Recommended Practices for Macerating Human Thyroid Cartilage

Visible abnormalities on the thyroid cartilage may be indicative of perimortem trauma including fractures or sharp force trauma. During autopsy, the thyroid cartilage must be freed of surrounding soft tissue before these abnormalities can be clearly observed. Several processing methods were first experimentally tested on pig ears to narrow down which might work best to process human thyroid cartilage. Several methods in the categories of hot water maceration, chemical/enzyme maceration, and dermestid beetles were then tested on a sample of 37 human thyroid cartilages. An adapted 22-point scoring system was used to score the efficacy of each processing method. Chemical maceration using bleach was found to be the best method for removing surrounding tissue from thyroid cartilage, with Arm & Hammer^TM powdered laundry detergent as a close second. These methods are inexpensive, quick, and easy to implement, making them a simple addition to busy medical examiner’s offices or forensic anthropology laboratories.     

磁力搅拌超声乳化微萃取用于血清中有机磷农药的检测

目的 将涡旋搅拌与超声乳化技术相结合,建立了磁力搅拌结合超声辅助乳化微萃取新方法 .方法 使用一套自制前处理装置,将所建立的方法 应用于血清中的7种有机磷农药的前处理过程中,并联用GC/MS对其进行了检测.考查了影响方法 萃取效率的若干因素,确定了最优条件:90μL甲苯作为萃取剂;磁力搅拌转速为200 rpm;超声萃取...     

液相微萃取-气相色谱法检测尿液中三唑仑药物

目的建立液相微萃取与气相色谱联用技术快速分析尿液中三唑仑的方法。方法本方法考察了萃取溶剂、体积、萃取时间、萃取振荡速度等条件对液相微萃取的影响,优化的实验条件为:萃取溶剂为0.5ml三氯甲烷,萃取时间25min,振荡速度200r/min。结果采用本文所建立的方法,三唑仑回收率为88.0%~90.5%。结论实验结果表明该方法简便、快速、灵敏、消耗有机溶剂少,是尿液中三唑仑检测的一种有效方法。     

A Procedure for Calculating the Vertical Space Height of the Sacrum When Determining Skeletal Height for Use in the Anatomical Method of Adult Stature Estimation

Estimating stature from skeletonized remains is one of the essential parameters in the development of a biological profile. A new procedure for determining skeletal height (SKH) incorporating the vertical space height (VSH) from the anterior margin of the sacral promontory to the superior margins of the acetabulae for use in the anatomical method of stature estimation is introduced. Regression equations for stature estimation were generated from measurements of 38 American males of European ancestry from the William M. Bass Donated Skeletal Collection. The modification to the procedure results in a SKH that is highly correlated with stature (r = 0.925–0.948). Stature estimates have low standard errors of the estimate ranging from 21.79 to 25.95 mm, biases from to 0.50 to 0.94 mm, and accuracy rates from 17.71 mm to 19.45 mm. The procedure for determining the VSH, which replaces “S1 height” in traditional anatomical method models, is a key improvement to the method.     

A rapid technique for preparation of human fetal and adult skeletal material.

The techniques described above have been found to provide a rapid method of human skeletal preparation, with advantages of speed and applicability to fresh, fixed, or partially decomposed skeletal materials. While other techniques which can be used include the traditional use of Dermestidae beetle colonies, a five-step anatomical procedure, and other combinations of chemical solvents, the antiformin technique appears to have advantages useful to those involved in forensic medicine.     

Development of a rapid, 96-well alkaline based differential DNA extraction method for sexual assault evidence

We present a rapid alkaline lysis procedure for the extraction of DNA from sexual assault evidence that generates purified sperm fraction extracts that yield STR typing results similar to those obtained from the traditional organic/dithiothreitol differential extraction. Specifically, a sodium hydroxide based differential extraction method has been developed in a single-tube format and further optimized in a 96-well format. The method yields purified extracts from a small sample set (∼2-6 swabs) in approximately 2 h and from a larger sample set (up to 96 swabs) in approximately 4 h. While conventional differential extraction methods require vigorous sample manipulation to remove the spermatozoa from the substrate, the method described here exploits the propensity of sperm to adhere to a substrate and does not require any manipulation of the substrate after it is sampled. For swabs, sample handling is minimized by employing a process where the tip of the swab, including the shaft, is transferred to the appropriate vessel eliminating the need for potentially hazardous scalpels to separate the swab material from the shaft. The absence of multiple handling steps allows the process to be semi-automated, however the procedure as described here does not require use of a robotic system. This method may provide forensic laboratories a cost-effective tool for the eradication of backlogs of sexual assault evidence, and more timely service to their client agencies. In addition, we have demonstrated that a modification of the procedure can be used to retrieve residual sperm-cell DNA from previously extracted swabs.     

