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.     

Evaluation of four deoxyribonucleic acid (DNA) extraction protocols for DNA yield and variation in restriction fragment length polymorphism (RFLP) sizes under varying gel conditions.

This study originated from discussions and recommendations of the Technical Working Group on DNA Analysis Methods (TWGDAM). Four bloodstain deoxyribonucleic acid (DNA) extraction protocols and five semen stain DNA extraction protocols were evaluated. Nine laboratories participated in the extraction of DNA from 20 bloodstains and 20 semen stains using each protocol. All blood and semen stains originated from a single donor and were prepared under uniform conditions to permit the direct comparison of DNA yields and restriction fragment lengths. The extracted DNA from approximately 600 bloodstains and 700 semen stains was quantified by yield gel analysis and a slot blot hybridization technique. The extracted DNA was digested and restriction fragment length polymorphism (RFLP) patterns were generated using three single-locus probes. The RFLP sizing data produced from the blood and semen stains were evaluated with respect to (1) DNA extraction method, (2) gel length, (3) agarose type, (4) presence or absence of ethidium bromide in the gel, and (5) fragment sizes obtained from DNA isolated directly from the donor's liquid blood. This study demonstrates conclusively that high-molecular-weight DNA can be isolated using either organic or nonorganic DNA extraction protocols and that the resulting RFLP sizes are highly reproducible regardless of gel length, agarose type, or presence/absence of ethidium bromide.     

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.     

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.     

PGD phenotyping in bloodstains, organ tissues, dental pulps and hair roots by isoelectric focusing

Polymorphism of PGD was investigated in bloodstains, organ tissues, dental pulps, hair roots and semen by isoelectric focusing. This technique provided much higher resolution of PGD isoenzymes than starch gel electrophoresis. Phenotyping was possible from bloodstains for 5 weeks, from organ tissues (except pancreas) for 1-3 weeks, from dental pulps for 2 weeks and from hair roots for 2 weeks when they were stored at room temperature. The method is simple, rapid, reliable and therefore useful in medicolegal individualization of bloodstains, organ tissues, teeth and hairs.     

Simultaneous identification of alpha-L-fucosidase and phosphoglucomutase (PGM) subtyping in semen stains

Seminal fluid and stains were analyzed by isoelectric focusing to determine the donor phenotype in the alpha-L-fucosidase (AlFuc) polymorphic system. The enzyme is found in both seminal fluid and spermatazoa. Three common phenotypes exist and can be identified in fluid specimens stored at 4 degrees C for more than a year. Untreated semen specimens display more than eight distinct bands of alpha-L-fucosidase activity with isoelectric points of pH 6.6 and below. Neuraminidase-treated specimens have enhanced banding patterns cathodally with a loss of activity in anodal bands making it easier to phenotype specimens. Semen stains maintained in dehumidified chambers at 25 or 37 degrees C retained activity for at least one month and could be accurately phenotyped. Activity was observed in semen specimens maintained at -20 degrees C in the dried state for a period of one year, whereas a complete loss of activity was observed after two weeks in similar specimens maintained at 25 or 37 degrees C under humid conditions. Of seventy-four semen stains analyzed, two had no apparent activity. Of the remaining seventy-two specimens 56, 32, and 12% were phenotyped as FUC 1-1, FUC 2-1, and FUC 2-2, respectively. Calculated gene frequencies are FUC1 = 0.72 and FUC2 = 0.28. Following analysis of alpha-L-fucosidase, the agarose gel can be chemically developed to reveal the PGM1 subtyping pattern. The ability to phenotype both systems in semen stains significantly improves the ability of the analyst to individualize this type of physical evidence. The probability of discrimination for these two combined systems is approximately 0.89.     

A silver staining method for the detection of polymorphic proteins in minute bloodstains after isoelectric focusing

A silver staining method has been developed to study polymorphic proteins in bloodstains after isoelectric focusing. This method is highly sensitive and permits the detection of polymorphic proteins (i.e. alpha 1-antitrypsin, Gc and Tf C subtypes) in bloodstains as small as 0.2 microliter or less. The method is simple and reproducible and can be used after immunofixation. Blood stains can be identified after longer storage periods than is possible by using conventional staining methods.     

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.     

Typing study of human semen DIA3 by isoelectric focusing--distribution in the Wuhan population, China

Human semen DIA3 typing was studied by isoelectric focusing on ultra-thin-layer polyacrylamide gel which resulted in a simpler and more definite separation of the products of DIA3 alleles than hitherto. In 198 semen samples collected from unrelated Chinese males four different phenotypes were observed. The DIA3 allele frequencies were calculated: DIA 3(1) = 0.7727, DIA 3(2) = 0.2172, DIA 3(3) = 0.0101. The results of the stability study of 12 laboratory-prepared semen stains stored at room temperature suggested that DIA3 in seminal strains is a relatively stable genetic marker. Our gene frequencies have been compared to those reported in other populations.     

