Ultrasound-guided removal of Implanon™ devices

Our study has shown that ultrasound-guided localisation and removal of Implanon™ rods is safe, practical and highly successful. Over a 4-year period, 119 patients had successful, uncomplicated removal of their subdermal devices.The technique is particularly useful for removal of the device when it is not palpable or when an attempt at removal of a palpable device has not been successful.     

Next generation sequencing of SNPs using the HID-Ion AmpliSeq™ Identity Panel on the Ion Torrent PGM™ platform

The HID-Ion AmpliSeq™ Identity Panel (the HID Identity Panel) is designed to detect 124-plex single nucleotide polymorphisms (SNPs) with next generation sequencing (NGS) technology on the Ion Torrent PGM™ platform, including 90 individual identification SNPs (IISNPs) on autosomal chromosomes and 34 lineage informative SNPs (LISNPs) on Y chromosome. In this study, we evaluated performance for the HID Identity Panel to provide a reference for NGS-SNP application, focusing on locus strand balance, locus coverage balance, heterozygote balance, and background signals. Besides, several experiments were carried out to find out improvements and limitations of this panel, including studies of species specificity, repeatability and concordance, sensitivity, mixtures, case-type samples and degraded samples, population genetics and pedigrees following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. In addition, Southern and Northern Chinese Han were investigated to assess applicability of this panel. Results showed this panel led to cross-reactivity with primates to some extent but rarely with non-primate animals. Repeatable and concordant genotypes could be obtained in triplicate with one exception at rs7520386. Full profiles could be obtained from 100 pg input DNA, but the optimal input DNA would be 1 ng–200 pg with 21 initial PCR cycles. A sample with ≥20% minor contributor could be considered as a mixture by the number of homozygotes, and full profiles belonging to minor contributors could be detected between 9:1 and 1:9 mixtures with known reference profiles. Also, this assay could be used for case-type samples and degraded samples. For autosomal SNPs (A-SNPs), F_ST across all 90 loci was not significantly different between Southern and Northern Chinese Han or between male and female samples. All A-SNP loci were independent in Chinese Han population. Except for 18 loci with H_e <0.4, most of the A-SNPs in the HID Identity Panel presented high polymorphisms. Forensic parameters were calculated as >99.999% for combined discrimination power (CDP), 0.999999724 for combined power of exclusion (CPE), 1.390 × 10¹¹ for combined likelihood ratio (CLR) of trios, and 2.361 × 10⁶ for CLR of motherless duos. For Y-SNPs, a total of 8 haplotypes were observed with the value of 0.684 for haplotype diversity. As a whole, the HID Identity Panel is a well-performed, robust, reliable and high informative NGS-SNP assay and it can fully meet requirements for individual identification and paternity testing in forensic science.     

Accuracy of the SenseWear Armband™ during ergocycling

The present study aims to show the accuracy of a portable motion sensor, the SenseWear Armband, for the estimation of energy expenditure vs. energy expenditure measured by indirect calorimetry during ergocycling. 31 healthy adults (52% women; age: 26.7±6.3 years; Body Mass Index: 23.9±3.3?kg/m2) completed a 45-min ergocycling session at 50% of their VO2(peak). Despite a significant underestimation of 18.7±13.2?kcal during the first 10?min of the activity (T=5.06; p<0.001), we observed an overall good agreement between energy expenditure estimated by the SenseWear Armband during ergocycling and indirect calorimetry (260.3±80.1 vs. 287.8±97.1?kcal, respectively) (T=-2.148; p=0.04) and a significant intra-class correlation (r=0.81; p<0.001). The results of the present study indicate that the SenseWear Armband underestimated energy expenditure during a 45-min ergocycling session at a 50% VO2(peak) intensity, mainly during the first 10?min. Underestimation at the onset of the activity warrants further research.     

