一阶导数光谱法直接测定血中一氧化碳成分的研究

本文采用一阶导数光谱法直接测定血中一氧化碳含量,线性范围10％～90％。该法无须添加还原剂,操作简便、快速,且稳定性好,不仅适于一氧化碳中毒死亡尸体的检验,而且适于一氧化碳中毒较轻患者的检验。     

甲醛对一氧化碳中毒血检验干扰的研究

目的探讨甲醛对一氧化碳中毒血的检测是否产生干扰,提高一氧化碳中毒鉴定的可靠性。方法采用常用的血中一氧化碳或碳氧血红蛋白饱和度的检测方法对未加甲醛和加甲醛的血样分别进行实验研究。结果甲醛对加热法、氢氧化钠法、氯化钯法、分光光度法等检测方法均可产生不同程度的干扰。结论经福尔马林灌注或固定的检材不宜用于一氧化碳中毒血检测,否则可能导致错误的鉴定结论。     

血尿肝中巴比妥类药物硅藻土提取紫外差示导数光谱测定

目的建立一种操作简单、快速、去除杂质能力强、提取率高的硅藻土提取血、尿、肝中巴比妥类药物的方法。方法尿液不经稀释、血液经稀释过硅藻土柱,乙醚洗脱;肝匀浆用6%高氯酸沉淀蛋白,上清液过硅藻土柱,二氯甲烷洗脱药物。洗脱液挥干用0.45mol/L氢氧化钠溶液溶解,将溶液等分为两份,分别用等量的0.45mol/L氢氧化钠溶液和0.6mol/L硼酸-氯化钾溶液稀释,得到pH10和pH14水溶液,以pH10溶液为参比,测定pH14溶液的紫外二阶导数光谱进行药物检测。结果该法提取率血98.6%~100.3%,尿99.7%~103.2%,肝78.4%~102%,检出限均低于1.0μg/g(m l),变异系数小于2.9%,线性范围0.5~5.0μg/m l。结论硅藻土提取血、尿、肝中巴比妥类药物、紫外差示导数光谱法进行测定,适合作为法医毒物常规检验方法。     

四阶导数光谱法测定血液碳氧血红蛋白饱和度

本文报导了两种利用四阶导数光谱测定血液 HbCO%的方法。一种需从检血制备一氧化碳饱和血溶液,操作稍繁但准确可靠,受仪器稳定性影响较小。另一种不需制备一氧化碳饱和血溶液,是一种操作简单,快速亦准确可靠的方法,但由于计算中使用预先测定的常数,测定的准确性乃受仪器稳定性波动的影响。     

自动化工作站与Chelex-100法对血样本检验效果比较

目的比较自动化工作站与Chelex-100法对不同浓度血液样本的检验效果。方法取新鲜血液,按倍量稀释法制成2~1 024倍10种不同浓度的血样本,分别采用自动化工作站和Chelex-100法提取模板DNA,应用Identifiler Plus试剂盒进行扩增及毛细管电泳检测,并对电泳结果进行比较。结果稀释2~32倍的血样本,采用两种方法的检验成功率均可达到100%;稀释64倍的样本,Chelex-100法成功率为41.3%、完整分型数为78.3%,自动化工作站成功率为13.0%、完整分型数为43.5%,两者比较,差异均有统计学意义(P0.01);稀释128倍的样本,成功率在Chelex-100法(8.7%)和自动化工作站(2.4%)之间比较,其差异无统计学意义(P0.05);稀释256倍以上,两种方法均无法获得分型结果。结论自动化工作站适用于一定浓度血样的检验,而对于微量血样,Chelex-100法的效果可能更优。     

微量体表脱落上皮细胞的DNA检验

目的建立微量体表脱落上皮细胞的DNA检验方法. 方法采用Chelex-100法提取DNA,以Microcon-100纯化柱纯化浓缩DNA,用Profiler Plus 试剂盒PCR扩增后用310基因分析仪检测. 结果 10种常见粘附有体表脱落上皮细胞的样本100个,其中7种70个样本成功检测到10个STR位点的分型,检出率为100%,其余3 种样本检出率分别为50%、20%、10%,将该方法应用于2例实际检案,取得满意效果. 结论所建立方法稳定可靠,易于操作,适用于多种检材,为微量体表脱落上皮细胞的DNA检验提供了确实可行的检验方法.     

