Flow Cytometric Method For Enumeration Research Articles

A flow cytometric method for enumeration and speciation of coccidia affecting broiler chickens

Production losses, mortality, and control measures associated with coccidiosis, caused by Eimera species, cost the broiler industry over $14 billion a year. Current means to distinguish Eimeria species such as oocyst morphology, pre-patent period and site of infection are subjective, labor intensive or unsuitable for high-throughput applications. Although Polymerase Chain Reaction (PCR) techniques have been validated, the target gene cannot differentiate relative abundance of each species in mixed infections. In this study, we developed a non-antibody-based flow cytometry high throughput method to simultaneously enumerate and speciate four Eimeria species, E. acervulina, E. mitis, E. maxima, and E. tenella, using commercial coccidia vaccine as well as field fecal samples. Our findings showed that the four Eimeria oocyst populations could be distinctly speciated based on their size and granularity (shape) via scatter plotting. These distinct populations were sorted and confirmed by quantitative real-time PCR assay. Finally, the flow cytometry findings were applied to enumerate and speciate oocysts from fecal samples collected from commercial broiler flocks vaccinated for coccidiosis at day of hatch and the results were validated against the conventional manual method of floatation and microscopic examination. Collectively, the findings of this study suggested that non-antibody based Flow Cytometry technique can be successful in the simultaneous enumeration and speciation of coccidia. Further development and validation is needed to make this diagnostic tool useful for field applications at a much larger scale as well as to speciate other Eimeria species.

Impact of UV/H2O2 advanced oxidation treatment on molecular weight distribution of NOM and biostability of water

The presence of natural organic matter (NOM) poses several challenges to the commercial practice of UV/H2O2 process for micropollutant removal. During the commercial application of UV/H2O2 advanced oxidation treatment, NOM is broken down into smaller species potentially affecting biostability by increasing Assimilable Organic Carbon (AOC) and Biodegradable Organic Carbon (BDOC) of water. This work investigated the potential impact of UV/H2O2 treatment on the molecular weight distribution of NOM and biostability of different water sources. A recently developed flow cytometric method for enumeration of bacteria was utilized to assess biological stability of the treated water at various stages through measurement of AOC. BDOC was also assessed for comparison and to better study the biostability of water. Both AOC and BDOC increased by about 3–4 times over the course of treatment, indicating the reduction of biological stability. Initial TOC and the source of NOM were found to be influencing the biostability profile of the treated water. Using high performance size exclusion chromatography, a wide range of organic molecule weights were found responsible for AOC increase; however, low molecular weight organics seemed to contribute more. Positive and meaningful correlations were observed between BDOC and AOC of different waters that underwent different treatments.

Further evaluation of a flow cytometric in vitro micronucleus assay in CHO-K1 cells: a reliable platform that detects micronuclei and discriminates apoptotic bodies

The in vitro micronucleus (MN) assay is widely used to assess genotoxic potential of the test substances by measuring frequency of MN in cultured mammalian cells. Traditionally, MN frequency has been determined by microscopy. In recent years, a flow cytometric method for enumeration of MN has been developed, which significantly shortens analysis time and enhances assay throughput. However, a major concern has been raised that the MN results obtained from flow cytometry can be impacted by chromatin bodies produced during apoptosis or necrosis. In this work, we further evaluated this flow cytometry-based in vitro MN assay with CHO-K(1) cells in a 24-well platform. Our results showed that the MN frequency determined using the flow cytometric method was highly correlated with the microscopy results. Importantly, several non-genotoxic apoptosis inducers or cytotoxins that have been previously reported to produce 'artificial positives' in various in vitro genotoxicity tests were evaluated in this system. As a result, these non-genotoxic cytotoxins did not produce false-positive MN response in the flow cytometric system in CHO-K(1) cells when cytotoxicity was <50 +/- 10%. Moreover, a total of 21 compounds were evaluated in this work, including direct or indirect clastogens, aneugens and non-genotoxic chemicals. A sensitivity of 83.3% and a specificity of 100% were obtained from the compounds we tested. Finally, significant increase of incidents in the hypodiploid region, an aneugenic signature, was confirmed in our evaluation. In conclusion, the flow cytometric in vitro MN assay is a reliable method that can be used to detect clastogenic or aneugenic potential of the test substances in CHO-K(1) cells.

