Performance evaluation of two multiplexes used in fluorescent short tandem repeat DNA analysis

The performance of two commercial multiplex kits that together amplify the 13 core short tandem repeat (STR) loci currently in use by forensic laboratories and the U.S. national Combined DNA Indexing System (CODIS) were evaluated. The typing systems examined were AmpFlSTR Profiler Plus and AmpFlSTR COfiler (PE Applied Biosystems, Foster City, CA). Electrophoretic separation and detection of the fluorescent PCR products was achieved by capillary electrophoresis (CE) using an ABI Prism 310 Genetic Analyzer. The studies addressed the on-site validation of the instrument, the software, and each typing system. These studies included instrument sensitivity, resolution, precision, binning, peak height ratios, mixtures, stutter, and the amplification of non-probative and simulated forensic samples. Other additional developmental-type work is also reported herein, such as species specificity testing and amplification of environmentally insulted samples. Amplification conditions were found to be robust and the primer sets shown to be specific to human DNA. Stutter and peak height ratios fell within limits published by the manufacturer and other laboratories. The data demonstrate that the CE instrument can consistently resolve fragments differing in length by one base and that the +/-0.5 base bin used by the Genotyper software is acceptable for making accurate allele calls. Correct typing results were obtained from non-probative and simulated case samples, as well as samples exposed to outdoor environmental conditions. The results support the conclusion that DNA extracted from biological samples routinely encountered in the forensic laboratory can be reliably analyzed with AmpFlSTR Profiler Plus and COfiler using CE.     

The application of an automated allele concordance analysis system (CompareCalls) to ensure the accuracy of single-source STR DNA profiles

A powerful method for validating a scientific result is to confirm specific results utilizing independent methodologies and processing pathways. Thus, we have designed, developed and validated an automated allele concordance analysis system (CompareCalls, patent pending) that performs comparisons between two independent DNA analysis platforms to ensure the highest accuracy for allele calls. Application of this system in a quality assurance role has shown the potential to eliminate greater than 90% of the STR analysis required of a DNA data analyst. While this system is broadly applicable for use with any two independent STR analysis programs, either prior to or following human data review, we are presenting its application to data generated with the ABI Prism Genotyper software system versus data generated with the SurelockID system. With the automated allele concordance analysis system, the GeneScan DNA fragment data generated from an ABI 377 gel image are analyzed in two independent pathways. In one analysis pathway, the GeneScan data are imported into Genotyper software where STR labels are assigned to the fragment data based upon the criteria of the Kazam 20% macro. The "Kazam" macro provided with the Genotyper program works by labeling all peaks in a category (or locus) and then filtering (or removing) the labels from peaks, such as those in stutter positions, that meet predefined criteria. In the second pathway, the GeneScan data are imported into the SurelockID analysis platform where STR labels and error messages are assigned to the fragment data based upon hard-coded allele calling criteria and quality parameters. The resulting STR allele calls for each analysis platform are then compared, utilizing the automated allele concordance analysis system. Any differences in the STR allele calls between the two systems are flagged in a discordance report for further review by a qualified DNA data analyst. The automated allele concordance analysis system guides the DNA data analyst to the discordant data generated by either analysis platform. Additionally, the analyst is also directed to data that are of less than pristine quality which may have an increased potential for errors in interpretation by either analysis platform or by a human DNA data analyst. Implementation of an automated allele concordance analysis system will yield high-quality data for CODIS and free the human DNA data analyst to perform other critical duties within the laboratory.     

Exploring the Impacts of Ordinary Laboratory Alterations During Forensic DNA Processing on Peak Height Variation,Thresholds, and Probability of Dropout,

Impacts of validation design on DNA signal were explored, and the level of variation introduced by injection, capillary changes, amplification, and kit lot was surveyed by examining a set of replicate samples ranging in mass from 0.25 to 0.008 ng. The variations in peak height, heterozygous balance, dropout probabilities, and baseline noise were compared using common statistical techniques. Data indicate that amplification is the source of the majority of the variation observed in the peak heights, followed by capillary lots. The use of different amplification kit lots did not introduce variability into the peak heights, heterozygous balance, dropout, or baseline. Thus, if data from case samples run over a significant time period are not available during validation, the validation must be designed to, at a minimum, include the amplification of multiple samples of varying quantity, with known genotype, amplified and run over an extended period of time using multiple pipettes and capillaries.     

