Dysfunctional mitochondria, disrupted levels of reactive oxygen species, and autophagy in B cells from common variable immunodeficiency patients.

Introduction: Common Variable Immunodeficiency (CVID) patients are characterized by hypogammaglobulinemia and poor response to vaccination due to deficient generation of memory and antibody-secreting B cells. B lymphocytes are essential for the development of humoral immune responses, and mitochondrial function, hreactive oxygen species (ROS) production and autophagy are crucial for determining B-cell fate. However, the role of those basic cell functions in the differentiation of human B cells remains poorly investigated. Methods: We used flow cytometry to evaluate mitochondrial function, ROS production and autophagy processes in human naïve and memory B-cell subpopulations in unstimulated and stimulated PBMCs cultures. We aimed to determine whether any alterations in these processes could impact B-cell fate and contribute to the lack of B-cell differentiation observed in CVID patients. Results: We described that naïve CD19+CD27- and memory CD19+CD27+ B cells subpopulations from healthy controls differ in terms of their dependence on these processes for their homeostasis, and demonstrated that different stimuli exert a preferential cell type dependent effect. The evaluation of mitochondrial function, ROS production and autophagy in naïve and memory B cells from CVID patients disclosed subpopulation specific alterations. Dysfunctional mitochondria and autophagy were more prominent in unstimulated CVID CD19+CD27- and CD19+CD27+ B cells than in their healthy counterparts. Although naïve CD19+CD27- B cells from CVID patients had higher basal ROS levels than controls, their ROS increase after stimulation was lower, suggesting a disruption in ROS homeostasis. On the other hand, memory CD19+CD27+ B cells from CVID patients had both lower ROS basal levels and a diminished ROS production after stimulation with anti-B cell receptor (BCR) and IL-21. Conclusion: The failure in ROS cell signalling could impair CVID naïve B cell activation and differentiation to memory B cells. Decreased levels of ROS in CVID memory CD19+CD27+ B cells, which negatively correlate with their in vitro cell death and autophagy, could be detrimental and lead to their previously demonstrated premature death. The final consequence would be the failure to generate a functional B cell compartment in CVID patients.

Facemask analyses for the non-invasive detection of chronic and acute P. aeruginosa lung infections using nanoparticle-based immunoassays.

Pseudomonas aeruginosa (P. aeruginosa) is a pathogen that persistently colonizes the respiratory tract of patients with chronic lung diseases. The risk of acquiring a chronic P. aeruginosa infection can be minimized by rapidly detecting the pathogen in the patient's airways and promptly administrating adequate antibiotics. However, the rapid detection of P. aeruginosa in the lungs involves the analysis of sputum, which is a highly complex matrix that is not always available. Here, we propose an alternative diagnosis based on analyzing breath aerosols. In this approach, nanoparticle immunosensors identify bacteria adhered to the polypropylene layer of a surgical facemask that was previously worn by the patient. A polypropylene processing protocol was optimized to ensure the efficient capture and analysis of the target pathogen. The proposed analytical platform has a theoretical limit of detection of 105 CFU mL-1 in aerosolized mock samples, and a dynamic range between 105 and 108 CFU mL-1. When tested with facemasks worn by patients, the biosensors were able to detect chronic and acute P. aeruginosa lung infections, and to differentiate them from respiratory infections caused by other pathogens. The results shown here pave the way to diagnose Pseudomonas infections at the bedside, as well as to identify the progress from chronic to acute infection.

Medial gastrocnemius rotational flap for the reconstruction of extensor mechanism disruption in periprosthetic knee infections: a retrospective series with minimum two year follow-up.

PURPOSE: Extensor mechanism disruption following total knee arthroplasty has a prevalence ranging from 0.3 to 3%. Its management is challenging, especially in case of associated infection of the prosthetic implant. Surgical options are limited due to the septic process, and the use of allograft or synthetic mesh are not recommended. The aim of this study was to report clinical outcomes, complications, survival, and surgical technique of medial gastrocnemius flap for the treatment of extensor mechanism disruptions associated with periprosthetic knee infection. METHODS: This is a retrospective study from a prospectively collected arthroplasty registry from 2012 to 2019. Patients who received the gastrocnemius flap in the setting of a two-stage knee replacement for periprosthetic infection were included. Results of physical examination, Knee Society Score, Oxford Knee Score, and measurement of the range of motion registered pre-operatively were compared to those obtained at last follow-up. Survival was analysed through Kaplan-Meier curve. RESULTS: A total of 15 patients were included, with a mean age of 63.4 years (range 36-77). The reconstruction of the extensor mechanism demonstrated a success rate of 73.3%. The mean extension lag at final follow-up was 7.5° (range, 0-30). The mean Knee Society Score and Oxford Knee Score improved from 29.0 (range, 21-36) and 17.5 (range, 13-22) respectively, to 82.9 (range, 74-89) and 36.0 (range, 33-39). CONCLUSION: Medial gastrocnemius rotational flap is a reliable option for joint and limb salvage in case of periprosthetic knee infection associated with wide soft tissue degeneration and extensor mechanism disruption. The technique and surgical protocol presented in this study are reproducible and guaranteed good clinical outcomes and infection control.

