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PubMed1989

Heterogeneity of fibrinogen receptor expression on platelets activated in normal plasma with ADP: analysis by flow cytometry.

Fibrinogen receptor expression of platelets activated in normal plasma by ADP was measured by flow cytometry after labelling bound fibrinogen with fluorescein-conjugated antifibrinogen antibody. The platelet response to ADP was heterogeneous both with respect to number of platelets binding fibrinogen and the amount of fibrinogen bound per platelet. The proportion of platelets showing positive antifibrinogen antibody binding increased with increasing ADP concentration; however, even at 10(-3) M ADP, usually about one-fifth of the platelets failed to demonstrate bound fibrinogen. The non-responsive platelets tended to be the smaller ones. The relative fluorescence intensity of individual platelets also increased as ADP concentration was increased, indicating that the average number of fibrinogen molecules bound was also related to agonist concentration. The amount of fibrinogen bound following platelet activation directly correlated with the quantity of surface glycoprotein IIb detected by Tab antibody and with platelet size. This study demonstrates that platelet response to ADP in native plasma is heterogeneous in both the proportion of platelets activated and in the number of available fibrinogen receptors per platelet. This heterogeneity is related to platelet size and glycoprotein IIb-IIIa content. These observations indicate that models of ligand interaction with membrane receptors on intact cells requiring an exposure step must take into account the heterogeneity of response within a cell population. In addition to providing new insights into the response of individual platelets to activation, these results suggest that study of platelet bound fibrinogen by flow cytometry may be useful for the detection of platelet activation in vivo.

Adenosine Diphosphate↗

PubMed1986

Quantitative analysis of cellular glutathione by flow cytometry utilizing monochlorobimane: some applications to radiation and drug resistance in vitro and in vivo.

An assay using a bimane derivative has been developed to detect free glutathione (GSH) in individual viable cells by flow cytometry. Monochlorobimane [syn-(ClCH2CH3)-1,5-diazabicycla[3.30]acta-3,6-diene-2,8-dio ne], itself nonfluorescent, reacts with GSH to form a highly fluorescent derivative. High pressure liquid chromatography analysis showed that, using specific staining conditions, the only low molecular weight fluorescent derivative formed in Chinese hamster ovary cells was that formed with GSH. Very little reaction with protein sulfhydryls was observed. Rates of GSH depletion in Chinese hamster ovary cells exposed to diethylmaleate were essentially the same, whether measured by relative fluorescence intensity, by flow cytometry or by enzymatic assay on cellular extracts. This method was shown to be useful for measurement of GSH resynthesis, uptake, and depletion by prolonged hypoxia and misonidazole treatment. Since measurements are made on individual cells, cell-to-cell variation and populational heterogeneity in GSH content are revealed by flow cytometry. Although under most conditions in vitro GSH content is relatively homogeneous, under certain circumstances, such as release from hypoxia, heterogeneity in populational GSH levels was observed. The significance of this heterogeneity is discussed in regard to the induction of gene amplification and drug resistance by transient hypoxia. Numerous subclones of Chinese hamster ovary cells selected by growth in Adriamycin or methotrexate-containing medium express elevated levels of GSH per cell. The method was extended to quantitate the GSH content of cells excised from EMT-6/SF mouse tumors that had been treated in vivo with L-buthionine-S-R-sulfoximine, an inhibitor of GSH synthesis. The bivariate analysis (forward angle light scatter versus monochlorobimane fluorescence) of cells derived from these tumors gave excellent resolution of normal and tumor cells and demonstrated extensive heterogeneity in the tumor cell population with respect to GSH content per cell.

Animals↗

PubMed2006

Noninvasive immune monitoring assessed by flow cytometry and real time RT-PCR in urine of renal transplantation recipients.

BACKGROUND: Monitoring recipient's alloreactivity has shown to be critical for limiting overimmunosuppression besides allowing preemptive treatment of acute rejection (AR). METHODS: Flow cytometry and real time RT-PCR were performed in urine of kidney transplant recipients with AR (n = 13) and compared with pyelonephritis (n = 10), chronic allograft nephropathy (n = 13), acute tubular necrosis (n = 13) and stable graft function (n = 11). Expression of CD3, CD4, CD8, HLA-DR, Fas-L, ICAM-1 and CD25 were assessed using flow cytometry. mRNA of perforin, granzyme B and Fas-L were quantified by real time RT-PCR. RESULTS: Frequencies of CD3+, HLA-DR+, Fas-L+, ICAM-1+ and CD25+ cells were significantly higher in AR group (p < 0.05). ROC curves showed sensitivity from 70% to 91% and specificity from 30% to 100%, whereas the highest sensitivity and specificity was 91% and 100% respectively, for Fas-L+ cells. Levels of mRNA of perforin, granzyme B and Fas-L were significantly augmented in AR, while the sensitivity and specificity ranged from 85% to 88% and from 55% to 100%, respectively. CONCLUSIONS: Analyses of immune activation markers by flow cytometry and real time RT-PCR are equally useful for noninvasive monitoring kidney allografts.

