Home About us Contact
|
Home About us Contact | ||
|
|
||
Single Tube (single + tube)
Selected AbstractsTotal nucleated cell differential for blood and bone marrow using a single tube in a five-color flow cytometer,,CYTOMETRY, Issue 2 2008Sven Björnsson Abstract Background: Flow cytometry allows the use of several antibodies in addition to light scatter, and most flow cytometers will provide at least seven measurements on each cell passing through the laser beam. A skilled microscopist will classify at least 14 cell classes in bone marrow or blood. Our goal was to use the seven parameters available in our flow cytometer to provide a reliable differential count using only one tube. Methods: Peripheral blood samples were analyzed on the Beckman Coulter LH750 cell counter, and the flagging and messages from the cell counter were used to select normal or pathological samples. Samples without flags (N = 50), with >2% erythroblasts (N = 80), or with "Blast" or "Verify diff" flags (N = 54) were investigated. We used a lyse-no-wash method to ensure minimal loss of fragile cells with live gating on DRAQ5-positive cells to acquire only nucleated cells. The FL-1 to FL-4 channels were used for the antibodies CD36-FITC, CD203-PE, CD138-PE, CD45-ECD, CD16-Pcy5, and CD56-Pcy5. FL-5 was used for the DNA-stain DRAQ5. Results: Using live gate acquisition on DRAQ5, we were able to classify total nucleated cells into 10 classes. We were unable to identify megakaryocytes, but platelets could be studied by rerunning the sample after dilution and gating on DRAQ5-negative CD36-posive events. Validation against digitized microscopy and cell counter showed linear correlations within each cell class with correlation coefficients that seem reasonable for cellular classification. The lowest correlation was found for basophil granulocytes. Flow cytometry detected twice as many immature neutrophils compared to microscopy. Conclusions: We have designed a one-tube immunophenotyping panel for classification of total nucleated cells and platelets in blood or bone marrow. The seven parameters available in one single tube in our cytometer seem to be enough for reliable differential count even in difficult pathological samples. The analytical imprecision of the flow cytometer differential was much lower than that obtained with microscopy or cell counter differentials. © 2007 Clinical Cytometry Society. [source] Diagnosing PNH with FLAER and multiparameter flow cytometryCYTOMETRY, Issue 3 2007D. Robert Sutherland Abstract Background: PNH is an acquired hematopoietic stem cell disorder leading to a partial or absolute deficiency of all glycophosphatidyl-inositol (GPI)-linked proteins. The classical approach to diagnosis of PNH by cytometry involves the loss of at least two GPI-linked antigens on RBCs and neutrophils. While flow assays are more sensitive and specific than complement-mediated lysis or the Hams test, they suffer from several drawbacks. Bacterial aerolysin binds to the GPI moiety of cell surface GPI-linked molecules and causes lysis of normal but not GPI-deficient PNH cells. FLAER is an Alexa488-labeled inactive variant of aerolysin that does not cause lysis of cells. Our goals were to develop a FLAER-based assay to diagnose and monitor patients with PNH and to improve detection of minor populations of PNH clones in other hematologic disorders. Methods: In a single tube assay, we combined FLAER with CD45, CD33, and CD14 allowing the simultaneous analysis of FLAER and the GPI-linked CD14 structure on neutrophil and monocyte lineages. Results: Comparison to standard CD55 and CD59 analysis showed excellent agreement. Because of the higher signal to noise ratio, the method shows increased sensitivity in our hands over single (CD55 or CD59) parameter analysis. Using this assay, we were able to detect as few as 1% PNH monocytes and neutrophils in aplastic anemia, that were otherwise undetectable using CD55 and CD59 on RBC's. We also observed abnormal FLAER staining of blast populations in acute leukemia. In these cases, the neutrophils stained normally with FLAER, while the gated CD33bright cells failed to express normal levels of CD14 and additionally showed aberrant CD45 staining and bound lower levels of FLAER. Conclusion: FLAER combined with multiparameter flow cytometry offers an improved assay for diagnosis and monitoring of PNH clones and may have utility in detection of unsuspected myeloproliferative disorders. © 2007 Clinical Cytometry Society [source] Effect of fins on forced convection heat transfer around a tube in an aligned-arranged tube bundleHEAT TRANSFER - ASIAN RESEARCH (FORMERLY HEAT TRANSFER-JAPANESE RESEARCH), Issue 8 2005Kenichi Hashizume Abstract The effect of fins on heat transfer around a tube in an aligned-arranged tube bundle was investigated experimentally, and the obtained results were compared for three arrangements, i.e., single tube, single tube row, and staggered-arrangement. It was found from the experiment that the effect of fins begins to appear in an aligned-arrangement with larger fin spacing than in a staggered-arrangement. The