Spacer exchange in persistent periprosthetic joint infection: microbiological evaluation and survivorship analysis.

PURPOSE: The purposes of this study were to determine demographics and characteristics of patients who underwent spacer exchange for persistent infection in the setting of two-stage arthroplasty for periprosthetic joint infection, to describe the microbiology of pathogens involved, to analyze survivorship free from infection in these patients. METHODS: The institutional prospectively collected database was reviewed to enroll patients with minimum 2 years follow-up. Patients who underwent two-stage procedure for septic arthritis were excluded, as were patients who had spacer fracture or dislocation. RESULTS: A total of 34 patients (41 procedures) were included. Mean age was 65.0 ± 12.8 years. Mean follow-up was 53.4 ± 24.8 months. Mean number of previous procedures was 3.6 ± 1.2. A total of 27 (79.4%) patients underwent final reimplantation. The most frequently isolated pathogen in spacer exchange was Staphylococcus epidermidis (10 cases, 28.6%). Polymicrobial cultures were obtained from 9 (25.71%) patients, 10 (28.6%) presented culture-negative infections. A total of 11 (32.4%) resistant pathogens were isolated, and 16 (47.0%) difficult to treat pathogens were detected. Eradication rate was 78.8%. Overall survivorship of implants after final reimplantation was 72.8% at 51.8 months. CONCLUSION: Surgeons should be aware that subjects necessitating spacer exchange often present multiple comorbidities, previous staged revision failures, soft-tissue impairment and difficult to treat infection. In these patients, spacer exchange provides good clinical results and infection eradication, preventing arthrodesis or amputation.

Clinical radiographic outcomes and survivorship of medial pivot design total knee arthroplasty: a systematic review of the literature.

BACKGROUND: Total knee arthroplasty is a reliable procedure able to reduce pain and disability in patients suffering from osteoarthritis. However, a considerable percentage of patients still experiences unsatisfactory results. Medial pivot total knee arthroplasty has been introduced in the clinical practice to overcome problems related with classic design implants and better mimic native knee kinematics. The aim of this study was to analyze survivorship and clinical and radiographic outcomes of medial pivot implants. METHODS: A systematic research was conducted in eight different databases. Thirty-four studies met the inclusion criteria and were included in the analysis. Data on objective and patients-reported outcomes, radiographic alignment, and survivorship were collected and analyzed. Revision rate was expressed as revision per 100 components years. RESULT: A total of 3377 procedures were included. Mean follow-up was 85.7 months (range, 12-182). The revision per 100 components years was 0.19, which corresponds to a revision rate of 1.9% after 10 years. Mean post-operative range of motion was 117.3 ± 0.4°. Mean clinical and functional Knee Society Score were, respectively, 85.9 ± 1.1 and 84.7 ± 3.5 at final follow-up. Post-operative femorotibial alignment was 177.1 ± 0.5°. Alfa and beta angles were 95.7 ± 0.1° and 89.2 ± 0.1°, respectively. Gamma and delta angles were 2.3 ± 0.6° and 86.7 ± 0.4°. CONCLUSION: Medial pivoting implants provided excellent survivorship and low revision rate, as well as good-to-excellent results in term of objective and patient-reported clinical outcomes, and reliable correction of radiographic parameters. More high-quality studies with long-term follow-up are needed to clarify the role of medial pivoting implants.

Shed CNTNAP2 ectodomain is detectable in CSF and regulates Ca2+ homeostasis and network synchrony via PMCA2/ATP2B2.

