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.     

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.     

Postmortem stability and interpretation of beta 2-agonist concentrations.

This paper describes a series of stability and redistribution studies aimed at understanding the presence and significance of beta 2-agonists in asthma deaths. Salbutamol and terbutaline were shown to be stable in postmortem blood at 23 degrees C for 1 week, 4 degrees C for 6 months and -20 degrees C for 1 to 2 years. However, fenoterol was shown to degrade at 23 degrees C (83% loss), 4 degrees C (93% loss) and -20 degrees C (66% loss) over the same time. Salbutamol concentrations detected in blood taken at the time of body admission to the mortuary were not significantly different from the concentrations detected in blood taken from the same cases at the time of autopsy (45 h later). This suggests that significant postmortem redistribution of salbutamol is unlikely to occur during this period. Postmortem blood concentrations of at least salbutamol are likely to reflect the concentration of these drugs in the body at the time of death.     

Intratracheal gas analysis for volatile substances by gas chromatography/mass spectrometry--application to forensic autopsies

Intratracheal gas analysis was carried out by gas chromatography/mass spectrometry (GC/MS) in 20 burned body cases (13 males and 7 females). Volatile aromatic and aliphatic hydrocarbons were detected by GC/MS using a GS-Q column with the intratracheal gas as well as the blood in 19 cases. The characteristic patterns of mass chromatograms for gasoline, kerosene (gas oil), and liquid petroleum gas could be differentiated from each other using the intratracheal gas. The burned body in one case showed no presence of volatile substances in the intratracheal gas, nor intratracheal soot, although high concentrations (1 microg/g and more) of volatile substances were detected on the clothes. The victim also had normal CO-Hb concentrations (0.1 to 0.2%) in the heart blood. The results of intratracheal gas analysis were consistent with signs of the vital reaction. In conclusion, intratracheal gas analysis provides a supportive method for diagnosing the cause of death in burned bodies, and yields for at least 48 hours valuable information on volatile hydrocarbons (being detected in deliberate or accidental fire cases) to which the body had been exposed just before death.     

Driving under the influence of toluene

Toluene is the most common volatile used for sniffing among adolescents. During 1983-1987, 114 drivers were arrested in Norway with blood toluene concentrations (BTCs) greater than 10 microM. Only four of these drivers were women. The age range was 15-34 years, and the mean age was 21. The mean BTC was 109 microM. There was no simple relation between blood toluene concentration and degree of impairment, however, most drivers with BTCs greater than 100 microM were considered as impaired or probably impaired by toluene. In a five year prospective study of rearrests among drivers arrested for driving after toluene sniffing, 12 out of 15 drivers were rearrested. They were responsible for 40 cases of suspected driving under influence of toluene, alcohol, or other drugs. The blood levels of toluene determined in this study must be regarded as minimum concentrations, since the toluene concentration fell rapidly in samples stored at 4 degrees C or 23 degrees C. Blood samples from drivers suspected of driving under influence of toluene must therefore be kept frozen.     

Gamma-hydroxybutyric acid stability and formation in blood and urine

Gamma-hydroxybutyric acid (GHB) can cause problems in interpretation of toxicological findings due to its endogenous nature, significant production in tissues after death and potential formation in stored samples. Our study was designed to determine the influence of storage conditions on GHB levels and its possible in vitro formation in blood and urine in cases where no exogenous use of GHB or its precursors was suspected. The samples were prepared by validated method based on liquid-liquid reextraction with adipic acid internal standard and MSTFA derivatization and assayed on a GC-MS operating in EI SIM mode. The first part of the study was performed with pooled blood and urine samples obtained from living and deceased subjects stored with and without NaF (1% w/v) at 4 and -20 degrees C over 8 months. In ante-mortem samples (both blood and urine) no significant GHB production was found. After 4 months of storage, the substantial GHB rise up to 100 mg/Lwas observed in post-mortem blood stored at 4 degrees C without NaF with subsequent gradual decrease in following months. The inhibition of GHB production was apparent during storage in NaF treated frozen blood samples. In post-mortem urine only slight temporary GHB levels were ascertained (up to 8 mg/L). The second part of our study was aimed to analyse 20 individual post-mortem blood samples stored at 4 degrees C for 16-27 days between autopsy and analysis without preservation followed by storage at 4 degrees C with NaF for 4 months. The temporary GHB production with maximum of 28 mg/Lwas detected in some samples.     

