Bloodstain characterization in the EAP, Hp, Hb, AK and Glo I typing systems using minigels and the PhastSystem

Application of minigels and the PhastSystem to obtain phenotyping results from bloodstains in the EAP, Hp, AK, and Glo I typing systems was investigated. Nonequilibrium isoelectric focusing with 4-6.5 PhastGel produced readily interpretable phenotypes in the EAP typing system. Both 4-6.5 and 5-8 PhastGel produced AK typing system phenotypes using nonequilibrium isoelectric focusing conditions. The 8-25% PAG PhastGel developed by two staining techniques allowed discrimination of phenotypes for the Hp typing system. Phenotypes from the Glo I typing system were also obtained with this gel type. Variant haemoglobins could be detected on pH 5-8 PhastGel using isoelectric focusing conditions. Much potential for standardized, rapid phenotyping of bloodstains was found to exist utilizing the PhastSystem.     

A hybrid ampholyte focusing technique for esterase D subtyping of evidentiary material

An ultrathin-layer polyacrylamide gel isoelectric focusing technique that uses a composite of ampholytes from three commercial sources is described for subtyping esterase D. All common allelic products of esterase D were separated clearly. The technique described in this paper provides a higher conclusive call rate on known blood specimens (95.8%) and questioned bloodstains (69.7%) compared with continuous zone electrophoresis in agarose gels (89.9 and 37.6%, respectively).     

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 degrees C and room temperature and 3-month-old bloodstains maintained at 37 degrees C.     

The use of isoelectric focusing (IEF) as a method for the combined phenotyping of erythrocyte acid phosphatase (EAP) and esterase D (EsD)

The simultaneous isoelectric focusing (IEF) in polyacrylamide gels (PAG) of erythrocyte acid phosphatase (EAP) and esterase D (EsD) allows the poor discriminating power (DP) of EsD to be usefully combined with a highly discriminating system EAP, such that a joint DP of 0.766 was achieved compared with PGM IEF DP 0.756. Focusing was carried out in a centrally flattened gradient containing ampholines (pH 4-6 and 6-8) and the chemical spacer 3-(N-morpholino) propanesulphinic acid (MOPS). It enabled the identification of six EsD phenotypes including the recently discovered EsD5 isozymes. The application of this method to casework bloodstains is discussed.     

The collaborative study on typing group-specific component by eight forensic science laboratories

This report describes a collaborative study on typing group-specific component (GC), conducted between the Central Research and Support Establishment and the forensic science laboratories of England, Wales and Northern Ireland. A population study (n=114) was performed. Fifty blood donors were selected to provide a distribution, slightly biased from normal, in favour of the GC 1F-1F and GC 1F-1S phenotypes. A protocol was devised for preparing large bloodstains. The strongest GC bands were obtained from the edge of a stain after the blood had been treated with K+/EDTA. Each laboratory received a representative portion of the large bloodstains for GC typing. Five of the eight laboratories correctly grouped all the bloodstains. No errors directly attitributable to the system were recorded in over 800 tests, indicating that GC in bloodstains can be typed reliably using the combination of isoelectric focusing in ultrathin narrow pH interval gels followed by immunofixation and silver staining.     

Subtyping phosphoglucomutase-1 in semen stains and bloodstains: a report on the method

A method is described for obtaining nondistorted, reproducible phosphoglucomutase-1 subtyping patterns from semen stains and bloodstains. Isoelectric focusing of phosphoglucomutase-1 was accomplished in 80 min in a 0.2-mm-thick polyacrylamide gel with an interelectrode wick distance of 8.0 cm. The gel contained 1.2% (w/v) N-(2-hydroxyethyl) piperazine-N-3-propanesulfonic acid (EPPS) and pH 5 to 7 ampholytes (4% w/v). When maintained at room temperature, laboratory-prepared bloodstains and semen stains could be typed for phosphoglucomutase-1 up to four months and three weeks, respectively. An evaluation of phosphoglucomutase-1 typing by isoelectric focusing and the Group I system was performed on casework samples submitted to the FBI Laboratory. In addition to the increased discriminating probability of phosphoglucomutase-1 when subtyped, isoelectric focusing yielded an increase in positive calls on questioned bloodstains (65.6 versus 36.2%) and dried seminal stains (16.4 versus 13.1%) compared with the Group I system.     

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.     

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.     

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.     

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.     

Haptoglobin phenotyping by polyacrylamide gel isoelectric focusing and its application to simultaneous typing of serum proteins

A simple isoelectric focusing method for haptoglobin (HP) typing is described. Serum was pretreated first with C. perfringens neuraminidase (CPN) and then with dithiothreitol (DTT). The treated serum was subjected to polyacrylamide gel isoelectric focusing (PAGIF), and the band patterns were detected by immunoblotting. The method could be successfully applied to HP typing of bloodstains as old as 2 months. A slight modification of it enabled HP, complement component C81, and factor I (IF) to be typed simultaneously. The immunoblotting facilitated preservation of HP patterns. Thus, the PAGIF method for HP typing is suitable for routine use in the forensic laboratory.     

