保存温度对血液酒精含量检测的影响

目的为筛选最佳的保存温度,准确检测酒驾血液酒精含量,为交管部门客观判断酒驾行为提供技术支撑。方法本研究选取EDTA-2真空抗凝采血管,采取酒后人体静脉鲜血后,分别在-20℃、4℃~8℃、25℃常温、35℃~42℃高温等4个温度条件下保存,GC法按0、3d、7d、14d、21d及28d后检测血液酒精含量,并对测试结果进行比较统计分析。结果在35℃~42℃和25℃温度下存储的血液酒精含量在0~3d内基本稳定,3d后显著下降(P0.05);4℃~8℃温度下存储的血液酒精含量在0~14 d内基本稳定,14d后显著下降(P0.05);-20℃温度保存条件下血液酒精含量测试28d统计结果间无显著差异。结论建议血样采集后低温保存,-20℃温度为血样的最佳保存温度。     

A method for the determination of MN antigens in dried blood

MN phenotypes of experimentally prepared dried blood samples, some as old as six months, were obtained using sodium dodecyl sulfate polyacrylamide gels, electroblotting, and monoclonal antibodies.     

Immunocytochemical determination of ABH and MN antigens on dried blood traces in the nanoliter range.

The absorption-elution test and the mixed cell agglutination reaction are both ultimately based on the ability of indicator cells to agglutinate. This agglutination reaction requires a relatively large amount of antigenic epitopes, and, in addition, a relatively high volume of blood traces. The immunocytochemical demonstration of epitopes requires a lower volume, which, however must be fixed for investigation, thus possibly causing damage to the epitopes and thereby preventing detection. An immunocytochemical method is presented which permits ABH and MN antigen determination on dried blood traces of nanoliter quantities without special fixation. This method is based on immunocytochemical demonstration of antigens directly on the cell membrane in combination with the use of a coated glass slide that ensures maximum economy of epitopes.     

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.     

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.     

On the detection of minor Rhesus antigens in blood stains

A new method for the detection of minor C, Cw, c, E, e-antigens of the Rhesus system in blood stains has been developed based on the absorption-elution technique with the use of anti-C, anti-Cw, anti-c, anti-E, and anti-e sera and standard erythrocyte preparations preliminarily treated with highly active proteases (protease C or papain). This method makes it possible to determine complete Rhesus phenotype in blood stains and substantially extend the possibilities of their differentiation on material objects (evidence) for the purpose of forensic-biological examination.     

Specific research of antigens of the ABO system in samples of decayed blood

Liquid blood and blood stains were examined after storage under different conditions and temperature regimes ranging from 18 to 26 degrees C. Blood stains were washed by distilled water or heated to 120 degrees C for as long as 4 hours. Then, blood groups were determined by absorption-elution.     

Long-term storage of blood samples as whole blood at extremely low temperatures for methemoglobin determination

Changes in methemoglobin (Met-Hb) concentrations during storage of whole blood and a hemolysate at refrigerated or various freezing temperatures were examined using experimentally prepared blood samples. When whole blood was stored at 3 degrees C, rapid reduction of Met-Hb was observed in the nitrite-treated blood whereas neither reduction nor formation of Met-Hb was observed in the untreated and heated blood within 7 days. When hemolysate was stored at 3 degrees C, Met-Hb concentrations were stable within a few days regardless of the initial values. However, slight autoxidation was observed 7 days after storage in the untreated and heated blood. When whole blood was stored at various freezing temperatures, Met-Hb concentrations were practically stable until at least 30 days at -80 degrees C or -196 degrees C regardless of the initial values, although considerable autoxidation was observed at -30 degrees C especially in the blood containing small amounts of Met-Hb. Based on the results obtained, a new method was devised for long-term storage of whole blood at extremely low temperatures for Met-Hb determinations.     

Simultaneous screening and detection of drugs in small blood samples and bloodstains

A gas chromatography-mass spectrometry (GC-MS) method is described for the screening and detection of morphine, codeine, cocaine, benzoylecgonine, methylecgonine, cocaethylene, delta-9-tetrahydrocannabinol (THC), 11-nor-9-carboxy-THC (THC-COOH), 11-hydroxy-THC (11-OH-THC), amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymetamphetamine (MDMA) and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) in small blood samples and bloodstains using solid phase SPE columns and a pipetting robot (Gilson Aspec XL). The detection limits are in the order of 1.62-4.10 ng/50 microl spot (amphetamines), 0.15-0.82 ng/50 microl spot (cannabinoids), 1.67-4.70 ng/50 microl spot (cocaine and derivatives) and 4.53-4.91 ng/50 microl spot (opiates) and the correlation factors are between 0.9957 and 0.9999. The method has proven useful in forensic cases with only small sample volumes or bloodstains.     

Stability of diazepam in blood samples at different storage conditions and in the presence of alcohol

Diazepam is one of the mostly used benzodiazepines and it is frequently analyzed in different biological samples, especially blood samples. The diazepam stability in the sample matrices is an important factor regarding reliable data obtaining. The storage is the main factor determining the stability of diazepam in blood samples and it is the object of the study presented. Remaining diazepam amount in spiked whole blood and plasma samples were tested at different storage temperatures, in the absence or presence of sodium fluoride as stabilizer as well as the influence of ethanol on diazepam stability was evaluated. The results of the study indicated that the temperature is the main storage factor affecting diazepam stability. In the fluoride stabilized blood samples the amount of diazepam decreases up to 85% of initial level when stored at -20° C for the period of testing (12 weeks). The presence of low (0.5 g/L) or high (3g/L) ethanol concentrations influences the stability of diazepam at -20 °C. In whole blood samples, the combination of sodium fluoride and ethanol decreases additionally (15-25%) the concentration of the analyte. Freeze-thaw experiments of whole blood samples show about 5-9% decrease in diazepam concentration after the first cycle. The freeze-thaw experiments on plasma samples, containing ethanol and/or fluoride show insignificant decreases of analyte concentration. Further experiments on benzodiazepines stability at different storage conditions or in combination of different factors should be undertaken in forensic toxicology to ensure the data quality, their reliability and reproducibility.     

