Creatine phosphokinase in serum and cerebrospinal fluid, and microscopic findings in brain and heart in hypothermic rabbits

Signs of hypothermia injury were studied in rabbits cooled to a core temperature of 30 degrees C by immersion in ice water and thereafter rewarmed to 35 degrees C. Anaesthetized control rabbits were kept normothermic (37 degrees C) for a corresponding time (4 h). Creatine phosphokinase (CPK) activity increased 24 h after hypothermia to 20-fold in serum. In cerebrospinal fluid the activity was already significantly (5-fold) increased after hypothermia and was still as high at 24 h. Smaller increase was also found in the control normothermic rabbits both in serum (10-fold) and cerebrospinal fluid (2-fold). The values had returned to the initial level after 1 week. Small haemorrhages were observed in the brain at 24 h and slight scarring was seen in the myocardium of some rabbits which had lived 4 weeks following hypothermia. The results indicate that CPK can be a useful marker in the diagnostics of hypothermia death, especially in cerebrospinal fluid, which is less affected than blood by autolysis.     

Investigation of the short-time autolysis of rat hearts by means of SDS. Polyacrylamide gel electrophoresis and electron microscopy.

The short-time autolysis of hearts was regarded as a model of ischaemic heart failure. Therefore, isolated rat hearts were subjected to 30--120 min autolysis in a Locke solution at 37 degrees C. Electron microscopic examinations and myofibrillar preparations were made from the autolysed heart ventricles. The myofibrillar proteins were resolved by SDS-polyacrylamide gel electrophoresis. After 30 min autolysis the amount of a protein of 192,000 daltons greatly increased. At the same time on the electron micrographs the focal destruction of filament destruction on the A filament area and the mitochondrial structure altered too. After 60 min autolysis another protein of 36,400 daltons appeared. On the electron micrographs the focal desintegration of Z membranes and the focal destruction of I filaments can be observed. After 120 min autolysis further proteolytic products could not be detected by gel electrophoresis but on the electron micrographs the destruction of Z membranes and I filaments became more pronounced.     

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.     

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.     

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.     

Peroxidase activity in traumatic skin lesions

Peroxidase activity was determined in experimental compression-excoriation lesions and incision wounds of rat skin after different periods of vital time. The peroxidase enzyme was extracted from the tissues by homogenization in 0.5% cetyltrimethylammoniumbromide, and the enzyme activity was measured from the supernatant by o-dianisidine-H2O2 assay. In the blood of the rats a mean activity of approx. 5.26 +/- 1.11 U/g dry weight was observed. In the control specimens of the skin the activity was very low and generally below the detection limit of the methods used. In 30-min-old compression-excoriation lesions the mean peroxidase activity was 0.38 +/- 0.21 U/g dry weight. In lesions older than 30 min the activity started to increase rapidly. In 4-h-old compression-excoriation lesions it was 10 times higher than the 30-min level and was 40 times higher in 12-h-old lesions and 70-100 times higher in 1-3-day-old compression-excoriation lesions, respectively. In 30-min-old incision wounds the mean peroxidase activity was 0.65 +/- 0.37 U/g dry weight. The increase of the activity compared with the 30-min level was even faster in the incision wounds: in 4-h-old wounds the mean activity was 50 times higher, in 12-h-old wounds 200 times higher and in those of 1-5 days it was several hundreds of times higher. Compression-excoriation lesions made after death showed activity similar to the control specimens. Postmortem autolysis at +22 degrees C resulted in a loss of the enzyme activity in 1-day-old compression-excoriation lesions so that after 3 days approx. 80% remained, and after 5 and 7 days approx. 40% was present. After 3 days of autolysis at +4 degrees C, nearly 100% of the activity remained and approx. 90% was present after 5 and 7 days of autolysis. Increased peroxidase activity was also detectable in human vital excoriations in the specimens which were taken in autopsies several days postmortem.     

A stability study on Gc subtyping in bloodstains: comparison by two different techniques

The limits of determination of Gc subtypes in bloodstains were compared between the immunofixation method and the sulfosalicylic acid precipitation method using isoelectric focusing on polyacrylamide gel. By the immunofixation method Gc subtyping in bloodstains was successfully made at 37 degrees C after 7 weeks, at room temperature after 17 weeks and at 4 degrees C even after 25 weeks storage. By the sulfosalicylic method Gc subtypes were no longer able to be determined a few weeks after stain formation. The superiority of the results obtained by the immunofixation method makes it the recommended method for the Gc subtyping from bloodstains in medicolegal practice.     