慢性乙醇中毒小鼠脑神经细胞IP3R1表达的变化

目的研究慢性乙醇中毒引起小鼠脑神经细胞Ⅰ型1,4,5-三磷酸肌醇受体（IP3R1）表达的变化。方法 40只小鼠随机分为90d、180d组,各组再分为正常对照组、10%、20%、30%乙醇组,每组5只,乙醇组给予相应浓度乙醇饮用至相应时间;取各组小鼠脑组织,分别采用免疫组化和Western blot方法检测脑皮质神经细胞IP3R1表达的变化;SPSS 13.0软件对实验数据进行单因素方差分析。结果正常IP3R1免疫组化染色物分布于神经细胞胞浆内。90d组随乙醇浓度的增加,IP3R1免疫组化阳性细胞数和阳性细胞率逐步增加,组间比较,差异显著（P〈0.05）;180d组中10%、20%乙醇组阳性细胞数和阳性细胞率逐步增加,组间比较,差异显著（P〈0.05）,而30%乙醇组阳性细胞数和阳性细胞率反而减少,且低于90d组的相同浓度组（P〈0.05）。Western blot与免疫组化检测结果基本一致。结论慢性乙醇中毒可引起小鼠大脑皮质神经细胞IP3R1表达增加,而高浓度（30%）、长时间（180d）乙醇使IP3R1表达降低,可能与神经细胞变性、坏死、数目减少有关。     

液基细胞学技术应用于血样羊水栓塞的诊断

目的：建立基于液基细胞学技术的血样羊水栓塞的诊断方法,并对其有效性进行研究。方法收集羊水栓塞者血液样本,分别用两种直接涂片法（上清涂片法、沉淀涂片法）和两种液基细胞涂片法（自动涂片法、人工涂片法）对血液样本中的羊水成分进行检测,对4种方法的羊水成分检出率进行比较。结果建立的两种液基细胞涂片法的羊水成分检出率（分别为84.6%、92.3%）明显高于直接涂片法（分别为53.8%、61.5%）。结论液基细胞涂片法可以提高羊水栓塞的检出率。     

顶空气质联用法测定腐败血、肝检材中CO

目的研究CO中毒腐败血、肝组织检材中CO的HS/GC/MS检测。方法用HS/GC/MS法分析碳氧血红蛋白(COHb)血的线性范围。配制10%、30%、50%、70%浓度COHb血样,分别在室温、冷藏、冷冻条件下保存,分别在当日、第4、14、45d进行测定,比较实验结果。腐败肝组织由雄性健康家兔通CO气体致死,当天解剖,家兔肝常温隔绝空气保存并放35d至腐败,期间进行不定期顶空测定分析。结果制备的COHb血在0-100%之间有良好的线性关系Y=2.4X+2.2(r=0.9995)。以此方法测定家兔CO中毒致死的COHb新鲜血的浓度和4℃下放置45dCOHb腐败血,结果表明温度对血样中COHb%的测定影响最大。采用HS/GC/MS法检测,每次只需0.25ml血样或1g肝脏,分析一次时间只需3min,均可检测出新鲜检材和常温放置45d的腐败肝组织检材CO的含量。结论HS/GC/MS法能检出CO中毒的腐败生物检材中CO。     

气相色谱法快速分析人血、尿中地芬尼多

目的建立人血、尿中地芬尼多的气相色谱快速分析方法。方法用氯仿提取血、尿中的地芬尼多,采用气相色谱法进行定性、定量分析。以正常人血浆、尿液为空白样本,分别添加标准地芬尼多及SKF525（内标物）对方法进行考察和优化,并对1例大剂量误服地芬尼多中毒者体液浓度进行快速测定和检测。结果所建方法分析血、尿中地芬尼多的线性范围均为5.0～200.0μg/mL;最低检测限均为1.0μg/mL（S/N≥3）;日内、日间精密度RSD≤5.6%（n=5）;回收率：血≤（106.23±2.05）%;尿≤（104.19±5.51）%。结论该分析方法操作便捷、实用,适用于地芬尼多的临床血药浓度快速监测和法医毒物鉴定。     

Sample pre-treatment for CO-oximetric determination of carboxyhaemoglobin in putrefied blood and cavity fluid

Blood or blood-containing cavity fluid samples recovered from decomposed bodies in suspected carbon monoxide (CO) poisoning cases often have low total haemoglobin (tHb) levels or contain significant amounts of oily droplets, methaemoglobin (MetHb) and sulphaemoglobin (SHb), all conditions which render the validity of the measurements of carboxyhaemoglobin (HbCO) by commercially available CO-oximeters doubtful. Two sample pre-treatment methods, namely, centrifugal evaporation and addition of a commercially available standard haemoglobin solution containing a known level of HbCO, were developed to render such putrefied samples amenable to analysis by CO-oximeters. The methods developed were validated with samples of various degrees of putrefaction and various HbCO levels. The relative uncertainty of the determined HbCO levels as compared to their corresponding reference levels for the centrifugal evaporation method was approximately 5% and that for the standard addition method was <20%.     

