Detection of thyroglobulin in blood stains as an aid in the diagnosis of mechanical asphyxia

Mechanical forces applied to the neck region are known to release certain amounts of thyroglobulin into circulation. In this experiment, an attempt was made to detect thyroglobulin in bloodstains as an aid in the diagnosis of mechanical asphyxia. Experimental bloodstains containing thyroglobulin at concentrations of 1, 2, 5 and 10 mu g/mL were prepared on a sheet of filter paper. Small pieces of bloodstains, measuring approximately 2.4 cm2 in area, were extracted with 0.1 mL of distilled water and the extracts were tested against an antihuman thyroglobulin serum by precipitation-electrophoresis. Bloodstains containing more than 1 mu g/mL of thyroglobulin formed distinct precipitin lines for up to one month of storage, while bloodstains containing more than 5 mu g/mL of thyroglobulin formed distinct precipitin lines for up to three months of storage. The present results suggest that the bloodstains can be utilized in the diagnosis of mechanical asphyxia.     

A sensitive enzyme-linked immunosorbent assay used to differentiate type AB specimen from a mixture of type A and type B specimens

A sensitive enzyme-linked immunosorbent assay (ELISA) for detection of ABO blood substances in human body fluids was devised. The ELISA plates coated with purified human anti-A or anti-B serum effectively captured the blood substances, and these were then analysed by the combination of rabbit anti-A and goat anti-B. This capture ELISA could differentiate the type AB specimen from a mixture of the type A and the type B specimens, and the method was applied to rape cases to make the ABO typing of the criminal.     

Green pigments in epidermal blisters of decomposed cadavers

A spectrophotometrical method for discrimination between sulfhemoglobin (SHb) and choleglobin was applied to analyses of the green pigments in epidermal blisters of decomposed cadavers. SHb appeared to be a predominant constituent of the pigments, although significant amounts of choleglobin, one third to one sixth of SHb, were also demonstrated. The present results indicate that the greenish discoloration of decomposed cadavers is due to formation of a mixture of SHb, choleglobin and possibly other denatured hemoglobin derivatives.     

Rapid isolation with Sep-Pak C18 cartridges and capillary gas chromatography of triazine herbicides in human body fluids.

A simple and rapid method, for the isolation of eight triazine herbicides from human serum and urine, using Sep-Pak C18 cartridges is presented. After mixing with distilled water, serum and urine samples containing the herbicides, were loaded on Sep-Pak C18 cartridges and eluted with either chloroform only or chloroform/methanol (9:1). The herbicides were detected by capillary gas chromatography with both flame ionization detection (FID) and nitrogen-phosphorus detection (NPD). Separation of eight triazine herbicides from each other and from impurities was generally satisfactory with the use of a non-polar DB-1 capillary column. Recovery of most compounds was excellent for both chloroform and chloroform/methanol (9:1) as elution solvents. Backgrounds were cleaner and evaporation time was shorter for the chloroform only than for the chloroform/methanol (9:1). The NPD gave sensitivity more than 10-20 times higher than that of FID.     

The ABO blood grouping of a minute hair sample by the immunohistochemical technique

The unlabeled antibody (PAP) immunoperoxidase technique was applied to the ABO blood grouping of human scalp hairs. Hair samples were subjected to longitudinal- or cross-sectioning, thus obtaining suitable samples for subsequent immunostaining. The immunostaining was carried out using rabbit anti-A and anti-B sera as the primary antibodies. With this technique, the group-specific staining which is revealed as a dark brown precipitate was clearly observed within the medullae of the hair shaft, and depending on the presence or absence of these precipitates, respective blood groups of unknown hair samples were determined. At the hair root, on the other hand, positive stainings were observed not only in medullary cells but also in some cortical cells of the keratogenous zone. From the present study, it can be safely said that this technique is of practical use for the ABO blood grouping from a minute (less than 3 mm) hair sample.     

Determination of total hemoglobin in forensic blood samples with special reference to carboxyhemoglobin analysis

For the determination of total hemoglobin (Hb) in blood containing elevated carboxyhemoglobin (COHb), a newly developed reagent containing a 100-fold concentration of ferricyanide (20 g/l) and a 2-fold concentration of Sterox SE was compared with a standard reagent (0.2 g/l ferricyanide), the reagent of van Kampen and Zijlstra, using forensic blood samples and experimentally heated blood samples. There were no significant differences between the spectra of hemiglobincyanide (HiCN) solution produced with our reagent and the van Kampen and Zijlstra reagent using experimentally heated blood samples. Although the spectra of HiCN changed gradually with increased heating time and with the passage of time after mixing, the absorbance at 540 nm (A540) did not change until at least 120 min for both the reagents. When forensic blood samples containing elevated COHb were mixed with the van Kampen and Zijlstra reagent, total-Hb concentrations determined 5 min after mixing were 10-20% higher than those determined after 180 min. The overestimates of total Hb determined after 5 min resulted in comparable underestimates of percentage saturation of COHb (COHb%) when COHb% was obtained from the ratio of COHb content, determined by gas chromatogrpahy, to total-Hb concentration in blood. However, there was an extremely good correlation between the values of total Hb in forensic blood samples determined with the van Kampen and Zijlstra reagent after 180 min and those determined with our reagent after 5 min. From the results obtained, our reagent proved to be suitable for the determination of total Hb in forensic science practice.     