Evaluation of eosin-fluorescence in the diagnosis of sudden cardiac death

The diagnostic significance of the eosin-fluorescence method was evaluated and compared with the enzymehistochemical beta-hydroxybutyrate-dehydrogenase method (beta-HBDH) and the degree of hyperchromasia in 568 samples from 24 bets-HBDH-negative and beta-HBDH-positive sudden cardiac deaths (SCD) and 23 non-cardiac deaths as controls. The effect of autolysis was investigated separately on isolated human hearts at +4 degrees C and +22 degrees C. All samples were examined without any knowledge of clinical or autopsy data. Normal olive-green fluorescence was observed in only five individuals of the control group and two in the beta-HBDH-negative one. The frequency of the yellow-fluorescence and hyperchromasia increased from the control subjects towards the beta-HBDH-positive-ones. The difference in frequency distribution of fluorescence between the groups was statistically highly significant (P less than 0.001). The change in fluorescence did not correlate with the loss of beta-HBDH-reaction or the distribution pattern of hyperchromasia. Postmortem autolysis did not change the fluorescence significantly but the 78% wrong positive subjects in the control group render this method too sensitive and unreliable for medicolegal purposes.     

Trial elution of semen stains and measurement of IgG level: a contribution to Gm typing

The conditions for the elution of IgG in seminal stains have been investigated systematically. The amount of IgG recovered could neither or hardly be influenced by variation of the time (15 minutes to 120 hours) and temperature (4 degrees C, 20 degrees C, 37 degrees C, 56 degrees C) of elution, nor by mechanical treatment (cutting in small pieces, crushing), ultrasonic treatment or addition of a detergent. For fresh traces and such of an age of several weeks an elution time of 30 minutes at room temperature is sufficient; for very old stains an elution up to 2 hours may be recommended for safety. The investigations were restricted to the measurement of the IgG concentration in the eluates. No statement can yet be given about the biological value of the IgG for Gm typing due to this investigation alone.     

Postmortem sera and cerebrospinal fluid enzymes

Antemortem and postmortem sera from 60 dogs were evaluated for lipase, amylase, alkaline phosphatase, gamma-glutamyltransferase, and alanine aminotransferase (AAT); cerebrospinal fluid was examined for AAT and alkaline phosphatase. The postmortem intervals were 3, 6, 12, 24, and 48 h at temperatures of 4, 20, and 37 degrees C. Amylase levels remained stable at 4 and 20 degrees C and may be beneficial for diagnosing pancreatitis. Lipase levels may be useful as an adjunct to amylase values. Serum alkaline phosphatase values increased with postmortem interval; values were higher at 37 degrees C than at 4 degrees C. Other enzymes were of little value for diagnosis.     

A new marker for estimation of bloodstain age by high performance liquid chromatography.

The applicability of a new marker for estimation of bloodstain age by reverse-phase high performance liquid chromatography (HPLC) is described. Using a microBondasphere C18 column with a two step linear gradient of 10.5-46.25% acetonitrile in 0.1% trifluoroacetic acid, an intriguing peak (unidentified) at a retention time of about 5 min was observed on chromatograms from human adult bloodstains and designated as 'X'. The area of this peak, which could be detected in extracts of bloodstains, but not in their fresh whole blood, increased with time. The ratios of the X area to heme area in bloodstains stored at room temperature and 4 degrees C for up to 52 weeks old linearly correlated with stain age by plotting on a double logarithmic scale. In bloodstains exposed to fluorescent light at room temperature, the regression equation calculated from the ratios (Rx) and the ages of stains in weeks (W) is ln(1000.Rx) = 1.1084 + 0.3937.ln(7.W), and the coefficient of correlation (r) is 0.9776 (n = 144, P < 0.001). When stains were stored at 37 degrees C, the ratio transformed into logarithms correlated linearly with stain age. The regression equation describing the relationship in bloodstains exposed to fluorescent light at 37 degrees C is ln(1000.Rx) = 2.4477 + 0.0866.W (r = 0.9826, n = 144, P < 0.001). The results of the present study suggest that the HPLC method may be applicable to the estimation of bloodstain age.     

Characterization of deoxyribonucleic acid (DNA) obtained from teeth subjected to various environmental conditions.

