Impact of blood storage and sample handling on quality of high dimensional flow cytometric data in multicenter clinical research

Obtaining reliable and reproducible high quality data in multicenter clinical research settings requires design of optimal standard operating procedures. While the need for standardization in sample processing and data analysis is well-recognized, the impact of sample handling in the pre-analytical phase remains underestimated. We evaluated the impact of sample storage time (approximate to transport time) and temperature, type of anticoagulant, and limited blood volume on reproducibility of flow cytometric studies. EDTA and Na-Heparin samples processed with the EuroFlow bulk lysis protocol, stained and stored at 4 degrees C showed fairly stable expression of cell surface markers and distribution of the major leukocyte populations for up to 72 h. Additional sample fixation (1% PFA, Fix & Perm) did not have any beneficial effects. Blood samples stored for < 24 h at room temperature before processing and staining seemed suitable for reliable immunophenotyping, although losses in absolute cell numbers were observed. The major losses were observed in myeloid cells and monocytes, while lymphocytes seemed less affected. Expression of cell surface markers and population distribution were more stable in Na-Heparin blood than in EDTA blood. However, storage of Na-Heparin samples was associated with faster decrease in leukocyte counts over time. Whole blood fixation strategies (Cyto-Chex, TransFix) improved long-term population distribution, but were detrimental for expression of cellular markers. The main conclusions from this study on healthy donor blood samples were successfully confirmed in EDTA clinical (patient) blood samples with different time delays until processing. Finally, we recognized the need for adjustments in bulk lysis in case of insufficient blood volumes. Despite clear overall conclusions, individual markers and cell populations had different preferred conditions. Therefore, specific guidelines for sample handling should always be adjusted to the clinical application and the main target leukocyte population

Bone Tissue Response to Porous and Functionalized Titanium and Silica Based Coatings

Background: Topography and presence of bio-mimetic coatings are known to improve osseointegration. The objective of this study was to evaluate the bone regeneration potential of porous and osteogenic coatings. Methodology: Six-implants [Control (CTR); porous titanium coatings (T1, T2); thickened titanium (Ti) dioxide layer (TiO2); Amorphous Microporous Silica (AMS) and Bio-active Glass (BAG)] were implanted randomly in tibiae of 20-New Zealand white rabbits. The animals were sacrificed after 2 or 4 weeks. The samples were analyzed histologically and histomorphometrically. In the initial bone-free areas (bone regeneration areas (BRAs)), the bone area fraction (BAF) was evaluated in the whole cavity (500 mm, BAF-500), in the implant vicinity (100 mm, BAF-100) and further away (100–500 mm, BAF-400) from the implant. Bone-to-implant contact (BIC-BAA) was measured in the areas where the implants were installed in contact to the host bone (bone adaptation areas (BAAs)) to understand and compare the bone adaptation. Mixed models were used for statistical analysis. Principal Findings: After 2 weeks, the differences in BAF-500 for different surfaces were not significant (p.0.05). After 4 weeks, a higher BAF-500 was observed for BAG than CTR. BAF-100 for AMS was higher than BAG and BAF-400 for BAG was higher than CTR and AMS. For T1 and AMS, the bone regeneration was faster in the 100-mm compared to the 400-mm zone. BIC-BAA for AMS and BAG was lower after 4 than 2 weeks. After 4 weeks, BIC-BAA for BAG was lower than AMS and CTR. Conclusions: BAG is highly osteogenic at a distance from the implant. The porous titanium coatings didn’t stimulate bone regeneration but allowed bone growth into the pores. Although AMS didn’t stimulate higher bone response, it has a potential of faster bone growth in the vicinity compared to further away from the surface. BIC-BAA data were inconclusive to understand the bone adaptation.status: publishe

