The effect of liquid-liquid extraction on metabolite detection and analysis using NMR spectroscopy in human synovial fluid.

The evaluation of joint disease using synovial fluid is an emerging field of metabolic profiling. The analysis is challenged by multiple macromolecules which can obscure the small molecule chemistry. The use of protein precipitation and extraction has been evaluated previously, but not in synovial fluid. We systematically review the published NMR spectroscopy methods of synovial fluid analysis and investigated the efficacy of three different protein precipitation techniques: methanol, acetonitrile and trichloroacetic acid. The trichloroacetic wash removed the most protein. However, metabolite recoveries were universally very poor. Acetonitrile liquid/liquid extraction gave metabolite gains from four unknown compounds with spectral peaks at δ = 1.91 ppm, 3.64 ppm, 3.95 ppm & 4.05 ppm. The metabolite recoveries for acetonitrile were between 1.5 and 7 times higher than the methanol method, across all classes of metabolite. The methanol method was more effective in removing protein as reported by the free GAG undefined peak (44 % vs 125 %). However, qualitative evaluation showed that acetonitrile and methanol provided good restoration of the spectra to baseline. The methanol extraction has issues of a gelatinous substrate in the samples. All metabolite recoveries had a CV of > 15 %. A recommendation of acetonitrile liquid/liquid extraction was made for human synovial fluid (HSF) analysis. This is due to consistency, effective protein precipitation, recovery of metabolites and additional compounds not previously visible.

The influence of sample collection, handling and low temperature storage upon NMR metabolic profiling analysis in human synovial fluid.

The impact of metabolism upon the altered pathology of joint disease is rapidly becoming recognized as an important area of study. Synovial joint fluid is an attractive and representative biofluid of joint disease. A systemic review revealed little evidence of the metabolic stability of synovial joint fluid collection, handling or storage, despite recent reports characterizing the metabolic phenotype in joint disease. We aim to report the changes in small molecule detection within human synovial fluid (HSF) using nuclear magnetic resonance (NMR) spectroscopy at varying storage temperatures, durations and conditions. HSF was harvested by arthrocentesis from patients with isolated monoarthropathy or undergoing joint replacement (n = 30). Short-term storage (0-12 h, 4°C & 18°C) and the effect of repeated freeze-thaw cycles (-80°C to 18°C) was assessed. Long-term storage was evaluated by early (-80°C, <21days) and late analysis (-80°C, 10-12 months). 1D NMR spectroscopy experiments, NOESYGPPR1D and CPMG identified metabolites and semi-quantification was performed. Samples demonstrated broad stability to freeze-thaw cycling and refrigeration of <4 h. Short-term room temperature or refrigerated storage showed significant variation in 2-ketoisovalerate, valine, dimethylamine, succinate, 2-hydroxybutyrate, and acetaminophen glucuronide. Lipid and macromolecule detection was variable. Long-term storage demonstrated significant changes in: acetate, acetoacetate, creatine, N,N-dimethylglycine, dimethylsulfone, 3-hydroxybutyrate and succinate. Changeable metabolites during short-term storage appeared to be energy-synthesis intermediates. Most metabolites were stable for the first four hours at room temperature or refrigeration, with notable exceptions. We therefore recommend that HSF samples should be kept refrigerated for no more than 4 hours prior to freezing at -80°C. Furthermore, storage of HSF samples for 10-12 months before analysis can affect the detection of selected metabolites.

Differences between infected and noninfected synovial fluid.