Although many neuronal membrane proteins undergo proteolytic cleavage, little is known about the biological significance of neuronal ectodomain shedding (ES). Here, we show that the neuronal sheddome is detectable in human cerebrospinal fluid (hCSF) and is enriched in neurodevelopmental disorder (NDD) risk factors. Among shed synaptic proteins is the ectodomain of CNTNAP2 (CNTNAP2-ecto), a prominent NDD risk factor. CNTNAP2 undergoes activity-dependent ES via MMP9 (matrix metalloprotease 9), and CNTNAP2-ecto levels are reduced in the hCSF of individuals with autism spectrum disorder. Using mass spectrometry, we identified the plasma membrane Ca2+ ATPase (PMCA) extrusion pumps as novel CNTNAP2-ecto binding partners. CNTNAP2-ecto enhances the activity of PMCA2 and regulates neuronal network dynamics in a PMCA2-dependent manner. Our data underscore the promise of sheddome analysis in discovering neurobiological mechanisms, provide insight into the biology of ES and its relationship with the CSF, and reveal a mechanism of regulation of Ca2+ homeostasis and neuronal network synchrony by a shed ectodomain.

Optimized detection of lung IL-6 via enzymatic liquefaction of low respiratory tract samples: application for managing ventilated patients.

Lung IL-6 is a promising biomarker for predicting respiratory failure during pulmonary infections. This biomarker is found in respiratory samples which need to be liquefied prior to analysis. Traditional liquefying methods use reducing agents such as dithiothreitol (DTT). However, DTT impairs immunodetection and does not liquefy highly viscous samples. We propose an enzymatic method that liquefies samples by means of generating O2 bubbles with endogenous catalase. Low respiratory tract specimens from 48 mechanically ventilated patients (38 with SARS-CoV-2 infection) were treated with DTT or with the enzymatic method. We used turbidimetry to compare the liquefaction degree and IL-6 was quantified with ELISA. Finally, we used AUC-ROC, time-to-event and principal component analysis to evaluate the association between respiratory compromise or local inflammation and IL-6 determined with both methods. Enzymatically treated samples were better liquefied than those reduced by DTT, which resulted in higher ELISA signals. Lung IL-6 levels obtained with the enzymatic procedure were negatively correlated with the oxygenation index (PaO2/FiO2) and the time of mechanical ventilation. The proposed enzymatic liquefaction method improves the sensitivity for lung IL-6 detection in respiratory samples, which increases its predictive power as a biomarker for evaluating respiratory compliance.

Nanoparticle transfer biosensors for the non-invasive detection of SARS-CoV-2 antigens trapped in surgical face masks.

Detecting SARS-CoV-2 antigens in respiratory tract samples has become a widespread method for screening new SARS-CoV-2 infections. This requires a nasopharyngeal swab performed by a trained healthcare worker, which puts strain on saturated healthcare services. In this manuscript we describe a new approach for non-invasive COVID-19 diagnosis. It consists of using mobile biosensors for detecting viral antigens trapped in surgical face masks worn by patients. The biosensors are made of filter paper containing a nanoparticle reservoir. The nanoparticles transfer from the biosensor to the mask on contact, where they generate colorimetric signals that are quantified with a smartphone app. Sample collection requires wearing a surgical mask for 30 min, and the total assay time is shorter than 10 min. When tested in a cohort of 27 patients with mild or no symptoms, an area under the receiving operating curve (AUROC) of 0.99 was obtained (96.2 % sensitivity and 100 % specificity). Serial measurements revealed a high sensitivity and specificity when masks were worn up to 6 days after diagnosis. Surgical face masks are inexpensive and widely available, which makes this approach easy to implement anywhere. The excellent sensitivity, even when tested with asymptomatic patient samples, along with the mobile detection scheme and non-invasive sampling procedure, makes this biosensor design ideal for mass screening.

Clinical outcomes and survivorship of two-stage total hip or knee arthroplasty in septic arthritis: a retrospective analysis with a minimum five-year follow-up.

