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.     

alpha-L-fucosidase phenotyping in human placentae, semen and seminal stains

The polymorphism of alpha-L-fucosidase (Fu) was investigated in a Japanese population from samples of placentae and semen, using isoelectric focusing. The gene frequencies of placental types were Fu1 = 0.748 and Fu2 = 0.252, and those of seminal types were Fu1 = 0.739 and Fu2 = 0.261. The coincidence in the distribution between the placental and seminal types suggests that the Fu types occurring in placentae and in semen are controlled by the same Fu alleles. The Fu typing was possible in seminal stains stored at 4 degrees C for up to 9 weeks, at room temperature for up to 7 weeks and at 37 degrees C for up to 4 weeks. The Fu types were still detectable at semen dilutions of up to 1:4. This polymorphism would provide a useful genetic marker for the medicolegal grouping of seminal stains.     

DIA3 phenotyping in human semen and seminal stains by isoelectric focusing

The polymorphism of DIA3 was investigated by isoelectric focusing in semen samples from 235 unrelated Japanese volunteers and patients. Besides the three common phenotypes seven samples of the type 3-1 were observed. However, readable isoenzyme patterns were not demonstrated in semen samples of oligospermia under about 10 X 10(6)/ml sperm cells. The allele frequencies were DIA3*1 = 0.821, DIA3*2 = 0.164, and DIA3*3 = 0.015. The DIA3*1 frequency in oligospermia (0.765) was lower than that in normospermia (0.836). The isoelectric focusing method was successfully applied to phenotyping DIA3 in seminal stains; each phenotype was demonstrated at 37 degrees C for up to 4 weeks, at room temperature for up to 8 weeks, and at 4 degrees C for over 12 weeks after stain formation. In vaginal swabs the isoenzyme bands were very faint and not identifiable.     

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.     

A sandwich enzyme-linked immunosorbent assay for ABO blood typing of semen by using anti-p 84 monoclonal antibody as a marker of blood group substance in semen

A blood group substance (BGS), a protein with ABH antigenic activity, was isolated from human seminal plasma and designated as p 84 (Sato, 1995). We have developed a method for determining the ABO blood type of semen by performing a sandwich enzyme-linked immunosorbent assay (ELISA) in which p 84 is captured with an anti-p 84 monoclonal antibody, and evaluated the specificity and sensitivity of this method. Although BGS activity was detected in semen sensitively by this method, it was not detected in saliva, urine, breast milk, blood or vaginal secretions. Since the concentration of p 84 in semen was independent of the secretion status, the status can be determined as non-secretor when p 84 but not BGS activity was detected. To determine the stability of BGS activity on p 84, dried stains of semen on filter paper were kept at 4, 26, and 37 degrees C for 8 months, 2 years and 1 month, respectively, and their BGS activities were examined. After 8 months at 4 degrees C, over 60% of the original BGS activity was recovered from the stain. The activity could be detected even from a square as small as 0.25 by 0.25 cm. After 1 month at 37 degrees C and 2 years at 26 degrees C, 31 and 20% of the BGS activity, respectively, still remained. It could be detected from the pieces of 1.0 by 1.0 cm and 0.5 by 0.5 cm squares, kept for 1 month at 37 degrees C and 2 years at 26 degrees C, respectively. Finally, semen was mixed with saliva or blood at varying volumetric ratios and used for the sources of dried stains. The BGS activity of p 84 could be detected in the stains until the ratio between semen and saliva or blood reached 1:4. We conclude that this sandwich ELISA offers a more sensitive and specific method for determining the ABO blood type of semen samples obtained from sexual assault victims than existing methods, such as the conventional absorption-elution and classical hemagglutination-inhibition tests.     