Postmortem formation of carbon monoxide

Since carbon monoxide (CO) production after death was suggested in a drowned body, CO and carboxyhemoglobin (HbCO) levels in blood and body cavity fluids of cadavers which were not exposed to fire and CO have been analyzed. CO released from the tissues was determined by gas chromatography and gas chromatography-mass spectrometry, and the total concentration of hemoglobin (Hb) was measured as cyanmethemoglobin (CNmHb). The HbCO level was calculated by the ratio of CO content and CO-binding capacity. CO levels (ml/100 g at STP) of the seven cases in which blood and body cavity fluids could be collected ranged from 0.13 to 0.87 in blood and 0.02 to 0.80 in body cavity fluids. HbCO levels in blood and body cavity fluids were from 0.3 to 6.0% and from 2.3 to 44.1%, respectively. In a typical case showing postmortem formation of CO, the CO levels in body cavity fluids were higher than that in blood. It is suggested that CO in a putrefied body is due to CO in blood prior to death and the CO formed by the decomposition of Hb, myoglobin and other substances during putrefaction. The significance of HbCO levels in body cavity fluids of cases with marked postmortem decomposition seems difficult to interpret without the value of HbCO in blood.     

A New Approach for the Carbon Monoxide (CO) Exposure Diagnosis: Measurement of Total CO in Human Blood Versus Carboxyhemoglobin (HbCO)

The aim of the study is to present the application of a headspace–gas chromatography–mass spectrometry (HS‐GC‐MS) method for the determination of the carbon monoxide (CO) blood concentration and to compare it with carboxyhemoglobin (HbCO) saturation. In postmortem cases, the HbCO measured by spectrophotometry frequently leads to inaccurate results due to inadequate samples or analyses. The true role of CO intoxication in the death of a person could be misclassified. The estimation of HbCO from HS‐GC‐MS CO measurements provides helpful information by determining the total CO levels (CO linked to hemoglobin (HbCO) and CO dissociated from hemoglobin). The CO concentrations were converted in HbCO saturation levels to define cutoff blood CO values. CO limits were defined as less than 1 μmol/mL for living persons, less than 1.5 μmol/mL for dead persons without CO exposure, and greater than 3 μmol/mL for dead persons with clear CO poisoning.     

Comparison of Different Analytical Methods for the Determination of Carbon Monoxide in Postmortem Blood

The determination of carbon monoxide (CO) and carboxyhemoglobin (COHb) is of utmost importance in forensic toxicology to determine the cause of death in cases of CO poisoning, fire, and explosions. To this end, reliable and updated analytical methods are required. In this paper, four different methods for the determination of carbon monoxide in postmortem blood samples were compared: (i) the spectrophotometric determination of COHb applying the method proposed by Rodkey and modified by Beutler–West, (ii) the spectrophotometric determination of CO using a micro-diffusion-based method, (iii) the determination of CO by gas chromatography coupled to a TCD detector, and (iv) the determination of COHb by blood gas analysis. Three postmortem blood samples were analyzed with all methods, and the results were comparable. The applied methodologies showed different features depending on the sensitivity, sample preparation, and volume. The HS-GC/TCD method in our hand was the most appropriate, on postmortem samples, and versatile to apply. Unfortunately, only a limited number of postmortem blood samples were available for this study due to the rarity of that kind of intoxication in our jurisdiction.     

Production of carbon monoxide in cadavers

Carbon monoxide (CO), total hemoglobin (Hb) and carboxyhemoglobin (HbCO) in the blood and reddish discolored body cavity fluids of cadavers which had not been exposed to fire and CO were analyzed. In 13 cadavers found on land, the maximum saturation of HbCO in the blood was 3.6%, and was 10.1% in the body cavity fluids. There was only one case in which the HbCO saturations in the body cavity fluids were more than 10%. In seven drowned bodies found in fresh water, the highest HbCO saturation in the blood was 6.1%, and was 44.1% in the body cavity fluids. There were three cases in which the HbCO saturations in the body cavity fluids were more than 10%. In 12 drowned bodies found in sea water, the HbCO saturations in the blood were not more than 6.2%, and the maximum saturation of HbCO in the body cavity fluids was 83.7%. There were eight cases in which the HbCO saturations in the body cavity fluids were more than 10%. The results seem to indicate that the interpretation of HbCO saturation in the blood would not be affected significantly by the postmortem formation of CO, and that body cavity fluids should not be used for CO determination.     