Biochemical measurements of glucose metabolism in relation to cause of death and postmortem effects

This study was performed to examine the relationship between postmortem biochemical values and cause of death. The follow samples were taken from 399 corpses: cerebrospinal fluid (CSF; n = 376, suboccipital), blood (n = 158, femoral vein), and urine (n = 101, at autopsy). (See Table 1 for causes of death) All samples were stored at -80 degrees C. A further 100 samples of blood were later taken and stored at +4 degrees C before testing. Biochemical determinations made were: glucose in CSF, blood, and urine (hexokinase method); lactate (LDH/GPT) and free acetone (HS-gas chromatography) in CSF; hemoglobin A1 in blood (microcolumn technique). In 34 cases fatal diabetic coma was considered verified by morphological and chemical findings. One hundred cases of sudden cardiac death were chosen as the main control group. In 32 of the 34 cases defined above, the value of the formula of Traub (glucose + lactate in CSF) exceeded 415 mg/dl. It is not influenced significantly by hyperglycemia or hyperlactatemia due to factors other than diabetes (i.e., carbon monoxide, asphyxia). After death the value rose till the 30th hpm, then remained stable for at least 1 week. Fatal coma was defined as the ketoacidotic form if free acetone in CSF ranged above 21 mg/l. In these cases, CSF glucose and free acetone correlated positively. Hemoglobin A1 remained stable after death. Its amount was independent from postmortem blood glucose, postmortem interval and total hemoglobin. Furthermore, the manner of storage (-80 degrees or +4 degrees C) had no significant influence on its values. In 29 of 34 cases of fatal coma, Hb A1 exceeded 12.1%. Analysis of urine glucose showed elevated levels (over 500 mg/dl) in diabetic comas. On conclusion, fatal diabetic coma seems indicated as the cause of death if measured values of postmortem biochemistry exceed the following limits: CSF-Traub 415 mg/dl, free acetone (CSF) 21 mg/l; Hb A1 12.1%; urine glucose 500 mg/dl. Most important are the Traub formula and hemoglobin A1. Usually, in fatal coma both values are elevated. If both of them are normal, diabetic coma can nearly be excluded. Combined evaluation of all values is absolutely necessary. Morphology must also always be taken into account. Consequently, a diagnosis of fatal coma can be obtained by a process of elimination.     

Changes in the concentration of carboxyhemoglobin in the blood under the action of high temperatures

Changes in carboxyhemoglobin concentrations are studied in 18 specimens of cadaveric blood 1-2 h and 3 days after heating to 50, 65, 70, 75, 80, and 90 degrees C for 5, 10, and 20 min. The concentration of carboxyhemoglobin decreased to 15% after heating at 70-75 degrees C for 10 and 20 min. Heating to 80-90 degrees makes the measurements impossible. Five-min heating at 50-65 degrees C did not change the concentration of carboxyhemoglobin in the blood.     

Cyanide distribution in five fatal cyanide poisonings and the effect of storage conditions on cyanide concentration in tissue

The cyanide distribution in five fatal cyanide poisonings was analyzed by the pyridine-pyrazolone method using a Conway diffusion cell. In order to study the effect of storage conditions on cyanide concentration in tissue samples, the cyanide concentrations were first measured immediately after collection of the samples at autopsy, then measured again after storage in a refrigerator (4 degrees C) or in a freezer (-20 degrees C) for periods ranging from 1 day to 3 weeks. Concentrations in all but three of the blood samples stored at 4 degrees C or -20 degrees C increased, with concentration ratios based on measurement made before and after storage ranging from 0.71 to 1.46. The concentrations in the liver, kidney, and brain samples either increased or decreased, with ratios of from 0.2 to 8.8. The concentrations in the stomach contents samples decreased rapidly at 4 degrees C, but hardly changed at all at -20 degrees C.     