Second-generation sequencing of forensic STRs using the Ion Torrent™ HID STR 10-plex and the Ion PGM™

Second-generation sequencing (SGS) using Roche/454 and Illumina platforms has proved capable of sequencing the majority of the key forensic genetic STR systems. Given that Roche has announced that the 454 platforms will no longer be supported from 2015, focus should now be shifted to competing SGS platforms, such as the MiSeq (Illumina) and the Ion Personal Genome Machine (Ion PGM™; Thermo Fisher). There are currently several challenges faced with amplicon-based SGS STR typing in forensic genetics, including current lengths of amplicons for CE-typing and lack of uniform data analysis between laboratories.Thermo Fisher has designed a human identification (HID) short tandem repeat (STR) 10-plex panel including amelogenin, CSF1PO, D16S539, D3S1358, D5S818, D7S820, D8S1179, TH01, TPOX and vWA, where the primers have been designed specifically for the purpose of SGS and the data analysis is supported by Ion Torrent™ software. Hence, the combination of the STR 10-plex and the Ion PGM™ represents the first fully integrated SGS STR typing solution from PCR to data analysis.In this study, four experiments were performed to evaluate the alpha-version of the STR 10-plex: (1) typing of control samples; (2) analysis of sensitivity; (3) typing of mixtures; and (4) typing of biological crime case samples. Full profiles and concordant results between replicate SGS runs and CE-typing were observed for all control samples. Full profiles were seen with DNA input down to 50 pg, with the exception of a single locus drop-out in one of the 100 pg dilutions. Mixtures were easily deconvoluted down to 20:1, although alleles from the minor contributor had to be identified manually as some signals were not called by the Ion Torrent™ software. Interestingly, full profiles were obtained for all biological samples from real crime and identification cases, in which only partial profiles were obtained with PCR-CE assays. In conclusion, the Ion Torrent™ HID STR 10-plex panel offers an all-in-one solution from amplification of STRs and amelogenin, and sequencing to data analysis.     

The imaging features of MACROLANE™ in breast augmentation

Macrolane? is an injectable, biocompatible, soft-tissue filler that has been available in the UK since 2008 and is promoted for use in breast augmentation. There are few data available on the long-term effects of this relatively new product and concerns have been raised about the implications for breast imaging, in particular breast screening. In this context we present a spectrum of imaging appearances and complications encountered to date.     

Direct electrophilic iodination of 2-thiouracil using iodo-gen™

5-Iodo-2-thiouracil (ITU) is of interest due to its ability to bind specifically to the pigment melanin during melanogenesis and is of potential value in the diagnosis and treatment of malignant melanoma. Radioiodinated ITU was prepared directly from 2-thiouracil in a two-phase reaction using Iodo-Gen™ in 0.05 M phosphate buffer pH 7.0. The identity radiochemical purity and stability of the product were checked by reversed-phase high pressure liquid chromatography (HPLC). ITU labeled with ¹²³I, ¹²⁵I or ¹³¹I has been produced in millicurie amounts and isolated on a semi-preparative reversed-phase HPLC column. Production time was 2–3 h, overall radiochemical yields averaged 80%; the radiochemical purity was greater than 98%. Specific activities on the order of 20 Ci/mmol have been obtained.     

Genetic data for PowerPlex 21™ autosomal and PowerPlex 23 Y-STR™ loci from population of the state of Uttar Pradesh,India

International Journal of Legal Medicine - In the present study, the statistical forensic parameters were evaluated for the loci present in PowerPlex 21 autosomal and PowerPlex 23 Y-STR multiplex...     

Determination of the variables affecting mixed MiniFiler™ DNA profiles

The interpretation of mixed DNA profiles presents additional challenges for the forensic scientist. There has been a broad based call for transparency in the process of interpretation of all evidence including mixed DNA profiles. This interpretation is greatly facilitated by a sound understanding of the variability in peak heights for the two peaks of a heterozygote, in the sizes of stutter peaks and in the variability in peak heights across loci.This study examines single source and mixed DNA profiles to assess this variability. The relative variability in peak height between the two peaks of a heterozygote and in the peak heights across loci becomes greater as the peaks themselves become smaller. This is consistent with findings from other multiplexes. This variability appears larger in the MiniFiler™ system at 30 cycles than, for example, in the Identifiler™ system at 28 cycles and this difference is largely explained by the two extra cycles of amplification.Stutter peaks appear no larger in the MiniFiler™ system at 30 cycles than in the Identifiler™ system at 28 cycles.     

