ACR Appropriateness Criteria® Myelopathy

Myelopathy is a problem that requires imaging to distinguish among numerous specifically treatable causes. The first priority is to determine mechanical stability after trauma. Next, it is crucial to distinguish intrinsic disease from extrinsic compression-for example, by epidural abscess. Osteophytes or disc extrusions and metastatic compression are the most common causes of extrinsic lesions. Imaging approaches rely on clinical features such as pain, fever, trauma, and pattern of progression. CT is preferred initially in acute trauma and MRI in all other circumstances. Contrast-enhanced MRI is added when tumor or infection is suspected or with slow or stepwise progression, especially when pain is not prominent. Vascular imaging is used when arteriovenous malformation, fistula, or occlusive disease is suspected. Because the treatment of myelopathy is often complex, treatment planning may require more than one imaging study or sequential examination to assess interval change. The ACR Appropriateness Criteria(?) are evidence-based guidelines for specific clinical conditions that are reviewed every 2 years by a multidisciplinary expert panel. The guideline development and review include an extensive analysis of current medical literature from peer-reviewed journals and the application of a well-established consensus methodology (modified Delphi) to rate the appropriateness of imaging and treatment procedures by the panel. In those instances in which evidence is lacking or not definitive, expert opinion may be used to recommend imaging or treatment.     

Developmental validation of the PowerPlex® ESX 16 and PowerPlex® ESX 17 Systems

We describe the developmental validation study performed on the PowerPlex(?) ESX 16 (European Standard Extended 16) and the PowerPlex(?) ESX 17 Systems, part of a suite of four new DNA profiling kits developed by Promega in response to the ENFSI and EDNAP groups' call for new STR multiplexes for Europe. The PowerPlex(?) ESX 16 System combines the 11 loci compatible with the UK National DNA Database, contained within the AmpFlSTR(?) SGM Plus(?) PCR Amplification Kit, with five additional loci: D2S441, D10S1248, D22S1045, D1S1656 and D12S391. The multiplex was designed to incorporate these five new loci as mini- and midi-STRs while maintaining the loci found in the AmpFlSTR(?) SGM Plus(?) kit as standard size. The PowerPlex(?) ESX 17 System amplifies the same loci as the PowerPlex(?) ESX 16 System, but with the addition of a primer pair for the SE33 locus. Tests were designed to address the developmental validation guidelines issued by the Scientific Working Group on DNA Analysis Methods (SWGDAM), and those of the DNA Advisory Board (DAB). Samples processed include DNA mixtures, PCR reactions spiked with inhibitors, a sensitivity series, and 306 United Kingdom donor samples to determine concordance with data generated with the AmpFlSTR(?) SGM Plus(?) kit. Allele frequencies from 242 white Caucasian samples collected in the United Kingdom are also presented. The PowerPlex(?) ESX 16 and ESX 17 Systems are robust and sensitive tools, suitable for the analysis of forensic DNA samples. Full profiles were routinely observed with 62.5 pg of a fully heterozygous single source DNA template. In mixture analysis, a range of 52-95% of unique minor contributor alleles was observed at 19:1 mixture ratios where only 25 pg of the minor component was present. Improved sensitivity combined with the robustness afforded by smaller amplicons has substantially improved the quantity of information obtained from degraded samples, and the improved chemistry confers exceptional tolerance to high levels of laboratory prepared inhibitors.     

Population genetic data of the AmpFℓSTR® Identifiler® Plus and PowerPlex® 16 HS STR loci in four Canadian populations

Allele frequencies and forensically relevant population statistics were estimated for the short tandem repeat (STR) loci of the AmpF?STR® Identifiler® Plus and PowerPlex® 16 HS amplification kits, including D2S1338, D19S433, Penta D, and Penta E, for three First Nations Aboriginal populations and for Caucasians in Canada. The cumulative power of discrimination was ≥0.999999999999984 and the cumulative power of exclusion was ≥0.999929363 for both amplification systems in all populations. No significant departure from Hardy–Weinberg equilibrium was detected for D2S1338, D19S433, Penta D, and Penta E or the 13 Combined DNA Index System core STR loci after correction for multiple testing. Significant genetic diversity was observed between these four populations. Comparison with published frequency data for other populations is also presented.     