PURPOSE: Septic arthritis of the native joint is challenging for orthopedic surgeons because it may lead to wide bone defects and severe impairment of joint function. This study aimed to analyze clinical functional outcomes, the rate of infection eradication, and survival of implants of patients who underwent two-stage arthroplasty for septic arthritis of the hip and knee. METHODS: A retrospective single-centre analysis was conducted of patients treated for septic arthritis of the hip and knee joints through a two-stage surgery between 2012 and 2015. Clinical and radiological records were gathered from the prospectively collected Institutional Arthroplasty Registry. Patients' pre-operative Harris hip scores and Knee Society scores were compared with those obtained at the latest follow-up. Kaplan-Meier curves were generated to assess survival of implants. RESULTS: Forty-seven patients were included. The mean follow-up was 85.2 ± 15.4 months. The Harris hip score improved from 39.4 ± 9.9 to 84.5 ± 10.8 points (p < 0.001). The Knee Society score improved from 40.7 ± 8.4 to 86.0 ± 7.8 points (p < 0.001). Knee Society score-function increased from 25.7 ± 14.2 to 85.4 ± 23.4 points (p < 0.001). The infection eradication rates were 92.0% and 90.9% in patients who underwent hip and knee operation, respectively (p = 0.891). Overall survivorship of implants after the second stage was 93.6%. CONCLUSIONS: Two-stage arthroplasty provides good to excellent clinical outcomes in cases of active septic arthritis of the hip and the knee, high rates of infection control, and implant survival.

Permanent Spacers Are a Reliable Solution for Peri-prosthetic Shoulder Infection: A Systematic Review.

BACKGROUND: Peri-prosthetic shoulder infection (PSI), a highly disabling complication of shoulder arthroplasty, often requires additional surgery and prolonged antibiotic therapy. Of strategies proposed to manage this devastating condition, the use of cement spacers, perhaps even as a definitive treatment, is debated. QUESTIONS/PURPOSES: We sought to systematically review the literature on antibiotic-loaded cement spacers as a viable, perhaps definitive, treatment for PSI, evaluating the eradication rates, mechanical reliability, and functional results related to its use. METHODS: We conducted a systematic review of studies published from January 1, 1980, through September 1, 2019. Following the Cochrane Handbook of Systematic Reviews of Interventions and Preferred Reporting Items for Systematic Review and Meta-analysis, we searched for studies reporting functional and clinical outcomes in patients with PSI treated with a permanent spacer of the shoulder. Two independent reviewers searched eight databases, as well as reference lists of the retrieved articles. RESULTS: After exclusion criteria were applied, 12 studies were included, involving a total of 143 patients. The mean age was 65.8 years; the mean follow-up was 37.4 months. A total of 133 patients (93%) were free from infection at latest follow-up. The mean post-operative active elevation of the shoulder ranged from 48.6 to 90°, the mean abduction ranged from 51 to 75°, and external rotation ranged from 3.6 to 29°. The mean Constant-Murley score ranged from 20.6 to 42 points (out of 100, from worst to best). CONCLUSION: The use of a permanent cement spacer is a reliable solution to PSI in low-demand, older patients with comorbidities, a population in whom it is desirable to avoid additional surgery. Our review found a high rate of infection eradication and moderate-to-good objective and subjective results. However, the overall level of evidence of included studies was very low, and higher-quality studies are needed to clarify the role of permanent spacers in the treatment of PSI.

IL-21 and anti-CD40 restore Bcl-2 family protein imbalance in vitro in low-survival CD27+ B cells from CVID patients.

Common variable immunodeficiency (CVID) is characterized by an abnormal B cell differentiation to memory and antibody-secreting B cells. The defective functionality of CVID patients' B cells could be the consequence of alterations in apoptosis regulation. We studied the balance of Bcl-2 family anti-/pro-apoptotic proteins to identify molecular mechanisms that could underlie B cell survival defects in CVID. We used flow cytometry to investigate Bcl-2, Bcl-XL, Bax, and Bim expression in B cells ex vivo and after anti-CD40 or anti-BCR activation with or without IL-21, besides to spontaneous and stimulation-induced Caspase-3 activation and viable/apoptotic B cell subpopulations. We found increased basal levels of Bax and Bim in CVID B cells that correlated with low viability and high Caspase-3 activation only in CD27+ B cells, particularly in a subgroup of apoptosis-prone CVID (AP-CVID) patients with low peripheral B cell counts and high autoimmunity prevalence (mostly cytopenias). We detected a broad B cell defect in CVID regarding Bcl-2 and Bcl-XL induction, irrespective of the stimulus used. Therefore, peripheral CVID memory B cells are prompted to die from apoptosis due to a constitutive Bcl-2 family protein imbalance and defective protection from activation-induced apoptosis. Interestingly, anti-CD40 and IL-21 induced normal and even higher levels of Bcl-XL, respectively, in CD27+ B cells from AP-CVID, which was accompanied by cell viability increase. Thus low-survival memory B cells from AP-CVID can overcome their cell death regulation defects through pro-survival signals provided by T cells. In conclusion, we identify apoptosis regulation defects as disease-contributing factors in CVID. B cell counts and case history of cytopenias might be useful to predict positive responses to therapeutic approaches targeting T-dependent signaling pathways.