Female↗

PubMed1991

Measurement of proliferation nuclear and membrane markers in tumor cells by flow cytometry.

Nuclear and membrane markers that have been related to proliferative activity were measured by flow cytometry. The markers studied were transferrin receptor (TR), Ki-67 antigen, and epidermal growth factor receptor (EGFR). Two-color analysis for DNA via propidium iodide binding and for antigen expression via either a direct or indirect immunofluorescence assay was performed on three different cell lines and a solid human tumor model. The three cell lines tested were MCF-7 (breast), K-562 (leukemia), and A431 (a squamous cell). The solid tumor was obtained by subcutaneous injection of A431 cells into an athymic nude mouse. Our results demonstrate that TR are cell-cycle specific and can be readily measured in the cell lines. Ki-67 antigen is also cell-cycle specific in the cell lines tested, but the mean channel specific fluorescence uptake varies in the cell types. Finally, the EGFR was observed only in the A431 cell line, with most cells equally expressing this receptor. A bimodal distribution of EGFR was observed in A431 cells obtained from a solid tumor grown in an athymic nude mouse system. This suggests that cell line analysis may not always represent what might be observed under in vivo conditions. There are advantages to flow cytometry measurements of these factors which might be useful in predicting how patients should be treated and possibly the prognosis of cancer patients.

Cell Cycle↗

PubMed1989

Simultaneous measurement of progesterone receptors and DNA indices by flow cytometry: analysis of breast cancer cell mixtures and genetic instability of the T47D line.

A flow cytometry assay that can simultaneously measure progesterone receptors (PR) and DNA indices in breast cancers would be a valuable clinical tool. We have developed a prototype assay that has proven useful in studies of the cell biology of breast cancer cell lines. The assay uses PR-specific monoclonal primary antibodies and fluorescein-conjugated secondary antibodies to measure PR, and propidium iodide to measure DNA. We find that the specific PR fluorescence generated by labeling PR-rich T47D human breast cancer cells is located predominantly in nuclei. The flow cytometry assay can quantitatively measure large fluctuations in intracellular PR levels: an apparent increase in PR following acute progestin treatment that cannot be documented by ligand binding assays; and the receptor down-regulation that follows chronic progestin treatment. The assay can identify fewer than 10% PR-positive cells in a population of PR-negative cells having the same DNA content, and it can sort PR-positive and PR-negative cells from cell mixtures having different DNA indices. Gating allows quantitative analysis of these mixed cell populations by ploidy, cell-cycle phase, and PR content. Finally, the assay has allowed us to monitor the gradual emergence of a stable hypertetraploid cell population, designated T47Dv, from the wild-type hyperdiploid T47Dco stocks. The new cells have unchanged estrogen receptors but even higher PR levels than the parental cells. They have five to ten copies of chromosome 11, site of the PR gene and other genes of interest in breast cancer.

Biomarkers, Tumor↗

PubMed1992

Differentiation and maturation of growth factor expanded human hematopoietic progenitors assessed by multidimensional flow cytometry.

