Temperature and time after urine collection affect the detection of phenethylamine, a substance prohibited in sports.

2-Phenylethylamine (phenethylamine) and its derivatives are stimulant drugs, which are prohibited in sports because of their potential performance-enhancing properties. If phenethylamine is detected in an athlete's urine, the athlete may be subjected to serious sanctions, such as disqualification for both domestic and international competitions. Given the serious consequences athletes face for phenethylamine detection, great care should be taken to avoid false positive tests. In forensic medicine, it is widely known that phenethylamine is produced by putrefactive bacteria in autopsy urine samples; it is possible that this process could also occur in an athlete's urine sample without proper storage. In this study, human urine samples were stored at -20, 4, or 22°C for 14 days, and phenethylamine in the samples was quantitatively analyzed using ultra-high-performance liquid chromatography-tandem mass spectrometry. No phenethylamine was detected in urine samples stored at -20°C throughout 14-day period. Nevertheless, phenethylamine was detected after 6 days in these samples stored at 4°C and after only 1 day in samples stored at 22°C. Additionally, the concentration of phenethylamine in these samples increased each day after detection. These results suggest that urine samples should be stored immediately at -20°C after collection when testing athletes for phenethylamine, especially if the sample must be stored for extended period before testing.

Influence of tibial plateau levelling osteotomy on the tensile forces sustained by ligaments in cranial cruciate ligament-intact canine stifles: An ex vivo pilot study.

BACKGROUND: Tibial plateau levelling osteotomy (TPLO) changes the anatomical tibial conformation and might alter the positional relationship of the ligaments comprising the stifle joint. As a result, it is expected to affect the tensile force of the ligaments. However, studies analyzing the details of the effect of osteotomy are limited. OBJECTIVES: To evaluate the influence of TPLO on the tensile force on the stifle ligaments in the intact canine stifle using a six-degree-of-freedom (6-DOF) robotic testing system. METHODS: Eight stifles were categorised into the reference group and nine stifles into the TPLO group. The stifles were then analysed using a 6-DOF robotic joint biomechanical testing system. The stifles were applied 30 N at cranial, caudal, and compression loads and 1 Nm at the internal and external torque loads (the load applied to the tibia relative to the femur) on extension, at 135° and 120°, respectively. The tensile force placed on the cranial cruciate ligament (CrCL), the caudal cruciate ligament, the medial collateral ligament, lateral collateral ligament and the total tensile force placed on the four ligaments was calculated under each load. RESULTS: For the caudal load applied to the tibia relative to the femur, the CrCL tensile force in the TPLO group was lower than that in the reference group at 120° (p = 0.02). The CrCL tensile force in the TPLO group was lower than that in the reference group at 120° (p < 0.01) for the compression load. Regarding the cranial, internal, and external load, the CrCL tensile force remains unchanged between both groups at each angle. CONCLUSIONS: TPLO reduces CrCL tensile force during compression and caudal force application. TPLO may reduce tensile forces contributing to CrCL rupture.

Effects of the Ankle Flexion Angle During Anterior Talofibular Ligament Reconstruction on Ankle Kinematics, Laxity, and In Situ Forces of the Reconstructed Graft.

BACKGROUND: This study aimed to evaluate the effects of the ankle flexion angle during anterior talofibular ligament (ATFL) reconstruction on ankle kinematics, laxity, and in situ force of a graft. METHODS: Twelve cadaveric ankles were evaluated using a 6-degrees of freedom robotic system to apply passive plantar flexion and dorsiflexion motions and multidirectional loads. A repeated measures experiment was designed using the intact ATFL, transected ATFL, and reconstructed ATFL. During ATFL reconstruction (ATFLR), the graft was fixed at a neutral position (ATFLR 0 degrees), 15 degrees of plantar flexion (ATFLR PF15 degrees), and 30 degrees of plantar flexion (ATFLR PF30 degrees) with a constant initial tension of 10 N. The 3-dimensional path and reconstructed graft tension were simultaneously recorded, and the in situ force of the ATFL and reconstructed grafts were calculated using the principle of superposition. RESULTS: The in situ forces of the reconstructed grafts in ATFLR 0 degrees and ATFLR PF 15 degrees were significantly higher than those of intact ankles. The ankle kinematics and laxity produced by ATFLR PF 30 degrees were not significantly different from those of intact ankles. The in situ force on the ATFL was 19.0 N at 30 degrees of plantar flexion. In situ forces of 41.0, 33.7, and 21.9 N were observed at 30 degrees of plantar flexion in ATFLR 0, 15, and 30 degrees, respectively. CONCLUSION: ATFL reconstruction with the peroneus longus (PL) tendon was performed with the graft at 30 degrees of plantar flexion resulted in ankle kinematics, laxity, and in situ forces similar to those of intact ankles. ATFL reconstructions performed with the graft fixed at 0 and 15 degrees of the plantar flexion resulted in higher in situ forces on the reconstructed graft. CLINICAL RELEVANCE: Fixing the ATFL tendon graft at 30 degrees of plantar flexion results in an in situ force closest to that of an intact ankle and avoids the excessive tension on the reconstructed graft.