Volatile substance abuse--post-mortem diagnosis

A substantial number of children and adolescents world-wide abuse volatile substances with the intention to experience an euphoric state of consciousness. Although the ratio of deaths to nonfatal inhalation escapades is low, it is an important and preventable cause of death in young people. In the analytical investigation of volatile substances proper sample collection, storage and handling are important in view of the volatile nature of the compounds. Volatile organic compounds in post-mortem matrices such as blood, urine and tissues are generally determined by gas chromatography after extracting the compounds with methods such as static and dynamic headspace or even with pulse-heating and solvent extraction. In post-mortem cases, metabolites in urine seem less relevant, however, trichloroethanol and trichloroacetic acid were determined in several cases. When interpreting qualitative and quantitative results, researchers should be aware of false conclusions. The main reason why scepticism is necessary is the occurrence of losses of analytes during sampling, sample handling and storage, which results in false quantitation.     

Stability of Morphine,Codeine, and 6‐Acetylmorphine in Blood at Different Sampling and Storage Conditions

The stability of drugs in biological specimens is a major concern during the evaluation of the toxicological results. The stability of morphine, codeine, and 6‐acetyl‐morphine in blood was studied after different sampling conditions: (i) in glass, polypropylene or polystyrene tubes, (ii) with addition of dipotassium ethylene diamine tetraacetic acid (K₂EDTA) or sodium oxalate (Na₂C₂O₄), and (iii) with or without the addition of sodium fluoride (NaF). Spiked blood samples were stored at two different temperatures (4 and ?20^°C), analyzed after different storage times and after three freeze–thaw cycles. Opiate concentrations were decreased in all conditions, but the most unstable was 6‐acetyl‐morphine. The addition of NaF as preservative improved the stability of opiates at all conditions studied, whereas the type of anticoagulant did not affect the stability of opiates. It was concluded that blood samples should be stored at ?20^°C in glass tubes containing oxalate and NaF for maximum stability.     

Detection of cocaine in blood stains]

Cocaine is rapidly degraded in blood samples, and its degradation was found to be highly dynamic in nature. The analysis of blood spots dried on filter paper may provide a method to minimize the break-down of cocaine and to largely preserve the analytical profile of the parent drug and its hydrolysis products at the time of sampling. The short term stability of cocaine in 100 microL blood spots prepared from unpreserved and preserved (sodium fluoride, 0.25%) blood samples was compared to the stability of the particular whole blood specimens stored in tubes at ambient temperature and at -20 degrees C. Due to dehydration, both the chemical and the enzymatic hydrolysis of cocaine and its products could be stopped in dried blood spots. More than 75% of the initial cocaine concentration could be detected in the blood spots, and the analytical profile was ensured for 17 days. Provided its practical suitability, the spot technology should offer a simple approach to detect actual impairment of motorists taken in police custody in the view of section 24a of the German traffic act as well as in cocaine associated criminal cases.     

Positive- and negative-ion mass spectrometry and rapid clean-up of some carbamate pesticides

Positive-ion electron impact (PIEI), positive-ion chemical ionization (PICI) and negative-ion chemical ionization (NICI) mass spectra of 9 carbamate pesticides are presented. In the PIEI mode, the spectra showed small molecular peaks, intense or base peaks due to M - CH3NHCO + H and peaks at m/z 58 due to CH3NHCO. In the PICI mode, peaks due to M + H, M + C2H5, M - CH3NHCO + 2H, CH3NHCO(m/z 58) and M-28 appeared. The cations at m/z 58 found in both PIEI and PICI modes seem very useful for screening of a carbamate. In the NICI mode, the spectra showed peaks due to M - CH3NHCO and characteristic anions appearing at mass numbers higher than molecular ones, which were probably due to dimerization of [M - CH3NHCO]-followed by hydrogen attachment. Carbamates, which had been added to urine, plasma, whole blood, the liver, kidney and brain, could be rapidly isolated by use of Sep-Pak C18 cartridges with chloroform as an elution solvent. They could be detected by wide-bore capillary gas chromatography with a SPB-5 column, with satisfactory separation from impurities in their underivatized forms.     