Non-adherent cord blood and bone marrow mononuclear cells were analyzed by multiparameter flow cytometry before and at day 2, 4, 7, and 11 of culture in recombinant interleukin 3 (IL-3) and granulocyte colony-stimulating factor (G-CSF, cord blood) or stem cell factor (SCF), IL3 and granulocyte-macrophage colony-stimulating factor (GM-CSF, BM) to assess the differentiation and maturational pathway of myeloid cells. Before cell culture cord blood contained progenitor cells (CD34+) in various differentiation stages (CD38(-)----CD38bright), mature lymphocytes, monocytes, and neutrophils, but no immature neutrophils and immature monocytes. During cell culture, all CD34+ cells acquired the CD38 antigen between day 2 and 5 of cell culture, the CD34 antigen was lost between day 5 and 11 of cell culture. Differentiation of cells into the myeloid cell lineage was characterized by the acquisition of both CD33 and CD71. The latter is indicative for the active proliferation of these cells. Maturation of the cells into the neutrophilic pathway was indicated by the acquisition of first the CD15 antigen followed by CD11b and CD16 respectively. Whereas maturation of the cells into the monocytic pathway was indicated by the acquisition of first CD11b followed by CD14 and a dim expression of both CD15 and CD16. In normal bone marrow, cells of various maturational stages are already present before cell culture. During cell culture differentiation of cells into the myeloid lineage and maturation of the cells along the monocyte and neutrophilic lineage followed identical pathways as was observed before cell culture. Differentiation and maturational pathways of cord blood and adult bone marrow were identical. The results confirm the surface-antigen-defined pathways of myeloid cell differentiation described previously for non-cultured normal bone marrow aspirates. The detailed assessment of cell maturation and differentiation of cultured cells by multidimensional flow cytometry permits the determination of the specific effects of various recombinant human growth factors on myeloid cells.

Antigens, CD↗

PubMed1989

Use of two-colour flow cytometry to assess killing of Giardia muris trophozoites by antibody and complement.

A two-colour flow cytometry technique was used to assess killing of Giardia muris trophozoites by rabbit anti-trophozoite antibodies and complement. Binding of rabbit antibody to trophozoites and killing of trophozoites were documented by flow cytometry, after incubating the organisms with fluorescein-conjugated anti-rabbit IgG and propidium iodide (PI). Percentages of PI+ (dead) trophozoites ranged from greater than 80%, after incubation with rabbit antiserum and complement, to less than 30%, after incubation with complement alone. The assay technique may be applicable to studies aimed at determining whether intestinal antibodies from Giardia-infected mammals can kill Giardia trophozoites.

Animals↗

PubMed2000

A multicenter study evaluating three methods for counting residual WBCs in WBC-reduced blood components: Nageotte hemocytometry, flow cytometry, and microfluorometry.

BACKGROUND: A multicenter study was conducted to evaluate the performance characteristics of flow cytometry and microfluorimetry for counting low concentrations of WBCs and to compare the results with Nageotte hemocytometry. STUDY DESIGN AND METHODS: A two-phase study involving 10 centers located in the United States and in Europe was performed. Coded samples of RBCs and platelets were distributed by 24-hour (Phase 1) or 2-day (Phase 2) courier service to each test site for analysis. Samples were prepared to include concentrations of WBCs slightly above and below the concentration corresponding to the threshold standards for WBC-reduced RBCs and platelets. All centers tested samples by Nageotte hemocytometry plus one or both of two automated methods. RESULTS: Both flow cytometry and microfluorometry gave better results than Nageotte hemocytometry in testing freshly prepared samples. At WBC concentrations >5 per microL (RBCs) or >3 per microL (platelets), the intersite CV was <20 percent for the automated methods but >30 percent for the Nageotte hemocytometer method (p<0.001). Accuracy was greater for the automated methods than for the Nageotte hemocytometer method (p<0. 001). Nageotte hemocytometry showed a bias to underestimation relative to the results obtained with the automated methods. All methods had poorer performance in testing samples that required > or =2 days' shipment than in testing of those requiring overnight shipment. CONCLUSION: Automated methods for counting residual donor WBCs in WBC-reduced cellular components offer advantages of improved precision and greater accuracy than are seen with the Nageotte hemocytometer method. Automated methods are less labor-intensive but more costly than microscopic methods. Preparation and shipping methods will need further refinement for samples to be counted more than 24 hours after sample collection.

Blood Platelets↗

PubMed1997

Detection of p53 in inflammatory tissue and lymphocytes using immunohistology and flow cytometry: a critical comment.

AIMS: To analyse the expression of p53 in lymphatic cells found in inflammatory tissues and the peripheral blood by immunological methods. METHODS: Immunohistological analysis of synovial tissues from patients with rheumatoid arthritis and flow cytometric analysis of peripheral blood lymphocytes were performed with anti-p53 antibodies from different sources. RESULTS: The anti-p53 antibodies PAb240, PAb421, and PAb1801 from one supplier bound to the cytoplasm of lymphocytes, fibroblasts, and endothelial cells in rheumatoid synovial tissue, while the same anti-p53 antibodies from other sources and the p53 specific antibodies PAb1620 and DO1 were negative. Using flow cytometry, the antibodies that labelled cells in inflammatory tissues were shown to bind also to peripheral lymphocytes, while the antibodies that were negative in immunohistology did not react with peripheral blood lymphocytes. p53 expression could be confirmed by western blot in rheumatoid synovial tissue, but not in peripheral blood lymphocytes using PAb421 and PAb240 antibodies from our own laboratory, which had been negative in immunohistology. CONCLUSIONS: Demonstration of p53 by western blot is more sensitive and reliable than immunohistology and flow cytometry. Western blot is the gold standard for the demonstration of p53 expression and should be used, whenever possible, to confirm p53 expression in normal tissue shown by immunohistology or flow cytometry. All other reports on p53 expression, especially those obtained using antibodies with an unusual staining pattern must be interpreted with caution.