Stereotactic radiotherapy for choroidal melanomas by means of HybridArc™

Purpose

Stereotactic radiotherapy (SRT) is suitable to treat ocular tumours. The optimal beam geometry for SRT, however, has not been defined. Here we evaluate a combination technique with dynamic conformal arcs (DCAs) and intensity-modulated static fields (IMRT), known as HybridArc? (HA).

Methods

For the first consecutive 25 cases with choroidal melanomas with volumes of 0.02 to 1.18?cm³ treated with 50?Gy in five fractions, the results with respect to dose conformity, homogeneity, and dose distributions were summarised. To describe the dose distribution at the planning target volume (PTV) boundary, we defined a spatially averaged dose gradient (SADG) and compared it with Paddick’s gradient index (GI). We made dosimetric comparisons between HA and other irradiation techniques.

Results

The PTVs ranged from 0.42 to 3.37?cm³. The conformity index (CI) was 1.25?±?0.15, and the homogeneity index (HI) 0.08?±?0.02. The SADG was (?3.5?±?0.5) Gy/mm or (?7.0?±?1.0) %/mm between the isodose levels 95 and 20%; local minima reached ?11.5?Gy/mm or ?22.9%/mm. The coefficient of determination for a nonlinear regression of GI on SADG was 0.072. After a median follow-up time of 19.6 months, local tumour control was 100% without any case of post-therapeutic enucleation. Two patients (8%) developed liver metastases.

Conclusion

SRT of ocular tumours by HA is highly appropriate, and HA is superior to intensity-modulated arc therapy (IMAT) concerning dose reduction in organs at risk (OARs). The novel gradient measure SADG is more informative than Paddick’s GI.

     

Autologous osteochondral mosaicplasty or TruFit™ plugs for cartilage repair

Purpose

Autologous osteochondral mosaicplasty and TruFit? Bone graft substitute plugs are methods used to repair symptomatic articular cartilage defects in the adult knee. There have been no comparative studies of the two techniques.

Methods

This retrospective study assessed functional outcome of patients using the EQ-5D, Knee Injury and Osteoarthritis Outcome Score (KOOS) and Modified Cincinnati scores at follow-up of 1–5 years.

Results

There were 66 patients in the study (35 TruFit and 31 Mosaicplasty): 44 males and 22 females with a mean age of 37.3 years (SD 12.6). The mean BMI was 26.8. Thirty-six articular cartilage lesions were due to trauma, twenty-six due to osteochondritis dissecans and three due to non-specific degenerative change or unknown. There was no difference between the two groups age (n.s.), sex (n.s.), BMI (n.s.), defect location (n.s.) or aetiology (n.s.). The median follow-up was 22 months for the TruFit cohort and 30 months for the mosaicplasty group. There was no significant difference in the requirement for re-operation (n.s). Patients undergoing autologous mosaicplasty had a higher rate of returning to sport (p = 0.006), lower EQ-5D pain scores (p = 0.048) and higher KOOS activities of daily living (p = 0.029) scores. Sub-group analysis showed no difference related to the number of cases the surgeon performed. Patients requiring re-operation had lower outcome scores regardless of their initial procedure.

Conclusion

This study demonstrated significantly better outcomes using two validated outcome scores (KOOS, EQ-5D), and an ability to return to sport in those undergoing autologous mosaicplasty compared to those receiving TruFit plugs.

Level of evidence

IV.     