Attentive processes,blood lactate and CrossFit®

Objectives: To analyze the influences of blood lactate produced during a specific session of CrossFit® on intensity and selectivity of attention. The first was evaluated by measuring the reaction time and the second by analyzing divided attention with a dual task.

Methods: Fifteen male professionals of CrossFit® volunteered in the study. The training session was the Workout Of the Day (WOD) called 15.5, marked as: 27–21–15–9 repetitions (without recovery) in term of calories measured by using a rowing ergometer (e.g. 27 rowed calories) and in term of barbell full squats (raising a weight of 43 kg for men and of 29.5 kg for women). Blood lactate, blood glucose, reaction time, execution time of a dual task, number of errors and number of omissions were measured at rest, at the conclusion of the session and 15 minutes after its end.

Results: The levels of the blood lactate before the start of the session were considerably higher than those which normally occur at rest (<2 mmol /L), with a mean value of 4.5 mmol /l (± 1.99 SD). At the end of the workout session the blood lactate exhibited a significant increase, reaching a mean value of 13.8 mmol /l (± 1.18 SD) and then returning to values similar to the initial ones after 15 minutes. Blood glucose did not exhibit any statistically significant differences during the session. Reaction time, execution time, number of errors and number of omissions exhibited a significant worsening concomitantly with the increase in blood lactate.

Conclusion: Athletes practicing CrossFit®, with high levels of blood lactate even at rest, should consequently have attentional performances somewhat limited.     

Optimization and validation of a fast amplification protocol for AmpFlSTR® Profiler Plus® for rapid forensic human identification

The goal of this work was to optimize and validate a fast amplification protocol for the multiplex amplification of the STR loci included in AmpFlSTR(?) Profiler Plus(?) to expedite human DNA identification. By modifying the cycling conditions and by combining the use of a DNA polymerase optimized for high speed PCR (SpeedSTAR? HS) and a more efficient thermal cycler instrument (Bio-RAD C1000?), we were able to reduce the amplification process from 4h to 26 min. No modification to the commercial AmpFlSTR(?) Profiler Plus(?) primer mix was required. When compared to the current Royal Canadian Mounted Police (RCMP) amplification protocol, no differences with regards to specificity, sensitivity, heterozygote peak height ratios and overall profile balance were noted. Moreover, complete concordance was obtained with profiles previously generated with the standard amplification protocol and minor alleles in mixture samples were reliably typed. An increase in n-4 stutter ratios (2.2% on average for all loci) was observed for profiles amplified with the fast protocol compared to the current procedure. Our results document the robustness of this rapid amplification protocol for STR profiling using the AmpFlSTR(?) Profiler Plus(?) primer set and demonstrate that comparable data can be obtained in substantially less time. This new approach could provide an alternative option to current multiplex STR typing amplification protocols in order to increase throughput or expedite time-sensitive cases.     

Evaluation and comparative analysis of direct amplification of STRs using PowerPlex® 18D and Identifiler® Direct systems