AIMS: The diagnosis of joint infections is an inexact science using combinations of blood inflammatory markers and microscopy, culture, and sensitivity of synovial fluid (SF). There is potential for small molecule metabolites in infected SF to act as infection markers that could improve accuracy and speed of detection. The objective of this study was to use nuclear magnetic resonance (NMR) spectroscopy to identify small molecule differences between infected and noninfected human SF. METHODS: In all, 16 SF samples (eight infected native and prosthetic joints plus eight noninfected joints requiring arthroplasty for end-stage osteoarthritis) were collected from patients. NMR spectroscopy was used to analyze the metabolites present in each sample. Principal component analysis and univariate statistical analysis were undertaken to investigate metabolic differences between the two groups. RESULTS: A total of 16 metabolites were found in significantly different concentrations between the groups. Three were in higher relative concentrations (lipids, cholesterol, and N-acetylated molecules) and 13 in lower relative concentrations in the infected group (citrate, glycine, glycosaminoglycans, creatinine, histidine, lysine, formate, glucose, proline, valine, dimethylsulfone, mannose, and glutamine). CONCLUSION: Metabolites found in significantly greater concentrations in the infected cohort are markers of inflammation and infection. They play a role in lipid metabolism and the inflammatory response. Those found in significantly reduced concentrations were involved in carbohydrate metabolism, nucleoside metabolism, the glutamate metabolic pathway, increased oxidative stress in the diseased state, and reduced articular cartilage breakdown. This is the first study to demonstrate differences in the metabolic profile of infected and noninfected human SF, using a noninfected matched cohort, and may represent putative biomarkers that form the basis of new diagnostic tests for infected SF. Cite this article: Bone Joint Res 2021;10(1):85-95.

Can joint fluid metabolic profiling (or "metabonomics") reveal biomarkers for osteoarthritis and inflammatory joint disease?: A systematic review.

AIMS: Metabolic profiling is a top-down method of analysis looking at metabolites, which are the intermediate or end products of various cellular pathways. Our primary objective was to perform a systematic review of the published literature to identify metabolites in human synovial fluid (HSF), which have been categorized by metabolic profiling techniques. A secondary objective was to identify any metabolites that may represent potential biomarkers of orthopaedic disease processes. METHODS: A systematic review was conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines using the MEDLINE, Embase, PubMed, and Cochrane databases. Studies included were case series, case control series, and cohort studies looking specifically at HSF. RESULTS: The primary analysis, which pooled the results from 17 published studies and four meeting abstracts, identified over 200 metabolites. Seven of these studies (six published studies, one meeting abstract) had asymptomatic control groups and collectively suggested 26 putative biomarkers in osteoarthritis, inflammatory arthropathies, and trauma. These can broadly be categorized into amino acids plus related metabolites, fatty acids, ketones, and sugars. CONCLUSION: The role of metabolic profiling in orthopaedics is fast evolving with many metabolites already identified in a variety of pathologies. However, these results need to be interpreted with caution due to the presence of multiple confounding factors in many of the studies. Future research should include largescale epidemiological metabolic profiling studies incorporating various confounding factors with appropriate statistical analysis to account for multiple testing of the data.Cite this article: Bone Joint Res. 2020;9(3):108-119.

Scapula Fractures in Elite Soccer and Rugby Players.

BACKGROUND: Scapula fractures are uncommon in sports and are poorly understood in this patient group. PURPOSE: To report on scapula fractures in contact and collision athletes and assess the injury patterns of different mechanisms of injury. STUDY DESIGN: Case series; Level of evidence, 4. METHODS: A retrospective case series was performed of all sports-related scapula fractures treated at a single institution between 2007 and 2015. The mechanisms of injury were divided into direct lateral impact, fall onto an outstretched arm, or abduction/external rotation. RESULTS: A total of 11 patients were identified: 9 professional rugby players, 1 professional soccer player, and 1 amateur soccer player. The mean age was 28 years (range, 18-35 years). The mean return to play was 127 days in those treated nonoperatively and 163 days in those treated operatively. A direct impact mechanism occurred in 7 patients, all of whom sustained glenoid neck and body fractures and were treated nonoperatively. Two rugby players had a concomitant suprascapular nerve injury. An outstretched arm mechanism occurred in 2 cases, leading to posterior and inferior glenoid fractures. Both patients were treated operatively. An abduction/external rotation mechanism occurred in 2 cases, resulting in an anteroinferior and an anterior glenoid rim fracture. One case was treated operatively and the other was treated nonoperatively. Of those with glenoid fractures, 75% were not visible on plain radiographs and required further imaging. CONCLUSION: Scapula fractures acquired in sports are a serious injury with a prolonged recovery period. The mechanism of injury can help predict the injury pattern and highlight the need for further imaging. There is a high association with suprascapular nerve injuries.

