Banner-TopLeft Banner-Mid Search barrettsinfo
Home Barrett's Basics Barrett's and Cancer Frequently Asked Question Print/Download

What is flow cytometry?

Flow Cytometry is a test that can be used to measure the amount of DNA in cells. By measuring the amount of DNA in cells, this test is able to identify the proportions of cells in different parts of the cell cycle (the growth cycle of a cell). It can also detect populations of cells that have abnormal amounts of DNA (cells that have a lot of gene abnormalities). In the past, flow cytometry has primarily been a research tool in the study of cancer and other conditions including Barrett's esophagus. This test is now widely used in the characterization of many different human cancers, with the information used by physicians to determine how well a cancer may respond to a particular therapy. At the present time, flow cytometry is not widely used to clinically manage patients with Barrett's esophagus.

To perform flow cytometry on an endoscopic biopsy, when the biopsy is removed from the esophagus it needs to be placed immediately into a special solution that protects the cells from breaking apart, and then frozen. What happens to the biopsy next depends on the type of flow cytometry performed. DNA content flow cytometry is the most commonly performed test in characterizing cancers and has been the test most commonly used in Barrett's esophagus.

DNA content flow cytometry

When DNA content flow cytometry is performed, the frozen biopsy is thawed and placed in a solution that ruptures the cells, leaving only the nuclei (the component of the cell that contains the DNA). The nuclei are stained with a fluorescent dye that binds to the DNA of the nuclei. The solution of stained nuclei is placed into a machine called a flow cytometer that has a focused light source, typically a laser, that excites the fluorescent dye bound to the nuclear DNA causing it to fluoresce (emit visible light). Because the  fluorescent dye is bound to the DNA in the nucleus of the cell, the intensity or brightness of the cell's fluorescence is proportional to the amount of DNA in the cell (the greater the amount of DNA, the greater the intensity of the fluorescence). Because cells contain different amounts of DNA depending on where they are in the cell cycle, it can thus be determined what percentage of cells are in the different parts of the cell cycle based on the intensity of fluorescence of the nuclei. The data are expressed as a flow cytometric histogram.

A typical flow cytometric histogram in Barrett's esophagus

A typical flow cytometric histogram in Barrett's esophagus

Most of the cells have a DNA content of 2N, seen here as a large peak on the flow histogram. The 4N peak is much smaller and in this case makes up only 3.4% of the cell cycle.

Normally, in a biopsy from Barrett's tissue, most of the cells are in the G0/G1 phase of the cell cycle and have a 2N DNA content, the content of most of the normal cells of our bodies. Normally, in a biopsy from Barrett's tissue, 6% or less of the cells have a 4N DNA content (twice the DNA of the G0/G1 cells).

Flow cytometric abnormalities

In Barrett's esophagus, flow cytometry can be used to identify patients who are at low- or high- risk for progression to high-grade dysplasia or cancer. Several studies have shown that in Barrett's esophagus, flow cytometric abnormalities can occur in patients BEFORE high-grade dysplasia or cancer develops. One study showed that patients who had 4N populations greater than 6% of the total cell cycle, or aneuploid cell populations (abnormal populations of cells with DNA content between 2N and 4N), were at increased risk of developing high-grade dysplasia or cancer. Another study in which patients were followed for a long time showed similar results. In this study, patients who did not have aneuploidy were in a low-risk group compared to those who had aneuploidy.

The most recent, and largest, flow cytometry study is one of more than 300 patients who had Barrett's esophagus followed for up to 13 years. In this study, patients had both flow cytometry and histology performed on their endoscopic biopsies and then were followed in the study. The chance of developing cancer over a 5 year period was 0% in patients who had biopsy results of negative, indefinite or low-grade dysplasia and normal flow cytometry upon entry into the study. However, the chance of developing cancer over a 5 year period was 28% in patients who had biopsy results of negative, indefinite or low-grade dysplasia and increased 4N fractions or aneuploid cells by flow cytometry at the time of entry into the study. In the group of patients with high-grade dysplasia, when they entered the study, the chance of developing cancer over a 5-year period was 59% regardless of their flow cytometry result, so having a diagnosis of high-grade dysplasia was an independent risk factor for developing cancer in this particular study. Based on these study results, for patients who DO NOT have high-grade dysplasia, flow cytometry is more useful than histologic analysis in separating those patients who have a low-risk of progression to cancer from those who have a much higher risk of progressing to cancer. Because not all patients who have high-grade dysplasia or flow cytometric abnormalities develop cancer, other biologic measurements or biomarkers are needed to better predict which patients in these groups will ultimately develop cancer.

Another, more recent, flow cytometry study of the same Barrett's esophagus study group above further characterized flow cytometric abnormalities and looked at the percentage of cells in the S phase fraction of the cell cycle as a predictor of who will get cancer in Barrett's esophagus. What this study found is that increased S phase fraction is NOT an independent predictor of who will develop cancer in Barrett's esophagus so is not helpful in determining patient risk. This study also divided aneuploid cell populations by DNA content and found that, of the 11 patients who had what is called a "near-diploid aneuploid cell population (a DNA content of less than 2.7N), the 5-year cancer risk was zero (none of these patients developed cancer). The numbers of patients who had aneuploid cell populations with DNA contents of less than 2.7N are too small to draw any definite conclusions and many more patients will need to be followed to confirm whether these patients' risk of cancer is much lower compared to those who have aneuploid cell populations with DNA contents of greater than 2.7N. Most patients who have Barrett's esophagus and who develop aneuploid cell populations have populations of greater than 2.7N DNA content.

Increased 4N cells in Barrett's esophagus

Increased 4N cells in Barrett's esophagus

There is an increased percentage (16.7%) of cells in this biopsy with a DNA content of 4N.

Aneuploidy in Barrett's esophagus

Aneuploidy in Barrett's esophagus

A large aneuploid (cells with abnormal DNA content) peak with a DNA content of 2.8N can be seen just to the right of the diploid (2N) peak. The aneuploid cell population makes up 78% of the cells in this Barrett's biopsy.

Clinical use of flow cytometry

Clinical guidelines have been developed for DNA content flow cytometric analysis. There is extensive literature on decreasing the causes of variation (disagreement in the analysis of results) among laboratories that perform flow cytometry. Recent studies have shown flow cytometry to be consistent from laboratory to laboratory when the guidelines are followed. For example, one large study found 94% agreement among laboratories in the interpretation of DNA content flow cytometry. There is now good evidence that flow cytometry is a more objective test than histologic analysis of biopsies. However, flow cytometry should be performed in a reference laboratory or at a center experienced in DNA content flow cytometry.

At the present time, treatment is not recommended based solely on flow cytometric abnormalities as many patients who have flow cytometric abnormalities DO NOT progress to high-grade dysplasia or cancer during long-term follow-up. However, flow cytometry may be clinically useful in separating patients who do NOT have a diagnosis of high-grade dysplasia into those who need more frequent endoscopic biopsy surveillance from those who need much less frequent surveillance.

Current page: What is flow cytometry?
Next Page: What are the treatments options for high-grade dysplasia in Barrett's?

Section References

nfl jerseysray ban sunglassestiffany jewelrymac cosmeticsrosetta stoneJordan shoes
Coach handbagsJordan shoesCHI flat iron
horizontal line
Home | Site Map | References | Site Developers / Reviewers | Site Disclaimer | Feedback
this site last reviewed and updated 1/31/2008 © 2008