Validation study of the TrueAllele automated data review system

The New York State Convicted Offender DNA Databank is the first U.S. lab to complete an internal validation of the TrueAllele expert data review system. TrueAllele is designed to assess short tandem repeat (STR) DNA data based on several key features such as peak height, shape, area, and position relative to a standard ladder and use this information to make accurate allele calls. The software then prioritizes the allele calls based on several user-defined rules. As a result, the user need only review low-quality data. The validation of this system consisted of an extensive optimization phase and a large concordance phase. During optimization, the rule settings were tailored to minimize the amount of high-quality data viewed by the user. In the concordance phase, a large dataset was typed in parallel with the ABI software Gene Scan and Genotyper (manual review) and TrueAllele (automated review) for comparison of allele calls and sample state assignment. Only one significant difference was discovered out of 2048 samples in the concordance study. In this case, TrueAllele revealed a spike in the profile that was interpreted as a DNA peak by the analyst in Genotyper. TrueAllele was designed to focus the review on poor data and to eliminate the need for complete reanalysis technical review. This validation project proved TrueAllele to be dependable for use at the NYS Convicted Offender DNA Databank.     

Run-specific limits of detection and quantitation for STR-based DNA testing

STR-based DNA profiling is an exceptionally sensitive analytical technique that is often used to obtain results at the very limits of its sensitivity. The challenge of reliably distinguishing between signal and noise in such situations is one that has been rigorously addressed in numerous other analytical disciplines. However, an inability to determine accurately the height of electropherogram baselines has caused forensic DNA profiling laboratories to utilize alternative approaches. Minimum thresholds established during laboratory validation studies have become the de facto standard for distinguishing between reliable signal and noise/technical artifacts. These minimum peak height thresholds generally fail to consider variability in the sensitivity of instruments, reagents, and the skill of human analysts involved in the DNA profiling process over the course of time. Software (BatchExtract) made publicly available by the National Center for Biotechnology Information now provides an alternative means of establishing limits of detection and quantitation that is more consistent with those used in other analytical disciplines. We have used that software to determine the height of each data collection point for each dye along a control sample's electropherogram trace. These values were then used to determine a limit of detection (the average amount of background noise plus three standard deviations) and a limit of quantitation (the average amount of background noise plus 10 standard deviations) for each control sample. Analyses of the electropherogram data associated with the positive, negative, and reagent blank controls included in 50 different capillary electrophoresis runs validate that this approach could be used to determine run-specific thresholds objectively for use in forensic DNA casework.     

Human height estimation from highly distorted surveillance image

Video surveillance camera (VSC) is an important source of information during investigations especially if used as a tool for the extraction of verified and reliable forensic measurements. In this study, some aspects of human height extraction from VSC video frames are analyzed with the aim of identifying and mitigating error sources that can strongly affect the measurement. More specifically, those introduced by lens distortion are present in wide-field-of-view lens such as VSCs. A weak model, which is not able to properly describe and correct the lens distortion, could introduce systematic errors. This study focuses on the aspect of camera calibration to verify human height extraction by Amped FIVE software, which is adopted by the Forensic science laboratories of Carabinieri Force (RaCIS), Italy. A stable and reliable approach of camera calibration is needed since investigators have to deal with different cameras while inspecting the crime scene. The performance of the software in correcting distorted images is compared with a technique of single view self-calibration. Both approaches were applied to several frames acquired by a fish-eye camera and then measuring the height of five different people. Moreover, two actual cases, both characterized by common low-resolution and distorted images, were also analyzed. The height of four known persons was measured and used as reference value for validation. Results show no significant difference between the two calibration approaches working with fish-eye camera in test field, while evidence of differences was found in the measurement on the actual cases.     