Knee Arthroscopy: A Simulation Demonstrating the Imperial Knee Arthroscopy Cognitive Task Analysis (IKACTA) Tool.

BACKGROUND: Virtual reality and cadaveric simulations are expensive and not readily accessible1. Innovative and accessible training adjuncts are required to help meet training needs. Cognitive task analysis (CTA) has been used extensively to train pilots and surgeons in other surgical specialties2-6. However, the use of CTA tools within orthopaedics is in its infancy. Arthroscopic procedures are minimally invasive and require a different skill set compared with open surgery. Residents often feel poorly prepared to perform this in the operating room because of the steep learning curve associated with acquiring basic arthroscopic skills. We designed the Imperial Knee Arthroscopy Cognitive Task Analysis (IKACTA) tool, which is, to our knowledge, the first CTA tool described in the orthopaedic literature, demonstrating significant objective benefits in training novices to perform diagnostic knee arthroscopy. DESCRIPTION: The IKACTA tool, which is the combination of the written description of the phases below and the videos (with superimposed audio recordings) of each phase, utilizes simultaneous written and audiovisual modalities to teach diagnostic knee arthroscopy. The procedure was divided into 7 phases: (1) operating room and patient setup, (2) preparation and draping, (3) anterolateral portal placement, (4) examination of the patellofemoral joint and the lateral gutter, (5) examination of the medial compartment and anteromedial portal placement, (6) examination of the intercondylar notch and the lateral compartment, and (7) postoperative care and rehabilitation.For each phase, there are sections on the technical steps, cognitive decision-making behind each technical step, and potential errors and solutions. Video clips recorded by an expert surgeon in the operating room specific to each phase and audio voice recordings explaining each phase superimposed on the video clips were combined with the written information to design the IKACTA tool. ALTERNATIVES: Not applicable. RATIONALE: This learning tool allows a trainee to learn each technical step, the cognitive decision-making underpinning each step, and potential errors and solutions relevant to each phase of the procedure. Furthermore, the learner can use written and audiovisual modalities simultaneously to learn this technique by reading the written component of the tool first and then watching the relevant video clips with the audio recordings for each phase of the procedure. Alternative training techniques currently include the traditional apprenticeship model, which is becoming increasingly insufficient in the current environment of reduced training hours7,8. Adjuncts to this model are essential to help meet training needs. The IKACTA tool has demonstrated significant objective benefits for novice trainees to learn diagnostic knee arthroscopy9. The idea behind this learning tool is for the trainee surgeon to use this tool independent of the trainer, prior to attending the operating room. The tool provides trainees with knowledge and cognitive understanding of the procedural steps before they perform this procedure on patients. They are aware of potential errors and methods to avoid or overcome these errors. We believe that this tool will reduce the initial difficult phase of the learning curve for junior residents and, therefore, will improve training efficiency in the operating room.

The Avon Patellofemoral Joint Replacement: Mid-Term Prospective Results from an Independent Centre.