degradation in the local heat transfer coefficient due to fins can be recognized not only on the rear region of the tube, as observed in other arrangements, but also on the frontal region. As a result of this phenomenon, the degradation in the average heat transfer coefficient in an aligned-arrangement becomes larger than in other arrangements with the same fin spacing. © 2005 Wiley Periodicals, Inc. Heat Trans Asian Res, 34(8): 555,563, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/htj.20091 [source] Heat transfer on tube bundles embedded horizontally in a liquid-fluidized bedHEAT TRANSFER - ASIAN RESEARCH (FORMERLY HEAT TRANSFER-JAPANESE RESEARCH), Issue 2 2005Kenichi Hashizume Abstract Heat transfer on tube bundles embedded horizontally in a liquid-fluidized bed was investigated experimentally. In the experiment, a total of 5 kinds of tube bundles in an equilateral triangular staggered arrangement, including a single tube, was used. Tested particles were of glass and ceramics, and their diameter range was from 2.1 to 6.0 mm. It was found that the distribution of local heat transfer coefficients around a tube depends not on the kind of particles, but on the tube pitch only, when a good fluidizing condition is maintained. Based on the experimental data, a new method was proposed to predict average heat transfer coefficient, which can be applicable for tube bundles having a tube pitch to diameter ratio of 1.2 to infinity (single tube). © 2005 Wiley Periodicals, Inc. Heat Trans Asian Res, 34(2): 85,98, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/htj.20048 [source] Condensation of steam in the presence of air on a single tube and a tube bankINTERNATIONAL JOURNAL OF ENERGY RESEARCH, Issue 4 2003Adrian Briggs Abstract Data are presented for condensation of steam in cross-flow with and without the presence of air on the outside of a single tube and a bank of tubes. The tube bank consisted of ten staggered rows of two and one tubes per row. For pure steam the experimental results for both the single tube and tube bank gave good agreement with single-tube theory when account was taken of the reduction in vapour velocity due to condensation. There was some evidence, however, that condensate inundation may play a minor role in reducing heat-transfer coefficients on the lower tubes in the bank. For the case of condensation from steam,air mixtures, the single-tube data gave good agreement with theory. For condensation from steam,air mixtures on the bank of tubes, the data were significantly under predicted by single-tube theory, possibly because of mixing and re-circulation due to the complex flow pattern around the tubes. Copyright © 2003 John Wiley & Sons, Ltd. [source] Ultrastructure and embryonic development of a syconoid calcareous spongeINVERTEBRATE BIOLOGY, Issue 3 2006Dafne I. Eerkes-Medrano Abstract. Recent molecular data suggest that the Porifera is paraphyletic (Calcarea+Silicea) and that the Calcarea is more closely related to the Metazoa than to other sponge groups, thereby implying that a sponge-like animal gave rise to other metazoans. One ramification of these data is that calcareous sponges could provide clues as to what features are shared among this ancestral metazoan and higher animals. Recent studies describing detailed morphology in the Calcarea are lacking. We have used a combination of microscopy techniques to study the fine structure of Syconcoactum Urban 1905, a cosmopolitan calcareous sponge. The sponge has a distinct polarity, consisting of a single tube with an apically opening osculum. Finger-like chambers, several hundred micrometers in length, form the sides of the tube. The inner and outer layers of the chamber wall are formed by epithelia characterized by apical,basal polarity and occluding junctions between cells. The outer layer,the pinacoderm,and atrial cavity are lined by plate-like cells (pinacocytes), and the inner choanoderm is lined by a continuous sheet of choanocytes. Incurrent openings of the sponge are formed by porocytes, tubular cells that join the pinacoderm to the choanoderm. Between these two layers lies a collagenous mesohyl that houses sclerocytes, spicules, amoeboid cells, and a progression of embryonic stages. The morphology of choanocytes and porocytes is plastic. Ostia were closed in sponges that were vigorously shaken and in sponges left in still water for over 30 min. Choanocytes, and in particular collar microvilli, varied in size and shape, depending on their location in the choanocyte chamber. Although some of the odd shapes of choanocytes and their collars can be explained by the development of large embryos first beneath and later on top of the choanocytes, the presence of many fused collar microvilli on choanocytes may reflect peculiarities of the hydrodynamics in large syconoid choanocyte chambers. The unusual formation of a hollow blastula larva and its inversion through the choanocyte epithelium are suggestive of epithelial rather than mesenchymal cell movements. These details illustrate that calcareous sponges have characteristics that allow