Population genetic analyses of the AmpFlSTR® NGM™ in Brazil

Population data of 15 short tandem repeat loci of the AmpFlSTR? next generation multiplex (NGM)? were obtained from a sample of 835 individuals. The loci are the ten short tandem repeats (STRs) in the SGM Plus? Kit plus the EDNAP- and ENSFI-recommended STRs D10S1248, D22S1045, D2S441, D1S1656, and D12S391. Allele frequency and other forensically relevant statistics data were generated for the NGM loci into five current country macroregions of Brazil (North, Northeast, Central West, Southeast, and South). All the analyzed loci meet Hardy-Weinberg equilibrium expectations and no linkage disequilibrium in all pairs of loci. The observed and expected heterozygosity, power of discrimination, polymorphic information content, and the other population-genetic indices were calculated. The overall power of discrimination was greater than 0.99999999999999999996 and the combined power of exclusion was greater than 0.9999998 in all Brazilian populations. Comparative analysis between populations from different Brazilian macroregions as well as between Brazil and Caucasian, African Americans, and Hispanic US populations are presented.     

Amplification volume reduction on DNA database samples using FTA™ Classic Cards

The DNA forensic community always strives towards improvements in aspects such as sensitivity, robustness, and efficacy balanced with cost efficiency. Therefore our laboratory decided to study the feasibility of PCR amplification volume reduction using DNA entrapped in FTA? Classic Card and to bring cost savings to the laboratory. There were a few concerns the laboratory needed to address. First, the kinetics of the amplification reaction could be significantly altered. Second, an increase in sensitivity might affect interpretation due to increased stochastic effects even though they were pristine samples. Third, statics might cause FTA punches to jump out of its allocated well into another thus causing sample-to-sample contamination. Fourth, the size of the punches might be too small for visual inspection. Last, there would be a limit to the extent of volume reduction due to evaporation and the possible need of re-injection of samples for capillary electrophoresis. The laboratory had successfully optimized a reduced amplification volume of 10 μL for FTA samples.     

SPERM HY-LITER™ for the identification of spermatozoa from sexual assault evidence

Accurate microscopic identification of human spermatozoa is important in sexual assault cases. We have compared the results of examinations with (1) a fluorescent microscopy method, SPERM HY-LITER™, and (2) Baecchi's method for identification of human spermatozoa. In 35 artificial, forensic type samples, spermatozoa were identified in 45.7% with SPERM HY-LITER™ in Copenhagen, in 54.3% in the laboratory of the manufacturer of SPERM HY-LITER™, and 40.0% of the samples with Baecchi's staining method. When differences occurred between the two methods, it was significantly more often that SPERM HY-LITER™ detected spermatozoa when Baecchi's method did not (t_s = 6.567, df = 1, P = 0.048). This trend was also seen in selected compromised or degraded samples and in selected adjudicative samples. The reactions with spermatozoa from dog, horse, pig and bull were negative with SPERM HY-LITER™, whereas Baecchi's method was non-selective. Data from forensic casework samples in Copenhagen from two years (2008 and 2009) are presented. The samples from 2008 were investigated using Baecchi's method, while those from 2009 were investigated using SPERM HY-LITER™. The frequencies of positive results were similar between the two methods for the two years (27.9% and 32.1% respectively). Analysis of acid phosphatase (ACP) activity for the positive results obtained for these two years does not support the use of a negative ACP result as a prescreen for microscopic analysis for spermatozoa.     