Simultaneous electrophoretic determination of phosphoglucomutase subtypes, adenosine deaminase, erythrocyte acid phosphatase, and adenylate kinase enzyme phenotypes

Many of the conventional agarose phosphoglucomutase (PGM) subtyping systems presently in use fail to provide a good separation between the 1 + and 2- bands as well as the 2+ band and the more anodic moving bands. Use of a 1-mm-thick gel composed of 1% ISO GEL (FMC Corp.) and phosphate-citric acid gel and tank buffers with a pH of 5.3 provided exceptionally good separation between all four of the major subtyping bands. The additional criteria for this procedure is a voltage of 21 V/cm and a run time of 4 h. Utilization of this procedure using case samples of varied ages proved the reliability of the procedure. Also examined were the effects of several reducing agents on the enzyme band patterns and the use of this system for the simultaneous determinations of the adenosine deaminase (ADA), erythrocyte acid phosphatase (EAP), and adenylate kinase (AK) enzyme phenotypes.     

The effects of chemical and heat maceration techniques on the recovery of nuclear and mitochondrial DNA from bone

Forensic anthropologists use a number of maceration techniques to facilitate skeletal analysis of personal identity and trauma, but they may unwittingly eliminate valuable DNA evidence in the process. This study evaluated the effect of 10 maceration methods on gross bone structure and the preservation of DNA in ribs of 12 pigs (Sus scrofa). A scoring system was applied to evaluate the ease of maceration and resulting bone quality while DNA purity was quantified by optical densitometry analysis, followed by polymerase chain reaction (PCR) amplification of three mitochondrial and three nuclear loci. The results demonstrated that while mitochondrial DNA could be amplified for all experiments, cleaning treatments using bleach, hydrogen peroxide, ethylenediaminetetraacetic acid/papain, room temperature water and detergent/sodium carbonate followed by degreasing had low DNA concentrations and failed to generate nuclear PCR products. In general, treatments performed at high temperatures (90 degrees C or above) for short durations performed best. This study shows that traditionally "conservative" maceration techniques are not necessarily the best methods to yield DNA from skeletal tissue.     

Rapid confirmation of enzyme multiplied immunoassay technique (EMIT) cocaine positive urine samples by capillary gas-liquid chromatography/nitrogen phosphorus detection (GLC/NPD)

A rapid gas-liquid chromatographic (GLC) method was developed for the confirmation of benzoylecgonine (BE) positive urine samples screened by the enzyme multiplied immunoassay technique (EMIT) assay. The procedure is performed by solvent extraction of BE from 0.1 or 0.2 mL of urine, followed by an aqueous wash of the solvent and evaporation. The dried residue was derivatized with 50 microL of pentafluoropropionic anhydride and 25 microL of pentafluoropropropanol at 90 degrees C for 15 min. The derivatizing reagents were evaporated to dryness, and the derivatized BE, and cocaine if present, were reconstituted and injected into the gas chromatograph. The column was a 15-m by 0.2-mm fused silica capillary column, coated with 0.25 micron of DB-1, terminating in a nitrogen phosphorus detector (NPD). Cocaine and the pentafluoro BE derivatives retention times were 3.2 and 2.6 min, respectively. Nalorphine was used as reference or internal standard with a retention time of 4.78 min. The complete procedure can be performed in approximately 1.5 h. The EMIT cutoff between positive and negative urine samples is 300 ng/mL of BE. The lower limit of sensitivity of this method is 25 ng of BE extracted from urine. Validation studies resulted in confirmation of 101 out of 121 EMIT cocaine positive urine samples that could not be confirmed by thin-layer chromatography (TLC). This represents 84% confirmation efficiency.     

单管一步甲基化可变位点分析技术

目的建立单管一步甲基化可变位点（methylationvariableposition,MVP）分析技术一单管消化后PCR链融解曲线分析（post—digestionPCR—meltingcurveanalysis,PDP—MCA）。方法以文献报道的差异甲基化区（differentiallymethylatedregion,DMR）为模型,在MVP两侧设计一组解链温度各不相同的引物。应用FastDigest甲基化敏感性限制酶（methylation—sensitiverestrictionenzyme,MSRE）,在同一反应管内顺次进行DNA的酶切、复合扩增和MCA检测．生成MCA图谱。同时用该方法和传统的MSRE—PCRMCA技术检测相同样品（外周静脉血、精液、阴道液各5份）,比较两种方法检测结果,验证其可行性,并分析比较不同样品的MCA／HRM图谱。结果解链温度相差2℃以上的片段,MCA峰分离良好,复合扩增后可以用MCA技术检测。应用单管PDP—MCA技术,可以集酶切、扩增和检测三步于一管,在2h内得到与传统方法一致的特异性图谱和数据,并实现样品的快速分类鉴别。结论单管PDP-MCA技术可以实现多个MVP的单管、闭管检测,具有简便、快速、易于自动化等优点,可用于样品DNA甲基化差异的检测。     