TWGDAM validation of AmpFlSTR PCR amplification kits for forensic DNA casework

Laboratory procedures used in short tandem repeat (STR) analysis were subjected to various scenarios that assessed reliability and identified potential limitations. These validation studies were designed as recommended by the Technical Working Group on DNA Analysis Methods (TWGDAM) and the DNA Advisory Board (DAB) (17,18). Various DNA samples were amplified by the polymerase chain reaction (PCR) using AmpFlSTR PCR Amplification Kits (i.e., AmpFlSTR Green I, Profiler, Profiler Plus, and COfiler kits), detected with ABI Prism instrumentation, and analyzed using GeneScan and Genotyper software. Data acquired in these studies reinforced an existing body of knowledge and expertise regarding application and interpretation of STR typing in the forensic science community. Consistent STR genotypes were detected in various body tissues and fluids. Inter-laboratory comparisons produced concordant genotype results. Quantitative interpretational aids for DNA mixtures were characterized. Ability of the typing systems to type potentially compromised samples reliably was evaluated. Nonprobative case evidentiary DNA was successfully amplified, genotyped, and interpreted. Potential limitations or cautionary factors in the interpretation of minimal fluorescence intensity were demonstrated. Differential amplification between loci was observed when PCR was inhibited; preferential amplification typically was not. Single AmpFlSTR locus amplification did not offer consistent benefit over AmpFlSTR multiplexing, even in cases of DNA degradation or PCR inhibition. During rigorous evaluation, AmpFlSTR PCR Amplification Kits reproducibly yielded sensitive and locus-specific results, as required in routine forensic analyses.     

Forensic Analysis of Explosives Using Isotope Ratio Mass Spectrometry (IRMS)—Part 1: Instrument Validation of the DELTAplusXP IRMS for Bulk Nitrogen Isotope Ratio Measurements

Abstract: A significant amount of research has been conducted into the use of stable isotopes to assist in determining the origin of various materials. The research conducted in the forensic field shows the potential of isotope ratio mass spectrometry (IRMS) to provide a level of discrimination not achievable utilizing traditional forensic techniques. Despite the research there have been few, if any, publications addressing the validation and measurement uncertainty of the technique for forensic applications. This study, the first in a planned series, presents validation data for the measurement of bulk nitrogen isotope ratios in ammonium nitrate (AN) using the DELTA^plusXP (Thermo Finnigan) IRMS instrument equipped with a ConFlo III interface and FlashEA? 1112 elemental analyzer (EA). Appropriate laboratory standards, analytical methods and correction calculations were developed and evaluated. A validation protocol was developed in line with the guidelines provided by the National Association of Testing Authorities, Australia (NATA). Performance characteristics including: accuracy, precision/repeatability, reproducibility/ruggedness, robustness, linear range, and measurement uncertainty were evaluated for the measurement of nitrogen isotope ratios in AN. AN (99.5%) and ammonium thiocyanate (99.99+%) were determined to be the most suitable laboratory standards and were calibrated against international standards (certified reference materials). All performance characteristics were within an acceptable range when potential uncertainties, including the manufacturer’s uncertainty of the technique and standards, were taken into account. The experiments described in this article could be used as a model for validation of other instruments for similar purposes. Later studies in this series will address the more general issue of demonstrating that the IRMS technique is scientifically sound and fit‐for‐purpose in the forensic explosives analysis field.     