This study was designed to determine the effects of various environmental factors on the deoxyribonucleic acid (DNA) obtained from dental pulp. Extracted teeth were subjected to the following conditions: varying pH (3,7,10); temperature (4 degrees C, 25 degrees C, 37 degrees C, incineration); humidity (20%, 66%, 98%); various types of soil (sand, potting soil, garden soil); seawater; burying the teeth outdoors, and aging (one week to six months). In addition, teeth that had been extracted and held at room temperature for 16 and 19 years were also examined. Following isolation of DNA, the samples were analyzed on yield gels to determine the concentration and integrity of the recovered DNA. Restriction digestion with Pst I was followed by electrophoresis of the generated fragments, Southern transfer to nylon membranes, and hybridization to both human and bacterial probes. It was determined that, aside from soil, the environmental conditions examined did not affect the ability to obtain high-molecular-weight human DNA from dental pulp. Restriction fragment length polymorphic (RFLP) analysis of selected samples was performed. Dental pulp patterns were compared with bloodstain exemplars, revealing matching patterns, although an increase in band-shifting was observed with extended exposure to elevated temperatures.     

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.     

Headspace gas chromatographic determination of ethanol: the use of factorial design to study effects of blood storage and headspace conditions on ethanol stability and acetaldehyde formation in whole blood and plasma

Our headspace gas chromatographic flame ionization detection (HS-GC-FID) method for ethanol determination showed slightly, but consistently, low ethanol concentrations in whole blood (blood) in proficiency testing programs (QC-samples). Ethanol and acetaldehyde were determined using HS-GC-FID with capillary columns, headspace equilibration temperature (HS-T degrees ) of 70 degrees C and 20 min equilibration time (HS-EqT). Full factorial designs were used to study the variables HS-T degrees (50 degrees -70 degrees C), HS-EqT (15-25 min), ethanol concentration (0.20-1.20 g/kg) and storage at room temperature (0-6 days) with three sample-sets; plasma, hemolyzed blood and non-hemolyzed blood. A decrease in the ethanol concentration in blood was seen as a nearly equivalent increase in the acetaldehyde concentration. This effect was not observed in plasma, indicating chemical oxidation of ethanol to acetaldehyde in the presence of red blood cells. The variables showed different magnitude of effects in hemolyzed and non-hemolyzed blood. A decrease in ethanol concentration was seen even after a few days of storage and also when changing the HS-T degrees from 50 to 70 degrees C. The formation of acetaldehyde was dependent on all the variables and combinations of these (interactions) and HS-T degrees was involved in all the significant interaction effects. Favorable instrumental conditions were found to be HS-T degrees of 50 degrees C and HS-EqT of 15-25 min. The ethanol concentrations obtained for the range 0.04-2.5 g/kg after analyzing authentic forensic blood samples with a HS-T degrees of 50 degrees C were statistically significantly higher than at 70 degrees C (+0.0154 g/kg, p < 0.0001, n = 180). In conclusion, chemical oxidation of ethanol to acetaldehyde in the presence of red blood cells has been shown to contribute to lowered ethanol concentrations in blood samples. Storage conditions before analysis and the headspace equilibration temperature during analysis were important for the determination of blood ethanol concentrations.     

The Stability of Prostate-Specific Antigen in Semen Under Various Temperatures

Prostate-specific antigen (PSA) is most commonly used for identifying semen, especially in the absence of sperm. However, PSA concentration varies according to storage temperature and duration, and little is known about its stability in semen. This study was therefore aimed to determine the stability under five different temperatures: −80, −20, 4, 25, and 37°C; and nine different durations: 1, 2, 3, 5, 7, 14, 30, 90, and 180 days. All samples were stored at −80°C after being secreted from the volunteers' body until analyzed. Results showed that the PSA concentration declined significantly over time under all temperatures studied except −80°C. At −20 and 4°C, PSA was still detectable on Day 180 with 50% and 70% decrease from its original concentration, respectively. At 25 and 37°C, PSA was detected up to Day 7 and 3, respectively. This information might assist forensic scientists understand more about PSA nature and integrate it into their works.     

