Correction of the alkylnitrite method for the measurement of ethanol in blood: direct experimental evidence

A coefficient (multiplier) for the correction of a systemic inaccuracy of ethanol detection in the blood by the alkylnitrite method arising from calibration against an aqueous solution has been improved. The experiment was carried out as a multilaboratory study (with the participation of six laboratories). The value of the coefficient was estimated at 0.82 +/- 0.014 (SD) instead of 0.95 computed earlier (CV = 1.7%). The main factor influencing the value of the coefficient turned out to be the time of preparation of the model ethanol-blood mixture for comparison with the aqueous solution. The coefficient of 0.82 was obtained using fresh blood samples that of 0.93 to 0.96 with blood samples previously stored during 3-10 days. The results of the study suggest different ethanol distribution patterns in vivo and in vitro in the aqueous phase of the blood stored for a few days.     

Estimation of ethanol concentrations in biological media. Comparison of alkylnitrite and direct vapor-phase methods

Ethanol assays in the blood, saliva and urine were made according to two methods: alkylnitrite gas-liquid chromatography and direct vapor-phase method without thermostating. N-propanol was used as an internal standard. For saliva and urine the methods produced similar results. For blood, the direct vapor-phase method detected significantly lower level of ethanol compared to alkylnitrite one (p = 0.001). 24-h storage of the samples in refrigerator does not deviate the results. Recommendations are provided for most effective usage of each method.     

A note on taking account of the automatic death penalty jurors

The California Supreme Court'sHovey opinion identified a separate group of “automatic death penalty” (ADP) persons whose exclusion had been overlooked in previous studies of death qualification. Using data unavailable at the timeHovey was decided, this brief article estimates the effect of excluding this group on the attitudinal skewing and conviction-proneness of death-qualified jurors. It concludes that the impact of excluding the ADPs is negligible.     

血液中乙醇保存稳定性研究

目的确定血液中乙醇最佳保存条件,探讨影响血液中乙醇含量稳定性的主要因素。方法对血液保存的温度(-20、4、20℃)、防腐剂(NaF、无防腐剂、Na2O2)、储存容器中空气所占比例(0%、25%、50%)和血醇质量浓度(0.2、0.8、2.0mg/mL)四个因素采用正交试验L9(34)方法分组,样本采用顶空气相色谱法进行测定,测定结果采用方差分析进行讨论。结果在20℃保存且不加入防腐剂的两组样本中血醇浓度变化明显,其余变化不明显。结论血液样本在4℃、储存容器中空气比例为50%和加防腐剂(NaF)的条件下保存,稳定性最佳;四个影响因素中温度为影响血液中乙醇含量稳定性的主要因素。     

A novel fluorescence-based method in forensic science for the detection of blood in situ

Full DNA profiles can be generated from just a few cells; however these profiles can be contaminated from other cell types present at the crime scene. We report here on the development of an immunofluorescent technique to spatially locate human-specific blood in situ and also on the ability of this technique to detect individual leukocytes and the DNA contained within them. Four monoclonal mouse anti-human antibodies were evaluated; anti-glycophorin A to detect erythrocytes and anti-CD45, anti-myeloperoxidase (MPO) and anti-histone H1 to detect the nucleated leukocytes. Each antibody was labeled with either Alexa Fluor 488 or 568 for direct application to blood smears which allowed the simultaneous detection of erythrocytes and leukocytes. Furthermore, because histones are DNA binding proteins, the application of anti-histone H1 allowed the detection of DNA within a blood smear. Importantly it was found that full DNA profiles could be achieved after using this method with similar peak area ratios compared to untreated cells. The fluorescent antibodies were found to be human-specific with the exception of anti-histone H1 due to its conserved sequence. However, used in combination with anti-CD45 or anti-MPO the location of DNA from human-specific leukocytes could be detected. The technique was also tested on older blood stains and was still found to be sensitive and cell-specific after 4 months. Following the optimization of the methodology, the fluorescent antibodies were applied to short lengths of black cotton fibres covered with human blood spots. Although the background fluorescence from the cotton was found to be high, erythrocytes and even individual leukocytes could easily be detected, indicating that this technique could be used to detect extremely minute amounts of blood. Used in combination with laser capture microdissection (LCM), this method could be used to pick off individual leukocytes for LCN DNA techniques.     

