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Agilent Develops Method for Single-Cell Analysis on its CGH Microarrays

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Agilent Technologies now claims its comparative genomic hybridization arrays can be used for single-cell analysis.

Anniek De Witte, Agilent's technical marketing manager for cytogenetics, said the firm's approach could be used in "clinical research applications where there are only a handful of cells." For example, tumor samples are "so heterogeneous, that it is important to look at one cell at a time," she said.

De Witte spoke with BioArray News at the European Society of Human Genetics conference, held in Nuremberg, Germany, last week. During the interview, she also discussed Agilent's recent acquisition of Dako.

Agilent described the single-cell analysis application in a conference poster. According to the poster, while the ability to characterize single cells isolated from solid tumors or pre-implantation samples represents an "important advancement for scientific research of genomic imbalances," other methods, including fluoresence in situ hybridization, PCR, and bacterial artificial chromosome array-based CGH all suffer from various limitations.

In the case of FISH and PCR, the approaches can "only measure a limited number of genetic loci simultaneously." BAC arrays, meantime, "only contain a few thousand probes and are prone to batch-to-batch variation in performance," Agilent maintains in the poster. Agilent's solution was to develop a method that combines single-cell whole-genome amplification with copy number analysis employing its SurePrint G3 8x60K Oligo CGH Microarrays.

The whole-genome amplification step is performed using Rubicon Genomics' PicoPlex WGA Kit.

"We are recommending the catalog 8x60K array because it has very good genome-wide coverage," De Witte said. She said that the firm does not recommend using a higher-resolution array because of the bias introduced with whole-genome amplification.

"Because you are starting from a number of single cells, the quality of the copy number will not be as good as what [you] are used to seeing when [you] start with higher amounts of DNA, so it doesn't make sense to go to a higher-resolution array," said De Witte. "Also it allows you to run eight samples at a time, so basically one can run eight cells at a time" on the array, she added.

To establish proof of principle, Agilent assayed the copy number difference between a reference sample and a test sample with a known aberration, each using amplified DNA that was diluted to single-cell levels. The firm then visualized the expected aberrations using its CytoGenomics software, according to the poster. Agilent then assayed the genomic aberrations in single cells biopsied from embryos, in which it not only detected whole-chromosome losses or gains, but also found smaller aberrations of portions of chromosome arms.

According to Agilent, this ability to detect abnormalities involving any of the 24 chromosomes following a 24-hour workflow "represents a major advantage over FISH and PCR-based methods" and "offers new possibilities for research on genetic analysis of single cells." De Witte added that the company already has a number of customers who are using the method and is collaborating with customers on projects as well.

Along with Agilent's new application, there are a number of array and similar platforms available for single-cell analysis. BlueGnome, for example, manufactures a bacterial artificial chromosome array for preimplantation genetic diagnosis called 24sure (BAN 5/28/2012). Oxford Gene Technology has provided stem cell researchers with cell arrays manufactured using an internally designed printer (BAN 5/27/2008). And Ambry Genetics sells its own CGH array for stem cell research, though Ambry's chip is manufactured by Agilent (BAN 1/19/2010).

Dako

Agilent's microarray business now falls under a new business segment within Agilent called the Diagnostics and Genomics Group. The segment contains Agilent's Genomic Solutions division and Dako, the Danish diagnostics firm that Agilent acquired earlier this month for $2.2 billion in cash. Agilent's array business formerly was part of the company's Life Sciences segment. Agilent has two other segments: Chemical Analysis and Electronic Measurement.

Through its buy of Dako, Agilent has gained the firm's immunohistochemistry products and a growing companion diagnostics business. According to De Witte, that could help the company's Genomics Solutions division, including its array business, in the future, as Dako has a "large regulatory department" and "a lot of experience" with submitting tests to the US Food and Drug Administration for clearance.

At the same time, she said that the Dako acquisition will not impact Agilent's own plans to have its array platform cleared for use in clinical constitutional cytogenetics.

"We have been in dialog with the FDA for quite some time now, long before the Dako acquisition," De Witte said.

She described the submission process as "positive," but declined to provide a timeline for when Agilent might submit or achieve clearance.

Affymetrix, Agilent's competitor in the cytogenetics market, last week affirmed its attention to submit its CytoScan HD product to the FDA for clearance by the end of this year (BAN 6/26/2012).

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