Alloimmunization in dogs after transfusion: A serial cross-match study.

BACKGROUND: Cross-matching is performed to determine the serological compatibility of donor and recipient blood. Current guidelines recommend that cross-matching should be performed in dogs when an initial transfusion was performed more than 4 days ago or when the transfusion history is unknown. HYPOTHESIS: Determination at what time point alloantibodies are detected in dogs after transfusion. The hypothesis was that dogs would form alloantibodies within 4 days after a transfusion. ANIMALS: Twenty-one anemic dogs were transfused and monitored for at least 4 subsequent days. Exclusion criteria were persistent red blood cell (RBC) agglutination and a previous transfusion. METHODS: Prospective observational study. Cross-matching was performed before the initial DEA 1-compatible transfusion and on days 1, 2, 3, and 4 and if possible, between day 5 and 28, using the tube method without enhancement (major cross-match, recipient controls); recipients were monitored for transfusion reactions. RESULTS: In 12/21 dogs a positive cross-match (microscopic degree of agglutination [AD] 1+ to 2+) was observed within 4 days after the transfusion. In a nonlinear regression model, no significant association was detected between type of anemia (P = .41), RBC storage time (P = .44), immunosuppressive treatment (P = .75) nor transfusion volume (P = .70) and the occurrence of positive cross-matches within 4 days after transfusion. Another 4 dogs developed a positive cross-match (microscopic AD 1+ to 2+) after 6 to 13 days. CONCLUSIONS AND CLINICAL IMPORTANCE: Because production of alloantibodies was detected as early as 1 day after transfusion, cross-matching should be performed before every subsequent transfusion.

Investigation of direct counting and sizing of DNA fragments in flow applying an improved data analysis and correction method.

Direct detection of single stained DNA fragments in flow is a very sensitive method for nucleic acid detection which does not need any amplification process. We have developed an instrument for direct counting and sizing of single DNA fragments (single or double stranded DNA) in flow with integrated sample volume measurement for concentration determination. As the method is a potential reference method for DNA quantification, processes affecting the measurement uncertainty are of major interest. Additionally, comparison of this method to the orthogonal method of digital PCR is useful with the restriction of low specificity of the direct detection method. In this study, we analysed raw detector signals and the sizing performance for target identification and the effect of coincidence detection concerning concentration measurements. We present data of purified artificial DNA samples measured with the home-built setup. Main emphasis was to develop an improved data analysis method to gain insight into and carefully correct for coincident detection of DNA fragments and for estimation of the amount of fragment dimers.

Label-free whole blood cell differentiation based on multiple frequency AC impedance and light scattering analysis in a micro flow cytometer.

We developed a microfluidic sensor for label-free flow cytometric cell differentiation by combined multiple AC electrical impedance and light scattering analysis. The measured signals are correlated to cell volume, membrane capacity and optical properties of single cells. For an improved signal to noise ratio, the microfluidic sensor incorporates two electrode pairs for differential impedance detection. One-dimensional sheath flow focusing was implemented, which allows single particle analysis at kHz count rates. Various monodisperse particles and differentiation of leukocytes in haemolysed samples served to benchmark the microdevice applying combined AC impedance and side scatter analyses. In what follows, we demonstrate that AC impedance measurements at selected frequencies allow label-free discrimination of platelets, erythrocytes, monocytes, granulocytes and lymphocytes in whole blood samples involving dilution only. Immunofluorescence staining was applied to validate the results of the label-free cell analysis. Reliable differentiation and enumeration of cells in whole blood by AC impedance detection have the potential to support medical diagnosis for patients with haemolysis resistant erythrocytes or abnormally sensitive leucocytes, i.e. for patients suffering from anaemia or leukaemia.

Microflow cytometers with integrated hydrodynamic focusing.

This study demonstrates the suitability of microfluidic structures for high throughput blood cell analysis. The microfluidic chips exploit fully integrated hydrodynamic focusing based on two different concepts: Two-stage cascade focusing and spin focusing (vortex) principle. The sample--A suspension of micro particles or blood cells--is injected into a sheath fluid streaming at a substantially higher flow rate, which assures positioning of the particles in the center of the flow channel. Particle velocities of a few m/s are achieved as required for high throughput blood cell analysis. The stability of hydrodynamic particle positioning was evaluated by measuring the pulse heights distributions of fluorescence signals from calibration beads. Quantitative assessment based on coefficient of variation for the fluorescence intensity distributions resulted in a value of about 3% determined for the micro-device exploiting cascade hydrodynamic focusing. For the spin focusing approach similar values were achieved for sample flow rates being 1.5 times lower. Our results indicate that the performances of both variants of hydrodynamic focusing suit for blood cell differentiation and counting. The potential of the micro flow cytometer is demonstrated by detecting immunologically labeled CD3 positive and CD4 positive T-lymphocytes in blood.