Peptide-MHC-based nanomedicines for autoimmunity function as T-cell receptor microclustering devices.

We have shown that nanoparticles (NPs) can be used as ligand-multimerization platforms to activate specific cellular receptors in vivo. Nanoparticles coated with autoimmune disease-relevant peptide-major histocompatibility complexes (pMHC) blunted autoimmune responses by triggering the differentiation and expansion of antigen-specific regulatory T cells in vivo. Here, we define the engineering principles impacting biological activity, detail a synthesis process yielding safe and stable compounds, and visualize how these nanomedicines interact with cognate T cells. We find that the triggering properties of pMHC-NPs are a function of pMHC intermolecular distance and involve the sustained assembly of large antigen receptor microclusters on murine and human cognate T cells. These compounds show no off-target toxicity in zebrafish embryos, do not cause haematological, biochemical or histological abnormalities, and are rapidly captured by phagocytes or processed by the hepatobiliary system. This work lays the groundwork for the design of ligand-based NP formulations to re-program in vivo cellular responses using nanotechnology.

Increased STAT3 phosphorylation on CD27(+) B-cells from common variable immunodeficiency disease patients.

Maturation and differentiation of B-cells are driven by T-cells' help through IL-21/STAT3 axis in GC centers or through extrafollicular pathways, in a T-independent manner. B-cell differentiation is defective in common variable immunodeficiency disease (CVID) patients. We investigated if IL-21/STAT3 axis alterations could influence B-cell fate. We activated purified CVID B-cells with surrogate T-dependent (anti-CD40), T-independent (TLR-9 ligand) stimuli or through B-cell receptor engagement (anti-IgM) with or without IL-21. IL-21 mediated STAT3 activation was greater on CD27(-) than CD27(+) B-cells depending on the stimulus. IL-21 alone induced STAT3 phosphorylation (pSTAT3) only on CD27(-) B-cells and IL-21 induced higher pSTAT3 levels on CD27(-) than CD27(+) B-cells after anti-IgM or anti-CD40 activation. CVID CD27(+) B-cells showed selective STAT3 hyperphosphorylation after activation with anti-IgM or anti-CD40 alone and anti-IgM, anti-CD40 or ODN combined with IL-21. Increased STAT3 activation during immune responses could result in B-cell differentiation defects in CVID.

Expansion of myeloid-derived suppressor cells in chronic obstructive pulmonary disease and lung cancer: potential link between inflammation and cancer.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a risk factor for lung cancer (LC). Myeloid-derived suppressor cells (MDSCs) down-regulate the T cell receptor ζ chain (TCR ζ) through L-arginine deprivation and lead to T cell dysfunction and deficient antitumor immunity. We hypothesized that abnormally high levels of MDSCs in COPD patients may alter tumor immunosurveillance. METHODS: We compared the proportion of circulating MDSCs (Lin-HLA-DR-/CD33+/CD11b+) (by flow cytometry), arginase I (ARG I) serum levels (by ELISA), and expression levels of TCR ζ on circulating lymphocytes (by flow cytometry) in 28 patients with LC, 62 subjects with COPD, 41 patients with both LC and COPD, 40 smokers with normal spirometry and 33 non-smoking controls. T cell proliferation assays were performed in a subgroup of participants (CFSE dilution protocol). RESULTS: We found that: (1) circulating MDSCs were up-regulated in COPD and LC patients (with and without COPD); (2) MDSCs expansion was associated with TCR ζ down-regulation in the three groups; (3) in LC patients, these findings were independent of COPD and tobacco smoking exposure; (4) TCR ζ down-regulation correlates with T cell hyporesponsiveness in COPD and LC patients. CONCLUSIONS: These results suggest that tumor immunosurveillance might be impaired in COPD and may contribute to the increased risk of LC reported in these patients.

[Traumatic lung hernia]. / Hérnia traumática do pulmão.

Traumatic lung herniation is an unusual clinical problem. We present a case of a large left post-traumatic lung hernia on the left, anterior, second intercostal space following blunt chest trauma. An important factor in the etiology of these lesions is the relative lack of muscular support of the anterior part of the chest. This report describes the diagnosis and management of a post-traumatic lung hernia.