Secreted blood group substances: distributions in semen and stabilities in dried semen stains

A sensitive microplate hemagglutination-inhibition technique has been used to ascertain the distributions of secreted blood group substances (BGS) in a population of 176 semen specimens and to characterize the stability of these substances in dried semen stains. The BGS concentrations in semen were found to vary throughout a wide range of titer. Despite this latitude of variation, the titers for the component BGS within the blood groups could be described by a log-normal distribution function. Studies of a number of sequential semen specimens obtained from the same donors revealed that the intraindividual variation in BGS titers was much more limited than the interindividual BGS titers. Attempts to correlate variations in titers between A and H in Group A semen or B and H in Group B semen indicated that the levels of these component substances vary independently. Studies of the stability of BGS in Groups A and O semen suggested that these substances were stable when the semen stains were stored at -20 degrees C, 4 degrees C, or at ambient laboratory temperature in a dry state. In contrast, stains stored at 37 degrees C under humid conditions suffered a dramatic loss in BGS titer, with the half-life of the BGS being on the order of 30 days.     

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.     

Polymorphism of EsD by isoelectric focusing: description of the new allele EsD*Kofu and phenotyping in bloodstains

The polymorphism of EsD was investigated in 1115 unrelated Japanese individuals by isoelectric focusing. Besides the three common phenotypes two heterozygotes EsD 7-1 and EsD 7-2 were observed. The gene frequencies were: EsD*1 = 0.6234, EsD*2 = 0.3663, and EsD*7 = 0.0103. In addition, a rare variant was detected in a probandus living in the city of Kofu. The family analysis suggested the hereditary occurrence of a new allele EsD*Kofu. The isoelectric focusing method was successfully applied to phenotyping EsD in bloodstains; each phenotype was demonstrated at 37 degrees C for up to 2 weeks, at room temperature for up to 9 weeks, and at 4 degrees C for over 20 weeks after stain formation.     

Studies on glyoxalase 1 isoenzymes in semen stains: polymorphism in Himachal (India) population

One hundred and ten pairs of blood and semen samples and their stains were studied to type glyoxalase 1 (GLO 1) isoenzymes using agarose-starch medium. A good agreement was observed between the phenotypes expressed in blood and semen samples of the same donor. No GLO 1 activity however could be demonstrated in the vaginal swabs tested. The gene frequencies of GLO 1 polymorphs in Himachal population has been worked out and their stability studies carried out at -12 degrees C and at room temperature.     

Detection of orosomucoid 1 phenotypes in semen and semen stains

Orosomucoid 1 phenotypes were detected in seminal plasma by isoelectric focusing and immunoprinting. The orosomucoid 1 phenotypes in seminal plasma correlated with the types found in the corresponding serum specimens. Semen stains stored for ten days could be typed for orosomucoid 1. The present work revealed that orosomucoid 1 is a useful genetic marker for the medicolegal grouping of semen stains.     

精浆r—谷氨酰转移酶多态性研究

本次实验采用聚丙烯酰胺阶段梯度凝胶电泳方法,对武汉地区135名无关男性精液γ—GT进行了遗传多态性调查,根据γ—GT的电泳差异,分为1型、2型、2—1型三种常见表现型,1型为靠近正极的单一带,2型为靠近负极的单一带,2—1型为1型带和2型带组成的杂合双带。表现型频率分别为0.156,0.356,0.488,基因频率为γ—GT~1=0.400,γ—GT~2=0.600(P>0.5)。室温下保存8周的精斑,也能检测出较清晰的谱带。本次实验还探讨了单纯阴道分泌物及精液、阴道分泌物混合斑进行γ—GT分型的可能性。     

A dot-blot-immunoassay for semen identification using a polyclonal antibody against semenogelin, a powerful seminal marker

Among various seminal plasma proteins, semenogelin (Sg), produced in the seminal vesicle, has been considered a candidate for demonstrating the presence of semen. Sg consists of two proteins, one 52 kDa (Sg-I) in size, and the other a mixture of 71 and 76 kDa proteins (Sg-II). Recombinant Sg-I and Sg-II proteins were obtained using a baculovirus system and then injected into a rabbit to produce the respective antibodies [Characterization of recombinant precursor proteins of the human seminal plasma sperm motility inhibitor synthesized in insect cells, Int. J. Mol. Med. 2 (1998) 693]. When liquefied seminal plasma was immunoblotted with the anti-Sg-I and Sg-II antibodies, the anti-Sg-II antibody identified a wider range of the polypeptides originating from Sg than did the anti-Sg-I antibody. A dot-blot-immunoassay using anti-Sg-II antibody revealed a clear immunoreactive spot even when the semen was diluted 6400-fold. However, this assay showed that the Sg antigen was undetectable in saliva, urine, vaginal secretions, sweat, nasal secretions and serum. To determine the stability of Sg antigenic activity, filter paper with dried semen stains were kept at 37, 4 and 22 degrees C for 1, 6 and 18 months, respectively, and the Sg antigenic activity was examined. The activity was detectable in an area not less than 0.5 cm x 0.5 cm under all of the above environmental conditions during each period. Finally, semen was mixed with saliva or blood at various volumetric ratios, and used as a source of dried stains. The Sg antigenic activity was detectable in the stains until the ratio of semen to saliva or blood reached 1:8. These results suggest that Sg may be useful as a marker for semen identification.     

