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

Summary 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.Supported in part by Grant-in-Aid for Encouragement of Young Scientest No. 62770408 from The Ministry of Education, Science and Culture     

Early increase in TNF-alpha-containing mast cells in skin lesions

We have evaluated the numbers and immunohistochemical positivity for tumor necrosis factor (TNF) alpha of the mast cells in (a) 40 skin samples collected at autopsy from subjects who had survived for a few seconds to 1 h, (b) 10 samples of post-mortem skin lesions and (c) 10 surgical biopsies of healthy skin. Sections were treated with fluoresceinated avidin, to tag mast cell granules, followed by indirect immunohistochemistry for TNF-alpha with polyclonal primary and rhodaminated secondary antibodies. We could confirm a progressive increase in mast cell numbers, which became significant 1 h after trauma. More important, the fraction of mast cells positive for TNF-alpha increased progressively in the same time period and became significantly higher than controls in specimens collected more than 15 min after trauma. Samples from post-mortem lesions had significantly fewer mast cells and fewer TNF-alpha-positive cells than any other group of samples. The results suggest that mast cells are quickly recruited to an injured site in response to trauma and upregulate their TNF-alpha content, which can play an early role in directing tissue response to injury. The forensic pathologist can take advantage from this behavior of mast cells for the evaluation of the timing of early vital lesions.     

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

Summary 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 blood-stains. The determination limits were: at 4°C 8 weeks, at room temperature 2 weeks and at 37°C only 2 days after storage. The BF subtyping is of practical use in medicolegal individualization of unknown bloodstains.     

DIA3 phenotyping in human semen and seminal stains by isoelectric focusing

Summary 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 10×10⁶/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°C for up to 4 weeks, at room temperature for up to 8 weeks, and at 4°C for over 12 weeks after stain formation. In vaginal swabs the isoenzyme bands were very faint and not identifiable.     

Haptoglobin subtyping by isoelectric focusing in ultrathin-layer polyacrylamide gels population genetic data for Hanover and Lower Saxony

Summary This report describes a method for subtyping haptoglobin by means of isoelectric focusing in 0.2-mm ultrathin-layer polyacrylamide gels. Haptoglobin (Hp) is purified by ion-exchange chromatography and reduced. The well-known advantages of ultrathin-layer gels combine high isoelectrophoretic resolution of the Hp subtypes with less demands for time and material and make sequential visualization by fixation and protein staining possible. The distribution of the Hp subtypes in 1500 unrelated adults from Hanover and Lower Saxony is presented. Allelic frequencies are calculated to be: Hp*2FF=0.0030; *2FS=0.5620; *2SS=0.0290; *1F=0.1537; *1S=0.2523. Segregation analysis for 68 matings shows an autosomal codominant mode of transmission in all cases. For the population investigated the chance of isolated paternity exclusion with the subtyped Hp system amounts to 33.91%.     

DIA3 phenotyping in human tissues,dental pulps,and hair roots by isoelectric focusing

Summary The polymorphism of DIA3 was investigated in tissues of various human organs, dental pulps, and hair roots by isoelectric focusing. DIA3 types were demonstrated from tissues of brain, prostate, testis, ovary, and uterus, but not from tissues of spleen, pancreas, heart, liver, muscle, lung, skin, and kidney. Determination was possible from dental pulps stored at room temperature for up to 2 weeks and from fresh hair roots. The results show that the DIA3 typing by isoelectric focusing is useful for medicolegal individualization of brain, reproductive organs, teeth, and hairs.     

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

Summary 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.