DNA ploidy

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 DNA ploidy is basically a test that measures the DNA content within cells. In the resting state, they contain one complete set of chromosomes (2n). after receiving signals for proliferation, diploid cells exit the resting state Gap 0 (G0) phase and enter the Gap 1 (G1) phase with two complete sets of chromosomes (2N). As the cells enter the synthesis (S) phase, DNA replication starts and DNA content reaches a tetraploid state (4N). Tetraploid cells in the G2 phase start preparing for division and enter the mitosis (M) phase when the cells divide into two identical diploid(2N) daughter cells. The daughter cells continue on to another division cycle or enter the resting stage (G0 phase). Based on DNA content alone, the M phase is indistinguishable from the G2 phase, and G0 is indistinguishable from G1. Therefore, when based on DNA content, cell cycle is commonly described by the G0/G1, S, and G2/M phases.  Due to anomalies in DNA replication, some cell populations such as cancer cells can have abnormal DNA content, and different ploidy.The DNA content of tumour cells in G1 of the cell cycle compared to that of normal cells. Normal cells have a DI of 1. DNA index of >= 1.16 and <= 1.6 is associated with hyperdiploidy.

                                                                                   

                                                                                    Flow cytometry can measure DNA content of cells and cell position in the cell cycle. The DNA content is generally 

                                                                                    expressed as a DNA index, which is the quantity of DNA in the test cell population in relation to that in normal

                                                                                   diploid cells. there are many nucleic acid dyes( PI, DAPI, Hoescht  33342, Fx Cycle, YOYO-1, TOTO-1 etc) used for

                                                                                   cell cycle analysis. Fix and permeabilisation procedure can be use for staining. 

                                                                                  

                                                                                   

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                             cell cycle

 

 

 

                                                                                                                         DNA ploidy analysis for B-ALL case

 

 

 

 

 

 

 

 

 

 

 

 

 

 

              

Singlet population opened in CD45/CD34 plot and gated for blasts and lymphocytes. blast and lymphocytes are opened for cell cycle analysis. DNA Index is calculated by the Mean of G1 phase of Blasts and lymphocytes. DNA index of >= 1.16 and <= 1.6 is associated with hyperdiploidy. (Diploid- 2n number of chromosomes, Haploid- Half the normal 2n number of chromosomes, Hyperdiploid- More than the normal 2n chromosomes, Hypodiploid- Less than the normal 2n chromosomes, Tetraploid- Double the normal number of 2n chromosomes, Aneuploid- An abnormal number of chromosomes).

 

 

Cells should be kept as concentrated as possible to allow the lowest sample pressure differential to be used. This will ensure that the core sample stream is as narrow as possible and give optimal CVs. The CV, or coefficient of variation, is a measure of spread of the data and is defined as the standard deviation (sd) divided by the mean (m) expressed as a percentage (sd/m x 100)

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Reference:

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• http://flowbook.denovosoftware.com/chapter-6-dna-analysis

• Flowcytometry (ploidy determination, cell cycle analysis, DNA content per nucleus) Sergio J. Ochatt

• Cell Cycle and DNA Content Analysis Using the BD Cycletest Assay on the BD FACSVerse™ System Yibing Wang, Catherine McIntyre, and Dev Mittar BD Biosciences, San Jos

• Ross, J.S. 1996. DNA Content analysis. In: DNA Ploidy and Cell Cycle Analysis in Pathology. Igaku-Shoin, NY.

• Bagwell, C.B. 1993. Theoretical aspects of data analysis.In: Clinical Flow Cytometry Principles and Application.Ed Bauer KD, Duque RE and Shankey TV. p. 41-61.

• Rabinovitch, P.S. 1993. DNA analysis guidelines. In:Practical Considerations for DNA Content and Cell CycleAnalysis. Ed

• Bauer KD, Duque RE and Shankey TV. p.117-142.Bauer, K.D.,et al.1993. Cell cycle antigens. In: Guidelinesfor Implementation of Clinical DNA Cytometry. Vol. 14,No. 5. p. 472-477.

• Bauer, K.B., and Jacobberger, J.W. 1994. Analysis of intracellular proteins. In: Methods in Cell Biology: Flow Cytometry Vol 41, 2nd edition, eds. Z. Darzynkiewicz, H.A. Crissman, J. P. Robinson, Academic Press, Inc., NY, pp351-376.

• Srivastava, P., Sladek, T.L., Goodman, M.N., and Jacobberger, J.W. 1992. Streptavidin-based quantitative staining of intracellular antigens for flow cytometric analysis. Cytometry. 13: 711-720.

• Sladek, T.L., and Jacobberger, J.W. 1992. Dependence of SV40 large T-antigen cell cycle regulation on T-antigen expression levels. Oncogene. 7: 1305-1313