F and Gc 1S bands is compared favorably with that obtained using separator isoelectric focusing in conventional polyacrylamide gels dimensions (interelectrode distance: 110 to 120 mm). The technique is rapid and economical, and the immunoblotting method described is more sensitive than immunofixation followed by silver staining.     

Determination of C3 phenotype in dried bloodstains using isoelectric focusing

Fresh whole blood and bloodstains were analyzed by isoelectric focusing (IEF) to determine the C3 phenotype of the blood donor. Three common phenotypes exist as a result of two autosomal alleles. The three phenotypes can be identified in fresh serum or in serum samples which had been stored at -20 degrees C for more than a year. Bloodstains maintained in a desiccator at 25 or at 37 degrees C retained the native form of C3 which could be detected for at least two weeks. Beyond two weeks of storage, stains became difficult to phenotype due to decreased banding intensity. Bloodstains aged longer than one month could not be phenotyped. C3 could not be detected in human semen by the serological methods employed.     

A comparison of the sensitivity of typing group-specific component (Gc) and the phosphoglucomutase (PGM1) locus in control and casework bloodstains by three isoelectric focusing methods

The performance of typing group-specific component (Gc) in bloodstains by two isoelectric focusing methods followed by its detection with silver staining has been compared with an established forensic system of typing phosphoglucomutase (PGM1) locus phenotypes by isoelectric focusing (IEF) in 1 mm gels. For Gc typing ultra-thin isoelectric focusing (UTIEF) gels and immobilized pH gradient (IPG) gels were used. Both laboratory prepared stains and casework stains were examined. The Gc UTIEF method is approximately eight times more sensitive than the existing PGM1 1 mm IEF method for control and casework stains. However, on average, a larger amount of stain was taken from casework stains than control stains for each typing system. A total of 53 casework stains were examined. Comparable success rates of 62% and 64% were obtained for typing Gc on UTIEF gels and PGM1 by 1 mm IEF, respectively. A success rate of 55% was obtained for typing Gc on IPGs. Bloodstains that were over 200 days old were successfully grouped by all three methods.     

Transferrin subtyping of bloodstains and semen using isoelectric focusing and immunoblotting

Transferrin (TF) subtyping was carried out on bloodstains that had been made on cotton sheeting and stored under a variety of conditions ranging from −20°C to +37°C. The time limit of detection was longer than 54 weeks after dry storage under each condition. Moreover the correlation between isoprotein types of the TF in blood and semen samples from the same individual was determined in 103 men. All three TF common types and two rare types in all semen samples correlated with the type found in the corresponding blood sample. A combination of isoelectric focusing separation and immuno-enzyme-linked detection may prove to be very useful for forensic TF subtyping.     

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.; while Gc data from northern U.S.A. black sample populations appeared to be heterogeneous compared with a southern United States black sample population.     

Transferrin subtyping of bloodstains and semen using isoelectric focusing and immunoblotting.

Transferrin (TF) subtyping was carried out on bloodstains that had been made on cotton sheeting and stored under a variety of conditions ranging from -20 degrees C to +37 degrees C. The time limit of detection was longer than 54 weeks after dry storage under each condition. Moreover the correlation between isoprotein types of the TF in blood and semen samples from the same individual was determined in 103 men. All three TF common types and two rare types in all semen samples correlated with the type found in the corresponding blood sample. A combination of isoelectric focusing separation and immuno-enzyme-linked detection may prove to be very useful for forensic TF subtyping.     

Esterase D typing of bloodstains by non-equilibrium focusing

Non-equilibrium focusing in a pH 4-6 gradient in ultra-thin polyacrylamide gels has been shown to be a reliable and reproducible method for detecting the six common esterase D phenotypes (EsD 1,2-1,2,5-1,5-2 and 5) in dried bloodstains. Successful typing is dependent on both the age and phenotype of the stain in question. The effects of age on the isozyme pattern of each phenotype are described and illustrated. In a comparative trial using 100 simulated and 300 authentic casework bloodstains, non-equilibrium focusing was shown to be more efficient than thin-layer starch gel electrophoresis for the typing of esterase D.     