Short tandem repeat (STR) analysis remains the primary forensic tool for DNA identification. Because of the success of forensic DNA typing and the use of database searches to develop investigative leads, there is an increased demand for populating forensic DNA databases. Reference samples tend to be of high quantity and quality and are somewhat standardized in format. Being more predictable in quality than unknown forensic casework samples, reference samples lend themselves to alternate methods of analysis such as direct amplification. Two commercially available direct amplification kits for processing reference samples were evaluated. The kits are PowerPlex^® 18D (Promega Corp., Madison, Wisconsin) and Identifiler^® Direct (Life Technologies, Carlsbad, CA). Both kits offer the core CODIS loci plus amelogenin, and the loci D2S1338, D19S433. The PP18D kit offers two additional loci, Penta E and Penta D. To determine the robustness and reliability of the PP18D and ID Direct amplification systems, buccal cell samples (deposited on FTA paper using the EasiCollect? device (Florham Park, NJ)) from 400 anonymous donors were analyzed under conditions to achieve a high rate of successful typing. First-pass success rates were 96.25%, 96.25%, and 95% for PP18D with a 5 s injection, ID Direct with a 10 s injection, and ID Direct with a 5 s injection, respectively. Profiles that could not be typed were not a result of the kits’ performance but were a result of the inherent variation in the amount of DNA obtained with the collection device and buccal cells. Low signal profiles can be re-analyzed by either re-injecting for a longer time or by re-amplification with an additional PCR cycle. Overloaded profiles can be re-analyzed by re-injecting for a shorter time or by re-amplification with one less cycle. All called typing results, when interpretable, were consistent under the prescribed conditions, different injection times, and 26-28 PCR cycles for both chemistries. Peak height ratios for both kits were well balanced with peaks ranging in height >2000 RFUs to those with one or more peaks with heights <100 RFUs. A change in the ILS morphology sloping downward to the right relative to a normal ILS profile for PP18D and ID Direct was an indication of a poor injection. Re-injection effectively overcame the effect manifested by a sloping ILS phenomenon. A subset of samples were subjected to direct amplification using the reagents in Identifiler^® Plus kit and successful typing results were obtained for the majority of samples. However, the profiles displayed increased amounts of non-adenylated products. The results of this study demonstrate that PP18D and ID Direct are both robust kits for direct amplification. The interpretation guidelines used for this study can form a basis for internal validation studies by databasing laboratories.     

Comprehensive mutation analysis of 17 Y-chromosomal short tandem repeat polymorphisms included in the AmpFlSTR® Yfiler® PCR amplification kit

The Y-chromosomal short tandem repeat (Y-STR) polymorphisms included in the AmpFlSTR® Yfiler® polymerase chain reaction amplification kit have become widely used for forensic and evolutionary applications where a reliable knowledge on mutation properties is necessary for correct data interpretation. Therefore, we investigated the 17 Yfiler Y-STRs in 1,730–1,764 DNA-confirmed father–son pairs per locus and found 84 sequence-confirmed mutations among the 29,792 meiotic transfers covered. Of the 84 mutations, 83 (98.8%) were single-repeat changes and one (1.2%) was a double-repeat change (ratio, 1:0.01), as well as 43 (51.2%) were repeat gains and 41 (48.8%) repeat losses (ratio, 1:0.95). Medians from Bayesian estimation of locus-specific mutation rates ranged from 0.0003 for DYS448 to 0.0074 for DYS458, with a median rate across all 17 Y-STRs of 0.0025. The mean age (at the time of son’s birth) of fathers with mutations was with 34.40 (±11.63) years higher than that of fathers without ones at 30.32 (±10.22) years, a difference that is highly statistically significant (p?<?0.001). A Poisson-based modeling revealed that the Y-STR mutation rate increased with increasing father’s age on a statistically significant level (α?=?0.0294, 2.5% quantile?=?0.0001). From combining our data with those previously published, considering all together 135,212 meiotic events and 331 mutations, we conclude for the Yfiler Y-STRs that (1) none had a mutation rate of >1%, 12 had mutation rates of >0.1% and four of <0.1%, (2) single-repeat changes were strongly favored over multiple-repeat ones for all loci but 1 and (3) considerable variation existed among loci in the ratio of repeat gains versus losses. Our finding of three Y-STR mutations in one father–son pair (and two pairs with two mutations each) has consequences for determining the threshold of allelic differences to conclude exclusion constellations in future applications of Y-STRs in paternity testing and pedigree analyses.     

Spektrographische Untersuchungen zur Strahlenresistenz von Miltex® und Miltefosin

Aim

With simultaneous application of Miltex^® and radiation therapy in the combined treatment of topical relapses and skin metastases in breast carcinoma patients the question arises, how radioresistant is the new cytotoxic agent. Because of the long penetration times of the active agent Miltefosine the answer is important with particular regard to the time of the external application of Miltex^®.