Directing Cluster Formation of Au Nanoparticles from Colloidal Solution

Discrete clusters of closely spaced Au nanoparticles can be utilized in devices from photovoltaics to molecular sensors because of the formation of strong local electromagnetic field enhancements when illuminated near their plasmon resonance. In this study, scalable, chemical self-organization methods are shown to produce Au nanoparticle clusters with uniform nanometer interparticle spacing. The performance of two different methods, namely electrophoresis and diffusion, for driving the attachment of Au nanoparticles using a chemical cross-linker on chemically patterned domains of polystyrene-block-poly(methyl methacrylate) (PS-b-PMMA) thin films are evaluated. Significantly, electrophoresis is found to produce similar surface coverage as diffusion in 1/6th of the processing time with an ~2-fold increase in the number of Au nanoparticles forming clusters. Furthermore, average interparticle spacing within Au nanoparticle clusters was found to decrease from 2-7 nm for diffusion deposition to approximately 1-2 nm for electrophoresis deposition, and the latter method exhibited better uniformity with most clusters appearing to have about 1 nm spacing between nanoparticles. The advantage of such fabrication capability is supported by calculations of local electric field enhancements using electromagnetic full-wave simulations from which we can estimate surface-enhanced Raman scattering (SERS) enhancements. In particular, full-wave results show that the maximum SERS enhancement, as estimated here as the fourth power of the local electric field, increases by a factor of 100 when the gap goes from 2 to 1 nm, reaching values as large as 10(10), strengthening the usage of electrophoresis versus diffusion for the development of molecular sensors

Alternating current electrophoretic deposition for the immobilization of antimicrobial agents on titanium implant surfaces

One prominent cause of implant failure is infection; therefore, research is focusing on developing surface coatings that render the surface resistant to colonization by micro-organisms. Permanently attached coatings of antimicrobial molecules are of particular interest because of the reduced cytoxicity and lower risk of developing resistance compared to controlled release coatings. In this study, we focus on the chemical grafting of bioactive molecules on titanium. To concentrate the molecules at the metallic implant surface, we propose electrophoretic deposition (EPD) applying alternating current (AC) signals with an asymmetrical wave shape. We show that for the model molecule bovine serum albumin (BSA), as well as for the clinically relevant antifungal lipopeptide caspofungin (CASP), the deposition yield is drastically improved by superimposing a DC offset in the direction of the high-amplitude peak of the AC signal. Additionally, in order to produce immobilized CASP coatings, this experimental AC/DC-EPD method is combined with an established surface activation protocol. Principle component analysis (PCA) of time-of-flight secondary ion mass spectrometry (ToF-SIMS) data confirm the immobilization of CASP with higher yield as compared to a diffusion-controlled process, and higher purity than the clinical CASP starting suspensions. Scratch testing data indicate good coating adhesion. Importantly, the coatings remain active against the fungal pathogen C. albicans as shown by in vitro biofilm experiments. In summary, this paper delivers a proof-of-concept for the application of AC-EPD as a fast grafting tool for antimicrobial molecules without compromising their activities.status: publishe

Alternating Current Electrophoretic Deposition for the Immobilization of Antimicrobial Agents on Titanium Implant Surfaces

One prominent cause of implant failure is infection; therefore, research is focusing on developing surface coatings that render the surface resistant to colonization by micro-organisms. Permanently attached coatings of antimicrobial molecules are of particular interest because of the reduced cytoxicity and lower risk of developing resistance compared to controlled release coatings. In this study, we focus on the chemical grafting of bioactive molecules on titanium. To concentrate the molecules at the metallic implant surface, we propose electrophoretic deposition (EPD) applying alternating current (AC) signals with an asymmetrical wave shape. We show that for the model molecule bovine serum albumin (BSA), as well as for the clinically relevant antifungal lipopeptide caspofungin (CASP), the deposition yield is drastically improved by superimposing a DC offset in the direction of the high-amplitude peak of the AC signal. Additionally, in order to produce immobilized CASP coatings, this experimental AC/DC-EPD method is combined with an established surface activation protocol. Principle component analysis (PCA) of time-of-flight secondary ion mass spectrometry (ToF-SIMS) data confirm the immobilization of CASP with higher yield as compared to a diffusion-controlled process, and higher purity than the clinical CASP starting suspensions. Scratch testing data indicate good coating adhesion. Importantly, the coatings remain active against the fungal pathogen <i>C. albicans</i> as shown by in vitro biofilm experiments. In summary, this paper delivers a proof-of-concept for the application of AC-EPD as a fast grafting tool for antimicrobial molecules without compromising their activities