Cell Counter (cell + counter)

Distribution by Scientific Domains

Selected Abstracts

Total nucleated cell differential for blood and bone marrow using a single tube in a five-color flow cytometer,,

CYTOMETRY, Issue 2 2008
Sven 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]

Performance evaluation of the PENTRA 60C+ automated hematology analyzer and comparison with the ADVIA 2120

Summary The PENTRA 60C+ hematology analyzer provides a complete blood cell (CBC) count, including a five-part differential (5-DIFF) count and two leukocyte subpopulations, i.e. large immature cells (LIC's) and atypical lymphocytes (ALY's). We evaluated its analytical performance and assessed agreement with the ADVIA 2120, in order to install the analyzer in a small satellite hematology laboratory. First we assessed repeatability, reproducibility and carry-over to evaluate the analytical performance. Then we used Pearson correlation coefficients, Passing and Bablok regression analysis and a graphical approach (n = 209) to evaluate agreement with the ADVIA 2120. Repeatability and reproducibility were excellent for the majority of CBC and 5-DIFF count parameters. Carry-over was negligible. Our data showed very good correlation for most CBC count parameters. Lower correlation coefficients were observed for red cell distribution width, mean corpuscular volume and mean platelet volume. As compared to the ADVIA 2120, the 5-DIFF count performed very well. Agreement was poorer for low-level eosinophils and basophils. Furthermore, the PENTRA 60C+ was equally able to identify pathological blood samples through the determination of LIC's and ALY's. Therefore, the PENTRA 60C+ is an eligible blood cell counter to be operational in a satellite laboratory setting. [source]

Hematopoietic progenitor cells (HPC) and immature reticulocytes evaluations in mobilization process: new parameters measured by conventional blood cell counter

J.F.A. Noronha
Abstract Monitoring the timing of leukapheresis in peripheral blood stem cells (PBSC) mobilization is an important clinical decision that requires an accurate analytical tool. The present study assessed hematopoietic progenitor cells (HPC) and immature reticulocyte fraction (IRF) counts provided by a routine automated blood counter as potential parameters for predicting the appropriate time for harvesting. The HPC and IRF values were compared with white blood cell (WBC) and CD34+ cell counts obtained by flow cytometry in 30 adult patients with hematological malignancies undergoing PBSC mobilization. It was observed that there was a significant correlation between HPC counts and CD34+ cells in peripheral blood counts (r=0.61, P=0.0003) and between the number of HPC and CD34+cells collected by leukapheresis (r=0.5733, P=0.0009). Comparing HPC, IRF, WBC, and CD34+ cells parameters as a sign of hematological recovery showed that the raise in immature reticulocytes counts preceded the increase of WBC (P=0.0002), HPC (P=0.0001), and CD34+ (P=0.0001) cells in peripheral blood counts. According to our results, HPC and IRF parameters may be integrated into clinical protocols to evaluate the timing of leukapheresis. IRF, as previously demonstrated in bone marrow transplantation, is the earliest sign of hematopoietic recovery in mobilization process. J. Clin. Lab. Anal. 20:149,153, 2006. © 2006 Wiley-Liss, Inc. [source]

Cell Subpopulation-related Volumetric Parameters: a Complementary Tool of the Modified Hypo-osmotic Swelling Test on Model of Boar Spermatozoa

A. Petrounkina
Content It is a general property of the intact animal cell to swell rapidly in response to hypo-osmotic conditions. The modified hypo-osmotic swelling test (HOS-test) is an indicative test to evaluate the integrity of the plasma membrane by means of an electronic cell counter, based on the relative increase of the cell volume in response to hypo-osmotic conditions. In this study the relationships between the osmotically induced changes of the cell volume of boar spermatozoa as determined by cell counter and the integrity of the membrane as determined by propidium iodide staining (PI) were studied. Boar sperm cell volume distributions were measured under iso-osmotic (300 mosmolar) conditions and after a hypo-osmotic stress (150 mosmolar). The relative volume shift of mean and modal volume were calculated as a proportion coefficient of modal and mean values of the cell volume distributions by transition from iso-osmotic to hypo-osmotic conditions. The volumetric parameters related to the different cell subpopulations were derived from the different peaks of cell volume distributions. PI-staining techniques were used for comparison. The values of the volume shift and of derived percentages of the osmotically inactive cells were correlated negatively and positively, respectively (p < 0.05) with the percentage of the PI-stained cells. This correlation indicates that a relationship exists between membrane functions of the different cell compartments (sperm head and tail) due to the circumstance that the increase of the cell volume in the HOS-test is associated with the morphological changes in the tail and the PI-staining is associated with the membrane integrity and permeability of the head region. The advantage of computer-assisted volume measurement is that a large number of cells (5000,50 000 spermatozoa) can be measured and evaluated during one procedure and in a very short time. The relative volume shift is a quantitative continuous parameter characterizing the osmotic reactivity and membrane functional competence of a cell population and of subpopulations within one ejaculate. This parameter could be useful to evaluate membrane functional competence rapidly and sensitively. Inhalt Es ist eine generelle Eigenschaft membranintakter tierischer Zellen, mit einer Volumenzunahme auf eine hypoosmotische Belastung zu reagieren. Der auf der relativen Vergrößierungdes Zellvolumens basierende modifizierte hypoosmotischeSchwelltest ist ein indikativer Test zur Beurteilung der Membranintegrität mittels eines elektronischen Partikelzählers. In dieser Studie wurden die Zusammenhänge zwischen der mittels der Propidiumjodid-Färbung bestimmten Zellmembranintegrität und den osmotisch induzierten Veränderungen des Zellvolumens von Eberspermien untersucht. Volumenverteilungen von Eberspermien wurden unter isoosmotischen (300 mosmolar) und hypoosmotischen (150 mosmolar) Bedingungen gemessen. Die relative Volumenverschiebung der modalen und mittleren Werte der Volumenverteilung wurde als Quotient aus Modalwerten der Zellvolumenverteilungen und des mittleren Zellvolumens beim Übergang von isotonen zu hypotonen Bedingungen berechnet. Die auf verschiedene Subpopulationen bezogenen volumetrischen Parameter werden aus den originalen Volumenverteilungen berechnet. Der Betrag der Zellvolumenzunahme und die aus den Volumenverteilungen bestimmten Anteile an Zellen mit beschädigter Geißielmembran korrelierten signifikant negativ bzw. positiv (p < 0,05) mit dem Anteil an den Zellen mit beschädigter Kopfmembran, der sich aus der Propidiumjodid-Färbung ergab. Es wird geschlossen, daßi im Verhalten zwischen den Membranen der verschiedenen Zellkompartimente (Spermienkopf und-Geißiel) ein Zusammenhang besteht. Die beschriebene Methode ermöglicht die Analyse großier Zellpopulationen (5.000,50.000 Zellen). Die relative Volumenverschiebung stellt einen quantitativen kontinuierlichen Parameter dar, der den Membranzustand der Eberspermien einer Spermatozoenpopulation und Subpopulationen innerhalb eines Ejakulates charakterisiert. Diese Parameter können zur schnellen und sensitiven Beurteilung der Membranzustandes eingesetzt werden. [source]