Material and Method

After the application of a single dose of 10 Gy we studied the stability of the commercial preparation and its active agent Miltefosine by means of absorption spectroscopy.

Results

Immediately following the irradiation no alterations in absorption spectra of Miltex^® and Miltefosine were found. However, 2 and 8 h post radiation the absorption curves of Miltex^® and Miltefosine solutions were distinctly changed. The radiation induced changes of Miltex^® dilutions were smaller than those of the Miltefosine solutions. For the commercial preparation the amount of the radiation-induced destruction is 0.10.

Conclusions

Consequently Miltex^® has shown a sufficient radioresistance or its decrease in the effectiveness is small. With daily single doses of 2 Gy in the radiotherapy of the topical relapses and skin metastases the destruction degree should be reduced to 0.02 assuming linear changes. Because of the distinct changes in the spectra and relative slow penetration of Miltefosine in various cell lines [10, 11, 14] we will propose an application of the commercial cytotoxic agent 5 h before the radiation fractions. The smaller effect on Miltex^® is discussed in relation to the solution mediators of the active agent.     

DNA extraction method from bones using Maxwell® 16

This paper describes the automated purification of DNA extracted from human bones using Maxwell® 16 bench top instrument. Analysis of nuclear short tandem repeats (STR) is invaluable in identification of human remains exhumed from mass graves in Croatia. Up to today 4683 skeletal remains have been recovered and for 897 human remains identity has not been determined. DNA has been extracted from 70% of all unidentified samples. For more than 90% of the samples nuclear STR profiles have been obtained using either organic phenol/chloroform method or silica-column purification for the extraction of DNA from bones or teeth. In order to evaluate a Maxwell® 16 DNA extraction performance 40 bone samples with different stage of decomposition were analyzed. The efficacy of manual silica based extraction and an automated purification was compared. The DNA yield per gram of starting material, removal of inhibitors and the quality of resulting STR profiles of the Maxwell extracts from duplicate amplifications were evaluated. The results show that Maxwell 16 platform can be used instead of manual DNA extraction procedures.     

ACR Appropriateness Criteria® Acute Trauma to the Knee

There are more than 1 million visits to the ER annually in the United States for acute knee trauma. Many of these are twisting injuries in young patients who can walk and bear weight, and emergent radiography is not required. Several clinical decision rules have been devised that can considerably reduce the number of radiographic studies ordered without missing a clinically significant fracture. Although fractures are seen on only 5% of emergency department knee radiographs, 86% of knee fractures result from blunt trauma. In patients with falls or twisting injuries who have focal tenderness, effusion, or inability to bear weight, radiography should be the first imaging study performed. If radiography shows no fracture, MRI is best for evaluating for a suspected meniscal or ligament tear or patellar dislocation. Patients with knee dislocation should undergo radiography and MRI, as well as fluoroscopic angiography, CT angiography, or MR angiography. The ACR Appropriateness Criteria(?) are evidence-based guidelines for specific clinical conditions that are reviewed every 2 years by a multidisciplinary expert panel. The guideline development and review include an extensive analysis of current medical literature from peer-reviewed journals and the application of a well-established consensus methodology (modified Delphi) to rate the appropriateness of imaging and treatment procedures by the panel. In those instances in which evidence is lacking or not definitive, expert opinion may be used to recommend imaging or treatment.     

Korean population genetic data and concordance for the PowerPlex® ESX 17, AmpFlSTR Identifiler®, and PowerPlex® 16 systems