Origin of blood ethanol in decomposed bodies

Problems related to blood contamination by other postmortem fluids in decomposed bodies (DB) make the interpretation of medicolegal blood alcohol levels (B EtOH) a very difficult task. So the aim of this paper is to show the utilization of vitreous humor (VH) as the biological fluid for an unequivocal determination of ethanol origin in DB for forensic purposes. Alcohol was determined in VH, blood (chest fluid-CF) and urine (Ur) collected from 27 DB in different states of putrefaction. A simple head-space gas-chromatographic method was used. In fifteen cases alcohol was found to be of endogenous production due to its absence in VH. In the twelve remainders, alcohol was detected in VH and CF in an atypical distribution. Examining the reliable scene and historical information together with the analytical data, ethanol origin in these cases was classified: endogenous production (3 cases), ingested (2 cases), both (2 cases), contaminated plus endogenous production (3 cases) and unable to determine (2 cases). According to the results obtained it was possible to conclude that alcohol analysis in VH is fundamental for determining the origin of ethanol detected in CF of DB.     

Brain acetaldehyde and ethanol: method of determination and diagnostic significance in ethanol poisoning

The content of ethanol and acetaldehyde in the limbic cortex and reticular formation of the brain was measured by gas-liquid chromatography in lethal ethanol poisoning. The content of acetaldehyde was significantly increased in the gyrus cinguli of the brain. Lethal poisonings occurred during any stage of ethanol intoxication. The data characterizing individual ethanol tolerance were obtained, which can be used for differential diagnosis of ethanol poisoning in practical forensic medicine.     

Micromethod for the detection of erythrocyte antigens in blood stains

A micromethod was developed to allow the analysis of blood stains of minor size by the absorption elution technique. The individual absorption, washing, and elution steps were carried out in Beckman tubes containing 5 microliter antiserum. The final agglutination reaction was read through the inverted microscope in microtest plates regularly used for HLA typing. For this final reaction, 2-4 microliter eluate was incubated with 2,000 red blood cells suspended in 1 microliter saline and supplement. For the purpose of standardization, the intensity of agglutination in the microtest plate had to be defined. In comparison to the standard method (tube test and centrifugation), the proposed method proved to be slightly more sensitive with regard to the Rhesus and slightly less sensitive with regard to the AB0 system. With the proposed method very small traces could be successfully analyzed. Thus, two cotton threads 1 mm in length were sufficient for testing antigens A and B, and two cotton threads 2.5 mm in length were enough to detect an Rh antigen.     

Blood typing of blood stains on sweat stains by the iso-agglutinins detection method

When a blood typing is made for mixed stains of sweat and blood, erroneous results may be obtained. The reason is that the blood group substance in the sweat is detected at the same time as that in the blood. In this paper the typing of the blood stain on the sweat stain is carried out by the detection of isoagglutinins which may give additional information to the forensic serologist.     

HS-GC内标曲线法测定血中乙醇含量的不确定度评估

目的建立自动顶空-气相色谱(HS-GC)内标曲线法测定血中乙醇含量的不确定评估方法。方法从分析测定程序着手,依据不确定度评定的指导性文件,分析不确定度来源,量化不确定度分量,计算检测结果的合成标准不确定度和扩展不确定度。结果各相对不确定度来自于检材重复性检测为3.4%,乙醇标准溶液为0.71%,检材为0.61%,叔丁醇内标溶液为0.41%,标准曲线为1.1%,气相色谱仪为1.3%,血液中乙醇的相对扩展不确定度为3.9%。结论血液中乙醇含量的不确定度主要来源于检材重复性检测、气相色谱仪、乙醇标准曲线。     