Arthritis, Rheumatoid↗

PubMed2005

Classifying antibodies using flow cytometry data: class prediction and class discovery.

Classifying monoclonal antibodies, based on the similarity of their binding to the proteins (antigens) on the surface of blood cells, is essential for progress in immunology, hematology and clinical medicine. The collaborative efforts of researchers from many countries have led to the classification of thousands of antibodies into 247 clusters of differentiation (CD). Classification is based on flow cytometry and biochemical data. In preliminary classifications of antibodies based on flow cytometry data, the object requiring classification (an antibody) is described by a set of random samples from unknown densities of fluorescence intensity. An individual sample is collected in the experiment, where a population of cells of a certain type is stained by the identical fluorescently marked replicates of the antibody of interest. Samples are collected for multiple cell types. The classification problems of interest include identifying new CDs (class discovery or unsupervised learning) and assigning new antibodies to the known CD clusters (class prediction or supervised learning). These problems have attracted limited attention from statisticians. We recommend a novel approach to the classification process in which a computer algorithm suggests to the analyst the subset of the "most appropriate" classifications of an antibody in class prediction problems or the "most similar" pairs/ groups of antibodies in class discovery problems. The suggested algorithm speeds up the analysis of a flow cytometry data by a factor 10-20. This allows the analyst to focus on the interpretation of the automatically suggested preliminary classification solutions and on planning the subsequent biochemical experiments.

Antibodies, Monoclonal↗

PubMedDate not supplied

Prospective malignancy grading and flow cytometry DNA distribution in biopsy specimens from invasive squamous cell carcinoma of the uterine cervix.

Flow cytometry was used for the investigation of the DNA distribution in biopsy specimens from 72 patients with squamous cell carcinoma of the uterine cervix. A prospective grading score system using tumor cell parameters and tumor host parameters was also applied. 50% of the tumors were aneuploid with up to 4 different tumor populations. The definition of DI +/- 8% was applied. The median age of the patients was 61 years with FIGO median value of 2B. Significant correlations were observed between ploidy and MGS-scores and stage stadium according to FIGO. Increasing MGS score was noted with increasingly distorted ploidy. No significant difference was found between DI below and above 1.5 for MGS, FIGO stage stadium, histology according to Ackerman, tumor parameters and tumor host parameters for the total material. However, for aneuploid tumors MGS and tumor cell parameters were related with DI below and above 1.5 (P = 0.05 and P = 0.02, respectively). No correlation between clinical stage according to FIGO and S-phase % was noted. It remains to be settled to what extent DNA flow cytometry and MGS-scoring in our ongoing prospective series of invasive squamous cell carcinoma of the uterine cervix can take their place in the multifactorial prognostic index suggested by Jacobsen et al (Am. J. Clin. Oncol. 8: 39, 1985).

Adult↗

PubMed1989

[Fluorescent formazans in flow cytometry. Studies of their oxygen sensitivity].

In order to supply the tetrazolium salt procedure to flow cytometry we have synthesized monotetrazolium salts which are converted into fluorescent formazans on reduction. In this preliminary study we investigated the "oxygen sensitivity" of four of our compounds and compared them to the commercially available nitrotetrazole blue (NBT). The amount of formazan produced by Ehrlich ascites tumour cells via the succinic dehydrogenase reaction during oxic and anoxic incubations was determined by elution technique. Under oxic conditions no NBT formazan but much cyano-di-chlorophenyl formazan (==CCPC) was generated. The yields of this formazan differed only slightly between oxic and anoxic incubations. Twice as much of the amount of the other three formazans cyano-ditolyl- (==CTC, Stellmach 1984), cyanodiphenyl- (==CPC), and cyano-dianisyl tetrazolium chloride (==CAC), were produced in nitrogen atmosphere as in oxygen. The amount of all formazans investigated increased both in oxic and in anoxic conditions if cyanide was present. All cyano-aryl formazans have the maximum of absorbance between 430 and 470 nm and fluoresce in the spectral range above 580 nm with CTC formazan being the brightest. The tetrazolium salts, i.e. in unreduced state, did not fluoresce in the visible part of the spectrum. The formazans could be applied to flow cytometry. After staining the nuclear DNA with the fluorochrome DAPI, we obtained detailed two-parametric distributions correlating the amount of formazan per cell with the DNA content. The results are a step to establish a method for automated identification of malignant cells.

