Potential for error when assessing blood cyanide concentrations in fire victims.

The present study explores toxicologic significance of blood cyanide concentrations in fire victims. Headspace gas chromatography was used for cyanide detection. Analysis of blood samples from ten fire victims (postmortem interval = 8 h to 3 to 5 d) detected zero to 11.9 mg/L of cyanide and a large difference in cyanide concentrations among victims. Carboxyhemoglobin (COHb) saturation was in the range of 24.9 to 84.2%. To examine the effects of methemoglobinemia and postmortem interval on blood cyanide concentrations in fire victims, an experiment was carried out using rabbits as the animal model. The rabbits were sacrificed by intramuscular injection of 1 mL/kg 2% potassium cyanide 5 min after intravenous injection of 0.33 mL/kg of 3% sodium nitrite (Group A, n = 3) or physiological saline (Group B, n = 6). Average methemoglobin contents immediately before potassium cyanide administration were 6.9 and 0.8% in Groups A and B, respectively. Average cyanide concentrations in cardiac blood at the time of death were 47.4 and 3.56 mg/L, respectively. When blood-containing hearts of the rabbits (n = 3 for Group B) were left at 46 degrees C for the first 1 h, at 20 to 25 degrees C for the next 23 h and then at 4 degrees C for 48 h, approximately 85 and 46% of the original amounts of blood cyanide disappeared within 24 h in Groups A and B, respectively. After the 72-h storage period, 37 and 10%, respectively, of the original amounts of cyanide remained in the blood. When the other three hearts in Group B were left at 20 to 25 degrees C for the last 48 h without refrigeration, cyanide had disappeared almost completely by the end of the experiment. The present results and those published in the literature demonstrate that the toxic effects of cyanide on fire victims should not be evaluated based solely on the concentration in blood.     

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.     

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.     

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.     

Experimental studies on death by fire in automobiles and exhaust gas poisoning

Studies were made on the acid-base balance, blood gases, and carbon monoxide (CO), cyanide, and sulfur dioxide concentrations in the blood of albino rabbits that died from automobile exhaust gas poisoning (group I) or fires in cars (complete combustion, group II; incomplete combustion, group III). In group I, the temperature and CO concentration increased gradually to 35 degrees C and 5.2% in 70 min. The animals died after 9 min, when the values were 20 degrees C and 5.2%, respectively. In group II the animals died after 9 min, when the values were 55 degrees C and 1.95%, respectively. In group III, the temperature was very high (870 degrees C), but the CO concentration was not (0.6-1.3%) after 4 min. The animals died after 5 min. In all experimental groups, marked acidosis and hypoxemia were seen, but the CO2 tension (PCO2) was high, in contrast to previous studies on pure CO poisoning. In group I, the level of carboxyhemoglobin (CO-Hb) was significantly higher (91.2 +/- 3.4% in arterial blood, 87.5 +/- 8.1% in venous blood; p less than 0.01) than in groups II and III. Although the O2 tensions of venous and arterial blood (PvO2, PaO2) were very low, that of arterial blood was higher, suggesting that O2 was still being utilized in the tissues at the time of death. In group II, CO-Hb was high (57.7 +/- 16.0% in arterial blood, 61.2 +/- 20.6% in venous blood) and the acid-base balance indicated marked acidosis. In group III, the CO-Hb, PCO2 and cyanide levels in the blood were very high.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time- and Temperature-Dependent Changes in Cytochrome c Oxidase Activity and Cyanide Concentration in Excised Mice Organs and Mice Cadavers

Postmortem stability of cyanide biomarkers is often disputed. We assessed the time and temperature-dependent changes in cytochrome c oxidase (CCO) activity and cyanide concentration in various organs of mice succumbing to cyanide. Immediately after death, excised mice organs and mice cadavers were stored at room temperature (35°C ± 5°C) or in frozen storage (−20°C ± 2°C). At various times after death, CCO activity and cyanide concentrations were measured in excised mice organs or organs removed from mice cadavers. The study revealed that (i) measuring both the biomarkers in mice cadavers was more reliable compared to excised mice organs, (ii) measuring temporal CCO activity and cyanide concentration in vital organs from mice cadavers (room temperature) was reliable up to 24 h, and (iii) CCO activity in the brain and lungs and cyanide concentration in organs from mice cadavers (frozen) were measurable beyond 21 days. This study will be helpful in postmortem determination of cyanide poisoning.     