Ossification of laryngeal structures as indicators of age

As the role of forensic anthropologists expands to the medical examiner setting, their expertise is being applied beyond the traditional dry skeletal material. In such scenarios radiographic techniques can be applied when maceration is not appropriate. This study explores the use of radiographic analysis of laryngeal structures for age-at-death determination. Isolated human laryngeal structures (n = 104) from individuals between the ages of 15 and 89 were removed at autopsy and radiographically examined. The cricoid and individual regions of the thyroid cartilage were scored according to degree of ossification, and the relationship between age and degree of ossification statistically examined. A previously published study on age-determination from thyroid ossification by Cerny was assessed for accuracy. The results of the study indicate that although a consistent sequence in the ossification of laryngeal structures exists, variations in timing does not permit narrow age range estimates. Consequently the method presented by Cerny is inaccurate and should not be used in the forensic setting.     

Medicare program; establishing procedures for transmitting information between Medicare carriers and Medicare supplemental insurers--HCFA. General notice

This notice announces and describes the procedure for the automatic transfer of claims information from Medicare carriers to Medicare supplemental (Medigap) insurers when a beneficiary has assigned his or her right of payment to a participating physician or supplier. It delineates the roles of the Medicare carriers, Medigap insurers, State insurance departments, beneficiaries, physicians and suppliers, and HCFA. The procedure for the automatic transfer of claims is required by section 1842(h)(3)(B) of the Social Security Act, as added by section 4081(a) of the Omnibus Budget Reconciliation Act of 1987 and is intended to speed payment of Medicare supplemental insurance benefits to participating physicians and suppliers.     

The Role of Site Variance in the American Judicature Society Field Study Comparing Simultaneous and Sequential Lineups

Objectives

Police departments often use photo lineups for eyewitness identification purposes. A widely adopted lineup reform designed to reduce eyewitness misidentifications involves switching from the standard simultaneous photo presentation format to a sequential format. These two lineup procedures were recently tested in the American Judicature Society (AJS) field study, which was conducted in four different police jurisdictions. The results from two phases of that investigation reached opposite conclusions as to which lineup procedure is superior, and the purpose of our current investigation was to elucidate the role of site variance in shaping those contrasting conclusions.

Methods

In previous analyses, the field study data were either (1) aggregated across all four study sites or (2) drawn from only one study site (Austin, Texas). Here, we analyze the data separately for the Austin study site, where 69 % of the eyewitnesses were tested, and the other three study sites combined, where 31 % of the eyewitnesses were tested.

Results

The results indicate significant site variance between the Austin and non-Austin study sites. In addition, the results suggest that aggregating the data across sites played a determinative role in creating the apparent disagreement about which lineup procedure is diagnostically superior.

Conclusions

Once large differences across the AJS study sites are taken into consideration, there is no longer any disagreement about which lineup procedure is superior. The simultaneous procedure is diagnostically superior to the sequential procedure, but the sequential procedure sometimes induces more conservative responding (a result that can and often does masquerade as diagnostic superiority).

     

Determination of the active principles of Catha Edulis: quali-quantitative analysis of cathinone, cathine, and phenylpropanolamine

In the last years, all the vegetable material supposed to belong to the Catha Edulis species, seized at the Malpensa and Orio al Serio airports, were analyzed in our laboratory on behalf of the Tribunals of Busto Arsizio and Bergamo, respectively. After a preliminary botanic examination, the quali-quantitative determination of the active principles cathinone, cathine and phenylpropanolamine (PPA) was carried out by means of GC/MS and GC/FID techniques, which meet the requirements of the forensic analyses. We developed a fast, effective and reliable derivatization procedure which allowed to simultaneously detect cathine and PPA, whose discrimination is mandatory since PPA is not a psychoactive agent. Cathine was distributed in the various parts of the plant (leaves and stems) and its quantity ranged from 0.03% to 0.17% of the weight of the vegetable material; PPA was not detected in the twigs and its quantity in the leaves ranged from 0.07% to 0.16%. The quantitative determination of cathinone was carried out directly on the methanol solution after maceration of the vegetable material, its quantity ranging from 0.02% to 0.10%. No significant difference in the content of the two active principles was found between the fresh and the dried material.     