Temperature-correction of abdominal impedance: improved relationship between impedance and postmortem interval

The relationship between extracellular abdominal impedance and postmortem interval (PMI) reflects the combined effects, on impedance, of postmortem cooling of the tissues and of autolysis per se. This study was performed in order to eliminate temperature change as a major factor contributing to the time course of postmortem change in abdominal impedance. Dissociation of thermal and autolytic influences was achieved by recording deep abdominal temperature at the time of impedance measurement, followed by correction of all measured impedances to their theoretically predicted values at an arbitrarily chosen temperature of 40 degrees C. Uncorrected abdominal impedance increased from 82+/-12 Ohmz, 1 h after death, to 108+/-21 Ohmz after 12 h. Impedance then decreased to 96+/-23, 89+/-22, 75+/-19, 66+/-21 and 59+/-19 Ohmz at postmortem intervals of 24, 36, 48, 60 and 72 h, respectively. In contrast, corrected abdominal impedance decreased progressively from 63+/-7 Ohmz, 1 h after death, to 61+/-9, 56+/-11, 51+/-10, 46+/-10, 39+/-11 and 35+/-10 Ohmz at postmortem intervals of 12, 24, 36, 48, 60 and 72 h, respectively. The improved relationship between (corrected) abdominal impedance and PMI is of potential value in estimating time since death.     

Development of a radioimmunoassay technique for the detection of human hemoglobin in dried bloodstains

A sensitive radioimmunoassay for the detection of human hemoglobin in dried bloodstains for the purpose of forensic science species identification has been developed. Bloodstains from 13 animal species were tested and found to be negative for human blood. A minimum volume of 0.8 microL of fresh blood is required to produce sufficient stain for successful testing. Bloodstains prepared from newborn and sickle-cell bloods were determined to be human. Bloodstains ranging in age from 1 month to 6 years which had been maintained desiccated at 20 to 25 degrees C were also successfully tested. Positive results were obtained on human bloodstains stored at 24 degrees C with relative humidity ranging from 0 to 98% for a period of 3 weeks. Absolute counts per minute (CPM) decreased with increased humidity. Human bloodstains exposed to bacterial contamination (gram positive or negative species) under humid conditions for 2 weeks also tested positive. Bacterial contamination caused a decrease in CPM, but insufficient to result in an erroneous conclusion as to species of origin. Positive results were also obtained on human bloodstains stored for 6 weeks at various temperatures ranging from -16 to 37 degrees C. No significant decreases in CPM were noted for any of the temperature conditions described.     

Vital reactions to frostbite of the ear and paw skin in guinea pigs exposed to the cold

Vital reactions to frostbite in the paw and ear skin of guinea pigs were studied in order to find an animal model for frostbite in cases of accidental hypothermia. One group of animals was rendered hypothermic (rectal temperature, 30 degrees C) by exposure to an ambient temperature of -20 degrees C, and samples were taken from the frozen skin. A second group was rendered hypothermic and rewarmed in warm air at 45 degrees C, and samples were taken from the thawed skin. The only vital reaction in the first group (freezing time, 4-5 h) was mild initial inflammation, which was expressed in granulocyte adhesion to the vessel wall and the migration of a few cells into the dermis. The inflammatory reaction was more distinct in the second group (freezing and thawing together 5-7 h), with a large number of granulocytes being present in the dermis. Oedema and hyperaemia were also present in the frostbitten tissue after thawing, but no signs of necrosis developed. The alkaline-phosphatase reaction demonstrated the presence of granulocytes more clearly than H & E or Masson trichrome staining. Vital reactions were more advanced in the ear skin. It is concluded that vital reactions are very scarce in cases of frostbite, even after several hours' exposure, unless the tissue is allowed to thaw.     

Bacterial Degradation of Risperidone and Paliperidone in Decomposing Blood

The stability of two benzisoxazole antipsychotics was determined in vitro in decomposing porcine blood inoculated with bacteria, utilizing a high‐performance liquid chromatography with ultraviolet and fluorescence detection method for drug quantitation. Stability experiments for risperidone and paliperidone were conducted at 7, 20 and 37°C for 4 days using sterile and bacterially inoculated porcine blood. The drugs were stable in sterile blood at each temperature and in inoculated blood at 7°C, but degraded significantly in inoculated blood at 20 and 37°C. Complete loss occurred within 2 days when incubated at 37°C. The benzisoxazole‐cleaved degradation products for both drugs were identified as 2‐hydroxybenzoyl‐risperidone and 2‐hydroxybenzoyl‐paliperidone utilizing liquid chromatography quadrupole‐time‐of‐flight mass spectrometry and accurate mass measurements. The degradation products have been found in postmortem case studies, including one case where risperidone and paliperidone were not detected, indicating complete conversion can occur in situ.     

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.