Biomechanical Effects of Tibial Plateau Levelling Osteotomy on Joint Instability in Normal Canine Stifles: An In Vitro Study.

OBJECTIVE: The aim of the study was to determine the changes in biomechanical characteristics following tibial plateau levelling osteotomy (TPLO) using simulated manual tests. STUDY DESIGN: Twenty-one stifles from healthy Beagle dogs that had undergone TPLO or had not (control) were first tested in the intact form, and then the cranial cruciate ligament (CrCL) was transected in each to provide four test situations: control-intact, control-CrCL-transected, TPLO-intact and TPLO-CrCL-transected. The stifles were then analysed using a robotic joint biomechanical testing system. The craniocaudal drawer, axial rotation and proximal compression tests were applied. RESULTS: The craniocaudal displacement during the drawer test was not significantly different between the control-intact and TPLO-intact. However, the displacement was significantly greater in the TPLO-CrCL-transected than in the control-intact. In the axial rotation test, the internal-external (IE) rotation was significantly greater in the TPLO-intact than in the control-intact. Similarly, the IE rotation was significantly greater in the TPLO-CrCL-transected than in the control-CrCL-transected. In the proximal compression test, craniocaudal displacement was not significantly different among the control-intact, TPLO-intact and TPLO-CrCL-transected. CONCLUSION: These findings suggest that TPLO influences the tension of the collateral ligaments and might generate laxity of the tibiofemoral joint. Instability after the osteotomy might be associated with the progression of osteoarthritis.

Effect of Initial Graft Tension During Anterior Talofibular Ligament Reconstruction on Ankle Kinematics, Laxity, and In Situ Forces of the Reconstructed Graft.

BACKGROUND: Although a variety of surgical procedures for anterior talofibular ligament (ATFL) reconstruction have been reported, the effect of initial graft tension during ATFL reconstruction remains unclear. PURPOSE/HYPOTHESIS: This study investigated the effects of initial graft tension on ATFL reconstruction. We hypothesized that a high degree of initial graft tension would cause abnormal kinematics and laxity. STUDY DESIGN: Controlled laboratory study. METHODS: Twelve cadaveric ankles were tested with a robotic system with 6 degrees of freedom to apply passive plantarflexion and dorsiflexion motions and a multidirectional load. A repeated measures experiment was designed with the intact ATFL, transected ATFL, and reconstructed ATFL at initial tension conditions of 10, 30, 50, and 70 N. The 3-dimensional path and reconstructed graft tension were simultaneously recorded, and the in situ forces of the ATFL and reconstructed graft were calculated with the principle of superposition. RESULTS: Initial tension of 10 N was sufficient to imitate normal ankle kinematics and laxity, which were not significantly different when compared with those of the intact ankles. The in situ force on the reconstructed graft tended to increase as the initial tension increased. In situ force on the reconstructed graft >30 N was significantly greater than that of intact ankles. The in situ force on the ATFL was 19 N at 30° of plantarflexion. In situ forces of 21.9, 30.4, 38.2, and 46.8 N were observed at initial tensions of 10, 30, 50, and 70 N, respectively, at 30° of plantarflexion. CONCLUSION: Approximate ankle kinematic patterns and sufficient laxity, even with an initial tension of 10 N, could be obtained immediately after ATFL reconstruction. Moreover, excessive initial graft tension during ATFL reconstruction caused excessive in situ force on the reconstructed graft. CLINICAL RELEVANCE: This study revealed the effects of initial graft tension during ATFL reconstruction. These data suggest that excessive tension during ATFL reconstruction should be avoided to ensure restoration of normal ankle motion.