“Recovery™” Vena Cava Filter: Experience in 96 Patients

The purpose of the study was to assess the clinical safety and efficacy of the “Recovery^TM” (Bard) inferior vena cava (IVC) filter. We retrospectively evaluated the clinical and imaging data of patients who had a “Recovery^TM” IVC filter placed between January 2003 and December 2004 in our institution. The clinical presentation, indications, and procedure-related complications during placement and retrieval were evaluated. Follow-up computed tomography (CT) examinations of the abdomen and chest were evaluated for filter-related complications and pulmonary embolism (PE), respectively. “Recovery” filters were placed in 96 patients (72 males and 24 females; age range: 16–87 years; mean: 46 years). Twenty-four patients presented with PE, 13 with deep vein thrombosis (DVT) and 2 with both PE and DVT. The remaining 57 patients had no symptoms of thromboembolism. Indications for filter placement included contraindication to anticoagulation (n = 27), complication of anticoagulation (n = 3), failure of anticoagulation (n = 5), and prophylaxis (n = 61). The device was successfully deployed in the infrarenal (n = 95) or suprarenal (n = 1) IVC through a femoral vein approach. Retrieval was attempted in 11 patients after a mean period of 117 days (range: 24–426). The filter was successfully removed in nine patients (82%). Failure of retrieval was due to technical difficulty (n = 1) and the presence of thrombus in the filter (n = 1). One of the nine patients who had the filter removed developed IVC thrombus after retrieval and another had an intimal tear of the IVC. Follow-up abdominal CT (n = 40) at a mean of 80 days (range: 1–513) showed penetration of the IVC by the filter arms in 11, of which 3 had fracture of filter components. In one patient, a broken arm migrated into the pancreas. Asymmetric deployment of the filter legs was seen in 12 patients and thrombus within the filter in 2 patients. No filter migration or caval occlusion was encountered. Follow-up chest CT (n = 27) at a mean of 63 days (range: 1–386) showed PE in one patient (3%). During clinical follow-up, 12 of 96 patients developed symptoms of PE and only 1 of the 12 had PE on CT. There was no fatal pulmonary embolism in our group of patients following “Recovery” filter placement. However, the current version of the filter is associated with structure weakness, a high incidence of IVC wall penetration, and asymmetric deployment of the filter legs.     

Competition for DNA binding sites using Promega DNA IQ™ paramagnetic beads

The Promega DNA IQ? system is easily amenable to automation and has been an integral part of standard operating procedures for many forensic laboratories including those of the Royal Canadian Mounted Police (RCMP) since 2004. Due to some failure to extract DNA from samples that should have produced DNA using our validated automated DNA IQ?-based protocol, the competition for binding sites on the DNA IQ? magnetic beads was more closely examined. Heme from heavily blooded samples interfered slightly with DNA binding. Increasing the concentration of Proteinase K during lysis of these samples did not enhance DNA recovery. However, diluting the sample lysate following lysis prior to DNA extraction overcame the reduction in DNA yield and preserved portions of the lysates for subsequent manual or automated extraction. Dye/chemicals from black denim lysates competed for binding sites on the DNA IQ? beads and significantly reduced DNA recovery. Increasing the size or number of black denim cuttings during lysis had a direct adverse effect on DNA yield from various blood volumes. The dilution approach was successful on these samples and permitted the extraction of high DNA yields. Alternatively, shortening the incubation time for cell lysis to 30 min instead of the usual overnight at 56 °C prevented competition from black denim dye/chemicals and increased DNA yields.     

Population data of the AmpFlSTR® NGM™ loci in South Portuguese population

Allele frequencies and other relevant forensic parameters for 15 loci studied with AmpFlSTR(R) NGM™ kit were calculated in a population of individuals residing in the south of Portugal. Blood stain samples were obtained from a total of 452 unrelated individuals involved in paternity testing casework. The kit has five loci – D10S1248, D22S1045, D2S441, D1S1656 and D12S391 not present in any other kit used in our laboratory (Powerplex 16 System and Identifiler Plus). In our laboratory, this new kit is used as a screening tool to solve deficient cases as fatherless paternity test, and to help in paternity investigations with only one genetic incompatibility after the use of routine seventeen loci. Furthermore, this five loci included in the European Standard Set are also recommended by the European Network of Forensic Science Institutes “ENFSI” and the European DNA Profiling group “EDNAP”. These studies are necessary to calculate statistical forensic parameters such as power of discrimination, power of exclusion, minimum allele frequency. Statistical parameters such as heterozigoty, homozigoty and combined power of exclusion were determinated. This kind of study is part of the Quality Program for Certificated Forensic Laboratories.