A Comparison of DNA Extraction Using AutoMate Express™ and EZ1 Advanced XL from Liquid Blood,Bloodstains, and Semen Stains

In this study, DNA was extracted using an AutoMate Express? and an EZ1 Advanced XL from liquid blood, fresh and aged bloodstains, and fresh and aged semen stains. Extracted DNA was quantified by real‐time PCR using the D17Z1 locus. Short tandem repeat typing was performed using an AmpF?STR^® Identifiler kit. The yields of DNA obtained by the AutoMate Express? were higher from fresh bloodstains and fresh semen stains, almost the same from aged bloodstains and aged semen stains, but slightly lower from liquid blood compared with those obtained by the EZ1 Advanced XL. The addition of dithiothreitol or the use of PrepFiler? lysis buffer improved the EZ1 Advanced XL results from fresh bloodstains, but not for liquid blood and aged bloodstains. Our results demonstrated that the PrepFiler? lysis buffer is the main contributor to the higher DNA yields of the AutoM ate Express? for fresh bloodstains.     

Usefulness of two methods of isoelectric focusing for the erythrocyte acid phosphatase phenotypes determination in human bloodstains

The authors tried to compare the usefulness of the isoelectric focusing of EAP in bloodstains on 0.2 mm polyacrylamide gel with their method of determination of the enzyme on 1 mm polyacrylamide gel. Both methods turned out to be useful but better results were obtained on 0.2 mm gel. Isoelectric focusing on the ultra-thin gel is more sensitive; it gives clear enzyme strips, takes less time (30 min) and demands about half the amount of material.     

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.     

Gm/Km determination in sperm traces

In the forensic laboratories of the Federal Republic of Germany and West-Berlin 23 different semen stains and in our own laboratory 20 semen stains were typed in the gm/km-system doing 125 and 61 (own) test respectively. Examination was carried out by means of the haemagglutination method, which has been used successfully in typing bloodstains. Our critical assessment based on earlier experiences with semen stains was now confirmed and statistically evaluated: typing was successful in about 35-50% of the tests, but besides false-negative results, there was also a considerable percentage (4-10%) of false-positive ones. Therefore for the present it seems best to exclude the gm/km-typing of secretion stains from forensic investigations in order to avoid false incriminations or exonerations of suspects.     

An alternative, effective substrate for erythrocyte acid phosphatase phenotype determinations

A method is described for obtaining nondiffusing, nonfading fluorogenic zymograms for erythrocyte acid phosphatase variants separated by isoelectric focusing. The synthetic substrate 4-trifluoromethylcoumarin phosphate was impregnated into cellulose diacetate membranes and air-dried overnight. After isoelectric focusing, the substrate overlay membrane was rehydrated in 0.05M citrate buffer, pH 4.0, lightly blotted, and overlayed on the gel. A 5- to 10-min incubation at 37 degrees C produced intensely fluorescing, light-blue bands on the diacetate membrane. Interaction of the trifluoromethyl group on the substrate with hydrophobic regions of the diacetate membrane impeded diffusion, yielding a permanent zymogram. There were no discrepancies in phenotype determinations using this method when compared with the 4-methylumbelliferyl phosphate assay approach. Further, an increased number of conclusive calls were obtained (91.8 versus 79.5% and 54.1 versus 34.9%) with this new assay when compared with the 4-methylumbelliferyl phosphate substrate on known liquid bloods and questioned dried bloodstains, respectively.     

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.     

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.     

The application of immunoblotting to the phenotyping of group-specific component

A sensitive immunoblotting method for the routine detection of group-specific component (GC) from fresh serum, and from control and casework bloodstains has been developed. GC phenotypes were separated in a thin layer polyacrylamide gel by isoelectric focusing, transferred to nitrocellulose by a rapid capillary blotting procedure, and detected using a double antibody enzyme immunoassay. This method is capable of phenotyping 8 ng of GC extracted from bloodstains, a four-fold increase in sensitivity when compared to immunofixation and silverstaining. A total of 2424 casework bloodstains have been analysed and GC phenotypes identified in 78% of samples. The method is suitable for use in routine laboratories and is more sensitive than other methods for GC phenotyping of casework bloodstains.     

A method to increase the volume of sample applied to isoelectric focusing gels

Isoelectric focusing of extracts from diluted or aged bloodstains may be more successfully accomplished with larger sample volumes applied to the gel. A technique is described using teflon tubing to apply larger sample volumes (up to 100 μl) to isoelectric focusing (IEF) gels. This method is reproducible and easy to perform.     

血痕中PGM_1亚型的检出及酶谱的保存方法

本文报告用等电聚焦方法,采用拉丁方设计,对血痕保存的温度、布质及含量,以及血痕中 PGM_1亚型检出时间进行了研究。保存在0℃(6个月)、4℃(2个月)、18℃(1个月)及30℃(3周)的6μL 血痕,PGM_1亚型均可检出。血痕的总量对 PGM_1亚型的检出时间也有一定的影响。不同布质对血痕中 PGM_1亚型的检出时间,无明显差异。另外,利用聚脂膜具有亲水膜面的特点,将 PGM_1原始酶谱贴附在聚酯膜上,可长期保存酶谱。     

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.