A microflow cytometer exploited for the immunological differentiation of leukocytes.

In this article, we demonstrate the potential of a microfluidic chip for the differentiation of immunologically stained blood cells. To this end, white blood cells stained with antibodies typically applied for the determination of the immune status were measured in the micro-device. Relative concentrations of lymphocytes and subpopulations of lymphocytes are compared to those obtained with a conventional flow cytometer. The stability of the hydrodynamic focusing and the optical setup was determined by measuring the variation of the signal pulse height of fluorescence calibration beads, being about 2% for the micro-device. This value and the overall performance of the micro-device are similar to conventional flow cytometers. It follows from our results that such microfluidic structures are well suited as modules in a compact, portable read-out instrument. The production process of the microflow cytometers, which we exploited for immunological cell differentiation, is compatible with mass production technology like injection molding and, hence, low cost disposable chips could be available in the future.

Bruchpilot in ribbon-like axonal agglomerates, behavioral defects, and early death in SRPK79D kinase mutants of Drosophila.

Defining the molecular structure and function of synapses is a central theme in brain research. In Drosophila the Bruchpilot (BRP) protein is associated with T-shaped ribbons ("T-bars") at presynaptic active zones (AZs). BRP is required for intact AZ structure and normal evoked neurotransmitter release. By screening for mutations that affect the tissue distribution of Bruchpilot, we have identified a P-transposon insertion in gene CG11489 (location 79D) which shows high homology to mammalian genes for SR protein kinases (SRPKs). SRPKs phosphorylate serine-arginine rich splicing factors (SR proteins). Since proteins expressed from CG11489 cDNAs phosphorylate a peptide from a human SR protein in vitro, we name CG11489 the Drosophila Srpk79D gene. We have characterized Srpk79D transcripts and generated a null mutant. Mutation of the Srpk79D gene causes conspicuous accumulations of BRP in larval and adult nerves. At the ultrastructural level, these correspond to extensive axonal agglomerates of electron-dense ribbons surrounded by clear vesicles. Basic synaptic structure and function at larval neuromuscular junctions appears normal, whereas life expectancy and locomotor behavior of adult mutants are significantly impaired. All phenotypes of the mutant can be largely or completely rescued by panneural expression of SRPK79D isoforms. Isoform-specific antibodies recognize panneurally overexpressed GFP-tagged SRPK79D-PC isoform co-localized with BRP at presynaptic active zones while the tagged -PB isoform is found in spots within neuronal perikarya. SRPK79D concentrations in wild type apparently are too low to be revealed by these antisera. We propose that the Drosophila Srpk79D gene characterized here may be expressed at low levels throughout the nervous system to prevent the assembly of BRP containing agglomerates in axons and maintain intact brain function. The discovery of an SR protein kinase required for normal BRP distribution calls for the identification of its substrate and the detailed analysis of SRPK function for the maintenance of nervous system integrity.

A human protein-protein interaction network: a resource for annotating the proteome.

Protein-protein interaction maps provide a valuable framework for a better understanding of the functional organization of the proteome. To detect interacting pairs of human proteins systematically, a protein matrix of 4456 baits and 5632 preys was screened by automated yeast two-hybrid (Y2H) interaction mating. We identified 3186 mostly novel interactions among 1705 proteins, resulting in a large, highly connected network. Independent pull-down and co-immunoprecipitation assays validated the overall quality of the Y2H interactions. Using topological and GO criteria, a scoring system was developed to define 911 high-confidence interactions among 401 proteins. Furthermore, the network was searched for interactions linking uncharacterized gene products and human disease proteins to regulatory cellular pathways. Two novel Axin-1 interactions were validated experimentally, characterizing ANP32A and CRMP1 as modulators of Wnt signaling. Systematic human protein interaction screens can lead to a more comprehensive understanding of protein function and cellular processes.