Nonspecific binding of monoclonal antibodies (mAbs) to Fc-receptors on leukocytes is an important cause of background fluorescence in flow cytometry, and failing to block such nonspecific binding can lead to erroneous results. A major part of previous studies on blocking reagents for flow cytometry have been done in mice, and published results are not completely in agreement. In humans, Fc-receptors are found on most leukocytes, with highest abundance on monocytes/macrophages. Therefore, in the present study our aim was to thoroughly investigate the efficiency of different commonly used blocking reagents regarding inhibition of nonspecific binding of mouse mAbs to human peripheral blood mononuclear cells (MNCs) and monocyte-derived macrophages (MDMs). Monocytes and MDMs showed strong nonspecific binding of IgG1 and IgG2a isotypes, but not IgG2b. In contrast, B-cells, T-cells, and NK-cells did not substantially bind any of the mouse isotype control antibodies evaluated (IgG1, IgG2a, and IgG2b). Importantly, we show that binding of IgG1 and IgG2a to monocytes and MDMs can be eliminated by blocking, either with a commercial Fc-blocking reagent, with mouse or human serum, or with mouse or human IgG in high concentration. Previously, isotype controls have been widely used in flow cytometry assays. However, we show that such controls may be highly unreliable, and we believe they should not be used as gating controls, or to determine background signal. Based on these results, as well as considerations of price and applicability, our recommendation is not to use isotypes as gating controls in flow cytometry, but instead to use 100 μg/mL of purified human IgG as blocking reagent for elimination of nonspecific binding of mouse mAbs to human MNCs and MDMs. © 2016 International Society for Advancement of Cytometry.

Fused real-time ultrasound and magnetic resonance imaging (MRI) may be used to improve the accuracy of advanced image guided procedures. However, its use in regional anesthesia is practically nonexistent. In this randomized controlled crossover trial, we aim to explore effectiveness, procedure-related outcomes, injectate spread analyzed by MRI, and safety of ultrasound/MRI fusion versus ultrasound guided Suprasacral Parallel Shift (SSPS) technique for lumbosacral plexus blockade. Twenty-six healthy subjects aged 21–36 years received two SSPS blocks (20 mL 2% lidocaine-epinephrine [1 : 200,000] added 1 mL diluted contrast) guided by ultrasound/MRI fusion versus ultrasound. Number (proportion) of subjects with motor blockade of the femoral and obturator nerves and the lumbosacral trunk was equal (ultrasound/MRI, 23/26 [88%]; ultrasound, 23/26 [88%]; p = 1.00). Median (interquartile range) preparation and procedure times (s) were longer for the ultrasound/MRI fusion guided technique (686 [552–1023] versus 196 [167–228], p < 0.001 and 333 [254–439] versus 216 [176–294], p = 0.001). Both techniques produced perineural spread and corresponding sensory analgesia from L2 to S1. Epidural spread and lidocaine pharmacokinetics were similar. Different compartmentalized patterns of injectate spread were observed. Ultrasound/MRI fusion guided SSPS was equally effective and safe but required prolonged time, compared to ultrasound guided SSPS. This trial is registered with EudraCT (2013-004013-41) and ClinicalTrials.gov (NCT02593370).

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