In case of paternity or maternity investigations with short tandem repeat (STR) analysis, deficient cases, missing person, or mutations are encountered and common STRs cannot provide sufficient forensic parameters. Thus, it is recommended that additional STRs are needed to complement conventional analysis for more reliable forensic information. We analyzed variation of 23 STRs contained in the new PowerPlex^® ESX 17 kit (Promega) and two conventional kits of the AmpFlSTR Identifiler^® (Applied Biosystems) and PowerPlex^® 16 systems (Promega) in 452 randomly chosen individuals from Korea to provide an expanded and reliable Korean database. Allele frequencies and forensic parameters were used to evaluate suitability and robustness of the new kit for forensic genetic analysis as well as in concordance studies. The combined probability of match for the 16 loci in the PowerPlex^® ESX 17 system was 2.76 × 10⁻²⁰. One genotyping discrepancy due to a null allele was observed at the D18S51 locus (the concordant rate = 99.99%), showing a primer-binding site mutation in the sequence of the locus (G-to-A substitution at position 146 of Genbank accession number JX018211). Thus, the new kit is a valuable forensic tool and is suitable to extend the Korean population genetic data obtained with well-established polymerase chain reaction multiplex-kits of the AmpFlSTR Identifiler^® and PowerPlex^® 16 systems.     

Prototype PowerPlex® Y23 System: A concordance study

The Prototype PowerPlex^® Y23 System (Promega Corporation, Madison, WI) is a polymerase chain reaction-based amplification kit that targets the 23 Y STR loci DYS19, DYS385a/b, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS481, DYS533, DYS549, DYS570, DYS576, DYS635, DYS643, and Y-GATA-H4. A total of 951 samples from six populations were typed to evaluate the kit and examine concordance for 17 of the loci that are in common with those that can be typed using the AmpFlSTR^® Yfiler™ kit (Life Technologies, Carlsbad, CA). A total of 16,167 loci were analyzed for each multiplex, and overall concordance was observed. Because of different kit designs, and although concordant for the genetic type, discordant calls can occur due to a deletion at the DYS448 locus. Users should take into consideration such nomenclature anomalies when comparing Y STR profiles. This new kit allows a large battery of Y STR loci to be analyzed using the same basic technologies already employed in forensic laboratories.     

搭建CTCT®线上平台助力创伤中心建设

中国创伤救治培训(CTCT?)自2016年7月启动,迄今已近5年,在全国同道的大力支持下高速推进,已经建设了标准版、基层版、护理版和主任版四位一体的课程体系,极大地促进了各地创伤中心和体系的建设.2020年,新冠病毒大流行,前所未有地改变了医院工作环境、学术交流和任职教育等,也增加了各地创伤中心建设的难度,作为极具中国特色的CTCT?,积极适应新形势、新常态,在CTCT?相关微信群的基础上,2020年10月推出了"创伤救治云端论道",并将于2021年上半年正式推出CTCT?在线学院.本文小结了CTCT?进展,并介绍了"微信CTCT?在线学院+腾讯会议云端论道案例讨论+CTCT?微信群"三位一体的CTCT?线上课程建设框架,为各位同道积极参与线上交流提供借鉴.     

ACR Appropriateness Criteria® Acute Respiratory Illness in Immunocompromised Patients

The respiratory system is often affected by complications of immunodeficiency, typically manifesting clinically as acute respiratory illness. Ongoing literature reviews regarding the appropriateness of imaging in these patients are critical, as advanced medical therapies such as stem cell transplantation, chemotherapy, and immunosuppressive therapies for autoimmune disease continue to keep high the population of immunosuppressed patients in our health care system today. This ACR Appropriateness Criteria(?) topic describes clinical scenarios of acute respiratory illness in immunocompromised patients with cough, dyspnea, chest pain, and fever; in those with negative, equivocal, or nonspecific findings on chest radiography; in those with diffuse or confluent opacities on chest radiography; and in those in whom noninfectious disease is suspected. The use of chest radiography, chest CT, transthoracic needle biopsy, and nuclear medicine imaging are all discussed in the contexts of these clinical scenarios. The ACR Appropriateness Criteria are evidence-based guidelines for specific clinical conditions that are reviewed every 2 years by a multidisciplinary expert panel. The guideline development and review include an extensive analysis of current medical literature from peer-reviewed journals and the application of a well-established consensus methodology (modified Delphi) to rate the appropriateness of imaging and treatment procedures by the panel. In those instances in which evidence is lacking or not definitive, expert opinion may be used to recommend imaging or treatment.