comparison with other metazoans,one of the reasons they have long been the focus of studies of evolution and development. [source] Detection of human sapovirus by real-time reverse transcription-polymerase chain reactionJOURNAL OF MEDICAL VIROLOGY, Issue 10 2006Tomoichiro Oka Abstract Sapovirus (SaV) is an agent of gastroenteritis for humans and swine, and is divided into five distinct genogroups (GI,GV) based on its capsid gene sequences. Typical methods of SaV detection include electron microscopy (EM), enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR). A novel TaqMan-based real-time RT-PCR assay was developed that is sensitive and has the ability to detect the broad range of genetically diverse human SaV strains. A nucleotide alignment of 10 full-length SaV genome sequences was subjected to similarity plot analysis, which indicated that the most conserved site was the polymerase-capsid junction in open reading frame 1 (ORF1). Based on multiple alignments of the 27 available sequences encoding this junction, we designed sets of primers and TaqMan MGB probes that detect human SaV GI, GII, GIV, and GV sequences in a single tube. The reactivity was confirmed with SaV GI, GII, GIV, and GV control plasmids, and the efficiency ranged from 2.5,×,107 to 2.5,×,101 copies per tube. Analysis using clinical stool specimens revealed that the present system was capable of detecting SaV GI, GII, GIV, and GV sequences, and no cross-reactivity was observed against other enteric viruses, including norovirus (NoV), rotavirus, astrovirus, and adenovirus. This is the first real-time RT-PCR system that could detect all genogroups of human sapoviruses. J. Med. Virol. 78:1347,1353, 2006. © 2006 Wiley-Liss, Inc. [source] Gene expression measurements in the context of epidemiological studiesALLERGY, Issue 12 2008C. Bieli Background:, Gene expression measurements became an attractive tool to assess biological responses in epidemiological studies. However, collection of blood samples poses various technical problems. We used gene expression data from two epidemiological studies to evaluate differences between sampling methods, comparability of two methods for measuring RNA levels and stability of RNA samples over time. Methods:, For the PARSIFAL study, PBLC of 1155 children were collected using EDTA tubes in two countries. In the PASTURE study, tubes containing RNA-stabilizing solutions (PAXgene® Blood RNA Tubes; PreAnalytiX) were used to collect cord blood leucocytes of 982 children in five countries. Real-time PCR (conventional single tube assay and high-throughput low density arrays) was used to quantify expression of various innate immunity genes. In 77 PARSIFAL samples, gene expression was measured repeatedly during prolonged storage. Results:, In PARSIFAL (EDTA tubes) the median RNA yield after extraction significantly differed between the two centres (70 and 34 ng/,l). Collecting blood into an RNA-stabilizing solution markedly reduced differences in RNA yield in PASTURE (range of medians 91,107 ng/,l). The agreement [Spearman rank correlation (r)] between repeated measurements of gene expression decreased with increasing storage time [e.g., for CD14: r (first/second measurement) = 0.35; r (first/third measurement) = 0.03]. RNA levels measured with either the conventional method or low-density arrays were comparable (r > 0.9). Conclusion:, Collecting blood samples into tubes containing an RNA-stabilizing solution increases RNA yield and reduces its variability. Long-term storage of samples may lead to RNA degradation, requiring special attention in longitudinal studies. [source] Triplex real-time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussisAPMIS, Issue 9 2010YINGHUA XU Xu Y, Xu Y, Hou Q, Yang R, Zhang S. Triplex real-time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis. APMIS 2010; 118: 685,91. A triplex real-time PCR assay for detection and differentiation of Bordetella pertussis and Bordetella parapertussis was developed. Three targets were used for amplification in a single tube: the insertion sequence IS481 and the pertussis toxin promoter region (ptxP) for B. pertussis, and the insertion sequence IS1001 for B. parapertussis. The performance of this PCR assay was evaluated in parallel in three single-target real-time PCR assays using DNA extracted from B. pertussis and B. parapertussis reference strains and nasopharyngeal swabs taken from 105 patients who had been coughing for more than 7 days. The minimum detection limit of the triplex PCR was one to five colony-forming units (CFU) of B. pertussis and 1 CFU of B. parapertussis per reaction, and the coefficients of both intra- and inter-assay variation were less than 7%. Results were available within 4 h. Of the 105 nasopharyngeal samples, seven were culture positive and 23 were PCR positive for B. pertussis. All culture-positive samples were also PCR positive. Our single-tube triplex real-time PCR assay proved to be sensitive, specific and suitable for simultaneous detection and discrimination of B. pertussis and B. parapertussis. [source] | ||||||||||||||||