Detection of ABH blood group antigens in the saliva of Koreans and their stability according to storage of saliva samples

The purpose of the present study was to identify salivary molecules carrying the ABH blood group antigens in Koreans and to investigate the changes in these antigens according to processing and storage of saliva samples. Secretor or non-secretor phenotypes and salivary components carrying the ABH antigens were identified in 90 subjects, 30 subjects in each ABO blood group, by SDS-PAGE and immunoblotting. Saliva samples were then obtained from 12 secretors-two males and two females in each ABO blood group and aliquots of both fresh saliva samples and their supernatants after centrifugation were stored at room temperature, 4, -20 and -70 degrees C. The same experiments were performed after 1, 3 and 6 months to investigate changes in the blood group antigens. In all 68 secretors, high-molecular-weight salivary mucin (MG1) was found to be the primary carrier of the ABH antigens. A salivary component of approximately 80 kDa also carried H antigen in seven saliva samples of 22 blood type O secretors. The blood group antigens were better detected in centrifuged samples. In saliva samples preserved at room temperature and 4 degrees C, the blood group antigens were either not detected or detected as degraded molecules. No change was found in the blood group antigens in saliva samples preserved at -20 and -70 degrees C for 6 months.     

Chlorpromazine-induced alterations in hypothalamic amine metabolism and stress responses in severe cold

To investigate the effects on the central nervous system of severe cold stress with and without chlorpromazine, guinea pigs were treated with chlorpromazine or 0.9% NaCl and exposed to -20 degrees C or +23 degrees C for 1 h. Hypothalamic noradrenaline (NA), dopamine (DA), 5-hydroxy-tryptamine (5-HT), 3-methoxy-4-hydroxyphenyl ethylene glycol (MHPG), homovanillinic acid (HVA) and 5-hydroxy-indoleacetic acid (5-HIAA) were determined by high-performance liquid chromatography. Serum, urinary and vitreous fluid catecholamines, muscle and liver glycogen, and blood glucose were also measured. Chlorpromazine caused distinct hypothermia at -20 degrees C and slight hypothermia at +23 degrees C. The rise in hypothalamic MHPG, 5-HIAA and MHPG/NA and in 5-HIAA/5-HT ratios in the cold indicate increased noradrenergic and serotonergic activity. The latter was inhibited by chlorpromazine and a drug-induced inhibition of noradrenergic neurons could not be ruled out. Chlorpromazine increased the turnover of DA at room temperature and the same tendency was seen in the cold. The hypothermic animals had low serum catecholamines, indicating diminished sympathetic activity. The chlorpromazine-treated cold-exposed animals did not react to the environmental stress by sympathetic activation, as urinary NA and adrenaline were not elevated, but DA was excreted by all the drug-treated animals. Vitreous fluid NA and DA were elevated as an indicator of cold stress, and no drug effect was seen in this fluid.     

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.     

Postmortem production of ethanol in different tissues under controlled experimental conditions

The aim of this study was to follow the postmortem ethanol production phenomenon under controlled experimental conditions (temperature, time interval) in different tissues. Specimens of blood, liver, skeletal muscle and kidney were taken from 30 corpses and no chemical preservatives were used in the specimens collected. Ethanol concentrations were detected by gas chromatography. All specimens stored at -20 degrees C and 4 degrees C did not show any change in ethanol concentration in an eight-day time interval. At 20 degrees C and 30 degrees C, all tissues, except blood, showed statistically significant ethanol production over the time interval tested. However, blood sample kept at 30 degrees C, showed statistically significant increase in ethanol production on the 2nd and 4th day comparing to the controls. Thus, we can state that postmortem ethanol production occurs in different tissues, and is increased at higher temperatures and, in general, it is in accordance with the course of time.     

