Seroprevalence of antibodies and chronic Q fever among post-mortal and living donors of tissues and cells from 2010 to 2015 in the Netherlands.

BackgroundAfter a large Q fever outbreak in the Netherlands in the period from 2007 to 2010, the risk of Q fever transmission through tissue and cell transplantation from undiagnosed chronic Q fever cases became a potential issue. We aimed to evaluate the risk of Q fever transmission through tissue and cell transplantation. We performed a retrospective observational cohort study among 15,133 Dutch donors of tissues and stem cells from 2010 to 2015 to assess seroprevalence of antibodies, to identify factors associated with presence of antibodies, and to assess the proportion of undiagnosed chronic Q fever cases.

Processing cord blood from premature infants into autologous red-blood-cell products for transfusion.

Background And Objectives: The use of umbilical cord blood (UCB) for transfusion purposes has gained interest the past years. UCB transfusion could serve premature infants, who often need transfusions early in life.

Material And Methods: We investigated the suitability of different storage media.

Development of Specific Recombinant Monoclonal Antibodies Against the Lipopolysaccharide of Ralstonia solanacearum Race 3.

ABSTRACT Recombinant single-chain antibodies (scFvs) against the lipopolysaccharide of Ralstonia solanacearum (biovar 2, race 3) were successfully selected by phage display from a large combinatorial antibody library. Characterization with regard to cross-reaction and use in routine immunoassays showed that the selected antibodies had improved characteristics when compared with the polyclonal antiserum that is currently used for brown rot diagnosis of potato in the Netherlands. The isolated monoclonal scFvs reacted in both enzyme-linked immunosorbent assay (ELISA) and immunofluorescence cell staining with all race 3 strains tested, but with only some strains belonging to other races.

Multiplex microsphere immuno-detection of potato virus Y, X and PLRV.

To monitor seed potatoes for potato virus X, Y and PLRV, a multiplex microsphere immunoassay (MIA) was developed based on the Luminex xMAP technology, as an alternative to ELISA. The xMAP technology allowed detection of a number of antigens simultaneously whereas ELISA only allowed simplex detection of antigens. The use of paramagnetic beads in the MIA procedure allowed efficient removal of excess sample compounds and reagents.

Multi-laboratory evaluation of procedures for reducing the volume of cord blood: influence on cell recoveries.

Background: Various procedures can be used to isolate stem and progenitor cells from cord blood. This study evaluated the hydroxyethyl starch sedimentation (HES) with two centrifugation steps, and the top and bottom (T&B) isolation of buffy coat following a single centrifugation, and two filter systems for processing cord blood, one developed by Asahi Kasei Medical (filter A) and the second by Terumo (filter B).

Methods: Each of seven laboratories was randomly assigned the evaluation of either the HES or T&B method and one of the filter methods (n=8 cord blood units, per laboratory, for each method).

Detection of Clavibacter michiganensis subsp. sepedonicus by AmpliDet RNA, a new technology based on real time monitoring of NASBA amplicons with a molecular beacon.

Aims: To develop a procedure for direct detection of viable cells of Clavibacter michiganensis subsp. sepedonicus (Cms), the causal organism of bacterial ring rot in potato, based on AmpliDet RNA, in which amplicons generated by nucleic acid sequence based amplification (NASBA) are monitored in real time with a molecular beacon.

Methods And Results: Five methods were evaluated and fine-tuned for extraction of RNA from Cms.

Amplification of RNA by NASBA allows direct detection of viable cells of Ralstonia solanacearum in potato.

Aims: The objective of this study was to develop a Nucleic Acid Sequence Based Amplification (NASBA) assay, targeting 16S rRNA sequences, for direct detection of viable cells of Ralstonia solanacearum, the causal organism of bacterial wilt. The presence of intact 16S rRNA is considered to be a useful indicator for viability, as a rapid degradation of this target molecule is found upon cell death.

Methods And Results: It was demonstrated by RNase treatment of extracted nucleic acids from R.

An internal control for the diagnosis of crown gall by PCR.

The addition of an internal control (IC) at the appropriate concentration enables recognize of false negatives in the detection of Agrobacterium tumefaciens and improves the reliability of PCR for crown gall diagnosis. Co-amplification of the IC and target sequence from A. tumefaciens ensures the attainment of at least one amplification product in every PCR reaction if the DNA extracted is of high enough quality to be amplified.

Microbial contamination of cord blood stem cells.

Background And Objectives: After storage, low levels of contaminating bacteria in standard blood components can reach bacteraemic levels, causing severe transfusion-associated sepsis. For cord blood (CB), the significance of low levels of contaminating bacteria and the optimal detection method is unknown and not supported by available guidelines.

Materials And Methods: Spiking experiments and testing of various subfractions of CB units were used to determine the behaviour of bacteria during centrifugation, freezing and thawing.