An improved method for semen alpha-L-fucosidase typing--distribution in the Wuhan population of China

An improved method for the separation of the genetic variants of alpha-L-fucosidase in human semen is described. The method involves, isoelectric focusing in ultrathin-layer PAG containing a mixture of ampholines, pH 5-7 and pH 3.5-9.5, and separator HEPES. The Fu pattern obtained is simple, easy to interpret, and reproducible. The occurrence of fucosidase phenotypes in 189 semen samples from the population of Wuhan was investigated, and the frequencies of the Fu alleles were calculated.     

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.     

Gc in bloodstains

Gc-subtyping was carried out on blood stains that had been made on cotton and glass and stored under a variety of conditions ranging from -20 degrees to +56 degrees C. The limits of detection ranged from 2 weeks at 56 degrees C up to 92 weeks at +4 degrees C and greater than 116 weeks at -20 degrees C. Additional bands that have been reported in other studies could not be detected during this study, and this difference is thought to be due to storage of the samples in the liquid state.     

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.     

Km(3) identification by enzyme-linked immunosorbent assay (ELISA) as an internal control for Km(1) activity determined by inhibition in dried bloodstains

A stability study comparing the identification of kappa marker Km(1), using the classical inhibition of agglutination, and the identification of Km(3), using an automated enzyme-linked immunosorbent assay (ELISA) technique, was done. Preliminary tests were performed to establish the specificity and sensitivity of the methods. Based on the results, the quantities of stain required to detect each marker were determined. Blood samples from 24 staff donors of known phenotype were aged at room temperature and at 37 degrees C in the dried stain and liquid forms. In addition, 192 stains from cases 1 to 7 months old and 76 staff-donor stains from 1 1/2 to 10 years old were tested in dried stain form. The known sensitivity of the ELISA technique was exploited by deliverately testing a decreased quantity of antigen. As control stains were aged beyond the detectable limits of sensitivity, results consistently showed an almost simultaneous success or failure to detect Km(1) and Km(3). This indicates that the interpretive criteria established for ELISA are sufficiently demanding to eliminate the danger of reporting false Km(-1) results but true Km(3) results.     

Polymorphism of EsD by isoelectric focusing: phenotyping in human tissues, dental pulps, hair roots, and semen

The polymorphism of EsD was investigated in tissues of various human organs, dental pulps, hair roots, and seminal stains by isoelectric focusing. The method yielded an excellent resolution of the isoenzyme components. The time limits of determination were: in organ tissues 3 weeks, in dental pulps 1 week, and in hair roots several days. The 7-1 type was less stable than the common types. Phenotyping was possible from fresh semen samples, but was unsuccessful from dried seminal stains after storage. The results show that the EsD typing by isoelectric focusing is of practical use for medicolegal individualization of organs, teeth, and hairs.     

精斑中磷酸葡萄糖变位酶(PGM_1)及其亚型的电泳分型

本文用淀粉凝胶电泳法和 PAGIEF 对精斑 PGM_1普通型及亚型进行了检测。169份精液斑的 PGM_1分型结果是:PGM_1 1—1 87例;PGM_1 2—1 66例;PGM_1 2—2 16例,其中31例同一个体红细胞及精液 PGM_1分型的结果完全一致。研究了138例不同精子数精斑的 PGM_1型,发现精子数的多少对分型无影响。亚型检测结果与红细胞一样可分10型。     

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.