B cells from common variable immunodeficiency patients fail to differentiate to antibody secreting cells in response to TLR9 ligand (CpG-ODN) or anti-CD40+IL21.

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterised by hypogammaglobulinaemia and antibody deficiency to T dependent and independent antigens. Patients suffer from recurrent respiratory infections and poor response to vaccination. Although the underlying molecular defect is unknown, most CVID patients show impaired late B cell differentiation. We investigated B cell differentiation and immunoglobulin secretion induced by two different stimuli: TLR9 specific ligand (CpG-ODN) and anti-CD40 combined with IL21. The contribution of BCR signalling (anti-IgM stimulation) was also evaluated. B cells from CVID patients produced low levels of IgG and IgA in response to both kinds of stimuli that was not restored by anti-IgM. Production of IgM was conserved when cells were stimulated with anti-CD40 and IL21. These results point to a wide signalling defect in B lymphocytes from CVID patients that may be related to their hypogammaglobulinaemia and poor response to vaccination.

Hérnia traumática do pulmão / Traumatic lung hernia

Traumatic lung herniation is an unusual clinical problem. We present a case of a large left post-traumatic lung hernia on the left, anterior, second intercostal space following blunt chest trauma. An important factor in the etiology of these lesions is the relative lack of muscular support of the anterior part of the chest. This report describes the diagnosis and management of a post-traumatic lung hernia.

Casein kinase 2 regulates the NR2 subunit composition of synaptic NMDA receptors.

N-methyl-D-aspartate (NMDA) receptors (NMDARs) play a central role in development, synaptic plasticity, and neurological disease. NMDAR subunit composition defines their biophysical properties and downstream signaling. Casein kinase 2 (CK2) phosphorylates the NR2B subunit within its PDZ-binding domain; however, the consequences for NMDAR localization and function are unclear. Here we show that CK2 phosphorylation of NR2B regulates synaptic NR2B and NR2A in response to activity. We find that CK2 phosphorylates NR2B, but not NR2A, to drive NR2B-endocytosis and remove NR2B from synapses resulting in an increase in synaptic NR2A expression. During development there is an activity-dependent switch from NR2B to NR2A at cortical synapses. We observe an increase in CK2 expression and NR2B phosphorylation over this same critical period and show that the acute activity-dependent switch in NR2 subunit composition at developing hippocampal synapses requires CK2 activity. Thus, CK2 plays a central role in determining the NR2 subunit content of synaptic NMDARs.

Brachial plexus blocks: a review of approaches and techniques.

PURPOSE: The purpose of this narrative review is to summarize the evidence derived from randomized controlled trials (RCTs) regarding established approaches and techniques for brachial plexus anesthesia. SOURCE: Using the MEDLINE (January 1966 to November 2006) and EMBASE (January 1980 to November 2006) databases, key words "brachial plexus", "nerve blocks", "interscalene", "cervical paravertebral", "suprascapular", "supraclavicular", "infraclavicular", "axillary", "brachial canal" and "humeral canal" were searched for full text articles pertaining to the evaluation of recognized approaches and techniques for brachial plexus anesthesia. The search was limited to RCTs involving human subjects and published in the English language. Seventy-six RCTs were identified. PRINCIPAL FINDINGS: Many of the published studies were underpowered and contained various methodological limitations. We found that, for shoulder and proximal humeral surgery, interscalene and cervical paravertebral approaches to the brachial plexus appear to provide equally effective surgical anesthesia. Intersternocleidomastoid supraclavicular blocks are not associated with improved postoperative analgesia despite eliciting more complete anesthesia of the brachial plexus. For surgery at or below the elbow, an infraclavicular block may result in decreased performance time and block-related pain while providing similar efficacy compared to (multiple-stimulation) axillary and brachial canal approaches. With respect to technique, it is unclear if nerve stimulation provides a more effective interscalene block than elicitation of paresthesiae. For supraclavicular blocks, nerve stimulation with a minimal threshold of 0.9 mA is recommended, whereas a double-stimulation technique is optimal for infraclavicular blocks. For the axillary approach, a triple-stimulation technique, involving injections of the musculocutaneous, median and radial nerves, is the most effective option. CONCLUSIONS: Published reports of RCTs provide evidence to formulate limited recommendations regarding optimal approaches and techniques for brachial plexus anesthesia. Further well-designed and meticulously executed RCTs are warranted, particularly in light of new techniques involving ultrasound or combining neurostimulation and echoguidance.