BACKGROUND: Approximately 10% of patients with osteoarthritis (OA) of the knee have unicompartmental OA confined to the patellofemoral joint (PFJ). The main surgical options are total knee replacement (TKR) and PFJ replacement (PFJR). PFJR has a number of advantages over TKR, including being less invasive, preserving the unaffected parts of the knee, allowing faster recovery and better range of motion and function. We report our prospective mid-term results of the Avon PFJR for established isolated PFJ arthritis in 61 consecutive procedures. METHODS: Sixty-one Avon PFJRs were performed in 57 patients. The outcome measures were the new Oxford knee score (OKS), Hungerford and Kenna score (HKS), and Crosby Insall knee scores. Only patients with severe isolated PFJ OA were included. The diagnosis was based on a combination of clinical, radiological and, where available, arthroscopic findings. RESULTS: Mean follow-up was 5.09 years (range, 12 to 124 years). There were 2 revisions in the first 5 years. The median HKS score was 80 (interquartile range, 70 to 95) and the mean OKS was 31.8 (± standard deviation, 8.7) at 5 years. These were significantly better (p < 0.001) than the preoperative scores. CONCLUSIONS: The Avon prosthesis gives good functional outcomes in the medium term and survives well. Our data support other studies in the literature and is the largest independent prospective study to date.

Open reduction internal fixation of lateral humeral condyle fractures in children. A series of 105 fractures from a single institution.

Lateral humeral condyle fractures account for 17 % of the distal humeral condyle fractures. Displaced and/or rotated fractures require appropriate reduction and stabilisation. There are, however, a number of controversies in the surgical management of these patients. The aim of the present study was to review the results of patients with a displaced lateral humeral condyle fracture treated with open reduction and internal fixation (ORIF). We retrospectively reviewed children treated with ORIF of lateral humeral condyle fractures at a single institution over a period of 13 years. All cases were identified through the trauma register. Case notes and radiographs were retrieved. Fracture classification, mode of fixation, time to union, and final outcomes at the latest follow-up were reviewed. One hundred and five lateral condyle fractures were identified in 76 male and 29 female patients. Average age was 6.2 years. Ninety-two were Milch type II and 13 Milch type I. According to the Jacob's classification, 38 were type II and 67 type III. All fractures were treated with open reduction and fixation with K-wires. Average time to radiological union was 33 days. Follow-up ranged between 2 and 8 years (average 3.2 years). Radiological hypertrophy of the lateral condyle was present in 45 cases (42 %). Three patients developed a pseudo-cubitus varus deformity. Further four patients developed a true cubitus varus. There was one case of superficial infection of the K-wires and one case of delayed union. At the latest follow-up, 96 % of the patients achieved an excellent final result and 4 % a good final result. Our results demonstrate that fracture union and excellent final outcomes can be expected in all patients using our protocol, whereby all patients with a displaced fracture are managed by ORIF with K-wire fixation, with the wires only being removed after there is evidence of radiological union. Compared to recent reports of closed reduction internal fixation, this series demonstrates good results with no complications directly relating to the open reduction technique. Level of evidence Case series, Level IV.

Hip pathology in Hutchinson-Gilford progeria syndrome: a report of two children.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder. The estimated incidence is one in 4 million births. Orthopaedic manifestations include abnormality of the hips occurring early in the disease process. Severe coxa valga can be apparent by the age of 2 years. We report two cases of HGPS, one in a 7-year-old girl with avascular necrosis of the left hip and the second in a 13-year-old girl with recurrent traumatic hip dislocations. We demonstrate the pathoanatomical changes in the hip with HGPS using a combination of imaging modalities including radiographic, computed tomographic and MRI scans. These include coxa magna, coxa valga and acetabular dysplasia. We also comment on how these would affect the surgical management of this high-risk group of patients.

Chylous ascites following radical nephrectomy: a case report.

INTRODUCTION: Chylous ascites may result from diverse pathologies. Ascites results either due to blockage of the lymphatics or leak secondary to inadvertent trauma during surgery. CASE PRESENTATION: We report the first case of chylous ascites following radical nephrectomy for a renal cell carcinoma involving the right half of a crossed fused renal ectopia. The patient was managed conservatively. CONCLUSION: Post-operative chylous ascites is a rare complication of retroperitoneal and mediastinal surgery. Most cases resolve with conservative treatment which aims at decreasing lymph production and optimizing nutritional requirements along with palliative measures. Refractory cases need either open or laparoscopic ligation of the leaking lymphatic channels. A review of the current literature on the management of post-operative chylous ascites is presented.