Cytometry

In tumour pathology, cytometric study consists in quantifying shape and/or density parameters for various markers like DNA content or proteins related to cell proliferation.

Two techniques may be used: flow cytometry and cytometry through image analysis.

Flow cytometry studies the cell cycle. Normal cell cycle includes 4 phases:

• phase G1: differentiated cells, under the influence of external signals and neighbouring cells, carry out all the specific tasks, according to their programming. During this period involving intense protein synthesis, DNA stock remains stable, related to 2N chromosomes.

• phase S: cells duplicate their DNA content which progressively increases from 2N to 4N at the end of phase S.

• phase G2: cells synthesise the proteins required for mitosis, for example tubulin necessary for the mitotic spindle.

• phase M: or mitotic phase during which a condensation of DNA occurs in 23 chromosome pairs, then a balanced distribution of chromosomes is made along the mitotic spindle leading to the formation of two daughter cells.

In opposition to these 4 phases related to cell cycle and cell division, there is another phase: phase G0 (with more or less the same characteristics as phase G1). The cell is either in a postmitotic transitory state (stem cells that can be recruited for division, such as lymphocytes) or in a permanent state (differentiated cells whose fate is programmed cell death or apoptosis, such as differentiated intestine cells or neurones).

During flow cytometry, cells are dissociated and labelled with a fluorescent dye (propidium iodine) in order to be counted by a laser beam. Optical signals are sensed by multiple photomultiplicators then converted into digital signals. More than 10,000 cells are analysed allowing the quantitative analysis of DNA, and the calculation of the different cell cycle phases using sophisticated software.

Results are visualized as histograms.

DIfferent histograms may be seen in tumours:

• diploid histogram - normal cell histogram (2N). Malignant cells may be diploid,

• hypoploidy - presence of a peak before 2N concerning more than 10 % of total cells,

• aneuploidy - presence of a different 2N peak concerning more than 10 % of total cells,

• tetraploidy - presence of a peak at position 4N concerning more than 15 % of total cells,

• multiploidy - presence of multiple 2N peaks.

Example of cytometry peaks

Cytometry with image analysis allows a quantification of DNA content from cellular nuclei which are dissociated and spread on slides, then stained with Feulgen dye. After automatic image scanning, 200 to 1,000 nuclei may be analysed by software for ploidy study.

Image analysis unit
Dissociated cells
Quantitative analysis of DNA content
Automatic distribution into cell cycle.

Image analysis may now be used in pathology to help the pathologist, offering the precise study of mathematical fractions (such as the percentage of receptor positive factors).