血液样品中防腐剂对碳氧血红蛋白稳定性的影响

目的研究临床上常用8种试剂对血液样品中碳氧血红蛋白(HbCO)稳定性的影响。方法将血液样品分为高、低两个HbCO浓度组,选用临床常用的甲醛、氟化钠、乙二胺四乙酸二钠、亚硝酸钠、草酸钾、肝素钠、柠檬酸钾及氟化钠与草酸钾混合物(1:3)8种试剂,按常用浓度分别添加到血样品中,并于添加后0h、2h、8h、24h、3d、7d用紫外可见光分光光度法检测其中HbCO饱和度,用统计学方法进行结果分析。结果本实验选用的8种试剂只有甲醛和亚硝酸钠对HbCO的稳定性影响较为显著,而其余6种对HbCO稳定性的影响无统计学意义。结论在检验疑似CO中毒并经甲醛或亚硝酸钠防腐的检材时应慎重,以免导致错误的鉴定结论。     

Experimental study on postmortem formation of carbon monoxide

Rats were drowned and kept immersed for 1 month in either boiled city water, or boiled or unboiled fresh water collected from a river. A small amount of carbon monoxide (CO) formed after death and a low carboxyhemoglobin (HbCO) saturation was found in blood and thoracic cavity fluid of the animals immersed in boiled city water and in boiled fresh water. A considerable amount of CO and a high HbCO saturation was observed in blood and thoracic cavity fluid in two out of three rats immersed in unboiled fresh water at 4-6 degrees C, and in one out of three at 6-16 degrees C. It is suggested that microorganisms in the water, in which the rats were drowned and kept immersed, and low temperatures of around 5 degrees C during storage, played an important role in the postmortem formation of carbon monoxide.     

Determination of carbon monoxide in blood by gas chromatography using a thermal conductivity detector

A method is described for the gas chromatographic quantitation of carbon monoxide by means of thermal conductivity detection. Carbon monoxide is released from blood samples as small as 0.02 mL using a unique extraction chamber. The method was compared to a standard gas chromatographic and spectrophotometric method of carbon monoxide quantitation. It was comparable to the former with all samples evaluated and apparently more reliable than the latter with decomposed samples.     

A simple spectrophotometric method for the determination of carboxyhemoglobin in blood

The spectrophotometric method for the determination of carboxyhemoglobin (HbCO) in blood reported by Fretwurst and Meinecke was modified so as to give the same values of percentage HbCO (HbCO%) as those determined by the oxygen electrode method. Values of HbCO% of nine practical samples determined by both the oxygen electrode method and the present method were nearly identical regardless of the presence of methemoglobin (Met-Hb) in blood. The present method is suitable for forensic practice.     

Stability of blood carbon monoxide and hemoglobins during heating

The effects of heating on hemoglobin (Hb) and carbon monoxide (CO) levels in human blood were investigated by in vitro experiments. Head-space gas chromatography (HS-GC) using a molecular sieve 5A stationary phase and thermal conductivity detection was adopted for the measurement of CO gas, and spectrophotometric methods were used for the measurement of various Hb forms, protein and heme contents. Deteriorated absorbance spectra were observed for heat-treated blood samples, and double wavelength spectrophotometry was proven to give wrong percent saturation of carboxyhemoglobin content (% CO-Hb). The blood sample taken from one fatal fire casualty gave significantly higher % CO-Hb measured spectrophotometrically, compared to that by HS-GC. Control blood or purified Hb solution, which was saturated with CO in designated extent, was heated in a sealed vial. Under the incubation below 54 degrees C, all Hb forms were stable, except for oxyhemoglobin (Hb-O(2)), which was partially oxidized to met-hemoglobin (Met-Hb). In contrast, under the incubation at 65 degrees C, Met-Hb was denatured completely to be insoluble, and Hb-O(2) was partially denatured via Met-Hb formation. CO-Hb was resistant against heating. The difference of heat susceptibility and precipitability among Hb forms resulted in artificial increase of % CO-Hb. During heating, spontaneous CO was produced from blood.