Physiological variations in blood ethanol measurements during the post-absorptive state

Specimens of arterial plasma and venous whole blood were obtained at 3-10 min intervals during the post-peak phase of ethanol metabolism in healthy volunteers. The concentrations of ethanol in blood and plasma were determined by headspace gas chromatography. This method had a standard deviation of 0.28 mg/dl for whole blood and 0.26 mg/dl for plasma and the coefficients of variation were 0.43% and 0.79% respectively. The physiological variation from time-to-time, expressed as the residual standard deviation after fitting the ethanol concentration-time regression relationships, ranged from 0.43-3.7 mg/dl (0.65-16%). The time-to-time variations in concentrations of ethanol were maximum when there were problems in getting an unimpeded flow of blood through the indwelling catheters. The results do not support the existence of sporadic fluctuations or spiking in the blood alcohol concentration-time profile during the post-absorptive state. Instead, this study underscores the need to control carefully the method of sampling blood and in this way keep pre-analytical sources of variation to a minimum.     

The distribution of ethanol in postmortem blood specimens.

Ethanol was determined by gas chromatography in a variety of tissues and body fluids secured at autopsy in 61 cases. The specimens tested included right and left heart blood, femoral blood, pericardial fluid, cerebrospinal fluid, vitreous humor, urine, stomach contents, and brain. Statistical analysis of the cases revealed no significant differences among the various blood sites tested. However, the variations in blood ethanol concentrations among the various sampling sites within each case were as follows: 40 cases showed differences of less than 25%; 16 cases revealed variability between 25% and 50%, 4 cases had differences exceeding 50%. In one case, satisfactory blood analyses could not be accomplished. The larger variances occurred especially in those instances in which stomach alcohol concentration was 0.50% or greater. In one case, the variability amongst the different blood sites exceeded 400% (femoral blood--0.043%, right atrium--0.070%, root of aorta--0.156%); the brain was 0.050%, and the stomach contents was 1.2%. For all 61 cases, variances in blood alcohol content among the different sampling sites in a single cadaver ranged from 1.8 to 428%.     

Current strategic approaches for the detection of blood doping practices

Aerobic sport performance may be strongly influenced by the number of red blood cells available for transport and delivery of oxygen from lungs to muscles. Often, athletes search for an acute increase in red blood cells by means of blood transfusions. This paper reviews the possibilities for detecting such prohibited practice. Flow cytometry methods are able to detect a double population of red blood cell membrane surface antigens, thus revealing an allogeneic transfusion. Other ingenious approaches for total hemoglobin mass measurements or to test for the metabolites of blood bag plasticizers in urine are new trends for facing the detection of autologous transfusions. Steady increase of red blood cell number may be obtained also by erythropoietic stimulant agents such as erythropoietin, analogs and mimetics. The challenge of detecting those substances has stimulated the development of indirect markers of altered erythropoiesis, leading to the consequent development of the hematological blood passport approach, which is gaining legal acceptance.     

Improved method for the detection of TATP after explosion

TATP in post explosion exhibits was reported earlier to be best recovered from vapor phase. A typical procedure includes its adsorption on Amberlite XAD-7, elution with acetonitrile and analysis by GC/MS. In this work, improved recovery of TATP from the vapor phase was achieved by SPME using PDMS/DVB fiber and immediate sampling to GC/MS. The recovery of TATP by SPME was compared with headspace and with adsorption on Amberlite XAD-7 by spiking onto filter paper put in a 100 mL beaker. The limit of detection of TATP was 6.4 ng in these conditions, few orders magnitude more than in the other tested methods. Recovery of TATP in the presence of various solvents was also studied. Acetone, water, and mixtures of water:alcohols (1:1) were found to reduce the recovery of TATP. Using SPME, TATP has been identified in dozens of post-explosion cases.