STR基因座等位基因阶梯制备方法尝试

提高PCR-STR分型技术的准确性。采用荧光标记dNTP掺入法,通过DNA自动测序仪分析不同等位基因片段的长度并确定其命名后,建立并比较扩增产物混合-纯化-重扩增法、琼脂糖凝胶电泳-切割回收纯化DNA-重扩增法、非变性聚丙烯酰胺凝胶电泳-切割回收纯化DNA-重扩增法等3种Ladder制备方法。扩增产物混合-纯化-重扩增法相对较简便、快速、经济。Ladder对照使基因分型数据化,判型准确,重复性好,有利于标准化的实现。     

Developmental validation of reduced-size STR Miniplex primer sets

This paper describes a developmental validation study of three Miniplex sets covering 12 of the 13 CODIS loci. As these new sets will be used for the analysis of degraded and low level DNA, the validation studies were performed using 100-125 pg of DNA, the lowest input level at which peak balance, peak intensity, and allele consistency were stable. To demonstrate the applicability of the Miniplex sets to forensic casework, these validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM). A range of tests were performed including studies of concordance with standard multiplex kits, sensitivity and reproducibility, and PCR amplification conditions. Additionally, studies of mixtures, nonhuman and environmentally degraded DNA, and simulated forensic samples were performed. Our results demonstrate that Miniplex STR amplification procedures are a robust and sensitive tool for the analysis of degraded DNA.     

Sugar and fatty acid analysis in ecstasy tablets

Sugars and stearates (composed of fatty acids) are both frequent components used in the production of ecstasy tablets. Their analysis can therefore provide supplementary information useful for drug intelligence. Links established using these substances would be very significant as they should give us information about the manufacturer of the tablets. Two methods have been developed for the analysis of sugars and fatty acids by GC-MS and were applied to 109 ecstasy tablets. Characterisation of the samples should allow the differentiation of a certain number of them and furthermore their classification into groups. This is obtained by analysing the raw data using chemometric methods. Several pre-treatments have been tested together with six similarity measures on a small number of ecstasy samples in order to determine which combination would best characterise one ecstasy sample and differentiate it from the others at the same time. Normalisation followed by the fourth square and applied together with the squared cosine function appeared to give the best results and has been applied to all samples. The correlation values obtained of each sample with all others express the probability of a presence of a link between two samples. In order to verify the signification of these values, and thus of a link, all samples have been compared considering the data visually according to three selected criterions. The 109 examined samples could be divided into 67 groups, with 43 of them containing only one sample. Considering the distribution of their correlation values, sample pairs showing a value below 0.23 can be considered as linked. As the excipients are necessarily related to the blending, which also includes the active substance, and variation in the excipient content has been proven by the grouping of the samples, a low similarity value does indicate a link with regard to the producer. In conclusion, it appears that the result obtained with the excipients is certainly very valuable, but all other available information has to be taken into account as well before making any conclusions.     

An innovative method for human height estimation combining video images and 3D laser scanning

Digitalization has increased the number of video surveillance systems that sometimes capture crime images. Traditional methods of human height estimation use projective geometry. However, sometimes they cannot be used because the video camera surveillance system is not available or has been moved and there are no reference lines on the frame. Scientific studies have developed a new method for human height estimation using 3D laser scanning. This model necessarily requires a series of approximations, which increase the final measurement error. To overcome this problem, in the present study, images of a subject are projected directly on the 3D model, estimating the height of the subject. This article describes the methodological approach adopted through the analysis of a real case study in a controlled environment executed by Carabinieri Forensic Investigation Department (Italy). The aim is to obtain a human anthropometric measure derived from frames extracted from the videos associated with the digital survey of the framed area obtained with 3D laser scanning and point cloud analysis. The result is the height estimation of five subjects filmed by a camera obtained through the combination of 2D images extracted by a DVR/surveillance systems with 3D laser scanning. Results show that most estimated measurements are less than the real measurement of the subject; it also depends on the posture of the subject while walking. Furthermore, results shows the differences between the real height and the estimated height with a statistical approach.     