Kinetics of ethanol degradation in forensic blood samples

To determine ethanol in human post-mortem blood samples is problematic, largely due to the inappropriate and variable methods of preserving and storing, which can cause decomposition and loss of alcohol concentration. In this study, four crucial parameters of sample conservation were studied: temperature (T), percentage of air chamber in container (%CA), ethanol concentration in blood and post-mortem time. Blood samples from post-mortem cases were stored under different conditions (ethanol levels were known in all cases); factorial design variables: (%CA) 0, 5, 20, 35, 65%; storage temperature: 25, 4 and -10 degrees C; in a total of 15 experiments. No preserving agent was used in samples. Quantification of ethanol in blood was carried out by gas chromatography with head-space FID detector. Initial ethanol concentration ranged from 0.50 to 4.30 g/L. The kinetics of degradation observed was pseudo-first-order. The parameter that characterised the kinetics of ethanol degradation (k(0)) ranged from (4 x 10(-4) and 5.0 x 10(-1) day(-1)), depending on storage conditions. A strong dependence between ethanol degradation and the content of the air chamber was observed and this dependence was found to be stronger than that between degradation and temperature; there was an experimental relation between (k(0)) and (%CA). Activation energy for different conditions, i.e. 0, 5, 20, 35 and 65 (%CA), were calculated and contour plots were made. A mathematical equation relating air chamber, temperature and ethanol concentration at a certain time was determined. This equation allowed estimation of initial concentrations of ethanol with minimal error. A good correlation between experimental data and data calculated with the equation was obtained (r(2) = 0.9998). The best storage conditions were: 0% CA and storage at -10 degrees C, obtaining an ethanol degradation of 0.01% after 15 days. However, 33% of ethanol degradation was obtained with 35% CA at 25 degrees C after 15 days. This equation is useful in forensic cases in which original concentration of ethanol has to be estimated under different sample storage conditions.     

Preservation of methanol in cadaveric material

Duration of preservation of methanol in cadaveric material at 20 +/- 3 degrees C during 4-13 months was studied on 19 model and expert objects of cadaveric material (muscle tissue, kidney, blood, urine). Exponential relationship between methanol concentration and duration of storage of the object was detected. Methanol concentration decreased by 30-70% during the first weeks, after which the process decelerated to 5-10% a month. Toxicologically significant concentrations of endogenous methanol were not revealed in any case.     

The effect of sodium fluoride on the stability of cyanide in postmortem blood samples from fire victims

Assigning a level of significance to cyanide concentrations found in the blood of fire victims is often hampered by the fact that cyanide is inherently unstable in cadavers and in stored blood samples. A few researchers have proposed that sodium fluoride can be used to minimize the instability of cyanide in blood samples; however, controlled studies have not been performed to support validation of this hypothesis. To test the sodium fluoride hypothesis, both treated and control blood samples from 14 autopsied fire victims were tested over a 25-30 day period. A 2% concentration of sodium fluoride was added to the blood samples at the start of testing and the samples were refrigerated between testing intervals. Cyanide concentrations in the treated and control samples were measured between 9 and 11 days post treatment and between 25 and 30 days post treatment. A statistically significant difference was not present between blood cyanide concentrations in treated and control samples between 9 and 11 days. During this time period, although there were small statistically significant increases in both treated and untreated samples the fluctuations were minor. Since the treated and control samples did not exhibit instability between 9 and 11 days, it is not surprising that the sodium fluoride appeared to have no effect. However, a statistically significant difference between blood cyanide concentrations in treated and control samples was observed between 25 and 30 days. Those samples treated with sodium fluoride showed a reduction in blood cyanide variability with virtually no overall change, over a 25-30 day period when compared to control samples, while unconditioned samples showed a significant, average increase of 35%. Based on the findings of this study, it is recommended that 2% sodium fluoride be added to blood samples obtained from fire victims to reduce cyanide instability due to bacteriological activity.     