A Fatal Case of Poisoning with Fentanyl Transdermal Patches in Japan.

Fentanyl transdermal patches have been used to treat cancer- and noncancer-related chronic pain. However, its inappropriate or illegal application may cause fatal poisoning. We herein present the case of a Japanese woman in her 40s who was found dead with seven 25-µg/h fentanyl transdermal patches on her body. We established a detailed toxicological analysis procedure to quantify fentanyl, and its metabolite norfentanyl, and other drugs (acetaminophen, allylisopropylacetylurea, celecoxib, estazolam, promethazine, and sertraline) in human whole blood by ultra-high-performance liquid chromatography-tandem mass spectrometry. The measured fentanyl and norfentanyl concentrations in the femoral and cardiac blood were 0.051 and 0.072 µg/mL and 0.033 and 0.076 µg/mL, respectively. The decedent's fentanyl concentrations were consistent with previously reported postmortem blood levels for fatal cases of poisoning by fentanyl transdermal patches. Based on the decedent's case history, autopsy findings, and toxicological analyses, the cause of death was identified as intoxication with transdermal fentanyl.

Forensic analysis using ultra-high-performance liquid chromatography-tandem mass spectrometry with solid-phase extraction of α-solanine and α-chaconine in whole blood.

PURPOSE: The potato glycoalkaloids (PGAs), α-solanine and α-chaconine can exert adverse effects on human health when consumed in excess. This study aimed to investigate the optimal extraction method for the quantitative analysis of PGAs in whole blood by using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and to apply this validated method to postmortem blood. METHODS: A total of 200 µL of human whole blood was prepared for PGA extraction. For validation, a solid-phase extraction (SPE) using Oasis® PRiME HLB, in which extraction could be performed in three simple steps (sample loading, washing, and elution) was used, with no need for both conditioning and equilibration of columns for sample preparation. RESULTS: In this method, the limit of detection and the lower limit of quantification (LLOQ) of both α-solanine and α-chaconine were 1 and 2 µg/L, respectively. The calibration curves of the two compounds were obtained with good linearity in the range of 2-100 µg/L. The recovery rates at the LLOQ of α-solanine and α-chaconine were ≥ 91.8% and ≥ 85.9%, respectively. The validation data (intra- and inter-day combined) for accuracy ranged from 93.5 to 106.6% for α-solanine and from 93.9 to 107.7% for α-chaconine. This validated method was successfully applied to one forensic autopsy case, and the concentrations of α-solanine and α-chaconine in the postmortem cardiac blood were 45.1 and 35.5 µg/L, respectively. CONCLUSIONS: This validated UHPLC-MS/MS with SPE for quantitative analysis of PGAs could be useful in forensic toxicology.

ACL Function in Bicruciate-Retaining Total Knee Arthroplasty.

BACKGROUND: Bicruciate-retaining total knee arthroplasty (BCR-TKA) is attracting attention because of the functional and satisfaction outcomes associated with keeping the anterior cruciate ligament (ACL) intact. However, knowledge of the functional importance of the ACL after BCR-TKA is limited. We performed a biomechanical investigation of ACL function following BCR-TKA compared with that in the intact knee. METHODS: We investigated 8 fresh-frozen human cadaveric knees using a 6-degrees-of-freedom robotic system that allowed natural joint motion. Three knee states-intact knee, BCR-TKA, and BCR-TKA with ACL transection (BCR-TKA + ACLT)-were evaluated. For each knee state, the kinematics during passive flexion-extension motion (from 0° to 120°) and anteroposterior laxity at 0°, 15°, 30°, 60°, and 90° of flexion in response to a 100-N load were investigated. The recorded knee motions of the intact and BCR-TKA knees during each test were repeated after ACLT to calculate the ACL in situ force. RESULTS: The femur in the BCR-TKA group translated posteriorly and rotated externally during passive knee flexion and was in an anterior position compared with the femur in the intact-knee state. After ACLT, the femur translated posteriorly, compared with the BCR-TKA group, at 0° and 10° (p < 0.05). The anteroposterior laxities of the BCR-TKA and intact knees were comparable at all flexion angles and increased 2-fold or more after ACLT (p < 0.01). The ACL in situ force in the BCR-TKA knees was 2-fold to 6-fold higher than that in the intact knees at 0°, 15°, 90°, and 120° during a passive path (p < 0.05) and equivalent to that in the intact knees under anterior loading. CONCLUSIONS: The preserved ACL in the BCR-TKA knees was functional, like the ACL in the intact knees, under anterior tibial loading and contributed to good anteroposterior stability. However, the kinematics and ACL in situ force differed between the intact and BCR-TKA knees during passive flexion-extension movements. CLINICAL RELEVANCE: Surgeons may not be able to prevent overtensioning of the ACL during a standardized BCR-TKA procedure, which could potentially limit range of motion.