An exploratory examination of the Spiritual Well-Being Scale among incarcerated black and white male drug users

A number of studies have examined the link between criminality and religiosity. However, only a limited number of studies have examined the relationship between spirituality and criminality. Because spirituality has been identified as a fundamental attribute of the personalities of Blacks, studies examining differences in the association between spirituality by ethnicity could provide information to understand the disparity of incarceration rates among Blacks and Whites. For this study, data were collected from 661 male prisoners with prior histories of drug use to examine spirituality that was assessed using two factors from a modified version of the Spiritual Well-Being Scale: relationship with a higher power and satisfaction with oneself in the world. Analyses revealed that White men reported significantly higher scores on both factors than Black men. The unexpected findings are discussed in light of the existing literature that identifies the significance of spirituality in the personality and coping style of Blacks.     

Optimization and validation studies of the Mentype Argus X-8 kit for paternity cases

The use of ChrX-STRs is enormous in forensic case as these have proven to be powerful tools, mainly in deficiency paternity cases when the disputed child is female, and also some special cases involving blood relatives, incest cases, fetal typing in abortion material. The Mentype^® Argus X-8 kit is a commercial multiplex system which contains Amelogenin for gender determination as well as gonosomal STR markers (DXS8378, HPRTB, DXS7423, DXS7132, DXS10134, DXS10074, DXS10101 and DXS10135). Validation studies were being performed on blood obtained from the volunteers in Turkish population. In this study, some parameters were taken under consideration for validation like DNA extraction using different protocols, quantitated by using commercially available Invitrogen Qubit Fluorometer, reaction volume validation of Master Mix and the analysis of female/male, female/female and male/male mixtures were performed. The conditions were optimized and validated using GenAmp 9700 and reducing reaction volume from 25 μl to 12.5 μl and 6.5 μl. After reducing the total volume of the reaction, the results were same and there was no effect on peak height and quality when analyzed on ABI 310 genetic analyzer. 2 paternity cases were also performed which gave the same power of discrimination as has been mentioned in Mentype^® Argus X-8 kit.     

国产Goldeneye~(TM) 20A试剂盒性能指标验证

目的测试国产GoldeneyeTM20A试剂盒技术性能指标,评估其法医学应用能力。方法从方法学验证、准确性、峰值均衡性、灵敏度、批次间试剂及稳定性测试、耐受性、不同检材的适应性与一致性、种属特异性、混合样本等9个方面对GoldeneyeTM20A试剂盒进行测试。结果阳性DNA样本分型正确,内标和等位基因分型标准物符合要求;等位基因间的均衡性≥83%,同一荧光标记基因座间的均衡性≥55%,不同标记物间的均衡性≥52%;0.125ng DNA阳性样本可检出全部STR基因座分型,不同批次间和反复冻融后试剂盒测试可以获得正确分型,对降解检材和混有抑制剂的样本等具有一定的耐受性,能对案件中多种检材进行分型且分型结果一致,具有一定的种属特异性和混合DNA样本检测能力。结论国产GoldeneyeTM 20A试剂盒可用于法庭科学实际检案与建库。     

Increased Hematocrit Due to Electrical‐Waveform Exposures in Splenectomized Sus scrofa,

In laboratory studies of the pig Sus scrofa, hematocrit has consistently increased after conducted‐electrical‐weapon (CEW) exposures, possibly due to contraction of the spleen. Splenectomized animals and intact sham control animals were exposed, each for 30 sec, to a benchtop‐produced electrical waveform of net charge levels similar to those of some CEWs. Changes in the blood were compared statistically. Hematocrit increased significantly in both splenectomized and sham animals. There were no significant main‐effect differences between values of hematocrit from the two groups. There were, however, significant interactive effects of time and splenectomy for hematocrit, red blood cell count, and hemoglobin. After peak values were reached for these variables, values returned toward baseline levels more slowly in splenectomized animals. This may have been due to the lack of a spleen to sequester red blood cells (thereby resulting in more cells remaining in the general circulation), unlike sham animals with intact spleens.     