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.     

A blood cyanide distribution study in the rabbits intoxicated by oral route and by inhalation

Blood cyanide concentration was determined in rabbits intoxicated orally or by inhalation. Experiments were carried out under urethane anaesthesia. In the inhalation experiments, rabbits inhaled a combustion product containing HCN via the tracheal cannula and in the oral studies animals were administered NaCN solution into the stomach. In addition to the carotid artery and jugular vein blood samples, postmortem samples were obtained from both sides of the heart and the descending vena cava. The arterial cyanide concentration in the inhalation group showed a close relationship with ventilation. After an initial rise, blood levels decreased a little, in some cases with transient apnea. At the last stage it again increased with gasping, reaching its maximal value. After ultimate apnea, the blood cyanide concentration declined. The blood cyanide values were higher in the oral group than in the inhalation group. The difference between the two groups became larger in the inferior order, the left heart blood--the right heart blood--blood in the descending vena cava. The left heart/right heart ratio of the inhalation group was significantly higher than that of the oral group (1.28+/- 0.28 vs. 0.95+/- 0.09). The coefficient of variation (c.v.) of the inhalation group was larger than that of the other group. Within the inhalation group, the left heart blood showed the largest c.v. values and this was probably due to redistribution of the cyanide by bloodstream after attainment of the maximal concentration.     

A micro-chemiluminescence determination of cyanide in whole blood

A reactant volume self-controlled micro-device was presented and applied to the flow injection chemiluminescence (CL) for determination of cyanide in whole blood. A mini distiller was fabricated for cyanide extraction from the blood samples with the extraction efficiencies of cyanide > or = 98%. A fluidic control platform with air driving was fabricated. The described system showed the features of easy fabrication, undiluted sample injection, safe analysis operation, and suitability for automatic cyanide analysis. The calibration curve showed linearity in the cyanide concentration range of 5.0 x 10(-7) to 5.0 x 10(-5) mol l(-1) with the detection limits (3sigma) of 2.3 x 10(-7) mol l(-1). CL peak-height precision was 1.9% R.S.D. (n = 11) at the 1.0 x 10(-6) mol l(-1) cyanide level. The new devices were applied to the analysis of cyanide in rabbit whole blood samples and the results agreed well with those obtained from official method.     

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.     

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.     

Brain concentrations of cocaine and benzoylecgonine in fatal cases

Since cocaine in blood rapidly hydrolyzes to benzoylecgonine, cocaine concentrations determined in postmortem blood may not reflect the presence or concentration of cocaine in the body at the time of death. The interpretative value of the determination of cocaine and benzoylecgonine in brain tissue was investigated. Cocaine and benzoylecgonine were quantitated by coextraction and formation of the propyl derivative of benzoylecgonine followed by selected ion monitoring gas chromatography/mass spectrometry (GC/MS) using electron ion impact ionization. Cocaine and benzoylecgonine were found to be evenly distributed throughout the brain. Cocaine and benzoylecgonine concentrations were stable in frozen brain tissue (-4 degrees C) on reanalysis after 1 to 3 months of storage, and in refrigerated tissue (10 degrees C) after 30 days of storage. Blood, brain, and liver concentrations of cocaine and benzoylecgonine in 37 cocaine overdose cases and 46 cases in which cocaine was incidental to the cause of death were reviewed. The ratios of cocaine/benzoylecgonine in the toxic cases (brain mean 14.7 and blood mean 0.64) were clearly different from those found in the incidental cases (brain mean 0.87 and blood mean 0.27). The brain/blood ratios of cocaine and benzoylecgonine concentrations generally were characteristic of the time elapsed since cocaine dosing. In cocaine overdose cases, the mean ratio was 9.6 for cocaine and 0.36 for benzoylecgonine. These are within the range found in animal studies for brain/blood ratios of cocaine and benzoylecgonine 0.5 to 2 h after cocaine administration. In incidental cases, the brain/blood ratios were mean 2.5 for cocaine and 1.4 for benzoylecgonine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of storage conditions on restriction fragment length polymorphism (RFLP) analysis of deoxyribonucleic acid (DNA) bound to positively charged nylon membranes.