Effect of Initial Graft Tension During Calcaneofibular Ligament Reconstruction on Ankle Kinematics and Laxity.

BACKGROUND: Although a variety of surgical procedures for lateral ankle ligament reconstruction have frequently been reported, little is known about the effects of initial graft tension. Purpose/Hypothesis: The purpose was to investigate the effects of initial graft tension in calcaneofibular ligament (CFL) reconstruction. It was hypothesized that a high degree of initial graft tension would cause abnormal kinematics, laxity, and excessive graft tension. STUDY DESIGN: Controlled laboratory study. METHODS: Twelve cadaveric ankles were tested with a 6 degrees of freedom robotic system to apply passive plantarflexion-dorsiflexion motion and multidirectional loads. A repeated-measures experiment was designed with the CFL intact, CFL transected, and CFL reconstructed with 4 initial tension conditions (10, 30, 50, and 70 N). The 3-dimensional path and reconstructed graft tension were simultaneously recorded. RESULTS: The calcaneus in CFL reconstruction with an initial tension of 70 N had the most eversion relative to the intact condition (mean eversion translations of 1.2, 3.0, 5.0, and 6.2 mm were observed at initial tensions of 10, 30, 50, and 70 N, respectively). The calcaneus also moved more posteriorly with external rotation as the initial tension increased. The reconstructed graft tension tended to increase as the initial tension increased. CONCLUSION: Ankle kinematic patterns and laxity after CFL reconstruction tended to become more abnormal as the initial graft tension increased at the time of surgery. Moreover, excessive initial graft tension caused excessive tension on the reconstructed graft. CLINICAL RELEVANCE: This study indicated the importance of initial graft tension during CFL reconstruction. Overtensioning during CFL reconstruction should be avoided to imitate a normal ankle.

Fatal Poisoning with Both Dichlorvos and Phenthoate.

Organophosphates are widely used as pesticides. However, organophosphates are occasionally orally ingested to commit suicide. In this case, a man in his late 80s committed suicide by ingesting both dichlorvos and phenthoate. Autopsy findings revealed a characteristic volatile odor from his mouth, stomach, lungs, liver, and kidneys. The esophageal mucosa was denatured and had lost elasticity. Serum cholinesterase activity was 9 IU/L. Toxicological analyses performed using high-performance liquid chromatography-tandem mass spectrometry revealed that dichlorvos concentrations in the left and right cardiac blood samples were 11.6 and 4.6 µg/mL, respectively. Phenthoate concentrations in the left and right cardiac blood samples were 5.8 and 0.51 µg/mL, respectively. The total amounts of dichlorvos and phenthoate in the stomach were 7.35 and 4.55 g, respectively. The case history, autopsy findings, and toxicological analyses indicated that the cause of death was acute fatal poisoning after oral ingestion of both dichlorvos and phenthoate.

Collection of intraoral findings in corpse with small-scale color dental scanner system.

Together with X-ray radiography and the description in the dental chart (odontogram), the collection of intraoral images is extremely important in dental identification. Recently, thanks to advances in digital devices for taking images in the oral cavity, problems with developing images and images being lost due to scanning errors have been minimized. However, in corpses where postmortem rigidity has firmly set in and burned bodies where the jaw has to be forced open, it is difficult to open the jaw enough to allow images to be taken. In addition, collection of intraoral images requires skill. Our goal was to determine the efficacy of a newly developed, small-scale color dental scanner in collecting intraoral images. The results showed that it was comparatively easy to obtain an entire image of the oral cavity with even a minimum degree of jaw opening. This should enable even a non-expert to perform oral image collection.