Detectability of group-specific component (Gc) in aged bloodstains

An improved method of group-specific component (Gc) typing was conducted electrophoretically on agarose gel. Individual bloodstains randomly collected from different individual donors over a five-year period at intervals of approximately one month were checked for Gc activity. Group-specific component was typed accurately in dried bloodstains stored at room temperature up to 43 months in age. From 100 different donors, bloodstains ranging in age from 38 to 43 months were tested by the methods described and 73% of the samples were interpretable for Gc.     

C7 typing of blood stains using isoelectric focusing and immunoblotting

By means of isoelectric focusing and immunoblotting C7 types were clearly demonstrated from bloodstains which had been stored at 37 degrees C for up to three weeks, at room temperature for up to six weeks and at 4 degrees C for over ten weeks. The C7 typing is practically useful in medicolegal individualization of unknown bloodstains.     

Factor B subtyping in sera and bloodstains by isoelectric focusing and immunoblotting

The polymorphism of BF was investigated in 765 unrelated Japanese individuals by isoelectric focusing and immunoblotting. Besides five common subtypes three rare variants were observed. The allele frequencies were: BF*S = 0.8078, BF*FA = 0.1797, BF*FB = 0.0105, BF*Var. = 0.0020. The above method was successfully applied to subtyping BF in stored bloodstains. The determination limits were: at 4 degrees C 8 weeks, at room temperature 2 weeks and at 37 degrees C only 2 days after storage. The BF subtyping is of practical use in medicolegal individualization of unknown bloodstains.     

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.     

Phenotyping of alpha-2-HS glycoprotein in bloodstains by isoelectric focusing and immunoblotting

A sensitive immunoblotting procedure has been applied to the detection of alpha-2-HS-glycoprotein (A2HS) phenotypes from control and casework bloodstains. A2HS phenotypes were separated by thin layer polyacrylamide gel isoelectric focusing (PAGIEF) in gels containing Pharmalyte pH 4.2-4.9. After transfer to nitrocellulose by a rapid capillary blot, the A2HS phenotypes were developed using a double antibody enzyme-immunoassay. The evaluation of A2HS phenotyping of casework material was undertaken in parallel with phosphoglucomutase (PGM) phenotyping by PAGIEF. A total of 598 water extracts from casework bloodstains have been tested. Positive results were obtained in 84% and 75% of samples for PGM and A2HS respectively. The A2HS gene frequencies A2HS*1 = 0.6420, A2HS*2 = 0.3530, and A2HS*3 = 0.0050 were determined from a survey of 1000 people in Brisbane.     

Orosomucoid (ORM) typing by isoelectric focusing: description of two new alleles in a German population and thermostability in bloodstains

The genetic polymorphism of serum orosomucoid (ORM) was studied in 168 unrelated German individuals using isoelectric focusing followed by immunoprinting. Two new alleles, tentatively designated ORM1*14 and ORM2*13, were identified. The method was successfully applied to demonstrate ORM1 types in dried bloodstains. Each type of ORM1 was also correctly determined in bloodstains heated at 130 degrees C for 30 min. The results indicated that ORM1 is a new powerful genetic marker system for the grouping of bloodstains.     

C6 phenotyping in sera and bloodstains by isoelectric focusing followed by electroimmunoblotting

The genetic polymorphism of C6 was investigated in 329 unrelated Japanese individuals using isoelectric focusing in polyacrylamide gels followed by an electroimmunoblotting technique. Besides six common phenotypes C6 A, AB, B, AB2, BB2 and B2, six rare variants were observed. The allele frequencies were: C6*A = 0.4422, C6*B = 0.4757, C6*B2 = 0.0714, C6*A3 = 0.0015, C6*M1 = 0.0046 and C6*B3 = 0.0046. The population data confirmed that the C6*B2 allele is the third common allele characterizing Japanese. The present electroimmunoblotting technique was applied to demonstrate C6 types in dried bloodstains. The C6 types were determined from bloodstains stored at 4 degrees C for up to 10 weeks, at room temperature for up to 2 weeks and at 37 degrees C for up to 4 days. The results show that this component system offers a new powerful means for the medico-legal grouping of bloodstains.     

Esterase D phenotyping of bloodstains and hair roots by low voltage isoelectric focusing

A new isoelectric focusing method is described for phenotyping of esterase D in blood stains and hair roots. It permitted easy and rapid discrimination of six phenotypes determined by ESD*1, ESD*2 and ESD*7. Experiments showed it to be practicable in forensic stain work. In addition, this technique was also usable in phenotyping of ESD 5.