唾液与血液中海洛因代谢物的检测时限

目的比较唾液与血液中海洛因代谢物吗啡和O6-单乙酰吗啡的检测时限,为实际检测时选择样本采集时间提供依据。方法将实验大耳白兔分为3组,通过耳缘静脉注射浓度为2.6mg/m L的海洛因溶液,给药量分别为0.5m L,1m L,2m L。给药后于0.5~48h期间采集静脉血液;给药后于3~48h采唾液。采用超高效液相色谱串联质谱法,分别检测各时间点,各组不同种类样本中的海洛因代谢产物吗啡和O6-单乙酰吗啡。结果 1O6-单乙酰吗啡在血液中的检测时间为6h(给药量2.6mg和5.2mg)和1.5 h(给药量1.3mg),9h后已检测不到;而唾液样本在24h时虽下降明显,但3种给药剂量下均仍可检出,至48h时均不能检出;2吗啡在血液和唾液中不能检出的时间分别为24h和48h。结论血液中O6-单乙酰吗啡和吗啡的检测时限随用药量的增加而延长,而在唾液的检测时限均明显比在血液中更长久。该结果可作为实际检测时根据样本种类和选择采集时间的依据。     

In vitro production of GHB in blood and serum samples under various storage conditions

The in vitro production of GHB was observed in freshly collected, untreated whole blood samples using glass BD-Vacutainers and polypropylene S-monovettes. GHB concentrations were determined daily over a period of one week and after 3, 6 and 9 weeks again. Furthermore, the GHB concentration in 40 untreated random whole blood samples stored at 4°C for a longer period of time (10 samples 12 month, 10 samples 24 month and 20 samples 36 month) was also determined. For comparison, the in vitro production of GHB in freshly collected and prepared serum samples was observed. GHB serum concentrations were determined three times over a period of one week and once again after six weeks. Sample preparation was performed by means of methanolic extraction following the precipitation of whole blood and serum samples. A methanolic standard calibration was done in a low range of 0.005-0.1 μg/mL (LOD: 0.004, LLOQ: 0.013). For quantification a spiked blood bank serum with a determined GHB concentration of 0.09 μg/mL was used. Corrected calibrations in the range of 0.09-5.09 μg/mL were used (LOD: 0.08 μg/mL, LLOQ: 0.30 μg/mL), recovery: 91.3% (high level: 4.09 μg/mL) 50.5% (low level: 0.19 μg/mL). RESULTS: Relevant elevation of GHB was observed in all whole blood samples stored in liquid form (4°C or room temperature). In two of the 40 whole blood samples stored over a longer period of time at 4°C, GHB concentrations in the range of 13 μg/mL were even determined. These findings constitute grounds for caution. Even a GHB cut-off level of 5 μg/mL cannot be considered as "absolutely positive" proof of a case of exogenous administration, at least in untreated liquid blood samples in long time storage. However, no significant elevations of GHB were otherwise observed in any of the serum samples independently of storage temperature nor in the whole blood samples that were frozen for storage. CONCLUSIONS: The results suggest that the cut-off for exogenous GHB of 5 μg/mL could be lowered significantly, with the consequence of winning valuable time for the potential victim, but only if serum is collected for GHB determination or if the whole blood sample is frozen immediately after collection and the procedure well documented.     

Determination of MN blood group from blood stains by electrophoresis and immunoblotting

The determination of blood groups from blood stains is extremely important in medicolegal practice, but there is the possibility of an error in the determination of MN phenotypes by the absorption-elution test. We investigated a new method applying electrophoresis and immunoblotting. As a consequence of various experiments, the most appropriate pretreatment of blood stains was as follows. Blood stains were immersed in physiological saline for 0.5 to 1 h and centrifuged. The supernatant was discarded. The sediment was dissolved in sample buffer (TRIS-buffered physiological saline containing 2% sodium dodecyl sulfate) and followed by thermodegradation. It was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After transfer to a nitrocellulose membrane by Western blotting, MN phenotypes could be determined accurately from blood stains by an enzyme immunoassay (EIA) using commercially available polyclonal anti-M and anti-N sera. For blood stains more than 1 month old it was not easy to determine the MN phenotypes.     

Stable isotope analysis of human hair and nail samples: the effects of storage on samples

When submitting samples for analysis, maintaining sample integrity is essential. Appropriate packaging must be used to prevent damage, contamination or loss of sample. This is particularly important for stable isotope analysis by isotope ratio mass spectrometry as this technique is capable of detecting subtle differences in isotopic composition with great precision. In a novel study, scalp hair and fingernail samples were placed in five different types of packaging, routinely used in forensic laboratories and stored for 6 weeks and 6 months. Samples were subsequently cleaned and submitted for (13)C/(12)C, (15)N/(14)N, (2)H/(1)H and (18)O/(16)O analysis. Results from (13)C analysis indicate that type of packaging can cause slight changes in (13)C abundance over time. Differences were noted in the (15)N isotope signatures of both hair and nail samples after 6-week storage, but not after 6 months. This apparent discrepancy could be a result of the packaging not being properly sealed in the 6 weeks study. Fewer differences were noted when analyzing samples for (2)H and (18)O abundance.