[Simple quantitation of arsenic by energy dispersive fluorescence X-ray spectrometer using Reinsch's test].

We examined the clinical usefulness of the Reinsch's test for the detection of the small amounts of the heavy metals such as arsenic and mercury using the fluorescence X-ray spectrometry. We tried t o measure various kinds of biological samples, including serum, urine, and gastric contents using this method. 0.4 ml or 1 ml of hydrochloric acid were added to 2 ml of serum or 6 ml urine and gastric content, respectively, and a copper plate (5.0x 0.8 cm) was immersed into this solution. The mixture heated at 90 degrees C by a heating block for 30 minutes. After heating, the copper plate was washed with water and dried. The copper arsenide that stuck to the copper plate due to Reinsch's test dissolved by methanol/ammonia (8:2) solution at 60 degrees C for 15 minutes. A drop gave the solution to a filter paper fluorescence X-ray analysis and completely dried the filter paper, and applied to the fluorescence X-ray spectrometer. As a result, this method showed about 20 times high sensitivity in comparison with the measurement with condition of solution. The minimal detectable limits of rsenic was 0.4 ppm in serum and were 0.2 ppm in rine and gastric content. The calibration curve could be made for 0.5 to 50 ppm. It will take about 90 min for the measurement using this method for the detection of arsenic in biological samples. We showed the usefulness of the Reinsch's test using the fluorescence X-ray spectrometry in the clinical toxicology.

DNA analysis of neonatal human remains wrapped and kept in a vinyl bag for 15 years.

DNA analysis of a newborn baby wrapped and kept in a vinyl bag for 15 years was performed. DNA isolated from the femur and humerus was used to determine the sex and kinship between the infant and the putative parents. Amplification of mtDNA, ABO, HLA, CST3, CST5, VWA, D12S66, D21S11, CSF1PO, TPOX, THO1 and 10Y polymorphisms and the amelogenin gene was carried out. Several mtDNA types were obtained, suggesting that the sample was contaminated by exogenous DNAs. One of the DNA samples obtained from the femur showed an identical mtDNA sequence to that of the mother except for one site, and this pattern was also found in another DNA sample. None of our laboratory personnel had that type, so we thought it was possible that this sample contained the target DNA. However, maternity was denied by the CST3 polymorphism. Finally, we concluded that the sample had been contaminated with exogenous DNA before we started to examine the body. Although it is difficult to determine the sources of this contamination, PCR amplification from highly degraded DNA is very sensitive to such contamination, and we must be even more careful in DNA analysis of such samples than in that of not so severely degraded specimens.

Evaluation of intraoral CCD camera for dental examination in forensic inspection.

This study was performed to assess the effectiveness of an intraoral CCD camera for dental examinations when sufficient jaw opening or adequate lighting cannot be obtained. A handpiece-type intraoral CCD camera (Crystal Cam; GC Corp., Japan) was used for the study. Because a full view taken by the intraoral CCD camera covers only one or two teeth, all the teeth were individually photographed and a view of the dentition assembled on a personal computer. Assuming that the jaw could not be opened widely enough to inspect an occlusal view, a dry skull and a volunteer were restricted to open the mouth and all the teeth were photographed with an intraoral CCD camera. These were compared to intraoral photographs taken by the conventional method using a single-lens reflex camera and mirror. When the intraoral CCD camera was used to photograph teeth, the color tone of metal restorations could be readily identified, but special care was required to identify carious lesions, discoloration of tooth structure, and esthetic restorations. The dentition photographs assembled from the original intraoral CCD images were transferred via the Internet as e-mail attachment files to allow preparation of the dental chart at the destination. Based on the transferred images, it was possible to prepare a dental chart agreeing satisfactorily with actual oral conditions. The easy transfer of digital images provides various advantages in evaluating and discussing certain cases in cooperation with other forensic odontologists via the Internet. The camera may be made more effective or useful through improvement of the tip portion of the camera and the entire system to achieve a more compact design and better portability.