Optimal storage conditions for highly dilute DNA samples: a role for trehalose as a preserving agent

DNA extraction from trace samples or noninvasively collected samples often results in the recovery of low concentration solutions of DNA that are prone to DNA degradation or other loss. Because of the difficulty in obtaining such samples, and their potentially high value in wildlife and forensic studies, it is critical that optimal methods are employed for their long-term storage. We assessed the amplification yield of samples kept under different storage conditions with the addition of potential preserving agents. We stored dilutions of known concentration human placental DNA, and gorilla fecal DNA, under four conditions (+4 degrees C, -20 degrees C, -80 degrees C, dry at room temperature), and with three additives (Tris EDTA (TE) buffer, Hind III digested Lambda DNA, trehalose). The effectiveness of the treatment methods was tested at regular intervals using qPCR to assess the quantity of amplifiable DNA, and a PCR assay of a larger 757 bp fragment to evaluate the quality of that remaining DNA. The highest quantity of DNA remained in samples stored at -80 degrees C, regardless of storage additives, and those dried at room temperature in the presence of trehalose. Surprisingly, DNA quality was best preserved in the presence of trehalose, either dried or at -80 degrees C; significant quality loss occurred with -20 degrees C and +4 degrees C storage.     

Stability of drugs in stored postmortem femoral blood and vitreous humor

The stability of 46 drugs in postmortem femoral blood stored for one year at -20 degrees C was investigated. The drugs included benzodiazepines, antidepressants, analgetics and hypnotics. For seven drugs we found a significant change in the concentration between the first and second analysis. Five substances; ethanol, desmethylmianserin, 7-amino-nitrazepam, THC and zopiclone showed a decrease in the concentration whereas the concentrations of two substances; ketobemidone and thioridazine increased. However, the changes observed were not of such an order that it would affect the interpretation in normal forensic casework. We also investigated the possible influence of potassium fluoride on the concentrations of the 46 drugs in vitreous humor after storage for one year. For two substances, ethanol and zopiclone, there were significantly lower concentrations in the samples without potassium fluoride. Furthermore, we also studied the correlation between the concentrations in femoral blood and vitreous humor. For 23 substances there was a significant difference between the concentrations in the vitreous humor and femoral blood. Significant correlations between the concentrations in these two specimens were found for 23 substances, indicating that vitreous humor can be an alternative specimen when blood samples are not available, provided that such correlation exists for the particular substance. Statistical analysis also revealed a correlation between the degree of protein binding of the different drugs and percentage of vitreous/femoral blood concentrations.     

Storage of blood for methemoglobin determination: comparison of storage with a cryoprotectant at -30 degrees C and without any additions at -80 degrees C or -196 degrees C

Changes in methemoglobin (Met-Hb) concentrations during storage of whole blood or mixtures of blood and a cryoprotectant at refrigerated or various freezing temperatures were examined using blood samples from nitrite-administered rats and from autopsy cadavers. When whole blood was stored at 3 degrees C, Met-Hb reduction was observed in blood samples from nitrite-administered rats and in the blood from a victim poisoned by a weed killer containing some oxidant. When samples were stored at -30 degrees C, Met-Hb formation by autoxidation was inevitably observed in blood samples stored as whole blood, whereas addition of a cryoprotectant to whole blood could prevent Met-Hb formation in all the blood samples. When whole blood was stored at -80 degrees C or -196 degrees C, Met-Hb concentrations were practically stable until at least 30 days regardless of the initial values except in the control rat blood samples stored at -80 degrees C which showed slight formation of Met-Hb. From the results obtained, both the storage with a cryoprotectant at -30 degrees C and that without any additions at -80 degrees C or -196 degrees C proved to be suitable for long-term storage of blood samples from autopsy cadavers for Met-Hb determination.