Flow cytometric method for enumeration and characterization of newly released polymorphonuclear leukocytes from the bone marrow using 5′-bromo-2′-deoxyuridine

Inflammation accelerates polymorphonuclear leukocyte (PMN) release from the bone marrow, and these PMNs are implicated in inappropriate tissue injury. We have previously developed a method using 5'-bromo-2'-deoxyuridine (BrdU) to study PMN kinetics using an immunocytochemical grading system of PMN on cytospin slides. The aim of this study was to develop a flow cytometric method to quantify the number of positively stained PMN and grade the intensity of staining for the transit time calculation of PMN through the marrow. Dividing myeloid progenitors in the marrow of rabbits were labeled with a pulse dosage of intravenous BrdU. BrdU-labeled PMN (PMN(BrdU)) were detected in the circulation using a FITC-conjugated anti-BrdU monoclonal antibody. The PMN(BrdU) were assigned to five groups according to their FITC intensity, and the transit times of PMN at different stages of development in the marrow were calculated. Results were compared using parallel immunocytochemical analysis of the same samples. In control animals, PMN(BrdU) in the circulation peaked at 72 h after BrdU labeling with 36.0% of PMN labeled. In normal rabbits, the transit times of PMN through the mitotic pool (49.5 +/- 4.2 h) and maturation pool (65.5 +/- 3.1 h) correlated well with immunocytochemical analysis and previously published values. Using this method, we demonstrated that exposure to air pollution particles accelerates the release of PMN(BrdU) from the marrow. We conclude that a flow cytometric approach for identifying BrdU-labeled leukocytes provides an objective and accurate method for studying leukocyte kinetics and behavior.

A flow cytometric method for enumeration of lymphocyte sub-populations in sample containing lysis-resistant red blood cells

Determination of lymphocyte sub-populations is usually carried out by flow cytometry using two-color immunophenotyping reagent. By this technique, however, the combination of FSC and SSC with CD45-FITC/CD14-PE is unable to identify the lymphocyte population in a sample containing lysis-resistant red blood cells (RBC). The actual values of lymphocyte sub-populations, therefore, cannot be determined in these RBC contaminated samples. To overcome this problem, we describe here the use of 7-aminoactinomycin D (7-AAD) to exclude lysis-resistant RBC from white blood cells (WBC). By adding 7-AAD, lymphocytes of samples containing RBC could be identified by using FL3/SSC, therefore, the actual number of lymphocyte sub-populations of the stained cells was obtained. We have proved that 7-AAD can be used to exclude contaminated RBC and has no effect on the measurement of lymphocyte sub-populations by using two-color immunophenotyping reagent. In routine blood samples that contain lysis-resistant RBC, 7-AAD markedly increased the purity of lymphocytes in the lymphocyte gate to >95% and the lymphocyte sub-populations therefore could be correctly determined. The described method is inexpensive, simple and gives successful analysis of lymphocyte sub-populations in a sample containing lysis-resistant RBC.

Serial Determinations of Platelet Counts in Mice by Flow Cytometry

Elucidation of the pathophysiological basis of platelet disorders in murine models requires a reliable method for the frequent determinations of platelet counts in individual mice. Here, we present a rapid, reproducible and accurate flow cytometric method for enumeration of platelets that involves fluorescent staining of platelets in whole blood with specific antibody and the addition of known numbers of fluorescent beads for standardization of the sample volume. Analysis of platelets obtained by tail bleeding indicated that this sampling procedure did not activate platelets, and that only five microliters of blood were required for platelet counting. Using this method, we followed platelet counts in mice infected with the relapsing fever spirochete Borrelia turicatae for 26 days, and found that this bacterium induces thrombocytopenia, a common manifestation of human relapsing fever. Therefore, this method can be used to follow the number and the activation state of circulating platelets from individual mice over extended periods of time and is applicable to a wide range of murine models of platelet disorders.

Three-colour, one-tube flow cytometric method for enumeration of CD4+ and CD8+ lymphocytes.

We investigated the usefulness of a one-tube, three-colour flow cytometric method for enumerating CD4+ and CD8+ lymphocytes. This method does not use any control antibodies and we compared it to the standard methods using either control and CD14/CD45 antibodies or control antibodies only on 38 blood samples from healthy and human immunodeficiency virus-infected patients. The one-tube method showed good agreement with the other, more complicated methods and is therefore suitable for reliable enumeration of CD4+ and CD8+ T-lymphocytes.

A flow cytometric technique for quantitation of B-cell activation

A flow cytometric method for enumeration of intracytoplasmic antibody-containing cells was developed. Flow cytometry was found to yield results comparable to traditional fluorescence microscopy in cells stimulated to produce intracytoplasmic antibody by either pokeweed mitogen or Epstein-Barr virus. This technique offers several advantages over traditional fluorescence microscopy, including objective measurement of fluorescence intensity and rapid analysis of large numbers of cells.