Application of Flow Cytometry for Rapid Determination of Ploidy Levels in Asparagus (Asparagus officinalis L.)

Urochloa (including Brachiaria, Megathyrus and some Panicum) tropical grasses are native to Africa and are now, after selection and breeding, planted worldwide, particularly in South America, as important forages with huge potential for further sustainable improvement and conservation of grasslands. We aimed to develop an optimized approach to determine ploidy of germplasm collection of this tropical forage grass group using dried leaf material, including approaches to collect, dry and preserve plant samples for flow cytometry analysis. Our methods enable robust identification of ploidy levels (coefficient of variation of G0/G1 peaks, CV, typically <5%). Ploidy of some 348 forage grass accessions (ploidy range from 2x to 9x), from international genetic resource collections, showing variation in basic chromosome numbers and reproduction modes (apomixis and sexual), were determined using our defined standard protocol. Two major Urochloa agamic complexes are used in the current breeding programs at CIAT and EMBRAPA: the ’brizantha’ and ’humidicola’ agamic complexes are variable, with multiple ploidy levels. Some U. brizantha accessions have odd level of ploidy (5x), and the relative differences in fluorescence values of the peak positions between adjacent cytotypes is reduced, thus more precise examination of this species is required. Ploidy measurement of U. humidicola revealed aneuploidy.

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Intraoperative flow cytometry analysis of glioma tissue for rapid determination of tumor presence and its histopathological grade

Journal of Neurosurgery ◽

10.3171/2013.1.jns12681 ◽

2013 ◽

Vol 118 (6) ◽

pp. 1232-1238 ◽

Cited By ~ 28

Author(s):

Takahiro Shioyama ◽

Yoshihiro Muragaki ◽

Takashi Maruyama ◽

Takashi Komori ◽

Hiroshi Iseki

Keyword(s):

Flow Cytometry ◽

Predictive Value ◽

Rapid Determination ◽

Characteristic Curve ◽

Biopsy Sample ◽

Surgical Biopsy ◽

Diagnostic Efficacy ◽

Histopathological Grade ◽

Number Of Cells

Object Intraoperative histopathological investigation plays an important role during surgery for gliomas. To facilitate the rapid characterization of resected tissue, an original technique of intraoperative flow cytometry (iFC) was established. The objective in this study was evaluation of this technique's efficacy for rapidly determining tumor presence in the surgical biopsy sample and WHO histopathological grade of the neoplasm. Methods In total, 328 separate biopsy specimens obtained during the resection of 81 intracranial gliomas were analyzed with iFC. The evaluated malignancy index (MI) was defined as the ratio of the number of cells with greater than normal DNA content to the total number of cells. The duration of iFC in all cases was approximately 10 minutes. Each sample was additionally investigated histopathologically on frozen and permanent formalin-fixed paraffin-embedded tissue sections. The latter process was used as a “gold standard” control for evaluation of the diagnostic efficacy of iFC analysis. Results The MI differed significantly between neoplastic and perilesional brain tissue (25.3% ± 22.0% vs 4.6% ± 2.6%, p < 0.01). Receiver operating characteristic curve analysis revealed a corresponding area under the curve value of 0.941. The optimal cutoff level of the MI for identification of tumor in the biopsy specimen was 6.8%, which provided 0.88 sensitivity, 0.88 specificity, 0.97 positive predictive value, 0.60 negative predictive value, and 0.88 diagnostic accuracy. Additionally, the MI showed a significant association with WHO histopathological grades of glioma (p < 0.01), but its values in Grade II, III, and IV tumors overlapped prominently and were on average 13.3% ± 11.0%, 35.0% ± 21.8%, and 46.6% ± 23.1%, respectively. Conclusions Results of this study demonstrate that iFC with the determination of the MI may be feasible for rapidly determining glioma presence in a surgical biopsy sample.

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Rapid determination of general cell status, cell viability, and optimal harvest time in eukaryotic cell cultures by impedance flow cytometry

Applied Microbiology and Biotechnology ◽

10.1007/s00253-019-10046-3 ◽

2019 ◽

Vol 103 (20) ◽

pp. 8619-8629 ◽

Cited By ~ 4

Author(s):

Christian Opitz ◽

Grit Schade ◽

Silvan Kaufmann ◽

Marco Di Berardino ◽

Marcel Ottiger ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Viability ◽

Cell Cultures ◽

Rapid Determination ◽

Eukaryotic Cell ◽

Harvest Time ◽

Optimal Harvest

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Non-invasive ploidy determination in live fish by measuring erythrocyte size in capillaries

Acta Ichthyologica Et Piscatoria ◽

10.3897/aiep.51.65718 ◽

2021 ◽

Vol 51 (3) ◽

pp. 275-280

Author(s):

Karel Halačka ◽

Karel Janko ◽

Lukáš Vetešník

Keyword(s):

Flow Cytometry ◽

Rapid Determination ◽

Minimum Size ◽

Gibel Carp ◽

Small Fish ◽

Reliable Determination ◽

Live Fish ◽

Blood Smears ◽

The Individual

Information about ploidy is important in both commercial and conservation aquaculture and fish research. Unfortunately, methods for its determination, such as karyology, determination of the amount of DNA in a cell using microdensitometry or flow cytometry and/or measuring erythrocytes in a blood smear can be stressful or even destructive. Some of these methods are also limited by the relatively large minimum size of the individual being measured. The aim of this study was to test a new low-stress method of determining ploidy by measuring the size of erythrocytes in the capillaries of a fish, including small individuals. First, we examined diploid and triploid loach (Cobitis sp.) and gibel carp, Carassius gibelio (Bloch, 1782), using flow cytometry and blood smears, with these results being used as a control. Subsequently, we measured the size of erythrocytes in the caudal fin capillaries of anesthetized fishes of known ploidy under a light microscope. For both the loaches and gibel carp, direct observation of the mean erythrocyte size in epithelial fin capillaries provided a consistent and reliable determination of ploidy when compared with the controls based on flow cytometry and blood smears. This new method allows for rapid determination of ploidy in living small fish, where collection of tissue using other methods may cause excessive stress or damage. The method outlined here simply requires the measurement of erythrocytes directly in the bloodstream of a live fish, thereby making it possible to determine ploidy without the need for blood sampling. The method described is sufficiently efficient, less demanding on equipment than many other procedures, can be used by relatively inexperienced personnel and has benefits as regards animal welfare, which is especially important for fish production facilities or when dealing with rare or endangered species.

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