Testing Discriminant Functions for Sex Determination from Deciduous Teeth*,†

Abstract: Three studies have proposed discriminant functions for sex determination from deciduous tooth crown dimensions, and this study tests the existing functions on a sample of 46 Portuguese immature skeletons of known sex, aged from birth to 10 years. Deciduous teeth were measured in their mesiodistal and faciolingual crown dimensions, and percentage of correct allocation accuracy in determining sex using each specific function was determined. Discriminant functions were also calculated from data collected for this study and tested using cross‐validation. Results show poor overall accuracy (33.3–75%) and poor cross‐validation (46.2–60.0%). This is related to low sexual dimorphism in deciduous tooth crown size, as well as differences in degree of sexual dimorphism and in overall tooth size between different samples. For these reasons, deciduous crown size does not seem to show significant forensic value as discriminator of sex, particularly when methods developed on one population are applied to individuals of another population.     

Comparisons of protocols for enhanced DNA profile quality from FTA®-deposited urine samples

Disputes over the identity of a urine sample donor have been reported, and urine authentication by genetic profiling has helped resolved the cases. However, since genotyping of urine is not always required, many drug-testing laboratories may face sample storage issues. Several studies have investigated the use of FTA® cards as a convenient tool for keeping specimen at room temperature for extended periods of time. However, generating complete STR profile from some FTA®-deposited urine samples remains challenging due to low levels of genetic material content, necessitating amendments to the laboratory’s standard protocols. This work therefore aims to evaluate the effects of two DNA template preparation methods, both employing FTA® cards as the storage medium, on the success rates of STR profiling from urine. Specimen from a female volunteer, representing a particularly low-yield sample, was employed. Aliquots of 1 and 2 mL were used as the starting material to evaluate DNA template preparation using the FTA® manufacturer’s protocol for disc purification against elution of DNA from the FTA® using Prepfiler™ Forensic DNA Extraction Kit. AmpFℓSTR™ Identifiler™ Plus PCR Amplification Kit was used to amplify the STR markers, and the PCR products were analysed using Applied Biosystems™ 3500xL Genetic Analyzer. The DNA profile qualities were examined in terms of number of loci detected and peak height balance. Comparisons with the profiles obtained from DNA isolated using QIAamp® DNA Micro Kit from 1 and 2 mL of the same batch of urine were also made. The optimised protocol was then tested on urine samples from three male volunteers. The results showed that the purification of FTA® punches according to the manufacturer’s protocol enabled full DNA profiles to be obtained from both 1 and 2 mL of urine from all samples tested, including male samples. In contrast, no DNA profile could be generated from the DNA eluted with the Prepfiler™ kit. When compared with the more conventional solid-phase DNA extraction method, the profiles generated from the FTA® punches exhibited similar reproducibility and quality to those from the template isolated by the QIAamp® Kit. This work further demonstrated the feasibility of FTA® cards as a tool for specimen storage and DNA template preparation from small volumes of urine for authentication by STR profiling. Full STR profiles could be generated from sample from both sexes without modification of the PCR conditions or injection time.     

Racial variation in the proximal and distal femur: heritability and forensic utility.

The femur has been studied successfully by physical anthropologists for many years. Such traits as femoral head diameter and bicondylar width have been examined extensively and are of great value to forensic anthropologists and other skeletal biologists in sex identification. A number of studies over the past decade by the author and his former students have shown marked racial differences in the shape of the proximal femur and in at least one trait of the distal femur--intercondylar notch height. Anterior-posterior (AP) diameter of the proximal femur is much greater among Whites and Blacks than among East Asians and American Indians. Blacks have slightly greater intercondylar notch height than Whites. Other features, such as torsion, also differ between the major geographic racial populations. Current analysis suggests that the East Polynesians fall close to the American Indians and East Asians in the degree of flatness of the proximal femur. One study has tracked the degree of change in flatness during individual development, and finds little change within major populations from the youngest to the oldest individuals. Temporal changes within populations are likewise minimal. Two studies have examined sex differences within populations, which are also found to be very slight. Racial differences, on the other hand, are quite significant, and individuals of admixed ancestry fall intermediate between the two parental populations. Such suggestions of high heritability in the shape of the proximal and distal femur make these traits very useful in assessing ancestry in forensic contexts.