Animals↗

PubMed1990

Fluorescent in-situ hybridization to detect cellular RNA by flow cytometry and confocal microscopy.

A fluorescent in-situ hybridization procedure was developed which is suitable for detection of specific cellular RNA in cells fixed in suspension. The procedure was originally developed for analysis of single-cell suspensions by flow cytometry. The resulting fluorescent cells proved to have their 3-D morphology perfectly preserved. The spatial distribution of specific ribosomal RNA and messenger RNA could then be analysed by confocal microscopy of individual cells. In the hybridization procedure, biotinylated single-stranded RNA probes were used that were produced by transcription from cloned DNA fragments. Detection of poly-(A)+ RNA was performed with a poly-biotin-d(U)-tailed oligo-d(T) probe. Bound probe was detected using streptavidin-fluorescein isothiocyanate. For flow cytometry, nuclear DNA was counterstained with di-amidine-2-phenyl indol. Multi-parameter flow cytometry was used to quantify the fluorescence intensity, i.e. the hybridization signal, of thousands of cells. Confocal scanning microscopy on individual cells revealed the intracellular distribution of the target RNA. The distribution of ribosomal RNA and poly-(A)+ RNA mRNA in mouse bone marrow and the human leukaemia cell line HL60 cells was investigated. Ribosomal RNA was confined to the cytoplasm of the cells, although in many cells the nucleoli could also be distinguished. Poly-(A)+ RNA in HL60 cells was found both in the cytoplasm and nucleus. A granular cytoplasmic distribution was seen in part of these cells.

Animals↗

PubMed1997

Flow cytometry in cervical adenocarcinoma. Correlating DNA content analysis with the clinical picture.

OBJECTIVE: DNA quantitation by flow cytometry was used in this study to determine prognosis in stage I adenocarcinoma of the uterine cervix with negative pelvic lymph nodes. STUDY DESIGN: DNA content and proliferation index were determined by flow cytometry on formalin-fixed, paraffin-embedded tissue from 29 patients with stage I, pelvic lymph node-negative adenocarcinoma of the cervix. Using Student's t test, the results were correlated with tumor recurrence after 57 +/- 8 months. RESULTS: Diploid DNA content predominated (79%), whereas only 21% of the tumors were aneuploid. There was recurrence of the tumor in five cases (17%); two tumors were aneuploid, and three were euploid. The recurrence rate among the aneuploid population of 33% (2 of 6) was not statistically significant as compared to 13% (3 of 23) in the diploid population (P > .2). Similarly, the mean proliferation index (percentage in S plus G2/M phases) of the tumors in patients with recurrence was 13.7 +/- 3.68%, which was not significantly different from those that did not recur (mean 15.6 +/- 3.49%). CONCLUSION: DNA content analysis and proliferative activity do not predict recurrence in stage I, pelvic lymph node-negative adenocarcinoma of the uterine cervix.

Adenocarcinoma↗

PubMedDate not supplied

[Microspheres, nanospheres and flow cytometry: from cellular to molecular analysis].

Micro- and nanospheres are tightly associated with the development of flow cytometry. They are indispensable tools to optimize diffraction and fluorescence signals as well as for fluorescence calibration and cellular purification (magnetic micro- and nanospheres). They are also usefull to evaluate phagocytosis and to detect slightly expressed antigens. Recently, developments of microspheres-based flow cytometric assays have raised to quantify soluble analytes in biological fluids, cellular and tissue samples. The technology utilizes spectrally distinct fluorescent microspheres as a solid support for a conventional immunoassay, affinity assay or DNA hybridisation assay which is subsequently analyzed on a flow cytometer. Several multiplexed bead systems are now available facilitating the development of multiplexed assays that simultaneously measure many different analytes in few microliters of sample. Some recent applications with fluorescent microspheres coated with antibodies or oligonucleotides include cytokines and PCR products quantitation and single nucleotide polymorphism genotyping. Thus, multiplex assays using microspheres and flow cytometry technologies are exciting techniques which have the potential to contribute to the development of efficient diagnostic and research methods.