Transferrin polymorphism detected in human urine using isoelectric focusing followed by immunoblotting

Genetic polymorphism of transferrin (TF) was revealed in human urine by isoelectric focusing and immunoblotting on thin-layer polyacrylamide gels. Using this technique more than 300 urine samples were examined, and correct TF typing from a small volume of urine (approx. 0.5 ml) was achieved, in comparison with the results of direct grouping for plasma. Three common phenotypes, TF C1, C2-1 and C2, were differentiated. In addition, the rare types TF C1D, C2D, and C1B were observed. The frequencies of the TF alleles in our samples were found to be: TF*C1 = 0.7265, TF*C2 = 0.2624, TF*D = 0.0083 and TF*B = 0.0028.     

Time-course changes in the expression of heme oxygenase-1 in human subcutaneous hemorrhage

To determine the time-course of human subcutaneous hemorrhage, heme oxygenase (HO)-1 expression and macrophage infiltration were observed using an immunohistochemical technique and semiquantitative analysis. The number of immunoreactive cells and the number of all infiltrating cells of each microscopic field were counted, and the ratio of the former to the latter was calculated as the positive cells ratio. An increase in the HO-1-positive cells ratio was observed starting at 3 h after injury, and the maximum ratio was observed 3 days after injury. The pattern of the increase in the macrophage ratio was similar to that of the HO-1-positive cells ratio in the early period after injury. Observation of serial sections revealed that the expression of HO-1 in the cells corresponded to the localization of macrophage. The present results suggest that the determination of HO-1 expression, as derived from macrophages, might be useful for the estimation of the time-course of subcutaneous hemorrhage.     

Ethanol production by Candida albicans in postmortem human blood samples: effects of blood glucose level and dilution

We present two cases in which the ethanol concentration in blood samples taken after death continued to increase in the absence of any remarkable increase in n-propanol concentration. Species of bacteria and yeasts, including Candida albicans were isolated from these samples. We then examined whether C. albicans, the most common yeast in the general environment, was able to produce ethanol in human blood stored at room temperature. Ethanol production increased as the glucose concentration increased, indicating that C. albicans produced ethanol from the glucose. Our results also suggested that C. albicans produced ethanol more easily in blood diluted by intravenous infusions that included glucose than in undiluted blood. These findings are useful for the evaluation of postmortem ethanol production in subjects whose blood has been diluted by infusions with glucose. Furthermore, there was no quantitative relationship between the amount of n-propanol detected and the amount of ethanol production: n-propanol appears to be an unreliable index of putrefaction and postmortem ethanol production by C. albicans. It is possible for the blood ethanol level to be high and n-propanol not to be detected, even if the subject has not been drinking alcohol. We reconfirmed the necessity of immediately adding sodium fluoride to samples for ethanol analysis to prevent postmortem ethanol production.     

Analyses of sexual dimorphism of reconstructed pelvic computed tomography images of contemporary Japanese using curvature of the greater sciatic notch, pubic arch and greater pelvis

Three-dimensional pelvic images were reconstructed from multi-slice CT data of contemporary Japanese (males: 124; females: 104, 25-92 years old), and curvature analysis to examine sexual dimorphism was carried out in the great sciatic notch (GSN), the pubic arch and the greater pelvis in the images. Reconstructed pelvic CT images were visualized fairly well and anatomical landmarks were easily recognizable. When calculating the radii (curvature radii) of the best-fit circles for the spline curve lines set along the edges of the GSNs and of the pubic arches, sexes from these regions were correctly identified in 89.1% (males: 93.8%; females: 83.7%) and 94.7% (males: 97.3%; females: 91.8%) of cases, respectively, by setting an appropriate cut-off value. Furthermore, sexing was possible even in deeper regions of the GSN which are relatively resistant to postmortem damage. Curvature radii of the best-fit spheres of greater pelves showed no significant difference between sexes. However, curvature of the best-fit sphere for the left iliac fossa was significantly larger than that of the right one (p<10(-24)) in males, and the ratios were >1.0 in 88% of all male specimens analyzed. Meanwhile, no significant difference was observed among female samples. Although some left-sided dominancy has been reported in 2-dimensional measurements of the human pelvis, this 3-dimensional laterality in males was much more significant, and is a potential index of sex difference.