Hemopoietic stem and precursor cell analysis in umbilical cord blood using the Sysmex SE-9000 IMI channel.

Circulating hematopoietic progenitor cells (HPCs) are routinely measured by flow cytometry using CD34 expression. As an alternative, the "immature information" (IMI) channel measurement of the automated hematology analyzer Sysmex SE machines was developed. We tested the IMI channel HPC method with umbilical cord blood specimens.

Association of stress during delivery with increased numbers of nucleated cells and hematopoietic progenitor cells in umbilical cord blood.

Objective: Umbilical cord blood can be used as a source of bone marrow repopulating cells for allogeneic stem cell transplantation. Large variations in the frequencies of white blood cells and hematopoietic progenitor cells have been found for umbilical cord blood. These variations may be due in part to specific circumstances during labor and delivery.

The number of nucleated cells reflects the hematopoietic content of umbilical cord blood for transplantation.

A single umbilical cord blood (UCB) collection may contain sufficient hematopoietic stem cells to achieve engraftment and repopulation of the hematopoietic system of children and adults after myeloablative therapy. The hematopoietic potential of a UCB unit is often defined by the number of CD34+ cells or the number of colony-forming units as measured in semisolid hematopoietic progenitor cell (HPC) cultures. However, these assays are relatively difficult to standardize between UCB banks.

Immunomagnetic separation of Erwinia carotovora subsp. atroseptica from potato peel extracts to improve detection sensitivity on a crystal violet pectate medium or by PCR.

Immunomagnetic separation (IMS) procedures for the selective separation of Erwinia carotovora subsp. atroseptica from potato peel extract were optimized for the recovery of target and removal of non-target bacteria. A streptomycin-resistant strain of Erw.

Polymerase chain reaction for verification of fluorescent colonies of Erwinia chrysanthemi and Pseudomonas putida WCS358 in immunofluorescence colony staining.

The potential of polymerase chain reaction (PCR) for verifying the identity of colonies stained by the immunofluorescence colony-staining (IFC) procedure was investigated. Using primers directed against conserved sequences of the pectate lyase-genes coding for isozymes PLa, PLd and PLe of Erwinia chrysanthemi, the authors confirmed the identity of 96% of 20 fluorescent target colonies, punched from IFC-stained samples with pure cultures. In pour plates with mixtures of Erw.

Verification of ELISA results by immunomagnetic isolation of antigens from extracts and analysis with SDS-PAGE and western blotting, demonstrated for Erwinia spp. in potatoes.

Isolation of antigens on immunomagnetic beads and subsequent analysis with SDS-PAGE and Western blotting (immunomagnetic isolation-Western blotting (IMI-WB)) was used to verify positive ELISA results for Erwinia chrysanthemi and Erw. carotovora subsp. atroseptica in potato peel extracts.

Reverse cholesterol transport: relationship between free cholesterol uptake and HDL3 in normolipidaemic and hyperlipidaemic subjects.

High density lipoproteins (HDL) are responsible for the Reverse Cholesterol Transport (RCT). The role of the composition of the HDL particle in RCT, involving free cholesterol (chol) uptake from cell membranes, is not completely understood. We have therefore studied the uptake capacity from subjects with a wide variety of plasma HDL cholesterol concentrations in an HDL-receptor free model consisting of bovine heart mitochondrial membranes labeled with [14C]cholesterol.

Increased cholesterol synthesis in Chinese hamster ovary cells deficient in peroxisomes.

In a previous study we have shown that Chinese hamster ovary (CHO) cells deficient in intact peroxisomes, lack the nonspecific lipid transfer protein (nsL-TP; sterol carrier protein 2) (van Heusden, G.P.H.

The subcellular distribution of the nonspecific lipid transfer protein (sterol carrier protein 2) in rat liver and adrenal gland.

The distribution of the nonspecific lipid transfer protein (i.e., sterol carrier protein 2) over the various subcellular fractions from rat liver and adrenal gland was determined by enzyme immunoassay and immunoblotting.

Analysis of aspartic acid racemization. Evaluation of a chiral capillary gas chromatographic and a diastereomeric high-performance liquid chromatographic method.

A recently developed chiral gas chromatographic method and a diastereomeric high-performance liquid chromatographic method for the analysis of aspartic acid enantiomers in protein hydrolyzates have been evaluated. Although both techniques are fast and convenient, the latter is preferred because of its higher reproducibility and shorter analysis time. Furthermore, this method offers the possibility of on-line derivatization and analysis.

Detection of aspartic acid enantiomers by chiral capillary gas chromatography. Determination of in vivo racemization and reduction of metal-induced background.

Racemization of aspartyl residues in proteins is a post-translational process, related to ageing. A method is presented for the detection of aspartic acid enantiomers in protein hydrolysates, based on chiral capillary gas chromatography. It is fast, easy and preferable to the usual diastereomeric dipeptide technique.