Size and Concentration of Cell-Free DNA Measured Directly from Blood Plasma, without Prior DNA Extraction.

Cell-free DNA in human blood plasma (cfDNA) is now widely used and studied as a biomarker for several physiological and pathological situations. In addition to genetic and epigenetic alterations that provide information about the presence and the nature of non-constitutive DNA in the body, cfDNA concentration and size distribution may potentially be independent biomarkers suitable for monitoring at-risk patients and therapy efficacy. Here, we describe a simple, in-line, method, which measures cfDNA concentration and size distribution from only a few microliters of plasma without the need to extract and/or concentrate the DNA prior to the analysis. This method is based on a dual hydrodynamic and electrokinetic actuation, adapted for samples containing salts and proteins such as biological fluids. The method provides analytical performances equivalent to those obtained after purification and concentration of cfDNA, with a precision of ∼1% for size features and of 10-20% for the concentrations of the different size fractions. We show that concentration and size distribution of cfDNA analyzed from plasma can differentiate advanced lung cancer patients from healthy controls. This simple and cost-effective method should facilitate further investigations into the potential clinical usefulness of cfDNA size profiling.

Immune-mediated thrombotic thrombocytopenic purpura plasma induces calcium- and IgG-dependent endothelial activation: correlations with disease severity.

Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is characterized by a severe ADAMTS13 deficiency due to the presence of anti-ADAMTS13 auto-antibodies, with subsequent accumulation of circulating ultra-large von Willebrand factor (VWF) multimers. The role of endothelial cell activation as a trigger of the disease has been suggested in animal models but remains to be demonstrated in humans. We prospectively obtained plasma from the first plasma exchange of 25 patients during iTTP acute phase. iTTP but not control plasma, induced a rapid VWF release and P-selectin exposure on the surface of dermal human micro-vascular endothelial cell (HMVEC-d), associated with angiopoietin-2 and endothelin-1 secretion, consistent with Weibel-Palade bodies exocytosis. Calcium (Ca2+) blockade significantly decreased VWF release, whereas iTTP plasma induced a rapid and sustained Ca2+ flux in HMVEC-d which correlated in retrospect, with disease severity and survival in 62 iTTP patients. F(ab)'2 fragments purified from the immunoglobulin G fraction of iTTP plasma mainly induced endothelial cell activation with additional minor roles for circulating free heme and nucleosomes, but not for complement. Furthermore, two anti-ADAMTS13 monoclonal antibodies purified from iTTP patients' B cells, but not serum from hereditary TTP, induced endothelial Ca2+ flux associated with Weibel-Palade bodies exocytosis in vitro, whereas inhibition of endothelial ADAMTS13 expression using small intering RNA, significantly decreased the stimulating effects of iTTP immunoglobulin G. In conclusion, Ca2+-mediated endothelial cell activation constitutes a "second hit" of iTTP, is correlated with the severity of the disease and may constitute a possible therapeutic target.

Comparative evaluation of the depletion-red cell exchange program with the Spectra Optia and the isovolemic hemodilution-red cell exchange method with the COBE Spectra in sickle cell disease patients.

BACKGROUND: This study aims to compare in patients with sickle cell disease (SCD), the technical performance and packed red blood cell unit consumption between the automated depletion/Red Blood Cell exchange (RBCx) program (Spectra Optia Apheresis System) with the isovolemic hemodilution (IHD)/RBCx procedure (COBE Spectra Apheresis System) in a routine clinical setting. METHODS: We retrospectively reviewed the data of 23 patients treated between October 2010 and August 2013 who underwent repeated RBCx on both apheresis systems for preventive indications. Each patient was their own control and had undergone two procedures on each system, totaling 46 sessions per group. On Spectra Optia, we performed the automated depletion/RBCx program. For COBE Spectra, we used a modified IHD/RBCx protocol. All patients had an initial 250 mL depletion offset by a 5% albumin prior to the exchange procedure, for the respective device, with leucodepleted Rh/Kell compatible and cross-matched RBC packs. RESULTS: All procedures were well tolerated except three mild febrile nonhemolytic reactions. Postprocedure hemoglobin S (HbS), fraction of cells remaining (FCR), procedure duration and processed blood and anticoagulant volumes were comparable in the two groups. However, the RBCx volume was significantly higher for the Spectra Optia group (+71 mL, P = 0.01), with no significant difference in the number of RBC units used. CONCLUSIONS: Technical performance and packed RBC unit consumption were not compromised when switching from the COBE Spectra IHD/RBCx protocol to the depletion/RBCx protocol on the Spectra Optia. Tolerability was equal for both protocols. J. Clin. Apheresis 31:429-433, 2016. © 2015 Wiley Periodicals, Inc.

Campylobacter fetus Bacteremia Revealed by Cellulitis without Gastrointestinal Symptoms in the Context of Acquired Hypogammaglobulinemia: A Report of Three Cases.

Campylobacter fetus bacteremia is rare and occurs mainly in patients with immunosuppression. This infection, which often involves secondary localizations has already been reported in some primary humoral immune deficiencies. We describe three cases of severe infection due to C. fetus with cellulitis at presentation, but without any gastrointestinal symptoms, occurring in patients with acquired hypogammaglobulinemia.