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Background: Circulating endothelial cells (CECs) are biomarkers for endothelial cell (EC) injury and are quantified using immunomagnetic bead extraction (IBE), or flow cytometry (FC). Reports suggest that there is good agreement between these methods for CEC quantification. Objectives: We examined levels of agreement between these techniques in children with systemic vasculitis. Methods: We added HUVEC or human pulmonary artery EC to whole blood to optimize FC gating strategies for EC. EC-optimized FC was then compared with IBE for CEC enumeration in 25 children with vasculitis and 20 healthy controls. Results: Using Bland–Altman analysis, agreement between IBE and EC-optimized FC was poor in children with vasculitis (n = 25) and healthy controls (n = 20): IBE consistently detected higher values than the EC-optimized FC method: the mean difference between the two techniques was 60 CECs mL−1, 95% CI ±374 CECs mL−1(paired analyses of 45 individuals). Agreement was poorest for vasculitis patients: mean difference (IBE – EC-optimized FC) 120 CECs mL−1, 95% CI ±460 CECs mL−1(P = 0.018). We identified three reasons for this discrepancy: (i) sub-optimal FC gating parameters previously used for detecting CECs; (ii) inherent lack of sensitivity of FC compared with IBE for CEC rare event detection; and (iii) use of lysis buffers required for FC causing CEC lysis. Conclusions: There was poor agreement between EC-optimized FC and IBE for the quantification of CECs from children with active vasculitis and controls. We emphasize that in this clinical setting the two techniques are not directly comparable when comparing results obtained using these different methodologies.

BackgroundRituximab is a chimeric IgG1 monoclonal antibody that depletes B cells for the treatment of several conditions including autoimmune diseases. It is not licensed for use in children but administered off-label. This study aimed to quantify the effect of rituximab on B cell depletion in children with autoimmune diseases and to optimise the dosing regimen.MethodsElectronic health record data were collected from a retrospective and anonymised study at Great Ormond Street Hospital in London. Dosing protocols of rituximab were two 750 mg/m2intravenous infusions or four weekly 375 mg/m2infusions. Serum concentrations of rituximab were not measured. CD19+ lymphocyte counts were taken before and after rituximab treatment. A turnover mechanism described the life cycle of CD19+ lymphocytes with rituximab increasing the death rate of CD19+ lymphocytes; a negative feedback was added on the production rate to examine the rebound effect. Rituximab was assumed to decay by first-order kinetics.Results258 measurements of CD19+ lymphocyte counts were collected from 39 children with 8 autoimmune diseases. The dose-response model well described the time course of CD19+ lymphocytes following rituximab administration. The elimination half-lives of rituximab and CD19+ lymphocytes were estimated to be 19 and 35 days, respectively, consistent with findings from other studies [1–5]. The rebound increase in CD19+ lymphocytes was found negligible. Methotrexate and cyclophosphamide increased the maximum death rate by 66% and 38% respectively. Age and gender were not significant covariates.Simulations from the model suggested that a single infusion of rituximab of 375 mg/m2can provide similar six-month suppression of CD19+ lymphocytes to the higher doses currently used. Methotrexate or cyclophosphamide added minimal suppression effect on CD19+ lymphocytes when taken concurrently with rituximab.ConclusionsOur results could be used in future to assess the effect of rituximab biosimilars and to inform biosimilar dosing in paediatric populations.ReferencesNg, et al. J Clin Pharmacol. 2005; 45:792–801Li, et al. Blood. 2007; 110:2371Li, et al. J Clin Pharmacol. 2012; 52:1918–26Fulcher and Basten. Immunol Cell Biol. 1997; 75:446–55Macallan, et al. Blood. 2005; 105:3633–40Disclosure(s)Nothing to disclose

Background: Patients with autoimmune diseases and latent tuberculosis infection(LTBI) are at risk of developing catastrophic TB disease following infliximab treatment. Quantiferon-TB gold in-Tube (QTB), an interferon gamma releasing assay, has proven a more accurate screening tool for TB than tuberculin skin test (TST) in adult populations.Objectives: To assess the utility and validity of QTB in children in detecting LTBI, prior to treatment with infliximab.Methods: Retrospective case note review of patients started on infliximab following introduction of QTB as a screening tool in Paediatric Rheumatology.Results: Twenty-three patients (12 females, 11 males) were included in the study. A chest radiograph (CXR) and QTB was performed prior to starting infliximab. Fourteen patients had a recorded negative TST result. Five patients were on methotrexate, and the remaining were on methotrexate and steroids. One patient had a positive QTB while two had indeterminate results. Their CXRs were not suggestive of TB and TSTs were negative. All three were on methotrexate and steroids. The patient with the positive QTB received 6 months anti TB treatment following which QTB became negative. The patients with indeterminate results were started on infliximab and had regular clinical assessment for TB disease. Repeat QTB was negative in one while remained indeterminate in the other. None of our 23 patients developed TB.Conclusion: QTB is a useful screen tool for LTBI. Our yield of indeterminate results is lower compared to previous studies. Indeterminate results warrant careful assessment and re-evaluation, but should not preclude from initiation of anti-TNFa treatment.