Cell culture monitoring via an auto-sampler and an integrated multi-functional off-line analyzer

Gayle E. Derfus
Abstract Mammalian cell-based bioprocesses are used extensively for production of therapeutic proteins. Off-line monitoring of such cultivations via manual sampling is often labor-intensive and can introduce operator-dependent error into the process. An integrated multi-functional off-line analyzer, the BioProfile FLEX (NOVA Biomedical, Waltham MA) has been developed, which combines the functionality of three off-line analyzers (a cell counter, an osmometer, and a gas/electrolyte & nutrient/metabolite bio-profile analyzer) into one device. In addition, a novel automated sampling system has also been developed that allows the BioProfile FLEX to automatically analyze the culture conditions in as many as ten bioreactors. This is the first report on the development and function of this integrated analyzer and an auto-sampler prototype for monitoring of mammalian cell cultures. Evaluation of the BioProfile FLEX was conducted in two separate laboratories and involved two BioProfile FLEX analyzers and two sets of reference analyzers (Nova BioProfile 400, Beckman-Coulter Vi-Cell AS, and Advanced Instruments Osmometer 3900), 13 CHO cell lines and over 20 operators. In general, BioProfile FLEX measurements were equivalent to those obtained using reference analyzers, and the auto-sampler did not alter the samples it provided to the BioProfile FLEX. These results suggest that the system has the potential to dramatically reduce the manual labor involved in monitoring mammalian cell bioprocesses without altering the quality of the data obtained, and integration with a bioreactor control system will allow feedback control of parameters previously available only for off-line monitoring. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source]

Co-inheritance of Hb Hershey [,70(E14) Ala,Gly] and Hb La Pommeraie [,133(H11)Val,Met] in a Sicilian subject

Antonino Giambona
Abstract Objectives:,This report represents the first observation in Sicily of two rare , -globin gene variants, Hb Hershey [,70(E14) Ala,Gly] and Hb La Pommeraie [,133(H11)Val,Met], found in a 35-year-old male patient from Messina, in the north-east of Sicily during population screening for hemoglobinopathies. Methods: The occurrence of the Hb variants was assessed by cation exchange chromatography while complete blood counts were obtained using automatic cell counters. Red cell lysates were analyzed by electrophoresis at alkaline and acid pH. Stability of hemoglobin was checked by the isopropanol precipitation test and by the heat tests while inclusion bodies and reticulocyte count were determined by incubation of blood samples with brilliant cresyl blue. Molecular analysis was performed by DNA sequencing of ,- and , -globin genes. Results: We observed an abnormally high performance liquid chromatography elution with a slight reduction in mean corpuscular volume and mean corpuscular haemoglobin parameters and mutations at codon 70 GCC,GGC (Hb Hershey) and at codon 133 GTG,ATG (Hb La Pommeraie) in , -globin gene. Conclusion: Family analysis of three generations demonstrated the presence of these two mutations in trans. So it was possible to describe the phenotypes of these variants in a heterozygous state and in double heterozygous state. [source]

Superiority of a functional leukocyte adhesiveness/aggregation test over the white blood cell count to discriminate between mild and significant inflammatory response in patients with acute bacterial infections

Ori Rogowski
Abstract Electronic cell counters may underestimate the white blood cell count (WBCC) in the presence of aggregated leukocytes. In the present study we focused on the possibility of using a functional, as opposed to an anatomic, count to circumvent this eventual underestimation. A model of bacterial infection was used because of the importance of leukocytosis in the physician's clinical decision-making process. There were 35 patients with low C-reactive protein (CRP) concentrations (0.5,4.9 mg/dL), 45 with intermediate (5,9.9 mg/dL), and 120 with relatively high (>10 mg/dL) CRP concentrations. A significant (P=0.008) difference was noted between the state of leukocyte adhesiveness/aggregation in the peripheral blood of individuals with low CRP concentrations (3.5%±4.3%) and those with high CRP concentrations (7.4%±8%), while there was no significant difference in the respective number of WBCs per cubic millimeter (cmm) (11,600 ± 5,500 and 14,000 ± 7,200, respectively). We raise the possibility that a functional test might be superior over an anatomic count in patients with acute bacterial infection and a significant acute phase response. © 2002 Wiley-Liss, Inc. [source]