Micromethod for MN antigen grouping of dried bloodstains

A micromethod based on the absorption elution technique was shown to be applicable to the detection of M and N blood groups of dried bloodstains on cotton cloth. Each antigen M and N was tested using two different types of antisera. Two hundred different bloodstains, stored up to six months, were analyzed. Conclusive results were obtained for M typing on 2.5-mm-long bloodstained threads. For N typing, some cross-reactivity of homozygous M stains with anti-N sera was observed. This may be explained by the structure of the M and N antigens on the red cell membrane.     

测定血痕MN型的血清制备

用免疫吸收方法制备的抗-M与抗-N血清,检测10天内的不相应的MN血型血痕和大约2年的相应MN型血痕,凡能获得特异性结果的抗血清才能用于血痕的MN血痕测定。否则,只能用于测定血液的MN血型。     

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.     

A novel approach to obtaining reliable PCR results from luminol treated bloodstains

In recent years the forensic scientist has been afforded great advances in technology both in the detection of latent bloodstains and in acquiring reliable DNA typing results from very small pieces of physical evidence. Scientists are now able to detect minute quantities of latent bloodstains by utilizing the luminol reagent, oftentimes indicating that an attempt has been made to conceal any evidence of bloodshed. With the introduction of PCR based technology to the forensic arena, scientists are now routinely able to obtain DNA typing results from previously insufficient amounts of biological material, items as small as a single hair, saliva on a cigarette butt, or a bloodstain the size of a pin head. We present here a merging of these two advances coupled with a new collection medium for post luminol treated latent bloodstains. The forensic scientist is now able to routinely isolate and recover an adequate amount of DNA suitable for PCR typing at all of the Promega GenePrint PowerPlex 1.1 loci. In this study, several dilutions of latent bloodstains were prepared in an effort to simulate transferred bloodstains that are routinely encountered in a crime scene setting. The latent bloodstains were treated with luminol and subsequently collected using conventional cotton tipped swabs as well as a Puritan sponge tipped swab. PCR typing at the Promega GenePrint PowerPlex 1.1 loci was then attempted upon all dilutions of the latent bloodstains for both collection mediums. The results clearly indicate that it is now routinely possible to recover adequate amounts of DNA suitable for PCR typing upon post luminol treated bloodstains.     

Gc subtyping in blood stains by isoelectric focusing in immobilized pH gradients

The applicability of isoelectric focusing in immobilized pH gradients for the Gc-subtyping in the forensic examination of bloodstains was tested. It is shown that due to the excellent separation of the Gc 1S and 1F bands subtyping of bloodstains can be done with high reliability.     

The esterase D polymorphism as revealed by isoelectric focusing in ultra-thin polyacrylamide gels

The polymorphism of human red cell esterase D (EsD) was studied using isoelectric focusing (pH 4-6) in ultra-thin polyacrylamide gels. Typing was possible without the EsD isozymes attaining true equilibrium focusing conditions. Using this single method, six phenotypes (EsD 1, 2-1, 2, 5-1, 5-2 and 5) could be recognized in the White population of south-east England. Family studies showed these to be controlled by three co-dominant alleles and the gene frequencies were calculated to be EsD1 0.8856; EsD2 0.0946 and EsD5 0.0198. For successful and reliable EsD typing by this method, the electrophoretic system must be carefully optimized with respect to the duration of electrophoresis and the temperature attained in the gel during the electrophoretic run.     

A new method for typing haptoglobin in bloodstains using immobilized allo A lectin

Allo A lectin from the beetle, which is beta-D-galactose specific, reacts to haptoglobin but not to hemoglobin. The use of allo A-Sepharose for typing haptoglobin in bloodstains helped eliminate hemoglobin from the bloodstain extract and presented highly resolved haptoglobin patterns by disc gel electrophoresis. This method is simple and rapid for typing haptoglobin in bloodstains and can be easily used in forensic science laboratories.     

Group specific component: isoelectric focusing subtyping and immunoblot detection

A method for the detection of group specific component (Gc) by immunoblotting, following isoelectric focusing (IEF), is described. This isoelectric focusing method resolves the six common phenotypes of Gc using a narrow range pH 4.5 to 5.4 ampholyte. The Gc proteins were passively transferred from the IEF gel to nitrocellulose and detected with goat anti-Gc followed by peroxidase labeled anti-goat immunoglobulin (Ig) antibody. The increased sensitivity of this technique results in the typing of stains older than one year and also those stains with minimal concentrations of the Gc protein. The polyacrylamide gel can also be used for the subtyping of esterase D.     

北京地区人群血清型α2HS频率调查与血痕中α2HS的检测

本文采用聚丙烯酰胺凝胶等电聚焦和免疫固定技术,调查北京地区随机人群的α2HS糖蛋白(α2HS)的频率分布。在185名无亲缘关系的健康人中,发现3种常见表型,即α2HS1-1型(99人)、2-1型(74人)、2-2型12人。未发现稀有型。基因频率为:α2HS~1=0.7351,α2HS~2=0.6490。室温中保存6个月的血痕,可检出其α2HS表型。