Cytological Techniques↗

PubMed2006

Residual disease monitoring in childhood acute myeloid leukemia by multiparameter flow cytometry: the MRD-AML-BFM Study Group.

PURPOSE: Monitoring of residual disease (RD) by flow cytometry in childhood acute myeloid leukemia (AML) may predict outcome. However, the optimal time points for investigation, the best antibody combinations, and most importantly, the clinical impact of RD analysis remain unclear. PATIENTS AND METHODS: Five hundred forty-two specimens of 150 children enrolled in the AML-Berlin-Frankfurt-Muenster (BFM) 98 study were analyzed by four-color immunophenotyping at up to four predefined time points during treatment. For each of the 12 leukemia-associated immunophenotypes and time points, a threshold level based on a previous retrospective analysis of another cohort of children with AML and on control bone marrows was determined. RESULTS: Regarding all four time points, there is a statistically significant difference in the 3-year event-free survival (EFS) in those children presenting with immunologically detectable blasts at 3 or more time points. The levels at bone marrow puncture (BMP) 1 and BMP2 turned out to have the most significant predictive value for 3-year-EFS: 71% +/- 6% versus 48% +/- 9%, P(Log-Rank) = .029 and 70% +/- 6% versus 50% +/- 7%, P(Log-Rank) = .033), resulting in a more than two-fold risk of relapse. In a multivariate analysis, using a combined risk classification based on morphologically determined blasts at BMP1 and BMP2, French-American-British classification, and cytogenetics, the influence of immunologically determined RD was no longer statistically significant. CONCLUSION: RD monitoring before second induction has the same predictive value as examining levels at four different time points during intensive chemotherapy. Compared with commonly defined risk factors in the AML-BFM studies, flow cytometry does not provide additional information for outcome prediction, but may be helpful to evaluate the remission status at day 28.

Adolescent↗

PubMedDate not supplied

Flow cytometry as a tool to identify Mycobacterium tuberculosis interaction with the immune system and drug susceptibility.

Flow cytometric analysis is a useful and widely employed tool to identify immunological alterations caused by different microorganisms, including Mycobacterium tuberculosis. However, this tool can be used for several others analysis. We will discuss some applications for flow cytometry to the study of M. tuberculosis, mainly on cell surface antigens, mycobacterial secreted proteins, their interaction with the immune system using inflammatory cells recovered from peripheral blood, alveolar and pleura spaces and the influence of M. tuberculosis on apoptosis, and finally the rapid determination of drug susceptibility. All of these examples highlight the usefulness of flow cytometry in the study of M. tuberculosis infection.

Antigens, Surface↗

PubMed2002

Ploidy Determination in Agrostis Using Flow Cytometry and Morphological Traits.

The taxonomic classification of the genus Agrostis is one of the most complicated of the grass genera. Classification based upon morphological and anatomical characters is difficult and complicated by the presence of intermediate forms and the misapplication of names. Determining ploidy levels of new germplasm can assist in species determination and is necessary before initiating breeding or genetics studies. The objectives of this study were to (i) evaluate the use of laser flow cytometry as a quick, reliable tool to determine ploidy level and aid in Agrostis species determination, and (ii) identify morphological characters associated with DNA content or ploidy level. The six Agrostis species evaluated were A. canina L. subsp. canina, A. canina L. subsp. montana (Hartm.) Hartm., A. palustris Huds. [= A. stolonifera var. palustris (Huds.) Farw.], A. tenuis Sibth. (= A. capillaris L.), A. castellana Boiss. & Reut., and A. alba L. Ploidy level was determined by flow cytometry and root tip chromosome counts. Plant height, panicle height, flag leaf length, flag leaf width, and highest internode length of mature field-grown spaced plants were measured. Significant differences in 2C DNA content were found between species (P < 0.01) differing in ploidy level. Flow cytometry was effective in differentiating between diploid, tetraploid, and hexaploid species. Chromosome numbers previously reported and those observed in this study were positively correlated with 2C nuclear DNA content (r = 0.98, P < 0.01). Flag leaf length was the only morphological measurement taken that was significantly positively correlated to DNA content (r = 0.98, P < 0.001). The results of this study indicate that laser flow cytometry is a quick, reliable tool to determine ploidy levels and infer certain species of AGROSTIS: This technique will aid breeders to quickly and accurately determine ploidy levels of new germplasm collections.

Journal Article↗