Spectrophotometric and tristimulus analysis of the colors of subcutaneous bleeding in living persons

Skeletal injuries are often strong indicators of child abuse and their detection is therefore crucial. The aim of this study was to compare the sensitivity of three diagnostic approaches, namely autopsy, traditional (conventional) radiology, and computed tomography on "battered" piglets, in order to verify the sensitivity of each method, with respect to the true number of bone fractures assessed once the piglet was skeletonised (osteological control). Four newborn cadaver piglets who had died from natural causes were severely beaten post-mortem in every district of the body. Traditional radiography, computed tomography (CT) and autopsy were performed. The piglet was then macerated until skeletonised and the number of all fractures present recorded (osteological control). On the cranium, traditional radiology revealed only 35% circa of actual fractures, autopsy detected only 31% (P<0.01 for both comparisons versus osteological control), whereas CT imaging detected all fractures actually present. For ribs, radiology detected only 47% of all fractures present, and autopsy 65% circa (P>0.05 for both comparisons versus osteological control), while CT scans detected 34% (P<0.01). In suspected cases of fatal child abuse, we suggest that the bones of specific districts be directly analysed either at autopsy or by collecting specific diagnostic sites, such as parts of the rib cage, and subjecting them to maceration. The removed areas could be replaced with artificial material for cosmetic purposes. The authors stress the importance of combined radiological, CT scan, autopsy and osteological survey in the detection of perimortem bone fractures.     

小鼠死后脑RNA降解与死亡时间的相关性

目的 探讨小鼠死后不同时间和温度下脑组织β-actin mRNA、18S rRNA的降解规律与死亡时间的关系.方法 将24只健康成年C57BL/6小鼠随机分为2组,每组12只,断颈法处死后分别置于不同的环境温度中(4℃、37℃),恒定环境湿度为80%.分别于死后即刻和0.5、2、6、24、48h取小鼠脑组织进行总RNA...     

Detergent: an alternative approach to traditional bone cleaning methods for forensic practice

Osteological assessment of human remains forms an essential part of forensic work, especially during the examination of extensively decomposed, dismembered, or burnt bodies. Currently employed methods for removal of adherent soft tissue reflect practices often used by museum curators, notably insect consumption, enzymatic maceration, or boiling of the bones, with subsequent manual removal of material. This study was designed to assess the effectiveness of detergents for the purpose of soft-tissue removal from animal-derived specimens. The results indicate that such a means is comparable to enzymatic maceration but with fewer health and safety issues and greater advantages regarding transportation and availability of materials when an investigator is in a fieldwork scenario.     

A simplified method for mitochondrial DNA extraction from head hair shafts

DNA isolation from hair shafts can involve a number of steps, each of which adds time to the procedure and increases the risk of contamination. A simple alkaline digestion procedure that directly dissolves hairs was developed and compared with a widely used glass grinding/organic extraction method, using samples collected from 30 volunteers with varying population ancestries, hair colors, and hair treatments. A 203 bp mtDNA product could be amplified from 90% of samples extracted by alkaline digestion and 73% of hairs extracted by glass grinding. DNA obtained from alkaline digested hair generated equal or greater amplification success for virtually all criteria examined, and mtDNA sequences matched buccal control sequences in all cases. The two methods were similar in DNA yield (amplification success at template dilution) and quality of DNA obtained (amplicon length). Alkaline digestion of hair shafts required 6-7 h to complete, compared to 22-24 h for glass grinding, and proved a less laborious yet equally robust method for mtDNA extraction.     

The application of immunoblotting to the phenotyping of group-specific component

A sensitive immunoblotting method for the routine detection of group-specific component (GC) from fresh serum, and from control and casework bloodstains has been developed. GC phenotypes were separated in a thin layer polyacrylamide gel by isoelectric focusing, transferred to nitrocellulose by a rapid capillary blotting procedure, and detected using a double antibody enzyme immunoassay. This method is capable of phenotyping 8 ng of GC extracted from bloodstains, a four-fold increase in sensitivity when compared to immunofixation and silverstaining. A total of 2424 casework bloodstains have been analysed and GC phenotypes identified in 78% of samples. The method is suitable for use in routine laboratories and is more sensitive than other methods for GC phenotyping of casework bloodstains.     

Fluorescence microscopy in the detection of early myocardial infarction

Fluorescent microscopy is amongst the many techniques devised for the post-mortem detection of early myocardial infarction. The method has the advantage of sensitivity, speed and simplicity, compared with more complex techniques such as enzyme histochemistry and electron microscopy. Disadvantages include the difficulty of permanent preservation of the sections and lack of sharp differentiation between normal and very early infarction--though the latter problem is common to most other methods, except enzyme histochemistry. There are two main techniques in fluorescent microscopy: The examination of sections specifically stained by fluorochrome dyes; The re-examination of haematoxylin-eosin sections for 'autofluorescence'.