The ability to generate an autoradiogram from deoxyribonucleic acid (DNA) immobilized on a positively charged nylon membrane could be compromised by the storage conditions of the membrane. HaeIII-digested human DNA was size fractionated and transferred to two types of positively charged nylon membranes. The membranes were stored at -20 degrees C, 4 degrees C, and ambient temperature and humidity for times ranging from 1 day to 13 weeks, then hybridized to variable number of tandem repeat (VNTR) probes to examine the effect of the storage conditions on the membrane-bound DNAs. It was shown that such membranes could be successfully hybridized and rehybridized if they were stored at -20 or 4 degrees C, but storage under ambient conditions reduced or eliminated the likelihood of successful hybridization.     

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.     

Review of factors susceptible of influencing post-mortem carbohydrate-deficient transferrin

Seric carbohydrate-deficient transferrin (CDT) is a biochemical marker of chronic alcohol abuse. Assessment of the influence of factors likely to modify CDT concentration is necessary to justify its use in the analysis of post-mortem blood samples. Hemolysis, site of collection and storage were tested. Hemolysed samples showed decreased CDT concentration. Statistical analysis of CDT concentration in cardiac blood versus femoral blood revealed no significant differences. Storage for fifteen days at +4 degrees C or +20 degrees C did not affect CDT concentration but repeated freezing and thawing resulted in decreased levels of CDT.     

Improper sealing caused by the Styrofoam integrity seals in leakproof plastic bottles lead to significant loss of ethanol in frozen evidentiary urine samples

Evidentiary urine samples (n = 345) stored frozen at -20 degrees C in their original containers (leakproof 100 mL plastic bottles) upon retesting for ethanol resulted in concentrations that were significantly lower (average loss = approximately 30%) than those prior to their storage at -20 degrees C (p < or = 0.0001). The observed loss of ethanol was independent of the method of thawing or the concentration of ethanol in the samples, but was dependent on the sample volume in the container, i.e., the larger the volume of sample the larger the magnitude of ethanol loss. The loss of ethanol was determined to be due to improper sealing by a Styrofoam integrity seal attached to the mouth of the container. Accordingly, adopting leakproof plastic containers that do not contain Styrofoam integrity seals, but rather an outside and across the cap tape integrity seal for evidence collection and long-term storage, will prevent loss of ethanol due to evaporation.     

Acute effects of carbon monoxide and cyanide on hepatic mitochondrial function

The effects of carbon monoxide and cyanide on the hepatic redox state and energy charge were investigated. Rats were used for the experiment under pentobarbital anesthesia. Immediately after laparotomy, a rat was placed in an animal chamber made of a transparent plastic box and exposed to a test gas for 3 min. Every test gas was produced in a gas chamber connected to the animal chamber with a flexible tube. HCN was produced from NaCN and H2SO4. In the CO inhalation experiment, various amounts of CO were introduced into the gas chamber. Immediately after an exposure, about 2 g liver was frozen in situ with a precooled clamp. Oozed blood from the wound surface was sampled. Concentrations of ATP, ADP, AMP, acetoacetate, and beta-hydroxybutyrate in hepatic mitochondria were determined, and the redox state and the energy charge were calculated. For cyanide as well as CO, significant negative correlations were found between the concentration in the blood and the redox state. The same held true for the energy charge. The redox state showed a slight increase at low concentrations of both gases; however, thereafter it began to decrease sharply with increases in concentrations. When concentrations of the toxicant in the blood reached certain levels, a kind of turning point, beyond which the redox state does not decrease any more, was observed. It was about 40% for HbCO and about 2.0 micrograms/ml for cyanide, and the points seemed to be related to the concentrations, beyond which cells are irreversibly damaged. On the other hand, the energy charge did not change at low concentrations. With an increase in toxicant concentrations, the energy charge decreased drastically. The rate of decrease in the energy charge became higher when blood concentrations exceeded certain levels. It was about 40% for HbCO and 2.0 micrograms/ml for cyanide. The presence of low levels of blood cyanide did not affect the relationship between the energy charge and the HbCO concentration.